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David Ammar, Omid Masihzadeh, Malik Kahook, Tim Lei; Imaging the Effects of Prostaglandins on Cultured Trabecular Meshwork Cells by Coherent Anti-Stokes Raman Scattering (CARS). Invest. Ophthalmol. Vis. Sci. 2013;54(15):3530. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To non-destructively monitor morphological changes to the lipid membranes of living primary cultures of human trabecular meshwork cells (hTMC) without the application of exogenous label.
hTMC were imaged using a non-linear optical technique, coherent anti-Stokes Raman scattering (CARS), after treatment with a commercial formulation of latanoprost. Untreated cells and cells treated with vehicle containing the preservative benzalkonium chloride (BAK) were imaged as controls. Cells were fixed, stained with the fluorescent lipid dye Nile Red and imaged by conventional confocal microscopy to verify lipid membrane structures.
Analysis of CARS images of hTMC treated for 24 hours with 0.5 µg/ml 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 membrane.
CARS microscopy detected changes in living hTMC morphology that was validated by subsequent histological stain. Prostaglandin-induced changes to hTMC involved rearrangement of lipid membrane within these cells. These in vitro results identify a novel biological response to a class of anti-glaucoma drugs, and further experiments are needed to establish how this effect is involved in the hypotensive action of prostaglandins in vivo.
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