Subtle alterations of vestibulomotor functioning in conductive hearing loss.

Introduction: Conductive hearing loss (CHL) attenuates the ability to transmit air conducted sounds to the ear. In humans, severe hearing loss is often accompanied by alterations to other neural systems, such as the vestibular system; however, the inter-relations are not well understood. The overall goal of this study was to assess vestibular-related functioning proxies in a rat CHL model. Methods: Male Sprague-Dawley rats (N=134, 250g, 2months old) were used in a CHL model which produced a >20dB threshold shift induced by tympanic membrane puncture. Auditory brainstem response (ABRs) recordings were used to determine threshold depth at different times before and after CHL. ABR threshold depths were assessed both manually and by an automated ABR machine learning algorithm. Vestibular-related functioning proxy assessment was performed using the rotarod, balance beam, elevator vertical motion (EVM) and Ferris-wheel rotation (FWR) assays. Results: The Pre-CHL (control) threshold depth was 27.92dB±11.58dB compared to the Post-CHL threshold depth of 50.69dB±13.98dB (mean±SD) across the frequencies tested. The automated ABR machine learning algorithm determined the following threshold depths: Pre-CHL=24.3dB, Post-CHL same day=56dB, Post-CHL 7 days=41.16dB, and Post-CHL 1 month=32.5dB across the frequencies assessed (1, 2, 4, 8, 16, and 32kHz). Rotarod assessment of motor function was not significantly different between pre and post-CHL (~1week) rats for time duration (sec) or speed (RPM), albeit the former had a small effect size difference. Balance beam time to transverse was significantly longer for post-CHL rats, likely indicating a change in motor coordination. Further, failure to cross was only noted for CHL rats. The defection count was significantly reduced for CHL rats compared to control rats following FWR, but not EVM. The total distance traveled during open-field examination after EVM was significantly different between control and CHL rats, but not for FWR. The EVM is associated with linear acceleration (acting in the vertical plane: up-down) stimulating the saccule, while the FWR is associated with angular acceleration (centrifugal rotation about a circular axis) stimulating both otolith organs and semicircular canals; therefore, the difference in results could reflect the specific vestibular-organ functional role. Discussion: Less movement (EVM) and increase time to transverse (balance beam) may be associated with anxiety and alterations to defecation patterns (FWR) may result from autonomic disturbances due to the impact of hearing loss. In this regard, vestibulomotor deficits resulting in changes in balance and motion could be attributed to comodulation of auditory and vestibular functioning. Future studies should manipulate vestibular functioning directly in rats with CHL.

Cholecystokinin receptor antagonist challenge elicits brain-wide functional connectome modulation with micronetwork hippocampal decreased calcium transients.

The cortical distribution and functional role of cholecystokinin (CCK) are largely unknown. Here, a CCK receptor antagonist challenge paradigm was developed to assess functional connectivity and neuronal responses. Structural-functional magnetic resonance imaging and calcium imaging were undertaken in environmental enrichment (EE) and standard environment (SE) groups (naïve adult male mice, n = 59, C57BL/B6J, P = 60). Functional connectivity network-based statistics and pseudo-demarcation Voronoi tessellations to cluster calcium signals were used to derive region of interest metrics based on calcium transients, firing rate, and location. The CCK challenge elicited robust changes to structural-functional networks, decreased neuronal calcium transients, and max firing rate (5 s) of dorsal hippocampus in SE mice. However, the functional changes were not observed in EE mice, while the decreased neuronal calcium transients and max firing rate (5 s) were similar to SE mice. Decreased gray matter alterations were observed in multiple brain regions in the SE group due to CCK challenge, while no effect was observed in the EE group. The networks most affected by CCK challenge in SE included within isocortex, isocortex to olfactory, isocortex to striatum, olfactory to midbrain, and olfactory to thalamus. The EE group did not experience network changes in functional connectivity due to CCK challenge. Interestingly, calcium imaging revealed a significant decrease in transients and max firing rate (5 s) in the dorsal CA1 hippocampus subregion after CCK challenge in EE. Overall, CCK receptor antagonists affected brain-wide structural-functional connectivity within the isocortex, in addition to eliciting decreased neuronal calcium transients and max firing rate (5 s) in CA1 of the hippocampus. Future studies should investigate the CCK functional networks and how these processes affect isocortex modulation. Significance Statement  Cholecystokinin is a neuropeptide predominately found in the gastrointestinal system. Albeit abundantly expressed in neurons, the role and distribution of cholecystokinin are largely unknown. Here, we demonstrate cholecystokinin affects brain-wide structural-functional networks within the isocortex. In the hippocampus, the cholecystokinin receptor antagonist challenge decreases neuronal calcium transients and max firing rate (5 s) in CA1. We further demonstrate that mice in environmental enrichment do not experience functional network changes to the CCK receptor antagonist challenge. Environmental enrichment may afford protection to the alterations observed in control mice due to CCK. Our results suggest that cholecystokinin is distributed throughout the brain, interacts in the isocortex, and demonstrates an unexpected functional network stability for enriched mice.

Environmental enrichment leads to behavioral circadian shifts enhancing brain-wide functional connectivity between sensory cortices and eliciting increased hippocampal spiking.

Environmental enrichment induces widespread neuronal changes, but the initiation of the cascade is unknown. We ascertained the critical period of divergence between environmental enriched (EE) and standard environment (SE) mice using continuous infrared (IR) videography, functional magnetic resonance imaging (fMRI), and neuron level calcium imaging. Naïve adult male mice (n = 285, C57BL/6J, postnatal day 60) were divided into SE and EE groups. We assessed the linear time-series of motion activity using a novel structural break test which examined the dataset for change in circadian and day-by-day motion activity. fMRI was used to map brain-wide response using a functional connectome analysis pipeline. Awake calcium imaging was performed on the dorsal CA1 pyramidal layer. We found the preeminent behavioral feature in EE was a forward shift in the circadian rhythm, prolongation of activity in the dark photoperiod, and overall decreased motion activity. The crepuscular period of dusk was seen as the critical period of divergence between EE and SE mice. The functional processes at dusk in EE included increased functional connectivity in the visual cortex, motor cortex, retrosplenial granular cortex, and cingulate cortex using seed-based analysis. Network based statistics found a modulated functional connectome in EE concentrated in two hubs: the hippocampal formation and isocortical network. These hubs experienced a higher node degree and significant enhanced edge connectivity. Calcium imaging revealed increased spikes per second and maximum firing rate in the dorsal CA1 pyramidal layer, in addition to location (anterior-posterior and medial-lateral) effect size differences between EE and SE. The emergence of functional-neuronal changes due to enrichment consisted of enhanced hippocampal-isocortex functional connectivity and CA1 neuronal increased spiking linked to a circadian shift during the dusk period. Future studies should explore the molecular consequences of enrichment inducing shifts in the circadian period.

Structural Alterations in a Rat Model of Short-Term Conductive Hearing Loss Are Associated With Reduced Resting State Functional Connectivity.

Conductive hearing loss (CHL) results in attenuation of air conducted sound reaching the inner ear. How a change in air conducted sound alters the auditory system resulting in cortical alterations is not well understood. Here, we have assessed structural and functional magnetic resonance imaging (MRI) in an adult (P60) rat model of short-term conductive hearing loss (1 week). Diffusion tensor imaging (DTI) revealed fractional anisotropy (FA) and axial diffusivity alterations after hearing loss that circumscribed the auditory cortex (AC). Tractography found the lateral lemniscus tract leading to the bilateral inferior colliculus (IC) was reduced. For baseline comparison, DTI and tractography alterations were not found for the somatosensory cortex. To determine functional connectivity changes due to hearing loss, seed-based analysis (SBA) and independent component analysis (ICA) were performed. Short term conductive hearing loss altered functional connectivity in the AC and IC, but not the somatosensory cortex. The results present an exploratory neuroimaging assessment of structural alterations coupled to a change in functional connectivity after conductive hearing loss. The results and implications for humans consist of structural-functional brain alterations following short term hearing loss in adults.

Hearing loss impacts gray and white matter across the lifespan: Systematic review, meta-analysis and meta-regression.

Hearing loss is a heterogeneous disorder thought to affect brain reorganization across the lifespan. Here, structural alterations of the brain due to hearing loss are assessed by using unique effect size metrics based on Cohen's d and Hedges' g. These metrics are used to map coordinates of gray matter (GM) and white matter (WM) alterations from bilateral congenital and acquired hearing loss populations. A systematic review and meta-analysis revealed m = 72 studies with structural alterations measured with magnetic resonance imaging (MRI) (bilateral = 64, unilateral = 8). The bilateral studies categorized hearing loss into congenital and acquired cases (n = 7,445) and control cases (n = 2,924), containing 66,545 datapoint metrics. Hearing loss was found to affect GM and underlying WM in nearly every region of the brain. In congenital hearing loss, GM decreased most in the frontal lobe. Similarly, acquired hearing loss had a decrease in frontal lobe GM, albeit the insula was most decreased. In congenital, WM underlying the frontal lobe GM was most decreased. In congenital, the right hemisphere was more negatively impacted than the left hemisphere; however, in acquired, this was the opposite. The WM alterations most frequently underlined GM alterations in congenital hearing loss, while acquired hearing loss studies did not frequently assess the WM metric. Future studies should use the endophenotype of hearing loss as a prognostic template for discerning clinical outcomes.

Lithium from breast-milk inhibits thyroid iodine uptake and hormone production, which are remedied by maternal iodine supplementation.

BACKGROUND: Lithium is especially taken as a maintenance medication for Bipolar Disorder. In women with bipolar disorder, lithium is often effective during postpartum period, but breast-feeding for medicated mothers is controversial because of harmful effects for her child. At present, the biological mechanisms of lithium are not well-understood, affecting its usage and overall health implications. PROCEDURE: We developed a rat lithium and breast-feeding model at human therapeutic levels to study the effects of lithium exposure through breast-milk on pups' thyroid function. Novel laser analytical spectroscopy, along with traditional blood and immunohistochemical tests, were applied to further investigate the mechanisms behind the thyroid dysfunction. Maternal iodine supplementation was evaluated as a therapeutic method to address the pups' thyroid dysfunction. RESULTS: Pups exposed to lithium via breastmilk, even with the dam on a sub-therapeutic level, experienced weight gain, reduced blood thyroxine (T4 ), and elevated blood urea nitrogen, indicating effects on thyroid and kidney function. We show that lithium inhibited iodine uptake by thyroid follicles, initiating a mechanism that reduced iodination of tyrosine, thyroglobulin cleavage, and thyroid hormone production. Importantly, infant thyroid function can be significantly improved by administering supplementary iodine to the medicated dam's diet during breast-feeding. CONCLUSION: These results elucidate the mechanisms of lithium in thyroid function, provide valuable information on use postpartum, and suggest a clinically applicable remedy to side-effects. The results are particularly important for patients (and their infants) who respond well to lithium and need, or choose, to breast-feed.

Structural and Functional Hippocampal Correlations in Environmental Enrichment During the Adolescent to Adulthood Transition in Mice.

Environmental enrichment is known to induce neuronal changes; however, the underlying structural and functional factors involved are not fully known and remain an active area of study. To investigate these factors, we assessed enriched environment (EE) and standard environment (SE) control mice over 30 days using structural and functional MRI methods. Naïve adult male mice (n = 30, ≈20 g, C57BL/B6J, postnatal day 60 initial scan) were divided into SE and EE groups and scanned before and after 30 days. Structural analyses included volumetry based on manual segmentation as well as diffusion tensor imaging (DTI). Functional analyses included seed-based analysis (SBA), independent component analysis (ICA), the amplitude of low-frequency fluctuation (ALFF), and fractional ALFF (fALFF). Structural results indicated that environmental enrichment led to an increase in the volumes of cornu ammonis 1 (CA1) and dentate gyrus. Structural results indicated changes in radial diffusivity and mean diffusivity in the visual cortex and secondary somatosensory cortex after EE. Furthermore, SBA and ICA indicated an increase in resting-state functional MRI (rsfMRI) functional connectivity in the hippocampus. Using parallel structural and functional analyses, we have demonstrated coexistent structural and functional changes in the hippocampal subdivision CA1. Future research should map alterations temporally during environmental enrichment to investigate the initiation of these structural and functional changes.

Spectroscopic and microscopic examination of teeth exposed to green tea at different temperatures.

Tea is a popular beverage consumed at different temperatures. The effect of tea on teeth at different temperatures has not been studied previously. The present study used an in vitro green tea immersed tooth model at different tea temperatures (hot and cold) compared to an in vivo tea administration model allowing rats to drink tea over the course of a week. The elements present in tea leaves were identified by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and compared to the elements in teeth (enamel surface) using Laser-Induced Breakdown Spectroscopy (LIBS). Here, LIBS demonstrated in vivo and in vitro green tea treatments resulted in a significant increase in the mineral elements found in enamel. For the in vitro assessment, elements in enamel varied based on cold-tea and hot-tea treatment; however, hot water reduced the elements in enamel. Atomic force microscopy found the in vivo tea group had a higher roughness average (RA) compared with the in vivo water group. Cold tea and hot tea in vitro groups demonstrated lower RA than in vitro water controls. Scanning electron microscopy found hot water induced cracks more than 1.3µm in enamel while cold tea and hot tea promoted the adhering of extrinsic matter to teeth. Overall, teeth treated to high temperature lost the mineral phase leading to demineralization. Our results indicate that green tea protects enamel, but its protective action in dental structures is enhanced at cold temperature.

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation.

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

Estrogen accelerates heart regeneration by promoting the inflammatory response in zebrafish.

Sexual differences have been observed in the onset and prognosis of human cardiovascular diseases, but the underlying mechanisms are not clear. Here, we found that zebrafish heart regeneration is faster in females, can be accelerated by estrogen and is suppressed by the estrogen-antagonist tamoxifen. Injuries to the zebrafish heart, but not other tissues, increased plasma estrogen levels and the expression of estrogen receptors, especially esr2a. The resulting endocrine disruption induces the expression of the female-specific protein vitellogenin in male zebrafish. Transcriptomic analyses suggested heart injuries triggered pronounced immune and inflammatory responses in females. These responses, previously shown to elicit heart regeneration, could be enhanced by estrogen treatment in males and reduced by tamoxifen in females. Furthermore, a prior exposure to estrogen preconditioned the zebrafish heart for an accelerated regeneration. Altogether, this study reveals that heart regeneration is modulated by an estrogen-inducible inflammatory response to cardiac injury. These findings elucidate a previously unknown layer of control in zebrafish heart regeneration and provide a new model system for the study of sexual differences in human cardiac repair.

Simple Surgical Induction of Conductive Hearing Loss with Verification Using Otoscope Visualization and Behavioral Clap Startle Response in Rat.

Conductive hearing loss (CHL) is a prevalent hearing impairment in humans. The goal of the protocol is to describe a simple surgical procedure for inducing CHL in rodents. The protocol demonstrates CHL by tympanic membrane puncture. Verification of CHL surgery was by otoscope examination and behavioral assessment by clap startle response, both replicable and reliable, and are simple methods to demonstrate hearing loss has occurred. The simple CHL procedure is advantageous due to its reproducibility and flexibility to different pursuits in hearing loss research. The limitations of inducing CHL by a surgical approach are associated with the learning curve to perform the surgical procedure and confidence in audiological examination. Inducing a hearing impairment by CHL allows one to readily study the neural manifestations and behavioral outcomes of hearing loss.

The human amygdala disconnecting from auditory cortex preferentially discriminates musical sound of uncertain emotion by altering hemispheric weighting.

How do humans discriminate emotion from non-emotion? The specific psychophysical cues and neural responses involved with resolving emotional information in sound are unknown. In this study we used a discrimination psychophysical-fMRI sparse sampling paradigm to locate threshold responses to happy and sad acoustic stimuli. The fine structure and envelope of auditory signals were covaried to vary emotional certainty. We report that emotion identification at threshold in music utilizes fine structure cues. The auditory cortex was activated but did not vary with emotional uncertainty. Amygdala activation was modulated by emotion identification and was absent when emotional stimuli were chance identifiable, especially in the left hemisphere. The right hemisphere amygdala was considerably more deactivated in response to uncertain emotion. The threshold of emotion was signified by a right amygdala deactivation and change of left amygdala greater than right amygdala activation. Functional sex differences were noted during binaural uncertain emotional stimuli presentations, where the right amygdala showed larger activation in females. Negative control (silent stimuli) experiments investigated sparse sampling of silence to ensure modulation effects were inherent to emotional resolvability. No functional modulation of Heschl's gyrus occurred during silence; however, during rest the amygdala baseline state was asymmetrically lateralized. The evidence indicates changing hemispheric activation and deactivation patterns between the left and right amygdala is a hallmark feature of discriminating emotion from non-emotion in music.

Uncertain Emotion Discrimination Differences Between Musicians and Non-musicians Is Determined by Fine Structure Association: Hilbert Transform Psychophysics.

Humans perceive musical sound as a complex phenomenon, which is known to induce an emotional response. The cues used to perceive emotion in music have not been unequivocally elucidated. Here, we sought to identify the attributes of sound that confer an emotion to music and determine if professional musicians have different musical emotion perception than non-musicians. The objective was to determine which sound cues are used to resolve emotional signals. Happy or sad classical music excerpts modified in fine structure or envelope conveying different degrees of emotional certainty were presented. Certainty was determined by identification of the emotional characteristic presented during a forced-choice discrimination task. Participants were categorized as good or poor performers (n = 32, age 21.16 ± 2.59 SD) and in a separate group as musicians in the first or last year of music education at a conservatory (n = 32, age 21.97 ± 2.42). We found that temporal fine structure information is essential for correct emotional identification. Non-musicians used less fine structure information to discriminate emotion in music compared with musicians. The present psychophysical experiments revealed what cues are used to resolve emotional signals and how they differ between non-musicians and musically educated individuals.

Prolonged neutrophil retention in the wound impairs zebrafish heart regeneration after cryoinjury.

Neutrophils are the first line defenders in the innate immune response, and rapidly migrate to an infected or injured area. Recently, bidirectional migration of neutrophils to the wound and the corresponding functions have become popular research pursuits. In zebrafish larvae, CXCR1/CXCL8 is the predominant chemoattractant pathway to recruit neutrophil to wound, while CXCR2/CXCL8 pathway mediate neutrophil dispersal in wound after injury. Here, we found that both CXCR1/CXCL8 and LTB4/BLT1 signals are activated in zebrafish heart after cryoinjury. And with a CXCR1/2 selective inhibitor (SB225002) treatment, the recruitment of neutrophils was not affected, but reverse migration of neutrophils was inhibited after cryoinjury of heart. We suggested that the neutrophil recruitment to cryoinjured area might be mediated by LTB4/BLT1 signals at the presence of SB225002. Therefore, SB225002 treatment resulted more accumulation and long retention of neutrophils in the injured heart. The long retention of neutrophils in the wound promoted revascularization in the injured heart; however, the AKT/mTOR pathway was inhibited and the regeneration was impaired. Our findings suggest that retention of neutrophils is a well-orchestrated process and might regulate regeneration by the AKT/mTOR pathway.

Sparse Sampling of Silence Type I Errors With an Emphasis on Primary Auditory Cortex.

Sparse sampling functional MRI (ssfMRI) enables stronger primary auditory cortex blood oxygen level-dependent (BOLD) signal by acquiring volumes interspersed with silence, reducing the physiological artifacts associated with scanner noise. Recent calculations of type I error rates associated with resting-state fMRI suggest that the techniques used to model the hemodynamic response function (HRF) might be resulting in higher false positives than is generally acceptable. In the present study, we analyze ssfMRI to determine type I error rates associated with whole brain and primary auditory cortex voxel-wise activation patterns. Study participants (n = 15, age 27.62 ± 3.21 years, range: 22-33 years; 6 females) underwent ssfMRI. An optimized paradigm was used to determine the HRF to auditory stimuli, which was then substituted for silent stimuli to ascertain false positives. We report that common techniques used for analyzing ssfMRI result in high type I error rates. The whole brain and primary auditory cortex voxel-wise analysis resulted in similar error distributions. The number of type I errors for P < 0.05, P < 0.01, and P < 0.001 for the whole brain was 7.88 ± 9.29, 2.37 ± 3.54, and 0.53 ± 0.96% and for the auditory cortex was 9.02 ± 1.79, 2.95 ± 0.91, and 0.58 ± 0.21%, respectively. When conducting a ssfMRI analysis, conservative α level should be employed (α < 0.001) to bolster the results in the face of false positive results.

Early Stage Alterations in White Matter and Decreased Functional Interhemispheric Hippocampal Connectivity in the 3xTg Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized in the late stages by amyloid-ß (Aß) plaques and neurofibrillary tangles. Nevertheless, recent evidence has indicated that early changes in cerebral connectivity could compromise cognitive functions even before the appearance of the classical neuropathological features. Diffusion tensor imaging (DTI), resting-state functional magnetic resonance imaging (rs-fMRI) and volumetry were performed in the triple transgenic mouse model of AD (3xTg-AD) at 2 months of age, prior to the development of intraneuronal plaque accumulation. We found the 3xTg-AD had significant fractional anisotropy (FA) increase and radial diffusivity (RD) decrease in the cortex compared with wild-type controls, while axial diffusivity (AD) and mean diffusivity (MD) were similar. Interhemispheric hippocampal connectivity was decreased in the 3xTg-AD while connectivity in the caudate putamen (CPu) was similar to controls. Most surprising, ventricular volume in the 3xTg-AD was four times larger than controls. The results obtained in this study characterize the early stage changes in interhemispheric hippocampal connectivity in the 3xTg-AD mouse that could represent a translational biomarker to human models in preclinical stages of the AD.

Excessive inflammation impairs heart regeneration in zebrafish breakdance mutant after cryoinjury.

Inflammation plays a crucial role in cardiac regeneration. Numerous advantages, including a robust regenerative ability, make the zebrafish a popular model to study cardiovascular diseases. The zebrafish breakdance (bre) mutant shares several key features with human long QT syndrome that predisposes to ventricular arrhythmias and sudden death. However, how inflammatory response and tissue regeneration following cardiac damage occur in bre mutant is unknown. Here, we have found that inflammatory response related genes were markedly expressed in the injured heart and excessive leukocyte accumulation occurred in the injured area of the bre mutant zebrafish. Furthermore, bre mutant zebrafish exhibited aberrant apoptosis and impaired heart regenerative ability after ventricular cryoinjury. Mild dosages of anti-inflammatory or prokinetic drugs protected regenerative cells from undergoing aberrant apoptosis and promoted heart regeneration in bre mutant zebrafish. We propose that immune or prokinetic therapy could be a potential therapeutic regimen for patients with genetic long QT syndrome who suffers from myocardial infarction.

Auditory-visual convergence at the superior colliculus in rat using functional MRI.

The superior colliculus (SC) of the midbrain has been a model structure for multisensory processing. Many neurons in the intermediate and deep SC layers respond to two or more of auditory, visual, and somatosensory stimuli as assessed by electrophysiology. In contrast, noninvasive and large field of view functional magnetic resonance imaging (fMRI) studies have focused on multisensory processing in the cortex. In this study, we applied blood oxygenation leveldependent (BOLD) fMRI on Sprague-Dawley rats receiving monaural (auditory) and binocular (visual) stimuli to study subcortical multisensory processing. Activation was observed in the left superior olivary complex, lateral lemniscus, and inferior colliculus and both hemispheres of the superior colliculus during auditory stimulation. The SC response was bilateral even though the stimulus was monaural. During visual stimulation, activation was observed in both hemispheres of the SC and lateral geniculate nucleus. In both hemispheres of the SC, the number of voxels in the activation area $( \mathrm {p}<10 -8$) and BOLD signal changes $( \mathrm {p}<0.01)$ were significantly greater during visual than auditory stimulation. These results provide functional imaging evidence that the SC is a site of auditoryvisual convergence due to its involvement in both auditory and visual processing. The auditory and visual fMRI activations likely reflect the firing of unisensory and multisensory neurons in the SC. The present study lays the groundwork for noninvasive functional imaging studies of multisensory convergence and integration in the SC.

Spectroscopic examination of enamel staining by coffee indicates dentin erosion by sequestration of elements.

The mechanism of coffee eliciting erosion on teeth is unclear as few studies have investigated the direct effect of coffee on enamel and dentin structures. The present study identified how coffee, the most popular beverage worldwide, induces staining and erosion on teeth. We show the grade of erosion of molars and incisors in Sprague Dawley rats from two different age groups, young (four weeks) and old (six months). We quantified the concentration of metals contained in coffee by mass spectrometry (ICP-MS). To determine elemental content in enamel (i.e. superficial) and dentin (i.e. substructure), we used Laser-induced Breakdown Spectroscopy (LIBS) and X-ray fluorescence (XRF) spectroscopy, respectively. For LIBS, a significant decrease of Ca, P, and Na was observed in the young coffee group relative to age-matched controls, whereas a significant increase in Mn, Fe, and K was observed. In the old coffee group, a significant increase of Mg, Fe, and K was observed along with a decrease of Mg, Ca, P, Na, Sr and Zn. For XRF, a significant decrease of the Ca/P ratio in the coffee group was observed. Spectroscopy results were correlated with scanning electron microscopy (SEM) and histological analysis. The SEM analysis showed pores and open spaces between young and old coffee groups, respectively. Thinning of enamel layers, loss of continuity in the enamel-dentin-junction, and wide spaces in dentin tubules with coffee use was found histologically. Coffee induces decalcification of teeth that corresponds to erosion, exposing the dentin structure by reducing enamel. Coffee immersion demonstrated an intrinsic staining in dentin by metal deposition.