Objective To study the differential expression profiling of the transcripts modified by m5C methylation in a rat model of N-methyl-D-aspartate (NMDA)-induced retinal excitotoxicity. MethodsA total of 65 Sprague Dawley male rats aged 7-8 weeks were randomly divided into two groups: normal control group and NMDA group. The right eye (model eye) of rats in the NMDA group were injected with 50.0 mmol/L of NMDA 3 μl in the vitreous cavity, while in the normal control group, equal volume of normal saline was injected into the vitreous cavity. After 1 week of the injection, the optic nerve conduction function of rats was detected by visual evoked potential. The whole structure of rat retina was observed by hematoxylin-eosin staining, and the thickness of each retinal layer and the number of retinal ganglion cell layer were detected. The number of β3 tubulin immunofluorescence positive cells was detected by immunofluorescence staining on retinal stretched preparation. Total RNA was extracted from the retinas of normal control group and NMDA group, and high-throughput m5C modified RNA was sequenced, and bioinformatics analysis was performed. The relative expression levels of SLFN3, PLXNB3, CD36 and HIC2 mRNA in retina were detected by real-time quantitative polymerase chain reaction. The comparison between the two groups was performed using an unpaired t test. ResultsThe P1 latency of control group and NMDA group were (117.86±6.48) and (148.46±3.78) ms, and the amplitudes were (42.57±2.41) and (8.68±0.63) μV, respectively. Compared with the normal control group, the latency period was prolonged and the amplitude was significantly decreased in the NMDA group, with statistical significance (P＜0.001). In normal control group, retinal ganglion cells (RGC) were uniformly arranged with large round nuclei. In NMDA group, the volume of retinal RGC was atrophied and the number of RGC was reduced. The total retinal thickness in the control group and NMDA group was (207.51±12.76) μm and (187.51±12.54) μm, respectively. The number of β3 tubulin positive cells was 79.86±6.56 and 29.36±2.16, respectively. Compared with normal control group, the total retinal thickness and the number of β3 tubulin positive cells in NMDA group were decreased, with statistical significance (P＜0.001). Compared with the control group, 576 differentially expressed m5C mRNA were screened in the NMDA group, among which 230 up-regulated and 346 down-regulated genes were detected, respectively. The results of biological information analysis showed that compared with the control group, the upregulated m5C mRNA in the NMDA group was mainly involved in biological processes such as perception and cell-cell adhesion, and was mainly concentrated in the cytokine-cytokine receptor interaction and neural active ligand-receptor interaction pathway. The biological processes in which down-regulated m5C mRNA was mainly involved in biological processes such as G-protein-coupled receptor signaling pathway and cell communication, which were mainly concentrated in primary immune deficiency pathway and neural active ligand-receptor interaction pathway. Real-time quantitative polymerase chain reaction detection results showed that compared with the normal control group, the relative expression levels of SLFN3 and PLXNB3 mRNA in the retina of rats in NMDA group were significantly increased, while the relative expression levels of CD36 and HIC2 mRNA were significantly decreased, with statistical significance (P＜0.05). ConclusionIn NMDA induced retinal excitatory toxicity rat models, m5C modified retinal transcriptome showed abnormal expression.
Objective To observe the retinal apoptosis of laser-induced retinal injury in mice after bone marrow mesenchymal stem cells transplantation. Methods Green fluorescent protein (GFP) labeled MSCs from C57BL/6 mice were cultured in vitro. A total of 135 C57BL/6 mice were divided into three groups including normal control group (15 mice), injured control group (60 mice) and MSCs treatment group (60 mice). Laser retinal injuries were induced by laser photocoagulation. One day after photocoagulation, 02 ml cell suspension, which contained 1times;106 GFP-MSCs, were injected into the mice in treatment group via tail vein, and the mice in injured control group were given equal volume of phosphate buffer solution. Animal were execute on three, seven, 14 and 21 days following laser damage. Hematoxylin and eosin (HE) staining was performed to assess the changes of injured retinas. The diameters of laser spots and areas with total loss of cells in outer nuclear layer (ONL) were analyzed by image processing software. The apoptosis of retinal cells was examined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining. The migration of GFP-MSCs into the retina was observed by fluorescence microscope. Results HE staining showed that the retinal structures were integrated in normal control group. Retinal damages were observed both in injured control group and MSCs treatment group, but milder in the latter. Though the average diameter of area with total loss of cells in ONL of MSCs treatment group was less than the injured control group (t=5.769, P＜0.05), the diameters of laser spots show no difference (t=0.964,P＞0.05) on day three. Both the average diameter of laser spots (t=5.180, 5.417, 2.381) and area with total loss of cells in ONL (t=3.530, 3.224, 3.162) were less in the MSCs treatment group on day seven, 14 and 21 (P＜0.05). TUNEL staining shows that the apoptosis were decreased after MSCs transplantation on day three, seven, 14 and 21 (t=11.142, 7.479, 6.678, 3.953,P＜0.05). No apoptosis was observed in normal control group. Very few GFP-MSCs were observed in the retina at all time-points. They were only seen in the subretinal and choroidal neovascularization occasionally on day seven and 14. Conclusion MSCs transplantation can effectively limit the range of retinal laser damage and inhibit cell apoptosis.
Objective To investigate the effect of peptidoglycan (PGN) on the secretion of pro-inflammatory cytokines by dendritic cells (DCs) and the regulation of T helper 17 (Th17) responses in experimental autoimmune uveitis. Methods Bone marrow cells from naive mice were cultured with granulocyte macrophage-colony-stimulating factor and interleukin (IL)-4 to induce DCs. DCs cultured for six days were randomly divided into two groups: PGNtreated group and control group. The DCs in PGNtreated group were stimulated with PGN and the same volume of phosphate buffered saline was added to the DCs as control group. The relative mRNA expression levels of IL-23, tumor necrotic factor alpha; (TNF-alpha;), IL-6,IL-1beta;were measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Peptide fragment of interphotoreceptor retinoidbinding protein (IRBP1-20)specific T cells, which were isolated from the spleen and draining lymph nodes of C57BL/6 mice immunized with IRBP1-20 peptide fragments 13 days earlier, were co-cultured with PGN-treated or untreated DCs, respectively. Total RNA from T cells cocultured for two days were isolated and the relative expression of retinoic acid receptor-related orphan receptor gamma;t (ROR-gamma;t), IL-17, T-box expression in T cells (T-bet), interferon gamma; (IFN-gamma;) mRNA were detected by realtime RT-PCR. On the second, the fifth and the seventh day, the cocultured T cells were analyzed by flow cytometry to detect the percentages of IFN-gamma;, IL-17 positive cells. Results The real-time RT-PCR results revealed that the level of IL-23, IL-1beta;, IL-6, TNF-alpha; mRNA from PGNstimulated DCs were significantly increased compared to the control group (t=-14.363, -5.627, -3.85, -28.151; P＜0.05). The level of RORgamma;t, IL-17 mRNA from the T cells cocultured with PGN-stimulated DCs were greatly increased compared with the control group (t=-5.601, -19.76；P＜0.05). However, the level of T-bet, IFN-gamma; mRNA from the T cells cocultured with PGNstimulated DCs were significantly decreased compared with the control group (t=4.717, 11.207; P＜0.05). Data of flow cytometry showed that at two days, five days, seven days after cocultured with PGN-treated DCs, the percentages of IL-17 positive T cells were increased compared to the control group (t=-2.944, -3.03, -4.81; P＜0.05), and the percentages of IFN-gamma; positive T cells had no remarkable change (t=-1.25, -0.18, -2.16; P＞0.05). Conclusion PGN can promote the secretion of Th17-related cytokines by DCs, which favors proliferation and differentiation of Th17 in experimental autoimmune uveitis.
ObjectiveTo investigate the relationship between the pathological and functional changes of the retina and the expression of monocyte chemoattractant protein (MCP)-1 after retinal laser injury in mice. MethodsA total of 116 C57BL/6 mice were randomly divided into the normal group (58 mice) and the injured group (58 mice). Retinal laser injuries were induced by Argon ion laser. At 1, 3, 7 days after laser injury, electroretinogram (ERG) responses were recorded to detect the function of the retina. Hematoxylin and eosin (HE) staining was performed to observe pathological changes. Quantitative real-time polymerase chain reaction (PCR) was performed to detect gene expression of MCP-1. Western blot was used to measure the protein expression of MCP-1. ResultsHE staining showed a progressive damage of the retinal structure. The results of ERG showed that the differences of dark-adaptive a wave (t=6.998, 9.594, 13.778) and b wave (t=12.089, 13.310, 21.989) amplitudes of 1, 3 and 7 day post-injury between normal group and injured group were statistically significant (P=0.000). At 1 day post-injury, the differences of light adaptive b wave amplitudes between the two groups were statistically significant (t=8.844, P=0.000). While the differences of light-adaptive a wave amplitudes were not (t=2.659,P=0.200). At 3, 7 days post-injury, the differences of a (t=3.076, 7.544) and b wave amplitudes (t=10.418, 8.485) between the two groups were statistically significant (P=0.000). In dark-adaptive ERG, the differences of a wave amplitudes between 1 day and 3 days (t=3.773), 1 day and 7 days (t=5.070) and b wave amplitudes between 1 day and 7 days (t=4.762) were statistically significant (P<0.01), while the differences of a wave amplitudes between the 3 days and 7 days (t=1.297) and b wave amplitudes between 1 day and 3 days (t=2.236), 3 day and 7 days (t=2.526) were not significant (P=0.660, 0.120, 0.060). In light-adaptive ERG, the differences of a wave amplitudes between 1 day and 7 days (t=2.992) and b wave amplitudes between 1 day and 3 days (t=3.570), 1day and 7 days (t=4.989) were statistically significant (P<0.05), while the differences of a wave amplitudes between 1 day and 3 days (t=0.516), the 3 days and 7 days (t=2.475) and b wave amplitudes between 3 days and 7 days (t=1.419) were not significant (P=1.000, 0.710, 0.070). Quantitative real-time PCR showed that the differences of MCP-1 gene expression at 1, 3 and 7 day post-injury between normal group and injured group were statistically significant (t=14.329, 16.861, 5.743; P<0.05). Western blot showed that the differences of MCP-1 protein expression at 1, 3 and 7 day post-injury between normal group and injured group were statistically significant (t=75.068, 54.145, 14.653; P<0.05). ConclusionIn the first 7 days after mice retinal laser injury, there are progressive pathological and functional damage of the retina, which might be correlated with MCP-1 expression.
ObjectiveTo study morphological characteristics and microRNA (miR) expression profiling in a mouse model of oxygen-induced retinopathy (OIR). MethodsHealthy C57BL/6J female mice and pups were randomly divided into normal and OIR group at postnatal day 7 (P7). The normal group was raised in a conventional cage and exposed to room air for 10 days. The OIR group was raised in a sealed chamber and exposed to (75±2)% oxygen. The moms were alternated between the two groups every day to promote their survival under hyperoxia. The OIR group was returned to the room air at P12. At P17, mice from either group were retro-orbitally injected with high molecular weight fluorescein isothiocyanate-dextran (FITC-dextran), the eye balls were fixed in 4% paraformaldehyde, and the retinal whole mounts were prepared. The retinal vessels labeled with FITC-dextran were observed under a fluorescence microscope; the eye balls were also processed for paraffin sections and Hematoxylin and Eosin (H&E) staining. The cell nucleus in the newly-formed vessels beyond the inner limiting membrane was quantified. The miR was extracted from the eyes, reverse transcribed, and subjected to a customized miR array analysis. The real-time PCR was preformed to verify the results of the miR array. ResultsRetinal whole mounts labeled with FITC-dextran showed that the peripheral retinal microvessels in the OIR group were tortuous, disorganized with neovascular buds, and the avascular area was prominent in central retina. In contrast, the vessels were smooth, organized, and evenly distributed in the retinas of normal group. The percentage of avascular area in total retina area in OIR group (25.81±2.12)% was 4-fold that in normal group (6.57±3.6)% (P < 0.01, normal group vs OIR group). H & E staining showed that the number of the cell nuclei beyond inner limiting membrane was (28.41±4.01) in OIR retina, which was substantially higher than that (0.16±0.31) in normal retina (P < 0.01, normal group vs OIR group). More interestingly, the results of miR array showed that 21 out of the 80 miRs examined exhibited more than 1.5-fold changes at expression level. Among these 21 miRs, 9 were up-regulated, 12 were down-regulated; 4 miRs showed more than 3-fold expression changes, 3 were down-regulated and 1 was up-regulated. The expression of the 4 miRs was verified by real-time PCR. The expression trends of miR-3078, miR-140, miR-29b and miR-29c were consistent with those revealed by the miR array. MiR-3078 was significantly up-regulated (t=-2.380, P < 0.05. normal group vs OIR group), and the other 3 miRs were significantly down-regulated (t=2.638, 2.323, 2.415, P < 0.05. normal group vs OIR group). ConclusionsThe OIR mouse model has been established in our study. Differential expression of the microRNAs, including miR-3078, 140, 29b and 29c, was detected in normal and OIR mouse retinas. These miR expression changes may be associated with retinal neovascularization. These results would provide the new leads for further studying pathogenic mechanisms and therapeutic targets for neovascular retinopathy.
ObjectiveTo observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Müller cells induced by hydrogen peroxide (H2O2).MethodsHuman retinal Müller cells cultured in vitro were divided into normal control group, model group (H2O2 group) and experimental group (H2O2+NBP group). The cells in the H2O2 group and H2O2+NBP group were cultured with 200 μmol/L H2O2 for 2 h. Then the culture solution of the H2O2 group replace with complete medium and the H2O2+NBP group replace with complete medium containing 1 μmol/L NBP. The normal control group was a conventional cultured cells. Müller cells were identified by immunofluorescence staining. Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes. MTT assay was used to detect the activity of of retinal Müller cells after after 24 h and 48 h of NBP intervention. Hoechst33258 staining was used to observe the apoptosis. LIVE/DEAD ® cell activity/cytotoxicity kit was used to detect cell viability. Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells. One-way ANOVA combined with Dunnett statistical method were used for data analysis.ResultsHE staining showed that the number of cells in H2O2+NBP group was higher than that in H2O2 group. MTT assay showed that after 24 h and 48 h of NBP intervention, the differences in cell viability between the normal control group and the H2O2 group, the H2O2 group and the H2O2+NBP group were statistically significant (t=28.96, 3.658, 47.58, 20.33; P＜0.001, 0.022). The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H2O2+NBP group was crescent-shaped and the nuclear fragmentation was reduced, and the blue fluorescence of the remaining cells was uniform. The LIVE/DEAD ® cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H2O2 group increased significantly, and the number of viable cells with green fluorescence decreased significantly. In the H2O2+NBP group, the number of viable cells with green fluorescence increased, and the number of dead cells with red fluorescence decreased. The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H2O2 group was significantly enhanced; the green fluorescence intensity of H2O2+NBP group was lower than that of H2O2 group.ConclusionNBP alleviates H2O2-induced apoptosis of human retinal Müller cells by inhibiting ROS production.
ObjectiveTo analyze the expression of miRNA involved in regulating retinal neovascularizationin in retinal tissue of oxygen-induced retinopathy (OIR) mice.MethodsEighty healthy C57BL/6J mice were randomly divided into control group and OIR group at postnatal day 7(P7). Control group were not received any treatment and then exposed to room air. The OIR group was exposed to (75±2)% oxygen and then under room air at P12. Mice of all groups were euthanized at P17. Retinal neovasculation (RNV) was evaluated by counting the number of pre-retinal neovascular cells and analysing no perfusion area by immunofluorescent staining of the mouse retina.Total RNA was extracted from retinal tissue,and miRNA microarrays was performed to identify differentially expressed miRNA in the two groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed differential microRNA.ResultsCompared with the control group,the retinal neovascular tufts and the no perfusion area were both significantly smaller than those in OIR group. The number of pre-retinal neovascular cell nuclei in retinas from control group were obviously lower than those in the retinas from OIR group (t=9.025, P＜0.05). MiRNA microarray analysis showed that 54 miRNA in OIR group showed statistically different expression in control group, 47 miRNA were up-regulated and 7 miRNA were down-regulated. The results of PCR were consistent with the trend of microarray. In GO analysis, 1112 items were significantly different (P＜0.05), and 65 items were significantly different in KEGG analysis of expression profile (P＜0.05).ConclusionsThe miRNA expression in retinal tissue of OIR mice is different from that of normal mice, and these miRNA may be involved in the development of RNV. There are 54 miRNA expression differences in retinal tissue of OIR compared with normal mouse retinal tissue.
ObjectiveTo investigate the protection and the corresponding molecular mechanisms of polypyramidine tract binding protein-associated splicing factor (PSF) overexpression on human retinal microvascular endothelial cells (hRMECs) induced by advanced glycation end-products (AGEs).MethodsThe hRMECs were divided into the normal group, the vector group, PSF group, zinc protoporphyrin (ZnPP) group and PSF+ZnPP group for experiment. Cells in the normal group were cultured in a DMEM medium containing 10% fetal calf serum, penicillin/streptomycin, and placed in a closed constant temperature incubator at 37 °C, 95% air, and 5% CO2. Cells in the vector group were infected with empty lentivirus. The cells in the PSF group were infected with overexpressing PSF lentivirus. Cells in the ZnPP group were treated with ZnPP (10 mol/L) for 2 h. The PSF+ZnPP group cells were infected with overexpressing PSF lentivirus, and then pretreated with ZnPP (10 mol/L) for 2 h. With the last four groups of cells stimulated with AGEs, HE, Hoechst33258 staining and flow cytometry were used to observe the protective effect of high expression of PSF on cell damage and the antagonistic effect of ZnPP on PSF. Western blot was used to detect the protein expression of heme oxygenase-1 (HO-1), phosphorylated (p) extracellular regulatory protein kinase (ERK), and Nrf2 in the cells. U0126, a specific antagonist of ERK pathway, was introduced, and Western blot verified the reversal effect of U0126 on the expression of HO-1 induced by PSF protein.ResultsHE staining and Hoechst33258 staining showed that the number of nuclei of damaged cells of PSF group were significantly increased compared with control group, while decreased compared with PSF+ZnPP group (F=27.5, 38.7; P＜0.05). The results of flow cytometry showed that the ROS produced by cells in the PSF group was significantly increased compared to the normal group, and significantly decreased compared to the PSF+ZnPP group, the difference was statistically significant (F=126.4, P＜0.05). Western blot results showed that HO-1 expression of PSF group was significantly increased compared with control and the vector group (F=70.1, P＜0.05). AGEs inducement of 30, 60, 120 and 240 min could significantly improve pERK expression compared with 15 min (F=474.0, P＜0.05). The expression of HO-1 and Nrf2 proteins in the PSF+/U0126－ group was significantly more than those in the PSF－/U0126－ group, the expression of HO-1 and Nrf2 proteins in the PSF+/U0126+ group was significantly lower than that in the PSF+/U0126－ group, and the differences were statistically significant (F=30.2, 489.4; P＜0.05).ConclusionOver expression of PSF can promote the HO-1 expression by activating ERK pathway and promoting the Nrf2 to the nucleus, thus protect hRMECs against AGEs-induced oxidative damage.
ObjectiveTo observe the inhibitory effect of lentiviral vector miR-191 (LV-191) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).MethodsEighty healthy 7-day-old C57BL/6J mice were randomly divided into 5 groups including normal group, non-intervention group, normal saline (NS) group, LV-191 group and LV-green fluorescent protein (GFP) group, 16 mice in each group. The OIR model was established in the non-intervention group, NS group, LV-191 group and LV-GFP group. NS group, LV-191 group and LV-GFP group were given an intravitreal injection of 1 μl of NS, LV-191 and LV-GFP at the age of 12 days. No injection was performed in the non-intervention group. In normal group,newborn mouse were maintained in room air form P0 to P17, and no treatment was performed. Mice in all five groups were euthanized at P17. Retinal neovasculation (RNV) was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR (RT-PCR) to detect miR -191 and P21 expression of retinal tissue.ResultsIn the LV-191 group, the non-perfusion area were both significantly smaller than those in non-intervention group, NS group and LV-GFP group (F=127.20, P＜0.001). The number of pre-retinal neovascular cell nuclei in retinas from LV-191 group were obviously lower than those in the retinas from non-intervention group, NS group and LV-GFP group (F=31.71, P＜0.05). RT-PCR showed that the LV-191 and P21 level of LV-191 group increased significantly than other groups (F=10.95, 15.60; P＜0.05).ConclusionIntravitreal injection of LV-191 inhibits RNV in mice model of OIR possibly through up-regulating p21.