B’Ann T. Gabelt

Changes in aqueous humor dynamics with age and glaucoma

Changes in aqueous humor dynamics with age and in glaucoma have been studied for several decades. More recently, techniques have been developed which confirm earlier studies showing that outflow facility decreases with age and in glaucoma and add the newer finding that uveoscleral outflow also decreases. Morphologic studies in aging and glaucoma eyes have shown an increase in accumulation of extracellular material in both the trabecular meshwork and ciliary muscle and a loss of trabecular meshwork cells, which contribute to this reduction in outflow and result in an increase in intraocular pressure. A reduction in hyaluronic acid and increases in fibronectin and thrombospondin contribute to the change in the extracellular environment. Imbalances in responses to age-related stresses such as oxidative damage to long-lived molecules, protein cross-linking and loss of elasticity could trigger excess production of factors such as transforming growth factor beta, interleukin-1 and CD44S that could stimulate pathways leading to increases in fibronectin, transformation of trabecular meshwork cells to a myoepithelial state and decrease the breakdown in extracellular matrix material, allowing excess to accumulate. Ultimately trabecular outflow and uveoscleral outflow are reduced and intraocular pressure becomes elevated, adding more stress and perpetuating the pathological condition. Future research to identify additional factors and clarify their roles in these processes could lead to alternative therapies for age and glaucoma related changes in the eye.

Differential expression of matrix metalloproteinases in monkey eyes with experimental glaucoma or optic nerve transection.

Extracellular matrix (ECM) remodeling after neuronal injury and reactive gliosis is carried out by activation of matrix metalloproteinases (MMPs) regulated by their tissue inhibitors (TIMPs). In glaucoma, there is a loss of retinal ganglion cells and extensive ECM remodeling (cupping) at the level of the optic nerve head, frequently associated with elevated intraocular pressure. To determine whether ECM remodeling in the glaucomatous optic nerve head occurs in response to loss of axons or to elevated intraocular pressure we compared the patterns of MMP and TIMP expression in the eyes of monkeys with laser-induced glaucoma or with optic nerve transection. MT1-MMP and MMP1 expression was markedly increased in reactive astrocytes in optic nerve heads with experimental glaucoma but not in the optic nerve head of transected eyes. In normal control eyes retinal ganglion cells expressed MMP2, TIMP1 and TIMP2 constitutively, and the proteins were detected in their axons. At the site of transection, MT1-MMP, MMP1, MMP2, TIMP1 and TIMP2 were expressed by reactive astrocytes. Inflammatory cells, fibroblasts and reactive astrocytes at the transected site expressed MMP3 and MMP9, which were undetectable in the retina and optic nerve head in any condition. Constitutive expression of MMP2, TIMP1 and TIMP2 in retinal ganglion cells suggests a role in maintenance of synaptic integrity and plasticity and maintenance of the periaxonal space. Increased MMP1 and MT1-MMP1 expression in the glaucomatous optic nerve head is specific to tissue remodeling due to elevated intraocular pressure and not secondary to loss of axons.

Eyelid lymphatics II: a search for drainage patterns in the monkey and correlations with human lymphatics.

Purpose To study the lymphatic drainage of the cynomolgus monkey through the use of lymphoscintigraphy. Methods Lymphoscintigraphy with 500 &mgr;Ci of 99mTechnetium sulfur colloid injected at specific sites around the eyelids was performed with five cynomolgus monkeys in lateral and ventral positions. Results Lymphoscintigraphy of the monkey eyelid and periocular tissue revealed lymphatic drainage to the parotid lymph nodes from the entire upper eyelid, medial canthus, and lateral lower eyelid and drainage to the submandibular lymph nodes from the medial and central lower eyelid. In addition to draining to the parotid lymph nodes, the central upper eyelid was also seen to drain to the submandibular lymph nodes. Conclusions Lymphoscintigraphy of the cynomolgus monkey eyelids reveals discrete lymphatic drainage pathways for the upper and lower eyelids and a dual pathway for the central upper eyelid. Future studies will help to clarify the lymphatic drainage pathways of human eyelids.

Introductory Comments on Neuroprotection

All current glaucoma therapy is directed at lowering intraocular pressure (IOP) by pharmacologic or surgical means. Intraocular pressure undoubtedly plays a causal role, albeit not necessarily an exclusive one, in many, if not most, cases of glaucomatous visual loss. 4 However, assaulting or bypassing the trabecular meshwork, ciliary muscle, and ciliary processes, which are the target tissues for current treatments, completely neglects the optic nerve/retinal ganglion cells, whose dysfunction and death are directly responsible for the visual loss. The release of excitotoxic substances from neighboring retinal ganglion cells or other neuroretinal cells (e.g., photoreceptors) 1 injured by elevated IOP or other adverse ambient factors may contribute to retinal ganglion cell death. 2,3 Conversely, interaction with neighboring cells, which have been altered to release protective factors, may be used to intervene favorably in the process of cell death. 5 Only recently has our knowledge of the mechanisms of neuronal death and its prevention, delay, or even reversal after a variety of insults reached the point where we can seriously entertain the possibility of glaucoma therapy directed at the retinal ganglion cell bodies or axons themselves. 6

Eyelid lymphatics I: histochemical comparisons between the monkey and human.

Purpose: To study the lymphatic drainage of the cynomolgus monkey and human eyelid and periocular tissue by means of histochemistry. Methods: Eyelid and periocular tissue from three cynomolgus monkeys undergoing sacrifice for glaucoma and retina research purposes and discarded tissue from a wedge resection of one human eyelid were used for histochemical analysis. Lymphatic capillaries were distinguished histochemically in monkey and human eyelids by light microscopy with a 5′-nucleotidase (5′-Nase) staining method. Identification of lymphatic vessels was based on strict morphologic criteria combined with specific 5′-Nase staining. Results: Histochemical analysis with 5′-nucleotidase revealed a subcutaneous and pretarsal lymphatic plexus in both the human and monkey. Conclusions: Histochemical results demonstrate similar lymphatic plexi in the monkey and human. Future studies will help to clarify the lymphatic drainage pathways of monkey and human eyelids.

A Perspective of Gene Therapy in the Glaucomas

Gene therapy in the anterior and posterior segment tissues may have the potential to favorably influence aqueous hydrodynamics and retinal ganglion cell biology, thereby preventing, delaying, or minimizing glaucomatous damage to the optic nerve. We demonstrated the feasibility of using a herpes viral vector (ribonucleotide reductase defective HSV-1, hrR3) to deliver the lacZ reporter gene to living cat and rat eyes. Cats received injections into the anterior chamber and rats into the vitreous cavity. In cats, lacZ expression was detectable at 1 to 2 days in the anterior outer portion of the ciliary muscle and the lining of the intertrabecular spaces of the corneoscleral and uveal meshwork. Rat eyes showed lacZ expression in the retinal pigment epithelium and photoreceptor outer segments 2 days after injection.

Effects of serotonergic compounds on aqueous humor dynamics in monkeys

Purpose. The effects of several serotonergic agonists on aqueous humor formation (AHF), total outflow facility (OF) and intraocular pressure (IOP) were investigated in living cynomolgus monkeys. Methods. We determined the effect of a single topical unilateral 300 µg or 3 mg dose of the 5-HT agonists serotonin, 5-carboxamidotryptamine (5-CT), sumatripan, gepirone, and 8-hydroxy-2(di-n-propylaminotetralin) (8-OH-DPAT) and a 450 µg dose of flesinoxan on IOP (Goldmann applanation tonometry), AHF (scanning ocular fluorophotometry) and total OF (8-OH-DPAT only, topically and intracamerally). Results. Serotonin, 5-CT, sumatripan or gepirone had no significant effect on IOP or AHF. 8-OH-DPAT caused an AHF increase of ~70% over 6 hr in both ipsilateral drug- and contralateral vehicle-treated eyes, but no significant change in IOP compared with baseline measured on a separate occasion in the same animals. 8-OH-DPAT did not increase protein levels or rate of entry of systemically administered fluorescein in the anterior chamber aqueous humor compared to historic controls, and no difference was seen between ipsilateral and contralateral eyes. Flesinoxan had no effect on IOP and produced an insignificant 25% increase in flow in treated eyes compared to baseline. Conclusion. The results for 8-OH-DPAT and possibly flexinoxan indicate the presence of a secretion-stimulating 5-HT1A receptor in monkey ciliary epithelium that has little effect on IOP. OF was unchanged following 8-OH-DPAT administered topically or following intracameral exchange.

1α,25-Dihydroxyvitamin D3 and its analog, 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD), suppress intraocular pressure in non-human primates

Ocular hypertension is the greatest known risk factor for glaucoma that affects an estimated 70 million people worldwide. Lowering intraocular pressure (IOP) remains the mainstay of therapy in the management of glaucoma. By means of microarray analysis, we have discovered that 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) regulates genes that are known to be involved in the determination of intraocular pressure (IOP). Topical administration of 1α,25-(OH)(2)D(3) or its analog, 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D(3) (2MD), markedly reduces IOP in non-human primates. The reduction in IOP is not the result of reduced aqueous humor formation, while a 35% increase in aqueous humor drainage by 1α,25-(OH)(2)D(3) was found but this increase did not achieve significance. Nevertheless, our results suggest that 1α,25-(OH)(2)D(3), or an analog thereof, may present a new approach to the treatment of glaucoma.

Effect of nitric oxide compounds on monkey ciliary muscle in vitro.

The effects of various nitric oxide compounds and their inhibitors on monkey ciliary muscle contraction in vitro were investigated in both the longitudinal and circular vectors. The responses to nitric oxide compounds in carbachol precontracted ciliary muscle consisted of an initial relaxation often followed by recovery to near carbachol precontracted levels while the compound was still present. Sodium nitroprusside produced the greatest relaxation responses (nearly 100% relaxation in both vectors at 10(-3) M). The highest concentrations of isosorbide dinitrate (10(-4) M) and L-arginine (10(-3) M) produced relaxation responses of approximately 50% in both vectors. 8-Bromo cyclic GMP produced the smallest relaxation responses (25-35%). Nitric oxide synthase inhibition enhanced carbachol contraction up to 20% in the longitudinal but not the circular vector. Phosphodiesterase inhibition did not further enhance the relaxation response to L-arginine. Guanylate cyclase inhibition partially attenuated the relaxation response to sodium nitroprusside. Nitric oxide generating compounds were effective in relaxing precontracted monkey ciliary muscle in vitro. Endogenous production of nitric oxide is likely involved in the regulation of the contractile response in monkey ciliary muscle. Nitric oxide generating compounds may have potential value in therapeutic areas where modulation of ciliary muscle tension is desirable.

Prospects for Lentiviral Vector Mediated Prostaglandin F Synthase Gene Delivery in Monkey Eyes In vivo

Abstract Currently, the most effective outflow drugs approved for clinical use are prostaglandin F2α analogues, but these require daily topical self-dosing and have various intraocular, ocular surface and extraocular side effects. Lentiviral vector-mediated delivery of the prostaglandin F synthase (PGFS) gene, resulting in long-term reduction of intraocular pressure (IOP), may eliminate off-target tissue effects and the need for daily topical PGF2α self-administration. Lentiviral vector-mediated delivery of the PGFS gene to the anterior segment has been achieved in cats and non-human primates. Although these results are encouraging, our studies have identified a number of challenges that need to be overcome for prostaglandin gene therapy to be translated into the clinic. Using examples from our work in non-human primates, where we were able to achieve a significant reduction in IOP (2 mm Hg) for 5 months after delivery of the cDNA for bovine PGF synthase, we identify and discuss these issues and consider several possible solutions.