A Study Of The Effect Of Exogenous Application Of All Trans-retinoic Acid On The Refractive Status And Posterior Eye Tissue Of Guinea Pig

Part IA study of the effect of exogenous application of all trans-retinoic acid on the refractive status and posterior eye tissue of guinea pigObjective To investigate the role of all-trans retinoic acid (ATRA) on the development of refractive status in guinea pig. Methods 48 pigmented guinea pig aged 4weeks participated in the experiment. Measurement of the refractive status after cycloplegia with streak retinoscopy, ocular length and vitreous depth detected with A-scan ultrasonography were calculated before experiment and 4 weeks after the treatment of ATRA to 24 guinea pigs. 0.1 mL of ATRA(0.4mg/mL) was injected posterior subtenon on one randomly selected eye, and the eye which taken as control was posterior subtenon injected with the same volume of saline and DMSO(0.001ml/L). The eyes were conducted light microscopy examination to evaluate the pathological changes of the ATRA treated group and control group. Data was presented as the mean ±SEM, differences between the ATRA treated and control group were analyzed by t -test. P<0.05 indicate that the variance has statistical significance. Results Eye elongation and vitreous depth gain could be observed after four weeks of ATRA treatment. There were statistically significantly variance of the refractive error, ocular length and vitreous depth between control group and ATRA treated group 4 weeks after ATRA treatment. Before experiment, the refractive status was 3.73D±0.75D in ATRA treated group and 3.70D±0.83D in control group; the vitreous depth was 3.09mm±0.67mm and 3.19mm±0.74mm respectively, and ocular length was 6.44mm±0.27mm and 6.50mm±0.38mm respectively. statistical analysis of the three parameter mentioned above indicate no statistically significance between them( P=0.96; P=0.68 and P =0.26) Four weeks later, the refractive status was 3.56D±0.80D in control group and 0.9D±1.25D in ATRA treated group; the vitreous depth was 3.57mm±0.54m and 3.98mm±0.68mm respectively, and ocular length was 7.8mm±0.26mm mm and 8.5mm±0.39mm respectively. Analysis of variance of the three parameters mentioned above indicates there is statistically significance between ATRA treated eye and control eye. (P<0.001) The ocular elongation of ATRA-treated eye is more rapid than control eye(2.04±0.06 mm and 1.24±0.04 mm, t-test: P<0.001) ; The gain of vitreous depth of ATRA-treated eye is larger than control(0.89±0.05 mm and 0.36±0.04mm ,t-test: P<0.001) Masked comparison of the light microscopy examination results revealed that choroids thickness decreased(P=8.34×10^-16,P<0.001) and sclera thickened(P=4.38×10^-14, P <0.001) in the ATRA treated eye compared with the similar position of controlled eye 4 weeks later, choroids thickness remained thinner than control eye(P=7.32×10^-15, P <0.001) but the sclera became thinner than control eye when the sclera of control eye developed with aging(P=2.77×10^-8, P<0.001). The data 4 week later shows that sclera thickness is 124.39±6.48μm, choroids thickness is 42.21 ±2.32μm in ATRA treated eyes and 83.59±5.28μm and 92.83±8.26μm in normal control eyes. The data 8 weeks later were 81.62±12.96μm and 44.53±4.16μm in ATRA treated eyes and 118.53±6.30μm and 162.98±21.87μm in normal eyes respectively. Conclusions Exogenous ATRA can accelerate the eye elongation of guinea pig and induce myopic shift. Certain concentration of ATRA might be a mediator of underlying eye growth. Exogenous ATRA might result in the thinner choroids which may induce the gain of vitreous depth and then lead to the pre-location of focus of subject on retina compared to control eye.Part IIA compare study on the expression of cone opsins in exogenous ATRA-treated and FDM retina of guinea pigObjective To investigate the effect of exogenous ATRA on the expression of co ne opsins in guinea pig, and compare to that of form-deprived myopia (FDM). Met hods 42 pigmented guinea pig aged 1 week participated in the experiment and divid ed into three group: ATRA treated group (n=14), FDM group (n=14) and control gr oup (n=14). 0.1mL of ATRA (0.4mg/mL) was posterior subtenon injected on one ran domly selected eye of ATRA group, white translucent vinyl diffuser attached to one randomly selected eye was used to deprive eyes of form-vision in FDM group, and the eye which taken as control was posterior subtenon injected with 0.1mL of DMS O(0.001 ml/L).opsin immunohistochemistry and RT-PCR assays were performed to co mpare the mRNA expression level in total and spatial density of photoreceptor opsinexpression in the ventral ,central and dorsal retinal areas of three groups using anti body against short and medium-wave-length-sensitive cone opsin.(CHEMICON interna tional,Inc). Slides were viewed using Leica confocal microscope and Leica light mic roscope. Results The distribution of opsin across the guinea pig retinae as determine d from antibody labeling of cone opsin is asymmetric, with the dorsal retina domina ted by M-opsin, and the ventral retinae by S-opsin. But the ventral area also showspositive for the M-opsin. Opsin spatial density decreased with increasing retinal ecc entricity for both of the two opsins. The spatial density of S-opsin decreased most s teeply for eccentricity up to one-third distance from optic disc to retinal edge. Thereis no sharp decrease in spatial density of M-opsin but in a gradually decreasing wa y. The spatial density of M-opsin expression is higher than that of S-opsin's in all t hree regions. The spatial density of S-opsin expression in normal control eye:805.0± 203.3mm^-2 (ventral);100.0±57.7mm^-2 (dorsal);1637.2±314.1mm^-2 (Central).ATRA eye: 499.4±147mm^-2 (ventral); 87.8±44.9 mm^-2 (dorsal); 968.4±210.2 mm^-2 (central). FDMeye: 640.9 ±196.8 mm^-2 (ventral); 1016.7 ±144.6 mm^-2 (central); 70.9 ±30.8 mm^-2 . The spatial density of M-opsin expression in normal control eye: 946.2±388.5mm^-2(dorsal); 1666.7 ±137.8 mm^-2 (central); 175.0±100.9 mm^-2 (ventral). ATRA eye: 1326. 1±267.0mm^-2 (dorsal); 2984.0 ±613.4 mm^-2 (central); 232.9±173.6 mm^-2 (ventral). FD M eye: 1436.7±366.0 mm^-2 (dorsal); 2780.0±180.5mm^-2 (central); 318.2 ±172.7 mm^-2(ventral). RT-PCR results: the relative optical density(OD) value of M-opsin: 1.25± 0.11(ATRA-treated eye), 1.06±0.07(FDM eye), 0.51±0.10(control eye), The relative OD value of S-opsin: 0.61±0.09(ATRA-treated eye), 0.70±0.07(FDM eye), 1.25±0.06(c ontrol eye). The analysis of the varience of the parameters mentioned above betweenthe ATRA -treated eye and control eye, FDM eye and control eye indicate satatical ly significance. (P<0.05) Conclusion Local exogenous ATRA posterior subtenon injec tion can increase the expression of M-opsin and decrease the S-opsin expression in guinea pig, which is similar to the change in the FDM eye. The findings indicate th e target effect of ATRA on the development of cones and the expression of cone o psin. The similarity of the mRNA expression and the distribution and density of thetwo opsins in ATRA treated group and FDM group provides evidence of RA beinga possible signal in eye development of guinea pig.Part IIIThe role of all-trans retinoic acid on the human RPE in vitroObjective to evaluate the role of ATRA on the morphology, proliferation and apoptosis and the function of secreting TGF-β₂ of cultured human retinal pigment epithelial cell (RPE). Methods RPE were plated into 96-well dishes at a density of 1*10⁵ well^-1 or plated into 250ml bottles at a density of 1*10⁶ well^-1. Forty-eight hours after seeding, the medium was changed to F-12 medium contained 0.5%FBS , twelve hours after the first medium change , the medium was changed to F-12 medium containing the above concentration of FBS plus 1 nM/mL,5 nM/mL ,10 nM/mL, 20 nM/mL , 30nM/mL of ATRA (Sigma) . The cell cultured in corresponding concentration of DMSO without ATRA was taken as controlled. 48 hours later, the morphology of RPE was investigated, cell proliferation and apoptosis were analyzed using the MTT assay and flow cytometer respectively. The study was repeated three times. The TGF-β₂ expressed by RPE was tested with immunohistochemistry method and ELISA. Results Different concentration of ATRA has different influence on the RPE cell morphology, proliferation and apoptosis. 1 nM/mL ,5 nM/mL of ATRA did not affect the proliferation of RPE and its morphology remained normal; but 10nM/mL, 20nM/mL, 30nM/mL of ATRA all inhibited the proliferation of RPE and the cell increased in size and become flattened, the process decreased in number and some cell split, the effect show concentration dependant. Flow cytometer analysis show apoptosis rate were 14.58%, 19.95.5% and 21.69% respectively, immunohistochemistry method and ELISA showed that the expression and secretion of TGF-β₂ increased with the ATRA concentration rising. Conclusion 1 nM/mL, 5 nM/mL of ATRA do not affect the proliferation of RPE and the cell morphology remained normal, but concentration more than 10 nM/mLof ATRA can induce pathologic change in RPE cell and increase its secretion of TGF-β₂...