Qi X, Guy J
Departments of Ophthalmology, College of Medicine, University of Florida, Gainesville 32610-0284, USA.
J Comp Neurol. 1996 Jul 1;370(3):396-404. doi: 10.1002/(SICI)1096-9861(19960701)370:3<396::AID-CNE9>3.0.CO;2-#.
The aim of this study is to determine the presence and subcellular distribution of NADPH diaphorase (NADPH-d)/nitric oxide synthase (NOS) in the optic nerve of the normal guinea pig. Optic nerve specimens were stained by NADPH-d histochemistry, and double labeled by combining NADPH-d histochemistry with immunostaining for (a) anti-glial fibrillary acidic protein (GFAP) antibody for recognition of astrocytes, (b) griffonia simplicifilia B4-isolectin (GSA-IB4) horse radish peroxidase (HRP)-conjugate for identification of microglia, or (c) oligodendrocyte-associated antibodies to carbonic anhydrase isoenzyme II (CA-II) or to galactocerebroside (GalC) for visualization of oligodendrocytes. In addition, constitutive NOS (cNOS) and inducible NOS (iNOS) immunostaining were used for colocalization with NADPH-d histochemistry. Light microscopy revealed NADPH-d reaction product in the blood vessels and neuroglia of the unmyelinated optic nerve head and myelinated retrobulbar optic nerve. Double labeling with GFAP immunoperoxidase combined with NADPH-d histochemistry revealed both activities in astrocytes. Microglia were labeled with GSA-IB4 isolectin HRP-conjugate, but they did not have NADPH-d activity. Oligodendroglia were immunolabeled with anti CA-II or anti GalC antibodies, but they did not have NADPH-d activity. Both iNOS and cNOS immunoperoxidase labeled astrocytes, but not microglia or oligodendroglia. Under transmission electron microscopy, NADPH-d reaction product appeared as electron-dense particles. These particles were seen in the cytoplasm of endothelial cells, perivascular smooth muscle cells and fibrous astrocytes. Axons and myelin were devoid of NADPH-d activity. This study demonstrates the existence and cellular distribution of NADPH-d/NOS activity in endothelial cells, perivascular smooth muscle cells and fibrous astrocytes of the optic nerve of the normal guinea pig. The presence of these non-neuronal sources of NOS in the optic nerve provides the foundation for future comparative studies of the functional role of reactive oxygen induced toxicity in disorders affecting the optic nerve.
本研究的目的是确定正常豚鼠视神经中NADPH黄递酶（NADPH-d）/一氧化氮合酶（NOS）的存在及其亚细胞分布。视神经标本采用NADPH-d组织化学染色，并通过将NADPH-d组织化学与免疫染色相结合进行双重标记，具体为：（a）抗胶质纤维酸性蛋白（GFAP）抗体识别星形胶质细胞；（b）格里菲斯豆凝集素B4异凝集素（GSA-IB4）辣根过氧化物酶（HRP）偶联物鉴定小胶质细胞；或（c）与碳酸酐酶同工酶II（CA-II）或半乳糖脑苷脂（GalC）相关的少突胶质细胞抗体可视化少突胶质细胞。此外，组成型NOS（cNOS）和诱导型NOS（iNOS）免疫染色用于与NADPH-d组织化学进行共定位。光学显微镜显示，在无髓鞘的视神经乳头和有髓鞘的球后视神经的血管和神经胶质中存在NADPH-d反应产物。GFAP免疫过氧化物酶与NADPH-d组织化学双重标记显示星形胶质细胞中同时存在这两种活性。小胶质细胞用GSA-IB4异凝集素HRP偶联物标记，但它们没有NADPH-d活性。少突胶质细胞用抗CA-II或抗GalC抗体进行免疫标记，但它们没有NADPH-d活性。iNOS和cNOS免疫过氧化物酶均标记星形胶质细胞，但不标记小胶质细胞或少突胶质细胞。在透射电子显微镜下，NADPH-d反应产物表现为电子致密颗粒。这些颗粒可见于内皮细胞、血管周围平滑肌细胞和纤维性星形胶质细胞的细胞质中。轴突和髓鞘缺乏NADPH-d活性。本研究证明了正常豚鼠视神经内皮细胞、血管周围平滑肌细胞和纤维性星形胶质细胞中存在NADPH-d/NOS活性及其细胞分布。视神经中这些非神经元来源的NOS的存在为今后比较研究活性氧诱导的毒性在影响视神经疾病中的功能作用奠定了基础。
