The Effect of Topical PGF2x on Uveoscleral Outflow and Outflow Facility in the Rabbit Eye
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Eq,. Eye Res. (1992) 54. 277-283
The Effect of Topical PGF,= on Uveoscleral Outflow and Outflow Facility in the Rabbit Eye
J O H N F. POYER, B 'ANN GABELT A N D P A U L L. K A U F M A N ' Department of Ophthalmology, University of Wisconsin Medical School, Madison, Wl53792, U.S.A.
(Received Lund 73 February 7997 and accepted in revised form 22 April 7997)
Prostaglandin F,, (PGF,,) is a ponrerful ocular hypotensive agent in rabbit, cat, dog, monkey and human. In cynomolgus monkeys, the intraocular pressure (IOP) lowering is due to increased uveoscleral outflow (F,). Because the anatomy of the rabbit outflorv apparatus differs significantly from that of the primate, we sought to determine whether the mechanism of the PGF2,-induced IOP fall was the same. PGF,, tromethamine salt (PGF2,-TS) (50 pg) applied to one eye of 14 conscious rabbits produced a significant IOP fall of 7.4k0.9 mmHg (P < 0.001). In untreated control eyes. F, determined from the quantity of intracamerally perfused albumin found in the ocular and periocular tissues accounted for 5 4 % of total aqueous outflow. In 15un!@erally PGF,,-treated rabbits, after 4-6 hr dosing F, was 49+ 14% higher in the treated than in the contralateral control eyes. Total outflow facility of outflow from the anterior chamber to the general circulation were measured concurrently in 11 rabbits using a two-level constant pressure perfusion and isotope accumulation technique. Both facilities tended to be higher in the treated eyes than in the controls. with a strong correlation between drug-induced changes in total facility and changes in facility of flow to blood (r = 0.85, P < 0.001). In eight rabbits treated unilaterally with 501cg PGF,,-TS, the fluorophotometrically determined aqueous formation rate was probably not decreased relative to control eyes. Protein levels in the aqueous humor were approximately eight-fold higher in PG-treated vs. control eyes. suggesting a drug-induced compromise of the blood-aqueous barrier. The hypotensive mechanism of PGF,, in the rabbit differs from that of the primate. perhaps due to significant differences in the outflow and orbital anatomy.
Keu words: aqueous humor formation: aqueous humor outflow: intraocular pressure: prostaglandin F,,; rabbit.
determine whether the mechanism of the PGF2,- 1. Introduction induced IOP fall was the same. PGF,, is a powerful ocular hypotensive agent in rabbit, cat,-dog, monkey, and human (cam& Bito and 2. Materials and Methods Eakins, 1977; Bito et al., 1983; Lee. Podos and Severin, 1984; Giuffre', 1985; Bito et al., 1989 ; Chenlictrls nr~d Drugs Camras et al., 1989; Groeneboer, Hoyng and Kuiz- enga, 1989; Villumsen and Alm, 1989). Rabbits develop tachyphylaxis of the IOP response with repeated dosing of PGF,, (Bito et al., 1983), and overt compromise of the blood-aqueous barrier (evidenced by biomicroscopically visible anterior chamber flare). after a single topical application of 50,~ig PGF,, tromethamine salt (PGF2,-TS) (Lee et al., 1984). By contrast, monkeys exhibit no IOP tachyphylaxis with repeated PGF,, dosing and comparatively less increase in blood-aqueous barrier permeability (Camras et al., 1987b; Crawford, Kaufman and Gabelt, 1987). In cynomolgus monkeys, the IOP lowering is due to increased uveoscleral outflolv (Gabelt and Kaufman, 1989; Nilsson et al.. 1989). The ocular hypotensive mechanism in the rabbit was unknown. Because the anatomy of the rabbit outfloiv apparatus differs significantly from that of the primate, we sought to
PGF2,-TS and fluorescein isothiocyanate-conjugated dextran (FITC-Dex, MW 46600 Da) were obtained from Pharmacia Ophthalmics AB, Uppsala, Sweden and dissolved in 0.9 % NaCl. ['251] and  were supplied by Dupont NEN (Boston, MA) and ICN Biomedicals, Inc. (Irvine, CA), respectively. Rabbit serum albumin and indornethacin were purchased from Sigma Chemical (St Louis, MO) and fluorescein sodium 2 % from IOLAB Pharmaceuticals (Claremont, CA). Immediately prior to use, indomethacin was dissolved in 0.9% NaCl by adjusting the pH of the solution to approximately 8.4 with sodium carbonate (Columbus Chemical Industries, Columbus, WI). Hep- arin sodium was obtained from SoloPak Laboratories (Franklin Park, IL). Rabbit albumin was iodinated using Iodo-Beads (Pierce Chemical Company, Rock- ford, IL) and purified on Econo-Pac lODG columns (Bio-Rad, Richmond, CA). Protein determinations were performed using Bio-Rad reagent and bovine
For correspondence and reprint requests at: Department of Serum albumin (Sigma Chemical) as standard. Ophthalmology. University of Wisconsin. Clinical Science Center. 600 Highland Avenue. hladison. \ V I 53792. U.S.A. 0014-4835/92/020277+07 903.00/0 O 1992 Academic Press Limited
PGF,, Treatrrlent Protocol All experiments were performed with female New
Zealand white rabbits weighing 2-3 kg. PGF,,-TS was given as two 5-it1 drops (total dose 50 jig) instilled into one randomly selected eye of conscious rabbits. Drug solution was applied to the central cornea using a micropipette while the eyelids were held open to prevent blinking, allowing 30 sec between drops. Dosing n7as done at 0630-0800 hr for all studies. The contralateral control eye was untreated.
Using a Digilab Model 30R pneumatonometer, IOP was determined in 14 conscious animals at 3 . 54 . 5 hr following PGF,, dosing. In three of these animals, IOP was measured every 30-60 min for 6 hr following PG dosing.
At 4.5-6 hr follorving PGF,,, 1 5 rabbits (1 1 from the above IOP group and four others) underwent determination of uveoscleral outfloiv as previously described (Gabelt and Kaufman. 1989). Briefly, ani- mals were anesthetized with an intramuscular dose of ketamine, xylazine, and acepromazine (30, 3, and 0.5 mg kg-', respectively). Anesthesia was maintained throughout the remainder of the experiment with 15 mg kg-' ketamine administered intramuscularly twice an hour. One hour before the eyes were to be cannulated, indomethacin was given intraperitoneally (40 mg kg-') follo~ved by heparin sodium (500 U) intravenously. Each eye (treated and contralateral control) was cannulated with three 23-gauge needles connected, via polyethylene tubing, stopcocks, and micro-T pieces to a pressure transducer, external reservoir, peristaltic pump (for mixing the contents of the anterior chamber), motorized coupled push-pull gas-tight infusion-withdrawal syringes (Hamilton Company, Reno. NV) and on-line gamma well counter (Gamma Products. Inc, Palos Hills, IL) (Sperber and Bill, 1984). The perfusand was Ba'ra'ny's solution with pH adjusted to 7.6 (BSrSny, 1964). The anterior chamber contents were then exchanged over approxi- mately 10 min with about 2 ml of the perfusand containing 5 x 10"pm ml-' of either "jI (right eyes) or I3'I (left eyes) labeled albumin (the final albumin concentration was adjusted to 0.1% with unlabeled albumin), or 2 x 10-a~r FITC-Dex (FITC-Dex was used in conjunction with other preliminary studies; five rabbits received FITC-Dex while ten received [I]-albumin). The infusion-~vithdraival rate was then reduced to 2-3 i t1 min-' by slowing the motorized syringes. For the next 30-60 min, the anterior chamber contents were continually mixed by the external mixing pump at 60jll min-'. The anterior chamber was then rinsed over approximately 10 min
J. F. POYER ET AL.
with 4 ml of perfusand without tracer, after which IOP was elevated to about 30 mmHg for 5 min to wash tracer from the outflow pathway. The animal was then exsanguinated by opening the chest cavity and severing the ascending aorta. The general circulation was not flushed in any way. The eyes were enucleated within 5 min of death, rinsed with saline and imme- diately dissected. All periocular tissues except the cornea and aqueous humor were used to calculate F,. The tissue remaining in the socket (including fat and vasculature) was removed, rinsed with saline, and designated as distal periocular tissue. Periocular tissue adhering to the globe during enucleation was removed and designated as proximal periocular tissue. The globe was placed in a plastic well and the aqueous humor aspirated transcorneally with a tuberculin syringe attached to a 30-gauge needle. The globe was then sectioned 4 mm posterior to the limbus and tissue was divided as follolvs: vitreous, posterior uvea (including retina), anterior uvea, posterior sclera, anterior sclera, and irislciliary body. The entire dissection process was completed within 1 5 min after death. The tissues and fluids, as well as aliquots of the infusion solution, were either counted in a Packard Instruments C-5330 Auto-Gamma Spectrometer "Y I3lI or, for FITC-Dex, homogenized (vortexed in the case of the fluids) in 10% ZnSO,.H,O. Next, the protein rvas precipitated at neutral pH with an equal volume of 0.5 N NaOH (Somogyi, 1930). and fluorescence determined using a Fluorotron Master Scanning Fluorophotometer (Coherent Medical. Palo Alto, CA). IOP during all perfusions was maintained at
18-20 mmHg via an external reservoir filled with labeled perfusand. F, was considered to be the volume (V,) of labeled anterior chamber fluid required to have deposited the amount of tracer recovered from the ocular and periocular tissues, divided by the duration (T)of the labeled infusion (Bill, 1966a). For each tissue and fluid compartment.
quantity of tracer in tissue or fluid (cpm or ng) vu=
concentration of tracer in perfusand (cpm or ng ml-l)
Aqtreoris Flo\v nrld Arlterior CIlnrrtber Volrrrrle ns Detert~lir~edbg Isotope Dilrttioti In the untreated rabbit, dilution rates immediately
following anterior chamber exchange (as described above) and for 1 hr after exchange provided data allorving the calculation of anterior chamber volume and aqueous flow rate, respectively. Briefly, the mixing circuit of knorvn volume was diluted upon anterior
PGF,, AND OUTFLOW I N THE RABBIT EYE
chamber content exchange. The extent of this dilution was indicative of the anterior chamber volume required to obtain such a dilution. The rate of dilution of the mixing circuit over the next hour provided the rate of aqueous flow since the circuit was diluted as newly formed 'cold' aqueous was mixed into the circuit.
Totnl Fncility nrld Facility of Flonl to the Genernl Circulntior~
Eleven rabbits were unilaterally PG-treated and anesthetized, and the anterior chambers cannulated and exchanged with iodinated rabbit albumin as described above. Total facility and facility of flow to the general circulation were measured from 4.5 to 6 hr follolving PG dose as previously described (Gabelt and Kaufman. 1990). Briefly. IOP was held at approxi- mately 15, 2 5, and 15 mmHg for three successive 30- min periods, during which blood samples were collected via a femoral artery every 5 min. Flow to the general circulation (c,,)was considered to be the volume (V,,,) of labeled anterior chamber fluid required to have deposited the amount of [12jI] or ['311]albumin found in the general circulation divided by the duration (T) of the labeled infusion (Bill, 1966a). Blood equivalent albumin space (BEAS) was assumed to be 7-5 % of body weight (Bill. 1964). A 10-min equilibration period was allowed between changes in pressure before blood collection was begun. Facility of flow to blood (C,,,) was considered to be the difference in flow to the general circulation at the two different intraocular pressures divided by the pressure difference. Total facility (C,,,) was the difference in flow from prelveighed reservoirs into the anterior chamber (Flo\v,,,) at the two different intraocular pressures divided by the pressure difference. All facility values were the average of the two values obtained from the three 30-min periods.'Thus,
BEAS x specific activity of blood v,,, = specific activity of perfusand '
F~o\v,,,~-Flo\vreS2 c,,, = IOP, -IOP, '
Aqueous Flo\v ns Deterttlirled by Fl~iorophotoll~etry Eight conscious rabbits underwent aqueous flow
determination by fluorophotometry in both eyes for 5-5 hr following unilateral PGF,, as previously de- scribed (Crawford et al., 1987). Briefly, anterior chamber fluorescence was determined follo\ving top- ical 2% fluorescein sodium using a Fluorotron Master Scanning Ocular Fluorophotometer equipped with an
anterior segment adapter. On the first afternoon, background fluorescence was measured. Topical 0.5 % proparacaine HCI (Ophthetic, Allergan) was applied to the central cornea, followed 5 min later by four 2 - 4 drops of 2% fluorescein sodium applied 30 sec apart with blinking prevented between drops. On the morning of the second day, baseline aqueous flow rates for all eyes were determined by moriitoring fluorescence for 5 hr, after which proparacaine and fluorescein were reapplied as described above. On the morning of the third study day, 50 jig PGF,,-TS was applied unilaterally as two 5-/[I drops 30 sec apart with blinking prevented. Fluorescence was then monitored from 0.5 to 5-5 h r following PG dose.
Aqueous humor was analysed for protein con-centration following the flow analyis in four of these PG-treated animals under ketamine plus xylazine plus acepromazine anesthesia, using Bio-Rad reagent and bovine serum albumin as standard. Aqueous humor samples were collected transcorneally with a 30-gauge needle attached to a tuberculin syringe. Approximately 100 /tl aqueous humor was with-drawn of which lOftl was analysed for protein content.
3. Results Anterior Chnrilber Volurr~e. Aqueous Huti~or Flo\v Rnte nrld Uveosclernl Outflo\v in Urltrented Arlit11nls The anterior chamber volume as determined by
isotope dilution in nine untreated rabbits was 139 f 15 ftl (mean+s.~.hr.) while the aqueous humor for- mation rate was 2.65f0.22 /tl min-'. The anterior chamber volume as determined from limbus-to-limbus corneal diameter, pachymetrically-determined anterior chamber depth, and keratometrically-determined corneal radius of curvature (Erickson et al., 1984) in both eyes of nine rabbits was 118 f 3 /tl. F,,, was 2.5 1f0-22i t1 min-'..while isotope accumulation in the ocular and periocular tissues revealed Fu to be 0.22f0.05 i t1 min-l. Fu calculated as the difference between the anterior chamber aqueous humor flow rate and the flow to the general circulation (Fu.indirect) was 0.14f 0.30 111 min-'. No correlation between Fu values obtained directly and indirectly was apparent (r = 0.04).
In three rabbits, IOP was monitored follo~ving unilateral topical application of PGF,, (Fig. 1).Initial ipsilateral hypertension was follo\ved by prolonged hypotension, as previously reported (Camras et al.. 1977). At 3 .54 .5 hr after similar dosing, the IOP for 14 animals was: treated (Tr) = 17.0f1.5 ; control
280 J. F. POYER ET AL.
C - 10 a 0 B IOP
-m* I I 0 2 4 6 8
Time (hr) FIG. 1. IOP follolving unilateral topical PGF,,-TS (50~ ig ) .
hleasurements were made by pneumatonometry. Data in (B) represents treated-control...