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Objective To investigate the causes for death in rats after spinal cord injury.Methods A total of 120 adult Wister rats were selected for the study. The animal model with acute spinal injury at T10 was established by using Allen' s combat (25 g · cm). The dissection analysis was performed in death rats. Results Of all, 25 patients died, with mortality rate of 21%. Of death rats, five rats were died before awakening, with no abnormal anatomy; 12 rats died within three days after injury and three died of injuries 3-7days injury. Anatomy found pulmonary bleeding and edema, even hematocele bladder in some rats. There were three rats died within 1-2 weeks, one died of injury only after 2-3 weeks, with lung infection and urinary tract infection. There was no death after three weeks. Conclusions The early causes for death of rats with spinal cord injury is mainly due to lung congestion and pulmonary edema, whereas the leading cause of late death of rats is pulmonary and urinary tract infection.
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Objective To observe biological effect of cardiotrophin-1(Adv-CT1)gene transfection mediated by adnovims on traumatic brain iniuries(TBI)in-vivo and discuss the role and mechanism of Adv-CT1 on TBI. Metheds A rat TBI model was established bv Allen method.After Adv-CT1 was transfefred into the iniured brain by adnovims,the effect of CT-1 on apoptosis and survival of neurons after TBI was determined by means of Nissl staining,TUNEL and flow cytometry apoptosis assay. Resuits Apoptotic cells were increased but the survived cells decreased in the injured cortical brain and hippocampus from 12 hours to 14 days after TBI in the control group.As compared with control group,Adv-CT1 treatment reversed this situation to some degrees. Conclusion CT-1 has neuropmtective effect on neurons after TBI by reducing apoptosis of neurons.
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Objective To probe the mechanism of anticonvulsant by melatonin from the angle of neurotransmitter.Methods Rat status epilepticus(SE) model was induced by pilocarpine(PILO).?-aminobutyric acid(GABA) content and glutamin acid decarboxylase(GAD)67 mRNA expression was detected at 6,48,72 h,and 7 d in the hippocampus of post-SE rats.The effect of melatonin on these changes was observed.Results GABA content was significantly lower in the hippocampus than in control(P
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Objective To construct human cardiotrophin 1(huCT 1) adenovirus vector for central nervous system(CNS) gene therapy in vivo . Methods The huCT 1 and eGFP genes were cloned into shuttle plasmid pDC316 to construct pDC316 huCT 1 and pDC316 eGFP. Virus genome plasmids pBHGloxdeltaE1,3Cre and pHG140 were purified by CsCl banding certification. Recombinant replication defective adenovirus vectors AdCMV huCT1 and AdCMV eGFP were rescued in 293 packaging cells by co transfection and Cre mediated recombination of both plasmids pDC316 huCT1 and pBHGloxdelta1,3Cre. The insert gene and its expression were identified by PCR, RT PCR and immunohistochemistry after recombinant adenovirus transfected 293 cells and NIH 3T3 cells. Recombinant adenovirus vectors were purified by CsCl banding and titrated by plaque forming test. AdCMV huCT1 expression in vivo was analyzed by RT PCR and immunohistochemistry after transfection of the cervical spinal cord in adult rats. Results We have constructed two recombinant adenoviral vectors: AdCMV huCT1 and AdCMV eGFP, containing MCMV promoter, foreign DNA and SV40 PolyA with deletions of E1 and E3 regions. The positive huCT 1 mRNA and protein were identified in AdCMV huCT1 transfected NIH 3T3 cells and rat cervical spinal cord. The titer of virus stocks was generally up to 3.0?10 10 plaque forming units(pfu) per milliliter. Conclusion Recombinant purified AdCMV huCT1 vectors can be highly expressed in vitro and in vivo and is suitable for CNS gene therapy in vivo .
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Objective To express recombinant human cardiotrophin 1 (huCT 1) protein with biological activity in E coli Methods huCT 1 open reading frame gene from plasmid pBSSK huCT1 was cloned into plasmid pMD18 T by PCR and T A cloning, and then cloned into prokaryotic GST fusion expression vector pGEX 2T to give pGEX2T huCT1 After pGEX2T huCT1 expression was induced by IPTG in E coli at different temperature and different time, the expression level and the proportion of the soluble protein were analyzed by SDA PAGE Then the soluble GST/huCT1 was purified by immobilized glutathione columns The GST fusion protein was cleaved by thrombin and purified again The recombinant huCT 1 with biological activity was identified according to the survival of axotomized sciatic motoneurons Results After the induction of pGEX2T huCT1 DH5? cells by IPTG at 29 ℃ for 4 h, the highest expression level of the recombinant protein was about 1/5 of total cell proteins, and the soluble portion was about 2/5 of fusion protein Purification of the soluble portion and thrombin cleaved fusion protein resulted in 85% and 80% purified recombinant GST fusion protein and huCT 1 protein, respectively Recombinant huCT 1 protected 55% motoneurons in spinal cord against sciatic axotomy in vivo in adult rats Conclusion Recombinant huCT 1 has biological activity in rat neurons
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Objective To compare the gene expression changes in astrocytes before and after injury. Methods The total RNA from the scratched spinal cord astrocytes two days after injury (injury group) and from the unscratched astrocytes (control group) were extracted for construction of cDNA difference library by means of Switching Mechanism At 5’ end of the RNA Transcript (SMART) and suppression subtractive hybridization (SSH) techniques. The change of the gene expression was identified in astrocytes two days after trauma and the sequences of different expressed segments were analyzed for searching their homology in NCBI BLAST database so as to know the whole change trend of the genes two days after scratch injury of rat spinal cord astrocytes. Results At day 2 after scratch injury of spinal cord astrocytes in newborn rats, expressions of 17 genes including heat shock protein 70, acidic fibroblast growth factor and calmodulin II were up-regulated but those of genes such as ?-actin down-regulated. Conclusion The gene expression is either up-regulated in proteins like heat shock protein 70 or down-regulated in proteins like ?-actin in the reactive astrocytosis after scratch injury of spinal cord astrocytes in rats.
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<p><b>OBJECTIVE</b>To explore the preparative method and study the degradation characteristics of bone repair composite of DL-polylactic acid (PDLLA)/hydroxyapatite(HA)/decalcifying bone matrix (DBM) in vitro.</p><p><b>METHODS</b>An emulsion blend method was developed to prepare the composite of PDLLA/HA/DBM in weight ratio of PDLLA:HA:DBM = 1.5-2:1-1.5:1. The dynamic changes of weight, biomechanical property and pH value of PDLLA/HA/DBM and PDLLA in phosphate buffered saline (PBS, pH 7.4) were studied respectively through degradation tests in vitro.</p><p><b>RESULTS</b>Without being heated, PDLLA, HA and DBM could be synthesized with the emulsion blend method as bone composite of PDLLA/HA/DBM, which had both osteoconductive and osteoinductive effects. The diameter of the aperture was 100-400 microm and the gap rate was 71.3%. During degradation, the pH value of PDLLA solution decreased lightly within 2 weeks, but decreased obviously at the end of 4 weeks and the value was 4.0. While the pH value of PDLLA/HA/DBM kept quite steady and was 6.4 at the end of 12 weeks. The weight of PDLLA changed little within 4 weeks, then changed obviously and was 50% of its initial weight at the end of 12 weeks. While the weight of PDLLA/HA/DBM changed little within 5 weeks, then changed obviously and was 60% of the initial weight at the end of 12 weeks. The initial biomechanical strength of PDLLA was 1.33 MPa, decreased little within 3 weeks, then changed obviously and kept at 0.11 MPa at the end of 12 weeks. The initial biomechanical strength of PDLLA/HA/DBM was 1.7 MPa, decreased little within 4 weeks, then changed obviously and kept at 0.21 MPa at the end of 12 weeks.</p><p><b>CONCLUSIONS</b>The emulsion blend method is a new method to prepare bone repair materials. As a new bone repair material, PDLLA/HA/DBM is more suitable for regeneration and cell implantation, and the environment during its degradation is advantageous to the growth of bone cells.</p>
Subject(s)
Biocompatible Materials , Biopolymers , Bone Density , Physiology , Bone Matrix , Metabolism , Bone Substitutes , Chemistry , Pharmacology , Bone and Bones , Physiology , Durapatite , Chemistry , Pharmacology , Fracture Healing , Physiology , Fractures, Bone , General Surgery , In Vitro Techniques , Lactic Acid , Chemistry , Pharmacology , Materials Testing , Osseointegration , Physiology , Polyesters , Polymers , Chemistry , Pharmacology , Sensitivity and Specificity , Tensile StrengthABSTRACT
<p><b>OBJECTIVE</b>To study the changes of excitatory amino acids (EAAs) and intracellular calcium ([Ca2+]i), and the protective effect of EAAs receptor antagonists in the tissues of rabbit lumbar spinal cord after 40-minues ischemia and 4-hours reperfusion.</p><p><b>METHODS</b>Thirty healthy rabbits were divided into six groups: sham-operation, 40-minues ischemia, 4-hour reperfusion, ketamine and MgSO4 treatment, ketamine treatment, and saline treatment groups. The contents of EAAs (glutamate and aspartate) and [Ca2+]i were measured.</p><p><b>RESULTS</b>The contents of glutamate and aspartate were decreased to 15.18 micromol/g+/-2.33 micromol/g and 9.99 micromol/g+/-0.69 micromol/g, respectively; 13.75 micromol/g+/-2.58 micromol/g and 6.49 micromol/g+/-1.39 micromol/g after reperfusion. In the ischemia group, the [Ca2+]i was elevated to 221.2 microg/g+/-4.27 microg/g, and elevated further to 298.3 microg/g+/-9.26 microg/g after reperfusion, being significantly higher than that of ischemia and control groups. Ketamine could obviously increase the level of glutamate and aspartate and decrease the level of [Ca2+]i during the ischemia and reperfusion injury.</p><p><b>CONCLUSIONS</b>The excitotoxicity of EAAs and the overload of calcium induced by EAAs play a harmful role in ischemia and reperfusion injury. Ketamine has an effective inhibitory effect.</p>
Subject(s)
Animals , Female , Male , Rabbits , Calcium , Excitatory Amino Acids , Blood , Ischemia , Blood , Random Allocation , Receptors, Amino Acid , Reperfusion Injury , Blood , Spinal CordABSTRACT
<p><b>OBJECTIVE</b>To investigate the apoptosis rules of the astrocytes and oligodendrocytes induced by Ca(2+) reperfusion.</p><p><b>METHODS</b>The apoptosis of purified cultured astrocytes and oligodendrocytes induced by Ca(2+) reperfusion and the relationship between the development of the cell apoptosis and post-reperfusion time was observed.</p><p><b>RESULTS</b>Both the astrocytes and oligodendrocytes were obviously in a time-dependent fashion, and the apoptosis ratios of the oligodendrocytes (39.73%+/-4.16%) were higher than the astrocytes (19.64%+/-4.67%) 24 hours after Ca(2+) reperfusion. The TUNEL positive cells were 13.6+/-1.82 and 21.4+/-1.95 at every visual field of astrocytes and oligodendrocytes respectively 24 hours after Ca(2+) reperfusion.</p><p><b>CONCLUSIONS</b>The astrocytes and oligodendrocytes are similar wi th the development rules on apoptosis and have different susceptiveness to the situation.</p>
Subject(s)
Animals , Rats , Apoptosis , Physiology , Astrocytes , Cell Biology , Pathology , Physiology , Calcium , Physiology , Cells, Cultured , Flow Cytometry , In Situ Nick-End Labeling , Oligodendroglia , Cell Biology , Pathology , Physiology , Rats, WistarABSTRACT
Objective To probe the changes of nitric oxide (NO) content and caspase-3 mRNA expression in hippocampus and the correlation between NO and caspase-3 mRNA expression post-status epilepticus (SE). Methods The models of SE were induced by pilocarpine. The nitric oxide content was detected by chromatometry and caspase-3 mRNA expression was observed by reverse transcription polymerase chain reaction (RT-PCR) method. Results The level of NO increased rapidly at 6~72 h post-SE,and was still significantly higher than that of the controls (all (P)0.05).Conclusions The peak expression of nitric oxide is earlier than caspase-3 mRNA post-SE, although they have the similar diversity tendency. Nitric oxide pathway is definitely involved in the seizure initiation and correlated with caspase-3 activation.