Localization of IgG in the normal and dystrophic rat retina after laser lesions

Purpose: To test the hypothesis that access to extravasated plasma pro-rein IgG may influence photoreceptor survival following laser photocoagulation and to determine whether this correlates with the retinal glial reaction. Methods: A total of 45 rats (18 Royal College of Surgeons (RCS) dystrophic and 18 RCS-rdy(+) congenic control) were used for this experiment. Nine non-lasered littermates of same age were used as controls. The superior retinas of postnatal day 23 rats were irradiated with a grid pattern of 40 argon green laser lesions of 50 mu m in diameter and two powers (150 and 300 mW) for 0.2 s. Ar various times after laser lesions (up to 14 days), animals were perfused, the retinas snap frozen and sectioned on a cryostat. A onestep immunohistochemical technique was used by incubating with rabbit anti-rat IgG conjugated directly to horseradish peroxidase. Adjacent sections were processed using an antibody to glial fibrillary acidic protein (GFAP) by the standard avidin-biotin complex method. Results: The labelling pattern for extravasated IgG after laser lesion was very similar in both RCS and RCS-rdy(+) rat retinas. At 6, 12 and 24 h after lesions, IgG immunoreactivity (IR) was very intense in the lesion core and flanks. The outer plexiform layer (OPL) and photoreceptor inner segments provided a ready pathway for lateral spread of IgG. However; in the outer nuclear layer (ONL), IgG localization was much more restricted. Despite very intense IgG IR in the ONL of the coagulated lesion core, there was always a very sharply delineated boundary where the label abruptly halted. The GFAP labelling in both RCS dystrophic and RCS-rdy(+) congenic control rat retinas showed that this boundary was between normal and necrotic cells because there was a core where GFAP was not produced by Muller cells. By 2 days after lesions, the coagulated cells in the lesion core were being removed by phagocytic cells that were IgG IR Labelled phagocytic cells were also found among the inner and outer segment region on the lesion flanks. There was still IgG IR in the lesion, but the label was faint. No IgG IR was found in the retina at 3, 4, 7 and 14 days after lesions. Absorption control with pure rat IgG showed the label to be specific. Conclusions: The extravasated IgG was derived from the choroidal circulation because at no stage was IgG localized around the retinal vasculature. The IgG labelling was surprisingly widespread and, therefore, did not correlate with photoreceptor sparing, al;hough it preceded the widespread Muller cell expression of GFAP and may, therefore, trigger glial reaction.