Expression of cyclinD1 in a rat model of oxygen-induced retinopathy

This study sought to elucidate the correlation between cyclinD1 expression and the emergence of neovascularization in oxygen-induced retinopathy （OIR）. OIR was induced in Sprague-Dawley 7-day-old neonatal rats exposed to hyperoxia （75% O2） for 5 days, and then returned to room air. Adenosine diphosphatase staining showed that the neovascularization started to emerge at rat age of day 14, and reached a peak at day 17, then gradually decreased and resolved by day 26. Immunohistochemical and immunofluorescence staining revealed that cyclinD1 protein expression was seen in the OIR rats at the age of day 12, then gradually increased and returned to normal levels by day 26. These experimental findings demonstrated that the temporal pattern of cyclinD1 protein expression is consistent with the emergence of retinal neovascularization.

Role of unc5b in retinal neovascularization in mice with oxygen-induced retinopathy

AIM: To explore the role of unc5b in retinal neovascularization in murine oxygen-induced retinopathy (OIR). METHODS: On postnatal 7 (P7), C57BL/6J mice were exposed to 75% +/- 2% oxygen for 5 days. On postnatal 12 (P12), the mice were brought back to the room air (21% oxygen) to induce retinal neovascularization. Western blot analysis was performed to examine the temporal expression of unc5b in murine retinas. Double staining for unc5b and isolectin B4 were employed to determine the location of unc5b in murine retinas. The effect of unc5b on retinal neovascularization was evaluated by intravitreal injection of unc5b-FC in mice with OIR. Retinal neovascularization was measured by counting neovascular cell nuclei above the internal limiting membrane and by angiography of flat-mounted retinas perfused with fluorescein dextran. o RESULTS: Compared to age-matched normal mice, the expression of unc5b was significantly increased in retinas of OIR mice on P17 and P21. Unc5b was apparently expressed in retinal vessels of OIR while being negative in normal retinal vessels. Retinal neovascularization in eyes injected with unc5b-FC was significantly reduced. CONCLUSION: Unc5b-FC can effectively inhibit retinal neovascularization induced by OIR. It may serve as a powerful and novel therapy for ischemia-induced retinal disease.

Activated complement classical pathway in a murine model of oxygen-induced retinopathy

AIM: To investigate whether the complement system is involved in a murine model of oxygen-induced retinopathy(OIR).METHODS: Forty C57BL/6J newborn mice were divided randomly into OIR group and control group. OIR was induced by exposing mice to 75% ±2% oxygen from postnatal 7d(P7) to P12 and then recovered in room air.For the control group, the litters were raised in room air.At the postnatal 17d(P17), gene expressions of the complement components of the classical pathway(CP),the mannose-binding lectin(MBL) pathway and the alternative pathway(AP) in the retina were determined by quantitative real-time polymerase chain reaction(RT-PCR). Retinal protein expressions of the key components in the CP were examined by Western blotting.· RESULTS: Whole mounted retina in the OIR mice showed area of central hypoperfusion in both superficial and deep layers and neovascular tufts in the periphery.The expressions of C1 qb and C4 b genes in the OIR retina were significantly higher than those of the controls. The expression of retinal complement factor B(CFB) gene in OIR mice was significantly lower than those of the controls. However, the expressions of C3 and complement factor H(CFH) genes were higher. The protein synthesis of the key components involved in the CP(C1q, C4 and C3) were also significantly higher in OIR mouse retina. Although MBL-associated serine protease 1(MASP1) and MASP2 were detected in both the OIR and the control groups, the expressions were weak and the difference between the two groups was not significant.CONCLUSION: Our data suggest that the complement system CP is activated during the pathogenesis of murine model of OIR.

Acetylcholinesterase inhibition ameliorates retinal neovascularization and glial activation in oxygen-induced retinopathy

AIM:To investigate whether inhibition of acetylcholinesterase(AChE)by donepezil ameliorate aberrant retinal neovascularization(RNV)and abnormal glial activation in oxygen-induced retinopathy(OIR).METHODS:A mouse model of RNV was induced in postnatal day 7(P7)mice by exposure to 75%oxygen.Donepezil was administrated to P12 mice by intraperitoneal injection.Expression and localization of AChE in mouse retinas were determined by immunofluorescence.RNV was evaluated by paraffin sectioning and hematoxylin and eosin(HE)staining.Activation of retinal M uller glial cells were examined by immunoblot of glial fibrillary acidic protein(GFAP).rMC-1.a retinal Muller cell line,was used for in vitro study.Expression of hypoxia-induced factor 1α(HIF-1α)and vascular endothelial growth factor(VEGF)were determined by Western-blot analysis,enzyme-linked immunosorbent assay(ELISA)or immunostaining.RESULTS:Aberrant RNV and glial activation was observed after OIR.Of note,retinal AChE was mainly expressed by retinal Muller glial cells and markedly increased in OIR mice.Systemic administration of donepezil significantly reduced RNV and abnormal glial activation in mice with OIR.Moreover,ischemia-induced HIF-1αaccumulation and VEGF upregulation in OIR mouse retinas and cultured rMC-1 were significantly inhibited by donepezil intervention.CONCLUSION:AchE is implicated in RNV with OIR.Inhibition of AChE by donepeizl is likely to be a potential therapeutic approach for retinal neovascular diseases.

Efficacy of intravitreal captopril on oxygen-induced retinopathy in mice

AIM: To study the inhibitory effect of intravitreal captopril on oxygen-induced retinopathy (OIR) in mice. METHODS: Eighty postnatal day (P)7 C57BL/63 mice were randomly divided into treated group and control group with forty mice in each group. The mice were exposed to 75% 2% oxygen for 5 days (P7-P11) and then returned to room air for 5 days (P12-P17) to induce retinal neovascularization (RNV). Beginning on P12, the mice in treated group received daily intravitreal injections of captopril(3.0mL/kg), while those in control group received daily intravitreal injections of phosphate-buffered saline (PBS) (3.0mL/kg) through P17. After anesthetized at P17, one eye was chosen randomly as experimental eye and were enucleated. RNV was examined by Adenosine diphosphate-ase (ADPase) stained retina flat-mounts and was quantitated histologically by counting the neovascular endothelial cell nuclei anterior to inner limiting membrane (ILM). The expressions of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) were measured by immunohistochemical method. RESULTS: Comparing with control group, more regular distributions, better branch and reduced density of RNV were observed in eyes of treated group. The number of neovascular cell nuclei was less in treated group than that in control group ( t =6.135, P <0.01). Stain of MMP-2 and VEGF was weaker in treated group than that in control group. CONCLUSION: The results indicate that captopril can significantly inhibit RNV in OIR mice.

AB017.Investigation of the effect of lymphocyte-derived microparticles on retinal macrophages in the oxygen-induced retinopathy model

Background:Retinopathy of prematurity(ROP)is the major cause of blindness in children,mainly caused by the retinal neovascularization(NV).Mounting of evidences shown that macrophage plays a pivotal role in the regulation of angiogenesis in ROP.Numerous studies confirmed that the deletion of macrophage significantly reduce the neovascularized areas in the oxygen-induced retinopathy(OIR)model.We have been studied the effect of lymphocyte derived-microparticles(LMPs)over ten years.LMPs are extracellular vesicles derived from apoptotic human CEM T lymphocytes.Our previous studies demonstrated that LMPs possess strong anti-angiogenic effect.Recently we observed that LMPs are capable to switch the phenotype of macrophage,thus to suppress the choroidal neovascularization(CNV).However,the role of LMPs on macrophage in ROP has not been clarified.Thus,my project is to disclose the relationship between LMPs and macrophage in ROP using the OIR model.Hypothesis:LMPs may inhibit retinal NV in the OIR model through targeting at macrophage by affecting the migration of macrophage,thus to inhibit pathological angiogenesis in ROP.Methods:Cell culture[RAW 264.7 and bone marrow-derived macrophage(BMDM)]for cell migration and viability assay.Generate the OIR model for in vivo detection of macrophage recruitment.Quantification of retinal NV,immunohistostaining of the macrophage in vivo,ex vivo retinal explants for cell migration and qPCR.Results:LMPs do not affect RAW 264.7 and BMDM cell viability(P>0.05).LMPs significantly decrease the BMDM cell migration indirectly(P<0.05).I successfully generate the OIR model and confirm that more macrophages infiltrate during retinal angiogenesis with counting the F4/80 immunostaining in the retinal flat mount.LMPs exert inhibiting effect on retinal angiogenesis through decreasing the migration of macrophages in vivo.Conclusions:LMPs have the negative effect on retinal angiogenesis via reducing the infiltrated macrophages to the neovascularized areas in the OIR model.

A comparing study of quantitative staining techniques for retinal neovascularization in a mouse model of oxygen-induced retinopathy

AIM: To explore an efficient, practical and objective quantitative method to evaluate the retinal neovascularization in mouse model of oxygen induced retinopathy (OIR). METHODS: Thirty C57BL/6J mice were explored in OIR model procedure. Eyes were removed for different staining methods including: (1) HE staining; (2) immunohistochemistry with Griffonia Simplicifolia Lectin (GSL); (3) Immunofluorescence with FITC labeled CD31 antibody; (4) Two-step immunofluorescence with purified-CD31 antibody; (5) FITC-Dextran perfusion combined with two-step purified-CD31immunofluorescence. Images of the retinal vasculature were analyzed by imaging software. ' RESULTS: GSL immunohistochemistry could clearly demonstrate the deep and superficial capillary beds. FITC labeled CD31 Immunofluorescence was blurring with high fluorescence background which was hard to distinguish retinal neovascularization in some area. Excellent detail of neovascularization and preexistent retinal vessels was provided in two-step Purified-CD31 immunofluorescence group. CONCLUSION: GSL immunohistochemistry can clearly demonstrate neovascularization tufts in deep and superficial capillary beds. Immunofluorescence of specific antigen CD31 on vascular endothelium can selectively label the neovascularization of mouse retina. When combined with computer analysis software, it is an effective and objective quantitative method to evaluate the retinal neovascularization in OIR mouse model.

The protective effect of 17 beta-estradiol on oxygen-induced retinopathy and its relation with the changes of malondialdehyde

Objective： Retinopathy of prematurity is becoming obvious with the improvement of neonatal ambulance. However there is still not a good treatment. The present study is to observe the effect of 17 beta-estradiol （E2） on oxygen-induced retinopathy （OIR）, and explore the relationship between the changes of avascular area and malondialdehyde （MDA） in retina. Methods： Newborn oxygen-exposed mice underwent subcutaneous injections of different dose of E2 （0.1 μg, 1.0 μg, 10.0 μg ）, tamoxifen or phosphate buffered saline （PBS; controls）everyday from post-natal day （p）7 to p17. At p17, retinal flat mounts were scored for the percentage of avascular/total retinal area, and pathological changes during revascularization. The MDA concentration in the retina was determined also. In the most efficacious E2 group （10.0 μg）, 100.0 μg tamoxifen was also administered, and the percentage of capillary-free/total retinal area determined, and the retinal malondialdehyde concentration assayed. Results： The mean percentage of capillary-free area over total retinal area was 0（PBS, in room air）, 34.197±1.301（PBS, in hyperoxia）, 23.685±0.407 （0.1 μg E2）, 14.648±0.355 （1.0 μg E2）, 4.693±0.450 （10.0 μg E2） and 32.240±0.654 （10.0 μg E2 ＋100.0 μg tamoxifen）. The difference was significant （F = 2778.759, P 〈 0.01）, and the difference between any two groups were also significant （all P value were less than 0.01）. The predilection of tufts and clusters during revascularization was mainly aggregated in zones 2 and 3, but the difference of retinal neovascular clusters and tufts in fourth zone among different groups were significant [clusters （F = 44.719, P 〈 0.01） vs tufts （F = 39.997,P 〈 0.01）]. The mean MDA concentration were 0.711 ±0.037（PBS, in room air）, 2.084±0.066 （PBS, in hyperoxia）, 1.829±0.091（0.1 μg E2）, 1.152± 0.067（1.0 μg E2）, 0.796 ±0.027（10.0 μg E2）, 1.988 ± 0.049（10.0μg E2 ＋100.0 μg tamoxifen） （F = 628.103, P 〈 0.01）. The difference between any two groups were also significant （all P value were less than 0.05）. The close relation between the percentage of avascular/total retinal area and MDA concentration was also verified （r = 0.981, P 〈 0.01）. Conclusion： Oxidative stress responses play a pivotal role in OIR, by means of receptor pathway. E2 can alleviate oxidative stress reaction, and thus ameliorate the severity of oxygen induced retinopathy.

Identification of altered microRNAs in retinas of mice with oxygen-induced retinopathy

AIM: To identify disease-related miRNAs in retinas of mice with oxygen-induced retinopathy(OIR), and to explore their potential roles in retinal pathological neovascularization. METHODS: The retinal miRNA expression profile in mice with OIR and room air controls at postnatal day 17(P17) were determined through miRNA microarray analysis. Several miRNAs were significantly up-and down-regulated in retinas of mice with OIR compared to controls by quantitative real-time reverse transcription-polymerase chain reaction(qRT-PCR). Two databases including Targetscan7.1 and MirdbV5 were used to predict target genes that associated with those significantly altered mi RNAs in retinas of mice with OIR. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analyses were also conducted to identify possible biological functions of the target genes. RESULTS: In comparison with room air controls, 3 and 8 miRNAs were significantly up-and down-regulated, respectively, in retinas of mice with OIR. The qRT-PCR data confirmed that mmu-miR-350-3 p and mmu-miR-202-3 p were significantly up-regulated, while mmu-miR-711 and mmu-miR-30 c-1-3 p were significantly down-regulated in mice with OIR compared to controls. GO analysis demonstrated that the identified target genes were related to functions such as cellular macromolecule metabolic process. KEGG pathway analysis showed a group of pathways, such as Wnt signaling pathway, transcriptionalmisregulation in cancer, Mucin type O-glycan biosynthesis, and mitogen-activated protein kinase(MAPK) signaling pathway might be involved in pathological process of retinal neovascularization. CONCLUSION: Our findings suggest that the differentially expressed miRNAs in retinas of mice with OIR might provide potential therapeutic targets for treating retinal neovascularization.

CCR7/p-ERK1/2/VEGF signaling promotes retinal neovascularization in a mouse model of oxygen-induced retinopathy

AIM： To investigate the role of CCR7/p-ERKI/2/VEGF signaling in the mouse model of oxygen-induced retinopathy （OIR）. METHODS： Neonatal C57BL/6J mice were evenly randomized into four groups： normoxia, OIR, OIR control （treated with scramble siRNA）, and OIR treated （treated with CCR7 siRNA）. Normoxia group was not specially handled. Postnatal day 7 （P7） mice in the OIR group were exposed to 75%±5% oxygen for 5d （P7-P12） and then maintained under normoxic conditions for 5d （P12-P17）. Mice in the OIR control and OIR treated groups were given injections of scramble or CCR7 siRNA plasmid on P12 before returning to normoxic conditions for 5d （P12-P17）. Retina samples were collected from all mice on P17, stained with adenosine diphosphatase （ADPase）, and retinal neovascularization （RNV） was assessed. Retinas were also stained with hematoxylin and eosin （H＆E） for RNV quantitation. The distribution and expression of CCR7, p-ERKI/2 and vas- cular endothelial growth factor （VEGF） were assessed via immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction （qRT-PCR）. RESULTS： High oxygen promoted retinal neovascularization （P〈0.05） and increased the number of endothelial nuclei in new vessels extending from the retina to the vitreous body; CCR7 promoted this process （P〈0.05）. CCR7 and VEGF mRNA were expressed at higher levels in the OIR and OIR control groups than in the normoxia and OIR treated groups. CCR7, p-ERK1/2, and VEGF protein were expressed in the retinas of mice in the OIR and OIR control groups. Intravitreal injection of CCR7 siRNA significantly reduced CCR7, p-ERKI/2, and VEGF expression in the OIR mouse model （all P〈0.05）. CCR7 significantly enhancedthe neovascularization and non-perfusion areas in the OIR group （P〈0,05）, CCR7 siRNA significantly reduced levels of p-ERK1/2 and VEGF as compared to OIR controls （P〈0.05）. CONCLUSION： These results suggest that CCR7/p-ERK I/2NEGF signaling plays an important role in OIR, CCR7 may be a potential target for the prevention and treatment of retinopathy of prematurity.

Effect of endothelial progenitor cells derived from human umbilical cord blood on oxygen-induced retinopathy in mice by intravitreal transplantation

AIM： To investigate the effect of endothelial progenitor cells（EPCs） labeled by carboxy fluorescein diacetate succinimidyl ester（CFSE） on murine oxygen-induced retinopathy（OIR） by intravitreal transplantation.METHODS： After isolated from human umbilical cord blood mononuclear cells, EPCs were cultivated and then labeled with CFSE in vitro. C57BL/6J mice were placed to75% hyperoxia chamber from P7 to P12 to establish OIR model. At P12, OIR mice were intravitreally injected with1 μL suspension contained 2×105EPCs（EPCs group） or isometric phosphate buffered saline（PBS group）. The contralateral eye of each mice received no injection（OIR group）. Evans blue angiography and frozen section were examined to track the labeled cells in OIR group at P15 and P19. Using retina paraffin sections and adenosinediphos phatase staining at P12 and P19, the effect of EPCs on OIR mice was evaluated quantitatively and qualitatively. RESULTS： The retinas from EPCs group with less non-perfusion area and fewer peripheral tufts wereobserved at P19, comparing with that from PBS or OIR group. The retinopathy in EPCs group receded earlier with less non-ganglion cells and neovascular nuclei,together with relatively regular distribution. The counts of the neovascular nuclei at P19 were reduced by 44% or45%, compared with those of OIR group or PBS group respectively. Three days after EPCs injection, a large number of EPCs appeared in the vitreous cavity and adhered to the retinal surface. While at one week, the cells gathered between the internal plexiform layer and the inner limiting membrane, and some EPCs appeared in retinal vessels.CONCLUSION： EPCs transplantation can participate in the reparative procedure of the neovascularization in OIR.

Effects of aminoguanidine on retinal apoptosis in mice with oxygen-induced retinopathy

AIM:To explore the protective effects of amino-guanidine(AG) on retinal apoptosis in mice with oxygeninduced retinopathy(OIR).·METHODS:A total of 80 C57BL/6J mice,aged 7 days,were randomly divided into four groups:normal,high oxygen,high oxygen saline and high oxygen treated with AG.In the normal group,mice were housed in normoxic conditions from postnatal day P7 to P17.Mice in the other 3 groups were placed under hyperoxic conditions(75 ±2% O2) in an oxygen-regulated chamber for 5 days and subsequently placed in normoxic conditions for 5days.Mice in the AG group were treated once daily,from P12 to P17,with AG hemisulfate(100mg/kg body weight,intraperitoneally) dissolved in physiological saline.An equivalent amount of 0.9% physiological saline was administered,as above,to mice in the high oxygen saline group.Ten mice were randomly selected from each group on P14 and on P17,euthanized and the retinas examined.Apoptotic cells in the retina were detected using the terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) method.The expression of nitric oxide synthase(iNOS) in the retina was detected by immunohistochemistry and changes in rod cells were observed using electron microscopy.·RESULTS:TUNEL-positive cells and iNOS immunoreactive neurons were present in the inner nuclear and ganglion cell retinal layers of mice in the high oxygen group.The number of TUNEL-positive cells was significantly greater in the high oxygen group compared with the normal group(t =-20.81,P14d【0.05;t =-15.05,P17d【0.05).However,the number of TUNEL-positive cells in the AG treatment group was significantly lower(t =-13.21,P14d【0.05;t =-6.61,P17d【0.05) compared with thehigh oxygen group.The expression of iNOS was significantly higher in the high oxygen group compared with the normal group(t =-21.95,P14d【0.05;t =-17.30,P17d【0.05).However,the expression of iNOS in the AG treatment group was significantly lower(t =-12.17,P14d【0.05;t =-10.30,P17d【0.05) compared with the high oxygen group.The outer segments of the rods were disorganized and short in the high oxygen group.Rod morphology appeared to be slightly improved in the AG group.·CONCLUSION:AG may protect retinal neurons in OIR by inhibiting apoptosis.The mechanism may be related to iNOS.

Effects of long non-coding RNA myocardial infarction-associated transcript on retinal neovascularization in a newborn mouse model of oxygen-induced retinopathy

Whether long non-coding RNA myocardial infarction-associated transcript is involved in oxygen-induced retinopathy remains poorly understood. To validate this hypothesis, we established a newborn mouse model of oxygen-induced retinopathy by feeding in an oxygen concentration of 75 ± 2% from postnatal day 8 to postnatal day 12, followed by in normal air. On postnatal day 11, the mice were injected with the myocardial infarction-associated transcript siRNA plasmid via the vitreous cavity to knockdown long non-coding RNA myocardial infarction-associated transcript. Myocardial infarction-associated transcript siRNA transcription significantly inhibited myocardial infarctionassociated transcript mRNA expression, reduced the phosphatidylinosital-3-kinase, phosphorylated Akt and vascular endothelial growth factor immunopositivities, protein and mRNA expression, and alleviated the pathological damage to the retina of oxygen-induced retinopathy mouse models. These findings suggest that myocardial infarction-associated transcript is likely involved in the retinal neovascularization in retinopathy of prematurity and that inhibition of myocardial infarction-associated transcript can downregulate phosphatidylinosital-3-kinase, phosphorylated Akt and vascular endothelial growth factor expression levels and inhibit neovascularization. This study was approved by the Animal Ethics Committee of Shengjing Hospital of China Medical University, China(approval No. 2016 PS074 K) on February 25, 2016.

Comparison of Dextran Perfusion and GSI-B4 Isolectin Staining in a Mouse Model of Oxygen-induced Retinopathy

Purpose: Oxygen-induced retinopathy(OIR) is a robust and widely used animal model for the study of retinal neovascularization(NV). Dextran perfusion and Griffonia simplicifolia isolectin B4(GSI-B4) staining are two common methods for examining the occurrence and extent of OIR. This study provides a quantitative comparison of the two for OIR detection.Methods: At postnatal day 7(PN7), fifteen C57 BL / 6J mice were exposed to a 75% hyperoxic condition for 5 days and then returned to room air conditions. At PN17, the mice received intravitreal injection of GSI-B4 Alexa Fluor 568 conjugate. After 10 hours, they were infused with FITC-dextran conjugate via the left ventricle. Retinal flat mounts were photographed by confocal microscopy. Areas with fluorescent signals and the total retinal areas were quantified by Image J software.Results:Both GSI-B4 and dextran detected the peripheral neovascular area. The mean hyper fluorescence area was 0.33 ±0.14% of whole retinal area determined by GSI-B4 staining and 0.25±0.28% determined by dextran perfusion. The difference between the two measures was 0.08%(95% CI:-0.59%,0.43%)..The Pearson correlation coefficient between the two methods was 0.386,P =0.035..The mean coincidence rates were 14.3 ±13.4% and 24.9 ±18.5% for GSI-B4 and dextran staining, respectively.Conclusion:.Both methods can complement each other indemonstrating and quantitatively evaluating retinal NV. A poor agreement was found between the two methods;.GSI-B4 isolectin was more effective than FITC-dextran perfusion in evaluating the extent of retinal NV in a mouse model of OIR.

AB043.Long-standing choroidal thinning in oxygen-induced retinopathy

Background:Retinopathy of prematurity(ROP),the most common cause of blindness in premature infants,has long been associated with pathologic retinal vasculature.However,recent studies reveal choroidal involution in adolescent patients formerly afflicted with ROP.We have recently demonstrated that choroidal thinning occurs early in retinopathy and persists into adulthood.Unlike retinal vessels,the damaged choroidal vasculature in ROP is incapably to regenerate.Herein,we investigated the molecular mechanism implicated in the lack of choroidal repair in ischemic retinopathy.Methods:The oxygen-induced retinopathy(OIR)model was used.Newborn Sprague-Dawley(albino)or Long-Evans rats(pigmented)rats were placed under oxygen concentration which cycles at 50%±1%or 10%±1%every 24 hours(hr)from postnatal day(P)0 to P14.On P14,all rats were returned to room air.Western blotting and qPCR were used to quantify protein and RNA abundances,respectively.The Dual-Luciferase®Reporter Assay System was used to confirm microRNA(miRNA)-mRNA interaction.Results:We detected a substantial oxidative stress in retinal pigment epithelium(RPE)and choroidal tissue,accompanied by a drastic reduction in insulin-like growth factor 1 receptor(IGF1R),a critical player in post-injury revascularization.The mechanism of decreasing IGF1R involves the over-activation of the p53 tumor suppressor that regulates miRNA let-7b,which subsequently silences Igf1r mRNA in the RPE/choroid complex of OIR subjects.Luciferase reporter assay confirmed that let-7b directly targets Igf1r mRNA at its 3’untranslated region(UTR).Indeed,silencing p53 resulted in a decreased let-7b expression,and re-established IGF1R abundance that promoted choroidal regeneration.Conclusions:Together,this study sets forth new mechanistic notion by uncovering the novel p53/let-7b/IGF1R axis;timely intervention of this pathway facilitates healthy choroidal revascularization.Future investigations on anti-angiogenic miRNAs can better our understanding on degenerative choroidopathy,such as geographic atrophy.

Long-lasting impairments in rodent oxygen-induced retinopathy measured by retinal vessel density and visual function

In mild or moderate retinopathy of prematurity(ROP), retinal vessels undergo obliteration, proliferation, and regression. Despite complete regression of vessel abnormalities, a variety of visual impairments have been reported. Rodent oxygen-induced retinopathy(OIR) is widely used as a model to study ROP. However, the long-term changes of OIR model remain unclear. The aim of this study is to examine long term changes of retinal vessel and visual function in a rodent OIR model resembling human mild or moderate ROP. In this study, after subjecting the animals to 80% oxygen(O_2) for 5–7 d, the retinal vessel density at postnatal day 12(P12) was approximately 30% lower than that in the age-matched control, but this difference was not significant between the groups. Vessel abnormalities, such as vessel tortuosity, neovascular tufts, and the number of vessels protruding into the vitreous, peaked between P17 and P20. Despite the regression of many abnormalities, vessel density in the OIR group was 36% and 32% lower than that in the control animals at 6 weeks and 4 months, respectively. The visual acuity and contrast sensitivity were impaired in the OIR group when measured at 2, 3 and 4 months. Therefore, the rodent OIR model exhibited long-lasting reduction in retinal vessel density and visual impairments, similar to those observed in mild or moderate human ROP. This study suggests that the rodent OIR model can be used to explore possible interventions for mild and moderate human ROP.

Construction and validation of a neovascular glaucoma nomogram in patients with diabetic retinopathy after pars plana vitrectomy

BACKGROUND Neovascular glaucoma(NVG)is likely to occur after pars plana vitrectomy(PPV)for diabetic retinopathy(DR)in some patients,thus reducing the expected benefit.Understanding the risk factors for NVG occurrence and building effective risk prediction models are currently required for clinical research.AIM To develop a visual risk profile model to explore factors influencing DR after surgery.METHODS We retrospectively selected 151 patients with DR undergoing PPV.The patients were divided into the NVG(NVG occurrence)and No-NVG(No NVG occurrence)groups according to the occurrence of NVG within 6 months after surgery.Independent risk factors for postoperative NVG were screened by logistic regression.A nomogram prediction model was established using R software,and the model’s prediction accuracy was verified internally and externally,involving the receiver operator characteristic curve and correction curve.RESULTS After importing the data into a logistic regression model,we concluded that a posterior capsular defect,preoperative vascular endothelial growth factor≥302.90 pg/mL,glycosylated hemoglobin≥9.05%,aqueous fluid interleukin 6(IL-6)≥53.27 pg/mL,and aqueous fluid IL-10≥9.11 pg/mL were independent risk factors for postoperative NVG in patients with DR(P<0.05).A nomogram model was established based on the aforementioned independent risk factors,and a computer simulation repeated sampling method was used to internally and externally verify the nomogram model.The area under the curve(AUC),sensitivity,and specificity of the model were 0.962[95%confidence interval(95%CI):0.932-0.991],91.5%,and 82.3%,respectively.The AUC,sensitivity,and specificity of the external validation were 0.878(95%CI:0.746-0.982),66.7%,and 95.7%,respectively.CONCLUSION A nomogram constructed based on the risk factors for postoperative NVG in patients with DR has a high prediction accuracy.This study can help formulate relevant preventive and treatment measures.

Diabetic retinopathy identification based on multi-sourcefree domain adaptation

AIM:To address the challenges of data labeling difficulties,data privacy,and necessary large amount of labeled data for deep learning methods in diabetic retinopathy(DR)identification,the aim of this study is to develop a source-free domain adaptation(SFDA)method for efficient and effective DR identification from unlabeled data.METHODS:A multi-SFDA method was proposed for DR identification.This method integrates multiple source models,which are trained from the same source domain,to generate synthetic pseudo labels for the unlabeled target domain.Besides,a softmax-consistence minimization term is utilized to minimize the intra-class distances between the source and target domains and maximize the inter-class distances.Validation is performed using three color fundus photograph datasets(APTOS2019,DDR,and EyePACS).RESULTS:The proposed model was evaluated and provided promising results with respectively 0.8917 and 0.9795 F1-scores on referable and normal/abnormal DR identification tasks.It demonstrated effective DR identification through minimizing intra-class distances and maximizing inter-class distances between source and target domains.CONCLUSION:The multi-SFDA method provides an effective approach to overcome the challenges in DR identification.The method not only addresses difficulties in data labeling and privacy issues,but also reduces the need for large amounts of labeled data required by deep learning methods,making it a practical tool for early detection and preservation of vision in diabetic patients.

Proteomic study of vitreous in proliferative diabetic retinopathy patients after treatment with aflibercept:a quantitative analysis based on 4D label-free technique

AIM:To identify different metabolites,proteins and related pathways to elucidate the causes of proliferative diabetic retinopathy(PDR)and resistance to anti-vascular endothelial growth factor(VEGF)drugs,and to provide biomarkers for the diagnosis and treatment of PDR.METHODS:Vitreous specimens from patients with diabetic retinopathy were collected and analyzed by Liquid Chromatography-Mass Spectrometry(LC-MS/MS)analyses based on 4D label-free technology.Statistically differentially expressed proteins(DEPs),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway representation and protein interactions were analyzed.RESULTS:A total of 12 samples were analyzed.The proteomics results showed that a total of 58 proteins were identified as DEPs,of which 47 proteins were up-regulated and 11 proteins were down-regulated.We found that C1q and tumor necrosis factor related protein 5(C1QTNF5),Clusterin(CLU),tissue inhibitor of metal protease 1(TIMP1)and signal regulatory protein alpha(SIRPα)can all be specifically regulated after aflibercept treatment.GO functional analysis showed that some DEPs are related to changes in inflammatory regulatory pathways caused by PDR.In addition,protein-protein interaction(PPI)network evaluation revealed that TIMP1 plays a central role in neural regulation.In addition,CD47/SIRPαmay become a key target to resolve anti-VEGF drug resistance in PDR.CONCLUSION:Proteomic analysis is an approach of choice to explore the molecular mechanisms of PDR.Our data show that multiple proteins are differentially changed in PDR patients after intravitreal injection of aflibercept,among which C1QTNF5,CLU,TIMP1 and SIRPαmay become targets for future treatment of PDR and resolution of anti-VEGF resistance.

Gut microbiota induced abnormal amino acids and their correlation with diabetic retinopathy

AIM:To explore the correlation of gut microbiota and the metabolites with the progression of diabetic retinopathy(DR)and provide a novel strategy to elucidate the pathological mechanism of DR.METHODS:The fecal samples from 32 type 2 diabetes patients with proliferative retinopathy(PDR),23 with nonproliferative retinopathy(NPDR),27 without retinopathy(DM),and 29 from the sex-,age-and BMI-matched healthy controls(29 HC)were analyzed by 16S rDNA gene sequencing.Sixty fecal samples from PDR,DM,and HC groups were assayed by untargeted metabolomics.Fecal metabolites were measured using liquid chromatographymass spectrometry(LC-MS)analysis.Associations between gut microbiota and fecal metabolites were analyzed.RESULTS:A cluster of 2 microbiome and 12 metabolites accompanied with the severity of DR,and the close correlation of the disease progression with PDR-related microbiome and metabolites were found.To be specific,the structure of gut microbiota differed in four groups.Diversity and richness of gut microbiota were significantly lower in PDR and NPDR groups,than those in DM and HC groups.A cluster of microbiome enriched in PDR group,including Pseudomonas,Ruminococcaceae-UCG-002,Ruminococcaceae-UCG-005,Christensenellaceae-R-7,was observed.Functional analysis showed that the glucose and nicotinate degradations were significantly higher in PDR group than those in HC group.Arginine,serine,ornithine,and arachidonic acid were significantly enriched in PDR group,while proline was enriched in HC group.Functional analysis illustrated that arginine biosynthesis,lysine degradation,histidine catabolism,central carbon catabolism in cancer,D-arginine and D-ornithine catabolism were elevated in PDR group.Correlation analysis revealed that Ruminococcaceae-UCG-002 and Christensenellaceae-R-7 were positively associated with L-arginine,ornithine levels in fecal samples.CONCLUSION:This study elaborates the different microbiota structure in the gut from four groups.The relative abundance of Ruminococcaceae-UCG-002 and Parabacteroides are associated with the severity of DR.Amino acid and fatty acid catabolism is especially disordered in PDR group.This may help provide a novel diagnostic parameter for DR,especially PDR.