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On April 9th 2013 took place at Vall d’Hebron Institut de Recerca (VHIR) the seminar ‘Multi-stage, multimodal approaches for regenerative stroke therapies’, conducted by Pr. Aurel Popa-Wagner, PhD, Professor of Experimental Neurology at the Department of Neurology and Head of the Research Department. Every six minutes a stroke occurs in Spain, the first cause of death in women and the second for men. In the world, it is estimated that there are 4.5 million deaths a year from stroke. Almost one in four men and nearly one in five women aged 45 years can expect to have a stroke if they live to their 85th year. It is estimated that by 2023 there will be an absolute increase in the number of patients experiencing a first ever stroke of about 30% compared with 1983. It is known that intracellular vessel occlusion has a strong age dependency. As long as aging is an important risk factor for stroke, aged animals are being used in the laboratory because of its relevance in clinical rehabilitation and cellular studies. In addition, Pr. Aurel Popa-Wagner and his research group have shown that potential mechanisms for self-repair also operate in the post-ischemic aged brain. Young and aged animals affected by stroke differ in their post-stroke response, being more pronounced at 14 days post-stroke, where downregulated genes decrease in young rats and increases in aged rats, and the opposite occurs with the upregulated genes. Referring to therapy, Pr. Aurel Popa-Wagner and his research group concluded that including both physical methods and methods of cellular therapy is more effective for improving recovery of function in aged rats after stroke than therapies aimed at only a single target system. This is, for example, combining neurogenesis, functional in the subventricular zone of the adult brain, with G-CSF (stem cell mobilisator granulocyte-colony stimulating factor), significantly effective in treatment of aged rats after stroke due to that it reduces the mortality rate in rats. In the first post-stroke hours, induced hypothermia with hydrogen sulfide diminish inflammation and improve neurorehabilitation in aged rats by simultaneously targeting multiple points of intervention. This technique could have a higher probability of success in treating the illness, as the Pr. Popa-Wagner affirms. The next step in post-stroke therapy, according to the speaker, might be the combination of nanotherapy with cell therapy.
Citation preview
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)