elevation of MCP-1 in AMD patients with CNV [22]. Mice deficient in CCR2 or MCP-1, have a marked impairment in macrophage influx into sites of inflammation [23–25] and are protected from inflammatory diseases in a range of animal models [26]–[][][29]. The reduction in the number of infiltrating macrophages and the following amelioration of CNV formation also occurs in CCR2 KO mice [16] or MCP-1 KO mice [30]. Therefore, the inhibition of macrophages by blockage of the CCR2/MCP-1 signal has emerged as a novel therapeutic target for CNV treatment. Recently, INCB3344, a potent and selective small molecule antagonist of CCR2, was discovered and was demonstrated to have a high ability to compete with MCP-1 [26]. INCB3344 rapidly binds both rodent [26] and human CCR2 [27] with a high affinity, inhibits CCR2 binding to MCP-1, and blocks MCP-1- induced signaling and functioning in CCR2-expressing cells. Blocking the CCR2/MCP-1 signal by INCB3344 suppresses macrophage recruitment and attenuates the signs and symptoms of a variety of inflammatory diseases such as peritonitis, delayed- type hypersensitivity, experimental autoimmune encephalomyeli- tis, atherosclerosis, arthritis and thermal hyperalgesia in animal models [26], [28], [29]. All of these lines of evidence suggest that INCB3344 acts as an effective and ideal tool for treating inflammatory diseases. Given the close relationship between inflammation and neovascularization, we hypothesized that INCB3344 might be of therapeutic value in treating CNV. In this study, we administered INCB3344 to mouse models with different phases of CNV to determine whether this compound has the ability to suppress and regress CNV. We also investigated the possible molecular mechanism of INCB3344 involved in CNV formation. Materials and Methods Animals Male wild-type C57BL/6 mice (Charles River, Japan) 8 weeks of age were used as the laser induced CNV mouse model. Cu, Zn- superoxide dismutase (SOD1)-deficient mice with a C57BL/6 background (Jackson Laboratory, U.S.A.) were generated as described [24] and used as an established CNV model. Anesthesia was induced by peritoneal injection of 50 mg/kg ketamine HCl (Sankyo, Tokyo, Japan) and 10 mg/kg xylazine (Bayer, Tokyo, Japan), and the pupils were dilated with topical 1% tropicamide (Santen, Osaka, Japan). The animals were cared for in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All animal experiments were carried out in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Osaka University (#20–094-0). Laser induced CNV and drug treatment Laser photocoagulation (514 nm Argon laser, 130 mW, 100 ms duration, 75 mm spot size; Ultima 2000 SE, Lumenis/Coherent, Palo Alto, CA, USA) was performed bilaterally in each wild-type C57BL/6 mouse. Laser spots were applied in a standard fashion around the optic nerve using a slit lamp delivery system (Carl Zeiss, Germany) and using a handheld cover slip as a contact lens. Only burns that produced a bubble, indicating rupture of the Bruch membrane, were included in the study. INCB3344 (PF-418725, MW577.6) was supplied by Pfizer (New York, USA) and shown to be safe to the mouse retina at concentrations of zero-1800 nM in previous toxicity experiments (Data not be shown here). Here, we selected the highest dose of INCB3344 (1800 nM) in the safe range in our experiment. CCR2 Blocker Inhibits Choroidal Neovascularization Immediately after laser photocoagulation, mice were randomized into two groups and received intravitreal injections of 1 ml INCB3344 (1800 nM) or 1 ml vehicle (dimethyl sulfoxide dissolved in phosphate buffered saline, PBS). Intravitreal injection was performed with the FemtoJet Microinjector System (Eppendorf, Germany) under a high magnification stereomicroscope (Leica MS5, Germany). Histological examination For histological examination, 3 mice in each group were sacrificed on Day 14 after the treatments. The eyes were enucleated and fixed with 4% PFA for 1 hour at 4uC. After removing the anterior segment, the eyecups were fixed again in 4% PFA overnight, dehydrated in 30% sucrose for 6 hours, and then embedded in Tissue-TekH O.C.T. Compound (Sakura Finetek, Japan). The eyecups were sectioned into 7 mm-thick slices and stained with haematoxylin and eosin. Sections were examined using an Olympus BX50 microscope (Olympus, Japan), and images were digitalized using an Axiocam HRc camera and Axiovision version 3.1 image capture software (Carl Zeiss, Germany). Measurement of laser-induced CNV size On Day 14 after laser photocoagulation, the sizes of CNV lesions were measured on RPE-choroid flat mounts by a similar method to that described previously [31]. In brief, mice were deeply anaesthetized and perfused with 1 ml of phosphate- buffered saline containing 50 mg/ml fluorescein-labelled dextran (#FD2000S-1G, Sigma, MO, USA). Then mice were sacrificed humanely, and the eyes were enucleated and fixed in 4% paraformaldehyde (PFA) for 1 hour. The anterior segment of the eye was cut off, and the vitreous and the entire retina were carefully removed from the eyecups. Four or five radial cuts in the remaining RPE-choroid-sclera were made from the edge to the equator, and the eyecups were flat-mounted in PermaFluorTM Aqueous Mounting Medium (Thermo, USA) with the retinal pigment epithelium (RPE) layer facing up. Those flat mounts were examined and recorded by the same microscope as before. 78 spots from 14 vehicle treated mice and 81 spots from 14 INCB3344 treated mice were examined, excluding eyes with hemorrhages. Image J for Windows (NIH, Bethesda, Maryland) analysis software was used to measure the area of CNV, with the operator blinded with respect to treatment groups. Immunohistochemistry of macrophages and VEGF To investigate possible cellular and molecular responses to INCB3344 administration to the CNV model, we examined the status of macrophages and related angiogenic cytokines. After laser photocoagulation, 3 mice in each group were sacrificed and eyes were enucleated on Day 3, and 7 mm cryosections were prepared for immunohistochemistry. The protocols for preparing the cryosections here were almost the same as those mentioned previously, except that the fixation time was shortened to 6 hours. The cryosections were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature. Primary antibodies against mouse F4/80 (1:500, Catalog No. BM40075, Monoclonal Anti- body to Mouse Macrophages: F4/80, Acris, Germany) and mouse VEGF (1:200, Catalog No. ab46154, Rabbit polyclonal to VEGF, Abcam, USA) were incubated overnight at 4uC. Those slides omitting primary antibodies were used as negative control. After three washes in PBS, the slides were incubated with fluorescent– tagged secondary antibodies (Alexa-Fluor 488 and Alexa-Fluor 546; Invitrogen, Carlsbad, CA) and DAPI for 1 hour at room PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28933