Exp. Eye Res. (1990)
The Effect
51, 87-91
87
of Prostaglandin F,, on Trabecular Cynomolgus Monkeys B’ANN
Department of Ophthalmology,
TRUE GABELT
AND
PAUL
Outflow
Facility
in
L. KAUFMAN*
University of Wisconsin Medical School, Madison WI 53792, U.S.A.
(Received 13 September 7989 and accepted 22 November 1989) Cynomolgusmonkeyswere treated topically in one eye with 2 ,ug of prostaglandinF,,-I-isopropylester (PGF,,-IE)twice daily. On day 4, 3 hr after the seventhdose,intraocular pressure(IOP) was 8.1f0.7 mmHglower in the treated than in the control eyes.On day 5, after the ninth PGF,, dose,grossfacility wasmeasuredin both eyesby determiningthe rate of fluid flow from an external reservoirinto the eye at two different IOP levels.Simultaneously,trabecular facility was measuredby determiningthe rate of accumulationof intracamerally infusedradioiodinatedalbumin in the generalcirculation. Grossfacility was 40-60x higher in PGF,, treated than in control eyes,but there was no differencein trabecular facility. The increasein grossfacility sometimes reportedfollowing PGF,, probably representsincreased pseudofacility or uveoscleral facility. rather than true facility of outflow through the trabecular meshworkand Schlemm’scanal. Keu words: aaueoushumor outfIow ; grossoutflow facility: intraocular pressure:Macacafascicularis: pro&glandin Fi, ; trabecular outflow facility.
1. Introduction Measurements of total outflow facility during prosta-
glandin (PG)-induced ocular hypotension in experimental animals have yielded variable results. In some studies, total facility was statistically significantly increased by approx. 2%lOO’% while in others no increase at all was found (cf. Kaufman and Crawford, 1989, for complete review). In the only human study thus far reported (Camras et al., 1989) total facility measured tonographically in patients with ocular hypertension or primary open angle glaucoma was increased by approx. 40% following PGF,,. However, in none of these studies was the facility increase sufficient to account for the decrease in intraocular pressure (IOP). Indeed, given these inconsistent and modest increases in total facility and the limitations of the measurement techniques employed (cf. Kaufman and Crawford, 1989), we questioned whether any increase in trabecular outnow facility (i.e. facility of aqueous humor flow from the anterior chamber through the trabecular meshwork into Schlemm’s canal and thence into collector channels, intra- and episcleral veins, and the general circulation (Kaufman, 1985) ever occurs. We report here the effect of topical PGF,, on trabecular outflow facility in the cynomolgus monkey.
2. Materials
and Methods
PGF,,-IE concentration of 0.02%. lz51 and 1311were obtained respectively from NEN Products (Boston, MA) and ICN Biomedicals, Inc. (Irvine, CA). Albumin was isolated from cynomolgus monkey (Mucacufusciculuris)
plasma on Blue Sepharose CL-6B (Pharmacia
Fine Chemicals, Piscataway, NJ) according to Travis et al. (1976), lyophiliid, and stored desiccated at 4°C until use. Two milligrammes of the purified monkey serum albumin (MSA) was iodinated with 0.5 mCi “‘1 or 1311using Enzymobeads (BioRad, Richmond, CA) or
Iodo-Beads (Pierce, Rockford, IL) and separated from free iodine on a Sephadex G-25 column (Pharmacia Fine Chemicals) or an EconoPac 10 DG disposable column (BioRad). Aliquots were frozen until the time of the experiment, thawed and again passedthrough
a similar column immediately Animals
prior to use.
and Treatment Protocol
Fourteen young adult female cynomolgus monkeys weighing 2.1-3.1 kg were anesthetized with intramuscular (i.m.) ketamine (10 mg kg-‘) and placed prone in a headholder with the eyes 4 cm higher than the heart. The animals were examined by slit-lamp biomicroscopy to assure that both eyes were normal and non-inflamed, and IOP was measured with a
ChemicaIs and Drugs PGF,,-1-isopropylester (PGF,,-IE) was obtained from * For reprint requests at: Department of Ophthalmology. University of Wisconsin, Clinical Science Center, 600 Highland Avenue, Madison, WI 53792, U.S.A.
00144835/90/070087+05
Pharmacia Ophthalmics AB (Uppsala, Sweden) as a 0.107% aqueous solutiin also containing 0.5 % Polysorbate 80 and 09% NaCl. This stock solution was further diluted with 09% NaCl to give a fmal
%03.00/O
minified Goldmann applanation tonometer (Kaufman and Davis, 1980). Beginning on the same or 1-S days later, one eye of the fully conscious monkey was treated topically twice daily with 2 ,ug of PGF,,-IE, given as two S-,ul drops of the 0.02% solution at 0800 hr and 1500 hr (Gabelt and Kaufman, 1989). 0 1990 AcademicPressLimited
B’A.T.
88
Equal numbers of right and left eyes were treated: opposite eyes did not receive the vehicle. IOP was measured with the minified Goldmann tonometer under i.m. ketamine anesthesia on the third or fourth treatment day at 30-min intervals between 2 hr and 4 hr after the morning (fifth or seventh) PGF,, dose. The ocular hypotensive response of each monkey thus verified, the facility experiment was conducted on day 5 after the morning (ninth) dose.
Facility Measurements The monkey was anesthetized initially with ketamine, the eyes examined by slit-lamp biomicroscopy, and IOPs determined with the minified Goldmann tonometer. Deep anesthesia was then induced with i.m. pentobarbital Na (30-35 mg kg-l). The anterior chamber of each eye was cannulated with three unbranched needles connected to a perfusion and mixing apparatus as described previously (Gabelt and Kaufman, 1989). Following a 5-10 min stabilization period at spontaneous IOP, the anterior chamber contents were exchanged over a 5-10 min period with approx. 1.5 ml of mock aqueous humor (Barany, 1964) containing 5 x lo6 cpm ml-’ of [lz51]- or [1311]MSA (OD and OS, respectively) and a total albumin concentration adjusted to 0.1% with unlabeled MSA. Blood samples were obtained in a heparinized syringe from a tail arterial line prior to anterior chamber exchange and at 5-min intervals for 90 min, beginning approx. 2.5 hr after the ninth PGF,, dose. During this time, intracameral infusion of labeled fluid was continued at a rate of 2.2 ,ul min’ while IOP was held constant for successive 30-min periods at approx. 15, 24 and 15 mmHg, respectively, via an external reservoir which also contained labeled perfusion solution. Ten to 15 min were allowed after each change in reservoir height before blood collection was initiated for that period. Flow of anterior chamber fluid into the general circulation was calculated separately for each 30-min period from the rate of isotope accumulation in the blood as determined by least-squares linear regression and the appropriate blood equivalent albumin space (Bill, 1964; Gabelt and Kaufman, 1989). Since fluid leaving the anterior chamber via the trabecular meshwork and Schlemm’s canal enters the general venous circulation almost immediately, while fluid leaving via the uveoscleral pathway requires several hours to reach the general circulation, the flow calculated from the isotope accumulation data represents trabecular outflow (Bill and Barany, 1966). Trabecular facility was therefore calculated as the change in flow to blood/change in IOP for two successive periods. The three periods thus gave two values which were averaged to give a single trabecular facility for that eye. Total facility for the same time period (C,,) was calculated as change in flow from the
open anterior PGF2a i
GABELT
P. L. KAUFMAN
reSer”Oll
chamber exchange
IOP 15
I/ N
Trobecular Gross 0 hr
AND
2.5
hr
(mmHg1 24 facility faulity
15
-_--___f
(Ctrab) Kg11
Gross
foclldy
(Cg2)
,
4.0
hr
4 75
hr
FIG. 1. Time line for perfusion experiments on day 5 of twice daily unilateral treatment with PGF,,-IE.
reservoir into the eye/change in IOP (B&-any, 1964; Bill and Barany, 1966). At the end of the 90-min period (4 hr after the ninth PGF dose) the infusion-withdrawal and mixing pumps were disconnected. Total facility (C,,) was then determined by two-level constant pressure perfusion at 15 and 24 mmHg for 3045 min, raising and lowering the reservoir every 4 min (B&-any, 1964). The data used in the calculations encompassed approx. 4.254.75 hr after PGF,,. Figure 1 schematizes the time sequence of the perfusion experiment. Blood-Equivalent Albumin Space (BEAS) BEAS was determined 14 months after the perfusion experiments in ten of the same animals by slow infusion (11.1 ,~l min-I) of 1251-MSA (5 x lo6 cpm mll’, 0.1% MSA) into the saphenous vein. Blood samples were collected from a taii arterial line at 15-min intervals for 2 hr. Trabecular facility was calculated using the BEAS corresponding to the time of blood sampling for that specific animal, or from the average of the pooled data in the case of the other four animals. This study conforms to the ARVO Resolution on the Use of Animals in Research (Association for Research in Vision and Ophthalmology, 1983). 3. Results
At slit-lamp examination, four animals exhibited an occasional cell in the anterior chamber aqueous of the treated eye on days 3, 4 or 5; this was never seen in the control eyes. One of these four animals also exhibited trace flare in the treated eye and another exhibited trace flare in both eyes. IOP was significantly lowered in treated as compared to controls at all times tested on days 3, 4 and 5 [Fig. 2(A)]. Three hours after the fifth or seventh PGF,, dose, on days 3 or 4, respectively, of twice daily unilateral treatment, IOP was 4.6 i: 0.9 mmHg (mean f s.E.M.) in treated eyes and 12.7 + 0.6 mmHg in contralateral controls. The difference was 8.1+ 0.7 mmHg, P < 0.001. A significant reduction in IOP relative to the pretreatment baseline was also observed in both treated and control eyes on days 3, 4 and 5 [Fig. 2(B)].
PGF,,AND
TRABECULAR
FACILITY
89
TABLE I
Gross and trabecular outflow facility on day 5 of twice daily unilateral treatment with PGF,,-IE
c,, cm c. c::
n
Treated
Control
Treated/Control
14 14 14 I3.t
0.527f0.081 0.391 f0.082 0.408 + 0.045 0.389+0.044
0.388+0.077 0.346 &- 0044 0.322 f 0.049 0.322 + 0.049
1.594_+0.244** 1.176+0.219 1.358f0.195* 1.4181frO.199*
C,, = gross oufflow facility (~1 min -r mmHg-‘) 34 hr after the ninth unilateral dose of PGF,,; C,,,, = trabecular outflow facility (same units) 34 hr after that same dose (i.e. during the same time period as C,r): C,, = gross facility (same units) 4.254.75 hr after that same dose: methods for determining Cgl and C,, differed slightly (see text). Data are mean i S.E.M. for n animals, each contributing one treated and one untreated eye. f One animal with unphysiologically low control eye value excluded. Significantly diierent from I.0 by the two-tailed paired t-test: *P
The Effect
51, 87-91
87
of Prostaglandin F,, on Trabecular Cynomolgus Monkeys B’ANN
Department of Ophthalmology,
TRUE GABELT
AND
PAUL
Outflow
Facility
in
L. KAUFMAN*
University of Wisconsin Medical School, Madison WI 53792, U.S.A.
(Received 13 September 7989 and accepted 22 November 1989) Cynomolgusmonkeyswere treated topically in one eye with 2 ,ug of prostaglandinF,,-I-isopropylester (PGF,,-IE)twice daily. On day 4, 3 hr after the seventhdose,intraocular pressure(IOP) was 8.1f0.7 mmHglower in the treated than in the control eyes.On day 5, after the ninth PGF,, dose,grossfacility wasmeasuredin both eyesby determiningthe rate of fluid flow from an external reservoirinto the eye at two different IOP levels.Simultaneously,trabecular facility was measuredby determiningthe rate of accumulationof intracamerally infusedradioiodinatedalbumin in the generalcirculation. Grossfacility was 40-60x higher in PGF,, treated than in control eyes,but there was no differencein trabecular facility. The increasein grossfacility sometimes reportedfollowing PGF,, probably representsincreased pseudofacility or uveoscleral facility. rather than true facility of outflow through the trabecular meshworkand Schlemm’scanal. Keu words: aaueoushumor outfIow ; grossoutflow facility: intraocular pressure:Macacafascicularis: pro&glandin Fi, ; trabecular outflow facility.
1. Introduction Measurements of total outflow facility during prosta-
glandin (PG)-induced ocular hypotension in experimental animals have yielded variable results. In some studies, total facility was statistically significantly increased by approx. 2%lOO’% while in others no increase at all was found (cf. Kaufman and Crawford, 1989, for complete review). In the only human study thus far reported (Camras et al., 1989) total facility measured tonographically in patients with ocular hypertension or primary open angle glaucoma was increased by approx. 40% following PGF,,. However, in none of these studies was the facility increase sufficient to account for the decrease in intraocular pressure (IOP). Indeed, given these inconsistent and modest increases in total facility and the limitations of the measurement techniques employed (cf. Kaufman and Crawford, 1989), we questioned whether any increase in trabecular outnow facility (i.e. facility of aqueous humor flow from the anterior chamber through the trabecular meshwork into Schlemm’s canal and thence into collector channels, intra- and episcleral veins, and the general circulation (Kaufman, 1985) ever occurs. We report here the effect of topical PGF,, on trabecular outflow facility in the cynomolgus monkey.
2. Materials
and Methods
PGF,,-IE concentration of 0.02%. lz51 and 1311were obtained respectively from NEN Products (Boston, MA) and ICN Biomedicals, Inc. (Irvine, CA). Albumin was isolated from cynomolgus monkey (Mucacufusciculuris)
plasma on Blue Sepharose CL-6B (Pharmacia
Fine Chemicals, Piscataway, NJ) according to Travis et al. (1976), lyophiliid, and stored desiccated at 4°C until use. Two milligrammes of the purified monkey serum albumin (MSA) was iodinated with 0.5 mCi “‘1 or 1311using Enzymobeads (BioRad, Richmond, CA) or
Iodo-Beads (Pierce, Rockford, IL) and separated from free iodine on a Sephadex G-25 column (Pharmacia Fine Chemicals) or an EconoPac 10 DG disposable column (BioRad). Aliquots were frozen until the time of the experiment, thawed and again passedthrough
a similar column immediately Animals
prior to use.
and Treatment Protocol
Fourteen young adult female cynomolgus monkeys weighing 2.1-3.1 kg were anesthetized with intramuscular (i.m.) ketamine (10 mg kg-‘) and placed prone in a headholder with the eyes 4 cm higher than the heart. The animals were examined by slit-lamp biomicroscopy to assure that both eyes were normal and non-inflamed, and IOP was measured with a
ChemicaIs and Drugs PGF,,-1-isopropylester (PGF,,-IE) was obtained from * For reprint requests at: Department of Ophthalmology. University of Wisconsin, Clinical Science Center, 600 Highland Avenue, Madison, WI 53792, U.S.A.
00144835/90/070087+05
Pharmacia Ophthalmics AB (Uppsala, Sweden) as a 0.107% aqueous solutiin also containing 0.5 % Polysorbate 80 and 09% NaCl. This stock solution was further diluted with 09% NaCl to give a fmal
%03.00/O
minified Goldmann applanation tonometer (Kaufman and Davis, 1980). Beginning on the same or 1-S days later, one eye of the fully conscious monkey was treated topically twice daily with 2 ,ug of PGF,,-IE, given as two S-,ul drops of the 0.02% solution at 0800 hr and 1500 hr (Gabelt and Kaufman, 1989). 0 1990 AcademicPressLimited
B’A.T.
88
Equal numbers of right and left eyes were treated: opposite eyes did not receive the vehicle. IOP was measured with the minified Goldmann tonometer under i.m. ketamine anesthesia on the third or fourth treatment day at 30-min intervals between 2 hr and 4 hr after the morning (fifth or seventh) PGF,, dose. The ocular hypotensive response of each monkey thus verified, the facility experiment was conducted on day 5 after the morning (ninth) dose.
Facility Measurements The monkey was anesthetized initially with ketamine, the eyes examined by slit-lamp biomicroscopy, and IOPs determined with the minified Goldmann tonometer. Deep anesthesia was then induced with i.m. pentobarbital Na (30-35 mg kg-l). The anterior chamber of each eye was cannulated with three unbranched needles connected to a perfusion and mixing apparatus as described previously (Gabelt and Kaufman, 1989). Following a 5-10 min stabilization period at spontaneous IOP, the anterior chamber contents were exchanged over a 5-10 min period with approx. 1.5 ml of mock aqueous humor (Barany, 1964) containing 5 x lo6 cpm ml-’ of [lz51]- or [1311]MSA (OD and OS, respectively) and a total albumin concentration adjusted to 0.1% with unlabeled MSA. Blood samples were obtained in a heparinized syringe from a tail arterial line prior to anterior chamber exchange and at 5-min intervals for 90 min, beginning approx. 2.5 hr after the ninth PGF,, dose. During this time, intracameral infusion of labeled fluid was continued at a rate of 2.2 ,ul min’ while IOP was held constant for successive 30-min periods at approx. 15, 24 and 15 mmHg, respectively, via an external reservoir which also contained labeled perfusion solution. Ten to 15 min were allowed after each change in reservoir height before blood collection was initiated for that period. Flow of anterior chamber fluid into the general circulation was calculated separately for each 30-min period from the rate of isotope accumulation in the blood as determined by least-squares linear regression and the appropriate blood equivalent albumin space (Bill, 1964; Gabelt and Kaufman, 1989). Since fluid leaving the anterior chamber via the trabecular meshwork and Schlemm’s canal enters the general venous circulation almost immediately, while fluid leaving via the uveoscleral pathway requires several hours to reach the general circulation, the flow calculated from the isotope accumulation data represents trabecular outflow (Bill and Barany, 1966). Trabecular facility was therefore calculated as the change in flow to blood/change in IOP for two successive periods. The three periods thus gave two values which were averaged to give a single trabecular facility for that eye. Total facility for the same time period (C,,) was calculated as change in flow from the
open anterior PGF2a i
GABELT
P. L. KAUFMAN
reSer”Oll
chamber exchange
IOP 15
I/ N
Trobecular Gross 0 hr
AND
2.5
hr
(mmHg1 24 facility faulity
15
-_--___f
(Ctrab) Kg11
Gross
foclldy
(Cg2)
,
4.0
hr
4 75
hr
FIG. 1. Time line for perfusion experiments on day 5 of twice daily unilateral treatment with PGF,,-IE.
reservoir into the eye/change in IOP (B&-any, 1964; Bill and Barany, 1966). At the end of the 90-min period (4 hr after the ninth PGF dose) the infusion-withdrawal and mixing pumps were disconnected. Total facility (C,,) was then determined by two-level constant pressure perfusion at 15 and 24 mmHg for 3045 min, raising and lowering the reservoir every 4 min (B&-any, 1964). The data used in the calculations encompassed approx. 4.254.75 hr after PGF,,. Figure 1 schematizes the time sequence of the perfusion experiment. Blood-Equivalent Albumin Space (BEAS) BEAS was determined 14 months after the perfusion experiments in ten of the same animals by slow infusion (11.1 ,~l min-I) of 1251-MSA (5 x lo6 cpm mll’, 0.1% MSA) into the saphenous vein. Blood samples were collected from a taii arterial line at 15-min intervals for 2 hr. Trabecular facility was calculated using the BEAS corresponding to the time of blood sampling for that specific animal, or from the average of the pooled data in the case of the other four animals. This study conforms to the ARVO Resolution on the Use of Animals in Research (Association for Research in Vision and Ophthalmology, 1983). 3. Results
At slit-lamp examination, four animals exhibited an occasional cell in the anterior chamber aqueous of the treated eye on days 3, 4 or 5; this was never seen in the control eyes. One of these four animals also exhibited trace flare in the treated eye and another exhibited trace flare in both eyes. IOP was significantly lowered in treated as compared to controls at all times tested on days 3, 4 and 5 [Fig. 2(A)]. Three hours after the fifth or seventh PGF,, dose, on days 3 or 4, respectively, of twice daily unilateral treatment, IOP was 4.6 i: 0.9 mmHg (mean f s.E.M.) in treated eyes and 12.7 + 0.6 mmHg in contralateral controls. The difference was 8.1+ 0.7 mmHg, P < 0.001. A significant reduction in IOP relative to the pretreatment baseline was also observed in both treated and control eyes on days 3, 4 and 5 [Fig. 2(B)].
PGF,,AND
TRABECULAR
FACILITY
89
TABLE I
Gross and trabecular outflow facility on day 5 of twice daily unilateral treatment with PGF,,-IE
c,, cm c. c::
n
Treated
Control
Treated/Control
14 14 14 I3.t
0.527f0.081 0.391 f0.082 0.408 + 0.045 0.389+0.044
0.388+0.077 0.346 &- 0044 0.322 f 0.049 0.322 + 0.049
1.594_+0.244** 1.176+0.219 1.358f0.195* 1.4181frO.199*
C,, = gross oufflow facility (~1 min -r mmHg-‘) 34 hr after the ninth unilateral dose of PGF,,; C,,,, = trabecular outflow facility (same units) 34 hr after that same dose (i.e. during the same time period as C,r): C,, = gross facility (same units) 4.254.75 hr after that same dose: methods for determining Cgl and C,, differed slightly (see text). Data are mean i S.E.M. for n animals, each contributing one treated and one untreated eye. f One animal with unphysiologically low control eye value excluded. Significantly diierent from I.0 by the two-tailed paired t-test: *P
