Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

The SD-OCT and ERG can be used to monitor noninvasively retinal morphologic and functional changes induced by ONC. Pattern ERG and pSTR are able to detect early RGC dysfunction, but pattern ERG exhibits higher sensitivity. Our results support the use of these tools in studies using the mouse ONC model.

PurposeThe trabecular meshwork (TM) is involved in the outflow of aqueous humor and intraocular pressure (IOP) regulation. Regulation of the extracellular matrix (ECM) by TGFβ2 signaling pathways in the TM has been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in liver, kidney, lung, and skin. Here, we investigated the role of TGFβ2–TLR4 signaling crosstalk in the regulation of the ECM in the TM and ocular hypertension.MethodsCross sections of human donor eyes, primary human TM cells in culture, and dissected mouse TM rings were used to determine Tlr4 expression in the TM. Trabecular meshwork cells in culture were treated with TGFβ2 (5 ng/mL), TLR4 inhibitor (TAK-242, 15 μM), and a TLR4 ligand (cellular fibronectin isoform [cFN]-EDA). A/J (n = 13), AKR/J (n = 7), BALBc/J (n = 8), C3H/HeJ (n = 20), and C3H/HeOuJ (n = 10) mice were injected intravitreally with adenovirus 5 (Ad5).hTGFβ2c226s/c228s in one eye, with the uninjected contralateral eye serving as a control. Conscious IOP measurements were taken using a TonoLab rebound tonometer.ResultsToll-like receptor 4 is expressed in the human and mouse TM. Inhibition of TLR4 signaling in the presence of TGFβ2 decreases fibronectin expression. Activation of TLR4 by cFN-EDA in the presence of TGFβ2 further increases fibronectin, laminin, and collagen-1 expression, and TLR4 signaling inhibition blocks this effect. Ad5.hTGFβ2c226s/c228s induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant (C3H/HeJ) mice.ConclusionsThese studies identify TGFβ2–TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data further explain the complex mechanisms involved in the development of glaucomatous TM damage.

TGFβ2 induces extracellular matrix (ECM) remodeling and alters the cytoskeleton by both the canonical Smad and non-canonical signaling pathways. TGFβ2 regulates the expression of ECM proteins in trabecular meshwork (TM) cells, increases intraocular pressure (IOP) in an ex vivo perfusion organ culture model, and induces ocular hypertension in rodent eyes. A necessary step in the canonical Smad signaling pathway is phosphorylation of receptor protein Smad3 by the TGF-β receptor complex. The purpose of this study was to determine whether TGFβ2 signals in vivo through the canonical Smad signaling pathway in the TM using Smad3 knockout (KO) mice. Ad5.hTGFβ2226/228 (2.5 × 107 pfu) was injected intravitreally into one eye of homozygous (WT), heterozygous (HET), and homozygous (KO) 129-Smad3tm1Par/J mice (n=9–10 mice/group), with the uninjected contralateral eye serving as the control. IOP measurements were taken using a rebound tonometer. To test the effect of TGFβ2 signaling on the ECM, fibronectin expression was determined by immunohistochemistry and qPCR analysis. Transduction of the TM with viral vector Ad5.hTGFβ2226/228 caused a statistically significant difference in IOP exposure between Smad3 genotypes: WT, 187.7 +/− 23.9 mmHg*day (n=9); HET, 95.6 +/− 24.5 mmHg*day (n=9); KO, 52.8 +/− 25.2 mmHg*day (n=10); (p<0.05 WT versus HET, p<0.01 WT versus KO). Immunohistochemistry and qPCR analysis showed that Ad5.hTGFβ2226/228 increased fibronectin expression in the TM of WT mice (2.23 +/− 0.24 fold) compared to Smad3 KO mice (0.99 +/− 0.19 fold), p<0.05. These results demonstrate Smad3 is a necessary signaling protein for TGFβ2-induced ocular hypertension and fibronectin deposition in the TM.

Two experiments were designed to investigate the endocrine mechanisms by which progestin administration induces puberty in heifers. In experiment 1, prepubertal heifers were randomly assigned by age to receive either a single norgestomet implant for 10 days (NORG; n = 15) or to serve as unimplanted controls (CONT; n = 14). Serial blood samples were obtained on Days -0.5, 8.5, and 10.5 (Day 1 = day of implant insertion) and were analyzed for concentrations of LH. On days 9 and 11, 4 heifers in each treatment were slaughtered, and the reproductive tracts were obtained. Weekly progesterone analyses were performed to estimate the day of puberty in heifers not slaughtered. Puberty was induced in 6 of 7 heifers in the NORG treatment, resulting in an earlier (p < 0.05) day of puberty in the NORG than in the CONT treatment. The frequency of LH pulses was higher (p < 0.05) on Days 8.5 and 10.5 in the NORG as compared to the CONT treatment. Although no difference (p > 0.10) was observed between treatments in follicular development, uterine weight was greater (p < 0.05) in NORG than in CONT heifers on Day 11. In experiment 2, prepubertal heifers (n = 47) were administered either 1 (1NORG; n = 16), 3(3NORG; n = 16), or 0 (CONT; n = 15) norgestomet implants for 10 days, and serial blood samples were obtained as described for experiment 1. Transrectal ultrasonography was used to determine the diameter of the largest follicle on Day 9. Plasma samples were obtained after each serial sample collection period and were analyzed for estradiol concentrations. Puberty was induced in 75% (12 of 16) and 81% (13 of 16) of heifers in the 1NORG treatments, respectively. Four heifers in the 1NORG treatment, from which serial blood samples were collected, ovulated before removal of the progestin implant, and the LH data for this treatment were deleted. In the 3NORG treatment, LH pulse frequency was suppressed (p < 0.05) on Day 8.5, but was greater (p < 0.05) 12 h after removal of the progestin (Day 10.5) than in the CONT treatment. We conclude that progestin administration hastens puberty by increasing LH secretion after progestin withdrawal and propose that progestin administration induces puberty by accelerating the peripubertal decrease of estradiol negative feedback on LH secretion.

Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 107 pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p<0.0001). In contrast, the C3H/HeJ mouse strain was resistant to Ad5.MYOC.Y437H induced IOP elevation for the 8-week time period. IOPs were stable (12–15 mm Hg) in the uninjected control eyes. We also determined whether there were any strain differences in pressure-induced optic nerve damage. Even though IOP was similarly elevated in three of the strains tested (BALB/cJ, C57BL/6J, and A/J ) only the A/J strain had considerable and significant optic nerve damage at the end of 8 weeks with optic nerve damage score of 2.64 +/− 0.19 (n=18, p<0.001) in the injected eye. There was no statistical difference in retinal ganglion cell death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage.

Glaucoma is a neurodegenerative disease with retinal ganglion cell (RGC) loss, optic nerve degeneration and subsequent vision loss. There are about 30 different subtypes of RGCs whose response to glaucomatous injury is not well characterized. The purpose of this study was to evaluate the response of 4 RGC subtypes in a mouse model of optic nerve crush (ONC). In this study, we also evaluated the pattern of axonal degeneration in RGC subtypes after nerve injury. We found that out of the 4 subtypes, transient-Off α RGCs are the most susceptible to injury followed by On–Off direction selective RGCs (DSGC). Non-image forming RGCs are more resilient with ipRGCs exhibiting the most resistance of them all. In contrast, axons degenerate irrespective of their retinal soma after ONC injury. In conclusion, we show that RGCs have subtype specific cell death response to ONC injury and that RGC axons disintegrate in an autonomous fashion undergoing Wallerian degeneration. These discoveries can further direct us towards effective diagnostic and therapeutic approaches to treat optic neuropathies, such as glaucoma.

PURPOSE. Transforming growth factor-b2 induces extracellular matrix (ECM) remodeling, which likely contributes to the defective function of the trabecular meshwork (TM) leading to glaucomatous ocular hypertension. Bone morphogenetic proteins (BMPs) inhibit these profibrotic effects of TGFb2. The BMP antagonist gremlin is elevated in glaucomatous TM cells and increases IOP in an ex vivo perfusion culture model. The purpose of this study was to determine whether gremlin regulates ECM proteins in the TM, signals through the Smad3-dependent pathway, and induces ocular hypertension in mice.METHODS. Ad5.Gremlin or Ad5.TGFb2 was injected intravitreally into one eye of each mouse. Intraocular pressure measurements were taken using a TonoLab tonometer. Gremlin, TGFb2, fibronectin (FN), and collagen-1 (Col-1) expression in the TM was determined by immunofluorescence, Western immunoblot, and quantitative (q)PCR analyses.RESULTS. Ad5.Gremlin or Ad5.TGFb2 each caused significant IOP elevation in mice.Immunofluorescence and Western blot analysis demonstrated that gremlin and TGFb2 reciprocally increased the expression of each other, and both increased FN expression in the TM and surrounding tissues. Ad5.Gremlin elevated IOP and increased Fn and Col-1 gene expression in the TM of Smad3 wild-type (WT) mice, but had no effect in Smad3 HET or Smad3 KO mice.CONCLUSIONS. Our results demonstrate that intravitreal injections of either Ad5.Gremlin or Ad5.TGFb2 elevate IOP and upregulate the ECM protein FN in the TM of mice. These data show that gremlin signals through the Smad3-dependent pathway in the TM to elevate IOP. We determined for the first time gremlin's role in inducing ocular hypertension in an in vivo model system. Keywords: gremlin, glaucoma, trabecular meshwork, TGFb2, intraocular pressure E levated IOP is a well-known causative risk factor for both the development and progression of glaucoma. Intraocular pressure is regulated by aqueous humor (AH) production in the ciliary body and drainage from the eye via the trabecular meshwork (TM) and uveoscleral pathway. The role of the TM and the surrounding extracellular matrix (ECM) in IOP regulation has been extensively studied. Primary open-angle glaucoma (POAG) is associated with changes in the ECM composition within the TM, increased AH outflow resistance, and elevated IOP. 1,2Transforming growth factor-b2 signaling pathway has been shown to be an important regulator of ECM proteins in the TM, 3-9 and TGFb2 has been found to be elevated in the AH and TM of POAG patients. 7,10-13 Transforming growth factor-b2 has also been shown to cause ocular hypertension in both ex vivo anterior segment perfusion organ culture models, 14,15 and by overexpression of a bioactivated form of TGFb2 in mouse eyes. 4,16 Transforming growth factor-b2 is known to regulate the expression of ECM proteins through the canonical Smad pathway as well as noncanonical signaling pathways. [17][18][19][20] We have previously demonstrated that TGFb2 signals through the canonical Smad and non-Smad pathways an...