Objective:To investigate the association between cytokines and ocular chronic graft-versus-host disease (cGVHD) and identify specific biomarkers for ocular cGVHD to enhance clinical diagnosis, treatment, and evaluation.Methods:A mouse model of cGVHD was established to explore the correlation between cGVHD and serum cytokines. Based on the findings from the animal experiments and literature review, a panel of 16 cytokine combinations was identified. Enzyme-linked immunosorbent assay (ELISA) was used to compare the cytokine concentrations in the serum and tear samples from patients who underwent allogeneic hematopoietic stem cell transplantation from June 2017 to March 2022 at the Medical Center of Hematology, Xinqiao Hospital, Army Medical University.Results:① Compared with the control group, mice with cGVHD exhibited elevated serum IL-1β, IL-6, IL-8, IL-17, IFN-γ, CX3CL1, CXCL11, CXCL13, CCL11, and CCL19 concentrations (all P<0.05). ② Analysis of the cytokine profiles of the serum and tear samples revealed that compared with patients without ocular cGVHD, those with ocular cGVHD exhibited increased serum IL-8 [ P=0.032, area under the curve (AUC) =0.678]; decreased serum IL-10 ( P=0.030, AUC=0.701) ; elevated IL-8, IFN-γ, CXCL9, and CCL17 in tear samples; and lower IL-10 and CCL19 in tear samples (all P<0.05, all AUC>0.7). Moreover, cytokines in tear samples showed correlations with ocular surface parameters related to ocular cGVHD. Conclusions:Tear fluid demonstrates greater specificity and sensitivity as a biomarker for diagnosing ocular cGVHD than serum biomarkers. Among the identified cytokines in tear samples, IL-8, IL-10, IFN-γ, CXCL9, CCL17, and CCL19 serve as diagnostic biomarkers for ocular cGVHD post-transplantation, offering practical reference value for diagnosis.

Objective To investigate the changes in G protein-coupled receptor 124 (Gpr124) expression in human retinal microvascular endothelial cells (HRMEC)under high-glucose condition and the regulatory effect of Gpr124 interference on HRMEC.Methods Immunofluorescence assay was used to observe the expression of Gpr124 in HRMEC.The cells were divided into a blank group and a high glucose group.CCK-8 assay and EdU cell proliferation assay were used to detect cell viability and proliferation,respectively.The Gpr124 expression was knocked down by transducting lentiviral virus carrying shRNA targeting Gpr124.Thus,the cells were divided into a blank control group,a high glucose control group,a high glucose+shRNA group,and a high glucose+Gpr124 shRNA group.Cell scratch test was performed to evaluate cell migration,and Matrigel plug assay was employed to assess the cell tubule formation.Western blotting was applied to detect the protein levels of Gpr124,VEGFA,MMP9,and β-catenin.Results The HRMEC from the high glucose group showed enhanced cell viability and increased number of proliferating cells compared to the cells of the blank group (P<0.01).High glucose treatment also induced increased expression levels of Gpr124,VEGFA and MMP9 (P<0.01),and larger migratory area and in vitro angiogenic area as well as longer tube diameter at all time points when compared with the conditions in the blank control group (P<0.01).However,knockdown of Gpr124 resulted in a significant decrease in HRMEC migration and in vitro angiogenic capacity,accompanied by decreases in protein expression of VEGFA,MMP9 and β-catenin (P<0.01).Conclusion Gpr124 can regulate the proliferation,migration,and tube formation ability of HRMEC under high-glucose condition,and also inhibit the overexpression of VEGFA and MMP9,which may be through regulating the classical Wnt/β-catenin pathway.

Objective:To observe the clinical and multimodel image features in patients of acute macular neuroretinopathy (AMN) associated with COVID-19.Methods:Clinical data and multimodal imaging results in 12 patients (24 eyes) of AMN associated with COVID-19 which were diagnosed in our Department of Ophthalmology, The Second Hospital of The Army Medical University ranging from December 2022 to January 2023 were included. All patients were examined by best-corrected visual acuity (BCVA), color fundus photography, optical coherence tomography (OCT), OCT angiography (OCTA), visual field tests, high-resolution computed tomography of the lungs and inflammation-related laboratory tests. After diagnosis, patients were reveived methylcobalamin and Ginaton therapy, 6 patients receiced intravenous dripping of dexamethasone. The follow-up time of this study was 4 weeks.Results:There were 1 male (2 eyes) and 11 females (22 eyes), with the mean age of (29.00±5.17) years. For all cases, decreased vision presented 1 to 2 days after patients being suffered from several flu-like symptoms of COVID-19. Ten eyes in 5 patients were at the acute phrase of AMN, which the vision decrease occurred 1-5 days after they were diagnosed with COVID-19; 14 eyes in 7 patients were at the progressive stage of AMN, which they were diagnosed with COVID-19 for more than 5 days. The BCVA of patients were 0.02-0.9, in company with one or more central/pericentral scotoma. Fundus ophthalmoscopy revealed reddish-brown lesion around macula, IR imaging demonstrated localized areas of hypo-shape lesions in the macula. For 16 eyes, their OCTA revealed a decreased density of blood flow in the deep layer of retinal capillary plexus, and OCT revealed that high reflex bands existed in the outer nuclear layer (ONL) and outer plexiform layer of the retina at the acute phrase of AMN, in accompany with hypo-reflection in ellipsoid zone (EZ) and interdigitation zone (IZ); for retina at the progressive stage of AMN, ONL became thinner in some cases and the continuation interruption occurred in EZ and IZ. For en-face OCT, high reflex bands with clear edges existed in the ONL and EZ layer of the retina at the acute phrase of AMN, but no high reflex bands existed in the ONL and EZ layers of the retina at the progressive stage of AMN. Instead, hypo-reflection with similar shape occurred in the EZ and IZ layers at the progressive stage of AMN. During treatment, 5 patients at the acute phrase reported a shrinking central scotomas and raised BCVA, otherwise the BCVA of 7 patients at the progressive stage changed slightly. For 10 eyes at the acute phrase of AMN, OCT revealed that the high reflex bands of ONL and OPL missed within 1 week. En-face OCT revealed the hyper-reflection in the ONL and EZ layers of retina in the acute phrase of AMN receded within 1 week, but hypo-reflection of ONL and EZ still presented.Conclusions:For AMN associated with COVID-19, IR imaging demonstrated localized areas of hypo-shape lesions in the macula. OCT revealed high reflex bands in ONL and OPL of the retina at the acute phrase of AMN, but hypo-reflection of ONL and EZ still presented. The continuation interruption occurred in EZ and IZ at the progressive phrase. En-face OCT revealed hyper-reflection in ONL and EZ of the retina at the acute phrase which receded within 1 week, and hypo-reflection in EZ and IZ of the retina presented at all phrases during AMN progression.

Objective:To analyze the clinical features in patients of Purtscher-like retinopathy (PLR) associated with COVID-19.Methods:A retrospective clinical study. Clinical data of 4 patients (7 eyes) of PLR associated with COVID-19 which was firstly diagnosed in Department of Ophthalmology, The Second Hospital of The Army Medical University ranging from December 2022 to January 2023 were included. All patients were examined by best-corrected visual acuity (BCVA), color fundus photography, optical coherence tomography (OCT), OCT angiography, fundus fluorescein angiography (FFA), multifocal electroretinogram (mf-ERG), visual field. Oral vasodilators and neurotrophic agents were applied after definite diagnosis. Three patients were treated by intravenous dexamethasone (10 mg) for 3 days. Follow-up time spans for 4 weeks. The multi-model images, clinical features and treatment response of patients were also retrospectively studied.Results:Among 4 patients (7 eyes), 2 cases were male (3 eyes), 2 cases were female (4 eyes), the mean age was (36.00±17.57) years; 3 cases were unilateral, 1 case were unilateral. The time from diagnosis of COVID-19 to the onset of eye symptoms was 2 to 3 days. The BCVA of the affected eye was finger counting for 20 cm to 0.5. Color fundus photography examination revealed that several retinal whitening of varying sizes distributed diffusely on the retina (cotton-wool spots and Purtscher flecken). OCT examination showed that the retinal nerve fiber layer was significantly thickened and the reflex was enhanced in the area corresponding to the gray and white lesions, the inner nuclear layer, internal and external plexus layer segmental and banded strong reflex was observed in 5 eyes. En-face image showed mottled strong reflex in the inner retinal layer and around the deep capillary plexus (DCP). For OCTA, the signal of superficial capillaries and cotton wool spots were lost, and blood flow of deep DCP was poorly distributed. FFA examination showed that the obscured fluorescence of choroid background of Purtscher spot and capillary non-perfusion area. mf-ERG examination showed decreased amplitude of retinal a-wave and b-wave. Visual field examination showed central and paracentral scotoma. During follow-up, the BCVA of diseased eyes were raised, scotomas were shrunk, cotton wool spots and Purtscher flecken became smaller. OCT examination showed that the thickness of the original thickened retinal nerve fiber layer became thinner, the area of the strong reflex area was reduced, and the inner layer of the original segmental strong reflex area was thinner. OCTA examination revealed partial recovery of blood flow signal.Conclusion:For PLR associated with COVID-19, OCT shows thickening of retinal nerve fiber layer, segmental and banding strong reflex in part of the inner layer and inner plexus layers; OCTA is manifested by ischemia in the middle retina and DCP.

High myopia macular hole (MH) is a serious complication of high myopia. The main treatment method is surgery. Because of axial growth, posterior scleral staphyloma, choroidal atrophy and other factors, the operation is difficult, the anatomic reduction rate is low, and the visual prognosis is poor. How to improve the reduction rate of surgical dissection and the recovery of visual function is a hot topic. At present, the most popular surgeries include parsplanavitrectomy (PPV) and posterior scleral reinforcement (PSR). However, there are many controversies regarding the treatment of internal limiting membrane in PPV, the selection of vitreous gapfiller, the choice of reinforcement materials and reinforcement methods of PSR, and whether it is necessary to combine PPV and PSR, etc. In recent years, many new surgical methods or techniques have emerged, which significantly increase the success rate of MH.

In the central nervous system (CNS), three types of myelin-associated inhibitors (MAIs) have major inhibitory effects on nerve regeneration. They include Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein. MAIs possess two co-receptors, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB). Previous studies have confirmed that the inhibition of NgR only results in a modest increase in regeneration in the CNS; however, the inhibitory effects of PirB with regard to nerve regeneration after binding to MAIs remain controversial. In this study, we demonstrated that PirB is expressed in primary cultures of retinal ganglion cells (RGCs), and the inhibitory effects of the three MAIs on the growth of RGC neurites are not significantly decreased after direct PirB knockdown using adenovirus PirB shRNA. Interestingly, we found that retinal Müller cells expressed PirB and that its knockdown enhanced the regeneration of co-cultured RGC neurites. PirB knockdown also activated the JAK/Stat3 signaling pathway in Müller cells and upregulated ciliary neurotrophic factor levels. These findings indicate that PirB plays a novel role in retinal Müller cells and that its action in these cells may indirectly affect the growth of RGC neurites. The results also reveal that PirB in Müller cells affects RGC neurite regeneration. Our findings provide a novel basis for the use of PirB as a target molecule to promote nerve regeneration.

Objective To observe the corneal nerve fibres damage in different stage of diabetic retinopathy (DR) with type 2 diabetes.Methods A cross-sectional study.One hundred and twenty eyes of 120 patients with type 2 diabetes served as diabetes group.According to International Clinical Diabetic Retinopathy Disease Severity Scales (2002),diabetes patients were classified into 4 subgroups:patients without diabetic retinopathy (NDR),patients with mild or moderate non-proliferative diabetic retinopathy (mNPDR),patients with severe non-proliferative diabetic retinopathy (sNPDR) and patients with proliferative diabetic retinopathy (PDR),each subgroup has 30 eyes of 30 patients.Another 30 eyes of 30 healthy participants served as control group.All eyes were scanned with HRT3 in vivo corneal confocal microscopy.Images of sub-basal nerve plexus were quantified including nerve fiber length (NFL),nerve fiber density (NFD),nerve fiber branch density (NFB),and nerve tortuosity (NT).The correlations of corneal nerve fiber with age,duration of diabetes and glycated hemoglobin (HbA1 c) were analyzed using Spearman correlation analysis.Results NFL,NFD and NFB were found to be significantly lower in diabetic patients (F=147.315,142.586,65.898;P=0.000,0.000,0.000),NT was significantly greater in diabetic patients (F=39.431,P=0.000),when compared to control group.In diabetic patients,NFL,NFD and NFB were gradually reduced with DR severity,NT was gradually increased with DR severity.While the difference ofNFL,NFD,NFB,NT was not statistically significant between sNPDR and PDR subgroups (P＞.0.05),but was statistically significant between other subgroups (P＜0.05).Spearman correlation analysis results showed that age (r=-0.071,-0.080,0.001,0.100;P=0.391,0.328,0.991,0.224) and HbAlc (r=-0.109,-0.115,-0.126,0.025;P=0.238,0.211,0.169,0.781) had no correlation with NFL,NFD,NFB,NT.Duration of diabetes was negatively correlated with the NFL,NFD (r=-0.212,-0.264;P=0.020,0.004),positive correlated with NT (r=0.261,P=0.004),and had no correlation with NFB (r=-0.119,P=0.194).Conclusions Corneal nerve fiber loss and nerve tortuosity increased were found in patients with type 2 diabetes,and even without diabetic retinopathy.The progress of corneal neuropathy was correlated with the severity of DR,but it was not change significantly between sNPDR and PDR.

Background Collapsin response mediator protein-2 (CRMP-2) can promote the growth of axons,but CRMP-2 occurs hyperphosphorylation under the induction of cyclin-dependent kinase-5 (CDK5) after central nervous system injury,which leads to the collapse of the growth cone and hinders the repair of nervous system.Being a central nervous system tissue,whether the expressions of CRMP-2 and its phosphorylated protein (p-CRMP-2) change after optic nerve injury are rarely studied.Objective This study was to investigate the dynamic changes of CRMP-2 and p-CRMP-2 expressions in injured optic nerve tissue.Methods Forty-eight 8-or 9-week-old BALB/c mice were randomly divided into the sham operation group and postoperative 3-,7-and 14-day group.Optic nerves were exposed and clamped at retrobulbar 2 mm for 10 seconds in the right eyes during the surgery in the postoperative 3-,7-and 14-day groups,and the same operation was performed except the clamp of optic nerve in the sham operation group.The optic nerve tissue was obtained from the eyes 3,7 and 14 days after surgery.The relative expression levels of CRMP-2 mRNA and CRMP-2,p-CRMP-2 and CDK5 proteins in the tissue were detected by real-time fluorescence quantitative PCR and Western blot,respectively.The use and care of the experimental animals complied with the Regulation for the Administration of Affair Concerning Experimental Animals of Third Military Medical University.Results No significant differences were found in the expression levels of CRMP-2 mRNA and CRMP-2 protein among the sham operation group and postoperative 3-,7-and 14-day groups (CRMP-2 mRNA:F =2.971,P =0.097;C RMP-2 protein:F=1.202,P =0.370).The relative expression levels of p-CRMP-2 protein in the optical nerve were 0.001±0.000,0.064±0.003,0.136±0.005 and 0.346±0.012,and those of CDK5 protein were 0.440±0.009,0.723±0.011,0.874±0.015 and 0.952±0.019 in the sham operation group and postoperative 3-,7-and 14-day groups respectively,showing statistically significant differences among them (p-CRMP-2:F=445.600,P ＜ 0.001;CDK5:F=186.600,P＜0.001),and the relative expression levels of p-CRMP-2 and CDK5 protein were evidently higher in the optical nerve tissue in the postoperative 3-,7-and 14-day groups than those in the sham operation group (all at P＜0.01).Conclusions There are not significant changes in the expression level of CRMP-2 in the BALB/c mice after optic nerve injury.However,the expression levels of p-CRMP-2 and CDK5 proteins are gradually upregulated as the extending of injured time.

Objective To determine the effect of paired immunoglobulin-like receptor B (PirB) on growth of retinal ganglion cells (RGCs) in vitro.Methods Expression of PirB in RGCs isolated from wild-type C57 BJ/6 mice was measured by immunofluorescence.Real-time PCR and Western blot were performed to detect PirB expressions in RGCs cultured for 1,3,5,7 and 9 days.Primary cultured RGCs were left untreated as controls,transfected with lentiviral-delivered PirB RNAi as Group A,lentiviral-delivered NgR RNAi as Group B,lentiviral-delivered PirB RNAi plus ciliary neurotrophic factor (CNTF) as Group C,lentiviral-delivered NgR plus CNTF as Group D,and only CNTF as Group E.Growth and development of RGCs were evaluated by MTT assay and morphological analysis.Results Immunofluorescence confirmed expression of PirB in RGCs,with its mRNA and protein levels changing from increase at first to decrease later with time,up to peak at days 5 and 7.PirB and NgR were respectively down-regulated in Groups A and B.Viability of RGCs was improved in Group A compared with control group [(40.2 ± 5.3) μm vs (29.1 ± 3.8) μm at day 3 ; (72.2 ± 4.2) μm vs (52.3 ± 8.2) μm at day 5,both P ＜ 0.05].By contrast,no significant difference was found in viability of RGCs among Groups B,D and E when compared to control group (P ＞ 0.05).Growth of RGCs between Groups A and C revealed insignificant difference (P ＞ 0.05).Conclusion Knockdown of PirB is beneficial for the growth of RGCs,suggesting a novel method to treat optic nerve injury.

ObjectiveTo investigate the expression of paired immunoglobulin-like receptor B (PirB) in optic nerve, visual cortex, cerebella, spinal cord and sciatic nerve of normal adult mice.MethodsTwelve healthy adult BALB/c mice were randomly and equally divided into two groups.The immunohistochemistry and Western blot were used to detect the expression of PirB in the tissues described above respectively.ResultsBoth the immunohistochemistry and Western blot test revealed that the expression of PirB was positive in the optic nerve, visual cortex, cerebella and spinal cord, but negative in the sciatic nerve.The positive signals in the sections were located in the cell bodies and the neurites were observed in some of them.Western blot showed the apparent positive band of PirB in the optic nerve, visual cortex, cerebella and spinal cord rather than in the sciatic nerve.The protein expression level of PirB was relatively high in the visual cortex (P ＜0.05) but relatively low in the optic nerves (P ＜0.01).ConclusionThe PirB expresses positively in the optic nerve, indicating that PirB protein may closely correlate with the poor regeneration of the optic nerve.