Hanqi Chu

Cochlear NMDA Receptors as a Therapeutic Target of Noise-Induced Tinnitus

Background: Accumulating evidence suggests that tinnitus may occur despite normal auditory sensitivity, probably linked to partial degeneration of the cochlear nerve and damage of the inner hair cell (IHC) synapse. Damage to the IHC synapses and deafferentation may occur even after moderate noise exposure. For both salicylate- and noise-induced tinnitus, aberrant N-methyl-d-aspartate (NMDA) receptor activation and related auditory nerve excitation have been suggested as origin of cochlear tinnitus. Accordingly, NMDA receptor inhibition has been proposed as a pharmacologic approach for treatment of synaptopathic tinnitus. Methods: Round-window application of the NMDA receptor antagonist AM-101 (Esketamine hydrochloride gel; Auris Medical AG, Basel, Switzerland) was tested in an animal model of tinnitus induced by acute traumatic noise. The study included the quantification of IHC ribbon synapses as a correlate for deafferentation as well as the measurement of the auditory brainstem response (ABR) to close-threshold sensation level stimuli as an indication of sound-induced auditory nerve activity. Results: We have shown that AM-101 reduced the trauma-induced loss of IHC ribbons and counteracted the decline of ABR wave I amplitude generated in the cochlea/auditory nerve. Conclusion: Local round-window application of AM-101 may be a promising therapeutic intervention for the treatment of synaptopathic tinnitus.

Simultaneously reduced NKCC1 and Na,K-ATPase expression in murine cochlear lateral wall contribute to conservation of endocochlear potential following a sensorineural hearing loss

The mechanisms of the response in the murine cochlear lateral wall following sensorineural hearing loss (SNHL) are poorly understood. We focused on comparing the endocochlear potential (EP) with morphological changes in the lateral wall and expression of four important potassium (K(+)) transporters in a mouse model of SNHL induced by co-administration of aminoglycoside and loop diuretic. The expression of the α1 and α2 isoforms of Na,K-ATPase, Na-K-2Cl-Cotransporter-1 (NKCC1) and potassium channel KCNQ1 was assessed. The EP showed a significant decline at 12h post-treatment followed by complete recovery by 2 days post-treatment. The EP was maintained at near normal levels in animals deafened for periods up to 112 days. Despite this recovery, there was a significant and progressive decrease in the thickness of the stria vascularis, which was predominantly due to atrophy of marginal cells. Both protein and mRNA expression of α1 and α2 isoforms of Na,K-ATPase and NKCC1 in the lateral wall were dramatically reduced following a long-term deafening. KCNQ1 expression remained unchanged. These observations provide insight into the detailed mechanisms of EP modulation following SNHL and may have crucial implications in the future treatment of aminoglycoside-induced hearing loss.

Downregulation of Cav1.3 calcium channel expression in the cochlea is associated with age-related hearing loss in C57BL/6J mice.

Age-related hearing loss (ARHL) is the most common human morbidity. However, the molecular mechanisms underlying ARHL are little known. In the present study, the expression of Cav1.3 calcium channels in the C57BL/6J ARHL mouse cochlea was investigated. The hearing threshold was assessed by auditory brainstem response and the expressions of Cav1.3 calcium channels at the protein and mRNA levels were detected by immunohistochemistry, western blot, and real-time RT-PCR. Associated with the auditory brainstem response threshold increased with age, the Cav1.3 expression was gradually decreased. In comparison with 4-week-old mice, Cav1.3 expressions in the cochlea at 14, 24, and 48 weeks of age were significantly and gradually decreased at both the protein and the mRNA levels. Immunohistochemistry showed that the expression of Cav1.3 was apparently reduced at the inner hair cells, outer hair cells, and stria vascularis in the cochlear lateral wall in the aged mice. Our findings indicate that Cav1.3 calcium channel expression in the cochlea is reduced in the ARHL mice and is associated with ARHL. The data also support a view that Cav1.3 calcium channel is a good target for prevention and therapy of ARHL.

Correlation of PDCD5 and apoptosis in hair cells and spiral ganglion neurons of different age of C57BL/6J mice

This study examined the expression pattern of programmed cell death 5 (PDCD5) in cochlear hair cells and spiral ganglion neurons (SGNs) and its association with age-related hearing loss in mice. Sixty C57BL/6J (C57) mice at different ages were divided into four groups (3, 6, 9 or 12 months). PDCD5 expression was detected by using immunohistochemistry, real-time PCR and Western blot. Morphological change of the cochleae was also evaluated by using immunoassay. The results showed that the expression of PDCD5 had a gradual increase with ageing in both protein and RNA levels in C57 mice, as well as gradually increased apoptosis of cochlear hair cells and SGNs. In addition, we also found that caspase-3 activity was enhanced and its expression was enhanced with ageing. It is implied that overexpression of PDCD5 causes the increase in caspase-3 activity and the subsequent increase of apoptosis in cochlear hair cells and SGNs, and thereby plays a role in the pathogenesis of presbycusis. Thus, PDCD5 may be a new target site for the treatment and prevention of age-related hearing loss.SummaryThis study examined the expression pattern of programmed cell death 5 (PDCD5) in cochlear hair cells and spiral ganglion neurons (SGNs) and its association with age-related hearing loss in mice. Sixty C57BL/6J (C57) mice at different ages were divided into four groups (3, 6, 9 or 12 months). PDCD5 expression was detected by using immunohistochemistry, real-time PCR and Western blot. Morphological change of the cochleae was also evaluated by using immunoassay. The results showed that the expression of PDCD5 had a gradual increase with ageing in both protein and RNA levels in C57 mice, as well as gradually increased apoptosis of cochlear hair cells and SGNs. In addition, we also found that caspase-3 activity was enhanced and its expression was enhanced with ageing. It is implied that overexpression of PDCD5 causes the increase in caspase-3 activity and the subsequent increase of apoptosis in cochlear hair cells and SGNs, and thereby plays a role in the pathogenesis of presbycusis. Thus, PDCD5 may be a new target site for the treatment and prevention of age-related hearing loss.

Age-related change in the expression of NKCC1 in the cochlear lateral wall of C57BL/6J mice

Abstract Conclusions: Na+-K+-2Cl– co-transporter isoform 1 (NKCC1) mRNA and protein decrease with increasing age in the cochlear lateral wall of C57BL/6J (C57) mice. The down-regulation of NKCC1 may influence the K+ transport efficiency and the homeostasis of ion transport cells, and cause the irreversible damage of cochlear cells in old C57 mice. Our results indicate that NKCC1 may play an important role in the pathogenesis of age-related hearing loss (AHL). Objectives: The aim of the present study was to investigate the relationship between the functional expression of NKCC1 transporter and the etiology of AHL. Methods: C57 mice were used and randomly divided into four groups according to age (4 weeks, 14 weeks, 26 weeks, and 52 weeks). Immunofluorescence technique, quantitative real-time PCR, and western blot were applied to detect the expression of NKCC1 in the cochlear lateral wall of C57 mice at the various ages. Results: In all four groups, the expression of NKCC1 was observed in the stria vascularis and type II fibrocytes of the spiral ligament. Also, the expression of NKCC1 appeared to decrease with age at both the transcriptional level and the protein level.

Conservation of endocochlear potential in mice with profound hearing loss induced by co-administration of kanamycin and furosemide

Research in mammalian hair cell regeneration is hampered by a lack of in vivo model of adult mouse inner ear injury. In the present study we investigated the effects of a combination of a single dose of aminoglycoside followed by a loop diuretic in adult mice. The auditory brainstem response threshold shift, extent and defining characteristics of the cochlear lesion were assessed and verified at different time points post-treatment. Our data indicated that this drug combination caused the rapid and extensive death of outer hair cells (OHCs). OHC death presented throughout the cochlea that commenced in the basal turn by 24 h and progressed apically. In contrast, inner hair cell (IHC) loss was delayed and mild. Terminal deoxynucleotidyl transferase dUTP nick end labelling-positive nuclei demonstrated that the majority of OHCs died via an apoptotic pathway. Auditory threshold shifts of up to 90 dB SPL indicated a profound hearing loss. In addition, the endocochlear potential (EP) in the drug-treated animals displayed a significant decline at 12 h post-treatment followed by recovery by 48 h post-treatment. Despite this recovery, there was a significant and progressive decrease in strial vascularis thickness, which was predominantly due to atrophy of marginal cells. The present study reproduced an adult mouse model of aminoglycoside-induced hearing loss. The mechanism underlying the recovered EP in the model with extensive hair cell death is discussed.

The expression of plasma membrane Ca2+-ATPase isoform 2 and its splice variants at sites A and C in the neonatal rat cochlea

OBJECTIVE To study the expression of plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) and its alternative splicing at sites A (the first intracellular loop) and C (the C-terminal region) in the neonatal rat cochlea. METHODS The cochleae from rats postnatal day 3 to postnatal day 4 (P3-P4) were dissected, fixed, embedded, and sectioned. Meanwhile, the cochlear coils from neonatal rats were isolated and fixed. Using immunofluorescence staining, the expression of PMCA2 was respectively examined in the cochlear sections and cochlear coils. In addition, the total RNAs of basilar membrane (BM, including the organ of corti, the same below), spiral ganglion (SG), spiral ligament (SL, including SV, the same below), and the whole cochlea from neonatal rats were respectively extracted and reverse transcribed to cDNAs, then subjected to primers flanking site A or C in the PMCA2 gene using reverse transcription polymerase chain reaction (RT-PCR). Western blot was also applied to detect the expression of PMCA2 isoforms in the cochlear tissues. RESULTS We found that PMCA2 is strongly expressed in outer hair cell (OHC) bundles, SG, and stria vascularis (SV), weakly expressed in Reissners membrane (RM), and occasionally expressed in inner hair cell (IHC) bundles. Moreover, w/a is the major splice form of PMCA2 present in hair cell bundles, z/b and z/c are the major splice forms of PMCA2 present in SG, and w/a and w/c are the major splice forms of PMCA2 present in SV. In the whole cochlea, variants w, y, and z were detected at site A, and variants a, b, and c were detected at site C. Using Western blot, variant a or b was also detectable in the same cochlear tissues mentioned above. CONCLUSIONS PMCA2 and its splice variants at sites A and C are differentially expressed in cochlear tissues of neonatal rat.

Expression patterns of CaV1.3 channels in the rat cochlea

Although Ca(V)1.3 channels are known to be essential for neuronal excitation and signal transduction in the auditory system, their expression patterns in the cochlea are still not fully understood, particularly in the regions where non-sensory cells are located. We performed immunohistochemistry, western blotting and reverse transcription-polymerase chain reaction (RT-PCR) to identify the expression and distribution of Ca(V)1.3 channels in the rat cochlea. Immunohistochemistry revealed that Ca(V)1.3 channels were localized in the outer hair cells (OHCs), inner hair cells (IHCs), limbus laminae spiralis, spiral ganglion cell, spiral ligament (SL), and stria vascularis (STV). The results of RT-PCR and western blotting demonstrated Ca(V)1.3 channels had a tissue-specific expression pattern. Ca(V)1.3 mRNA and protein were intensively expressed in the basilar membrane and spiral ganglion while moderate level of Ca(V)1.3 channels was observed in SL and STV. Our study preliminarily revealed the expression patterns of Ca(V)1.3 channels in the rat cochlea, providing a theoretical basis for further research on the role of Ca(V)1.3 channels in the periphery auditory system.

Lessons Learned From 35 Cases of Laryngeal Foreign Bodies Undergoing Misdiagnosis in Pediatric Population

Objectives: To present 35 cases of laryngeal foreign bodies (FBs) in pediatric population undergoing misdiagnosis so as to draw on our lessons to improve early diagnosis. Methods: A retrospective analysis over 15 years was conducted of 35 cases of laryngeal FBs undergoing misdiagnosis in children. Meanwhile, a control group, including 42 cases of laryngeal FBs without misdiagnosis in children, was set. These patients’ clinical data were collected and analyzed to identify the risk factors for misdiagnosis. Results: The results of chi-square test and univariate analysis both showed a significant difference in time elapsed between discomforts and admission, witnessed foreign body (FB) aspiration history, biphasic stridor, aphonia, roentgenologic findings, and type and size of FBs between the misdiagnosed group and control group. Multivariate analysis further identified delayed doctor visits, unwitnessed FB aspiration history, nonspecific symptoms, and negative roentgenologic manifestations as independent risk factors for misdiagnosis. Conclusions: Diagnosis of laryngeal FBs, especially small, thin, and radiolucent FBs, remains a challenge. We emphasized the importance of timely doctor visits, careful clinical history inquisition, and prompt performance of radiographic or endoscopic examinations for diagnosis.

Downregulation of inwardly rectifying potassium channel 5.1 expression in C57BL/6J cochlear lateral wall

SummaryAge-related hearing loss (AHL) is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1 (Kir5.1) plays a vital role in regulating cochlear K+ circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The mRNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions (RT-PCR) and Western blotting respectively. Kir5.1 was detected in the type II and IV fibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 mRNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.Age-related hearing loss (AHL) is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1 (Kir5.1) plays a vital role in regulating cochlear K+ circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The mRNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions (RT-PCR) and Western blotting respectively. Kir5.1 was detected in the type II and IV fibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 mRNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.