Bender_whole_thesis.pdf (14.65 MB)
Investigations of excitotoxic injury in the visual system of mice
thesisposted on 2023-05-28, 12:29 authored by James BenderJames Bender
A principal feature of neurodegenerative conditions is the loss of nerve cells and altered neuronal circuitry by means of axonal degeneration. Neurodegeneration and alterations to the neuronal cytoskeleton are key pathological features associated with neurodegenerative conditions and nervous system trauma. Since the pathological processes that result in neurodegeneration may vary between diseases, identifying and understanding the underlying mechanisms of neurodegeneration in different pathological conditions may reveal pathways worthy of investigation for therapeutic intervention. Excitotoxicity is a pathological process that occurs in a variety of neurodegenerative diseases and following trauma to the nervous system. Currently, little is known about how excitotoxicity drives neurodegeneration, and in particular the degeneration of the axon and whether these mechanisms are similar or different to well-described neurodegenerative pathways such as Wallerian degeneration. Furthermore, it is unclear whether these pathways can be targeted to protect against or prevent the resultant pathology. Traditionally, investigating the in vivo effects of excitotoxicity and excitotoxic insult to the central nervous system (CNS) has been carried out either using invasive methods or has presented difficulties in analysis of changes to downstream circuitry. This thesis employed the visual system as a model to investigate mechanisms of neurodegeneration following excitotoxicity, as the visual system is an extension of the CNS with well-defined anatomy and function that is accessible with relatively minimal intervention. As such, this thesis presents a model of excitotoxic injury in the visual system of mice and uses this model to interrogate the mechanisms of excitotoxic insult and axonal degeneration. To do this, mice were subjected to unilateral intravitreal administration of the excitotoxin, kainic acid (KA), to elicit an excitotoxic insult that was subsequently investigated using behavioural, histological, and immunohistochemical techniques. Before interrogation of the underlying pathological processes of excitotoxic insult was performed, initial development and characterisation of the injury was carried out. Using immunohistochemical techniques, a qualitative analysis of cellular changes in the retina was performed over a 7-day time course and with varying doses of KA, along with assessments of visual acuity as a measure of function. Of particular interest were the observations of distinct changes to the immunolabelling of the cytoskeletal components, the neurofilament and microtubules, in the retinal ganglion cells (RGCs), and of pathological features associated with the processes of axonal degeneration. These pathological changes were associated with ablation to reflexes of visual acuity following exposure to the excitotoxin at concentrations ‚Äöv¢‚Ä¢1mM in the 1˜í¬¿m that was intravitreally administered. As changes to the cytoskeletal profile were a common feature in the tissue exposed to excitotoxic insult, subsequent research investigating the modulation of cytoskeletal elements was performed using the visual system model. First, I investigated whether stabilizing the microtubule network with the microtubule stabilising drug, Epothilone D, prior to intravitreal administration of 1mM KA, 2mM KA, or PBS, would have an effect on any of the observed functional or pathological outcomes 7-days after treatment. Tissue from animals treated with KA exhibited a profound increase in the presence of dystrophic and swollen axonal profiles that was associated with the ablation of performance in the visual acuity behavioural testing. These data demonstrated a limited protective capacity of microtubule stabilisation with Epothilone D against the histopathological features of neuronal loss or axonal degeneration profiles in this model of excitotoxic insult. However, the data from the optomotor behavioural test of visual function did suggest that Epothilone D treatment may facilitate an accelerated recovery from the loss of visual acuity that was associated with a moderate (1mM) dose of KA. Based on my qualitative analysis of alterations to neurofilament proteins, I next examined the role of the neurofilament cytoskeleton in neuron and axon degeneration following excitotoxicity. To do this I used a mouse model with a knockout of the neurofilament light chain protein. As in the previous study, mice were injected with 1mM KA, 2mM KA or PBS and visual function was assessed prior to tissue collection and histopathological assessment. Interestingly, these mice demonstrated a significant retention of visual acuity 7-days after injury, where the wildtype equivalents exhibited near-uniform ablation of this behavioural response. This retention of functional performance was not associated with any significant differences in the histopathological features of cell body loss and dystrophic axonal profiles in this model, which were equally present in both the transgenic and wildtype tissue following excitotoxic insult. As the retention of visual acuity in NFL-KO animals occurs independently of any observable retention of ganglion cell layer nuclei or axonal integrity (relative to WT) it may be that the changes to visual function observed in this model are not solely associated with the observed pathological alterations. Finally, I used a mouse model with genetic modulation of mechanistic pathways associated with axonal degeneration. The model of excitotoxic injury developed in this thesis was applied to a mouse line with a knockout of the Toll/interleukin receptor protein and pro-degenerative factor in the axon degeneration pathway, SARM1. The knockout animals used in this study did not exhibit any significant alterations in the loss of visual acuity induced by the excitotoxic injury, nor were there any differences in cell body loss relative to the wildtype equivalents. However, the transgenic mice did have a significant reduction in the presence of degenerative axonal profiles following excitotoxic insult. Additionally, analysis of the optic nerves of the SARM1-KO mice relative to wildtype mice demonstrated that these animals displayed cross-sectional axonal profiles that were larger and more varied in size than wildtype animals independently of excitotoxic insult. This suggests that the model of injury applied in this thesis can result in a functional deficit independently of distinct changes to axonal morphology. As the SARM1-KO mice exhibited the same degree of cell body loss as the WT, it may be that SARM1 is necessary for the processes specific to axonal degeneration to be carried out, but this does not mean that neuronal health or function is upheld. In summary, the research in this thesis presents an in vivo model of excitotoxic insult in the visual system of mice. This injury model results in a demonstrable functional deficit and observable pathological features associated with the mechanisms of axonal degeneration. The modifications that are applied to the model of injury presented in this thesis do not individually confer systemic protection against the presented model of injury but do each exhibit unique features of protection or recovery from the insult.
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