Objective To observe whether theograde axial flow of retinal ganglion cells (RGC) in diabetic rats at the early stage was damaged. Methods Diabetic model was induced by streptozotocin in 6 adult male Sprague-Dawley (SD)rats. Fluorogold (FG) was injected to the superior colliculi 4 weeks later.Streched preparation of retina was made 12 and 72 hours after the injection, and was stained after photographed by fluorescent microscope. The proportion of RGC with different sizes labeled by FG was calculated. Other 6 normal adult male SD rats were in the control group. Results Twelve hours after injection with FG, there was no difference of the total number of RGC in experimental and control group, but the ratio of small RGC was lower in experimental group than that in the control group; 72 hours after injection with FG, The number of RGC, especially the small RGC, decreased obviously in experimental group compared with the control group. Conclusion The speed of the retrograde axial flow of RGC in diabetic rats at the early stage is affected, and the small RGC are damageable. (Chin J Ocul Fundus Dis, 2006, 22: 4-6)
Objective To observe the morphological changes of dendrite and soma in retinal ganglion cells (RGCs) which subsisted in early diabetic rats. Methods The RGCs of 3-months-course diabetic rats and coeval normal rats were marked by gene gun techniques. To collect RGCs photographs by Leica microscope with Z axis and CCD camera；to observe the changes of diameter, variance of structural features in dendritic field and somata after classification which according to the size and morphology. Thy-1 antibody marks on the retinal RGCs, taking a photograph under fluorescent microscope, counting the changes of retinal RGCs density in early diabetic rat. Results In three-month diabetic rats,the density of retinal RGCs was decreased obviously. Morphological changes of RGCs in the dendritic fields were observed with gene gun technique. There was no severe variation in all kinds of the bole of cell dendrite, in which some only showed crispation partially and sparseness also twisting in the dendritic ramus. The mean diameter of dendritic field and soma in class A of diabetic rats was (401plusmn;86) mu;m, the mean diameter of dendritic field in control group was (315plusmn;72) mu;m,compared with each other, there is statistically significant differences (t=21.249，Plt;0.001); the mean diameter of soma in class A of diabetic rats was (24plusmn;6) mu;m, the mean diameter of soma in control group was (22plusmn;5) mu;m, compared with each other, there is no statistically significant differences (t=0.927，Pgt;0.05); the mean diameter of dendritic field and soma in class B of diabetic rats were (170plusmn;36)、(14plusmn;2) mu;m respectively, in control group were (165plusmn;36)、(16plusmn;2) mu;m, the mean diameter of dendritic field and soma in class C of diabetic group were(265plusmn;78)、(17plusmn;5) mu;m respectively, in control group were (251plusmn;57)、(17plusmn;4) mu;m , compared with each other, there are on statistically significant differences(t=1.357，0.798，0.835，1.104，Pgt;0.05). Conclusions In short-term diabetes, the survived RGCs show good plasticity in adult diabetic rats, especially in class A. The changes of dendrites were more sensitive than the soma, which could be the leading index of the morphologic changes of RGCs in the early stage. The good plasticity showed by the RGCs and the time window from changing in dendrite to cell death provide us many evidences not only for the research but also for the nerve protection in clinic. (Chin J Ocul Fundus Dis,2008,24:249-254)
ObjectiveTo construct a lentiviral vector carrying rat sirt1 gene and observe the expression of sirt1 in retinal ganglion cell (RGC) of rat. MethodsRat sirt1 cDNA was inserted into pLV5 vector. After identification by sequencing analysis and PCR, the recombinant sirt1expressinglentivirus vector was packaged by cotransfecting 293T cells with packaged plasmid.Then pLV5-sirt1 was used to infect the cultured Sprague-Dawley rat RGC cell in vitro.The expressions of sirt1 protein and mRNA in infected rat RGC were detected by quantitative real-time PCR and Western blot. ResultsThe sirt1 expression vector pLV5 was successful constructed and sequence was proved to be correct. The expression of sirt1 protein and mRNA in RGC was significantly increased than that in cells infected with control lentiviruses(P < 0.05). ConclusionWe have successful constructed a sirt1 expression lentivirus vector pLV5-sirt1 and it can increase the expression of sirt1 protein and mRNA in the rat retinal ganglion cells.
Objective To study the effect of down-regulation of Claudin-3 mediated by adeno-associated virus (AAV) of shRNA on the cultured retinal ganglion cells (RGCs) in vitro. Methods RGCs isolated from mouse eyes were divided into normal control group, AAV-shScramble group, and AAV-shClaudin-3 group. The RGCs in AAV-shScramble group and AAV-shClaudin3 group were treated with AAV-shScramble and AAV-shClaudin-3 respectively 24 hours after cell seeding. Dynamic live cell fluorescence microscopy was used to observe the transfection efficiency 96 hours after transfection. Immunofluorescent staining of β-tubulin was used to measure the length of RGCs′ axon. 4′, 6-diamidino-2-phenylindole staining was used to observe the nuclei of apoptotic cells. The mRNA level of Claudin-3 and VEGF was measured by real-time polymerase chain reaction. The protein levels of Claudin-3, vascular endothelial growth factor (VEGF), Bcl-2 and Caspase-3 was determined by Western blot. Results The positive transfection rate was more than 50% in both AAV-shScramble group and AAV-shClaudin-3 group. The length of RGCs' axon in AAV-shClaudin-3 group was shorter than that in normal control group and AAV-shScramble group (F=22 363.274,P＜0.05). Down-regulation of Claudin-3 accelerated RGCs' apoptosis with nuclei shrinkage, tapering, and nucleolus formation of apoptotic bodies. The mRNA levels of Claudin-3 and VEGF in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=257.408, 160.533;P＜0.05). The protein levels of Claudin-3, VEGF and Bcl-2 in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=129.671, 420.552, 62.669;P＜0.05), while the protein level of Caspase-3 in AAV-shClaudin-3 group was higher than that in normal control group and AAV-shScramble group (F=231.348,P＜0.05). Conclusion Down-regulation of Claudin-3 increases the expression of Caspase-3, reduces the expression of VEGF and Bcl-2, accelerates RGCs' apoptosis and inhibit the RGCs' axon growth.
ObjectiveTo investigate the effects of form deprivation on the morphology of different types of RGC in mice.MethodsSixty B6.Cg-Tg (Thy1-YFP) HJrs/J transgenic mice were randomly assigned to form-deprived group (n=28) and control group (n=32). The right eyes of mice in the form-deprived group were covered by an occluder for 2 weeks as experimental eyes. The right eyes of mice in the control group were taken as control eyes. Before and 2 weeks after form deprivation, the refraction and ocular biometrics were measured; RGC were stained with Bra3a antibody and counted; the morphology of RGC was reconstructed with Neuroexplore software after immunohistochemical staining. The data was compared among experimental eyes, fellow eyes and control eyes by one-way analysis of variance.ResultsTwo weeks after form deprivation, the axial myopia was observed in the experimental eyes (refraction: F=15.009, P＜0.001; vitreous chamber depth: F=3.360, P=0047; ocluar axial length: F=5.011, P=0013). The number of RGC in central retina of the experimental eyes was decreased compared with the fellow eyes and the control eyes (F=4.769, P=0.035). The reconstructed RGC were classified into 4 types according to their dendritic morphology. Form deprivation affected all 4 types of RGC but in a different way. Among them, 3 types of RGC were likely contribute to form vision perception. Form deprivation increased the dendrite branches in these types of ganglion cells. However, form deprivation decreasd dendrite segment numbers in both eyes and the intersection and length insholl analyse type 4 ganglion cells which were morphologically identified as ipRGC.ConclusionForm deprivation distinguishingly affects the morphology of different types of RGC, indicating that form vision and non-form vision play different role in myopia development.
ObjectiveTo explore the light response, retinal inflammation and apoptosis of the retinal ganglion cells (RGCs) 1 year after the new type of channelrhodopsin PsCatCh2.0 was transfected into the retina of rd1 mice. MethodsTwenty-four male rd1 mice were randomly divided into rd1 experimental group and rd1 control group, 12 mice in each group. 1.5 μl of recombinant adeno-associated virus (rAAV)2/2-cytomegalovirus (CMV)-PsCatCh2.0-enhanced green fluorescent protein (EGFP) was injected into the vitreous cavity 1 mm below the corneoscleral limbus of mice in the rd1 experimental group, and the same dose of recombinant virus was injected 2 weeks later at temporal side 1 mm below the corneoscleral limbus. One year after virus injection, the light response of RGCs expressing PsCatCh2.0 was recorded by patch clamp technique; the expression of PsCatCh2.0 in the retina was evaluated by immunofluorescence staining; the transfection efficiency of recombinant virus was evaluated by the transfection efficiency of virus and the number of RGCs. Hematoxylin-eosin staining was performed to measure the inner retinal thickness. Western blotting was used to detect the protein expression of nuclear factor (NF)-κB p65 in retina; real-time quantitative polymerase chain reaction was used to detect the relative expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and Bax mRNA. Terminal deoxynucleotidyl transferase kit was used to observe the apoptosis of retinal cells in each group of mice. ResultsOne year after the intravitreal injection of recombinant virus, PsCatCh2.0-expressing RGCs can still generate 30 pA photocurrent. The virus PsCatCh2.0-EGFP was mainly transfected into RGCs, and partly transfected into amacrine cells, almost no transfection was seen in bipolar and horizontal cells. There were no significant differences in the number of RGCs and thickness of the inner retina between the rd1 experimental group and the rd1 control group (F=14.35, 0.05; P＞0.05), while the rd1 experimental group NF-κB p65 protein expression, TNF-α and IL-6 mRNA quantification were significantly lower than those of rd1 control group (F=4.61, 5.91, 5.78; P＜0.05). The number of red fluorescent apoptotic cells in the retina of mice in the rd1 experimental group was less than that in the rd1 control group, and the Bax mRNA expression was lower than that in the rd1 control group, and the difference was statistically significant (F=7.52, P＜0.01). ConclusionOne year after intravitreal injection of recombinant virus, the PsCatCh2.0 expressing RGCs can still generate photocurrent. Long term transfection and expression of PsCatCh2.0 has no obvious cytotoxic effect on RGCs, nor it increases the inflammatory effect of the retina of rd1 mice with retinal degeneration.
ObjectiveTo investigate the protective effects of different concentrations of chloroquine on RGC in n-methyl-d-aspartate (NMDA) injured mice and its possible mechanisms.MethodsFifty-four healthy male C57/BL6 mice were randomly divided into three groups, 18 in each group. The mice in low-dose chloroquine group were intraperitoneally injected with chloroquine solution at a dose of 10 mg/kg daily. Mice in high-dose chloroquine group were intraperitoneally injected with chloroquine solution at a dose of 100 mg/kg, and the mice in control group were intraperitoneally injected with the same volume of PBS. NMDA intravitreal injection was performed 2 days after intraperitoneal injection, 5 nmoles NMDA was injected into the left eye, and the same volume of PBS was injected into the right eye as a control. The RGC staining of retinal plaques were performed 7 days after NMDA injection, and the number of alive RGC was calculated. The visual acuity and electroretinogram were used to evaluate the electrophysiological functions of RGC at 9 and 10 days after modeling. Real-time quantitative PCR and retinal frozen sections and glial fibrillary acidic protein (GFAP) immunofluorescence staining were performed 11 days after NMDA injection to evaluate the glial activation of the retina. The density, visual acuity, and the amplitude of PhNR-wave of RGC between groups were compared by one-way analysis of variance.ResultsAt 7 days after NMDA injection, the density of RGC in retinal patch of low-dose chloroquine group was significantly higher than that of intraperitoneal injection of PBS control group (F=54.41, P＜0.01). The density of RGC in retinal patch of high-dose chloroquine group was lower than that of control group (F=1.18, P＞0.05). The visual acuity was higher than control group, and the difference was statistically significant (F=9.10, P＜0.05). The amplitude of PhNR-wave was significantly higher in low-dose chloroquine group than that of the control group (F=17.60, P＜0.01). The mRNA level of inflammatory factor and GFAP positive signal was also significantly lower than that of the control group (F=23.66, P＜0.05). The amplitude of PhNR-wave, the expression of GFAP (F=110.20, P＜0.01) and the mRNA level of inflammatory factors (F=167.60, 17.78; P＜0.01) in the high-dose chloroquine group were higher than the other two groups, and the differences were statistically significant.ConclusionsIn NMDA injury retinal model, low-dose chloroquine significantly increased the survival and physiological function of RGC, and the mechanism may be related to the inhibition of glial activation and inflammatory response. High-dose of chloroquine would aggravate the apoptosis of RGC.
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.