Weiwei Guo

Correlation of Cochlear Blood Supply with Mitochondrial DNA Common Deletion in Presbyacusis

Objectives To study the relationships between cochlear hypoxia, mitochondrial (mt) DNA4977 deletion and metabolic features of mtDNA in presbyacusis. Material and Methods Sixty-seven temporal bones from a presbyacusis group, an age-matched control group and a young and middle-aged control group were involved in the experiment. Nested and tri-nested polymerase chain reactions (PCRs) were applied to test for the presence of the mtDNA4977 deletion. Computer imaging processing was used to measure blood vessel parameters in the internal acoustic meatus (IAM). Results The mtDNA4977 deletion was detected in 17/34 ears in the presbyacusis group, 4/19 ears in the age-matched control group and 0/14 ears in the young and middle-aged control group. In the presbyacusis group, the lumen of the vasa nervorum of the IAM showed a more severe narrowing in cases with than without the mtDNA4977 deletion. Conclusions The high incidence of the mtDNA4977 deletion in the temporal bones of presbyacusis patients suggests a correlation between the mtDNA4977 deletion and presbyacusis. Hypoxia of the cochlea may cause the mtDNA4977 deletion and other mtDNA mutants and furthermore may cause a reduction in mitochondrial oxidative phosphorylation and decreased function of the acoustic neural system. The symptoms of presbyacusis may occur when the function of the acoustic neural system is impaired as a result of abnormal mtDNA metabolism reaching a particular threshold.

Fate of Mammalian Cochlear Hair Cells and Stereocilia after Loss of the Stereocilia

Cochlear hair cells transduce mechanical stimuli into electrical activity. The site of hair cell transduction is the hair bundle, an array of stereocilia with different height arranged in a staircase. Tip links connect the apex of each stereocilium to the side of its taller neighbor. The hair bundle and tip links of hair cells are susceptible to acoustic trauma and ototoxic drugs. It has been shown that hair cells in lower vertebrates and in the mammalian vestibular system may survive bundle loss and undergo self-repair of the stereocilia. Our goals were to determine whether cochlear hair cells could survive the trauma and whether the tip link and/or the hair bundle could be regenerated. We simulated the acoustic trauma-induced tip link damage or stereociliary loss by disrupting tip links or ablating the hair bundles in the cultured organ of Corti from neonatal gerbils. Hair-cell fate and stereociliary morphology and function were examined using confocal and scanning electron microscopies and electrophysiology. Most bundleless hair cells survived and developed for ∼2 weeks. However, no spontaneous hair-bundle regeneration was observed. When tip links were ruptured, repair of tip links and restoration of mechanotransduction were observed in <24 h. Our study suggests that the dynamic nature of the hair cells transduction apparatus is retained despite the fact that regeneration of the hair bundle is lost in mammalian cochlear hair cells.

Molecular Epidemiology and Functional Assessment of Novel Allelic Variants of SLC26A4 in Non-Syndromic Hearing Loss Patients with Enlarged Vestibular Aqueduct in China

Background Mutations in SLC26A4, which encodes pendrin, are a common cause of deafness. SLC26A4 mutations are responsible for Pendred syndrome and non-syndromic enlarged vestibular aqueduct (EVA). The mutation spectrum of SLC26A4 varies widely among ethnic groups. To investigate the incidence of EVA in Chinese population and to provide appropriate genetic testing and counseling to patients with SLC26A4 variants, we conducted a large-scale molecular epidemiological survey of SLC26A4. Methods A total of 2352 unrelated non-syndromic hearing loss patients from 27 different regions of China were included. Hot spot regions of SLC26A4, exons 8, 10 and 19 were sequenced. For patients with one allelic variant in the hot spot regions, the other exons were sequenced one by one until two mutant alleles had been identified. Patients with SLC26A4 variants were then examined by temporal bone computed tomography scan for radiological diagnosis of EVA. Ten SLC26A4 variants were cloned for functional study. Confocal microscopy and radioisotope techniques were used to examine the membrane expression of pendrin and transporter function. Results Of the 86 types of variants found, 47 have never been reported. The ratio of EVA in the Chinese deaf population was at least 11%, and that in patients of Han ethnicity reached at least 13%. The mutational spectrum and mutation detection rate of SLC26A4 are distinct among both ethnicities and regions of Mainland China. Most of the variants caused retention of pendrin in the intracellular region. All the mutant pendrins showed significantly reduced transport capability. Conclusion An overall description of the molecular epidemiological findings of SLC26A4 in China is provided. The functional assessment procedure can be applied to identification of pathogenicity of variants. These findings are valuable for genetic diagnosis, genetic counseling, prenatal testing and pre-implantation diagnosis in EVA families.

The differentiation of mesenchymal stem cells into inner ear hair cell-like cells in vitro

Abstract Conclusion. Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into hair cells, and this method of culturing MSCs provides a useful tool for studies on mammalian cochlear hair cell regeneration. Objective: To investigate a method to induce bone marrow MSCs to differentiate into inner ear hair cells. Methods: Rat bone marrow MSCs were isolated from healthy rats and cultured in vitro. To make sure that the cultured cells were bone marrow MSCs, the expression of MSC markers such as SH2, CD31, CD34, and CD44 genes on the cultured cells was assessed by RT-PCR. Adipogenic cells and osteogenic cells were induced by the differentiation of the cultured cells, respectively, suggesting that the cultured cells have the characteristic of pluripotent differentiation. Then they were induced to differentiate into neural stem cells and hair cell progenitor cells. Immunohistochemistry experiments were carried out to detect the expression of molecular markers. Scanning electron microscope samples were prepared for observation of the morphology of the cells. Results: Rat bone marrow MSCs were successfully isolated, purified, cultured, and identified in vitro. They were also successfully induced to differentiate into neural progenitor cells and then hair cell-like cells that expressed myosin VIIa.

Expression and clinical significance of cathepsin B and stefin A in laryngeal cancer.

The aim of this study was to investigate the roles of the cathepsin B cysteine protease and its endogenous inhibitor stefin A in laryngeal cancer. Immunohistochemistry was employed to detect the expression of cathepsin B and stefin A in 84 patients with laryngeal cancer, respectively. The protein expression of stefin A was negatively associated with lymphatic metastasis, recurrence of laryngeal cancer and the survival rate, which was not observed with cathepsin B protein expression. Both down-regulation of cathepsin B and up-regulation of stefin A in vitro significantly inhibited the migration, invasion and proliferation of laryngeal cancer cells, respectively. Our results strongly suggest that stefin A may be a potential predictor of laryngeal cancer and may be used in the molecular diagnosis and gene therapy of laryngeal cancer. Cathepsin B may be used as a promising therapeutic target in the treatment of laryngeal cancer.

De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafness-onychodystrophy syndrome

De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafness-onychodystrophy syndrome

The Morphology and Electrophysiology of the Cochlea of the Miniature Pig

To report the cochlear morphology and electrophysiology of Chinese experimental miniature pigs. Twenty Chinese experimental miniature pigs were used in this study. Auditory brainstem responses (ABR), cochlear endolymphatic potentials (EP), and the potassium concentrations of cochlear endolymph were recorded. Hair cell morphology was examined using electron microscopy. The capsule of cochlea of the miniature pig has three and one‐half turns which contains a 39‐mm long membranous labyrinth. The organ of Corti in the labyrinth encompasses three rows of outer hair cells and one row of inner hair cells. The stereocilia of the hair cells in the apical turn of the cochlea were significantly longer than those in the basal turn. The vestibular apparatus consists of three semicircular canals and the otolith organs. The average threshold of the ABR was 35–45 dB SPL (n = 20) from 4 to 32 kHz. There was no significant difference in the threshold or latency of the ABR between 1‐day‐old and 30‐day‐old miniature pigs. The average EP value was 77.3 ± 14 mV (n = 9) and the average potassium concentration was 147.1 ± 13 mM (n = 5) recorded from the second turn of the cochlea. These studies on the cochlear morphology and electrophysiology of the miniature pigs help to establish the Chinese experimental miniature pig as an animal model for future studies in otology and audiology. Anat Rec, 298:494–500, 2015.

Migration and differentiation of mouse embryonic stem cells transplanted into mature cochlea of rats with aminoglycoside-induced hearing loss

Abstract Conclusion: Mouse embryonic stem cells (ESCs) transplanted into the scala tympani are able to migrate in the cochlea of rats deafened with aminoglycoside and partly restore the structure of sensory epithelia of the inner ear. Objectives: To explore the migration and differentiation of enhanced green fluorescence protein (EGFP)-expressing ESCs by transplanting them into the scala tympani of rats with amikacin sulfate-induced hearing loss. Methods: Adult Sprague-Dawley (SD) rats were deafened with amikacin sulfate. Mouse ESCs expressing EGFP (EGFP-ESCs) were transplanted into the scala tympani. The migration and differentiation were observed at different time points. Results: EGFP-ESCs transplanted into normal cochlea did not migrate, but those in the amikacin-damaged cochlea could survive and migrate into the scala media and the vestibular cisterna. For the first time, we observed that the EGFP-ESCs migrated into the scala media, took the place of the organ of Corti, and formed a structure just like the cochlear tunnel. Some grafted stem cells even expressed myosin VIIa, the molecular marker of hair cells. Some nerve fibers reached to the bottom of the hair cell-like cells. The ESCs migrated into the vestibule and restored the sensory epithelia of the ampullary crest. The number of the transplanted ESCs reduced over the 6 week period of the study.

Spontaneous and Partial Repair of Ribbon Synapse in Cochlear Inner Hair Cells After Ototoxic Withdrawal

Ototoxicity is one of the major causes of sensorineural deafness. However, it remains unclear whether sensorineural deafness is reversible after ototoxic withdrawal. Here, we report that the ribbon synapses between the inner hair cells (IHCs) and spiral ganglion nerve (SGN) fibers can be restored after ototoxic trauma. This corresponds with hearing restoration after ototoxic withdrawal. In this study, adult mice were injected daily with a low dose of gentamicin for 14 consecutive days. Immunostaining for RIBEYE/CtBP2 was used to estimate the number and size of synaptic ribbons in the cochlea. Hearing thresholds were assessed using auditory brainstem responses. Auditory temporal processing between IHCs and SGNs was evaluated by compound action potentials. We found automatic hearing restoration after ototoxicity withdrawal, which corresponded to the number and size recovery of synaptic ribbons, although both hearing and synaptic recovery were not complete. Thus, our study indicates that sensorineural deafness in mice can be reversible after ototoxic withdrawal due to an intrinsic repair of ribbon synapse in the cochlea.

Hydrogen-saturated saline protects intensive narrow band noise-induced hearing loss in guinea pigs through an antioxidant effect.

The purpose of the current study was to evaluate hydrogen-saturated saline protecting intensive narrow band noise-induced hearing loss. Guinea pigs were divided into three groups: hydrogen-saturated saline; normal saline; and control. For saline administration, the guinea pigs were given daily abdominal injections (1 ml/100 g) 3 days before and 1 h before narrow band noise exposure (2.5–3.5 kHz 130 dB SPL, 1 h). The guinea pigs in the control group received no treatment. The hearing function was assessed by the auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recording. The changes of free radicals in the cochlea before noise exposure, and immediately and 7 days after noise exposure were also examined. By Scanning electron microscopy and succinate dehydrogenase staining, we found that pre-treatment with hydrogen-saturated saline significantly reduced noise-induced hair cell damage and hearing loss. We also found that the malondialdehyde, lipid peroxidation, and hydroxyl levels were significantly lower in the hydrogen-saturated saline group after noise trauma, indicating that hydrogen-saturated saline can decrease the amount of harmful free radicals caused by noise trauma. Our findings suggest that hydrogen-saturated saline is effective in preventing intensive narrow band noise-induced hearing loss through the antioxidant effect.