Title: Collagen Gene Expression and the Altered Accumulation of Scleral Collagen during the Development of High Myopia
Abstract: The development of high myopia is associated with reduced scleral collagen accumulation, scleral thinning, and loss of scleral tissue, in both humans and animal models. Reduced collagen fibril diameter is also observed in the sclera of eyes with high myopia. The present study investigated aspects of scleral collagen synthesis and degradation, in a mammalian model of high myopia, to elucidate the factors underlying scleral changes. General synthesis and degradation of scleral collagen was investigated in monocularly deprived tree shrews, through the in vivoadministration of [3H]proline and subsequent assay of scleral tissue for [3H]collagen. In addition, PCR enriched cDNA, produced from tree shrew scleral mRNA, was used to synthesize probes for hybridization to custom gene arrays consisting of partial sequences for 11 collagen subtypes. Finally, real-time reverse transcriptase-PCR was employed to investigate collagen type I, III, and V mRNA expression in the sclera of myopic, contralateral control, and normal tree shrew eyes. Scleral [3H]proline incorporation was reduced at the posterior pole of myopic eyes following 5 days of monocular deprivation (−36 ± 47), whereas [3H]proline content was similar in treated and control eyes before myopia induction (−1 ± 87) but was reduced in myopic eyes following 5 (−8 ± 27), 12 (−15 ± 47), and 24 (−10 ± 47) days of myopia induction. The majority of the collagens investigated were found to be expressed in the sclera, with 11 subtypes being identified. Collagen type I mRNA expression was reduced in the sclera of myopic eyes (−20 ± 77), however, collagen type III (+2 ± 97) and type V (−1 ± 67) expression was unchanged relative to control, resulting in a net increase in the ratio of expression of collagen type III/type I and collagen type V/type I (22 and 257, respectively). These results show that reduced scleral collagen accumulation in myopic eyes is a result of both decreased collagen synthesis and accelerated collagen degradation. Furthermore, changes in collagen synthesis are driven by reduced type I collagen production. Short term increases in the ratio of newly synthesized collagen type III/type I and type V/type I are likely to be important in the increasing frequency of small diameter scleral collagen fibrils observed in high myopia and may be important in the subsequent development of posterior staphyloma in humans with pathological myopia. The development of high myopia is associated with reduced scleral collagen accumulation, scleral thinning, and loss of scleral tissue, in both humans and animal models. Reduced collagen fibril diameter is also observed in the sclera of eyes with high myopia. The present study investigated aspects of scleral collagen synthesis and degradation, in a mammalian model of high myopia, to elucidate the factors underlying scleral changes. General synthesis and degradation of scleral collagen was investigated in monocularly deprived tree shrews, through the in vivoadministration of [3H]proline and subsequent assay of scleral tissue for [3H]collagen. In addition, PCR enriched cDNA, produced from tree shrew scleral mRNA, was used to synthesize probes for hybridization to custom gene arrays consisting of partial sequences for 11 collagen subtypes. Finally, real-time reverse transcriptase-PCR was employed to investigate collagen type I, III, and V mRNA expression in the sclera of myopic, contralateral control, and normal tree shrew eyes. Scleral [3H]proline incorporation was reduced at the posterior pole of myopic eyes following 5 days of monocular deprivation (−36 ± 47), whereas [3H]proline content was similar in treated and control eyes before myopia induction (−1 ± 87) but was reduced in myopic eyes following 5 (−8 ± 27), 12 (−15 ± 47), and 24 (−10 ± 47) days of myopia induction. The majority of the collagens investigated were found to be expressed in the sclera, with 11 subtypes being identified. Collagen type I mRNA expression was reduced in the sclera of myopic eyes (−20 ± 77), however, collagen type III (+2 ± 97) and type V (−1 ± 67) expression was unchanged relative to control, resulting in a net increase in the ratio of expression of collagen type III/type I and collagen type V/type I (22 and 257, respectively). These results show that reduced scleral collagen accumulation in myopic eyes is a result of both decreased collagen synthesis and accelerated collagen degradation. Furthermore, changes in collagen synthesis are driven by reduced type I collagen production. Short term increases in the ratio of newly synthesized collagen type III/type I and type V/type I are likely to be important in the increasing frequency of small diameter scleral collagen fibrils observed in high myopia and may be important in the subsequent development of posterior staphyloma in humans with pathological myopia. hypoxanthine-guanine phosphoribosyltransferase glyceraldehyde-3-phosphate dehydrogenase reverse transcriptase analysis of variance 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 (1Curtin B.J. The Myopias: Basic Science and Clinical Management. Harper ' Row, Philadelphia, PA1985Google Scholar). 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 (2Sorsby A. Leary G.A. Med. Res. Counc. (G. B.) Spec. Rep. Ser. 1969; 309: 1-41PubMed Google Scholar, 3Wiesel T.N. Raviola E. Nature. 1977; 266: 66-68Crossref PubMed Scopus (505) Google Scholar). The prevalence of human high myopia (usually defined as eyes with >6 dioptres (D) of myopia, or >26 mm in length) is ∼27 in the general population (1Curtin B.J. The Myopias: Basic Science and Clinical Management. Harper ' Row, Philadelphia, PA1985Google Scholar) and it is well documented that individuals with high myopia have a greatly increased risk of ocular pathology (4Grossniklaus H.E. Green W.R. Retina. 1992; 12: 127-133Crossref PubMed Scopus (423) Google Scholar). 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 (5Curcio C.A. Sloan K.R. Packer O. Hendrickson A.E. Kalina R.E. Science. 1987; 236: 579-582Crossref PubMed Scopus (450) Google Scholar,6Müller B. Peichl L. J. Comp. Neurol. 1989; 282: 581-594Crossref PubMed Scopus (117) Google Scholar). In some individuals, the thinned posterior sclera precipitates local ectasic change or staphyloma (7Curtin B.J. Teng C.C. Trans. Am. Acad. Ophthalmol. Oto-laryngol. 1957; 62: 777-788Google Scholar, 8Curtin B.J. Trans. Am. Ophthalmol. Soc. 1977; 75: 67-86PubMed Google Scholar). 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 (7Curtin B.J. Teng C.C. Trans. Am. Acad. Ophthalmol. Oto-laryngol. 1957; 62: 777-788Google Scholar, 9Curtin B.J. Iwamoto T. Renaldo D.P. Arch. Ophthalmol. 1979; 97: 912-915Crossref PubMed Scopus (204) Google Scholar). 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 (10McBrien N.A. Cornell L.M. Gentle A. Invest. Ophthalmol. Vis. Sci. 2001; 42: 2179-2187PubMed Google Scholar). Collagen accounts for 907 of scleral dry weight, the majority of this being collagen type I (11Norton T.T. Miller E.J. Invest. Ophthalmol. Vis. Sci. 1995; 36: S760Google Scholar, 12Zorn N. Hernandez M.R. Norton T.T. Yang J. Ye H.O. Invest. Ophthalmol. Vis. Sci. 1992; 33: S1053Google Scholar). Mammalian sclera also contains small amounts of other fibrillar and fibril-associated collagens (13Marshall G.E. Konstas A.G. Lee W.R. Curr. Eye Res. 1993; 12: 143-153Crossref PubMed Scopus (34) Google Scholar, 14Tamura Y. Konomi H. Sawada H. Takashima S. Nakajima A. Invest. Ophthalmol. Vis. Sci. 1991; 32: 2636-2644PubMed Google Scholar, 15Wessel H. Anderson S. Fite D. Halvas E. Hempel J. SundarRaj N. Invest. Ophthalmol. Vis. Sci. 1997; 38: 2408-2422PubMed Google Scholar) and studies have shown that scleral fibrils are heterologous, comprising collagen types I, III, and V (13Marshall G.E. Konstas A.G. Lee W.R. Curr. Eye Res. 1993; 12: 143-153Crossref PubMed Scopus (34) Google Scholar). To date, at least 21 different collagen subtypes have been identified (16Fitzgerald J. Bateman J.F. FEBS Lett. 2001; 505: 275-280Crossref PubMed Scopus (67) Google Scholar), 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 (17Birk D.E. Fitch J.M. Babiarz J.P. Doane K.J. Linsenmayer T.F. J. Cell Sci. 1990; 95: 649-657Crossref PubMed Google Scholar). Mammalian models of myopia have shown that scleral thinning and tissue loss occur rapidly in myopia development (10McBrien N.A. Cornell L.M. Gentle A. Invest. Ophthalmol. Vis. Sci. 2001; 42: 2179-2187PubMed Google Scholar). Scleral tissue loss occurs in conjunction with increased expression and activation of collagen-degrading enzymes, such as matrix metalloproteinase-2 (18Guggenheim J.A. McBrien N.A. Invest. Ophthalmol. Vis. Sci. 1996; 37: 1380-1395PubMed Google Scholar), decreased production of glycosaminoglycans (19McBrien N.A. Lawlor P. Gentle A. Invest. Ophthalmol. Vis. Sci. 2000; 41: 3713-3719PubMed Google Scholar, 20Rada J.A. Nickla D.L. Troilo D. Invest. Ophthalmol. Vis. Sci. 2000; 41: 2050-2058PubMed Google Scholar) and decreased proliferative activity of scleral cells (21Gentle A. McBrien N.A. Exp. Eye Res. 1999; 68: 155-163Crossref PubMed Scopus (65) Google Scholar). The net result is a decrease in scleral collagen content (22Norton T.T. Rada J.A. Vision Res. 1995; 35: 1271-1281Crossref PubMed Scopus (226) Google Scholar), as also found in human high myopia (23Avetisov E.S. Savitskaya N.F. Vinetskaya M.I. Iomdina E.N. Metab. Pediatr. Syst. Ophthalmol. 1984; 7: 183-188Google Scholar). 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 (24Phillips J.R. McBrien N.A. Ophthalmic Physiol. Opt. 1995; 15: 357-362Crossref PubMed Scopus (98) Google Scholar, 25Siegwart J.T. Norton T.T. Vision Res. 1999; 39: 387-407Crossref PubMed Scopus (194) Google Scholar). The majority of scleral thinning and tissue loss occur well before the increased frequency of smaller collagen fibrils (10McBrien N.A. Cornell L.M. Gentle A. Invest. Ophthalmol. Vis. Sci. 2001; 42: 2179-2187PubMed Google Scholar), 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 (26Verzijl N. DeGroot J. Thorpe S.R. Bank R.A. Shaw J.N. Lyons T.J. Bijlsma J.W. Lafeber F.P. Baynes J.W. TeKoppele J.M. J. Biol. Chem. 2000; 275: 39027-39031Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar), 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 (27McBrien N.A. Norton T.T. Vision Res. 1992; 32: 843-852Crossref PubMed Scopus (147) Google Scholar). 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. 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 (28Siegwart J.T. Norton T.T. Lab. Anim. Sci. 1994; 44: 213-215Google Scholar). 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 (27McBrien N.A. Norton T.T. Vision Res. 1992; 32: 843-852Crossref PubMed Scopus (147) Google Scholar, 29Siegwart J.T. Norton T.T. Vision Res. 1998; 38: 3505-3515Crossref PubMed Scopus (121) Google Scholar). 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 (30McBrien N.A. Moghaddam H.O. Reeder A.P. Invest. Ophthalmol. Vis. Sci. 1993; 34: 205-215PubMed Google Scholar). 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 (19McBrien N.A. Lawlor P. Gentle A. Invest. Ophthalmol. Vis. Sci. 2000; 41: 3713-3719PubMed Google Scholar). 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. In the group of animals that provided information regarding collagen production, an intraperitoneal injection of 300 ॖCi ofl-[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 ∼47 of non-collagenous proteins and 147 of collagen (31Laurent G.J. Sparrow M.P. Bates P.C. Millward D.J. Biochem. J. 1978; 176: 393-401Crossref PubMed Scopus (51) Google Scholar). However, when proline is assayed in a tissue such as the sclera, which comprises around 907 collagen (11Norton T.T. Miller E.J. Invest. Ophthalmol. Vis. Sci. 1995; 36: S760Google Scholar), 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 ॖCi ofl-[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 (32Lee R.E. Davison P.F. Exp. Eye Res. 1981; 32: 737-745Crossref PubMed Scopus (39) Google Scholar). At the time of fitting of the occluder, a dose of “cold” proline (7 mmol/kg l-proline in 0.97 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 (33Laurent G.J. Biochem. J. 1982; 206: 535-544Crossref PubMed Scopus (87) Google Scholar). MultiScreen filtration plates (0.65 ॖm 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, andp-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; [α-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. 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 (34Newsome D.A. Gross J. Hassell J.R. Invest. Ophthalmol. Vis. Sci. 1982; 22: 376-381PubMed Google Scholar), 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 (35Koyano Y. Hammerle H. Mollenhauer J. BioTechniques. 1997; 22 (710–712, 714): 706-708PubMed Google Scholar). Filters of the multiwell plate were wetted with 100 ॖl of 257 trichloroacetic acid. Triplicates (100 ॖl) of each corneal, anterior/equatorial, or posterior scleral sample were added to the wells with 100 ॖl of 507 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 ॖl of 107 trichloroacetic acid to remove the unincorporated label. The filters were allowed to air dry before being punched into plastic scintillation vials containing 500 ॖl of 4 m guanidine hydrochloride in 337 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 (36Reddy G.K. Enwemeka C.S. Clin. Biochem. 1996; 29: 225-229Crossref PubMed Scopus (1100) Google Scholar). Standards of hydroxyproline were prepared (0.5–8.0 ॖg in 40 ॖl) and 10 ॖl of sodium hydroxide (10 m) was mixed with 40 ॖl 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 107 1-propanol and acetate citrate buffer, 450 ॖl) 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 mp-dimethylaminobenzaldehyde in 1-propanol/perchloric acid, 2:1, v/v) was freshly prepared and added to each vial (500 ॖl) 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. 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 α-subunit clones were available for a given collagen subtype, the most common naturally occurring α-subunit, usually α1, 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 controlsHPRT1 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. Tissues were homogenized (4m guanidine thiocyanate, 25 mm sodium citrate) in a freezer mill, then total RNA was isolated using a standard method (37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (65695) Google Scholar). 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 707 ethanol. Glycogen (10 ॖg) 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. SMART cDNA synthesis was carried out using 1 ॖg 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. Radiolabeled probes were prepared from 100 ng of the PCR-enriched cDNA library using the Megaprime DNA labeling system, [α-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, 0.17 SDS. The array was visualized using a PhosphorImager. Gene array hybridizations were screened using an RT-PCR and sequencing protocol developed in this laboratory (38Gentle A. Anastasopoulos F. McBrien N.A. BioTechniques. 2001; 31 (504–506, 508): 502Crossref PubMed Scopus (70) Google Scholar). Briefly, primers were designed against areas of the coding regions of COL1A1,COL2A1, COL3A1, COL5A1, andCOL8A1 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. 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 (39Gentle A. McBrien N.A. Cytokine. 2002; 18: 344-348Crossref PubMed Scopus (47) Google Scholar). 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 ॖl) were mixed with primers (1 ॖm) and the FastStart DNA master mixture containing FastStart Taq polymerase, SYBR gre