小鼠软脑膜淋巴管内皮细胞的发育特点及其对行为功能的影响

The Development and Function of Leptomeningeal Lymphatic Endothelial Cells in Mouse

【目的】观察小鼠软脑膜淋巴内皮细胞的空间时间分布及其对小鼠行为的影响。【方法】免疫荧光检测1周,2周,4周,10周,15周,15月龄野生型C57BL/6小鼠背侧,颞侧,腹侧,侧脑室旁软脑膜中Lyve-1+和CD68+细胞数量,并将15周龄野生型C57BL/6小鼠和APP/PS1转基因小鼠不同软脑膜区域中Lyve-1+和CD68+细胞数量进行比较。将26只2周龄C57BL小鼠随机进行如下分组:PBS注射组(n=10)、anti-Lyve-1中和抗体注射组(n=9)、SAR131675小分子抑制剂注射组(n=7),3组注射剂量均为2μL。注射后2周(即4周龄)对两组小鼠进行旷场实验,三箱社交实验,新物体识别实验。随后免疫荧光检测三组小鼠软脑膜淋巴内皮细胞的比例。【结果】软脑膜淋巴内皮细胞数量在不同区域分布的差异无统计学意义(F=0.8700,P=0.4668,df=3)。小鼠软脑膜Lyve-1+淋巴管内皮细胞数量呈增龄性下降(F=17.30,P<0.0001,df=5)。不同年龄小鼠背侧软脑膜Lyve-1+和CD68+细胞占Lyve-1+细胞比例差异无统计学意义(F=0.2686,P=0.9244,df=5)。15周龄APP/PS1转基因小鼠软脑膜Lyve-1+细胞比例小于同周龄野生型C57BL/6小鼠(t=6.381,P=0.0078)。中和抗体注射组抑制剂组小鼠软脑膜Lyve-1+细胞占比均低于对照组,差异具有统计学意义(均P<0.001)。旷场实验中,抑制剂注射组小鼠的探索运动能力下降,但对旷场中央区域的探索意愿增强;中和抗体注射组小鼠仅对旷场中央区域的探索意愿增强。三箱社交实验中,中和抗体注射小鼠未表现出社交行为减少和社交偏好。抑制剂注射组小鼠在接触频次表现出社交行为减少,但未表现出社交偏好,说明抑制剂注射小鼠存在社交行为的减少。新物体识别实验中,中和抗体注射小鼠在接触频次,时间与路程上与对照组相比没有变化;但小分子抑制剂注射小鼠在新物体识别实验中表现出短期记忆能力的下降。【结论】软脑膜淋巴内皮细胞的发育可能调控小鼠生命早期的行为功能,可能与其对脑脊液中大分子物质的吞噬有关。

【Objective】To observe the spatial-temporal distribution of mouse leptomeningeal lymphatic endothelial cells and their effects on behavior.【Methods】Immunofluorescence was used to detect the number of Lyve-1+and CD68+cells in the dorsal,temporal,ventral,and lateral paraventricular leptomeningeal of 1-week,2-week,4-week,10-week,15-week,and 15-month-old wild-type C57BL mice and 15-week-old APP/PS1 transgenic mice.Two-week-old C57BL mice were randomly grouped as follows:PBS-injected group,anti-Lyve-1-injected group and SAR131675-inject⁃ed group,which were injected with corresponding reagents into lateral ventricle.Two weeks after injection(i.e.,4 weeks old),the three groups of mice were subjected to the open field experiment,the three-chamber social interaction experi⁃ment,and the novel object recognition experiment.Then their ratio of leptomeningeal lymphatic cells were detected by im⁃munofluorescence.【Results】There was no statistical difference in the distribution of leptomeningeal lymphatic endothelial cells in different regions(F=0.8700,P=0.4668,df=3).The percentage of Lyve-1+cells in the leptomeninges of mice de⁃creased with ages(F=17.30,P<0.0001,df=5).There was no statistical difference in the proportion of Lyve-1+and CD68+cells in the dorsal leptomeninges of mice of different ages(F=0.2686,P=0.9244,df=5).Proportion of Lyve-1+cells in the leptomeninges of 15-week-old APP/PS1 transgenic mice was lower than that of wild-type C57BL/6 mice(t=6.381,P=0.0078).The ratio of Lyve-1+cells was lower in the leptomeningeal of anti-Lyve-1-injected mice than that in the control group(MPBS=0.4513,Manti-Lyve-1=0.2692,q=8.726,P<0.0001).The ratio of Lyve-1+cells in the leptomeningeal of SAR131675-injected mice was lower than that in the control group(MSAR131675=0.3230,q=5.588,P=0.0006).In the open field tests,SAR131675-injected mice showed reduced exploratory locomotion,but increased willingness to explore the central area.The anti-Lyve-1-injected mice showed an increased willingness to explore the central area.In the social in⁃teraction tests,the anti-Lyve-1-injected mice showed no reduction in social behavior or social preference.The SAR131675-injected mice showed reduced social behavior in terms of frequency of interaction but no social preference,suggesting that the SAR131675-injected mice had a social interaction decrease.In the novel object recognition tests,the anti-Lyve-1-injected mice showed no change in frequency,time and distance,indicating that the anti-Lyve-1-injected mice showed no change in short-term memory.The SAR131675-injected mice showed a decrease in short-term memory.【Conclusion】Leptomeningeal lymphatic endothelial cells play an important role in the early development of mice,which can be related to their phagocytosis of macromolecular substances.