Nanofibrous scaffolds for the guidance of stem cell-derived neurons for auditory nerve regeneration.

Impairment of spiral ganglion neurons (SGNs) of the auditory nerve is a major cause for hearing loss occurring independently or in addition to sensory hair cell damage. Unfortunately, mammalian SGNs lack the potential for autonomous regeneration. Stem cell based therapy is a promising approach for auditory nerve regeneration, but proper integration of exogenous cells into the auditory circuit remains a fundamental challenge. Here, we present novel nanofibrous scaffolds designed to guide the integration of human stem cell-derived neurons in the internal auditory meatus (IAM), the foramen allowing passage of the spiral ganglion to the auditory brainstem. Human embryonic stem cells (hESC) were differentiated into neural precursor cells (NPCs) and seeded onto aligned nanofiber mats. The NPCs terminally differentiated into glutamatergic neurons with high efficiency, and neurite projections aligned with nanofibers in vitro. Scaffolds were assembled by seeding GFP-labeled NPCs on nanofibers integrated in a polymer sheath. Biocompatibility and functionality of the NPC-seeded scaffolds were evaluated in vivo in deafened guinea pigs (Cavia porcellus). To this end, we established an ouabain-based deafening procedure that depleted an average 72% of SGNs from apex to base of the cochleae and caused profound hearing loss. Further, we developed a surgical procedure to implant seeded scaffolds directly into the guinea pig IAM. No evidence of an inflammatory response was observed, but post-surgery tissue repair appeared to be facilitated by infiltrating Schwann cells. While NPC survival was found to be poor, both subjects implanted with NPC-seeded and cell-free control scaffolds showed partial recovery of electrically-evoked auditory brainstem thresholds. Thus, while future studies must address cell survival, nanofibrous scaffolds pose a promising strategy for auditory nerve regeneration.

The Role of Glia in the Peripheral and Central Auditory System Following Noise Overexposure: Contribution of TNF-α and IL-1ß to the Pathogenesis of Hearing Loss.

Repeated noise exposure induces inflammation and cellular adaptations in the peripheral and central auditory system resulting in pathophysiology of hearing loss. In this study, we analyzed the mechanisms by which noise-induced inflammatory-related events in the cochlea activate glial-mediated cellular responses in the cochlear nucleus (CN), the first relay station of the auditory pathway. The auditory function, glial activation, modifications in gene expression and protein levels of inflammatory mediators and ultrastructural changes in glial-neuronal interactions were assessed in rats exposed to broadband noise (0.5-32 kHz, 118 dB SPL) for 4 h/day during 4 consecutive days to induce long-lasting hearing damage. Noise-exposed rats developed a permanent threshold shift which was associated with hair cell loss and reactive glia. Noise-induced microglial activation peaked in the cochlea between 1 and 10D post-lesion; their activation in the CN was more prolonged reaching maximum levels at 30D post-exposure. RT-PCR analyses of inflammatory-related genes expression in the cochlea demonstrated significant increases in the mRNA expression levels of pro- and anti-inflammatory cytokines, inducible nitric oxide synthase, intercellular adhesion molecule and tissue inhibitor of metalloproteinase-1 at 1 and 10D post-exposure. In noise-exposed cochleae, interleukin-1ß (IL-1ß), and tumor necrosis factor α (TNF-α) were upregulated by reactive microglia, fibrocytes, and neurons at all time points examined. In the CN, however, neurons were the sole source of these cytokines. These observations suggest that noise exposure causes peripheral and central inflammatory reactions in which TNF-α and IL-1ß are implicated in regulating the initiation and progression of noise-induced hearing loss.

Antioxidant vitamins and magnesium and the risk of hearing loss in the US general population.

BACKGROUND: The protective effects of antioxidant vitamins on hearing loss are well established in animal studies but in few human studies. Recent animal studies suggest that magnesium intake along with antioxidants may act in synergy to prevent hearing loss. OBJECTIVE: We examined associations between intake of antioxidant vitamins (daily ß-carotene and vitamins C and E) and magnesium and hearing thresholds and explored their joint effects in US adults. DESIGN: We analyzed cross-sectional data from 2592 participants aged 20-69 y from NHANES 2001-2004. Hearing thresholds as pure tone averages (PTAs) at speech (0.5, 1, 2, and 4 kHz) and high frequencies (3, 4, and 6 kHz) were computed. RESULTS: When examined individually, modeled as quartiles, and after adjustment for potential confounders, higher intakes of ß-carotene, vitamin C, and magnesium were associated with lower (better) PTAs at both speech and high frequencies. High intakes of ß-carotene or vitamin C combined with high magnesium compared with low intakes of both nutrients were significantly associated with lower (better) PTAs at high frequencies (-14.82%; 95% CI: -20.50% to -8.74% for ß-carotene + magnesium and -10.72%; 95% CI: -16.57% to -4.45% for vitamin C + magnesium). The estimated joint effects were borderline significantly larger than the sums of the individual effects [high ß-carotene/low magnesium (-4.98%) and low ß-carotene/high magnesium (-0.80%), P-interaction = 0.08; high vitamin C/low magnesium (-1.33%) and low vitamin C/high magnesium (2.13%), P-interaction = 0.09]. CONCLUSION: Dietary intakes of antioxidants and magnesium are associated with lower risks of hearing loss.

Nestin-expressing cells in the developing, mature and noise-exposed cochlear epithelium.

The auditory sensory epithelium in non-mammalian vertebrates can replace lost hair cells by transdifferentiation of supporting cells, but this regenerative ability is lost in the mammalian cochlea. Future cell-based treatment of hearing loss may depend on stem cell transplantation or on transdifferentiation of endogenous cells in the cochlea. For both approaches, identification of cells with stem cell features within the mature cochlea may be useful. Here we use a Nestin-ß-gal mouse to examine the presence of Nestin positive cells in the mature auditory epithelium, and determine how overstimulation of the ear impacts these cells. Nestin positive cells were found in the apical turn of the cochlea lateral to the outer hair cell area. This pattern of expression persisted into mature age. The area of Nestin positive cells was increased after the noise lesion. This increase in area coincided with an increase in expression of the Nestin mRNA. The data suggest that cells with potential stem cell features remain in the mature mammalian cochlea, restricted to the apical turn, and that an additional set of signals is necessary to trigger their contribution to cell replacement therapy in the ear. As such, this population of cells could serve to generate cochlear stem cells for research and potential therapy, and may be a target for treatments based on induced transdifferentiation of endogenous cochlear cells.

Wnt signaling promotes neuronal differentiation from mesenchymal stem cells through activation of Tlx3.

Wnt/ß-catenin signaling promotes neural differentiation by activation of the neuron-specific transcription factors, Neurogenin1 (Ngn1), NeuroD, and Brn3a, in the nervous system. As neurons in cranial sensory ganglia and dorsal root ganglia transiently express Ngn1, NeuroD, and Brn3a during embryonic development, we hypothesized that Wnt proteins could instructively promote a sensory neuronal fate from mesenchymal stem cells (MSCs) directed to differentiate into neurons. Consistent with our hypothesis, Wnt1 induced expression of sensory neuron markers including Ngn1, NeuroD, and Brn3a, as well as glutamatergic markers in neurally induced MSCs in vitro and promoted engraftment of transplanted MSCs in the inner ear bearing selective loss of sensory neurons in vivo. Given the consensus function of T-cell leukemia 3 (Tlx3), as a glutamatergic selector gene, we postulated that the effects of canonical Wnt signaling on sensory neuron and glutamatergic marker gene expression in MSCs may be mediated by Tlx3. We first confirmed that Wnt1 indeed upregulates Tlx3 expression, which can be suppressed by canonical Wnt inhibitors. Next, our chromatin immunoprecipitation assays revealed that T-cell factor 3/4, Wnt-activated DNA binding proteins, interact with a regulatory region of Tlx3 in MSCs after neural induction. Furthermore, we demonstrated that forced expression of Tlx3 in MSCs induced sensory and glutamatergic neuron markers after neural induction. Together, these results identify Tlx3 as a novel target for canonical Wnt signaling that confers somatic stem cells with a sensory neuron phenotype upon neural induction.

Safety of ciprofloxacin and dexamethasone in the guinea pig middle ear.

OBJECTIVE: To investigate the ototoxic potential of ciprofloxacin hydrochloride, 0.3%, plus dexamethasone, 0.1%, after administration to the guinea pig middle ear. DESIGN: Fifty guinea pigs were randomly assigned to 4 test groups of 10 animals each and 2 control groups of 5 animals each. The 4 test groups were treated twice daily for 4 weeks with 10 muL of (1) ciprofloxacin hydrochloride, 0.3%, plus dexamethasone, 0.1%; (2) ciprofloxacin hydrochloride, 1.0%, plus dexamethasone, 0.3%; (3) ciprofloxacin hydrochloride, 0.3%, or (4) vehicle. The positive and negative control groups were treated with neomycin sulfate, 10%, or isotonic sodium chloride solution, respectively. SETTING: Academic research laboratory. INTERVENTIONS: Study animals were implanted with a drug delivery cannula to the middle ear, terminating in the round window niche for direct delivery to the round window membrane. MAIN OUTCOME MEASURES: Auditory brainstem responses were collected at baseline and following 2 and 4 weeks of dosing. At the termination of the study, inner ear tissues were evaluated microscopically. RESULTS: No biologically relevant hearing losses were observed after either 2 or 4 weeks of treatment with vehicle, ciprofloxacin alone, or combinations of ciprofloxacin plus dexamethasone. Examination of the organ of Corti revealed normal hair cell counts in all animals that received isotonic sodium chloride solution, vehicle, ciprofloxacin, or combinations of ciprofloxacin and dexamethasone. Conversely, the neomycin sulfate positive control group demonstrated a significant elevation in hearing threshold and profound hair cell loss (P <.001, P = .02, and P <.001 at 2, 8, and 16 kHz, respectively). CONCLUSION: The results of this preclinical study support the safety of ciprofloxacin hydrochloride, 0.3%, plus dexamethasone, 0.1%, for clinical use in the open middle ear cavity.

Functional evaluation of a cell replacement therapy in the inner ear.

HYPOTHESIS: Cell replacement therapy in the inner ear will contribute to the functional recovery of hearing loss. BACKGROUND: Cell replacement therapy is a potentially powerful approach to replace degenerated or severely damaged spiral ganglion neurons. This study aimed at stimulating the neurite outgrowth of the implanted neurons and enhancing the potential therapeutic of inner ear cell implants. METHODS: Chronic electrical stimulation (CES) and exogenous neurotrophic growth factor (NGF) were applied to 46 guinea pigs transplanted with embryonic dorsal root ganglion (DRG) neurons 4 days postdeafening. The animals were evaluated with the electrically evoked auditory brainstem responses (EABRs) at experimental Days 7, 11, 17, 24, and 31. The animals were euthanized at Day 31, and the inner ears were dissected for immunohistochemistry investigation. RESULTS: Implanted DRG cells, identified by enhanced green fluorescent protein fluorescence and a neuronal marker, were found close to Rosenthal canal in the adult inner ear for up to 4 weeks after transplantation. Extensive neurite projections clearly, greater than in nontreated animals, were observed to penetrate the bony modiolus and reach the spiral ganglion region in animals supplied with CES and/or NGF. There was, however, no significant difference in the thresholds of EABRs between DRG-transplanted animals supplied with CES and/or NGF and DRG-transplanted animals without CES or NGF supplement. CONCLUSION: The results suggest that CES and/or NGF can stimulate neurite outgrowth from implanted neurons, although based on EABR measurement, these interventions did not induce functional connections to the central auditory pathway. Additional time or novel approaches may enhance functional responsiveness of implanted cells in the adult cochlea.

Glutamatergic neuronal differentiation of mouse embryonic stem cells after transient expression of neurogenin 1 and treatment with BDNF and GDNF: in vitro and in vivo studies.

Differentiation of the pluripotent neuroepithelium into neurons and glia is accomplished by the interaction of growth factors and cell-type restricted transcription factors. One approach to obtaining a particular neuronal phenotype is by recapitulating the expression of these factors in embryonic stem (ES) cells. Toward the eventual goal of auditory nerve replacement, the aim of the current investigation was to generate auditory nerve-like glutamatergic neurons from ES cells. Transient expression of Neurog1 promoted widespread neuronal differentiation in vitro; when supplemented with brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), 75% of ES cell-derived neurons attained a glutamatergic phenotype after 5 d in vitro. Mouse ES cells were also placed into deafened guinea pig cochleae and Neurog1 expression was induced for 48 h followed by 26 d of BDNF/GDNF infusion. In vivo differentiation resulted in 50-75% of ES cells bearing markers of early neurons, and a majority of these cells had a glutamatergic phenotype. This is the first study to report a high percentage of ES cell differentiation into a glutamatergic phenotype and sets the stage for cell replacement of auditory nerve.

Stem cell transplantation for auditory nerve replacement.

The successful function of cochlear prostheses depends on activation of auditory nerve. The survival of auditory nerve neurons, however, can vary widely in candidates for cochlear implants and influence implant efficacy. Stem cells offer the potential for improving the function of cochlear prostheses and increasing the candidate pool by replacing lost auditory nerve. The first phase of studies for stem cell replacement of auditory nerve has examined the in vitro survival and differentiation as well as in vivo differentiation and survival of exogenous embryonic and tissue stem cells placed into scala tympani and/or modiolus. These studies are reviewed and new results on in vivo placement of B-5 mouse embryonic stem cells into scala tympani of the guinea pig cochleae with differentiation into a glutamatergic neuronal phenotype are presented. Research on the integration and connections of stem cell derived neurons in the cochlea is described. Finally, an alternative approach is considered, based on the use of endogenous progenitors rather than exogenous stem cells, with a review of promising findings that have identified stem cell-like progenitors in cochlear and vestibular tissues to provide the potential for auditory nerve replacement.

Glial cell line-derived neurotrophic factor and antioxidants preserve the electrical responsiveness of the spiral ganglion neurons after experimentally induced deafness.

Cochlear implant surgery is currently the therapy of choice for profoundly deaf patients. However, the functionality of cochlear implants depends on the integrity of the auditory spiral ganglion neurons. This study assesses the combined efficacy of two classes of agents found effective in preventing degeneration of the auditory nerve following deafness, neurotrophic factors, and antioxidants. Guinea pigs were deafened and treated for 4 weeks with either local administration of GDNF or a combination of GDNF and systemic injections of the antioxidants ascorbic acid and Trolox. The density of surviving spiral ganglion cells was significantly enhanced and the thresholds for eliciting an electrically evoked brain stem response were significantly reduced in GDNF treated animals compared to deafened-untreated. The addition of antioxidants significantly enhanced the evoked responsiveness over that observed with GDNF alone. The results suggest multiple sites of intervention in the rescue of these cells from deafferentation-induced cell death.

Bcl-2 genes regulate noise-induced hearing loss.

Proteins of the Bcl-2 family have been implicated in control of apoptotic pathways modulating neuronal cell death, including noise-induced hearing loss. In this study, we assessed the expressions of anti- and proapoptotic Bcl-2 genes, represented by Bcl-xL and Bak following noise exposures, which yielded temporary threshold shift (TTS) or permanent threshold shift (PTS). Auditory brainstem responses (ABRs) were assessed at 4, 8, and 16 kHz before exposure and on days 1, 3, 7, and 10 following exposure to 100 dB SPL, 4 kHz OBN, 1 hr (TTS) or 120 dB SPL, 4 kHz OBN, 5 hr (PTS). On day 10, subjects were euthanized. ABR thresholds increased following both exposures, fully recovered following the TTS exposure, and showed a 22.6 dB (4 kHz), 42.5 dB (8 kHz), and 44.9 dB (16 kHz) mean shift on day 10 following the PTS exposure. PTS was accompanied by outer hair cell loss progressing epically and basally from the 4-kHz region. Additional animals were euthanized for immunohistochemical assessment. BcL-xL was robustly expressed in outer hair cells following TTS exposure, whereas Bak was expressed following PTS exposure. These results indicate an important role of the Bcl-2 family proteins in regulating sensory cell survival or death following intense noise. Bcl-xL plays an essential role in prevention of sensory cell death following TTS levels of noise, and PTS exposure provokes the expression of Bak and, with that, cell death.

Delayed neurotrophin treatment following deafness rescues spiral ganglion cells from death and promotes regrowth of auditory nerve peripheral processes: effects of brain-derived neurotrophic factor and fibroblast growth factor.

The extent to which neurotrophic factors are able to not only rescue the auditory nerve from deafferentation-induced degeneration but also promote process regrowth is of basic and clinical interest, as regrowth may enhance the therapeutic efficacy of cochlear prostheses. The use of neurotrophic factors is also relevant to interventions to promote regrowth and repair at other sites of nerve trauma. Therefore, auditory nerve survival and peripheral process regrowth were assessed in the guinea pig cochlea following chronic infusion of BDNF + FGF(1) into scala tympani, with treatment initiated 4 days, 3 weeks, or 6 weeks after deafferentation from deafening. Survival of auditory nerve somata (spiral ganglion neurons) was assessed from midmodiolar sections. Peripheral process regrowth was assessed using pan-Trk immunostaining to selectively label afferent fibers. Significantly enhanced survival was seen in each of the treatment groups compared to controls receiving artificial perilymph. A large increase in peripheral processes was found with BDNF + FGF(1) treatment after a 3-week delay compared to the artificial perilymph controls and a smaller enhancement after a 6-week delay. Neurotrophic factor treatment therefore has the potential to improve the benefits of cochlear implants by maintaining a larger excitable population of neurons and inducing neural regrowth.

Creatine and tempol attenuate noise-induced hearing loss.

To define the role of free radical formation and potential energy depletion in noise induced hearing loss (NIHL), we measured the effectiveness of tempol (free radical scavenger) and creatine (enhances cellular energy storage) alone and in combination to attenuate NIHL. Guinea pigs were divided into four treatment groups: controls, 3% creatine diet (2 weeks prior to noise exposure), tempol (3 mM in drinking water 2 weeks prior to exposure), and creatine plus tempol and exposed to 120 dB SPL one-octave band noise centered at 4 kHz for 5 h. The noise-only control group showed frequency-dependent auditory threshold shifts (measured by auditory brainstem response, ABR) of up to 73 dB (16 kHz) on day 1, and up to 50 dB (8 kHz) on day 10. Creatine-treated subjects had significantly smaller ABR threshold shifts on day 1 and on day 10. Tempol alone significantly reduced ABR threshold shifts on day 10 but not on day 1. ABR shifts after combination treatment were similar to those in the creatine group. Hair cell loss on day 10 was equally attenuated by creatine and tempol alone or in combination. Our results indicate that the maintenance of ATP levels is important in attenuating both temporary and permanent NIHL, while the scavenging of free radicals provides protection from permanent NIHL.

Regeneration of human auditory nerve. In vitro/in video demonstration of neural progenitor cells in adult human and guinea pig spiral ganglion.

Time lapse video recordings of cultured adult human and guinea pig spiral ganglion (hSG and gpSG) show that mitogen responsive progenitor/stem cells develop in the form of spheres that proliferate and differentiate into mature neurons and glia cells. Neurospheres, cultured with EGF and bFGF showed expression of nestin and incorporation of 5'-Bromo-2-deoxyuridine (BrdU). Newly formed BrdU labelled cells were positive for beta-tubulin, and also for GFAP demonstrating that neuronal cells were derived from a dividing population of progenitor cells. Dissociated spheres cultured either with glia cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), induced differentiation of the progenitor cells. Video microscopy showed that neurons develop from subcultured spheres maintained for up to four weeks. Neurons showed fasciculation and migration with a speed of 10-30 microm/h, and some cells had up to 6 mm long neurites coexpressing TrkB and TrkC receptors. Precise dissection suggests that the neurons formed are cochlea-specific. The results suggest that the mammalian auditory nerve has the capability for self-renewal and replacement. Transplantation of progenitor cells together with established means to induce neural differentiation and fiber growth may facilitate strategies for better repair and treatment of auditory neuronal damage.

Calcineurin activation contributes to noise-induced hearing loss.

Acoustic overstimulation increases Ca(2+) concentration in auditory hair cells. Because calcineurin is known to activate cell death pathways and is controlled by Ca(2+) and calmodulin, this study assessed the role of calcineurin in auditory hair cell death in guinea pigs after intense noise exposure. Immediately after noise exposure (4-kHz octave band, 120 dB, for 5 hr), a population of hair cells exhibited calcineurin immunoreactivity at the cuticular plate, with a decreasing number of positive-stained cells on Days 1-3. By Day 7, the levels of calcineurin immunoreactivity had diminished to near control, non-noise exposed values, concomitant with an increasing loss of hair cells. Staining of hair cell nuclei with propidium iodide (PI), restricted to calcineurin-immunopositive cells, indicated breakdown of cell membranes symptomatic of incipient cell death. The local application of the calcineurin inhibitors, FK506 and cyclosporin A, reduced the level of noise-induced auditory brain stem response threshold shift and hair cell death, indicating that calcineurin is a factor in noise-induced hearing loss. The results suggest that calcineurin inhibitors are of potential therapeutic value for long-term protection of the morphologic integrity and function of the organ of Corti against noise trauma.

Comparison of the protective efficacy of neurotrophins and antioxidants for vibration-induced trauma.

BACKGROUND: Patients undergoing temporal bone surgery or subjects working with vibrating tools may develop vibration-induced hearing loss (VHL). The aim of this study was to characterize the effects of pretreatment with N-acetylcysteine (NAC) or the neurotrophic factors, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), on VHL in an animal model. METHODS: Trauma to the cochlea was created with a vibrating probe placed on the bone of the external ear canal. BDNF and CNTF(Ax1) were delivered into the cochlea with mini-osmotic pumps. NAC was delivered into the cochlea by round window membrane (RWM) injection, by RWM permeation, or by oral administration. Hearing was evaluated with electrocochleography (ECoG). RESULTS: For control animals, vibration resulted in an average immediate threshold shift of 42 +/- 26 dB. NAC provided no protective benefit in animals subjected to VHL, regardless of the delivery method, with average threshold shifts varying from 38 to 56 dB across groups. NAC injection through the round window membrane was toxic, causing a ECoG threshold shift of >25 dB. In BDNF+CNTF(Ax1)-treated animals, immediate hearing loss was similar to that in control animals. There was a trend of threshold recovery by 1 day after vibration; however, the improvement was not statistically significant, nor was there a significant difference in 1-day thresholds across groups. CONCLUSIONS: Local infusion of BDNF and CNTF(Ax1) may enhance the rate of recovery from VHL, compared to control animals. In contrast, NAC had no effect on VHL, and when delivered by RWM injection, was actually toxic to the inner ear.

8-iso-prostaglandin F(2alpha), a product of noise exposure, reduces inner ear blood flow.

Noise exposure induces the formation in the cochlea of 8-isoprostaglandin F(2alpha) (8-iso-PGF(2alpha)), a marker for reactive oxygen species [Ohinata et al., 2000a] and a potent vasoconstrictor, raising the possibility that 8-iso-PGF(2alpha) may be responsible for noise-induced reductions in cochlear blood flow (CBF). To test this hypothesis, CBF was assessed in the guinea pig in response to 'local' (via the anterior inferior cerebellar artery) and systemic (i.v.) delivery of 8-iso-PGF(2alpha) using laser Doppler flowmetry. Local 8-iso-PGF(2alpha) induced a clear reduction in CBF. With systemic infusion, vascular conductance (VC), the ratio of CBF to systemic blood pressure, decreased in a dose-dependent manner up to 30%, consistent with an 8-iso-PGF(2alpha)-induced constriction of the cochlear vasculature. Infusion of SQ29548, a specific antagonist of 8-iso-PGF(2alpha), appropriately blocked an 8-iso-PGF(2alpha)-induced CBF response. Similarly, noise-induced changes in CBF and VC were prevented by infusion of SQ29548 during noise exposure or by antioxidant treatment (glutathione monoethyl ester) prior to exposure. Prevention of isoprostane-mediated vasoconstriction may have clinical utility in the protection from noise-induced hearing loss.

Systemic co-treatment with alpha-melanocyte stimulating hormone delays hearing loss caused by local cisplatin administration in guinea pigs.

It has previously been demonstrated that ototoxicity induced by systemic administration of cisplatin is reduced by concomitant administration of melanocortins, like alpha-melanocyte stimulating hormone (alpha-MSH). However, these experiments were hampered by large interanimal variability. Therefore, we re-investigated the effects of systemically administered alpha-MSH during local (intracochlear) administration of cisplatin. Guinea pigs, implanted with a round-window electrode, allowing daily monitoring of the compound action potentials (CAPs), and a mini-osmotic pump, pumping either 0.5 microl/h physiological saline or cisplatin solution (15 microg/ml), were co-treated daily with a subcutaneous bolus injection of either alpha-MSH (75 microg/kg) or physiological saline for 1 week or until the electrocochleogram showed a persistent decrease in CAP amplitude (40 dB threshold shift at 8 kHz). Next, the animals were sacrificed and the cochleas were processed for histology. After 2-3 days, cisplatin alone caused a threshold shift at all frequencies (2-16 kHz). Co-administration with alpha-MSH consistently delayed the criterion threshold shift by 1 day. When the 40 dB criterion had been reached, similar outer hair cell losses in both the cisplatin/alpha-MSH- and cisplatin/saline-treated groups were observed. This experiment confirms that direct administration of cisplatin into the cochlea results in considerably less interanimal variability than systemic administration and that co-treatment with alpha-MSH delays cisplatin ototoxicity. Since cisplatin was delivered directly to the cochlea, the ameliorating effect of alpha-MSH probably involves a cochlear target.

Protection from noise-induced lipid peroxidation and hair cell loss in the cochlea.

In order to delineate mechanisms of noise-induced hearing loss, we assessed noise trauma and its pharmacological modulation in the guinea pig. Auditory threshold shifts (measured by auditory brainstem responses), hair cell loss and lipid peroxidation (8-isoprostane formation) were determined in the absence or presence of agents known to influence the formation or action of reactive oxygen species (ROS): the non-specific N-methyl-D-aspartate (NMDA) receptor antagonist (+)-MK-801, its inactive isomer (-)-MK-801, the selective NR1/2B NMDA receptor antagonist PD 174494, the nitric oxide synthase (NOS) inhibitor L-N(omega)-Nitroarginine methyl ester (L-NAME) and the anti-oxidant N-acetylcysteine (NAC). (+)-MK-801 and NAC attenuated threshold shifts and hair cell loss effectively while PD 174494 did so partially. L-NAME attenuated threshold shifts at 2 kHz but increased them at 20 kHz, and (-)-MK-801 was ineffective. Noise-induced elevation in 8-isoprostane in the cochlea was significantly attenuated by (+)-MK-801 and PD 174494 in the organ of Corti and modiolar core, by L-NAME in the lateral wall and modiolar core, and by NAC in all three regions. (-)-MK-801 did not influence noise-induced 8-isoprostane formation. There was a significant correlation between threshold shifts at 4 kHz, hair cell loss and the level of 8-isoprostane formed in the organ of Corti, but not in the lateral wall tissues. This finding suggests a causal relationship between ROS formation and functional and morphological damage. NMDA receptors and, to some extent, NOS may be involved in noise-induced ROS formation. The data also indicate that lipid peroxidation in the lateral wall tissues does not influence permanent threshold shifts.

Neonatal deafening causes changes in Fos protein induced by cochlear electrical stimulation.

The influence of neonatal deafness on cochlear electrically evoked Fos expression in the auditory brainstem was examined. Newborn rats were deafened by systemic injection of kanamycin, 1 mg/g daily for 12 days. At 4, 5, 6 or 8 weeks of age, these animals received cochlear electrical stimulation with a basal monopolar electrode for 90 minutes. Age-matched untreated control animals received similar stimulation. Experimental and control animals were assessed for spiral ganglion cell densities and Fos immunoreactive staining in the central nucleus of the inferior colliculus. Spiral ganglion cell assessments showed significant decreases in spiral ganglion cell densities in deafened rats compared to age-matched controls, at 5 weeks of age in lower turns and 6 and 8 weeks in all turns. Cochlear electrical stimulation induced Fos immunoreactive staining in the nucleus of auditory brain stem neurons in treatment and control groups. A significantly greater number of Fos immunoreactive neurons was found in the contralateral central nucleus of inferior colliculus in 5, 6 and 8 week old deafened animals compared to age-matched controls. The increases were larger with a longer duration of deafness. These results suggest that there are changes in auditory processing as a consequence of neonatal deafness.