Permeability Of PPTA To Blood-labyrinth Barrier And Protective Mechanism Of PPTA Against Sudden Sensorineural Hearing Loss In Guinea Pig

The concept of sudden sensorineural hearing loss was fist proposed by De Kleyn in 1944.It is defined as a disease of over 30 dB hearing loss in three adjacent frequency and lasting more than three days. Sudden sensorineural hearing loss can be diagnosed by clinical manifestation and hearing test. Sudden sensorineural hearing loss is a common disease, the morbidity of which is various with all kinds of large-scale surveys. De Kleyn found the morbidity of sudden sensorineural hearing loss is about 0.05‰ to 0.2‰. The data from global survey is about 0.03% per year which is different from the 1.6‰ of the German research. Vascular factor plays an important role in the pathogenesis of sudden sensorineural hearing loss. The cochlear ischemia injury was thought to be a major pathogenesis in sudden sensorineural hearing loss, but the ischemia-reperfusion injury take the main position.The methods of making the ischemia-reperfusion models is various. There are two major way of making the ischemia reperfusion model. Kuaskari made the ischemia-reperfusion model through pressing the vertebral artery from Labyrinthine artery. Another mothed is pressing the vertebral artery through neck incision. There is not a perfect mothed up to present. We make the ischemia reperfusion model through freeing and bilateral vertebral artery and right carotid atery and pressing them for 1 hour and then release them. This method is is simple to achieve, easy to observe. What’s more,the survival rate after surgery is the highest among these method.The treatment of sudden sensorineural hearing loss is mainly systemic or local administration of glucocorticoids. Vasodilators, such as thrombolytic therapy, are also be used. These medications are not useful for ischemia-reperfusion injury in cochlear. Thus, how to stop ischemia-reperfusion injury has become a key issue in the treatment of sudden sensorineural hearing loss.Lidocaine piperphentonamine hydrochloride (PPTA) is independently invented by Guangzhou City Public Institute for Biotechnology Co., Ltd. and the Chinese Academy of Medical Sciences cooperation. It is a calcium sensitizer cardiac drugs and myocardial protective agent and synthesized for the first time. The pharmaceutical substances and synthesis process of the drug and other three related patents have been authorized. It is belongs to class 1.1 chemical drugs. Numerous studies show that Lidocaine piperphentonamine hydrochloride has a good protective effect on the brain, heart, cochlear ischemia-reperfusion injury. And 126 cases in Chinese healthy subjects I clinical trials has completed wiich showed good tolerance, It is now preparing to do the first phase Ⅱ clinical trials. This study is divided into two parts.Part Ⅰ Application of a UPLC-MS/MS method for the analysis of PPTA in the perilymphSudden sensorineural hearing loss is a disease which can affect the health and life quality of people a lot. Recent studies have shown that ischemia-reperfusion injury plays an important role in the oneset of sudden sensorineural hearing loss. What’s more, PPTA can release the injury caused by ischemia-reperfusion process. But we still don’t know whether it could pass the blood-labyrinth barrier. This is the first time using Ultra Performance Liquid Chromatography/Mass Spectrometry to analysis PPTA in perilymph that established within a fast, accurate, efficient and sensitive cochlea perilymph analysis. After extraction and enrichment by Oasis(?) MCX (30um) solid phase, the perilymph was send to Waters-ACQUITY UPLC BEH C18 (1.7 um,2.1mm id×50mm) separation column for further extraction. Acetonitrile-water were used for mobile phase. The process of analysis could be done in 4 minutes. The concentration of PPTA has a good linear correlation, r>0.99. The recoveries were 81.4%,85.3%,89.6% and the relative standard deviation was 7.3%,7.2%,4.1% respectively. (RSD) was<10%, the stability test RSD<10%. The method is rapid, accurate, sensitive and specific, suitable for guinea pig cochlea hydrochloride perilymph pepper benzophenone amine concentration detection.Part Ⅱ The affect of PPTA on the expression of IL-1β and TNF-α mRNA in sudden sensorineural hearing lossThe objective of this part is to investigate the protective mechanisms of PPTA on sudden sensorineural hearing loss caused by ischemia-reperfusion injury. The ischemia-reperfusion injury model was made by freeing and bilateral vertebral artery and right carotid atery and pressing them for 1 hour and then release them.54 guinea pigs were randomly divided into nine groups. ABR RT test were detected before all surgery. Another ABR RT test was done after administration of PPTA or saline. The modiolus were carefully stripped from cochlea and were used for RT-pcr test. All data were analyzed by SPSS 13.0. P<0.05 was considered statistically significant. The ABR RT threshold shift of PPTA treated group significantly reduced compared with ischemia-reperfusion injury group. Quantitative PCR product melting curve analysis dissolution curve ofβ-actin, IL-1β, TNF-αmRNA showed a single peak, indicating specific amplification product is good. The expressions of IL-1βand TNF-amRNA in normal group are significantly lower than that of the treated group(P <0.001).Comparing with the I/R 1 day group, intravenously immediately after reperfusion, IL-1β and TNF-αmRNA expression of PPTA treated group was significantly lower (P<0.001). And as the dose increased, the expression of IL-1β and TNF-αmRNA gradually decreased (P<0.001). Comparing with the I/R 3d group, intravenous injection of PPTA 2 days after I/RI,mRNA expression levels of IL-1β and TNF-α in each group is significantly reduced(P<0.001). The expression of IL-1β mRNA in lOmg/Kg PPTA3d group is significantly lower than that of the rest group(P <0.05). No significant difference was found between the expression of IL-1β mRNA in 2.5mg/Kg PPTA3d group and 5mg/Kg PPTA3d group. The expression of TNF-amRNA in 2.5mg/Kg PPTA3d group is significantly higher than that of the rest group(P<0.05). There is no significant difference was found between the expression of TNF-αmRNA in 5 mg/Kg PPTA3d group and 10 mg/Kg PPTA3d group.In summary, PPTA has decreased the expression of IL-1β and cochlear tissue TNF-αmRNA rather antagonistic ischemia-reperfusion injury and hearing protection.