Analysis of death cause in rats with spinal cord injury / 中华创伤杂志

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.

Protective effects of cardiotrophin-1 gene transfection mediated by adnovirns on traumatic brain injuries / 中华创伤杂志

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.

Expression and purification of human cardiotrophin-1 in E.coli and identification of its biological activity / 第三军医大学学报

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

Construction and purification of human cardiotrophin-1 adenovirus vector and its expression in vitro and in vivo / 第三军医大学学报

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 .

Construction of subtractive cDNA library and expression changes following scratch injury of rat spinal cord astrocytes / 中华创伤杂志

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.

Effect of melatonin on GAD67 mRNA expression in post-SE rat hippocampus induced by pilocarpine / 第三军医大学学报

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

Experimental study on the apoptosis of the astrocytes and the oligodendrocytes induced by Ca(2+) reperfusion / 中华创伤杂志(英文版)

<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>

Functional recovery of the spinal cord following ischemia and reperfusion injury / 中华创伤杂志(英文版)

<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>

Preparation and degradation characteristic study of bone repair composite of DL-polylactic acid/hydroxyapatite/decalcifying bone matrix / 中华创伤杂志(英文版)

<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>

Dynamic changes of nitric oxide content and caspase-3 mRNA expression in hippocampus in epileptic rats induced by pilocarpine / 临床神经病学杂志

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.

Effects of Melatonin on hippocampal neuron apoptosis and caspase-3 expression in epileptic rats / 临床神经病学杂志

Objective To observe the effects of Melatonin on hippocampal neuron apoptosis and caspase-3 expression in epileptic rats. Methods Using model of status epilepticus (SE) induced by Pilocarpine, the rats were administered with melatonin (melatonin group) or saline (Pilocarpine group) before Pilocarpine injection. The apoptotic neuron was detected by TdT-mediated dUTP Nick End Labeling (TUNEL) and caspase-3 expression was observed by immunocytochemistry method, respectively.Results In Pilocarpine group, the number of TUNEL positive neurons began to increase at 6 h post-SE, peaked at 72 h post-SE, and decreased at 7 d post-SE. Caspase-3 immunoreactivity began to increase at 6 h post-SE, peaked at 48 h post-SE, especially in CA1 and CA3 region of hippocampus, began to decrease at 72 h post-SE and dropped to normal level at 7 d post-SE. The numbers of TUNEL positive neurons and caspase-3 expression in Melatonin group were significantly decreased compared to Pilocarpine group at different time points (all P

Convergence of Visceral and Somatic Nociceptive Inputs onto Hippocampal Units in Cats / 第三军医大学学报

Ten cats lightly anesthetized and D-tubocurarine immobilized were employed.The responses of a same unit to the train stimuli of square wave applied to the saphenous nerve,the greater splanchic nerve,and the two nerves simultaneously were observed.Among the 80 units recorded on the 10 cats,43 went through all the experimental procedures,33 out of the 43 could be activated by splanchic inputs.Convergence of the splanchic and saphenous inputs occurred to 26(79％) out of the 33.These 26 units could respond to the noxious stimuli on the saphenous and greater splanchic nerves in turn or simultaneously.Furthermore,they could be divided into 3 types;15 units showed similar pattern and duration of discharge only when the greater splanchic nerve was stimulated,6 showed similar discharge only when the saphenous nerve was stimulated,and 5 showed smaller responses when either of the two nerves was stimulated.When the interval between stimulations was shortened to 5 seconds,37(86％) out of the 43 units showed a decrease in discharge,which was apparently an after-inhibitory effect.The results indicate that the inputs from visceral and somatic C or A-delta afferent fibers can converge onto the units of the dorsal hippocampal CA1 area,and evoke blockage and inhibition of these units.

The modulation of met-enkephalin and naloxone on the excitatory effects of kainic acid and glutamic acid in hippocampus of rats / 第三军医大学学报

Objective To observe the modulation of met enkephalin (ME) and naloxone (NLX) on the excitatory effects of kainic acid (KA) and glutamic acid (GLU) in hippocampus of rats and to explore the possible mechanism of the effects of ME in the KA epilepsy model. Methods A total of 45 healthy Wistar rats were anesthetized with 1% sodium pentobarbital (3 ml/kg intraperitoneal injection). After endotracheal intubation, the animal was mounted onto a SN 1stereotaxic apparatus (Narishige) and routine craniotomy was performed. Then the animal was immobilized by 0.01% curare (1 mg/kg) after it woke up. The peripheral pipettes of a multiple pipette microelectrode were used for microelectrophoretic application of drugs; the central pipette was used for extracellular recording of the hippocampal unit discharges (HUDs). Results ①ME excited most of the HUDs; opioid receptor antagonist NLX could reverse the excitatory effects of met enkephalin. NLX applied alone had no effects on the firing rate of HUDs. ②KA and GLU excited the HUDs intensively but ME and NLX could modulate their excitatory effects. Conclusion ME may promote the development of epilepsy by modulating the excitatory effects of KA and GLU in hippocampus.

Effects of electroacupuncture on hippocampal epileptic burst discharges induced by intracerebroventricular injection of kainic acid in the rat / 第三军医大学学报

Epilepsy induced by intracerebroventricular injection of kainic acid in the rat could be attenuated by electroacupuncture(EA)at the "Du Mo point" since EA resulted in a reduction of the frequency and amplitude of the hippocampal EEG especially in the 1st min after EA.No unsynchronization of the hippocampal EEG occurred before or after EA.The hippocampal unit discharges after intracerebroventricular injection of kainic acid were classified into positive units,negative units and indifferent units,The positive units were inhibited by EA while the negative units,on the contrary,were activated.The positive units(21%)and the indifferent units(33%)were converted into negative units by EA.At the same time,EA reduced the NSEB value and IBSF value of hippocampal epileptic burst discharges.On the basis of these findings,it is believed that the inhibition of the amplitude of hippocampal epileptic EEG by EA plays an important role of the antiepileptic action of EA,hippocampal negative units are one of the factors of antiepileptic action,and EA can regulate the intrinsic characteristics of epileptic neurones.

The effects of indomethacin on thromboxane A2/Prostacyclin ratio after spinal cord injury in rats / 第三军医大学学报

The changes of the spinal cord blood flow(SCBF),the content of TXB2 and 6-keto-PGF1a of the injured segment of the cord and other neurological manifestations were observed on a rat model of spinal cord injury(SCI)established with Allen's weight drop(50g-cm)method and the effects of indomethacin on these changes were studied.It was found that SCBF was significantly reduced in the first 2 hours after SCI and further reduced in the 4th ~ 8th hour.Increase of TXB2 was observed in the 1st hour and reached the peak in the 4th hour.The level of 6-keto-PGF1a was also increased in the 1st hour and maintained at that level for 24 hours.The changes of TXB2/6-keto-PGF1a was similar to those of TXB2.There was a negative correlation of SCHF with TXB2 content and TXB2/6-keto-PGF1a ratio.The intravenous injection of 10mg/kg indomethacin could inhibit the increase of TXB2 content and increase 6-keto-PGF1a content relatively.It could also alleviate or retard the decrease of SCBF after SCI and improve the motar function of the hind limbs of the rats.These findings suggest that indomethacin can improve SCBF and promote the recovery of neurological functions through its regulatory effects on the levels of TXB2 and 6-keto-PGF1a