| Background:Cardiac arrest is one of common emergency in emergency medicine. The incidence of cardiac arrest could reach 50-55/10 million per annum in America and Canada emergency medical system, among those people, almost 86 percent of patients had accepted cardiopulmonary resuscitation. The sign of initial assessment of successful resuscitation is restoration of spontaneous circulation(CPR). There were 25 to 50 percent of patient who needing CPR can return to spontaneous circulation. however, only 2 to 10 percent of patients can recover nerve function. As the brain cells is particularly vulnerable under the condition of hypoxia and global brain is ischemia after cardiac arrest, it can occur unrecoverable neurologic impairment and reperfusion injury followed ROSC after circulation terminate for 4-6 minutes. We try and find out the way or medicine to improve the quality of brain resuscitation, and achieve a certain degree, but we still lack of right treatment plan to cerebral resuscitation. Therefore, the exploration of brain resuscitation is significant, bone marrow mescenchymal stem cells(BMSCs) has been hot area in recent years, which can open up broad prospect for brain function after CPR.Objective: To investigate the improvement of bone marrow mesenchymal stem cells on ischemic hypoxic injury of global brain after we transplant the BMSCs via tail vein injection, and prove the IGF-1 secreted by BMSCs acts as a main benefited mechanism on hippocampus.Methods:1. The study about BMSCs’ transplantation improves the brain function via tail vein injection.(1) BMSCs’ abstraction in vitro, cultivation, identification.The research choosed SPF SD rats between the body weight of 100 and 110 g,whose bone marrow of long bone was selected as donor. We separated and cultivated BMSCs by adherence method. The 3rd generation cells were used for further testing or transplantation. BMSCs were measured by flow cytometry analysis of CD 29, CD 90, CD45, CD11 b. Before BMSCs intravenous transplantation, we marked BMSCs by green fluorescent protein(GFP) and observed the distribution of BMSCs in hippocampus by frozen section.(2) The rat model of global brain ischemia after cardiac arrest induced by asphyxia.Thirty adult male Sprague-Dawley rats were divided into three groups randomly:sham operation group, cardiac arrest group and BMSCs treatment group, 10 rats in each group. The model of cardiac arrest was induced by asphyxia. One hour after ROSC, BMSCs were transplanted by injecting into the vein of the tails. The total number of the cells was approximately 1×106. The 3rd day and the 7th day were selected as the time point to be observed, each time point had 5 rats.(3) The measurement of brain water content and pathological examination of HE staining.These rats were sacrificed at day 3 and day 7 after intervention, and we also made frozen sections in hippocampal and performed hematoxylin-eosin staining. The rest of brain tissue was weighed by electronic balance, then dried to constant weight by putting it into drying oven remained 95℃ for 72 hours, after that we weighed it again. We expressed it as a percentage: brain water content(%)=( wet weight- dry weight) / wet weight×100%.2. The potential efficacy of transplanted BMSCs in treating the neurological outcome and prognosis of rat after CPR.(1) The experimental group.Adult male Sprague-Dawley rats were selected and divided into three group randomly: sham operation group(n=15), cardiac arrest group(n=15) and BMSCs treatment group(n=18), we made the same model induced by asphyxia. The 1st day, the 3rd day and the 7th day were chosen as the time point to be observed after ROSC. There were 6 rats on each time point in BMSCs treatment group with 5 rats on each time point in the other group.(2) The score of behavioral functions and the measurement of expression of IGF-1 by Real-time quantitative PCR and immunohistochemical staining.After the animal model was established, the rats’ neurological status was assessed by modified neurological severity score(m NSS) tests on the time point, both Real-time quantitative PCR and immunohistochemical staining were used to detect the expression of IGF-1. Moreover, the double fluorescent labeling of GFP and IGF-1 was used to detect the expression of IGF-1 in BMSCs.(3) The serum level of S100BSerum level of S100 B was examined by Enzyme-linked immunosorbent assay( ELISA) after ROSC via drawing 5ml blood from inferior vena vein.Results:1. A small quantity of BMSCs anchored the test tubes after cultivating 24 hours, and then presented sparsely spindle-shape clusters, cells multiplicated significantly in 5-6 days, sticked to each other closely. At 7 days, 80-90% BMSCs were emerged, at the moment, the cells were subcultured for the first time. The cells were subcultured 4 days apart. The 3rd generation cells were tested by flow cytometry. The expressions of CD29, CD90 were 97.15%, 99.02%. The expressions of CD45, CD11 b were 0.36%, 1.45%. It confirmed that BMSCs had been successfully isolated, cultured and subcultured by attachment growth method, which can be used for further research. 2. The rats after cardiac arrest induced by asphyxia were done chest compressions, mechanical ventilation and injected the adrenaline to strengthen the heart. Which the mean arterial pressure over 60 mm Hg for 10 minutes proved the model is success. 3. BMSCs were observed in hippocampus after transplanted at 1st day, the 3rd day and the 7th day in fluorescent microscopy. 4. Compared with sham operation group and BMSCs treatment group, brain water content of cardiac arrest group was higher(P<0.05). HE dyeing results showed BMSCs transplantion could reduce hypoxia ischemia brain damage. BMSCs treatment could decrease significantly the number of neuronal death and degeneration in hippocampus, the extent lightened.5. Through the m NSS test, the m NSS score of cardiac arrest group was higher than sham operation group significantly after ROSC at 1st day, 3rd day and 7th day(p<0.05), the m NSS score of BMSCs treatment group was reduced significantly(p<0.05). Compared with sham operation group, the m NSS score of BMSCs treatment group was no statistical differences at 7th day(p>0.05). 6. Real-time quantitative PCR analysis confirmed IGF-1 m RNA relative amount of BMSCs treatment group was higher than that of sham operation group and cardiac arrest group at each time point significantly( p<0.05). The double fluorescent labeling results showed that the BMSCs expressed IGF-1 positive in hippocampus. Immunohistochemistry results demonstrated the hippocampus expressed IGF-1 positive,which were found in cell-transplanted group, there was a little expression in cardiac arrest group, both group were higher than that of sham operation group(p<0.05),but the expression in cardiac arrest group was no statistical difference with sham operation group at 7th day(p>0.05). 7. Serum level of S100 B of cell-transplanted group and cardiac arrest group was higher than sham operation group and cardiac arrest group significantly after ROSC at each time point(p<0.05). Cell-transplanted group decreased significantly after ROSC at 1st, 3rd, 7th day than cardiac arrest group at the content of S100B(p<0.05).Conclusion: 1. BMSCs separated and cultivated by adherence method in vitro were high purity, which can be used to transplant experiment. 2. BMSCs reduced the global brain damage inducing by cardiac arrest and promoted neurological function by settling in the damage area of hippocampus across the blood-brain-barrier after transplanted into the rat’ bodies. 3. We found BMSCs treatment could slow down the cell apoptosis and promote recovery of cerebral function by encouraging themselves secretion of IGF-1, which showed one of the neuroprotective mechanisms of BMSCs could be implemented by secreting IGF-1. |
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