Oxygen-induced Retinopathy Group Research Articles

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

To investigate whether the complement system is involved in a murine model of oxygen-induced retinopathy (OIR). 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. 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 C1qb and C4b 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. Our data suggest that the complement system CP is activated during the pathogenesis of murine model of OIR.

Optimization of reference genes for microRNA expression analysis in a mouse model of oxygen-induced retinopathy

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. Key words: Oxygen-induced retinopathy; Quantitative real-time PCR; Reference gene; MicroRNA; GeNorm

MicroRNA expression profiling in a mouse model of oxygen-induced retinopathy

Objective To study morphological characteristics and microRNA (miR) expression profiling in a mouse model of oxygen-induced retinopathy (OIR). Methods Healthy C57BL/6J female mice and pups were randomly divided into normal and OIR group at postnatal day 7 (P7). The normal group was raised in a conventional cage and exposed to room air for 10 days. The OIR group was raised in a sealed chamber and exposed to (75±2)% oxygen. The moms were alternated between the two groups every day to promote their survival under hyperoxia. The OIR group was returned to the room air at P12. At P17, mice from either group were retro-orbitally injected with high molecular weight fluorescein isothiocyanate-dextran (FITC-dextran), the eye balls were fixed in 4% paraformaldehyde, and the retinal whole mounts were prepared. The retinal vessels labeled with FITC-dextran were observed under a fluorescence microscope; the eye balls were also processed for paraffin sections and Hematoxylin and Eosin (H&E) staining. The cell nucleus in the newly-formed vessels beyond the inner limiting membrane was quantified. The miR was extracted from the eyes, reverse transcribed, and subjected to a customized miR array analysis. The real-time PCR was preformed to verify the results of the miR array. Results Retinal whole mounts labeled with FITC-dextran showed that the peripheral retinal microvessels in the OIR group were tortuous, disorganized with neovascular buds, and the avascular area was prominent in central retina. In contrast, the vessels were smooth, organized, and evenly distributed in the retinas of normal group. The percentage of avascular area in total retina area in OIR group (25.81±2.12)% was 4-fold that in normal group (6.57±3.6)% (P<0.01, normal group vs OIR group). HE 4 miRs showed more than 3-fold expression changes, 3 were down-regulated and 1 was up-regulated. The expression of the 4 miRs was verified by real-time PCR. The expression trends of miR-3078, miR-140, miR-29b and miR-29c were consistent with those revealed by the miR array. MiR-3078 was significantly up-regulated (t=-2.380, P<0.05. normal group vs OIR group), and the other 3 miRs were significantly down-regulated (t=2.638, 2.323, 2.415, P<0.05. normal group vs OIR group). Conclusions The OIR mouse model has been established in our study. Differential expression of the microRNAs, including miR-3078, 140, 29b and 29c, was detected in normal and OIR mouse retinas. These miR expression changes may be associated with retinal neovascularization. These results would provide the new leads for further studying pathogenic mechanisms and therapeutic targets for neovascular retinopathy. Key words: Retinal diseases/etiology; MicroRNAs; Animal experimentation

Erythropoietin promotes retinal angiogenesis in a mouse model.

This study aimed to investigate the effect and potential mechanisms of exogenous administration of recombinant human erythropoietin (rhEPO) on retinal angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). Postnatal day 7 (P7) mice (n=132) were randomly assigned to one of six groups: Control group (n=22), OIR group (n=22), OIR + vehicle control group (n=22), OIR + rhEPO 10 IU group (n=22), OIR + rhEPO 50 IU group (n=22), and OIR + rhEPO 100 IU group (n=22). OIR was induced by exposing mice to 75±2% O2 for five days, followed by exposure to room air for a further five days. Animals in groups 3-6 (the OIR + vehicle control group and OIR + rhEPO 10 IU, 50 IU, and 100 IU groups) received an intraperitoneal injection of saline, or rhEPO 10 IU, 50 IU and 100 IU, respectively, which were administered daily from P7-P12. Immunofluorescent and hematoxylin-eosin staining were used to detect retinal neovascularization (RNV) in retinal whole mounts. Quantitative polymerase chain reaction and western blot analysis were used to detect the expression levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS). RNV occurred in the OIR groups and was accompanied by dilated, twisted and occluded blood vessels. rhEPO treatment resulted in an increase in the number of newly formed and severely dilated vessels. rhEPO increased RNV in a dose-dependent manner, which was accompanied by an increase in the messenger RNA and protein expression of VEGF, eNOS and nNOS. Thus, exogenous use of rhEPO promotes the RNV in a mouse model of OIR and is accompanied by increased expression levels of VEGF, eNOS and nNOS.

Inhibitory effects of 5-lipoxygenase on oxygen-induced retinal neovascularization in mice

Objective To investigate the inhibitory effects of 5-lipoxygenase (5-LOX) on oxygeninduced retinal neovascularization in mice and to explore its possible mechanisms.Methods 7-day-old C57BL/6J mice were randomly divided into normal group,oxygen induced retinopathy (OIR) model group,large-dose group,small-dose group and control group with 12 mice in each group.The mice with their mothers were kept in (75±2)％ of oxygen environment for 5 days and then returned to normoxia for 5 days to establish the OIR model except for normal group.From postnatal day 12 to 17,the large-dose group and small-dose group received intravitreous injection of 5-LOX at dose of 100 mg/kg and 50 mg/kg respectively,while the control group received the same volume of 1 ％ dimethyl sulfoxide.The mice in the OIR group received no treatment.The number of endothelium cell nuclei breaking through the inner limiting membrane (ILM) was counted on hematoxylin and eosin-stained retinal section.The mRNA expression of 5 LOX,vascular endothelial growth factor (VEGF) a,VEGF receptor 2 (VEGFR-2) on retinal tissue were detected by reverse transcription polymerase chain reaction (RT-PCR).The protein expression of 5 LOX,VEGF a,VEGFR-2 and phosphorylation extracellular signal-regulated kinase (P-ERK) 1/2 on retinal tissue were detected by Western blot.Results The number of vascular cell nuclei breaking through the ILM in the large-dose group and small dose group decreased significantly compared with the OIR group and control group (F=73.390,P＜0.05).The mRNA expression and protein expression of 5-LOX,VEGFa,VEGFR 2 on retinal tissue were decreased significantly in the large-dose group and small dose group as compared with the OIR group and control group (F =92.668,P＜ 0.05).The difference of VEGFR-2 protein expression between large-dose group and small dose group was not significant (F=2.118,P＞0.05).The differences of 5-LOX,VEGF a,P-ERK 1/2 protein expression between large-dose group and small-dose group were significant (F=86.490,165.128,139.424; P＜0.05).Conclusion Hypoxia may induce 5 LOX expression in the retina.Retinal neovascularization was significantly inhibited by selective inhibition of 5 LOX. Key words: Retinal neovascularization/prevention & control; Arachidonate 5-lipoxygenase; Extracellular signal-regulated MAP kinases; Animal experimentation

Lentivirus mediated small interference RNA targeting cyclic adenosine monophosphate responsive element binding protein 1 suppress retinal neovascularization in mice

Objective To observe the inhibitory effect of lentivirus mediated small interference RNA (siRNA) targeting cyclic adenosine monophosphate responsive element binding protein 1 (CREB1) on retinal neovascularization (RNV) in mice.Methods CREB1 siRNA construct was created,screened and packaged to produce CREB1 RNAi-lentivirus.One hundred and forty (5-day-old) C57BL/6J mice were randomly divided into 4 groups including normal group,oxygen induced retinopathy (OIR) group,empty vector group and CREB1 therapy group with 35 mice in each group.Mice in the normal group were kept in normal room air,while in the other three groups retinal neovascularization was induced by hypoxia on postnatal day 7 (P7).The mice in the OIR group were not treated.The mice in the vector group received intravitreal injection of lentivirus-green fluorescent protein (lenti-GFP,1 μl),and the CREB1 therapy group received CREB1 RNAi-lentivirus (1 μl) on P5.The proliferative neovascular response was quantified by counting the vascular cell nuclei extending breaking through the internal limiting membrane (ILM) and fluorescent angiography.The areas of RNV and non-perfusion region were calculated.The expression of CREB1,phosphorylated-CREB1 (P-CREB1) and vascular endothelial growth factor (VEGF)-A levels,Akt and phosphoinositide 3-kinases (PI3K) in retinas were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot.Results The number of vascular cell nuclei breaking through the ILM of the OIR group and the empty vector group increased significantly compared with the normal group (P＜0.05),while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group(P＜0.05).The area of RNV and non-perfusion region of the OIR group and the empty vector group increased significantly compared with the normal group,while obviously decreased in the CREB1 therapy group compared with the OIR group and the empty vector group.The difference of area of RNV and non-perfusion region among 4 groups were significant (F=67.220,110.090; P＜0.05).The mRNA expression of CREB1 and protein expression of P-CREB1,the mRNA and protein expression of VEGF-A,Akt,PI3K in the retina were increased significantly in the OIR group and the empty vector group as compared with the normal group,while decreased significantly in the CREB1 therapy group as compared with the OIR group and the empty vector group.The difference of mRNA expression of CREB1,VEGF-A,Akt,PI3K in the retina among 4 groups were significant (F=6.087,5.464,6.191,8.627; P＜0.05).The protein expression of P-CREB1,VEGF-A,Akt,PI3K in the retina among 4 groups were significant (F=162.944,13.861,19.710,22.827; P＜0.05).Conclusion RNV in the mice is significantly inhibited by intravitreal injection of lentivirus-mediated CREB1 down-regulation. Key words: Retinal neovascularization/prevention & control; Gene transfer techniques; Transfection; Animal experimentation

The effect of semaphorin 3A in the process of apoptosis in oxygen induced retinopathy in rats

To observe the change of semaphorin 3A (SEMA3A) expression in the retina of oxygen induced retinopathy (OIR) in rats and to investigate its influence on retinal degeneration in OIR. Experimental study. Forty-eight newborn pups were randomly classified into four groups and only one eye was examined in each pup. Thirty-six pups was induced into OIR model through aspirating 50% and 10% oxygen every 24 hours alternatively while the control group (12 pups) was raised under normal atmosphere. OIR+SEMA3Aab group accepted intravitreous injection of rabbit anti-rat SEMA3A antibody (Abcam Co.LTD, 40 mg/L, 2 µl) while OIR+IgG group was injected the same amount of non-active rabbit IgG at postnatal day 7. And the OIR group had no injections. All the pups were executed at postnatal day 18. Histological changes of retinas were examined through HE stain while Immunoflurecence staining and TUNEL procedure were used to detect focal expression of SEMA3A and apoptosis respectively. Total tissue protein was extracted and the expression of SEMA3A and Caspase-3 p17subunit were examined by Western blot. The data including retinal thickness, cell counting of retinal ganglion cells layers (RGCL) , endothelia outside inner limiting membrane, retinal apoptosis index (AI), the relative expression of SEMA3A and Caspase3 p17 subunit were analyzed by One-way ANOVA and followed LSD-t test to compare group differences. There were significant differences of retinal thickness, cell counting of RGCL and endothelia outside inner limiting membrane among each groups respectively (F = 13.222, F = 22.537, F = 14.478;P < 0.01) . The retinal thickness was (117.07 ± 8.13) µm in normal control group, (70.93 ± 5.68) µm in OIR group, (91.28 ± 4.58) µm in OIR+SEMA3Aab group, and (67.27 ± 10.15) µm in OIR+IgG group; the cell counting of RGCL was 42.7 ± 3.6 in normal control group, 24.3 ± 3.1 in OIR group, 35.0 ± 6.2 in OIR+SEMA3Aab group, and 22.8 ± 4.3 in OIR+IgG group while the endothelia outside inner limiting membrane was 1.0 ± 0.3 in normal control group, 14.2 ± 3.2 in OIR group, 9.6 ± 1.1 in OIR+SEMA3Aab group, and 10.8 ± 1.6 in OIR+IgG group. The retinal thickness and cell counting of RGCL in OIR+SEMA3Aab group were significantly lower than those in the normal control group (P < 0.01) , but were apparently higher than the data of OIR group and OIR+IgG group respectively (P < 0.01) . There were no more endothelia on the vitreal side of the inner limiting membrane in OIR+SEMA3Aab group than OIR group or OIR+IgG group (P > 0.05) although these three groups had much more endothelia than it in the normal control group (P < 0.01). The retinal AI detected by TUNEL staining was 27.67 ± 2.51 in normal control, 58.33 ± 8.50 in OIR group, 37.33 ± 5.03 in OIR+SEMA3Aab group and 61.67 ± 6.65 in OIR+IgG group. There was significant difference of the retinal AI among the four groups (F = 19.250, P = 0.001) . Apoptotic cells were significantly reduced in OIR+SEMA3Aab group compared with OIR + IgG group or OIR group (P < 0.01). The difference of SEMA3A protein expression among the groups detected by Western blot was significant (F = 38.59, P = 0.000) . SEMA3A expression in OIR group was 0.97 ± 0.05, which was significantly upregulated compared with the normal control group (0.64 ± 0.03) ( P < 0.01) . And its expression was successfully neutralized in OIR + SEMA3Aab group (0.41 ± 0.02) with comparison with OIR+IgG group (1.03 ± 0.15) through Western blot (P < 0.01) . SEMA3A was detected apparently in the photoreceptors layer in the normal control while its fluorescence was stronger and much more scattered in the whole retina. However, anti-SEMA3Aab intravitreous injection successfully reduced its fluorescence compared with the IgG injection. The cleavage of Caspase-3 was not detected in the normal control while the relative expression of Caspase-3 p17 subunit was significantly different in the other three groups (F = 304.619, P < 0.01). OIR + SEMA3Aab group had much less Caspase-3 p17 subunit (0.12 ± 0.01) than OIR group (0.30 ± 0.02) or OIR +IgG group (0.27 ± 0.02) (P < 0.01) . The over regulation of SEMA3A probably enhances the aggravation of apoptosis in OIR rats. Inhibition of SEMA3A is helpful to protect neuroretina.

Adenosine A2A receptor and retinal pathological neovascularization in mice

Objective To investigate the role of adenosine A2A receptor plays in retinal pathological neovascularization in mice.Methods A total of 202 mice were divided into room-air group (n =66) and oxygen induced retinopathy (OIR) group (n=136).Among room-air group,there were 18 A2A knock-out (KO) mice (KO subgroup) and 24 C57BL/6 mice as wide type (wide type subgroup).OIR group were divided into OIR control subgroup (n =48),A2A-OIR subgroup (n =24) and Caffeine-OIR subgroup (n =64).The retinal neovascularization of OIR group was induced by oxygen.The pathological neovascularization was determined by retinal sections.Fluorescent quantitative polymerase chain reaction (PCR) was used to measure the mRNA expression of A2A and vascular endothelial growth factor (VEGF).0.1,0.3,1.0 g/L Caffeine was dissolve in drinking water of lactating females in Caffeine-OIR subgroup,non-perfusion areas of retina in mice at the age of 0-17,0-7,7-17,7-12,and 12-17 days were analyzed in different dosage and when the dosage as 1.0 g/L.Results Compared with OIR control subgroup,the retinal non-perfusion areas and the numbers of endothelial cell nuclei breaking through the internal limiting membrane in A2A-OIR subgroup were reduced significantly (t =7.694,7.747; P ＜0.001).Compared with wide type subgroup,the level of A2A and VEGF mRNA in OIR control subgroup increased significantly (t=4.036,2.230; P＜0.05).Compared with OIR control subgroup,the level of VEGF mRNA in A2A-OIR subgroup decreased significantly (t=3.122,P＜0.01).Compared with OIR control subgroup,the retinal non perfusion areas in mice at the dosage of 0.1 and 1.0 g/L (t=2.397,4.533) and at the age of 0-17,0-7 days when the dosage as 1.0 g/L (t=4.070,2.399) were reduced significantly (P＜0.05).Conclusions The expression of adenosine A2A receptor increases in oxygeninduced retinal pathological neovascularization..Adenosine A2A receptor may regulate the expression of VEGF.A2A receptor inactivation can inhibit oxygen-induced retinal pathological neovascularization. Key words: Retinal neovascularization/physiopathology; Receptor, adenosine A2A; Caffeine

Differential expression of claudin-23 in retina of oxygen-induced retinopathy mice

Background Researches showed that some claudin subtypes are closely related to the integrity of blood retina barrier and participate in pathological angiogenesis.Ciaudin-23 is a novel discovered claudin subtype.Understanding its expression in the retina and its relationship with retinal vascular lesion is very important for the targeting treatment of retinal vascular diseases.Objective The aim of this study was to investigate the differential expression of claudin-23 in the retina of mice with oxygen-induced retinopathy(OIR).Methods OIR mice models were established by putting postnatal day 7 (P7)SPF C57BL/6J mice in the enclosed container with oxygen concentration of 75％ ±3％ together with mother mice for 5 days.The age-matched normal mice were used as controls.Fluorescein isothiocyanate dextran (FITC-dextran) was retro-orbitally injected to mice to confirm the models by whole-mount retina.Expressions of claudin-23 mRNA and protein in P12,P17 and P25 mice retinas were examined using real-time PCR and Western blot.lmmunofluorescent staining of frozen section was used to identify the expression and location of claudin-23 in the normal and OIR mice.Results Tortuous and engorged retinal vessels were found in P17 mice retina of the OIR group.Nonperfusion zones,abnormal capillary network and lots of pathological neovascular tufts were observed on the whole-mount retinas.The level of claudin-23 mRNA was slowly increased with the aging,reaching 2.3 fold in P25 normal mice more than P7 mice.However,that in OIR mice demonstrated 12.5-fold elevation from P7 to P25.There were no significant change in the mRNA and protein levels of claudin-23 in P7 mice retina between the normal group and OIR group(P＞0.05).But the mRNA level of claudin-23 in P12,P17 and P25 mice retinas was significantly higher in the OIR group than that in normal group(P＜0.05).The protein level of claudin-23 in P25 mice retina was significantly higher in the OIR group than that in normal group(P＜0.05).Claudin23 expressed mainly in inner nuclear cell layer and retinal ganglion cell layer of the normal mice,but claudin-23 expression was obviously increased in OIR mice.No colocalization of claudin-23 and Isolectin B4 was found in the retina in both normal and OIR mice by double immunofluorescent staining.Conclusions Claudin-23 expression upregulates in the retina of OIR mice.Claudin-23 locates in retinal neurons but not vascular endothelial cells. Key words: Claudin-23; Retina; Oxygen-induced retinopathy

Expression of integrin linked kinase in retina with oxygen-induced retinopathy

Background It has been well-known that integrin linked kinase(ILK) plays an important role in the pathogenesis of neorascularization.But there are few researches to elucidate the relationship between ILK and retinal nevascularization. Objective This study was to explore the expression and significance of ILK on retinal neovascularization and new vessels regression in the oxygen-induced retinopathy(OIR) mouse model. Methods One hundred and twenty-eight 7-day-old C57BL/6J mice were randomly divided into the normal group and model group.The mice were rose in(75±2)％ O2 environment with mother mice together for 5 days and then in normal environment for 5 days to establish the OIR models.The mice in normal group were rose in the normal environment for 21 days.The 17-day-old mice were sacrificed and retinal sections were prepared for histopathological examination and the numbers of the cellular nuclei of vascular endothelium breaking retinal internal limiting membrane were calculated.Retinal sections and sheeting were prepared in 12,14,17 and 21 -day-old mice to examine the formation and regression of new blood vessel using ADP histochemistry staining,and then immunochemistry,real-time PCR and Western-blotting were used to detect the expressions of ILK and its mRNA in retina. Results The numbers of the cellular nuclei of vascular endothelium breaking retinal internal limiting membrane were (45.64 ± 12.17 )in OIR group,and those of the normal group were( 0.35±0.14 )with a significant difference between two groups (t =22.85,P＜0.05).Retinal new blood vessel appeared in 14-day mice,and peaked in 17-day mice and then regression in 21-day mice.ILK protein was expressed mainly in retinal ganglion cell layer,inner nuclear layer,inner plexiform layer and photoreceptor layer.Real-time PCR showed that ILK mRNA expressing levels in retina in model group were( 1.00±0.22),(1.85±0.17),(1.58±0.43) and(1.53±0.36) respectively in 12-,14-,17- and 21-day mice.Westernblotting determined that the A value of the ILK/β-actin was increased in 12-,14-,17-day mice and decreased in 21-day mice,and the A values were significantly higher in model group than the control group in various aged mice ( t =2.97,P＜0.05 ;t =11.88,P＜0.01 ;t =16.84,P＜0.01 ;t =13.00,P＜0.01 ). Conclusions These results indicate a space-time corresponding relation between the expression of ILK and retinal neovascularization.The obvious positive expression of ILK may be highly correlated with retinal neovascularization. Key words: Integrin linked kinase; Oxygen; Retinopathy; Retinal neovascularization

Expression of pro- and anti-angiogenic isoforms of VEGF in the mouse model of oxygen-induced retinopathy

Retinopathy of prematurity (ROP) has become one of the leading causes of blindness and visual loss in children over the last half century. Vascular Endothelial growth Factor (VEGF-A) is the principal stimulator of angiogenesis. Recently, it has been identified that VEGF was differentially spliced from Exons 8 to Exons 8a and 8b to form two families: the pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxxb family. This alternate splicing produced VEGFxxxb proteins of the same length as VEGFxxx family, but with different C terminal amino acid sequences. VEGFxxxb appeared to be able to inhibit VEGFxxx-dependent angiogenesis. In our study, we investigated the protein expression course of VEGFxxx and VEGFxxxb by Western-blot in a mouse model of Oxygen-induced Retinopathy (OIR) from postnatal day 1 (P1) to postnatal day 21 (P21). We also analyzed the relative protein expression level of VEGF165b isoform in the OIR mouse model. We found that both VEGFxxx and VEGFxxxb were present in the mouse retina, among which, VEGF164 and VEGF165b appeared to be predominant VEGFxxx and VEGFxxxb isoforms respectively in the mouse retina. We also found that the two family had different expression pattern correlated with neovascularization development and that the relative expression level of VEGF165b isoform switched during the neovascularization development in the OIR mouse model. In OIR group, the protein level of total VEGF isoforms (a mix of VEGF164 and VEGF165b, detected by pan-VEGF antibody) continuously increased and peaked at P17 while VEGF165b continuously decreased from P9 which was well related with the vessel obliteration and neovascularization development in the mouse model of OIR. The neovascularization development correlates with an increase of total VEGF isoforms and the decrease of VEGF165b, indicating that there is a pro-angiogenic VEGF shift. Therefore, anti-angiogenic therapy that could alter the ratio of VEGFxxxb/VEGFxxx may be more effective.

Expression of stromal cell-derived factor-1 in mouse retina with oxygen-induced retinopathy

Background The development of retinopathy of prematurity(ROP) is associated with many regulatory cytokines related to neovascularization;however,the retinal expression and regulated mechanism of stromal cell-derived factor-1 (SDF-1) in mouse model of oxygen-induced retinopathy (OIR) remain uncertain.Objective This study was to investigate the expression of SDF-1 in retina of mouse model of OIR.Methods Forty 7-day-old C57BL/6J mice were divided into OIR group and control group.In OIR group,20 mice were exposed to 75% oxygen for 5 days and then to room air for 5 days.In control group,20 mice were raised in room air.The expression of SDF-1 in retina of mice was studied by immunochemistry and quantified by real time reverse transcriptase polymerase chain reaction (RT-PCR).Results The positive immunohistochemical staining for SDF-1 was found mainly locating at the ganglion cell layer in 12-day-old mice of OIR group;the stronger positive immunohistochemical staining for SDF-1 was noted mainly locating at the ganglion cell layer,vascular endothelial cells of inner retina,neovascular endothelial cells in 17-day-old mice of OIR group;the delicate positive immunohistochemical staining for SDF-1 was both found mainly locating at the inner retina and being around the retinal vascular in 12-day-old mice of control group and 17-day-old mice of control group.The expression of SDF-1 mRNA in 17-day-old mice of OIR group was higher than that of 12-day-old mice of OIR group (t=8.072,P＜0.05)and 17-day-old mice of control group(t=10.026,P＜0.05),respectively.The expression of SDF-1 mRNA in 12-day-old mice of OIR group was lower than that of 12-day-old mice of control group (t=4.336,P＜0.05).Conclusion SDF-1 might improve the onset of retinal neovascularization of OIR. Key words: Stromal cell-derived factor-1; Oxygen-induced retinopathy; Disease models/mouse; Retinal neovascularization

Experimental study of (Pro)renin receptor siRNA inhibiting retinal neovascularization

To observe the inhibit effect of (pro)renin receptor siRNA on mice retinal neovascularization, and investigate its possible mechanism. Experimental study. 72 new born C57BL/6J mouse were randomly divided into six groups: group A to group E were exposed to hyperoxia, and then returned to normoxia to induce retinal neovascularization. Group A were treated with PRR siRNA plasmid, group B with control plasmid, group C with PRR siRNA and Losartan, and group D with Losartan. Group E were not treated. Group F was control group. Mouse were sacrificed at postnatal day 17, retinal perfusion stretched preparation and HE dyeing method were used to observe the status of retinal neovascularization. PRR expression and the activation of extracellular regulated protein kinases 1/2 (ERK1/2) were detected by Western Blot. And Real Time PCR was used to detect the expression of transforming growth factor-β1 (TGF-β1) in group A, B, E and F. Analysis of one way variance (LSD) was used and statistical difference was considered significant at a P value less than 0.05. The mouse treated with PRR siRNA, Losartan and combined therapy could significantly reduce retina neovascularization and vessel leakage compared with oxygen-induced retinopathy group and control plasmid group. Average counts of vascular endothelial cells which break through the inner limiting membrane performed at postnatal day 17 in PRR siRNA group (4.47 ± 1.96), Losartan group (5.94 ± 2.54) and combined therapy group (4.49 ± 2.53) were significantly lower than oxygen-induced retinopathy group (32.73 ± 6.38) (P < 0.05) and control plasmid group (21.04 ± 5.39). Western Blot showed that PRR protein express in hyperoxia induced group was significantly higher than normal (P = 0.007). After treated with PRR siRNA or combined therapy, the expression of PRR protein was significantly lower than hyperoxia induced group (P < 0.05). There are no significantly differences between control plasmid group, Losartan group and hyperoxia induced group (P > 0.05). The activated ERK1/2 lever in hyperoxia group was significantly higher than normal (P = 0.003). After treated with PRRsiRNA, Losartan or combined therapy, activated ERK1/2 lever was significantly lower than hyperoxia induced group (P < 0.05). And the effect of PRR siRNA group and combined therapy group seems more obviously, compared with Losartan group, the difference was significantly (P < 0.05). Real Time PCR showed that the lever of TGF-β1 in hyperoxia group was significantly higher than normal (P = 0.001). After treated with PRR siRNA, the TGF-β1 was significantly reduced (P = 0.004), and there was no significantly difference between control plasma group and hyperoxia induced group (P = 0.222). PRR combined with prorenin or renin could activate ERK1/2 signal transduction passageway, and promote cell proliferation, differentiation and migration, thus promote retinal neovascularization. PRR siRNA could obviously reduce PRR expression, inhibit ERK1/2 signal transduction passageway activation, and diminish retinal neovascularization.

Expression of FLT4 in hypoxia-induced neovascular models in vitro and in vivo.

Toinvestigatetheexpression of FLT4in retina with oxygen induced retinopathy (OIR) and in brain endothelial cell lines (bEnd3) under hypoxia conditions in mice. Fifty-two one-week-old C57BL/6J mice were divided into control group and hypoxia group. The mice of hypoxia group were exposed to 75% oxygen for 5 days and then returned to the room air to induce retinal neovascularization. Mice in control group were raised in the environment of room air at the same time. The expressions of FLT4 mRNA and protein were checked with RT-PCR and Western Blot analysis at postnatal day 14, 17 and 21 (P14, P17 and P21) respectively. 125mmol/L CoCl(2) were added to the culture medium of bEnd3 cell, proteins were extracted in 12, 24, 48 and 72 hours and FLT4 levels were examined by Western Blot analysis. The mRNAand protein level ofFLT4expressedinP14 and P17 OIR mice retina statistically up-regulated as compared with those in control group, but there was no statistical difference between OIR group and control group at P21. FLT4 levels increased significantly in 12, 24 and 48 hours hypoxia intervened bEnd3 cells, its levels in 72 hours increased mildly but showed no significance. FLT4 levels increase in OIR mice retinas and bEnd3 cells in hypoxia. It may play an important role in endothelial cells proliferation in hypoxia and retinal neovascularization in OIR mice.

Inhibitory effects of gene transfer of eanstatin on retinal neovascularization

Objective To observe the inhibitory effects of gene transfer of canstatin on retinal neovascularization in mice. Methods Fifty-six 7-day-old C57BL/6J mice were randomly divided into control group, oxygen-induced retinopathy (OIR) group, empty vector group and treated group, 14 mices in each group. Except for the control group, the mice in the other groups were exposed to (75±2)% oxygen for 5 days and then back to the normal air to establish the model of OIR. On postnatal 12 day, the treated group was received intravitreal injection of canstatin pCMV-HA, while the empty vector group was received the same volume of empty plasmid. The changes of retinal vessels were observed by Evans blue angiography on postnatal 17 day. With parafin section which stained by hematoxylin and eosin, then the number of endotheliocyte nuclei breaking throuhgh the internal limiting membrane(ILM) was observed and counted by optical microscope. Results Retinal blood vessels distributed regularly in treated group compared with OIR group and empty vector group. The differences of the number of endotheliocyte nuclei breaking throuhgh ILM in treated group was significant compared with the other two groups(F= 39. 006, P< 0. 001).Conclusion The canstatin pCMV-HA can effectively inhibit the retinal neovascularization in OIR. Key words: Retinal neovascularization/prevention & control; Cytokines; Gene transfer techniques; Animal experimentation

Triamcinolone Reduces Neovascularization, Capillary Density and IGF-1 Receptor Phosphorylation in a Model of Oxygen-Induced Retinopathy

To study the effects of intravitreous triamcinolone acetonide (TA) on neovascularization (NV), capillary density, and retinal endothelial cell (REC) viability in a model of oxygen-induced retinopathy (OIR). Newborn rats exposed to OIR underwent intravitreous injections (right eye) at day 14 to achieve intravitreous concentrations of: dexamethasone (DEX) (0.3 mg/mL), triamcinolone (TA; 0.4-4 mg/mL), or PBS. Animals were removed to room air and at day 18, retinal flatmounts were assayed for clock hours of NV, percent peripheral avascular retina, capillary density, apoptosis, and VEGF protein. At day 15, retinas were assayed for insulin-like growth factor (IGF)-1 receptor phosphorylation (IGF-1Rphos). Human RECs exposed to TA were assayed for trypan blue exclusion or activated caspase-3. TA but not DEX or PBS reduced NV (ANOVA, P < 0.001), capillary density (ANOVA, P < 0.001), and systemic weight gain (ANOVA, P = 0.002). VEGF protein was not different between TA- and PBS-injected or noninjected groups. Apoptosis was not increased in vivo or in vitro between groups, but there was a dose-dependent toxic effect of TA on cultured RECs (P < 0.001). At day 15, retinas from the 4 mg/mL TA-injected OIR group had a trend toward reduced IGF-1Rphos compared with room air-raised PBS- or non-injected OIR groups. TA caused dose-dependent reductions in NV, retinal vascularization, and systemic weight gain associated with a reduction in IGF-1Rphos. Long-term studies are needed to assess TA toxicity in vivo. TA doses should be carefully considered before administering the drug in diseases with ongoing retinal vascular development, such as retinopathy of prematurity.