Baohe Tian

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.

Outflow of aqueous humor.

Advances over the past decade in understanding the pathophysiologic features of outflow abnormalities in glaucoma include structural changes in the trabecular meshwork (TM), the role of cytokines and growth factors in normal and abnormal TM function, and the relationship of TM and optic nerve changes. Advances in enhancing aqueous humor outflow include pharmacologically altering the extracellular matrix (ECM) of the ciliary muscle (CM) and the shape, adhesion and contractility of TM cells, and inserting genes into glaucomarelevant ocular cells to produce these and other therapeutically useful changes.

Advances in Glaucoma Diagnosis and Therapy for the Next Millennium: New Drugs for Trabecular and Uveoscleral Outflow

Advances in our understanding of the physiology and molecular biology of the trabecular and uveoscleral outflow pathways of the eye will lead to the development of new approaches for glaucoma therapy. Therapies of the future will target the structures and enzymes involved in maintaining cell shape and cell-cell and cell-extracellular matrix interactions. Altering the extracellular matrix in the ciliary muscle has been important in the intraocular pressure lowering effects of prostaglandins and will be developed further as an approach to enhancing outflow through the trabecular meshwork. Gene therapy may be used to enhance or suppress the endogenous targets that are ultimately responsible for the outflow enhancement triggered by these agents.

Acute effects of H-7 on ciliary epithelium and corneal endothelium in monkey eyes.

Purpose. Topical or intracameral administration of H-7 doubles outflow facility and reduces intraocular pressure in cynomolgus monkeys, by relaxing and expanding the trabecular meshwork (TM) and Schlemm’s canal (SC). Since H-7 may have anti-glaucoma potential, we determined its effects on the corneal endothelium and ciliary epithelium for safety considerations. Methods. Following topical H-7, aqueous humor flow (AHF), corneal endothelial transfer coefficient (k a) and anterior chamber (AC) entry of i.v. fluorescein were measured by fluorophotometry; AC aqueous protein concentration ([Protein] AC) was determined by Lowry assay; and corneal thickness and endothelial cell density and morphology were measured by ultrasonic pachymetry and specular microscopy respectively. Following intracameral H-7, specular and/or light and electron microscopy of the corneal endothelium or ciliary epithelium were performed. Results. Following unilateral topical H-7: (1) AHF and k a were essentially unchanged at 0.5–3.0, 3.5–6.0, and 0.5–6.0 hr, with an insignificant increase from 0.5–1.5 hr; (2) [Protein] AC was insignificantly increased at 1–1.5 hr but had returned to baseline by 2.5 hr; (3) entry of i.v. fluorescein into aqueous or cornea was modestly and transiently increased; (4) the central cornea thickened significantly at 1–2.5 hr, gradually returning to baseline 2.5 hr after H-7, while peripheral corneal thickness was less affected; (5) corneal endothelial cell borders became indistinct by 1 hr, but cell morphology was recovering by 3–5 hr and had completely returned to normal by 24 hr; (6) corneal endothelial cell density was unchanged at 5–24 hr. Following intracameral H-7, no significant changes were observed in corneal endothelial cell density or morphology by specular microscopy, nor in corneal endothelial or ciliary epithelial morphology by light and electron microscopy. Conclusions. A facility-effective intracameral dose of H-7 had no discernible structural effect on the corneal endothelium or ciliary epithelium. It is not yet clear whether carefully chosen topical doses of H-7 or analogues can enhance outflow facility without meaningfully affecting the cornea and ciliary processes.

Comparisons of actin filament disruptors and Rho kinase inhibitors as potential antiglaucoma medications

Dynamics of the actin cytoskeleton in the trabecular meshwork play a crucial role in the regulation of trabecular outflow resistance. The actin filament disruptors and Rho kinase inhibitors affect the dynamics of the actomyosin system by either disrupting the actin filaments or inhibiting the Rho kinase-activated cellular contractility. Both approaches induce similar morphological changes and resistance decreases in the trabecular outflow pathway, and thus both have potential as antiglaucoma medications. Although the drugs might induce detrimental changes in the cornea following topical administration, lower drug concentrations in larger volumes as used clinically, but not higher drug concentrations in smaller volumes as used experimentally, could minimize corneal toxicity. Additionally, developments of trabecular meshwork-specific actin filament disruptors or Rho kinase inhibitors, prodrugs and new drug-delivery methods might avoid the drugs’ toxicity to the cornea. Gene therapies with cytoskeleton-modulating proteins may mimic the effects of the cytoskeleton-modulating agents and have the potential to permanently decrease trabecular outflow resistance.

Effect of daily prolonged ketamine anesthesia on intraocular pressure in monkeys.

Purpose: To determine if repeated intramuscular ketamine in monkeys on consecutive days affects intraocular pressure (IOP) and if the ketamine-induced IOP change has any relationship to systemic dehydration and/or changes in mean arterial pressure (MAP) of the animals. Methods: Nine monkeys were studied per four protocols. IOP was determined hourly for 6 hr by Goldmann tonometry under ketamine anesthesia on 3 (protocol 1) or 5 (protocols 2 and 3) consecutive days, or on alternating days 1, 3, and 5 (protocol 4). Monkeys in protocols 3 and 4, but not in protocols 1 and 2, received subcutaneous Ringers fluids at the end of each 6-hr session on days 1–4 or days 1, 3, and 5; monkeys in protocols 2 and 3 received intravenous fluid infusion throughout the experiment on day 5. In protocols 2–4, MAP was measured hourly following each IOP measurement. Results: Monkeys receiving ketamine but no Ringers fluids in protocol 1 or 2 showed significant IOP declines on days 2–3 or 2–4. The IOP declines were greater in magnitude in protocol 1 than in protocol 2. Daily subcutaneous Ringers fluids appeared to delay IOP declines in protocol 3. Continuous intravenous fluid infusion on day 5 variably prevented IOP declines in protocols 2 and 3. Monkeys receiving ketamine and subcutaneous fluids on alternate days in protocol 4 showed no decline in IOP. No significant relationship between IOP and MAP was observed. Conclusions: Anesthesia induced by repeated intramuscular ketamine on consecutive days may produce significant IOP declines. Systemic dehydration during the anesthesia seems to be the predominant factor contributing to the IOP reduction. However, inter-individual differences in monkeys indicate that multiple factors may be involved. This study also suggests that fluid supplementation plus alternating anesthesia with recovery days may prevent IOP reduction in monkeys resulting from daily prolonged ketamine anesthesia.

Application of canaloplasty in glaucoma gene therapy: where are we?

PURPOSE Schlemms canal (SC) inner wall is adjacent to the juxtacanalicular trabecular meshwork (TM) over their entire circumference. We seek to transfer reporter and therapeutic genes to these outflow-modulating tissues via canaloplasty surgery in live monkeys. METHODS A standard canaloplasty surgical approach was performed in cynomolgus monkeys using flexible canaloplasty catheters, modified for monkey eyes with a 175-μm outer diameter and an LED-lighted tip. A 6-0 prolene suture was used for the exact localization of SC. Trypan blue was injected during catheter withdrawal to document catheter placement within SC and to determine ease of injecting fluid into SC. Before, during, and after the injection, the position of the catheter and the anatomic details were video-captured with an externally positioned noncontact endoscopic imaging system and 50 mHz ultrasound biomicroscopy (UBM). RESULTS A 360° catheterization and injection of dye into SC was achieved. Suture, catheter, and trypan blue were imaged with the endoscope camera system and the catheter was also visualized with UBM. Trypan blue was seen in the SC over 5 clock hours after a 1 clock-hour insertion of the catheter. CONCLUSIONS A modified canaloplasty catheter device might be used for gene delivery to the SC/TM area without circumferential catheterization. Further studies comparing different delivery methods of the vector/transgene into the SC using canaloplasty are needed.

Effect of H-7 on Secondary Cataract After Phacoemulsification in the Live Rabbit Eye

PURPOSE This study is aimed to determine if the serine-threonine kinase inhibitor H-7 inhibits secondary cataract after phacoemulsification in the live rabbit eye. METHODS Eighteen rabbits underwent extracapsular lens extraction by phacoemulsification in 1 eye. The eye was treated with intravitreal H-7 (300 or 1,200 μM; n = 6 or 5) or balanced salt solution (BSS) (n = 7) immediately after the surgery and twice weekly for 10 weeks. Each eye received slit lamp biomicroscopy once a week, during which posterior capsule opacification (PCO) was evaluated. The eye was then enucleated and the lens capsule was prepared, fixed, and imaged. PCO was evaluated again on the isolated lens capsule under a phase microscope. Soemmerings ring area (SRA) and the entire lens capsule area were measured from capsule images on a computer and the percentage of SRA (PSRA) in the entire capsule area was calculated. Wet weight of the capsule (WW) was determined on a balance. RESULTS No significant difference in PCO was observed in any comparison. No significant differences in SRA, PSRA, and WW were observed between the 300 μM H-7-treated eye and the BSS-treated eye. However, SRA, PSRA, and WW in the 1,200 μM H-7-treated eye were significantly smaller than those in the BSS-treated eye [28.3 ± 16.2 vs. 61.4 ± 8.86 mm(2) (P = 0.001), 33% ± 20% vs. 65% ± 15% (P = 0.01), and 65.6 ± 27.9 vs. 127.0 ±37.3 mg (P = 0.01)]. CONCLUSIONS Intravitreal H-7 (1,200 μM) significantly inhibits Soemmerings ring formation in the live rabbit eye, suggesting that agents that inhibit the actomyosin system in cells may prevent secondary cataract after phacoemulsification.

Combined effects of H7 and pilocarpine on anterior segment physiology in monkey eyes.

Purpose: To determine, in monkey eyes in vivo if low doses of the cholinergic agonist pilocarpine (PILO) can enhance the outflow facility responses to a maximal dose of the serine-threonine kinase inhibitor H7 without producing apparent miosis and/or excessive accommodation. Methods: Outflow facility was determined by two-level constant pressure perfusion in living monkeys after 24.5 μM phenylephrine (PE) bilaterally (stimulates the iris dilator without influencing the iris sphincter, ciliary muscle, or outflow facility, and facilitates the measurement of miosis and accommodation), 24.5 μM PE + 300 μM H7 (maximal outflow facility-effective dose) bilaterally, or 24.5 μM PE + 300 μ M H7 ± 2 or 10 μM PILO (2 μM PILO alone does not significantly increase outflow facility or accommodation but moderately constricts the pupil; 10 μM PILO is a threshold outflow facility-effective dose) in opposite eyes. Pupil diameter (Vernier calipers) and accommodation (coincidence refractometer) were measured essentially concurrently. Results: Outflow facility in the PE + H7 + 10 μM PILO eyes was 73% higher than that in the PE + H7 eyes (n = 6; p < 0.05). Accommodation was greater (n = 4; 2.6 vs. 0.6 D; p < 0.05) and pupil diameter was smaller (n = 6; 3.4 vs. 7.6 mm; p < 0.02) in the former than in the latter. No significant difference in outflow facility, accommodation or pupil diameter was observed between the PE + H7 + 2 μM PILO eyes and the PE + H7 eyes. Conclusions: In living monkeys, 10 μM PILO, but not 2 μM PILO, enhances the 300 μM H7-induced increase in outflow facility with only ∼ 3 diopters of accommodation, whereas 300 μM H7 partially inhibits the 2 μM PILO-induced, but not the 10 μM PILO-induced miosis. This suggests that, although the miosis following the threshold facility-effective dose of PILO cannot be reduced by combination with the maximal facility-effective dose of H7, the combination may at least benefit young glaucoma or ocular hypertension patients who are bothered by PILO-induced myopia more than by miosis.

Effects of Vitrectomy and Lensectomy on Older Rhesus Macaques: Oxygen Distribution, Antioxidant Status, and Aqueous Humor Dynamics

Purpose The purpose of this study is to evaluate effects of vitrectomy (PPV) and lens extraction with intraocular lens implantation (PE/IOL) on molecular oxygen (pO2) distribution, aqueous humor antioxidant–oxidant balance, aqueous humor dynamics, and histopathologic changes in the trabecular meshwork (TM) in the older macaque monkey. Methods Six rhesus monkeys underwent PPV followed by PE/IOL. pO2, outflow facility, and intraocular pressure (IOP) were measured. Aqueous and vitreous humor specimens were analyzed for antioxidant status and 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of oxidative damage. TM specimens were obtained for immunohistochemical and quantitative PCR analysis. Results pO2 at baseline revealed steep gradients in the anterior chamber and low levels in the posterior chamber (PC) and around the lens. Following PPV and PE/IOL, pO2 significantly increased in the PC, around the IOL, and angle. IOP increased following both surgical interventions, with no change in outflow facility. Histopathologic analysis did not show changes in TM cell quantification, but there was an increase in 8-OHdG. Quantitative PCR did not reveal significant differences in glaucoma-related gene expression. Aqueous and vitreous humor analysis revealed decreased ascorbate and total reactive antioxidant potential and increased 8-OHdG in the aqueous humor only in the surgical eyes. Conclusions Oxygen distribution in the older rhesus monkey is similar to humans at baseline and following surgical interventions. Our findings of histopathologic changes of TM oxidative damage and alterations in the oxidant-antioxidant balance suggest a potential correlation of increased oxygen exposure with oxidative stress/damage and the development of open angle glaucoma.