Inner hair cell ribbon synapse plasticity might be molecular basis of temporary hearing threshold shifts in mice.

Recent studies have reported that noise exposure at relatively low intensities can cause temporary threshold shifts (TTS) in hearing. However, the mechanism underlying the TTS is still on debate. Here, we report that an acoustic stimulation (100 dB SPL, white noise) induced TTS in mice, with the maximal ABR threshold elevations seen on the 4(th) day after noise exposure. On the other hand, there were no significant morphological changes in the cochlea. Further, there were paralleled changes of pre-synaptic ribbons in both the number and postsynaptic density (PSDs) during this noise exposure. The numbers of presynaptic ribbon, postsynaptic density (PSDs), and colocalized puncta correlated with the shifts of ABR thresholds. Moreover, a complete recovery of ABR thresholds and synaptic puncta was seen on the 14(th) day after the noise stimulations. Thus, our study may indicate that noise exposure can cause a decline in cochlear ribbon synapses and result in consequent hearing loss. The reduction of synaptic puncta appears reversible and may contribute to hearing restoration in mice after noise exposure.

Effect evaluation of repeated debridement after endoscopic sinus surgery.

OBJECTIVE: To investigate the effect of early phase debridement by the different intervention frequencies on postoperative symptoms recovery and turnover of mucosa after functional endoscopic sinus surgery (FESS). METHODS: 67 patients undergone FESS were divided into intervention group and control group. Intranasal corticosteroids, macrolides antibiotics and postoperative saline douching were used in both groups. Debridement was performed on the 1(st), 4(th), 8(th) postoperative week on patients of invention group, while once per week on patients of control group. The primary outcome measure was visual analogue scale (VAS) and Lund-Kennedy Endoscopic Score (LKES) Results: On the 4(th) week, the control group presented more release on nasal block, the VAS of the two groups is 3.45 ± 1.16 and 4.83 ± 1.47 in the control group and intervention group respectively which was significantly different. The LKES on crust decreased more in the control group (1.12 ± 0.64 in the control group and 1.90 ± 0.47 in the intervention group). However, the control group complained more sever facial pain and uncomfortable; the VAS of two groups is 5.92 ± 0.91 and 2.74 ± 1.41 respectively. On the 8(th) week, there were no significant difference between the two groups on all domains of VAS and LKES except lower scar was shown in the control group. CONCLUSIONS: Benefit of frequent debridement during the early postoperative was not in positive correlation with patients recovering from ESS. Excessive debridement may induce more surgical trauma and cause more facial pain to patients. Therefore, in terms of subjective recovery and health care costs, appropriate extending postoperative management time and decreasing intervention frequencies will not affect the therapeutic effect of endoscopic surgery for chronic sinusitis.

[Culture, identification and label of embryonic rat neural stem cells].

OBJECTIVE: To explore the methods of culture, identification and label of embryonic rat neural stem cells. METHOD: The cells isolated from fetal rat hippocampus were identified with nestin immunocytochemical fluorescent staining. The cellular multiplication was observed by immunocytochemical fluorescence co-label after accession of BrDU. The neural stem cells (NSCs) were marked with fluorescent dye, bisbenzimide (Hoechest33342) and induced to differentiate. The differentiated cells were detected with Neuron Specific Enolase (NSE) and Glial Fibrillary Acidic Protein (GFAP) immunocytochemical fluorescent staining respectively. RESULT: Nest-like clusters of neural stem cells were obtained in suspension and the cells could be differentiated into neurons and astrocytes which maintaining the main characteristics of NSCs after 8 passages of culture. The label efficiency of cells with Hoechest33342 was 97% and no attenuation of fluorescent brightness was observed after 8 passages of culture. The cellular fluorescence was observed in the NSCs and the differentiated cells. CONCLUSION: The cells from embryonic rat hippocampus possessed the abilities of division, multiplication and self-renew, which were believed to be the main characteristics of NSCs of the central nervous system. The cells could be efficiently labeled with fluorescent dye and could be used as donor cells in experimental research on NSCs transplantation.

[Neurofibromatosis type 2].

OBJECTIVE: To recognise the predisposing factors, clinical manifestations, diagnosis and treatment of neurofibromatosis type 2 (NF2). METHOD: The clinical data of one NF2 case was reported and the literatures were also reviewed. RESULT: The patient was diagnosed at a much later stage than onset. Progressive hearing loss and tinnitus were the initial symptoms. MRI scan indicated space-occupying lesions in the bilateral cerebellopontine angles, bilateral cavernous sinuses and bilateral cervical parts of the patient. The patient was diagnosed as NF2 according to the National Institutes of Health (NIH) criteria, and received operation on the left acoustic tumor. The tumor was proved to be schwannomas by pathological test. The hearing loss and the facial nerve paralysis (House-Brackmann II) had appeared after operation. CONCLUSION: NF2 is an autosomal dominant, highly penetrant disease which is characterized by bilateral vestibular schwannomas. Early diagnosis and management for tumor is very important for survival and hearing preservation. The "golden standard" in terms of diagnostic precision is the magnetic resonance imaging (MRI) scan with gadolinium enhancement.

[Excitotoxic effects of glutamate on the in vitro spiral ganglion cells].

OBJECTIVE: To explore the excitotoxic effects of glutamate on the in vitro primarily cultured spiral ganglion cell (SGC) of rat. METHOD: Spiral ganglion cells (SGCs) were cultured in vitro for 4 days, and exposed to 1 mmol/L glutamate for 24 hours. Damaged cells double-labeling with Hoechest33258 and PI were observed by fluorescence microscope. Ffluo-3 and CLSM for measurement of intracellular calcium levels were also utilized. RESULT: Most cells were damaged and the intracellular calcium increased and after exposure to 1 mmol/L glutamate (P < 0.05). CONCLUSION: Glutamate excitotoxicity was associated with free intracellular calcium ion concentration elevation in SGC.

[Application of rat tail collagen in the primary culture of rats marginal cells of stria vascularis].

OBJECTIVE: To study the feasibility of application of rat-tail collagen in the primary culture of rats marginal cells of stria vascularis. METHOD: The effect of self-made rat-tail collagen on the primary culture of rats marginal cells of stria vascularis was observed and estimated. RESULT: When cochlear stria vascularis fragment was isolated and cultured for 24 h, a few culture cells appeared around the fragments. About 48 to approximately 72 h later, clusters of culture cells could be seen,these cells showed the cobblestone shape under the microscope. The immunohistochemistry and the transmission electron microscopy showed the epithelial origination of these cells. CONCLUSION: It is feasible to the primarily culture the rats marginal cells of stria vascularis with self-made rat-tail collagen.

[Experimental study of primary culture methods for spiral ganglion cells of newborn rats].

OBJECTIVE: To optimize culture condition of spiral ganglion cells (SGCs) in vitro and to obtain highly purified SGCs. METHOD: The spiral ganglions from newborn rats were digested in 0.25% trypsin and 0.001% DNase. The SGCs suspension was plated at a density of 10(9) cells/L. The cells were kept in serum free medium (DMEM/F12+B27 supplement). And 5 micromol/L cytosine arabinoside (Ara-C) was added to the medium on the second day and maintained for 48 hours. Serum medium with or without Ara-C was set as a control. The morphology and the purity of SGCs were observed under microscope. RESULT: Highly purified SGCs can be harvested in serum free medium (DMEM/F12+B27 supplement) with Ara-C. The SGCs were identified with mouse anti neurofilament protein antibody by immunohistochemistry methods. CONCLUSION: The method used in this study is an optimal means to culture and purify SGCs that can meet the needs of further study.

[Expression of perlecan in laryngeal carcinoma cell and its significance].

OBJECTIVE: To study the expression of perlecan in human laryngeal carcinoma cells and its significance. METHOD: The expression of perlecan mRNA in human laryngeal carcinoma cells, Hep-2 cells were investigated by using the techniques of semi-quantify RT-PCR. The expression of perlecan in the Hep-2 cells was investigated by using the techniques of immunohistochemistry. RESULT: It showed that the expression level of perlecan and perlecan mRNA significantly increased in Hep-2 cells as compared with the normal cells, Hacat cells (P < 0.01). CONCLUSION: These data raise the possibility that perlecan many play key roles in the growth,invasion and metastasis of human laryngeal carcinoma cells through either a paracrine or an autocrine mechanism.

[Suppression of proliferation of Hep-2 cells by stable expression of anti-sense perlecan cDNA].

OBJECTIVE: To study the suppression of proliferation of Hep-2 cells by stable expression of anti-sense perlecan cDNA. METHOD: In this study, the plasmids of recombination eukaryotic expression vector perlecan anti-sense cDNA (pAP) were transfected into Hep-2 cells by using cationic liposome (lipofectamine 2000) and divided into three groups: non-transfected group, WT group; transfection with no load carrier, neo group; and transfection with the pAP plasmid, pAP group. Semi quantify RT-PCR, western blot assay and MTT assay were used to detected the expression of perlecan mRNA and protein in the three groups; the level of cell proliferation; and the responsivity of basic fibroblast growth factor (bFGF). RESULT: It was showed that the expression of perlecan mRNA and protein were significantly reduced in the pAP group compared with WT group and ph beta Apr-neol transfected group ( P < 0.01). In the presence of 1 microg/L of bFGF in low serum (0.1% FCS), the pAP transfected cells showed a reduced proliferation rate (MTT assay) while the wild type cells and ph beta Apr-neol transfected cells grew rapidly. CONCLUSION: The growth of Hep-2 cells could be inhibited significantly by perlecan anti-sense cDNA plasmids transfection.

[The effects of sodium salicylate on the expression of GABAalpha, NR1 and hearing response properties of inferior colliculus neurons in mice].

AIM: To study the effects of sodium salicylate on the expression of GABAalpha NR1 and hearing response properties of inferior colliculus neurons in mice. METHODS: Thirty-six kunming mice were divided into three groups (A, B, C,). The expression of GABAalpha NR1 were measured by using RT-PCR. The intensity-rates functions, intensity-latency functions and frequency-turning curves were recorded by extracellular electrophysiological recording techniques. RESULTS: (1) The expression of GABAalpha mRNA of B group was decreased remarkably than the control group (A group, P < 0.05), there weren't noticeable differences between A group and C group (P > 0.05). The expression of NR1 mRNA of B group was increased remarkably than the control group (A group, P < 0.01), there were noticeable differences between A group and C group P < 0.05). (2) The intensity-rates functions, intensity-latency functions were monotonic while the frequency-turning curves were more broad when sodium salicylate was given. (3) The intensity-rates functions, intensity-latency functions were non-monotonic while the frequency-turning curves were sharpened after lidocaine was given. CONCLUSIONS: (1) The results suggested that administration of sodium salicylate decreased the expression of GABAalpha while increased the expression of NR1mRNA. (2) The intensity-rates functions, intensity-latency functions were monotonic, the frequency-turning curves were more broad when salicylate was given and the changes above could be reversed by given lidocaine.

[Expression of vascular endothelial growth factor and its receptors in laryngeal carcinoma cell and its significance].

OBJECTIVE: To study the expression of vascular endothelial growth factor (VEGF) and both the Flt-1 and KDR high affinity VEGF receptors in human laryngeal carcinoma cells and its significance. METHOD: In this study, we investigated the expression of VEGF mRNA and both the Fit-1 mRNA and KDR mRNA high affinity VEGF receptors in human laryngeal carcinoma cells using techniques of semi-quantify RT-PCR. RESULT: It was showed that the expression level of VEGF mRNA was significantly increased in human laryngeal carcinoma cells as compared with the normal cells, Hacat cells (P <0.05), and there was the high expression level of Flt-1 mRNA in the Hep-2 cells, but not KDR mRNA. CONCLUSION: These data raise the possibility that VEGF and its receptors many play key roles in the growth, invasion and metastasis of human laryngeal carcinoma cells through either a paracrine or an autocrine mechanism.

[Construction of recombinant adenovirus containing human Bcl-2 and its expression in the spiral ganglion cells].

OBJECTIVE: To construct the adenoviral vector containing human Bcl-2 gene and to study the expression of the gene in the spiral ganglion cells (SGC) in vitro. METHODS: Human Bcl-2 cDNA obtained from the plasmid pUC-CAGGS/Bcl-2 was cloned into the plasmid pAdTrack-CMV. Then, pAdTrack/Bcl-2 was cotransferred with adenoviral backbone vector into E. coli strain BJ5183. The recombinant adenoviral plasmid was identified by restriction analysis with Pac I and transfected into HEK293 cells to package and amplify recombinant adenovirus particles which would be identified by Electron microscope. After the adenovirus infected the rat spiral ganglion cells, the expression of Bcl-2 gene was detected by Western Blot and RT-PCR. RESULTS: The recombinant AdEGFP/Bcl-2 plasmid was correctly constructed and confirmed by restriction endonuclease analysis. The viral particles in HEK293 cells were identified by Electron microscope. RT-PCR and Western blot showed that Bcl-2 gene was exactly transcripted and expressed in transgene SGC. CONCLUSIONS: The method based on homologous recombination in bacteria is simple and high efficient. The recombinant adenoviral vector containing human Bcl-2 cDNA was constructed and the transgene SGC expressed human Bcl-2 gene in vitro successfully. It provided foundation for the further study of protection for the impaired SGC by hBcl-2 gene.

Effects of neuroglobin gene transfer in vivo on hearing response properties of neurons in the inferior colliculus in mice after administration of sodium salicylate.

The effects of neuroglobin (NGB) gene transfer in vivo mediated by GeneJamer on the hearing response properties of the inferior colliculus (IC) neurons in mice after administration of sodium salicylate were studied. Forty-eight Kunming mice were divided into 4 groups (n=12 in each group): Group A1 (negative control);Group A2 (positive control);Group B, sodium salicylate (450 mg/kg every day) + pEGFP-C1;Group C, sodium salicylate (450 mg/kg every day) + pEGFP-NGB. The GeneJamer and pEGFP-NGB were mixed and injected into IC neurons in mice. The expression of NGB mRNA and protein of IC neurons in mice was detected by using RT-PCR and Western blot methods. The intensity-rate functions, intensity-latency functions and frequency-turning curves in IC neurons were recorded by extracellular electrophysiological recording techniques and the effects of pEGFP-NGB transfer following injection of sodium salicylate on them were studied. It was found that: (1) The GeneJamer-mediated pEGFP-NGB could be effectively transferred into the IC brain tissues in mice and NGB could be expressed intensively. (2) The intensity-rate functions of IC neurons were raised after administration of sodium salicylate. The non-monotonic styles of intensity-rate functions in groups A1, A2 and C were accounted for 74.6%, 72.2 %, 59.3 %, respectively, and the function in group B for 47%. There were significant differences between group B and groups A1, A2 or C (P<0.01, P<0.01, P<0.05). (3) The intensity-latency functions in IC neurons were reduced after administration of sodium salicylate. The non-monotonic styles of intensity-latency functions in groups A1, A2 and C were accounted for 3.2 %, 5.1 %and 21 %, respectively, and that in group B for 45.5 %. There were significant differences between group B and groups A1, A2 or C (P<0.01, P<0.01, P<0.05, respectively). (4) The frequency-turning curves in groups A1 and A2 were sharpened. In 72 acoustic neurons recorded in the group B, the frequency-turning curves from 53 neurons were broadened while those of the rest were sharpened. In group C the frequency-turning curves recorded from 12 of 67 acoustic neurons were broadened while those of the remaining were sharpened. These results suggest that in vivo transfer of NGB gene is highly expressed in IC neurons in mice. In vivo transfer of NGB gene reverses the change of intensity-rate functions, intensity-latency functions and the code styles after administration of sodium salicylate in IC neurons in mice.