Oxygen-induced Retinopathy Control Research Articles

Proteomics Analysis of R-Ras Deficiency in Oxygen Induced Retinopathy.

Small GTPase R-Ras regulates vascular permeability in angiogenesis. In the eye, abnormal angiogenesis and hyperpermeability are the leading causes of vision loss in several ischemic retinal diseases such as proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP). Oxygen-induced retinopathy (OIR) is the most widely used experimental model for these ischemic retinopathies. To shed more light on how the R-Ras regulates vascular permeability in pathological angiogenesis, we performed a comprehensive (>2900 proteins) characterization of OIR in R-Ras knockout (KO) and wild-type (WT) mice by sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics. OIR and age-matched normoxic control retinas were collected at P13, P17, and P42 from R-Ras KO and WT mice and were subjected to SWATH-MS and data analysis. The most significant difference between the R-Ras KO and WT retinas was an accumulation of plasma proteins. The pathological vascular hyperpermeability during OIR in the R-Ras KO retina took place very early, P13. This led to simultaneous hypoxic cell injury/death (ferroptosis), glycolytic metabolism as well compensatory mechanisms to counter the pathological leakage from angiogenic blood vessels in the OIR retina of R-Ras deficient mice.

Antiproliferative and Mitochondrial Protective Effects of Apigenin in an Oxygen-Induced Retinopathy In Vivo Mouse Model.

Purpose: To investigate the effects of a common dietary flavonoid apigenin on retinal endothelial cell proliferation, retinal morphological structure, and apoptotic cell death in an oxygen-induced retinopathy (OIR) mouse model to evaluate the possibility of the use of apigenin in the treatment of ocular neovascular diseases (ONDs). Methods: Ninety-six newborn C57BL/6J mice were included. Eight groups were randomized, each including 12 mice. Two negative control groups were kept in room air: the first without any injection and the second received intravitreal (IV) dimethyl sulfoxide (DMSO), which is the solvent we used. The OIR groups were exposed to 75% ± 2% oxygen from postnatal days (PD) 7 to 12. On PD 12, the mice were randomly assigned to 6 groups: 2 OIR control groups (1 received no injection, 1 received IV-DMSO), 2 IV-apigenin groups (10 and 20 μg/mL), and 2 intraperitoneal (IP)-apigenin groups (10 and 20 mg/kg). We quantified retinal endothelial cell proliferation by counting neovascular tufts in cross-sections and examined histological and ultrastructural changes through light and electron microscopy. We evaluated apoptosis by terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL). Results: We detected a significant increase in endothelial cell proliferation in the OIR groups. Groups receiving apigenin, both IP and IV, had significant decreases in endothelial cells, atypical mitochondrion count, and apoptotic cells compared with the groups receiving no injections. None of the apigenin-injected groups revealed cystic degeneration or cell loss. Conclusions: Apigenin suppresses neovascularization, has antiapoptotic and antioxidative effects in an OIR mouse model, and can be considered a promising agent for treating OND. Clinical trial (Project number: DA15/19).

Effect of Autophagy Modulators on Vascular, Glial, and Neuronal Alterations in the Oxygen-Induced Retinopathy Mouse Model.

Hypoxia is one of the main insults in proliferative retinopathies, leading to neovascularization and neurodegeneration. To maintain homeostasis, neurons require efficient degradation and recycling systems. Autophagy participates in retinal cell death, but it is also a cell survival mechanism. Here, we analyzed the role of autophagy at the three characteristic time periods in the oxygen-induced retinopathy (OIR) mouse model and determined if its modulation can improve vascular and non-vascular alterations. Experiments were performed with chloroquine (CQ) in order to monitor autophagosome accumulation by lysosomal blockade. Post natal day (P)17 OIR mouse retinas showed a significant increase in autophagy flux. In particular, an intense LC3B and p62 staining was observed in inner layers of the retina, mainly proliferating endothelial cells. After a single intraocular injection of Rapamycin at P12 OIR, a decreased neovascular area and vascular endothelial growth factor (VEGF) protein expression were observed at P17 OIR. In addition, whereas the increased expression of glial fibrillary acidic protein (GFAP) was reversed at P26 OIR, the functional alterations persisted. Using a similar therapeutic schedule, we analyzed the effect of anti-VEGF therapy on autophagy flux. Like Rapamycin, VEGF inhibitor treatment not only reduced the amount of neovascular tufts, but also activated autophagy flux at P17 OIR, mainly in ganglion cell layer and inner nuclear layer. Finally, the effects of the disruption of autophagy by Spautin-1, were evaluated at vascular, glial, and neuronal levels. After a single dose of Spautin-1, Western blot analysis showed a significant decrease in LC3B II and p62 protein expression at P13 OIR, returning both autophagy markers to OIR control levels at P17. In addition, neither gliosis nor functional alterations were attenuated. In line with these results, TUNEL staining showed a slight increase in the number of positive cells in the outer nuclear layer at P17 OIR. Overall, our results demonstrate that all treatments of induction or inhibition of the autophagic flux reduced neovascular area but were unable to completely reverse the neuronal damage. Besides, compared to current treatments, rapamycin provides a more promising therapeutic strategy as it reduces both neovascular tufts and persistent gliosis.

Soluble Tei2 fusion protein inhibits retinopathy of prematurity occurrence via regulation of the Ang/Tie2 pathway.

The aim of the present study was to investigate the potential mechanism of retinopathy of prematurity (ROP) using an oxygen-induced retinopathy (OIR) mouse model. For experiments, mice were divided into either the OIR group or control group. Fluorescein isothiocyanate-dextran cardiac perfusion and stretched retina preparation were performed. The total retina area, area of instillation, density of microvascular network, area of new blood vessels, vein width and the tortuosity of arteries were measured. Next, mice were randomly assigned into the PBS, soluble TEK receptor tyrosine kinase (sTie2)-fusion protein (Fc), angiopoietin 1 (Ang1), ranibizumab, ranibizumab + sTie2-Fc and ranibizumab + Ang1 treatment groups. Following housing for 5 days, the body weight of each mouse was recorded. Mice in the OIR group presented smaller total retina area and larger area of instillation, larger area of new blood vessels, and higher microvascular network density compared with the control PBS group. Obvious retinal vein dilatation and arterial tortuosity were identified in the OIR group. The amount of endotheliocyte nuclei of new vessels beyond the inner limiting membrane was larger in the OIR group compared with the control group. Furthermore in the next set of experiments, a larger area of instillation, smaller area of new blood vessels and decreased amount of endotheliocyte nuclei of new vessels were observed in the sTie2-Fc group, Ang1 group, ranibizumab group, ranibizumab + sTie2-Fc group and ranibizumab + Ang1 group compared with the PBS group. Specifically, the ranibizumab + sTie2-Fc group and ranibizumab + Ang1 group demonstrated markedly reduced retina instillation area and microvascular network density in the instillation area. Total retina area and body weight following 10 days of the experiment for the ranibizumab group were significantly lower compared with other groups. In conclusion, the combined regulation of the Ang/Tie2 and the vascular endothelial growth factor (VEGF)/VEGF receptor pathways markedly increased the efficacy of treatment with retinal neovascularization (RNV). Regulation of these pathways has a potential for treating RNV, in particular ROP.

β‑elemene inhibits oxygen‑induced retinal neovascularization via promoting miR‑27a and reducing VEGF expression.

The present study aimed to investigate the significant role of β-elemene in mouse models of oxygen-induced retinopathy (OIR). C57BL/6J neonatal mice were used to establish OIR models. They were divided into four groups: Normoxia, OIR, OIR control and OIR-treated. Mice in the OIR group were exposed to 75±5% oxygen for 5 days and returned to a normal oxygen environment on postnatal day 12 (P12). The OIR treated group was intravitreally injected with 1 µl β-elemene on P12 and subsequently returned to a normal oxygen environment for 5 days (P12-P17). Retinas were obtained on P17. Retinal neovascularization (RNV) was detected using adenosine diphosphatase staining and analyzed by counting the nuclei of neovascular endothelial cells. Vascular endothelial growth factor (VEGF) expression was determined by reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. MicroRNA (miRNA/miR) microarrays were used to screen out differentially expressed miRNAs between the OIR and β-elemene-treated groups. Binding the 3′-untranslated region (UTR) of VEGF and miR-27a was confirmed using luciferase assays. It was found that high oxygen concentrations accelerated RNV and increased the number of preretinal neovascular cells; β-elemene treatment reduced these effects. VEGF mRNA and protein expression was higher in the OIR and OIR control groups, compared with the normoxia and OIR-treated groups. Further, it was shown that miR-22, miR-181a-1, miR-335-5p, miR-669n, miR-190b, miR-27a and miR-93 were upregulated in the OIR-treated group, and downregulated in the OIR group. The prediction websites TargetScan and miRanda revealed that VEGF contained a potential miR-27a binding site in its 3′-untranslated region (UTR). Luciferase assays demonstrated that miR-27a directly bound to the 3′-UTR of VEGF. In vitro experiments demonstrated that miR-27a inhibited VEGF expression. In addition, β-elemene treatment upregulate miR-27a expression in vivo and in vitro. When miR-27a expression was depleted by miR-27a inhibitor, the protective effect of β-elemene on RNV was eliminated. The present study demonstrated that β-elemene reduced RNV in mouse OIR models via miR-27a upregulation, leading to reduced VEGF expression. This finding may contribute to the development of novel therapeutic strategies for human retinopathy.

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/μL), 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.

Mini-peptide RPL41 attenuated retinal neovascularization by inducing degradation of ATF4 in oxygen-induced retinopathy mice

Endoplasmic reticulum (ER) stress signaling is activated in retinal degeneration disease. Activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response (UPR), is a key element that maintains cell survival and proliferation in hypoxic conditions. Our previous studies showed that a small ribosomal protein L41 (RPL41) inhibits ATF4 by inducing its phosphorylation and degradation. In the present study, the effects of mini-peptide RPL41 on retinal neovascularization (RNV) in oxygen-induced retinopathy (OIR) mice was investigated. We induced OIR in C57BL/6 mice and obtained retinas from normoxia, OIR, OIR control (treated with PBS), and OIR treated (treated with RPL41) mice. Our results showed that ER stress signaling was activated and ATF4 was overexpressed in the retinas of OIR mice. After intravitreal injection of RPL41, the size of RNV and vaso-obliteration, and the number of preretinal neovascular cell nuclei in the retinas of OIR mice were significantly decreased. Western blot analysis and quantitative real-time polymerase chain reaction (qPCR) showed ATF4 and VEGF expression decreased after intravitreal injection of RPL41. Furthermore, the expression levels of inflammatory genes including TNF-α, IL-1β, and IL-6 were significantly decreased compared with the OIR control mice. In conclusion, RPL41 prevented pathologic neovascularization and exerted anti-inflammatory effects by degrading the important ER stress factor ATF4, thus, RPL41 could be a promising therapeutic agent for the treatment of neovascular eye diseases, especially retinopathy of prematurity (ROP).

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

To investigate the role of CCR7/p-ERK1/2/VEGF signaling in the mouse model of oxygen-induced retinopathy (OIR). 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-ERK1/2 and vascular endothelial growth factor (VEGF) were assessed via immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). 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-ERK1/2, and VEGF expression in the OIR mouse model (all P<0.05). CCR7 significantly enhanced the 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). These results suggest that CCR7/p-ERK 1/2/VEGF signaling plays an important role in OIR. CCR7 may be a potential target for the prevention and treatment of retinopathy of prematurity.

The characteristics of the full-field flash electroretinogram in rats with oxygen induced retinopathy

Objective To observe the characteristics of the full-field flash electroretinogram (F-ERG) in rats with oxygen induced retinopathy (OIR). Methods Twenty-four neonatal Sprague Dawley rats were divided into OIR group and control group. In OIR group, 12 rats were exposed to (75±2)% oxygen for 7 days and then to room air for 7 days; in control group, 12 rats were raised in room air for 14 days. At postnatal day 21, F-ERG tests were performed to examine the rod response , the maximum mixing reaction and the cone reaction. Results Compared with the control group, the b-wave amplitudes decreased (t=3.650) and the implicit times increased (t=2.410) in rod response in OIR group, the differences were statistically significant (P 0.05). 0.001 cd.s/m2 light intensity stimulation on rats F-ERG wave almost no response. 0.010 cd.s/m2 light intensity stimulation on rats can be recorded to the rod response waveform, with the increase of light intensity, the amplitude of b-wave increases. Conclusions F-ERG of OIR rat showed that the amplitude and sensitivity of the rod response and maximal rod-cone response was decreased. The intensity of light had effect on the OIR rod cells, and the amplitude of b-wave increased with the increase of light intensity. Key words: Retinal diseases/physiopathology; Electroretinography; Animal experimentation

Matrix metalloproteinase-9 and vascular endothelial growth factor expression change in experimental retinal neovascularization.

To investigate the signal transduction mechanism of matrix metalloproteinase-9 (MMP-9) mediated- vascular endothelial growth factor (VEGF) expression and retinal neovascularization (RNV) in oxygen-induced retinopathy (OIR) model. C57BL/6J mice were divided into four groups: control group, OIR group, OIR control group (phosphate-buffered saline by intravitreal injection) and treated group [tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) by intravitreal injection]. OIR model was established in C57BL/6J mice exposed to 75%±2% oxygen for 5d. mRNA level and protein expression of MMP-9, TIMP-1 and VEGF were measured by real-time polymerase chain reaction and Western blotting, and located by immunohistochemistry. Levels of MMP-9 and VEGF in retina were significantly increased in animals with OIR and OIR control group. Levels of TIMP-1 in retina was significantly reduced in animals with OIR and OIR control group. Furthermore, a significant correlation was found between MMP-9 and VEGF. Intravitreal injection of TIMP-1 significantly reduced MMP-9 and VEGF expression of the OIR mouse model (all P<0.05). These results demonstrate that MMP-9-mediated up-regulation of VEGF promotes RNV in retinopathy of prematurity (ROP). TIMP-1 may be a potential target for the prevention and treatment of ROP.

The mechanism of CCN1-enhanced retinal neovascularization in oxygen-induced retinopathy through PI3K/Akt-VEGF signaling pathway.

BackgroundCCN1 (also called Cyr 61) is an extracellular matrix signaling molecule that has been implicated in neovascularization through its interactions with several endothelial integrin receptors. The roles of vascular endothelial growth factor (VEGF) in angiogenesis are well described. The aim of this study was to investigate the signal transduction mechanism of CCN1–PI3K/Akt–VEGF in retinopathy of prematurity (ROP), and the effects of CCN1 knockdown on ROP.MethodsThe oxygen-induced retinopathy (OIR) model was established in C57BL/6J mice exposed to a high concentration of oxygen. Retinas were obtained from the normoxia, OIR, OIR control (treated with scramble siRNA) and OIR treated (with CCN1 siRNA) groups. Retinal neovascularization (RNV) was qualitatively analyzed with ADPase staining and quantitatively analyzed by counting neovascular endothelial cell nuclei at postnatal day 17 when RNV reached a peak. mRNA level and protein expression of CCN1, p-Akt, and VEGF were measured by real-time PCR and Western blotting, and located with immunohistochemistry.ResultsCCN1 depletion resulted in less neovascularization clock hour scores in the number of preretinal neovascular cells compared with the OIR treated group (1.28±0.83 versus 4.80±0.82; and 7.12±2.50 versus 23.25±2.35, respectively, both P<0.05). Furthermore, CCN1, p-Akt and VEGF mRNA, and protein were significantly expressed in the retina of the OIR and OIR control groups. Intravitreal injection of CCN1 siRNA significantly reduced PI3K/Akt–VEGF pathway expression of the OIR mouse model (all P<0.05). CCN1 siRNA significantly enhanced the avascular area and avascular diameter of OIR model (P<0.05). CCN1 siRNA decreased the levels of IL-1β, IL-6, and TNF-α significantly compared to the OIR group (P<0.05).ConclusionThese results suggest that CCN1 plays an important role in RNV via the PI3K/Akt–VEGF signaling pathway. CCN1 may be a potential target for the prevention and treatment of ROP.

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.

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

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

TMP Prevents Retinal Neovascularization and Imparts Neuroprotection in an Oxygen-Induced Retinopathy Model

To evaluate the effects of tetramethylpyrazine (TMP) on retinal neovascularization (NV) and neuroprotection in an oxygen-induced retinopathy (OIR) model. Neonatal C57BL/6J mice were subjected to 75% oxygen from postnatal day 7 (P7) to P12 and then returned to room air. TMP (200 mg/kg) or normal saline was given daily from P12 to P17. Immunostaining, HE staining, TUNEL assay, and RT-PCR were used to assess the effects of TMP on retinal neurovascular repair. TMP effectively prevented pathologic NV and accelerated physiologic revascularization by enhancing the formation of endothelial tip cells at the edges of the repairing capillary networks and preserving the astrocytic template in the avascular retina. TMP also prevented morphologic changes and significantly decreased TUNEL-positive cells in the avascular retina by rescuing neurons such as amacrine, rod bipolar, horizontal, and Müller cells. In TMP-treated mice retinas, there was a less obvious loss of amacrine cell bodies and their distinct bands; the number of both rod bipolar and horizontal cell bodies, as well as the density of their dendrites in the outer plexiform layer, was greater than that in OIR control mice. TMP not only decreased the loss of alignment of Müller cell bodies and distortion of processes but reduced the reactive expression of GFAP in Müller cells. Furthermore, HIF-1α and VEGF mRNA expression were downregulated in TMP-treated mice retinas. TMP improved neurovascular recovery by preventing NV and protecting retinal astroglia cells and neurons from ischemia-induced cell death partially due to its downregulation of HIF-1α and VEGF mRNA expression.

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

Tetramethylpyrazine inhibits retinal cell death in oxygen-induced retinopathy

Objective To observe the inhibitory effect of tetramethylpyrazine(TMP)on apoptosis of retinal neurons in oxygen-induced retinopathy(OIR).Methods 48 C57BL/6 mice were randomly divided into normal control group(n=18),OIR control group(n=18)and OIR TMP group(n=12).The mice of normal control group were raised in room air.From the postnatal day 7(P7),mice of the other two groups mice of OIR TMP group received intraperitoneal injection of TMP(200 mg/kg)once a day from P12 to P16.meanwhile the mice of normal control group and OIR control group were iniected with the same volume of normal saline containing of 0.1% DMSO.At P12,P14 and P17,the morphologic changes in retinal avascular zone and the number of retinal apoptotic cell were observed by HE staining and TUNEL assay.Results At P1 2.there were a few of chromatin condensation and pycnic nuclei in the inner nuclear layer (INL)of OIR control group.At P14,a great quantity(OIR control group)or some(OID TMP treated group)chromatin condensation and pycnic nuclei in the central INL were observed.At P17,the thickness of INL,inner plexiform layer(IPL)and outer plexiform layer(OPL)in the OIR control group were reduced;the thickness of INL,IPL and OPL in the OIR TMP group weas thinner than those in the normal control group and thicker than those in the OIR control group.At P12,the TUNEL-positive cells in the OIR control group was 6 times of the normal eontrol group(F=587.217,P＜0.001).At P14,the difference of TUNEL-positive cells in three groups was significant(F=587.217,P＜0.001);the TUNEL-positive cells in the OIR control group was 28 times of the normal control group(t=49.813,P＜0.001);the TUNEL-positive cells in the OIR TMP group has reduced 50% compared with the OIR controI group(t=42.434,P＜0.00 1).At P17,there was no significant difference in TUNEL-positive cells among the three groups (F=587.217,P>0.05).Conclusions TMP can inhibit apoptosis of retinal cells in OIR significantly. Key words: Retinal diseases/drug therapy; Tetramethylpyrazine/therapeutic use; Apoptosis/drug effects; Animal experimentation

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.