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.

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.

In established cases of multiple sclerosis (MS), the normal-appearing white matter (NAWM), as defined for magnetic resonance imaging (MRI), is abnormal in the majority of cases. The clinical significance of these NAWM abnormalities is the subject of debate, but there is strong correlation with degree and progression of disability. New lesions form in NAWM before blood-brain barrier breakdown, as evidenced by gadolinium enhancement. The pathological basis of these neuroimaging abnormalities is largely unknown. Definitive pathological studies on the NAWM are few and are often based on small numbers of samples and of cases. Despite a variety of MS NAWM pathological studies, major research questions, of importance to our understanding of basic pathogenetic mechanisms and consequent rational therapies, remain unanswered. These relate to the frequency and extent of oligodendrocyte/myelin and axonal abnormalities in MS NAWM, and to the cellular basis of very early MS lesions detected by neuroimaging. In a pilot study of MS NAWM, microglial activation was demonstrated in 9 of 10 MS cases. We are currently testing the hypothesis that microglial activation, as defined by altered phenotype and HLA-DR positivity, will act as a marker for oligodendrocyte/myelin and axonal pathology in MS NAWM.