Color Image Analysis of Lymphocyte Subsets in Frozen Section in Experimental Uveitis

我们试图阐明s抗原（S-Ag）诱导的实验性葡萄膜视网膜炎中淋巴细胞亚群的动态变化。牛S-Ag的分离：采用低渗法提取粗牛视网膜的可溶性组分，用饱和硫酸铵进行盐分馏。离心去除不溶性物质后，将清上清溶液应用于Sephadex G-200和SDS凝胶电泳。经微孔过滤器冷凝后，用吸附色谱法纯化S-Ag馏分，逐步增加磷酸摩尔浓度。视网膜S-Ag对大鼠实验性葡萄膜视网膜炎的影响：将50 μ g S-Ag和完全的弗氏佐剂注射到Lewis大鼠的后脚垫中免疫。免疫后12 ~ 18 d出现葡萄膜炎。免疫后，在第1、2、3、7、14、21和28.3天立即进行眼去核处理。淋巴细胞亚群单克隆抗体和免疫过氧化物酶法染色：经正常痛风血清阻断后，冷冻切片与小鼠杂交瘤来源的抗四种大鼠淋巴细胞单克隆抗体、过氧化物标记的山羊抗小鼠IgG、底物溶液反应。彩色图像分析：开发的程序检测颜色向量的IHP （I：强度，H：色相，P：纯度），并以色相为主要鉴定指标，以识别、二值化和测量底物与过氧化物酶反应的红棕色。该程序还控制了显微镜的自动舞台，扫描所有领域的标签，没有遗漏或重复。一种抗原在淋巴细胞表面的体积(s)表示为面积率（s/ s）与背景组织面积(s)的比值。

We have tried to elucidate the dynamic changes of lymphocyte subsets in experimental uveoretinitis induced by S-antigen (S-Ag).1. Isolation of bovine S-Ag: Soluble fraction of crude bovine retinas was collected by hypotonic processing and submitted salt fractionation by saturated ammonium sulfate. After the removal of insolubel material by centrifugation the clear supernatant solution was applied to a Sephadex G-200 and to a SDS gel electrophoresis. After condensation by a millipore filter, the S-Ag fraction was purified by adsorption chromatography with stepwise increases in phosphate molarity.2. Production of experimental uveoretinitis in rats by retinal S-Ag: Lewis rats were immunized with 50 micrograms of S-Ag with complete Freund's adjuvant injected into the hind footpads. Uveitis was produced 12-18 days after immunization. Eyes were enucleated and processed immediately after immunization, and on Days 1, 2, 3, 7, 14, 21, and 28.3. Staining of lymphocyte subsets by monoclonal antibodies and immunoperoxidase method: After blocking process by normal gout serum, frozen sections were reacted with mouse hybridoma-derived monoclonal antibokies against four kinds of rat lymphocytes, with peroxidaselabeled goat anti-mouse IgG, and with the substrate solution.4. Color image analysis: The developed program detects IHP (I:intensity,H:hue, P:purity) of a color vector and makes the hue the main index of identification, in order to identify, to binarize, and to measure the red-brown, the color of the substrate reacting on peroxidase. The program also controls an autostage of a microscope, scanning labels in all fields without omission or duplication. The volume (s) of a kind of antigens on the surface of lymphocytes is expressed as an area rate (s/S) to the area of the background tissue (S).