Johnson P T, Geller S F, Lewis G P, Reese B E
Neuroscience Research Institute, University of California, Santa Barbara 93106-5060, USA.
Exp Eye Res. 1997 May;64(5):759-66. doi: 10.1006/exer.1996.0270.
Cellular retinaldehyde binding protein (CRALBP) is present in Müller glia and in cells of the retinal pigment epithelium, but we have recently observed CRALBP-like immunoreactivity near the inner limiting membrane in the newborn mouse retina. The present study has examined whether this protein is present in developing retinal astrocytes. Retinal tissue was collected at various embryonic and postnatal ages and in adulthood. Tissue for immunohistochemistry was fixed by immersion in 4% paraformaldehyde and immunostained using rabbit polyclonal antisera to CRALBP or glial fibrillary acidic protein (GFAP), while fresh tissue was homogenized for Western analysis. Specificity of the antiserum for the 33 kDa protein was shown in retinal homogenates by immunoblotting, with expression of the protein increasing steadily from E15.5 through adulthood. Immunostaining of sections from fetal eye-cups revealed faint labeling of cells in the optic nerve, with progressive migration of CRALBP-immunoreactive cells into the retina at the inner limiting membrane during the perinatal period. By the day of birth, these cells were intensely immunoreactive, showing a morphology characteristic of migrating astrocytes. These CRALBP-immunoreactive cells mimicked the progressive infiltration of GFAP-positive astrocytes which are known to migrate into the retina from the optic nerve head, many of which were double-labeled with GFAP. Their distribution across the retina is distinct from that of the lighter-staining Müller glial somata during these stages, and they are not misidentified Müller glial endfeet. Astrocytes are only transiently CRALBP-immunoreactive, no longer containing the protein after the second post-natal week. Preincubation of the antiserum with purified CRALBP abolished all staining of astrocytes. Coupled with the fact that only a single (approximately 33 kDa) molecular weight protein is labeled by the antiserum, it was concluded that retinal astrocytes contain CRALBP during a limited period of development.
细胞视黄醛结合蛋白（CRALBP）存在于穆勒胶质细胞和视网膜色素上皮细胞中，但我们最近在新生小鼠视网膜的内界膜附近观察到了类似CRALBP的免疫反应性。本研究检测了这种蛋白是否存在于发育中的视网膜星形胶质细胞中。在不同的胚胎期、出生后各年龄段以及成年期收集视网膜组织。用于免疫组织化学的组织通过浸入4%多聚甲醛固定，并用抗CRALBP或胶质纤维酸性蛋白（GFAP）的兔多克隆抗血清进行免疫染色，同时将新鲜组织匀浆用于蛋白质印迹分析。通过免疫印迹在视网膜匀浆中显示了抗血清对33 kDa蛋白的特异性，该蛋白的表达从胚胎第15.5天到成年期稳步增加。对胎儿眼杯切片的免疫染色显示视神经中的细胞有微弱标记，围产期期间CRALBP免疫反应性细胞逐渐迁移到视网膜内界膜。到出生时，这些细胞具有强烈的免疫反应性，呈现出迁移星形胶质细胞的形态特征。这些CRALBP免疫反应性细胞模仿了已知从视神经乳头迁移到视网膜的GFAP阳性星形胶质细胞的逐渐浸润，其中许多细胞与GFAP双标。在这些阶段，它们在视网膜中的分布与染色较浅的穆勒胶质细胞胞体不同，并且它们不是被误认的穆勒胶质细胞终足。星形胶质细胞只是短暂地具有CRALBP免疫反应性，出生后第二周后不再含有该蛋白。抗血清与纯化的CRALBP预温育消除了星形胶质细胞的所有染色。再加上抗血清仅标记单一（约33 kDa）分子量蛋白这一事实，得出结论：视网膜星形胶质细胞在发育的有限时期内含有CRALBP。
