Lindquist T D, Ingoglia N A, Gould R M
Brain Res. 1981 Dec 28;230(1-2):181-94. doi: 10.1016/0006-8993(81)90400-5.
Experiments were designed to determine if following injection of [3H]uridine into the lumbar spinal cord of the rat, [3H]RNA could be demonstrated within axons of the sciatic nerve, and if 4S RNA is the predominant RNA species present in these axons. In one experiment the left sciatic nerve of a rat was crushed. Two days later 170 microCi of [3H]uridine was injected into the vicinity of the lumbar ventral horn cells. Ten days after injection, rats were sacrificed and sciatic nerves were prepared for autoradiography. Photomicrographs were taken of labeled areas of intact and regenerating nerves and grains were counted over Schwann cells, myelin, axons and other unspecified areas. In both intact and regenerating sciatic nerves more than 20% of the silver grains were associated with motor axons and approximately 40% were found over cytoplasm of Schwann cells surrounding these axons. These data indicate an intra-axonal localization of RNA in sciatic nerve axons, as well as an active transfer of RNA precursors from axons to their surrounding Schwann cels. In separate studies, the left sciatic nerve was crushed and 10 days later [3H]uridine was bilaterally injected intraspinally into 6 rats. Four control rats were sacrificed at 14 or 20 days after injection. In the remaining 2 rats the sciatic nerve was cut 14 days after injection and the distal part of the nerve was allowed to degenerate for 6 days before sacrificing the rat. Thus, the distal portion of the nerve contained Schwann cells labeled by axonal transport but lacked intact axons. RNA was isolated from experimental and control nerve segments by hot phenol extraction and ethanol precipitation. RNA species (28S, 18S and 4S) were separated by polyacrylamide gel electrophoresis and radioactivity was measured in a liquid scintillation counter. Control groups had RNA profiles similar to those already described, with greater than 30% of the radioactivity present as 4S RNA. The proximal portions of nerve taken from the group in which nerves were cut, had a similar amount of radioactivity present as 4S RNA. However, in the distal segments of these nerves (in which the axons had degenerated thus creating an 'axon-less' nerve) the amount of radioactivity in the 4S peak decreased to approximately 15% of the total RNA, suggesting that 4S RNA is the predominant if not the only RNA present in these axons. These results strongly indicate that both intact and regenerating sciatic nerves of rats selectively transport 4S RNA along their motor axons.
实验旨在确定向大鼠腰脊髓注射[3H]尿苷后，坐骨神经轴突内是否能显示出[3H]RNA，以及4S RNA是否是这些轴突中存在的主要RNA种类。在一项实验中，将一只大鼠的左侧坐骨神经切断。两天后，向腰腹角细胞附近注射170微居里的[3H]尿苷。注射后十天，处死大鼠并制备坐骨神经用于放射自显影。对完整和再生神经的标记区域拍摄显微照片，并对雪旺细胞、髓鞘、轴突和其他未明确区域的银粒进行计数。在完整和再生的坐骨神经中，超过20%的银粒与运动轴突相关，约40%的银粒出现在围绕这些轴突的雪旺细胞的细胞质上。这些数据表明坐骨神经轴突内存在RNA的轴突内定位，以及RNA前体从轴突向其周围雪旺细胞的活跃转移。在单独的研究中，将左侧坐骨神经切断，十天后向6只大鼠双侧脊髓内注射[3H]尿苷。4只对照大鼠在注射后14或20天处死。在其余2只大鼠中，注射后14天切断坐骨神经，在处死大鼠前让神经的远端部分退化6天。因此，神经的远端部分含有通过轴突运输标记的雪旺细胞，但缺乏完整的轴突。通过热酚提取和乙醇沉淀从实验和对照神经段中分离RNA。通过聚丙烯酰胺凝胶电泳分离RNA种类（28S、18S和4S），并在液体闪烁计数器中测量放射性。对照组的RNA谱与已描述的相似，超过30%的放射性以4S RNA形式存在。从神经被切断的组中取出的神经近端部分，以4S RNA形式存在的放射性量相似。然而，在这些神经的远端段（其中轴突已经退化，从而形成“无轴突”神经），4S峰中的放射性量降至总RNA的约15%，这表明4S RNA即使不是这些轴突中唯一存在的RNA，也是主要的RNA。这些结果有力地表明，大鼠完整和再生的坐骨神经都沿其运动轴突选择性地运输4S RNA。
