PAPER DE LA BARRERA HEMATOENCEFÀLICA EN LES … · PAPER DE LA BARRERA HEMATOENCEFÀLICA EN LES MALALTIES NEURODEGENERATIVES Jaume dldel VllValle i MiàMacià Grup de Barrera Hematoencefàlica

PAPER DE LA BARRERA HEMATOENCEFÀLICA EN LES MALALTIESHEMATOENCEFÀLICA EN LES MALALTIES

NEURODEGENERATIVESJ d l V ll i M iàJaume del Valle i Macià

Grup de Barrera HematoencefàlicaDepartament de FisiologiaFacultat de Farmàcia, UB

0‐ Introduction

0a‐ The blood‐brain barrier

•The BBB

•Components

•Role

0‐ Introduction

0b‐ BBB and neurodegenerative diseases

Vascular origin

1‐ Circulating neurotoxins

1‐ Proinflammatory signals from the

vascular cellsvascular cells

1‐ Reduced capillary blood flow

2‐ Activation of microglia, endothelium

and astrocytes by neurons

3‐ Activated endothelium, microglia, and

astrocytes aggravate the injuryastrocytes aggravate the injury

0‐ Introduction

0c‐ BBB and neurodegenerative diseases

Neuronal origin

1‐ Activation of microglia, endothelium

and astrocytes by neurons

2‐ Activated endothelium, microglia, and

astrocytes signal back to neurons

3‐ Circulating neurotoxins

3‐ Proinflammatory signals from the

vascular cells

3‐ Reduced capillary blood flow3 Reduced capillary blood flow

BBB group’s projects

Alzheimer’sDisease

Huntington’sDisease

BBB substancedeliverydelivery

BBB

Senescence Cell Death

BLOOD‐BRAIN BARRIER ALTERATIONS AND BRAIN AMYLOID PATHOLOGYAND BRAIN AMYLOID PATHOLOGY

IN SAMP8 MICE

Summary

1‐ Introduction: The SAMP8 mice

2‐ Objectives

3‐ BBB status in SAMP8 mice, article 1

5‐ Time‐course of blood–brain barrier disruption in SAMP8 mice article 3

4‐Method for determining BBB integrity, article 2

5‐ Time‐course of blood–brain barrier disruption in SAMP8 mice, article 3

6‐ Early amyloid accumulation in the hippocampus of SAMP8 mice, article 4

7‐ Cerebral amyloid angiopathy, amyloid deposition and BBB disruption in SAMP8, article 5

1‐ The SAMP8 mice

1a‐ The SAMP8 mice

Accelerated senescence+ activity loss+ hair loss & lack of glossiness+ periophthalmic lesions+ periophthalmic lesions+ increased lordokyphosis+ early death

Most ‘‘age‐dependent’’ geriatric disorders in humans, described as ‘‘a direct consequence of physiologic senescence’’ are included in the pathobiological phenotypes in the SAMP model: ‐Osteoporosis‐OsteoarthritisOsteoarthritis‐Cataracts‐Hearing impairment‐Hyperinflation of the lungs

1‐ The SAMP8 mice

1b‐ Characteristics and neuropathology of the SAMP8 mice

Behavioral changes‐Learning and memory deficits‐Reduced anxiety‐like behavior‐Alterations in circadian rhythms

Neuropathological and neurochemical changes‐Reduction of dendritic spines in the hippocampusp pp p‐Astrogliosis‐Increase of β‐amyloid protein‐Cerebellar degeneration‐Microglial cell proliferationg p‐Spongyform degeneration‐Local accumulation of excitatory amino acids ‐Increased oxidative stress‐Phosphorylation of tau and Cdk5 expression

Neurovascular hypothesis

p y p‐Mitochondrial dysfunction‐Blood‐brain barrier alterations (BBB)‐…

Alzheimer’sAlzheimer sdisease

Auguste D.

1‐ The SAMP8 mice

1c‐ Neurovascular hypothesis in SAMP8

‐Increase of β‐amyloid protein

Very few, unclear and Takemura et al., 1993

Fukunari et al 1994

‐BBB alterations

contradictory dataFukunari et al., 1994

Morley et al., 2000

Contradictory data

Takemura et al., 1993

Banks et al 2000

Moinuddin et al., 2000

Contradictory dataBanks et al., 2000

Ueno et al., 1993

Hosokawa i Ueno, 1999

Summary

1‐ Introduction: the SAMP8 mice

2‐ Objectives



4‐ A new method for determining BBB integrity, article 2




2‐ Objectives

2‐Main objectives

1. To determine the existence and temporal progression of any changes in thepermeability of the BBB in the hippocampus and the cerebral cortex of SAMP8 mice.

2. To characterize the Aβ presence and its temporal progression in the hippocampus andb i t f SAMP8 ibrain cortex of SAMP8 mice.

3 To study the possible spatial and temporal relationship between an increase in Aβ and3. To study the possible spatial and temporal relationship between an increase in Aβ andthe BBB disruption in SAMP8 mice.

Summary


2‐ Objectives







3‐ BBB status in SAMP8 mice, study 1.

Animals Age ExperimentAnimals Age Experiment

SAMP8 3, 7 and 12

Analysis of cerebral IgG extravasationmonths

Analysis of cerebral IgG extravasation

SAMR1


PECAMIgG

Pelegrí et al., 2007. Mech. Ag. Dev. 128, 522‐8




*



3‐Conclusions

•Neither SAMP8 nor SAMR1 mice aged 3 and 7 months showed alterations in thepermeability of BBB to IgG.

•No differences can be seen between strains in the vessel diameter.

•12‐month‐old SAMP8 mice show significantly higher levels of IgG extravasation in thehippocampus than SAMR1 animals.


Summary


2‐ Objectives







4‐ A new method for determining BBB integrity

Group1Anesthesia Cryolesion PBS Perfusion Cocktail Perfusion Brain extraction

p

PerfusionCryolesion

1 Anesthesia

Cocktail

1 EB 1%1. Anesthesia

2. Right frontoparietal

cranial exposure

3. Lesion with a 5mm dry

1. EB 1%

2. Hoechst 0,01%.

3. P‐Formaldehid 4%

4. PBS 50 ml

ice (‐78ºC) pellet.


EB Hoechst

Scale Bar = 750µm

del Valle et al., 2009. J. Neurosci. Meth. 174, 42‐9


Nucleus Peripheral ring Peripheral ring

Lesion

Lesion

Scale bar= 150µm



PECAM EB

GFAP

EB

CD11b

NeuN

EBCD11b EB

Scale bar = 50µm



4‐Conclusions

•Intracardiac cocktail administration allows BBB disruption localization by EB and Hoechst

fluorescence visualization.

• Cocktail perfusion method also allows immunohystochemical studies to characterize

different processes in the BBB disruption.


Summary


2‐ Objectives







5‐ Time‐course of BBB disruption in SAMP8 mice

Animals Age ExperimentAnimals Age Experiment

SAMP8

3, 6, 9, 12 and 15 months

Analysis of cerebral cocktailextravasation

SAMR1

ICR‐CD1


**

* **

*

**

*

While in ICR‐CD1 EB extravasation values remain stable, in both SAMP8 and SAMR1 theyincrease in CA1While in ICR‐CD1 EB extravasation values remain stable, in both SAMP8 and SAMR1 theyincrease in CA1 and hippocampal fissure.While in ICR‐CD1 EB extravasation values remain stable, in both SAMP8 and SAMR1 theyincrease in CA1, hippocampal fissure and cortex.

Both SAMP8 and SAMR1 mice show higher extravasation values in CA1 and hippocampalfissure than ICR‐CD1

del Valle et al., 2009. Int. J. Devl. Neuroscience. 27, 47‐52


5‐Conclusions

•While BBB integrity remains stable throughout the life of ICR‐CD1 mice, SAMP8 andSAMR1 show a progressive increase of disruption from 6 to 15 months of age in cortex, CA1SAMR1 show a progressive increase of disruption from 6 to 15 months of age in cortex, CA1and hippocampal fissure.

•Results obtained at 3 months of age are unexpected, as all strains show higher EBextravasation values than those obtained at 6 months of age.

del Valle et al., 2009. Int. J. Devl. Neuroscience. 27, 47‐52

Summary


2‐ Objectives








Animals Age Experiment

SAMP8SAMP8

3, 6, 9, 12 and 15

hAnalysis of A depositionSAMR1

months

ICR‐CD1


Py OrRad

3m 6m 9m 12 m 15m

C

P

R

del Valle et al., 2010. J. Alz. Dis. 27, 47‐52



In SAMP8 there is a progressive increase in the number of amyloid


‐In SAMP8, there is a progressive increase in the number of amyloid clusters in the hippocampus from 3 months onwards.

‐ In the other two strains, the number of amyloid clusters keep low y pand stable up to 15 months of age, when numbers increase; although they do not reach the levels shown by SAMP8 mice.

‐In SAMP8, there is a progressive increase in the number of β42clusters in the hippocampus from 3 months onwards.

I th th t t i th b f β l t i l d‐ In the other two strains, the number of β42 clusters remain low and stable up to 15 months of age, when numbers increase; although they do not reach the levels shown by SAMP8 mice.

‐ In SAMP8, there is an increase in the hippocampus in the number of β40 clusters compared to the other two strains from 6 months β40 ponwards.



SAMP8 i h k d l id d iti f 6 th d d ith

6‐Conclusions

•SAMP8 mice show a marked amyloid deposition from 6 months onwards compared withSAMR1 and ICR‐CD1 control strains.

•SAMP8 mice become a useful tool to study the mechanisms involved in the formation ofAβ deposition in spontaneous Alzheimer’s disease.


Summary


2‐ Objectives







7‐ CAA, amyloid deposition and BBB disruption in SAMP8

Animals Age Experiment

SAMP8 3, 6, 9 and 12 months

Analysis of vessels with CAA, BBB disruption and A40 aggregation

ICR CD1ICR‐CD1


Amyloid positive vessels (CAA)

14161820

y p ( )vessels

**

*

*

†

†

468

101214

ICR

SAMP8

†

024

3 6 9 12 months

‐SAMP8 mice exhibit higher levels of CA1 amyloid positive vessels than age‐matched ICR mice. ‐Old mice of 12 months show more amyloid vessels than younger, say 3 months, or d lt f 6 t 9 th i b th t iadult ages from 6 to 9 months in both strains

del Valle et al., pendent


IgG positive vessels

35

40

45

g pvessels

†

†*

10

15

20

25

30ICR

SAMP8

†

0

5

10

3 6 9 12 months

‐At 12 months of age, IgG positive vessels appear in a regular pattern in both strains and very few IgG positive vessels are seen in the CA1 subzone of the hippocampus at 3, 6 y g p pp p ,and 9 months of age.‐Old mice of 12 months show more IgG positive vessels than young and adult mice.‐At 12 months of age old SAMP8 mice seem to have a more disrupted BBB with more I G iti l th ld ICR i (P 0 075)IgG positive vessels than old ICR mice (P=0,075).


βPECAM


β40IgG

PECAMβ40

β40IgG

PECAMβ40



F 3 th d SAMP8 i t hi h l l f β l id iti l

7‐Conclusions

•From 3 months onwards, SAMP8 mice present higher levels of β40 amyloid positive vesselsthan age‐matched ICR mice.

•Both SAMP8 and ICR strains show an increase of CAA at 12 months of age compared toyoung and adult mice.

•There is no direct association between the location amyloid clusters and:• blood vesselsblood vessels• BBB disrupted vessels• Amyloid positive vessels

•All the vessels with amyloid deposition show a disruption in the BBB with IgG deposition.In addition, several capillaries with IgG deposition can be found with no amyloidaccumulation within their walls.

BLOOD‐BRAIN BARRIER ALTERATIONS AND BRAIN AMYLOID PATHOLOGYAND BRAIN AMYLOID PATHOLOGY

IN SAMP8 MICE

Jaume del Valle i Macià

Grup de Barrera HematoencefàlicaDepartament de FisiologiaFacultat de Farmàcia, UB

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PAPER DE LA BARRERA HEMATOENCEFÀLICA EN LES … · PAPER DE LA BARRERA HEMATOENCEFÀLICA EN LES MALALTIES NEURODEGENERATIVES Jaume dldel VllValle i MiàMacià Grup de Barrera Hematoencefàlica