Objective To observe the changes of electrophysio logical results in rabbits with normal and injured photoreceptor due to subretinal implantation of chip. Methods Photoreceptor damage was induced by injection with NaIO3 solution in 22 out of 30 rabbits. A chip with the diameter of 3 mm made by the array composed of 90 microelectrodes photodiode and conjoint electrode was implanted into subretinal space or choroid of the right eyes of 22 rabbits with photoreceptor and 4 normal rabbits, and the left eyes were the control. The examinations of local flash-visual evoked potential (F-VEP), local flash-electroretinogram (F-ERG), full-field F-ERG and full-filed F-VEP were measured respectively.Another 4 rabbits underwent biocular extirpation for path ological examination . Results In 22 rabbits with photo-receptor damage, the amplitude of the main wave of local ERG was obviously higher in 11 eyes with chips than that in the control ones, and was also higher in 2 eyes with chips of the 4 mormal rabbits than that in the control eyes. No wave was found in an eye with retinal hole on the surface of the chip. The repeataility of main amplitude of local-VEP and full-field F-VEP is not satisfactory; no significant changes were observed between chip-implanted eyes and the control eyes examined by full-filed F-ERG. Conclusion The implanted chip may stimulate local retina and induce electrical activities after stimulated by light. (Chin J Ocul Fundus DIs, 2006, 22: 324-327)
ObjectiveTo analyze the early changes of gene expression levels and signaling pathways in 661W cell line under hypoxic conditions and to find potential functional target genes.MethodsThe cultured mouse 661W cells were divided into hypoxia treatment group and normoxia control group. Cells in the hypoxia treatment group were cultured in a three-gas incubator with volume fraction of 1% and 5% CO2 at 37 ℃. Cells in the normoxia control group were cultured in an incubator at 37 ℃ with volume fraction of 5% CO2. High-throughput sequencing technology was used to sequence the transcriptome of 661W cell treated with hypoxia and normoxia for 4 hours to screen for differentially expressed genes (DEG). Clustering heat map analysis, gene ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network (PPI) analysis were performed. The reverse transcription-polymerase chain reaction (RT-PCR) was used to verify the accuracy of the sequencing results.ResultsA total of 506 differentially expressed genes were screened, including 459 up-regulated genes and 47 down-regulated genes. GO functional enrichment analysis showed that the main biological processes of DEG were the cell's response to hypoxia, glycolysis, negative regulation of cell proliferation and apoptosis. hypoxia inducible factor (HIF)-1α pathway, glycolysis, Forkhead box O (FoxO) pathway, Insulin signaling pathway and Adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway were involved in the above process. PPI analysis results showed that hub genes related to hypoxia were Aldoa, Aldoc, Gpi1, Hk2, Hk1, Pfkl, Pfkp, Vhl, Fbxo10 and Fbxo27. The RT-PCR results showed that the relative expression levels of 15 DEG mRNA in the hypoxic treatment group were higher than that of the normoxic control group, and the difference was statistically significant (P＜0.05). The mRNA expression levels of N-myc downstream-regulated gene-1 (Ndrg1), Mt1, and vascular endothelial growth factor A (VEGFA) were time-dependent on hypoxia.ConclusionsUnder hypoxia, DEG is mainly related to glucose metabolism, cell response to hypoxia, regulation of proliferation and apoptosis. HIF-1α pathway, glycolysis, FoxO pathway and AMPK pathway are involved in the early changes of 661W cells under hypoxia. Aldoa, Aldoc, Gpi1, Hk2, Hk1, Pfkl, Pfkp, Vhl, Fbxo10, Fbxo27 may play key roles in the response of 661W cells to hypoxia. Ndrg1, Mt1 and VEGFA could be potential functional target genes for the study of ischemia and hypoxia-related fundus diseases.
Objective To investigate the protective effect of recombinant erythropoietin (EPO) on the photoreceptor cells in rat with retinal detachment (RD).Methods One hundred and sixtytwo normal male rats were randomly divided into normal control (NC) group, RD model group, RD+phosphate buffer solution （RD+PBS） group, RD+EPO 100 ng group, RD+EPO 200 ng group and RD+EPO 400 ng group. Three days after RD, activated caspase3 and bclXL were detected by Western blot and/or immunofluorescence, and apoptosis were measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick-end labeling（TUNEL）. Fourteen and 28 days and two months after RD, the outer nuclear layer （ONL）thickness was measured by histopathologic method.Results Western bolt indicated that the protein level of activated caspase-3 and bcl-XL between six groups were statistically significant（F=35.96, 30.75；P＜0.01）. The number of TUNEL positive cells and activated caspase-3 positive cells are consistent with each other in different groups. Fourteen days and two months after RD，the differences of ONL thickness between six groups were statistically significant（F=21.52，96.25；P＜0.01）.Conclusion Supplement of EPO after RD can alleviate apoptosis by inhibiting of the caspase-3 activity and increasing the expression of bcl-XL,thus exerts protective effect on photoreceptor cells.
Objective To investigate the effect of astragaloside A (AS-A) on the photoreceptor degeneration induced by sodium iodate (NaIO3) and its related mechanism. MethodsSixty healthy male C57BL/6J mice, aged 6-8 weeks, were randomly divided into normal control (NC) group, NaIO3 group, and AS-A group, with twenty mice in each group. 30 min before modeling, AS-A group mice were intraperitoneally injected with 100 μl AS-A at a dose of 100 mg/kg body weight. 30 min later, mice in NaIO3 group and AS-A group were intraperitoneally injected with 100 μl NaIO3 at a dose of 30 mg/kg body weight. Subsequently, AS-A group mice were administered AS-A twice daily at 12 h intervals until the end of the experiment. On day 1 post-modeling, zonula occludens-1 (ZO-1) immunohistochemistry was performed to observe the structure of retinal pigment epithelium (RPE) cells; real-time quantitative polymerase chain reaction (qPCR) was conducted to detect the mRNA expression of various retinal chemokine ligand-2 (Ccl2), interleukin-1 beta (Il-1β), mixed lineage kinase domain-like protein (Mlkl), receptor-interacting protein kinase 3 (Ripk3), and tumor necrosis factor (Tnf). On day 3 post-modeling, immunohistochemistry was performed to observe the expression of ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acid protein (GFAP) in the retina; TdT-mediated dUTP nick-end labeling (TUNEL) assay was used to detect photoreceptor cell death in each group. On day 4 post-modeling, fundus morphology of mice in each group was observed by fundus color photography and optical coherence tomography (OCT). Hematoxylin-eosin staining (HE) was used to observe the morphological structure of the retina in each group. Inter-group comparisons between two groups were conducted using independent samples t-test, while comparisons among three groups were performed using one-way ANOVA. ResultsFundus color photography and OCT examination showed that a large number of scattered yellow-white subretinal nodular structures in the fundus of NaIO3 group mice, and a large number of strong reflection areas in the RPE layer. The number of strong reflection areas in the RPE layer was reduced in the AS-A group. Immunohistochemical analysis of ZO-1 showed that ZO-1 was largely lost on the RPE cell membrane in that NaIO3 group; whereas in the AS-A group, ZO-1 was evenly distributed on the RPE cell membrane. HE staining results showed circular black deposits were visible in the RPE layer of the NaIO3 group, and the inner and outer segments of photoreceptors were severely damaged, with a significant decrease in the number of outer nuclear layer (ONL) cell nuclei; whereas in the AS-A group, the RPE layer pigments were orderly, the inner and outer segments of photoreceptors were intact, and the number of ONL cell nuclei significantly increased. The results of TUNEL staining show that numerous TUNEL-positive cell nuclei were observed in the ONL of the retina in the NaIO3 group, while the number of TUNEL-positive cell nuclei in the ONL of the retina was significantly reduced in the AS-A group, with statistically significant differences (t=2.66, P＜0.05). The analysis of qPCR data showed that compared with the AS-A group, the relative expression levels of Mlkl, Ripk3, Ccl2, Il-1β and Tnf mRNA in the retina were significantly increased in the NaIO3 group, with statistically significant differences (F=39.18, 10.66, 53.51, 41.40, 24.13; P＜0.001). Immunohistochemical staining results showed that compared with NC group and AS-A group, the positive expression of GFAP in retina of NaIO3 group was significantly increased, and the difference was statistically significant (F=9.62, P＜0.05). ConclusionAS-A antagonizes NaIO3-induced photoreceptor degeneration in part by inhibiting photoreceptor cell death and neuroinflammation. Meanwhile, AS-A treatment protects against NaIO3-triggered perturbation of retinal homeostasis.
Objective To observe the relationship between the size of idiopathic macular hole (IMH) and the healing types of postoperative photoreceptor layer after vitrectomy. Methods This prospective uncontrolled study included 33 eyes of 31 consecutive patients who underwent vitrectomy for IMH. There were 9 males (9 eyes) and 22 females (22 eyes), with the mean age of (58.16±9.10) years. The mean duration of symptoms was (4.97±5.97) months. The best corrected visual acuity (BCVA) and optical coherence tomography (OCT) were measured for all patients. BCVA was measured with international standard visual acuity chart and then converted to logarithm of the minimum angle of resolution (logMAR). The mean logMAR BCVA was 1.07± 0.38. The mean intraocular pressure was (14.05±0.54) mmHg (1 mmHg=0.133 kPa). The minimum size of the macular hole (MIN), the base diameter of the macular hole (BASE), the average width of the macular hole (AWMH) and the average height of the macular hole (AHMH) were (465.19±232.84), (943.63±389.26), (704.72±292.64), (443.84±72.47) μm, respectively. According to the MIN value, the hole size were divided into small, medium and large group which had 9 eyes, 15 eyes, 9 eyes, respectively. According to the postoperative OCT characteristics, the healing types of the photoreceptor layer were divided into 0 - Ⅳ types. All patients underwent pars plana vitrectomy (25G or 27G standard three-incision) with internal limiting membrane peeling with tamponade agents. The mean follow-up was (326.42±157.17) days. The first postoperative OCT characteristics were defined as the early period. The therapy results were evaluated according to the last follow-up time point. BCVA and intraocular pressure before and after operation were compared by paired t test. The postoperative BCVA were compared with preoperative BCVA, MIN, AWMH, AHMH and follow-up using Pearson correlation analysis. Results At the last follow-up, the LogMAR BCVA was 1.52 - 1.40 in 3 eyes, 1.30 - 0.52 in 22 eyes and 0.40 - −0.07 in 8 eyes. Compared with preoperative that, the difference was statistically significant (t=−6.023, P＜0.001). The photoreceptor healing was type 0 in 10 eyes (30.3%), type Ⅰ in 4 eyes (12.1%), typeⅡ in 10 eyes (30.3%), type Ⅲ in 9 eyes (27.3%) at the early postoperative period. The photoreceptor healing was type 0 in 5 eyes (15.2%), type Ⅰ in 5 eyes (15.2%), type Ⅲ in 12 eyes (36.4 %), type Ⅳ in 11 eyes (33.3%) at the last follow-up. The preoperative size of IMH was negatively correlated to the photoreceptor healing types at early postoperative period (r=−0.590, P＜0.01) and the last follow-up (r=−0.768, P＜0.01), respectively. The correlation analysis showed that the postoperative BCVA associated with the preoperative BCVA, the stage of the macular hole, the size of the macular hole, MIN, BASE, AWMH, AHMH, the healing types of photoreceptor layer of the early and the last follow-up after surgery (r=0.500, 0.370, 0.470, 0.435, 0.533、0.505, 0.462, −0.442, −0.656, P＜0.05). There was no correlation between age, visual decreasing times and follow-up times (r=0.285, 0.234, −0.310, P＞0.05). Conclusion The preoperative sizes of IMH were associated with the postoperative healing types of photoreceptor layer.