| BackgroundHearing disorder is a common disease in Otorhinolaryngology Head and Neck Surgery, according to the world health organization (who) reported in 2013, there were 360 million people suffer from crippling hearing loss wordwise, accounting for 5.3% of the world’s population, and two-thirds of them in developing countries, this is a huge number. China is the largest developing country, hearing disabled person has 20.57 million, accounts for the population of 16.79%o, for all kinds of disabilities,severely affecting the healthy and living quality of human race. So,now,we are facing the challenge that can we find effective methods of prevention and treatment of hearing impairment.Hearing loss include conduction deafness, sensorineural hearing loss and mixed deafness, the vast majority of patients are sensorineural hearing loss. Now as we know that reasons are hereditary, ischemia, ototoxicity, genetic,senile,noise,virus infection, autoimmune disease, auditory neuropathy, nerve centre disease, tumor, etc. Sensorineural hearing loss caused by the lesion of the inner ear and auditory neural pathway. Pathological changes basically due to the damage to hair cells of cochlea, the physical changes of spiral ganglion, support cells and nerve endings, and lesions or damage of cochlear nerve and auditory pathway and center of brainstem.There are two types of sensory cells in inner eaninner hair cells(IHCs) and outer hair cells (OHCs).which can convert mechanical energy (sound energy, gravity or acceleration) to electrical energy (cell receptor potential), which are passed through the spiral ganglion neurons(SGCs) to the auditory brainstem pathways, spiral ganglion cells is afferent neurons. Inner hair cells and outer hair cells connect I, II afferent neuron dendrites of SGCs, respectively.Inner hair cells transform different frequency and intensity voice signal into electrical signal, which can stimulate the presynaptic membrane and release neurotransmitters into the corresponding spiral neurons, signal is transmited to central nervous system by spiral ganglion. Outer hair cells can magnify the voice signal,so, the cochlea has high sensitivity and selectivity to the sound frequency,OHCs assist IHCs to transmit the voice signals.The abnormal structure%function and shape of afferent synapses all can lead to significant sensorineural hearing loss. Any injury of hair cells and afferent nerve will result in sensorineural hearing loss.Cochlea is energy intensive organization, ischemia and hypoxia will cause hair cells necrosis and afferent nerve swelling, which result in hearing impairment, finally;noise is one of the reasons.noise, drug are the common causes of sensorineural hearing loss, but there are reports say that afferent nerve was swelling after ischemia 5 mins.Sensorineural deafness pathological phenomena of the covers the spiral ganglion, hair cells, nerve endings, and support the physical changes, the common pathogenic factors including virus infection, ischemia, senile degeneration and ototoxic drug poisoning, etc., including ischemia is one of the most common factors causing sensorineural deafness, but blood disorder characterized by ischemia reperfusion injury after generally, few to pure ischemia injury. Such as sudden deafness is generally believed that the inner ear microcirculation disorder caused by hearing impairment. Statistics show simple significantly smaller than that of ischemic reperfusion after ischemia caused by the loss caused by the loss, visible study of inner ear ischemia/reperfusion (I/RI) of the related mechanism is very key to the prevention and treatment of ear disease.Among mammals, now think that hair cell damage occurs, it is hard to regenerate, is still in research approaches for treating sensorineural deafness, such as renewable cochlea hair cells or transplantation of stem cells for treatment. A key factor of sensorineural deafness afferent nerve system damage the pathological change, nerve endings, spiral ganglion cells and complex synapses are afferent nerve system, synaptic structure under the influence of the external environment will hurt its functionality, and abnormal, but it is difficult to repair damaged spiral ganglion cells. Have experiments to study, can afferent nerve regeneration after injury, nerve fibers and hair cells can be associated with regeneration of nerve fibers; Schwann cells, meanwhile, will be in after death spiral ganglion cells proliferation, but not to the normal function of neurons differentiation, therefore cannot play their effects. At present, in order to make patients with sensorineural deafness can hear the voice of the outside world generally to wear hearing AIDS or cochlear transplant, however, because of the cochlear price is higher, people also can’t adapt to wear hearing AIDS, so there is no in a wide range of application, so drugs are most of the patients with primary treatment. So, the way should explore the induction of nerve deafness on drug therapy.The new compound named peperphentonamine hydrochl-oride,PPTA, which is first synthesized creatively by China, is the synthesized medicine with completely independent intellectual property screened by the Materia Medica Institute in Chinese Academy of Medical Sciences. This new synthesized medicine has alreasy authorized three invention patent,and PPTA had won 11 kinds of chemical medicines clinical test approved documents and even completed 127 clinical phase I tests PPTA as the advanced international calcium sensitizer original innovation chemical medicine, preclinical research proves it can clear free radical of injury cells, as well restrain the overload of peroxidatic reaction of lipid. According to the research say that, PPTA has a clear-up function to oxygen free radicals produced by the damage of brain cells,so it protect cranial nerve and improve animal cognitive impairment; as well increase SOD activity and GSH content Experiment results show that the PPTA has the function of resisting calcium overload, as well obvious protective effects on mitochondria of myocardial injury. PPTA can also remain the membrane fluidity of mitochondria normal, significantly reduce the damage to the ultrastructure of mitochondria. Overload Oxygen free radicals and calcium often leads to the damage of major tissue and cell, and mitochondrial injury can activate the Caspase-3 to start the apoptosis of cell. Studies have shown that PPTA can significantly reduce the expression of Caspase-1 mRNA, exhibit good resistance to apoptosis. Like cardio and cerebral tissue damage, calcium overload and free radical, apoptosis are common causes of afferent nerve damage, this research is base on protection mechanism of PPTA.This experiment is divided into two parts, the first part of successful guinea pigs cochlear ischemia/reperfusion injury model was established. Temporary arteriole clip clip through bilateral vertebral artery and the right common carotid artery, clip 1 hour ischemia model, loosen the three manufacturing artery reperfusion model. Can effectively reduce the inner ear blood perfusion, the success of the ischemia reperfusion model was established, the model of small trauma surgery, intraoperative animal mortality is low, the postoperative survival rate is high, suitable for a large building, provides good foundation for the next experiment. The second part aims to from apoptosis (using TUNEL technology) and the change of Fas proteins related to apoptosis and caspase 1 mRNA expression to explore pepper benzene ketone amine of protection to the inner ear ischemia/reperfusion injury and the possible mechanism, to enlarge the clinical indications of pepper benzene ketone amine, for pepper benzene ketone amine provide pharmacological basis for the treatment of ischemic inner ear disease, is expected to develop protective to the inner ear ischemia reperfusion injury of new drugs.Part I Cochlear Ischemia/Reperfusion (I/R) Model in Guinea PigsObjectiveThe purpose of this study was to establish an animal model of cochlea ischemia/reperfusion(I/R) injury in guinea pigs.MethodsTwenty-four healthy guinea pigs with normal Preyer’S reflex weighed from 200 to 250g were used in this study. They were randomly divided into 3 groups:the normal control group, blank control group、and ischemia reperfusion groups. The ischemia/reperfusion model by transient blocking bilateral vertebral artery and unilateral cephalic artery for 60min,the cochlear blood flow(CoBF)was monitored continuously with Laser Doppler flowmeter.ResultsIschemia model success:noninvasive arteriole clip clip bilateral vertebral artery and the right common carotid artery,5-10 minutes after each animal CoBF fell to a level of 30%, before ischemia after maintain at this level, the ischemia model set up successfully.I/R model:after 1 hour ischemia, lift the right common carotid artery arteriole clip, and gently remove the bilateral vertebral artery arteriole clip, in case of vertebral artery break away from the testing each animal CoBF, before 10 minutes back to ischemia reperfusion level of 70%, then remain stable, reperfusion model was established.ConclusionWe had successfully established an animal model by blocking bilateral vertebral artery and unilateral cephalic artery in guinea pigs. It can effectively reduce the inner ear blood perfusion, the success of the ischemia reperfusion model was established, the model of small trauma surgery, intraoperative animal mortality is low, the postoperative survival rate is high, suitable for a large building, provides good foundation for the next experiment.Part 2 Effect of mRNA Expression of Caspase-1 on Cochlear Ischemia Reperfusion Injury in Guinea Pigs and Protection of PPTAObjective:1. To observe the cell apoptosis in cochlear after ischemia reperfusion in different time.2. To analysis the relationship between expression of transcription of Caspase-1 and the cell apoptosis in cochlear after ischemia reperfusion.3. To investigate the application of PPTA.Methods:48 healthy guinea pigs were devided into the normal group, negative control group, IRI control group after the application of 0.9%NaCl,Group with PPTA (10mg /kg) were given by intraperitoneal injection after onset of ischemia-reperfusion(60min after ischemia). Every groups have 12 guinea pigs and we would take samples 24h after reperfusion. The cell apoptosis was studied by TUNEL method, The difference between every groups was analyzed by apoptotic index (AI).. The mRNA of caspase 1 in cochlear was detected by RT-PCR.The data was compared between groups.Statistical analysis Was performed with one-way ANOVA of SPSS 13.0 statistical package and statistical significance was defined as P<0.05.Result:No or only individual cell apoptosis appeared in the each part of cochlear in the normal group, control group and the ischemia-reperfusion group with the PPTA with TUNEL method.In the ischemia-reperfusion control group the cochlea apoptotic cells increased obviously.PPTA could significantly reduce apoptotic cells, AI in I/RI group is significantly higher than the normal group, negative control group and PPTA group(P<0.001)..The expression of Caspase-1 mRNA of cochlear in ischemia-reperfusion group is significantly higher than normal group, control group and ischemia-reperfusion group with PPTA (P< 0.001).In ischemia reperfusion group with the PPTA the expression rate of Caspase-1 mRNA of cochlear was significantly higher than it in normal group (P< 0.001). The difference of expression of Caspase-1 mRNA of cochlear tissue is not significant between the normal group and the control group (P= 0.825).Conclusion:There were almost no cell apoptosis in the normal group and the control groups. But there were more cell apoptosis in the ischemia group and the reperfusion groups, The numbers of apotosis cells in the drug(PPTA) groups were smaller than the groups without drug(PPTA)。In the ischemia group and reperfusion groups, transcription level of Caspase-1 mRNA increased significantly compared with the normal group and the control groups. Compared with the model groups, transcription level of Caspase-1 mRNA in cochlear decreased in drug(PPTA) groups.. All of above indicates the ischemia reperfusion injury can cause the cell apoptosis in cochlear. The therapy of PPTA may prevent the cochlear from cellapoptosis in the ischemia reperfusion injury through blocking the expression of transcription of Caspase-1. |
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