Juan Carlos Alvarado

Normal variations in the morphology of auditory brainstem response (ABR) waveforms: a study in wistar rats

Auditory brainstem evoked responses (ABR) have been used for decades to assess auditory function. Surprisingly, despite the fact that rats are one of the most widely used experimental models in hearing, there have been no studies that have characterized in detail the normal morphological variations that occur in ABR waves. Therefore, the goal of this study was to characterize the patterns of ABR waves in rats to establish baseline criteria that could be used to identify abnormalities. Rats were stimulated with pure tone sounds at different frequencies and ABR waves were classified based on morphology. The most definitive finding was that, unlike what is observed in human ABRs, wave II of the rat ABR was the most prominent. Additionally, wave III was the smallest and, in many cases, was not apparent at low frequencies. Wave III was frequently involved in the formation of complexes, often appearing as a small wave or adjoining primarily wave IV. Complexes were common at low and medium frequencies and rare at high frequencies. These results indicate that knowledge of the different wave patterns in normal rats is fundamental to understanding how the wave morphology changes in pathological conditions that could lead to hearing impairment.

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.

Upregulation of insulin-like growth factor and interleukin 1β occurs in neurons but not in glial cells in the cochlear nucleus following cochlear ablation

One of the main mechanisms used by neurons and glial cells to promote repair following brain injury is to upregulate activity‐dependent molecules such as insulin‐like growth factor 1 (IGF‐1) and interleukin‐1β (IL‐1β). In the auditory system, IGF‐1 is crucial for restoring synaptic transmission following hearing loss; however, whether IL‐1β is also involved in this process is unknown. In this study, we evaluated the expression of IGF‐1 and IL‐1β within neurons and glial cells of the ventral cochlear nucleus in adult rats at 1, 7, 15, and 30 days following bilateral cochlear ablation. After the lesion, significant increases in both the overall mean gray levels of IGF‐1 immunostaining and the mean gray levels within cells of the cochlear nucleus were observed at 1, 7, and 15 days compared with control animals. The expression and distribution of IL‐1β in the ventral cochlear nucleus of ablated animals was temporally and spatially correlated with IGF‐1. We also observed a lack of colocalization between IGF‐1 and IL‐1β with either astrocytes or microglia at any of the time points following ablation. These results suggest that the upregulation of IGF‐1 and IL‐1β levels within neurons—but not within glial cells—may reflect a plastic mechanism involved in repairing synaptic homeostasis of the overall cellular environment of the cochlear nucleus following bilateral cochlear ablation. J. Comp. Neurol. 521:3478‐3499, 2013.

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.

Long-term interaction between microglial cells and cochlear nucleus neurons after bilateral cochlear ablation

The removal of afferent activity has been reported to modify neuronal activity in the cochlear nucleus of adult rats. After cell damage, microglial cells are rapidly activated, initiating a series of cellular responses that influences neuronal function and survival. To investigate how this glial response occurs and how it might influence injured neurons, bilateral cochlear ablations were performed on adult rats to examine the short‐term (16 and 24 hours and 4 and 7 days) and long‐term (15, 30, and 100 days) changes in the distribution and morphology of microglial cells (immunostained with the ionized calcium‐binding adaptor molecule 1; Iba‐1) and the interaction of microglial cells with deafferented neurons in the ventral cochlear nucleus. A significant increase in the mean cross‐sectional area and Iba‐1 immunostaining of microglial cells in the cochlear nucleus was observed at all survival times after the ablation compared with control animals. These increases were concomitant with an increase in the area of Iba‐1 immunostaining at 24 hours and 4, 7, and 15 days postablation. Additionally, microglial cells were frequently seen apposing the cell bodies and dendrites of auditory neurons at 7, 15, and 30 days postablation. In summary, these results provide evidence for persistent glial activation in the ventral cochlear nucleus and suggest that long‐term interaction occurs between microglial cells and deafferented cochlear nucleus neurons following bilateral cochlear ablation, which could facilitate the remodeling of the affected neuronal circuits. J. Comp. Neurol. 520:2974–2990, 2012.

The Role of Glia in the Peripheral and Central Auditory System Following Noise Overexposure: Contribution of TNF-α and IL-1β 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-1β (IL-1β), 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-1β are implicated in regulating the initiation and progression of noise-induced hearing loss.

Synergistic effects of free radical scavengers and cochlear vasodilators: a new otoprotective strategy for age-related hearing loss

The growing increase in age-related hearing loss (ARHL), with its dramatic reduction in quality of life and significant increase in health care costs, is a catalyst to develop new therapeutic strategies to prevent or reduce this aging-associated condition. In this regard, there is extensive evidence that excessive free radical formation along with diminished cochlear blood flow are essential factors involved in mechanisms of other stress-related hearing loss, such as that associated with noise or ototoxic drug exposure. The emerging view is that both play key roles in ARHL pathogenesis. Therapeutic targeting of excessive free radical formation and cochlear blood flow regulation may be a useful strategy to prevent onset of ARHL. Supporting this idea, micronutrient-based therapies, in particular those combining antioxidants and vasodilators like magnesium (Mg2+), have proven effective in reducing the impact of noise and ototoxic drugs in the inner ear, therefore improving auditory function. In this review, the synergistic effects of combinations of antioxidant free radicals scavengers and cochlear vasodilators will be discussed as a feasible therapeutic approach for the treatment of ARHL.

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.

Validation of Reference Genes for RT-qPCR Analysis in Noise-Induced Hearing Loss: A Study in Wistar Rat.

The reverse transcriptase–quantitative polymerase chain reaction (RT–qPCR) requires adequate normalization in order to ensure accurate results. The use of reference genes is the most common method to normalize RT–qPCR assays; however, many studies have reported that the expression of frequently used reference genes is more variable than expected, depending on experimental conditions. Consequently, proper validation of the stability of reference genes is an essential step when performing new gene expression studies. Despite the fact that RT–qPCR has been widely used to elucidate molecular correlates of noise–induced hearing loss (NIHL), up to date there are no reports demonstrating validation of reference genes for the evaluation of changes in gene expression after NIHL. Therefore, in this study we evaluated the expression of some commonly used reference genes (Arbp, b–Act, b2m, CyA, Gapdh, Hprt1, Tbp, Tfrc and UbC) and examined their suitability as endogenous control genes for RT–qPCR analysis in the adult Wistar rat in response to NIHL. Four groups of rats were noise–exposed to generate permanent cochlear damage. Cochleae were collected at different time points after noise exposure and the expression level of candidate reference genes was evaluated by RT–qPCR using geNorm, NormFinder and BestKeeper software to determine expression stability. The three independent applications revealed Tbp as the most stably expressed reference gene. We also suggest a group of top–ranked reference genes that can be combined to obtain suitable reference gene pairs for the evaluation of the effects of noise on gene expression in the cochlea. These findings provide essential basis for further RT–qPCR analysis in studies of NIHL using Wistar rats as animal model.

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.