Endocannabinoid (eCB) system is composed of endogenous CB ligands including anandamide (AEA) and 2-Arachidonyl glycerol (2-AG), enzymes involved in their biosynthesis and degradation such as diacylglycerol lipase-α (DAGL- α), and CB receptors. Primarily, there are three types of CB receptors - CB receptor 1(CB1), CB receptor 2 (CB2) and non CB1 non CB2 types of CB receptor (e.g. GPR, TRPV1) and they belong to G-protein (Gi/o) coupled receptors (GPCR) family.CB1 receptors are abundant in the brain where they modulate neuronal activities. On the other hand, CB2 receptors are predominantly expressed in the immune cells and regulate the growth and proliferation of different immune cells and modulate the activities of cytokines network and anti-oxidant machinery in stress conditions. Inflammation plays a central role in hearing loss (HL) caused by different ototoxic insults including anti-neoplastic agents such as cisplatin, aminoglycosides and acoustic trauma. These insults can trigger chronic production of reactive oxygen species (ROS) in regions of cochlea such as organ of Corti, stria vascularis (SVA), spiral ligament (SL) and spiral ganglion neurons (SG). This leads to increased synthesis of pro-inflammatory cytokines, disruption of mitochondrial membrane integrity, activation of DNA damage/repair pathways and activation of pro-apoptotic enzymes. Jeong et al. (2007) have shown that CB2 receptor specific agonist (JWH-015) protects the HEI-OC1 hair cell cultures against cisplatin-induced cytotoxicity in-vitro. The goal of the current study was to examine the distribution and function of CB receptors (mainly CB2) in the cochlea and determine whether activation of these receptors could protect the cochlea by altering the expression of ROS generating proteins, along with pro-inflammatory and pro-apoptotic proteins. This study also investigated whether inhibition of eCB synthesis can causes HL. Aim 1 of the current study investigated the expression of CB receptors in the cochlea using different in-vivo models such as male Wistar rat and knock-in mice with GFP-tagged CB2 receptors, in-vitro models such as organotypic culture of neonatal mouse (C57BL/6) cochlea and University of Bristol organ of Corti (UB/OC1) cells. We show that both CB1 and CB2 receptors are expressed in the outer and the inner hair cells (OHCs and IHCs), SV, SG and supporting cells (SCs) included outer and inner pillar cells. The distribution of DAGL- α was also examined in the male Wistar rats and we found the similar distribution pattern of this enzymes as CB2. DAGL- α catalyzes the hydrolysis of DAG to synthesize 2-AG, which acts as a chief endogenous CB2 ligand. Our initial studies suggested a role of CB2 and not CB1 in protection, leading us to focus on CB2 receptors for subsequent studies. Aim 2 examined the otoprotective role of trans-tympanic application of CB2 specific agonist (JWH-015) against cisplatin-induced hearing loss in male Wistar rats. Activation of CB2 receptors restored cisplatin-induced elevations in ABR thresholds which was significantly reversed by CB2 antagonist AM-630. Pre-treatment with JWH-015 protected against cisplatin-induced loss of hair cell and synaptic ribbons. In-vitro studies in UB/OC-1 cells demonstrated that pre-treatment of JWH-015 modulates the activities of signal transducer and activator of transcription 1 and 3 (STAT1 and STAT3), increases the expression of anti-apoptotic protein Bcl-xL, indicating its role in regulating the apoptosis Activation of CB2 also abrogated cisplatin-induced decrease in Na+/K+ATPase- α in the SV and SL fibrocytes and ameliorated the expression of different pro-inflammatory genes including TRPV1, COX2, NOX3, KIM1, iNOS and TNF- α. We also found that blocking of CB2 by AM630 itself resulted in hearing loss and loss in CB2 receptors, indicating eCB system is tonically active and could be important for physiological function of the cochlea. Indeed, we observed that inhibition of DAGL- α by RHC80267 results in HL. Aim 3 of this current study investigated whether pre-treatment of CB2 agonist will interfere with anti-cancer efficacy of cisplatin against various cancer cell lines head and neck cancer cells (UMSCC10B), and colon cancer cells (HCT116). Our data indicate that JWH-015 did not interfere with cisplatin-induced apoptosis in these cells. Overall, this study provides novel insights into the essential role eCBs plays in protection the cochlea under non-stressed conditions and following exposure to ototoxic agents. It also demonstrates that application of exogenous CB2 agonist (JWH-015) could serve as an effective protective agent against cisplatin ototoxicity These data suggest that localized delivery of CB2 agonists should be studied in human for protection against hearing loss.

Regulators of G-protein signaling (RGS) are a multifunctional and highly diverse group of proteins that negatively regulate G-protein coupled receptor (GPCR) signaling pathways. The common mechanism of RGS is to act as GTPase accelerating proteins (GAP) to accelerate the hydrolysis of active GTP bound G proteins and terminate the actions of the associated GPCR. In addition to the traditional function of inhibiting G-protein signaling, recent studies have highlighted the role of RGS proteins in modulating GPCRs in GAP-independent way. There are more than 30 RGS proteins, and depending upon cell/tissue type, they interact and associate with different G proteins and GPCRs to modulate various physiological functions. RGS17, a member of the RGS-RZ subfamily, commonly targets GTP bound Gαz, Gαi, and Gαo for hydrolysis and signal termination. RGS17 is abundantly expressed in the central nervous system and is highly associated with opioid, dopamine and cannabinoid receptors in the brain. RGS17 is also upregulated in many malignant tumors such as lung, prostate and breast cancers. Analysis of whole cochlea transcriptome data from our lab revealed higher levels of RGS17 in the cochlea after cisplatin treatment. This highlights a possible role of RGS17 (and probably other RGS proteins) in cisplatin ototoxicity. Activation of endogenous, otoprotective GPCRs such as adenosine (A1AR) and cannabinoid (CB2) receptor is beneficial for promoting protection against cisplatin-induced hearing loss. Taking all this together, the underlying hypothesis for this study is that cisplatin could possibly mediate ototoxicity by increasing the expression of RGS17, which reduces the otoprotective effect of endogenous receptors such as cannabinoid receptor 2. The main objective of the study is to examine the expression and function of RGS17 in the cochlea and determine if inhibition of RGS17 could protect against cisplatin ototoxicity.The expression of RGS17 was observed in the both in vitro and in vivo models of cochlear cell types. Immunofluorescence study, western blot analysis, and RT-qPCR results showed the presence of RGS17 in UB\OC-1, as well as Wistar rat cochlea; expression levels increased after cisplatin treatment. To determine the role of RGS17 in hearing, first, it was overexpressed in the cochlea using adenoviral vector that was found to significantly increase ABR threshold shifts and decrease ABR Wave I amplitude. Conversely, knockdown of RGS17 (by siRGS17) decreased cisplatin-induced elevations in ABR thresholds along with increased wave I amplitude and latency. Furthermore, siRGS17 pretreatment prevented cisplatin-mediated synapse loss at inner hair cells. This indicates inhibition of RGS17 can preserve the functional and physiological integrity of the cochlea, which is essential for hearing. Cochleae that were treated with siRGS17, followed by cisplatin, showed fewer TUNEL-positive cells and reduced loss of Outer hair cells (OHC) as compared to cisplatin-treated rats. Moreover, overexpression of RGS17 increased the ratio of the transcription factors, pSTAT1/pSTAT3, which may indicate initiation of the apoptotic pathway. Moreover, UB\OC-1 cells treated with Celastrol, a RGS17 inhibitor, showed an increase in cell viability against cisplatin toxicity. In addition to apoptosis, overexpression of RGS17 also elevated ROS production and oxidative stress. But, the inhibition of RGS17 attenuated the cisplatin-induced increase in transcripts for oxidative and inflammatory stress markers, such as NOX3, iNOS, KIM1, TNF-α, and COX2, whereas the mRNA level of antioxidant genes such as Nrf2 and SOD2 were increased. Activation of CB2 via JWH-015 (a CB2 agonist) prior to cisplatin administration significantly reduced the cisplatin-induced elevated levels of RGS17, while knockdown of CB2 increased RGS17 expression in the cochlea. siRGS17 treatment boosted endogenous CB2R-Gα expression. Additionally, cisplatin decreased the expression of Gαi/o and Gαz in vitro, but the activation of CB2 increased the expression of these G proteins. Furthermore, JWH-015 treatment alleviated RGS17-dependent cell death. This study suggests that RGS17 could serve as a mediator of cisplatin ototoxicity by reducing the duration of active CB2R-G protein signaling, which normally suppresses cochlear oxidative stress, inflammation and hair cell apoptosis, and thereby preserves normal hearing. These data also indicate the existence of tonic reciprocal inhibition between RGS17 and CB2 mediated via the G proteins. Thus, we propose that RGS17 inhibitors could serve as an effective treatment against cisplatin ototoxicity when used alone and can potentiate the actions of CB2 agonists when used in combination therapy against cisplatin-induced hearing loss.

D-methionine (D-met) has demonstrated otoprotection from noise-, aminoglycoside-, and cisplatin-induced hearing loss in animal studies. As a result, D-met is currently progressing through translational "bench to bedside" research. However, D-met's exact otoprotective mechanisms have not been fully elucidated. This study investigated relationships between dose- and time-dependent D-met otoprotection from noise- and aminoglycoside-induced hearing loss. Further, the study correlated protective D-met dose to endogenous antioxidant enzyme activity and lipid peroxidation. Specific aim 1 tested D-met dose response protection by auditory brainstem response (ABR) threshold shift analysis and outer hair cell (OHC) quantification. D-met doses ranging from 25-200 mg/kg/dose were administered to chinchillas every 12 hours five times each before and after steady state noise exposure totaling 10 D-met doses. Results demonstrated optimal, sub-optimal, and supra-optimal bi-phasic D-met otoprotective dose response. Optimal D-met protection from steady state noise occurred at the 50 mg/kg/dose level. OHC quantification confirmed electrophysiological assessment. Specific aim 2 measured D-met rescue protection from steady state noise exposure by ABR threshold shift analysis and OHC quantification. Five intraperitoneal (ip) D-met injections were administered every 12 hours beginning 3, 5, 7, 9, 12, 18, 24, 36, or 48 hours after steady state noise exposure. Results measured full D-met protection when administration began as late as 24 hours after noise secession. Significant partial protection was also measured for the 36 hour delay. OHC quantification confirmed electrophysiological assessment. Specific aim 3 measured D-met preloading protection from steady state noise exposure by ABR threshold shift analysis and OHC quantification. Five ip D-met injections were administered every 12 hours beginning 2, 2.5, or 3 days prior to steady state noise exposure. Results measured significant D-met protection when administration ended as early as 24 hours prior to noise exposure. OHC quantification confirmed electrophysiological assessment. Specific aim 4 tested dose-dependent D-met influence on antioxidant enzyme activity and oxidative stress in steady state noise-exposed chinchillas. One ip D-met injection, ranging from 25 to 200 mg/kg/dose, was administered every 12 hours beginning 2 days prior to steady state noise exposure for a total of 5 injections. Two hours post-noise exposure, animals were sacrificed and serum, liver, and cochleae were collected for endogenous antioxidant analysis. Glutaredoxin 2 (Grx2) was also analyzed 21 days post-noise exposure. Lower D-met doses (25 and 50 mg/kg/dose) increased superoxide dismutase and catalase activity. Glutathione reductase and glutathione peroxidase activities significantly increased with D-met doses but only at high concentrations (200 mg/kg/dose). At 21 days post-noise, Grx2 activity was significantly decreased in liver but greatly increased in the cochlea with high D-met doses (200 mg/kg/dose). The endogenous enzyme studies suggest optimal protective D-met dose determined in specific aims 1 through 3 may be secondary to increased superoxide dismutase and catalase activity which may result from D-met's free radical scavenging characteristics. Glutathione pathway activity increased only with high D-met doses that resulted in less optimal protection in specific aim 1. Thus, D-met-induced glutathione pathway enhancement may be a compensatory or saturation mechanism rather than the primary protective mechanism. Further, the extended pre-loading and rescue protection may be a result of significantly increased s-glutathionylation activity in the cochlea. Specific aim 5 tested D-met protection from impulse noise exposures. D-met dose response, rescue, and antioxidant enzyme assay protocols, similar to those in specific aims 1, 3, and 4 in steady state animals, were performed on impulse noise-exposed chinchillas. D-met provided dose- and time-dependent optimal protection from impulse noise similar to the steady-state noise studies. Optimal D-met protection was measured at the 100 mg/kg/dose level with complete rescue protection as late as 24 hours post-noise exposure. Endogenous enzyme activity measures demonstrated significant superoxide dismutase, catalase, and glutathione peroxidase activity increases which correlated with optimal D-met protective dose (100 mg/kg/dose) and catalase and superoxide dismutase activity decreases at the higher doses (200 mg/kg/dose). Specific aim 6 tested dose-dependent D-met protection from tobramycin, amikacin, kanamycin, and gentamicin aminoglycoside antibiotics. Guinea pig animal models were normalized to achieve a 30-40 dB ABR threshold shift with the lowest possible aminoglycoside dose. D-met and the aforementioned single aminoglycoside were administered for 21, 28, 23, or 14 days, respectively. ABRs were collected and assessed at baseline, 2, 4, and 6 weeks after drug administration initiation. After the 6-week ABR data collection, cochleae were collected and prepared for OHC quantification. ABR threshold shifts and OHC quantifications demonstrate significant bi-phasic D-met-induced protection from each aminoglycoside type with different D-met doses. OHC quantification confirmed electrophysiological assessment. This study identified optimal protective D-met dose for aminoglycoside- and noise- induced ototoxicity. It also identified optimal protective D-met dose timing for steady state and impulse noise-induced hearing loss. Further, this study has identified dose-dependent D-met-induced endogenous antioxidant changes and Grx2 enhancement, and therefore s-glutathionylation, as a potential mechanism for D-met protection. Thus, dose- and time-dependent D-met protection influences endogenous antioxidant activity, but exact optimal D-met protection will continue to warrant further investigation.

As investigações sobre os efeitos da oxigenoterapia hiperbárica (OHB) em lesão por ruído são escassas e apontam para diferentes efeitos em função do momento de intervenção. Dentre os trabalhos já descritos foi observado efeito lesivo da OHB quando aplicada imediatamente ao trauma acústico, contudo, efeito positivo foi observado quando aplicada após 2 e 6 horas. Com relação aos tratamentos usados para trauma acústico, alguns estudos descrevem o uso de corticoides como melhor alternativa, mas recentemente estudos apontam para a sua combinação com OHB como a terapêutica com maior beneficio nas lesões por ruído. O presente estudo teve como objetivos avaliar o momento da intervenção pela OHB após 2, 4 e 6 horas de repouso auditivo após exposição ao ruído e avaliar a associação terapêutica entre a OHB e corticoterapia (CT). Cobaias albinas foram expostas a um ruído branco na faixa de 4 kHz com intensidade igual a 110dB NPS por 72h e divididas em cinco grupos terapêuticos: OHB com início após 2, 4 e 6h de repouso auditivo após exposição ao ruído, CT isolada e OHB após 6 horas de repouso associada a CT. O tratamento durou 5 dias, sendo uma sessão terapêutica por dia. Todos os animais tiveram a função auditiva avaliada pelo Potencial Evocado Auditivo de Tronco Encefálico (PEATE) e pelas Emissões Otoacústicas Produtos de Distorção (EOAPD) em três momentos: pré-ruído, pós-ruído e pós-tratamento. Após a eutanásia dos animais e preparação dos espécimes cocleares, todas as cócleas foram analisadas através de Microscopia Eletrônica de Varredura (MEV). Não houve diferença estatística significativa entre os momentos de intervenção pela OHB após 2, 4 e 6 horas, contudo, os dados de MEV demonstraram que uma maior otoproteção ocorreu quando a intervenção foi realizada após um maior repouso auditivo. Apesar da não diferença estatística significativa, os achados anatômicos e funcionais permitiram concluir que a associação terapêutica entre a OHB e a corticoterapia desempenhou um melhor efeito otoprotetor e terapêutico se comparada a essas mesmas terapias isoladas. / Investigations on the effects of hyperbaric oxygen therapy (HBOT) in noise injury are scarce and point to different effects depending on the time of intervention. Among the work already described has been observed damaging effect of HBOT when applied immediately after the acoustic trauma, however, positive effect was observed when applied after 2 and 6 hours of rest after the trauma. Studies describe the use of corticosteroids as the best alternative to treat acoustic trauma, but recent studies point to their combination with HBOT as the major benefit in lesions by noise. This study aimed to evaluate the time of intervention by HBOT after 2, 4 and 6 hours of rest after hearing noise exposure and to evaluate the association between HBOT and corticoid. Female guinea pigs were exposed to a white noise on 4kHz at 110dB SPL during 72 hours and divided into five treatment groups: HBOT after 2, 4 and 6 hours of rest after the noise exposure, corticosteroid therapy and HBOT combined with corticoid. The treatment lasted five days, being a therapy session per day. All animals were exposed to Distortion Product Otoacoustic Emissions (DPOAE) and Auditory Brainstem evoked Response (ABR) in three moments: before and after exposure to the noise and after the treatment. All cochleae were examined by scanning electron microscopy (SEM). There was no statistically significant difference between the moments of intervention by HBOT after 2, 4 and 6 hours, however, the SEM data showed that a greater otoprotection occurred when the intervention was performed after a higher auditory rest. Although not statistically significant, the anatomical and functional findings concluded that the association between HBOT and corticosteroid therapy played a better otoprotective and therapeutic effect compared to those same therapies alone.

As investigações sobre os efeitos da oxigenoterapia hiperbárica (OHB) em lesão por ruído são escassas e apontam para diferentes efeitos em função do momento de intervenção. Dentre os trabalhos já descritos foi observado efeito lesivo da OHB quando aplicada imediatamente ao trauma acústico, contudo, efeito positivo foi observado quando aplicada após 2 e 6 horas. Com relação aos tratamentos usados para trauma acústico, alguns estudos descrevem o uso de corticoides como melhor alternativa, mas recentemente estudos apontam para a sua combinação com OHB como a terapêutica com maior beneficio nas lesões por ruído. O presente estudo teve como objetivos avaliar o momento da intervenção pela OHB após 2, 4 e 6 horas de repouso auditivo após exposição ao ruído e avaliar a associação terapêutica entre a OHB e corticoterapia (CT). Cobaias albinas foram expostas a um ruído branco na faixa de 4 kHz com intensidade igual a 110dB NPS por 72h e divididas em cinco grupos terapêuticos: OHB com início após 2, 4 e 6h de repouso auditivo após exposição ao ruído, CT isolada e OHB após 6 horas de repouso associada a CT. O tratamento durou 5 dias, sendo uma sessão terapêutica por dia. Todos os animais tiveram a função auditiva avaliada pelo Potencial Evocado Auditivo de Tronco Encefálico (PEATE) e pelas Emissões Otoacústicas Produtos de Distorção (EOAPD) em três momentos: pré-ruído, pós-ruído e pós-tratamento. Após a eutanásia dos animais e preparação dos espécimes cocleares, todas as cócleas foram analisadas através de Microscopia Eletrônica de Varredura (MEV). Não houve diferença estatística significativa entre os momentos de intervenção pela OHB após 2, 4 e 6 horas, contudo, os dados de MEV demonstraram que uma maior otoproteção ocorreu quando a intervenção foi realizada após um maior repouso auditivo. Apesar da não diferença estatística significativa, os achados anatômicos e funcionais permitiram concluir que a associação terapêutica entre a OHB e a corticoterapia desempenhou um melhor efeito otoprotetor e terapêutico se comparada a essas mesmas terapias isoladas. / Investigations on the effects of hyperbaric oxygen therapy (HBOT) in noise injury are scarce and point to different effects depending on the time of intervention. Among the work already described has been observed damaging effect of HBOT when applied immediately after the acoustic trauma, however, positive effect was observed when applied after 2 and 6 hours of rest after the trauma. Studies describe the use of corticosteroids as the best alternative to treat acoustic trauma, but recent studies point to their combination with HBOT as the major benefit in lesions by noise. This study aimed to evaluate the time of intervention by HBOT after 2, 4 and 6 hours of rest after hearing noise exposure and to evaluate the association between HBOT and corticoid. Female guinea pigs were exposed to a white noise on 4kHz at 110dB SPL during 72 hours and divided into five treatment groups: HBOT after 2, 4 and 6 hours of rest after the noise exposure, corticosteroid therapy and HBOT combined with corticoid. The treatment lasted five days, being a therapy session per day. All animals were exposed to Distortion Product Otoacoustic Emissions (DPOAE) and Auditory Brainstem evoked Response (ABR) in three moments: before and after exposure to the noise and after the treatment. All cochleae were examined by scanning electron microscopy (SEM). There was no statistically significant difference between the moments of intervention by HBOT after 2, 4 and 6 hours, however, the SEM data showed that a greater otoprotection occurred when the intervention was performed after a higher auditory rest. Although not statistically significant, the anatomical and functional findings concluded that the association between HBOT and corticosteroid therapy played a better otoprotective and therapeutic effect compared to those same therapies alone.

Brazil is among the world's largest consumers of pesticides and their increased use is given in agriculture, especially monoculture in large areas. Research shows that the ototoxic agents, in addition to compromising the peripheral auditory and vestybular systems, they may cause changes in central auditory pathways. O objetivo deste estudo foi verificar os aspectos ultra-estruturais de cócleas de cobaias expostas a agrotóxico e ginkgo biloba, utilizando doses comprovadamente ototóxicas, lesivas às células ciliadas externas, avaliando-se as alterações anatômicas através da microscopia eletrônica de superfície. The aim of this study was to evaluate the ultrastructural aspects of the cochlea of guinea pigs exposed to pesticides and ginkgo biloba, using ototoxic doses, damaging the outer hair cells, to evaluate the anatomic changes by surface electron microscopy. This is a prospective experimental study, conducted in guinea pigs, wich the inclusion criterion was the presence of DPOAE. The sample was divided into five groups, in which they administered 0.9% saline solution (group 1 - control), pesticides - 0.3 mg / kg / day (group 2), Ginkgo biloba - 100mg/kg/day, 90 minutes after a pesticide 0.3 mg / kg / day (group 3), pesticides - 3 mg / kg / day (group 4) and Ginkgo biloba - 100mg/kg/day, 90 minutes after a pesticide 3.0 mg / kg / days (group 5) for seven consecutive days. The pesticide used was that of methamidophos from Fersol ®. The anatomical evaluation was performed with scanning electronic microscopy. The guinea pigs subjected to pesticides showed morphological cochlear lesions in the three turns analyzed by electronic microscopy, intensified according to the dosage received from the pesticide agent. Guinea pigs treated with pesticides and Ginkgo biloba showed a maintenance of ciliary architecture in outer hair cells in all cochlear turns, whereas in the group treated only with pesticides, there was disappearance of the cilia of outer hair cells and distortion in the architecture of cilia remaining the MoU. We conclude that the greater the exposure to pesticides increased the damage in outer hair cells of the cochlea of albino guinea pigs, while those who had previously received ginkgo biloba keep outer hair cells cytoarchitecture preserved. / O Brasil está entre os principais consumidores mundiais de agrotóxicos e sua maior utilização se dá na agricultura, especialmente na monocultura, em grandes extensões. Pesquisas demonstram que os agentes ototóxicos, além de comprometer os sistemas auditivo e vestibular periféricos, provocam ainda alterações nas vias auditivas centrais. O objetivo deste estudo foi verificar os aspectos ultra-estruturais de cócleas de cobaias expostas a agrotóxico e ginkgo biloba, utilizando doses comprovadamente ototóxicas, lesivas às células ciliadas externas, avaliando-se as alterações anatômicas através da microscopia eletrônica de superfície. Trata-se de um estudo experimental prospectivo, realizado em cobaias albinas, cujo critério de inclusão foi à presença de OEAPD. A amostra foi dividida em cinco grupos, nos quais se administrou soro fisiológico 0,9% (grupo 1 - controle), agrotóxico - 0,3mg/Kg/dia (grupo 2), Ginkgo biloba - 100mg/kg/dia, 90 minutos após, agrotóxico 0,3 mg/Kg/dia (grupo 3), agrotóxico 3 mg/Kg/dia (grupo 4) e Ginkgo biloba - 100mg/kg/dia, 90 minutos após, agrotóxico 3,0 mg/Kg/dia (grupo 5) durante sete dias consecutivos. O agrotóxico utilizado foi o metamidofós da Fersol®. A avaliação anatômica foi realizada com Microscopia Eletrônica de Varredura. As cobaias submetidas ao agrotóxico apresentaram alterações morfológicas cocleares, com lesões nas três espiras analisadas na microscopia eletrônica, intensificadas de acordo com a dosagem recebida do agente. As cobaias tratadas com agrotóxico e Ginkgo biloba, apresentaram uma manutenção da arquitetura ciliar nas células ciliadas externas em todas as espiras da cóclea, enquanto que nos grupo tratado somente com agrotóxico, houve desaparecimento dos cílios das células ciliadas externas e distorção na arquitetura dos cílios remanescentes à ME. Concluímos que quanto maior a exposição ao agrotóxico maior o dano nas CCE da cóclea de cobaias albinas, enquanto que aquelas que recebem previamente o ginkgo biloba mantêm a citoarquitetura das CCE preservadas.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This paper aimed to investigate the apoptosis modulation pathway and the otoprotection mechanism of N-acetylcysteine (NAC) through the analysis of the glutathione peroxidase (GSH-Px) enzyme and the Bcl-2 protein expression in outer hair cells (OHCs) of rats treated with cisplatin. The listening function was also assessed in mice under the effect of different doses of cisplatin and NAC. Two experiments were performed, named A and B, the first being over an experimental period of five days, and the second during three days. Each experiment comprised four groups, under the following protocols: group A1 (negative control): intraperitoneally saline solution 0,9%, in the same volume corresponding to cisplatin dose; group A2 (positive control): 100mg/kg/day of NAC, oral administration by gavage; group A3 (ototoxic): 3mg/kg/day of intraperitoneally cisplatin; group A4 (ototoxic with otoprotection): 100 mg/kg/day of NAC oral administration by gavage, one hour before the administration of 3 mg/kg/day of intraperitoneally cisplatin; group B1 (negative control): intraperitoneally saline solution 0,9% in the same volume corresponding to the cisplatin dose (8mg/kg/day); group B2 (positive control): 300 mg/kg/day of NAC, oral administration by gavage; group B3 (ototoxic): 8 mg/kg/day of intraperitoneally cisplatin; group B4 (ototoxic with otoprotection): 300 mg/kg/day of NAC orally by gavage, one hour before the administration of 8 mg/Kg/day of intraperitoneally cisplatin. The animals in experiment A underwent otoscopy, distortion-product otoacustic emissions (DPOAEs) and brainstem auditory evoked potential (BAEP), before and after the administration of drugs. The animals in experiment B underwent the same testing in pre- and post-treatment, their tympanic bulla being removed ant their cochleae prepared for anatomical assessment with scanning electron microscopy and immunofluorescence for labeling the GSH-Px enzyme and the Bcl-2 protein. In experiment A, it was verified that there was no significant decrease in the signal-to-noise ratio of DPOEAs, but there was a significant increase in the electrophysiologic threshold obtained through BAEP in groups A3 e A4. In experiment B, it was verified that: there was no significant increase in the electrophysiologic threshold obtained through BAEP; the OHCs remained anatomically intact; the GSH-Px enzyme showed immunostaining absent in group B1 and immunostaining present in groups B2, B3 and B4; the Bcl-2 protein showed immunostaining absent in all groups. From the results, it was concluded that the listening function was more impaired by the exposure to a subdose of cisplatin over a longer period, the apoptosis modulation pathway in outer hair cells of mice treated with cisplatin is related to the expression of the GSH-Px enzyme and not expression of the Bcl-2 protein. / Este trabalho teve o objetivo de investigar o mecanismo de otoproteção da N-acetilcisteína (NAC) e a via de modulação da apoptose por meio da análise da expressão da enzima glutationa peroxidase (GSH-Px) e da proteína Bcl-2 em células ciliadas externas (CCEs) de ratos tratados com cisplatina. Também foi avaliada a função auditiva de ratos sob efeito de diferentes doses de cisplatina e NAC. Foram realizados dois experimentos, denominados de A e B, sendo o primeiro com um período experimental de cinco dias e o segundo de três dias. Cada experimento foi composto por quatro grupos, submetidos aos seguintes protocolos: grupo A1 (controle negativo): solução fisiológica 0,9%, via intraperitoneal, no mesmo volume correspondente à dose de cisplatina; grupo A2 (controle positivo): 100mg/Kg/dia de NAC, via oral por gavagem; grupo A3 (ototóxico): 3mg/Kg/dia de cisplatina via intraperitoneal; grupo A4 (ototóxico com otoproteção): 100 mg/Kg/dia de NAC via oral por gavagem, uma hora antes da administração de 3 mg/Kg/dia de cisplatina via intraperitoneal; grupo B1 (controle negativo): solução fisiológica 0,9% via intraperitoneal no mesmo volume correspondente à dose de cisplatina (8mg/Kg/dia); grupo B2 (controle positivo): 300 mg/Kg/dia de NAC via oral por gavagem; grupo B3 (ototóxico): 8 mg/Kg/dia de cisplatina via intraperitoneal; grupo B4 (ototóxico com otoproteção): 300 mg/Kg/dia de NAC via oral por gavagem, uma hora antes da administração via intraperitoneal de 8 mg/Kg/dia de cisplatina. Os animais do experimento A realizaram otoscopia, emissões otoacústicas produto de distorção (EOAPD) e potencial evocado auditivo de tronco encefálico (PEATE), antes e depois da administração das drogas. Os animais do experimento B realizaram estas mesmas avaliações também no pré e pós-tratamento, além de terem suas bulas timpânicas removidas e suas cócleas preparadas para a avaliação anatômica com microscopia eletrônica de varredura e imunofluorescência para a marcação da enzima GSH-Px e da proteína Bcl-2. No experimento A, verificou-se que não houve diminuição significativa da relação sinal-ruído das EOAPD, porém houve aumento significativo do limiar eletrofisiológico obtido por PEATE nos grupos A3 e A4. No experimento B, verificou-se que: não houve aumento significativo do limiar eletrofisiológico obtido por PEATE; as CCEs mantiveram-se anatomicamente íntegras; a enzima GSH-Px apresentou imunomarcação ausente no grupo B1 e imunomarcação presente nos grupos B2, B3 e B4; a proteína Bcl-2 apresentou imunomarcação ausente em todos os grupos estudados. A partir dos resultados, concluiu-se que a função auditiva foi mais prejudicada com a exposição de uma subdose de cisplatina durante um período mais prolongado, a via de modulação da apoptose nas células ciliadas externas de ratos tratados com cisplatina está relacionada com a expressão da enzima GSH-Px e não expressão da proteína Bcl-2.