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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / January 1987
References
1. Santonastaso A: La rifrazione oculare nei primi anni di vita.
Ann Ottal Clin Ocul 58:852, 1930.
2. Gwiazda J, Scheiman M, Mohindra I, and Held R: Astigmatism
in children: changes in axis and amount from birth to six years.
Invest Ophthalmol Vis Sci 25:88, 1984.
3. Dobson V, Fulton AB, and Sebris SL: Cycloplegic refractions of
infants and young children: the axis of astigmatism. Invest
Ophthalmol Vis Sci 25:83, 1984.
4. Howland HC and Sayles N: Photorefractive measurements of
astigmatism in infants and young children. Invest Ophthalmol
Vis Sci 25:93, 1984.
5. Mohindra I: A non-cycloplegic refraction technique for infants
and young children. J Am Optom Assoc 48:518, 1977.
6. Owens DA, Mohindra I, and Held R: The effectiveness of a
retinoscope beam as an accommodative stimulus. Invest
Ophthalmol Vis Sci 19:942, 1980.
7. Mohindra I, Held R, Gwiazda J, and Brill S: Astigmatism in
infants. Science 202:329, 1978.
8. Mohindra I and Molinari JF: Near retinoscopy and cycloplegic
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retinscopy in early primary grade school children. Am J Optom
Physiol Opt 56:34, 1979.
Howland HC: Infant eyes: optics and accommodation. Curr Eye
Res 2:217, 1982.
Doxanas MT and Anderson RL: Oriental eyelids: an anatomic
study. Arch Ophthalmol 102:1232, 1984.
Miller D: Pressure of the lid on the eye. Arch Ophthalmol 78:
328, 1967.
Vihlen FS and Wilson G: The relation between eyelid tension,
corneal toricity, and age. Invest Ophthalmol Vis Sci 24:1367,
1983.
Wilson G, Bell C, and Chotai S: The effect of lifting the lids on
corneal astigmatism. Am J Optom Physiol Opt 59:670, 1982.
Nisted M and Hofstetter HW: Effect of chalazion on astigmatism.
Am J Optom Physiol Opt 51:579, 1974.
Robb RM: Refractive errors associated with hemangiomas of
the eyelids and orbit in infancy. Am J Ophthalmol 83:52, 1977.
Atkinson J and Braddick O: Vision screening and photo-refraction—the relation of refractive errors to strabismus and amblyopia. Behav Brain Res 10:71, 1983.
Chronic Stimulation of Ocular Sympathetic Fibers in Unanesthetized Rabbits
Carlos Belmonre, Esreban Perez,* Laura G. Lopez-Driones, and Juana Gallar
The goal of this study was to devise a technique to implant
permanent electrodes in the cervical sympathetic trunk, to
stimulate the ocular adrenergic fibers for periods of hours or
days in awake, unrestrained rabbits. Electrodes were made
of a silver wire soldered to a multistranded wire and enclosed
in silicone. Two of these electrodes were wrapped around the
preganglionic sympathetic nerve, their leads emerging through
a hole in the back of the neck. Success of the procedure was
confirmed by the mydriasis elicited by electrical stimulation
of the nerve following surgery; threshold voltages for the pupillary response varied between 5-10 volts. In eight rabbits,
suprathreshold sympathetic stimulation was performed on
the following days by means of a portable stimulator using
increasing frequencies (1, 3, 5, 8, and 10 Hz) during a 20-hr
period. Dilation of the ipsilateral pupil and vasoconstriction
in the ear, measured by the fall in temperature of the ear's
surface, was observed as long as stimulation was maintained.
Both effects were proportional to the frequency of stimulation.
Maximal mydriasis was obtained at 8 Hz, whereas full vasoconstriction was elicited with 5 Hz. Intraocular pressure,
measured in 10 rabbits with a Perkins tonometer at the end
of a 24-hr stimulation period, did not differ from pre-stimulation values. It was concluded that chronic stimulation of
the sympathetic nerve allows to maintain known levels of
adrenergic activity in the eye, and may be a useful method to
study the actions of the adrenergic system on various ocular
functions in unanesthetized animals. Invest Ophthalmol Vis
Sci 28:194-197, 1987
Electrical stimulation of the cervical sympathetic
nerve has been frequently used to determine the influence of the adrenergic system on various ocular func-
tions. For instance, acute stimulation of the sympathetic reduces intraocular pressure,1'2 blood flow
through the uveal vessels,3 and aqueous humor production.2
However, little is known about the circumstances in
which peripheral adrenergic mechanisms are physiologically activated in the eye, and what is the role played
by continuous sympathetic activity, the sympathetic
tone, in the maintenance of such functions. Experimental approaches to these questions have used surgical
or chemical denervation or blocking drugs to abolish
sympathetic effects. These drastic procedures totally
suppress adrenergic activity, but, with denervation, the
normal physiology of the target organ is altered.
The aim of our work was to devise a technique using
chronic stimulation of the cervical sympathetic nerve
that would allow us to adjust, for long periods of time,
the degree of sympathetic activity that reaches the eye
in awake, unrestrained animals. Preliminary results
have been reported elsewhere.4
Materials and Methods. Electrodes: Implantable
electrodes based on those described by Sweet and
Bourassa5 were constructed. The electrodes consisted
of a rectangle of 6 mm X 2 mm of silicone sheeting
(silastic sheeting, 500-3, Dow Corning Corp. Medical
Products, Midland, MI) in which a 0.125 mm diameter
silver wire was pierced through two small holes and
soldered to the lead wire. This lead wire was introduced
within a silastic tube to cover the wire up to the soldering point with the silverfilament.A second, thinner
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silicone sheet (silastic sheeting, Dow Corning, 500-1)
was glued to the external face of the electrode and to
the silastic tube containing the lead wire with adhesive
(Dow Corning 891 medical adhesive) to secure the
electrode in a flat silicone block and to ensure electrical
isolation (Fig. 1A-C).
Surgical procedures: Adult pigmented and albino
rabbits were anesthetized with intravenous sodium
pentobarbitone (Nembutal, 30 mg/Kg). Animals were
treated according to the ARVO Resolution on the Use
of Animals in Research. The preganglionic sympathetic
trunk was identified in the neck and carefully dissected
for a 2 cm length using a stereomicroscope. Two electrodes were introduced and wrapped around the nerve
trunk, closing the electrode borders with a 8-0 suture.
The electrodes were further secured in place with sutures to the neighboring muscles; electrodes were separated from each other by a distance of 5-10 mm (Fig.
1D-F). Care was taken to maintain the nerve in its
natural position without being twisted by the electrodes.
The lead wires were carried subcutaneously to the back
of the neck and passed to the outside through a hole
in the skin. The integrity of the cervical sympathetic
trunk was ascertained at the end of surgery by the induction of mydriasis with electrical stimulation (see
Results).
Stimulation: Electrical stimulation was performed
using 1 ms square pulses of variable amplitude (3-15
V) and frequency (1-15 Hz) delivered by an Ortec 4710
electrical stimulator (Ortec Inc., Oak Ridge, TN) connected to a stimulus isolation unit in acute trials, and
by a portable stimulator when prolonged stimulation
in the unrestrained animal was desired. This batterydriven portable stimulator produced continuous trains
of 0.5-1 ms duration pulses at adjustable frequencies
ranging from 0.1-20 Hz and amplitudes of 0-15 volts;
due to its reduced dimensions ( 5 X 3 X 2 cm), the
rabbits could carry the stimulator for several days in a
bag attached to their backs without apparent discomfort.
Pupillary, intraocular pressure and ear temperature
measurements: The rabbits were placed in restraint
boxes during the measurement periods. Pupillary diameter was measured with a ruler under uniform illumination. Intraocular pressure (IOP) was determined
using a Perkins hand-held tonometer (Clement Clark,
London, England). Tonometric readings were converted to mm Hg using a curve constructed with data
obtained from 21 cannulated rabbit eyes, in which IOP
was measured with a transducer and with the tonometer and artificially varied between 10 and 50 mm Hg
in 10 mm Hg steps. Topical 0.5 propacaine was administered as local anesthetic. Temperature of the ear
was gauged with a thermistor probe (YSI model 427)
attached with adhesive tape to the internal surface of
195
Fig. 1A-F. Schematic diagram of the procedure used to build
wrapping electrodes. A silver wire is passed through two holes made
3 mm apart in a silicone sheet; the silver wire is soldered to a multistranded, teflon-coated wire introduced within a silicone tube. Silicone glue is used to attach a second sheet that covers and isolates
one of the sides of the electrode (A). After the glue has cured, the
silicone block is cut (B) to give the electrode the desired shape (C).
For surgical implantation, the electrode is wrapped around the cervical
sympathetic nerve (D), and its ends are closed with an 8-0 suture
(E). Electrodes are secured in place by suturing their wires to neighboring muscles (F).
the ear and connected to an electronic thermometer
(YSI model 42TD) and to a polygraph for continuous
paper recordings of temperature.
Results. Every day during the week that followed
surgery, the pupil response to sympathetic stimulation
was tested using 10 Hz, 1 ms pulses to determine the
threshold voltage at which a distinct mydriasis could
be obtained. Threshold values stabilized usually between 5 and 10 volts; stimulating amplitudes over 1215 volts were often accompanied by overt muscle contractions in the neck due to current spread, and the
animal showed signs of unrest and discomfort. Hence,
rabbits with thresholds over 12 volts were discarded.
With stimulating voltages 1-2 volts over threshold,
stimulation could be maintained for hours or days
without apparent manifestations of displeasure or
changes in the feeding or resting behavior of the rabbits.
Failures occurred in about 30% of the operated animals, and appeared to be due to mechanical damage
of the nerve due to displacement of the electrode or to
tight fitting; it is apparently necessary to leave enough
lumen to allow initial slippage of the nerve and space
for invading connective tissue. Successful experiments
were performed in 10 rabbits that exhibited stable
threshold voltages of 10 volts or less during the 2-month
period of this study.
Figures 2 and 3 show the time course of pupil dilation
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Vol. 28
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / January 1987
37
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20
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40
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Fig. 2. Time course of average pupil diameter change following a
suprathreshold, continuous stimulation of the ipsilateral sympathetic
nerve during a 20-hr period at 1,3, 5, and 10 Hz. Each point is the
mean value of six rabbits. Stimulation was started at zero time.
and ear temperature during a continuous suprathreshold stimulation lasting for 20 hr. Data are mean values
of six rabbits stimulated at increasing frequencies with
a 4-8 day interval between each stimulation. Maximal
levels of pupillary and vascular responses were attained
10 min after the beginning of stimulation, and both
remained stabilized for the following 6 hours. At 20
hr, some return of basal values could be observed both
in the pupillary response and the ear's temperature.
There was correlation between the magnitude of the
pupillary and vascular responses and frequency of
stimulation. Figure 4 depicts the mean temperature
and pupil diameter values of eight rabbits measured
every hour during 6 hr of stimulation at different frequencies. For the vessels, maximal effects were attained
with frequencies of 3-5 Hz with a clear response occurring at 1 Hz. For the pupil, more gradual changes
were observed with increasing stimulation frequencies;
maximal effects appeared at 8 Hz.
Intraocular pressure was measured in ten rabbits before and at the end of 24-hr stimulation periods at 3,
5, and 10 Hz. No significant variations of IOP were
found at any of the stimulus frequencies explored (initial IOP: 15.6 ± 0.3 mm Hg, mean ± S.E., n = 10;
IOP after a 24-hr stimulation: 16.2 ± 0.6 mm Hg,
mean ± S.E., n = 9).
Discussion. The technique described here is based
on implanting permanent, wrapping electrodes on the
preganglionic cervical sympathetic trunk to chronically
stimulate ocular adrenergic fibers and to reproduce the
effects of their natural activation. This technique seems
to offer satisfactory results in terms of integrity of the
nerve fibers, responsiveness during prolonged stimulation periods, and reproducibility of threshold voltages
after several weeks.
Pupillary dilation and decreased ear temperature
upon stimulation were the results of well-known ad-
10
20
30
Minutes
40
50
4
6
Hours
60
20
Fig. 3. Average time course of ipsilateral ear's temperature following
continuous suprathreshold stimulation of the cervical sympathetic
during 20 hr at 1, 3, 5, and 10 Hz. Each point is the mean of six
rabbits (same experiments of Figure 2). Stimulation was started at
zero time.
renergic effects on the iris dilator muscle and vascular
smooth muscle.6 Maximal effects at a given frequency
were obtained with voltages slightly over threshold. At
these voltages, recruitment of all the sympathetic neurons in the ganglion probably occurs, even if not all
the preganglionic fibers are excited, because these have
a high degree of convergence; each ganglion neuron
receives, on average, synaptic contacts from a minimum often different preganglionic fibers.7 Thus, even
if a small percentage of the preganglionic fibers are
damaged as a consequence of surgery, the synchronous
activation of the remaining fibers should be sufficient
to depolarize virtually all neurons in the ganglion. Furthermore, antidromic invasion of the somata of the
preganglionic neurons by each stimulus should reset
their membrane potential and reduce to near zero their
spontaneous activity.
We found the expected correlation between stimulus
frequency and magnitude of the pupillary and vascular
response.8'9 Full pupillary dilation and vasoconstriction
were obtained at frequencies over 3 Hz. In the cat, the
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Fig. 4. Relationship between stimulus frequency and amplitude
of the pupillary and ear vessels response during continuous sympathetic stimulation of 6 hr duration. Data are the mean + S.E. of the
pupil diameters (right) and of the ear temperature (left) measured
every hour in eight rabbits.
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Reporrs
average spontaneous activity of preganglionic sympatheticfibersis 1.4 impulses/sec, reaching maximal frequencies of 9 impulses/sec, probably for only short periods of time, during stress conditions, such as acute
hemorrhage.10 Hence, we can assume that an artificially
imposed train of electrical pulses, which evokes a synchronous discharge in practically all postganglionic
neurons, releases large amounts of neurotransmitter,
which will probably produce maximal effects on the
target organ at relatively low frequencies. Acute stimulation of the sympathetic produces a reduction of IOP
in the anesthetized rabbit,1'2 but did not vary it substantially after 24 hr of stimulation in the awake animal. IOP is the final result of a complex balance between aqueous humor production and outflow, vascular resistance, and blood volume. Adrenergic
influences on these parameters vary in magnitude and
time course, and may produce antagonistic effects on
IOP; thus, it is not surprising that drastic changes in
basal IOP were not observed after long-term stimulation.
The present experiments demonstrate that chronic
stimulation of the cervical sympathetic in awake, unrestrained animals can be used at desired frequencies
to reproduce known adrenergic effects in the eye. This
technique will allow setting of the ocular sympathetic
tone at pre-established levels, and will, therefore, be
useful for expanding our knowledge of the role of the
adrenergic system in the modulation of ocular functions, as well as the study of the action of drugs that
mimic or interfere with ocular adrenergic effects.
Key words: adrenergic stimulation, ocular sympathetic, pupil,
intraocular pressure, vasoconstriction
Acknowledgments. The authors wish to thank Mr. Alfonso
Perez, Departamento de Fisiologia, Universidad de Alicante,
and Mr. J. Fisher, FT Electrotek, 5386 Colter Drive, Kearns,
UT 84118, for designing and building the portable stimulator;
to Mr. S. Moya for technical assistance; and to Drs. A. Neufeld
and C. Eyzaguirre for critical reading of the manuscript.
From the Departamento de Fisiologia and *Departamento de Oftalmologia, Facultad de Medicina, Universidad de Alicante, Alicante,
Spain. Supported by grant number 1382/82 from the CAICYT Spain,
and by the Pain Research Laboratory, Matrix Biology Institute,
Bridgefield, New Jersey, USA. Submitted for publication: January
22, 1986. Reprint requests: Dr. C. Belmonte, Depto de Fisiologia,
Facultad de Medicina, Universidad de Alicante, Alicante, Spain.
References
1. Davson H and Matchett PA: The control of the intraocular pressure in the rabbit. J Physiol 113:387, 1951.
2. Langham ME and Rosenthal AR: Role of cervical sympathetic
nerves in regulating intraocular pressure and circulation. Am J
Physiol 210:786, 1966.
3. Bill A: Autonomic nervous control of uveal blood flow. Acta
Physiol Scand 56:70, 1981.
4. Belmonte C, Perez E, and Gallar J: Chronic stimulation of the
ocular sympathetic in unanesthetized rabbits. ARVO Abstracts.
Invest Ophthalmol Vis Sci 26 (Suppl):234, 1985.
5. Sweet JF and Bourassa CM: Electrical stimulation of peripheral
nerve. In Electrical Stimulation Research Techniques. Patterson
MM and Kesner RP, editors. New York, Academic Press, Inc,
1981, pp. 243-295.
6. Bernard C: Lecons sur la Physiologie et la Pathologie du Systeme
Nerveaux. Paris, Baillere, 1958.
7. Nja A and Purves D: Specific innervation of guinea-pig superior
cervical ganglion cells by preganglionic fibres arising from different levels of the spinal cord. J Physiol 264:565, 1977.
8. Rosenblueth A: The chemical mediation of autonomic nervous
impulses as evidenced by summation of responses. Am J Physiol
102:12, 1932.
9. Folkow B: Impulse frequency in sympathetic vasomotor fibers
correlated to the release and elimination of the transmitter. Acta
Physiol Scand 25:49, 1952.
10. Polosa C: Spontaneous activity of sympathetic preganglionic
neurons. Can J Physiol Pharmacol 46:887, 1968.
Chorionic Gonadotropin Decreases Intraocular Pressure
and Aqueous Humor Flow in Rabbit Eyes
Joseph Elman, Joseph Caprioli, Marvin Sears, Alden Mead, and Peter Rubin
The effect of human chorionic gonadotropin (hCG) on the
rabbit eye was studied. Inrravitreal injections of hCG in albino
rabbits provoked a reduction of intraocular pressure (IOP).
Intravenous administration of hCG in single doses of 5,00010,000 units in male pigmented rabbits caused a significant
reduction in IOP from 1.5-5 hr after injection. When two
successive intravenous doses of 5,000 units of hCG were given
at 0 and 3 hr to pigmented rabbits, a significant reduction of
net aqueous flow occurred, as measured by scanning fluorophotometry. These results indicate that the decrease in
aqueous flow rate in the rabbit eye after administration of
hCG can account for the reduction in IOP. Invest Ophthalmol
Vis Sci 28:197-200, 1987
Gonadotropins are a class of glycoprotein hormones
which include human chorionic gonadotropin (hCG),
follicle stimulating hormone (FSH), and luteinizing
hormone (LH). Each of these is comprised of two polypeptide subunits, an alpha and a beta chain. The alpha
chains possess nearly identical peptide sequences,
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