Effect of hypocholesterolemic drug AY9944 on cultured nervous tissue: morphologic and biochemical studies.

Abstract

The effects of different concentrations of the hypocholesterolemic drug AY9944, an inhibitor of delta7-reductase, on organotypic cultures of fetal mouse spinal cord, were studied by light and electron microscopy. Exposure to 10(-6)M produced no observable changes. After 6 hours exposure to 10(-4)M, dense membrane-bound inclusions were occasionally observed in neurons. After 24 hours exposure to 10(-4)M, numerous cytoplasmic inclusions occurred in neurons, glia and macrophages. The form of these inclusions varied but were predominantly of two types; concentric, loosely-packed lamellae resembling membranous cytoplasmic bodies (MCB) of Tay-Sachs disease and irregular dense bodies. They were identical to those observed in our previous in vivo study. Prolonged exposure to the drug at 10(-4)M caused an increased number of inclusions in all cell types. Eventually the cultures degenerated. The number of inclusions increased for at least 38 days following a 2-5 day exposure to AY9944 at 10(-4)M. However, by 70 days, although inclusions persisted, the cultures were mostly astrocytic. In long-term cultures, in addition to these inclusions, curved or straight electron-dense paired profiles were seen in some cells, presumably macrophages. Biochemical analysis of cultures exposed to 10(-4)M revealed the continuous presence of delta7, 24-cholesta-diene-3beta-ol and 7-dehydrocholesterol even after the drug wa removed from the cultures. In our previous animal experiments, intracytoplasmic inclusions and abnormal sterols with a double bond at the 7 position disappeared quickly after discontinuation of the drug. Therefore, the results obtained in our present in vitro experiments are different in this regard from the in vivo studies of AY9944.