Vulnerability of white matter structure and function to chronic cerebral hypoperfusion and the effects of pharmacological modulation

Abstract

The structural integrity of the white matter is required for neuronal communication within the brain which is essential for normal cognitive function. Post-mortem and clinical imaging studies of elderly individuals have demonstrated that white matter integrity is weakened with increasing age which is proposed to underlie age-related cognitive decline. Whilst the exact mechanisms are unknown it is thought that modest age-related reductions in cerebral blood flow, termed chronic cerebral hypoperfusion, may contribute to white matter disruption and impaired cognition with ageing. Investigating the effects of white matter integrity in humans is limited as it is difficult to definitively ascertain a cause and effect relationship. Indeed, elderly individuals with cerebral hypoperfusion often have co-existing disease such as hypertension thus the effects of hypoperfusion in isolation cannot be determined. This has led to the development of a mouse model of chronic cerebral hypoperfusion which provides the opportunity to directly assess whether cerebral hypoperfusion results in disruption to white matter and cognitive impairment. This is achieved by applying small wire coils around both common carotid arteries of the mouse resulting in a global reduction in cerebral blood flow. Importantly the extent of blood flow reduction is dependent on the internal diameter of the coils meaning that differing severities of hypoperfusion can be studied. Previous studies using this model have demonstrated diffuse white matter pathology in white matter tracts including the corpus callosum, internal capsule and optic tract following 1 month of hypoperfusion which is accompanied by impaired spatial working memory. This thesis sought to test the hypothesis that chronic cerebral hypoperfusion would influence the structural integrity of nodal and paranodal domains of myelinated axons of the white matter and result in decreased numbers of oligodendroglial cells. It was additionally hypothesised that treatment with the anti-inflammatory and antioxidant drug dimethyl fumarate (DMF) would ameliorate structural and functional alterations to white matter following hypoperfusion. Aim 1 – To determine the impact of chronic cerebral hypoperfusion on the structural integrity of nodal and paranodal domains of myelinated axons The first aim of this thesis was to investigate the effects of chronic cerebral hypoperfusion on the structural integrity of nodal and paranodal domains of myelinated axons. This was addressed by examining key myelin and axonal proteins found at nodal, paranodal and internodal domains. This revealed significant alterations to the distribution of voltage-gated sodium (Nav1.6) channels at nodes of Ranvier which were differentially altered in response to increasing durations of chronic cerebral hypoperfusion. Specifically an increase in the Nav1.6+ domain length was observed in the corpus callosum following 3 days (p < 0.0001) and 1 month (p < 0.001) of chronic cerebral hypoperfusion but was not significantly different from sham controls following 6 weeks of hypoperfusion (p = 0.066). A significant decrease in Nav1.6 domain length was observed following 3 months of hypoperfusion (p = 0.003). Assessment of paranodal integrity was carried out by measuring nodal gap length and by ultrastructural analysis of paranodal domains. This revealed pronounced alterations to nodal gap length, loss of paranodal septate-like junctions and abnormal morphology of paranodal loops. Furthermore this study revealed a significant loss of myelin associated glycoprotein, a key protein involved in the maintenance of axon-glial integrity, as early as 3 days following the onset of hypoperfusion. A further aim of this study was to examine potential mechanisms underlying the observed alterations to nodal and paranodal domains following cerebral hypoperfusion. It was hypothesised that increased inflammation and accumulation of mitochondria at nodes of Ranvier would be observed following hypoperfusion. The extent of inflammation was assessed by counting numbers of microglia which revealed no significant difference between groups following 3 days of hypoperfusion (p = 0.425) but a significant increase in microglial number was observed following 1 month of hypoperfusion (p = 0.001). In addition, assessment of mitochondrial distribution along myelinated axons revealed decreased numbers of nodes containing mitochondria following 6 weeks of hypoperfusion (p = 0.03) with no difference between groups observed following 3 months (p = 0.742). Taken together the results from this study provide evidence that chronic cerebral hypoperfusion results in dynamic alterations in the localisation of Nav1.6 channels which are accompanied by disruption to paranodal domains and impaired axon-glial integrity. Furthermore microglial number does not appear to mediate nodal and paranodal disruption following 3 days but may contribute to ongoing pathology following 1 month of chronic cerebral hypoperfusion. Aim 2 – To determine the effects of chronic cerebral hypoperfusion on oligodendroglial populations The second aim of this thesis was to determine the effect of chronic cerebral hypoperfusion on numbers of mature oligodendrocytes and oligodendrocyte precursor cells (OPCs). This revealed a significant decrease in numbers of both populations following 3 days of cerebral hypoperfusion however following 1 month numbers of OPCs were restored and a significant increase in mature oligodendrocyte number was observed. Assessment of OPC proliferation demonstrated low numbers of proliferating cells but revealed that a proportion of newly generated cells had differentiated into mature oligodendrocytes. To determine a potential mechanism involved in OPC differentiation following cerebral hypoperfusion the expression of the GPR17 receptor was examined which has recently been reported to mediate OPC differentiation in response to injury. The results demonstrated decreased expression of GPR17 following 3 days of hypoperfusion (p = 0.007) with no difference between groups observed following 1 month (p = 0.362) indicating that this receptor is not involved in differentiation of OPCs following hypoperfusion. Taken together the results from this study show that mature oligodendrocytes and OPCs are lost early in response to hypoperfusion but that these cells recover over time, highlighting the regenerative capacity of the white matter following cerebral hypoperfusion.Aim 3 – To investigate whether modulation of inflammation and oxidative stress could ameliorate alterations to white matter structure and function following severe chronic cerebral hypoperfusion The third and final aim of this thesis was to determine whether treatment with the anti-inflammatory and antioxidant drug DMF could ameliorate structural and functional alterations to white matter following severe chronic cerebral hypoperfusion. This was achieved by examining myelin and axonal integrity in addition to numbers of oligodendrocytes and OPCs following 7 days of severe chronic cerebral hypoperfusion. This revealed that myelin integrity was significantly decreased in vehicle-treated hypoperfused animals as compared to shams (p = 0.005). However no differences in myelin integrity were observed between sham and hypoperfused mice treated with DMF (p = 0.312). In contrast to the previous study, numbers of oligodendrocytes and OPCs were not altered following severe hypoperfusion however DMF treatment led to significantly increased numbers of oligodendrocytes in sham animals (p = 0.003). Assessment of white matter function using electrophysiology revealed that the conduction velocity of myelinated axons was significantly increased in DMF-treated hypoperfused animals as compared to those treated with vehicle (p = 0.04). Taken together the results of this study demonstrate that modulation of inflammation and oxidative stress may improve structural and functional white matter alterations following chronic cerebral hypoperfusion. Conclusions The results presented in this thesis demonstrate that chronic cerebral hypoperfusion results in structural alterations to myelinated axons and to oligodendroglial populations within the white matter which are accompanied by impaired spatial working memory. Whilst previous studies using the model have reported that cerebral hypoperfusion results in diffuse white matter pathology, this study has highlighted the vulnerability of nodal and paranodal domains of myelinated axons as regions which are altered early in response to hypoperfusion. Furthermore, characterisation of oligodendroglial populations has revealed that these cells are replaced over time despite ongoing hypoperfusion which demonstrates the regenerative capacity of the white matter following cerebral hypoperfusion. Critically the results presented in this thesis demonstrate that treatment with DMF improved the function of myelinated axons in response to severe reductions in cerebral blood flow and thus may represent an appropriate therapeutic strategy for chronic cerebral hypoperfusion.