Glucocorticoid-Induced Ocular Hypertension and Glaucoma.

Glucocorticoid (GC) therapy is indicated in many diseases, including ocular diseases. An important side-effect of GC therapy is GC-induced ocular hypertension (GIOHT), which may cause irreversible blindness known as GC-induced glaucoma (GIG). Here, we reviewed the pathological changes that contribute to GIOHT including in the trabecular meshwork and Schlemm's canal at cellular and molecular levels. We also discussed the clinical aspects of GIOHT/GIG including disease prevalence, risk factors, the type of GCs, the route of GC administration, and management strategies.

The application of lentiviral vectors for the establishment of TGFß2-induced ocular hypertension in C57BL/6J mice.

Elevated levels of TGFß2 in the aqueous humor is associated with the pathological changes in the trabecular meshwork (TM). These changes lead to ocular hypertension (OHT), the most important risk factor for the development and progression of primary open angle glaucoma (POAG), a leading cause of blindness worldwide. Therefore, TGFß2 is frequently used to develop OHT models including in perfusion cultured eyes and in mouse eyes. Adenovirus-mediated overexpression of human mutant TGFß2 has demonstrated great success in increasing intraocular pressure (IOP) in mouse eyes. However, adenoviruses have limited capacity for a foreign gene, induce transient expression, and may cause ocular inflammation. Here, we explored the potential of using lentiviral vectors carrying the mutant human TGFß2C226S/C228S (ΔhTGFß2C226S/C228S) gene expression cassette for the induction of OHT in C57BL/6J mice. Lentiviral vectors using CMV or EF1α promoter to drive the expression of ΔhTGFß2C226S/C228S were injected into one of the mouse eyes and the fellow eye was injected with the same vector but expressing GFP/mCherry as controls. Both intravitreal and intracameral injection routes were tested in male and female mice. We did not observe significant IOP changes using either promoter or injection route at the dose of 8 × 105 PFU/eye. Immunostaining showed normal anterior chamber angle structures and a slight increase in TGFß2 expression in the TM of the eyes receiving intracameral viral injection but not in those receiving intravitreal viral injection. At the dose of 2 × 106 PFU/eye, intracameral injection of the lentiviral vector with the CMV-ΔhTGFß2C226S/C228S cassette induced significant IOP elevation and increased the expression of TGFß2 and fibronectin isoform EDA in the TM. Our data suggest that lentiviral doses are important for establishing the TGFß2-induced OHT model in the C57BL/6J strain.

Cross-linked actin networks (CLANs) affect stiffness and/or actin dynamics in transgenic transformed and primary human trabecular meshwork cells.

Cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells may contribute to increased IOP by altering TM cell function and stiffness. However, there is a lack of direct evidence. Here, we developed transformed TM cells that form spontaneous fluorescently labelled CLANs. The stable cells were constructed by transducing transformed glaucomatous TM (GTM3) cells with the pLenti-LifeAct-EGFP-BlastR lentiviral vector and selection with blasticidin. The stiffness of the GTM3-LifeAct-GFP cells were studied using atomic force microscopy. Elastic moduli of CLANs in primary human TM cells treated with/without dexamethasone/TGFß2 were also measured to validate findings in GTM3-LifeAct-GFP cells. Live-cell imaging was performed on GTM3-LifeAct-GFP cells treated with 1 µM latrunculin B or pHrodo bioparticles to determine actin stability and phagocytosis, respectively. The GTM3-LifeAct-GFP cells formed spontaneous CLANs without the induction of TGFß2 or dexamethasone. The CLAN containing cells showed elevated cell stiffness, resistance to latrunculin B-induced actin depolymerization, as well as compromised phagocytosis, compared to the cells without CLANs. Primary human TM cells with dexamethasone or TGFß2-induced CLANs were also stiffer and less phagocytic. The GTM3-LifeAct-GFP cells are a novel tool for studying the mechanobiology and pathology of CLANs in the TM. Initial characterization of these cells showed that CLANs contribute to at least some glaucomatous phenotypes of TM cells.

Age and sex affect TGFß2-induced ocular hypertension in C57BL/6J mice.

Glaucoma is a leading cause of blindness worldwide. The loss of vision in glaucoma patients is due to optic nerve damage. The most important risk factor of glaucoma is elevated intraocular pressure (IOP) which is due to glaucomatous changes in the trabecular meshwork. Animal models, especially mouse models for ocular hypertension (OHT), are important for studying glaucoma. Published studies showed that 2.5 × 107 PFU adenoviral vectors expressing the biologically active form of human TGFß2 elevate IOP in female C57BL/6J mice when they are intravitreally delivered. In this study, we found that 2.5 × 107 PFU adenoviral TGFß2 vector did not elevate IOP in 3- or 5-month old male C57BL/6J mice. In contrast, 5 × 107 PFU of the same viral vectors elevated IOP in both 3- and 5-month old male C57BL/6J mice. Also, 5-month old mice showed earlier OHT and higher IOP compared to 3-month old mice. In summary, our data showed that age and sex play roles in adenoviral vector-mediated TGFß2-induced OHT in C57BL/6J mice.

An ex vivo model of human corneal rim perfusion organ culture.

The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 µl/min, the outflow facility was 0.359 ± 0.216 µl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.

Using CRISPR Interference as a Therapeutic Approach to Treat TGFß2-Induced Ocular Hypertension and Glaucoma.

Purpose: Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide with elevated intraocular pressure (IOP) as the most important risk factor. POAG IOP elevation is due to pathological changes in the trabecular meshwork (TM). Elevated TGFß2 contributes to these changes and increases IOP. We have shown that histone hyperacetylation is associated with TGFß2 elevation in the TM. In this study, we determined if clustered regularly interspaced short palindromic repeats (CRISPR) interference could specifically deacetylate histones and decrease TGFß2 in the TM. Methods: We tested the efficiency of different promoters in driving KRAB-dCAS9 expression in human TM cells. We also screened and determined the optimal sgRNA sequence in the inhibition of TGFß2. Chromatin immunoprecipitation-qPCR was used to determine the binding of KRAB-dCAS9. An adenovirus-mediated TGFß2-induced ocular hypertension (OHT) mouse model was used to determine the effect of the CRISPR interference system in vivo. Results: We found that the CRISPR interference system inhibited TGFß2 expression in human TM cells, and properly designed sgRNA targeted the promoter of the TGFß2 gene. Using sgRNA targeting the CMV promoter of the Ad5-CMV-TGFß2 viral vector, we found that lentivirus-mediated KRAB-dCAS9 and sgRNA expression was able to inhibit Ad5-CMV-TGFß2-induced OHT in C57BL/6J female and male mice eyes. This inhibition of OHT was associated with decreased levels of TGFß2 and extracellular matrix proteins in the mouse eye. Conclusions: Our results indicate that CRISPR interference is a useful tool for gene inhibition and may be a therapeutic approach to treat TGFß2-induced OHT.

The Canonical Wnt Signaling Pathway Inhibits the Glucocorticoid Receptor Signaling Pathway in the Trabecular Meshwork.

Glucocorticoid-induced glaucoma is a secondary open-angle glaucoma. About 40% of the general population may develop elevated intraocular pressure on prolonged glucocorticoid treatment secondary to damages in the trabecular meshwork (TM), a tissue that regulates intraocular pressure. Therefore, identifying the key molecules responsible for glucocorticoid-induced ocular hypertension is crucial. In this study, Dickkopf-related protein 1 (Dkk1), a canonical Wnt signaling inhibitor, was found to be elevated in the aqueous humor and TM of glaucoma patients. At the signaling level, Dkk1 enhanced glucocorticoid receptor (GR) signaling, whereas Dkk1 knockdown or Wnt signaling activators decreased GR signaling in human TM cells as indicated by luciferase assays. Similarly, activation of the GR signaling inhibited Wnt signaling. At the protein level, glucocorticoid-induced extracellular matrix was inhibited by Wnt activation using Wnt activators or Dkk1 knockdown in primary human TM cells. In contrast, inhibition of canonical Wnt signaling by ß-catenin knockdown increased glucocorticoid-induced extracellular matrix proteins. At the physiological level, adenovirus-mediated Wnt3a expression decreased glucocorticoid-induced ocular hypertension in mouse eyes. In summary, Wnt and GR signaling inhibit each other in the TM, and canonical Wnt signaling activators may prevent the adverse effect of glucocorticoids in the eye.

A smartphone based method for mouse fundus imaging.

Noninvasive in vivo imaging of the mouse retina is essential for eye research. However, imaging the mouse fundus is challenging due to its small size and requires specialized equipment, maintenance, and training. These issues hinder the routine evaluation of the mouse retina. In this study, we developed a noncontact imaging system consisting of a smartphone, a 90D condensing lens, a homemade light diaphragm, a tripod, and a Bluetooth remote. With minimal training, examiners were able to capture fundus images from the mouse retina. We also found that fundus images captured using our system from wild type mice, mice with laser-induced retinal injury, and a mouse model of retinitis pigmentosa showed a quality similar to those captured using a commercial fundus camera. These images enabled us to identify normal structures and pathological changes in the mouse retina. Additionally, fluorescein angiography was possible with the smartphone system. We believe that the smartphone imaging system is low cost, simple, accessible, easy to operate, and suitable for the routine screening and examination of the mouse eye.

The Role of the Ocular Tissue in SARS-CoV-2 Transmission.

The current global pandemic of coronavirus disease 2019 (COVID-19) has affected over 21 million people and caused over half a million deaths within a few months. COVID-19 has become one of the most severe public health crises in recent years. Compared to other pathogenic coronaviruses, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly infectious. Due to the lack of specific and effective treatment or vaccines, disease prevention and early detection are essential for establishing guidelines to mitigate further spread. The potential role of the ocular system in COVID-19 is still not clear but it has gained increasing attention. Here, we reviewed both clinical and research evidence on the ocular manifestations associated with COVID-19, the presence of SARS-CoV-2 in ocular surface tissues and tears, and the potential role of the eye in contracting SARS-CoV-2.

Evidence that cannabinoid CB1 receptors regulate intraocular pressure via two opposing mechanisms.

The cannabinoid signaling system regulates intraocular pressure (IOP) in the mouse via a complex system that includes three receptors: CB1, GPR18 and GPR119. In each case, activating the receptor lowers IOP, but CB1 receptors are found both at sites of aqueous humor inflow and outflow. As such, knockout mice for any of these receptors would be expected to have higher-than average, or at least unchanged, intraocular pressure. The current study investigates the unexpected observation that CB1 knockout mice have lower pressure than wild type counterparts by testing various regulators of cannabinoid signaling in murine models of IOP. We now report that a CB1 antagonist has differential effects on IOP: SR141716 raises IOP in standard light cycle (SLC) but lowers IOP in reverse light cycle (RLC). This is mimicked by ABD1085, a negative allosteric modulator of CB1. CB1 inhibitors lower IOP in both normotensive and hypertensive mouse eyes. The pressure-lowering effect is absent in CB1 knockout mice. IOP rebounds after the end of treatment but shows no sign of desensitization with daily treatment for a week. Unlike the positive cannabinoid effect, antagonist effects are not sex-dependent. We propose that there are two mechanisms of action for CB1, one that lowers IOP upon activation and a second with inverse sign that lowers IOP when CB1 is antagonized. The relatively lower pressure in CB1 knockout mouse eyes suggests that this second negative regulation of IOP is dominant.