B'Ann T. Gabelt

Prostaglandin F2α increases uveoscleral outflow in the cynomolgus monkey

Cynomolgus monkeys were treated topically in one eye twice daily with prostaglandin F2 alpha-l-isopropylester (PGF2 alpha-IE) for nine doses. On treatment day 4, 3 hr after the seventh dose, intraocular pressure (IOP) in the treated eye was reduced by 65% compared to the controls, to less than 5 mmHg. On treatment day 5, 3 hr after the ninth dose, total outflow facility was determined by two-level constant pressure perfusion of the anterior chamber. Immediately thereafter, uveoscleral outflow was determined by intracamerally infusing [125I]- or [131I]-albumin and fluoresceinated dextran, and calculating the volume of anterior chamber fluid required to have deposited the quantity of tracer recovered from the various ocular and periocular tissues. Simultaneously, trabecular outflow was determined by calculating the volume of anterior chamber fluid required to have deposited the quantity of tracer recovered from the general circulation. Total facility was approximately 50% higher in treated than in control eyes, but the effect was variable, of marginal statistical significance, and perhaps due to increased pseudofacility or uveoscleral facility. Uveoscleral outflow was approximately two to three-and-a-half times higher in treated than in control eyes, the magnitude of the effect being dependent upon the timing and pressure at which the perfusion was conducted. Trabecular outflow was reduced by approximately 75% in the treated eyes relative to control so that the proportion of total outflow comprised by trabecular outflow in the treated eyes was only one third that in the controls. Total aqueous flow was slightly (approximately 20%) but not significantly reduced in the treated eyes. The IOP lowering effect of PGF2 alpha in the cynomolgus monkey is due largely if not exclusively to an increase in uveoscleral outflow of aqueous humor, with aqueous outflow being redirected from the trabecular to the uveoscleral route.

Update on the Mechanism of Action of Topical Prostaglandins for Intraocular Pressure Reduction

A decade has passed since the first topical prostaglandin analog was prescribed to reduce intraocular pressure (IOP) for the treatment of glaucoma. Now four prostaglandin analogs are available for clinical use around the world and more are in development. The three most efficacious of these drugs are latanoprost, travoprost, and bimatoprost, and their effects on IOP and aqueous humor dynamics are similar. A consistent finding is a substantial increase in uveoscleral outflow and a less consistent finding is an increase in trabecular outflow facility. Aqueous flow appears to be slightly stimulated as well. Prostaglandin receptors and their associated mRNAs have been located in the trabecular meshwork, ciliary muscle, and sclera, providing evidence that endogenous prostaglandins have a functional role in aqueous humor drainage. Earlier evidence found that topical PG analogs release endogenous prostaglandins. One well-studied mechanism for the enhancement of outflow by prostaglandins is the regulation of matrix metalloproteinases and remodeling of extracellular matrix. Other proposed mechanisms include widening of the connective tissue-filled spaces and changes in the shape of cells. All of these mechanisms alter the permeability of tissues of the outflow pathways leading to changes in outflow resistance and/or outflow rates. This review summarizes recent (since 2000) animal and clinical studies of the effects of topical prostaglandin analogs on aqueous humor dynamics and recent cellular and molecular studies designed to clarify the outflow effects.

Effects of prostaglandins on the aqueous humor outflow pathways.

Topical treatments with certain prostaglandins (PGs), including FP receptor agonists, lower intraocular pressure by increasing uveoscleral outflow. Although the precise mechanism for the increased uveoscleral outflow is not known, there appears to be activation of a molecular transduction cascade and an increase in the biosynthesis of certain metalloproteinases. This leads to reduction of extracellular matrix components within the ciliary muscle, iris root, and sclera. It is possible that this reduction of extracellular matrix present within portions of the uveoscleral pathway may contribute to the mechanism of increased uveoscleral outflow. Additional mechanisms that may contribute to the PG-mediated increase of uveoscleral outflow include relaxation of the ciliary muscle, cell shape changes, cytoskeletal alteration, or compaction of the extracellular matrix within the tissues of the uveoscleral outflow pathway. Future studies should clarify the importance of these various responses that may contribute to increased uveoscleral outflow. At present, there is no compelling evidence for a substantial facility-increasing effect on the trabecular meshwork outflow for any of these compounds.

Effects of topical PGF2α on aqueous humor dynamics in cynomolgus monkeys

Single topical applications of prostaglandin F2 alpha (PGF2 alpha) tromethamine salt to living cynomolgus monkey eyes reduced intraocular pressure (IOP). Twice daily topical application was far more effective, so that after the 7th 50 micrograms or 100 micrograms dose on day 4, IOP fell 40-50%, to 8-10 mm Hg. Following twice daily application of 50 or 100 micrograms for greater than 3 days: (1) no increase in total outflow facility could be demonstrated by 2-level constant pressure perfusion or Schiotz tonography; (2) no decrease in aqueous humor formation rate could be demonstrated by fluorophotometry--rather, aqueous flow may have increased; (3) anterior chamber aqueous humor protein concentration was unaltered, but entry of intravenously injected fluorescein into the cornea and anterior chamber tended to increase; (4) there was a weak but sometimes statistically significant miosis of up to approximately 0.5 mm. We conclude that in the cynomolgus monkey: (1) PGF2 alpha is a potent ocular hypotensive agent with only very weak miotic and blood-aqueous barrier-disrupting effects; (2) the ocular hypotensive action of PGF2 alpha is definitely not due to increased conventional outflow facility or decreased aqueous production, but probably to increased uveoscleral drainage of aqueous humor.

The role of the actomyosin system in regulating trabecular fluid outflow

Abnormally high resistance to aqueous humor drainage via the trabecular meshwork and Schlemms canal is highly correlated with the development of primary open-angle glaucoma. Contractility of the actomyosin system in the trabecular cells or inner wall endothelium of Schlemms canal is an important factor in the regulation of outflow resistance. Cytoskeletal agents, affecting F-actin integrity or actomyosin contractility, or gene therapies, employing overexpression of caldesmon or Rho-A inhibition, can decrease outflow resistance in the drainage pathway. In this review, we discuss the mechanisms underlying these and similar effects on trabecular outflow resistance in living animals and/or in cultured ocular anterior segments from enucleated animal or human eyes.

The effect of prostaglandin F2α on trabecular outflow facility in cynomolgus monkeys

Abstract Cynomolgus monkeys were treated topically in one eye with 2 μg of prostaglandin F 2α -I-isopropylester (PGF 2α -IE) twice daily. On day 4, 3 hr after the seventh dose, intraocular pressure (IOP) was 8·1±0·7 mmHg lower in the treated than in the control eyes. On day 5, after the ninth PGF 2α dose, gross facility was measured in both eyes by determining the rate of fluid flow from an external reservoir into the eye at two different IOP levels. Simultaneously, trabecular facility was measured by determining the rate of accumulation of intracamerally infused radioiodinated albumin in the general circulation. Gross facility was 40–60% higher in PGF 2α treated than in control eyes, but there was no difference in trabecular facility. The increase in gross facility sometimes reported following PGF 2α probably represents increased pseudofacility or uveoscleral facility, rather than true facility of outflow through the trabecular meshwork and Schlemms cannal.

Non-invasive observation of repeated adenoviral GFP gene delivery to the anterior segment of the monkey eye in vivo.

Glaucoma is a group of chronic eye diseases often associated with an elevated intraocular pressure (IOP). If not controlled, the condition leads to blindness. The eye tissue responsible for maintaining aqueous humor resistance and thus normal IOP is the trabecular meshwork (TM). Adenoviral vectors are capable of transducing the TM in several rodent species. Because of the relevance of the non‐human primate model in the study of glaucoma, gene transfer to the eyes of cynomolgus monkeys was investigated.

The Effect of Topical PGF2x on Uveoscleral Outflow and Outflow Facility in the Rabbit Eye

Prostaglandin F2x (PGF2x) is a powerful ocular hypotensive agent in rabbit, cat, dog, monkey and human. In cynomolgus monkeys, the intraocular pressure (IOP) lowering is due to increased uveoscleral outflow (Fu,). Because the anatomy of the rabbit outflorv apparatus differs significantly from that of the primate, we sought to determine whether the mechanism of the PGF2x-induced IOP fall was the same. PGF2x tromethamine salt (PGF2x-TS) (50 μg) applied to one eye of 14 conscious rabbits produced a significant IOP fall of 7.4±0.9 mmHg (P

Long-term activation of c-Fos and c-Jun in optic nerve head astrocytes in experimental ocular hypertension in monkeys and after exposure to elevated pressure in vitro.

This study investigates whether the immediate early gene (IEG) products c-Fos and c-Jun are activated in vivo in monkeys with experimental glaucoma, and in vitro in cultured human ONH astrocytes exposed to hydrostatic pressure (HP). Three Rhesus monkeys with mild glaucomatous damage (mean intraocular pressure (IOP) 27 +/- 1.3 mm Hg approximately 42 weeks) and three with moderate glaucomatous damage (mean IOP 44 +/- 6.7% mm Hg approximately 11 weeks) were used for this study; the contralateral eye served as normal control (mean IOP 18.6 +/- 1.7 mm Hg). ONH tissues were stained with GFAP, DAPI, and c-Jun or c-Fos, and transcription factor positive and negative nuclei were counted to determine nuclear localization. Cultured human normal and glaucomatous ONH astrocytes exposed to elevated HP served as the in vitro model of elevated pressure. Activation and nuclear localization of c-Fos and c-Jun increased significantly in the monkeys with elevated IOP. These data correlated with axonal loss, reactive astrocytes, and remodeling of the optic disc. Cultured human ONH astrocytes showed increased nuclear localization of c-Fos and c-Jun under exposure to HP. Immunohistochemistry demonstrated that the upstream regulators of c-Fos and c-Jun, ERK-MAPK and MAPKp38 localized to the nuclei of ONH astrocytes in monkeys with experimental glaucoma. Taken together, these results demonstrate c-Fos and c-Jun activation in ONH astrocytes in vivo and in vitro, and that activation of both transcription factors is associated with ERK and MAPKp38 activation in experimental glaucoma, suggesting that activation of transcription factors may participate in the induction and maintenance of the reactive astrocyte phenotype in glaucomatous optic neuropathy.

Gene therapy targeting glaucoma: where are we?

In a chronic disease such as glaucoma, a therapy that provides a long lasting local effect with minimal systemic side effects, while circumventing the issue of patient compliance, is very attractive. The field of gene therapy is growing rapidly and ocular applications are expanding. Our understanding of the molecular pathogenesis of glaucoma is leading to greater specificity in ocular tissue targeting. Improvements in gene delivery techniques, refinement of vector construction methods, and development of better animal models combine to bring this potential therapy closer to reality.