Salicylate decreases the spontaneous firing rate of guinea pig auditory nerve fibres.

Tinnitus has similarities to chronic neuropathic pain where there are changes in the firing rate of different types of afferent neurons. We postulated that one possible cause of tinnitus is a change in the distribution of spontaneous firing rates in at least one type of afferent auditory nerve fibre in anaesthetised guinea pigs. In control animals there was a bimodal distribution of spontaneous rates, but the position of the second mode was different depending upon whether the fibres responded best to high (> 4 kHz) or low (≤4 kHz) frequency tonal stimulation. The simplest and most reliable way of inducing tinnitus in experimental animals is to administer a high dose of sodium salicylate. The distribution of the spontaneous firing rates was different when salicylate (350 mg/kg) was administered, even when the sample was matched for the distribution of characteristic frequencies in the control population. The proportion of medium spontaneous rate fibres (MSR, 1≤ spikes/s ≤20) increased while the proportion of the highest, high spontaneous firing rate fibres (HSR, > 80 spikes/s) decreased following salicylate. The median rate fell from 64.7 spikes/s (control) to 35.4 spikes/s (salicylate); a highly significant change (Kruskal-Wallis test p < 0.001). When the changes were compared with various models of statistical probability, the most accurate model was one where most HSR fibres decreased their firing rate by 32 spikes/s. Thus, we have shown a reduction in the firing rate of HSR fibres that may be related to tinnitus.

Effects of Kainic Acid-Induced Auditory Nerve Damage on Envelope-Following Responses in the Budgerigar (Melopsittacus undulatus).

Sensorineural hearing loss is a prevalent problem that adversely impacts quality of life by compromising interpersonal communication. While hair cell damage is readily detectable with the clinical audiogram, this traditional diagnostic tool appears inadequate to detect lost afferent connections between inner hair cells and auditory nerve (AN) fibers, known as cochlear synaptopathy. The envelope-following response (EFR) is a scalp-recorded response to amplitude modulation, a critical acoustic feature of speech. Because EFRs can have greater amplitude than wave I of the auditory brainstem response (ABR; i.e., the AN-generated component) in humans, the EFR may provide a more sensitive way to detect cochlear synaptopathy. We explored the effects of kainate- (kainic acid) induced excitotoxic AN injury on EFRs and ABRs in the budgerigar (Melopsittacus undulatus), a parakeet species used in studies of complex sound discrimination. Kainate reduced ABR wave I by 65-75 % across animals while leaving otoacoustic emissions unaffected or mildly enhanced, consistent with substantial and selective AN synaptic loss. Compared to wave I loss, EFRs showed similar or greater percent reduction following kainate for amplitude-modulation frequencies from 380 to 940 Hz and slightly less reduction from 80 to 120 Hz. In contrast, forebrain-generated middle latency responses showed no consistent change post-kainate, potentially due to elevated "central gain" in the time period following AN damage. EFR reduction in all modulation frequency ranges was highly correlated with wave I reduction, though within-animal effect sizes were greater for higher modulation frequencies. These results suggest that even low-frequency EFRs generated primarily by central auditory nuclei might provide a useful noninvasive tool for detecting synaptic injury clinically.

Trk agonist drugs rescue noise-induced hidden hearing loss.

TrkB agonist drugs are shown here to have a significant effect on the regeneration of afferent cochlear synapses after noise-induced synaptopathy. The effects were consistent with regeneration of cochlear synapses that we observed in vitro after synaptic loss due to kainic acid-induced glutamate toxicity and were elicited by administration of TrkB agonists, amitriptyline, and 7,8-dihydroxyflavone, directly into the cochlea via the posterior semicircular canal 48 hours after exposure to noise. Synaptic counts at the inner hair cell and wave 1 amplitudes in the auditory brainstem response (ABR) were partially restored 2 weeks after drug treatment. Effects of amitriptyline on wave 1 amplitude and afferent auditory synapse numbers in noise-exposed ears after systemic (as opposed to local) delivery were profound and long-lasting; synapses in the treated animals remained intact 1 year after the treatment. However, the effect of systemically delivered amitriptyline on synaptic rescue was dependent on dose and the time window of administration: it was only effective when given before noise exposure at the highest injected dose. The long-lasting effect and the efficacy of postexposure treatment indicate a potential broad application for the treatment of synaptopathy, which often goes undetected until well after the original damaging exposures.

Caspase inhibitor z-VAD-FMK increases the survival of hair cells after Actinomycin-D-induced damage in vitro.

Actinomycin-D (Act-D) is a highly effective chemotherapeutic agent that induces apoptosis in systemic tissues. Act-D combined with other chemotherapeutic agents exhibits ototoxic effects and causes hearing impairment. To investigate the potential toxic effects of Act-D in the inner ear, we treated cochlear organotypic cultures with varying concentrations of Act-D for different durations. For the first time, we found that Act-D specifically induced HC loss and apoptosis in a dose- and time-dependent manner but not neuronal degeneration. Co-treatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK), a pan cysteine protease inhibitor, significantly reduced HC loss and apoptosis induced by Act-D, indicating increased cell survival. Taken together, Act-D exposure has ototoxic effects on the auditory system, while z-VAD-FMK prevents Act-D-induced hair cell damage.

LSP5-2157 a new inhibitor of vesicular glutamate transporters.

Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

Long-term effects and potential limits of intratympanic dexamethasone-loaded hydrogels combined with dexamethasone-eluting cochlear electrodes in a low-insertion trauma Guinea pig model.

Cochlear implantation has become the most effective hearing restoration method and is one of the great advances in modern medicine. Early implants have been continuously developed into more efficient devices, and electro-acoustic stimulation is increasingly expanding the indication criteria for cochlear implants to patients with more residual hearing. Therefore, protecting the cochlear structures and maintaining its intrinsic capacities like residual hearing has become more important than ever before. In the present study, we aimed to assess the long-term protective effects of a dexamethasone-eluting electrode combined with the preoperative intratympanic application of a dexamethasone-loaded thermoreversible hydrogel in a cochlear implant guinea pig model. 40 normal-hearing animals were equally randomized into a control group receiving an unloaded hydrogel and a non-eluting electrode, a group receiving a dexamethasone-loaded hydrogel and a non-eluting electrode, a group receiving an unloaded hydrogel and a dexamethasone-eluting electrode and a group receiving both a dexamethasone-loaded hydrogel and a dexamethasone-eluting electrode. Residual hearing and impedances were investigated during a period of 120 days. Tissue response and histological changes of cochlear structures were analyzed at the end of the experiments. Treatment with dexamethasone did not show a significant protective effect on residual hearing independent of treatment group. Although the majority of the cochleae didn't exhibit any signs of electrode insertion trauma, a small degree of tissue response could be observed in all animals without a significant difference between the groups. Foreign body giant cells and osteogenesis were significantly associated with tissue response. Hair cells, synapsin-1-positive cells and spiral ganglion cells were preserved in all study groups. Cochlear implantation using a dexamethasone-eluting electrode alone and in combination with a dexamethasone-loaded hydrogel significantly protected auditory nerve fibers on day 120. Post-implantation impedances were equal across study groups and remained stable over the duration of the experiment. In this study we were able to show that use of a dexamethasone-eluting electrode alone and in combination with preoperative application of dexamethasone-loaded hydrogel significantly protects auditory nerve fibers. Furthermore, we have shown that a cochlear implantation-associated hearing threshold shift and tissue response may not be completely prevented by the sole application of dexamethasone.

The Effects of Riluzole on Cisplatin-induced Ototoxicity

Abstract Introduction Riluzole (2-amino-6-trifluoromethoxy benzothiazole) is known as a neuroprotective, antioxidant, antiapoptotic agent. It may have beneficial effects on neuronal cell death due to cisplatin-induced ototoxicity. Objective To evaluate the effect of riluzole on cisplatin-induced ototoxicity in guinea pigs. Methods Twenty-four guinea pigs, studied in three groups, underwent auditory brainstem response evaluation using click and 8 kHz tone burst stimuli. Subsequently, 5 mg/kg of cisplatin were administered to all animals for 3 days intraperitoneally (i.p.) to induce ototoxicity. Half an hour prior to cisplatin, groups 1, 2 and 3 received 2 ml of saline i.p., 6 mg/kg of riluzole hydrochloride i.p., and 8 mg/kg of riluzole hydrochloride i.p., respectively, for 3 days. The auditory brainstem responses were repeated 24 hours after the last drug administration. The cochleae were analyzed by transmission electron microscopy (TEM). Results After drug administiration, for 8,000 Hz stimulus, group 1 had significantly higher threshold shifts when compared with groups 2 (p < 0.05) and 3 (p < 0.05), and there was no significant difference in threshold shifts between groups 2 and 3 (p > 0.05). Transmission electron microscopy findings demonstrated the protective effect of riluzole on the hair cells and the stria vascularis, especially in the group treated with 8 mg/kg of riluzole hydrochloride. Conclusion We can say that riluzolemay have a protective effect on cisplatin- induced ototoxicity. However, additional studies are needed to confirm these results and the mechanisms of action of riluzole.

Lead Induced Ototoxicity and Neurotoxicity in Adult Guinea Pig.

Lead exposure causes or aggravates hearing damage to human or animal, but the detailed effects of lead exposure on auditory system including injury sites of the cochlea in mammal remain controversy. To investigate the effect of chronic lead exposure on auditory system, 40 adult guinea pigs with normal hearing were randomly divided into five groups. They were fed 2 mmol/L lead acetate in drinking water for 0, 15, 30, 60, and 90 days (n = 8), respectively. Lead concentrations in blood, cochlea, and brainstem were measured. Auditory function was measured by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE). The morphology of cochlea and brainstem was observed, and expression of autophagy-related protein in brainstem was also assessed. The blood lead concentration reached a high level at the 15th day and kept stable, but the lead level in brainstem and cochlear tissue increased obviously at the 60th day and 90th day of lead exposure, respectively. There was no significant difference in the morphology of hair cells and stria vascularis (SV) among these five groups, but the number of spiral ganglion neuron (SGN) gradually decreased after 60 days. The differences of ABR thresholds and DPOAE amplitudes were not statistically significant among each group, but I wave latency, III latency, and I-III wave interval of ABR were delayed with the prolonging of time of lead exposure. The expressions of autophagy-related protein ATG5, ATG6, and LC3B in brainstem were increased after 30 days. These results suggest that the key target of lead toxicity was the auditory nerve conduction pathway including SGNs and brainstem, rather than cochlear hair cells and SV. Autophagy may play a very important role in lead toxicity to auditory nervous system.

Effects of selective auditory-nerve damage on the behavioral audiogram and temporal integration in the budgerigar.

Auditory-nerve fibers are lost steadily with age and as a possible consequence of noise-induced glutamate excitotoxicity. Auditory-nerve loss in the absence of other cochlear pathologies is thought to be undetectable with a pure-tone audiogram while degrading real-world speech perception (hidden hearing loss). Perceptual deficits remain unclear, however, due in part to the limited behavioral capacity of existing rodent models to discriminate complex sounds. The budgerigar is an avian vocal learner with human-like behavioral sensitivity to many simple and complex sounds and the capacity to mimic speech. Previous studies in this species show that intracochlear kainic-acid infusion reduces wave 1 of the auditory brainstem response by 40-70%, consistent with substantial excitotoxic auditory-nerve damage. The present study used operant-conditioning procedures in trained budgerigars to quantify kainic-acid effects on tone detection across frequency (0.25-8 kHz; the audiogram) and as a function of duration (20-160 ms; temporal integration). Tone thresholds in control animals were lowest from 1 to 4 kHz and decreased with increasing duration as in previous studies of the budgerigar. Behavioral results in kainic-acid-exposed animals were as sensitive as in controls, suggesting preservation of the audiogram and temporal integration despite auditory-nerve loss associated with up to 70% wave 1 reduction. Distortion-product otoacoustic emissions were also preserved in kainic-acid exposed animals, consistent with normal hair-cell function. These results highlight considerable perceptual resistance of tone-detection performance with selective auditory-nerve loss. Future behavioral studies in budgerigars with auditory-nerve damage can use complex speech-like stimuli to help clarify aspects of auditory perception impacted by this common cochlear pathology.

Bilirubin-induced neurotoxic and ototoxic effects in rat cochlear and vestibular organotypic cultures.

Exposure to high levels of bilirubin in hyperbilirubinemia patients and animal models can result in sensorineural deafness. However, the mechanisms underlying bilirubin-induced damage to the inner ear, including the cochlear and vestibular organs, remain unknown. The present analyses of cochlear and vestibular organotypic cultures obtained from postnatal day 3 rats exposed to bilirubin at varying concentrations (0, 10, 50, 100, or 250 µM) for 24 h revealed that auditory nerve fibers (ANFs) and vestibular nerve endings were destroyed even at low doses (10 and 50 µM). Additionally, as the bilirubin dose increased, spiral ganglion neurons (SGNs) and vestibular ganglion neurons (VGNs) exhibited gradual shrinkage in conjunction with nuclei condensation or fragmentation in a dose-dependent manner. The loss of cochlear and vestibular hair cells (HCs) was only evident in explants treated with the highest concentration of bilirubin (250 µM), and bilirubin-induced major apoptosis most likely occurred via the extrinsic apoptotic pathway. Thus, the present results indicate that inner ear neurons and fibers were more sensitive to, and exhibited more severe damage following, bilirubin-induced neurotoxicity than sensory HCs, which illustrates the underlying causes of auditory neuropathy and vestibulopathy in hyperbilirubinemia patients.

Clustered Ca2+ Channels Are Blocked by Synaptic Vesicle Proton Release at Mammalian Auditory Ribbon Synapses.

A Ca2+ current transient block (ICaTB) by protons occurs at some ribbon-type synapses after exocytosis, but this has not been observed at mammalian hair cells. Here we show that a robust ICaTB occurs at post-hearing mouse and gerbil inner hair cell (IHC) synapses, but not in immature IHC synapses, which contain non-compact active zones, where Ca2+ channels are loosely coupled to the release sites. Unlike ICaTB at other ribbon synapses, ICaTB in mammalian IHCs displays a surprising multi-peak structure that mirrors the EPSCs seen in paired recordings. Desynchronizing vesicular release with intracellular BAPTA or by deleting otoferlin, the Ca2+ sensor for exocytosis, greatly reduces ICaTB, whereas enhancing release synchronization by raising Ca2+ influx or temperature increases ICaTB. This suggests that ICaTB is produced by fast multivesicular proton-release events. We propose that ICaTB may function as a submillisecond feedback mechanism contributing to the auditory nerve's fast spike adaptation during sound stimulation.

Persistent Auditory Nerve Damage Following Kainic Acid Excitotoxicity in the Budgerigar (Melopsittacus undulatus).

Permanent loss of auditory nerve (AN) fibers occurs with increasing age and sound overexposure, sometimes without hair cell damage or associated audiometric threshold elevation. Rodent studies suggest effects of AN damage on central processing and behavior, but these species have limited capacity to discriminate low-frequency speech-like sounds. Here, we introduce a new animal model of AN damage in an avian communication specialist, the budgerigar (Melopsittacus undulatus). The budgerigar is a vocal learner and speech mimic with sensitive low-frequency hearing and human-like behavioral sensitivity to many complex signals including speech components. Excitotoxic AN damage was induced through bilateral cochlear infusions of kainic acid (KA). Acute KA effects on cochlear function were assessed using AN compound action potentials (CAPs) and hair cell cochlear microphonics (CMs). Long-term KA effects were assessed using auditory brainstem response (ABR) measurements for up to 31 weeks post-KA exposure. KA infusion immediately abolished AN CAPs while having mild impact on the CM. ABR wave I, the far-field AN response, showed a pronounced 40-75 % amplitude reduction at moderate-to-high sound levels that persisted for the duration of the study. In contrast, wave I latency and the amplitude of wave V were nearly unaffected by KA, and waves II-IV were less reduced than wave I. ABR thresholds, calculated based on complete response waveforms, showed no impairment following KA. These results demonstrate that KA exposure in the budgerigar causes irreversible AN damage, most likely through excitotoxic injury to afferent fibers or synapses as in other species, while sparing ABR thresholds. Normal wave V amplitude, assumed to originate centrally, may persist through compensatory mechanisms that restore central response amplitude by downregulating inhibition. Future studies in this new animal model of AN damage can explore effects of this neural lesion, in isolation from hair cell trauma and threshold elevation, on central processing and perception of complex sounds.

Antenatal Corticosteroid Exposure Disrupts Myelination in the Auditory Nerve of Preterm Sheep.

BACKGROUND: Antenatal corticosteroids (ACS) improve preterm neonatal outcomes. However, uncertainty remains regarding the safety of ACS exposure for the developing fetus, particularly its neurosensory development. OBJECTIVES: We investigated the effect of single and multiple ACS exposures on auditory nerve development in an ovine model of pregnancy. METHODS: Ewes with a single fetus (gestational age [GA] 100 days) received an intramuscular injection of 150 mg medroxyprogesterone-acetate, followed by intramuscular (i) betamethasone (0.5 mg/kg) on days 104, 111, and 118 GA; (ii) betamethasone on day 104 and saline on days 111 and 118 GA; or (iii) saline on days 104, 111, and 118 GA, with delivery on day 125 GA. Transmission electron microscope images of lamb auditory nerve preparations were digitally analyzed to determine auditory nerve morphology and myelination. RESULTS: Relative to the control, mean auditory nerve myelin area was significantly increased in the multiple-treatment group (p < 0.001), but not in the single-treatment group. Increased myelin thickness was significantly changed only in a subgroup analysis for those axons with myelin thickness greater than the median value (p < 0.001). Morphological assessments showed that the increased myelin area was due to an increased likelihood of decompacted areas (p = 0.005; OR = 2.14, 95% CI 1.26-3.63; 31.6 vs. 18.2% in controls) and irregular myelin deposition (p = 0.001; OR = 5.91, 95% CI 2.16-16.19; 49.0 vs. 16.8% in controls) in the myelin sheath. CONCLUSIONS: In preterm sheep, ACS exposure increased auditory nerve myelin area, potentially due to disruption of normal myelin deposition.

The maturation state of the auditory nerve and brainstem in rats exposed to lead acetate and supplemented with ferrous sulfate / Estado de maturação do nervo auditivo e tronco encefálico em ratos expostos a acetato de chumbo e suplementados com sulfato ferroso

Abstract Introduction The literature has reported the association between lead and auditory effects, based on clinical and experimental studies. However, there is no consensus regarding the effects of lead in the auditory system, or its correlation with the concentration of the metal in the blood. Objective To investigate the maturation state of the auditory system, specifically the auditory nerve and brainstem, in rats exposed to lead acetate and supplemented with ferrous sulfate. Methods 30 weanling male rats (Rattus norvegicus, Wistar) were distributed into six groups of five animals each and exposed to one of two concentrations of lead acetate (100 or 400 mg/L) and supplemented with ferrous sulfate (20 mg/kg). The maturation state of the auditory nerve and brainstem was analyzed using Brainstem Auditory Evoked Potential before and after lead exposure. The concentration of lead in blood and brainstem was analyzed using Inductively Coupled Plasma-Mass Spectrometry. Results We verified that the concentration of Pb in blood and in brainstem presented a high correlation (r = 0.951; p < 0.0001). Both concentrations of lead acetate affected the maturation state of the auditory system, being the maturation slower in the regions corresponding to portion of the auditory nerve (wave I) and cochlear nuclei (wave II). The ferrous sulfate supplementation reduced significantly the concentration of lead in blood and brainstem for the group exposed to the lowest concentration of lead (100 mg/L), but not for the group exposed to the higher concentration (400 mg/L). Conclusion This study indicate that the lead acetate can have deleterious effects on the maturation of the auditory nerve and brainstem (cochlear nucleus region), as detected by the Brainstem Auditory Evoked Potentials, and the ferrous sulphate can partially amend this effect.

Resumo Introdução A literatura relatou a associação entre o chumbo e os efeitos auditivos, com base em estudos clínicos e experimentais. No entanto, não há consenso em relação aos efeitos do chumbo no sistema auditivo, ou sua correlação com a concentração do metal no sangue. Objetivo Investigar o estado de maturação do sistema auditivo, especificamente do nervo auditivo e do tronco encefálico, em ratos expostos ao acetato de chumbo e suplementados com sulfato ferroso. Método 30 ratos machos desmamados (Rattus norvegicus, Wistar) foram distribuídos em seis grupos de cinco animais e expostos a uma de duas concentrações de acetato de chumbo (100 ou 400 mg/L) e suplementados com sulfato ferroso (20 mg/kg). O estado de maturação do nervo auditivo e do tronco encefálico foi analisado pelo Potencial Evocado Auditivo do Tronco Encefálico antes e após a exposição ao chumbo. A concentração de chumbo no sangue e tronco encefálico foi analisada utilizando-se Espectrometria de Massa com Plasma Indutivamente Acoplado. Resultados Verificamos que as concentrações de Pb no sangue e no tronco encefálico apresentaram alta correlação (r = 0,951, p < 0,0001). Ambas as concentrações de acetato de chumbo afetaram o estado de maturação do sistema auditivo, a maturação foi mais lenta nas regiões correspondentes à porção do nervo auditivo (onda I) e dos núcleos cocleares (onda II). A suplementação com sulfato ferroso reduziu significativamente a concentração de chumbo no sangue e no tronco encefálico no grupo exposto à menor concentração de chumbo (100 mg/L), mas não para o grupo exposto à maior concentração (400 mg/L). Conclusão Esse estudo indica que o acetato de chumbo pode ter efeitos deletérios na maturação do nervo auditivo e do tronco encefálico (região do núcleo coclear), como detectado pelos potenciais evocados auditivos do tronco encefálico, e que o sulfato ferroso pode diminuir parcialmente esse efeito.

Drug-Induced Ototoxicity: Diagnosis and Monitoring.

Ototoxicity diagnosis and management has historically been approached using a variety of methods. However, in recent years a consensus on useful and practical approaches has been developed through clinical guidelines of the American Speech Language Hearing Association, the American Academy of Audiology, and multiple clinical trials published in peer-reviewed literature. Some of the guidelines and approaches are used to detect and monitor ototoxicity, while others are used to grade adverse events. Some of the audiologic measures are primary, while others are adjunct measures and may be tailored to the specific needs of the patient or clinical trial. For some types of monitoring, such as drug-induced tinnitus or dizziness, validated paper survey instruments can be both sensitive and easy for fragile patients. This review addresses the characteristics of some of the most common clinical ototoxins and the most common methods for detecting and monitoring ototoxicity in clinical practice and clinical trials.

Effect of granulocyte colony-stimulating factor on the cochlear nuclei after creation of a partial nerve lesion: an experimental study in rats.

OBJECTIVE The risk of injury of the cochlear nerve during angle (CPA) surgery is high. Granulocyte colony-stimulating factor (G-CSF) has been found in various experimental models of peripheral and CNS injury to have a neuroprotective effect by inhibiting apoptosis and inflammation. However, to the authors' knowledge, the influence of G-CSF on cochlear nerve regeneration has not been reported. This study investigated the neuroprotective effect of G-CSF after a partial cochlear nerve lesion in rats. METHODS A lesion of the right cochlear nerve in adult male Sprague-Dawley rats was created using a water-jet dissector with a pressure of 8 bar. In the first group (G-CSF-post), G-CSF was administrated on Days 1, 3, and 5 after the surgery. The second group (G-CSF-pre/post) was treated with G-CSF 1 day before and 1, 3, and 5 days after applying the nerve injury. The control group received sodium chloride after nerve injury at the various time points. Brainstem auditory evoked potentials (BAEPs) were measured directly before and after nerve injury and on Days 1 and 7 to evaluate the acoustic function of the cochlear nerve. The animals were sacrificed 1 week after the operation, and their brains were fixed in formalin. Nissl staining of the cochlear nuclei was performed, and histological sections were analyzed with a light microscope and an image-processing program. The numbers of neurons in the cochlear nuclei were assessed. RESULTS The values for Waves 2 and 4 of the BAEPs decreased abruptly in all 3 groups in the direct postoperative measurement. Although the amplitude in the control group did not recover, it increased in both treatment groups. According to 2-way ANOVA, groups treated with G-CSF had a significant increase in BAEP Wave II amplitudes on the right side (p = 0.0401) after the applied cochlear nerve injury. With respect to Wave IV, a trend toward better recovery in the G-CSF groups was found, but this difference did not reach statistical significance. In the histological analysis, higher numbers of neurons were found in the G-CSF groups. In the statistical analysis, the difference in the numbers of neurons between the control and G-CSF-post groups reached significance (p = 0.0086). The difference in the numbers of neurons between the control and G-CSF-pre/post groups and between the G-CSF-post and G-CSF-pre/post groups did not reach statistical significance. CONCLUSIONS The use of G-CSF improved the function of the eighth cranial nerve and protected cochlear nucleus cells from destruction after a controlled partial injury of the nerve. These findings might be relevant for surgery that involves CPA tumors. The use of G-CSF in patients with a lesion in the CPA might improve postoperative outcomes.

The maturation state of the auditory nerve and brainstem in rats exposed to lead acetate and supplemented with ferrous sulfate.

INTRODUCTION: The literature has reported the association between lead and auditory effects, based on clinical and experimental studies. However, there is no consensus regarding the effects of lead in the auditory system, or its correlation with the concentration of the metal in the blood. OBJECTIVE: To investigate the maturation state of the auditory system, specifically the auditory nerve and brainstem, in rats exposed to lead acetate and supplemented with ferrous sulfate. METHODS: 30 weanling male rats (Rattus norvegicus, Wistar) were distributed into six groups of five animals each and exposed to one of two concentrations of lead acetate (100 or 400mg/L) and supplemented with ferrous sulfate (20mg/kg). The maturation state of the auditory nerve and brainstem was analyzed using Brainstem Auditory Evoked Potential before and after lead exposure. The concentration of lead in blood and brainstem was analyzed using Inductively Coupled Plasma-Mass Spectrometry. RESULTS: We verified that the concentration of Pb in blood and in brainstem presented a high correlation (r=0.951; p<0.0001). Both concentrations of lead acetate affected the maturation state of the auditory system, being the maturation slower in the regions corresponding to portion of the auditory nerve (wave I) and cochlear nuclei (wave II). The ferrous sulfate supplementation reduced significantly the concentration of lead in blood and brainstem for the group exposed to the lowest concentration of lead (100mg/L), but not for the group exposed to the higher concentration (400mg/L). CONCLUSION: This study indicate that the lead acetate can have deleterious effects on the maturation of the auditory nerve and brainstem (cochlear nucleus region), as detected by the Brainstem Auditory Evoked Potentials, and the ferrous sulphate can partially amend this effect.

Kanamycin Damages Early Postnatal, but Not Adult Spiral Ganglion Neurons.

Although aminoglycoside antibiotics such as kanamycin are widely used clinically to treat life-threatening bacterial infections, ototoxicity remains a significant dose-limiting side effect. The prevailing view is that the hair cells are the primary ototoxic target of aminoglycosides and that spiral ganglion neurons begin to degenerate weeks or months after the hair cells have died due to lack of neurotrophic support. To test the early developmental aspects of this issue, we compared kanamycin-induced hair cell and spiral ganglion pathology in rat postnatal day 3 cochlear organotypic cultures with adult whole cochlear explants. In both adult and postnatal day 3 cultures, hair cell damage began at the base of the cochleae and progressed toward the apex in a dose-dependent manner. In postnatal day 3 cultures, spiral ganglion neurons were rapidly destroyed by kanamycin prior to hair cell loss. In contrast, adult spiral ganglion neurons were resistant to kanamycin damage even at the highest concentration, consistent with in vivo models of delayed SGN degeneration. In postnatal day 3 cultures, kanamycin preferentially damaged type I spiral ganglion neurons, whereas type II neurons were resistant. Spiral ganglion degeneration of postnatal day 3 neurons was associated with upregulation of the superoxide radical and caspase-3-mediated cell death. These results show for the first time that kanamycin is toxic to postnatal day 3 spiral ganglion neurons, but not adult neurons.

Time-dependent activity of primary auditory neurons in the presence of neurotrophins and antibiotics.

In vitro cultures provide a valuable tool in studies examining the survival, morphology and function of cells in the auditory system. Primary cultures of primary auditory neurons have most notably provided critical insights into the role of neurotrophins in cell survival and morphology. Functional studies have also utilized in vitro models to study neuronal physiology and the ion channels that dictate these patterns of activity. Here we examine what influence time-in-culture has on the activity of primary auditory neurons, and how this affects our interpretation of neurotrophin and antibiotic-mediated effects in this population. Using dissociated cell culture we analyzed whole-cell patch-clamp recordings of spiral ganglion neurons grown in the presence or absence of neurotrophins and/or penicillin and streptomycin for 1-3 days in vitro. Firing threshold decreased, and both action potential number and latency increased over time regardless of treatment, whilst input resistance was lowest where neurotrophins were present. Differences in firing properties were seen with neurotrophin concentration but were not consistently maintained over the 3 days in vitro. The exclusion of antibiotics from culture media influenced most firing properties at 1 day in vitro in both untreated and neurotrophin-treated conditions. The only difference still present at 3 days was an increase in input resistance in neurotrophin-treated neurons. These results highlight the potential of neurotrophins and antibiotics to influence neural firing patterns in vitro in a time-dependent manner, and advise the careful consideration of their impact on SGN function in future studies.