Anterior Chamber Perfusion Research Articles

Effects of Latrunculin-B on Outflow Facility and Trabecular Meshwork Structure in Human Eyes

To determine the effect of the F-actin-disrupting agent latrunculin-B on aqueous outflow facility and trabecular meshwork architecture in human eyes. After baseline facility measurement in human eye bank eyes (n = 9 pairs), one eye of each pair received anterior chamber exchange and continued perfusion with medium containing 1 microM latrunculin-B. Contralateral eyes were treated in a similar manner with vehicle. Eyes were fixed by anterior chamber exchange and perfusion with universal fixative at 8 mm Hg (corresponding to a physiologic pressure of 15 mm Hg in vivo), and outflow pathway tissues were examined by transmission and scanning electron microscopy. Perfusion of eyes with 1 microM latrunculin-B caused a continuous and ongoing increase in outflow facility, resulting in a net facility difference of 64% 2 hours after drug administration (P < 0.006). Transmission electron microscopy showed subtle and focal detachment of the inner wall of Schlemm's canal, rarefaction of the juxtacanalicular tissue (JCT), and cell-cell and cell-matrix detachment. Scanning electron microscopy showed collapsed vacuoles in the inner wall of Schlemm's canal and a marked increase in the number and size of border (paracellular) pores in the inner wall. Latrunculin-B increases outflow facility in postmortem human eyes. The mechanism of facility increase is most likely due to loss of mechanical integrity of the trabecular meshwork as a consequence of reduction in cell-cell and cell-matrix adhesion. The facility increase and the extent of inner wall separation from the JCT that we observed were both qualitatively similar to that reported in living monkey eyes, but the magnitude of the facility increase and morphologic changes were much less than in the living monkey. This supports the idea that inner wall separation from the JCT may modulate outflow facility.

Aquaporins in endothelia

Aquaporin-1 (AQP1) water channels are expressed widely in microvascular endothelia outside of the central nervous system, including renal vasa recta and tumor microvessels, as well as in non-vascular endothelia in pleura, peritoneum, cornea, and lymphatics. In kidney, AQP1-facilitated water transport in outer medullary descending vasa recta is likely an important component of the urinary concentrating mechanism. However, in most vascular endothelia outside of kidney, it remains uncertain whether AQP1 expression and high water permeability are physiologically important. AQP1 in non-vascular endothelia at the inner corneal surface is involved in the maintenance of corneal transparency. Recently, a new role of AQP1 in endothelial cell migration was discovered in analyzing the cause of defective tumor angiogenesis in AQP1-deficient mice. AQP1 facilitates endothelial cell migration by a mechanism that may involve facilitated water transport across cell protrusions (lamellipodia). AQP1 inhibitors may thus have aquaretic and antiangiogenic activity.

Effect of age on outflow resistance washout during anterior chamber perfusion in rhesus and cynomolgus monkeys

When aqueous humour outflow resistance is measured by two-level constant pressure perfusion in non-human primate eyes, a progressive decrease in outflow resistance, known as the ‘washout effect’ occurs with time. The effect of age on total outflow resistance washout (the reciprocal of outflow facility (OF)) was determined in rhesus and cynomolgus monkeys. In cynomolgus monkeys, the effect of time between exchange of the anterior chamber (AC) contents and post-exchange OF measurements on outflow resistance was also examined. Total OF was determined at baseline in one eye of 35 rhesus monkeys aged 4–29 yrs, and in 27 cynomolgus monkeys, aged 3–17 yrs, at baseline and after 10 min AC exchange with 1 or 2 ml Bárány's perfusand or in Bárány's containing 0.01–1% dimethyl sulfoxide (DMSO) or Tris base. Resistance washout did not differ with age at baseline in rhesus or cynomolgus monkeys. Similarly, no changes were found when comparing post-exchange resistance washout vs. age in cynomolgus monkeys that had undergone AC exchange with Bárány's perfusand only or Bárány's containing 0.01–1% DMSO or Tris base. Rate of resistance washout decreased with increased length of time between exchange of the AC contents and post-exchange outflow facility readings (−0.0004±0.0092 mmHg min −1 μl −1 yr −1; p=0.016). Several explanations for these findings are plausible.

Aqueous humor dynamics in monkeys after topical 8-iso PGE(2).

To determine in normotensive cynomolgus monkeys, the effects of topical 8-iso prostaglandin (PG)E(2) on intraocular pressure (IOP), aqueous humor formation (AHF), uveoscleral outflow (Fu), and total and trabecular outflow facility. IOP was measured by Goldmann applanation tonometry under ketamine anesthesia after single or twice-daily topical treatments with 8-iso PGE(2). With animals under pentobarbital anesthesia, AHF and flow to blood (equated to trabecular outflow) were determined by anterior chamber perfusion with radioactively labeled albumin solution. Fu and trabecular outflow facility were calculated from these measurements. Total outflow facility was measured by two-level, constant-pressure perfusion. IOP was not significantly changed after single or multiple 10- micro g doses of 8-iso PGE(2). The 25- micro g dose significantly decreased IOP by 2 to 3 mm Hg compared to the contralateral vehicle-treated control 4 to 6 hours after a single dose and by 3 to 5 mm Hg within 1.5 hours after twice-daily treatments for 4 to 5 days. Total outflow facility corrected for control eye washout was increased by an apparent 37% (P < 0.02, n = 7) from 2 to 3.5 hours after the ninth dose, largely due to outlier values obtained in one monkey. Isotope studies performed after twice-daily treatments totaling 9 to 29 doses showed no change in AHF, trabecular outflow facility, or total outflow facility. Relative to AHF, trabecular outflow was significantly decreased, and the calculated Fu was significantly increased when all data were analyzed. The present findings are consistent with lowering of IOP by 8-iso PGE(2), primarily by increasing Fu. A direct effect on the trabecular meshwork was not indicated by these in vivo studies.

Influence of molecular weight on intracameral dextran movement to the posterior segment of the mouse eye.

Uveoscleral outflow provides a potential pathway to the posterior segment for drug delivery. In this study, the influence of molecular weight on the intraocular distribution of dextran molecules after intracameral injection in the mouse eye was investigated. The anterior chambers of the eyes of 64 anesthetized NIH Swiss mice were perfused with various fluorescent dextran solutions (10, 40, 70, and 500 kDa) at 500 nL/min for 10 minutes. At 10, 20, or 60 minutes after the initiation of the anterior chamber perfusion, the mice were killed and tissue obtained for evaluation by fluorescence microscopy. Each of the different molecular weight dextrans were visible in the anterior chamber of the mouse eye after the termination of the experiments. The 10-kDa dextran was observed in the supraciliary space and the supraciliary sclera after 10 minutes and in the anterior sclera after 60 minutes of perfusion. The 40-kDa dextran was detected in the supraciliary space and the anterior sclera after 10 minutes and in the anterior choroid and sclera after 20 and 60 minutes, but not in the posterior segment. The 70-kDa dextran was observed in the supraciliary space and anterior choroid after 10 minutes. After 20 minutes, it was visible in the equatorial choroid. After 60 minutes, it was observed in the posterior choroid. The 500-kDa dextran was observed in the supraciliary space and the anterior choroid after 10 minutes and in the supraciliary sclera at 20 minutes. At 60 minutes, 500-kDa dextran was observed in the equatorial choroid, but not farther toward the posterior. The influence of molecular weight on the redistribution of macromolecules from the anterior chamber to the posterior globe in the mouse eye appears to be similar to primate eyes. These similarities include passage of all size dextrans through the proximal uveoscleral pathway, the dependence of the extent of posterior movement on the size of the dextran, and the absence of large dextran entry into the distal uveoscleral pathway.

Optineurin Gene Expression Level in Human Trabecular Meshwork Does Not Change in Response to Pressure Elevation

Mutations in the gene optineurin (OPTN) have been associated with primary-open angle glaucoma. Here we present a study on the level of OPTN gene expression in the human trabecular meshwork in response to increased perfusion pressure in the anterior chamber perfusion model of the human eye. Perfusion pressure was raised from 10 to 30 mm Hg for periods ranging between 1 and 24 h. OPTN transcript levels in the trabecular meshwork were determined using real-time quantitative polymerase chain reaction. The results show no statistically significant alteration of the OPTN transcript level after raising the pressure. Moreover, no changes were detected in the transcript levels of the 3 known OPTN isoforms. This result shows that enhanced pressure levels do not lead to rapid changes in gene expression levels of OPTN in human trabecular meshwork. This suggests that alterations in OPTN gene expression are not involved in the mechanisms regulating aqueous humor outflow after an increase in intraocular eye pressure.

Aquaporin Deletion in Mice Reduces Corneal Water Permeability and Delays Restoration of Transparency after Swelling

Two aquaporin (AQP)-type water channels are expressed in mammalian cornea, AQP1 in endothelial cells and AQP5 in epithelial cells. To test whether these aquaporins are involved in corneal fluid transport and transparency, we compared corneal thickness, water permeability, and response to experimental swelling in wild type mice and transgenic null mice lacking AQP1 and AQP5. Corneal thickness in fixed sections was remarkably reduced in AQP1 null mice and increased in AQP5 null mice. By z-scanning confocal microscopy, corneal thickness in vivo was (in microm, mean +/- S.E., n = 5 mice) 123 +/- 1 (wild type), 101 +/- 2 (AQP1 null), and 144 +/- 2 (AQP5 null). After exposure of the external corneal surface to hypotonic saline (100 mosm), the rate of corneal swelling (5.0 +/- 0.3 microm/min, wild type) was reduced by AQP5 deletion (2.7 +/- 0.1 microm/min). After exposure of the endothelial surface to hypotonic saline by anterior chamber perfusion, the rate of corneal swelling (7.1 +/- 1.0 microm/min, wild type) was reduced by AQP1 deletion (1.6 +/- 0.4 microm/min). Base-line corneal transparency was not impaired by AQP1 or AQP5 deletion. However, the recovery of corneal transparency and thickness after hypotonic swelling (10-min exposure of corneal surface to hypotonic saline) was remarkably delayed in AQP1 null mice with approximately 75% recovery at 7 min in wild type mice compared with 5% recovery in AQP1 null mice. Our data indicate that AQP1 and AQP5 provide the principal routes for corneal water transport across the endothelial and epithelial barriers, respectively. The impaired recovery of corneal transparency in AQP1 null mice provides evidence for the involvement of AQP1 in active extrusion of fluid from the corneal stroma across the corneal endothelium. The up-regulation of AQP1 expression and/or function in corneal endothelium may reduce corneal swelling and opacification following injury.

3Alpha,5beta-tetrahydrocortisol effect on outflow facility.

3Alpha,5beta-Tetrahydrocortisol (THF) was administered topically and intracamerally to ocular normotensive cynomolgus monkeys to determine whether it affects outflow facility. Monkeys received THF either topically at a dose of 2 x 5 microl drops of 300 microg/10 microl twice daily for 4 days (n = 4) or 3 times daily for 10 days (n = 4) with 10% DMSO as vehicle to the control eye, or intracamerally via 2 ml anterior chamber (AC) exchange of 30 microg/ml THF with vehicle, 0.1% DMSO, to the control eye followed by a second AC exchange using 300 microg/ml THF with vehicle to the control eye. Outflow facility was measured by a two-level constant pressure AC perfusion after administration of eye drops or after baseline outflow facility measurement and AC exchange with THF solution. The results showed no effect on outflow facility in normotensive cynomolgus monkeys.

Involvement of nitric oxide in the ocular hypotensive action of nipradilol

Purpose. To evaluate the role of nitric oxide (NO) in the mechanism of the ocular hypotensive action of nipradilol, a ß-blocker with a 1 -blocking activity. Methods. Change in intraocular pressure (IOP) of albino rabbits was measured after a single application of carboxy-PTIO (c-PTIO), an NO trapping agent. Next, IOP change was measured every hour for 5 hours after the instillation of 0.25% nipradilol into one of the eyes with and without c-PTIO pretreatment of both eyes. IOP change induced by desnitro-nipradilol was also examined. The outflow facility and uveoscleral outflow were determined by two-level constant pressure and anterior chamber perfusion methods before and at 3 hours after the application of nipradilol with and without c-PTIO pretreatment. Results. Topical administration of c-PTIO showed no significant effect on IOP. Unilateral instillation of nipradilol reduced IOP significantly compared with control eyes with a maximum reduction of 3.6 mmHg and effect duration of 3 hours. Pretreatment with c-PTIO partially inhibited the reduction during an earlier period (1~2 hours) and completely at 3 hours. IOP change by desnitro-nipradilol was similar to that by nipradilol with c-PTIO pretreatment. Nipradilol increased both outflow facility and uveoscleral outflow at 3 hours, whereas pretreatment with c-PTIO inhibited both of these outflows. Conclusions. Results indicate that ocular hypotensive action by nipradilol during the relatively late period may be mainly due to enhancement of aqueous humor outflow by NO at least in the rabbits.

Effects of Topical Epinephrine on Aqueous Humor Dynamics in the Cat

The purpose of this study was to investigate, in cats, the effects of topical epinephrine on aqueous humor dynamics as measured by the non-invasive method of fluorophotometry and by other methods. Measurements were carried out on 12 cats before and after one week of twice daily treatment with 2% epinephrine hydrochloride to one eye. Aqueous flow and outflow facility were determined using fluorophotometry. Uveoscleral outflow was calculated from these results and was evaluated with anterior chamber perfusion of FITC-dextran. Outflow facility also was measured by tonography. Epinephrine-treated eyes, compared with their baseline values, showed a 31% reduction in intraocular pressure (P<0.001), a 23% reduction in aqueous flow (P<0.05), a 60% increase in fluorophotometric outflow facility (P<0.05), and a 43% increase in tonographic outflow facility (P<0.05). Treated eyes, compared with contralateral control eyes, showed a 27% reduction in IOP (P<0.005), a 25% reduction in aqueous flow (P<0.005), a 38% increase in fluorophotometric outflow facility (P<0.05), and a 34% increase in tonographic outflow facility. When evaluated by both fluorophotometry and FITC-dextran tracer methods, epinephrine had no significant effect on uveoscleral outflow. It was concluded that, in cats treated with topical epinephrine twice daily for a week, a reduction in intraocular pressure is induced by an increase in outflow facility and decrease in aqueous flow.

Effects of Adenosine Agonists on Intraocular Pressure and Aqueous Humor Dynamics in Cynomolgus Monkeys

The effects of single or multiple topical doses of the relatively selective A1adenosine receptor agonists (R)-phenylisopropyladenosine (R-PIA) and N6-cyclohexyladenosine (CHA) on intraocular pressure (IOP), aqueous humor flow (AHF) and outflow facility were investigated in ocular normotensive cynomolgus monkeys. IOP and AHF were determined, under ketamine anesthesia, by Goldmann applanation tonometry and fluorophotometry, respectively. Total outflow facility was determined by anterior chamber perfusion under pentobarbital anesthesia. A single unilateral topical application of R-PIA (20–250 μg) or CHA (20–500 μg) produced ocular hypertension (maximum rise=4.9 or 3.5 mmHg) within 30 min, followed by ocular hypotension (maximum fall=2.1 or 3.6 mmHg) from 2–6 hr. The relatively selective adenosine A2antagonist 3,7-dimethyl-1-propargylxanthine (DMPX, 320 μg) inhibited the early hypertension, without influencing the hypotension. Neither 100 μg R-PIA nor 500 μg CHA clearly altered AHF. Total outflow facility was increased by 71% 3 hr after 100 μg R-PIA. In conclusion, the early ocular hypertension produced by topical adenosine agonists in cynomolgus monkeys is associated with the activation of adenosine A2receptors, while the subsequent hypotension appears to be mediated by adenosine A1receptors and results primarily from increased outflow facility.

Effect of DMSO and exchange volume on outflow resistance washout and response to pilocarpine during anterior chamber perfusion in monkeys

PURPOSE. To determine the dependence of outflow resistance washout on anterior chamber (AC) fluid exchange volume (EV); the minimum EV required for complete AC mixing, and the effect of 0.01–5% DMSO in the exchange solution on out-flow resistance. METHODS. Total outflow facility was determined in 63 pento-barbital-anesthetized cynomolgus monkeys, before and after AC exchange, for 10 min, with 1, 2, and 4 ml of Bárány's perfusand, containing 0.01–5% DMSO or containing 1 µg/ml or 5 µg/ml pilocarpine HCL (pilo), each volume. RESULTS. Post-exchange facility increased by 21% (p < 0.05) vs. baseline for both the 1 and 2 ml Bárány's volumes and by 50% (p < 0.001) for the 4-ml volume. Washout-corrected post-exchange facility was unchanged from baseline, following 1 or 2 ml exchange with 1 µg/ml pilo; 5 µg/ml pilo increased facility by 130 ± 41% and 174 ± 40% respectively, relative to baseline. Exchange with 4 ml of 1 µg/ml pilo increased facility 72% more than the 2 ml (p < 0.029) and 100% more than the 1 ml (p < 0.014) volumes. When corrected for their respective baselines, 2 ml of 5 µg/ml pilo increased facility 12.7 ± 4.4% more than the 1 ml volume (p < 0.05). Post exchange facility was not significantly increased over baseline in eyes exchanged with 2 ml of 0.01–4% DMSO or in eyes exchanged with Bárány's alone. When corrected for their respective baselines, post-exchange facility was significantly lower in eyes exchanged with 5% DMSO vs. Bárány's (p = 0.022). CONCLUSIONS. One- and 2-ml EVs preserve outflow resistance equally well, and substantially better than 4 ml. Two ml is the minimum volume necessary for adequate mixing of AC drug solutions, to achieve the full facility effect. Concentrations of DMSO up to 4% in a 2-ml EV can solubilize compounds without additionally decreasing outflow resistance.

Total iridectomy does not alter outflow facility responses to cyclic AMP in cynomolgus monkeys

Total outflow facility was measured by two-level constant pressure anterior chamber perfusion in surgically untouched and totally iridectomized cynomolgus monkey eyes receiving bolus intracameral infusions of cyclic AMP, dibutyryl cyclic AMP, or 5'AMP. Cyclic AMP doses of 50 and 100 micrograms (corresponding to initial intracameral concentrations of 1.5- and 3 mM) increased facility by approximately 20 and 40% respectively in both surgically untouched and aniridic eyes. Higher doses produced no greater effect, and lower doses were ineffective. Dibutyryl cyclic AMP and 5'AMP were ineffective in doses up to 100 micrograms (2- and 3 mM respectively). These findings indicate that the iris plays no mechanical role in mediating the facility response to cyclic AMP, and that the iridectomy procedure per se does not compromise the ability of the outflow pathways to respond to cyclic AMP. Since untouched and aniridic eyes exhibit similar facility responses to epinephrine and norepinephrine, the present findings also suggest that the iris is not the source of cyclic AMP mediating facility responses to catecholamines.

Isoproterenol dose-outflow facility response relationships in the vervet monkey.

Total outflow facility was measured by two-level constant pressure anterior chamber perfusion in surgically untouched vervet monkeys receiving intracameral infusions of mock aqueous humor without or with isoproterenol. Bolus dl-isoproterenol sulfate doses of 1 to 10 micrograms, corresponding respectively to initial intracameral base concentrations of 2 X 10(-5) M and 2 X 10(-4) M, increased facility 30-50%; higher and lower bolus doses were ineffective, as were 0.1 and 1 g doses of 1-isoproterenol hydrochloride (4 X 10(-6) M and 4 X 10(-5) M base). Relatively maintained intracameral dl-isoproterenol concentrations of 2 X 10(-7) M and 2 X 10(-6) M or l-isoproterenol concentrations of 4 X 10(-7) M to 4 X 10(-5) M did not significantly increase facility, although there was great variability with the latter agent. Possible reasons for species, investigator, technique, and dosage-associated differences in results are discussed.

Effect of hypothermic perfusion on corneal endothelial morphology.

The effect of moderate in-vivo hypothermic perfusion on corneal endothelial integrity was studied in the cat. Eleven cats underwent in-vivo anterior chamber perfusion for 30 minutes with either normothermic (23 degrees C) or hypothermic (5 degrees C) perfusate. Corneas were then evaluated clinically (biomicroscopy), functionally (vital staining), and morphologically (scanning electron microscopy) for changes attributable to hypothermic perfusion. All 3 modes of evaluation suggested no difference in corneal endothelial integrity under the 2 experimental perfusion conditions. At the clinical and scanning electron microscope levels hypothermic perfusion does not show any effects on the corneal endothelium. Regional hypothermia is of theoretical and potential utility in procedures involving prolonged intraocular perfusion.

PILOCARPINE‐INDUCED SUBSENSITIVITY TO CARBACHOL AND PILOCARPINE OF CILIARY MUSCLE IN VERVET AND CYNOMOLGUS MONKEYS

Vervet monkeys were given unilateral treatment for two weeks with one 2% pilocarpine eye drop three times daily between 8 a.m. and 6 p.m. (night interval 14h) and were then subjected to anterior chamber perfusion 20 mug pilocarpine intracamerally caused similar and substantial increases in outflow facility in both eyes. Cynomolgus monkeys were unilaterally treated with continuous release of 33 mug/h of pilocarpine for 5-6 days. The facility response to 1 mg/kg pilocarpine iv was small or absent on the treated side. Iridectomized cynomolgus monkeys responded with 12.6+/-5.2 (SD) diopters accommodation to 1.5 mg/kg pilocarpine im, with very similar responses in the two eyes. During continuous release of 30 mug/h pilocarpine, accommodation of the treated eye dropped grdually and after 4-8 days treatment the accommodative response was markedly reduced to pilocarpine 1.5 mg/kg im or 100 mug topically. The degree of subsensitivity was much less when tested with either systemic or topical carbachol. This was also the case in a few vervet experiments. Recovery of full pilocarpine sensitivity took several weeks in the cynomolgus monkey. As an explanation for the excessive subsensitivity and large interindividual differences found with pilocarpine, an individually variable, nonmuscarinic relaxant action counteracting the muscarinic excitatory action is suggested. Clinical implications of this hypothesis are discussed.

The effect of trabeculotomy in normal eyes of rhesus and cynomolgus monkeys studied by anterior chamber perfusion.

Trabeculotomy ab externo was performed in 5 rhesus and 18 cynomolgus monkeys. Prae- and postoperative (2 weeks and later) anterior chamber perfusions were made to estimate intraocular pressure and outflow facility. The operation caused little or no consistent changes in intraocular pressure measured in the anesthetized monkey. In rhesus monkeys facility increased, in cynomolgus monkeys it decreased on an average. A mathematical analysis of the perfusion data was made under the assumption that the operation had produced a hole in the trabecular meshwork but that simultaneously parts of the filter had been clogged. The part of the scleral wall of the canal perfused from the hole was estimated to be of the order of 10 to 30° while in some cases the capacity for filtration was greatly reduced over much of the remaining meshwork. The combined effect of these opposite changes can be a slight increase in facility or a decrease, depending on the prae-operative state of the meshwork. If resistance is high to begin with, as in glaucoma, the operation may normalize overall outflow facility. The present paper shows that by an almost superhuman effort it is still just barely possible to write about matters of glaucoma without quoting our admired and beloved friend and teacher Goldmann.

Effects of Pilocarpine on Ocular Tension Dynamics

Pilocarpine has been used since 1877 in the treatment of glaucoma because of its effect in lowering intraocular pressure. How­ ever, it is only since the advent of tonography and anterior chamber perfusion tech­ niques that the mechanism of this effect has been investigated. It was the purpose of this study to evalu­ ate further the effects of pilocarpine by means of diurnal tonometric and tonographic testing of normal and glaucomatous sub­ jects.