Daogong Zhang

Curcumin attenuates peroxynitrite-induced neurotoxicity in spiral ganglion neurons.

The present study was designed to investigate the effect of curcumin on peroxynitrite (ONOO(-))-induced damage in rat spiral ganglion neurons (SGNs). The primary cultured rat SGNs were exposed to ONOO(-) with or without curcumin pretreatment. Cell viability was measured by MTT assay. Apoptosis was determined by Ho.33342 and propidium iodide (PI) double staining and flow cytometry. The cellular glutathione (GSH) content, superoxide dismutase (SOD) activity and malonaldehyde (MDA) levels were evaluated by spectrophotometer. The mRNA expressions of Apaf-1, Caspase-9, Caspase-3, Bcl-2, and Bax were examined by RT-PCR, while, the protein expressions of mitochondrial and cytosolic cytochrome c, Caspase-9, Caspase-3, Bcl-2 and Bax proteins were determined by Western blot respectively. The cell viability was markedly reduced, while, the apoptotic rate increased significantly after exposure of ONOO(-) (100μM) to SGNs. The activity of SOD and level of GSH were notably reduced, whereas, the MDA level was significantly increased. Pretreatment with curcumin protected SGNs against ONOO(-)-induced cell damage, declined the apoptotic rate, and improved the levels of SOD and GSH, decreased the elevation of MDA. ONOO(-) induced cytochrome c release from the mitochondria of SGNs and subsequently activated Caspase-9, Caspase-3 and cell apoptosis. Meanwhile, pretreatment with curcumin abrogated cytochrome c release, blocked activation of Caspase-3, and altered the expression of Bcl-2 family triggered by ONOO(-). Our data indicate that curcumin can attenuate ONOO(-)-induced damage in SGNs by the anti-oxidative activity as well as protect mitochondria from oxidative stress.

Benign paroxysmal vertigo of childhood: Diagnostic value of vestibular test and high stimulus rate auditory brainstem response test

OBJECTIVE To investigate the diagnostic value of vestibular test and high stimulus rate auditory brainstem response (ABR) test and the possible mechanism responsible for benign paroxysmal vertigo of childhood (BPVC). METHODS Data of 56 patients with BPVC in vertigo clinic of our hospital from May 2007 to September 2008 were retrospectively analyzed in this study. Patients with BPVC were tested with pure tone audiometry, high stimulus rate auditory brainstem response test (ABR), transcranial Doppler sonography (TCD), bithermal caloric test, and VEMP. The results of the hearing and vestibular function test were compared and analyzed. RESULTS There were 56 patients with BPVC, including 32 men, 24 women, aged 3-12 years old, with an average of 6.5 years. Among 56 cases of BPVC patients, the results of pure tone audiometry were all normal. High stimulus rate ABR was abnormal in 66.1% (37/56) of cases. TCD showed 57.1% abnormality in 56 cases, including faster flow rate in 28 cases and slower flow rate in 4 cases. High stimulus rate ABR and TCD were both abnormal in 48.2% (27/56) of cases. Bithermal caloric test was abnormal in 14.3% (8/56) of cases. VEMP showed 32.1% abnormality, including amplitude abnormality in 16 cases and latency abnormality in 2 cases. The abnormal rate of VEMP was much higher than that of caloric test. CONCLUSION Vascular mechanisms might be involved in the pathogenesis of BPVC and there is strong evidence for close relationship between BPVC and migraine. High stimulus rate ABR is helpful in the diagnosis of BPVC. The inferior vestibular pathway is much more impaired than the superior vestibular pathway in BPVC.

Inferior vestibular neuritis: a novel subtype of vestibular neuritis.

OBJECTIVE To report eight cases of inferior vestibular neuritis, in order to raise awareness of this new subtype of vestibular neuritis. MATERIALS AND METHODS We retrospectively analysed 216 patients (104 males and 112 females; age range 10-64 years; mean age 38.4 years) with full clinical documentation who had attended our hospitals vertigo clinic between May 2007 and December 2008. All patients underwent systematic investigation, including hearing tests, radiology, caloric testing and vestibular evoked myogenic potential testing. RESULTS Of 216 patients with vestibular neuritis, eight cases were diagnosed as inferior vestibular neuritis, based on comprehensive analysis of test data. The clinical features of these eight patients were consistent with the characteristics of vestibular neuritis. The results of pure tone audiometry and caloric testing were normal, and the possibility of central lesions was excluded by cerebral computed tomography or magnetic resonance imaging on admission. Six cases had unilateral loss of vestibular evoked myogenic potentials, whereas two had a unilateral lower amplitude of vestibular evoked myogenic potentials. CONCLUSIONS Inferior vestibular neuritis is a novel subtype of vestibular neuritis, which involves the inferior vestibular nerve alone. Vestibular evoked myogenic potential testing is a useful aid to the diagnosis of inferior vestibular neuritis.

The Three-Dimensional Culture System with Matrigel and Neurotrophic Factors Preserves the Structure and Function of Spiral Ganglion Neuron In Vitro

Whole organ culture of the spiral ganglion region is a resourceful model system facilitating manipulation and analysis of live sprial ganglion neurons (SGNs). Three-dimensional (3D) cultures have been demonstrated to have many biomedical applications, but the effect of 3D culture in maintaining the SGNs structure and function in explant culture remains uninvestigated. In this study, we used the matrigel to encapsulate the spiral ganglion region isolated from neonatal mice. First, we optimized the matrigel concentration for the 3D culture system and found the 3D culture system protected the SGNs against apoptosis, preserved the structure of spiral ganglion region, and promoted the sprouting and outgrowth of SGNs neurites. Next, we found the 3D culture system promoted growth cone growth as evidenced by a higher average number and a longer average length of filopodia and a larger growth cone area. 3D culture system also significantly elevated the synapse density of SGNs. Last, we found that the 3D culture system combined with neurotrophic factors had accumulated effects in promoting the neurites outgrowth compared with 3D culture or NFs treatment only groups. Together, we conclude that the 3D culture system preserves the structure and function of SGN in explant culture.

c-Myb knockdown increases the neomycin-induced damage to hair-cell-like HEI-OC1 cells in vitro

c-Myb is a transcription factor that plays a key role in cell proliferation, differentiation, and apoptosis. It has been reported that c-Myb is expressed within the chicken otic placode, but whether c-Myb exists in the mammalian cochlea, and how it exerts its effects, has not been explored yet. Here, we investigated the expression of c-Myb in the postnatal mouse cochlea and HEI-OC1 cells and found that c-Myb was expressed in the hair cells (HCs) of mouse cochlea as well as in cultured HEI-OC1 cells. Next, we demonstrated that c-Myb expression was decreased in response to neomycin treatment in both cochlear HCs and HEI-OC1 cells, suggesting an otoprotective role for c-Myb. We then knocked down c-Myb expression with shRNA transfection in HEI-OC1 cells and found that c-Myb knockdown decreased cell viability, increased expression of pro-apoptotic factors, and enhanced cell apoptosis after neomycin insult. Mechanistic studies revealed that c-Myb knockdown increased cellular levels of reactive oxygen species and decreased Bcl-2 expression, both of which are likely to be responsible for the increased sensitivity of c-Myb knockdown cells to neomycin. This study provides evidence that c-Myb might serve as a new target for the prevention of aminoglycoside-induced HC loss.

Apoptosis-Inducing Factor Is Involved in Gentamicin-Induced Vestibular Hair Cell Death

Aim: Vestibular hair cell loss in response to different stimuli may be attributable to the occurrence of apoptosis, in which apoptosis-inducing factor (AIF) is an important regulator mediating apoptotic process independent of caspases. This study was designed to investigate the possible involvement of AIF in gentamicin (GM)-induced vestibular hair cell death. Methods: Vestibular organs from postnatal day 3 or 4 rats were maintained in tissue culture and were exposed to 2 mg/ml GM for up to 72 h. Vestibular hair cell viability was quantified by MTT assay. Apoptosis was determined by flow cytometry. AIF activation was examined by RT-PCR. The expressions of the mitochondrial protein and cytoplasm protein of AIF were detected by Western blot. Results: GM could significantly inhibit the cell viability of vestibular hair cells in a dose- and time-dependent manner. The number of apoptotic cells treated with GM was higher than that of cells not treated with GM. RT-PCR showed upregulation of AIF mRNA under GM. Western blot showed that AIF from mitochondria was decreased, whereas AIF from cytoplasm was increased after GM exposure. Conclusions: AIF participates in GM-induced apoptosis of vestibular hair cells.

GJB2, SLC26A4, and mitochondrial DNA12S rRNA hot-spots in 156 subjects with non-syndromic hearing loss in Tengzhou, China

Abstract Conclusion: In this cohort of 156 non-syndromic hearing-impaired subjects of Tengzhou area, the most common deafness-associated genes GJB2, SLC26A4 and mtDNA 12S rRNA were investigated by SNPscan efficiently. GJB2 c.235delC and SLC26A4 c.IVS7-2A > G were the most common mutation sites. Objectives: Until now, there is no systematic gentic analysis in patients with non-syndromic hearing loss for Tengzhou area, so we evaluated the molecular etiology to investigate the hot-sports. Methods: Peripheral blood samples were obtained from 156 patients with severe-to-profound non-syndromic deafness in Tengzhou. The SNP scan assay technique was performed for a rapid multiplex genetic screening to detect the 115 mutations of the most common three genes. All results were statistically analyzed with SPSS software. Results: Among the 156 analyzed patients, 60 patients were demonstrated with deafness genes, accounting for 38.46% (60/156), including GJB2 (22.44%, 35/156), SLC26A4 (13.66%, 22/156), and mtDNA 12S rRNA (2.56%, 4/156). In this study, we confirmed 23 deafness-causing mutations and 27 different allelic combinations including GJB2 (eight variants, 11 allelic combinations), SLC26A4 (13 variants, 16 allelic combinations) and mtDNA 12S rRNA (two variants). The occurrence rates of these deafness-causing mutations GJB2 c.235delC and SLC26A4 c.IVS7-2A > G were significantly higher than other mutation sites (p < 0.01).

Intranuclear localization of apoptosis-inducing factor and endonuclease G involves in peroxynitrite-induced apoptosis of spiral ganglion neurons

Abstract Objectives: The present study was designed to determine whether or not the caspase-independent apoptotic pathway participated in the cellular death of spiral ganglion neurons (SGNs) after exposure to peroxynitrite (ONOO−), with particular attention given to the intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in this process. Methods: The rat SGNs were isolated and primary cultured in vitro and were exposed to ONOO− with pre-treatment of pan-caspase inhibitor. Morphological changes of SGNs were observed by acridine orange cytochemistry staining, and apoptosis was examined by flow cytometry. The translocation of mitochondrial AIF and Endo G was detected by immunocytochemistry and Western blot. The protein expressions of Bcl-2 family in SGNs exposed to ONOO− were determined by Western blot. Results: Treatment of SGNs with ONOO− resulted in the occurrence of caspase-independent apoptosis as evidenced by acridine orange staining and flow cytometry analysis. The immunocytochemical analysis showed that AIF and Endo G labeling were marked in neuronal nuclei, while the Western blot demonstrated the intranuclear localization of AIF and Endo G in SGNs treated with ONOO−. Western blot analysis demonstrated that ONOO− increased the Bax expression while reducing Bcl-2 expression, which was not prevented by pre-treatment with caspase inhibitor. Conclusion: These data indicate that ONOO− can trigger caspase-independent apoptosis in SGNs associated with mitochondrial AIF and Endo G intranuclear localization.

PARP-1-modulated AIF translocation is involved in streptomycin-induced cochlear hair cell death

Abstract Conclusion SM-induced dose- and location-dependent cochlear hair cell death in vitro. AIF might be translocated from mitochondria to nucleus and cytoplasm within SM-treated hair cells. The translocation of AIF might be modulated by PARP-1. Objective Streptomycin (SM), one of the widely used aminoglycoside nowadays, is still causing significant permanent sensorineural hearing loss owing to sensory hair cell death. This study was designed to investigate the role of apoptosis-inducing factor (AIF), an important mitochondrial cell death regulator, in SM ototoxicity within neonatal rat cochleae and HEI-OC1 cells. Methods The viability of HEI-OC1 cells was quantified by MTT assay. AIF, PARP-1, and myosin VIIa distributions were achieved by immunofluorescence. mRNA and protein expression of AIF and PARP-1 were examined by q-PCR and Western-blot. Results The hair cell loss was concomitant with the SM concentration variation, and aggravated from apical to basal turn. AIF was detected in nuclear region and AIF mRNA was up-regulated after SM incubation. Besides, AIF protein expression in mitochondria was decreased, whereas in cytosol it was increased. PARP-1 mRNA and protein were also up-regulated. 3-AB could attenuate the cell death and reverse the changes of AIF distribution by blocking PARP-1.

Allicin protects auditory hair cells and spiral ganglion neurons from cisplatin - Induced apoptosis

&NA; Cisplatin is a broad‐spectrum anticancer drug that is commonly used in the clinic. Ototoxicity is one of the major side effects of this drug, which caused irreversible sensorineural hearing loss. Allicin, the main biologically active compound derived from garlic, has been shown to exert various anti‐apoptotic and anti‐oxidative activities in vitro and in vivo studies. We took advantage of C57 mice intraperitoneally injected with cisplatin alone or with cisplatin and allicin combined, to investigate whether allicin plays a protective role in vivo against cisplatin ototoxicity. The result showed that C57 mice in cisplatin group exhibited increased shift in auditory brainstem response, whereas the auditory fuction of mice in allicin + cisplatin group was protected in most frequencies, which was accordance with observed damages of outer hair cells (OHCs) and spiral ganglion neurons (SGNs) in the cochlea. Allicin markedly protected SGN mitochondria from damage and releasing cytochrome c, and significantly reduced pro‐apoptosis factor expressions activated by cisplatin, including Bax, cleaved‐caspase‐9, cleaved‐caspase‐3and p53. Furthermore, allicin reduced the level of Malondialdehyde (MDA), but increased the level of superoxide dismutase (SOD). All data suggested that allicin could prevent hearing loss induced by cisplatin effectively, of which allicin protected SGNs from apoptosis via mitochondrial pathway while protected OHCs and supporting cells (SCs) from apoptosis through p53 pathway. HighlightsAllicin prevents hearing loss induced by cisplatin in C57 mice.Allicin increases survival numbers of outer hair cells and spiral ganglion neurons.Allicin prevents spiral ganglion neurons from apoptosis via mitochondrial pathway.Allicin prevents OHCs and supporting cells from apoptosis through p53 pathway.Allicin reduce the oxidative stress response caused by cisplatin in the cochlea.