Background: Glaucoma is a leading neurodegenerative disease affecting over 70 million individuals worldwide. Early pathological events of axonal degeneration and retinopathy in response to elevated intraocular pressure (IOP) are limited and not well-defined due to the lack of appropriate animal models that faithfully replicate all the phenotypes of primary open angle glaucoma (POAG), the most common form of glaucoma. Glucocorticoid (GC)-induced ocular hypertension (OHT) and its associated iatrogenic open-angle glaucoma share many features with POAG. Here, we characterized a novel mouse model of GC-induced OHT for glaucomatous neurodegeneration and further explored early pathological events of axonal degeneration in response to elevated IOP. Methods: C57BL/6 J mice were periocularly injected with either vehicle or the potent GC, dexamethasone 21-acetate (Dex) once a week for 10 weeks. Glaucoma phenotypes including IOP, outflow facility, structural and functional loss of retinal ganglion cells (RGCs), optic nerve (ON) degeneration, gliosis, and anterograde axonal transport deficits were examined at various stages of OHT. Results: Prolonged treatment with Dex leads to glaucoma in mice similar to POAG patients including IOP elevation due to reduced outflow facility and dysfunction of trabecular meshwork, progressive ON degeneration and structural and functional loss of RGCs. Lowering of IOP rescued Dex-induced ON degeneration and RGC loss, suggesting that glaucomatous neurodegeneration is IOP dependent. Also, Dex-induced neurodegeneration was associated with activation of astrocytes, axonal transport deficits, ON demyelination, mitochondrial accumulation and immune cell infiltration in the optic nerve head (ONH) region. Our studies further show that ON degeneration precedes structural and functional loss of RGCs in Dex-treated mice. Axonal damage and transport deficits initiate at the ONH and progress toward the distal end of ON and target regions in the brain (i.e. superior colliculus). Most of anterograde transport was preserved during initial stages of axonal degeneration (30% loss) and complete transport deficits were only observed at the ONH during later stages of severe axonal degeneration (50% loss). Conclusions: These findings indicate that ON degeneration and transport deficits at the ONH precede RGC structural and functional loss and provide a new potential therapeutic window for rescuing neuronal loss and restoring health of damaged axons in glaucoma.

Purpose: We evaluated differences in aqueous humor dynamics (AHD) among several mouse strains within younger and older age groups. Methods: Albino (A/J, BALB/cJ) and pigmented (C3H/HeJ, C57-BL/6J) mice (young [2½–4½ months] and aged [10–12 months]) were studied. Intraocular pressure (IOP) was measured. In cannulated eyes, episcleral venous pressure (Pe) was assessed (blood reflux). Other AHD parameters (outflow facility [C], aqueous humor formation rate [Fin]) were assessed (constant flow infusion). Uveoscleral outflow rate (Fu) was obtained by calculation (Fu_(calc)) using the modified Goldmann equation, and in additional eyes (for comparison), by FITC-dextran perfusion (Fu_(FITC-dex)). Results: Intraocular pressure was higher in pigmented strains, but did not exhibit age-dependence, except in the C57-BL/6J strain. Fu_(calc) decreased with age in BALB/cJ (↓83.3%), C3H/HeJ (↓78.0%), and C57-BL/6J (↓85.0%) strains. In the A/J strain, Fu_(calc) decreased with age (↓70.0%), but not significantly. Fin decreased with age in the C3H/HeJ (↓53.6%) strain. In C57-BL/6J and A/J strains, Fin decreased with age, but not significantly. C in the BALB/cJ strain increased with age (↑62.5%). In C3H/HeJ and C57-BL/6J strains, C increased with age, but not significantly. Episcleral venous pressure ranged from 6.0 to 6.6 mm Hg (albino strains) to 8.5 to 8.9 mm Hg (pigmented strains). Pe was not age dependent, but was higher in pigmented animals. Conclusions: In mouse, Fu and Fin diminish with age. C tends to increase as animals progress to middle life. There are strain differences in Fu, IOP, C, Fin, and Pe. The current findings provide an important foundation for comparisons among different strains in different study reports.

Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) β2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFβ2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFβ2 in the TM. We developed an LV vector-encoding active hTGFβ2^C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFβ2^C226,228S. We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFβ2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFβ2^C226,228S exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFβ2^C226,228S that induces TM dysfunction and outflow resistance.

We tested five chemically and metabolically stable prostaglandin (PG) receptor agonists in a mouse model of dexamethasone-induced ocular hypertension (OHT). Whilst all compounds significantly (p < 0.05, ANOVA) lowered intraocular pressure (IOP) after twice-daily bilateral topical ocular dosing (5 µg/dose) over three weeks, the time course and magnitude of the responses varied. The onset of action of NS-304 (IP-PG receptor agonist) and rivenprost (EP4-PG receptor agonist) was slower than that of misoprostol (mixed EP2/EP3/EP4-PG receptor agonist), PF-04217329 (EP2-PG receptor agonist), and butaprost (EP2-PG receptor agonist). The rank order of IOP-lowering efficacies aligned with the onset of actions of these compounds. Peak IOP reductions relative to vehicle controls were as follows: misoprostol (74.52%) = PF-04217329 (74.32%) > butaprost (65.2%) > rivenprost (58.4%) > NS-304 (55.3%). A literature survey indicated that few previously evaluated compounds (e.g., latanoprost, timolol, pilocarpine, brimonidine, dorzolamide, cromakalim analog (CKLP1), losartan, tissue plasminogen activator, trans-resveratrol, sodium 4-phenyl acetic acid, etc.) in various animal models of steroid-induced OHT were able to match the effectiveness of misoprostol, PF-04217329 or butaprost. Since a common feature of the latter compounds is their relatively high affinity and potency at the EP2-PG receptor sub-type, which activates the production of intracellular cAMP in target cells, our studies suggest that drugs selective for the EP2-PG receptor may be suited to treat corticosteroid-induced OHT.