IL-17A mediates inflammation-related retinal pigment epithelial cells injury via ERK signaling pathway

AIM:To investigate whether interleukin-17A(IL-17A)gets involved in the mechanisms of inflammation-related retinal pigment epithelium(RPE)cells injury and its significance in age-related macular degeneration(AMD).MRTHODS:A sodium iodate(NaIO3)mouse model as well as IL-17A-/-mice were established.The effects of inflammatory cytokines in RPE cells and retinal microglia before and after NaIO3 modeling in vivo and in vitro,were investigated using immunofluorescence,immunoprotein blotting,and quantitative real-time fluorescence polymerase chain reaction(qRT-PCR),respectively.Interventions using recombinant IL-17A protein(rIL-17A)or IL-17A neutralizing antibody(IL-17A NAb)were used to observe the subsequent differences in fundus,fundus photography and optical coherence tomography(OCT),cell viability,and expression of oxidative stress-related markers before and after modeling,and to screen for key signaling pathways.RESULTS:In the scenario of NaIO3 stimulation,RPE cells obviously tended to degenerate.Simultaneously proliferation and activation of retinal microglia was confirmed in NaIO3-stimulated mice,whereas such effects induced by NaIO3 were significantly ameliorated with IL-17A NAb intervention or in IL-17A-/-mice.In addition,IL-17A promoted the proliferation and activation of microglia as well as oxidative damage and the secretion of inflammatory cytokines alongside NaIO3-induced damage in RPE cells in vivo and ex vivo.Meanwhile,the extracellular signalregulated kinase(ERK)signaling pathway was shown to be participated in the regulation of NaIO3-induced RPE cells injury mediated by IL-17A in vivo and ex vivo,as IL-17A induced inflammatory cytokines release in the NaIO3 model was alleviated after blocking the ERK pathway.CONCLUSION:IL-17A probably promotes the NaIO3-induced RPE cells injury through exacerbating inflammation in terms of retinal microglia activation and inflammatory cytokines release via ERK signaling pathway.Inhibition of IL-17A may be a new potential target for dry AMD treatment.

Establishing an experimental rat model of photo-dynamically-induced retinal vein occlusion using erythrosin B

AIM:To develop a reliable,reproducible rat model of retinal vein occlusion(RVO)with a novel photosensitizer(erythrosin B)and study the cellular responses in the retina.METHODS:Central and branch RVOs were created in adult male rats via photochemically-induced ischemia.Retinal changes were monitored via color fundus photography and fluorescein angiography at 1 and 3h,and 1,4,7,14,and 21d after irradiation.Tissue slices were evaluated histopathologically.Retinal ganglion cell survival at different times after RVO induction was quantified by nuclear density count.Retinal thickness was also observed.RESULTS:For all rats in both the central and branch RVO groups,blood flow ceased immediately after laser irradiation and retinal edema was evident at one hour.The retinal detachment rate was 100%at 3h and developed into bullous retinal detachment within 24h.Retinal hemorrhages were not observed until 24h.Clearance of the occluded veins at 7d was observed by fluorescein angiography.Disease manifestation in the central RVO eyes was more severe than in the branch RVO group.A remarkable reduction in the ganglion cell count and retinal thickness was observed in the central RVO group by 21d,whereas moderate changes occurred in the branch RVO group.CONCLUSION:Rat RVO created by photochemicallyinduced ischemia using erythrosin B is a reproducible and reliable animal model for mimicking the key features of human RVO.However,considering the 100%rate ofretinal detachment,this animal model is more suitable for studying RVO with chronic retinal detachment.

Activation of autophagy in photoreceptor necroptosis after experimental retinal detachment

AIM:To investigate whether photoreceptor necroptosis induced by z-VAD-FMK(pan caspase inhibitor) was involved the activation of autophagy and whether Necrostatin-1, a specific necroptosis inhibitor, could inhibit this induction of autophagy after experimental retinal detachment.METHODS:Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate and subretinal injections of z-VAD-FMK, vehicle or z-VAD-FMK plus Necrostatin-1.Three days after retinal detachment, morphologic changes were observed by transmission electron microscopy. In other animals, retinas were subjected to immunoprecipitation and Western Blotting, then probed with anti-RIP1, phosphoserine, LC-3II or caspase 8antibody.RESULTS:It was proved by immunoprecipitation and western blotting, that photoreceptor necroptosis was mediated by caspase-8 inhibition and receptor interacting protein kinase(RIP1) phosphorylation activation. Transmission electron microscope and western blotting results indicated that photoreceptornecroptosis was involved the LC-3II and autophagosomes induction. We also discovered Necrostatin-1 could inhibit RIP1 phosphorylation and LC-3II induction.CONCLUSION:These data firstly indicate photoreceptor necroptosis is associated with the activation of autophagy. Necrostatin-1 protects photoreceptors from necroptosis and autophagy by down-regulation of RIP1 phosphorylation and LC-3II.

Macular changes of neuromyelitis optica through spectral-domain optical coherence tomography

AIM： To evaluate the thickness of the retinal layers in the macula using spectral-domain optical coherence tomography（SD-OCT） in patients with neuromyelitis optica（NMO）. METHODS： Spectralis SD-OCT, utilizing automated macular layer segmentation, was performed in 26 NMO patients and 26 healthy controls. Visual function including visual field tests and pattern visual evoked potential were recorded in study subjects.RESULTS： Forty-one eyes from 26 NMO patients and52 eyes from 26 age- and sex-matched healthy controls were included. Besides total macular volume, peri-paipillary retinal nerve fiber layer（RNFL） thickness, the thickness of macular RNFL, ganglion cell layer（GCL） and inner plexiform layer（IPL） were also significantly reduced in NMO patients compared to those inhealthy controls（P 〈0.000）. No differences were found in the thickness of macular inner nuclear layer（INL）, outer plexiform layer（OPL）, and outer nuclear layer（ONL） between the two groups. Reversely, the outer retinal layer（ORL） was shown to be thicker in NMO than controls（P 〈0.05）.Compared with the peri-papillary RNFL thickness, the GCL thickness was demonstrated to correlate with visual function better.CONCLUSION： The study provides in vivo evidence of retinal neural loss in NMO patients and demonstrates abetter structure-function correlation between retinal ganglion cell and visual function than peri-papillary RNFL does. In addition, no evidence of primary neural damage is found. Besides, the photoreceptor cells and retinal pigments epithelial（RPE） cells presumably proliferated in compensation in NMO after retinal neural loss.

Improvement of human embryonic stem cell-derived retinal pigment epithelium cell adhesion, maturation, and function through coating with truncated recombinant human vitronectin

AIM:To explore an xeno-free and defined coating substrate suitable for the culture of H9 human embryonic stem cell-derived retinal pigment epithelial(hES-RPE)cells in vitro,and compare the behaviors and functions of h ESRPE cells on two culture substrates,laminin521(LN-521)and truncated recombinant human vitronectin(VTN-N).METHODS:hES-RPE cells were used in the experiment.The abilities of LN-521 and VTN-N at different concentrations to adhere to hES-RPE cells were compared with a high-content imaging system.Quantitative real-time polymerase chain reaction was used to evaluate RPE-specific gene expression levels midway(day 10)and at the end(day 20)of the time course.Cell polarity was observed by immunofluorescent staining for apical and basal markers of the RPE.The phagocytic ability of hES-RPE cells was identified by flow cytometry and immunofluorescence.RESULTS:The cell adhesion assay showed that the ability of LN-521 to adhere to hES-RPE cells was dosedependent.With increasing coating concentration,an increasing number of cells attached to the surface of LN-521-coated wells.In contrast,VTN-N presented a strong adhesive ability even at a low concentration.The optimal concentration of LN-521 and VTN-N required to coat and adhesion to hES-RPE cells were 2 and 0.25μg/cm^(2),respectively.Furthermore,both LN-521 and VTN-N could facilitate adoption of the desired cobblestone cellular morphology with tight junction and showed polarity by the hES-RPE cells.However,hES-RPE cells cultivated in VTN-N had a greater phagocytic ability,and it took less time for these hES-RPE cells to mature.CONCLUSION:VTN-N is a more suitable coating substrate for cultivating hES-RPE cells.

Deletion of prominin-1 in mice results in disrupted photoreceptor outer segment protein homeostasis

AIM:To illustrate the underlying mechanism how prominin-1(also known as Prom1)mutation contribute to progressive photoreceptor degeneration.METHODS:A CRISPR-mediated Prom1 knockout(Prom1-KO)mice model in the C57BL/6 was generated and the photoreceptor degeneration phenotypes by means of structural and functional tests were demonstrated.Immunohistochemistry and immunoblot analysis were performed to reveal the localization and quantity of related outer segment(OS)proteins.RESULTS:The Prom1-KO mice developed the photoreceptor degeneration phenotype including the decreased outer nuclear layer(ONL)thickness and compromised electroretinogram amplitude.Immunohistochemistry analysis revealed impaired trafficking of photoreceptor OS proteins.Immunoblot data demonstrated decreased photoreceptor OS proteins.CONCLUSION:Prom1 deprivation causes progressive photoreceptor degeneration.Prom1 is essential for maintaining normal trafficking and normal quantity of photoreceptor OS proteins.The new light is shed on the pathogenic mechanism underlying photoreceptor degeneration caused by Prom1 mutation.

Beta-alanine promotes angiogenesis in laser-induced choroidal neovascularization mice models

AIM:To investigate the effect ofβ-alanine(BA)on laserinduced choroidal neovascularization(CNV)mice models.METHODS:Laser-induced CNV mice models were established,and BA was administrated for one week and two weeks in advance,separately.Furthermore,retinal pigment epithelium(RPE)-choroid flat mounts were separated,and immunohistochemical staining was performed.The laser-induced CNV lesion areas were measured and compared.In addition,liver and kidney morphologies were observed to identify potential hepatorenal toxicity.RESULTS:Enlarged CNV lesion areas were observed in the BA treated group.No significant differences were observed in the liver and kidney sections between groups.CONCLUSION:BA treatment increase CNV lesion areas,suggesting the detrimental effects of BA as a nutritional supplement in age-related macular degeneration(AMD)population.

GSK3β inhibits epithelial-mesenchymal transition via the Wnt/β-catenin and PI3K/Akt pathways

AIM： To investigate the regulatory mechanism of glycogen synthase kinase 3β（GSK3β） in epithelialmesenchymal transition（EMT） process after proliferative vitreoretinopathy（PVR） induction. METHODS： Experimental PVR was induced by intravitreal injection of retinal pigment epithelium（RPE） cells in the eyes of rabbits. A PI3 K/Akt inhibitor（wortmannin） and a GSK3β inhibitor（Li Cl） were also injected at different time during PVR progress. Electroretinogram（ERG）, ocular fundus photographs, and B-scan ultrasonography were used to observe the PVR progress. Western blot test on the extracted retina were performed at 1, 2, 4 wk. The expression of the mesenchymal marker vimentin was determined by immunohistochemistry. Toxicity of wortmannin and Li Cl were evaluated by ERG and Td Tmediated d UTP nick-end labeling（TUNEL） assay. The vitreous was also collected for metabolomic analysis. RESULTS： Experimental PVR could significantly lead to EMT, along with the suppressed expression of GSK3β and the activation of Wnt/β-catenin and PI3 K/Akt pathways. It was verified that upregulating the expression of GSK3β could effectively inhibit EMT process by suppressing Wnt/β-catenin and PI3 K/Akt pathways. CONCLUSION： GSK3β effectively inhibits EMT via the Wnt/β-catenin and PI3 K/Akt pathways. GSK3β may be regarded as a promising target of experimental PVR inhibition.

Association between exposure to a mixture of organochlorine pesticides and hyperuricemia in U.S.adults:A comparison of four statistical models

The association between the exposure of organochlorine pesticides(OCPs)and serum uric acid(UA)levels remained uncertain.In this study,to investigate the combined effects of OCP mixtures on hyperuricemia,we analyzed serum OCPs and UA levels in adults from the National Health and Nutrition Examination Survey(2005–2016).Four statistical models including weighted logistic regression,weighted quantile sum(WQS),quantile g-computation(QGC),and bayesian kernel machine regression(BKMR)were used to assess the relationship between mixed chemical exposures and hyperuricemia.Subgroup analyses were conducted to explore potential modifiers.Among 6,529 participants,the prevalence of hyperuricemia was 21.15%.Logistic regression revealed a significant association between both hexachlorobenzene(HCB)and trans-nonachlor and hyperuricemia in the fifth quintile(OR:1.54,95%CI:1.08–2.19;OR:1.58,95%CI:1.05–2.39,respectively),utilizing the first quintile as a reference.WQS and QGC analyses showed significant overall effects of OCPs on hyperuricemia,with an OR of 1.25(95%CI:1.09–1.44)and 1.20(95%CI:1.06–1.37),respectively.BKMR indicated a positive trend between mixed OCPs and hyperuricemia,with HCB having the largest weight in all three mixture analyses.Subgroup analyses revealed that females,individuals aged 50 years and above,and those with a low income were more vulnerable to mixed OCP exposure.These results highlight the urgent need to protect vulnerable populations from OCPs and to properly evaluate the health effects of multiple exposures on hyperuricemia using mutual validation approaches.

A two-year longitudinal observational study of the peripapillary microvasculature in pediatric type 1 diabetes mellitus patients without visual impairment or diabetic retinopathy

Dear Editor,Approximately 5 million children worldwide have type 1 diabetes mellitus(T1DM),and this number increases by 80000 people per year.1 Diabetic children have a younger age of onset and a longer survival period than adult DM patients;almost all of them develop diabetic reti-nopathy(DR)by the time their DM duration reaches 20 years.DR is the most common microvascular complication in DM patients and the lead-ing cause of blindness and vision loss in working-age people.

Correlation of Vascular Endothelial Growth Factor Production with Photochemical Reaction-induced Retinal Edema

Background： Retinal edema is the major complication of retinal vein occlusion and diabetic retinopathy; it can damage visual function by influencing macular region. This study was to establish a rat retinal edema model and explore the related VEGF expression and observe the responses to anti-VEGF drugs in this model. Methods： A rat retinal edema model was established by inducing photochemical reaction using a 532 nm laser after the intravenous injection of Erythrosin B. Immediately after the laser treatment, models were given intravitreal injections of Ranibizumab or Conbercept to inhibit VEGF expression, and the changes of retinal thickness were measured. Retinal edema was observed using fundus photography （FP）, optical coherence tomography （OCT）, and fluoresce in fundus angiography （FFA） at 0, 1, 2, 4, 7 and 14 days after intervention. The retinal VEGF expression was measured using enzyme-linked immunosorbent assay （ELISA） and western blotting at each time point. The rat retinal edema model was also used to verify the function of anti-VEGF polypeptide ZY1. Results： Both retinal edema and vascular leakage were clearly observed at 1, 2 and 4 days after photochemical induction and the retinal thickness increased notably over the same period. The retinal VEGF expression peaked at day 1 and retina became thickening simultaneously. After the interventions, the VEGF expression of the Ranibizumab and Conbercept groups decreased at each time point compared to the edema group （26.90 ± 3.57 vs. 40.29 ± 6.68, F = 31.269 on day 1 and 22.36 ± 1.12 vs. 29.92 ± 0.93 F = 163.789 on day 2, both P 〈 0.01）; the mean RT （278 ± 4 vs. 288 ± 3, F = 134.190 on day 1 and 274 ± 7 vs. 284 ± 6, F = 64.367 on day 2, both P 〈 0.05） and vascular leakage in these groups also decreased. The same results were observed in the ZY1 group, particularly at day 2 （P 〈 0.05）. Conclusions： This retinal edema model induced by a photochemical reaction is reliable and repeatable. Induced edema increases expression of VEGF. This model can be used to test new drugs.

Predictors of anti-vascular endothelial growth factor treatment responses in macular edema following central vein occlusion

Retinal vein occlusion （RVO） ranged the second common cause of retinal vascular disease, consisting of central retinal vein occlusion （CRVO） and branch retinal vein occlusion （BRVO） Although the prevalence of CRVO was one-third of BRVO, it associated with worse prognosis both before and after the anti-vascular endothelial growth factor （VEGF） regimes came into clinic use. A recent pooled age- and sex-standardized analysis showed that the prevalence was 0.8 per 1 000 （CI： 0.61-0.99） for CRVO around the world. Similarly, in our country, the 10-year incidence of CRVO in Beijing （2001-2011） was 0.3±0.1 per 100 persons.

Age-related macular degeneration： vision challenge of old age

Age-related macular degeneration （AMD） is the leading cause of legal blindness in the developed world.

Conducting gene therapy clinical trials based on natural history studies for inherited retinal diseases in Chinese population

A breakthrough in the treatments of patients with inherited retinal diseases(IRDs)have been achieved with the development of gene sequencing and gene editing technologies.Luxturna,the world's first IRD gene therapy drug for the treatment of Leber's Congenital Amaurosis(LCA)was launched in 2017.1 It indicates that IRD gene therapy had entered the new stage of clinical application.The new era of gene therapy is coming.However,IRD has a low prevalence and a high degree of genetic and clinical heterogeneity.Most of the existing gene therapy-related researches and drugs were based on the genetic backgrounds of western people.

乙醇胺可作为血糖控制良好的糖尿病患者发生糖尿病视网膜病变的潜在生物标志物和基于生物标志物的防治策略

Diabetic retinopathy(DR)is the leading cause of blindness among the working-age population.Although controlling blood glucose levels effectively reduces the incidence and development of DR to less than 50%,there are currently no diagnostic biomarkers or effective treatments for DR development in glucose-wellcontrolled diabetic patients(GW-DR).In this study,we established a prospective GW-DR cohort by strictly adhering to glycemic control guidelines and maintaining regular retinal examinations over a median 2-year follow-up period.The discovery cohort encompassed 71 individuals selected from a pool of 292 recruited diabetic patients at baseline,all of whom consistently maintained hemoglobin A1c(HbA1c)levels below 7%without experiencing hypoglycemia.Within this cohort of 71 individuals,21 subsequently experienced new-onset GW-DR,resulting in an incidence rate of 29.6%.In the validation cohort,we also observed a significant GW-DR incidence rate of 17.9%.Employing targeted metabolomics,we investigated the metabolic characteristics of serum in GW-DR,revealing a significant association between lower levels of ethanolamine and GW-DR risk.This association was corroborated in the validation cohort,exhibiting superior diagnostic performance in distinguishing GW-DR from diabetes compared to the conventional risk factor HbA1c,with AUCs of 0.954 versus 0.506 and 0.906 versus 0.521 in the discovery and validation cohorts,respectively.Furthermore,in a streptozotocin(STZ)-induced diabetic rat model,ethanolamine attenuated diabetic retinal inflammation,accompanied by suppression of microglial diacylglycerol(DAG)-dependent protein kinase C(PKC)pathway activation.In conclusion,we propose that ethanolamine is a potential biomarker and represents a viable biomarker-based therapeutic option for GW-DR.

Artificial intelligence for diabetic retinopathy

Diabetic retinopathy(DR)is an important cause of blindness globally,and its prevalence is increasing.Early detection and intervention can help change the outcomes of the disease.The rapid development of artificial intelligence(AI)in recent years has led to new possibilities for the screening and diagnosis of DR.An AI-based diagnostic system for the detection of DR has significant advantages,such as high efficiency,high accuracy,and lower demand for human resources.At the same time,there are shortcomings,such as the lack of standards for development and evaluation and the limited scope of application.This article demonstrates the current applications of AI in the field of DR,existing problems,and possible future development directions.