Download Anterior Segment Chemical Sympathectomy by 6-Hydroxy

Anterior segment chemical sympathectomy
by 6-hydroxy-dopamine
I. Effect on intraocular pressure and facility
of outflow
Monte G. Holland and James L. Minis, III
Histoflnorometric techniques have confirmed that topical ocular application of 6-hydroxydopamine, a norepinephrine congener, causes a selective and reversible destruction of
syynpathetic nerve terminals in the anterior segment. An investigation of the effects of
"chemical sympatheclomy" on the pupil, intraocular pressure, and facility of outflow showed:
the pupil underwent a sequence of changes characteristic of surgical sympathetic denervation, but with a different time course; the intraocular pressure .was significantly lowered,
transiently in rabbits and of longer duration in monkeys; the facility of outflow was
transiently increased in monkeys and probably in rabbits; the episcleral venous pressure was
unchanged in both species. It was concluded that the lowered intraocular pressure and
lowered outflow pressure were the result of a reduction of aqueous inflow. There was no
unequivocal experimental demonstration of supersensitization to topical norepinephrine or
isoproterenol following chemical sympathetic denervation; however, the experiments were
not conclusive on this important point. It was concluded that chemical sympathectomy with
6-hydroxy-dopamine reproduces many of the ocular phenomena of surgical sympathectomy.
6-Hydroxy-dopamine is a useful drug for experimental ophthalmology, and may be useful
clinically.
Key words: chemical sympathectomy, surgical sympathectomy, 6-hydroxy-dopamine,
sympathetic denervation effects, denervation supersensitization, intraocular pressure,
facility of outflow, episcleral venous pressure, norepinephrine.
S,
'everal investigators1"5 have reported
that the norepinephrine congener 6-hydroxy-dopamine, produces sympathectomy
by selective destruction of adrenergic nerve
endings, leaving parasympathetic and other
nerve terminals unaffected.
The possibility of producing ocular sympathectomy by chemical means is interesting and potentially useful. Since Homer's6
early observation of lowered intraocular
pressure as a part of his well-known syndrome, there have been attempts to lower
intraocular pressure in glaucoma patients
From the Department of Ophthalmology, Tulane
University School of Medicine, New Orleans,
La.
Supported in part by National Eye Institute
Research Grant EY-00207-11 and Training
Grant EY-00007-15; National Institutes of
Health Research Grant 1 ROl HE 13616-01;
and Research to Prevent Blindness, Inc.
Manuscript submitted Oct. 6, 1970; revised manuscript accepted Dec. 18, 1970.
Reprint requests: Dr. Monte G. Holland, Department of Ophthalmology, Tulane University
School of Medicine, 1430 Tulane Ave., New
Orleans, La. 70112.
120
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Volume 10
Number 2
Anterior segment chemical sympathectomy
by superior cervical ganglionectomy or
cervical sympathectomy.7'8 However, because of the unpredictable results from
this surgical intervention and its attendant
risks, the procedure has been abandoned.9
More recently there has been a renaissance of interest in the effects of ocular
sympathetic denervation and the mechanisms of action of sympathetic drugs on
aqueous secretion and outflow. Because
6-hydroxy-dopamine has the unique effect
of producing a reversible degeneration of
sympathetic nerve terminals, an investigation of the effects of its topical administration on the intraocular pressure,
facility of outflow, and episcleral venous
pressure of owl monkeys and New Zealand
rabbits was carried out.
Methods
Experimental animals. Both sexes of adult owl
monkeys (Aotus trivirgatus), weighing 0.6 to
0.9 kilograms, and adult New Zealand strain
rabbits, weighing 2.4 to 3.6 kilograms, were used.
Three of the eight rabbits were pigmented.
Anesthesia. Owl monkeys were anesthetized
with 15 to 20 mg. per kilogram of sodium pentobarbital given intramuscularly and supplemented
as necessary by intravenous or intramuscular administration. Other agents were tried and found
to be less satisfactory.
Rabbits were anesthetized with sodium pentobarbital by marginal ear vein.
Drugs. 6-Hydroxy-dopamine* was prepared as
a ten per cent aqueous solution, buffered to
pH 7 with sodium carbonate. Sodium bisulfite
was added as a reducing agent in equimolar
concentration to 6-hydroxy-dopamine in the final
solution.
DL-Isoproterenolf hydrochloride and norepinephrine (L-Arterenol bitartratef) were prepared as
two per cent aqueous solutions.
All drugs were dissolved just prior to their
topical administration.
Techniques of topical administration. As 6-hydrdxy-dopamine is a highly polar compound, it
would be anticipated that its lipid solubility and
comeal penetrance would be low. This was verified experimentally utilizing the pupillary responses to the drug as criteria. To enhance corneal penetration, 0.5 per cent topical proparacaine (one to two drops) (Ophthetic, Allergan
Pharmaceuticals, Irvine, Calif.), was given prior
°Regis Chemical Co., Chicago, 111.
fSagma Chemical Co., St. Louis,' Mo.
121
to tonography in monkeys. Ten per cent 6-hydroxy-dopamine (0.2 ml.) was dropped onto the
cornea after tonography, making use of the slight
epithelial damage to enhance penetration.
In rabbit experiments, where tonography was
not done, 6-hydroxy-dopamine (0.2 ml.) was
applied after six applications of proparacaine
spaced over 5 minute intervals.
It should be noted that the control eyes in
all experiments were treated identically to the
treated eyes, except that the 6-hydroxy-dopamine
was omitted from the solution.
Experimental techniques
Measurement of intraocular pressure. The intraocular pressure was measured by three methods:
electronic Schi0tz tonometry (Crescent Instrument Co., El Monte, Calif.); applanation tonometry (Draeger hand applanation tonometer,
Edward Week & Company, Long Island City,
N. Y.); and by cannulation with the use of
pressure transducers (Statham Instrument Co.,
Oxnard, Calif.). Appropriate technical details are
given below.
Electronic Schi0tz tonometry. The Crescent
electric tonometer was utilized to make intraocular pressure measurements in anesthetized monkeys just prior to tonography. In a separate set
of experiments,10 an open manometric calibration
of this Schi0tz tonometer was done in the cannulated eyes of anesthetized monkeys. The P t
values of the owl monkey eye were sufficiently
close to the 1955 Schi0tz calibration tables11
for human eyes, so that these tables could be
utilized for tonometric and tonographic calculations.
Tonometric measurements were taken with 5.5,
7.5, and 10 gram weights in each experimental
animal on several occasions; the msan value of
ocular rigidity in each animal was used to correct intraocular pressure estimates and facility
of outflow. Average ocular rigidity in the owl
monkey (16 eyes) was 0.0173 (± 0.0015).
Applanation tonometry. The Draeger hand applanation tonometer was modified so that the
doubling prism was set for a 4 mm. diameter
of applanation, as recommended by Schmidt.12
A series of standardization measurements in cannulated monkey eyes yielded a regression line
for a 4 mm. diameter of applanation, which related tonometer readings to manometrically determined intraocular pressure.10
In a series of measurements in 16 eyes of
anesthetized monkeys, it was found that the
Schi0tz pressures averaged 0.77 (±0.47) mm.
Hg higher than the applanation pressures.
Cannulation and pressure transducers. In seven
monkeys the intraocular pressure was measured
in the control and treated eyes by cannulating
the anterior chambers with 25 gauge needles
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Investigative Ophthalmology
February 1971
122 Holland and Mims
connected to Statham, Model P23BB, pressure
transducers.* Schi0tz pressures averaged 1.6
(-1.6) mm. Hg higher than the transducer
pressure, whereas the applanation pressure averaged 1.1 (±2.3) mm. Hg below transducer pressures.10
Applanation tonometry in conscious topically
anesthetized rabbits was attempted. However, in
a large series of measurements the standard
errors were large and the technique gave no
reliable information about the effects of 6-hydroxydopamine on the intraocular pressure. Applanation measurements on conscious rabbits after intraperitoneal water loading had smaller standard
errors and were usable.
Applanation tonometry in the anesthetized rabbit was highly satisfactory and was the method
used in rabbits to determine intraocular pressure.
Facility of outflow. The total facility of outflow was measured by tonography and by perfusion, utilizing the two-pressure perfusion method
of Barany.13
Tonographic facility. Tonography was carried
out on the anesthetized monkey by placing the
animal in a specially constructed box for stabilization. The tonometer was also stabilized upon
the animal's eye by a mechanical support; the
latter made it possible to acquire technically
superb tonograms. The average P t was calculated by averaging the P t 's of the midpoints of
each minute of the 4 minute tonography. Scleral
rigidity was measured in each animal, and the
C values appropriately corrected.
The average radius of corneal curvature was
determined for the experimental group of monkey eyes and was found to be 7.32 mm.
(±0.09). Appropriate corrections in C were introduced in each facility calculation by using this
average value.
Tonography in anesthetized rabbits was not
technically satisfactory in our hands and was not
used in these experiments.
Facility of outflow by perfusion. The twolevel constant pressure perfusion technique described by Barany13 was used to measure total
facility of outflow in ten monkey eyes. The
femoral artery and vein were cannulated to monitor blood pressure and administer anesthesia.
A type SC-II Beckman dynagraph simultaneously
recorded intraocular pressures, systemic blood
pressure, and the weights from hanging reservoirs supplying fluid to each eye (Barany's13
polyelectrolyte fluid). An integrating amplifier
for each hanging reservoir recorded the change
in the fluid weight over the 5 minute intervals
of perfusion. Each eye was perfused six times,
alternating between 5 and 10 mm. Hg above
°The cannulation was accomplished by a needle gun
kindly supplied by M. F. Armaly.
Po (steady-state or baseline intraocular pressure).
After each pair of perfusions, the eyes were
allowed to come to a new equilibrium Po for
5 minutes. This new Po was used for the next
pair of perfusion measurements.
The facility of outflow was calculated from
the perfusion pressure and change in weight of the
reservoirs for each minute. The average of three
different perfusion pairs at 5 and 10 mm. above
Po were used to calculate the facility.
Perfusion facility estimates were also done in
a small series of rabbit eyes. The technical quality of the experiments was poor and are not included in this report.
Episcleral venous pressure. The episcleral
venous pressure was measured in a series of
three treated and three control monkey eyes and
in nine treated and nine control rabbit eyes to
determine whether chemical sympathectomy altered
the episcleral venous pressure. The method was
a modification of that described by Brubaker.14
A pelotte pressure chamber* was connected to
a calibrated Statham pressure transducer and a
500 /A. micrometer syringe. Adjustment of the
micrometer made it possible to change the pressure in the saline-filled system from 0 to 40
mm. Hg. The chamber pressure was continuously
recorded. Frog pericardial membranes were used
initially, but it was found that a thin, highly
elastic, transparent polyurethane membrane f was
superior to the frog pericardial membranes. The
characteristics of this system and experimental
techniques are described in a separate report.10
The episcleral vessel selected in the rabbits
was approximately 2 to 3 mm. from the nasal
limbus. In the owl monkey, because of the
anatomical features of the episcleral vessels, a
point of measurement 1 to 1.5 mm. from the
temporal limbus was selected.
Intraperitoneal water loading. McDonald and
associates15 reported that water loading, by gavage, in the anesthesized rabbit was a sensitive
test for measuring the pressure lowering effects
of drugs. However, intraperitoneal water loading (75 ml. per kilogram) in the conscious
rabbit, proved to be a safer, more reproducible
and more human procedure than the gavage
method.
Standard intravenous equipment was used to
give the water intraperitoneally within a period
of five to seven minutes. Five applanation pressure measurements were taken on each eye
every ten minutes for the following hour. The
pressures were then plotted as a function of time,
and the difference in intraocular pressures (con-
*Supplied through the courtesy of Richard Brubaker.
\ Produced for this purpose by Dr. Elias Klein of Gulf
South Research Institute, Inc., 3525 N. Causeway Blvd.,
Metairie, La.
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Volume 10
Number 2
Anterior segment chemical sympathectomy
trol minus the treated) was integrated over the
entire hour and expressed as average intraocular
pressure difference per minute. This technique
was found to be very sensitive and reliable in
the conscious animals.
Pupils. Due to the varying excitation of the
animals during handling, it was found that quantitatively precise pupillary measurements were
not possible with the equipment available; however, a consistent pattern of pupillary changes
could be seen after repeated handling and observation of the animals.
Experimental protocols and results
The effects of topical application of 6hydroxy-dopamine on the pupils, intraocular pressure, and facility varied with the
time elapsed following treatment. Accordingly, experimental protocols were designed
to take into consideration early and late
effects.
Pupillary changes. The pupillary responses could be grouped into three different types which are illustrated in Fig. 1.
Phase 1 pupils were observed 30 minutes to 3 hours after the first treatment.
The pupil of the treated eye was widely
dilated and did not appear to react to
light. Our interpretations of this Phase 1
pupil were that it probably corresponded
to "denervation contraction" from release
of endogenous norepinephrine resulting
from chemical sympathectomy. It could
not be elicited on repeated treatments of
the animal, unless the animal had been
allowed to recover for at least two full
weeks after the last treatment.
In Phase 2 (3 to 48 hours after treatment), the pupil of the treated side remained miotic at all times. When the
animal was excited or angry, the control
pupil would dilate as indicated in the
figure, whereas the treated pupil would
remain miotic.
Phase 3 pupils occurred as early as
24 hours and lasted as long as five weeks.
The pupil of the treated eye would dilate
slowly and remain dilated when the animal was excited. Under conditions of rest,
however, the pupil appeared to be approximately the same as the control pupil
(whether or not it was slightly miotic
123
TREATED CONTROL
O
REST
ANGER
O
PHASE
I
30MIN 3HR
-
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REST -i
( PHASE TJ
ANGER 3 3HR.-48HR.
O
O
O o
-O
REST
PHASE I E
ANGER
24HR.-5WEEKS
Fig. 1. Pupillary changes following 6-hydroxydopamine vary with time after drug application
and the state of excitation of the animal. Phase
1 pupils probably represent a denervation contraction mydriasis; Phase 2, a typical Homer's
miosis; and Phase 3, a dilation in excitation, possibly from sensitization to circulating catecholamines.
could not be ascertained under the conditions of these observations).
The Phase 2 pupil might be interpreted
as a phase of Horner's miosis and the
Phase 3 pupils, in excitation, might represent a denervation supersensitivity to
circulating endogenous catecholamines.
Phase 1 and 2 pupils could be seen
in rabbits, but Phase 3 pupils were seen
only in monkeys. These changes were
characteristic and appeared in every monkey after sufficient treatment. They were
used as a guideline as to whether sufficient
amounts of the drug had been transferred
through the cornea.
Intraocular pressure following topical appication of 6-hydroxy-dopamine.
Schi0tz tonometry in monkey eyes following topical 6-hydroxy-dopamine. Seven
owl monkeys were subjected to Schi0tz
tonometry and tonography three times to
establish control values for each eye. Before each animal awoke after the tonogram, the eye which had the higher Schi0tz
pressure in the control measurements was
selected for topical treatment. Five ani-
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124 Holland and Minis
|
CONTROL
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Investigative Ophthalmology
February 1971
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Fig. 2. Intraocular pressure (IOP) differences obtained by Schi0tz tonometry expressed as
per cent difference between control and treated eyes show a significant lowering of IOP
lasting approximately two weeks after the last treatment.
mals were treated with 10 per cent 6hydroxy-dopamine, one was treated with
5 per cent, and one with 2V2 per cent.
Because of limitations imposed by repetitive anesthesia, the intraocular pressure
was measured at weekly intervals; therefore, the time of the earliest occurrence of
an effect on the intraocular pressure is
not known precisely.
The intraocular pressure of all control
measurements averaged 14.8 (± 0.5) mm.
HgOne week following treatment, the intraocular pressure measured by Schi0tz tonometry averaged 3.8 mm. Hg or 29.5 per
cent lower in the treated eyes. This is indicated in Fig. 2 at days six through nine.
The statistical significance of this difference, based upon a paired sample t test,
is shown below the bar graph. The effect
on intraocular pressure appeared to be
somewhat greater the following week
when the intraocular pressure difference
was 5.0 mm. Hg or 35.2 per cent lower
in the treated eyes. Two weeks after the
last treatment, the effect was diminished,
although still significant (2.9 mm. Hg or
19.2 per cent lower in the treated eyes).
The intraocular pressure returned to con-
trol levels three weeks after the last treatment and remained at normal levels.
An attempt was made to determine if
the response of the pupil and intraocular
pressure were dose dependent. It was
determined quickly that, because of the
variable penetrance of the drug, the response to 2.5 per cent 6-hydroxy-dopamine
was at least as good as the 10 per cent
dose in several cases, depending upon the
penetration of the drug and its degree
of enhancement by proparacaine and/or
tonography.
Intraocular pressure measurements by
applanation tonometry in monkey eyes follotoing 6-hydroxy-dopamine treatment. The
intraocular pressure in this same group of
animals was measured with the applanation tonometer. Five measurements were
made on the treated and control eyes of
each animal immediately preceding tonography.
Fig. 3 illustrates applanation intraocular
pressure differences taken at the same
time as the Schi0tz measurements. The per
cent differences are somewhat less, but
the statistical significance is the same for
the pressure-lowering effect by either
method.
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Anterior segment chemical sympathectomy 125
Volume 10
Number 2
-
5 -
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TREATMENTS
t
NO TREATMENT
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Fig. 3. Applanation tonometry in the same animals showed a smaller pressure difference,
but a similar time course and statistical significance.
The mean intraocular pressure in the
control eyes by applanation tonometry was
11.6 (±0.7) mm. Hg.
The average pressure differences measured by applanation were 1.4, 1.6, and 1.2
mm. Hg lower in the treated eyes which
correspond to 9.4, 15.2, and 11.6 per cent
lowering of intraocular pressure. The difference between the applanation and
Schi0tz pressures were greater than an
earlier series10; the higher Schi0tz pressures were probably the result of errors in
estimating ocular rigidity.
Intraocular pressure measurements by
cannulation in eyes treated topically with
6-hydroxy-dopamine. Five additional monkeys were treated topically in one eye
with 6-hydroxy-dopamine after tonography.
Four to six days after the first treatment,
a measurement of intraocular pressure by
cannulation was done in the control and
treated eye of each animal. Initial pressure
after equilibrium (about 1 minute after
-10-
+ 10-
+20-
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Fig. 4. Measurement of IOP by pressure transducers and cannulation of the anterior chambers
in a similarly treated, but separate group of
monkeys, confirmed the pressure lowering effect
of topical 6-hydroxy-dopamine.
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Investigative Ophthalmolugy
February 1971
126 Holland and Mims
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20-22
27-29
13-15
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Fig. 5. Tonographic facility was transiently increased one week after a topical 6-hydroxydopamine treatment. One week after a second treatment no statistically significant difference
between treated and control eyes was present.
DAY
cannulation) was recorded as the Po value.
Another group of five monkeys was
treated three times at weekly intervals
after tonography. The intraocular pressure
was measured by cannulation 20 to 22
days after the initial treatment.
In this separately treated series of monkey eyes, the pattern of intraocular pressure
change was the same as observed in the
group measured by Schi0tz and applanation methods.
The intraocular pressure measured by
cannulation in the control eyes averaged
11.8 (± 1.1) mm. Hg. The treated eyes averaged 2.5 (±0.5) mm. Hg or 26.2 per cent
lower than controls on approximately the
fifth day (Fig. 4). One week following the
last treatment (three weeks after the first
treatment), the intraocular pressure difference was less (i.e., 19.1 per cent ± 4.3
lower) in the treated eyes. On the twentyfirst day after the first treatment, the intraocular pressure averaged 2.8 (± 0.9) mm.
Hg lower in the treated eyes. These results
were of high statistical significance.
COMMENT. Using three different techniques of measuring intraocular pressure, it
was demonstrated that approximately one
week following an initial treatment with
6-hydroxy-dopamine, the mean intraocular
pressure in the treated eyes was about 25
to 30 per cent lower than the control eyes.
If the treatment were continued weekly,
by the second week the effects seemed to
be greater, and by the third week the effects on the intraocular pressure seemed
to be definitely diminishing. The long-term
duration of the lowered intraocular pressure following continued weekly applications has not been explored. One might
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Volume 10
Number 2
Anterior segment chemical sympathectomy
127
>- -30_l
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NO.EXPS.
DAY
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(5)
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4-6
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20-22
Fig. 6. Measurement of facility by perfusion showed an increase in the treated eyes one
week after treatment; because of the small number of animals and large variance, the change
was not significant statistically.
expect that it would resemble surgical
sympathectomy in its duration.
Facility of outflow following topical 6hydroxy-dopamine
Tonographic facility. In the group of
seven owl monkeys used for Schi0tz and
applanation Po measurements, three control tonograms were done in each eye prior
to treatment. This control series showed
there was an average difference in facility
between the two eyes (randomly occurring
between right and left) of about 33 per
cent. The eye with the higher facility was
usually associated with a higher Po. Because of these differences in control measurements, the eye with higher Po was
selected for treatment and its opposite for
control. A tonogram was always done prior
to treatment. Measurements continued for
two weeks following the last treatment.
Fig. 5 summarizes this series of experiments. The number of animals in each
group is included in parenthesis below the
bar graph in the figure. The average control facility was 1.12 (± 0.27) ^.1 per minute
per millimeter of Hg (hereafter called
facility units). A week after a single treatment, the mean difference in facility between treated and control eyes was 66.7
per cent (an increase in facility of treated
eyes by approximately 33 per cent, 0.01 <
p < 0.05). One week after the second
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Investigative Ophthalmology
Februartj 1971
128 Holland and Mims
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Fig. 7. Intraperitoneal water loading of rabbits following topical 6-hydroxy-dopamine
showed a significantly lower pressure rise per minute in the treated eyes, probably reflecting
a transiently increased facility of outflow (ganglionectomy effect). This change was not
demonstrable one and two weeks later.
treatment, the facility difference was approximately the same as the control difference prior to treatment. Subsequent weekly
measurements without treatment showed a
variation about the control difference in
facility.
COMMENT. Thus, it can be seen that
tonographic facility of outflow was increased significantly following topical 6hydroxy-dopamine, but the effect did not
persist through the second week, even
though the intraocular pressure was
lowered significantly at this time.
Perfusion facility. Fig. 6 summarizes the
results of facility measurements by Barany's
technique in ten owl monkeys treated with
6-hydroxy-dopamine. Five animals were
perfused approximately five days after the
first treatment. The average per cent difference in facility (control minus treated
eye) was 28.9 per cent (± 23.6) higher
in the treated eyes. One week after the
last treatment, the difference in facility
was 7.2 per cent. The average total facility
of all controls was 0.55 (± 0.07) facility
units. The average increase in facility in
the treated eyes on the fifth day was 0.29
facility units. Because of the large standard errors of these measurements and
the small sample size, none of the differences in perfusion facility were statistically
significant. However, the differences in Po in
these same animals measured by cannulation at the same time were statistically
significant.
COMMENT. The pattern of facility change
after treatment was similar to the group
measured tonographically in which the facility change one week after treatment was
statistically significant.
It should be noted that the "C" values
obtained by tonography in control eyes
averaged 0.57 facility units higher than
those obtained by perfusion. This difference is probably explainable by the combination of errors in using human calibra-
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Volume 10
Number 2
Anterior segment chemical sympathectomy
tion tables, as well as possibly greater
pseudofacility effects during tonography.
Results of studies of 6-hydroxy-dopamine
on the rabbit eye
Water provocative testing. Eight rabbits
were treated topically twice a day for
seven days with 6-hydroxy-dopamine in one
eye, the opposite eye receiving buffer solution. On the third and seventh days after
treatment, and also seven days after discontinuing treatment, intraperitoneal water
loading was done in the conscious animal.
The mean of five applanation readings was
taken every five minutes in both eyes as
the pressure rose from intraperitoneal
water absorption.
Fig. 7 illustrates the results. Three days
after the institution of daily treatment,
the difference in pressure rise per minute
(control minus treated eye) was 11.2 (±
3.8) mm. Hg lower in the treated eyes.
This difference was statistically significant
(p < 0.01). On the seventh day of continued treatment, the average difference
had dropped to 4.8 mm. Hg per minute,
and one week after discontinuing treatment it was 2.2 mm. Hg per minute higher
in the treated eye. The latter differences
were not statistically significant.
COMMENT. Topical treatment with 6hydroxy-dopamine produced a lower intraocular pressure rise after intraperitoneal
water loading, which was significant three
days after daily treatment, but not thereafter. The lower pressure rise at this time is
suggestive of the ganglionectomy effect on
facility in rabbits as measured by constantrate perfusion techniques, where equilibrium pressures are much lower on the side
of the sympathectomy.10
Applanation pressures. Under pentobarbital anesthesia, applanation tonometry
was done on a separate group of nine rabbits. The mean intraocular pressure in all
control eyes was 21.9 (± 1.3) mm. Hg.
These eyes averaged 1.1 mm. Hg higher
than those eyes which were selected for
treatment (Fig. 8). Following three days
of daily treatment, the intraocular pressure
was 3.3 (± 1.3) mm. Hg lower in the
129
treated eyes, a total change of 4.4 mm. Hg
(0.01 < p < 0.05). Seven days after
initiating daily treatment, the pressure difference between the control and treated
eyes was not statistically significant.
EpiscJeral venous pressure in the rabbit
treated with 6-hydroxy-dopamine. Immediately after applanation tonometry in the
nine rabbits as described above, episcleral
venous pressure measurements were made.
The results are given in Fig. 9. The
average episcleral venous pressure in the
control eyes was 9.4 (± 0.3) mm. Hg,
which is almost identical to that reported
by Linner17 in the rabbit. The treated eyes
averaged 0.7 (± 0.4) mm. Hg lower episcleral venous pressure three days after a
single treatment, while the intraocular
pressure averaged 3.3 mm. Hg lower in
the treated eyes. This change in episcleral
venous pressure is slight and not statistically significant. Seven days following a
single treatment with 6-hydroxy-dopamine,
the mean difference in episcleral venous
pressure was 0.5 (± 0.3) mm. Hg lower
in treated eyes, which also was not statistically significant.
COMMENT. It is evident that the episcleral venous pressure is not significantly
affected by chemical sympathectomy by
6-hydroxy-dopamine, even at a time when
the effects on intraocular pressure are easily
demonstrated. This is in agreement with
the measurements of episcleral venous pressure in rabbits by Linner17 following surgical sympathectomy.
The effect of topical norepinephrine
and isoproterenol on the intraocular pressure of monkeys pretreated with topical
6-hydroxy-dopamine
Norepinephrine—effects on intraocular
pressure. Five monkeys were used in this
experiment (Fig. 10). One week after a
single treatment with 6-hydroxy-dopamine
the intraocular pressures averaged 23.1
per cent (± 4.9) lower in the treated eyes
(p < < 0.01). The eyes were treated each
week for three weeks. One week after the
last treatment, three applications of 2 per
cent norepinephrine (0.1 ml.) were given
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130 Holland and Minis
-2
-
-I
-
Investigative Ophthalmology
February 1971
CONTROL Rx
NO TREATMENT
a:
14
+1 LJ
UJ
U.
U.
5 +2
z
<
UJ
+3 H
+4
-
• 0KP<.05
DAY
Fig. 8. Applanation pressure differences showed a significantly lowered IOP in treated eyes
of rabbits approximately three days after a single treatment. Seven days later no significant
difference could be detected.
to both eyes after proparacaine. Ninety
minutes later, the intraocular pressure and
facility were measured. After norepinephrine, the eyes previously treated with 6hydroxy-dopamine showed an average of
only 0.1 (± 0.2) mm. Hg lower pressure
than the control eyes, a result which was
not statistically significant.
COMMENT. The possibility of sensitization
to topical norepinephrine after chemical
sympathectomy was not demonstrable
under these experimental conditions, even
though the 6-hydroxy-dopamine had
exerted an intraocular pressure-lowering
effect the preceding week. It is interesting
to note, however, that the intraocular pressure of control eyes averaged 20.7 (± 1.1)
mm. Hg after norepinephrine and 16.3
mm. Hg one week prior. This increase in
pressure was statistically significant. It
would be easily explainable by an increase
in blood pressure. However, similar experiments done in anesthetized monkeys, in
which the intraocular and blood pressures
were monitored by cannulas in the anterior
chamber and femoral artery, showed that
there was no increase in blood pressure following topical application of norepinephrine.
Norepinephrine—effects on facility of
outflow. Control measurements of facility
on these five monkeys averaged 0.88 (±
0.09) facility units (Fig. 11). One week
following the first treatment with 6-hydroxy-dopamine, the per cent difference in
facility of outflow (control minus treated
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Anterior segment chemical sympathectomy
Volume 10
Number 2
-2
131
CONTROL Rx
NO TREATMENT
ui
o
z
UJ
cc
UJ
+3
-
+4
-
DAY
T—i—r
0
S
1
1
4
5
1
6
1
7
Fig. 9. Episcleral venous pressure measurements at the same time as the IOP measurements
showed no significant change.
eye) averaged 64 per cent (±21) higher
on the treated side (0.01 < p < 0.05).
This reconfirmed the previous measurements. One week after the second treatment, the facility was slightly lower in the
treated eyes by 1.6 per cent. These eyes
were given a third treatment with 6-hydroxy-dopamine and treated a week later
with topical norepinephrine three times
as described above. The facility of outflow
after norepinephrine was 13 per cent higher
in the treated eyes, but the difference between the control and treated eyes was
not statistically significant.
COMMENT. When one considers control
eyes alone, it is interesting to observe that
prior to any treatment the facility averaged 0.88 (± 0.09) units, and three weeks
later with norepinephrine the facility aver-
aged 1.47 (± 0.31) units which is statistically significant (0.01 < p < 0.05).
Also, it is interesting to note that the
average difference between the facility of
control eyes on approximately day 14 before norepinephrine and the facility of control eyes on approximately day 21 after
norepinephrine (0.43 ± 0.36 units) is almost a statistically significant difference (p
< 0.1; t = 2.0) when compared to the
average difference between the facility of
6-hydroxy-dopamine-treated eyes on day
14 and the facility of these same treated
eyes on day 21 after norepinephrine (0.71
± 0.34). This difference suggests, but does
not demonstrate, the possibility that 6hydroxy-dopamine may produce supersensitivity to the facility-increasing effect of
norepinephrine.
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132 Holland and Mims
-20
-
-10 —
E
Investigative Ophthalmology
February 1971
CONTROL
TREATMENTS
NOREPINEPHRINE
1
11
o
LLJ
O
z
UJ
2 *10
+ 20
-
30 —
P«.OI
P<.OI
6-8
13-15
20-22
DAY
Fig. 10. Chemical sympathectomy reproduced the lowered IOP seen in other experiments,
but topical application of norepinephrine did not produce an additional IOP lowering from
supersensitization.
Perfusion measurements of facility of
outflow folloioing 6-hydroxy-dopamine and
norepinephrine. Four monkeys were
treated topically with 2 per cent norepinephrine (0.1 ml.) at the end of three
pairs of control perfusions (at 5 and 10
mm. Hg above P o ). The norepinephrine
treatment was repeated four times at 5
minute intervals. One hour after the first
treatment, three additional pairs of perfusion were done.
The facility of outflow in control eyes
prior to norepinephrine treatment averaged 0.603 (± 0.14) units and after norepinephrine the facility had increased to
0.873 (± 0.13) units. This difference was
not significant (t = 1.68).
The facility of outflow of 6-hydroxydopamine-treated eyes prior to norepineph-
rine application averaged 0.625 (± 0.08)
units and after norepinephrine the facility
had increased to 1.08 (± 0.34) units. This
difference also was not significant (t =
1.31).
The facility of eyes treated with 6hydroxy-dopamine and norepinephrine
averaged 18.6 per cent higher than the
control eyes also treated with norepinephrine. These eyes had a mean facility of
0.213 (± 0.32) units higher than control
eyes, but this difference was not statistically significant.
COMMENT. A statistically significant increase in facility after topical treatment
with norepinephrine was not seen in the
6-hydroxy-dopamine-treated eyes. However, in comparing facility of control eyes
before and after topical norepinephrine,
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Volume 10
Number 2
Anterior segment chemical sympathectomy
133
-90 -80 -
-70 -60 -50 -40 2
-30 -20 -10 -
.0KP<.05
+ 10 -
CONTROL
DAY
NOREPINEPHRINE O.U.
TREATMENT
6-8
I
7
13-15
20-22
Fig. 11. The increased tonographic facility was reproduced in a separate series of animals,
but no augmentation of facility was seen following topical norepinephiine.
it appeared that there was a facility increase, although further control measurements are needed.
Perfusion facility folloiving 6-hydroxydopamine and isoproterenol. A pilot experiment was done in three monkeys in which
control perfusion facilities were measured
prior to topical isoproterenol application.
The mean facility in control eyes was
0.503 (± 0.056) units. Two per cent isopreterenol was applied topically to both
eyes in the same manner described for
norepinephrine. Sixty minutes after isoproterenol, the facility in the control eyes
averaged 1.09 (± 0.22) units. The blood
pressure was 146 (±16) mm. Hg before
and 97 (± 18) mm. Hg after topical isoproterenol.
In the eyes treated with 6-hydroxydopamine, the facility of outflow following
isoproterenol averaged 5.2 per cent ( ± 9 )
lower. These differences are not statistically significant.
Episcleral venous pressure measurements
in the monkey eye. As treatment with 6hydroxy-dopamine lowered the intraocular
pressure, it was important to determine
whether a lower episcleral venous pressure
could account for the lowering of intraocular pressure.
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Investigative Ophthalmology
Februanj 1971
134 Holland and Minis
In three monkeys treated in one eye with
6-hydroxy-dopamine, episcleral venous
pressure and applanation pressures were
measured the second, third, and fourth
weeks of treatment.
One week after the first 6-hydroxydopamine treatment, the applanation intraocular pressure in the treated eyes averaged 11.0 (± 0.5) and 12.7 (±0.5) mm.
Hg in the control eyes. The episcleral
venous pressure (Pe) in these treated eyes
averaged 11.1 (± 0.4) and in the control
eyes 11.4 (± 1.1) mm. Hg. The end point
used for these episcleral venous pressure
measurements (complete collapse of the
blood column) produced higher Pe values
than the end point utilized in subsequent
measurements (a 50 per cent reduction
in blood column10). These Pe values are
estimated to be 0.5 to 2 mm. Hg higher.is
However, the end points selected were the
same for the treated and control eyes and
there was no difference in episcleral venous
pressure in spite of a 1.7 mm. Hg lower
intraocular pressure in treated eyes.
One week after the second treatment
with 6-hydroxy-dopamine, the applanation
pressure in treated eyes averaged 16.2 (±
4.4) mm. Hg and 18.1 (±3.7) mm. Hg in
control eyes. The episcleral venous pressure in these treated eyes averaged 9.9 (±
1.1) mm. Hg while control eyes averaged
10.0 (± 0.6) mm. Hg (using 50 per cent
collapse of the blood column as the end
point10).
Thus, there was no significant difference
in Pe between the 6-hydroxy-dopaminetreated and control eyes, even though the
intraocular pressure effects were quite significant.
Ninety minutes prior to the fourth measurement, 2 per cent norepinephrine (0.1
ml.) was given topically to both eyes
after pretreating with proparacaine. Two
additional 0.1 ml. doses of norepinephrine
were given at 5 minute intervals.
Measurements of Pe in control eyes
treated with norepinephrine, and in eyes
treated with both 6-hydroxy-dopamine and
norepinephrine, similarly showed no sig-
nificant difference (12.3 and 13.2 mm. Hg,
respectively).
Discussion
Our knowledge of the effects of ocular
sympathectomy on aqueous inflow-outflow
dynamics is woefully incomplete in the
primate eye. This is probably a result of
the relative expense of studying the primate, the technical difficulty in accomplishing superior cervical ganglionectomy
in the monkey,10 and the small number of
patients available for study with postganglionic Horner's syndrome. Indeed, part
of the motivation for the present investigation was to explore the possibility of the
production of chemical sympathectomy by
topical applications of 6-hydroxy-dopamine,
which would obviate the surgical difficulties of sympathectomy, as well as possibly
open up new therapeutic avenues in the
treatment of human disease.
Several points to be raised in this discussion are: (1) a review of the present
knowledge of the effects of superior
cervical ganglionectomy on aqueous inflowoutflow dynamics of the rabbit eye and a
comparison with the primate eye; (2) a
comparison of the effects of chemical sympathectomy with surgical sympathectomy;
(3) consideration of the question of the presence of supersensitization to adrenergic
amines following chemical sympathectomy;
(4) and finally, the pharmacologic mechanism of action of 6-hydroxy-dopamine, its
side effects, toxicology, and its relationship
to other sympatholytic agents will be discussed.
Summary of the effect of superior cervical ganglionectomy on the inflow-outflow
dynamics of aqueous humor in the rabbit
eye. Our understanding of the mechanisms
of lowering of intraocular pressure following superior ganglionectomy in the rabbit
("ganglionectomy effects") was initiated
by Linner and Prijot,17 and developed in
an elegant series of studies by a number
of investigators, but particularly by
Barany16>19-20"24; Sears16'25-30; Langham3139
; Eakins40"43; and their co-workers. Excel-
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Volume 10
Number 2
Anterior segment cliemical sympathectomy
lent summaries of this work can be found
in the papers of Sears and Rosser,30 Kramer
and Potts,44'45 and Treister and Barany.23'2i
Approximately 14 hours after ganglionectomy, neurotransmitter begins to leak
from storage sites in degenerating sympathetic nerve terminals stimulating the
adjacent iris dilator muscle and producing
a degeneration mydriasis. The duration and
course of the pupillary changes are a function of the rate of release of norepinephrine and the rate of development of supersensitization of the dilator muscle. Norepinephrine accumulates in the aqueous humor, reaching 25 per cent of its maximum
concentration four hours after the onset
of degeneration mydriasis. At this time, the
intraocular pressure begins to fall (reaching a maximum five hours later), a result of an increased facility of outflow
produced by norepinephrine acting on
sensitized alpha-adrenergic receptors in
the outflow pathway.23'24> 29 The exact site
of the latter is unknown; however, opinion
favors a vascular structure located close
to the chamber angle.26
Denervation supersensitization to norepinephrine gradually occurs during the
phase of degeneration mydriasis, and correlates temporally with the loss of norepinephrine and the failure of normal uptake inactivation.26' 29>44> 45 Several investigators have demonstrated lower intraocular
pressure and facility augmentation from
supersensitization to exogenously applied
norepinephrine.20' 34> 35>40"42
The intraocular pressure returns to normal levels in two to three days17'23> 24; but,
curiously, in the absence of nonadrenergic
tone the resistance to outflow becomes
higher than normal.20
Review of the effects of ocular sympathetic denervation on the aqueous inflowoutflow dynamics of the primate eye. There
is, of course, no a priori reason to expect
the same result from sympathetic denervation of the primate eye because of wellknown anatomical and innervational differences.40
19
MONKEY. Barany
observed that adre-
135
nergic effects on the facility in the rabbit
are easy to demonstrate, but clear-cut
similar effects are difficult to establish in
the monkey.
Langham34 found an alpha-receptor
response in the monkey 7 to 14 days after
sympathetic denervation. The intraocular
pressure dropped after topical adrenalin,
but there was no facility change. Langham
(with David Honey) 39 has also repeatedly
made reference to unpublished rhesus
monkey experiments in which a prolonged
intraocular pressure-lowering effect has
been seen following superior cervical
ganglionectomy; a sustained fall in intraocular pressure associated with an increase
in outflow facility was also mentioned.
Sensitization to topical norepinephrine (intraocular pressure and pupil) was said to
be increased a thousandfold.
Hoffman47 found in the Cynomolgus
monkey that the intraocular pressure was
not significantly lower in the side of
sympathetic denervation. One per cent
norepinephrine applied topically produced
a pressure rise in control eyes, but not
denervated eyes, and it was without statistical significance. Injection of norepinephrine intracamerally caused a fall in intraocular pressure in the denervated and control eyes. At high perfusion pressures (but
not at low), norepinephrine caused a significant increase in facility of outflow.
Hoffman concluded that superior cervical
ganglionectomy in the monkey caused only
a slight drop in intraocular pressure from
the ganglionectomy; facility was slightly
higher in the denervated eye, and with high
perfusion pressure, facility was increased
and secretion was stimulated by intracameral norepinephrine.
RESUME OF OBSERVATIONS IN HUMAN SUBJECTS. Sears a n d Sherk 2 0 mentioned t h a t
a greater facility of outflow occurred on
the homolateral side of patients with
Homer's syndrome and that the resistance to outflow further decreased after
topical norepinephrine. Sears28 also reported supersensitization to adrenalin in
these patients; the magnitude of these ef-
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Investigative Ophthalmology
February 1971
136 Holland and Mims
fects was reported to be inversely proportional to the duration of the sympathetic denervation in Horner's syndrome
patients.
Swegmark48 reported a case of postganglionic Horner's syndrome in which
the intraocular pressure was 35 per cent
lower on the affected side; the difference
in intraocular pressure was presumed due
to diminished secretion as measured by
suction cup techniques. There was no
difference in facility of outflow by tonography with several measurements. Topical
treatment of the normal eye with guanethidine reproduced the same changes seen
in the eye with Horner's syndrome. Sensitization of the denervated pupil to adrenalin 1/1,000 was demonstrated.
Langham and Weinstein3S reported
three patients with Horner's syndrome
(one preganglionic and two postganglionic). Measurements of pressure, facility,
and flow, before and after topical norepinephrine, were incomplete. The authors
concluded that the outflow facilities measured by Schi0tz tonography were not
significantly different in the three patients.
They assumed episcleral venous pressures
were unchanged and inferred that aqueous
inflow was diminished by sympathetic
denervation. In one patient, topical epinephrine produced a lower intraocular
pressure in both eyes and a greater aqueous humor formation in the denervated
eye, along with an increase in outflow
facility.
Weinstein and Langham49 reported a
case of Homer's syndrome, in association
with bilateral glaucoma, in which the eye
with Horner's syndrome had a relatively
lower intraocular pressure and less visual
field loss. Supersensitivity to topical norepinephrine was claimed for the pupil
and the pressure on the denervated side.
There was no change in facility of outflow
following norepinephrine in this patient.
Drance in the Gilston Glaucoma Symposium34 reported that Christansen's follow-up studies of six patients with cervical
sympathectomy demonstrated a marked
fall in intraocular pressure which was of
short duration and gradually became normal.
Bron50 studied a patient with postganglionic Horner's syndrome and a normal
patient treated with topical guanethidine.
Intraocular pressure was lower in the
sympathetically denervated eye due to a
lower aqueous humor production. Guanethidine did not significantly lower the
intraocular pressure, but did lower aqueous
humor formation. Topical norepinephrine
restored flow to normal levels in both eyes.
Pupillary supersensitization to norepinephrine was demonstrated following guanethidine application. There was no intraocular
pressure-lowering effect produced by
topical norepinephrine in either the Horner's patient or the guanethidine-treated
eye, an observation which is similar to that
reported by Drance (see above).
COMMENT. The quality and quantity of
data on the primate eye concerning the
effects of ocular sympathectomy are not
sufficient to form a complete and convincing picture. Trends which have emerged
from the existing studies are: (1) a lower
intraocular pressure on the side of sympathetic denervation which may persist indefinitely or disappear (depending on
duration); (2) no change in facility of
outflow in chronically denervated eyes; (3)
lower aqueous secretion (accounting for
the lower intraocular pressure if it is assumed episcleral venous pressure is unchanged); (4) denervation supersensitization to topical catecholamines sometimes
occurs, but it is not a regularly observed
event in all investigations. Supersensitization of pupils is a frequent observation.
Intraocular pressure may remain unchanged following topical adrenergic
amines because of mutually opposing influences of increased facility of outflow in
sensitized outflow pathways and increased
aqueous humor secretion. The intraocular
pressure may fall following topical application of adrenergic amines when a marked
increase in the outflow facility occurs.
It seems likely that the variable results
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Volume 10
Number 2
Anterior segment chemical sympathectomy 137
of these investigations of supersensitivity
phenomena may be due to different thresholds of the different ocular tissues, as
well as differences in catecholamine concentrations reaching the target sites after
topical applications.
Comparison of the effects of ocular
chemical sympathectomy by 6-hydroxydopamine with surgical sympathectomy.
There are many similarities between these
methods of producing ocular sympathectomy, but, also, there are some outstanding differences.
Pupil. Degeneration contraction (mydriasis) is seen one half to three hours
following topical application of 6-hydroxydopamine, but occurs approximately 14
hours after superior cervical ganglionectomy. This difference is probably related
to the great rapidity of uptake of 6hydroxy-dopamine by sympathetic nerve
terminals compared to the relatively large
time interval required for anatomic degeneration and release of endogenous norepinephrine.
Intraocular pressure. Two effects can be
observed in the 6-hydroxy-dopaminetreated eyes which depend on the species.
RABBIT. In the rabbit, the intraocular
pressure is significantly lower on the third
day after 6-hydroxy-dopamine treatment.
At this time, the pressure rise in the treated
eyes on water loading is much lower, probably because of an increased facility of
outflow. After surgical sympathectomy, the
intraocular pressure and facility effects occur between 19 and 36 hours following
ganglionectomy. Since our experiments
were done every two days, because of
anesthesia limitations, the earliest occurrence of the ganglionectomy effect from
6-hydroxy-dopamine is unknown.
One week after treatment, the ganglionectomy effect of 6-hydroxy-dopaminetreated rabbits is not demonstrable. This
is reminiscent of the transient (lasting two
to three days) effects on intraocular pressure and facility produced by surgical ganglionectomy.
MONKEY. In monkeys, the intraocular
pressure-lowering effect of 6-hydroxydopamine persists at least two to three
weeks following topical treatment. This
agrees with the available data on surgical
sympathectomy. One outstanding difference of the effects of surgical sympathectomy on rabbits and monkeys is the longer
duration of effects on the latter species.
The exact duration of the 6-hydroxy-dopamine effect on intraocular pressure has not
been investigated.
TONOGRAPHIC FACILITY. The tonographic
facility is increased transiently by 6-hydroxy-dopamine approximately one week
following treatment. This increase in facility, however, disappears by the second
week. The time of earliest occurrence of
this increased facility has not been investigated. The transient increase in facility seen in the monkey is certainly reminiscent of the similar effect observed in
rabbits following surgical sympathectomy.
EPISCLERAL VENOUS PRESSURE. T h e e p i -
scleral venous pressure is unchanged by
chemical sympathectomy in both monkeys
and rabbits, agreeing well with the single
measurement reported in the literature by
Linner and Prijot17 in the rabbit. This permits the inference to be made that aqueous
secretion is reduced following chemical
sympathectomy by 6-hydroxy-dopamine,
agreeing well with the reported effects of
surgical sympathectomy.
Haeusler, Haefely, and Thoenen51 have
compared the effects of chemical sympathectomy in the cat produced by 6-hydroxy-dopamine to the effect of surgical
sympathetic denervation. The norepinephrine content of smooth muscles and the
nictitating membranes denervated surgically was reduced to almost zero, whereas
chemically sympathectomized membranes
average 8 per cent of the control norepinephrine levels. Similar results were
obtained in the iris in which corresponding values for norepinephrine were zero
and 3 per cent, respectively. The adrenergic
sensitization of the surgically denervated
membranes was increased by a factor of
approximately 96 compared to the con-
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Investigative Ophthalmology
February 1971
138 Holland and Mims
RATE LIMITING
CH.-CHNHL-COOH
CH2CHNH2COOH
HO —
-CH2CH2NH2
TYROSINE HYDROXYLASE-
f
[OJ
L-TYROSINE
HO(DOPA)
C02
3,4-DIHYDROXYPHENYLALANINE
DOPAMINE
i
r
MELANIN
[0]
-CHOH-CH 2 NH 2
HO —
HO —
NOREPINEPHRINE
HO-I
-OH
\T' 'ZW:
6-HYDR0XYD0PAMINE
-CHOH-CH-NH
2
I
Fig. 12. Metabolic pathway involved in synthesis of norepinephrine and epinephrine. The
rate-limiting enzyme, tyrosine hydroxylase, is presumed to be inhibited by 6-hydroxy-dopamine.
The similarity in structure between 6-hydroxy-dopamine and norepinephrine is apparent.
trol values, whereas chemically sympathectomized membranes were increased in
sensitivity by a factor of approximately 71.
Supersensitization phenomena. Our experiments failed to demonstrate an increased pressure-lowering response to topically applied adrenergic amines in chemically sympathectomized eyes. There was
no statistically significant change in facility in the few experiments which were
designed to explore this. There was an
increase both in facility and intraocular
pressure in the control eyes and a smaller
increase in the 6-hydroxy-dopamine-treated
eyes, similar to the observations of Bron50
and Hoffman.47 However, there was no
increased effect due to chemical sympathectomy as might have been anticipated.
We wish to emphasize that these experiments are preliminary and of a pilot nature, and we conjecture that under other
experimental conditions, supersensitization
effects of topically applied adrenergic
amines may be demonstrable. Possibly,
the dose delivered to the target sites may
be the critical issue. Nonetheless, our present experiments do not demonstrate a
facility-increasing or additional pressurelowering effect of topically applied adrenergic amines in the 6-hydroxy-dopamine
chemically sympathectomized eye.
Pharmacologic mechanisms of action of
6-hydroxy-dopamine. 6-Hydroxy-dopamine
(2,4,5-trihydroxyphenylethylamine) is a
congener of norepinephrine (Fig. 12). It
has the same formula weight and similar
structure, except that the -OH group attached to the beta-carbon of norepinephrine
is transposed to the 2 position of the benzyl
ring of 6-hydroxy-dopamine. 6-Hydroxydopamine is very similar to norepinephrine
in its chemical properties,52 including its
propensity for rapid oxidation and rapid
uptake into sympathetic nerve terminals.
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Volume 10
Number 2
Anterior segment chemical sympathectomy
A single injection of 6-hydroxy-dopamine
markedly reduces the norepinephrine content of sympathetically innervated organs of
several species for days or weeks, and restoration of norepinephrine may not be complete for as long as 80 to 90 days.1'3-51"55
Thoenen and Tranzer3 developed a
treatment regimen which led to a generalized and almost complete destruction of adrenergic nerve terminals. Adrenergic nerve
cell bodies, stem axons, and other nerve
endings (e.g., cholenergic) are unaffected
by 6-hydroxy-dopamine. These authors proposed the term "chemical sympathectomy"
to describe these unique effects.
Thoenen (personal communication)
points out that the sympathetic nerve
terminals accumulate the amine very efficiently. Oxidation products of 6-hydroxydopamine undergo covalent bonding to
nucleophilic groups of macrornolecules
which leads to destruction of the nerve
terminals. Miller, Thoenen, and Axelrod50
postulate that the rate-limiting enzyme in
norepinephrine synthesis, tyrosine hydroxylase (Fig. 12), is destroyed by 6-hydroxydopamine since the enzyme's disappearance
from the heart is coincident with the destruction of cardiac sympathetic nerve
endings.
Electron microscopic studies of nerve
terminals following 6-hydroxy-dopamine
treatment show sympathetic nerve terminal degeneration.4'5> 55 Using the histofluorometric technique of Falck, Malmfors
and Sachs53 and Knyihar and associates5
have shown a depletion of catecholamines
from sympathetic axonal tenninals with
relative preservation of catecholamines in
nerve cell bodies and stem axons. This
depletion of endogenous norepinephrine
was due to a destruction of nerve terminals
by 6-hydroxy-dopamine or some metabolite, making the terminals unable to retain
endogenous norepinephrine. Inhibition of
norepinephrine uptake occurred simultaneously with the disappearance of the
fluorescence of sympathetic nerve terminals.
Studies in our laboratory with the use
139
of the Falck histofluorometric technique
(in collaboration with Dr. Jeffrey Ellison)
have demonstrated a loss of sympathetic
nerve terminals in the anterior segment
following topical 6-hydroxy-dopamine (Fig.
13). These observations have been correlated with electron microscopic observation of the effects of topical administration
of 6-hydroxy-dopamine in a report now
in preparation.
The concept of "chemical sympathectomy" is not a new one in ophthalmology.
Depletion of endogenous norepinephrine
has been accomplished experimentally by
reserpine,20 cocaine,57 tyramine analogs,30
and guanethidine. It is the latter that
6-hydroxy-dopamine most closely resembles.
It may be informative to compare the
effects of guanethidine with those of 6hydroxy-dopamine.
In rabbits, guanethidine has been reported to lower the intraocular pressure
and to increase the facility of outflow following intracameral injection.43 In humans,
guanethidine has been reported to lower
the intraocular pressure following intravenous58 and topical application.48'50'59> 60
Topical application in normal eyes lowers
the pressure 1 to 3 mm. Hg. Topical
or intravenous administration in glaucomatous eyes lowers the pressure about 8
mm. Hg.
Four to 12 hours after the first administration of topical 10 per cent guanethidine, a significant increase in facility
of outflow has been reported.60 This increase in normal eyes is about 0.045 /xl
per millimeter of mercury per minute. Beyond 12 hours after the first administration,
there is no effect on facility but the
intraocular pressure is still significantly
lowered. Anselmi, Bron, and Maurice59 have
demonstrated directly, with a fluorophotometric technique, that eyes treated twice
a day with 10 per cent guanethidine for
3 to 36 days ("chronic pharmacological
sympathectomy") have a significantly decreased rate of aqueous flow and increased
vascular permeability. The effect of guan-
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Inoc$tit>atioe Ophthalmology
February 1971
140 Holland and Mims
Fig. 13. (Right) Sections of control albino rabbit iris treated by Falck histofluorometric
method showing fluorescence of catecholamine containing axons. (Left) Three days following a single treatment with 6-hydroxy-dopamine, there is a marked diminution of terminal
and preternu'nal sympathetic axons, demonstrating "sympathectomy" by chemical means.
(Slides courtesy of Dr. Jeffrey Ellison.)
ethidhie on episcleral venous pressure has
not been reported.
Like guanethidine, 6-hydroxy-dopamine
produces a transient increase in outflow
facility followed (with continued treatment) by a decrease in intraocular pressure due to diminished aqueous inflow.
The transient increase in facility caused
by 6-hydroxy-dopamine lasts much longer
(at least six to seven days) than that
caused by guanethidine. Also, the duration of the effect is considerably longer
than that of guanethidine.
Other significant differences exist between 6-hydroxy-dopamine and guanethidine. For example, there is a slightly
greater potency of 6-hydroxy-dopamine in
depleting cardiac norepinephrine. Reserpine, guanethidine, and tyramine reversibly release norepinephrine from the tissues, whereas 6-hydroxy-dopamine destroys or irreversibly alters the norepinephrine binding sites. The slow recovery of
norepinephrine after 6-hydroxy-dopamine
is a consequence of the rate required for
regeneration of binding sites.01
Histofluorometric experiments by CsillikG2 showed that guanethidine caused a
reduction of axonal fluorescence, but did
not produce a complete depletion of catecholamines, which is in marked contrast to
the complete loss of axonal terminals produced by 6-hydroxy-dopamine.
Toxicology of topically applied 6-hydroxy-dopamine. There were no animal
deaths in any experiments performed in
this investigation which could be attributed
to the use of 6-hydroxy-dopamine.
In early experiments, adrenochrome-like
staining of the deep corneal lamellae occurred when an ascorbate was used as an
antioxidant at pH 5.2. However, when
bisulfite was substituted at a pH of 7, no
corneal staining or oxidation products were
observed on repeated slit lamp examinations.
Iris vasodilation was a regular occurrence in the 6-hydroxy-dopamine-sympa-
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Volume 10
Number 2
Anterior segment chemical sympathectomy
thectomized eyes. Slit lamp examinations
of eight monkeys showed no increased
aqueous flare compared to control eyes.
The semiquantitative trichloroacetic acid
test of Langham and Taylor 31 showed no
significant elevation of protein in the aqueous humor of control and treated eyes in
four monkeys used for perfusion studies.
Histology of these eyes has shown no
abnormalities in rabbits and a slight vacuolization of the pigment epithelium of the
iris in monkeys.
It is our impression that this drug is
reasonably safe for clinical investigation.
Limited clinical investigations are in progress and will be reported on in the near
future.
The authors wish to acknowledge the assistance
of Mrs. Parveen Walia and Mr. Fred Clark and
the collaborative assistance and efforts of Dr.
Jeffrey Ellison.
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