Anti-inflammatory effect of Celastrol in the ocular tissues of mice with exper-imental autoimmune uveitis and its impact on microglia polarization / 眼科新进展

Objective To investigate the anti-inflammatory action of Celastrol in the ocular tissues of mice with ex-perimental autoimmune uveitis(EAU)and its effect on microglia polarization.Methods A total of 36 healthy B10.RⅢmice at 6-8 weeks of age were selected and randomly divided into the normal control group,EAU solvent control group and Celastrol intervention group,with 12 mice in each group.The interphotoreceptor retinoid-binding protein(IRBP)161-180 and Freund's complete adjuvant were mixed by thorough emulsification and injected subcutaneously into the bilateral thighs and tails of mice in the EAU solvent control group and the Celastrol intervention group with a total volume of 200 μL and 50 μg IRBP 161-180 in each mouse.On 7-14 days after immunization,mice in the Celastrol intervention group received a daily intraperitoneal injection of 0.5 mg·kg-1 Celastrol,and mice in the EAU solvent control group were injected with an equivalent dose of sterile Phosphate Buffered Saline solution.On the 14th day after immunization,the anterior segment of mice in each group was observed by slit-lamp microscope and Hematoxylin and Eosin(HE)staining of tissue sections was performed;the clinical and histopathological scores of mice in each group were obtained by reference to the Caspi grading standards;immunofluorescence staining was used to observe the activation of microglia in the eyes of mice;Western blot was used to detect the protein expression levels of inducible nitric oxide synthase(iNOS)and arginase-1(Argl)in the reti-na;quantitative real-time PCR was used to detect the relative mRNA expression of inflammatory factors in the retina,such as tumor necrosis factor(TNF)-α,interleukin(IL)-1β and IL-6.GraphPad Prism 9.0 was used for data analysis.Results On the 14th day after immunization,it was observed by the slit-lamp microscope that the anterior segment of mice in the EAU solvent control group was markedly congested with dilated iris blood vessels,corneal edema,and anterior chamber exudation;the inflammation in the anterior segment of mice in the Celastrol intervention group was markedly at-tenuated,and the iris blood vessels were seen to be mildly congested.Compared with the normal control group,the clini-cal scores of mice in the EAU solvent control group and the Celastrol intervention group were significantly elevated(both P＜0.05);the clinical scores of mice in the Celastrol intervention group were lower than those in the EAU solvent control group(P＜0.05).HE staining results showed that on the 14th day after immunization,mice in the EAU solvent control group showed severe retinal folds and detachment with diffuse infiltration of inflammatory cells,while mice in the Celastrol intervention group showed slight structural damage to the retina and a small amount of inflammatory cell infiltration.Com-pared with the normal control group,the histopathological scores of mice in the EAU solvent control group and the Celas-trol intervention group were significantly elevated(both P＜0.05);the histopathological scores of mice in the Celastrol in-tervention group were lower than those in the EAU solvent control group(P＜0.05).The intraocular Iba1+cell densities of mice in the normal control,EAU solvent control and Celastrol intervention groups were(1.00±0.12)％,(36.07± 4.57)％,and(1.83±0.36)％,respectively.Compared with the normal control group,the number of Iba1+cells in the eyes of mice in the EAU solvent control group and the Celastrol intervention group significantly increased(both P＜0.05);compared with the EAU solvent control group,the number of Iba1+cells in the eyes of mice in the Celastrol intervention group was significantly reduced(P＜0.05).Compared with the normal control group,the expression levels of iNOS and Arg1 proteins in the retinas of mice in the EAU solvent control group were significantly elevated(both P＜0.01);compared with the EAU solvent control group,the expression of iNOS protein in the retinas of mice in the Celastrol intervention group was significantly reduced(P＜0.01).Compared with the normal control group,the relative mRNA expressions of TNF-α.IL-1β,and IL-6 in the retinas of mice in the EAU solvent control group was significantly elevated(all P＜0.05);compared with the EAU solvent control group,the relative mRNA expressions of TNF-α,IL-1 β,and IL-6 in the retinas of mice in the Celastrol intervention group significantly decreased(all P＜0.05).Conclusion Celastrol inhibits Ml microglia activation and reduces the production of retinal inflammatory factors TNF-α,IL-1 β and IL-6 in EAU mice,thereby attenuating the in-flammatory reaction.

Research progress of retinal neurovascular unit injury in glaucoma / 国际眼科杂志(Guoji Yanke Zazhi)

Glaucoma is one of the leading causes of vision loss worldwide. More and more studies have suggested that glaucoma is a complicated retinal neurovascular disease. The homeostasis imbalance of retinal neurovascular unit(RNVU)composed of neurons, glial cells and microvascular cells not only induces changes in microvascular structure and glial cells, but also affects the nerve tissue of the retina, resulting in vision loss, which there is no effective treatment to reverse, currently. Exploring the cellular composition and molecular structure of RNVU and investigating the destruction mechanism of normal cellular environment and intercellular connections in glaucoma are of great significance in exploring the pathogenesis and the treatment of glaucoma. The research progress on structural changes and dysfunction of RNVU in glaucoma are reviewed, hoping to provide new ideas for the treatment of glaucoma.

Protective effect of COG1410 on the survival of retinal ganglion cells in mice with retinal ischemia-reperfusion injury and its mechanism / 中华实验眼科杂志

Objective:To explore the effects of apolipoprotein E-mimetic peptide COG1410 on M1/M2 microglia polarization and retinal ganglion cells (RGCs) survival after ischemia-reperfusion (IR) injury in the mouse retina and its possible mechanisms.Methods:Eighteen 8-week-old C57BL/6J male mice were divided into control group (6 mice), IR 3 days group (6 mice), IR 7 days group (3 mice), and IR 14 days group (3 mice) according to the randomized number table method.Mice in IR group were perfused in the anterior chamber using saline, and the intraocular pressure (IOP) was raised to 100 mmHg (1 mmHg=0.133 kPa) and maintained for 1 hour in order to establish a model of IR injury in the retina.Three mice from control group and 3 mice from IR 3 days group were taken to observe the distribution of retinal microglia by immunofluorescence staining of retinal frozen sections.Three mice were taken from normal control, IR 3 days, IR 7 days, and IR 14 days groups respectively to observe the changes of retinal M1-type and M2-type microglial cells with time after IR injury by immunofluorescence staining of retina.Another 91 C57BL/6J mice were randomly divided into normal control group (19 mice), IR group (24 mice), saline group (24 mice), and COG1410 group (24 mice) according to the random number table method.Mice in normal control group maintained a normal IOP, and the IR injury model was established in the other three groups.In addition, COG1410 group and saline group were injected with 1 mg/kg COG1410 and an equal volume of saline by tail vein injection, respectively.The microglia phenotype and survival rate of RGCs were observed by immunofluorescence staining of retinal wholemount.The relative expressions of retinal tumor necrosis factor-ɑ (TNF-ɑ) and interleukin-1β (IL-1β) mRNA were detected by real-time fluorescence quantitative PCR.The apoptosis of retinal neuronal cells was observed by the TUNEL assay.The expression levels of retinal nuclear factor-κB (NF-κB), B lymphocyte-2 (Bcl-2), and Bcl-2-associated X protein (Bax) proteins were detected by Western blot.Use and care of animals strictly complied with the Hubei Provincial Regulations on the Management of Laboratory Animals and the experiment was approved by the Animal Ethics Committee of the Renmin Hospital of Wuhan University (No.WDRM20190113).Results:Retinal microglia in normal control group and IR 3 days group were mainly distributed in the ganglion cell layer, inner plexiform layer, and outer plexiform layer.There were statistically significant differences in the comparison of the proportions of M1-type and M2-type microglia among normal control, IR 3 days, IR 7 days, and IR 14 days groups ( F=29.83, 57.62; both at P<0.001). Compared with normal control group, the number of M1-type microglia was higher in IR 3 days group, and the number of M2-type microglia was higher in IR 7 days group, and the differences were statistically significant (all at P<0.05). The proportions of M1-type microglia in normal control group, IR group, saline group, and COG1410 group were (4.25±0.57)%, (65.26±10.43)%, (63.01±4.93)%, and (33.13±4.46%), respectively, and the proportions of M2-type microglia in the four groups were (4.50±0.20)%, (11.47±0.24 )%, (11.75±0.17)%, and (38.93±4.26)%, showing statistically significant differences among them ( F=23.33, 50.82; both at P<0.001). The proportions of M1-type microglia decreased while the proportions of M2-type microglia increased in COG1410 group when compared with IR group, and the differences were statistically significant (both at P<0.05). There were statistically significant differences in RGCs survival rate, relative expression of retinal TNF-ɑ and IL-1β mRNA, retinal apoptotic cell count, retinal NF-κB and Bax protein expression levels, and Bax/Bcl-2 ratio among the four groups ( F=30.77, 12.52, 6.74, 28.72, 13.02, 7.94, 7.58; all at P<0.05). Compared with normal control group, there were significant decreases in the survival rate of RGCs and increases in retinal apoptotic cell number, TNF-ɑ and IL-1β mRNA expression, retinal NF-κB and Bax protein expression levels, and Bax/Bcl-2 ratio in IR group (all at P<0.05). Compared with IR group, the COG1410 group had increased retinal RGCs survival rate, decreased TNF-ɑ and IL-1β mRNA expression levels, decreased TUNEL-positive cells, decreased NF-κB and Bax proteins expression levels, and decreased Bax/Bcl2 ratio, and the differences were statistically significant (all at P<0.05). Conclusions:Three days after retinal IR modeling, COG1410 promotes the polarization of M1-type microglia to M2-type, inhibits the expression of retinal NF-κB and downstream inflammatory factors, and attenuates the retinal inflammatory response, as well as inhibits the expression of apoptosis-related proteins, which promotes the survival of RGCs.

New advances of basic research on non-arteritic anterior ischemic optic neuropathy / 中华实验眼科杂志

Non-arteritic anterior ischemic optic neuropathy (NAION) is an optic neuropathy that usually occurs in people over 50 years old.The pathogenesis of NAION remains unknown, and there is no recognized effective treatment.The animal model of NAION established by photodynamic method has similar fundus and electrophysiological changes to clinical NAION.In recent years, studies on the pathological mechanisms of NAION based on animal models have found that axonal structure destruction, demyelination and inflammatory cells infiltration in the region of optic nerve infarction, accompanied by secondary retinal ganglion cells apoptosis.There are a wide range of drugs for NAION based on animal models, including glucocorticoids, granulocyte colony-stimulating factor, prostaglandin J2, anti-vascular endothelial growth factor, neurotrophic factors, effective drugs for glaucoma or central nervous system damage, etc.Routes of administration include systemic administration, intravitreal injection or topical application of eye drops.The neuroprotective effects of some drugs in animal models provide a basis for clinical screening of new therapeutic drugs.In this review, the animal models of NAION, pathophysiology and treatment based on animal models were summarized.

The efficacy of platelet-rich fibrin membrane insertion combined with air filling for giant macular hole / 中华眼底病杂志

Objective:To observe the efficacy of platelet-rich fibrin (PRF) membrane tamponade combined with air filling for giant macular hole (MH).Methods:A 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. Results:Six 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 mm 2; 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). Conclusion:PRF 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.

Platelet-rich fibrin membrane packing and air filling in the treatment of refractory macular holes / 中华眼底病杂志

Objective:To observe and explore the feasibility and effectiveness of platelet-rich fibrin (PRF) membrane packing and air filling in the treatment of refractory macular holes.Methods:A 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. Results:Among 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. Conclusion:Minimally 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.

Research progress of β-adrenergic receptor antagonists in the treatment of ocular neovascular diseases / 中华眼底病杂志

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.

Warburg effect of retinal energetic metabolism and its regulatory mechanism / 中华实验眼科杂志

Retinal metabolism includes material metabolism and energetic metabolism.Retina is one of the most energy-consuming nerve tissue in human body and mainly relies on glycolysis for energy production, which is similar to very fast-growing tumor tissue.This process is known as Warburg effect.Warburg effect is of great significance, which is demonstrated that glucose is metabolized via glycolysis in a more rapid approach in comparison with oxidative phosphorylation pathway.In addition, glucose also supplies neoplastic tissue with carbon source or metabolic intermediates due to biosynthesis.The produced energy of retina is a summation of different retinal cells and tissue, such as photoreceptors, retinal pigment epithelium (RPE), Müller cells and retinal capillary endothelial cells etc.To understand the underlying mechanism contributing to Warburg effect and provide insight into metabolic coupling between neuron and glia is of important significance.Since key glycolysis enzymes (HK2, PFKFB3 and PKM2) take a pivotal role in controlling retinal cell proliferation and neovascularization, bioenergetic strategy targeting these enzymes suggests new idea in the treatment of retinal diseases where energy failure is part of the pathogenesis.Investigating underlying mechanism of retinal energy metabolism can provide new ideas for the treatment of age-related macular degeneration (AMD) and other diseases related to disordered retinal energy metabolism.The Warburg effect of retinal energetic metabolism and its regulatory mechanism were reviewed in this article.

Survival rate change of retinal ganglion cells following optic nerve injuries in mice / 中华创伤杂志

Objective:To investigate the survival rate change of retinal ganglion cells（RGCs）in a mouse of optic nerve crush（ONC）.Methods:Ninety-seven male C57BL/6J mice（6 to 8 weeks）were selected and divided into normal group（ n=5）, sham-operation group（ n=5）and ONC group（ n=5）according to the random number table. In normal group, both eyes of the mice did not receive any intervention. In sham-operation group, the right eye of the mice received sham operation, while the left eye reveived no intervention. In ONC group, the left eye received ONC, and the right eye received sham operation. In normal group, the density of RGCs in both eyes was quantified and compared. In sham-operatioin group, the density of RGCs in the sham operation eye was calculated and then compared to the average density of RGCs in normal group. In ONC group, the survival rate of RGCs was set as the ratio between the left eye（ONC eye）and the right eye（sham-operation eye）. The survival rate of RGCs in ONC group was compared after crush injury for 5, 10, 20, 30 seconds）（the sacrifice time was set at day 7）, and was compared after sampling on days 3, 4, 5, 7, 14, 30, 60, 90, 180（the duration of crush injury was set as 20 seconds）. Results:In normal group, the density of RGCs in the right eye was（5, 167.3±55.6）cell/mm 2, with no statistical difference from that in the left eye［（5, 199.6±44.8）cell/mm 2］（ P>0.05）. The density of RGCs in normal group and sham-operation group was（5, 183.5±33.4）cell/mm 2 and（5, 151.5±87.6）cell/mm 2, showing no statistical difference（ P>0.05）. The survival rate of RGCs in ONC group after crush injury for 5, 10, 20, 30 seconds was（37.6±1.1）%,（34.0±0.9）%,（33.6±1.6）% and（30.3±0.6）%（ P<0.01）. In comparison, there was statistical difference in the survival rate of RGCs between crush injury for 5 seconds and for 30 seconds（ P<0.01）, but not among other duration of crush injury（ P>0.05）. The survival rate of RGCs in ONC group after sampling on days 3, 4, 5, 7, 14, 30, 60, 90, 180 was（85.4±2.0）%,（67.6±3.1）%,（43.0±1.0）%,（33.6±1.6）%,（22.7±2.0）%,（12.8±0.6）%,（10.4±0.8）%,（8.6±0.5）% and（6.7±0.2）%（ P<0.01）, showing the most obvious drop from day 3 to day 5. Additionally, the curve became flattened after 30 days. Conclusions:In a mouse model of ONC, varying durations of crushing will lead to different damage to RGCs in a progressive mode, indicating that following the primary injury（ONC）, the RGCs suffer secondary injury as well. Therefore, effectively controlling the secondary injury may be the key point of treating optic nerve injuries.

Repair effect of platelet-rich plasma on traumatic optic nerve injury in rabbits and its mechanism / 中华实验眼科杂志

Objective:To study the therapeutic effect and mechanism of autologous platelet-rich plasma (PRP) on rabbit traumatic optic neuropathies (TON) and retina.Methods:Forty adult New Zealand white rabbits were selected to establish the optic nerve clamp injury model in their right eyes.According to the random number table method, 36 New Zealand white rabbits with effective model were randomly divided into model control group, normal saline control group and PRP group, 12 for each group.Another 12 healthy rabbits served as the normal control group.Rabbit autologous blood was collected to prepare PRP.The retrobulbar 20 μl PRP/20 μl saline solution injection was administered every two days near the injury after modeling according to grouping.The injection was carried out for 10 times.There was no other interference administrated to the model control group except the normal anti-infective treatment.No interference was given to the normal control group.At 30 and 60 days after modeling, the eyeballs and optic nerves of right eyes were harvested through sacrificing the animals by anesthetic overdose, three eyes for each time.Histopathological assessments were performed to observe the morphological changes of retina and optic nerve, and to evaluate the changes of retinal ganglion cells (RGCs) density and retinal nerve fiber layer (RNFL) thickness.Immunohistochemistry was used to assess the expressions of apoptosis factors caspase-3 and B cell lymphoma-2(Bcl-2). Quantitative real-time PCR and Western blot were used to detect the mRNA and protein expressions of brain-derived neurotrophic factor (BDNF) and growth associated protein-43 (Gap-43). This study protocol was approved by the Experimental Animal Ethics Committee of Wuhan University (No.E2019072805). The use and care of animals complied with ARVO statement.Results:The thickness of RNFL and number of RGCs at 30 days and 60 days after modeling were (6.60±1.16) μm, (6.89±1.21) μm, (13.00±1.00)/field of vision, (20.00±2.65)/field of vision in the PRP group, respectively, and were (4.80±0.43)μm, (2.18±0.23)μm, (6.33±0.58)/field of vision, (10.33±1.53)/field of vision in the model control group, respectively.The number of RGCs in the PRP group at 60 days was higher than that at 30 days after modeling, the number of RGCs in the PRP group was higher than that in the model control group, the thickness of RNFL in the PRP group was higher than that in the model control group; and the differences were statistically significant (all at P<0.05). At 30 and 60 days after modeling, the positive expression A value of caspase-3 protein in the normal saline group and model control group were higher than those in the normal control group and PRP group, while the positive expression A value of Bcl-2 protein in the PRP group was higher than those in the model control group and normal saline group, and the differences were statistically significant (all at P<0.05). The mRNA level and protein content of BDNF and Gap-43 in the retina and optic nerves at 30 days and 60 days after modeling in the PRP group were higher than those in the model control group, and the differences were statistically significant (all at P<0.05), but the mRNA and protein expression levels of BDNF and Gap-43 in different tissues in the PRP group at 60 days after modeling were lower than those at 30 days after modeling ( P<0.05). Conclusions:PRP can effectively inhibit the apoptosis of RGCs and the secondary injury of the retina after optic nerve injury, promote cell anti-apoptosis effect of RGCs, thereby retard the damage of the retina and optic nerve after TON, and also promote the repair of optic nerve and retina through upregulating the expression of nerve growth factors.

Downregulation of heat shock protein B8 protects retinal ganglion cell after optic nerve injury in mice / 中华眼底病杂志

Objective:To 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).Methods:Adeno-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. Results:Compared 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). Conclusions:Expression 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.

Effects of DJ-1 protein on retinal ganglion cells and visual function in mice after optic nerve crush injury / 中华眼底病杂志

Objective:To 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.Methods:Thirty-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 μl 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.Results:Compared 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/m 2. 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). Conclusion:The 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.

Application of optical coherence tomography in the eye disease screening and health management of primary and secondary school teachers in Wuchang District, Wuhan City / 中华健康管理学杂志

Objective:To explore the value of applying optical coherence tomography (OCT) in the physical examination of the eyes of primary and secondary school teachers in Wuchang District, Wuhan.Methods:A total of 3,495 teachers in primary and secondary schools in Wuchang District, who were examined at Physical Examination Center of Renmin Hospital of Wuhan University from September to December, 2018, were enrolled, including 1 130 men and 2 365 women, aged 22-93 years. All enrolled subjects underwent visual acuity assessment, slit lamp examination, direct ophthalmoscopy, and OCT of the macula. The chi-square test was used to analyze differences between different age groups for the ten most common eye diseases and positive findings, as well as the difference in the detection rate of macular disease between OCT and direct ophthalmoscopy.Results:The detection rate of eye diseases was 70.76% (2 473/3 495 people) in this study; 551 people <40 years old, 775 people aged 40-64 years old, and 1 147 aged ≥65 years old had abnormalities. There were significant differences in the detection rates of ametropia, cataract, maculopathy, postoperative cataract, pathological myopic fundus change, pterygium, trichiasis, retinal arteriosclerosis, and cup disk ratio abnormality between the three groups (χ2=100.24, 1037.23, 507.61, 232.50, 14.46, 54.92, 21.48, 84.24, 17.73, respectively, all P<0.05), but there was no significant difference in conjunctivitis between the three age groups (χ 2=0.58, P>0.05). The detection rate of OCT for maculopathy was higher than that for direct ophthalmoscopy (χ2=36.357, P<0.05), and the detection rate of OCT for macular anterior membrane, macular degeneration, pathological myopia, retinochoroidopathy, and macular split was higher than that for direct ophthalmoscope; the difference was statistically significant (χ2=10.065, 4.932, 19.836, 12.010, respectively, all P<0.05). Conclusion:Primary and secondary school teachers have different distributions of eye diseases depending on their age group. Health education and physical examination of the eye can be provided according to the age of the teacher, which is conducive to early prevention and treatment of eye diseases. OCT has important application value in physical examination and the screening of macular diseases.

The macular vascular density in hypertensive patients without obvious hypertensive retinopathy / 中华眼底病杂志

Objective:To observe the changes of macular vascular density of hypertensive patients without obvious hypertensive retinopathy (HRP).Methods:Twenty-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. Results:Significant 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. Conclusions:The 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.

Effects of Krüppel-like factor 7 on the survival of retinal ganglion cells and electroretinogram after retinal ischemia-reperfusion injury / 中华眼底病杂志

Objective:To 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.Methods:A 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 1.0×10 12 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. Results:Compared 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). Conclusion:Overexpression of KLF7 can improve RGCs’ survival rates and preserve the electrophysiological function.

Application of optical coherence tomography in the eye disease screening and health management of primary and secondary school teachers in Wuchang District, Wuhan City / 中华健康管理学杂志

Objective@#To explore the value of applying optical coherence tomography (OCT) in the physical examination of the eyes of primary and secondary school teachers in Wuchang District, Wuhan.@*Methods@#A total of 3,495 teachers in primary and secondary schools in Wuchang District, who were examined at Physical Examination Center of Renmin Hospital of Wuhan University from September to December, 2018, were enrolled, including 1 130 men and 2 365 women, aged 22-93 years. All enrolled subjects underwent visual acuity assessment, slit lamp examination, direct ophthalmoscopy, and OCT of the macula. The chi-square test was used to analyze differences between different age groups for the ten most common eye diseases and positive findings, as well as the difference in the detection rate of macular disease between OCT and direct ophthalmoscopy.@*Results@#The detection rate of eye diseases was 70.76% (2 473/3 495 people) in this study; 551 people <40 years old, 775 people aged 40-64 years old, and 1 147 aged ≥65 years old had abnormalities. There were significant differences in the detection rates of ametropia, cataract, maculopathy, postoperative cataract, pathological myopic fundus change, pterygium, trichiasis, retinal arteriosclerosis, and cup disk ratio abnormality between the three groups (χ²=100.24, 1037.23, 507.61, 232.50, 14.46, 54.92, 21.48, 84.24, 17.73, respectively, all P<0.05), but there was no significant difference in conjunctivitis between the three age groups (χ2=0.58, P>0.05). The detection rate of OCT for maculopathy was higher than that for direct ophthalmoscopy (χ²=36.357, P<0.05), and the detection rate of OCT for macular anterior membrane, macular degeneration, pathological myopia, retinochoroidopathy, and macular split was higher than that for direct ophthalmoscope; the difference was statistically significant (χ²=10.065, 4.932, 19.836, 12.010, respectively, all P<0.05).@*Conclusion@#Primary and secondary school teachers have different distributions of eye diseases depending on their age group. Health education and physical examination of the eye can be provided according to the age of the teacher, which is conducive to early prevention and treatment of eye diseases. OCT has important application value in physical examination and the screening of macular diseases.

Inhibitory effects of resveratrol on high glucose-induced apoptosis of retinal cells / 中华实验眼科杂志

Objective:To investigate the inhibitory effects of resveratrol (Res) on the apoptosis of retinal neurons of diabetic rats and ARPE-19 cells induced by high glucose.Methods:Streptozotocin was intraperitoneally injected to establish a diabetes model in 25 adult male SD rats, and 20 successful models were randomized into diabetic model group and Res-administered group according to random number table method.Another 10 matched normal rats were served as normal control group.Res was intragastrically given to the rats in the Res-administered group with the dose of 40 mg/(kg·d), and an equivalent volume of normal saline solution was used in the same way in the diabetic model group and normal control group.The body weight and blood glucose level were measured on the 0th, 4th, 8th, 12th week of administration.The rats were sacrificed on the 12th week by over-anesthesia and the eyeballs were enucleated.The ultrastructure of the retinal ganglion cells (RGCs) were examined under the transmission electron microscope.The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay was to assess the apoptosis of retinal neurons.ARPE-19 cells were divided into normal control group, high glucose group and Res-treated group and cultured for 48 hours in medium containing 5.5 mmol/L glucose, 30 mmol/L glucose, 30 mmol/L glucose and 10 mol/L Res, respectively.Apoptosis rate was detected by a flow cytometry.The distribution and expression of bax and bcl-2 in the cells was detected by immunofluorescence and Western blot assay, respectively.This study protocol was approved by an Experimental Animal Ethic Committee of Hubei University of Science and Technology, and the use and care of the animals complied with the Statement of ARVO.Results:Compared with the diabetic model group, the body weight was increased at 4-12 weeks and the blood glucose level was lowered at 8-12 weeks of Res administration in the Res-administered group (both at P<0.01). The chromatin condensation, mitochondrial swelling and vacuolation in cytoplasm were obviously slight and the apoptosis rate was reduced in the Res-administered group in comparison with the diabetic model group.The apoptosis indexes of the retinal ganglion cell layer cells and inner nuclear layer cells in the Res-administered group were (18.36±3.37)% and (23.67±8.98)%, respectively, which were significantly lower than (83.91±9.8)% and (64.26±10.66)% in the diabetic model group (both at P<0.01). The apoptosis rate of the ARPE-19 cells in the normal control group, high glucose group and Res-treated group was (3.11±0.26)%, (11.41±1.06)% and (5.38±0.58)%, respectively, and the apoptosis rate in the Res-treated group was significantly lower than that in the high glucose group (all at P<0.01). Immunofluorescence assay showed that the fluorescence of bax in the cell nucleus of Res-treated group was obviously enhanced in comparison with the normal control group and weaker in comparison with the high glucose group.The fluorescence of bcl-2 protein in the Res-interfered group was weaker in comparison with the normal control group and enhanced in comparison with the high glucose group.The relative expressions level of bax protein in the Res-treated group was 0.21±0.08, which was significantly higher than 0.15±0.06 in the normal control group and lower than 0.31±0.09 in the high glucose group (both at P<0.05). The relative expressions of bcl-2 protein was 0.66±0.25 in the Res-treated group, which was significantly lower than 0.80±0.14 in the normal control group and higher than 0.23±0.09 in the high glucose group (both at P<0.05). The bcl-2/bax ratio in the Res-treated group was significantly higher than that in the high glucose group ( P<0.01). Conclusions:Res can inhibit the apoptosis of RGCs of diabetic rats and high glucose-induced RPE cells in vitro.

Applications of artificial intelligence in the diagnosis of eye diseases / 中华眼底病杂志

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.

Treatment of inverted internal limiting membrane flap technique combined with phacoemulsification for idiopathic macular hole with cataract / 中国医师进修杂志

Objective@#To analyze the efficacy and safety of inverted internal limiting membrane flap technique combined with phacoemulsification and intraocular lens implantation for idiopathic macular hole (IMH) with cataract.@*Methods@#From January 2018 to July 2018, fifty-seven patients (57 eyes) with IMH treated with 23G vitrectomy in Renmin Hospital of Wuhan University, were included. The patients were divided into 2 groups. 41 eyes underwent inverted internal limiting membrane flap technique (A group), and 16 eyes underwent inverted internal limiting membrane flap technique combined with phacoemulsification and intraocular lens implantation (B group). The visual acuity recovery, macular hole changes and the incidence of complications were analyzed retrospectively.@*Results@#The best corrected visual acuity of the two groups was significantly improved after 6 months (P < 0.01), and there was no significant difference between group A and group B (P= 0.43). The closure rate of macular hole was 100.0% in both groups. There were 11 eyes (26.8%, 11/41) of group A and 5 eyes (5/16) of group B with temporarily increased intraocular pressure (P= 0.75), and 4 eyes (9.8%, 4/41) of group A and 6 eyes (6/16) of group B with temporary corneal edema (P < 0.01). Complicated cataract was found in 7 eyes of group A (17.1%, 7/41). Concurrently, posterior capsular opacity occurred in 1 case (1/16) of group B. No iatrogenic retinal breaks, macular edema, choroidal neovascularization or infective endophthalmitis occurred in both groups.@*Conclusions@#Inverted internal limiting membrane flap technique combined with phacoemulsification and intraocular lens implantation for IMH with cataract can achieve higher anatomical reduction rate and hole healing rate, and significantly improve the visual acuity of patients while avoiding secondary surgery. It is a safe and effective surgical operation.

Effect of resveratrol on the expression of Bcl-2 and Bax and in the retina of diabetic rats / 实用医学杂志

Objective To observe the effect of resveratrol(Res)on the expression of Bcl-2 and Bax in the retina of streptozotocin(STZ)induced diabetic rats.Methods Among 35 adult healthy male SD rats,10 were ran-domly divided into normal control group(Control),25 were used for the diabetic animal model with STZ.Success-fully modeled 20 rats were randomly divided into diabetes mellitus group(DM group)and resveratrol treated group (DM+Res group)equally. The DM+Res group were administrated with intragastric Res daily. After 12 weeks of Res administration,the eyes of three groups were dissected. Retinal histology was examined,and the thickness of the retina was measured.And the expression of Bcl-2 and Bax in the retinal tissues was determined by immunohisto-chemical staining. Results Compared with the control group,the thickness of the retina in DM group significant-ly decreased(P < 0.01),while the thickness of the retina in DM+Res group significantly increased(P < 0.05) compared with the DM group.Consistent with these effects,Res treatment increased the Bcl-2 level and decreased Bax expression level,at the same time,increased the ratio of Bcl-2-to-Bax(P<0.01). Conclusion Res is able to reduce apoptosis in retinal neurons caused by diabetes through up-regulating Bcl-2 expression and inhibiting Bax expression,as a result of increasing the ratio of Bcl-2-to-Bax.