The optic nerve belongs to the central nervous system (CNS). Because of the lack of neurotrophic factors in the microenvironment of the CNS and the presence of myelin and glial scar-related inhibitory molecules, and the inherent low renewal potentials of CNS neurons comparing to the peripheral nerve system, it is difficult to spontaneously regenerate the optic nerve after injury. Protecting damaged retinal ganglion cells (RGCs), supplementing neurotrophic factor, antagonizing axon regeneration inhibitory factor, and regulating the inherent regeneration potential of RGCs can effectively promote the regeneration and repair of optic nerve. Basic research has made important progress, including the restoration of visual function, but there are still a lot of unsolved problems in clinical translation of these achievements, so far there is no ideal method of treatment of optic nerve injury. Therefore, it is rather urgent to strengthen the cooperation between basic and clinical research, to promote the transformation of basic research to the clinical applications as soon as possible, which will change the unsatisfactory clinical application status.
Objective To compare the effects of olfactory ensheathing cell (OEC)-containing and pre-degenerated peripheral nerve (PN) transplantation on the axonal regeneration of axotomized retinal ganglion cells (RGC) in adult rats. Methods Twenty-four Sprague-Dawley rats were randomly divided into 4 groups with 6 rats in each group. A segment of the normal (group A) or 10mu;l-OEC-injected (group B) autogenetic sciatic nerve was sutured onto the ocular stump of the left transected optic nerve (ON). In another 2 groups, the removed sciatic nerve was cultured (group C) or co-cultured with OEC (group D) in vitro for 5 days before transplantation. All animals were executed 4 weeks after transplantation, and the number of Fluoro-goldlabeled RGC in each group was counted. Results The averages of regenerating RGC in group B (1481plusmn;268), C (1235plusmn;266) and D (1464plusmn;285) were significantly higher than that in group A (799plusmn;109; P=0.0002, 0.0010 and 0.0003, respectively). No significant difference was found among group B, C and D (P=0.3644, 0.9167 and 0.4344). Conclusion OEC can promote the axonal regeneration of axotomized RGC in fresh PN graft, which doesnprime;t differ much from the effect of the pre-degenerated PN graft. No additive effect of OEC and the pre-degenerated PN graft can be detected. (Chin J Ocul Fundus Dis, 2007, 23: 130-132)
Objective To investigate the influnce of L-arginine (L-Arg) and L-nitro-arginine-methyl-ester(L-NAME) to purified retinal ganglion cells(RGCs) apoptosis of rats cultured in different consistencies of L-Arg and L-NAME. Method RGCs from Sprague Dawley (SD) neonatal rats(postnatal 1~5 day) were cultured in assimilative culture solution in vitro and RGCs were purified by Thy1.1 with sheep anti rat FITC monoclonal antibody. RGCs were cultured in different consistencies of L-Arg and L-NAME: 1×10-6, 1×10-5,1×10-4, 1×10-3, 1×10-2 and 1×10-1 mol/L for 24 hours and 48 hours, respectively. The changes of bcl-2, bax and p53 mRNA in RGCs in different consistencies of L-Arg and L-NAME were demonstrated qualitatively and quantitatively by in situ hybridization, and their apoptosis were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) method, respectively. Results After 24 hours in vitro, the purification rate of RGCs in the experiment arrived at 97 %. After 48 hours, there were a few apoptotic cells expression in the control group. Apoptotic cells expression in L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L groups increased that had a significant difference with the control group (Plt;0.05). In the group of L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L, the expression of bcl-2 mRNA in RGCs became weaker and weaker as the consistencies were increased, but the expression of bax and p53 mRNA in RGCs became higher and higher and had a significant difference with control group (Plt;0.05). Conclusion Lower concentration of L-Arg can promote the growth of purified RGCs in vitro and higher concentration of L-Arg can promote the apoptosis of RGCs. (Chin J Ocul Fundus Dis, 2002, 18: 137-139)
Objective To explore the effects of drugs on functions of mitochondria in retinal nerve cells, and to lay a foundation of the investigation of drug protection for retinal nerve cells. Methods Cultivation of the retinal nerve cells of 8 eyes of neonatal calves was performed. The changes of fluorescent density of the mitochondria of cultured cells labeled by dye rhodamine 123 (Rh123) before and after the activation of the medicines, including ferulic acid (FA), arginine, glycine,taurine, vitamine E and brain derived neurotrophic factor( BDNF) respectively, were detected by laser-scanning confocal microscopy. Results FA with the concentration of 500 μg/ml led the diphasic variation of the fluorescent intensity of mitochondria. After scanning for 60.772 seconds when treated with FA firstly, the fluorescent intensity decreased rapidly (from 45.425±4.153 to 22.135±5.293); while after 112.774 seconds when treated secondly, the in tensity increased obviously (from 19.655±4.383 to 28.247±4.764), and after 168.773 seconds when treated thirdly the intensity still increased. After scanning for 56.457 seconds when treated with vitamin E (12.5 mg/ml), the fluorescent in tensity increased obviously (from 88.255±5.039 to 111.273±4.529), which suggested that vitamin E with the concentration of 12.5 mg/ml strengthen the fluorescent intensity. After scanning for 58.147 and 134.148 seconds when treated with BDNF(50 ng/ml) respectively, the fluorescent intensity increased obviously (from 69.115±5.038 to 77.225±5.131) which suggested that BDNF with the concent ration of 50 ng/ml led the increase of the fluorescent intensity. Glycine (2.5 mg/ml) and arginine(30 mg/ml) didn’t affect the fluorescent intensity of mitochondria, and taurine (6.25 mg/ml) caused the appreciable decrease of the fluorescent intensity . Conclusion FA, BDNF and vitamin E may promote the metabolism of retinal nerve cells via the path of mitochondria, while amino acids may adjust the activation of retinal nerve cells through other ways. (Chin J Ocul Fundus Dis，2004，20：229-232)
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 observe the correlation between the thickness of foveal ganglion cell-inner plexiform layer (GCIPL) and visual field mean defect before and after gamma knife treatment in patients of sellar region tumors with optic chiasmal compression. MethodsThis was a prospective case series. 72 eyes of 37 consecutive patients suffering from optic chiasmal compression of sellar region tumors treated with gamma knife were enrolled in the study. According to the change of visual field before and after gamma knife treatment, the patients were divided into three groups. There were 13 eyes of 7 patients in group 1 with no vision defect pre-and post-treated, 34 eyes of 17 patients in group 2 with improvement of visual field defect after treatment, 25 eyes of 13 patients in groups 3 with no improvement or reorganization of visual field defect after treatment. Overall average thickness of GCIPL, and of the superotemporal, superior, superonasal, inferonasal, inferior, and inferotemporal retina were measured with the Cirrus high-definition spectral domain optical coherence tomography, and mean deviation (MD) with the Humphrey field analyzer before and 6 months after treatment. There was no significant difference in MD values between group 2 and 3 pre-treated (t=1.471, P=0.084). There was significant difference between all the groups in total average value of GCIPL thickness and the 6 quadrant GCIPL thickness values pre-treated (P < 0.05). Logistic regression model was applied to analysis of the correlation between GCIPL thickness and the improvement of visual field after treatment. ResultsThe MD values of the group 1, 2 and 3 were (-2.96 ±0.75), (-10.24 ±1.31), (-20.2 ±5.88) dB at 6 months after treatment. There was significant difference between group 2 and 3 of MD value after treatment (t=6.974, P=0.000). In group 1, there was no significant difference in mean GCIPL thickness and the 6 quadrant GCIPL thickness values between pre-and post-treated (t=0.882, P=0.395).The mean thickness of GCIPL, superonasal and inferonasal GCIPL was increased than pre-treated in group 2, and the difference was statistically significant (t=2.438, 4.630, 4.457; P=0.035, 0.001, 0.001). The mean thickness of GCIPL, superonasal and inferonasal GCIPL was decreased than pre-treated in group 3, and the difference was statistically significant (t=-2.387, -4.603, -4.975; P=0.041, 0.002, 0.001).Logistic regression analysis showed that the greater of the value of average GCIPL thickness of patients with visual field defect pre-treated, the higher of the proportion of patients with improvement of visual field defect post-treated. There was a significant correlation between the value of superonasal or inferonasal GCIPL and the improvement of the visual field post-treated (OR=5.374, 4.693; P=0.000, 0.000). There was no significant correlation between the value of superotemporal or upper or lower or inferotemporal GCIPL and the improvement of the visual field post-treated (OR=1.058, 1.101, 1.074, 1.056; P=0.183, 0.080, 0.162, 0.186). ConclusionsIn patients with optic chiasmal compression of sellar region tumor, the greater of the average GCIPL thickness pre-treated, the higher of the proportion of patients with improvement of visual field defect post-treated. There was a significant correlation between superonasal or inferonasal value of the GCIPL thickness and the improvement of visual field defect post-treated.
Objective To observe the effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell (RGC). Methods Four short hairpin (sh) RNA interference sequences targeting rat Sirt1 gene were designed. The target sequences of Oligo DNA were synthesized and annealed to double strand DNA, which was subsequently connected with pGLV3 lentivirus vector to build the lentiviral vector. The positive clones were identified by polymerase chain reaction (PCR) and DNA sequencing. The lentiviral vector construct and lentiviral packaging plasmids were co-transfected into 293T cells, then the titer of lentivirus were determined. The RGC were divided into 6 groups including blank group, negative control group and si-Sirt1-1, si-Sirt1-2, si-Sirt1-3, si-Sirt1-4 groups. Real-time PCR and Western blotting were used to detect the expression of Sirt1 mRNA and protein in the RGC cells. Results PCR and DNA sequencing analysis confirmed that the shRNA sequence was successfully inserted into the lentivirus vector. The concentrated titer of virus suspension was 8×108 TU/ml after the recombinant lentiviral vector successfully transfected and harvested in 293T cells. Comparing with NC group, the expression of Sirt1 mRNA and protein were significantly decreased in the si-Sirt1-1, si-Sirt1-2, si-Sirt1-3 and si-Sirt1-4 groups (F=27.682, 1 185.206; P=0.000, 0.000). The si-Sirt1-2 group had the strongest effect in reducing the expression of Sirt1 mRNA and protein. Conclusion The 4 lentiviral vectors harboring RNAi targeting rat Sirt1 gene can effectively down regulate the expression of Sirt1 mRNA and protein in RGC cells.
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.
ObjectiveTo investigate the effect of DJ-1 encoded by Park7 gene on retinal ganglion cells (RGC) and visual function after optic nerve crush injury (ONC) in mice.MethodsThirty-seven and 116 healthy male C57BL/6J mice were randomly divided into group normal, group ONC 2d, group ONC 5d, group ONC 7d and group control, group Park7, group Park7-ONC, group ONC and group green fluorescent protein (GFP)-ONC. Group ONC 2d, group ONC 5d and group ONC 7d were sacrificed on the 2nd, 5th and 7th day after the establishment of ONC model, and the follow-up experiments were carried out. The mice in group Park7 and group Park7-ONC were injected 1 μ recombinant adeno-associated virus (rAAV) with knocking down Park7 gene into vitreous cavity, and 1 μ l rAAV with only GFP was injected into vitreous cavity of mice in group GFP- ONC, and virus transfection was observed 4 weeks after injection. The injury of ONC was perfomed at 23 days after vitreous injection in group ONC, group Park7-ONC and group GFP-ONC, and the samples were taken for follow-up experiment 5 days after modeling. The average density of RGC was observed by immunofluorescence staining, the latencies and amplitudes of a-wave, b-wave and photopic negative response (phNR) and the amplitude of oscillatory potential (OPs)were detected by full-field flash electroretinogram,and the visual acuity of mice was measured by optomotor response (OMR). The relative expression levels of DJ-1, Bax and B lymphoblastoma / leukemia-2 (Bcl-2) protein in the retina of mice in each group were detected by Western blot. One-way ANOVA was used to compare the data between groups, and t-test was used for pairwise comparison between groups.ResultsCompared with the normal group, the relative expression of DJ-1 protein in the retina of the ONC 2 d group and ONC 5 d group increased significantly, and the difference was statistically significant (t=16.610, 5.628, P＜0.01,＜0.05). Four weeks after virus transfection, strong GFP expression was seen in the RGC layer and inner plexiform layer of the retina of mice in the Park7 group. Compared with the control group, the RGC density of the retina in the ONC group decreased significantly, and the difference was statistically significant (t=16.520, P＜0.000); compared with the ONC group, the RGC density of the retina in the Park7-ONC group decreased significantly, and the difference was statistically significant (t=6.074, P＜0.01). With the increase of stimulus light intensity, the dark adaptation a wave and b wave latency of the mice in the control group gradually shortened, and the amplitude gradually increased. The stimulus light intensity was 3 cd·s/m2. There was no statistically significant difference in the dark adaptation a wave and b wave latency and amplitude of the control group, Park7 group, Park7-ONC group, ONC group, and GFP-ONC group (Incubation period: F=0.503, 2.592; P=0.734, 0.068. Amplitude: F=0.439, 1.451; P=0.779, 0.247). Compared with the control group, the Ops and PhNR amplitudes of the ONC group mice were significantly decreased (t=15.07, 12.80; P＜0.000,＜0.001). Compared with the ONC group, the Ops and PhNR amplitudes of the mice in the Park7-ONC group were significantly decreased (t=4.042, 5.062; P＜0.05,＜0.01); there was no statistically significant difference in the PhNR latency of the mice in each group (F=1.327, P=0.287). Compared with the control group, the visual acuity of the mice in the ONC group was significantly decreased, and the difference was statistically significant (t=23.020, P＜0.000); compared with the ONC group, the visual acuity of the mice in the Park7-ONC group was significantly decreased, and the difference was statistically significant (t=3.669, P＜0.05). Compared with the control group, Park7-ONC group and ONC group, the relative expression of DJ-1 protein in the mouse retina was significantly down-regulated, and the difference was statistically significant (t=47.140, 26.920; P＜0.000,＜0.000). There was no significant difference between ONC group and GFP-ONC group (t=0.739, P=0.983). Compared with the ONC group, the relative expression of Bax protein in the mouse retina of the Park7-ONC group was significantly increased, and the relative expression of Bcl-2 protein was significantly reduced. The differences were statistically significant (t=5.960, 9.710; P＜0.05,＜0.05); the relative expression ratio of Bcl-2/Bax in the Park7-ONC group was significantly lower than that in the ONC group, and the difference was statistically significant (t=13.620, P＜0.01).ConclusionThe expression of DJ-1 encoded by Park7 gene is down-regulated after Park7 gene was knocked down, which aggravates the RGC damage and the decrease of retinal electrophysiological response and visual function in ONC injury mice.