title: Immunofluorescence method for quantifying the trabecular meshwork glucocorticoid response (TIGR) protein in trabecular meshwork and Schlemm's canal cells.
abstract: OBJECTIVE
  Decreased flow of aqueous humor through the trabecular meshwork (TM) and into Schlemm's canal (SC) is believed to be a predominant factor in the development of the elevated intraocular pressure found in primary open-angle and steroid glaucoma. Recent biochemical and genetic evidence has suggested that alterations in the expression of the TM glucocorticoid response (TIGR) protein within the chamber angle may play a role in the development of these glaucomas. To understand the process of TIGR induction in outflow pathway cells we developed an assay for TIGR expression that could distinguish both individual cell responses and also provide a semi-quantitative comparison between cell cultures. The present study demonstrates this approach, using digital image capture of immunofluorescently stained human TM and SC cells after treatment with dexamethasone (Dex).
  
  
  METHODS
  Confluent cultures of human TM and SC cells were treated with 1 M Dex or vehicle control for 10 days. The cells were then fixed, permeabilized, and stained using polyclonal antibodies produced against recombinant TIGR. Digital images of fluorescently stained cells, using the same exposure time within an experiment, were evaluated by tabulating the staining intensity of all cells on each image. Between 10 and 40 cells were evaluated per image, 8-10 frames/ sample, 2-3 samples per treatment. Each cell was ranked as either 0 (background), 1+ (minor), 2+ (moderate) or 3+ (very bright staining).
  
  
  RESULTS
  TM cells showed a significant basal level of TIGR staining. About 20% of control cells showed appreciable levels of TIGR staining, with intensity levels evenly distributed between 1, 2 and 3+. Dex treatment increased the number of TM cells expressing TIGR to 60-80%, with the majority of cells showing 2+ to 3+ staining throughout the cytoplasm. SC cells showed no basal TIGR staining, but Dex-treated cells exhibited TIGR staining in 6-15% of cells. SC cell TIGR staining varied between 1+ to 2+ in intensity, and showed a distribution different from TM cells. Staining in SC cells was localized to a ribbon-like compartment adjacent to the nucleus. Such perinuclear localization was rarely seen in TM cells.
  
  
  CONCLUSIONS
  The low standard errors of the mean TIGR responses within each experiment, and the reproducibility between experiments for each cell type, suggests good reliability for this method. At the same time, the consistent, marked contrast between TM and SC cells in their response to glucocorticoids demonstrates that the assay can distinguish significant differences between cell types. The data support the view that Dex has a cell type specific effect on TIGR induction. The different extent, pattern and localization of TIGR staining between cell types suggests that TIGR expression in SC cells could play a functional role in the outflow pathway, but one that may be distinct from that played in TM cells.
authors.affiliation: Department of Biology, University of North Carolina,Chapel Hill 27599, USA. etobrien@email.unc.edu
authors.forename: T E
authors.initials: TE
authors.lastname: O'Brien
authors.forename: C D
authors.initials: CD
authors.lastname: Metheney
authors.forename: J R
authors.initials: JR
authors.lastname: Polansky
mesh_terms: D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D003907:Dexamethasone / Q000494:pharmacology; D005136:Eye Proteins / Q000032:analysis* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D012590:Sclera / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D014129:Trabecular Meshwork / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D003907:Dexamethasone; D005136:Eye Proteins; D006023:Glycoproteins; D000099179:Myocilin