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Possible Mechanism of Spiral Ganglion Neuron Damage Induced by Salicylate

Author: DengLiLi
Tutor: SuJiPing
School: Guangxi Medical University
Course: Department of Otolaryngology Head and Neck Surgery
Keywords: Salicylate spiral ganglion superoxide apoptosis NMDAreceptor endocytosis
CLC: R764.43
Type: PhD thesis
Year: 2013
Downloads: 31
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Abstract


Deafness is not only a common disease, but also a refractory disease in otolaryngology department. Hearing impairment became the most common of all kinds of disable in China. And due to the abuse of antibiotics, acute or chronic otitis media and sudden deafness without timely and effective treatment, increasing noise pollution and an ageing population and many other factors, the incidence rate of deafness increases year by year. Therefore, exploring the pathogenesis of deafness in depth, developing treatment and medical drugs to control and treat deafness has become one of the urgent need in the clinic practise. Salicylate, the active ingredient of aspirin, is currently one of the most widely used anti-inflammatory analgesic drugs. However, there are many side effects associated with the use of salicylates, especially the ototoxicity which is most common, mainly characterized by tinnitus and hearing loss. However, its mechanism is still not clear.The spiral ganglion neurons transfer sound signal from the sensory hair cells to the brain, which is an important acting site for salicylate-induced ototoxicity. Moreover, recent studies indicate that after several days of post treatment, salicylate selectively destroys the spiral ganglion neurons and auditory nerve fibers,but the mechanism is remain unclear. To understand the processes that initiate SGN death, cochlear organ cultures were prepared from postnatal day3rats and treated with different concentrations of salicylate. We observed the morphology change of SGN by immunofluorescence staining to clarify the damage types; Monitored superoxide generation in the cochlear culture to investigate the relationship between superoxide generation and the apoptosis of SGN induced by salicylate, using DHE fluorescent probes; Characterized the early stages of caspase-mediated SGN death to explore the signaling pathways of SGN apoptosis induced by salicylate,using CaspGLOW Red Active Caspase-3, Caspase-8and Caspase-9Staining Kits. Through above experiments,we try to explore the mechanism of SGN apoptosis induced by salicylate. Moreover,it takes several days for salicylate to induce a significant morphological damage to SGN,so we guess there should be anothor pathway involved in the early stage post salicylate treatment. To verify this hypothesis, we studied the expression of surface and total NMDAR1in SGN during the early stage of the salicylate treatment.Meanwhile, using western blot techniques to quantify the expression of surface and total NMDAR1protein on SGN,to explore the role of receptor trafficking in the the early stage of salicylate treatment. Our research will help to reveal the mechanism of saliclyate ototoxicity in the early and late stage which appears SGN apoptosis, to provides new theoretical basis for the further study of salicylate ototoxicity. Our research contains two parts:[Part1] The study of the mechanism of spiral ganglion neurons apoptosis induced by salicylate[Objectives] To identify the damage type of SGN induced by salicylate and to investigate the relationship between the superoxide generation and SGN damage.[Methods]Cochlear organotypic cultures were prepared from postnatal day2-3Sprague Dawley rats. Cochlear organotypic cultures were divided into control group,3mM,5mM,10mM salicylate treatment groups, maintained for48h or96h;50μM Z-VAD-FMK group,50μM Z-VAD-FMK+3mM、5mM、10mM salicylate treatment groups;100μM PyP group,100μMPyP+3mM、5mM、1OmM salicylate treatment groups;0.2mM AP5group,0.2mM AP5+3mM,5mM、10mM salicylate treatment grousp; maintained for48h. Hair cells,SGN and nuclear were labeled with TRITC-conjugated phalloidin, monoclonal primary antibody against neuronal class Ⅲ β-tubulin and To-Pro-3iodide respectively. Superoxide generation was detected with dihydroethidium, a fluorescent probe. Initiator caspase8and caspase9and downstream executioner caspase3activity were detected with Fluorengenic caspase-specific probes.[Results](1) There was no evidence of hair cells loss or damage after3mML5mM、10mM48h or96h post-salicylate treatment.(2) Exposing cochlear cultures to3MM、5mM、10mM salicylate for48h or96h resulted in the fragmentation, blebbing and destruction of the fiber bundles that increased with the dose and duration of salicylate treatment.(3) Exposuring cochlear cultures to3mM、5mM、10mM salicylate for48h or96h, many SGN soma were shrunken and their nuclei were condensed, fragmented and marginated, morphological features apoptosis, Soma shrinkage and nuclear fragmentation increased with the dose and duration of salicylate treatment. When cultured for48h, the percentages of apoptotic SGN in control group was20.61±6.165%,but increased to68.34±7.193%,83.80±8.481%,88.14±7.424%(P<0.05),after3mm,5mm,10mm salicylate treatment. When cultured for96h, the percentages of apoptotic SGN were further increased, in control group and3mm,5mm,10mm salicylate treatment groups respectively were20.76±6.690%,85.57±9.137%,95.42±4.280%,97.97±2.987%(P<0.05)(4) The percentage of positive caspase-3,-8and-9labeled SGN in12h control group were5.91±5.517%,6.870±3.064%,6.62±3.492%respectively and in10mM salicylate treatment group were6.69±6.445%,7.67±2.141%,5.70±3.307%, respectively, there were no difference between these two conditions (p>0.05).After it treated with10mM salicylate for24h, the percentage of positive caspase-3,-8and-9labeled SGN were14.42±6.392%,18.76±12.030%,13.04±7.129%respectively,the increase was subtle (p>0.05).When cultured for48h, the percentage of positive caspase-3,-8and -9labeled SGN in control group were10.23±4.810%,12.15±6.660%,8.610±2.636%respectively; All three caspases labeled abruptly increased to75.19±10.420%,79.40±11.290%after treated with10mM salicylate for48h (p<0.05).A pan-caspase inhibitor Z-VAD-FMK was used to protect SGN. The percentage of positive caspase-3,-8and-9labeled SGN in50μM Z-VAD-FMK group,50μM Z-VAD-FMK+3mM、5mM、10mM salicylate treatment groups were20.32±7.526%,65.55±11.190%,80.13±7.867%,85.69±6.984%respectively, There were no obvious protective effect for SGN (p>0.05)(5) Superoxide generation was detected in the culture after10mM salicylate treatment for48h using DHE staining. DHE stainning was selectively appeared in SGN, but not observed in the hair cells or support cells of the organ of Corti.The percentage of DHE positive SGN in control group was11.07±3.523%,after10mM salicylate treatment, increasing to56.12±8.316%(p<0.05).The percentage of DHE positive SGN in the group treated with100μM superoxide inhibitor PyP alone was10.40±2.620%,in PyP plus salicylate was25.04±6.107%,this difference was statistically significant (p<0.05). PyP greatly reduced the level of superoxide in SGN after the salicylate treatment, and largely prevented the degeneration of SGN and their peripheral nerve fibers. the nerve fibers in cultures treated with3mM SS plus PyP were nearly normal while those treated with5or10mM SS plus PyP exhibited only slight blebbing. PyP plus3mM、5mM、10mM salicylate treatment decrease percentage of apoptotic SGN to31.86±8.101%,32.68±7.166,35%,71±10.040%(p<0.05)(6) A NMD A receptor inhibitor AP5was used to protect SGN, the percentages of apoptotic SGN in0.2mM AP5group,0.2mM AP5+3mM,5mM.10mM salicylate treatment groups were14.95±5.420%,61.39±9.535%,75.56±11.710%,82.58±9.074%respectively, there was no obvious protection for SGN (p>0.05)[Summary] In vitro cochlea culture, Salicylate selectively destroyed SGN and nerve fibers in a dose-dependent manner. Both caspase-dependent and caspase-independent pathway were involved in SGN apoptosis, and caspase-dependent pathway induced both intrinsic and extrinsic apoptosis pathways.The selective destruction of SGN in cochlear cultures treated with salicylate is due to the buildup of the toxic superoxide radical exclusively in SGN. Salicylate induced destruction of SGN and auditory nerve fibers can be prevented by scavenger of superoxide. Part2The study of mechanism of salicylate treatment on spiral ganglion neurons in the early stage[Objectives] To explore the mechanism of NMDA receptor trafficking in the early stage post salicylate treatment in SGN.[Methods] SGN organotypic cultures were prepared from postnatal day2-3 Sprague Dawley rats. SGN organotypic cultures were divided into control group,3mM salicylate treatment group (15min and6h). Hair cells,SGN and nuclear were labeled with TRITC-conjugated phalloidin, monoclonal primary antibody against neuronal class Ⅲ β-tubulin and To-Pro-3iodide respectively in explants. Same primary antibody against NMDARlwas used to label surface and total NMDAR1on SGN, but the secondary antibody conjugated with Alexa Fluor488was used to stain the surface NMDAR1and the secondary antibody conjugated with Cy-3was used to stain the total NMDAR1. Observing the protein expression changes of surface and total NMDAR1on SGN induced by salicylate with biotin labeling surface protein and Western blot techniques.[Results](1) There was no apparent morphology damage on SGN after3mM salicylate treatment for15min or6h.(2) The expression of surface NMDAR1on SGN was significantly decreased after3mM salicylate treatment for15min, but the total NMDAR1remained the same level as the control group.(3) The expression of surface NMDAR1protein was significantly decreased by38.15%(p<0.05) after3mM salicylate treatment for15min when compared to control group; But it reached a new equilibrium by6h after salicylate treatment (p>0.05).(4) There was no significant difference of the expression of total NMDAR1on SGN between3mM salicylate treatment group (treated for15min)and the control group.(p>0.05). But the expression of total NMDAR1on SGN increased two-fold when treated with3mM salicylate for6h compared to the control group (p<0.05).[Summary]The endocytosis of the surface NMDARlin SGN was induced by salicylate in early stage of post treatment before the apparent morphology damage in SGN was observed. However, the expression of total NMDARl in SGN increased in the late stage of post salicylate treatment.[Conclusion](1) Salicylate selectively induced SGN apoptosis in a dose-and time-dependent manner.(2) Both caspase-dependent and caspase-independent pathway were involved in SGN apoptosis, and caspase-dependent pathway induced both intrinsic and extrinsic apoptosis pathways(3) Superoxide radical plays an important role in the SGN apoptosis induced by salicylate, scavenger of superoxide can prevent this salicylate-induced destruction of SGN and auditory nerve fibers.(4) The endocytosis of the surface NMDARlin SGN was induced by salicylate in early stage of post treatment before the apparent morphology damage in SGN was observed. However, the expression of total NMDARl in SGN increased in the late stage of post salicylate treatment.

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CLC: > Medicine, health > Otorhinolaryngology > Otology,ear disease > Ear nervous system diseases > Deaf
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