title: Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  Excised rabbit and human corneoscleral rims maintained in organ culture were infected using 108cells 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: University of Sheffield, Sheffield, S10 2TA, UK.
authors.forename: Abigail
authors.initials: A
authors.lastname: Pinnock
authors.affiliation: LV Prasad Eye Institute, Banjara Hills, Hyderabad, 500034, India.
authors.forename: Nagaveni
authors.initials: N
authors.lastname: Shivshetty
authors.affiliation: LV Prasad Eye Institute, Banjara Hills, Hyderabad, 500034, India.
authors.forename: Sanhita
authors.initials: S
authors.lastname: Roy
authors.affiliation: University of Bradford, Bradford, BD7 1DP, UK.
authors.forename: Stephen
authors.initials: S
authors.lastname: Rimmer
authors.affiliation: University of Sheffield, Sheffield, S10 2TA, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Douglas
authors.affiliation: University of Sheffield, Sheffield, S10 2TA, UK. s.macneil@sheffield.ac.uk.|The Kroto Research Institute, North Campus, University of Sheffield, Broad Lane, Sheffield, S3 7HQ, UK. s.macneil@sheffield.ac.uk.
authors.forename: Sheila
authors.initials: S
authors.lastname: MacNeil
authors.affiliation: LV Prasad Eye Institute, Banjara Hills, Hyderabad, 500034, India.
authors.forename: Prashant
authors.initials: P
authors.lastname: Garg
mesh_terms: D000818:Animals; D001419:Bacteria / Q000302:isolation & purification; D015169:Colony Count, Microbial; D003315:Cornea / Q000382:microbiology* / Q000473:pathology; D004195:Disease Models, Animal; D015818:Eye Infections, Bacterial / Q000175:diagnosis* / Q000382:microbiology; D015821:Eye Infections, Fungal / Q000175:diagnosis* / Q000382:microbiology; D005658:Fungi / Q000302:isolation & purification; D006801:Humans; D007634:Keratitis / Q000175:diagnosis* / Q000382:microbiology; D008856:Microscopy, Fluorescence; D009924:Organ Culture Techniques; D011817:Rabbits
keywords: Colony-forming units; Corneal model; Ex vivo cornea; Microbial keratitis