The intrinsic stiffness of human trabecular meshwork cells increases with senescence.

Dysfunction of the human trabecular meshwork (HTM) plays a central role in the age-associated disease glaucoma, a leading cause of irreversible blindness. The etiology remains poorly understood but cellular senescence, increased stiffness of the tissue, and the expression of Wnt antagonists such as secreted frizzled related protein-1 (SFRP1) have been implicated. However, it is not known if senescence is causally linked to either stiffness or SFRP1 expression. In this study, we utilized in vitro HTM senescence to determine the effect on cellular stiffening and SFRP1 expression. Stiffness of cultured cells was measured using atomic force microscopy and the morphology of the cytoskeleton was determined using immunofluorescent analysis. SFRP1 expression was measured using qPCR and immunofluorescent analysis. Senescent cell stiffness increased 1.88±0.14 or 2.57±0.14 fold in the presence or absence of serum, respectively. This was accompanied by increased vimentin expression, stress fiber formation, and SFRP1 expression. In aggregate, these data demonstrate that senescence may be a causal factor in HTM stiffening and elevated SFRP1 expression, and contribute towards disease progression. These findings provide insight into the etiology of glaucoma and, more broadly, suggest a causal link between senescence and altered tissue biomechanics in aging-associated diseases

Ocular Pathology Relevant to Glaucoma in a Gja1(Jrt) Mouse Model of Human Oculodentodigital Dysplasia

PURPOSE. Oculodentodigital dysplasia (ODDD) is a human disorder caused by mutations in the gap junction alpha 1 (GJA1) gene encoding the connexin43 (Cx43) gap junction protein. Causal links between GJA1 mutations and glaucoma are not understood. The purpose in this study was to examine the ocular phenotype for Gja1(Jrt/+) mice harboring a Cx43 G60S mutation. METHODS. In young Gja1(Jrt/+) mice, Cx43 abundance was assessed with a Western blot, and Cx43 localization was visualized using immunohistochemistry and confocal microscopy. Intraocular pressure (IOP) was measured by rebound tonometry, and eye anatomy was imaged using ocular coherence tomography (OCT). Hematoxylin and eosin (H&E)-stained eye sections were examined for ocular histopathology related to the development of glaucoma. RESULTS. Decreased Cx43 protein levels were evident in whole eyes from Gja1(Jrt/+) mice compared with those of wild-type mice at postnatal day 1 (P = 0.005). Cx43 immunofluorescence in ciliary bodies of Gja1(Jrt/+) mice was diffuse and intracellular, unlike the gap junction plaques prevalent in wildtype mice. IOP in Gja1(Jrt/+) mice changed during postnatal development, with significantly lower IOP at 21 weeks of age in comparison to the IOP of wild-type eyes. Microphthalmia, enophthalmia, anterior angle closure, and reduced pupil diameter were observed in Gja1(Jrt/+) mice at all ages examined. Ocular histology showed prominent separations between the pigmented and nonpigmented ciliary epithelium of Gja1(Jrt/+) mice, split irides, and alterations in the number and distribution of nuclei in the retina. CONCLUSIONS. Detailed phenotyping of Gja1(Jrt/+) eyes offers a framework for elucidating human ODDD ocular disease mechanisms and evaluating new treatments designed to protect ocular synaptic network integrity

Management of Ocular Siderosis: Visual Outcome and Electroretinographic Changes

Purpose. Ocular siderosis (OS) is a sight threatening complication of retained iron-containing Intraocular Foreign Body (IOFB). Successful localization of the IOFB and timely removal are crucial to its management. The purpose of this study was to review the presentation, management, and outcome of OS at our institution. Methods. A retrospective case series of eyes with OS that underwent IOFB removal from January 2009 to March 2015 at our institution. Results. OS was seen in 9 eyes of 9 patients during the study period. There were 8 males and 1 female with an age range of 31.6 years. An IOFB was in all the eyes. The most common features of siderosis were cataract and pigmentary retinopathy seen in 6 (67%) and 4 (44%) eyes, respectively. Electroretinogram (ERG) readings were reduced in the 9 eyes. The IOFB was removed by pars plana vitrectomy in all the cases with improvement in ERG amplitudes occurring postoperatively in 7 (78%) eyes. Conclusion. A retained iron-containing IOFB can manifest itself after several years with features of OS. A careful clinical and radiologic evaluation is imperative in patients with history suggestive of penetrating ocular injury to rule out retained or occult IOFB and thus prevent this catastrophic condition

Effect of intraocular scattering in macular pigment optical density measurements

Fundus reflectometry is a common in-vivo, noninvasive method to estimate the macular pigment optical density (MPOD). The measured density, however, can be affected by the individual’s intraocular scattering. Scattering causes a reduction in the contrast of the fundus image, which in turn leads to an underestimation of the measured density. Intraocular scattering was measured optically in a group of seven young, healthy subjects using the method of optical integration and was subsequently used to correctly estimate the MPOD from fundus images. It was shown that when scattering is not considered, the measured optical density using fundus reflectometry can be underestimated by as high as 16% for our group of subjects

GLIS1 regulates trabecular meshwork function and intraocular pressure and is associated with glaucoma in humans.

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P = 4.73 × 1

Doctor of Philosophy

dissertationProgressive retinal ganglion cell (RGC) degeneration in glaucoma, the leading cause of permanent vision loss, is commonly caused by elevated intraocular pressure (IOP). Neuroprotective treatments complementing current IOP-reducing therapies could improve glaucoma management (Chapters 1, 3). IOP elevations induce glial reactivity (Chapter 4) and dysregulate RGC calcium (Chapters 2-3, 5-7), contributing to RGC degeneration (Chapters 5, 7), but it is unknown how glaucomatous forces perturb RGC and glial Ca2+ homeostasis. We discovered that mouse RGCs and Müller glia respond to osmotic pressure and tensile stretch with a cytosolic Ca2+ elevation that is primarily mediated by opening of the mechanosensitive cation channel transient receptor potential vanilloid 4 (TRPV4; Chapters 5-7). We therefore hypothesized that TRPV4 activation by glaucomatous forces drives RGC excitotoxicity. Consistent with this, intraocular injection of a selective TRPV4 agonist (GSK1016790A) induced mouse RGC loss (Chapter 7). This was prevented by systemic administration of a selective TRPV4 antagonist (HC-067047). Sustained exposure to glaucomatous mechanical strain caused RGC apoptosis, which was rescued by Ca2+ chelation or pharmacological/genetic TRPV4 antagonism, indicating that Ca2+ influx via TRPV4 is required for mechanical excitotoxicity (Chapter 7). Furthermore, RGCs and Müller glia swell during the progression of glaucoma and other blinding conditions, indicating the presence of aberrant osmotic gradients and loss of volume control. We found that RGC and Müller iv cell swelling is exacerbated by TRPV4-dependent Ca2+ influx. Swelling differentially activated TRPV4 in neurons and glia, the later of which required phospholipase A2- dependent production of 5,6-EET, an endogenous TRPV4 agonist. The water channel aquaporin 4 (AQP4) facilitated water entry, which enhanced glial TRPV4 activation (Chapter 6). Finally, we found that TRPV4 antagonism in mouse and primate glaucoma models lowered IOP to normal levels, potentially by promoting fluid drainage from the eye via the trabecular meshwork (TM). Although IOP elevation for eight weeks caused mouse RGC loss, this was prevented by daily treatment with a TRPV4 antagonist (Chapter 7). TRPV4 inhibition, therefore, simultaneously lowers IOP and increases RGC resilience. This, together with our finding that TRPV4 is expressed in human RGCs, Müller glia and TM cells (Chapters 6, 7), makes TRPV4 an attractive therapeutic target for prevention of glaucoma