Publications using the SigGen

Significance: Quantitative measurements of cerebral hemodynamic changes due to functional activation are widely accomplished with commercial continuous wave (CW-NIRS) instruments despite the availability of the more rigorous multi-distance frequency domain (FD-NIRS) approach. A direct comparison of the two approaches to functional near-infrared spectroscopy can help in the interpretation of optical data and guide implementations of diffuse optical instruments for measuring functional activation. Aim: We explore the differences between CW-NIRS and multi-distance FD-NIRS by comparing measurements of functional activation in the human auditory cortex. Approach: Functional activation of the human auditory cortex was measured using a commercial frequency domain near-infrared spectroscopy instrument for 70 dB sound pressure level broadband noise and pure tone (1000 Hz) stimuli. Changes in tissue oxygenation were calculated using the modified Beer–Lambert law (CW-NIRS approach) and the photon diffusion equation (FD-NIRS approach). Results: Changes in oxygenated hemoglobin measured with the multi-distance FD-NIRS approach were about twice as large as those measured with the CW-NIRS approach. A finite-element simulation of the functional activation problem was performed to demonstrate that tissue oxygenation changes measured with the CW-NIRS approach is more accurate than that with multi-distance FD-NIRS. Conclusions: Multi-distance FD-NIRS approaches tend to overestimate functional activation effects, in part due to partial volume effects.

Noise-induced hearing loss (NIHL) is one of the most prevalent forms of acquired hearing loss, and it is associated with aberrant microglial status and reduced hippocampal neurogenesis; however, the nature of these associations is far from being elucidated. Beyond its direct effects on the auditory system, exposure to intense noise has previously been shown to acutely activate the stress response, which has increasingly been linked to both microglial activity and adult hippocampal neurogenesis in recent years. Given the pervasiveness of noise pollution in modern society and the important implications of either microglial activity or hippocampal neurogenesis for cognitive and emotional function, this study was designed to investigate how microglial status and hippocampal neurogenesis change over time following acoustic exposure and to analyze the possible roles of the noise exposure-induced stress response and hearing loss in these changes. To accomplish this, adult male C57BL/6J mice were randomly assigned to either a control or noise exposure (NE) group. Auditory function was assessed by measuring ABR thresholds at 20 days post noise exposure. The time-course profile of serum corticosterone levels, microglial status, and hippocampal neurogenesis during the 28 days following noise exposure were quantified by ELISA or immunofluorescence staining. Our results illustrated a permanent moderate-to-severe degree of hearing loss, an early but transient increase in serum corticosterone levels, and time-dependent dynamic alterations in microglial activation status and hippocampal neurogenesis, which both present an early but transient change and a late but enduring change. These findings provide evidence that both the stress response and hearing loss contribute to the dynamic alterations of microglia and hippocampal neurogenesis following noise exposure; moreover, noise-induced permanent hearing loss rather than noise-induced transient stress is more likely to be responsible for perpetuating the neurodegenerative process associated with many neurological diseases.

Pathogenic variants in GJB2, the gene encoding connexin 26, are the most common cause of autosomal-recessive hereditary deafness. Despite this high prevalence, pathogenic mechanisms leading to GJB2-related deafness are not well understood, and cures are absent. Humans with GJB2-related deafness retain at least some auditory hair cells and neurons, and their deafness is usually stable. In contrast, mice with conditional loss of Gjb2 in supporting cells exhibit extensive loss of hair cells and neurons and rapidly progress to profound deafness, precluding the application of therapies that require intact cochlear cells. In an attempt to design a less severe Gjb2 animal model, we generated mice with inducible Sox10iCre ERT2 -mediated loss of Gjb2. Tamoxifen injection led to reduced connexin 26 expression and impaired function, but cochlear hair cells and neurons survived for 2 months, allowing phenotypic rescue attempts within this time. AAV-mediated gene transfer of GJB2 in mature mutant ears did not demonstrate threshold improvement and in some animals exacerbated hearing loss and resulted in hair cell loss. We conclude that Sox10iCre ERT2 ;Gjb2 flox/flox mice are valuable for studying the biology of connexin 26 in the cochlea. In particular, these mice may be useful for evaluating gene therapy vectors and development of therapies for GJB2-related deafness.

Mitochondrial oxidative stress is involved in hair cell damage caused by noise-induced hearing loss (NIHL). Sirtuin-3 (SIRT3) plays an important role in hair cell survival by regulating mitochondrial function; however, the role of SIRT3 in NIHL is unknown. In this study, we used 3-TYP to inhibit SIRT3 and found that this inhibition aggravated oxidative damage in the hair cells of mice with NIHL. Moreover, 3-TYP reduced the enzymatic activity and deacetylation levels of superoxide dismutase 2 (SOD2). Subsequently, we administered adeno-associated virus-SIRT3 to the posterior semicircular canals and found that SIRT3 overexpression significantly attenuated hair cell injury and that this protective effect of SIRT3 could be blocked by 2-methoxyestradiol, a SOD2 inhibitor. These findings suggest that insufficient SIRT3/SOD2 signaling leads to mitochondrial oxidative damage resulting in hair cell injury in NIHL. Thus, ameliorating noise-induced mitochondrial redox imbalance by intervening in the SIRT3/SOD2 signaling pathway may be a new therapeutic target for hair cell injury.

Calcitonin-gene-related peptide (CGRP) is a lateral olivocochlear (LOC) efferent neurotransmitter. Depression of sound-driven auditory brainstem response amplitude in CGRP-null mice suggests the potential for endogenous CGRP release to upregulate spontaneous and/or sound-driven auditory nerve (AN) activity. We chronically infused CGRP into the guinea pig cochlea and evaluated changes in AN activity as well as outer hair cell (OHC) function. The amplitude of both round window noise (a measure of ensemble spontaneous activity) and the synchronous whole-nerve response to sound (compound action potential, CAP) were enhanced. Lack of change in both onset adaptation and steady state amplitude of sound-evoked distortion product otoacoustic emission (DPOAE) responses indicated CGRP had no effect on OHCs, suggesting the origin of the observed changes was neural. Combined with results from the CGRP-null mice, these results appear to confirm that endogenous CGRP enhances auditory nerve activity when released by the LOC neurons. However, infusion of the CGRP receptor antagonist CGRP (8–37) did not reliably influence spontaneous or sound-driven AN activity, or OHC function, results that contrast with the decreased ABR amplitude measured in CGRP-null mice.

Auditory brainstem response (ABR) serves as an objective indication of auditory perception at a given sound level and is nowadays widely used in hearing function assessment. Despite efforts for automation over decades, ABR threshold determination by machine algorithms remains unreliable and thereby one still relies on visual identification by trained personnel. Here, we described a procedure for automatic threshold determination that can be used in both animal and human ABR tests. The method terminates level averaging of ABR recordings upon detection of time-locked waveform through cross-correlation analysis. The threshold level was then indicated by a dramatic increase in the sweep numbers required to produce "qualified" level averaging. A good match was obtained between the algorithm outcome and the human readouts. Moreover, the method varies the level averaging based on the cross-correlation, thereby adapting to the signal-to-noise ratio of sweep recordings. These features empower a robust and fully automated ABR test.

Mitochondrial dynamics are essential for maintaining the physiological function of the mitochondrial network, and the disorder of mitochondrial dynamics leads to neurodegenerative diseases. However, how mitochondrial dynamics affects auditory function in the inner ear remains unclear. FAM73a and FAM73b are mitochondrial outer membrane proteins that mediate mitochondrial fusion. Here, we found that FAM73a or FAM73b deficiency resulted in elevated oxidative stress and apoptosis of hair cells. Additionally, mitochondrial fission also causes an increase expression of IL-12 in basilar membrane macrophages through accumulating IRF1. As a bridge between innate and adaptive immune responses, hyperproduction of IL-12 further promoted the polarization of Th1 and tissue damage. Our data highlighted an important role of mitochondrial dynamics in maintaining cochlear homeostasis and hair cell survival. Mitochondrial dynamics not only disturbed hair cell function, but also induced the disorder of immune responses.

Hearing is one of the most important senses needed for survival, and its loss is an independent risk factor for dementia. Hearing loss (HL) can lead to communication difficulties, social isolation, and cognitive dysfunction. The hippocampus is a critical brain region being greatly involved in the formation of learning and memory and is critical not only for declarative memory but also for social memory. However, until today, whether HL can affect learning and memory is poorly understood. This study aimed to identify the relationship between HL and hippocampal-associated cognitive function. Mice with complete auditory input elimination before the onset of hearing were used as the animal model. They were first examined via auditory brainstem response (ABR) to confirm hearing elimination, and behavior estimations were applied to detect social memory capacity. We found significant impairment of social memory in mice with HL compared with the controls ( p < 0.05); however, no significant differences were seen in the tests of novel object recognition, Morris water maze (MWM), and locomotion in the open field ( p > 0.05). Therefore, our study firstly demonstrates that hearing input is required for the formation of social memory, and hearing stimuli play an important role in the development of normal cognitive ability.

The application of ultrasound microbubbles (USMBs) enhances the permeability of the round window membrane (RWM) and improves drug delivery to the inner ear. In this study, we investigated the efficiency of USMB-aided delivery of chitosan-coated gold nanoparticles (CS-AuNPs) and the mechanism of USMB-mediated enhancement of RMW permeability. We exposed mouse inner ears to USMBs at an intensity of 2 W/cm 2 and then filled the tympanic bulla with CS-AuNPs or fluorescein isothiocyanate-decorated CS-AuNPs (FITC-CS-AuNPs). The membrane uptake of FITC-CS-AuNPs and their depth of permeation into the three-layer structure of the RWM, with or without prior USMB treatment, were visualized by z-stack confocal laser scanning microscopy. Ultrastructural changes in the RWM due to USMB-mediated cavitation appeared as sunburn-like peeling and various degrees of depression in the RWM surface, with pore-like openings forming in the outer epithelium. This disruption of the outer epithelium was paralleled by a transient reduction in tight junction (TJ)-associated protein levels in the RWM and an enhanced delivery of FITC-CS-AuNPs into the RWM. Without prior USMB exposure, the treatment with CS-AuNPs also caused a noticeable reduction in TJ proteins of the RWM. Our findings indicated that the combined treatment with USMBs and CS-AuNPs represents a promising and efficient drug and gene delivery vehicle for a trans -RWM approach for inner ear therapy. The outer epithelial layer of the RWM plays a decisive role in controlling the transmembrane transport of substances such as CS-AuNPs following the administration of USMBs. Most importantly, the enhanced permeation of AuNPs involved the transient disruption of the TJ-created paracellular barrier in the outer epithelium of the RWM.

Deafness is known to occur in more than 400 syndromes and accounts for almost 30% of hereditary hearing loss. The molecular mechanisms underlying such syndromic deafness remain unclear. Furthermore, deafness has been a common feature in patients with three main syndromes, the BÖrjeson-Forssman-Lehmann syndrome, Wildervanck syndrome, and Congenital Generalized Hirsutism, all of which are characterized by loss-of-function mutations in the Fgf13 gene. Whether the pathogenesis of deafness in these syndromes is associated with the Fgf13 mutation is not known. To elucidate its role in auditory function, we generated a mouse line with conditional knockout of the Fgf13 gene in the inner ear ( Fgf13 cKO). FGF13 is expressed predominantly in the organ of Corti, spiral ganglion neurons (SGNs), stria vascularis, and the supporting cells. Conditional knockout of the gene in the inner ear led to sensorineural deafness with low amplitude and increased latency of wave I in the auditory brainstem response test but had a normal distortion product otoacoustic emission threshold. Fgf13 deficiency resulted in decreased SGN density from the apical to the basal region without significant morphological changes and those in the number of hair cells. TUNEL and caspase-3 immunocytochemistry assays showed that apoptotic cell death mediated the loss of SGNs. Further detection of apoptotic factors through qRT-PCR suggested the activation of the mitochondrial apoptotic pathway in SGNs. Together, this study reveals a novel role for Fgf13 in auditory function, and indicates that the gene could be a potential candidate for understanding deafness. These findings may provide new perspectives on the molecular mechanisms and novel therapeutic targets for treatment deafness.

The application of insulin-like growth factor 1 (IGF-1) to the round window membrane (RWM) is an emerging treatment for inner ear diseases. RWM permeability is the key factor for efficient IGF-1 delivery. Ultrasound microbubbles (USMBs) can increase drug permeation through the RWM. In the present study, the enhancing effect of USMBs on the efficacy of IGF-1 application and the treatment effect of USMB-mediated IGF-1 delivery for noise-induced hearing loss (NIHL) were investigated. Forty-seven guinea pigs were assigned to three groups: the USM group, which received local application of recombinant human IGF-1 (rhIGF-1, 10 µg/µL) following application of USMBs to the RWM; the RWS group, which received IGF-1 application alone; and the saline-treated group. The perilymphatic concentration of rhIGF-1 in the USM group was 1.95- and 1.67- fold of that in the RWS group, 2 and 24 h after treatment, respectively. After 5 h of 118 dB SPL noise exposure, the USM group had the lowest threshold shift in auditory brainstem response, least loss of cochlear outer hair cells, and least reduction in the number of synaptic ribbons on postexposure day 28 among the three groups. The combination of USMB and IGF-1 led to a better therapeutic response to NIHL. Two hours after treatment, the USM group had significantly higher levels of Akt1 and Mapk3 gene expression than the other two groups. The most intense immunostaining for phosphor-AKT and phospho-ERK1/2 was detected in the cochlea in the USM group. These results suggested that USMB can be applied to enhance the efficacy of IGF-1 therapy in the treatment of inner ear diseases.

Aging-related cognitive decline is an emerging health crisis; however, no established unifying mechanism has been identified for the cognitive impairments seen in an aging population. A vascular hypothesis of cognitive decline has been proposed but is difficult to test given the contradictory radiologic needs of high-fidelity microvascular imaging resolution and a broad and deep brain imaging field of view. Super-resolution ultrasound localization microscopy (ULM) offers a potential solution by exploiting circulating microbubbles to achieve a vascular resolution approaching the capillary scale without sacrificing imaging depth. In this report, we apply ULM imaging to a mouse model of aging and quantify differences in cerebral vascularity, blood velocity, and vessel tortuosity across several brain regions. We found significant decreases in blood velocity, and significant increases in vascular tortuosity, across all brain regions in the aged cohort, and significant decreases in blood volume in the cortex. These data provide the first-ever measurements of subcortical microvascular dynamics in vivo and reveal that aging has a major impact on these measurements.

Collagens are major constituents of the extracellular matrix (ECM) that play an essential role in the structure of the inner ear and provide elasticity and rigidity when the signals of sound are received and transformed into electrical signals. LOXL3 is a member of the lysyl oxidase (LOX) family that are copper-dependent amine oxidases, generating covalent cross-links to stabilize polymeric elastin and collagen fibers in the ECM. Biallelic missense variant of LOXL3 was found in Stickler syndrome with mild conductive hearing loss. However, available information regarding the specific roles of LOXL3 in auditory function is limited. In this study, we showed that the Col2a1-Cre-mediated ablation of Loxl3 in the inner ear can cause progressive hearing loss, degeneration of hair cells and secondary degeneration of spiral ganglion neurons. The abnormal distribution of type II collagen in the spiral ligament and increased inflammatory responses were also found in Col2a1–Loxl3 –/– mice. Amino oxidase activity exerts an effect on collagen; thus, Loxl3 deficiency was expected to result in the instability of collagen in the spiral ligament and the basilar membrane, which may interfere with the mechanical properties of the organ of Corti and induce the inflammatory responses that are responsible for the hearing loss. Overall, our findings suggest that Loxl3 may play an essential role in maintaining hearing function.

Hearing loss is a debilitating disease that affects 10% of adults worldwide. Most sensorineural hearing loss is caused by the loss of mechanosensitive hair cells in the cochlea, often due to aging, noise, and ototoxic drugs. The identification of genes that can be targeted to slow aging and reduce the vulnerability of hair cells to insults is critical for the prevention of sensorineural hearing loss. Our previous cell-specific transcriptome analysis of adult cochlear hair cells and supporting cells showed that Clu, encoding a secreted chaperone that is involved in several basic biological events, such as cell death, tumor progression, and neurodegenerative disorders, is expressed in hair cells and supporting cells. We generated Clu-null mice (C57BL/6) to investigate its role in the organ of Corti, the sensory epithelium responsible for hearing in the mammalian cochlea. We showed that the deletion of Clu did not affect the development of hair cells and supporting cells; hair cells and supporting cells appeared normal at 1 month of age. Auditory function tests showed that Clu-null mice had hearing thresholds comparable to those of wild-type littermates before 3 months of age. Interestingly, Clu-null mice displayed less hair cell and hearing loss compared to their wildtype littermates after 3 months. Furthermore, the deletion of Clu is protected against aminoglycoside-induced hair cell loss in both in vivo and in vitro models. Our findings suggested that the inhibition of Clu expression could represent a potential therapeutic strategy for the alleviation of age-related and ototoxic drug-induced hearing loss.

Hair cells detect sound, head position or water movements when their mechanosensory hair bundle is deflected. Each hair bundle has an asymmetric architecture that restricts stimulus detection to a single axis. Coordinated hair cell orientations within sensory epithelia further tune stimulus detection at the organ level. Here, we identify GPR156, an orphan GPCR of unknown function, as a critical regulator of hair cell orientation. We demonstrate that the transcription factor EMX2 polarizes GPR156 distribution, enabling it to signal through Gαi and trigger a 180° reversal in hair cell orientation. GPR156-Gαi mediated reversal is essential to establish hair cells with mirror-image orientations in mouse otolith organs in the vestibular system and in zebrafish lateral line. Remarkably, GPR156-Gαi also instructs hair cell reversal in the auditory epithelium, despite a lack of mirror-image organization. Overall, our work demonstrates that conserved GPR156-Gαi signaling is integral to the framework that builds directional responses into mechanosensory epithelia.

Piccolo is a presynaptic protein with high conservation among different species, and the expression of Piccolo is extensive in vertebrates. Recently, a small fragment of Piccolo (Piccolino), arising due to the incomplete splicing of intron 5/6, was found to be present in the synapses of retinas and cochleae. However, the comprehensive function of Piccolo in the retina and cochlea remains unclear. In this study, we generated Piccolo knockout mice using CRISPR-Cas9 technology to explore the function of Piccolo. Unexpectedly, whereas no abnormalities were found in the cochlear hair cells of the mutant mice, significant differences were found in the retinas, in which two layers (the outer nuclear layer and the outer plexiform layer) were absent. Additionally, the amplitudes of electroretinograms were significantly reduced and pigmentation was observed in the fundoscopy of the mutant mouse retinas. The expression levels of Bassoon, a homolog of Piccolo, as well as synapse-associated proteins CtBP1, CtBP2, Kif3A, and Rim1 were down-regulated. The numbers of ribbon synapses in the retinas of the mutant mice were also reduced. Altogether, the phenotype of Piccolo-/- mice resembled the symptoms of retinitis pigmentosa (RP) in humans, suggesting Piccolo might be a candidate gene of RP and indicates Piccolo knockout mice are a good model for elucidating the molecular mechanisms of RP.

Purpose. Slc26a4-/- mice exhibit severer defects in the development of the cochlea and develop deafness, while the underlying mechanisms responsible for these effects remain unclear. Our study was to investigate the potential mechanism linking SLC26A4 deficiency to hearing loss. Materials and Methods. RNA sequencing was applied to analyze the differential gene expression of the stria vascularis (SV) from wildtype and Slc26a4-/- mice. GO and KEGG pathway analysis were performed. Quantitative RT-PCR was applied to validate the expression of candidate genes affected by Slc26a4. ELISA and immunofluorescence technique were used to detect the homocysteine (Hcy) level in serum, brain, and SV, respectively. Results. 183 upregulated genes and 63 downregulated genes were identified in the SV associated with Slc26a4 depletion. Transcriptomic profiling revealed that Slc26a4 deficiency significantly affected the expression of genes associated with cell adhesion, transmembrane transport, and the biogenesis of multicellular organisms. The SV from Slc26a4-/- mice exhibited a higher expression of Bhmt mRNAs, as well as altered homocysteine (Hcy) metabolism. Conclusions. The altered expression of Bhmt results in a dramatic change in multiple biochemical reactions and a disruption of nutrient homeostasis in the endolymph which may contribute to hearing loss of Slc26a4 knockout mouse.

Our previous study demonstrated rapamycin added to diet at 4 months of age had significantly less age-related outer hair cell loss in the basal half of the cochlea at 22 months of age compared to mice without rapamycin. The present study tested adding rapamycin to diet later in life, at 14 months of age, and added a longitudinal assessment of auditory brain stem response (ABR). The present study used UMHET4 mice, a 4 way cross in which all grandparental strains lack the Cdh23 753A allele that predisposes to early onset, progressive hearing loss. UMHET4 mice typically have normal hearing until 16–17 months, then exhibit threshold shifts at low frequencies/apical cochlea and later in more basal high frequency regions. ABR thresholds at 4, 12, 24, and 48 kHz were assessed at 12, 18, and 24 months of age and compared to baseline ABR thresholds acquired at 5 months of age to determine threshold shifts (TS). There was no TS at 12 months of age at any frequency tested. At 18 months of age mice with rapamycin added to diet at 14 months had a significantly lower mean TS at 4 and 12 kHz compared to mice on control diet with no significant difference at 24 and 48 kHz. At 24 months of age, the mean 4 kHz TS in rapamycin diet group was no longer significantly lower than the control diet group, while the 12 kHz mean remained significantly lower. Mean TS at 24 and 48 kHz in the rapamycin diet group became significantly lower than in the control diet group at 24 months. Hair cell counts at 24 months showed large loss in the apical half of most rapamycin and control diet mice cochleae with no significant difference between groups. There was only mild outer hair cell loss in the basal half of rapamycin and control diet mice cochleae with no significant difference between groups. The results show that a later life addition of rapamycin can decrease age-related hearing loss in the mouse model, however, it also suggests that this decrease is a delay/deceleration rather than a complete prevention.

Noise-induced hearing loss (NIHL) is a common inner ear disease but has complex pathological mechanisms, one of which is increased oxidative stress in the cochlea. The high-mobility group box 1 (HMGB1) protein acts as an inflammatory mediator and shows different activities with redox modifications linked to the generation of reactive oxygen species (ROS). We aimed to investigate whether manipulation of cochlear HMGB1 during noise exposure could prevent noise-induced oxidative stress and hearing loss. Sixty CBA/CaJ mice were divided into two groups. An intraperitoneal injection of anti-HMGB1 antibodies was administered to the experimental group; the control group was injected with saline. Thirty minutes later, all mice were subjected to white noise exposure. Subsequent cochlear damage, including auditory threshold shifts, hair cell loss, expression of cochlear HMGB1, and free radical activity, was then evaluated. The levels of HMGB1 and 4-hydroxynonenal (4-HNE), as respective markers of reactive nitrogen species (RNS) and ROS formation, showed slight increases on post-exposure day 1 and achieved their highest levels on post-exposure day 4. After noise exposure, the antibody-treated mice showed markedly less ROS formation and lower expression of NADPH oxidase 4 (NOX4), nitrotyrosine, inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) than the saline-treated control mice. A significant amelioration was also observed in the threshold shifts of the auditory brainstem response and the loss of outer hair cells in the antibody-treated versus the saline-treated mice. Our results suggest that inhibition of HMGB1 by neutralization with anti-HMGB1 antibodies prior to noise exposure effectively attenuated oxidative stress and subsequent inflammation. This procedure could therefore have potential as a therapy for NIHL.

Attenuation of noise-induced hair cell loss and noise-induced hearing loss (NIHL) by treatment with FK506 (tacrolimus), a calcineurin (CaN/PP2B) inhibitor used clinically as an immunosuppressant, has been previously reported, but the downstream mechanisms of FK506-attenuated NIHL remain unknown. Here we showed that CaN immunolabeling in outer hair cells (OHCs) and nuclear factor of activated T-cells isoform c4 (NFATc4/NFAT3) in OHC nuclei are significantly increased after moderate noise exposure in adult CBA/J mice. Consequently, treatment with FK506 significantly reduces moderate-noise-induced loss of OHCs and NIHL. Furthermore, induction of reactive oxygen species (ROS) by moderate noise was significantly diminished by treatment with FK506. In agreement with our previous finding that autophagy marker microtubule-associated protein light chain 3B (LC3B) does not change in OHCs under conditions of moderate-noise-induced permanent threshold shifts, treatment with FK506 increases LC3B immunolabeling in OHCs after exposure to moderate noise. Additionally, prevention of NIHL by treatment with FK506 was partially abolished by pretreatment with LC3B small interfering RNA. Taken together, these results indicate that attenuation of moderate-noise-induced OHC loss and hearing loss by FK506 treatment occurs not only via inhibition of CaN activity but also through inhibition of ROS and activation of autophagy.