Author: Wai Ping Yew
Yew, Wai Ping, 2016 Peri-infarct Glial Cell Responses and Functional Recovery After Stroke, Flinders University, School of Medicine
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Recovery of function following stroke depends, at least in part, on the re-organisation of neurons within the viable tissue immediately surrounding the infarct. The processes underlying neuronal plasticity are critically influenced by the responses of microglia and astroglia within this tissue region, also known as the "peri-infarct" tissue. However, the development of these responses and the nature of their influence are not well understood. The investigations described in this thesis aimed to (i) further characterise the changes induced in peri-infarct glial cells, (ii) develop novel approaches for evaluating key changes in these cells in the brain and in a cell culture model and (iii) assess the consequences of early treatment with the anti-inflammatory drug, minocycline, on the peri-infarct glial cell responses and functional recovery.
In the first part of this thesis, quantitative measures were developed based on the changes in morphology, proliferation and migration of glial cells that occur after stroke. These measures were applied to the characterisation of the photothrombotic model of stroke and enabled the detection of activation in peri-infarct microglia by 3 hours following infarction and the novel observation of a loss of these cells at 24 hours. Using the characterised model, the influence of the early post-stroke glial cell responses on functional recovery was investigated by time-targeted treatment with minocycline, a drug widely reported to inhibit microglial activation. Surprisingly, minocycline treatment had limited effects on the quantitative measures of microglial activation following stroke. Despite this, the treatment resulted in improved functional recovery that was associated with increased astroglial reactivity as assessed by GFAP and vimentin expression. Crucially, the early treatment did not result in changes to the infarct volume indicative of neuroprotection. These results provided evidence that minocycline treatment can improve functional recovery after stroke via non-neuroprotective mechanisms, including the enhancement of aspects of the astroglial response that are beneficial to recovery. However, the limited effects on microglial activation suggested the possibility that either microglia were not the primary targets of minocycline, or there were subtle changes that were not detected by the measures used.
In the second part of the thesis, a cell culture model of focal injury was developed based on a method of inducing cell death by rapid, localised cooling. Characterisation of the model in primary mixed glial cultures revealed that it recapitulated key features of the peri-infarct glial cell responses following stroke. Using the model, the role of microglial activation in the development of astrogliosis was investigated by (i) depleting microglial cells from the cultures and, (ii) pre-treatment with minocycline. Depletion of microglial cells led to an increase in the baseline astroglial reactivity as measured by nestin expression, but did not prevent the development of the astroglial response to focal injury. Minocycline treatment did not result in changes to the microglial and astroglial response. The results suggested that the development of the astroglial response is not dependent on microglial activity but may be modulated by it. The lack of effects of minocycline treatment further suggested that microglia may not be its primary target.
Keywords: Stroke, Photothrombosis, Cerebral Ischemia, Microglia, Astrocytes, Astrogliosis, Neuroplasticity, Neurological Recovery
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
Completed: 2016
School: School of Medicine
Supervisor: Professor Neil Sims