To Establish Auditory Deprivation Modal And Explore The Relationship Between BDNF/TrkB Signaling And The Plasticity Of Auditory Cortex

Objective:To reveal the molecular mechanism participating in the plasticity of central auditory system by the aid of auditory deprivation (AD) modal. With the variation of neuronal numbers in auditory cortex (AC), the expression change of BDNF and TrkB in AC were studied. It indicated the important role of BDNF/TrkB signaling in the regulation of central auditory plasticity which provided reference for cochlear implant or even brainstem implant.Methods:Male Sprague-Dawley (SD) rats of two weeks old were used in this study. They were divided into two groups including experimental group and control group. After anaesthetized with intraperitoneal injection of5%chloral hydrate (8ml/kg body weight), the experimental rats were received bilateral cochlear ablation through retroauricular incision. Then in control group, the skin was incised and the otic vesicle was opened, but the cochlea was not ablated. The hearing threshold of each rat was determined by auditory brainstem response (ABR) before and after operation. At2W,4W,6W and8W, animals were perfused intracardially. Their brain tissues were removed and fixed in4%paraformaldehyde, immersed in gradient alcohol, dimethylbenzene, and paraffin to make serial sections. At the same time point, they were decapitated, and bilateral auditory cortex was extracted to put into Trizol solution and stored at-80℃. HemateinEosin (HE) staining and Nissl’s staining were used to detect morphology and number changes of neurons. Immunohistochemistry method and Real-Time polymerase chain reaction (Real-Time PCR) technology were used to detect the expressions of BDNF and TrkB in AC. Results:1. Establishment of animal modal:The modal of auditory deprivation was established by bilateral cochlear ablation through retroauricular incision. The average threshold of ABR in control group after operation was20.21±4.29dB SPL, while in experimental group, it was91.33±5.32dB SPL. There was significantly statistical difference between the two group (P<0.05)2. HE staining:The morphology of normal cortical neuron was irregular with large volume, abundant cytoplasm which showed laky, large nucleus which was dark blue. While in AD group, the volume of cortical neuron was shrunken, cytoplasm was cavity sample variable, and nucleus was pyknosis, fragmentation or even disappeared.3. Nissl’s staining and the count of neurons in AC:The morphology of normal cortical neurons was irregular with large volume and obvious Nissl body near the membrane. After cochlear ablation, it became variably-sized with disorded distribution. The numbers of neuron in AD group were less than those in control. The rate of reduction kept slow before4W postablation, while at the later period, it got fast to reduce, which lead to significant difference between each group after4W (P<0.05). Using T-test analysis, it was significantly statistical different between each experimental group and age-matched control group.4. The expression change of BDNF and TrkB protein in AC:The normal auditory cortex of rats contained6leyers which showed:molecular layer, outer granulose cells layer, outer vertebral somatic cells layer, inner granulose cells layer, inner vertebral somatic cells layer and polymorphic cells layer from exterior to interior of the auditory cortex. Except of the molecular layer, the expression of BDNF and TrkB was located in cell membrane and axon. At the early period postablation, the protein expression of BDNF and TrkB were significantly reduced compared to the normal control group (P<0.05). Auditory deprivation induced parallel regulation of BDNF and TrkB in the auditory cortex, which peaked at4W postablation, and then decreased gradually after4W. While in age-matched control groups, it reached maximum at2W, and decreased afterward. It was significantly different between each experimental group (P<0.05). Except for6W group, there was significantly difference between each experimental group and age-matched control group (P<0.05).5. The gene expression change of BDNF and TrkB:The gene expression tendency of both BDNF and TrkB from AD groups was similar, which was consistent with the result of immunohistochemistry. A positive corretation was observed between the gene expression of BDNF and TrkB. For BDNF, comparing with age-matched control group, it showed0.444-fold decrease in expression at2W postablation,0.869-fold increase at4W. For TrkB, it was0.367-fold decrease at2W,0.541-fold increase at4W. Except for6W group, it was significantly different between each group (P<0.05).Conclusion:In the early postnatal stage, it was found high expression level of BDNF along with the development of AC. As the functional receptor of BDNF, TrkB showed a similar expression change with BDNF. The number of cortical neurons was decreased after bilateral cochlear ablation. The rate of reduction was slow at early stage but increased afterward. The expression of BDNF and TrkB was positive correlation. They decreased at initial stage postablation, increased afterward, and decreased again later. The expression of both BDNF and TrkB peaked at4W postablation. BDNF participates in the plasticity change of auditory cortex through BDNF/TrkB signal passway which may play critical role in the impairment of neuron. BDNF and TrkB could be acted as significant markers for auditory plasticity.