The pathology of multiple sclerosis (MS) is characterised by breakdown of the blood-brain barrier accompanied by infiltration of macrophages and T cells into the central nervous system (CNS). Myelin is degraded and engulfed by the macrophages, producing lesions of demyelination. Some or all of these mechanisms might involve proteinases, and here we have studied the cellular localisation and distribution of two matrix metalloproteinases (MMPs), MMP-7 (matrilysin) and MMP-9 (92-kDa gelatinase), in the normal human CNS and active demyelinating MS lesions. Cryostat sections of CNS samples were immunostained with antisera to MMP-7 and MMP-9. In addition, non-radioactive in situ hybridisation (ISH) was performed using a digoxygenin-labelled riboprobe to detect the expression of MMP-7. MMP-7 immunoreactivity was weakly detected in microglial-like cells in normal brain tissue sections, and was very strong in parenchymal macrophages in active demyelinating MS lesions. This pattern of expression was confirmed using ISH. MMP-7 immunoreactivity was not detected in macrophages in spleen or tonsil, indicating that it is specifically induced in infiltrating macrophages in active demyelinating MS lesions. MMP-9 immunoreactivity was detected in a few small blood vessels in normal brain tissue sections, whereas many blood vessels stained positive in CNS tissue sections of active demyelinating MS lesions. The up-regulation of MMPs in MS may contribute to the pathology of the disease.

A solid-phase, indirect β-galactosidase-linked immunoassay (ELISA) is described for screening large numbers of monoclonal antibodies that recognize cell surface antigens of primary monolayer cerebellar cultures. Target cultures were prepared from perikaryal suspensions of postnatal rodent cerebellum seeded into poly-l-lysine pre-coated, flat-bottom microtiter wells and fixed with glutaraldehyde after growth in vitro. Hybridoma supernatants were then incubated on these cultures. After the addition of β-galactosidase-linked anti-mouse IgG F(ab′)2 fragments, antigen-positive supernatants were detected with the enzyme substrate o-nitrophenyl-β-d-galactopyranoside. Using a monoclonal antibody specific for rat brain Thy-1 glycoprotein, this solid-phase ELISA was found to be useful in quantifying changes in the developmental expression of cerebellar surface antigens in these cultures.

A perfusion chamber is described for studying the efflux of putative neurotransmitters from CNS cells maintained in monolayer culture. We have used this apparatus to investigate the efflux of newly accumulated [3H]GABA from cell cultures of the early postnatal rat cerebellum.