title: In vitro model of infectious crystalline keratopathy: tissue architecture determines pattern of microbial spread.
abstract: OBJECTIVE
  To develop an in vitro model of infectious crystalline keratopathy using human corneal buttons and to test the hypothesis that the compactness of the corneal stroma determines the pattern of microbial spread.
  
  
  METHODS
  Twenty human corneal buttons obtained after penetrating keratoplasty for keratoconus (KC) and eight human corneal buttons obtained from eye bank (EB) donor eyes were maintained in organ culture. Fourteen buttons (10 KC and 4 EB donors) were maintained in a turgid state (swollen, edematous) and 14 in a nonturgid state (compact, normal state of deturgescence) by the omission or addition of 5% dextran to the culture medium. Eight KC and four EB nonturgid buttons and eight KC and four EB turgid buttons were inoculated with Streptococcus viridans (Lancefield group G, gram-positive) organisms. Two KC nonturgid and two KC turgid buttons were inoculated with Klebsiella oxytoca (gram-negative) organisms. Bacterial migration and spread in the tissue were observed by light and electron microscopy.
  
  
  RESULTS
  Of the nonturgid buttons, six KC buttons and all four EB buttons inoculated with S. viridans and both KC buttons inoculated with K. oxytoca demonstrated an arborizing, crystallike pattern of bacterial spread. In the turgid buttons, five KC and all four EB buttons inoculated with S. viridans and both KC buttons inoculated with K. oxytoca demonstrated globular, amorphous colonies. This was in complete contrast to the needlelike branching appearance seen in nonturgid corneal buttons. Electron microscopy confirmed an interlamellar spread of the bacterial colonies.
  
  
  CONCLUSIONS
  This is the first in vitro model of bacterial keratitis. It demonstrates that the pattern of spread of bacteria within corneal tissue is largely determined by the compactness of the corneal stroma. Altering tissue architecture changed the pattern of bacterial migration and spread. This model has considerable potential in further understanding host-microbe interactions and microbial spread that occurs during infection.
authors.affiliation: Larry A. Donoso Laboratory for Eye Research, Division of Ophthalmology and Vision Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom.
authors.forename: T K
authors.initials: TK
authors.lastname: Butler
authors.forename: H S
authors.initials: HS
authors.lastname: Dua
authors.forename: R
authors.initials: R
authors.lastname: Edwards
authors.forename: J S
authors.initials: JS
authors.lastname: Lowe
mesh_terms: D000328:Adult; D003319:Corneal Stroma / Q000382:microbiology* / Q000648:ultrastructure; D005260:Female; D006801:Humans; D007634:Keratitis / Q000382:microbiology* / Q000473:pathology; D007640:Keratoconus / Q000382:microbiology / Q000601:surgery; D015948:Keratoplasty, Penetrating; D007709:Klebsiella / Q000472:pathogenicity*; D008297:Male; D008855:Microscopy, Electron, Scanning; D008954:Models, Biological; D009924:Organ Culture Techniques; D013291:Streptococcus / Q000472:pathogenicity*