ObjectiveTo observe the morphological and functional changes of retinal degeneration in mice with CLN7 neuronal ceroid-lipofuscinosis, and the therapeutic effects of glial cell derived neurotrophic factor (GDNF) and/or ciliary neurotrophic factor (CNTF) based on neural stem cells (NSC) on mouse photoreceptor cells. MethodsA total of 100 CLN7 mice aged 14 days were randomly divided into the experimental group and the control group, with 80 and 20 mice respectively. Twenty C57BL/6J mice aged 14 days were assigned as wild-type group (WT group). Mice in control group and WT group did not receive any interventions. At 2, 4, and 6 months of age, immunohistochemical staining was conducted to examine alterations in the distribution and quantity of cones, rod-bipolar cells, and cone-bipolar cells within the retinal of mice while electroretinography (ERG) examination was utilized to record scotopic a and b-waves and photopic b-wave amplitudes. At 14 days of age, the mice in the experimental group were intravitreally injected with 2 μl of CNTF-NSC, GDNF-NSC, and a 1:1 cell mixture of CNTF-NSC and GDNF-NSC (GDNF/CNTF-NSC). Those mice were then subdivided into the CNTF-NSC group, the GDNF-NSC group, and the GDNF/CNTF-NSC group accordingly. The contralateral eyes of the mice were injected with 2 μl of control NSC without neurotrophic factor (NTF) as their own control group. At 2 and 4 months of age, the rows of photoreceptor cells in mice was observed by immunohistochemical staining while ERG was performed to record amplitudes. At 4 months of age, the differentiation of grafted NSC and the expression of NTF were observed. Statistical comparisons between the groups were performed using a two-way ANOVA. ResultsCompared with WT group, the density of cones in the peripheral region of the control group at 2, 4 and 6 months of age (F=285.10), rod-bipolar cell density in central and peripheral retina (F=823.20, 346.20), cone-bipolar cell density (F=356.30, 210.60) and the scotopic amplitude of a and b waves (F=1 911.00, 387.10) in central and peripheral retina were significantly decreased, with statistical significance (P＜0.05). At the age of 4 and 6 months, the density of retinal cone cells (F=127.30) and b-wave photopic amplitude (F=51.13) in the control group were significantly decreased, and the difference was statistically significant (P＜0.05). Immunofluorescence microscopy showed that the NSC transplanted in the experimental group preferentially differentiated into astrocytes, and stably expressed CNTF and GDNF at high levels. Comparison of retinal photoreceptor nucleus lines in different treatment subgroups of the experimental group at different ages: CNTF-NSC group, at 2 months of age: the whole, central and peripheral regions were significantly different (F=31.73, 75.06, 75.06; P＜0.05); 4 months of age: The difference between the whole area and the peripheral region was statistically significant (F=12.27, 12.27; P＜0.05). GDNF/CNTF-NSC group, 2 and 4 months of age: the whole (F=27.26, 27.26) and the peripheral area (F=16.01, 13.55) were significantly different (P＜0.05). In GDNF-NSC group, there was no statistical significance at all in the whole, central and peripheral areas at different months of age (F=0.00, 0.01, 0.02; P＞0.05). ConclusionsCLN7 neuronal ceroid-lipofuscinosis mice exhibit progressively increasing degenerative alterations in photoreceptor cells and bipolar cells with age growing, aligning with both morphological and functional observations. Intravitreal administration of stem cell-based CNTF as well as GDNF/CNTF show therapeutic potential in rescuing photoreceptor cells. Nevertheless, the combined application of GDNF/CNTF-NSC do not demonstrate the anticipated synergistic protective effect. GDNF has no therapeutic effect on the retinal morphology and function in CLN7 neuronal ceroid-lipofuscinosis mice.
Objective To investigate the possibility of constructing eukaryotic expression vector for human glial derived neurotrophic factor （hGDNF）, transfecting it to spinal cord tissue of rats so as to treat acute spinal cord injury. Methods The eukaryotic expression vector pcDNA3-hGDNF was constructed by recombinant DNA technique, transfected into glial cell and neuron of spinal cord by liposome DOTAP as experimental group. In control group, mixture of empty vector and liposome was injected. The mRNA and protein expressions of hGNDF were detected by RT-PCR and Western blot. Results After the recombinant eukaryotic expression vector for hGDNF was digested with Hind III and XbaⅠ, electrophoresis revealed 400 bp fragment for hGDNF gene and 5 400 bp fragment for pcDNA3 vector. In the transfected spinal cord tissue, the mRNA and protein expressions of hGDNF gene were detected with RT-PCR and Western blot. Conclusion The constructed eukaryotic expression vector pcDNA3hGDNF could be expressed in the transfected spinal cord tissue of rat, so it provide basis for gene therapy of acute spinal cord injury.
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 review the research progress on the role of Schwann cells in regulating bone regeneration. MethodsThe domestic and foreign literature about the behavior of Schwann cells related to bone regeneration, multiple tissue repair ability, nutritional effects of their neurotrophic factor network, and their application in bone tissue engineering was extensively reviewed. ResultsAs a critical part of the peripheral nervous system, Schwann cells regulate the expression level of various neurotrophic factors and growth factors through the paracrine effect, and participates in the tissue regeneration and differentiation process of non-neural tissues such as blood vessels and bone, reflecting the nutritional effect of neural-vascular-bone integration. ConclusionTaking full advantage of the multipotent differentiation ability of Schwann cells in nerve, blood vessel, and bone tissue regeneration may provide novel insights for clinical application of tissue engineered bone.
OBJECTIVE To study the protective effects of Schwann cell derived neurotrophic factor (SDNF) on motoneurons of spinal anterior horn from spinal root avulsion induced cell death. METHODS Twenty SD rats were made the animal model of C6.7 spinal root avulsion induced motoneuron degeneration, and SDNF was applied at the lesion site of spinal cord once a week. After three weeks, the C6.7 spinal region was dissected out for motoneuron count, morphological analysis and nitric oxide synthase (NOS) enzyme histochemistry. RESULTS 68.6% motoneurons of spinal anterior horn death were occurred after 3 weeks following surgery, the size of survivors was significantly atrophy and NOS positive neurons increased. However, in animals which received SDNF treatment, the death of motoneurons was significantly decreased, the atrophy of surviving motoneurons was prevented, and expression of NOS was inhibited. CONCLUSION SDNF can prevent the death of motoneurons following spinal root avulsion. Nitric oxide may play a role in these injury induced motoneuron death.
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.
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.
ObjectiveTo investigate the protective effects of carboxymethylated chitosan (CMCS) on oxidative stress induced apoptosis of Schwann cells (SCs), and the expressions of brain derived neurotrophic factor (BDNF) and gl ial cell line derived neurotrophic factor (GDNF) in oxidative stress induced SCs. MethodsTwenty-four 3-5 days old Sprague Dawley rats (weighing 25-30 g, male or female) were involved in this study. The bilateral sciatic nerves of rats were harvested and SCs were isolated and cultured in vitro. The purity of SCs was identified by immunofluorescence staining of S-100. SCs were treated with different concentrations of hydrogen peroxide (H2O2, 0.01, 0.10, and 1.00 mmol/L) for 3, 6, 12, and 24 hours to establ ish the apoptotic model. The cell counting kit 8 (CCK-8) and flow cytometry analysis were used to detect the cell viabil ity and apoptosis induced by H2O2, and the optimal concentration and time for the apoptotic model of SCs were determined. The 2nd passage SCs were divided into 5 groups and were treated with PBS (control), with 1.00 mmol/L H2O2, with 1.00 mmol/L H2O2+50 μg/mL CMCS, with 1.00 mmol/L H2O2+100 μg/mL CMCS, and with 1.00 mmol/L H2O2+200 μg/mL CMCS, respectively. After cultured for 24 hours, the cell viabil ity was assessed by CCK-8, cell apoptosis was detected by flow cytometry analysis, the expressions of mRNA and protein of BDNF and GDNF were detected by real-time quantitative PCR and Western blot. ResultsThe immunofluorescence staining of S-100 indicated the positive rate was more than 95%. CCK-8 and flow cytometry results showed that H2O2 can inhibit the proliferation of SCs and induce the SCs apoptosis with dose dependent manner, the effect was the most significant at 1.00 mmol/L H2O2 for 24 hours; after addition of CMCS, SCs exhibited the increased proliferation and decreased apoptosis in a dose dependent manner. Real-time quantitative PCR and Western blot analysis showed that 1.00 mmol/L H2O2 can significantly inhibit BDNF and GDNF expression in SCs when compared with control group (P<0.05), 50-200 μg/mL CMCS can reverse the oxidative stress-induced BDNF and GDNF expression in SCs in a dose dependent manner, showing significant difference compared with control group and 1.00 mmol/L H2O2 induced group (P<0.05). There were significant differences among different CMCS treated groups (P<0.05). ConclusionCMCS has the protective stress on oxidative stress induced apoptosis of SCs, and may promote the BDNF and GDNF expressions of neurotrophic factors in oxidative stress induced SCs.
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.
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.