| Spinal cord injury is a common clinical disease, and most of them occur atthe thoracic and lumber level. Because of the irreversible growth of spinal cord,the clinical treatment of SCI is very difficult, and the cost of treating anindividual spinal cord injury is high. Published incidence rates for traumaticspinal-cord injury in the USA range between28and55per million people, withabout10000new cases reported every year. Nowadays, some progression havebeen made for the pathomechanism of SCI and spinal restoration andregeneration, but the mechanism of constitutional spinal cord injury is still notclear. So, the research of the mechanism of acute mechanical spinal cord injuryand its ultrastructural changes is very important, also neural protection at theearly stage of injury to lighten the further injury of spinal cord is critical.Objective:Our research aims at exploring the mechanism of mechanical injury ofcultured dorsal root ganglion (DRG) cells in vitro, using the equipment ofmechanical pressure administration(national patent, NO. ZL200820030390.7), giving the DRG cells mechanical stimulation, to investigate the cytoskeletonchanges, especially the changes of cortical cytoskeleton and its effects, alsoinvestigation of the relationship among cytoskeleton, calpain and apoptosis. Atthe same time, the new type of calpain inhibitor-PD150606was used to observethe neural protective effects.Materials and Methods:Our research use the newly born S-D rat to culture the DRG cells, utilizingthe equipment of mechanical pressure administration(national patent, NO.ZL200820030390.7), excluding other injury factors of spinal cord injury,exerting the mechanical pressure injury. The mechanical pressure value isdivided into4groups, including the blank contrast, the0.3mpa group, the0.5mpa group and the0.7mpa group. We choose the time point of8hours and24hours after injury to execute the observation, detection and analysis. TheLaser confocal microscope, immunohistochemistry, atomic force microscopeand Tunel technique were used to investigate the cytoskeleton changes,especially the changes of cortical cytoskeleton under different injury level,also investigate the mechanism of mechanical injury and the occurrence ofapoptosis. At the same time, the new type of calpain inhibitor-PD150606wasused to observe the neural protective effects, which further interpreted themechanism of mechanical cell injury, and made the basic knowledge of neuralprotection at the early stage of SCI.Results:(1) the cultured DRG cells in vitro adherenced4hours after implantation,showed roundlet shape at first but24hours later the cells formed as the typical neuron shape, which is long synapses and ellipse cytoplasm.(2) the growth ofnon-neuron cells was apparently inhibited by the cytarabine and the purity ofneurons could achieve to95%.(3) the distribution of β-tubulinⅢ in the controlgroup was continuous, no beading and disruption was found. Coomassiebrilliant blue stainings presented the same distribution and no marginaldistribution was found.(4) some floating cells in the0.5mpa group was foundby observation of phase contrast microscope, the disruption of the cytoskeletonwas also apparent and the amount of apoptosis cell was largest in this group.(5)The3-dimensional picture of the cell membrane skeleton at the nano scale leveland its information could be obtained by the AFM, which made it possible toanalysis the changes of cortical skeleton protein.(6) In comparison with thecontrol group, different membrane particle data was found in the injured group.With the increase of the mechanical pressure value, the height of membraneparticles was found increasing accordingly, seemed statistical difference withcontrol group.(7) cell membrane in the0.7mpa injury group was found someporous channel structure,0.7mpa was the limitation of the mechanical injuryvalue of neural cells.(8) with the preconditioning of100μmol/L PD150606inDRG cells, the protective effects of calpain inhibitor was apparent;Conclusion:(1) Our research establish the mechanical pressure injury model of neuralcell in vitro, found that the cell injury characterized by changes of cytoskeletonand cortical membrane skeleton, these structure possibly participate in theprocess of mechanotrasduction.(2) the DRG cells has low tolerance tomechanical pressure injury, so0.5mpa mechanical injury value can lead toobvious cell injury. Moreover,0.7mpa mechanical injury value can lead to membrane porous channel structure and cell death.(3) the application of calpaininhibitor in the early stage of mechanical pressure injury of spinal cord hasneural protective effects.(4) the evidence about the participation ofcytoskeleton and calpain in mechanical pressure injury established foundationsfor further investigations of constitutional SCI mechanism and spinal cordprotection. |
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