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Experimental Study on the Expression of Three Kinds of Myelin-associated Proteins in Rat Auditory Cortex after Auditory Deprivation and Their Effects on Regulating the Cortex Plasticity

Author: HuZuoZuo
Tutor: LiuYanXing
School: Hebei Medical University
Course: Pharmacology
Keywords: Auditory Deprivation myelin-associated protein Nogo-66receptor Auditory Cortex Plasticity
CLC: R965
Type: Master's thesis
Year: 2014
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Abstract


Objective:Nogo-A, oligodendrocyte myelin glycoprotein (Omgp) andmyelin-associated glycoprotein (MAG) were known as the kind of proteinsthat inhibit axon growth by binding a common receptor, the Nogo-66receptor(NgR). Our study aimed at observing the expression of the three types ofprotein after auditory deprivation and exploring the relationship betweenauditory deprivation and auditory cortex plasticity. Our study also aimed toprovide theoretical basis for the further study of mechanism of the auditorycortex plasticity.Methods:A total of7414days old SD rats were selected whose auricle reflectionwere normal. The rats were randomly divided into2groups (36each group):experimental group and control group. The experimental group were givenbilateral cochlear damage after anesthetic by intraperitoneal injection, whilethe control groups just got a incision behind the ear. The experimental groupand control group were both divided into four groups (9each group) accordingto the survival time after cochlear ablation:2week group,4week group,6week group and8week group. The rats were fed in the same condition fordifferent time according to the need of experiment. Give the ABR tests to therats of experimental groups and control groups two weeks after cochlearablation. The judgment whether animal model was successfully establishedwas leading to the minimum stimulus intensity as the ABR threshold reaction,such as Ⅲ wave repetitive waveforms. We used western blot technique、real-time fluorescent quantitative PCR technology and frozen sectionimmunofluorescence technique to detect the expression level of Nogo-A, MAG, Omgp, NgR mRNA and proteins in the rat auditory cortex inexperimental group and control group.Result:1Results of cochlear ablation surgery:The results showed that after cochlear ablation, the rats ate and drunk asnormal. After two weeks no one had head and neck askew, or had performanceof vestibular function damage, such as couldn’t keep balance when carry thetail dangling, circleed repeatedly in situ, and so on. Compared to controlgroups, the rats of experimental group acted slower, and were not sensitive tonoise stimulation. The auricle reflection was also disappeared.2ABR test results:The results showed that in the control groups auditory threshold of40SPL dB could lead to repetitive waveforms, and in the experimental groupsauditory threshold of90SPL dB could not lead to repetitive waveforms.According to the classification standard of deafness set by WHO in1980,people who had the auditory threshold above90SPL dB could be regarded astotal deafness. It was proved that the stable and reliable model of auditorydeprivation in rat was successfully established.3Frozen section immunofluorescence histochemical detection results:The results showed that Nogo-A, MAG, Omgp expressed in the neuronand nerve fiber myelin of the auditory cortex. The positive cells were inmulticellular forms such as neuron forms, funicular nerve fiber myelin formsor both together. The positive signals of Nogo-A and Omgp distributed in thecell membrane and nerve fiber myelin and had neuron forms, funicular nervefiber myelin forms. MAG positive signals distributed in the nerve fiber andhad funicular nerve fiber myelin forms. NgR positive signals distributed in thecell membrane, cytoplasm and had only neurons forms. We use ImagePro Plus6.0to count the number of positive cells of experimental and control2,4,6,8week group. The AOD of Nogo-A protein in experimental groups was0.254,0.368,0.591,0.510and in control groups was0.233,0.313,0.427,0.368. TheAOD of NgR protein in experimental groups was0.390,0.445,0.602,0.507 and in control groups was0.346,0.416,0.529,0.452. The AOD of MAGprotein in experimental groups was0.366,0.471,0.642,0.513and in controlgroups was0.318,0.418,0.603,0.442. The AOD of Omgp protein inexperimental groups was0.304,0.481,0.644,0.520and in control groups was0.270,0.415,0.560,0.424. Laser confocal scanning imaging showed that bothin the experimental groups and control groups, the number of positive cells ofMAG, Omgp, NgR and Nogo-A were extended over time. MAG, OmgpNogo-A and NgR protein expression had similar increasing trends, and thenumber of positive cells reached the peak for about six weeks after surgery.The protein expression of the AOD value in the the experimental group weresignificantly higher than that in the control group.4Real-time fluorescent quantitative PCR detection results:From the melting curve of the Nogo-A, MAG, Omgp and its receptorNgR, we could see that there were two strong single unimodal of target genesand GAPDH in each figure, no impurity peak and other products ofnonspecific fluorescence. Based on this, we concluded that the quantificationwas accurate. In both experimental and control groups, the original Ct value ofNogo-A, MAG, Omgp and NgR had an obviously downward trend (The lowvalue of the Ct represent the high expression of protein). The expression ofNogo-A and Omgp mRNA in experimental6week group was the highest. Theexpression of MAG and NgR mRNA in experimental4weeks group was thehighest. The expression of Nogo-A mRNA in each experimental group issignificantly higher than the control group, The expression of NgR and MAGin experimental2,4week group is higher than that in the the control group.The expression of Omgp2,4,6week group was significantly higher than thatin the the control group. Set2w control group as calibration, the average of2-Ctof Nogo-A in experimental2,4,6,8week group were1.323,2.085,2.792,2.077. The average of2-Ctof NgR were2.028,2.276,1.833,1.688. Theaverage of2-Ctof MAG were1.351,2.809,1.906,1.331. The average of2-Ctof Omgp were1.298,1.616,2.389,1.878. We can saw that whethersurgery or not Nogo-A, MAG, Omgp and NgR mRNA were extended over time. The expression of MAG and NgR mRNA reached the peak for aboutfour weeks while Omgp and Nogo-A mRNA for about six weeks after surgeryand then cut. The mRNA expression of the experimental groups weresignificantly higher than control groups.5Western blot detection results:The specificity protein expression stripes of Nogo-A, MAG, Omgp, NgRwere respectively in200kD,100kD,25kD,66kD. Set GAPDH (37kD) asthe the internal calibration to keep the consistency of the sample amount, theaverage gray value of Nogo-A in experimental2,4,6,8week groups were0.275,0.389,0.460,0.343and were0.110,0.131,0.162,0.122in controlgroups. The average gray value of NgR in experimental groups were0.097,0.107,0.149,0.120, and were0.065,0.070,0.081,0.072in control groups.The average gray value of MAG in experimental groups were0.248,0.365,0.478,0.327and were0.195,0.257,0.309,0.276in control groups. Theaverage gray value of Omgp in experimental groups were0.173,0.320,0.405,0.3445and in control groups were0.092,0.257,0.309,0.276. Results showedthat the protein expression of Nogo-A, MAG, Omgp, NgR increased with agegrowing. They all have similar trends over the time and reached peak at6week and then the expression of both experimental groups and control groupsreduced. The protein expression of Nogo-A, Omgp, NgR in each experimentalgroup was significantly increased compared to control groups. The proteinexpression of MAG in the experimental4,6week group was higher comparedto the control groups.Conclusion:1Bilateral cochlear damage surgery was a stable and reliable method tobuild up the auditory deprivation model in rat.2The three kind of axon growth inhibition protein Nogo-A, MAG, Omgp,and its receptor NgR expressed in the auditory cortex.3In the critical period of auditory development, axon growth inhibitionprotein Nogo-A, MAG, Omgp, and its receptor NgR expressed in theauditory cortex of the normal development rats. The expression of axon growth inhibition protein increased over time and peaked in4~6weeksafter birth. This phenomenon might be because in the early postnatalperiod and the critical period of auditory development, the axon growthinhibition protein and its receptor mediated elongation of axon,impairment of nerve fibers and axon. The promoted function of was givenpriority. But along with the development to adulthood, the function ofaxon growth inhibition protein and its receptor gradually turned to inhibitthe excessive growth of axons.4After auditory deprivation of rats caused by cochlea damage and injury ofperipheral auditory organ,axon growth inhibition protein Nogo-A, MAG,Omgp, and its receptor NgR in auditory cortex had an significantlyincreased expression compared to the control rats in the same process ofdevelopment. The experimental group had a similar time to the peakcompared to the control group, but the expression was significantly higher.The reason may be that in the early time after injury of peripheral auditoryorgan, Nogo-A, MAG, Omgp, and its receptor NgR mainly participant inthe process of promote the impairment of nerves and the growth of neuron.When the new synapses formed to myelin, and the auditory cortexgradually establish a new neural network, the expression began to reduce.The function of inhibiting excessive growth of the axon and nerve fiberswas given priority. It suggested that peripheral auditory organ damage canlead to the change of senior auditory center plasticity. Themyelin-associated protein and its receptor had a double impact on axongrowth inhibition and promotion which showed that they participated inthe regulation of auditory cortex neuron plasticity. It also indirectly provedthat the rat auditory cortex was a specific functional area of brain cortexwith the high capacity of plasticity.

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