Gene Expression, Network Analysis, and Drug Discovery of Neurofibromatosis Type 2-Associated Vestibular Schwannomas Based on Bioinformatics Analysis.

Neurofibromatosis Type 2- (NF2-) associated vestibular schwannomas (VSs) are histologically benign tumors. This study aimed to determine disease-related genes, pathways, and potential therapeutic drugs associated with NF2-VSs using the bioinformatics method. Microarray data of GSE108524 were downloaded from the Gene Expression Omnibus (GEO) database, and differentially expressed genes (DEGs) were screened using GEO2R. The functional enrichment and pathway enrichment of DEGs were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes Genomes (KEGG). Furthermore, the STRING and Cytoscape were used to analyze the protein-protein interaction (PPI) network of all differentially expressed genes and identify hub genes. Finally, the enriched gene sets belonging to the identified pathways were queried against the Drug-Gene Interaction database to find drug candidates for topical use in NF2-associated VSs. A total of 542 DEGs were identified, including 13 upregulated and 329 downregulated genes, which were mainly enriched in terms of focal adhesion, PI3K-Akt signaling pathway, ECM-receptor interaction, Toll-like receptor signaling pathway, Rap1 signaling pathway, and regulation of actin cytoskeleton. 28 hub genes were identified based on the subset of PPI network, and 31 drugs were selected based on the Drug-Gene Interaction database. Drug discovery using bioinformatics methods facilitates the identification of existing or potential therapeutic drugs to improve NF2 treatment.

[Protective effects of N-acetylcysteine on cochlear damage occur in guinea pigs exposed to irradiation].

OBJECTIVE: To explore the protective effects of N-acetylcysteine (NAC) on cochlear damage occurring in irradiated guinea pigs. METHODS: Seventy-two guinea pigs were randomly divided into 4 groups (n = 18 each). Control group received neither NAC nor irradiation, irradiation group received total cranium irradiation of 70 Gy, irradiation & saline group cranium irradiation of 70 Gy and saline solution through a round window and NAC group cranium irradiation of 70 Gy and NAC through a round window. The right ear received radiation. The animals were sacrificed at Day 14 post-irradiation. The specimens were dehydrated, embeded in paraffin and serially cut into 5-µm slices. Sections were stained with immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The cochlear basal membranes were observed for malondialdehyde (MDA) and superoxide dismutase (SOD) with scanning electron microscope. RESULTS: The cilium of hair cells had no clear loss and apoptotic number of spiral ganglion cells decreased in NAC group. The average optical density value of Caspase 3 in spiral ganglion in NAC group significantly decreased versus the irradiation group (0.08 ± 0.02 vs 0.10 ± 0.01, P < 0.01). The level of MDA of NAC group also decreased versus the irradiation group (0.33 ± 0.05 vs 0.84 ± 0.13, P < 0.05). The level of SOD in the NAC group increased versus the irradiation group (10.7 ± 3.0 vs 8.7 ± 1.3, P < 0.05). The ratio of apoptotic cell in SGC in the NAC group at Day 14 (7.8% ± 1.8%) decreased versus the irradiation group (32.0% ± 8.7%) at Day 14. CONCLUSION: MDA and SOD may be involved in the pathogenesis of cochlear cell damage. And NAC protects the irradiated cochlear cell.

[Analysis of sniffing test in otitis media with effusion].

OBJECTIVE: To explore the roles of the closure disorder of Eustachian tube in occurrence and development of otitis media with effusion (OME). METHODS: Fifty-six adults with OME, 16 children with OME, 66 health adults and 20 health children were selected according to diagnosis criteria. Sniffing test was measured by Tubo-tymanoaerodynamic graphy and the self-designed questionnaires were surveyed in all cases. RESULTS: The positive levels in sniffing test were regarded as external auditory canal press 10dapa lower than baseline of the pressure. The positive rate was 64.86% in adults with OME, which was higher than health adults (P < 0.01). The positive rate was 70.83% in children with OME which was higher than health children (P < 0.05). CONCLUSIONS: The higher positive rate of sniff test in OME patients suggests that closure disorder in Eustachian tube playing an important role in the occurrence and development of OME.

[Clinical analyses of sudden sensorineural hearing loss in 14 nasopharyngeal carcinomas following radiotherapy].

OBJECTIVE: To investigate the clinical characteristics and effects of sudden sensorineural hearing loss in nasopharyngeal carcinoma (NPC) following radiotherapy. METHODS: The clinical characteristics and effects in 14 NPC patients (15 ears) with sudden sensorineural hearing loss following radiotherapy were retrospectively analyzed. RESULTS: The sudden sensorineural hearing loss happened more in male subjects than female subjects and more in the left ear than the right ear. Its occurrence time was averaged 6.6 years following radiotherapy. Most of the patients suffered hearing loss prior to the sudden sensorineural hearing loss. The 250, 500, 1000, 2000, 4000 Hz average hearing thresholds: sudden hearing loss ears (78.5 ± 24.7) dBHL, none-sudden hearing loss ears (57.0 ± 32.4) dBHL, among which, 73.33% (11/15) for sensorineural hearing loss, 26.67% (4/15) for mixed hearing loss. 12 cases had complications following radiotherapy. At least one case had posterior circulation barrier. The total effective rate was 26.67% (4/15) and four cases had relapsed and in vain thereafter. CONCLUSIONS: In NPC patients who received radiotherapy, it caused more serious sudden sensorineural hearing loss and the treatment effects were poor and hearing loss was susceptible to relapse. The pathogenesis may be related to the radiation caused posterior circulation disorders.

Salicylate initiates apoptosis in the spiral ganglion neuron of guinea pig cochlea by activating caspase-3.

Salicylate-induced ototoxicity leading to sensorineural hearing loss (SNHL) and tinnitus is well documented. However, the exact mechanisms are poorly defined. Caspase-3 is a member of the class of effector caspases and has been activated in nearly every model of apoptosis. To examine its role in salicylate-induced injury, we subjected guinea pigs to treatment with a specific inhibitor zDEVD-FMK via the round window niche (RWN) followed by a systemic injection of salicylate at a dose of 200 mg · kg(-1) · d(-1) i.p. for 10 consecutive days. For those animals administered with salicylate, immunohistochemical studies revealed that caspase-3 was activated in the spiral ganglion neurons (SGNs) and method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) to identify neuronal apoptosis showed that fragmented nuclei were distributed in Rosenthal's canal. Topical administration of the zDEVD-FMK at a concentration of 500 mM blocked caspase-3 activation and had an effect in reducing the number of TUNEL-positive auditory neurons. In contrast, the inhibitor at a concentration of 125 or 250 mM caused no variation in the expression of activated caspase-3, or in the ratio of TUNEL-positive neurons. These results indicate that caspase-3 is a crucial mediator of apoptosis induced by salicylate in the primary auditory neuron in vivo, and suggest that the specific inhibitor at a relatively high concentration may be therapeutically beneficial in salicylate-induced apoptosis.

Blocking caspase-3-dependent pathway preserves hair cells from salicylate-induced apoptosis in the guinea pig cochlea.

In the present study, we aim to explore whether the caspase-3-dependent pathway is involved in the apoptotic cell death that occurs in the hair cells (HCs) of guinea pig cochlea following a salicylate treatment. Guinea pigs received sodium salicylate (Na-SA), at a dose of 200 mg·kg(-1)·d(-1) i.p., as a vehicle for 5 consecutive days. In some experiments, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-FMK), a specific apoptosis inhibitor, was directly applied into the cochlea via the round window niche (RWN) prior to salicylate treatment for determination of caspase-3 activation. Alterations in auditory function were evaluated with auditory brainstem responses (ABR) thresholds. Caspase-3 activity was determined by measuring the proteolytic cleavage product of caspase-3 (N-terminated peptide substrate). DNA fragmentation within the nuclei was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Ultrastructure variation in the target cell was assessed by electron microscopy (EM). Salicylate treatment initiated an obvious elevation in ABR thresholds with a maximum average shift of 60 dB sound pressure level (SPL), and caused significant apoptosis in both inner (IHCs) and outer (OHCs) hair cells resulted from an evident increasing in immunoreactivity to caspase-3 protease. Transmission electron microscopy (TEM) displayed chromatin condensation and nucleus margination accompanied by cell body shrinkage in the OHCs, but not in the IHCs. Scanning electron microscopy (SEM) showed breakdown, fusion, and loss in the stereociliary bundles at the apex of OHCs rather than IHCs. zDEVD-FMK pretreatment prior to salicylate injection substantially attenuated an expression of the apoptotic protease and protected HCs against apoptotic death, followed by a moderate relief in the thresholds of ABR, an alleviation in the submicroscopic structure was also identified. In particular, disorientation and insertion in the hair bundles at the apex of OHCs was exhibited though no classic apoptotic change found. The above changes were either prevented or significantly attenuated by zDEVD-FMK. These findings indicate that salicylate could damage cochlear hair cells via inducing apoptosis associated with caspase-3 activation.

Caspase-3 activation in the guinea pig cochlea exposed to salicylate.

In the current study, we explored whether chronic salicylate exposure could induce apoptosis in outer hair cells (OHCs) and spiral ganglion neurons (SGNs) of the cochlea. Guinea pig received sodium salicylate (400 mg/kg/d) or saline vehicle for 10 consecutive days. Programmed cell death (PCD) executioner was evaluated with immunohistochemistry detection of activated caspase-3. Apoptosis was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Repeated salicylate administration activated caspase-3 and caused apoptosis in OHCs and SGNs (p<0.01 vs. saline control for both measures and in both cell types). Cell counting showed a significant loss in OHCs (p<0.01 vs. saline control), but not in inner hair cells (IHCs). Transmission electron microscopy (TEM) revealed chromatin condensation and nucleus margination in salicylate-treated cochlea. Scanning electron microscopy (SEM) demonstrated stereociliary bundles breakdown and fusion at the apical of OHCs, villous matter was discovered to attach on the surface of SGNs. These findings suggest that long-term administration of high-dose salicylate can activate caspase-3 pathway to induce OHC and SGN apoptosis.

[Effect of sodium salicylate on the auditory brain stem response threshold and expression of glutamic acid decarboxylase in spiral ganglion of juvenile and adult guinea pigs].

OBJECTIVE: To study the differences of regulation of sodium salicylate on the auditory brain stem response (ABR) threshold and expression of glutamic acid decarboxylase (GAD) protein in spiral ganglion of juvenile and adult guinea pigs. METHODS: Fourty juvenile guinea pigs which were born just four days and fourty adult guinea pigs which were born thirty days were selected. They were divided four groups (group A; group B; group C; group D). ABR threshold was detected before administration, after administration for 15 days and after administration stopped for 30 days. The protein expression of GAD were measured after administration for 15 days and after administration stopped for 30 days by the method of immunohistochemistry. RESULTS: ABR threshold of juvenile sodium salicylate groups (group C) was increased remarkably than that of before administration and the control after administration for 15 days (P < 0.001). ABR threshold of group C was returned to the level of that of before administration and after administration stopped for 30 days. ABR threshold of adult sodium salicylate groups (group D) was increased remarkably than that of before administration and the control after administration for 15 days (P < 0.001). ABR threshold of group D was kept the high level after administration stopped for 30 days. The protein expression of GAD of sodium salicylate groups (group C and D) was decreased than that of the control after administration for 15 days. The protein expression of group C was more visible regression than that of group D (t = 4.7, P < 0.001). The protein expression of group C was returned the level of before administration after administration stopped for 30 days, but the protein expression of group D was kept the high level. CONCLUSIONS: The results suggest that sodium salicylate can regulate differently ABR threshold and expression of GAD protein in spiral ganglion of juvenile and adult guinea pigs. The effects of sodium salicylate on ABR threshold and expression of GAD protein in spiral ganglion of juvenile pigs are more noticeable than that of adult guinea pigs, but these changes are easier to return the normal than that of adult guinea pigs.

[Effects of sodium salicylate on the expressions of gamma-aminobutyricacid and glutamate and auditory response properties of the inferior colliculus neurons].

The effects of sodium salicylate (NaSA) on the expressions of gamma-aminobutyricacid (GABA) and glutamate (Glu), and auditory response properties of the inferior colliculus neurons in mice were studied. Thirty-six Kunming mice were divided into three groups: control group (saline injection); NaSA group (NaSA 450 mg/kg, i.p., each day for 15 d); NaSA + lidocaine group (NaSA 450 mg/kg + lidocaine 10 mg/kg, i.p., each day for 15 d). The expressions of GABA and Glu were examined with immunohistochemical method. The intensity-rate function, intensity-latency function and frequency-tuning curve were determined with extracellular electrophysiological recording. Results are as follows: (1) The expression of GABA in the NaSA and NaSA + lidocaine groups decreased remarkably compared with that in the control group; there was no noticeable difference between the NaSA and NaSA + lidocaine groups. The expression of Glu in the NaSA group increased significantly compared with that in the control and NaSA + lidocaine groups. No difference in the expression of Glu was found between the control and NaSA + lidocaine groups. (2) In NaSA group, the intensity-rate function displayed a non-monotonic pattern, rising at low intensity and descending at high intensity; the tip of frequency-tuning curves became broad after administration of NaSA. (3) The changes in intensity-rate function and intensity-latency function were not evident and the tips of the frequency-tuning curves sharpened in the NaSA + lidocaine group. These results suggest that administration of NaSA increases the expression of Glu-positive neurons and reduces that of GABA-positive neurons in the inferior colliculus. NaSA changes the auditory response properties of the inferior colliculus and lidocaine can reverse these changes.

[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.

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.

[Apoptosis in the immune response of inner ear].

OBJECTIVE: To investigate whether apoptosis is one of the mechanism in the immune response of inner ear,and to detect the expression of Fas, FasL, Bcl-2 and Bax in the inner ears. METHODS: Sixteen healthy, female guinea pigs were employed in the experiment. Sensitized systematically with keyhole limpet hemocyanin (KLH), the KLH-immunized animals were inoculated with the same antigen, and the control animals were injected PBS through cochlea basal turn. The animals were sacrificed at 7 day after inner ear vaccination. Transmission electron microscopy was used to detect inner ear apoptotic cells, and paraffin sections of cochlea from animals were stained using a terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay to identify inner ear cells undergoing apoptosis. Immunohistochemistry method was used to detect the expression of Fas, FasL, Bcl-2 and Bax in the inner ears. RESULTS: The observation of electron microscopy had shown the features characteristic of apoptotic cells in the KLH-immunized inner ears but not in the control inner ears. TUNEL-positive cells were found in the KLH-immunized inner ears but not in the control inner ears. The positive cells were the hair cells in Corti's organ, and the marginal cells in the stria vascularis and the neurons in the spiral ganglion. Moreover under morphological analysis by light microscope, these cells had the features characteristic of apoptosis. High expression of Fas and FasL could be detected in Corti's organ, the stria vascularis, the spiral ligament and the neurons of the spiral ganglion in the KLH-immunized inner ears. A low expression of Fas could be detected in the stria vascularis and the neurons of the spiral ganglion in the control inner ears, but no cells staining positive for FasL were found in the control inner ears. No cells staining positive for Bcl-2 were found in the KLH-immunized animals but moderate expression of Bcl-2 could be detected in Corti's organ, the lateral wall and the neurons of the spiral ganglion in the control inner ears. High expression of Bax could be detected in Corti's organ, the lateral wall and the neurons of the spiral ganglion in the KLH-immunized inner ears. A low expression of Bax could be detected in the neurons of the spiral ganglion and no cells staining positive for Bax were found in Corti's organ, the lateral wall in the control inner ears. CONCLUSIONS: These findings suggest apoptosis is involved in the pathogenesis of the immune response of inner ear and Fas- FasL pathway is one of important signal transportation of the course and Bcl-2 and Bax have a critical role in the regulation of apoptotic cell death induced by the immune response of inner ear.