John Kirk

Tight junctional abnormality in multiple sclerosis white matter affects all calibres of vessel and is associated with blood-brain barrier leakage and active demyelination.

Blood–brain barrier (BBB) hyperpermeability in multiple sclerosis (MS) is associated with lesion pathogenesis and has been linked to pathology in microvascular tight junctions (TJs). This study quantifies the uneven distribution of TJ pathology and its association with BBB leakage. Frozen sections from plaque and normal‐appearing white matter (NAWM) in 14 cases were studied together with white matter from six neurological and five normal controls. Using single and double immunofluorescence and confocal microscopy, the TJ‐associated protein zonula occludens‐1 (ZO‐1) was examined across lesion types and tissue categories, and in relation to fibrinogen leakage. Confocal image data sets were analysed for 2198 MS and 1062 control vessels. Significant differences in the incidence of TJ abnormalities were detected between the different lesion types in MS and between MS and control white matter. These were frequent in oil‐red O (ORO)+ active plaques, affecting 42% of vessel segments, but less frequent in ORO− inactive plaques (23%), NAWM (13%), and normal (3.7%) and neurological controls (8%). A similar pattern was found irrespective of the vessel size, supporting a causal role for diffusible inflammatory mediators. In both NAWM and inactive lesions, dual labelling showed that vessels with the most TJ abnormality also showed most fibrinogen leakage. This was even more pronounced in active lesions, where 41% of vessels in the highest grade for TJ alteration showed severe leakage. It is concluded that disruption of TJs in MS, affecting both paracellular and transcellular paths, contributes to BBB leakage. TJ abnormality and BBB leakage in inactive lesions suggests either failure of TJ repair or a continuing pathological process. In NAWM, it suggests either pre‐lesional change or secondary damage. Clinically inapparent TJ pathology has prognostic implications and should be considered when planning disease‐modifying therapy. Copyright

Abnormal Endothelial Tight Junctions in Active Lesions and Normal-appearing White Matter in Multiple Sclerosis

Blood‐brain barrier (BBB) breakdown, demonstrable in vivo by enhanced MRI is characteristic of new and expanding inflammatory lesions in relapsing‐remitting and chronic progressive multiple sclerosis (MS). Subtle leakage may also occur in primary progressive MS. However, the anatomical route(s) of BBB leakage have not been demonstrated. We investigated the possible involvement of interendothelial tight junctions (TJ) by examining the expression of TJ proteins (occludin and ZO‐1) in blood vessels in active MS lesions from 8 cases of MS and in normal‐appearing white (NAWM) matter from 6 cases. Blood vessels (10–50 per frozen section) were scanned using confocal laser scanning microscopy to acquire datasets for analysis. TJ abnormalities manifested as beading, interruption, absence or diffuse cytoplasmic localization of fluorescence, or separation of junctions (putative opening) were frequent (affecting 40% of vessels) in oil‐red‐O‐positive active plaques but less frequent in NAWM (15%), and in normal (<2%) and neurological controls (6%). Putatively “open” junctions were seen in vessels in active lesions and in microscopically inflamed vessels in NAWM. Dual fluorescence revealed abnormal TJs in vessels with pre‐mortem serum protein leakage. Abnormal or open TJs, associated with inflammation may contribute to BBB leakage in enhancing MRI lesions and may also be involved in subtle leakage in non‐enhancing focal and diffuse lesions in NAWM. BBB disruption due to tight junctional pathology should be regarded as a significant form of tissue injury in MS, alongside demyelination and axonopathy.

Persistent endothelial abnormalities and blood?brain barrier leak in primary and secondary progressive multiple sclerosis

Epithelial and endothelial tight junctions are pathologically altered in infectious, inflammatory, neoplastic and other diseases. Previously, we described such abnormalities, associated with serum protein leak, in tight junctions of the blood–brain barrier endothelium, in lesional and normal‐appearing white matter (NAWM) in secondary progressive (SP) and acute multiple sclerosis (MS). This work is extended here to lesions and NAWM in primary progressive multiple sclerosis (PPMS) and to cortical grey matter in PPMS and SPMS. Immunocytochemistry and semiquantitative confocal microscopy for the tight junction protein zonula occludens 1 (ZO‐1) was performed on snap‐frozen sections from PPMS (n = 6) and controls (n = 5). Data on 2103 blood vessels were acquired from active lesions (n = 10), inactive lesions (n = 15), NAWM (n = 42) and controls (n = 20). Data on 1218 vessels were acquired from normal‐appearing grey matter (PPMS, 5; SPMS, 6; controls, 5). In PPMS abnormal ZO‐1 expression in active white matter lesions and NAWM, was found in 42% and 13% of blood vessels, respectively, comparable to previous data from acute and SPMS. In chronic white matter plaques, however, abnormalities were considerably more frequent (37%) in PPMS than in SPMS. Abnormality was also more frequent in normal‐appearing grey matter in SPMS (23%) than in PPMS (10%). In summary, abnormal tight junctions in both SPMS and PPMS are most frequent in active white matter lesions but persist in inactive lesions, particularly in PPMS. Abnormal tight junctions are also common in normal‐appearing grey matter in SPMS. Persistent endothelial abnormality with leak (PEAL) is therefore widespread but variably expressed in MS and may contribute to disease progression.

Absence of aquaporin-4 expression in lesions of neuromyelitis optica but increased expression in multiple sclerosis lesions and normal-appearing white matter

Aquaporin-4 (AQP4) has recently been implicated in the pathogenesis of neuromyelitis optica (NMO) where it has been identified as the first defined autoantigen pertinent to an inflammatory demyelinating disorder of the human CNS. Furthermore, a recent case report has shown a lack of AQP4 expression in the spinal cord lesions of NMO. However, the pattern of AQP4 expression in multiple sclerosis (MS) tissues has not been well-defined. In the present investigation we have confirmed a lack of expression of AQP4 in optic and spinal cord lesions in NMO which contrasted sharply with the increased levels of AQP4 expression seen in MS lesions. Furthermore a detailed immunohistochemical and semi-quantitative analysis is used to describe the expression pattern of AQP4 on well-characterized tissue microarray samples of MS and control white matter. Anatomically AQP4 was more highly expressed in all categories of MS tissue compared to normal control tissues with the most abundant expression in active lesions. Within active lesions AQP4 expression was significantly correlated with expression of the pro-inflammatory cytokine osteopontin. At the cellular level dual-labeling immunofluoresence demonstrated that increased expression of AQP4 was most pronounced at the astrocytic endfeet but was also associated with the cell bodies of astrocytes in the tissue parenchyma. The finding of increased AQP4 expression in MS lesions in contrast to the lack of expression in NMO lesions may suggest different mechanisms of initiation and progression between the two disease states.

Up-regulation of osteopontin and alphaBeta-crystallin in the normal-appearing white matter of multiple sclerosis: an immunohistochemical study utilizing tissue microarrays.

Tissue microarrays assembled from control and multiple sclerosis (MS) brain tissue have been used to assess the expression patterns and cellular distribution of two antigens, the proinflammatory cytokine osteopontin and the inducible heat shock protein αΒ‐crystallin, which have previously been implicated in MS pathogenesis. Tissue cores were taken from paraffin‐embedded donor blocks containing chronic active or chronic inactive plaques and normal‐appearing white matter (NAWM) in seven MS cases, and white matter (WM) in five control cases. Expression patterns of both proteins were assessed against myelin density and microglial activation in the different tissue categories. Both proteins showed increased expression in all categories of MS tissue compared with control WM. The results indicate progressive up‐regulation of expression of osteopontin with increased plaque activity, while elevation of αΒ‐crystallin expression in MS tissue was independent of demyelination. In MS NAWM a significant correlation was observed between high levels of expression of osteopontin and αΒ‐crystallin. Osteopontin expression was predominantly confined to astrocytes throughout MS tissues. αΒ‐crystallin was expressed on astrocytes, oligodendrocytes and occasionally on demyelinated axons. Taken together, these data indicate a wider distribution of osteopontin and αΒ‐crystallin in MS tissues than previously described and support their proposed role in MS pathogenesis.

Cerebral endothelial cell infection by measles virus in subacute sclerosing panencephalitis : ultrastructural and in situ hybridization evidence

Infection of vascular endothelium plays a central role in the pathogenesis of acute measles virus infection outside the central nervous system (CNS) but has not been described in the human CNS. An ultrastructural survey was made of blood vessels in five cases of subacute sclerosing panencephalitis (SSPE) to determine whether or not infection of cerebral vascular endothelium occurred in this persistent fatal CNS disease caused by measles virus. Morbillivirus nucleo‐capsids were found in a few endothelial cells in three necropsy cases but not in the limited tissue available from two biopsies. In a severe parenchymal lesion in one necropsied case, endothelial cells hybridized in situ with a biotinylated probe specific for the N genomic RNA of measles virus. It is concluded that human cerebral endothelium is susceptible to measles virus infection.

Association of measles virus with neurofibrillary tangles in subacute sclerosing panencephalitis: a combined in situ hybridization and immunocytochemical investigation

Neurofibrillary tangle formation, a cardinal characteristic of Alzheimers disease, is also a feature of several other neurodegenerative disorders, including subacute sclerosing panencephalitis (SSPE). In the present study the association of measles virus genome with neurofibrillary tangle formation has been studied in five cases of SSPE, using in situ hybridization (measles genome) and immunocytochemistry (tau, ubiquitin and p7A4 amyloid). In two cases with duration of disease less than one year, neurofibrillary tangle formation was not observed. However, in those cases in which the disease was of several years duration, numerous tau– and ubiquitin positive neurofibrillary tangles were demonstrated. In the two cases of longest duration, double–labelling techniques demonstrated the frequent association of neurofibrillary tangle formation with neuronal measles virus genome positivity. Immunocytochemistry for p7A4 amyloid failed to demonstrate amyloid in any of the five cases. These findings support the hypothesis that neurofibrillary tangle formation can occur independently of amyloid formation and that this mechanism may operate in both Alzheimers disease and in virallyinduced disease.

Measles virus infection of cells in perivascular infiltrates in the brain in subacute sclerosing panencephalitis: confirmation by non-radioactive in situ hybridization, immunocytochemistry and electron microscopy

SummaryAs part of continuing multidisciplinary studies on the neuropathogenesis of subacute sclerosing panencephalitis (SSPE), in situ hybridisation, immunocytochemistry and electron microscopy were used to detect measles virus nucleic acid, protein and nucleocapsids in brain perivascular infiltrates of three cases. Perivascular cuffing cells which contained measles virus nucleic acid and antigens were found in all cases. Infected cuffs occurred predominantly in areas of general parenchymal cell infection and in many of these a high proportion of the infiltrating cells were infected. Other cuffs in these areas were either uninfected or contained only a few infected cells. Occasional infected cells were also seen in cuffs in non-infected areas. In contrast, no specific immunocytochemical reactions or in situ hybridisation for measles virus was observed in brain tissue from a patient with herpes encephalitis. By electron microscopy viral nucleocapsid, consistent with measles virus, was found within the cytoplasm of plasma cells in the inflammatory cuffs in SSPE brain tissue. Possible explanations for our results are that infiltrates become infected on arrival in the CNS or alternatively, that the infected infiltrates reflect a generalised infection of the reticuloendothelial system. The frequent presence of uninfected cuffs favours the former explanation.

An ultrastructural study of experimental high velocity penetrating head injury

SummaryExperimental high velocity missile brain injury in the rhesus monkey produces widespread swelling of perivascular astrocytes within 30 min of injury. Possible mechanisms for this lesion include a direct effect of force, chemical mediation secondary to the extravasation of blood, alterations in the permeability of the blood brain barrier and ischaemia. The implications of this finding for the function of the blood brain barrier, for neurotransmission and for neuronal survival are discussed.

THE FINE STRUCTURE OF THE CNS IN MULTIPLE SCLEROSIS. I. INTERPRETATION OF CYTOPLASMIC PAPOVAVIRUS-LIKE AND PARAMYXOVIRUS-LIKE INCLUSIONS

During an electron microscopic study of the white matter in multiple sclerosis (MS), spheroidal reticular particles were found both in MS and in control brains. These particles have previously been described in the brain in MS and in brain‐derived cell cultures in subacute sclerosing panencephalitis. In both cases they were interpreted as papovaviruses, but in size, morphology and distribution they are identical to the reticulosomes and related particles which occur as proteinaceous artefacts in a variety of tissues and in subcellular fractions. Inclusions in endothelial cell cytoplasm, previously reported from the CNS in MS as paramyxovirus similar to measles, have also been found in the present study. They were present both in MS and in control brains and are identified as ‘rod‐shaped tubular bodies’, normally occurring organelles of endothelial cells. The necessity for a cautious interpretation of virus‐like inclusions is emphasized.