Developmental Brain Research, 54 (1990) 71-79 71 Elsevier BRESD 51086 Gomori-positive astrocytes in primary culture: effects of in vitro age and cysteamine exposure Hyman M. Schipper 1, David E. Scarborough 2, Ronald M. Lechan 3 and Seymour Reichlin 3 1Department of Neurology, McGill University, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis --Jewish General Hospital, Montreal, (Que.) (Canada), 2Louisiana State University Medical Center, Dept. of Internal Medicine, Section on Endocrinology and Metabolism, Shreveport, LA (U.S.A.) and 3Dept. of Medicine, Division of Endocrinology, New England Medical Center and Tufts University, Boston, MA (U.S.A.) (Accepted 23 January 1990) Key words: Astrocyte; Gomori stain; Cell culture; Cysteamine; Peroxidase Gomori-positive astrocytes have been identified in the periventricular brain in situ and in diencephalic explants on the basis of their endogenous peroxidase activity, affinity for chrom alum hematoxylin, and orange-red autofluorescence. To facilitate analyses of their functional properties, we sought to identify these cells in dissociated fetal rat brain cultures. Astrocytes containing cytoplasmic inclusions with the above tinctorial and fluorescent properties represented less than 1% of cultured astrocytes at day 10 in vitro (DIV). There was a marked increase in the fraction of Gomori-positive astrocytes and their granule content between 10 and 46 DIV. As in situ, the peroxidase activity appeared to be non-enzyme-mediated insofar as it catalyzed diaminobenzidine oxidation over a wide range of pH (3-11) and could not be inhibited by tissue preheating or the catalase inhibitor, aminotriazole. Metalloporphyrins probably mediate both the pseudoperoxidase activity and autofluorescence in these cells. Cysteamine and cystamine, but not ethanolamine or L-cysteine, induced a massive accumulation of Gomori-positive astrocytes when administered from DIV 6-18. Alterations of the redox microenvironment or induction of porphyrin/heme biosynthetic enzymes may be the mechanisms responsible for this cyst(e)amine effect. Dissociated rat brain culture enriched for Gomori astroglia should provide ample opportunity to investigate the functional properties of these cells. INTRODUCTION A sub-population of astrocytes with distinct morpho- logical and histochemical characteristics has been de- scribed in the periventricular brain of many vertebrate species 39 including humans 37. These cells contain abun- dant cytoplasmic granules which exhibit an affinity for the Gomori stains, chrome alum hematoxylin (CAH) and aldehyde fuchsin. These tinctorial properties may be due to the high sulfhydryl content of the astrocytic inclusions 39. The absence of visible pigment, lipids, and lysosomal enzymes 14"39 and their unique orange-red autofluorescence 15 exclude lysosomes, lipofuscin, and neuromelanin as primary components of these gliosomes. The granules stain intensely with diaminobenzidine (DAB), a marker for endogenous peroxidase activity. This peroxidase activity is presumed to be non-enzyme- mediated because it persists over a very wide pH range and cannot be suppressed by tissue preheating or by the catalase inhibitor, aminotriazole (AT) 36. We have re- cently suggested that metalloporphyrins may be respon- sible for both the pseudoperoxidase activity and specific autofluorescence which characterize these cells36. The function of the Gomori astrocytes is unknown. Srebro 38, after observing an increase in numbers of Gomori astrocytes adjacent to the third ventricle of X-irradiated rats, hypothesized that the endogenous peroxidase may neutralize toxic lipoperoxides generated by the irradiation. Based on the finding that Gomori gliosomes are increased by estrogen administration, we have suggested that astrocytes rich in peroxidase activity may oxidize neutral substances, such as catecholestrogens and catecholamines, to potentially neurotoxic free radical intermediates 36. If Gomori astrocytes do in fact form free radical intermediates, it is reasonable to postulate that their activity may in turn be regulated by free radicals. To test this hypothesis, we established the fact that these cells are demonstrable in dispersed cerebral brain cell cultures and determined the effect of exposure to cysteamine, a well-characterized scavenger of free radi- cals which has been widely used as a radioprotective agent 2. Contrary to our expectation, cysteamine expo- sure was found to markedly stimulate the accumulation of peroxidase-positive astrocytes in vitro. Correspondence: H.M. Schipper, Department of Neurology, McGill University, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis -- Jewish General Hospital, 3755 Cote St. Catherine Road, Montreal, (Que.) H3T 1E2, Canada. O16~-gR06/q0/$03.50 f~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)