The Expression Of Neuritin MRNA And Protein In The Visual Cortex Of Normal And Monocular Form Deprived Rats

ObjectiveTo detect the expression of Neuritin mRNA and protein in the visual cortex of normal and monocular form deprived rats by SYBRgreenâ… real-time fluorescent polymerase chain reaction and immunohistochemistry stain methods.To explore the associatiion Neuritin with visual experience and ages,and influence by Abnormal vision.To provide the molecular mechanism of the visual plasticity.Methods1.Fifty healthy Wistar rats were divided into 10 groups:5 in every group,in which 1-6 groups for normal group(N),7-10 groups for monocular deprivation group (MD).The eyelids of MD groups were sutured right eyelid at 14 days.Rats were housed in alike environment,the time-point for normal group is postnatal 7,14,21,28,45,90 days.The time-point for MD group is postnatal 21,28,45,90 days.The rats were killed at the design time-point,the procedur is anesthesia,decapitation,get the brain,extract total RNA,detect the quality and density of total RNA,reverse transcription reaction,common PCR reaction,SYBRgreenâ… real-time fluorescent polymerase chain reaction,then apply computer self-software compared the sample with standard preparation,and combine with references to obtain the Neuritin mRNA quantity in rats' visual cortex.Analyzed and compared the Neuritin mRNA quantity,experiment data was indicated with mean±standard deviation((?)±s). Analyzed data with SPSS16.0,adopted t-test in normal and MD groups,ANOVA in the different stages of same group.2.the rats' groups and animal model the same to the part one.The procedur is anesthesia,perfusion,fixation and opening cranium,cut the posterior brain coronally, putted it in fixative solution for 24-48h,then imbedded with paraffin routinely.Sliced the prepared tissue in 4μm,carried out routine HE stain and immunohistochemistry stain.Observed the expression of Neuritin in visual cortex under the light microscope.Pictures were Collectted with Olympus micro-image system,integrated optical density(IOD) were determined with image analysis system.Analyzed and compared the IOD,experiment data was in dicated with(?)±s.Analyzed data with SPSS16.0,adopted t-test in normal and MD groups,ANOVA in the different stage of same group.Results1.(1)To analysis the standard curve:from the solubility curve we know that Neuritin and GAPDH amplification single PCR production in any density ranges, without primer dimer and hairpin;From the amplification curve and standard curve we know that there is a well linear correlation,there could obtain exact quantitation.(2) The expression of Neuritin mRNA in normal rat visual cortex: before open the eyes there is a small quantity expression,P7 was 0.152±0.011,then increased gradually,P14 was 0.234±0.052,P21 was 0.527±0.047,then reached its climax value in P28,it was 0.846±0.067,then decreased slightly and maintained stabiy in adults,P45 was 0.445±0.020,P90 was 0.410±0.017.Significant deviation existed among different ages with one-factor analysis of variance(F=177.52, P＜0.01).3.The MD groups:the expression is obviously less than normal groups,at MD21 the quantitation was 0.412±0.015(t₂₁=5.262,P＜0.01),MD28 was 0.501±0.019(t₂₈=11.223,P＜0.00),MD45 was 0.381±0.030(t₄₅=3.970,P＜0.01) and MD90 was 0.384±0.015(t₉₀=2.492,P＜0.05)。2.(1).HE stain results:The neurons of visual cortex in rats distributed in layers,from superficial to the deep layer was molecular layer,external granular layer,external pyramidal layer,internal granular layer,internal pyramidal layer and polymorphous cell layer,the neurons in every layer lined in order.There was not obviously abnormality in the visual cortex of monocular deprivation group.(2).The expression of Neuritin in rat visual cortex in different stages:In light microscope, Neuritin positive reaction product was yellow granulous,main located between neurons.Neuron cell body,blood vessel and callosum were not labeled.The Neuritin positive granula were seen in every lays of visual cortex.The expression of Neuritin increased developmentally in the normal groups.At P7 the IOD was 6.48±0.62,P14 the IOD was14.56±2.37,at P21 it increased to23.36±4.28,at p28 reach to the peaking the IOD was 67.94±5.98,then decreased P45 was 26.85±2.33, maintained at higher level.The expression of Neuritin in visual cortex of monocular deprivation rats decreased,the IOD at MD21 was18.17±1.42(t=5.571,P＜0.05), 30.92±2.17 at MD28(t=6.802,P＜0.01),19.33±2.08at P45(t=5.379,P＜0.01).Conclusion1.The expression of Neuritin mRNA and protein in any ages of rats visual cortex,as well the both to show the synchronism.To suppose may that the Neuritin gene to participate in developing visual system,and play an important roles in the growth and maturation of visual cortical neuron.2.The age factor effect the expression of Neuritin mRNA and protein,to accord with the wave change of the visual development critical.3.Visual experience regulats the expression of Neuritin mRNA and protein in development stage of visual cortex,to suppose may that affect Synapse development and maturation in the visual cortical neuron.In order to provide theory for the pathogenetic of amblyopia.