Objective To study the effects of several neurotrophic factors and growth factors on the survival of human retinal ganglion cells(RGC)in vitro. Methods RGC were isolated from donor eyes and cultured.RGC in cell culture were identified by morphologic criteria and immunocytochemical staining.Various neurotrophic factors and growth factors were added individually to the cultures.Numbers of RGC in wells in which these agents had been added were compared with those from control wells(cultures without supplements). Results No or very few RGC were present in cell cultures containing medium without supplements or those supplemented with neurotrophin-3(NT-3),nerve growth factor (NGF),epidermal growth factor(EGF)amd plateletderived growth factor(PDGF).Numbers of RGC(per 10 fields)in cell cultures containing brain derived neurotrophic factor(BDNF),ciliary neurotrophic factor(CNTF),neurotrophin-4/5(NT-4/5)and basic fibroblast growth factor(bFGF)wer 4.08,1.23,2.63 and 2.65,respectively,significantly more than found in the control cultures. Conclusions BDNF,NT-4/5,bFGF,CNTF improve survival of human RGC in vitro,while NGF,NT-3,EGF and PDGF do not. (Chin J Ocul Fundus Dis, 1999, 15: 149-152)
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.
ObjectiveTo observe the effects of human umbilical cord mesenchymal stem cells (hUCMSCs) on blood glucose levels and diabetic retinopathy in diabetes mellitus (DM) rats. MethodA total of 45 healthy male Sprague-Dawley rats were randomly divided into normal control group (group A, 10 rats) and DM group (33 rats). Diabetic model was established in DM group by tail vein injection of streptozotocin.The DM group was further randomly divided into 3 groups (11 rats in each group), including group B (no transplantation), group C (hUCMSC was injected through tail vein) and group D (hUCMSC was injected into the vitreous). Blood glucose, retina wholemont staining and expression of brain derived neurotrophic factor (BDNF) in the retina were measured at 2, 4, 6, 8 weeks after hUCMSC injection. The blood glucose was significantly different between A-D groups before injection (t=-64.400, -60.601, -44.065, -43.872; P=0.000) BDNF expression was studied by real time fluorescence quantitative polymerase chain reaction (RT-PCR) and immunohistochemistry staining. ResultsThe blood glucose was significantly different between A-D groups after hUCMSC injection (F=400.017, 404.410, 422.043, 344.109; P=0.000), and between group C and group B/D (t=4.447, 4.990; P < 0.01). Immuno-staining shown that BDNF was positive in ganglion cell layer (RGC) of group A, weak in group B while BDNF expression increased in group C/D. BDNF mRNA expression was significantly different between group B, C and D at 4, 6 and 8 weeks after hUCMSC injection (F=29.372, 188.492, 421.537; P=0.000), and between group B and C/D (t=66.781, 72.401, 63.880, 88.423, 75.120, 83.002; P < 0.01) by RT-PCR analysis. The BDNF mRNA expression was significantly different between C and D groups only at 8 weeks after hUCMSC injection (t=127.321, P=0.005). ConclusionsTail vein injection of hUCMSCs can significantly reduce the blood glucose levels of rats. Intravenous and intravitreal injection of hUCMSCs can increase the expression of BDNF.
Objective To observe the effects of neural stem cells(NSCs) transplantation on the glial cell line-derived neurotrophic factor (GDNF) and growth associated protein 43(GAP-43) after the spinal cord injury(SCI), and to investigate the mechanism of repairing the SCI by NSCs transplantation. Methods The neural stem cells from the hippocampus of rats’ embryo were cultured and identified by immunocytochemistry. The SCI model was made by the modified Allen device. Sixty adult Wistar rats were randomly divided into three groups: spinal cord injury was treated with transplantation of NSCs (group A, n=24), with DMEM solution(group B, n=24) and normal control group without being injured(group C, n=12). Seven days after the operation of SCI, the NSCs were transplanted into the injured site. Then GAP-43 and GDNF expressions were tested by RT-PCR and immunohistochemistry. Results Compared with group B, the GDNF mRNA expression of group A increased by 23.3％ on the 1st day, by 26.8% on the 3rd day and by 32.7% on the 7th day; the GAP-43 mRNA expression increased by 19.5% on the 1st day, 21.6% on the 3rd day and 23.1% on the 7th day. There were statistically significant differences(Plt;0.05). Conclusion The transplantation of NSCs can change the microenvironment injured site and promote the regeneration of axon by enhancing the expressions of GDNF mRNA and GAP-43 mRNA. It is one of the mechanisms of repairing the SCI by NSCs transplantation.
Objective To construct human brain-derived neurotrophic factor retroviral vector-pLXSN (hBDNFpLXSN), and to evaluate the bioactivity of hBDNF. Methods The genome mRNA was extracted from embryonic brain tissue of a 5-month-old infant, the hBDNF gene sequence was obtained with RT-PCR technology, and hBDNF-pLXSN constructed in vitro was used to infect the fibroblasts (NIH/3T3). The expression of hBDNF was identfied by the immunohistochemistry method, and the NIH/3T3 and BDNF biological activities were determined by culture of the PC12 cells and dorsal root gangl ia. Results The hBDNF-pLXSN was constructed successfully by sequencing analyses. The infected NIH/3T3 showed positive expression of hBDNF. The infected NIH/3T3 could product hBDNF. Bioactivity of the products could support the PC12cell survival and neurite growth in the primary cultures of dorsal root gangl ia neurons of mice. Conclusion hBDNF-pLXSNvirus has the abil ity to infect NIH/3T3 and make it expressed and secreted hBDNF with the biological activity. It can be used to treat facial paralysis as a gene therapy.
ObjectiveTo investigate the effect of intravitreal injection of neural stem cells (NSC) derived from human umbilical cord mesenchymal stem cells (hUCMSC) on the expression of brain-derived neurotrophic factor (BDNF) and the number of retinal ganglion cells (RGC). MethodsFifty-two adult male Sprague-Dawley rats were randomly divided into normal group (group A) and diabetes mellitus group which received intraperitoneal injection of streptozocin to make diabetic rat models. One month after the diabetic rat models were confirmed successfully, diabetic rats were randomly divided into diabetic group (group B), hUCMSC group (group C) and hUCMSC-induced NSC group (group D). And thirteen diabetic rats were included in each group. Immuno-cytochemistry was applied to observe BDNF and thymosin-1(Thy-1) staining in the retina. Then mean integrated absorbance of the staining region on the retina slices were analyzed by Image-Pro Plus 6.0. The number of Thy-1 labeled RGC was record. ResultsBDNF and Thy-1 were positive on the retina slices from group A. The staining intensity from group B became weak and the expression of BDNF and Thy-1 gradually decrease with time (P < 0.05), and those from group C and group D were positively (P < 0.05), especially in group D (P < 0.05). The BDNF expression and Thy-1 labeled RGC were the same between group B and C (P > 0.05) at 2 weeks after injection, but were significant different for other time points (P < 0.05).Significant positive correlation between the expression of BDNF and the number of RGC were found by the Pearson correlation analysis (r=0.964, P < 0.05). ConclusionIntravitreal injection of hUCMSC-derived NSC to diabetic rat may protect the retina by promoting the expression of BDNF and increasing the number of RGC.
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.
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.