Lidong Zhao

Variants in mitochondrial tRNAGlu, tRNAArg, and tRNAThr may influence the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in three Han Chinese families with hearing loss†

We report here on the clinical, genetic, and molecular characterization of three Han Chinese pedigrees with aminoglycoside‐induced and nonsyndromic hearing loss. Clinical evaluation revealed the variable phenotype of hearing loss including severity, age‐at‐onset, audiometric configuration in these subjects. Penetrances of hearing loss in BJ107, BJ108, and BJ109 pedigrees are 35%, 63%, and 67%, respectively. Mutational analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mitochondrial DNA (mtDNA) variants belonging to haplogroups N, F, and M, respectively. Of these variants, the A14693G mutation in the tRNAGlu, the T15908C mutation in the tRNAThr, and the T10454C mutation in the tRNAArg are of special interest as these mutations occur at positions which are highly evolutionarily conserved nucleotides of corresponding tRNAs. These homoplasmic mtDNA mutations were absent among 156 unrelated Chinese controls. The A14693G and T10454C mutations occur at the highly conserved bases of the TψC‐loop of tRNAGlu and tRNAArg, respectively. Furthermore, the T15908C mutation in the tRNAThr disrupts a highly conserved A‐U base‐pairing at the D‐stem of this tRNA. The alteration of structure of these tRNAs by these mtDNA mutations may lead to a failure in tRNA metabolism, thereby causing impairment of mitochondrial translation. Thus, mitochondrial dysfunctions, caused by the A1555G mutation, would be worsened by these mtDNA mutations. Therefore, these mtDNA mutations may have a potential modifier role in increasing the penetrance and expressivity of the deafness‐associated 12S rRNA A1555G mutation in those Chinese pedigrees.

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.

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.

Type I hair cell regeneration induced by Math1 gene transfer following neomycin ototoxicity in rat vestibular sensory epithelium

Abstract Conclusion: In the current study, hair cells of vestibular terminal organs in rats were completely eliminated with trans-scala vestibuli injection of neomycin, and then the Math1 gene was transferred. It was shown that type I vestibular hair cells were regenerated and synapses were formed. Objectives: The objective of this study was to identify the cell type of the regenerated vestibular hair cells and relative innervation and synaptic linkage after hair cells of vestibular terminal organs in rats were completely eliminated. Methods: Neomycin injection was used to eliminate all the vestibular terminal organs, and then the animals were treated with an injection of Ad-Math1-EGFP in the scala vestibuli of the cochlea. Results: Math1 gene transfer into the inner ear induced type I hair cell regeneration and synaptic formation. However, neither the number nor the appearance of the hair cells was normal.

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.

The diagnosis and surgical treatment of occult otogenic CSF leakage

Abstract Conclusions: The diagnosis of occult otogenic cerebrospinal fluid (CSF) leakage is challenging and it can easily be misdiagnosed. Some characteristics of clinical presentation can supply important clues and confirmed diagnosis should be obtained according to these clues and suitable imaging studies before meningitis develops. Different surgical techniques should be adopted to treat the CSF leakage according to different leakage etiologies, and good results can be obtained. Objective: The aim of the study was to evaluate the diagnosis and surgical treatment of occult otogenic CSF leakage, including the characteristics of clinical presentation, imaging studies, and operation methods in order to decrease the rate of misdiagnosis and obtain a good curative effect. Methods: We performed a retrospective review of 11 cases of CSF leakage that were all misdiagnosed and accompanied by meningitis, operated in our department from 2007 to 2012 after a mean follow-up of 3 years. In this context, the characteristics of clinical presentation, imaging studies, and management of CSF leakage were studied. Results: The CSF leakage had arisen traumatically (n = 9) or congenitally (n = 2). The medical history and special clinical presentation such as repeated otorrhea or rhinorrhea, fever, headache, and unilateral deafness can supply important diagnostic clues. Imaging studies including high-resolution noncontrast CT (HRCT), CT cisternography, and magnetic resonance imaging (MRI) are very important diagnostic methods. The surgical repairs were performed via a transmastoid approach (n = 8), packing the vestibule (n = 1) or a translabyrithine approach (n = 2). Recurrent leakage did not occur.

The morphological and functional development of the stria vascularis in miniature pigs

The purpose of this study was to examine the morphological and functional development of the lateral wall of the scala media of the cochlea in miniature pigs; light and transmission electron microscopy and electrophysiology were used for this purpose. We showed that the lateral wall of the scala media of the cochlea appears at embryonic Day 21 (E21) when the cochlear duct begins to form. From E28 to E49, the lateral wall can be distinguished according to its position along the cochlea. At E56, cells in the lateral wall begin to differentiate into three different types. At E70, three cell types, marginal, intermediate and basal, can be clearly distinguished. At E91, the stria vascularis is adult-like and the organ of Corti is also morphologically mature. The average endocochlear potential measured from the second turn of the cochlea (at E98, postnatal Day 1 (P1), P13 and P30) was 71.4 ± 2.5 (n = 7), 78.8 ± 1.5 (n = 10), 77.3 ± 2.3 (n = 10) and 78.0 ± 2.1 mV (n = 10), respectively. Our results suggest that in miniature pigs the stria vascularis develops during the embryonic period, concurrent with maturation of the organ of Corti. The magnitude of the endocochlear potential reached its mature level when the stria vascularis was morphologically adult-like at E98. These findings provide a morphological and functional basis for future animal studies using the miniature pig model concerning the pathogenesis of various inner-ear diseases.The purpose of this study was to examine the morphological and functional development of the lateral wall of the scala media of the cochlea in miniature pigs; light and transmission electron microscopy and electrophysiology were used for this purpose. We showed that the lateral wall of the scala media of the cochlea appears at embryonic Day 21 (E21) when the cochlear duct begins to form. From E28 to E49, the lateral wall can be distinguished according to its position along the cochlea. At E56, cells in the lateral wall begin to differentiate into three different types. At E70, three cell types, marginal, intermediate and basal, can be clearly distinguished. At E91, the stria vascularis is adult-like and the organ of Corti is also morphologically mature. The average endocochlear potential measured from the second turn of the cochlea (at E98, postnatal Day 1 (P1), P13 and P30) was 71.4±2.5 (n=7), 78.8±1.5 (n=10), 77.3±2.3 (n=10) and 78.0±2.1 mV (n=10), respectively. Our results suggest that in miniature pigs the stria vascularis develops during the embryonic period, concurrent with maturation of the organ of Corti. The magnitude of the endocochlear potential reached its mature level when the stria vascularis was morphologically adult-like at E98. These findings provide a morphological and functional basis for future animal studies using the miniature pig model concerning the pathogenesis of various inner-ear diseases.

The role of molecular margins as prognostic factors in laryngeal carcinoma in Chinese patients

Abstract Conclusion: Molecular margins were a more important prognostic factor in laryngeal carcinoma in Chinese patients than histopathological margins. eIF4E was the most sensitive molecular index of those that we tested for in these patients. Objectives: Safe surgical margins are closely related to prognosis. The aim of the present study was to investigate the role of molecular margins, not traditional histopathological margins, as prognostic factors in laryngeal carcinoma in Chinese patients. An additional aim of the study was to investigate the prognostic significance of tumor markers in the primary site of laryngeal carcinoma. Methods: From January 1992 to January 2000, the data for 321 Chinese patients with laryngeal carcinoma who were divided into a recurrent laryngeal carcinoma group and a non-recurrent group were analyzed. Tumor markers in surgical margins and primary site, such as cyclin D1, p53, and eIF4E, were detected in the two groups with immunohistochemical staining. Results: There was a significant difference in the expression of cyclin D1, p53, and eIF4E in surgical margins between the recurrent laryngeal carcinoma group and the non-recurrent group. The eIF4E-positive rate in surgical margins was higher than that for the other two factors. There was a significant difference in cyclin D1 and p53 in the primary site of laryngeal carcinoma and no significant difference in eIF4E in the two groups.