Fish glial cells were obtained from cultivated segments of the optic nerve and raised in vitro. Two types of cells were identified as astrocyte-and oligodendrocyte-like glia by the monoclonal antibody Mab O~ (specific for oligodendrocytes) and the rabbit serum anti-goldfish glial fibrillary acidic protein (anti-G-GFAP). ('ells of compact morphology were rare, and anti-G-GFAP positive and O1 negative. Multipolar cells in 5-day-old cultures were anti-G-GFAP but rarely O~ positive. In 5-week-old cultures, however, roughly 75% of the multipolar cells were double-labeled with both anti-G-GFAP and O1; 10% were single labeled with Mab O~ and 15% with anti-G-GFAP, respectively.Since goldfish retinal axons regenerate readily after injury it has been supposed that the glial cells of the fish optic nerve possess growth-promoting properties [5, 8]. Compared to mammals very little is known about glial cells in fish, one reason being that it has been difficult to identify fish astrocytes by immunohistochemistry [3,5]. To test the interaction of regenerating axons with glial cells in vitro requires that glial cells can be raised and identified in culture. This was the goal of the present study. We developed and describe here a simple method how to obtain and raise fish glial cells of goldfish optic nerves in culture. Of those cells two types are astrocyte-and oligodendrocyte-like cells.Previously transected and normal optic nerves from goldfish (7-10 cm) were isolated, divided into small pieces (1 mm 3) and incubated in Ca2+-free phosphate-buffered saline (PBS) for 5 h at 4°C. These pieces were transferred onto polylysine/laminin-coated coverslips, covered with Leibowitz medium (L 15, complemented with 10% fetal calf serum) and kept at 22°C. Cells began to emigrate and were cultured up to 6 weeks.Immunostaining of cultured cells: living cells were incubated with the monoclonal Correspondence: C.A.O. Stuermer, Friedrich-Miescher
Regeneration of injured central nervous system axons is largely dependent on the response of the associated nonneuronal glial cells to injury. Glial cells of the mammalian central nervous system, unlike those of fish, are apparently not conducive to axonal regeneration. While the lineage of rat glial cells is well characterized and its role in the support or inhibition of regenerative growth is beginning to be understood, little is known about fish glial cells. Accordingly, glial cells in cultures of adult goldfish brain and of newly hatched goldfish larvae were studied in an attempt to establish their lineage. The cells were identified by means of indirect immunofluorescence, using antibodies against fish astrocytes and oligodendrocytes. The cell count in the cultures increased from a small number of cells at 24 h after plating to a large number of both astrocytes and oligodendrocytes after 1 week in culture. Both of these cell types had originated from proliferating cells, as shown by their uptake of tritiated thymidine and by the inhibition of cell proliferation by 5-fluoro-2'-deoxyuridine. Both astrocytes, i.e., glial fibrillary acidic protein-positive cells, and oligodendrocytes, i.e., 6D2-positive cells, were positively labeled also by A2B5 antibodies, which are known to label progenitors of type-2 astrocytes and oligodendrocytes in the rat optic nerve. The results suggest that A2B5 positive progenitor cells in the goldfish central nervous system, as in the rat optic nerve, might be a common progenitor of astrocytes and oligodendrocytes.
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