ObjectiveTo observe the morphological characteristics of photoreceptor necroptosis in experimental retinal detachment, and explore the mechanism. MethodsA total of 60 Sprague-Dawley male rats were included in this study. Retinal detachment were induced in the right eyes with 1% sodium hyaluronate (50 μl) injection (experimental group), while the left eyes received no treatment (control group). At 3 days after modeling, the morphological characteristics of photoreceptor cell were observed by electron microscopy. Cleaved Caspase 8 and phosphorylation receptor-interacting protein 1 (p-RIP1) were measured by Western blot and immunoprecipitation. ResultsAt 3 days after modeling, photoreceptor necroptosis showed the following morphological features: chromatin condensation, severe vacuolation, early loss of plasma membrane integrity, and many autophagosomes. Western blot showed that the protein expression of cleaved Caspase 8 were 0.78±0.03, 0.06±0.01 in experimental group and control group respectively, which was significantly different (F=4 023.21, P < 0.05). Immunoprecipitation showed that the protein expression of p-RIP1 were 0.23±0.03, 0.14±0.02 in experimental group and control group respectively, which was significantly different (F=56.44, P < 0.05). ConclusionsPhotoreceptor necroptosis showed chromatin condensation, severe vacuolation, early loss of plasma membrane integrity, and many autophagosomes. Necroptosis activation was associated with the increase of RIP1 phosphorylation.
ObjectiveTo observe the frequency domain optical coherence tomography (SD-OCT) features of Henle fiber layer (HFL) of health adults in china by changing the angle of the measurement beam. Methods Twenty-four subjects (28 eyes) who showed no abnormalities on routine eye examination were included in the study, including 15 males (16 eyes) and 9 females (12 eyes) with an average age of (35.51±3.54) years old, and mean refraction power of (-0.89±1.15) D. All subjects underwent corrected visual acuity, intraocular pressure, slit lamp microscope, direct ophthalmoscope, visual field and SD-OCT examination. The macular area was scanned by Zeiss Cirrus SD-OCT (5 HD line) single line scan mode. Based on the entry position of the SD-OCT beam through the pupil, the subjects were divided into 3 groups, including group A (center of the pupil), group B (near the temporal edge of the pupil) and group C (near the nasal edge of the pupil). The thickness of outer plexiform layer (OPL), HFL, and outer nuclear layer (ONL) were measured at 0.75 mm, 1.50 mm from the fovea. ResultsWhen entry position of the SD-OCT beam was near the temporal edge of the pupil (group B); there were two layer structures with different signal intensities in the weak reflectivity zones in front of the external limiting membrane (ELM). The signal of the inner layer was slightly higher than the outer layer. The OPL thickness at the decreased side (nasal) increased significantly compared with the other side, but the ONL thickness was significantly thinner than other side. When entry position of the SD-OCT beam was near the nasal edge of the pupil (group C), there were also two layer structures with different signal intensities in the weak reflectivity zones in front of the ELM. The signal of the outer layer was slightly higher than the inner layer. The OPL thickness at the decreased side (temporal) increased significantly compared with the other side, but the ONL thickness was significantly thinner than other side. The OPL thickness at the decreased side was significantly different between these 3 groups (P < 0.01). ConclusionsSD-OCT provided the possibility of distinguishing HFL from the actual ONL by changing the angle of the measurement beam. This finding has great clinical significance for related diseases affecting HFL or ONL.
ObjectiveTo evaluate the photoreceptor-protective effects of Cdk5 inhibitor Roscovitine on retinal degeneration in Royal College of Surgeons (RCS) rat. MethodsThe RCS rats were divided into three groups according to postnatal days: the early (17 days), medium (25 days) and late intervention group (35 days). Cdk5 inhibitor Roscovitine were used in the right eyes by intravitreal injection as experimental eyes and Roscovitine solvent dimethylsulfoxide were used in the left as control at postnatal 17, 25, 35 days. Hematoxylin-eosin (HE) staining was used to observe the thickness of outer nuclear layer. The expression of Cdk5 P25 and cleave-caspase 3 in the retina was evaluated by immunohistochemistry. The protein expression of cleave-caspase 3 in the retina was determined by Western blot. The apoptosis of retinal cells was examined by terminal-deoxynucleotidyl transferase mediated nick end labeling. ResultsHE staining showed that thickness of outer nuclear layer in the early and medium intervention groups were significantly thicker than that in the control group (P < 0.05), particularly in the early intervention group. And there was no significant change in the late intervention group (P > 0.05). The expression level of Cdk5, p25, cleave-caspase 3 in the outer nuclear layer in three intervention groups were lower than that in the control group (P < 0.05), especially in the early intervention group. ConclusionCdk5 inhibitor Roscovitine can delay the retinitis pigmentosa process in RCS rats by early, medium interventional therapy and may have a certain degree of photoreceptor-protective effects.
ObjectiveTo investigate the effects of FTY720 on retinal photoreceptor cells and microglial following light-induced degeneration in rat retina. Methods120 Sprague-Dawley rats were randomly divided into four groups including FTY720 group, solvent control group, model group and normal group. The rats of normal group were not intervened. The FTY720 group, solvent control group and model group establish retinal light injury mode. FTY720 was injected into abdominal cavity of the rats in FTY720 group 0.5 hours before light exposure. 50% dimethylsulfoxide was injected into abdominal cavity of the rats in solvent control group. The expressions of microglial cells in rat retinal were quantified using flow cytometry, the expressions of interleukin (IL)-1βwere examined by enzyme-linked immuno sorbent assay at 6 hours, 1 day, 3 days, 7 days after light exposure. The apoptosis of retinal photoreceptor cells were measured by terminal-deoxynucleoitidyl transferase mediated nick end labeling at 1 day after light exposure. The morphological change of retinal were viewed by haematoxylin and eosin staining at 7 days after light exposure. ResultsThe expressions of microgilal and IL-1βbegan to rise at 1 day after light exposure, reached at peak at 3 days and decreased at 7 days. The expressions of IL-1βand microglial in FTY720 group were significantly lower than solvent control group and model group, but higher than normal group (P < 0.05).One day after exposure to light, the apoptosis cell ratio in normal group, model group, solvent control group and FTY720 group were 0, (87.66±2.50)%, (86.00±2.44)%, (49.66±2.80)%. The apoptosis cell in FTY720 group were higher than normal group, lower than solvent control group and model group (P < 0.05). Seven days after exposure to light, the retinal in normal group was structured and the cell was arranged well, the cell in solvent control group and model group was irregular arrangement and the outer nuclear layer (ONL) was thin after light exposure. The thickness of the ONL in FTY720 group was significantly higher than solvent control group and model group, below normal group. ConclusionFTY720 can prevents retinal photoreceptor cells from apoptosis and inhibits activation of microglial.
Objective To observe the effects of Crumbs (Crb) proteins on different types of zebrafish photoreceptors. Methods The retinal cell population dynamics of adult wild-type zebrafish and Tg (RH2-2:Crb2b-sf-EX/RH2-2:GFP)pt108b transgenic zebrafish (called pt108b zebrafish for short) were evaluated by monitoring the densities of three categories of retinal photoreceptors (rod cells, UV cone cells and RGB cone cells) in different retinal regions, which were visualized by Feulgen nuclear staining histology technique. Results The wild-type zebrafish retinal photoreceptor cell densities are generally higher in the central region than the peripheral regions. Compared with wild-type zebrafish, pt108b zebrafish had much less RGB cone cells at the top of outer nuclear layer, and no RGB cone cells at the central and intermediate regions of retina. While pt108b zebrafish had normal density of UV cone cells at the top of rods and the bottom of outer limiting membrane, they had much higher density of rods. Conclusions Crb proteins may affect the zebrafish retinal cell densities of different photoreceptor types.
Photoreceptor cells are special retinal neurons with photo-transformation ability. Loss of photoreceptors in age-related macular degeneration (AMD) is secondary to RPE loss, leakage of serum components from the neovascularization and scar formation, which is one of the main mechanisms of irreversible visual impairment in patients with AMD. Many studies have shown that inflammatory environment is involved in the process of photoreceptor cell death. Aging, photooxidation injury and other factors affects the retinal microenvironment through different levels of mechanisms such as retinal pigment epithelial cells, retinal glial cells, hematogenous macrophages and inflammatory factors, which results in photoreceptor injuries and participates in the progression of AMD by drusen formation and neovascularization. This study reviews the research status and progress of inflammation and photoreceptor cell death, and provides new ideas for exploring the blinding mechanism and treatment strategies of AMD.
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.
ObjectiveTo observe the effect of metformin on the polarization state and photoreceptor cell activity of microglia (BV2 cells) in a high glucose environment. MethodsAn experimental study. BV2 cells were divided into a control group, a high glucose group, and a metformin+high glucose group. The cells in the high glucose group were cultured with 75 mmol/L glucose in the medium; the cells in the metformin+high glucose group were pretreated with 2 mmol/L metformin for 12 h and then placed in 75 mmo/L glucose concentration medium. The relative expression of M1 marker inducible nitric oxide synthase (iNOS), CD86 and M2 markers arginase 1 (Arg-1), and CD206 protein were detected by Western blot. Interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-4 were detected by enzyme-linked immunosorbent assay (ELISA). BV2 cells were co-cultured with mouse retinal photoreceptor cells (661W cells) for 24 h. The proliferation rate of 661W cells in each group was measured by methyl thiazolyl tetrazolium (MTT) colorimetric assay; the apoptosis rate of 661W cells in each group was measured by flow cytometry and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). An independent sample t-test was used for comparison between groups. ResultsWestern blot assay showed that the relative expression of iNOS and CD86 protein was increased and the relative expression of Arg-1 and CD206 protein was decreased in BV2 cells in the high glucose group compared with the control group, and the differences were all statistically significant (t=－16.783, －11.605, 4.325, 4.649; P＜0.05); compared with the high glucose group, the relative expression of iNOS and CD86 protein was decreased and the relative expression of Arg-1 and CD206 protein was increased in BV2 cells in the metformin + high glucose group compared with the high glucose group, and the differences were all statistically significant (t=7.231, 5.560, －8.035, －8.824; P＜0.01). ELISA results showed that compared with the control group, the BV2 cells in the high glucose group had increased IL-6, TNF-α content and IL-4 content was decreased in BV2 cells in the high glucose group compared with the control group, and the differences were all statistically significant (t=－64.312, －127.147, 71.547; P＜0.001); compared with the high glucose group, IL-6 and TNF-α content was significantly decreased and IL-4 content was significantly increased in BV2 cells in the metformin+high glucose group, and the differences were all statistically significant (t=44.426, 83.232, －143.115; P＜0.001). After co-culture of BV2 cells with 661W cells for 24 h, the results of MTT colorimetric assay showed that compared with the control group, the activity of 661W cells in the high glucose group was significantly reduced, and the difference was statistically significant (t=7.456, P＜0.01); compared with the high glucose group, the activity of 661W cells in the metformin+high glucose group was increased (t=－3.076, P＜0.05). TUNEL method and flow cytometry showed that the apoptosis rate of 661W cells in the high glucose group was significantly higher compared with the control group, and the differences were both statistically significant (t=－22.248, －22.628; P＜0.001); compared with the high glucose group, the apoptosis rate of 661W cells in the metformin+high glucose group was significantly decreased, and the difference was statistically significant (t=11.767, 6.906; P＜0.001, 0.01). ConclusionIn the high glucose environment, metformin inhibited the inflammatory response and attenuated the apoptosis of photoreceptor cells by regulating the polarization of microglia toward the M2 type.