Studies Of Molecular Mechanisms Underlying The Enhancement Of Cochlear Amplification Induced By Long-term Administration Of Sodium Salicylate

Large dose of salicylate can cause reversible, moderate hearing loss and tinnitus. Some clinic study reported that reversible decrease of otoacoustic emissions (OAEs) were found in those who received acute administration of salicylate. Acute administration of salicylate can reversibly eliminated spontaneous otoacoustic emissions (SOAEs) and reduced the amplitude of distortion product otoacoustic emissions (DPOAEs), which reflect the outer hair cells (OHCs) electromotility. Several neurophysiological and histological studies suggested that salicylate mainly acts on cochlear especially the vascular supply and the OHCs which may lead to hearing loss.However, our previously study showed that sodium salicylate paradoxically raised the amplitude of DPOAEs in guinea pigs under chronic administration condition. The mechanisms contributing to this paradoxical enhancement are unknown. OAEs are generated by the active cochlear. OHC somatic electromotility are known as the most important contributors to the cochlear amplifier. Prestin is a key novel membrane motor protein which is considered to be the essential for outer hair cell electromotility. The protein is located in the OHCs'basolateral membrane and is not expressed in the inner hair cells. It has also been found that Cl" works as an external voltage sensor of prestin to trigger OHC electromotility; salicylate can competitively bind prestin with Cl- to eliminate OHC electromotility. However, long-term administration of salicylate increased the DPOAEs. Several mechanisms could be underlying this enhancement: long-term use of salicylate may induce prestin up-regulation and/or relative increase in affinity of prestin with Cl-. Long-term use of salicylate may also result in increase the intracellular Cl-. When more Cl" combined to the binding sites, conformational changes in the molecule may be triggered causing the enhancement of OHC electromotility.Na-K-2Cl co-transporter family (NKCC) and K-Cl co-transporter family (KCC) are two important cation-coupled chloride cotransporters in the regulation of cell volume and intracellular chloride concentration during osmotic disturbances.In this study, the same animal mode was used which was proved to be an effective mode before. The prestin mRNA and protein expression and also the cation-coupled chloride cotransporters NKCCi and KCC1-4 mRNA were detected using the real-time PCR.The present study is composed of two parts.Part1 Different expression of prestin in rat cochlear outer hair cells induced by acute and chronic sodium salicylate injectionTo investigate the correlation between the different expression of prestin and the outer hair cell motility alteration in rat cochlear induced by acute and chronic sodium salicylate injection, forty-eight adult Wistar rats were divided into four groups (A: chronic sodium salicylate injected, B:normal rats, C:acute sodium salicylate injected and D:chronic anastatic group). The mRNA levels of prestin in each group were detected by Taqman real-time RT-PCR. Western-Blot was used to study the different expression of prestin protein in the fours groups. Results showed that the expression of prestin mRNA and protein level in chronic sodium salicylate injected group was significantly increased (p<0.05) as compared with that of normal control group), of which the mRNA level is 2.25 fold than normal group; There were no significant changes among the chronic anastatic group, normal control group and acute sodium salicylate injected group (p>0.05). It suggested that the elevation of the prestin expression in rat cochlear outer hair cells might be one of the causes to the enhance of the OHC motility induced by chronic sodium salicylate injection. It also indicated that the up-regulating of prestin mRNA and protein might be one of the mechanisms for the tinnitus induced by chronic sodium salicylate injection.Part 2 Different expression of NKCC and KCC family in rat cochlear induced by acute and chronic sodium salicylate injection 1. The expression of NKCC and KCC family members in normal adult rat cochlearFourteen adult rats of either sex with normal pinna reflex and were free from middle ear infections were used in the experiments. RT-PCR was use to detect the NKCC and KCC mRNA expressions in the cochlea. The sense and antisense primers specific for rat NKCC 1-2 and KCC1-4 were designed and used. The brains were extracted as positive control for KCC2 and the kidneys were used for other members positive control. Furthermore, immunohistochemical staining methods were explored to detect the distribution of the neuron-specific KCC2 protein in the rat cochlea. It was showed that PCR products with lengths corresponding to the KCC2 cDNA fragment (268bp) were obtained from the rat cochlea and brains. Products with lengths corresponding to the NKCC1?KCC1?KCC3 and KCC4cDNA fragment (401 bp?314 bp?128 bp and 246bp) were obtained from the rat cochlea and kidney. Products corresponding to the NKCC2 (408bp) were only obtained from rat kidney but not from rat cochlea. With immunohistochemical staining, anti-KCC2 primary antibody produced and intense immunostaining reaction in the spiral ganglion neurons and tectorial membrane. Less intense reactions were observed on the outer hair cells, inner hair cells, Deiters's cells, stria vascularis, and fibrocytes of spiral ligament. This study suggested that KCC1 and KCC1-4 might be important crediates to maintain the ionic homeostasis, especially the K+ and Cl-, in cochlear.2. Different expression of NKCC1 and KCC1-4 in rat cochlear induced by acute and chronic sodium salicylate injectionTwenty-four normal Wistar rats were selected, and then divided into four groups (Chronic sodium salicylate injected, normal rats, acute sodium salicylate injected and chronic anastatic group). Each has 6 rats. Animals were deeply anesthetized and sacrificed immediately. The mRNA levels of NKCC1 and KCC1-4 in the four groups of rats were detected by Taqman real-time RT-PCR.28s rRNA, a house-keeping gene, was chosen as a referred gene. Results showed that the expression of NKCC1 and KCC2 mRNA level in chronic sodium salicylate injected group was significantly increased (p<0.05, as compared with that of normal control group); The expression of KCC3 and KCC4 mRNA level in chronic sodium salicylate injected group was significantly decreased (p<0.05, as compared with that of normal control group); The expression of KCC1 mRNA level was significantly increased (p<0.05), but the KCC1-4 and NKCC1 m RNA levels were significantly decreased (p<0.05) in acute sodium salicylate injected group. It suggested that the ionic homeostasis in the cochlea might be destroyed after the different administration of salicylate through changing the expression of NKCC1 and KCC1-4 mRNA expression way. Intracellular Cl- levels of OHC might be changed which would lead to the abnormal motility of OHCs.In conclusion, this study shows that long-term administration of salicylate can up-regulate the prestin expression and simultaneously induce the changes of the cation-coupled chloride cotransporters (NKCC1, KCC1-4) mRNA expression. That may be the underlying mechanisms by which long-term administration of salicylate enhances active cochlear mechanics.