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Species Variation in Facility of Aqueous Outflow*
SPECIES VARIATION IN FACILITY O F AQUEOUS O U T F L O W * BERNARD BECKER, M.D.,
AND MARGUERITE A.
CONSTANT, P H . D .
Saint Louis, Missouri
The perfusion of enucleated eyes permits the evaluation of the facility of outflow (C) of aqueous from the anterior chamber of the eye.1,2 This method can be applied to eyes regardless of size, shape, corneal curva ture, or scleral rigidity. When modified for use in the living animal, estimates of intra ocular pressure ( P 0 ) can also be obtained.3 The in vivo data for rabbit and human eyes agree well with the in vitro results as well as with the findings of tonography for the same eyes. A comparison of the perfusion values for the very different sized eyes of cats, guinea pigs, and rabbits revealed strik ing variations in outflow facility. The differ ences observed suggested a relationship be tween facility of outflow and volume of the anterior chamber. The results of studies on outflow facility and its variation with an terior chamber volume and species differ ences are presented in this report. METHOD
Rabbits weighing two to three kg., cats weighing three to four kg., and guinea pigs weighing 600 to 700 gm. were anesthetized with Nembutal or urethane and heparinized. Cannulation and perfusion were carried out in the living animal as well as in the freshly enucleated eyes as previously described.3 Human eyes were obtained at autopsy and were perfused eight to 144 hours after death. At each infusion pressure ( P i ) , the rate of buffered saline* inflow ( I ) was measured * From the Department of Ophthalmology and the Oscar Johnson Institute, Washington University School of Medicine. This investigation was sup ported in part by a research grant, B-621, from the National Institute of Neurological Diseases and Blindness of the National Institutes of Health, Public Health Service. Presented in part at the First Conference on Glaucoma of the Josiah Macy, Jr., Foundation, Princeton, New Jersey, Decem ber 5-7, 1955. 189
until a constant value was obtained. Testicular hyaluronidase (Wydase®) 15 T R U / ml. or bacterial hyaluronidase (Hyason®)* 15 U/ml. were used in some infusions. The in vivo data were analyzed graphically by plotting I, the rate of inflow in cu. mm./min. against Pi, the inflow pressure in mm. Hg. Straight lines were obtained with slope equal to C, the facility of outflow, and which intercepted the pressure axis (zero inflow) at the initial intraocular pressure. (Po). This method of interpretation was based upon the equation: I = C (Pi-Po) (1) where 1= inflow rate (cu. mm./min.) corrected to 37°C. Pi = inflow pressure (mm. Hg) Po = intraocular pressure in the undisturbed eye C = facility of outflow
In the enucleated eye the equation simplified to: I = C x Pi RESULTS
The data obtained from the in situ eyes of living cats indicated an average intra ocular pressure of 22 mm. Hg and facility of outflow of 1.54. The in vitro data for enucleated cat eyes revealed a facility aver aging 1.62 and differed from the in vivo values on the same eyes by an average of only 10 percent (table 1). Similar results for guinea pigs are summarized in Table 2. Here the intraocular pressure averaged 20 mm. Hg with a facility of outflow of 0.08 in vivo and 0.09 in the enucleated eye. In the 18 experiments where data were available' t Isotonic sodium phosphate and potassium dihydrogen phosphate, pH 7.4, diluted 1:10 with iso tonic saline. t The Hyason used in this study was kindly sup plied by Dr. Kenneth W. Thompson of Organon, Inc., Orange, New Jersey.
190
BERNARD BECKER AND MARGUERITE A. CONSTANT TABLE 1
TABLE 2
COMPARISON OF IN VIVO AND IN VITRO ESTIMATES OF FACILITY OF OUTFLOW IN CATS
COMPARISON OF IN VIVO AND IN VITRO ESTIMATES OF FACILITY OF OUTFLOW IN GUINEA PIGS
Vivo
Vitro
Vivo
Vitro
Po
C
C
AC
Po
C
25 27 20 23 25 20 24 24 23 22 20 25 25 20 19 18 15 15
1.20 1.55 1.12 1.25 1.20 1.15 1.10 2.00 1.70 1.45 1.30 0.91 1.40 2.20 2.10 1.88 2.25 2.00
1.14 1.80 1.27
0.06 0.25 0.15
1.40 1.25 1.40
0.20 0.10 0.30
1.68
0.02
0.95 1.44
0.04 0.04
19 15 15 21 18 20 20 25 15 17 24 26 17
0.05 0.17 0.11 0.10 0.11 0.10 0.07 0.08 0.07 0.08 0.08 0.05 0.07
2.35 1.94 2.30 2.10
0.25 0.06 0.05 0.10
Mean 22 No. Eyes 18
1.54 18
1.62 13
0.13 13
AC=absoIute difference between in vivo and in vitro values for C Po=intraocular pressure (mm.Hg) C = facility of outflow on the same eyes, the in vivo and in vitro values for facility differed by only some 15 percent. Data obtained previously 3 for rab bits revealed an average pressure of 19 mm. H g with facility of 0.35. Representative eyes of each species in vivo are presented graphi cally in Figure 1. T h e mean data for all three species are summarized in Table 3. It was apparent at once that although the eyes of all three species had similar average intraocular pressures, there were enormous (almost 20-fold) differences in facility of outflow. This comparison could be carried one step further by computing the species differences in rate of aqueous secretion.* Mean values for cats, rabbits, guinea pigs, and humans are summarized in Table 4. It is evident that * Flow (F) in cu. mm./min. was calculated from the equation: F = C (Po-Pv) (2) where Pv is the episcleral venous pressure; Po is the intraocular pressure; and C = facility of out flow. Assuming a mean value of 10 mm. Hg for
24 15 23 25 22 19 19 20 19 20 20 18 22 17 20 22 20 17 19 17 20
0.09 0.14 0.06 0.05 0.05 0.07 0.07 0.04 0.08 0.09 0.06 0.09 0.08 0.06 0.05 0.10 0.06 0.10 0.06 0.05 0.09
Mean 20 No. Eyes 34
0.08 34
C
AC
0.15
0.02
0.11
0.01
0.13
0.03
0.10
0.02
0.09 0.05
0.01 0.0
— —
0.08 0.08 0.12
0.03
0.06 0.06
0.0 0.01
0.08 0.07
0.01 0.03
0.09
0.0
0.10 0.08 0.08 0.08 0.09
0.01 0.0 0.02 0.03 0.01
0.11
0.01
0.09 20
0.014 18
AC=absolute difference between in vivo and in vitro values for C Po = intraocular pressure (mm. Hg) C = facility of outflow F, the rate of secretion of aqueous, varied enormously in these species from an average of only 0.8 cu. mm./min. for the guinea pig to 18.5 cu. mm./min. in the cat eye. However, when flow was expressed as perPv, flow rates were determined from the data in Table 3 or estimated graphically from the inflow vs. pressure plots (the flow value at a pressure of 10 mm. H g ) . Average tonographic values for intraocular pressure and facility of outflow in a series of 175 normal human eyes were included for comparison.
191
FACILITY OF AQUEOUS OUTFLOW 12
TABLE 4 Cannulation in vivo
SPECIES VARIATION
X = cat B - rabbit
10
0 = guinea pig
*
o
?4 C=0. 09
10
'Zu' 30 40 ^ - Inflow pressure ( mm. Hg. )
50
Fig. 1 (Becker and Constant). Graphic represen tation of species variation in facility of outflow (C) and intraocular pressure.
cent of the volume of the anterior chamber per minute ( K f ) , it became apparent that all four species had very similar values for the flow constant K t (1.3 to 1.6 percent). That the values for K f were similar for the eyes of different species was noted by Barany. 4 Using the para-amino hippuric acid method for measuring the flow constant K f , he reported values of 1.4 to 1.7 percent for guinea pigs, 1.4 percent for cats, and 1.1 to 1.3 percent for American rabbits. Further more, Goldmann5 found a value of 1.3 per cent for human eyes from turnover studies with fluorescein. The agreement of estimates of aqueous flow obtained by such different TABLE 3 PERFUSION MEASUREMENTS
Animal Guinea pig Rabbit Cat
C
F
20 19 22 17»
0.08 0.3S 1.54 0.31»
0.8 3.2 18.5 2.2
V
Kf
0.050 1.6% 0.200 1.6% 1.200 1.5% 0.175 b 1.3%
Po = intraocular pressure (mm. Hg). C = facility of outflow. F=aqueous flow (cu.mm./min.), assuming episceral venous pressure 10 mm. Hg. V = volume anterior chamber (ml.) (estimated from volume obtained from anterior chamber puncture). p — = flow constant (percent of anterior Kf =
C=0. 33
0
Guinea pig Rabbit Cat Man
Po
SPECIES VARIATION
No Eyes
Po
^vivo
c.
34 53 18
20 19 22
0.08 0.35 1.54
0.09 0.37 1.62
^-vitro
Po = intraocular pressure (mm. Hg). Cvivo = facility of outflow in vivo. Cvitro = facility of outflow in enucleated eye.
chamber per minute) a = Mean tonographic values for 175 normal human eyes. b = Value for volume of anterior chamber of hu man eyes is average of Goldmann's measure ments.6
approaches as turnover studies, tonography, and cannulation perfusion, each with its own errors and assumptions, is supportive evi dence for the validity of the methods of measurement. The similarity of average values for intra ocular pressure and the coefficient of aqueous flow, Kf, for different species indicated a direct correlation of facility of outflow with anterior chamber volume.* From Table 4 it is apparent that in the four species con sidered in spite of the large differences in facility of outflow (C) and anterior chamber volume ( V ) , the average values for C/V all fell between 1.3 and 1.8. These findings raised entertaining specu lations as to the stability of the parameter Kf in an individual eye. The question of the * If the volume of the anterior chamber (V) is expressed in ml., from equation (2) it is apparent that: F (P0_PV) C K, = = — (3) 10 V 10 V Thus, if Kf were to remain constant in a given eye without producing alterations in intraocular pressure (Po) or episcleral venous pressure (Pv), the fa cility of outflow (C) must vary directly with the volume of the anterior chamber (V) or C — = constant (4) V
192
BERNARD BECKER AND MARGUERITE A. CONSTANT
constancy of C/V for any one eye was, therefore, subjected to experimental investi gation. This relationship could be approached most readily by perfusion of the enucleated eye. Under these conditions, the volume of the anterior chamber could be varied at will by moving the iris lens diaphragm forward or backward. This was accomplished by the introduction of a measured amount of saline into the vitreous cavity as described by Purnell 6 or by a clamp about the sclera posteriorly. The alteration in anterior cham ber volume was measured readily by the rapid change in the site of the meniscus in the anterior chamber pipet. In Figure 2 are plotted the variations of facility of outflow (C) with alterations in volume of the anterior chamber in two representative enucleated human eyes. It is readily seen that reasonably straight line plots were obtained for each eye with slopes (C/V) equal to 1.2 and 1.5 respectively. In Table 5 the estimates obtained by such facility vs. volume plots for 32 enucleated human eyes are summarized. Values for C/V varied from 0.9 to 2.5 in this series of eyes, averaging 1.5. This was in reason able agreement with the in vivo value of 1.8 0.5B VARIATION O F OUTFLOW FACILITY WITH ANTERIOR CHAMBER VOLUME Human e y e s :
0
x R. W. 380* fclR. R 5]rf"
0.1 0.2 0.3 0.4 Volume r e m o v e d from A. C. ( m l . )
Fig. 2 (Becker and Constant). Variation of fa cility of outflow with alterations in volume of an terior chamber in two representative enucleated eyes.
TABLE 5 THE RATIO* C/V
Patient
IN 32 ENUCLEATED HUMAN EYES
Age & Sex
O.D. A.P.
89 M 45 M
W.P.
49 M
R.W.
38 M
J.R. E.S. S.S.
51 F 44 M 64 F
G.W.
42 F
j.n.
63 M
A.M.
79 F
J.F.
87 M
W.H. K.P.
39 F 60 F
V.H.
58 M
E.H. W.C.
68 F 70 M
V.H.
58 F
K.P.
7 F
R.R.
51 M
Average (32 eyes)
Time (hr.)t
9
C/V 1.0
44 46 50 52 25 28
1.3 1.2 1.1 1.2 1.2 1.3
5 8 22 70 20 72 20 20 44 44
1.6 1.8 1.4 1.4 1.7 1.6 1.8 1.9 1.2 1.4
43 70
66
1.9 1.4
1.3
8 144 70 70
1.1 1.1 2.1 2.0
40 40 40 40 140 140
2.2 2.5 1.8 2.0 1.0 0.9
76
30
1.7
1.5 1.5
* Ratio C/V determined from slope of straight line plot of variation of facility of outflow (C) with volume of anterior chamber in ml. (V) in vitro. t Approximate time after death.
calculated from average tonography and estimates of mean anterior chamber volume of normal eyes.5 Alterations in outflow re sistance with variations of depth of the anterior chamber in enucleated human eyes have been reported recently by Francois.7 Although the data of these workers cannot be quantitated as to variation of resistance with changes in volume of the anterior cham ber, they do appear to agree qualitatively with the above findings. Similar studies of the variation with anterior chamber volume of the outflow facility in the perfused rabbit eye were carried out. The same linear relationship of C and V was readily demonstrated. Barany and Woodin 8 had reported alterations in out-
FACILITY OF AQUEOUS OUTFLOW flow facility following perfusion with hyaluronidase. The question arose as to the inter relationships of the hyaluronidase and vol ume induced alterations in facility of out flow. In Figure 3 are presented a selected facility vs. volume plot in a rabbit eye per fused with buffered saline without and then with the addition of hyaluronidase 15 TRU/ml. It is apparent that linear relations of C and V were obtained before and after hyaluronidase. Of particular importance was the finding that the hyaluronidase effect on facility was not the result of change in anterior chamber volume, but a true in crease in the ratio C/V. Data recorded in Table 6 revealed an average value for C/V of 1.7 for 17 enucleated rabbit eyes and 2.4 for eight of these after hyaluronidase. Thus, at least two independent mechanisms for al teration in outflow facility appear to be avail able in enucleated rabbit eyes—the reversible change with anterior chamber volume and the permanent effects of hyaluronidase. These findings on enucleated eyes are of 0.6
VARIATION OF OUTFLOW FACILITY WITH ANTERIOR CHAMBER VOLUME Rabbit #412 X before hyaluronidase ■ after hyaluronidaae
C/y. = 2. 1
0
0. 1 0.2 " 0. 3 0.4 Volume removed from A. C. (ml.)
Fig. 3 (Becker and Constant). The effects of hyaluronidase upon the variation of outflow facility ( C ) with anterior chamber volume ( V ) in a single rabbit eye.
193
TABLE 6 T H E RATIO C / V IN 17 ENUCLEATED RABBIT EYES AND EFFECT OF HYALURONIDASE
Before Hyaluronidase C/V
After Hyaluronidase C/V
0.8 0.9 1.8 1.5 1.6 1.2 1.0 1.0 1.8 2.1 1.7 2.2 2.4 2.6 2.0 2.2 2.5
1.5
Average 1.7 No. Eyes 17
—
2.2
—
2.2
—
2.1
—
3.0
—
2.6
—
2.4
—
3.1
— —
2.4 8
C / V = slope of plot of outflow facility (C) vs. an terior chamber volume in ml. (V).
considerable theoretical interest for they suggest the possibility that in the normal living eye alterations in anterior chamber depth or volume may also be associated with changes in outflow facility. The role of this potential factor in some of the induced and spontaneous changes in facility observed in vivo deserves exploration. If these facility modifications occurred at constant levels of intraocular pressure and in similar fashion to those in enucleated eyes, this would offer a mechanism for maintaining a relatively constant coefficient of flow, K f . Thus, the rate of aqueous secretion expressed in per cent of the volume of the anterior chamber may well remain reasonably constant not only among different species, but for the same eye under different conditions. This would provide a unique method for main taining a uniform concentration rather than total amount of a constituent of the aqueous humor. The clinical significance of such speculation remains obscure, however. Tonographic data do not indicate any gross correlations in patient populations of facility of outflow and volume of the anterior cham-
194
BERNARD BECKER AND MARGUERITE A. CONSTANT
ber. The large anterior chamber of aphakic eyes as well as the very small volume of anterior chamber in some "narrow angle" eyes do not appear to be associated with significant alterations in outflow facility un less synechiae are present. However, Fran cois9 has recently noted in one human eye increasing values of C associated with deepening of the anterior chamber in vivo. It is clear that more data are needed as to the relationship of facility of outflow and volume of the anterior chamber in the living eye. In the enucleated eye, the mechanism whereby outflow facility is altered with changes in anterior chamber volume should prove a fruitful field for investigation. SUMMARY
1. Perfusion measurements of facility of outflow (C) in vivo reveal average values for the cat of 1.54, the rabbit 0.35, and the
guinea pig 0.08 in spite of very similar values for intraocular pressure in all three species. 2. In vivo and in vitro values for C on the same eyes differ by only some 10 to 15 percent. 3. Although absolute rates of aqueous flow vary greatly in the eyes of the guinea pig (0.8 cu. mm./min.), the rabbit (3.2 cu. mm./min.), man (2.2 cu. mm./min.), and the cat (18.5 cu. mm./min.), the rate of secretion of aqueous expressed as percent of the volume of the anterior chamber is re markably constant at 1.3 to 1.6 percent for all four species. 4. Facility of outflow varies in linear fashion with the volume of the anterior chamber not only among different species, but even more precisely in the individual enucleated human or animal eye. 640 South Kingshighway (10).
REFERENCES
1. Barany, E. H., and Scotchbrook, S.: Influence of testicular hyaluronidase on the resistance to flow through the angle of the anterior chamber. Acta physiol. scandinav., 30:240, 1954. 2. Grant, W. M., and Trotter, R. R.: Tonographic measurements in enucleated eyes. Arch. Ophth., 53:191,1955. 3. Becker, B., and Constant, M. A.: The facility of outflow. Arch. Ophth., 55 :305, 1956. 4. Barany, E. H.: Modern Trends in Ophthalmology by A. Sorsby. New York, Hoeber, 1955, pp. 13-20. 5. Goldmann, H.: Abflussdruck, minutenvolumen und widerstand der kammerwasserstromung des menschen. Documenta Ophthalmologica, 5-6:278, 1951. 6. Purnell, E. W., Melton, C. E., Jr., and Adams, E. Q.: Measurements of aqueous outflow in enucleated eyes. Presented at the East Central Section of the Association for Research in Ophthalmology, Toronto, January 9, 1955. 7. Frangois, J., Rabaey, M., and Neetens, A.: Perfusion studies on the outflow of aqueous humor in human eyes. Arch. Ophth., 55:193, 1956. 8. Barany, E., and Woodin, A. M.: Hyaluronic acid and hyaluronidase in the aqueous humor and the angle of the anterior chamber. Acta physiol. scandinav., 33:257, 1955. 9. Francois, J., and Neetens, A.: Hypertension oculaire et zona ophtalmique. Acta Ophthalmologica, 34:35, 1956. DISCUSSION DR. ELMER J. BALLINTINE (Cleveland) : The au
thors sent me a copy of this paper over a month before this meeting so that we had plenty of time to study it and actually repeat some of the experi ments. The authors are modest for the experiments are more difficult than they appear to be from the description in the text. However, we were able to confirm, in general, the results. The authors emphasize the similarity of "C" when measured by perfusion in vivo and after enucleation. It should also be emphasized that while
the "C's" are similar, the values measured in vitro are consistently higher than in vivo by about seven to 12 percent, depending on the species. The reason for this discrepancy requires further investigation. In our experiments on freshly enucleated eyes, the linear relationship between "C" and volume of the anterior chamber were studied only after the perfusion had been continued for about one-half hour. In the first half-hour the value of "C" was erratic, and lower than the values obtained sub sequently. The reasons for this initial low "C" are not apparent.
AND MARGUERITE A.
CONSTANT, P H . D .
Saint Louis, Missouri
The perfusion of enucleated eyes permits the evaluation of the facility of outflow (C) of aqueous from the anterior chamber of the eye.1,2 This method can be applied to eyes regardless of size, shape, corneal curva ture, or scleral rigidity. When modified for use in the living animal, estimates of intra ocular pressure ( P 0 ) can also be obtained.3 The in vivo data for rabbit and human eyes agree well with the in vitro results as well as with the findings of tonography for the same eyes. A comparison of the perfusion values for the very different sized eyes of cats, guinea pigs, and rabbits revealed strik ing variations in outflow facility. The differ ences observed suggested a relationship be tween facility of outflow and volume of the anterior chamber. The results of studies on outflow facility and its variation with an terior chamber volume and species differ ences are presented in this report. METHOD
Rabbits weighing two to three kg., cats weighing three to four kg., and guinea pigs weighing 600 to 700 gm. were anesthetized with Nembutal or urethane and heparinized. Cannulation and perfusion were carried out in the living animal as well as in the freshly enucleated eyes as previously described.3 Human eyes were obtained at autopsy and were perfused eight to 144 hours after death. At each infusion pressure ( P i ) , the rate of buffered saline* inflow ( I ) was measured * From the Department of Ophthalmology and the Oscar Johnson Institute, Washington University School of Medicine. This investigation was sup ported in part by a research grant, B-621, from the National Institute of Neurological Diseases and Blindness of the National Institutes of Health, Public Health Service. Presented in part at the First Conference on Glaucoma of the Josiah Macy, Jr., Foundation, Princeton, New Jersey, Decem ber 5-7, 1955. 189
until a constant value was obtained. Testicular hyaluronidase (Wydase®) 15 T R U / ml. or bacterial hyaluronidase (Hyason®)* 15 U/ml. were used in some infusions. The in vivo data were analyzed graphically by plotting I, the rate of inflow in cu. mm./min. against Pi, the inflow pressure in mm. Hg. Straight lines were obtained with slope equal to C, the facility of outflow, and which intercepted the pressure axis (zero inflow) at the initial intraocular pressure. (Po). This method of interpretation was based upon the equation: I = C (Pi-Po) (1) where 1= inflow rate (cu. mm./min.) corrected to 37°C. Pi = inflow pressure (mm. Hg) Po = intraocular pressure in the undisturbed eye C = facility of outflow
In the enucleated eye the equation simplified to: I = C x Pi RESULTS
The data obtained from the in situ eyes of living cats indicated an average intra ocular pressure of 22 mm. Hg and facility of outflow of 1.54. The in vitro data for enucleated cat eyes revealed a facility aver aging 1.62 and differed from the in vivo values on the same eyes by an average of only 10 percent (table 1). Similar results for guinea pigs are summarized in Table 2. Here the intraocular pressure averaged 20 mm. Hg with a facility of outflow of 0.08 in vivo and 0.09 in the enucleated eye. In the 18 experiments where data were available' t Isotonic sodium phosphate and potassium dihydrogen phosphate, pH 7.4, diluted 1:10 with iso tonic saline. t The Hyason used in this study was kindly sup plied by Dr. Kenneth W. Thompson of Organon, Inc., Orange, New Jersey.
190
BERNARD BECKER AND MARGUERITE A. CONSTANT TABLE 1
TABLE 2
COMPARISON OF IN VIVO AND IN VITRO ESTIMATES OF FACILITY OF OUTFLOW IN CATS
COMPARISON OF IN VIVO AND IN VITRO ESTIMATES OF FACILITY OF OUTFLOW IN GUINEA PIGS
Vivo
Vitro
Vivo
Vitro
Po
C
C
AC
Po
C
25 27 20 23 25 20 24 24 23 22 20 25 25 20 19 18 15 15
1.20 1.55 1.12 1.25 1.20 1.15 1.10 2.00 1.70 1.45 1.30 0.91 1.40 2.20 2.10 1.88 2.25 2.00
1.14 1.80 1.27
0.06 0.25 0.15
1.40 1.25 1.40
0.20 0.10 0.30
1.68
0.02
0.95 1.44
0.04 0.04
19 15 15 21 18 20 20 25 15 17 24 26 17
0.05 0.17 0.11 0.10 0.11 0.10 0.07 0.08 0.07 0.08 0.08 0.05 0.07
2.35 1.94 2.30 2.10
0.25 0.06 0.05 0.10
Mean 22 No. Eyes 18
1.54 18
1.62 13
0.13 13
AC=absoIute difference between in vivo and in vitro values for C Po=intraocular pressure (mm.Hg) C = facility of outflow on the same eyes, the in vivo and in vitro values for facility differed by only some 15 percent. Data obtained previously 3 for rab bits revealed an average pressure of 19 mm. H g with facility of 0.35. Representative eyes of each species in vivo are presented graphi cally in Figure 1. T h e mean data for all three species are summarized in Table 3. It was apparent at once that although the eyes of all three species had similar average intraocular pressures, there were enormous (almost 20-fold) differences in facility of outflow. This comparison could be carried one step further by computing the species differences in rate of aqueous secretion.* Mean values for cats, rabbits, guinea pigs, and humans are summarized in Table 4. It is evident that * Flow (F) in cu. mm./min. was calculated from the equation: F = C (Po-Pv) (2) where Pv is the episcleral venous pressure; Po is the intraocular pressure; and C = facility of out flow. Assuming a mean value of 10 mm. Hg for
24 15 23 25 22 19 19 20 19 20 20 18 22 17 20 22 20 17 19 17 20
0.09 0.14 0.06 0.05 0.05 0.07 0.07 0.04 0.08 0.09 0.06 0.09 0.08 0.06 0.05 0.10 0.06 0.10 0.06 0.05 0.09
Mean 20 No. Eyes 34
0.08 34
C
AC
0.15
0.02
0.11
0.01
0.13
0.03
0.10
0.02
0.09 0.05
0.01 0.0
— —
0.08 0.08 0.12
0.03
0.06 0.06
0.0 0.01
0.08 0.07
0.01 0.03
0.09
0.0
0.10 0.08 0.08 0.08 0.09
0.01 0.0 0.02 0.03 0.01
0.11
0.01
0.09 20
0.014 18
AC=absolute difference between in vivo and in vitro values for C Po = intraocular pressure (mm. Hg) C = facility of outflow F, the rate of secretion of aqueous, varied enormously in these species from an average of only 0.8 cu. mm./min. for the guinea pig to 18.5 cu. mm./min. in the cat eye. However, when flow was expressed as perPv, flow rates were determined from the data in Table 3 or estimated graphically from the inflow vs. pressure plots (the flow value at a pressure of 10 mm. H g ) . Average tonographic values for intraocular pressure and facility of outflow in a series of 175 normal human eyes were included for comparison.
191
FACILITY OF AQUEOUS OUTFLOW 12
TABLE 4 Cannulation in vivo
SPECIES VARIATION
X = cat B - rabbit
10
0 = guinea pig
*
o
?4 C=0. 09
10
'Zu' 30 40 ^ - Inflow pressure ( mm. Hg. )
50
Fig. 1 (Becker and Constant). Graphic represen tation of species variation in facility of outflow (C) and intraocular pressure.
cent of the volume of the anterior chamber per minute ( K f ) , it became apparent that all four species had very similar values for the flow constant K t (1.3 to 1.6 percent). That the values for K f were similar for the eyes of different species was noted by Barany. 4 Using the para-amino hippuric acid method for measuring the flow constant K f , he reported values of 1.4 to 1.7 percent for guinea pigs, 1.4 percent for cats, and 1.1 to 1.3 percent for American rabbits. Further more, Goldmann5 found a value of 1.3 per cent for human eyes from turnover studies with fluorescein. The agreement of estimates of aqueous flow obtained by such different TABLE 3 PERFUSION MEASUREMENTS
Animal Guinea pig Rabbit Cat
C
F
20 19 22 17»
0.08 0.3S 1.54 0.31»
0.8 3.2 18.5 2.2
V
Kf
0.050 1.6% 0.200 1.6% 1.200 1.5% 0.175 b 1.3%
Po = intraocular pressure (mm. Hg). C = facility of outflow. F=aqueous flow (cu.mm./min.), assuming episceral venous pressure 10 mm. Hg. V = volume anterior chamber (ml.) (estimated from volume obtained from anterior chamber puncture). p — = flow constant (percent of anterior Kf =
C=0. 33
0
Guinea pig Rabbit Cat Man
Po
SPECIES VARIATION
No Eyes
Po
^vivo
c.
34 53 18
20 19 22
0.08 0.35 1.54
0.09 0.37 1.62
^-vitro
Po = intraocular pressure (mm. Hg). Cvivo = facility of outflow in vivo. Cvitro = facility of outflow in enucleated eye.
chamber per minute) a = Mean tonographic values for 175 normal human eyes. b = Value for volume of anterior chamber of hu man eyes is average of Goldmann's measure ments.6
approaches as turnover studies, tonography, and cannulation perfusion, each with its own errors and assumptions, is supportive evi dence for the validity of the methods of measurement. The similarity of average values for intra ocular pressure and the coefficient of aqueous flow, Kf, for different species indicated a direct correlation of facility of outflow with anterior chamber volume.* From Table 4 it is apparent that in the four species con sidered in spite of the large differences in facility of outflow (C) and anterior chamber volume ( V ) , the average values for C/V all fell between 1.3 and 1.8. These findings raised entertaining specu lations as to the stability of the parameter Kf in an individual eye. The question of the * If the volume of the anterior chamber (V) is expressed in ml., from equation (2) it is apparent that: F (P0_PV) C K, = = — (3) 10 V 10 V Thus, if Kf were to remain constant in a given eye without producing alterations in intraocular pressure (Po) or episcleral venous pressure (Pv), the fa cility of outflow (C) must vary directly with the volume of the anterior chamber (V) or C — = constant (4) V
192
BERNARD BECKER AND MARGUERITE A. CONSTANT
constancy of C/V for any one eye was, therefore, subjected to experimental investi gation. This relationship could be approached most readily by perfusion of the enucleated eye. Under these conditions, the volume of the anterior chamber could be varied at will by moving the iris lens diaphragm forward or backward. This was accomplished by the introduction of a measured amount of saline into the vitreous cavity as described by Purnell 6 or by a clamp about the sclera posteriorly. The alteration in anterior cham ber volume was measured readily by the rapid change in the site of the meniscus in the anterior chamber pipet. In Figure 2 are plotted the variations of facility of outflow (C) with alterations in volume of the anterior chamber in two representative enucleated human eyes. It is readily seen that reasonably straight line plots were obtained for each eye with slopes (C/V) equal to 1.2 and 1.5 respectively. In Table 5 the estimates obtained by such facility vs. volume plots for 32 enucleated human eyes are summarized. Values for C/V varied from 0.9 to 2.5 in this series of eyes, averaging 1.5. This was in reason able agreement with the in vivo value of 1.8 0.5B VARIATION O F OUTFLOW FACILITY WITH ANTERIOR CHAMBER VOLUME Human e y e s :
0
x R. W. 380* fclR. R 5]rf"
0.1 0.2 0.3 0.4 Volume r e m o v e d from A. C. ( m l . )
Fig. 2 (Becker and Constant). Variation of fa cility of outflow with alterations in volume of an terior chamber in two representative enucleated eyes.
TABLE 5 THE RATIO* C/V
Patient
IN 32 ENUCLEATED HUMAN EYES
Age & Sex
O.D. A.P.
89 M 45 M
W.P.
49 M
R.W.
38 M
J.R. E.S. S.S.
51 F 44 M 64 F
G.W.
42 F
j.n.
63 M
A.M.
79 F
J.F.
87 M
W.H. K.P.
39 F 60 F
V.H.
58 M
E.H. W.C.
68 F 70 M
V.H.
58 F
K.P.
7 F
R.R.
51 M
Average (32 eyes)
Time (hr.)t
9
C/V 1.0
44 46 50 52 25 28
1.3 1.2 1.1 1.2 1.2 1.3
5 8 22 70 20 72 20 20 44 44
1.6 1.8 1.4 1.4 1.7 1.6 1.8 1.9 1.2 1.4
43 70
66
1.9 1.4
1.3
8 144 70 70
1.1 1.1 2.1 2.0
40 40 40 40 140 140
2.2 2.5 1.8 2.0 1.0 0.9
76
30
1.7
1.5 1.5
* Ratio C/V determined from slope of straight line plot of variation of facility of outflow (C) with volume of anterior chamber in ml. (V) in vitro. t Approximate time after death.
calculated from average tonography and estimates of mean anterior chamber volume of normal eyes.5 Alterations in outflow re sistance with variations of depth of the anterior chamber in enucleated human eyes have been reported recently by Francois.7 Although the data of these workers cannot be quantitated as to variation of resistance with changes in volume of the anterior cham ber, they do appear to agree qualitatively with the above findings. Similar studies of the variation with anterior chamber volume of the outflow facility in the perfused rabbit eye were carried out. The same linear relationship of C and V was readily demonstrated. Barany and Woodin 8 had reported alterations in out-
FACILITY OF AQUEOUS OUTFLOW flow facility following perfusion with hyaluronidase. The question arose as to the inter relationships of the hyaluronidase and vol ume induced alterations in facility of out flow. In Figure 3 are presented a selected facility vs. volume plot in a rabbit eye per fused with buffered saline without and then with the addition of hyaluronidase 15 TRU/ml. It is apparent that linear relations of C and V were obtained before and after hyaluronidase. Of particular importance was the finding that the hyaluronidase effect on facility was not the result of change in anterior chamber volume, but a true in crease in the ratio C/V. Data recorded in Table 6 revealed an average value for C/V of 1.7 for 17 enucleated rabbit eyes and 2.4 for eight of these after hyaluronidase. Thus, at least two independent mechanisms for al teration in outflow facility appear to be avail able in enucleated rabbit eyes—the reversible change with anterior chamber volume and the permanent effects of hyaluronidase. These findings on enucleated eyes are of 0.6
VARIATION OF OUTFLOW FACILITY WITH ANTERIOR CHAMBER VOLUME Rabbit #412 X before hyaluronidase ■ after hyaluronidaae
C/y. = 2. 1
0
0. 1 0.2 " 0. 3 0.4 Volume removed from A. C. (ml.)
Fig. 3 (Becker and Constant). The effects of hyaluronidase upon the variation of outflow facility ( C ) with anterior chamber volume ( V ) in a single rabbit eye.
193
TABLE 6 T H E RATIO C / V IN 17 ENUCLEATED RABBIT EYES AND EFFECT OF HYALURONIDASE
Before Hyaluronidase C/V
After Hyaluronidase C/V
0.8 0.9 1.8 1.5 1.6 1.2 1.0 1.0 1.8 2.1 1.7 2.2 2.4 2.6 2.0 2.2 2.5
1.5
Average 1.7 No. Eyes 17
—
2.2
—
2.2
—
2.1
—
3.0
—
2.6
—
2.4
—
3.1
— —
2.4 8
C / V = slope of plot of outflow facility (C) vs. an terior chamber volume in ml. (V).
considerable theoretical interest for they suggest the possibility that in the normal living eye alterations in anterior chamber depth or volume may also be associated with changes in outflow facility. The role of this potential factor in some of the induced and spontaneous changes in facility observed in vivo deserves exploration. If these facility modifications occurred at constant levels of intraocular pressure and in similar fashion to those in enucleated eyes, this would offer a mechanism for maintaining a relatively constant coefficient of flow, K f . Thus, the rate of aqueous secretion expressed in per cent of the volume of the anterior chamber may well remain reasonably constant not only among different species, but for the same eye under different conditions. This would provide a unique method for main taining a uniform concentration rather than total amount of a constituent of the aqueous humor. The clinical significance of such speculation remains obscure, however. Tonographic data do not indicate any gross correlations in patient populations of facility of outflow and volume of the anterior cham-
194
BERNARD BECKER AND MARGUERITE A. CONSTANT
ber. The large anterior chamber of aphakic eyes as well as the very small volume of anterior chamber in some "narrow angle" eyes do not appear to be associated with significant alterations in outflow facility un less synechiae are present. However, Fran cois9 has recently noted in one human eye increasing values of C associated with deepening of the anterior chamber in vivo. It is clear that more data are needed as to the relationship of facility of outflow and volume of the anterior chamber in the living eye. In the enucleated eye, the mechanism whereby outflow facility is altered with changes in anterior chamber volume should prove a fruitful field for investigation. SUMMARY
1. Perfusion measurements of facility of outflow (C) in vivo reveal average values for the cat of 1.54, the rabbit 0.35, and the
guinea pig 0.08 in spite of very similar values for intraocular pressure in all three species. 2. In vivo and in vitro values for C on the same eyes differ by only some 10 to 15 percent. 3. Although absolute rates of aqueous flow vary greatly in the eyes of the guinea pig (0.8 cu. mm./min.), the rabbit (3.2 cu. mm./min.), man (2.2 cu. mm./min.), and the cat (18.5 cu. mm./min.), the rate of secretion of aqueous expressed as percent of the volume of the anterior chamber is re markably constant at 1.3 to 1.6 percent for all four species. 4. Facility of outflow varies in linear fashion with the volume of the anterior chamber not only among different species, but even more precisely in the individual enucleated human or animal eye. 640 South Kingshighway (10).
REFERENCES
1. Barany, E. H., and Scotchbrook, S.: Influence of testicular hyaluronidase on the resistance to flow through the angle of the anterior chamber. Acta physiol. scandinav., 30:240, 1954. 2. Grant, W. M., and Trotter, R. R.: Tonographic measurements in enucleated eyes. Arch. Ophth., 53:191,1955. 3. Becker, B., and Constant, M. A.: The facility of outflow. Arch. Ophth., 55 :305, 1956. 4. Barany, E. H.: Modern Trends in Ophthalmology by A. Sorsby. New York, Hoeber, 1955, pp. 13-20. 5. Goldmann, H.: Abflussdruck, minutenvolumen und widerstand der kammerwasserstromung des menschen. Documenta Ophthalmologica, 5-6:278, 1951. 6. Purnell, E. W., Melton, C. E., Jr., and Adams, E. Q.: Measurements of aqueous outflow in enucleated eyes. Presented at the East Central Section of the Association for Research in Ophthalmology, Toronto, January 9, 1955. 7. Frangois, J., Rabaey, M., and Neetens, A.: Perfusion studies on the outflow of aqueous humor in human eyes. Arch. Ophth., 55:193, 1956. 8. Barany, E., and Woodin, A. M.: Hyaluronic acid and hyaluronidase in the aqueous humor and the angle of the anterior chamber. Acta physiol. scandinav., 33:257, 1955. 9. Francois, J., and Neetens, A.: Hypertension oculaire et zona ophtalmique. Acta Ophthalmologica, 34:35, 1956. DISCUSSION DR. ELMER J. BALLINTINE (Cleveland) : The au
thors sent me a copy of this paper over a month before this meeting so that we had plenty of time to study it and actually repeat some of the experi ments. The authors are modest for the experiments are more difficult than they appear to be from the description in the text. However, we were able to confirm, in general, the results. The authors emphasize the similarity of "C" when measured by perfusion in vivo and after enucleation. It should also be emphasized that while
the "C's" are similar, the values measured in vitro are consistently higher than in vivo by about seven to 12 percent, depending on the species. The reason for this discrepancy requires further investigation. In our experiments on freshly enucleated eyes, the linear relationship between "C" and volume of the anterior chamber were studied only after the perfusion had been continued for about one-half hour. In the first half-hour the value of "C" was erratic, and lower than the values obtained sub sequently. The reasons for this initial low "C" are not apparent.