JOURNAL OF OCULAR PHARMACOLOGYAND THERAPEUTICSVolume 15, Number 1, 1999Mary Ann Liebert, Inc.

Aqueous Humor Dynamicsin a-Chymotrypsin-InducedOcular Hypertensive Rabbits

JOSÉ MELENA, JUAN SANTAFÉ,JOSÉ SEGARRA-DOMÉNECH, and GUSTAVO PURAS

Departamento de Farmacología, Facultad de Farmacia,Universidad del País Vasco, Paseo de la Universidad, No. 7, Vitoria, Spain

ABSTRACT

Aqueous humor dynamics were studied in a-chymotrypsin-induced ocular hypertensive rabbitseither by tonographic or two-level constant pressure perfusion techniques. A significant correlation wasobtained between the values of outflow facility in a-chymotrypsin-induced ocular hypertensive rabbitsas determined by tonography and constant pressure perfusion. The mean value of tonographic outflowfacility in ocular hypertensive rabbits was not statistically different from that found in ocularnormotensive rabbits. On the contrary, the estimated rate of aqueous inflow in ocular hypertensiverabbits was about 1.5-fold higher than that of ocular normotensive ones. While topical timolol loweredintraocular pressure and aqueous humor inflow in ocular hypertensive rabbits, pilocarpine did not

produce any significant effect. Aqueous humorproteinwas significantly increased in ocularhypertensiveeyes. The results of this study show that accurate measurements of outflow facility can be obtained ina-chymotrypsin-induced ocular hypertensive rabbits by tonographic technique. Our data suggest thatthe long-term ocular hypertension induced by a-chymotrypsin in albino rabbits may be secondary to an

increase in the rate of aqueous humor inflow, likely produced by a breakdown of the blood-aqueousbarrier. This finding strongly conflicts with the hypothesis of trabecular blockage as the cause of a-chymotrypsin-induced ocular hypertension in this species.

INTRODUCTION

Animal models for glaucoma, or more accurately for ocular hypertension, permit investigations notpossible on the human eye and offer a valuable mean for the screening of antiglaucoma drugs, a-

Chymotrypsin rabbit glaucoma is one of the animal models more widely used for the study of ocularhypotensive drugs. Observation of a dose-related, transient rise in intraocular pressure accompanied bya decrease in aqueous outflow facility in patients (1-3) who had undergone intracameral a-chymotrypsininjection during cataract surgery led to use of this enzyme to induce experimental ocular hypertensionboth in rabbits (4-6) and monkeys (7,8).

In rabbits, a sustained increase in intraocular pressure is produced by injection ofa-chymotrypsininto the posterior chamber of the eye that allows the study of long-term effects of elevated intraocular

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pressure and the screening ofantiglaucoma drugs (5,6). However, despite the considerable body ofworkobtained by using this experimentalmodel for such purposes, nothing is known about the consequencesof a-chymotrypsin injection on aqueous humor dynamics in this species, its ocular hypertensive effectbeing classically ascribed to a decrease in aqueous outflow resulting from trabecular blockage caused bylysed zonular material on the basis of experiments largely carried out both in monkeys and humans(2,7,9,10).

In order to determine the characteristics of this glaucoma model for further studies on new ocularhypotensive drugs, the aim of the present work was to examine the aqueous humor dynamics in rabbitsmade ocular hypertensive by intracameral injection ofa-chymotrypsin either by tonographic or constantpressure perfusion techniques. Tonographic values were compared with those obtained in ocularnormotensive rabbits to ascertain the mechanism of the ocular hypertension caused by a-chymotrypsin.Furthermore, changes induced on this experimental model by two currently used antiglaucoma drugs,pilocarpine and timolol, are also described in this work.

MATERIALS AND METHODS

Animals

Experiments were carried out in adult albino New Zealand rabbits, weighing 3-4 kg, which were

previously trained to be handled and restrained in boxes in the laboratory environment. This studyconformed to the ARVO Resolution on the use of Animals in Research.

Induction ofOcular HypertensionOcular hypertension was induced in the left eye of 10 albino rabbits as previously described (4-6).

Briefly, the animals were anesthetized with intramuscular ketamine hydrochloride (40 mg/kg) andacepromazine maléate (0.1 mg/kg). Under topical anesthesia with tetracaine hydrochloride (1.66 x 102M), left eyes were treated by injection (syringes Micro-Fine®, 29G x 1/2, Becton Dickinson, Dublin,Ireland) into the anterior chamber of 60 units ofa-chymotrypsin (Quimotrase oftálmico®, LaboratoriosCusí, Barcelona, Spain) dissolved in 0.2 ml saline. To avoid efflux of aqueous and a-chymotrypsinthrough the injection hole, the syringe was kept for two minutes in the eye before being removed. Onlyanimals exhibiting intraocular pressures above 25 mmHg two months after a-chymotrypsin injection (10out 14 treated rabbits) were included in this study.

Measurement of Intraocular Pressure and Aqueous Humor Dynamics

Intraocular pressure measurements and tonographies were done using a Mentor Model 30 classicpneumatonograph (Norwell,MA, USA) that was calibratedby directmanometry in anesthetized rabbits.The reliability of the intraocular pressure readings in a-chymotrypsin-treated eyes was also checked bydirect manometry.

In order to perform tonographies, a 10 g weight was attached to the pneumatic plunger accordingto the instructions of the manufacturer. The coefficient ofoutflow facility was calculated from Grant'sequation C = AV/AP x T, where AFis the fluid volume lost in 7minutes and AP is the average intraocularpressure elevation induced during the test. /IFwas evaluated using mean pressure-volume relation ofliving rabbit eyes published by Eisenlohr and Langham (11). The rate of aqueous humor inflow was

calculated according to the Goldman's equation F = C(P0- Pev), where F is the rate of aqueous humorinflow, C is the tonographic facility of aqueous humor outflow, P0 is the intraocular pressure and P„ isthe episcleral venous pressure. A value of 9 mmHg was estimated for episcleral venous pressure (12).

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Measurement of outflow facility by constant pressure perfusion was performed according to themethod ofBárány (13). The anterior chamber of the rabbit eye was cannulated under topical anesthesiawith one 50 pi drop of tetracaine hydrochloride (1.7 x 10'2 M) to avoid corneal reflexes with a 25-gaugeneedle introduced through the limbus, parallel to the plane of the iris. The needle was connected via a

polyethylene tube to a perfusion system consisting in a microflowmeter (Gilmont Instruments, Inc.,Barrington, IL, USA) attached to a suspended reservoir of Balanced Salt Solution (BSS, AlconLaboratories, Fort Worth, TX, USA). Prior to cannulation, any air bubbles were flushed out of theperfusion system. Following cannulation and a 5 minute stabilization period at the resting IOP withoutinfusion, the eyes were successively perfused for 5 minutes at inflow pressure of 10 and 20mmHg abovenormal intraocular pressure to obtain accurate measurements of the fluid flow. During each 5 minuteperiod, fluid flow was measured for 3 minutes beginning 2 minutes after the pressure change was

induced. The total outflow facility was calculated from the difference between the successive flow ratesdivided by the difference between the pressures.

For the measurement of outflow facility, ocular hypertensive rabbits were anesthetized withintramuscular ketamine hydrochloride and acepromazine maléate at the doses given above. In order toavoid any bearing of circadian variations of the intraocular pressure on the results, outflow facility was

always measured at the same time of the day (12 a.m). Baseline intraocular pressure was recorded 2hours (10 a.m.) before determination ofoutflow facility. At 15 minutes after intramuscular anesthesia,intraocular pressure was measured again, and one 50 pi drop of tetracaine hydrochloride (1.66 x 10~2 M)was instilled into the left eye ofthe rabbit to prevent possible discomfort. Thereafter, the outflow facilityin the left eye of the rabbits was consecutively determined by two-minute tonography and two-levelconstant pressure perfusion as described above. Acceptable measurements of outflow facility by theconstant pressure perfusion technique were obtained in 9 out of 10 ocular hypertensive rabbits studied.

In order to compare the aqueous humor dynamics in a-chymotrypsin-induced ocular hypertensiverabbits with that ofnormotensive rabbits, tonographies were also performed in 10 normal albino rabbitsusing the same experimental procedure.

Effects of Timolol and Pilocarpine

In a second stage, the effect of equimolar doses (4.0 x 102 M) of topical timolol maléate andpilocarpine hydrochloride (both coming from Sigma Chemical Co., St. Louis, MO, USA) on intraocularpressure and aqueous humor dynamics in the ocular hypertensive rabbits was examined. For thispurpose, one 50 pi drop of the drug tested was instilled in the middle of the inferior cul-de sac ofthe lefteye, followed by lid closure, immediately after determination ofbaseline intraocular pressure ( 10 a.m.).At two hours postdrug, intraocular pressure was measured, and two-minute tonographies were recordedas described above. At least 10 days lapsed between treatments for a washout period on individualrabbits.

Determination ofAqueous Humor Protein Concentration

Six months after having completed these experiments, aqueous humor samples were obtained byanterior chamber paracentesis under ketamine hydrochloride/acepromazine maléate intramuscularanesthesia experiments in order to assess the integrity of the blood-aqueous barrier in a-chymotrypsin-induced ocular hypertensive rabbits. The protein concentration ofaqueous humor samples from 5 ocularhypertensive and 5 ocular normotensive eyes wasmeasured usingBiuret and Folin phenol reagents (TotalProtein Kit, No. 690-A, Sigma Chemical Co., St. Louis, MO, USA).

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Analysis of Data

Results are expressed as mean ± S.E.M. Statistical analyses were done using the two-way Student's/-test, with a P value of less than 0.05 considered statistically significant. The outflow facility as

determined by tonography was compared to that found with constant pressure perfusion using linearregression and Pearson correlation coefficient.

RESULTS

The values of outflow facility derived from tonography and the constant pressure perfusion in 9 a-chymotrypsin-induced ocular hypertensive rabbits are shown in Table 1. Themean valuewas 0.20 ± 0.01pl/min/mmHg using tonography and 0.19 ± 0.01 pl/min/mmHg using two-level constant pressureperfusion. There was a significant correlation between values ofoutflow facility calculated by these twotechniques (Pearson correlation coefficient r = 0.87, P < 0.01).

TABLE 1.Outflow Facility in a-Chymotrypsin-induced Ocular Hypertensive Rabbits

by Tonography and Constant Pressure Perfusion Technique.

Outflow facility (pl/min/mmHg)Rabbit No. Tonography Constant pressure perfusion

123456789

Mean ± S.E.M

0.280.200.180.200.190.150.200.230.13

0.20 ±0.01

0.230.210.180.190.180.170.180.180.16

0.19 ±0.01

The values of facility of aqueous humor outflow and aqueous inflow rate as determined bytonographic techniqueboth innormal anda-chymotrypsin-inducedocularhypertensive rabbits are shownin Table 2. The mean value of tonographic outflow facility in ocular hypertensive rabbits (0.21 ± 0.02pl/min/mmHg) was not statistically different from that observed in normal rabbits (0.24 ± 0.01pl/min/mmHg). On the other hand, both the starting intraocular pressure and the rate of aqueous humorinflow in ocular hypertensive rabbits were shown to be statistically different from those observed innormal rabbits. The calculated aqueous inflow in ocular hypertensive rabbits was found to be about 1.5-fold (5.4 ± 0.6 pl/min) higher than that obtained in ocular normotensive rabbits (3.1 ± 0.2 pl/min).Whereas a statistically significant rise in intraocular pressure was noted in normal rabbits at 15 minutespostanesthesia, a decrease in intraocular pressure was found in ocular hypertensive rabbits which lackedstatistical significance (Table 2).

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TABLE 2.Aqueous Humor Dynamics in Ocular Normotensive and a-Chymotrypsin-induced

Ocular Hypertensive Rabbits.

Starting Intraocular Facility of Calculated rateintraocular pressure aqueous humor of aqueouspressure postanesthesia outflow humor inflow(mmHg) (mmHg) (pl/min/mmHg) (pl/min)

Ocular hypertensiverabbits (« = 10) 39.3 ± 2.5 34.4 ±1.5 0.21 ±0.02 5.4 ±0.6

Ocular normotensiverabbits (« = 10) 20.1 ±0.8 21.8 ±0.7* 0.24 ±0.01 3.1 ±0.2

P (unpaired /-test) O.0001 O.0001 N.S. <0.01Data represent the mean ± S.E.M. 'Significantly different from starting intraocular pressure bypaired /-test (P < 0.05).

TABLE 3.Effects of Topical Pilocarpine (4.0 x 10"2 M) and Timolol (4.0 x 10"2 M) on Intraocular Pressure (IOP)

and Aqueous Humor (AH) Dynamics in a-Chymotrypsin-induced Ocular Hypertensive Rabbits.

Baseline IOP(mmHg)

IOP at 2 hourspostdrug(mmHg)

IOPpostanesthesia

(mmHg)

AH outflowfacility

(ul/min/mmHg)

Calculated rateof AH inflow(ul/min)

Untreated

Pilocarpine-treatedTimolol-treated

39.8 ±2.541.7 ±2.940.1 ±3.1

39.3 ±2.538.7 ±2.634.3 ± 2.5+

34.4 ±1.535.3 ±2.030.2 ± 1.6s

0.21 ± 0.020.24 ± 0.030.18 ±0.02

5.4 ±0.66.5 ±1.13.7 ± 0.4*

Data represent the mean ± S.E.M of 10 individual measurements. ' Value significantly different fromthat of the untreated group by unpaired r-test (P < 0.001);f Significantly different from baseline intra-ocular pressure by paired /-test (P < 0.05);§ Significantly different from preanesthesia intraocularpressure by paired /-test (P < 0.05).

The effects ofpilocarpine hydrochloride and timolol maléate both on intraocular pressure and aqueoushumor dynamics in a-chymotrypsin-induced ocular hypertensive rabbits are shown in Table 3. Neitherthe intraocular pressure nor the aqueous humor inflow and outflow were found to significantly changeat two hours after the instillation of 4.0 x 102 M pilocarpine. On the contrary, topical timolol atequimolar dose was shown to significantly lower both the intraocular pressure and the rate of aqueoushumor inflow (3.7 ± 0.4 pl/min; -31%) in a-chymotrypsin-induced ocular hypertensive rabbits.Although a slight decrease in aqueous outflow was noted after timolol treatment (0.18 ± 0.02pl/min/mmHg; -14%), it lacked statistical significance. While the intraocular pressure was notsignificantly modified by intramuscular anesthesia in pilocarpine-treated ocular hypertensive rabbits, astatistically significant fall in intraocular pressure was found in timolol-treated rabbits (Table 3).

Aqueous humor protein concentration in a-chymotrypsin-induced ocular hypertensive eyes (106.7± 36.8 mg/dl; mean intraocular pressure = 38.5 ± 3.6 mmHg, n = 5) was significantly greater (P < 0.05)than that of the ocular normotensive ones (52.5 ± 6.4 mg/dl; mean intraocular pressure = 19.9 ± 1.4mmHg, n = 5).

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DISCUSSION

The results of this study suggest that accurate measurements of the outflow facility can be obtainedin a-chymotrypsin-induced ocular hypertensive rabbits by tonographic technique, because a significantcorrelation was found between the values ofoutflow facility as determined by tonographic and two-levelconstant pressure perfusion techniques (Pearson correlation coefficient r = 0.87, P < 0.01). Such a

correlation coefficient, which was similar to those previously reported when comparing these twotechniques in ocular normotensive rabbits (14,15), seems to rule out any bearing ofpseudofacility on thevalues ofoutflow facility obtained by tonography in a-chymotrypsin-induced ocular hypertensive eyes.

On the other hand, since no significant difference was observed between the mean tonographicoutflow facility in a-chymotrypsin-induced ocularhypertensive rabbits and ocular normotensive rabbits,our data clearly conflict with the classical hypothesis on the mechanism of the ocular hypertensionobserved in rabbits following intracameral injection of a-chymotrypsin, that states that a decrease inaqueous humor outflow is responsible for the rise in intraocular pressure (4). On the contrary, ourfindings suggest that ocular hypertension following a-chymotrypsin injection may be caused by an

increase in the rate of aqueous humor inflow, as it was estimated to be about 1.5-fold higher than that ofocular normotensive rabbits. The tonographic outflow facility observed here for normal rabbits was verysimilar to that previously reported by Fourman and Fourman (15) using the same anesthesia (0.26 ± 0.02pl/min/mmHg).

An unexpected finding of our study was the increase in aqueous humor protein observed in a-

chymotrypsin-induced ocular hypertensive eyes, suggesting that the blood-aqueous barrier may havebecome damaged in an irreversible way after the intracameral injection of this enzyme, a fact that couldexplain the high values of aqueous humor inflow noted in these rabbits. However, if that is the case,values of aqueous humor inflow as estimated from Goldman's equation should be interpreted withcaution. The aqueous humor protein concentration obtained here for ocular normotensive eyes was verysimilar to those previously reported using the same analytical method (16).

Classically, ocular hypertension following a-chymotrypsin injection has been ascribed to trabecularblockage produced by tissue debris resulting from enzymatic hydrolysis of the zonules. First evidencefor such hypothesis was obtained by Kirsch (2) who found a decrease in tonographic outflow facility inhuman eyes showing transient ocular hypertension after a-chymotrypsin treatment. Furthermore,scanning electron microscopy both in monkey (9) and in human (10) eyes revealed the existence oftrapped zonular fibers in the trabecular meshwork after a-chymotrypsin injection into the posteriorchamber of the eye. Injection ofunfiltered aqueous humor frommonkeys that had received intracamerala-chymotrypsinwas also found to increase the intraocular pressure in untreatedmonkeys, suggesting thatlysed zonularmaterial caused the aqueous outflow reduction (7). Nevertheless, neither tonographic norconstant pressure perfusion studies have been carried out in rabbit or monkey eyes in order to confirmthe impaired aqueous outflow after a-chymotrypsin injection as the cause of ocular hypertension.

Although the above-mentioned findings suggest that a-chymotrypsin-induced ocular hypertensionis caused by reduced aqueous outflow, they do not completely rule out the existence ofother mechanismsthat may be responsible for ocular hypertension, such as the potential breakdown of the blood-aqueousbarrier and/or the production of physiological modulators. In this regard, concentration of atrialnatriuretic factor (ANF) has been recently found to correlate with the degree of intraocular pressureelevation in rabbits injectedwith a-chymotrypsin, suggesting that thismodulator may play a role in suchan ocular hypertensive effect (17). Moreover, Sears and Sears (5) noted that systemic administration ofindomethacin, which is known to stabilize the blood-aqueous-barrier, prevented the long-lasting rise inintraocular pressure produced by the intracameral injection of a-chymotrypsin in rabbit eyes, althougha transient ocular hypertension was also observed during the first two weeks following a-chymotrypsintreatment. Therefore, we may speculate that a-chymotrypsin ocular hypertension could comprise twodifferent stages: an initial transient phase, produced by trabecularmeshwork blockage and not susceptibleto indomethacin pretreatment, and a second long-lasting phase, blunted by indomethacin pretreatment,which may involve the breakdown ofblood-aqueous barrier, as our data indicate, and/or the productionofpharmacological mediators. This approachmay explain the discrepancies found among species in the

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ocular hypertensive response to a-chymotrypsin. In humans, where the ocular hypertension followinga-chymotrypsin administration seems to be transient, it is likely that only the first phase takes place,whereas in the rabbit, whose blood-aqueous barrier is very susceptible to disruption, both phases mayplay a role in the rise in intraocular pressure caused by this enzyme. Furthermore, the need of injectinga-chymotrypsin into the posterior chamber of the eye to induce ocular hypertension would be alsosatisfactorily supported by this approach, since both actions would be produced at this locus. In thisregard, some studies have reported changes in the structure of the ciliary body after the injection of a-chymotrypsin into the posterior chamber of rabbit eyes (4).

Another remarkable finding ofour studywas the different response ofa-chymotrypsin-induced ocularhypertensive rabbits to two well-known antiglaucoma drugs, pilocarpine and timolol. Topical applicationof pilocarpine (4.0 x 1O"2 M) did not significantly modify either the intraocular pressure or aqueoushumor dynamics in a-chymotrypsin ocular hypertensive rabbits. This diminished response to pilocarpinein a-chymotrypsin-treated rabbits had been previously reported by other workers (18), being ascribedto an enzyme-inducedmodification ofthe locus ofaction ofpilocarpine in the trabecular meshwork. So,it appears that a-chymotrypsin affects in someway to the normal functioning ofthe trabecularmeshwork,but this action must not be confused with the mechanism ofthe ocular hypertensive effect of this enzymethat, in view of our results, may be secondary to an increase in the rate of aqueous inflow. On thecontrary,we have found that equimolar timolol significantly lowers both the intraocularpressure and therate of aqueous inflow in a-chymotrypsin ocular hypertensive rabbits.

We also must point out the different response observed in ocular normotensive and ocularhypertensive rabbits to ketamine/acepromazine intramuscular anesthesia. Whereas a rise in theintraocular pressure was noted in ocular normotensive rabbits after such an anesthesia, no significantchange was shown in ocular hypertensive rabbits. Such a discrepancy is difficult to explain from our

scarce knowledge both of the mechanisms of the ocular hypertensive effect of a-chymotrypsin and ofthe effects of this anesthetic mixture on the intraocular pressure, but this different response may reflecta higher dependence of the intraocular pressure in ocular hypertensive rabbits on hemodynamic supplyto the ciliary body. While the increase in intraocular pressure found in ocular normotensive rabbits afteranesthesia may result from a ketamine-induced contraction of extraocular muscles (19), the decreaseobserved in ocular hypertensive animals may be secondary to a reduction of the infusion pressure in theciliary arteries, that could bemore prominent in ocularhypertensive rabbits due to themajor contribution,as our results show, of the rate of aqueous inflow to the intraocular pressure in these animals, especiallyif the blood-aqueous barrier, as our data indicate, would have become damaged after a-chymotrypsininjection. On the other hand, a significant decrease in intraocular pressure after intramuscular anesthesiawas noted in ocular hypertensive rabbits receiving timolol, likely resulting from a synergistic effect bothof anesthesia and timolol on aqueous humor inflow.

In summary, our results suggest that ocular hypertension following injection ofa-chymotrypsin intothe posterior chamber of albino rabbits may be produced by an increase in the rate of aqueous inflow,likely secondary to the breakdown ofthe blood-aqueous barrier, rather than by a reduction ofthe outflowfacility. Hence, this animal model for ocular hypertension seems to be based on an aqueous inflowincrease, as in water-loading, rather than on an impaired aqueous outflow, as happens in human ocularhypertension. The ocular hypertension induced by a-chymotrypsin was shown to be sensitive to timololbut not to pilocarpine, suggesting that the injection ofthis enzyme may alter the normal pharmacologicalresponse of the trabecular meshwork. Further fluorophotometric and biochemical studies would berequired to confirm these findings and to ascertain the intimate mechanism of the ocular hypertensiveeffect of a-chymotrypsin in rabbits.

ACKNOWLEDGMENTS

This research work has been carried out in the frame ofproject PI3894, funded by the Gobierno Vasco

(Spain). J. Melena and G. Puras were supported by fellowships from the Gobierno Vasco (Spain) andthe Universidad del País Vasco (Spain), respectively.

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REFERENCES

1. Kirsch, R.E. Use ofalpha-chymotrypsin in cataract extraction followed by transient glaucoma. Arch.Ophthalmol 72:612-620, 1964.

2. Kirsch, R.E. Further studies on glaucoma following cataract extraction associated with the use ofalpha-chymotrypsin. Trans. Am. Acad. Ophthalmol. Otolaryngol. 69:1011-1023, 1965.

3. Kirsch, R.E. Dose relationship of alpha-chymotrypsin in production of glaucoma after cataractextraction. Arch. Ophthalmol. 75:774-775, 1966.

4. Best, M., Rabinovitz, A.Z. and Masket, S. Experimental alpha-chymotrypsin glaucoma. Ann.Ophthalmol. 7:803-810, 1975.

5. Sears, D. and Sears, M. Blood-aqueous barrier and alpha-chymotrypsin glaucoma in rabbits. Am. J.Ophthalmol. 77:378-383, 1974.

6. Vareilles, P., Durand, G, Siou, G. and Le Douarec, J.C. Le modèle de glaucoma expérimental àl'aipha-chymotrypsine chez le lapimétude histologique. J. Fr. Ophtalmol. 10:561-568, 1979.

7. Chee, P. and Hamasaki, D.I. The basis for chymotrypsin-induced glaucoma. Arch. Ophthalmol.85:103-106, 1971.

8. Lessell, S. and Kuwabara, T. Experimental alpha-chymotrypsin glaucoma. Arch. Ophthalmol.81:853-864, 1969.

9. Anderson, D. Experimental alpha-chymotrypsin glaucoma studied by scanning electronmicroscopy.Arch. Ophthalmol. 11:410-416, 1971.

10. Worthen, DM. Scanning electron-microscopy after alpha-chymotrypsin perfusion in man. Am. J.Ophthalmol. 73:637-642, 1972.

11. Eisenlohr, J.E. and Langham, R.F. The relationship between pressure and volume changes in livingand dead rabbit eyes. Invest. Ophthalmol. 1:63-67, 1962.

12. Green, K. Models and methods for testing toxicity of aqueous humor, iris and ciliary body. In:Manual of Oculotoxicity Testing of Drugs, Hockwin, O., Green, K. and Rubin, L.F., eds. GustavFischer Verlag, Stuttgart, 1992, pp. 219-242.

13. Bárány, E.H. Simultaneous measurement ofchanging intraocular pressure and outflow facility in thevervet monkey by constant pressure infusion. Invest. Ophthalmol. 3:135-143, 1964.

14. Becker, B. and Constant, M.A. The facility of aqueous outflow. A comparison of tonography andperfusion measurements in vitro and in vivo. Arch. Ophthalmol. 55:305-312, 1956.

15. Fourman, S. and Fourman, M.B. Correlation of tonography and constant pressure perfusionmeasurements of outflow facility in the rabbit. Curr. Eye Res. 9:963-969, 1989.

16.Wallace, I., Krupin, T., Stone, R.A. and Moolchandani, J. The ocular hypotensive effects ofdemeclocycline, tetracycline and other tetracycline derivatives. Invest. Ophthalmol. Vis. Sei. 30:1594-1598,1989.

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17. Femández-Durango, R., Ramírez, J.M., Trivino, A., Sánchez, D., Paraíso, P., García de Lacoba, M.,Ramírez, A., Salazar, J.J., Fernández-Cruz, A. and Gutkowska, J. Experimental glaucomasignificantly decreases atrial natriuretic factor (ANF) receptors in the ciliary processes of the rabbiteye. Exp. Eye Res. 53:591-596, 1991.

18.Diepold, R., Kreuter, J., Himber, J., Gumy, R., Lee, V.H.L., Robinson, J.R., Saettone, M.F. andSchnaudigel, O.E. Comparison of different models for the testing of pilocarpine eyedrops usingconventional eyedrops and a novel depot formulation (nanoparticles). Graefe's Arch. Clin. Exp.Ophthalmol. 227:188-193, 1989.

19. Schütten, W. and Van Horn, D.L. The effects of ketamine sedation and ketamine-pentobarbitalanaesthesia upon the intraocular pressure of the rabbit. Invest. Ophthalmol. Vis. Sei. 16:531-534,1977.

Received: March 27, 1998Accepted for Publication: June 17, 1998

Reprint Requests: Dr. Juan SantaféDepartamento de FarmacologíaFacultad de FarmaciaPaseo de la Universidad No. 7E-01006 Vitoria, Spain

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