Objective：To observe the protective effect of ginkgo bilo ba extrac t (EGb 761), a free radical scavenger, on the photoreceptor cells after lighti nduced retinal damage. Methods：Seventytwo female SpragueDa wley (SD) rats we re randomly divided into 4 groups: normal control group, lightinduced retinal da m age model group, model+physiological saline group, and model+EGb 761 group, with 18 rats in each group. All of the rats except the ones in the control group were exposed to white light at (2740plusmn;120) lx for 6 hours after the dark adap tation for 24 hours to set up the lightinduced retinal damage model. Rats in m o del + physiological saline group and model+EGb 761 group were intraperitoneall y injected daily with physiological saline and 0.35% EGb 761 (100 mg/kg), respec tively 7 days before and 14 days after the light exposure. Apoptosis of photorec eptor cells was detected 4 days after light exposure; 7 and 14 days after light exposure, histopathological examination was performed and the layer number of ou ter nuclear layers (ONL) on the superior and inferior retina was counted. Results：Four days after light exposure, the apoptosis of photorecep tor cells was fou nd on ONL in model, model+ physiological saline and model+EGb 761 group, and w as obviously less in model + EGb 761 group than in model and model+physiologic al saline group. Seven days after light exposure, the layers of ONL on the super ior retina were 3 to 4 in model and model+physiological saline group, and 7 to 8 in model+EGb 761 group; the mean of the layer number of ONL in model+EGb 761 group (6.92plusmn;0.82) was less than that in normal control group (8.40plusmn;0.95) (t=-1.416, P＜0.05), but significantly more than that in model (5.96 plusmn;1.36 ) and model+physiological saline group (5.90plusmn;1.40）（t=1.024, 1.084; P＜0.05). Fourteen days after light exposure, the layers of ONL on the superior retina were 0 to 1 in model and model+physiological saline group, and 3 to 4 i n model+EGb 761 group. The mean of the layer number of ONL in model+EGb 761 group (5.5 2plusmn;1.06) was significantly more than that in model (3.44plusmn;2.15) and model + physiological saline group (3.37plusmn;1.91) (t=2.082, 2.146, P＜0.05). Conclusion：EGb 761 can partially inhibit the apoptosis of pho toreceptor cells, thus exert protective effect on photoreceptor cells.
Objective To observe the effect of high glucose on the expression of activating transcription factor 4 (ATF4) in cultured retinal Muuml;ller glia cells. Methods The retinal tissue of Sprague-Dawley (SD) rats was collected, and Muuml;ller cells were isolated and cultured. The glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) of Muuml;ller cells were identified by streptavidin-biotin-peroxidase complex. Cultured rat Muuml;ller cells were divided into control group (5.5 mmol/L glucose), group A (20 mmol/L glucose), group B (30 mmol/L glucose) and group C (40 mmol/L glucose). ATF4 protein expressions in Muuml;ller cells of four groups were measured by Western blot four days after cultured. Results GFAP and GS expressed in more than 95% of Muuml;ller cells. Over 95% of Muuml;ller cells of group A, B and C were positive for GFAP and GS. Western blots indicated that ATF4 protein in group A, B and C increased obviously compared with the control group (q=0.293, 0.754,0.484;P＜0.05). Conclusion High glucose can increase the expression of ATF4 protein and cause endoplasmic reticulum stress in retinal Muuml;ller glia cells in vitro.
ObjectiveTo observe the effects of Rhodiola on the rat retinal tissue morphology and the hypoxia-inducible factor (HIF)-1α at simulated hypoxia at different altitudes. Methods Forty-eight adult female Sprague Dawley rats were randomly divided into the Rhodiola Intervention group (intervention group) and the control group, each group had 24 rats. The intervention group rats were treated with intraperitoneal injection of 10 ml/kg of large plants Rhodiola solution, and the control group rats were injected with same volume of saline. One hour after the injection, six rats were randomly selected from both of the two groups and reared in the plateau environment simulation laboratory modules with the oxygen partial pressure of 17.4, 14.6, 11.3 and 7.4 kPa, which simulated the altitudes of 1500, 3000, 5000 and 8000 meters indoor respectively. Six hours later the rat eyeballs were harvested for paraffin sections and analyzed by hematoxylin and eosin staining, and immunohistochemical staining to observe the expression of HIF-1α and p53. ResultsIn the control group, the rat retinal layers were edema and loose, the retinal thickness increased, the retinal structure was disorganized, the ganglion cells were swollen and degenerated, and some can observe the karyopyknosis, karyolysis and the reduced cells number. As the altitude increased, the pathological changes of retinal became more obvious. In the intervention group, the characteristics of rat retinal morphology were same with the control group, while the degree of morphology changes was lighter than the control group. HIF-1α and p53 expressed mainly in the ganglion cell layer and inner nuclear layer of rat retina in the control group. As altitude increased, the expression of HIF-1α and p53 were increased too, which was positive correlated (r=0.9846, P < 0.05). Compared with the control group, the rat retinal expression of HIF-1α increased, while expression of p53 decreased in the intervention group, and the differences were statistically significant (P < 0.05). ConclusionRhodiola can reduce the retinal tissue pathology damage caused by high altitude hypoxia, and its mechanism may be related to the increasing expression of HIF-1α and reducing expression of p53.
ObjectiveTo study the inhibitory effects of pigment epithelium derived factor (PEDF) on oxygen-induced retinal neovascularization in mice, and to investigate the possible involvement of interleukin-1β (IL-1β) in the neovascular-inhibitory function of PEDF. Methods A total of 140 postnatal day (P)7 C57BL/6 mice were randomly divided into normal control group, oxygen-induced retinopathy (OIR) model group, PEDF treatment group and PBS treatment control group. All mice except normal control group with their mothers were exposed to (75±2)% oxygen environment for 5 days and then kept in room air for another 5 days to establish the OIR model. Mice in normal control group were kept in room air only. At P12 and P14, respectively, mice in PEDF treatment group received intravitreous injections of 1 μl PEDF (2 μg/μl), while PBS treatment control group received the same volume of PBS (10 mmol/L, pH7.4).All mice were euthanized at P17 and eyes were isolated. The changes of retinal vessels were observed on retinal flat mounts and cryosections by fluorescence microscopy. Retinal specimens were prepared for IL-1β protein and mRNA analysis by Western blot and real time fluorescence quantitative reverse transcription-polymerase chain reaction (Real-time RT-PCR). ResultsChanges of retinal vessels had been viewed by fluorescence microscopy on flat-mounted retina, the relative retinal neovascularization areas were significantly increased in OIR model group compared with normal control group (t=15.02, P < 0.01), and the relative retinal neovascularization areas were obviously smaller in PEDF treatment group than those in PBS treatment control group (t=5.96, P < 0.01). Fluorescence staining revealed that retinal vascular tufts were extending from outer plexiform layer (OPL) to ganglion cell layer (GCL) of the retina along with multiple interconnections; Neovascular tufts in OIR model group and PBS treatment control group were presenting distinctly more than those of normal control group and PEDF treatment group. The specific expression levels of IL-1β protein in retinas of OIR mice by Western-blot analysis were higher than those of normal control group(t=3.35, P < 0.05), While these of PEDF treatment group showed a considerable decline in comparison with PBS treatment control group (P < 0.01), and there were no difference in normal control group and PEDF-treated group (F=11.764, P > 0.05). Similarly, expression levels of IL-1β mRNA tested by Real-time RT-PCR were obviously increased in the OIR model group when compared to normal control group(t=4.43, P < 0.01). After treated with PEDF, expression levels of IL-1β mRNA showed a considerable decrease when compared to PBS treatment control group (P < 0.01), and there were no difference in normal control group and PEDF-treated group (F=11.15, P > 0.05). ConclusionsPEDF can inhibit oxygen-induced retinal neovascularization. The mechanism may be related to that PEDF can downregulate the expression of IL-1β in retina.
Objective To observe the interferon-gamma; (IFN-gamma;), interleukin-2 (IL-2) levels of Th1 cytokine and IL-4、IL-10 levels of Th2 cytokine in serum and culture supernatants of splenic cells of the rats in the prevention of experimental autoimmune uveoretinitis（EAU）by oral tolerance. Methods 72 Lewis rats were randomly divided into EAU group，oral tolerance group (which including 10 mu;g、100 mu;g、1 mg、10 mg of S antigen group respectively) and control group，12 rats in each group. The animal model of EAU was induced by immunization with S antigen（50 mu;g）and Freundrsquo;s complete adjuvant. Oral tolerance 10 mu;g、100 mu;g、1 mg and 10 mg group were fed with 1 ml mixture of 10 mu;g、100 mu;g、1 mg、10 mg S antigen and 1 mg trypsin inhibitor respectively by intubation，once the other day，totally 7 times，and then induced EAU according to above methods；control group was fed with 1 ml mixture of phosphate buffered saline and 1 mg trypsin inhibitor，once the other day，totally 7 times，and then induced EAU. The clinical manifestation of EAU in the eye were recorded,the eyeballs were enucleated at the peak of EAU，followed by pathological grading. Meanwhile the serum was colleced; splentic cells were separated and cultured to collect the supernatant. Cytokine levels of IFN-gamma;, IL-2, IL-4 and IL-10 in serum, cultured supernatant of splenic cells were determined by enzyme-linked immunosorbent assay (ELISA). Results Compared with EAU and control group, the levels of IFN-gamma; and IL-2 (Th1 cytokine) in the serum in 100 mu;g and 1 mg group were decreased while the levels of IL4 and IL10 (Th2 cytokine) were increased，the differences were statistically significant（F=51.9, 68.8, 35.7,7.5,P＜0.01). Compared the levels of Th1 and Th2 cytokines in the serum in 10 mu;g, 10 mg group with EAU and control group, the differences were not statistically significant. In 100 mu;g、1 mg group, the levels of IFN-gamma; and IL-2 (Th1 cytokine) in the culture supernatant of splenic cells were decreased while the levels of IL-4 and IL-10 (Th2 cytokine) were increased, compared with EAU and control group, the differences were statistically significant（F=57.1,15.6,33.1,167.7, P＜0.01）. Compared the levels of Th1 and Th2 cytokine in the culture supernatant of splenic cells in 10 mu;g、10 mg groups with EAU and control group, the difference are not statistically significant. Conclusions In the process to prevent EAU by oral intake, the levels of IFN-gamma; and IL-2 (Th1 cytokine ) were decrease while the levels of IL-4 and IL-10 (Th2 cytokine). Oral administration with too high or low dose of the antigen can not prevent EAU as well as the cytokine levels do not change obviously. Cytokines has played an important role in the prevention of EAU.
Objective To investigate the effect of proanthocyanidins (PC) on retina of rats with acute ocular hypertension. Methods The SpraqueDawley (SD) rats were randomly divided into the normal group, the model group and PC high or lowdosage group. The PC high or low dose group received 300mg/kg.d or 100mg/kg.d of PC in suspension solution for 5 days respectively. The normal group and the model group fed with distilled water for 5 days. Then acute ocular hypertension was induced in the model group and all PC groups, and after 48h of ocular hypertension the eyeballs were analyzed by electron microscope and UV spectrophotometer to measure the levels of superoxide dismutase(SOD), malonaldehyde (MDA), nitrogen monoxidum (NO), glutamic acid (Glu) and calcium ion(Ca2+). Results PC could raise the activity of SOD and reduced the levels of MDA,NO,Glu and Ca2+ in retina tissue. Electron microscope examination revealed that PC reduced retinal edema and ganglion cell apoptosis. PC also enhanced the SOD activity and suppressed the levels of MDA, NO, Glu and Ca2+. Conclusions PC can protect retina from acute ocular hypertension. The main mechanism might relate to anti-free radical oxidation, antagonizing calcium overloading, reducing toxicity of NO and Glu on the retina.
ObjectiveTo develop an experimental model of gastroesophageal reflux-induced esophageal stricture in rats and explore the mechanism of esophageal stricture. MethodsA total of 30 male Sprague-Dawley (SD) rats by random number table method were randomly divided into three groups as follows: an operation+acid perfusion group, first the models of lower esophageal sphincter relaxation and hiatal hernia were made, and then the rats’ esophagus were perfused with hydrochloric acid-pepsin; acid perfusion group, the rats’ esophagus were directly perfused with hydrochloric acid-pepsin; and control group, rats’ esophagus were perfused with normal saline. After 4 weeks of continuous perfusion, the esophageal mucosal injury of SD rats in each group were observed, and the concentrations of inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-18] in esophageal tissues were detected by enzyme-linked immunosorbent assay. ResultsIn the operation+acid perfusion group, esophageal stricture was formed in 2 SD rats, but no esophageal stenosis was found in the acid perfusion group and the control group. The body weight of rats in the operation+acid perfusion group and the acid perfusion group were lower than that in the control group (P<0.05). The esophageal mucosal injury scores of rats in the operation+acid perfusion group and the acid perfusion group were higher than that in the control group (P<0.001), and the operation+acid perfusion group was higher than that in the acid perfusion group (P=0.014). The concentrations of TNF-α, IL-1β and IL-18 in esophageal tissues were higher in the operation+acid perfusion group and the acid perfusion group than that in the control group (P<0.001), and the operation+acid perfusion group was higher than that in the acid perfusion group (P<0.001). ConclusionsThe anti-reflux barrier is an important part of preventing gastroesophageal reflux disease. The destruction of anti-reflux barrier, hydrochloric acid-pepsin perfusion and inflammatory cytokines jointly induced esophageal inflammation and injury, and even caused esophageal stricture.
Objective To observe the expression of miRNA in retinal tissue of mice with oxygen-induced retinopathy (OIR), and screen miRNAs related to p21 and retinal neovascularization (RNV) formation. MethodsA experimental study. Forty healthy 7-day-old C57BL/6J mice were randomly divided into normal group and OIR group, with 20 mice in each group. The oxygen induced RNV model was constructed in the OIR group, and no treatment was performed in the normal group. At the age of 17 days, the mice were killed and the RNV of mice was observed by retinal fluorescence; the nuclei of vascular endothelium that broke through the inner limiting membrane of retina were counted under light microscope. The retinal tissues were taken for miRNA chip analysis to detect the differentially expressed miRNAs between the normal group and the OIR group. The resulting differential miRNA target genes were subjected to enrichment analysis based on gene annotation (GO) and Kyoto Encyclopedia of genes and genomes (KEGG); miRNAs and pathways that may be related to p21 were screened through Targetscan, MiRanda and MicroT-CDs database alignment. Independent sample t-test was used for pairwise comparison between groups. ResultsCompared with the normal group, the area of nonperfusion area, RNV and the number of vascular endothelial nuclei that broke through the inner limiting membrane of the retina in the OIR group increased significantly, differences were statistically significant (t=18.800, 9.025; P＜0.05). Compared with the normal group, there were 54 miRNAs that were statistically differentially expressed in the OIR group, of which 47 were up-regulated and 7 were down-regulated. A total of 13 miRNAs related to p21 were screened from the alignment results of the three databases with the obtained differential miRNAs. According to the difference multiples, they were miR-7218-5p, miR-322-5p, miR-224-5p, miR-335-5p, miR-329-3p, miR-362-3p, miR-532-5p, miR-20b-5p, miR-20a-5p, miR-195a-5p, miR-423-5p, miR-497a-5p, and miR-129-5p. Differential miRNA target gene enrichment analysis yielded 1 112 go entries and 50 KEGG pathways, of which 50 go entries and 13 KEGG pathways were related to p21. Conclusion13 miRNAs related to p21 were screened out in the OIR model.
Objective To observe the toxic effects of intravitreal injection of etoposide on retinal ganglion cells (RGC) in rabbit eyes. Methods Twenty-four healthy rabbits were divided into 8 groups randomly, including one normal group without any treatment, and 7 experimental groups with intravitreal injection of different dosages of etoposide (0.0, 1.5, 15.0, 150.0, 375.0, 750.0, 1500.0 mu;g).Twelve days after the injection, the animals were killed by air embolism, and the eyeballs were prepared for routine pathological examination with hematoxylineosin staining and transmission electron microscopy. Results Four experimental groups (0.0, 1.5, 15.0, 150.0 mu;g etoposide) had normal retinal structure and normal shape of ganglion cells while other 3 experimental groups with higher dosage of etoposide (375.0, 750.0 and 1500.0 mu;g) showed thinner and disorganized retina. The number of RGC of the normal group and 0.0, 1.5, 15.0, 150.0, 375.0, 750.0, 1500.0 mu;ggroups were 61.66plusmn;3.72, 61.83plusmn;4.59, 60.00plusmn;7.07, 62.00plusmn;4.43, 62.67plusmn;3.98, 7.33plusmn;1.37, 0.00 and 0.00 per 400times;visual field, with statistical difference among groups （F=145.04，P=0.00）. There was no statistical difference between the normal group and 0.0, 1.5, 15.0, 150.0 mu;g groups （F=0.244，P＞0.05）. The number of RGC of 375.0 mu;g group was decreased obviously compared with normal group and 0.0, 1.5, 15.0, 150.0 mu;g groups with statistical difference（F=145.04，P＜0.05）. Conclusion Etoposide has a dosage-dependent toxicity on rabbit RGCs.
Objective To investigate if lactic acid can promote the expression of vascular endothelial growth factor (VEGF) in the rat retinal explants.Methods The retinas of two-week neonatal SD rats were placed onto the culture plate inserts and incubated with Dulbeccoprime;s modified Eagleprime;s medium （DMEM） plus 2% fetal bovine serum (FBS) containing 10,20,30 mmol/L of lactic acid, respectively. Each group had 24 retinas. At 24 hours after incubation, the retinas were sectioned for light microscopy and the expression of VEGF was measured by real time PCR and Western blot. Results The cultured retinas maintained intact construction, and no cytolysis and apoptosis were observed under light microscope. RT-PCR showed the levels of VEGF mRNA were 0.74plusmn;0.06 for 10 mmol/L lactic acid group, 0.99plusmn;0.12 for 20 mmol/L group, and 1.45plusmn;0.17 for 30 mmol/L group respectively. VEGF expression was 0.34plusmn;0.15 for 10 mmol/L, 0.54plusmn;0.16 for 20 mmol/L, and 0.93plusmn;0.23 for 30 mmol/L group respectively by Western blot. Both PCR and Western blot showed 30 mmol/L of lactic acid significantly increased the levels of VEGF mRNA and VEGF expression. Conclusion The induction of retinal VEGF by lactic acid is concentration-dependent.