The relationship of pressure and aqueous outflow in enucleated human eyes*

The relationship of pressure and aqueous outflow in enucleated human eyes* Bruce A. Ellingsen and W. Morton Grant The influence of intraocular pressure on resistance to aqueous outflow was evaluated in enucleated normal and glaucomatous human eyes by quantitative aqueous perfusion of the anterior chamber. When provision was made for free communication from anterior to posterior chamber to avoid artificial deepening of the anterior chamber, and the intraocular pressure was raised stepwise from 5 to 50 mm. Hg, the resistance to outflow progressively increased. The resistance to outflow increased more steeply in eyes with open-angle glaucoma than in nonglaucomatous eyes. When no communication was provided for flow of perfusion fluid from anterior to posterior chamber, and the pressure was raised in the same manner, the anterior chamber progressively deepened, but above 20 mm. Hg the resistance to outflow became nearly independent of intraocular pressure. Effects of reducing intraocular pressure, repeating perfusions, prolonging perfusions, and modifying the perfusion fluid were also evaluated. We postulate that intraocular pressure has direct influence on aqueous outflow channels. In openangle glaucomatous eyes the aqueous outflow channels appear to be abnormally susceptible to this influence. Key words: intraocular pressure, outflow facility, outflow resistance, enucleated eyes, aqueous perfusion, glaucoma. hen the aqueous outflow characteristics of the eye are being evaluated and analyzed clinically or experimentally it is important to examine and take into account the influence of intraocular pressure on From the Howe Laboratory of Ophthalmology, Harvard Medical School, and the Massachusetts Eye and Ear Infirmary, Boston, Mass. This investigation was supported by National Institutes of Health Center Grant 5-PO1-EY00292 and National Institutes of Health Research grant 5-RO1-EY00002 from the National Eye Institute. Manuscript submitted March 22, 1971; manuscript accepted April 30, 1971. Reprint requests: Dr. Bruce A. Ellingsen, Spokane Eye Clinic, S. 427 Bernard St., Spokane, Wash. 99204. "Presented at the ARVO meeting Sarasota, Fla., May, 1970. 430 resistance to outflow of aqueous humor. This deserves attention, for instance, in analysis of individual and consecutive Schi0tz tonographies in which the basic assumption generally has been made, but not conclusively validated, that outflow resistance remains constant at different pressures. Similarly, possible changes in resistance to outflow secondary to changes in pressure need to be considered in evaluating perfusion methods used for estimating outflow resistance, aqueous formation, and pseudofacility. Several investigators have concluded from studies in vivo and in vitro that outflow resistance of primate eyes may not be affected by variation in pressure within physiologic intraocular pressure ranges, 1 " 3 whereas others have reported that outflow

Volume 10 Number 6 10? and resistance to aqueous outflow 431 resistance may be increased by increasing intraocular pressure. 4 " 7 The suggestion also has been made that an increase in resistance with increased pressure is a property of the living eye and reflects certain unspecified homeostatic mechanisms that are not operative in the enucleated eye. 8 The present series of experiments was undertaken to establish the facts more definitely if possible, and to re-examine some of the factors that affect the pressure-outflow relationship in normal and glaucomatous eyes. Methods Measurements were made on enucleated normal and glaucomatous human eyes by means of a modification of the constant pressure perfusion technique of Barany. 9 Except for five eyes which have been clearly identified as glaucomatous, the eyes which were used had no history of glaucoma, and examination of the anterior segment using an operating microscope revealed no abnormalities. The glaucomatous eyes had been diagnosed clinically as having primary open-angle glaucoma, which was under treatment with various medications at the time of death. All eyes were obtained post mortem and stored at 4 C. in a moist environment until approximately one-half hour prior to perfusion. Perfusions were started 4 to 58 hours post mortem and were carried out at room temperature. The eyes were routinely set or encased in silicone rubber, enveloping the posterior segment to just anterior to the equator. The encased eye was mounted on an adjustable ball-bearing fitting which allowed easy manipulation of the preparation. Exposed episclera and conjunctiva were covered with strips of tissue paper saturated with perfusing solution. Control eyes perfused without encasement in silicone rubber gave results that were not appreciably different from those obtained with encasement. The constant pressure perfusion technique of Barany was selected after trial and comparison of other established methods, because it offers the advantage of requiring a very short time to achieve a steady state, and this facilitates multiple determinations of outflow facility. The eyes were perfused through either a previously described corneal fitting 10 or a 23 gauge needle. The fitting or needle was attached to polyethylene tubing connecting to a reservoir. The height of the reservoir was adjustable and its weight was constantly monitored with an electric strain gauge and recorder. A radial iridotomy was performed prior to perfusion, or perfusion was accomplished through a needle with its tip located specifically in either the posterior chamber or the anterior chamber, depending upon the purpose of the experiment. The perfusing solution in most experiments was a sterile phosphate-buffered, balanced salt solution containing glucose as previously described by Barany, 11 passed through a micropore filter before using. In certain experiments, sterile unbuffered 0.9 per cent sodium chloride solution commercially prepared without preservative for intravenous administration was used for comparison. Results -flow relationship. In 32 normal and 5 glaucomatous eyes multiple determinations of outflow facility were made at specific pressures increasing step wise from 5 to 50 mm. Hg. Then multiple determinations were made in the reverse order with decreasing pressures. An attempt was made to maintain uniform periods of perfusion. The usual time allowed for reaching steady conditions and for measurement of flow at each pressure was 4 to 5 minutes, although occasionally technical diflbculty necessitated somewhat longer perfusion times. When pressure was reduced to 5 or 10 mm. Hg, 7 to 8 minutes were required to reach steady conditions. Determinations of the average pressureoutflow relationship in normal eyes (Fig. 1) indicated a curvilinear relationship in which facility of outflow decreased, or resistance increased, with increasing pressure. With decreasing pressure a fairly straight line relationship was found, and facility of outflow was more nearly constant, except for a small decrease in facility between 50 and 40 mm. Hg and a larger decrease between 10 and 5 mm. Hg. Outflow facility calculated for increasing pressure (Table I) through the range from 5 to 50 mm. Hg decreased progressively from 0.42 to 0.16 ju,l per minute per millimeter of mercury. The percentage change in total facility per millimeter of mercury of pressure between various pressure levels (listed in Table I) decreased with increasing pressure. To determine more about what happened to outflow at very low pressures, seven eyes were perfused at pressures be-

432 Ellingsen and Grant Investigative Ophthalmology June 1971 10 20 30 40 mmhg 2 SD. below normal Open angle glaucoma Fig. 1. Data from enucleated human eyes in which anterior and posterior chambers were in free communication, and aqueous perfusion flow rates were measured at a series of steady states as the intraocular pressure was raised stepwise to 50 mm. Hg, and again as the pressure was reduced (as indicated by the arrows). The upper loop of solid lines represents mean values from 32 normal eyes and the lower loop of solid lines represents 5 open-angle glaucomatous eyes. The dashed line represents values calculated as 2 S.D. below the means of the normal eyes. tween 0 and 5 mm. Hg. This showed that as pressure was raised from 0 the onset of flow began between 0.5 and 2 mm. Hg, and that with decreasing pressure the flow ceased between 5 and 3 mm. Hg. The glaucomatous eyes differed from the normal in that at low pressure the initial values of outflow facility were lower than normal and that as pressure was raised between 10 and 30 mm. Hg the rate of decrease in facility was greater than normal (Table II). At 20 mm. Hg and at higher pressures the mean outflow rates of glaucomatous eyes were calculated to be less than the mean of normal eyes by more than two standard deviations (Fig. 1). At lower pressures the means of the facility measurements of the glaucomatous and the normal differed less than this. 50 It is noteworthy that a similar abnormally steep increase of resistance to outflow with elevated pressure in glaucomatous eyes has been reported by Kleinert 5 from perfusion measurements on a series of patients. Successive determinations of pressureflow relationship. Four eyes were perfused two or more times up and down the pressure range in order to determine the response of eyes subjected to successive determinations of the pressure-flow relationship. Between successive determinations the eyes were allowed to remain at reduced pressure with no inflow for 10 to 20 minutes. Results suggest that under these conditions there is a tendency during the rest period for restoration of outflow facility that had become reduced during elevation of pressure. The characteristic form of the pressure-flow relationship is maintained, but with facility values at most pressures 30 to 40 per cent below those of the initial determination (Table III). Also, in this experiment in successive cycles the facility of outflow became higher at 10 mm. Hg than at 5 mm. Hg, in contrast to the initial determination. Effect of anterior-posterior chamber communication. In order to determine the effect of establishing a communication between anterior and posterior chamber, one glaucomatous and six normal eyes were perfused with and without a communication between anterior and posterior chamber. In one group of four normal eyes and one glaucomatous eye the initial perfusion measurements were made with flow into the anterior chamber and no artificial communication with the posterior chamber. Comparative measurements were then made after establishing communication between the two chambers by means of a radial iridotomy, or by placing a small polyethylene tube under the pupillary margin, or by placing the tip of the perfusing needle in the posterior chamber. In two other normal eyes the initial perfusion was done with the anterior-posterior chamber communication already established,

Volume 10 Number 6 IOP and resistance to aqueous outflow 433 Table I. Outflow facility (mean of 32 normal eyes) at each pressure level, reading left to right Facility Per cent facility change (per mm. Hg) 10 20 30 40 1 50 1 40 30 20 \ 10 5 0.424 0.360 0.268 0.218 0.182 0.160 0.150 0.155 0.155 0.153 0.124-2.9-2.6-1.9-1.7-1.2-0.6 +0.3 0-0.1-3.8 Table II. Outflow facility (mean of 5 open-angle glaucomatous eyes) at each pressure level Facility Per cent facility change (per mm. Hg) 10 20 30 40 50 1 40 1 30 1 20 JO 5 0.286 0.245 0.146 0.117 0.095 0.078 0.073 0.072 0.071 0.068 0.030-2.9-4.0-2.0-1.9-1.8-0.6-0.1-0.1-0.4-5.6 Table III. Outflow facility (mean of 4 normal eyes) at each pressure level going up and down the pressure cycle of 5 to 50 mm. Hg the first time and the second time Facility first Facility second Per cent decrease 10 20 30 40 50 40 30 20 10 0.435 0.377 0.284 0.260 0.203 0.177 0.164 0.171 0.173 0.177 0.072 0.202 0.246 0.180 0.157 0.146 0.124 0.119 0.126 0.127 0.118 0.060 53.5 33.0 36.6 39.5 28.0 30.0 27.5 26.3 26.5 30.5 16.7 and the subsequent comparative perfusion was done after the iris was allowed to go back into place against the lens. When the perfusion fluid was introduced into the anterior chamber without a communication from anterior to posterior chamber, an artificial deepening of the anterior chamber was easily observable with an operating microscope as the pressure was raised. The change in depth was most conspicuous in the lower portion of the pressure range. When a communication from anterior to posterior chamber was provided, or when the perfusing needle was introduced into the posterior chamber, the anterior chamber did not deepen appreciably as pressure was raised. In Fig. 2, the mean pressure-flow relationships with and without anterior-posterior chamber communications are plotted. It is evident that with no communication, and with the anterior chamber becoming deepened as the pressure increased, the facility of outflow remained essentially constant above 20 mm. Hg (Table IV). When a communication was established avoiding the deepening of anterior chamber, the facility of outflow decreased as the pressure was raised. With decreasing pressure, when there is no anterior-posterior communication, there appears to be a tendency for outflow facility to decrease (Table IV). The one glaucomatous eye perfused as described above reacted like the normal eyes, but facility values were lower than normal. Prolonged perfusion time. In six normal eyes the effect of prolonging perfusion was examined by determining perfusion flow rates every 10 minutes during a period of 40 minutes at pressures maintained at 10,

434 Ellingsen and Grant Investigative Ophthalmology June 1971 I I8r 16 14 12 10 2- Without 10 O O With 20 40 50 mmhg Fig. 2. Mean values from enucleated human eyes in which steady-state rates of perfusion of the anterior chamber were determined at ascending and descending intraocular pressures, either as in Fig. 1 with free communication between anterior and posterior chambers to minimize variations in depth of the anterior chamber (lower loop), or without this communication, leaving the lens-iris diaphragm intact so that the anterior chamber deepened automatically as the pressure in the anterior chamber was raised (upper loop). 20, and 30 mm. Hg. At each pressure the outflow facility was found to decrease slowly but continuously during the extended perfusions (Table V). This effect was particularly notable at the lower pressures. Most of the decrease of outflow facility occurred during the first two or three measurements. When the pressure was reduced stepwise through the same range the facility values were more constant during the extended periods of perfusion. When pressure was reduced to 10 mm. Hg, facility tended to increase during the period of perfusion. Comparison of Bdrdny's solution and normal saline. In order to determine if the outflow resistance characteristics of enucleated eyes might be affected by using a different perfusion solution, five pairs of normal eyes were perfused between 5 and 50 mm. Hg with the use of unbuffered 0.9 per cent sodium chloride in one eye of each pair, and solution in the other eye. In another five pairs these solutions were compared according to the prolonged perfusion protocol. Although differences were slight, the results indicate that the outflow facility was more affected by increase of pressure and prolonged perfusion with sodium chloride than with solution (Tables VI and VII). Discussion In considering the results obtained from a conventional perfusion technique, as used in these experiments, it must be kept in mind that the flow actually measured is inflow rather than outflow. Also, when facility of outflow is calculated from rate of inflow at a steady pressure, it is assumed that the intraocular volume is constant during the measurements. Enucleated eyes have an advantage of eliminating from consideration the variations of intraocular volume that are associated in vivo with aqueous formation and with blood flow, but enucleated eyes are not free from a volume variable which is associated with the viscoelastic properties of the eye. Unfortunately, the published data that are available concerning the slow creep or plastic properties of the eye do not provide a quantitative assessment directly applicable to the conditions of the present perfusion studies. 12 However, we estimate from the data that are available that the increase of volume which may be occurring from very slow stretching of the eye during the 4 or 5 minutes involved in measurements of flow would probably account for only a fraction of the calculated variations

Volume 10 Number 6 IOP and resistance to aqueous outflow 435 Table IV. Outflow facility (mean of 6 normal eyes) at each pressure level during cycles with and without free communication from anterior to posterior chamber Facility with communication Facility without communication Per cent increase 10 20 30 40 50 40 30 20 10 0.322 0.289 0.209 0.179 0.152 0.137 0.129 0.133 0.140 0.130 0.124 0.412 0.403 0.361 0.367 0.361 0.360 0.356 0.311 0.291 0.261 0.241 28 40 73 105 137 163 176 134 108 101 95 Table V. Outflow facility (mean of 6 normal eyes), changing with time of perfusion, at each pressure level Facility, 10 mm. Hg Facility, 20 mm. Hg Facility, 30 mm. Hg Facility, 20 mm. Hg Facility, 10 mm. Hg 0.306 0.227 0.161 0.153 0.181 Time (min.) 10 20 30 0.253 0.194 0.151 0.152 0.202 0.239 0.181 0.144 0.152 0.214 0.234 0.183 0.140 0.159 0.215 Change (%) -30.4-19.3-13.0 +3.9 +18.8 in outflow facility. Furthermore, it seems to us that difference in creep would be hard to imagine as a factor in explaining the fundamental difference in pressure-flow relationship with and without deepening of the anterior chamber. Osmotic movement of water through the cornea and sclera presumably was a negligible factor in these experiments, since the outer surfaces of the eyes were bathed with the same solutions as used for the perfusions. We have considered the question of whether flow characteristics of geometrically rigid or fixed outflow channels could account for pressure-flow relationships indicated by our experiments. According to advisors in fluid mechanics from the Massachusetts Institute of Technology, the fluid viscosity, flow rates, and approximate dimensions of the outflow system are such as to give a Reynolds number of sufficiently low order that one should expect inertia-free outflow in the human eye. With inertia-free flow in a geometricallyfixed system the resistance to flow (i.e., ratio of driving pressure difference to flow) is constant and independent of flow or pressure. 13 In the human eye it appears, therefore, that to account for the increase in resistance found experimentally the outflow system must not be geometrically fixed and it must be altered physically by increasing flow and pressure. The relative constancy of resistance with decreasing pressure suggests that the postulated physical alteration is not immediately reversible by reducing pressure for a prolonged period. It appears that artificial deepening of the anterior chamber tends to counteract the influence of increasing flow and pressure and to stabilize the outflow system physically. An obvious explanation would be that the forcible retrodisplacement of lens-iris diaphragm tends to stabilize facility of outflow by its traction on scleral spur and trabecular meshwork, while intraocular pressure acting directly upon the aqueous outflow channel tends to reduce facility of outflow in the absence of this stabilizing force. When we think about the mechanism by which intraocular pressure and aqueous outflow may directly alter the resistance to flow through the outflow system our attention is drawn to Schlemm's canal,

436 Ellingsen and Grant Investigative Ophthalmology June 1971 Table VI. Outflow facility (means of 5 pairs of normal eyes) at each pressure level, with solution in one eye of each pair and 0.9 per cent sodium chloride solution in the other Facility, Facility, saline Per cent different from 0.506 0.502 10 20 30 40 50 40 30 20 10 0.422 0.410 0.270 0.275 0.229 0.216 0.209 0.177 0.187 0.155 0.182 0.146 0.185 0.143 0.194 0.147 0.196 0.129 0.174 0.066-0.4-2.4 +1.8-5.7-15.3-17.1-19.8-22.7-24.2-34.1-62.1 Table VII. Outflow facility (mean of 5 pairs of normal eyes) at each pressure level and for different times of perfusion, with solution in one eye of each pair and 0.9 per cent sodium chloride solution in the other 0 Time 10 (min.) 20 30 Change (%) Facility, 10 mm. Hg 0.309 0.328 0.248 0.265 0.243 0.255 0.230 0.244-25.6-25.6 Facility, 20 mm. Hg 0.228 0.206 0.193 0.164 0.180 0.154 0.182 0.144-20.2-30.1 Facility, 30 mm. Hg 0.160 0.135 0.152 0.120 0.143 0.115 0.140 0.114-12.5-15.5 Facility, 20 mm. Hg 0.153 0.119 0.150 0.128 0.150 0.126 0.155 0.131 + 1.3 + 9.9 Facility, 10 mm. Hg 0.176 0.144 0.198 0.155 0.213 0.167 0.213 0.174 +21.0 +20.8 particularly the inner wall, and to its relationship to trabecular meshwork and the outer wall of Schlemm's canal, as a conceivable site for compression or geometric alteration, but at this time we can only speculate. At the same time it is tempting to speculate that the site or mechanism of abnormal resistance to outflow in glaucomatous eyes may be the same location as the compression and variable resistance mechanism since the perfusions of glaucomatous eyes have indicated that they differed not only in having an abnormally high resistance to outflow but also in responding with abnormally steep increase of resistance to elevated pressure. In design of perfusion experiments it appears from our findings that among the factors deserving attention are anteriorposterior chamber communication, level of intraocular pressure, direction of change of pressure, duration of perfusion, and type of perfusing solution, since each of these may influence experimental results. The experiments were carried out in the Howe Laboratory of Ophthalmology of Harvard Medical School at the Massachusetts Eye and Ear Infirmary. REFERENCES 1. Grant, W. M.: Tonographic method for measuring the facility and rate of aqueous flow in human eyes, Arch. Ophthal. 44: 204, 1950. 2. Becker, B., and Constant, M.: The facility of aqueous outflow, Arch. Ophthal. 55: 305, 1956. 3. Bill, A., and Barany, E.: Gross facility, facility of conventional routes and pseudo facility of aqueous humor outflow in the Cynomolgus monkey, Arch. Ophthal. 75: 665, 1966.

Volume 10 Number 6 IOP and resistance to aqueous outflow 437 4. Frangois, J., Rabaey, M., Neetens, A., and Evens, L.: Further perfusion studies on the outflow of aqueous humor in human eyes, Arch. Ophthal. 59: 683, 1958. 5. Kleinert, H.: Abflussdruck und Abflusswiderstand, Ber. Dtsch. Ophthal. Ges. 64: 57, 1961. 6. Nihard, P.: Influence de la pression oculaire sur la resistance a l'ecoulement de l'humeur aqueuse, Acta Ophthal. (Kbh.) 40: 12, 1962. 7. Levene, R., and Hyman, B.: The effect of intraocular pressure of the facility of outflow, Exp. Eye Res. 8: 116, 1969. 8. Langham, M.: Manometric, pressure cup and tonographic procedures in the evaluation of intraocular dynamics, in Leydhecker, W., editor: Glaucoma: Tutzing symposium, Basel, 1967, Karger AG. 9. Barany, E. H.: The mode of action of pilocarpine on outflow resistance in the eye of a primate (Cercopithicus ethiops), INVEST. OPHTHAL. 1: 712, 1962. 10. Grant, W. M.: Further studies on facility of flow through trabecular meshwork, Arch. Ophthal. 60: 523, 1958. 11. Barany, E. H.: Simultaneous measurement of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion, INVEST. OPHTHAL. 3: 135, 1964. 12. McEwen, W. K.: Difficulties in measuring ocular pressure and ocular rigidity, in Leydhecker, U., editor: Glaucoma: Tutzing symposium, Basel, 1967, Karger, AG. 13. Shapiro, A. H.: Personal communication, 1970.