Veronica Rodriguez Fernandez, Rosario Amato, Simona Piaggi, Barbara Pinto, Giovanni Casini, Fabrizio Bruschi
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At different times, starting at 12 h up to 10 days of incubation, the explants were analyzed with immunofluorescence and Western blot to investigate their responses to parasite infection. <i>T. gondii</i> invasion of the retinal thickness was evident after 3 days in culture, where parasites could be detected around retinal cell nuclei. This was paralleled by putative cyst formation and microglial activation. At the same time, an evident increase in inflammatory and oxidative stress markers was detected in infected explants compared to controls. Cell death also appeared to occur in retinal explants after 3 days of <i>T. gondii</i> infection, and it was characterized by increased necroptotic but not apoptotic markers. The proposed model recapitulates the main characteristics of <i>T. gondii</i> retinal infection within 3 days of incubation and, therefore, allows for studying the very early events of the process. 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引用次数: 0
摘要
眼弓形虫病是弓形虫感染最常见的临床表现,会造成不可逆的视网膜损伤。目前已开发出不同的实验模型来研究这种病理现象。本研究提出了一种新的体外模型,以帮助阐明疾病机制和可能的治疗方案。按照既定方案从小鼠视网膜上制备的体外视网膜外植体与维持在 Vero 细胞中的淋病双球菌瞬生体一起孵育。从孵育 12 小时到孵育 10 天的不同时间,用免疫荧光和 Western 印迹分析外植体,研究它们对寄生虫感染的反应。培养 3 天后,淋球菌明显侵入视网膜厚度,在视网膜细胞核周围可检测到寄生虫。与此同时,寄生虫还可能形成囊肿并激活小胶质细胞。同时,与对照组相比,在受感染的外植体中检测到炎症和氧化应激标记物明显增加。淋球菌感染 3 天后,视网膜外植体中也出现了细胞死亡,其特征是坏死标志物增加,而不是凋亡标志物增加。所提出的模型再现了淋病双球菌在培养 3 天内感染视网膜的主要特征,因此可以对这一过程的早期事件进行研究。此外,该模型只需要有限数量的动物,易于操作,便于设置不同的实验条件和评估治疗药物的效果。
A New Ex Vivo Model Based on Mouse Retinal Explants for the Study of Ocular Toxoplasmosis.
Ocular toxoplasmosis is the most prevalent clinical manifestation of T. gondii infection, which causes irreversible retinal damage. Different experimental models have been developed to study this pathology. In the present study, a new, ex vivo model is proposed to contribute to the elucidation of disease mechanisms and to possible therapeutic solutions. Ex-vivo retinal explants, prepared from mouse retinas following established protocols, were incubated with T. gondii tachyzoites maintained in Vero cells. At different times, starting at 12 h up to 10 days of incubation, the explants were analyzed with immunofluorescence and Western blot to investigate their responses to parasite infection. T. gondii invasion of the retinal thickness was evident after 3 days in culture, where parasites could be detected around retinal cell nuclei. This was paralleled by putative cyst formation and microglial activation. At the same time, an evident increase in inflammatory and oxidative stress markers was detected in infected explants compared to controls. Cell death also appeared to occur in retinal explants after 3 days of T. gondii infection, and it was characterized by increased necroptotic but not apoptotic markers. The proposed model recapitulates the main characteristics of T. gondii retinal infection within 3 days of incubation and, therefore, allows for studying the very early events of the process. In addition, it requires only a limited number of animals and offers easy manipulation and accessibility for setting up different experimental conditions and assessing the effects of putative drugs for therapy.
期刊介绍:
Pathogens (ISSN 2076-0817) publishes reviews, regular research papers and short notes on all aspects of pathogens and pathogen-host interactions. There is no restriction on the length of the papers. Our aim is to encourage scientists to publish their experimental and theoretical research in as much detail as possible. Full experimental and/or methodical details must be provided for research articles.