(Investigative Ophthalmology and Visual Science. 2000;41:2684-2688.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Sympathetic Nervous System Plays a Role in Postnatal Eyeball Enlargement in the Rabbit
John H. K. Liu1,
Hamed Farid1 and
David H. Rapaport2
From the Departments of
1 Ophthalmology and
2 Surgery (Division of Anatomy), University of California, San Diego, La Jolla.
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Abstract
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PURPOSE. To examine the role of ocular sympathetic activity in the enlargement
of the rabbit eyeball during postnatal growth.
METHODS. Fourteen New Zealand albino rabbits aged 5 weeks underwent unilateral
surgical transection of the cervical sympathetic trunk caudal to the
superior cervical ganglion. Postoperative enlargement of both eyeballs
was monitored by measuring the axial length and corneal diameters every
2 weeks for 22 weeks (7–27 weeks of age). Rabbits were housed under a
12-hour light/12-hour dark cycle, and the measurements were made in the
middle of the light period. At a final age of 30 to 31 weeks, the
refractive state of the whole eye was determined on both sides by
measurement through the central cornea with a refractometer. Rabbits
were then killed, eyeballs enucleated, and their ocular volumes
determined.
RESULTS. From 9 weeks of age the axial length and corneal diameters were
significantly shorter (P < 0.05) in the decentralized
eye (surgical side) compared with the intact eye. This reduction
remained statistically significant throughout the study period.
However, the final refractive states of the two eyes were found not to
be different. The mean ocular volume determined after postmortem
enucleation was 4.5% less in the decentralized eye than in the intact
eye (P < 0.05).
CONCLUSIONS. Sympathetic nervous system activity is involved in the normal
enlargement of the rabbit eyeball during postnatal growth. However,
removal of the ocular sympathetic tone at the age of 5 weeks does not
significantly alter the refractive state of the eye when measured in
young adulthood.
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Introduction
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When young adult rabbits were entrained under a 12-hour
light/12-hour dark cycle, a consistent 24-hour variation in axial
length (the distance between the center of the cornea and the posterior
pole of the retina) was observed.1
This variation of axial
length was endogenous and independent of the level of illumination
because these data were collected during an acute constant dark period.
The physiological basis for the 24-hour variation in axial length is
largely unknown, except that ocular sympathetic activity probably plays
a role. When the eye’s sympathetic tone was removed by surgical
transection of the cervical sympathetic trunk in young adult rabbits,
the magnitude of the nocturnal elongation of axial length was smaller
on the operated side than the unoperated, control side.1
The eye, in concert with the body, grows considerably during postnatal
development. Given that ocular sympathetic activity is probably
involved in the nocturnal elongation of axial length,1
we
hypothesized that ocular sympathetic activity might play a role in the
postnatal enlargement of the rabbit eyeball. Such a positive
relationship between the sympathetic nervous system and eyeball
enlargement has not been previously observed in humans or any other
animal species. Human patients with congenital Horner’s syndrome, in
which sympathetic signals to the head (including the eye) are
compromised early in life, have not been described as having a gross
abnormality in eyeball size.2
Experimental data from
chicks, which are frequently used to study eye growth, indicate a
negative correlation between ocular sympathetic activity and eyeball
enlargement. Superior cervical ganglionectomy in neonatal chicks (2
days post hatching) potentiates the developmental increases in eye
weight3
and axial length.4
In the present study, we examined the enlargement of the rabbit eyeball
for approximately 6 months after surgically removing its sympathetic
tone at 5 weeks of age. Postoperatively, the sizes of the eyeballs on
the operated side and on the unoperated side were compared at regular
intervals until the rabbit grew to young adulthood. Eyes were refracted
before euthanasia, and ocular volumes were measured after postmortem
enucleation.
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Methods
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New Zealand albino rabbits were used in accordance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research.
Fourteen rabbits, 4 weeks of age, were purchased from a local supplier.
At this time they were approximately 0.7 kg in body weight and were
weaned from their mothers. Rabbits were housed in individual cages in a
room kept at a constant temperature (21°C). The lighting was adjusted
to go on at 6 AM and off at 6 PM providing a daily cycle of 12 hours of
light and 12 hours of dark. Food and water were available ad libitum.
Approximately 1 week after arrival, each rabbit underwent unilateral
transection of the cervical sympathetic trunk.5
The
cervical sympathetic trunk was sectioned caudal to the superior
cervical ganglion in the neck area. The postganglionic nerve fibers
remained intact while not receiving their normal preganglionic drive
from cells of the intermediolateral column of the thoracic spinal cord
(decentralization). The transection was performed on the right side in
8 rabbits and on the left side in 6 rabbits. Rabbits were allowed to
recuperate postoperatively in the accustomed daily light–dark cycle.
Between 1 to 2 weeks after surgery the rabbits were examined for the
appearance of miosis and ptosis on the operated side to verify the loss
of sympathetic tone. In addition, decentralization of the ocular
sympathetic nerves was verified by two tests involving either topical
hydroxyamphetamine or topical cocaine.6
These tests are a
way of confirming that the sympathetic nerves (i.e., postganglionic
fibers) were intact in both eyes, but that the sympathetic tone was
lost in the decentralized eye.
Selected parameters of the eyeball (axial length, anterior chamber
depth, lens thickness, vitreous chamber depth, and horizontal and
vertical corneal diameters) were determined before surgery (i.e., at 5
weeks of age) and subsequently every 2 weeks (±1 day) for 22 weeks
postoperatively. At each measurement session, the individual rabbit was
removed from its cage at noon (±1 hour), weighed, and placed in a
shallow tray in a designated area with constant illumination. No
further restraint was necessary. In all cases the right eye was
measured first. One or two drops of 0.1% proparacaine (a 1:5 dilution
of 0.5% commercial preparation) were applied to the eye. An ultrasonic
sensor probe (Humphrey Ultrasonic Biometer, model 810; San Leandro, CA)
was gently placed on the corneal surface with care taken to measure
from the central-most region and to hold the probe perpendicular to the
surface. Values of axial length, anterior chamber depth, and lens
thickness were obtained from the Ultrasonic Biometer. Three
measurements were taken from each eye, and the average was used in data
analyses. The depth of the vitreous chamber was calculated by
subtracting the anterior chamber depth and lens thickness from the
axial length. The horizontal and vertical corneal diameters were
measured manually with a Castroviejo caliper. After data had been
collected from each rabbit, the values of the decentralized eyes and
the intact eyes were grouped and comparisons were made using the paired
t-test. A difference of P < 0.05 was
regarded as statistically significant.
It is known that sympathetic decentralization reduces the nocturnal
elongation of axial length1
and the nocturnal elevation of
intraocular pressure (IOP)5
at 3 to 4 weeks after the
operation. However, the long-term effects of sympathetic
decentralization on axial length and IOP have not been determined. For
12 of the 14 postoperative rabbits in the present study, light–dark
variations in axial length and IOP were studied at weeks 23 to 24
postoperatively (28–29 weeks of age). To eliminate any influence of
environmental light, the measurements were performed in constant
darkness.1
5
All light in the rabbit holding room was
extinguished at 11 AM, and the first set of measurements was made at
noon. Subsequent readings were made every 4 hours, on the hour (±15
minutes), until 8 AM the next day. A dim red photograph-safe light
(wavelength > 600 nm; intensity < 5 lux) was turned on as needed
to assist with the measurements. For each measurement session, axial
length was determined first using the Ultrasonic Biometer as described
previously, and then the IOP was measured with a modified
pneumatonometer previously calibrated for the rabbit eye.5
The average axial length and average IOP in the "subjective" light
period (pooled from the 3 measurements of 8 AM, noon, and 4 PM) and the
dark period (pooled from 8 PM, midnight, and 4 AM) were calculated for
each eye. The changes in the average axial length from the subjective
light period to the dark period between the two eyes were compared
using the paired t-test (n = 12). Similarly,
elevations of average IOP from the subjective light period to the dark
period were compared between the two eyes.
All 14 rabbits were killed at a final age of 30 to 31 weeks, by which
age we were having difficulty performing ultrasonography in a timely
manner due to frequent movements of the rabbit’s head. Immediately
before euthanasia the refractive state of each eye was measured in the
rabbits while conscious. Readings were taken through the central cornea
using a Jena Coincidence Refractometer (model 110; Seiler Instrument &
Manufacturing, St. Louis, MO). At least 3 consecutive measurements were
made for each eye, and the average was calculated. Rabbits were then
killed by intravenous injection of 1 ml Beuthanasia-D
(Schering–Plough, Kenilworth, NJ). The eyes were enucleated, the right
eye first, and rinsed thoroughly with saline. The extraocular muscles
and connective tissue were carefully removed from the globe, and the
optic nerve was cut as close to the sclera as possible. The globe was
cleared of excess fluid by dabbing with filter paper, and it was
carefully immersed in a container (approximately 30 mm in diameter)
half-filled with saline. The height of the fluid column in the
container was measured (to an accuracy of 0.01 mm) using the Ultrasonic
Biometer sensor attached to a stereotaxic micromanipulator (model 1460;
David Kopf Instruments, Tujunga, CA). The sensor was slowly lowered
until it touched the surface of the fluid. The height of the fluid
column was determined before and after immersion of the eyeball. The
change in the height of the fluid column was converted to the volume of
the eyeball according to a preestablished relationship between the
height of fluid column and the volume of saline in the container.
Ocular volumes of the decentralized eyes and the intact eyes were
compared using the paired t-test.
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Results
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Hydroxyamphetamine and cocaine tests confirmed that the unilateral
sympathetic decentralization performed at 5 weeks of age was successful
in all 14 rabbits. All rabbits continued growing during the course of
the study with an increase of body weight from 0.7 ± 0.1 kg
(mean ± SD) initially to 4.5 ± 0.4 kg at the time of
euthanasia. Based on the steady increase in individual body weight and
normal cage behavior, we ascertained that all rabbits recovered from
the surgery normally and remained healthy.
The mean values of axial length, horizontal corneal diameter, lens
thickness, and anterior chamber depth in the decentralized eye and in
the intact eye during the study period are summarized in Figure 1
. Differences between the decentralized eye and the intact eye are
presented in Figure 2
. Beginning at 9 weeks of age, the axial length of the decentralized eye
was significantly less than that of the intact eye. Horizontal and
vertical corneal diameters were also significantly less in the
decentralized eye from 9 weeks of age. The reduction of axial length in
the decentralized eye was from both the anterior and vitreous chamber
components. Anterior and vitreous chamber depths were both less in the
decentralized eyes, although a consistent reduction was not apparent
until slightly different ages (i.e., 13 and 11 weeks, respectively).
The effect of sympathetic decentralization on lens thickness was
relatively small. However, the lens was consistently thicker in the
decentralized eye from 15 weeks of age. These differences in eyeball
parameters between the operated and control sides remained
statistically significant until at least 27 weeks of age.
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Figure 1. The developmental increase of eyeball parameters in the postnatal
rabbit. The mean derived from 14 rabbits is plotted for the control eye
(nonsurgical side; closed circles), and the eye
underwent decentralization of the ocular sympathetic nerves at 5 weeks
of age (open circles). Three parameters (axial length,
horizontal corneal diameter, and anterior chamber depth) showed a
difference between the decentralized eye and the control eye, with the
former consistently being smaller. Only for lens thickness was the
decentralized eye larger than the control eye.
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The light–dark variations in axial length and IOP appeared in 12
rabbits postoperatively at 28 to 29 weeks of age. The pattern of
variation in axial length, as well as IOP, with time was generally
similar in both eyes (Table 1)
; it troughed in the subjective light period and peaked in the dark
period. At all 6 time points, the mean value of either axial length or
IOP was less in the decentralized eye than in the contralateral, intact
eye. For both eyes, the average axial length and IOP in the dark period
were greater than their respective values in the subjective light
period. The elongation of average axial length from the subjective
light period to the dark period in the decentralized eye, 0.13 ±
0.02 mm (mean ± SEM, n = 12), showed no
statistical difference from that of the intact eye, 0.14 ± 0.05
mm. However, the light–dark elevation of average IOP in the
decentralized eye (2.0 ± 0.4 mm Hg) was significantly less
(P < 0.05) than in the intact eye (3.6 ± 0.7 mm
Hg).
The final refractive state of each eye was successfully determined for
13 of the 14 rabbits (1 rabbit presented no clear refractive images).
Measurements were performed at 30 to 31 weeks of age, immediately
before euthanasia. Mean refractive power was -0.1 ± 0.3 D
(mean ± SEM, n = 13) for the decentralized eye
and 0.4 ± 0.4 D for the intact eye. The difference between the
two eyes was neither consistent nor statistically significant (paired
t-test). The mean ocular volume determined postmortem was
2.69 ± 0.06 cm3 (n = 14)
for the decentralized eyes, which was significantly less
(P < 0.05) than that for the intact eyes (2.81 ±
0.05 cm3). The average reduction in the
decentralized eye was 4.5% compared with the intact eye.
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Discussion
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Effects on Eyeball Parameters and Refractive State
Previous studies in rabbits have demonstrated that postnatal
eyeball enlargement can be retarded by damaging ocular structures using
transcleral cryotherapy of the ciliary body7
and laser
photocoagulation or transcleral cryotherapy of the anterior choroid and
retina.8
When an eye is injured it is difficult to
separate normal/physiological and nonphysiological (due to eye
perturbation) mechanisms that might influence ocular growth. In the
present study, the preganglionic sympathetic nerve trunk was sectioned,
whereas the postganglionic sympathetic nerves were not touched. This
would not lead to any direct damage of ocular tissues, although there
might eventually be some anterograde, transsynaptic degeneration of the
postganglionic sympathetic nerves. A transsynaptic degeneration seems
unlikely to develop within a few weeks after the sectioning of the
cervical sympathetic trunk,9
when the reductions of
eyeball parameters begin to appear. The likelihood that transsynaptic
degeneration of the ocular sympathetic nerves would lead to damage of
other ocular tissues is low.
In addition to signals to the eye, unilateral transection of the
cervical sympathetic trunk removes sympathetic signals to other parts
of the head. For example, the pineal gland receives bilateral
postganglionic innervation from the superior cervical ganglia.
Unilateral superior cervical ganglionectomy reduces pineal
function.10
Therefore, unilateral sectioning of the
cervical sympathetic trunk may lead to a centrally mediated change
affecting the whole body including the eyes. By comparing eyeball
parameters between the two eyes, we are able to evaluate only the
direct effects of reducing ocular sympathetic activity. Whether there
is a centrally mediated postoperative influence on eyeball enlargement
cannot be evaluated in an individual rabbit because the effect would be
similar in both eyes.
Our results support the hypothesis that sympathetic input to the eye
plays a role in the postnatal enlargement of the rabbit eyeball.
Decentralization of the ocular sympathetic nerves at the age of 5 weeks
significantly slowed the normal postnatal, developmental increases in
axial length and corneal diameters by 9 weeks of age. The magnitude of
the reduction of these parameters was relatively stable after 11 to 13
weeks of age. It is generally believed that at an early age the rabbit
eyeball is more susceptible to induced growth change than in
adulthood.11
Exogenous forces (an increase of IOP plus a
rise of body temperature) can change the refractive state of the eye (a
reflection of ocular size) in 5- to 6-week-old
rabbits11
12
13
but not in 6- to 8-month-old
rabbits.11
In the present study, the physiological changes
brought about by the sectioning of the cervical sympathetic trunk at
the age of 5 weeks were translated into differences in axial length and
corneal diameters at the age of 9 weeks. But, the same surgical
procedure performed in young adult rabbits in a previous
study1
did not cause a change in axial length, measured in
the subjective light period from 3 to 4 weeks postoperatively.
Lens thickness seemed to be less affected during the early
postoperative period. At a later age (>15 weeks), the lens in the
decentralized eye was thicker than the lens in the intact eye, whereas
the other eyeball parameters remained reduced in the decentralized eye.
Because the equatorial dimension of the eyeball was reduced in the
decentralized eye, there would be less stretch of the lens in its
equatorial direction and, consequently, thickness in the axial
direction might increase.
Although the rabbit eyeball grows considerably during the age interval
of 5 to 31 weeks, it maintains a relatively constant refractive
state.14
The refractive state is determined by the axial
length and the refractive powers of the cornea and lens. In the present
study, both the decentralized and intact eyes had a normal final
refractive state. This suggests that emmetropization took place in the
decentralized eye despite the loss of sympathetic tone. However, it
should be noted that our refractive data were obtained through the
central cornea. We cannot rule out the possibility that loss of
sympathetic tone affects the refractive state through the nasal,
peripheral cornea that is used by rabbits for near
vision.15
How Sympathetic Tone Might Influence Eyeball Enlargement
One possible explanation for our finding that removal of
sympathetic tone slows the normal enlargement of the rabbit eyeball
involves the daily fluctuation of IOP. Light–dark entrained laboratory
rabbits exhibit circadian variation in IOP, with it being high in the
dark period.16
Although several physiological factors are
likely to be involved in this circadian IOP fluctuation, the nocturnal
increase of ocular sympathetic activity is the most important
factor.16
17
A circadian IOP fluctuation of several
millimeters of mercury is within the range for a reversible stretch of
the rabbit eyeball.18
It is possible that the decrease in
the magnitude of daily IOP fluctuation brought about by the sympathetic
decentralization leads to less dynamic stretch on the elastic ocular
coats and less eyeball enlargement. Previously, a 24-hour variation of
IOP was suggested to play a role in normal eye enlargement in
chicks.19
Clinical observations suggest that excessive ocular parasympathetic
tone during visual accommodation may lead to myopia,20
which is usually accompanied by an increase of axial length. Visual
accommodation in humans is an active process determined by the balance
between parasympathetic and sympathetic inputs to the eye. In the
present study, sympathetic inputs to the rabbit eye are irreversibly
lost after sectioning of the cervical sympathetic trunk. Although the
rabbit’s eye has little ability to accommodate,21
it is
possible that the effect of sympathetic decentralization on eyeball
enlargement is related to the now unopposed parasympathetic tone,
independent from accommodation. However, this unopposed parasympathetic
tone causes a decrease in axial length.
Besides the mechanical factor of IOP fluctuation, changes in
biochemical processes in the eye, such as the process of growth
signals, also need to be considered as causative in the retardation of
eyeball enlargement after sympathetic decentralization. Removal of
ocular sympathetic tone in young adult rabbits leads to a rapid and
long-lasting reduction in tyrosinase activity in the choroid and iris,
and this affects iris color in pigmented rabbits.22
23
Whether or not there is a correlation between postnatal eyeball
enlargement and iris pigmentation and whether or not tyrosinase or any
other biochemical process plays a role in the enlargement of rabbit
eyeball warrant further investigation. In chicks, eyeball enlargement
is correlated with biosynthetic activity of the
sclera,24
25
and the ocular growth may be regulated by
various neural mechanisms.26
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Acknowledgements
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The authors thank Huan Li and Roger Kash for their technical
assistance.
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Footnotes
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Supported by NIH Grants EY07544 (JHKL) and EY11875 (DHR).
Submitted for publication August 6, 1999; revised December 20, 1999 and March 10, 2000; accepted March 22, 2000.
Commercial relationships policy: N.
Corresponding author: John Liu, Department of Ophthalmology, University of California, San Diego, La Jolla, CA 92093-0946. joliu{at}ucsd.edu
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Top
Abstract
Introduction
), anterior chamber depth
(
), vitreous chamber depth (
), and lens thickness
(
). (B) shows the parameters of horizontal
corneal diameter (•) and vertical corneal diameter
(
). The error bars represent SEM (n =
14). Differences that reached statistical significance with the paired
t-test are indicated by asterisks
(*P < 0.05; **P < 0.01).