Anticonvulsant effects of omega-3 polyunsaturated fatty acids in rodents By Ameer … · 2013-09-27 · Ameer Y. Taha Department of Pharmacology and Toxicology University of Toronto

Anticonvulsant effects of omega-3 polyunsaturated fatty acids in rodents

By

Ameer Y. Taha

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Pharmacology and Toxicology University of Toronto

© Copyright by Ameer Taha 2009

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Anticonvulsant effects of omega-3 polyunsaturated fatty acids in rodents

Ameer Y. Taha

Department of Pharmacology and Toxicology University of Toronto

Doctor of Philosophy, 2009

ABSTRACT

The present research examined the hypothesis that omega-3 polyunsaturated fatty

acids would increase seizure threshold in rats in vivo, and reduce neuronal excitability in

mouse hippocampal slices. Seizure thresholds were measured in rats using the maximal

pentylenetetrazol and electrical stimulation seizure tests following α-linolenic acid (ALA)

or docosahexaenoic acid administration. ALA raised seizure threshold in the maximal

PTZ seizure test, but this effect probably occurred because ALA displaced DHA from

liver to the brain. DHA itself was therefore tested in the PTZ and electrical stimulation

seizure tests. Direct administration of DHA by subcutaneous injection raised seizure

thresholds in the PTZ seizure test, which models tonic-clonic attacks in humans. Dietary

enrichment with DHA raised afterdischarge seizure thresholds in the cortex and

amygdala, which model simplex and complex partial seizures in humans, although this

effect took some time to occur. In vitro, the application of DHA also reduced the

incidence of excitatory sharp waves in mouse hippocampal slices. This effect did not

appear to be due to either an increase in GABAergic inhibitory tone, nor to a decrease in

glutamatergic drive. The fatty acid composition of phospholipids and unesterified fatty

acids were measured in the brain following microwave fixation in order to determine

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whether the effects of DHA on seizure thresholds were due to its de-esterification from

the phospholipid membrane. The assay surprisingly revealed that subcutaneous

administration of DHA at a dose that raised seizure threshold, increased unesterified

arachidonic acid, but not unesterified DHA concentrations during seizures. The results of

these studies support the hypothesis that DHA raises seizure threshold in rats, and

reduces neuronal excitability in vitro. The effects of DHA on seizure threshold are

possibly mediated by the de-esterification of arachidonic acid, which is known to have

effects on the voltage-dependent sodium channel.

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Acknowledgements

I would like to extend my gratitude and utmost appreciation and admiration to my

supervisor, Dr. W. McIntyre Burnham, for his support, dedication and scientific rigour.

Working with him over the years has been an honour, and an intellectually rewarding

pleasure. My gratitude also extends to our collaborator and colleague, Dr. Richard

Bazinet for his constructive criticism, help and support of this work. Dr. Liang Zhang is

also thanked for his feedback and pivitol role in the electrophysiology project presented

in the appendix.

I am also grateful to my committee members, Drs. Richard Bazinet, Elizabeth

Donner, Carol Greenwood and Jane Mitchell for their insights, invaluable feedback and

contructive criticism. Their guidance helped shape this thesis.

Special thanks to all the lab members and project students who have assisted with

the projects. In particular, Marc-Olivier Trepanier, Brian Scott and Flaviu Ciobanu were

instrumental in their advice and technical assistance with several projects.

The Canadian Institutes of Health Research (Fredrick Banting and Charles Best

Canada Graduate Scholarships) and the Department of Pharmacology (University of

Toronto) are thanked for funding my work over the years.

Finally, I would like to acknowledge my family and friends for their

unconditional support. This thesis is dedicated to my beloved parents, Mohamed Yassin

Taha and Sadia Abdul Rahman.

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Table of contents 1 Literature review......................................................................................................... 2

1.1 Overview............................................................................................................. 2 1.2 Epilepsy............................................................................................................... 3 1.3 Causes of Epilepsy.............................................................................................. 4 1.4 Types of seizures................................................................................................. 5

1.4.1 Partial seizures ............................................................................................ 5 1.4.2 Generalized seizures ................................................................................... 6

1.5 Therapy for epilepsy ........................................................................................... 6 1.5.1 Anti-seizure drugs....................................................................................... 7 1.5.2 Surgery........................................................................................................ 8 1.5.3 Ketogenic diet ........................................................................................... 10 1.5.4 Vagus nerve stimulation ........................................................................... 12

1.6 Drawbacks of current treatment options ........................................................... 14 1.7 Alternative treatment for epilepsy .................................................................... 14 1.8 About fatty acids ............................................................................................... 15

1.8.1 Definition and structure ............................................................................ 15 1.8.2 Types of PUFA ......................................................................................... 17 1.8.3 Sources of PUFA ...................................................................................... 19

1.9 Mechanisms of fatty acid uptake by the gut and transport to the brain ............ 21 1.9.1 Physiological components ........................................................................ 22 1.9.2 Lipid carrier molecules ............................................................................. 22 1.9.3 Lipid molecules that bind to the carrier proteins ...................................... 23 1.9.4 Pathways involved in fatty acid absorption, transport and uptake............ 24 1.9.5 Uptake of fatty acids by the brain ............................................................. 28 1.9.6 PUFA incorporation into the brain ........................................................... 29

1.10 Biological role of PUFA in the brain................................................................ 31 1.10.1 Role of AA in the brain............................................................................. 32 1.10.2 Role of DHA in the brain.......................................................................... 33

1.11 Behavioral effects of n-3 PUFA in vivo............................................................ 36 1.11.1 Learning and memory ............................................................................... 36 1.11.2 Anxiety...................................................................................................... 38 1.11.3 Mood - aggression..................................................................................... 38 1.11.4 Mood - depression..................................................................................... 38

1.12 N-3 PUFA and epilepsy .................................................................................... 39 1.12.1 Antiarrhythmic effects .............................................................................. 39

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1.12.2 Possible anticonvulsant effects of the n-3 PUFA – in vitro studies.......... 40 1.12.3 Anticonvulsant effects of the n-3 PUFA – animal studies........................ 41 1.12.4 Anticonvulsant effects of the n-3 PUFA – clinical trials.......................... 42

1.13 Unanswered questions ...................................................................................... 42 1.14 Hypothesis and objectives................................................................................. 46 1.15 Animal models used to test the anticonvulsant effects of n-3 PUFA ............... 46

2 Experiment 1: Lack of benefit of linoleic and α-linolenic polyunsaturated fatty acids on seizure latency, duration, severity or incidence in rats ................................................ 49

2.1 Abstract ............................................................................................................. 52 2.2 Introduction....................................................................................................... 53 2.3 Materials and methods ...................................................................................... 54

2.3.1 SR-3 preparation ....................................................................................... 54 2.3.2 Subjects and treatments............................................................................. 55 2.3.3 Seizure testing........................................................................................... 55 2.3.4 Fatty acid analysis..................................................................................... 56 2.3.5 Statistical analysis..................................................................................... 57

2.4 Results............................................................................................................... 58 2.4.1 Body weights ............................................................................................ 58 2.4.2 Fatty acid profile of SR-3 constituents ..................................................... 58 2.4.3 Seizure latency .......................................................................................... 58 2.4.4 Seizure duration ........................................................................................ 58 2.4.5 Seizure severity......................................................................................... 59 2.4.6 Seizure incidence within each seizure category........................................ 59

2.5 Discussion......................................................................................................... 60 2.6 References......................................................................................................... 68

3 Experiment 2: Dose-dependent anticonvulsant effects of linoleic and α-linolenic polyunsaturated fatty acids on pentylenetetrazol induced seizures in rats ....................... 72

3.1 Abstract ............................................................................................................. 76 3.2 Introduction....................................................................................................... 77 3.3 Materials and methods ...................................................................................... 79

3.3.1 SR-3 preparation ....................................................................................... 79 3.3.2 Subjects and treatments............................................................................. 79 3.3.3 Seizure testing........................................................................................... 81 3.3.4 Brain lipid analysis ................................................................................... 82 3.3.5 Fatty acid composition of the SR-3 compound......................................... 83 3.3.6 Fatty acid methyl ester analysis by gas-chromatography ......................... 83

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3.3.7 Data presentation and statistical analysis.................................................. 84 3.4 Results............................................................................................................... 85

3.4.1 Fatty acid profile of the SR-3 constituents ............................................... 85 3.4.2 Body weights ............................................................................................ 85 3.4.3 Food intake................................................................................................ 86 3.4.4 Possible physiological signs of toxicity – Liver weight and percent liver of body weight............................................................................................................... 86 3.4.5 Seizure occurence ..................................................................................... 86 3.4.6 Seizure latency .......................................................................................... 87 3.4.7 Seizure severity......................................................................................... 87 3.4.8 Brain phospholipid fatty acid composition ............................................... 88 3.4.9 Brain unesterified fatty acid composition ................................................. 88 3.4.10 Correlation between seizure latency and n-3 PUFA levels within the unesterified fatty acid fraction .................................................................................. 89

3.5 Discussion......................................................................................................... 89 3.6 References....................................................................................................... 103

4 Experiment 3: Assessing the metabolic and toxic effects of anticonvulsant doses of polyunsaturated fatty acids on the liver in rats ............................................................... 109 Forward ........................................................................................................................... 109

4.1 Abstract ........................................................................................................... 113 4.2 Introduction..................................................................................................... 114 4.3 Materials and methods .................................................................................... 117

4.3.1 Measurements taken................................................................................ 117 4.3.2 SR-3 preparation ..................................................................................... 117 4.3.3 Animals and treatments........................................................................... 118 4.3.4 Euthanasia and liver harvesting .............................................................. 119 4.3.5 Liver lipid analysis.................................................................................. 119 4.3.6 Fatty acid methyl ester analysis by gas-chromatography ....................... 120 4.3.7 mRNA expression analysis by quantitative real time PCR .................... 121 4.3.8 Data presentation and statistical analysis................................................ 122

4.4 Results............................................................................................................. 123 4.4.1 Liver concentrations of TL, PL and TG (expressed as mg per g of liver tissue) …………………………………………………………………………..123 4.4.2 Liver absolute levels of TL, PL and TG (expressed as mg) ................... 123 4.4.3 HMG-CoA lyase mRNA expression ...................................................... 125 4.4.4 Catalase mRNA expression .................................................................... 125

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4.4.5 GST A1 and A4 mRNA expression........................................................ 125 4.5 Discussion....................................................................................................... 126 4.6 References....................................................................................................... 134

5 Experiment 4: Acute administration of docosahexaenoic acid increases resistance to pentylenetetrazol-induced seizures ................................................................................. 140 Forward ........................................................................................................................... 140


5.3.1 Drug preparation ..................................................................................... 145 5.3.2 Subjects ................................................................................................... 146 5.3.3 Seizure tests and sedation scoring in Experiment 3................................ 147 5.3.4 Assays in Experiment 3 .......................................................................... 148 5.3.5 Plasma total lipid analysis in Experiment 3............................................ 149 5.3.6 Brain phospholipid and unesterified fatty acid analysis in Experiment 3 …………………………………………………………………………..149 5.3.7 Fatty acid methyl ester analysis by gas-chromatography in Experiment 3 …………………………………………………………………………..150 5.3.8 Data presentation and statistical analysis................................................ 151

5.4 Results............................................................................................................. 151 5.4.1 Experiment 1........................................................................................... 151 5.4.2 Experiment 2........................................................................................... 152 5.4.3 Experiment 3........................................................................................... 153

5.5 Discussion....................................................................................................... 155 5.6 References....................................................................................................... 166

6 Experiment 5: Dietary fish oil supplementation elevates seizure threshold in the cortex and amygdala of rats ............................................................................................ 171 Forward ........................................................................................................................... 171


6.3.1 Subjects and treatments........................................................................... 179 6.3.2 Procedure for surgery.............................................................................. 180 6.3.3 Afterdischarge threshold and seizure score measurements ................... 180 6.3.4 Diets and diet administration .................................................................. 182 6.3.5 Body weight and food intake measurements .......................................... 183

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6.3.6 Sacrifice and tissue fixation .................................................................... 184 6.3.7 Histological confirmation of electrode placement.................................. 184 6.3.8 Dietary fatty acid analysis....................................................................... 185 6.3.9 Data presentation and statistical analysis................................................ 185

6.4 Results............................................................................................................. 186 6.4.1 Fatty acid composition of the diets ......................................................... 186 6.4.2 Body weight gain .................................................................................... 187 6.4.3 Food intake.............................................................................................. 187 6.4.4 Dietary fish oil supplementation raises seizure threshold in the cortex and amygdala …………………………………………………………………………..187 6.4.5 Dietary fish oil supplementation does not alter seizure score................. 188


7 Experiment 6: Seizures increase unesterified arachidonic acid but not unesterified docosahexaenoic acid concentrations in the micro-wave fixated brain of rats............... 208 Forward ........................................................................................................................... 208


7.3.1 Drug preparation ..................................................................................... 214 7.3.2 Subjects ................................................................................................... 214 7.3.3 Brain phospholipid and unesterified fatty acid analysis ......................... 217 7.3.4 Fatty acid methyl ester analysis by gas-chromatography ....................... 218 7.3.5 Data presentation and statistical analysis................................................ 219

7.4 Results............................................................................................................. 219 7.4.1 DHA delays latency to seizure onset (Experiment 1)............................. 219 7.4.2 DHA delays latency to seizure onset (Experiment 2)............................. 220 7.4.3 Unesterified AA concentrations increase during seizures, particularly in DHA-treated subjects (Experiment 2) .................................................................... 221 7.4.4 Unesterified DHA concentration decrease pre-seizure, regardless of fatty acid pre-treatment (Experiment 2) .......................................................................... 222 7.4.5 Phospholipid-bound AA and DHA concentrations are not altered by fatty acid treatment or seizures (Experiment 2) .............................................................. 222


8 Discussion............................................................................................................... 237

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8.1 General discussion .......................................................................................... 237 8.2 Future studies .................................................................................................. 246 8.3 Collected hypotheses related to future studies................................................ 250 8.4 Clinical relevance............................................................................................ 251 8.5 Conclusions..................................................................................................... 251

9 References............................................................................................................... 254 10 Appendix 1: Assessing the anti-seizure effects of eicosapentaenoic acid in rats 277

10.1 Background..................................................................................................... 277 10.2 Methods........................................................................................................... 277 10.3 Results............................................................................................................. 278 10.4 Discussion....................................................................................................... 279 10.5 References....................................................................................................... 282

11 Appendix 2 (the following manuscript has not been submitted for publication) 284 11.1 Abstract ........................................................................................................... 285 11.2 Introduction..................................................................................................... 286 11.3 Materials and methods .................................................................................... 287

11.3.1 Drugs and solutions................................................................................. 288 11.3.2 Procedure for obtaining thick hippocampal slices .................................. 288 11.3.3 Extracellular recordings .......................................................................... 289 11.3.4 DHA composition of slices..................................................................... 290 11.3.5 Data analyses .......................................................................................... 291

11.4 Results............................................................................................................. 292 11.4.1 DHA and CBZ, but not NPD-1, reduced the incidence of hippocampal SPWs …………………………………………………………………………..292 11.4.2 DHA does not alter the incidence of inhibitory, spontaneous rhythmic filed potentials (SRFPs) .......................................................................................... 293 11.4.3 Effect of DHA and CBZ on population field EPSPs .............................. 293 11.4.4 DHA is incorporated into the phospholipid membrane .......................... 294


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Table of tables

Chapter 1 Table 1: Clinical efficacy and side-effects of treatment options for epilepsy .................. 14 Chapter 2 Table 1: Percentage of rats experiencing stage 1, stage 2, stage 3, stage 4 and stage 3+4 in control and SR-3 groups ................................................................................................... 67 Chapter 3 Table 1: Brain phospholipid fatty acid composition, expressed as a percentage of total fatty acids, within the phospholipid pool ....................................................................... 101 Table 2: Brain unesterified free fatty acid composition, expressed as a percentage of total fatty acids, within the free fatty acid lipid pool ............................................................. 102 Chapter 4 Table 1: Fatty acid levels in liver total lipids ................................................................. 131 Table 2: Fatty acid levels in liver phospholipids ........................................................... 132 Table 3: Fatty acid levels in liver triglycerides .............................................................. 133 Chapter 6 Table 1: Fatty acid composition of the AIN-93G control and fish oil experimental diets (mg per g of diet) ........................................................................................................... 202 Chapter 8 Table 1: Summary of key findings of Chapters 2 to 7 and appendices 1 and 2 ............. 252 Table 2: Summary of measured changes in PUFA levels in phospholipids and unesterified fatty acids ................................................................................................... 252

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Table of figures Chapter 1 Figure 1: Structure of fatty acids ...................................................................................... 16 Figure 2: Structure of omega-3 and omega-6 fatty acids.................................................. 18 Figure 3: The n-3 and n-6 PUFA synthetic pathways....................................................... 21 Figure 4: Modulation of brain tissue PUFA composition by diet..................................... 30 Chapter 2 Figure 1: Body weight gain over time .............................................................................. 63 Figure 2: Seizure latency .................................................................................................. 64 Figure 3: Seizure duration................................................................................................. 65 Figure 4: Seizure severity ................................................................................................. 66 Chapter 3 Figure 1: Effect of treatment on body weight gain ........................................................... 95 Figure 2: Effect of treatment on food intake..................................................................... 96 Figure 3-A: Liver weight ................................................................................................. 97 Figure 3-B: % liver of total body weight ......................................................................... 97 Figure 4: Seizure latency following PTZ administration.................................................. 99 Figure 5: Correlation between seizure latency and brain n-3 PUFA composition ......... 100 Chapter 5 Figure 1-A: Latency to the onset of myoclonic jerks over time in rats treated with OA or DHA (400 mg/kg) Effect of treatment on body weight gain .......................................... 160 Figure 1-B: Latency to the onset of tonic-clonic seizures over time in rats treated with OA or DHA (400 mg/kg) Effect of treatment on body weight gain............................... 161 Figure 2-A: Sedation scores of subjects that were seizure tested 1-hour following drug administration ................................................................................................................. 161 Figure 2-A: Sedation scores of subjects that were decapitated 1-hour following drug administration ................................................................................................................. 162 Figure 3-A: DHA concentrations in brain phospholipids following saline, OA or DHA (400mg/kg) subcutaneous injections............................................................................... 164 Figure 3-B: DHA concentrations in brain unesterified fatty acids following saline, OA or DHA (400mg/kg) subcutaneous injections..................................................................... 165 Chapter 6 Figure 1: Study design .................................................................................................... 193 Figure 2-A: Body weight gain over time in cortex-implanted subjects.......................... 195

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Figure 2-B: Body weight gain over time in amygdala-implanted subjects .................... 196 Figure 3-A: Change in ADT over time in cortex-implanted subjects............................. 197 Figure 3-B: Change in ADT over time in amygdala-implanted subjects ....................... 198 Figure 4-A: Seizure score over time in cortex-implanted subjects................................. 200 Figure 4-B: Seizure score over time in amygdala-implanted subjects ........................... 201 Chapter 7 Figure 1-A: Latency to the onset of myoclonic jerks following OA or DHA subcutaneous injections ......................................................................................................................... 227 Figure 1-B: Latency to the onset of tonic-clonic seizures following OA or DHA subcutaneous injections .................................................................................................. 228 Figure 2-A: Brain unesterified AA concentrations following OA, AA or DHA in saline or PTZ treated rats............................................................................................................... 229 Figure 2-B: Brain unesterified DHA concentrations following OA, AA or DHA in saline or PTZ treated rats .......................................................................................................... 230 Appendix 1 Figure 1-A: Effect of acute EPA administration on the latency to myoclonic jerks ...... 281 Figure 1-B: Effect of acute EPA administration on the latency to tonic-clonic seizures 281 Appendix 2 Figure 1-A: Effect of DHA on the incidence of SPWs................................................... 298 Figure 1-B: Effect of CBZ on the incidence of SPWs.................................................... 299 Figure 1-C: Effect of NPD-1 on the incidence of SPWs ................................................ 300 Figure 1-D: Representation of CA3 SPWs from a slice before and after 100 μM of DHA treatment ......................................................................................................................... 300 Figure 2: Effect of DHA on the incidence of SRFPs...................................................... 303 Figure 3: Effect of DMSO and DHA on the ratio of DHA to AA in phospholipids isolated from brain slices ............................................................................................................. 304

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List of abbreviations

ALA: Alpha-linolenic acid

AA: Arachidonic acid

CBZ: Carbamazepine

CMs: Chylomicrons

DHA: Docosahexaenoic acid

EPA: Eicosapentaenoic acid

EEG: Electroencepharlogram

FAMEs: Fatty acid methyl esters

GABA: Gamma-Aminobutyric acid

GC: Gas-chromatography

HDLs: High-density lioproteins

ICV: Intracerebroventivular

IP: Intraperitoneal

IV: Intravenous

LDLs: Low-density lipoproteins

MES: Maximal electroconvulsive shock

Mg: Milligrams

Ml: Millilitres

NPD1: Neuroprotectin D1

n-3 PUFA: Omega-3 polyunsaturated fatty acids

PTZ: Pentylenetetrazol

PUFA: Polyunsaturated fatty acids

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RD1: Resolvin D1

SPWs: Sharp Waves

SC: subcutaneous

VDSC: Voltage-dependent sodium channels

VLDLs: Very low-density lipoproteins

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CHAPTER 1

LITERATURE REVIEW

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1 Literature review

1.1 Overview

Section 1.1 presents a general overview of the topics covered in this literature

review. Subsequesnt sections will provide details and references.

Epilepsy is a serious neurological disorder that involves spontaneous, recurrent

seizures. It affects about one in a hundred individuals worldwide. People with

uncontrolled epilepsy often face lives of social and economic hardship, and are at a

higher risk of developing seizure-related psychiatric co-morbidities or dying prematurely.

Epilepsy-related seizures can be controlled by anti-seizure drugs, surgery, vagus

nerve stimulation or the high-fat ketogenic diet. Seizure control is achieved in

approximately 70% of the patients treated with anti-seizure drugs. The remaining 30% of

patients are said to have “drug-resistant” or “intractable” epilepsy. These patients are

treated by surgically removing the seizure focus (if one exists), by vagus nerve

stimulation or with the high-fat ketogenic diet. The efficacy of these treatments, however,

ranges between 40-70%. Despite the best anticonvulsant therapy, one in ten individuals

with epilepsy continues to live with uncontrolled seizures.

Even individuals who respond to anti-seizure therapies, however, often achieve

seizure freedom at a considerable cost. The anti-seizure medications, for instance, often

produce side-effects such as sedation and gastro-intestinal problems. New, safe and more

tolerable therapies are therefore needed for the treatment of people with epilepsy.

Omega-3 polyunsaturated fatty acids (n-3 PUFA) are dietary fatty acids that have

been proposed to have anti-seizure effects. They are structural components of neuronal

membranes that are involved in neurotransmission, cell signaling and gene regulation in

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the brain. They have no reported side-effects, even at high doses, which suggests that – if

therapeutically successful - they might provide seizure control without toxicity.

The following review will provide background information on the nature, types,

causes and treatment of epilepsy, and on the limitations of the current treatment options

(sections 1.2 to 1.7). Because this thesis will address the role of n-3 PUFA in modulating

seizure threshold, the general properties of fatty acids, and the physiological and

biochemical processes involved in regulating their incorporation into the brain will be

reviewed (sections 1.8 to 1.9). This will be followed by an overview of the biochemical

and behavioral effects of n-3 PUFA (sections 1.10 to 1.11), with a particular focus on

studies that have examined their possible anti-seizure effects (section 1.12).

1.2 Epilepsy

Epilepsy is a neurological disorder that is characterized by spontaneous and

recurrent seizures, which manifest as disruptions of mental function, consciousness and /

or motor activity (Burnham, 2007). A seizure is a brief episode of self-sustained neuronal

hyperexcitation. A person is diagnosed with epilepsy after experiencing two or more

seizures. Epilepsy affects approximately 1% of the population (Annegers et al., 1999;

Theodore et al., 2006), although a single seizure episode occurs in up to 10% of the

population (Burnham, 2007).

A seizure may be convulsive or non-convulsive. A “convulsive” seizure involves

behavioural muscle spasms, whereas a “non-convulsive”seizure occurs without muscle

spasms, although there may be other disturbances in behaviour (ILAE, 1981). Both

convulsive and non-convulsive seizures, however, involve brain hyperexcitability, which

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can be detected in electroencephalographic (EEG) recordings (ILAE, 1981).

1.3 Causes of Epilepsy

The basic pathophysiological problem in people with epilepsy is that they have a

constant, low seizure threshold in one or more parts of the brain (Abdelmalik et al., 2005;

Burnham, 2007). Therefore external stimuli, such as light or sound, or internal changes in

brain neurochemistry or electrical activity might provoke a seizure episode in these

patients (Burnham, 2007).

The causes for low seizure threshold may be described as symptomatic or

idiopathic (Luders et al., 2009). Seizures are described as symptomatic when there is a

clear-cut abnormality in the brain that is causing the seizures. Approximately 30-40% of

the patients with epilepsy have the symptomatic form, with a clear-cut brain abnormality

such as a scar, a neoplasm, a vascular anomaly, or an area of cortical dysplasia (Annegers,

1994; Engel, 1998). In the remaining 60-70% of cases of epilepsy, the brain appears to

be completely normal, and the seizures are described as idiopathic. Idiopathic epilepsy is

believed to have genetic causes (Annegers, 1994; Engel, 1998).

The causes of idiopathic epilepsies are multi-factorial, but broadly speaking, they

are thought to be caused by mutations in one or several genes involved in brain excitation

and / or inhibition. For instance mutations in the α-subunit of the voltage-dependent

sodium channel (Mulley et al., 2005; Okumura et al., 2007; Zucca et al., 2008) or a

subunit of the gamma-aminobutyric acid (GABA)A (Dibbens et al., 2009) receptor are

associated with epileptic seizures. Angelicheva et al. (2009) have recently discovered a

mutation on chromosome 5q (5q31.1-q32) that contains a yet-to-be identified gene

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associated with temporal lobe epilepsy in a Gypsy family (Angelicheva et al., 2009).

1.4 Types of seizures

Seizures are classified into types according to the extent of brain involvement in

epileptic activity (ILAE, 1981). A seizure that involves only a part of the brain is called a

partial or focal seizure. A seizure that involves both hemispheres (or the whole brain) is

called a generalized seizure (Engel, 1998). Genralized seizures can occur either without a

focal onset (primary generalized), or they can start at a specific focus and then spread to

the whole brain (secondary generalized) (Engel, 1998).

1.4.1 Partial seizures

Partial seizures can be classified into two disctinct types – simple partial and

complex partial. The patient is fully conscious during a simple partial seizure. He / she

may experience sensory symptoms, such as a flashing light, or contralateral convulsive

jerking if the seizure occurs in a motor area of the brain. Later, the patient will have full

memory for the period of the attack (ILAE, 1981).

During a complex partial seizure, the patient appears to be conscious, but

consciousness is impaired and the patient appears to be detached from the surrounding

environment (ILAE, 1981). Certain purposeless movements may be made (automatisms),

but convulsions do not occur. Later, the patient will have no memory for the period of the

attack. Complex partial seizures were formally called ‘temporal lobe’ seizures because

they often originate from limbic structures within the temporal lobe (Burnham, 2007).

A partial seizure evolving to secondary generalization refers to the spreading of a

partial seizure from the original focus to other parts of the brain (ILAE, 1981; Luders et

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al., 2009). For example, if a cortical simple partial seizure spreads or generalizes to the

temporal lobe, it may become a complex partial seizure. In other cases, a simple or a

complex partial seizure can spread to the whole brain, resulting in a secondary

generalized seizure. As will be discussed in the following section, generalized seizures

can be convulsive or non-convulsive.

1.4.2 Generalized seizures

There are two common types of generalized seizures, absence seizures and tonic-

clonic seizures (ILAE, 1981; Luders et al., 2009). Absence seizures are non-convulsive,

and are characterized by epileptiform activity that consists of three spike and wave

complexes per second on the electroencephalogram (EEG). Tonic-clonic seizures are

convulsive and involve stiffening (tonus) and jerking (clonus) of the whole body. They

are characterized by constant spiking in the EEG. Notably, consciousness is lost during

both sorts of generalized seizures, and the patient will later have no memory of the attack.

1.5 Therapy for epilepsy

Epilepsy may be associated with premature death (Hauser et al., 1980; Nilsson et

al., 1997; Nilsson et al., 1999; Walczak et al., 2001; Carpio et al., 2005; Forsgren et al.,

2005; Langan et al., 2005) and a higher risk of metabolic, hormonal, cognitive and

psychiatric co-morbidities (Kobau et al., 2004; Strine et al., 2005; Caplan et al., 2008;

Picot et al., 2008; Walpole et al., 2008). People with epilepsy are also likely to face lives

of economic and social hardship (O'Donoghue et al., 1999; Ronen et al., 2003; Elliott et

al., 2005). Seizure control is therefore necessary in order to improve the quality of life of

people with epilepsy.

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There are four treatment options for epilepsy patients (Shorvon, 1996; Brodie and

French, 2000; Brodie, 2001). These are anti-seizure drugs, brain surgery, the ketogenic

diet and vagus nerve stimulation. Anti-seizure drugs are the most common, and almost

always the first treatment option for epileptic seizures. Surgery is considered if the

seizures are non-responsive to anti-seizure medications and the epileptiogenic focus is

identified. The ketogenic diet and vagus nerve stimulation may also be used to treat drug-

resistant epilepsies. The ketogenic diet is commonly used in children, whereas vagus

nerve stimulation is used in children or adults.

1.5.1 Anti-seizure drugs

Anti-seizure drugs are the most common, and almost always the first treatment

option for epileptic seizures (Burnham, 2007). About 15-20 anticonvulsant drugs are

currently available (Bazil, 2002). While these drugs may have side effects, such as skin

rashes, naseau, sedation and memory loss (Hirsch et al., 2008; Arif et al., 2009), they

provide seizure control in about 70% of the cases of epilepsy (Kwan and Brodie, 2000).

The choice of an anticonvulsant primarily depends on the seizure type. A number

of additional factors, however, play a role in the choice of an anticonvulsant regimen.

These include safety, tolerability, the possibility of drug–drug interactions, the speed of

titration, the frequency of administration and the cost of treatment.

There are currently three well-established mechanisms for the anticonvulsant

drugs: 1) a number of agents, such as the barbiturates and benzodiazepines, decrease

neuronal excitability by enhancing the chloride flux associated with GABA-A receptors

(Bowser et al., 2002; Mercado and Czajkowski, 2008; Sharkey and Czajkowski, 2008);

2) anticonvulsants such as phenytoin and carbamazepine act as negative allosteric

8

modulators of the α-subunit of the voltage-gated sodium channel (Francis and Burnham,

1992); 3) drugs such as ethosiximude act as negative allosteric modulators of T-type

voltage-gated calcium channels (Meldrum and Rogawski, 2007).

Depsite the proven efficacy of anticonvulsant medications, they have considerable

limitations. The older drugs, such as phenytoin and sodium phenobarbital, have

numerous side-effects, including sedation, fatigue, dizziness and gastro-intenstinal upsets

(Zimmerman and Ishak, 1982; Carroll et al., 2001; Anderson, 2002). Less common side-

effects are skin rashes, hepatotoxicity, personality changes, cognitive deficits and

photophobia (Aman et al., 1987; Aman et al., 1990; Aman et al., 1994; Aihara et al.,

2001; Anderson, 2002; Hessen et al., 2007a; Hessen et al., 2007b). These effects are less

common with the drugs approved more recently (since 1992), such as gabapentin and

topimarate, but these are considerably more expensive (Bazil, 2002; Snead and Donner,

2007).

About 30% of the people with epilepsy have seizures that resist the anticonvulsant

drugs (Shorvon, 1996; Kwan and Brodie, 2000). These patients are said to have

refractory or intractable seizures. They are treated by seizure surgery, with the ketogenic

diet or by vagus nerve stimulation.

1.5.2 Surgery

Surgery is considered if the seizures are non-responsive to anticonvulsant

medications and if an epileptiogenic focus can be identified (Penfield and Steelman,

1947). Surgery is also considered when there is an active pathology, such as an infection

or an expanding tumour, is detected in the brain (Penfield and Steelman, 1947). The

most common surgery is removal of the anterior two-thirds of a temporal lobe which

9

contains a seizure focus. Extra-temporal lobe foci may also be removed and, in certain

cases, the corpus collosum may be cut (Penfield and Steelman, 1947).

Surgery has been shown to be effective for the treatment of epilepsies of temporal

lobe origin or those caused by lesions, infections, cysts or tumours in the brain.

Approximately 70% of the patients with these sorts of epilepsies become seizure free

following surgical lobectomy (Awad et al., 1989; Katz et al., 1989; Kuzniecky et al.,

1992; Sirven et al., 2000a; Benifla et al., 2006; Guarnieri et al., 2009). In a recent meta-

analysis, Schmidt and Staven (2009) calculated a relative risk of 4.26 for seizure freedom

following surgery, relative to anticonvulsant drug therapy in patients with drug-resistant

epilepsy (Schmidt and Stavem, 2009).

Although seizure-freedom is attained in most cases following surgery, long-term

follow-up studies of 10 or more years reported that seizures recurr in approximately 20%

of the patients who become seizure-free during the first few years following surgery

(McIntosh et al., 2004; Benifla et al., 2008). Also, quality of life is not significantly

improved post-surgery, as compared to pre-surgery or to healthy controls (Gilliam et al.,

1997), possibly because surgery does not improve some of the psychiatric co-morbidities

associated with epilepsy (Guarnieri et al., 2009).

Despite the risk of seizure remittance and a reported lack of improvement in the

quality of life following surgery, ‘social outcomes’ such as employment and school

enrollment are generally improved following surgery. Benifla et al., for instance, reported

a signigicantly greater incidence of employment and school enrollment in seizure-free

patients versus patients with recurrent seizures, following temporal lobe surgery (Benifla

et al., 2008).

10

Little is known about the possible behavioral complications associated with

surgical lobectomy. Bladin (1992) reported anexiety symptoms in more than 50% of the

patients that had undergone surgerical leboctomy at 12 months post-surgery (Bladin,

1992). Although suggestive that anexiety may be associated with unilateral removal of

the temporal lobe, this conclusion cannot be supported in the absence of a control group.

Cognitive performance and IQ, however, are usually maintained following surgery, as

compared to pre-surgery (Gilliam et al., 1997; Cukiert et al., 2008).

1.5.3 Ketogenic diet

The ketogenic diet may be tried for intractable seizures that are not amenable to

seizure surgery. It is usually administered to children. This non-drug therapy will be

discussed in some detail, since the diet may elevate brain n-3 PUFA (Taha et al., 2005), a

major topic of the present thesis.

The high-fat ketogenic diet was developed by Wilder Penfield in 1921 in order to

mimic the effects of fasting, which had been reported since biblical times to suppress

seizures. The ‘classic’ version of the diet contains 80% fat, 16% protein and 4%

carbohydrates by weight. The diet raises plasma ketone bodies (acetoacetate, β-

hydroxybutyrate [β-HBA] and acetone) because fatty acid oxidation is activated when

plasma levels of insulin are suppressed by the low carbohydrate content of the diet

(Likhodii et al., 2002; Musa-Veloso et al., 2002a; Musa-Veloso et al., 2002b). The fatty

acid β-oxidation pathway becomes saturated and free fatty acids are consequently

shunted into ketogenesis, resulting in a sustainable state of mild to moderate ketosis

(Likhodii et al., 2002; Musa-Veloso et al., 2002a; Musa-Veloso et al., 2002b).

The mechanism of action of the ketogenic diet is unknown, although several

11

theories have been suggested (Rho and Sankar, 2008). One possibility is that the

anticonvulsant effects of the ketogenic diet are related to the elevation of the ketone body,

acetone, which has been shown to suppress seizures in animal seizure models (Likhodii

and Burnham, 2002; Likhodii et al., 2003).

The diet has also been shown to raise brain n-3 PUFA concentrations, due to the

mobilization of n-3 fatty acids from adipose tissue to the brain, and this has been

suggested as another possible mode of action of the ketogenic diet - one involving n-3

PUFA (Cunnane et al., 2002; Taha et al., 2005). The possible anticonvulsant effects of

the n-3 PUFA will be discussed below (section 1.12).

Prospective and randomized trials have demonstrated the anticonvulsant efficacy

of the ketogenic diet, particularly in children. Overall, these studies indicate that the diet

benefits approximately two-thirds of the children with intractable epilepsy. Full seizure

control is attained in approximately 3% of the patients, while 31% exhibit more than a

90% reduction in seizure frequency and 26% show a 50-90% reduction in seizure

frequency following treatment with the ketogenic diet (Lefevre and Aronson, 2000).

These observations are consistent with a recent, non-blinded, randomized, controlled trial

which showed that, in contrast to control patients on their usual anticonvulsant regimen,

subjects who were randomized to the ketogenic diet while maintaining their

anticonvulsant regimen showed a mean reduction of 38% in seizure frequency after 3

months (Neal et al., 2008a). Out of 73 patients on the diet, 7% (versus 0% of controls)

showed a >90% reduction in seizure frequency, and 38% (versus 6% of controls) showed

a reduction of >50% (Neal et al., 2008a).

There is some clinical evidence suggesting that the ketogenic diet works in adults

12

as well as children; but, the ‘true’ efficacy of the diet in adults cannot be accurately

assessed due to the high drop-out rates of adult patients and their unwillingness to

participate in clinical trials (Sirven et al., 1999; Mosek et al., 2008). The high drop-out

rates are mainly due to poor compliance or to a lack of efficacy of the diet. It appears,

however, that the the ketogenic diet reduces seizure frequency by at least 50% in

approximately 55% of adults with intractable epilepsy (Barborka, 1929; Sirven et al.,

1999; Mosek et al., 2008).

Despite the efficacy of the ketogenic diet, it is associated with a number of

physiological and biochemical side-effects. Up to 25% of the patients on the diet report

gastrointestinal discomfort or hunger (Neal et al., 2008a). In children, it has been reported

to lead to stunted growth (Neal et al., 2008b) and increase the risk of kidney stones (~ 4-

10% of subjects) (Kinsman et al., 1992; Ballaban-Gil et al., 1998; Hassan et al., 1999). In

adults, the diet elevates plasma levels of risk factors that have been associated with the

formation of atherosclerotic vascular lesions, such as cholesterol, LDL-cholesterol and

triglycerides (Sirven et al., 1999; Kwiterovich et al., 2003; Fuehrlein et al., 2004; Mosek

et al., 2008).

An interesting facet of the ketogenic diet is that it has been reported to improve

quality of life, cognitive performance and mood in both children and adults (Kinsman et

al., 1992). In both cases, the improvement in quality of life has been related to improved

mood and cognitive performance, even in the absence of seizure control, suggesting a

separate mechanism of action (Kinsman et al., 1992).

1.5.4 Vagus nerve stimulation

Vagus nerve stimulation is another therapy used to treat drug-resistant seizures. It

13

is used in both children and adults. It involves implanting a device similar to a cardiac

pace-maker which stimulates the left vagus nerve (Penry and Dean, 1990; Uthman et al.,

1990). For reasons that are not clear, this tends to reduce the incidence of complex-partial

seizures (Penry and Dean, 1990; Uthman et al., 1990).

The clinical efficacy of vagus nerve stimulation is lower than the efficacy of

surgery or the ketogenic diet. Clinical studies have shown that vagus nerve stimulation

reduces seizure frequency by at least 50% in approximately 40-50% of the patients

(Penry and Dean, 1990; Uthman et al., 1990; Ben-Menachem et al., 1994; Amar et al.,

1998; Sirven et al., 2000b). Smaller reductions in seizure frequency of about 30% may be

seen in the remaining patients.

A few studies have examined the tolerability and possible side-effects of vagus

nerve stimulation. The stimulation is well tolerated by most patients (Uthman et al., 1990;

Ramsay et al., 1994). Coughing and hoarseness do occur in one-third to two-thirds of

patients (Uthman et al., 1990; George et al., 1994; Ramsay et al., 1994; Sirven et al.,

2000b), but this is tolerable if seizure control is improved. Since the left vagus nerve

inervates the left ventricle of the heart and the gastro-intestinal system, it had been feared

that vagus nerve stimulation might cause changes in cardiac rhythm or the formation of

gastric ulcers. Long-term studies (up to 18 months), however, have shown no significant

changes in cardiac rhythm or in the incidence of ulcers or of acid reflux due to vagus

nerve stimulation (George et al., 1994; Ramsay et al., 1994; Sirven et al., 2000b).

Quality of life is improved in some people with epilepsy by vagus nerve

stimulation (Sirven et al., 2000b). Unlike the ketogenic diet, however, the improvement

in quality of life is only seen in individuals whose seizures are suppressed. This suggests

14

that seizure-relief is necessary for an increase in the well-being of patients.

1.6 Drawbacks of current treatment options

Table 1.2 summarizes the efficacy and drawbacks of the current treatment options

for epilepsy. As indicated, despite the proven clinical efficacy of the various treatments,

none works in every case, and they are often less than ideal due to their side-effects.

There is still a need for novel and well tolerated therapies for the treatment of

patients with epilepsy.

Table 1: Clinical efficacy and drawbacks / side-effects of treatment options for epilepsy Clinical efficacy

Side-effects / Drawbacks Benefits

Anticonvulsants 70% Nausea, fatigue, sedation, dizziness, gastro-intestinal upsets, skin rashes, hepatotoxicity, photo-phobia, personality changes, memory loss

Improved seizure control

Surgery 70% Not fully investigated Improved seizure control

Ketogenic diet 60-70% Hyperlipidemia, gastrointestinal, hunger, stunted growth in children

Improved seizure control, improved cognitive performance and mood

Vagus nerve stimulation 40-50% Coughing, hoarseness Improved seizure control

1.7 Alternative treatment for epilepsy

Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been proposed as a

possible new treatment for epilepsy (Cunnane et al., 2002; Yuen and Sander, 2004). N-3

15

PUFA are dietary derived fatty acids that play a crucial role in brain development and

function. As will be discussed in section 1.9, interest in the possible anticonvulsant

effects of the n-3 PUFA derives in part from their known effects on the cardiac

arrhythmias. The cardiac arrythmias involve hyper-excitability in the heart, just as

seizures involve hyper-excitatability in the brain.

The following sections will review the structural properties of the PUFA, their

sources, their metabolism and their impact on brain neurochemistry and function, with a

particular focus on the n-3 PUFA and their impact on the brain.

1.8 About fatty acids

1.8.1 Definition and structure

A fatty acid is a hydrocarbon molecule with a carboxylic terminal on one end and

a methyl terminal on the other end (Sprecher, 2000). Figure 1-a shows an example of a

fatty acid. The methyl and carboxylic terminals are indicated.

There are three classes of fatty acids: saturated, monounsaturated and

polyunsaturated fatty acids. The classification is based on the number of double bonds in

the molecule (Burr and Burr, 1930; Sprecher and Lee, 1975; Sprecher, 2000). A saturated

fatty acid has no double bonds. Figure 1-a shows an example of an eighteen-carbon

saturated fatty acid, called “stearic” acid. A monounsaturated fatty acid has one double

bond, which usually lies on the 5th, 7th, 9th or 11th carbon molecule relative to the methyl

terminal. Figure 1-b illustrates a monounsaturated fatty acid, “oleic” acid. A

polyunsaturated fatty acid has more than one double bond. Typically, polyunsaturated

fatty acids have two to six double bonds. Their position is measured from the methyl

16

terminal. The example provided in Figure 1-c is linoleic acid, which has two double

bonds, at the omega-6 and omega-9 positions.

Figure 1: Structure of fatty acids Figure 1-a: Example of a saturated fatty acid

Figure 1-b: Example of a monounsaturated fatty acid

Carboxylic terminal

Methyl terminal

Double bondDouble bond

17

Figure 1-c: Example of a polyunsaturated fatty acid

1.8.2 Types of PUFA

There are two types of PUFA, omega-3 and omega-6 PUFA (Burr and Burr,

1930; Sprecher and Lee, 1975; Sprecher, 2000). The “omega” nomenclature refers to the

position of the first double bond relative to the methyl terminal. Thus, the first double

bond in an omega-3 fatty acid occurs at the third carbon relative to the methyl terminal,

whereas the first double bond on an omega-6 fatty acid starts from the sixth carbon

(Sprecher and Lee, 1975). Figure 2-a shows an example of an omega-3 fatty acid,

docosahexaenoic acid (DHA). Figure 2-b shows an example of an omega-6 fatty acid,

arachidonic acid (AA). As indicated, the first double bond from the methyl terminal is on

the third carbon of the fatty acid chain in DHA, and on the sixth carbon in AA.

Traditionally, omega-3 is usually abbreviated as “n-3” and omega-6 is usually

abbreviated as “n-6”.

The most abundant n-3 PUFA in mammalian tissues are α-linolenic acid (ALA)

and DHA (Bernert and Sprecher, 1975; Taha et al., 2005; Igarashi et al., 2007a; Stark,

2008). Eicosapentaenoic acid (EPA), another n-3 PUFA, is also present in most tissues,

Double bonds

18

but to a smaller extent. Linoleic acid (LA) and arachidonic acid (AA) are the most

abundant n-6 PUFA (Bernert and Sprecher, 1975; Taha et al., 2005; Igarashi et al.,

2007a; Stark, 2008).

Figure 2: Structure of omega-3 and omega-6 fatty acids Figure 2-a: Example of docosahexaenoic acid, an omega-3 (n-3) fatty acid

Figure 2-b: Example of arachidonic acid, an omega-6 (n-6) fatty acid

Double bond on the third carbon from the methyl terminal

Double bond on the sixth carbon from the methyl terminal

19

1.8.3 Sources of PUFA

LA and ALA cannot be synthesized by mammals and must, therefore, be obtained

from the diet (Burr and Burr, 1930). They are primarily found in plants and vegetable oils

(Burr and Burr, 1930). LA is highly abundant in canola oil, safflower oil, corn oil and

sunflower oil (Beadle et al., 1965; Davison and Dutton, 1967). ALA is enriched in

flaxseed and almonds (Sathe et al., 2008; Metherel et al., 2009).

In rodents and humans, AA and DHA, the longer-chain PUFA, can be synthesized

endogenously from their n-6 and n-3 fatty acid precursors, LA and ALA respectively

(Bernert and Sprecher, 1975; Sprecher and Lee, 1975; Sprecher, 2000; Igarashi et al.,

2006; Igarashi et al., 2007b; Lin and Salem, 2007; Gao et al., 2009). Figure 3 shows the

synthetic pathway involved. As shown in Figure 1-3, AA is the end-product of the n-6

PUFA synthesis pathway and DHA is the end-product of the n-3 PUFA synthesis

pathway (Lin and Salem, 2007). AA is synthesized in several steps from LA, whereas

DHA is synthesized from ALA. EPA is an earlier step in the synthetic chain that leads to

DHA (Sprecher, 2000; Lin and Salem, 2007; Gao et al., 2009).

The synthesis of AA and DHA takes place in the liver via elongase and desaturase

enzymes (Brenner and Peluffo, 1966; Bernert and Sprecher, 1977; Igarashi et al., 2007c).

Elongase enzymes elongate the fatty acid carbon chain from 18 carbons (LA or ALA) to

20 (EPA or AA) or 22 (DHA) carbons (Bernert and Sprecher, 1977; Carreau et al., 1981;

Vasireddy et al., 2007). Desaturases insert a double bond between the carbon molecules

following elongation (Brenner and Peluffo, 1966; Carreau et al., 1981). Thus, in contrast

to LA and ALA precursors which have 3 double bonds, AA, EPA and DHA have 4, 5 and

6 double bonds respectively.

20

Although AA, EPA and DHA can be synthesized in the liver from LA or ALA,

the conversion efficiency of LA to AA and ALA to DHA is very low and amounts to less

than 1% and 7% in rodents and humans, respectively (Burdge and Wootton, 2002;

Burdge et al., 2003; Lin and Salem, 2005; Burdge, 2006; Lin and Salem, 2007; Pawlosky

et al., 2007). In practice, therefore, most of the longer n-3 and n-6 PUFA in mammals are

derived from the diet. Dietary sources of AA include red meat, poultry and dairy products

(Astorg et al., 2004). The naturally occurring forms of EPA and DHA are found only in

marine sources, such as fish and shell-fish (Mozaffarian and Rimm, 2006).

The North American diet is characterized by an abundance of omega-6 fatty acids

in the form of LA and AA, and a limited supply of omega-3 fatty acids (Denomme et al.,

2005; Fratesi et al., 2009; Harris et al., 2009; Lucas et al., 2009). As a result, it is

relatively difficult for North Americans to achieve the daily intakes of omega-3 fatty

acids recommended by nutritionists - 1.2 g of ALA and 0.5 g of DHA (Brenna et al.,

2009; Harris et al., 2009). Recent estimates suggest that less than one-third of the North

American population, consume the recommended daily amounts of ALA and DHA

(Denomme et al., 2005; Fratesi et al., 2009; Harris et al., 2009; Lucas et al., 2009).

21

Figure 3: The n-3 and n-6 PUFA synthetic pathways N-3 pathway N-6 pathway

D5D = Detla-5-desaturase, D6D = Detla-6-desaturase

1.9 Mechanisms of fatty acid uptake by the gut and transport to the brain

Since the present thesis relates to the effects of injected or dietary n-3 PUFA on n-

3 PUFA concentrations in the brain and on seizure threshold, this section will provide an

overview of the physiological and biochemical processes involved in fatty acid uptake by

the gut and fatty acid transport to the brain. Figure 4 summarizes these processes. As

shown, the key physiological factors involved in regulating brain fatty acid composition

α-linolenic acid (18:3n-3)

Elongase

D5D

Elongase

Elongase, D6D, β-oxidation

D6D

Linoleic acid (18:2n-6)

D6D

Octadectetranoic acid (18:4n-3) Gamma-linoleic acid (18:3n-6)

Dihomo-gamma-linolenic acid (18:4n-6)

Elongase

Arachidonic acid (20:4n-6)

D5D

Adrenic acid (22:4n-6)

Elongase

n-6 Docosapentaenoic acid (22:5n-6)

Eicosapentaenoic acid (20:5n-3)

Eicosatetranic acid (20:4n-3)

n-3 Docosapentaenoic acid (22:5n-3)

Docosahexaenoic acid (22:6n-3)

Elongase, D6D, β-oxidation

22

in the brain relate to the gut, the liver, adipose tissue and the plasma. The key molecules

involved in PUFA transport between these tissues and the brain, via plasma, are the

chylomicrons (CMs), very low-density lipoproteins (VLDLs), low-density lipoproteins

(LDLs), high-density lioproteins (HDLs) and albumin. The lipid molecules that

chemically bind to the PUFA and are incorporated into one of the transport molecules,

are triglycerides and phospholipids (Goldstein et al., 1974; Hussain et al., 2000).

1.9.1 Physiological components

The composition of PUFA in the brain is primarily regulated by dietary fatty acid

consumption. PUFA absorption takes place in the gut. The distribution of dietary PUFA

to the brain and other organs is then modulated by the liver, which is responsible for the

packaging and export of PUFA in lipid carrier protein molecules, such as “lipoproteins”

or albumin, to other tissues such as adipose and the brain (Rodbell et al., 1964; Polozova

and Salem, 2007). Adipose tissue is the primary storage site of PUFA. Under conditions

of dietary PUFA deficit, PUFA are mobilized from adipose tissue to other organs

including the brain (Conner et al., 1996; Zimmermann et al., 2004; Taha et al., 2005).

The transport of PUFA from the gut to other tissues is mediated by lipoprotein protein

carrier molecules through the plasma (Rodbell et al., 1959; Levy et al., 1966; Fredrickson

et al., 1967).

1.9.2 Lipid carrier molecules

There are several types of carrier molecules involved in the transport of lipids

between tissues. These are CM, VLDL, LDL, HDL and albumin. These are proteins that

carry lipid molecules through the plasma. They differ from each other in protein

23

composition.

CM, LDL, VLDL and HDL are composed of one or more proteins referred to as

“apolipoproteins” (Rodbell, 1958; Rodbell and Frederickson, 1959; Rodbell et al., 1959;

Shelburne and Quarfordt, 1974; Schittmayer and Birner-Gruenberger, 2009). There are

at least 9 types of apolipoproteins, specifically A-I, A-II, A-IV, B-48, C-1, C-II, C-III, D

and E (Rodbell, 1958; Rodbell and Frederickson, 1959; Rodbell et al., 1959; Shelburne

and Quarfordt, 1974; Schittmayer and Birner-Gruenberger, 2009). Apolipoprotein

synthesis takes place mainly in the liver and gut (Luskey et al., 1974). CMs, for instance,

are formed by the association of eight different apolipoproteins (A-I, A-II, A-IV, B-48,

C-1, C-II, C-III and E). Apolipoproteins B-100, C-I, C-II and C-III aggregate in the liver

to form VLDL particles. LDLs are mainly composed of apolipoprotein B-100 (Shelburne

and Quarfordt, 1974; Shelness et al., 1999). HDL particles contain a mixture of

apolipoproteins A-I, A-II, A-IV, C-1, C-II, C-III, D and E (Schittmayer and Birner-

Gruenberger, 2009). Albumin, a plasma protein, does not contain apolipoproteins, and is

synthesized in the liver (Marsh and Drabkin, 1958; Braun et al., 1962).

1.9.3 Lipid molecules that bind to the carrier proteins

There are several types of lipid molecules that non-covalently bind to the carrier

proteins (Hussain et al., 2000). These include triglycerides, phospholipids, cholesterol

esters, unesterified fatty acids and cholesterol (Goldstein et al., 1974; Hussain et al.,

2000).

A triglyceride is a lipid molecule that is formed by the chemical binding of a

glycerol to three fatty acids. The chemical bond that joins the alcohol group (-OH) of the

glycerol to each fatty acid (-COOH), is called an “ester” bond (-C-O-C-). In essense, it is

24

the bond formed between an oxygen atom and two carbon atoms.

A phospolipid molecule consists of a glycerol that is bound to two fatty acids.

Each fatty acid is bound to the glycerol molecule by an ester bond. The first bound fatty

acid is said to be at the “sn-1” position. The second, consecutive bound fatty acid is said

to be at the “sn-2” position. A phospholipid molecule also contains a phosphate head

group at the “sn-3” position.

Cholesterol is a sterol lipid, and a cholesterol-ester is a cholesterol molecule that

is bound to a fatty acid via an ester bond.

Unesterified fatty acids are “free” fatty acids, which are not chemically bound to

any molecule.

Dietary lipids occur mainly in the form of triglycerides, phospholipids or

cholesterol. CMs are involved in the transport of ingested triglycerides, phospholipids

and cholesterol from the intestines to the liver (Rodbell et al., 1959; Rodbell, 1960;

Rodbell and Scow, 1965; Quarfordt and Goodman, 1967; Quan et al., 2003). VLDLs

preferentially carry triglycerides from the liver to other tissues such as muscle (Quarfordt

and Goodman, 1967). LDL particles transport cholesterol, cholesterol esters and to a

lesser extent, unesterified fatty acids from the liver to other tissues (Quarfordt and

Goodman, 1967; Osono et al., 1995; Quan et al., 2003). HDL particles transport

cholesterol and phospholipids from the other tissues back to the liver (Chung et al., 2009).

Albumin is the main carrier of unsterified fatty acids (Rodriguez de Turco et al., 2002;

Belayev et al., 2005; Ouellet et al., 2009).

1.9.4 Pathways involved in fatty acid absorption, transport and uptake

Section 1.9.4 will describe the biochemical processes involved in fatty acid

25

absorption, transport and uptake by tissues.

Absorption:

The absorption of dietary fat takes place in the gut. It is a highly efficient process.

Approximtely > 98% of the ingested dietary lipids are transported into the body

(Cunnane and Anderson, 1997). The absorption of lipids in the gut is facilitated by bile

salts, lipase enzymes and CMs (Garfinkel et al., 1967; Quarfordt and Goodman, 1967;

Dietschy, 1969; Dietschy et al., 1971; Sallee and Dietschy, 1973).

The bile salts emulsify the lipids so that they can easily be broken down by gut

lipases (Wilson et al., 1971). Lipases then break down the triglycerides and

phospholipids into glycerol and unesterified fatty acids, which cross through the luminal

surface of the intestenial walls by both passive and facilitated diffusion (Dietschy, 1969;

Dietschy et al., 1971; Sallee and Dietschy, 1973). CMs then transport the fatty acids from

the gut to other tissues via the lymphatic system and the blood circulation (Garfinkel et

al., 1967; Quarfordt and Goodman, 1967; Quarfordt and Hilderman, 1970).

Bile salts are released post-prandially from the gallbladder in order to facilitate

the digestion of dietary lipids, by causing them to form “micelles” (Dietschy, 1967;

Dietschy, 1968). Micelles are spherical structures that consist of a hydrophobic lipid core

and a hydrophilic surface consisting of the polar head groups of phosphlipids (Dietschy,

1967; Dietschy, 1968; Wilson et al., 1971; Sallee and Dietschy, 1973). Micelle structures

increase the surface area of the ingested fats, so that they are readily accessible for

digestion by gut lipase enzymes that are supplied by the pancreas (Dietschy, 1967;

Dietschy, 1968; Wilson et al., 1971; Sallee and Dietschy, 1973).

Once inside the intestinal cells, the fatty acids are re-esterified to glycerol. The

26

formed triglycerides and phospholipids are then incorporated into the CMs (Rodbell et al.,

1964; Rodbell and Scow, 1965). Since dietary fat occurs predominantly in the form of

triglycerides, the CMs tend to be rich in triglycerides. Cholesterol, which also enters the

intestinal cells by passive diffusion, is also esterified to a fatty acid by a cholesteryl-

transferase, before it is incorporated into the CM (Dietschy, 1969; Zilversmit and Hughes,

1974). The CM, which is structurally large and lipid-dense, is then transported through

the basal side of the intestines to the lymphatic system, by vesicle-mediated exocytosis.

CMs that leave the intestinal cells enter the lymphatic system by endocytosis. They are

subsequently transferred from the lymphatic system to the blood circulation by

exocytosis (Zilversmit and Hughes, 1974; Kortz et al., 1984).

Transport:

CMs are soluble in plasma, owing to the hydrophilic nature of the apolipoprotein

sections that protrude through the surface of the CM (Rodbell, 1958; Rodbell and

Frederickson, 1959). This facilitates their transport to tissues. The clearance of CMs from

plasma is rapid, with a half-life of approximately one hour (Quarfordt and Goodman,

1967). Approximately 80-90% of the CMs that enter the circulation from the lymphatic

system are distributed via the liver to metabolically active sites, such as the muscle and

heart, and to adipose tissue for storage (Quarfordt and Goodman, 1967). The remaning

particles are degraded and recycled into other liporprotein molecules (Quarfordt and

Goodman, 1967).

Uptake by tissues:

Fatty acids in CM triglycerides or phospholipids are released from their glycerol

backbone by lipase enzymes (Rodbell, 1964; Garfinkel et al., 1967). These enzymes are

27

present in endothelial walls of blood vessels and in most tissues (Rodbell, 1964;

Garfinkel et al., 1967). They release the bound fatty acids from the triglycerides and

phospholipids in the CM, for energy utilization or storage (Rodbell, 1960; Rodbell et al.,

1964). Notably, the fatty acids released by endothelial lipase are solubilized in the plasma

by non-covalently associating with albumin. Tissues such as the brain, then extract the

unesterified, albumin-bound fatty acids from the plasma by passive diffusion (Ouellet et

al., 2009). The remaining CM, which contains less triglycerides and phospholipids, is

referred to as the “chylomicron remnant” (CM-remnant) (Andersen et al., 1977; Ross and

Zilversmit, 1977).

The CM-remnant enters the liver by receptor-mediated endocytosis (Wade et al.,

1986), where it is partially degraded by hepatic lipases (Rodbell, 1964; Rodbell et al.,

1964). The CM-remnant degredation process involves the release of esterified fatty acids

and dissociation of the lipoproteins (Rodbell et al., 1959; Rodbell, 1964; Rodbell et al.,

1964). This process, however, is insufficient to completely break down the esterified fatty

acids (Rodbell, 1964; Rodbell et al., 1964). Thus, other types of liver lipoproteins such as

VLDLs and LDLs incorporate the triglycerides of the CM-remnant, which contain

esterified fatty acids (Rodbell et al., 1959).

VLDL and LDL molecules serve as reservoirs and vehicles for the transport of

triglycerides to extra-hepatic tissues (Rodbell et al., 1964; Polozova and Salem, 2007).

These molecules are rich in triglycerides and are smaller than CM-remnant particles.

They enter tissues by receptor-mediated endocytosis (Wade et al., 1986). Unesterified

fatty acids are released from VLDL and LDL particles by the action of tissue or

endothelial lipases (Shearer and Newman, 2008).

28

In tissues, the fatty acids that are released from VLDL and LDL particles are used

to supply the structural and metabolic requirements of the tissue (Garfinkel et al., 1967;

Shearer and Newman, 2008). The unesterified fatty acids that are released from VLDL

and LDL particles by endothelial lipase, however, bind to plasma albumin carrier proteins

(Polozova et al., 2006). As will be discussed in the following section, albumin-bound

fatty acids serve as the key supply of fatty acids to the brain (Chen et al., 2008a).

HDL molecules, which are also synthesized in the liver, transport ‘excess’ lipid

molecules such as triglycerides, phospholipids, cholesterol esters and cholesterol, from

extra-hepatic tissues to the liver (Rodbell et al., 1959; Chung et al., 2009). HDL particles

are smaller than VLDL and LDL particles because of their lower lipid content (Rodbell

and Frederickson, 1959; Chung et al., 2009).

1.9.5 Uptake of fatty acids by the brain

The brain obtains its fatty acids almost exclusively from albumin-bound fatty

acids (Chen et al., 2008a). Fatty acids enter the brain by passive diffusion (Ouellet et al.,

2009). Thus, a concentration gradient between the plasma and blood-brain-barrier likely

facilitates the dissociation of the albumin-bound fatty acids and subsequent uptake by the

brain (Ouellet et al., 2009).

In contrast to other tissues, the brain does not rely on lipoprotein particles for its

supply of fatty acids (Chen et al., 2008b). The reason for this biological anomaly is not

clear. However, the VLDL and LDL molecules indirectly contribute to brain fatty acid

concentrations, since they supply the plasma albumin with fatty acids (Polozova et al.,

2006).

29

1.9.6 PUFA incorporation into the brain

The brain obtains its PUFA from albumin-bound, unesterified PUFA in the

plasma (Ouellet et al., 2009). All fatty acids, including PUFA, readily cross the blood-

brain-barrier by passive diffusion. Once in the blood-brain-barrier, fatty acids including

PUFA are esterified to an acyl-CoA group by the action of acyl-CoA synthetase (also

known as fatty acid transport protein) (Milger et al., 2006; Jia et al., 2007). The addition

of an acyl-CoA group makes the fatty acid more water soluble and therefore easier to

transport to various brain regions via the cerebrospinal fluid (Lee et al., 2007).

Although all PUFA cross the blood-brain-barrier, only AA and DHA are

incorporated into the phospholipid bilayer of neuronal membranes (Giovacchini et al.,

2004; Bazinet et al., 2006; Chen et al., 2009). This is achieved enzymatically by a fatty

acyl transferase (MacDonald and Sprecher, 1991). In contrast, more than 99% of LA,

ALA and EPA that enter the brain are immediately utilized by β-oxidation or recycling

into saturated fatty acids, monounsaturated fatty acids and cholesterol (Demar et al.,

2005; DeMar et al., 2006a; Taha et al., 2006b; Chen et al., 2009). Less than 1 % of the

LA or ALA that crosses the blood-brain-barrier is elongated and desaturated into AA or

DHA (Demar et al., 2005; DeMar et al., 2006a; Igarashi et al., 2007a).

The differences in incorporation amongst the various PUFA are reflected in the

fatty acid composition of the brain. AA and DHA constitute up to 30% of total fatty acids

in the rodent brain, whereas LA, ALA and EPA amount to less than 5% of total brain

fatty acids (Chen et al., 2008b; Taha et al., 2008b).

30

Figure 4: Modulation of brain tissue PUFA composition by diet

Liver – repackaging & distribution of VLDL & LDL

Brain – PUFA incorporation

Gut – absorption of dietary fat

CM

Lymphatic system

Blood

CM

CM FFA

CM FFA

CM-remnant

VLDL / LDL FFA

Adipose – storage Muscle – oxidation

31

Figure legend

Figure 1-4: A schematic outline illustrating the regulation of brain fatty acid composition by diet. Dietary fatty acids are absorbed in the gut, where they are packaged into protein carriers called chylomicrons (CMs). CMs enter the lymphatic system and then the blood circulation. They are transported to tissues (excluding the brain) via blood plasma. As they pass through the blood circulation, endothelial lipase acts on the CMs to release free fatty acids (FFAs) which associate with albumin. These FFAs can be utilized by the tissues such as adipose and muscle for energy or storage (dark, dashed line), or they can be uptaken by the brain (blue dotted line). In tissues such as muscle or adipose, the CMs are further degraded by tissue lipases to release FFAs and CM-remnant particles. The CM-remnants are released back into the circulation and go to the liver, where they are used to make VLDL and LDL particles. These particles are then exported out of the liver, into the circulation, where endothelial lipases degrade them to release FFAs. These FFAs enter tissues such as the brain by passive diffusion. The VLDL and LDL particles are uptaken by muscle and adipose, where they are further broken down by lipases to release fatty acids for energy or storage. Thus, the brain exclusively obtains its FFAs from albumin-bound FFAs in plasma, whereas other tissues obtain FFAs from albumin, CMs, CM-remnants, VLDL or LDL particles.

1.10 Biological role of PUFA in the brain

AA and DHA are highly concentrated in the brain, in contrast to other tissues. As

noted above, they represent 30% of the brain’s total fatty acids. It is not surprising,

therefore, to suspect that these PUFA play an important role in brain physiology and

function.

AA is known to play a crucial role in mediating inflammatory reponses in the

brain. DHA on the other hand, is known to regulate neurotransmitter release and synaptic

transmission, to antagonize neuroinflammation through its oxygenated derivatives, and to

modulate the nuclear receptor-mediated transcription of genes. A brief review of the

actions of AA and DHA in the brain will be provided in the following sections.

32

1.10.1 Role of AA in the brain

AA is a precursor for the compounds that mediate inflammatory responses in the

brain, and which play a role in vasoconstriction. In order for AA to produce its pro-

inflammatory products, it has to be released from the phospholipid membrane. AA is

selectively released from membrane phospholipids by: 1) group IV calcium-dependent

cytosolic phospholipase A2 and 2) secretory phospholipase A2 (Kolko et al., 2005; Kolko

et al., 2006). Cytosolic phospholipase A2 releases AA into the the cytosol, whereas

secretory phospholipase A2 releases AA on the outer side of the cell membrane. Both

cytosolic and secretory AA-selective phospholipases are activated by excitatory signaling

in the brain, such as the activation of NMDA receptors (Rao et al., 2007b).

Neuroinflammation can also activate these AA-selective phospholipases (Rao et al.,

2009).

Only a small fraction of the AA which is released from the membrane plays a role

in the inflammatory process. Studies involving radiolabeled AA tracers have shown that

approximately 97% of the AA that is released from the phospholipid membrane is re-

esterified into the membrane by fatty acyl transferase following addition of an acyl-Co-A

group by acyl-CoA synthetase (Robinson et al., 1992; Rapoport, 2003; Bazinet et al.,

2005b; Bazinet et al., 2006). A small fraction of the remaining 3% undergoes β-oxidation

or is metabolized into prostaglandins and other oxygenated AA-metabolites via

cyclooxygenases, lipoxygenase, cytochrome P450 or epoxygenase enzymes (Zeldin,

2001; Rapoport, 2003; Bazinet et al., 2005b; Bazinet et al., 2006; Lee et al., 2007). The

majority of AA-derived metabolites have pro-inflammatory actions, although recent

studies suggest that some are anti-inflammatory (Bazan, 1989a; Bazan, 1989b; Panetta et

33

al., 1989). Despite the antagonizing effects of AA metabolites, the net effect of activating

the release of AA via phospholipase A2 is an increase in neuroinflammation (Rao et al.,

2007b; Rao et al., 2009).

1.10.2 Role of DHA in the brain

DHA plays several roles in the brain. It is involved in regulating

neurotransmission, preventing neuroinflammation and in gene expression.

DHA and neurotransmitters:

Evidence for the modulatory effects of DHA on neurotransmission and

neurotransmitter levels in the brain has come from studies involving dietary n-3 PUFA

deficiency or supplementation. These studies have shown that both n-3 PUFA deficiency

and supplementation alter neurotransmitter levels and drug-induced neurotransmitter

release. Overall, DHA depletion reduces the release or receptor binding of

neurotransmitters such as acetylcholine, serotonin and dopamine, whereas enrichment

increases them.

In particular, n-3 PUFA deficiency has been shown to affect cholinergic,

serotonergic and dopaminergic neurotransmitter concentrations and release in the brain.

Aid et al., for instance, demonstrated that dietary depletion of brain DHA increased

acetylcholine release, and reduced binding of acetylcholine to the muscurinic receptor in

rat hippocampus but not in the frontal cortex (Aid et al., 2003).

Chronic n-3 PUFA deficiency has also been shown to impair amphetamine-

induced dopamine release in the frontal cortex and nucleus accumbens (Zimmer et al.,

2002). This observation is consistent with a study which showed that n-3 PUFA

deficiency reduced levels of dopamine-carrying pre-synaptic vesicles in rat cortex

34

(Zimmer et al., 2000).

Rats deprived of n-3 PUFA have also been reported to have higher basal levels of

serotonin in the hippocampus, but impaired release of serotonin following the stimulation

of serotonin release by fenfluramine (Kodas et al., 2004). Serotonin receptor (5HT-2)

density has also been reported to be higher in the frontal cortex of n-3 PUFA deprived

animals, as compared to non-deprived controls, reflecting an adaptation to low serotonin

function in the n-3 PUFA deprived rats (Kodas et al., 2004).

A smaller number of studies have examined the effects of chronic DHA

supplementation on neurotransmitter levels and release in the brain. Favreliere et al., for

instance, reported that a DHA-enriched diet increased spontaneous and potassium

chloride-evoked acetylcholine release in rat hippocampus (Favreliere et al., 2003).

Chalon et al. reported an increase in basal dopamine levels in rat frontal cortex following

fish oil supplementation, which was associated with a significant reduction in the

dopamine-degrading monoamine oxidase enzymes in the frontal cortex (Chalon et al.,

1998). Chronic administration of DHA via gavage has recently been shown to increase

neurotransmitter levels of 3,4-dihydroxyphenylacetic acid in the frontal cortex, while

marginally reducing serotonin and 5-hydroxyindolacetic acid levels in the hippocampus

of rats (Vancassel et al., 2008).

DHA and neuroinflammation:

DHA also antagonizes neuroinflammation. It does so through its biologically

active, oxygenated metabolites, such as resolvin D1 (RD1) and neuroprotectin D1

(NPD1) (Hong et al., 2003; Marcheselli et al., 2003). RD1 and NPD1 are formed from

unesterified DHA, which is released from the sn-2 position of a phospholipid molecule

35

by the action of a DHA-specific phospholipase (Rao et al., 2007a; Strokin et al., 2007).

The identity of the particular phospholipase that releases DHA from the cell membrane to

the extracellular space is still in question, but group VI calcium-independent

phospholipase A2 is thought to initiate the release of DHA from the membrane into the

cytosol (Rao et al., 2007a; Strokin et al., 2007).

Similar to AA, most of the DHA (~90%) that is released from the membrane is

rapidly re-esterified into the membrane via acyl-CoA transferase (DeMar et al., 2004).

The small fraction of released, unesterified DHA that does not rejoin the membrane is

converted into bioactive resolvins or docosanoids via the actions of 15-lipoxygenase or

cyclooxygenases. DHA metabolites such as RD1 and NPD1 have been shown to suppress

neuroinflammation induced by ischemia-reprefusion or liposaccharaide administration

both in vitro and in vivo in mice. The mechanism by which these DHA-derived

metabolites act to suppress neuroinflmmation is not known.

DHA and gene expression:

DHA also plays an important role in regulating gene expression in the brain.

Unesterified DHA is an endogenous ligand for the peroxisome proliferator-activated

receptor-alpha (PPAR-α) (Lin et al., 1999; Hostetler et al., 2005), which is a transcription

factor that induces the expression of genes involved in fatty acid oxidation. Using

microarray gene expression assays, Kitajka et al. have shown that dietary

supplementation of fish oil (containing DHA) in rats induces the mRNA expression of 55

genes in brain, and suppresses the expression of 47 others (Kitajka et al., 2002). In

general, the genes that are induced are genes related to the expression of functional

proteins involved in brain energy metabolism, synaptic transmission, cytoskeleton

36

formation, signal transduction and neuronal cell survival (Kitajka et al., 2002; Puskas et

al., 2003).

1.11 Behavioral effects of n-3 PUFA in vivo

The following sections will review the evidence linking changes in brain PUFA

levels to behavioral outcome in animal models. In particular, the modulatory effects of n-

3 PUFA on behavior will be reviewed.

N-3 PUFA have been shown to alter behavior in animal models. Dietary DHA

supplementation appears to improve cognitive performance, anxiety-like behavior and

mood in rodents, whereas dietary DHA-deficiency appears to adversely affect these

parameters. The effects of DHA on behavior may be related both to its various roles in

regulating neurotransmitter release and gene expression, and to antagonizing the pro-

inflammatory n-6 PUFA synthesis pathway.

1.11.1 Learning and memory

A number of studies have examined the link between DHA and learning and

memory. Most of these have studied the effects of DHA deprivation. They have clearly

shown that chronic n-3 PUFA deficiency impairs spatial learning and memory in rats.

Limiting n-3 PUFA in the diet for three generations, for instance, has been shown

to reduce brain DHA levels by 87%, and also to impair procedural spatial learning

performance and memory in the Morris Water Maze test in 9- and 13-week old third

generation rats (Moriguchi et al., 2000; Moriguchi and Salem, 2003). Compared to n-3

PUFA sufficient rats, the DHA-deprived subjects took approximately 67% longer to

reach a position-set, hidden platform during the first and second days of the Morris water

37

maze test (Moriguchi et al., 2000; Moriguchi and Salem, 2003). The DHA-depleted

animals also spent more time swimming randomly in quadrants that did not contain the

platform, indicating that short-term memory was compromised (Moriguchi et al., 2000;

Moriguchi and Salem, 2003). The impairment in learning and memory was no longer

seen after the n-3 PUFA deficient offspring had been placed on a diet containing DHA

for six weeks. This behavioral change was accompanied by a full recovery of the brain

DHA levels, indicating that brain DHA repletion can reverse the learning and memory

deficits caused by a dietary-induced reduction of brain DHA (Moriguchi and Salem,

2003).

DHA supplementation has also been shown to improve learning and memory.

Jiang et al. (2008) have recently reported that daily gavage of 50 or 100 mg/kg of DHA to

female mice for 7 weeks, reduced the number of errors in the step-through performance

and passageway water maze tests by at least 20%, and in a dose-dependent fashion (Jiang

et al., 2008). The background diet of the mice, however, was not reported in the study of

Jiang et al. (2008). In a recent study Chung et al. (2008) have reported that daily

gavaging of fish oil containing 180 mg of EPA and 120 mg of DHA for 11.4 weeks in

rats on an n-3 PUFA deficient or an n-3 PUFA adequate diet, improved spatial learning

performance in the Morris water maze test, as evidenced by a significant reduction in

escape latencies of at least 16% (Chung et al., 2008). The benefits of the EPA and DHA

supplement was seen in both rats fed an n-3 PUFA deficient diet and in rats fed a normal

diet. The fish oil-supplemented rats also spent significantly less time finding the platform

when they were re-introduced to the pool three weeks after the spatial learning

performance test, indicating that fish oil improved long-term memory and retention of

38

spatial cues (Chung et al., 2008). These findings are also consistent with other studies

which have reported that chronic, oral administration of oils containing DHA exerts a

beneficial effect on learning and memory performance, independent of the dietary

background.

1.11.2 Anxiety

The elevated plus maze is used to measure anxiety-like behavior in rats, which is

indicated by the amount of time spent in closed arm of the maze versus the open arm. So

far, only the effects of n-3 PUFA deficiency on anxiety have been studied. The effects of

high n-3 PUFA diets have not yet been examined.

Chronic n-3 PUFA deficiency has been reported to increase anxiety-like behavior

in rats. This has been seen only in conditions of high stress induced by bright light or the

intracerebroventicular infusion of corticotrophin-releasing hormone. In these conditions,

n-3 PUFA deficient rats have demonstrated increased anxiety by spending less time in the

open arms of the elevated plus maze and by reducing the frequency of rearing, sniffing

and feeding, as compared to n-3 PUFA sufficient animals.

1.11.3 Mood - aggression

The effects of n-3 PUFA deprivation on aggression have also been examined in a

recent study. DeMar et al. have shown that n-3 PUFA deprivation for 15 weeks, which

reduced brain DHA levels by approximately 36%, increased aggression scores in rats

(DeMar et al., 2006b).

1.11.4 Mood - depression

The forced swim test is used to model depression-like behavior in rats, which is

39

indicated by immobile floating rather than active swimming. Two animal studies have

shown an increase in depression-like behavior in this test in rodents fed an n-3 PUFA

deficient diet. Frances et al. (1996) for instance, have reported an increase in immobility

scores in mice deprived of n-3 PUFA for 15 weeks, relative to non-deprived control

animals (Frances et al., 1996). Recently, DeMar et al. (2006) have also reported a 30%

increase in immobility in rats fed an n-3 PUFA deficient diet for 15 weeks, as compared

to those that were on an adequate n-3 PUFA diet (DeMar et al., 2006b). In both studies,

brain DHA levels were reduced by 36-40%.

Consistent with the observation that dietary-induced depletion of brain DHA

increases scores for depression in rodents, dietary supplementation with fish oil for two

generations in rats, reduces the immobility time in the forced swim test, relative to both

controls on an n-3 PUFA adequate diet and to rats on an n-3 PUFA deficient diet .

1.12 N-3 PUFA and epilepsy

In addition to their effects on learning, anxiety and mood, n-3 PUFA may also

raise seizure thresholds in the brain. This idea was first suggested by the discovery of the

stabilizing effect of the DHA on cardiac arrythmias. The cardiac arrythmias involve

hyper-excitability in the heart, just as seizures involve hyper-excitatability in the brain.

1.12.1 Antiarrhythmic effects

The antiarrythmic effects of the n-3 PUFA were first studied in cardiac myocytes

(heart muscle cells). It was discovered that the n-3 PUFA reduced the excitability of

myocytes, via inhibitory effects on the voltage-dependent sodium and calcium currents

that initiate the cardiac action potential (Kang et al., 1995; Xiao et al., 1995). Since the n-

40

3 PUFA appeared to “stabilize” the membranes (Kang et al., 1995), it seemed likely that

the n-3 PUFA would have an antiarrhythmic effect in whole animals (Kang and Leaf,

1994). Such an effect was first demonstrated in dogs infused or fed a diet enriched with

n-3 fatty acids (Billman et al., 1994; Billman et al., 1999). Similar effects were later

demonstrated in humans on n-3 PUFA-enriched diets (Leaf, 1995; Leaf and Kang, 1996;

Leaf et al., 2005; Tavazzi et al., 2008).

These effects are believed to relate to the partial inhibition of voltage-dependent

sodium channels (VDSCs) (Xiao et al., 1995). They appear to be mediated by the n-3

PUFA in their unesterified, free form rather than by the n-3 PUFA bound to

phospholipids (Kang et al., 1995; Weylandt et al., 1996).

1.12.2 Possible anticonvulsant effects of the n-3 PUFA – in vitro studies

Since the n-3 PUFA stabilize cardiac cell membranes, it seemed possible that they

might also stabilize neuronal membranes - which also contain VDSC - and that they

therefore might be anticonvulsant as well as antiarrhythmic. A number of anticonvulsant

drugs are partial inhibitors of VDSC - including phenytoin, carbamazepine, lamotrigine

and zonisamide (Bazil, 2002).

Single-cell studies performed in vitro have offered preliminary support for the

idea that the n-3 PUFA might have anticonvulsant actions (Vreugdenhil et al., 1996; Xiao

and Li, 1999; Young et al., 2000; Borjesson et al., 2008). Vreugdenhil et al. (1996), for

instance, studied the effects of DHA and EPA in dissociated cells from rat CA1, and

discovered that both of the n-3 compounds favored the “inactivated” state of the VDSC

(Vreugdenhil et al., 1996). Xiao and Li (1999) likewise found that n-3 PUFA reduced the

frequency of electrically induced action potentials in hippocampal slices isolated from

41

rats (Xiao and Li, 1999).

1.12.3 Anticonvulsant effects of the n-3 PUFA – animal studies

In agreement with the single-cell studies that suggested that the n-3 PUFA ought

to have anticonvulsant effects, past animal studies have reported that n-3 PUFA raise

seizure thresholds in animal seizure models. These studies, however, have not been

widely accepted.

Yehuda’s group, for instance, has reported that ALA, administered to rats in

combination with LA in a 1 to 4 ratio (the “SR-3 mixture”), raises brain levels of DHA

(Yehuda et al., 1996) and increases resistance to pentylenetetrazol (PTZ) induced

seizures (Yehuda et al., 1994; Rabinovitz et al., 2004). ALA was administered in

combination with LA because the conversion of ALA to DHA is thought to be optimized

when ALA is co-administered with LA (Yehuda et al., 1996).

Voskuyl and colleagues have also reported the anticonvulsant effects of the

longer-chain n-3 PUFA, EPA and DHA, in a rodent cortical stimulation model. They

found that the acute intravenous administration of either EPA or DHA over a 30 minute

period resulted in an increase in both focal and generalized seizure thresholds (Voskuyl et

al., 1998). Willis et al, however, found no protective effects following one month of

dietary supplementation with EPA or DHA in various seizure models, including the

pentylenetetrazol threshold test (Willis et al., 2008).

While suggestive, these studies have not been widely accepted – perhaps because

Voskuyl’s work involves an unusual, little-used seizure model, and because Yehuda’s

work could not be replicated.

42

1.12.4 Anticonvulsant effects of the n-3 PUFA – clinical trials

A few clinical trials of the anticonvulsant effects of the n-3 PUFA’s have been

done, but these have produced ambiguous results. Schlanger et al. (2002), in an open trial

that lasted for 6 months, found marked anticonvulsant effects of an n-3 PUFA enriched

diet containing 3.2g of DHA and EPA (Schlanger et al., 2002). Yuen et al. (2005),

however, in a better controlled trial, found only transient effects at a lower dose of 1.7 g

per day for 3 months, whereas Bromfield et al. (2008) and DeGiorgio et al. (2008) found

no effects after 3 months at doses of 2.2 and 2.9 g per day, respectively. These trials only

lasted for 3 months.

In evaluating these results, it must be noted that the duration of n-3 PUFA

treatment in the Yuen et al., Bromfield et al. and DeGiorgio et al. studies was short (12

weeks) (Yuen et al., 2005; Bromfield et al., 2008; Degiorgio et al., 2008b). The trial with

the best results (Schlanger et al.) was the trial that had the longest duration of treatment

(Schlanger et al., 2002). The findings of this trial, however, need to be interpreted with

caution since it was an open-label study (Schlanger et al., 2002).

1.13 Unanswered questions

In assessing the above studies, it remains unclear as to whether n-3 PUFA have a

clear-cut effect on seizure threshold. Yehuda and colleagues have shown that ALA raises

seizure threshold (Yehuda et al., 1994; Rabinovitz et al., 2004); however, the conversion

efficiency of ALA to DHA is inefficient in rodents (<0.5%) (Igarashi et al., 2006). Also,

ALA is found in very low amounts in the brain (<1%). Voskuyl et al. suggested that both

EPA and DHA raise seizure threshold in a cortical stimulation model (Voskuyl et al.,

43

1998). Recent evidence, however, suggests that the majority of EPA that enters the brain

is oxidized and not incorporated (Chen et al., 2009). Finally, some studies have

demonstrated an acute effect of the n-3 PUFA on seizure threshold (Voskuyl et al., 1998),

whereas others have failed to reproduce an effect following chronic supplementation

(Willis et al., 2009).

Upon consideration of the existing literature, it was clear that a number of

questions needed to be addressed:

1) Does ALA raise seizure threshold and stop seizures? Yehuda and

colleagues have reported that the administration of 40 mg/kg of dietary ALA for 21 days

raises seizure threshold and stops seizures (Yehuda et al., 1994; Rabinovitz et al., 2004).

This is somewhat confusing, since 40 mg/kg represents a very small increase in a rat’s

daily intake of ALA. The question of ALA’s effects on seizures at 40 mg/kg (Yehuda’s

dose) was addressed in the first experiment in this thesis (Experiment 1), whereas the

effect of higher doses was addressed in the second experiment (Experiment 2) (Chapters

2 and 3).

2) Does ALA raise levels of DHA in the brain? ALA is found in very low

amounts in the brain (<1%), and is unlikely that it raises seizure thresholds directly. It is

possible, however, that chronic ALA administration raises brain levels of DHA, the final

product of the omega-3 synthesis pathway. (DHA is the only PUFA found in abundance

in the brain.) This was the belief of Yehuda et al. (1994), who assayed brain PUFA after

chronic ALA administration and reported that chronic ALA significantly raised brain

phospholipid levels of DHA (Yehuda et al., 1996). They suggested that it was this

elevation in DHA that caused a rise in seizure threshold (Rabinovitz et al., 2004). This

44

question of whether administration of ALA actually does raise levels of DHA in the brain

was addressed in Experiment 2.

3) If ALA raises brain levels of DHA, how does it do so? The question of how

ALA raises brain DHA levels because the conversion efficiency of ALA to DHA is very

low in rodents (<0.5%) (Igarashi et al., 2006). This would seem to be too low a rate of

conversion to raise brain DHA levels via the normal synthesis pathway. The possibility

exists, however, that chronic ALA administration results in the migration of endogenous

DHA from other tissues into the brain. This possibility was addressed in Experiment 3

(Chapter 4).

4) Does acutely administered EPA raise seizure thresholds?

Voskuyl et al. (1998) have reported that EPA, acutely administered, raises

seizure threshold in a cortical stimulation model (Voskuyl et al., 1998). This is confusing,

since recent evidence suggests that the majority of the infused EPA that enters the brain

is immediately oxidized (Chen et al., 2009). It is hard to see, therefore, how it could raise

seizure thresholds. The question of whether acutely administered EPA raises seizure

thresholds was addressed in a pilot study which is presented in Appendix 1.

5) Does acutely administered DHA raise seizure thresholds? If DHA is the

PUFA that raises seizure thresholds, then thresholds should go up after the acute

administration of DHA. Voskuyl et al. (1998) in fact, have reported that acutely

administered DHA does raise seizure thresholds in a cortical stimulation seizure model

(Voskuyl et al., 1998). This finding, however, has yet to be replicated in a well-validated

seizure model. The effect of acute DHA - administered subcutaneously – was addressed

in Experiment 4 (Chapter 5).

45

6) Does dietary DHA raise seizure thresholds? If DHA is to be used in the

chronic treatment of epilepsy, it will probably be taken by mouth - as are the

anticonvulsant drugs. The question then arises as to whether the administration of DHA

by mouth will raise seizure thresholds. This question was addressed in Experiment 5

(Chapter 6). The question is not a trivial one since the pharmacokinetics of dietary DHA

are very different from the pharmacokinetics of DHA given by injection.

Experiment 5 involved a change in the animal model employed. Experiments 1-4

used the PTZ seizure model, the model originally used by Yehuda et al. (1994) In

Experiment 5, however, seizures were elicited via electrical brain stimulation - the

stimulus being delivered through chronically implanted depth electrodes in the brain.

This model allowed for the repeated testing of threshold. Repeated threshold testing

proved to be necessary in the dietary administration studies.

7) Does the hyperactivity preceding seizures release DHA from the neural

membrane? The results to Experiment 2 led us to hypothesize that it is the free

(unesterified) form of DHA that raises seizure thresholds. This hypothesis presented

some problems, however, since DHA in the brain is normally found only in its esterified

(phospholipid) form. This led to the further hypothesis that the hyperactivity in the brain

that precedes seizures (which would have occurred in both the PTZ and electrical

stimulation models) leads to the release of unesterified DHA – the released free DHA

then acting as a sort of endogenous anticonvulsant. This hypothesis was tested in

Experiment 6 (Chapter 7).

8) What is DHA’s mechanism of anticonvulsant action? Experiment 7

(Appenidix 2) represents an initial attempt to address DHA’s molecular mechanism of

46

anticonvulsant action. DHA was applied to hippocampal slices, and measurements of

neural excitation and inhibition were made.

1.14 Hypothesis and objectives

The central hypothesis of the present thesis was that DHA, the end product of the

n-3 PUFA synthesis pathway, would raise seizure thresholds. This hypothesis was based

on DHA’s demonstrated effects on neural excitability in vitro, and on its documented

anti-arrhythmic effects. Subordinate hypotheses are described in the chapters that

describe specific experiments.

The overall objective was to demonstrate that DHA would raise seizure thresholds

in rodent seizure models.

1.15 Animal models used to test the anticonvulsant effects of n-3 PUFA

There are several animal seizure models used to screen for anticonvulsants. The

five commonly used seizure models are the electrical stimulation model for simple and

complex partial seizures (Albright, 1983), the amygdala kindling model for complex-

partial seizures (Albright and Burnham, 1980), the maximal electroconvulsive shock test

for generalized tonic-clonic seizures (Edwards et al., 2002), the subcutaneous

pentylenetetrazol (PTZ) test for absence seizures (Depaulis et al., 1989) and the maximal

PTZ threshold test for generalized tonic-clonic seizures (Krall et al., 1978). These models

are commonly used for anticonvulsant drug screening because they have predictive

validitiy, which means that the response seen in animals is likely to predict the response

in humans.

47

Other seizure models exist, such as the kianic acid, pilocarpine, flourothyl and 6

Hz tests (Hartman et al., 2008; Willis et al., 2009). These tests, however, either lack

predictive validity, or their predictive validity has not been extensively confirmed in

pharmacological studies.

The present thesis assessed the effect of n-3 PUFA in only two seizure models –

the the maximal PTZ model (Chapters 2, 3 and 5) and the electrical stimulation seizure

threshold test (Chapter 6). Both models have predictive validity for anticonvulsant drugs.

PTZ induces tonic-clonic seizures when injected to rats, by antagonizing the GABAA

receptors (Macdonald and Barker, 1977). The electrical stimulation model involves the

stimulation of a brain focus such as the amygdala, with an incremental electrical current

until an afterdischarge (i.e. seizure) is evoked and visualized on the EEG (Goddard,

1967).

48

CHAPTER 2

LACK OF BENEFIT OF LINOLEIC AND α-LINOLENIC POLYUNSATURATED FATTY ACIDS ON SEIZURE LATENCY,

DURATION, SEVERITY OR INCIDENCE IN RATS

49

2 Experiment 1: Lack of benefit of linoleic and α-linolenic polyunsaturated fatty acids on seizure latency, duration, severity or incidence in rats

Forward

The purpose of Experiment 1 was to replicate the work of Yehuda and

collaborators, who had reported that the n-3 PUFA have anticonvulsant properties in rats

(Yehuda et al., 1994; Rabinovitz et al., 2004). These workers had chronically injected a 4

to 1 mixture of linoleic and α-linolenic acids (the “SR-3” formula) into rats via the

intraperitoneal (i.p.) route, and then seizure tested the rats using the maximal

pentylenetetrazol (PTZ) model. This combination of short-chain n-3 PUFA was designed

to raise docosahexaenoic acid (DHA) levels in the brain, since α-linolenic acid is

converted into DHA in the liver, and DHA is transported to the brain where it is

incorporated into phospholipids (Yehuda et al., 1996). Linoleic acid was provided in

addition to α-linolenic to optimize the conversion of α-linolenic into DHA (Yehuda et al.,

1996).

Yehuda and colleagues had reported that 40 mg/kg of the SR-3 mixture, injected

for 21 days, increased seizure latency and decreased seizure duration and severity in the

maximal PTZ model in Long-Evans rats (Yehuda et al., 1994; Rabinovitz et al., 2004).

The SR-3 mixture was also reported to have raised brain levels of the n-3 PUFA DHA

(Yehuda et al., 1996), which was assumed to have caused the anticonvulsant effects

(Yehuda et al., 1994; Rabinovitz et al., 2004).

Based on the work by Yehuda and colleagues, the hypothesis of Experiment 1

was that chronic administration of 40 mg/kg of the SR-3 compound would raise seizure

threshold and decrease duration and severity in rats. No assays were performed in

50

Experiment 1.

As will be presented below, the findings of Yehuda et al (1994) and Rabinovitz et

al. (2004) were not replicated. Chronic administration of 40 mg/kg of the SR-3 mixture

neither increased seizure latency nor reduced duration and severity in rats (Taha et al.,

2006a). Seizure occurrence was also recorded. As will be shown, control and SR-3

treated rats did not differ significantly in the incidence of myoclonic jerks, forelimb and

hindlinb clonus, forelimeb and hindlimb tonus or running fits.

The published manuscript in Epilepsy Research begins on the next page (Taha et

al., 2006a). The co-authors, Bogdan Baghiu Richard Lui and Kirk Nylen provided a

great deal of assistance with the i.p. injections and PTZ testing. Dr. David W.L. Ma

collaborated in the project and provided the gas-chromatography system for assessing the

composition of the stock solution containing α-linolenic and linoleic acids. Dr. W. M.

Burnham was the principal investigator.

51

Lack of benefit of linoleic and α-linolenic polyunsaturated fatty acids on seizure

latency, duration, severity or incidence in rats

Ameer Y. Taha1,2,3, Bogdan M. Baghiu1, 3, Richard Lui1, 3, Kirk Nylen1,3, David W.L. Ma2

and W. McIntyre Burnham1,3*

Departments of Pharmacology1 and Nutritional Sciences2, and University of Toronto

Epilepsy Research Program3, Faculty of Medicine, University of Toronto, Toronto,

Canada, M5S-1A8

*Address for correspondence:

Dr. W. McIntyre Burnham

Department of Pharmacology

Medical Sciences Building

University of Toronto

1 King’s College Circle

Toronto, ON. M5S 1A8

Canada

e-mail: [email protected]

52

2.1 Abstract

BACKGROUND: Polyunsaturated fatty acids have been reported to increase seizure

threshold, and to reduce seizure duration and severity in rats.

OBJECTIVE: The purpose of the present study was to test the anticonvulsant effects of

an essential fatty acid mixture containing linoleic and α-linolenic acids at a 4 to 1 ratio

(SR-3 compound), using the pentylenetetrazol seizure model in Long-Evans hooded rats.

RESULTS: There were no significant effects of SR-3 on seizure latency, duration or

severity (P>0.05). There were also no significant differences in the incidence of

myoclonic jerks, forelimb and hindlimb clonus, forelimb and hindlimb tonus or running

fits in rats that received SR-3, as compared to control rats (P>0.05).

CONCLUSION: Linoleic and α-linolenic polyunsaturated fatty acids have no beneficial

effects on seizure latency, duration, average severity or incidence.

53

2.2 Introduction

Epilepsy is a neurological disorder characterized by spontaneous, recurrent

seizures (Burnham, 1998). Although 60-70% of patients respond to conventional

anticonvulsant drug treatment, 30-40% of patients continue to experience seizures despite

the best anticonvulsant therapy (Vining, 1999). New therapies are required to help these

patients with drug-resistant seizures.

The high-fat ketogenic diet is a commonly used treatment for drug-resistant

epilepsy (Strafstorm, 1999). The classic ketogenic diet contains 80% fat, mainly in the

form of saturated fatty acids, derived from butter. Despite the diet’s efficacy (Vining,

1999), there is some concern regarding its unfavorable, atherogenic effect on plasma lipid

profiles. It elevates triglycerides, LDL-cholesterol and total cholesterol (Kwiterovich et

al., 2003; Fuehrlein et al., 2004).

Polyunsaturated fatty acids (PUFAs), which have anti-atherogenic properties,

have been considered as a potential alternate therapy for drug-resistant seizures

(Schlanger et al., 2002; Fuehrlein et al., 2004). PUFAs, such as docosahexaenoic acid and

arachidonic acid, are essential for normal brain function, due to their role as structural

components of membranes and their involvement in neurotransmission, cell signaling and

gene regulation (Rapoport, 2003; Kitajka et al., 2004). They are synthesized in the liver

from dietary linoleic and α-linolenic acids, or obtained directly from the diet (Sprecher,

2000).

Studies have reported that dietary or infused PUFAs, such as linoleic, α-linolenic,

arachidonic, eicosapentaenoic and docosahexaenoic acids, confer seizure protection in

cell cultures (Fraser et al., 1993; Vreugdenhil et al., 1996; Keros et al., 1997; Lauritzen et

54

al., 2000; Young et al., 2000), animal models (Yehuda et al., 1994; Voskuyl et al., 1998;

Blondeau et al., 2002; Rabinovitz et al., 2004), and, most recently, human cases of drug-

resistant epilepsy (Schlanger et al., 2002). In particular, Yehuda et al. (1994) have

reported that a mixture of linoleic and α-linolenic acids in a 4 to 1 ratio (i.e. the “SR-3

compound”) reduces latency and severity in young rats in the maximal pentylenetetrazol

(PTZ) seizure model. This observation has recently been replicated by Rabinovitz et al.

(2004).

Although these studies are promising, they are somewhat flawed, in that they

compared the anticonvulsant properties of the SR-3 PUFA mixture in experimental rats,

to control rats that were injected with saline as a vehicle, rather than the mineral oil

which was used to dissolve the PUFA mixture. Thus, it is not clear whether the reported

anticonvulsant effects of the SR-3 compound in the PTZ seizure model were due to its

PUFA content (i.e. - linoleic and α-linolenic acids), or to an anticonvulsant effect of the

mineral oil. The present study was, therefore, conducted to determine whether a PUFA-

based mixture, containing linoleic and α-linolenic acids in a 4 to 1 ratio (SR-3), confers

protection against PTZ induced seizures in rats, as compared to controls treated with

mineral oil.

2.3 Materials and methods

2.3.1 SR-3 preparation

The SR-3 mixture was prepared as described by Rabinovitz et al. (2004). Briefly,

0.05 ml of non-esterified α-linolenic acid (0.92 g/ml) and 0.2 ml of non-esterified linoleic

acid (0.90 mg/ml; Sigma-Aldrich, St. Louis, Missouri, USA) were dissolved in 0.73 ml of

55

mineral oil (Sigma-Aldrich, St. Louis, Missouri, USA), containing 0.02 ml of α-

tocopherol. The SR-3 mixture was stored at -20ºC until use. PTZ (Sigma-Aldrich, St.

Louis, Missouri, USA) was dissolved in 0.9% saline on the day of seizure testing.

2.3.2 Subjects and treatments

The following experiments were conducted according to the guidelines of the

Canadian Council of Animal Care, and approved by the Animal Care Committee of the

Faculty of Medicine of the University of Toronto. One-month-old male Long-Evans

hooded rats (Charles River, La Prairie, QC, Canada), weighing on average 116 g, served

as subjects. Subjects were individually housed in plastic cages with corn-cob bedding and

maintained in a 12 h light, 12h dark cycle (lights on at 7am) at 21ºC.

Water and Purina® rat chow were available ad libitum to both control and

experimental groups. The Purina rat chow contained (g/kg diet) 234.0 protein, 45.0 fat,

623.5 carbohydrates, 58.0 fiber, 0.3 vitamins and 39.2 minerals. After 7 days in the

facility, subjects were randomly divided into experimental (n=12) and control (n=8)

groups. The experimental subjects received daily intraperitoneal injections of 40 mg/kg

SR-3 in mineral oil. The control subjects received intraperitoneal injections of an equal

volume of vehicle (mineral oil). Subjects were injected daily for 21 consecutive days as

previously done by Rabinovitz et al. (2004), and were weighed each day prior to

receiving the injections.

2.3.3 Seizure testing

On experimental day 22, subjects were weighed and then seizure tested using the

PTZ procedure (Krall et al., 1978). Eighty mg/kg of PTZ were injected intraperitoneally.

56

The subjects were then placed in an open field and videotaped for 30 minutes. Videotapes

were subsequently scored by two independent “blinded” observers. Latency (seconds)

was measured between PTZ injection and the onset of: 1) myoclonic jerks, 2) forelimb

and hindlimb clonus, 3) forelimb and hindlimb tonus and 4) running fits. “Seizure

duration” was also measured. It was defined as the time from seizure onset (myoclonic

jerks, clonus, tonus or running fits) until the cessation of convulsions, unless the rat

exhibited severe running fits, in which case the rat was immediately sacrificed by an

intra-peritoneal injection of sodium pentobarbital (MCT Pharmaceuticals, Cambridge,

ON) and excluded from the seizure duration analysis. “Seizure severity” was scored

according to the following scale: stage 1, myoclonic jerks; stage 2, forelimb or hindlimb

clonus; stage 3, forelimb or hindlimb tonus; stage 4, running fits. Scores were averaged in

order to yield a measure of “seizure severity” (out of 4). “Seizure incidence”, defined as

the percentage of rats experiencing stage 1, stage 2, stage 3 and stage 4 seizures was also

determined. It was calculated by dividing the number of rats experiencing convulsions at

a certain seizure stage by the total number of rats, and multiplying by 100%.

2.3.4 Fatty acid analysis

The fatty acid composition of each component of the SR-3 mixture (ie - linoleic

acid, α-linolenic acid and mineral oil) was verified by gas chromatography as previously

described (Taha et al., 2005). Briefly, total fatty acids from each compound were

extracted from 4 - 5 samples, and derivitized according to the method of Folch et al

(1957). The resulting fatty acid methyl esters were quantified on a HP6890 gas

chromatograph (Agilent Technologies, Mississauga, ON), equipped with a flame

ionization detector, and separated on a fused silica capillary SP2560 100 m column

57

(Supelco, Bellefonte, PA) with 0.2 µm film thickness and 0.25 mm internal diameter.

One µl of fatty acid methyl esters from each sample was injected into the column in

splitless mode, using helium gas as a carrier at a constant flow rate of 1.3 ml per minute.

A 5-stage temperature program was used to acquire the fatty acid methyl ester profile.

The initial temperature was 60ºC. This was followed by a ramp up at 10ºC per minute to

170ºC and a 5 minute hold, a 5ºC per minute ramp up to 175ºC, a 2ºC per minute ramp up

to 185ºC, a 1ºC per minute ramp up to 190ºC, and a final 10ºC per minute ramp up to

240ºC and an 18 minute hold (total run time = 50 minutes). Fatty acid peaks were

identified by comparing the retention time of each peak against the retention times of a

fatty acid standard of known composition (GLC463, NuCheck Prep., ON, Can).

2.3.5 Statistical analysis

The data are presented as means ± SE. Data analysis was performed on Statistical

Analysis Software (version 8.02, SAS Institute, Cary, NC) and Sigma Stat v.3.2 (Jandel

Corporation). A 2-way analysis of variance was used to determine the effects of treatment

and time on body weight gain. Seizure threshold and duration were analyzed using an

unpaired t-test after verifying the normality of the data. The data for seizure duration and

severity did not have a normal distribution, and, therefore, the Mann-Whitney U test was

used. Outliers falling more than 2 standard deviations from the mean were excluded

from the statistical analyses. Fisher’s exact test was used to compare the incidence of

seizures at each stage in the control and SR-3 groups. Statistical significance was

accepted at P<0.05.

58

2.4 Results

2.4.1 Body weights

Body weights of control and experimental subjects are presented in Figure 1. All

subjects gained weight over time (P<0.05). There was no significant difference in body

weights, however, between control and experimental subjects at any time point (P>0.05).

2.4.2 Fatty acid profile of SR-3 constituents

The purity of the SR-3 constituents was verified by gas-chromatography. The

purities of linoleic and α-linolenic acids, on a percent composition basis, were 96.2 ± 1.6

and 91.3 ± 1.4 respectively. As expected, mineral oil, being a petroleum hydrocarbon

chain, contained no fatty acids.

2.4.3 Seizure latency

All animals in the control and experimental groups exhibited seizure activity after

PTZ administration. The data related to seizure latency are presented in Figure 2. As

indicated by Figure 2, latencies in most subjects were in the range of 60-70 seconds.

Outliers that were excluded from the analysis included one rat from the control group

which seized at 15 minutes post PTZ injection, and two rats from the SR-3 group which

respectively seized at 10 and 25 minutes after PTZ administration. With the outliers

excluded, mean seizure latency did not differ significantly between the control and the

SR-3 groups (69.7±2.8 s in controls versus 67.9±2.5 s in SR-3 the group; P>0.05).

2.4.4 Seizure duration

The data for seizure duration are presented in Figure 3. As indicated by Figure 3,

59

seizure duration in most subjects fell in the range of 80-100 seconds. One out of the eight

control rats and two out of the twelve experimental rats were excluded from the seizure

duration analysis because they had severe running fits, and were therefore euthanized

immediately. As a result, their seizure duration was not determined. The data for seizure

duration was not normally distributed, and therefore, the Mann-Whitney U test was used

to detect significance in ranking between the two groups. The results showed that seizure

duration did not differ significantly between the control and SR-3 groups (81.9±19.0 s in

controls versus 96.5±15.8 s in SR-3 group, P>0.05).

2.4.5 Seizure severity

The data for seizure severity are presented in Figure 4. As indicated, most

subjects had clonic seizures, that were ranked between 2 and 2.5. Because the data for

seizure severity were not normally distributed, the Mann-Whitney U test was used to

analyze the data. The results showed that there were no significant differences in seizure

severity between the control and SR-3 groups (P>0.05).

2.4.6 Seizure incidence within each seizure category

The incidence of seizures within each seizure score category is shown in Table 1.

There were no significant differences between the percentage of rats experiencing

myoclonic jerks (stage 1), forelimb and hindlimb clonus (stage 2), forelimb and hindlimb

tonus, running fits (stage 4), or forelimb and hindlimb tonus and running fits combined

(stage 3 + stage 4).

60

2.5 Discussion

The primary objective of the present study was to determine the potential

anticonvulsant properties of a PUFA-based mixture containing linoleic and α-linolenic

acids. This has been termed the “SR-3 mixture” (Yehuda et al., 1994). Our results

indicate that the SR-3 PUFA mixture did not alter seizure latency, duration or severity, as

compared to controls that received a mineral oil vehicle. It also did not alter the incidence

of myoclonic jerks, forelimb and hindlimb clonus, forelimb and hindlimb tonus or

running fits in SR-3 treated subjects, as compared to control subjects.

The lack of effect of the SR-3 mixture on seizure latency, duration, severity or

incidence contrasts with the reports of Yehuda et al. (1994) and Rabinovitz et al. (2004).

A similar experimental design and the same seizure model were used in this study, so

differences in design or seizure model can not explain the differences in the results.

The differing results, however, may relate to the fact that we injected our control

subjects with mineral oil (the SR-3 vehicle) instead of saline. This raises the possibility

that the mineral oil may possess anticonvulsant properties. Mineral oil is a petroleum

hydrocarbon containing n-alkanes and cyclic paraffin (Christensen et al., 2005). Previous

research has demonstrated that after 5 hours of oral administration of H3 labeled mineral

oil to rats, 80% of the label appeared in faeces, 1-5% was absorbed and stored in liver,

kidney and adipose tissue, whereas 15% was detected in brain, liver and other tissues as

an unidentified H3 labeled mineral oil metabolite (Ebert et al., 1966). It is possible that a

metabolite of the mineral oil accumulated in brain after 21 days of daily administration,

and increased seizure latency in the control group, thereby masking any potential

anticonvulsant properties of the SR-3 compound.

61

It is also possible that SR-3 did not raise brain PUFA composition to the

necessary threshold for detecting seizure protection following 3 weeks of SR-3

administration. SR-3 contains linoleic and α-linolenic acids, which are converted

primarily in the liver to their elongation / desaturation products, arachidonic and

docosahexaenoic acids, respectively (Sprecher, 2000). Arachidonic and docosahexaenoic

acids are considered to be the bioactive products of SR-3 in the brain, because 1) they

constitute more than 50% of brain total lipids (versus < 2% for linoleic and α-linolenic

acids), and 2) they have been reported to confer seizure protection in animal models and

humans (Voskuyl et al., 1998; Schlanger et al., 2002).

Brain PUFAs were not measured in the present study due to the possibility that

brain fatty acid composition would be altered after seizure induction (Kulagina et al.,

2000). SR-3, however, has been previously shown to significantly raise brain

arachidonate and docosahexaenoate composition (Yehuda et al., 1996). New evidence

suggests that the seizure protective effects of the ketogenic diet may in part be attributed

to its ability to increase brain PUFA composition, particularly arachidonic acid and

docosahexaenoic acid by at least 15% each (Fraser et al., 2003; Taha et al., 2005). Thus,

the possibility remains that the duration of the trial was too short to achieve the desired

threshold concentrations of arachidonic and docosahexaenoic acids (>15%) in brain, that

may lead to seizure protection.

We have suggested in previous publications that the elevation of ketones,

particularly acetone, may contribute to the anticonvulsant effects of the ketogenic diet in

humans (Likhodii et al., 2002). It is not clear, however, that the ketogenic diet produces

significant elevations of ketone bodies in rats (Nylen et al., 2005; Taha et al., 2005). Thus,

62

it is tempting to speculate that the previously reported alterations in brain PUFA

composition observed in rats fed a ketogenic diet (Taha et al., 2005) may potentially be

partially or fully responsible for the diet’s ability to ameliorate seizure severity (Cunnane,

2004; Taha et al., 2005).

We conclude that SR-3 did not alter seizure latency, duration, severity or

incidence in young rats. The lack of benefit of SR-3 on seizures may be due to the

possibility that PUFAs have no measurable influence on these outcomes at the dose used

in this study. Alternatively, a mineral oil metabolite may have raised seizure threshold

and reduced duration and severity in the control group to the extent of masking any

potential benefits of SR-3. Finally, it is possible that the duration of the trial was not long

enough to produce an effect on these parameters. Further studies assessing the potential

anticonvulsant effects of PUFAs at higher doses and prolonged periods of intake are

warranted.

Acknowledgements

The authors would like to acknowledge Mr. Jerome Cheng for his help in scoring the

seizures. NSERC provided financial support for this study.

63

Figure 1: Body weight gain

0

50

100

150

200

250

300

350

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Day

Wei

ght (

g)

ControlSR-3

Body weight gain in control and SR-3 subjects (n=8-12 per group).

64

Figure 2: Seizure latency

Seizure Latency

0

10

20

30

40

50

60

70

80

Control SR-3

Treatment

Late

ncy

(s)

Effect of SR-3 on seizure latency (n=7-9 per group)

65

Figure 3: Seizure duration

Seizure Duration

0.0

20.0

40.0

60.0

80.0

100.0

120.0

Control SR-3

Treatment

Dur

atio

n (s

)

Effect of SR-3 on seizure duration (n=7-10 per group)

66

Figure 4: Seizure severity

Seizure Severity

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Control SR-3

Treatment

Sco

re (a

rbitr

ary

units

)

Effect of SR-3 on seizure severity (n=8-12 per group)

67

Table 1: Percentage of rats experiencing stage 1, stage 2, stage 3, stage 4 and stage 3+4 in control and SR-3 groups Control SR-3

Stage 13 0%1 [0 / 8] 2 8% [1 / 12]

Stage 2 63% [5 / 8] 75% [9 / 12]

Stage 3 25% [2 / 8] 0% [0 / 12]

Stage 4 13% [1 / 8] 17% [2 / 12]

Stage 3+4 38% [3 / 8] 17% [2 / 12]

1Values represent the percentage of rats experiencing stage 1, stage 2, stage 3, stage 4 and the sum of stages 3 and 4 (stage 3+4) seizures in control and SR-3 groups. 2Number of rats experiencing seizures at a certain stage were divided by the total number of rats. 3Stage 1 = Myoclonic jerks Stage 2 = Forelimb / hindlimb clonus Stage 3 = Forelimb / hindlimb tonus Stage 4 = Running fits

68

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Rapoport, S.I., 2003. In vivo approaches to quantifying and imaging brain arachidonic and docosahexaenoic acid metabolism. J Pediatr. 143(4), S26-34.

Schlanger, S., Shinitzky, M., Yam, D., 2002. Diet enriched in omega-3 fatty acids alleviates convulsion symptoms in epilepsy patients. Epilepsia. 43(1), 103-4.

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Stafstrom, C.E. 2004. Dietary approaches to epilepsy treatment: old and new options on the menu. Epilepsy Curr. 4(6), 215-22.

Taha, A.Y., Ryan, M.A., Cunnane, S.C., 2005. Despite transient ketosis, the classic high-fat ketogenic diet induces marked changes in fatty acid metabolism in rats. Metabolism 54(9), 1127-32.

Vining, E.P.G., 1999. Clinical efficacy of the ketogenic diet. Epilepsy Res. 37, 181-90.

Voskuyl, R.A., Vreugdenhil, M., Xang, J.X., Leaf, A., 1998. Anticonvulsant effects of polyunsaturated fatty acids in rats, using the cortical stimulation model. Eur J Pharmacol. 341, 145-52.

Vreugdenhil, M., Bruehl, C., Voskuyl, R.A., Xang, J.X., Leaf, A., 1996. Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons. PNAS 93, 12559-63.

Yehuda, S., Carasso, R.L., Mostofsky, D.I., 1994. Essential fatty acid preparation (SR-3) raises seizure threshold in rats. Eur J Pharmacol. 254: 193-8.

Yehuda, S., Brandys, Y., Blumenfeld, A., Mostofsky, D.I., 1996. Essential fatty acid preparation reduces cholesterol and fatty acids in rat cortex. Int J Neurosci. 86(3-4), 249-56.

Young, C., Gean, P.W., Chiou, L.C., Shen, Y.Z., 2000. Docosahexanoic acid inhibits synaptic transmission and epileptiform activity in the rat hippocampus. Synapse 37, 90-4.

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CHAPTER 3

DOSE-DEPENDENT ANTICONVULSANT EFFECTS OF LINOLEIC AND α-LINOLENIC POLYUNSATURATED FATTY ACIDS ON

PENTYLENETETRAZOL INDUCED SEIZURES IN RATS

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3 Experiment 2: Dose-dependent anticonvulsant effects of linoleic and α-linolenic polyunsaturated fatty acids on pentylenetetrazol induced seizures in rats

Forward

Although the SR-3 fatty acid mixture did not alter seizure latency, duration,

severity or incidence in rats in Experiment 1, we realized that 40 mg/kg of the SR-3

mixture represents only a small fraction of a rat’s normal daily dietary intake of

polyunsaturated fatty acids (approximately 1.2%). Higher doses of the SR-3 mixture

were therefore attempted in Experiment 2.

The hypothesis was that chronic administration of the SR-3 compound at higher

doses would increase latency to seizure onset and reduce seizure severity in rats. Seizure

duration was not measured, in accordance with new guidelines proposed by the Animal

Care Committee at the University of Toronto. These guidelines indicate that subjects

must be euthanized immediately following the beginning of a generalized seizure.

Long-Evans male rats were therefore injected with 40 mg/kg, 400 mg/kg or 1000

mg/kg of the SR-3 fatty acid mixture containing α-linolenic acid. The 400 mg/kg dose

was equivalent to an increase in daily intake of linoleic and α-linolenic acid of 12%,

while the 1000 mg/kg was equivalent to an increase of 30%.

These higher doses were compared to a 40 mg/kg dose (original dose), and to

both saline and mineral oil control groups. The two control groups were used because

Yehuda et al. (1994) had used saline alone as their only control, whereas we had used

mineral oil, the vehicle for the SR-3 mixture (Taha et al., 2006a).

The doses were injected i.p. for 21 consecutive days, as in Yehuda’s experiments,

and the maximal PTZ model was used to seizure test the subjects.

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Unfortunately, by day 10, the 1000 mg/kg doses proved to be toxic, apparently

due to an inhibition of peristalsis caused by direct effects of DHA on the gastrointestinal

tract. Testing of the 1000 mg/kg was therefore discontinued, and the subjects were

sacrificed.

Symptoms of reduced food intake and impaired peristalsis were also evident to a

lesser extent in the group that received the 400 mg/kg dose. This group was therefore

taken off the SR-3 mixture and injected with saline for 5 consecutive days, after which

their food intake had returned to normal. The subjects were then placed on a lower dose

of 200 mg/kg for the remainder of the experiment. Two hundred mg/kg amounts to 6%

of a rat’s daily intake of linoleic and α-linolenic acids. Following seizure testing, all

subjects were sacrificed and their brains were assayed for n-3 PUFA levels.

N-3 PUFA levels were expressed as a paercentage of total fatty acids instead of

absolute concentrations (mg per g of tissue), because the variability (based on the

standard error) in the concentration data was higher than that of the percent composition

data (0.1 ± 0.02, 0.1 ± 0.01, 0.3 ± 0.1 and 0.2 ± 0.1 mg per g in the saline, mineral oil,

SR-3 40 mg/kg and SR-3 200 mg/kg groups respectively; P>0.05 by one-way ANOVA).

As indicated in the discussion of the following manuscript (section 3.5), the variability in

the unesterified fatty acid concentration data is probably due to the effects of ischemia

following decapitation. This could increase the chances of a type II error (see ‘data

presentation and statistical analysis’ section; section 3.3.7).

Notably, the percent composition data is considered to accurately reflect the

concentration data in the absence of changes in total fatty acid concentrations (Taha and

McIntyre Burnham, 2007). As indicated in the following manuscript, total fatty acid

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concentration in the unesterified fatty acid pool did not differ significantly between the

groups (Table 2).

As indicated below, the 200 gm/kg dose caused a significant increase in seizure

latency, although seizure severity was not significantly altered. The 200 mg/kg dose also

raised brain n-3 PUFA levels, expressed as a percentage of total fatty acids, in the

unesterified lipid pool.

The increase in seizure latency and brain DHA levels was not seen in the group

that received the SR-3 at 40 mg/kg. Although n-3 PUFA concentrations Subsequent

correlation analysis showed that in the 200 mg/kg group, seizure latencies were

significantly correlated with brain levels of unesterified n-3 PUFA.

The published manuscript in Epilepsia (Taha et al., 2009c) starts on the next page.

The co-authors of the paper are Elvis Filo, David W.L. Ma and W. McIntyre Burnham.

Elvis Filo helped with the animal work (i.p. injections and euthanasia). Dr. Ma was our

collaborator, and helped in the interpretation of the fatty acid data. Dr. Burnham was the

principal investigators in the study.

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Dose-dependent anticonvulsant effects of linoleic and α-linolenic polyunsaturated

fatty acids on pentylenetetrazol induced seizures in rats

Ameer Y. Taha1,2*, Elvis Filo1, David W.L. Ma3,4 and W. McIntyre Burnham1,2

1Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto,

Canada, M5S 1A8

2University of Toronto Epilepsy Research Program, Faculty of Medicine, University of

Toronto, Toronto, Canada, M5S 1A8

3Department of Nutritional Sciences, Faculty of Medicine, University of Toronto,

Toronto, Canada, M5S 3E2

4Department of Human Health and Nutritional Sciences, College of Biological Science,

University of Guelph, Guelph, Canada, N1G 2W1


Ameer Y. Taha

Department of Pharmacology





Canada


Running title: Anticonvulsant effects of polyunsaturated fatty acids

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3.1 Abstract

Purpose: Linoleic and α-linolenic polyunsaturated fatty acids, derived from plant oils,

have been reported to reduce neuronal excitability ex-vivo and in cell culture. The

evidence derived from animal seizure models, however, has been contradictory. The goal

of the present study was to assess the dose-dependent anticonvulsant effects of a fatty

acid mixture containing linoleic and α-linolenic acids in a 4 to 1 ratio (the “SR-3”

compound).

Methods: The maximal pentylenetetrazol seizure model and Long-Evans hooded rats

were used.

Results: Daily intraperitoneal injection of SR-3 for 21 consecutive days raised omega-3

polyunsaturated fatty acid (n-3 PUFA) composition in the unesterified fatty acid fraction

of brain lipids (P<0.05), and increased latency to seizure onset, when administered at 200

mg/kg (P<0.05), but not at 40 mg/kg (P>0.05). There were no significant effects of SR-3

on seizure occurrence or on seizure severity (P>0.05). A toxic effect of the SR-3

compound on peristalsis was observed at a dose of 400 mg/kg and above.

Conclusion: Linoleic and α-linolenic polyunsaturated fatty acids in a 4 to 1 ratio raises n-

3 PUFA composition of unesterified fatty acids in the brain and increases resistance to

pentylenetetrazol induced seizures.

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3.2 Introduction

Epilepsy is a neurological disorder characterized by spontaneous, recurrent

seizures (Burnham, 2006), which can be controlled in 60-70% of patients by the use of

anticonvulsant medications (Vining, 1999; Shorvon, 1996). Patients using

anticonvulsants often experience drug-related side-effects, such as fatigue, sedation and

nausea (Vining, 1999). Thus, new and less toxic therapies are required for people with

epilepsy.

N-3 polyunsaturated fatty acids (n-3 PUFA), derived from seafood and plants

such as flax, have been considered as a complementary to drug treatment for patients

with epilepsy (Schlanger et al., 2002; Yuen et al., 2005; Bromfield et al., 2008). N-3

PUFA are diet-derived lipids, which are essential for normal brain function and

development (Clandinin et al., 1980; Crawford et al., 2002). They are important structural

components of neuronal membranes, and are involved in modulating neurotransmission,

cell signaling and gene regulation (Rapoport, 2003; Kitajka et al., 2004).

The most abundant n-3 PUFA in the brain is docosahexaenoic acid (DHA, 22:6n-

3). DHA, destined for the brain, can be synthesized in the liver from α-linolenic acid. It

has been suggested that DHA synthesis is optimal when the n-6 PUFA linoleic acid is

also present at a specific ratio. In particular, it has been proposed that a 4 to 1 ratio of

linoleic acid and α-linolenic acid is best for raising brain DHA levels in rats. Linoleic

and α-linolenic acid in a 4 to 1 ratio has been termed the “SR3 compound” (Yehuda et al.,

1996).

The best evidence suggesting possible anticonvulsant properties for n-3 PUFA

including α-linolenic, eicosapentaenoic and docosahexaenoic acids has come from

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studies involving cell cultures and ex-vivo preparations (Vreugdenhil et al., 1996; Xiao

and Li, 1999; Lauritzen et al., 2000; Young et al., 2000). These studies have shown that

n-3 PUFA confer protection against seizures by increasing the threshold for action

potentials and by extending the refractory period in neurons. This action appears to result

from a partial inhibition of sodium and calcium voltage-gated channels.

The evidence derived from in vivo studies in animal seizure models, however, has

been contradictory. It has been reported that co-administration of linoleic acid with α-

linolenic acid in a 4 to1 ratio (i.e. the “SR-3 compound”) raises brain DHA levels in rats

(Yehuda et al., 1996), and increases resistance to pentylenetetrazol (PTZ) induced

seizures, when it is injected intraperitoneally (i.p.) at 40 mg/kg for 21 consecutive days

(Yehuda et al., 1994; Rabinovitz et al., 2004). These findings, however, have failed to be

replicated in a recent study which used the same dose of the SR-3 mixture and the same

seizure model (Taha et al., 2006).

Actually, it would be surprising if a dose of 40 mg/kg of the SR-3 mixture could

increase resistance to PTZ induced seizures, because the 40 mg/kg dose represents an

increase of only 1.2% in a rat’s daily intake of these fatty acids. Higher SR-3 doses would

be more likely to achieve a physiologically relevant rise in brain DHA, and this might be

accompanied by a significant increase in seizure threshold.

The goal of the present study was to assess the possible anticonvulsant effects of

the SR-3 mixture in a dose-response paradigm involving higher doses of the compound.

N-3 PUFA composition in brain phospholipids and unesterified fatty acids was also

determined.

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The SR-3 compound was prepared by mixing non-esterified linoelic acid (0.90

g/ml; Sigma-Aldrich, St. Louis, Missouri, USA) and non-esterified α-linolenic acid (0.92

mg/ml; Sigma-Aldrich, St. Louis, Missouri, USA) at a 4 to 1 ratio, in a vehicle that

consisted of 0.73 ml of mineral oil (Sigma-Aldrich, St. Louis, Missouri, USA ) and 0.02

ml of α-tocopherol (Sigma-Aldrich, St. Louis, Missouri, USA). Four different doses of

the SR-3 compound were prepared – 40 mg/kg, 200 mg/kg, 400 mg/kg and 1000 mg/kg.

Each dose was dissolved in the same fixed volume of mineral oil (0.73 ml) and α-

tocopherol (0.02 ml). After preparation, the SR-3 mixture was stored at -20ºC until

further use, in order to minimize oxidation of the linoleic and α-linolenic polyunsaturated

fatty acids.


All experimental procedures were conducted in accordance to the guidelines of

the Canadian Council of Animal Care, and approved by the Animal Care Committee of

the Faculty of Medicine of the University of Toronto.

One-month-old male Long Evans Hooded rats (Charles River, La Prairie, QC,

Canada), weighing on average 151 g at the start of the experiment, served as subjects.

Subjects were individually housed in plastic cages with corn-cob bedding in a vivarium

maintained on a 12 h light, 12 h dark cycle (lights on at 7am) and at a temperature of

21ºC. Water and rat chow (Teklad Global, 2018 18% Protein Rodent Diet) were available

ad libitum. The rat chow contained (g/kg diet) 189 protein, 60 fat, 554 carbohydrates, 38

80

fiber, 59 ash and 100 moisture. The fat component of the diet mainly contained (% of

total fatty acids) palmitate (13.5%), stearate (2.7%), oleate (22.3%), linoleate (55.5%)

and α−linolenate (4.9%).

After 7 days in the facility, subjects were randomly divided into five groups

which initially received daily i.p. injections (starting at 11 a.m.) of: 1) 0.9% saline (0.035

ml; n=10), 2) mineral oil vehicle (0.035 ml; n=7), 3) SR-3 40 mg/kg (in 0.035 ml vehicle;

n=8) 4), SR-3 400 mg /kg (in 0.035 ml vehicle; n=8) or 5) SR-3 1000 mg/kg (in 0.035 ml

vehicle; n=8). The saline and mineral oil groups served as controls. All subjects were

intended to be injected with their respective treatments for 21 consecutive days. By the

10th day of the experiment, however, it was clear that the injections were causing toxicity

in the 1000 mg/kg group. The symptoms consisted of low weight gain, low food intake

and bloating, which appeared to be caused by impaired peristalsis. This group was

therefore terminated, and the subjects were euthanized with CO2. These symptoms were

also evident to a lesser extent in the group that received the 400 mg/kg daily dose.

Therefore, from days 10 to 15, these rats were injected with saline and not the SR-3. The

bloating was gone, and food intake had returned to normal by day 16. The injections were

therefore resumed, but at a lower dose of 200 mg/kg. These animals then became the 200

mg/kg group. Overall, they received 16 days of SR-3 injections, the last 9 days at 200

mg/kg.

All subjects were weighed each day prior to receiving the injections. Food intake

was also measured every day by measuring the difference in weight in the stainless steel

dish which contained the food.

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3.3.3 Seizure testing

On day 22, after 21 days of treatment, the subjects were weighed and

subsequently seizure tested using the maximal PTZ procedure (Fisher, 1989), starting at

11 a.m. At high (“maximal”) doses, PTZ models tonic-clonic generalized seizure attacks

in humans (Fisher, 1989). As previously described (Taha et al., 2006), subjects received

eighty mg/kg of PTZ via the i.p route. Subjects were then placed in an open field and

videotaped for 30 minutes. Videotapes were subsequently scored by two independent

“blinded” observers. Seizure latency, severity and occurrence were scored. “Seizure

latency” (seconds) was scored as the interval between injection and the onset of a

myclonic jerk or forelimb clonus. “Seizure severity” was scored according to the

following scale: stage 1 - myoclonic jerks; stage 2 - forelimb or hindlimb clonus; stage 3

- forelimb or hindlimb tonus; and stage 4 - running fits. Scores were averaged based on

the maximum seizure score displayed by the subject over the half-hour observation

period, in order to yield a measure of “seizure severity” (out of 4). Subjects that displayed

a running fit, however, were immediately euthanized using a lethal injection of sodium

pentobarbital (MTC Pharmaceuticals, Cambridge, ON), and their seizure score was

ranked as stage 4. Accordingly, 50%, 25%, 14% and 13% of the saline, mineral oil, SR-3

40 mg/kg and SR-3 200 mg/kg groups were respectively euthanized due to a stage 4

running fit. “Seizure occurrence” was scored according to the severity scale, seizures

being scored as “present” if any of the stages described above was present, and “absent”

if none of them was present.

At the end of the 30-minute seizure test, subjects were euthanized via a lethal

intraperitoneal injection of sodium pentobarbital, following which the whole brain and

82

liver were excised and snap frozen in liquid nitrogen. The samples were stored at –80o C

for later analysis.

3.3.4 Brain lipid analysis

The left hemisphere of the brains was used for phospholipid and unesterified free

fatty acid analysis, the right hemisphere being reserved for possible future analyses. Total

lipids were extracted according to the extraction method of Folch et al. (1957), following

the addition of diheptadecanoyl L-α-phosphatidylcholine (1 mg) and non-esterified

heptadecaenoic acid (1.5 mg) (Sigma, St. Louis, Mo) in chloroform as internal standards,

to approximately 0.8 g of brain tissue. The samples were then homogenized in

chloroform/methanol (2:1, v/v) in order to extract total lipids. Saline (0.9%) was added an

hour later in order to separate the polar phase. Following distinct separation of the phases,

the lower chloroform phase was transferred to new 15 ml glass screw cap tubes with

TeflonR lined caps, dried under a gentle stream of nitrogen and reconstituted in 2 ml of

hexane.

Phospholipids and free fatty acids in the brain total lipid extracts were

fractionated by lipid thin-layer chromatography (TLC) using 20 x 20 cm silica gel plates

(Whatman LK6D plates, precoated with 250 μm of Silica Gel 60A). Separate lanes were

spotted with phospholipids or free fatty acid standards. The plates were developed using

hexane, diethyl ether, and acetic acid (80:20:1 by volume) in covered glass tanks for 35

minutes. Bands corresponding to phospholipids and free fatty acids were viewed under

ultraviolet light, after lightly spraying with 8-anilino-1-naphthalenesulfonic acid. The

bands were scraped off each plate, into 15 ml glass screw cap tubes with Teflon lined

caps, and directly methylated by incubating with hexane (2 mL) and 14% methanolic BF3

83

(2 mL) at 100°C for 1 hour. Deionized water (2 mL) was then added to separate the

phases. The upper hexane phase was extracted, dried under nitrogen and reconstituted in

hexane for analysis by gas chromatography.

3.3.5 Fatty acid composition of the SR-3 compound

The fatty acid composition of each component of the SR-3 mixture (ie - linoleic

acid, α-linolenic acid and mineral oil) was verified by gas chromatography.

Approximately 0.1 ml of each compound was dissolved in 2 ml of hexane and directly

derivatized in 14% boron trifluoride in methanol (2 mL, Sigma) for 1 hour at 100oC.

Deionized water (2 mL) was then added to separate the phases. The upper hexane phase

was extracted, dried under nitrogen and reconstituted in hexane for analysis by gas

chromatography.

3.3.6 Fatty acid methyl ester analysis by gas-chromatography

Fatty acid methyl esters (FAME) in phospholipids and unesterified fatty acids of

brain were analyzed using an Agilent 6890 gas-chromatography system equipped with a

flame ionization detector and a SP2560 fused silica capillary column (Supelco; 100 m,

0.25 µm film thickness, 0.25 mm ID, Pennsylvania, USA). One µL of fatty acid methyl

esters from each sample were injected in splitless mode. The injector and detector ports

were set at 250˚C. Methyl esters were eluted using a temperature program set initially at

60˚C for 5 min, 10˚C/min until 170˚C, 5˚C/min until 175˚C, 2˚C/min until 185˚C,

1˚C/min until 190˚C, and 10˚C/min until 240˚C. Helium was used as a carrier gas, at a

constant flow rate of 1.3 mL/min. Fatty acid peaks were identified by comparing the

retention time of each peak against the retention times of an authentic fatty acid standard

84

of known composition (GLC463, NuCheck Prep., ON, Can).

Fatty acid profiles of the SR-3 constituents was determined on a 30m x 25mm

capillary column (J and W Scientific, DB-23, Folsom, CA) in the Agilent 6890 gas-

chromatography system equipped with a flame ionization detector. One µl of FAME

from each sample was injected into the column in splitless mode, using helium gas as a

carrier, at a constant flow rate of 0.7 ml per minute. A three stage temperature program

was used to acquire the fatty acid methyl ester profile. Initial temperature setting was at

50ºC with a 2 minute hold, followed by a ramp up at 20ºC per minute to 170ºC and a 1

minute hold, and a final 3ºC per minute ramp up to 212ºC followed by a 10 minute hold.

Fatty acid peaks were identified by comparing the retention time of each peak against the

retention times of authentic fatty acid standards of known composition (GLC463,

NuCheck Prep., ON, Can).

3.3.7 Data presentation and statistical analysis

All data are presented as means ± SEM. The fatty acid profile data for brain

phospholipids and unesterified fatty acids are expressed as a percentage of total fatty

acids and not absolute concentrations (mg per g of wet tissue). This is because 1) no

significant differences between the groups were observed in the total pool of

phospholipids and unesterified fatty acids, and therefore the fatty acid percent

composition data in general, reflected the absolute concentration data, and 2) the

variability in the percent composition data is lower as compared to the absolute

concentration data, and therefore the chances of a type II error are minimized. Data

analysis was performed on Statistical Analysis Software (version 8.02, SAS Institute,

Cary, NC). The group which received the SR-3 at 1000 mg/kg was excluded from the

85

statistical analyses, because it was discontinued. A 2-way repeated measures analysis of

variance was used to determine the effects of treatment and time on body weight gain and

food intake. A 1-way ANOVA was used to determine the effect of treatment on seizure

latency, severity and fatty acid concentrations of phospholipids and free fatty acids.

Outliers falling more than 2 standard deviations from the mean were excluded from the

statistical analyses. Post-hoc Tukey t-tests were applied when appropriate. The chi-square

test was used to assess differences in seizure occurrence. Statistical significance was

accepted at P<0.05.

3.4 Results

3.4.1 Fatty acid profile of the SR-3 constituents

The fatty acid composition of the components used to mix the SR-3 compound

was determined in each stock bottle by gas-chromatography. The composition of linoleic

acid and α-linolenic acid, expressed as a percentage of total fatty acids, was 95.8 ± 1.9

(n=3) and 99.7 ± 0.3 (n=3) respectively. As expected, no fatty acids were detected in the

mineral oil (n=2).

3.4.2 Body weights

Body weights of control and experimental subjects are presented in Figure 1. All

subjects gained weight over time (P<0.05). There was a significant difference in body

weights, however, between controls and the experimental subjects that received the SR-3

at 200 mg/kg (P<0.05). Weight gain in the SR-3 200 mg/kg rats was significantly lower

than weight gain in the control saline and mineral oil subjects or the subjects that

86

received the SR-3 at 40 mg/kg (P<0.05). In the SR-3 200 mg/kg group, the lower body

weight was evident on day 4 and persisted until the end of the experiment.

3.4.3 Food intake

The data related to food intake are presented in Figure 2. All rats consumed more

food over time (P<0.05). Food intake, however, in subjects that received the SR-3 200

mg/kg was significantly lower, as compared to food intake in subjects that were injected

with saline, mineral oil or SR-3 40 mg/kg (P<0.05). The differences in food intake

between the SR-3 200 mg/kg and the other groups were no longer statistically different

during the last 10 days of the experiment.

3.4.4 Possible physiological signs of toxicity – Liver weight and percent liver of

body weight

Liver weight and liver weight as a percentage of total body weight are indirect

markers of treatment-induced toxicity, with greater liver mass being indicative of

potential treatment-induced stress. The data for liver weight and liver percentage of body

weight are presented in Figures 3-A and 3-B, respectively. As shown in Figure 3A, liver

weight was significantly lower in rats that received the SR-3 200 mg/kg treatment, as

compared to those that received the saline, mineral oil or SR-3 40 mg/kg treatments

(P<0.05). As indicated by Figure 3B, however, no significant differences were observed

between the groups when the liver weight was expressed as a percentage of total body

weight (P>0.05).

3.4.5 Seizure occurence

All animals in the control and experimental groups exhibited seizure activity after

87

PTZ administration, except for one rat in the saline group and one rat in the SR-3 200

mg/kg group. There were no statistically significant differences between the groups

(P>0.05).

3.4.6 Seizure latency

The data related to seizure latency are presented in Figure 4. Outliers that were

excluded from the statistical analysis included one rat in the saline group, one rat in the

mineral oil group and one rat in the SR-200 mg/kg group, with latencies of 5.8 minutes,

3.4 minutes and 12.3 minutes respectively. It is not surprising to have a few rats that do

not seize within the first few minutes following PTZ injection (Krall et al., 1978; Taha et

al., 2006). A dose titration was not performed in a subset of rats, and it is therefore likely

that the PTZ dose used in this study was below the ED100.

As indicated by Figure 4, subjects that received the SR-3 at 200 mg/kg exhibited

an increase in latency to seizure onset. Their latency was approximately three-fold higher,

as compared to latencies in the other three groups, which had similar, shorter latencies.

Statistical analysis showed that seizure latency in the SR-3 200 mg/kg group was

significantly greater than latencies in the saline, mineral oil and SR-3 40 mg/kg groups

(P<0.05).

3.4.7 Seizure severity

Seizure scores were obtained for all the subjects that exhibited seizure activity,

and were averaged within each experimental group. Averaged seizure scores were similar

in all of the four experimental groups, with group averages being 3.2 ± 0.3, 3.1 ± 0.2, 3.3

± 0.2 and 2.3 ± 0.4 for the saline, mineral oil, SR-3 40 and SR-3 200 groups respectively.

88

Statistical analysis revealed no significant differences among the groups (P=0.2),

although a strong tendency towards a reduction in seizure severity was noticeable in the

SR-3 200 mg/kg group.

3.4.8 Brain phospholipid fatty acid composition

In order to define differences in the localization of n-3 PUFA within brain lipids,

total lipid extracts were subjected to thin layer chromatography to separate membrane

phospholipids and unesterified free fatty acids.

The data for brain phospholipid fatty acid profile are shown in Table 1. Total

brain phospholipid concentrations, expressed as mg per g of tissue, as well as

phospholipid fatty acid percent composition, expressed as a percentage of total

phospholipids, did not differ significantly between the groups (P>0.05).

3.4.9 Brain unesterified fatty acid composition

The data related to brain fatty acid profile of the unesterified fatty acid fraction

are presented in Table 2. Total concentrations of unesterified fatty acids did not

significantly differ between the groups (P>0.05). There were no significant differences in

the percent composition of total saturated, monounsaturated and polyunsaturated fatty

acids within the unesterified fatty acid fraction (P>0.05). Total n-3 PUFA percent

composition, however, was highest in the SR-3 200 mg/kg group, as compared to the

saline, mineral oil or SR-3 40 mg/kg groups. Statistical analysis showed that n-3 PUFA

percent composition in the SR-3 200 mg/kg group was significantly different from n-3

PUFA percent composition in the saline group (P<0.05), but not the mineral oil and SR-3

40 mg/kg groups. Total n-6 PUFA percent composition were significantly lower in the

89

SR-3 40 mg/kg group, as compared to the saline and mineral oil groups (P<0.05), but not

the SR-3 200 mg/kg group. The n-6 to n-3 PUFA ratio was significantly lower in the SR-

3 40 mg/kg and SR-3 200 mg/kg groups as compared to the saline and mineral oil groups

(P<0.05). This was due to the increase in the percent composition of n-3 PUFA in the

SR-3 200 mg/kg group, and the slight decrease in n-6 PUFA percent composition in the

SR-3 40 mg/kg group.

3.4.10 Correlation between seizure latency and n-3 PUFA levels within the

unesterified fatty acid fraction

Pearson’s correlation analysis was performed in order to determine whether the

observed changes in seizure latency were correlated with n-3 PUFA levels in the

unesterified fatty acid fraction. As shown in Figure 5, seizure latency was positively

correlated to n-3 PUFA percent composition within the unesterified fatty acid fraction (R

= 0.65, P<0.001).

3.5 Discussion

The findings of the present study suggest that the chronic administration of the

SR-3 compound, a mixture of linoleic and α-linolenic and acids, significantly increases

seizure latency in the maximal PTZ model at a dose of 200 mg/kg. No effect was seen at

the lower dose of 40 mg, whereas toxicity was seen at the higher doses of 400 and 1000

mg/kg.

The finding of increased seizure latency at 200 mg/kg suggests that chronic

administration of n-3 PUFA has the ability to increase seizure threshold. These data are

90

in general agreement with past studies that have shown anticonvulsant properties of n-3

PUFA in cell cultures and ex-vivo preparations (Vreugdenhil et al., 1996; Xiao and Li,

1999; Lauritzen et al., 2000; Young et al., 2000).

The failure to find anticonvulsant effects at a dose of 40 mg/kg of the SR-3

preparation is in agreement with the past findings of our own research group (Taha et al.,

2006), and in contrast to past reports by Yehuda and colleagues (Yehuda et al., 1994;

Rabinovitz et al., 2004). The reasons for these differing findings are not clear, since

similar experimental paradigms and the same strain of rats were used in the different

experiments (Rabinovitz et al., 2004). The higher dose of 200 mg/kg achieved a larger

rise in brain n-3 PUFA composition within the unesterified fatty acid fraction, and this

was accompanied by a significant increase in seizure latency.

Brain n-3 PUFA percent composition in the unesterified fatty acid, but not the

phospholipid fraction was highest in the animals that received the SR-3 200 mg/kg (Table

2). This was associated with longer seizure latency (Figure 5), suggesting that n-3 PUFA,

in their unesterified, as opposed to their incorporated form, protect against seizures.

These findings are consistent with a previous study which showed that tail vein infusion

of unesterified n-3 PUFA, protects against focal and generalized seizures induced by

electrical stimulation in the cortex (Voskuyl et al., 1998).

Brain concentrations of the unesterified fatty acid fraction in this study, exceeded

values reported in the literature by at least 34 fold (Deutsch et al., 1997). These higher

unesterified fatty acid concentrations probably reflect ischemia-induced release of free

fatty acids from phospholipid membranes, due to decapitation (Rapoport, 1995; Deutsch

et al., 1997; Bazinet et al., 2005). Deutsch et al. (1997) reported that the contribution of

91

ischemia-induced release of free fatty acids overestimates the actual unesterfied fatty acid

pool in brain by at least 7-fold. The magnitude of increase in the unesterified fatty acids

reported by Deutsch et al. (1997) is still lower than what we observed (34 fold increase),

but this is likely due to the additional effects of PTZ on free fatty acid release from

membrane phospholipids (Bazan, 1971). Although microwaving the brains prior to

decapitation would provide a more accurate estimate of the amount of unesterified fatty

acid concentrations in brain (Rapoport, 1995; Deutsch et al., 1997; Bazinet et al., 2005),

the contribution of ischemia and PTZ induced release of free fatty acids does not explain

the differing n-3 PUFA profiles between the four groups (Table 2).

The differing fatty acid profiles observed in the unesterified fatty acid pool most

likely reflect differences in the release of free fatty acids from phospholipid membranes

due to PTZ administration. PTZ, in addition to being excitatory, has been shown to

increase neuroinflammation by increasing the production of pro-inflammatory

prostaglandins (Seregi et al., 1990). N-3 PUFA such as eicosapentaenoic (20:5n-3) and

docosahexaenoic acids (22:6n-3) have been reported to protect against

neuroinflammation through their autacoid metabolites, which are eicosanoids and

docosanoids respectively (Hong et al., 2003; Marcheselli et al., 2003; Lukiw et al., 2005).

The increased composition of n-3 PUFA in the SR-3 200 mg/kg group is most likely

indicative of increased utilization of n-3 PUFA for eicosanoid and docosanoid production,

to counteract the pro-inflammatory effects of PTZ. There is some evidence indicating that

preventing or reducing neuroinflammation in brain by using anti-inflammatory agents

such as aspirin, can protect against PTZ induced seizures (Tandon et al., 2003; Tu and

Bazan, 2003; Dhir et al., 2006; Oliveira et al., 2008). Thus, the release of anti-

92

inflammatory lipid mediators derived from n-3 PUFA is a possible explanation for the

observed anticonvulsant effects of the SR-3 mixture.

We cannot exclude the possibility that ketone bodies such as acetone, acetoacetate

and β-hydroxybutyrate, which also have anticonvulsant properties (Likhodii and

Burnham, 2002; Rho et al., 2002; Likhodii et al., 2003; Ma et al., 2007), may have

contributed to the observed anticonvulsant effects of the SR-3 compound. This is because

the lower body weight gain in the SR-3 200 mg/kg group (Figure 1), which occurred

despite similar food intake from days 12 to 21 relative to the other three groups (Figure 2),

is suggestive of enhanced β-oxidation and possibly ketosis (Cunnane, 2004).

Polyunsaturated fatty acids such as α-linolenic acid have been reported to decrease

weight gain in mice (Cunnane et al., 1986) by activating the transcription of genes

involved in β-oxidation (Ide et al., 1996). The increase in β-oxidation may have resulted

in an elevation of ketone bodies. Levels of ketone bodies such as acetone, were not

measured in this study because all subjects received PTZ, which has previously been

shown to increase basal levels of acetone in plasma (Nylen, 2005). It is worth noting,

however, that in contrast to humans, rats are incapable of chronically sustaining or

achieving clinical levels of ketosis (>2 mM) after being placed on a high-fat ketogenic

diet, which is reported in several studies to chronically raise plasma ketone bodies in

humans, but only transiently in rats (Likhodii et al., 2000; Taha et al., 2005; Musa-Veloso

et al., 2006). Thus, the anticonvulsant effects of the SR-3 are unlikely due to elevated

ketone bodies.

It is not surprising that the observed increase in total unesterified fatty acid

concentrations was not reflected by a decrease in the concentration of total phospholipids.

93

This is because, on a relative basis, phospholipids make up the majority of total brain

lipids. On a quantitative basis, they exceeded the concentration of unesterified fatty acids

by approximately 8-20 fold (Tables 1-2). Thus, it would be difficult to account for the

release of free fatty acids from phospholipids, by measuring changes in phospholipid

concentrations. Alternatively, the use of radiolabeled fatty acid tracers in future studies

may provide more insight into the release of free fatty acids from membrane

phospholipids (Rapoport, 2003).

Toxicity was found at the doses of 400 and 1000 mg/kg. The symptoms consisted

of low weight gain, low food intake and bloating. These symptoms appeared to be

caused by impaired peristalsis, which may have been caused by a direct effect of the

injected fatty acids on the gastrointestinal tract following their diffusion through the

peritoneal cavity. Future experiments will involve injection of the SR-3 compound via

the subcutaneous (s.c.) route, in order to avoid localized exposure of the gastrointestinal

tract to high doses of the SR-3. It is possible that higher SR-3 doses will be tolerated

when administered via s.c. or oral routes, and that these may have larger effects on

seizure severity and occurrence as well as on seizure latency. Administering the SR-3

through the oral route is of considerable importance, as this will have practical

implications for future clinical studies.

While the present findings need to be confirmed by further studies, they do

provide support for the idea that n-3 PUFA might provide a treatment for patients with

epilepsy (Schlanger et al., 2002; Yuen et al., 2005; Bromfield et al., 2008). A diet rich in

n-3 PUFA might supplement the anticonvulsant effects of antiepileptic drugs or the

ketogenic diet (Fuehrelein et al., 2005; Dahlin et al., 2007; Taha and Burnham, 2007), or

94

even possibly offer an alternative to anticonvulsant drug therapy.

ACKNOWLEDGEMENTS

We would like to thank Mr. Jerome Cheng for assisting in scoring the seizures. Funding

for this study was provided by the Bahen Chair in Epilepsy Research grant to Dr. W.M.

Burnham, the Natural Sciences and Engineering Research Council grant to Dr. D.W.L.

Ma and the Canadian Institutes of Health Research doctoral research award (Fredrick

Banting and Charles Best Canada Graduate Scholarships) to A.Y. Taha.

DISCLOSURES

The authors declare that there are no competing personal or financial interests. The work

described within is consistent with the journal’s guidelines for ethical publication.

95

Figure 1: Effect of treatment on body weight gain

100

150

200

250

300

350

400

1 4 6 8 10 12 14 16 18 20 22

Time (days )

Wei

ght (

g)

S a lineMinera l OilS R-3 40 mg /kgS R-3 200 mg /kgS R-3 1000 mg /kg

The effects of daily saline (dark circle, solid line), mineral oil (dark square, solid line),

SR-3 40 mg/kg (Open diamonds, dotted line), SR-3 200 mg/kg (open triangle, dotted

line) and SR-3 1000 mg/kg (open circle, dotted line) injections on body weight gain.

Subjects that received the SR-3 at 1000 mg/kg were excluded because they were

terminated before the study ended. Body weight gain of the SR-3 200 mg/kg group was

significantly lower over time, as compared to the saline, mineral oil and SR-3 40 mg/kg

groups: P<0.05 for significant main effect of treatment and time on body weight gain, by

2-way repeated measures ANOVA

96

Figure 2: Effect of treatment on food intake

0

5

10

15

20

25

30

35

40

2 6 8 10 12 14 15 16 18 20 22

Time (days )

Am

ount

(g)

S a lineMinera l OilS R-3 40 mg /kgS R-3 200 mg /kgS R-3 1000 mg /kg

The effects of daily (dark circle, solid line), mineral oil (dark square, solid line), SR-3 40

mg/kg (Open diamonds, dotted line), SR-3 200 mg/kg (open triangle, dotted line) and

SR-3 1000 mg/kg (open circle, dotted line) injections on food intake. Subjects that

received the SR-3 at 1000 mg/kg were excluded because they were terminated before the

study ended. Food intake of the SR-3 200 mg/kg group was significantly lower during the

first 12 days, as compared to the saline, mineral oil and SR-3 40 mg/kg groups: P<0.05

for significant main effect of treatment and time on food intake, and an interaction

between treatment and time, by 2-way repeated measures ANOVA

97

Figure 3-A: Liver weight

0

2

4

6

8

10

12

14

16

18

20

Saline Mineral Oil SR-3 40 mg/kg SR-3 200 mg/kg

Treatment

Live

r w

eigh

t (g

) b

aa a

Figure 3-B: % liver weight of total body weight

0

1

2

3

4

5

6


Treatment

% L

iver

of B

ody

Wei

ght

98

Data are mean ± SEM of = n=7-9 for each group. Bars marked with different letters

differed significantly from each other, as determined by 1-way ANOVA and Tukey’s

post-hoc test.

A) Liver weight: The difference between the SR-3 200 mg/kg group and the other groups

was statistically significant.

B) % Liver weight of total body weight: There were no significant differences among the

different groups.

99

Figure 4: Seizure latency following PTZ administration

0

20

40

60

80

100

120

140

160

180

200


Treatment

Seiz

ure

Late

ncy

(s)

a a a

b

Data are mean ± SEM of = n=6-8 for each group. Bars marked with different letters

differed significantly from each other, as determined by 1-way ANOVA and Tukey’s

post-hoc test. The difference between the SR-3 200 mg/kg group and the other groups

was statistically significant.

100

Figure 5: Correlation between seizure latency and brain n-3 PUFA composition

0

50

100

150

200

250

300

350

0 5 10 15 20 25

Total n-3 PUFA (% of total fatty acids)

Seiz

ure

Late

ncy

(s)

Correlation between seizure latency and n-3 PUFA composition of brain unesterified

fatty acids. Dark circles represent the saline group, dark squares represent the mineral oil

group, open diamonds represent the SR-3 40 mg/kg group and open triangles represent

the SR-3 200 mg/kg group. Seizure latency was positively correlated to n-3 PUFA

composition of brain unesterified fatty acids (R=0.65, P<0.001 by Pearson’s correlation).

101

Table 1: Brain phospholipid fatty acid composition, expressed as a percentage of total fatty acids, within the phospholipid lipid pool

Saline

Mineral Oil

SR-3 40 mg/kg

SR-3 200 mg/kg

14:0 0.2 ± 0.03 0.1 ± 0.01 0.1 ± 0.01 0.1 ± 0.01 16:0 22.9 ± 0.8 22.3 ± 1.0 21.8 ± 1.4 22.0 ± 0.8 18:0 18.7 ± 0.5 17.1 ± 0.6 16.5 ± 0.5 17.1 ± 0.5 19:0 0.2 ± 0.1 0.5 ± 0.2 0.8 ± 0.1 0.8 ± 0.1 20:0 1.0 ± 0.2 2.0 ± 0.3 2.4 ± 0.4 2.6 ± 0.4 22:0 1.0 ± 0.3 0.9 ± 0.2 1.0 ± 0.3 0.9 ± 0.3 24:0 0.9 ± 0.3 0.6 ± 0.1 0.4 ± 0.05 0.5 ± 0.1 16:1 n-9 0.02 ± 0.005 0.02 ± 0.01 0.02 ± 0.01 0.01 ± 0.01 16:1 t-9 0.3 ± 0.03 0.3 ± 0.01 0.3 ± 0.02 0.2 ± 0.01 18:1 t-9/t11 0.1 ± 0.1 0.3 ± 0.1 0.1 ± 0.1 0.2 ± 0.1 18:1 n-9 19.6 ± 0.4 18.1 ± 0.5 18.0 ± 0.4 17.8 ± 0.5 18:1 n-11 4.2 ± 0.1 4.1 ± 0.1 4.2 ± 0.1 4.0 ± 0.1 20:1 n-5 0.1± 0.1 0.1 ± 0.1 0.3 ± 0.2 0.1 ± 0.1 20:1 n-8 0.2 ± 0.1 0.9 ± 0.4 0.6 ± 0.2 0.5 ± 0.2 20:1 n-11 2.2 ± 0.1 2.1 ± 0.2 2.2 ± 0.2 2.2 ± 0.2 22:1 n-9 0.3 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 24:1 n-9 0.4 ± 0.04 0.5 ± 0.1 0.3 ± 0.04 0.3 ± 0.04 18:2 n-6 2.4 ± 0.5 4.4 ± 0.6 5.2 ± 0.8 4.7 ± 0.6 20:2 n-6 0.5 ± 0.1 0.6 ± 0.2 1.1 ± 0.3 0.7 ± 0.3 20:3 n-6 0.5 ± 0.1 0.7 ± 0.2 0.8 ± 0.2 0.6 ± 0.1 20:4 n-6 8.9 ± 0.1 8.7 ± 0.2 8.3 ± 0.3 8.7 ± 0.3 22:2 n-6 0.2 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 22:4 n-6 2.9 ± 0.1 2.7 ± 0.1 2.6 ± 0.1 2.7 ± 0.1 22:5 n-6 0.4 ± 0.02 0.4 ± 0.03 0.4 ± 0.02 0.4 ± 0.02 18:3 n-3 0.8 ± 0.1 1.1 ± 0.2 1.2 ± 0.3 1.2 ± 0.2 20:3 n-3 0.2 ± 0.1 0.2 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 20:5 n-3 0.03 ± 0.02 0.005 ± 0.003 0.02 ± 0.01 ND 22:3 n-3 ND 0.1 ± 0.05 0.1 ± 0.03 0.1 ± 0.02 22:5 n-3 0.1 ± 0.01 0.1 ± 0.01 0.1 ± 0.01 0.1 ± 0.01 22:6 n-3 10.7 ± 0.3 10.5 ± 0.5 10.3 ± 0.4 10.7 ± 0.5 Total Saturates 44.9 ± 0.7 43.4 ± 0.9 43.1 ± 1.2 44.1 ± 0.5 Total Monounsaturates 27.4 ± 0.6 26.7 ± 0.4 26.3 ± 0.4 25.6 ± 0.5 Total Polyunsaturates 27.7 ± 0.5 29.9 ± 0.7 30.6 ± 0.9 30.3 ± 0.7 Total n-6 Polyunsaturates 15.9 ± 0.5 17.8 ± 0.8 18.5 ± 1.0 18.0 ± 0.7 Total n-3 Polyunsaturates 11.9 ± 0.2 12.1 ± 0.2 12.1 ± 0.2 12.3 ± 0.4 n-6 / n-3 1.3 ± 0.1 1.5 ± 0.1 1.5 ± 0.1 1.5 ± 0.1 Total phospholipids (mg/g) 19.7 ± 1.3 20.5 ± 1.2 23.4 ± 1.9 20.4 ± 2.1

Data are mean ± SEM of n=8-10 / group. Values with different superscipts are significantly different at P<0.05 by 1-way ANOVA and Tukey’s post-hoc test

102

Table 2: Brain unesterified free fatty acid composition, expressed as a percentage of total fatty acids, within the free fatty acid lipid pool

Saline

Mineral Oil

SR-3 40 mg/kg

SR-3 200 mg/kg

14:0 1.2 ± 0.4 0.6 ± 0.2 0.8 ± 0.3 2.1 ± 1.0 16:0 15.7 ± 0.9 14.3 ± 0.5 18.2 ± 1.2 13.5 ± 2.1 18:0 24.7 ± 1.1 23.6 ± 0.7 24.3 ± 1.2 21.2 ± 2.9 19:0 1.3 ± 0.4 0.8 ± 0.2 0.6 ± 0.1 1.4 ± 0.5 20:0 3.0 ± 1.3 a 9.0 ± 1.4 b 3.8 ± 0.9 a 3.8 ± 0.6 a 22:0 3.9 ± 0.8 1.8 ± 0.7 1.6 ± 0.6 2.3 ± 0.5 24:0 3.9 ± 0.9 1.8 ± 0.7 1.8 ± 0.6 2.9 ± 0.6 16:1 n-9 ND ND 0.01 ± 0.01 ND 16:1 t-9 0.3 ± 0.05 0.4 ± 0.02 0.3 ± 0.01 0.3 ± 0.03 18:1 t9/t11 0.1 ± 0.04 0.1 ± 0.1 0.1 ± 0.02 0.4 ± 0.2 18:1 n-9 11.2 ± 0.8 12.6 ± 0.5 14.6 ± 1.4 11.3 ± 1.5 18:1 n-7 2.9 ± 0.2 a 3.1 ± 0.1 ab 3.6 ± 0.2 b 3.2 ± 0.1 ab 20:1 n-5 0.03 ± 0.03 ND 0.01 ± 0.01 ND 20:1 n-8 0.1 ± 0.1 ND 0.03 ± 0.02 0.01 ± 0.01 20:1 n-11 1.5 ± 0.4 1.8 ± 0.8 1.3 ± 0.2 2.5 ± 0.8 22:1 n-9 0.3 ± 0.2 0.2 ± 0.01 0.2 ± 0.01 0.5 ± 0.2 24:1n-9 0.2 ± 0.1 0.2 ± 0.03 0.2 ± 0.03 1.1 ± 0.6 18:2 n-6 9.1 ± 1.7 a 6.5 ± 0.9 ab 2.5 ± 0.4 b 3.8 ± 0.5 b 20:2 n-6 0.1 ± 0.04 0.1 ± 0.03 0.2 ± 0.02 1.1 ± 0.6 20:3 n-6 0.4 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 20:4 n-6 13.0 ± 1.6 14.9 ± 0.7 14.5 ± 1.1 14.2 ± 1.1 22:2 n-6 0.1 ± 0.03 0.1 ± 0.03 0.1 ± 0.02 0.03 ± 0.02 22:4 n-6 1.1 ± 0.1 1.3 ± 0.2 1.7 ± 0.3 2.1 ± 0.4 22:5 n-6 0.2 ± 0.1 0.2 ± 0.1 0.3 ± 0.04 1.3 ± 0.7 18:3 n-3 0.4 ± 0.1 0.6 ± 0.1 0.6 ± 0.1 0.8 ± 0.2 20:3 n-3 0.1 ± 0.04 0.1 ± 0.02 0.1 ± 0.02 0.9 ± 0.5 20:5 n-3 0.1 ± 0.03 0.1 ± 0.03 0.1 ± 0.03 2.1 ± 1.3 22:3 n-3 1.1 ± 0.3 0.7 ± 0.1 1.4 ± 0.4 1.2 ± 0.2 22:5 n-3 0.4 ± 0.1 0.3 ± 0.1 0.4 ± 0.1 0.9 ± 0.5 22:6 n-3 3.8 ± 0.6 4.4 ± 0.6 6.1 ± 1.1 4.8 ± 1.0 Total Saturates 53.6 ± 1.9 51.8 ± 1.5 51.1 ± 1.7 47.2 ± 3.1 Total Monounsaturates 16.6 ± 1.2 18.3 ± 1.1 20.5 ± 1.8 19.3 ± 1.0 Total Polyunsaturates 29.8 ± 1.3 29.8 ± 1.1 28.5 ± 0.7 33.5 ± 2.8 Total n-6 Polyunsaturates 24.0 ± 0.9 a 23.6 ± 0.6 a 19.8 ± 1.1 b 22.9 ± 0.9 ab Total n-3 Polyunsaturates 5.8 ± 0.6a 6.2 ± 0.6 ab 8.6 ± 0.8 ab 10.6 ± 2.2 b n-6 / n-3 4.4 ± 0.3 a 4.1 ± 0.4 ab 2.5 ± 0.3 b 2.8 ± 0.5 b Total fatty acids (mg/g) 1.8 ± 0.5 1.0 ± 0.1 2.8 ± 1.1 1.3 ± 0.3

Data are mean ± SEM of n=8-10 / group. Value with different superscipts are significantly different at P<0.05 by 1-way ANOVA and Tukey’s post-hoc test

103

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Taha AY, McIntyre Burnham W (2007) Commentary on the effects of a ketogenic diet enriched with omega-3 polyunsaturated fatty acids on plasma phospholipid fatty acid profile in children with drug-resistant epilepsy. Epilepsy Res. 76(2-3):148-9.

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Vining EPG (1999) Clinical efficacy of the ketogenic diet. Epilepsy Res. 37: 181-90.

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CHAPTER 4

ASSESSING THE METABOLIC AND TOXOC EFFECTS OF ANTICONVULSANT DOSES OF POLYUNSATURATED FATTY

ACIDS ON THE LIVER IN RATS

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4 Experiment 3: Assessing the metabolic and toxic effects of anticonvulsant doses of polyunsaturated fatty acids on the liver in rats

Forward

Upon reflection, it seemed unlikely that the increase in brain n-3 fatty acid levels

in the SR-3 200 mg/kg group was due to increased synthesis of longer-chain n-3 fatty

acids from ALA - since the conversion efficiency of ALA into other n-3 PUFA, such as

DHA, amounts to less than 0.5% in rats (Igarashi et al., 2006).

The 200 mg/kg SR-3 dose contains 50 mg of ALA, which means that only 0.075

mg of the administered ALA dose to a 0.3 kg rat would have been converted into DHA

by the liver per day. This amount seems unlikely to have caused a significant elevation in

brain n-3 PUFA levels.

A possible explanation for the increase in brain n-3 PUFA levels following ALA

administration would relate to increased transport of n-3 PUFA from other tissues to the

brain. It has been proposed that diets that promote fatty acid oxidation, such as the high-

fat ketogenic diet, increase the mobilization of PUFA from liver and adipose to the brain

(Taha et al., 2005; Taha et al., 2009d). Under dietary conditions of increased fatty acid

oxidation, PUFA and other fatty acids are mobilized from liver and adipose to

metabolically active tissues such as muscle and brain in order to replenish their fatty acid

supply. ALA and LA are inducers of fatty acid oxidation and possibly ketosis (Cunnane,

2004). It is, therefore possible that chronic administration of LA and ALA may have

increased brain n-3 PUFA levels by promoting selective mobilization of PUFA from liver

and adipose tissue to the brain. If so, then n-3 PUFA levels would be expected to be

depleted in the livers of the SR-3 subjects that were treated with 200 mg/kg of the SR-3

110

mixture. This was addressed in Experiment 3 by determining fatty acid concentrations in

liver.

An alternate possibility is that the longer seizure latencies observed in the SR-3

200 mg/kg group in Expeiment 2 were due to an increase in the levels of brain ketone

bodies (fat metabolites) rather than to the n-3 fatty acids per se. This is because LA and

ALA are inducers not only of fatty acid oxidation but also possibly of ketosis (Dell et al.,

2001; Cunnane, 2004). The induction of ketosis is controlled by HMG-CoA lyase, which

synthesizes ketone bodies such as acetone, from its HMG-CoA substrate (Cullingford et

al., 1998a; Cullingford et al., 1998b). Acetone has been shown to suppress seizures in

animal seizure models (Likhodii and Burnham, 2002; Likhodii et al., 2003). Liver HMG-

CoA lyase gene expression was therefore also measured in Experiment 3, in order to

assess the possible involvement of ketone bodies in the observed effect of the SR-3

compound on seizure latency.

Finally, the hepatic expression of catalase, glutathione-S-transferase (GST) A1

and GST A4 were assessed in Experiment 3 in order to confirm that the SR-3 mixture did

not cause liver toxicity at the 200 mg/kg dose. These enzymes are involved in antioxidant

defence and phase II xenobiotic metabolism (Xie et al., 1998; Pool-Zobel et al., 2005;

Romero et al., 2006).

As indicated below, it appears that the longer latencies observed in the SR-3 200

mg/kg group were most probably due to an increase in the mobilization of n-3 fatty acids

from liver to the brain, since PUFA liver concentrations were lower in the SR-3 200

mg/kg group, as compared to the saline, mineral oil and SR-3 40 mg/kg groups. HMG-

CoA lyase gene expression did not differ significantly between the saline, mineral oil,

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SR-3 40 mg/kg and 200 mg/kg groups, suggesting that the increase in seizure latency of

the SR-3 200 mg/kg group was not due to the production of ketone bodies. The catalase,

GST A1 and GST A4 mRNA assays indicated that the SR-3 compound did not induce

markers of oxidative stress or xenobiotic metabolism.

It is important to note that, while the findings reported in the following

manuscript are suggestive of a lack of involvement of HMG-CoA lyase in seizure

protection, other relevant genes or pathways that might have been altered due to the SR-3

mixture were not assessed. PUFA are known to alter the expression of over 100 genes

within the brain (Kitajka et al., 2002). A key gene that might be involved in seizure

protection for instance, is the peroxisome proliferator-alpha gene (PPAR-α). PPAR-α is a

transcription factor that is involved in the regulation of genes involved in fatty acid

oxidation and mitochondrial utilization of fatty acid energy substrates (Cullingford et al.,

2002a; Cullingford et al., 2002b). There is some evidence suggesting that drugs that

induce PPAR-α, such as fenofibrate, have anticonvulsant activity in animal seizure

models (Porta et al., 2009). Future studies using microarray techniques could be used to

explore the possible involvement of other pathways that might explain the effects of the

SR-3 on seizure threshold.

The paper published in the Journal of Toxicology and Environmental Health

(Part A), starts here (Taha et al., 2009a). The co-authors are Solmaz Alizadeh, Qiudi

Zeng, Elvis Filo, Peter McPherson and W.M. Burnham. Solmaz Alizadeh assisted with

the mRNA analysis. Quidi Zeng assisted with the lipid analysis and Elvis Filo assisted

with the animal work and tissue harvesting. Drs. McPherson and Burnham were the

principal investigators in this study.

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Assessing the metabolic and toxic effects of anticonvulsant doses of polyunsaturated fatty acids on the liver in rats

Ameer Y. Taha, Solmaz Alizadeh, Qiudi H. Zeng, Elvis Filo, J. Peter McPherson and

W.M. Burnham

Department of Pharmacology and Toxicology, Faculty of Medicine, University of



Ameer Y. Taha

Department of Pharmacology and Toxicology

Faculty of Medicine






Running title: Metabolic and toxic effects of polyunsaturated fatty acids

Key words: Linoleic acid, α-linolenic acid, omega-3 polyunsaturated fatty acids,

pentylenetetrazol, metabolic, toxic, adverse, liver, anticonvulsant doses, seizures

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4.1 Abstract

Polyunsaturated fatty acids (PUFA), at high doses, have been demonstrated to possess

anticonvulsant properties in animal seizure models. Little is known, however, about the

possible metabolic or adverse effects of PUFA at these high, anticonvulsant doses. The

goal of the present study was to assess the metabolic and potential adverse effects of

high-dose PUFA administration to rats. Adult male rats received a fatty acid mixture

containing α-linolenic and linoleic acid in a 1 to 4 ratio, intraperitoneally, for 3 weeks.

After sacrifice, livers were isolated and analyzed for fatty acid composition and for

mRNA expression of HMG-CoA lyase, catalase and glutathione-S-transferases A1 and

A4 markers for ketosis, antioxidant defense and phase II xenobiotic metabolism,

respectively. Chronic administration of the PUFA mixture decreased hepatic levels of

total lipids - and several fatty acids within total lipids - without altering mRNA

expression of HMG-CoA lyase, a metabolic marker of ketosis. The PUFA mixture did

not affect mRNA expression of catalase or glutathione-S-transferases A1 and A4, which

are involved in antioxidant defense and phase II xenobiotic metabolism. These findings

suggest that PUFA, given for 3 weeks at anticonvulsant doses, result in significant

changes in liver lipid metabolism, but do not alter measured genetic markers of liver

toxicity.

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4.2 Introduction

Polyunsaturated fatty acids (PUFA), and particularly the omega-3 (n-3) PUFA,

have been proposed as a therapy - complementary to anticonvulsant medications - in the

treatment of epilepsy (Cunnane et al., 2002; Yuen and Sander, 2004). PUFA were

reported to reduce neuronal excitability in cell cultures (Vreugdenhil et al., 1996;

Lauritzen et al., 2000; Young et al., 2000), and to raise seizure threshold in animal

seizure models at high doses (Yehuda et al., 1994; Voskuyl et al., 1998; Rabinovitz et al.,

2004; Porta et al., 2009; Taha et al., 2008; Taha et al., 2009). Clinical studies of the n-3

PUFA showed mixed, but promising, results (Schlanger et al., 2002; Yuen et al., 2005;

Bromfield et al., 2008; Degiorgio et al., 2008).

Our own lab recently demonstrated that chronic intraperitoneal (i.p.)

administration of a fatty acid mixture containing linoleic acid (LA) and α-linolenic

(ALA), at a 4 to 1 ratio (termed the “SR-3” compound) for 21 days, elevated seizure

threshold in rats, and raised brain n-3 PUFA levels within the unesterified fatty acid lipid

fraction, following seizure induction by pentylenetetrazol (PTZ) (Taha et al., 2009). LA

and ALA are 18-carbon fatty acids, derived from plant oils, that are converted into longer

chain PUFA via elongation and desaturation enzymes in the liver (Sprecher, 2000).

Although PUFA appear to provide a possible adjunctive therapeutic strategy for

the future management of epilepsy, little is known about their metabolic or possible

adverse effects at high, anticonvulsant doses, which were calculated to be 150-fold higher

than normal plasma levels. In the present study, these were studied in liver tissue

removed from the rats in our 2008 experiment (Taha et al., 2009). The assay of liver

tissue from these animals could potentially answer a number of important questions.

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A first question to be addressed was related to the source of the elevated n-3

PUFA found in our rats’ brains following three weeks of treatment with the “SR-3”

mixture. ALA and LA themselves are not abundant in brain tissue (<6% of total fatty

acids), so it is possible that the elevation of n-3 PUFA seen in the brains of our rats was

due to the conversion of ALA into longer chain n-3 PUFA (Taha et al., 2009).

ALA may be converted into longer chain PUFA such as eicosapentaenoic acid

(20:5n-3), n-3 docosapentaenoic acid (22:5n-3), and docosahexaenoic acid (22:6n-3;

DHA) via a series of metabolic steps (Sprecher, 2000). Such conversions are thought to

be optimal when LA is present at 4 times the concentration of ALA (Yehuda et al., 1996).

The conversion of ALA to other n-3 PUFA, however, is poor in rodents, being estimated

to be less than 1% (Igarashi et al., 2006). This suggests that the elevated n-3 PUFA found

in the brains of our animals may not have entirely resulted from the conversion of ALA

to DHA.

Another possible source for the elevated brain n-3 PUFA found after “SR-3”

treatment is transport of n-3 PUFA from liver to brain. PUFA such as LA and ALA are

potent inducers of fatty acid oxidation (Lin et al., 1999; Hostetler et al., 2005; Hostetler et

al., 2006) and possibly ketosis (Cunnane, 2004), and, under chronic conditions of

increased fatty acid oxidation, PUFA become preferentially mobilized from adipose

tissue or liver, to other parts of the body, including the brain (Taha et al., 2005). It is

therefore likely that LA and ALA administration, at high, anticonvulsant doses, may

increase brain PUFA levels by promoting selective mobilization of PUFA from liver to

brain. If so, we should find depleted levels of n-3 PUFA in the livers of our “SR-3”

treated rats.

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A second question that can be addressed by assaying the livers of our “SR-3”

treated rats relates to possible changes in the levels of reactive oxygen species (ROS).

Despite their anticonvulsant effects, a possible drawback to the use of high-dose PUFA as

an adjunctive therapy for epilepsy is the potential for the production of ROS. PUFA, such

as ALA and LA, are considered to be substrates for the production of ROS, due to the

presence of unsaturated carbon bonds in their chemical structures. Auto-oxidation of

polyunsaturated fatty acids could therefore yield ROS, such as hydrogen peroxide, which

could subsequently engage in secondary reactions to produce 4-hydroxy-2-nonenal

(Uchida, 2003). 4-Hydroxy-2-nonenal is a potent inducer of catalase (Zhu et al., 2006),

which is involved in antioxidant defence (Scott et al., 1991; Zhu et al., 2006). It also

induces glutathione-S-transferases A1 and A4 (Raza and John, 2006; Zhu et al., 2006;

Malone and Hernandez, 2007), which are involved in both antioxidant defence and phase

II xenobiotic metabolism (Xie et al., 1998; Pool-Zobel et al., 2005; Romero et al., 2006).

If chronic treatment with the “SR-3” mixture elevated ROS species, this should be

reflected in increased levels of catalase and glutathione-S-transferase A1 and A4 in our

liver samples. Our assays allowed us to investigate this.

The present study, therefore, assessed the possible metabolic and adverse effects

of the SR-3 PUFA compound at high, anticonvulsant doses. Our objectives were to

determine whether chronic administration of the SR-3 mixture might increase

mobilization of PUFA from liver, and also to monitor their possible effects on liver

antioxidant and xenobiotic enzymes.

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4.3.1 Measurements taken

Liver fatty acid levels of total lipids (TL), phospholipids (PL) and triglycerides

(TG) were measured by gas-chromatography in order to determine whether there was a

net loss of fatty acids from liver. Liver expression of HMG-CoA lyase mRNA, the

enzyme involved in ketone body production from HMG-CoA substrate, was also

measured in order to determine whether the predicted ‘loss’ of fatty acids in liver was due

to excessive fatty acid oxidation. Finally, hepatic mRNA expression of catalase,

glutathione-S-transferase (GST) A1 and GST A4 were also assessed, since these enzymes

are involved in antioxidant defence and phase II xenobiotic metabolism. Since acute PTZ

administration was previously reported to affect protein activity and basal levels, enzyme

activity and protein expression assays were not performed in this study (Akbas et al.,

2005).


Four doses of the SR-3 compound (40 mg/kg, 400 mg/kg and 1000 mg/kg) were

prepared, as previously described (Taha et al., 2009). Non-esterified LA (0.90 mg/ml;

Sigma-Aldrich, St. Louis, Missouri, USA) and non-esterified ALA (0.92 mg/ml; Sigma-

Aldrich, St. Louis, Missouri, USA) were mixed at a 4 to 1 ratio, in a vehicle that

consisted of 0.73 ml of mineral oil (Sigma-Aldrich, St. Louis, Missouri, USA) and 0.02

ml of α-tocopherol (Sigma-Aldrich, St. Louis, Missouri, USA). Each dose was dissolved

in the same fixed volume of mineral oil (0.73 ml) and α-tocopherol (0.02 ml). Later,

when it became clear that the 400 and 1000 mg/kg doses were too high (discussed later),

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a 200 mg/kg dose of the SR-3 compound was prepared using the same methodology.

4.3.3 Animals and treatments

All experimental procedures were approved by the Animal Care Committee of the

Faculty of Medicine of the University of Toronto and were in accordance to the

guidelines described by the Canadian Council of Animal Care. The measurements were

performed on livers of rats that had previously been injected for 21 days with the SR-3

mixture, and then treated acutely with PTZ and sacrificed within 30 min following PTZ

administration (Taha et al., 2009).

All procedures utilized male Long Evans hooded rats (Charles River, La Prairie,

QC, Canada). The rats were one month of age and weighed an average of 151 g upon

arrival, and were individually housed in plastic cages with corn-cob bedding in a

vivarium maintained on a 12 hr light / dark cycle and at a temperature of 21ºC. The rats

had ad libitum access to water and food (Teklad Global 2018, 18% Protein Rodent Diet).

After 7 days of acclimatization to the facility, the animals were randomized into 5

groups which initially received daily i.p. injections of: 1) 0.9% saline (n=10), 2) mineral

oil vehicle (n=7), 3) SR-3 40 mg/kg (n=8) 4), SR-3 400 mg /kg (n=8) or SR-3 1000

mg/kg (n=8). Weight gain was monitored on a daily basis throughout the study, and food

intake was measured once every two days.

All animals had been intended to be injected with their respective treatments for

21 consecutive days. By the 10th day of the experiment, however, it was clear that the

injection of the highest PUFA dose was producing low weight gain, low food intake and

bloating in the group that received the 1000 mg/kg dose. This group was therefore

euthanized and excluded from the remainder of the study. The bloating symptoms were

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evident, but to a much lesser extent, in the group that received the SR-3 at 400 mg/kg.

This group was therefore injected with saline from days 10 to 15, at which point food

intake returned to normal and the bloating was gone. The injections were subsequently

resumed, but at a lower dose of 200 mg/kg. Thus, this group became the 200 mg/kg

group.

4.3.4 Euthanasia and liver harvesting

On day 22, all animals were seizure tested with 80 mg/kg of PTZ (i.p.) and

subsequently observed in the open field for 30 min. The rats were then euthanized with

sodium pentobarbital (i.p.), with the exception of one or two rats from each group that

had experienced a running fit or died spontaneously from the tonic-clonic seizures before

the end of the half-hour. Rats that showed a running fit were immediately euthanized

with sodium pentobarbital, due to the severity of their seizures. Livers were excised

immediately following euthanization, weighed, snap frozen in liquid nitrogen and stored

at -80°C for future fatty acid and mRNA analysis.

4.3.5 Liver lipid analysis

Approximately 0.5 g of liver tissue were weighed and homogenized in 10 ml of

chloroform / methanol (2:1 v/v), following the addition of diheptadecanoyl L-α-

phosphatidylcholine and triheptadecanoic acid (NuCheck Prep) as internal standards for

the quantification of phospholipids (PL) and triglycerides (TG), respectively. The internal

standards were also used to quantify fatty acid levels in total lipids (TL). Saline (0.9%,

2.2 ml) was added in order to separate the aqueous polar phase from the non-polar phase

containing total lipids. The samples were left for 24 hrs at 4°C under nitrogen in order for

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the layers to separate. The non-polar phase, containing total lipids, was then dried under

nitrogen and re-constituted in 4 ml of chloroform.

Approximately 0.5 ml of total lipid extract was transferred to new tubes and

methylated with 14% methanolic BF3 (2 mL) and hexane (2 mL) at 100°C for 1 hr. The

reaction was terminated with deionized water (2 mL), and the resulting fatty acid methyl

esters from the TL extract were separated (top layer) following vortexing and

centrifugation at 1600 rpm.

PL and TG in total lipid liver extracts were fractionated by thin-layer

chromatography (TLC), on 20 x 20 cm silica gel plates (Whatman LK6D plates, pre-

coated with 250 μm of Silica Gel 60A). Separate lanes were spotted with PL and TG

standards in order to identify the bands based on their migration on the plate relative to

the standard lane. The bands were resolved for 35 min in covered glass tanks containing

petroleum ether, diethyl ether, and acetic acid (80:20:1 by volume). Bands corresponding

to the PL and TG standard lanes were identified under ultraviolet light, after spraying the

plates with 8-anilino-1-naphthalenesulfonic acid. The bands were scraped off and directly

methylated in 14% methanolic BF3 (2 mL) and hexane (2 mL) at 100°C for 1 hr.

Deionized water (2 mL) was then added. The mixture was vortexed and centrifuged at

1600 rpm in order to separate the phases. The upper hexane phase was separated, dried

under nitrogen and reconstituted in hexane for analysis by gas chromatography.


Fatty acid methyl esters (FAME) of liver PL and TG were analyzed on an Agilent

6890 gas-chromatography system equipped with a flame ionization detector and a DB-23

capillary column (30m x 25mm ID; J and W Scientific, DB-23, Folsom, CA, USA) . One

121

µl of FAME from each sample was injected into the column in splitless mode, using

helium gas as a carrier, at a constant flow rate of 0.7 ml per min. A three stage

temperature program was used to acquire the fatty acid methyl ester profile. The initial

temperature setting was at 50ºC with a 2 min hold, followed by a ramp up at 20ºC per

min to 170ºC and a 1 min hold, and then a final 3ºC per min ramp up to 212ºC followed

by a 10 min hold. Fatty acid peaks were identified by comparing the retention time of

each peak to the retention times of fatty acid standards of known composition (GLC463,

NuCheck Prep., ON, Can).

4.3.7 mRNA expression analysis by quantitative real time PCR

RNA was prepared from approximately 100 mg of liver homogenate per sample

using TrizolTM (Invitrogen) according to the manufacturer’s directions. First-strand

cDNA was prepared from 2 μg of total RNA using Superscript III reverse transcriptase

(Invitrogen). Quantitative PCR was done in triplicate assays using the Power SYBR

Green PCR master mix (Applied Biosystems) for each target gene as well as an internal

control Glyceraldehyde-3-Phosphate dehydrogenase (GAPDH). PrimerExpress (Applied

Biosystems) software was used to design the following primers: HmgCoA sense 5’-TCA

GAA GTT TCC CGG CAT CA-3’, HmgCoA antisense 5’-TGT GTA CAC CCA ACT

CCC AC-3’, Catalase sense 5’-TGA CCA GGG CAT CAA AAA CTT-3’, Catalase

antisense 5’-ACT GGC GAT GGC ATT GAA A-3’, Gst-A1 sense 5’-CTC TAT GGG

AAG GAC ATG AAG GA-3’, Gst-A1 antisense 5’-TGC CAA CCC TTC CGA ATA CA,

Gst-A4 sense 5’-TAT GGG AAG GAC ATG AAG GAG AGA-3’, Gst-A4 antisense 5’-

CAG GTG GGT CAA ATG GGT AGA-3’, Gapdh sense 5’-GGG CAT CTT GGG CTA

CAC TG-3’, Gapdh antisense 5’-AGC CGT ATT CAT TGT CAT ACC-3’. Reactions

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were performed in 25 μl final mixture containing 12.5μl PCR master mix, 2.5μl cDNA,

25 pmol of each primer and 9.5μl water. PCR amplification reactions were performed in

a 7500 Real Time PCR System (Applied Biosystems) as follows: 40 cycles with

denaturation at 95°C for 15 sec, annealing at 50°C for 2 min and extension at 60°C for 1

min. The expression of target genes was normalized by the internal reference gene,

Gapdh. A no template negative control was also included in all experiments. Each

reaction was subjected to melting point analysis to confirm single amplified products.

Moreover a control cDNA dilution series was created for each gene to establish a

standard curve and check the PCR efficiency. Fold changes in gene expression were

determined using the 2-ΔΔCt method (Livak and Schmittgen, 2001).


All data are presented as means ± SEM. Data analysis was performed on

Statistical Analysis Software (version 8.02, SAS Institute, Cary, NC). A one-way analysis

of variance (ANOVA) was used to determine the effects of treatment on the fatty acid

profiles of liver lipid fractions and mRNA expression. The means were then compared

with Tukey’s post-hoc test, if the one-way ANOVA was statistically significant. A p

value of equal to or less than 0.05 was accepted as statistically significant.

The fatty acid data that was derived from the liver samples of the animals that

died spontaneously or had to be euthanized before the end of the 30-min observation

period (1-2 rats per group) was assessed in order to determine whether it fell within the

outlier range of two standard deviations. None of the animals outlied the standard

deviation limit and therefore none were excluded from the statistical analyses.

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4.4 Results

4.4.1 Liver concentrations of TL, PL and TG (expressed as mg per g of liver

tissue)

Gas-chromatography was used to quantify TL, PL and TG concentrations,

expressed as mg per g of wet tissue, in liver. There were no statistically significant

differences between the groups in TL (saline, 38.9 ± 2.2; mineral oil, 36.6 ± 1.0; SR-3 40

mg/kg, 34.0 ± 1.2; SR-3 200 mg/kg, 34.6 ± 1.1), PL (saline, 23.3 ± 2.8; mineral oil, 21.0

± 0.9; SR-3 40 mg/kg, 22.1 ± 1.2; SR-3 200 mg/kg, 22.0 ± 1.9), or TG (saline, 13.5 ± 2.1;

mineral oil, 14.4 ± 2.0; SR-3 40 mg/kg, 10.0 ± 2.0; SR-3 200 mg/kg, 11.0 ± 2.9)

concentrations, as determined by one-way ANOVA.

There were minor changes in fatty acid concentrations within each of these

fractions (data not shown). In particular, vaccenic acid (18:1n-7) was lowest in the TL (-

33% relative saline), PL (-29% relative saline) and TG (-75% relative saline) of the group

that received the SR-3 at 200 mg/kg, relative to the saline, mineral oil and SR-3 40 mg/kg

groups. One-way analysis of variance, followed by Tukey’s post-hoc test, revealed that

the differences between the SR-3 200 mg/kg and control saline and mineral oil groups

were statistically significant for vaccenic acid in TL, TG and PL. Vaccenic acid

concentrations in TL, TG and PL of the SR-3 40 mg/kg group were intermediate between

the control and SR-3 200 mg/kg groups, but these differences did not reach statistical

significance.

4.4.2 Liver absolute levels of TL, PL and TG (expressed as mg)

Previously a significant reduction was noted in the liver weight of rats that

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received the SR-3 mixture at 200 mg/kg, in comparison to the saline, mineral oil and SR-

3 40 mg/kg groups (Taha et al., 2009). This suggested that despite the lack of differences

in total fatty acid concentrations (in mg/g) between the SR-3 200 and the other groups,

the absolute amount of fatty acids (in mg) in the SR-3 200 mg/kg group might be reduced

as a result of the lower liver mass. Thus, the TL, PL and TG concentrations were

multiplied by liver weights in order to yield absolute fatty acid levels (in mg) within the

TL, PL and TG of liver.

The data for absolute total fatty acids within TL, PL and TG are presented in

Tables 1 to 3. As shown in Table 1, TL levels were lower by 22-30% in the SR-3 200

mg/kg group, as compared to the saline, mineral oil and SR-40 mg/kg groups. Statistical

analysis by one-way ANOVA and Tukey’s post hoc test showed that the differences

between the saline and SR-3 200 mg/kg groups were significant. The decrease in TL

levels in the SR-3 200 mg/kg group was mainly due to significant decreases in palmitate

(16:0), stearate (18:0), vaccinate (18:1n-7), arachidonate (20:4n-6) and docosahexaenoate

(22:6n-3) levels in the SR-3 200 mg/kg group.

Absolute levels of total PL and TG are shown in Tables 2 and 3. These showed a

“trend” similar to the TL, but the differences were not statistically significant by one-way

ANOVA. However, there were some changes in fatty acids levels within the PL and TG

fractions. Palmitate (16:0), stearate (18:0), linoleate (18:2n-6), and adrenate (22:4n-6) in

the PL fraction were lower in the SR-3 200 mg/kg relative to saline, mineral oil and SR-3

40 mg/kg groups. The differences between the SR-3 200 mg/kg and saline groups

reached statistical significance for palmitate and oleate. The changes in linoleic and

adrenic acids were statistically different in the SR-3 200 mg/kg relative to the SR-3 40

125

mg/kg group. Within the TG fraction, oleic acid only was significantly lower in the SR-3

200 mg/kg group, as compared to the saline, mineral oil and SR-3 40 mg/kg groups.

4.4.3 HMG-CoA lyase mRNA expression

It has been suggested that PUFA may act as both inducers and substrates for

ketone body production (Cunnane, 2004). HMG-CoA lyase is the rate-limiting enzyme

which is involved in the synthesis of ketone bodies from HMG-CoA (Cullingford et al.,

1998b). One-way analysis of variance revealed no significant differences in the hepatic

mRNA expression of HMG-CoA lyase among the four groups (saline, 1.0 ± 0.1; mineral

oil, 1.0 ± 0.1; SR-3 40 mg/kg, 0.9 ± 0.2; SR-3 200 mg/kg, 1.1 ± 0.1), suggesting that

chronic administration of the SR-3 PUFA for 3 weeks did not induce ketosis in rats.

4.4.4 Catalase mRNA expression

Catalase is primarily involved in antioxidant defense in the phospholipid

membranes (Perichon and Bourre, 1996). An increase in catalase expression due to

PUFA injections would signify an increase in oxidative stress. There were no significant

differences in catalase mRNA levels among the groups (saline, 1.0 ± 0.1; mineral oil, 1.5

± 0.05; SR-3 40 mg/kg, 1.1 ± 0.3; SR-3 200 mg/kg, 1.0 ± 0.1), as analyzed by one-way

analysis of variance.

4.4.5 GST A1 and A4 mRNA expression

GST A1 and A4 are also involved in antioxidant defense, as well as

glucuronidation of 4-hydroxy-2-nonenal, a metabolite of excessive PUFA peroxidation

(Xie et al., 1998). The mRNA expression levels of GST A1 (saline, 0.9 ± 0.1; mineral oil,

0.6 ± 0.02; SR-3 40 mg/kg, 0.7 ± 0.3; SR-3 200 mg/kg, 0.5 ± 0.1) and GST A4 (saline,

126

0.9 ± 0.1; mineral oil, 0.5 ± 0.05; SR-3 40 mg/kg, 0.6 ± 0.2; SR-3 200 mg/kg, 0.5 ± 0.1)

did not differ significantly among the groups, as determined by one-way analysis of

variance.

4.5 Discussion

This is the first study to assess the metabolic and possible adverse effects of

chronic LA and ALA administration to rats at high, anticonvulsant doses that may exceed

plasma levels by at least 150-fold. Our results indicate that chronic, high-dose

administration of the SR-3 compound, which contains LA and ALA, decreased absolute

levels of total fatty acid content of liver, without altering the expression of genes

involved in ketosis, antioxidant defense and xenobiotic metabolism.

The decrease in liver TL is likely due to increased oxidation and transport of fatty

acids from liver to other tissues, such as muscle and brain. The relative contributions of

oxidation and transport to the total net loss of fatty acids within the TL fraction cannot be

determined from the present data. An increase in fatty acid transport from liver to brain,

however, would be consistent with the previous finding that n-3 PUFA levels in brain

were elevated in the SR-3 200 mg/kg group (Taha et al., 2009). Notably, despite the

reduction in several n-6 fatty acids in liver TL (Table 1), n-6 PUFA levels did not

increase in the brain (Taha et al., 2009), suggesting preferential incorporation of n-3 fatty

acids into the brain.

An increase in fatty acid mobilization from liver to brain might be attributed to

the metabolic effects of LA and ALA on fatty acid oxidation. It is well established that

LA and ALA are potent inducers of PPAR-α, a transcription factor which is known to

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enhance the expression of genes involved in fatty acid oxidation (Lin et al., 1999;

Hostetler et al., 2005; Hostetler et al., 2006). Under conditions of enhanced fatty acid

oxidation, PUFA are preferentially mobilized from adipose and liver to the brain and

other tissues (Taha et al., 2005). Thus, an increase in the net transport of fatty acids from

liver to the brain, as evidenced by a net reduction of fatty acids in liver TL and increased

incorporation of n-3 PUFA in the brain, would be a consequence of enhanced fatty acid

oxidation. Unfortunately, it was not possible to directly measure fatty acid transport in

our present study, because the rats were not injected with a radiolabelled tracer that can

be imaged or quantified by isotope-ratio-mass-spectrometry.

The induction of genes involved in hepatic fatty acid oxidation by PUFA (Lin et

al., 1999; Hostetler et al., 2005; Hostetler et al., 2006) might also lead to the activation of

genes involved in ketone body production (Cunnane, 2004). However, no significant

changes in HMG-CoA lyase expression were found, suggesting that chronic, high-dose

administration of LA and ALA enhanced fatty acid oxidation without increasing the

production of ketone bodies. This finding also suggests that the previously reported

anticonvulsant effects of the SR-3 mixture (Taha et al., 2009) were probably due to

increased mobilization of PUFA from liver and subsequent incorporation into brain,

rather than due to the increased synthesis of ketone bodies such as acetone, which was

reported to raise seizure threshold in animal seizure models (Likhodii and Burnham,

2002; Likhodii et al., 2003). Notably, ketone bodies (such as acetone) were not measured

in this study, due to previous work from our lab indicating that acute administration of

PTZ alters basal levels of acetone in plasma (Nylen, 2006).

Fatty acid concentrations within the TL, TG or PL fractions were not markedly

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altered when expressed as mg per g of liver. This is not unexpected, since the total liver

weight was significantly lower in the SR-3 200 mg/kg group relative to the saline,

mineral oil and SR-40 mg/kg groups (Taha et al., 2009). After correcting for the changes

in liver weight, the data showed a significant reduction in the amount of TL in liver of the

SR-3 200 mg/kg group when compared to the saline, mineral oil and SR-40 mg/kg

groups. The decrease in TL was quantitatively reflected in the PL and TG lipid fractions

although the differences did not reach statistical significance. This suggests that the

decrease in liver TL fatty acids was likely attributed to a decrease in fatty acid levels

within the TG and PL fractions.

Some, but not all animal studies reported that the chronic consumption of the n-3

PUFA increases the production of ROS (Demoz et al., 1992; Benito et al., 1997; Schimke

et al., 1997; Sarkadi-Nagy et al., 2003; Kang et al., 2005; Dimitrova-Sumkovska et al.,

2006; Hatanaka et al., 2006; Brooks et al., 2008). In order to assess this possibility,

mRNA expression of candidate genes that were reported to be elevated in response to

lipid peroxidation was measured. Chronic administration of the SR-3 PUFA at high doses

did not affect mRNA levels of catalase, GST A1 or GST A4, as compared to saline or

mineral oil injected rats. This suggests that chronic, high dose administration of LA and

ALA PUFA does not induce oxidative stress in liver.

GST A1 and A4 are also involved in phase II xenobiotic metabolism. In vitro

studies demonstrated that the GST enzymes are involved in the elimination of 4-

hydroxy-2-nonenal, a by product of excessive PUFA peroxidation (Xie et al., 1998). No

change in either GST A1 or A4 mRNA expression was found, suggesting that LA and

ALA did not produce sufficient amounts of 4-hydroxy-2-nonenal to lead to GST gene

129

induction. Our results also demonstrate that LA and ALA are not involved in the direct

regulation of GST A1 and A4. This is a pharmacologically important observation, since

the SR-3 compound would not be expected to interact with drugs that are eliminated via

the GST A1 and A4 pathways.

The highest dose of PUFA provided in this study (200 mg/kg) only amounts to

approximately 6% of a rat’s daily intake. Although this dose may appear to be relatively

low, the bioavailability of the PUFA is much higher when they are administered i.p. in

their unesterified form, as compared to oral administration. This is because fatty acids are

packaged into chylomicrons in the gut when given orally, which means that they are not

readily available in their unesterified, active form. As a result, a higher oral dose or a

longer duration of treatment of the SR-3 fatty acids may be required in order to induce

changes in lipid metabolism. By contrast, exogenous administration ensures direct

delivery of the unesterified PUFA to the liver. Despite the higher bioavailability of the

injected PUFA, no changes in catalase and GST A1 and A4 were detected, suggesting

that high dose administration of LA ands ALA does not induce the expression of genes

involved in antioxidant defense in liver.

Although the livers were harvested 30 min after the rats were injected with PTZ,

this period is not sufficient to compromise the reliability of our mRNA and lipid data.

This is because previous studies showed that a period of 30 min is not sufficient time to

alter mRNA levels of the enzymes that were measured in this study (Zhu et al., 2006).

Although PTZ might enhance the release of unesterified fatty acids from PL and TG in

order to produce prostaglandins or possibly ROS (Hayashi et al., 1987; Akbas et al.,

2005), this time-frame is unlikely to mask treatment effects on fatty acid composition of

130

PL and TG. This is because prostaglandins and thiobarbituric acid-reactive substances

(TBARS) products of fatty acid oxidation occur in the nano- to pico- molar concentration

range, and therefore an increase in prostaglandin or TBARS turnover due to the PTZ is

unlikely to affect PL and TG fatty acid concentrations, which are highly enriched in liver

and occur at millimolar concentrations. As such, both our mRNA expression

measurements and fatty acid composition determinations reflect an actual effect of the

chronic SR-3 PUFA treatment and not the acute PTZ treatment.

In conclusion, the findings of the present study suggest that the chronic,

exogenous administration of LA and ALA at high, anticonvulsant doses, might increase

brain levels of n-3 PUFA by promoting their mobilization from liver to brain, and that the

SR-3 PUFA compound does not induce oxidative stress or phase II xenobiotic

metabolism enzymes in the liver. The lack of effect of the PUFA on phase II xenobiotic

metabolizing genes suggests that LA and ALA may be administered with anticonvulsant

drugs without producing interactions mediated by GST A1 and A4. Thus, the PUFA may

be used to provide a healthy, safe adjunct treatment for the management of epilepsy – a

treatment that may potentially alleviate seizure symptoms without the induction of

oxidative stress or phase II xenobiotic enzymes.

Acknowledgements

Funding for this study was provided by the Canadian Institutes of Health Research

(CIHR) to W.M.B, the CIHR New Investigator Award to J.P.M. and the CIHR doctoral

research award to A.Y.T.

131

Table 1: Fatty acid levels (mg) in liver total lipids (TL)

Saline Mineral oil

SR-3 40 mg/kg SR-3 200 mg/kg

6:0 30.4 ± 6.0a 26.4 ± 3.4a 12.5 ± 1.3ab 8.4 ± 2.7b 7:0 0.3 ± 0.3 0.3 ± 0.3 0.6 ± 0.2 0.5 ± 0.1 8:0 1.0 ± 1.0 ND ND ND 9:0 1.4 ± 1.4 3.4 ± 2.2 1.0 ± 0.9 ND 10:0 ND ND ND ND 11:0 ND ND ND ND 12:0 ND ND ND ND 13:0 ND ND ND 0.05 ± 0.05 14:0 1.2 ± 0.2 1.3 ± 0.2 1.3 ± 0.1 1.0 ± 0.2 15:0 0.8 ± 0.1 1.2 ± 0.2 1.2 ± 0.2 1.1 ± 0.3 16:0 97.3 ± 6.1a 88.6 ± 5.0ab 91.6 ± 1.1ab 70.6 ± 4.0b 18:0 126.6 ± 8.6a 114.7 ± 7.9ab 118.6 ± 3.8ab 90.0 ± 7.3b 19:0 0.9 ± 0.3 0.9 ± 0.2 0.9 ± 0.2 0.9 ± 0.1 20:0 0.1 ± 0.1 ND 0.2 ± 0.1 0.2 ± 0.1 22:0 0.7 ± 0.2 2.3 ± 0.8 0.7 ± 0.2 1.2 ± 0.4 24:0 1.0 ± 0.4 2.0 ± 0.7 1.2 ± 0.2 1.8 ± 0.4 Total saturates 261.9 ± 20.3a 241.1 ± 10.8ab 229.9 ± 3.3ab 175.8 ± 12.2b 15:1 n-10 0.8 ± 0.2 1.0 ± 0.3 1.0 ± 0.2 1.3 ± 0.3 16:1 n-9 2.7 ± 0.4 2.9 ± 0.4 2.4 ± 0.2 1.8 ± 0.3 18:1 t-9 0 ± 0 0.1 ± 0.1 0.2 ± 0.1 0.3 ± 0.1 18:1 t-11 0 ± 0 0 ± 0 0 ± 0 0 ± 0 18:1 n-9 21.6 ± 1.1 23.8 ± 3.2 22.5 ± 0.8 17.4 ± 1.1 18:1 n-7 18.2 ± 1.7a 18.7 ± 1.2a 15.8 ± 0.4a 9.8 ± 0.9b 19:1 n-7 0 ± 0 0 ± 0 0.1 ± 0.05 0.03 ± 0.03 20:1 n-8 0 ± 0 0 ± 0 0 ± 0 0 ± 0 20:1 n-11 0 ± 0 0 ± 0 0 ± 0 0 ± 0 22:1 n-9 0 ± 0 0.5 ± 0.5 0.5 ± 0.3 0.8 ± 0.4 Total MUFA 43.4 ± 3.0a 46.9 ± 4.2a 42.4 ± 1.4ab 31.3 ± 2.2b 18:2 n-6 64.6 ± 3.2 61.8 ± 4.9 68.0 ± 2.8 52.6 ± 3.5 18:3 n-6 0.8 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 0.8 ± 0.03 20:2 n-6 7.2 ± 0.8 6.1 ± 0.4 5.6 ± 0.4 4.4 ± 0.2 20:3 n-6 4.6 ± 0.3 4.2 ± 0.2 3.9 ± 0.2 2.8 ± 0.2 20:4 n-6 164.9 ± 9.1a 147.1 ± 8.2ab 148.2 ± 4.8ab 113.7 ± 6.7b 22:2 n-6 0 ± 0 0 ± 0 0.4 ± 0.3 0.7 ± 0.4 22:4 n-6 6.0 ± 1.2 8.0 ± 1.6 4.5 ± 0.6 6.3 ± 1.0 22:3 n-6 0 ± 0 0 ± 0 0 ± 0 0 ± 0 22:5 n-6 3.8 ± 0.7 4.5 ± 1.1 2.6 ± 0.5 3.3 ± 0.5 Total n-6 PUFA 251.8 ± 11.9 232.6 ± 8.1 234.1 ± 7.1 184.7 ± 8.8 18:3 n-3 1.5 ± 0.2 1.6 ± 0.2 1.7 ± 0.1 1.6 ± 0.2 18:4 n-3 0 ± 0 0 ± 0 0 ± 0 0 ± 0 20:3 n-3 2.0 ± 0.2 1.8 ± 0.2 1.3 ± 0.03 1.2 ± 0.1 20:5 n-3 1.0 ± 0.4 1.4 ± 0.4 0.9 ± 0.1 1.1 ± 0.1 22:5 n-3 4.7 ± 0.2 4.7 ± 0.4 4.1 ± 0.3 4.1 ± 0.3 22:6 n-3 42.3 ± 3.0a 36.9 ± 3.6ab 35.1 ± 1.2ab 28.2 ± 1.9b Total n-3 PUFA 51.5 ± 3.2a 46.4 ± 3.5a 43.1 ± 1.3ab 36.1 ± 1.9b Total fatty acids 608.6 ± 37.1a 567.0 ± 22.9ab 549.4 ± 12.3ab 427.9 ± 23.5b

Data are mean ± SEM of n = 7-8 per group. Values with different superscripts denote significant differences between the means, as determined by one-way ANOVA and Tukey’s post-hoc test.

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Table 2: Fatty acid levels (mg) in liver phospholipids (PL)

Saline Mineral oil


12:0 1.7 ± 0.7 0.9 ± 0.3 1.2 ± 0.3 4.2 ± 3.1 13:0 1.3 ± 0.6 0.7 ± 0.3 1.0 ± 0.4 0.7 ± 0.3 14:0 5.2 ± 2.5 2.1± 0.4 2.2 ± 0.6 1.4 ± 0.5 15:0 6.3 ± 3.3 4.3 ± 1.5 5.2 ± 1.8 3.4 ± 1.2 16:0 54.0 ± 6.5a 45.5 ± 2.0ab 46.4 ± 2.1ab 36.8 ± 2.9b 18:0 81.9 ± 9.3a 74.2 ± 4.7ab 74.2 ± 2.0ab 57.5 ± 3.9b 19:0 3.8 ± 1.4 2.2 ± 0.6 4.1 ± 1.4 4.1 ± 1.8 20:0 2.3 ± 1.0 0.8 ± 0.3 2.2 ± 0.9 1.0 ± 0.3 22:0 11.6 ± 5.4a 0.2 ± 0.2ab 0.1 ± 0.1b 0.3 ± 0.1ab 24:0 3.1 ± 1.2 2.2 ± 0.4 4.3 ± 1.0 2.9 ± 1.0 Total SFA 171.2 ± 26.7 133.3 ± 6.5 140.9 ± 8.4 112.3 ± 9.4 14:1 n-9 9.3 ± 5.3 5.9 ± 2.5 7.3 ± 2.8 4.7 ± 1.9 15:1 n-10 6.9 ± 3.9 4.3 ± 1.8 5.3 ± 2.0 3.4 ± 1.3 16:1 t-9 0 ± 0 0 ± 0 0 ± 0 0 ± 0 16:1 n-9 2.3 ± 0.6 1.6 ± 0.2 1.8 ± 0.4 1.2 ± 0.3 18:1 t-9 3.7 ± 1.1 2.7 ± 0.4 3.2 ± 1.2 2.9 ± 1.3 18:1 t-11 1.9 ± 1.0 1.1 ± 0.6 1.1 ± 0.6 0.9 ± 0.4 18:1 n-9 14.7 ± 2.8 12.2 ± 1.0 11.9 ± 1.5 10.3 ± 1.0 18:1 n-7 10.5 ± 0.9a 10.7 ± 0.5a 9.1 ± 0.5a 5.9 ± 0.5b 19:1 n-7 0 ± 0 0 ± 0 0 ± 0 0 ± 0 20:1 n-5 0 ± 0 0 ± 0 0 ± 0 0.5 ± 0.5 20:1 n-8 0.6 ± 0.3 0.4 ± 0.2 1.1 ± 0.4 0.5 ± 0.3 22:1 n-9 4.1 ± 1.5 3.7 ± 1.8 5.9 ± 2.2 1.9 ± 0.8 Total MUFA 15.3 ± 7.1 12.9 ± 5.3 10.3 ± 3.7 8.3 ± 2.5 18:2 n-6 25.8 ± 1.6ab 24.7 ± 1.1ab 28.0 ± 1.0a 22.6 ± 1.2b 18:3 n-6 1.2 ± 0.4 0.9 ± 0.1 1.7 ± 0.4 1.2 ± 0.3 20:2 n-6 4.9 ± 1.2 4.3 ± 0.2 6.7 ± 1.2 4.8 ± 1.2 20:3 n-6 3.0 ± 0.4 2.8 ± 0.2 3.7 ± 0.5 2.4 ± 0.5 20:4 n-6 59.7 ± 10.5 73.3 ± 4.5 71.6 ± 3.2 52.6 ± 2.8 22:2 n-6 13.7 ± 9.2 7.2 ± 2.2 13.7 ± 4.9 10.3 ± 4.0 22:4 n-6 4.6 ± 1.2ab 6.4 ± 1.1ab 12.6 ± 2.8a 7.4 ± 2.3b 22:3 n-6 0 ± 0 0 ± 0 0 ± 0 0 ± 0 22:5 n-6 5.3 ± 2.4 3.3 ± 0.6 7.2 ± 1.8 3.5 ± 1.1 Total n-6 PUFA 118.3 ± 18.8 122.8 ± 5.0 145.1 ± 8.9 104.7 ± 11.4 18:3 n-3 1.5 ± 0.3 0.9 ± 0.3 2.1 ± 0.6 1.7 ± 0.7 20:3 n-3 6.3 ± 2.1 5.3 ± 0.9 8.7 ± 2.5 5.2 ± 1.5 20:5 n-3 1.2 ± 0.7 0.8 ± 0.2 1.5 ± 0.3 0.7 ± 0.3 22:5 n-3 4.0 ± 1.2 3.4 ± 0.5 5.7 ± 1.1 4.0 ± 1.0 22:6 n-3 17.0 ± 1.8 17.9 ± 1.9 19.2 ± 0.9 14.8 ± 1.7 Total n-3 PUFA 30.0 ± 5.1 28.2 ± 2.7 37.2 ± 4.8 26.4 ± 4.9 Total area 373.5 ± 59.8 326.9 ± 15.4 370.1 ± 30.8 275.8 ± 31.4


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Table 3: Fatty acid levels (mg) in liver triglycerides (TG)

Saline

Mineral oil


12:0 3.5 ± 1.3 2.6 ± 0.8 1.3 ± 0.3 1.7 ± 0.3 13:0 1.7 ± 0.6 1.2 ± 0.7 0.3 ± 0.2 1.3 ± 0.4 14:0 2.9 ± 0.7 2.9 ± 0.6 1.5 ± 0.3 2.2 ± 0.5 15:0 4.0 ± 1.6 3.8 ± 1.7 1.5 ± 0.5 4.1 ± 1.9 16:0 29.9 ± 3.8 42.7 ± 16.1 21.2 ± 2.3 18.0 ± 2.7 18:0 24.4 ± 9.2 24.1 ± 8.5 12.7 ± 2.2 14.6 ± 3.4 19:0 4.7 ± 1.9 5.3 ± 2.5 6.9 ± 3.2 6.1 ± 3.2 20:0 2.5 ± 1.9 1.2 ± 0.7 0.2 ± 0.1 0.4 ± 0.2 22:0 1.5 ± 0.7 0 ± 0 0.1 ± 0.1 4.9 ± 4.9 24:0 0.7 ± 0.4 1.7 ± 1.1 1.4 ± 1.3 0.6 ± 0.3 Total SFA 75.9 ± 14.3 85.5 ± 26.1 47.1 ± 6.8 53.7 ± 13.3 14:1 n-9 4.9 ± 2.8 5.2 ± 2.8 1.8 ± 0.7 6.0 ± 3.0 15:1 n-10 3.0 ± 1.7 3.4 ± 1.9 1.2 ± 0.5 4.1 ± 2.1 16:1 t-9 0 ± 0 0.4 ± 0.3 0.1 ± 0.1 0.3 ± 0.1 16:1 n-9 2.2 ± 0.7 3.1 ± 0.8 1.5 ± 0.2 2.0 ± 0.6 17:1 n-10 0.6 ± 0.3 0.4 ± 0.2 0.1 ± 0.1 0.9 ± 0.4 18:1 t-9 2.9 ± 0.7 4.3 ± 1.2 3.0 ± 0.9 4.4 ± 1.8 18:1 t-11 0.6 ± 0.5 0 ± 0 0 ± 0 0 ± 0 18:1 n-9 31.1 ± 4.6a 30.2 ± 7.4a 21.8 ± 3.2a 10.9 ± 2.4b 18:1 n-11 6.0 ± 0.9 6.1 ± 1.7 3.5 ± 0.7 1.7 ± 0.3 19:1 n-7 0.1 ± 0.1 0.2 ± 0.2 0.02 ± 0.01 0.1 ± 0.1 20:1 n-5 1.9 ± 1.9 0 ± 0 0 ± 0 0 ± 0 20:1 n-8 0.6 ± 0.3 0.9 ± 0.4 1.2 ± 0.4 0.2 ± 0.1 20:1 n-11 0.4 ± 0.3 0.7 ± 0.4 0.1 ± 0.1 0.1 ± 0.1 22:1 n-9 1.0 ± 0.4 1.5 ± 0.8 2.8 ± 2.0 4.8 ± 4.4 24:1 n-9 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Total MUFA 55.2 ± 8.5 56.5 ± 10.4 37.1 ± 6.0 35.5 ± 12.0 18:2 n-6 27.2 ± 5.6 29.9 ± 8.5 18.6 ± 2.7 14.4 ± 3.9 18:3 n-6 1.0 ± 0.4 1.4 ± 0.4 1.2 ± 0.5 1.1 ± 0.5 20:2 n-6 4.0 ± 1.6 4.1 ± 1.0 3.1 ± 1.1 1.9 ± 0.6 20:3 n-6 1.3 ± 0.4 1.1 ± 0.5 1.7 ± 0.6 1.4 ± 0.5 20:4 n-6 7.1 ± 3.1 8.3 ± 1.7 8.2 ± 3.4 4.4 ± 1.6 22:2 n-6 3.7 ± 3.1 5.5 ± 2.2 19.0 ± 7.3 9.4 ± 6.1 22:4 n-6 7.3 ± 2.1 7.7 ± 2.5 6.4 ± 2.4 4.3 ± 1.8 22:3 n-6 0 ± 0 1.3 ± 0.8 1.3 ± 0.9 0.4 ± 0.4 22:5 n-6 3.1 ± 0.9 4.1 ± 1.0 3.8 ± 1.4 2.1 ± 0.9 Total n-6 PUFA 54.7 ± 12.2 63.5 ± 11.3 63.3 ± 16.7 39.3 ± 12.8 18:3 n-3 2.9 ± 0.9 4.1 ± 1.1 3.0 ± 0.9 2.4 ± 0.8 18:4 n-3 0.2 ± 0.2 0.4 ± 0.4 0.6 ± 0.5 0.2 ± 0.2 20:3 n-3 7.9 ± 3.9 9.1 ± 2.9 9.2 ± 4.0 5.4 ± 2.6 20:5 n-3 0.7 ± 0.3 0.6 ± 0.5 0.7 ± 0.6 0.2 ± 0.1 22:5 n-3 3.7 ± 1.7 4.5 ± 1.2 3.9 ± 1.5 1.8 ± 0.7 22:6 n-3 4.4 ± 1.4 5.8 ± 1.1 7.2 ± 3.1 3.1 ± 1.5 Total n-3 PUFA 19.7 ± 7.4 24.6 ± 5.4 24.4 ± 10.1 13.1 ± 5.1 Total area 205.6 ± 26.6 230.1 ± 39.9 171.9 ± 37.9 141.6 ± 41.2


134

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CHAPTER 5

ACUTE ADMINISTRATION OF DOCOSAHEXAENOIC ACID INCREASES RESISTANCE TO PENTYLENETETRAZOL-

INDUCED SEIZURES

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5 Experiment 4: Acute administration of docosahexaenoic acid increases resistance to pentylenetetrazol-induced seizures

Forward

It seemed likely that the anticonvulsant effects of the n-3 PUFA seen in

Experiment 2 were mediated by docosahexaenoic acid (DHA), the final compound in the

n-3 PUFA synthetic pathway, and the most abundant n-3 PUFA found in the brain. ALA

and EPA, the precursors to DHA, are not likely to have anticonvulsant effects, because

they are immediately oxidized or recycled into saturated fatty acids and cholesterol upon

entering the brain (Demar et al., 2005; Chen et al., 2009). This is consistent with their

negligible levels in brain phospholipids (<1% of total fatty acids) (Demar et al., 2005;

Chen et al., 2009).

Appendix 1 shows the results of a pilot study that tested the effects of acutely

administered EPA to rats, on PTZ-induced seizure thresholds. As expected, EPA did not

raise seizure threshold.

In Experiment 4, therefore, the anticonvulsant effects of direct DHA

administration were tested in male Wistar rats using the PTZ model. DHA was

administered via the subcutaneous route. Sedation was also measured in order to

investigate possible toxic effect of DHA. In addition, we measured levels of DHA post-

injection in blood and brain in order to confirm that DHA was elevated after acute

subcutaneous injections.

The rationale for using Wistar rats instead of Long Evans was that Wistar rats

have been regularly used in the past by our group to screen for for novel anticonvulsant

compounds (Albright and Burnham, 1980; Likhodii et al., 2003). Long Evans rats were

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used in Experiment 1 and 2 only because Yehuda and colleagues had used them (Yehuda

et al., 1994; Rabinovitz et al., 2004). For the rest of our experimental work, therefore, we

switched to working with Wistar rats.

The hypothesis of Experiment 4 was that acute administration of DHA to male

Wistar rats would increase the latency to seizure onset - and also raise DHA

concentrations in plasma and brain.

Subsequent to the initial dose-response study, a subsequent time-response study

was performed in order to establish the optimum time for testing DHA’s anticonvulsant

effects.

In Experiment 4, unlike Experiment 2, DHA levels were assayed in the plasma

and brains of a separate, parallel group of subjects that had been injected with DHA but

had not been seizure tested.

As indicated below, it was found that acute, subcutaneous administration of DHA

increased latency to seizure onset in the maximal PTZ seizure model, and did so without

causing sedation. DHA administration, however, did not alter plasma and brain

phospholipid and unesterified fatty acid DHA concentrations. Overall, these findings

suggest that DHA increases resistance to PTZ-induced seizures.

The related manuscript starts on the following page. It is currently under review

by Epilepsy Research. The co-authors, Melanie Jeffrey and Saimir Bala assisted with the

PTZ studies. Nadeen Taha assisted with the lipid analysis. Dr. W. McIntyre Burnham

was the principal investigator of the study.

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Acute administration of docosahexaenoic acid increases resistance to

pentylenetetrazol-induced seizures in rats

Ameer Y. Taha, Melanie A. Jeffrey, Nadeen M.Y. Taha, Saimir Bala and W. McIntyre

Burnham

Department of Pharmacology and Toxicology, Faculty of Medicine, University of


University of Toronto Epilepsy Research Program, Faculty of Medicine, University of



Ameer Y. Taha





Canada


Running title: Anticonvulsant effects of DHA

Key words: Docosahexaenoic acid, omega-3 polyunsaturated fatty acids,

pentylenetetrazol, anticonvulsant, seizures, epilepsy

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5.1 Abstract

Purpose: Docosahexaenoic acid (DHA), an omega-3 fatty acid, has been proposed to

raise seizure threshold. The purpose of the present study was to test the acute

anticonvulsant effects of DHA in rats, using the maximal pentylenetetrazol (PTZ) seizure

model, and to confirm DHA incorporation and distribution in plasma total lipids and

brain phospholipids and unesterified fatty acids. Sedation was also measured in order to

monitor for potential toxicity of the DHA.

Methods: Male Wistar rats received a subcutaneous injection of saline, oleic acid (OA) or

DHA (400mg/kg) and seizure tested using the maximal PTZ seizure test at several time-

points. Another batch of rats received saline, OA or DHA (400 mg/kg) and were either

seizure tested or sacrificed for plasma and brain DHA analysis at one hour. Sedation was

measured during the one-hour period prior to seizure testing or sacrifice.

Results: Acute administration of DHA increased latency to seizure onset by one-hour, as

compared to the saline or OA controls (P<0.05), which did not differ significantly from

each other (P>0.05). There were no significant effects of treatment on plasma total lipids

or brain phospholipid and unesterified fatty acid profiles (P>0.05), or on measures of

sedation (P>0.05).

Conclusion: DHA increases resistance to PTZ-induced seizures without altering

measures of sedation.

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5.2 Introduction

Seizures are self-sustained, usually time-limited, episodes of neuronal

hyperexcitability, involving synchronous discharges in large neuronal populations

(Burnham, 2007). In people with epilepsy, spontaneous seizures occur because of a

chronically low seizure threshold in some part of the brain (Burnham, 2007). Epilepsy is

treated with anticonvulsant medications, which control seizures in approximately 60% of

patients (Shorvon, 1996).

Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been reported to raise

seizure thresholds in rodents. Yehuda and colleagues, for instance, have reported that the

chronic administration of the n-3 PUFA α-linolenic acid, with linoleic acid in a 1 to 4

ratio (i.e. the “SR-3 mixture”), increases resistance to pentylenetetrazol (PTZ)-induced

seizures in rats (Yehuda et al., 1994), possibly by raising brain levels of the n-3 PUFA,

docosahexaenoic acid (DHA), the final compound in the n-3 PUFA synthetic pathway

and the most abundant n-3 PUFA in the brain (Yehuda et al., 1996). A subsequent study

that used the same SR-3 dose and seizure model failed to replicate these findings (Taha et

al., 2006), A recent study, however, has shown that the SR-3 PUFA mixture does

increase latency to seizure onset when chronically administered at higher doses (Taha et

al., 2009). The increase in seizure latency was associated with an increase in brain total n-

3 PUFA levels within the unesterified fatty acid fraction (Taha et al., 2009).

To date, only one study has examined the direct effects of DHA on seizure

occurrence. Voskuyl and colleagues (Voskuyl et al., 1998) have reported that acute

intravenous infusion of DHA increases seizure threshold in a cortical stimulation model.

While suggestive, this study did not use a well-validated pharmacological seizure model.

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The goal of the present study was to examine the acute anticonvulsant effects of

DHA, injected subcutaneously (s.c.) in the maximal PTZ seizure model - and also to

investigate levels of DHA in plasma and brain following acute injection. Maximal PTZ

seizures model generalized tonic-clonic attacks in humans, and have been used to screen

for new anticonvulsant drugs (Fisher, 1989). DHA levels in plasma and brain were

measured after acute administration and (possible) sedation was measured using a non-

invasive rating scale.

Three different experiments were conducted in order to determine the

anticonvulsant effects of DHA in rats. Experiment 1 was a pilot dose-response study that

was designed to determine the dose of DHA that would increase seizure latency in the

maximal PTZ seizure test. Experiment 2 was a time-response study that measured the

effects of DHA on seizure latency from 15 to 480 minutes post injection, using the dose

derived from Experiment 1. In Experiment 3, we explored the dose—response effects of

DHA on seizure latency and sedation in a larger group of subjects - and also measured

the distribution of DHA in plasma and brain lipids following acute injection.


5.3.1 Drug preparation

Saline containing albumin, oleic acid (OA) and DHA stock solutions were

prepared on the day of the experiment. Saline-albumin was prepared by dissolving 90 mg

of albumin per ml of 0.9% saline. Unesterified OA and DHA (Sigma-Aldrich, St. Louis,

Missouri, USA) were each dissolved in 0.9% saline containing 90 mg of albumin per ml,

at a concentration of 140 µl per ml. All stock solutions were sonicated for 5 minutes, and

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kept on ice throughout the experiment in order to minimize oxidation of the fatty acids.

The final pH of the fatty acid mixtures was approximately 5.65.

PTZ (Sigma-Aldrich, St. Louis, Missouri, USA) was prepared by dissolving 50

mg of PTZ per ml of 0.9% saline. The PTZ solution was also kept on ice throughout the

experiment.

5.3.2 Subjects

All experimental protocols were approved by the Animal Care Committee of the

Faculty of Medicine of the University of Toronto, and were conducted in accordance with

the guidelines of the Canadian Council on Animal Care.

Male Wistar rats (Charles River, La Prairie, QC, Canada), aged 53 days, served as

subjects for all experiments. Subjects were housed individually in plastic cages with

corn-cob bedding in a vivarium maintained on a 12 h light, 12 h dark cycle (lights on at

7am) and at a temperature of 21ºC. The subjects were allowed access to water and regular

rat chow ad libitum (Teklad Global, 2018 18% Protein Rodent Diet). Before testing, each

subject was handled daily for 6 consecutive days, starting on the second day after arrival

and continuing until the day prior to the experiments.

Experiment 1:

The goal of Experiment 1 was to establish an approximate anticonvulsant dose of

DHA for subsequent studies. After 7 days in the facility, the subjects were weighed in

order to calculate the injection doses of PTZ and DHA. The subjects were then randomly

allocated to the following treatment groups – saline (n=3), DHA 200 mg/kg (n=3), DHA

400 mg/kg (n=3) and DHA 800 mg/kg (n=2). At the time of testing, subjects were

injected s.c. with an appropriate dose of the DHA or saline (volume of injection ~ 0.5 to 2

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ml). Ten minutes following the saline or DHA injection, subjects received an

intraperitoneal (i.p.) injection of eighty mg/kg of PTZ. Subjects were then placed in an

open field for a 30-minute observation period, and latency to the onset of the first

myclonic jerk was scored by two independent observers. Following testing, all subjects

were euthanized with a lethal i.p. injection of sodium pentobarbital (100 mg/kg).

Experiment 2:

Our first experiment indicated that a dose of 400 mg/kg of DHA was most

effective in inceasing latency to seizure onset. Experiment 2 was a time-response study,

carried out in a separate group of rats using that dose. Subjects were obtained and housed

as decribed above. After 7 days in the vivarium, they received s.c. injections of oleic acid

(OA) (isoclaoric control) or DHA at a dose of 400 mg/kg (volume of injection ~ 1 ml).

They were then injected with 105 mg/kg of PTZ (i.p.) at the following post DHA time-

points: 15, 30, 60, 120, 240 and 480 minutes (n=8 rats per treatment for each time-point).

Latencies to the onset of myoclonic jerks and tonic-clonic seizures were recorded.

Experiment 3

Our second experiment indicated the DHA was maximally effective 1 hour after

injection. Experiment 3 was designed to confirm the effects of DHA on seizure latency

and to to explore its effects on sedation - and also to measure the distribution of DHA in

plasma and brain lipids following acute administration.

5.3.3 Seizure tests and sedation scoring in Experiment 3

A new batch of 53-day-old male Wistar rats was obtained, housed and handled as

described above. After 7 days in the vivarum, they were randomly allocated to three

groups, which received s.c. injections of: 1) saline (n=11), 2) 400 mg/kg of OA (n=8)

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(isocaloric control), or 3) 400 mg/kg of DHA (n=12) (volume of injection ~ 1 ml). One

hour following DHA or control treatment, the subjects were injected with with 80 mg/kg

of PTZ (i.p.) and then placed in an open field for a 60-minute observation period. Latency

to the onsets of the first myclonic jerk and the first tonic-clonic seizure was scored by two

independent observers. Following the seizure tests, the subjects were euthanized with a

lethal i.p. injection of sodium pentobarbital (100 mg/kg).

Sedation was also scored in these subjects by two independent observers during

the 60-minute observation period prior to seizure testing. Scores were obtained once

every minute for the first twenty minutes, and then once every 5 minutes for the

remaining forty minutes. Sedation was scored using the Loscher sedation scale, which

involves the following categories: stage 1, slightly reduced forward locomotion; stage 2,

reduced locomotion with rest periods in between; stage 3, reduced locomotion with

frequent rest periods and partly closed eyes; 4, no forward locomotion, with closed eyes

(Loscher et al., 1987).

5.3.4 Assays in Experiment 3

Assays were done on a separate group of 22 subjects, that were obtained, housed

and handled as described above, but not seizure tested. After acclimatization to the

vivarium, subjects were randomized to the same DHA or control treatments (n=8 saline,

n=8 OA and n=6 DHA), and then placed in an open field for one hour of observation.

Sedation was scored during the one hour period as described above. The subjects were

then injected with a lethal dose of sodium pentobarbital and decapitated. Whole blood

was collected immediately following decapitation and was placed on ice. The blood was

then centrifuged at 400 x g (3000 rpm) for 20 minutes and the top plasma layer was

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pipetted into centrifuge tubes and stored at -80°C. Brains were rapidly collected, weighed

(average weight of 1.8g), flash-frozen in liquid nitrogen, and stored at -80°C, for future

lipid analysis.

5.3.5 Plasma total lipid analysis in Experiment 3

Non-esterified heptadecaenoic acid (Sigma, St. Louis, Mo), dissolved in

chloroform, was added to 1 ml of plasma as an internal standard. Ten ml of chloroform /

methanol (2:1 v/v) were then added to all plasma samples, followed by 2.5 ml of 0.9%

saline. The samples were capped under nitrogen, manually shaken for 10 seconds and

placed in cold room set at 4°C for forty-eight hours to allow for separation of the phases.

The bottom layer containing total lipids was transferred to 15 ml glass screw cap

tubes with Teflon lined caps, dried under nitrogen, and directly methylated in 2 ml of

14% methanolic BF3 and 2 ml of hexane at 100 °C for 1 hour. Deionized water (2 ml)

was then added to terminate the reaction. The upper hexane layer was extracted, dried

under nitrogen and reconstituted in 100 ul of hexane for analysis by gas chromatography.

5.3.6 Brain phospholipid and unesterified fatty acid analysis in Experiment 3

Diheptadecanoyl L-α-phosphatidylcholine and non-esterified heptadecaenoic acid

(Sigma, St. Louis, Mo) in chloroform were added as internal standards to whole brain

samples. The brain tissues were homogenized for approximately 30 seconds in 10 ml of

2:1 chloroform / methanol (v/v). Saline (0.9%, 2.2ml) was then added to separate the

polar phase. All samples were capped under nitrogen and shaken manually. The total

lipids were allowed to extract for forty-eight hours at 4°C. Following the formation of

two distinct phases, the lower chloroform / methanol layer containing the total lipids was

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transferred to 15 ml glass screw cap tubes with Teflon-lined caps. The extract was dried

under a gentle stream of nitrogen and reconstituted in 300 μl of chloroform /methanol

(1:1 v/v).

Thin layer chromatography (TLC) was used to separate the phospholipid and free

fatty acid fractions from brain total lipids, using 20 x 20 cm silica gel TLC plates

(Whatman LK6D plates, precoated with 250μm of Silica Gel 60A). Separate lanes were

spotted with phospholipids or free fatty acid standards. The plates were developed using

hexane, diethyl ether, and acetic acid (80:20:1 by volume) in covered glass tanks for 35

minutes. The plates were lightly sprayed with 8-anilino-1-naphthalenesulfonic acid, and

bands corresponding to phospholipid and free fatty acid standards were viewed under

ultraviolet light. The bands were scraped off each plate, into 15 ml glass screw cap tubes

with Teflon lined caps, and directly methylated in 14% methanolic BF3 (2 mL) and

hexane (2 ml) at 100°C for 1 hour. Deionized water (2 ml) was then added to terminate

the reaction and separate the phases. The upper hexane layer was extracted, dried under

nitrogen, and reconstituted in 100 µl of hexane for analysis by gas chromatography.

5.3.7 Fatty acid methyl ester analysis by gas-chromatography in Experiment 3

Fatty acid methyl esters (FAME) in brain phospholipids and unesterified fatty

acids, and in plasma total lipids were analyzed on an Agilient 6890 gas-chromatography

system equipped with a 30m x 25mm capillary column (J and W Scientific, DB-23,

Folsom, CA) and a flame ionization detector. One µl of fatty acid methyl esters from

each sample was injected into the column in splitless mode, using helium gas as a carrier,

at a constant flow rate of 0.7 ml per minute. A three stage temperature program was used

to acquire the fatty acid methyl ester profile. Initial temperature setting was at 50ºC with

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a 2 minute hold, followed by a ramp up at 20ºC per minute to 170ºC and a 1 minute hold,

and a final 3ºC per minute ramp up to 212ºC followed by a 10 minute hold. The fatty acid

peaks were identified using authentic fatty acid standards of known composition

(GLC463, NuCheck Prep., ON, Can).


All data are presented as mean ± SEM. Data analysis was performed using Sigma

Stat v.3.2 (Systat Software, Inc.). A one-way ANOVA followed by Tukey’s post-hoc

comparisons was used to determine the effect of treatment on seizure latency

(Experiments 1 and 3), plasma fatty acid concentrations (Experiment 3), and brain

phospholipid and unesterified fatty acid concentrations (Experiment 3). A two-way

repeated measures analysis of variance was used to determine the effects of treatment and

time on seizure latency (Experiment 2) and on sedation (Experiment 3). Outliers falling

more than 2 standard deviations from the mean were excluded from all statistical

analyses. The chi-square test was used to assess differences in seizure occurrence.

Statistical significance was accepted at P<0.05.

5.4 Results

5.4.1 Experiment 1

Experiment 1 was a pilot study that was designed to establish a working dose of

the DHA. All animals in both control and experimental groups of Experiment 1 exhibited

seizure activity after PTZ administration. Seizure latency (to myoclonic jerks) averaged

39.7 ± 2.3 seconds in the saline group, and 53.3 ± 6.9, 57.7 ± 9.8 and 34.5 ± 0.5 seconds

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in subjects that received the DHA at 200 mg/kg, 400 mg/kg and 800 mg/kg respectively

(n=2-3 per group). Analysis of variance did not show these differences to be significant

(P=0.12), most likely due to the small number of subjects that were used. A dose of 400

mg/kg of DHA was chosen for use in the following experiments, however, because

seizure latency was highest in the subjects that received this dose.

5.4.2 Experiment 2

Most, but not all, rats of Experiment 2 exhibited seizure activity following PTZ

injection. One rat did not seize at one hour post OA injection, two and three rats from the

OA and DHA groups, respectively, did not seize at four hours and two and one rat from

the OA and DHA groups respectively, did not seize at eight hours. There were no

statistically significant differences between the groups in the number of rats that failed to

seize at the time-points tested (P> 0.05, Chi Square test). Subjects that failed to seize

were excluded from the latency analyses (below).

The data related to the latency to onset of myoclonic jerks and tonic-clonic

seizures are presented in Figures 1-A and 1-B, respectively. As indicated, at one hour

after treatment subjects that were injected with DHA had a 3-fold increase in the latency

of both myoclonic jerks and tonic-clonic seizures, as compared to subjects injected with

OA. Two-way repeated analyses of variance showed that treatment, but not time, was a

significant factor affecting latency to the onset of both myoclonic jerks (P<0.05) and

tonic-clonic seizures (P<0.05).

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5.4.3 Experiment 3

Seizure occurrence and latency

All of the animals in the saline, OA and DHA groups exhibited seizure activity

after PTZ administration, except for two rats in the saline group and four rats in the DHA

group. There were no statistically significant differences in seizure occurrence among the

groups (P>0.05). Two outliers were excluded from the seizure latency statistical analyses,

including one subject in the saline group and one subject in the DHA group, with

myoclonic jerk or tonic-clonic latencies over 2.4 minutes.

The latency to the onset of myoclonic jerks was 38.8 ± 1.7, 39.8 ± 2.9 and 51.6 ±

4.2 seconds in the saline, OA and DHA treated groups, respectively (n=7-8/ treatment).

One-way analysis of variance showed that there were significant differences among the

group means (P<0.05). Tukey’s post-hoc test indicated that the average latency in the

DHA group was significantly longer than latencies in the saline and OA groups (P <

0.05), which did not differ from each other (P > 0.05).

DHA-treated subjects took 25% longer to exhibit a tonic-clonic seizure than

subjects that received the saline or OA. ± 2.1, 45 ± 3.4 and 59 ± 6.4 seconds in saline,

OA and DHA treated subjects, respectively (n=7-8/ treatment). One-way analysis of

variance showed that there were significant differences among the group means (P<0.05).

Tukey’s post-hoc test indicated that the average latency in the DHA group was

significantly longer than latencies in the saline and OA groups (P < 0.05), which did not

differ from each other (P > 0.05).

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Sedation scores

Average sedation scores for the subjects that were seizure tested and decapitated

are presented in Figures 2-A and 2-B, respectively. As indicated, sedation scores

increased over time for all groups (P<0.05), possibly because the subjects had habituated

to the test environment. Two-way repeated analysis of variance showed no significant

differences in sedation among the saline, OA and DHA treated subjects, although the

DHA treated rats displayed the greatest sedation scores between 40-60 minutes (P>0.05).

Plasma fatty acid concentrations in total lipids

DHA treatment did not significantly alter plasma DHA concentrations, measured

at one-hour post-injection (24.0 ± 3.4, 19.4 ± 5.4 and 25.5 ± 6.1 μg / ml in saline, OA and

DHA treated subjects), as determined by one-way analysis of variance. Concentrations of

other fatty acids in plasma, including saturated, monounsaturated and polyunsaturated

fatty acids, did not differ significantly among the groups (P>0.05, data not shown), as

indicated by a one-way analysis of variance. Also, total fatty acid concentrations were not

altered significantly (780 ± 93.5, 647 ± 145.0 and 691.1 ± 134.9 μg / ml in saline, OA

and DHA – treated subjects respectively).

Brain phospholipid fatty acid concentrations

The data related to brain DHA concentrations are presented in Figure 3-A. DHA

levels were not significantly different among the groups, as determined by one-way

analysis of variance (P>0.05). The concentration of other fatty acids within total

phospholipids, and total phospholipid concentrations did not differ significantly among

the three groups (P > 0.05, data not shown).

155

Brain unesterified fatty acid concentrations

Figure 3-B shows DHA concentrations in the unesterified fatty acid fraction of

brain total lipids. One-way analysis of variance indicated that DHA (Figure 3-B) and

other fatty acids within the unesterified fatty acid pool (data not shown) did not differ

significantly among the three groups (P > 0.05). No significant differences were detected

in total unesterified fatty acids (P>0.05).

5.5 Discussion

The results of the present study suggest that acute administration of DHA

increases latency to seizure onset without producing significant sedation. To our

knowledge, this is the first study to demonstrate that acute administration of DHA can

increase latency to seizure onset in the maximal PTZ seizure model.

The increase in seizure latency in the DHA treated group cannot be attributed to

an increase in caloric load caused by DHA, since administration of isocaloric amounts of

OA caused no change in seizure latency. Previous studies have shown that some of the

appetite-regulatory hormones, such as Neuropeptide Y and ghrelin, increase in plasma

following acute exposure to a caloric load (Okada et al., 1993) and that this increase

raises seizure thresholds in rats (Woldbye, 1998; Morris et al., 2007; Obay et al., 2007;

Noe et al., 2008). No such effect was seen in the present study, because no significant

differences in seizure latency were observed between subjects that received the OA,

which has a caloric value similar to DHA, and subjects that received saline, which has no

caloric value.

Previous work in our laboratory has shown that chronic administration of the

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“SR-3” mixture, which contains α-linolenic acid, raises total n-3 PUFA composition

(Taha et al., 2009). Interestingly, in that study, increases were seen in the brain

unesterified fatty acid pool, whereas no differences in n-3 PUFA levels were seen in the

present study (Taha et al., 2009). The difference in the results of these two studies may

relate to whether or not the subjects had been seizure tested. In the 2009 study, brain

fatty acid levels within the unesterified fatty acid fractions were measured post-seizure

(Taha et al., 2009). In the present study, the fatty acid measurements were performed in

rats that had not received PTZ. This suggests that seizures may cause brain n-3 PUFA to

shift from the phospholipid pool into the unesterified fatty acid pool.

It seems possible, then, that hyperexcitability in the brain may cause the release of

n-3 PUFA from phospholipid membranes (Bazan, 1970; Bazan, 1971; Rodriguez de

Turco and Bazan, 1983). The unesterified PUFA may then be utilized for energy and/or

act to reduce neuronal excitability. A reduction in neuronal excitability caused by

unesterified n-3 PUFA has been demonstrated in vitro (Xiao and Li, 1999; Lauritzen et

al., 2000; Young et al., 2000). Future studies should test the involvement of DHA de-

esterification in seizure protection.

The mechanism by which n-3 PUFA reduce neuronal excitation is not fully

understood. It might involve the partial inhibition of voltage-gated ion channels, or

protection against neuroinflammation. Unesterified DHA has been shown to reduce

neuronal excitability by partially inhibiting sodium and calcium voltage-gated channels

(Vreugdenhil et al., 1996). DHA, once released from brain phospholipids, might also

reduce neuronal excitability through its anti-inflammatory metabolites, such as

neuroprotectin D1 (Bazan, 2007). Previous studies have shown that neuroinflammation

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in the brain can lower seizure thresholds (Akarsu et al., 2006; Auvin et al., 2009), and

that anti-inflammatory agents can raise them (Tandon et al., 2003; Tu and Bazan, 2003;

Dhir et al., 2006; Oliveira et al., 2008). DHA concentrations in brain phospholipids did

not differ significantly between the groups. The lack of expected increase in phospholipid

DHA concentration following DHA injection does not necessarily imply that DHA was

not incorporated into brain phospholipids, since previous studies have shown that

radiolabeled DHA is incorporated into brain phospholipids when injected into rats

(Polozova and Salem, 2007). It is likely, however, that small changes in DHA

concentrations following a bolus injection of DHA would not have been detected by our

assays, due to the variability resulting from the large pool of DHA in the brain.

Plasma levels of DHA and OA were not significantly higher in the subjects that

received the DHA and OA injections, respectively. This is not an unexpected finding,

since the half-life of these free fatty acids in plasma is less than one minute (Robinson et

al., 1992). Their short half-life is due to their rapid uptake, and incorporation or

utilization by tissues such as adipose and brain (Polozova and Salem, 2007).

In the present study, DHA delayed latency to seizure onset within one-hour post-

injection (Figure 1). This suggests that DHA raised seizure threshold, since an increase in

seizure latency – at a time when brain levels of PTZ should be rising – suggests an

increase in seizure threshold. This observation requires further confirmation in other

seizure models that allow a direct measurement and quantification of threshold, such as

the focal electrical stimulation model (Albright, 1983).

Sedation was scored in the present study in order to determine whether DHA

would cause this type of toxicity at anticonvulsant doses. Although the DHA-treated

158

subjects showed the highest sedation scores (Figures 2-A and 2-B), this effect was not

statistically significant. The lack of significant sedative effects of DHA in rats at

anticonvulsant doses suggests that DHA may raise seizure threshold without producing

the sedative side-effects seen with many anticonvulsant drugs (Burnham, 2007).

We found acute anticonvulsant effects of the DHA within one hour following

injection. These findings appear to conflict with the findings of a recent study which

showed that dietary supplementation of DHA did not raise seizure threshold in rats

(Willis et al., 2009). It is not surprising, however, to observe no change in seizure

threshold following one month of dietary supplementation with DHA. DHA, when taken

by mouth, is packaged into chylomicrons and low-density lipoproteins, which keep the

DHA in the bloodstream and out of the brain for a few weeks (Polozova et al., 2006). It is

now believed that the brain obtains its DHA directly from plasma albumin (Chen et al.,

2008a; Ouellet et al., 2009), and that lipoproteins are not a major source of DHA for the

brain (Chen et al., 2008b). Thus, the rise in brain DHA levels is likely to be gradual and

more prolonged when provided by mouth, as compared to when it is directly injected into

the bloodstream. This has implications for clinical trials involving DHA, which to date

have produced mixed results (Schlanger et al., 2002; Yuen et al., 2005; Bromfield et al.,

2008; DeGiorgio et al., 2008), possibly due to their short duration and the low doses of

the supplements used in some trials (Schlanger et al., 2002; Yuen et al., 2005; Bromfield

et al., 2008; DeGiorgio et al., 2008).

In summary, the findings of the present study provide evidence that DHA raises

seizure threshold in rats within one-hour of subcutaneous administration. Further, in

contrast to several anticonvulsant medications, DHA appears to raise seizure threshold

159

without causing marked sedation (Albright, 1983). It seems possible that the n-3 PUFA

– in combination with anticonvulsant drugs – might be useful in the therapy of epilepsy.

ACKNOWLEDGEMENTS

We are deeply indebted to Dr. David W.L. Ma for providing us with the gas-

chromatography system to perform the fatty acid analyses, and to Dr. Richard P. Bazinet

for his valuable input and critical insight. Funding for this study was provided by the

Michael Bahen Chair in Epilepsy Research grant to Dr. W.M. Burnham and the Canadian

Institutes of Health Research doctoral award (Fredrick Banting and Charles Best Canada

Graduate Scholarships) to A.Y. Taha. We confirm that we have read the Journal’s

position on issues involved in ethical publication and affirm that this report is consistent

with those guidelines.

DISCLOSURES The authors declare that there are no competing personal or financial interests.

160

Figure 1-A: Latency to the onset of myoclonic jerks over time in rats treated with OA or DHA (400 mg/kg)

0

50

100

150

200

250

OA DHA OA DHA OA DHA OA DHA OA DHA OA DHA

15 30 60 120 240 480

Time (minutes)

Late

ncy

to m

yocl

onic

jerk

s (s

econ

ds)

161

Figure 1-B: Latency to the onset of tonic-clonic seizures over time in rats treated with OA or DHA (400 mg/kg)

0

50

100

150

200

250

OA DHA OA DHA OA DHA OA DHA OA DHA OA DHA

15 30 60 120 240 480

Time (minutes)

Late

ncy

to to

nic-

clon

ic s

eizu

res

(sec

onds

)

Data are mean ± SEM of n=5-8 / group.

Figure 1-A: Latency to the onset of myoclonic jerks over time in rats treated with OA or

DHA (400 mg/kg). Two-repeated analysis of variance showed a significant effect of

treatment (P<0.05), when the analysis was performed from 15 to 120 minutes. There was

no main effect of time (P>0.05).

Figure 1-B: Latency to the onset of tonic-clonic seizures over time in rats treated with

OA or DHA (400 mg/kg). Two-repeated analysis of variance showed a significant effect

of treatment (P<0.05), when the analysis was performed from 15 to 120 minutes. There

was no main effect of time (P>0.05).

162

Figure 2-A: Sedation score of subjects that were seizure tested 1-hour following drug administration

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 25 30 35 40 45 50 55 60Time (minutes)

Seda

tion

scor

e (o

ut o

f 4)

SalineOADHA

Figure 2-B: Sedation score of subjects that were decapitated 1-hour following drug administration

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 25 30 35 40 45 50 55 60Time (minutes)

Seda

tion

scor

e (o

ut o

f 4)

SalineOADHA

163

Data are mean ± SEM of n=6-8 / group.

Figure 2-A: Sedation score for subjects that were seizure tested 1-hour following drug

administration. Effect of saline, OA and DHA treatment on sedation score over time.

Sedation score significantly increased over time for all treatments, relative to baseline

(time 0): P<0.05 for significant main effect of time by 2-way repeated measures ANOVA.

Figure 2-B: Sedation score for subjects that were decapitated 1-hour following drug

administration. Effect of saline, OA and DHA treatment on sedation score over time.

Sedation score significantly increased over time for all treatments: P<0.05 for significant

main effect of time by 2-way repeated measures ANOVA.

164

Figure 3-A: DHA concentrations in brain phospholipids following saline, OA or DHA (400 mg/kg) subcutaneous injections

0.0

0.5

1.0

1.5

2.0

2.5

Saline OA DHA

Treatment

DH

A c

once

ntra

tion

in b

rain

PL

(mg

per g

)

165

Figure 3-B: DHA concentrations in brain unesterified fatty acids following saline, OA or DHA subcutaneous injections

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Saline OA DHA

Treatment

DH

A c

once

ntra

tion

in b

rain

une

ster

ified

fatty

aci

ds

(mg

per g

)

Data are mean ± SEM of n=6-8 for each group.

Figure 3-A: DHA concentrations in brain phospholipids following saline, OA or DHA

(400 mg/kg) subcutaneous injections. DHA concentrations in brain phospholipids were

measured at one-hour post saline, OA or DHA (400 mg/kg) subcutaneous injections.

There were no significant differences among the three groups, by one-way ANOVA

(P>0.05).

Figure 3-B: DHA concentrations in brain unesterified fatty acids following saline, OA or

DHA (400 mg/kg) subcutaneous injections. DHA concentrations in brain unesterified

fatty acids were measured at one hour post saline, OA or DHA (400 mg/kg) subcutaneous

injections. There were no significant differences among the three groups, by one-way

ANOVA (P>0.05).

166

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Albright, P.S., 1983. Effects of carbamazepine, clonazepam, and phenytoin on seizure threshold in amygdala and cortex. Exp Neurol 79, 11-17.

Auvin, S., Porta, N., Nehlig, A., Lecointe, C., Vallee, L., Bordet, R., 2009. Inflammation in rat pups subjected to short hyperthermic seizures enhances brain long-term excitability. Epilepsy Res. Epub ahead of print.

Bazan, N.G., 2007. Omega-3 fatty acids, pro-inflammatory signaling and neuroprotection. Curr Opin Clin Nutr Metab Care 10, 136-141.

Bazan, N.G. Jr., 1970. Effects of ischemia and electroconvulsive shock on free fatty acid pool in the brain. Biochim Biophys Acta 218, 1-10.

Bazan, N.G. Jr., 1971. Changes in free fatty acids of brain by drug-induced convulsions, electroshock and anaesthesia. J Neurochem 18, 1379-1385.

Bromfield, E., Dworetzky. B., Hurwitz, S., Eluri, Z., Lane, L., Replansky, S., Mostofsky, D., 2008. A randomized trial of polyunsaturated fatty acids for refractory epilepsy. Epilepsy Behav 12, 187-190.

Burnham, W.M., 2007. Antiseizure drugs. In: Principles of Medical Pharmacology Kalant, H., Grant, D.M. and Mitchell, J., eds., 223-235.

Chen, C.T., Green, J.T., Orr, S.K., Bazinet, R.P., 2008a. Regulation of brain polyunsaturated fatty acid uptake and turnover. Prostaglandins Leukot Essent Fatty Acids 79, 85-91.

Chen, C.T., Ma, D.W., Kim, J.H., Mount, H.T., Bazinet, R.P., 2008b. The low density lipoprotein receptor is not necessary for maintaining mouse brain polyunsaturated fatty acid concentrations. J Lipid Res 49, 147-152.

DeGiorgio, C.M., Miller, P., Meymandi, S., Gornbein, J.A., 2008. n-3 fatty acids (fish oil) for epilepsy, cardiac risk factors, and risk of SUDEP: clues from a pilot, double-blind, exploratory study. Epilepsy Behav 13, 681-684.

Dhir, A., Naidu, P.S., Kulkarni, S.K., 2006. Effect of cyclooxygenase inhibitors on pentylenetetrazol (PTZ)-induced convulsions: Possible mechanism of action. Prog Neuropsychopharmacol Biol Psychiatry 30, 1478-1485.

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Lauritzen, I., Blondeau, N., Heurteaux, C., Widmann, C., Romey, G., Lazdunski, M., 2000. Polyunsaturated fatty acids are potent neuroprotectors. Embo J 19, 1784-1793.

Loscher, W., Honack, D., Hashem, A., 1987. Anticonvulsant efficacy of clonazepam and the beta-carboline ZK 93423 during chronic treatment in amygdala-kindled rats. Eur J Pharmacol 143, 403-414.

Morris, M.J., Gannan, E., Stroud, L.M., Beck-Sickinger, A.G., O'Brien, T.J., 2007. Neuropeptide Y suppresses absence seizures in a genetic rat model primarily through effects on Y receptors. Eur J Neurosci 25, 1136-1143.

Noe, F., Pool, A.H., Nissinen, J., Gobbi, M., Bland, R., Rizzi, M., Balducci, C., Ferraguti, F., Sperk, G., During, M.J., Pitkanen, A., Vezzani, A., 2008. Neuropeptide Y gene therapy decreases chronic spontaneous seizures in a rat model of temporal lobe epilepsy. Brain 131(Pt 6), 1506-1515.

Obay, B.D., Tasdemir, E., Tumer, C., Bilgin, H.M., Sermet, A., 2007. Antiepileptic effects of ghrelin on pentylenetetrazole-induced seizures in rats. Peptides 28, 1214-1219.

Okada, K., Sugihara, H., Minami, S., Wakabayashi, I., 1993. Effect of parenteral administration of selected nutrients and central injection of gamma-globulin from antiserum to neuropeptide Y on growth hormone secretory pattern in food-deprived rats. Neuroendocrinology 57, 678-686.

Oliveira, M.S., Furian, A.F., Royes, L.F., Fighera, M.R., Fiorenza, N.G., Castelli, M., Machado, P., Bohrer, D., Veiga, M., Ferreira, J., Cavalheiro, E.A., Mello, C.F., 2008. Cyclooxygenase-2/PGE2 pathway facilitates pentylenetetrazol-induced seizures. Epilepsy Res 79, 14-21.

Ouellet, M., Emond, V., Chen, C.T., Julien, C., Bourasset, F., Oddo, S., Laferla, F., Bazinet, R.P., Calon, F., 2009. Diffusion of docosahexaenoic and eicosapentaenoic acids through the blood-brain barrier: An in situ cerebral perfusion study. Neurochem Int. Epub ahead of print.

Polozova, A., Gionfriddo, E., Salem, N. Jr., 2006 Effect of docosahexaenoic acid on tissue targeting and metabolism of plasma lipoproteins. Prostaglandins Leukot Essent Fatty Acids 75, 183-190.

Polozova, A., Salem, N. Jr., 2007. Role of liver and plasma lipoproteins in selective transport of n-3 fatty acids to tissues: a comparative study of 14C-DHA and 3H-oleic acid tracers. J Mol Neurosci 33, 56-66.

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Robinson, P.J., Noronha, J., DeGeorge, J.J., Freed, L.M., Nariai, T,. Rapoport, S.I., 1992. A quantitative method for measuring regional in vivo fatty-acid incorporation into and turnover within brain phospholipids: review and critical analysis. Brain Res Brain Res Rev 17, 187-214.

Rodriguez de Turco, E.B., Bazan, N.G., 1983. Changes in free fatty acids and diglycerides in mouse brain at birth and during anoxia. J Neurochem 41, 794-800.

Schlanger, S., Shinitzky, M., Yam, D., 2002. Diet enriched with omega-3 fatty acids alleviates convulsion symptoms in epilepsy patients. Epilepsia 43, 103-104.

Shorvon, S.D., 1996. The epidemiology and treatment of chronic and refractory epilepsy. Epilepsia 37 Suppl 2, S1-S3.

Taha, A.Y., Baghiu, B.M., Lui, R., Nylen, K., Ma, D.W., Burnham, W.M., 2006. Lack of benefit of linoleic and alpha-linolenic polyunsaturated fatty acids on seizure latency, duration, severity or incidence in rats. Epilepsy Res 71, 40-46.

Taha, A.Y., Filo, E., Ma, D.W., McIntyre Burnham, W., 2009. Dose-dependent anticonvulsant effects of linoleic and alpha-linolenic polyunsaturated fatty acids on pentylenetetrazol induced seizures in rats. Epilepsia 50, 72-82.

Tandon, M., Anuradha, K., Pandhi, P., 2003. Evaluation of antiepileptic activity of aspirin in combination with newer antiepileptic lamotrigine in mice. Methods Find Exp Clin Pharmacol 25, 607-610.

Tu, B., Bazan, N.G., 2003. Hippocampal kindling epileptogenesis upregulates neuronal cyclooxygenase-2 expression in neocortex. Exp Neurol 179, 167-175.

Voskuyl, R.A., Vreugdenhil, M., Kang, J.X., Leaf, A., 1998. Anticonvulsant effect of polyunsaturated fatty acids in rats, using the cortical stimulation model. Eur J Pharmacol 341, 145-152.

Vreugdenhil, M., Bruehl, C., Voskuyl, R.A., Kang, J.X., Leaf, A., Wadman, W.J., 1996. Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons. Proc Natl Acad Sci U S A 93, 12559-12563.

Willis, S., Samala, R., Rosenberger, T.A., Borges, K., 2009 Eicosapentaenoic and docosahexaenoic acids are not anticonvulsant or neuroprotective in acute mouse seizure models. Epilepsia 50, 138-142.

Woldbye, D.P., 1998. Antiepileptic effects of NPY on pentylenetetrazole seizures. Regul Pept 75-76, 279-282.

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raises the seizure threshold in rats. Eur J Pharmacol 254, 193-198. Young, C., Gean, P.W., Chiou, L.C., Shen, Y.Z., 2000. Docosahexaenoic acid inhibits

synaptic transmission and epileptiform activity in the rat hippocampus. Synapse 37, 90-94.

Yuen, A.W., Sander, J.W., Fluegel, D., Patsalos, P.N., Bell, G.S., Johnson, T., Koepp, M.J., 2005. Omega-3 fatty acid supplementation in patients with chronic epilepsy: a randomized trial. Epilepsy Behav 7, 253-258.

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CHAPTER 6

DIETARY FISH OIL SUPPLEMENTATION ELEVATES SEIZURE THRESHOLD IN THE CORTEX AND AMYGDALA OF RATS

171

6 Experiment 5: Dietary fish oil supplementation elevates seizure threshold in the cortex and amygdala of rats

Forward

The findings of Experiment 4 suggest that DHA, the final compound in the n-3

PUFA synthetic pathway and the most abundant n-3 PUFA found in the brain, elevates

seizure threshold in the PTZ seizure model.

The PTZ test is a pharmacological seizure model for generalized, tonic-clonic

attacks in humans (Fisher, 1989). A problem with pharmacological seizure models,

however, is that pro-convulsant drugs such as PTZ might interact with the drug that is

hypothesized to raise seizure threshold. Such an interaction could occur at the receptor or

pharmacokinetic level, thereby potentiating or masking the potential anti-seizure effects

of the drug of interest.

PTZ, for instance, induces seizures by non-competitively antagonizing GABA-A

receptors (Macdonald and Barker, 1977). DHA has recently been shown to increase the

binding capacity of GABA-A receptors, by altering the elastic properties of the

phospholipid membrane (Sogaard et al., 2006). Such an effect, therefore, might

counteract the effects of PTZ.

In order to exclude the possibility of a PTZ-DHA interaction, the effects of DHA

on seizure threshold were examined using an electrical stimulation threshold test. The test

involves implanting bipolar electrodes into a specific site within the brain, and

stimulating at incrementing currents until a simple partial seizure is detected on an EEG

machine.

Thus, the main purpose of Experiment 5 was to confirm the anti-seizure effects of

172

DHA using the electrical stimulation seizure test.

Another objective of Experiment 5 was to identify the types of seizures that the

DHA is likely to suppress. Seizures originating from limbic structures such as the

amygdala tend to be difficult to suppress with anti-seizure medications, whereas seizures

originating from extra-limbic structures such as the neocortex, are generally responsive to

anti-seizure medications (Albright, 1983). In order to determine whether the DHA is

effective against cortical and amygdaloid seizures, bipolar electrodes were implanted in

the motor cortex or the amygdala of rats. The subjects were then randomized to a diet

containing fish oil, a source of DHA, or to a soybean oil (control) diet. Seizure thresholds

were then measured once every few weeks in the cortex and the amygdala.

A final goal of experiment 5 was to determine whether DHA would have

anticonvulsant effects when administered by mouth. Previous studies had involved direct

injection of the DHA to rats. People with epilepsy, however, would likely consume

DHA-containing supplements by mouth. Experiment 5, therefore, involved dietary

administration of DHA using fish oil.

The findings of Experiment 5 suggest that a diet enriched with DHA does elevate

seizure thresholds in both the cortex and the amygdala. A higher dose of DHA was

required to elevate seizure threshold in the amygdala. Interestingly, threshold elevations

only occurred after prolonged administration of DHA in the diet.

The manuscript for this experiment begins on the next page. It has not been

submitted for publication. Flaviu Ciobanu and Bryan Ip helped with the electrode

placement assessment. Nadeen Taha helped with the dietary fatty acid analysis and tissue

dissections. Muaz Ahmed and Qiudi Zeng helped with the perfusions. Waiyin Cheuk,

173

helped with the threshold measurements. Elvis Filo helped with the surgeries. Brian Scott

(PhD candidate) provided expert advice on various aspects of the study design. Drs.

Richard P. Bazinet and W. McIntyre Burnham were the principal investigators in the

study.

174

Dietary fish oil supplementation elevates seizure threshold in the cortex and

amygdala of rats

Ameer Y. Taha1,2, Flaviu A. Ciobanu1,2, Nadeen M.Y. Taha1,2, Muaz Ahmed1,2, Qiudi

Zeng1,2, Waiyin I. Cheuk1,2, Bryan Ip1,2, Elvis Filo1,2, Brian W. Scott1,2, Richard P.

Bazinet2,3 and W. McIntyre Burnham1,2

1Departments of Pharmacology and Toxicology, and 2Nutritional Sciences, Faculty of

Medicine, University of Toronto, Toronto, ON, Canada, M5S 1A8


Toronto, Toronto, ON, Canada, M5S 1A8


Ameer Y. Taha






Canada


175

Running title: Fish oil supplementation elevates cortical and amygdaloid seizure

thresholds

Key words: Docosahexaenoic acid, fish oil, omega-3 polyunsaturated fatty acids, cortex,

amygdala, afterdischarge threshold, seizures, epilepsy, brain

176

6.1 Abstract

Background: Omega-3 polyunsaturated fatty acids (n-3 PUFA) have been suggested as

a treatment for patients with epileptic seizures. Simple partial seizures originating in the

frontal, parietal or occipital lobes are generally responsive to anti-seizure medications,

whereas complex-partial seizures, which often originate in limbic structures such as the

amygdala, are often resistant to anti-seizure medications.

Objective: To examine the effects of dietary omega-3 fatty acid supplementation with

fish oil on frontal cortical and amygdaloid seizure thresholds in rats.

Procedures: Male Wistar rats were surgically implanted with chronic bipolar,

stimulating/recording electrodes in the frontal cortex or amygdala, and subsequently

randomized to the AIN-93G diet supplemented with either soybean oil (control subjects)

or n-3-PUFA-containing fish oil (experimental subjects) for up to 34 weeks. Seizure

thresholds and seizure scores in the cortex and amygdala were measured every 2-4 weeks.

Results: Fish oil supplementation elevated seizure threshold in the cortex and amygdala

by 36% and 64% respectively from baseline (P<0.05), while soybean oil had no effect

(P>0.05). Seizure scores did not differ between the diet groups in either the cortex or

amygdala (P>0.05).

Conclusions: These observations indicate that dietary supplementation with fish oil,

which contains n-3 PUFA, can raise seizure threshold in the cortex and amygdala,

without altering seizure scores.

177

6.2 Introduction

Epilepsy is the most common of the serious neurological disorders, affecting

about 1% of the North American population (Theodore et al., 2006). If uncontrolled,

epileptic seizures disrupt life, and may lead to injury or death (Hauser et al., 1980;

O'Donoghue et al., 1999).

The most common therapy for epilepsy is treatment with anticonvulsant drugs.

These provide seizure control in approximately 60-70% of the patients (Shorvon, 1996;

Kwan and Brodie, 2000). The remaining 30-40% of patients, however, are “drug-

resistant”. Many of these patients have complex partial seizures.

Complex partial seizures, often of limbic origin, are usually difficult to control

with anticonvulsant medications (Oles et al., 1989; Stephen et al., 2001). By contrast,

simple-partial seizures, originating from extra-limbic brain structures such as the cortex,

are generally responsive to anticonvulsant drug treatment (Brodie, 2001; Stephen et al.,

2001).

In 1980, Albright and Burnham developed a pharmacological model in rats

designed to screen for anticonvulsant drugs that might be effective against simple and

complex partial seizures in humans (Albright and Burnham, 1980). Using kindled

subjects (Goddard et al., 1969; Racine, 1972b), Albright and Burnham reported that

anticonvulsants such as phenytoin, carbamazepine and valproate were effective at

suppressing cortical focal and generalized seizures, but had much less efficacy against

amygdala focal seizures (Albright and Burnham, 1980). They proposed that the cortical

focus might serve as a model for drug-responsive, simple-partial seizures in humans,

whereas the amygdala focus might serve as a model for drug-resistant complex-partial

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seizures (Albright, 1983).

Seizures that resist control by anticonvulsant drugs may sometimes be controlled

by the high-fat ketogenic diet, which reduces seizure frequency in up to two-thirds of the

patients who follow it (Cross and Neal, 2008; Neal et al., 2008a). The ketogenic diet,

however, is hard to maintain, and is nutritionally unbalanced and pro-atherogenic, since it

raises plasma LDL-cholesterol and triglyceride levels (Kwiterovich et al., 2003;

Fuehrlein et al., 2004). New healthier treatments are therefore necessary to treat patients

with simple partial or complex partial seizures.

It has recently been proposed that a normal diet, enriched by omega-3

polyunsaturated fatty acids (n-3 PUFA), might provide a healthier form of dietary seizure

control (Cunnane et al., 2002; Yuen and Sander, 2004). In agreement with this

suggestion, we have recently shown that chronic administration of the n-3 PUFA, α-

linolenic acid to rats, raises seizure thresholds in the maximal pentylenetetrazol (PTZ)

seizure test, possibly by increasing brain docosahexaenoic acid (DHA) levels (Taha et al.,

2009b; Taha et al., 2009c). Also, acute administration of DHA raised seizure thresholds

in the PTZ seizure model (Taha et al., under review). These effects on seizure threshold

were not associated with sedation (Taha et al., under review), increased weight gain or

any alteration in the expression of enzymatic markers of hepatotoxicity (Taha et al.,

2009a).

The maximal PTZ seizure test models tonic-clonic, generalized attacks in humans

(Fisher, 1989), and it is used to screen for drugs effective against generalized convulsive

seizures (Krall et al., 1978). It still remains to be determined whether a diet enriched in

the n-3 PUFA, particularly DHA, would be successful at raising focal seizure thresholds

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in the cortex or the amygdala.

The present study, therefore, investigated the effects of a diet enriched with n-3

PUFA on cortical and amygdala focal seizure thresholds. Male Wistar rats were

surgically implanted with chronic bipolar, stimulating/recording electrodes in the frontal

cortex or amygdala, and subsequently randomized to the AIN-93G diet supplemented

with either soybean oil (control subjects) or n-3-PUFA-containing fish oil (experimental

subjects) for up to 34 weeks. Seizure thresholds and scores in the cortex and amygdala

were measured every 2-4 weeks.

We demonstrate for the first time that dietary fish oil supplementation elevates

seizure threshold in both the cortex and the amygdala, but does not alter seizure scores,

which are a measure for seizure severity.



All experimental procedures were approved by the Animal Care Committee of the

Faculty of Medicine of the University of Toronto, and followed the guidelines of the

Canadian Council on Animal Care.

Male Wistar rats (Charles River, La Prairie, QC, Canada), aged 60 days, were

used as subjects in this study. Subjects were housed individually in transparent plastic

cages with corn-cob bedding in a vivarium maintained on a 12 h light-dark cycle (lights

on at 7am), and at a temperature of 21ºC. The subjects were allowed ad libitum access to

water and rat chow (Teklad Global, 2018 18% Protein Rodent Diet). Before surgery,

subjects were handled for one week (minimum), starting the second day after arrival from

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the breeding farm.

6.3.2 Procedure for surgery

Approximately 10 days after arrival in the vivarium, electrodes were implanted in

40 subjects in either the cortex (n=20) or the right amygdala (n=20). For implantation,

subjects were anesthetized with intraperitoneal injections of ketamine hydrochloride (90

mg/kg) and xylazine (10 mg/kg). They were then implanted with stainless steel bipolar

electrodes (MS303/1, Plastics One, Roanoke, VA, USA) aimed at the right forelimb

cortex (n=20) or right basolateral amygdala (n=20). The following coordinates were used

for the cortex (mm): anterior-posterior, +0.2 (bregma); medial-lateral, 3.3 (bregma); and

dorsal-ventral, -2.5. The amygdala coordinates were as follows (mm): anterior-posterior,

-2.8; medial-lateral, 4.6; and dorsal-ventral, -8.6. The incisor bar was set at -3.3, and

horizontal alignment of the skull was confirmed by aligning bregma and lambda to the

horizontal plane. Additional injections of pentobarbital were used to maintain the rats

under anesthesia, if needed (~10 mg/kg). The electrodes were fixed to the skull with 3 to

4 stainless steel anchor screws and acrylic, dental cement (Nuweld, LD, Caulk). All

subjects received subcutaneous injections of buprenorphine analgesic (0.05 mg/kg) and

physiological saline (1 ml/kg) for rehydration following surgery. Following surgery,

subjects were allowed at least one-month to recover before the start of experimental

procedures.

6.3.3 Afterdischarge threshold and seizure score measurements

One month following surgery, baseline afterdischarge thresholds (ADTs) in the

cortex and amygdala were measured using the ascending series method. Subjects

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received a one-second train of stimulation pulses at a frequency of 60 Hz. Stimulation

pulses were composed of a 1 ms positive and 1 ms negative phase separated by 0.5 ms.

The stimulation was generated by a Grass model S-88 stimulator (Grass Intruments,

Quincy, MA, USA). Electroencephalographic (EEG) activity at the stimulated focus was

recorded on a Grass model 6 electroencephalograph (Grass Intruments, Quincy, MA,

USA), using a device that switched the electrode circuit to the electroencephalograph

immediately following stimulation. Afterdischarges were digitally recorded using an IBM

compatible personal computer with a National Instruments digital acquisition board (AT-

MIO-16E) and the National Instruments Labview 7.0 graphical programming

environment.

Electrical stimulations started at 60 µA for amygdala-implanted subjects, and at

100 µA for the cortex-implanted subjects. In the amygdala, thresholds were determined

by increasing the current in steps of 20 µA up to 400 µA, and then in steps of 40 µA from

400 µA upwards, until an afterdischarge of at least five seconds was evoked. In the cortex,

thresholds were determined by increasing the current in steps of 100 µA up to 1000 µA,

and 200 µA from 1000 µA upwards, until an afterdischarge of five or more seconds was

evoked. The interval between stimulations was 5 minutes for both the cortex and

amygdala subjects.

Baseline ADT was measured twice. The interval between the first and second

threshold measurements was one week. Since thresholds tend to be very high when first

measured, and drop significantly after the first measurement, we used the second ADT

measurement as our reference baseline point.

Subjects that did not display an ADT, or had a baseline amygdala ADT of >500

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µA or a cortex ADT >1000 µA were excluded from the study. Also, subjects that

developed an infection or lost their implanted electrodes were excluded from the study.

Following the initial measurement, cortical and amygdala ADTs were measured

every 2 weeks for the first 12 weeks of the study, and then once every 4 weeks for the

remainder of the study.

Seizures were also scored at the time an afterdischarge was seen on the EEG, in

order to determine the severity of the seizures. Amygdala seizures were scored as follows

– stage 1, lip smacking; stage 2, head nodding; stage 3, forelimb clonus; stage 4,

forelimb clonus with rearing; stage 5, loss of postural control. Stages 1 and 2 are

considered focal seizures, whereas stages 3 to 5 are considered to be generalized and

more severe than stages 1 and 2 (Goddard et al., 1969; Racine, 1972b). Cortical seizures

were scored as follows - stage 1, contralateral forelimb clonus; stage 2, ipsilateral loss of

postural control; stage 3, bilateral folrelimb clonus. Stages 1 and 2 are focal seizures, and

stage 3 is generalized (Albright and Burnham, 1980).

6.3.4 Diets and diet administration

The experimental design is presented in Figure 1. Amygdaloid and cortical

subjects were started on the control and experimental diets on the day after the second

preliminary ADT measurement. Fresh diets were mixed every two to three days in our

laboratory, and stored at 4 ºC in order to minimize the oxidation of vitamins and fatty

acids. The control diet consisted of an AIN-93G diet that contained (g/kg): casein (200),

cornstarch (530), sucrose (100), soybean oil (70), cellulose (50), vitamin mix (10),

mineral mix (35), L-cysteine (3), choline bitartrate (2.5) and tertbutyl hydroquinone

(0.014) The cornstarch, cellulose, vitamin mix, mineral mix, L-cysteine, choline bitartrate

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and tertbutyl hydroquinone were obtained from Dyets Inc. (Bethlehem, PA, USA).

Cornstarch and sucrose were obtained from Disley Food Services (Toronto, ON, Canada),

and soybean oil was purchased from local stores (Loblaws, Toronto, ON, Canada). The

composition of the isocaloric experimental diets was similar to that of the control diet,

except that 20% or 40% of the soybean oil was replaced by Manhedan fish oil (Dyets Inc.,

Bethlehem, PA, USA), which is a source of n-3 PUFA. The fatty acid composition of the

diets was confirmed by gas-chromatography, and is presented in Table 1.

The amygdala experimental subjects were initially placed on the 20% fish oil diet.

After thresholds had failed to rise by week 19, the subjects were placed on the control

diet for 4 weeks (washout period) and then on a 40% fish oil diet in order to determine

whether a higher dose of n-3 PUFA would elevate seizure threshold in the amygdala.

The cortically-implanted experimental subjects were maintained on the 20% fish

oil diet for 12 weeks, and then switched to the control diet for another 8 weeks, after

thresholds had increased by the eighth week of supplementation. The rationale for

switching to the control diet was to determine whether discontinuation of the fish oil diet

would lower seizure threshold in the cortex.

6.3.5 Body weight and food intake measurements

Body weights and food intake were measured on a monthly basis. Since the food

was in powder form, most of the rats tended to spill the food. At least 4 to 5 subjects per

dietary treatment, however, did not spill their food. Food intake was therefore measured

in the respective cages by measuring the weight difference in the amount of food

provided and amount of food remaining over a 3-day period.

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6.3.6 Sacrifice and tissue fixation

Prior to sacrifice, the subjects were weighed, deeply anesthetized with sodium

pentobarbital (100 mg/kg), and subjected to a direct current of 200μA for 30 seconds in

order to lesion the site of the electrode implant for subsequent histological evaluation of

the position of the electrode tip. The subjects were then perfused intracardially, through

the left ventricle, with 200 ml of ice-cold phosphate buffered saline (0.9% NaCl in 0.1M

phosphate buffer, pH 7.2), followed by 200 ml of ice-cold, 4% phosphate buffered

paraformaldehyde as a fixative. The brains were excised and stored overnight in 4%

phosphate buffered paraformaldehyde at 4°C, to ensure complete fixation of the tissue.

The tissues were subsequently dehydrated by replacing the paraformaldehyde with 20%

phosphate buffered sucrose solution containing 0.1% sodium azide to prevent bacterial

degradation of the brain samples, and stored at 4°C.

6.3.7 Histological confirmation of electrode placement

The right hemisphere from the kindled animals was used to confirm the position

of the electrode. Each hemisphere was chilled in isopentane on dry ice and sectioned

using a cryostat (Leica Instruments, Willowdale, Ontario, Canada) maintained at − 25 °C.

The samples were allowed to equilibrate for 30 minutes. Coronal sections were obtained

at a thickness of 40μm and mounted onto gelatin coated glass slides. Since the electrode

tract was clearly visible, sections were collected close to where the tract ended. The

position at the end of the tract was confirmed under light microscopy (Research Analysis

System Model 421251; Amersham, MI). Only subjects with properly positioned

electrodes were included in the subsequent data analysis.

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6.3.8 Dietary fatty acid analysis

The fatty acid composition of the control and fish oil diets was determined by gas-

chromatography. Total lipids were first extracted from approximately 0.5 g of diet in

chloroform / methanol (2:1 v/v) after adding 2 mg of unesterified heptadecaenoic acid as

an internal standard (Sigma, St. Louis, Mo). Saline (0.9% w/v, 2 ml) was added to

separate the aqueous phase. The bottom layer containing total lipids was transferred to

test-tubes. A portion of the extract was dried under nitrogen, reconstituted in 2 ml of

hexane, and directly methylated with 2 ml of 14% boron triflouride in methanol at 100°C

for one hour. The reaction was terminated by adding 2 ml of deionized water. The

samples were then centrifuged at 1600 rpm for 4 minutes. The methylated fatty acids

were separated from the aqueous phase, reconstituted with hexane and analyzed on an

Agilent 6890 gas-chromatography system equipped with a 30m x 25mm capillary column

(J and W Scientific, DB-23, Folsom, CA) and a flame ionization detector. Fatty acids

were injected into the column (1 μl) in splitless mode. The gas carrier was helium, which

was set at a constant flow rate of 0.7 ml per minute. The fatty acid methyl ester profile

was acquired by setting the temperature at 50ºC for 2 minutes, followed by a ramp up at

20ºC per minute to 170ºC and a 1 minute hold at 170ºC, and a final 3ºC per minute ramp

up to 212ºC followed by a 10 minute hold. The fatty acid peaks were identified using

fatty acid standards of known composition (GLC463, NuCheck Prep., ON, Can).


The data are presented as means ± SEM. Data analysis was performed using

Sigma Stat v.3.2 (Jandel Corporation). A one-way analysis of variance was used to

compare the fatty acid composition of the control, 20% fish oil and 40% fish oil diets. A

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two-way repeated measures analysis of variance was used to determine the effects of diet

and time on body weight, ADT and seizure score during the 20% fish oil supplementation

period for both the cortex and amaygdala implanted subjects. A separate two-way

analysis of variance was used to assess the effects diet and time on body weight, ADT

and seizure score during the fish oil deprivation period in the cortically-implanted rats,

and the 40% fish oil supplementation period of the amygdala subjects (i.e. after the 20%

fish oil supplementation period). Statistical significance was accepted at P<0.05.

6.4 Results

6.4.1 Fatty acid composition of the diets

The fatty acid percent composition of the diets is presented in Table 1. As

expected, the composition of eicoosapentaenoic acid (EPA, 20:5 n-3) and

docosahexaenoic acid (DHA, 22:6n-3) was significantly higher in the fish-oil

supplemented diets relative to the control diet, as determined by one-way analysis of

variance (P<0.05). Tukey’s post-hoc test revealed that the differences between the 40%

fish oil diet and the control and 20% fish oil diets to be significant (P<0.05). The

differences between the 20% and 40% fish oil diets were also statistically significant

(P<0.05).

Linoleic (18:2n-6) and α-linolenic (18:3n-3) acids, which are the major

constituents of soybean oil, were significantly lower in the 20% fish oil diet, followed by

the 40% fish oil diet, relative to the control diet (P<0.05).

The percent composition of total saturated and monounsaturated fatty acids was

significantly higher in the 20% and 40% fish oil diets as compared to the control diet

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(P<0.05).

6.4.2 Body weight gain

Figures 2-A and 2-B show the body weight data over time in cortex and amygdala

implanted subjects, respectively. The effect of diet over time for each of the cortex and

amygdala implanted subjects was determined by a repeated measures two-way analysis

of variance. As shown in Figures 2-A and 2-B, all rats gained weight over time, during

and after the 20% fish oil supplementation period (P<0.05). There was no significant

difference in body weights between control and fish oil treated subjects at any time point

during and after the 20% fish oil supplementation period (P>0.05).

6.4.3 Food intake

Food intake between the control and fish oil treated subjects did not differ

significantly at any time-point, as determined by two-way repeated measures analysis of

variance (P>0.05; data not shown). The average food intake was 34 g per day.

Accordingly, the amount of EPA and DHA consumed throughout the study period, based

on the composition data, ranged between 102-184 mg/day and 58-119 mg per day for

EPA and DHA respectively. The amount of DHA ingested is therefore equivalent to an

approximate daily dose of at least 82 mg/kg per day (for a 700 g rat).

6.4.4 Dietary fish oil supplementation raises seizure threshold in the cortex and

amygdala

The data for cortical ADT are presented in Figure 3-A. As indicated, cortical

thresholds increased by the seventh week, and continued to rise until week 11 at which

time the fish oil was discontinued from the experimental group. After the discontinuation

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of the fish oil, thresholds in the experimental group dropped back toward baseline.

Repeated measures two-way analysis of variance during the 20% fish oil supplementation

period showed a significant main effect of the 20% fish oil diet (P<0.01), but no effect of

time (P>0.05). Subsequent analysis by two-way repeated measures ANOVA during the

20% fish oil deprivation period showed a significant effect of diet removal on seizure

threshold (P<0.01), but no effect of time (P>0.05).

Figure 3-B presents the mean changes from baseline in amygdala ADT in rats fed

a diet enriched with fish oil or rats fed a normal control diet. After amygdaloid thresholds

had failed to rise after 10 weeks (P>0.05 for main effect of diet and time by two-way

repeated measures ANOVA), the percentage of fish oil in the experimental group was

increased from 20% to 40% of total dietary fat content. Subsequently, a rise in

amygdaloid thresholds was seen, which was maintained for at least 4 weeks. Repeated

measures two-way analysis of variance during the 40% fish oil supplementation period

revealed no significant main effect of time nor treatment (P>0.05), probably because

seizure thresholds rose transiently. However, post-hoc statistical comparison by unpaired

t-test, on the change in thresholds between weeks 25 (when threshold was highest in the

fish oil group) and 17 (when the 40% fish oil diet was started), revealed a “trend”

towards statistical significance (P=0.06).

6.4.5 Dietary fish oil supplementation does not alter seizure score

Rats began to show behavioral seizures in response to the ADT stimulation over

time. The behavioral seizure score is a measure of seizure severity. Seizures were scored

when the ADT was reached, in order to determine the effects of the fish oil treatment on

seizure severity. The data for seizure score of cortex and amygdala subjects are shown on

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Figures 4-A and 4-B, respectively. Two-way repeated measures analysis of variance

revealed that seizure score in the cortex-stimulated subjects did not increase significantly

over time, nor differ between the control and fish oil treated subjects during or after the

20% fish oil supplementation period (P>0.05; Figure 4-A). Two-way repeated measures

analysis of variance indicated that seizure score increased over time in the amygdala-

stimulated subjects during the 20% fish oil enrichment period (P<0.05). There was no

significant difference in seizure score, however, between control and fish oil treated

subjects at any time point (P>0.05; Figure 4-B). No significant effects of diet or time

were detected on amygdaloid seizure scores during the 40% fish oil treatment period, by

two-way analysis of variance (P>0.05).

6.5 Discussion

The results of the present study demonstrate that chronic consumption of n-3

PUFA derived from fish oil elevates seizure threshold in the cortex and amygdala of rats,

without altering measures of seizure severity.

Our findings related to the increase in threshold are consistent with previous

reports which had indicated that n-3 PUFA raise seizure thresholds in animal seizure

models (Yehuda et al., 1994; Voskuyl et al., 1998; Rabinovitz et al., 2004; Porta et al.,

2008; Taha et al., 2008a; Taha et al., 2008b).

The elevation in the amygdala seizure threshold required a higher dose of fish oil

than elevation of the cortical threshold – a finding consistent with our classic finding that

higher doses of anticonvulsants are required to elevate amygdala focal seizures (Albright,

1983; Albright and Burnham, 1983).

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Albright and Burnham (1980) proposed the amygdala focal as an animal model of

complex partial seizures (Albright and Burnham, 1980). If this hypothesis is valid, the

fact that dietary n-3 PUFA elevated thresholds in the amygdala suggests that they might

also help to control complex partial seizures in humans. Complex partial seizures are the

most common type of seizure in the adult population, and they are often resistant to

anticonvulsant drug therapy(Oles et al., 1989; Brodie, 2001; Stephen et al., 2001).

Our findings do not appear to be consistent with some clinical studies that have

reported a lack of beneficial effect of dietary fish oil supplementation (1 to 3g per day for

12 weeks) on drug-resistant seizures (Yuen et al., 2005; Bromfield et al., 2008;

DeGiorgio et al., 2008a). Our finding in rats that is takes a long time and a high dose of

dietary fish oil to achieve an elevation in threshold in the amygdala suggests that the

doses of fish oil and the time of exposure may have been too low in past clinical studies.

Higher doses and a longer duration of treatment may be necessary to achieve seizure

control in the clinical setting.

The effect of the fish oil diet on amygdaloid thresholds, however, appeared to be

transient. This could be related to the modulatory effects of repeated electrical

stimulations on amygdaloid afterdischarge seizure thresholds in the fish oil group.

Previous studies have reported a significant drop in seizure thresholds following repeated

stimulations, particularly in the amygdala (Racine, 1972a; Ng et al., 2006). The

interaction between repeated electrical stimulations and dietary fish oil in limbic

structures requires further examination in future studies.

The seizure control achieved with anticonvulsant drugs may be associated with

significant side effects (Burnham, 2007). We did not formally measure side effects in the

191

present study, but - based on our food intake, weight gain, and general observation –

subjects appeared to thrive on the high PUFA diet. It should be noted that even the

highest fish oil dose (40% of total dietary fat) used in the present study was equivalent to

approximately 82 mg/kg of DHA per day per rat. This is still below the 200-400 mg/kg

dose of DHA that we have recently shown to be safe and non-toxic in rat subjects (Taha

et al, under review).

We were able to detect an increase in cortical and amygdaloid thresholds

following a minimum of 8 weeks of fish oil supplementation. Previous studies have

reported an acute anticonvulsant effect of injected or intravenously administered EPA or

DHA (Voskuyl et al., 1998). It has also been shown that it is possible to achieve an

increase in the amount of DHA incorporated into brain phospholipids within one hour

following acute intravenous administration (Polozova and Salem, 2007). This rapid

anticonvulsant effect cannot be achieved through dietary administration, however,

because dietary fatty acids become incorporated into plasma lipoprotein molecules

following oral administration (Polozova et al., 2006). DHA incorporated into plasma

lipoproteins is not readily available to the brain (Chen et al., 2008b).

In contrast to most tissues, the brain relies on albumin-bound n-3 fatty acids

instead of lipoproteins for maintaining DHA concentrations (Chen et al., 2008b; Ouellet

et al., 2009). Chronic intake of DHA may cause the DHA to be released from the

lipoproteins into the plasma, where they non-covalently bind to albumin, and become

available to the brain. Thus, the processes involved in elevating brain n-3 PUFA

concentrations may take some weeks. For instance, a recent study showed no effect of

dietary EPA or DHA on brain DHA levels and seizure threshold, following four weeks of

192

supplementation (Willis et al., 2008).

It seems probable that the increase in seizure threshold in the cortex and amygdala

is mediated by DHA - although EPA has been suggested to also play a role (Voskuyl et

al., 1998). Both fatty acids have been reported to reduce neuronal excitability in vitro, by

acting on voltage-dependent sodium and calcium channels (Vreugdenhil et al., 1996;

Xiao and Li, 1999; Young et al., 2000). EPA, however, is not present in the brain, mainly

because it is extensively β-oxidized by the brain (Chen et al., 2009).

Although fish oil raised seizure threshold, it did not alter seizure severity in either

the cortex or amygdala. The lack of effect of the fish oil on seizure severity is consistent

with our previous findings related to the failure of n-3 PUFA to decrease seizure severity

in PTZ-induced seizures (Taha et al., 2008b; Taha et al., 2009c).

In conclusion, we have demonstrated for the first time that dietary

supplementation with fish oil elevates seizure threshold in the cortex and amygdala of

rats. The increase in seizure threshold in the amygdala suggests that the n-3 PUFA might

be effective against complex-partial seizures.

Acknowledgement

Funding for this study was provided by the Bahen Chair in epilepsy grant to W.M.B., and

the Canadian Institutes of Health Research doctoral award to A.Y.T.

.

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Figure 1: Study design

Rats 60d old

Electrode implantation in

cortex or amygdala

Baseline ADT measured twice

2 weeks handling

1 month recovery

cortex and amygdala subjects were randomized to control or 20% fish oil diet

Randomization

Experimental subjects

switched to control diet (“fish

oil deprivation” period)

11 wks


switched to control diet

17 wks

Cortex subjects Amygdala subjects

4 wks

Sacrifice Sacrifice


switched to 40% fish oil diet

194

Male rats were implanted with electrodes in either the cortex or amygdala, allowed to

recover, and then randomized to a control or 20% fish oil diet. The experimental cortex

subjects were maintained on the 20% fish oil diet for 11 weeks, and then the control diet

for 8 weeks. The experimental amygdala subjects were maintained on the 20% fish oil

diet for 17 weeks, washout phase for 4 weeks and then the 40% fish oil diet for the

remainder of the study.

195

Figure 2-A: Body weight gain over time in cortex-implanted subjects

0

100

200

300

400

500

600

700

800

0 3 9 13 17Time (weeks)

(wei

ght (

g)

ControlFish oil

196

Figure 2-B: Body weight gain over time in amygdala-implanted subjects

0

100

200

300

400

500

600

700

800

900

0 2 4 8 13 17 22 25 29 32Time (weeks)

Wei

ght (

g)

ControlFish oil

Body weight gain in cortex and amygdala subjects on a control or fish oil diet. Data are

mean ± SEM of n=7-11 for cortex rats and n=6-10 for amygdala rats.

Figure 2-A: Body weight gain in the cortex implanted subjects during and post 20% fish

oil supplementation. Two-way analysis of variance revealed a significant effect of time,

but no effect of treatment during the 20% fish oil supplementation period and during the

fish oil deprivation period.

Figure 2-B: Body weight gain in the amygdala implanted subjects before and during

kindling. Two-way analysis of variance revealed a significant effect of time, but no effect

of treatment during the 20% and 40% fish oil supplementation.

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Figure 3-A: Change in ADT over time in cortex-implanted subjects

-200

-100

0

100

200

300

400

0 3 7 9 11 13 17

Time (weeks)

Cha

nge

in A

DT

from

bas

elin

e (µ

A)

CtrFish oil

20% fish oil diet

20% fish oil deprivation

198

Figure 3-B: Change in ADT over time in amygdala-implanted subjects

-200

-150

-100

-50

0

50

100

150

200

0 2 4 6 8 13 17 22 25 29 32

Time (weeks)

Cha

nge

in A

DT

from

bas

elin

e (µ

A)

CtrFish oil

20% fish oil diet

40% fish oil dietwashout

Change in afterdischarge threshold (ADT) from baseline in cortex and amygdala subjects

on a control or fish oil diet. Data are mean ± SEM of n=5-11 for cortex rats and n=5-10

for amygdala rats.

Figure 3-A: Change in ADT in cortex-stimulated subjects. Two-way analyses of

variance revealed no significant effect of time, but a significant effect of dietary treatment

during the 20% fish oil supplementation period (P<0.01) and during the fish oil

deprivation period (P<0.01) , but no significant effect of time for either periods (P>0.05).

Figure 3-B: Change in ADT in amygdala-stimulated subjects. Two-way analyses of

variance revealed no significant effect of treatment nor time during the 20% and 40% fish

oil supplementation periods (P>0.05). Post-hoc statistical comparison by unpaired t-test

on the change in threshold between weeks 25 (when threshold was highest in the fish oil

199

group) and 17 (when the 40% fish oil diet was started), revealed a “trend” towards

statistical significance (P=0.06).

200

Figure 4-A: Seizure score over time in cortex-implanted subjects

0

1

2

3

0 3 7 9 11 13 17

Time (weeks)

Seiz

ure

scor

e (o

ut o

f 3)

Ctr fish oil

20% fish oil diet

20% fish oil deprivation

201

Figure 4-B: Seizure score over time in amygdala-implanted subjects

0

1

2

3

4

5

0 2 6 8 13 17 22 25 29 32Time (weeks)

Seiz

ure

scor

e (o

ut o

f 5)

Ctr fish oil

20% fish oil diet

40% fish oil dietWashout

Seizure score in cortex and amygdala subjects on a control or fish oil diet. Data are mean

± SEM of n=5-11 for cortex rats and n=5-10 for amygdala rats.

Figure 4-A: Seizure score (out of 3) in cortex-stimulated subjects. Two-way analysis of

variance revealed no significant effect of time or treatment on seizure score during the

20% fish oil supplementation period and the fish oil deprivation period.

Figure 4-B: Seizure score (out of 5) in amygdala-stimulated subjects. Two-way analysis

of variance revealed a significant effect of time during the 20% fish oil supplementation

period (P<0.05), but no significant effect of dietary treatment (P>0.05). Two-way

analysis of variance revealed no significant effects of diet or time on seizure scores

during the the 40% fish oil treatment period (P>0.05).

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Table 1: Fatty acid composition of the AIN-93G control and fish oil experimental diets (% of total fatty acids)

Control (AIN-93G) 20% fish oil 40% fish oil 14:0 0.2 ± 0.01a 2.2 ± 0.04b 4.1 ± 0.2c 16:0 12.3 ± 0.1a 15.6 ± 0.4b 16.9 ± 0.7b 18:0 3.7 ± 0.1a 5.2 ± 0.04b 4.8 ± 0.4b Total* saturates 18.8 ± 0.8a 25.0 ± 0.5 b 28.5 ± 1.2b 16:1 n-9 0.04 ± 0.04a 2.0 ± 0.04b 4.2 ± 0.2c 18:1 n-9 16.3 ± 0.1 17.1 ± 0.4 15.4 ± 0.5 18:1 n-7 1.2 ± 0.01a 1.6 ± 0.02b 2.0 ± 0.1c Total monounsaturates 17.8 ± 0.1a 20.7 ± 0.4b 21.8 ± 0.4b 18:2 n6 53.6 ± 0.5a 39.5 ± 1.1b 31.0 ± 0.7c Total n-6 polyunsaturates 53.9 ± 0.6a 43.1 ± 1.7b 33.9 ± 1.0c 18:3 n3 9.5 ± 0.1a 5.7 ± 0.2b 4.6 ± 0.1c 20:5 n3 0 ± 0a 3.0 ± 0.1b 5.4 ± 0.2c 22:6 n3 0 ± 0a 1.7 ± 0.1b 3.5 ± 0.1c Total n-3 polyunsaturates 9.5 ± 0.1a 11.3 ± 0.7a 15.8 ± 0.6b

Data are mean ± SEM of n=2-3 samples per diet. *Indicates that totals include other minor fatty acids that are not included in the Table. Values with different superscipts (eg:a versus b) are significantly different at P<0.05 by 1-way ANOVA and Tukey’s post-hoc test.

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6.6 References

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a high-fat ketogenic diet on plasma levels of lipids, lipoproteins, and apolipoproteins in children. Jama 2003;290: 912-20. [20] Cunnane SC, Musa K, Ryan MA, Whiting S, Fraser DD. Potential role of polyunsaturates in seizure protection achieved with the ketogenic diet. Prostaglandins Leukot Essent Fatty Acids 2002;67: 131-5. [21] Yuen AW, Sander JW. Is omega-3 fatty acid deficiency a factor contributing to refractory seizures and SUDEP? A hypothesis. Seizure 2004;13: 104-7. [22] Taha AY, Filo E, Ma DW, McIntyre Burnham W. Dose-dependent anticonvulsant effects of linoleic and alpha-linolenic polyunsaturated fatty acids on pentylenetetrazol induced seizures in rats. Epilepsia 2009;50: 72-82. [23] Taha AY, Ciobanu FA, Saxena A, McIntyre Burnham W. Assessing the link between omega-3 fatty acids, cardiac arrest, and sudden unexpected death in epilepsy. Epilepsy Behav 2009;14: 27-31. [24] Taha AY, Filo E, Ma DW, McIntyre Burnham W. Dose-dependent anticonvulsant effects of linoleic and alpha-linolenic polyunsaturated fatty acids on pentylenetetrazol induced seizures in rats. Epilepsia 2008. [25] Taha AY, Huot PS, Reza-Lopez S, Prayitno NR, Kang JX, Burnham WM, Ma DW. Seizure resistance in fat-1 transgenic mice endogenously synthesizing high levels of omega-3 polyunsaturated fatty acids. J Neurochem 2008;105: 380-8. [26] Taha AY, Alizadeh S, Zeng QH, Filo E, McPherson JP, Burnham WM. Assessing the metabolic and toxic effects of anticonvulsant doses of polyunsaturated fatty acids on the liver in rats. J Toxicol Environ Health A. 2009;In Press. [27] Fisher RS. Animal models of the epilepsies. Brain Res Brain Res Rev 1989;14: 245-78. [28] Krall RL, Penry JK, White BG, Kupferberg HJ, Swinyard EA. Antiepileptic drug development: II. Anticonvulsant drug screening. Epilepsia 1978;19: 409-28. [29] Edwards HE, Burnham WM, Mendonca A, Bowlby DA, MacLusky NJ. Steroid hormones affect limbic afterdischarge thresholds and kindling rates in adult female rats. Brain Res 1999;838: 136-50. [30] Racine RJ, Burnham WM, Gartner JG, Levitan D. Rates of motor seizure development in rats subjected to electrical brain stimulation: strain and inter-stimulation interval effects. Electroencephalogr Clin Neurophysiol 1973;35: 553-6. [31] Goddard GV. Development of epileptic seizures through brain stimulation at low intensity. Nature 1967;214: 1020-1. [32] Porta N, Bourgois B, Galabert C, Lecointe C, Cappy P, Bordet R, Vallee L, Auvin S. Anticonvulsant effects of linolenic acid are unrelated to brain phospholipid cell membrane compositions. Epilepsia 2008. [33] Rabinovitz S, Mostofsky DI, Yehuda S. Anticonvulsant efficiency, behavioral performance and cortisol levels: a comparison of carbamazepine (CBZ) and a fatty acid compound (SR-3). Psychoneuroendocrinology 2004;29: 113-24. [34] Voskuyl RA, Vreugdenhil M, Kang JX, Leaf A. Anticonvulsant effect of polyunsaturated fatty acids in rats, using the cortical stimulation model. Eur J Pharmacol 1998;341: 145-52. [35] Yehuda S, Carasso RL, Mostofsky DI. Essential fatty acid preparation (SR-3) raises the seizure threshold in rats. Eur J Pharmacol 1994;254: 193-8. [36] Albright PS, Burnham WM. Effects of phenytoin, carbamazepine, and

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clonazepam on cortex- and amygdala-evoked potentials. Exp Neurol 1983;81: 308-19. [37] Bromfield E, Dworetzky B, Hurwitz S, Eluri Z, Lane L, Replansky S, Mostofsky D. A randomized trial of polyunsaturated fatty acids for refractory epilepsy. Epilepsy Behav 2008;12: 187-90. [38] DeGiorgio CM, Miller P, Meymandi S, Gornbein JA. n-3 fatty acids (fish oil) for epilepsy, cardiac risk factors, and risk of SUDEP: clues from a pilot, double-blind, exploratory study. Epilepsy Behav 2008;13: 681-4. [39] Yuen AW, Sander JW, Fluegel D, Patsalos PN, Bell GS, Johnson T, Koepp MJ. Omega-3 fatty acid supplementation in patients with chronic epilepsy: a randomized trial. Epilepsy Behav 2005;7: 253-8. [40] Racine RJ. Modification of seizure activity by electrical stimulation. I. After-discharge threshold. Electroencephalogr Clin Neurophysiol 1972;32: 269-79. [41] Ng MS, Hwang P, Burnham WM. Afterdischarge threshold reduction in the kindling model of epilepsy. Epilepsy Res 2006;72: 97-101. [42] Abdelmalik PA, Burnham WM, Carlen PL. Increased seizure susceptibility of the hippocampus compared with the neocortex of the immature mouse brain in vitro. Epilepsia 2005;46: 356-66. [43] Krupp E, Heynen T, Li XL, Post RM, Weiss SR. Tolerance to the anticonvulsant effects of lamotrigine on amygdala kindled seizures: cross-tolerance to carbamazepine but not valproate or diazepam. Exp Neurol 2000;162: 278-89. [44] Polozova A, Salem N, Jr. Role of liver and plasma lipoproteins in selective transport of n-3 fatty acids to tissues: a comparative study of 14C-DHA and 3H-oleic acid tracers. J Mol Neurosci 2007;33: 56-66. [45] Polozova A, Gionfriddo E, Salem N, Jr. Effect of docosahexaenoic acid on tissue targeting and metabolism of plasma lipoproteins. Prostaglandins Leukot Essent Fatty Acids 2006;75: 183-90. [46] Chen CT, Ma DW, Kim JH, Mount HT, Bazinet RP. The low density lipoprotein receptor is not necessary for maintaining mouse brain polyunsaturated fatty acid concentrations. J Lipid Res 2008;49: 147-52. [47] Ouellet M, Emond V, Chen CT, Julien C, Bourasset F, Oddo S, Laferla F, Bazinet RP, Calon F. Diffusion of docosahexaenoic and eicosapentaenoic acids through the blood-brain barrier: An in situ cerebral perfusion study. Neurochem Int 2009. [48] Willis S, Samala R, Rosenberger TA, Borges K. Eicosapentaenoic and docosahexaenoic acids are not anticonvulsant or neuroprotective in acute mouse seizure models. Epilepsia l2008. [49] Vreugdenhil M, Bruehl C, Voskuyl RA, Kang JX, Leaf A, Wadman WJ. Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons. Proc Natl Acad Sci U S A l1996;93: 2559-63. [50] Xiao Y, Li X. Polyunsaturated fatty acids modify mouse hippocampal neuronal excitability during excitotoxic or convulsant stimulation. Brain Res 1999;846: 112-21. [51] Young C, Gean PW, Chiou LC, Shen YZ. Docosahexaenoic acid inhibits synaptic transmission and epileptiform activity in the rat hippocampus. Synapse 2000;37: 90-4. [52] Poling JS, Vicini S, Rogawski MA, Salem N, Jr. Docosahexaenoic acid block of neuronal voltage-gated K+ channels: subunit selective antagonism by zinc. Neuropharmacology 1996;35: 969-82. [53] Chen CT, Liu Z, Ouellet M, Calon F, Bazinet RP. Rapid beta-oxidation of

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eicosapentaenoic acid in mouse brain: an in situ study. Prostaglandins Leukot Essent Fatty Acids 2009;80: 157-63.

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CHAPTER 7

SEIZURES INCREASE UNESTERIFIED ARACHIDONIC BUT NOT ESTERIFIED DOCOSAHEXAENOIC ACID CONCENTRATIONS IN

MICROWAVE-FIXATED BRAINS OF RATS

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7 Experiment 6: Seizures increase unesterified arachidonic acid but not unesterified docosahexaenoic acid concentrations in the micro-wave fixated brain of rats.

Forward

Expeirments 4 and 5 provided evidence that DHA raises seizure threshold in the

PTZ and electrical stimulation seizure models. The mechanism of action of DHA,

however, is not clear.

Upon entering the brain, DHA is incorporated into membrane phospholipids, from

which it can be released in free form (de-esterified). Once de-esterified, the majority

(>90%) of DHA is quickly re-incorporated into the membrane, but the remaining

‘unesterified’ DHA may act as a signaling molecule, interact with receptor proteins,

and/or become converted into bioactive metabolites that play a role in suppressing

neuroinflammation (Marcheselli et al., 2003; DeMar et al., 2004).

Previous studies have reported that DHA is released in free form from the

phospholipid membrane during periods of seizure-induced hyperexcitatability (Rodriguez

de Turco and Bazan, 1983; Birkle and Bazan, 1987; Visioli et al., 1993). It is perhaps

possible that a pre-seizure increase in brain unesterified DHA, may represent a built-in

anticonvulsant mechanism, designed to raise seizure threshold.

Experiment 6 tested the hypothesis that de-esterified DHA levels increase in the

hyperexcitable state preceding seizures, and that this increase is associated with a rise in

seizure threshold. The n-6 PUFA arachidonic acid (AA) was also tested in Experiment 6

because, like DHA, it reduces neuronal excitability in vitro (Fraser et al., 1993), and is

released from the phospholipid membrane during seizure-induced hyperexcitability

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(Bazan et al., 1982; Rodriguez de Turco and Bazan, 1983; Birkle and Bazan, 1987;

Visioli et al., 1993). It is possible, therefore, that the anti-seizure effects of DHA could

also be mediated by AA released from the membrane.

Male Wistar rats received subcutaneous injections of oleic acid control, AA or

DHA, and were sacrificed one-hour later, following intraperitoneal administration of

saline or PTZ. The PTZ-injected subjects were sacrificed during seizures, or ten seconds

prior to seizure induction. Head-focused microwave fixation was used to euthanize the

subjects before decapitation in order to the inactivate phospholipase enzymes that would

de-esterify membrane-bound AA and DHA following decapitation (Farias et al., 2008).

Microwave fixation denatures these enzymes without altering basal levels of de-esterified

AA and DHA in the brain (Farias et al., 2008)

The findings of Experiment 6 do not support the hypothesis that unesterified DHA

levels increase pre-seizure. The results indicate that DHA treatment increased

unesterified AA but not unesterified DHA concentrations during seizures, in the brains of

microwave-fixated rats. It appears, therefore, that the rise in seizure threshold in the

DHA-treated rats may be related to an increase in unesterified AA levels.

The manuscript for this experiment begins on the next page. It has not been

submitted for publication. Marc-Olivier Trepanier, Flaviu Coibanu and Chuck Chen

assisted with the lipid analysis. Drs W. McIntyre Burnham and Richard Bazinet were the

principal investigators of the study.

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Seizures increase unesterified arachidonic acid but not unesterified docosohexanoic

acid concentrations in the microwave-fixed brains of rats

Ameer Y. Taha1,3, Marc-Olivier Trepanier1,3, Flaviu A. Coibanu1,3,

Chuck T. Chen2, W.M. Burnham1,3 and Richard P. Bazinet2,3

Departments of 1Pharmacology and Toxicology, and 2Nutritional Sciences, Faculty of

Medicine, University of Toronto, Toronto, ON, Canada, M5S 1A8


Toronto, Toronto, ON, Canada, M5S 1A8


Dr. Richard P. Bazinet

Department of Nutritional Sciences


FitzGerlad Bldg.

150 College St.


Canada


Running title: Effect of pentylenetetrazol-induced seizures on the release of free fatty

acids following head-focused microwave fixation

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7.1 Abstract

Background: Docosahexaenoic acid (DHA) has been reported to raise seizure threshold

in animal seizure models and in vitro, an effect which might be related to the release of

DHA and possibly arachidonic acid (AA) from the phospholipid membrane during

periods of seizure-induced hyperexcitatability.

Purpose: To test the hypothesis that AA and DHA are de-esterified from the

phospholipid membrane both by the hyperactivity that precedes pentylenetetrazol (PTZ)-

induced seizures and also by the hyperactivity that occurs during seizures, following oleic

acid, AA or DHA treatment.

Methods: De-esterified AA and DHA levels were measured in brains of rats that were

injected with oleic acid, AA or DHA, and then euthanized one-hour later by head-focused

microwave fixation after saline or PTZ injections. The PTZ-injected subjects were

euthanized shortly before (i.e. pre-seizure) and after seizure induction (i.e. during

seizures).

Results: AA and DHA unesterified levels decreased pre-seizure, regardless of fatty acid

treatment (P<0.05). AA levels increased during seizures, but the increase was greatest in

the subjects that were pre-treated with DHA (P<0.05).

Conclusion: DHA treatment increased unesterified AA but not unesterified DHA

concentrations during seizures, in the brains of microwave-fixated rats.

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7.2 Introduction

Arachidonic acid (AA) and decosahexanoic acid (DHA) are long-chain

polyunsaturated fatty acids (PUFA) that are important components of neuronal

phospholipid membranes (Kitajka et al., 2002). In the brain, both AA and DHA play a

role in regulating membrane fluidity, gene expression and signal transduction (Kitajka et

al., 2002; Rapoport, 2003; Innis, 2007). AA is also a substrate for the production of pro-

inflammatory or anti-inflammatory prostaglandins, whereas DHA is a substrate for the

production of anti-inflammatory compounds called “docosanoids” (Marcheselli et al.,

2003; Farias et al., 2008).

Upon entering the brain, DHA and AA are immediately incorporated into

membrane phospholipids, where they may interact with membrane receptor proteins

(Contreras and Rapoport, 2002; Ma, 2007). These fatty acids are also continuously

released in free form (de-esterified) from membrane phospholipids. Once de-esterified,

the majority (>90%) of AA and DHA are quickly re-incorporated into the membrane, but

the remaining ‘unesterified’ DHA and AA may act as signaling molecules and/or become

converted into bioactive metabolites that play a role in promoting or suppressing

neuroinflammation (Rapoport, 2003). AA and DHA are released from the phospholipid

membrane by the enzymes calcium-dependent phospholipase A2 and calcium-

independent phospholipase A2, respectively (Green et al., 2008).

AA and DHA have been reported to reduce neuronal excitability in cell-culture

studies (Fraser et al., 2003; Xiao and Li, 1999), and DHA has been reported to raise

seizure threshold in studies involving whole animals (Voskuyl et al., 1998; Taha et al.,

2008b). DHA in particular has been reported to reduce neuronal excitability and raise

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seizure thresholds by raising the depolarization threshold for action potentials

(Vreugdenhil et al., 1996). AA’s effects have not been investigated in whole animals,

although in vitro studies have shown that it reduces neuronal excitability in hippocampal

slices (Fraser et al., 1993).

Previous studies have reported that AA and DHA are released in free form from

the phospholipid membrane during periods of seizure-induced hyperexcitatability

(Rodriguez de Turco and Bazan, 1983; Birkle and Bazan, 1987; Visioli et al., 1993).

There is also evidence suggesting that these fatty acids may be released by the increased

neural excitation preceding seizures. Bazan et al. for instance, have reported that

unesterified AA concentrations increase progressively prior to the tonic-clonic phase of

bicuculline-induced seizures (Bazan et al., 1982).

It is perhaps possible that a pre-seizure increase in brain unesterified AA, and

possibly DHA, may represent some sort of built-in anticonvulsant mechanism, designed

to raise seizure threshold and prevent seizures. AA or DHA might raise seizure threshold

either by inhibiting voltage-dependent ion channels (Vreugdenhil et al., 1996; Xiao and

Li, 1999; Lauritzen et al., 2000; Young et al., 2000; Danthi et al., 2005; Borjesson et al.,

2008) or through their anti-inflammatory metabolites (Chen et al., 2008c; Fabene et al.,

2008). Brain inflammation is thought to play a role in lowering seizure thresholds (Tu

and Bazan, 2003; Akarsu et al., 2006; Oliveira et al., 2008).

The purpose of the present study was to test the hypothesis that AA and DHA are

freed from the phospholipid membrane both by the hyperactivity that precedes

pentylenetetrazol (PTZ)-induced seizures (Depaulis et al., 1989; Visioli et al., 1993) and

also by the hyperactivity that occurs during seizures (Depaulis et al., 1989; Visioli et al.,

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1993). We tested this hypothesis in rats that had been pre-treated with 300 mg/kg of oleic

acid (control), DHA or AA. It was hoped that pre-treatment with DHA or AA might

increase the release of unesterified AA or DHA. Head-focused microwave fixation was

used to euthanize the subjects before decapitation in order to the inactivate phospholipase

enzymes that are normally induced by decapitation (Farias et al., 2008). (These data are

presented as Experiment 2 below.)

A dose of 300 mg/kg DHA was chosen based a preliminary experiment

(Experiment 1), which showed that this dose increases latency to seizure onset in PTZ-

treated rats. Doses of 300 mg/kg OA and AA were used in order to match the DHA dose.


7.3.1 Drug preparation

Stock solutions were prepared by dissolving each of 140 μl of OA, AA and DHA

(Nu-Check Prep, Elysian, MN, USA) in 0.9% saline containing 90 mg per ml of bovine

serum albumin (Sigma-Aldrich, St. Louis, Missouri, USA). All fatty acid stock solutions

were sonicated for 5 minutes. PTZ (Sigma-Aldrich, St. Louis, Missouri, USA) was

prepared by dissolving 50 mg of PTZ per ml of 0.9% saline. All solutions were kept on

ice throughout the experiment.

7.3.2 Subjects

The present experiments were conducted in accordance with the standards of the

Canadian Council on Animal Care and were approved by the Animal Care Committee of

the Faculty of Medicine of the University of Toronto. Male Wistar rats (Charles River,

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La Prairie, QC, Canada), aged 53 days, served as subjects for all experiments. All

subjects were individually housed in a vivarium maintained on a 12 h light-dark cycle

(lights on at 7am) and at a temperature of 21ºC. Rat chow (Teklad Global, 2018 18%

Protein Rodent Diet) and water were available ad libitum. All subjects were handled for 6

consecutive days, starting on the second day after arrival and continuing until the day

prior to the experiments.

Experiment 1:

A preliminary dose-response study was performed in order to establish the dose of

DHA that would maximally increase latency to seizure onset in the PTZ seizure model.

Subjects were randomly sorted into the following treatment groups (n=7-8 per treatment)-

300 mg/kg OA (control), 100 mg/kg DHA, 200 mg/kg DHA, 300 mg/kg DHA and 400

mg/kg DHA. The fatty acid treatments were injected subcutaneously one hour before

PTZ administration. One hour after the injections of OA or DHA, the subjects received

an intraperitoneal injection of 105 mg/kg of PTZ. In a pilot study, this dose of PTZ had

been shown to reliably induce tonic-clonic convulsions (n=7). Following the PTZ

injection, the subjects were placed in the open field and observed for five minutes. The

latencies to the first myoclonic jerk and and the first tonic-clonic seizure were scored by

two independent observers. Following seizure testing, all subjects were euthanized with a

lethal i.p. injection of sodium pentobarbital (100 mg/kg).

This preliminary dose-response study revealed that a DHA dose of 300 mg/kg

maximally increased latency to onset of both myoclonic jerks and tonic-clonic seizures.

This dose was therefore used in Experiment 2, the microwave study.

Experiment 2:

216

The purpose of Experiment 2 was to test the effect of OA, AA and DHA, injected

subcutaneously, on brain unesterified AA and DHA concentrations in rats sacrificed

before and after the onset of PTZ-induced seizures - and also in non-seizing rats. DHA

and AA concentrations in membrane phospholipids were also measured, to determine the

specific effects of OA, AA and DHA treatment on membrane fatty acid composition. All

subjects in this experiment were sacrificed with head-focused microwave fixation in

order to inactivate enzymes involved in the release of AA and DHA from the

phospholipid membrane. The experiment was conducted over a 3-day period. The

subjects were sacrificed during PTZ-induced seizures on the first day. On the second day,

the subjects were sacrificed 10 seconds prior to seizure induction by PTZ. On the third

day, subjects were injected with saline instead of PTZ, and then sacrificed (no-seizure

group).

The purpose of the test on the first day was to determine the levels of AA and

DHA in subjects sacrificed during PTZ-induced seizures. On day 1, therefore, subjects

were divided into 3 treatment groups, which received pre-treatment subcutaneous

injections of 300 mg/kg OA, 300 mg/kg AA, or 300 mg/kg DHA (n=6-8 per group). One

hour after the injections of OA, AA or DHA, the subjects were injected intraperitoneally

with 105 mg/kg of PTZ and observed for five minutes in an open field. The latency of

the first tonic-clonic convulsion was recorded. These animals were then sacrificed by

microwave fixation within 20 seconds after the onset of tonic-clonic convulsions, and the

brains were saved for assays. The average latency to seizure onset was approximately 40

seconds.

The purpose of the tests on the second and third day was to determine the assay

217

levels of AA and DHA in animals sacrificed before seizure onset – and also in non-

seizing animals. On day 2, therefore, the subjects received subcutaneous pre-treatment

injections of 300 mg/kg OA, 300 mg/kg AA, or 300 mg/kg DHA, Following that, the

subjects were injected intraperitoneally one hour later with 105 mg/kg of PTZ. The

subjects were then sacrificed at 30 seconds post-PTZ injection using head-focused

microwave fixation. (This time of sacrifice was approximately 10 seconds prior to the

estimated time of seizure onset).

On the third day, the subjects were also pre-treated with 300 mg/kg OA, 300

mg/kg AA, or 300 mg/kg DHA, but were injected with saline (instead of PTZ) one hour

later. This ‘non-seizing’ group of subjects was injected one hour after pre-treatment with

physiological saline (0.9%), volume matched to an equivalent dose of 105 mg/kg PTZ.

The subjects were sacrificed 30 seconds after saline injection with head-focused

microwave fixation.

All subjects were decapitated immediately following microwave fixation and the

heads were cooled on dry ice. The brains were then excised and the hemispheres were

separated and frozen on dry ice. The right hemisphere was used for assays, the left was

stored for possible future analysis. Brain samples were stored in a -80 °C freezer.

7.3.3 Brain phospholipid and unesterified fatty acid analysis

Total lipids were extracted from the right hemisphere by the method of Folch et al.

(Folch et al., 1957). The right hemisphere was weighed, grinded in 6.5 ml of 0.9% KCl

using a glass-grinder, and washed once with 5 ml methanol twice with 10 ml of

chloroform and once with 20 ml of chloroform. The total lipid extract was then dried

under nitrogen and reconstituted in 2 ml of chloroform. Diheptadecanoyl L-α-

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phosphatidylcholine or non-esterified heptadecaenoic acid (Sigma, St. Louis, Mo) in

chloroform were used as internal standards.

Brain phospholipids and unesterified fatty acids were isolated from 10 μl of the

total lipid extract, by thin-layer chromatography (TLC). TLC plates (Whatman LK6D

plates, precoated with 250μm of Silica Gel 60A) were washed in chloroform and

methanol (2:1) and activated by heating at 100oC for 1 hour prior to use. Phospholipids

and unesterified fatty acids were resolved in heptane: diethyl ether: glacial acetic acid

(60:40:2 by volume), alongside authentic standards (Avanti, Alabaster, AL). The plates

were lightly sprayed with 0.1% (w/v) 8-anilino-1-naphthalenesulfonic acid, and the bands

corresponding to phospholipid and free fatty acid standards were identified under

ultraviolet light. The bands were scraped off the plates into 15 ml glass screw cap tubes

with Teflon lined caps, and directly methylated in 14% methanolic BF3 (2 mL) and

hexane (2 ml) at 100°C for 1 hour. The samples were allowed to cool at room

temperature for 10 minutes and centrifuged at 1600 rpm following the addition of

deionized water (2 ml). The upper hexane layer was extracted and dried under nitrogen

and reconstituted in 25-50 µl and 100 µl of hexane for fatty acid methyl ester (FAME)

analysis of unesterified fatty acids and phospholipids respectively.


FAMEs were analyzed on a Varian-430 gas chromatograph (Varian, Lake Forest,

CA, USA) equipped with a Varian FactorFour capillary column (VF-23ms; 30 m x 0.25

mm i.d. x 0.25 μm film thickness) and a flame-ionization detector. 1.5 μl of unesterified

fatty acid FAMEs and 1 μl of phospholipid FAMEs were injected in splitless mode. The

carrier gas was helium, set to a constant flow rate of 0.7 ml/min. The injector and

219

detector ports were set at 250oC. FAMEs were eluted using a temperature program set

initially at 50oC for 2 min, increased at 20oC/min and held at 170oC for 1 min, then at

3oC/min and held at 212oC for 5 min. Peaks were identified by retention times of

authentic FAME standards of known composition (Nu-Chek-Prep, Elysian, MN).


All data are expressed as mean ± SEM. Data were analyzed using Sigma Stat

v.3.2 (Systat Software, Inc.). A one-way ANOVA followed by Tukey’s post-hoc

comparisons was used to determine the effect of treatment on seizure latency if the data

was normally distributed (Experiments 1 and 2). One-way ANOVA on ranks, followed

by Tukey’s post-hoc test, was used for skewed data. One-way analysis of variance was

also used to compare the effects of fatty acid treatments on phospholipid DHA and AA

levels. A two-way analysis of variance was used to determine the effects of fatty acid

treatment and seizure status on brain unesterified fatty acid concentrations. Outliers

falling more than 2 standard deviations from the mean were excluded from all statistical

analyses. Statistical significance was accepted at P<0.05.

7.4 Results

7.4.1 DHA delays latency to seizure onset (Experiment 1)

The data related to latencies to the onset of myoclonic jerks and tonic-clonic

seizures following PTZ injection (Experiment 1) are presented in Figures 1-A and 1-B,

respectively. One subject from each of the OA, DHA 200 mg/kg and DHA 300 mg/kg

groups was excluded from the statistical analyses because they were outliers (more that 2

220

standard deviations from the mean). As indicated by the figures, the dose of of 300 mg/kg

of DHA appeared to cause the maximal increase in latency to seizure onset, yielding

latencies two fold longer for myoclonic jerks and more than 2 fold longer for tonic-clonic

convulsions. A one-way ANOVA on ranks was used to compare the effects of treatment

on seizure latency, since the data were not normally distributed. The one-way analysis of

variance, however, did not reveal a significant difference among the groups in mean

latency to myoclonic jerks (P=0.2) or tonic-clonic seizures (P=0.2), perhaps due to the

variability of the data.

The latencies between the OA control group and the DHA dose that appeared to

cause the highest increase in threshold was also compared by a Mann-Whitney U test.

The mean latencies to myoclonic jerks and tonic-clonic seizures were significantly higher

in the DHA 300 mg/kg group relative to the OA 300 mg/kg group, as determined by the

Mann-Whitney U test (P < 0.05).

7.4.2 DHA delays latency to seizure onset (Experiment 2)

Experiment 2 involved the quantification of brain unesterified and phospholipid-

bound AA and DHA shortly prior to PTZ-induced seizures, during PTZ-induced seizures,

and in animals that were injected with physiological saline instead of PTZ.

Determinations of seizure thresholds were done following pre-treatment with OA, AA or

DHA administered via the subcutaneous route. The seizure latency data in the OA and

DHA groups followed similar trends as in Experiment 1, whereas AA did not appear to

delay latency to seizure onset (myoclonic jerk latencies of 39.0 ± 4.0 s, n=5; 43.2 ± 4.0 s,

n=7; and 42.3 ± 1.9 s, n=4 for OA, DHA and AA treated rats respectively; tonic-clonic

latiencies of 44.4 ± 1.2 s, n=5; 48.7 ± 0.5 s, n=7; 45.3 ± 2.7 s; n=4 for OA, DHA and AA

221

treated rats respectively; P>0.05 by 1-way ANOVA for myoclonic jerks and tonic-clonic

seizures).

7.4.3 Unesterified AA concentrations increase during seizures, particularly in

DHA-treated subjects (Experiment 2)

The data related to unesterified concentrations of AA are shown in Figure 2-A.

There was little change in free concentrations of unesterified AA before the onset of

seizure activity. Unesterified AA concentrations increased by at least 10-fold during

seizures, relative to non-seizure and pre-seizure values. This was greater in the AA pre-

treated group than the OA pretreated group, and greater in the DHA pre-treated group

than in either of the other two groups.

A two-way analysis of variance revealed a significant effect of seizures (P<0.01)

and a significant interaction between seizures and fatty acid treatment (P<0.05), on

unesterified AA concentrations. Tukey’s post-hoc comparisons with the pre-treatment

groups lumped together indicated that the differences in unesterified AA concentrations

between the seizing subjects and the no-seizure and pre-seizure subjects, were

statistically significant (P<0.05). No significant differences were detected between the

no-seizure and pre-seizure subjects (P>0.05). Individual post-hoc comparison of means

in the different pre-treatment groups revealed that AA release during seizures was

significantly higher in the DHA pre-treated group than in the OA and AA pre-treated

group (P<0.05), which did not differ significantly from each other.

222

7.4.4 Unesterified DHA concentration decrease pre-seizure, regardless of fatty

acid pre-treatment (Experiment 2)

Figure 2-B shows the effects of seizures and fatty acid pre-treatment on

unesterified DHA concentrations before and during seizures and in non-seizing rats. As

indicated by the figure, mean free DHA concentrations were relatively similar in all three

pre-treatment groups. In all three groups, free DHA concentrations dropped dramatically

just before seizure onset (as compared to non-seizing levels), but returned to normal or

somewhat higher during seizures. As indicated by a two-way analysis of variance, there

were significant differences among the groups related to the seizures factor (P<0.01), but

no significant differences related to fatty acid pre-treatment. Tukey’s post-hoc

comparisons with the pre-treatment groups lumped together showed that free DHA

concentrations were significantly lower in the pre-seizure subjects, as compared to both

the non-seizing and seizing subjects (P<0.05), which did not significantly differ from

each other.

7.4.5 Phospholipid-bound AA and DHA concentrations are not altered by fatty

acid treatment or seizures (Experiment 2)

We previously reported a lack of change in phospholipid-bound DHA following

acute injection (Taha et al., under review). Consistent with previous observations, there

was no effect of fatty acid injection on DHA or AA concentrations within total

phospholipids, regardless of seizure state, as determined by one-way analysis of variance

(P>0.05, data not shown).

223

7.5 Discussion

The purpose of the present study was to test the hypothesis that AA and DHA are

freed from the phospholipid membrane both by the hyperactivity that precedes

pentylenetetrazol (PTZ)-induced seizures as well as by the hyperactivity that occurs

during seizures. We tested this hypothesis in rats that had been pre-treated with 300

mg/kg of oleic acid (control), AA or DHA, since it was hoped that pre-treatment with AA

or DHA might increase the release of unesterified AA or DHA. Head-focused microwave

fixation was used to euthanize the subjects before decapitation in order to inactivate

phospholipase enzymes that are normally induced by decapitation, and which could,

therefore produce artificially high concentrations of free fatty acids.

The major findings of the present study were not in agreement with our

hypothesis. There was no increase in the level of free AA in the PTZ animals before

seizure onset, and there was, unexpectedly, a significant drop in free DHA at that time.

The same patterns were seen in all three pre-treatment groups, indicating that pre-

treatment had no significant effect in PTZ-injected animals before the onset of seizures.

With regard to AA and DHA during seizures, the expected increases in free AA

and DHA were seen in both cases. The most striking increases were seen in AA, where

increases from 100 to 500% occurred, depending on pretreatment. In the case of AA

release, pretreatment was an important factor, with DHA pretreatment causing

significantly more AA release than either of the other pre-treatments. With regard to

DHA, free DHA was higher during seizures in two of the pre-treatment groups, the OA

and DHA pre-treated groups - but these increases were less than 100% and they did not

reach statistical significance. No increase was seen in the AA pre-treatment groups

224

during seizure activity.

To our knowledge, this is the first study to assess brain unesterified

concentrations of non-seizing and seizing brains that have been fixed by head-focused

microwave fixation. Previous studies have measured brain unesterified fatty acid

concentrations pre- and post-seizure, in rats that were euthanized by decapitation (Bazan,

1970; Bazan, 1971), or in rats that were microwave-fixed immediately after decapitation

(Visioli et al., 1993). Microwave-fixation is necessary in order to inactivate

phospholipase enzymes involved in the de-esterification of fatty acids from the

phospholipid membrane (Farias et al., 2008). These phospholipases are immediately

activated following decapitation, due to the effects of ischemia (Bazan, 1970; Bazinet et

al., 2005a). In the present study, microwave fixation resulted in basal unesterified and

phospholipid-bound AA and DHA concentrations that were similar to previous studies

that used microwave-fixation prior to decapitation (Farias et al., 2008).

The increase in brain unesterified AA concentrations during seizures is consistent

with previous studies that measured free AA levels in the brain of seizing rats, although

they were done following decapitation (Bazan et al., 1982; Rodriguez de Turco and

Bazan, 1983; Birkle and Bazan, 1987; Visioli et al., 1993). All of these studies reported

an increase in free AA during seizure activity. Notably, however, the rise in AA in these

reports was most likel y confounded by the effects of decapitation.

The increase in unesterified AA was highest is seizing rats that received DHA as a

pre-treatment at a dose that raises seizure thresholds. Smaller increases were seen during

seizures in OA and AA pre-treated subjects. The mechanism by which DHA increased

the concentration of unesterified AA during seizures is not clear. This could be a topic of

225

future experiments. It is possible, however, that unesterified AA may play a role in

seizure protection. This would be consistent with in vitro studies which have reported that

direct application of unesterified AA reduces neuronal excitability by partially inhibiting

voltage-dependent ion channels (Fraser et al., 1993; Keros and McBain, 1997), or

possibly through some of its anti-inflammatory metabolites, such as prostaglandin E2

(Rosenkranz and Killam, 1979; Rosenkranz and Killam, 1981).

In contrast to previous reports (Rodriguez de Turco and Bazan, 1983; Birkle and

Bazan, 1987; Visioli et al., 1993), unesterified DHA concentrations did not increase

significantly during seizures, although “trends” toward increase were seen in the OA and

DHA pre-treatment groups. The difference between our data – showing a lack of increase

– and the data previously reported by other groups may be related to the method of

euthanization. Previous studies have measured unesterified DHA levels in non-

microwaved brain samples, following decapitation (Rodriguez de Turco and Bazan,

1983; Birkle and Bazan, 1987; Visioli et al., 1993; Taha et al., 2009c). It is possible

therefore, that the previously reported rises in unesterified DHA during seizures, was an

artifact produced by the effects of ischemia. Ischemia has been shown to increase brain

unesterified DHA levels (Farias et al., 2008).

It might be noted, however, that while free DHA levels during seizures did not

differ from those in non-seizing animals, they were significantly elevated from the

unexpectly low levels of free DHA seen just before seizures.

Unexpectedly, unesterified DHA concentrations were significantly lower than

baseline during the period just before seizure onset. This was true regardless of the type

of fatty acid pre-treatment. The decrease in free DHA before seizures could be caused by

226

a decrease in the release of DHA from the phospholipid membrane, or by an increased

utilization of free DHA for some unknown activity. following its release from the

membrane. Further study of this phenomenon will be required, to assess its significance,

if any, to seizure protection.

We conclude that DHA treatment, increased unesterified AA but not unesterified

DHA concentrations during seizures, in the brains of microwave-fixated rats. The rise in

seizure threshold in the DHA-treated rats may, therefore, be related to an increase in

unesterified AA levels.

ACKNOWLEDGEMENT

This study was funded by a grant from the Canadian Institutes of Health Research

(CIHR) to W.M.B. and R.P.B., and the CIHR Doctoral Research Award to A.Y.T.

227

Figure 1-A: Latency to the onset of myoclonic jerks following OA or DHA subcutaneous injections

0

20

40

60

80

100

120

OA 300 mg/kg DHA 100 mg/kg DHA 200 mg/kg DHA 300 mg/kg DHA 400 mg/kg

Treatment

Late

ncy

to m

yocl

onic

jerk

s (s

econ

ds)

228

Figure 1-B: Latency to the onset of tonic-clonic seizures following OA or DHA subcutaneous injections

0

50

100

150

200

250

OA 300 mg/kg DHA 100 mg/kg DHA 200 mg/kg DHA 300 mg/kg DHA 400 mg/kg

Treatment

Late

ncy

to to

nic-

clon

ic s

eizu

res

(sec

onds

)

Data are mean ± SEM of n=7-8 per treatment.

Figure 1-A: DHA appeared to delay latency to the onset of myoclonic jerks at a dose of

300 mg/kg. One way analysis of variance, however, revealed no significant effects of

treatment on myoclonic jerks latency (P=0.2).

Figure 1-B: DHA appeared to delay latency to the onset of myoclonic jerks at a dose of

300 mg/kg. One way analysis of variance, however, revealed no significant effects of

treatment on the latency to tonic-clonic seizures (P=0.2).

229

Figure 2-A: Brain Unesterified AA concentrations following OA, AA or DHA treatment in saline or PTZ treated rats

0

5

10

15

20

25

30

35

40

45

No-seizure Pre-seizure During seizure

Une

ster

ified

AA

con

cent

ratio

n (n

mol

/g) OA 300

AA 300DHA 300

a

a

b***

230

Figure 2-B: Brain Unesterified DHA concentrations following OA, AA or DHA treatment in saline or PTZ treated rats

0

2

4

6

8

10

12

No-seizure Pre-seizure During seizure

Une

ster

ified

DH

A c

once

ntra

tions

(nm

ol/g

) OA 300AA 300DHA 300 *

**

Data are mean ± SEM of n=4-8 per fatty acid treatment per seizure state (no seizure, pre-

seizure and during seizure).

Figure 2-A: Two-way analysis of variance showed a significant effect of seizures

(P<0.01) and a significant interaction between seizures and fatty acid treatment (P<0.05),

on unesterified AA concentrations. Tukey’s post-hoc comparisons with the pre-treatment

groups lumped together indicated significant differences in unesterified AA

concentrations between the seizing subjects and the no-seizure and pre-seizure subjects

(P<0.05). No significant differences were detected between the no-seizure and pre-

seizure subjects (P>0.05). Individual post-hoc comparison of means in the different pre-

treatment groups revealed that AA concentrations during seizures was significantly

231

higher in the DHA pre-treated group than in the OA and AA pre-treated group (P<0.05),

which did not differ significantly from each other.

Figure 2-B: Two-way analysis of variance showed significant differences among the

groups related to the seizures factor (P<0.01), but no significant differences related to

fatty acid pre-treatment. Tukey’s post-hoc comparisons with the pre-treatment groups

lumped together showed that free DHA concentrations were significantly lower in the

pre-seizure subjects, as compared to both the non-seizing and seizing subjects (P<0.01),

which did not significantly differ from each other (P<0.05).

232

7.6 References

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Danthi S. J., Enyeart J. A. and Enyeart J. J. (2005) Modulation of native T-type calcium channels by omega-3 fatty acids. Biochemical and biophysical research communications 327, 485-493. Depaulis A., Snead O. C., 3rd, Marescaux C. and Vergnes M. (1989) Suppressive effects of intranigral injection of muscimol in three models of generalized non-convulsive epilepsy induced by chemical agents. Brain Res 498, 64-72. Fabene P. F., Navarro Mora G., Martinello M., Rossi B., Merigo F., Ottoboni L., Bach S., Angiari S., Benati D., Chakir A., Zanetti L., Schio F., Osculati A., Marzola P., Nicolato E., Homeister J. W., Xia L., Lowe J. B., McEver R. P., Osculati F., Sbarbati A., Butcher E. C. and Constantin G. (2008) A role for leukocyte-endothelial adhesion mechanisms in epilepsy. Nature medicine 14, 1377-1383. Farias S. E., Basselin M., Chang L., Heidenreich K. A., Rapoport S. I. and Murphy R. C. (2008) Formation of eicosanoids, E2/D2 isoprostanes, and docosanoids following decapitation-induced ischemia, measured in high-energy-microwaved rat brain. Journal of lipid research 49, 1990-2000. Folch J., Lees M. and Sloane Stanley G. H. (1957) A simple method for the isolation and purification of total lipides from animal tissues. The Journal of biological chemistry 226, 497-509. Fraser D. D., Hoehn K., Weiss S. and MacVicar B. A. (1993) Arachidonic acid inhibits sodium currents and synaptic transmission in cultured striatal neurons. Neuron 11, 633-644. Green J. T., Orr S. K. and Bazinet R. P. (2008) The emerging role of group VI calcium-independent phospholipase A2 in releasing docosahexaenoic acid from brain phospholipids. Journal of lipid research. Innis S. M. (2007) Dietary (n-3) fatty acids and brain development. The Journal of nutrition 137, 855-859. Keros S. and McBain C. J. (1997) Arachidonic acid inhibits transient potassium currents

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and broadens action potentials during electrographic seizures in hippocampal pyramidal and inhibitory interneurons. J Neurosci 17, 3476-3487. Kitajka K., Puskas L. G., Zvara A., Hackler L., Jr., Barcelo-Coblijn G., Yeo Y. K. and Farkas T. (2002) The role of n-3 polyunsaturated fatty acids in brain: modulation of rat brain gene expression by dietary n-3 fatty acids. Proceedings of the National Academy of Sciences of the United States of America 99, 2619-2624. Lauritzen I., Blondeau N., Heurteaux C., Widmann C., Romey G. and Lazdunski M. (2000) Polyunsaturated fatty acids are potent neuroprotectors. The EMBO journal 19, 1784-1793. Ma D. W. (2007) Lipid mediators in membrane rafts are important determinants of human health and disease. Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 32, 341-350. Marcheselli V. L., Hong S., Lukiw W. J., Tian X. H., Gronert K., Musto A., Hardy M., Gimenez J. M., Chiang N., Serhan C. N. and Bazan N. G. (2003) Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression. The Journal of biological chemistry 278, 43807-43817. Oliveira M. S., Furian A. F., Royes L. F., Fighera M. R., Fiorenza N. G., Castelli M., Machado P., Bohrer D., Veiga M., Ferreira J., Cavalheiro E. A. and Mello C. F. (2008) Cyclooxygenase-2/PGE2 pathway facilitates pentylenetetrazol-induced seizures. Epilepsy research 79, 14-21. Rapoport S. I. (2003) In vivo approaches to quantifying and imaging brain arachidonic and docosahexaenoic acid metabolism. The Journal of pediatrics 143, S26-34. Rodriguez de Turco E. B. and Bazan N. G. (1983) Changes in free fatty acids and diglycerides in mouse brain at birth and during anoxia. J Neurochem 41, 794-800. Rosenkranz R. P. and Killam K. F., Jr. (1979) Effects of intracerebroventricular administration of prostaglandins E1 and E2 on chemically induced convulsions in mice. The Journal of pharmacology and experimental therapeutics 209, 231-237.

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Rosenkranz R. P. and Killam K. F., Jr. (1981) Anticonvulsant effects of PGE2 on electrical, chemical and photomyoclonic animal models of epilepsy. Progress in lipid research 20, 515-522. Taha A. Y., Filo E., Ma D. W. and McIntyre Burnham W. (2009) Dose-dependent anticonvulsant effects of linoleic and alpha-linolenic polyunsaturated fatty acids on pentylenetetrazol induced seizures in rats. Epilepsia 50, 72-82. Taha A. Y., Huot P. S., Reza-Lopez S., Prayitno N. R., Kang J. X., Burnham W. M. and Ma D. W. (2008) Seizure resistance in fat-1 transgenic mice endogenously synthesizing high levels of omega-3 polyunsaturated fatty acids. J Neurochem 105, 380-388. Tu B. and Bazan N. G. (2003) Hippocampal kindling epileptogenesis upregulates neuronal cyclooxygenase-2 expression in neocortex. Experimental neurology 179, 167-175. Visioli F., Rihn L. L., Rodriguez de Turco E. B., Kreisman N. R. and Bazan N. G. (1993) Free fatty acid and diacylglycerol accumulation in the rat brain during recurrent seizures is related to cortical oxygenation. J Neurochem 61, 1835-1842. Voskuyl R. A., Vreugdenhil M., Kang J. X. and Leaf A. (1998) Anticonvulsant effect of polyunsaturated fatty acids in rats, using the cortical stimulation model. European journal of pharmacology 341, 145-152. Vreugdenhil M., Bruehl C., Voskuyl R. A., Kang J. X., Leaf A. and Wadman W. J. (1996) Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons. Proceedings of the National Academy of Sciences of the United States of America 93, 12559-12563. Xiao Y. and Li X. (1999) Polyunsaturated fatty acids modify mouse hippocampal neuronal excitability during excitotoxic or convulsant stimulation. Brain Res 846, 112-121. Young C., Gean P. W., Chiou L. C. and Shen Y. Z. (2000) Docosahexaenoic acid inhibits synaptic transmission and epileptiform activity in the rat hippocampus. Synapse (New York, N.Y 37, 90-94.

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CHAPTER 8

DISCUSSION

237

8 Discussion

8.1 General discussion

The overall purpose of the present experiments was to test the hypothesis that

n-3 PUFA, and especially DHA, would have anticonvulsant effects (raise seizure

thresholds). This hypothesis was suggested by the fact that the n-3 PUFA, including

DHA, have anti-arrhythmic effects.

In general, the results of the present experiments support the overall hypothesis.

The n-3 PUFA do raise seizure thresholds in rats, and the effects appear to be mediated

by DHA, the end-product of the n-3 PUFA synthesis pathway.

The key findings of each experiment are summarized in Table 10-1. Table 10-2

summarizes the changes in DHA levels in phospholipids and unesterified fatty acids.

Since the specific experiments have all been discussed in the specific Discussion Sections

that conclude each chapter, the present General Discussion will focus on questions that

will need to be addressed by future studies.

Why does ALA work despite its limited conversion? Yehuda et al. (1994)

suggested that ALA works by elevating brain DHA. We now know, however, that the

conversion of ALA to DHA is very poor in rats. Still, ALA (200 mg/kg) increased the

latency to seizure onset in our hands despite its limited conversion to DHA (Experiment

2). Is there some other ways that ALA could increase brain DHA? Experiment 3

demonstrated that the effects of ALA are possibly due to an increase in PUFA

mobilization from liver to the brain. This possibility might be investigated in future

experiments by using 13C-DHA tracers to measure the distribution of 13C-DHA in rats

(Taha et al., 2005) following chronic ALA injections (as in Experiment 2), in non-seizing

238

rats.

Why didn’t ALA produce an elevation in esterified brain DHA? When the

fatty acid composition measurements were performed in Experiment 2, no significant

increases in esterified brain DHA levels were seen in the rats that had received an

anticonvulsant dose of the SR-3 mixture (200 mg/kg). This finding does not appear to fit

very well with our overall hypothesis.

The failure to find an elevation in brain esterified DHA (to phospholipids) may

possibly relate to the variability introduced by the background DHA levels in the brain,

and the sensitivity of the assays used in Experiment 2. The assays used involved thin

layer and gas chromatography, which enable the separation and detection of total DHA in

membrane phospholipids. However, since brain phospholipids are normally enriched

with DHA, it may be difficult to detect a small change in DHA levels following chronic

ALA administration, due to the variability introduced by the background levels of DHA.

Future experiments could employ a more sensitive assay involving radiolabeled tracers

that would be able to detect very slight changes in DHA incorporation.

Why didn’t esterified DHA concentrations in membrane phospholipids

increase following acute DHA injection? DHA concentrations in brain phospholipids

were also not found to be increased in non-seizure tested animals following acute DHA

administration (Experiments 4 and 6, Table 2). The lack of change in esterified

phospholipid DHA is puzzling, and suggests some problem with our measurement

techniques. Previous studies using radiolabeled injected DHA have shown that the

majority (>90%) of intravenously-administered, radiolabeled DHA is rapidly

incorporated into the phospholipid membrane (DeMar et al., 2004). Once again, it is

239

possible that the gas-chromatography assays used to measure esterified DHA levels in the

present study were not sensitive enough to detect small increases in the amount of brain

DHA, due to the variability associated with the relatively large pool of incorporated DHA

in the brain. Therefore more sensitive assays involving radiolabeled DHA could be used

in future experiments to measure the relative amount of DHA incorporated into brain

phospholipids following acute DHA administration.

Why did total n-3 fatty acids increase during / post – seizures following ALA

treatment (Experiment 2), but not after DHA treatment (Experiment 6)? ALA

injections for 3 weeks (in the SR-3 mixture) raised seizure threshold and increased total,

unesterified n-3 PUFA composition in the brains of rats following PTZ administration.

The rise in n-3 PUFA composition is not in agreement with Experiment 6 (Table 2),

which showed that unesterified AA concentrations, but not unesterified DHA

concentrations, increased during PTZ-induced seizures. The discrepancy between the

findings of the two experiments may have two possible explanations. One possible

explanation is that there was a difference in the time that elapsed before sacrifice, a factor

which might have caused a differential effect on unesterified fatty acid concentrations

between the two experiments. In Experiment 2, n-3 PUFA levels were measured in

subjects that had been sacrificed 30 minutes following PTZ injection – and about 29

minutes after seizure onset - unless the subjects died spontaneously while seizing. In

contrast, the subjects in Experiment 6 were sacrificed within 20 seconds after the

beginning of convulsions.

A second possible explanation, relates to the methods of euthanasia used in the

two experiments. In Experiment 2, the subjects were euthanized by a lethal dose of

240

sodium pentobarbital followed by decapitation. In contrast, in Experiment 6 subjects

were euthanized by head-focused microwave fixation and then decapitated. The observed

increase in unseterified n-3 PUFA composition in Experiment 2 might be due to the

effects of ischemia following decapitation. Ischemia induces the release of n-3 PUFA via

selective phospholipases (Farias et al., 2008). Previous studies that have shown that

unesterified n-3 PUFA concentrations increase during or post seizures when the animals

are sacrificed without microwave fixation prior to decapitation (Birkle and Bazan, 1987;

Visioli et al., 1993). This is consistent with the findings of Experiment 2, in which the

animals were sacrificed by decapitation following sodium pentobarbital euthanasia. The

confounding effects of ischemia were avoided in Experiment 6, by microwave-fixating

the brains prior to decapitation. Head-focused microwave fixation denatures the

phospholipase enzymes that release fatty acids from the membrane during ischemia,

which is induced by decapitation (Farias et al., 2008). The findings of Experiment 2,

therefore, are likely artifactual, because they are confounded by both the effects of time

of sacrifice and the method of sacrifice post PTZ-induced seizures.

Does DHA raise seizure thresholds via a direct effect on the brain? Regardless

of the ambiguity in the results of our assay studies, acute DHA does raise seizure

thresholds, as demonstrated in Experiment 4. Does this elevation in thresholds involve

an effect on the brain? A limitation of the present experimental series is that DHA has

never been administered intraventricularly. This means that the possibility that DHA

may raise seizure thresholds via some peripheral action cannot be ruled out. This is not

an impossibility. Several studies, for instance, have shown that peripheral DHA produces

bioactive metabolites that antagonize the production of pro-inflammatory molecules, and

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that these metabolites enter the brain (Belayev et al., 2005). A recent study has shown

that chronic brain inflammation is associated with seizures, and that the pharmacological

blockade of pro-inflammatory molecules reduces the incidence of seizures (Fabene et al.,

2008). Thus, it is possible that DHA acts peripherally by modulating the levels of pro-

inflammatory markers in the plasma.

This question could be settled in future experiments by administering DHA

intracerebroventrically (i.c.v.), and then applying our standard seizure tests. The

expected result – according to our central hypothesis - would be that i.c.v. DHA would

have anticonvulsant effects.

Why Does DHA Act Rapidly When Injected Subcutaneously, but Slowly

When Given by Mouth? An apparent paradox appears when the times of onset of

anticonvulsant actions are compared in Experiments 4 and 5. The effects of DHA in rats

are seen within 30 minutes when DHA is injected subcutaneously (Experiment 4),

whereas it takes more than 8 weeks to raise seizure thresholds when DHA is taken by

mouth. This time discrepancy may relate to the different routes of administration

involved in the two experiments. When DHA is directly injected, it probably remains in

its unesterified, “free’ form, then binds to albumin and quickly enters the brain (Polozova

and Salem, 2007). When it is taken by mouth, however, it is probably packaged and

trapped in chylomicrons (CMs) and low-density lipoproteins (LDLs) (Polozova et al.,

2006). This would keep DHA in the bloodstream and out of the brain for some weeks.

CMs, LDLs and related particles, such as very low density lipoproteins (VLDLs),

are involved in the transport of DHA from the gut, liver and adipose tissue to a variety of

tissues - but not to the brain (Rodbell, 1960; Quarfordt and Goodman, 1967; Polozova

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and Salem, 2007; Chen et al., 2008b). Although the brain contains LDL and VLDL

receptors, it does not rely on LDL or VLDL particles for its uptake of DHA (Chen et al.,

2008b). Instead, the brain extracts free DHA from plasma albumin (Chen et al., 2008a;

Ouellet et al., 2009).

Thus, in the chronic feeding paradigm, free DHA probably begins to bind to

plasma albumin (and enter the brain), only when the chylomicron / LDL / VLDL pool

becomes saturated with DHA over a period of a several weeks.

This hypothesis could be tested in future experiments that would administer

dietary DHA and test levels in blood and brain at different intervals after the start of

administration. The expected result would be that brain levels of DHA would not begin

to rise until four or more weeks after the start of dietary administration.

Why does DHA injection not increase DHA concentrations in plasma? Total

(bound and unbound) DHA concentrations in plasma were measured at one hour

following subcutaneous DHA injection (Experiment 4). There was no significant change,

however, in DHA concentrations following DHA injection. This is probably related to the

short half-life of DHA in plasma (less than 40 seconds) (Robinson et al., 1992). Another

possibility, however, is that the concentration of albumin-bound, “free” DHA did

increase gradually following subcutaneous injection (slow release into the plasma), but

the change in concentration was masked by measuring both the albumin-bound and

lipoprotein-bound DHA levels in plasma. Since DHA was injected in its albumin-bound

form, the concentration of albumin-bound DHA would be expected to increase at one

hour post subcutaneous injection. This could be addressed by measuring albumin-bound

DHA concentration in plasma at one hour post DHA injection.

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Why did DHA loose its effect over time following subcutaneous

administration? This question is hard to answer without knowing whether brain levels

go up after injection. (Our assay data are ambiguous due to the analytical limitations of

our assays.) Assuming that the anticonvulsant effects are central and do relate to brain

DHA, the loss of effect over time following subcutaneous injection might be related to

the turnover of DHA within the brain. Upon entering the brain, DHA is rapidly

incorporated into the phospholipid membrane (DeMar et al., 2004). Phospholipid DHA

is then regularly de-esterified by membrane phospholipases (Robinson et al., 1992;

DeMar et al., 2004; Strokin et al., 2007). When that happens, approximately 90% of the

DHA that is de-esterified is rapidly re-incorporated into the membrane, whereas the

remaining de-esterified 10% is utilized for gene regulation, signaling or energy

(Robinson et al., 1992; DeMar et al., 2004). This rate of turnover into this pool (de-

esterified) has been shown to be modulated by the amount of DHA available to the brain.

For instance, low dietary DHA intake has been shown to decrease the rate of DHA de-

esterification from the phospholipid membrane (DeMar et al., 2004). Although no studies

have addressed the effects of increased DHA supply to the brain on DHA turnover, it is

possible that a greater availability of DHA to the brain might increase the amount of de-

esterified DHA that does not get re-incorporated. Although increasing the supply of DHA

to the brain in Experiment 6 did not increase brain unesterified DHA concentrations, the

assays used did not measure turnover rate. This could be measured in future studies using

radiolabelled DHA tracers.

Is the dose-response curve for DHA’s anticonvulsant effects truly an

“Inverted U”? A consistent observation in the acute injection studies involving DHA

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(Experiments 4and 6) was that the effect of the DHA on seizure latency appeared to be

lost at higher doses, resulting in an “inverted U” dose-response curve. This effect was not

statistically significant, although future studies involving a larger number of animals

would likely achieve statistical significance. There are two possible explanations for the

inverted U dose-response curve. The first possibility is that high doses of DHA really do

produce smaller effects, as seen with some of the steroid hormones (Diamond et al.,

1992).

A second possibility, perhaps more probable, is that DHA comes out of solution

after the injection of higher doses, producing lower blood levels. This suggests that the

DHA would form fatty globules under the skin at the point of injection due to its

lipophilic nature. These low-surface-area globules would only slowly release DHA

molecules into the bloodstream, producing subtherapeutic DHA-albumin levels. A

somewhat similar phenomenon has been reported with phenytoin, which is also poorly

water soluble (McNamara et al., 1989).

This hypothesis could be tested in future experiments by injecting different levels

of DHA, and assaying the resulting unesterified, albumin-bound DHA in plasma. The

expected result would be that higher doses of injected DHA would produce lower levels

of DHA in the plasma.

Why doesn’t DHA stop seizure occurrence? DHA has raised seizure threshold

but has failed to stop seizure occurrence - as an anticonvulsant drug would. This is likely

because of our “inverted U” dose-response curves, and the possibility that at higher doses,

DHA may come out of solution. Because of that, the effects of high doses that might

actually stop seizures occurrence, have not been tested.

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In future experiments, a way to get around this problem might be to administer

DHA via the intraperitoneal or intravenous routes. Administering the DHA through the

intraperitoneal or intravenous routes would allow for higher injection volumes (at lower

DHA concentrations) than the subcutaneous route, and possibly produce high plasma

levels following the administration of high doses. This will probably normalize the

“inverted U” curves, and increase plasma DHA concentrations. Higher plasma

concentrations following higher doses might stop seizure occurrence.

What are the Possible Mechanisms of Action of DHA? A first possible

mechanism to consider is genomic effects, since PUFA are known to affect gene

expression (Kitajka et al., 2002). The anticonvulsant effects of DHA seen in the present

experiments probably do not involve genomic effects, since the threshold elevations

occur within an hour after administration. These considerations apply, at least, to the

acute experiments. Whether genomic effects play role in the long-lasting chronic

experiments might be addressed in future experiments.

A second mechanism to consider is that DHA may work directly – perhaps

through the inhibition of VDSCs – even in its esterified form. Experiment 6 (Chapter 7)

did not show a release of unesterified DHA during seizure activity, so free DHA may not

be involved in the observed anticonvulsant actions of DHA. Conceivably, however,

esterified DHA might play a role. Even in phospholipid form, DHA might bind to and

inhibit ion channels by finding binding sites within the phospholipid bilayer. It is known

that some of the toxins that bind to the VDSC bind to sites in the lipophilic parts of the

molecule (Catterall, 1980).

The findings of Experiment 6 (Chapter7) suggest a third possibility - the

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possibility that the anticonvulsant effects of DHA may relate to the release of unesterified

AA during seizure activity. An unexpected finding of Experiment 6 was that seizure

activity did not cause an increase of unesterified DHA (as hypothesized), but rather

caused a large increase in unesterified AA. In in vitro experiments, AA, like DHA, has

been shown to inhibit the VDSC in heart cells (Kang and Leaf, 1996). To our knowledge,

the anticonvulsant effects of AA have never been shown in vivo. An attempt to show

such effects is currently underway in our laboratory.

Notably, unesterified AA might act directly – as postulated – or it could also act

through some of its prostaglandin metabolites - which are known to have anticonvulsant

effects in animal seizure models (Rosenkranz and Killam, 1979; Rosenkranz and Killam,

1981). Testing the anticonvulsant effects of AA in the presence of antagonists that block

its conversion to prostaglandins (eg – cox-2 inhibitors) would provide evidence that AA

itself is an anticonvulsant.

8.2 Future studies

The present research has provided descriptive evidence that DHA raises seizure

threshold in vivo and reduces the incidence of excitatory sharp waves in vitro (Appendix

2). It also suggests a possible mechanism for the action of the DHA, involving the

release of AA from the membrane.

A great deal of research, however, will be required to improve our understanding

of DHA and its anticonvulsant actions. A number of possible future experiments were

proposed in the discussion above. Some of the most important issues that need to be

addressed are:

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1) Measuring DHA mobilization from liver to brain following ALA treatment

ALA raised seizure threshold, possibly by increasing mobilization of DHA from

liver to the brain. This hypothesis needs to be tested in non-seizing animals, and by

injecting radiolabeled DHA to rats receiving ALA intraperitoneally for a minimum of 3

weeks. If ALA works by mobilizing DHA to the brain, less of the radiolabeled DHA

should partition into liver or possibly adipose in the ALA-treated rats relative to controls,

and more radiolabeled DHA would be detected in brain membrane phospholipids.

2) Measuring DHA incorporation into membrane phospholipids

The assays used in Experiments 2, 4 and 6 did not detect a change in DHA

incorporation into brain phospholipids following ALA or DHA treatment. The limitation

of the assays used in these experiments could be overcome by measuring the amount of

radiolabeled DHA incorporated into brain phospholipids, following acute subcutaneous

injection. It is expected that after subcutaneous administration of a radiolabeled dose of

DHA, an increase in radiolabeled DHA would be found in brain phospholipids.

3) Direct versus peripheral effects of DHA on seizure thresholds

It is not known whether the observed effects of DHA on seizure thresholds are

due to the actions of DHA within the brain, or to some peripheral action involving DHA

or its metabolites. Intracranial administration of DHA should confirm that the effects of

acute DHA injections on seizure threshold are directly related to the actions of DHA in

the brain. They should rule out the possibility that DHA acts via some peripheral action.

4) Plasma unesterified DHA levels following acute and chronic administration of

DHA

DHA raised seizure thresholds within one hour following acute administration,

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but took a few weeks to raise thresholds when administered orally. This is likely because

DHA is `trapped` in lipoproteins when taken by mouth. To test this, unesterified,

albumin-bound DHA concentrations in plasma could be measured following

subcutaneous DHA injection or gavauge. Direct injection of DHA should increase

unesterified DHA levels in plasma, whereas no changes in unesterified DHA levels

would be detected when DHA is administered by gavauge.

5) Anti-seizure effects of arachidonic acid

Experiment 6 indicated that DHA might possibly work by increasing unesterified

AA concentrations. The anticonvulsant effects of unesterified AA could by tested by

increasing unesterified AA levels using a calcium dependent phospholipase A2 agonist,

which is an AA-selective phospholipase involved in de-esterifying AA from the

membrane. Injecting AA to rats would not likely raise seizure threshold, because like

DHA, it is mostly esterified in the brain. Also, preliminary evidence from Experiment 6

suggests that AA does not delay latency to seizure onset, nor does it increase unesterified

AA concentrations, although higher doses of AA should be attempted. Notably, in vitro

studies have reported that AA reduces neuronal excitability (Fraser et al., 1993); however,

perfusing ex-vivo tissue or cell cultures with unesterified AA in the absence of an intact

blood-brain-barrier could increase unesterified AA concentrations and reduce

hyperexitability.

Although testing an AA-selective phospholipase agonist to promote the release of

AA from the membrane would be a good strategy, at present, selective agonists for

calcium-dependent phospholipase A2 are not available commercially.

6) Loss of anti-seizure effects over time

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The effect of DHA on seizure threshold was lost by 2 hours following

subcutaneous injection. The loss of the DHA effect could be due to an increase in the

turnover rate of DHA (i.e. de-esterification and subsequent utilization). If this is the case,

then unesterified AA concentrations should not be elevated in DHA-treated subjects at 2

hours post subcutaneous injection. This could be tested by repeating Experiment 6, and

euthanizing the animals by head-focused microwave fixation at 2 hours post DHA

administration. It would be expected that unesterified AA concentrations during seizures

would be similar to control levels.

7) Determining the maximal efficacy of DHA.

DHA appears to loose efficacy at higher doses, producing “inverted U” dose-

reponse curves. It is not clear whether this is related to the precipitation of DHA at higher

doses, or due to a real effect of DHA at high doses. The “inverted U” curve might

normalize if we increased the injection volume and decreased the DHA concentrations

administered. This would be possible if we administered, DHA through the

intraperitoneal or intravenous routes, both of which should allow for higher injection

volumes.

Expected results would be that DHA would have a stronger effect on seizure

threshold and possibly suppress seizure occurrence when administered intraperitoneally

or intravenously.

8) Testing the anti-seizure effects of DHA in other animal seizure models.

Anti-seizure drugs are typically validated in several different animal seizure

models before undergoing clinical testing. The present thesis assessed the effect of n-3

PUFA in only two seizure models – the maximal PTZ model and the electrical

250

stimulation seizure threshold test.

The effects of the n-3 PUFA, therefore, should be tested in the other models used

to screen for effects on different types of seizures. The common animal seizure models

that are used for screening anti-seizure medications are: 1) the maximal electroconvulsive

shock (MES) test, 2) the amygdala kindling model, and 3) the subcutaneous PTZ test.

The MES test models tonic-clonic seizures in humans, the amygdala kindling test models

complex-partial seizures in humans, and the subcutaneous (minimal) PTZ test models

absence seizures in humans (Fisher, 1989; Likhodii et al., 2003; Borges, 2008).

Other models that might be considered are the 6Hz seizure test, kainic acid,

pilocarpine and flourothyl models (Willis et al., 2009). The predictive validity of some of

these tests, however, has yet to be fully established.

8.3 Collected hypotheses related to future studies

1) When ALA is administered, it raises seizure threshold by increasing

mobilization of DHA from liver (and possibly adipose) to the brain.

2) When DHA is injected, it raises seizure threshold by increasing unesterified

AA concentrations during seizures.

3) When DHA is injected intraperitoneally or intravenously, DHA will stay in

solution and possibly suppress seizures or provide better seizure protection than when

administered subcutaneously.

4) When DHA is taken as part of the diet, it takes several weeks to obtain an

effect on seizure threshold, because DHA is trapped in lipoprotein molecules.

5) DHA raises seizure threshold by increasing unesterified AA concentrations.

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8.4 Clinical relevance

The findings of the present experiments suggest that DHA could potentially be

used to control seizures in humans without causing toxicity. Co-administration of DHA

with anticonvulsant drugs in humans might offer improved seizure control. This, however,

needs to be proven in a clinical trial.

A clinical trial involving DHA would probably use high doses of 6 grams per day

for a minimum period of 6 months. This long duration of study is because DHA is

trapped in lipoproteins when taken by mouth, and it might take several months for it to

reach the brain. Previous clinical trials have used low doses of 1 to 3 grams per day and

found no effects. Schalnager et al., however, in an open clinical trial used 3 grams for 6

months and found significant effects. Thus, a dose of 6 grams per day, which remains

within the range of intake of human populations that regularly consume seafoods, is

likely to reduce seizure frequency within 6 months. (Such a clinical trial is currently in

the planning stages.)

The use of DHA for seizure control could represent a major step forward in the

management of epilepsy in humans. This is because: 1) DHA is cheaper than many

anticonvulsant drugs; 2) DHA is readily available in health food stores without a

prescription; 3) DHA has no known side-effects in humans at higher doses of intake (6 –

10 grams per day); and 4) is beneficial to overall health, mainly due to its hypolipidemic

and cardioprotective effects (Leaf, 1995; Leaf et al., 2005; Holub, 2007).

8.5 Conclusions

The present exploratory research provided preliminary evidence that: 1) n-3

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PUFA raise seizure threshold in rats; 2) the effects of the n-3 PUFA on seizure threshold

are likely mediated by DHA; 3) the anti-seizure effects of DHA might be related to an

increase in unesterified AA concentrations during or shortly before seizures.

Table 10-1: Summary of key findings of Chapters 2 to 7 and appendices 1 and 2.

Experiment 1 (Chapter 2) Dietary SR-3 at 40 mg/kg does not alter seizure threshold

Experiment 2 (Chapter 3) Dietary SR-3 raises seizure threshold at a higher dose of 200 mg/kg

Experiment 3 (Chapter 4) Dietary SR-3 at 200 mg/kg raises threshold possibly by inducing the redistribution of PUFA from liver to brain

Experiment 4 (Chapter 5) Injected DHA, the end product of the n-3 fatty acid synthesis pathway, raises seizure threshold within one hour

Experiment 5 (Chapter 6) Dietary fish oil containing DHA raises cortical and amygdaloid thresholds after more than a month

Experiment 6 (Chapter 7) DHA increases unesterified AA concentrations during seizures.

Appendix 1 Injected EPA does not raise seizure thresholds within one hour

Appendix 2 DHA reduces the incidence of excitatory sharp waves in hippocampal slices without increasing inhibitory GABAergic rhythms.

Table 10-2: Summary of measured changes in PUFA levels in phospholipids and unesterified fatty acids Experiment 1 (Chapter 2) NM NM Experiment 2 (Chapter 3) ↔ ↑ in total n-3 PUFA Experiment 3 (Chapter 4) NM NM Experiment 4 (Chapter 5) ↔ ↔ Experiment 5 (Chapter 6) NM NM Experiment 6 (Chapter 7) ↔ ↑ in AA Appendix 1 NM NM Appendix 2 NM NM NM, Not measured; ↔, No change; ↑, increase

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CHAPTER 9

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APPENDIX 1

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10 Appendix 1: Assessing the anti-seizure effects of eicosapentaenoic acid in rats

10.1 Background

Eicosapentaenoic acid is the metabolic precursor of docosahexaenoic acid

(Depaulis et al.). The synthesis of DHA from EPA takes place mainly in the liver (Lin

and Salem, 2005; Igarashi et al., 2006; Lin and Salem, 2007).

Voskuyl and colleagues (1998) have reported that the acute administration of

EPA raises seizure threshold in a cortical stimulation model involving rats (Voskuyl et

al., 1998). Recent studies, however, suggest that EPA is not present in the brain to any

extent because it is immediately oxidized as soon as it enters the brain (Chen et al.,

2008b; Chen et al., 2009). It is unlikely, therefore, that EPA would have anti-seizure

effects.

The following pilot study tested the effects of EPA in the maximal PTZ seizure

test, which is a pharmacological model for tonic-clonic seizures in humans (Krall et al.,

1978; Fisher, 1989). The hypothesis was that EPA would not raise seizure thresholds

when acutely administered.

10.2 Methods

Male rats, aged 53 days were individually housed and handled once daily for 6

consecutive days. On the seventh day, the subjects were seizure tested using the maximal

pentylenetetrazol (PTZ) seizure test.

On the day of the test, the subjects were first given a subcutaneous injection of

300 mg/kg of oleic acid (OA) or EPA at 100, 200, 300 and 400 mg/kg (n=9-11 / group).

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Both drugs were dissolved in 0.9% saline containing 90 mg of albumin per ml, at a

concentration of 140 μl per ml of saline-albumin.

One hour after the injections of OA or EPA, PTZ was injected intraperitoneally at

a dose of 105 mg/kg. This PTZ dose induces tonic-clonic convulsions in animals.

Subjects were then observed in an open field for 5 minutes. Latency to the onset of

myoclonic jerks and tonic-clonic seizures was determined by two independent observers.

Differences between the treatment groups were compared by one-way analysis of

variance, followed by Tukey’s post-hoc t-test. Outliers that fell beyond two standard

deviations from the mean were excluded.

10.3 Results

All subjects displayed seizures following the PTZ injections. One outlier from

each of the OA 300 mg/kg, EPA 200 mg/kg and EPA 400 mg/kg groups was excluded

from the data analysis because its latency fell beyond two standard deviations from the

mean.

Figures 1-A and 1-B show mean latencies to the onset of myoclonic jerks and

tonic-clonic seizures, respectively. Curiously, latencies were shorter than control

latencies in the 100 mg/kg group, longer in the 200 mg/kg group and not much different

from controls in the 300 and 400 mg/kg groups.

A one-way analysis of variance indicated a significant effect of treatment on the

latencies of both myoclonic jerks and tonic-clonic seizures (P<0.05). Post-hoc

comparisons using Tukey’s test indicated that latencies to the onset of myoclonic jerks

and tonic-clonic seizures in the EPA 100 mg/kg group were significantly shorter than in

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the EPA 200 and 300 mg/kg groups, but no the 400 mg/kg group (P<0.05). The mean

latencies to myoclonic jerks and tonic-clonic seizures in the EPA 100, 200, 300 and 400

mg/kg groups, however, did not differ significantly from the OA controls. The 200, 300

and 400 mg/kg EPA groups did not differ signicantly from each other (P>0.05).

10.4 Discussion

The findings of this pilot study indicate that, compared to OA controls, EPA

appeared to lower seizure thresholds at a low dose of 100 mg/kg (non-significant), but

had no statistically significant effects at higher doses. Higher doses caused a slight

increase in seizure threshold, but this effect was much smaller than the increases

previously seen with DHA, and was not statistically significant. These observations

suggest that EPA does not raise seizure thresholds in rats, at the doses tested.

The lack of a strong effect of EPA on seizure threshold is not consistent with the

previous findings of Voskuyl et al. (1998), but is in good agreement with biochemical

evidence suggesting that EPA is rapidly oxidized upon entering the brain (Chen et al.,

2009).

The differences between the results of this study and those of Voskuyl and

colleagues may possibly be related to the seizure models used. Voskuyl et al. used a non-

validated seizure model involving cortical stimulation, whereas the present study

involved the maximal PTZ test, which is a validated pharmacological tool for screening

anticonvulsants that suppress tonic-clonic seizures in humans (Krall et al., 1978; Fisher,

1989).

The slight (but non-significant) decrease in seizure thresholds following EPA

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administration at the lower dose of 100 mg/kg is rather surprising. These effects might

conceivably be related to the actions of EPA-derived metabolites that have been reported

to form in plasma following the acute administration of EPA (Dona et al., 2008). It is not

known whether these metabolites cross the blood-brain-barrier, however, or have

neuromodulatory effects in the brain.

In conclusion, EPA does not appear to have strong anticonvulsant effects in the

PTZ seizure model.

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Figure 1-A: Effect of acute EPA administration on the latency to myoclonic jerks

0

20

40

60

80

100

120

OA 300 mg/kg EPA 100 mg/kg EPA 200 mg/kg EPA 300 mg/kg EPA 400 mg/kg

Treatment

Late

ncy

to m

yocl

onic

jerk

s (s

econ

ds)

ab

a

b

b

ab

Figure 1-B: Effect of acute EPA administration on the latency to tonic-clonic seizures

0

20

40

60

80

100

120

140

160

180

200

OA 300 mg/kg EPA 100mg/kg

EPA 200mg/kg

EPA 300mg/kg

EPA 400mg/kg

Treatment

Late

ncy

to to

nic-

clon

ic s

eizu

res

(sec

onds

)

ab

a

b

bab

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Data are mean ± SEM of n= 9-11 subjects per treatment. Bars with different letter superscripts are statistically different from each other, as determined by one-way analysis of variance (P<0.05).

10.5 References

Chen CT, Liu Z, Ouellet M, Calon F and Bazinet RP (2009) Rapid beta-oxidation of eicosapentaenoic acid in mouse brain: an in situ study. Prostaglandins Leukot Essent Fatty Acids 80:157-163.

Chen CT, Ma DW, Kim JH, Mount HT and Bazinet RP (2008) The low density lipoprotein receptor is not necessary for maintaining mouse brain polyunsaturated fatty acid concentrations. J Lipid Res 49:147-152.

Depaulis A, Snead OC, 3rd, Marescaux C and Vergnes M (1989) Suppressive effects of intranigral injection of muscimol in three models of generalized non-convulsive epilepsy induced by chemical agents. Brain Res 498:64-72.

Dona M, Fredman G, Schwab JM, Chiang N, Arita M, Goodarzi A, Cheng G, von Andrian UH and Serhan CN (2008) Resolvin E1, an EPA-derived mediator in whole blood, selectively counterregulates leukocytes and platelets. Blood 112:848-855.

Fisher RS (1989) Animal models of the epilepsies. Brain Res Brain Res Rev 14:245-278.

Igarashi M, Ma K, Chang L, Bell JM, Rapoport SI and DeMar JC, Jr. (2006) Low liver conversion rate of alpha-linolenic to docosahexaenoic acid in awake rats on a high-docosahexaenoate-containing diet. J Lipid Res 47:1812-1822.

Krall RL, Penry JK, White BG, Kupferberg HJ and Swinyard EA (1978) Antiepileptic drug development: II. Anticonvulsant drug screening. Epilepsia 19:409-428.

Lin YH and Salem N, Jr. (2005) In vivo conversion of 18- and 20-C essential fatty acids in rats using the multiple simultaneous stable isotope method. J Lipid Res 46:1962-1973.

Lin YH and Salem N, Jr. (2007) Whole body distribution of deuterated linoleic and alpha-linolenic acids and their metabolites in the rat. J Lipid Res 48:2709-2724.

Voskuyl RA, Vreugdenhil M, Kang JX and Leaf A (1998) Anticonvulsant effect of polyunsaturated fatty acids in rats, using the cortical stimulation model. Eur J Pharmacol 341:145-152.

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APPENDIX 2

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11 Appendix 2 (the following manuscript has not been submitted for publication)

Docosahexaenoic acid but not its docosanoid metabolite reduces the incidence of hippocampal sharp waves in vitro

Ameer Y. Taha1,2,5, Tariq Zahid1,3, Tina Epps1,3,5, Richard P. Bazinet4,5, W. McIntyre

Burnham2,3,5, and Liang Zhang1,3,5*

1Division of Fundamental Neurobiology, Toronto Western Research Institute, University

Health Network, Toronto, Ontario, Canada, M5T 2S8

2Departments of Pharmacology and Toxicology, 3Medicine and 4Nutritional Sciences,

Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada, M5S 1A8


Toronto, Toronto, Ontario, Canada, M5S 1A8


Dr. Liang Zhang

Room 13-311, Toronto Western Hospital

399 Bathurst St.

Toronto, ON. M5T 2S8

Canada


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11.1 Abstract

BACKGROUND: Excitatory sharp waves (SPWs) originating from the hippocampus are

considered to model the interictal “spikes” that occur in people with temporal lobe

epilepsy. Docosahexaenoic acid, an omega-3 polyunsaturated fatty acid, has been

reported to reduce neuronal excitability in vitro. The effects of DHA on hippocampal

SPWs, however, have not been reported. Also, it is not known whether DHA reduces

excitability directly, or though its neuroprotectin D1 (NPD-1) metabolite.

OBJECTIVE: To determine whether DHA or its NPD-1 metabolite suppresses SPWs in

hippocampal slices, and to compare the effects of these compounds to the effects of the

standard anticonvulsant carbamazepine.

RESULTS: Extracellular CA1 and CA3 recordings from hippocampal slices revealed that

DHA reduced the incidence of SPWs, as did carbamazepine (P<0.05), without altering

the amplitude of excitatory post-synaptic potentials (EPSPs). Oleic acid (control) and

DMSO alone (vehicle control) had no effect on SPWs. The DHA metabolite NPD-1 also

had no effect (P>0.05). An examination of extracellular recordings of inhibitory

GABAergic field potentials revealed that the effect of DHA on excitatory SPWs was not

related to an increase in inhibitory GABAergic tone. Fatty acid quantification of the

slices by gas-chromatography indicated that slices exposed to DHA had an increased

DHA to arachidonic acid ratio in phospholipid membranes of the slice.

CONCLUSION: DHA, but not its neuroprotectin D1 metabolite, reduces the incidence

of excitatory SPWs in the mouse hippocampus. This reduction in activity may explain the

anticonvulsant effects of DHA that have been observed in animal seizure models.

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11.2 Introduction

The rodent hippocampus is known to exhibit electroencephalographic (EEG)

sharp waves (SPWs) during slow wave sleep and consummatory behavior (Buzsaki,

1986; Buzsaki et al., 1989b; Buzsaki et al., 1992; Buzsaki et al., 2003; Clemens et al.,

2003; Fabo et al., 2008). These SPWs originate from the hippocampal CA3 region, and

are thought to result from the cooperative network activity of the CA3 recurrent circuitry.

SPWs are characterized by an excitatory glutaminergic drive, and are frequently observed

in patients with temporal lobe epilepsy and in related in vitro (Buzsaki et al., 1989a;

Cohen et al., 2002; He et al., 2009) and in vivo animal models (Bauer et al., 2008; Fabo et

al., 2008).

Several past studies have attempted to characterize the factors involved in SPW

generation (Behrens et al., 2005; Wu et al., 2005a). In particular, Behrens et al. (2005)

have described large-amplitude, intermittent and self-sustained in vitro SPWs (~1Hz) in

conventional rat hippocampal slices following repeated high frequency or theta burst

stimulation (Behrens et al., 2005). Wu et al (Wu et al., 2005a) have also reported that

SPWs occur following extra-afferent stimulation of the CA3 region in thick mouse

hippoampal slices, and are similar in their waveform, amplitude and intermittent

occurrence to those seen in vivo. These SPWs, in vitro, serve as a useful model for

examining the network activities of the isolated CA3 circuitry, which is often involved in

epileptic seizures in humans (Koutroumanidis et al., 2004; Fabo et al., 2008).

Docosahexaenoic acid is an omega-3 fatty that has been reported to raise seizure

threshold in rats and mice (Taha et al., 2008b), and to raise the threshold for action

potential in vitro (Xiao and Li, 1999; Young et al., 2000). DHA is thought to raise the

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depolarization threshold by inhibiting voltage-dependent sodium channels in isolated

neurons (Vreugdenhil et al., 1996). However, the actions of DHA on spontaneous SPWs,

which are thought to predict seizure predisposition in humans, remain unknown. This was

investigated in the present study.

In addition to its direct effects, DHA has metabolites that might contribute to its

actions. In the brain, DHA is converted by lipoxyganse enzymes into “docosanoid”

metabolites, such as neuroprotectin D1 (NPD-1) (Hong et al., 2003). It has been

suggested that these “docosanoid” metabolites may reduce epileptiform activity and raise

seizure threshold by suppressing neuroinflammation (Tu and Bazan, 2003). To date, no

studies have examined the influence of these “docosanoid” metabolites on brain network

activities, such as those modeled in the hippocampal slice.

The goal of the present study was to examine the effects of DHA and the DHA

metabolite, NPD-1, on in vitro SPWs, in mouse hippocampal slices. We also compared

the actions of DHA and NPD-1 to the standard anticonvulsant carbamazepine (CBZ).

Finally, fatty acid quantification of the slices by gas-chromatography was done to

determine whether slices exposed to DHA would incorportate it into their phospholipid

membranes.

Our data show that DHA was incorporated into membrane phospholipids of

whole slices, and that, like CBZ, it reduced the incidence of hippocampal SPWs. The

DHA metabolite neuroprotectin D1 had no measurable effect on SPWs.


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11.3.1 Drugs and solutions

Artificial cerebrospinal fluid (ACSF) was made with distilled, de-ionized water

and contained (mM): 3.5 KCl, 1.25 NaH2PO4, 125 NaCl, 25 NaHCO3, 10 glucose, 2

CaCl2 and 1.3 MgSO4. The pH of ACSF was 7.4 when aerated with 95% O2-5% CO2.

Docosahexaenoic acid (DHA) and oleic acid (OA) (Nu-Check Prep, Elysian, MN, USA)

were each dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 μM. The

stocks for fatty acids were prepared in a nitrogen fumehood in order to minimize their

oxidation, and were subsequently stored in a -20°C freezer until further use. NPD-1 came

dissolved in ethanol at 0.1 mM (Cayman Chemicals, Ann Arbor, Michigan

USA). Carbamazepine (CBZ; Sigma, Ontario, Canada) was dissolved in DMSO at a

concentration of 200 mM and stored at room temperature (22-23°C).

11.3.2 Procedure for obtaining thick hippocampal slices

Male C57BL/6N mice (Charles River Laboratory, Quebec, Canada; aged 21-49

days) were used in the present experiments. Animals were anesthetized with sodium

pentobarbital (70mg/Kg, intra-peritoneal injection) and intracardially perfused with cold

(4°C) artificial cerebrospinal fluid (ACSF). Subjects were then decapitated, and, after

decapitation, the brain was quickly excised, hemi-sectioned and subsequently maintained

in ice-cold, oxygenated (95% O2-5% CO2) ACSF for a few minutes before further

dissection.

The thalamus and brainstem were then removed from the excised brains. The

dentate gyrus was separated from the adjacent CA1 area under a dissecting microscope,

by using a fine glass probe along the hippocampal fissure. We have previously shown

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that separating the dentate gyrus ensures sufficient oxygenation of thick hippocampal

slices, without disrupting the connections within the CA3-CA1 regions (Wu et al., 2005a;

Wu et al., 2005b). After separating the dentate gyrus, the brain tissue was glued onto an

aluminum block, and transverse hippocampal slices were obtained in ice-cold,

oxygenated ACSF using a vibratome. The slice thickness was 600-700 µm for the ventral

hippocampus or 800-900 µm for the dorsal hippocampus. After obtaining vibratome

sections, the thick slices were stabilized for 30 minutes in a beaker that contained

warmed ACSF (at 35°C) and 2 mM kynurenic acid. The purpose of using warmed ACSF

and the non-specific glutamate receptor blocker kynurenic acid was to reduce the

possibility of dissection-related excitotoxicity, and to facilitate post-dissection recovery.

After the stabilizing period, the slices were washed with, and maintained, in the standard

ACSF at room temperature (22-23°C) for 30 minutes to 6 hours before being used for

recordings.

11.3.3 Extracellular recordings

During recordings, the slice was placed in a sub-merged chamber and perfused

with oxygenated (95% O2-5% CO2), warmed (at 35°C) ACSF (Wu et al., 2002). Under

our recording conditions, warmed (at 35°C) and humidified stream of 95% O2 and 5%

CO2 was also allowed to pass above the perfusate in order to increase local oxygen

tension. DHA, 10,17S docosanoid or CBZ were applied by adding the drug to the

perfusate at the desired concentrations.

Recording electrodes were made from thin wall glass tubes (1.5 mm OD; World

Precision Instruments, Sarasota, FL). The resistance of these electrodes was

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approximately 2MΩ after being filled with a solution containing 150 mM NaCl and 2mM

HEPES, with a pH of 7.4. Extracellular signals were recorded using a dual channel

amplifier (700B) and analogue-digital converter (Digidata 1300, Axons/Molecular

Devices, CA, US). Data acquisition, storage and analyses were done using Pclamp

software (version 9, Axon/Molecular Devices). To evoke synaptic field potentials, a

bipolar electrode made of tungsten wire (50 μm diameter) was positioned at the CA3

stratum oriens region. Paired constant-current pulses at intervals of 350 – 400 ms and

maximal intensities of 100-150 μA (duration of 0.1 ms) were generated by a Grass

stimulator (Model S88) and delivered through an isolation unit every 30 seconds, for a

period of 2.5 to 3 minutes. Each slice was evoked pre- and post- drug treatment, and after

washout. Population excitatory post-synaptic potentials (EPSPs) were measured offline.

Spontaneous rhythmic field potentials (SRFPs), which reflect inhibitory GABAergic

activity (Wu et al., 2005b), were also measured before, during and after DHA treatment,

in slices that did not exhibit SPWs spontaneously, or following stimulation (at 80 Hz with

maximal intensities of 100-150 uA, and duration of 0.1 ms).

11.3.4 DHA composition of slices

In order to determine whether DHA would be incorporated into the phospholipid

membrane of whole brain slices, transverse sections were incubated in oxygenated ACSF

containing either DMSO (100 ul per 100 ml) or DHA (100 ul per 100 ml) for a period of

10 minutes. The slices were then immediately frozen on dry ice and subsequently stored

at -80 degrees °C until further analysis.

Fatty acid composition within total phospholipids was determined in each slice as

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previously described (Taha et al., under review). In brief, total lipids were extracted using

chloroform / methanol (10 ml, 2:1 v/v) and 0.9% potassium chloride over 48 hours.

Phospholipids were then separated from the total lipid extract using thin layer

chromatography. The phospholipid bands were scraped and methylated with 14%

methanolic boron triflouride at 100oC, and subsequently analyzed on Varian gas-

chromatography system equipped with flame ionization detector (Varian, Lake Forest,

CA, USA) and a Varian FactorFour capillary column (VF-23ms; 30 m x 0.25 mm i.d. x

0.25 μm film thickness). The samples were injected in splitless mode, with injector and

detector ports set at 250oC. FAMEs were eluted using a temperature program set initially

at 50oC for 2 min, increased at 20oC/min and held at 170oC for 1 min, then at 3oC/min

and held at 212oC for 5 min to complete the run at 28 min. The carrier gas was helium,

set to a constant flow rate of 0.7 ml/min. Peaks were identified by retention times of

FAME standards (Nu-Chek-Prep, Elysian, MN).

11.3.5 Data analyses

All data are expressed as mean ± SEM. The peak amplitudes of evoked field

population EPSPs and somatic population spikes were measured as previously described

(Wu et al., 2005a). The measurements were made from an average of 5-6 consecutive,

evoked responses. SPWs were measured using the event detection function of Pclamp

software (Clampfit, threshold method). Detected SPW events were visually inspected and

false events were rejected. In each slice, the inter-event intervals of spontaneous SPWs

were calculated from ≥30 SPW events before or during baseline recordings or at the end

of drug application. For group comparisons, the incidence of SPWs and inter-event

292

interval between the SPWs were normalized as % of the baseline control in individual

slices. A two-way analysis of variance (ANOVA) was used to determine the effect of

drug treatment and time on the incidence of SPWs. If treatment was a significant factor, a

one-way ANOVA followed by Tukey’s post-hoc test was used to compare differences in

the means during and after drug treatment. Differences in EPSPs were also assessed with

a one-way ANOVA. A P < 0.05 was considered statistically significant.

11.4 Results

11.4.1 DHA and CBZ, but not NPD-1, reduced the incidence of hippocampal SPWs

Extracellular recordings from mouse hippocampus were performed before, during

and after the application of DHA, CBZ or NPD-1 to the slice. The data related to

incidence of SPWs during the treatment and post-treatment (washout) periods, expressed

as a percentage of baseline, are presented in Figures 1-A (DHA), 1-B (CBZ) and 1-C

(NPD-1), respectively. A two-way repeated measures analyses of variance revealed a

significant effect of treatment and time on the incidence of SPWs for DHA (P<0.05) and

CBZ (P<0.05), but not for NPD-1 (P>0.05).

As shown in Figure 1-A, 100 μM DHA reduced the incidence of SPWs during the

10 minute treatment period, but not during the washout period (P=0.1). The differences

between DHA at 100 μM and the OA and DMSO controls were statistically significant at

the P<0.05 level. At 50 μM, a “trend” toward reduction was seen but the mean SPW

occurrence did not differ significantly from the OA and DMSO controls. A representation

of the effects of DHA on CA3 SPWs is depicted in Figure 1-D.

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As shown in Figure 1-B, CBZ also reduced the incidence of SPWs during the

treatment period. Tukey’s post-hoc comparisons revealed a significant reduction of

SPWs at both 50 uM and 100 uM as compared to the DMSO controls (P<0.05). A

significant effect of 100 uM CBZ was also seen during the washout period (P<0.05).

Figure 1-C shows the changes in SPW incidence following NPD-1 treatment. As

indicated, NPD-1 did not significantly alter the incidence of SPWs during or after

treatment (P>0.05).

11.4.2 DHA does not alter the incidence of inhibitory, spontaneous rhythmic filed

potentials (SRFPs)

SRFPs represent population activity thought to be mediated by the inhibitory

GABAergic system (Wu et al., 2006). Since DHA reduced the incidence of excitatory

SPWs (Figure 1-A), we conducted a separate experiment in hippocampal slices to

determine whether the reduction in SPW incidence was related to an increase in the

incidence of inhibitory SRFPs.

The data related to the effects of DHA on SRFPs are presented in Figure 2. As

shown, application of the DHA at a dose that reduced the incidence of SPWs (100 μM )

did not significantly alter the incidence of SRFPs. (P>0.05).

11.4.3 Effect of DHA and CBZ on population field EPSPs

The amplitude of the population field EPSP and the paired pulse depression,

expressed as a change from baseline, was determined in DMSO, OA, DHA and CBZ–

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treated slices, at a dose of 100 μM. Population EPSP amplitudes did not differ

significantly amongst the groups (0.03 ± 0.1, 0.1 ± 0.1, 0.5 ± 0.5, 0.1 ± 0.1 for DMSO,

OA, DHA and CBZ, respectively; n=3-4 per treatment), as determined by one-way

analysis of variance (P>0.05).

11.4.4 DHA is incorporated into the phospholipid membrane

DHA competes with AA for incorporation into membrane phospholipids. A

greater DHA to AA ratio, therefore, is reflective of greater incorporation of DHA into the

phospholipid pool (Tocher and Dick, 2001).

The data for the DHA/AA ratio in slices that were incubated in DMSO or 100 uM

DHA for 10 minutes are presented in Figure 3. As shown, the ratio of DHA to AA was

significantly higher in DHA-incubated slices, as compared to DMSO-incubated slices

(P<0.05).

11.5 Discussion

To our knowledge, this is the first study to assess the effect of DHA and its main

metabolite, NPD-1, on SPWs in vitro. Our results suggest that DHA reduces the

incidence of excitatory SPWs in a similar manner to CBZ, and that it does so without

altering the incidence of inhibitory SRFPs. The effect of DHA on SPWs is not likely to

be mediated by its NPD-1 metabolite – which proved to be inactive - but may be related

to its incorporation into the slice.

The observed reduction in SPWs does not appear to be related to changes in the

population EPSP amplitude. The population EPSP is essentially an evoked potential,

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elicited by stimulation of the CA3 region, related to glutamate-mediated activation of

AMPA and NMDA receptors (Wu et al., 2005a). A change in the EPSP amplitude is

suggestive of changes in neural conduction, or, possibly, the number of neurons

generating action potentials. The lack of effect of DHA on EPSPs suggests that the

observed decrease in SPW incidence during DHA treatment is not related to changes in

the conduction of spaced neuronal impulses, or changes in glutamatergic receptor

function. It is still possible that there would be changes in neural conduction if impulses

were delivered at higher frequencies, as is the case during population activity comprising

SPWs. This was not tested in the present experiment.

Sogaard et al. have reported that DHA, at concentrations of 1 to 100 μM,

increases the binding capacity of tritiated muscimol to the GABAA receptors, by altering

the membrane characteristics of neuronal cells (Sogaard et al., 2006). SRFPs were

measured, therefore, in separate slices in order to assess whether the decrease in

excitatory SPW incidence was related to an increase in inhibitory GABAergic activity

during DHA treatment. Our findings suggest that the decrease in SPW incidence is not

related to changes in inhibitory SRFPs. Our findings do not refute the possibility that

DHA may have potentially increased the binding capacity of GABA to GABAA receptors

(Sogaard et al., 2006). The lack of change in SRFPs, however, suggests that the possible

changes in GABA binding were not sufficient to alter SRFPs in the present experiment.

The effect of DHA on SPW incidence appeared to mimic that of CBZ. Similar to

DHA, CBZ treatment did not alter the population EPSP or paired pulse depression,

suggesting that it is unlikely to raise seizure threshold by acting on the glutamatergic

system or the GABAergic inhibitory system.

296

Both DHA and CBZ are thought to raise seizure thresholds by acting on voltage-

dependent ion channels (Poling et al., 1996; Vreugdenhil et al., 1996; Xiao and Li, 1999;

Lauritzen et al., 2000; Young et al., 2000; Seebungkert and Lynch, 2002; Danthi et al.,

2005; Borjesson et al., 2008). The observed reduction in the SPW incidence caused by

DHA and CBZ suggests that both compounds reduced the excitability properties of the

slice, an effect that may involve voltage-dependent ion channels. Further patch clamp

studies will be required, however, to determine whether the actions of DHA and CBZ on

the incidence of SPWs is directly related to an effect on voltage-gated ion channels.

The concentrations at which DHA reduced the incidence of SPWs in vitro are

likely to be within the physiological range. The concentration of DHA in the plasma

unesterified fatty acid pool - which contains most of the DHA in the brain, ranges

between 2 to 37 μM, depending on the DHA composition of the diet and the duration of

DHA supplementation (Bazinet et al., 2005b; Taha et al., 2005; Bazinet et al., 2006). The

in vitro concentration of DHA which appeared to reduce the incidence of SPWs was

between 50-100 μM. Although our in vitro concentrations were slightly higher than

previously reported plasma unesterified DHA concentrations, it may be possible to

achieve plasma levels this high with chronic dietary DHA supplementation at high doses.

This requires confirmation in future studies.

The DHA to AA ratio was measured in slices in order to determine the relative

incorporation of DHA into the slice. Our results indicate that the DHA to AA ratio

increased following DHA (dissolved in DMSO) incubation, relative to slices that were

incubated with DMSO alone, suggesting that DHA was incorporated into the slice. It was

not possible to obtain quantitative (mg DHA per g of brain slice) estimates of DHA

297

incorporation into the slice, since it was difficult to accurately measure the weight of the

wet slice following incubation with ACSF containing DHA. The DHA to AA ratio was

therefore used as a surrogate estimate, since DHA displaces AA when incorporated into

the membrane (Tocher and Dick, 2001).

NPD-1 is one of the DHA metabolites that plays a role in antagonizing

neuroinflammation (Marcheselli et al., 2003; Bazan, 2007). It has been suggested that

neuroinflammation lowers seizure threshold (Akarsu et al., 2006). Drugs that block the

cox-1 or cox-2 proinflmmatory pathways, or stop leukocyte infiltration, have been

reported to raise seizure threshold (Tu and Bazan, 2003; Dhir et al., 2006b; Dhir et al.,

2006a; Fabene et al., 2008; Oliveira et al., 2008). Although the NPD-1 doses ranged from

physiological to pharmacological doses, we found no significant effect of NPD-1

administration on the incidence of SPWs. This could be due to a true lack of effect of

NPD-1 on SPWs, or because SPWs do not induce significant neuroinflammation.

In summary, the findings of the present study suggest that DHA reduced the

incidence of excitatory SPWs. This effect appears to be unrelated a reduction in the

glutamatergic drive or an increase in inhibitory GABAergic response, and is not related

to the DHA metabolite NPD-1.

ACKNOWLEDGEMENTS

This study was funded by the Canadian Institutes of Health Research (CIHR). A.Y.T is a

recipient of the Canada Graduate Scholarships CIHR doctoral research award.

298

Figure 1-A: Effect of DHA on the incidence of SPWs

-50

-40

-30

-20

-10

0

10

20

30

40%

Cha

nge

from

bas

elin

eDMSOOA 100 uMDHA 50 uMDHA 100 uM

Treatment

DMSOOA 100uM

DHA 50uM

DHA 100uM

Washout

DMSO OA 100uM

DHA 50uM

DHA 100uM

a

a

ab

b

299

Figure 1-B: Effect of CBZ on the incidence of SPWs

-100

-80

-60

-40

-20

0

20

40

60%

Cha

nge

from

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eDMSOCBZ 50 uMCBZ 100 uM

a

b

b

a a

b

WashoutTreatment

300

Figure 1-C: Effect of NPD-1 on the incidence of SPWs

-30

-25

-20

-15

-10

-5

0

5

10

15

20%

Cha

nge

from

bas

elin

eEthanol NPD1 20μMNPD1 40μM

Ethanol EthanolNPD-1 20nM

NPD-1 20nM

NPD-1 40nM

NPD-1 40nM

Treatment Washout

Figure 1-D: Representation of CA3 SPWs from a slice before, during and after 100 μM of DHA treatment

301

Effect of DHA, CBZ and NPD-1 treatment on the incidence of SPWs, expressed as a

percent change from baseline

Figure 1-A: The effects of 100μl DMSO, 100 μM OA, 50 μM DHA and 100 μM DHA

on the incidence of SPWs, expressed as a percent change from baseline. Data are mean ±

SEM of = n=4-7 per treatment. Two-way repeated measures analysis of variance revealed

a significant effect of treatment and time on the incidence of SPWs (P<0.05). A one way

analysis of variance was then used to compare the means during the drug treatment

period and the washout period. Different superscripts denote significant differences

between the means. The means differed significantly during the treatment period

(P<0.05), but not the washout period (P>0.05). Tukey’s post-hoc comparison of the

means during the drug treatment period showed that the differences between DHA at 100

μM and the OA and DMSO controls were statistically significant at the P<0.05 level.

The differences between DHA at 50 μM and OA, DMSO and DHA at 100 μM did not

differ significantly.

Figure 1-B: The effects of 100μl DMSO, 50 μM CBZ and 100 μM CBZ on the incidence

of SPWs, expressed as a percent change from baseline. Data are mean ± SEM of = n=4-7

per treatment. Two-way repeated measures analysis of variance revealed a significant

effect of treatment and time on the incidence of SPWs (P<0.05). A one way analysis of

variance was then used to compare the means during the drug treatment period and the

washout period. Different superscripts denote significant differences between the means.

The means differed significantly during the treatment and washout period (P<0.05).

Tukey’s post-hoc comparison of the means during the drug treatment period showed that

the differences between CBZ at 50 and 100 μM and DMSO, were statistically significant

302

at the P<0.05 level. During the washout period, CBZ at 100 μM differed significantly

from DMSO and CBZ at 50 μM; CBZ at 50 μM did not differ significantly from DMSO.

Figure 1-C: The effects of 40 μl ethanol, 20 μM NPD-1 and 40 μM NPD-1 on the

incidence of SPWs, expressed as a percent change from baseline. Data are mean ± SEM

of = n=2-4 per treatment. Two-way repeated measures analysis of variance revealed no

significant effects of treatment or time on the incidence of SPWs (P>0.05).

Figure 1-D: Representation of CA3 SPWs from a slice before, during and after 100 μM

of DHA treatment, based on extracellular recordings.

303

Figure 2: Effect of DHA on the incidence of SRFPs

0

1

2

3

Baseline DHA 100uM Washout

SRFP

inci

denc

e

Effect of 100 μM DHA treatment on the incidence of SRFPs. Data are mean ± SEM of

n=4. One way repeated measures analysis of variance revealed no significant differences

amongst the means at pre, during and post 100 μM of DHA treatment (P>0.05).

304

Figure 3: Effect of DMSO and DHA on the ratio of DHA to AA in phospholipids isolated from brain slices

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

DMSO 100 μM DHA

Treatment

DH

A/A

A ra

tio

*

Effect of DMSO or 100 μM DHA on the ratio of DHA to AA in membrane phospholipids

of brain slices. Data are mean ± SEM of n=4-7 per group. *P<0.05 by unpaired student’s

t-test.

305

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Anticonvulsant effects of omega-3 polyunsaturated fatty acids in rodents By Ameer … · 2013-09-27 · Ameer Y. Taha Department of Pharmacology and Toxicology University of Toronto