S,'everal diverse methods have been introduced

Transcription

1 Measurement of aqueous humor formation rates by posterior-anterior chamber perfusion with inulin: Normal values and the effect of carbonic anhydrase inhibition W. W. Oppelt* Aqueous humor (AH) formation rates in anesthetized cats were measured by continuous posterior-anterior chamber perfusion with an inulin-containing, AH-like buffer. AH formation rates were calculated, with the knoioledge of inulin concentration in inflow, outflow, and rate of infusion. They averaged about 14 fil per minute, which represents a turnover rate of about 1.4 per cent per minute. Insignificant inulin left the system either by diffusion into blood, or by uptake by ocular tissues. Moderate elevations of outflow protein concentrations occurred; hoioever, there was no correlation between protein concentrations and measured AH formation rates, unless protein concentrations rose above 600 mg. per cent. AH formation rates were stable over a six hour experimental period. There was adequate mixing of the isotope in the anterior chamber fluids, as measured by changing the position of the outflow needle during the experiment. Acetazolamide, to 20 mg. per kilogram, was given in a single intravenous injection after a three hour control period. Maximum reduction in AH formation rates of about 45 per cent occurred when either or 20 mg. per kilogram acetazolamide was used, while smaller decreases were noted with smaller doses. There was no physiologically significant effect at the lowest dose. It is concluded that posterior-anterior chamber perfusion with inulincontaining buffer is a valid, direct method of measuring AH formation rates which allows one to use each eye as its own control, and which can be used to measure AH formation rates after a great variety of pharmacological or physiological manipulations. The carbonic anhydrase inhibitor, acetazolamide, at doses which lower intraocular pressures (?), acts by directly reducing AH formation rates. S,'everal diverse methods have been introduced to estimate formation rate of aqueous humor (AH). Some of these are From the Departments of Pharmacology and Therapeutics and Medicine, University of Florida College of Medicine, Gainesville, Fla. Read at the Meeting of the Association for Research in Ophthalmology, Chicago, 111., October 13 to 15, This investigation was supported in part by National Institutes of Health Grant GM 760. "Burroughs Wellcome Scholar in Clinical Pharmacology. 76 based on tonography and tonometry, 1 ' 3 some are based on the transport of organic anions out of the anterior chamber after they have accumulated there following vitreous injection, 4 some directly measure AH formation rate after the anterior chamber has been filled with oil, 5 and others measure the turnover of various materials after they have been injected into the anterior chamber. The latter group of methods usually involves either single or slow continuous injections of materials such as inulin, 0 Na + and PAH 7 or iodo-

2 Volume 6 Number 1 Measurement of aqueous humor formation rates 77 pyracet. 8 After such anterior chamber injections, ocular fluids and tissues are analyzed for the material injected, and with the knowledge of the original amount introduced, a single turnover rate of the material can then be used to estimate AH formation rates. These turnover methods have the disadvantage that only single estimates can be made, and therefore, it is not possible to use the same eye as its own control, if one wishes to determine the effect of drugs or metabolic manipulations on AH formation rates. These same criticisms can be applied to some of the other methods listed. Some of these, in addition, involve serious alterations in the normal physiology of the eye, as well as the necessity to make important assumptions, in order to accurately calculate AH formation rates. This report describes a method of measuring AH formation rates by continuously perfusing an inulin- 14 C-containing, AHlike buffer into the posterior and out of the anterior chamber of the cat eye. Dilution of inulin, as it passes through both of the eye chambers, and the rate of inflow are used to calculate AH formation rates. This method allows for continuous determination of AH formation rate, and thus each eye may be used as its control in studies involving drugs or metabolic alterations. The theory of this method is based on the ventriculo-cisternal perfusion technique of measuring cerebrospinal fluid (CSF) production introduced by Heisey and associates 9 and Pappenheimer and his associates, and since then has been used by others. 11 In addition to a detailed description of the method and determination of normal AH formation rates, this report contains studies on the effect of intravenous acetazolamide on AH formation rates. Materials and methods Male and female cats, of mixed breed, weighing 2 to 3.5 kilograms, were anesthetized with 30 mg. per kilogram intrahepatic pentobarbital, and anesthesia was maintained with occasional intravenous drug. The head was mounted in a stereotaxic headholder for stabilization. The lateral, postlimbal conjunctiva was grasped with a small hemostat to stabilize the eye. A 24 gauge, stainless steel, hubless needle, attached to polyethylene tubing, was put through the cornea by hand at a 45 angle to the iris, using a drilling motion. The needle was advanced through the pupil, behind and parallel to the iris, so that its tip would lie in the posterior chamber, halfway between the pupillary margin and the lateral margin of the posterior chamber. The hemostat was then removed from the eye, and intraocular pressure was measured through the posterior chamber needle, with Sanborn pressure transducers which had previously been attached to the tubing leading from the posterior chamber needle. The eye was then again fixed with the hemostat, and another 24 gauge needle was introduced into the anterior chamber, so that its tip lay 180 from the tip of the needle in the posterior chamber (Fig. 1). Infusion of an AHlike buffer,* containing inulin-^c, was then started through the posterior chamber needle, and the outflow was collected from the anterior chamber needle into a closed vial, minimizing evaporation. Continuous, posterior to anterior chamber perfusion was thus accomplished. Either a Holter, microbilateral, or a Harvard syringe type pump, both previously calibrated, was used. For the initial half hour of the experiment, perfusion rate was about 60 ML per minute to facilitate mixing and equilibration. This was then slowed to 30 j«l per minute for the rest of the experiment. The pressure of the system was continuously monitored through the inflow needle by Sanborn pressure transducers. The height of the outflow tubing was adjusted, so that the pressure in the system corresponded to the initial measured intraocular pressure. Outflow was collected in half hour periods, and the total duration of the experiment was usually six hours. Inulin radioactivity in inflow and outflow was determined using a liquid scintillation system. AH formation rates were calculated using the following formula: (C, - Co) r, formation rate = Co where Ci and C o are inulin concentrations in inflow and outflow, respectively, and r is the rate of inflow in /*L per minute. Total protein was determined in all outflow samples using the method described by Lowry and associates. 12 AH formation rates were measured in 11 cats for six hours without drugs. These animals served as general controls, particularly to determine how stable AH formation rates are over the total experimental period. In these experiments it was "Buffer contents: NaCl, 127 meq. per liter; KC1, 4.3 meq. per liter; NaHCOa, 24 meq. per liter; Ca(Cl);, 2.5 meq. per liter; Mg(Cl)s, 1.6 meq. per liter; NaHjPOi, 1 meq. per liter; NasHOP*, 1 meq.'per liter; heparin, units per milliliter; inulin" 14 C, 2 p.c (0.7 mg.) per 0 ml.

3 78 Oppelt Investigative Ophthalmology February 1967 Fig. 1. Diagrammatic representation of the experimental preparation. also tried to correlate AH formation rates with outflow protein contents. In another series of cats, total recovery of i mil in, during abnormally low intraocular pressure, was measured. This was done to determine if significant inulin would diffuse either into the blood or into the ocular tissues. The experiment was run under normal intraocular pressure for the first three hours, then the outflow tubing was lowered to reduce the pressure of the system to close to zero. Total inulin pumped into the eye was then compared with total amounts leaving the eye. In other cats, the location of the tip of the outflow needle was changed several times during the experiment to determine if proper mixing of inulin in the anterior chamber had occurred. One would expect differences in inulin concentration of the outflow, if there were a systematic lack of mixing. The effect of intravenous acetazolamide was determined in another series of cats. A three hour control AH production period was run in each eye of each cat. Then various doses of acetazolamide ( to 20 mg. per kilogram) were given in single intravenous injections, and AH formation rates were measured for another three hours. The effect of this drug was thus determined, using each eye as its own control. Results The average control AH formation rate in 21 eyes of 11 cats, using the entire six hour experimental period, was 13.6 /.il per minute. In these eyes, the highest rate was 24.5 /.il per minute, while the lowest was 8.3 fjil per minute. An hour to hour breakdown of these results is noted in Fig. 2. Here we can see that there is a tendency for AH formation rate to slightly decrease during the latter part of the six hour experimental period. During the first three hours, AH formation rates were 13.5, 14.1, and 13.4 /.il per minute, while during the TIME - HOURS Fig. 2. Aqueous humor formation rates in control cats. Six hour perfusions were performed in 21 eyes of 11 cats, and each hour's average aqueous humor formation rate is graphed -2 standard errors of the mean. It can be seen, although there is a slight tendency for the rate to decrease in the latter hours of the experiment; formation rate is quite stable for the six hours. 600 PROTEIN-mg % Fig. 3. Outflow protein concentrations as a function of aqueous humor formation rates. Each point represents a single observation period with its outflow protein concentration. The 11 control cats, represented in Fig. 2, were used. The open circle is the average formation rate at the average protein concentration. next three hours they were 14.5, 11.5, and 12.6 fxl per minute. As indicated in Fig. 2, the hourly differences are not statistically significant, and one can say that control AH formation rate is quite stable during a six hour experimental period. It is thus valid to use the first three hours of an experiment as a control for pharmacological manipulations performed during the second three hours.

4 Volume 6 Number 1 Measurement of aqueous humor formation rates 79 Fig. 3 shows the correlation between outflow protein concentration and AH formation rates. Here we first see that some elevation of protein concentration occurs in all experiments. The variation in this, however, is tremendous: Some of the veiy high values were noted after obvious injury to the iris during the introduction of the needle. There has also been a tendency for the proteins to be lower in our more recent experiments, as we get more practiced in the technique. There seems to be a tendency for a slight increase in AH formation rates with veiy high protein concentrations; however, clearly there is no correlation when protein concentrations in outflow are below 600 mg. per cent. Now, therefore, we are discarding any experiments in which any outflow protein concentration is above that figure. Table I shows total inulin recoveiy in five eyes of three cats, where the first three hours were rim with normal intraocular pressure, and the next three hours with the pressure in the system close to zero. We can see that inulin recovery using normal pressure was around 40 per cent, while, when the pressure was decreased, as expected, inulin recovery averaged around 0 per cent. This suggests that when the normal AH outflow channels are not used, no significant quantity of inulin either diffuses into the blood or is taken up by ocular tissues. Inulin concentration in outflow, when the tip of outflow needle was moved to various parts of the anterior chamber during the experiment, did not change significantly, suggesting that adequate mixing of inulin had taken place (Table II). The data in Table III summarize experiments with intravenous acetazolamide. Here, each eye is used as its own control, the drag having been injected after a three hour control period. The values are the average of six half hourly periods before, and six after drug injection. As can be seen, full drug effect, approximately a 45 per cent decrease in AH formation rate, is noted after mg. per kilogram acetazolamide. No further effect is seen when this dose is doubled, and smaller effects are seen at lower doses. Discussion of methodology This method of estimating AH formation rates is based on a continuous estimation of the dilution of inulin by newly formed AH. The perfusate, containing inulin, is introduced into the posterior chamber, and in its passage through the eye, is mixed with newly formed AH, which lowers the inulin concentration of the perfusate. Part of the perfusate will then pass into the blood, through the normal AH outflow channels, and part out of the outflow cannula. Knowing the rate of infusion, and inulin concentration in in- Table I. Inulin recovery from outflow during normal (16 to 18 mm. Hg) and low (3 mm. Hg) pressure Experiment 42 right eye 43 right eye 46 right eye Total inulin infused (radioactivity counts/ 3 hrs.) 144, , , , ,756 Total inulin in outflow (radioactivity counts/3 hrs.) Normal pressure Low pressure 60,380 72,881 71,732 22,685 72,747 (42%) (50%) (46%) (25%) (52%) 43% 152, , , , ,814 (6%) (1%) ( 94%) (2%) (117%) 4% Each eye was perfused at its normal pressure, as described in text. Outflow volume and inulin concentration were determined, and inflow rate and inulin concentration were known. After three hours, the level of the outflow catheter was lowered to decrease pressure in the system. Total inulin counts in outflow were again determined, and compared to those at normal intraocular pressure. The percentages in parentheses refer to percentage of inflow inulin recovered from outflow.

5 80 Oppelt Investigative Ophthalmology Febmanj 1967 Table II. Inulin concentration in outflow during successive time periods in six eyes with different positions of the tip of the outflow needle Experiment Position of tip of outflow cannula in anterior chamber 61 Ventral half Dorsal half Nasal half Temporal half Inulin concentration in outflow (c.p.m./ ml.) 49,072 48,872 46,897 46, Ventral nasal quadrant 53,368 Ventral temporal quadrant 56,097 Dorsal half " 53,319 right eye Ventral nasal quadrant 56,021 Ventral temporal quadrant 52,517 Dorsal half 54, Temporal half 18,683 Nasal half, near iris 17,357 Nasal half, near cornea 19, right eye Ventral half Temporal half Nasal half Center of anterior chamber Ventral half Temporal half Nasal half Center of anterior chamber 17,824 18,627 17,215 18,005 16,095 17,623 17,215 18,321 Perfusion was performed as described in text. The inflow needle remained in the same position. The outflow needle was moved (without drawing it from the anterior chamber) so that its tip would lie in the indicated positions. Outflow inulin radioactivity concentration is expressed in counts per minute per milliliter. flow and outflow, it is then possible to calculate net AH formation rates. It should be noted that this calculation does not require knowledge of the rate of outflow. However, several assumptions have to be made to make this a valid method: (1) Puncture of the cornea and perfusion with an AH-like buffer does not alter the normal physiology of the eye to such an extent as to make any estimate of AH formation rate invalid. (2) Inulin does not bind to ocular tissues, is not metabolized in the eye, does not diffuse into ocular tissues to a significant degree, is not actively transported from the eye, and does not diffuse into blood through channels other than the normal bulk outflow channels for AH. (3) There is adequate mixing Table III. Aqueous humor formation rates after intravenous acetazolamide Cat 38 right eye 39 right eye 45 right eye 53 right eye 54 right eye 32 right eye 33 right eye 34 right eye 26 right eye 28 right eye 29 right eye 21 right eye right eye 011 right eye 012 right eye right eye 12 right eye 14 right eye 15 right eye Drug dose ( g./ kr.) Control production (fil/ min.) Postdrug production (nl/ min.) Percentage of change Aqueous humor formation rates were measured during 6 half hour periods, and values were averaged. Drug was then given in single intravenous injection, and aqueous humor formation rates were measured for 6 more half hour periods and were averaged.

6 Volume 6 Number 1 Measurement of aqueous humor formation rates 81 of the inulin-containing inflow with the newly formed AH. Some of the experiments performed bear on these assumptions. In general it should be said that the present method is similar to the ventricular perfusion method of determining CSF production rates, as introduced by Heisey and colleagues 9 and Pappenheimer and his group. The assumptions in the CSF system are similar to those for the AH system, and, as the excellent work of Pappenheimer has indicated, they are adequately met for the CSF system. 9 Some confidence regarding the validity of the presently described method can be obtained from this, as the basic physiology of the two systems is, in many ways, quite similar. The first assumption is probably the most difficult one. There certainly is some injury to the eye from puncture and perfusion. This is indicated by the rise in protein concentrations of the outflow, which in some cases was considerable (Fig. 3). In some instances one could explain this rise as some obvious, albeit slight, injury which was done to the iris as the inflow needle was passed under it. However, in most experiments the punctures of the cornea seemed clean, there was no leakage of AH, and no obvious injury to any intraocular tissues. In spite of this, there was significant protein elevation. In other experiments which seemed identical, very little protein elevation in outflow occurred. It is difficult to explain the variability, except to say that there was a tendency for outflow protein concentrations to decrease with the greater experience that we had with the method. Thus in the future, greater skill may reduce this variability factor. To determine if there would be any systematic effect that injury had on the estimation of AH formation rates, we attempted to correlate protein concentration with this rate. Assuming that higher protein concentrations mean greater injury, there might then be a reproduceably noticeable effect on AH formation rate. As can be seen in Fig. 3, such correlation does not exist when protein concentrations are below 600 mg. per cent. Above that there seems to be a tendency for slightly increased formation rates. Because of this, we now discard all experiments where any of the protein values are above that amount. Thus it seems fair to say that when outflow protein concentrations are below 600 mg. per cent, there is no systematic effect on AH formation rates, as estimated by this method. As indicated in Fig. 2, AH formation also seems to be stable over the total six hour experimental period. Hence, it seems valid to compare a three hour control period to a subsequent three hour period after a drug or other physiological manipulation has been introduced. Should any part of the second assumption be incorrect, inulin concentration in outflow would be smaller than that which is due to dilution by newly formed AH. This would cause an overestimation of AH formation rates. However, there is evidence that the assumption is a valid one. In our experiments using an abnormally low pressure, one would not expect recovery of all radioactivity from the outflow if inulin would bind to ocular tissues, would diffuse into ocular tissues to a significant degree, or would diffuse into the plasma through channels other than the normal bulk AH outflow channels. As we recovered about 0 per cent of infused inulin (Table I), it seems that the above-mentioned possibilities are not occurring to a significant extent. The question of in vivo metabolism was not directly investigated. However, there is no evidence that this occurs in any mammalian tissue; consequently, this possibility may be discounted. Thus, it follows that the second assumption is met, at least within the accuracy of the experimental procedure, and that there is not significant overestimation of AH formation rates by this method. The third assumption involves proper mixing of the infused inulin with the newly formed AH. Should this not occur, one would expect irregular, constantly changing inulin concentrations in outflow, causing

7 82 Oppelt Investigative Ophthalmology February 1967 highly irregular values for AH formation rates. This is not the case, as is indicated in Fig. 2, which shows control AH formation rates as a function of time after the beginning of experiments. A more direct indication of adequate mixing is found in the experiments where position of the tip of the outflow cannula in the anterior chamber was changed during the experiment (Table II). As inulin concentration in outflow remained the same with change in position, adequate mixing of the isotope with newly formed AH is suggested. Hence, it seems that the major assumptions which have to be made in order for the method to be a valid one have been met, and that intraocular perfusion with an inulin-containing buffer is a valid estimation of net AH formation rates. Discussion of results Normal AH formation rates: In control cats the average AH formation rate was about 14 JXL per minute. Assuming an anterior chamber volume of about 1,000 (xl in cats (estimated by aspiration of cat anterior chamber, and from data of Macri and associates, 0 ) this represents an AH turnover rate of about 1.4 per cent per minute. This is identical to the value estimated in cats by Davson and Spaziani, 7 using turnover of 21 Na, and close to the value of 1.5 per cent per minute quoted by Barany. 13 Slightly higher turnover rates of 2.1 per cent per minute were found by Macri and associates, 0 using inulin turnover in cats. Our value for the AH turnover of cats is quite close to the 1.3 per cent per minute turnover reported in Cynomolgus monkeys by Bill and Hellsing, 8 who used the turnover of intraocular iodopyracet. Similar turnover values, arrived at with various methods, were found in rabbits, chickens, guinea pigs, and man by other investigators. 14 " 17 Thus, the more direct estimation of AH formation rates discussed in this paper closely agree with values of other investigators who used many different methods, further indicating the validity of this technique. Results with acetazolamide The IOP-lowering effect of carbonic anhydrase inhibitors, such as acetazolamide, has been found in many species, and these drugs are currently in wide use in the treatment of human glaucoma. The studies of many authors are well summarized by Becker ls and by Maren. 19 It has generally been assumed that carbonic anhydrase inhibitors lower IOP by directly reducing the flow rate of AH, 5-1S - 20 > 21 which is, at least in part, regulated by carbonic anhydrase activity. However, there has been some disagreement about this proposed mechanism of action of carbonic anhydrase inhibitors. Davson and Spaziani 7 found no change in the turnover of intraocularly injected 21 Na after the intravenous injection of adequate doses of acetazolamide and use this finding as an argument against the theory that the fall in IOP after acetazolamide is caused by a decrease of AH secretion rates. Macri and his co-workers 0 came to a similar conclusion. They used the turnover rate of intracamerally injected inulin to estimate AH formation rates. Using veiy large doses of intravenous acetazolamide, they could not detect changes in AH turnover rates after 0 mg. per kilogram intravenous acetazolamide, but found some decrease in this rate beginning at 250 mg. per kilogram. The interpretation of their results, however, is complicated by the fact that they did not observe a regular decrease in IOP after the high acetazolamide doses which produced a decrease in AH turnover rate and that acetazolamide in the usual therapeutic doses was not tried. Our experiments, using the inulin perfusion technique, support the arguments that carbonic anhydrase inhibition produces a decrease in IOP by decreasing AH formation rate. Studies by Wistrand 20 and Wistrand and associates 22 indicate that full carbonic anhydrase inhibitory effects on IOP are reached after single injection of mg. per kilogram acetazolamide. Our studies indicate an approximately 45 per cent reduction in AH formation rates after

8 Volume 6 Number 1 Measurement of aqueous humor formation rates 83 either or 20 mg. per kilogram acetazolamide, thus closely agreeing with the above-cited studies. A lesser decrease is noted after 5 mg. per kilogram, and 1 mg. per kilogram, while there is no physiologically significant response after mg. per kilogram. Thus there is a dose-response curve indicating decrease in AH formation rates at doses when one would expect the effect, judging from data where IOP is the criterion for drug effect. I wish to acknowledge valuable technical assistance by Miss Rebecca Embry and Mr. Dalton White. In addition I wish to thank Dr. T. H. Maren for critical review of the manuscript. REFERENCES 1. Chandler, M. R.: Aqueous flow measurements in man by the perilimbal suction cup technique. I. Observations in normal subjects and cases of glaucoma, Brit. J. Ophth. 48: 423, Becker, B., and Friedenwald, J. S.: Clinical aqueous outflow, Arch. Ophth. 50: 557, Moses, R. A., and Becker, B.: Clinical tonography: The scleral rigidity correction, Am. J. Ophth. 45: 196, Forbes, M., and Becker, B.: The transport of organic anions by the rabbit ciliary. IV. Acetazolamide and rate of aqueous flow, Am. J. Ophth. 51: 47, Langham, M. E.: Specificity and comparative activity of the carbonic anhydrase inhibitors Neptazane and Diamox on animal and human eyes, Brit. J. Ophth. 42: 577, Macri, F. J., Dixon, R. L., and Rail, D. P.: Aqueous humor turnover rates in the cat. I. The effect of acetazolamide, INVEST. OPHTH. 4: 927, Davson, H., and Spaziani, E.: The fate of substances injected into the anterior chamber of the eye, J. Physiol. 151: 202, Bill, A., and Hellsing, K.: Production and drainage of aqueous humor in the Cynomolgus monkey (Macaca irus), INVEST. OPHTH. 4: 920, Heisey, S. R., Held, D., and Pappenheimer, J. R.: Bulk flow and diffusion in the cerebrospinal fluid system of the goat, Am. J. Physiol. 203: 775, Pappenheimer, J. R., and Heisey, S. R.: 1st International Pharmacological Meeting, Vol. 4, Hogben, C. A., editor, Drugs and Membranes, Oxford, 1963, Pergamon Press, p Oppelt, W. W., Patlak, C. S., and Rail, D. P.: Effect of certain drugs on cerebrospinal fluid production in the dog, Am. J. Physiol. 206: 247, Lowiy, O. H., Rosebrough, N. J., Fan-, A. L., and Randall, R. J.: Protein measurements with the Folin phenol reagents, J. Biol. Chem. 193: 265, Barany, E.: The measurement of aqueous flow in the experimental animal, Modern Trends in Ophthalmology, 3rd series, London, 1955, Butterworth Publishing Company. 14. Kinsey, V. E., and Barany, E.: The rate of flow of aqueous humor. II. Derivation of rate of flow and its physiologic significance, Am. J. Ophth. 32: (Part 2): 189, Barany, E.: Rate of flow of the aqueous humor in the chicken (Gallus domesticus), Acta physiol. scandinav. 22: 340, Barany, E.: Rate of flow of aqueous humor in normal and scorbutic guinea pigs, Arch. Ophth. 46: 326, Goldmann, H.: Das Minutenvolumen der menschlichen Vorderkammer bei Normalen und bei Fallen von primarem Glaukom, Ophthalmologica 120: 150, Becker, B.: Carbonic anhydrase and the formation of aqueous humor: The Friedenwald Memorial Lecture, Am. J. Ophth. 47 (Part II): 342, Maren, T. H.: Carbonic anhydrase: Chemistry, physiology, and inhibition, Physiol. Rev. In press. 20. Wistrand, P.: The effect of carbonic anhydrase inhibitor on intraocular pressure with observations on the pharmacology of acetazolamide in the rabbit, Acta pharmacol. toxicol. 16: 171, Kronfeld, P. C: Effects of acetazolamide on human aqueous dynamics, Arch. Ophth. 68: 442, Wistrand, P. J., Rawls, J. A., Jr., and Maren, T. H.: Sulphonamide carbonic anhydrase inhibitors and intraocular pressure in rabbits, Acta pharmacol. toxicol. 17: 337, 1961.