The family of Toll-like receptors (TLRs) plays a key role in controlling innate immune responses to a wide variety of pathogen-associated molecules. In this study we investigated expression of TLRs in vitro by purified human microglia, astrocytes, and oligodendrocytes, and in vivo by immunohistochemical examination of brain and spinal cord sections. Cultured primary microglia were found to express mRNA encoding a wide range of different TLR family members while astrocytes and oligodendrocytes primarily express TLR2 and TLR3. Comparisons between microglia derived from a series of control subjects and neurodegenerative cases indicate distinct differences in levels of mRNA encoding the different TLRs indifferent microglia samples. Interestingly, expression of TLR proteins in cultured microglia as revealed by immunocytochemistry was restricted to intracellular vesicles, whereas in astrocytes they were exclusively localized on the cell surface. Finally, in vivo expression of TLR3 and TLR4 was examined by immunohistochemical analysis of brain and spinal cord sections from both control and multiple sclerosis brains, revealing enhanced expression of either TLR in inflamed CNS tissues. Together, our data reveal broad and regulated expression of TLRs both in vitro and in vivo by human glia cells.

The primary progressive form of multiple sclerosis is characterized by accrual of neurological dysfunction from disease onset without remission and it is still a matter of debate whether this disease course results from different pathogenetic mechanisms compared with secondary progressive multiple sclerosis. Inflammation in the leptomeninges has been identified as a key feature of secondary progressive multiple sclerosis and may contribute to the extensive cortical pathology that accompanies progressive disease. Our aim was to investigate the extent of perivascular and meningeal inflammation in primary progressive multiple sclerosis in order to understand their contribution to the pathogenetic mechanisms associated with cortical pathology. A comprehensive immunohistochemical analysis was performed on post-mortem brain tissue from 26 cases with primary progressive multiple sclerosis. A variable extent of meningeal immune cell infiltration was detected and more extensive demyelination and neurite loss in the cortical grey matter was found in cases exhibiting an increased level of meningeal inflammation. However, no tertiary lymphoid-like structures were found. Profound microglial activation and reduction in neuronal density was observed in both the lesions and normal appearing grey matter compared with control cortex. Furthermore, cases with primary progressive multiple sclerosis with extensive meningeal immune cell infiltration exhibited a more severe clinical course, including a shorter disease duration and younger age at death. Our data suggest that generalized diffuse meningeal inflammation and the associated inflammatory milieu in the subarachnoid compartment plays a role in the pathogenesis of cortical grey matter lesions and an increased rate of clinical progression in primary progressive multiple sclerosis.

Multiple sclerosis is the major inflammatory condition affecting the central nervous system (CNS) and is characterised by disseminated focal immune-mediated demyelination. Demyelination is accompanied by variable axonal damage and loss and reactive gliosis. It is this pathology that is thought to be responsible for the clinical relapses that often respond well to immunomodulatory therapy. However, the later secondary progressive stage of MS remains largely refractory to treatment and it is widely suggested that accumulating axon loss is responsible for clinical progression. Although initially thought to be a white matter (WM) disease, it is increasingly apparent that extensive pathology is also seen in the grey matter (GM) throughout the CNS. GM pathology is characterised by demyelination in the relative absence of an immune cell infiltrate. Neuronal loss is also seen both in the GM lesions and in unaffected areas of the GM. The slow progressive nature of this later stage combined with the presence of extensive grey matter pathology has led to the suggestion that neurodegeneration might play an increasing role with increasing disease duration. However, there is a paucity of studies that have correlated the pathological features with clinical milestones during secondary progressive MS. Here, we review the contributions that the various types of pathology are likely to make to the increasing neurological deficit in MS.

Disease progression in multiple sclerosis occurs within the interface of glial activation and gliosis. This study aimed to investigate the relationship between biomarkers of different glial cell responses: (i) to disease dynamics and the clinical subtypes of multiple sclerosis; (ii) to disability; and (iii) to cross-validate these findings in a post-mortem study. To address the first goal, 51 patients with multiple sclerosis [20 relapsing remitting (RR), 21 secondary progressive (SP) and 10 primary progressive (PP)] and 51 neurological control patients were included. Disability was assessed using the ambulation index (AI), the Expanded Disability Status Scale score (EDSS) and the 9-hole PEG test (9HPT). Patients underwent lumbar puncture within 7 days of clinical assessment. Post-mortem brain tissue (12 multiple sclerosis and eight control patients) was classified histologically and adjacent sites were homogenized for protein analysis. S100B, ferritin and glial-fibrillary acidic protein (GFAP) were quantified in CSF and brain-tissue homogenate by ELISA (enzyme-linked immunosorbent assay) techniques developed in-house. There was a significant trend for increasing S100B levels from PP to SP to RR multiple sclerosis (P < 0.05). S100B was significantly higher in RR multiple sclerosis than in control patients (P < 0.01), whilst ferritin levels were significantly higher in SP multiple sclerosis than in control patients (P < 0.01). The S100B : ferritin ratio discriminated patients with RR multiple sclerosis from SP, PP or control patients (P < 0.05, P < 0.01 and P < 0.01, respectively). Multiple sclerosis patients with poor ambulation (AI > or =7) or severe disability (EDSS >6.5) had significantly higher CSF GFAP levels than less disabled multiple sclerosis or control patients (P < 0.01 and P < 0.001, respectively). There was a correlation between GFAP levels and ambulation in SP multiple sclerosis (r = 0.57, P < 0.01), and between S100B level and the 9HPT in PP multiple sclerosis patients (r = -0.85, P < 0.01). The post-mortem study showed significantly higher S100B levels in the acute than in the subacute plaques (P < 0.01), whilst ferritin levels were elevated in all multiple sclerosis lesion stages. Both GFAP and S100B levels were significantly higher in the cortex of multiple sclerosis than in control brain homogenate (P < 0.001 and P < 0.05, respectively). We found that S100B is a good marker for the relapsing phase of the disease (confirmed by post-mortem observation) as opposed to ferritin, which is elevated throughout the entire course. GFAP correlated with disability scales and may therefore be a marker for irreversible damage. The results of this study have broad implications for finding new and sensitive outcome measures for treatment trials that aim to delay the development of disability. They may also be considered in future classifications of multiple sclerosis patients.

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Results:The cortical LB (CLB) burden was the only independent predictor of dementia (OR: 4.12, p<0.001). The total cortical Aβ plaque burden was an independent predictor of a shorter latency to dementia from onset of motor signs (p=0.001). DLB cases had a higher LB burden in the parietal and temporal cortex, compared to PDD. Carrying at least one APOE ϵ4 allele was associated with a higher cortical LB burden (p=0.02), particularly in the neocortical frontal, parietal, and temporal regions. Conclusions:High CLB burden is a key neuropathological substrate of dementia in LBD.Elevated cortical LB pathology and Aβ plaques are both correlated with a faster progression to dementia. The higher LB load in the temporal and parietal regions, which distinguishes DLB cases, could have a role in the shorter latency to dementia and may be mediated by the APOE ϵ4 allele.

Components of the plasminogen activator (PA) and matrix metalloprotease (MMP) cascade have been characterized in multiple sclerosis lesions by immunohistochemistry, enzyme-linked immunosorbent assay and enzyme activity assays in order to establish a functional role for the enzyme sequence in lesion development. Highly significant quantitative increases in urokinase PA (uPA), urokinase receptor (uPAR) and plasminogen activator inhibitor-1 were detected in acute multiple sclerosis lesions (P < 0.0001) and in uPAR in normal-appearing white matter (P < 0.0001) compared with control tissue. All three proteins were immunolocalized to mononuclear cells in perivascular cuffs and to macrophages in the lesion parenchyma. MMP-9 and the tissue inhibitor of metalloprotease-1 also increased during lesion development but the enzyme was present largely in the inactive pro-form. In contrast to uPA, the concentration and activity of tissue PA (tPA), the most abundant plasminogen activator in normal control brain, were reduced in multiple sclerosis specimens. In acute lesions tPA co-localized with fibrin(ogen) on large diameter axons also stained with SMI-32, an immunohistochemical marker of axonal damage. The uPA-uPAR complex, concentrated on inflammatory cells in the perivascular zone of the evolving lesion, may facilitate cellular infiltration into the CNS which is amplified by MMP- mediated degradation of blood vessel matrix. tPA localization on injured axons may be a marker of axonal damage or represent a protective mechanism aimed at removal of fibrin deposits and restoration of axonal function.

Tissue plasminogen activator (tPA), a neuronal as well as the key fibrinolytic enzyme, is found concentrated on demyelinated axons in multiple sclerosis lesions together with fibrin(ogen) deposits. The decreased tPA activity in normal-appearing white and grey matter and lesions of multiple sclerosis is reflected in diminished fibrinolysis as measured by a clot lysis assay. Nonetheless, peptide products of fibrin, including D-dimer, accumulate on demyelinated axons-the result of fibrinogen entry through a compromised blood-brain barrier (BBB). Analysis of tissue samples on reducing and non-reducing polyacrylamide gels demonstrates complexes of tPA with plasminogen activator inhibitor-1 (PAI-1) but not with neuroserpin, a tPA-specific inhibitor concentrated in grey matter. As total tPA protein remains unchanged in acute lesions and the concentration of PAI-1 rises several fold, complex formation is a probable cause of the impaired fibrinolysis. Although the tPA-plasmin cascade promotes neurodegeneration in excitotoxin-induced neuronal death, in inflammatory conditions with BBB disruption it has been demonstrated to have a protective role in removing fibrin, which exacerbates axonal injury. The impaired fibrinolytic capacity resulting from increased PAI-1 synthesis and complex formation with tPA, which is detectable prior to lesion formation, therefore has the potential to contribute to axonal damage in multiple sclerosis.