Tissue microarray methodology identifies complement pathway activation and dysregulation in progressive multiple sclerosis

BACKGROUND: The complement pathway has potential contributions to both white (WM) and grey matter (GM) pathology in Multiple Sclerosis (MS). A quantitative assessment of complement involvement is lacking. OBJECTIVE: Tissue MicroArray methodology was used in conjunction with immunohistochemistry to investigate the localization of complement pathway proteins in progressive MS cortical GM and subcortical WM. METHODS: Antibodies targeting complement proteins C1q, C3b, regulatory proteins C1 inhibitor (C1INH), complement receptor 1 (CR1), clusterin, factor H (FH) and the C5a anaphylatoxin receptor (C5aR) were utilised alongside standard markers of tissue pathology. All stained slides were digitised for quantitative analysis. RESULTS: Numbers of cells immunolabelled for HLA-DR, GFAP, C5aR, C1q and C3b were increased in WM lesions (WML) and GM lesions (GML) compared to normal appearing WM (NAWM) and GM (NAGM), respectively. The complement regulators C1INH, CR1, FH and clusterin were more abundant in WM lesions, while the number of C1q+ neurons were increased and the number of C1INH+, clusterin+, FH+ and CR1+ neurons decreased in GM lesions. The number of complement component positive cells (C1q, C3b) correlated with complement regulator expression in WM, but there was no statistical association between complement activation and regulator expression in the GM. CONCLUSION: Tissue microarray methodology and quantitative analysis provides evidence of complement dysregulation in MS GML, including an association of the numerical density of C1q+ cells with tissue lesions. Our work confirms that complement activation and dysregulation occur in all cases of progressive MS and suggest that complement may provide potential biomarkers of the disease.

Contribución al estudio de la colpotomía posterior : Tesis presentada para optar al grado de doctor en medicina

A la cabeza de portada: Universidad Nacional de Buenos Aires. Facultad de Ciencias Médicas. - Incluye nómina de Catedráticos y Asignaturas. Tesis con dedicatoria

Cartilage viability after repetitive loading: A preliminary report

Objective: To assess matrix changes and chondrocyte viability during static and continuous repetitive mechanical loading in mature bovine articular cartilage explants. Methods: Cartilage explants were continuously loaded either statically or cyclically (0.5 Hz) for 1-72 h (max. stress 1 megapascal). Cell death was assessed using fluorescent probes and detection of DNA strand breakage characteristic of apoptosis. Cell morphology and matrix integrity were evaluated using histology and transmission electron microscopy. Results: Repetitive loading of articular cartilage at physiological levels of stress (1 megapascal) was found to be harmful to only the chondrocytes in the superficial tangential zone (STZ) and depended on the characteristics (static vs cyclic) and duration (1-72 h) of the applied load. The chondrocytes in the middle and deep zone remained viable at all times. Static loads caused cell death at an early time (3 h) as compared with cyclic loads (sinusoidal, 0.5 cycles per s for 6 h). The amount and extent of cell death peaked at 6 h of cyclic loading, and did not change in subsequent experiments run for longer periods of time (up to 72 h). There was no indication of fragmented nuclear DNA but there was evidence of injurious cell death (necrosis) by electron microscopy. Morphological analysis of cartilage repetitively loaded for 24 h showed matrix damage only in the uppermost superficial layer at the articular surface, reminiscent of the early stages of osteoarthritis. Conclusions: Cell death in mature cartilage explants occurred after 6 hours of continuous repetitive load or 3 h of static load. Cell death was directly related to the mechanical load, as control (free-swelling) explants remained viable at all times. The excessive, repetitive loading conditions imposed are not physiological, and demonstrate the deleterious effects of mechanical overload resulting in morphological and cellular damage similar to that seen in degenerative joint disease. © 2002 OsteoArthritis Research Society International