中性粒细胞在不同品系正常小鼠角膜的分布及其与角膜创伤修复的关系

摘要

Background C57BL/6J,BALB/c and nude mice are commonly used models in laboratory study of eye immunology,and neutrophil is a natural immune barrier.Previous studies showed that there are no vessel and neutrophils in mouse cornea.Therefore,whether there are neutrophils,the key natural immune cells,in mouse cornea is worth studying.Objective This study was to illuminate the distribution traits of neutrophils in normal cornea in C57BL/6J mice,BALB/c mice and nude mice,and to investigate the relation between corneal neutrophil and wound repair.Methods Male SPF C57BL/6J,BALB/c and nude mice with a BALB/c background were enrolled in this study.The whole mount mouse cornea was prepared and divided into 3 concentric zones.Gr-1-FITC and CD31/ PECAM-1-PE antibodies were used to label the neutrophils and vessels.Corneal wound models were established using scratching method.Fluorescine sodium was used to observe the corneal wound area.The whole mount cornea was prepared and corneal vessels and neutrophils were quantified by Gr-1-FITC and CD31/PECAM-1-PE antibody staining,respectively.The use and care of the animals complied with the Statement of Association for Research in Vision and Ophthalmology (ARVO).Results The number of neutrophils was (1 733±237),(353±96) and (1 601±223)/cornea in C57BL/6J,BALB/c and nude mice,respectively,and the number of neutrophils in cornea was significantly reduced in BALB/c mice compared with C57BL/6J and nude mice (both at P＜0.01).The blood vessels were seen in limbal with the red fluorescence for CD31-PE antibody in three strains of mice,and vessel area was smaller in the BALB/c mice than those in C57BL/6J and nude mice (both at P＜0.01).The corneal vessel area was the largest in the C57BL/6J mice.Significantly positive correlations were found between the corneal vessel area and number of neutrophils in three strains of mice (C57BL/6J mice:r=0.936,P=0.001 ;BALB/c mice:r=0.939,P =0.001 ; nude mice:r =0.935,P =0.001).In 24 hours after corneal scratching,the wound areas were larger in the C57BL/6J mice and nude mice than those in the BALB/c mice (both at P =0.000),and the corneal wound area was the largest in the nude mice.The number of neutrophils was (3 340±417),(1 235±337) and (4 680 ±450)/cornea in the C57BL/6J,BALB/c and nude wound mice,showing significant differences between the BALB/c models and C57BL/6J models or nude models (all at P=0.000).In addition,positive correlations were seen between the corneal wound area and the number of neutrophils in all the three model groups (C57BL/6J models:r =0.805,P =0.016 ; BALB/c models:r=0.943,P=0.000;nude models:r=0.916,P=0.001).Conclusions The number of corneal neutrophils depends on the distribution and area of corneal vessels in three strains of mice.The number of neutrophils is associated with corneal wound healing.These results suggest that the distribution and number of neutrophils in cornea are associated with the infection susceptibility and early inflammatory response.%背景 以往人们通常认为角膜无血管、无髓系来源的免疫细胞存在.C57BL/6J小鼠、BALB/c小鼠和裸鼠是眼科免疫学基础研究的常用模型,这些小鼠的角膜是否存在天然免疫的关键细胞——中性粒细胞是值得关注的问题. 目的 研究实验室常用的C57BL/6J鼠、BALB/c鼠和裸鼠正常角膜的中性粒细胞分布特征及其与角膜创伤修复的关系,为相关研究提供依据. 方法 选取角膜正常、10 ～ 12周龄的SPF级雄性C57BL/6J鼠、BALB/c鼠和BALB/c背景的裸鼠各16只,取3种小鼠各8只制备成中央区相连的4瓣角膜铺片,并以角膜周边血管缘为界向内以3个同心圆分区.分别用Gr-1-FITC抗体和CD31/PECAM-1-PE抗体对角膜中性粒细胞和血管进行免疫荧光染色,用免疫荧光显微镜和AR软件测量角膜血管面积并计数中性粒细胞.选取3种小鼠各8只制备角膜创伤模型,用无菌手术刀片以角膜中央为中心做十字划痕,深度至角膜前弹力层.创伤后即刻(0 h)、12h、24 h用2 g/L荧光素钠点眼,荧光显微镜下以AR软件计算不同时间点的创伤面积.于划痕后24 h取小鼠角膜制备铺片,用Gr-1-FITC抗体和CD31/PECAM-1-PE抗体行荧光染色,比较3种创伤小鼠角膜的血管分布、中性粒细胞的数量和分布情况.实验动物的饲养与使用均遵循美国视觉与眼科研究协会制定的科研动物使用规范. 结果 正常C57BL/6J小鼠、BALB/c小鼠和裸鼠角膜中性粒细胞总数分别为(1 733±237)、(353±96)和(1 601 ±223)个/角膜,BALB/c小鼠角膜中性粒细胞数明显少于C57BL/6J小鼠和裸鼠,差异均有统计学意义(P＜0.01).3种正常小鼠角膜缘均可见血管分布,BALB/c小鼠角膜缘血管面积明显小于C57BL/6J小鼠和裸鼠,C57BL/6J小鼠角膜缘血管面积明显大于裸鼠,差异均有统计学意义(P＜0.01).3种正常小鼠中性粒细胞的数量与血管面积均呈明显正相关(C57BL/6J:r=0.936,P=0.001;BALB/c:r =0.939,P=0.001;裸鼠:r=0.935,P=0.001).角膜划痕后24 h,C57BL/6J小鼠和裸鼠的创伤面积均明显大于BALB/c小鼠(均P=0.000),且以裸鼠的创伤面积最大.角膜划痕后C57BL/6J模型鼠、BALB/c模型鼠和模型裸鼠的角膜上中性粒细胞数目分别为(3 340±417)、(1 235±337)和(4 680±450)个/角膜,BALB/c模型鼠角膜上中性粒细胞数目明显少于C57BL/6J模型鼠和模型裸鼠,差异均有统计学意义(均P=0.000).3种小鼠角膜创伤面积与中性粒细胞数目均呈明显正相关(C57BL/6J小鼠:r=0.805,P=0.016;BALB/c小鼠:r=0.943,P=0.000;裸鼠:r=0.916,P=0.001). 结论 不同品系的正常小鼠角膜中性粒细胞分布的数目取决于角膜血管的分布和面积,角膜损伤后小鼠角膜中性粒细胞数目增加,角膜中性粒细胞数目较少的小鼠其角膜创伤修复速度较快,提示角膜血管网部位中性粒细胞的基础状态可能与角膜对感染的易感性以及感染后的早期反应有关.