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INTRODUCTION |
The refractive power of the eye is correlated with ocular axial
length and it is well established that myopia is caused by increased
axial eye size (1). Studies, in humans and animal models, have shown
how this increased axial length is predominantly a consequence of
increased vitreous chamber depth, rather than marked changes in any
other ocular component parameter such as anterior chamber depth or lens
thickness (2, 3). The prevalence of human high myopia (usually defined
as eyes with >6 dioptres (D) of myopia, or >26 mm in length) is
~2% in the general population (1) and it is well documented that
individuals with high myopia have a greatly increased risk of ocular
pathology (4). Current thinking holds that excessive elongation of the
vitreous chamber is the causative factor in the development of
chorioretinal pathology, likely because of the increased biomechanical
stresses that are placed on the retina and choroid in an enlarged eye.
The development of high myopia in humans is associated with marked
thinning of the sclera, the tough outer coat of the eye that
facilitates any change in eye size. Scleral thinning is greatest at the
posterior pole of the eye, the anatomical region of greatest retinal
photoreceptor density and vital to detailed visual discrimination (5,
6). In some individuals, the thinned posterior sclera precipitates
local ectasic change or staphyloma (7, 8). This altered scleral
morphology is associated with local changes in collagen fibril
ultrastructure and increased numbers of small diameter collagen
fibrils. In addition, there is a more lamellar organization of
posterior scleral collagen fibril bundles (7, 9). Similar
characteristics are reported in the sclera of mammalian models of
myopia progression, such as the tree shrew, where scleral thinning and
tissue loss precede the appearance of more small diameter collagen
fibrils and altered fibril bundle morphology (10).
Collagen accounts for 90% of scleral dry weight, the majority of this
being collagen type I (11, 12). Mammalian sclera also contains small
amounts of other fibrillar and fibril-associated collagens (13-15) and
studies have shown that scleral fibrils are heterologous, comprising
collagen types I, III, and V (13). To date, at least 21 different
collagen subtypes have been identified (16), with subtype expression
patterns tissue- and structure-specific. As a result, it is likely that
additional collagen subtypes to those already identified are expressed
in the mammalian sclera. Of particular note is the fact that in tissues
such as the cornea, minor fibrillar collagen subtypes, particularly
collagen type V, are important in regulating lateral accretion of
collagen, thus controlling fibril diameter (17).
Mammalian models of myopia have shown that scleral thinning and tissue
loss occur rapidly in myopia development (10). Scleral tissue loss
occurs in conjunction with increased expression and activation of
collagen-degrading enzymes, such as matrix metalloproteinase-2 (18), decreased production of glycosaminoglycans (19, 20) and
decreased proliferative activity of scleral cells (21). The net result
is a decrease in scleral collagen content (22), as also found in human
high myopia (23). However, there has to date been no explicit
demonstration of the process of collagen degradation in eyes with
progressive myopia. Scleral remodeling accompanies alteration in its
material properties, with the sclera becoming more extensible in myopic
eyes. Studies show that increased extensibility is not solely accounted
for by scleral thinning, suggesting that altered scleral biochemistry
results in changes to the physical properties of the matrix (24, 25).
The majority of scleral thinning and tissue loss occur well before the
increased frequency of smaller collagen fibrils (10), suggesting that changes in fibril diameter are secondary to the initial process of
scleral thinning in myopic eyes. However, given that collagen turns
over relatively slowly in mature tissues (26), it is likely that the
immediate effects of altered scleral biochemistry on collagen fibril
morphology do not manifest for some time.
This study investigated two important issues relating to the role of
scleral collagen in the development of high myopia, using the well
characterized tree shrew model of myopia (27). First, it was
established whether general collagen synthesis, collagen degradation,
or a combination of the two, underlies reduced scleral collagen
accumulation and tissue loss in eyes with progressive myopia. Second,
expression of a range of collagen subtypes was demonstrated in the
sclera, before short term expression patterns of collagen subtypes III
and V, in conjunction with those of type I, were investigated in myopic
eyes, to determine their possible role in fibril diameter changes.
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EXPERIMENTAL PROCEDURES |
Experimental Subjects and in Vivo Procedures--
Tree shrew
(Tupaia belangeri) pups were maternally reared in our
breeding colony before being transferred to individual cages and
assigned to one of the experimental groups. Following the commencement
of experimental procedures, animals were maintained on a 15/9 h
light/dark cycle and food and water were available ad
libitum. Myopia was induced monocularly, using a translucent occluder attached to a head-mounted durilium goggle, which was fitted
to animals under anesthesia (ketamine, 90 mg/kg; xylazine, 10 mg/kg)
using the procedure previously described (28). The unoccluded eye
served as a paired control. The myopia-inducing goggle was always
fitted 15 days after natural eye-opening, the start of the period where
tree shrews have been found to be most susceptible to the inducement of
myopia (27, 29). Numbers of right or left eye treatments were as
balanced as possible within each group. All of the procedures were
carried out in accordance with the National Health and Medical Research
Council of Australia's Guidelines for the Care and Use of Animals in Research.
General scleral collagen production was investigated in one group of
animals (n = 6) that had myopia induced for 5 days
before newly synthesized collagen was labeled via delivery of a
radiolabeled collagen precursor ([3H]proline) and the
scleral tissue was collected and assayed. Four groups of animals
(n = 5 each group) were used to investigate the
elimination of collagen from the sclera. Animals were injected with the
radiolabeled collagen precursor, allowed a suitable period for collagen
labeling (24 h), then scleral tissue was collected (time 0) or myopia
was induced for periods of 5, 12, or 24 days before the tissue was
collected. Scleral tissue was then assayed for the amount of labeled
collagen remaining in the sclera.
One group of untreated animals (n = 2) was used to
determine collagen expression profiles in the sclera. Whole scleral
tissue was collected from these animals 20 days after natural eye
opening, representing an equivalent age to the animals that underwent 5 days of myopia induction. Scleral collagen mRNA expression in myopia was investigated in one group of animals that had myopia induced
for 5 days (n = 8), whereas one group of age-matched
untreated animals (n = 4) were employed as a control
for this study.
Ocular axial dimensions and ocular refraction were measured at the end
of the treatment period in all animals, using A-scan ultrasonography
and streak retinoscopy, respectively, as has previously been reported
(30). Measurements were taken after the animals had been placed under
anesthesia (ketamine, 90 mg/kg; xylazine, 10 mg/kg). Following ocular
measurements, eyes were enucleated with animals under terminal
anesthesia (120 mg/kg sodium pentobarbital).
In animals used to investigate general collagen synthesis and
degradation, both eyes were enucleated and the left eye was dissected
before the right eye, thus randomizing whether the treated or control
eye was dissected first. Following enucleation, extraneous orbital
tissue was cleaned away and the anterior structures were removed with
scissors cutting close to the limbus. The posterior segment was
flat-mounted and a 7-mm trephine, centered on the posterior pole of the
eye, was used to separate the sample into posterior and
anterior/equatorial samples. These samples were cleaned of retina and
choroid. The optic nerve head was removed from the posterior sample
using a 1.5-mm trephine. The lens and ciliary body were removed from
the anterior ocular structures and a 5-mm trephine was used to isolate
the central cornea for use as a control. The corneal stroma is a
collagenous extracellular matrix that is continuous with the sclera but
does not alter its rate of matrix turnover during myopia progression
(19). All samples were snap frozen in liquid nitrogen and then stored
at
80 °C until assayed. In animals utilized for mRNA
expression studies, the dissection process was essentially identical,
except the fact that the left eye was removed and dissected while the animal was maintained under deep anesthesia on a heating pad, following
that the right eye was removed and dissected. The scleral samples were
the only tissues collected from these animals and all samples were snap
frozen in liquid nitrogen within 6 min of enucleation.
Delivery of [3H]Proline--
In the group of
animals that provided information regarding collagen production, an
intraperitoneal injection of 300 µCi of L-[2,3,4,5-3H]proline (1 mCi/ml, 3.7 TBq/mmol; PerkinElmer Life Sciences) was administered on the
final morning of the procedure. Proline is a relatively specific
collagen precursor, accounting for ~4% of non-collagenous proteins
and 14% of collagen (31). However, when proline is assayed in a tissue
such as the sclera, which comprises around 90% collagen (11), almost
all of that assayed will represent collagen. A period of 9 h was
allowed for incorporation of the label into newly synthesized collagen
before ocular measurements were taken and the tissue was collected for
assay. Pilot studies suggested that peak label incorporation was
reached by this point. In the four groups of animals that provided
information of general collagen degradation, an intraperitoneal
injection of 300 µCi of
L-[2,3,4,5-3H]proline was delivered 24 h
prior to the onset of monocular occlusion, the literature suggesting
that even in a rapidly remodeling tissue such as the neonatal cornea,
24 h is sufficient for peak incorporation of label incorporation
to have passed (32). At the time of fitting of the occluder, a dose of
"cold" proline (7 mmol/kg L-proline in 0.9% saline)
was administered, the dose being 100 times that of the radiolabeled
proline, with the intention of diluting intracellular proline precursor
pools and limiting the potential for reutilization of label (33).
Materials--
MultiScreen filtration plates (0.65 µm pore
size, Durapore membrane, number MANP N6510) were obtained from
Millipore (Bedford, MA); cytoscint liquid scintillation fluid from ICN
(Irvine, CA); pepsin, L-hydroxyproline, chloramine T, and
p-dimethylaminobenzaldehyde were from Sigma; Macro arrays
were custom made to our design by the Australian Genome Research
Foundation (AGRF, Melbourne, Australia); SMART cDNA synthesis kit
and the Advantage 2 PCR kit were obtained from
Clontech (Palo Alto, CA); Micro Bio-Spin 30 columns
were obtained from Bio-Rad; [
-32P]dCTPs, Rapid Hyb
buffer, and Megaprime labeling kits were obtained from Amersham
Biosciences; PCR primers were obtained from Genset (South La Jolla,
CA); guanidine thiocyanate, DNase I, Moloney murine leukemia virus
reverse transcriptase, dNTPs, and RNasin were obtained from Promega;
FastStart DNA Master Mix was obtained from Roche Molecular Biochemicals
(Mannheim, Germany); QIAquick PCR purification kits were obtained from
Qiagen (Valencia, CA); CEQ DTCS Quick Start sequencing kits were
obtained from Beckman; All other reagents were purchased from Sigma.
Assay of Radiolabeled Collagen--
Tissue was homogenized in
sterile de-ionized water using a freezer mill. The homogenate was then
mixed with pepsin and acetic acid to give final concentrations of 2 mg/ml and 0.5 M, respectively. The samples were gently
agitated at 4 °C for 48 h. The digested samples were then
centrifuged to pellet insoluble material and the supernatant was
collected for assay. Connective tissues contain collagen fractions that
are variously soluble under specific conditions. Our pilot studies and
previous reports indicated that the above method gave the best recovery
of collagen for minimal processing (34), however, to control for
variations in extraction efficiency, raw data were always normalized to
hydroxyproline content of the homogenate.
The [3H]proline assay was adapted from a previous report
with slight modifications to suit this experiment (35). Filters of the
multiwell plate were wetted with 100 µl of 25% trichloroacetic acid.
Triplicates (100 µl) of each corneal, anterior/equatorial, or
posterior scleral sample were added to the wells with 100 µl of 50%
trichloroacetic acid. The plates were then incubated at 4 °C for
1 h with gentle agitation to precipitate the macromolecules. The
precipitate was collected on the filter membrane by
draining thoroughly on a vacuum manifold, then washed 3 times with 300 µl of 10% trichloroacetic acid to remove the unincorporated label. The filters were allowed to air dry before being punched into plastic
scintillation vials containing 500 µl of 4 M guanidine hydrochloride in 33% isopropyl alcohol. The vials were incubated overnight with gentle agitation to dissociate the collagen from the
filter. The radiolabel was quantified through the addition of 10 ml of
cytoscint and scintillation counting. Data (disintegrations/min) were
corrected for isotope counting efficiency using the internal standard
and isotope library of the machine.
Hydroxyproline content, a highly specific indicator of collagen content
in each sample, was determined using a previously described assay (36).
Standards of hydroxyproline were prepared (0.5-8.0 µg in 40 µl)
and 10 µl of sodium hydroxide (10 M) was mixed with 40 µl of sample or standard in autoclavable tubes. Each sample and
standard was hydrolyzed by autoclaving at 120 °C for 20 min.
Chloramine-T solution (0.056 M in 10% 1-propanol and
acetate citrate buffer, 450 µl) was added to each hydrolysate after
cooling and, after mixing gently, the oxidation reaction proceeded for
25 min at room temperature. Ehrlich's aldehyde reagent (1 M
p-dimethylaminobenzaldehyde in 1-propanol/perchloric
acid, 2:1, v/v) was freshly prepared and added to each vial (500 µl) and mixed gently. All samples and standards were then incubated in a
water bath at 65 °C for 20 min to develop the color change. The
color density was quantified at 550 nm using a spectrophotometer.
Collagen Gene Array Design and Validation--
The 40,000 Human Unigene clone library at the AGRF was searched to find available
IMAGE clone sequences for the various collagen subtypes. Where multiple
-subunit clones were available for a given collagen subtype, the
most common naturally occurring
-subunit, usually
1, was
selected. The sequence associated with the accession number of each
selected clone was confirmed using the BLAST facility at the NCBI
(www.ncbi.nlm.nih.gov:80/BLAST). Once clones had been validated
(subunits representing collagens I-IX, XI, and XIII-XVIII) the custom
array was produced and included two
ubiquitously expressed genes as positive controls
HPRT1 and GAPDH. The final
array had each selected clone represented in duplicate. Colonies were
grown, transferred onto membranes, and lysed at the AGRF. The colony
plates were supplied with the arrays to allow sequence validation.
Following delivery of the arrays, it was found that the colonies
containing COL2A1 and COL14A1 had not grown and
further investigation, through colony PCR, gel electrophoresis, and
sequencing with the Beckman Coulter CEQ 8000, revealed that
"COL4A1," "COL5A2," and
"COL11A1" were not the sequences anticipated. As a
result of these findings, positive hybridization signals to any of
these positions on the array could not be considered to represent the
collagen subtype of interest.
Total RNA Extraction--
Tissues were homogenized (4 M guanidine thiocyanate, 25 mM sodium citrate)
in a freezer mill, then total RNA was isolated using a standard method
(37). Phenol:chloroform reagent (Sigma number P1944) was added to the
samples, RNA was precipitated from the aqueous phase using isopropyl
alcohol, then the resultant pellet was washed twice with 70% ethanol.
Glycogen (10 µg) was added prior to isopropyl alcohol precipitation
to maximize RNA recovery. The resuspended pellet was protected with
RNasin and treated with DNase I to remove potential genomic DNA
contamination. The RNA was repurified as before. The RNA content of the
resuspended pellet was quantified and checked for purity and condition
by spectrophotometry and gel electrophoresis.
Reverse Transcription--
SMART cDNA synthesis was carried
out using 1 µg of total RNA isolated from whole sclera. SMART
technology produces cDNA libraries with common primer sites, thus
allowing amplification of the library via PCR. The reaction was carried
out using the reagents, and in accordance with the protocol, supplied
with the kit. The cDNA library generated was then enriched through
PCR with the Advantage 2 PCR system and the SMART primer. PCR products
were monitored through agarose gel electrophoresis and 30 cycles of PCR
was found to be optimal for the amplification of this library. PCR
products were purified using the QIAquick PCR purification kit,
quantified, and stored for use as probe templates.
Posterior scleral sample mRNA from myopic and normal animals was
reverse transcribed using a standard technique. Amounts of 100-500 ng
of total RNA were used depending on the amount recovered from each eye.
Identical amounts of RNA were always reverse transcribed from scleral
RNA samples of paired treated and control eyes. Standard reverse
transcriptase reactions for paired samples were carried out from the
same reagent master mix (containing Moloney murine leukemia virus
reverse transcriptase, RNasin, and dNTPs) and at the same time.
Gene Array Hybridization--
Radiolabeled probes were prepared
from 100 ng of the PCR-enriched cDNA library using the Megaprime
DNA labeling system, [
-32P]dCTPs, and the protocol
supplied. The synthesized probes were purified by application to Micro
Bio-spin P-30 columns, checked for activity by scintillation counting,
and stored at
20 °C before use. Using a hybridization oven,
macroarrays were prehybridized for 30 min at 45 °C in Rapid Hyb
buffer, then the probes were denatured and added to the hybridization
bottle at 68 °C. The hybridization was carried out for 24 h and
was followed by one wash at room temperature (20 min), then two washes
at 68 °C (15 min) in 2× SSC, 0.1% SDS. The array was visualized
using a PhosphorImager.
Gene array hybridizations were screened using an RT-PCR and sequencing
protocol developed in this laboratory (38). Briefly, primers were
designed against areas of the coding regions of COL1A1, COL2A1, COL3A1, COL5A1, and
COL8A1 that were found to be well conserved across species.
Each primer was also checked to ensure there was little or no
cross-subtype specificity. These primers were then used to perform PCR
reactions on scleral SMART cDNA and amplified fragments were
purified and sequenced to confirm their identity.
Collagen mRNA Expression--
Scleral tissue samples were
processed for gene expression analysis using real-time PCR. Data was
collected from treated and control eyes of 8 myopic and 4 age-matched
normal animals. Semiquantitative real-time PCR was carried in paired
treated and control or right and left eyes for COL1A1,
COL3A1, and COL5A1, and HPRT, a
housekeeping gene whose expression was found not to be changed during
the development of, or recovery from, myopia (39). Tree shrew
sequence-specific primers were generated from the sequence information
obtained in previous experiments. Final primer sequences and product
sizes are given in Table I. The PCR
assays were carried out in triplicate to improve the accuracy of
estimates. Templates (2-4 µl) were mixed with primers (1 µM) and the FastStart DNA master mixture containing
FastStart Taq polymerase, SYBR green I dye, dNTPs, and PCR
buffer. Additional magnesium chloride was added as appropriate. Reactions were carried out in the LightCycler (Roche Molecular Biochemicals) real-time PCR machine. Standard cycling conditions for
this machine were used (10 min initial denaturation at 95-97 °C,
then 40 cycles of denaturation 95-97 °C for 0-10 s, annealing at
58-60 °C for 10 s, extension at 72 °C for 8-20 s,
dependent on product size) following which detection was carried out.
Melting curves were then used to ensure the purity of the amplified
product.
Data Analysis--
Ocular biometric data was presented either as
absolute values or as the mean interocular difference between eyes
(treated-control) ± S.E. Values of [3H]proline
incorporation (disintegrations/min) were normalized to hydroxyproline
content (dpm/µg) and either analyzed as absolute values or as the
mean percentage difference between eyes
((treated-control)/control) ± S.E. Comparisons were made between
the mean of triplicate PCR cycle curves of treated and control eyes, in
the log linear phase of the PCR reaction, using GraphPad Prism software
(GraphPad Software Inc., San Diego, CA) as previously reported (38).
Fold difference between eyes was normalized to that of the housekeeping
gene, then converted to a percentage difference. Data are presented as
the mean percentage difference ± S.E. Statistical comparisons were performed either using a paired t test or a general
linear model ANOVA with Tukey's post-hoc analysis for comparison
across groups. All analyses was performed on Minitab software (Minitab Inc. State College, PA), unless stated.
 |
RESULTS |
Ocular Biometry and Refraction--
In the two groups of myopic
animals (n = 14) used to investigate general and
subtype-specific collagen production, 5 days of monocular deprivation
produced
8.7 ± 0.9 dioptres (p < 0.001) of
relative myopia between the treated and control eyes. This resulted
from a relative increase in ocular axial length of 0.18 ± 0.02 mm
(p < 0.001), which was predominantly caused by a
vitreous chamber depth increase of 0.16 ± 0.02 mm
(p < 0.001) in the treated eye. No other ocular
parameter altered significantly between myopic and control eyes, as has
previously been reported (19).
In animal groups used to evaluate collagen degradation, after the
statistical method had allowed for processing order effects, there was
a significant treatment effect on refractive error (F (3,15) = 178.7, p < 0.001), vitreous chamber depth (F
(3,15) = 88.3, p < 0.001), and axial length (F
(3,15) = 76.2, p < 0.001), as expected. No
significant changes were found in any other ocular parameter over the
treatment period (Table II). In addition
to the treatment effects, control eye data shows there is a general increase in lens thickness and axial length with age, which was also as
expected (27).
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Table II
Ocular biometry in tree shrews during the development of induced axial
myopia
Absolute values represent the group means of treated and control or
right and left eyes ± SE. Interocular differences are expressed
as the group mean of the differences between treated and control or
right and left eyes ± SE.
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General Collagen Synthesis--
The mean hydroxyproline level in
posterior scleral extracts of control eyes in the present study was
59.7 ± 3.5 µg, which we estimate represents ~40% of the
collagen present, assuming a collagen hydroxyproline content of 10%
and a scleral collagen content of 90% (11, 40). This finding is in
keeping with a previous report in corneal tissue (34). Similar
extraction efficiencies were obtained for both corneal (48.0 ± 4.0 µg) and anterior/equatorial scleral samples (65.1 ± 5.4 µg). There was found to be no significant difference in the amount of
hydroxyproline extracted from paired treated or control eyes for any
tissue. Labeled proline incorporation (dpm/µg) into newly synthesized
collagen was reduced in the posterior sclera (
36 ± 4%,
p < 0.001 paired t test) and total sclera
(
28 ± 5%, p < 0.01) after 5 days of myopia
induction (Fig. 1). No statistical
difference in proline incorporation was found in the anterior/equatorial sclera or the cornea of the same eyes (Fig. 1).
There was no significant correlation between the difference in axial
eye size and the magnitude of change in proline incorporation in these
animals.

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Fig. 1.
Collagen synthesis, as estimated by the
interocular difference in [3H]proline content, is
significantly reduced in the posterior and total sclera, but not
significantly altered in the anterior/equatorial sclera or cornea, of
young tree shrews following 5 days of myopia development.
n = 6. **, p < 0.01; *,
p < 0.05.
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General Collagen Degradation--
After statistical analysis had
accounted for processing order effects there was a statistically
significant treatment effect in the posterior sclera of treated eyes (F
(3,16) = 7.1 p < 0.001), demonstrating that the
elimination of labeled proline (dpm/µg) from eyes developing myopia
was more rapid than from contralateral control eyes. Furthermore,
pairwise comparison showed there to be a significant interocular
difference between the 12-day MD group and the time 0 control group
(p < 0.05), however, this was not significant in other
groups, despite the fact that a paired t test showed there
to be a significant difference between treated and control eyes at 5 (
8 ± 2%, p < 0.05) and 12 (
15 ± 4%,
p < 0.05) days (Fig.
2A). In addition, there was a
significant overall treatment effect in the total sclera of the treated
groups (F (3,16) = 5.3, p < 0.01), suggesting
there was also a more rapid elimination of proline from the whole
sclera of eyes developing myopia, although the pairwise comparisons
between the 5-, 12-, and 24-day MD groups and the time 0 control group
were not significant (p = 0.08; Fig. 2B). No
significant changes were found with time in the treated eye of the
anterior sclera alone or of the cornea.

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Fig. 2.
A, posterior scleral collagen
degradation is significantly increased in eyes developing myopia, as
indicated by general linear model ANOVA (p < 0.001).
The graph shows the percentage difference in [3H]proline
content between treated and control eyes of animals monocularly
deprived for 0, 5, 12, or 24 days. B, total scleral collagen
degradation is significantly increased in eyes developing myopia, as
indicated by general linear model ANOVA (p < 0.01).
The graph shows the percentage difference in [3H]proline
content between treated and control eyes of animals monocularly
deprived for 0, 5, 12, or 24 days. n = 5 each group. *,
p < 0.05 by ANOVA pairwise comparison.
|
|
Collagen Gene Array Findings--
Positive hybridizations were
obtained for 9 of the 11 validated collagen
-subunits investigated
using the macroarray (Fig. 3,
A and B), the two subunits not hybridizing being
COL8A1 and COL18A1. The ubiquitously expressed
genes HPRT and GAPDH showed positive
hybridization signals, as expected.

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Fig. 3.
A, phosphorimage of representative
collagen gene array following hybridization with radiolabeled SMART
cDNA probes, generated from scleral mRNA. Clones containing
specific collagen subtype fragments and validated by sequencing are
indicated. Duplicates of each clone were present, related clones being
indicated using lines. The ovals indicate the
positive hybridizations where sequencing had shown the clone sequences
were not collagen subtypes. B, the tabulated data indicates
the collagen subtypes that were to be investigated, the accession
numbers of the clones immobilized on the array, clone sequence
verification data, whether a positive hybridization was found on both
membranes, if the array result was confirmed by PCR, and sequencing and
whether a particular collagen subtype was found to be present in the
sclera. Check mark indicates an affirmative response to the
question stated as the column title, cross indicates a
negative response, question mark indicates that from the
data collected it was not possible to give either an affirmative or
negative response, and dash indicates that no data was
collected in this respect.
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|
Of the selected collagen subtypes that were screened using RT-PCR and
sequencing, COL1A1 and COL3A1 were found to be
present in the sclera, thus validating the positive array finding in
this respect. However, COL8A1 was also found to be present
in the sclera, despite the fact that it did not hybridize to the array,
suggesting that the technique may generate false negative results. In
addition, of the collagen species not represented on the array,
COL5A1 was found in the sclera, whereas COL2A1
was not present (Fig. 4). The gene array
findings were replicated in whole sclera from a second tree shrew, thus
demonstrating the robustness of the technique in this respect. The
results are summarized in Fig. 3, A and B, and
show that the mammalian sclera expresses message for at least 11 collagen subtypes.

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Fig. 4.
Gel electrophoresis of PCR products following
amplification with primers to collagen type II. The first
lane shows no amplification from tree shrew scleral SMART
cDNA, whereas a product of the expected size was obtained from tree
shrew ear cartilage cDNA (second lane). The product
shown in the second lane was sequenced and its identity
confirmed as type II collagen.
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|
Collagen Subtype mRNA Expression--
Analysis of collagen
gene expression, normalized to the expression of the housekeeping gene
HPRT, showed there to be a significant decrease in posterior scleral
collagen type I mRNA expression in the myopic, relative to the
contralateral control, eye (
20 ± 7%, p < 0.05) as previously reported for the whole sclera (Fig. 5A). However, the mRNA
expression patterns of collagen type III (+2 ± 9%,
p = 0.84) and collagen type V (
1 ± 6%,
p = 0.74) were not significantly altered by 5 days of
myopia induction (Fig. 5A). These findings differed from
those in the normal animals assessed, where there was found to be no
significant change in subtype expression (Fig. 5A). The
reduction in type I collagen gene expression in myopic eyes resulted in
a significant relative increase in the ratio of type III/type I (+22%)
and type V/type I (+25%) collagen expression (p = 0.05; Fig. 5B). No change was observed in normal eyes when
this ratio was calculated using a within animal comparison (Fig.
5B).

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Fig. 5.
A, type I collagen mRNA expression
is reduced in the posterior sclera of myopic, relative to contralateral
control, eyes following 5 days of myopia development. Expression levels
of types III and V collagen were found to be unaltered relative to
control. No significant changes were found in age matched normal eyes
for any of the subtypes investigated. n = 8 in the
myopic group and n = 4 in the normal group. *,
p < 0.05 by paired t test. B,
when a within animal comparison was made of the type III/type I and
type V/type I collagen expression ratios, the ratio was found to be
elevated in myopic, but not normal, animals. n = 8 in
the myopic group and n = 4 in the normal group.
n = 8 in the myopic group and n = 4 in
the normal group. *, p = 0.05 by paired
comparison.
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|
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DISCUSSION |
Scleral thinning, tissue loss, and altered tissue morphology are
associated with the development of high myopia in humans and animals
models of myopia and the current study makes three major contributions
to the literature in this area. First, this study shows that reduced
collagen accumulation in the myopic eye results from both reduced
collagen synthesis and accelerated collagen degradation in the sclera,
which until now could only be speculated upon from previous findings.
Second, this study shows that scleral thinning early in high myopia
development is associated with differential changes in collagen subtype
expression and provides the first evidence of a mechanism that may
underlie the longer term changes in fibril diameter reported in the
sclera of humans with pathological myopia and animal models of high
myopia. Third, this study represents a comprehensive demonstration of
many of the collagen subtypes for which message is expressed in the
mammalian sclera.
Collagen Accumulation in Myopia--
Reduced proline incorporation
in the sclera of eyes following 5 days of myopia development is
consistent with the conclusion that general collagen production is
reduced in the sclera of eyes developing myopia. Indeed, although
proline is not exclusive to collagen in the body, previous studies have
found that only 4% of non-collagenous proteins comprise proline (31).
Furthermore, some 90% of the scleral dry weight is collagen (11) and
the vast majority of this is fibrillar collagen type I (12), indicating that nearly all of the radiolabeled proline assayed in the sclera during the present study would have been incorporated in collagen type
I. Reduced collagen synthesis in eyes developing myopia is consistent
with the results of both this and a previous study (41), in that
collagen type I mRNA production is reduced following different
periods of myopia development. The findings of the present study
confirm that reduced accumulation of collagen in the sclera of myopic
eyes, as determined from the reported reduction in scleral dry weight
and reduced scleral hydroxyproline content (19, 22), is partly
accounted for by reduced collagen synthesis.
In addition, the current study employed a modification of the
experimental paradigm to radiolabel scleral collagen, prior to the
induction of myopia, to demonstrate that labeled collagen was
eliminated more rapidly from the sclera of eyes in which myopia was
developing. This is, to our knowledge, the first study to directly show
that accelerated collagen degradation is associated with scleral
thinning and tissue loss in mammalian eyes with progressive myopia.
Furthermore, this is consistent with studies that show an increase in
the activity of collagen degrading enzymes, such as matrix
metalloproteinase-2, during myopia progression in animal models of
myopia (18).
The findings of the current study show that the elimination of collagen
from the posterior region of the sclera is most rapid during the first
12 days of myopia development. This is consistent with previous
findings that indicate that the majority of posterior scleral tissue
loss in high myopia development in this mammalian model occurs over the
first 12 days (Fig. 6) (10, 19). These studies also demonstrate that the lost tissue is not replaced if myopia
continues to progress (10). Indeed, when considered in the context of
human high myopia, these findings imply that scleral tissue loss occurs
much earlier than the clinical signs of pathological myopia, which only
usually manifest after high levels of refractive error have been
present for some time.

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Fig. 6.
Collagen degradation in the posterior sclera,
as indicated by the interocular difference between treated and control
eyes with time, follows a similar pattern to posterior scleral dry
weight loss. The patterns of scleral dry weight loss are plotted
for 0, 5, and 12 days of myopia development and projected out to 8 months using previously published data (10, 19).
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The rapid manifestation of altered collagen accumulation in the sclera
of eyes developing myopia suggests that it may contribute to the rapid
alterations that have been reported in the mechanical properties of the
sclera in myopic eyes (25). Such a finding is expected given the major
role that collagen plays in determining the biomechanical properties of
many different tissue systems (42).
Fibrillar Collagen Synthesis in Myopia--
To evaluate the role
that modulation of fibrillar collagen subtype-specific gene expression
plays in the long term alterations to the sclera in myopia, the present
study investigated changes in the production of mRNA encoding the
fibril-forming collagen types I, III, and V in posterior scleral tissue
in tree shrews. Each of the three collagen subtypes has previously been
shown to contribute to the scleral collagen fibrils of the sclera of other mammalian species (13), and it is widely reported that variation
in fibril composition of these subtypes is critical to fibril diameter
(17). Analysis of mRNA levels in the sclera of myopic, relative to
contralateral control, eyes showed a significant reduction in collagen
type I mRNA expression, as previously reported (41). Importantly,
however, there was no evidence of a similar reduction in the expression
of collagen type III and type V. Indeed, unlike collagen type I, no
significant difference in mRNA expression was observed between
treated and fellow control eyes for these quantitatively minor, but
important components of the scleral collagen fibrils.
The differential expression patterns observed for the fibril-forming
collagens investigated demonstrate that the relative ratio of collagen
type III/type I and collagen type V/type I mRNA is increased in the
sclera of eyes with progressive myopia, with significant increases of
some 22 and 25% in these respective ratios. This relative change in
the ratio of collagen subtype production occurs during one of the most
rapid phases of collagen synthesis and tissue remodeling in the
developing tree shrew eye. Smaller diameter collagen fibrils have been
observed in the sclera of highly myopic human (9) and monkey eyes (43)
and the accumulation of a significant number of small diameter collagen
fibrils has also been reported in the sclera of highly myopic tree
shrew eyes following relatively long (3 to 9 months) periods of myopia
development (10). Studies in other connective tissue systems have
examined the role co-assembly of different collagen types plays in
regulating fibrillogenesis and such studies have provided evidence to
suggest that fibril diameter is modulated according to the specific
composition of the fibrils (17). The effects of collagen type I/type V
interactions on fibril diameter regulation have been best demonstrated
using in vitro self-assembly assays, where the presence of
increasing amounts of collagen type V was found to result in decreased
fibril diameters (17). Of particular note is the fact that for a
relative increase in collagen type V of 20%, a value comparable with
the relative increase in the type V/type I ratio observed in the
present study, there was a relative decrease in collagen fibril
diameter of 36% (relative to homotypic collagen type I fibrils), which is consistent with the approximate 34% decrease observed in the collagen fibril diameters of the outer posterior scleral "layers" of myopic tree shrew eyes (10). It is hypothesized that larger diameter
collagen fibrils develop through lateral accumulation of molecular
collagen on small diameter immature fibrils, and that the presence of
collagen type V inhibits this lateral accumulation. Indeed, studies in
the corneal stroma, an ocular tissue closely associated with the sclera
and containing particularly high concentrations of type V collagen,
suggest it is this collagen subtype that is responsible for the small
and regular collagen fibrils that are characteristic of this tissue
(17).
We propose that the increase in expression of fibrillar collagen type
V, and perhaps collagen type III, which is also associated with reduced
fibril diameter (44), relative to that of type I collagen in the
present study, is indicative of a similar mechanism of fibril diameter
control in the sclera of highly myopic tree shrew eyes. Inhibition of
the lateral growth of newly synthesized fibrils during this early stage
would potentially lead to the gradual accumulation and predominance of
the smaller diameter fibrils, observed in long-term myopia. Given that
this observed alteration in collagen expression is associated with a
period of rapid eye growth (27), it seems likely that even a relatively short period of aberrant collagen synthesis might have profound effects
on the fibril diameter distributions observed later in axial myopia
development. Indeed, data from a number of connective tissue systems
suggests that collagen residency times are protracted in the body (26).
This may explain why the impact of early changes in fibril constituents
may not be apparent until later in development. Presumably, a continued
predominance of small diameter fibrils would require that collagen
synthesis be altered to some extent over a longer time period and this
remains to be demonstrated.
Collagen Subtype Expression in the Sclera--
The present study
demonstrates that the modified gene array approach reported can be
highly specific in identifying mRNA sequences of known genes in
novel species. This is evidenced by the fact that of the positive array
hybridizations that were screened using RT-PCR (collagen types I and
III), each subtype was confirmed as present in the sclera. However,
findings also suggest that the technique has a limited capacity to
identify a negative result as evidenced by the finding that collagen
type VIII did not hybridize to the array but was found to be present in
the scleral cDNA when screened using RT-PCR. Collagen type II was
not found to be present in the mammalian sclera, however, problems with
the array production in respect of this clone prevent us from
commenting on the likelihood of false positive hybridizations. We
conclude that this array approach represents a useful tool in the
identification of mRNA populations in novel species.
Data from the current study serves to demonstrate that the collagen
subtype expression profile in the mammalian sclera is wider than
reported to date. Collagen subtypes I, III, V, VI, VIII, and XIII were
found to be present in the tree shrew sclera, as expected given
previous reports of their presence in the human sclera (13, 45, 46). In
addition, collagen subtypes VII, IX, XV, XVI, and XVII were identified
in the tree shrew sclera and these are also likely to be present in the
human sclera. RT-PCR and sequencing results suggest that collagen type
II is absent from the tree shrew sclera and, although type II collagen
was recently identified in the adult rodent sclera (47), this is consistent with a previous report of its absence from the human sclera
(13), suggesting that the tree shrew sclera is much closer in structure
to primates than that of rodents.
In summary, the current study is the first to demonstrate that reduced
collagen accumulation in the sclera of highly myopic eyes is a result
of reduced collagen synthesis and increased collagen degradation. These
changes occur very early on in the process of myopia development. In
addition this study has shown that previous reports of reduced fibril
diameter in long term myopic eyes are linked to differential expression
patterns between the quantitatively major and the more minor fibrillar
collagens of the sclera, during the early stages of myopia development.
Finally, the current study represents a comprehensive demonstration of
the collagen subtypes expressed in the mammalian sclera.