Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21735
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Browsing Eye / Vision by Author "McDowell, Colleen M."
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Item Crosstalk of transforming growth factor beta-2 and toll-like receptor 4 in the trabecular meshwork(2015-03) Hernandez, Humberto; Medina-Ortiz, Wanda E.; Clark, Abbot F.; McDowell, Colleen M.Purpose: Glaucoma is characterized by progressive optic neuropathy that is associated with elevated intraocular pressure (IOP) and extracellular matrix (ECM) remodeling. The trabecular meshwork (TM) is involved in the outflow of aqueous humor and IOP regulation. Glaucomatous eyes show elevated levels of transforming growth factor-β 2 (TGF-β2) and its signaling pathways in the ECM of the TM have been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in the liver, kidney, lung and skin. Based on the potential for shared signaling pathways, we hypothesize that endogenous TLR4 ligands activate TLR4 and augment TGF-β2 signaling sensitivity by downregulating BAMBI, leading to increase ECM production in the TM and increase IOP. Methods: Cross-sections of human donor eyes and dissected mouse TM rings were used to determine TLR4 expression in the TM. Primary human TM cells were used to test for the expression of BAMBI. To study the role of TGF-β2 and TLR4 crosstalk in the expression of ECM proteins, four primary human TM cell strains were treated with a selective TLR4 inhibitor (TAK-242, 15µM), TGFβ2 (5ng/ml), and a TLR4 ligand (Fibronectin-EDA isoform). In-vivo studies were carried out to examine the induction of ocular hypertension in wild-type (A/J, AKR/J, BALBc/J, and C3H/HeOuJ) or Tlr4 mutant strains of mice (C3H/HeJ) by intravitreally injecting Ad5.hTGFβ2226/228 (2.5x107 pfu) in one eye while the contralateral uninjected eye was used as negative controls. Results: Our studies reveal the expression of TLR4 in the human and mouse TM. BAMBI is expressed in human TM cells and its expression is significantly decreased in the presence of TGF-β2. Inhibition of TLR4 in the presence of TGF-β2 decreases fibronectin and collagen-1 expression. Activation of TLR4 in the presence of TGF-β2 increases fibronectin and collagen-1 expression and TLR4 inhibition blocks this effect. Our in-vivo studies show that Ad5.hTGF-β2 induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant mice. Conclusions: These studies identify TGFβ2 – TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data provide potential new targets to lower IOP and further explain the mechanisms involved in the development of glaucomatous TM damage.Item Retina ganglion cell subtype specific cell death following optic nerve crush in mice(2015-03) Daniel, Steffi; Huberman, Andrew; Clark, Abbot F.; McDowell, Colleen M.Purpose: Glaucoma is an optic neuropathy that causes cupping of the optic disc, retina ganglion cell (RGC) loss, and characteristic visual field defects. Published literature suggests differential RGC susceptibility to damage. However, the mechanisms by which the optic nerve and RGCs become more susceptible to injury and damage are largely unknown. We investigated individual RGC subtypes’ susceptibility to damage after optic nerve crush (ONC). Methods: We utilized two mouse strains that selectively express GFP in individual RGC subtypes: CB2-GFP strain (selectively expresses GFP in transient OFF-alpha RGCs) and TRHR-GFP strain (selectively expresses GFP in On-Off direction selective RGCs). ONC was performed unilaterally, with the contralateral eye serving as a control. RGC subtype specific damage was evaluated at 0, 1, 3, 7, and 14 days post ONC. RGC damage was assessed by immunofluorescence of labeled retinal flat mounts using the GFP biomarker for the specific RGC subtypes and NeuN for total RGCs. Results: Throughout the 14 day time course, GFP positive RGCs in the CB2-GFP strain died faster than the GFP positive RGCs in the TRHR-GFP strain, with similar rates of total RGC death in each strain. The half-life (T1/2) of GFP positive cells in the TRHR-GFP strain was T1/2=7.11 days with total RGC death T1/2=9.65 days. The half-life of GFP positive cells in the CB2-GFP strain was T1/2=4.19 days with total RGC death T1/2=10.77 days. There was a significant difference in percent cell survival of each individual RGC subtype at 3 days (TRHR-GFP, 61.3 +/- 7.4%; CB2-GFP, 38.2 +/- 14.3%; n=4, p=0.029), 7 days (TRHR-GFP, 63.1 +/- 26.4%; CB2-GFP, 22.2 +/- 5.3%; n=4-5, p=0.011) and 10 days (TRHR-GFP, 38.0 +/- 2.9%; CB2-GFP, 3.5 +/- 3.5%; n=4-5, p<0.001) post-crush. There was no significant difference in percent cell survival of each individual RGC subtype at 1 day (TRHR-GFP, 76.2 +/- 20.9%; CB2-GFP, 70.3 +/- 10.0%; n=4-5, p=0.621) and 14 days (TRHR-GFP, 5.0 +/- 6.5%; CB2-GFP, 2.6 +/- 3.3%; n=3-4, p=0.556) post-crush. Conclusions: These studies demonstrate differences in individual RGC subtype susceptibilities to ONC. These data provide valuable information to develop new targets to slow and/or prevent the progression of RGC damage and new methods to detect early damage in diseases such as glaucoma.