去小胶质细胞化对糖尿病小鼠视网膜光感受器细胞的影响

目的观察去小胶质细胞化对早期糖尿病小鼠视网膜光感受器细胞的影响。方法选取6～8周龄的SPF级雄性C57BL/6J小鼠作为实验动物,未经处理的5只作为空白对照组(A组),10只采用链脲佐菌素(STZ)腹腔注射法成功诱导出糖尿病后的小鼠随机等分为B、C两组。A、B组继续喂养标准实验饲料4周,C组在喂养1周标准实验饲料后添加含290 mg·kg^-1 PLX3397(集落刺激因子1受体拮抗剂)的AIN-76A(标准饮食配制的啮齿类实验动物纯化饲料)3周以去除小胶质细胞。4周后于同一时间点处死各组动物,眼球标本均于处死后即刻获取并固定。制备视网膜石蜡切片,HE染色后光学显微镜下观察视网膜结构;小胶质细胞特异性抗体P2ry12免疫荧光化学法检测小胶质细胞在视网膜上的分布,并测算平均吸光度(D)值以间接代表小胶质细胞的数量;TUNEL法测定光感受器细胞的凋亡情况;将上述观察指标在三组间进行比较。结果光镜观察视网膜结构显示,与A组相比,B组神经纤维层变水肿,内丛状层、内核层及外核层排列变疏松;C组也出现上述改变,但程度较轻。小胶质细胞的免疫荧光化学检测结果显示:A组可在视网膜内层...

Effects of deletion microglia on retinal photoreceptor cells in diabetic mice

Objective　To observe the effect of deleted microglia on retinal photoreceptor cells in early diabetic mice.Methods　Six-week-old to eight-week-old SPF male C57BL/6J mice were selected as experimental animals. Five untreated mice were served as blank control group (group A). Ten animals with diabetes induced by intraperitoneal injection of streptozotocin (STZ) were randomly divided into group B, and group C with 5 rats in each. Group A and group B was fed standard laboratory chow for 4 weeks. Group C was fed standard laboratory chow for 1 week followed with AIN-76A (purified feed for rodent experimental animals prepared with standard diet) containing 290 mg?kg^-1 PLX3397 (colony stimulating factor 1 receptor antagonist) for 3 weeks for depletion of microglia. Animals in group A, B and C were killed in the 4th week, and all the eyeball were obtained and fixed immediately after execution. The retinal structure was observed under light microscope after HE staining, and the distribution of the microglia specific antibody P2ry12 on the retina was detected by immunofluorescence methods, the immunofluorescence average optical density (D value) was calculated to represent the number of microglia cells indirectly, and the apoptosis of photoreceptor cells was determined by TUNEL methods. These observations and indicators were compared across groups.Results　The retinal structure under light microscope showed that, compared with the group A, the nerve fiber layer of group B became edema, the arrangement of inner plexiform layer and inner and outer nuclear layer became loose. Although these changes were also found in group C, the degree was slighter than those in group B. The immunofluorescence detection of microglia showed that a small amount of microglia can be detected in the inner layer of the retina in group A, while the microglia were distributed in the whole layer of the retina in group B , which suggested that microglia migrated from the inner retinal layer to the whole retinal layer in diabetic status. The D value of P2ry12 in group B was significantly higher than that in group A and group C (t=3.478,8.166,both P<0.001). The D value of P2ry12 in group C was significantly lower than that in group A (t=30.409, P<0.001). The average number of apoptosis of photoreceptor cells in group A, group B, and group C was (0.67±0.87) cell, (9.22 ±1.56) cells, and (2.22 ±0.97) cells, respectively. There were significant differences between group B and group A (t=14.360，P<0.001) and between group B and C (t=11.408，P<0.001), and group A was less than group C, with significant difference (t=-3.585，P=0.02).Conclusion　In the early stage of diabetes, microglia are involved in the damage process of diabetic retinal photoreceptor cells, and the early depletion treatment of microglia can alleviate retinal edema and apoptosis of outer nuclear layer cells.