title: Imaging the effects of prostaglandin analogues on cultured trabecular meshwork cells by coherent anti-stokes Raman scattering.
abstract: OBJECTIVE
  The aim of this study was to nondestructively monitor morphological changes to the lipid membranes of primary cultures of living human trabecular meshwork cells (hTMC) without the application of exogenous label.
  
  
  METHODS
  Live hTMC were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). The hTMC were treated with a commercial formulation of latanoprost (0.5 μg/mL) for 24 hours before imaging. Untreated cells and cells treated with vehicle containing the preservative benzalkonium chloride (BAK; 2 μg/mL) were imaged as controls. After CARS/TPAF imaging, hTMC were fixed, stained with the fluorescent lipid dye Nile Red, and imaged by conventional confocal microscopy to verify lipid membrane structures.
  
  
  RESULTS
  Analysis of CARS/TPAF images of hTMC treated with latanoprost revealed multiple intracellular lipid membranes absent from untreated or BAK-treated hTMC. Treatment of hTMC with sodium fluoride or ouabain, agents shown to cause morphological changes to hTMC, also did not induce formation of intracellular lipid membranes.
  
  
  CONCLUSIONS
  CARS microscopy detected changes in living hTMC morphology that were validated by subsequent histological stain. Prostaglandin-induced changes to hTMC involved rearrangement of lipid membranes within these cells. These in vitro results identify a novel biological response to a class of antiglaucoma drugs, and further experiments are needed to establish how this effect is involved in the hypotensive action of prostaglandin analogues in vivo.
authors.affiliation: Department of Electrical Engineering, University of Colorado Denver, Denver, Colorado, USA.
authors.forename: Tim C
authors.initials: TC
authors.lastname: Lei
authors.forename: Omid
authors.initials: O
authors.lastname: Masihzadeh
authors.forename: Malik Y
authors.initials: MY
authors.lastname: Kahook
authors.forename: David A
authors.initials: DA
authors.lastname: Ammar
mesh_terms: D000959:Antihypertensive Agents / Q000494:pharmacology; D002478:Cells, Cultured; D006801:Humans; D007429:Intraocular Pressure; D011465:Prostaglandins, Synthetic / Q000494:pharmacology*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013194:Staining and Labeling; D014129:Trabecular Meshwork / Q000737:chemistry / Q000166:cytology / Q000187:drug effects*
chemical_list: D000959:Antihypertensive Agents; D011465:Prostaglandins, Synthetic
keywords: coherent anti-Stokes Raman scattering (CARS); prostaglandin analogs; trabecular meshwork cells
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