Research Article Increased Expression of CCN2 in the Red...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 761823 7 pageshttpdxdoiorg1011552013761823

Research ArticleIncreased Expression of CCN2 in the Red FlashingLight-Induced Myopia in Guinea Pigs

Hong Wang Kang Zhuang Lei Gao Linna Zhang and Hongling Yang

Department of Ophthalmology Qilu Hospital Shandong University 107 Wenhua Xi Road Jinan 250012 China

Correspondence should be addressed to Hong Wang deltabiotechsd126com

Received 6 April 2013 Revised 11 June 2013 Accepted 18 June 2013

Academic Editor Youhua Liu

Copyright copy 2013 Hong Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Visual environment plays an important role in the occurrence of myopia We previously showed that the different flashing lightscould result in distinct effects on the ocular growth and development ofmyopia CCN2has been reported to regulate various cellularfunctions and biological processes However whether CCN2 signaling was involved in the red flashing light-induced myopia stillremains unknown In the present study we investigated the effects of the red flashing lights exposure on the refraction and axiallength of the eyes in vivo and then evaluated their effects on the expression of CCN2 and TGF-120573 in sclera tissues Our data showedthat the eyes exposed to the red flashing light became more myopic with a significant increase of the axial length and decrease ofthe refraction Both CCN2 and TGF-120573 as well as p38 MAPK and PI3K were highly expressed in the sclera tissues exposed to thered flashing light Both CCN2 and TGF-120573 were found to have the same gene expression profile in vivo In conclusion our findingsfound that CCN2 signaling pathway plays an important role in the red flashing light-induced myopia in vivo Moreover our studyestablishes a useful animal model for experimental myopia research

1 Introduction

Myopia is a vision condition in which the visual imagescome to a focus in front of the retina of the eye becauseof defects in the refractive media of the eye or of abnormallength of the eyeball resulting especially in defective visionof distant objects It has emerged as a significant public healthissue in the world because of its increasing prevalence Theprevalence of myopia has been reported as high as 70ndash90in Asia 30ndash40 in Europe and United States and 10ndash20in Africa [1] More and more studies revealed that earlymyopia might affect ocular growth and refractive statusMyopia is still a challenging disease to treat because themechanisms of the development of myopia and the hostresponse of myopic genes are not yet fully understood Animproved understanding of the effects of vision environmenton the development ofmyopia is urgently required to identifypotential preventative and therapeutic strategies

In addition to the well-known cause of myopia genes andbad posture habits the flashing lights may be directly associ-ated with myopia with the growing popularity of computersand television More andmore studies have shown that many

individuals who work at a computer experience high level ofeye or vision disorders such as the visual fatigue [2] Ourprevious study revealed that flashing light could cause axialelongation and further induce myopia in pigmented guineapigs Moreover different wavelength of unique flashing lightshad distinct influences and the most effective light is the redlight (750 nm) [3] Therefore in this study we will furtherfocus on the effects of the red flashing light on the oculargrowth and refraction in guinea pigs

Connective tissue growth factor (CTGFCCN2) is anextracellular matrix protein composed of four domainswhich belongs to the CCN protein family CCN2 has beenidentified to be involved in various cellular functions andbiological processes including fibrosis angiogenesis differ-entiation and wound healing in various cell types [4ndash7]Transforming growth factor-120573 (TGF-120573) is a multifunctionalcytokine involved in diverse cellular processes such as cellproliferation apoptosis differentiation and migration [8 9]Its dysfunctions could lead to various kinds of diseases suchas tissue fibrosis and cancer [10] Previous study reported thatTGF-120573 functioned as the direct inducer of CCN2 expressionin vitro and in vivo studies [11] Our previous study revealed

2 BioMed Research International

that the flashing light could induce the cell number andactivity of posterior sclera cells which resulted in an abnormalproliferation status of sclera in guinea pigs [12] indicatingthat active sclera remodeling plays a significant role in theflashing light-induced ocular growth and vision impairmentHowever it is still unknown whether CCN2 and TGF-120573 arealso involved in the red flashing light-induced vision changeand myopia The exact role of CCN2 in the progression ofexperimental myopia has yet to be determined

In the present study we exposed the guinea pigs under thered flashing light condition and further evaluated its effectson the changes of refractive error and axial length We alsoinvestigated the expression of CCN2 andTGF in sclera tissuesof the red flashing light-induced myopia model to exploretheir role in the pathogenesis of experimental myopia

2 Materials and Methods

21 Myopic Animal Model The animal research proceduresin this study were approved by the Animal Care and EthicsCommittee at Shandong University School of Medicine Thetreatment and care of animals were conducted according tothe ARVO statement for the Use of Animals in Ophthalmicand Vision Research Thirty guinea pigs (aged from 15to 20 days) weighting from 70 to 90 grams had similarrefractive error (from +200 to +350D) and were obtained atExperimental Animal Center of Shandong University Theywere randomly assigned to 3 groups (119899 = 10 each group)Group 1 was in a tightly closed carton and exposed to thered flashing light (see Supplementary Figure S1A availableonline at httpdxdoiorg1011552013761823) Group 2 wasin a tightly closed around carton and exposed to the whiteflashing light (Supplementary Figure S1B) Group 3 wasin a sight-widen cage and exposed to the natural light(Supplementary Figure S1C) For exposure to the red flashinglight a PS-I programmable flash stimulator (SupplementaryFigure S1D) was performed with 100 red diode and circuitboard to produce the red light All the animals were exposedon a cycle of 12 h illumination (800 lux) and 12 h darknessdaily during the experimental periodThe 800 lux lamps wereturned on and off with a 2 sec-2 sec on-off cycle The animalswere sacrificed at the end of light exposure

22 Refraction and Axial Length Measurement The refrac-tion and axial length of eyes were checked in the guineapigs at 8 weeks after light exposure One drop of 05tropicamide was administered to the eyes every 5min fortwice to achieve a completely dilated pupil After 30minretinoscopy was performed (accuracy 025D) in a dark roomusing a streak retinoscope The refraction was evaluated bythe mean value of the horizontal and vertical meridiansThe animal specific A-scan ultrasonography was used forthe axial length measurement Before the ultrasound mea-surement 05 oxybuprocaine hydrochloride eye drops wasadministered for twice for topical anesthesia The ultrasoundprobe directly contacted with the cornea during the axiallength measurement All the data represented averages from10 repeated measurements

23 Tissue Preparation All the animals were euthanized withan overdose of 3pentobarbital sodiumand the eyeballswereenucleated with the sclera immediately The cornea centralhorizontal diameter at the nasal and temporal limbus weremarked and then the bulbar conjunctiva along the limbuswas cut off to remove the anterior segment of the organizationand vitreous washed with saline choroidal pigment residueson the sclera Part of sclera tissues were then stored in minus80∘Clow temperature refrigerator for further mRNA and proteindetection and another part of sclera tissues were fixed inbuffered 4 formalin solution (PH = 74) and embedded inparaffin

24 Immunohistochemistry Assay Slides were dewaxed andrehydrated using xylene and graded alcohols followed by theincubation in the 3 H

2O2solution for 10min to quench

the endogenous peroxidise activity The tissue section washeated by amicrowave oven to repair the antigenNonspecificbinding was blocked with normal goat serum for 30minThe slides were incubated with the diluted primary antibody(rabbit anti-mouse CCN2 antibody Boster BioengineeringLimited Company China) at 4∘C overnight The followingmorning the biotinylated goat anti-rabbit IgG antibody wasadded to the tissue section and incubated for 30min NextSABC peroxidase and DAB chromogenic kit were appliedDAB developing time was controlled based on observationunder bright-field light microscopy The tissue sections wereslightly counterstained with haematoxylin and then washedwith distilled water to clean haematoxylin Finally the tissuesection was dried by baking and sealed by a drop of resin witha cover slide PBS was applied as negative control

25 Quantitative Real-Time PCR Total RNA was extractedwith trizol reagent purchased from Shanghai Sangon Biolo-gical Engineering Technology Company Briefly the conce-ntration and purity were determined by ZF spectropho-tometry (Shanghai Kang Huasheng instrument factory)The RNA integrity was verified by electrophoresis in 3denaturing formaldehyde agarose gel Subsequently 1120583g oftotal RNA was used to synthesize cDNA by SuperScript IIIkit (Invitrogen) according to the manufacturersquos instructionsReal-time PCR was performed on SDS 7500 instrument(Applied Biosystems) with the Taq polymerase and primersPrimers of PCR were designed using Primer Express 30software (Applied Biosystems) and synthesized by ShanghaiBioon Biotechnology The primers were as follows CCN2forward 51015840-TCTCCACCCGAGTTACCAATG-31015840 reverse51015840-CACCCCGCAGAACTTAGCC-31015840 TGF-120573 forward 51015840-TTGAACTCAGAGACGTAAGCGT-31015840 reverse 51015840-AGC-GCCAGGAATTGTTGCT-31015840 p38MAPK forward 51015840-AAG-TTCCTGTCCACATTGCC-31015840 reverse 51015840-TGGATTCAG-TGTCAAGCTGC-31015840 PI3 K forward 51015840-CGAGAGTGT-CGTCACAGTGTC-31015840 reverse 51015840-TGTTCGCTTCCACAA-ACACAG-31015840 120573-Actin forward 51015840-CTGTTGCTCGCG-TCGCTATA-31015840 reverse 51015840-AACGATGCCGTGCTCAATG-31015840 The amplification process was as follows predenaturationfor 5min at 95∘C followed by 40 cycles of denaturation at95∘C for 15 s annealing at 60∘C for 30 s extension at 72∘C

BioMed Research International 3

minus10

minus8

minus6

minus4

minus2

0

2

4Re

frac

tion

(D)

Unexposed Exposed

lowast

ControlWhite lightRed light

lowastlowast

(a)

0

2

4

6

8

10

Axi

al le

ngth

(mm

)

Unexposed Exposed


⋆

(b)Figure 1 Effects of red flashing light on refraction and axial length in guinea pigs (a)The refraction was detected using a streak retinoscope(b)The axial lengthwas evaluated byA-scan ultrasonography Data shown are from three independent experiments lowast and ⋆119875 lt 005 indicatesignificant differences from unexposed groups

119875 lt 005 indicates significant difference from white flashing light group

for 30 s and a final extension at 72∘C for 5min Fluorescencereading was taken during 60∘C step Equal volume of PCRproducts was electrophorezed and photographed underultraviolet illumination The relative mRNA expression levelof each gene was calculated with the ΔΔ119862

119905method 120573-Actin

was used as control in the same sample

26 Western Blot Analysis The sclera tissues were homog-enized and lysed with ice-cold radioimmunoprecipitation(RIPA) buffer containing protease inhibitors Protein con-centration was then quantified by Bradford Protein AssayKit Equal amounts of protein were boiled loaded on a10 SDS-PAGE gel and transferred onto a nitrocellulosemembrane The membrane was blocked with 5 nonfat drymilk in tris-buffered saline containing 01 Tween20 (TBST)for 1 h exposed to rabbit anti-mouse CCN2 antibody (BosterBio-engineering Limited Company China) and incubatedovernight at 4∘C The same blots were stripped and rean-alyzed using anti-Actin antibody as the internal controlMembranes were washedwith TBST and then incubatedwiththe horseradish-peroxidase- (HRP-) labeled antibody for 1 hThe protein bands were visualized using ImmobilonWesternChemiluminescent HRP Substrate reagents and exposed toautoradiography film developed and fixed The film wasscanned and analyzed with Quantity One Analyzer SoftwareIndependent experiments were repeated three times

27 Statistical Analysis All datawere expressed as themeanplusmnSEM from at least three independent experiments Statisticalcomparisons between the groups including corneal radiusof curvature eye refractive error axial length the TGF-120573and CCN2 expression levels were performed by a one-wayANOVA using SPSS (version 110) Statistical comparisonsamong CCN2 immunohistochemical positive rates were

performed by chi-square test 119875 lt 005 was considered to bestatistically significant

3 Results

31 Confirmation of Phenotypic Changes Induced by RedFlashing Light There was no significant difference of refrac-tion and axial length between the two eyes of the same animalat the beginning of the experimentThe eyes after red flashinglight exposure becamemoremyopic by minus1129D (Figure 1(a))and had an increase of axial length by 132mm (Figure 1(b))In white flashing light exposure group the refraction was alsoclearly decreased by +235D (Figure 1(a)) with an elongationof axial length by 041mm (Figure 1(b)) compared to theeyes under natural light exposure (Figures 1(a) and 1(b))Interestingly the refraction and axial length data showed thatthe natural light control eyes also had a myopic shift

32 CCN2 Protein Expression Was Enhanced in PosteriorSclera after Red Flashing Light Exposure To determine theeffect of flashing light exposure on CCN2 expression theprotein level of CCN2wasmeasured byWestern blot analysisThe data showed that the protein levels of CCN2 were thehighest in the red flashing light irradiated eyes followed bythe eyes of the white flashing light exposure group and thelowest in the eyes of the natural light controls (Figure 2(a))The differences of the protein levels between any two groupsare shown as in Figure 2(b) and the protein levels in redflashing light exposure group were remarkably increased by20-fold and by 13-fold in the white flashing light exposuregroup

33 CCN2 ImmunostainingWas Increased after Flashing LightExposure To further confirm the effect of flashing light


Red White Control

CCN2 expression in posterior sclera

CCN2

TGF-120573

(a)

CCN

2120573

-act

in

Red White Control

lowast

lowastlowast

00

04

08

12

(b)

Figure 2 CCN2 protein expression in posterior sclera tissues afterlight exposure (a) The protein level of CCN2 was detected byWestern blot analysis (b) The relative CCN2120573-actin protein levelwas evaluated in posterior sclera tissues lowast119875 lt 005 indicatessignificant differences from control lowastlowast119875 lt 001 indicates significantdifference from control

119875 lt 001 indicates significant differencefrom white light group

exposure on CCN2 expression the levels of CCN2 in thesclera tissues of guinea pigs exposed to the natural lightwhite flashing light and red flashing light were comparedwith immunohistochemistry The data showed that the levelof CCN2 immunostaining was detectable in control scleratissues (Figure 3(a)) increased in white flashing light group(Figure 3(b)) and significantly further increased in the redflashing light group (Figure 3(c))TheCCN2positive rate was(1036 plusmn 078) (1504 plusmn 059) and (4021 plusmn 065) inthe eyes of natural light white light and red light exposuregroups respectively (Figure 3(d)) These data indicated thatthe red flashing light exposure could significantly induceCCN2 expression in sclera tissue of guinea pigs

34 Both TGF-120573 and CCN2 Were Overexpressed in FlashingLight-Induced Myopia Model As CCN2 is a direct targetgene of TGF-120573 signaling the contribution of TGF-120573 inred flashing light-induced CCN2 expression was analyzedin sclera tissues The mRNA levels of TGF-120573 and CCN2were detected by RT-PCR The data demonstrated that theexpression of TGFmRNA in both the red light andwhite lightexposed eyes increased compared to the natural light exposedeyes and such an increase in the red flashing light exposedeyes was more obvious than in white flashing light exposedeyes (Figures 4(a) and 4(b))The expression of CCN2mRNAwas lower in natural light control eyes but increased in thewhite light exposed eyes andmore apparently increased in thered flashing light exposed eyesThese data indicated that both

TGF and CCN2 were coexpressed in sclera tissues in flashinglight-induced myopia model suggesting that TGF-120573-CCN2signaling might play an important role in the ocular growthand development of myopia

4 Discussion

In this study we used the flashing light to induce myopia inthe guinea pigs and compared the refraction and axial lengthamong the red flashing light and white flashing light exposedeyes of the experimental animals and natural light controlanimals The eyes under red flashing light exposure becamemore myopic and had an obvious increase of axial lengthsuggesting that flashing light is related to ocular growthand further development of myopia This result establishesa general and useful animal model for experimental myopiaresearchWe further compared CCN2 and TGF-120573 expressionlevels in sclera among the experimental and control animalsThe eyes exposed to red flashing light had the highest expres-sion level followed by the eyes exposed to white flashinglight and eyes of natural light control animals had the lowestexpression levels We also noticed that both CCN2 and TGF-120573 have the same gene expression profile in sclera tissuesof myopic model These data indicated that TGF-120573-CCN2signaling might play an important role in the developmentof myopia From the CCN2 and TGF-120573 expression data andthe change of refraction and axial length the red flashing lightcould clearly promote ocular growth and induce myopia Wethink that the red flashing lightmight inducemyopia throughthe emmetropization interruption in both eyes of animals andthe physiologic control of gene expression

Guinea pigs have been increasingly considered to be analternative model in the study of experimental myopia sincethe biometric changes of guinea pig eye are similar to thoseof other species and are relatively susceptible to developmyopia [13ndash15] Moreover they are very cooperative lessexpensive easily handled readily responded [16 17] andborn with a well-developed vision system [18] than most ofother small mammals In this study the red flashing light-induced myopic animal model completely meets four basicrequirements of myopic animal models Firstly it strictlyfollows the replacement of the 12 h circadian rhythm Thered flashing light has a similar intensity of 800 lux with thenormal natural light The visual environment alters betweenlight and dark during red light exposure and the imageon the retina is constantly changing from clear to blurredSecondly it maintains the integrity of the retinal anatomicstructures There was no toxicity to the eyes exposed to thered flashing light intensity of 800 lux in this experimentThirdly the starting time of the experiment begins at thevisual sensitive period of the guinea pig All the animalsthat aged from 15 to 20 days began to accept the lightstimulation at the sensitive period Finally it maintains thecontinuity of the experiment In this study all the guineapigs were continuously exposed to red flashing light for eightweeks Therefore our study for the first time verified thatred flashing light-inducedmyopia in guinea pig represents animportant animal model to study ocular growth and myopicdevelopment


(a) (b)

(c)

CCN

2 po

sitiv

e rat

e (

)

Control White Red0

10

20

30

40

50

lowast

lowastlowast

(d)

Figure 3 CCN2 immunostaining in posterior sclera tissues after light exposure ((a)ndash(c)) CCN2 expression was stained in control white lightand red light exposed sclera tissues respectively (d) CCN2 positive rate in posterior sclera tissues after light exposure lowast119875 lt 005 indicatessignificant differences from control lowastlowast119875 lt 001 indicates significant difference from control

119875 lt 001 indicates significant difference fromwhite light group


Relat

ive m

RNA

leve

l

00

05

10

15

20

⋆

lowast

⋆

CCN2 TGF-120573

(a)

p38 MAPK PI3K

Relat

ive m

RNA

leve

l


00

05

10

15

20

lowast

⋆lowast⋆

(b)Figure 4 The expression of CCN2 and downstream pathway genes in posterior sclera tissues after light exposure was evaluated by RT-PCR(a) The relative CCN2 and TGF-120573 mRNA level were analyzed in posterior sclera tissues (b) The relative p38 MAPK and PI3K mRNA levelwere also examined in posterior sclera tissues lowast119875 lt 005 indicates significant differences from control

119875 lt 001 indicates significantdifference from control ⋆119875 lt 005 indicates significant differences from control


Human sclera has been considered as a dynamic tissuethat can alter extracellular matrix (ECM) components andtheir biomechanical characters in response to changes in thevisual environment to regulate ocular refraction and axiallength [19] It has been reported that active sclera ECMremodeling could result in the development of myopia [20]Our previous study found that flashing light could lead toan increase of sclera cells number and its biological activityin guinea pigs [12] suggesting that the sclera remodelingwas associated with flashing light-induced myopia Howeverwe still have relatively little understanding of the cellularsignaling factors that drive the changes of ocular growth andmyopia development

CCN2 was first identified as a profibrotic mediator whichwas upregulated both in vitro and in vivo models of diabeticnephropathy [21]More andmore studies revealed that CCN2largely functions as a matricellular protein regulating andintegrating the role of other growth factor signaling cellmotility and differentiation in tissue remodeling [22] CCN2can bind to multiple receptors and activate divergent signal-ing pathways including TGF-120573 p38 MAPK [23] PI3K [24]and Rho GTPase [25] CCN2 is expressed in a variety of celltypes including fibroblasts endothelial cells vascular smoothmuscle cells and epithelial cells [26ndash28] Previous studyreported that the expression of CCN2 was lower in sclerafibroblasts than in corneal fibroblasts and the sclera appearednormal in the eyes of CCN2 knockout mouse [29] Our studyfor the first time found that CCN2 as well as its downstreamsignaling pathway genes including p38MAPK and PI3K waslower in sclera tissue in guinea pigs but increased after whiteflashing light exposure and significantly increased under redflashing light exposure suggesting that CCN2 might playan important role in sclera remodeling in the developmentof myopia However to what extent CCN2 gene of smalleffect and gene-environment interactions contribute to thedevelopment of myopia remains to be elucidated

In conclusion the refractive and axial length data indi-cated that the red flashing light could successfully inducemyopia in guinea pigs providing a useful animal modelfor the study of experimental myopia We also identified asignificant change of CCN2 TGF-120573 p38 MAPK and PI3Kexpression levels in the eyes of the guinea pigs exposed tothe red flashing light CCN2 signaling pathway might playa significant role in flashing light-induced ocular growthand myopic development Exploration of CCN2 signalingpathways will help us reveal the molecular mechanismsunderlying sclera remodeling and ocular growth

AbbreviationsCCN2 Connective tissue growth factorTGF-120573 Transforming growth factor-120573RIPA RadioimmunoprecipitationRT-PCR Reverse transcription-polymerase chain reactionHRP Horseradish peroxidase

Authorsrsquo Contribution

Hong Wang and Kang Zhuang equally contributed to thisstudy

Acknowledgments

This work was supported by the Promotive Research Fundfor Young- andMiddle-Aged Scientists of Shandong Province(no 2007BS03014) and the Science-TechnologyDevelopmentProject of Shandong Province of China

References

[1] D R Fredrick ldquoMyopiardquo British Medical Journal vol 324 no7347 pp 1195ndash1199 2002

[2] J Ma X M Zhang H J Wang et al ldquoThe effect of flicker fromtelevision screen on the function of visual accommodationrdquoChinese Journal of Preventive Medicine vol 2 no 3 pp 204ndash206 2001

[3] H Wang K Zhuang Y Tao et al ldquoEffects of light pollution ondevelopment of myopia in guinea pigsrdquo Journal of Environmen-tal Health vol 24 no 6 pp 388ndash390 2007

[4] B Perbal ldquoCCN proteins multifunctional signalling regula-torsrdquoThe Lancet vol 363 no 9402 pp 62ndash64 2004

[5] P de Winter P Leoni and D Abraham ldquoConnective tissuegrowth factor structure-function relationships of a mosaicmultifunctional proteinrdquo Growth Factors vol 26 no 2 pp 80ndash91 2008

[6] I Cicha and M Goppelt-Struebe ldquoConnective tissue growthfactor context-dependent functions and mechanisms of regu-lationrdquo BioFactors vol 35 no 2 pp 200ndash208 2009

[7] J A Arnott A G Lambi C Mundy et al ldquoThe role of conne-ctive tissue growth factor (CTGFCCN2) in skeletogenesisrdquoCritical Reviews in Eukaryotic Gene Expression vol 21 no 1 pp43ndash69 2011

[8] F Huang and Y G Chen ldquoRegulation of TGF-120573 receptoractivityrdquo Cell amp Bioscience vol 2 p 9 2012

[9] X Guo and S Y Chen ldquoTransforming growth factor-120573 andsmooth muscle differentiationrdquo World Journal of BiologicalChemistry vol 3 no 3 pp 41ndash52 2012

[10] J Massague S W Blain and R S Lo ldquoTGF120573 signaling ingrowth control cancer and heritable disordersrdquo Cell vol 103no 2 pp 295ndash309 2000

[11] A Igarashi H Okochi D M Bradham and G R GrotendorstldquoRegulation of connective tissue growth factor gene expressionin human skin fibroblasts and during wound repairrdquoMolecularBiology of the Cell vol 4 no 6 pp 637ndash645 1993

[12] Z-Y Cheng J-H Li R Li and Y-B Xie ldquoEffects of flashinglight on ocular growth and development of myopia in pig-mented guinea pigsrdquo Chinese Journal of Ophthalmology vol 40no 9 pp 601ndash604 2004

[13] S A McFadden M H C Howlett and J R Mertz ldquoRetinoicacid signals the direction of ocular elongation in the guinea pigeyerdquo Vision Research vol 44 no 7 pp 643ndash653 2004

[14] X Zhou J Ye M D P Willcox et al ldquoChanges in proteinprofiles of guinea pig sclera during development of formdeprivation myopia and recoveryrdquoMolecular Vision vol 16 pp2163ndash2174 2010

[15] H L Zhao R Q Wang M Q Wu et al ldquoDynamic changesof ocular biometric parameters a modified form-deprivationmyopia model of young guinea pigsrdquo International Journal ofOphthalmology vol 4 no 5 pp 484ndash488 2011

[16] M H C Howlett and S A McFadden ldquoForm-deprivationmyopia in the guinea pig (Cavia porcellus)rdquoVisionResearch vol46 no 1-2 pp 267ndash283 2006


[17] M H C Howlett and S A McFadden ldquoSpectacle lens compen-sation in the pigmented guinea pigrdquoVision Research vol 49 no2 pp 219ndash227 2009

[18] L Jiang K Long F Schaeffel et al ldquoDisruption of emmetro-pization and high susceptibility to deprivationmyopia in albinoguinea pigsrdquo Investigative Ophthalmology and Visual Sciencevol 52 no 9 pp 6124ndash6132 2011

[19] J A Summers Rada S Shelton and T T Norton ldquoThe scleraand myopiardquo Experimental Eye Research vol 82 no 2 pp 185ndash200 2006

[20] J A Rada D L Nickla and D Troilo ldquoDecreased proteoglycansynthesis associated with form deprivation myopia in matureprimate eyesrdquo Investigative Ophthalmology and Visual Sciencevol 41 no 8 pp 2050ndash2058 2000

[21] M Murphy C Godson S Cannon et al ldquoSuppression subtrac-tive hybridization identifies high glucose levels as a stimulus forexpression of connective tissue growth factor and other genesin human mesangial cellsrdquo Journal of Biological Chemistry vol274 no 9 pp 5830ndash5834 1999

[22] X Shi-Wen A Leask and D Abraham ldquoRegulation and func-tion of connective tissue growth factorCCN2 in tissue repairscarring and fibrosisrdquo Cytokine and Growth Factor Reviews vol19 no 2 pp 133ndash144 2008

[23] F Furlong J Crean L Thornton R OrsquoLeary M Murphy andF Martin ldquoDysregulated intracellular signaling impairs CTGF-stimulated responses in humanmesangial cells exposed to highextracellular glucoserdquo American Journal of PhysiologymdashRenalPhysiology vol 292 no 6 pp F1691ndashF1700 2007

[24] J K Crean F Furlong D Finlay et al ldquoConnective tissuegrowth factor [CTGF]CCN2 stimulates mesangial cell migra-tion through integrated dissolution of focal adhesion complexesand activation of cell polarizationrdquo The FASEB Journal vol 18no 13 pp 1541ndash1543 2004

[25] J K Crean F Furlong D Mitchell E McArdle C Godsonand F Martin ldquoConnective tissue growth factorCCN2 stimu-lates actin disassembly through Aktprotein kinase B-mediatedphosphorylation and cytoplasmic translocation of p27(Kip-1)rdquoThe FASEB Journal vol 20 no 10 pp 1712ndash1714 2006

[26] E E-D A Moussad and D R Brigstock ldquoConnective tissuegrowth factor whatrsquos in a namerdquo Molecular Genetics andMetabolism vol 71 no 1-2 pp 276ndash292 2000

[27] J G Browne S L Ho R Kane et al ldquoConnective tissue growthfactor is increased in pseudoexfoliation glaucomardquo InvestigativeOphthalmology amp Visual Science vol 52 no 6 pp 3660ndash36662011

[28] J G Abreu N I Ketpura B Reversade and E M de RobertisldquoConnective-tissue growth factor (CTGF) modulates cell sig-nalling by BMP and TGF-120573rdquo Nature Cell Biology vol 4 no 8pp 599ndash604 2002

[29] E J Kuiper P Roestenberg C Ehlken et al ldquoAngiogenesis is notimpaired in Connective Tissue Growth Factor (CTGF) knock-out micerdquo Journal of Histochemistry and Cytochemistry vol 55no 11 pp 1139ndash1147 2007

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


that the flashing light could induce the cell number andactivity of posterior sclera cells which resulted in an abnormalproliferation status of sclera in guinea pigs [12] indicatingthat active sclera remodeling plays a significant role in theflashing light-induced ocular growth and vision impairmentHowever it is still unknown whether CCN2 and TGF-120573 arealso involved in the red flashing light-induced vision changeand myopia The exact role of CCN2 in the progression ofexperimental myopia has yet to be determined

In the present study we exposed the guinea pigs under thered flashing light condition and further evaluated its effectson the changes of refractive error and axial length We alsoinvestigated the expression of CCN2 andTGF in sclera tissuesof the red flashing light-induced myopia model to exploretheir role in the pathogenesis of experimental myopia

2 Materials and Methods

21 Myopic Animal Model The animal research proceduresin this study were approved by the Animal Care and EthicsCommittee at Shandong University School of Medicine Thetreatment and care of animals were conducted according tothe ARVO statement for the Use of Animals in Ophthalmicand Vision Research Thirty guinea pigs (aged from 15to 20 days) weighting from 70 to 90 grams had similarrefractive error (from +200 to +350D) and were obtained atExperimental Animal Center of Shandong University Theywere randomly assigned to 3 groups (119899 = 10 each group)Group 1 was in a tightly closed carton and exposed to thered flashing light (see Supplementary Figure S1A availableonline at httpdxdoiorg1011552013761823) Group 2 wasin a tightly closed around carton and exposed to the whiteflashing light (Supplementary Figure S1B) Group 3 wasin a sight-widen cage and exposed to the natural light(Supplementary Figure S1C) For exposure to the red flashinglight a PS-I programmable flash stimulator (SupplementaryFigure S1D) was performed with 100 red diode and circuitboard to produce the red light All the animals were exposedon a cycle of 12 h illumination (800 lux) and 12 h darknessdaily during the experimental periodThe 800 lux lamps wereturned on and off with a 2 sec-2 sec on-off cycle The animalswere sacrificed at the end of light exposure

22 Refraction and Axial Length Measurement The refrac-tion and axial length of eyes were checked in the guineapigs at 8 weeks after light exposure One drop of 05tropicamide was administered to the eyes every 5min fortwice to achieve a completely dilated pupil After 30minretinoscopy was performed (accuracy 025D) in a dark roomusing a streak retinoscope The refraction was evaluated bythe mean value of the horizontal and vertical meridiansThe animal specific A-scan ultrasonography was used forthe axial length measurement Before the ultrasound mea-surement 05 oxybuprocaine hydrochloride eye drops wasadministered for twice for topical anesthesia The ultrasoundprobe directly contacted with the cornea during the axiallength measurement All the data represented averages from10 repeated measurements

23 Tissue Preparation All the animals were euthanized withan overdose of 3pentobarbital sodiumand the eyeballswereenucleated with the sclera immediately The cornea centralhorizontal diameter at the nasal and temporal limbus weremarked and then the bulbar conjunctiva along the limbuswas cut off to remove the anterior segment of the organizationand vitreous washed with saline choroidal pigment residueson the sclera Part of sclera tissues were then stored in minus80∘Clow temperature refrigerator for further mRNA and proteindetection and another part of sclera tissues were fixed inbuffered 4 formalin solution (PH = 74) and embedded inparaffin

24 Immunohistochemistry Assay Slides were dewaxed andrehydrated using xylene and graded alcohols followed by theincubation in the 3 H

2O2solution for 10min to quench

the endogenous peroxidise activity The tissue section washeated by amicrowave oven to repair the antigenNonspecificbinding was blocked with normal goat serum for 30minThe slides were incubated with the diluted primary antibody(rabbit anti-mouse CCN2 antibody Boster BioengineeringLimited Company China) at 4∘C overnight The followingmorning the biotinylated goat anti-rabbit IgG antibody wasadded to the tissue section and incubated for 30min NextSABC peroxidase and DAB chromogenic kit were appliedDAB developing time was controlled based on observationunder bright-field light microscopy The tissue sections wereslightly counterstained with haematoxylin and then washedwith distilled water to clean haematoxylin Finally the tissuesection was dried by baking and sealed by a drop of resin witha cover slide PBS was applied as negative control

25 Quantitative Real-Time PCR Total RNA was extractedwith trizol reagent purchased from Shanghai Sangon Biolo-gical Engineering Technology Company Briefly the conce-ntration and purity were determined by ZF spectropho-tometry (Shanghai Kang Huasheng instrument factory)The RNA integrity was verified by electrophoresis in 3denaturing formaldehyde agarose gel Subsequently 1120583g oftotal RNA was used to synthesize cDNA by SuperScript IIIkit (Invitrogen) according to the manufacturersquos instructionsReal-time PCR was performed on SDS 7500 instrument(Applied Biosystems) with the Taq polymerase and primersPrimers of PCR were designed using Primer Express 30software (Applied Biosystems) and synthesized by ShanghaiBioon Biotechnology The primers were as follows CCN2forward 51015840-TCTCCACCCGAGTTACCAATG-31015840 reverse51015840-CACCCCGCAGAACTTAGCC-31015840 TGF-120573 forward 51015840-TTGAACTCAGAGACGTAAGCGT-31015840 reverse 51015840-AGC-GCCAGGAATTGTTGCT-31015840 p38MAPK forward 51015840-AAG-TTCCTGTCCACATTGCC-31015840 reverse 51015840-TGGATTCAG-TGTCAAGCTGC-31015840 PI3 K forward 51015840-CGAGAGTGT-CGTCACAGTGTC-31015840 reverse 51015840-TGTTCGCTTCCACAA-ACACAG-31015840 120573-Actin forward 51015840-CTGTTGCTCGCG-TCGCTATA-31015840 reverse 51015840-AACGATGCCGTGCTCAATG-31015840 The amplification process was as follows predenaturationfor 5min at 95∘C followed by 40 cycles of denaturation at95∘C for 15 s annealing at 60∘C for 30 s extension at 72∘C


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Acknowledgments


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Disease Markers



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Journal of

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Control White Red0

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Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

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2 po

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Control White Red0

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Relat

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Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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