Morcos Y, Chan-Ling T
Department of Anatomy and Histology, University of Sydney, NSW, Australia.
Glia. 1997 Oct;21(2):163-82.
Whole-mount techniques have been applied to the myelinated axons of the rabbit retina in order to study oligodendrocytes in the developing and adult central nervous system in vivo. Retinas from rabbits on embryonic day (E) 26 to postnatal day (P) 50 and from adults were subjected to immunohistochemistry with antibodies to glial fibrillary acidic protein (GFAP), vimentin, glycolipid O4, myelin basic protein (MBP), galactocerebroside (Gal-C), 4D6, and Griffonia simplicifolia isolectin, markers chosen for their specificity for astrocytes, cells of the oligodendrocyte lineage, Müller cells, and microglia. The first glial cells labelled within the myelinated streak (MS) were vimentin+ astrocytes. These cells were first apparent near the optic disc at E26. Their numbers and distribution increased markedly between E26 and E30, but between E29 and P3, vimentin expression was replaced by GFAP expression. Between P3 and P4, O4+, Gal-C-, MBP oligodendrocyte precursor cells, O4+, Gal-C-, MBP- pre-oligodendrocytes, and O4+, Gal-C-, MBP- immature oligodendrocytes appeared in low numbers in the region adjacent to the optic nerve head. O4-/+, Gal-C+, MBP+ mature oligodendrocytes appeared soon after at P4 to P5. With age, the outer extent of Gal-C and MBP immunoreactivity expanded, with positive cells forming a continuous sheath around nerve fibre bundles. The sequence of gliogenesis in the (MS) of the rabbit retina thus appears similar to that in the rat optic nerve. In adult rabbits, a large population of O4+ cells was distributed across the MS, the outer limit of the cells coinciding with the outer limit of retinal vessels. These O4+ cells could be classified into three stages of differentiation on the basis of expression of developmental markers and morphology: O4+, Gal-C-, MBP- oligodendrocyte precursor cells with a bipolar, unipolar, or simple morphology; O4+, Gal-C-, MBP- pre-oligodendrocytes with a complex multipolar morphology; and O4+, Gal-C+, MBP- immature oligodendrocytes with a complex multipolar morphology. The final stage of differentiation was characterized as O4-, Gal-C+, MBP+ mature oligodendrocytes with multiple parallel processes aligned along nerve fibre bundles. These results provide in vivo evidence for the existence of a substantial population of oligodendrocyte precursor cells and non-myelin-producing immature oligodendrocytes in the MS of the adult rabbit retina. This preparation makes possible in situ patch clamping and determination of other functional responses of these cell types in vivo.
为了在体内研究发育中和成年中枢神经系统中的少突胶质细胞，全装技术已应用于兔视网膜的有髓轴突。对胚胎第（E）26天到出生后第（P）50天的兔视网膜以及成年兔视网膜进行免疫组织化学检测，使用针对胶质纤维酸性蛋白（GFAP）、波形蛋白、糖脂O4、髓鞘碱性蛋白（MBP）、半乳糖脑苷脂（Gal-C）、4D6和简氏相思子凝集素的抗体，这些标志物因其对星形胶质细胞、少突胶质细胞系细胞、穆勒细胞和小胶质细胞的特异性而被选用。在有髓条纹（MS）内最早标记的胶质细胞是波形蛋白阳性的星形胶质细胞。这些细胞在E26时首先出现在视盘附近。它们的数量和分布在E26和E30之间显著增加，但在E29和P3之间，波形蛋白表达被GFAP表达所取代。在P3和P4之间，O4阳性、Gal-C阴性、MBP少突胶质细胞前体细胞、O4阳性、Gal-C阴性、MBP阴性前少突胶质细胞和O4阳性、Gal-C阴性、MBP阴性未成熟少突胶质细胞在视神经乳头附近区域少量出现。O4阴性/阳性、Gal-C阳性、MBP阳性成熟少突胶质细胞在P4到P5之后不久出现。随着年龄增长，Gal-C和MBP免疫反应性的外层范围扩大，阳性细胞围绕神经纤维束形成连续的鞘。兔视网膜（MS）中的胶质细胞生成序列因此似乎与大鼠视神经中的相似。在成年兔中，大量O4阳性细胞分布在MS中，细胞的外层界限与视网膜血管的外层界限一致。根据发育标志物的表达和形态，这些O4阳性细胞可分为三个分化阶段：具有双极、单极或简单形态的O4阳性、Gal-C阴性、MBP阴性少突胶质细胞前体细胞；具有复杂多极形态的O4阳性、Gal-C阴性、MBP阴性前少突胶质细胞；以及具有复杂多极形态的O4阳性、Gal-C阳性、MBP阴性未成熟少突胶质细胞。分化的最后阶段特征为O4阴性、Gal-C阳性、MBP阳性成熟少突胶质细胞，其多个平行突起沿神经纤维束排列。这些结果为成年兔视网膜MS中存在大量少突胶质细胞前体细胞和不产生髓鞘的未成熟少突胶质细胞提供了体内证据。这种制备方法使得在体内对这些细胞类型进行原位膜片钳记录和其他功能反应的测定成为可能。
