Stable topical application of antimicrobials using plumbing rings in an ex vivo porcine corneal infection model

Abstract

Microbial keratitis (MK) is a substantial cause of clinical blindness worldwide. Pseudomonas aeruginosa is an opportunistic gram-negative bacterium and is the leading cause of MK. Infection models are vital tools in understanding host-pathogen interactions and the development of novel therapies. As well as ethical and practical advantages, ex vivo infection models can allow researchers to better investigate host-pathogen interactions more accurately than traditional cell culture systems. The versatility of porcine corneal ex vivo models have been employed to study various pathogens (for example Staphylococcus aureus and Acanthamoeba) and has enabled innovation of novel MK therapies. Here, we describe an improved porcine corneal ex vivo protocol, which uses plumbing rings and medical adhesive to circumvent several distinct limitations and challenges. The application of a 10 mm plumbing ring to the center of the cornea allows localized inoculation of pathogens of interest, maintaining them at the site of infection, rather than running the risk of "run off" of topically added aqueous solutions. The second important advantage is that topically applied therapeutic agents can be properly maintained on the cornea within the plumbing ring reservoir, allowing more accurate study of antimicrobial effects. In this contextualized protocol, we infected porcine corneas with the P. aeruginosa strain PA103 with topical treatments of moxifloxacin. PA103 colony forming unit (CFU) deduction, quantification of corneal opacity and histology analysis (hematoxylin and eosin staining), was used to assess infection over 48h, which was alleviated by moxifloxacin in a dose-dependent manner.