Four rats in each group were killed with sodium pentobarbital (intravenous; Wako Pure Chemicals) 7 days after the intravenous injection described above. Eyes were enucleated and immersion fixed for 10 minutes in 4% paraformaldehyde in a 0.1 N sodium cacodylate buffer (pH 7.4; Wako Pure Chemicals). After the cornea and lens were removed, the eyecup was cut in half. One half of the tissue was stored in the fixative solution, and small areas of retina were excised and embedded in low-melting-point agarose for immunocytochemical analysis by confocal microscopy. Sections (100 μm) were cut on a vibratome, blocked overnight in normal donkey serum (1:20; Jackson Immunoresearch Laboratories, West Grove, PA) at 4°C, and then incubated with primary antibodies overnight at 4°C on a rotator. The primary antibodies were mouse monoclonal antibodies to IL-6 (1:100; R&D Systems, Minneapolis, MN), IL-17 (1:100; R&D Systems), and glutamine synthetase (1:100; Chemicon, Temecula, CA) and a rabbit polyclonal antibody to VEGF (1:100; Santa Cruz Biotechnology, Santa Cruz, CA). Mouse and rabbit immunoglobulin G (IgG; Sigma) were used as controls. All antibody solutions were made in PBTA (0.1 M phosphate-buffered saline containing 0.5% bovine serum albumin [Fisher Scientific, Pittsburgh, PA], 0.1% Triton X-100 [Boehringer-Mannheim, Indianapolis, IN], and 0.1% sodium azide [Sigma]). After rinsing with PBTA, the sections were incubated with Cy3-conjugated donkey anti-mouse IgG (IL-17), Cy5-conjugated donkey anti-mouse IgG (glutamine synthetase) or Cy2-conjugated donkey anti-rabbit IgG (IL-6, VEGF; Jackson Immunoresearch Laboratories) overnight at 4°C on a rotator. The sections were mounted in medium for fluorescence (Vectashield, Vector Laboratories, Burlingame, CA) and viewed by laser scanning confocal microscopy.
For high-resolution transmitted light microscopy, the other half of the eyecup was fixed in 1% glutaraldehyde (Wako Pure Chemicals) and 1% paraformaldehyde (Wako Pure Chemicals) in a 0.086 M sodium phosphate buffer (pH 7.3) overnight at 4°C, fixed in 2% phosphate-buffered osmium tetroxide (Nisshin-EM Co., Tokyo, Japan) for 1 hour, and then embedded in epoxy resin (Nisshin-EM Co.). Sections of 1 μm were prepared and stained with toluidine blue (Wako Pure Chemicals). The histologic severity of EAU was graded in five histologic sections from each animal in a blinded fashion by two ophthalmologists (TS and GA) using a previously described semiquantitative system.
18 : 0, no destruction, no cell infiltration, and 1–7, limited or total destruction of the various layers of the retina: grades 1–2; destruction of outer segments of rods and cones; grades 3–4, destruction of the outer nuclear layer; grades 5–6, destruction of the inner nuclear layer; grade 7, destruction of the ganglion cell layer. Severity of disease was calculated as the mean value for all rats from three separate experiments.