Significant differential (deprived eyes versus control eyes) changes in mRNA levels were found in four of the mRNAs examined, but not in the fifth (decorin core protein). The direction and time course of the differential mRNA changes are of interest because, if mRNA changes are the initial step in the tissue-remodeling process that produces the scleral and refractive changes, then the mRNA changes must precede, or at least accompany those changes. Changes in mRNA levels that occur after the scleral changes begin may be secondary responses not directly related to the initial signals that start the tissue-remodeling process. Binocular changes were also found in the levels of the four mRNAs that showed differential changes. The binocular changes are of interest, because they indicate significant effects on the control eye sclera, yet seem not to be reflected in any alteration in refractive state or creep rate in the control eyes compared with the normal eyes. The changes in mRNA levels during recovery are of interest, because many of the biochemical and creep rate changes that occurred during MD were reversed during recovery. Therefore, mRNA level changes involved in the scleral remodeling during MD might be expected to reverse during recovery.
Timing of mRNA Changes
Early Differential Changes during MD
The levels of three of the mRNAs studied showed significant differential changes that occurred as early as detectable increases in vitreous chamber depth () and as early as creep rate changes () and biochemical changes that have been reported in other studies. The mRNA levels of MMP-2 increased, and the levels of MMP-3 and TIMP-1 decreased, in the deprived eyes compared with the control eyes, after 1 or 2 days of lens wear, whereas a difference in vitreous chamber depth was first detectable after between 2 and 4 days of MD.
The onset of these mRNA changes also appeared to match reasonably well with the onset of changes in scleral creep rate () from a previous study.20
It should be noted that Siegwart and Norton20
did not measure creep rate until after 4 days of MD. However, when axial elongation was induced with a –5-D lens, which produces a similar effect, an increase in creep rate was present after 2 days, but was not statistically significant until 4 days.20
In previous biochemical studies, it has been found that scleral glycosaminoglycan (GAG) content is decreased by the fourth day of minus-power lens treatment25
and that a decrease in sulfate incorporation into GAGs is detectable after 5 days of MD, with an elevation after 3 days of recovery from MD.17
A significant reduction in scleral dry weight after 5 days of MD has also been reported.17
Thus, biochemical changes in the sclera are present after 4 days, and possibly as early as after 2 days, of treatment.
Because most studies use a limited number of animals, which limits statistical power, it is difficult to determine exactly when a change begins. Within the limits of the temporal resolution of currently available data, the timing of the early, differential mRNA level changes found in MMP-2, MMP-3, and TIMP-1 in this study suggests that these changes in gene expression could be causally related to the physical changes.
Late Differential Changes during MD
Our previous study27
found differential changes in mRNA levels for collagen after 11 days of MD and a reversed pattern after 4 days of recovery. In the present study, the time course information from groups with shorter periods of MD suggests that the differential changes in collagen mRNA levels occurred after physical and biochemical changes had begun in the sclera. These late differential changes in collagen mRNA levels could have been a secondary response to physical changes within the sclera38
as the tissue remodeling proceeded, rather than a direct response to the initial signal. Previous studies have shown that a change in mechanical stress can produce changes in the expression of a variety of genes, including those of collagen and MMPs,39,40
and the tissue-remodeling process in the sclera probably alters the local stresses within the sclera. It is interesting to note, however, that most studies report that an increase in tension induces an increase in collagen expression, the opposite of what was found after 11 days of MD. This suggests that the decrease in collagen mRNA is not due to an increase in tension in the deprived eye sclera. The dissipation of the difference in MMP-3 mRNA level from 4 to 11 days of MD () could also be a secondary response to a change in scleral stress induced by the tissue remodeling.
Binocular Changes during MD
In addition to differential changes, some mRNA levels were significantly altered in the control eyes, compared with normal eyes, at various points during MD (). In all cases, the changes in the control eye mRNA levels were in the same direction, compared with levels in normal eyes, as the changes in the deprived eyes. Two points are of interest regarding the mRNA level changes in the control eyes: One is how they occur. The other is why, given that the mRNA changes were in the same direction as the changes in the deprived eye, they did not appear to the affect creep rate or other physical characteristics of the sclera in a similar fashion.
Previous studies in tree shrew and other species have shown that MD or monocular treatment with a minus-power lens can produce alterations in the open control eye.20,26,41
It is currently not known how monocular treatment produces an effect in the open control eye. Possible mechanisms include systemic effects such as altered choroidal blood flow, CNS feedback to both eyes, and modified visual input to the control eyes resulting from modified visual behavior. In the present study, the 24-, 28-, and 35-day normal animals all underwent a surgical procedure to install a goggle pedestal at 21 days of VE, as did the deprived animals, which eliminates the possibility of a systemic effect from the surgical procedure. In addition, there was an open goggle frame, rather than a plano lens, in front of the control eyes, and the effect in the control eyes therefore cannot be a lens effect, such as an increase in temperature. In a recent study,42
the investigators found that decreased illuminance in one eye produces a decrease in choroidal blood flow in both eyes. A similar binocular reduction was found in the chick after MD.43
How a change in blood flow, or other binocular effects might alter mRNA levels in the control eye is not known. However, these effects on mRNA levels in the control eyes emphasize the importance of also obtaining measurements in completely normal eyes. It clearly could be misleading to draw conclusions about the effects of monocular treatment based solely on relative differences between the treated and control eyes.
It is not clear why the changes in mRNA levels in the control eyes, which moved generally in the same direction as the changes in the deprived eyes, did not appear to cause similar physical changes. The vitreous chamber appeared to become slightly smaller in the control eyes during MD, and the creep rate in the previous study20
was slightly lower (not statistically significant), but these changes were in the opposite direction from the changes in the deprived eyes. This could indicate that there are gene products that were not examined in this study that have expression changes only in the deprived eye. It could also suggest that the differences between the treated and control eyes are functionally more important than the absolute levels.
Differential Changes during Recovery
During recovery, there is an abrupt reversal in creep rate (), so that the deprived eyes switch from having a much higher creep rate than the control eyes to having a creep rate that is significantly lower than in control eyes.20
A relative change in sulfate incorporation, from lower in the deprived eye after 5 days of MD to higher in the recovering eye after 3 days of recovery, has also been reported in tree shrews.17
A similar reversal occurred in several of the mRNAs that showed significant differential changes during MD. The level of mRNA for type I collagen, which was significantly lower in the treated eyes after 11 days of MD, was not different after 2 days of recovery. After 4 days of recovery, the levels were higher in the recovering eyes. The effect of recovery on MMP-2 mRNA levels was similar, but in the opposite direction. The levels were higher in the deprived eyes after 11 days of MD, were not different from the control eyes after 2 days of recovery, and were significantly lower after 4 days of recovery. mRNA levels of TIMP-1 were transiently lower in the deprived eye after 1 day of MD and then were not significantly different for the rest of the MD period. During recovery, TIMP-1 levels in the recovering eyes were at approximately normal levels after 2 days and were significantly higher in the recovering eye after 4 days. Although MMP-3 mRNA levels became higher in the recovering eye after 2 and 4 days of recovery, the differences were not statistically significant.
Binocular Changes during Recovery
In general, the binocular effects produced by MD receded rapidly during recovery. MMP-3 mRNA levels, which were significantly lower than normal in both eyes after 11 days of MD rapidly returned to normal levels during recovery. Collagen mRNA levels in both eyes moved back toward normal levels during recovery, but the level in the control eye remained significantly below normal. MMP-2 mRNA levels in both eyes transiently moved back to normal levels after 2 days of recovery, but then decreased back below normal after 4 days.
Relation of mRNA Levels to Specific Creep Rates
A pattern that might be expected to occur if an mRNA level is causally related to the scleral remodeling would be for the mRNA level to change in unison with the changes in creep rate and/or other scleral changes throughout MD and recovery. This type of pattern did not occur among the mRNAs examined in the present study. In general, there appeared to be a stronger association between initial changes in mRNA levels and initial changes in creep rate than between sustained mRNA levels and sustained creep rates. The closest to a continuous correlation was the mRNA level for MMP-2, except that there was a binocular decrease in mRNA levels by 11 days of MD that was not paralleled by creep rate or other scleral changes. Two factors should be considered with regard to there being no perfect correlation between any mRNA level and, for instance, creep rate. One is that there may be other mRNA levels, not examined in this study, that change in unison with creep rate. Another is that it is not necessarily the case that mRNA changes that initiate scleral changes must remain altered. For example, a transient change in the MMP-2 mRNA level may contribute to tissue remodeling that changes the creep rate, which then remains constant until another transient change in an mRNA level, such as an increase in TIMP-1 during recovery contributes to a decrease in the creep rate. Further, an elevated mRNA level and excessive production of a degradative enzyme such as MMP-2 may produce excessive tissue degradation if it were maintained too long.
Possible Functional Significance of Changes in mRNA Level
The changes in mRNA levels found in this study should be considered in light of known changes in protein levels during MD or minus-power lens wear, and with general principles of ECM remodeling from other tissue systems. A change in the level of a protein during tissue remodeling can be achieved through different combinations of synthesis and degradation and a key to understanding the remodeling process is to determine the relative contribution of the two distinct processes. Comparing changes in mRNA level with changes in protein level can help determine whether synthesis of the proteins is altered, and further, whether it can be controlled at the transcriptional level or at a later stage of gene expression.
Previous studies have found reduced amounts of type I collagen in the deprived-eye sclera compared with the control-eye sclera after 21 days of MD.14,18
This suggests that a reduction in the synthesis of type I collagen, the primary structural component of the sclera, could play an important role in scleral remodeling during MD. It is not known how quickly collagen protein levels change or whether there is a difference between the deprived and control eyes after several days of treatment when anatomic changes are first apparent. Therefore, it is not clear whether the early binocular and late differential mRNA changes are consistent with protein changes. The absence of an early difference in collagen mRNA level, and possibly protein level, between the deprived and control eyes does not necessarily mean that a change in collagen expression cannot play a role in the increase in creep rate in the deprived-eye sclera that is apparent by 4 days of MD.20
The rapid decrease in α1(I) collagen mRNA (compared with normal eyes) that occurs in both the deprived and control eyes, could be essential to the biomechanical changes that occur in the deprived-eye sclera without being sufficient to cause any changes in the control-eye sclera. An analogy is the effect of systemic lathyritic treatment in tree shrews, which undoubtedly affects collagen cross-linking in both the deprived and control eyes, but only enhances the effect of MD in the deprived eyes, without affecting the control eyes.44
The absence of an early difference in collagen mRNA between the deprived and control eyes suggests that a change in collagen expression alone is not sufficient to produce the early differential biomechanical effects between the deprived and control eyes and that another component, such as proteoglycans, may be involved. The substantial increase in the α1(I) collagen mRNA level in the deprived eyes after 2 days of recovery suggests that an increase in expression of type I collagen plays a role in the rapid decrease in creep rate that occurs during recovery.
That decorin mRNA levels did not change was an unexpected result. Proteoglycans, which consist of a core protein with attached GAG chains, are known to influence the mechanical properties of biological tissue.45
Decorin, a small proteoglycan with a single GAG chain, is the most abundant proteoglycan in the sclera46
making it a potentially important component in the scleral creep rate changes that occur during MD and recovery. Several previous studies have shown that MD and recovery alter the levels of sulfated and unsulfated GAGs in the sclera.12–14,24
The amount of 35
S sulfate that is incorporated into sulfated GAGs is reduced in the deprived eyes compared with the control eyes, suggesting that MD and recovery may alter GAG or proteoglycan core protein synthesis. In the present study, MD and recovery did not alter decorin core protein mRNA levels in the deprived eyes compared with the control eyes. This suggests that the synthesis of the most abundant proteoglycan (core protein) in the sclera was not altered. This finding is not necessarily inconsistent with previous findings, because other mechanisms, including a change in the rate of proteoglycan degradation and modification of GAG change, length, and extent of sulfation could produce the observed changes in GAG levels and 35
S sulfate incorporation. Data on GAG chain length and extent of sulfation and separate measures of proteoglycan core protein and GAG chain synthesis and degradation are needed to learn conclusively what mechanism is responsible for the observed changes in GAGs. In addition, other less abundant scleral proteoglycans, such as biglycan, lumican, and aggrecan should be examined.
The changes in MMP-2 mRNA levels generally matched the known changes in MMP-2 protein during MD and recovery.26
MMP-2 activity can be controlled at a number of levels, including transcription, activation, and inhibition by TIMPs. Although a change in mRNA stability cannot be ruled out, these data suggest that control at the transcriptional level is important in this system. In addition, the early onset of the MMP-2 mRNA level changes suggests that a change in MMP-2 transcription may be one of the initial responses of the scleral fibroblasts to signals from the retina.
The decrease in MMP-3 mRNA during MD was also an unexpected finding. Other studies23
suggest that one role of MMP-3 in tissue remodeling is to degrade proteoglycans by cleaving the core protein. Therefore, based on reports that MD produces a loss of GAGs in the deprived-eye sclera compared with the control eye sclera,14
we hypothesized that MMP-3 might degrade proteoglycans during MD and therefore that there might be an increase MMP-3 mRNA in the deprived-eye sclera during MD. Instead, the data suggest that MMP-3 mRNA levels decrease during MD and increase during recovery. Given this unexpected result, it is interesting to speculate the role that MMP-3 may play. It has been reported that MMP-3–null mice display delayed wound healing47
and that MMP-3–null dermal fibroblasts in culture contract collagen gels significantly less than wild-type fibroblasts.48
These data suggest that MMP-3 plays a role in scleral remodeling other than degrading proteoglycan core proteins.
Current evidence suggests that the primary role of the TIMPs is to inhibit MMPs, and TIMP-1 is thought to be the primary inhibitor of MMP-3. In many but not all systems that have been studied, TIMP levels tend to decrease under conditions of increased tissue degradation and to increase under conditions of decreased degradation.39,49
The changes in TIMP-1 mRNA levels in the present study were in general agreement with these findings in that TIMP-1 levels decreased during MD and increased during recovery. However, the changes in TIMP-1 mRNA levels were in the same direction as the changes in MMP-3 mRNA levels, which does not necessarily fit if the primary role of TIMP-1 is to inhibit MMP-3, and it should be kept in mind that inhibiting MMPs may be but one role that TIMPs can play.50
In conclusion, this study provides quantitative data on the changes in mRNA levels that occur during MD and recovery in a sample of important ECM proteins. Within the limits of resolution that these data provide, some of the mRNA changes appeared to occur at least as early as the physical changes, making it plausible that a change in gene expression initiates the scleral tissue remodeling. It is likely that a number of other genes play a role in the observed tissue remodeling. Together, the opposite responses of specific mRNA levels to MD and recovery, and the temporal correspondence between mRNA, biochemical, and biomechanical changes suggest that the visually guided emmetropization mechanism modulates scleral gene expression to control axial elongation rate and the refractive state of the eyes.