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This review presents many but not all the major historical events that have led to our current understanding of gene and stem cell therapies for the treatment of hearing and balance disorders in animal models of these disorders. In order to better understand the application of these emerging therapies to the treatment of inner ear disorders in a clinical setting, it has been necessary to provide some genetic and pathobiology backgrounds from both animal models and clinical disorders. The current focus and goal of gene and stem cell therapies are directed toward understanding the effective treatment of animal models that mimic human disorders of hearing and balance. This approach not only addresses the most effective ways to deliver the gene or stem cell therapies to affected inner ears, it also provides an assessment of the efficacy of the applied therapy(s) in achieving either partial or full restoration of either hearing and/or balance within the animal models receiving these therapeutic interventions. This review also attempts to present a realistic assessment of how close the research fields of gene and stem cell therapies are to application for the treatment of human disorders in a clinical setting. Progress made in developing these novel therapies toward clinical applications would not have been possible without the many pioneering studies and discoveries achieved by the investigators cited in this review. There were also many other excellent studies performed by gifted investigators that were not able to be included within this review. Anat Rec, 303:390-407, 2020. © 2019 American Association for Anatomy.

The crosstalk between TGF-β1 and WNT pathways has been proven to regulate aspects of the development and tissue homeostasis processes. Recently, it has been demonstrated this collaboration also takes place during fibrotic diseases, where TGF-β1 activates the WNT/β-catenin pathway that results in dedifferentiation of fibroblasts into myofibroblasts, increased production of extracellular matrix components and fibrosis. Independent studies show the functions of these molecules during the development of the inner ears in several different species. However, little is known about the collaboration between TGF-β1 and WNT in the injured inner ear and particularly how this collaboration affects the fibrotic process that often occurs following cochlear implantation. First, we used a cochlear explant model to study the effect of electrode insertion trauma and TGF-β1 signaling in activation of the WNT pathway. Finally, adult TopGal mutant mice were used in vivo to track the activation of the WNT/β-catenin in response to EIT. A chronic inflammatory response, increased cell proliferation and tissue remodeling are hallmarks of fibrotic disease. This study explores and highlights the collaboration between the TGF-β1 and WNT pathways in the trauma-initiated fibrotic process within the implanted cochlea. WNT signaling is involved in the development of the inner ear and therefore a potential target in hair cell regeneration therapies. However, in light of our observations from the current study, manipulation of the WNT pathway by gene therapy techniques in the pathological ear seems a very complex process with an increased risk of inducing excessive fibrosis thereby compromising the efficacy of implant function. Anat Rec, 303:608-618, 2020. © 2019 American Association for Anatomy.

HYPOTHESIS Dexamethasone (DXM) protects against cisplatin-induced auditory hair cell (HC) loss in rat organ of Corti (OC) explants in vitro by reducing levels of oxidative stress and NADPH-Oxidase-3 (NOX-3). BACKGROUND Intratympanic DXM has demonstrated protective effects against cisplatin-induced hearing loss in a few animal studies and one clinical trial. However, levels of protection with intratympanic DXM vary significantly between studies, which may not be a result of the intrinsic properties of DXM but rather reflect the diffusion of DXM into the cochlea. The molecular mechanisms and degree of DXM protection against cisplatin ototoxicity are currently unknown. METHODS OC explants from 3-day-old rats were cultured with no treatment or various concentrations of cisplatin (2, 5, or 10 μM) and DXM (75, 150, or 300 μg/mL) in vitro. HC viability and TUNEL assay were performed after 72 hours in vitro and levels of oxidative stress and NOX-3 were evaluated with confocal microscopy after 48 hours in vitro. Analysis of variance with Tukey's post hoc testing was performed. RESULTS Cisplatin initiated dose-dependent losses of outer HCs (OHCs) in the basal turns of exposed explants (p

Programmed cell death (PCD) initially starts in the support cells (SCs) after electrode insertion trauma (EIT), followed by PCD in hair cells (HCs). Activation of caspase-3 was observed only in SCs. Protecting both SCs and HCs with selective otoprotective drugs at an early stage post implantation may help to preserve residual hearing.Cochlear implant EIT can initiate sensory cell losses via necrosis and PCD within the organ of Corti, which can lead to a loss of residual hearing. PCD appears to be a major factor in HC loss post-EIT. The current study aimed to: (1) determine the onset of PCD in both SCs and HCs within the traumatized organ of Corti; and (2) identify the molecular mechanisms active within the HCs and SCs that are undergoing PCD.Adult guinea pigs were assigned to one of two groups: (1) EIT and (2) unoperated contralateral ears as controls. Immunostaining of dissected organ of Corti surface preparations for phosphorylated-Jun, cleaved caspase-3, and 4-hydroxy-2,3-nonenal (HNE) were performed at 6, 12, and 24 h post-EIT and for contralateral control ears.At 6 h post-EIT the SCs immunolabeled for the presence of phosphorylated-Jun and activated caspase-3. Phosphorylated p-Jun labeling was observed at 12 h in both the HCs and SCs of middle and basal cochlear turns. Cleaved caspase-3 was not observed in HCs of any cochlear turn at up to 24 h post-EIT. Lipid peroxidation (HNE immunostaining) was first observed at 12 h post-EIT in both the HCs and SCs of the basal turn, and reached the apical turn by 24 h post-EIT.

We have studied aminoglycoside-induced vestibular hair-cell renewal using long-term culture of utricular macula explants from 4-day-old rats. Explanted utricles were exposed to 1 mM of gentamicin for 48 h, during 2nd and 3rd days in vitro (DIV), and then recovering in unsupplemented medium. Utricles were harvested at specified time points from the 2nd through the 28th DIV. The cellular events that occurred within hair cell epithelia during the culture period were documented from serial sectioned specimens. Vestibular hair cells (HCs) and supporting cells (SCs) were systematically counted using light microscopy (LM) with the assistance of morphometric software. Ultrastructural observations were made from selected specimens with transmission electron microscopy (TEM). After 7 DIV, i.e. four days after gentamicin exposure, the density of HCs was 11% of the number of HCs observed in non-gentamicin-exposed control explants. At 28 DIV the HC density was 61% of the number of HCs observed in the control group explant specimens. Simultaneously with this increase in HCs there was a corresponding decline in the number of SCs within the epithelium. The proportion of HCs in relation to SCs increased significantly in the gentamicin-exposed explant group during the 5th to the 28th DIV period of culture. There were no significant differences in the volume estimations of the gentamicin-exposed and the control group explants during the observed period of culture. Morphological observations showed that gentamicin exposure induced extensive loss of HCs within the epithelial layer, which retained their intact apical and basal linings. At 7 to 14 DIV (i.e. 3-11 days after gentamicin exposure) a pseudostratified epithelium with multiple layers of disorganized cells was observed. At 21 DIV new HCs were observed that also possessed features resembling SCs. After 28 DIV a new luminal layer of HCs with several layers of SCs located more basally characterized the gentamicin-exposed epithelium. No mitoses were observed within the epithelial layer of any explants. Our conclusion is that direct transdifferentiation of SCs into HCs was the only process contributing to the renewal of HCs after gentamicin exposure in these explants of vestibular inner ear epithelia obtained from the labyrinths of 4-day-old rats.

Evaluate the effectiveness of short interfering RNA against Bax (Bax siRNA) as a treatment for tumor necrosis factor α (TNFα)-induced auditory hair cell (HC) loss in rat organ of Corti (OC) explants.Basic science.Basic science laboratory, University of Miami Ear Institute.Organ of Corti explants dissected from 3-day-old rats were cultured in control media, TNFα, and TNFα + Bax siRNA at 0, 48, and 72 hours in vitro. Real-time polymerase chain reaction, enzyme-linked immunosorbent assay, HC viability studies, immunofluorescence, and confocal microscopy were performed. Analysis of variance with post hoc testing was used with P.05 considered significant.The TNFα-damaged explants demonstrated significant decreases in viable HCs in the basal turn with corresponding increased levels of Bax gene and protein expression, compared with control explant levels. The levels of viable HCs and Bax gene and protein expression in TNFα + Bax siRNA-treated explants approached levels measured in control explants. Immunolocalization studies showed increased Bax protein expression in basal turn HCs in TNFα-treated explants, whereas control explants and TNFα + Bax siRNA-treated explants had low levels of Bax expression.TNFα initiates the programmed cell death of auditory HCs in OC explants through upregulation of proapoptotic Bax gene and protein expression. Bax siRNA blocks TNFα-induced apoptosis of HCs by decreasing the TNFα-induced levels of Bax mRNA and protein expression in treated explants. Bax siRNA is an effective treatment for TNFα-induced ototoxicity in OC explants in vitro and has great potential to be a therapeutic agent against trauma/inflammation-induced hearing loss.

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-1β 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.

This review of local drug delivery for inner ear therapy covers the topics of: noise-induced hearing loss (NIHL); vibration-induced hearing loss (VHL); cisplatin ototoxicity; aminoglycoside ototoxicity; and mechanical trauma-induced hearing loss that can occur during the process of cochlear implantation. The cellular, biochemical and molecular mechanisms involved in the causation of the hearing losses that result from exposure to these diverse traumas to the cochlea and its auditory sensory epithelium are explored as well as the efficacy of different drug therapies in their ability to either prevent or lessen the damage to the cochlear sensory epithelium and ameliorate the level of hearing loss. This review concludes with a section that explores future strategies for unique methods of drug delivery to the cochlea (e.g. biorelease from hydrogels via the round window membrane) and the development of novel inner ear therapies (e.g. short interfering ribonucleic acids, siRNAs) to conserve hearing against trau...

The programmed cell death of stress-damaged auditory hair cells can occur through a variety of signal pathways, and therapeutic modalities that block pro-cell-death pathways are being developed and evaluated for hearing preservation. Because of their ability to have both anti-inflammatory and anti-apoptotic actions, corticosteroids have long been used to protect against several types of acute sensorineural hearing loss. Other anti-apoptotic drugs that target the mitogen-activated protein kinase (MAPK)/c-Jun-N terminal kinase (JNK) signal cascade, such as D-JNKI-1 (AM-111) and SP600125, have produced promising results both in vitro and in laboratory animal studies, with AM-111 showing promise in preliminary clinical trials. Antioxidant drugs, e.g. sodium thiosulfate, N-acetylcysteine, and D-methionine, have been shown in animal studies to attenuate permanent threshold shifts in hearing by reducing oxidative stress. In addition to reviewing selected therapeutic trends for the conservation of hearing, we review our experiences with dexamethasone and D-JNKI-1 and report results from our current research.

Objective: Determine the molecular mechanism(s) behind tumor necrosis factor-alpha (TNFα)–induced loss of auditory hair cells and the ability of dexamethasone base (DXMb) to protect against TNFα ototoxicity. Methods: Hair cell counts: Three-day-old rat organ of Corti explants were cultured under three different conditions: 1) untreated-control; 2) TNFα (2 μg/ml); and 3) TNFα (2 μg/ml)+DXMb (70 μg/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 s-actin (housekeeping gene), TNFR1, Bcl-2, Bax, and Bcl-xl. Results: DXMb protected explant hair cells from TNFα-induced loss. Bax gene expression was greater in TNFα-exposed explants compared with TNFα+DXMb-treated explants at 48 h (P=0.023), confirmed by the increase in the Bax/Bcl-2 ratio at 48 h (P

The progressive loss of hearing that develops after electrode insertion trauma (EIT) can be attenuated by local dexamethasone (DXM) therapy.Hearing loss (HL) that develops after cochlear implant EIT occurs in two stages in laboratory animals, that is, an immediate loss followed by a progressive loss. Direct infusion of DXM into the guinea pig cochlea can attenuate both ototoxin- and noise-induced HL.Auditory-evoked brainstem responses (ABRs) of guinea pigs were measured for 4 frequencies (i.e., 0.5, 1, 4, and 16 kHz) before, immediately after, and more than 30 days post-EIT for experimental (EIT,EIT + artificial perilymph, and EIT + DXM) and for the contralateral unoperated cochleae of each group. An electrode analog of 0.14-mm diameter was inserted through a basal turn cochleostomy for a depth of 3 mm and withdrawn. DXM in artificial perilymph was delivered immediately post-EIT into the scala tympani via a miniosmotic pump for 8 days.The ABR thresholds of EIT animals increased progressively post-EIT. Contralateral unoperated cochleae had no significant changes in ABR thresholds. Immediately post-EIT, that is, Day 0, the DXM-treated animals exhibited a significant HL at 1, 4, and 16 kHz, but this HL was no longer significant by Day 30 compared with contralateral control ears.The results from immediate local treatment of the cochlea with DXM in an animal model of EIT-induced HL suggest a novel therapeutic strategy for hearing conservation by attenuating the progressive HL that can result from the process of electrode array insertion during cochlear implantation.

International audience; Neomycin ototoxicity and electrode insertion trauma both involve activation of the mitogen activated protein kinase (MAPK)/c-Jun-N-terminal kinase (JNK) cell death signal cascade. This article discusses mechanisms of cell death on a cell biology level (e.g. necrosis and apoptosis) and proposes the blocking of JNK signaling as a therapeutic approach for preventing the development of a permanent hearing loss that can be initiated by either neomycin ototoxicity or electrode insertion trauma. Blocking of JNK molecules incorporates the use of a peptide inhibitor (i.e. D-JNKI-1), which is specific for all three isoforms of JNK and has been demonstrated to prevent loss of hearing following either electrode insertion trauma or loss of both hearing and hair cells following exposure to an ototoxic level of neomycin. We present previously unpublished results that control for the effect of perfusate washout of aminoglycoside antibiotic by perfusion of the scala tympani with an inactive form of D-JNKI-1 peptide, i.e. JNKI-1(mut) peptide, which was not presented in the original J. Neurosci. article that tested locally delivered D-JNKI-1 peptide against both noise- and neomycin-induced hearing loss (i.e. Wang, J., Van De Water, T.R., Bonny, C., de Ribaupierre, F., Puel, J.L., Zine, A. 2003a. A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss. J. Neurosci. 23, 8596-8607). D-JNKI-1 is a cell permeable peptide that blocks JNK signaling at the level of the three JNK molecular isoforms, which when blocked prevents the increases in hearing thresholds and the loss of auditory hair cells. This unique therapeutic approach may have clinical application for preventing: (1) hearing loss caused by neomycin ototoxicity; and (2) the progressive component of electrode insertion trauma-induced hearing loss.

Localized cochlear hypothermia was induced in a rat model, demonstrating the feasibility of modulating cochlear temperature without affecting core body temperature.Systemic hypothermia has been demonstrated to protect the rat cochlea against electrode insertion trauma-induced hearing loss. Due to potential adverse effects of systemic hypothermia, we set out to demonstrate the feasibility of inducing localized cochlear hypothermia and compared the efficacy of three cooling techniques.Twenty-four ears were prepared by sealing a temperature micro-probe into the basal turn of the cochlea. Cochleae were then cooled by cold saline irrigation of the external auditory canal (EAC) or bulla or by direct application of ice over the bulla. Cochlear temperature measurements were recorded every 30 s during the cooling period until stable. Rectal temperature was monitored continuously and maintained at 36 degrees C.All techniques resulted in cochlear hypothermia without a concomitant change in rectal temperature. EAC irrigation (14 degrees C and 11 degrees C) decreased cochlear temperature on average by 1.1 degrees C and 1.6 degrees C, respectively. Bulla irrigation (14 degrees C and 11 degrees C) decreased cochlear temperature on average by 3.3 degrees C and 4.1 degrees C, respectively. The ice produced an average cochlear temperature decrease of 4.1 degrees C. In all cases, a cochlear temperature nadir was reached in 5-6 min with no significant differences between groups with respect to time.

Objective: To test the hypothesis that bcl-2 prevents oxidative stress-induced apoptosis of auditory sensory cells in explants of the organ of Corti and dissociated cell cultures of the spiral ganglion. Methods: Organ of Corti explants and dissociated spiral ganglion cell cultures obtained from 3-day-old (P3) rats or adult spiral ganglion cell cultures from 28-day-old (P28) rats were transduced with vectors containing a human bcl-2 gene. Cultures were then exposed to neomycin, cisplatin or subjected to withdrawal of neurotrophin supplementation. Outcome measures included hair cell and neuron counts, mitochondrial membrane potential and a histological measure of apoptosis. Results: Expression of bcl-2 in the organ of Corti explants and neuronal cell cultures provided a significant level of protection against cell death. Bcl-2 expression in the organ of Corti explants also protected mitochondria from loss of membrane potential and blocked an early step in the commitment of hair cells to apoptosis. Conclusion: Expression of bcl-2 in cochlear tissues protects sensory cells from a variety of insults that have been demonstrated to damage the inner ear.

Pharmacological manipulation of the labyrinth for therapeutic purposes is becoming a reality for the treatment of inner ear disorders. The mitogen-activated protein kinase (MAPK)/c-Jun-N-terminal kinase (JNK) intracellular signal cascade is used by injured auditory sensory cell to initiate their elimination via apoptosis (programmed cell death). This intracellular signal cascade can be inhibited and/or down-regulated by the application of inhibitory peptides and through the use of antisense oligonucleotides directed against specific RNA message sequences. This approach has been shown to be highly effective in preventing the apoptotic cell death of auditory sensory cells that are under oxidative stress because of either ototoxin exposure or exposure to damaging levels of sound. The insertion of an electrode array into the cochlea has been postulated to generate a high level of oxidative stress and to have a progressive component of hearing loss that occurs for several days after the initial electrode insertion. It is probable that this ongoing loss of hearing due to trauma caused by electrode insertion may be preventable and may be due to apoptosis of sensory cells damaged by oxidative stress and signalling via the MAPK/JNK signal cascade. Pharmacological manipulation of the MAPK/JNK signal cascade during cochlear implantation may be an effective therapy for the conservation of residual hearing during and following cochlear implantation. Other promising treatment options include the use of antioxidant molecules, corticosteroids, and blocking antisense oligonucleotides or inhibitory RNAi sequences.

The Hmx homeobox gene family appears to play a conserved role in CNS development in all animal species examined, and in higher vertebrates has an additional role in sensory organ development. Here, we show that murine Hmx2 and Hmx3 have both overlapping and distinct functions in the development of the inner ear's vestibular system, whereas their functions in the hypothalamic/pituitary axis of the CNS appear to be interchangeable. As in analogous knockin studies of Otx and En function, Drosophila Hmx can rescue conserved functions in the murine CNS. However, in contrast to Otx and En, Drosophila Hmx also rescues significant vertebrate-specific functions outside the CNS. Our work suggests that the evolution of the vertebrate inner ear may have involved (1) the redeployment of ancient Hmx activities to regulate the cell proliferation of structural components and (2) the acquisition of additional, vertebrate-specific Hmx activities to regulate the sensory epithelia.

In this article, we describe an efficient method for testing both auditory receptor and auditory nerve function in a single recording session. Auditory receptor function is tested in response to pure tone, tone burst and click acoustic stimuli (i.e. distortion products of otoacoustic emissions, DPOAE; and auditory-evoked brainstem responses, ABR). The function of the auditory neurons and nerve is measured in response to direct electric current stimulation (i.e. electrically evoked auditory brainstem responses, eABR). All measurements were obtained from anesthetized laboratory rats during single recording sessions using hardware and software stimulation and analysis programs developed by Intelligent Hearing Systems, Miami, FL.

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, Go6976 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 4β-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.

We describe the methodology and rationale behind the delivery of therapeutic medicines to the inner ear. The inner ear has long been impervious to pharmacologic manipulation. This is most likely the result of a protective mechanism called the blood-labyrinth barrier, whose function closely resembles that of the blood-brain barrier. This protective barrier impedes the clinician's ability to treat inner ear diseases with systemically administered medications. Since 1935, otolaryngologists have attempted to manipulate the inner ear with transtympanically injected medicines. Success has varied widely, but medicinal ablation of vestibular function can be achieved in this manner. Unfortunately, the auditory system is also at great risk from any medicine that is delivered to the inner ear via the middle ear. Over the past 10 years, significant improvements in drug delivery have allowed for more “titratable” treatment, which has reduced (but not eliminated) the risk of permanent hearing loss. In this article, we discuss both novel and time-tested methods of delivering medicines to the inner ear. We also review the classes of medications that alter inner ear function and the attendant risks of such treatments.

Salicylate has recently been demonstrated to protect against the auditory and vestibular side effects of aminoglycoside antibiotics. Similarities in the toxic mechanisms suggest salicylate as a treatment strategy to prevent the ototoxic side effects of cisplatin (CDDP). We first tested protection of the inner ear in Wistar rats receiving a single infusion of 16 mg CDDP/kg body weight with or without treatment with 100 mg/kg salicylate (bid) for 5 days beginning one day before the CDDP infusion. Cisplatin induced a threshold shift of more than 30 dB (at 14 kHz; measured by auditory evoked brain stem response) that was significantly reduced by salicylate. We then examined the protective potential of salicylate on the cochlea, peripheral nerves, and kidney in a rat model of breast cancer--Fisher344 rats implanted with highly metastatic MTLn3 breast cancer cells. Animals received 3 x 5 mg CDDP/kg (given every third day), and salicylate was administered at 100 mg/kg (bid) from 2 days before to 3 days after CDDP treatment. Salicylate significantly attenuated the CDDP-induced threshold shift from approximately 20 dB (at 16 and 24 kHz) to approximately 5 dB, and drastically reduced the loss of cochlear outer hair cells. Likewise, salicylate protected kidney function (measured as plasma blood urea nitrogen and creatinine levels) from CDDP toxicity. Protection of nerve conduction velocities of both sensory and motor nerves was minimal. The chemotherapeutic efficacy of CDDP on suppression of tumor mass and cancer cell metastasis remained unaffected by salicylate. The results suggest that administration of salicylate may become the basis of an effective therapeutic intervention against the ototoxic and nephrotoxic side effects associated with CDDP chemotherapy.

In order to understand the growth and compaction behaviour of chalcopyrite (copper concentrate), batch granulation tests were carried out using a rotating drum. The granule growth exhibited induction-type behaviour, as defined by Iveson and Litster [AIChE J. 44 (1998) 15 10]. There were two consecutive stages during granulation: the induction stage, during which the granules are gradually being compacted and little or no growth occurs, and the rapid growth stage, which starts when the granules have become surface wet and are rapidly growing. In agreement with earlier findings. an increased amount of binder liquid shortened the induction time. The compaction behaviour was also investigated. A displaced volume method was adopted to determine the porosity of the granules. It was shown that this technique had a limitation as it was unable to detect the reduction of the volumes of the granule pores after the granules had become surface wet. Due to this, some of the measurements were not suited for fitting a three-parameter empirical model. Attempts were made to determine whether the rapid growth stage started with the pore saturation exceeding a certain critical value, but due to the scatter in the porosity measurements and the fact that some of the measurements could not be used, it was not possible to determine a critical pore saturation, However, the porosity measurements clearly demonstrated that the porosity of the granules decreased during the induction stage of an experiment and that when rapid growth occurred, the granules had a pore saturation was around 0.85. This value was slightly lower than unity, which is most likely due to trapped air bubbles. (C) 2002 Published by Elsevier Science B.V.

Objectives:(1) Describe the otoprotective mechanisms involved in common otoprotective drugs (ie, NAC, dexamethasone, and mannitol). (2) Describe the otoprotective effect of combining these drugs to prevent hair cell (HC) losses.Methods:Cochlear explants were dissected from P-3 rats and placed in serum-free media. Explants were divided into 3 groups: (1) untreated controls; (2) electrode insertion trauma (EIT); (3) Tri-therapy (L-NAC + DXM + mannitol). Cochlea of groups 2 and 3 were implanted and HC counts, oxidative stress and cleaved caspase-3 markers were studied in all explants post EIT.Results:There was a significant increase of HC loss in the EIT explants as opposed to control or the tri-therapy cochlea. The implantation resulted in an increased production of the total reactive oxygen species (ROS) in both HCs and the supporting cells (SCs) of only EIT group. There is also cleaved caspase-3 activation in EIT cochlea as compared to the control or tri-therapy cochlea.Conclusions:The tri-therapy combini...

Objectives: Evaluate the efficacy of combined antistaphylococcal and anti-pseudomonal preoperative antibiotics for preventing surgical site infections following tympanoplasty with mastoidectomy for contaminated cholesteatoma. Methods: Retrospective chart review of medical records of patients who underwent tympanoplasty with mastoidectomy for cholesteatoma were reviewed. Only cases considered to have contaminated or dirty/infected surgical fields were included. The primary outcome measure was postoperative perichondritis, pinna abscess, periotic cellulitis, and periotic abscess requiring systemic antibiotics or surgical intervention. Results: Charts of 362 patients who underwent tympanoplasty with mastoidectomy were reviewed, and 195 met inclusion criteria. Preoperative antibiotics included single doses of clindamycin and ceftazidime or gentamicin prior to incision. Patients that received no preoperative antibiotics had an infection rate of 11% and those given perioperative antibiotics had a rate of 1% (P = .02). Conclusions: Use of preoperative antibiotics covering both staphylococcus and pseudomonas species appears to be efficacious for the prevention of surgical site infections following tympanoplasty with mastoidectomy for contaminated cholesteatoma.

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.

CONFOCAL analysis of early postnatal auditory neurones in a bicompartmental culture system was used to test for chemoattractant properties of NGF, BDNF and NT-3 on neuronal process outgrowth. NT-3 exerted a strong tropic effect on neuritic outgrowth from auditory neurones in this system. BDNF and NGF did not have any tropic activity that directed processes outgrowth from auditory neurones. However, BDNF was important for the support of neuronal survival in NGF-treated cultures and for neuritogenesis in NT-3-treated cultures. Since NT-3 has been identified as both a survival factor and a chemotropic agent for auditory neurones, it is likely that this neurotrophin will be a useful therapeutic agent in the treatment of damaged cochleae for the recovery of hearing.

SUMMARY The neuronal survival promoting ability of brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), individually and in combination, was evaluated in dissociated cell cultures of postnatal day 5 (P5) rat acoustic ganglia. The neuritogenic promoting effect of these same neurotrophic factors was examined in organotypic explants of P5 rat acoustic ganglia. The results showed that BDNF was maximally effective at a concentration of 10 ng /mL in promoting both survival and neuritogenesis of these postnatal auditory neurons in vifro. CNTF was maximally effective at a concentration of 0.01 ng/mL at promoting both survival and neuritogenesis in the acoustic ganglion cultures. BDNF had its strongest effect on neuronal survival while CNTF was most effective in stimulating neurite outgrowth. These two neurotrophic factors, when added together at their respective maximally effective concentrations, behave in an additive manner for promoting both survival and neu

Destruction of auditory hair cells results in a subsequent loss of auditory neurons. In situ hybridization and neuronal cell culture studies as well as analyses of the inner ears of neurotrophin and neurotrophin receptor gene knockout mice have shown that NT-3 and BDNF mediate both the development and survival of auditory neurons. In this study guinea pigs were exposed to the ototoxic combination of an aminoglycoside antibiotic and a loop diuretic and then received 8 weeks of intracochlear infusion of either NT-3, BDNF or NT-3 + BDNF to determine whether site-specific application of these neurotrophins could prevent the loss of auditory neurons that follows a loss of auditory hair cells. Infusion of either NT-3 or NT-3 + BDNF into the scala tympani resulted in a > 90% survival of auditory neurons while BDNF infusion yielded a 78% survival rate, compared with a 14-24% neuronal survival rate in untreated ototoxin-exposed cochleae. These results show that loss of auditory neurons that occurs subsequent to a loss of auditory hair cells can be prevented by in vivo neurotrophin therapy with either NT-3 or BDNF.

Loss of auditory neurons is commonly associated with sensorineural deafness, and may result from either direct neuronal injury or be a consequence of sensory hair cell loss (i.e. loss of a source of trophic factors). Developmental studies and in vitro studies of adult auditory neurons have begun to identify growth factors important for the development, maintenance, and rescue/repair of auditory neurons. Specific neurotrophic factors have been shown to enhance the auditory neurons' ability to withstand traumatic loss of target tissue connections and toxic injury. Promising initial in vivo studies confirm that specific neurotrophic are able to support neuronal survival and promote neuronal repair in an intact animal following injury to the cochlea. Further study into unique methods and routes of growth factor delivery will provide insights into the possibility of neurotrophic growth factors to act as drugs for the treatment of injured or stressed auditory neurons.

Objective: 1) Understand the role of Bain tumor necrosis factor alpha (TNFα)-induced auditory hair cell (HC) death in rat organ of Corti (OC) explants in vitro 2. Determine the effectiveness of short interfering RNA that targets Bax mRNA (Bax siRNA) against TNFα-induced HC loss.Method: OC explants dissected from 3-day-old rats were cultured in control media, TNFα, TNFα+BasiRNA, or Bax siRNA for 0, 48, and 96 hours in vitro. Real-time PCR, ELISA, HC viability studies, immunofluorescence, and confocal microscopy were performed. ANOVA with post hoc testing was used with P value set

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-1β 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.

Sensorineural hearing loss resulting from the loss of auditory hair cells is thought to be irreversible in mammals. This study provides evidence that retinoic acid can stimulate the regeneration in vitro of mammalian auditory hair cells in ototoxic-poisoned organ of Corti explants in the rat. In contrast, treatment with retinoic acid does not stimulate the formation of extra hair cells in control cultures of Corti's organ. Retinoic acid-stimulated hair cell regeneration can be blocked by cytosine arabinoside, which suggests that a period of mitosis is required for the regeneration of auditory hair cells in this system. These results provide hope for a recovery of hearing function in mammals after auditory hair cell damage.

Neuronotrophic factors (e.g. basic fibroblast growth factor, bFGF and nerve growth factor, NGF) have been demonstrated to respectively promote survival and neuritogenesis in cultures of dissociated adult rat spiral ganglia. Transforming growth factor beta (TGF beta 1) has been shown to modulate the response of cultured auditory neurons to bFGF through the induction of high affinity receptors for bFGF in the neurons. In this study, we show that TGF beta is expressed in situ by adult auditory neurons in response to traumatic injury (i.e. transection of the eighth cranial nerve). Based on these in vivo results and on the results from our previous in vitro studies, we propose that TFG beta 1 acts as an early autocrine signal involved in the response to injury by neurons of the peripheral auditor system.

Triamcinolone acetonide crystalline suspension (e.g. Volon A) was not ototoxic to the auditory hair cells present within organ of Corti explants and protected them from an ototoxic molecule, i.e. 4-hydroxy-2,3-nonenal (HNE), that is produced within the organ of Corti as a result of oxidative stress-induced damage.To test the corticosteroid, triamcinolone acetonide, for ototoxicity and otoprotective capacity in organ of Corti explants.Organ of Corti explants excised from 4-day-old rats were the test system, HNE was the ototoxin challenge. Hair cell integrity counts were performed with fluorescent microscopy on fixed explants stained with FITC-labeled phalloidin. Statistical significance was set at p0.05.Triamcinolone acetonide did not affect hair cell integrity in the organ of Corti explants and it provided a high level of protection of hair cells against the ototoxic effects of a damaging level of HNE as determined by hair cell density counts.

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.

RT-PCR was used to assay for growth factors and receptors from seven different protein families in cochlea tissues of the juvenile rat. There was a broad representation of the growth factor families in all the cochlea tissues examined, though the organ of Corti and stria vascularis expressed a greater variety than the spiral ganglion. This broad expression suggests that a variety of known growth factors play significant roles in the development, maintenance, and repair of the inner ear. The results of this survey serve as a basis for the design of future in vitro experiments that will address the ability of growth factors to protect hair cells from damage and to evoke a repair-regeneration response by injured hair cells.

The neuronal survival promoting ability of brain derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF), individually and in combination, was evaluated in dissociated cell cultures of postnatal day 5 (P5) rat acoustic ganglia. The neuritogenic promoting effect of these same neurotrophic factors was examined in organotypic explants of P5 rat acoustic ganglia. The results showed that BDNF was maximally effective at a concentration of 10 ng/mL in promoting both survival and neuritogenesis of these postnatal auditory neurons in vitro. CNTF was maximally effective at a concentration of 0.01 ng/mL at promoting both survival and neuritogenesis in the acoustic ganglion cultures. BDNF had its strongest effect on neuronal survival while CNTF was most effective in stimulating neurite outgrowth. These two neurotrophic factors, when added together at their respective maximally effective concentrations, behave in an additive manner for promoting both survival and neuritic outgrowth by the auditory neurons.

The objective of this study was to explore the hypothesis (Van De Water, 1976) that differentiating sensory receptors of the inner ear may attract ingrowing neurites of the statoacoustic (VIIIn) ganglion by chemotaxis. Co-cultured embryonic inner ears which shared a single VIIIn ganglion were grown "in vitro" to the equivalent of 20 days gestation and then processed histologically to show both cytodifferentiation of sensory structures and the presence of neural elements. Specimens of both 11- and 12.5-day-old co-cultured otocysts showed that VIIIn ganglion neurites grew into sensory receptors of both(+) with and (-) without ganglion inner ear explants. Fourteen-day-old co-cultured inner ears revealed that only the (+) ganglion inner ear explants received VIIIn ganglion neurites into the sensory areas, and that neurites were not attracted into the (-) ganglion explants. The results were found to support the hypothesis of a limited period of chemotaxis as being a possible mechanism for the establishment of the pattern of innervation of inner ear sensory receptors by its VIIIn ganglion.

The 16th day mouse embryo inner ear was ex-planted and cultured “in vitro” for 6 days, which corresponds to 1 day post-partum development “in vivo”. A normal development occurred both when the inner ear anlage was divided into a vestibular and a cochlear portion and when it was cultured as a whole. The present model system may provide a helpful tool in the study of both normal development and inner ear pathology.

Preliminary observations of the effect of surgical excision of the endolymphatic duct and sac anlage in normal mouse embryo inner ear embryogenesis "in vitro" are reported. Otocysts of twelfth and thirteenth gestation day mouse embryos were grown in organ culture for 9 and 8 days respectively. The organ culture specimens of each gestational age group were further divided into otocysts with intact endolymphatic duct and sac anlages and otocysts that had their endolymphatic duct and sac anlages surgically excised prior to explantation. The resultant histological quantification of inner ear embryogenesis did not reveal any significant differences in morphogenesis, histodifferentiation of inner ear sensory structures or presence of endolymphatic hydrops in any of the organ culture specimens. It was concluded that in the system studied the absence of the endolymphatic duct and sac anlage did not have an effect on the organogenesis of the inner ear with the exception of the resultant lack of development of an endolymphatic duct and sac.

The embryonic development of the crista ampullaris in the CBA/CBA mouse was followed comparatively in vivo and in vitro after explanation of the 13th gestational day otocyst. Special attention was paid to hair cell differentiation. In general the in vivo and the in vitro groups of inner ears showed a similar morphological maturation of hair cells on the ultrastructural level. The first sign of hair cell differentiation occurred on the 14th day in vivo and after 1 day in culture showing a thickening of microvilli presumably indicating the development of future sensory hairs. On the 16th day or after 3 days of in vitro culture the sensory hairs were regularely arranged with the kinocilium located at the cell periphery. Afferent nerve endings were identified on the 17th gestational day and some nerve terminals were suspected to be of efferent character appearing on the 18th day. On the day of birth there did not occur complete nerve chalices but a differentiation of hair cells into type I and II was indicated on the 19th gestational day by a slight difference in hair cell configuration and extension of innervation.