The immediate effect on outflow resistance of intravenous pilocarpine in the vervet monkey, Cercopithecus ethiops. Ernst H. Bdrdny

Transcription

1 The immediate effect on outflow resistance of intravenous pilocarpine in the vervet monkey, Cercopithecus ethiops Ernst H. Bdrdny The experiments were made to see whether a direct action of pilocarpine on the tissue close to Schlemm's canal is essential for a maximum reduction in outflow resistance. Anterior chambers of anesthetized vervet monkeys were connected to pressure transducers and reservoirs suspended on strain gauges. One eye ivas kept at a low pressure, the other 8 to 10 mm. Hg above the first. From the difference in inflow rate and pressure, an apparent resistance can be calculated which would he correct if the eyes were identical. Intravenous injection of 1 ing. per kilogram pilocarpine HCl caused a precipitous resistance drop which was virtually complete after 2 to 3 and as deep as after a supramaximum intracameral dose. The amount of pilocarpine in the anterior chamber after such an injection was measured with tritiated drug. It was quite ineffective. Thus in normal vervet eyes no direct action of pilocarpine on the tissue of the inner wall of Schlemm's canal is necessary for a very marked effect. This means that the total resistance of the fully utilized filtering tissue in a healthy vervet eye is either quite small or that the conductance of the tissue per unit area, its specific facility, is insensitive to pilocarpine. A,.fter Tomqvist 1 had shown that systemic pilocarpine given to vervet monkeys caused marked accommodation in sublethal doses, the effect of intramuscular pilocarpine on outflow resistance was studied. A dissociation between the effects on resistance and on accommodation was found. Intracameral pilocarpine was able to cause a resistance drop in doses which hardly caused any accommodation. Systemic pilocarpine affected resistance much From the Department of Pharmacology, University of Uppsala, Uppsala, Sweden. This study was supported by Grant 14X733 from the Swedish Medical Research Council; and Grant NB3637 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service. 373 less per unit accommodation than did intracameral pilocarpine. 2 ' 3 The dissociation would be understandable if pilocarpine had two synergistic points of attack, one on the ciliary muscle and one directly on the tissue of the inner wall of Schlemm's canal.' 1 Changes in the endothelium of the canal under the action of pilocarpine were looked for and found, but their functional importance could not be assessed. 5 If a pilocarpine action directly on the wall of Schlemm's canal were essential for a maximum reduction of outflow resistance, even a high concentration of pilocarpine in the bloodstream might fail to cause a maximal effect until the drug had accumulated in the anterior chamber in sufficient concentration. The present experiments were designed

2 374 Bdrdny Investigative Ophthalmology August 1967 to test this point. They make use of two technical improvements. A modification of the two levelconstant pressure infusion technique 0 permits the resistance effect of systemic pilocarpine to be studied during the first after intravenous injection, and the availability of tritiated pilocarpine allows a check on the amounts of drug present in the aqueous humor. The results are, that systemic pilocarpine is capable of reducing the resistance virtually to rock bottom before appreciable amounts of the drug have entered the bulk of the aqueous. Nonetheless, a direct action of the miotic on the resistance properties of the inner wall of the canal has not been excluded. If it exists, it might be of some importance under pathological conditions where the amount of filtering area is reduced. Methods Ostensibly healthy adult vervets (Cercopithecus ethiops), body weight 3.5 to 4.5 kilograms, were used. They were anesthetized with intravenous pentobarbital and kept prone between heating pads. The head was fixed in a head holder and two needles shot into each anterior chamber. One needle led via polyethylene tubing to a pressure transducer, the other to a small reservoir with buffered perfusion fluid 0 suspended on a strain gauge. The reservoir and its gauge could be raised and lowered. The pressures and the weight of the reservoirs were recorded about every 15 seconds. In some experiments one plastic 1 ml. syringe per eye was connected to the tubing from the pressure transducer very close to the needle. These syringes served for removing the aqueous for analysis at a certain stage of the experiment. The dead space of the connectors was about 3 fil and was taken into account. While aqueous was withdrawn inflow from the reservoirs was blocked. In some experiments blood pressure was stabilized by a servo arrangement. The femoral artery was connected to one side of a differential transducer (Sanborn 267 BC) and air under a preselected pressure, usually 100 mm. Hg, to the other side. The output voltage was amplified and recorded and also used to drive an electrolytic pump which infused /?angiotensin II (Ciba Ba) 7 into the caval vein through a polyethylene catheter pushed up through the femoral vein until 10 cm. from the heart. Tritiated pilocarpine was obtained from New England Nuclear Corporation, lot number It was diluted with nonradioactive drug to a specific activity of 0.02 me. per milligram of the hydrochloride and used as a 1 mg. per milliliter solution made isotonic with NaCl. It was estimated by liquid scintillation counting (about 10,000 counts were counted). The experiment was so short that it was thought unnecessary to prove that the radioactivity present in the aqueous was undegraded pilocarpine. The basis of outflow resistance measurement is to estimate the change AF in rate of inflow when the level of the reservoir is shifted so as to change pressure by AP. Resistance R is AP/AF. In the single eye a period of at least 3, preferably 4, at each pressure is needed for a measurement of an inflow rate after a pressure change, and thus 6 to 8 of recording are needed to derive a single resistance value. If resistance is gradually changing, successive values of this kind fall on a sawtooth curve. This artefact is eliminated by the formation of successive means 0 and this is always done, yielding conventional resistance. The use of conventional resistance values means that in fact three recording periods, a total of 9 to 12, contribute to any point. Greatly increased temporal resolution can be achieved, albeit at a cost, by using both eyes as if they were identical. If they were in fact identical, one kept at a higher pressure by its reservoir and one at a lower would furnish two inflow rates from the reservoirs and the difference in pressure divided by the difference in inflow rate at any moment would give a record of resistance. The effect of drugs given systemically and presumably acting equally on both eyes could then be studied with high time resolution. It is evident that if the two eyes are not identical, because of differences in any of the parameters determining intraocular pressure, then the apparent resistance R app calculated in this manner will differ from the correct one. On the other hand, an idea of the temporal course of a change in parameters can still be had. In order to obtain correct values for the resistance of the individual eyes and at the same time values of R np p with high time resolution, the experiment was conducted as illustrated in Fig. 1. Successive 4 minute periods at 2.5 and 11.9 mm. Hg above resting pressure were used but 180 degrees out of phase in the two eyes. After pilocarpine injection, the reservoirs were kept constant for 8 to 10 whereafter the changes were resumed for an additional six 4 minute periods. The successive periods in each eye furnished conventional Rvalues for each eye separately while a comparison between the two eyes during each period furnished a value of R!1PP. Fig. 1 is not only a diagram of the protocol of the initial part of an experiment but summarizes some features of a typical result (except

3 Volume 6 Number 4 Effect of pilocarpine on outflow resistance MIN Fig. 1. Protocol of typical experiment. Above the time scale: course of pressure in the two eyes I and II. Below the time scale: course of reservoir weights. Pilocarpine injection at minute 16. From minute 16 to 24 readings for each minute were utilized separately for estimation of R IU,i). Periods marked A were also utilized for estimation of that it has been constructed for the case of identical eyes). Alternating periods of high and low pressure cause alternating periods of high and low slope in the graph showing reservoir weight as a function of time. When pilocarpine is injected, intraocular pressure rises as it usually does in these anesthetized monkeys. 3 The reservoir at the lower level actually gains weight while the higher reservoir now loses weight faster than before. The full effect is shown as established in about 2. Because of the rapid flow when the reservoir is high more of a pressure drop occurs in the tubing and needle. This explains the shape of the pressure curves. During the 8 to 10 following pilocarpine, the slopes and hence the inflow rates were averaged over 1 minute periods separately, yielding 8 to 10 individual AF values by comparing the eyes. Similarly the pressure difference between the eyes was averaged over every individual minute. These 1 minute averages were used for tlie calculation of the 1 minute values of R,, PP. Conventional values for R were also derived from successive 4 minute periods of each eye separately and then averaged, R nv. Since little pressure change occurred in each eye during the 8 to 10 following pilocarpine, no individual R values for the two eyes were obtained for this part of the experiment. For comparison R,,,,,, was also calculated in the periods marked A where the relative pressure relation between the eyes was the same as in the 8 to 10 minute period after pilocarpine. Results Time course of the drop in outflow resistance. Fig. 2 shows an experiment typical for a group of eight. This experiment was done without artificial blood pressure stabilization. The eyes are not identical and the apparent resistance R npp at the outset was below R av, the average of the two eyes. At ATR 0.1 mg. per kilogram atropine sulphate was given intravenously to prevent too much upset of the circulation by a subsequent injection of pilocarpine. T6rnqvist s has shown that atropine doses of this size prevent or greatly reduce the circulatory effects of systemic cholinergics without preventing their effect on the eye. The atropine evidently has very little effect on outflow resistance. Starting at time 28, 1 mg. per kilogram pilocarpine HC1 was given intravenously in a solution 1 mg. per milliliter made isotonic with NaCl. The injection took 15 seconds. There was an immediate reaction. No value was obtained for the very first minute in this experiment, but already during the second minute R npp had dropped far and during the continued course little more happened. The time scale is expanded for the first 8 following pilocarpine. In the later course which is quite flat it is seen that the averages of the conventional resistance values R ttv of the two eyes and the apparent resistance values R npp from the combined use of the two eyes virtually coincide. This shows that 10 to 20 after pilocarpine the parameters determining inflow from the reservoirs are veiy similar in the two eyes. It is probable, therefore, that also the apparent resistance values in the first following pilocarpine are not

4 376 Bdrdntj In oestigative Ophthalmology August ATR Pa MINUTES Fig. 2. Effect of intravenous pilocarpine on outflow resistance. Filled circles R app, open circles R»v, both in millimeters Hg per microliter per minute. At ATR intravenous injection of 0.1 mg. per kilogram atropine sulphate, at P1L intravenous injection (in 15 seconds) of 1 mg. per kilogram pilocarpine hydrochloride. Time scale expanded for 8 after start of injection. (Experiment 2,427.) Fig. 3. Effect of intravenous pilocarpine on outflow resistance. Filled circles R np p, open circles Rnv, both in millimeters Hg per microliter per minute. At PIL intravenous injection (in 2 ) of 1 mg. per kilogram pilocarpine HC1. Time scale expanded for 8 after start of injection. Blood pressure stabilized at 100 mm. Hg. There was no counterpart in systemic arterial pressure to the fluctuations in R np p. (Experiment 2,441.) too different from the real ones. Only in one experiment out of 8 was there a marked difference between R npp and R nv in the later course after the injection of pilocarpine. This experiment was, therefore, not used in Table I. The almost immediate and deep resistance decrease was seen in every one of these 8 experiments and in an additional 4 short ones following a similar protocol but with radioactive pilocarpine. It made no difference if 0.05 or 0.1 mg. per kilogram atropine had been given, nor if the blood pressure was stabilized or not. Sometimes even the first minute after the start of pilocarpine injection could be evaluated, sometimes however the circulatory upset was too large and the record too irregular during this first minute. The course of R ni)p as a rule was smooth and fairly regular, except for Experiment 2,441 shown in Fig. 3. This was an experiment without atropine but with stabilized blood pressure. The resistance fluctuated markedly before it settled down. The fluctuations are very similar to the fluctuations in accommodation seen by Tornqvist 1 after systemic pilocarpine. This experiment also is the only one of the 8 long ones where there is a marked gradual fall in resistance over an extended period of time. The reason for this is not clear. In all other experiments, the later course was very similar to that of Fig. 2. Table I has been constructed to show the degree of completeness of resistance drop a few after intravenous pilocarpine. Comparison between postpilocarpine and prepilocarpine values of R app can be misleading since before pilocarpine R npp can differ markedly from the true values. Therefore, the individual postpilocarpine values for the successive ; R x, R 2, etc., have been compared with the value R 3 derived from the third minute after start of the pilocarpine injection. The table shows that R t R 3 still is not negligible, indicating that during the first minute the effect was not complete. But already during the second minute, several

5 Volume 6 Number 4 Effect of pilocarpine on outflow resistance 377 animals showed almost complete effect, R 2 R M was quite small. In later very little happened. It is evident, therefore, that in 2 or 3 intravenous pilocarpine was able to reduce the resistance to a very low level. The question now is whether this reduction was partly due to the presence of pilocarpine in the anterior chamber. The amount of pilocarpine in the anterior chamber during the first after intravenous injection. Six experiments were conducted in the same manner as those described above but for the difference that tritiated pilocarpine was used and that aqueous samples were taken. This latter of course prevented further measurements of R. The aqueous was removed from the two eyes at an interval of a few and the samples weighed and counted. The specific gravity of the aqueous was taken as 1.0. Table II shows the results. It is seen that the concentrations present a few after intravenous injection of 1 mg. per kilogram are of the order of 0.1 /j,g per milliliter. Earlier data are available where known amounts of pilocarpine were injected into the anterior chamber and the chamber stirred with a roller pump. 3 ' In order to Table I. Time course of resistance drop after intravenous pilocarpine HCl, 1 mg. per kilogram at time 0. Subscripts of R l5 R a, etc., refer to successive 1 minute periods after start of injection. Last R av after drug is 20 to 24 later than first. First R av after drug is derived from individual values in Table IV Experiment No. Apparent resistance R opp from comparison of the two eyes R 3 R,R,\RtR*\RrRi R,R 5 \R 3 R , , , Conventional resistance R av, mean of the two eyes Last before drug First after drug Last after drug Intravenous atropine sulphate (mg./kg.) Blood pressure stabilized Table II. Appearance of pilocarpine in aqueous humor after intravenous injection of 1 mg. per kilogram. Duration of injection was 1 minute. The anterior chambers were not completely emptied. The samples marked with an asterisk were smaller than the rest Experiment No. 2,443 2,444 2,445 2,448 2,449 2, Pilocarpine HCl, fig/ml, aqueous humor (time of removal of sample after start of injection) "

6 378 Bdrdny Investigative Ophthalmology August 1967 make a comparison possible, tritiated pilocaqjine was injected into the anterior chamber and stirred in exactly the same manner. After 2 of stirring the aqueous of one eye was removed. Three doses, 0.5, 2, and 20 /xg of pilocarpine HC1 and one animal for each dose were used. The concentrations resulting from the injection of known doses yielded distribution volumes at 2. These were 0.59, 0.90, and 0.87 ml. Thus, a concentration of 0.1 /.ig per milliliter would have corresponded to doses of 0.059, 0.09, and fig injected and stirred. Such small doses would not be expected to have any measurable effect on outflow resistance. To make sure, however, the dose was given to 5 vervet monkeys. One eye received 0.1 /xg pilocarpine HC1 in 10 /.il into the anterior chamber, the other eye received only the solvent. Both anterior chambers were stirred by the usual roller pump for 2. Table III shows, that no appreciable effect of the pilocarpine could be detected. Thus, the bulk concentration is negligible early after intravenous injection. A direct action of the intravenously injected pilocarpine on the meshwork tissue can still not be excluded. The meshwork is so close to the vascularized ciliary muscle, especially in the vervet 10 that it is entirely possible for pilocarpine coming from the vessels of the muscle to build up a higher concentration in the mesh Table III. Lack of effect of 0.1 /.ig pilocarpine HC1 injected into the anterior chamber of vervet eye. All resistance values in millimeters Hg per microliter per minute. The control eye injected with solvent Mean 1.71 Conventional outflow resistance Experimental eye Control eye Before After Before After Ratio after/before Experimental Control Table IV. Total inflow from reservoirs and first conventional resistance value after drug. Inflow volume is the total during first 16 after start of intravenous pilocarpine injection except in No. 2,434, where the figure is 18. Resistance values derive from the period from 4 to 16 after injection, except in 2,434, where the figures are 6 to 18. Eye I had lower pressure during the period immediately following pilocarpine injection. Volume in microliters, outflow resistance in millimeters Hg per microliter per minute Experiment No. 2,426 2,427 2,433 2,434 2,438 2,439 2,441 Average Inflow from reservoirs Eye I Eye II I/Il Outflow resistance Eye I Eye II

7 Volume 6 Number 4 Effect of pilocarpine on outflow resistance 379 work than in the bulk. The excess concentration would depend markedly on the rate of fluid flow through the meshwork. Referring to Fig. 1, it is evident that around the 24 minute mark there should be more drug in the meshwork of eye I than in eye II. If the excess amount had any effect, resistance around time 24 should be different in the two eyes. Table IV lists the amoimts of perfusion fluid which had entered into the two eyes between the injection of pilocarpine (corresponds to minute 16 in Fig. 1) and the end of the period utilized for estimating the first value of conventional resistance after pilocarpine (corresponds to minute 32 in Fig. 1). On an average, twice as much had entered eye II than eye I. The amount of fluid flowing through the meshwork differs somewhat from that flowing into the eye, but not much. One would therefore expect that on an average about twice as much pilocarpine was acting on the meshwork of eye I than of eye II. The table however shows that the resistance values were very similar in the two eyes. It seems, therefore, certain that penetration of pilocarpine into the aqueous has not played any part in the rapid and deep resistance decrease seen after intravenous injection of the drug. Discussion There can be no doubt that the large and rapid effect of intravenous pilocarpine is due to its action on a vascularized tissue. By far the most probable candidate is the ciliary muscle. The possibility that the vessels beyond the canal of Schlemm are responsible for the effect is so remote that it will be disregarded in the further discussion. The resistance values reached are very low. In order to compare them with values previously found after intracameral injections of supramaximum doses of pilocarpine, the first conventional R postpilocarpine in each experiment and eye from Table IV, R pp have been plotted in Fig. 4 against the last conventional R before the drug, R s tnrt The figure also contains previously obtained 3 data for 90 monkeys of the same species, each individual contributing one eye where 20 /xg pilocarpine HCl had been injected into the anterior chamber. These 90 animals but not the present ones had received hexamethonium after the starting resistance had been measured, but the consequent lack of nervous tone to the ciliary muscle should make little difference in the presence of the large dose of pilocarpine. It is seen that intravenous injection has yielded values qiute as low as those found with intracameral pilocarpine. Even if during the early stages after intravenous pilocarpine R npp may have differed from the correct value R uv, there cannot have been any considerable amount of gradual change in resistance after the first precipitous drop. Thus, the present experiments show that in normal vervet eyes no direct action of pilocarpine on the meshwork tissue is necessary for a full resistancelowering effect. Does this mean that the hypothesis of a synergistic direct action on the meshwork tissue can now be discarded? Un 5.0 Rstart Fig. 4. Comparison between die effects of pilocarpine HCl 1 mg. per kilogram intravenously (open circles) and 20 fig intracamerally to ganglionblocked animals (dots). Ordihates: conventional resistance after pilocarpine, R PJ). Abscissa: conventional resistance before drug, Rstart. The line is an orthogonal regression line fitted to the dots. 3 Its equation is Rp P = R s tart (n = 90).

8 380 Bdrdny Investigative Ophthalmology August 1967 fortunately this is not so for the following reason. The relevant property of the inner wall tissue is hydrodynamic conductivity per unit area, which perhaps might be dubbed specific facility. This is the property which might be pilocarpine sensitive. It has been pointed out 10 that the state of contraction of the ciliary muscle can hardly affect specific facility but could increase the fraction of the total area involved in filtration. Recent experiments have furnished direct evidence for this view. 11 Assume for the sake of argument that the resistance of the total aggregate of scleral and postscleral channels in the healthy vervet eye is 0.4 and that the resistance of the inner wall tissue when fully exposed by ciliary muscle contraction is 0.1. Even if pilocarpine were to increase specific facility by 100 per cent, this would only cause an additional drop in resistance of 0.05, a hardly significant and barely measurable figure. Now consider a case of trabecular disease, with a considerable reduction in the total available area for filtration. Say that the available area of healthy tissue even with the ciliary muscle fully contracted is 10 per cent of that of the healthy eye. Its resistance would then be 1.0 before and 0.5 after pilocarpine. With the scleral channels intact, there would be a resistance drop from 1.4 to 0.9. The present experiments have neither shown nor excluded a direct pilocarpine action on the inner wall tissue. But they have shown that the total resistance of the fully exposed tissue in a healthy vervet eye either must be quite small or else, that the specific facility of the tissue is quite insensitive to pilocarpine. The expert assistance of Mrs. Ingalill Dalhagen Wersall is gratefully acknowledged. The /3angiotensin II was kindly donated by CIBA Produkter AB, Vallingby. REFERENCES 1. Tornqvist, G.: Effect on refraction of intramuscular pilocarpine in two species of monkey (Cercopithecus aethiops and Macaca irus), INVEST. OPHTH. 4: 211, Barany, E. H.: Dissociation of accommodation effects from outflow effects of pilocarpine, in Drug mechanisms in glaucoma, London, 1966, J. & A. Churchill, Ltd., p Barany, E. H.: Doyne memorial lecture. The mode of action of miotics on outflow resistance. A study of pilocarpine in the vervet monkey Cercopithecus ethiops, Tr. Ophth. Soc. U.K. 86: 539, Barany, E. H.: The mode of action of pilocarpine on outflow resistance in the eye of a primate (Cercopithecus ethiops), INVEST. OPHTH. 1: 712, Holmberg, A., and Barany, E. H.: The effect of pilocarpine on the endothelium forming the inner wall of Schlemm's canal: An electronmicroscopic study in the monkey Cercopithecus aethiops, INVEST. OPHTH. 5: 53, Barany, E. H.: Simultaneous measurement of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion, INVEST. OPHTH. 3: 135, Imhof, P., Brunner, H., Quitt, J., Steinmann, B., and Jacono, A.: Experimentelle und klinische Untersuchungen mit /6Angiotensin IE (Praparat CIBA 33902Ba), einem neuen AngiotensinIIAnalogen mit verstiirkter und verlangerter pressorischer Wirkung, Schweiz. med. Wchschr. 94: 1199, Tornqvist, G.: Accommodation in monkeys. Some pharmacological and physiological aspects, Acta ophth. 45: 1, Barany, E. H.: Relative importance of autonomic nervous tone and structure as determinants of outflow resistance in normal monkey eyes (Cercopithecus ethiops and Macaca irus), In Rohen, J. W., editor: Eye structure, II. Symp., Stuttgart 1965, SchattauerVerlag, p Rohen, J. W., Liitjen, E., and Barany, E.: The relation between the ciliary muscle and the trabecular meshwork and its importance for the effect of miotics on aqueous outflow resistance. A study in two contrasting monkey species, Macaca irus and Cercopithecus aethiops, Albrecht v. Graefes Arch. klin. exper. Ophth. 172: 23, Barany, E. H., and Rohen, J. W.: Control of the effective filtration area of the trabecular meshwork by the ciliary muscle. A study in the vervet monkey Cercopithecus ethiops. In preparation.