Hearing Loss Induced By Electrode Insertion Trauma: Mechanisms And The Effect Of Retinoic Acid In Inner Ear

Trauma-related heating loss caused by insertion of a cochlear implant electrode is thought to result from a combination of direct acute trauma to cochlear structures and the delayed cell death of injured hair cells.This delayed cell death of auditory hair cells may be attributed to oxidative stress initiated formation of reactive oxygen species(ROS)within traumatized cochlear sensory epithelium.ROS would cause damage to vital cellular components such as lipids,proteins and DNA,leading to subsequent cell death.DNA strand breakage activates poly(ADP-ribose)polymerase(PARP),a constitutive nuclear enzyme which is implicated in the DNA repair process,genomic stabilityand apoptosis.Activated PARP catalyses the addition of long branched chains of poly(ADPribose)from its substrate nicotinamide adenine dinucleotide(NAD)to a set of nuclear proteins.However,PARP overactivation leads to NAD depletion,resulting in a loss of ATP as it is used to synthetize new NAD,and finally to cell death.Reading the central nervous system,in vitro studies demonstrate PARP activation following oxidative stress.It was reported that overactivation of PARP was associated with hearing loss induced by noise in inner ear.The mitochondria play an important role in apoptosis.Apoptosis-inducing factor(AIF)is sequestered within the mitochondrial intermembrane space in cells. Upon apoptotic challenge,however,a rapid release of these factors through the outer mitochondrial membrane into the cytoplasm initiates the apoptotic process. AIF translocates in the nucleus,where it induces peripheral chromatin condensation and high molecular weight DNA fragmentation.The general view is that excess intracellular ROS results in the production of peroxynitrite to initially cause DNA damage,which allows for the activation of PARP.Overactivation of PARP triggers a poly(ADP-ribosyl)ation-dependent mechanism that results in the relocation of AIF from the mitochondria to the nucleus,where AIF promotes DNA fragmentation and nuclear condensation to result in apoptosis.JNK is associated with the mitochondrila dysfunciton and cell death pathway which was initiated by PARP overactivation,JNK is also involved in the auditory nruon apoptosis induced by oxidative stress.The aim of this study was to investigate the expression of PARP,JNK,and AIF in cochleae of electrode insertion trauma animal model and explain the mechanisms which were involved in the injury of inner ear.We also tested the effects of ATRA on trauma-induced heating loss and evaluated the otoprotective of ATRA on cochlear hair cells.PartⅠDeleterious Activation of Poly(ADP-Ribose)Polymerase in Electrode Insertion Trauma-related Hearing LossObjective:Oxidative stress has been shown to be implicated in the pathogenesis of EIT-related hearing loss.In vitro studies show that oxidative stress, particularly peroxynitrite,could trigger DNA strand breaks,which lead to the activation of repairing enzymes including Poly(ADP-ribose)Polymerase(PARP). We investigated the role of PARP in cochlea in an animal model of EIT.Methods:Hearing function was tested by auditory brainstem responses(ABRs). Expression of PARP and NT was detected by western blot and immunofluorescence staining 3h after EIT.Results:There was no increase in the hearing thresholds of unoperated contralateral control ears.There was an increase in ABR thresholds after electrode insertion trauma in untreated and in 3-aminobenzamide-injected cochleae.Immunofluorescence staining showed that NT,an indicator of nitrosative stress,and PARP,were present in auditory hair cells after EIT.The PARP inhibitor,3-aminobenzamide,significantly inhibited PARP activation and NT expression,and reduced the increase of ABR thresholds. Conclusions:These results demonstrate that oxidative stress induced in vivo in cochlea leads to the activation of PARP,which contributes to EIT-related hearing loss.Administration the inhibitor of PARP(3-AB)significantly inhibited the PARP activity and NT expression,prevented the trauma-related heating loss.PartⅡPARP could Induce AIF Translocation in Auditory Hair Cell Via JNK Activation in a Cochlea Implantation Trauma ModelObjective:Overactivation of PARP would trigger a poly(ADP-ribosyl)ationdependent mechanism that result in the relocation of AIF from the mitochondria to the nucleus,where AIF promotes DNA fragmentation and nuclear condensation.It was suggested that JNK was required for PARP-induced mitochondrial dysfunction,AIF translocation,and subsequent cell death.We examined whether such a cascade also occurs in the inner ear of EIT.Methods:Expression of PARP,p-JNK and AIF was detected by western blot and immunofluorescence staining 3h after EIT.Results:We observed PARP activation,JNK activation(phospho- JNK)and AIF translocation significantly increased in auditory hair cells of EIT animals. Inhibition of PARP activity reduced the p-JNK.The neuclea translocation of AIF was significantly attenuated by inhibition of PARP or JNK activity.Conclusions:These results suggested that JNK would be mediated the AIF translocation which was regulated by PARP in the hair cells of EIT animals.The PARP activation→JNK activation→AIF translocation pathway would play a critical role in injured hair cells. PARTⅢEffects of Retinoic Acid on Electrode Insertion Traumainduced hearing loss in guinea pigsObjective:EIT-related hearing loss occurs in two stages in laboratory animals, that is,an immediate loss followed by a progressive loss.JNK could be activated by EIT-induced inner ear injury.All-trans retinoic acid(ATRA),an active metabolite of vitamin A,could inhibit the activation of JNK to preserve the hearing when cochleae exposed to noise.In this study,we attempted to protect guinea pigs from developing HL by administering ATRA.Methods:ABR of guinea pigs were measured before,immediately after,3 days and 7 days posttrauma for experimental(EIT and EIT + ATRA)and for the contralateral unoperated cochleae of each group.An electrode analog of 0.14-mm diameter was inserted through a basal turn cochleostomy for a depth of 3 mm and withdrawn.Morphological changes of EIT-damaged hair cell nuclei was observed by Hoechst staining.Results:Hoechst 33342 staining showed significantly enhanced preservation of the auditory hair cells and relatively low injured nuclei,in animal-injected ATRA than in animal-injected sesame oil.Phospho-JNK immunohistochemistry showed that ATRA inhibited the activation of JNK.Conclusions:These results suggest that ATRA has an preservation on auditory hair cells and hearing loss in EIT animals.