title: Scleral fibroblast response to experimental glaucoma in mice.
abstract: OBJECTIVE
  To study the detailed cellular and molecular changes in the mouse sclera subjected to experimental glaucoma.
  
  
  METHODS
  Three strains of mice underwent experimental bead-injection glaucoma and were euthanized at 3 days and 1, 3, and 6 weeks. Scleral protein expression was analyzed with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using (16)O/(18)O labeling for quantification in 1- and 6-week tissues. Sclera protein samples were also analyzed with immunoblotting with specific antibodies to selected proteins. The proportion of proliferating scleral fibroblasts was quantified with Ki67 and 4',6-diamidino-2-phenylindole (DAPI) labeling, and selected proteins were studied with immunohistochemistry.
  
  
  RESULTS
  Proteomic analysis showed increases in molecules involved in integrin-linked kinase signaling and actin cytoskeleton signaling pathways at 1 and 6 weeks after experimental glaucoma. The peripapillary scleral region had more fibroblasts than equatorial sclera (p=0.001, n=217, multivariable regression models). There was a sixfold increase in proliferating fibroblasts in the experimental glaucoma sclera at 1 week and a threefold rise at 3 and 6 weeks (p=0.0005, univariate regression). Immunoblots confirmed increases for myosin, spectrin, and actinin at 1 week after glaucoma. Thrombospondin-1 (TSP-1), HINT1, vimentin, actinin, and α-smooth muscle actin were increased according to immunohistochemistry.
  
  
  CONCLUSIONS
  Scleral fibroblasts in experimental mouse glaucoma show increases in actin cytoskeleton and integrin-related signaling, increases in cell division, and features compatible with myofibroblast transition.
authors.affiliation: Glaucoma Center of Excellence, Wilmer Ophthalmological Institute Department of Ophthalmology, Johns Hopkins University, Baltimore, MD.
authors.forename: Ericka N
authors.initials: EN
authors.lastname: Oglesby
authors.affiliation: Department of Ophthalmology, Columbia University, New York, NY.
authors.forename: Gülgün
authors.initials: G
authors.lastname: Tezel
authors.affiliation: Glaucoma Center of Excellence, Wilmer Ophthalmological Institute Department of Ophthalmology, Johns Hopkins University, Baltimore, MD.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Cone-Kimball
authors.affiliation: Glaucoma Center of Excellence, Wilmer Ophthalmological Institute Department of Ophthalmology, Johns Hopkins University, Baltimore, MD.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Steinhart
authors.affiliation: Glaucoma Center of Excellence, Wilmer Ophthalmological Institute Department of Ophthalmology, Johns Hopkins University, Baltimore, MD.
authors.forename: Joan
authors.initials: J
authors.lastname: Jefferys
authors.affiliation: Glaucoma Center of Excellence, Wilmer Ophthalmological Institute Department of Ophthalmology, Johns Hopkins University, Baltimore, MD.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Pease
authors.affiliation: Glaucoma Center of Excellence, Wilmer Ophthalmological Institute Department of Ophthalmology, Johns Hopkins University, Baltimore, MD.
authors.forename: Harry A
authors.initials: HA
authors.lastname: Quigley
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D049109:Cell Proliferation / Q000502:physiology; D002853:Chromatography, Liquid; D004195:Disease Models, Animal; D005136:Eye Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D005901:Glaucoma / Q000378:metabolism / Q000503:physiopathology*; D015151:Immunoblotting; D007211:Indoles / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D040901:Proteomics; D012590:Sclera / Q000166:cytology*; D053719:Tandem Mass Spectrometry; D000099200:Scaffold Protein ILK
chemical_list: D000199:Actins; D005136:Eye Proteins; D007211:Indoles; D019394:Ki-67 Antigen; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK; C007293:DAPI