Translating from Macro-scale to Micro-scale in Multiple Sclerosis: Histological underpinnings of macroscopic structural and functional alterations in MS

Concluding remarks 1. What are the cellular substrates of DTI measured diffusivity alterations in normal appearing and lesioned cortex in MS? - Chapter 2.1 showed that axonal orientation may be an important driver of diffusivity measures, such as FA, not only in white matter but also in grey matter in MS and in control cortex. - Chapter 2.1 showed that the typical diffusivity pattern of MRI measured low FA in NAGM compared to controls and increased FA in grey matter lesions compared to NAGM in MS is associated with cortical axonal degeneration. 2. What are the cellular substrates of DTI based network measures of integration and segregation in MS? - Chapter 2.2 showed that cortical regions with segregative properties are characterized by small neurons and a low axonal density, while regions with integrative properties contain large neurons and high axonal densities. - Chapter 2.2 showed that MS patients with a higher whole brain WM lesion volume showed higher more pronounced segregative properties and less integrative properties and correspondingly also smaller neurons and a lower axonal densities on average. 3. To what extent do remote white matter and local grey matter pathology explain cortical neuroaxonal degeneration in MS? - Chapter 3 showed that both white matter damage and cortical demyelination contribute to cortical axonal loss. Variance in cortical axonal density in NAGM is explained for 22.6% by remote axonal diffusivity in WML and for 12.6% by axonal diffusivity in NAWM. Furthermore, local cortical demyelination was associated with additional axonal loss in lesioned cortex and explained 34.4% of variance in axonal loss, while microglia showed no association. 4. What are possible histological substrates of altered brain function in MS? - Chapter 4 showed that GAD67 immuno-reactivity is increased in WM astrocytes, inhibitory interneurons and synapses in inflamed and demyelinated hippocampi of MS patients. This is possibly related to cognitive functioning according to the medical history of the donors. - Preliminary data showed that inhibitory synaptic and excitatory synaptic densities are reduced in layer 6 of the cortex. This may reflect ongoing processes such as synaptic displacement, synaptic stripping and neurodegeneration

Enhanced GABAergic Immunoreactivity in Hippocampal Neurons and Astroglia of Multiple Sclerosis Patients

Cognitive dysfunction occurs frequently in multiple sclerosis (MS). Research suggests that hippocampal lesions and GABAergic neurotransmitter changes contribute to cognitive dysfunction. In the present study, we aim to determine the cellular changes in GABAergic expression in MS hippocampus related to inflammation and demyelination. To this end, the presence and inflammatory activity of demyelinating lesions was determined by immunohistochemistry in human postmortem hippocampal tissue of 15 MS patients and 9 control subjects. Subsequently, GABAergic cells were visualized using parvalbumin (PV) and glutamate acid decarboxylase 67 (GAD67) markers. Fluorescent colabeling was performed of GAD67 with neuronal nuclei, PV, astrocytic glial fibrillary acidic protein, or vesicular GABA transporter. We observed increased GAD67-positive (GAD67+) neuron and synapse numbers in the CA1 of MS patients with active hippocampal lesions, not due to neurogenesis. The number and size of PV-positive neurons remained unchanged. GAD67+ astrocytes were more numerous in hippocampal white matter than grey matter lesions. Additionally, in MS patients with active hippocampal lesions GAD67+ astrocyte surface area was increased. Disturbed cognition was most prevalent in MS patients with active hippocampal lesions. Summarizing, increased GAD67 immunoreactivity occurs in neurons and astrocytes and relates to hippocampal inflammation and possibly disturbed cognition in MS

Axonal degeneration as substrate of fractional anisotropy abnormalities in multiple sclerosis cortex

Cortical microstructural abnormalities are associated with clinical and cognitive deterioration in multiple sclerosis. Using diffusion tensor MRI, a higher fractional anisotropy has been found in cortical lesions versus normal-appearing cortex in multiple sclerosis. The pathological substrates of this finding have yet to be definitively elucidated. By performing a combined post-mortem diffusion tensor MRI and histopathology study, we aimed to define the histopathological substrates of diffusivity abnormalities in multiple sclerosis cortex. Sixteen subjects with multiple sclerosis and 10 age- and sex-matched non-neurological control donors underwent post-mortem in situ at 3T MRI, followed by brain dissection. One hundred and ten paraffin-embedded tissue blocks (54 from multiple sclerosis patients, 56 from non-neurological controls) were matched to the diffusion tensor sequence to obtain regional diffusivity measures. Using immunohistochemistry and silver staining, cortical density of myelin, microglia, astrocytes and axons, and density and volume of neurons and glial cells were evaluated. Correlates of diffusivity abnormalities with histological markers were assessed through linear mixed-effects models. Cortical lesions (77% subpial) were found in 27/54 (50%) multiple sclerosis cortical regions. Multiple sclerosis normal-appearing cortex had a significantly lower fractional anisotropy compared to cortex from non-neurological controls (P = 0.047), whereas fractional anisotropy in demyelinated cortex was significantly higher than in multiple sclerosis normal-appearing cortex (P = 0.012) but not different from non-neurological control cortex (P = 0.420). Compared to non-neurological control cortex, both multiple sclerosis normal-appearing and demyelinated cortices showed a lower density of axons perpendicular to the cortical surface (P= 0.012 for both) and of total axons (parallel and perpendicular to cortical surface) (P= 0.028 and 0.012). In multiple sclerosis, demyelinated cortex had a lower density of myelin (P= 0.004), parallel (P= 0.018) and total axons (P= 0.029) versus normal-appearing cortex. Regarding the pathological substrate, in non-neurological controls, cortical fractional anisotropy was positively associated with density of perpendicular, parallel, and total axons (P = 0.031 for all). In multiple sclerosis, normal-appearing cortex fractional anisotropy was positively associated with perpendicular and total axon density (P= 0.031 for both), while associations with myelin, glial and total cells and parallel axons did not survive multiple comparison correction. Demyelinated cortex fractional anisotropy was positively associated with density of neurons, and total cells and negatively with microglia density, without surviving multiple comparison correction. Our results suggest that a reduction of perpendicular axons in normal-appearing cortex and of both perpendicular and parallel axons in demyelinated cortex may underlie the substrate influencing cortical microstructural coherence and being responsible for the different patterns of fractional anisotropy changes occurring in multiple sclerosis cortex

Inhibitory synaptic loss drives network changes in multiple sclerosis: An ex vivo to in silico translational study

Background: Synaptic and neuronal loss contribute to network dysfunction and disability in multiple sclerosis (MS). However, it is unknown whether excitatory or inhibitory synapses and neurons are more vulnerable and how their losses impact network functioning. Objective: To quantify excitatory and inhibitory synapses and neurons and to investigate how synaptic loss affects network functioning through computational modeling. Methods: Using immunofluorescent staining and confocal microscopy, densities of glutamatergic and GABAergic synapses and neurons were compared between post-mortem MS and non-neurological control cases. Then, a corticothalamic biophysical model was employed to study how MS-induced excitatory and inhibitory synaptic loss affect network functioning. Results: In layer VI of normal-appearing MS cortex, excitatory and inhibitory synaptic densities were significantly lower than controls (reductions up to 14.9%), but demyelinated cortex showed larger losses of inhibitory synapses (29%). In our computational model, reducing inhibitory synapses impacted the network most, leading to a disinhibitory increase in neuronal activity and connectivity. Conclusion: In MS, excitatory and inhibitory synaptic losses were observed, predominantly for inhibitory synapses in demyelinated cortex. Inhibitory synaptic loss affected network functioning most, leading to increased neuronal activity and connectivity. As network disinhibition relates to cognitive impairment, inhibitory synaptic loss seems particularly relevant in MS

Cortical axonal loss is associated with both gray matter demyelination and white matter tract pathology in progressive multiple sclerosis: Evidence from a combined MRI-histopathology study

Background: Neuroaxonal degeneration is one of the hallmarks of clinical deterioration in progressive multiple sclerosis (PMS). Objective: To elucidate the association between neuroaxonal degeneration and both local cortical and connected white matter (WM) tract pathology in PMS. Methods: Post-mortem in situ 3T magnetic resonance imaging (MRI) and cortical tissue blocks were collected from 16 PMS donors and 10 controls. Cortical neuroaxonal, myelin, and microglia densities were quantified histopathologically. From diffusion tensor MRI, fractional anisotropy, axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) were quantified in normal-appearing white matter (NAWM) and white matter lesions (WML) of WM tracts connected to dissected cortical regions. Between-group differences and within-group associations were investigated through linear mixed models. Results: The PMS donors displayed significant axonal loss in both demyelinated and normal-appearing (NA) cortices (p < 0.001 and p = 0.02) compared with controls. In PMS, cortical axonal density was associated with WML MD and AD (p = 0.003; p = 0.02, respectively), and NAWM MD and AD (p = 0.04; p = 0.049, respectively). NAWM AD and WML AD explained 12.6% and 22.6%, respectively, of axonal density variance in NA cortex. Additional axonal loss in demyelinated cortex was associated with cortical demyelination severity (p = 0.002), explaining 34.4% of axonal loss variance. Conclusion: Reduced integrity of connected WM tracts and cortical demyelination both contribute to cortical axonal loss in PMS

Meningeal inflammation in multiple sclerosis induces phenotypic changes in cortical microglia that differentially associate with neurodegeneration

Meningeal inflammation strongly associates with demyelination and neuronal loss in the underlying cortex of progressive MS patients, thereby contributing significantly to clinical disability. However, the pathological mechanisms of meningeal inflammation-induced cortical pathology are still largely elusive. By extensive analysis of cortical microglia in post-mortem progressive MS tissue, we identified cortical areas with two MS-specific microglial populations, termed MS1 and MS2 cortex. The microglial population in MS1 cortex was characterized by a higher density and increased expression of the activation markers HLA class II and CD68, whereas microglia in MS2 cortex showed increased morphological complexity and loss of P2Y12 and TMEM119 expression. Interestingly, both populations associated with inflammation of the overlying meninges and were time-dependently replicated in an in vivo rat model for progressive MS-like chronic meningeal inflammation. In this recently developed animal model, cortical microglia at 1-month post-induction of experimental meningeal inflammation resembled microglia in MS1 cortex, and microglia at 2 months post-induction acquired a MS2-like phenotype. Furthermore, we observed that MS1 microglia in both MS cortex and the animal model were found closely apposing neuronal cell bodies and to mediate pre-synaptic displacement and phagocytosis, which coincided with a relative sparing of neurons. In contrast, microglia in MS2 cortex were not involved in these synaptic alterations, but instead associated with substantial neuronal loss. Taken together, our results show that in response to meningeal inflammation, microglia acquire two distinct phenotypes that differentially associate with neurodegeneration in the progressive MS cortex. Furthermore, our in vivo data suggests that microglia initially protect neurons from meningeal inflammation-induced cell death by removing pre-synapses from the neuronal soma, but eventually lose these protective properties contributing to neuronal loss