Kanamycin and Cisplatin Ototoxicity: Differences in Patterns of Oxidative Stress, Antioxidant Enzyme Expression and Hair Cell Loss in the Cochlea.

Kanamycin and cisplatin are ototoxic drugs. The mechanisms are incompletely known. With subcutaneous kanamycin (400 mg/kg, 15 days), auditory threshold shifts were detected at days 12-13 at 16 and 32 kHz, extending to 8 and 4 kHz at days 14-15. The outer hair cell (OHC) loss was concentrated past day 12. The maximum cochlear length showing apoptotic cells, tested with TUNEL, was at day 13. At day 15, 1/5 of the apical cochlea contained preserved OHCs. 3-nitrotyrosine (3-NT) immunolabeling, showing oxidative stress, was found in surviving OHCs and in basal and middle portions of the stria vascularis (SV). The antioxidant Gpx1 gene expression was decreased. The immunocytochemistry showed diminished Gpx1 in OHCs. With intraperitoneal cisplatin (16 mg/kg, single injection), no evoked auditory activity was recorded at the end of treatment, at 72 h. The basal third of the cochlea lacked OHCs. Apoptosis occupied the adjacent 1/3, and the apical third contained preserved OHCs. 3-NT immunolabeling was extensive in OHCs and the SV. Gpx1 and Sod1 gene expression was downregulated. Gpx1 immunostaining diminished in middle and basal SV. More OHCs survived cisplatin than kanamycin towards the apex, despite undetectable evoked activity. Differential regulation of antioxidant enzyme levels suggests differences in the antioxidant response for both drugs.

Oral Antioxidant Vitamins and Magnesium Limit Noise-Induced Hearing Loss by Promoting Sensory Hair Cell Survival: Role of Antioxidant Enzymes and Apoptosis Genes.

Noise induces oxidative stress in the cochlea followed by sensory cell death and hearing loss. The proof of principle that injections of antioxidant vitamins and Mg2+ prevent noise-induced hearing loss (NIHL) has been established. However, effectiveness of oral administration remains controversial and otoprotection mechanisms are unclear. Using auditory evoked potentials, quantitative PCR, and immunocytochemistry, we explored effects of oral administration of vitamins A, C, E, and Mg2+ (ACEMg) on auditory function and sensory cell survival following NIHL in rats. Oral ACEMg reduced auditory thresholds shifts after NIHL. Improved auditory function correlated with increased survival of sensory outer hair cells. In parallel, oral ACEMg modulated the expression timeline of antioxidant enzymes in the cochlea after NIHL. There was increased expression of glutathione peroxidase-1 and catalase at 1 and 10 days, respectively. Also, pro-apoptotic caspase-3 and Bax levels were diminished in ACEMg-treated rats, at 10 and 30 days, respectively, following noise overstimulation, whereas, at day 10 after noise exposure, the levels of anti-apoptotic Bcl-2, were significantly increased. Therefore, oral ACEMg improves auditory function by limiting sensory hair cell death in the auditory receptor following NIHL. Regulation of the expression of antioxidant enzymes and apoptosis-related proteins in cochlear structures is involved in such an otoprotective mechanism.

Expression and Localization of Kv1.1 and Kv3.1b Potassium Channels in the Cochlear Nucleus and Inferior Colliculus after Long-Term Auditory Deafferentation.

Deafness affects the expression and distribution of voltage-dependent potassium channels (Kvs) of central auditory neurons in the short-term, i.e., hours to days, but the consequences in the expression of Kvs after long-term deafness remain unknown. We tested expression and distribution of Kv1.1 and Kv3.1b, key for auditory processing, in the rat cochlear nucleus (CN), and in the inferior colliculus (IC), at 1, 15 and 90 days after mechanical lesion of the cochlea, using a combination of qRT-PCR and Western blot in the whole CN, along with semi-quantitative immunocytochemistry in the AVCN, where the role of both Kvs in excitability control for accurate auditory timing signal processing is well established. Neither Kv1.1/Kv3.1b mRNA or protein expression changed significantly in the CN between 1 and 15 days after deafness. At 90 days post-lesion, however, mRNA and protein expression for both Kvs increased, suggesting that expression regulation of Kv1.1 and Kv3.1b is part of cellular mechanisms for long-term adaptation to auditory input deprivation in the CN. Consistent with these findings, immunocytochemical localization showed increased labeling intensity for both Kvs in the AVCN at day 90 after cochlear lesion, further supporting that up-regulation of Kv1.1 and Kv3.1b in neurons of this CN division, over a long term after auditory deprivation, may be required to adapt intrinsic excitability to altered input. Contrary to findings in the CN, in the IC, expression levels of Kv1.1 and Kv3.1b did not undergo major changes after cochlear lesion. In particular, there was no evidence of long-term up-regulation of neither Kv1.1 or Kv3.1b, supporting that such post-lesion adaptive mechanism may not be needed in the IC. This suggests that post-lesion plastic adaptations to auditory input deprivation are not stereotypical along the auditory pathway.

Neuroglial Involvement in Abnormal Glutamate Transport in the Cochlear Nuclei of the Igf1 -/- Mouse.

Insulin-like growth factor 1 (IGF-1) is a powerful regulator of synaptic activity and a deficit in this protein has a profound impact on neurotransmission, mostly on excitatory synapses in both the developing and mature auditory system. Adult Igf1 -/- mice are animal models for the study of human syndromic deafness; they show altered cochlear projection patterns into abnormally developed auditory neurons along with impaired glutamate uptake in the cochlear nuclei, phenomena that probably reflect disruptions in neuronal circuits. To determine the cellular mechanisms that might be involved in regulating excitatory synaptic plasticity in 4-month-old Igf1 -/- mice, modifications to neuroglia, astroglial glutamate transporters (GLTs) and metabotropic glutamate receptors (mGluRs) were assessed in the cochlear nuclei. The Igf1 -/- mice show significant decreases in IBA1 (an ionized calcium-binding adapter) and glial fibrillary acidic protein (GFAP) mRNA expression and protein accumulation, as well as dampened mGluR expression in conjunction with enhanced glutamate transporter 1 (GLT1) expression. By contrast, no differences were observed in the expression of glutamate aspartate transporter (GLAST) between these Igf1 -/- mice and their heterozygous or wildtype littermates. These observations suggest that congenital IGF-1 deficiency may lead to alterations in microglia and astrocytes, an upregulation of GLT1, and the downregulation of groups I, II and III mGluRs. Understanding the molecular, biochemical and morphological mechanisms underlying neuronal plasticity in a mouse model of hearing deficits will give us insight into new therapeutic strategies that could help to maintain or even improve residual hearing when human deafness is related to IGF-1 deficiency.

An Oral Combination of Vitamins A, C, E, and Mg++ Improves Auditory Thresholds in Age-Related Hearing Loss.

The increasing rate of age-related hearing loss (ARHL), with its subsequent reduction in quality of life and increase in health care costs, requires new therapeutic strategies to reduce and delay its impact. The goal of this study was to determine if ARHL could be reduced in a rat model by administering a combination of antioxidant vitamins A, C, and E acting as free radical scavengers along with Mg++, a known powerful cochlear vasodilator (ACEMg). Toward this goal, young adult, 3 month-old Wistar rats were divided into two groups: one was fed with a diet composed of regular chow ("normal diet," ND); the other received a diet based on chow enriched in ACEMg ("enhanced diet," ED). The ED feeding began 10 days before the noise stimulation. Auditory brainstem recordings (ABR) were performed at 0.5, 1, 2, 4, 8, 16, and 32 kHz at 3, 6-8, and 12-14 months of age. No differences were observed at 3 months of age, in both ND and ED animals. At 6-8 and 12-14 months of age there were significant increases in auditory thresholds and a reduction in the wave amplitudes at all frequencies tested, compatible with progressive development of ARHL. However, at 6-8 months threshold shifts in ED rats were significantly lower in low and medium frequencies, and wave amplitudes were significantly larger at all frequencies when compared to ND rats. In the oldest animals, differences in the threshold shift persisted, as well as in the amplitude of the wave II, suggesting a protective effect of ACEMg on auditory function during aging. These findings indicate that oral ACEMg may provide an effective adjuvant therapeutic intervention for the treatment of ARHL, delaying the progression of hearing impairment associated with age.

Noise-Induced "Toughening" Effect in Wistar Rats: Enhanced Auditory Brainstem Responses Are Related to Calretinin and Nitric Oxide Synthase Upregulation.

An appropriate conditioning noise exposure may reduce a subsequent noise-induced threshold shift. Although this "toughening" effect helps to protect the auditory system from a subsequent traumatic noise exposure, the mechanisms that regulate this protective process are not fully understood yet. Accordingly, the goal of the present study was to characterize physiological processes associated with "toughening" and to determine their relationship to metabolic changes in the cochlea and cochlear nucleus (CN). Auditory brainstem responses (ABR) were evaluated in Wistar rats before and after exposures to a sound conditioning protocol consisting of a broad-band white noise of 118 dB SPL for 1 h every 72 h, four times. After the last ABR evaluation, animals were perfused and their cochleae and brains removed and processed for the activity markers calretinin (CR) and neuronal nitric oxide synthase (nNOS). Toughening was demonstrated by a progressively faster recovery of the threshold shift, as well as wave amplitudes and latencies over time. Immunostaining revealed an increase in CR and nNOS levels in the spiral ganglion, spiral ligament, and CN in noise-conditioned rats. Overall, these results suggest that the protective mechanisms of the auditory toughening effect initiate in the cochlea and extend to the central auditory system. Such phenomenon might be in part related to an interplay between CR and nitric oxide signaling pathways, and involve an increased cytosolic calcium buffering capacity induced by the noise conditioning protocol.

Glia-related mechanisms in the anteroventral cochlear nucleus of the adult rat in response to unilateral conductive hearing loss.

Conductive hearing loss causes a progressive decline in cochlear activity that may result in functional and structural modifications in auditory neurons. However, whether these activity-dependent changes are accompanied by a glial response involving microglia, astrocytes, or both has not yet been fully elucidated. Accordingly, the present study was designed to determine the involvement of glial related mechanisms in the anteroventral cochlear nucleus (AVCN) of adult rats at 1, 4, 7, and 15 d after removing middle ear ossicles. Quantitative immunohistochemistry analyses at light microscopy with specific markers of microglia or astroglia along with immunocytochemistry at the electron microscopy level were used. Also, in order to test whether trophic support by neurotrophins is modulated in glial cells by auditory activity, the expression and distribution of neurotrophin-3 (NT-3) and its colocalization with microglial or astroglial markers was investigated. Diminished cochlear activity after middle ear ossicle removal leads to a significant ipsilateral increase in the mean gray levels and stained area of microglial cells but not astrocytes in the AVCN at 1 and 4 d post-lesion as compared to the contralateral side and control animals. These results suggest that microglial cells but not astrocytes may act as dynamic modulators of synaptic transmission in the cochlear nucleus immediately following unilateral hearing loss. On the other hand, NT-3 immunostaining was localized mainly in neuronal cell bodies and axons and was upregulated at 1, 4 and 7 d post-lesion. Very few glial cells expressed this neurotrophin in both control and experimental rats, suggesting that NT-3 is primarily activated in neurons and not as much in glia after limiting auditory activity in the AVCN by conductive hearing loss.

Hearing impairment in the P23H-1 retinal degeneration rat model.

The transgenic P23H line 1 (P23H-1) rat expresses a variant of rhodopsin with a mutation that leads to loss of visual function. This rat strain is an experimental model usually employed to study photoreceptor degeneration. Although the mutated protein should not interfere with other sensory functions, observing severe loss of auditory reflexes in response to natural sounds led us to study auditory brain response (ABR) recording. Animals were separated into different hearing levels following the response to natural stimuli (hand clapping and kissing sounds). Of all the analyzed animals, 25.9% presented auditory loss before 50 days of age (P50) and 45% were totally deaf by P200. ABR recordings showed that all the rats had a higher hearing threshold than the control Sprague-Dawley (SD) rats, which was also higher than any other rat strains. The integrity of the central and peripheral auditory pathway was analyzed by histology and immunocytochemistry. In the cochlear nucleus (CN), statistical differences were found between SD and P23H-1 rats in VGluT1 distribution, but none were found when labeling all the CN synapses with anti-Syntaxin. This finding suggests anatomical and/or molecular abnormalities in the auditory downstream pathway. The inner ear of the hypoacusic P23H-1 rats showed several anatomical defects, including loss and disruption of hair cells and spiral ganglion neurons. All these results can explain, at least in part, how hearing impairment can occur in a high percentage of P23H-1 rats. P23H-1 rats may be considered an experimental model with visual and auditory dysfunctions in future research.

Wistar rats: a forgotten model of age-related hearing loss.

Age-related hearing loss (ARHL) is one of the most frequent sensory impairments in senescence and is a source of important socio-economic consequences. Understanding the pathological responses that occur in the central auditory pathway of patients who suffer from this disability is vital to improve its diagnosis and treatment. Therefore, the goal of this study was to characterize age-related modifications in auditory brainstem responses (ABR) and to determine whether these functional responses might be accompanied by an imbalance between excitation and inhibition in the cochlear nucleus of Wistar rats. To do so, ABR recordings at different frequencies and immunohistochemistry for the vesicular glutamate transporter 1 (VGLUT1) and the vesicular GABA transporter (VGAT) in the ventral cochlear nucleus (VCN) were performed in young, middle-aged and old male Wistar rats. The results demonstrate that there was a significant increase in the auditory thresholds, a significant decrease in the amplitudes and an increase in the latencies of the ABR waves as the age of the rat increased. Additionally, there were decreases in VGLUT1 and VGAT immunostaining in the VCN of older rats compared to younger rats. Therefore, the observed age-related decline in the magnitude of auditory evoked responses might be due in part to a reduction in markers of excitatory function; meanwhile, the concomitant reduction in both excitatory and inhibitory markers might reflect a common central alteration in animal models of ARLH. Together, these findings highlight the suitability of the Wistar rat as an excellent model to study ARHL.

Role of Salmonella enterica exposure in Chilean Crohn's disease patients.

AIM: To study the association between exposure to Salmonella enterica (SE) and Crohn's disease (CD) and its clinical implications in Chilean patients. METHODS: Ninety-four unrelated Chilean CD patients from CAREI (Active Cohort Registry of Inflammatory Bowel Disease) presenting to a single inflammatory bowel disease (IBD) unit of a University Hospital were prospectively included in this study. A complete clinical evaluation, including smoking history, was performed at the initial visit, and all the important data of clinical evolution of CD were obtained. Blood samples from these CD patients and 88 healthy sex- and age-matched control subjects were analyzed for exposure to SE and for their NOD2/CARD15 gene status using the presence of anti-Salmonella lipopolysaccharide antibodies [immunoglobulin-G type (IgG)] and polymerase chain reaction (PCR), respectively. We also evaluated exposure to SE in 90 sex- and age-matched patients without CD, but with known smoking status (30 smokers, 30 non-smokers, and 30 former smokers). RESULTS: CD patients comprised 54 females and 40 males, aged 35.5 ± 15.2 years at diagnosis with a mean follow-up of 9.0 ± 6.8 years. CD was inflammatory in 59 patients (62.7%), stricturing in 24 (25.5%) and penetrating in 15 (15.5%). Thirty cases (31.9%) had lesions in the ileum, 29 (30.8%) had ileocolonic lesions, 32 (34.0%) had colonic lesions and 23 (24.4%) had perianal disease. Sixteen CD patients (17%) were exposed to SE compared to 15 (17%) of 88 healthy control subjects (P = 0.8). Thirty-one CD patients (32.9%) were smokers, and 7 (7.4%) were former smokers at diagnosis. In the group exposed to SE, 10 of 16 patients (62.5%) were active smokers compared to 21 of 78 patients (26.9%) in the unexposed group (P = 0.01). On the other hand, 10 of 31 smoking patients (32%) were exposed to SE compared to 5 of 56 nonsmoking patients (9%), and one of the seven former smokers (14%) (P = 0.01). In the group of 90 patients without CD, but whose smoking status was known, there was no difference in exposure to SE [3 of 30 smokers (10%), 5 of 30 non-smokers (16%), and 5 of 30 former smokers (16%); P = 0.6]. There were no differences in disease severity between CD patients with and those without anti-SE IgG antibodies, estimated as the appearance of stricturing [2 (12.5%) vs 22 (28.2%); P = 0.2] or penetrating lesions [2 (12.5%) vs 13 (16.6%); P = 1.0]; or the need for immunosuppressants [11 (68.7%) vs 55 (70.5%); P = 1.0], anti-tumor necrosis factor therapy [1 (6.2%) vs 7 (8.9%); P = 1.0], hospitalization [13 (81.2%) vs 58 (74.3%); P = 0.7], or surgery [3 (18.7%) vs 12 (15.3%); P = 0.3), respectively]. No other factors were associated with SE, including NOD2/CARD15 gene status. Seventeen CD patients (18%) had at least one mutation of the NOD2/CARD15 gene. CONCLUSION: Our study found no association between exposure to SE and CD. We observed a positive correlation between SE exposure and cigarette smoking in Chilean patients with CD, but not with disease severity.

Long-term evolution of brainstem electrical evoked responses to sound after restricted ablation of the auditory cortex.

INTRODUCTION: This study aimed to assess the top-down control of sound processing in the auditory brainstem of rats. Short latency evoked responses were analyzed after unilateral or bilateral ablation of auditory cortex. This experimental paradigm was also used towards analyzing the long-term evolution of post-lesion plasticity in the auditory system and its ability to self-repair. METHOD: Auditory cortex lesions were performed in rats by stereotactically guided fine-needle aspiration of the cerebrocortical surface. Auditory Brainstem Responses (ABR) were recorded at post-surgery day (PSD) 1, 7, 15 and 30. Recordings were performed under closed-field conditions, using click trains at different sound intensity levels, followed by statistical analysis of threshold values and ABR amplitude and latency variables. Subsequently, brains were sectioned and immunostained for GAD and parvalbumin to assess the location and extent of lesions accurately. RESULTS: Alterations in ABR variables depended on the type of lesion and post-surgery time of ABR recordings. Accordingly, bilateral ablations caused a statistically significant increase in thresholds at PSD1 and 7 and a decrease in waves amplitudes at PSD1 that recover at PSD7. No effects on latency were noted at PSD1 and 7, whilst recordings at PSD15 and 30 showed statistically significant decreases in latency. Conversely, unilateral ablations had no effect on auditory thresholds or latencies, while wave amplitudes only decreased at PSD1 strictly in the ipsilateral ear. CONCLUSION: Post-lesion plasticity in the auditory system acts in two time periods: short-term period of decreased sound sensitivity (until PSD7), most likely resulting from axonal degeneration; and a long-term period (up to PSD7), with changes in latency responses and recovery of thresholds and amplitudes values. The cerebral cortex may have a net positive gain on the auditory pathway response to sound.

Axon morphologies and convergence patterns of projections from different sensory-specific cortices of the anterior ectosylvian sulcus onto multisensory neurons in the cat superior colliculus.

Corticofugal projections to the thalamus reveal 2 axonal morphologies, each associated with specific physiological attributes. These determine the functional characteristics of thalamic neurons. It is not clear, however, whether such features characterize the corticofugal projections that mediate multisensory integration in superior colliculus (SC) neurons. The cortico-collicular projections from cat anterior ectosylvian sulcus (AES) are derived from its visual, auditory, and somatosensory representations and are critical for multisensory integration. Following tracer injections into each subdivision, 2 types of cortico-collicular axons were observed. Most were categorized as type I and consisted of small-caliber axons traversing long distances without branching, bearing mainly small boutons. The less frequent type II had thicker axons, more complex branching patterns, larger boutons, and more complex terminal boutons. Following combinatorial injections of 2 different fluorescent tracers into defined AES subdivisions, fibers from each were seen converging onto individual SC neurons and indicate that such convergence, like that in the corticothalamic system, is mediated by 2 distinct morphological types of axon terminals. Nevertheless, and despite the conservation of axonal morphologies across different subcortical systems, it is not yet clear if the concomitant physiological attributes described in the thalamus are directly applicable to multisensory integration.