John T. Siegwart

Regulation of the mechanical properties of tree shrew sclera by the visual environment

Experiments in several species have shown that the axial elongation rate of the developing eye can be increased or decreased by manipulating the visual environment, indicating that a visually guided emmetropization mechanism controls the enlargement of the vertebrate eye during postnatal development. Previous studies in tree shrews (Tupaia glis belangeri) suggest that regulation of the mechanical properties of the sclera may be an important part of the mechanism that controls the axial elongation rate in this mammal. To learn whether the mechanical properties of the sclera change when the axial elongation rate is increased or decreased under visual control, uniaxial mechanical tests were performed on 3-mm wide strips of tree shrew sclera. The creep rate was measured under 1, 3, and 5 g of tension, maintained for 30 min at each level. The modulus of elasticity was calculated from the elastic extension that occurred when the force was increased from 0 to 1 g, 1 to 3 g, and 3 to 5 g. Both were measured in the sclera of both eyes from animals exposed to four experimental conditions: (1) Normal development, at intervals from the day of natural eyelid opening (day 1 of visual experience [VE]) to greater than 5 years of age; (2) Monocular form deprivation (MD), for varying lengths of time; (3) Recovery from MD; (4) Monocular -5 D lens treatment. The creep rate was low in normal animals (1-2% elongation/h), did not change significantly between day 1 and day 75 of VE, and was not significantly different between the two eyes. Four days of MD produced a 200-300% increase in creep rate in the sclera from deprived eyes. Creep rate remained similarly elevated after 11 and 21 days of MD. After 2 days of recovery, which followed 11 days of MD, the creep rate of sclera from the recovering eyes was below normal levels. In animals that wore a monocular -5 D lens for up to 21 days, creep rate increased, and then decreased, in concert with the increase, and decrease, in axial elongation rate as the eyes compensated for the lens. The modulus of elasticity of the sclera was not significantly affected by any manipulation. The temporal correspondence between changes in axial elongation rate and changes in creep rate support the hypothesis that regulation of the time-dependent mechanical properties of fibrous mammalian sclera plays a role in controlling axial elongation rate during both normal emmetropization and the development of refractive errors.

Effect of interrupted lens wear on compensation for a minus lens in tree shrews.

BACKGROUND When a young animal wears a monocular minus (concave) lens that shifts the focal plane away from the cornea, the vitreous chamber elongates over a period of days, shifting the retinal location to compensate for the altered focal plane. We examined the effect of removing the lens for a portion of each day on the amount of compensation in tree shrews. METHODS Starting 24 days after natural eye opening, juvenile tree shrews wore a goggle frame that held a -5 D lens in front of one eye, with an open frame around the fellow control eye. The goggle was removed for 0, 0.5, 1, 2, or 7 h each day (N = 5, 5, 5, 5, and 3 animals per group, respectively), starting 0.5 h after the start of each 14 h light-on period. After 21 days of treatment, measures were made of the cycloplegic refractive state (streak retinoscopy) and the ocular component dimensions (A-scan ultrasound). Normal animals that experienced 14 h each day with no lens (N = 3) were also examined. RESULTS The treated eyes of the 0 h group developed full refractive compensation for the lens (treated eye - control eye, mean +/- SEM = -5.8+/-1.1 D) and had increased vitreous chamber depth (0.13+/-0.02 mm) and axial length (0.12+/-0.02 mm) relative to the untreated control eye. The groups in which the lens was removed for 0.5 and 1 h each day showed partial compensation for the -5 D lens, both in refractive state (-4.2+/-0.4 D; -2.9+/-1.6 D) and in vitreous chamber depth (0.12+/-0.02 mm; 0.09+/-0.02 mm). The 2, 7, and 14 h (normal) groups showed no significant refractive or axial compensation. In the 0.5 and 1 h groups, A-scan ultrasound showed a thinning of the region between the front of the retina and back of the sclera. CONCLUSIONS The eyes of tree shrews can tolerate altered monocular visual stimulation produced by a minus lens worn for 12 h of a 14-h light cycle without developing an induced myopia. However, when the lens is worn more than 12 of 14 h each day, compensation appears to increase linearly with decreased lens-off time. If the eyes of human children respond similarly to defocus from near work or other sources, it would seem that the defocus must be present almost all the time to induce myopia. If defocus contributes to human myopia through a compensation mechanism, then an increase in the amount of time that focused images are present should reduce myopic progression.

The susceptible period for deprivation-induced myopia in tree shrew

To examine the susceptible period for deprivation-induced myopia, six groups of tree shrew pups (Tupaia glis belangeri) were monocularly deprived for 12 days with an opaque occluder starting 7, 15, 21, 33, 48, or 63 days after natural eyelid opening. Compared to the untreated fellow control eye, significant myopia and vitreous chamber elongation were produced by the deprivation in all six groups. The effect was greater in the middle three groups in comparison with the youngest and the two oldest groups and the amount of induced myopia and axial elongation was not proportional to the normal rate of axial growth. The peak period of susceptibility was between approximately 15 and 45 days after eye opening during the juvenile, slow-elongation phase of ocular development when the eye is within 7% of its adult axial length. Significant myopia and axial elongation were also induced in adult animals by 70 days of monocular deprivation. To examine recovery from deprivation-induced myopia, the occluder was removed at the end of the 12 day deprivation period. After an additional 48 days of binocular visual experience, no significant myopia was present in the previously deprived eyes in any experimental group. During the recovery period, the elongation rate of the previously deprived eyes was reduced in comparison with the control eyes while normal corneal flattening and lens development continued, thus reducing the myopia. No difference in corneal curvature, relative to the untreated control eyes, was found after deprivation or after the recovery period. Data are presented which suggests that changes in the thickness of the choroid may occur in this mammal during deprivation and recovery that are in the same direction, but of smaller magnitude, than those reported in the chicken. The results of this study provide evidence that visually guided emmetropization occurs in this mammalian species during a period of ocular development analogous to the juvenile period in humans.

Light levels, refractive development, and myopia – A speculative review

Recent epidemiological evidence in children indicates that time spent outdoors is protective against myopia. Studies in animal models (chick, macaque, tree shrew) have found that light levels (similar to being in the shade outdoors) that are mildly elevated compared to indoor levels, slow form-deprivation myopia and (in chick and tree shrew) lens-induced myopia. Normal chicks raised in low light levels (50 lux) with a circadian light on/off cycle often develop spontaneous myopia. We propose a model in which the ambient illuminance levels produce a continuum of effects on normal refractive development and the response to myopiagenic stimuli such that low light levels favor myopia development and elevated levels are protective. Among possible mechanisms, elevation of retinal dopamine activity seems the most likely. Inputs from intrinsically-photosensitive retinal ganglion cells (ipRGCs) at elevated light levels may be involved, providing additional activation of retinal dopaminergic pathways.

The effect of age on compensation for a negative lens and recovery from lens-induced myopia in tree shrews (Tupaia glis belangeri).

We examined in tree shrews the effect of age on the development of, and recovery from, myopia induced with a negative lens. Starting at 11, 16, 24, 35 or 48days after natural eye-opening (days of visual experience [VE]), juvenile tree shrews (n=5 per group) wore a monocular -5D lens for 11days. A long-term lens-wear group (n=6) began treatment at 16days of VE and wore the lens for 30days. A young adult group (n=5) began to wear a -5D lens between 93 and 107days of VE (mean+/-SD, 100+/-6days of VE) and wore the lens for 29-54days (mean+/-SD, 41.8+/-9.8days). The recovery phase in all groups was started by discontinuing -5D lens wear. Contralateral control eyes in the three youngest groups were compared with a group of age-matched normal eyes and showed a small (<1D), transient myopic shift. The amount of myopia that developed during lens wear was measured as the difference between the treated and control eye refractions. After 11days of lens wear, the induced myopia was similar for the four younger groups (near full compensation: 11days, -5.1+/-0.4D; 16days, -4.7+/-0.3D; 24days, -4.9+/-0.4D; 35days, -4.0+/-0.02) and slightly less in the oldest juvenile group (48days, -3.3+/-0.5D). The young adult animals developed -4.8+/-0.3D of myopia after a longer lens-wear period. The rate of compensation (D/day) was high in the 4 youngest groups and decreased in the 48-day and young adult groups. The refractions of the long-term lens-wear juvenile group remained stable after compensating for the -5D lens. During recovery, all animals in the youngest group recovered fully (<1D residual myopia) within 7days. Examples of both rapid (<10days) and slow recovery (>12days) occurred in all age groups except the youngest. Every animal showed more rapid recovery (higher recovery slope) in the first 4days than afterward. One animal showed extremely slow recovery. Based on the time-course of myopia development observed in the youngest age groups, the start of the susceptible period for negative-lens wear is around 11-15days after eye opening; the rate of compensation remains high until approximately 35days of VE and then gradually declines. Compensation is stable with continued lens wear. The emmetropization mechanism, both for lens compensation and recovery, remains active into young adulthood. The time-course of recovery is more variable than that of compensation and seems to vary with age, with the amount of myopia (weakly) and with the individual animal.

Gene expression signatures in tree shrew choroid during lens-induced myopia and recovery

Gene expression in tree shrew choroid was examined during the development of minus-lens induced myopia (LIM, a GO condition), after completion of minus-lens compensation (a STAY condition), and early in recovery (REC) from induced myopia (a STOP condition). Five groups of tree shrews (n = 7 per group) were used. Starting 24 days after normal eye-opening (days of visual experience [DVE]), one minus-lens group wore a monocular -5 D lens for 2 days (LIM-2), another minus-lens group achieved stable lens compensation while wearing a monocular -5 D lens for 11 days (LIM-11); a recovery group also wore a -5 D lens for 11 days and then received 2 days of recovery starting at 35 DVE (REC-2). Two age-matched normal groups were examined at 26 DVE and 37 DVE. Quantitative PCR was used to measure the relative differences in mRNA levels in the choroid for 77 candidate genes that were selected based on previous studies or because a whole-transcriptome analysis suggested their expression would change during myopia development or recovery. Small myopic changes were observed in the treated eyes of the LIM-2 group (-1.0 ± 0.2 D; mean ± SEM) indicating eyes were early in the process of developing LIM. The LIM-11 group exhibited complete refractive compensation (-5.1 ± 0.2 D) that was stable for five days. The REC-2 group recovered by 1.3 ± 0.3 D from full refractive compensation. Sixty genes showed significant mRNA expression differences during normal development, LIM, or REC conditions. In LIM-2 choroid (GO), 18 genes were significantly down-regulated in the treated eyes relative to the fellow control eyes and 10 genes were significantly up-regulated. In LIM-11 choroid (STAY), 10 genes were significantly down-regulated and 12 genes were significantly up-regulated. Expression patterns in GO and STAY were similar, but not identical. All genes that showed differential expression in GO and STAY were regulated in the same direction in both conditions. In REC-2 choroid (STOP), 4 genes were significantly down-regulated and 18 genes were significantly up-regulated. Thirteen genes showed bi-directional regulation in GO vs. STOP. The pattern of differential gene expression in STOP was very different from that in GO or in STAY. Significant regulation was observed in genes involved in signaling as well as extracellular matrix turnover. These data support an active role for the choroid in the signaling cascade from retina to sclera. Distinctly different treated eye vs. control eye mRNA signatures are present in the choroid in the GO, STAY, and STOP conditions. The STAY signature, present after full compensation has occurred and the GO visual stimulus is no longer present, may participate in maintaining an elongated globe. The 13 genes with bi-directional expression differences in GO and STOP responded in a sign of defocus-dependent manner. Taken together, these data further suggest that a network of choroidal gene expression changes generate the signal that alters scleral fibroblast gene expression and axial elongation rate.

Refractive State of Tree Shrew Eyes Measured with Cortical Visual Evoked Potentials

Purpose. To determine the refractive state of tree shrew eyes using visual evoked potentials (VEP’s) recorded from primary visual cortex and compare the values with those obtained with streak retinoscopy and with an autorefractor. Methods. VEP’s were recorded in seven normal tree shrews and three animals in which ∼5 D of myopia (relative to control eye) was induced by monocular −5 D lens wear. While the animals were awake, refractive correction was measured with an autorefractor before and after cycloplegia (1% atropine and 2.5% phenylepherine). When anesthetized, cycloplegic refractive correction was measured with streak retinoscopy. Then VEP’s were produced with square-wave counterphased (1 Hz) high-contrast checkerboard patterns near the animals’ high spatial frequency cutoff. Spherical lenses (2 D steps) were placed before the eye, and the VEP (average of 128 sweeps) was measured to determine the lens that produced the largest first positive peak (P1). Results. VEP’s were obtained over a broad range of trial lenses. Tuning was narrower when check sizes were small. In normal and control eyes, the P1 amplitude was largest, on average, for a trial lens of (mean ± SD) −0.6 ± 1.6 D (corrected for working distance but not vertex distance). The mean streak retinoscopy value (spherical equivalent at the corneal plane) was 7.0 ± 0.8 D, and mean autorefractor values were 4.0 ± 1.1 D (cycloplegic) and 3.7 ± 1.2 D (noncycloplegic). In the eyes that compensated for a −5 D lens, the largest P1 values occurred with lenses with a power of −6.3 ± 3.2 D. Thus, the VEP measure showed a similar treated vs. control eye difference as did streak retinoscopy (treated eyes, 4.7 ± 0.4 D myopic) and the autorefractor (treated eyes, 4.8 ± 0.5 D myopic). Conclusions. Normal tree shrew eyes are approximately emmetropic. The hyperopic values obtained with streak retinoscopy and the autorefractor are consistent with the presence of a “small-eye artifact” in tree shrews. Eyes that have compensated for a −5 D lens are myopic by approximately the value of the lens.

Perspective: How Might Emmetropization and Genetic Factors Produce Myopia in Normal Eyes?

Substantial evidence has emerged over the past decades for a role of genetics in the development of human refractive error. There is also an emmetropization mechanism that uses visual signals to match the axial length to the focal plane. There has been little discussion of how these two important factors might interact. We explore here ways in which genetic factors driving axial growth may interact with the emmetropization mechanism, mostly to produce emmetropic eyes but often to produce myopia. An important factor may be a normal, yet reduced ability of juvenile eyes to use myopia to restrain genetically driven axial elongation. Reduced ability to respond to myopia by slowing axial elongation may contribute to the development of myopia in cases where genetics alone would make the axial length longer than the focal plane.

Changing Material Properties of the Tree Shrew Sclera During Minus Lens Compensation and Recovery

PURPOSE To estimate two collagen-specific material properties (crimp angle and elastic modulus of collagen fibrils) of the remodeling tree shrew sclera during monocular -5 diopter (D) lens wear and recovery. METHODS Tensile tests were performed on scleral strips obtained from juvenile tree shrews exposed to three different visual conditions: normal, monocular -5 D lens wear to induce myopia, and recovery. Collagen fibrils are crimped in the unloaded sclera and uncrimp as the tissue stiffens under load. Inverse numerical analyses were performed to estimate the (unloaded) crimp angle and elastic modulus of collagen fibrils using a microstructure-based constitutive model. RESULTS Compared with the control eye, the crimp angle was significantly higher in the treated eye after 2 days and remained significantly higher until 21 days of lens wear (P < 0.05). The difference between the crimp angle of the treated and control eye rapidly vanished during recovery in concert with the changes in axial elongation rate. A rapid and extensive increase in the elastic modulus was seen in both eyes after starting and stopping the lens wear. CONCLUSIONS The estimated change in the crimp of scleral collagen fibrils is temporally associated with the change in axial elongation rate during myopia development and recovery. This finding suggests that axial elongation may be controlled by a remodeling mechanism that modulates the collagen fibril crimp. The observed binocular changes in scleral stiffness are not temporally associated with the axial elongation rate, indicating that scleral stiffening may not be causally related to myopia.

Intravitreally-administered dopamine D2-like (and D4), but not D1-like, receptor agonists reduce form-deprivation myopia in tree shrews

We examined the effect of intravitreal injections of D1-like and D2-like dopamine receptor agonists and antagonists and D4 receptor drugs on form-deprivation myopia (FDM) in tree shrews, mammals closely related to primates. In eleven groups (n = 7 per group), we measured the amount of FDM produced by monocular form deprivation (FD) over an 11-day treatment period. The untreated fellow eye served as a control. Animals also received daily 5 µL intravitreal injections in the FD eye. The reference group received 0.85% NaCl vehicle. Four groups received a higher, or lower, dose of a D1-like receptor agonist (SKF38393) or antagonist (SCH23390). Four groups received a higher, or lower, dose of a D2-like receptor agonist (quinpirole) or antagonist (spiperone). Two groups received the D4 receptor agonist (PD168077) or antagonist (PD168568). Refractions were measured daily; axial component dimensions were measured on day 1 (before treatment) and day 12. We found that in groups receiving the D1-like receptor agonist or antagonist, the development of FDM and altered ocular component dimensions did not differ from the NaCl group. Groups receiving the D2-like receptor agonist or antagonist at the higher dose developed significantly less FDM and had shorter vitreous chambers than the NaCl group. The D4 receptor agonist, but not the antagonist, was nearly as effective as the D2-like agonist in reducing FDM. Thus, using intravitreally-administered agents, we did not find evidence supporting a role for the D1-like receptor pathway in reducing FDM in tree shrews. The reduction of FDM by the dopamine D2-like agonist supported a role for the D2-like receptor pathway in the control of FDM. The reduction of FDM by the D4 receptor agonist, but not the D4 antagonist, suggests an important role for activation of the dopamine D4 receptor in the control of axial elongation and refractive development.