PURPOSE: To characterize the distribution of white matter neurons (WMNs) positive for perineuronal nets (PNNs) in the adult monkey. WMNs are a mixed population of excitatory and inhibitory neurons. They have an important role in axon guidance during cortical development, but their role in the adult brain is less understood. In vitro and in vivo experiments provide evidence that WMNs are incorporated into cortical circuitry. The majority of investigations in the adult, however, have focused on regional variations in overall density, or on characterization of morphological and neurochemical subtypes. The present study was motivated by the observation that some WMNs exhibit PNNs in adult monkey. Since PNNs are associated with plasticity in younger animals, their occurrence with some WMNs might be functionally significant. METHODS: PNNs were visualized, at the light microscopic level, by WFA staining in three adult macaque brains. Density of WFA positive WMNs was scored at three anterior-posterior levels (frontal, mid-hemispheric, and occipital), and compared with overall density of WMNs, as visualized by immunocytochemistry for NeuN. Quantitation of WFA+ neurons and neuron morphology were analyzed via light microscopy. Soma size and appearance, and dendritic length were recorded and measured. RESULTS: On the basis of soma size and proximal dendritic shape, several types of WFA+ WMNs were provisionally identified, consistent with previous reports in the literature. Subpopulation densities were of highest density in mid-cortical areas and lowest quantities at occipital, matching previous studies. Morphological measurements suggested a heterogeneous neuron population through soma measurements and dendrite orientation. Soma sizes exhibited a range of circularity and size (10 µm – 30 µm). Dendrites were stained beyond the “proximal” area, including intermediate areas beyond the first branch, and up to 500 µm. CONCLUSIONS: A small population of WMNs are coated by PNNs in adult monkey. On the basis of morphology, these might be further subdivided, but combined studies with other markers would be needed. Future studies might investigate age- or pathology-related changes in the density and subtypes of WMNs that express PNNs in human or nonhuman primates. We speculate that these WMNs might have functional specializations, perhaps similar to the plasticity effects documented for PNNs in early development.

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CADASIL est une forme héréditaire, autosomique dominante, de maladie des petits vaisseaux cérébraux dans laquelle surviennent précocement des lésions de la substance blanche cérébrale qui progressent avec le temps, mais dont la nature histopathologique demeure très mal connue. La maladie est causée par des mutations très stéréotypées du récepteur Notch3. Une des signatures de CADASIL est la présence, dans les vaisseaux, d’une accumulation du domaine extracellulaire de NOTCH3 (Notch3ECD). Un faisceau d’arguments suggère que le processus pathogène de CADASIL résulte d’un effet toxique de ces dépôts de Notch3ECD, qui passerait par l’accumulation avec le NOTCH3ECD d’autres protéines de la matrice extracellulaire. Il a cependant été montré que des mutations CADASIL affectent les capacités de signalisation du récepteur, de manière constitutive ou avec le temps, ce qui a conduit à formuler l’hypothèse qu’une perte de fonction Notch3 pourrait également constituer un déterminant important du processus pathogène.Nous avons réalisé une analyse détaillée des lésions de la substance blanche dans un modèle murin de la maladie CADASIL obtenu par surexpression d’un allèle Notch3 avec la mutation R169C / R170C, qui en récapitule les stades précliniques (TgPACNotch3R169C). Ceci a permis de mettre en évidence aux stades précoces un oedème intramyélinique associé à une dégradation / décompaction de la myéline détectable en immunohistochimie dès l’âge de 6 mois. L’analyse de l’intégrité axonale au sein des lésions de la myéline suggère une perte secondaire. Une méthode de quantification semi-automatisée des débris myélinique a été élaborée.Nous avons ensuite testé l’hypothèse qu’une perte de fonction Notch3 pourrait constituer un déterminant majeur dans le processus pathogène de CADASIL. Nous avons pour cela identifié un set de gènes dont l’expression est sensible à la quantité de Notch3, capable de détecter une diminution de moitié de la dose de Notch3. La mesure de l’expression de ces gènes chez des souris Knock-in pour la mutation R170C, hétérozygotes ou homozygotes, a montré que l’activité Notch3 n’était pas diminuée dans ce modèle. Nous avons ensuite étudié l’impact de la suppression des copies endogènes de Notch3 sur les lésions de la substance blanche chez les souris TgPACNotch3R169C, qui n’apparaissent pas aggravées. Ces résultats plaident contre un effet hypomorphe commun à toutes les mutations CADASIL et suggèrent que les lésions de la substance blanche ne sont pas secondaires à un tel effet.Nous avons enfin étudié le rôle pathogène de l’excès de TIMP3 et vitronectine, deux protéines dont il a été démontré qu’elles s’accumulent précocement avec le NOTCH3ECD. En utilisant des approches d’interaction génétique (diminution et/ou augmentation de la quantité de TIMP3 et vitronectine chez les souris TgPACNotch3R169C), nous avons observé un effet différent de l’excès des deux protéines sur les anomalies de la réactivité cérébrovasculaire et celles de la substance blanche. En effet, la réduction de la quantité de vitronectine limite les lésions de la substance blanche sans effet sur la réactivité cérébrovasculaire alors que la réduction de TIMP3 corrige les anomalies vasculaires fonctionnelles sans effet sur la substance blanche. Ces résultats apportent la preuve de concept du rôle pathogène de l’accumulation des protéines TIMP3 et vitronectine dans le processus pathogène de CADASIL et remettent en question les dogmes faisant de l’hypoperfusion le facteur promoteur des lésions de la substance blanche dans la maladie. / CADASIL is an autosomal dominant, hereditary, small vessel disease of the brain causing early and progressive white matter lesions. The histopathological characteristics of these lesions remain poorly known. The disease is caused by stereotyped mutations in the gene coding for the NOTCH3 receptor. One of CADASIL hallmarks is the presence in vessels of an abnormal accumulation of NOTCH3 extracellular domain (NOTCH3ECD). Data suggest that CADASIL pathophysiological process may be caused by a toxic effect resulting from NOTCH3ECD deposits, due to an abnormal recruitment of other extracellular matrix components. However, it has been shown that CADASIL mutations differentially affect Notch3 signaling, constitutively or progressively. The latter observations led scientists to propose the hypothesis that Notch3 loss of function may play an important role in CADASIL pathogenesis.We conducted a detailed white matter analysis in a CADASIL mouse model that overexpresses a Notch3 allele with the R169C/R170C mutation and that recapitulates the preclinical stages of the disease (TgPACNotch3R169C). In this model, we observed intramyelinic edema associated with myelin degradation / decompaction detectable by immunochemistry in the brain of mice as young as 6 months of age. Axonal integrity analysis in myelin lesions suggested that axonal loss may appear secondarily. A semi-quantitative method for the quantification of myelin debris has been developed.Next, we tested the hypothesis that Notch3 loss of function might play a key role in CADASIL pathophysiology. We first identified a set of genes that are sensitive to a reduction in Notch3 dosage by half. Quantification of these genes expression in both heterozygous and homozygous mice Knock-in for the R170C mutation showed that Notch3 activity was not lowered in this model. In addition, we analyzed the effect of a suppression of endogenous Notch3 copies on white matter lesions observed in TgPACNotch3R169C mice and observed no worsening of these lesions. Together these results suggest that hypomorphism is not a feature common to all CADASIL mutations, and that white matter lesions in CADASIL do no result from Notch3 loss of function.Finally, we studied the pathogenic effect of Timp3 and vitronectine accumulation, both proteins having been shown to accumulate with NOTCH3ECD early in the course of the disease. By the use of genetic interaction approaches (lowering and increase in Timp3 and vitronectine in TgPACNotch3R169C mice), we observed differential effects of the proteins on white matter lesions and cerebrovascular reactivity impairment. Indeed, vitronectine lowering improves white matter lesions without any effect on cerebrovascular reactivity while Timp3 diminution restores cerebrovascular reactivity without any effect on white matter lesions. These results provide proof of concept for the implication of TIMP3 and vitronectin excess in CADASIL pathogenesis and questions the dogma that make hypoperfusion the main determinant of white matter lesions in CADASIL.

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Background. Lacunar strokes account for 25% of all ischaemic stroke but the exact nature of the causative cerebral small vessel abnormality remains unknown. Pathological studies are technically difficult and brain imaging cannot adequately characterise the cerebral small vessels. The retinal blood vessels are of similar size and physiology to the cerebral small vessels and may act as a surrogate marker for these cerebral small vessels. We therefore investigated retinal microvascular abnormalities in lacunar stroke. Methods. We performed a systematic review of retinal microvascular abnormalities in lacunar stroke to clarify associations and identify where further research was required. We then established a cohort of patients presenting with lacunar stroke with cortical stroke controls to investigate differences in retinal microvascular abnormalities between stroke subtypes. All patients had MRI brain at presentation and digital retinal photography of both eyes. We investigated the prevalence of retinopathy (hard and soft exudates or haemorrhages/microaneurysms), focal arteriolar narrowing and arteriovenous nicking . We developed, validated and used novel semi-automated techniques for measuring retinal arteriolar and venular widths, retinal arteriolar geometry (branching co-efficients (change in arteriolar cross sectional area across a bifurcation) and branching angles) and fractal dimensions (reflecting branching complexity) of the vasculature. We also assessed MRI parameters in lacunar stroke. We used multivariable analysis to correct for baseline imbalances in vascular risk factors. Results. From the systematic review we demonstrated that retinal microvascular abnormalities are associated with incident and prevalent stroke but that in general, strokes were inadequately characterised and there were no data regarding retinal microvascular abnormalities in ischaemic stroke subtypes. We recruited 253 patients, 129 lacunar strokes and 124 cortical strokes, mean age 68 years. We found no difference in the prevalence of retinopathy, arteriovenous nicking, focal arteriolar narrowing or arteriolar widths between lacunar and cortical stroke subtypes. We found that venules were wider in lacunar stroke. We found no differences in arteriolar branching co-efficients or arteriolar branching angles between lacunar and cortical strokes but found that deep white matter white matter hyperintensities on MRI were associated with increased branching co-efficients and periventricular white matter hyperintensities associated with decreased branching co-efficients. We found that the fractal dimension of the vascular tree was decreased in lacunar stroke. Furthermore we found that enlarged perivascular spaces on MRI are associated with lacunar stroke and white matter disease. Conclusions. We have clearly demonstrated that retinal microvascular abnormalities differ between lacunar and cortical stroke suggesting that a distinct small vessel vasculopathy may cause lacunar stroke. We have also identified MR markers of lacunar stroke. These results suggest that venular disease (a hitherto underresearched area) may play a role in the pathophysiology of lacunar stroke. Retinal microvascular abnormalities can act as markers for cerebral small vessel disease. We plan collaborative analyses with colleagues who have performed similar studies to further assess retinal abnormalities in lacunar stroke.

It has been known for almost two decades that the water proton NMR signal of diffusing water molecules in brain white matter undergoes a non-monoexponential decay with increasing diffusion gradient factor b. With the help of numerical simulations and analytical expressions, much effort has been directed to describing the signal decay and to extracting relevant biophysical features of the system under investigation. However, the physical basis of such nonmonoexponential behavior is still not properly understood. The primary difficulty in characterizing this phenomenon is the variation in behavior in the different directions of diffusion measurement. A combined framework that accounts for the diffusion process in all directions requires several parameters. Addition of many such parameters renders a model to be unwieldy and over-complicated, but over-simplifications can be shown to miss crucially relevant information in the data. In this thesis, I have attempted to handle this problem with simple measurements that span a wide range of parameter space. Compared to often-performed measurements that probe diffusion over a time-scale of 50-100 ms with relatively low diffusion weighting, the measurements here have been done for very short diffusion times of 2 ms and also very long diffusion times up to 2 s. The temperature dependence of the diffusion coefficients has also been extensively probed. To avoid problems related to gross tissue heterogeneity, diffusion-weighted MR imaging in vivo was performed with ultra-high resolution. These simple measurements allowed sequential assessment of many possible arguments that could have led to such non-monoexponential decay curves. Finally, it was concluded that the water in the glial processes was the major contributor to the non-exponential decay, giving rise to a \'slow\' component both along the axonal fibers and transverse to them.

Contact and collision sports (CCS) expose athletes to countless repetitive head impacts (RHI) across a single season, potentially leading to increased risk of long-term difficulties in cognition and the development of neurodegenerative disease. There is mixed literature on whether RHI from CCS result in changes to white matter and long-term neurobehavioral outcomes, therefore this research project seeks to provide supporting evidence by comparing the total volume of fluid-attenuated inversion recovery (FLAIR) white matter lesions in individuals with a history of RHI from CCS to those without a history of RHI from the Boston University Alzheimer’s Disease Research Center (BU ADRC). The RHI participants were matched to a group of non-RHI participants based on age (+/- 5 years). Effects of RHI on white matter hyperintensities (WMHs) are evaluated, while considering hippocampal volume across RHI and non-RHI groups. When controlling for age, sex, education, and total hippocampal volume, those with a history of football were found to have a significantly greater WMH volume (p=.02) compared to those without a history of football play. Compared to the non-RHI group, the RHI group including all athletes (n=42) had a greater WMH volume, although it did not reach a level of significance (p=.91). This investigation provided preliminary evidence for a link between high RHI exposure and WMHs in football players, and a non-significant relationship between RHI and increased WMHs in those with a history of CCS compared to individuals in the non-RHI group. Future research should expand upon this investigation, by examining RHI exposure and WMH consequences in a diverse assortment of sports, follow athletes longitudinally for repeated in vivo MRIs and post-mortem neuropathological confirmation, and include more female athletes.

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.