ACTA O P H T H A L M O L O G I C A 65 (1987) 503-506

Regional ocular blood flow after chronic topical glaucoma drug treatment

Keith GreenlIz and Thomas L. Hatchett'

Department of Ophthalmology1 (Head: M. N. Luxenberg), Department of Physiology and Endocrinology2. (Head: V. B. Mahesh),

Medical College of Georgia, Augusta, GA, USA

Abstract. The effects of a chronic three times a day treatment over a 5 to 6 week period of rabbit eyes with 1 of 5 topically applied glaucoma drugs on ocular blood flow was determined using a radioactive microsphere technique. The drugs employed were timolol (0.5%), pilocarpine (4%), epinephrine (2%), norepinephrine (2%), and ecothiophate iodide 0.125%). The results showed that epinephrine statistically decreased blood flow to the iris and ciliary processes while not to the posterior uveal tissues or optic nerve head. Pilocarpine also showed this same trend, while the differences were not statistically significant. Other drugs were without effect on regional ocular blood flow.

Key words: rabbit - ocular blood flow - glaucoma drugs - chronic treatment - epinephrine - microspheres.

The sub-chronic treatment of aphakic albino rabbit eyes with each of 5 commonly prescribed glaucoma drugs has been shown to cause no significant change in blood flow to ocular tissues and the proximal 2 to 3 mm of the optic nerve (Green & Schermerhorn 1985). This study, how- ever, administered thrice-daily drops only 5 days per week and, although the blood flow determi- nations were done after at least three continuous days of treatment, no drops were given on the day of the experiment thereby leaving a significant gap in the dosing regimen.

The present study had as its purpose to admi- nister drops in a more uniform fashion (3 times daily, 7 days per week) over a 5 to 6 week period with the blood flow determinations being made 1 h after the last drop was given. By this method a

more accurate comparison can be made to both a real-life treatment regimen, and to acute studies which have shown epinephrine and other adre- nergic agonists to decrease anterior uveal blood flow in phakic rabbit eyes (Morgan et al. 1981; Jay et al. 1984) and timolol to cause no change in blood flow Uay et al. 1984). If variations in blood flow occur in these tissues after a sustained treat- ment, the treatment itself could be deleterious to an already compromised eye.

Materials and Methods

Each of 5 groups of 10 adult albino rabbits (2-3 kg) were treated three times daily in one eye every day for 5 to 6 weeks with one of the following drugs: timolol (0.5%), pilocarpine (4%), d, l-epi- nephrine ( 2 % ) , I-norepinephrine (2%), and eco- thiophate iodide (0.125%). The contralateral eye was untreated. All of the drugs were available in commercial ophthalmic solutions except l-nore- pinephrine which was prepared twice weekly from the anhydrous salt in 0.9% NaCl solution. The drops were administered normally at 08.00 h, 12.00 h, and 16.00 h. On the day of the blood flow determination, if the procedure was to be done before 12.00 h, the 08.00 h drop was omitted, and a drop was given 1 h prior to the blood flow measurement. If the procedure was to be done after 12.00 h, the 08.00 h drop was given and the 12.00 h drop omitted with a drop administered 1 prior to the measurement. Usually, either 3 or 4 animals were done per day following the chronic

503

treatment; thus, for the morning determinations, no drop was administered between 17 and 19 h following the last drop on the preceding day (16.00 to 09.00 or 11.00 h), while for the after- noon measurements, only 5 to 7 h passed from the time of the last drop (08.00 to 13.00 or 15.00 h) to the time of the drop given 1 h before the meas- urement.

The animals were initially anaesthetized with an intramuscular injection of 1 ml ketamine HCl(l00 mgiml) and 1 ml xylazine (20 mgiml). An iv solution of 25% urethane in heparinized saline was administered throughout the remainder of the procedure via a marginal ear vein. The urethane was generally given in 2 ml increments before each surgical procedure.

A femoral artery was cannulated with PE50 tubing, and the animal was placed on a respirator (Harvard Small Animal Respirator) initially set at a stroke volume of 15 ml and rate of 20 cycles per min after a tracheotomy. An arterial blood sample was collected for immediate analysis of PCOz and pH using a Radiometer blood-gas analyzer. The respirator was then adjusted as needed until PCOz, and pH values were similar to those in an unanaesthetized rabbit (PCOz, 32; pH, 7.4) (Green et al. 1984; Altman & Dittmer 1971). The chest was then opened, the heart exposed, and a 22-gauge needle, attached to PE50 tubing and a 3-way stopcock, inserted into the left ventricular wall until pulsations were noted in the tubing. The needle was secured to the heart wall with a drop of Histoacryl Blue tissue adhesive. Heparin (2 ml; 1000 Uiml) was injected iv 10 min before the blood flow determination. Another blood sample was taken and analyzed immediately preceding the blood flow measurement. Blood was allowed to flow through the femoral artery cannula until it reached a constant rate before microsphere in- jection. Blood flow to the ocular tissues was meas- ured using a modified version of the technique of Alm & Bill (1973) and Alm et al. (1973). Following the injection of ssSr-labelled (15 pm) micro- spheres in 0.9% NaCl and collection of 10 second blood samples, over 90 seconds, the rabbit was killed with an intracardiac injection of saturated KCI (5 ml). The microspheres were injected in 1 ml, a volume unlikely to alter arterial blood pres- sure as injected over a 5-second time frame. Any minor effects would be reflected in both the experimental eye and the contralateral control eye.

504

The eyes were quickly removed and cleaned, and the desired tissues (iris, scraped ciliary pro- cesses, choroid, retina, and optic nerve head) were dissected and placed in tared vials. The blood and tissue samples were subsequently weighed and then analyzed for Sr-85 activity using a Tracor Gamma Trac 1191. For each animal, an experi- mental and a contralateral control eye were ob- tained.

The blood flow through each tissue (mg blood/ minuteimg tissue wet weight) was determined from the femoral artery reference and the re- spective activities. Mean blood flows through the treated and untreated tissues for each drug group were compared using the paired T-test with P less than 0.005 representing statistical significance.

Results

The ciliary processes and irides of the eyes treated with epinephrine showed a statistically lower blood flow when compared to the paired control eyes of that group, while the choroid showed a non-significant reduction and retinal and optic nerve blood flows tended to be increased (Table 1). The pilocarpine-treated eyes also showed a trend toward reduction in anterior segment blood flow; however, this was not statistically significant. All tissues in the norepinephrine-treated eyes tended to have reduced blood flow, while no obvious changes were noted in the timolol and ecothiophate iodide groups. A comparison of eyes treated in the morning (only one drop preceding the measurement of blood flow by 1 h), and those that received a morning drop followed by a drop 1 h before blood flow measurements in the after- noon showed no difference in the data.

Discussion

While the values for blood flow in the control eyes were slightly lower than other values reported in the literature (Green & Schermerhorn 1985; Morgan et al. 1981; Jay et al. 1984; Green et al. 1979), there was an internal consistency in this study as well as a constant order in magnitude of values (choroid > ciliary processes > iris > retina) which is identical to that found in other studies (Green & Schermerhorn 1985; Morgan et al. 1981; Jay et al. 1984; Alm & Bill 1973; Alm et

Scraped

processes Iris ciliary

Controls (20) 0.52 f 0.07 Timolol(8) C 0.68 f 0.13

T 0.70 t 0.12 Pilocarpine (9) C 0.58 f 0.13

T 0.32 t 0.07 Epinephrine (8) C 0.78 t 0.13

T 0.27 f 0.07* Norepinephrine (10) C 0.57 f 0.17

T 0.44 f 0.09

0.79 f 0.15 T 0.47 f 0.08 Ecothiophate iodide (8) C

Optic nerve head Choroid Retina

1.09 f 0.12 2.05 f 0.48 1.89 f 0.53 1.08 f 0.2 1 0.62 t 0.19 1.81 t 0.25 0.50 f 0.14* 1.38 f 0.35 1.04 t 0.14 0.46 f 0.08 1.39 f 0.25

6.03 f 0.7 1 9.25 f 0.94

10.18 f 2.92 6.65 f 1.60 4.54 t 0.68 9.57 f 1.23 7.02 f 0.76 5.2 1 f 0.9 1 4.59 f 0.60

6.20 t 1.27 1.28 f 0.26

1.22 f 0.29 0.3 1 f 0.10 0.21 f 0.04 0.15 f 0.05 0.13 f 0.04 0.17 f 0.04 0.33 f 0.11 0.27 f 0.09 0.23 f 0.05 5.77 f 0.91

0.14 f 0.05

1.22 f 0.29 1.46 f 0.30 1.47 f 0.70 1.38 f 0.4 1 0.81 .f 0.3 1 1.50 k 0.2 1 2.08 f 1.06 1.61 f 0.46 0.93 f 0.37 0.29 f 0.14 0.66 f 0.09

Values are the mean f SEM in mg blood/minute/mg tissue. Number of animals in parentheses after drug names, except for Controls,where number of eyes are indicated. C = untreated, contralateral eye; T = treated eye. P < 0.005 using paired t-test.

al. 1973; Green et al. 1979; O’Day et al. 1971; Caprioli et al. 1984). The control series run in parallel with these studies indicates the internal consistency of the data (Table 1). These relation- ships indicate that the technique used was valid. Discussions of the distribution, and quantitative measurements, of microspheres have been made previously (Green & Schermerhorn 1985; Alm & Bill 1973; O’Day et al. 1971).

While the sub-chronic study performed earlier showed no significant changes in blood flow after treatment over a 3 week period (Green & Scher- merhorn 1985), this study did not treat the ani- mals for period up to 23 h (range, 16 to 23 h) before blood flow measurements were per- formed. The results of that study, while indicating certain trends were possibly caused by different drugs, showed that no statistically significant changes occurred save with ecothiophate iodide increasing choroidal blood flow. The present study (Table 1) shows that epinephrine caused a significant decrease in iris and ciliary process blood flow. These changes support previous acute studies that also demonstrated marked vaso- constrictive effects in the anterior uvea in rabbits (Morgan et al. 1981; Jay et al. 1984). The lack of an effect on blood flow of norepinephrine in the present study, however, differs from the decrease in anterior uveal blood flow seen after acute administration (Morgan et al. 1981). Although the

trend noted with pilocarpine in the present study in rabbits does not coincide with the results of Alm et al. (1973) in primates, where a vasodilation was found 45 to 60 min after a single drop administration, a chronic study in primates of the effects of these drugs (epinephrine and pilo- carpine) as well as other glaucoma drugs could resolve both the apparent species differences and their response to these drugs. Primate eyes do, however, respond to acute administration of epi- nephrine with a decrease in anterior segment blood flow after acute topical drug administration (Caprioli et al. 1984).

The fact that there was a response to epine- phrine and not to I-norepinephrine may reflect the relative effectiveness of adrenergic agonists in reducing anterior uveal blood flow (epinephrine > phenylephrine > norepinephrine) reported in an acute study (Morgan et al. 1981). This, in turn, may reflect the alpha adrenergic agonist activities of epinephrine versus norepinephrine. Additio- nally, the different effects of epinephrine and norepinephrine, when given acutely without pre- treatment (Morgan et al. 1981) compared to the effects seen in the present study, may indicate that the eye adapts to norepinephrine but not epi- nephrine after continued treatment. Timolol has been shown not to induce a change in blood flow on an acute basis (Jay et al. 1984).

Both timolol and ecothiophate iodide failed to

505

produce significant changes in blood flow in a previous sub-chronic treatment study, a finding confirmed here with a more rigorous dosing regimen. Indeed, the same albino rabbit model was used in the present experiments for direct comparison with the previous data (Green & Schermerhorn 1985). The lack of an effect of ecothiophate iodide in the present study, using a more regimented dosing schedule, compared to the significantly increased choroidal blood flow found earlier is probably related to the use of phakic animals in the present study. Aphakic eyes are known to allow more of' a topically applied drug to pass posteriorly beyond the iris (Morgan et al. 1983; Green et al. 1983), and the previous ecothiophate effect on choroidal blood flow (Green & Schermerhorn 1985) could have oc- curred through this mechanism. Perhaps this also explains why epinephrine caused an effect in the anterior segment in the present experiments, since the diffusion barrier of the lens would prevent loss of the drug posteriorly compared to the previous experiments using aphakic rabbits (Green & Schermerhorn 1985).

I t is doubtful that the lack of effects of the drugs in this study, or the effects of epinephrine (Table l), could be attributed to changes in IOP and/or systemic blood pressure as discussed in detail previously (Green & Schermerhorn 1985). Nonetheless, it is obvious that continual cyclic ischemia to the iris and ciliary processes of an eye already prone to the effects of glaucoma, in- cluding possible reductions in blood flow, would cause additional damage. The decrease in blood flow, as demonstrated here in the rabbit, and acutely in the albino rabbit (Morgan et al. 1983) and the primate (Caprioli et al. 1984), indicates that the presence of melanin has little effect on the initial pharmacological response.

Acknowledgments

This investigation was supported in part by PHS research grant EYO4559 from the National Eye In- stitute, in part by National Glaucoma Research, a program of the American Health Assistance Founda- tion (both to KG), in part by a departmental award from Research to Prevent Blindness, Inc., and in part by the American Medical Association, Education and Research Foundation (TH).

We thank Rajeev Saxena for his excellent secretarial assistance and Mrs Sylvia Catravas for her excellent secretarial assistance.

References

Alm A & Bill A (1973): Ocular and optic nerve blood flow at normal and increased intraocular pressures in monkeys (Macaca irus): a study with radioactively labelled microspheres including flow determinations in brain and some other tissues. Exp Eye Res 15: 15-29.

Alm A, Bill A & Young F A (1973): The effects of pilocarpine and neostigmine on the blood flow through the anterior uvea in monkeys. A study with radioactively labelled microspheres. Exp Eye Res 15: 31-36.

Altman P L & Dittmer D S (197 1): Blood and other body fluids. Fed Am Soc Exp Biol, p 186. Bethesda, MD.

Caprioli J, Sears M & Mead A (1984): Ocular blood flow in phakic and aphakic monkey eyes. Exp Eye Res 39: 1-7.

Green K, Bowman K, Luxenberg M N & Friberg T (1983): Penetration of indomethacin into phakic and aphakic rabbit eyes. Arch Ophthalmol 101: 284-288.

Green K, Cheeks K E, Bowman K A, Deutsch H M, Hodges L C & Zalkow L H (1984): Marihuana-- derived material; distribution and systemic effects after systemic administration. Curr Eye Res 3: 75 1-761.

Green K, Hull D S & Bowman K (1979): Cyclocryo- therapy and ocular blood flow. Glaucoma 1:

Green K & Schermerhorn J (1985): Blood flow in aphakic rabbit eyes after sub-chronic glaucoma drug treatment. Curr Eye Res 4: 667-670.

Jay W M, Aziz M & Green K (1984): Effect of topical epinephrine and timolol on ocular and optic nerve blood flow in phakic and aphakic rabbit eyes. Curr Eye Res 3: 1199- 1202.

Morgan T R, Green K & Bowman K (1981): Effects of adrenergic agonists upon regional ocular blood flow in normal and ganglionectomized rabbits. Exp Eye Res 32: 691-697.

Morgan T R, Mirate D, Bowman K & Green K (1983): Topical epinephrine and regional ocular blood flow in aphakic rabbits. Arch Ophthalmol 101: 112-116.

O'Day D M, Fish B, Aronson S B, Pollycove M &Coon A (1971): Ocular blood flow measurements by nuclide labelled microspheres. Arch Ophthalmol 86: 205-209.

14 1 - 144.

Received on February 4th, 1987.

Author's address:

Keith Green, Ph.D., D.Sc., Department of Ophthalmology, Medical College of Georgia, MCG Box 3059, Augusta, GA 30912-0300, USA.

506