Samar Betmouni

EVIDENCE FOR AN EARLY INFLAMMATORY RESPONSE IN THE CENTRAL NERVOUS SYSTEM OF MICE WITH SCRAPIE

In Alzheimers disease, the most prevalent of the neurodegenerative diseases, inflammation of the CNS contributes to the pathology and is a target for therapy. In contrast, the group of neurodegenerative conditions known as the Prion Diseases have been widely reported as lacking any inflammatory elements despite the many similarities between the pathologies of Alzheimers Disease and Prion Diseases We have found evidence for an inflammatory component in mouse scrapie, characterized by microglial activation and T-lymphocyte recruitment, which appears long before any clinical signs of the disease and spreads along well-defined anatomical pathways. These observations emphasize the potential value of murine scrapie as a model for studying the inflammatory pathology of other neurodegenerative diseases.

Elevated activity and microglial expression of myeloperoxidase in demyelinated cerebral cortex in multiple sclerosis.

Recent studies have revealed extensive cortical demyelination in patients with progressive multiple sclerosis (MS). Demyelination in gray matter lesions is associated with activation of microglia. Macrophages and microglia are known to express myeloperoxidase (MPO) and generate reactive oxygen species during myelin phagocytosis in the white matter. In the present study we examined the extent of microglial activation in the cerebral cortex and the relationship of microglial activation and MPO activity to cortical demyelination. Twenty‐one cases of neuropathologically confirmed multiple sclerosis, with 34 cortical lesions, were used to assess microglial activation. HLA‐DR immunolabeling of activated microglia was significantly higher in demyelinated MS cortex than control cortex and, within the MS cohort, was significantly greater within cortical lesions than in matched non‐demyelinated areas of cortex. In homogenates of MS cortex, cortical demyelination was associated with significantly elevated MPO activity. Immunohistochemistry revealed MPO in CD68‐positive microglia within cortical plaques, particularly toward the edge of the plaques, but not in microglia in adjacent non‐demyelinated cortex. Cortical demyelination in MS is associated with increased activity of MPO, which is expressed by a CD68‐positive subset of activated microglia, suggesting that microglial production of reactive oxygen species is likely to be involved in cortical demyelination.

Elevated myeloperoxidase activity in white matter in multiple sclerosis

Recent studies have revealed extensive axonal damage in patients with progressive multiple sclerosis (MS). Axonal damage can be caused by a plethora of factors including the release of proteolytic enzymes and cytotoxic oxidants by activated immune cells and glia within the lesion. Macrophages and microglia are known to express myeloperoxidase (MPO) and generate reactive oxygen species during myelin phagocytosis in the white matter. In the present study we have measured MPO levels in post-mortem homogenates of demyelinated and non-demyelinated regions of white matter from nine patients with MS and seven controls, and assessed MPO immunoreactivity within MS brain. In homogenates of MS white matter, demyelination was associated with significantly elevated MPO activity when compared to controls. Immunohistochemistry showed MPO to be expressed mainly by macrophages within and adjacent to plaques. Demyelination in MS is associated with increased activity of MPO, suggesting that this production of reactive oxygen species may contribute to axonal injury within plaques.

An integrated, temporal study of the behavioural, electrophysiological and neuropathological consequences of murine prion disease.

We have conducted an integrated study of ME7 prion disease by examining the electrophysiological and neuropathological features of hippocampal slices from behaviourally characterised C57Bl/6J mice 12, 14, 16, 18, 20 and 24 weeks after intracerebral micro-injection of ME7 or normal brain homogenate. We describe the pathogenesis of ME7 as a three-stage process. STAGE ONE: PrPSc deposition, synaptic pathology and abnormal synaptic plasticity. STAGE TWO: Onset of behavioural changes, exemplified by an increase in open-field activity, enhancement of the slow AHP and development of vacuolation. Membrane depolarisation is also an early feature, but its exact timing remains to be confirmed. STAGE THREE: Clinical disease, substantial neurodegeneration and further disruption of the action potential profile. We suggest that the mechanisms underlying the electrophysiological changes of Stages one and two may provide novel approaches to treatment of prion disease, and that those seen in Stage three may be relevant to neurodegenerative diseases more generally.

Behavioral consequences of prion disease targeted to the hippocampus in a mouse model of scrapie

Sheep scrapie is an archetypal member of a group of chronic neurodegenerative diseases that can afflict both humans and animals and that are known collectively as the transmissible spongiform encephalopathies or prion diseases. The study of these diseases has been facilitated by the experimental transmission of sheep scrapie to laboratory rodents, and this has led to an ever-increasing literature on the pathogenesis and molecular biology of this unusual group of diseases. There is relatively little known about the relationship between the chronic neurodegenerative process and the development of clinical signs, the latter occurring very late in the course of the disease after a prolonged incubation period. We have previously shown that following intrahippocampal injection of scrapie brain homogenate, there is an early inflammatory response in the brains of affected mice. This develops long before there is neuronal loss and also before the mice develop any overt clinical signs of disease. It was surprising that despite the marked and evolving inflammatory response in the brains of scrapie-affected mice, indicative of underlying pathology, there were no outward signs that the mice had any form of central nervous system pathology. The present study shows that by using a number of behavioral tasks, including multitrial passive avoidance, open-field motor activity, and tests of muscle strength, it is possible to detect more subtle clinical signs much earlier during the course of disease in scrapie-affected mice. The relevance of this finding to the investigation of mouse scrapie is discussed.

Inflammatory response and retinal ganglion cell degeneration following intraocular injection of ME7

Scrapie is a prion disease which occurs naturally in sheep and which can be transmitted experimentally to rodents. After intracerebral injection of ME7 into mouse, an atypical inflammatory response, characterized by T‐lymphocytes and activated microglia is present early in the course of the disease. In the present work, we have investigated the relationship between this inflammatory response, astrocytosis and neuronal loss along the visual pathway after intraocular injection (intraocular) of ME7 in C57BL/6J mice. We have demonstrated that microglia activation and T‐lymphocyte recruitment accompanies the spread of prion pathology along the visual pathway and in the early stages of the disease is restricted to the subcortical visual pathway. Inflammation was also present in non‐visual areas in association with PrPsc deposition at late stages of the disease, possibily indicating that diffusion of the scrapie agent also contributes to the spread of the disease. After intraocular injection of the prion agent, the disease is believed to be transported into the brain via axons of retinal ganglion cells (RGCs). Despite the high levels of infectivity reported to be present in the retina early in the disease after intraocular injection of ME7, retinal pathology has not been extensively investigated. We have studied the RGCs response in whole mount retinas after intraocular injection of ME7. We have shown that RGCs degenerate after intraocular injection of ME7 whereas amacrine cells, retinal interneurones, are more resistant. Our results suggest that two distinct population of neurones, exposed in vivo at the same time to the same agent scrapie strain, show different susceptibility to the toxic effects of PrPsc .

Elevated Matrix Metalloproteinase-9 and Degradation of Perineuronal Nets in Cerebrocortical Multiple Sclerosis Plaques

Matrix metalloproteinases (MMPs) degrade extracellular matrix; MMP activity, particularly of MMP-9, is elevated in the white matter in multiple sclerosis (MS) patients. The cerebral cortical extracellular matrix includes perineuronal nets (PNs) that surround parvalbumin-positive neurons (PV-positive neurons) and are important for their function. We measured active and total MMP-9 levels in postmortem homogenates of demyelinated and nondemyelinated cerebral cortical regions from 9MS and 7 control cases and assessed Wisteria floribunda agglutin (WFA)-positive PNs in paraffin sections from 15 MS and 6 controls and PV-positive neurons in sections from 26 MS and 6 controls. Active MMP-9 levels were higher in demyelinated than in nondemyelinated or control cortex (p < 0.05). The area fraction positive for WFA was lower in demyelinated than nondemyelinated MS or control cortex; the latter difference was significant (p < 0.05). Most PV-positive neurons in demyelinated but not intact cortex lackeda PN, and some showed perikaryal phosphorylated neurofilament protein accumulation. Loss of WFA-labeled PNs was not associated with reduced PV-positive neurons numbers. Thus, elevated MMP-9 in cortical plaques is associated with loss of PNs; PV-positive neurons are preserved but show abnormal neurofilament accumulations. Matrix metalloproteinase-mediated degradation of PNs in cortical plaques may, therefore, contribute to neuronal dysfunction and degeneration in MS patients.

Loss of Perineuronal Net in ME7 Prion Disease

Microglial activation and behavioral abnormalities occur before neuronal loss in experimental murine prion disease; the behavioral changes coincide with a reduction in synaptic plasticity. Because synaptic plasticity depends on an intact perineuronal net (PN), a specialized extracellular matrix that surrounds parvalbumin (PV)-positive GABAergic (&ggr;-aminobutyric acid [GABA]) inhibitory interneurons, we investigated the temporal relationships between microglial activation and loss of PN and PV-positive neurons in ME7 murine prion disease. Anesthetized C57Bl/6J mice received bilateral intracerebral microinjections of ME7-infected or normal brain homogenate into the dorsal hippocampus. Microglial activation, PrPSc accumulation, the number of PV-positive interneurons, and Wisteria floribunda agglutinin-positive neurons (i.e. those with an intact PN) were assessed in the ventral CA1 and subiculum at 4, 8, 12, 16, and 20 weeks postinjection. Hippocampal areas and total neuron numbers in the ventral CA1 and subiculum were also determined. Loss of PN coincided with early microglial activation and with a reduction in synaptic plasticity. No significant loss of PV-positive interneurons was observed. Our findings suggest that the substrate of the earliest synaptic and behavioral abnormalities in murine prion disease may be inflammatory microglia-mediated degradation of the PN.

The Bath Advanced Neurology Course 2003: Progressive Neurological Decline in Pregnancy

THE CASE The story A young right-handed woman 30 weeks into her third pregnancy presented to the accident and emergency department with a 14-day history of headache; 7 days of progressive left-sided visual blurring, speech difficulties and vomiting; and 2 days of memory impairment, agitation and confusion. The early pregnancy had been uneventful, but at 22 weeks gestation she had been admitted with low back pain and urinary incontinence; MRI of the brain and spine were normal. There was a previous history of asthma, appendicectomy, and a miscarriage 3 years before, with a strong maternal family history of recurrent deep venous thromboses. Her only child had recently contracted chickenpox. Examination On initial examination she was poorly co-operative, confused, distressed and agitated. Registration and recall were reduced and she had nominal dysphasia. She was afebrile without meningism. Visual acuity was 6/6 on the right, but reduced to finger counting on the