Objective To observe the expression of related proteins of retina after subretinal implantation with inactive chips.Methods A total of 27 healthy adult New Zealand white rabbits were randomly divided into three groups: operation group (12 rabbits) in which the rabbits were implanted with inactive chips into the interspace beneath retina;shamoperation group (12 rabbits) in which the rabbits were implanted with inactive chips into the interspace beneath retina which was taken out immediately;the control group (3 rabbits). Animals were sacrified for immunohistological study 7,15,30 and 60 days after surgery.The rabbits in control group group were sacrified for immunohistological study after bred for 30 days.The expressions of glial fibrillary acidic protein (GFAP) and brain derived neurotrophic facor (BDNF) were observed.Results In operation group, the outer nulear layer of retina thinned, and the cells in the inner nulear layer was disorganized 7,15,and 30 days after the surgery;glial cells proliferated 60 days after surgery; the positive expression of BDNF and GFAP was more than that in the shamoperation and control group.In shamoperation group, the positive expression of BDNF and GFAP was more than that in the control group.No obvious difference of expression of BDNF and GFAP between each time point groups was found.Conclusions The expression of neroprotective related proteins increased after subretinal implantation with inactive chips suggests that limited neuroprotective effects might be led by the implantation.
ObjectiveThe aim of this study was to evaluate the repair effect of spontaneous reinnervation in rats underwent recurrent laryngeal nerve (RLN) transection. MethodsThirty male Wistar rats (340-360 g) were divided into experiment group (n=15) and blank control group (n=15), and then 15 rats of these 2 groups were divided into 3 time point groups equally:4 weeks group, 8 weeks group, and 12 weeks group. Fifteen rats of experiment group underwent right RLN transection with excision of a 5 mm segment, and other 15 rats of blank control group exposed RLN only, without transection. Grade of vocalization, maximum angle of arytenoid cartilage, axon number of distal part of RLN, and expression of the brain-derived neurotrophic factor (BDNF) in right thyroarytenoid muscle were evaluated at different time points, including 4, 8, and 12 weeks after operation. ResultsGrade of vocalization, maximum angle of arytenoid cartilage, axon numbers of distal part of RLN, and the expression of BDNF in the right thyroarytenoid muscle of experiment group were all lower than those corresponding index of blank control group (P < 0.05), and these indexes of experiment group were restored gradually with time, but failed to reach normal level during the observed time. ConclusionsEven though spontaneous reinnervation is presented after RLN injury, but the effect is unsatisfactory.
ObjectiveTo evaluate the effect of the combination of collagen scaffold and brain-derived neurotrophic factor (BDNF) on the repair of transected spinal cord injury in rats.MethodsThirty-two Sprague-Dawley rats were randomly divided into 4 groups: group A (sham operation group), T9, T10 segments of the spinal cord was only exposed; group B, 4-mm T9, T10 segments of the spinal cord were resected; group C, 4-mm T9, T10 segments of the spinal cord were resected and linear ordered collagen scaffolds (LOCS) with corresponding length was transplanted into lesion site; group D, 4-mm T9, T10 segments of the spinal cord were resected and LOCS with collagen binding domain (CBD)-BDNF was transplanted into lesion site. During 3 months after operation, Basso-Beattie-Bresnahan (BBB) locomotor score assessment was performed for each rat once a week. At 3 months after operation, electrophysiological test of motor evoked potential (MEP) was performed for rats in each group. Subsequently, retrograde tracing was performed for each rat by injection of fluorogold (FG) at the L2 spinal cord below the injury level. One week later, brains and spinal cord tissues of rats were collected. Morphological observation was performed to spinal cord tissues after dehydration. The thoracic spinal cords including lesion area were collected and sliced horizontally. Thoracic spinal cords 1 cm above lesion area and lumbar spinal cords 1 cm below lesion area were collected and sliced coronally. Coronal spinal cord tissue sections were observed by the laser confocal scanning microscope and calculated the integral absorbance (IA) value of FG-positive cells. Horizontal tissue sections of thoracic spinal cord underwent immunofluorescence staining to observe the building of transected spinal cord injury model, axonal regeneration in damaged area, and synapse formation of regenerated axons.ResultsDuring 3 months after operation, the BBB scores of groups B, C, and D were significantly lower than those of group A (P<0.05). The BBB scores of group D at 2-12 weeks after operation were significantly higher than those of groups B and C (P<0.05). Electrophysiological tests revealed that there was no MEP in group B; the latencies of MEP in groups C and D were significantly longer than that in group A (P<0.05), and in group C than in group D (P<0.05). Morphological observation of spinal cord tissues showed that the injured area of the spinal cord in group B extended to both two ends, and the lesion site was severely damaged. The morphologies of spinal cord tissues in groups C and D recovered well, and the morphology in group D was closer to normal tissue. Results of retrograde tracing showed that the gray matters of lumbar spinal cords below the lesion area in each group were filled with FG-positive cells; in thoracic spinal cords above lesion sites, theIA value of FG-positive cells in coronal section of spinal cord in group A was significantly larger than those in groups B, C, and D (P<0.05), and in groups C and D than in group B (P<0.05), but no significant difference was found between groups C and D (P>0.05). Immunofluorescence staining results of spinal cord tissue sections selected from dorsal to ventral spinal cord showed transected injured areas of spinal cords which were significantly different from normal tissues. The numbers of NF-positive axons in lesion center of group A were significantly larger than those of groups B, C, and D (P<0.05), and in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05).ConclusionThe combined therapeutic approach containing LOCS and CBD-BDNF can promote axonal regeneration and recovery of hind limb motor function after transected spinal cord injury in rats.
Objective To transplant intravenously human brain-derived neurotrophic factor (hBDNF) genemodified bone marrow mesenchymal stem cells (BMSCs) marked with enhanced green fluorescent protein (EGFP) to injured spinal cord of adult rats, then to observe the viabil ity of the cells and the expressions of the gene in spinal cord, as well as theinfluence of neurological morphological repairing and functional reconstruction. Methods Ninety-six male SD rats weighing (250 ± 20) g were randomly divided into 4 groups: hBDNF-EGFP-BMSCs transplantation group (group A, n=24), Ad5-EGFPBMSCs transplantation group (group B, n=24), control group (group C, n=24), and sham operation group (group D, n=24). In groups A, B, and C, the spinal cord injury models were prepared according to the modified Allen method at the level of T10 segment, and after 3 days, 1 mL hBDNF-EGFP-BMSCs suspension, 1 mL Ad5-EGFP-BMSCs suspension and 1 mL 0.1 mol/L phosphate buffered sal ine (PBS) were injected into tail vein, respectively; in group D, the spinal cord was exposed without injury and injection. At 24 hours after injury and 1, 3, 5 weeks after intravenous transplantation, the structure and neurological function of rats were evaluated by the Basso-Beattie-Bresnahan (BBB) score, cortical somatosensory evoked potential (CSEP) and transmission electron microscope. The viabil ity and distribution of BMSCs in the spinal cord were observed by fluorescent inverted phase contrast microscope and the level of hBDNF protein expression in the spinal cord was observed and analyzed with Western blot. Meanwhile, the expressions of neurofilament 200 (NF-200) and synaptophysin I was analyzed with immunohi stochemistry. Results After intravenous transplantation, the neurological function was significantly improved in group A. The BBB scores and CSEP in group A were significantly higher than those in groups B and C (P lt; 0.05) at 3 and 5 weeks. The green fluorescence expressions were observed at the site of injured spinal cord in groups A and B at 1, 3, and 5 weeks. The hBDNF proteinexpression was detected after 1, 3, and 5 weeks of intravenous transplantation in group A, while it could not be detected in groups B, C, and D by Western blot. The expressions of NF-200 and synaptophysin I were ber and ber with transplanting time in groups A, B, and C. The expressions of NF-200 and synaptophysin I were best at 5 weeks, and the expressions in group A were ber than those in groups B and C (P lt; 0.05). And the expressions of NF-200 in groups A, B, and C were significantly ber than those in group D (P lt; 0.05), whereas the expressions of synaptophysin I in groups A, B, and C were significantly weaker than those in group D (P lt; 0.05). Ultramicrostructure of spinal cords in group A was almost normal. Conclusion Transplanted hBDNF-EGFP-BMSCs can survive and assemble at the injured area of spinal cord, and express hBDNF. Intravenous implantation of hBDNF-EGFP-BMSCs could promote the restoration of injured spinal cord and improve neurological functions.
Objective To investigate the memory amelioration of the Alzheimer disease (AD)model rat after being transplanted the single neural stem cells(NSC) and NSC modified with human brain-derived neurotrophic factor（hBDNF） gene. Methods Forty SD rats were divided evenly into 4 groups randomly. The AD model rats were made by cutting unilaterallythe fibria fornix of male rats. Ten to twelve days after surgery, the genetically modified and unmodified NSC were implanted into the lateral cerebral ventricle of group Ⅲ and group Ⅳ respectively. Two weeks after transplantation, theamelioration of memory impairment of the rats was detected by Morris water maze. Results The average escaping latency of the group Ⅲ and group Ⅳ (41.84±21.76 s,25.23±17.06 s respectively) was shorter than that of the group Ⅱ(70.91±23.67 s) (Plt;0.01). The percentage of swimming distance inthe platform quadrant in group Ⅲ (36.9%) and in group Ⅳ(42.0%) was higherthan that in the group Ⅱ(26.0%) (Plt;0.01). More marginal and random strategies were used in group Ⅱ.The percentage of swimming distance in the platform quadrant in group Ⅳ was also greater than that in group Ⅲ(Plt;0.05). There were no significant differences in the average escaping latency, the percentage of swimming distance in the platform quadrant and the probe strategy between group Ⅳ and group Ⅰ(Pgt;0.05).More lineal and oriented strategies were used in group Ⅳ. Conclusion The behavioral amelioration of AD model rat was obtained by transplanting single NSC and hBDNF-gene-modified NSC. The effect of the NSC group modified with hBDNF gene is better than that of the groupⅢ.
摘要:目的:观察阿托伐他丁对脑梗死大鼠脑保护的作用以及对脑源性神经营养因子(braindeprived neurotrophic factor,BDNF)的影响。方法: 线栓法制备SD大鼠大脑中动脉梗死(middle cerebral artery occlusion,MCAO)再灌注模型。将大鼠随机分为:假手术组;MCAO组的2 h、24 h、3 d、5 d组;阿托伐他丁组的2 h、24 h、3 d、5 d组。MCAO组和阿托伐他丁组的各时程组再分别分为脑梗死体积亚组、免疫组化亚组,每亚组及假手术组各6只大鼠。在不同时间点观察阿托伐他丁组和MCAO组大鼠神经行为评分、脑梗死体积,用免疫组化法检测BDNF阳性细胞数。结果: 神经行为评分和脑梗死体积在阿托伐他丁组和MCAO组的2 h组之间无显著性差异(Pgt;0.05),在阿托伐他丁24 h、3 d、5 d组均显著低于对应时程的MCAO组(Plt;0.05);各组缺血半暗带BDNF阳性细胞数均增高,但阿托伐他丁组的阳性细胞数显著高于对应时程的MCAO组(Plt;0.05)。结论:阿托伐他丁能提高大鼠局灶脑缺血半暗带BDNF的表达水平,促进神经元的修复。Abstract: Objective: To observe the effect of atorvastatin in cerebral protection and braindeprived neurotrophic factor(BDNF) in rats. Methods: Ischemic reperfusion model of rats as established by an intraluminal filament and recirculation at different time point respectively. One hundred and two healthy SD rats were randomly assigned into three groups for different preconditioning, including the sham surgery group (SS, n=6), the sham and middle cerebralartery occlusion (MCAO) group (MCAO, n=48), and the atorvastatin and MCAO group (atorvastatin +MCAO, n=48). The latter two groups were further divided into two subgroups on different time points of tests. Each subgroup hase six rats. In the atorvastatin +MCAO group, intragastric administration of atorvastatin was given for five days, then the MCAO followed. In the MCAO group, the MCAO was given directly. The neurophysical marks and the volume of the cerebral infarction in atorvastatin group and MCAO group were determined at different time point. The expression of BDNF was valued by immunohistochemitry respectively. Results: At 2 h, there were no differences in the neurophysical marks and volume of the cerebral infarction between atorvastatin group and MCAO group (Pgt;0.05). At 24 h,3 d,5 d, the neurophysical marks and volume of the cerebral infarction of atorvastatin group were lower than that of MCAO group in the corresponding time (Plt;0.05). Around the necrotic areas,BDNF positive neurons were increased in both groups, but they were higher in atorvastatin group than in MCAO group in the corresponding time (Plt;0.05). Conclusion: Atorvastatin could increase the expression level of BDNF and promote the ischemic neuron to revive.
Objective To observe the protective effect of ultrasound microbubble contrast agentmediated transfection of brain-derived neurotrophic factor(BDNF) into the retina and visual cortex on retinal ganglion cells (RGC) after optic nerve injury. Methods A total of 88 male Sprague-Dawley (SD) rats were randomly divided into normal group (group A, eight rats), sham operation group (group B, 16 rats), control group (group C, 16 rats), eyes transfection group (group D, 16 rats), brain transfection group (group E, 16 rats), combined transfection group (group F, 16 rats). The optic nerve crush injury was induced, and then the groups B to F were divided into one-week and two-week after optic nerve injury subgroup with eight rats each, respectively. The rats in group B and C underwent intravitreal and visual cortex injection with phosphate buffered solution respectively. The rats in group D and E underwent intravitreal and visual cortex injection with the mixture solution of microbubbles and BDNF plasmids respectively. The rats in group F underwent both intravitreal and visual cortex injection with the mixture solution of microbubbles and BDNF plasmids at the same time. The ultrasound exposure was performed on the rats in group D to F after injection with the mixture solution of microbubbles and BDNF plasmids. One and two weeks after optic nerve injury, RGC were retrogradely labeled with Fluorogold; active caspase-3 protein was observed by immunohistochemistry and the N95 amplitude was detected by pattern electroretinogram (PERG). Results Golden fluorescence can be observed exactly in labeled RGC in all groups，the difference of the number of RGC between the six groups and ten subgroups were significant（F=256.30，65.18；P＜0.01）. Active caspase-3 in ganglion cell layer was detected in group C to F, but not in group A and B. The difference of the N95 amplitude between the six groups and ten subgroups were significant（F=121.56，82.38；P＜0.01）.Conclusion Ultrasound microbubble contrast agent-mediated BDNF transfection to the rat retina and visual cortex can inhibit the RGC apoptosis after optic nerve injury and protect the visual function.