RH株弓形虫颅内定位注射建立急性弓形虫脑炎小鼠模型的研究

Construction of an acute toxoplasma encephalitis mouse model by the stereotaxic surgery with RH toxoplasma trophozoites

目的观察RH株弓形虫定位感染小鼠海马引起的组织病变,以构建急性弓形虫脑炎小鼠模型。方法利用颅内定位注射技术,将定量(100、500、1000个)RH株弓形虫滋养体,定位注射入小鼠海马CA1区,观察小鼠的生存状况;Giemsa染色方法观察小鼠腹水和脑组织匀浆中弓形虫数量的变化;尼氏染色和HE染色观察小鼠海马神经组织的病理改变;用免疫组化ABC法观察弓形虫在脑组织的分布。结果RH株弓形虫感染各组小鼠在感染第4天表现出明显的弓背、竖毛、腹胀、头颅细微震颤、偏瘫等症状。定量100感染组小鼠生存时间较长,腹水中未发现弓形虫滋养体,脑组织匀浆在96 h后发现少量的假包囊,小鼠死亡后则有较多滋养体;尼氏染色和HE染色则在144 h发现较多的组织坏死灶,CA1区神经细胞缺失;随感染时间的延长而脑组织损伤加重。而定量500感染组和定量1000感染组小鼠腹水和脑匀浆中均发现弓形虫滋养体;尼氏染色发现海马区神经元丢失和大量坏死;HE染色发现组织坏死灶及炎性细胞浸润,脑组织损伤均较定量100感染组小鼠显著加重;免疫组化证实坏死灶中有弓形虫的分布。结论定位感染RH株弓形虫100个滋养体小鼠存活时间较长,脑组织病理变化逐渐加重,损伤局限于脑组织内,小鼠表现典型的弓形虫脑炎症状。因此定位感染100个弓形虫滋养体可以构建急性弓形虫脑炎小鼠模型,可为弓形虫对脑神经的损伤机制研究奠定基础。

Objective To construct an acute toxoplasma encephalitis mouse model by observing the pathological changes in the hippocampus of mice infected with Toxoplasma gondii strain RH.Methods The quantitative RH Toxoplasma gondii(100,500,and 1000 trophozoites)were injected into the hippocampal CA1 region of mice by the stereotaxic surgery;the survival status of mice was observed.Giemsa staining was used to observe the changes of toxoplasma in mouse ascites and brain tissue homogenates.Nissl staining and HE staining were used to observe the pathological changes of hippocampal nerve tissue.The distribution of Toxoplasma gondii in brain tissue was observed by immunohistochemical ABC method.Results The RH Toxoplasma gondii infected mice showed obvious symptoms such as arched back,bristling hair,abdominal distension,subtle tremor and hemiplegia on the fourth day of infection.The survival of mice in 100 trophozoites group was longer,no trophozoites of Toxoplasma gondii were found in ascites,a few pseudocysts were found in brain tissue homogenates after infected for 96 hours,and more trophozoites were found after death.Nysl staining and HE staining showed more tissue necrosis foci and loss of nerve cells in CA1 area after infected 144 h.The injury aggravated with the prolongation of infection time.Toxoplasma trophozoites were found in ascites and brain homogenates of mice in 500 and 1000 trophozoites groups.Nissl staining revealed neuronal loss and massive necrosis in the hippocampus.HE staining showed necrosis and inflammatory cell infiltration.The brain tissue injury significantly aggravated compared with 100 trophozoites group.The distribution of Toxoplasma gondii in the necrotic foci was confirmed by immunohistochemistry.Conclusion The survival of 100 trophozoite mice infected with Toxoplasma gondii strain RH was longer,and the pathological changes of brain tissue gradually aggravated.The damage was relatively confined to the brain tissue,and the mice showed typical symptoms of toxoplasma encephalitis.Therefore,the mouse model of acute toxoplasma encephalitis can be constructed by localized infection of 100 toxoplasma trophozoites,which can lay a foundation for future research on the mechanism of toxoplasma injury to cranial nerves.