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TSPO activation modulates the effects of high pressure in a rat ex vivo glaucoma model.

ABSTRACT: We previously reported that elevated pressure induces axonal swelling and facilitates the synthesis of the neurosteroid, allopregnanolone (AlloP), in the ex vivo rat retina. Exogenously applied AlloP attenuates the axonal swelling, suggesting that the neurosteroid plays a neuroprotective role against glaucomatous pressure-induced injuries, although mechanisms underlying neurosteroidogenesis have not been clarified. The aim of this study was to determine whether AlloP synthesis involves activation of translocator protein 18 kD (TSPO) and whether TSPO modulates pressure-induced retinal injury. Ex vivo rat retinas were exposed to various pressures (10, 35, or 75 mmHg) for 24 h. Expression of TSPO, 5?-reductase (5aRD), and AlloP was examined by quantitative real-time RT-PCR, ELISA, immunohistochemistry, and LC-MS/MS. We also examined the effects of TSPO ligands on AlloP synthesis and retinal damage. In this acute model, quantitative real-time RT-PCR and ELISA analyses revealed that elevated pressure facilitated TSPO expression. Similarly, these methods also detected enhanced 5aRD (mostly type II), which was observed in retinal ganglion cells (RGC) and the inner nuclear layer (INL). Atriol, a TSPO antagonist, suppressed pressure mediated AlloP synthesis and induced more severe histological changes in the inner retina when combined with elevated pressure. PK11195, a TSPO ligand that facilitates AlloP synthesis by itself, remarkably diminished pressure-mediated retinal degeneration. These results suggest that AlloP synthesis is induced by sequential activation of TSPO and 5aRD in an ex vivo glaucoma model, and that TSPO agonists may serve as potential therapeutic agents for the prevention of pressure-induced retinal damage.

SUBMITTER: Ishikawa M

PROVIDER: S-EPMC5473616 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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TSPO activation modulates the effects of high pressure in a rat ex vivo glaucoma model.

Ishikawa Makoto M Yoshitomi Takeshi T Covey Douglas F DF Zorumski Charles F CF Izumi Yukitoshi Y

Neuropharmacology 20160903

We previously reported that elevated pressure induces axonal swelling and facilitates the synthesis of the neurosteroid, allopregnanolone (AlloP), in the ex vivo rat retina. Exogenously applied AlloP attenuates the axonal swelling, suggesting that the neurosteroid plays a neuroprotective role against glaucomatous pressure-induced injuries, although mechanisms underlying neurosteroidogenesis have not been clarified. The aim of this study was to determine whether AlloP synthesis involves activatio ...[more]

PMID: 27596950

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Project description:Glaucoma is the second leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG), the most common form of glaucoma, is often associated with elevation of intraocular pressure (IOP) due to the dysfunction of trabecular meshwork (TM) tissues. Currently, an ex vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors and disease modifying drugs on physiological parameters like aqueous humor (AH) dynamics and IOP homeostasis. This system requires the use of freshly enucleated intact human eyes, which are sparsely available at very high cost. In this study, we explored the feasibility of using human donor corneoscleral segments for modeling morphological and biochemical changes associated with POAG. Among the number of corneas donated each year, many are deemed ineligible for transplantation due to stringent acceptance criteria. These ineligible corneoscleral segments were obtained from the Lions Eye Bank, Tampa, Florida. Each human donor anterior corneoscleral segment was dissected into four equal quadrants and cultured for 7 days by treating with the glaucoma factors dexamethasone (Dex) or recombinant transforming growth factor (TGF) β2 or transduced with lentiviral expression vectors containing wild type (WT) and mutant myocilin. Hematoxylin and Eosin (H&E) staining analysis revealed that the TM structural integrity is maintained after 7 days in culture. Increased TUNEL positive TM cells were observed in corneoscleral quadrants treated with glaucoma factors compared to their respective controls. However, these TUNEL positive cells were mainly confined to the scleral region adjacent to the TM. Treatment of corneoscleral quadrants with Dex or TGFβ2 resulted in glaucomatous changes at the TM, which included increased extracellular matrix (ECM) proteins and induction of endoplasmic reticulum (ER) stress. Western blot analysis of the conditioned medium showed an increase in ECM (fibronectin and collagen IV) levels in Dex- or TGFβ2-treated samples compared to control. Lentiviral transduction of quadrants resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis of conditioned medium revealed decreased secretion of mutant myocilin compared to WT myocilin. Moreover, increased ECM deposition and ER stress induction was observed in the TM of mutant myocilin transduced quadrants. Our findings suggest that the ex-vivo cultured human corneoscleral segment model is cost-effective and can be used as a pre-screening tool to study the effects of glaucoma factors and anti-glaucoma therapeutics on the TM.

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TSPO activation modulates the effects of high pressure in a rat ex vivo glaucoma model.

Publications

TSPO activation modulates the effects of high pressure in a rat ex vivo glaucoma model.

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