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Type: Thesis
Title: Molecular and Cellular Investigations Relating to Neuroplasticity in Stroke
Author: Winderlich, Joshua Nathan
Issue Date: 2018
School/Discipline: Adelaide Medical School
Abstract: Stroke is a leading cause of death and the leading cause of adult neurological disability in Australia. The interventions currently available for the management of stroke include intravenous thrombolysis, thombectomy, decompressive hemicraniectomy, antithrombotic therapy, stroke unit care and rehabilitation. The aims of these interventions are to reduce the amount of damage caused by stroke and to support recovery. Currently, there is no method to reverse the damage caused by stroke. The pattern of functional recovery following stroke reveals a critical period of enhanced neuroplasticity. This period is a candidate therapeutic target for post-stroke neurological repair, either by increasing the degree of neuroplasticity that occurs or by extending the duration of enhancement. Experimental interventions that promise to enhance post-stroke neuroplasticity are being investigated. There is extensive evidence that functional recovery can be improved through interruption of endogenous inhibitory mechanisms, which include perineuronal nets (PNNs) and Nogo signalling. Chapter one outlines a review of the literature concerning the pathophysiology of ischaemic stroke, evidence in support of enhanced post-stroke neuroplasticity and evidence in support of potential therapies that target post-stroke neuroplasticity. This review forms the basis for the body of work described in this thesis. Cell-based therapy is one potential way of enhancing recovery from ischaemic stroke. Stem cell transplantation following stroke has resulted in functional improvements in pre-clinical studies. The mechanism of action underlying this effect is unclear, though it is thought to be through the paracrine secretion of neurotrophic cytokines and not through replacement of lost tissue. Intriguingly, both direct intracerebral transplantation and intravascular transplantation are efficacious. For transplanted stem cells to enter the brain from circulation, they must cross the blood-brain barrier (BBB). While this has been shown to occur, the mechanism through which stem cells cross the BBB has not been fully elucidated. Chapter two demonstrates that human dental pulp stem cells (DPSC) can increase BBB permeability through the expression of vascular endothelial growth factor. DPSC conditioned medium caused an increase in permeability of an in vitro model of the BBB and this effect was reversed by blocking the VEGF receptor. These results support the further investigation of intravascular DPSC administration as a treatment for ischaemic stroke. One of the hypothetical mechanisms of action for cell-based therapy is the interruption of PNNs. These are a specialised form of dense ECM within the adult brain and spinal cord that form part of an endogenous system for the inhibition of neuroplasticity. Digestion of PNNs through administration of a bacterial enzyme has been shown to improve outcomes following neurological insults, including stroke. Chapter three demonstrates that DPSC express soluble products that digest PNNs. Application of DPSC conditioned media to in vitro PNN models and brain slices resulted in decreased staining of PNNs. Additionally, DPSC were shown to express active matrix metalloproteinase-2, which digested aggrecan, one of the main PNN components. These results suggest that interruption of PNNs may be a mechanism through which cell-based therapy enhances recovery in the setting of ischaemic stroke. As PNNs are a target for future stroke therapies, it is important to understand the endogenous response of PNNs to stroke. There is evidence in the literature that PNNs are temporarily downregulated after stroke. Numerous studies have demonstrated the temporary downregulation of PNNs after stroke through general staining for the carbohydrate components of PNNs and some of the protein components. However, PNNs are not homogeneous throughout the central nervous system and variations in their composition affect their ability to inhibit neuroplasticity. The response of PNNs to stroke has not yet been fully described. Chapter four addresses this by characterising the expression profile of several PNN components in a mouse stroke model. Following photochemical infarction, there was a temporary decrease in staining of cartilage link protein-1, aggrecan and WFAbinding glycans in the cortex. This effect was more pronounced in the region of the cortex contralateral to the lesion. Additionally, 4-O-sulfated chondroitin, which is the most inhibitory PNNassociated carbohydrate component, was temporarily enriched in the ischaemic border zone. This pattern of regulation may underlie the post-stroke critical period enhanced neuroplasticity. The development of new stroke treatment strategies targeting neuroplasticity is dependent on preclinical in vivo studies. To ensure that pre-clinical studies are mutually comparable, there is a need for a standardised protocol for modelling human stroke. The requirements of this model are that it be reproducible, technically accessible, it must minimise animal suffering and it must accurately model chronic stroke in humans. Many studies use young animals, which recover rapidly and more completely from ischaemic stroke. This is not appropriate for testing stroke therapies for humans as stroke risk is correlated with age. Additionally, middle cerebral artery occlusion is still considered the gold standard for modelling stroke. This is biologically accurate, but is technically difficult, results in variable infarcts and is associated with relatively high mortality and suffering. Chapter five outlines a preliminary study towards the development of a standardised protocol for chronic stroke in aged mice. A photochemical induction method was used, which resulted in reproducible, targeted lesions that were detectable by MRI. This method produced detectable dysfunction of the affected limb at early time points only. These results suggest that further optimisation needs to be done in developing a standardised model of chronic stroke.
Advisor: Koblar, Simon
Kremer, Karlea
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2018
Keywords: Stroke
dental pulp stem cells
blood-brain barrier
perineuronal nets
animal stroke models
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
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