The Study Of Effect Of VEGF-A On The Expression Of TGF-β1 And CTGF In The Rat Retina

ObjectiveTo observe the effect of vascular endothelial growth factor-A(VEGF-A) on the expression of transforming growth factor-β1(TGF-β1) and connective tissue growth factor(CTGF) in the rat retina.After the injection,the expressions of TGF-β1 and CTGF in the rat retina of different time are observed.To discuss the effect of TGF-β1 and CTGF on the mechanism of diseases of retinal neovascularization,and provide the basic rationale of treatment of diseases of retinal neovascularization.Method1.Thirty three wistar rats aged 12-14 weeks,male and female unlimited,weighed approximately 250g,which were all healthy and no eye diseases conformed by slit lamp and ophthalmoscope.24 rats were selected as treatment groups and the left 9 rats as blank group by random.Three microliters of a solution of 0.1%bovine serum albumin(BSA) in phosphate buffered solution(PBS),containing 100ng recombinant rat VEGF-A was injected into the vitreous in the right eye of each rat of the 24 (GroupA),whereas the veichle alone was injected into the vitreous in the left eye of the 24 rats(GroupB) using a 10μl syringe.Blank group(GroupC) was given no treatment and divided into 3 subgroups by random.The eyes which vitreous haemorrhage occurred when injecting were excluded.Every treatment group was divided into three subsets seperately:group A1-C1(24 hour),groupA2-C2(48 hour), groupA3-C3(72 hour).2.At the 24,48,72 hour,the rats were anesthetized by the 10%chloral hydrate,and then the eyes were enucleated.When making the frozen sections,sections were obtained parallel optic nerve with a thickness of 10μm.Hematoxylin-eosin(HE) staining was used to observed the morphological changes of the retina and immunohistochemistry staining was used to observe the expression of TGF-β1 and CTGF in the rat retina.Two sections with integrate retina structure were selected from each eye and 5 fields from each section were selected and photographed by random(×200 times).Then we analyzed the pictures using Image-Pro Plus 6.0.Mean integrated optic density(IOD) was used as the main statistic index. 3.The results were input the spss11.5 statistic software.Different treatment groups and subsets of different time between identical group were analyzed by one-way ANOVA.Then using SNK test and Tamhane's T2 test in Post Hoc to compare each group,P<0.05 was regarded as the standard of statistical significance.Result1.HE staining displayed that the layers of the rat retina of GroupAl,B and C were regular,the photoreceptor cells were regular,the nuclears stained deep violet,the boundary was sharp.While in GroupA2,the retina was all thickening and GCs swelling,the photoreceptor cells were irregular and displayed a ambiguous structure.RPE cells swelled and turned to be round,but number not changed.There were no obvious morphological difference between GroupA3 and A2.2.In the eyes of GroupC,CTGF mainly located in the ganglion cell layer(GCL) and a weak postivity in the inner plexiform layer(IPL).A slight increase in labeling intensity was observed for CTGF throughout all layers of the retina in eyes of GroupB.In addition to this slight panretinal increase in staining,eyes of Group A showed a stronger staining intensity for CTGF in the GCL.A staining of small vessels of inner nuclear layer(INL) was visible in the eyes of Group A.TGF-β1 staining was observed only in the retinal vascularture.TGF-β1 staining was somewhat enhanced in the eyes of GroupB and significantly increase in the eyes of GroupA when compared to that in the untreated eyes.Immuno-histochemistry staining of TGF-β1 and CTGF showed that under different time after injection,mean IOD as main statistic index.The difference between different subgroups of group A was significant at the 0.05 level analyzed by statistics software spssll.5(F=41.361,P=0.000).Futher more,we compared each subgroup with SNK test,the difference of mean IOD of CTGF among every subgroup were all significant(P<0.05).So did the different time of group B(F=25.284,P=0.000),the difference between Bland B2,B2 and B3 were significant (P<0.05).The difference of mean IOD of CTGF among every subgroup of Group C had no statistical significance(F=0.002,P=0.998).The difference of mean IOD of TGF-β1 between different subgroups of group A were significant at the 0.05 level(F=26.783,P=0.000).Then,we compared each subgroup with SNK test,the difference among every subgroup were all significant(P<0.05).The difference of the mean IOD between different subgroups of group B were significant at the 0.05 level (F=27.644,P=0.000).We compared each subgroup onwards with Tamhane's T2 test of Post Hoc,the difference between Bland B2,B2 and B3 were significant(P< 0.05).The difference of mean IOD of TGF-β1 among every subgroup of Group C had no statistical significance(F=1.308,P=0.300).There were no statistic significance of the difference of mean IOD of CTGF and TGF-β1 between the different treatment group at 24 hours after treatment(F1=3.482,P1=0.053;F2=0.604,P2=0.558).So we think that the expression of CTGF and TGF-β1 had not been induced to increase 24 hours after the injection of VEGF-A..Whereas after 48 hours of injection,the expression of the two factors arrived the peak and there was a decrease of expression after 72 hours.The mean IOD of positive area of CTGF and TGF-β1 among subgroupA2,B2,C2 and A3,B3,C3 were all significantly different(P<0.05).Then we compared the mean IOD of positive area of CTGF and TGF-β1 every subgroup horizontal and found that there were statistic significance among every group(P< 0.05).ConclusionIn our reseach,we used a method of intravitreal injection to study the effect of exogenous VEGF-A on the expression of CTGF and TGF-β1 in the rat retina and observed the expression of CTGF and TGF-β1 by the way of immunohistochemistry staining.We compared the difference of different treatment and different time after treatment through the main index of mean IOD of the photographs and we found that exogenous VEGF-A could induce the expression of CTGF and TGF-β1.The expression arrived the peak at 48 hours after injection of VEGF-A.We concluded that VEGF might participate the pathogenesis of thickening of the basement membrane of diseases of retinal neovascularization by inducing the increase of expression of profrotic factor such as CTGF,and further more provided a foundation for retinal neovascularization.