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.

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.

Objective:To investigate the role of platelet-rich fibrin (PRF) membrane tamponade combined with silicone oil filling in the treatment of high myopia macular hole (HMMH) with retinal detachment (RD).Methods:A randomized controlled study was conducted.A total of 52 patients (52 eyes) with HMMH with RD were enrolled at the Eye Center, Renmin Hospital of Wuhan University from September 2021 to April 2023.The patients were randomly divided into three groups according to the random number table method.All patients in the three groups underwent standard three-channel 23-gauge pars plana vitrectomy.In the internal limiting membrane (ILM) peeling group including 18 cases (18 eyes), the ILM was peeled intraoperatively.In the ILM tamponade group including 16 cases (16 eyes), the ILM flap was inverted and filled into the macular hole (MH).In the PRF tamponade group including 18 cases (18 eyes), the MH was filled with PRF.Intraocular pressure measurement, best corrected visual acuity (BCVA) measurement, and central macular thickness (CMT) measured by optical coherence tomography were performed preoperatively and 1 week, 1 month, 3 months, and 6 months postoperatively.Superficial retinal vessel density (SVD) and deep retinal vessel density (DVD) were determined by optical coherence tomography angiography before surgery and 6 months after surgery.The efficacy of the procedure was determined at six months postoperatively, and the rates of MH closure and retinal reattachment were compared among the three groups.This study adhered to the Declaration of Helsinki and the study protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University (No.2022-1-X-53).Written informed consent was obtained from each subject before any medical examination.Results:At 6 months postoperatively, the MH closure rates in the ILM peeling, ILM tamponade, and PRF tamponade groups were 83.3%(15/18), 87.5%(14/16), and 94.4%(17/18), respectively, without significant differences among them (fit χ2=1.180, P>0.05).Furthermore, the retinas of all groups were reattached.The postoperative BCVA of ILM peeling, ILM tamponade, and PRF tamponade groups were elevated compared with before surgery, and the differences were statistically significant (all at P<0.05).At 6 months after surgery, the CMT of the PRF tamponade group was significantly thicker than that of the ILM peeling and ILM tamponade groups (both at P<0.05).The CMT of the three groups at different time points after surgery was significantly decreased compared with before surgery, with statistically significant differences (all at P<0.05).SVD and DVD of the three groups at 6 months postoperatively were higher compared with before surgery, with statistically significant differences (all at P<0.05).No serious complications such as endophthalmitis and vitreous haemorrhage occured during treatment and follow-up.Müller cell gliosis was observed in 4 eyes in the ILM tamponade group, and no Müller cell gliosis eyes were seen in the remaining two groups. Conclusions:PRF tamponade combined with silicone oil filling can promote MH healing and retinal reattachment, improve visual acuity and blood flow density in patients suffering from HMMH with RD, and is a safe and effective surgical procedure.

Objective:To observe the effects of overexpression of S100A4 protein on retinal capillary cells and retinal ganglion cells (RGC) after retinal ischemia-reperfusion injury (RIRI).Methods:One 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.Results:Three 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). Conclusions:Pericytes 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.

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.

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.

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.

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.

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.

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.