Protective effect of melatonin against oxygen-induced retinopathy: a study based on the HMGB1/NF-κB/NLRP3 axis / 中国当代儿科杂志

OBJECTIVES@#To study the protective effect of melatonin (Mel) against oxygen-induced retinopathy (OIR) in neonatal mice and the role of the HMGB1/NF-κB/NLRP3 axis.@*METHODS@#Neonatal C57BL/6J mice, aged 7 days, were randomly divided into a control group, a model group (OIR group), and a Mel treatment group (OIR+Mel group), with 9 mice in each group. The hyperoxia induction method was used to establish a model of OIR. Hematoxylin and eosin staining and retinal flat-mount preparation were used to observe retinal structure and neovascularization. Immunofluorescent staining was used to measure the expression of proteins and inflammatory factors associated with the HMGB1/NF-κB/NLRP3 axis and lymphocyte antigen 6G. Colorimetry was used to measure the activity of myeloperoxidase.@*RESULTS@#The OIR group had destruction of retinal structure with a large perfusion-free area and neovascularization, while the OIR+Mel group had improvement in destruction of retinal structure with reductions in neovascularization and perfusion-free area. Compared with the control group, the OIR group had significant increases in the expression of proteins and inflammatory factors associated with the HMGB1/NF-κB/NLRP3 axis, the expression of lymphocyte antigen 6G, and the activity of myeloperoxidase (P<0.05). Compared with the OIR group, the OIR+Mel group had significant reductions in the above indices (P<0.05). Compared with the control group, the OIR group had significant reductions in the expression of melatonin receptors in the retina (P<0.05). Compared with the OIR group, the OIR+Mel group had significant increases in the expression of melatonin receptors (P<0.05).@*CONCLUSIONS@#Mel can alleviate OIR-induced retinal damage in neonatal mice by inhibiting the HMGB1/NF-κB/NLRP3 axis and may exert an effect through the melatonin receptor pathway.

Inhibitory effect of gamma-secretase inhibitor on retinal neovascularization and regulative mechanism of Notch1 signal pathway / 中华实验眼科杂志

Objective:To investigate the inhibitory effect and underlying mechanism of gamma-secretase inhibitor blocking Notch1 signaling on retinal neovascularization caused by oxygen-induced retinopathy (OIR) in mice.Methods:To establish the OIR model, 7-day-old pups of C57BL/6J mice were exposed to 75% oxygen together with their mother until postnatal day (P)12.On P12, the mice were transferred to room air.All the mice were randomly divided into three groups, OIR group as control group, OIR+ DAPT group and OIR+ DMSO group receiving 1 μl intravitreal injection of gamma-secretase inhibitor (DAPT, 10 mmol/L) and 1∶20 DMSO dilution respectively.The right eye was taken as experimental eye.The mice were euthanized on P17 and the eyes were harvested to obtain retinas for further investigation.The total proteins were extracted from the retinas.The relative expression levels of Notch1 signal pathway and its downstream Hes1, the markers of M1 phenotype inducible nitric oxide synthase (iNOS) and M2 phenotype arginase-1 (Arg-1) microglia were measured by western blot.Retinal flat mounts were made and the retinal vessels were stained with isolectin B4 (IB4) to investigate the relative retinal neovascularization areas which was calculated as the ratio of neovascularization area/retinal area.The mumber of the neovascular endothelium cells beyond the inner limiting membrane was observed by hematoxylin-eosin staining.The use and care of animals complied with ARVO statement.This study protocol was approved by the Animal Ethics Committee of Guangdong Provincial People's Hospital (No.KY-Z-2021-2015-01).Results:The relative protein expression levels of Notch1 and Hes1 in OIR+ DAPT group, OIR group, and OIR+ DMSO group were 0.68±0.06 and 0.70±0.08, 1.00±0.00 and 1.00±0.00, 1.03±0.08 and 1.02±0.07, respectively, with statistically significant differences among them ( F=70.62, 53.65; both at P<0.01). Compared with the OIR group and OIR+ DMSO group, the expressions of Notch1 and Hes1 were significantly reduced in OIR+ DAPT group (all at P<0.01). The relative protein expression levels of iNOS and Arg-1 in OIR+ DAPT group, OIR group, and OIR+ DMSO group were 0.74±0.07 and 1.49±0.12, 1.00±0.00 and 1.00±0.00, 1.04±0.10 and 0.94±0.07, respectively, showing statistically significant differences ( F=31.63, 89.32; both at P<0.01). Compared with OIR group and OIR+ DMSO group, the expression of iNOS in OIR+ DAPT group was significantly reduced, and the expression of Arg-1 was significantly increased (all at P<0.01). The relative neovascularization area and the number of neovascular endothelium cells beyond the inner limiting membrane in OIR+ DAPT group, OIR group, and OIR+ DMSO group were (8.82±2.71)% and 38.17±3.29, (22.32±5.34)% and 60.83±5.11, (20.27±3.36)% and 58.67±4.75, respectively, showing statistically significant differences ( F=33.72, 39.44; both at P<0.01). The relative neovascularization area and the number of neovascular endothelium cells in OIR+ DAPT group were significantly reduced in comparison with OIR group and OIR+ DMSO group (all at P<0.01). Conclusions:Intravitreal injection of DAPT can inhibit the retinal neovascularization in OIR mice through blocking Notch1 signaling activation and promoting retinal microglia polarization from M1 to M2 phenotype.

Effect of intravitreal and intraperitoneal cyanidin-3-glucoside injection in oxygen-induced retinopathy mouse model

Purpose: To evaluate the effect of cyanidin-3-glucoside (C3G) in oxygen-induced retinopathy (OIR) mouse model. Methods: In this experimental study, 10 C57BL / 6J type mice exposed to room air comprised two control groups (n = 5 each; a negative control and a group receiving intravitreal sterile dimethyl sulfoxide [IVS DMSO]). Thirty C57BL / 6J type mice exposed to 75% ± 2% oxygen from postnatal day 7 to postnatal day 12 comprised the OIR groups. On postnatal day 12, these mice were randomized into six groups (n = 5 each): two OIR control groups (negative control and IVS DMSO), two intravitreal C3G groups (300 and 600 ng/?L), and two intraperitoneal C3G groups (0.05 and 0.1 mg/kg). We quantified neovascularization by counting endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina and examined histological and ultrastructural changes via light and electron microscopy and apoptosis by terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling. Results: The intravitreal C3G groups yielded lower endothelial cell counts compared with the intravitreal DMSO group. The intraperitoneal high-dose group had lower cell counts compared with the OIR control groups. Electron microscopy revealed significantly less mitochondrial dysmorphology in intravitreal groups and the high-dose intraperitoneal mice. We noted no difference in apoptotic cell count between the controls, low-dose intravitreal, and both intraperitoneal groups. However, apoptotic cell count was significantly higher in the high-dose intravitreal group. Conclusion: C3G suppresses endothelial cell proliferation in an OIR mouse model, leads to a reduced hyperoxia-induced mitochondrial dysmorphology, but increases apoptotic cell death in high concentrations.

Effects of PEDF on the expression of MCP-1 in mice retina of oxygen-induced retinopathy / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To observe the effect of pigment epithelium-derived factor(PEDF)on retinal neovascularization(RNV)and monocyte chemoattractant protein-1(MCP-1)expressions in mice retina of oxygen-induced retinopathy(OIR), and to investigate the protective effect of PEDF on ischemia hypoxia retinopathy and the possible mechanism. <p>METHODS: A total of 160 postnatal day(P)7 C57BL/6 mice were obtained. All mice except normal control group were exposed to(75±2)% oxygen environment for 5d and then kept in room air for another 5d to establish the OIR mice model. All mice in normal control group(40 mice)were exposed to room air only. At P12 and P14, respectively, mice in PEDF treatment group were injected intravitreously with recombinant human PEDF(2μg/eye,1μL)in the right eye, while mice in treatment control group were injected intravitreously with the same volume of vehicle [1μL, 10mmol/L phosphate buffered saline(PH7.4), PBS] in the right eye. All mice were euthanized at P17. Eyes were whole mounted and stained with Lectin to observe the growth of abnormal RNV; And retinal specimens were prepared for PEDF, MCP-1 protein and mRNA analysis by Western blot and real time RT-PCR respectively. <p>RESULTS: Changes of retinal vessels had been detected by fluorescence microscopy on flat-mounted retina. The relative RNV areas were significantly increased in OIR model group compared with those in normal control group(<i>P</i><0.01). However, the relative RNV areas were significantly reduced in PEDF treatment group compared with those in PBS treatment control group(<i>P</i><0.01). The specific expression of MCP-1 protein and mRNA in the OIR model group were higher than those of normal control group, presenting a statistically significance(both <i>P</i><0.05). The specific expression of PEDF protein and mRNA in the OIR model group showed a considerable decline in comparison with normal control group, presenting a statistically significance(both <i>P</i><0.01). And the specific expression of MCP-1 protein and mRNA in those of PEDF-treated group showed a considerable decline in comparison with PBS-treated group, and the differences were statistically significant(both <i>P</i><0.05). However, there were increase of the expression of MCP-1 protein and mRNA between normal control group and PEDF-treated group, presenting no statistically significance(both <i>P</i>>0.05). <p>CONCLUSION: PEDF could inhibit oxygen-induced retinal neovascularization and down-regulate retinal MCP-1 expression under hypoxia, which may underlie its anti-neovascularization effects and play a role of protection in ischemic retinopathy.

Relationship between Pericytes and Endothelial Cells in Retinal Neovascularization: A Histological and Immunofluorescent Study of Retinal Angiogenesis

PURPOSE: To evaluate the relationship between pericytes and endothelial cells in retinal neovascularization through histological and immunofluorescent studies. METHODS: C57BL/6J mice were exposed to hyperoxia from postnatal day (P) 7 to P12 and were returned to room air at P12 to induce a model of oxygen-induced retinopathy (OIR). The cross sections of enucleated eyes were processed with hematoxylin and eosin. Immunofluorescent staining of pericytes, endothelial cells, and N-cadherin was performed. Microfluidic devices were fabricated out of polydimethylsiloxane using soft lithography and replica molding. Human retinal microvascular endothelial cells, human brain microvascular endothelial cells, human umbilical vein endothelial cells and human placenta pericyte were mixed and co-cultured. RESULTS: Unlike the three-layered vascular plexus found in retinal angiogenesis of a normal mouse, angiogenesis in the OIR model is identified by the neovascular tuft extending into the vitreous. Neovascular tufts and the three-layered vascular plexus were both covered with pericytes in the OIR model. In this pathologic vascularization, N-cadherin, known to be crucial intercellular adhesion molecule, was also present. Further evaluation using the microfluidic in vitro model, successfully developed a microvascular network of endothelial cells covered with pericytes, mimicking normal retinal angiogenesis within 6 days. CONCLUSIONS: Pericytes covering endothelial cells were observed not only in vasculature of normal retina but also pathologic neovascularization of OIR mouse at P17. Factors involved in the endothelial cell-pericyte interaction can be evaluated as an attractive novel treatment target. These future studies can be performed using microfluidic systems, which can shorten the study time and provide three-dimensional structural evaluation.

Animal models of oxygen-induced retinopathy / 中华实验眼科杂志

Models of oxygen-induced retinopathy (OIR) have been built on several animals,including kitten,mouse,rat,rabbit,puppy and zebrafish.These models play an important role in studying the mechanisms and treatments of retinal neovascularization,which is partially mimicking the pathological process of retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR).As the extensive application of transgenic technology,animal models of OIR will provide more evidences on investigating the retinal angiogenic diseases.However,none of these OIR models is perfect to simulate human ROP completely,which still needs more comprehensive and profound exploration.

Inhibitory effects of PI3K/AKT inhibitor LY294002 on retinal neovascularization in mice / 眼科新进展

Objective To explore the effects of phosphatidylinositol-3-kinase (PI3K)/serine-threonine kinase (AKT) signal pathway inhibitor LY294002 on retinal neovascularization (RNV) in oxygen-induced retinopathy (OIR) mice.Methods Totally 60 C57BL/6J mice were collected and randomly divided into the experimental group and control group,with 30 mice in each group.Then OIR model was induced by Smith methods.Rats in the experimental group were intravitreally injected with 0.5 μL LY294002,while the control group was given the same amount of phosphate buffer saline (PBS) one day before out of the incubator.Retinal sections with HE staining were applied to count the number of neovascular cell nuclei breaking through the inner limiting membrane,as well as the protein and mRNA expression of pAKT and vascular endothelial growth factor (VEGF) were detected by immunohistochemistry and RT-PCR.Results The number of retinal neovascular cell nucleus in the experimental group was obviously smaller than that in the control group (P ＜ 0.05).The protein expression of pAKT and VEGF was weakly expressed,and the absorbance (A) value of the positive cells was decreased in the experimental group compared with the control group (all P ＜ 0.05).The mRNA expression of AKT and VEGF was obviously decreased in the experimental group compared with the control group (all P ＜ 0.05).Conclusion The development of RNV in OIR mice can be markedly inhibited by LY294002 inhibiting PI3K/AKT pathway,and therefore LY294002 is expected to be an effective method for preventing vascular proliferative retinopathy.

Expression and function of P16 in a rat model of oxygen-induced retinopathy / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effect of p16 protein expression on the proliferation of retinal neovascularization in oxygen-induced retinopathy(OIR).<p>METHODS: Totally 60 SD rats aged 7d were randomly divided into 4 groups: normal group, model group, intervention group and NS control group. Normal group was raised in a normal air feeding; model group at 75% high oxygen for 5d to establish the model of oxygen induced retinopathy; intervention group was given anti p16 methylation drug 5-aza-CdR(0.25 mg/kg)intraperitoneal injection; NS control group was given the same volume NS intraperitoneal injection. The eyes were taken from each group and the left eyes were removed for observation of retinal neovascularization by HE staining, and immunohistochemistry and immunofluorescence were taken for observations of p16 protein expression. Right retina had been performed real time-PCR to analysis p16mRNA expression. <p>RESULTS: The normal group were not found retinal neovascularization breaking through internal limiting mebrane. In model group and NS control group, the retinal tissue was obviously thickened, and a large number of new blood vessels were found. In the intervention group, a small amount of new blood vessels were found in the retina. The expression of p16 was low in the model group, the positive cell number was 19.52±2.67, and the number of the positive cells was 36.38±3.16 in the intervention group, the difference was statistically significant(<i>P</i><0.001). Real time-PCR showed decreased expression of p16 mRNA in the model group(2^-△△ct=0.14±0.01), the expression of p16 mRNA in the intervention group rats retina was significantly higher than that of NS control group rat retina, there was significant difference between two groups(2^-△△ct=0.68±0.08, <i>P</i><0.001). <p>CONCLUSION: The abnormal expression of P16 may be closely related to the proliferation of retinal neovascularization. Inhibition of p16 methylation can decrease the proliferation of retinal neovascularization.

Effect of intravitreal injection of celecoxib on neovascularization in rats / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: Through intravitreal injection of celecoxib in oxygen-induced retinopathy(OIR)rat model, to investigate the effect and mechanism of celecoxib on neovascularization of OIR.<p>METHODS: Ninety-six 7-day-old SD rats were randomly divided into 6 groups: Group Z:Normal group; Group O: OIR; Group A: OIR + vehicle control group; Group B: OIR+5μg celecoxib group; group C: OIR + 20μg celecoxib group; group D: OIR + 80μg celecoxib group. In addition to Z group in the normal environment, the other groups were established the OIR model. The neonatal rats were given intravitreal injections of dimethyl sulfoxide(DMSO)and the corresponding doses of celecoxib on the 12th day after birth. The rats were sacrificed on day 17 after birth. HE staining were employed to count the vascular endothelial cells which were breakthrough within the internal limiting membrane of retina. Immunohistochemistry staining were utilized to probe the expression of VEGF protein. <p>RESULTS: HE staining showed that, the number of the endothelial cells in the retina was 0.44±0.18, 30.60±5.36, 28.05±4.68, 19.58±4.58, 10.13±1.93, 7.58±2.68 in Group Z, O, A, B, C and D. In addition to Group O and Group A, there were significant differences between the two groups(<i>P</i><0.05). After treatment with celecoxib, breakthrough of the internal limiting membrane of the vascular endothelial cell nucleus was significantly reduced, and positively correlated with the dose. Immunohistochemical results showed, the expression of VEGF protein in Group Z was negative, the expression rate was 10%,the positive expression of VEGF protein in Group A was higher than that in Group B, C and D, and the positive rate was 86%, which was higher than that of Group B, C and D, as 68%,42%, 30%.<p>CONCLUSION: Celecoxib can inhibit the OIR model of rat retinal angiogenesis, and the effect of suppressing a positive correlation with the dose,its action may inhibit VEGF expression.

α-Melanocyte-stimulating hormone inhibits retinal neovascularization in a mouse model of oxygen-induced retinopathy / 中华实验眼科杂志

Background Retinal neovascularization (RNV) occurs in multiple eye diseases,which can lead to bleeding and retinal detachment.Therefore,inhibition of pathological RNV is becoming crucial to the treatment of ocular diseases.Research has shown that α-melanocyte-stimulating hormone (α-MSH) inhibits retinal angiogenesis during physiological development;however,the effects of α-MSH on pathological RNV remain unknown.Objective This study was to investigate the effects of intravitreal injection of α-MSH at different concentrations on pathological RNV in a mouse model of oxygen-induced retinopathy (OIR).Methods Forty healthy clean C57BL/6J mice were randomly divided into OIR+0.33 μg/μl α-MSH,OIR+ 1.67 μg/μl α-MSH,OIR+3.30 μg/μl α-MSH,OIR,and normal control groups at postnatal day 7 (P7),with 8 pups in each group.The α-MSH intervention groups and OIR group were exposed to high oxygen (75 ±2)％ for 5 days,then maintained under normal air condition for another 5 days;whereas the normal control group was raised under normoxia for 10 days.Retro-orbital injection of high molecular weight fluorescein isothiocyanate-dextran (FITC-dextran) was performed on P17 mice.The retina whole mounts were prepared to reveal retinal vasculature and quantify relative area of vessel obliteration.The mouse eyeballs were subjected to paraffin sections and hematoxylin-eosin staining,and the average number of pre-retinal nuclei per section was quantified.Results FITC-dextran labeled retinal whole mounts showed that the relative vessel obliteration area in normal control,OIR,OIR+0.33 μg/μl α-MSH,OIR+ 1.67 μg/μl α-MSH,and OIR+3.30 μg/μl α-MSH groups were (0.00±0.00) ％,(23.01 ±3.39) ％,(18.14±7.20) ％,(15.64±7.07) ％,and (7.62±6.52) ％,respectively.There was a statistical significance in the relative avascular area among the groups (F=19.635,P＜0.05).The relative avascular area in the OIR group was significantly higher than that in the OIR+3.30 μg/μl α-MSH group (t=4.293,P＜0.01).The results of histopathological examinations showed that the average number of pre-retinal nuclei per section in normal control,OIR,OIR+0.33 μg/μl α-MSH,OIR+1.67 μg/μl α-MSH,and OIR+3.30 μg/μl α-MSH groups were 0.00±0.00,11.45 ±4.26,6.35 ±2.34,4.96 ± 1.79 and 1.03 ± 1.25,respectively.There was a statistical significance in the average number of pre-retinal nuclei per section among the groups (F =147.87,P＜0.05).The average number of pre-retinal nuclei per section in the OIR group was significantly higher than that in the OIR+0.33 μg/μl α-MSH,OIR+ 1.67 μg/μl α-MSH,and OIR+3.30 μg/μl α-MSH groups,the differences between the groups had statistical significances (all at P＜0.001).Conclusions α-MSH reduces the relative area of vessel obliteration and the average number of pre-retinal nuclei in the retinas of OIR mouse model.The inhibitory effects of α-MSH on the pathological RNV are dose-dependent.

Expressions of VEGFR-1 and VEGFR-2 in mouse retinas with oxygen-induced retinopathy / 中华实验眼科杂志

Background Retinopathy of prematurity (ROP) causes blindness due to retinal vasculopathy caused by abnormal oxygen dynamics.Studies have clarified the pivotal role of vascular endothelial growth factor (VEGF) in the development of ROP,while the studies on the role of VEGF receptor (VEGFR) in ROP are fewer.Objective This study was to evaluate the relationship between the expressions of VEGFR-1 and VEGFR-2 in retina and post-birth time in the mice with oxygen-induced retinopathy (OIR).Methods Sixty 7-day-old (P7) SPF C57BL/6J mice together with lactating female mice were fed in the environment with (75±2)％ oxygen concentration for 5 days and then returned to normal air to establish OIR models,and other 60 matched mice were kept in normal air environment as the control group.At P8,P11,P12,P13,P14,and P17,both eyes of each mouse were enucleated to prepare the retinal sections.Retinal blood vessels were examined by hematoxylin and eosin stain under the light microscope.Real-time fluorescence quantitative PCR and immunohistochemistry were used to detect the expressions of VEGFR1 mRNA and VEGFR2 mRNA and their proteins in mouse retinas,respectively.The use and care of the animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Many vascular endothelial cell nucleus broke through the internal limiting membrane were seen with disorganized cell proliferation under the internal limiting membrane and retinal neovascularization bud in the P13,P14 and P18 mice in the OIR group,but no similar manifestation was found in the mice of the normal control group.The relative expression values of VEGFR-1 mRNA in retinas were significantly higher only in P12,P13,P14 and P18 mice in the OIR group than those in the normal control group (P=0.046,0.000,0.000,0.042),but the expression values of VEGFR-2 mRNA were all increased in the retinas of P8,P11,P12,P13,P14 and P18 mice in the OIR group,showing significant differences in comparison with the normal control group (all at P=0.000).No considerable difference was found in the expression level of VEGFR-1 protein in P8,P11,P12,P13,P14 and P18 mice between the two groups (M=50.00,36.00,41.00,31.00,28.00,36.00,all at P＞ 0.05).The expression levels of VEGFR-2 protein in retinas of P8 and P11 were close between the two groups (all at P＞0.05).Gradually attenuated expressions were seen in VEGFR-2 protein in P12,P13 mice of the normal control group,however,the expressions were enhanced in the OIR group,with significant differences between the two groups (all at P＜0.01).Enhanced expressions of VEGFR-2 protein were found in P14 mice in both groups,but stronger expressions were in the OIR group (P＜0.01).The positive response of the expression of VEGFR-2 protein was weaker in P18 mice in the normal control group but peaked in the OIR group,showing significant difference between them (M=20.11,P＜0.01).Conclusions VEGFR participates in the neovascularization of OIR mouse,and the effect of VEGFR-2 is stronger than that of VEGFR-1 in the development of OIR neovascularization.

Optimization of reference genes for microRNA expression analysis in a mouse model of oxygen-induced retinopathy / 中华实验眼科杂志

Background Retinal neovascularization varies with the change of microRNA (miRNA) expression level.Quantitative real-time PCR (qRT-PCR) is a common method for the analysis of miRNA expression profiling.However,the housekeeping genes selected as references may exhibit differential expression levels under the distinctive experimental conditions.The accuracy of the levels of target gene expression often is affected if the selected housekeeping genes with significantly fluctuating expression as references.Determining a reference gene with stable expression level is the premise to consecutive studies.Objective This study was to compare the expression levels and stability of the frequently used reference genes for miRNA expression analysis in normally developing eyes and in eyes of a mouse model of oxygen-induced retinopathy (OIR),and select the optimal reference gene (s) exhibiting stable ocular expression under both hypoxia and normal development conditions.Methods P7,P12,P17,P37 and 8-week-old clean C57BL/6J mice were selected randomly.q-PCR was used to detect the dynamic changes of relative expressions of 5 kinds of reference genes in different ages of mice,including snRNAU6 (RNU6),5S ribosomal RNA (5s rRNA),snoRNA U68 (U68),snoRNA U70 (U70),snoRNA U49A (U49A).Other 40 P7 mice were randomized into the normal control group and the OIR group.The mice of the normal control group were fed in the normal oxygen environment,and those in the OIR group were raised in the (75-±2)％ oxygen environment for 10 days.Fluorescine was injected via ritrobulbar vein for retinal stretched preparation,and the histopathological examination of mouse retinas was performed to identified the models.The expressing difference of 5 kinds of reference genes in the retinas were detected to compare the differences of 5 kinds of genes expression between the two groups.GeNorm program was employed to compare the stability of the expressing genes.This study were approved and granted by Ethical Committee of Tianjin Medical University and met the requirements stipulated in the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.Results Across the developmental ages,the expression levels of U68 in the eye exhibited the greatest variability,and then coming with U49A,U70,RNU6 and 5s rRNA.Significant differences were seen across the developmental ages for the expression levels of U68,U49A,U70,and RNU6 (U70:F =7.005,P =0.000 ; U68:F =10.189,P =0.000 ; U49A:F =21.134,P=0.000;RNU6:F=4.968,P=0.004).Expression of 5s rRNA showed the least variability across the developmental ages (F=2.099,P =0.107).In P17 mice of the OIR group,tortuous access vessels and non-perfusion area were found in the retinal section.Hematoxylin-eosin stain showed the neovascular sprout through across the inner limiting membrane.The relative expression of U70 exhibited the greatest variability and then were U49A,U68 and 5s rRNA in turn in P17 OIR mice,and significantly elevated expressions were found in U70,U49A,U68 and 5s rRNA genes between the OIR group and the normal control group (t =5.174,P =0.000;t =3.376,P =0.012;t =4.802,P =0.000 ;t=2.856,P=0.029).Expression of RNU6 showed the least variability between the two groups (t =2.104,P=0.065).As analyzed by GeNorm program,the stability for the five reference genes across developmental ages was 5s rRNA/RNU6＞ U70 ＞ U49A ＞ U68,and the optimal reference combination was 5s rRNA and RNU6.Whereas the stability for the OIR model was 5s rRNA/RNU6＞U68＞ U49A＞U70,and the optimal reference combination was 5s rRNA and RNU6.Conclusions Reference genes should be selected based on specific subjects and experimental conditions when qRT-PCR is used to analyze miRNA expression levels.In the OIR model,both developmental and hypoxic factors need to be considered.5s rRNA and RNU6 reference combination is the preferred option for the qRT-PCR analysis of ocular miRNA expression if available.

Aggressive posterior retinopathy of prematurity in infants ≥1500 g birth weight.

In this retrospective case series, we report the spectrum and outcomes of aggressive posterior retinopathy of prematurity (APROP) in infants ≥1500 g birth weight. Twenty‑nine eyes of 15 infants are included. All infants were referred from level I or II nurseries, received supplemental unmonitored oxygen for prolonged duration (>1 week) and had multiple systemic co‑morbidities. Of the 29 eyes, 10 (34.5%) had zone 1 and 19 (65.5%) had posterior zone 2 disease. Twenty‑five (86.2%) eyes had flat neovascularization and 4 (13.8%) eyes had brush like proliferation. We noticed large vascular loops in 10 (34.5%) eyes. After confluent laser photocoagulation, 22 (75.9%) eyes had a favorable outcome. The study concludes that APROP in heavier (≥1500 g birth weight) premature infants occurs mostly in posterior zone 2 with flat neovascularization and atypical features like large vascular loops. Supplemental unmonitored oxygen for prolonged duration and multiple systemic co‑morbidities could be a contributing factor.

Expression of transcriptional factor lslet-1 in retina with experimental retinal neovascularization induced by oxygen / 国际眼科杂志(Guoji Yanke Zazhi)

AlM:To evaluate the expression of transcriptional factor lslet-1 in retina in experimental retinal neovascularization induced by oxygen. METHODS: The murine retinal neovascularization were induced by hyperoxia exposure. The morphological observation of retinal neovascularization was performed using angiography by fluorescein dextran injection under the fluorescence microscope, and the new blood vessels were quantified after 5d in room air (17-day-old) by counting the vascular epithelial cell nuclei protruding into viteous cavity using HE stain. Realtime PCR and Western blot were used to examine retinal lslet-1 level in postnatal 7,12, 14,17 and 26d respectively. RESULTS: A lots of new blood vessels were demonstrated in the mouse retina in hyperoxic group by fluorescein angiography and histological method. Moreover, no significant difference was found in retinal lslet-1 level in postnatal 7d between hyperoxic group and control group, but was significantly higher in postnatal 12, 14 and 17d mice compared with control mice. However, mice at postnatal 26d, expression of lslet-1 in retina decreased to normal level. CONCLUSlON: ln processing mouse model of retinal neovascularization, sustained hypoxia retinal tissue induce retinal neovascularization by increas the expression of transcription factor lslet-1.

Effect of Curcumin in a Mouse Model of Oxygen-Induced Retinopathy

PURPOSE: To investigate the effect of curcumin, known to inhibit hypoxia-inducible factor-1, on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). METHODS: OIR was induced by exposing C57BL/6 mice on postnatal day 7 (P7) to 75% hyperoxia for 5 days, followed by 5 days in a room with normal oxygen level. Curcumin was administered intraperitoneally once a day for 5 days from P12 or intravitreally once on P13. Mice retinas on P17 were analyzed for retinal neovascularization, which was compared between curcumin-treated and control mice. RESULTS: After intraperitoneal and intravitreal administration of curcumin, qualitative assessment of retinal neovascularization of flat-mounted retina showed no significant difference compared to control retinas. Quantitative assessment of retinal neovascularization also showed no significant difference between curcumin-treated and control mice. CONCLUSIONS: Both intraperitoneal and intravitreal administration of curcumin did not reduce retinal neovascularization in an OIR mouse model. Further investigation including development of new formulations is required for the use of curcumin as an anti-angiogenic agent for retinal neovascularization.

Expression and signiifcance of thrombonspondin-1 in oxygen-induced retinopathy in mice / 中南大学学报(医学版)

Objective:To examine the expression and function of thrombonspondin-1 (TSP-1) in oxygen-induced retinopathy in new-born mice, and to investigate its role in retinal neovascularization. Methods:A total of 40 C57BL/6J newborn mice were divided equally into a model group (n=20)andanormalcontrolgroup(n=20).Miceinthemodelgroupwereexposedto75%oxygentoestablish the oxygen-induced retinopathy (OIR) model. On the 7th, 9th, and 11th day after the birth of mice, 5 mice were randomly selected each time from the 2 groups to examine the expression of TSP-1 mRNA with reverse transcription polymerase chain reaction (RT-PCR). After that, 5 mice were selected on the 11th day to observe the retinal neovascularization by fluorescein angiography retinal flatmount. Results:On the 11th day, fluorescein angiography retinal flatmount showed that the retinal blood vessels presented mean network distribution in the normal control group, while in the model group, a lot of dilatated areas in the retinal main vessels surrounded the optic disc. Meanwhile lots of new blood vessels were found surrounding the optic disc with irregular distribution but well distributed peripherial retinal small vessels, which was typical of early stage OIR. There was no signiifcant difference in the retinal TSP-1 mRNA level between the model group and the normal control group in the postnatal 7-day mice (P>0.05). Compared with the normal control group, the expression of TSP-1 mRNA in the model group was signiifcantly lower in postnatal 9-day and 11-day mice (P Conclusion:In the early stage of OIR model (retinal vascular growth and development stage), the expression of TSP-1 mRNA in the retinal tissue is gradually decreased, implying that TSP-1 (as a negative regulatory factor) may be involved in the formation of retinal neovascularization in the early stage.

Inhibitory effects of small interference RNA targeting vascular endothelial growth factor on oxygen-induced retinal neovascularization / 中华实验眼科杂志

Background Arresting the overexpression of vascular endothelial growth factor (VEGF) will be a new approach to the inhibition of neovascularization.RNA interference (RNAi) can inhibit the expression of specific gene,and its application in eye has little interference to other gene expression.Objective This study was to investigate the effect of small interference RNA (siRNA) targeting VEGF on the expression of VEGF and retinal neovascularization in oxygen-induced retinopathy (OIR) model.Methods psi-HITM/enhanced green fluorescent protein (EGFP)/VEGF siRNA was designed and prepared in vitro.Mouse endothelioma (EOMA) were cultured in DMEM without antibiotic and divided into 5 groups.The cells were incubated in DMEM only in the blank control group;while 1 μl of LipofectamineTM 2000 + psi-HITM/EGFP,1 μl LipofectamineTM 2000 + 40,50 or 60 nmol/L of psi-HITM/EGFP/VEGF siRNA was added into DMEM in the negative control group and siRNA groups,respectively.The expression of VEGF mRNA and protein was detected by real time PCR (RT-PCR) and Western blot.The optimal effective concentration of VEGF siRNA was assessed.OIR models were established in 48 7-day-old C57BL/6J mice by raising them at an oxygen concentration of (75±2) ％ for 5 days and then to normal air.The mice were randomized into the model group,null vector group and VEGF siRNA group.1 μl of a mixture of psi-HITM/EGFP or VEGF siRNA (60 nmol/L) and LipofectamineTM 2000 was intravitreally injected,respectively,in the null vector group and VEGF siRNA group.The normal mice were used as the normal control group.Expression of VEGF mRNA and protein in the mouse retinas was detected by RT-PCR and Western blot,respectively,and FITC-dextran stretched retinal preparation was examined to evaluate the neovascularization,and retinal sections were examined to quantify the number of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM).Results The in vitro transfection test showed that the expression of VEGF mRNA and protein in the EOMA cells was significantly different among the blank control group,negative control group and 40,50,60 nmol/L VEGF siRNA groups (F =148.890,P ＜ 0.001; F =306.960,P ＜ 0.001),and the expression of VEGF mRNA was lower in different concentrations of VEGF siRNA groups than that in the blank control group (t=73.950,119.890,156.480,all at P＜0.001).Also,the expression of VEGF protein was less in different concentrations of VEGF siRNA groups than that in the blank control group (t =15.452,23.344,42.119,all at P＜0.001).The optimal inhibitory concentration of VEGF siRNA was 60 nmol/L.In vivo study determined that compared to the model group and null vector group,the non-perfusion zones and neovascular net in the retina were decreased,and the number of vascular endothelial cell nuclei extending beyond the ILM was less in the VEGF siRNA group.The relative expression level of VEGF mRNA in the retinas was 1.23±0.18,4.02±0.16,3.98±0.19 and 1.98±0.12 in the normal control group,model group,null vector group and VEGF siRNA group,respectively,with a significant difference among them (F=369.780,P＜0.001),and the relative expression levels of VEGF mRNA in the model group and null vector group were higher than that in the normal control group (t=37.880,37.336,both P＜0.001),and the expression of VEGF mRNA in the VEGF siRNA group declined by 50.8％ (t=10.183,P＜0.001).The difference in the expression levels of VEGF protein also was assayed among the various groups (F=408.980,P＜0.001),and VEGF level in the retina was lowered by 68.0％ in the VEGF siRNA group compared to the model group (t =11.473,P＜0.001).However,VEGF level in the VEGF siRNA group remained at a high level in comparison with the normal control group (t =2.422,P＜0.001).Conclusions Intravitreal injection of VEGF siRNA can attenuate retinal neovascularization by effectively downregulate the expression VEGF mRNA and protein in the retina.

The effect of Delta-like ligand 4 monoclonal antibody on retinal neovascularization and vascular endothelial growth factor expression / 中华实验眼科杂志

Background Studieshowed thaDelta-like ligand 4 (Dll4) participatein the deveopmenof retinal celland angiogenesis.The Dll4-Notch pathway and vasculaendothelial growth facto(VEGF) are thoughto be critical mediatorof neovascularization undehypoxiconditions.The relationship between Dll4 and VEGF inovery cleaand furtheresearch ineeded.Objective Thistudy wato observe the inhibition of Dll4 on experimental retinal neovascularization and VEGF expression.MethodThe retinal neovascularization animal model wainduced by oxygen-induced retinopathy (OIR) in 5-day-old SPF SD ratby rearing the new postnatal ratwith the motherattogethein closed box with oxygen level a(80±2) ％ till 12-day-old.The ratwere then raised in normal aifo5 days.Aftethat,2.5μl (0.5 μg) of Dll4 monoclonal antibody wainjected into the mid-vitreoucavity in the righeye(Dll4 injected group) and PBwaused in the same way in the fellow eye(PBcontrol group) in the 12-day-old rats.Retinawere isolated in the 17-day-old rats,and retinal vasculamorphology waexamined by adenosine diphosphatease (ADPase) staining of retinal flatmounts,and the endotheliocyte nuclei above the internal limiting membrane were counted in the retinal tissue-slices.Reverse transcription PC(RT-PCR) waused to detecthe mRNexpression level of Dll4,VEGF,VEGF receptor-1 (VEGFR-1),VEGFR-2 and neuropilin-1 mRNin the retinas.Statistical analysiwaperformed by the paired t-test.The care and use of the animalcomplied with the Guidance Suggestion issued by the Ministry of Science and Technology of Chinin 2006.ResultThe Dll4 mRNexpression in the retin(Dll4 mRNA/β-actin mRNA) wa0.22± 0.06 and 0.98 ± 0.13 in the Dll4 injected group and the PBcontrol group,respectively,with statistically significandifference (=21.839,P =0.000).No significandifferencewere found in the expression of the VEGF mRNA,VEGFR-1 mRNand VEGFR-2 mRNin the retinabetween the two group(t=0.463,P=0.649;=1.687,P=0.109;=-1.674,P=0.111).Compared with the PBcontrol group,the expression of neuropilin-1 mRNwasignificantly elevated in the Dll4-injected group (0.73±0.08 vs.0.64±0.07) (t=-2.677,P=0.015).ADPase staining showed thathere were much more new blood vesselin the Dll4 injected group than those of the PBcontrol group.The numbeof nuclei structurally adjacento the vitreal side of the internal limiting membrane wa(63.6± 11.6)/slide in the Dll4 injected group,which wamore than thaof the PBcontrol group a(35.1±5.2)/slide (=-7.879,P =0.000).ConclusionDll4 playan essential role in the procesof pathological angiogenesiin the retina.Dll4 ithoughto be feedback regulatoof VEGFR,which participatein the procesof restraining pathological vasculogenesis.

Effect of Triamcinolone on Retinal Vessel-Related Factors in Oxygen-Induced Retinopathy Rats

PURPOSE: The present study investigated the effects of triamcinolone acetonide (TA) on retinal vessel-related factors and retinal vascular leakage in the retina of oxygen-induced retinopathy (OIR) rats. METHODS: Sprague-Dawley rats used for OIR were exposed to hyperoxia from postnatal day 2 to day 14, then returned to normoxia from day 15 to day 30 and compared with control rats. On postnatal day 16, 2 microl of TA (4 mg/kg) was injected into the vitreous of the right eye in control and OIR rats. The Evans blue method was used for evaluating vascular leakage and RT-PCR was performed for confirmation of mRNA expression. RESULTS: In OIR rats exposed to hyperoxia, retinal vascular permeability increased when returned to normoxia. After intravitreal injection of TA, vascular permeability was decreased in OIR rats, but no changes were found in control rats. In OIR rats, mRNAs of HIF-1alpha, VEGF, SDF-1 and ICAM-1 were more expressed and down-regulated after intravitreal injection of TA. Occludin mRNA level was decreased in the hypoxic condition and up-regulated after TA treatment. CONCLUSIONS: The present study suggests the ability of TA to inhibit retinal vascular leakage in OIR rats and a possibility that TA controls expression of the HIF-1, VEGF, SDF-1, ICAM-1 and Occludin, which may protect retinal vascular destruction from hypoxic conditions; further studies are necessary to confirm changes in protein levels.

Effect of Triamcinolone on Retinal Vessel-Related Factors in Oxygen-Induced Retinopathy Rats

PURPOSE: The present study investigated the effects of triamcinolone acetonide (TA) on retinal vessel-related factors and retinal vascular leakage in the retina of oxygen-induced retinopathy (OIR) rats. METHODS: Sprague-Dawley rats used for OIR were exposed to hyperoxia from postnatal day 2 to day 14, then returned to normoxia from day 15 to day 30 and compared with control rats. On postnatal day 16, 2 microl of TA (4 mg/kg) was injected into the vitreous of the right eye in control and OIR rats. The Evans blue method was used for evaluating vascular leakage and RT-PCR was performed for confirmation of mRNA expression. RESULTS: In OIR rats exposed to hyperoxia, retinal vascular permeability increased when returned to normoxia. After intravitreal injection of TA, vascular permeability was decreased in OIR rats, but no changes were found in control rats. In OIR rats, mRNAs of HIF-1alpha, VEGF, SDF-1 and ICAM-1 were more expressed and down-regulated after intravitreal injection of TA. Occludin mRNA level was decreased in the hypoxic condition and up-regulated after TA treatment. CONCLUSIONS: The present study suggests the ability of TA to inhibit retinal vascular leakage in OIR rats and a possibility that TA controls expression of the HIF-1, VEGF, SDF-1, ICAM-1 and Occludin, which may protect retinal vascular destruction from hypoxic conditions; further studies are necessary to confirm changes in protein levels.