Understanding the molecular mechanism of glaucoma and development of neuroprotectants is significantly hindered by the lack of a reliable animal model that accurately recapitulates human glaucoma. Here, we sought to develop a mouse model for the secondary glaucoma that is often observed in humans after silicone oil (SO) blocks the pupil or migrates into the anterior chamber following vitreoretinal surgery. We observed significant intraocular pressure (IOP) elevation after intracameral injection of SO, and that SO removal allows IOP to return quickly to normal. This simple, inducible and reversible mouse ocular hypertension model shows dynamic changes of visual function that correlate with progressive retinal ganglion cell (RGC) loss and axon degeneration. It may be applicable with only minor modifications to a range of animal species in which it will generate stable, robust IOP elevation and significant neurodegeneration that will facilitate selection of neuroprotectants and investigating the pathogenesis of ocular hypertension-induced glaucoma.

PurposeIncreased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation.MethodsCultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFβ receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4′,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test.ResultsTGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFβ receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption.ConclusionsTGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.

Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughput in vivo screening in both male and female mouse eyes and identified the mouse c-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.

The most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student’s t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs.