All experimental procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and to the guidelines of the facility Animal Resource Center. Before the procedure, rabbits were sedated with an intramuscular injection of xylazine (3 mg/kg)/ketamine (15 mg/kg; Henry Schein, Inc., Melville, NY), and proparacaine was applied topically to the eye (Henry Schein, Inc.). Approximately one half of the inferior retina was detached in the right eyes of adult New Zealand Red pigmented rabbits by infusing hyaluronate sodium (Healon; Pharmacia, Piscataway, NJ) in 0.25% balanced salt solution via a glass pipette between the neural retina and RPE (sodium hyaluronate was used to prevent retinal reattachment). In three eyes with retinal detachments, 1 mg JSM6427 in 50 μL PBS was intravitreally injected with a 30-gauge needle via the same hole as the pipette; three control rabbits were injected with PBS (vehicle) alone. Both 3- and 7-day time points were examined. In the 3-day group, JSM6427 or vehicle injections were administered on either day 0 (immediately after creating the detachment) or 24 hours later (day 1). On postdetachment day 3, 10 μg BrdU (Sigma-Aldrich) in 50 μL BSS was injected intravitreally and euthanatization (120 mg/kg intravenous sodium pentobarbital; Henry Schein, Inc.) was performed 4 hours later, after which retinal tissues were harvested. These experiments established that injection on postdetachment day 1 was more effective than on day 0; therefore, in the 7-day experiment, injections were administered on postdetachment day 1, bromodeoxyuridine (BrdU) was again administered on day 3, and euthanatization was performed on day 7.
The rabbit eyes were enucleated and fixed (4% paraformaldehyde/0.1 M sodium cacodylate buffer, pH 7.4; Electron Microscopy Sciences, Fort Washington, PA). Retinal tissue (∼3 mm2) was excised from three detached regions of the JSM6427- and vehicle-treated eyes and from three regions from the nondetached control eyes. Tissues were rinsed in PBS, embedded in low-melt agarose (5%; Sigma-Aldrich), and sectioned at 100 μm (Vibratome; Technical Products International, Warrington, PA). The sections were incubated in normal donkey serum (1:20) in PBS/0.5% BSA/0.1% Triton X-100/0.1% sodium azide (PBTA) and pretreated with 2 N HCl for 1 hour to facilitate detection of the BrdU. After the sections were rinsed in PBTA, pooled primary antibodies or lectin were added in PBTA overnight at 4°C. Anti-BrdU (1:200; Accurate Chemical and Scientific Corp., Westbury, NY) was used to label dividing cells, anti-vimentin (1:500; Dako) was used to label Müller cells, and isolectin B4 Griffonia simplicifolia (1:50; Vector Laboratories, Burlingame, CA) was used to label microglia and macrophages. After the sections were washed in PBTA, they were incubated with pooled secondary antibodies (streptavidin CY5, donkey anti-rat CY3, and donkey anti-mouse CY2, all at 1:200 in PBTA; Jackson ImmunoResearch, West Grove, PA) then washed in PBTA. They were mounted with 5% n-propyl gallate/glycerol, and single-plane images were taken from at least four sections from three different regions within each eye (Fluoview 500 laser scanning confocal microscope; Olympus, Tokyo, Japan) to quantify BrdU-labeled cells or subretinal scars and to measure the length of subretinal scars. Labeled cells (either anti-BrdU/vimentin or isolectin B4) were counted per millimeter of retina from the stored images by using an embedded magnification bar. For each experimental group, at least 24 section areas were quantified. To reveal the morphology of the retina, high-quality images were taken as a Z series of five planes and collapsed as a projection. All values are expressed as the mean ± SEM. Statistical comparisons between treatment groups were analyzed using the paired Student's t-test; P < 0.05 was considered significant.