Vijaya Prakash Krishnan Muthaiah

Tinnitus and hyperacusis involve hyperactivity and enhanced connectivity in auditory-limbic-arousal-cerebellar network

Hearing loss often triggers an inescapable buzz (tinnitus) and causes everyday sounds to become intolerably loud (hyperacusis), but exactly where and how this occurs in the brain is unknown. To identify the neural substrate for these debilitating disorders, we induced both tinnitus and hyperacusis with an ototoxic drug (salicylate) and used behavioral, electrophysiological, and functional magnetic resonance imaging (fMRI) techniques to identify the tinnitus–hyperacusis network. Salicylate depressed the neural output of the cochlea, but vigorously amplified sound-evoked neural responses in the amygdala, medial geniculate, and auditory cortex. Resting-state fMRI revealed hyperactivity in an auditory network composed of inferior colliculus, medial geniculate, and auditory cortex with side branches to cerebellum, amygdala, and reticular formation. Functional connectivity revealed enhanced coupling within the auditory network and segments of the auditory network and cerebellum, reticular formation, amygdala, and hippocampus. A testable model accounting for distress, arousal, and gating of tinnitus and hyperacusis is proposed. DOI: http://dx.doi.org/10.7554/eLife.06576.001

N-acetyl-cysteine prevents age-related hearing loss and the progressive loss of inner hair cells in γ-glutamyl transferase 1 deficient mice

Genetic factors combined with oxidative stress are major determinants of age-related hearing loss (ARHL), one of the most prevalent disorders of the elderly. Dwarf grey mice, Ggt1dwg/dwg, are homozygous for a loss of function mutation of the γ-glutamyl transferase 1 gene, which encodes an important antioxidant enzyme critical for the resynthesis of glutathione (GSH). Since GSH reduces oxidative damage, we hypothesized that Ggt1dwg/dwg mice would be susceptible to ARHL. Surprisingly, otoacoustic emissions and cochlear microphonic potentials, which reflect cochlear outer hair cell (OHC) function, were largely unaffected in mutant mice, whereas auditory brainstem responses and the compound action potential were grossly abnormal. These functional deficits were associated with an unusual and selective loss of inner hair cells (IHC), but retention of OHC and auditory nerve fibers. Remarkably, hearing deficits and IHC loss were completely prevented by N-acetyl-L-cysteine, which induces de novo synthesis of GSH; however, hearing deficits and IHC loss reappeared when treatment was discontinued. Ggt1dwg/dwgmice represent an important new model for investigating ARHL, therapeutic interventions, and understanding the perceptual and electrophysiological consequences of sensory deprivation caused by the loss of sensory input exclusively from IHC.

Prolonged noise exposure-induced auditory threshold shifts in rats

Noise-induced hearing loss (NIHL) initially increases with exposure duration, but eventually reaches an asymptotic threshold shift (ATS) once the exposure duration exceeds 18-24 h. Equations for predicting the ATS have been developed for several species, but not for rats, even though this species is extensively used in noise exposure research. To fill this void, we exposed rats to narrowband noise (NBN, 16-20 kHz) for 5 weeks starting at 80 dB SPL in the first week and then increasing the level by 6 dB per week to a final level of 104 dB SPL. Auditory brainstem responses (ABR) were recorded before, during, and following the exposure to determine the amount of hearing loss. The noise induced threshold shift to continuous long-term exposure, defined as compound threshold shift (CTS), within and above 16-20 kHz increased with noise level at the rate of 1.82 dB threshold shift per dB of noise level (NL) above a critical level (C) of 77.2 dB SPL i.e. CTS = 1.82(NL-77.2). The normalized amplitude of the largest ABR peak measured at 100 dB SPL decreased at the rate of 3.1% per dB of NL above the critical level of 76.9 dB SPL, i.e., %ABR Reduction = 3.1%(NL-76.9). ABR thresholds measured >30 days post-exposure only partially recovered resulting in a permanent threshold shift of 30-40 dB along with severe hair cell loss in the basal, high-frequency region of the cochlea. In the rat, CTS increases with noise level with a slope similar to humans and chinchillas. The critical level (C) in the rat is similar to that of humans, but higher than that of chinchillas.

Neuroprotective role of naringenin on carbaryl induced neurotoxicity in mouse neuroblastoma cells

Objective: Neuroprotective effect of naringenin against carbaryl toxicity was studied in mouse neuroblastoma cell line. Materials and Methods: Mouse neuroblastoma cells (Neuro 2A) obtained from National Center for Cell Sciences, Pune, India were either exposed to carbaryl or pre-treated with naringenin (a flavonoid prepared from grape fruit) before their exposure to carbaryl. Results were analyzed using MTT [3-4,5-Dimethylthiazol-2-yl)-2,5-diphenltetrazolium bromide] assay for cell viability, FACS (fluorescence assisted cell sorting) analysis for apoptotic and necrotic cell populations, DCFH-DA (2`,7`-dichlorofluorescin-diacetate) assay for Reactive Oxygen Species (ROS) visualization, JC-1 staining for determining mitochondrial membrane potential and real-time PCR for quantifying pro and anti-apoptotic gene expression. Results: Exposure to naringenin resulted in better survival of Neuro 2A cells which were subsequently subjected to carbaryl toxicity. Treatment with naringenin was found to reduce the oxidative stress by decreasing the ROS and was found to maintain the integrity of mitochondrial membrane potential. It was also found to downregulate pro-apoptotic genes (BAX and Caspase-3) while upregulating anti-apototic gene (Bcl2). Conclusion: The results of this pilot study underline the potential of naringenin in treating carbaryl induced neurotoxicity and further studies are warranted to establish the effect of naringenin in vivo conditions.

Frequency-specific alternations in the amplitude of low-frequency fluctuations in chronic tinnitus.

Tinnitus, a phantom ringing, buzzing, or hissing sensation with potentially debilitating consequences, is thought to arise from aberrant spontaneous neural activity at one or more sites within the central nervous system; however, the location and specific features of these oscillations are poorly understood with respect to specific tinnitus features. Recent resting-state functional magnetic resonance imaging (fMRI) studies suggest that aberrant fluctuations in spontaneous low-frequency oscillations (LFO) of the blood oxygen level-dependent (BOLD) signal may be an important factor in chronic tinnitus; however, the role that frequency-specific components of LFO play in subjective tinnitus remains unclear. A total of 39 chronic tinnitus patients and 41 well-matched healthy controls participated in the resting-state fMRI scans. The LFO amplitudes were investigated using the amplitude of low-frequency fluctuation (ALFF) and fractional ALFF (fALFF) in two different frequency bands (slow-4: 0.027–0.073 Hz and slow-5: 0.01–0.027 Hz). We observed significant differences between tinnitus patients and normal controls in ALFF/fALFF in the two bands (slow-4 and slow-5) in several brain regions including the superior frontal gyrus (SFG), inferior frontal gyrus, middle temporal gyrus, angular gyrus, supramarginal gyrus, and middle occipital gyrus. Across the entire subject pool, significant differences in ALFF/fALFF between the two bands were found in the midbrain, basal ganglia, hippocampus and cerebellum (Slow 4 > Slow 5), and in the middle frontal gyrus, supramarginal gyrus, posterior cingulate cortex, and precuneus (Slow 5 > Slow 4). We also observed significant interaction between frequency bands and patient groups in the orbitofrontal gyrus. Furthermore, tinnitus distress was positively correlated with the magnitude of ALFF in right SFG and the magnitude of fALFF slow-4 band in left SFG, whereas tinnitus duration was positively correlated with the magnitude of ALFF in right SFG and the magnitude of fALFF slow-5 band in left SFG. Resting-state fMRI provides an unbiased method for identifying aberrant spontaneous LFO occurring throughout the central nervous system. Chronic tinnitus patients have widespread abnormalities in ALFF and fALFF slow-4 and slow-5 band which are correlated with tinnitus distress and duration. These results provide new insights on the neuropathophysiology of chronic tinnitus; therapies capable of reversing these aberrant patterns may reduce tinnitus distress.

Carbaryl-induced ototoxicity in rat postnatal cochlear organotypic cultures.

Carbaryl, a widely used carbamate‐based insecticide, is a potent anticholinesterase known to induce delayed neurotoxicity following chronic exposure. However, its potential toxic effects on the cochlea, the sensory organ for hearing that contains cholinergic efferent neurons and acetylcholine receptors on the hair cells (HC) and spiral ganglion neurons has heretofore not been evaluated. To assess ototoxic potential of carbaryl, cochlear organotypic cultures from postnatal day 3 rats were treated with doses of carbaryl ranging from 50 to 500 μM for 48 h up to 96 h. Carbaryl damaged both the sensory HC and spiral ganglion neurons in a dose‐ and duration‐dependent manner. HC and neuronal damage was observed at carbaryl concentrations as low as 50 μM after 96‐h treatment and 100 μM after 48‐h treatment. Hair cell was greatest in the high frequency basal region of the cochlea and progressively decreased towards the apex consistent with the majority of ototoxic drugs. In contrast, damage to the spiral ganglion neurons was of similar magnitude in the basal and apical regions of the cochlea. Carbaryl damage was characterized by soma shrinkage, nuclear condensation and fragmentation, and blebbing, morphological features of programmed cell death. Carbaryl upregulated the expression of executioner caspase‐3 in HC and spiral ganglion neurons indicating that cellular damage occurred primarily by caspase‐mediated apoptosis. These results suggest that chronic exposure to carbaryl and other carbamate anticholinesterases may be ototoxic.

JNK1 and JNK3 play a significant role in both neuronal apoptosis and necrosis. Evaluation based on in vitro approach using tert-butylhydroperoxide induced oxidative stress in neuro-2A cells and perturbation through 3-aminobenzamide

In spinal cord injury (SCI), oxidative stress in the penumbra of the injury site is a characteristic feature. The predominance of necrosis over apoptosis in the ensuing delayed cell death results in progressive waves of necrosis affecting neighboring cells and thus exaggerates the severity of the lesion. Necrosis has been classified into subtypes based on the active molecular players and parthanatos is one among them, which is characterized by the over activation of PARP1 as the pre-mitochondrial event that triggers necrosis. Parthanatos being the necrosis mode reported in SCI, we intended to study the molecular players in the elusive pre-mitochondrial events of PARP1 over activation using an in vitro model. tert-Butylhydroperoxide (tBuOOH) was reported to induce oxidative stress in various cell types including Neuro-2A cells. Using a tailored protocol, a predominantly PARP1 mediated necrotic mode of cell death was obtained in Neuro-2A cells using tBuOOH. By perturbing the progress of necrosis using 3-amniobenzamide, a known PARP1 inhibitor, it was found that JNK1 and JNK3 but not JNK2 were involved in pre-mitochondrial stages of PARP1 mediated cell death. Given that JNK1 and JNK3 play a role in apoptosis also, they may serve as common targets to counter both apoptosis and necrosis. The in vitro model used in the present study may be useful in delineating molecular mechanisms in necrosis.

Effect of manganese and manganese plus noise on auditory function and cochlear structures.

The degenerative actions of Mn caused by persistent exposure to high atmospheric levels not only provokes irreversible damage to the CNS with symptoms comparable to that of Parkinsons disease but also may have deleterious consequences to other organs including the auditory system. The putative deleterious consequences of prolonged Mn overexposure on hearing, however, is confounded by the fact that chronically-exposed individuals often work in high noise environments where noise by itself is known to cause hearing loss. Thus, the question as to whether Mn alone is actually ototoxic and whether exposure to Mn when combined with noise increases the risk of hearing loss and cochlear pathology has never been examined. To examine whether noise effects Mn ototoxicity, we exposed rats to a moderate dose of Mn (10mg MnCl2/liter water) alone, a high level of noise (octave band noise, 8-16kHz, presented at 90dB SPL for 8h/d) alone or the combination of Mn plus noise and measured the changes in auditory function and the cochlear histopathologies. Results of these studies, based on various measures of hearing including histological examination of cochlear tissue suggest that noise alone produced significant hearing deficits whereas semi-chronic exposure to moderate levels of Mn in drinking water for 90days either in the presence or absence of noise had, at best, only a minor effect on hearing.

Impact of occupational health hazards on serum markers of bone formation in spray painters of Chennai region in Tamil Nadu.

Context: The association between spray paint exposure and bone remodeling received little attention despite the high usage of spray paints in automobile industries, steel furniture workshops etc. Aim: The present study was aimed at investigating the level of serum markers of bone formation in spray painters. The spray painting subjects were selected from automobile body repair workshops in Chennai region of TamilNadu which constitutes 30% of Indias automobile industry. Setting and Design: All the study subjects, exposed to spray paint were working in a workshop without standard spraying room and did not wore any aerosol removing respirator. The controls were selected from random population irrespective of occupation. Data relevant to the socioeconomic features and personal history was collected using a questionnaire. The current study included 50 spray painters and 25 control subjects of same age group. Materials and Methods: We examined the level of serum calcium, serum phosphorus, serum differentiation markers of bone such as alkaline phosphatase (bone specific) and serum osteocalcin in which these levels were found to be high in serum of spray painters. Conclusion: The current study concludes dysregulation in bone remodeling of spray painters exposed to chronic solvents and paint pigments.

Effects of cochlear-synaptopathy inducing moderate noise exposure on auditory-nerve-fiber responses in chinchillas

It has been hypothesized that selective loss of low-spontaneous-rate (low-SR) auditory-nerve (AN) fibers following moderate noise exposure may underlie perceptual difficulties some people experience in noisy situations, despite normal audiograms. However, the finding of selective low-SR-fiber loss has not been replicated in an animal model with behavioral thresholds similar to humans. We recently established a behavioral chinchilla model for which neural and behavioral AM-detection thresholds are in line with each other and similar to humans. Here, we report physiological AN-fiber response properties from anesthetized chinchillas exposed to noise that produced cochlear synaptopathy, as confirmed by immunofluorescence histology. Auditory-brainstem responses, distortion-product otoacoustic emissions, and compound action potentials confirmed no significant permanent threshold shift. Stimuli included both simple (pure tones, as studied previously) and complex (broadband noise) sounds. Low-SR fibers were reduc...