Study Of Rho Kinase Regulating Neurite Growth And Microtubule Rearrangement In Cultured Hippocampal Neurons Of Rats

Objective: Investigating regulation of Rho kinase to neurites growth and microtubule rearrangement.Materials and methods: Cultured rat hippocampal neurons treated with LPA and Y27632, accelerator and inhibitor of Rho kinase, were utilized to make neurites extension and microtubule rearrangement. Neurites extension were analyzed by atomic force microscopy and a quantification assay kit, meanwhile, the images of microtubules in immunofluorescence stained neurons were obtained by using a confocal laser scanning microscopy.Results: (1) Neurobasal had shown excellent long-term viability and nearly pure of postnatal rat hippocampal neurons. When culturing during 6-11 days, hippocampal neurons could be adapted for some other experiments. (2) Observed with atomic force microscopy: Treated with LPA, the number of neurites was decreased. The number of 1St-3th order neurites was decreased significantly compared with the control cells (P＜0.05). Treated with Y27632 after LPA incubation, the important difference from control cells is that more 1st neuritis. The radiatiform 2nd neurites' number was increased significantly (P＜0.05). Optical density of neurites extraction was decreased after treated with LPA (P＜0.05), while it was contrast to LPA treated cells after incubation with Y27632. The neurites' outgrowth was super to the control cells, but no significant difference. (3) Observed with confocal laser scanning microscopy: Visible microtubules were viewed in cell bodies, neurites and growth cones in cultured hippoampal neurons. There were no ramified and bird nest like microtubules in LPA treated neurons cell bodies substituted by irregular microtubules, accompanying with short neurites which were stained superficial fluorescence in distal of them. In neurites the arrangement of microtubules just liked a braid. The growth cones were to be missing and just left a tiny tip at the end of neurites where microtubules were almost disappeared. Y27632 treated neurons recuperated more and more visible ramified microtubules in their bodies. These microtubules extended to the membranes and grew into neurites. Meanwhile, microtubules in neurites were still had some crevices but better than in LPA treated cells. Ends of neurites kept growth cones to be untypical shaped where visible straight and snaked microtubules extended into the inner of growth cones. Conclusions: (1) Hippocampal neurons could be isolated from postnatal rat, which had shown excellent lorig-term viability in neurobasal. (2) Activating Rho kinase could induce neurites to be collapsed and outgrowth to be retarded, and the results were the decreased number of neurites and shortened length. Contrast to activation, inhibition of Rho kinase induced neurites to branch and outgrow. (3) The results suggested that Rho kinase not only participated in regulating neurites growth, but also regulating neurites branch formation by rearranging microtubules. Inhibiting activity of Rho kinase could reverse the function of LPA to retard neurite growth, otherwise, only inhibiting Rho kinase could not reverse the rearrangement of microtubules induced by LPA.