Chu Y, Alder V A, Humphrey M F, Constable I J
Lions Eye Institute and Centre for Ophthalmology and Visual Science, University of Western Australia, Nedlands, Australia.
Aust N Z J Ophthalmol. 1999 Apr;27(2):117-25. doi: 10.1046/j.1440-1606.1999.00164.x.
To test the hypothesis that access to extravasated plasma protein IgG may influence photoreceptor survival following laser photocoagulation and to determine whether this correlates with the retinal glial reaction.
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 microm in diameter and two powers (150 and 300 mW) for 0.2 s. At various times after laser lesions (up to 14 days), animals were perfused, the retinas snap frozen and sectioned on a cryostat. A one-step 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.
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 Müller 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.
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, although it preceded the widespread Müller cell expression of GFAP and may, therefore, trigger glial reaction.
验证以下假说，即外渗的血浆蛋白IgG的存在可能会影响激光光凝后光感受器的存活，并确定这是否与视网膜胶质反应相关。
本实验共使用了45只大鼠（18只皇家外科学院（RCS）营养不良大鼠和18只RCS-rdy+同基因对照大鼠）。选取9只相同年龄未进行激光照射的同窝大鼠作为对照。对出生后第23天大鼠的视网膜上半部分进行照射，采用直径50微米的40个氩绿激光损伤的网格图案，设置两种功率（150和300毫瓦），照射时间为0.2秒。在激光损伤后的不同时间点（最长14天），对动物进行灌注，将视网膜速冻并在低温恒温器上切片。采用一步免疫组织化学技术，用直接与辣根过氧化物酶偶联的兔抗大鼠IgG进行孵育。相邻切片采用标准抗生物素蛋白-生物素复合物法，用抗胶质纤维酸性蛋白（GFAP）抗体进行处理。
激光损伤后，RCS和RCS-rdy+大鼠视网膜中外渗IgG的标记模式非常相似。损伤后6、12和24小时，IgG免疫反应性（IR）在损伤核心和边缘非常强烈。外网状层（OPL）和光感受器内段为IgG的侧向扩散提供了一条现成的途径。然而，在外核层（ONL）中，IgG的定位受到更多限制。尽管在凝固损伤核心的ONL中IgG IR非常强烈，但始终存在一个非常清晰的边界，标记在此突然停止。RCS营养不良大鼠和RCS-rdy+同基因对照大鼠视网膜中的GFAP标记表明，这个边界位于正常细胞和坏死细胞之间，因为存在一个Müller细胞不产生GFAP的核心区域。损伤后2天，损伤核心中凝固的细胞被IgG IR的吞噬细胞清除。在损伤边缘的内段和外段区域也发现了标记的吞噬细胞。损伤部位仍有IgG IR，但标记很淡。损伤后3、4、7和14天在视网膜中未发现IgG IR。用纯大鼠IgG进行吸收对照显示标记具有特异性。
外渗的IgG来源于脉络膜循环，因为在任何阶段IgG都未定位在视网膜血管周围。IgG标记出人意料地广泛，因此，尽管它先于GFAP在Müller细胞中的广泛表达，可能因此触发胶质反应，但它与光感受器的保留无关。
