Zhaomin Fan

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.

Identification of a novel missense mutation in the WFS1 gene as a cause of autosomal dominant nonsyndromic sensorineural hearing loss in all‐frequencies

Hearing loss is the most common sensory disorder affecting 278 million people in the world, and more than 60% of hearing loss patients can be attributed to genetic causes. Although many loci have been linked to hereditary hearing impairment, most of the causative genes have not been identified as yet. The goal of this study was to investigate the cause of dominantly inherited sensorineural all‐frequency hearing loss in a six‐generation Chinese family. We performed exome sequencing to screen responsible candidate genes in three family members with all‐frequency hearing loss and one member with normal hearing. Sanger sequencing was employed to examine the variant mutations in the members of this family and 200 healthy persons. PCR‐RFLP was performed to further confirm the nucleotide mutation. A novel missense mutation c.2389G > A (GAC → AAC) in WFS1 gene was identified, which was co‐segregated with the hearing loss phenotype. No mutation was found in 200 controls and the family members with normal hearing in this site. The present study identifies, for the first time, a novel mutation in WFS1 gene that causes non‐syndromic hearing loss in all, rather than in low or high, frequencies.

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.

Expression of Surfactant Protein-A during LPS-Induced Otitis Media with Effusion in Mice.

Objective The objective of this study was to investigate the expression and role of surfactant protein (SP) in the middle ear throughout lipopolysaccharide (LPS)-induced otitis media with effusion (OME). Study Design Randomized case-controlled animal model. Setting Shandong University, Jinan, China. Subjects and Methods SP expression was monitored using reverse transcription polymerase chain reaction (PCR) in normal mice (n = 5). Two groups, control phosphate-buffered saline–injected mice (n = 5) and LPS-injected mice (n = 5), were euthanized 5 days following injection. RNA was extracted for reverse transcription PCR and real-time PCR, and temporal bone samples were used for hematoxylin and eosin staining. LPS was injected into mice, and 5 mice per test were euthanized at 0, 12, 24, 48, 72, and 96 hours following injection. For mRNA expression quantification, reverse transcription PCR and real-time PCR were performed, and proinflammatory cytokine levels were measured by enzyme-linked immunosorbent assay. Results SP-A and SP-D expression was detected in normal murine Eustachian tubes. SP-A expression was up-regulated after LPS-induced OME (P = .01). At various time points after LPS injection, concentrations of proinflammatory cytokines (tumor necrosis factor–α [TNF-α], interleukin (IL)–1β, and IL-6) in the middle ear increased significantly (P < .05) and correlated with changes in SP-A expression. Conclusion SP-A and SP-D exist in the murine middle ear. The expression of SP-A and TNF-α, IL-1β, and IL-6 was up-regulated in the middle ear of the LPS-induced OME animal model.

Complications in retrosigmoid cranial nerve surgery

Conclusions: Although microvascular decompression (MVD), facial nerve splitting (FNS) and neurectomy procedures were safe treatments for hemifacial spasm (HFS), trigeminal neuralgia (TN) and glossopharyngeal neuralgia (GPN) in retrosigmoid cranial nerve surgery, fatal and severe complications may occur. It is essential to pay great attention to the entire procedure to avoid these complications. Objective: To report the complications of cranial nerve surgery via the retrosigmoid approach. Patients and methods: We reviewed 516 cases of cranial nerve surgery via the retrosigmoid approach for HFS, TN and GPN. There were 208 cases of HFS, of which 117 cases underwent FNS alone and 91 cases underwent combined MVD and FNS. There were 273 cases of TN treated by MVD and selective neurectomy. There were 35 cases of GPN treated by neurectomy. Results: Of the cases with complications, two (0.4%) died. Hearing impairment ranging from mild to severe occurred in 31 (6.0%) patients; 4 of these (0.8%) presented total hearing loss. Postoperative cerebrospinal fluid leakage occurred in 29 (5.6%) cases.

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.