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Drug effects on aqueous humor formation and pseudofacility in sympathectomized rabbit eyes
Eq.
Eye Res. (1982) 34, 14
Drug Effects Pseudofacility KEITH
Departments
on Aqueous Humor Formation in Sympathectomized Rabbit
GREEN*?,
DAVID
of Ophthalmology* Augusta,
ELIJAH*
AND
GAYLE
and Physiology?, Medical Georgia 30912, [J.S.A.
(Received 13 August 1979 and accepted 1 January
and Eyes
LOLLIS*
College ,of Georgia,
1981, New York)
The effects of the adrenergic agonists, norepinephrine, epinephrine, isoproterenol and phenylephrine have been assessed on aqueous humor formation rate (AHFR) and pseudofacility (C,,) in the ganglionectomized rabbit eye. The drug concentrations used were @l o/0 as a topical drop. Norepkephrine increased AHFR by 50 y0 and doubled C,, with no change in intraocular press& (IOPI. EDineDhrine caused a 10 % decrease in AHFR and an increase in C,.. e-. also with little chanw in IdP. Isopioterenol decreased AHFR and increased C,,, with a fall in IOP. Phenylephrine reduced AHFR and C,, was unchanged, yet IOP barely changed. Together with in vitro data which indicated that ciliary epithelial permeability does not increaae after ganglionectomy the data can be used to estimate other effects of the drugs. It is apparent that ganglionectomy alone must cause an increase in the perfused area of ciliary epithelium and that two major effects of the drugs employed are on true outflow facility and on blood flow through the anterior uvea. Key words: adrenergic agonists; aqueous humor formation: pseudofacility; sympathectomy ; rabbit.
1. Introduction Previous studies (Green and Padgett, 1979) have been made on rabbit eyes to investigate the effects of adrenergic drugs on aqueous humor formation rate (AHFR) and pseudofacility (C,,). The data were used in conjunction with determination of the effect of the drugs on the passive fluid permeability (LpA) of the isolated ciliary epithelium (Green and Griffin, 1978) to allow an estimation of direction of change of the pressure index of the capillaries. Previous results suggested that the hemodynamic effects of the drugs were relatively small compared to the effects on total outflow facility (Green and Padgett, 1979). The experiments were performed on normal eyes, however, with an intact adrenergic innervation, yet the eye is known to become supersensitive to adrenergic agents following superior cervical ganglionectomy (Eakins and Eakins, 1964; Eakins and Ryan, 1964). The present studies, therefore, represent an examination of the effects of adrenergic agonists on aqueous dynamics in ganglionectomized eyes, and on the values of Lp in ciliary epithelium from ganglionectomized eyes.
2. Materials
and Methods
Adult albino rabbits (2-3 kg) were used unless otherwise indicated. While some animals were normal others were ganglionectomized. Rabbits were anesthetized with urethane (25 yc w/v in @9 O/c heparinized NaCl) or a combination of xylazine and ketamine and one superior
cervical ganglion was removed (usually the right) in a manner described previously (Green 1976). At least 10 days after surgery, supersensitivity to adrenergic agonists was tested by the topical administration of a 50 ~1 drop of @1 o/0 norepinephrine. All animals showing a mydriasis in the operated eye were kept for at least three further days prior to their experimental use.
and Kim,
Address for correspondence: Keith Green, Ph.D., Department Georgia, Augusta, Georgia 30912, U.S.A. 0014/4835/82/010001+06$01.00/0 1
of Ophthalmology,
Medical College of
0 1982 Academic Press Inc. (London) Limited EBH34
control 6 6 5 5 6 6 6
n 277+064 446 f 1.32 2%X*651 217&O-80 166+@32 296+@72 2.69fO-89
Aqueous humor formation W/mm)
-
< 04x n.8. n.8. < O-05
P + 1.91 -632 -021 - 1.03
Change in aqueous humor flow (@/mm) 0~10+@011 02.5 * 0022 018_+0912 - @18*0011 067+0906 o-07 @09
Pseudofacility &l/min/mmHg)
in the ganglionectomized
< 0.01 < 0.01 < 0.01 n.8.
P
eye
1&17+@97 19~08+_0%4 1867fOM 16+)0+063 17+0+093
Steady state IOP (mmHg)
= = = 1
z,
Number of eyes, n. Values are the mean + S.E.M. Steady state IOP is that at the time of measurement of AHFR and C,,. The P-values compare each treatment with the control values. For explantion of corrected change in AHFR, see text. The direction of change of zc (calculated as xc = C,,/LpA) is also given.
Ganglionectomised Norepinephrine Epinephrine Isoproterenol Phenylephrine Normal control I Normal control II
Drug
I
Drug effects on aqueous humor dynamics
TABLE
GANGLIONECTOMY
AND
DRUG
EFFECTS
3
In vivo. Rabbits were anesthetized with urethane and the anterior chamber cannulated with 22-gauge needles as described previously (Green and Padgett, 1979; Green and Elijah, 1981). The inulin dilution technique of Macri (1967) was used to determine AHFR and Cp, (Green and Padgett, 1979; Green and Elijah, 1981). Inulin samples were counted using a Nalgene liquid scintillation system. Drugs, and their concentrations, used in ganglionectomized eyes were as follows: L-norepinephrine (L-arterenol), 50 pl of a @I y!, solution applied 60 min prior to sampling; L-epinephrine, 50 yl of a 6 1 y0 solution applied 60 min prior to sampling ; isoproterenol, 50 ~1 of a 61 o/0solution applied 30 min before sampling; L-phenylephrine, 50 ~1 of a 61 0/0solution applied 30 min before sampling; if the pupil began to lose dilation an extra drop of phenylephrine was placed on the eye between the 2nd and 3rd sample. The AHFR was subjected to a correction factor to take into account the change in IOP between the experimental and control eyes. This factor (C,, x change in IOP) takes into account the change in pressure gradients and their effect on ultrafiltration (Kupfer. Gaasterland and Ross, 1971) thus providing the real change in AHFR. In vitro. The ciliary body-iris was removed from animals and mounted between waterjacketed chambers at 37% as previously described (Green and Pederson, 1972; Green, Hensley and Lollis, 1979). Both one normal and the contralateral ganglionectomized tissue were used simultaneously as paired tissues: if paired tissues could not be used, the experiment was discontinued. Fluid permeability (L,) with an applied hydrostatic pressure of 10 mmHg was measured following protocols described previously (Pederson and Green, 1975), following the serial addition of adrenergic agonists (Green and Griffin, 1978; Green, Griffin and Hensley, 1978). Agonist concentrations from 10-l* to 10-e M were serially applied at hourly intervals with determinations of L/p at 30 and 60 min for two one-minute periods.
3. Results In viva. The values for control and treated ganglionectomized previously obtained normal eye control data (Green and Elijah, Table I. TABLE
Drug effects on permeability
eyes, together with 1981), are shown in
II
of isolated ciliury epithelium ganglionectomized eyes
from normal and
Drug
n
C
10-l’ M
io-lo
Norepinephrine
8
1.20 f O-05 1.03 *ON
1.26 f006 1.08 *of6
1.31 &-907 1.25 kO.08
172 *O-o7 1.38 + 046
206 kO.08 1.47 $- 606
1.13 f 004 1.16 * 0.05
1.26 +om 1.22 * 0.05
1.39 +0@6 140 *0@6
144 f907 l-61 + 0.08
1.61 + 007 1.91 +912
N
1.28 + 908 1,29 f 009
1.81 f017 1.37 iOl0
2.25 +026 1.55 +010
2.62 kO.23 1.57 + 0.08
2.92 +919 1.72 + 907
N
1.54 f 007 1.23 +0X%
1.49 + 605 1.26 + 907
1.55 f 0.08 1.31 *@of5
1.55 + 0.07 1.47 + 0.07
160 +907 1.59 f 908
N
8 Epinephrine
9 9
Isoproterenol
6 6
Phenylephrine
8 8
M
lo-’
M
lo--’
M
N Gx
Gx
Gx
Gx
Normal, N; ganglionectomized, Gx; number of tissues, n; control, C; lo-‘*+ 10-O M, agonist concentration. Values are the mean +s.E.M, units are &min/lO mmHg and represent data obtained from paired tissues.
4
K. GREEK ET AL
Superior cervical ganglionectomy had no effect on AHFR, although there was a slight increase in C,, which was not significantly different from normal controls (Table I). Norepinephrine greatly increased AHFR and also approximately doubled C,, as compared to the nontreated ganglionectomized animals. Epinephrine, however. marginally (10%) decreased AHFR and increased C,, compared to t.he controls. Isoproterenol decreased AHFR and increased C,,, although there was a fall in IOP. Phenylephrine caused a marked fall in AHFR but C,, was unaltered and the IOP fell by only a small amount. While changes in IOP were relatively slight, it must be recalled that the concentrations used were only 0.1 Oh, compared to the 8 or 4 y0 concentration used in the normal eye (Green and Padgett, 1979) to elucidate similar changes. The use of higher concentrations in perfused eyes, with two needles inserted into them, frequently led to a breakdown of the blood-aqueous barrier. In vitro. The data on the isolated ciliary body-iris preparation fluid permeability is shown in Table II. It is noticeable that there is no significant differencebetween the L, values from paired normal and ganglionectomized tissues following treatment with the same adrenergic agonists as used in the in vivo experiments. 4. Discussion A key to understanding not only the changes after ganglionectomy, but also after drug treatment is a knowledge of the site of the known supersensitivity to adrenergic drugs after ganglionectomy (Eakins and Eakins, 1964; Eakins and Ryan, 1964). Studies on the response of the isolated ciliary epithelium to adrenergic agents after ganglionectomy have revealed that the effects on these drugs are identical to those found in normal tissues (Table II and Green and Lollis, 1981). The site of the supersensitivity, therefore, does not reside in the ciliary epithelium but elsewhere. The data obtained in the present in vivo studies coupled to the in vitro data suggest that the supersensitivity possibly resides in the vasculature and the outflow pathways. In vitro measurements of LpA have a fixed A, since the latter is the surface area of the ciliary epithelium exposed to bathing solution. The use of the term LpA in the equation x, = C,,/LpA does, therefore, lead to a denominator smaller than in effect in the living eye. Substitution of in vitro LpA values obtained from tissues treated with the same agonists does, however, offer a qualitative estimate of the direction of change of x, (Table I). In addition the values obtained, with the caveats listed above, indicate that the numerical values of x, are much greater for ganglioneetomized eyes than those calculated for normal eyes under the same experimental conditions. Despite the assumptions in the calculation, there is an indication from this information that the perfused area of tissue (control as well as treated) is greater following ganglionectomy. The adrenergic agonist, norepinephrine, released from the synapse is known to influence the tone of vascular muscle (Bhagat, 1979) and without such an influence, such as after superior cervical ganglionectomy, the maintenance of the tone of ocular vessels, normally receiving innervation, would be lost. Under these cirumstances it is likely that the perfused area of the ciliary processes would be increased since the whole vasculature would behave more passively through vessels which could become more dilated. Within 39-60 min after application of these adrenergic agonists blood flow in the anterior uvea is markedly decreased (Morgan, Green and Bowman, 1981) even though IOP is not reduced.
GANGLIONECTOMY
AND
DRUG
EFFECTS
5
Phenylephrine (exclusively an a-agonist) causes a small increase in Lp, and a small reduction in C,,. Isoproterenol (primarily a b-agonist) has a relatively large effect on Lp and C,, is increased. Epinephrine (predominantly a b-agonist) causes an increase in Lp and an increase in Cps; while, norepinephrine (primarily an a-agonist, with some /?-agonist activity) causes a larger increase in Lp and Cps, which suggests that the effects onx, are minimal. It isevident, therefore, that each agonist exerts quantitatively different effects on Lp, and presumably x,, leading to a compound value for C,,. Most drugs caused no alteration x, in the ganglionectomized eye, although phenylephrine reduced x, (with the caveats listed above regarding this calculation). In the normal eye, phenylephrine was also the only drug to show such an effect (Green and Padgett, 1979). The correlation of these drugs with their effect on C,, reflects their conjoint effects on Lp and x, (since C,, = LpAx,). The effects on the vasculature may have a further influence on A which adds another factor to be considered in the total effect of a drug. In addition, increased effects on true outflow facility (trabecular flow) must prevail after drug treatment since the changes in IOP are small and, indeed, since the adrenergic agents are known to increase total outflow facility part of this latter increase may also be due to C,, changes. This must be true in the present experiments
since the increase in LpA (and concurrently C,,) should lead to an increase in IOP. Since there is a fall in IOP, then total outflow facility must be increased. From this, and previous data (Green and Elijah, 1981) it is evident that drug effects within the eye (normal or ganglionectomized) cannot be considered as simple responses of one particular effector site. There are suggestions from both the present studies, and previous data (Langham and Rosenthal, 1966; Cole and Rumble, 1970), that adrenergic agents, particularly a-agonists, may alter blood flow through the anterior uvea. Thus, each individual component of the drug-induced response must be identified and considered as an integral part of the total response. ACKNOWLEDGMENTS Supported in part by Public Health Service Research Grant EY 01329 from the National Eye Institute and in part by the American Health Assistance Foundation Glaucoma Research Fund. We thank Mrs I. Prior and Mn M. Green for their valuable secretarial assistance. Calculations of data were made using a Wang 2209 Computer, made available through a Research to Prevent Blindness, Inc., award. REFERENCES Barany, E. H. (1963). A mathematical formulation of intraocular pressure as dependent upon secretion, ultrafiltration, bulk outflow, and osmotic reabsorption of fluid. Invest. Ophth&wl. 2, 584-W. Bhagat, B. D. (1979). Mode of Action of Autonomic Drugs. Graceway, New York. Cole, D. F. and Rumble, R. (1970). Effects of catecholamines on circulation in the rabbit iris. Exp. Eye Res. 9, 21S32. Eakins, K. E. and Eakins, H. M. T. (1964). Adrenergic mechanisms and the outflow of aqueous humor from the rabbit eye. J. Pharmad 144, 60-5. Eakins, K. E. and Ryan, S. J. (1964). The action of sympathetic amines on the outflow of aqueous humor from the eye. Br. J. Pharmacol. 23, 374-82. Green, K. and Elijah, D. (1981). Drug effects on aqueous humor formation and pseudofacility in the normal rabbit eye. Exp. Eye Re8. 33, 23945. Green, K. and Griffin, C. (1978). Adrenergic effects on the isolated ciliary epithelium. Exp. Eye Res. 27, 14359. Green, K., Griffin, C. and Hensley, A. (1978). Effect of parasympathetic and vasoactive drugs on ciliary epithelium. Exp. Eye Rea. 27, 533-8.
6
K. (:REEN
ET AL
Green, K., Hensley, A. and Lollis, G. (1979). Dopamine stimulation of passive permeability and secretion in the isolated rabbit ciliary epithelium. Exp. Eye Res. 29, 423-7. Green, K. and Kim, K. (1976). Mediation of ocular tetrahydrocannabinol effects by adrenergic nervous system. Exp. Eye Res. 23, 443-8. Green, K. and Lollis, G. (1981). Response of the isolated rabbit ciliary epithelium to adrenergic drugs following superior cervical ganglionectomy. Curr. Eye Res. 1, 217-23. Green, K. and Padgett, D. (1979). Effect of various drugs on pseudofacility and aqueous humor formation in the rabbit eye. Exp. Eye Res. 28, 23946. Green, K. and Pederson, J. E. (1972). Contribution of secretion and filtration to aqueous formation. Am. J. Physiol. 222, 121826. Kupfer, C., Gaasterland, D. E. and Ross, K. (1971). Studies of aqueous dynamics in man. II. Measurements in young normal subjects using acetazolamide and L-epinephrine. Invest. Opthalmol. 10, 523-33. Langham, M. E. and Rosenthal, A. R. (1966). Role of cervical sympathetic nerve in regulating intraocular pressure and circulation. Am. J. Physiol. 210, 786-94. Macri, F. J. (1967). The pressure dependence of aqueous humor formation. Invest. Ophthalmol. 78, 629-33. Morgan, T. R., Green, K. and Bowman, K. (1981). Effects of adrenergic agonists upon regional ocular blood flow in normal and ganglionectomized rabbits. Exp. Eye Res. 32, 691-98. Pederson, J. E. and Green, K. (1973). Aqueous humor dynamics: a mathematical approach to the measurement of facility, pseudofacility, secretion, capillary pressure and xc. Exp. Eye Res. 15, 265-76. Pederson, J. E. and Green, K. (1975). Solute permeability of the normal and prostaglandinstimulated ciliary epithelium and the effect of ultrafiltration on active transport. Exp. Eye Res. 21, 569-80.
Eye Res. (1982) 34, 14
Drug Effects Pseudofacility KEITH
Departments
on Aqueous Humor Formation in Sympathectomized Rabbit
GREEN*?,
DAVID
of Ophthalmology* Augusta,
ELIJAH*
AND
GAYLE
and Physiology?, Medical Georgia 30912, [J.S.A.
(Received 13 August 1979 and accepted 1 January
and Eyes
LOLLIS*
College ,of Georgia,
1981, New York)
The effects of the adrenergic agonists, norepinephrine, epinephrine, isoproterenol and phenylephrine have been assessed on aqueous humor formation rate (AHFR) and pseudofacility (C,,) in the ganglionectomized rabbit eye. The drug concentrations used were @l o/0 as a topical drop. Norepkephrine increased AHFR by 50 y0 and doubled C,, with no change in intraocular press& (IOPI. EDineDhrine caused a 10 % decrease in AHFR and an increase in C,.. e-. also with little chanw in IdP. Isopioterenol decreased AHFR and increased C,,, with a fall in IOP. Phenylephrine reduced AHFR and C,, was unchanged, yet IOP barely changed. Together with in vitro data which indicated that ciliary epithelial permeability does not increaae after ganglionectomy the data can be used to estimate other effects of the drugs. It is apparent that ganglionectomy alone must cause an increase in the perfused area of ciliary epithelium and that two major effects of the drugs employed are on true outflow facility and on blood flow through the anterior uvea. Key words: adrenergic agonists; aqueous humor formation: pseudofacility; sympathectomy ; rabbit.
1. Introduction Previous studies (Green and Padgett, 1979) have been made on rabbit eyes to investigate the effects of adrenergic drugs on aqueous humor formation rate (AHFR) and pseudofacility (C,,). The data were used in conjunction with determination of the effect of the drugs on the passive fluid permeability (LpA) of the isolated ciliary epithelium (Green and Griffin, 1978) to allow an estimation of direction of change of the pressure index of the capillaries. Previous results suggested that the hemodynamic effects of the drugs were relatively small compared to the effects on total outflow facility (Green and Padgett, 1979). The experiments were performed on normal eyes, however, with an intact adrenergic innervation, yet the eye is known to become supersensitive to adrenergic agents following superior cervical ganglionectomy (Eakins and Eakins, 1964; Eakins and Ryan, 1964). The present studies, therefore, represent an examination of the effects of adrenergic agonists on aqueous dynamics in ganglionectomized eyes, and on the values of Lp in ciliary epithelium from ganglionectomized eyes.
2. Materials
and Methods
Adult albino rabbits (2-3 kg) were used unless otherwise indicated. While some animals were normal others were ganglionectomized. Rabbits were anesthetized with urethane (25 yc w/v in @9 O/c heparinized NaCl) or a combination of xylazine and ketamine and one superior
cervical ganglion was removed (usually the right) in a manner described previously (Green 1976). At least 10 days after surgery, supersensitivity to adrenergic agonists was tested by the topical administration of a 50 ~1 drop of @1 o/0 norepinephrine. All animals showing a mydriasis in the operated eye were kept for at least three further days prior to their experimental use.
and Kim,
Address for correspondence: Keith Green, Ph.D., Department Georgia, Augusta, Georgia 30912, U.S.A. 0014/4835/82/010001+06$01.00/0 1
of Ophthalmology,
Medical College of
0 1982 Academic Press Inc. (London) Limited EBH34
control 6 6 5 5 6 6 6
n 277+064 446 f 1.32 2%X*651 217&O-80 166+@32 296+@72 2.69fO-89
Aqueous humor formation W/mm)
-
< 04x n.8. n.8. < O-05
P + 1.91 -632 -021 - 1.03
Change in aqueous humor flow (@/mm) 0~10+@011 02.5 * 0022 018_+0912 - @18*0011 067+0906 o-07 @09
Pseudofacility &l/min/mmHg)
in the ganglionectomized
< 0.01 < 0.01 < 0.01 n.8.
P
eye
1&17+@97 19~08+_0%4 1867fOM 16+)0+063 17+0+093
Steady state IOP (mmHg)
= = = 1
z,
Number of eyes, n. Values are the mean + S.E.M. Steady state IOP is that at the time of measurement of AHFR and C,,. The P-values compare each treatment with the control values. For explantion of corrected change in AHFR, see text. The direction of change of zc (calculated as xc = C,,/LpA) is also given.
Ganglionectomised Norepinephrine Epinephrine Isoproterenol Phenylephrine Normal control I Normal control II
Drug
I
Drug effects on aqueous humor dynamics
TABLE
GANGLIONECTOMY
AND
DRUG
EFFECTS
3
In vivo. Rabbits were anesthetized with urethane and the anterior chamber cannulated with 22-gauge needles as described previously (Green and Padgett, 1979; Green and Elijah, 1981). The inulin dilution technique of Macri (1967) was used to determine AHFR and Cp, (Green and Padgett, 1979; Green and Elijah, 1981). Inulin samples were counted using a Nalgene liquid scintillation system. Drugs, and their concentrations, used in ganglionectomized eyes were as follows: L-norepinephrine (L-arterenol), 50 pl of a @I y!, solution applied 60 min prior to sampling; L-epinephrine, 50 yl of a 6 1 y0 solution applied 60 min prior to sampling ; isoproterenol, 50 ~1 of a 61 o/0solution applied 30 min before sampling; L-phenylephrine, 50 ~1 of a 61 0/0solution applied 30 min before sampling; if the pupil began to lose dilation an extra drop of phenylephrine was placed on the eye between the 2nd and 3rd sample. The AHFR was subjected to a correction factor to take into account the change in IOP between the experimental and control eyes. This factor (C,, x change in IOP) takes into account the change in pressure gradients and their effect on ultrafiltration (Kupfer. Gaasterland and Ross, 1971) thus providing the real change in AHFR. In vitro. The ciliary body-iris was removed from animals and mounted between waterjacketed chambers at 37% as previously described (Green and Pederson, 1972; Green, Hensley and Lollis, 1979). Both one normal and the contralateral ganglionectomized tissue were used simultaneously as paired tissues: if paired tissues could not be used, the experiment was discontinued. Fluid permeability (L,) with an applied hydrostatic pressure of 10 mmHg was measured following protocols described previously (Pederson and Green, 1975), following the serial addition of adrenergic agonists (Green and Griffin, 1978; Green, Griffin and Hensley, 1978). Agonist concentrations from 10-l* to 10-e M were serially applied at hourly intervals with determinations of L/p at 30 and 60 min for two one-minute periods.
3. Results In viva. The values for control and treated ganglionectomized previously obtained normal eye control data (Green and Elijah, Table I. TABLE
Drug effects on permeability
eyes, together with 1981), are shown in
II
of isolated ciliury epithelium ganglionectomized eyes
from normal and
Drug
n
C
10-l’ M
io-lo
Norepinephrine
8
1.20 f O-05 1.03 *ON
1.26 f006 1.08 *of6
1.31 &-907 1.25 kO.08
172 *O-o7 1.38 + 046
206 kO.08 1.47 $- 606
1.13 f 004 1.16 * 0.05
1.26 +om 1.22 * 0.05
1.39 +0@6 140 *0@6
144 f907 l-61 + 0.08
1.61 + 007 1.91 +912
N
1.28 + 908 1,29 f 009
1.81 f017 1.37 iOl0
2.25 +026 1.55 +010
2.62 kO.23 1.57 + 0.08
2.92 +919 1.72 + 907
N
1.54 f 007 1.23 +0X%
1.49 + 605 1.26 + 907
1.55 f 0.08 1.31 *@of5
1.55 + 0.07 1.47 + 0.07
160 +907 1.59 f 908
N
8 Epinephrine
9 9
Isoproterenol
6 6
Phenylephrine
8 8
M
lo-’
M
lo--’
M
N Gx
Gx
Gx
Gx
Normal, N; ganglionectomized, Gx; number of tissues, n; control, C; lo-‘*+ 10-O M, agonist concentration. Values are the mean +s.E.M, units are &min/lO mmHg and represent data obtained from paired tissues.
4
K. GREEK ET AL
Superior cervical ganglionectomy had no effect on AHFR, although there was a slight increase in C,, which was not significantly different from normal controls (Table I). Norepinephrine greatly increased AHFR and also approximately doubled C,, as compared to the nontreated ganglionectomized animals. Epinephrine, however. marginally (10%) decreased AHFR and increased C,, compared to t.he controls. Isoproterenol decreased AHFR and increased C,,, although there was a fall in IOP. Phenylephrine caused a marked fall in AHFR but C,, was unaltered and the IOP fell by only a small amount. While changes in IOP were relatively slight, it must be recalled that the concentrations used were only 0.1 Oh, compared to the 8 or 4 y0 concentration used in the normal eye (Green and Padgett, 1979) to elucidate similar changes. The use of higher concentrations in perfused eyes, with two needles inserted into them, frequently led to a breakdown of the blood-aqueous barrier. In vitro. The data on the isolated ciliary body-iris preparation fluid permeability is shown in Table II. It is noticeable that there is no significant differencebetween the L, values from paired normal and ganglionectomized tissues following treatment with the same adrenergic agonists as used in the in vivo experiments. 4. Discussion A key to understanding not only the changes after ganglionectomy, but also after drug treatment is a knowledge of the site of the known supersensitivity to adrenergic drugs after ganglionectomy (Eakins and Eakins, 1964; Eakins and Ryan, 1964). Studies on the response of the isolated ciliary epithelium to adrenergic agents after ganglionectomy have revealed that the effects on these drugs are identical to those found in normal tissues (Table II and Green and Lollis, 1981). The site of the supersensitivity, therefore, does not reside in the ciliary epithelium but elsewhere. The data obtained in the present in vivo studies coupled to the in vitro data suggest that the supersensitivity possibly resides in the vasculature and the outflow pathways. In vitro measurements of LpA have a fixed A, since the latter is the surface area of the ciliary epithelium exposed to bathing solution. The use of the term LpA in the equation x, = C,,/LpA does, therefore, lead to a denominator smaller than in effect in the living eye. Substitution of in vitro LpA values obtained from tissues treated with the same agonists does, however, offer a qualitative estimate of the direction of change of x, (Table I). In addition the values obtained, with the caveats listed above, indicate that the numerical values of x, are much greater for ganglioneetomized eyes than those calculated for normal eyes under the same experimental conditions. Despite the assumptions in the calculation, there is an indication from this information that the perfused area of tissue (control as well as treated) is greater following ganglionectomy. The adrenergic agonist, norepinephrine, released from the synapse is known to influence the tone of vascular muscle (Bhagat, 1979) and without such an influence, such as after superior cervical ganglionectomy, the maintenance of the tone of ocular vessels, normally receiving innervation, would be lost. Under these cirumstances it is likely that the perfused area of the ciliary processes would be increased since the whole vasculature would behave more passively through vessels which could become more dilated. Within 39-60 min after application of these adrenergic agonists blood flow in the anterior uvea is markedly decreased (Morgan, Green and Bowman, 1981) even though IOP is not reduced.
GANGLIONECTOMY
AND
DRUG
EFFECTS
5
Phenylephrine (exclusively an a-agonist) causes a small increase in Lp, and a small reduction in C,,. Isoproterenol (primarily a b-agonist) has a relatively large effect on Lp and C,, is increased. Epinephrine (predominantly a b-agonist) causes an increase in Lp and an increase in Cps; while, norepinephrine (primarily an a-agonist, with some /?-agonist activity) causes a larger increase in Lp and Cps, which suggests that the effects onx, are minimal. It isevident, therefore, that each agonist exerts quantitatively different effects on Lp, and presumably x,, leading to a compound value for C,,. Most drugs caused no alteration x, in the ganglionectomized eye, although phenylephrine reduced x, (with the caveats listed above regarding this calculation). In the normal eye, phenylephrine was also the only drug to show such an effect (Green and Padgett, 1979). The correlation of these drugs with their effect on C,, reflects their conjoint effects on Lp and x, (since C,, = LpAx,). The effects on the vasculature may have a further influence on A which adds another factor to be considered in the total effect of a drug. In addition, increased effects on true outflow facility (trabecular flow) must prevail after drug treatment since the changes in IOP are small and, indeed, since the adrenergic agents are known to increase total outflow facility part of this latter increase may also be due to C,, changes. This must be true in the present experiments
since the increase in LpA (and concurrently C,,) should lead to an increase in IOP. Since there is a fall in IOP, then total outflow facility must be increased. From this, and previous data (Green and Elijah, 1981) it is evident that drug effects within the eye (normal or ganglionectomized) cannot be considered as simple responses of one particular effector site. There are suggestions from both the present studies, and previous data (Langham and Rosenthal, 1966; Cole and Rumble, 1970), that adrenergic agents, particularly a-agonists, may alter blood flow through the anterior uvea. Thus, each individual component of the drug-induced response must be identified and considered as an integral part of the total response. ACKNOWLEDGMENTS Supported in part by Public Health Service Research Grant EY 01329 from the National Eye Institute and in part by the American Health Assistance Foundation Glaucoma Research Fund. We thank Mrs I. Prior and Mn M. Green for their valuable secretarial assistance. Calculations of data were made using a Wang 2209 Computer, made available through a Research to Prevent Blindness, Inc., award. REFERENCES Barany, E. H. (1963). A mathematical formulation of intraocular pressure as dependent upon secretion, ultrafiltration, bulk outflow, and osmotic reabsorption of fluid. Invest. Ophth&wl. 2, 584-W. Bhagat, B. D. (1979). Mode of Action of Autonomic Drugs. Graceway, New York. Cole, D. F. and Rumble, R. (1970). Effects of catecholamines on circulation in the rabbit iris. Exp. Eye Res. 9, 21S32. Eakins, K. E. and Eakins, H. M. T. (1964). Adrenergic mechanisms and the outflow of aqueous humor from the rabbit eye. J. Pharmad 144, 60-5. Eakins, K. E. and Ryan, S. J. (1964). The action of sympathetic amines on the outflow of aqueous humor from the eye. Br. J. Pharmacol. 23, 374-82. Green, K. and Elijah, D. (1981). Drug effects on aqueous humor formation and pseudofacility in the normal rabbit eye. Exp. Eye Re8. 33, 23945. Green, K. and Griffin, C. (1978). Adrenergic effects on the isolated ciliary epithelium. Exp. Eye Res. 27, 14359. Green, K., Griffin, C. and Hensley, A. (1978). Effect of parasympathetic and vasoactive drugs on ciliary epithelium. Exp. Eye Rea. 27, 533-8.
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Green, K., Hensley, A. and Lollis, G. (1979). Dopamine stimulation of passive permeability and secretion in the isolated rabbit ciliary epithelium. Exp. Eye Res. 29, 423-7. Green, K. and Kim, K. (1976). Mediation of ocular tetrahydrocannabinol effects by adrenergic nervous system. Exp. Eye Res. 23, 443-8. Green, K. and Lollis, G. (1981). Response of the isolated rabbit ciliary epithelium to adrenergic drugs following superior cervical ganglionectomy. Curr. Eye Res. 1, 217-23. Green, K. and Padgett, D. (1979). Effect of various drugs on pseudofacility and aqueous humor formation in the rabbit eye. Exp. Eye Res. 28, 23946. Green, K. and Pederson, J. E. (1972). Contribution of secretion and filtration to aqueous formation. Am. J. Physiol. 222, 121826. Kupfer, C., Gaasterland, D. E. and Ross, K. (1971). Studies of aqueous dynamics in man. II. Measurements in young normal subjects using acetazolamide and L-epinephrine. Invest. Opthalmol. 10, 523-33. Langham, M. E. and Rosenthal, A. R. (1966). Role of cervical sympathetic nerve in regulating intraocular pressure and circulation. Am. J. Physiol. 210, 786-94. Macri, F. J. (1967). The pressure dependence of aqueous humor formation. Invest. Ophthalmol. 78, 629-33. Morgan, T. R., Green, K. and Bowman, K. (1981). Effects of adrenergic agonists upon regional ocular blood flow in normal and ganglionectomized rabbits. Exp. Eye Res. 32, 691-98. Pederson, J. E. and Green, K. (1973). Aqueous humor dynamics: a mathematical approach to the measurement of facility, pseudofacility, secretion, capillary pressure and xc. Exp. Eye Res. 15, 265-76. Pederson, J. E. and Green, K. (1975). Solute permeability of the normal and prostaglandinstimulated ciliary epithelium and the effect of ultrafiltration on active transport. Exp. Eye Res. 21, 569-80.