Regulation Of Rho Kinase To Neurite Growth And Cytoskeleton Distribution In Cultured Hippocampal Neurons Of Rats

Objective:Investigating regulation of Rho kinase to neurite growth and cytoskeleton distribution in hippocampus neuronsMaterials and methods:Primarily cultured neurons from hippocampus of postnatal rat were treated with Rho kinase activator LPA or inhibitor Y27632 to change Rho kinase's activity.Then the dynamics of neurites outgrowth were surveyed by time-lapse microscopy, and the distribution of cytoskeleton in neurons were observed by laser confocal microscopy.Results:(1) Observed by time-lpase microscopy:The new first-level and second-level neurites in control neurons contiuously developed and extended,meanwhile the third-level neurites were formed at the end of second-level neurites.After LPA exposure,numbers of neurites were decreased,most neurites collapsed progressively and became smaller.While many first-level neurites from the cell body of neurons treated with Y-27632 extended longer, the branch phenomenon were obviously showed on most of neurites.The distal second-level and third-level neurites grew more rapidly compared with the control group.Treated with Y27632 after LPA incubation,some small neurites retracted and disappeared,however the remaining neurites continued to branch and extend,and the number and length of the first-level,second-level,third-level neurites increased compared with the LPA group.The neurons acceptted the opposite treatment,treated with LPA after Y27632 incubation,the first-level neurites stopped collapsing gradually,and then extended outward.The second-level neurites grew rapidly and the third-level and fourth neurites were formed in its distal.(2) Observed by laser confocal microscopy:The neuron body and neurites had clear microfilaments and microtubules distribution.Microfilaments were much more than microtubules compared with the control group in the small neurites,terminal and filopodia. All neurites especially the sub-neurites in LPA group shorten in the length and reduced in the number.The distribution of microtubules and microfilaments in neurites decreased significantly,and filopodia were formed less.Neurons in Y-27632 group had more branches, neurites elongated for a long distance.Microtubules and microfilaments extended along the cell bodies and neurites,meanwhile more microfilaments distributed in terminal and small branches,and a number of slender filopodia were created on neurites.Neurites in the LPA+Y-27632 and Y-27632+LPA group of neurons were rich,on which the expression of microfilaments and microtubules had the distinct improvement compared with the LPA group, and more filopodia were formed in the distal.Conclusions:(1) Inhibition of Rho kinase could promote neurites at all levels to increase in the number and the length.(2) Activation of Rho kinase could induce neurites collapsed, displaying in all levels of neurites the number reduce and the length decrease.(3) Inhibition after the activation of Rho kinase or the contrary,Rho kinase function induing neurites retraction declined,and the neurites continued to grow including all levels of branches.(4) Rho kinase could regulate the cytoskeletal distribution that was involved in neurites growth and branch formation.