Browsing by Subject "Trabecular meshwork"
Now showing 1 - 3 of 3
- Results Per Page
- Sort Options
Item DETERMINATION OF PROTEINS INVOLVED IN THE FORMATION OF CROSS-LINKED ACTIN NETWORKS IN THE TRABECULAR MESHWORK(2013-04-12) Bermudez, Jaclyn Y.Purpose: Glaucoma is a leading cause of blindness worldwide and the primary risk factor of glaucoma is increased intraocular pressure (IOP). IOP is determined by the equilibrium of aqueous humor (a fluid that fills the anterior segment of the eye) production and outflow. In glaucoma patients, the outflow resistance through the trabecular meshwork (TM), a special tissue located at the angle between the cornea and iris, is abnormally elevated. Inside TM cells, actin proteins form cross-linked actin networks (CLANs). Excessive formation of these unique structures can be found in the glaucomatous TM. They can also be induced by dexamethasone (DEX) and TGFβ2. It is suggested that CLANs increase cell rigidity and therefore elevate aqueous humor outflow resistance. However, the proteins that are involved in CLANs formation are not fully identified. We hypothesize that by comparing CLANs enriched and un-enriched TM cells, we will be able to identify the proteins that are involved in CLANs formation. Methods: We treated cultured mouse TM cells with DEX (100 nM), TGFβ2, (5 ng/mL), or ethanol (as vehicle control), evaluated CLANs formation, and separated the cell proteins by 2D gel electrophoresis. Differentially expressed proteins were analyzed by the Redfin software, and gel spots were picked and assessed by spectrometry analysis. Western blotting was used to confirm 2D gel results. Results: We found a subset of proteins that are differentially expressed in CLANs-enriched TM cells, compared to non-enriched samples. Among these proteins, ferritin heavy chain, glial fibrillary acidic protein (GFAP), and integrin alpha V, were confirmed by mass spectrometry and Western immunoblot. Conclusions: We found a subset of proteins that are differentially expressed in CLANs-enriched mouse TM cells. Their contributions and involvements in CLANs formation and regulation of TM cell morphology and functions are being investigated. They may provide new insight of the pathogenesis of glaucoma.Item EFFECTS OF TGF-BETA2 ON THE ELASTIC MODULUS OF TRABECULAR MESHWORK TISSUE(2013-04-12) Baradia, HusseinPurpose: The primary risk factor for developing glaucoma is elevated intraocular pressure (IOP.) The key outflow passage for aqueous humor (AH) in the human eye involves the trabecular meshwork (TM) and the Schlemm's canal (SC) with the main regulator of IOP being at the junction of the two. Elevated IOP has been attributed to increased resistance to flow at the TM/SC junction. Aqueous humor levels of transforming growth factor beta-2 (TGF-b2) are elevated in glaucoma patients, suggesting its contribution to the progression of the disease. Studies using cultured TM cells as well as ex vivo tissue have shown that TGF-b2 induces extracellular matrix (ECM) remodeling. A separate study used atomic force microscopy (AFM) to measure the elastic modulus (e.g. stiffness) of TM tissue obtained from glaucoma patients compared to age-matched controls. A marked increase in stiffness in the glaucoma tissue was observed compared to non-glaucomatous controls. Taken together, these findings imply that an elevated level of TGF-b2 may lead to ECM remodeling and increased stiffness thus reducing AH outflow and elevating IOP. The hypothesis of this study was that increased expression of TGF-b2 will cause increased stiffness (e.g. increased elastic modulus) of the TM. The goal of our study was to compare the stiffness of the TM cultured overnight with or without TGF-b2. Methods: Bovine eyes were obtained from an abattoir and the TM dissected from the anterior segment. The dissected TM was then cut into halves. One half was cultured in control media and the other half cultured in media containing TGF-b2 (5 ng/ml). Both TM halves were incubated at 37oC for a period of 72 hours. The TM was then loaded onto a force transducer and contraction induced with carbachol to measure stiffness. Results: The raw data obtained indicated that the TM exposed to TGF-b2 contracted with greater force than control indicating that TGF-b2 reduced the stiffness of the TM. However, using a Students T test at a p of 0.05 the results showed no statistical significance. Conclusions: The results obtained are from one experiment with an N =8 and since no statistical significance was observed, the data has to be considered inconclusive. The force transducer used was actually that designed for skeletal muscles which may not be the best system to test trabecular meshwork which has some smooth muscle components. The future goal of the study is to actually use force microscopy, the current standard for such experiments.Item Fibronectin extra domain A (FN-EDA) causes glaucomatous trabecular meshwork, retina, and optic nerve damage in mice(BioMed Central Ltd., 2022-05-26) Mavlyutov, Timur A.; Myrah, Justin J.; Chauhan, Anil K.; Liu, Yang; McDowell, Colleen M.BACKGROUND: Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage. Here, we investigate the role of an endogenous Toll-like receptor 4 (TLR4) ligand, FN-EDA, in the development of glaucoma utilizing a transgenic mouse strain (B6.EDA(+/+)) that constitutively expresses only FN containing the EDA isoform. METHODS: Eyes from C57BL6/J (wild-type), B6.EDA+/+ (constitutively active EDA), B6.EDA-/- (EDA null) mice were processed for electron microscopy and consecutive images of the entire length of the TM and Schlemm's canal (SC) from anterior to posterior were collected and montaged into a single image. ECM accumulation, basement membrane length, and size and number of giant vacuoles were quantified by ImageJ analysis. Tlr4 and Iba1 expression in the TM and ONH cells was conducted using RNAscope in situ hybridization and immunohistochemistry protocols. IOP was measured using a rebound tonometer, ON damage assessed by PPD stain, and RGC loss quantified in RBPMS labeled retina flat mounts. RESULTS: Ultrastructure analyses show the TM of B6.EDA(+/+) mice have significantly increased accumulation of ECM between TM beams with few empty spaces compared to C57BL/6 J mice (p < 0.05). SC basement membrane is thicker and more continuous in B6.EDA(+/+) mice compared to C57BL/6 J. No significant structural differences are detected in the TM of EDA null mice. Tlr4 and Iba1 expression is increased in the TM of B6.EDA(+/+) mice compared to C57BL/6 J eyes (p < 0.05). IOP is significantly higher in B6.EDA(+/+) mice compared to C57BL/6 J eyes (p < 0.001), and significant ON damage (p < 0.001) and RGC loss (p < 0.05) detected at 1 year of age. Tlr4 mRNA is expressed in mouse ONH cells, and is present in ganglion cell axons, microglia, and astrocytes. There is a significant increase in the area occupied by Iba-1 positive microglia cells in the ONH of B6.EDA(+/+) mice compared to C57BL/6 J control eyes (p < 0.01). CONCLUSIONS: B6.EDA(+/+) mice have increased ECM accumulation in the TM, elevated IOP, enhanced proinflammatory changes in the ONH, loss of RGCs, and ONH damage. These data suggest B6.EDA(+/+) mice recapitulate many aspects of glaucomatous damage.