RESUMEN

Cochlear fibrosis is a common finding following cochlear implantation. Evidence suggests that cochlear fibrosis could be triggered by inflammation and epithelial-to-mesenchymal cell transition (EMT). In this study, we investigate the mechanisms of cochlear fibrosis and the risk/benefit ratio of local administration of the anti-inflammatory drug dexamethasone (DEX) and antimitotic drug aracytine (Ara-C). Cochlear fibrosis was evaluated in cochlear fibrosis models of rat cochlear slices in vitro and in KLH-induced immune labyrinthitis and platinum wire cochlear implantation-induced fibrosis in vivo. Cochleae were invaded with tissue containing fibroblastic cells expressing α-SMA (alpha smooth muscle actin), which along with collagen I, fibronectin, and laminin in the extracellular matrix, suggests the involvement of a fibrotic process triggered by EMT in vitro and in vivo. After perilymphatic injection of an adenoviral vector expressing GFP in vivo, we demonstrated that the fibroblastic cells derived from the mesothelial cells of the scalae tympani and vestibuli. Activation of inflammatory and EMT pathways was further assessed by ELISA analysis of the expression of IL-1ß and TGF-ß1. Both markers were elevated in vitro and in vivo, and DEX and Ara-C were able to reduce IL-1ß and TGF-ß1 production. After 5days of culture in vitro, quantification of calcein-positive cells revealed that Ara-C was 30-fold more efficient in preventing fibrosis, and provoked less sensory hair cell loss, than DEX. In KLH-induced immune labyrinthitis and platinum wire-implanted models, Ara-C was more efficient in preventing proliferation of fibrosis with less side effects on hair cells and neurons than DEX. In conclusion, DEX and Ara-C both prevent fibrosis in the cochlea. Analysis of the risk/benefit ratio favors the use of Ara-C for preventing cochlear fibrosis.

Asunto(s)

Antiinflamatorios/farmacología , Cóclea , Citocinas/metabolismo , Heridas y Lesiones/complicaciones , Adyuvantes Inmunológicos/toxicidad , Animales , Cóclea/efectos de los fármacos , Cóclea/lesiones , Cóclea/patología , Cóclea/ultraestructura , Colágeno/metabolismo , Dexametasona/farmacología , Modelos Animales de Enfermedad , Electrodos Implantados/efectos adversos , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Fibronectinas/metabolismo , Fibrosis/tratamiento farmacológico , Fibrosis/etiología , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas/patología , Hemocianinas/toxicidad , Técnicas In Vitro , Laminina/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Células Receptoras Sensoriales/efectos de los fármacos , Factores de Tiempo

RESUMEN

BACKGROUND AND PURPOSE Exposure to an ototoxic level of an aminoglycoside can result in hearing loss. In this we study investigated the otoprotective efficacy of dexamethasone (DXM), melatonin (MLT) and tacrolimus (TCR) in gentamicin (GM)-treated animals and cultures. EXPERIMENTAL APPROACH Wistar rats were divided into controls (treated with saline); exposed to GM only (GM); and three GM-exposed groups treated with either DXM, MLT or TCR. Auditory function and cochlear surface preparations were studied. In vitro studies of oxidative stress, pro-inflammatory cytokine mRNA levels, the MAPK pathway and caspase-3 activation were performed in organ of Corti explants from 3-day-old rats. KEY RESULTS DXM, MLT and TCR decreased levels of reactive oxygen species in GM-exposed explants. The mRNA levels of TNF-α, IL-1ß and TNF-receptor type 1 were significantly reduced in GM + DXM and GM + MLT groups. Phospho-p38 MAPK levels decreased in GM + MLT and GM + TCR groups, while JNK phosphorylation was reduced in GM + DXM and GM + MLT groups. Caspase-3 activation decreased in GM + DXM, GM + MLT and GM + TCR groups. These results were consistent with in vivo results. Local treatment of GM-exposed rat cochleae with either DXM, MLT or TCR preserved auditory function and prevented auditory hair cell loss. CONCLUSIONS AND IMPLICATIONS In organ of Corti explants, GM increased oxidative stress and initiated an inflammatory response that led to the activation of MAPKs and apoptosis of hair cells. The three compounds tested demonstrated otoprotective properties that could be beneficial in the treatment of ototoxicity-induced hearing loss.

Asunto(s)

Antibacterianos/efectos adversos , Dexametasona/uso terapéutico , Gentamicinas/efectos adversos , Pérdida Auditiva/tratamiento farmacológico , Melatonina/uso terapéutico , Sustancias Protectoras/uso terapéutico , Tacrolimus/uso terapéutico , Animales , Catalasa/metabolismo , Dexametasona/farmacología , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/metabolismo , Interleucina-1beta/genética , Masculino , Melatonina/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Órgano Espiral/metabolismo , Sustancias Protectoras/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Tipo I de Interleucina-1/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Superóxido Dismutasa/metabolismo , Tacrolimus/farmacología , Factor de Necrosis Tumoral alfa/genética

Asunto(s)

Cóclea , Dexametasona/administración & dosificación , Sistemas de Liberación de Medicamentos , Potenciales Evocados Auditivos/efectos de los fármacos , Glucocorticoides/administración & dosificación , Pérdida Auditiva/prevención & control , Animales , Modelos Animales de Enfermedad , Cobayas , Células Ciliadas Auditivas/efectos de los fármacos , Pérdida Auditiva/etiología , Pérdida Auditiva/patología

RESUMEN

The objective was to determine the role of nuclear factor kappa B (NFκB) in dexamethasone base (DXMb) protection of auditory hair cells from tumor necrosis factor-alpha (TNFα)-induced loss on gene expression and cell signaling levels. Organ of Corti (OC) explants from 3-day-old rats were cultured under one of the following conditions: (1) media only--no treatment; (2) media+TNFα; (3) media+TNFα+DXMb; (4) media+TNFα+DXMb+NFκB-Inhibitor (NFκB-I); or (5) media+TNFα+DXMb+NFκBI-Scrambled control (NFκBI-C). A total of 60 organ of Corti explants (OC) were stained with FITC-Phalloidin after 96 h in culture (conditions 1-5) for hair cell counts and imaging of surface characteristics. A total of 108 OC were used for gene expression studies (i.e. B-actin, Bax, Bcl-2, Bcl-xl, and TNFR1) after 0, 24, or 48 h in vitro (conditions 1-4). A total of 86 OC were cultured (conditions 1-3) for 48 h, 36 of which were used for phosphorylated NFκB (p-NFκB) ELISA studies and 50 for whole mount anti-p-NFκB immunostain experiments. TNFα+DXMb exposed cultures demonstrated significant upregulation in anti-apoptotic Bcl-2 and Bcl-xl genes and downregulation in pro-apoptotic Bax gene expression; DXMb treatment of TNFα explants also lowered the Bax/Bcl-2 ratio and inhibited TNFR1 upregulation. After inhibiting NFκB activity with NFκB-I, the gene expression profile following TNFα+DXMb treatment now mimics that of TNFα-challenged OC explants. The levels of p-NFκB and the degree of nuclear translocation are significantly greater in TNFα+DXMb exposed OC explants than observed in the TNFα and control groups in the middle+basal turns of OC explants. These findings were supported by the results of the hair cell counts and the imaging results obtained from the whole mount OC specimens. DXMb protects against TNFα-induced apoptosis of auditory hair cells in vitro via activation of NFκB signaling in hair cell nuclei, and regulation of the expression levels of anti- and pro-apoptotic genes and a pro-inflammatory gene.

Asunto(s)

Antiinflamatorios/farmacología , Dexametasona/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , FN-kappa B/metabolismo , Órgano Espiral/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Expresión Génica/efectos de los fármacos , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patología , Técnicas de Cultivo de Órganos , Órgano Espiral/metabolismo , Órgano Espiral/patología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/toxicidad

RESUMEN

OBJECTIVE: Determine the molecular mechanism(s) behind tumor necrosis factor-alpha (TNFalpha)-induced loss of auditory hair cells and the ability of dexamethasone base (DXMb) to protect against TNFalpha ototoxicity. METHODS: Hair cell counts: Three-day-old rat organ of Corti explants were cultured under three different conditions: 1) untreated-control; 2) TNFalpha (2 mug/ml); and 3) TNFalpha (2 mug/ml)+DXMb (70 mug/ml) for 4 days, fixed, and stained with FITC-phalloidin. Hair cells were counted in the basal and middle turns. Gene expression: total RNA was extracted from the three different groups of explants at 0, 12, 24 and 48 h. Using quantitative real-time RT-PCR, mRNAs were transcribed into cDNAs and amplification was performed using primers for rat ss-actin (housekeeping gene), TNFR1, Bcl-2, Bax, and Bcl-xl. RESULTS: DXMb protected explant hair cells from TNFalpha-induced loss. Bax gene expression was greater in TNFalpha-exposed explants compared with TNFalpha+DXMb-treated explants at 48 h (P=0.023), confirmed by the increase in the Bax/Bcl-2 ratio at 48 h (P<0.001). These results correlated with increased TNFR1 expression at 24 h (P=0.038). DXMb otoprotection in TNFalpha-exposed cultures was accompanied by an up-regulation of Bcl-xl at both the 24 (P<0.001) and 48 h time points (P=0.030) and up-regulation of Bcl-2 expression at 24 h (P=0.018). DXMb treatment also prevented increases in the expression levels of Bax, TNFR1, and the Bax/Bcl-2 ratio that occurred in untreated TNFalpha-exposed explants. CONCLUSIONS: TNFalpha's ototoxicity may be mediated through an up-regulation of Bax and TNFR1 expression as well as an increase in the Bax/Bcl-2 ratio. DXMb protects the organ of Corti against TNFalpha ototoxicity by up-regulating Bcl-2 and Bcl-xl expression and by inhibiting TNFalpha-induced increases in Bax, TNFR1, and the Bax/Bcl-2 ratio. These results support the use of local dexamethasone treatment to conserve hearing following a trauma.

Asunto(s)

Apoptosis/efectos de los fármacos , Dexametasona/farmacología , Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Órgano Espiral/citología , Análisis de Varianza , Animales , Animales Recién Nacidos , Apoptosis/genética , Técnicas de Cultivo de Órganos , Órgano Espiral/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/toxicidad , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo

RESUMEN

The c-Jun N-terminal kinases (JNKs) are also called stress activated protein kinases (SAPKs) and are members of the family of mitogen activated protein kinases (MAPKs). While the functions of the JNKs under physiological conditions are diverse and not completely understood, there is increasing evidence that JNKs are potent effectors of apoptosis of oxidative stress-damaged cells in both the brain and the mammalian inner ear following a trauma. The activation of the inducible transcription factor c-Jun by N-terminal phosphorylation is a central event in JNK-mediated apoptosis of oxidative stress-damaged auditory hair cells following exposure to either acoustic trauma or a toxic level of an aminoglycoside antibiotic and also the apoptosis of auditory neurons as a consequence of a loss of the trophic support provided by the auditory hair cells. In this review, we summarise what is known about the expression and activation of G-proteins, JNKs, c-Jun and c-Fos under oxidative stress conditions within the mammalian cochlea. A particular focus is put on a new peptide conjugate that is a promising protective agent(s) and pharmacological strategies for preventing cochlear damage induced by both acoustic trauma and aminoglycoside ototoxic damage.

Asunto(s)

Apoptosis/fisiología , Sordera/enzimología , Sistema de Señalización de MAP Quinasas/fisiología , Degeneración Nerviosa/enzimología , Estrés Oxidativo/fisiología , Péptidos/farmacología , Aminoglicósidos/antagonistas & inhibidores , Aminoglicósidos/toxicidad , Animales , Apoptosis/efectos de los fármacos , Sordera/tratamiento farmacológico , Sordera/fisiopatología , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas/enzimología , Células Ciliadas Auditivas/patología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/fisiopatología , Ruido/efectos adversos , Estrés Oxidativo/efectos de los fármacos , Péptidos/uso terapéutico

RESUMEN

This review covers the general roles of members of the cysteine protease family of caspases in the process of apoptosis (programmed cell death) looking at their participation in both the "extrinsic" cell death receptor and the "intrinsic" mitochondrial cell death pathways. It defines the difference between initiator and effector caspases and shows the progression of caspase activations that ends up in the apoptotic cell death and elimination of a damaged cell. The review then presents what is currently know about the participation of caspases in the programmed cell death of inner ear sensory cells during the process of normal development and maturation of the inner ear and their importance in this process as illustrated by the results of caspase-3 gene knockout experiments. The participation of specific caspases and the sequence of their activation in the elimination (apoptosis) of damaged sensory cells from adult inner ears after an injury that generates oxidative stress are reviewed. Both the possibility and the potential efficacy of caspase inhibition with a broad-spectrum pancaspase inhibitor as an interventional therapy to treat and rescue oxidative stress-damaged inner ear sensory cells from apoptosis are presented and discussed.

Asunto(s)

Apoptosis/fisiología , Caspasas/fisiología , Células Ciliadas Auditivas/patología , Estrés Oxidativo/fisiología , Adulto , Envejecimiento/patología , Envejecimiento/fisiología , Animales , Inhibidores de Caspasas , Caspasas/genética , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Gerbillinae , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva Provocada por Ruido/patología , Humanos , Ratones , Ratones Noqueados , Mitocondrias/fisiología

RESUMEN

Hearing loss can be caused by a variety of insults, including acoustic trauma and exposure to ototoxins, that principally effect the viability of sensory hair cells via the MAP kinase (MAPK) cell death signaling pathway that incorporates c-Jun N-terminal kinase (JNK). We evaluated the otoprotective efficacy of D-JNKI-1, a cell permeable peptide that blocks the MAPK-JNK signal pathway. The experimental studies included organ cultures of neonatal mouse cochlea exposed to an ototoxic drug and cochleae of adult guinea pigs that were exposed to either an ototoxic drug or acoustic trauma. Results obtained from the organ of Corti explants demonstrated that the MAPK-JNK signal pathway is associated with injury and that blocking of this signal pathway prevented apoptosis in areas of aminoglycoside damage. Treatment of the neomycin-exposed organ of Corti explants with D-JNKI-1 completely prevented hair cell death initiated by this ototoxin. Results from in vivo studies showed that direct application of D-JNKI-1 into the scala tympani of the guinea pig cochlea prevented nearly all hair cell death and permanent hearing loss induced by neomycin ototoxicity. Local delivery of D-JNKI-1 also prevented acoustic trauma-induced permanent hearing loss in a dose-dependent manner. These results indicate that the MAPK-JNK signal pathway is involved in both ototoxicity and acoustic trauma-induced hair cell loss and permanent hearing loss. Blocking this signal pathway with D-JNKI-1 is of potential therapeutic value for long-term protection of both the morphological integrity and physiological function of the organ of Corti during times of oxidative stress.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Pérdida Auditiva Provocada por Ruido/prevención & control , Pérdida Auditiva/prevención & control , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Órgano Espiral/efectos de los fármacos , Péptidos/farmacología , Estimulación Acústica , Aminoglicósidos/antagonistas & inhibidores , Aminoglicósidos/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Evaluación Preclínica de Medicamentos , Cobayas , Células Ciliadas Auditivas/citología , Pérdida Auditiva/inducido químicamente , Pruebas Auditivas , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos , Ligandos , Ratones , Fármacos Neuroprotectores/farmacología , Órgano Espiral/citología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

In the current study, we have investigated the ability of substance P (SP) to protect 3-day-old (P3) rat spiral ganglion neurons (SGNs) from trophic factor deprivation (TFD)-induced cell death. The presence of SP high affinity neurokinin-1 receptor (NK1) transcripts was detected in the spiral ganglion and the NK1 protein localized to SGNs both ex vivo and in vitro. Treatment with SP increased cytoplasmic Ca2+ in SGNs, further arguing for the presence of functional NK1 on these neurons. Both SP and the agonist [Sar9,Met(O2)11]-SP significantly decreased SGN cell death induced by TFD, with no effect on neurite outgrowth. The survival promoting effect of SP was blocked by the NK1 antagonist, WIN51708. Both pan-caspase inhibitor BOC-D-FMK and SP treatments markedly reduced activation of caspases and DNA fragmentation in trophic factor deprived-neurons. The neuroprotective action of SP was antagonised by specific inhibitors of second messengers, including 1.2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, the protein kinase C (PKC) inhibitors bisindolylmaleimide I, Gö6976 and LY333531 and the MAPK/ERK inhibitor U0126. In contrast, nifedipine, a specific inhibitor of l-type Ca2+ channel, and LY294002, a phosphatidylinositol-3-OH kinase (PI3K) inhibitor, had no effect on SP trophic support of SGNs. Moreover, activation of endogenous PKC by 4 beta-phorbol 12-myristate 13-acetate (PMA) also reduced the loss of trophic factor-deprived SGNs. Thus, NK1 expressed by SGNs transmit a survival-promoting regulatory signal during TFD-induced SGN cell death via pathways involving PKC activation, Ca2+ signalling and MAPK/ERK activation, which can be accounted for by an inhibition of caspase activation.

Asunto(s)

Apoptosis/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neuronas/efectos de los fármacos , Ganglio Espiral de la Cóclea , Sustancia P/farmacología , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Quelantes/farmacología , Citosol/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Crecimiento Nervioso/farmacología , Neuronas/citología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Nifedipino/farmacología , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/metabolismo , Sustancia P/biosíntesis , Sustancia P/genética

RESUMEN

The organ of Corti is a complex structure containing a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs), supported respectively by one row of inner phalangeal cells and three rows of Deiters' cells. When fetal rat organ of Corti explants are cultured, supernumerary OHCs and supernumerary Deiters' cells are produced, without any additional cell proliferation. Analysis of semi- and ultrathin sections revealed that supernumerary OHCs are produced at the distal edge of the organ of Corti. Quantitative analysis of cell types present in the organ of Corti demonstrates that when the number of OHCs increases: (i) the total number of cells remains constant; (ii) the number of Deiters' cells increases; (iii) the number of tectal cells decreases and of Hensen's cells decreases. Using specific HC markers, i.e. jagged2 (Jag2) and Math1, we showed that in addition to existing OHCs, supernumerary OHCs, tectal cells and Hensen's cells expressed these markers in embryonic day 19 organ of Corti explants after 5 days in vitro. The results of this study suggest that Hensen's cells retain the capacity to differentiate into either tectal cells, which differentiate into OHCs, or into undertectal cells which differentiate into Deiters' cells.

Asunto(s)

Diferenciación Celular/fisiología , Desarrollo Embrionario y Fetal/fisiología , Células Epiteliales/citología , Células Ciliadas Auditivas Externas/embriología , Órgano Espiral/embriología , Núcleo Vestibular Lateral/embriología , Animales , Células Epiteliales/fisiología , Femenino , Células Ciliadas Auditivas Externas/citología , Técnicas de Cultivo de Órganos , Órgano Espiral/citología , Embarazo , Ratas , Ratas Wistar , Núcleo Vestibular Lateral/citología

RESUMEN

Presbycusis is a complex of high frequency hearing loss and disproportionate loss of speech discrimination that is seen concomitantly with physical signs of aging. Among the most extensively characterized strains of mice that show an early hearing loss is the C57B16/J strain, a strain that shows early onset of high frequency hearing loss at age 6 months and complete hearing loss by 1 year of age. The histopathology of this strain consists of loss of hair cells and spiral ganglion neurons in the basal turn, with a progression of loss of hair cells and ganglion neurons towards the apical portion of the cochlea as the animal ages. The process of aging has been extensively studied and although details differ in various organisms the consensus today is that oxidative stress, i.e. free radical-mediated tissue damage, is one of the core mechanisms of aging. Aerobic metabolism results in the creation of hydrogen peroxide and reactive oxygen species. These are normally detoxified by a variety of enzymes and free radical scavengers, including superoxide dismutase (SOD), catalase and glutathione. To determine whether oxidative stress plays a role in the pathophysiology of hearing loss in this mouse model of presbycusis we determined the relative change in mRNA production for selected free radical detoxifying enzymes in the C57B16/J mouse cochlea. Using semi-quantitative RT-PCR with tubulin mRNA as a control, relative levels of antioxidant enzyme mRNAs were determined. There was an overall increase in SOD1 mRNA levels when comparing 1 and 9 month time points, and a transient increase in the expression level of catalase mRNA. B6.CAST+ Ahl mice, which carry the C57B16/J genome but receive their Ahl gene from CAST mice, do not show these alteractions in antioxidant enzyme production. Our results suggest that at an age of 9 months, at which point significant hearing loss has developed, the C57B16/J mouse cochlea is exposed to increased levels of free radicals and that the Ahl gene of the C57B16/J mouse mediates this decrease in protective enzymes and therefore increase in levels of oxidative stress.

Asunto(s)

Envejecimiento/fisiología , Cóclea/metabolismo , Estrés Oxidativo/fisiología , Animales , Catalasa/metabolismo , Cóclea/citología , Cóclea/fisiopatología , Glutatión Peroxidasa/metabolismo , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Trastornos de la Audición/fisiopatología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1

RESUMEN

OBJECTIVES: The aim of this study is to determine the efficacy of L-N-acetyl-cysteine (L-NAC) as a protectant for inner ear auditory sensory cells against the toxic effects of cisplatin. STUDY DESIGN: Prospective laboratory study of the otoprotective effect of L-NAC on auditory neurons and hair cells in vitro. METHODS: The study has two arms. The first arm evaluated the neuroprotective effect of L-NAC on early postpartum auditory ganglion cell cultures. Two culture media were used. The two media differed in that one of them was enhanced by the addition of neurotrophins (neurotrophin type 3 and brain-derived neurotrophic factor) and a growth factor (transforming growth factor-beta1). Then the survival of cisplatin-treated auditory neurons was studied before and after pretreatment with protective levels of L-NAC. The second arm of the study evaluated the effect of L-NAC on cisplatin damage initiated to auditory hair cells. Early-postpartum organ of Corti explants were grown in culture. Their rate of survival was studied after exposure to toxic levels of cisplatin. Then, survival of cisplatin-damaged hair cells was studied after they were pretreated with L-NAC. RESULTS: Pretreatment of cultures with L-NAC protected both auditory neurons and hair cells from the effects of exposure to toxic levels of cisplatin. This observed otoprotective effect was dose dependent. CONCLUSIONS: Our in vitro studies have demonstrated that L-NAC protected both auditory neurons and hair cells from the toxic effects of cisplatin. Because it protects both of these inner ear structures, L-NAC may be potentially useful in protecting hearing, in general, from cisplatin-induced damage. In addition, L-NAC has low systemic and mucosal toxicity. It also has a low molecular weight that may allow it to readily cross the round window membrane. All these characteristics make it potentially suitable for transtympanic application for the prevention of the ototoxicity of cisplatin in vivo.

Asunto(s)

Acetilcisteína/farmacología , Antineoplásicos/toxicidad , Cisplatino/toxicidad , Nervio Coclear/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Células Ciliadas Auditivas Internas/efectos de los fármacos , Neuronas/efectos de los fármacos , Análisis de Varianza , Animales , Células Cultivadas , Sordera/inducido químicamente , Sordera/prevención & control , Audición/efectos de los fármacos , Humanos , Órgano Espiral/efectos de los fármacos , Estudios Prospectivos , Ratas , Factores de Tiempo

RESUMEN

Cisplatin (cis-diamminedichloroplatinum(II) (CDDP)) is a widely used, highly effective, oncolytic agent that has serious ototoxic side-effects. To test the effectiveness of local delivery, of L-methionine (L-Met) as an otoprotective agent against CDDP ototoxicity, we used a rat model of a highly metastatic breast cancer tumor, i.e. Fisher 344 rats implanted with MTLn3 breast cancer cells. Four experimental groups were evaluated--I: untreated; II: CDDP-treated (three dosages); III: systemically-delivered L-Met + CDDP-treated; IV: locally delivered L-Met + CDDP-treated. The integrity of the outer hair cells (OHCs) was determined using scanning electron microscopy (SEM); hearing was assessed by recording auditory brainstem responses (ABRs) at multiple frequencies. The chemotherapeutic effectiveness of CDDP was quantified by measuring changes in tumor mass and the presence of tumor metastasis. L-Met provided otoprotection of the OHCs against CDDP toxicity in the cochleae of rats following either systemic (III) or local (IV) administration. The ABRs were unchanged in each of the L-Met protection Groups (III and IV) and in the untreated animals of Group I. Treatment with CDDP only (II) induced significant hearing losses at both 16 and 18 kHz when compared to ABRs of untreated rats(I). CDDP was effective in controlling the MTLn3 initiated breast cancer tumors in the CDDP-treated (II) and the local L-Met protection, CDDP-treated (IV) Groups. In contrast, the tumors in the systemic L-Met protection, CDDP-treated Group (III) were not controlled by the CDDP treatment regime. This study demonstrates that local delivery of L-Met to the scala tympani of the cochlea via the round window membrane (IV) provides effective protection against CDDP ototoxicity without compromising its ability to control a highly metastatic form of cancer.

Asunto(s)

Antineoplásicos/toxicidad , Cisplatino/antagonistas & inhibidores , Cisplatino/toxicidad , Trastornos de la Audición/inducido químicamente , Trastornos de la Audición/prevención & control , Metionina/administración & dosificación , Metionina/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Animales , Antineoplásicos/uso terapéutico , Cisplatino/uso terapéutico , Implantes de Medicamentos , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Femenino , Células Ciliadas Auditivas Externas/patología , Trastornos de la Audición/patología , Inyecciones Intraperitoneales , Membranas Artificiales , Microscopía Electrónica de Rastreo , Trasplante de Neoplasias , Ratas , Ratas Endogámicas F344 , Células Tumorales Cultivadas

RESUMEN

OBJECTIVE: To evaluate the efficacy of esterified hyaluronic acid (MeroGel) as a middle ear (ME)-packing material. DESIGN: Randomized controlled trial. MATERIAL: Twenty-four guinea pigs. INTERVENTION: Group 1, MeroGel-treated animals (n = 10), bilateral wounding of ME mucosa with 5 of the animals receiving the MeroGel packing in the left ME and 5 of the animals receiving MeroGel in the right ME; group 2, absorbable gelatin sponge-treated animals (n = 10), with the same experimental protocol as in group 1 except that the absorbable gelatin sponge was the packing material; group 3, untreated animals (n = 4), unilateral wounding of the left ME mucosa in 2 animals and in 2 animals in the right ME, with no packing material. Auditory brainstem recordings were performed for all groups before the ME operation and 5 days and 6 weeks after the operation. RESULTS: Auditory brainstem response recordings at postoperative day 5 showed that all ears with ME packing had hearing losses in the frequency range of 500 to 4000 Hz. The recovery of hearing acuity at postoperative week 6 was significantly better in group 1 (MeroGel-treated) guinea pigs compared with group 2 (the absorbable gelatin sponge-treated) animals. In group 2 animals, 20% of the packing material remained in the ME cavities and new bone formation was observed, while in group 1 animals, there was less packing material in the ME and no formation of new bone. CONCLUSIONS: MeroGel is a nonototoxic packing material with a high level of biocompatibility for ME mucosa; it is an effective supportive material following ME surgery and is easily expelled from the ME cavity.

Asunto(s)

Materiales Biocompatibles/farmacocinética , Oído Medio/efectos de los fármacos , Ácido Hialurónico/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Materiales Biocompatibles/toxicidad , Oído Medio/lesiones , Oído Medio/cirugía , Esterificación , Esponja de Gelatina Absorbible , Cobayas , Audición/efectos de los fármacos , Ácido Hialurónico/toxicidad , Membrana Mucosa/efectos de los fármacos , Distribución Aleatoria

RESUMEN

The vestibules of adult guinea pigs were lesioned with gentamicin and then treated with perilymphatic infusion of either of two growth factor mixtures (i.e., GF I or GF II). GF I contained transforming growth factor alpha (TGFalpha), insulin-like growth factor type one (IGF-1), and retinoic acid (RA), whereas GF II contained those three factors and brain-derived neurotrophic factor. Treatment with GF I significantly enhanced vestibular hair cell renewal in ototoxin-damaged utricles and the maturation of stereociliary bundle morphology. The addition of brain-derived neurotrophic factor to the GF II infusion mixture resulted in the return of type 1 vestibular hair cells in ototoxin-damaged cristae, and improved vestibular function. These results suggest that growth factor therapy may be an effective treatment for balance disorders that are the result of hair cell dysfunction and/or loss.

Asunto(s)

Sustancias de Crecimiento/farmacología , Células Ciliadas Vestibulares/efectos de los fármacos , Animales , Gentamicinas/administración & dosificación , Cobayas , Células Ciliadas Vestibulares/fisiología , Células Ciliadas Vestibulares/ultraestructura , Microscopía Electrónica

RESUMEN

Successful delivery of genes to the inner ear has been demonstrated using a variety of vectors and animal models. As our understanding of the molecular pathophysiology of hearing and balance disorders increases, the delivery of genes is becoming central to our ability to manipulate the function of the inner ear. This study evaluates the efficacy of gene transfer and the distribution of three different vector types within the inner ear. Adenovirus vectors, herpes virus vectors and liposomes carrying a plasmid with the green fluorescent protein or beta galactosidase marker genes and a CMV promoter were introduced into the inner ear of 3-month-old mice. The temporal bones and brain were then removed from the animals and examined for transgene expression. Distribution of staining in the treated ear was compared with distribution of staining in the contralateral inner ear. Staining for T cell markers was also carried out to determine inner ear immune response to gene transfer. Herpes virus vectors appear to target neurons most efficiently. Liposome vectors were least efficient in terms of gene transfer. Adenovirus vectors accomplished gene transfer to the widest variety of inner ear cells including auditory and vestibular hair cells. Newer generation adenovirus vectors promise less immune reaction and toxicity than traditional vectors and will be useful for both research and future clinical applications.

Asunto(s)

Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos , Pérdida Auditiva Sensorineural/terapia , Animales , Cóclea/patología , Femenino , Expresión Génica/fisiología , Marcadores Genéticos/genética , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/patología , Ratones , Neuronas/patología , beta-Galactosidasa/genética

RESUMEN

During the development of the mammalian inner ear, the number of hair cells produced is highly regulated and remains constant throughout life. The mechanism underlying this regulation is beginning to be understood although many aspects still remain obscure. When late embryonic or early postnatal rat organs of Corti were cultured, the production of supernumerary hair cells was observed. This overproduction of sensory cells could be modulated by the addition of several growth factors. In this study, we examined explants of rat organs of Corti that produced supernumerary hair cells. In the supernumerary hair cell region, up to two rows of inner hair cells and five rows of outer hair cells were observed. Morphological evaluation of these specimens revealed that less mature hair cells were located in the most external rows of these sensory cells. When a supernumerary hair cell was produced, a supporting cell (i.e. Deiters' cell) was also produced, strongly suggesting that the conversion of a Deiters' cell into a hair cell was not the mechanism that produced these extra hair cells. Based on these results, we propose that prosensory cells located at the external edge of the organ of Corti retain a capacity to form hair cells and that it is these prosensory cells that differentiate into supernumerary hair cells and Deiters' cells.

Asunto(s)

Diferenciación Celular/fisiología , Células Ciliadas Auditivas Externas/citología , Órgano Espiral/citología , Animales , Recuento de Células , Femenino , Edad Gestacional , Células Ciliadas Auditivas Internas/citología , Embarazo , Ratas , Ratas Wistar

RESUMEN

Congenital deafness is a very frequent disorder occurring in approximately I in 1000 live births. Mutations in GJB2 encoding for gap junction protein connexin-26 (Cx26) have been established as the basis of autosomal recessive non-syndromic hearing loss and proposed in some rare cases of autosomal dominant form of deafness. Connexin are gap-junction proteins which constitute a major system of intercellular communication important in the exchange of electrolytes, second messengers and metabolites. In the inner ear, connexin 26 expression was demonstrated in the stria vascularis, basement membrane, limbus and the spiral prominence of the human cochlea. The loss of connexin 26 in the gap junction complex would expect to disrupt the recycling of potassium from the synapses at the base of hair cells through the supporting cells and fibroblasts of potassium ions back to the high potassium containing endolymph of the cochlear duct and therefore would result in a local intoxication of the Corti s organ by potassium, leading to the hearing loss. The discovery of the genes responsible of hearing loss in particular the identification of mutations in the gene coding for connexin 26 allows to hope some tremendous help in genetic counseling. The possible implication of the mutation of the connexin gene in the pathophysiology of some progressive adult deafness opens new prospects in the fine diagnostic of the ear diseases and eventually may lead to new therapeutic strategies applied to the cochlea.

Asunto(s)

Conexinas/genética , Sordera/genética , Sordera/fisiopatología , Uniones Comunicantes/fisiología , Audición/fisiología , Mutación , Adulto , Conexina 26 , Humanos

RESUMEN

The development of the mammalian cochlea is an example of patterning in the peripheral nervous system. Sensory hair cells and supporting cells in the cochlea differentiate via regional and cell fate specification. The Notch signaling components shows both distinct and overlapping expression patterns of Notch1 receptor and its ligands Jagged1 (Jag1) and Jagged2 (Jag2) in the developing auditory epithelium of the rat. On embryonic day 16 (E16), many precursor cells within the Kölliker's organ immunostained for the presence of both Notch1 and Jag1, while the area of hair cell precursors did not express either Notch1 and Jag1. During initial events of hair cell differentiation between E18 and birth, Notch1 and Jag1 expression predominated in supporting cells and Jag2 in nascent hair cells. Early after birth, Jag2 expression decreased in hair cells while the pattern of Notch1 expression now included both supporting cells and hair cells. We show that the normal pattern of hair cell differentiation is disrupted by alteration of Notch signaling. A decrease of either Notch1 or Jag1 expression by antisense oligonucleotides in cultures of the developing sensory epithelium resulted in an increase in the number of hair cells. Our data suggest that the Notch1 signaling pathway is involved in a complex interplay between the consequences of different ligand-Notch1 combinations during cochlear morphogenesis and the phases of hair cell differentiation.

Asunto(s)

Diferenciación Celular , Células Ciliadas Auditivas/embriología , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular , Transducción de Señal , Factores de Transcripción , Animales , Animales Recién Nacidos , Proteínas de Unión al Calcio , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/fisiología , Cóclea/citología , Cóclea/embriología , Cóclea/ultraestructura , Técnicas de Cultivo , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestructura , Péptidos y Proteínas de Señalización Intercelular , Proteína Jagged-1 , Proteína Jagged-2 , Mamíferos , Proteínas de la Membrana/biosíntesis , Oligonucleótidos Antisentido/farmacología , Biosíntesis de Proteínas , Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Receptor Notch1 , Proteínas Serrate-Jagged

RESUMEN

Gap junctions play a pivotal role in embryonic development by forming specialized regions of cell-cell communication. In this study, we demonstrate the temporal-spatial distribution of connexin 26 in the embryonic and early postnatal mouse cochlea. Our results show localization of this gap junction protein to specific cochlear structures, including the inner and outer sulcus cells, the supporting cells of the inner hair cells, the mesenchyme derived portion of the stria vascularis, and the cells of the spiral ligament that interface with the basal cells of the stria vascularis. This suggests that this gap junction protein of served patterns of connexin 26 distribution is important for the differentiation and development of these structures (e.g., the role of the inner sulcus cells in producing the tectorial membrane).

Asunto(s)

Conexinas/análisis , Uniones Comunicantes/química , Células Ciliadas Auditivas Internas/química , Células Ciliadas Auditivas Internas/embriología , Células Ciliadas Auditivas Externas/química , Células Ciliadas Auditivas Externas/embriología , Animales , Diferenciación Celular/fisiología , Conexina 26 , Conexinas/biosíntesis , Femenino , Uniones Comunicantes/metabolismo , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Externas/citología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Embarazo