Marcoci, Cristina MD; (2022) Overcoming tissue hypoxia – a new approach to the therapy of optic neuritis studied in an experimental model. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background: Multiple sclerosis (MS) is an inflammatory demyelinating and degenerative disease of the central nervous system (CNS) that causes severe neurological deficits, including sensory and motor deficits, and visual impairment. The available therapies aim to interfere with the action of the acquired immune response in worsening disease severity, but the therapies do not primarily aim to protect the nervous system from the mechanisms mainly responsible for effecting loss of function and causing tissue damage. Recent research in the host laboratory reveals an important role for hypoperfusion and tissue hypoxia in causing neurological deficits and tissue damage, and the protective benefit of strategies to protect tissue oxygenation. The tissue hypoxia can be sufficiently severe to impair mitochondrial function, and the consequent energy deficits directly contribute to loss of neuronal function and tissue damage. Notably, the vasodilating drug nimodipine has been found promptly to improve CNS blood flow and tissue oxygenation, and promptly to restore function lost due to inflammation, and protect tissue integrity. We now wish to monitor the success of the therapy in also protecting visual function. Initial experiments (Chapter 2) examined different animal models of inflammation within the visual system, and different methods for retinal examination and detecting mitochondrial failure. Experimental autoimmune uveitis was induced and examined, but the retinal damage was judged to be too severe for reliable observations regarding blood flow and mitochondrial function. The model was further complicated by the appearance of bright spots of autofluoresence induced by concurrent inflammation, probably reflecting autofluoresence in recruited inflammatory cells. The autofluorescence of retinal flavoprotein (produced by functioning mitochondria) was assessed as a biomarker of retinal mitochondrial function, especially when the signal was enhanced by spectral unmixing. The intensity of flavoprotein fluorescence was found to be modulated by changes in the inspired oxygen concentration, and the intensity change was especially clear around veins. We then examined the model of experimental autoimmune encephalomyelitis (EAE) induced in Dark Agouti rats by immunisation with recombinant myelin oligodendrocyte glycoprotein (rMOG) (Chapter 3). The model has been described as the best model of multiple sclerosis, and it has been reported to exhibit experimental autoimmune optic neuritis. Our histological examination of the optic nerves confirmed this result, revealing inflammatory demyelination in at least one optic nerve in all animals expressing a neurological deficit in the hindlimbs and tail. Indeed, optic nerve pathology was present even in some animals that were asymptomatic for EAE. This model was chosen for detailed histological and electrophysiological examination as a basis for our proposed studies with nimodipine (examined in Chapter 4). The inflammation and demyelination started from the retro-orbital end of the optic nerve and progressed towards the chiasm, so that animals with involvement of the chiasm had the most severe disease. The inflammatory cells would appear first around the circumference of the optic nerve, with a predilection to surround veins. Methods for the electrophysiological examination of visual function under general anaesthesia were developed, and they revealed that the optic neuritis was accompanied by a reduction in the amplitude of the visual evoked potential (VEP), which was sometimes ‘flat’, coupled with a delayed latency and changes in its waveform, indicating that the optic neuritis resulted in axonal conduction block and/or conduction slowing, in common with observations in humans. rMOG EAE was chosen as the model for examination of the efficacy of nimodipine in acutely improving electrophysiological function in optic neuritis. Animals were anaesthetised for terminal electrophysiological examination at different stages during the course of disease (before immunisation as baseline, then at day 5 or day 7 after immunisation, at the onset of neurological deficit, at peak of disease, and at relapse), and the VEP was serially recorded in response to unilateral and bilateral flash stimulation, before and after (at 30 minutes, and at 1, 2, 3 and 4 hours) the systemic (intramuscular) administration of nimodipine. Nimodipine caused a significant reduction in the latency of the VEP at 3 and 4 hours after administration (p<0.05). Blind assessment of the efficacy of nimodipine in improving the VEP revealed that the improvement was most pronounced in animals examined at the onset of disease, and also at remission and relapse. Nimodipine had less effect in animals examined at the peak of disease, perhaps because blood flow is restricted by physical compression of the blood vessels due to oedema, preventing their dilation by nimodipine. When animals were divided into nimodipine responders and non-responders, nimodipine caused a significant decrease in latency as early as 30 minutes (p<0.05) after administration, which persisted at 3 hours (p<0.05) and 4 hours (p<0.01). Vehicle administration had no significant effect on latency in either animals with EAE or IFA controls, and nimodipine had no significant effect on latency in IFA controls. At the end of electrophysiological recording, the animals were fixed by perfusion for histological examination, which revealed the presence of optic neuritis in all animals with EAE. We conclude that nimodipine promptly improves function along the visual pathway in experimental optic neuritis, and that the drug should be considered in a clinical trial for the acute treatment of optic neuritis.

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