Abstract: Objective　To investigate the changes in eosinophil counts and the activation of microglial cells in the brain tissues of mice at different stages of Angiostrongylus cantonensis infection, and to examine the role of microglia in regulating the progression of angiostrongyliasis and unravel the possible molecular mechanisms. Methods　Fifty BALB/c mice were randomly divided into the control group and the 7⁃d, 14⁃d, 21⁃day and 25⁃d infection groups, of 10 mice in each group. All mice in infection groups were infected with 30 stage Ⅲ A. cantonensis larvae by gavage, and animals in the control group was given an equal amount of physiological saline. Five mice were collected from each of infection groups on days 7, 14, 21 d and 25 d post⁃infection, and 5 mice were collected from the control group on the day of oral gavage. The general and focal functional impairment was scored using the Clark scoring method to assess the degree of mouse neurological impairment. Five mice from each of infection groups were sacrificed on days 7, 14, 21 d and 25 d post⁃infection, and 5 mice from the control group were sacrificed on the day of oral gavage. Mouse brain tissues were sampled, and the pathological changes of brain tissues were dynamically observed using hematoxylin and eosin (HE) staining. Immunofluorescence staining with eosinophilic cationic protein (ECP) and ionized calcium binding adaptor molecule 1 (Iba1) was used to assess the degree of eosinophil infiltration and the counts of microglial cells in mouse brain tissues in each group, and the morphological parameters of microglial cells (skeleton analysis and fractal analysis) were quantified by using Image J software to determine the morphological changes of microglial cells. In addition, the expression of M1 microglia markers Fcγ receptor Ⅲ (Fcgr3), Fcγ receptor Ⅱb (Fcgr2b) and CD86 antigen (Cd86), M2 microglia markers Arginase 1 (Arg1), macrophage mannose receptor C⁃type 1 (Mrc1), chitinase⁃like 3 (Chil3), and phagocytosis genes myeloid cell triggering receptor expressed on myeloid cells 2 (Trem2), CD68 antigen (Cd68), and apolipoprotein E (Apoe) was quantified using real⁃time quantitative reverse transcription PCR (RT⁃qPCR) assay in the mouse cerebral cortex of mice post⁃infection. Results　A large number of A. cantonensis larvae were seen on the mouse meninges surface post⁃infection, and many neuronal nuclei were crumpled and deeply stained, with a large number of bleeding points in the meninges. The median Clark scores of mouse general functional impairment were 0 (interquartile range, 0), 0 (interquartile range, 0.5), 6 (interquartile range, 1.0), 14 (interquartile range, 8.5) points and 20 (interquartile range, 9.0) points in the control group and the 7⁃d, 14⁃d, 21⁃d and 25⁃d groups, respectively (H = 22.45, P < 0.01), and the median Clark scores of mouse focal functional impairment were 0 (interquartile range, 0), 2 (interquartile range, 2.5), 7 (interquartile range, 3.0), 18 (interquartile range, 5.0) points and 25 (interquartile range, 6.5) points in the control group and the 7⁃d, 14⁃d, 21⁃d and 25⁃d groups, respectively (H = 22.72, P < 0.01). The mean scores of mice general and focal functional impairment were all higher in the infection groups than in the control group (all P values < 0.05). Immunofluorescence staining showed a significant difference in the eosinophil counts in mouse brain tissues among the five groups (F = 40.05, P < 0.000 1), and the eosinophil counts were significantly higher in mouse brain tissues in the 14⁃d (3.08 ± 0.78) and 21⁃d infection groups (5.97 ± 1.37) than in the control group (1.00 ± 0.28) (both P values < 0.05). Semi⁃quantitative analysis of microglia immunofluorescence showed a significant difference in the counts of microglial cells among the five groups (F = 17.66, P < 0.000 1), and higher Iba1 levels were detected in mouse brain tissues in 14⁃d (5.75 ± 1.28), 21⁃d (6.23 ± 1.89) and 25⁃d infection groups (3.70 ± 1.30) than in the control group (1.00 ± 0.30) (all P values < 0.05). Skeleton and fractal analyses showed that the branch length [(162.04 ± 34.10) μm vs. (395.37 ± 64.11) μm; t = 5.566, P < 0.05] and fractal dimension of microglial cells [(1.30 ± 0.01) vs. (1.41 ± 0.03); t = 5.266, P < 0.05] were reduced in mouse brain tissues in the 21⁃d infection group relative to the control group. In addition, there were significant differences among the 5 groups in terms of M1 and M2 microglia markers Fcgr3 (F = 48.34, P < 0.05) , Fcgr2b (F = 55.46, P < 0.05), Cd86 (F = 24.44, P < 0.05), Arg1 (F = 31.18, P < 0.05), Mrc1 (F = 15.42, P < 0.05) and Chil3 (F = 24.41, P < 0.05), as well as phagocytosis markers Trem2 (F = 21.19, P < 0.05), Cd68 (F = 43.95, P < 0.05) and Apoe (F = 7.123, P < 0.05) in mice brain tissues. Conclusions　A. cantonensis infections may induce severe pathological injuries in mouse brain tissues that are characterized by massive eosinophil infiltration and persistent activation of microglia cells, thereby resulting in progressive deterioration of neurological functions. 

Key words: Angiostrongylus cantonensis, Brain tissue, Eosinophil, Microglia, Cell activation, Mouse

摘要： 目的　观察广州管圆线虫不同感染阶段小鼠脑组织中嗜酸性粒细胞数量变化以及小胶质细胞活化状态，探究小胶质细胞在调节广州管圆线虫病进程中的作用和可能的分子机制。方法　50只6～8周龄雌性BALB/c小鼠随机分为对照组和感染7、14、21、25 d组，每组10只。各感染组小鼠均通过灌胃方式感染30条广州管圆线虫Ⅲ期幼虫，对照组以等量生理盐水灌胃。感染7、14、21、25 d组分别于灌胃感染后7、14、21、25 d随机各取5只小鼠，对照组于灌胃当天随机取5只小鼠，采用Clark评分法对小鼠神经功能损伤程度进行一般功能和局灶功能损伤评分。感染7、14、21、25 d组分别于感染后7、14、21、25 d处死5只小鼠，对照组于灌胃当天处死5只小鼠，并取各组小鼠脑组织，经苏木素⁃伊红（hematoxylin and eosin，HE）染色后动态观察脑组织病理改变。此外，采用嗜酸性粒细胞阳离子蛋白（eosinophilic cationic protein，ECP）和离子钙结合衔接分子 1（ionized calcium binding adaptor molecule 1，Iba1）免疫荧光染色，分别评估各组小鼠脑组织中嗜酸性粒细胞浸润程度和小胶质细胞数量，采用Image J软件通过骨架分析、分形分析等量化小胶质细胞形态，并判断其形态变化。采用实时荧光定量PCR（real⁃time quantitative reverse transcription polymerase chain reaction，RT⁃qPCR）检测感染后小鼠大脑皮质中小胶质细胞M1型标志物Fcγ受体Ⅲ（Fc receptor Ⅲ，Fcgr3）、Fcγ受体Ⅱb（Fc receptor IIb，Fcgr2b）、CD86抗原（Cd86），M2型标志物精氨酸酶1（arginase 1，Arg1）、巨噬细胞甘露糖受体1（mannose receptor C⁃type 1，Mrc1）、几丁质酶样蛋白3（chitinase⁃like 3，Chil3）以及吞噬功能基因髓细胞触发受体2（triggering receptor expressed on myeloid cells 2，Trem2）、CD68抗原（Cd68）和载脂蛋白E（apolipoprotein E，Apoe）等基因表达水平。结果　感染广州管圆线虫后，小鼠脑膜表面可见大量广州管圆线虫幼虫，较多神经元胞核皱缩且深染，脑膜内出现大量出血点。对照组及感染7、14、21、25 d组小鼠一般功能损伤Clark评分中位数（四分位间距）［M（QR）］分别为0（0）、0（0.5）、6（1.0）、14（8.5）、20（9.0）分，差异有统计学意义（H = 22.45，P < 0.01）；各组小鼠局灶功能损伤Clark评分分别为0（0）、2（2.5）、7（3.0）、18（5.0）、25（6.5）分，差异有统计学意义（H = 22.72，P < 0.01）。各感染组小鼠一般功能损伤和局灶功能损伤评分均高于对照组（P 均< 0.05）。免疫荧光染色结果显示，5组小鼠脑组织中嗜酸性粒细胞数量差异有统计学意义（F = 40.05，P < 0.000 1），感染14 d组（3.08 ± 0.78）、21 d组（5.97 ± 1.37）小鼠脑组织中嗜酸性粒细胞数量高于对照组（1.00 ± 0.28）（P 均< 0.05）；5组小鼠脑组织中小胶质细胞数量差异有统计学意义（F = 17.66，P < 0.000 1），其中感染14 d组（5.75 ± 1.28）、21 d组（6.23 ± 1.89）和25 d组（3.70 ± 1.30）小鼠脑组织中Iba1水平高于对照组（1.00 ± 0.30）（P 均 < 0.05）。细胞骨架分析和分形分析结果显示，感染21 d组小鼠脑组织中小胶质细胞分支长度［（162.04 ± 34.10） μm vs.（395.37 ± 64.11） μm；t = 5.566，P < 0.05］、分形维数［（1.30 ± 0.01）vs.（1.41 ± 0.03）；t = 5.266，P < 0.05］均低于对照组。5组小鼠脑组织中小胶质细胞M1、M2标志物Fcgr3、Fcgr2b、Cd86、Arg1、Mrc1、Chil3和吞噬细胞标志物Trem2、Cd68、Apoe相对表达量差异均有统计学意义（F = 48.34、55.46、24.44、31.18、15.42、24.41、21.19、43.95、7.123，P 均 < 0.05）。结论　广州管圆线虫感染可导致小鼠脑组织发生严重病理损伤，伴随嗜酸性粒细胞大量浸润和小胶质细胞持续活化，进而导致神经功能进行性恶化。

关键词: 广州管圆线虫；脑组织；嗜酸性粒细胞；小胶质细胞；细胞活化；小鼠