Integrated bioinformatics analysis of retinal ischemia/reperfusion injury in rats with potential key genes.

The tissue damage caused by transient ischemic injury is an essential component of the pathogenesis of retinal ischemia, which mainly hinges on the degree and duration of interruption of the blood supply and the subsequent damage caused by tissue reperfusion. Some research indicated that the retinal injury induced by ischemia-reperfusion (I/R) was related to reperfusion time.In this study, we screened the differentially expressed circRNAs, lncRNAs, and mRNAs between the control and model group and at different reperfusion time (24h, 72h, and 7d) with the aid of whole transcriptome sequencing technology, and the trend changes in time-varying mRNA, lncRNA, circRNA were obtained by chronological analysis. Then, candidate circRNAs, lncRNAs, and mRNAs were obtained as the intersection of differentially expression genes and trend change genes. Importance scores of the genes selected the key genes whose expression changed with the increase of reperfusion time. Also, the characteristic differentially expressed genes specific to the reperfusion time were analyzed, key genes specific to reperfusion time were selected to show the change in biological process with the increase of reperfusion time.As a result, 316 candidate mRNAs, 137 candidate lncRNAs, and 31 candidate circRNAs were obtained by the intersection of differentially expressed mRNAs, lncRNAs, and circRNAs with trend mRNAs, trend lncRNAs and trend circRNAs, 5 key genes (Cd74, RT1-Da, RT1-CE5, RT1-Bb, RT1-DOa) were selected by importance scores of the genes. The result of GSEA showed that key genes were found to play vital roles in antigen processing and presentation, regulation of the actin cytoskeleton, and the ribosome. A network included 4 key genes (Cd74, RT1-Da, RT1-Bb, RT1-DOa), 34 miRNAs and 48 lncRNAs, and 81 regulatory relationship axes, and a network included 4 key genes (Cd74, RT1-Da, RT1-Bb, RT1-DOa), 9 miRNAs and 3 circRNAs (circRNA_10572, circRNA_03219, circRNA_11359) and 12 regulatory relationship axes were constructed, the subcellular location, transcription factors, signaling network, targeted drugs and relationship to eye diseases of key genes were predicted. 1370 characteristic differentially expressed mRNAs (spec_24h mRNA), 558 characteristic differentially expressed mRNAs (spec_72h mRNA), and 92 characteristic differentially expressed mRNAs (spec_7d mRNA) were found, and their key genes and regulation networks were analyzed.In summary, we screened the differentially expressed circRNAs, lncRNAs, and mRNAs between the control and model groups and at different reperfusion time (24h, 72h, and 7d). 5 key genes, Cd74, RT1-Da, RT1-CE5, RT1-Bb, RT1-DOa, were selected. Key genes specific to reperfusion time were selected to show the change in biological process with the increased reperfusion time. These results provided theoretical support and a reference basis for the clinical treatment.

Role of caspase-11 non-canonical inflammasomes in retinal ischemia/reperfusion injury.

BACKGROUND: Retinal ischemia/reperfusion (IR) injury is a common pathological process in many ophthalmic diseases. Interleukin-1ß (IL-1ß) is an important inflammatory factor involved in the pathology of retinal IR injury, but the mechanism by which IL-1ß is regulated in such injury remains unclear. Caspase-11 non-canonical inflammasomes can regulate the synthesis and secretion of IL-1ß, but its role in retinal IR injury has not been elucidated. This study aimed to evaluate the role of caspase-11 non-canonical inflammasomes in retinal IR injury. METHODS: Retinal IR injury was induced in C57BL/6J mice by increasing the intraocular pressure to 110 mmHg for 60 min. The post-injury changes in retinal morphology and function and in IL-1ß expression were compared between caspase-11 gene knockout (caspase-11-/-) mice and wild-type (WT) mice. Morphological and functional changes were evaluated using hematoxylin-eosin staining and retinal whole mount staining and using electroretinography (ERG), respectively. IL-1ß expression in the retina was measured using enzyme-linked immunosorbent assay (ELISA). The levels of caspase-11-related protein were measured using western blot analysis. The location of caspase-11 in the retina was determined via immunofluorescence staining. Mouse type I astrocytes C8-D1A cells were used to validate the effects of caspase-11 simulation via hypoxia in vitro. Small-interfering RNA targeting caspase-11 was constructed. Cell viability was evaluated using the MTT assay. IL-1ß expression in supernatant and cell lysate was measured using ELISA. The levels of caspase-11-related protein were measured using western blot analysis. RESULTS: Retinal ganglion cell death and retinal edema were more ameliorated, and the ERG b-wave amplitude was better after retinal IR injury in caspase-11-/- mice than in WT mice. Further, caspase-11-/- mice showed lower protein expressions of IL-1ß, cleaved caspase-1, and gasdermin D (GSDMD) in the retina after retinal IR injury. Caspase-11 protein was expressed in retinal glial cells, and caspase-11 knockdown played a protective role against hypoxia in C8-D1A cells. The expression levels of IL-1ß, cleaved caspase-1, and GSDMD were inhibited after hypoxia in the si-caspase-11 constructed cells. CONCLUSIONS: Retinal IR injury activates caspase-11 non-canonical inflammasomes in glial cells of the retina. This results in increased protein levels of GSDMD and IL-1ß and leads to damage in the inner layer of the retina.

Deletion of myeloid HDAC3 promotes efferocytosis to ameliorate retinal ischemic injury.

Ischemia-induced retinopathy is a hallmark finding of common visual disorders including diabetic retinopathy (DR) and central retinal artery and vein occlusions. Treatments for ischemic retinopathies fail to improve clinical outcomes and the design of new therapies will depend on understanding the underlying disease mechanisms. Histone deacetylases (HDACs) are an enzyme class that removes acetyl groups from histone and non-histone proteins, thereby regulating gene expression and protein function. HDACs have been implicated in retinal neurovascular injury in preclinical studies in which nonspecific HDAC inhibitors mitigated retinal injury. Histone deacetylase 3 (HDAC3) is a class I histone deacetylase isoform that plays a central role in the macrophage inflammatory response. We recently reported that myeloid cells upregulate HDAC3 in a mouse model of retinal ischemia-reperfusion (IR) injury. However, whether this cellular event is an essential contributor to retinal IR injury is unknown. In this study, we explored the role of myeloid HDAC3 in ischemia-induced retinal neurovascular injury by subjecting myeloid-specific HDAC3 knockout (M-HDAC3 KO) and floxed control mice to retinal IR. The M-HDAC3 KO mice were protected from retinal IR injury as shown by the preservation of inner retinal neurons, vascular integrity, and retinal thickness. Electroretinography confirmed that this neurovascular protection translated to improved retinal function. The retinas of M-HDAC3 KO mice also showed less proliferation and infiltration of myeloid cells after injury. Interestingly, myeloid cells lacking HDAC3 more avidly engulfed apoptotic cells in vitro and after retinal IR injury in vivo compared to wild-type myeloid cells, suggesting that HDAC3 hinders the reparative phagocytosis of dead cells, a process known as efferocytosis. Further mechanistic studies indicated that although HDAC3 KO macrophages upregulate the reparative enzyme arginase 1 (A1) that enhances efferocytosis, the inhibitory effect of HDAC3 on efferocytosis is not solely dependent on A1. Finally, treatment of wild-type mice with the HDAC3 inhibitor RGFP966 ameliorated the retinal neurodegeneration and thinning caused by IR injury. Collectively, our data show that HDAC3 deletion enhances macrophage-mediated efferocytosis and protects against retinal IR injury, suggesting that inhibiting myeloid HDAC3 holds promise as a novel therapeutic strategy for preserving retinal integrity after ischemic insult.

Short peptides derived from pigment epithelium-derived factor attenuate retinal ischemia reperfusion injury through inhibition of apoptosis and inflammatory response in rats.

Pigment epithelium-derived factor (PEDF) is widely recognized as a neuroprotective factor expressed in the retina and has shown therapeutic potential in several retinal diseases. Our study aimed to identify the neuroprotective fragment in PEDF and investigate its protective activity in retinas under ischemia-reperfusion (IR) condition. We synthesized a series of shorter synthetic peptides, 6-mer (Ser93-Gln98) and its d-form variant (6 dS) derived from the 44-mer (Val78-Thr121; a PEDF neurotrophic fragment), to determine their cytoprotective activity in IR injury, which was induced in rat retinas by injection of saline into the anterior chamber to increase the intraocular pressure (IOP) followed by reperfusion. We found the cytoprotective effect of 6-mer on glutamate-treated Neuro-2a cells and tert-butyl hydroperoxide (tBHP)-treated 661W cells were 2.6-fold and 1.5-fold higher than the 44-mer, respectively. The cytoprotective effect was blocked by a chemical inhibitor atglistatin and blocking antibody targeting PEDF receptor (PEDF-R). IR induced several impairments in retina, including cell apoptosis, activation of microglia/macroglia, degeneration of retinal capillaries, reduction in electroretinography (ERG) amplitudes, and retinal atrophy. Such IR injuries were ameliorated by treatment with 6-mer and 6 dS eye drops. Also, the neuroprotective activity of 6-mer and 6 dS in ischemic retinas were dramatically reversed by atglistatin preconditioning. Taken together, our data demonstrate smallest neuroprotective fragment of PEDF has potential to treat retinal degeneration-related diseases.

N-Acetylserotonin Alleviates Retinal Autophagy via TrkB/AKT/Nrf2 Signaling Pathway in Retinal Ischemia-Reperfusion Injury Rats.

INTRODUCTION: The objective of this study was to investigate the impact of N-acetylserotonin (NAS) on the autophagy of retinal cells in rats with retinal ischemia-reperfusion injury (RIRI) and to explore the mechanisms by which NAS administration can alleviate RIRI through the tropomyosin-related kinase receptor B (TrkB)/protein kinase B (Akt)/nuclear factor erythroid-derived factor 2-related factor (Nrf2) signaling pathway. METHODS: Healthy adult male rats were randomly assigned to four groups: sham, RIRI, RIRI+NAS, and RIRI+NAS+ANA-12. The RIRI group was induced by elevating intraocular pressure, and changes in retinal structure and edema were assessed using H&E staining. The RIRI+NAS and RIRI+NAS+ANA-12 groups received intraperitoneal injections of NAS before and after modeling. The RIRI+NAS+ANA-12 group was also administered ANA-12, a TrkB antagonist. Immunohistochemical staining and Western blot analysis were used to evaluate phosphorylated TrkB (p-TrkB), phosphorylated Akt (p-Akt), Nrf2, sequestosome 1 (P62), and microtubule-associated protein 1 light chain 3 (LC3-II) levels in the retinas of each group. Electroretinogram was recorded to detect retinal function in each group of rats 24 h after modeling. RESULTS: The RIRI+NAS group had a thinner retina and more retinal ganglion cells (RGCs) than RIRI and RIRI+NAS+ANA-12 groups (p < 0.05). Immunohistochemical staining and Western blot results showed that p-TrkB, p-Akt, n-Nrf2, and P62 levels in the RIRI+NAS group were higher compared with those in RIRI and RIRI+NAS+ANA-12 groups (p < 0.05). Also, lower LC3-II levels were observed in the RIRI+NAS group compared with that in RIRI and RIRI+NAS+ANA-12 groups (p < 0.05). Electroretinogram recording results showed that 24 h after retinal ischemia-reperfusion, the magnitude of b-wave changes was attenuated in the RIRI+NAS group compared with the RIRI group (p < 0.05). CONCLUSION: The administration of NAS activates the TrkB/Akt/Nrf2 signaling pathway, reduces autophagy, alleviates retinal edema, promotes the survival of retinal ganglion cells (RGCs), and provides neuroprotection against retinal injury.

PDGFRB upregulation contributes to retinal damages in the rat model of retinal ischemia-reperfusion.

Retinal ischemic disease is a major type of retinal diseases causing vision loss. Identifying the molecular mechanisms mediating the retinal ischemia-reperfusion (RIR) is the key to targeted intervention. In this study, we performed RNA-seq analysis of the retinal tissues of a retinal ischemia-reperfusion model of Sprague-Dawley (SD) rats, followed by differential gene expression analysis, gene ontology (GO) enrichment analysis, and protein-protein interaction (PPI) analysis. After studying we found that: The major biological processes affected after RIR was the regulation of vascular development. PPI analysis unveiled a regulatory module in which Platelet Derived Growth Factor Receptor Beta (PDGFRB) was upregulated. In the RIR cell model of human retinal microvascular endothelial cells (HRCEC) induced by oxygen-glucose deprivation/reperfusion (OGD/R), silencing PDGFRB at least partially rescued the detrimental effect on cell proliferation and in vitro angiogenic ability. In the rat model of RIR, the administration of PDGFR inhibitor alleviated the damages in the retinal microvascular system. Besides, we further demonstrated the protective effect of procyanidin against RIR induced damages in both the cell and animal model by dampening the overexpression of PDGFRB. Together, our data indicate that the upregulation of PDGFRB contributes to RIR-induced damages in retinal microvascular system, which provides a targetable strategy for therapeutic intervention.

Geranylgeranylacetone-induced heat shock protein70 expression reduces retinal ischemia-reperfusion injury through PI3K/AKT/mTOR signaling.

Retinal ischemia-reperfusion (I/R) injury is a common pathophysiological stress state connected to various diseases, including acute glaucoma, retinal vascular obstruction, and diabetic retinopathy. Recent studies have suggested that geranylgeranylacetone (GGA) could increase heat shock protein70 (HSP70) level and reduce retinal ganglion cells (RGCs) apoptosis in a rat retinal I/R model. However, the underlying mechanism remains unclear. Moreover, the injury caused by retinal I/R includes not only apoptosis but also autophagy and gliosis, and the effects of GGA on autophagy and gliosis have not been reported. Our study established a retinal I/R model by anterior chamber perfusion pressuring to 110 mmHg for 60 min, followed by 4 h of reperfusion. The levels of HSP70, apoptosis-related proteins, GFAP, LC3-II, and PI3K/AKT/mTOR signaling proteins were determined by western blotting and qPCR after treatment with GGA, HSP70 inhibitor quercetin (Q), PI3K inhibitor LY294002, and mTOR inhibitor rapamycin. Apoptosis was evaluated by TUNEL staining, meanwhile, HSP70 and LC3 were detected by immunofluorescence. Our results demonstrated that GGA-induced HSP70 expression significantly reduced gliosis, autophagosome accumulation, and apoptosis in retinal I/R injury, indicating that GGA exerted protective effects on retinal I/R injury. Moreover, the protective effects of GGA mechanistically relied on the activation of PI3K/AKT/mTOR signaling. In conclusion, GGA-induced HSP70 overexpression has protective effects on retinal I/R injury by activating PI3K/AKT/mTOR signaling.

Various Forms of Programmed Cell Death Are Concurrently Activated in the Population of Retinal Ganglion Cells after Ischemia and Reperfusion.

Retinal ischemia-reperfusion (IR)-which ultimately results in retinal ganglion cell (RGC) death-is a common cause of visual impairment and blindness worldwide. IR results in various types of programmed cell death (PCD), which are of particular importance since they can be prevented by inhibiting the activity of their corresponding signaling cascades. To study the PCD pathways in ischemic RGCs, we used a mouse model of retinal IR and a variety of approaches including RNA-seq analysis, knockout animals, and animals treated with an iron chelator. In our RNA-seq analysis, we utilized RGCs isolated from retinas 24 h after IR. In ischemic RGCs, we found increased expression of many genes that regulate apoptosis, necroptosis, pyroptosis, oxytosis/ferroptosis, and parthanatos. Our data indicate that genetic ablation of death receptors protects RGCs from IR. We showed that the signaling cascades regulating ferrous iron (Fe2+) metabolism undergo significant changes in ischemic RGCs, leading to retinal damage after IR. This data suggests that the activation of death receptors and increased Fe2+ production in ischemic RGCs promote the simultaneous activation of apoptosis, necroptosis, pyroptosis, oxytosis/ferroptosis, and parthanatos pathways. Thus, a therapy is needed that concurrently regulates the activity of the multiple PCD pathways to reduce RGC death after IR.

Single-cell RNA sequencing reveals a landscape and targeted treatment of ferroptosis in retinal ischemia/reperfusion injury.

BACKGROUND: The aim of this study was to establish a complete retinal cell atlas of ischemia-reperfusion injury by single-cell RNA sequencing, and to explore the underlying mechanism of retinal ischemia-reperfusion injury in mice. METHODS: Single-cell RNA sequencing was used to evaluate changes in the mouse retinal ischemia reperfusion model. In vivo and in vitro experiments were performed to verify the protective effect of inhibiting ferroptosis in retinal ischemia-reperfusion injury. RESULTS: After ischemia-reperfusion injury, retinal cells were significantly reduced, accompanied by the activation of myeloid and a large amount of blood-derived immune cell infiltration. The IFNG, MAPK and NFKB signaling pathways in retinal neuronal cells, together with the TNF signaling pathway in myeloid give rise to a strong inflammatory response in the I/R state. Besides, the expression of genes implicating iron metabolism, oxidative stress and multiple programed cell death pathways have changed in cell subtypes described above. Especially the ferroptosis-related genes and blocking this process could apparently alleviate the inflammatory immune responses and enhance retinal ganglion cells survival. CONCLUSIONS: We established a comprehensive landscape of mouse retinal ischemia-reperfusion injury at the single-cell level, revealing the important role of ferroptosis during this injury, and targeted inhibition of ferroptosis can effectively protect retinal structure and function.

7,8-Dihydroxyflavone alleviates apoptosis and inflammation induced by retinal ischemia-reperfusion injury via activating TrkB/Akt/NF-kB signaling pathway.

Retinal ischemia-reperfusion injury (RIRI) is of common occurrence in retinal and optic nerve diseases. The BDNF/TrkB signaling pathway has been examined to be neuroprotective in RIRI. In this study, we investigated the role of a potent selective TrkB agonist 7,8-dihydroxyfavone (DHF) in rat retinas with RIRI. Our results showed that RIRI inhibited the conversion of BDNF precursor (proBDNF) to mature BDNF (mBDNF) and increased the level of neuronal cell apoptosis. Compared with RIRI, DHF+RIRI reduced proBDNF level and at the same time increased mBDNF level. Moreover, DHF administration effectively activated TrkB signaling and and downstream Akt and Erk signaling pathways which increased nerve cell survival. The combined effects of mBDNF/proBDNF increase and TrkB signaling activation lead to reduction of apoptosis level and protection of retinas with RIRI. Moreover, it was also found that astrocytes labeled by GFAP were activated in RIRI and NF-kB mediated the increased expressions of inflammatory factors and these effects were partially reversed by DHF administration. Besides, we also used RNA sequencing to analyze the differently expressed genes (DEGs) and their enriched (Kyoto Encyclopedia of Genes and Genomes) KEGG pathways between Sham, RIRI, and DHF+RIRI. It was found that 1543 DEGs were differently expressed in RIRI and 619 DEGs were reversed in DHF+RIRI. The reversed DEGs were typically enriched in PI3K-Akt signaling pathway, Jak-STAT signaling pathway, NF-kB signaling pathway, and Apoptosis. To sum up, the DHF administration alleviated apoptosis and inflammation induced by RIRI via activating TrkB signaling pathway and may serve as a promising drug candidate for RIRI related ophthalmopathy.

Role of Curcumin in Retinal Diseases-A review.

PURPOSE: To review the role of curcumin in retinal diseases, COVID era, modification of the molecule to improve bioavailability and its future scope. METHODS: PubMed and MEDLINE searches were pertaining to curcumin, properties of curcumin, curcumin in retinal diseases, curcumin in diabetic retinopathy, curcumin in age-related macular degeneration, curcumin in retinal and choroidal diseases, curcumin in retinitis pigmentosa, curcumin in retinal ischemia reperfusion injury, curcumin in proliferative vitreoretinopathy and curcumin in current COVID era. RESULTS: In experimental models, curcumin showed its pleiotropic effects in retinal diseases like diabetic retinopathy by increasing anti-oxidant enzymes, upregulating HO-1, nrf2 and reducing or inhibiting inflammatory mediators, growth factors and by inhibiting proliferation and migration of retinal endothelial cells in a dose-dependent manner in HRPC, HREC and ARPE-19 cells. In age-related macular degeneration, curcumin acts by reducing ROS and inhibiting apoptosis inducing proteins and cellular inflammatory genes and upregulating HO-1, thioredoxin and NQO1. In retinitis pigmentosa, curcumin has been shown to delay structural defects of P23H gene in P23H-rhodopsin transgenic rats. In proliferative vitreoretinopathy, curcumin inhibited the action of EGF in a dose- and time-dependent manner. In retinal ischemia reperfusion injury, curcumin downregulates IL-17, IL-23, NFKB, STAT-3, MCP-1 and JNK. In retinoblastoma, curcumin inhibits proliferation, migration and apoptosis of RBY79 and SO-RB50. Curcumin has already proven its efficacy in inhibiting viral replication, coagulation and cytokine storm in COVID era. CONCLUSION: Curcumin is an easily available spice used traditionally in Indian cooking. The benefits of curcumin are manifold, and large randomized controlled trials are required to study its effects not only in treating retinal diseases in humans but in their prevention too.

Mitoquinone intravitreal injection ameliorates retinal ischemia-reperfusion injury in rats involving SIRT1/Notch1/NADPH axis.

Retinal ischemia-reperfusion injury (RIRI) is an important pathological process of many ocular diseases. Mitoquinone (MitoQ), a mitochondrial targeted antioxidant, is a potential compound for therapeutic development of RIRI. This study observed the effect of MitoQ on RIRI, and further explored its possible molecular mechanism. Temporary increase in intraocular pressure was used to establish rat model of RIRI to observe the effect of MitoQ treatment on retinal function, pathological injury, oxidative stress, inflammation and apoptosis. Immunohistochemistry and Western blot were used to detect expressions of cleaved caspase 3, B cell leukemia/lymphoma 2 associated X (Bax), nicotinamide adenine dinucleotide phosphate oxidase (NOX1), NOX4, cleaved-Notch 1, hairy and enhancer of split 1 (Hes1), and sirtuin 1 (SIRT 1) in retina were detected by immunohistochemistry and Western blot. MitoQ treatment significantly improved retinal function and pathological injury, inhibited the over-production of reactive oxygen species, increased the expression of superoxide dismutase 1 (SOD 1), suppressed the releases of inflammatory cytokines, and inhibited retinal cells apoptosis. MitoQ also down-regulated the expressions of cleaved caspase 3, Bax, NOX 1, NOX 4, cleaved-Notch 1, and Hes 1, increased the expression of SIRT 1 protein and its activity. These effects were significantly reversed by SIRT1 inhibitor EX527. Our data suggests that MitoQ, as a potentially effective drug for improving RIRI, may act through the SIRT1/Notch1/NADPH signal axis.

Nicotinamide Mononucleotide Prevents Retinal Dysfunction in a Mouse Model of Retinal Ischemia/Reperfusion Injury.

Retinal ischemia/reperfusion (I/R) injury can cause severe vision impairment. Retinal I/R injury is associated with pathological increases in reactive oxygen species and inflammation, resulting in retinal neuronal cell death. To date, effective therapies have not been developed. Nicotinamide mononucleotide (NMN), a key nicotinamide adenine dinucleotide (NAD+) intermediate, has been shown to exert neuroprotection for retinal diseases. However, it remains unclear whether NMN can prevent retinal I/R injury. Thus, we aimed to determine whether NMN therapy is useful for retinal I/R injury-induced retinal degeneration. One day after NMN intraperitoneal (IP) injection, adult mice were subjected to retinal I/R injury. Then, the mice were injected with NMN once every day for three days. Electroretinography and immunohistochemistry were used to measure retinal functional alterations and retinal inflammation, respectively. The protective effect of NMN administration was further examined using a retinal cell line, 661W, under CoCl2-induced oxidative stress conditions. NMN IP injection significantly suppressed retinal functional damage, as well as inflammation. NMN treatment showed protective effects against oxidative stress-induced cell death. The antioxidant pathway (Nrf2 and Hmox-1) was activated by NMN treatment. In conclusion, NMN could be a promising preventive neuroprotective drug for ischemic retinopathy.

Krüppel-like factor 7 protects retinal ganglion cells and promotes functional preservation via activating the Akt pathway after retinal ischemia-reperfusion injury.

OBJECTIVE: The purpose of this study is to investigate the effects of Krüppel-like factor 7 (KLF7) on retinal ganglion cells (RGCs) and retinal function after retinal ischemia-reperfusion (RIR) injury in mice. METHODS: Male C57BL/6J mice were intravitreally injected with recombinant adeno-associated vectors (rAAV-KLF7-EGFP or rAAV-EGFP), and subsequently used to induce RIR injury. Retinal cryosections were used to access the efficacy of virus transfection, 1, 2, 3, and 4 weeks after rAAV-KLF7-EGFP transfer. RGCs survival rate was observed and quantified by immunofluorescent staining, 7 days after RIR injury. Meanwhile, electroretinogram (ERG) and optomotor response were used to evaluate the electrophysiological functions and visual acuity. Apoptosis was evaluated by TUNEL staining 1 day after RIR injury. Expression of KLF7, Akt, phospho-Akt, Bcl-2, and Bax were further detected by western blot to excavate the underlying mechanism. RESULTS: The transfection efficiency of rAAV-KLF7-EGFP was increased in a time-dependent manner, and the number of EGFP-positive cells was increased significantly 3 weeks after rAAV-KLF7-EGFP transfer. RGCs survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR, and visual acuity of mice were decreased after RIR injury. 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. RGCs survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR, and visual acuity of mice were increased after rAAV-KLF7-EGFP transfer. The protein level of KLF7 was up-regulated after rAAV-KLF7-EGFP transfer. Up-regulation of KLF7 significantly inhibited cells apoptosis, increased phospho-Akt and Bcl-2 expression, and decreased Bax expression. There were no significant changes in Akt expression. CONCLUSION: Overexpression of KLF7 can not only prevent the loss of RGCs, but also preserve the electrophysiological function. In addition, overexpression of KLF7 can ameliorate the retinal dysfunction after RIR injury, and ultimately improve the visual acuity of mice. The activation of Akt pathway and the suppression of the mitochondrial apoptotic pathway contribute to the neuroprotection of KLF7.

Involvement of the NLRC4 inflammasome in promoting retinal ganglion cell death in an acute glaucoma mouse model.

PURPOSE: To explore the role of nucleotide-binding oligomerization domain-like receptors (NLRs) family caspase-activation and the recruitment domain containing 4 (NLRC4) inflammasome in retinal ganglion cell (RGC) injury induced by an acute glaucoma mouse model. METHOD: A mouse model of acute ocular hypertension, which can lead to retinal ischemia-reperfusion (I/R) injury, was established. The expression level of NLRC4 was detected by polymerase chain reaction and western blotting. Localized expression of NLRC4 was detected by examining immunofluorescence in eyeball sections. Intravitreal adeno-associated virus 2(AAV2) administration was used to knockdown retinal Nlrc4. Fluoro-Gold labeled RGCs and TdT-mediated dUTP nick end labeling were used to evaluate the survival and apoptosis of RGCs. Tlr4-/- mice were utilized to explore whether NLRC4 inflammasome is influenced by Toll-like receptor4 (TLR4). RESULTS: NLRC4, expressed in RGCs and microglial cells, was actively involved in mouse retinal I/R injury. Knockdown of Nlrc4 using an AAV2 vector caused an obvious reduction in the generation of IL-1ß led by the rapidly elevated intraocular pressure, and thereby improved the RGC survival. In addition, activation of the NLRC4 inflammasome could influence the phosphorylation of p38 and Jun N-terminal kinase, which was largely dependent on TLR4 signaling. CONCLUSION: Our study demonstrated the role of NLRC4 inflammasome in promoting RGC damage in mouse retinal I/R injury. Inhibition of NLRC4 might be leveraged as a potential therapeutic target in glaucomatous retinopathy.

N-acetylserotonin alleviated the expression of interleukin-1ß in retinal ischemia-reperfusion rats via the TLR4/NF-κB/NLRP3 pathway.

This study aimed to explore the effects of N-acetylserotonin (NAS) on the expression of interleukin-1ß (IL-1ß) in the retina of retinal ischemia-reperfusion injury (RIRI) rats via the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB)/nod-like receptor pyrin domain containing 3 (NLRP3) signaling pathway. In this study, adult male Sprague Dawley rats were randomly divided into the sham, RIRI, RIRI + NAS and RIRI + TAK-242 + NAS groups. The rats in the RIRI + NAS and RIRI + TAK-242 + NAS groups were intraperitoneally injected with NAS 30 min before and after modeling. TAK-242, a selective TLR4 inhibitor, was administered by intraperitoneal injection in RIRI + TAK-242 + NAS group. The RIRI rat model was established by elevating the intraocular pressure to 110 mmHg for 60 min. The retinal structure and edema were assessed by H&E staining. The expression levels of TLR4, phosphorylated NF-κB (p-NF-κB), NLRP3, cleaved Caspase-1, and IL-1ß in the retina of each group were detected using immunohistochemistry and Western blot. The correlations of the differences of TLR4+ and cleaved Caspase-1+ with IL-1ß+ cells (between the NAS and the RIRI groups) were analyzed, using linear regression in the RIRI + NAS group. Results showed that thinner retina, more RGCs, and less TLR4+, p-NF-κB+, NLRP3+, cleaved Caspase-1+, and IL-1ß+ cells in the retina were observed in the RIRI + NAS and RIRI + TAK-242 + NAS groups compared with the RIRI group 12 h after RIRI (all P < 0.01). Western blot analysis results showed that the expression of IL-1ß in the RIRI + NAS group began to increase 6 h after RIRI, and it reached a high level 12 h after RIRI, and then decreased. And it was lower at each time point in the RIRI + NAS group than in the RIRI group, and there existed significant difference (all P < 0.01). Besides, the expression levels of TLR4, p-NF-κB, NLRP3, and cleaved Caspase-1 proteins in the RIRI + NAS and RIRI + TAK-242 + NAS groups decreased 12 h after RIRI compared with those in the RIRI group (all P < 0.01). The difference in IL-1ß+ cells was significantly correlated with those of TLR4+ and cleaved Caspase-1+ cells in the RIRI + NAS group (r2 = 0.9054 or 0.7431, P < 0.01). In conclusion, NAS could attenuate the expression of IL-1ß by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway, reduce the retina edema, and promote the survival of RGCs, thereby alleviating the retinal injury and exert its neuroprotective effect.

Inactivation of Endothelial ADAM17 Reduces Retinal Ischemia-Reperfusion Induced Neuronal and Vascular Damage.

Retinal ischemia contributes to visual impairment in ischemic retinopathies. A disintegrin and metalloproteinase ADAM17 is implicated in multiple vascular pathologies through its ability to regulate inflammatory signaling via ectodomain shedding. We investigated the role of endothelial ADAM17 in neuronal and vascular degeneration associated with retinal ischemia reperfusion (IR) injury using mice with conditional inactivation of ADAM17 in vascular endothelium. ADAM17Cre-flox and control ADAM17flox mice were subjected to 40 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Albumin extravasation and retinal leukostasis were evaluated 48 h after reperfusion. Retinal morphometric analysis was conducted 7 days after reperfusion. Degenerate capillaries were assessed by elastase digest and visual function was evaluated by optokinetic test 14 and 7 days following ischemia, respectively. Lack of ADAM17 decreased vascular leakage and reduced retinal thinning and ganglion cell loss in ADAM17Cre-flox mice. Further, ADAM17Cre-flox mice exhibited a remarkable reduction in capillary degeneration following IR. Decrease in neurovascular degeneration in ADAM17Cre-flox mice correlated with decreased activation of caspase-3 and was associated with reduction in oxidative stress and retinal leukostasis. In addition, knockdown of ADAM17 resulted in decreased cleavage of p75NTR, the process known to be associated with retinal cell apoptosis. A decline in visual acuity evidenced by decrease in spatial frequency threshold observed in ADAM17flox mice was partially restored in ADAM17-endothelial deficient mice. The obtained results provide evidence that endothelial ADAM17 is an important contributor to IR-induced neurovascular damage in the retina and suggest that interventions directed at regulating ADAM17 activity can be beneficial for alleviating the consequences of retinal ischemia.

Epigallocatechin 3-Gallate Has a Neuroprotective Effect in Retinas of Rabbits with Ischemia/Reperfusion through the Activation of Nrf2/HO-1.

Retinal ischemia-reperfusion (rI/R) generates an oxidative condition causing the death of neuronal cells. Epigallocatechin 3-gallate (EGCG) has antioxidant and anti-inflammatory properties. Nonetheless, its correlation with the pathway of nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) for the protection of the retina is unknown. We aimed to evaluate the neuroprotective efficacy of single-doses of EGCG in rI/R and its association with Nrf2/Ho-1 expression. In albino rabbits, rI/R was induced and single-doses of EGCG in saline (0-30 mg/kg) were intravenously administered to select an optimal EGCG concentration that protects from retina damage. To reach this goal, retinal structural changes, gliosis by glial fibrillary acidic protein (GFAP) immunostaining, and lipid peroxidation level by TBARS (thiobarbituric acid reactive substance) assay were determined. EGCG in a dose of 15 mg/kg (E15) presented the lowest levels of histological damage, gliosis, and oxidative stress in the studied groups. To determine the neuroprotective efficacy of E15 in a timeline (6, 24, and 48 h after rI/R), and its association with the Nrf2/HO-1 pathway, the following assays were done by immunofluorescence: apoptosis (TUNEL assay), necrosis (high-mobility group box-1; HMGB1), Nrf2, and HO-1. In addition, the Ho-1 mRNA (qPCR) and lipid peroxidation levels were evaluated. E15 showed a protective effect during the first 6 h, compared to 24 and 48 h after rI/R, as revealed by a decrease in the levels of all damage markers. Nuclear translocation Nrf2 and HO-1 staining were increased, including Ho-1 mRNA levels. In conclusion, a single dose of E15 decreases the death of neuronal cells induced by oxidative stress during the first 6 h after rI/R. This protective effect is associated with the nuclear translocation of Nrf2 and with an elevation of Ho-1 expression.

Protective effects of autophagy inhibitor 3-methyladenine on ischemia-reperfusion-induced retinal injury.

OBJECTIVE: To investigate the protective effects of autophagy inhibitor 3-methyladenine (3-MA) in a rat model of ischemic-reperfusion injury (IRI). METHODS: Forty Sprague-Dawley male rats (weight 220-250 g) were randomly divided into four groups: a control group (NC, n = 10), a Sham surgery group (n = 10), an IRI group (n = 10), and a 3-MA-treated IRI group [10 µL 3-MA (10 mmol/L) was injected in vitreous after the injury, n = 10]. The retinal IRI was induced by elevating the intraocular pressure to 110 mmHg for 60 min. Hematoxylin and eosin (HE) staining was used to calculate the number of retinal ganglion cells (RGCs). The level of microtubule-associated protein 1A/1B light chain 3 (LC3), Beclin-1, and Caspase-3 in the retina was detected using the immunofluorescence staining method. The LC3, Beclin-1, B-cell lymphoma/leukemia-2 (Bcl-2), and Caspase-3 protein levels were examined by Western blotting. RESULTS: The number of RGCs in IRI group was significantly lower than that in NC group (P < 0.05), demonstrated by HE staining. Western blotting results indicated that the protein expression of LC3 and Beclin-1 in the IRI group was significantly elevated compared with those in the NC group (P < 0.05). However, with 3-MA treatment, the number of RCGs in 3-MA-treated IRI group was elevated and protein levels of LC3, Beclin-1 were down-regulated, compared with those in the IRI group (P < 0.05). Further immunohistochemistry staining and Western blot showed that 3-MA-treated IRI group presented down-regulated Caspase-3 and up-regulated Bcl-2 protein expression with comparison of IRI group (P < 0.05). CONCLUSIONS: Retina IRI-caused RGCs loss involved activated autophagy pathway and apoptosis, which could be prevented by autophagy inhibitor 3-MA. Autophagy inhibitor 3-MA may act as a potent therapeutic tool in attenuating retina IRI.

Myeloid differentiation protein 2-dependent mechanisms in retinal ischemia-reperfusion injury.

Retinal ischemia-reperfusion (I/R) injury is a common pathological process in many eye disorders. Oxidative stress and inflammation play a role in retinal I/R injury. Recent studies show that toll-like receptor 4 (TLR4) is involved in initiating sterile inflammatory response in retinal I/R. However, the molecular mechanism by which TLR4 is activated is not known. In this study, we show that retinal I/R injury involves a co-receptor of TLR4, myeloid differentiation 2 (MD2). Inhibition of MD2 prevented cell death and preserved retinal function following retinal I/R injury. We confirmed these findings using MD2 knockout mice. Furthermore, we utilized human retinal pigment epithelial cells (ARPE-19 cells) to show that oxidative stress-induced cell death as well as inflammatory response are mediated through MD2. Inhibition of MD2 through a chemical inhibitor or knockdown prevented oxidative stress-induced cell death and expression of inflammatory cytokines. Oxidative stress was found to activate TLR4 in a MD2-dependent manner via increasing the expression of high mobility group box 1. In summary, our study shows that oxidative stress in retinal I/R injury can activate TLR4 signaling via MD2, resulting in induction of inflammatory genes and retinal damage. MD2 may represent an attractive therapeutic target for retinal I/R injury.