RESUMO
Introduction: Preservation of residual hearing remains a great challenge during cochlear implantation. Cochlear implant (CI) electrode array insertion induces changes in the microvasculature as well as nitric oxide (NO)-dependent vessel dysfunction which have been identified as possible mediators of residual hearing loss after cochlear implantation. Methods: A total of 24 guinea pigs were randomized to receive either a CI (n = 12) or a sham procedure (sham) by performing a cochleostomy without electrode array insertion (n = 12). The hearing threshold was determined using frequency-specific compound action potentials. To gain visual access to the stria vascularis, a microscopic window was created in the osseous cochlear lateral wall. Cochlear blood flow (CBF) and cochlear microvascular permeability (CMP) were evaluated immediately after treatment, as well as after 1 and 2 h, respectively. Finally, cochleae were resected for subsequent immunohistochemical analysis of the iNOS expression. Results: The sham control group showed no change in mean CBF after 1 h (104.2 ± 0.7%) and 2 h (100.8 ± 3.6%) compared to baseline. In contrast, cochlear implantation resulted in a significant continuous decrease in CBF after 1 h (78.8 ± 8.1%, p < 0.001) and 2 h (60.6 ± 11.3%, p < 0.001). Additionally, the CI group exhibited a significantly increased CMP (+44.9% compared to baseline, p < 0.0001) and a significant increase in median hearing threshold (20.4 vs. 2.5 dB SPL, p = 0.0009) compared to sham after 2 h. Intriguingly, the CI group showed significantly lower iNOS-expression levels in the organ of Corti (329.5 vs. 54.33 AU, p = 0.0003), stria vascularis (596.7 vs. 48.51 AU, p < 0.0001), interdental cells (564.0 vs. 109.1 AU, p = 0.0003) and limbus fibrocytes (119.4 vs. 18.69 AU, p = 0.0286). Conclusion: Mechanical and NO-dependent microvascular dysfunction seem to play a pivotal role in residual hearing loss after CI electrode array insertion. This may be facilitated by the implantation associated decrease in iNOS expression. Therefore, stabilization of cochlear microcirculation could be a therapeutic strategy to preserve residual hearing.
RESUMO
In the vertebrate cochlea, the reticular lamina seals the organ of Corti against the endolymph filled scala media. After noise exposure, fast alterations in the endothelial nitric oxide synthase (eNOS) expression level were identified in this cochlear structure. Minor amounts of nitric oxide (NO) produced by eNOS or applied by NO donors such as S-nitroso-N-acetyl-penicillamine (SNAP) might protect this vulnerable part of the organ of Corti, on the line of gap junctions of supporting cells and cochlear microcirculation. In n=5 anesthetized guinea pigs, SNAP was intravenously applied in two concentrations. Six untreated animals served as controls. The cochleae were removed and prepared for immunoelectron microscopy using specific gold-labeled anti-eNOS antibodies. The density of the gold particles was quantified for seven cellular regions in the reticular lamina at the ultrastructural level. Following SNAP application, a significant increase in eNOS expression (+176%) was detected compared with controls (p=0.012). The increase occurred mainly in actin-rich cuticular structures and the prominent microtubules bundles. Correlation analysis revealed three clear and five moderate cellular associations for controls, whereas only one clear and one moderate after SNAP application. Thus, application of the NO donor SNAP resulted in an increase in eNOS expression in distinct regions of the reticular lamina.
Assuntos
Doadores de Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Órgão Espiral/metabolismo , Lâmina Espiral/metabolismo , Animais , Cobaias , MasculinoRESUMO
Constitutively expressed endothelial nitric oxide synthase (eNOS) is supposed to play a role in noise-induced nitric oxide (NO)-production. It is commonly known that intense noise exposure results in inducible NOS (iNOS) expression and increased NO-production, but knowledge about a contribution of the eNOS isoform is still lacking. Effects of noise exposure on eNOS immunolabeling were determined in male guinea pigs (n=24). For light microscopic analysis, 11 animals were exposed to 90 dB for 1 hr and 6 animals were used as controls. After exposure, eNOS immunostaining was performed on paraffin sections, and the staining intensities were quantified for 4 cochlear regions. For electron microscopic analysis, 2 animals were exposed for 2 hr to 90 dB and 5 animals were used as controls. The intensity of eNOS immunolabeling was found to be already comprehensively increased 1 hr after noise exposure to 90 dB. At the ultrastructural level, a clear increase in eNOS immunolabeling was found in microtubules-rich areas of cochlear cuticular structures. Hence, our findings indicate that the reticular lamina forming the endolymph-perilymph barrier at the apical side of the organ of Corti is involved in a fast intrinsic otoprotective mechanism of the cochlea.
Assuntos
Cóclea/metabolismo , Cobaias/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ruído/efeitos adversos , Animais , Cóclea/ultraestrutura , Perda Auditiva Provocada por Ruído/metabolismo , Imuno-Histoquímica , Masculino , Óxido Nítrico Sintase Tipo III/análiseRESUMO
OBJECTIVES/HYPOTHESIS: Dexamethasone is widely used in the treatment of various inner ear diseases. However, knowledge about its direct impact on glucocorticoid receptor (GR) expression is still limited. STUDY DESIGN: Prospective animal study in male guinea pigs. METHODS: A therapeutic concentration of dexamethasone (8 mg/mL) or a physiological concentration of NaCl (0.9% solution) were intratympanically injected into the ears of guinea pigs (n = 10 in each case) 14 hours prior to 90 dB noise exposure (1 hour). Eighteen ears were exposed to noise only. Seven untreated ears were used as controls. Auditory brainstem responses were recorded prior to noise exposure or treatment and 2 hours thereafter. The cochleae were removed from the bullae, transferred to fixative, and embedded in paraffin. GR expression was identified immunohistochemically in the cochlea. Local staining intensities were quantified for seven regions by a computer. RESULTS: Dexamethasone application significantly lowered noise-induced hearing loss. Statistically significant alterations in the average GR expression levels were identified exclusively in the spiral ligament. Comparing GR expression at the level of individual ear, numerous highly significant local associations were identified in the other six cochlear regions. CONCLUSIONS: The intratympanic application of dexamethasone is suitable for supporting cochlear homeostasis under stress conditions. The lateral wall, mainly responsible for potassium recycling, seems to be the main target in glucocorticoid therapy.
Assuntos
Cóclea/metabolismo , Dexametasona/administração & dosagem , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Receptores de Glucocorticoides/biossíntese , Animais , Cóclea/efeitos dos fármacos , Modelos Animais de Doenças , Glucocorticoides/administração & dosagem , Cobaias , Perda Auditiva Provocada por Ruído/metabolismo , Perda Auditiva Provocada por Ruído/fisiopatologia , Injeção Intratimpânica , Masculino , Estudos Prospectivos , Receptores de Glucocorticoides/efeitos dos fármacosRESUMO
The potential toxicity of nanoparticles has currently provoked public and scientific discussions, and attempts to develop generally accepted handling procedures for nanoparticles are under way. The investigation of the impact of nanoparticles on human health is overdue and reliable test systems accounting for the special properties of nanomaterials must be developed. Nanoparticular zinc oxide (ZnO) may be internalised through ambient air or the topical application of cosmetics, only to name a few, with unpredictable health effects. Therefore, we analysed the determinants of ZnO nanoparticle (NP) genotoxicity. ZnO NPs (15-18 nm in diameter) were investigated at concentrations of 0.1, 10 and 100 µg mL(-1) using the cell line A549. Internalised NPs were only infrequently detectable by TEM, but strongly increased Zn(2+) levels in the cytoplasm and even more in the nuclear fraction, as measured by atom absorption spectroscopy, indicative of an internalised zinc and nuclear accumulation. We observed a time and dosage dependent reduction of cellular viability after ZnO NP exposure. ZnCl2 exposure to cells induced similar impairments of cellular viability. Complexation of Zn(2+) with diethylene triamine pentaacetic acid (DTPA) resulted in the loss of toxicity of NPs, indicating the relevant role of Zn(2+) for ZnO NP toxicity. Foci analyses showed the induction of DNA double strand breaks (DSBs) by ZnO NPs and increased intracellular reactive oxygen species (ROS) levels. Treatment of the cells with the ROS scavenger N-acetyl-l-cysteine (NAC) resulted in strongly decreased intracellular ROS levels and reduced DNA damage. However, a slow increase of ROS after ZnO NP exposure and reduced but not quashed DSBs after NAC-treatment suggest that Zn(2+) may exert genotoxic activities without the necessity of preceding ROS-induction. Our data indicate that ZnO NP toxicity is a result of cellular Zn(2+) intake. Subsequently increased ROS-levels cause DNA damage. However, we found evidence for the assumption that DNA-DSBs could be caused by Zn(2+) without the involvement of ROS.
Assuntos
Nanopartículas Metálicas/química , Óxido de Zinco/química , Acetilcisteína/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cloretos/química , Cloretos/toxicidade , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Humanos , Nanopartículas Metálicas/toxicidade , Microscopia Eletrônica de Transmissão , Espécies Reativas de Oxigênio/metabolismo , Compostos de Zinco/química , Compostos de Zinco/toxicidadeRESUMO
Intratympanic gentamicin therapy has become a popular treatment modality for Ménière's disease (MD) through controlled elimination of vertigo spells caused by the balance organ. However, the known ototoxic properties of aminoglycosides lead to cochlear damage. In order to gain more information about cellular preferences for aminoglycoside accumulation within the cochlea, gentamicin was immuno histochemically localized by light microscopy in male guinea pigs 1 and 7 days after intratympanic application (n = 8 ears/incubation time). Differences in the gentamicin-specific cellular storage capacities were quantified by determination of the local immuno staining intensities. Gentamicin was detected in every cochlear cell type, but with spatiotemporal variability. One day after application, an intense staining reaction was found in all cell types except the spiral ganglion cells and the stria vascularis. Six days later, gentamicin staining intensities were additionally reduced in the nerve fibers and the spiral ligament. Statistic analysis revealed strong cellular associations in respect to aminoglycoside accumulation. Furthermore, associations with recorded hearing losses were identified comparing the cellular gentamicin content in the organ of Corti, in the stria vascularis, in the spiral ganglion cells and in fibrocytes of the Limbus. In the lateral wall, clear differences in cellular gentamicin accumulation were found between type I fibrocytes of the spiral ligament compared with basal and intermediate cells of the stria vascularis. This finding was unexpected as these three cell types belong to a well-developed gap-junction system which normally enables unhampered cell communication. Cellular differences in local gentamicin storage capacities, transport processes and inherent diffusion barriers are discussed.
Assuntos
Cóclea/metabolismo , Gentamicinas/farmacocinética , Animais , Transporte Biológico Ativo , Cóclea/citologia , Cóclea/efeitos dos fármacos , Junções Comunicantes/metabolismo , Gentamicinas/administração & dosagem , Gentamicinas/toxicidade , Cobaias , Humanos , Imuno-Histoquímica , Injeção Intratimpânica , Masculino , Doença de Meniere/tratamento farmacológico , Modelos Animais , Distribuição TecidualRESUMO
Survivin (BIRC5) is an acknowledged cancer therapy-resistance factor and overexpressed in head and neck squamous cell carcinomas (HNSCC). Driven by its nuclear export signal (NES), Survivin shuttles between the nucleus and the cytoplasm, and is detectable in both cellular compartments in tumor biopsies. Although predominantly nuclear Survivin is considered a favorable prognostic disease marker for HNSCC patients, the underlying molecular mechanisms are not resolved. Hence, we performed immunohistochemical and mutational analyses using laser capture microdissection on HNSCC biopsies from patients displaying high levels of nuclear Survivin. We found somatic BIRC5 mutations, c.278T>C (p.Phe93Ser), c.292C>T (p.Leu98Phe), and c.288A>G (silent), in tumor cells, but not in corresponding normal tissues. Comprehensive functional characterization of the Survivin mutants by ectopic expression and microinjection experiments revealed that p.Phe93Ser, but not p.Leu98Phe inactivated Survivin's NES, resulted in a predominantly nuclear protein, and attenuated Survivin's dual cytoprotective activity against chemoradiation-induced apoptosis. Notably, in xenotransplantation studies, HNSCC cells containing the p.Phe93Ser mutation responded significantly better to cisplatin-based chemotherapy. Collectively, our results underline the disease relevance of Survivin's nucleocytoplasmic transport, and provide first evidence that genetic inactivation of Survivin's NES may account for predominantly nuclear Survivin and increased therapy response in cancer patients.
Assuntos
Carcinoma de Células Escamosas/diagnóstico , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteínas Inibidoras de Apoptose/genética , Transporte Ativo do Núcleo Celular , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cisplatino/uso terapêutico , Citoplasma/genética , Citoplasma/metabolismo , Modelos Animais de Doenças , Ácidos Graxos Insaturados/farmacologia , Feminino , Humanos , Imuno-Histoquímica , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Mutação , Sinais de Exportação Nuclear/genética , Prognóstico , SurvivinaRESUMO
AIMS: To quantify the procedure-related complication rate after using modified technique of amniocentesis with a 29-gauge (29-G) pencil-point needle. METHODS: This is a prospective, descriptive study of 316 amniocenteses that were performed by means of atraumatic 29-G pencil-point needle under ultrasound control. RESULTS: A total of 316 amniocenteses were observed through the postprocedural period. The median time needed to retrieve 15 mL of amniotic fluid was 4 min. A total of 19 pregnancies were terminated after genetic testing. No case was regarded as procedure-related fetal loss. No other complications were observed. Seventeen children were born before 37 completed weeks of gestation and five children had a birth weight <2000 g. CONCLUSIONS: Amniocentesis with the 29-G atraumatic pencil-point needle seems to be a safe procedure with extremely low risk of complications and is a good alternative to the traditional 22-G Quincke needle.
Assuntos
Amniocentese/efeitos adversos , Amniocentese/instrumentação , Agulhas , Adulto , Amniocentese/métodos , Aberrações Cromossômicas , Feminino , Morte Fetal/etiologia , Idade Gestacional , Humanos , Recém-Nascido , Cariotipagem , Gravidez , Resultado da Gravidez , Gravidez de Gêmeos , Estudos Prospectivos , Ultrassonografia Pré-NatalRESUMO
Nitric oxide (NO) is a signaling molecule which can generally be formed by three nitric oxide synthases (NOS). Two of them, the endothelial nitric oxide synthase (eNOS) and the neural nitric oxide synthase (nNOS), are calcium/calmodulin-dependent and constitutively expressed in many cell types. Both isoforms are found in the vertebrate cochlea. The inducible nitric oxide synthase (iNOS) is independent of calcium and normally not detectable in the un-stimulated cochlea. In the inner ear, as in other tissues, NO was identified as a multitask molecule involved in various processes such as neurotransmission and neuromodulation. In addition, increasing evidence demonstrates that the NO-dependent processes of cell protection or, alternatively, cell destruction seem to depend, among other things, on changes in the local cochlear NO-concentration. These alterations can occur at the cellular level or within a distinct cell population both leading to an NO-imbalance within the hearing organ. This dysfunction can result in hearing loss or even in deafness. In cases of cochlear malfunction, regulatory systems such as the gap junction system, the blood vessels or the synaptic region might be affected temporarily or permanently by an altered NO-level. This review discusses potential cellular mechanisms how NO might contribute to different forms of hearing disorders. Approaches of NO-reduction are evaluated and the transfer of results obtained from experimental animal models to human medication is discussed.
Assuntos
Cóclea/metabolismo , Transtornos da Audição/metabolismo , Óxido Nítrico/metabolismo , Animais , Antioxidantes/metabolismo , Ácido Ascórbico/farmacologia , Cóclea/efeitos dos fármacos , Junções Comunicantes/metabolismo , Gentamicinas/efeitos adversos , Humanos , CamundongosRESUMO
Novel strategies of cancer therapy combine irradiation and anti-angiogenic active compounds. However, little is known concerning the undesired cellular and molecular effects caused by this novel treatment concept. We used a mouse squamous cell carcinoma (SCC) xenotransplantation model to evaluate the potential undesired effects which compromise the success of this therapeutic combination. SCCs were subcutanously implanted in nude mice. Animals were treated with a fractionated irradiation scheme (5x4 Gy) alone or in combination with daily injections of anti-vascular endothelial growth factor (VEGF) antibodies. Controls remained untreated. Before and after treatment, resonance imaging (MRI), ultrasound and near-infrared spectrometry were used to evaluate tumor vessel integrity. Finally, tumors were explanted and VEGF, basic fibroblast growth factor (bFGF), vessel density, proliferation and apoptotic activity were analyzed by immunohistochemistry. Irradiation caused VEGF release and we found evidence for VEGF-mediated vessel protection. In the tumors derived from the combined treatment, blood volume was decreased, and apoptotic indices were increased. Remarkably, bFGF levels and proliferative indices were also increased. Combined irradiation/anti-VEGF treatment resulted in the desired VEGF depletion and increased tumor cell apoptosis. Nonetheless, bFGF and proliferation also increased, possibly suggesting a compensatory response. The application of additional targeted drugs may help develop more effective SCC treatments.
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Carcinoma de Células Escamosas/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Hemodinâmica , Humanos , Camundongos , Camundongos Nus , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature of primary cancer cells. However, to date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined. In the present study, we analyzed the promoter methylation status of the genes mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of the three proteins was quantified by semi-quantitative immunohistochemical analysis. The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing. Proliferation was determined by Alamar blue staining. We found that the MGMT promoter in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated. The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples while in the examined cell line the MLH promoter was unmethylated. The RASSF1A promoter showed methylation in 13% of the tumor samples and in the cell line. MGMT expression in the group of tumor samples with a hypermethylated promoter was statistically significantly lower compared to the group of tumors with no measured hypermethylation of the MGMT promoter. After treatment of the cell line with the demethylating agent 5-Aza no demethylation of the methylated MGMT and RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results, however, increased numbers of unmethylated CpG islands in the promoter region of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05) reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this epigenetic inactivation of TSGs may play a role in the development of HNSCC. 5-Aza application resulted in partial demethylation of the MGMT and RASSF1A TSGs and reduced proliferation of the tumor cells suggesting further evaluation of 5-Aza for HNSCC treatment.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Azacitidina/farmacologia , Carcinoma de Células Escamosas/genética , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Inibidores Enzimáticos/farmacologia , Genes Supressores de Tumor , Neoplasias de Cabeça e Pescoço/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Ilhas de CpG , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Remoção de Radical Alquila , Relação Dose-Resposta a Droga , Regulação para Baixo , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismoRESUMO
OBJECTIVE: To compare perforation characteristics of standard 22 G (0.7 mm) to 29 G needle (0.34 mm) for amniocentesis. METHODS: Seventeen human chorio-amnion membranes were perforated immediately after cesarean section using 22 G needle for spinal anesthesia and 29 G "pencil-point" needles for amniocentesis under in-vitro conditions. Area of perforation was determined using a microscope and volume of fluid leakage was measured over a period of 5 min. RESULTS: Membrane perforation with the 22 G needle resulted in a mean damaged area of 225,147.4 µm(2), a hole with a mean area of 50,154 µm(2) and amniotic fluid volume passage of 17.5 mL/5 min, whereas the 29 G needle generated a mean damaged area of 114,812.4 µm(2), a hole with an average area of 1382.5 µm(2) and volume passage of 0.28 mL/5 min. These differences were significant. CONCLUSION: The hole formed by membrane perforation with 29 G "pencil-point" needle for amniocentesis is 36 times smaller, and the amniotic fluid loss is 61 times less than that measured with the 22 G standard needle for spinal anesthesia. Significant reduction of complications following amniocentesis is expected with the 29 G needle.
Assuntos
Amniocentese/instrumentação , Seringas , Amniocentese/efeitos adversos , Âmnio/lesões , Córion/lesões , Feminino , Humanos , Técnicas In Vitro , GravidezRESUMO
Malignant triton tumour (MTT) is a rare, highly malignant neoplasm, characterized by a mixture of cells with nerve sheath and skeletal muscle differentiation. Cytogenetic analyses of this neoplasm are rare to date and none comparative genomic hybridisation (CGH) analysis has been published. In the present study we report about the genomic imbalances of a MMT analysed by CGH, in a 39-year-old male patient without neurofibromatosis. We observed the amplifications at chromosomal location 1p, 6p, 16p, 16q, 17p, 17q, 19p, 19q, 20p, and 22q. Comparing our results with those of previous studies, we found evidence for recurrent genomic aberrations at the chromosomes 1, 16, 17, 19, and 22 suggesting the involvement of several oncogenes in the genesis of MTT.
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Aberrações Cromossômicas , Hibridização Genômica Comparativa/métodos , Neuroma Acústico/genética , Neuroma Acústico/patologia , Adulto , Biópsia por Agulha , Terapia Combinada , Análise Citogenética/métodos , Progressão da Doença , Evolução Fatal , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética/métodos , Masculino , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Neuroma Acústico/terapia , Procedimentos Neurocirúrgicos/métodos , Radioterapia Adjuvante , Tomografia Computadorizada por Raios XRESUMO
OBJECTIVE: Gentamicin application is an important therapeutic option to control vertigo spells in Ménière's disease. However, even in the case of low-dose intratympanic application, gentamicin might contribute to a pathological NO-increase leading to cochlear damage and hearing impairment. The study was performed to evaluate the nitric oxide (NO) reducing capacity of doxycycline in the inner ear after NO-induction by gentamicin. METHODS: In a prospective animal study, a single dose of gentamicin (10mg/kg body weight) was injected intratympanically into male guinea pigs (n=48). The auditory brainstem responses (ABRs) were recorded prior to application and 3, 5 and 7 days afterwards. The organ of Corti and the lateral wall of 42 animals were isolated after 7 days and incubated separately for 6h in cell culture medium. Doxycycline was adjusted to organ cultures of 5 animals. Two NOS inhibitors, N(G)-Nitro-l-arginine methyl ester (l-NAME) and NG-monomethyl-l-arginine monoacetate (l-NMMA), were applied in three different concentrations to the organ cultures of 30 animals in total (5 animals per concentration). As controls, seven animals received no further substance except gentamicin. The NO-production was quantified by chemiluminescence. Additional six gentamicin-treated animals were used for immunohistochemical studies. RESULTS: The ABRs declined continuously from the first to the seventh day after gentamicin application. Doxycycline reduced NO-production in the lateral wall by 54% (p=.029) comparable to the effect of the applied nitric oxide inhibitors. In the organ of Corti, NO-production was reduced by about 41% showing no statistical significance in respect to great inter-animal variations. CONCLUSION: The application of doxycycline might offer a new therapeutic approach to prevent NO-induced cochlea damage through ototoxic substances.
Assuntos
Doxiciclina/farmacologia , Orelha Interna/metabolismo , Óxido Nítrico/biossíntese , Animais , Citoproteção/efeitos dos fármacos , Orelha Interna/efeitos dos fármacos , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Gentamicinas/farmacologia , Cobaias , Imuno-Histoquímica , Luminescência , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Técnicas de Cultura de Órgãos , Órgão Espiral/metabolismo , Estudos Prospectivos , Regulação para Cima , ômega-N-Metilarginina/farmacologiaRESUMO
OBJECTIVES/HYPOTHESIS: Intratympanic application of gentamicin is an important therapeutic option to control vertigo spells in Ménière's disease. Low doses eliminate the function of semicircular canal ampullae (SCCA) and saccule in most patients, although utricular function is maintained in many cases. Local alteration in free radical production might be responsible for these differences. Therefore, the gentamicin-induced nitric oxide (NO) production was determined in an animal model using separate organ cultures of SCCA, saccule, and utricle. STUDY DESIGN: Prospective pilot study in male guinea pigs. METHODS: SCCA, saccule, and utricle of 28 guinea pigs were isolated and incubated separately for 6 hours in cell culture medium. Gentamicin was administered in two different concentrations (0.4 mg/mL and 0.8 mg/mL) to organ cultures of 16 animals. Tissues from 12 animals were used as controls. Nitric oxide was quantified by chemiluminescence. RESULTS: Gentamicin led to an NO increase of about 70% in the saccule, an NO reduction of more than 70% in SCCA, and an NO reduction of 36% in the utricle. CONCLUSIONS: The selective effects of gentamicin on the NO production in the different sensory areas of the vestibular organ have to be taken into account in the therapy of Ménière's disease.
Assuntos
Gentamicinas/farmacologia , Óxido Nítrico/metabolismo , Membrana dos Otólitos/efeitos dos fármacos , Canais Semicirculares/efeitos dos fármacos , Animais , Cobaias , Modelos Lineares , Masculino , Membrana dos Otólitos/metabolismo , Projetos Piloto , Estudos Prospectivos , Canais Semicirculares/metabolismoRESUMO
BACKGROUND: The guinea pig is widely used as a model to study (patho)physiological processes, such as neurodegenerative disorders. Survivin's dual function as an apoptosis inhibitor and a mitotic regulator is crucial not only for ordered development but its modulation seems crucial also under disease conditions. However, data on the expression and function of the guinea pig Survivin protein (Survivin(Gp)) are currently lacking. RESULTS: Here, we here report the cloning and functional characterization of Survivin(Gp). The respective cDNA was cloned from spleen mRNA, containing a 426 bp open reading frame encoding for a protein of 142aa. Survivin(Gp) displays a high homology to the human and murine orthologue, especially in domains critical for function, such as binding sites for chromosomal passenger complex (CPC) proteins and the nuclear export signal (NES). Notably, phylogenetic analyses revealed that Survivin(Gp) is more related to humans than to rodents. Ectopic expression studies of a Survivin(Gp)-GFP fusion confirmed its dynamic intracellular localization, analogous to the human and murine counterparts. In interphase cells, Survivin(Gp)-GFP was predominantly cytoplasmic and accumulated in the nucleus following export inhibition with leptomycin B (LMB). A typical CPC protein localization during mitosis was observed for Survivin(Gp)-GFP. Microinjection experiments together with genetic knockout demonstrated that the NES is essential for the anti-apoptotic and regulatory role of Survivin(Gp) during cell division. In vivo protein interaction assays further demonstrated its dimerization with human Survivin and its interaction with human CPC proteins. Importantly, RNAi-depletion studies show that Survivin(Gp) can fully substitute for human Survivin as an apoptosis inhibitor and a mitotic effector. Immunohistochemistry, immunofluorescence, and western blotting were employed to detect Survivin expression in guinea pig tissues. Besides its expression in proliferating tissues, such as spleen and liver, we also found Survivin in terminally differentiated cell types. Importantly, Survivin was detectable also in the cochlea, suggesting a potential role for Survivin in the auditory system. CONCLUSIONS: We provide the first experimental evidence for the expression of Survivin in the guinea pig. As Survivin(Gp) can substitute for known functions of human Survivin, the guinea pig model will now also allow investigating Survivin's (patho)physiological role and to test Survivin-directed potential therapeutic strategies.
Assuntos
Cobaias/genética , Proteínas Inibidoras de Apoptose/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Orelha Interna/metabolismo , Proteínas Associadas aos Microtúbulos , Mitose , Modelos Animais , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Survivina , TransfecçãoRESUMO
Hearing impairment is a worldwide health problem. Employing semi-quantitative immunological detection methods, we found that the apoptosis inhibitor protein Birc5 is expressed in cell types critical for hearing perception. In the guinea pig model, moderate noise exposure causing only a temporary mean hearing impairment of 33dB significantly enhanced Birc5 expression in the spiral ligament, nerve fibers and the organ of Corti. In contrast, intratympanic gentamicin injection inducing permanent cell damage and mean hearing loss of 24dB correlated with a significant Birc5 downregulation in the ligament, nerve fibers and the organ of Corti. The cytoprotective activity of the guinea pig and human Birc5 protein was confirmed by cloning of the gene and by subsequent ectopic expression and challenging studies against the ototoxin gentamicin in epithelial and auditory cell models. As the mammalian cochlea is unable to regenerate upon damage, these data suggest that modulation of Birc5 expression may represent a novel physiological mechanism to protect the inner ear against stress-induced cell damage. Hence, the targeted modulation of Birc5 levels may lead to novel otoprotective therapeutic strategies.
Assuntos
Citoproteção , Orelha Interna/fisiologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Células Cultivadas , Orelha Interna/anatomia & histologia , Feminino , Gentamicinas/toxicidade , Cobaias , Células HeLa , Perda Auditiva/induzido quimicamente , Humanos , Proteínas Inibidoras de Apoptose , Masculino , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Ruído/efeitos adversos , Inibidores da Síntese de Proteínas/toxicidade , SurvivinaRESUMO
CONCLUSION: Changes in the metabolism of arachidonic acid (AA) might be part of a noise-induced compensatory mechanism with regional specificity. OBJECTIVES: The released imbalance of prostaglandins and leukotrienes, both AA metabolites, might result in altered blood flow regulation in the inner ear and probably contributes to noise-induced hearing loss. The aim of this study was to gain further information about noise-dependent changes in AA metabolism in the mammalian cochlea. METHODS: In this prospective animal study, 10 male guinea pigs were exposed to tone bursts for 1 h at 70 dB sound pressure level (SPL) (n = 5) or 90 dB SPL (n = 5). Five animals were used as controls. Alterations in cyclooxygenase 1 (COX-1) and 5-lipoxygenase (5-LO) expression were determined by quantitative immunohistochemical analysis in 11 cochlear regions. RESULTS: COX-1 expression was decreased after both 70 dB SPL and 90 dB SPL exposure in most cell types of the organ of Corti and increased in the nerve fibers of the osseous spiral lamina. 5-LO was lowered after 90 dB SPL exposure, preferentially in the third cochlear turn in the organ of Corti, in the first and second turn in spiral ganglion cells, and in all turns in the stria vascularis.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Cóclea/patologia , Ciclo-Oxigenase 1/metabolismo , Perda Auditiva Provocada por Ruído/patologia , Animais , Ácido Araquidônico/metabolismo , Regulação para Baixo/fisiologia , Cobaias , Humanos , Técnicas Imunoenzimáticas , Masculino , Fibras Nervosas/patologia , Órgão Espiral/patologia , Estudos Prospectivos , Células de Schwann/patologia , Gânglio Espiral da Cóclea/patologia , Lâmina Espiral/patologia , Ligamento Espiral da Cóclea/patologia , Estria Vascular/patologia , Regulação para Cima/fisiologiaRESUMO
OBJECTIVES/HYPOTHESIS: Gentamicin application is an important therapeutic option for Ménière's disease. However, even if given at intervals, a destruction of the cochlea was often observed in various animal models together with an increased content of nitric oxide (NO) and reactive oxygen species. The present study was undertaken to identify the correlation between hearing threshold alteration and the NO production in the lateral wall and organ of Corti of the guinea pig in response to gentamicin application. STUDY DESIGN: Prospective animal study in guinea pigs. METHODS: A single dose of gentamicin (10 mg/kg body weight) was injected intratympanally into male guinea pigs and the auditory brainstem responses were recorded before treatment and 1, 2, and 7 days after application. The organ of Corti and the lateral wall were removed from the bulla, incubated separately for 6 hours in cell culture medium and the amount of NO production was determined by chemiluminescence. RESULTS: Gentamicin application resulted in a hearing threshold shift beginning on the second day after gentamicin application. This hearing impairment correlates simultaneously with an increased NO2(-) content--the stable oxidation product of NO--in the lateral wall. In the organ of Corti, a slight increase in NO2(-) production was seen as early as on day 1 after gentamicin injection. CONCLUSIONS: The correlation between hearing threshold shift and NO production in the cochlea leads to the assumption that increased NO contributes to gentamicin-induced hearing impairment.
Assuntos
Cóclea/metabolismo , Gentamicinas/toxicidade , Perda Auditiva/induzido quimicamente , Perda Auditiva/metabolismo , Óxido Nítrico/biossíntese , Animais , Potenciais Evocados Auditivos do Tronco Encefálico , Cobaias , Masculino , Órgão Espiral/metabolismo , Estudos ProspectivosRESUMO
OBJECTIVES: Noise-induced hearing loss can be caused, among other causes, by increased nitric oxide (NO) production in the inner ear leading to nitroactive stress and cell destruction. Some studies in the literature suggest that the degree of hearing loss (HL) could be reduced in an animal model through ascorbic acid supplementation. To identify the effect of ascorbic acid on tissue-dependent NO content in the inner ear of the guinea pig, we determined the local NO production in the organ of Corti and the lateral wall separately 6 hours after noise exposure. STUDY DESIGN: Prospective animal study in guinea pigs. METHODS: Over a period of 7 days, male guinea pigs were supplied with minimum (25 mg/kg body weight/day) and maximum (525 mg/kg body weight/day) ascorbic acid doses, and afterwards exposed to noise (90 dB sound pressure level for 1 hour). The acoustic-evoked potentials were recorded before and after noise exposure. The organ of Corti and the lateral wall were incubated differently for 6 hours in culture medium, and the degree of NO production was determined by chemiluminescence. RESULTS: Ascorbic acid treatment reduced the hearing threshold shift after noise exposure depending on concentration. When the maximum ascorbic acid dose was substituted, NO production was significantly reduced in the lateral wall after noise exposure and slightly reduced in the organ of Corti. CONCLUSIONS: Oral supplementation of the natural radical scavenger ascorbic acid reduces the NO-production rate in the inner ear in noisy conditions. This finding supports the concept of inner ear protection by ascorbic acid supplementation.