Expanding the Window of Opportunity: Intransal Delivery of Wnt 3a and Whisker Stimulation in the Ischemic Stroke Mouse Model Open Access

Sharpe, Michka G. (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/gh93h0462?locale=en



Expanding the Window of Opportunity: Intransal Delivery of Wnt 3a and Whisker
Stimulation in the Ischemic Stroke Mouse Model

Each year, one out of six individuals will suffer a stroke. Despite extensive basic and clinical
investigations, stroke remains to be the leading threat to human life and health in the US and
worldwide. Up to now, there are very few effective treatments for acute stroke patients. Novel
neuroprotective therapy and regenerative therapy are urgently needed for clinical applications.
The purpose of this investigation was to test a combination therapy of a canonical Wnt signaling
protein involved in neurodevelopment, Wnt 3a and whisker stimulation in the enhancement of
neurogenesis and angiogenesis following focal ischemic stroke in Nestin-GFP mice. In an
ischemic barrel cortex stroke mouse model induced by ligations of distal branches of the middle
cerebral artery (MCA) in Nestin-GFP mice, Wnt 3a was administrated intranasally to stimulate
the Wnt signaling pathway while peripheral whisker stimulation was applied as a central specific
rehabilitation treatment after stroke. The combination therapy showed increased expression of
brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in the
penumbra region 14 days after stroke. Co-labeling of 5-Bromo-2-deoxyuridine (BrdU) and NeuN
positive cells in the penumbra demonstrated augmented neurogenesis, while increased co-
labeling of BrdU and Collagen IV suggested increased angiogenesis in these mice. Wnt 3a paired
with whisker stimulation also increased local blood flow compared to the saline control. In
addition, the SDF-1 receptor CXCR4 expression increased when Wnt 3a was paired with
peripheral stimulation, suggesting enhanced sensitivity to chemoattractant directed migration that
may promote homing of neuroblasts from the subventricular zone to the infarct site. Cellular
migration was confirmed with positive Doublecortin (DCX) and BrdU co-labeling.

These data support that the combination therapy targeting the Wnt pathway paired with peripheral signals
may be beneficial for long-term tissue repair and functional recovery after ischemic stroke.

Table of Contents

Table of Contents

Acknowledgements ...1

Terminology and Abbreviations ...2

Abstract ...3 Introduction ...4

i. Stroke

ii. Ischemic Cascade

iii. Penumbra as a therapeutic target region in stroke

iv. Limitations of Current Stroke Therapy

v. Peripheral Whisker Stimulation

vi. Wnt 3a and Canonical Wnt Signaling

vii. The Adult Brain and Neurogenesis

Specific Aims and Hypotheses ...14 Materials and Methods ...15

i. Focal Ischemia in Animals

ii. Wnt 3a Intranasal Delivery

iii. BrdU Injections

iv. Whisker Stimulation

v. Measurement of Cerebral Blood Flow

vi. Immunohistochemistry

vii. Western Blot Analysis

viii. Sterological Cell Counting

ix. Statistical Analysis

Results ...20 Figures ...25

Figure 1. Wnt 3a+Stimulation enhances blood flow to the penumbra 14 days after stroke.

Figure 2. Stimulation and not Wnt 3a+Stimulation enhances cerebral blood flow 21 days after stoke.

Figure 3. Immunohistochemistry for vessels, neuronal nuclei and newly proliferating cells.

Figure 4. Combination therapy further enhances neurogenesis in the penumbra 14 days after stroke.

Figure 5. Increased BDNF expression with combination of Wnt 3a and whisker stimulation.

Figure 6. Combination treatment of Wnt 3a+Stimulation further enhances angiogenesis 14 days after ischemic stroke.

Figure 7A. Wnt 3a+Whisker stimulation demonstrates cell migration towards the penumbra 14 days after ischemic stroke.

Figure 7B. Wnt 3a+Stimulation has increased CXCR4 expression compared to Wnt 3a and saline groups but not stimulation only group.

Figure 8. Increased expression of VEGF when stimulation is paired with Wnt 3a.

Figure 9. XAV939 Mechanism of Action.

Figure 10A. Ischemic stroke in saline control C57/B6 mouse.

Figure 10B. XAV939 Wnt antagonist truncates de novo neurogenesis 14 days following ischemic stroke.

Figure 11. XAV939 blocks Wnt 3a activity in vivo.

Discussion ...36 References ...45

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