Experimental Choroidal Neovascularization In Rats Research Articles

The Effect of CM082, an Oral Tyrosine Kinase Inhibitor, on Experimental Choroidal Neovascularization in Rats.

The aims of this study were to evaluate the effects of CM082 on the development of choroidal neovascularization (CNV) in a laser-induced CNV rat model and to determine the drug concentration in the ocular tissues. After the laser-induced CNV model was established in rats, CM082 was orally administered. The effects of CM082 on the CNV lesions were assessed using fundus fluorescein angiography (FFA), CNV histology, and retinal pigment epithelium- (RPE-) choroid-sclera eyecup analysis. The concentrations of CM082 in the plasma and eye tissues were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results of FFA, histology, and RPE-choroid-sclera eyecup analysis demonstrated that the CM082-treated (10 mg/kg/d or 30 mg/kg/d) rats exhibited significantly less neovascularization than did the control group. The total concentration of CM082 in the eyes (172.86 ± 57.11 ng/g) was similar to that in the plasma (196.87 ± 73.13 ng/ml). Within the eye, the concentrations of CM082 and its metabolites were highest in the retina-sclera. The orally administered CM082 thus effectively passed through the blood-retina barrier (BRB) to reach the retina in the Brown Norway rats. Therefore, at both 10 mg/kg/d and 30 mg/kg/d, CM082 was able to reduce CNV lesions in the laser-induced CNV rat model.

Inhibitory Effect of Chrysin (5,7-Dihydroxyflavone) on Experimental Choroidal Neovascularization in Rats

Purpose: The aim of this study was to evaluate the effect of chrysin on laser-induced experimental choroidal neovascularization (CNV) in a rat model. Methods: Male brown Norway rats were anesthetized, and a diode laser was used to break Bruch's membrane. One week later, each rat was intravitreally injected with 5 µl of 15 mg/ml chrysin, and CNV development was determined by fluorescein angiography at 2 weeks. The effect of chrysin on experimental CNV was assessed by fluorescein angiography and histology. Results: Two weeks after laser treatment, the intensity of fluorescein leakage from the photocoagulated lesions decreased significantly compared with the control group (p = 0.044). When the lesions were categorized into low- and high-leakage groups, there was a significant correlation between chrysin treatment and degree of leakage (p = 0.028). Compared with the chrysin-treated group, the relative risk of developing high-leakage lesions in the control group was 3.18. The mean CNV thickness was significantly thinner in chrysin-treated eyes than in control eyes (34.13 ±.0.88 vs. 37.76 ± 0.90 μm, p = 0.005). Conclusion: Chrysin has an inhibitory effect on CNV in an experimental rat model, indicating that chrysin should be further evaluated for its potential as a therapy for CNV in age-related macular degeneration and in other vision-threatening conditions associated with CNV.

Inhibitory Effect of YC-1, 3-(5′-Hydroxymethyl-2′-Furyl)-1-Benzylindazole, on Experimental Choroidal Neovascularization in Rat

Purpose: It was the aim of this study to evaluate the effects of YC-1, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole, one of the hypoxia-inducible factor 1 (HIF-1) inhibitors, on laser-induced choroidal neovascularization (CNV) in rats. Methods: Thirty female Brown Norway rats underwent laser photocoagulation to induce CNV. Twenty of them (treatment group) were treated with a single intravitreal injection of 5 µg YC-1, and the remaining 10 (control) were sham-treated with a single intravitreal injection of 2.5 mg/ml dimethyl sulfoxide 2 weeks after laser photocoagulation. The expression of HIF-1α and vascular endothelial growth factor (VEGF) in CNV was evaluated by immunofluorescence staining. Fluorescein angiography was performed 2 weeks before and 2 weeks after single intravitreal YC-1 (5 µg) and dimethyl sulfoxide (2.5 mg/ml) injection, grading fluorescein leakage from 0 to 3. The size of the CNV was measured on histologic sections. Results: Both HIF-1α and VEGF were expressed in CNV lesions in the control group 4 weeks after laser photocoagulation, whereas the expressions of HIF-1α and VEGF were not observed in the intravitreally YC-1-treated group. The mean fluorescein leakage score decreased from 2.56 ± 0.49 to 0.79 ± 0.71 in the intravitreally YC-1-injected group and from 2.62 ± 0.49 to 1.58 ± 0.60 in the control group. Sixty-eight (71.6%) out of 95 CNV lesions of intravitreally YC-1-treated eyes (71.4%) and 12 (21.8%) out of 55 lesions in DMSO-treated eyes showed a decreased fluorescein leakage score of 2 or more. The mean difference of fluorescein leakage scores between the intravitreally YC-1-treated group and the control group was significant (p = 0.004). The mean thickness of the CNV lesions in the intravitreally YC-1-treated group (27.30 ± 6.47 µm) was smaller than that of the control group (64.36 ± 8.26 µm, p < 0.001). There was no ocular inflammation, retina hemorrhage or systemic toxicity induced by YC-1 treatment. Conclusion: These results suggest that YC-1 inhibits the HIF-1 expression after photocoagulation and suppresses the development of laser-induced CNV formation.

Novel Drug Delivery System for Age-related Macular Degeneration Using Nanotechnology

Age-related macular degeneration (AMD) is a leading cause of legal blindness in developed countries. Even with the recent advent of several treatment options such as photodynamic therapy (PDT) and anti-vascular endothelial growth factor (VEGF) therapy for the treatment of exudative AMD, characterized by choroidal neovascularization (CNV), their efficacy is still limited. Thus, in this review article, we investigated novel drug delivery system for AMD using nanotechnology. Polyion complex (PIC) micelle has a size range of several tens of nanometers formed through electrostatic interaction, and accumulates in solid tumors through enhanced permeability and retention (EPR) effect. The distribution of the PIC micelle encapsulating fluorescein isothiocyanate-labeled poly-l-lysine (FITC-P(Lys)) in experimental CNV in rats was investigated. PIC micelle accumulates in the CNV lesions and is retained in the lesion for as long as 168 h after intravenous administration. PIC micelles can be used for achieving effective drug delivery system to CNV. Although PDT is a main treatment option for CNV, most patients require repeated treatments. For effective PDT against AMD, the selective delivery of photosensitizer to the CNV lesions and an effective photochemical reaction at the CNV site are necessary. The characteristic dendritic structure of the photosensitizer prevents aggregation of its core sensitizer, thereby inducing a highly effective photochemical reaction. A supramolecular nanomedical device, i.e., a novel dendritic photosensitizer encapsulated by a polymeric micelle formulation was employed for an effective PDT for AMD. With its highly selective accumulation on CNV lesions, this treatment resulted in a remarkably efficacious CNV occlusion with minimal unfavorable phototoxicity. Our results will provide a basis for an effective approach to PDT for AMD.

Expression of CD105 in laser-induced experimental choroidal neovascularization in rat

目的 观察激光诱导BN大鼠脉络膜新生血管(CNV)中CD105的表达情况.方法 532 nm倍频Nd:YAG激光诱导生成BN大鼠CNV,于光凝后7、14、21、28、56 d行荧光素眼底血管造影(FFA)检查,处死动物后摘取眼球,进行组织病理学观察.激光后7、14、28 d以免疫组织化学染色法观察CNV中CD105的表达情况.结果 激光后7 d,CNV开始形成,7～14 d为迅速增生期,此后CNV增生平稳.CD105在激光后7 d初步表达于CNV组织,于激光后14 d时表达明显.激光后28 d,新生血管形成趋于静止,CD105在CNV的表达较早期和中期明显减弱.结论 CD105在CNV形成过程中可能起重要作用,并且可以作为增生内皮细胞的一种标记物。

Effect of pitavastatin on experimental choroidal neovascularization in rats

The association between the use of statins and age-related macular degeneration (AMD), a leading cause of blindness, has been evaluated in many clinical studies; however, the results have been contradictory. We evaluated the effect of pitavastatin administration on laser-induced experimental choroidal neovascularization (CNV) in rats. Brown Norway rats received pitavastatin (1.0 mg/kg per day) for 1 day prior to laser-induced CNV and continued to receive the drug for 14 days. Fluorescein angiograms were graded by masked observers. CNV area and thickness were assessed by fluorescein isothiocyanate-labeled dextran angiography and histology, respectively. Vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (Ccl-2; also known as MCP-1), and intercellular adhesion molecule-1 (ICAM-1) mRNA levels were measured using reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR. Pitavastatin-treated rats had significantly less fluorescence leakage compared with the vehicle-treated rats estimated by CNV score using fluorescein angiography. Both the area and the thickness of CNV in pitavastatin-treated rats were significantly reduced compared with the vehicle-treated rats. Gene expression of VEGF, Ccl-2, and ICAM-1 were significantly decreased by pitavastatin administration in experimental CNV. Thus, we demonstrated that the therapeutic dose of pitavastatin for human hypocholesterolemia effectively suppressed experimental CNV in rats. The use of pitavastatin may be helpful in preventing CNV development in AMD patients.

Therapy with Ribozyme to the Proliferating Cell Nuclear Antigen-Ribozyme and 5-Fluorouracil of Experimental Choroidal Neovascularization in Rats

The aim of this study was to evaluate the efficacy of hammerhead ribozyme to the proliferating cell nuclear antigen (PCNA-Rz) and 5-fluorouracil (5-FU) in experimental choroidal neovascularization (CNV) model in rats. Laser was used to induce CNV in each eye of 44 rats. For angiography studies, injections of either a mixture of PCNA-Rz 10 microg/microL and 5-FU 1.5 microg/microL, versus the same dose of either drug alone versus a control injection of Hanks' Balanced Salt Solution (HBSS) were performed. We also studied this regimen to evaluate scar size and volume. There was significantly less angiographic leakage for the treated eyes compared to the controls by 3.53 grading points (P = 0.0005); CNV leakage was reduced in the combination group compared to 5-FU alone by 1.75 grading units (P = 0.04) and compared to PCNARz by 2.22 grading units (P = 0.07). The scar size and volume were smaller (diameter 354.6 +/- 174.2 microm vs 477.3 +/- 157.0 microm), (thickness 52.7 +/- 43.0 microm versus 79.6 +/- 46.2 microm) with a reduction in scar volume of 44.8%. Subretinal injection of PCNA-Rz and 5-FU mixture is more effective as treatment of laser-induced CNV, than either drug alone. The majority of the antiangiogenic effect is a result of 5-FU activity with a contribution by the PCNA ribozyme.

Inhibition of experimental choroidal neovascularization in rats by an av-integrin antagonist

Purpose. Integrin a v ß 3 is predominantly expressed on endothelial cells in choroidal neovascularization (CNV). We evaluated the efficacy of cyclic RGD (Arg-Gly-Asp) peptide, an a v -integrin antagonist, in a rat model of laser-induced CNV. Methods. We evaluated the effect of cyclic RGD on the adhesion and cell viability of bovine choroidal endothelial cells (BCECs) by cell counting and the trypan blue dye exclusion test. CNV was induced by laser photocoagulation in female Long Evans rats (day 0), followed by intravitreal administration of one dose of cyclic RGD of 200 (n = 9), 100 (n = 10), 50 (n = 4), or 0µg (n = 9) on days 9 and 11. We assessed the area of CNV by fluorescein angiography and the thickness microscopically on histologic sections. Neovascular vessels were detected by an antibody against factor VIII. Results. Cyclic RGD (0.02 to 200µg/ml) inhibited adhesion of BCECs in a dose-dependent manner without changing the cell viability (p < 0.01). In eyes treated with two injections of 200 or 100µg of cyclic RGD peptide, the development of CNV was significantly (p < 0.01) inhibited in the area of leakage on fluorescein angiography. Histologically, the CNV membrane was observed beneath the retina and the factor VIII-positive cells and red blood cells were involved. The thickness of the lesions was significantly (p < 0.01) reduced in eyes that received 200 or 100µg of RGD. Conclusions. Cyclic RGD effectively inhibited CNV progression in a rat model of laser-induced CNV, suggesting that this a v -integrin antagonist may be beneficial in the treatment of CNV.

Effective accumulation of polyion complex micelle to experimental choroidal neovascularization in rats

Exudative age-related macular degeneration, characterized by choroidal neovascularization (CNV), is a major cause of visual loss. In this study, we examined the distribution of the polyion complex (PIC) micelle encapsulating FITC-P(Lys) in blood and in experimental CNV in rats to investigate whether PIC micelle can be used for treatment of CNV. We demonstrate that PIC micelle has long-circulating characteristics, accumulating to the CNV lesions and is retained in the lesion for as long as 168 h after intravenous administration. These results raise the possibility that PIC micelles can be used for achieving effective drug targeting to CNV.

Long-term effectiveness of photodynamic therapy by using a hydrophilic photosensitizer ATX-S10(Na) against experimental choroidal neovascularization in rats.

We previously demonstrated that a hydrophilic photosensitizer ATX-S10 had a potent photodynamic effect. This study was designed to reveal the long-term effectiveness of photodynamic therapy (PDT) with this agent in occluding choroidal neovascularization (CNV) and its selectivity in the neovascular tissue. Experimental CNV was induced by intense photocoagulation in rat eyes. Immediately or 2 hours after intravenous injection of 8 mg/kg body weight of ATX-S10(Na), a cis isomer of ATX-S10, eyes were irradiated by a diode laser at the radiance of 3.25-65.3 J/cm(2) Vascular occlusion was identified by fundus photography, fluorescein angiography, and histology at 1, 3, 7, 14, and 28 days after PDT. As controls, non-neovascular eyes were subjected to PDT and similarly analyzed. By using the following treatment parameters, PDT with ATX-S10(Na) successfully occluded CNV without causing occlusion of retinal capillaries for 28 days; 7.4 and 19.6 J/cm(2) immediately after dye injection and 36.7 and 65.3 J/cm(2) 2 hours after injection. Although these conditions also caused occlusion of normal choriocapillaries and mild injuries of retinal vessels, retinal pigment epithelium, and photoreceptors at 1 day, retinal vessels and pigment epithelial cells recovered from damages by 28 days. No injuries were found in the inner retina. In optimal treatment conditions, PDT with ATX-S10(Na) can induce long-term, selective occlusion of CNV without causing irreversible damages in the inner retina.