Objective To investigate the inhibitory effects and possible related mechanism of OTX008 [a selective inhibitor of galectin-1 (Galectin-1)] on retinal neovascularization (RNV) in mouse model of oxygen-induced retinopathy (OIR). Methods 7-day-old (P7) C57BL/6J mice were randomly (according to random number table) divided into 4 groups including normal group, OIR group, OIR-OTX008 group and OIR-phosphate buffered saline (PBS) group. To establish the OIR mouse model, mice from all groups except normal group were expose to (75±2)% oxygen for 5 days and then to room air. OIR-OTX008 group received an intravitreal injection of 1 μl (0.25 μg/μl) OTX008 at P12, OIR-PBS group received the equal volume (1 μl) of PBS injection. Mice from 4 groups were euthanized at P17, and retinas were collected for molecular biological analysis and morphological study. RNV was evaluated by counting the number of pre-retinal neovascular nuclei and the whole-mount immunofluorescent staining of mouse retina. Cyrosections of retinas were imaged via confocal microscopy to observe the enrichment of staining of Galectin-1. Protein levels of Galectin-1, Neuropilin-1 and phosphorylation of vascular endothelial growth factor receptor 2 (pVEGFR2) were determined with Western blot. Results At P17, Galectin-1 expressed higher in retinal ganglion cell layer, inner plexiform layer and inner nuclear layer from OIR group and OIR-PBS group than normal group. Galectin-1 expressed less in cryosection retinas from OIR-OTX008 group than OIR group and OIR-PBS group. The numbers of pre-retinal neovascular cell nuclei from OIR group and OIR-PBS group were obviously more than that from normal group (t=9.314,P＜0.05). The number of pre-retinal neovascular cell nuclei from OIR-OTX008 group were obviously lower than those from OIR group and OIR-PBS group (t=8.038, 7.774;P＜0.05). The RNV tufts area (t=13.250, 12.570), non-perfusion area (t=15.590, 12.430) and hypoxic area (t=9.542, 9.928) from OIR-OTX008 group were significantly smaller than those in OIR group and OIR-PBS group (P＜0.05). Protein levels of Galectin-1 (t=24.800, 23.060), Neuropilin-1 (t=4.120, 3.530) and pVEGFR2 (t=25.880, 15.480) in the OIR-OTX008 group were significantly down-regulated than those from OIR group and OIR-PBS group (P＜0.05). Conclusion Intravitreal injection of OTX008 inhibits RNV and ameliorates retinal hypoxia in mice model of OIR possibly through down-regulating Galectin-1, Neurolinpin-1 and pVEGFR2.
Ophthalmic imaging examination is the main basis for early screening, evaluation and diagnosis of eye diseases. In recent years, with the improvement of computer data analysis ability, the deepening of new algorithm research and the popularization of big data platform, artificial intelligence (AI) technology has developed rapidly and become a hot topic in the field of medical assistant diagnosis. The advantage of AI is accurate and efficient, which has great application value in processing image-related data. The application of AI not only helps to promote the development of AI research in ophthalmology, but also helps to establish a new medical service model for ophthalmic diagnosis and promote the process of prevention and treatment of blindness. Future research of ophthalmic AI should use multi-modal imaging data comprehensively to diagnose complex eye diseases, integrate standardized and high-quality data resources, and improve the performance of algorithms.
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.
Ocular neovascularization is a pathological change in various ocular diseases such as diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion and age-related macular degeneration, which seriously affects patient's vision. β receptors are expressed in conjunctiva, corneal epithelial cells, corneal endothelial cells, extraocular muscles, trabecular meshwork, ciliary muscle, lens and retina. β adrenergic receptor antagonists bind to β receptors to exert anti-angiogenic effects by inhibiting the expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1, interleukin-6 and other angiogenic cytokines; reducing macrophage-related inflammatory response; increasing the expression of anti-angiogenic factors. In the treatment of corneal neovascularization, choroidal neovascularization, and retinopathy of prematurity, it can significantly reduce the area of neovascularization and delay disease progression. Co-administration of anti-VEGF drugs can reduce the frequency of administration of anti-VEGF drugs. At effective therapeutic concentrations, β-adrenergic receptor antagonists are well tolerated; they have broader targets than anti-VEGF drugs, which offers new treatment strategies for ocular neovascularization such as corneal, choroidal and retinal neovascularization.
ObjectiveTo observe the efficacy of platelet-rich fibrin (PRF) membrane tamponade combined with air filling for giant macular hole (MH). MethodsA prospective case-control study. From January 2019 to February 2021, 56 patients (56 eyes) diagnosed with giant MH from Eye Center of Renmin Hospital of Wuhan University were enrolled. Among them, there were 17 males with 17 eyes and 39 females with 39 eyes. The average age of the patients was 64.23±9.30 years old. The average MH minimum diameter was 827.36±83.16 μm. The best corrected visual acuity (BCVA) and optical coherence tomography angiography (OCTA) examination were performed before surgery. The Chinese version of 25-item National Eye Institute visual functioning questionnaire (NEI VFQ-25) was used to investigate patient's visual-related quality of life. There were 28 eyes of 28 cases receiving PRF membrane covering, as PRF group, another 28 eyes of 28 cases receiving inverted internal limiting membrane (ILM) insertion into giant MH, as ILM group. The differences of the age (t=－1.588), sex ratio (χ2=0.760), BCVA (Z=－0.400), macular hole minimum diameter (t=－0.604), choriocapillary blood flow area (CBFA) (t=1.331) and NEI VFQ-25 score (t=0.921) were not statistically significant (P＞0.05). All eyes underwent 23G minimally invasive vitrectomy. In the PRF group, PRF membrane was used to fill the hole, and in the ILM group, the hole was filled with ILM inversion, and filled with sterile air after full gas-liquid exchange. The follow-up time after surgery was ≥6 months. The same equipment and methods as before surgery were used to conduct related examinations, and the changes of BCVA, the shape of hole closure, CBFA and the improvement of vision-related quality of life were compared between the two groups. For comparison between groups, independent samples t-test was used for data with normal distribution, and Mann-Whitney U test was used for data with non-normal distribution. For intra-group comparisons, paired-samples t-test was used for data with normal distribution, and Wilcoxon rank-sum test was used for non-normally distributed data. ResultsSix months after surgery, in the eyes of PRF group and ILM group, the hole of 27 (96.4%, 27/28) and 26 (92.6%, 26/28) eyes were closed; the median BCVA was 0.70 and 0.70, respectively; CBFA were 1.99±0.20 and 1.91±0.18 mm2; NEI VFQ-25 scores were 81.36±12.39 and 78.39±10.12, respectively. Compared with before surgery, the BCVA (Z=－4.636,－4.550) and CBFA (t=－27.115,－31.135) of the affected eyes in the PRF group and ILM group were significantly improved after surgery, and the NEI VFQ-25 scores (t=－15.557, －10.675) was significantly increased, and the difference was statistically significant (P＜0.05). There was no significant difference in BCVA (Z=－0.167), CBFA (t=1.554), and NEI VFQ-25 scores (t=0.980) between the two groups after interocular surgery (P=0.726, 0.126, 0.331). ConclusionPRF membrane insertion with air filling has the same efficacy as ILM insertion in the treatment of giant MH, which can improve the closure rate of MH, patients' vision and vision-related quality of life, and increase choroidal blood perfusion.
ObjectiveTo observe the effects of overexpression of S100A4 protein on retinal capillary cells and retinal ganglion cells (RGC) after retinal ischemia-reperfusion injury (RIRI). MethodsOne hundred healthy adult male C57BL/6 mice were randomly divided into normal control group (group C), RIRI group, adeno-associated virus (AAV2)-S100A4 green fluorescent protein (GFP) intravitreal injection group (group S), RIRI+AAV2-GFP intravitreal injection group (group GIR), and RIRI+AAV2-S100A4-GFP intravitreal injection group (group SIR), with 20 mice in each group. The RIRI model was established using the high intraocular pressure anterior chamber method in the RIRI, GIR and SIR groups of mice. Eyes were enucleated 3 days after modelling by over anaesthesia. The number of retinal capillary endothelial cells and pericytes in the retinal capillaries of mice in each group was observed by retinal trypsinised sections and hematoxylin-eosin and periodic acid-Schiff staining; immunofluorescence staining was used to observe endothelial cell, pericyte coverage and RGC survival; The relative expression of Toll-like receptor 4 (TLR4), p38 MAPK and nuclear factor erythroid 2-related factor 2 (NRF2) in retinal tissues was measured by Western blot. One-way analysis of variance was used to compare data between groups. ResultsThree days after modeling, the endothelial cell to pericyte ratio in group C was compared with group S and SIR, and the difference was not statistically significant (F=106.30, P＞0.05); the SIR group was compared with group RIRI and GIR, and the difference was statistically significant (F=106.30, P＜0.000 1). Comparison of endothelial cell coverage in each group, the difference was not statistically significant (F=3.44, P＞0.05); compared with the pericyte coverage in group C, the RIRI group and the GIR group were significantly lower, and the difference was statistically significant (F=62.69, P＜0.001). Compared with the RGC survival rate in group C, it was significantly lower in RIRI and GIR groups, and the difference was statistically significant (F=171.60, P＜0.000 1); compared with RIRI and GIR groups, the RGC survival rate in SIR group was significantly higher, and the difference was statistically significant (F=171.60, P＜0.000 1). The relative expression levels of TLR4, p38 and NRF2 proteins were statistically significant among all groups (F=42.65, 20.78, 11.55; P＜0.05). ConclusionsPericytes are more sensitive to ischemia than endothelial cells after retinal RIRI in mice, and early vascular cell loss is dominated by pericytes rather than endothelial cells. The overexpression of S100A4 protein protects against loss of pericytes and RGC after RIRI by inhibiting the TLR4/p38/NRF2 signaling pathway.
ObjectiveTo observe the changes of macular vascular density of hypertensive patients without obvious hypertensive retinopathy (HRP).MethodsTwenty-three patients (hypertension group) diagnosed as grade 2 or grade 3 essential hypertension in Cardiology Department of Renmin Hospital of Wuhan University from January to April 2019 were enrolled in the study. Among them, there were 13 males and 10 females. The mean age was 61.6 ± 5.6 years, and the mean BCVA was 0.74 ± 0.16. The course of hypertension was more than 7 years; Keith Wagener (K-W) grade was 0 or 1. Fifteen age-matched people without hypertension were selected as the control group, among which included 8 males and 7 females. Their average age was 59.7 ± 4.4 years and the average BCVA was 0.79 ± 0.17, the K-W grades were 0. There was no significant difference (P=0.265, 1.000, 0.563) between the two groups in age (t=1.739), sex ratio (χ2=0.036) or BCVA (t=0.585). All subjects were examined by BCVA, fundus photography and OCTA. OCTA scanned the macular area in the range of 3 mm × 3 mm. The software automatically divided the image into two concentric circles with the macular fovea as the center, which are the inner ring with a diameter of 1 mm (foveal area) and the outer ring with a diameter of 1-3 mm. The blood flow density of the whole, temporal, upper, nasal and lower capillary layers within 3 mm of the macular area, and foveal avascular zone (FAZ) area, central foveal retinal thickness (CFT) were measured.ResultsSignificant differences were observed in the vascular densities of total, temporal, nasal and inferior area of maculas (t=2.188, 2.472, 5.105, 2.734; P=0.037, 0.020, 0.000, 0.010) between the two groups, while no significant differences were evidenced in foveal vascular densities and superior vascular densities (t=0.575, 0.140; P=0.570, 0.889). There was no significant difference in FAZ area or CFT between the two groups (t=0.367, 0.753; P=0.714, 0.457). Macular arches were intact in all hypertension patients.ConclusionsThe vascular densities of total, temporal, nasal and inferior area of maculas in the hypertension patients without HRP decreased. The area of FAZ did not expand, and the structures of macular arch ring were normal.
ObjectiveTo observe and explore the feasibility and effectiveness of platelet-rich fibrin (PRF) membrane packing and air filling in the treatment of refractory macular holes. MethodsA retrospective clinical study. From January 2019 to January 2020, 17 patients with refractory macular hole (17 eyes) who diagnosed in Renmin Hospital of Wuhan University were included in the study. Among them, there were 7 males (7 eyes) and 10 females (10 eyes), with the age of 55.18±7.91 years. All eyes underwent 23G minimally invasive vitrectomy combined with internal limiting membrane stripping and PRF packing, and air filling was performed at the end of the operation. The best corrected visual acuity (BCVA) and optical coherence tomography angiography were performed in all eyes before surgery and at 1 week and 1, 3 months after surgery. The BCVA examination was performed using a international standard logarithmic visual acuity chart, which was converted into logarithm of the minimum angle of resolution visual acuity during statistics. Taking 3 months after surgery was as the time point to judge the efficacy, the changes of BCVA, superficial retinal vascular density (SVD), foveal avascular zone (FAZ) area and central foveal thickness (CFT) before and after surgery were compared. Paired t-test was used to compare the indicators before and after surgery. ResultsAmong the 17 eyes, there were 6, 7, and 4 eyes with giant macular hole, high myopia macular hole, and recurrent macular hole, respectively; the hole diameter was 723.94±38.30 μm. Three months after surgery, all holes were closed. Compared with before surgery, the BCVA (t=4.458) and SVD (t=2.675) increased, and the CFT (t=6.329) and FAZ area (t=4.258) decreased at 3 months after surgery, and the differences were statistically significant (P<0.05). At the last follow-up, there was no complications such as intraocular hypertension and retinal detachment in all eyes.ConclusionMinimally invasive vitrectomy combined with internal limiting membrane stripping and PRF tamponade in the treatment of refractory macular holes can increase the closure rate, improve visual acuity and retinal blood perfusion.
ObjectiveTo investigate the effects of Krüppel-like factor 7 (KLF7) on the survival of retinal ganglion cells (RGCs) and electroretinogram (ERG) after retinal ischemia-reperfusion (RIR) injury in mice.MethodsA total of 126 male C57BL/6J mice were randomly divided into normal group, RIR group, normal-KLF7 group, normal-green fluorescent protein (GFP) group, RIR-KLF7 group and RIR-GFP group. At the age of 8 weeks, mice of normal-KLF7 group and RIR-KLF7 group were intravitreally injected 1ul of 1.0×1012 vg/ml adeno-associated virus overexpressing KLF7 (AAV2-KLF7-GFP). Mice of normal-GFP group and RIR-GFP group were injected adeno-associated virus of AAV2-GFP with the same titer. At the age of 11 weeks, RIR injury was induced in mice of RIR group, RIR-KLF7 group and RIR-GFP group, and intraocular pressure was measured. Retinal cryosections were used to access the efficacy of virus transfection 4 weeks after AAV2-KLF7-GFP transfer. 7 days after RIR injury, RGCs’ survival rate was observed and quantified by immunofluorescent staining. ERG was performed to observe the differences in amplitudes and incubation period of scotopic ERG a-, b-wave, oscillatory potentials (Ops), photopic negative responses (PhNR). Optomotor response was performed to observe the differences of visual acuity. Expression of KLF7 was detected by western blot 4 weeks after AAV2-KLF7-GFP transfer.ResultsCompared with normal group, RGCs’ survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR and visual acuity of mice in RIR group were decreased, and the differences were statistically significant (t=12.860, 7.157, 5.735, 8.953, 4.744, 9.887; P＜0.05). With the increase of light intensity, the amplitudes of scotopic ERG a- and b-wave were gradually increased while the incubation period was gradually shortened. Compared with RIR group, RGCs’ survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR and visual acuity of mice in RIR-KLF7 group were increased, and the differences were statistically significant (t=6.350, 3.253, 3.695, 5.825, 5.325, 4.591; P＜0.05). Protein level of KLF7 was up-regulated in normal-KLF7 group than those in normal group, and the difference was statistically significant (t=4.105, P＜0.01).ConclusionOverexpression of KLF7 can improve RGCs’ survival rates and preserve the electrophysiological function.
ObjectiveTo investigate the effect of heat shock protein B8 (HspB8) downregulation on retinal ganglion cell (RGC) and retinal function in the mice model of optic nerve injury (ONC).MethodsAdeno-Associated Virus (AAV) 2 AAV2-shHspB8-GFP was constructed to knockdown HspB8. 66 adult male C57/BL6 mice were randomly divided into the control group, the ONC group, the AAV2-shHspB8 group, the ONC+AAV2-shHspB8 group, and the ONC+AAV2- GFP group. There were 10, 20, 16, 10 and 10 mice respectively, and both eyes were used as experimental eyes. Western blot was used to evaluate the expression of HspB8 on day 3 and 7 after ONC. By GFP immunofluorescence staining, the efficacy of AAV2-shHspB8-GFP transfer was accessed. Moreover, it was possible to identify functional and RGC survival differences between groups by optomotor response (OMR), dark adapted full-field flash electroretinogram (ff-ERG), oscillatory potentials (OPs), photopic negative response (PhNR) and retinal flat-mount RGC counting 5 days after ONC. Comparisons between two groups were made using Mann-Whitney U test, unpaired t-test, unpaired t-test with Welch’s correction, one-way ANOVA, and Bonferroni t test.ResultsCompared with the control group, the expression of HSPB8 protein in the retina of mice in ONC3 group was significantly increased, and the difference was statistically significant (F=43.63, P＜0.01). Compared with the control group, the ONC group showed obviously lower visual acuity (P＜0.01), lower a-wave, b-wave, OPs, PhNR amplitude, longer b-wave latency (P＜0.05), and the survival rates of RGC in ONC3 group, ONC5 group and ONC7 group decreased in a time-dependent manner(F=384.90, P＜0.01). Transfection of AAV2 efficiency was highest on 4 weeks after IVT. Besides, there was no significant differences between the control group and the AAV2-shHspB8 group on visual acuity, ff-ERG, OPs, PhNR and RGC survival (P>0.05). In comparison of the control group, we found that RGC survival of the ONC5+AAV2-shHspB8 group was significantly elevated (F=10.62, P＜0.01).ConclusionsExpression of HspB8 on the retina can be induced by ONC. The investigation of RGC counting, visual acuity, and ff-ERG revealed that optic nerve injury destructed functionality of mice retina and resulted to RGC death ultimately. The Most crucial finding of this research is that HspB8 knockdown had a neuroprotective effect in RGC after ONC.