Steady State mRNA Levels in Tree Shrew Sclera with Form-Deprivation Myopia and during Recovery

Abstract

purpose. In tree shrews, visual form deprivation induces myopia and tissue remodeling in the sclera, characterized by decreased levels of collagen and glycosaminoglycans (GAGs) and increased levels of matrix metalloproteinases (MMPs). Removal of the visual deprivation allows recovery. This study investigated whether these changes are accompanied by changes in steady state mRNA levels in the sclera.

methods. Quantitative competitive reverse transcription–polymerase chain reaction (RT-PCR) was used to measure steady state levels of mRNA for collagen (α1(I) chain), decorin (core protein), gelatinase-A (MMP-2), stromelysin-1 (MMP-3), and a tissue inhibitor of metalloproteinase (TIMP-1) in the scleras of tree shrews that received either 11 days of monocular form deprivation (MD) or 11 days of MD followed by 4 days of recovery. A group of age-matched normal animals was also measured.

results. After 11 days of MD, α1(I) collagen mRNA levels were 34% lower, and MMP-2 mRNA levels were 66% higher in the deprived eyes than in the control eyes. After 4 days of recovery, collagen mRNA levels were 33% higher, MMP-2 levels were 20% lower, and TIMP-1 levels were 43% higher in the recovering eyes than in the control eyes. Decorin and MMP-3 mRNA levels were not significantly different between the treated and control eyes after MD or after recovery.

conclusions. The tissue remodeling in mammalian sclera induced by altering the visual environment is accompanied by modulation of mRNA levels in the sclera. The levels of collagen and MMP-2 mRNA were modulated in a pattern generally consistent with observed changes in protein levels, suggesting that visual regulation of the levels of these scleral proteins may involve modulation of gene expression at the transcriptional level.

Axial elongation and myopia are readily induced in juveniles of a number of animal species by visual form deprivation or minus-power lens wear. ¹ If the deprivation or lens wear is removed, eyes recover from the induced myopia by slowing the axial elongation rate. Visually induced myopia in both avian and mammalian models involves active remodeling of the scleral extracellular matrix (ECM) rather than passive stretching of the scleral shell. In chicks, which have a two-layered sclera consisting of an inner cartilaginous layer and an outer fibrous layer, growth of the cartilaginous layer of the sclera appears to be the dominant physical mechanism that enlarges the scleral shell. ^{2 3} In tree shrews, which, similar to other eutherian mammals, have an all-fibrous sclera, ⁴ the active remodeling does not produce a net increase in the amount of scleral tissue, but rather alters the mechanical properties (increases creep rate) of the sclera. ^{5 6} This may contribute to the enlargement of the globe by reducing scleral resistance to expansion by normal intraocular pressure. Thus, although the physical mechanism producing the vitreous chamber enlargement appears to be somewhat different in avians and mammals, in both species active tissue remodeling appears to underlie the enlargement of the scleral shell and the resultant myopia. 

Tissue remodeling can involve a number of ECM proteins and enzymes. These include structural components, such as collagen and proteoglycans; degradative enzymes, such as the matrix metalloproteinases (MMPs) that degrade ECM proteins; and tissue inhibitors of metalloproteinases (TIMPs) that bind to and inhibit the activity of the MMPs. In tree shrews, scleral remodeling in eyes with induced myopia is characterized by decreased levels of collagen, sulfated and unsulfated glycosaminoglycans (GAGs), and increased levels of gelatinase-A(MMP-2). ^{7 8 9 10 11 12} Levels of GAGs and MMP-2 are also altered in chick sclera by form deprivation and recovery. ^{13 14} The currently unanswered question is how the protein levels are modulated. 

The levels of many proteins, including MMPs, ¹⁵ are significantly influenced at the level of transcription. The types of mRNAs present are an important indicator of which proteins are produced, and an increase or decrease in the steady state level of an mRNA usually, although not always, indicates that the production of the protein is being increased or decreased. Very few studies have examined mRNA levels in sclera during experimentally induced myopia. Rada et al. ¹⁶ and Seko et al. ¹⁷ examined scleral mRNA levels in form-deprived chicks, but, to our knowledge, no study has examined mRNA levels in mammalian sclera during experimentally induced myopia. In this study, we measured steady state levels of mRNA for collagen, decorin, MMP-2, MMP-3, and TIMP-1. Collagen, decorin, and MMP-2 have been implicated by other studies as being involved in scleral tissue remodeling in tree shrew, MMP-3 is known to degrade proteoglycans, and TIMP-1 is the primary TIMP that binds to and inhibits MMP-3. We did not include MMP-1 in this study, because a homologue to human MMP-1 does not appear to be expressed in tree shrew sclera. 

Methods

Experimental Groups and Ocular Measurements

Three groups of tree shrews (Tupaia glis belangeri), with five animals in each group, were included in the study: MD group, MD/Recovery group, normal group. Starting at 21 ± 1 days after natural eyelid opening (days of visual experience [VE]), the animals in the two treated groups received a pedestal that held a goggle frame. ¹⁸ The animals were allowed 3 days to recover from the minor surgical procedure required to install the goggle pedestal before visual treatment was begun. At 24 ± 1 days of VE, the animals in the MD group began 11 days of monocular visual deprivation (MD) with a translucent diffuser held in the goggle frame. 

At the end of the treatment period, the refractive state of the treated and control eyes was measured with an autorefractor (Nidek, Gamagori, Japan) ¹⁹ and A-scan ultrasonography was performed as previously described. ²⁰ The animals in the MD/Recovery group also received 11 days of MD starting 3 days after pedestal installation. This was followed by 4 days of recovery, with the diffuser removed. In the MD/Recovery group, only refractive state was measured at the end of MD, whereas both refractive state and axial component dimensions (by A-scan ultrasonography) were measured after recovery. The animals in the normal group, which did not receive a pedestal or any treatment, were measured at 39 days of VE, the same VE as the recovery animals and 4 days of VE older than the MD animals. Refractive state was measured while the animals were awake with no ophthalmic or systemic atropine sulfate administered at any time because of concerns that atropine could alter the effect of MD in tree shrews. ²¹ A previous study on tree shrews found that noncycloplegic Nidek refractions were only slightly less hyperopic (<1 D) than cycloplegic Nidek refractions and that treated-control eye differences were very similar. ¹⁹ Eyes in all animals were enucleated between 10:00 and 11:30 AM. All procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Association for Assessment and Accreditation of Laboratory and Animal Care (AAALAC) regulations for the use of laboratory animals. 

RNA Isolation

In animals under deep, terminal pentobarbital sodium anesthesia, the eyes were enucleated, placed immediately into room-temperature solution (RNA Later; Ambion, Austin, TX) and dissected to obtain sclera free of other tissues. The scleras were then snap frozen in liquid nitrogen and stored at −80°C until the RNA was extracted. In each animal, the eye chosen to be enucleated first was randomly varied. Total RNA was extracted from individual scleras (SV Total RNA Isolation System; Promega, Madison, WI). RNA concentration and purity were determined by spectrophotometry at 260 nm and 280 nm. The A260/A280 absorbance ratio was consistently at approximately 2.0, indicating high-purity RNA. An average of 2.4 μg of total RNA was obtained from each sclera. 

Primers

Tree shrew–specific primers were designed for mRNAs of α1(I) collagen, decorin (core protein), gelatinase-A (MMP-2), stromelysin-1 (MMP-3), TIMP-1, and 18s. Beginning with sequences for other species available in GenBank (National Center for Biotechnology Information, Bethesda, MD; available in the public domain at http://www.ncbi.nlm.nih.gov), an initial set of primers corresponding to the human sequence was chosen in regions of high cross-species homology, by using a primer design program (Primer Premier; Premier Biosoft, Palo Alto, CA). The polymerase chain reaction (PCR) products produced from the initial human primers and tree shrew RNA were cloned (pGEM-T Easy; Promega) and sequenced (University of Alabama at Birmingham [UAB] sequencing facility) to verify the identity of the products and to obtain tree shrew sequences. Tree shrew–specific primers were then designed from the tree shrew sequences. Cloning and sequencing then verified the identity of the PCR product produced by each tree shrew–specific primer pair. ²² All primers are located within the coding region of the mRNA, and all primer pairs span at least two introns. Table 1 gives information about each primer. 

Competitive RT-PCR

Competitive reverse transcription–polymerase chain reaction (RT-PCR) can be used to obtain quantitative information on mRNA levels comparable to traditional RNA blot techniques ²³ but requires much less RNA. This is a critical advantage when the amount of sample RNA is limited, as in this study. Competitive RT-PCR allows the study of many mRNAs in the RNA from an individual sclera, which would be impossible with traditional RNA blot techniques. Competitive RT-PCR uses a synthetic competitor RNA molecule that is added to the RT-PCR reaction in known copy numbers. The competitor RNA is reverse transcribed and amplified by the same primers used to reverse transcribe and amplify the native mRNA so that both are amplified at approximately the same efficiency. The competitor RNA has an internal deletion (∼50 bases) that allows its PCR product to be distinguished from the native product by size on an electrophoretic gel. When the densities of the native product and the competitor product are equal in a gel, the number of copies of native mRNA in the RT-PCR reaction is approximately equal to the number of copies of competitor added. 

Competitor RNA

Each competitor RNA (cRNA) for this study was made with a kit (Competitor Construction Kit; Ambion) that uses modified nucleotides that render the cRNA molecules RNase resistant. The RNase-resistant cRNA produces results comparable to cRNA made with nonmodified nucleotides. ²⁴ The nucleotide sequence of the cRNA between the primer sites is identical with the native mRNA except for the ∼10% deletion. In addition, the cRNA molecules have a tail of at least 50 nucleotides after the antisense primer site, which also has a sequence identical with the native mRNA sequence. These features increase the similarity of the secondary structure between the native mRNA and the cRNA—particularly, the structure of the antisense binding site, which was used to prime the RT. This in turn should increase the similarity of reverse transcription efficiency of the native mRNA and the cRNA. Each cRNA was gel purified and quantified with spectrophotometry, and PCR only (no RT step) was run on a sample to ensure that the cRNA was not contaminated with the DNA template from which it was made. 

Procedure for RT-PCR

Hot-start single-tube RT-PCR reactions containing 5 ng of total RNA and a known number of copies of cRNA in a total volume of 50 μl were run (GeneAmp 2400; Perkin Elmer, Norwalk, CT). The RT-PCR conditions were: buffer (20 mM Tris-acetate, 10 mM ammonium acetate sulfate, 75 mM potassium acetate, and 0.05% Tween 20), 1.5 mM MgSO₄, 10 mM each dNTP, 0.5 units RNAsin (Promega), 50 picomoles each primer, 5 U avian myeloblastosis virus (AMV) reverse transcriptase, and 2.5 U Thermus flavus (tfl) DNA polymerase. The RT step consisted of an initial denaturation at 60°C for 2 minutes followed by 45 minutes at 48°C. The antisense primer primed the RT. The PCR cycle parameters were: 30 seconds of denaturation at 94°C, 1 minute of annealing at 60°C, and 1 minute 15 seconds of extension at 72°C, with a final 5-minute extension at 72°C. From 26 to 40 PCR cycles were performed, depending on the abundance of the particular message. A master mix that contained all ingredients except cRNA, primers, and enzymes was made for each eye. Total RNA was included in each master mix, so that one 43-μl aliquot contained 5 ng total RNA. The level of each mRNA was measured in aliquots from the same master mix by adding the appropriate cRNA and primers. 

Quantification Procedure

To quantify the RT-PCR products, 15-μl aliquots of each RT-PCR product were run on 2% high-resolution agarose gels and stained with ethidium bromide. The band densities were then measured with a video imaging system (Eagle Eye II Still Video System; Stratagene, La Jolla, CA). After initial RT-PCR runs to determine the approximate number of copies of each mRNA, three two-fold dilutions of competitor RNA were run versus constant total RNA (5 ng) to find the competitor copy numbers that were higher and lower than the copies of native mRNA in the sample. Figure 1 shows a representative gel demonstrating the results of this procedure. To calculate the cRNA copy number at which equal band densities would occur, band density versus cRNA copy number was plotted on a log–log scale for the native product (Fig. 1 , top band) and the cRNA product (Fig. 1 , bottom band). The crossing point of the two lines was then determined by solving simultaneous equations. 

To compensate for any difference in the quantity or the quality of total RNA in each RT-PCR reaction, the target mRNAs were normalized to the number of copies of 18s rRNA. Results were expressed as copies of target mRNA per copies of 18s rRNA. For each sclera, the RT-PCR reactions for 18s rRNA and all the target mRNAs were performed from the same master mix. 

It should be kept in mind that the estimate of copy number from competitive RT-PCR, compared with the true number of copies, is subject to error, due to any difference in RT efficiency between the cRNA and the native mRNA as well as to any difference in efficiency between different mRNAs. Therefore, caution is advised when drawing conclusions concerning the relative abundance of the different mRNAs. This source of error affects only the accuracy of the relative abundance of the different mRNAs in a sample. It does not affect the relative difference in levels of the same mRNA in different samples. 

Validation of the Competitive RT-PCR Technique

Several experiments were performed to determine the ability of this technique, as applied in these experiments, to detect mRNA changes. When competitive RT-PCR was performed four times on the same master mix, the SD was ±2.3%. The theoretical variability of quantification as copies per copies of 18s rRNA is therefore twice that, or approximately ±5%, suggesting that this technique can measure a difference in mRNA levels as small as 10%. 

Similar to previous investigators, ²⁵ we found that cycle number did not significantly affect quantification. Once the copies of cRNA that were just higher and just lower than the number of native copies in the sample were found, the sample copy number stayed in the cRNA bracket over a fairly large cycle range. For example, estimates of 18s rRNA copy number in the same sample quantified after 22 and 28 PCR cycles were within 1.4%, confirming that cycle number does not significantly affect competitive RT-PCR. This technique also was effective at high cycle numbers required with lower abundance mRNAs. For example, Figure 1 is an example of competitive RT-PCR on MMP-3 that required 40 cycles of amplification. 

There was not a significant difference in the amount of total RNA or the A260/A280 ratio of the RNA extracted from right or left eyes, treated or control. In addition, the number of copies of 18s rRNA per 5 ng of total RNA was not significantly different between eyes in any of the groups, indicating that there was no systematic effect on the quality of the RNA in general or the expression of 18s rRNA. Therefore 18s rRNA is a valid internal control for these experiments. The average number of copies of 18s rRNA per 5 ng of total RNA across all groups was within a factor of two of the number that should theoretically be in that amount of total RNA, assuming that approximately one fifth of total RNA is 18s rRNA. This suggests that any error due to a difference in the efficiency of RT between the native RNA and the cRNA is low and that this technique provides a reasonably accurate estimate of the number of copies in the sample. 

Finally, there was not a significant difference in the levels of any of the mRNAs studied in right and left eyes of the normal animals, demonstrating that their levels are similar in the two eyes of normally developing animals. 

Statistical Tests

Paired t-tests were used to determine whether differences between the treated and fellow control eyes were statistically significant. Analyses of variance (ANOVAs) and least-significant difference (LSD) post hoc tests were used to test differences between normal eyes and the treated or control eyes. Average normal values were calculated by averaging the right and left eye values of each normal animal and then taking the average of those values for the group. 

Results

Refractive and A-Scan Results

In the MD group, MD for 11 days produced (mean ± SEM)− 9.0 ± 0.8 D of myopia and 0.23 ± 0.03 mm of vitreous chamber elongation in the treated eye compared with the control eye. In the MD/Recovery group, 4 days of recovery reduced the relative myopia from −10.8 ± 1.1 D to −7.9 ± 1.4 D, and the difference in vitreous chamber depth was 0.16 ± 0.02 mm after 4 days of recovery. The refractions and axial lengths of the control eyes were not significantly different from the normal eye values. 

Competitive RT-PCR Results

Treated Eye Versus Control Eye.

Figure 2 A summarizes the competitive RT-PCR results for the MD group. Compared with their fellow control eyes, deprived-eye scleras contained 34% less collagen mRNA (P < 0.01) and 66% more MMP-2 mRNA (P < 0.01). Decorin, MMP-3, and TIMP-1 mRNA levels were not significantly different between the deprived and control eyes (P > 0.05). Figure 2B summarizes the results for the recovery group. Recovering-eye scleras contained 33% more collagen mRNA (P < 0.05), 20% less MMP-2 mRNA (P < 0.05), and 43% more TIMP-1 mRNA (P < 0.05). Decorin and MMP-3 mRNA levels were not significantly different between the recovering and control eyes (P > 0.05). Figure 2C shows the copies of mRNA in the treated eye as percentage more or less than the number of copies in the control eye. 

Treated and Control Eyes Versus Normal Eyes.

In Figure 3 , the mRNA levels in the treated and control eyes are compared with the mRNA levels in the normal eyes. In the deprived eyes of the MD group (Fig. 3A) , collagen mRNA levels were significantly lower (by 58%; P < 0.01) than in normal eyes (39 days of VE). Decorin, MMP-2, MMP-3, and TIMP-1 mRNA levels were not significantly different from normal (P > 0.05). In the control eyes in the MD group, collagen, decorin, MMP-2, and MMP-3 mRNA levels were significantly lower than in normal eyes: by 36% (P < 0.05), 38% (P < 0.01), 50% (P < 0.01), and 58% (P < 0.05), respectively. TIMP-1 was not different (P > 0.05). 

In the recovering eyes in the MD/Recovery group (Fig. 3B) , the only mRNA that was significantly different from normal eyes was MMP-2 (44% lower; P < 0.05). Collagen mRNA levels, which were significantly lower after MD in the MD group and were presumably lower after MD in the MD/Recovery group, were not different after 4 days of recovery, which suggests that the levels had returned to normal during 4 days of recovery. In the control eyes in the MD/Recovery group, collagen and MMP-2 mRNA levels were significantly lower than in normal eyes (33% lower, P < 0.05; and 30% lower, P < 0.05, respectively). 

Deprived Eyes Versus Recovering Eyes.

Figure 4 compares the mRNA levels in the MD group after 11 days of MD with the mRNA levels in the MD/Recovery group after 4 days of recovery. This comparison examines how the mRNA levels may have changed from the end of 11 days of MD to the 4th day of recovery. As shown in Figure 4A , MMP-2 levels were 32% lower (P < 0.05) in the recovering eye scleras than in the deprived-eye scleras, indicating that MMP-2 mRNA levels may have dropped in the treated eyes during recovery. Collagen and TIMP-1 mRNA levels were 112% (P < 0.05) and 49% (P < 0.05) higher in the recovering eyes than in the deprived eyes, indicating that levels of these mRNAs may have increased during recovery. In the control eyes (Fig. 4B) , MMP-3 mRNA levels were 83% (P < 0.05) higher in the control eyes of the MD/Recovery group than in the control eyes of the 11-day MD group, indicating that the level of this mRNA may have increased in the control eyes during 4 days of recovery (Fig. 4B) . 

Discussion

In this study, we found that mRNA levels in the sclera of tree shrews were altered after 11 days of MD and after 4 days of recovery from MD. Compared with their fellow control eyes, deprived-eye scleras had less collagen mRNA and more MMP-2 mRNA, whereas recovering eye scleras had more collagen mRNA, less MMP-2 mRNA, and more TIMP-1 mRNA. These changes show opposite and bidirectional modulation of mRNA levels for collagen, the major structural component of the sclera, and MMP-2, a degradative enzyme. This pattern is consistent with the observation that form deprivation produces a more extensible sclera, as evidenced by an increase in creep rate, and that recovery reduces the extensibility of the sclera, as evidenced by a decrease in creep rate. ^{5 6}  

Modulation of scleral mRNA levels by the visual environment had been demonstrated previously in chick but not in a mammal. In form-deprived chicks, Rada et al. ¹⁶ found that MMP-2 mRNA levels were higher in the deprived-eye sclera than in the control eye sclera after 10 days of MD and lower after 24 hours of recovery. TIMP-2 levels were lower in the deprived eye after MD and similar in the deprived and control eyes after recovery. They also showed that most of the MMP-2 mRNA was localized to the fibrous sclera. Thus, MMP-2 mRNA levels in fibrous sclera respond similarly in chicks and tree shrews. In chicks that were form deprived for 12 days, Seko et al. ¹⁷ found that the level of mRNA for the retinoic acid receptor RARβ was higher in the deprived-eye sclera than in the control eye sclera. These findings in chick, along with our findings in tree shrew, support the hypothesis that modulation of mRNA levels in the sclera plays a role in scleral remodeling during experimentally induced myopia and recovery in avians and mammals. 

The levels of the mRNAs we studied were generally modulated in the same direction as the corresponding protein levels measured in other studies. The percentage change in MMP-2 mRNA was similar to the percentage change in MMP-2 protein (active+latent) reported by Guggenheim and McBrien ⁷ in tree shrew sclera, with a similar treatment suggesting that a change in mRNA level could have produced the change in protein level. They also found an increase in the ratio of active versus latent MMP-2 during MD and a decrease in the ratio during recovery. Together, these data suggest that MMP-2 activity in tree shrew sclera may be controlled both at the transcriptional level and at the level of activation. 

The lower level of α1(I) collagen mRNA in deprived-eye scleras compared with control eyes is consistent with the lower level of hydroxyproline that has been found after deprivation, ^{8 9} suggesting that the lower collagen protein level may also be the result of lower mRNA levels. The reversed pattern of collagen mRNA levels after 4 days of recovery suggests that collagen protein levels should increase in the recovering eye compared with the control eye during recovery. However, Norton and Miller ⁹ did not find a relative increase in collagen in the recovering eye sclera after 30 days of recovery after 21 days of MD. This difference may be due to the difference in the treatment the animals received in the two studies. The animals in this study were measured at 39 days of VE (4 days of recovery after 11 days of MD), whereas the animals in the Norton and Miller study were measured at 75 days of VE (30 days of recovery after 21 days of MD). The higher collagen mRNA levels in the recovering eye after 4 days of recovery may not be maintained for as long as 30 days of recovery, and the normal level of collagen expression may be lower at 45 days of VE than at 35 days of VE when MD ended. 

It is interesting that we did not find a significant difference in the amount of decorin core protein mRNA between deprived and control eyes, given that relative differences have been found in GAG levels after deprivation. ^{8 10} Both total GAG content and the incorporation of sulfate are lower in the scleras of tree shrew eyes with induced myopia than in the fellow control eyes. ^{10 11 12} The amount of radioactive sulfate incorporated into newly synthesized GAGs is generally interpreted as a measure of the rate of proteoglycan synthesis, which in tree shrew sclera should be strongly influenced by decorin synthesis. In tree shrew sclera, 55% of the sulfated GAGs are dermatan sulfate, ¹¹ which is most likely associated with decorin (approximately 74% of the sulfated proteoglycans in human sclera are decorin ²⁶ ). The similarity of the decorin mRNA levels in the deprived and control eyes suggests that the lower GAG levels and levels of incorporated sulfate in deprived eyes are not primarily due to a decrease in the rate of synthesis of decorin core protein. The lower GAG levels and the lower incorporated sulfate levels could be due to an increase in the rate of destruction of GAGs without a change in the rate of synthesis. If newly synthesized GAGs that incorporate the radioactive sulfate are then rapidly destroyed, fewer intact labeled GAGs would be measured at the end of the experiment than were actually synthesized, giving the false impression that the rate of synthesis was lower. Alternately, a reduction in the expression of other scleral proteoglycans that contain sulfated GAGs (aggrecan, biglycan, lumican), or alterations in the length of sulfated GAG chains that do not involve core protein changes could produce the observed changes in sulfated GAG levels. 

Because MMP-3 is known to degrade proteoglycan core proteins, ²⁶ we had hypothesized that the lower levels of GAGs measured in other studies might be due to degradation of proteoglycans by MMP-3. Therefore, we expected to see an increase in MMP-3 mRNA during MD and a decrease during recovery, similar to the changes that we found in MMP-2 mRNA levels. These changes in MMP-3 mRNA levels did not occur at the periods measured. The level of MMP-3 mRNA was not different in the treated and control eyes after 11 days of MD or after 4 days of recovery, was lower (not statistically significant) in both the treated and control eyes after 11 days of MD than in 39-day normal animals and was significantly higher (P < 0.05) in the control eyes after 4 days of recovery than it was after 11 days of MD. This suggests that MMP-3 mRNA levels are not regulated in the same temporal pattern as MMP-2 levels. Further measurements at shorter periods are necessary to determine whether there are significant changes in MMP-3 mRNA levels. 

In addition to the effects on the treated eyes compared with their fellow control eyes, there were also significant effects on the control eyes compared with the normal eyes (Fig. 3) . In the MD group, the levels of four of the five mRNAs studied were lower in the control eyes than in the normal eyes. These effects on the control eye are consistent with other studies that have shown various effects on the fellow control eyes of monocularly treated animals, including creep rate ⁵ and MMP-2 protein levels, ⁷ which emphasizes the need to include normal animals in studies of induced myopia. In general, mRNA levels in both the treated and control eyes tended to be lower than levels in the normal eyes. A more extensive investigation of expression patterns in normally developing animals is currently under way to determine the extent to which form deprivation and recovery alter control eye mRNA levels. 

We did not determine whether changes in mRNA stability contributes to the changes in steady state mRNA levels. A change in mRNA stability has been shown to contribute to the modulation of mRNA levels under some conditions. ²⁸ Regardless of the cause, steady state mRNA levels for MMP-2 and α1(I) collagen were altered by MD and recovery in a pattern similar to that demonstrated for the corresponding protein. Thus, the altered steady state mRNA levels may underlie the observed changes in protein levels. 

The largest change in mRNA level found in this study was approximately twofold. Given that the emmetropization process lasts for several months in tree shrews (years in humans) and that the physical changes in eye size are modest, even in the most rapidly developing myopia, it might be expected that the modulation of gene expression that occurs would be relatively subtle. The changes in mRNA levels may have been greater had we studied the posterior sclera separately, given that other studies have shown that the effects of experimentally induced myopia are more pronounced in that region. ^{6 8 29} Because of the relatively small amount of total RNA present in a tree shrew sclera (∼2 μg), we extracted total RNA from the whole sclera and therefore measured the average mRNA level across the entire sclera. 

Finally, it should be kept in mind that these data are a snapshot of mRNA levels at specific time points, whereas emmetropization and the development of myopia are dynamic processes. We are currently investigating the time course of changes in scleral mRNA levels in normally developing animals and in animals with an altered visual environment. 

 Supported by Grant RO1 EY-05922 and Core Grant P30 EY-03909 from the National Institutes of Health and a University of Alabama Birmingham Provost’s Faculty Development Grant.

 Submitted for publication October 30, 2000; revised January 16, 2001; accepted February 7, 2001.

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 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked“ advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Primer Information

mRNA	Primer Sequence (5′–3′)	Product Length	Nucleotide Homology (%)	Amino Acid Homology (%)
Collagen	S CCC GAC TGG AAG AGC GGA GAA TA	386	94	95
	A ATC TCG TTG GAG CCC TGG AGG A
Decorin	S CAA AGA TGG GGA CTT TAA GAA CCT	393	92	96
	A TGT AAT TCA GTA AGG GAA GGA GGA
MMP-2	S GAC GAT ATC AAG GGC ATT CAA GA	425	91	97
	A TTC TTG CTC CAG TTA AAA GCA GC
MMP-3	S CAG GAC ACC TGA ACC TGA ATT TC	363	89	85
	A ACA GCA TCA ACC TTT GGT TCA AC
TIMP-1	S CCT GGT CAT CAG AGC CAA GTT CA	330	87	80
	A TCC TCA CAG CCA GCA GCG TAG
18s	S GTT TTC GGA ACT GAG GCC ATG A	320	99	NA
	A TGC CCT TCC GTC AAT TCC TTT A

 Homology is to human. S, sense primer; A, antisense primer; NA, not applicable.

Figure 1.

 

Example of competitive RT-PCR used to quantify mRNA levels. This example shows quantification of MMP-3 mRNA levels in the right and left eye scleras of a normal tree shrew. Lanes 1, 2, and 3: competitive RT-PCR products using right eye scleral RNA; lanes 4, 5, and 6: products using left eye scleral RNA. In each lane, the top band is PCR product from the native mRNA, and the bottom band is PCR product from the competitor RNA (cRNA). For each eye, three twofold dilutions of cRNA (4, 2, and 1 × 10⁴ copies, as indicated by the number below each cRNA band) were run versus 5 ng of total RNA. Data show the integrated pixel density of the native mRNA product band and the cRNA product band versus the number of copies of cRNA added to each reaction. The data points correspond to the bands directly above in the gel photograph. In both eyes, the second dilution of cRNA (2 × 10⁴ copies) resulted in approximately equal band densities. Calculating the equivalence point mathematically estimated there were 2.197 × 10⁴ copies of MMP-3 in 5 ng of right eye RNA and 2.270 × 10⁴ copies of MMP-3 in 5 ng of left eye RNA.

Figure 2.

 

Competitive RT-PCR results comparing mRNA levels in the treated and control eyes. (A) Steady state levels of mRNA at the end of 11 days of MD; (B) steady state levels of mRNA after 4 days of recovery after 11 days of MD; (C) steady state mRNA levels in the treated eye shown as percentage higher or lower than levels in the control eye. There are no error bars in (C) because the ratios shown are the ratios of the average copy numbers shown in (A) and (B). In Figures 2 3 and 4 , mRNA levels are normalized as copies per 10⁶ copies of 18s rRNA except for MMP-3, which is copies per 10⁹ copies of 18s rRNA. Data are mean ± SEM. *Statistically significant difference (paired t-test, P < 0.05).

Figure 3.

 

Competitive RT-PCR results comparing mRNA levels in treated and control eyes with levels in normal eyes. (A) Deprived eyes and their fellow control eyes compared with normal eyes; (B) recovering eyes and their fellow control eyes compared with normal eyes. *Significantly different from the average normal value (ANOVA, LSD test, P < 0.05).

Figure 4.

 

Competitive RT-PCR results comparing mRNA levels in MD animals with levels in MD/Recovery animals. Data show how mRNA levels may have changed in the treated and control eyes during recovery. (A) Deprived eyes compared with recovering eyes; (B) MD animal control eyes compared with MD/Recovery animal control eyes. *mRNA level in the MD/Recovery group after 4 days of recovery was significantly different from the mRNA level in the MD group after 11 days of MD (ANOVA, LSD test, P < 0.05).

The authors thank Margot E. Andison who participated in the early stages of this study; Joel Robertson for excellent technical assistance; Andrew Ju, who developed the primers for TIMP-1 during a National Aeronautic and Space Administration Sharp Summer program; and Yi Pang, who developed the primers for α1(I) collagen during a laboratory rotation.