title: Prolonged hypoxia induces lipid raft formation and increases Pseudomonas internalization in vivo after contact lens wear and lid closure.
abstract: OBJECTIVE
  To investigate the effects of hypoxia on lipid raft formation and Pseudomonas aeruginosa internalization by the corneal epithelium with and without the physical effects of contact lens wear.
  
  
  METHODS
  One eye of each rabbit was randomly fitted with a low-Dk rigid gas-permeable contact lens (LDCTL) or closed with sutures, with the other as a control. After 1 day or 3 days, the rabbits were killed and bacterial invasion was assessed by gentamicin survival assay. Lipid rafts were identified by staining with FITC-conjugated beta subunit of cholera toxin. Corneal epithelial Bcl-2 expression was detected by Western blotting; surface epithelial cell size and thickness (epithelium and stroma) were measured by confocal microscopy.
  
  
  RESULTS
  One-day hypoxia induced no significant changes in P. aeruginosa internalization, Bcl-2 expression, or lipid raft formation except in one of four eyelid-closed eyes. After 3 days, P. aeruginosa internalization was increased significantly (P < 0.05) in LDCTL-wearing eyes and not significantly (P = 0.10) increased in eyelid-closed eyes. Both 3-day test conditions also induced lipid raft-forming cells that bound P. aeruginosa, albeit in different regions of the cornea (peripherally in LDCTL-wearing eyes and centrally in closed eyes); did not alter epithelial thickness or surface cell size; and appeared to decrease epithelial Bcl-2 expression.
  
  
  CONCLUSIONS
  This is the first direct comparison in vivo between two different methods inducing hypoxia on the corneal surface. Association of P. aeruginosa internalization with lipid raft formation in both conditions suggests a critical link among prolonged hypoxia, lipid raft formation, and susceptibility to P. aeruginosa infection. However, different distribution patterns of lipid raft-forming cells suggest physical effects of contact lens wear may direct localization of lipid raft-associated P. aeruginosa internalization on the corneal surface.
authors.affiliation: Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, 75039-9057, USA.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Naoka
authors.initials: N
authors.lastname: Yamamoto
authors.forename: James V
authors.initials: JV
authors.lastname: Jester
authors.forename: W Matthew
authors.initials: WM
authors.lastname: Petroll
authors.forename: H Dwight
authors.initials: HD
authors.lastname: Cavanagh
mesh_terms: D000818:Animals; D015169:Colony Count, Microbial; D003261:Contact Lenses / Q000009:adverse effects*; D019573:Epithelium, Corneal / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D015818:Eye Infections, Bacterial / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D005260:Female; D005500:Follow-Up Studies; D000860:Hypoxia / Q000378:metabolism*; D066298:In Vitro Techniques; D007634:Keratitis / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D021962:Membrane Microdomains / Q000502:physiology*; D018613:Microscopy, Confocal; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011552:Pseudomonas Infections / Q000378:metabolism / Q000382:microbiology* / Q000473:pathology; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D011817:Rabbits; D013997:Time Factors
chemical_list: D019253:Proto-Oncogene Proteins c-bcl-2