Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis.
Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis.
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DOI：
10.1007/s00417-016-3546-0
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发表时间：
2017-02
期刊：
影响因子：
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通讯作者：
Garg P
中科院分区：
文献类型：
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作者：
Pinnock A;Shivshetty N;Roy S;Rimmer S;Douglas I;MacNeil S;Garg P
在微生物角膜炎的研究中，体内动物模型往往需要大量动物，而体外单层细胞培养不能维持体内模型组织的三维结构或细胞间的通讯。在这里，我们提出可重复的单感染和双重感染角膜炎离体模型作为体内和体外模型的替代方案。使用108个金黄色葡萄球菌、铜绿假单胞菌、白色念珠菌或茄病镰刀菌细胞感染维持在器官培养物中的离体兔和人角巩膜边缘。通过用手术刀受伤并将角膜暴露于微生物悬浮液或通过基质内注射来引入感染。接种后，角膜在 37°C 下保存 24 和 48 小时。孵育后，将角膜均质化以确定菌落形成单位（CFU）/角膜，或使用常规染色方法进行处理以进行组织学检查。比较了单一物种和混合物种感染。我们观察到，与 24 小时相比，48 小时后金黄色葡萄球菌和铜绿假单胞菌的 CFU 显着增加。然而，在感染白色念珠菌或茄镰孢的角膜中没有观察到这种增加。注射方法使来自受感染角膜的大多数生物体增加了大约 2 至 100 倍 (p<0.05)。手术刀损伤模型和注射模型的组织学表明，铜绿假单胞菌在整个角膜中广泛浸润，而金黄色葡萄球菌、白色念珠菌和茄镰孢的浸润较少。该模型还支持双重感染。手术刀损伤和注射方法均适合诱导离体兔和人角膜模型的感染。这些简单且可重复的模型将可作为体外和体内模型的替代方案，用于研究微生物角膜炎的检测和治疗，特别是当这可能是由两种感染性生物体引起时。
In the study of microbial keratitis, in vivo animal models often require a large number of animals, and in vitro monolayer cell culture does not maintain the three-dimensional structure of the tissues or cell-to-cell communication of in vivo models. Here, we propose reproducible ex vivo models of single- and dual-infection keratitis as an alternative to in vivo and in vitro models. Excised rabbit and human corneoscleral rims maintained in organ culture were infected using 108 cells of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans or Fusarium solani. The infection was introduced by wounding with a scalpel and exposing corneas to the microbial suspension or by intrastromal injection. Post-inoculation, corneas were maintained for 24 and 48 h at 37 °C. After incubation, corneas were either homogenised to determine colony-forming units (CFU)/cornea or processed for histological examination using routine staining methods. Single- and mixed-species infections were compared. We observed a significant increase in CFU after 48 h compared to 24 h with S. aureus and P. aeruginosa. However, no such increase was observed in corneas infected with C. albicans or F. solani. The injection method yielded an approximately two- to 100-fold increase (p < 0.05) in the majority of organisms from infected corneas. Histology of the scalpel-wounded and injection models indicated extensive infiltration of P. aeruginosa throughout the entire cornea, with less infiltration observed for S. aureus, C. albicans and F. solani. The models also supported dual infections. Both scalpel wounding and injection methods are suitable for inducing infection of ex vivo rabbit and human cornea models. These simple and reproducible models will be useful as an alternative to in vitro and in vivo models for investigating the detection and treatment of microbial keratitis, particularly when this might be due to two infective organisms.