Choroidal neovascularization (CNV) is the main complication of type I and type III age-related macular degeneration (AMD), characterized by abnormal neovascularization originating from the choroid. Approximately 10--15% of all AMD cases are found to have CNV. Although the exact mechanism of CNV has not been elucidated yet, it is now clear that AMD and CNV are closely related diseases.[@b1-dddt-11-2939] Anti-vascular endothelial growth factor (VEGF) treatment using ranibizumab has revolutionized the management of CNV. However, the recurrence of CNV can occur even after three monthly ranibizumab injections. Therefore, many anti-VEGF therapeutic strategies have been tried, including combining anti-VEGF treatment with photodynamic therapy,[@b2-dddt-11-2939] switching to another anti-VEGF agent, such as aflibercept[@b3-dddt-11-2939] or a novel anti-VEGF agent,[@b4-dddt-11-2939] and switching to other drugs, including a nonselective, small molecule VEGFR1--3 inhibitor, cediranib (AZD2171),[@b5-dddt-11-2939],[@b6-dddt-11-2939] the anti-lymphocyte antigen 4, cytotoxic T lymphocyte antigen 4 (CTLA4) antibody ipilimumab,[@b7-dddt-11-2939] and a humanized anti-platelet-derived growth factor receptor β antibody, ripasudil.[@b8-dddt-11-2939] Ripasudil ophthalmic solution 0.4% (hereinafter, ripasudil; Kowa Company Ltd., Tokyo, Japan) has been approved in Japan as a treatment option for glaucoma. Ripasudil is a nonselective Rho kinase inhibitor. It lowers intraocular pressure by decreasing the production of aqueous humor and improving the outflow facility by relaxing the trabecular meshwork, trabecular meshwork cells, and ciliary muscle. Recent studies have revealed that ripasudil has pleiotropic effects on the vitreous body in glaucoma[@b9-dddt-11-2939] and CNV.[@b10-dddt-11-2939] However, there are no reports on its effects in the CNV animal model of the mouse eye. In the current study, we investigated the effect of ripasudil ophthalmic solution 0.4% on experimental CNV in the mouse eye. Materials and methods ===================== Ethics statement ---------------- All procedures were carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of Kobe Gakuin University (Approval number: H25-009). Animals ------- We used the female BALB/c mice. The mice were given free access to food and water and maintained on a 12 h light--dark cycle. Experimental CNV model ---------------------- We used experimental CNV models that were developed by a bleomycin injection as previously reported.[@b11-dddt-11-2939],[@b12-dddt-11-2939] In short, intraperitoneal injections of saline or bleomycin sulfate (4 mg/kg; Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) were given every other day for 5 days, starting 2 weeks after subconjunctival injection with 5 mg/mL of 5-fluorouracil. The bleomycin-injected eyes were selected for each experiment as CNV models. The concentration of bleomycin was adjusted to 5 mg/mL with 0.9% sodium chloride solution. The mice were anesthetized with an intraperitoneal injection of 40 mg/kg of pentobarbital. Anesthesia was confirmed by checking the loss of righting reflex. Immediately after confirming the loss of righting reflex, one group of mice was intravitreally injected with 0.4% ripasudil ophthalmic solution (0.4 mg) into the bleomycin-injected eye under a microscope, according to previously reported methods.[@b9-dddt-11-2939] The other group was intravitreally injected with an equal volume of balanced salt solution as a control. Funduscopy and histological examinations ---------------------------------------- The experimental CNV was confirmed by an indirect ophthalmoscopic examination on day 7 after bleomycin injection. To confirm the effects of ripasudil, fluorescein-assisted funduscopy was performed on day 7 after bleomycin injection. The mouse eyes were enucleated, embedded in optimum cutting temperature compound, and cut into slices at −5°C in the vertical axis. Tissue slices were cut into 6 μm-thick sections and stained with hematoxylin and eosin (HE). Immunofluorescence staining --------------------------- The sections were deparaffinized with xylene, rehydrated in a graded series of alcohols, and washed with phosphate-buffered saline (PBS). Antigen retrieval was performed by heating the sections in a citrate buffer (pH 6.0) for 30 minutes, and then we blocked the samples with 5% normal donkey serum for 1 hour at room temperature. We then incubated the samples with primary antibodies, including mouse monoclonal antibody against CD31 (1:200; BD Pharmingen, San Jose, CA, USA), mouse monoclonal antibody against Ki67 (1:100; Invitrogen/Life Technologies, Carlsbad, CA, USA), and rabbit polyclonal antibody against VEGF (1:100; Abcam, Cambridge, UK). The sections were incubated at 4°C overnight, washed with PBS, incubated for 1 hour with secondary antibodies, including Alexa Fluor^®^ 568 goat anti-mouse IgG (1:200; Invitrogen/Life Technologies) and Alexa Fluor^®^ 488 goat anti-rabbit IgG (1:200; Invitrogen/Life Technologies), and observed under a fluorescence microscope. The number of CD31-positive cells and Ki67-positive cells was counted using the image J 1.45s program. The intensity of CD31 in the CNV area was measured using the image J 1.45s program. Statistics ---------- We used GraphPad Prism 4 software for statistical analyses. The unpaired *t*-test was used for comparison between the experimental groups. A *P*-value of \<0.05 was considered statistically significant. Results ======= Improvement of CNV development in the bleomycin-injected eye by treatment with ripasudil ophthalmic solution 0.4% ------------------------------------------------------------------------------------------------------------------ The CNV area was measured on day 7 after subconjunctival injection with bleomycin ([Figure 1A](#f1-dddt-11-2939){ref-type="fig"}). Although the treatment with 0.4% ripasudil ophthalmic solution decreased CNV area ([Figure 1B](#f1-dddt-11-2939){ref-type="fig"}) compared to the vehicle group ([Figure 1A](#f1-dddt-11-2939){ref-type="fig"}), this decrease was not significant compared to the control group ([Figure 1C](#f1-dddt-11-2939){ref-type="fig"}; *P*=0.6245). The CNV area was also measured on day 7 after subconjunctival injection with bleomycin ([Figure 2A](#f2-dddt-11-2939){ref-type="fig"}). To confirm the antiangiogenic effects of ripasudil, we performed immunofluorescence staining on day 7 after bleomycin injection. In the ripasudil-treated group, the CNV area was smaller than that in the vehicle group ([Figure 2B and C](#f2-dddt-11-2939){ref-type="fig"}; *P*\<0.0001) and the control group ([Figure 2C](#f2-dddt-11-2939){ref-type="fig"}; *P*\<0.0001). We also counted the CD31-positive cells, which were vascular endothelial cells. CD31-positive cells ([Figure 2D](#f2-dddt-11-2939){ref-type="fig"}) were also counted and quantified by intensity using an image J program. We found that the number of CD31-positive cells in the ripasudil-treated group was significantly lower than that in the vehicle group ([Figure 2E](#f2-dddt-11-2939){ref-type="fig"}; *P*\<0.0001) and the control group ([Figure 2E](#f2-dddt-11-2939){ref-type="fig"}; *P*\<0.0001). Although these parameters did not show a significant difference between the vehicle group and the control group, the number of CD31-positive cells in the control group was lower than that in the vehicle group ([Figure 2E](#f2-dddt-11-2939){ref-type="fig"}; *P*=0.0142). Reduced CNV area in the bleomycin-injected eye after treatment with ripasudil ophthalmic solution 0.4% ------------------------------------------------------------------------------------------------------- To confirm the antiproliferative effects of ripasudil, we performed immunofluorescence staining on day 7 after bleomycin injection ([Figure 3A](#f3-dddt-11-2939){ref-type="fig"}). The numbers of