Characterisation of endogenous repair mechanisms following endothelin-1 induced stroke in rats and the effects of human adult stem cell transplant to support brain reconstruction

Abstract

Brain injury from stroke often results in permanent damage and disability due to neurons failing to re-establish lost connections. Potential for brain regeneration relies heavily on the surrounding microenvironment. Contributions from inflammatory cells, angiogenesis and stem cells all require collective consideration when investigating treatment options. Understanding this paradigm is critical to developing therapies that promote recovery. Cell-based therapies offer hope in rescuing the stroke affected brain and restoring function. However, differentiation of transplanted cells into significant neuronal populations is yet to be realised. The work presented in this thesis investigates cellular responses to brain injury and repair mechanisms activated following stroke using the endothelin-1 model of focal cerebral ischemia in conscious rats. Additionally these studies explore the potential of human adult neural progenitor cells to support brain repair.

First, we investigated aspects of brain remodelling initiated following stroke, including the impact of lesion size on angiogenesis, cell responses within the subventricular zone (SVZ), inflammation, and scar formation. Immunohistochemical analysis revealed a positive correlation between stroke severity and the degree of pathological responses to recovery after stroke. Stroke severity was found to increase cell proliferation and migration from the SVZ, with many of these cells positive for GFAP and incorporated into the glial scar. Therefore, we highlight this as an important factor to consider when developing treatment strategies that stimulate cell responses within the neurogenic niche.

Long term survival and success of non-autologous stem cell transplants requires use of immunosuppressive agents such as cyclosporine A (CsA). The influence of CsA on stroke outcome required investigation prior to commencing the intended stem cell transplant studies. We explored the effects of CsA administration on neurological and histological outcomes 7 days after stroke. Findings indicated CsA treatment significantly reduced the development of neurological deficits after stroke but did not affect infarct volume, activation of microglia/macrophages, or events within the neurogenic niche. CsA treatment did however attenuate reactive gliosis after stroke and retained pro-survival astrocytic phenotypes important for supporting neuronal rescue.

Finally, we investigated the use of cell-based therapies to promote brain repair. SVZ-derived human neural progenitor cells (hNPCs) were isolated, characterized, and differentiated into GABAergic neurons. Pre-differentiated GABAergic neurons, undifferentiated SVZ-hNPCs or media alone were transplanted into the rat brain 7 days after stroke during angiogenesis as it was hoped exogenous transplants would benefit from a redeveloped microvascular bed. GABAergic cell transplants were observed to accelerate functional recovery, showed evidence of maturation and promoted endogenous neurogenesis 28 days post-transplant. Undifferentiated hNPC transplants were predominantly GFAP positive and incorporated into the glial scar. These results suggest techniques aimed at differentiating cells into a neural lineage prior to transplant may be a favourable alternative for stroke treatment. Furthermore, targeting angiogenesis for cell-based treatments may offer greater survival of cells and support graft maturation for functional recovery.

In conclusion, this work contributes significantly towards characterising endogenous cellular responses to stroke injury and recovery, and provides preclinical evidence for the benefits of directing exogenous stem cell differentiation towards neural lineage cells prior to transplant, resulting in accelerated recovery.