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Objective:To evaluate the effect of propofol anesthesia on autophagy in hippocampal neurons of newborn rats.Methods:Thirty-nine healthy Sprague-Dawley rats, aged 7 days, weighing 10-12 g, were divided into 3 groups ( n=13 each) using a random number table method: control group (group C), fat emulsion group (group F) and propofol group (group P). Normal saline 8 ml/kg was intraperitoneally injected for 5 consecutive days in group C. Medium-/long-chain fatty emulsion injection 8 ml/kg was intraperitoneally injected for 5 consecutive days in group F. Medium-/long-chain propofol injection 80 mg/kg was intraperitoneally injected for 5 consecutive days in group P. Five rats were sacrificed on 1st day after the end of propofol anesthesia, and hippocampal tissues were taken for determination of the expression of microtubule-associated protein 1 light chain 3B (LC3B) and Beclin-1 (by Western blot). The remaining rats in each group underwent the Morris water maze test on 19th day after the end of propofol anesthesia (30 days after birth), and the escape latency, percentage of time of staying at the target quadrant and the number of crossing the original platform were recorded. Results:Compared with group C, no significant change was found in the expression of hippocampal LC3B and Beclin-1, escape latency, percentage of time of staying at the target quadrant, and the number of crossing the original platform in group F ( P>0.05), and the expression of hippocampal LC3B and Beclin-1 was significantly up-regulated, the escape latency was prolonged, percentage of time of staying at the target quadrant was decreased, and the number of crossing the original platform was decreased in group P ( P<0.05 or 0.01). Conclusion:The mechanism by which propofol anesthesia causes long-term cognitive dysfunction may be related to promoting autophagy in hippocampal neurons of newborn rats.
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Objective:To evaluate the effects of exogenous biliverdin (BV) on the expression of Litaf in PC12 cells subjected to oxygen-glucose deprivation and restoration (OGD/R).Methods:PC12 cells were seeded in a 96-well cell culture plate at a density of 1×10 4 cells/well for 3 days and were divided into 3 groups ( n=18 each) by a random number table method: control group (group C), OGD/R group, and biliverdin group (BV group). Group C was incubated in a 37 ℃ incubator (95% air+ 5%CO 2) for 6 h. To establish the OGD/R model, cells were incubated with sugar-free medium in a 37 ℃ incubator (95% air+ 5%CO 2) for 2 h, and the medium was then replaced with normal medium and cells were continuously incubated in a 37 ℃ incubator (95% N 2+ 5% CO 2). In BV group, 2 μg/ml biliverdin was added immediately after oxygen-glucose restoration.Cells in 6 wells in each group were selected at 6 h of restoration for determination of the expression of Litaf protein and mRNA (by real-time polymerase chain reaction) and tumor necrosis factor-alpha (TNF-α) concentration (by enzyme-linked immunosorbent assay). Results:Compared with group C, the expression of Litaf protein and mRNA was significantly up-regulated, and TNF-α concentration in supernatant was increased in group OGD/R ( P<0.05). Compared with group OGD/R, the expression of Litaf protein and mRNA was significantly down-regulated, and TNF-α concentration in supernatant was decreased in group BV ( P<0.05). Conclusion:The mechanism by which exogenous biliverdin reduces OGD/R damage to PC12 cells is related to inhibiting up-regulated expression of Litaf and alleviating the inflammatory responses.
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Objective To observe the effects of different modes treadmill training on cognitive func-tion and transforming growth factor β1 ( TGF-β1 ) expression in cerebral cortex of rats. Methods Two months old rats were divided into the control group,piecewise training group and intermittent training group ( n=10 in each group) . The training was performed five times a week for 6 weeks. Learning and memory a-bility of all rats was detected by water maze at 6 weeks after the training. TGF-β1 expression and localization in cerebral cortex was tested by QRT-PCR and immunofluorescence, respectively. Results The platform time in piecewise group ((30±28) s) and intermittent group ((25±23)s) was both significantly shorter than that in control group ((58±50)s). In the space exploration,the time around Ⅳ quadrant platform in piecewise group((23.6±3.9)s) and intermittent group ((24.3±8.9)s) was significantly higher than that in the control group((17.7±2.0)s). The expression of TGF-β1 mRNA in cerebral cortex in intermittent group (0.0067±0.0043)was obviously higher than that in piecewise group (0.0035±0.0006) and control group (0.0041±0.001). TGF-β1 was located in cell membrane and cytoplasm,and the relative optical density of intermittent group (0.0045±0.0017) was significantly higher than that of control group (0.0019±0.0004) and staging group (0.00175±0.00045). Conclusion (1)Learning and memory function both were im-proved after treadmill six weeks with piecewise and intermittent training models. ( 2) The level of TGF-β1 gene and protein was significantly increased after interval training in cortex of rats.
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<p><b>OBJECTIVE</b>To study the effects of a Gold Belt (GB, a traditional Chinese herbal medicine) combined with methyl-prednisolone (MP) on the motor function and brain-derived neurotrophic factor (BDNF) expression in rats with contusive spinal cord injury (SCI).</p><p><b>METHODS</b>Thirty adult female SD rats were randomly divided into 5 equal groups, namely the sham-operated group, SCI group, SCI with MP treatment group (MP group, with intramuscular injection of 50 mg/kg MP within 8 hours after SCI and then dosage reduced 10 mg/kg daily), SCI with GB treatment group (GB group, with intragastric gavage of GB 50 mg/kg once daily for 7 days), and combined GB and MP treatment group. The Basso, Beattie and Bresnahan (BBB) locomotor scale was used to evaluate the hindlimb motor function of the rats on days 1, 3, 7, 14, 21 and 28 after the injury. After the last evaluation the rats were sacrificed for immunohistochemistry to observe the localization of BDNF in the ventral and dorsal horn of spinal cord.</p><p><b>RESULTS</b>BDNF were distributed mainly in neurons in the spinal cord grey matter ventral horn and dorsal horn of the rats. The number of BDNF-positive neurons and BBB scores in the combined treatment group were significantly higher than those in the other 4 groups (P<0.05).</p><p><b>CONCLUSION</b>GB combined with MP produces better therapeutic effects for treating SCI than GB or MP used alone, and such effects are probably related with enhanced BDNF expression in the spinal cord.</p>
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Animals , Female , Rats , Brain-Derived Neurotrophic Factor , Metabolism , Drugs, Chinese Herbal , Pharmacology , Immunohistochemistry , Methylprednisolone , Pharmacology , Neurons , Metabolism , Rats, Sprague-Dawley , Spinal Cord Injuries , Drug Therapy , MetabolismABSTRACT
BACKGROUND:There are several routes for stem cel transplantation;however, it is stil unable to determine which one is the best. As for the different individuals with brain injury, the type of transplanted cel s, transplantation route and time wil affect the therapeutic effects. OBJECTIVE:To investigate the effect of bone marrow mononuclear cel s transplanted via different approaches on neurological function of rats with traumatic brain injury. METHODS:Bone marrow mononuclear cel s of rats were administered gradient centrifugation with Ficol lymphocyte separation medium, and were labeled with CFDA-SE in vitro as standby. Rat models of traumatic brain injury were established by the method of freefal . After successful establishment of rat models, bone marrow mononuclear cel s labeled with CFDA-SE were immediately transplanted into rats via injured area, lateral ventricle and internal carotid artery. One control group was designated for each transplantation route (bone marrow mononuclear cel s were replaced with the same volume of DMEM). The degree of neurological deficits was evaluated using mNSS scores at different time points after treatment. The brain tissue was harvested after the last neurobehavioral evaluation. The survival and migration of bone marrow mononuclear cel s in the injured area were observed under an inverted fluorescent microscope. RESULTS AND CONCLUSION:At 7, 10, and 14 days after treatment, the mNSS scores of rats in al groups were al lower than those at 1 and 3 days (P<0.05). At 7 and 10 days, the mNSS scores of rats in the internal carotid artery transplantation group were significantly lower than those in the control group (P<0.05). At 14 days after treatment, the number of fluorescence-labeled cel s of rats in the internal carotid artery transplantation group was greater than that in the other groups (P<0.05) and these labeled cel s were widely distributed. The results demonstrate that the neurological function of rats can be improved by transplanting bone marrow mononuclear cel s via the internal carotid artery, and a large number of transplanted cel s can survive and migrate in the injured area.
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ObjectiveTo explore the effect of swimming on motor behavior and expression of brain derived neurotrophic factor(BDNF) and trkB in adrenal body.MethodsSD adult rats were divided into normal cotrol group and swimming group ( n=10 in each group).All rats in swimming group were subjected 6 weeks swimming for 1h each day.Motor performances including swimming speed and distance were recorded and expression of BDNF and trkB in adrenal body was measured.ResultsThere was a significant increase in swimming speed ( ( 157 ± 60) m/min) and distance ( (283.36 ±49.50)m) in swimming group,compared with control group ( (283± 60) m/min,( 156.92 ± 29) m) (P ＜ 0.05 ).Simultaneously,expression of BDNF in adrenal body had been significantly unregulated (0.93 ± 0.09 vs 0.56 ± 0.19 ) (P ＜ 0.05 ),while expression of trkB kept to be not changed.ConclusionSwimming increases notor ability in rats,and the possible mechanism may be related the upregulation of BDNF in adrenal body.
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ObjectiveTo explore the effects of Jinyaodai on neurological behavior and Akt expression in the cortex of rats following spinal cord contusion injury.MethodsRats were randomly divided into control group,spinal cord contusion group and Jinyaodai group.The weight-drop device was employed to prepare the spinal cord injury(SCI) model.Jinyaodai was administrated every day by using a stomach tube.Rats were performed the BBB assessment,and the detection of Akt expression and count of Neun positive neurons in cortex following SCI.ResultsCompared with control group,deficit of motor function in hindlimbs was seen at 3 dpo following cord contusion,and partial functional recovery could be seen from 7 dpo to 1 m.Treatment of Jinyaodai greatly increased the BBB scores ( 14.1 ± 1.4 ) more than SCI group ( 7.8 ± 1.3 ) at 1 month (P ＜ 0.05 ) ; Simultaneously,compared with SCI rats,treatment of Jinyaodai significantly increased the expression of Akt (0.53 ± 0.05,0.68 ± 0.07,P ＜0.05 ) and the number of neurons ( 11 ± 2, 15 ± 1 ; P ＜ 0.05 ) in the lesion-induced cortex of rats.Conclusion Jinyaodai may play an essential roles in functional recovery after spinal cord injury,in which the underlying mechanism may be involved in the expression of Akt in cortex.
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ObjectiveTo study the effect of bone mesenchymal stem cells (BMSC) transplantation into traumatic brain injury(TBI) rats by the external carotid artery on neurological function and learning and memory.MethodsTen adult SD rats were randomly divided into TBI group ( n =5 ) and BMSC transplantation group ( n=5).Feeney free falling method was used to establish TBI models.The experimental rats were administrated with BMSC via external carotid artery (ECA),while TBI rats were injected with sterile liquid medium of equal volume via right ECA.Neurological function were evaluated according to the modified neurological severity score (NSS) at 1,3,7,15 days.Morris water maze test was used to observe the animal capabilities of place navigation and space exploration at 15 days,then animals were sacrificed.Survival and migration of implanted BMSC in brains under fluorescence microscope. ResultAfter traumatic brain,varying degrees convulsions,paralysis,loss of balance function in rats were found.Compared with TBI group,BMSC transplantation decreased significantly NSS (P ＜0.01 ).BMSC transplantation significantly decreased on escape latency ( ( 20.48 ± 2.29 ) s ) than the TBI group ( ( 85.93 ± 47.48 ) s) (P ＜ 0.01 ).Moreover,BMSC group in the target quadrant dwell time ( ( 28.62 ± 1.72) ％ )and distance ( (29.05 ± 3.08 )％ ) as well as the number of passing the platform (8.00 ± 2.45 ) were significantly higher than the TBI group ( ( 19.37 ± 2.81 ) ％,(21.78 ± 3.06) ％,(2.00 ± 1.87) respectively,P ＜ 0.01 ).Transplanted BMSC could survive and migrate around injury brain through Hochest mark immunofluorescence.ConclusionBMSC can survive and migrate around injury brain by transplantation of external carotid artery,which results in a significant neurological function improvement and learning and memory increase in rats with traumatic brain injury.
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ObjectiveTo explore the effects of hyperbaric oxygen on neurological behavior and vascular endothelial growth factor (VEGF) in rats with traumatic brain injury (TBI). MethodsThirty rats were randomly divided into three groups, ie, control group, TBI group ( a 50 g weight-drop device was employed and fell from 30 cm height to induce the injury) and hyperbaric oxygen group ( HBO group,treated with hyperbaric oxygen once per day for seven days after TBI), 10 rats per group. Neurological severity score (NSS) was used to evaluate the movement and balance impairment in all groups. Expression of VEGF was detected by means of immunocytochemical staining.ResultsAfter TBI, the rats presented different degrees of convulsions, paralysis and balance dysfunction. The NSS score was (5.6 ±1.1 ) points in the TBI group and (0.3 ± O. 1 ) in the control group, with statistical difference ( P ＜0.05). While NSS score was (3.7 ± O. 7) points in the HBO group, showing a significant decrease compared with that in the TBI group (P ＜ 0. O1 ). Immunohistochemical staining showed 15 ± 3 positive neurons of VEGF in the TBI group, significantly less than 27 ± 2 in the control group ( P ＜ 0.05 ). There were 21 ±2 positive neurons of VEGF in the HBO group, significantly less than 21 ±2 in the TBI group (P ＜0.05). Conclusion Hyperbaric oxygen may attenuate experimental traumatic brain injury by stimulating production of VEGF.
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Objective To observe olfactory ensheathing cells transplantation on brain injury recovery nerve function and to explore its mechanism. Methods After purification of the olfactory ensheathing cells cultured for NGFRp75 immunocytochemical identification and preparation of cell suspension for transplantation, some cells were pre-labeled for bservation of survival after transplantation. 48 adult SD rats were randomly divided into three groups:sham operation without injury group (A group),cerebral cortex motor area injury group (B group) , the same brain injury and the olfactory ensheathing cell transplantation group (C group). At postoperative day,3 d, 7 d,14 d the neurological severity score (NSS) of rats were assessed; 14 d after injury of brain tissues were taken for NeuN immunohistochemistry host the number of neurons change. The data were statistically analyzed using SPSS17.0 software. Results (1) Cultured olfactory ensheathing cells showed NCFRp75 positive,the positive rate was 90%. (2) 14 d after transplantation of nuclear fluorescence labeling of olfactory ensheathing cells survived well in the host body. (3) 14 d after NSS score of B group( 2.00 ± 0.53) and C group ( 1.25 ± 0.46) were significantly better than the B group (P<0.05). (4) NeuN positive cells in B group (39.2 ±7. 1) and C group(45, 8 ± 6.0) were significantly better than B group (P<0.05). Conclusions Olfactory ensheathing cell transplantation can promote the recovery of neurological function in rats brain injury,which may be related with olfactory ensheathing cells to promote neuron survival in the host.
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Objective To research the effects of neural stem cells (NSCs) transplantation on the neurological function improvement and neural survive and axonal regeneration in traumatic rat brain. Methods NSCs were cultured in vitro, labeled with hochest and transplanted into rat brain injured area by weight-dropping. Neurological severity scores(NSS) tested the functions at the 0,3,7,14 day post-injury. Immunofluorescence was used to detect the expressions of NeuN cells and GAP-43. Results There was significant difference in NSS between NSCs transplantion group and brain injury group(4.38 ±0.74 vs 5.50 ± 1.07, P＜0.01) at 7th day. There were a significant increase in neural number (51.46 ± 3. 303 vs 42.83 ± 5. 401, P ＜ 0.01 ) ), and GAP-positive axons ( 13.3 ± 1.7 vs 8.7 ± 1.1, P＜0.01 ) in NSCs transplantion group than in control group. Conclusion NSCs have an effects on the neurological improvement in brain injured rat. This may be associated to the increase in number of the neurons and local axons.
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Objective To observe the effect of neural stem cell (NSC)transplantation on the hindlimb motor function and caspase-3 expression of motor cortex (MC) in spinal cord transected (SCT) rats. Methods S prague-Dawley (SD) rats were randomly divided into sham operated group, operation group( T9 transection), and subacute NSC transplantation group. The MC of each group( n= 8 )was harvested 7 days post operation (dpo), then western blot was employed to detect the level of caspase-3( β-actin was used as control ). The left 5 animals of each group were subjected to BBB score evaluation at 16th week,then the animals were sacrificed and the MC was harvested and performed immunostain by using caspase-3 rabbit antibody. Results Following NSC transplanta tion, BBB scores( ( 7.58 ± 0. 99 ) ) increased significantly than seen in the SCT animals( ( 5.16 ± 1.19) ). The expressional level of caspase-3 at 7day post operation was( (0.89 + 0.12)) in MC of SCT rats,while it decreased significantly to( (0.76 + 0.11 ) ) in NSC transplantation rats(P＜0.05). The immunoreactive stain of caspase-3 was seen in the cytoplasm of pyramidal neuron in the cortex. Conclusion NSC engraft can downregulate the expression of caspase-3, corresponding to a significant improvement in hindlimb motor function. These findings indicate that NSC transplantation probably regulate the expression of apoptosis genes in MC to promote neurological function recovery in rats subjected SCT.
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Objective To explore the effect and injury mechanism of reactive oxygen species (ROS) after spinal cord injury (SCI) through detecting the dynamic changes of malonyldialdehyed (MDA)content in spinal cord and observing neurocyte apoptosis and correlation apoptosis factor expression after SCI. Methods Totally 132 adult SD male rats were randomly divided into three groups: sham group, SCI group, methylprednisolone (MPSS) group. The SCI of SD rats was performed by Allen's weight dropping way to impact on the posteriors of spinal cord T_(10). The contents of MDA were determined by chromatometry, the expression of Caspase-3 and Bcl-2 family in the injured spinal cord was detected by immunohistochemical staining;Apoptotic cells were detected by using fluorometric terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (fluorometric TUNEL) staining. Results The content of MDA in the injured cord increased significantly after SCI;R3eached the peak at 6 hours and 3 days post-injury, then dropped down gradually, then was back to the normal level after 7 days. The number of TUNEL labeling positive cells of SCI group increased at 6 hours post-injury;R3eached the peak at 3 days, then dropped down gradually;Bcl-2, Bax protein began to increase at 6 hours post-injury;R3eached the peak at 5 days after injury, then dropped down gradually. Caspase-3 protein began to increase at 6 hours post-injury;R3eached the peak at 3 days after injury, then dropped down gradually. The content of MDA, the number of TUNEL labeling positive cells, the expression of Caspase-3 and Bax of MPSS group decreased significantly than that of SCI group at the same time;R3espectively, while Bcl-2 protein was up-regulated after administration of MPSS.Conclusion ROS could promote the expression of Caspase-3 and degrade the ratio of Bcl-2/Bax to induce apoptosis of neurocyte, which might play significantly role in the process of secondary SCI. In addition, MPSS exerts neuroprotective effects against ROS toxicity, which might be of importance and might contribute to their clinical efficacy for the treatment of SCI.
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BACKGROUND: Many studies showed that neural stem cells (NSC) transplantation can promote functional improvements in rats subjected to spinal cord injury. However, the underlying molecular mechanisms remain poorly understood.OBJECTIVE: To observe the effects of NSC transplantation on expressions of Bax, Bcl-2 and Caspase-3 in the motor cortex in rats subjected to spinal cord transection.DESIGN, TIME AND SETTING: The randomized controlled animal experiment was performed at Institute of Neuroscience,Kunming Medical College from July 2007 to December 2008.MATERIALS: Five green fluorescent protein transgenic mice with 14-15 embryonic days were prepared for NSC. Additionally 88 adult female Sprague-Dawley (SD) rats were randomly divided into sham operation group (n=8), model group (n=40) and NSC transplantation group (n=40).METHODS: Model of spinal cord transection was established by cut transversely Sprague-Dawley (SD) rats T_9 segment. Rats in the sham operation group were subjected to laminectomy at T_8, without spinal cord injury. After the spinal cord was exposed at the lesion site, a small piece of 2 mm~3 gel foam soaked with 3×10~5 NSC were implanted into the gap to fill the lesion site of the T_9 level in rats of the NSC transplantation group. The measurements of relative indexes were performed at the days 3, 7,14, 21 and 28 after transplantation.MAIN OUTCOME MEASURES: Changes of Bax, Bcl-2 and Caspase-3 expressions were detected by RT-PCR.RESULTS: Compared to the sham operation group, the Bax expression in the model group had no significant difference (P >0.05), the expression of Bcl-2 was decreased obviously at days 14 and 28 after operation (P < 0.05), while a significant increase on the expression of Caspase-3 at day 3 (P < 0.05). Compared to the model group, the expression of Bax was dramatically decreased in the NSC transplantation group at day 3 (P < 0.05), with a notably increased Bcl-2 expression at days 14 and 21 after transplantation (P < 0.05), but the expression of Caspase-3 presented a significant decrease at days 3 and 7 after transplantation (P < 0.05).CONCLUSION: NSC transplantation probably regulates the expression of apoptosis genes (Bax, Bcl-2 and Caspase-3 mRNA) to promote neurological function recovery in rats subjected to cord transection.
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The present study investigated different types of eoexpression of brain derived neumtmphic factor(BDNF),nerve growth factor(NGF)and neutmphin-3(NT-3)mRNA and/or proteins in the left sixth lumbar dorsal root ganglion(DRG)of cats and discuss themechanism of coexpression in order to provide foundation for elucidating the relationship between the expression of neurotrophic factors andspinal cord plasticity.The eats used in this study were normal animals without any interventional treatment.They were subjected to renloveof the left L6 DRG and their DRG were processed for immunohistechemistry and in situ hybridization double staining to observe whetherthere are coexpression of mRNA and proteins of BDNF,NGF and NT-3.The results showed that the pmteios and mRNA of BDNF,NGFand NT-3 were all expressed in the DRG of cats,but the types of coexpression of mRNA and proteins were different and diverse amongthese three neumtrophic factors.The results of immunohiatochemistry showed that BDNF immunoreactivities were mainly observed in thecytoplasm and nucleus,and the staining of nucleus was weaker than that of cytoplasm;NGF immunoreactivities were mainly observed innucleus while NT-3 mainly in cytoplasm.The results of in situ hybridization showed that BDNF and NGF positive signals mostly distributedin the cytoplasm,NT-3 positive signals were observed in both the cytoplasm and nucleus.Our results suggest that the proteins and mRNAof BDNF,NGF and NT-3 have different types of coexpression which indicate they may have autocrine and/or paracrine mechanism contrib-uting to the plasticity of spinal cord in the left L6 DRG of cats.
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Objective To explore the engrafted effect of NSCs combined with OECs on the motor function repair of hindlimbs in rats subjected to spinal cord transected injury.Methods The NSCs and OECs were isolated from the Transgenic Green Fluorescent Protein(GFP) embryo mouse' hippocamp and neonatal mouse' olfactory bulb.Rats were divided into NSCs group,OECs group and co-transplantation group as well as control group.The recovery of posterior limb motor function in rats subjected to spinal cord transected injurywas evaluated by the BBB locomotion score respectively in each weekend among eight weeks.This was followed by a histochemiscal observation to know the engrafted cells survival and migration in the spinal cord of host under the fluorescence microscope.Results The NSCs and OECs cells survived and migrated in the spinal cord of transplantation groups:the motor functional improvement of hindlimb in rats was seen in all cell transplanted groups;NSCs group,OECs group and co-transplantation group demonstrated a significant increase than that of control group at 3 and 4 weeks post operation(P 0.05).Conclusions The engrafted NSCs,OECs and co-transplantation can improve partly the motor function recovery of hindlimb in rats at 3 to 4 weeks following cord transection;The OECs group and co-transplantation have better effects than that of NSCs group,but there is no obvious difference between the OECs group and co-transplantation group at observed time point in this investigation.
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Objective To explore the changes on BDNF mRNA in transected spinal cord and associated motor cortex and skeleton muscle following cord injury in rats.Methods 20 adult Sprague Dawleys rats were performed spinal cord transected operation at T11 level,then rats in each group(n=5) were sacrificed on 1,3,7 and 14 days post operation respectively.Other 5 rats were used as normal control without operation.The tissues from the rostral,caudal segments near injury site,cerebral cortex and linea obliqua tibiae were harvested.Total RNA was extracted with Trizol reagent separately.The BDNF mRNA expression in each group was detected by RT-PCR.Results(1)BDNF positive bands were seen in the tissues of the rostral,caudal segments near injury site,cerebral cortex and linea obliqua tibiae.Moreover,BDNF level in cerebral cortex is more than in the spinal cord at normal control(P
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Objective Amniotic membrane as the carrier to culture the vascular endothelial cells was investigated in this study in order to explore whether the corneal endothelial cells superseded by the vascular endothelial cells is feasible on the account of directing a kind of the original method to handle the bullous keratopathy.Methods The vascular endothelial cells adhering to the sphagitides lumen of the experimental rabbits were digested and gained by means of the perfusion method with 0.25%tripsin plus 0.02%EDTA.Fresh amniotic membrane without the amniotic epithelial cells was cut into the square tissue about 1.5 cm?1.5 cm and spread evenly in the 24-well culture dish.Primary cultured cells were subcultured on the amniotic membrane.We would not transplant the vascular endothelial cells feeding on the amniotic membrane until cells is full of the whole amniotic membrane surface.One to two months after the operation,the change of corneal diaphaneity was observed.Results About 12 days since the cell transplantation was performed,the corneal transparency alteration between the experimental groups and the control one is different.The corneal buttons in the experimental group show the severe edema and opacity,and the anterior chamber couldn't be seen unclearly.But,10 days after the operation,the corneal oedema which begins to extenuate was judged through the indicatrix that the corneal edema in the experimental group has been recovering slowly,among which the anterior chamber tissue of 7 animals was visible through the implant.The corneal edema in the control groups intensified evidently,even,the part of these appeared the ulcerous necrosis as result of the corneal severely edema.There is the existence of difference between two groups(P
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Objective To establish a serial of stable and mature methods of primary culture hippocampus neurons of GFP-transgene embryonic mice,to get the morphologic data of cultured neurons.Based on this research,in future,we can give an important theoretical and practical support for the therapy of nervous system diseases by transplanting with hippocampus neurons of GFP-transgene embryonic mice.Methods We primarily cultured the hippocampus neurons derived from the GFP-transgene embryonic mice in vitro.Under the microscope,we found cultured hippocampus neurons could live for more than one month and appeared to be the best status in 5～7 d after culture.During this time,the processes of the neurons are thick and the neurons connected one another to form the"cells-net" through their processes.After 14 d,the growth of the hippocampus neurons became slow.Results A serial of culture methods of hippocampus neurons had been successfully established.These cultured neurons were identified by the immuno-histochemical methods.They grow well in different phases before 14 d after culture.Conclusion Culturing hippocampus neurons of GFP-transgene embryonic mice is a simple,stable and effective method which can be applied to scientific research by other researchers.
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Objective To isolate and identify the cells from the cerebral cortex,cerebellar cortex,hippocampus,sub-ventricle region,brain stem and the spinal cord of neonatal rats and observe their growth morphology in vitro.Methods The cells were incubated from different regions of the CNS of neonatal rats by using DMEM/F12 media added with 10% bovine serum,and their growth morphology was observed by using inverted phase contrast microscope;then in the 10th day after incubation cells were fixed and immunocytochemical method was used to detect specific NSE antigen of the neurons and specific GFAP antigen of the astrocytes.Results Both neurons and astrocytes were studied in each region and they bloomed in the 10th day after incubation.Neurons had big triangular or ellipsoidal cell bodies surrounded with a halo and had robust nervous process which interlaced each other around cell bodies.The astrocytes had an ellipsoidal nucleus located at one side of the cell body and they had abundant processes branching profusely.Conclusion A method of culturing cells from different regions of the CNS of neonatal rats was described.A comparative morphology study of their growth was made and neurons and astrocytes of all the regions studied were identified.