急性甲醇中毒大鼠视网膜神经胶质细胞的免疫组织化学观察

发布时间：2018-06-28 03:44

本文选题：甲醇中毒 + 视网膜　；参考：《昆明医科大学》2013年硕士论文

【摘要】：目的探索急性甲醇中毒大鼠视网膜神经胶质细胞的病理变化,为进一步研究急性甲醇中毒导致视觉功能损害提供实验依据。材料与方法将雌雄不限、体重(280+30)g的100只健康成年SD(Sprague-Dawley)大鼠随机分为吸入等比混合气体(N20/02)高剂量组(A组)、低剂量组(B组)、生理盐水对照组(C组)及空气生理盐水对照组(D组),每组又随机分为2h、12h、24h、72h、1w共五个时间点,每个时间点5只,给予大鼠并分别灌胃高剂量甲醇溶液(2g/kg)、低剂量甲醇溶液(Jg/kg)和生理盐水。采用气相色谱法检测大鼠血液中甲醇浓度。应用免疫组织化学单标和荧光双标染色观察大鼠甲醇中毒后视网膜AQP4(水通道蛋白4)、GS(谷氨酰胺合成酶,视网膜Muller细胞标记物)、GFAP(胶质纤维酸性蛋白,星形胶质细胞标记物)、lectin(凝集素,小胶质细胞标记物)的病理学变化规律。结果(1)大鼠血液中甲醇浓度检测结果：大鼠血液甲醇浓度在A组2h、12h、24h,B组2h、12h分别显著高于C、D组(p0.05),以2h浓度最高,A、B组72h、1w及C、D组血液中甲醇浓度未检出。(2)大鼠视网膜病理学变化：①AQP4在A组2h、12h、24h表达增强,A组视网膜AQP4阳性反应平均光密度值分别高于B组及C、D组(p0.05,p0.01),特别是在2h,随着时间延长逐渐降低。72h和1w与C、D组间差异无显著性(p0.05)。AQP4在B组2h、12h表达增强,B组视网膜AQP4阳性反应平均光密度值显著高于C、D组(p0.01,p0.05),24h后反应逐渐下降,与C、D组之间是没有显著性差异(p0.05)。组织学上视网膜分为内层(包括神经纤维层、神经节细胞层、内丛状层、内核层)和外层(包括外丛状层、外核层、外界膜、感光细胞层),在A组甲醇中毒后2h AQP4阳性反应在视网膜内层显著增高,但在外层明显降低。在A组内层和外层间AQP4阳性反应与其它组相比均有显著性差异(p0.01,p0.05)。A组12h AQP4阳性反应在视网膜内层显著增强,与B、C、D组相比均有显著性差异(p0.01,p0.05)；在视网膜外层A组12h AQP4阳性反应明显增强,与C、D组相比有显著性差异(p0.05),但是A、B组间无显著性差异。24h、72h和1w AQP4阳性反应在视网膜内层和外层A、B、C组间芜显著性差异。②视网膜GS免疫阳性细胞在A组2h表达增强、在12h组达到高峰,与C组相比,有显著统计学差异(F=6.592,p0.01),24h组GS表达逐渐减弱,但仍高于C组(p0.01)。大鼠急性甲醇中毒后GS免疫阳性细胞突起不连续,细胞结构紊乱。A组12h、24h,GS免疫阳性细胞表达高于B组(p0.05),72h、1w GS表达逐渐恢复至正常水平(p0.05)。在B组GS免疫阳性细胞表达2h开始增强,12h达到高峰,高于C、D组(p0.05),24h以后GS表达逐渐恢复至正常。在视网膜内层,GS免疫阳性细胞在A组12h表达最强,高于C、D组(p0.05)；在视网膜外层,GS免疫阳性细胞与各组间相比,差异无统计学意义(p0.05)。③视网膜GFAP免疫表达在生理盐水对照组主要表达于神经纤维层和节细胞层,甲醇中毒A组2h可见GFAP免疫阳性细胞突起增粗增长,表达高于C、D组(p0.05),GFAP免疫阳性细胞在12h、24h沿内丛状层和内核层一直延伸,以12h最为明显,平均光密度值高于C、D组和B组(p0.01,p0.05),随着时间的推移,GFAP免疫阳性细胞由内核层伸出突起在内、外界膜之间的表达逐渐减弱,A组72h和1w与C、D组间没有显著性差异(p0.05)。B组各时间点GFAP的表达变化与A组相对应时间点相似,但阳性反应较弱。在视网膜内层,A组GFAP免疫阳性细胞表达2h增多、12h达到高峰、24h下降,但均高于C、D组(p0.05)；B组视网膜内层GFAP免疫阳性细胞12h、24h表达高于C、D组(p0.05)。GFAP表达在A、B组72h和1w逐渐降低,GFAP免疫阳性细胞伸出的突起较长,直达外界膜。④GS与GFAP,AQP4与GS免疫荧光双标染色显示：在视网膜内层内界膜到内核层可见GS与GFAP有丝状的共表达；Muller细胞胞体位于的内核层,突起贯穿内、外界膜,可见胞体及突起均与AQP4有共表达,表明Muller细胞表达AQP4。⑤视网膜lectin荧光染色显示：A组12h、24h、72h及B组24h、72h,lectin日性细胞数量逐渐增多,在72h达高峰。一个显著的特点是,它们从视网膜内层向视网膜外层迁移突起增粗变短,与C、D组有显著性差异(F=17.664,p0.01),A、B组间差异具有统计学意义(p0.05)。在视网膜内层,A组12h、24h、72h,B组72h可见lectin阳性细胞表达在神经节细胞层,内丛状层,内核层,数量以72h组最多,与生理盐水对照组相比有显著性差异(p0.01)。在视网膜外层,A组24h、72h,B组72h lectin阳性细胞数量较生理盐水对照组增多(p0.01),其余各组间差异无显著性。结论(1)大鼠吸入等比混合气体(N2O/O2)并分别灌胃高、低剂量甲醇溶液能成功复制急性甲醇中毒高、低剂量大鼠模型。(2)大鼠视网膜内层Muller细胞早期谷氨酰胺合成酶增多,水通道蛋白4表达异常增高及异常表达胶原纤维酸性蛋白,可能与视网膜水肿有关,程度与甲醇中毒剂量有关；小胶质细胞被激活,从视网膜内层迁移到外层,一方面可能对视网膜Muller细胞和其它细胞具有保护作用,另一方面也可能加重视网膜炎症反应,造成神经损害,有待进一步研究。
[Abstract]:Objective to explore the pathological changes of retinal glial cells in rats with acute methanol poisoning, and to provide an experimental basis for further study of visual impairment caused by acute methanol poisoning.
Materials and methods 100 healthy adult SD (Sprague-Dawley) rats of body weight (280+30) g were randomly divided into high dose group of inhaled isometric mixed gas (N20/02), low dose group (A group), low dose group (B group), normal saline control group (C group) and air physiological saline group (D group), each group was randomly divided into 2h, 12h, 24h, and every five time points, each At a time point, 5 rats were given a high dose of methanol solution (2g/kg), a low dose of methanol solution (Jg/kg) and normal saline. The concentration of methanol in the blood of rats was detected by gas chromatography. The retinal AQP4 (water channel protein 4) and GS (glutamine) were observed by immunohistochemistry and fluorescence double staining. Synthetase, retina Muller cell marker), GFAP (glial fibrillary acidic protein, astrocyte marker), and pathological changes of lectin (lectin, microglia markers).
Results (1) the test results of the concentration of methanol in the blood of rats: the concentration of methanol in the blood of rats in group A 2h, 12h, 24h, B group 2h, 12h was significantly higher than that of C, D group (P0.05), 2h concentration was the highest, and the concentration of methanol in the blood was not detected. (1) the pathological changes in retina of rats: (1) The average optical density of sexual response was higher than that of B group and C, group D (P0.05, P0.01), especially in 2H, and gradually decreased.72h and 1W and C with time, and there was no significant difference between D groups (P0.05). There is no significant difference between the groups (P0.05). Histologically, the retina is divided into the inner layer (including the nerve fiber layer, the ganglion cell layer, the inner plexiform layer, the core layer) and the outer layer (including the outer plexiform layer, outer nucleus, external membrane, photoreceptor layer). In the group A, the positive reaction of 2H AQP4 in the inner layer of the retina significantly increases after the methanol intoxication, but the outer layer is obviously reduced. The positive reaction of AQP4 between the inner layer and the outer layer of the A group was significantly different from the other groups (P0.01, P0.05), and the positive reaction of 12h AQP4 in the.A group was significantly enhanced in the retina, and there were significant differences (P0.01, P0.05) with B, C and D groups. 05), however, there was no significant difference between A and B groups.24h, 72h and 1W AQP4 positive reactions in the inner and outer layers of A, B, and C group. 2. The expression of GS immunoreactive cells in the retina was enhanced in A group, and reached a peak in the group of 2H. 0.01. After acute methanol poisoning, GS immunoreactive cells were discontinuous, and the expression of 12h, 24h, GS immunoreactive cells in group.A was higher than that in group B (P0.05), and the expression of 72h and 1W GS gradually recovered to the normal level (P0.05). In the inner layer of the retina, the expression of GS immunoreactive cells in the A group was the strongest, higher than that of C, D group (P0.05), and in the outer retina, the GS immunoreactive cells were not significantly different from each other (P0.05). (3) the GFAP immune expression in the retina was mainly expressed in the nerve fiber layer and the ganglion layer, and the methanol in the saline control group was mainly expressed in the nerve fiber layer and the ganglion cell layer. 2H positive cells of GFAP immunoreactive cells grew thicker than C, D group (P0.05), GFAP immunoreactive cells in 12h, 24h along the inner plexiform layer and core layer, and 12h most obvious, the average optical density value was higher than C, the D group and the group (GFAP) protruded out with the kernel layer over time. There was no significant difference between the outer membrane and the 72h and 1W in the A group (P0.05). The expression of GFAP in the.B group was similar to that in the A group, but the positive reaction was weak. In the inner layer of the retina, the GFAP immunoreactive cells in the A group expressed more 2H, reaching the peak and decreasing. The expression of GFAP immunoreactive cells in the inner retina of the retina was higher than that of C, and the expression of 24h in group D (P0.05).GFAP expressed in A, B group 72h and 1W gradually decreased, and the protuberance extended by the GFAP immunoreactive cells was longer and directly reached the outer membrane. The expression of Muller cell body is located in the core layer, penetration, external membrane, visible cell body and protuberance are co expressed with AQP4, indicating that Muller cells express AQP4. retinal lectin fluorescence staining: A group 12h, 24h, 72h and B 24h, 72h, the number of daily cells in the peak. The migration of the retina from the inner retina to the outer layer of the retina became thicker and shorter. There was a significant difference between the group of C and D (F=17.664, P0.01), A, and B group had statistical significance (P0.05). In the inner retina, A group 12h, 24h, 72h. There was a significant difference in the saline control group (P0.01). In the outer retina, the number of 72h lectin positive cells in group A, 24h, 72h and B increased (P0.01) in the saline control group (P0.01), and there was no significant difference between the other groups.
Conclusion (1) rats inhaled the isometric mixture of gas (N2O/O2) and gavage respectively. The low dose methanol solution could successfully replicate the high and low dose rat models of acute methanol poisoning. (2) the increase of glutamine synthetase in the early Muller cells in the inner retina of the rats, the abnormal increase of the aquaporin 4 and the abnormal expression of the collagen fibrillary acidic protein, may be associated with the abnormal expression of collagen fibrillary acidic protein. The degree of retinal edema is related to the dose of methanol poisoning. Microglia is activated and migrated from the inner layer of the retina to the outer layer. On the one hand, it may have protective effect on the retinal Muller cells and other cells. On the other hand, it may also aggravate the retinal inflammation and cause nerve damage, which needs further study.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R595.6

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