Geert J. Schenk

Grey matter damage in multiple sclerosis: a pathology perspective.

Over the past decade, immunohistochemical studies have provided compelling evidence that gray matter (GM) pathology in multiple sclerosis (MS) is extensive. Until recently, this GM pathology was difficult to visualize using standard magnetic resonance imaging (MRI) techniques. However, with newly developed MRI sequences, it has become clear that GM damage is present from the earliest stages of the disease and accrues with disease progression. GM pathology is clinically relevant, as GM lesions and/or GM atrophy were shown to be associated with MS motor deficits and cognitive impairment. Recent autopsy studies demonstrated significant GM demyelination and microglia activation. However, extensive immune cell influx, complement activation and blood-brain barrier leakage, like in WM pathology, are far less prominent in the GM. Hence, so far, the cause of GM damage in MS remains unknown, although several plausible underlying pathogenic mechanisms have been proposed. This paper provides an overview of GM damage in MS with a focus on its topology and histopathology.

What drives MRI-measured cortical atrophy in multiple sclerosis?

Background: Cortical atrophy, assessed with magnetic resonance imaging (MRI), is an important outcome measure in multiple sclerosis (MS) studies. However, the underlying histopathology of cortical volume measures is unknown. Objective: We investigated the histopathological substrate of MRI-measured cortical volume in MS using combined post-mortem imaging and histopathology. Methods: MS brain donors underwent post-mortem whole-brain in-situ MRI imaging. After MRI, tissue blocks were systematically sampled from the superior and inferior frontal gyrus, anterior cingulate gyrus, inferior parietal lobule, and superior temporal gyrus. Histopathological markers included neuronal, axonal, synapse, astrocyte, dendrite, myelin, and oligodendrocyte densities. Matched cortical volumes from the aforementioned anatomical regions were measured on the MRI, and used as outcomes in a nested prediction model. Results: Forty-five tissue blocks were sampled from 11 MS brain donors. Mean age at death was 68±12 years, post-mortem interval 4±1 hours, and disease duration 35±15 years. MRI-measured regional cortical volumes varied depending on anatomical region. Neuronal density, neuronal size, and axonal density were significant predictors of GM volume. Conclusions: In patients with long-standing disease, neuronal and axonal pathology are the predominant pathological substrates of MRI-measured cortical volume in chronic MS.

Neuronal and Axonal Loss in Normal-Appearing Gray Matter and Subpial Lesions in Multiple Sclerosis

Abstract Multiple sclerosis (MS) is a demyelinating and neurodegenerative disease of the CNS. Multiple sclerosis lesions include significant demyelination of the gray matter, which is thought to be a major contributor to both physical and cognitive impairment. Subpial (Type III) lesions are the most common demyelinated cortical lesions. We investigated neurodegenerative features of subpial lesions in cerebral cortex samples from 11 patients with MS and 6 nondemented non-MS controls. There were no significant differences in neuron and axon density between normally myelinated normal-appearing gray matter (NAGM) and Type III MS lesions. Neurons were 11.2% smaller in Type III lesions than in NAGM in the cingulate cortex only; Type III lesions contained 25.4% fewer NeuN-positive neurons compared with control cortex. Neurons in MS NAGM were 13.6% smaller than those in control cortex. Finally, the same regions, immunostained with anti-SMI312 antibodies, showed reduced axon densities in Type III lesions (−31.4%) and NAGM (−33.0%) compared with controls. In conclusion, both NAGM and Type III lesions showed neurodegenerative changes, but they had no consistent differences in neuronal and axonal alterations. This suggests that neurodegeneration in the cerebral cortex of patients with MS may be independent of cortical demyelination.

Roles for HB-EGF and CD9 in multiple sclerosis.

Early events in multiple sclerosis (MS) lesion formation are loss of blood–brain barrier (BBB) integrity, immune cell trafficking into the central nervous system, and demyelination. To date, the molecular mechanisms underlying these pathogenic events are poorly understood. Heparin‐binding epidermal growth factor (HB‐EGF) is a trophic factor that is induced by inflammatory stimuli and has previously been shown to interact with tetraspanins (TSPs), a family of transmembrane proteins that are involved in cellular migration and adhesion. Given the known roles of TSPs and HB‐EGF, we hypothesized that HB‐EGF and TSPs may play a role in the processes that underlie MS lesion formation. We examined the expression of HB‐EGF and the TSPs CD9 and CD81 in MS brain and found that HB‐EGF was highly induced in reactive astrocytes in active lesions. TSPs were constitutively expressed throughout normal appearing white matter and control white matter. In contrast, CD9 was reduced in demyelinated lesions and increased on blood vessels in lesion areas. In vitro studies revealed that expression of HB‐EGF and TSPs is regulated during inflammation. Importantly, blocking either HB‐EGF or CD9 significantly reduced the migration of monocytes across brain endothelial cell monolayers. Moreover, blocking CD9 strongly enhanced the barrier function of the BBB in vitro. Together, we demonstrate that these molecules are likely implicated in processes that are highly relevant for MS lesion formation, and therefore, HB‐EGF and TSPs are promising therapeutic targets. GLIA 2013;61:1890–1905

Altered blood–brain barrier transport in neuro-inflammatory disorders

During neurodegenerative and neuroinflammatory disorders of the central nervous system (CNS), such as Alzheimers disease (AD) and multiple sclerosis (MS), the protective function of the blood-brain barrier (BBB) may be severely impaired. The general neuro-inflammatory response, ranging from activation of glial cells to immune cell infiltration that is frequently associated with such brain diseases may underlie the loss of the integrity and function of the BBB. Consequentially, the delivery and disposition of drugs to the brain will be altered and may influence the treatment efficiency of such diseases. Altered BBB transport of drugs into the CNS during diseases may be the result of changes in both specific transport and non-specific transport pathways. Potential alterations in transport routes like adsorptive mediated endocytosis and receptor-mediated endocytosis may affect drug delivery to the brain. As such, drugs that normally are unable to traverse the BBB may reach their target in the diseased brain due to increased permeability. In contrast, the delivery of (targeted) drugs could be hampered during inflammatory conditions due to disturbed transport mechanisms. Therefore, the inventory of the neuro-inflammatory status of the neurovasculature (or recovery thereof) is of utmost importance in choosing and designing an adequate drug targeting strategy under disease conditions. Within this review we will briefly discuss how the function of the BBB can be affected during disease and how this may influence the delivery of drugs into the diseased CNS.

Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis

Cellular injury and death are ubiquitous features of disease, yet tools to detect them are limited and insensitive to subtle pathological changes. Acridine orange (AO), a nucleic acid dye with unique spectral properties, enables real-time measurement of RNA and DNA as proxies for cell viability during exposure to various noxious stimuli. This tool illuminates spectral signatures unique to various modes of cell death, such as cells undergoing apoptosis versus necrosis/necroptosis. This new approach also shows that cellular RNA decreases during necrotic, necroptotic, and apoptotic cell death caused by demyelinating, ischemic, and traumatic injuries, implying its involvement in a wide spectrum of tissue pathologies. Furthermore, cells with pathologically low levels of cytoplasmic RNA are detected earlier and in higher numbers than with standard markers including TdT-mediated dUTP biotin nick-end labeling and cleaved caspase 3 immunofluorescence. Our technique highlights AO-labeled cytoplasmic RNA as an important early marker of cellular injury and a sensitive indicator of various modes of cell death in a range of experimental models.

Demyelination induces transport of ribosome-containing vesicles from glia to axons: evidence from animal models and MS patient brains

Glial cells were previously proven capable of trafficking polyribosomes to injured axons. However, the occurrence of such transfer in the general pathological context, such as demyelination-related diseases, needs further evidence. Since this may be a yet unidentified universal contributor to axonal survival, we study putative glia–axonal ribosome transport in response to demyelination in animal models and patients in both peripheral and central nervous system. In the PNS we investigate whether demyelination in a rodent model has the potential to induce ribosome transfer. We also probe the glia–axonal ribosome supply by implantation of transgenic Schwann cells engineered to produce fluorescent ribosomes in the same demyelination model. We furthermore examine the presence of axonal ribosomes in mouse experimental autoimmune encephalomyelitis (EAE), a well-established model for multiple sclerosis (MS), and in human MS autopsy brain material. We provide evidence for increased axonal ribosome content in a pharmacologically demyelinated sciatic nerve, and demonstrate that at least part of these ribosomes originate in the transgenic Schwann cells. In the CNS one of the hallmarks of MS is demyelination, which is associated with severe disruption of oligodendrocyte–axon interaction. Here, we provide evidence that axons from spinal cords of EAE mice, and in the MS human brain contain an elevated amount of axonal ribosomes compared to controls. Our data provide evidence that increased axonal ribosome content in pathological axons is at least partly due to glia-to-axon transfer of ribosomes, and that demyelination in the PNS and in the CNS is one of the triggers capable to initiate this process.

Insights into the Mechanisms That May Clarify Obesity as a Risk Factor for Multiple Sclerosis

Purpose of ReviewThe proportion to which genetic and environmental factors contribute to the etiology of multiple sclerosis (MS) is still incompletely understood. An interesting association between MS etiology and obesity has recently been shown although the mechanisms underlying this association are still unknown. We propose deregulated gut microbiota and increased leptin levels as possible mechanisms underlying MS etiology in obese individuals.Recent FindingsAlterations in the human gut microbiota and leptin levels have recently been established as immune modulators in both MS patients and obese individuals. A resemblance between pro-inflammatory bacterial profiles in MS and obese individuals was observed. Furthermore, elevated leptin levels push the immune system towards a more pro-inflammatory state and inhibit the regulatory immune response.SummaryDeregulated gut microbiota and elevated leptin levels may explain the increased risk of developing MS in obese individuals. Further research to confirm causality is warranted.

Quantitative biochemical investigation of various neuropathologies using high-resolution spectral CARS microscopy

The pathology of multiple sclerosis involves the gray and white matter regions of the brain and spinal cord often characterized by various combinations of demyelination, inflammatory infiltration, axonal degeneration, and later gliosis in chronic lesions. While acute and chronic white matter lesions are well characterized and easily identified, evidence indicates that the CNS of MS patients may be globally altered, with subtle abnormalities found in grossly normal appearing white matter (NAWM) with histochemical stains and magnetic resonance imaging only indicating a general alteration in tissue composition at best. Thus, the prototypical acute inflammatory lesion may merely represent the most obvious manifestation of a chronic widespread involvement of the CNS, which is difficult to examine reliably. The current study deals with the microstructure and biochemistry of demyelination, remyelination and axonal loss in various regions in post-mortem human MS brain, especially NAWM areas around more typical acute and chronic lesions. The myelin sheath, neuroglia and perivascular spaces were investigated through changes in the intrinsic molecular vibrational signatures of lipid biochemistry using a novel, label-free Coherent anti-Stokes Raman Scattering (CARS) microscope. The biochemistry of myelin lipids can be probed by detecting subtle changes to phospholipids and the intra-molecular disorder of their fatty acid acyl chains, various oxidation products and general protein contributions. NAWM regions surrounding pathological MS lesions were shown to reveal abnormalities despite morphological classifications indicating otherwise. CARS data were correlated with immunohistochemical stains and lipophilic dyes. Spectral data were analyzed using a unique non-linear algorithm, which allows quantification and classification through gated parameters and displayed through bivariate histograms. Our CARS microscopy system provides high-resolution, detailed morphological and unique biochemical information regarding CNS pathology in human MS examples and may be applicable to a broad range of other white matter centric neurological disorders.

Disease Influence on BBB Transport in Inflammatory Disorders

During diseases of the central nervous system (CNS), such as Alzheimer’s, Parkinson’s, epilepsy, stroke and multiple sclerosis (MS), the protective function of the blood–brain barrier (BBB) is significantly impaired. The inflammatory response that is frequently associated with such brain diseases may underlie the loss of the integrity and function of the BBB. Consequentially, the delivery and disposition of drugs to the brain will be altered and may influence the treatment efficiency of CNS diseases. Altered BBB transport of drugs into the CNS during diseases may be the result of changes in both specific transport and non-specific transport pathways. Potential alterations in transport routes like adsorptive-mediated endocytosis and receptor-mediated endocytosis may affect drug delivery to the brain. As such, drugs that normally are unable to traverse the BBB may reach their target in the diseased brain due to increased permeability. On the contrary, the delivery of (targeted) drugs could be hampered during inflammatory conditions due to disturbed transport mechanisms. Therefore, the inventory of the neuro-inflammatory status of the neurovasculature (or recovery thereof) is of utmost importance in choosing and designing an adequate drug targeting strategy under disease conditions. Within this chapter we discuss how the function of the BBB can be affected during disease and how this may influence the delivery of drugs into the diseased CNS.