ISOLATION OF PSEUDOPEROXIDASE-POSITIVE ASTROCYTE GRANULES FROM INTACTRAT-BRAIN AND CYSTEAMINE-TREATED NEUROGLIAL CULTURES

A subpopulation of astrocytes in periventricular regions of aging brai n and in cysteamine (CSH)-treated glial cultures contain autofluoresce nt cytoplasmic granules that exhibit an affinity for Gomori's chrome a lum hematoxylin (CAH), and non-enzymatic peroxidase activity. Although shown to be histochemically distinct from lipofuscin, the lack of pur e preparations of these glial inclusions has hindered the elucidation of their precise chemical constituents. Using sucrose gradient fractio nation and density centrifugation on percoll, we obtained enriched pre parations of astrocyte cytoplasmic granules from intact rat brain and CSH-treated astrocyte cultures. The presence and relative purity of th ese inclusions were confirmed by laser scanning confocal microscopy fo r red autofluorescent granules, diaminobenzidine histochemistry for no n-enzymatic peroxidase activity and chrome alum hematoxylin (Gomori) s taining. In the enriched fractions, the smaller granules (0.5-4.0 mum) were spherical and weakly autofluorescent, whereas larger inclusions (5.0-10.0 mum) tended to be intensely autofluorescent and pleomorphic. As in situ, the purified material was argyrophilic and did not stain for lipids. Isolation of these astrocytic inclusions should permit a m ore thorough characterization of their biochemical contents.