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From the Department of Biological Science and Disease, The New England College of Optometry, Boston, Massachusetts.
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Abstract |
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METHODS. Thirty-three common marmosets were used. Eyes in 26 monkeys served as untreated control eyes, and eyes in 7 received 3 weeks of monocular lid suture to induce changes in eye growth and refractive state. Refractive errors were measured using refractometry and retinoscopy, and axial ocular dimensions, including choroidal thickness, were measured using high-frequency A-scan ultrasonography. Eyes were measured before the lids were sutured and at frequent intervals after lid opening.
RESULTS. In the marmoset, choroidal thickness ranges from 88 to 150 µm and increases significantly during the first year of life. Monocular lid suture initially results in short, hyperopic eyes that then become elongated and myopic. In these animals the choroids of both the experimental and the fellow control eyes also increase in thickness with age but additionally show interocular differences that vary significantly with the relative changes in vitreous chamber depth and refraction. In eyes that are shorter and more hyperopic than control eyes the choroids are thicker, and in eyes that are longer and more myopic than control eyes the choroids are thinner.
CONCLUSIONS. In marmosets, the thickness of the choroid increases during postnatal eye growth. Superimposed on this developmental increase in choroidal thickness there are changes in thickness that are correlated with the induced changes in eye size. These changes are small (<50 µm) in comparison with those observed in the chick, contributing to less than a diopter change in refractive error.
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Introduction |
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In chicks, compensation for imposed defocus occurs through two mechanisms that affect vitreous chamber depth. One involves modulation of the growth of the sclera,7 8 9 10 11 and the other involves changes in the thickness of the choroid.12 13 Specifically, eyes wearing positive lenses, in which the image plane is in front of the retina, show a thickening of the choroid that pushes the retina forward toward the image plane, which is followed by changes in scleral extracellular matrix synthesis and decreased eye growth. Conversely, eyes wearing negative lenses, in which the image plane is behind the retina, show a thinning of the choroid that pulls the retina back toward the image plane, followed by changes in scleral extracellular matrix synthesis and increased eye growth.
There is circumstantial evidence from the chick model suggesting that the choroidal thickness and scleral growth mechanisms are causally related. First, the compensatory changes in choroidal thickness occur within a few hours and precede the compensatory axial growth changes.14 Second, the phase relationships between diurnal rhythms in choroidal thickness and axial length differ, depending on the rate of eye growth; in normal and fast-growing eyes, the two are in antiphase with one another, whereas in slow-growing eyes the two are in phase with one another.15 Third, in tissue culture experiments,16 the condition of the choroid (thin from fast-growing form-deprived myopic eyes or thick from slow-growing eyes recovering from form deprivation myopia) was found to alter the rate of scleral proteoglycan synthesis. Although these findings suggest a causal relationship between choroid thickness, eye growth, and, ultimately, refractive state, it is also possible that choroidal thickness change in chick eyes is a form of long-term accommodation12 17 and is unrelated to the mechanism underlying the visually dependent changes in eye growth.
It is unknown whether choroidal thickness changes occur in mammalian and primate eyes during development and how they relate to the control of ocular growth. Similar to chicks, mammalian scleras undergo changes in proteoglycan synthesis that are associated with induced changes in refractive state. Proteoglycan synthesis in tree shrews and marmoset monkeys is reduced during form deprivation myopia18 19 and is increased in tree shrews during recovery from form deprivation myopia.20 If changes in the choroid are causally related to changes in scleral proteoglycan synthesis and, consequently, eye growth and refractive state, we would expect to see changes in the thickness of primate choroids after experimentally induced refractive errors.
We report on the choroidal thickness changes of the marmoset monkey measured in vivo during normal ocular development and experimentally manipulated eye growth. We show that choroidal thickness in the marmoset can be reliably measured and that small but significant changes are correlated with induced changes in eye growth and refractive state.
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Methods |
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Thirty-three common marmosets (Callithrix jacchus) were used in this study. Untreated eyes in 26 animals provided normal control data for various ages (control eyes). Refractive errors were induced monocularly in the remaining seven animals by lid suture (experimental eyes). The contralateral untreated eyes of these animals served as interocular controls (fellow control eyes).
All marmosets were housed in family groups in our marmoset breeding colony. Artificial lighting was provided by daylight-balanced fluorescent lamps (Vita-Light, Fairfield, NJ) on a 12-hour light–12-hour dark cycle. Temperature was maintained at 75 ± 2°F and humidity at 45% ± 5%. Food and water were provided ad libitum. Food consisted of formulated dry pellets (Marmoset Lite; Animal Spectrum, North Platte, NE) with fresh fruit and protein supplements. Vitamin D3 supplement was provided twice weekly.
Treatments and Measurements
Monocular lid suture was performed at 10 to 17 days of age (mean,
13 days). The lids were reopened 14 to 28 days later (mean, 23 days).
After this manipulation, axial hyperopia (experimental eye shorter than
normal) is typically observed at lid opening and changes over time to
axial myopia (experimental eye longer than normal) without
recovery.21
Lid suture surgery was performed on anesthetized animals (0.2 ml/100 g; alphaxalone (0.9%), alphadolone acetate (0.3%) [Saffan]; Pitman-Moore, UK) as previously described.21 The lid margins were trimmed, and the tarsal plate was separated from the lid. The upper and lower tarsii were sutured together with 7-0 polyvicryl, and the upper and lower lids were then sutured together with 5-0 silk, leaving a small (1–2 mm) drainage opening at the nasal canthus. The outer sutures were removed 5 to 7 days after lid fusion. To conclude the deprivation period, the lids were reopened along the fusion line, with animals under Saffan anesthesia, and healed without notable lid dysfunction.
Refractive errors were measured by refractometry and streak retinoscopy in animals under Saffan anesthesia. A lid retractor was used to hold the lids open. Cycloplegia was induced with 2 drops of 1% cyclopentolate applied topically 5 to 10 minutes apart; refractions were measured 60 minutes later. This procedure reliably produces maximal cycloplegia in young marmosets.22 Retinoscopy was performed first, followed by refractometry with a Hartinger Coincidence Refractometer (Carl Zeiss, Oberkochen, Germany). The two measures were always performed independently by the same two investigators. All refraction data presented are the means of the retinoscopic and refractometry data expressed as equivalent spherical refractive errors derived by averaging the two principal meridians in each case.
Axial ocular dimensions, including choroidal thickness, were measured using high-frequency A-scan ultrasonography. Our system is based on that developed by Wildsoet and Wallman13 (Department of Biology, City College of New York, NY). We used a 33-MHz piezoelectric immersion transducer (model PZ25-0.25-SU-R1.00; Panametrics, Waltham, MA) driven by an ultrasound pulser receiver (model 5072 PR-15U; Panametrics). The transducer was coupled to the eye with a 16-mm water-filled stand-off that positions the focal zone of the sound wave inside the vitreous chamber of marmoset eyes at all ages. The ultrasound signal was digitized for analysis using a 100-MHz analog-to-digital conversion board (model STR-8100; Sonix, Springfield, VA). The high-frequency transducer coupled with the sampling rate of the digitizing board provided good resolution of the echoes returning from the posterior ocular tunics. Figure 1 shows an example of a typical ultrasound trace from a marmoset eye. The echoes from the posterior ocular tunics are expanded to show the thicknesses of the retina, choroid, and sclera. Each echo gives rise to a complex of peaks. The specific peaks used as measurement reference points were selected based mainly on their consistent appearance within repeated measurements and between different individuals (see Fig. 2 ). In chicks, peaks similarly identified have also been cross-referenced to histology and other in vivo measurements of the posterior ocular tunics.23 Echo latencies arising from the different ocular surfaces were converted to distances between surfaces using estimates of the velocity of sound in the different media of chick eyes.24 The thicknesses of the retina, choroid, and sclera were calculated using the velocity of sound in vitreous humor (1.534 m/sec).
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Ultrasound data for each eye are the average of a minimum of eight individual traces. The eye was typically realigned with the ultrasound probe several times over the course of measurement. We used as a criterion for proper alignment echoes of approximately equal amplitude from the anterior and posterior lens surface. On average, the standard errors of the mean of repeated measurements were less than 10 µm for vitreous chamber depth, and less than 5 µm for choroidal thickness. The precision25 26 of the ultrasound measures was determined by measuring one eye of an individual marmoset eight times during a 15-minute period. The SD of the mean difference between repeated measurements was ±36 µm for vitreous chamber depth and ±11 µm for choroidal thickness.
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Results |
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Choroidal Thickness in Eyes with Induced Refractive Errors
It has been reported in young marmosets that 3 weeks of visual
form deprivation by lid suture results in axial hyperopia that
eventually becomes axial myopia over time after the opening of the
lids.21
The present results confirm this observation. At
the time of lid opening, experimental eyes were, on average, more
hyperopic and shorter than their fellow control eyes. By the end of the
monitoring period, the experimental eyes had become more myopic and
longer than the control eyes. Figure 5
shows, plotted against time, the refractive error (top) and vitreous
chamber depth (bottom) expressed as the interocular difference between
experimental eyes and their contralateral control eyes. The changes
over time in both refractive error and vitreous chamber depth were
statistically significant (ANOVA, P < 0.01).
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Discussion |
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Normal Development of Choroidal Thickness
In untreated marmosets the thickness of the choroid increases by
approximately 28% from birth to puberty, when the eye is nearly adult
size.27
Similar results have been reported for
macaques.28
In marmosets, most of the increase in
thickness occurs early in development, during the period of rapid
ocular growth and emmetropization from infant hyperopia. Because this
emmetropization involves the elongation of the back of the eye toward
the focal plane, the observed increase in choroidal thickness is in the
wrong direction to be a compensatory component of the emmetropization.
Furthermore, the increase in thickness cannot be accounted for by a
passive mechanical change associated with increasing the size of the
eye, which would cause a thinning of the choroid.
We speculate that the normal choroidal thickening during the juvenile period of rapid eye growth may function to slow the growth of the eye, either by providing a diffusion barrier to a putative retinally derived growth signal, or as a mechanical buffer for the sclera, as proposed years ago by van Alphen,29 30 who showed in experiments on enucleated eyes that the choroid could withstand the effects of significant increases in force, such as that exerted by intraocular pressure (IOP). If a change in force on the sclera could, either by stretching the tissue or by causing a change in scleral biosynthesis, cause a change in eye size, then preventing that force from reaching the sclera could be a means of controlling the effects of that force. Relevant to this hypothesis, Friedman31 showed that the IOP-related stress on the posterior ocular tunics was inversely proportional to the thicknesses of the tunics. The developmental thickening of the choroid could, in effect, provide a breaking mechanism to slow axial growth of the eye at the appropriate stage in development. Conversely, thinner choroids and weaker (more compliant) scleras may be associated with eyes growing abnormally fast, as discussed in the next section.
Changes in Choroid Thickness Associated with Refractive Errors
If the choroid acts as a modulator of scleral growth, either as a
mechanical buffer as just described or, alternatively, as a diffusion
barrier to a retinally derived factor, then thinner choroids would be
associated with longer eyes, and changing scleras. In fact, there is
some circumstantial evidence in support of this view. In human myopia,
for example, thinner than normal choroids32
are associated
with weaker scleras.33
34
35
In primates with form
deprivation myopia, thinner scleras and changes in extracellular matrix
have also been reported.19
36
37
In tree shrews,
treatments that alter scleral collagen cross-linking exacerbate form
deprivation myopia,38
and greater scleral compliance has
been measured in the sclera of form deprived eyes relative to control
eyes.39
If choroidal thickness changes are responsible for
such changes in the sclera, and consequently, increased eye growth and
myopia, we should see choroidal thickness changes with induced
refractive errors.
In our study, we used short-duration lid suture to ascertain whether changes in refractive error are associated with changes in choroidal thickness in marmosets. This paradigm results in an initial hyperopia that progresses to myopia.21 We found that, superimposed on the developmental increase in choroidal thickness, the transient hyperopia seen after lid opening was associated with thicker choroids, and the progression toward myopia was associated with thinner choroids. In this respect, the results in marmosets seem qualitatively similar to those in chicks: Small, hyperopic eyes have thicker choroids, and large, myopic eyes have thinner choroids.12 13 40
At first glance, our results appear to support a causal role between choroid thickness and axial growth rate, whereby thick choroids inhibit growth and thin ones accelerate it. However, further examination indicates that the relationship between choroid thickness and eye size in the marmoset is not consistent with a true compensatory mechanism as seen in chicks. First, the choroidal thickness changes in the marmoset are approximately an order of magnitude smaller than those described in the chick. The largest changes would account for less than a diopter of refractive change. Second, and more important, in chicks the smaller hyperopic eyes and thicker choroids occur as the result of compensation for myopic defocus. In marmosets, the initial hyperopia and thick choroids are the result of form deprivation (an open-loop visual condition), which, by definition, is not a compensation but probably represents a default growth pattern. It is interesting, however, that form deprivation produces opposite responses in axial length and choroidal thickness in both species. In the chick, form deprivation produces axial length increases with choroidal thinning, in the marmoset it produces reduced axial growth and choroidal thickening. Restoring form vision results in changes in both choroidal thickness and axial length that are in the appropriate direction for compensation in both species. It could be argued that the thinning of the thick choroids in hyperopic marmosets after the end of deprivation is indeed a compensatory adjustment for the hyperopic defocus. However, that these eyes eventually overshoot emmetropia to become myopic, with thinner than normal choroids, argues against the choroid’s ability to respond actively to a defocus, at least bidirectionally, as seen in chicks. It is possible that the choroidal response to defocus in primates is unidirectional thinning, in the same way that the stronger of the compensatory eye growth responses to spectacle lenses in macaques seem to favor elongation in response to negative lenses rather than slowing growth in response to positive ones.4 That the marmoset choroid is able to become thicker during normal development and lid suture argues that the unidirectionality of the response is at the level of the signal detector, as opposed to the response generator.
It could be argued that the overshoot of emmetropia to myopia is a consequence of an unchecked "grow" signal that is initiated by the hyperopic defocus, but it is equally plausible that it is a delayed manifestation of earlier form deprivation.21 In either case, it is possible that the thinning of the choroid over the period after lid opening may be merely a mechanical consequence of an eye elongating abnormally rapidly. If we model the choroid as a simple isovolumetric shell of a sphere, we find that an increase in radius comparable with that seen in induced axial myopia in marmosets predicts approximately 24 µm of thinning, which compares closely with the observed thinning of approximately 20 µm. Conversely, decreasing the size of the eye by an amount seen, on average, in our hyperopic eyes resulted in a predicted increase in thickness of approximately 33 µm compared with the 12 µm observed. Thus, a mechanical effect could account for the observed choroidal thinning with axial myopia but is insufficient to account for the thicker choroids associated with axial hyperopia. In addition, this model does not take into account the increasing choroidal thickness observed with age. If it did, the disparity between the observed and predicted choroidal thickness change in shorter hyperopic eyes would be even greater.
In this study we showed that small changes in choroidal thickness occur in a primate model for eye growth. Exactly how the changes in choroid thickness are achieved is a matter for speculation. Several hypotheses with varying amounts of supporting evidence have been suggested. These include changes in one or several of the following: choroidal blood flow,41 choroidal extracellular matrix synthesis,12 uveoscleral flow,42 lymphatic or capillary fenestrations,43 or nonvascular choroidal smooth muscle tone.44 45 46
From research using chickens to investigate eye growth, it has been postulated that changes in choroidal thickness directly effect changes in scleral growth. The existence in chicks, tree shrews, and monkeys of a similar relationship between choroidal thickness and changes in eye growth (thicker choroids being associated with slower growing eyes and thinner choroids with faster growing eyes) appears to give additional support to a causal relationship. However, that the magnitude of the choroidal thickness changes in marmosets, tree shrews,47 48 and macaques28 are so small compared with those seen in chick implies that large changes in choroidal thickness are not required for altered eye growth in the primate. This does not discount, however, the possibility that the primate choroid is involved in growth regulation, but to a much lesser degree. Alternatively, the choroidal changes in chicks may be part of a local retinal focusing mechanism12 that is not necessary in a species with foveal retinas in which eye movements and accommodation obviate the need to adjust refraction more peripherally.
In conclusion, we found that the marmoset choroid undergoes small, but significant, changes in thickness that are correlated with the refractive state and size of the eye. Small hyperopic eyes were associated with thicker choroids, and large myopic eyes were associated with thinner choroids. Although the thinning response may be visually guided by the hyperopic defocus initially experienced, we cannot rule out alternative explanations.
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Acknowledgements |
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Footnotes |
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Submitted for publication June 16, 1999; revised August 17, 1999; accepted September 3, 1999.
Commercial relationships policy: N.
Corresponding author: David Troilo, New England College of Optometry, 424 Beacon Street, Boston, MA 02115. troilod{at}ne-optometry.edu
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