The Research Of The Effect Of Retinoic Acid On The Formation Of Form Deprivation Myopia In Chicken

PurposeTo Discuss the effect of retinoic acid on the formation of FDM in chicken.Experimental FDM has been used as the animal model for the research of humanmyopia. FDM is induced by the deprivation of normal imagination on theretina in newborn animal. Its structure and refractive feature is very similar tothat of spontaneous human myopia.The growth of eyeball is regulated by the developmental mechanism, andthe feedback mechanism of vision input signal as well. Many research haveshown that vision on the retina leads to the change in eye length, which is inthe form of sclera growth. So, it is reasonable to say, that there are certainfactors secreted by retain, changed with the environment, which may serve assignal to regulate the growth and metabolism of sclera cells.RA is thought to be related to it. RA, also know as VA acid, is the mostactive metabolite of Vitamin. A. It is already well known that RA has importanteffect on the cell proliferation. Difference and maintenance of cellular pheno-type. For example, amacrine cell on inner retina take a port in the metabolismof RA, which is located at the output end of retinal image processing. So isthis procession related to the control of eyeball growth? RA may be a messengerof experimental eyeball growth regulation. Whatsmore, RA, a nonsteroid in vivohormone, can play a role in the transcription of different gene by combined withnuclear receptors directly, activate the cellular difference. Because thereare RA receptors in sclera cells, RA may has effect on the regulation of sclera cell growth and difference. So it has theoretical and experimental basis to think that RA is a signalmolecule and growth regulator of eyeball axis changes in FDM.RALDH2 is the key enzyme in RA synthesis. The analysis of expression ofRA and RALDH2 in retina. Choroids and sclera can be used to judge the sit ofRA synthesis and whether RA has taken a part in the formation of FDM. RARβ,a nuclear receptor, comhined with RA, Can regulate the transcription of gene,cell growth difference and metabolism. The effect of RA on formation of FDMcan be further determined by the detection of RARβin posterior pole sclera. Thesclera active play on important role in the formation and recovery of FDM. Thereis delicate balance between the matrix synthesis and degeneration in sclera remodeling ting. MMP-2 and its inhibitor TIMP-2 are important in the synthesisand degeneration of matrix. So the expression of these enzymes, and their relation to RARβmay provide a clue to understand the relation of change in visualenvironment and sclera remodeling.MethodsExperiment 1:To observe the relation of change in visual environment to therefraction, axis length, sclera morphology.Newborn children 25, weight 30～40g. Left eye covered by semi-trans-parent eye pack at 2 days. FDM group: covered 14days. FDM recovery group:covered days, 3 day in normal vision condition. Control group: The other eye.The refraction and axis length were examined. Then the eyeballs were recov-ered. Prepared for pathology exam. The morphological change was observed byHE staining. The thickness of sclera cartilage and fiber were measured by meta-morph software.Experiment 2: Detection of RA in retain. Choroid and sclera. Specimenpreparation a tissue block in diameter 8 mm was removed from the posteriorpole, retina, choroid, and sclera were separated. The fiber and cartilage layerof sclera were separated under microscope. Then the tissue were put into Eppen-dorf tube, put in fluid nitrogen. Detection Method: The RA level in retina, cho-roids and sclera was detected by HPLC. Experiment 3: Detection of RALDH2 in Retina, Choroid, and Sclera.RT-PCR technique was used to detect the expression of RALDH2, mRNAlevel was calculated for statistical analysis.Experiment 4: Detection of RARβ, MMP-2 and TIMP-2 in posteriorpole sclera. The specimen were same as that in ExperimentⅡ. (1)RT-PCRwas used to detect the level of RARβin sclera tissue, calculate mRNA. (2)RT-PCR was used to detect the level of MMP-2. TIMP-2 in fiber and cartilagelayer of sclera. Calculate the mRNA. (3) Analysis the relatively between mRANof MMP-2 and mRNA of RARβ. MRNA of RARβin posterior pole sclera andRA in retina and choroids respectively.Statistical Analysis: t Test was used to determine the difference betweengroups, linear relativity was analyzed. Statistical soft are: SPSS 10.0.ResultsRefraction, axis length and morphorlogical change in sclera. (1)Ai 14days of form deprivation, the refraction was 12.1+-4.3D,axis length was 9.86+-0.38 mm, compared with the control group, which was +2.7+-0.5Dand 8.71+-0.28 mm, the difference was significant (p＜0.01);days afterrecovery from FDM, the refraction was less (-5.5+- 1.2D, p＜0.05), whilethe axis length no difference (p＜0.05), but the increase rate of axis lengthwas slower (0.003 mm/d Vs 0.196 mm/d), even slower than the posterior partof eye. (2) By HE staining, the cartilage sclera of FDM was thicker (p＜0.05), fibrous sclera thinner (p＜0.05); Compared with FDM eyes, the recovery group has thinner cartilage sclera (p＜0.05), fibrous sclera thicker (p＜0.05), which was close to that of the control group.Chang of RA in retina choroids and sclera:In retina: Ai 14 days, RA of FDM eyes increased markedly (0.769+-0.051 Vs 0.249+-0.031 ng/mg ww) (p＜0.01), Ai 3 days afar recovery fromFDM, RA decreased (p＜0.01,0.125+-0.038 Vs 0.769+-0.051 ng/mgww), even lower than that of control group (0.244+- 0.030 ng/mg ww). Nosignificant change between control groups. In choroids: Ai 14days, RA of FDM eyes decreased markedly (0.385+-0.01 Vs 1.306+-0.273, p＜0.01), at 3 days after recovery from FDM, RAincreased (2.725+-0.108), which was t times of that of FDM eyes and 2times of the control group. No significant change between control groups.In sclera: Ai 14 days, RA in cartilaginous sclera and fibrous sclera werebeth detected (p＜0.01). In which, RA in fibrous sclera were both increasedagain (p＜0.01), and higher than the control eyes. So the change in visual en-vironment can lead to the change of RA in sclera, in which, the change of RAin fibrous sclera was more obvious.Expression of RALDH2 in retina, choroids and sclera.There were expression of RACDH2 in retina and choroids, none in sclera.Ai 14 days of FDM, RALDH2 in retina increased (mRNA: 64.52+-0.27 Vs38.71+-0.19. p＜0.01) while RALDH2 in choroids (p＜0.01). No signif-icant difference between the control groups.RALDH2 in FDM retina was increased (p＜0.01), at 3 days after recov-ery from FDM, it decreased (p＜0.01), even lower than the control eyes. Thisindicated that the change in visual environment lead to the change in RALDH2in retina.RALDH2 in FDM choroids was decreased (p＜0.01), at 3 days after re-covery from FDM, it increased (p＜0.01), even higher than control eyes, thisindicated that the change in visual environment lead to the change in RALDH2in choroids.Expression of RARβ, MMP-2 and TIMP-2 in the posterior sclera.There was RARβin normal posterior sclera, the content was same in fibrous and cartilage sclera. At 14 days of FDM, RARβincreased (p＜0.01),more obvious in the fibrous sclera (mRNA: 81.08+-0.42 Vs 60.21+-0.43, p＜0.01). In cartilage sclera, [RARβincrease markedly ay 14 days, (p＜0.01), decreased at 3 days after recovery from FDM, (p＜0.01), no signif-icant difference compared with control group.] In fibrous sclera, the same as a-bove.There was strong expression of MMP-2 to TIMP-2 in fibrous selera,while none in cartilaginous sclera. While in fibrous sclera, MMP-2 was in- creased, TIMP-2 was decreased at 14 days of FDM; Ai 3 days after recovery from FDM, MMP-2 was decreased (p＜0.01), even lower than that of control group (p＜0.01) and TIMP - 2 was increased ( p＜0.05), but no difference with the control group.There was positive relatively between mRNA of RARβand MMP-2 in posterior fibrous sclera(r=0.944). There was negative relatively between mRNA of RARβand TIMP-2 (r=-0.863 ).Conclusions1. After from deprivation, the RA level in retina, choroids and sclera were changed markedly. So RA is related to the formation of FDM.2. There was the expression of RA receptor-RARβin posterior sclera. Its expression had close relation to the change of RA level in retina and choroids. So the effect of RA on FDM manifested as the function on sclera.3. In FDM, the expression of MMP-2 in posterior fibrous sclera increase, TIMP-2 decreased, which indicated the extracellular matrix degeneration increased in fibrous sclera. So the matrix degeneration was the cause of fibrous sclera thinning in FDH.4. There was positive relativity between RARβand MMP-2, and negative relativity between RARβand IMMP-2 in posterior fibrous sclera, which indicated that RA had regulation affect on extra cellular matrix of fibrous sclera.