Single-cell RNA sequencing analysis of the retina under acute high intraocular pressure

High intraocular pressure causes retinal ganglion cell injury in primary and secondary glaucoma diseases,yet the molecular landscape characteristics of retinal cells under high intraocular pressure remain unknown.Rat models of acute hypertension ocular pressure were established by injection of cross-linked hyaluronic acid hydrogel(Healaflow■).Single-cell RNA sequencing was then used to describe the cellular composition and molecular profile of the retina following high intraocular pressure.Our results identified a total of 12 cell types,namely retinal pigment epithelial cells,rod-photoreceptor cells,bipolar cells,Müller cells,microglia,cone-photoreceptor cells,retinal ganglion cells,endothelial cells,retinal progenitor cells,oligodendrocytes,pericytes,and fibroblasts.The single-cell RNA sequencing analysis of the retina under acute high intraocular pressure revealed obvious changes in the proportions of various retinal cells,with ganglion cells decreased by 23%.Hematoxylin and eosin staining and TUNEL staining confirmed the damage to retinal ganglion cells under high intraocular pressure.We extracted data from retinal ganglion cells and analyzed the retinal ganglion cell cluster with the most distinct expression.We found upregulation of the B3gat2 gene,which is associated with neuronal migration and adhesion,and downregulation of the Tsc22d gene,which participates in inhibition of inflammation.This study is the first to reveal molecular changes and intercellular interactions in the retina under high intraocular pressure.These data contribute to understanding of the molecular mechanism of retinal injury induced by high intraocular pressure and will benefit the development of novel therapies.

Dynamic changes of inducible nitric oxide synthase expression in rat's retina and its role on blood-retinal barrier injury after acute high intraocular pressure

AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized into 7 groups [control(Cont), 3, 6, 12, 24, 48, and 72 h, n=6]. Except Cont group, other groups’ retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of i NOS and tight junction protein zonula occludens(ZO)-1 was detected by Western blotting. Evans blue(EB;3%) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6 h, 12 h groups, the expression of i NOS was localized by immunofluorescence. In order to verify the role of i NOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor(Inh), 6 h and 6 h+Inh, n=9]. After treatment with the i NOSspecific inhibitor 1400 W, the expression of i NOS and ZO-1 and the leakage of BRB were detected again.RESULTS: The immunofluorescence results showed that the expression of i NOS was observed in the Cont group and 6 h group, but not in the 12 h group. i NOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker Neu N but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period(3 h, 6 h) after acute high IOP, the expression of i NOS was upregulated, then the down-regulation of i NOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous downregulation after 6 h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3 h after acute high IOP. At 6 h, the leakage of EB was lower, but at 12 h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of i NOS was down-regulated after 1400 W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6 h group, and significantly down-regulated in the 6 h+Inh group, and the leakage of EB was significantly increased after 1400 W treatment.CONCLUSION: These results suggest that the upregulation of i NOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.

Effect of Qingguang'anⅡon expressions of OX42 protein and IL-1βmRNA of retinal microglia cells of rats with chronic high intraocular pressure

The study investigated the effects of Qingguang＇an Ⅱ（a Chinese medicinal preparation） on expressions of OX42 protein and interleukin-1β（IL-1β） mRNA of retinal microglia cells of rats with chronic high intraocular pressure（IOP）. SD rats were randomly divided into 6 groups, that were： A： blank group; B： model group; C： Qingguang＇an Ⅱ low dose group; D： Qingguang＇an Ⅱ medium dose group; E： Qingguang＇an Ⅱ high dose group; F： Yimaikang disket（a Chinese medicinal preparation） group. Experimental rats in B, C, D, E, F groups were established the model of chronic high IOP by cauterizing of superficial scleral vein. Tissues of eyes were obtained after intragastric administration for 2 and 4 wk. At the time-point of 2 wk, OX42 protein and IL-1β mRNA in group B were statistically expressed in higher level comparing with other groups（P〈0.05）. Moreover, at the time-point of 4 wk, OX42 protein and IL-1β mRNA in groups C, D and E were statistically expressed in lower level comparing with group F（P〈0.05）. Besides, OX42 protein and IL-1β mRNA in groups C and D were statistically expressed in higher level comparing with group E（P〈0.05）. OX42 protein and IL-1β mRNA in groups C and D were expressed in similar level（P〉0.05）. The study indicated that, in the protection of optic nerve of rats with chronic high IOP, the high dose of Qingguang＇an Ⅱ at the time-point of 4 wk was the better choice.

TrkB and p-trkB expression in brain-derived neurotrophic factor-pretreated rat retina following acute high intraocular pressure

BACKGROUND： Exogenous brain-derived neurotrophic factor （BDNF） promotes retinal ganglion cell survival. However, the protective mechanisms remain unclear. OBJECTIVE： To investigate changes in retinal tyrosine kinase receptor B （trkB） expression and effects of exogenous BDNF on trkB activation in a rat model of acute high intraocular pressure （HtOP）. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Department of Anatomy and Neurobiology, Xiangya Medical School, Central South University from January 2004 to August 2006. MATERIALS： Rabbit anti-BDNF and anti-trkB.FL（full-length） polyclonal antibodies were purchased from Santa Cruz Biotechnology, USA; rabbit anti-p-trkB polyclonal antibodies were purchased from Cellsignal, USA. METHODS： A total of 48 healthy, adult, Sprague Dawiey rats were randomly assigned to acute HIOP （without BDNF pre-treatment） and BDNF pre-treated groups, with 24 animals in each group. In the BDNF pre-treated group, the left eyes were intravitreally injected with 3 pg/kg BDNF 2 days prior to HIOP. Rats in the acute HIOP group were not pre-treated with BDNE HIOP models were established by increased intraocular pressure in the left eyes until the b-wave of flash electroretinogragh disappeared and pressure was maintained for 60 minutes. The right eyes of all rats were not treated and served as the normal controls. MAIN OUTCOME MEASURES： Retinal structure and cell numbers in the ganglion cell layer （GCL） were detected by Nissl staining; expression of trkB and phosphorylated trkB in the rat retina were determined by immunohistochemistry. RESULTS： A greater number of GCL neurons were observed in the pre-treated group compared to the acute HIOP group （P 〈 0.05）. TrkB expression was significantly increased following HIOP at days 1 and 3 （P 〈 0.05）, but expression varied between retinal areas. Although trkB expression decreased at 7 days, phosphorylated trkB dramatically decreased with increasing time （P 〈 0.05）. TrkB expression in BDNF pre-treated rats was similar to the acute HIOP group at early injury time points. Nevertheless, trkB expression was significantly decreased compared to the acute HIOP group at 7 days （P 〈 0.05）, and phosphorylated trkB expression was significantly greater compared to the acute HIOP group at each time point （P〈 0.05）. CONCLUSION： TrkB expression displayed temporal and spatial changes in the rat retina following acute HIOP, and trkB up-regulation suggested that more BDNF was required for treating the injured retina. Exogenous BDNF partially ameliorated decreased expression of phosphorylated trkB and provided protection to the injured retina, to a certain degree, following HIOP.

Bioinformatics analysis of potential essential genes that response to the high intraocular pressure on astrocyte due to glaucoma

AIM: To study the gene expression response and predict the network in cell due to pressure effects on optic nerve injury of glaucoma.METHODS: We used glaucoma related microarray data in public database [Gene Expression Omnibus(GEO)] to explore the potential gene expression changes as well as correspondent biological process alterations due to increased pressure in astrocytes during glaucoma development.RESULTS: A total of six genes were identified to be related with pressure increasing. Through the annotation and network analysis, we found these genes might be involved in cell morphological remodeling, angiogenesis,mismatch repair.CONCLUSION: Increasing pressure in glaucoma on astrocytes might cause gene expression alterations,which might induce some cellular responses changes.

Vascular endothelial growth factor-165b protects the blood-retinal barrier from damage after acute high intraocular pressure in rats

AIM:To elucidate the role of vascular endothelial growth factor-165b(VEGF-165b)in blood-retinal barrier(BRB)injury in the rat acute glaucoma model.METHODS:In this study,the rat acute high intraocular pressure(HIOP)model was established before and after intravitreous injection of anti-VEGF-165b antibody.The expression of VEGF-165b and zonula occludens-1(ZO-1)in rat retina was detected by double immunofluorescence staining and Western blotting,and the breakdown of BRB was detected by Evans blue(EB)dye.RESULTS:The intact retina of rats expressed VEGF-165b and ZO-1 protein,which were mainly located in the retinal ganglion cell layer and the inner nuclear layer and were both co-expressed with vascular endothelial cell markers CD31.After acute HIOP,the expression of VEGF-165b was up-regulated;the expression of ZO-1 was down-regulated at 12h and then recovered at 3d;EB leakage increased,peaking at 12h.After intravitreous injection of anti-VEGF-165b antibody,the expression of VEGF-165b protein was no significantly changed;and the down-regulation of the expression of ZO-1 was more obvious;EB leakage became more serious,peaking at 3d.EB analysis also showed that EB leakage in the peripheral retina was greater than that in the central retina.CONCLUSION:The endogenous VEGF-165b protein may protect the BRB from acute HIOP by regulating the expression of ZO-1.The differential destruction of BRB after acute HIOP may be related to the selective loss of retinal ganglion cells.

Does high intraocular pressure exclude an open globe injury?

Dear Sir,Ocular trauma is a topic of unresolved controversies and there are continuous controversial and debatable diagnostic and management strategies for open-globe injuries[1].Amongst many types of ocular trauma,the open globe injury is the most serious due to its very poor visual prognosis and young population of patients are mostly affected[2].The treatment outcome may be improved by prompt diagnosis,and immediate surgical repair performed to high standard[3].Amongst the other clinical signs the intraocular pressure(IOP)is found to be particularly reduced and conventionally this is considered as a very reliable indicator of occult globe

NMI,POLR3G and APIP are the key molecules connecting glaucoma with high intraocular pressure:a clue for early diagnostic biomarker candidates

AIM:To understand the molecular connectivity between the intraocular pressure(IOP)and glaucoma which will provide possible clues for biomarker candidates.METHODS:The current study uncovers the important genes connecting IOP with the core functional modules of glaucoma.An integrated analysis was performed using glaucoma and IOP microarray datasets to screen for differentially expressed genes(DEGs)in both conditions.To the selected DEGs,the protein interaction network was constructed and dissected to determine the core functional clusters of glaucoma.For the clusters,the connectivity of IOP DEGs was determined.Further,enrichment analyses were performed to assess the functional annotation and potential pathways of the crucial clusters.RESULTS:The gene expression analysis of glaucoma and IOP with normal control showed that 408 DEGs(277 glaucoma and 131 IOP genes)were discovered from two GEO datasets.The 290 DEGs of glaucoma were extended to form a network containing 1495 proteins with 9462 edges.Using ClusterONE,the network was dissected to have 12 clusters.Among them,three clusters were linked with three IOP DEGs[N-Myc and STAT Interactor(NMI),POLR3G(RNA Polymerase Ⅲ Subunit G),and APAF1-interacting protein(APIP)].In the clusters,ontology analysis revealed that RNA processing and transport,p53 class mediators resulting in cell cycle arrest,cellular response to cytokine stimulus,regulation of phosphorylation,regulation of type Ⅰ interferon production,DNA deamination,and cellular response to hypoxia were significantly enriched to be implicated in the development of glaucoma.Finally,NMI,POLR3G,and APIP may have roles that were noticed altered in glaucoma and IOP conditions.CONCLUSION:Our findings could help to discover new potential biomarkers,elucidate the underlying pathophysiology,and identify new therapeutic targets for glaucoma.

Protective limb remote ischemic post-conditioning against high-intraocular-pressure-induced retinal injury in mice

AIM: To determine whether limb remote ischemic postconditioning(LRIC) protects against high-intraocularpressure(IOP)-induced retinal injur y, and to identify underlying molecular mechanisms. METHODS: In mice, IOP was increased to 110 mm Hg for 50 min and LRIC applied to the unilateral leg for three occlusion cycles(5 min/release). Three animal groups(control, high IOP, and high IOP+LRIC) were arranged in this study. Plasma was collected from LRIC treated mice. Retinal histology, oxidative stress were determined by histological section staining and chemical kit. C/EBP homologous protein(CHOP), and Iba-1 parameters were evaluated by immunofluorescent staining and Western blot. RESULTS: The data showed that LRIC treatment alleviated the retinal histological disorganization and ganglion cell loss induced by high IOP. The CHOP, Iba-1 expression and oxidative stress marker also were inhibited by LRIC treatment. To further explore underlying mechanisms, plasma from LRIC treated animals was intravenously transfused into high-IOP animals. The results showed plasma injection decreased caspase 9 expression and DHE staining signals compared with that in high IOP retinas. CONCLUSION: These data suggest that LRIC treatments exert retinal protective effects against high-IOP injury.Endogenous humoral factors release into the circulation by LRIC may contribute to homeostatic protection by reducing monocyte infiltration and/or microglia activation.

Influence of transient intraocular pressure elevation during laser in situ keratomileusis on rabbit retina thickness

AIMTo utilize tissue micro measurement to study the effect of transient high intraocular pressure (IOP) induced by different durations of suction during laser in situ keratomileusis (LASIK) on rabbit retina thickness.METHODSSixty healthy New Zealand white rabbits were randomly divided into a control group, and 3 negative-pressure suction groups (20s group, 45s group, and 3min group) and each group was comprised of 15 rabbits (30 eyes); the latter 3 groups were the transient high IOP models. The retinal tissue around the papilledema was separated. Hematoxylin and eosin (HE) staining was carried out to generate slices for light microscopy. The changes in the retina thickness values of each layer were measured for all animals in each group at different postoperative recovery periods and compared with the values recorded for the animals in the control group. The thickness of the retinal tissue showed a normal distribution. The ANOVA was performed by using SPSS13.0 statistic software.RESULTSIn the comparison between the 20s and 45s negative-pressure suction groups and the control group, no significant differences were observed, except at 14d. Significant difference was observed between the 3min negative-pressure suction group and the control group, and the retina thickness value of each layer reached a peak at 14d after repair.CONCLUSIONConventional negative suction during LASIK may not lead to significant changes in retinal tissue thickness; however, if the suction duration is increased to 3min, it will cause significant changes in retinal tissue thickness.