Rodriguez Fernandez Veronica, Amato Rosario, Piaggi Simona, Pinto Barbara, Casini Giovanni, Bruschi Fabrizio
Department of Translational Research, School of Medicine, University of Pisa, 56126 Pisa, Italy.
Department of Infectious Diseases and Public Health, La Sapienza University, 00185 Rome, Italy.
Pathogens. 2024 Aug 19;13(8):701. doi: 10.3390/pathogens13080701.
Ocular toxoplasmosis is the most prevalent clinical manifestation of 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 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. 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 infection, and it was characterized by increased necroptotic but not apoptotic markers. The proposed model recapitulates the main characteristics of 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.
眼部弓形虫病是感染最常见的临床表现，可导致不可逆的视网膜损伤。人们已开发出不同的实验模型来研究这种病理情况。在本研究中，我们提出了一种新的体外模型，以有助于阐明疾病机制和探索可能的治疗方案。按照既定方案从小鼠视网膜制备的体外视网膜外植体，与在Vero细胞中培养的速殖子一起孵育。在不同时间，即从孵育12小时到10天，通过免疫荧光和蛋白质印迹分析外植体，以研究它们对寄生虫感染的反应。培养3天后，可见寄生虫侵入视网膜全层，在视网膜细胞核周围可检测到寄生虫。与此同时，出现了假定的包囊形成和小胶质细胞活化。同时，与对照相比，在感染的外植体中检测到炎症和氧化应激标志物明显增加。感染3天后，视网膜外植体中也似乎发生了细胞死亡，其特征是坏死性凋亡标志物增加，而凋亡标志物未增加。所提出的模型在孵育3天内概括了视网膜感染的主要特征，因此，可用于研究该过程的早期事件。此外，它只需要有限数量的动物，并且便于操作，易于设置不同的实验条件和评估假定治疗药物的效果。
