Uttenthal L O, Alonso D, Fernández A P, Campbell R O, Moro M A, Leza J C, Lizasoain I, Esteban F J, Barroso J B, Valderrama R, Pedrosa J A, Peinado M A, Serrano J, Richart A, Bentura M L, Santacana M, Martínez-Murillo R, Rodrigo J
Department of Comparative Neuroanatomy, Instituto Cajal, C.S.I.C., Madrid, Spain.
Microsc Res Tech. 1998 Oct 1;43(1):75-88. doi: 10.1002/(SICI)1097-0029(19981001)43:1<75::AID-JEMT11>3.0.CO;2-0.
Neuronal and inducible nitric oxide synthase (nNOS and iNOS) and nitrotyrosine immunoreactivities were localized and semiquantitatively assessed in the cerebral cortex of aged rats by means of light microscopic immunocytochemistry and Western blotting, using a new series of specific polyclonal antibodies. In the aged rats the strongly nNOS-immunoreactive multipolar neurons found in layers II-VI of the cortex of young rats were seen in similar numbers, but showed varicose, vacuolated, and fragmented processes, with an irregular outline and loss of spines. A large number of more weakly nNOS-positive neurons, characterized by a ring of immunoreactive cytoplasm, and not seen in young rats, were observed in layers II-VI of aged rat cortex. While no iNOS-immunopositive neurons were found in the cortex of young rats, a large number of such neurons appeared throughout the aged rat cortex. Nitrotyrosine-positive cells outnumbered total NOS-positive neurons in the cortex of young rats, but this relation was inverted in the aged rats, although these showed a slight increase in the number and staining intensity of nitrotyrosine-positive cells. Western blots of brain extracts showed a several-fold increase in both nNOS- and iNOS-immunoreactive bands in the aged rat, but a less marked increase in nitrotyrosine-containing proteins. The results suggest that while nNOS and iNOS expression is substantially increased in the aged rat cortex, this is not necessarily accompanied by a proportionate increase in nitric oxide synthesis. The mechanisms underlying the increased expression of nNOS and iNOS, and the functional implications of this increase, require elucidation.
运用一系列新型特异性多克隆抗体，通过光学显微镜免疫细胞化学和蛋白质免疫印迹法，对老年大鼠大脑皮层中的神经元型和诱导型一氧化氮合酶（nNOS和iNOS）以及硝基酪氨酸免疫反应性进行了定位和半定量评估。在老年大鼠中，年轻大鼠皮层II-VI层中发现的强nNOS免疫反应性多极神经元数量相似，但呈现出曲张、空泡化和碎片化的突起，轮廓不规则且棘突缺失。在老年大鼠皮层的II-VI层中观察到大量较弱的nNOS阳性神经元，其特征为一圈免疫反应性细胞质，而在年轻大鼠中未见。虽然在年轻大鼠皮层中未发现iNOS免疫阳性神经元，但在整个老年大鼠皮层中出现了大量此类神经元。在年轻大鼠皮层中，硝基酪氨酸阳性细胞数量超过总NOS阳性神经元，但在老年大鼠中这种关系颠倒，尽管老年大鼠中硝基酪氨酸阳性细胞的数量和染色强度略有增加。脑提取物的蛋白质免疫印迹显示，老年大鼠中nNOS和iNOS免疫反应条带均增加了数倍，但含硝基酪氨酸的蛋白质增加不太明显。结果表明，虽然老年大鼠皮层中nNOS和iNOS表达大幅增加，但一氧化氮合成不一定随之成比例增加。nNOS和iNOS表达增加的潜在机制及其功能影响需要阐明。
