Study On In Vitro Infection Of Tachyzoites Of Toxoplasma Gondii RH Strain In Rat Brain Astrocytes

Objective Infected Toxoplasma gondii can lead to many pathological changes of histologies and organs, and the most severe influence happens to the brain.Because T.gondii can cross the human being's blood brain barrier and make toxoplasmic encephalitis happen.The aim of our study is just to research the machanism that T. gondii infects astrocyres,which is an innate immune cell in brain. So we establish a infection model in vitro for T. gondii RH strain and study the action of autophagy in course of T. gondii invading astrocytes.Methods (1) Primary brain cell were isolated from cerebral tissue neonatal rat. And mixed cell culture were shaken on the orbital shaker , then deserted the supernatant, the left was astrocytes. GFAP(glial fibrillary acidic protein) polyclonal antibody was used to identify astrocytes.(2) Tachyzoites stored were recoveried and injected in the enterocoelia of female mice. When the mice got toxoplasmosis ,the peritoneal fluid were extracted. And the virulence of tachyzoites were observed by microscope. Until it was planted 3 passage in mice, tachyzoites'virulence can be prefect.(3) Astrocytes were plated in culture plates,and they were infected by purified tachyzoites. The change of astrocytes and tachyzoites was observed after Giemsa staining and under microscopy.(4) Time-lapse video was used to analyze the interaction between astrocytes and tachyzoites.(5) MDC(monodansylcadaverine)was used to study the action of autophagy in the process of tachyzoites invading astrocytes. And the variation of MDC-labeled vesicles were analyzed by fluorescence microscopy.Results (1) Purified astrocytes were passaged 3 old for use, and preparations appeared >95% pure.(2) T.gondii can be cultured in mice and be passaged by 2-3 d. (3) By Giemsa staining, the results showed that after 2h, tachyzoites entered astrocyte. 48h later tachyzoites formed pseudo-cyst and were proliferating in astrocyte bodies. And most astrocyte were destroyed in 72h.(4)Time-lapse video analysis showed that tachyzoites were active to invade astrocytes,while astrocytes seemed to be slience toward infection and in 1h TA had entered astrocytes body.(5) By MDC staining, the autophagosomes appeared in 1h. At 4h they were increasing pronouncedly, however after 12h,the autophagosomes were shrinked in company with the cells'injure. And 48h later, autophagosomes disappeared and astrocytes were destroyed totally.Conclusion Rat's astrocytes can be invaded by tachyzoites of T. gondii RH strain. And the infected cycle from tachyzoites entering astrocytes to astrocytes broken is 72 h. Moreover tachyzoites were active to invade astrocytes.Otherwise, it indicated that autophagy is inhibited in defense against T. gondii in astrocytes.