Safety And Efficiency Of YC-1 On Experimental Central Retinal Vein Occlusion In Rhesus Monkey

Central retinal vein occlusion( CRVO) is the common vascular disease secondary to diabetic retinopathy, the prevalence of which was about 0.7-1.6%, and often cause serious vision loss and even blindness. Although the exact mechanism is not clear, the increased vascular permeability caused of the retinal extensive hemorrhage and macular edema(cystoids macular edema, CME) is one of the main reason. At present, many evidences supported that vascular endothelial growth factor(VEGF) play an important role in the onset of retinal vascular disease. In terms of Molecular levels, VEGF regulated proliferation and migration of vascular endothelial cells and increased permeability of the vascular vessels. Clinical studies have shown that anti VEGF vitreous injection can inhibit VEGF binding to its receptors, there by decreased choroid and retinal vascular leakage and neovascularization, and improved the macular edema and vision acuity. For many retinal vascular disease including age-related macular degeneration(AMD), retinal vein occlusion(RVO) and diabetic retinopathy(RD), anti VEGF drugs vitreous injection has become a first-line treatment. With the widely application of the anti VEGF drugs, however, there are still many problems to be solved: the short half-life need frequent vitreous injection for stabilizing the visual acuity, which could increase the potential risks of endophthalmitis; visual acuity and macular edema does not improve or even worse in some cases, which was the so-called "rebound" effect; the atrophy of retinal pigment epithelial and photoreceptor cell layer was observed after long term treatment.Additionally, Inflammatory factors may also play a kind of role in the CRVO progression. Many clinical studies demonstrated that various cytokines and chemokines such as: the expression of IL1 beta, IL6 and IL8 and IL10, IL12, IL13, monocyte chemokine(MCP-1), soluble cell adhesion factor 1(Si CAM 1), and interferon were significantly increased in the vitreous and aqueous humor. Through the vitreous injection of triamcinolone acetonide(TA), efficiently inhibit both VEGF and inflammatory cytokines, reduce macular edema and improve vision acuity in CRVO patients. however, serious side effects of steroids limited its application, research showed that 48 to 67% of patients had various kind of cataract, 40% had elevated intraocular pressure, and take 0.1% risk of endophthalmitis after vitreous injection of TA. So looking for other potential therapeutic agents is imperative.During the process of retinal vascular disease, hypoxia inducing factor 1(HIF-1) played a important role according to previous studies. HIF-1 is a transcription factor that regulate about 40 hypoxia inducible genes including V a variety of blood vessel related factor such as VEGF and EOP et al., YC-1, as inhibitors of HIF-1a, blocked HIF-1complex and downstream target genes. Additionally, through regulating NF-k B signaling pathway, which played a central role in immune response, YC-1 can indirectly down regulated cytokines and chemokines. Even YC-1 has dual function both anti-inflammatory and anti VEGF expression, It may be potential therapeutic treatment for CRVO patients. In this paper, we used rhesus monkey as experimental subjects which have macular structure similar to that of human, and set up CRVO model by argon laser photocoagulation. Then we observed that the function and structure changes of retina after vitreous injection of YC-1in vivo. inflammatory factors including IL-6,IL-8,MCP-1 and vascular related genes including HIF-1a,VEGF in vitreous were also examined using Cytometric Bead Array(CBA)or ELASA methods. At the end of time points of follow up period, we explored the HIF-1 a and VEGF expression pattern in the retina by immuno-histochemistry.The first part YC 1 drug pharmacokinetics study in normal macaques glass body cavityObjective: to observe the pharmacokinetics of YC-1 in vitreous body of adult rhesus monkeyMethod: three normal rhesus monkeys with 6 eyes were recruited in this study. All eyes were administered with a single vitreous injection of YC- 1 90 μl with concentration of 200 μM by vehicle of 0.01% DMSO(dimethyl sulfoxide).after vitreous injection, we collected vitreous 100 ul at 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5h, 6 h, 8 h, and 10 h respectively. Then we employed high performance liquid chromatography(HPLC) technology to detected YC 1 concentration at each time point. DAS 3.0 pharmacokinetic software and PKSolver 2.0 software were used to analysis YC-1 pharmacokinetics including half-life parameters.Results: the results showed that the vitreous humor itself did not interfered with the examination for YC-1contentration. the maintain time of vitreous humor in the test tube were 1.25 and 1.80 min. the curve of result shows that this method has a good specificity and sensitivity. The value of correlation efficient were>0.995, which indicates that this method has a higher linearity. Pharmacokinetics analysis shows that after treatment, the drug concentration is 1.217μM at 0.25 hours, 0.824 atμM 0.5 hours, 0.497μM at1 hour, 0.368μM at 2 hours, 0.326μM at 3 hours, 0.265μM at 4 hours, 0.196μM at 5 hour, 0.145 μM at 6 hours, 0.083μM at 8 hours. using the PKSolver 2.0 software, we calculated the half-life of YC 1 in the vitreous of rhesus monkey is 2.79 hours.Conclusion: high performance liquid chromatography(HPLC) and mass spectrometric analysis is the specific and sensitive method for pharmacokinetics analysis of YC-1 in vitreous cavity. Though observing the concentration of YC-1 in vitreous during the time, the result showed that the concentration of YC 1 in vitreous drops rapidly with the extension of time. the elimination half-life of YC- 1 in the vitreous cavity is 2.79 hours.The second part the toxicity and safety for vitreous injection of YC 1 in rhesus monkey eyeObjective: to observe the influence of YC-1 vitreous injection for retina structure and functions of rhesus monkey.Methods: three adult rhesus monkeys were included in this studies, the right eye of each monkey in the YC-1 group was treated with intravitreal injection of YC-1 90 μl(200μM). The left eye of each monkey in DMSO control group was treated with intravitreal injection of 0.01% DMSO 90μl. Then the check time point included preoperative and postoperative 6 hrs, 1 week, 2 weeks and 1 month. Observing parameters are as follows: eye conjunctiva reaction, intraocular pressure, anterior chamber inflammation and corneal transparency, fundus lesions, the macular area structure by Coherent optical tomography(optic coherence tomography, OCT), vascular lesions by fundus fluorescence angiography(FFA). Dark adaptation and light adaptation ERG examination, and histological examination at 1 month after intravitreal injection.Results: there are no complications such as vitreous hemorrhage, retinal detachment for any eyes after intravitreal injections in any time points. The result also showed that there are not any anterior chamber reactions in both YC-1 group and DMSO control group. The IOP has not significantly changes in both groups at all time points(P>0.05). the central macular thickness has also no obvious difference(P>0.05) for both groups. FFA studies did not show any vascular changes for both groups. ERG displayed that the amplitude of rod b wave and cones a and b wave in YC-1 group were no significantly different compared with control group(P>0.05). for histological examination, the three layers of retina did not show obvious different between YC-1 group and DMSO control group.Conclusion: Intravitreal injection of YC-1 90μl(200μM) in the normal rhesus monkey have not affect the retina and anterior segment structure at all time point in vivo. The function of retina detected by ERG did not show obvious retina function damages, at the 1 month after intravitreal injection of YC-1, histology research showed that the three cell layers of retina had no significant changes compared with that of DMSO control group.The third part the study of retina structure, functions and vitreous cyto-kines in the experimental CRVO of rhesus monkeyObjective: to study the feasibility for argon laser establishing experimental central retinal vein occlusion of rhesus monkey, and to observe the retinal structure and function changes, inflammatory factors and HIF, VEGF factors expression changes in vitreous in this CRVO model.Methods: 6 adult rhesus monkeys, a total of 6 eyes were recruited as the research object. Regular examination before study was administered for making sure no any eye diseases. Direct laser coagulation was performed on all branch retinal vein in the right eyes of six rhesus monkeys to establish a CRVO model. then immediately fundus fluorescence angiography was operated to confirm all veins completely obstructed. At 6 hours,1 day, 1 week, 2 weeks, 1 month after laser coagulation, we performed the slit lamp examination for observing to anterior segment, fundus photography and FFA for observing of retina vascular diseases, OCT for evaluation of central macular thickness and ERG examination for retinal function evaluation. Meanwhile, the vitreous fluids(200μl) were collected by 23 gauge needle with 1 ml syringe at before and after laser coagulation in above 5 time points. The concentration of IL6, IL8,MCP-1in vitreous fluids were analysis by CBA test. the concentration of VEGF and HIF-1α in vitreous fluids were detected by ELASA test. At 1 months after laser coagulation, the retina of each monkey(3 right eyes and 3 left eyes) was removed and underwent HE staining and immunohistochemical staining for evaluation of VEGF, HIF-1α and Caspase3 expression.Results: CRVO were successfully established in all right eyes in six rhesus monkeys by argon laser coagulation. the results showed that intraocular pressure was mindedly elevated but not significantly at 1 day and 1 week after coagulation compared with that of control group. the transiently new vascular in iris was observed in early period, the central macular thickness in 1 day, 1 week, 2 weeks was significantly increased compared with that of control group(P<0.05), and recovered to preoperative levels in 1 month(P>0.05). Flash ERG result display that rod b wave and cones a and b wave decreased significantly at all time points after coagulation(P<0.05), FFA proved that vascular leakage of retinal, perfusion area and new blood vessels in retina, which similar with the basic changes of CRVO of patients. Through the CBA and ELASA analysis, the concentration of inflammatory factors including IL-6, IL-8( since 1 day) and MCP 1(since 2 weeks) in vitreous fluids was found to be significantly unregulated compared with that of control group(P<0.05). The expression of VEGF and HIF-1 were also consisted increased at all time points compared with control group. in immuno histochemical staining, we calculated optical density of HIF- 1 α and VEGF expression in the retina, both which were significantly increased at one month, meanwhile the expression of Caspase3, a key apoptosis marker was also increased at the same time.(P< 0.05).Conclusion: argon laser induced CRVO in rhesus monkey eye is feasible. This model displayed typical macular edema, retinal hemorrhage and non perfusion area and have similar signs with that of CRVO patients. The expression of vascular related factors, HIF-1 α and VEGF and inflammatory factors, IL-6,IL-8 and MCP-1 in vitreous or retina were significantly increased in CRVO model demonstrated that both factors involved in the onset of experimental CRVO process. Additionally, the ERG test shows the significant correlations between different components and VEGF and cytokines during the experimental CRVO progression. The ERG can be use to evaluate the severity of retina function damaged. This CRVO model of rhesus monkey can simulate human CRVO disease process, and to be a candidate object for further new treatment study.The fourth part the treatment study of intravitreal injection of YC-1 for experimental central retinal vein occlusion of rhesus monkeyObjective: to observing the potential treatment value of intravitreal injection of YC-1 for experimental CRVO of rhesus monkey.Methods: 6 adult rhesus monkeys, a total of 6 eyes were recruited as the research object. Regular examination before study was administered for making sure no any eye diseases. Direct laser coagulation was performed on all branch retinal vein in the both eyes of six rhesus monkeys to establish a CRVO model. At 1 week after coagulation, intravitreal injection of YC-1 90μl(200μM) was administrated in right eye as the YC-1 treatment group, intravitreal injection of 0.01% DMSO 90μl was administrated in left eye as the DMSO control group. All eyes of monkeys were underwent regular examinations including slit lamp, FFA, OCT, IOP and ERG et.al., at pre and post intravitreal injection 1 day, 1 week, 2week and 1 month. Vitreous fluids(200μl) were collected at all above time points and be used to test VEGF, HIF-1α,and inflammatory factors including IL-6,IL-8 and MCP-1 by CBA or ELASA kit, Flash ERG retinal electric diagram were used to check function of the retina. Then 3 right eyes and 3 left eyes(one eye each monkey) were removed to be used for HE staining and immunohistochemical staining for VEGF, HIF-1 and Caspase3 expression. To qualify the expression density of three factors in the retina, the optical density value by specific software were employed to evaluate their expression in the retina.Results: the new vascular in iris, retinal hemorrhage, no perfusion area and new blood vessels were displayed in both YC-1 treatment group and DMSO control group. Intraocular pressure(IOP) showed no significant difference between two groups(P>0.05). OCT examination showed macular edema was obvious in both group at 1 week after argon coagulation, but after intravitreal injection of YC-1, the central macular displayed less thickness than that of DMSO control group at the same time point. In YC 1 treatment group, the macular area edema faded much quickly. ERG examination showed that the amplitude of rod b wave and cone a and b wave were significantly increased in the YC-1 group compared with that of DMSO group after 1 week of injection. After application of YC-1, IL-6, IL-8 were obvious down regulated from the initial period, however, MCP-1 were slightly down regulated in YC-1 group compared with that of DMSO control group. In addition, HIF-1 α and VEGF were signficantly inhibited after YC-1 vitreous injection at all time points, HE staining showed that all three cell layers of retina looks thinner than normal retina in both groups. Immunohistochemical staining showed that HIF-1α, VEGF and Caspase3 expression were signif- icantly suppressed in YC-1 treatment group compared that of DMSO control group at one month after injection. the optical density value of three factors expressed in the retina confirmed that HIF-1α, VEGF and Caspase3 expre- ssion were significantly reduced in YC-1 treatment compared with that of the DMSO group.Conclusion: intravitreal injection of YC-1 can significantly improve the thickness of macular, accelerate the absorption of macular edema, and increased amplitude of rod b wave and cone a and b wave amplitude. The concentration of IL-6, IL-8, VEGF, HIF-1 α in the vitreous was also decreased, the expression of VEGF and HIF-1 α in the retina were down regulated compare with that of DMSO control group. The Caspase3 expression was inhibited in the retina of YC-1 treatment group suggested that YC-1 may have protective effect for retinal cells.Based on the above research, we can draw the following conclusions: 1 application of YC-1 in vitreous of rhesus monkey eyes has no obvious toxic effects, and had no significant influence on function and structure of retina. 2 it is reliable that argon laser induced CRVO animal models of rhesus monkey can simulate the performance of the human eye CRVO. It can be used as an ideal experimental model for further new drug related study. 3 intravitreal injection of YC-190ul(200μM) can reduce the duration of the macular edema, improve the function of the retina, rapidly suppressed inflammatory factors in the vitreous and VEGF and HIF-1 α both in vitreous and retina for experimental CRVO of rhesus monkeys, meanwhile, YC-1 also inhibited the apoptosis of retinal cells. All these result suggested that YC-1 has potential therapeutic value for CRVO patients.