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Scleral fibroblast response to experimental glaucoma in mice.

ABSTRACT: PURPOSE: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 ?-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.

SUBMITTER: Oglesby EN

PROVIDER: S-EPMC4734151 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Scleral fibroblast response to experimental glaucoma in mice.

Oglesby Ericka N EN Tezel Gülgün G Cone-Kimball Elizabeth E Steinhart Matthew R MR Jefferys Joan J Pease Mary E ME Quigley Harry A HA

Molecular vision 20160129

<h4>Purpose</h4>To study the detailed cellular and molecular changes in the mouse sclera subjected to experimental glaucoma.<h4>Methods</h4>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 imm ...[more]

PMID: 26900327

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Project description:PurposeTo determine if oral losartan treatment decreases the retinal ganglion cell (RGC) death caused by experimental intraocular pressure (IOP) elevation in mice.MethodsWe produced IOP increase in CD1 mice and performed unilateral optic nerve crush. Mice received oral losartan, spironolactone, enalapril, or no drug to test effects of inhibiting angiotensin receptors. IOP was monitored by Tonolab, and blood pressure was monitored by tail cuff device. RGC loss was measured in masked axon counts and RGC bodies by ?-tubulin labeling. Scleral changes that could modulate RGC injury were measured including axial length, scleral thickness, and retinal layer thicknesses, pressure-strain behavior in inflation testing, and study of angiotensin receptors and pathways by reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry.ResultsLosartan treatment prevented significant RGC loss (median loss = 2.5%, p = 0.13), while median loss with water, spironolactone, and enalapril treatments were 26%, 28% and 43%; p < 0.0001). The lower RGC loss with losartan was significantly less than the loss with spironolactone or enalapril (regression model p = 0.001; drug treatment group term p = 0.01). Both losartan and enalapril significantly lowered blood pressure (p< 0.001), but losartan was protective, while enalapril led to worse than water-treated RGC loss. RGC loss after crush injury was unaffected by losartan treatment (difference from control p = 0.9). Survival of RGC in cell culture was not prolonged by sartan treatment. Axonal transport blockade after 3 day IOP elevations was less in losartan-treated than in control glaucoma eyes (p = 0.007). Losartan inhibited effects of glaucoma, including reduction in extracellular signal-related kinase activity and modification of glaucoma-related changes in scleral thickness and creep under controlled IOP.ConclusionsThe neuroprotective effect of losartan in mouse glaucoma is associated with adaptive changes in the sclera expressed at the optic nerve head.

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Scleral fibroblast response to experimental glaucoma in mice.

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Scleral fibroblast response to experimental glaucoma in mice.

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