Circadian rhythm of intraocular pressure.

Intraocular pressure (IOP) plays a crucial role in glaucoma development, involving the dynamics of aqueous humor (AH). AH flows in from the ciliary body and exits through the trabecular meshwork (TM). IOP follows a circadian rhythm synchronized with the suprachiasmatic nucleus (SCN), the circadian pacemaker. The SCN resets peripheral clocks through sympathetic nerves or adrenal glucocorticoids (GCs). IOP's circadian rhythm is governed by circadian time signals, sympathetic noradrenaline (NE), and GCs, rather than the local clock. The activity of Na+/K+-ATPase in non-pigmented epithelial cells in the ciliary body can influence the nocturnal increase in IOP by enhancing AH inflow. Conversely, NE, not GCs, can regulate the IOP rhythm by suppressing TM macrophage phagocytosis and AH outflow. The activation of the ß1-adrenergic receptor (AR)-mediated EPAC-SHIP1 signal through the ablation of phosphatidylinositol triphosphate may govern phagocytic cup formation. These findings could offer insights for better glaucoma management, such as chronotherapy.

Nitric oxide-donating compounds for IOP lowering in glaucoma. / Donadores de óxido nítrico como hipotensores en glaucoma.

INTRODUCTION: An elevated intraocular pressure (IOP) remains the main risk factor for progression of glaucoma upon which we can efficiently act. Pharmacological strategies to reduce IOP are directed towards the reduction of aqueous humour (AH) production and/or the increase in AH drainage through the uveoscleral pathway. However, there are no drugs currently available as first-line treatment to increase AH outflow primarily via the conventional route. Ocular nitric oxide (NO) production takes place in AH outflow pathways and in the ciliary muscle, modulating the cellular response to elevated IOP. METHODS: This review describes the mechanism of action of endogenous NO and NO-donating compounds that are under research. It includes information regarding pre-clinical and clinical studies previously conducted with these compounds, discussing their role and therapeutic potential in the pharmacological treatment of ocular hypertension in glaucoma. RESULTS: The topical ocular administration of NO-donating compounds significantly lowered IOP and maintained it in animal models of glaucoma and subjects with ocular hypertension. CONCLUSIONS: The mechanism of action of these compounds is novel and scientific evidence that shows promising results. However, there is a need for more comprehensive studies to assess long-term safety and tolerability in order to properly evaluate their use in chronic therapies.

Lymphatic drainage from the eye: A new target for therapy.

Lowering intraocular pressure (IOP) has been central to glaucoma care for over a century. In order to prevent sight loss from disease, there has been considerable focus on medical and surgical methods to improve fluid drainage from the eye. In spite of this, our understanding of exactly how aqueous humor leaves the eye is not complete. Recently, lymphatic vessels have been discovered in the human uvea, with studies showing lymphatic fluid outflow in several models, in addition to evidence for their pharmacological enhancement. The presence of a lymphatic outflow system points to an exciting, expanded understanding of how fluid and particulate materials such as proteins move out of the eye, and how IOP may be regulated. We coin the term "uveolymphatic pathway"-to reflect a comprehensive and compelling new target for glaucoma and an exciting opportunity for future investigations to better understand the eye in health and disease.

Effects of novel ethacrynic acid derivatives on human trabecular meshwork cell shape, actin cytoskeletal organization, and transcellular fluid flow.

To determine efficacy and therapeutic index in the context of ocular hypotensive activity of the new ethacrynic acid (ECA) derivatives of the series (SA8,248 and SA8,389), 9,000 series (SA9,000, SA9,622 and SA9,995) and ticrynafen, we undertook a comparative evaluation of the dose-dependent effects of these compounds on human trabecular meshwork (HTM) cell shape, actin cytoskeletal organization, focal adhesions and transcellular fluid flow. Responses were either scored using an arbitrary scale of 1-5 or quantified. Compounds of the 9000 series (SA9,995>SA9,000>SA9,622) were found to be 14- to 20-fold more potent than ECA, ticrynafen or analogs from the 8,000 series (SA8,389>SA8,248) in terms of ability to induce cell shape alterations in HTM cells. Similarly, compounds of the 9,000 series (SA9,995>SA9,622>SA9,000) were found to be much stronger (2 to 20 fold) than ECA, ticrynafen or analogs of the 8000 series in terms of affecting decreases in actin stress fiber content in HTM cells. Analogs of the 9000 series (SA9,622>SA9,995>SA9,000) were also observed to be 8 to 10 fold more potent than ECA (SA8,389>ECA>SA8,248>ticrynafen) at eliciting decreases in cellular focal adhesions. Interestingly, analogs of the 9000 series (SA9,000>SA9,622>SA9,995) and SA8,248 demonstrated a huge increase (by many folds) in transcellular fluid flow of HTM cell monolayers as compared to ECA and ticrynafen. Collectively, these analyses revealed that the structural modification of ECA improves its ocular hypotensive efficacy, indicating that the SA9,000 series compounds might be promising novel ocular hypotensive drugs.

Primary trabecular meshwork cells incubated in human aqueous humor differ from cells incubated in serum supplements.

PURPOSE: To determine whether aqueous humor, the in vivo source of nutrients for trabecular meshwork cells, alters cellular and molecular characteristics in primary trabecular monolayer cell cultures when compared with standard culture conditions. METHODS: Human primary trabecular meshwork cell cultures were grown in DMEM supplemented with 50% human aqueous humor (DMEM-AH), heat-denatured DMEM-AH, 10% fetal bovine serum (DMEM-FBS, the standard culture supplement), or heat-denatured DMEM-FBS. Confluent trabecular cells were assayed for cell propagation and morphology for 21 days. Protein expression profiles of trabecular cell lysates were analyzed by two-dimensional polyacrylamide gel electrophoresis. Western blot analysis was used to determine the protein expression of myocilin and TIMP-1 in conditioned media collected from trabecular cells at 5, 10, 15, and 21 days. Myocilin expression was also analyzed by Western immunoblots after addition of dexamethasone (10(-7) M) or ascorbic acid (29 mg/dL). RESULTS: Trabecular cells supplemented with DMEM-AH for 21 days showed decreased cell proliferation when compared with DMEM-FBS (11% vs. 141%). Cellular morphology was also altered: Trabecular cells incubated in DMEM-AH showed larger-, broader-, and flatter-appearing cells than did the more spindle-shaped cells grown in DMEM-FBS. Protein profiles of trabecular cell lysates isolated from cells incubated in DMEM-AH differed from those incubated in DMEM-FBS. In DMEM-AH-conditioned medium, myocilin expression was increased and TIMP-1 expression was decreased at day 21. Induction of myocilin by dexamethasone was observed in conditioned medium isolated from cells treated with DMEM-FBS (442%), but only a 10% increase in myocilin was observed beyond the normal induction in DMEM-AH. Daily administration of ascorbic acid to DMEM-AH failed to increase myocilin expression beyond that obtained with DMEM-AH. CONCLUSIONS: Addition of human aqueous humor rather than FBS to trabecular monolayer cell cultures triggers significant changes in cellular and molecular characteristics. The protein component of aqueous humor is responsible for these changes. Aqueous humor supplementation may maintain cultured trabecular cells in a more physiologic state.

Ocular hypotension induced by electroacupuncture.

The purpose of this study was to investigate the effects of electroacupuncture (EA) on aqueous humor dynamics in rabbits. EA stimulation was performed through two acupuncture needles placed in close proximity to the sciatic nerve. The sites of needle entry were anesthetized. After 1 hr of EA stimulation, intraocular pressure (IOP) decreased and was accompanied by reductions of blood pressure and aqueous humor flow rate. The maximum reduction of IOP was 9 mmHg at 3 hr and decreases in norepinephrine and dopamine levels in aqueous humor occurred simultaneously. In addition, EA stimulation induced an 8-fold increase of endorphin levels in aqueous humor. Ocular hypotension induced by EA lasted for more than 9 hrs and was antagonized by naloxone pretreatment. Furthermore, the EA-induced ocular hypotension was reduced markedly in sympathetically denervated eyes compared with the response of intact, normal eyes. Antagonism of EA-induced ocular hypotension by naloxone, suppression of aqueous humor flow and catecholamine levels by EA and elevation of endorphin levels in aqueous humor by EA indicate that opioids/opiate receptors are involved in modulating ocular hydrodynamics in response to EA.

Effects of kakkon-to and sairei-to on aqueous flare elevation after complicated cataract surgery.

We evaluate prospectively the effects of traditional herbal medicines on elevation of aqueous flare after complicated cataract surgery. Twenty-seven patients with bilateral complicated cataract undergoing phacoemulsification with intraocular lens implantation were studied. The patients received no herbal medicine when the right eyes underwent cataract surgery. Fifteen patients were given kakkon-to (ge-gen-yang in Chinese) granules (7.5 g daily) and 12 patients were given sairei-to (cai-ling-tang in Chinese) granules (9.0 g daily), for 3 days before surgery, the day of surgery, and for 7 days after surgery when the left eyes underwent cataract surgery. Diclofenac eyedrops were instilled in all patients. Aqueous flare was measured before and after surgery. The differences in preoperative flare intensities between groups treated with Kakkon-to and Sairei-to were not significant. In the untreated right eyes of the kakkon-to and Sairei-to groups, the flare was 99.1 and 89.6 photon counts/msec, respectively, on day 1, and then gradually decreased. The flare intensities on days 1, 3, and 5 in the kakkon-to treated left eyes were significantly lower than in those of the untreated right eyes (Fig. 1). The flare intensities in the Sairei-to treated left eyes were the same as those in the untreated right eyes. Kakkon-to contributed to a reduction of aqueous flare elevation after surgery for complicated cataract.

Effects of experimental ocular inflammation on ocular immune privilege.

PURPOSE: To determine whether the inflammation of endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU) alters key in vivo and in vitro parameters of ocular immune privilege. METHODS: For EIU induction, C3H/HeN mice received 200 microg lipopolysaccharide (LPS). For EAU induction, B10.A mice were immunized with 50 microg interphotoreceptor retinoid-binding protein (IRBP) mixed with complete Freund's adjuvant. Aqueous humor (AqH) was collected at periodic intervals and assayed for leukocyte content and the ability to suppress or enhance T-cell proliferation. Eyes with EAU were assessed for the capacity to support anterior chamber (AC)-associated immune deviation (ACAID) induction after injection of ovalbumin (OVA). RESULTS: Inflammation within the anterior segment in EIU peaked at 12 to 24 hours and was detected from 10 days onward in EAU. In AqH of EIU, protein content rose within 4 hours, followed by infiltrating leukocytes. EIU AqH promptly lost its capacity to suppress T-cell proliferation and became mitogenic for T cells. In AqH of EAU, protein and leukocyte content rose at 11 days and continued to remain elevated thereafter. Whereas 11-day EAU AqH failed to suppress T-cell proliferation, AqH at later time points reacquired immunosuppressive properties. Injection of OVA into the AC of eyes of mice with EAU failed to induce ACAID. CONCLUSIONS: The intraocular inflammation of EIU and EAU disrupted important parameters of immune privilege, ranging from breakdown of the blood- ocular barrier, to loss of an immunosuppressive microenvironment, to abrogation of ACAID. Because AqH from inflamed EAU reacquired the ability to suppress T-cell proliferation, the authors conclude that the capacity to regulate immune expression and inflammation can be a property even of inflamed eyes.

[Effect of ciliary muscle electrical stimulation on ocular hydrodynamics and visual function in patients with glaucoma]. / Vliianie élektrostimuliatsii tsiliarnoí myshtsy na pokazateli gidrodinamiki glaza i zritel'nye funktsii u bol'nykh glaukomoí.

A course of transscleral electrostimulation of the ciliary muscle (10 sessions) using ESOF-1 device was administered to 20 patients (23 eyes) with bilateral primary open-angle glaucoma. A lens with 4 electrodes was placed on the eyeball in the ciliary body projection. Rectangular bipolar pulses up to 15 msec long were generated in the pack mode with current amplitude up to 10 mA for 3 to 7 min. Control group consisted of 17 "paired" eyes. After electrostimulation the accommodation volume increased by 54%, relative accommodation reserve by 34%, coefficient of discharge by 42%, and vision acuity by 0.1 to 0.4 diopters in 6 out of 7 eyes in which vision was lower than 1.0 diopter; intraocular pressure was 16% decreased and the number of absolute and relative scotomas in the central visual field (Humphry, 24-2) dropped by 40%. Improvement of the visual functions and hydrodynamic parameters of the eye was gradual, reaching the maximum by the end of the follow-up (6 months). In control eyes no positive changes were observed. The results indicate a positive effect of electrostimulation of the ciliary body and posterior ocular structures on intraocular fluid circulation and visual functions.

Aceclidine effects on outflow facility after ciliary muscle disinsertion.

Aceclidine increases outflow facility with little accommodative effect. To determine whether this dissociation resides in the ciliary muscle (CM) or trabecular meshwork (TM), we measured aceclidine effects on perfusion outflow facility in both eyes of 8 rhesus monkeys after unilateral disinsertion of the CM from the TM. Facility in the control eyes increased by approximately 250% following intravenous pilocarpine and by an additional approximately 250% following intracameral pilocarpine, relative to baseline and uncorrected for washout. In CM-disinserted eyes, the facility response to intravenous and intracameral pilocarpine averaged approximately 25% of that in contralateral controls. Cytochalasin B, which acts directly on the TM to increase facility but is not additive to maximal pilocarpine doses in normal eyes, had no additional effect beyond that of pilocarpine in control eyes but induced an additional 100% facility increase relative to baseline in CM-disinserted eyes. The accommodative response to carbachol in CM-disinserted eyes was approximately 80% of that in contralateral controls, consistent with retention of CM contractility and the gonioscopic appearance of shallow CM disinsertion. Intracameral aceclidine HCl doses of 5 and 50 micrograms increased outflow facility by approximately 80 and 250%, respectively, in control eyes, and by approximately 0 and 80% in CM-disinserted eyes. Either the low aceclidine dose affected facility via the CM, while the high dose exerted an additional effect on the TM, or aceclidine acted only via the CM, with the low dose being ineffective and the high dose modestly effective in CM-disinserted eyes because only a few CM-TM attachments remained.

Regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye.

Contractile properties of isolated trabecular meshwork strips have recently been described. In the present paper we characterize the regulation of the outflow pathway in the isolated perfused anterior segment of the bovine eye. Anterior segments of bovine eyes with detached iris, ciliary body and ciliary muscle were perfused at constant pressure of 8.8 mmHg. A constant outflow of approximately 6-8 microliters min-1 could be obtained for at least 3 hr. The calculated outflow resistance was in the range 1.1-1.4 mmHg min microliter-1. The relative outflow was significantly reduced after application of carbachol, reaching a maximal inhibition of 30%. EC50 for carbachol was 3 x 10(-8) mol l-1. Atropin completely blocked the effect of carbachol on outflow. Morphological examination of perfused anterior segments which were perfused with carbachol revealed an intact fine structure of the meshwork cells. Pilocarpine at 10(-5) mol l-1 reduced outflow by 15%. Epinephrine at 10(-5) mol l-1 reduced outflow, while epinephrine at 10(-6) mol l-1 slightly increased the outflow rate. This effect could be blocked by metipranolol. Endothelin-1 in concentrations of 2 x 10(-9) and 2 x 10(-8) mol l-1 inhibited relative outflow by > 30%. Carbachol, pilocarpine, endothelin and a high dose of epinephrine, which have been shown to induce contractions in isolated bovine trabecular meshwork and ciliary muscle strips, induced a reduction of outflow rate and an increase of outflow resistance of the anterior segment. Thus, at least in the bovine eye, the trabecular meshwork per se is directly involved in the regulation of aqueous humor outflow.

Molecular cloning of the human volume-sensitive chloride conductance regulatory protein, pICln, from ocular ciliary epithelium.

Chloride channels in the ocular ciliary epithelium are believed to play a key role in aqueous humor formation. We isolated a cDNA clone from a lambda Uni-ZAP cDNA library of human nonpigmented ciliary epithelial (NPE) cells encoding the swelling-induced chloride channel/channel regulator pICln. The human clone contains an open reading frame of 237 amino acids (M(r) 26,293). The deduced human amino-acid sequence shows 90.2% and 92.7% identity with counterparts isolated from rat kidney and the canine kidney epithelial cell line MDCK. Human NPE cell lines exhibited significant levels of pICln transcripts. Complementary perforated-patch, whole-cell patch clamping demonstrated that swelling activates Cl- channels of the NPE cells, as suggested by ruptured-patch measurements. The results document the molecular isolation and identification of a human cDNA clone of a Cl- conductance regulator from ocular cells displaying volume-activated Cl-channels.

Platelet aggregation on the endothelium of Schlemm's canal.

By using monkey eyes and light microscopy, transmission electron microscopy and scanning electron microscopy we have studied the role of platelets in Schlemm's canal. In eyes connected to a reservoir and subjected to an elevated intraocular pressure, there was break-down of the cell membranes facing the invaginations into the endothelial cells, resulting in local protrusions from the invaginations and ruptures. Other unphysiological openings in the inner wall were due to separation of the intercellular junctions. Aggregates of platelets were observed at both these types of openings. Small aggregates were observed also in massaged eyes and in eyes which had not been touched before fixation. It seems likely that the intercellular junctions are fragile, tending to disrupt even under normal conditions and that platelets play a role in their repair. It is also suggested that platelets tend to restrict the size of the physiological pores.

Laser-induced glaucoma in rabbits.

Argon laser energy was applied to the trabecular meshwork of pigmented rabbits in an attempt to develop an animal model of 'glaucoma'. Laser energy was varied to determine the optimal level needed to produce sustained ocular hypertension. An initial response of ocular hypertension followed by hypotension was observed in all of the animals tested. Approximately half of the laser-treated rabbits developed a secondary buphthalmus and sustained ocular hypertension. In these animals outflow facility was decreased by approximately 60%. Histologic examination at 4- and 8 weeks after laser treatment demonstrated a wound-healing response resulting in closure of the intertrabecular spaces and obstruction of outflow to injected carbon particles. Optic nerve cupping and a loss of ganglion cells were also observed. Topical application of L-timolol (0.5%), pilocarpine (2.0%) and forskolin (1.0%) were found to be effective in decreasing intraocular pressure in the laser-treated, hypertensive eye with no significant effect in control non-laser-treated eyes, suggesting that this model can be a useful tool for screening potential antiglaucoma medications.

Morphology of tight junctions in the ciliary epithelium of rabbits during arachidonic acid-induced breakdown of the blood-aqueous barrier.

A reversible breakdown of the blood-aqueous barrier in the iridial processes of rabbits has been induced by arachidonic acid as demonstrated by the passage of horseradish peroxidase at places through the tight junctions. Freeze-fracture images reveal very discontinuous P-face ridges. However, the analysis of complementary replicas demonstrates that discontinuities of P-face ridges are always complemented by particles or short bars found in the E-face furrows. Though the problem exists of correlating freeze-fracture images of the junctional structure to the focal passage of horseradish peroxidase, the data suggest that the discontinuities of P-face ridges cannot be the structural counterpart of the passage of horseradish peroxidase. Alternative pathways of horseradish peroxidase are discussed in context with the offset bifibrillary model of the junction.

Physiology and pharmacology of aqueous humor inflow.

Recent physiological and pharmacological data pertinent to aqueous humor inflow regulation have been reviewed. New anatomical and electrophysiological data are presented, particularly related to aqueous humor secretion. The action of adrenergic agonists and antagonists is discussed in relation to changes in intraocular pressure, and the effects of a variety of experimental perturbations is presented. The multiple factors which affect aqueous humor inflow are discussed in the context of an evaluation of recent pertinent literature.

Ocular hypertension. I. The clinical course during ten years without therapy. Aqueous humour dynamics.

An initial group of 152 subjects with moderate ocular hypertension constituted the basic material of the present study. Antiglaucoma treatment was started in 8 men and in 6 women. A remaining group of 92 subjects were kept under clinical observation for ten years without antiglaucomatous therapy and without any evidence of progressive disc cupping or field defects. The intraocular pressure as well as the outflow facility showed a tendency to decrease with time, which was assumed to indicate a reduction in aqueous flow with increasing age. The tomographic findings support the view that the moderately elevated intraocular pressure is mainly due to an increased rate of aqueous flow.

[The role of the autonomic nervous system in regulating the hydrodynamics of the intact eye]. / Rol' vegetativnoi nervnoi sistemy v mekhanizme reguliatsii gidrodinamiki intaktnogo glaza

In cats, the low-molecular indicator: radioactive phosphorus, and the tonography revealed that the local application of acetylcholine accelerates the fluid exchange in the intact eye, considerably facilitating the outflow. Noradrenaline and the threshold stimulation of the cervical sympathetic nerve slow down the intraeye fluid exchange, decreasing the outflow. The data obtained suggest that the neural regulation of the eye hydrodynamics involves, mainly, a direct effect of the vegetative innervation on the eye drainage system.