| BackgroundTraumactic spinal cord injury (SCI) is a common cause of disability around the wordPatients offen suffered the dysfunction of motor and sensory below the injury sites after SCI,,which seriously impaires the quality of patients’life.Both in the developmental or developed countries,the incidence of SCI still remains relative high and the absolute number of SCI patients is increasing during the past years.Enormous medical and rehabilitative cost of SCI has brought an economic burden to the patients family and our society. The pathophysiology of SCI including primary and secondary injury. Primary was induced by mechanical trauma directly destroyed the spinal cord. Sequentially, ischaemia,release of toxic chemicals from disrupted neural membranes, and electrolyte shifts trigger a secondary injury cascade which leads to harming or killing the neighbouring cells.Though large efforts has been made to the research of SCI therapeutic method,however,we haven’t achieved an efficient and trustworthy treatment for clinical patients at present.During the past decades,most of the studies are focused on the research which takes measures to promote the regeneration of injured spinal cord. But recent studies found that atrophy was determined in the motor cortex of the brain after SCI and was closely related to functional recovery of patients with SCI. SCI animal studies has confirmed that SCI may induce neuron apoptosis in the motor cortex.Therefore, we speculate that motor cortex atrophy or apoptosis would have a significant impact on the recovery of motor function in patients with SCI. Supposed the situation that the neural cell body wich connected with the axon has undergone a structural and functional damage in the central neural system and effective synaptic connections corresponding can’t be formed to the brain, it is hard to achieve the goal of complete recovery of limb function even the the spinal cord axon at the injuried site regenerated completely. However,the mechanism of atrophy and apoptosis in motor cortex after SCI is still unclear. Thus, it is worthful for the treatment of SCI that making efforts to understanding the mechanism of the motor cortex atrophy after SCI.Previous studies showed that the way of neuron apoptosis including caspase-dependent apoptosis pathway and caspase-independent pathway. Activating cleaved caspase-3 plays a key role in caspase-dependent apoptosis pathway, which means the cell ultimately to apoptosis.Caspase-independent pathway is mediated by apoptosis inducing factor(AIF). AIF is a flavoprotein, which located in the mitochondrial intermembrane space, with the dual but de-coupling function of oxidoreductase activity and apoptosis-inducing activity.If activated by hydrolases, AIF rapidly penetrates mitochondrial membrane into the cytoplasm and then combines to the nuclear chromatin condensation, which can hydrolyze the DNA into small pieces and promote apoptosis. Mitochondrial dysfunction is an important factor leading to the hydrolysis of AIF. Tau is a microtubule-associated protein proteins and is essential to maintain the stability of microtubule. Tau is combined with the microtubule, however, while phosphorylation catalyzed by glycogen synthasekinase-3β (GSK-3β),tau will shed from the microtubule and cause the stability decreasing or microtubules depolymerization. Maitaining the balance between Phosphorylation and dephosphorylation balance of tau is crucial for microtubule stability that ensure the normal fuction of axonal transport. Thus,organelles such as mitochondria transport and distribute to the right position in neural axoplasm. In the study on traumatic brain injury, Hyperphosphorylation of tau protein induces microtubule depolymerization, abnormal distribution of mitochondria and mitochondrial dysfunction, eventually leading to neuronal degeneration or apoptosis. However, it still unclear whether SCI could cause hyperphosphorylation of tau protein in motor cortex after SCI and its relationship between the apoptosis of neurons in the motor cortex. Further study on the mechanism of apoptosis in motor cortex neurons after SCI is meaningful for the perfecting pathophysiological mechanisms and develop comprehensive measures for SCI.ObjectiveThe aim of this study was to explore the apoptotic pathway of motor cortex neurons in rat with SCI and whether hyperphosphorylation of tau protein played a key role in the apoptotic precess initiation.In this research,by using different grade of injury severity of SCI model and combining different detecting method, we have explored the apoptotic pathway in motor cortex neurons of rat with SCI, dynamic expression changes of p-tau protein and the apoptosis related protein. After inhibitting tau protein phosphation by application of LiCl, we investigate whether LiCl could decrease the apoptosis in motor cortex and promote the movement function. We tried to illustrate mechanism on the apoptosis of neurons in motor cortex after SCI and provide a new theoretical and experimental basis for the treatment of SCI.MethodsThis study includes two parts. In first part, one hundred and twenty aldult female SD rats were randomly divided into four groups:Sham group, mild SCI group moderate and severe SCI. For rats in Sham group, we conducted the process of removal T8 of vertebra and exposed ecal sac, not injued the spinal cord; For the rats in SCI groups, they received a contusion at T8 using NYU-MASCIS impactor with a 10 g of metal rod dropping from impact heights of 12.5mm(Mil SCI),25mm (Mod SCI) or 50mm (Sev SCI). We evaluated the motor function of all rats at Id,3d,7d, 14d and 28d according to BBB sore scale. And then the rats were sacrificed via intraperitoneal injection of overdoses 10% chloralhydrate, we harvested the spinal cord and cerebral motor cortex (M1) tissue according to the experimental requirements and stored them at different conditions. Main detecing method and index:(1)Hematoxylin-eosin(HE) staining for the injuried site of spinal cord was used to the observation of structure damage in different grade of injury severity; TUNEL staining for motor cortex was to the observation of apoptosis in motor cortex and culation apoptosis index (AI) by counting the TUNEL labeling positive of nucleus. (2). The expression of cleaved caspase-3, AIF and p-tau protein in motor cortex of rats was detected by western blot at each time after SCI; then we use the software of "image pro plus 6.0" to analyze the relative content of each proteins and compare the differences among groups. (3) Immunofluorescence staining method was used to label the AIF and p-tau colocalizational neurons, then we calculate the double labeling positive neurons and compare the differences among groups. (4) We explored the relationship between each indications by correlation analysis according Pearson or Spearman’s method. In the second part, thirty SD male rats with the average weight of about 230g were randomly divided into Sham group, SCI+LiCl group and SCI+NaCl group. Rats in Sham group model only received T8 laminectomy removal, didn’t damage the spinal cord. After T8 laminectomy removal, rats in SCI groups were fixed appropriately in the NYU-MASICS, then they were received injury at T8 with a 10 g of metal rod dropping from impact heights of 25mm. SCI+LiCl group immediate received a dose of 20mg/kg.b.w LiCl solution via intraperitoneal injection and SCI+NaCl group received the same volume of normal saline, then they were received the same dose for three consecutive days after SCI. Outcome measures and methods:(1) At 1W after injury,we sacrificed 18 rats from each groups and collected the motor cortex tissue for sequentially experimenal test. TUNEL staining was applied to the observation of apoptosis in the motor cortex and western blot was adopted for the detection of expression of p-tau and AIF. (2) BBB sore evaluation was conducted at Id,3d,7d,14d and 28d for the assessment of motor function.ResultsThe first part of our study showed:(1) HE staining of spinal cord tissue showed different grade of injury severity resulted in different degrees of destruction at 1d after SCI, which suggested successfully graded SCI model had been produced. (2) Apoptosis could be detected in the cerebral cortex in the early stage after SCI, and the AI reached the peak at 7 day after SCI. Severe injury of SCI leads to a higher AI and significant differences were determined among groups(P<0.05). The AI of motor cortex at 7d was negative correlated to the BBB score at 28d after SCI (r=0.873,P<0.05). Decreased from 7d, AI still remained at a high level. (3)Western blot showed that the expression p-tau and AIF in motor cortex changes with an spatio-temporal relationship after SCI. The expression of Caspase-3 showed no significant change in all four groups at each time point(P>0.05); AIF and p-tau in mortor cortex of rats in sham group also showed no significant changes by western blot at each time point after sugery. However,p-tau and AIF were significanlty increased at the early stage after SCI and reach the peak at 7d. The expression of p-tau and AIF still remained at a high level while decreased from 7d to the end of our study. We found that p-tau and AIF were significantly higher when compared to other groups at Id,3d and 7d. We also found that the expression of AIF were positively correlated with p-tau (r=0.827,P<0.01). Double immunofluorescence labeling of p-tau and AIF also showed the relationship of AIF and p-tau colocalized to the same neurons. As for the count of AIF and p-tau colocalized neurons, results showed the 7 day’s count was the maximum value.In the second of our study, results showed:(1)Supressing tau protein phosphorylation by the application of LiCl significantly reduced the apoptosis in motor cortex in the early stage after SCI, as showed by western blot in motor cortex that the expression of p-tau and AIF was significantly dercreased in SCI+LiCl group when compared to SCI+NaCl group (P<0.05). We also found a lower of AI in SCI+LiCl group in comparison with SCI+NaCl group (P<0.05). (2) Supressing tau protein phosphorylation can promote the motor function of rats with SCI.BBB score in SCI+LiCl group were higher than SCI+NaCl group, and showed a significant difference at 3W and 4W(P<0.05).Conclusion1. Apoptosis in motor cortex could be induced at the early stage after traumatic SCI and the apoptosis index was positively related to the severity of spinal cord injury. Severely injuried of SCI caused more apoptosis of motor cortex neurons after spinal cord injury and apoptosis may be the main cause of brain cortex atrophy after SCI.2. The process of apoptosis in the motor cortex after SCI may be the caspase independent pathway mediated by AIR Hyperphosphorylation of tau protein in motor cortex followed SCI may play an important role in the activation of the AIR3. Supress the hyperphosphorylation of tau protein can reduce the apoptosis in motor cortex of rats with SCI, and also can promote the recovery of motor function. Thus, take measures to precisely reduce phosphorylation of tau protein in motor cortex following SCI may become a new target for the treatment of SCI.However,we still need further study on the mechanism of hyperphosphorylation of tau protein in motor cortex activating the apoptotic pathway after SCI. Thus, SCI patients could recieve the accurate and effective therapeutic interventions to maximize recovery their function. |
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