The document discusses the challenges and strategies in stroke rehabilitation, particularly highlighting the importance of studying aged animal models to understand the age-dependent responses to stroke. It outlines findings from various studies on gene expression changes, treatment approaches including G-CSF and stem cell therapies, and the potential role of hypothermia in stroke recovery. The conclusion emphasizes the need for multimodal treatment strategies that address the complexity of stroke pathology and aging effects on neurogenesis and recovery.

1. MULTIMODAL APPROACHES FOR
REGENERATIVE STROKE THERAPIES:
Aurel Popa-Wagner
Medical University Rostock, Germany
University of Medicine and Pharmacy
Craiova, Romania

2. Intracerebral vessel occlusion has a strong AGE
dependency

3. Brain of a Patient after MCA Occlusion

4. 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.

5. STAIR Criteria
UKM (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

6. Beam- Young Stroke

7. Beam- Old Stroke

8. Young Stroke- Table

9. Old Stroke- Table

11. 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

12. 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%).
A
Buga..Popa-Wagner, JCBFM 2012
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

13. VENN-Diagram. Note that at 14 days post-stroke, the differences between
the age groups were more pronounced

14. Transcriptomics of Stroke:
Divergent gene expression
after 2 weeks post-stroke

15. 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

16. Patterns of gene expression after stroke. Patterns of gene expression for stroke-releva
Most classes of these new genes were upregulated, with the exception of “CNS physiolog
and “Neurogenesis & synaptic plasticity” which also displayed a large number of downregu

17. 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

18. Transcriptomics of Stroke. Disregulation of gene expression
required for Neurogenesis & Synaptic plasticity

19. Transcriptomics of Stroke. Disregulation of gene expression
required for Embryonic development & CNS regeneration

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

21. Pilot studies aimed at improving recovery of
function in aged rats after stroke
Stimulation of endogenous neurogenesis

22. Rat Modell for Cerebral Ischemia

23. Summary of functional tests after stroke
Rotarod T-Maze Neurol.
Status
Radial Maze i-Plane Cylinder Test

24. G-CSF, multimodale Mechanismen in der Schlaganfalltherapie
UKM
Schäbitz and Schneider 2007

25. G-CSF treatment after stroke significantly improved mortality rate and
performance in several but not all functional tests.

26. 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

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

28. 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.

29. Stroke Cell Therapy: flow diagram
Therapies Behavioral Analysis
Training
MCAO
MRI#1 MRI#2 Tissue Analysis
-14d 0 6h 3d 45d 60d

30. Multimodal Approaches for
Regenerative Stroke Therapies (MARS)
Animal Model and Treatment; 80 ♂ Sprague-Dawley (SD) –Ratten
Groups:
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

31. ROUTE of ADMINISTRATION: Jugular Vein

32. Validity of the administration pathway
Perilesional Area Some cell enter the brain via the ventricle

33. The Mortality Rate was low for all treatments
Group C: Survival proportions
105 Group No Deaths
CTRL 2
100 G-CSF 2
Percent survival
G-CSF + BM MNC2
95 G-CSF + BM MSC 3
90
85
80
0 20 40 60 80
Days after stroke

34. Cell therapy does not reduce the volume of the infarct

35. Water Maze
1
4
3 2

36. G-CSF Treatment led a significant improvement of spatial memory

37. G-CSF Treatment alone improves temporarily sensory function
(Adhesive Tape Removal Test)
0.9
0.8
0.7
Score
0.6 Group CTRL
Group A
0.5
* P<0.05
0.4
0 3 7 14 21 28 35 42 49 56
Day
0.9 0.9
0.8 0.8
0.7 0.7
Score
Score
0.6 0.6
Group CTRL Group C
0.5 0.5
Group B Group CTRL
0.4 0.4
0 3 7 14 21 28 35 42 49 56 0 3 7 14 21 28 35 42 49 56
Day Day

38. Beam-walking test: Both G-CSF alone and the combination therapy
improved performance vs controls
6 * P<0.04
5
4
Score
3
2
GROUP A
1 CTRL
0 3 7 14 21 28 35 42 49 56
Day
6
* P<0.05 * P<0.05 6
5
5
* P<0.05
4
4
Score
3 Score 3
2 2
GROUP B GROUP C
1 1
CTRL CTRL
0 3 7 14 21 28 35 42 49 56 0 3 7 14 21 28 35 42 49 56
Day Day

39. 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

40. At two months post-stroke, G-CSF + BM MSC therapy promotes
the growth of lymphatic vessel

41. Pilot studies aimed at improving recovery of
function in aged rats after stroke
Stimulation of endogeneous neurogenesis
by chemical and by small electrical currents

42. PTZ before Stroke
1 st PTZ 2nd PTZ
0
Day -48 -24 2- Start behavioral testing 48
3x BrdU 3x BrdU
End
PTZ after Stroke 0
2 7 1 st PTZ 31 2nd PTZ 48
Or ECS Or ECS
3x BrdU 3x BrdU
End
Control Groups
1 st Saline 2nd Saline
0
Day -48 -24 2 48
3x BrdU 3x BrdU
End
0
2 7 31 48
3x BrdU 3x BrdU
End

43. Neurogenesis does not significantly improved performance
in
Adhesive Tape Removal Test
0,6
0,4
0,2
time quotient
0,0
-0,2
-0,4
PTZ before stroke
-0,6 Ctrl
PTZ after stroke
-0,8
0 10 20 30 40 50
days

44. Neurogenesis after stroke significantly improved performance
on the
Inclined Plane
40
PTZ before stroke
Ctrl
PTZ after stroke
38
36
Angle
34
32
30
0 10 20 30 40 50
days

45. Neurogenesis after stroke significantly improved performance
in
Radial Maze
0,0
0,2
0,4
score
0,6
0,8
1,0 PTZ before
Ctrl
PTZ after
1,2
0 10 20 30 40 50
days

46. Stroke Volume of all Groups in the NGS Project
30
x10 25
Volume [mm3]
20
15
10
5
0
Stroke Volume
Control 13,38
PTZ before Stroke 15,69
PTZ after Stroke 17,86
ES after Stroke 18,16
Sham 5,49

47. Number of Doublecortin positive Cells in Hippocampus
10
8
Cell Number
6
4
2
0
Hippocampus ipsilateral Hippocampus contralateral
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
Tissue

48. Semiquantitative Evaluation of Doublecortin positive Cells in
Subventricular Zone
3
2
Cell Score
1
0
Subventricular Zone ipsilateral Subventricular Zone contralateral
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
Tissue

49. Number of PSA-NCAM positive cells in Hippocampus
10
8
Cell Number
6
4
2
0
Hippocampus ipsilateral Hippocampus contralateral
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
Tissue

50. Electric Stimulation is efficient in increasing the number of early
neurogenesis like Doublecortin (shown in green) in the subven

51. Increased number of axons in the perilesional area of aged rats following
neurogenesis enhancement

52. Accelerated scar buildup in aged rodents after stroke

53. (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)

54. Neuroepithelial cells contributing to the glial scar originate
mostly in the corpus callosum

55. Neuroepithelial cells contributing to the glial (D, H) scar
emanate from desintegrated the blood vessel walls; Green:
BrdU nuclei; Violett: Laminin

56. Early formation of the growth-inhibiting SCAR and late
outgrowth of axons after stroke in aged subjects
SCAR
AXONS

57. Microglia immunoreactivity is precipitously increased after cerebral ischemia in
pH3 rats. Could anti-inflammatory Drugs Improve Recovery of Function after
old pH10
Stroke?
3 days 7 days 14 days 28 days
young
aged
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).

58. Eight weeks after stroke the glial scar (RED) region is heavely populated
with infammatory cells (Blue)

59. H2S-induced Hypothermia Diminish Inflammation and
Improve Neurorehabilitation after Stroke in Aged
Rats

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

61. H2S is efficient to induce long-term hypothermia after stroke in aged rats
80 ppm H2S
Baltromejus et al., NSL, 2008

62. Baseline of Temperature and EEG under Normothermic
and Hypothermic Conditions
40 ppm H2S
Popa-Wagner et al., J Cereb Blood Flow & Met, 2012

63. Schematic overview of the experimental design. (B): Timecourse of whole body
cooling after stroke in rats immersed in an atmosphere containing 50 ppm H2S.

64. 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

65. EEG telemetry data. A representative telemetric recording
for a rat subjected to H2S is presented in a-f.

66. 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
Popa-Wagner et al., J Cereb Blood Flow & Met, 2012

67. Independent identification of annexin a1 by 2D
electrophoresis and Western Blotting

68. 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

69. What is next?
Combine Nanotherapy with Cell therapy?
Day 2 Day 7
+
BV
magnetic/plasmonic moderate hyperthermia Cell therapy under mild hyperthermia
(41.8°C). IC = infarct core; PI = perinfarct (38°C); IC = infarct core; PI = perinfarct;
BV = blood vessel

70. Take home message (I)
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

71. Take home message (II)
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.

72. Take home message (III)
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.