Focal ischemic reperfusion stroke model in rats and the …liu.diva-portal.org/smash/get/diva2:416160/FULLTEXT01.… · · 2011-05-10Focal ischemic reperfusion stroke model in rats

Focal ischemic reperfusion stroke model in rats and the role of galanin

Linköping University Medical DissertationsNo. 1242

Lovisa Holm

Linköping University, Faculty of Health Sciences, Department of Clinical and Experimental Medicine,

Clinical Chemistry

2011

The cover illustration depicts the overall structure of the rat brain

Published articles have been reprinted with permission of the copyright holders.

Linköping University Medical Dissertations: No. 1242ISBN: 978-91-7393-185-4ISSN: 0345-0082Copyright© 2011, Lovisa Holm

Printed in Sweden by LiU-tryck, Linköping, Sweden, 2011

To my beloved family, Peter, Mattias and Daniel

To the memory of my father Lennart Petersson

5

Abstract

AbstractStroke is the third most common cause for mortality in industrialised countries and amongst the major causes of long- time morbidity. While the mortality due to myocardial infarction has been dramatically reduced during the last 10-15 years, mortality due to stroke remains almost the same, despite the fact that the two share similar basic pathogenic mechanisms including atherosclero-sis, hypertension and diabetes. Treatment modalities of reperfusion therapy for acute ischemic stroke, including the use of tissue plasminogen activator for thrombolysis and endovascular treatments, are eff ective if applied early after onset of the irst symptoms. The more frequent use of reperfusion therapy, es-pecially in the most common type of stroke aff ecting the middle cerebral artery (MCA), increase the clinical relevance and demand for experimental models of temporary and focal ischemia of the brain. The primary goal of the present work was to develop a model in rats for studying the mechanisms underlying focal and temporary ischemia in brain regions supplied by the MCA.

We have modi ied the intracranial method of occluding the MCA originally de-scribed by Tamura et al. in the early 1980es by introducing a microclip to oc-clude the artery and induce reperfusion under direct visual control through an operating microscope. The goal was to create a mild ischemia model with low morbidity and mortality, optimizing conditions for the animals postoperatively and allowing long-term (weeks) observation periods of high relevance for hu-man stroke. Morbidity and mortality in experimental stroke models are crucial confounders. Change of anesthesia from intraperitoneally administrated chlo-ral hydrate to iso lurane inhalation anesthesia with endotracheal intubation and controlled ventilation reduced mortality markedly from 25% to ~10%. Improved overall skills in anesthesia and surgical techniques further reduced mortality to <3%.

Hypothermia reduces brain lesions caused by ischemia not only when admi-nistered before and during the ischemic episode, but also afterwards. Several studies have shown that galanin concentrations are increased in response to various types of lesions to the nervous system, and galanin may be amongst the factors supporting neuronal survival and functions. We therefore investiga-ted whether or not hypothermia-induced alterations in galanin concentrations could constitute a part of the established neuroprotective eff ect of hypothermia in our rat stroke model. Hypothermia induced an overall increase in the con-centrations of immunoreactive galanin (p < 0.001). The elevated galanin levels were predominantly found in the non-ischemic control hemisphere. The gala-nin concentrations were lower in the ischemic hemisphere in both the normo- and hypothermic animals compared to the corresponding contralateral intact hemisphere (p = 0.049). The hypothermia and not the ischemic/reperfusion lesions explained the major part of the observed changes in galanin concen-trations. Hypothermia-induced elevation in galanin concentration is therefore

Abstract

6

not likely to be amongst the major protective mechanisms of hypothermia. Our results support the notion that hypothermia-induced increase in tissue con-centrations of galanin in the brain are the result of changes from optimal ho-meostatic conditions – the hypothermia-induced stress – rather than the ische-mic/reperfusion lesion- induced changes in galanin concentrations.

Whether the lesion-induced increase in galanin concentrations is primarily a signal that a lesion has occurred, a consequence of the lesion or a mechanism for facilitating neuronal survival is an open question. We therefore infused th-ree diff erent concentrations of galanin intracerebroventricularly in a direct at-tempt to investigate whether or not galanin has neuroprotective properties in a rat model of MCA occlusion. Furthermore, we infused the GalR2/3 agonist Gal(2-11) (AR-M1896) shown to subserve neuroprotective functions. The le-sion was 98% larger seven days after a 60 min transient MCA occlusion and continuous administration of the GalR2/3 agonist Gal(2-11). No diff erences were found after seven days in the groups treated with galanin in three dif-ferent concentrations (0.24, 2.4 and 24 nmol/day; p = 0.939, 0.715 and 0.977, respectively). There was also no diff erence in the size of the ischemic lesion measured after three days in the galanin-treated group (2.4 nmol/d) compared to artificial cerebrospinal luid (p = 0.925).

The expression of the galanin, GalR1, GalR2 and GalR3 receptor genes were investigated in the female rat brain seven days after a 60 min unilateral occlusi-on/reperfusion of the MCA. Galanin gene expression showed a 2.5-fold increase and GalR1 a 1.5-fold increase in the locus coeruleus of the ischemic hemisphere compared to the control side, and the GalR1 mRNA levels decreased by 35% in the cortex of the ischemic hemisphere. Thus, stroke-induced forebrain lesion upregulates synthesis of galanin and GalR1 in the locus coeruleus, a noradren-ergic cell group projecting to many forebrain areas, including cortex and the hippocampal formation, supporting the notion that galanin may play a role in the response of the central nervous system to injury and have trophic eff ects.

7

List of the papers

List of the papers

Ι. Theodorsson A, Holm L, Theodorsson E. Modern anesthesia and peropera-tive monitoring methods reduce per- and postoperative mortality during transient occlusion of the middle cerebral artery in rats. Brain Research Protocols 2005;14(3):181-90.

ΙΙ. Theodorsson A, Holm L, Theodorsson E. Hypothermia-induced increase in galanin concentrations and ischemic neuroprotection in the rat brain. Neuropeptides 2008; 42(1):79-87.

ΙΙΙ. Holm L, Theodorsson E, Hökfelt T, Theodorsson A. Eff ects of intracerebro-ventricular galanin or a galanin receptor 2/3 agonist on the lesion induced by transient occlusion of the middle cerebral artery in female rats. Neuro-peptides 2011; 45(1):17-23.

ΙV. Holm L, Hilke S, Theodorsson E, Hökfelt T, Theodorsson A. Changes in ga-lanin and GalR1 gene expression in discrete brain regions after transient occlusion of the middle cerebral artery in female rats. Manuscript.

8

Contents

9

ContentsAbstract 5

List of the papers 7

Abbreviations 11

Introduction 13Stroke 13Models of focal cerebral ischemia 21Neuropeptides 31Galanin 33

Aims of the thesis 41

Material and methods 43Animals and surgery 43Brain biopsies 50Measuring the size of the ischemic brain lesions 51Galanin measurements 53Statistical methods 54

Results and discussion 55Stroke model – methodological aspects (paper I) 55Anesthesia model – methodological aspects (paper I) 57General methodological aspects (paper I) 59Eff ects of hypothermia on the ischemic brain lesion and on tissue concentrations of galanin in the stroke model (paper II) 61Eff ects of intracerebroventricular galanin and galanin receptor 2/3 agonist on the ischemic brain lesion (paper III) 63The eff ects of ischemic injury on galanin and galanin receptor gene expression in discrete brain regions (paper IV) 65

Conclusions 67

Acknowledgements 69

References 71

Paper I 93Modern anesthesia and peroperative monitoring methods reduce per- and postoperative mortality during transient occlusion of the middle cerebral artery in rats

Contents

10

Paper II 105Hypothermia-induced increase in galanin concentrations and ischemic neuroprotection in the rat brain

Paper III 117Eff ects of intracerebroventricular galanin or a galanin receptor 2/3 agonist on the lesion induced by transient occlusion of the middle cerebral artery in female rats

Paper IV 127Changes in galanin and GalR1 gene expression in discrete brain regions after transient occlusion of the middle cerebral artery in female rats

11

Abbreviations

Abbreviations

CCA Common carotid artery

CNS Central nervous system

DRG Dorsal root ganglion

CSF Cerebrospinal luid

ECA External carotid artery

Gal Galanin

GALP Galanin-like peptide

GalR1, -R2, -R3 Galanin receptors 1,2,3

Gi G-protein (inhibitory)

HiFo Hippocampal formation

ICA Internal carotid artery

ICH Intracerebral haemorrhage

LC Locus coeruleus

LDCV Large dense core vesicle

MCA Middle cerebral artery

MCAo Middle cerebral artery occlusion

NO Nitric oxide

RT-PCR Reverse Transcription Polymerase Chain Reaction

SAH Subarachnoid haemorrhage

SD Spraque Dawley

SHR Spontaneously Hypertensive Rats

TTC 2,3,5- Triphenyltetrazolium hydrochloride

12

13

Introduction

Introduction

Stroke is the third leading cause of death in Sweden (Socialstyrelsen 2010) as in other industrialised countries (Goetz et al. 1999; Gorelick et al. 1999; Bradley 2008). It is currently the somatic disease category resulting in

the largest number of patient days spent in hospital and a major cause of long-lasting disability in the workplace, since about a ifth of the cases occur before retirement.

According to the World Health Organization (WHO) stroke is “the rapidly de-veloping loss of brain function(s) due to disturbance in the blood supply to the brain” (WHO 1978). The time dimension was later included in the de inition “ra-pidly developed clinical signs of focal or global disturbance of cerebral function, lasting more than 24 hours or until death, with no apparent non-vascular cause” (WHO 1988).

Stroke is caused by ischemia resulting from obstruction to blood low to the brain by thrombosis or arterial embolism, or by haemorrhage. The brain is amongst the tissues of the body most vulnerable to ischemia due to its high oxygen de-mand, partly needed for nerve impulse propagation and chemical neurotrans-mission. The human brain represents only 2% of the total body weight, but uses 15% of the cardiac output, 25% of the total oxygen consumption of the body at rest, 75 L of molecular oxygen and 120g glucose daily (Bradley 2008).

Since brain damage is an important cause of mortality and morbidity in society, more reliable therapeutic options are needed, that can minimize the neuronal damage caused by cerebrovascular diseases and traumatic brain injury.

StrokeTypes of strokeStroke consists of two pathological subtypes: ischemic and haemorrhagic (Figu-re 1) (Bradley 2008). Ischemic stroke constitutes 80% of the cases and is caused either by a local blood clot (thrombus) which blocks blood low in an artery or by a wandering clot or some other particle (an embolus) which forms away from the brain, usually from the heart or from the bifurcation of the carotid artery and is transported to the brain by the blood. Haemorrhagic stroke consists of intrace-rebral haemorrhage (ICH) or subarachnoid haemorrhage (SAH). ICH are caused either by a defective artery in the brain parenchyma which bursts, looding the surrounding tissue with blood or by a weakness in the wall of a medium sized artery (aneurysm) which bursts, sending blood to the subarachnoidal room co-vering the brain. Several of the patients suff ering from haemorrhagic strokes die before reaching a hospital due to increased intracranial pressure which in itself causes brain ischemia due to the external pressure exerted mainly on minor ves-sels. However, survivors of haemorrhagic stroke usually enjoy a more favourable recovery than the suff erers of ischemic stroke. The reason is that when a blood vessel is blocked from within, a part of the brain dies and is not regenerated. In

14

Introduction

haemorrhagic stroke the pressure and the brain dysfunctions it causes are reli-eved as time passes and many of the lost brain functions are thereby regained.

Cerebrovascular diseases

Ischemic stroke 80% Haemorrhagic stroke 20%

Focal/multifocal DiffuseFocal/Parenchymatous

HypertensionAmyloidosisArterio-venous malformations

Diffuse/Subarachno-idal (10%)

Arterial Venous Cardiacarrest

Hypoxia/hypoperfusion

Figure 1 Types of stroke

Vital neurosurgical procedures e.g. as a result of vascular events, tumours or ac-cidents sometimes necessitate temporary occlusion of the arterial blood low to parts of the brain risking ischemic brain damage.

Risk factors for strokeAtherosclerosis is the main risk factor in ischemic stroke, and its risk factors are thus shared with all other disease states caused by atherosclerosis including myocardial infarction (Gorelick et al. 1999). Several risk factors for stroke have been identi ied including hypertension, atrial ibrillation, myocardial infarction, diabetes mellitus, smoking, elevated blood lipids and asymptomatic carotid ar-tery disease. Bradley (2008) classi ies the risk factors according to whether they are modi iable or not (Table I):

Table INonmodifi able and modifi able risk factors for stroke (Bradley 2008)

Nonmodifi able Modifi ableAge Arterial hypertensionGender Transient ischemic attacksRace/ethnicity Prior strokeFamily history Asymptomatic carotid bruit/stenosisGenetics Cardiac disease

Aortic arch atheromatosisDiabetes mellitusDyslipidemiaCigarette smokingAlcohol consumption

15

Introduction

Nonmodifi able Modifi ableIncreased ibrinogenElevated homocystineLow serum folateElevated anticardiolipin antibodiesOral contraceptive useObesity

Successful modi ication of the risk factors for stroke has substantial impact on the risk of stroke. Lowering diastolic blood pressure by as little as 5 mmHg can reduce stroke risk by 42% (Gorelick et al. 1999). At least 25% of adults suff er from hypertension de ined as diastolic blood pressure of more than 90 mmHg or systolic blood pressure of 140 mmHg or more (Bradley 2008). This excel-lent opportunity for prevention is unfortunately as yet not fully and properly exploited even in countries such as Sweden with health care systems that should have suf icient resources to cope with the problem. Statin treatment of hyperlipi-daemia and vascular in lammation is also very eff ective in preventing stroke, as exempli ied by the study of the Scandinavian Simvastatin Survival Study Group which showed a 28% reduction in fatal or nonfatal stroke and transient ischemic attacks (Pedersen et al. 1998).

Stroke symptomsDue to the multitude and complexity of brain functions and the many locations in the brain aff ected by stroke, it causes a wide range of symptoms, each cor-responding to the aff ected location.

The most common symptom is sudden weakness or paralysis of the face, arm or leg, most often aff ecting one side of the body. Other symptoms are confusion, trouble speaking or understanding, dif iculty seeing with one or both eyes, dif-iculty in walking, and loss of balance or coordination (Bradley 2008). A stroke

survivor is frequently also prone to emotional instability and sudden moods swings, even after long periods of time.

Treatment of strokeMost stroke survivors are left with lifelong disability. With the exception of early administered thrombolytic therapy by means of tissue-type plasminogen acti-vator (t-PA) and rare opportunities for endovascular interventions, no clinically proven, practical and causal therapy exists as yet for the management of acute ischemic stroke (Stapf et al. 2002; Benchenane et al. 2004). Since brain damage is an important cause of mortality and morbidity in society, additional reliable therapeutic options are needed. However, the development and use of multiple endovascular modalities of reperfusion therapy for acute ischemic stroke has reported promising results, i. a. intra-arterial thrombolysis and/or stent deploy-ment increase the chance of recanalization (Gupta et al. 2011).

16

Introduction

Stroke outcomesThe severity of the hypoxia-ischemia and the ability of the brain including its collateral circulation determines the extent of the lesions and its neurological consequences as shown in Figure 2.

Hypoxia-ischemia

Mild Moderate Severe

Necrosis

Homeostaticmachinery is

damaged

Adaptive homeostasis

Homeostaticmachinery fully

engaged

Tissue survivesInsult intact andfunctional with

blood flow restored

Tissue survival Cell survival Apoptosis

↓ Energy demand↓ Protein synthesis↓ Protein degradation↓ Membrane potential↓ Neuronal firing rate

↑ Gene expressionRestoration of cellularATP/glucosemetabolism

Irraparable damageto cell constituentsdue to initial insult orprolonged ischemia

Dysfunctional neurons

Figure 2Possible outcomes when the brain is affected by ischemic lesions. The fate of brain tissues is partially determined by the severity of the initial insult. Mild or short ischemic conditions engage compensatory mechanisms in the cells including the activation or inhibition of pre-existing proteins and new gene ex-pression. The possibility of viable tissue is thereby improved. When the ischemia is moderate adaptive homeostasis is again engaged succeeding only partially (e.g. in the penumbra part of the ischemic tissues). Apoptosis occurs in neurons that sustained irreparable damage at the initial insult thereby removing nonfunctional neurons. Severe ischemia occurs in the center of the infarction resulting in necrosis Adapted from (Fisher et al. 2003).

The brain has homeostatic mechanisms able to deal with mild ischemic attacks. Larger ischemic challenges result in a mixture of damaged and surviving cells, whereas severe ischemia overwhelms the homeostatic defences and causes cell death in smaller or larger parts of the brain.

Types of cerebral ischemiaIschemia of the brain occurs in several varieties (Goetz 2003; Bradley 2008)(Fi-gure 3). Global ischemia reduces blood low to the entire brain. It occurs in car-diac arrest, severe hypotension, or occasionally during surgical procedures that alter blood low. Focal ischemia aff ects circumscribed part(s) of the brain e.g.

17

Introduction

the internal capsule, caudoputamen or the cortex commonly from occlusion of the middle cerebral artery (MCAo). Focal ischemia occurs in response to trans-ient or permanent MCAo. The degree of brain damage in response to ischemia depends on duration of occlusion, site along the MCAo, and amount of collate-ral blood low into the middle cerebral artery (MCA) territory. Characteristic of focal ischemia is an ischemic core, where cell death is most extensive – or com-plete surrounded by a penumbra zone, of partially damaged but still surviving brain cells with an undecided long-time fate. The ischemic core is surrounded by the penumbra zone where cells suff er from the consequences of hypoxia (Astrup et al. 1981), but where the inal fate of the cells is not yet decided (Arvidsson et al. 2002). Extensive ischemia in the brain causes cell death within minutes. Permanent ischemia is caused e.g. by an embolus occluding the MCA for exten-ded periods of time (several hours), suf iciently to cause cell death. Temporary ischemia is e.g. caused when an embolus is dissolved by ibrinolytic therapy, re-moved using endovascular technique or when a vessel is occluded during neu-rosurgical procedures.

Types of cerebral ischemia

Global Focal

2-vesselocclusion

4-vesselocclusion

Circulatoryarrest

Crani-ectomy

NoCraniectomy

TranscientSurgical clipreperfusionIn situthromboembolic/lysis

PermanentSurgicalcauterizationIn situthromboembolic

TranscientMCAo/reperfusionThrombo-embolic/lysisEndothelin-1 induced

PermanentMCAoThrombo-embolicNon-clotembolicPhoto-thrombosis

Figure 3Types of experimental models of cerebral ischemia. Several types of global and focal ischemia are illustrated.

Experimental stroke modelsThe increased use of thrombolytic therapy for treating patients suff ering from cerebral ischemia and temporary ischemia during neurosurgical procedures in-creases the clinical relevance and demand for experimental models of tempora-ry and focal ischemia of the brain. Previous experimental paradigms described in the recent literature consist mainly of permanent MCAo. The primary goal of the present work was to develop a model in rats for studying focal and tempora-ry ischemia, since this is a state of considerable clinical importance. Temporary MCAo was chosen, since it is the vessel most commonly aff ected in human stroke (Goetz et al. 1999).

18

Introduction

Ten years ago when we started developing the present method we experienced rat mortality of 25% when performing temporary clipping of the MCA in the rat. This – in our opinion – high mortality prompted us to abandon the intraperito-neal anesthesia by chloral hydrate and ventilation by tracheotomy in favour of intubation, and iso lurane anesthesia (1% iso lurane in 30%/70% O2/N2O) in order to favour better survival rates and recovery after surgery.

We have worked strenuously to develop and perfect a model for temporary MCAo by micro clip applied on the MCA exposed by craniotomy. This paradigm caters for a visually controlled application of a micro clip and visual control that the blood- low in the MCA is inhibited. The method results in an atraumatic pressure on the artery with minimal risk for post occlusion thrombosis. Our ex-perimental paradigm thus caters for clear-cut but limited damage, allowing the rat to feed and thrive well for days to weeks in order for the long-time eff ects of the temporary ischemia to be studied. Even if the surgical methods and skills as such are important for the success of this kind of experiments, we have found the anesthetic procedures to be just as important.

Most experimental paradigms of stroke in experimental animals cater for obser-vations done during a short period of only 1-3 days. To study perhaps more re-levant observation of the end of outcome of brain ischemia, we set out to design a model of a mild reperfusion ischemic damage to the rat brain compatible with survival for days and weeks.

Many details in the basic mechanisms causing cell death in brain ischemia/stro-ke are preferably studied in individual cells in culture. However, the brain is the most extensively integrated and communicative organ in the body, and a com-prehensive assessment of its integrated functions can therefore only be studied in intact organisms/animals. Human brain tissues are – for natural reasons – not available for this type of studies except in cases, where noninvasive imaging techniques can be used and when microdialysis probes can be inserted in condi-tions, when that type of monitoring is deemed bene icial for the patient.

It is therefore crucial to have ready access to experimental animal models mi-micking human stroke in order to investigate stroke mechanisms and discover new treatment options. Animal models of stroke have been established in seve-ral species including mice, rats, cats, dogs, rabbits, monkeys (Sundt et al. 1966; Hudgins et al. 1970; Suzuki et al. 1980; Lyden et al. 1987). Rodents, in particular rats and mice, are the most commonly used species. Small animals are easy to maintain, entail comparatively low costs for storage and feeding, and have pro-ven less controversial from an ethical point of view than higher animals inclu-ding primates. The anatomy of the arterial and nerve supply to the rat cerebral hemispheres is similar to that of humans, and several aspects of the biochemical and molecular mechanisms of injury are also similar (Yamori et al. 1976). Ho-wever, it is crucial to realize that the brain of rats and mice have diff erent details in their anatomy and physiology compared to humans, and it should not be ex-pected that all mechanisms and therapeutic opportunities discovered in rodents

19

Introduction

automatically will also be useful in humans. Rats have very little white matter compared to humans, and their grey matter is not gyrated (Hoyte et al. 2004). It should also be realized that the highest cognitive brain functions in humans are absent from animals and may be aff ected by drugs proven neuroprotective in animals without cognitive side-eff ects in the animals.

In contrast to some other rodents (e. g. gerbils), rats and mice have a complete circle of Willis (Dirnagl et al. 1999). Rats have more eff ective collaterals between large cerebral vessels than humans, and suff er severe ischemia when proximal occlusion models are used (Maeda et al. 2000). In the common Spraque-Dawley (SD) rats, the wild type strain shows most consistent infarct, compared to, for example, the spontaneously hypertensive rats (SHR) which develop large and more variable-sized infarcts (Ginsberg 2003).

Since stroke is a disease of the highly integrated and complex brain, treatment options found valuable in cell cultures may not work when tested in intact ani-mals or in humans for that matter. Treatment modalities found valuable in ani-mal models of stroke may also not work in humans.

Several variables that may aff ect the experimental outcome need to be control-led in experimental animal models. Male animals are commonly used instead of females to avoid the eff ects of varying concentrations of female sex hormones during the ovarial cycle. Young and healthy animals are commonly used in ex-perimental studies, in contrast to humans suff ering from stroke, who are com-monly elderly and hypertensive, suff er from generalized atherosclerosis and/or suff er from diabetes. Even design features which could have been built into stroke studies in experimental animals, including extended observation periods of more than 1-3 days are seldom included in the study design. There is the-refore a multitude of reasons, why treatment strategies proven eff ective in the laboratory have failed when put to the test in the clinic (Howells et al. 2010a; Howells et al. 2010b; van der Worp et al. 2010).

Since the original description of a stroke model in dogs (Hill et al. 1955), a plet-hora of experimental stroke models in animals have been described and used in a variety of study paradigms (Stefanovich 1983; del Zoppo 1990; Overgaard 1994; Wang-Fischer 2009; Dirnagl 2010).

Ischemic stroke models are basically either global or focal models or models which create permanent ischemia or induce reperfusion after ischemic lesions (Table II). Furthermore, they include opening of the skull or not.

20

Introduction

Table IIRodent models of cerebral ischemia Modifi ed from (Ginsberg et al. 1989).

Models of global cerebral ischemia in rats

Two-vessel occlusion model of forebrain ischemia Transient bilateral common carotid artery (CCA) occlusions plus hypotension

Four-vessel occlusion model of forebrain ischemia Transient bilateral CCA occlusions plus permanent vertebral artery occlusions

Ischemia models involving elevated cerebrospinal fluid pressure Bihemispheral forebrain compression-ischemia Unihemispheral forebrain ischemia

Miscellaneous global ischemia-producing strategies Neck tourniquet Decapitation

Levine preparation of hypoxia-ischemia and its modifications Models of focal cerebral ischemia in rats

Middle cerebral artery occlusion and its variants (Laing et al. 1993) Direct occlusion of the MCA through craniotomy by electro cauterization or by a

clip (Tamura et al. 1981a; Bederson et al. 1986b) Inserting a poly-lysine coated suture thread into the carotid artery in the neck

until it occludes the MCA (Koizumi 1986; Longa et al. 1989; Belayev et al. 1996) By introducing an embolus (blood clot or synthetic embolus) to the carotid artery

in the neck making it travel to and occlude the MCA (Hill et al. 1955; Kudo et al. 1982; Kaneko et al. 1985; Zhang et al. 1997b).

Using light of a specific wavelength to activate a polymer injected into the blood, thus occluding the artery (Futrell et al. 1989; Matsuno et al. 1993; Zhao et al. 2002)

Stroke in the SHR Miscellaneous models of cerebral embolism and thrombosis

Blood clot embolization Microsphere embolization Photochemically initiated thromboembolism Arachidonate-induced thrombosis

Models of cerebral ischemia in gerbils

Unilateral CCA occlusion Bilateral CCA occlusions

21

Introduction

Models of focal cerebral ischemiaThe most common stroke model, due to its relevance to human stroke, is focal MCAo. It may, as listed above, be induced by several diff erent approaches inclu-ding temporary or permanent, proximal or distal occlusion of the artery. MCAo is sometimes combined with carotid artery occlusion (ipsi-, contra-, or bilate-ral; temporary or permanent) in order to increase the extent of the ischemic lesion(s). The MCA can be occluded in several diff erent ways, including direct clipping, intraluminal suture or by an embolus (blood clot). A widely used in-vasive permanent occlusion technique is cauterization of the MCA through cra-niectomy (Tamura et al. 1981a). A craniectomy technique allowing reperfusion is occlusion by means of micro clip (Theodorsson et al. 2005b) or ligature which can be released. Even some methods for photothrombic occlusion of the vessel have been claimed to result in reperfusion.

Middle cerebral artery occlusion (MCAo) through craniectomyMCAo in rats through a craniectomy has been used in experimental models of cerebral ischemia since 1975 (Robinson et al. 1975; Robinson 1979; Bederson et al. 1986b). The technique of directly and permanently (by electro coagula-tion) occluding the MCA has been optimized and characterized by Tamura et al. (1981a), and has been used for several recent studies of focal cerebral ischemia (Tamura et al. 1981b). Clips and ligatures have also been used to permanently or transiently occlude the MCA (Shigeno et al. 1985; Buchan et al. 1992; van Brug-gen et al. 1999; Theodorsson et al. 2005b).

The advantages of the craniectomy techniques are the ability to visually identify the artery to be occluded (Figure 4), and to visually verify that it has been oc-cluded. Furthermore, reperfusion of the artery can also be verified, when the clip is removed. The disadvantage of the method is its technical complexity and steep learning curve and the mortality due to the operation itself.

22

Introduction

Figure 4The rat MCA seen through an operation microscope by means of a drill hole in the skull. The use of the optic tract to locate the MCA is apparent.

Thromboembolic stroke modelIn 1955, Hill and colleagues pioneered in using injection of homologous blood clots into the carotid artery as an experimental model for cerebral ischemia in dogs (Hill et al. 1955). The thromboembolic stroke models in rats are the most frequently used models for studies of experimental thrombolytic therapies (Kudo et al. 1982; Kaneko et al. 1985). To induce microembolization, Kudo and co-workers used suspension of blood clots (≤ 100 μm) injected into the CCA, while Kaneko et al. used larger blood clots averaging between 100 and 200 μm in diameter. It is, however, dif icult to exert detailed/suf icient control of the size of the blood clots. Depending on the size of the clot lesions of diff erent sizes are induced. The smallest blood clots cause miroembolization, whereas the big clots run the risk of occluding even the entire targeted arterial circulation.

Zhang and co-workers 1997 inserted a modi ied PE50 catheter close to the MCA origin through internal carotid artery (ICA), and then occluded the MCA with injection of a single clot (Zhang et al. 1997b). This technique was improved by Busch et al. (1997) by inserting a PE50 catheter to the ICA through the external carotid artery (ECA), injection of a number (12) of clots (350 x 1500 μm), small enough to reach the MCA origin, during which the CCA was temporarily closed.

23

Introduction

Photochemical thrombotic stroke model��(� ��)��

�� !�"��#��$�

��%��"�� &'�� '��!�*��+��%��

��*�� *��

Intraluminal suture stroke model,��*�� -.��

�� /�� +�� !�

��0��1�� 2!��,��-.��*��

3.��*��..�$��*�� +��"��4.�$�

�� +��"��4.��-.��,��

��%��,��

��"��*�� -.��$��*��

3.��*��,��/��"��+��5��

��"�� !�� 6�'��"��

�� +��$�"�� 0��1�� 6�'��

��7�� 2!��+��*��*�5��

�� !�� *�� "��5��

�� ,��5�

�� *��"��-�1��

� 8�!$�� *��4��

�*��*�9�:��

,��#��;��*�*��"��

��9�� "��$�� <��

-.��9=��6�>��*��$��&6>��*��$��

�'>�"��9:��?>�5�� 3��$�+��$��?>��*�

�� @��@�&66��=��?''2!��=��"��

�� <��;�� *� ��-.��

<�� *� �� @��&66$� 9=� ��

��,��@��&66��"��6'>!��*�/��'>!�

��*�9�:� �� 9=$��"��"��

��,�� *�� "��"��

�� -��!��;��*��4.�$��

�� @��A��

�=��?''2!�

Staining methods for infarct volume determination9��%��*��*��

��,�� *��-��

�� "��;��*��

��%�� ,��?$&$��,��

��1��,,.!��

��*��"��"��*��*��

��

24

Introduction

Triphenyltetrazolium hydrochloride stainingTTC was irst synthesized in 1894 (von Pechman et al. 1894) and initially used for testing the viability of seeds (Glenner 1969). Since 1958 TTC is used to detect by staining ischemic lesions in human tissues, i. a. the myocardium (Sandritter et al. 1958). TTC, a water soluble, colourless salt reacting with intact oxidative enzymes, dehydrogenases, of the inner mitochondrial membrane, is reduced and forms a fat soluble compound (formazan) that turns normal tissue deep red and thereby delineates abnormal areas (Glenner 1969; Orten et al. 1975). Staining by TTC has been used to determine the location and extent of the infarcted areas in cerebral tissue after ischemic injury (Altman 1976; Bederson et al. 1986a; Lundy et al. 1986; Goldlust et al. 1996).

Pathophysiological mechanisms in cerebral ischemia The extraordinary vulnerability of cerebral tissues to ischemic damage re lects: 1) its high metabolic rate and oxygen demand, varying between diff erent cere-bral regions (Rosner et al. 1986) and 2) the unique pathological mechanisms that the normal neurotransmission/neurochemical mechanisms in the brain ex-ert in ischemic conditions damaging eff ects on its own and other cells.

A more or less orderly row of events – the ischemic cascade – has been establis-hed as the backbone in the pathogenesis of stroke. Each of the steps in this cas-cade needs to be studied as potential target for treatment. The currently most favoured excitotoxic mechanism of permanent ischemic damage in the brain is that hypoxia releases excitatory amino acids, in particular glutamate, which in-luences all cells in the vicinity (Siesjö et al. 1989; Siesjö 1992; Siesjö et al. 1998;

Siegel et al. 1999; Ginsberg 2003; Fawcett 2006). Glutamate is an excitatory amino acid normally of crucial importance for, among others, memory and is the neurotransmitter present in highest concentrations in the brain. When present in excessive concentrations, it causes depolarisation of cell membranes and in-creased intracellular calcium levels which trigger the cell damage. Glutamate in high concentrations is therefore toxic to neurons, and an important part in the mechanisms of excitotoxicity (Siesjö et al. 1989). Glutamate increases the entry of calcium into the cells which induces cell death, e.g. through apoptosis. The primary reason for the particular vulnerability of the brain in cerebral ischemia is the fact that the inter- and intra-cellular signalling mechanisms crucial for normal functions of the brain, become harmful under ischemic conditions; en-ergy failure is accelerated enhancing the inal pathways underlying ischemic cell death, including free radical production, activation of catabolic enzymes, mem-brane failure, apoptosis and in lammation (Calabresi et al. 2000; Centonze et al. 2001).

A fundamental pathophysiological mechanism of cell death in brain ischemia is lack of energy supply to the cells due to lack of oxygen leading within minutes to insuf icient cell respiration and to depletion of the cells ATP supplies (Siesjö et al. 1998). ATP is i. a. needed to power the ion pumps of the cell membranes which uphold the membrane potentials required i. a. for neurotransmission and

25

Introduction

for the synthesis of chemical neurotransmitters. Lacking ATP, the cells enter into a state of anoxic depolarization which opens up voltage-sensitive ion channels al-lowing pathological entry of calcium, sodium and chloride ions into the cells. Pas-sive and excessive entry of water into the cells subsequently results in cytotoxic oedema (Siesjö 1992).

Temporary brain ischemia is characterized by hypoxia during a short period of time followed by a period of hyper perfusion of the tissue with well-oxygenated blood. The reperfusion generates huge amounts of free radicals in the tissue, which are particularly damaging to macromolecules including proteins, nucleic acids and cell membranes. The pathophysiological end result is variable depending on the cell organelle or macromolecules aff ected.

The increased intracellular calcium levels cause cell death by various mechanisms, including activation of proteases and lipases, formation of free radicals, lipid per-oxidation and formation of nitric oxide (NO) and arachidonic acid. The generation of high levels of NO is results in free radicals which damage important biomolecu-les, including membrane lipids, enzymes and DNA.

The various mechanisms that normally protect the neurons against excitotoxicity are the calcium transport systems/ion pumps, mitochondrial function and radical scavengers. Transport systems are not able to counteract the increase in calcium concentrations, when ATP is missing. The mitochondrial function is disrupted, when mitochondrial stores are overloaded with calcium, and this results in even further reduced ATP synthesis.

When reperfusion occurs and the oxygenation is restored, it can lead to even further damage because of generation of reactive oxygen species. They include e.g. superoxide, hydroxyl radicals and hydrogen peroxide which are generated as side products in mitochondrial ATP synthesis. When oxygenation is restored, reactive oxygen species accumulate and can cause damage to important macromolecules in the cell.

The cerebral circulation in ratsThe brain is supplied through four primary arteries; the anterior and posterior circulation, connected through the circle of Willis formed by the anterior cerebral and posterior communicating arteries. From the arch section of aorta the inno-minate, left common carotid and left subclavian arteries arise. The innominate is divided to the right subclavian and right CCA. The ICA and ECA are derived from respective CCA. The ICA – anterior circulation - branches intracranially into several arteries; the posterior communicating artery, anterior cerebral artery and MCA are the major. The posterior cerebral artery is a branch of the posterior communica-tion artery (Figure 5).

The vertebral arteries – the posterior circulation – arise from the subclavian arte-ries, enters the skull through foramen magnum, and form the basilar artery which is a component of the circle of Willis (Wang-Fischer 2009).

26

Introduction

Anterior Cerebral artery (0.28 mm in diameter) Middle Cerebral artery (0.24 mm in diameter) Posterior cerebral artery (0.26 mm in diameter) Internal carotid artery (0.71 mm in diameter) Basilar artery (0.36 mm in diameter) Vertebral artery (0.34 mm in diameter) External carotid artery (0.77 mm in diameter) Common carotid artery (0.90 mm in diameter)

Figure 5 The cerebrovascular anatomy of the brain of rats. The cerebrovascular hemispheres are supplied th-rough four primary arteries, the right and left internal carotid arteries and the right and left vertebral arteries. The anterior, middle, and posterior cerebral arteries are derived mainly from the internal carotid arteries to form a modifi ed circle of Willis (modifi ed by kind permission from (Longa et al. 1989)).

Hypothermia and brain ischemiaThe neuroprotective properties of hypothermia in brain ischemia have long been suggested (O’Keeff e 1977; Chinard 1978). People suff ering from global ce-rebral hypoxia during near-drowning events have been reported to experience remarkable neurological recovery, if hypothermia is present through cold water drowning (Young et al. 1980; Nunney 2008). Hypothermia in humans also im-proves the neurological outcome in survivors of cardiac arrest which has resul-ted in global cerebral hypoxia (Bernard et al. 2002; Group 2002). The use of hy-pothermia is today recommended after cardiac arrest, i. a. by the International Liaison Committee on Resuscitation (ILCOR) (Nolan et al. 2003). Furthermore, whole-body hypothermia has been shown to reduce mortality and to improve the neurodevelopmental outcome in neonates suff ering from hypoxic-ischemic encephalopathy (Shankaran et al. 2005).

The eff ects of hypothermia in acute ischemic stroke in humans have as yet been tested only in a few clinical studies, i. a. by the COOL-AID study group in two clinical trials using surface cooling (Krieger et al. 2001) and by endovascular cooling (De Georgia et al. 2004). However, both studies are too small to allow comprehensive conclusions. There is i. a. an uncertainty about the optimal depth and duration of the hypothermia. Furthermore, cooling below 35oC, which is used in most experimental animal models, requires controlled mechanical ven-

27

Introduction

tilation and sedation only available in intensive care units. This limits the enrolling of patients in clinical trials as well as the practical implementation of any therapy shown to be eff ective. On the other hand, prospective observational studies have reported that elevated body temperatures are associated with poor outcome after stroke (Reith et al. 1996; Castillo et al. 1998).

Ischemia

Hypothermia

Glutamate

Aspartate

Dopamine

NMDA

Astroglia

Glycine

S-100 BActivation Fagocytosis

Ca+2

Inhibition

Inhibition

Stimulation

Stimulation

Hypothermia

Inhibition

Ca+2 Mitochondrialdysfunction

Celldeath

Cytokines

Free radicals

DNA lesions

Inhibition

Figure 6The role of hypothermia in maintaining the integrity and functional status of neurons affected by cerebral ischemia. Adapted from (Gonzalez-Ibarra et al. 2011).

Hypothermia inhibits the release of excitatory amino acids and calcium ions released by the ischemic insult. This inhibits elevation of intracellular concentrations of calcium ions and the mitochondrial dysfunction/cell death. Morover, hypothermia decreases DNA lesions in the cells, thereby improving the chance that cells in the penumbra zone survive. Finall, hypothermia also inhibits the activation of astroglia and thus release of protein S-100 B, cytokines and free radicals.

Several studies in experimental animals have been made on the eff ects of induced hypothermia in focal cerebral ischemia (Ginsberg 1997). van der Worp and co-workers (2007) recently reported a systematic review and meta-analysis of the evidence for ef icacy of hypothermia in animal models of ischemic stroke, iden-tifying 101 publications on the eff ects of hypothermia on infarction size or func-tional outcome, including data from a total of 3353 animals. Overall, hypothermia reduced infarction size by 44%. The eff ect was most pronounced when cooling to temperatures even below 31oC, when hypothermia was induced before or at the onset of the ischemic insult, and in transient stroke models compared to perma-nent ischemic models. A 30% reduction in infarction volume was reported with cooling to 35oC and with initiation of hypothermia between 90 and 180 min after the onset of the ischemic insult. The eff ects of hypothermia were also more pro-

28

Introduction

nounced in hypertensive animals compared to normotensives.

The neuroprotective mechanisms of hypothermia are most likely multiple and act in synergy on a broad spectrum of biochemical pathways (Figures 6 and 7, Table III). The brain metabolism decreases by hypothermia by slowing down the rate of oxygen and glucose utilization, and of ATP breakdown (Erecinska et al. 2003). The brain oxygen consumption is reduced by approximately 5% per every degree of fall in body temperature in the temperature range of 22-37oC (Hagerdal et al. 1975).

Excitatory amino acids including glutamate are toxic to neurons in high extra-cellular concentrations and are amongst the most important mediators of ex-citotoxicity and the damage caused by cerebral ischemia (Siesjö et al. 1989). Hypothermia reduces the excitotoxic damage by reducing glutamate release (Nakashima et al. 1996). In addition, hypothermia impairs glutamate-mediated calcium in lux (Takata et al. 1997). This reduces damage due to uncontrolled rise in the concentrations of intracellular calcium ion which activate enzymes (i. a. proteases and nucleases) that degrade proteins, nucleic acids and enzymes synthesizing NO.

Hypothermia also inhibits free radical formation (Yenari et al. 2002). The neu-roin lammatory response is attenuated (Inamasu et al. 2001), and sustained re-sponses as late as one week after hypothermia have been reported (Wang et al. 2002). Furthermore, hypothermia is reported to limit brain oedema formation and alter necrosis/apoptosis (Eberspacher et al. 2005; Wang et al. 2005).

The eff ect of an ischemic insult on markers of the biological activity of the neu-ropeptide galanin is still a matter of debate, since the galanin response has been reported to be increased (Barbelivien et al. 2004; De Michele et al. 2006), de-creased (Raghavendra Rao et al. 2002; Theodorsson et al. 2005c), as well as bi-phasic (Hwang et al. 2004). Since galanin has been claimed neuroprotective in models of cerebral ischemia, a possible component in the neuroprotective ef-fects of hypothermia-induced alterations in galanin concentrations was studied in the current thesis (Theodorsson et al. 2008).

Table IIINeuroprotective mechanisms of hypothermia from i. a. (Sinclair et al. 2010; Gonzalez-Ibarra et al. 2011)

Mechanism Explanation Time framePrevention of apopto-sis

Ischemia induces apoptosis and calpain-mediated proteolysis which both are re-duced or even prevented by hypother-mia.

Hours, days to weeks

29

Introduction

Mechanism Explanation Time frameReduced mitochondrial dysfunction and impro-ved energy homeosta-sis

Ischemia leads to mitochondrial dysfun-ction and apoptosis. Hypothermia redu-ces metabolic demands and improves energy homeostasis and mitochondrial functions.

Hours to days

Reduced free radical production

Mild to moderate hypothermia (30oC to 35oC) reduces the production of free radicals including superoxide peroxyni-trate, hydrogen peroxide and hydroxyl radicals.

Hours to days

Mitigation of reperfu-sion injury

Reperfusion- related reactions are in-hibited by hypothermia, including free radical production.

Hours to days

Reduced permeability of the blood-brain bar-rier and the vascular wall and reduced oedema formation

Blood-brain barrier disruptions, vascu-lar permeability and capillary leakage induced by trauma or ischemia are de-creased by hypothermia.

Hours to days

Reduced permeability of cellular membranes

Decreased membrane leakage, resulting in improved cellular homeostasis, mi-tigation of DNA injury and decrease in intracellular acidosis.

Hours to days

Improved ion ho-meostasis

Ischemia induces the excitotoxic cas-cade including release of calcium and excitatory neurotransmitters including glutamate. This cascade is inhibited by hypothermia.

Minutes to 72 hours

Reduced metabolism The requirements of the cells for oxygen and glucose are reduced 5 - 8% by each centigrade decrease in core body tem-perature.

Hours to days

Decrease in potentially harmful immune- and pro-in lammatory responses

Hypothermia blocks destructive in lam-matory reactions and secretion of pro-in lammatory cytokines in response to ischemic damage of the brain.

Hours to days

Reduction in cerebral thermopooling

Hyperthermia increases damage to inju-red brain cells. Hypothermia blocks the increase in brain temperatures in certain injured brain regions of up to 3oC which can be induced by ischemic damage.

Minutes to days

Anticoagulant eff ects Hypothermia exert anticoagulant eff ects which prevent microthrombus forma-tion, adding to brain ischemia.

Minutes to days

30

Introduction

Mechanism Explanation Time frameSuppression of seizu-res

Seizures after ischemic injury increase brain injury and hypothermia mitigates them.

Hours to days

Ischemia

Hypothermia

Increased TXA2 Platelet aggregationVasoconstriction

Vesselocclusion

Hypoxia

Decreased metabolismDecreased glucose reserve

Decreased ATP

Membrane pores

Mitochondrialdysfunction

Celldeath

Inhibition

Hypothermia

Inhibition

LactateHydrogen phosphate

DNA fragmentation

Anaerobic metabolism

Acidosis

Inhibition

Ischemia

Freeradicals

Inhibition

Figure 7The effects of therapeutic hypothermia on the oxidative stress and neuronal metabolism induced by cerebral ischemia Adapted from (Gonzalez-Ibarra et al. 2011).

Hypothermia inhibits the increase in thromboxin A2 induced by ischemia and thereby the subsequent platelet aggregation and vessel occlusion. Hypothermia reduces metabolic demands, reducing glucose use, generation of lactate and the subsequent acidosis with its detrimental effects on the mitochondria.

31

Introduction

NeuropeptidesNeuropeptides constitute the oldest neurotransmitter system known, found al-ready in the phylogenetically ancient Hydra (Grimmelikhuijzen 1983). Currently more than hundred neuropeptides have been characterized and studied, crea-ting an innovative ield of scienti ic enquiry – neuropeptide research (Klavdieva 1995; Klavdieva 1996a; Klavdieva 1996b; Klavdieva 1996c; Strand 1999; Hök-felt et al. 2000; Hökfelt et al. 2003; Kastin 2006; Burbach 2010).

Neuropeptides are synthesized in, and released from neurons of the central (CNS) and peripheral (PNS) nervous system - hence the name neuropeptides. They are regularly co-expressed with at least one classical neurotransmitter e.g. a monoamine and/or an amino acid, and often with more than one other neu-ropeptide (Hökfelt et al. 1980). Many neurons in the CNS are able to release a ‘cocktail’ of chemical messengers, including a fast-acting, excitatory transmitter amino acid such as glutamate together with a monoamine and even one or more neuropeptides. This caters for a more ’ef icient’ signalling suited for the purpose than the simple on/off signal that would be available, if neurons had one trans-mitter only.

Neuropeptides are actually a subgroup within the broader group of regulatory peptides which in addition to neuropeptides also include peptides present in and released from widely distributed endocrine cells. The concept of APUD cells was put forward already in the 1960’s by A. G. Pearse. The concept groups together seemingly unrelated endocrine cells having in common 1) high Amine content, 2) substantial Precursor Uptake, 3) and the enzyme amino acid Decarboxylase (Pearse 1969; Pearse 1974). In addition to amines, APUD cells also contain re-gulatory peptides which exert their eff ects in three diff erent ways: 1) by being released into the bloodstream (endocrine transmission); 2) via local diff usion to adjacent cells (paracrine transmission) or to the cell which released the pep-tide (autocrine transmission); 3) through modulation of signal transmission in or outside nerve cell synapses (neurocrine or synaptic/non-synaptic transmis-sion). Regulatory peptides in endocrine cells of the gut participate i. a. in the regulation of gut secretion and motility and in the absorption and utilization of nutrients.

Neuropeptides are 2-100 amino acids in length and have up to 100 times higher molecular weight than the classical neurotransmitters. However, they are smal-ler than regular proteins, including e.g. common metabolic enzymes and have a less complex three dimensional structure (Hökfelt et al. 2003). They expose more numerous binding sites and thereby convey ‘more’ chemical information to their receptors than classical transmitters and bind more slowly but more tightly than smaller neurotransmitters. The binding af inity between neuropep-tides and their receptors is in the nmol/L or higher range, 1000 times higher than classical transmitters which have binding af inity in the μmol/L range.

32

Introduction

Neuropeptide biosynthesis and releaseThere are several diff erences between neuropeptides and classical transmitters with regard to their synthesis, storage and release mechanisms (Figure 8).

Synaptic vesicle with classic transmitter

GPCR for peptide or classic tranmitter

Transporter for classic transmitter

Ionotropic receptor for classic transmitter

LDCV

NeuropeptideClassictransmitter

High frequencyburst firing

Low frequency

Extrasynapticrelease

Lowfrequency

Dendriticpeptidesynthesis

Dendriticrelease

Glialcells Break

down

Somaticrelease

Highfrequencyor burst firing

Synapse

Postsynapticdensity

1

2

3

4

5

6

7

8

Figure 8The synthesis, neuronal transport, release and effect of neuropeptides from (Hökfelt et al. 2003), with permission from the copyright holders. Neuropeptides are mainly synthetized as peptide precursors in the cell body, packaged into dense-core vesicles which also can contain and co-release classic/monoamine transmitters. The vesicle also contains convertases cleaving the bioactive peptide from its precursor. The peptide receptors contain seven transmembrane spanning regions of the G-protein-coupled type, and are present on cell soma, dendrites, axons, and nerve endings. Classic/monoamine transmitters are synthetised in the nerve terminal and released upon lower frequency stimulus than the neuropeptide transmitters. Classic/monoamine transmitters, in contrast to the neuropeptides, have reuptake mechanisms resulting in termination of their action and their re-cycling. Neuropeptides, on the other hand, are inactivated by extracellular proteases and only replaced by axonal transport from the cell body which can take up to days when long axons are involved. Therefore classical/monoamine neurotransmission has very large capacity and is not exhausted, whereas neuropeptides are selectively released and have comparatively low capacity over time.

Neuropeptides are produced by ribosomes in the cell body of their neuron as precursor peptides (prepropeptides), and subsequently packaged into large dense core vesicles (LDCVs, 90-250 nm in diameter) for further processing. They reach the nerve endings by fast calcium ion dependent active, fast transport in the axons and dendrites and are released extrasynaptically (Gainer 1981; Lund-

33

Introduction

berg et al. 1986a). In contrast classical neurotransmitters are produced locally and presynaptically by dedicated enzyme mechanisms and stored in small clear synap-tic vesicles (40-60 nm in diameter) located in nerve endings close to the release site of the synapse.

The neuropeptides are released by high frequency iring in the synapse, whereas the classical neurotransmitters are released into the synaptic cleft during low fre-quency activity (Lundberg et al. 1986b; Tallent 2008). The classical transmitters, in contrast to neuropeptides, have dedicated reuptake mechanisms located in the presynaptic cell membrane, wherefrom they are incorporated into synaptic vesic-les using vesicular transporter molecules. In contrast, peptides are cleaved and their activity terminated by extracellular peptidases. They are only re-supplied to the site of release through axonal transport. Taken together - the peptides are pro-duced far from the site of release and their transport takes long time. Therefore peptidergic neurotransmission is much more easily exhausted compared to classic monoaminergic transmission.

In some instances neuropeptides are present in high concentrations and ‘functio-nal’ all the time. In other instances neuropeptides are expressed in low or undetec-table concentrations, or not at all and then upregulated under certain conditions, for example in response to nerve injury. Neuropeptides may also be expressed ear-ly during development, often only prenatally, and then downregulated postnatally.

The biological functions of neuropeptides range from neurotransmitter to growth factor. They are hormones in the endocrine system, and are messenger in the im-mune system. Much evidence indicates that neuropeptides play a role mainly when the nervous system is challenged by diff erent physiological/pathophysiological processes (Hökfelt et al. 2003) (e. g. by stress, nociception, mood, feeding, injury, drug addiction, learning and memory or diseases).

It is dif icult to use neuropeptides as medicines because they decompose rapidly in the gastrointestinal tract and the bloodstream. Instead, research has focused on the identi ication of their receptors and exploits the knowledge of how these and neuropeptides are structured. The idea is to produce substances called anta-gonists, which cancels the eff ects of a neuropeptide by blocking the receptor and which, preferably, are small and pass the blood-brain barrier.

GalaninGalanin was extracted from porcine small intestines based on a novel isolation method (Tatemoto et al. 1978). Galanin consists of 29 amino acids Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-His-Ala-Ile-Asp-Asn-His-Arg-Ser-Phe-His-Asp-Lys-Tyr-Gly-Leu-Ala-NH2, and is C-terminally amidated. Its name stems from the fact that it contains an N-terminal glycine residue and a C-terminal ala-nine (Tatemoto et al. 1983). The sequence of amino acids in human galanin was determined in 1991. It consists of 30 amino acids, lacks the C-terminal amide but includes the additional amino acid serine (Evans et al. 1991).

It was early noted that galanin does not share any structural features with any oth-

34

Introduction

er biological active neuropeptides (Vrontakis et al. 1987) and exerts its biologi-cal eff ects by its N-terminal end, and it was long assumed to constitute a peptide family of its own (Bedecs et al. 1995). Galanin is phylogenetically old and highly conserved among diff erent species, showing over 85% homology between rat, mouse, porcine, bovine and human sequences (Bedecs et al. 1995). In all species (the tuna ish being the exception), the irst 15 amino acids from the N-terminal are identical, but amino acids diff er at several positions at the C-terminal end of the peptide (Kask et al. 1995). These slight diff erences in protein structure have far reaching implications on their biological eff ects. For example, porcine and rat galanin inhibit glucose-induced insulin secretion in rats and dogs but have no eff ect on insulin secretion in humans. This demonstrates that it is essential to study the eff ects of galanin, and other regulatory peptides, in their autologous species (Bersani et al. 1991). Galanin is produced from the cleavage of a 123 amino acid long precursor, known as preprogalanin, which is produced from the preprogalanin gene.

Figure 9Galanin – containing neuronal pathways in the rat brain. From Kang Zheng 2011 with permission.

The galanin family of neuropeptides consists of four members. After galanin it-self, galanin message associated protein (GMAP), a 59 or 60 amino acid peptide formed from the cleavage of preprogalanin, was characterized in 1986 (Rökaeus et al. 1986). The two remaining peptides, galanin-like peptide (GALP) and alarin, were identi ied relatively recently and are both encoded for by the same gene, the preproGALP gene. GALP and alarin are produced by diff erent post-transla-tional splicing of this gene (Lang et al. 2007).

35

Introduction

Expression of galanin Galanin is widely expressed in the central and peripheral as well as in the endo-crine nervous system and co-exists with a number of classical neurotransmitters (Melander et al. 1986b) and has strong inhibitory actions on synaptic transmis-sion by reducing the release of these neurotransmitters, for example acetylcholine (Fisone et al. 1987) and noradrenaline (Morilak et al. 2003), and also interacts with other neuromodulators including neuropeptide Y, substance P and vasoactive intestinal polypeptide (Figure 9).

The inhibitory actions of galanin result in a diverse range of physiological/pathop-hysiological functions, such as reproduction, memory and food intake (Liu et al. 2002; Taylor et al. 2009; Merchenthaler 2010; Crawley 2010; Barson et al. 2010), it also has roles in development and as a trophic factor (Hobson et al. 2010).

Galanin is also thought to play a role in a number of diseases including pain (Xu et al. 2010), Alzheimer’s disease (Counts et al. 2010), epilepsy (Lerner et al. 2010) as well as depression (Kuteeva et al. 2010), and cancer (Rauch et al. 2010).

Galanin coexists with choline acetyltransferase in basal forebrain cell bodies in se-veral species. In the rat, galanin is expressed after colchicine treatment in 50–70% of cholinergic choline acetyltransferasepositive neurons in the medial septal nu-cleus and diagonal band of Broca area, some of which project to the hippocampus, i.e. a septohippocampal projection (Melander et al. 1985; Senut et al. 1989). Howe-ver, there are important species diff erences. In humans, galanin is not co-localized in cholinergic neurons of the nucleus basalis of Meynert (Kordower et al. 1990), the main source of cortical cholinergic innervation in humans. In the rat, the majo-rity of hippocampal galanin-containing cholinergic neurons project to the ventral hippocampal region. It is important to note that a substantial number of galanin nerve terminals within the hippocampal formation (HiFo) are noradrenergic, de-rived from locus coeruleus (LC) somata (Melander et al. 1986c; Xu et al. 1998a). Galanin is also expressed after colchicine treatment in a population of 5-hydrox-ytryptamine (5-HT) neurons in the dorsal raphe (Melander et al. 1986b; Xu et al. 1998b).

Galanin binding sites have been detected in the ventral HiFo, septum, and ventral aspect of the amygdala complex and entorhinal and perirhinal areas with relatively low binding in the dorsal cortex and in the striatum (Sko itsch et al. 1986b; Melan-der et al. 1988). In the HiFo the binding sites are concentrated to the most ventral part with medium dense labelling in CA3, CA1 and CA2 regions, with a high density labelling in the subiculum.

Galanin and painNumerous studies have demonstrated that galanin and its receptors are involved in the transmission and modulation of nociceptive information at spinal levels (Zhang et al. 2000; Liu et al. 2002; Hua et al. 2004; Wiesenfeld-Hallin et al. 2005; Xu et al. 2010). In the brain, studies have demonstrated that galanin plays an antinocieptive role in the hypothalamic arcuate nucleus in intact rats, in rats with in lammation and in rats with chronic neuropathic pain (Sun et al. 2003; Gu et al. 2007).

36

Introduction

Galanin in the central nervous system (CNS)Galanin is distributed throughout the CNS of several species, including rat (Sko-itsch and Jacobowitz 1985; Melander et al. 1986a; Sko itsch and Jacobowitz

1986a; Ryan et al. 1996), where it co-exists with classical neurotransmitters (Melander et al. 1986b; Merchenthaler et al. 1993; Jacobowitz et al. 2004).

Galanin mRNA is most abundant in hypothalamus and brainstem of rats (Jaco-bowitz et al. 1990; Jacobowitz et al. 2004), with very high levels in the preoptic, periventricular, and dorsomedial hypothalmic nuclei, bed nucleus of the stria terminalis, medial and lateral amygdala, LC, and nuclues of the solitary tract. Low to medium galanin mRNA levels are observed in olfactory bulb, septal nu-clei, thalamus, parabrachial nucleus, and the spinal trigeminal tract nucleus. Shen et al. (2003) have demonstrated galanin mRNA in the proliferative zones of developing and adult brain – the subventricular zone and subgranular zone of hippocampus, and in oligodendrocyte precursor cells in the corpus callosum.

Ligand Receptors Site of action

Galanin

Highly inducibleneuropeptide withsynaptic andneurotrophic effects

GalR1

GalR2

GalR3

PeripheralNerve injury/painPancreatic function

CNSSeizuresDepressionAgingAlzheimer Disease

Figure 10Galanin exerts its effects through three G-protein coupled receptors widely distributed both in the central and peripheral nervous system (adapted from (Lundström et al. 2005a)).

Galanin and neuronal injuryGalanin has been shown to be markedly upregulated after injury, both in the cen-tral and peripheral nervous system and both mRNA and peptide levels. Examp-les of such lesion studies include the upregulation of galanin in (1) dorsal root ganglion (DRG) neurons after peripheral axotomy (Hökfelt et al. 1987; Villar et al. 1989); (2) trigeminal ganglion neurons after damage of the vibrissae of rats (White et al. 1994); (3) medial septum-vertical diagonal band neurons after (i) electrocoagulation lesions of the ventral hippocampus or decortication (Cortes et al. 1990), (ii) transection of the septohippocampal pathway (Agoston et al. 1994) or (iii) tetrodotoxin injections into the vertical diagonal band (Agoston et al. 1994); (4) LC neurons after olfactory bulbectomy (Holmes et al. 1996); and

37

Introduction

(5) magnocellular hypothalamic neurons after hypophysectomy, a procedure that transects the axons of these neurons (Villar et al. 1994).

As much as a 120-fold increase has been seen in dorsal root ganglia after nerve injury (Hökfelt et al. 1987; Villar et al. 1989). Galanin transcription is regulated in a tissue- speci ic manner both by enhancer and silencer sequences under the control of several transcription factors (see (Vrontakis 2002)).

These studies have led a number of investigators to suggest that galanin might play a cell survival or growth promoting role in addition to its classical neuromodula-tory eff ects.

To test this hypothesis, transgenic animals were generated, bearing loss- or gain-of-function mutations in the galanin gene (Bacon et al. 2002; Holmes et al. 2000; Steiner et al. 2001; Blakeman et al. 2001). Phenotypic analysis of galanin knockout animals demonstrated that, surprisingly, the peptide acts as a survival factor to subsets of neurons in the developing peripheral and central nervous system (Hol-mes, 2000; O’Meara et al. 2000). It has also been demonstrated that this neuro-nal survival role is also relevant to the adult DRG. Sensory neurons are dependent upon galanin for neurite extension after injury, mediated by activation of the se-cond galanin receptor subtype in a protein kinase C-dependent manner (Mahoney et al. 2003). There are several studies providing evidence that galanin might also act in a similar manner in the CNS, reducing cell death in animal models of brain injury, damage or disease.

Galanin receptorsIt took more than ten years after the galanin discovery for the irst galanin receptor (GalR1) to be cloned, by Habert-Ortoli and collaborators (1994). Then the remain-ing two (GalR2 and GalR3) were cloned fairly soon after that (Branchek et al. 2000).The distribution of galanin receptors was irst studied in the rat brain, originally by ligand binding autoradiography (Sko itsch et al. 1986b; Melander et al. 1988), then all three galanin receptors subtypes by in situ hybridization in particular by Dajan O’Donnell and associates at AstraZeneca, Montreal (see (O’Donnell et al. 1999; Burazin et al. 2000; Waters et al. 2000; Mennicken et al. 2002; O´Donnell et al. 2003; Xu et al. 2005) and others. Galanin receptors are expressed in the CNS in peripheral tissues, including in the pancreas as well as on solid tumours (Figure 10). The level of expression of the diff erent receptors varies at each location, and this distribution changes after injury to neurons (Figure 11).

38

Introduction

InflammationTissue concentrations of galanin

Nerve injury

GalR1 GalR2 GalR3

Neurodegeneration in Alzheimers disease

EstrogenSeizures

Neuronaldevelopment

-++

+

+-

InflammationOpiate

withdrawalAntidepressants

Inflammation

Nerve injuryNerve injury--

++- +

Figure 11Galanin expression is increased after peripheral nerve injury, in the basal forebrain in Alzheimers di-sease, during neuronal development and after stimulation with estrogen. Infl ammation suppresses ex-pression, and seizure activity depletes galanin in the hippocampus (adapted from (Lundström et al. 2005b)).

The biological eff ect of galanin are mediated by the activation of one or more of the three known, cloned G-protein-coupled galanin receptor subtypes, desig-nated GalR1, GalR2 and GalR3 (Branchek et al. 2000) which are all part of the G-protein-coupled receptor (GPCR) super family. The receptors show high in-terspecies homology and moderate homology to each other. All three receptors couple to Gi/0 and inhibit adenylyl cyclase (Habert-Ortoli et al. 1994; Smith et al. 1998) but GalR2 can in addition signal via Gq/11 to activate phospholipase C and protein kinase C (Wang et al. 1998b; Wittau et al. 2000). Many galanin receptor-speci ic ligands exist (Mitsukawa et al. 2010). One instrumental tool has been the galanin fragment Gal(2-11) (Liu et al. 2001).

Lu et al. (2005) has demonstrated binding of 125I-galanin and Gal(2–11) to re-ceptor subtypes on isolated membranes, showing for example high GalR1 ex-pression in the hypothalamic paraventricular nucleus, and predominantly GalR2 in the dorsal raphe, HiFo and the amygdala. Rafael Rodriguez-Puertas’ group used the [35S] GTPγS assay and autoradiography to analyse galanin receptor-coupling to G-proteins (Barreda-Gomez et al. 2005). In most areas agreement with earlier 125I-galanin binding studies and with GalR1 mRNA distribution was reported, but apparent discrepencies were also found The same group used a similar approach to study the eff ect of intraventricularly administered galanin

39

Introduction

on muscarinic and galaninergic G-protein coupling and found, for example, that this treatment increases the coupling of both galanin and muscarine type of re-ceptor in the medial amygdala nucleus, whereas in other areas only one type of receptor-coupling was modulated (Barreda-Gomez et al. 2005).

The third galanin receptor was irst described by Wang et al. (1997). There are only a few studies describing the tissue expression pro ile of GalR3 in the rat with using a variety of RNA pro iling techniques and certain discrepancies have emerged, particularly with respect to its CNS distribution.

By Northern blot, Wang and co-workers detected GalR3 mRNA in heart, spleen and testis but not in brain; isolation of GalR3 from a rat hypothalamic cDNA li-brary, however indicates that it is present in rat CNS at low abundance. Indeed, using the more sensitive RNase protection assay, Smith et al. (1998) detected GalR3 transcripts in discrete regions of the rat CNS with highest levels in the hy-pothalamus, lower levels in the olfactory bulb, cerebral cortex, medulla oblong-ata, caudate putamen, cerebellum, and spinal cord, and no signi icant detection in hippocampus or substantia nigra.

In the peripheral nervous system the highest levels of GalR3 mRNA were found in the rat pituitary gland. More recently, Waters and Krause (2000) described a similar GalR3 distribution pro ile using both reverse transcription/polymerase chain reaction (RT/PCR) and RNase protection assays. However, these authors observed GalR3 expression in the rat hippocampus, whereas Smith et al. did not. Mennicken et al. (2002) used in situ hybridization (ISH) with a cDNA riboprobe to study the cellular distribution of GalR3 within the rat CNS. They displayed low and discrete labelling throughout the CNS. GalR3 expression is most prominent in the preoptic/hypothalamic area and the subfornical organ, but is also evident in discrete regions of the basal forebrain, pons, medulla and dorsal horn of the spinal cord. The regions in which Mennicken et al. (2002) detected GalR3 mRNA are also known to contain high levels of galanin and galanin binding sites, repre-senting targets for the central action of galanin.

Galanin agonistsThe introduction of Gal(2-11) which acts as an agonist with 500-fold selectivity for GalR2 (Liu et al. 2001) compared with GalR1, was an important advance in the ield, although a latter publication showed that Gal(2-11) also binds and activates GalR3 in a transfected cell line with similar af inity to GalR2 (Lu et al. 2005). Gal(2-11) has since then been employed in several studies, as a non-GalR1 ligand, as no ligand with higher selectivity has been available (Jimenez-Andrade et al. 2006; Alier et al. 2008).

GalR2, along with the other galanin receptor subtypes, could in the future be an important target in several disease states such as epileptic seizure, Alzheimers disease, mood disorders, anxiety, alcohol intake in addiction, metabolic disease, pain and solid tumors (Mitsukawa et al. 2010). If so, a subtype speci ic ligand is needed, to downsize unwanted side-eff ects.

40

Introduction

The GalR2/3 agonist Gal(2-11) has been evaluated in several studies (Liu et al. 2001; Elliott-Hunt et al. 2004; Hua et al. 2005; Mazarati et al. 2005; Pirondi et al. 2005; Ding et al. 2006; Kuteeva et al. 2008) and its eff ects have in most cases been considered as mediated via GalR2, when interpreting the result. The exclu-sion of eff ects mediated via GalR3 are somewhat doubtful, as the expression pat-tern of GalR3 in the CNS is disparate in the literature (Smith et al. 1998; Agranoff et al. 1999; Waters et al. 2000; Mennicken et al. 2002). The introduction of a small molecule GalR3 antagonist has provided possibilties to study presence and importance of GalR3 in the CNS (Swanson et al. 2005; Barr et al. 2006).

Galanin receptors have been characterized in expression models. Sebastian Wirz in Bartfai’s group (2005) analysed GalR1 dimerization and internalization using luorescence resonance energy transfer (FRET) technique and Chinese hamster

ovarian cells (CHO) (Wirz et al. 2005). No evidence for dimerization was obtai-ned, but internalization was demonstrated using time lap luorescence imaging. Zhi-Qing David Xu from the Karolinska group has presented studies on GalR1 and GalR2 receptors transfected to PC12 cells, demonstrating both constitutive and ligand induced internalization for the GalR2 receptor (Xia et al. 2004; Xia et al. 2005; Xia et al. 2008). The data strongly suggest that GalR2 receptor traf-icking utilizes the clathrin-dependent endocytic recycling pathway, which also

seems to be the case for the GalR1 receptor, whereas no evidence for constitutive internalization was found for the latter receptor.

41

Aims of the thesis

Aims of the thesisThe main focus on the present thesis was to develop an experimental stroke model catering for focal and transient ischemia, modern anesthesia and in-traoperative monitoring with low mortality.

Using this reperfusion stroke model in rats the eff ects on the neuropeptide galanin and its receptors were studied in order to

• investigate the eff ects of hypothermia on the size of the ischemic lesions and on galanin concentrations in brain tissues after transient MCAo in naïve female rats.

• investigate the eff ects of intracerebroventricular galanin and galanin recep-tor 2/3 agonist on the ischemic brain lesions after transient MCAo in naïve female rats.

• investigate the eff ects on galanin and its three receptors gene expression in discrete brain regions after transient MCAo in naïve female rats.

42

43

Material and Methods

Material and methodsAnimals and surgeryEthical aspectsThe guidelines issued by the National Committee for Animal Research in Swe-den and Principles of Laboratory Animal Care (NIH publication No 86-23, re-vised 1985) were followed. All studies and their experimental protocol were approved by the Local Ethics Committee for Animal Care and Use at Linköping University.

HousingFemale SD rats were used in all studies. Animals from the breeder B&K Univer-sal (Sollentuna, Sweden) were used in paper I-III and from Taconic (Germany) in paper IV. Adult females with a body weight of 216-350g were housed at the Animal Department of Linköping University for at least 1 week before the start of the experiments. The rats were kept two in each cage (Macrolone, MAC3, 974 cm2 at the bottom, and 15,5 cm high and containing 300g aspen material (Tapvei, Finland)) at constant room temperature (21 ± 1°C), with free access to water and standard rat chow (Lactamine, Vadstena, Sweden), and with 12 hours light-dark (light on at 8.00 am).

Anesthetic proceduresThe animal was kept in its own cage with free access to water and standard rat chow until induction of anesthesia. The rat was placed in an induction chamber (50216 Stoelting, Wood Dale, IL, USA) and anesthesia was induced by 4% iso lu-rane (Forene; Abbott Scandinavia, Kista, Sweden) in a mixture (30%/70%) of oxygen/nitrous oxide.

Endotracheal intubationA specially designed sloping board was used to position the anaesthetized rat with the teeth’s on the upper jaw ixed to extend the head in order to identify the epiglottis and to intubate the trachea. The tube, a ven lon 14G (Ven lon Vig-go, Helsingborg, Sweden), without its mandrin, was introduced into the trachea with a specially modi ied laryngoscope for neonates under direct visualization (Figure 12).

44


A

B

C

D

Figure 12The technique of endotracheal intubation. A. Anesthesia is induced in the induction chamber. B. The neck is extended with the animal placed on a sloping board. C. Epiglottis and trachea opening is visualized. D. The tube is positioned.

The tube in the trachea was connected to an animal ventilator (Zoovent, CWC 600AP, ULV, Newport, UK), and the anesthesia was continued with 1% iso lu-rane in a mixture (30%/70%) of oxygen/nitrous oxide. The animals were kept normoventilated and the tidal volume and the frequency were carefully regu-lated according the results of repeated blood gas analysis. Normally a peak air-way pressure of 10-12 cmH2O and a rate of 80-85 breaths per minute were required to maintain appropriate blood gas tensions.

The depth of anesthesia was determined by sensory and motor responses. The absence of foot withdrawal when pinched, and the absence of eye blink when the eyelid was touched, were used as indices of an adequate anesthesia.

45


Preparation of rat for surgery and intraoperative monitoringEye gel (Lubrithal 15 g Leo laboratories, Dublin, Ireland) was applied to pro-tect the rat´s eyes during the surgery. Saline (NaCl 9 mg/mL, B. Braun Medi-

cal AB, Bromma, Sweden), 6 mL/kg body weight, was administered sub-cutaneously at the start of operation for substituting loss of luid. A long-acting analgetic (Rimadyl® 50 mg/mL, P izer, Dundee, Scotland) was given at the same time in a dose of 0.1 mL/kg. A surgical head holder was used to position the head during surgery by means of an adjustable nose clamp (Figure 13).To keep the animal’s body tempera-ture at the desired levels according the study protocols (37.0 ± 0.5oC paper I – IV and 33.0 ± 0.5oC pa-per II) the rat was placed on a hea-

ting pad (Homeothermic Blanket System Harvard, Edenbridge, UK) (tempera-ture controller) to keep the core temperature at intended level, and monitored through a rectal probe.

Catheterizering of the femoral artery Before insertion the catheter (Micro-Renathane® tubing 36 ft [MRE-25 Brain-tree Scienti ic, Inc., MA, USA]) was prepared with a needle (27 G) and lushed with Heparin (100 IU/mL, Lövens Läkemedel, Malmö, Sweden). The left femoral triangle was shaved carefully to avoid abrading the skin and thoroughly clea-

ned with 70% ethanol. A 1-2 cm long skin incision was made over the femoral vessels, and the fascia was opened to expose the femoral sheath. Through blunt dissection the artery was separated from the nerve and the vein (Figure 14).

Figure 14 Using a specially design vessel dilatators the catheter is inserted into the femoral artery and subse-quently tied in place.

Figure 13 The rat’s head was positioned and fi xed during sur-gery with a surgical head holder.

46


The femoral artery was gently retracted with a pair of sutures (Ethicon Vicryl 4/0, Johnson & Johnson Intl., Sollentuna, Sweden), irst “downstream” then “upstream” to the area into which the catheter was introduced. A micro scissor was used to cut a minute hole in the vessel. The catheter was inserted 1-1½cm in the artery, and the catheter was secured to the artery with ligatures. To inhi-bit coagulation the vessel was lushed with heparin (4 IU/mL) (Figure 15).

Data registrationAll data from the monitors of rectal temperature, arterial blood pressure and pulse were transferred through an interface module (UIM 100, BIOPAC Sys-tems, Inc., Goleta, CA, USA) and an acquisition unit (MP 100, BIOPAC Systems, Inc.) to the computer (IBM PC, Dublin, Irland). The data were processed by AcqKnowledge® software (BIOPAC Systems, Inc.), displaying parameters grap-hically on-line. All events during the operation were documented in real time in a surgical protocol included in the AcqKnowledge® computer for that speci ic operation.

Occlusion of the middle cerebral artery The rat was carefully shaved on the left side of the head, between the eye and the ear, to avoid abrading the skin and thoroughly cleaned with 70% ethanol. Care was taken to avoid abscising or shortening the rat´s whiskers when shaving the operation area, since this would have major impact on the behavior of the animal. The left MCA was exposed through a subtemporal approach, using an operating microscope (Figure 16). Transection of the facial nerve was avoided during exposure of the temporalis muscle, which was divided posteriorly and retracted in the anterior– inferior direction. The zygoma was partially removed (4–5 mm). A 3 mm craniectomy was drilled up 3 mm anterior and 1 mm lateral to the foramen ovale, where the mandibular nerve exits. To prevent heating of the underlying cortex physiological saline was lushed at the drill site. By a ine mi-cro hook the dura was opened by a cruciate incision. The MCA was visualized as it runs forwards initially, than turning laterally running over the olfactory tract.

Figure 15The intra-arterial catheter tied to the blood pressure manometer couplings

47


Figure 16 A general overview from the operating room.

Olfactorytract

Distal Trunkof MCA

Arterialbranch to

RhinalCortex

Proximal Trunkof MCA

OpenedDura

Site ofocclusion

Openmicroclip

in positionjust before

clipping

Lenticulo-striate artery

Figure 17 The MCA and the microclip positioned just before clipping the artery as visualized through the operating microscope.

48


The arachnoid membrane was opened on either side of the artery by a ine needle. The MCA was occluded with a microclip between the rhino cortical branch and the lenticulostriate artery (Figure 17). After 1h occlusion of the MCA the microclip was carefully removed. The craniectomy was covered by a small piece of surgical soft tissue (Surgicel, Johnson & Johnson, Intl.) and the wound was closed using re-absorbable suture (4-0 Vicryl, Ethicon, Inc., John-son & Johnson, Intl.).

Application of hypothermiaBy the application of icepacks around the animal it was cooled at an average rate of 0.25oC/min beginning after intubation. The temperature of 33oC was therefore achieved after an average of 15 min. The rats were maintained at the desired level of core/rectum temperature at 33.0 ± 0.5oC for the duration of the experiment by means of a thermostatic heating pad. In case the body tem-perature of a rat deviated more than 0.5oC from the set point for more than 5 min, that rat was excluded from the study. The hypothermic animals were re-warmed to 37oC with a heating lamp during a 1-hour session after termination of the MCAo operation and before being returned to a clean cage with free ac-cess to water and standard rat chow (paper II).

Figure 18Stererotactic application of the catheter för intracerebroventricular administration

Intracerebroventricular administration of galanin and galanin receptor 2/3 agonistAn Alzet® brain infusion kit including a 28 G stainless steel cannula and a height adjustment spacer (Alzet® Infusion Kit II, 3-5 mm, Durect Corporations, Cupertino, CA, USA) was used together with an Alzet® osmotic pump, (volume 100 μL, releasing rate 0.5 μL/h, lasting 1 week) (Alzet® Osmotic Pump 1007D, Durect Corporations) (Figure 18 and 19). The cannula was operated into the rats left ventricle using the stereotactic coordinates: Bregma (B) -1.3 mm an-terior/posterior, B -3.8 mm dorsal/ventral and +1.8 mm lateral to midline. The

49


height adjustment spacer was 0.5 mm. The cannula was secured with three stainless steel screws and glue (Dental® plus, Heraeus Kutzer, Dormagen, Ger-many) to secure lasting position. The Alzet® osmotic pump was inserted in the rat neck and connected to the brain infusion cannula. The osmotic pump was illed with rat galanin (SC 936, Neosystem, Strasbourg, France) dissolved in the isotonic Ringer-Acetate (Braun) to a concentration of 8.4 μg/100μL, 84 μg/100μL or 840 μg/100μL releasing a dose of 0.77 μg/day (0.24 nmol/day), 7.68 μg/day (2.4 nmol/day) or 76.8 μg/day (24 nmol/day). The GalR2/3 ago-nist Gal(2-11), (AR-M1896, Tocris Cookson Ltd., Bristol, UK), was dissolved as above to a concentration of 84 μg/100μL, releasing a dose of 7.68 μg/day (2.4 nmol/day) and processed as mentioned above (paper ΙΙΙ).

CatheterTube

BrainInfusionCannula

Removable Tab

Depth-AdjustmentSpacers

Flow Moderator

OsmoticPump

Figure 19 The Alzet® brain infusion kit with a stainless cannula using one height adjustment spacer was ope-rated into the left ventricle by stereotactical technique for intracerebroventricular administration of galanin, galanin R2/3 agonist or artifi cial CSF. The osmotic pump was inserted subcutaneously in the rat neck and connected to the brain infustion cannula by a thin tube.

Termination of the surgeryThe catheter in the femoral artery used for measuring the pulse and blood pressure was carefully removed and permanently ligated (Ethicon Vicryl 4/0, Johnson & Johnson Intl.), and the skin was sutured. The low of iso lurane and nitrous oxide to the respirator was stopped, and the rat was allowed to breathe oxygen until spontaneous breathing was regained. The rat was subsequently transferred to a clean cage with free access to water and standard rat chow. The next day, a new dose of analgesic Rimadyl® (50 mg/mL, P izer) 0.1 mL/kg was administered. Members of the staff of the Animal Department made daily post-operative controls of the rats.

50


Brain biopsiesBrain slices and biopsiesThe rats were sacri iced after anesthesia by pure carbon dioxide using a guil-lotine (Small animal decapitator, AH 55-0012, Harvard Apparatus, Edenbridge, UK) and the brain was carefully dissected out from the skull, avoiding damage from sharp pieces of bone and the meninges and cooled in +4oC saline (paper I – IV).After a period (2-4 min) of cooling in a solution of 0.9% NaCl the brain was transferred to a rat brain matrix (RBM-4000, ASI Instrument, Inc., Warren, MI, USA). The brain was cut into 2 mm thick coronary slices, by razor blades, using bregma as position zero (Paxinos 1995)(Figure 20). In paper II ive slices were cut at bregma and 2, 4, 6 and 8 mm posterior to bregma. In paper I, III and IV two more slices were added, 2 and 4 mm anterior to bregma. Furthermore, in paper IV two more slices were added, 10 and 12 mm posterior to bregma.

Figure 20Slicing the rat brain using a brain matrix and razor blades

Punch biopsiesPunch biopsies were taken for measurement of the concentrations of galanin (paper II) and galanin/galanin receptor gene expression (paper IV). The biop-sies were taken with a biopsy needle (Punch tip, 2 mm, ASI Instrument Inc.) and were 2 mm in diameter and 2 mm thick. The location of punch biopsies (paper II) are shown in Figure 21, and were taken from both the lesioned and the contra lateral control side; at bregma – frontal cortex, bregma minus 2 mm – parietal cortex and inferior striatum, bregma minus 4 mm – parietal cortex and medial thalamus, bregma minus 6 mm – medial thalamus, bregma minus 8 mm – dorsal and ventral hippocampus. For galanin gene expression punch biopsies were taken from from LC (bregma minus 12 mm), cortex (bregma mi-nus 2 mm) and dorsal hippocampus (bregma minus 8 mm) (paper IV). The biopsies were rapidly collected and snap-frozen on dry ice and stored at -70oC prior to analysis.

51


Bregma Bregma - 2 mm

Bregma - 4 mm

Bregma - 6 mm

Bregma - 8 mm

Figure 21Location of punch biopsies in the brain slices from both the lesioned and the contra-lateral control side for measurement of galanin or galanin receptor gene expression.

Measuring the size of the ischemic brain lesionsEach brain slice was carefully transferred to a Petri dish. The 2 mm thick coro-nary slices were freed from dura mater and soaked for 10 minutes in a solution of 2% TTC in 0.1 mol/L PBS (pH 7.4) in a Petri dish, maintained at 37°C in a heater. Gentle stirring of the slices was used to ensure even exposure of the surfaces to staining. Excess TTC was then drained, and the slices were scanned (ScanJet IIc, Hewlett-Packard, Cupertino, CA, USA) into a computer ile (Micro-soft Windows) for image analysis.

The size of the brain lesion was measured using the image analysis software SigmaScan Pro version 5 (Systat Software Inc., Richmond, CA, USA). The image was divided into its red, green and blue colour spectra. The red spectrum was used to measure the total area of the slice and the green spectrum for measur-ing the ischemic lesion area. To sharpen the boundary between the slice itself and its surroundings the intensity of the red spectra was maximized before the measurement. In the green spectra the outer boundaries of the ischemic area were marked in the “overlay draw mode” to demark the area from normal structures in the brain. To mark the ischemic area a threshold of 40% in the

52


green spectrum was used in the function “ ill mode”. The lesioned area was measured as percentage of the total area of each slice (Bederson et al. 1986a; Bederson et al. 1986b; Goldlust et al. 1996) (Figure 22) (papers I-IV).

Figure 22Measuring the size of the ischemic brain lesions. The original scanned image (A). The red spectrum of the original image (B) is used to estimate the size of the total area (C). The green spectrum of the original image (D) is used to estimate the size of the ischemic brain lesion (E).

53


Galanin measurementsExtraction of brain punch biopsiesThe punch biopsies from the brain slices were extracted in 2 mL of boiling 1 mol/L acetic acid (MERCK, Darmstadt, Germany) and boiled for 10 min. The tissues were homogenized with a polytron (CAT X520D, Scienti ic Industries, New York, NY, USA) and centrifuged at 1500 g in 4oC for 10 min. The super-natant was collected, and a second extraction was immediately performed of the supernatants with 10 mL of distilled water per gram tissue to increase the recovery. The supernatants from each sample were combined, lyophilized and stored at -70oC prior to analysis (paper II).

Measuring galanin concentrations in brain tissue extractsGalanin was analysed using antiserum RatGal4 raised against conjugated synt-hetic rat galanin(1-29). The antiserum does not cross react with neurokinin A, neuropeptide K, substance P, neurokinin B, neuropeptide Y, gastrin, pancreatic polypeptide, glucagon or neurotensin. HPLC-puri ied 125I rat galanin was used as radioligand and rat galanin as standard. The detection limit of the assay was 8 pmol/L. Intra- and interassay coef icients of variation were 6% and 10%, re-spectively (Theodorsson et al. 2000) (paper II).

Real-time reverse transcriptase-polymerase chain reaction in brain punch biopsiesGene expression was analysed by RT-PCR in punch biopsies from the brain sli-ces from LC (bregma minus 12 mm), cortex (bregma minus 2 mm) and the dorsal hippocampus (bregma minus 8 mm). The biopsies were rapidly collec-ted using a micro-dissection protocol and snap-frozen on dry ice and stored at -70oC. Total RNA was extracted using the RNeasy Micro kit (Qiagen, Sollentuna, Sweden), according to the manufacturer`s instructions, including deoxyribonu-clease treatment with ribonuclease-free deoxyribonuclease set (RNase-Free DNase set, Qiagen). The concentrations and purity of the RNA were measured with NanoDrop ND-1000 Spectrophotometer (Saveen & Werner AB, Limhamn, Sweden). RNA samples were kept at -70oC prior to analysis.

A sample of 80 ng of RNA from LC, dorsal HiFo and cortex, were reversely tran-scribed to single-stranded cDNA by random hexamer priming using High Capa-city cDNA RT kit (Applied Biosystem, Stockholm, Sweden), in a volume of 20 μL, using the following program: 25oC for 10 min, 37oC for 60 min, 85oC for 5 sec, 4oC ∞. The samples were diluted 1:10 and kept at -70oC prior to analysis.

For RT-PCR, 7 μL of cDNA was added to TaqMan® Fast Universal PCR Master Mix (2X), RNase-free water and TaqMan® Gene Expression Assay primer (App-lied Biosystems) to achieve a inal reaction volume of 20 μL. The PCR protocol consisted of: initiation at 1 cycle at 95oC for 20 sec, followed by ampli ication for 40 cycles at 95oC for 1 sec and 60oC for 20 sec.

The following TaqMan® Gene Expression assays were used: Galanin: Rn

54


00583681 _ m1 (Applied Biosystems); GalR1: FP: TGGCGCGCAGCAAAC, RP: GGCCAGGGTGAAGATGCT, probe: CCGTGTCCATGCTCG (Invitrogen, AB, Lidingö, Sweden); GalR2: Rn 01773918 _ m1 (Applied Biosystems); GalR3: FP: CT-GGACGTGGCCACCTT, RP: TAGGCCAGGCTCACCAC, probe: CCGCGGGCTACCTG (Invitrogen, AB).

Ywahz Rn 00755072_m1 and Sdha Rn 00590475_m1 (Applied Biosystems) were used as endogenous reference genes for study of the eff ects of lesions (Gubern et al. 2009) and of the estrous cycle (Hvid, Ekström et al. 2010).

The reactions were carried out in 96-well plates covered with optical adhesive ilm (Applied Biosystems). Each sample was analyzed in duplicate and aver-

age values were calculated. No-template reactions with water instead of cDNA were included as negative controls on all plates.

Gene expression was calculated using the ΔΔCt method (Ct = threshold cycle). Each gene was normalized with the corresponding average of Sdha and Ywahz expression in the same animal and expressed as the fold-diff erence in relation to the control group (paper IV).

Statistical methodsPaper II

The mean and standard error of the mean were used as measures of central tendency and variation respectively throughout the study. Multivariate analy-sis of variance (ANOVA) was used for signi icance testing (SYSTAT version 11, Systat Software Inc.) using galanin concentration and infarction size, respec-tively, as dependent variable and body temperature, observation periods and ischemia as independent factors. We refrained from making detailed multiple comparisons e.g. between regions and groups, since they were not an inherent part of the original hypothesis. p < 0.05 were considered signi icant.

Paper III

The mean and standard error of the mean were used as measures of central tendency and variation, respectively, throughout the study. Multivariate analy-sis of variance (ANOVA) and Tukey´s post hoc test were used for signi icance testing (SYSTAT version 11, Systat Software Inc.). p < 0.05 were considered sig-ni icant.

Paper IV

Data were analyzed using two-tailed t test for independent samples, only of the groups deemed of interest when the study was designed. A p < 0.05 was consi-dered statistically signi icant. Data are reported as mean± SEM.

55

Results and discussion

Results and discussionStroke model – methodological aspects (paper I)��

��

��

�� !��

��

��

��"#$o��%��%��

� ��"#$��&��

��'�� !�� "#$��

��(��

��)�� *++,�� -�� .��(��*+/0&�

1�� *+/+�� 2 �� *++3�&�2 ��

�� *++3��

� ��

��

�� 4��

��

� �� 5��"#$�� -��6��

��*+3,��6�� *+3,� �$�

��

��*+/*��

�� "#$��(��

�� -�� &��

��

�� !��

��

��

��

��(��

��

7�'��

8��$9"��:� �*��#��;� ��<�;� �� 4��

��

�� %�� %� ��

�� 7��"��=��

��>��4��1��?��8��

��"��-��(��/��+/@+3$��6��

��A��4��7�� A��B��C@� �

$�� "#$��

56


lenticulostriate artery and the arterial branch of the rhinal cortex, causing an ischemic damage to cortex and striatum. Since it is of uttermost importance that the microclip maintains its mechanical stability and strength, it has been regularly checked for its biomechanical properties at the Department of Medi-cal Engineering.

Using a stroke model with craniectomy and microclip occlusion of the MCA in-volves several challenges and risks for complications speci ic for this model. A subtemporal approach may cause bleedings from the structures passed to reach the MCA, temperature elevation of the cortex when drilling a hole in the skull, brain contusion, changes in intracranial pressure, damage to and blee-ding from the MCA (0.24 mm in diameter), leakage of cerebrospinal luid and infections. After acquiring the necessary technical skills, bleeding is the most common complication. We have not observed any complications of infection post operatively, nor cerebrospinal luid leakage.

An advantage of a direct microclipping method is that occlusion of the artery is visually veri ied, compared to the intraluminal ilament method, where occlu-sion of other cerebral vessel ori ices and insuf icient MCAo are complications described (Schmid-Elsaesser et al. 1998; Furuya et al. 2004), as well as SAH and endothelial lesions causing thrombotic complication. A mortality rate up to 92.7% within 48h after MCAo have been reported when a silicone rubber cylin-der is attached to the ilament (Nagasawa et al. 1989). Spontaneous hyperther-mia up to 40oC early after MCAo with the ilament method has been observed (Li et al. 1999; Gerriets et al. 2003). Increased body temperature is associated with damage to the hypothalamus and is likely the result of reduced blood low to this brain region due to incorrect placed ilament.

The visually controlled temporary MCAo between the rhino-cortical branch and the lenticulostriate artery results in small ischemic brain lesions and short-term (hours) neurological de icits allowing the animals to thrive well for at least 2 weeks. The size of the ischemic lesions are small – less than 10% of the total slice area and variably engaging cortex and/or striatum, predominantly the lateral part. The lesions are largest at bregma and 2 mm anterior and pos-

Figure 23The specially designed and modifi ed reusable microclip.

57


terior to bregma. There is a diff erence in collateral circulation between species, and in the rat – the animal used in the current stroke model – the variability in MCA and its branches including collateral anastomoses is well known (Oliff et al. 1995a; Oliff et al. 1995b; Oliff et al. 1997). These circumstances are likely to play an important role in the observed variation in the size of the ischemic lesions observed in our studies.

Anesthesia model – methodological aspects (paper I)When we started developing the present method most studies in the ield used intraperitoneal anesthesia (Bederson et al. 1986b; Goldlust et al. 1996; Simp-kins et al. 1997; Dubal et al. 1998; Wise 2000; Wise et al. 2000; Ardehali et al. 2003) and spontaneous respiration. This reason, in combination with the fact that no other anesthetic techniques were available at our Local Animal Department at that time, led us to an initial use of intraperitoneal anesthesia by chloral hydrate. However, we used oxygen ventilation by tracheotomy. With this method we experienced dif iculties in maintaining control of anesthesia during the surgical procedure, both concerning the time from induction to suf-icient anesthetic depth and the period of time when an additional dose of the

anesthetic agent was needed. Furthermore, the rats – in our experience - re-covered slowly from anesthesia, and often developed respiratory depression and hypotension after the surgery and extubation. Contributing factors to this could be inadvertent injections into the gut and urinary bladder (Zausinger et al. 2002). Thus, using intraperitoneal drug administration increased the risk of over as well as under dosage. Considerable dif iculties in controlling the anes-thesia and a mortality of about 25% in the 40 initially operated rats prompted us to abandon intraperitonela anesthesia by chloral hydrate and ventilation by tracheotomy in favour of endotracheal intubation and iso lurane anesthesia (1% iso lurane in 30%/70% O2/N2O).

The techniques for endotracheal intubation was described in the literature (Jaff e et al. 1973; Pena et al. 1980). However, the crucial equipment needed was not commercially available. Therefore we designed, and the Department of Medical Engineering produced the modi ied laryngoscope (initially designed for use in neonates) and the necessary sloping board extending the rat neck, facilitating the insertion of the tube into trachea (Figure 24).

The use of face-mask and spontaneously ventilated rats was not chosen, since this method critically aff ects the outcome after cerebral ischemia through in-traoperative physiological parameters like blood pressure (Patel et al. 1991; Chileuitt et al. 1996), arterial blood content of O2 and CO2 (Browning et al. 1997; Smrcka et al. 1998) and pH in blood (Siesjö et al. 1996). Zausinger and co-workers (2002) reported a signi icantly increased ischemic damage and a higher mortality rate in spontaneously breathing SD male rats (using inhalation of halothane administered with a face mask, as well as intraperitoneal chloral hydrate anesthesia) compared to mechanically ventilated rats with halothane

58


anesthesia in a transient ilament stroke model. In addition to the apparent risk of hypercapnia, acidosis and low blood pressure in luencing the inal outcome after an ischemic insult, there are other draw backs of the face mask method; e.g. inadequate itting masks and interference of the mask with the proper ac-cess to the operating area. Furthermore, there is an apparent risk of waste of anesthetic gases and hazardous pollution of the operating area, if an operation mask is used.

Modern available inhalational agents at the time of our start up were iso lu-rane, haltothane and sevo lurane, all reported to exert a degree of neuropro-tection similar to that of barbiturates in the setting of focal cerebral ischemia (Zausinger et al. 2002). The underlying mechanisms of neuroprotection may be related to the excitotoxic glutamate response (Martin et al. 1995), prostanoid production (Moore et al. 1994) and NO synthesis (Tobin et al. 1994). Our choi-ce was iso lurane and we use it in our experimental stroke model throughout all studies.

To avoid stress prior anesthesia the rats were kept in their own cages with free access to water and standard chow until transferred to the induction chamber for the anesthesia and subsequent anesthesia induction. Other groups report pre operative fasting (Yang et al. 1994) or only access to water (Zausinger et al. 2002). An important reason for preanesthesia fasting is to reduce the risk of vomiting and aspiration of vomit. Since rats do not vomit there is no risk for these complications (Wang-Fischer 2009). This is also easily veri ied in prac-tice.

Figure 24Specially designed sloping intubation board (A) and the modifi ed neonate laryngoscope (B). The size of the instruments is detailed (mm) in the fi gure. The lump of metal behind the sloping board itself gives the instrumen-tation the necessary weight to stand still during the intubation procedure.

59


General methodological aspects (paper I)Intra- and postoperative mortalityWhen we started developing the present stroke model, we experienced an overall rat mortality of 25% when performing MCAo. Morbidity and mortality in studies employing experimental stroke models are crucial confounding fac-tors, which unfortunately are rarely comprehensively reported. This – in our opinion – high mortality of 25% prompted us to abandon this anesthesia met-hod and to develop a method of endotracheal intubation and iso lurane anes-thesia. These anesthetic techniques reduced the intraoperative and irst 24 hours post-operative mortality from 25% to 10.6%. Improved overall skills in the anesthesia and operation methods further reduced the mortality to 2.7%.

Blood glucoseThe level of blood glucose is an important factor to be monitored and maintai-ned in the normal range in experimental animal stroke models, since hypergly-cemia is reported to impair outcome of stroke both in humans and in animal studies (Kagansky et al. 2001; Johan Groeneveld et al. 2002). When we desig-ned our stroke model we measured blood glucose in about 30-40 rats using the Hemocue® apparatus, and all samples were found within normal range (He-mocue, Ängelholm, Sweden). A reasonable explanation to this was that the rats were allowed free access to rat chow until anesthesia was induced. Unfortuna-tely the current blood gas analyzer (AVL OPTI 1 Medical Nordic AB, Stockholm, Sweden) we are using does not measure blood glucose and is the main reason, why this parameter is not reported in the studies.

Animal sexThe majority of experimental animal stroke studies are performed in male rats, avoiding the impact of the cyclic variation in ovarial hormone concentrations in females known to in luence the ischemic brain damage (Ström et al. 2010). When submitting manuscripts we have, surprisingly enough and at repeated occasions, got the question why our studies were done on female rats. Spec-trum, concentrations and cyclicity of steroid hormones are evidently widely diff erent in female and male rats. However, all our previous experience in the ield relates to female rats (Hilke et al. 2005; Theodorsson et al. 2005a; Theo-

dorsson et al. 2005c; Ström et al. 2008a; Ström et al. 2008b; Ström et al. 2009; Ström et al. 2010), which prompted us to use female rats. It is evident that a direct comparison of on our own data with studies made on male rats cannot be made.

Effect of animal strain and vendorThe rat is the species most frequently used in experimental animal stroke mo-dels, even though mice and gerbils are studied as well. It is known that genetic factors in luence the ischemic brain damage in animal stroke models. For ex-ample, using SHR and stroke-prone SHRs; e.g. genetically hypertensive strains, usually results in larger ischemic brain lesions and lower infarct size variabi-

60


lity than reported in normotensive strains (Brint et al. 1988; Duverger et al. 1988). Furthermore, intra-strain diff erences in cerebral infarction after MCAo depending on the animals’ origin/vendor is reported (Oliff et al. 1995a). We have used the same strain (SD females) in all studies and from the same vendor, except for paper IV where the animals were delivered from another breeder.

Age of animalsYoung adult healthy animals are routinely used in animal stroke models (Glad-stone et al. 2002), including ours, despite the importance of aging in cerebro-vascular disease in humans. Davis et al. (1995) report an age-dependent in-crease in the cerebral ischemic damage in a focal stroke model in male Wistar rats, indicating the relevance and need for further studies in old rats.

The staining method for measuring the size of the ischemic lesionThe TTC method of measuring the brain lesion (Bederson et al. 1986a; Goldlust et al. 1996) is carefully validated for use up to 24 hours after the occlusion, delineating both the ischemic core and its surrounding penumbra (Astrup et al. 1981), and partially validated after 3 days (Lin et al. 1993; Vergouwen et al. 2000). The aff ected area is continuous and well delineated after few hours, when the lesions are extensive, and becomes more irregular in shape as the days pass and the surviving cells in the penumbra are revitalized. These irregu-lar shapes preclude the use of volume-morphometric methods, and therefore area measurements were used. Brain oedema, of cytotoxic origin, developing the irst days after the ischemic insult, may arti icially and markedly increase the infarct volume (Brint et al. 1988). Swanson and co-workers (1990) have proposed a method compensating for the oedema when measuring the infarct volume, but this approach was not used in the current method.

61


Effects of hypothermia on the ischemic brain lesion and on tissue concentrations of galanin in the stroke model (paper II)Hypothermia is known to reduce brain lesions caused by ischemia (Churchill-Davidson 1954; Holzer et al. 2010), not only when administered before and during the ischemic episode, but also afterwards (Rosomoff et al. 1954). The neuroprotective role of moderate hypothermia has been well established in humans (Hemmen et al. 2009; Yenari et al. 2010), and in experimental animals (Kawai et al. 2000; Yanamoto et al. 2001; Miyazawa et al. 2003), and clinical use of hypothermia dates back to the 1960s (Westin 2006).

Several studies have shown that galanin concentrations are increased in re-sponse to various types of lesions to the central and peripheral nervous sys-tems (Hökfelt et al. 1987; Villar et al. 1989; Cortes et al. 1990) and, as mainly shown by Wynick and collegues (2001), galanin may be amongst the factors contributing to maintenance of neuronal survival and functions (Xu et al. 1996; Holmes et al. 2000; O’Meara et al. 2000; Elliott-Hunt et al. 2004). We therefore considered it of interest to investigate whether or not hypothermia-induced alterations in galanin concentrations could constitute a part of the established neuroprotective eff ect of hypothermia in a focal and transient rat stroke model (Theodorsson et al. 2008).

Female rats were allocated to hypothermia (33°C) and normothermia (37°C) treatment during a 60 min microclip MCAo. The ischemic lesions were visua-lized using the TTC method and quanti ied after observation periods of 2 and 7 days, respectively. Concentrations of galanin were measured using a speci ic competitive radioimmunoassy in extracts from punch biopsies from various relevant brain regions. After 2 days at 33°C the ischemic lesions were generally larger than in the 37°C group, but after 7 days a signi icant reduction (more than 50%) was observed in the ischemic brain of the 33°C group lesions (p = 0.011).

This results corroborate those of Maier et al (2001) who showed that intralu-minal transient (1.5 h) MCAo during intra-ischemic hypothermia (33°C) in rats resulted in a decreased size of the brain lesions monitored after 3 days (-37% [initiated at the onset of ischemia], -48% [initiated with 90 min delay from the onset of the ischemia] and -56% [initiated with 180 min delay from the onset of ischemia]).

Most studies reporting a hypothermia-induced decrease in ischemic brain le-sions used observation periods of 1-3 days (Xue et al. 1992; Karibe et al. 1994; Maier et al. 1998; Zausinger et al. 2000) in contrast to the 7 days used in our study (Theodorsson et al. 2008). The reason for the prolonged observation pe-riod used in our study was our interest in investigating whether galanin was a reactant acting for short period of time, e.g. 2 days, or whether galanin also was

62


a long time reactant/neuroprotective/neurotrophic factor likely to act over ex-tended periods of time, e.g. 7 days.

Hypothermia induced an overall increase in the concentrations of immuno-reactive galanin (p < 0.001). The elevated galanin levels were predominantly found in the non-ischemic control hemisphere, in the HiFo, thalamus and the posterior part of parietal cortex. The galanin concentrations were lower in the ischemic hemisphere in both the normo- and hypothermic animals compared to the corresponding contra lateral intact hemisphere (p = 0.049). The factor of time, 2 respectively 7 days, did not show any signi icant diff erence regarding the galanin concentrations (p = 0.844). Multivariate analyses of variance revea-led signi icant eff ect of ischemia on the size of the ischemic brain lesions (p = 0.001) but no overall eff ect of temperature, when data from both 2 and 7 days observation periods were analysed together. Thus, the hypothermia and not the ischemic/reperfusion lesions, may explain the major part of the observed changes in galanin concentrations. Hypothermia-induced elevation in galanin concentration is therefore not likely to be amongst the major protective mecha-nisms of hypothermia.

The ischemic lesion created in our focal and transient stroke model leads to decreased galanin concentrations in the lesioned hemisphere, indicating that diff erent mechanisms of tissue damage induce opposite responses in the tis-sue concentrations of galanin. Our data support the notion that hypothermia-induced increase in tissue concentrations of galanin in the brain are the result of changes from optimal homeostatic conditions – the hypothermia-induced stress – rather than the ischemic/reperfusion lesion-induced changes in gala-nin concentrations.

A study in gerbils by transient (5min) global ischemia, showed increased gala-nin mRNA expression and galanin in the CA1-region of hippocampus 12h after ischemia, but after 4 days the galanin mRNA and glanin peptide levels were lower than in the controls (Hwang et al. 2004). However, focal transient (intr-aluminal, 1 h) MCAo in SHR resulted in upregulation of galanin mRNA in the ipsilateral vs. contralateral cortex after 24 h, but not after 6 h (Raghavendra Rao et al. 2002). In a permanent MCAo model in rats, a delayed upregulation of galanin in the peri-infarction area was reported 3 days after the ischemic epi-sode. This upregulation was barely detectable after 24 h and was not seen after 1 and 4 h (De Michele et al. 2006). Unfortunately, De Michele and co-workers do not report the changes in galanin levels in the ischemic hemisphere com-pared to the unlesioned contra lateral control hemisphere (De Michele et al. 2006). Thus, diff erent stroke models (global vs. focal, permanent vs. transient) and methods used to measure galanin responses (immunohistochemical analy-sis, in situ hybridization, quantitative radioimmunoassay methods) apparently result in diff erent conclusions regarding the responses of the brain galanin sys-tems to injury.

63


Effects of intracerebroventricular galanin and galanin receptor 2/3 agonist on the ischemic brain lesion (pa-per III)Several studies have shown upregulation of the expression of galanin mRNA and increased concentrations of galanin itself in neurons of the central and pe-ripheral nervous systems exposed to various types of lesions. An early example was the dramatic increase in the expression of galanin mRNA in the DRG neu-rons after peripheral axotomy (Hökfelt et al. 1987).

Several studies have corroborated this inding in several species and locations of the nervous system (Villar et al. 1989; White et al. 1994). Whether this lesi-on-induced increase in galanin concentrations is primarily a signal that a lesion has occurred or a consequence of the lesion or – more interestingly – a mecha-nism for limiting tissue damage or facilitating neuronal survival and repair is still an open question. The upregulation of galanin in response to injury has logically prompted several investigators to hypothesize that galanin may play a neuroprotective role (Branchek et al. 2000; Holmes et al. 2000; O’Meara et al. 2000; Elliott-Hunt et al. 2004), an eff ect primarily mediated by activation of the GalR2 receptor (Mahoney et al. 2003; Pirondi et al. 2005; Elliott-Hunt et al. 2007).

Several studies have addressed the question of whether galanin is neuropro-tective, however indirectly. We therefore considered it of interest to infuse va-rious concentrations of galanin intracerebroventricularly in a direct attempt to address the question of whether galanin has neuroprotective properties in a rat model of MCAo. We infused three diff erent concentrations of galanin into the cerebral ventricles expecting to reduce the brain lesions in response to mild ischemia – reperfusion lesions in the female rat brain. Furthermore we infu-sed, in one concentration, the GalR2/3 agonist Gal(2-11) (AR-M1896) shown to preferably bind to galanin receptor subtypes subserving neuroprotective functions; that is to elucidate whether or not the possible neuroprotective pro-perties of galanin are dependent on eff ects on a speci ic receptor subtype (Liu et al. 2001), as visualized in the current experimental paradigm.

The biological eff ects of galanin are generally inhibitory, in particular when acting through the GalR1- and -3 subtypes. Since neuronal lesions are caused by excessive release of excitatory amino acids, a possible mechanism which galanin may subserve in neuronal lesions could be to decrease release of glu-tamate at noxious concentrations. The general inhibitory properties of galanin may however also hamper the upregulation of molecules needed for neuronal repair and metabolic activity in the neurons crucial for survival. Both of these perspectives may actually be too simplistic, and what really matters may be the known direct neurotrophic eff ects exerted by galanin, superseding possible inhibitory eff ects on cell growth.

64


Biological eff ects often, but not always, show linear dose-response relations, but non-linear dose/response relation also occurs, including U-shaped curves. We therefore considered it of interest to study the dose/response relationship of the eff ect of intracerebroventricularly infused galanin on ischemic brain le-sions over a wide range of concentrations.

In the present stroke model using female rats the lesion was 98% lager se-ven days after 60 min transient MCAo and continuous intracerebroventricular administration of the GalR2/3 agonist Gal(2-11), as compared to controls. No diff erences were found after seven days in the groups treated with galanin in three diff erent concentrations (0.24, 2.4 and 24 nmol/day; p = 0.939, 0.715 and 0.977, respectively). There was also no diff erence in the size of the ischemic lesions measured after three days in the galanin-treated group (2.4 nmol/d) compared to artificial CSF (p = 0.925).

In view of the many studies cited above showing neuroprotective eff ects of ga-lanin, mostly exerted via GalR2, we expected to ind that the GalR2/3 agonist Gal(2-11) (and actually galanin – for that matter) would decrease the size of the ischemic lesion. However, the results of the current study were contrary to our initial hypothesis. Thus further studies are needed to clarify underlying mechanisms.

65


The effects of ischemic injury on galanin and galanin receptor gene expression in discrete brain regions (paper IV)Injury to neurons results in upregulation of galanin in some central and pe-ripheral systems (Hökfelt et al. 1987; Villar et al. 1989; Cortes et al. 1990), and it has been suggested that this neuropeptide may play a protective and trophic role (Holmes et al. 2000; Kerr et al. 2000; O’Meara et al. 2000; Zigmond 2001; Pirondi et al. 2005; Hobson et al. 2008). The eff ects of galanin are mediated by the activation of three G-protein-coupled galanin receptor subtypes, GalR1, GalR2 or GalR3 (Habert-Ortoli et al. 1994; Wang et al. 1998a; O’Donnell et al. 1999; Branchek et al. 2000; Waters et al. 2000). In addition, the galanin frag-ment Gal(2–11) (AR-M1896) (Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-NH2) acts as an agonist with 500-fold af inity for GalR2 versus GalR1 (Liu et al. 2001). However, Gal(2–11) can also bind and activate GalR3 as shown in transfected cell lines, with a similar af inity as to GalR2 (Lu et al. 2005).

The objective of the present study was to investigate whether temporary and focal ischemic lesions (Theodorsson et al. 2005b) to the brain in luence gene expression of galanin, GalR1, GalR2 and GalR3 in various brain regions. Quanti-tative real-time PCR was employed to study gene expression in punch-biopsies from the LC, cortex and dorsal HiFo, including sham-operated female rats as controls.

We selected three brain regions, the parietal cortex which is partly lesioned by the occlusion (extending from the main lesion in the striatum), the HiFo which is apparently unaff ected, and the LC which provides noradrenergic/galaniner-gic innervations of both these regions, probably partly representing collaterals (Room et al. 1981). This means that one and the same neuron can send aff e-rents to both parietal cortex and HiFo. A 259% increase in the galanin transcript levels and a 45% increase in GalR1 mRNA levels were found in the LC in the ischemic hemisphere compared to the intact control side seven days after a 60-min-microclip MCAo in female rats. In contrast, expression of GalR1 mRNA decreased by 35% in the parietal cortex. However, in this area the Ct values were close to - or at detection limit (average Ct 35) and caution should be made when interpreting the signi icance of these results. These low values of galanin and galanin receptor mRNA in this brain region is in accordance with previous studies using in situ hybridization. Thus, under normal circumstances transcripts for galanin (Jacobowitz et al. 1990) and galanin receptors (O´Donnell et al. 2003) could not be detected within cor-tex or in the HiFo. However, after treatment with cholchicine, a toxic natural product inhibiting microtubule polymerization and therefore resulting in an accumulation of axonally transported neurotransmitters (Dahlström 1968; Kreutzberg 1969), galanin- positive cells can be seen in cortex and hippocam-pus, as well as in striatum (Sko itsch et al. 1985; Melander et al. 1986c), and

66


in glia (Xu et al. 1992; Calza et al. 1998; Shen et al. 2003). In addition, when measuring the galanin peptide by means of radioimmunoassay, low levels of galanin were found in cortical areas (Sko itsch et al. 1986a). In the LC, on the other hand, galanin levels are around 4- and 3-fold higher compared to parietal cortex and HiFo, respectively. Immunohistochemical studies suggest that un-der normal circumstances galanin in dorsal cortical and hippocampal areas is mainly in aff erent from the brain stem, especially in NA ibers originating in the LC (Melander et al. 1986c; Gabriel et al. 1995; Xu et al. 1998a).

Several earlier studies in male rats involving diff erent stroke models and time periods have reported on expression of the galanin system after MCAo. Gen-der, species, dissection protocols and/or time schedules however, make direct comparison with the present results dif icult (Bond et al. 2002; Raghavendra Rao et al. 2002; Hwang et al. 2004; Lee et al. 2005; De Michele et al. 2006). Two array studies monitored transcripts, that is similar to our own study. Thus, Raghavendra Rao et al. (2002) performed the occlusion for 60 min followed by reperfusion for 6 or 24 h and carefully dissected cortical “MCA territory” in SHR. They found a 12.5-fold increase in galanin mRNA at 24 h but no change at 6h. They also observed a 3.2-fold increase in GalR1 at 6h and a 2.6-fold increase at 24 h. Bond et al. (2002) used normotensive SD rats and occluded the MCA for 90 min followed by reperfusion for 24 h. They found that galanin mRNA was increased in the ipsilateral versus the contra-lateral “hemisphere” (“no local dissection was performed in order to minimize any variance due to anatomy or sampling”). However, the results were dependent on reference gene. When using cyclophilin as reference gene, the galanin transcript increase was 133% and signi icant, whereas with actin there was a 54%, not signi icant (“border-line”) increase. Even if actin was stable across treatment groups, also this gene had shortcomings. Thus, arrays of MCAo brains appear dif icult.

Is there a relation between the expression of the GalR2 demonstrated in the present study and our earlier indings of increased ischemic brain lesions cau-sed by the continuous infusion of a galanin agonist, Gal(2-11), for three or se-ven days (Holm et al. 2011)? This agonist mainly acts through GalR2, which in many other studies has been shown to exert trophic eff ects (see above). Inte-restingly, in the present study there was a trend towards lower GalR2 mRNA levels in the HiFo. This may indicate that stimulation of the GalR2 increases ischemic lesions. Decreased expression of the receptor might thus contribute to the amelioration of ischemic lesions.

Taken together our data support the notion that galanin may play a role in the response of the CNS to injury, but its relation to mechanisms underlying galanin’s trophic eff ects remains to be elucidated.

67

Conclusions

Conclusions• Modern anesthesia methods using endotracheal intubation and controlled

ventilation is a crucial determinant of morbidity and mortality in the present experimental animal stroke in rats. Since morbidity and mortality are important confounders in this kind of experimental paradigms, methods for administrating anesthesia are crucial.

• Mild per-operative hypothermia during transient MCAo decreased the size of the ischemic lesions and increased the concentrations of immunoreactive galanin in the non-ischemic control hemisphere. The galanin concentrations in the ischemic hemisphere, in both normo- and hypothermic animals, were lower compared to the contralateral control hemisphere.

• Continuous administration of galanin or galanin receptor 2/3 agonist intrace-rebroventricularly increases the ischemic lesions seven days after transient MCAo.

• Transient MCAo upregulates galanin and GalR1 gene expression in the locus coeruleus.

68

69

Acknowledgements

Acknowledgements

I would like to express my sincere gratitude to everyone who has supported me and contributed to the work leading to my thesis. In particular, thanks go to:

Annette Theodorsson, my supervisor. For giving me the opportunity to work as a PhD student, and for introducing me to the fascinating microsurgical world, and shared with you to me of your great experience in neurosurgery. Thank you stood up for me in the beginning, when I had many, many questions and thoughts, which you always responded to. Thank you for trusting me when we got the opposite result, and for all the encouragement over all these years. For giving me complete freedom, but has allowed me to work hard. I have learned a lot about many things during these years, but most important – never give up!

Elvar Theodorsson, my co-supervisor. Thanks for giving me the opportunity to become a member of your team and for your trust; you have given me great freedom and also a friendly environment in the lab and for that you introduced me in the neuroscience ield. You always have time, anytime I needed it.

Tomas Hökfelt, my co-supervisor. Thank you for your immense knowledge and outstanding mind, for all your help in the exciting world of galanin and in my writing process. Thank you for introducing me to your lab.

Susanne Hilke, co-author in paper IV. My best friend! – by wet and dry – th-rough good and tough times. You have inspired me with your huge energy sour-ce and for your enthusiastic support while I needed it. Perhaps (soon) we are colleagues again.

Lisbeth Hjälle, for your support throughout this project, and for valuable dis-cussions and friendship and of course for a very nice workout buddy!

Jakob Ström, room-mate, PhD student in our research group, for all discus-sions and sometimes computer-related assistance.

Jan Szymanowski, Dan Linghammar, Linda, Cissi, Nettan, Anders, Andreas, for taking so good care of the animals and thanks for all help and advices over the years and of course thanks to the car park!

Late Katarina Åman, for having taught me the transcardic perfusion method, and also Blanca Silva-Lopez to have learned immunohistochemistry and in situ hybridization, and how to work with cryostat sections.

Agneta and Tomas at AgnTho´s AB, for giving me/shared with you your expe-rience and excellent service.

Staff at the Department of Medical Engineering, for excellent collaboration and skills in developing of instruments.

Åsa Shippert and Anette Molbaek at Cellkärnan, Core facility, for having

70

Acknowledgements

taught me up to work with the pipetting robot and the use of real time PCR machine.

Tomas Johansson, for excellent photographs.

Ludmila Mackerlova, for interesting discussions about research and technical skills, and all pep-talks.

Lena Svensson, I really appreciate your friendship and our lunch meetings, and thanks for your support during this time.

Annika Thorsell, my new friend. Thank you for your support and pushing the end of my thesis.

All staff at the Clinical Chemistry, for having made the level 11 for such a nice atmosphere to work in, nice chats during coff ee-breaks and all parties.

I would also like to thank all my friends outside the laboratory (no board and no one forgotten) for all the fun and support in daily life.

Birgitta and Bengt Lindbro, for your tremendous generosity, and for all the nice moments together within your home in Trelleborg and some memorable days in Polen.

My parents in law, Olof and Ingegerd Holm, for your love and support and that I always feel welcome within your home and for all dinners and for your helpfulness and that you are always there. My brother-in-law Michael Holm with family, for nice times.

I wish to thank my mother Carin Petersson, for your caring support and your never ending love. Thank you also for never hesitating to lend a helping hand and for all good cakes, and my brother Magnus Petersson and his family for support.

My husband Peter – the love of my life! You are my best support, friend and give encouragement. I love you! And our children Mattias and Daniel for being the joy of my life.

71

References

ReferencesAgoston, D. V., S. Komoly and M. Palkovits (1994). ”Selective up-regulation of

neuropeptide synthesis by blocking the neuronal activity: galanin expres-sion in septohippocampal neurons.” Exp Neurol 126(2): 247-255.

Agranoff , B. W. and G. J. Siegel (1999). Basic neurochemistry : molecular, cellu-lar, and medical aspects. Philadelphia, Lippincott-Raven Publishers.

Alier, K. A., Y. Chen, U. E. Sollenberg, U. Langel and P. A. Smith (2008). ”Selective stimulation of GalR1 and GalR2 in rat substantia gelatinosa reveals a cellu-lar basis for the anti- and pro-nociceptive actions of galanin.” Pain 137(1): 138-146.

Altman, F. P. (1976). ”Tetrazolium salts and formazans.” Prog Histochem Cyto-chem 9(3): 1-56.

Ardehali, M. R. and G. Rondouin (2003). ”Microsurgical intraluminal middle ce-rebral artery occlusion model in rodents.” Acta Neurol Scand 107(4): 267-275.

Arvidsson, A., T. Collin, D. Kirik, Z. Kokaia and O. Lindvall (2002). ”Neuronal replacement from endogenous precursors in the adult brain after stroke.” Nat Med 8(9): 963-970.

Astrup, J., B. K. Siesjö and L. Symon (1981). ”Thresholds in cerebral ischemia - the ischemic penumbra.” Stroke 12(6): 723-725.

Bacon, A., F. E. Holmes, C. J. Small, M. Ghatei, S. Mahoney, S. Bloom and D. Wynick (2002). ”Transgenic over-expression of galanin in injured primary sensory neurons.” Neuroreport 13(16): 2129-2132.

Barbelivien, A., C. Vaussy, Y. Marchalant, E. Maubert, N. Bertrand, A. Beley, S. Roussel, E. T. Mackenzie and F. Dauphin (2004). ”Degeneration of the ba-salocortical pathway from the cortex induces a functional increase in gala-ninergic markers in the nucleus basalis magnocellularis of the rat.” J Cereb Blood Flow Metab 24(11): 1255-1266.

Barr, A. M., J. W. Kinney, M. N. Hill, X. Lu, S. Biros, J. Rebek, Jr. and T. Bartfai (2006). ”A novel, systemically active, selective galanin receptor type-3 li-gand exhibits antidepressant-like activity in preclinical tests.” Neurosci Lett 405(1-2): 111-115.

Barreda-Gomez, G., M. T. Giralt and R. Rodriguez-Puertas (2005). ”G protein-coupled galanin receptor distribution in the rat central nervous system.” Neuropeptides 39(3): 153-156.

Barson, J. R., I. Morganstern, et al. (2010). ”Galanin and consummatory beha-vior: special relationship with dietary fat, alcohol and circulating lipids.” EXS 102: 87-111.

Bedecs, K., M. Berthold and T. Bartfai (1995). ”Galanin--10 years with a neuro-endocrine peptide.” Int J Biochem Cell Biol 27(4): 337-349.

72

References

Bederson, J. B., L. H. Pitts, S. M. Germano, M. C. Nishimura, R. L. Davis and H. M. Bartkowski (1986a). ”Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quanti ication of experimental cerebral infarction in rats.” Stroke 17(6): 1304-1308.

Bederson, J. B., L. H. Pitts, M. Tsuji, M. C. Nishimura, R. L. Davis and H. Bartkow-ski (1986b). ”Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.” Stroke 17(3): 472-476.

Belayev, L., O. F. Alonso, R. Busto, W. Zhao and M. D. Ginsberg (1996). ”Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model.” Stroke 27(9): 1616-1622; discussion 1623.

Benchenane, K., J. P. Lopez-Atalaya, M. Fernandez-Monreal, O. Touzani and D. Vivien (2004). ”Equivocal roles of tissue-type plasminogen activator in stroke-induced injury.” Trends Neurosci 27(3): 155-160.

Bernard, S. A., T. W. Gray, M. D. Buist, B. M. Jones, W. Silvester, G. Gutteridge and K. Smith (2002). ”Treatment of comatose survivors of out-of-hospital car-diac arrest with induced hypothermia.” N Engl J Med 346(8): 557-563.

Bersani, M., A. H. Johnsen, P. Hojrup, B. E. Dunning, J. J. Andreasen and J. J. Holst (1991). ”Human galanin: primary structure and identi ication of two mole-cular forms.” FEBS Lett 283(2): 189-194.

Blakeman, K. H., K. Holmberg, J. X. Hao, X. J. Xu, U. Kahl, U. Lendahl, T. Bartfai, Z. Wiesenfeld-Hallin and T. Hökfelt (2001). ”Mice over-expressing galanin have elevated heat nociceptive threshold.” Neuroreport 12(2): 423-425.

Bond, B. C., D. J. Virley, N. J. Cairns, A. J. Hunter, G. B. Moore, S. J. Moss, A. W. Mudge, F. S. Walsh, E. Jazin and P. Preece (2002). ”The quanti ication of gene expression in an animal model of brain ischaemia using TaqMan real-time RT-PCR.” Brain Res Mol Brain Res 106(1-2): 101.

Bradley, W. G. (2008). Neurology in clinical practice. Philadelphia, PA, Butter-worth-Heinemann/Elsevier.

Branchek, T. A., K. E. Smith, C. Gerald and M. W. Walker (2000). ”Galanin recep-tor subtypes.” Trends Pharmacol. Sci. 21: 109-116.

Brint, S., M. Jacewicz, M. Kiessling, J. Tanabe and W. Pulsinelli (1988). ”Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral com-mon carotid arteries.” J Cereb Blood Flow Metab 8(4): 474-485.

Browning, J. L., M. L. Heizer, M. A. Widmayer and D. S. Baskin (1997). ”Eff ects of halothane, alpha-chloralose, and pCO2 on injury volume and CSF beta-endorphin levels in focal cerebral ischemia.” Mol Chem Neuropathol 31(1): 29-42.

Buchan, A. M., D. Xue and A. Slivka (1992). ”A new model of temporary focal neocortical ischemia in the rat.” Stroke 23(2): 273-279.

73

References

Burazin, T. C., J. A. Larm, M. C. Ryan and A. L. Gundlach (2000). ”Galanin-R1 and -R2 receptor mRNA expression during the development of rat brain sug-gests diff erential subtype involvement in synaptic transmission and plasti-city.” Eur J Neurosci 12(8): 2901-2917.

Burbach, J. P. (2010). ”Neuropeptides from concept to online database www.neuropeptides.nl.” Eur J Pharmacol 626(1): 27-48.

Busch, E., K. Kruger and K. A. Hossmann (1997). ”Improved model of thrombo-embolic stroke and rt-PA induced reperfusion in the rat.” Brain Res 778(1): 16-24.

Calabresi, P., D. Centonze and G. Bernardi (2000). ”Cellular factors controlling neuronal vulnerability in the brain: a lesson from the striatum.” Neurology 55(9): 1249-1255.

Calza, L., L. Giardino and T. Hökfelt (1998). ”Thyroid hormone-dependent regu-lation of galanin synthesis in neurons and glial cells after colchicine admi-nistration.” Neuroendocrinology 68(6): 428-436.

Castillo, J., A. Davalos, J. Marrugat and M. Noya (1998). ”Timing for fever-related brain damage in acute ischemic stroke.” Stroke 29(12): 2455-2460.

Centonze, D., G. A. Mar ia, A. Pisani, B. Picconi, P. Giacomini, G. Bernardi and P. Calabresi (2001). ”Ionic mechanisms underlying diff erential vulnerability to ischemia in striatal neurons.” Prog Neurobiol 63(6): 687-696.

Chileuitt, L., K. Leber, T. McCalden and P. R. Weinstein (1996). ”Induced hyper-tension during ischemia reduces infarct area after temporary middle cere-bral artery occlusion in rats.” Surg Neurol 46(3): 229-234.

Chinard, F. P. (1978). ”Accidental hypothermia-a brief review.” J Med Soc N J 75(9): 610-614.

Churchill-Davidson, H. C. (1954). ”Hypothermia; a review of the present posi-tion.” Postgrad Med J 30(346): 394-398.

Cortes, R., M. J. Villar, A. Verhofstad and T. Hökfelt (1990). ”Eff ects of central nervous system lesions on the expression of galanin: a comparative in situ hybridization and immunohistochemical study.” Proc Natl Acad Sci U S A 87(19): 7742-7746.

Counts, S. E., S. E. Perez, et al. (2010). ”Neuroprotective role for galanin in Alzheimer’s disease.” EXS 102: 143-162.

Crawley, J. N. (2010). ”Galanin impairs cognitive abilities in rodents: relevance to Alzheimer’s disease.” EXS 102: 133-141.

Dahlström, A. (1968). ”Eff ect of colchicine on transport of amine storage granu-les in sympathetic nerves of rat.” Eur J Pharmacol 5(1): 111-113.

Davis, M., A. D. Mendelow, R. H. Perry, I. R. Chambers and O. F. James (1995). ”Experimental stroke and neuroprotection in the aging rat brain.” Stroke 26(6): 1072-1078.

De Georgia, M. A., D. W. Krieger, A. Abou-Chebl, T. G. Devlin, M. Jauss, S. M. Davis,

74

References

W. J. Koroshetz, G. Rordorf and S. Warach (2004). ”Cooling for Acute Ische-mic Brain Damage (COOL AID): a feasibility trial of endovascular cooling.” Neurology 63(2): 312-317.

De Michele, M., G. Sancesario, D. Toni, A. Ciuff oli, G. Bernardi and G. Sette (2006). ”Speci ic expression of galanin in the peri-infarct zone after permanent fo-cal cerebral ischemia in the rat.” Regul Pept 134(1): 38-45.

del Zoppo, G. J. (1990). ”Relevance of focal cerebral ischemia models. Expe-rience with ibrinolytic agents.” Stroke 21(12 Suppl): IV155-160.

Ding, X., D. MacTavish, S. Kar and J. H. Jhamandas (2006). ”Galanin attenuates beta-amyloid (Abeta) toxicity in rat cholinergic basal forebrain neurons.” Neurobiol Dis 21(2): 413-420.

Dirnagl, U. (2010). Rodent models of stroke. [Totowa, N.J.], Humana.Dirnagl, U., C. Iadecola and M. A. Moskowitz (1999). ”Pathobiology of ischaemic

stroke: an integrated view.” Trends Neurosci 22(9): 391-397.Dittmar, M. S., B. Vatankhah, N. P. Fehm, G. Schuierer, U. Bogdahn, M. Horn and

F. Schlachetzki (2006). ”Fischer-344 rats are unsuitable for the MCAO i-lament model due to their cerebrovascular anatomy.” J Neurosci Methods 156(1-2): 50-54.

Dubal, D. B., M. L. Kashon, L. C. Pettigraw, J. M. Ren, S. P. Finkelstein, S. W. Rau and P. M. Wise (1998). ”Estradiol protects against ischemic injury.” J. Cereb. Blood Flow Metab. 18: 1253-1258.

Duverger, D. and E. T. MacKenzie (1988). ”The quanti ication of cerebral infar-ction following focal ischemia in the rat: in luence of strain, arterial pres-sure, blood glucose concentration, and age.” J Cereb Blood Flow Metab 8(4): 449-461.

Eberspacher, E., C. Werner, K. Engelhard, M. Pape, L. Laacke, D. Winner, R. Holl-weck, P. Hutzler and E. Kochs (2005). ”Long-term eff ects of hypothermia on neuronal cell death and the concentration of apoptotic proteins after incomplete cerebral ischemia and reperfusion in rats.” Acta Anaesthesiol Scand 49(4): 477-487.

Elliott-Hunt, C. R., B. Marsh, A. Bacon, R. Pope, P. Vanderplank and D. Wynick (2004). ”Galanin acts as a neuroprotective factor to the hippocampus.” Proc Natl Acad Sci U S A 101(14): 5105-5110.

Elliott-Hunt, C. R., R. J. Pope, P. Vanderplank and D. Wynick (2007). ”Activation of the galanin receptor 2 (GalR2) protects the hippocampus from neuronal damage.” J Neurochem 100(3): 780-789.

Erecinska, M., M. Thoresen and I. A. Silver (2003). ”Eff ects of hypothermia on energy metabolism in Mammalian central nervous system.” J Cereb Blood Flow Metab 23(5): 513-530.

Evans, H. F. and J. Shine (1991). ”Human galanin: molecular cloning reveals a unique structure.” Endocrinology 129(3): 1682-1684.

75

References

Fawcett, J. W. (2006). ”The glial response to injury and its role in the inhibition of CNS repair.” Adv Exp Med Biol 557: 11-24.

Fisher, M. and R. Ratan (2003). ”New perspectives on developing acute stroke therapy.” Ann Neurol 53(1): 10-20.

Fisone, G., C. F. Wu, S. Consolo, O. NordStröm, N. Brynne, T. Bartfai, T. Melander and T. Hökfelt (1987). ”Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vi-tro studies.” Proc Natl Acad Sci U S A 84(20): 7339-7343.

Furuya, K., N. Kawahara, K. Kawai, T. Toyoda, K. Maeda and T. Kirino (2004). ”Proximal occlusion of the middle cerebral artery in C57Black6 mice: re-lationship of patency of the posterior communicating artery, infarct evolu-tion, and animal survival.” J Neurosurg 100(1): 97-105.

Futrell, N., C. Millikan, B. D. Watson, W. D. Dietrich and M. D. Ginsberg (1989). ”Embolic stroke from a carotid arterial source in the rat: pathology and clinical implications.” Neurology 39(8): 1050-1056.

Gabriel, S. M., P. J. Knott and V. Haroutunian (1995). ”Alterations in cerebral cor-tical galanin concentrations following neurotransmitter-speci ic subcorti-cal lesions in the rat.” J Neurosci 15(8): 5526-5534.

Gainer, H. (1981). ”The biology of neurosecretory neurons.” Adv Biochem Psy-chopharmacol 28: 5-20.

Gerriets, T., F. Li, M. D. Silva, X. Meng, M. Brevard, C. H. Sotak and M. Fisher (2003). ”The macrosphere model: evaluation of a new stroke model for permanent middle cerebral artery occlusion in rats.” J Neurosci Methods 122(2): 201-211.

Ginsberg, M. D. (1997). Hypothermic neuroprotection in cerebral ischemia. Pri-mer on Cerebrovascular diseases. K. M. A. Welch, L. Caplan and D. J. Reis. San Diego, Academic Press: 272-275.

Ginsberg, M. D. (2003). ”Adventures in the pathophysiology of brain ischemia: penumbra, gene expression, neuroprotection: the 2002 Thomas willis lec-ture.” Stroke 34(1): 214-223.

Ginsberg, M. D. and R. Busto (1989). ”Rodent models of cerebral ischemia.” Stroke 20(12): 1627-1642.

Gladstone, D. J., S. E. Black and A. M. Hakim (2002). ”Toward wisdom from fai-lure: lessons from neuroprotective stroke trials and new therapeutic direc-tions.” Stroke 33(8): 2123-2136.

Glenner, G. G. (1969). Tetrazolium salts. H.J. Conn´s Biological Stains. R. D. Little. Baltimore, Williams & Wilkins: 154-162.

Goetz, C. G. (2003). Textbook of Clinical Neurology. New York, Elsevier Science.Goetz, C. G. and E. J. Pappert (1999). Textbook of clinical neurology. Philadel-

phia, W.B. Saunders.Goldlust, E. J., R. P. Paczynski, Y. Y. He, C. Y. Hsu and M. P. Goldberg (1996). ”Auto-

76

References

mated measurement of infarct size with scanned images of triphenyltetra-zolium chloride- stained rat brains.” Stroke 27: 1657-1662.

Gonzalez-Ibarra, F. P., J. Varon and E. G. Lopez-Meza (2011). ”Therapeutic hy-pothermia: critical review of the molecular mechanisms of action.” Front Neurol 2: 4.

Gorelick, P. B., R. L. Sacco, D. B. Smith, M. Alberts, L. Mustone-Alexander, D. Ra-der, J. L. Ross, E. Raps, M. N. Ozer, L. M. Brass, et al. (1999). ”Prevention of a irst stroke: a review of guidelines and a multidisciplinary consensus state-

ment from the National Stroke Association.” JAMA 281(12): 1112-1120.Grimmelikhuijzen, C. J. (1983). ”Coexistence of neuropeptides in hydra.” Neu-

roscience 9(4): 837-845.Group, H. a. C. A. S. (2002). ”Mild therapeutic hypothermia to improve the neu-

rologic outcome after cardiac arrest.” N Engl J Med 346(8): 549-556.Gu, X. L., Y. G. Sun and L. C. Yu (2007). ”Involvement of galanin in nociceptive

regulation in the arcuate nucleus of hypothalamus in rats with mononeuro-pathy.” Behav Brain Res 179(2): 331-335.

Gubern, C., O. Hurtado, R. Rodriguez, J. R. Morales, V. G. Romera, M. A. Moro, I. Lizasoain, J. Serena and J. Mallolas (2009). ”Validation of housekeeping ge-nes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia.” BMC Mol Biol 10: 57.

Gupta, R., A. H. Tayal, E. I. Levy, E. Cheng-Ching, A. Rai, D. S. Liebeskind, A. J. Yoo, D. P. Hsu, M. M. Rymer, O. O. Zaidat, et al. (2011). ”Intra-arterial thromboly-sis or Stent Placement during Endovascular Treatment for Acute Ischemic Stroke Leads to the Highest Recanalization Rate: Results of a Multi-center Retrospective Study.” Neurosurgery Article in Press.

Habert-Ortoli, E., B. Amiranoff , I. Loquet, M. Laburthe and J. F. Mayaux (1994). ”Molecular cloning of a functional human galanin receptor.” Proc Natl Acad Sci U S A 91(21): 9780-9783.

Hagerdal, M., J. Harp, L. Nilsson and B. K. Siesjö (1975). ”The eff ect of indu-ced hypothermia upon oxygen consumption in the rat brain.” J Neurochem 24(2): 311-316.

Hemmen, T. M. and P. D. Lyden (2009). ”Hypothermia after acute ischemic stro-ke.” J Neurotrauma 26(3): 387-391.

Hilke, S., A. Theodorsson, S. Fetissov, K. Aman, L. Holm, T. Hökfelt and E. Theo-dorsson (2005). ”Estrogen induces a rapid increase in galanin levels in female rat hippocampal formation--possibly a nongenomic/indirect eff ect.” Eur J Neurosci 21(8): 2089-2099.

Hill, N. C., C. H. Millikan, K. G. Wakim and G. P. Sayre (1955). ”Studies in cere-brovascular disease. VII. Experimental production of cerebral infarction by intracarotid injection of homologous blood clot; preliminary report.” Mayo Clin Proc 30(26): 625-633.

77

References

Hobson, S. A., A. Bacon, C. R. Elliot-Hunt, F. E. Holmes, N. C. Kerr, R. Pope, P. Van-derplank and D. Wynick (2008). ”Galanin acts as a trophic factor to the cen-tral and peripheral nervous systems.” Cell Mol Life Sci 65(12): 1806-1812.

Hobson, S. A., A. Bacon, et al. (2010). ”Galanin acts as a trophic factor to the central and peripheral nervous systems.” EXS 102: 25-38.

Hökfelt, T., T. Bartfai and F. Bloom (2003). ”Neuropeptides: opportunities for drug discovery.” Lancet Neurol 2(8): 463-472.

Hökfelt, T., C. Broberger, Z. Q. Xu, V. Sergeyev, R. Ubink and M. Diez (2000). ”Neu-ropeptides--an overview.” Neuropharmacology 39(8): 1337-1356.

Hökfelt, T., O. Johansson, A. Ljungdahl, J. M. Lundberg and M. Schultzberg (1980). ”Peptidergic neurones.” Nature 284(5756): 515-521.

Hökfelt, T., Z. Wiesenfeld-Hallin, M. Villar and T. Melander (1987). ”Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after pe-ripheral axotomy.” Neurosci Lett 83(3): 217-220.

Holm, L., E. Theodorsson, T. Hökfelt and A. Theodorsson (2011). ”Eff ects of in-tracerebroventricular galanin or a galanin receptor 2/3 agonist on the le-sion induced by transient occlusion of the middle cerebral artery in female rats.” Neuropeptides 45(1):17-23.

Holmes, P. V. and J. N. Crawley (1996). ”Olfactory bulbectomy increases prepro-galanin mRNA levels in the rat locus coeruleus.” Brain Res Mol Brain Res 36(1): 184-188.

Holmes, F. E., S. Mahoney, V. R. King, A. Bacon, N. C. Kerr, V. Pachnis, R. Curtis, J. V. Priestley and D. Wynick (2000). ”Targeted disruption of the galanin gene reduces the number of sensory neurons and their regenerative capacity.” Proc Natl Acad Sci U S A 97(21): 11563-11568.

Holzer, A. J., M. Holzer, H. Herkner and M. Müller (2010). Hypothermia for neu-roprotection in adults after cardiopulmonary resuscitation, The Cochrane Collaboration.

Howells, D. W. and G. A. Donnan (2010a). ”Where will the next generation of stroke treatments come from?” PLoS Med 7(3): e1000224.

Howells, D. W., M. J. Porritt, S. S. Rewell, V. O’Collins, E. S. Sena, H. B. van der Worp, R. J. Traystman and M. R. Macleod (2010b). ”Diff erent strokes for dif-ferent folks: the rich diversity of animal models of focal cerebral ischemia.” J Cereb Blood Flow Metab 30(8): 1412-1431.

Hoyte, L., J. Kaur and A. M. Buchan (2004). ”Lost in translation: taking neuro-protection from animal models to clinical trials.” Exp Neurol 188(2): 200-204.

Hua, X. Y., C. S. Hayes, A. Hofer, B. Fitzsimmons, K. Kilk, U. Langel, T. Bartfai and T. L. Yaksh (2004). ”Galanin acts at GalR1 receptors in spinal antinociception: synergy with morphine and AP-5.” J Pharmacol Exp Ther 308(2): 574-582.

Hua, X. Y., K. F. Salgado, G. Gu, B. Fitzsimmons, I. Kondo, T. Bartfai and T. L. Yaksh

78

References

(2005). ”Mechanisms of antinociception of spinal galanin: how does gala-nin inhibit spinal sensitization?” Neuropeptides 39(3): 211-216.

Hudgins, W. R. and J. H. Garcia (1970). ”Transorbital approach to the middle ce-rebral artery of the squirrel monkey: a technique for experimental cerebral infarction applicable to ultrastructural studies.” Stroke 1(2): 107-111.

Hvid, H., C. T. Ekström, S. Vienberg, M. B. Oleksiewicz and R. Klop leisch (2010). ”Identi ication of stable and oestrus cycle-independent housekeeping ge-nes in the rat mammary gland and other tissues.” Vet J. Article in Press

Hwang, I. K., K. Y. Yoo, D. S. Kim, S. G. Do, Y. S. Oh, T. C. Kang, B. H. Han, J. S. Kim and M. H. Won (2004). ”Expression and changes of galanin in neurons and microglia in the hippocampus after transient forebrain ischemia in gerbils.” Brain Res 1023(2): 193-199.

Inamasu, J., S. Suga, S. Sato, T. Horiguchi, K. Akaji, K. Mayanagi and T. Kawase (2001). ”Intra-ischemic hypothermia attenuates intercellular adhesion molecule-1 (ICAM-1) and migration of neutrophil.” Neurol Res 23(1): 105-111.

Jacobowitz, D. M., A. Kresse and G. Sko itsch (2004). ”Galanin in the brain: che-moarchitectonics and brain cartography--a historical review.” Peptides 25(3): 433-464.

Jacobowitz, D. M. and G. Sko itsch (1990). ”Localization of galanin cell bodies in the brain by immunocytochemistry and in situ hybridization. In: Galanin – A new multifunctional peptide in the neuro-endocrine system.” Hökfelt, T., Bartfai, D., Jacoboswitz and D., Ottoson, eds., Wenner-Gren International Symposium Series 58, Macmillan Press Houndsmill, London.: pp. 69-92.

Jaff e, R. A. and M. J. Free (1973). ”A simple endotracheal intubation technic for inhalation anesthesia of the rat.” Lab Anim Sci 23(2): 266-269.

Jimenez-Andrade, J. M., L. Lundström, U. E. Sollenberg, U. Langel, G. Castane-da-Hernandez and S. M. Carlton (2006). ”Activation of peripheral galanin receptors: diff erential eff ects on nociception.” Pharmacol Biochem Behav 85(1): 273-280.

Johan Groeneveld, A. B., A. Beishuizen and F. C. Visser (2002). ”Insulin: a won-der drug in the critically ill?” Crit Care 6(2): 102-105.

Kagansky, N., S. Levy and H. Knobler (2001). ”The role of hyperglycemia in acu-te stroke.” Arch Neurol 58(8): 1209-1212.

Kaneko, D., N. Nakamura and T. Ogawa (1985). ”Cerebral infarction in rats using homologous blood emboli: development of a new experimental model.” Stroke 16(1): 76-84.

Karibe, H., J. Chen, G. J. Zarow, S. H. Graham and P. R. Weinstein (1994). ”Delayed induction of mild hypothermia to reduce infarct volume after temporary middle cerebral artery occlusion in rats.” J Neurosurg 80(1): 112-119.

Kask, K., U. Langel and T. Bartfai (1995). ”Galanin--a neuropeptide with inhibi-

79

References

tory actions.” Cell Mol Neurobiol 15(6): 653-673.Kastin, A. J. (2006). Handbook of biologically active peptides. Amsterdam ; Lon-

don, Academic Press.Kawai, N., M. Okauchi, K. Morisaki and S. Nagao (2000). ”Eff ects of delayed in-

traischemic and postischemic hypothermia on a focal model of transient cerebral ischemia in rats.” Stroke 31(8): 1982-1989; discussion 1989.

Kerr, B. J., W. B. Caff erty, Y. K. Gupta, A. Bacon, D. Wynick, S. B. McMahon and S. W. Thompson (2000). ”Galanin knockout mice reveal nociceptive de icits following peripheral nerve injury.” Eur J Neurosci 12(3): 793-802.

Klavdieva, M. M. (1995). ”The history of neuropeptides I.” Front Neuroendocri-nol 16(4): 293-321.

Klavdieva, M. M. (1996a). ”The history of neuropeptides II.” Front Neuroendo-crinol 17(1): 126-153.

Klavdieva, M. M. (1996b). ”The history of neuropeptides III.” Front Neuroendo-crinol 17(2): 155-179.

Klavdieva, M. M. (1996c). ”The history of neuropeptides IV.” Front Neuroendo-crinol 17(3): 247-280.

Koizumi, J. Y., Y. Nakaqawa, T. Ooneda, G. (1986). ”Experimental studies of ische-mic brain edema: 1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area.” Jpn J Stroke 8: 1-8.

Kordower, J. H. and E. J. Mufson (1990). ”Galanin-like immunoreactivity within the primate basal forebrain: diff erential staining patterns between humans and monkeys.” J Comp Neurol 294(2): 281-292.

Kreutzberg, G. W. (1969). ”Neuronal dynamics and axonal low. IV. Blockage of intra-axonal enzyme transport by colchicine.” Proc Natl Acad Sci U S A 62(3): 722-728.

Krieger, D. W., M. A. De Georgia, A. Abou-Chebl, J. C. Andrefsky, C. A. Sila, I. L. Katzan, M. R. Mayberg and A. J. Furlan (2001). ”Cooling for acute ischemic brain damage (cool aid): an open pilot study of induced hypothermia in acute ischemic stroke.” Stroke 32(8): 1847-1854.

Kudo, M., A. Aoyama, S. Ichimori and N. Fukunaga (1982). ”An animal model of cerebral infarction. Homologous blood clot emboli in rats.” Stroke 13(4): 505-508.

Kuteeva, E., T. Hökfelt, T. Wardi, T. Ögren, S. O. (2010). ”Galanin, galanin receptor subtypes and depression-like behaviour.” EXS 102: 163-181.

Kuteeva, E., T. Wardi, L. Lundström, U. Sollenberg, U. Langel, T. Hökfelt and S. O. Ögren (2008). ”Diff erential role of galanin receptors in the regulation of depression-like behavior and monoamine/stress-related genes at the cell body level.” Neuropsychopharmacology 33(11): 2573-2585.

Laing, R. J., J. Jakubowski and R. W. Laing (1993). ”Middle cerebral artery occlu-

80

References

sion without craniectomy in rats. Which method works best?” Stroke 24(2): 294-297; discussion 297-298.

Lang, R., A. L. Gundlach and B. Ko ler (2007). ”The galanin peptide family: re-ceptor pharmacology, pleiotropic biological actions, and implications in health and disease.” Pharmacol Ther 115(2): 177-207.

Lee, H. Y., I. K. Hwang, D. H. Kim, J. H. Kim, C. H. Kim, B. O. Lim, T. C. Kang, K. H. Bang, N. S. Seong, H. J. Lee, et al. (2005). ”Ischemia-related changes in gala-nin expression in the dentate hilar region after transient forebrain ischemia in gerbils.” Exp Anim 54(1): 21-27.

Lerner, J. T., R. Sankar, et al. (2010). ”Galanin and epilepsy.” EXS 102: 183-194.Li, F., T. Omae and M. Fisher (1999). ”Spontaneous hyperthermia and its mecha-

nism in the intraluminal suture middle cerebral artery occlusion model of rats.” Stroke 30(11): 2464-2470; discussion 2470-2461.

Lin, T.-N., Y. Y. He, G. Wu, M. Khan and C. Y. Hsu (1993). ”Eff ect of Brain Edema on Infarct Volume in a Focal Cerebral Ischemia Model in Rats.” Stroke 24: 117-121.

Liu, H. X., P. Brumovsky, R. Schmidt, W. Brown, K. Payza, L. Hodzic, C. Pou, C. God-bout and T. Hökfelt (2001). ”Receptor subtype-speci ic pronociceptive and analgesic actions of galanin in the spinal cord: selective actions via GalR1 and GalR2 receptors.” Proc Natl Acad Sci U S A 98(17): 9960-9964.

Liu, H. X. and T. Hökfelt (2002). ”The participation of galanin in pain processing at the spinal level.” Trends Pharmacol Sci 23(10): 468-474.

Longa, E. Z., P. R. Weinstein, S. Carlson and R. Cummins (1989). ”Reversible middle cerebral artery occlusion without craniectomy in rats.” Stroke 20(1): 84-91.

Lu, X., L. Lundström and T. Bartfai (2005). ”Galanin (2-11) binds to GalR3 in transfected cell lines: limitations for pharmacological de inition of receptor subtypes.” Neuropeptides 39(3): 165-167.

Lundberg, J. M. and T. Hökfelt (1986a). ”Multiple co-existence of peptides and classical transmitters in peripheral autonomic and sensory neurons--func-tional and pharmacological implications.” Prog Brain Res 68: 241-262.

Lundberg, J. M., A. Rudehill, A. Sollevi, E. Theodorsson-Norheim and B. Ham-berger (1986b). ”Frequency- and reserpine-dependent chemical coding of sympathetic transmission: diff erential release of noradrenaline and neuro-peptide Y from pig spleen.” Neurosci Lett 63(1): 96-100.

Lundström, L., A. Elmquist, T. Bartfai and U. Langel (2005a). ”Galanin and its re-ceptors in neurological disorders.” Neuromolecular Med 7(1-2): 157-180.

Lundström, L., X. Lu, U. Langel and T. Bartfai (2005b). ”Important pharmacop-hores for binding to galanin receptor 2.” Neuropeptides 39(3): 169-171.

Lundy, E. F., B. S. Solik, R. S. Frank, P. S. Lacy, D. J. Combs, G. B. Zelenock and L. G. D’Alecy (1986). ”Morphometric evaluation of brain infarcts in rats and

81

References

gerbils.” J Pharmacol Methods 16(3): 201-214.Lyden, P. D., J. A. Zivin, M. Soll, M. Sitzer, J. F. Rothrock and J. Alksne (1987). ”In-

tracerebral hemorrhage after experimental embolic infarction. Anticoagu-lation.” Arch Neurol 44(8): 848-850.

Maeda, K., G. Mies, L. Olah and K. A. Hossmann (2000). ”Quantitative measure-ment of local cerebral blood low in the anesthetized mouse using intrape-ritoneal [14C]iodoantipyrine injection and inal arterial heart blood samp-ling.” J Cereb Blood Flow Metab 20(1): 10-14.

Mahoney, S. A., R. Hosking, S. Farrant, F. E. Holmes, A. S. Jacoby, J. Shine, T. P. Iismaa, M. K. Scott, R. Schmidt and D. Wynick (2003). ”The second galanin receptor GalR2 plays a key role in neurite outgrowth from adult sensory neurons.” J Neurosci 23(2): 416-421.

Maier, C. M., K. Ahern, M. L. Cheng, J. E. Lee, M. A. Yenari and G. K. Steinberg (1998). ”Optimal depth and duration of mild hypothermia in a focal model of transient cerebral ischemia: eff ects on neurologic outcome, infarct size, apoptosis, and in lammation.” Stroke 29(10): 2171-2180.

Maier, C. M., G. H. Sun, D. Kunis, M. A. Yenari and G. K. Steinberg (2001). ”De-layed induction and long-term eff ects of mild hypothermia in a focal mo-del of transient cerebral ischemia: neurological outcome and infarct size.” J Neurosurg 94(1): 90-96.

Martin, D. C., M. Plagenhoef, J. Abraham, R. L. Dennison and R. S. Aronstam (1995). ”Volatile anesthetics and glutamate activation of N-methyl-D-as-partate receptors.” Biochem Pharmacol 49(6): 809-817.

Matsuno, H., T. Uematsu, K. Umemura, Y. Takiguchi, Y. Asai, Y. Muranaka and M. Nakashima (1993). ”A simple and reproducible cerebral thrombosis model in rats induced by a photochemical reaction and the eff ect of a plasminogen-plasminogen activator chimera in this model.” J Pharmacol Toxicol Methods 29(3): 165-173.

Mazarati, A. M., R. A. Baldwin, S. Shinmei and R. Sankar (2005). ”In vivo interac-tion between serotonin and galanin receptors types 1 and 2 in the dorsal raphe: implication for limbic seizures.” J Neurochem 95(5): 1495-1503.

Mazia, D., G. Schatten and W. Sale (1975). ”Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy.” J Cell Biol 66(1): 198-200.

Melander, T., T. Hökfelt and A. Rökaeus (1986a). ”Distribution of galaninlike im-munoreactivity in the rat central nervous system.” J Comp Neurol 248(4): 475-517.

Melander, T., T. Hökfelt, A. Rökaeus, A. C. Cuello, W. H. Oertel, A. Verhofstad and M. Goldstein (1986b). ”Coexistence of galanin-like immunoreactivity with catecholamines, 5-hydroxytryptamine, GABA and neuropeptides in the rat CNS.” J Neurosci 6(12): 3640-3654.

Melander, T., C. Köhler, S. Nilsson, T. Hökfelt, E. Brodin, E. Theodorsson and T.

82

References

Bartfai (1988). ”Autoradiographic quantitation and anatomical mapping of 125I-galanin binding sites in the rat central nervous system.” J Chem Neu-roanat 1(4): 213-233.

Melander, T., W. A. Staines, T. Hökfelt, A. Rökaeus, F. Eckenstein, P. M. Salvaterra and B. H. Wainer (1985). ”Galanin-like immunoreactivity in cholinergic neu-rons of the septum-basal forebrain complex projecting to the hippocampus of the rat.” Brain Res 360(1-2): 130-138.

Melander, T., W. A. Staines and A. Rökaeus (1986c). ”Galanin-like immunoreac-tivity in hippocampal aff erents in the rat, with special reference to choliner-gic and noradrenergic inputs.” Neuroscience 19(1): 223-240.

Mennicken, F., C. Hoff ert, M. Pelletier, S. Ahmad and D. O’Donnell (2002). ”Res-tricted distribution of galanin receptor 3 (GalR3) mRNA in the adult rat central nervous system.” J Chem Neuroanat 24(4): 257-268.

Merchenthaler, I. (2010). ”Galanin and the neuroendocrine axes.” EXS 102: 71-85.

Merchenthaler, I., F. J. Lopez and A. Negro-Vilar (1993). ”Anatomy and physio-logy of central galanin-containing pathways.” Prog Neurobiol 40(6): 711-769.

Mitsukawa, K., X. Lu and T. Bartfai (2010). ”Galanin, galanin receptors, and drug targets.” EXS 102: 7-23.

Miyazawa, T., A. Tamura, S. Fukui and K. A. Hossmann (2003). ”Eff ect of mild hypothermia on focal cerebral ischemia. Review of experimental studies.” Neurol Res 25(5): 457-464.

Moore, L. E., J. R. Kirsch, M. A. Helfaer, J. R. Tobin, R. W. McPherson and R. J. Traystman (1994). ”Nitric oxide and prostanoids contribute to iso lurane-induced cerebral hyperemia in pigs.” Anesthesiology 80(6): 1328-1337.

Morilak, D. A., M. Cecchi and H. Khoshbouei (2003). ”Interactions of norepi-nephrine and galanin in the central amygdala and lateral bed nucleus of the stria terminalis modulate the behavioral response to acute stress.” Life Sci 73(6): 715-726.

Nagasawa, H. and K. Kogure (1989). ”Correlation between cerebral blood low and histologic changes in a new rat model of middle cerebral artery occlu-sion.” Stroke 20(8): 1037-1043.

Nakashima, K. and M. M. Todd (1996). ”Eff ects of hypothermia on the rate of excitatory amino acid release after ischemic depolarization.” Stroke 27(5): 913-918.

Nolan, J. P., P. T. Morley, T. L. Vanden Hoek, R. W. Hickey, W. G. Kloeck, J. Billi, B. W. Bottiger, K. Okada, C. Reyes, M. Shuster, et al. (2003). ”Therapeutic hypothermia after cardiac arrest: an advisory statement by the advanced life support task force of the International Liaison Committee on Resuscita-tion.” Circulation 108(1): 118-121.

83

References

Nunney, R. (2008). ”Inadvertent hypothermia: a literature review.” J Perioper Pract 18(4): 148, 150-142, 154.

O’Donnell, D., S. Ahmad, C. Wahlestedt and P. Walker (1999). ”Expression of the novel galanin receptor subtype GALR2 in the adult rat CNS: distinct distri-bution from GALR1.” J Comp Neurol 409(3): 469-481.

O´Donnell, D., F. Mennicken, C. Hoff ert, D. Hubatxch, M. Pelletier, P. Walker and S. Ahmad, Eds. (2003). Localization of galanin receptor subtypes in the rat CNS. Handbook of Chemical Neuroanatomy. Amsterdam, Elsevier.

O’Keeff e K, M. (1977). ”Accidental hypothermia: a review of 62 cases.” JACEP 6(11): 491-496.

O’Meara, G., U. Coumis, S. Y. Ma, J. Kehr, S. Mahoney, A. Bacon, S. J. Allen, F. Hol-mes, U. Kahl, F. H. Wang, et al. (2000). ”Galanin regulates the postnatal sur-vival of a subset of basal forebrain cholinergic neurons.” Proc Natl Acad Sci U S A 97(21): 11569-11574.

Oliff , H. S., P. Coyle and E. Weber (1997). ”Rat strain and vendor diff erences in collateral anastomoses.” J Cereb Blood Flow Metab 17(5): 571-576.

Oliff , H. S., E. Weber, G. Eilon and P. Marek (1995a). ”The role of strain/vendor diff erences on the outcome of focal ischemia induced by intraluminal midd-le cerebral artery occlusion in the rat.” Brain Res 675(1-2): 20-26.

Oliff , H. S., E. Weber, B. Miyazaki and P. Marek (1995b). ”Infarct volume varies with rat strain and vendor in focal cerebral ischemia induced by transcra-nial middle cerebral artery occlusion.” Brain Res 699(2): 329-331.

Orten, J. M. and O. W. Neuhaus (1975). Human Biochemistry. St Louis, CV Mos-by.

Overgaard, K. (1994). ”Thrombolytic therapy in experimental embolic stroke.” Cerebrovasc Brain Metab Rev 6(3): 257-286.

Patel, P. M., J. C. Drummond and D. J. Cole (1991). ”Induced hypertension during restoration of low after temporary middle cerebral artery occlusion in the rat: eff ect on neuronal injury and edema.” Surg Neurol 36(3): 195-201.

Paxinos, G. (1995). The rat nervous system. San Diego, Academic Press.Pearse, A. G. (1969). ”The cytochemistry and ultrastructure of polypeptide hor-

mone-producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept.” J Histochem Cytochem 17(5): 303-313.

Pearse, A. G. (1974). ”The APUD cell concept and its implications in pathology.” Pathol Annu 9(0): 27-41.

Pedersen, T. R., J. Kjekshus, K. Pyorala, A. G. Olsson, T. J. Cook, T. A. Musliner, J. A. Tobert and T. Haghfelt (1998). ”Eff ect of simvastatin on ischemic signs and symptoms in the Scandinavian simvastatin survival study (4S).” Am J Cardiol 81(3): 333-335.

Pena, H. and C. Cabrera (1980). ”Improved endotracheal intubation technique

84

References

in the rat.” Lab Anim Sci 30(4 Pt 1): 712-713.Pirondi, S., M. Fernandez, R. Schmidt, T. Hökfelt, L. Giardino and L. Calza (2005).

”The galanin-R2 agonist AR-M1896 reduces glutamate toxicity in primary neural hippocampal cells.” J Neurochem 95(3): 821-833.

Raghavendra Rao, V. L., K. K. Bowen, V. K. Dhodda, G. Song, J. L. Franklin, N. R. Gavva and R. J. Dempsey (2002). ”Gene expression analysis of sponta-neously hypertensive rat cerebral cortex following transient focal cerebral ischemia.” J Neurochem 83(5): 1072-1086.

Rauch, I. and B. Ko ler (2010). ”The galanin system in cancer.” EXS 102: 223-241.

Reith, J., H. S. Jorgensen, P. M. Pedersen, H. Nakayama, H. O. Raaschou, L. L. Jep-pesen and T. S. Olsen (1996). ”Body temperature in acute stroke: relation to stroke severity, infarct size, mortality, and outcome.” Lancet 347(8999): 422-425.

Robinson, R. G. (1979). ”Diff erential behavioral and biochemical eff ects of right and left hemispheric cerebral infarction in the rat.” Science 205(4407): 707-710.

Robinson, R. G., W. J. Shoemaker, M. Schlumpf, T. Valk and F. E. Bloom (1975). ”Eff ect of experimental cerebral infarction in rat brain on catecholamines and behaviour.” Nature 255(5506): 332-334.

Rökaeus, A. and M. J. Brownstein (1986). ”Construction of a porcine adrenal medullary cDNA library and nucleotide sequence analysis of two clones en-coding a galanin precursor.” Proc Natl Acad Sci U S A 83(17): 6287-6291.

Room, P., F. Postema and J. Korf (1981). ”Divergent axon collaterals of rat locus coeruleus neurons: demonstration by a luorescent double labeling techni-que.” Brain Res 221(2): 219-230.

Rosner, G., R. Graf, K. Kataoka and W. D. Heiss (1986). ”Selective functional vul-nerability of cortical neurons following transient MCA-occlusion in the cat.” Stroke 17(1): 76-82.

Rosomoff , H. L. and D. A. Holaday (1954). ”Cerebral blood low and cerebral oxygen consumption during hypothermia.” Am J Physiol 179(1): 85-88.

Ryan, M. C. and A. L. Gundlach (1996). ”Localization of preprogalanin mes-senger RNA in rat brain: identi ication of transcripts in a subpopulation of cerebellar Purkinje cells.” Neuroscience 70(3): 709-728.

Sandritter, W. and R. Jestadt (1958). ”Triphenyltetrazolium chloride (TTC) als reduktionsindikator zur makroskopischen diagnoses des frischen herzin-farktes.” Verh Dsch Ges Pathol 41(165).

Schmid-Elsaesser, R., S. Zausinger, E. Hungerhuber, A. Baethmann and H. J. Reu-len (1998). ”A critical reevaluation of the intraluminal thread model of focal cerebral ischemia: evidence of inadvertent premature reperfusion and sub-arachnoid hemorrhage in rats by laser-Doppler lowmetry.” Stroke 29(10):

85

References

2162-2170.Senut, M. C., D. Menetrey and Y. Lamour (1989). ”Cholinergic and peptidergic

projections from the medial septum and the nucleus of the diagonal band of Broca to dorsal hippocampus, cingulate cortex and olfactory bulb: a com-bined wheatgerm agglutinin-apohorseradish peroxidase-gold immunohis-tochemical study.” Neuroscience 30(2): 385-403.

Shankaran, S., A. R. Laptook, R. A. Ehrenkranz, J. E. Tyson, S. A. McDonald, E. F. Donovan, A. A. Fanaroff , W. K. Poole, L. L. Wright, R. D. Higgins, et al. (2005). ”Whole-body hypothermia for neonates with hypoxic-ischemic encephalo-pathy.” N Engl J Med 353(15): 1574-1584.

Shen, P. J., J. A. Larm and A. L. Gundlach (2003). ”Expression and plasticity of galanin systems in cortical neurons, oligodendrocyte progenitors and pro-liferative zones in normal brain and after spreading depression.” Eur J Neu-rosci 18(6): 1362-1376.

Shigeno, T., G. M. Teasdale, J. McCulloch and D. I. Graham (1985). ”Recirculation model following MCA occlusion in rats. Cerebral blood low, cerebrovascu-lar permeability, and brain edema.” J Neurosurg 63(2): 272-277.

Siegel, G. J. and B. W. Agranoff (1999). Basic neurochemistry : molecular, cellu-lar, and medical aspects. Philadelphia, Lippincott-Raven Publishers.

Siesjö, B. K. (1992). ”Pathophysiology and treatment of focal cerebral ischemia. Part I: Pathophysiology.” J. Neurosurg. 77: 169-184.

Siesjö, B. K. and F. Bengtsson (1989). ”Calcium luxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: a unifying hypothesis.” J Cereb Blood Flow Metab 9(2): 127-140.

Siesjö, B. K., K. Katsura and T. Kristian (1996). ”Acidosis-related damage.” Adv Neurol 71: 209-233; discussion 234-206.

Siesjö, B., T. Kristián and K.-I. Katsura (1998). Overview of bioenergetic failure and metabolic cascades in brain ischemia. Cerebrovascular Disease. Pat-hophysiology, diagnosis, and management. M. B. Ginsberg, J. Malden, MA, Blackwell Science.

Simpkins, J. W., G. Rajakumar, Y.-Q. Zhang, C. E. Simpkins, D. Greenwald, C. J. Yu, N. Bodor and A. L. Day (1997). ”Estrogens may reduce mortality and ische-mic damage caused by middle cerebral artery occlusion in the female rat.” J. Neurosurg. 87: 724-730.

Sinclair, H. L. and P. J. Andrews (2010). ”Bench-to-bedside review: Hypothermia in traumatic brain injury.” Critical Care 14(1): 204.

Sko itsch, G. and D. M. Jacobowitz (1985). ”Immunohistochemical mapping of galanin-like neurons in the rat central nervous system.” Peptides 6(3): 509-546.

Sko itsch, G. and D. M. Jacobowitz (1986a). ”Quantitative distribution of gala-

86

References

nin-like immunoreactivity in the rat central nervous system.” Peptides 7(4): 609-613.

Sko itsch, G, M.A. Sills, D. M. Jacobowitz. (1986b) Autoradiographic distribution of 125I-galanin binding sites in the rat central nervous system. Peptides 7(6):1029-1042.

Smith, K. E., M. W. Walker, R. Artymyshyn, J. Bard, B. Borowsky, J. A. Tamm, W. J. Yao, P. J. Vaysse, T. A. Branchek, C. Gerald, et al. (1998). ”Cloned human and rat galanin GALR3 receptors. Pharmacology and activation of G-protein in-wardly rectifying K+ channels.” J Biol Chem 273(36): 23321-23326.

Smrcka, M., C. S. Ogilvy, R. J. Crow, K. I. Maynard, T. Kawamata and A. Ames, 3rd (1998). ”Induced hypertension improves regional blood low and protects against infarction during focal ischemia: time course of changes in blood low measured by laser Doppler imaging.” Neurosurgery 42(3): 617-624;

discussion 624-615.Socialstyrelsen (2010). Dödsorsaker 2008. Stockholm, Socialsyrelsen.Stapf, C. and J. P. Mohr (2002). ”Ischemic stroke therapy.” Annu Rev Med 53:

453-475.Stefanovich, V. (1983). Stroke: Animal Models. Oxford, Pergamon Press.Steiner, R. A., J. G. Hohmann, A. Holmes, C. C. Wrenn, G. Cadd, A. Jureus, D. K.

Clifton, M. Luo, M. Gutshall, S. Y. Ma, et al. (2001). ”Galanin transgenic mice display cognitive and neurochemical de icits characteristic of Alzheimer’s disease.” Proc Natl Acad Sci U S A 98(7): 4184-4189.

Strand, F. L. (1999). Neuropeptides. Regulators of physiological processes. Cambridge, Massachusettes, The MIT Press.

Ström, J. O., E. Theodorsson, L. Holm and A. Theodorsson (2010). ”Diff erent methods for administering 17beta-estradiol to ovariectomized rats result in opposite eff ects on ischemic brain damage.” BMC Neurosci 11: 39.

Ström, J. O., A. Theodorsson and E. Theodorsson (2008a). ”Substantial discre-pancies in 17beta-oestradiol concentrations obtained with three diff erent commercial direct radioimmunoassay kits in rat sera.” Scand J Clin Lab In-vest 68(8): 806-813.

Ström, J. O., E. Theodorsson and A. Theodorsson (2008b). ”Order of magnitude diff erences between methods for maintaining physiological 17beta-oestra-diol concentrations in ovariectomized rats.” Scand J Clin Lab Invest 68(8): 814-822.

Ström, J. O., A. Theodorsson and E. Theodorsson (2009). ”Dose-related neu-roprotective versus neurodamaging eff ects of estrogens in rat cerebral ischemia: a systematic analysis.” J Cereb Blood Flow Metab. 29(8):1359-1372.

Sun, Y. G., X. L. Gu, T. Lundeberg and L. C. Yu (2003). ”An antinociceptive role of galanin in the arcuate nucleus of hypothalamus in intact rats and rats with

87

References

in lammation.” Pain 106(1-2): 143-150.Sundt, T. M., Jr. and A. G. Waltz (1966). ”Experimental cerebral infarction: retro-

orbital, extradural approach for occluding the middle cerebral artery.” Mayo Clin Proc 41(3): 159-168.

Suzuki, J., T. Yoshimoto, S. Tnanka and T. Sakamoto (1980). ”Production of vari-ous models of cerebral infarction in the dog by means of occlusion of intra-cranial trunk arteries.” Stroke 11(4): 337-341.

Swanson, C. J., T. P. Blackburn, X. Zhang, K. Zheng, Z. Q. Xu, T. Hökfelt, T. D. Wolin-sky, M. J. Konkel, H. Chen, H. Zhong, et al. (2005). ”Anxiolytic- and antidepres-sant-like pro iles of the galanin-3 receptor (Gal3) antagonists SNAP 37889 and SNAP 398299.” Proc Natl Acad Sci U S A 102(48): 17489-17494.

Swanson, R. A., M. T. Morton, G. Tsao-Wu, R. A. Savalos, C. Davidson and F. R. Sharp (1990). ”A semiautomated method for measuring brain infarct vo-lume.” J Cereb Blood Flow Metab 10(2): 290-293.

Takata, T., M. Nabetani and Y. Okada (1997). ”Eff ects of hypothermia on the neuronal activity, [Ca2+]i accumulation and ATP levels during oxygen and/or glucose deprivation in hippocampal slices of guinea pigs.” Neurosci Lett 227(1): 41-44.

Tallent, M. K. (2008). ”Presynaptic inhibition of glutamate release by neuro-peptides: use-dependent synaptic modi ication.” Results Probl Cell Diff er 44: 177-200.

Tamura, A., D. I. Graham, J. McCulloch and G. M. Teasdale (1981a). ”Focal cere-bral ischaemia in the rat: 1. Description of technique and early neuropatho-logical consequences following middle cerebral artery occlusion.” J Cereb Blood Flow Metab 1(1): 53-60.

Tamura, A., D. I. Graham, J. McCulloch and G. M. Teasdale (1981b). ”Focal ce-rebral ischaemia in the rat: 2. Regional cerebral blood low determined by [14C]iodoantipyrine autoradiography following middle cerebral artery oc-clusion.” J Cereb Blood Flow Metab 1(1): 61-69.

Tatemoto, K. and V. Mutt (1978). ”Chemical determination of polypeptide hor-mones.” Proc Natl Acad Sci U S A 75(9): 4115-4119.

Tatemoto, K., A. Rökaeus, H. Jornvall, T. J. McDonald and V. Mutt (1983). ”Gala-nin - a novel biologically active peptide from porcine intestine.” FEBS Lett 164(1): 124-128.

Taylor, A., F. N. Madison and G. S. Fraley (2009). ”Galanin-like peptide stimula-tes feeding and sexual behavior via dopaminergic ibers within the medial preoptic area of adult male rats.” J Chem Neuroanat 37(2): 105-111.

Theodorsson, A., S. Hilke, O. Rugarn, D. Linghammar and E. Theodorsson (2005a). ”Serum concentrations of 17beta-estradiol in ovariectomized rats during two times six weeks crossover treatment by daily injections in com-parison with slow-release pellets.” Scand J Clin Lab Invest 65(8): 699-705.

88

References

Theodorsson, A., L. Holm and E. Theodorsson (2005b). ”Modern anesthesia and peroperative monitoring methods reduce per- and postoperative mortality during transient occlusion of the middle cerebral artery in rats.” Brain Res Brain Res Protoc 14(3): 181-190.

Theodorsson, A., L. Holm and E. Theodorsson (2008). ”Hypothermia-induced increase in galanin concentrations and ischemic neuroprotection in the rat brain.” Neuropeptides 42(1): 79-87.

Theodorsson, E. and O. Rugarn (2000). ”Radioimmunoassay for rat galanin: im-munochemical and chromatographic characterization of immunoreactivity in tissue extracts.” Scand J Clin Lab Invest 60(5): 411-418.

Theodorsson, A. and E. Theodorsson (2005c). ”Estradiol increases brain lesions in the cortex and lateral striatum after transient occlusion of the middle ce-rebral artery in rats: No eff ect of ischemia on galanin in the stroke area but decreased levels in the hippocampus.” Peptides 26(11): 2257-2264.

Tobin, J. R., L. D. Martin, M. J. Breslow and R. J. Traystman (1994). ”Selective anesthetic inhibition of brain nitric oxide synthase.” Anesthesiology 81(5): 1264-1269.

van Bruggen, N., H. Thibodeaux, J. T. Palmer, W. P. Lee, L. Fu, B. Cairns, D. Tumas, R. Gerlai, S. P. Williams, M. van Lookeren Campagne, et al. (1999). ”VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain.” J Clin Invest 104(11): 1613-1620.

van der Worp, H. B., D. W. Howells, E. S. Sena, M. J. Porritt, S. Rewell, V. O’Collins and M. R. Macleod (2010). ”Can animal models of disease reliably inform human studies?” PLoS Med 7(3): e1000245.

van der Worp, H. B., E. S. Sena, G. A. Donnan, D. W. Howells and M. R. Macleod (2007). ”Hypothermia in animal models of acute ischaemic stroke: a syste-matic review and meta-analysis.” Brain 130(Pt 12): 3063-3074.

Vergouwen, M. D., R. E. Anderson and F. B. Meyer (2000). ”Gender diff erences and the eff ects of synthetic exogenous and non-synthetic estrogens in focal cerebral ischemia.” Brain Res 878(1-2): 88-97.

Villar, M. J., R. Cortes, E. Theodorsson, Z. Wiesenfeld-Hallin, M. Schalling, J. Fah-renkrug, P. C. Emson and T. Hökfelt (1989). ”Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin.” Neuroscience 33(3): 587-604.

Villar, M. J., B. Meister and T. Hökfelt (1994). ”Reorganization of neural pepti-dergic systems in the median eminence after hypophysectomy.” J Neurosci 14(10): 5996-6012.

von Pechman, H. and P. Runge (1894). ”Oxydation der formazyluerbindungen.” Ber Deutsch Chem Ges 27: 2920-2930.

Vrontakis, M. E. (2002). ”Galanin: a biologically active peptide.” Curr Drug Tar-gets CNS Neurol Disord 1(6): 531-541.

89

References

Vrontakis, M. E., L. M. Peden, M. L. Duckworth and H. G. Friesen (1987). ”Iso-lation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA.” J Biol Chem 262(35): 16755-16758.

Wang, G. J., H. Y. Deng, C. M. Maier, G. H. Sun and M. A. Yenari (2002). ”Mild hy-pothermia reduces ICAM-1 expression, neutrophil in iltration and microg-lia/monocyte accumulation following experimental stroke.” Neuroscience 114(4): 1081-1090.

Wang, S. and E. L. Gustafson (1998a). ”Galanin receptor subtypes.” Drug News Perspect 11(8): 458-468.

Wang, S., T. Hashemi, S. Fried, A. L. Clemmons and B. E. Hawes (1998b). ”Diff e-rential intracellular signaling of the GalR1 and GalR2 galanin receptor sub-types.” Biochemistry 37(19): 6711-6717.

Wang, S., C. He, T. Hashemi and M. Bayne (1997). ”Cloning and expressional characterization of a novel galanin receptor. Identi ication of diff erent phar-macophores within galanin for the three galanin receptor subtypes.” J Biol Chem 272(51): 31949-31952.

Wang, L. M., Y. Yan, L. J. Zou, N. H. Jing and Z. Y. Xu (2005). ”Moderate hypother-mia prevents neural cell apoptosis following spinal cord ischemia in rab-bits.” Cell Res 15(5): 387-393.

Wang-Fischer, Y. (2009). Manual of stroke models in rats, CRC Press, Taylor & Francis Group.

Waters, S. M. and J. E. Krause (2000). ”Distribution of galanin-1, -2 and -3 re-ceptor messenger RNAs in central and peripheral rat tissues.” Neuroscience 95(1): 265-271.

Watson, B. D., W. D. Dietrich, R. Busto, M.S. Wachtel and M. D. Ginsberg (1985). ”Induction of reproducible brain infarction by photochemically initiated th-rombosis”. Ann Neurol 17(5): 497-504.

Wester, P., B. D. Watson, R. Prado and W. D. Dietrich (1995). ”A photothrombotic ’ring’ model of rat stroke-in-evolution displaying putative penumbral inver-sion.” Stroke 26(3): 444-450.

Westin, B. (2006). ”Hypothermia in the resuscitation of the neonate: a glance in my rear-view mirror.” Acta Paediatr 95(10): 1172-1174.

White, F. A., B. F. Hoe linger, N. L. Chiaia, C. A. Bennett-Clarke, R. S. Crissman and R. W. Rhoades (1994). ”Evidence for survival of the central arbors of trigeminal primary aff erents after peripheral neonatal axotomy: experi-ments with galanin immunocytochemistry and Di-I labelling.” J Comp Neu-rol 350(3): 397-411.

WHO (1978). Cerebrovascular disorders : a clinical and research classi ication., World Health Organization.

WHO (1988). ”The World Health Organization MONICA Project (monitoring

90

References

trends and determinants in cardiovascular disease): a major international collaboration. WHO MONICA Project Principal Investigators.” J Clin Epide-miol 41(2): 105-114.

Wiesenfeld-Hallin, Z., X. J. Xu, J. N. Crawley and T. Hökfelt (2005). ”Galanin and spinal nociceptive mechanisms: Recent results from transgenic and knock-out models.” Neuropeptides 39(3): 207-210.

Wirz, S. A., C. N. Davis, X. Lu, T. Zal and T. Bartfai (2005). ”Homodimerization and internalization of galanin type 1 receptor in living CHO cells.” Neuro-peptides 39(6): 535-546.

Wise, P. M. (2000). ”Estradiol: a protective factor in the adult brain.” J. Pediatr. Endocrinol. Metab. 13: 1425-1429.

Wise, P. M. and D. B. Dubal (2000). ”Estradiol protects against ischemic brain injury in middle-aged rats.” Biol. Reprod. 63: 982-985.

Wittau, N., R. Grosse, F. Kalkbrenner, A. Gohla, G. Schultz and T. Gudermann (2000). ”The galanin receptor type 2 initiates multiple signaling pathways in small cell lung cancer cells by coupling to G(q), G(i) and G(12) proteins.” Oncogene 19(37): 4199-4209.

Wynick, D., S. W. Thompson and S. B. McMahon (2001). ”The role of galanin as a multi-functional neuropeptide in the nervous system.” Curr Opin Pharma-col 1(1): 73-77.

Xia, S., X. P. Dun, P. S. Hu, S. Kjaer, K. Zheng, Y. Qian, C. Solen, T. Xu, B. Fredholm, T. Hökfelt, et al. (2008). ”Postendocytotic traf ic of the galanin R1 receptor: a lysosomal signal motif on the cytoplasmic terminus.” Proc Natl Acad Sci U S A 105(14): 5609-5613.

Xia, S., S. Kjaer, K. Zheng, P. S. Hu, T. Xu, T. Hökfelt and Z. Q. Xu (2005). ”Consti-tutive and ligand-induced internalization of EGFP-tagged galanin R2 and Rl receptors in PC12 cells.” Neuropeptides 39(3): 173-178.

Xia, S., S. Kjaer, K. Zheng, P.S. Hu, L. Bai, J.Y. Jia, R. Rigler, A. Pramanik, T. Xu, T. Hökfelt, Z.Q. Xu (2004). Visualization of a functionally enhanced GFP-tagged galanin R2 receptor in PC12 cells: constitutive and ligand-induced interna-lization. Proc Natl Acad Sci U S A 101(42):15207-15212.

Xu, Z., R. Cortes, M. Villar, P. Morino, M. N. Castel and T. Hökfelt (1992). ”Evi-dence for upregulation of galanin synthesisin rat glial cells in vivo after col-chicine treatment.” Neurosci Lett 145(2): 185-188.

Xu, X. J., T. Hökfelt, et al. (2010). ”Galanin and spinal pain mechanisms: past, present, and future.” EXS 102: 39-50.

Xu, Z. Q., T. J. Shi and T. Hökfelt (1996). ”Expression of galanin and a galanin receptor in several sensory systems and bone anlage of rat embryos.” Proc Natl Acad Sci U S A 93(25): 14901-14905.

Xu, Z. Q., T. J. Shi and T. Hökfelt (1998a). ”Galanin/GMAP- and NPY-like immu-noreactivities in locus coeruleus and noradrenergic nerve terminals in the

91

References

hippocampal formation and cortex with notes on the galanin-R1 and -R2 receptors.” J Comp Neurol 392(2): 227-251.

Xu, Z. Q., X. Zhang, V. A. Pieribone, S. Grillner and T. Hökfelt (1998b). ”Galanin-5-hydroxytryptamine interactions: electrophysiological, immunohistochemi-cal and in situ hybridization studies on rat dorsal raphe neurons with a note on galanin R1 and R2 receptors.” Neuroscience 87(1): 79-94.

Xu, Z. Q., K. Zheng and T. Hökfelt (2005). ”Electrophysiological studies on ga-lanin eff ects in brain--progress during the last six years.” Neuropeptides 39(3): 269-275.

Xue, D., Z. G. Huang, K. E. Smith and A. M. Buchan (1992). ”Immediate or delayed mild hypothermia prevents focal cerebral infarction.” Brain Res 587(1): 66-72.

Yamori, Y., R. Horie, H. Handa, M. Sato and M. Fukase (1976). ”Pathogenetic si-milarity of strokes in stroke-prone spontaneously hypertensive rats and humans.” Stroke 7(1): 46-53.

Yanamoto, H., I. Nagata, Y. Niitsu, Z. Zhang, J. H. Xue, N. Sakai and H. Kikuchi (2001). ”Prolonged mild hypothermia therapy protects the brain against permanent focal ischemia.” Stroke 32(1): 232-239.

Yang, S. C., M. Ito, Y. Furukawa and S. Kimura (1994). ”Comparative study of alcohol metabolism in stroke-prone spontaneously hypertensive rats and Wistar-Kyoto rats fed normal or low levels of dietary protein.” J Nutr Sci Vitaminol (Tokyo) 40(6): 547-555.

Yenari, M. A. and T. M. Hemmen (2010). ”Therapeutic hypothermia for brain ischemia: where have we come and where do we go?” Stroke 41(10 Suppl): S72-74.

Yenari, M. A., S. Iwayama, D. Cheng, G. H. Sun, M. Fujimura, Y. Morita-Fujimura, P. H. Chan and G. K. Steinberg (2002). ”Mild hypothermia attenuates cytoch-rome c release but does not alter Bcl-2 expression or caspase activation after experimental stroke.” J Cereb Blood Flow Metab 22(1): 29-38.

Young, R. S., E. L. Zalneraitis and E. C. Dooling (1980). ”Neurological outcome in cold water drowning.” Jama 244(11): 1233-1235.

Zausinger, S., A. Baethmann and R. Schmid-Elsaesser (2002). ”Anesthetic met-hods in rats determine outcome after experimental focal cerebral ischemia: mechanical ventilation is required to obtain controlled experimental condi-tions.” Brain Res Brain Res Protoc 9(2): 112-121.

Zausinger, S., E. Hungerhuber, A. Baethmann, H. Reulen and R. Schmid-Elsaesser (2000). ”Neurological impairment in rats after transient middle cerebral artery occlusion: a comparative study under various treatment paradigms.” Brain Res 863(1-2): 94-105.

Zhang, R. L., M. Chopp, Z. G. Zhang, Q. Jiang and J. R. Ewing (1997a). ”A rat model of focal embolic cerebral ischemia.” Brain Res 766(1-2): 83-92.

92

References

Zhang, Y. P., T. Lundeberg and L. C. Yu (2000). ”Interactions of galanin and morp-hine in the spinal antinociception in rats with mononeuropathy.” Brain Res 852(2): 485-487.

Zhang, Z., R. L. Zhang, Q. Jiang, S. B. Raman, L. Cantwell and M. Chopp (1997b). ”A new rat model of thrombotic focal cerebral ischemia.” J Cereb Blood Flow Metab 17(2): 123-135.

Zhao, B. Q., Y. Suzuki, K. Kondo, K. Kawano, Y. Ikeda and K. Umemura (2002). ”A novel MCA occlusion model of photothrombotic ischemia with cyclic low reductions: development of cerebral hemorrhage induced by heparin.”

Brain Res Brain Res Protoc 9(2): 85-92.Zigmond, R. E. (2001). ”Can galanin also be considered as growth-associated

protein 3.2?” Trends Neurosci 24(9): 494-496; discussion 496.

Documents

Focal ischemic reperfusion stroke model in rats and the …liu.diva-portal.org/smash/get/diva2:416160/FULLTEXT01.… · · 2011-05-10Focal ischemic reperfusion stroke model in rats