MULTIMODAL APPROACHES FOR

REGENERATIVE STROKE THERAPIES:

Aurel Popa-Wagner

Medical University Rostock, Germany

University of Medicine and Pharmacy

Craiova, Romania

Intracerebral vessel occlusion has a strong AGE

dependency

Brain of a Patient after MCA Occlusion

Why use aged animals to study rehabilitation after stroke?

Although it is well known that aging is a risk factor for stroke,

the majority of experimental studies of stroke have been performed

on young animals, and therefore may not fully replicate the effects

of ischemia on neural tissue in aged subjects.

In this light, the aged post-acute animal model is clinically

most relevant to stroke rehabilitation and cellular studies,

a recommendation done by the STAIR committee and more

recently by the Stroke Progress Review Group.

UKM

STAIR Criteria

(Stroke Academic and Industry Round Table)

• Reproducibility of results proven in many different laboratories wordlwide

• Efficiency in many different species

• Testing on aged animals

• Efficiency in both transient and permanent ischemia

• Establish the therapeutic time-window

• Establish the dose-efficiency relationship

• Monitoring of physiologic parameters during the experiments

• Does the infarct volume correlate with functional recovery ?

• Longterm studies of the above parameters (min. 4 Weeks)

STAIR 1999

Beam- Young Stroke

Beam- Old Stroke

Young Stroke- Table

Old Stroke- Table

Legend (what it is based on), significance in terms of gene expression

A

Changes of gene expression in response to stroke.

(A) Dendrogram of two-dimensional hierarchical clustering

analysis of all 9,494 transcript-specific probe sets which

indicate differentially expressed genes

Legend (what it is based on), significance in terms of gene expression

A

Correspondence analysis. ( left panel) Eigenvalues of the correspondence analysis

and shows that the major factors contributing to the variance of stroketomics analysis

were stroke (52%), post-stroke time (25%) and age (12%).

Sources of variabilty: stroke and post-stroke time. Samples from young (green) and

aged (red) animals particularly differ in their post-stroke response (illustrated by ellipses.

Transcripts with characteristic expression in naive samples are encircled in black

Buga..Popa-Wagner, JCBFM 2012

VENN-Diagram. Note that at 14 days post-stroke, the differences between

the age groups were more pronounced

Transcriptomics of Stroke: Divergent gene expression

after 2 weeks post-stroke

Patterns of gene expression after stroke. Aged animals showed larger

numbers than young of genes that were late-upregulated, persistently

upregulated and persistently downregulated.

The young rats, in contrast, had a much larger number of transiently upregulated and delayed downregulated genes

Patterns of gene expression after stroke. Patterns of gene expression for stroke-relevant processes.

Most classes of these new genes were upregulated, with the exception of “CNS physiology & homeostasis”

and “Neurogenesis & synaptic plasticity” which also displayed a large number of downregulated genes

To date, all monotherapeutic attempts to prevent or

lessen brain damage following stroke have failed. Since

stroke impacts a wide range of systems in an age-

dependent manner, from CNS physiology to CNS

regeneration and plasticity, the failure of therapies aimed

at only a single target system is perhaps inevitable

Transcriptomics of Stroke. Disregulation of gene expression

required for Neurogenesis & Synaptic plasticity

Transcriptomics of Stroke. Disregulation of gene expression

required for Embryonic development & CNS regeneration

Neurogenesis is fully functional in the subventricular zone of the adult brain

Stimulation of endogenous neurogenesis

Pilot studies aimed at improving recovery of

function in aged rats after stroke

Rat Modell for Cerebral Ischemia

Summary of functional tests after stroke

Rotarod

Cylinder Test

T-Maze

Radial Maze i-Plane

Neurol.

Status

G-CSF, multimodale Mechanismen in der Schlaganfalltherapie

Schäbitz and Schneider 2007

UKM

G-CSF treatment after stroke significantly improved mortality rate and

performance in several but not all functional tests.

Pilot studies aimed at improving recovery of

function in aged rats after stroke

Daily treatment with G-CSF of aged rats after stroke

significantly reduces the mortality rate

Effect of the G-CSF treatment on cellular proliferation and neurogenesis.

First combination therapy of stroke: G-CSF + Stem Cells

Hypothesis: Our consortium tested the hypothesis that such

a combination is superior to G-CSF or stem cell treatment alone.

Stroke Cell Therapy: flow diagram

-14d 0 6h 3d 45d 60d

MRI#2 MRI#1 Tissue Analysis

Behavioral Analysis

Tra

inin

g

MC

AO

Th

era

pie

s

Animal Model and Treatment; 80 ♂ Sprague-Dawley (SD) –Ratten

n=20 G-CSF (30 µg/kg BW) given daily and for 28 days after stroke

n=20 G-CSF + BM MNC (single, 1x106/kg BW) given at 3 hrs after reperfusion

n=20 G-CSF + BMSC (single, 1x106/kg BW) given at 3 hrs after reperfusion

n=20 Glucose (Vehicle, 5%) given daily and for 28 days after stroke

Groups:

Multimodal Approaches for

Regenerative Stroke Therapies (MARS)

ROUTE of ADMINISTRATION: Jugular Vein

Validity of the administration pathway

Perilesional Area Some cell enter the brain via the ventricle

The Mortality Rate was low for all treatments

Group C: Survival proportions

0 20 40 60 8080

85

90

95

100

105

Days after stroke

Pe

rce

nt s

urv

iva

l

Group No Deaths

CTRL 2

G-CSF 2

G-CSF + BM MNC 2

G-CSF + BM MSC 3

Cell therapy does not reduce the volume of the infarct

Water Maze

1

2 3

4

G-CSF Treatment led a significant improvement of spatial memory

G-CSF Treatment alone improves temporarily sensory function

(Adhesive Tape Removal Test)

0 3 7 14 21 28 35 42 49 560.4

0.5

0.6

0.7

0.8

0.9

Group CTRL

Group A

* P<0.05

Day

Score

0 3 7 14 21 28 35 42 49 560.4

0.5

0.6

0.7

0.8

0.9

Group CTRL

Group B

Day

Score

0 3 7 14 21 28 35 42 49 560.4

0.5

0.6

0.7

0.8

0.9

Group CTRL

Group C

Day

Score

Beam-walking test: Both G-CSF alone and the combination therapy

improved performance vs controls

0 3 7 14 21 28 35 42 49 56

1

2

3

4

5

6

CTRL

GROUP A

* P<0.04

Day

Score

0 3 7 14 21 28 35 42 49 56

1

2

3

4

5

6

CTRL

GROUP B

* P<0.05 * P<0.05

Day

Score

0 3 7 14 21 28 35 42 49 56

1

2

3

4

5

6

CTRL

GROUP C

* P<0.05

Day

Score

Neurogenesis was not impaired in neither group

DCX immunofluorescence (red) is well visible in the Lateral Ventricle of all groups, both ipsilaterally and

contralaterally. BrdU (green) was mainly incorporated into a network of capillaries

At two months post-stroke, G-CSF + BM MSC therapy promotes

the growth of lymphatic vessel

Pilot studies aimed at improving recovery of

function in aged rats after stroke

Stimulation of endogeneous neurogenesis

by chemical and by small electrical currents

0 -24 -48 2- Start behavioral testing 48

End

PTZ before Stroke

1 st PTZ 2nd PTZ

3x BrdU 3x BrdU

Day

0 2 48

End

PTZ after Stroke

3x BrdU

0 -24 -48 2 48

End

Control Groups

1 st Saline 2nd Saline

3x BrdU 3x BrdU

Day

3x BrdU

1 st PTZ

Or ECS

2nd PTZ

Or ECS 7 31

0 2 48

End 3x BrdU 3x BrdU

7 31

Adhesive Tape Removal Test

days

0 10 20 30 40 50

tim

e q

uotient

-0,8

-0,6

-0,4

-0,2

0,0

0,2

0,4

0,6

PTZ before stroke

Ctrl

PTZ after stroke

Neurogenesis does not significantly improved performance

in

Inclined Plane

days0 10 20 30 40 50

Angle

30

32

34

36

38

40

PTZ before stroke

Ctrl

PTZ after stroke

Neurogenesis after stroke significantly improved performance

on the

Radial Maze

days0 10 20 30 40 50

score

0,0

0,2

0,4

0,6

0,8

1,0

1,2

PTZ before

Ctrl

PTZ after

Neurogenesis after stroke significantly improved performance

in

Stroke Volume of all Groups in the NGS Project

0

5

10

15

20

25

30

Vo

lum

e [

mm

3]

Control 13,38

PTZ before Stroke 15,69

PTZ after Stroke 17,86

ES after Stroke 18,16

Sham 5,49

Stroke Volume

x10

Number of Doublecortin positive Cells in Hippocampus

0

2

4

6

8

10

Tissue

Cell

Nu

mb

er

Control 1,68 1,85

PTZ before Stroke 6,89 6,21

PTZ after Stroke 0,85 0,42

ES after Stroke 5,48 3,00

Sham 2,13 1,72

Hippocampus ipsilateral Hippocampus contralateral

Semiquantitative Evaluation of Doublecortin positive Cells in

Subventricular Zone

0

1

2

3

Tissue

Cell

Sco

re

Control 2,19 1,12

PTZ before Stroke 2,09 1,69

PTZ after Stroke 1,43 1,12

ES after Stroke 2,06 1,84

Sham 1,52 1,36

Subventricular Zone ipsilateral Subventricular Zone contralateral

Number of PSA-NCAM positive cells in Hippocampus

0

2

4

6

8

10

Tissue

Cell

Nu

mb

er

Control 1,84 1,58

PTZ before Stroke 6,71 4,29

PTZ after Stroke 2,10 1,28

ES after Stroke 4,31 4,31

Sham 2,00 1,68

Hippocampus ipsilateral Hippocampus contralateral

Electric Stimulation is efficient in increasing the number of early markers of

neurogenesis like Doublecortin (shown in green) in the subventricular zone

Increased number of axons in the perilesional area of aged rats following

neurogenesis enhancement

Accelerated scar buildup in aged rodents after stroke

(i) Neuroepithelial cells contributing to the glial scar emanate

from desintegrated the blood vessel walls (E, F); (ii) but do

not pass the CC barrier (M)

Neuroepithelial cells contributing to the glial scar originate

mostly in the corpus callosum

Neuroepithelial cells contributing to the glial (D, H) scar

emanate from desintegrated the blood vessel walls; Green:

BrdU nuclei; Violett: Laminin

Early formation of the growth-inhibiting SCAR and late

outgrowth of axons after stroke in aged subjects

AXONS

SCAR

pH3 pH10 Microglia immunoreactivity is precipitously increased after cerebral ischemia in

old rats. Could anti-inflammatory Drugs Improve Recovery of Function after

Stroke?

Note the early activation of microglia in the brains of aged rats (E, F) as well

as persistent expression of activated microglia in the brains of young rats (C,D).

3 days 7 days 14 days 28 days

yo

un

g

ag

ed

Eight weeks after stroke the glial scar (RED) region is heavely populated

with infammatory cells (Blue)

H2S-induced Hypothermia Diminish Inflammation and

Improve Neurorehabilitation after Stroke in Aged

Rats

Long-term exposure to hydrogen sulfide induces a torpor-like state

H2S is efficient to induce long-term hypothermia after stroke in aged rats

Baltromejus et al., NSL, 2008

80 ppm H2S

Baseline of Temperature and EEG under Normothermic

and Hypothermic Conditions

40 ppm H2S

Popa-Wagner et al., J Cereb Blood Flow & Met, 2012

Schematic overview of the experimental design. (B): Timecourse of whole body

cooling after stroke in rats immersed in an atmosphere containing 50 ppm H2S.

On MRI, the ischemic lesion appeared as a hyperintense area on T2-

weighted images. The limits of the lesion are indicated by arrows

Popa-Wagner et al., J Cereb Blood Flow & Met, 2012

EEG telemetry data. A representative telemetric recording

for a rat subjected to H2S is presented in a-f.

Popa-Wagner et al., J Cereb Blood Flow & Met, 2012

Oligo DNA array analysis of RNA from aged rats subjected to MCA occlusion

and hypothermia identified annexin a1 as one of the downregulated mRNAs

in the peri-infarct area of hypothermic animals

Independent identification of annexin a1 by 2D

electrophoresis and Western Blotting

Phenotype and function of ANXA1 in the rat brain after stroke Phenotypically, ANXA1-

positive cells (red), co-localized with polymorphonuclear-like cells (D). Exposure to

hypothermia led to a large reduction in the number of co-localizations

What is next?

Combine Nanotherapy with Cell therapy?

magnetic/plasmonic moderate hyperthermia

(41.8°C). IC = infarct core; PI = perinfarct

Day 7

Cell therapy under mild hyperthermia

(38°C); IC = infarct core; PI = perinfarct;

BV = blood vessel

+

Day 2

BV

Our present results suggest that H2S-induced

hypothermia, by simultaneously targeting multiple points

of intervention, could have a higher probability of success

in treating stroke.

However, many questions still must be answered

regarding the use of therapeutic hypothermia for ischemia

in clinical practice, such as the H2S concentration, optimal

target temperature and duration, the therapeutic window

in humans, and cost-effectiveness

Take home message (I)

Our findings indicate that the aged brain has still

the capability to mount a neurogenic response to

stroke but this needs to be stimulated for

therapeutic purposes.

Cell therapy of stroke is a promising avenue of

further research. However, it is not cleat how to

overcome the barrier imposed by the fibrotic scar.

Take home message (II)

Our study has identified a number of potential therapeutic targets

acting both during the acute phase and in the post-stroke recovery

phase.

Our results suggest that a multi-stage, multimodal treatment in aged

animals may be more likely to produce positive results.

While a multi-modal therapeutic approach is promising, one

particularly difficult hurdle will be to offset the post-stroke

downregulation of genes such as those involved in normal physiology

and brain plasticity.

Take home message (III)