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Pulmonary fibrosis is a progressive interstitial lung disease with poor prognosis. Anemarrhenae Rhizoma is a traditional Chinese herbal medicine and has been applied in clinical practice for a long history. Recently, components of Anemarrhenae Rhizoma were reported to possess anti-inflammatory and immunomodulatory features; however, the effect of them on pulmonary fibrosis remains unknown. In this study, we explored the therapeutic effect of total extract of Anemarrhenae Rhizoma (TEAR) on bleomycin-induced pulmonary fibrosis. Pulmonary fibrosis rat model was established by a single intratracheal instillation of bleomycin, three doses of TEAR were intragastrically administered for consecutive 28 days. Subsequent to sacrificing of rats, pulmonary fibrosis was observed in rats treated with bleomycin, but administration of TEAR attenuated lung fibrosis, as evidenced by the improved lung histopathological damage and decreased weight loss and lung index. Moreover, TEAR treatment inhibited the inflammatory response in lung fibrosis, which was shown by the reduced nitrogen oxide level and myeloperoxidase activity. Furthermore, TEAR modulated the redox balance in lung tissue by alleviated lipid peroxidation and enhanced enzymatic antioxidants activity. Meanwhile, TEAR protected the rats from fibrosis in a dose-dependent manner, and the anti-fibrotic activity of TEAR may be related to the modulation of TGF-β1/Smad signaling pathway. Collectively, TEAR alleviates bleomycin-induced pulmonary fibrosis, indicating perspectives for development of a potential agent for lung fibrosis therapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: This study was to investigate the effect of high-flux hemodialysis (HD) combined with levocarnitine on vascular calcification, microinflammation, hepcidin, and malnutrition in elderly patients on maintenance HD (MHD)., Methods: 75 MHD elderly patients admitted to hospital between 1st September 2017 and 31st August 2019 were selected as the study subjects. They were randomly divided by digital table into three groups: low-flux group (n=25), high-flux group (n=25) and joint group (n=25). In the low-flux group, dialyzer had an ultrafiltration coefficient 12 mL/(h·mmHg) and effective surface area of 1.4 m2 compared with 59 mL/(h·mmHg) and 1.8 m2 in the high-flux group. After treatment, the calcification of blood vessels was examined by lateral X-ray, pelvic plain film and bilateral positive position. For patients in all groups, the concentrations of parathyroid hormone (PTH) and β 2-microglobulin (β 2-MG) in serum were measured by automatic chemiluminescence; levels of interleukin-6, C-reactive protein (CRP), and tumor necrosis factor alpha (TNF-α) were measured by ELISA before and after treatment; and the level of hepcidin was measured by ELISA. Before and 12 weeks after the treatment, the nutritional status of the patients was evaluated by modified quantitative subjective global assessment (MQSGA), hemoglobin (Hb) and red blood cell count (RBC). Complications in the three groups were recorded, including nausea, chest pain, hypotension, hypertension, pruritus, dry heat, muscle spasm, arrhythmia, and restless legs., Results: Vascular calcification in the joint group was better than the low-flux and high-flux groups (P<0.05). After treatment, the serum PTH and β 2-mg concentrations in the joint group were lower than those in the other two groups (P<0.05), and the levels of IL-6, CRP, TNF-α and hepcidin in the joint group were significantly lower than those before treatment (P<0.05). After treatment, the MQSGA scores in the joint group were lower than those in the low-flux and high-flux groups (P<0.05), and Hb and RBC were higher (P<0.05)., Conclusions: The combination of high-flux HD and levocarnitine in elderly patients on MHD can increase the clearance of medium and large molecular toxins, effectively correct malnutrition, alleviate microinflammation, delay the progress of vascular calcification, and is safe.

Objective: To measure the cochlear compound action potential (CAP) and the densities of hair cells (HCs) along the whole length of the basilar membrane (BM) in adult chinchillas. And to investigate the relationship between the severity of inner hair cells (IHCs) loss and the changes of CAP by using carboplatin-cochlear lesion model. Methods: Totally 18 chinchillas were recruited after ontological evaluation. They were randomly divided into three groups (with 6 subjects in each), A: control, B and C: legion groups treated with one or two shot(s) of carboplatin respectively (76 mg/kg in one shot, i.p., one-week interval between the two shots). Endpoint tests were performed 30 days after the carboplatin treatment in groups B and C, and matched time in group A. A sliver-ball electrode was placed into round window niche via hypotympanic approach in anesthetized chinchilla. CAP was measured in response to clicks and tone burst of 0.5, 1, 2, 4, 8, 16 kHz respectively under anesthesia. CAP amplitudes and thresholds were measured and compared across the groups. After the recording, the whole cochlea surface preparation was made and the HCs were stained in histochemistry against substrate of succinate dehydrogenase (SDH). Images were taken with high-resolution digital camera under light microscope and across the whole cochlea. The length of the basilar membrane (BM) and the number of both IHCs and OHCs were counted. The HC density was calculated as the number of HCs per 10% BM length. Results: The CAP thresholds were (7.1±2.6), (25.4±5.0), (24.6±5.4), (10.4±5.0), (0.4±1.4), (4.2±6.3) and (17.1±14.1) dB SPL (from 6 subjects in group A, n= 12 ears) corresponding to stimuli of Click and 0.5, 1, 2, 4, 8, 16 kHz tone bursts respectively. The total number of cochlear HCs were measured as (8 936±643) ( x ± s ) and the average length of the BMs was (17.73±1.012) mm from the six subjects in the group A ( n= 12 ears). The HC density was found to be varied slightly across the BM. There was no significant CAP threshold difference between the control (group A) and the group B, which received one shot of carboplatin. However, the maximal CAP amplitude was reduced by 40% in the group B and compared with group A. Correspondingly, approximately 40% loss of IHCs were seen. In contrast, a significant CAP threshold shift was seen in subjects receiving two shots of carboplatin (group C), which was accompanied by a loss of 90% IHCs. Conclusions: The CAP thresholds of adult chinchillas show typical open-V shape with the lowest values at 2, 4, and 8 kHz. IHC loss by carboplatin in certain degree is well correlated with CAP amplitude reduction, but does not change the threshold when inner hair cell loss reaches 40%, however, if inner hair cell loss exceeds 80%, the threshold shift of CAP will be inevitable.

This study aimed to investigate the effect of miR-671-5p on metastasis of clear cell renal cell carcinoma (ccRCC) and underlying mechanism involved. The migration and invasion of ccRCC cells were determined by transwell and boyden assays in vitro and in vivo. Genes mRNA and protein expression were detected by quantitative polymerase chain reaction (qPCR) and western blot analysis, respectively. The target gene of miRNA was confirmed by luciferase reporter assays. Transcriptional regulation of miRNA by transcription factor was detected by chromatin immunoprecipitation assay (ChIP). The expression of miRNA in clinical specimens were detected by in situ hybridization (ISH). miR-671-5p promoted migration and invasion of ccRCC in vivo and in vitro. Moreover, miR-671-5p directly targeted APC to activate Wnt signaling, thus inducing the epithelial-mesenchymal transition (EMT) in ccRCC. Intriguingly, miR-671-5p expression was transcriptionally enhanced by HMGA1. Consistently, bioinformatics analysis suggested that HMGA1 was positively correlated with miR-671 expression, however, miR-671 was negatively correlated with APC. In situ hybridization analysis showed that miR-671-5p was upregulated in ccRCC compared with paracarcinoma and correlated with poor prognosis of ccRCC patients. In addition, univariate and multivariate analysis indicated that miR-671-5p expression was an independent prognostic factor for overall survival in ccRCC patients. Our data suggest that miR-671-5p is a tumor enhancer in regulating of ccRCC metastasis, and miR-671-5p may be utilized as a factor for the clinical diagnosis and prognosis of ccRCC.

Objective: To explore the clinical characteristics and microsurgical strategies of intracranial posterior circulation aneurysms. Methods: The clinical manifestations, imaging data, surgical approaches and follow-up results of 35 patients with circulating aneurysms (37 aneurysms) treated by microsurgery in the First Affiliated Hospital of Zhengzhou University from January 2013 to January 2018 were analyzed retrospectively. Results: A total of 22 aneurysms were clipped, 13 were clipped and resected, 1 case was clipped and together with AVM resection and 1 case was isolated. Of 37 aneurysms in 35 patients, 11 aneurysms were at the basilar artery apexes, 10 at the posterior cerebral arteries, 6 at the posterior inferior cerebellar arteries, 3 at the basilar arteries, 3 at the vertebral arteries (including 1 case of vertebral arterial dissecting aneurysm), 2 at the anterior inferior cerebellar arteries and 2 at the superior cerebellar arteries. The surgical approaches included pterional approach, extensive pterional approach, infratemporal fossa approach, retrosigmoid approach and far-lateral approach. The Glasgow Outcome Scale (GOS) scores showed good recovery in 24 cases, moderate neurological dysfunction in 6 cases, severe neurological dysfunction in 2 cases, persistent vegetative state in 1 case and 2 cases of death 6 months after their discharge from hospital. Conclusions: Posterior circulation aneurysms are adjacent to important structures. They are deep in position, with small operation space and difficult to operate. Full preoperative evaluation of the condition, selection of appropriate surgical methods are the key factors to benefit the patients.

In this study, Paeonia suffruticosa roots and rhizospheric soil in five geographic regions which were harvested in October were utilized as experimental materials, then the diversity of endophytic and rhizospheric actinomycetes were investigated by High-throughput sequencing technique. The 1 754 operational taxonomic units (OTUs) were obtained from 129 954 high quality sequences, 1 311 OTUs were detected in rhizospheric actinomycetes and belonged to four classes, four orders, twenty-seven families and ninety-seven genera, thirty-three genera such as Ilumatobacter were found in the five regions rhizospheric soil while three genera such as Longispora were only detected in the Dao-di regions, the dominant genera were Mycobacterium, Nocardioides, Streptomyces. 443 OTUs were obtained in roots and distributed in three classes, three orders, twenty-four families and fifty genera, thirteen genera such as Cryptosporangium were found in the five regions roots while Planosporangium, Luteococcus were only detected in the Dao-di regions, the dominant genera were Nocardioides. Alpha diversity analysis showed that the Shannon and Chao1 index in rhizospheric actinomycetes in Bozhou, Tongling and Nanling region were higher than Heze and Luoyang. Based on principal co-ordinates analysis (PCoA) analysis, the rhizospheric actinomycetes formations were similar in Tongling and Nanling region, at the same in Tongling and Luoyang endophytic actinomycetes. According to heatmap analysis, Bozhou, Tongling and Nanling region rhizospheric actinomycetes showed a close similarity in actinomycetes community structures on phylogenetic analysis, while Tongling, Luoyang and Nanling endophytic actinomycetes showed the same. Our results not only suggested that the rich and diverse actinomycetes resources in P. suffruticosa roots and rhizospheric soil but also revealed rhizospheric actinomycetes in the Dao-di regions had high similarity., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Objective To observe decreased hearing in aged C57BL/6J mice, and to study pro- tective effects of Jian' erji ( JEJ ) for age-related hearing loss (AHL) and its possible mechanism. Methods Totally 36 C57BL/6J mice were randomly divided into four groups, i.e., the normal control group (n =6) , the AHL control group (n =12) , the high dose JEJ group (n =12) , the low dose JEJ group (n =6). Mice in the normal control group drank tap water from ablactation till 2 months old. Mice in the AHL control group drank tap water from ablactation till 7 months old. Mice in high and low dose JEJ groups drank JEJ at the daily dose of 3. 65 g/kg and 0. 91 g/kg respectively from ablactation till 7 months old. Six mice were selected from each group for auditory brainstem response (ABR) using brainstem evoked potentiometer on the day of ending the test. The cochlear tissue, auditory cortex, and liver were immediately collected from 6 mice of the high dose JEJ group and 6 of the AHL control group at the same ages. Contents of malondialdehyde (MDA) , end product of lipid peroxidation were detected by UV spectrophotometer using MDA coomassie blue kit. Results ABR thresholds evoked by short-pure tone from 4 to 48 KHz were in the normal range of 2 months old mice in the normal control group. Compared with 2 months old mice in the normal control group, ABR thresholds were significantly elevated in 7 months old mice of the AHL control group (P <0. 05). Significant differences also existed in ABR thresholds from 8 to 48 KHz in the high dose JEJ group (P <0. 05). Compared with 7 months old mice of the AHL control group, MDA contents in cochlear tissue, auditory cortex, and liver were obviously reduced in the high dose JEJ group (P <0. 01). Conclusions C57BL/6J mice showed significant symptoms of AHL in high frequency range at 7 months old. Daily drinking of high dose JEJ could significantly delay the occurrence and progress of AHL. Its protection might be related to antioxidant effects JEJ contained.

Single-nucleotide polymorphisms (SNPs) of microRNAs (miRNAs) are considered potential markers of cancer risk and prognosis in various cancers. In the current study, the primary aim is to determine whether the miR-492G>C polymorphism (rs2289030) altered hepatocellular carcinoma (HCC) prognosis. The SNP rs2289030 of miR-492 was genotyped using DNA from blood samples of 362 HCC patients that had undergone surgical resection of a HCC tumor. The associations between overall survival and demographic characteristics, clinical features, and the SNP rs2289030 were estimated using the Cox proportional hazards model. Results showed that patients who carried the CG genotype (P  = 0.015, hazard ratio [HR] = 0.704, 95 % confidence interval [CI] 0.530-0.934) and CG+GG genotype (P = 0.011, HR = 0.703, 95 % CI 0.536-0.924) had significantly decreased risk of death compared to those with the CC genotype. Similar results were found in the multivariate analysis adjusted by tumor size and venous invasion. Further stratification analysis indicated that the effect of rs2289030 had more prominence in patients ≤50 years old and that reported ever using alcohol, male gender, a family history of HCC, being HbsAg or alpha fetoprotein (AFP) positive, differentiation I + II, presence of venous invasion or cirrhosis, multiple tumors, and pTNM stage I + II. Results from this study illustrate the potential use of miR-492 rs2289030 as a prognostic marker for HCC patients that have undergone a surgical resection of the tumor.

Single nucleotide polymorphisms (SNPs) within microRNAs (miRNAs) are considered potential markers for risk and prognosis of various cancers. In the current study, we aimed to determine whether miR-608 rs4919510 affected hepatocellular carcinoma (HCC) prognosis. We genotyped rs4919510 using DNA from blood samples of 362 HCC patients receiving surgical resection of HCC tumor. Associations between rs4919510 and overall survival (OS) and demographic characteristics and clinical features were estimated using the Cox proportional hazards model. Results showed that HCC patients who carried the rs4919510 CC genotype had a significantly longer OS compared to those who carried the GG genotype (P = 0.013, hazard ratio [HR] = 0.600, 95 % confidence interval [CI] 0.402-0.897) and the CG + GG genotype (P = 0.033, HR = 0.681, 95 % CI 0.479-0.970) in univariate analysis. Similar results were obtained in multivariate analysis. Further stratification analysis indicated that rs4919510 was significantly associated with OS in patients who were satisfied with one of the following criteria: male gender, HbsAg-positive, α-fetoprotein (AFP)-positive, tumor size >5 cm, cirrhosis, solitary tumor, I + II pTNM stage, or no tumor capsule. Finally, a significantly higher frequency of rs4919510 CC genotype was observed in patients with cirrhosis (22.9 %, 55/240) than those without cirrhosis (14.0 %, 17/121) (P = 0.047). In conclusion, our results illustrated the potential role of miR-608 rs4919510 as a prognostic marker for HCC patients undergoing surgical resection of the tumor.

Recent genome-wide associated studies (GWASs) have revealed several common loci associated with the risk of hepatitis B virus (HBV)- or hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). We selected 15 single nucleotide polymorphisms (SNPs) identified through GWASs on HBV- or HCV-related HCC, and genotyped them in two independent Chinese cohorts of chronic HBV carriers, including 712 LC cases and 2601 controls. The association of each SNP with the risk of HBV-related LC was assessed by meta-analysis of the two cohorts. Of the 12 SNPs reported in HBV-related HCC GWASs, five SNPs (rs7574865 in STAT4, rs9267673 near C2, rs2647073 and rs3997872 near HLA-DRB1 and rs9275319 near HLA-DQ), were found to be significantly associated with the risk of HBV-related LC (rs7574865: P = 1.79 × 10(-2), OR = 1.17, 95% CI = 1.03-1.34; rs9267673: P = 4.91 × 10(-4), OR = 1.37, 95% CI = 1.15-1.63; rs2647073: P = 3.53 × 10(-5), OR = 1.63, 95% CI = 1.29-2.06; rs3997872: P = 4.22 × 10(-4), OR = 1.86, 95% CI = 1.32-2.62; rs9275319: P = 1.30 × 10(-2), OR = 1.32, 95% CI = 1.06-1.64). However, among the three SNPs associated with the risk of HCV-related HCC in previous GWASs, none of them showed significant association with the risk of HBV-related LC. Our results suggested that genetic variants associated with HBV-related hepatocarcinogenesis may already play an important role in the progression from CHB to LC.

The apoptotic pathway is important in the control of vital processes of hepatocellular carcinoma (HCC). In the current study, we aimed to determine whether apoptotic gene-related polymorphisms modified HCC prognosis. We genotyped 16 single nucleotide polymorphisms (SNPs) in 10 core genes (TP53, TP53INP1, TP53BP1, CDKN2A, CDKN1A, CDKN1B, MDM2, BAX, CCDN1 and BCL2) in the apoptotic pathway by using DNA from blood samples of 362 HCC patients receiving surgical resection of HCC tumor. The associations between genotypes/haplotypes of the 10 genes and overall survival (OS) of HCC patients were assessed using the Cox proportional hazards model. We found one CDKN1B haplotype CCT/ACT (constructed by rs36228499 C>A, rs34330 C>T and rs2066827 T>G) significantly associated with decreased OS of HCC patients, compared to the common haplotype ACT/CTT both in univariate analysis (P=0.013, HR=1.198, 95% CI: 1.039-1.381) and multivariate analysis (P=0.006, HR=1.224, 95% CI: 1.059-1.413). We also find two SNPs (rs560191 G>C and rs2602141 T>G) in TP53BP1 shown to be marginally significantly associated with decreased OS of HCC patients. However, none of the other SNPs or haplotypes were significantly associated with HCC OS. Our results illustrated the potential use of CDKN1B haplotype as a prognostic marker for HCC patients with surgical resection of tumor.

Unlabelled: Hepatitis B virus affects more than 2 billion people worldwide, 350 million of which have developed chronic hepatitis B (CHB). The genetic factors that confer CHB risk are still largely unknown. We sought to identify genetic variants for CHB susceptibility in the Chinese population. We undertook a genome-wide association study (GWAS) in 2,514 CHB cases and 1,130 normal controls from eastern China. We replicated 33 of the most promising signals and eight previously reported CHB risk loci through a two-stage validation totaling 6,600 CHB cases and 8,127 controls in four independent populations, of which two populations were recruited from eastern China, one from northern China and one from southern China. The joint analyses of 9,114 CHB cases and 9,257 controls revealed significant association of CHB risk with five novel loci. Four loci are located in the human leukocyte antigen (HLA) region at 6p21.3, including two nonsynonymous variants (rs12614 [R32W] in complement factor B [CFB], Pmeta =1.28 × 10(-34) ; and rs422951 [T320A] in NOTCH4, Pmeta  = 5.33 × 10(-16) ); one synonymous variant (rs378352 in HLA-DOA corresponding to HLA-DOA*010101, Pmeta  = 1.04 × 10(-23) ); and one noncoding variant (rs2853953 near HLA-C, Pmeta  = 5.06 × 10(-20) ). Another locus is located at 20q13.1 (rs1883832 in the Kozak sequence of CD40, Pmeta  = 2.95 × 10(-15) ). Additionally, we validated seven of eight previously reported CHB susceptibility loci (rs3130542 at HLA-C, rs1419881 at TCF19, rs652888 at EHMT2, rs2856718 at HLA-DQB1, rs7453920 at HLA-DQB2, rs3077 at HLA-DPA1, and rs9277535 at HLA-DPA2, which are all located in the HLA region, 9.84 × 10(-71)  ≤ Pmeta  ≤ 9.92 × 10(-7) )., Conclusion: Our GWAS identified five novel susceptibility loci for CHB. These findings improve the understanding of CHB etiology and may provide new targets for prevention and treatment of this disease., (© 2015 by the American Association for the Study of Liver Diseases.)

The EGFR signaling pathway is important in the control of vital processes in the carcinogenesis of hepatocellular carcinoma (HCC), including cell survival, cell cycle progression, tumor invasion and angiogenesis. In the current study, we aim to assess if genetic variants in the genes of the EGFR signaling pathway are associated with the prognosis of HCC. We genotyped 36 single nucleotide polymorphisms (SNP) in four core genes (EGF, EGFR, VEGF, and VEGFR2) by using DNA from blood samples of 363 HCC patients with surgical resection. The associations between genotypes and overall survival (OS) and disease-free survival (DFS) were estimated using the Kaplan-Meier method. Hazard ratios (HRs) and 95% confident intervals (CIs) were estimated for the multivariate survival analyses by Cox proportional hazards regression models, adjusting for age, gender, family history, HBsAg and AFP. We found that five SNPs in the VEGFR2 gene were significantly associated with clinical outcomes of HCC patients. Among them, four SNPs (rs7692791, rs2305948, rs13109660, rs6838752) were associated with OS (p=0.035, 0.038, 0.029 and 0.028, respectively), and two SNPs (rs7692791 and rs2034965) were associated with DFS (p=0.039 and 0.017, respectively). Particularly, rs7692791 TT genotype was associated with both reduced OS (p=0.037) and DFS (p=0.043). However, only one SNP rs2034965 with the AA genotype was shown to be an independent effect on DFS (p=0.009) in the multivariate analysis. None of the other 31 polymorphisms or 9 haplotypes attained from the four genes was significantly associated with OS or DFS. Our results illustrated the potential use of VEGFR2 polymorphisms as prognostic markers for HCC patients.

Aims: This meta-analysis was performed to evaluate the correlations between atrial natriuretic peptide (ANP) genetic polymorphism and its serum ANP levels with the risk of ischemic stroke., Methods: The PubMed, CISCOM, CINAHL, Web of Science, Google Scholar, EBSCO, Cochrane Library, and CBM databases were searched for relevant articles published before October 1st, 2013 without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. Crude odds ratios (ORs) or standardized mean difference (SMD) with their 95% confidence interval (95% CI) were calculated. Twelve case-control studies that met all inclusion criteria were included in this meta-analysis. A total of 1285 patients with ischemic stroke and 1088 healthy control subjects were involved in this meta-analysis. Three common single-nucleotide polymorphisms (1837 G/A, 2238 T/C, and 664 G/A) in the ANP gene were assessed., Results: Our meta-analysis results revealed that ANP 2238 T/C polymorphism might increase the risk of ischemic stroke (C allele vs. T allele: OR=2.26, 95% CI: 1.59-3.23, p<0.001; TC+CC vs. TT: OR=2.26, 95% CI: 1.34-3.81, p=0.002; respectively). However, we found no correlations of ANP 1837 G/A and 664 G/A polymorphisms with ischemic stroke risk (all p>0.05). Furthermore, ischemic stroke patients had higher levels of serum ANP than those of healthy control subjects (SMD=3.12, 95% CI: 1.16-5.07, p=0.002). Our study revealed no publication bias in this meta-analysis (all p>0.05)., Conclusion: Our findings indicate that ANP genetic polymorphism and serum ANP levels may contribute to the development of ischemic stroke. Thus, the ANP genetic polymorphism and serum ANP levels could be potential biomarkers for early detection of ischemic stroke.

Bilirubin can cause temporary or permanent sensorineural deafness in newborn babies with hyperbilirubinemia. However, the underlying targets and physiological effects of bilirubin-induced damage in the peripheral auditory system are unclear. Using cochlear functional assays and electron microscopy imaging of the inner ear in neonatal guinea pigs, we show here that bilirubin exposure resulted in threshold elevation in both compound action potential (CAP) and auditory brainstem response (ABR), which was apparent at 1 hr and peaked 8 hr after drug administration. The threshold elevation was associated with delayed wave latencies and elongated interwave intervals in ABR and CAP. At 72 hr postinjection, these measures returned to control levels, except for the CAP amplitude. Cochlear microphonics remained unchanged during the experiment. Morphological abnormalities were consistent with the electrophysiological dysfunction, revealing fewer auditory nerve fibers (ANFs) in the basal turn, myelin sheath lesions of spiral ganglion neurons (SGNs) and ANFs, and loss of type 1 afferent endings beneath inner hair cells (IHCs) without loss of hair cells at 8 hr posttreatment. Similar to the electrophysiological findings, morphological changes were mostly reversed 10 days after treatment, except for the ANF reduction in the basal turn. These results suggest that hyperbilirubinemia in neonatal guinea pigs impaired auditory peripheral neuromechanisms that targeted mainly the IHC synapses and the myelin sheath of SGNs and their fibers. Our observations indicate a potential connection between hyperbilirubinemia and auditory neuropathy., (Copyright © 2012 Wiley Periodicals, Inc.)

It is well known that ethacrynic acid (EA) can potentiate the ototoxicity of aminoglycoside antibiotics (AmAn) such as kanamycin (KM), if they were applied at the same time. Currently, to create the model of EA-KM-induced cochlear lesion in rats, adult rats received a single injection of EA (75 mg/kg, intravenous injection), or followed immediately by KM (500 mg/kg, intramuscular injection). The hearing function was assessed by auditory brainstem response (ABR) measurement in response to click and/or tone bursts at 4, 8, 12, 16, 20, 24, and 32 kHz. The static microcirculation status in the stria vascularis after a single EA injection was evaluated with eosin staining. The pathological changes in cochlear and vestibular hair cells were also quantified after co-administration of EA and KM. After a single EA injection, blood flow in vessels supplying the stria vascularis rapidly diminished. However, the blood supply to the cochlear lateral wall partially recovered 5 h after EA treatment. Threshold changes in ABR were basically parallel to the microcirculation changes in stria vascularis after single EA treatment. Importantly, disposable co-administration of EA and KM resulted in a permanent hearing loss and severe damage to the cochlear hair cells, but spared the vestibular hair cells. Since the cochlear lateral wall is the important part of the blood-cochlea barrier, EA-induced anoxic damage to the epithelium of stria vascularis may enhance the entry of KM to the cochlea. Thus, experimental animal model of selective cochlear damage with normal vestibular systems can be reliably created through co-administration of EA and KM.

Objective: To explore the pattern of hair cell injury and expression of P53 apoptosis protein in intensive impulse noise injured cochlear hair cells in guinea pigs., Methods: Twelve adult guinea pigs were exposed to a series of 40 pairs of impulse noise (2 second intervals) at the intensity of 168 dB (SPL). Animals were terminated at 3, 6 and 12 hours after noise exposure, respectively. Cochlear surface preparations were performed with a double staining of FITC-conjugated phalloidin and propidium iodide for the observations of the stereocilia and the nucleus. P53 immunochemical staining was also performed 12 hours post-noise exposure to observe if there was expression of p53 protein in injured hair cells. Results Three hours after noise exposure, the outer hair cells at the end of basal turn and beginning of second turn were destroyed first with a character of nuclear condensation. Six hours post-noise exposure, many hair cells in the center of damage region had nuclear fragmentations, and the damaging area expanded towards to basal turn and apical turn. Twelve hours after noise exposure, the nucleus in most outer hair cells and inner hair cells at the region of damage center were missing. The nuclear condensation and fragmentation were appeared in hair cells in both sides of the center region of degeneration. P53 immunoreactive products were also found in damaged hair cells, not only in the central damage area, but also in the basal turn and the third turn., Conclusions: Intensive impulse noise resulted in apoptosis of cochlear hair cells that initiated between the end of basal turn and the beginning of second turn. Hair cell degeneration spread to basal and third turn along the basilar membrane. P53 may play an important role in impulse noise induced-hair cell apoptosis.

Several studies have investigated whether the CCR2-Val64Ile polymorphism affects susceptibility to human immune deficiency virus type-1 (HIV-1), with inconclusive results. Here, we performed a meta-analysis of the literature aiming to clarify the relationship between the polymorphism of CCR2-Val64Ile and the risk of HIV-1 infection. Twelve studies with a total of 6,599 patients, including infants, were selected for inclusion in the analysis. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were assessed after the collected data were pooled for analysis. The risk estimates (OR) of HIV-1 infection were calculated in a homozygote comparison (OR=1.10, 95% CI 0.79-1.53), a heterozygote comparison (OR=0.98, 95% CI 0.70-1.37), a dominant model (OR=1.06, 95% CI 0.77-1.47) and a recessive model (OR=0.98, 95% CI 0.77-1.27, by random effects model) from among the total population. Taking into account the effect of sample size, ethnicity and control population, further stratified analyses were performed. The results showed no statistically significant difference in any genetic model, with the exception of the sub-analysis of mixed ethnicity (OR=0.33, 95% CI 0.11‑0.98) using heterozygote comparison. The meta-analysis clarified that the CCR2-Val64Ile polymorphism has no effect on susceptibility to HIV-1 infection in the total population.

Objective: To study the effects of Erlong Zuoci Pill (, ELZCP) and its disassembled: prescriptions on gentamicin (GM)-induced ototoxicity model in vitro., Methods: After the spiral organ of cochleae: of newborn mice (postnatal days: 2-3) cultured for 24 h, GM alone or combined with water extracting-alcohol precipitating solution of ELZCP or with its disassembled prescriptions was added. Hair cells were observed under a fluorescence microscope after TRITC-phalloidin staining, and the cochlear hair cell loss rate was calculated by counting the whole cochlear hair cells and analyzed by whole cochlear hair cells analyzing software., Results: GM induced cochlear outer hair cells (OHCs) and inner hair cells (IHCs) injuries in a dose-dependent manner, and they were significantly different as compared with those in the normal control group (P<0.05, P<0.01). ELZCP at the concentration of 0.003-3 mg/mL could decrease the hair cells loss induced by the 0.3 mmol/L GM (P<0.05, P<0.01), the effects was in a dose-dependent manner, and the concentration of 0.3 mg/mL showed the optimal protective effect. For the ELZCP disassembled prescriptions, Liuwei-Dihuang could decrease OHC loss rate than that in the 0.3 mmol/L GM model group (P<0.05), but the OHC loss rate was still higher than that in the ELZCP group (P<0.01), which indicated that the protective effect of hair cells by Liuwei-Dihuang was not better than that of ELZCP. Poria decreased OHC loss rate from 72.1 % +/-3.7 % to 58.8 %+/- 8.2 % (P<0.05)., Conclusions: ELZCP could play a role in antagonizing the injury of cochlear hair cells induced by GM ototoxicity,: and its disassembled prescriptions, Liuwei-Dihuang was the main component to protect the cochlear hair cells from GM-induced ototoxicity, and Magnetitum combined with Radix Bupleurui could strengthen the action of the whole prescription; Poria could reduce GM-induced OHC loss.

Objective: To evaluate if caspase pathway was involved in streptomycin-induced cell apoptosis in cochlear hair cells., Methods: F344 rats at postnatal day 3 or 4 were used for the study in cochlear organotypic cultures. The cochlear basilar membrane was micro-dissected out and cultured overnight, and then treated with 1 mmol/L streptomycin for 24 hours. Before the termination, the activity of caspase-8, 9 or 6 were detected with FAM-peptide-FMK labeled caspase-8, 9 or 6, respectively. The stereocilia and cuticular plate of hair cells were stained with TRITC conjugated phalloidin, and the nuclei were stained with Topro-3 DNA probe. The specimens were observed and photographed under confocal fluorescent microscope., Results: Streptomycin with 1 mmol/L causes about 80% cochlear hair cells missing in the basal turn and 10% hair cell loss in the apex. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in most cochlear hair cells, and the caspase-8, caspase-9 and caspase-6 were greatly activated., Conclusions: Apoptosis is involved in the cochlear hair cells death induced by Streptomycin in vitro. The caspase activities in upstream and downstream are maybe the major apoptotic pathway.

Objective: To explore the quantitative relationship between the reduction of distortion product otoacoustic emission (DPOAE) and the percentage of outer hair cell loss., Methods: Coadministration of cisplatin (0.2 mg/kg) and ethacrynic acid (40 mg/kg) were used to establish a cochlear lesion model in chinchillas. DPOAE was measured before and 1 week, 2 weeks, and 3 weeks later respectively after cisplatin and ethacrynic acid treatment. Animals were terminated 3 weeks after the treatment. Cochlear surface preparations were performed, and the cochlear hair cells were counted through entire length of the cochlea. The correlation between DPOAE reduction and outer hair cell missing was analyzed using Pearson correlation analysis., Results: Cisplatin and ethacrynic acid treatment induced cochlear hair cell lesion that the outer hair cell loss in the cochlea developed in a stereotypic pattern; damage began in the base of the cochlea and progressed towards the apex. Reduction of DPOAE was relatively consistent with outer hair cells loss. On the average, 1% outer hair cells loss may result in 0.24 dB reduction in DPOAE levels. Pearson analysis showed a positive correlation between the reduction in DPOAE and missing of outer hair cells (r = 0.796, P < 0.05)., Conclusions: It may be helpful to evaluate missing percentage of outer hair cells from reduction in DPOAE levels.

Objective: To investigate the ototoxic effects of streptomycin in vestibular organotypic cultures., Methods: F344 rats with age at postnatal day three or four were used for this study. The maculae of saccule and utricle were routinely dissected out and cultured with serum-free medium containing various dose of streptomycin for 24 hours. The ciliary turf of vestibular hair cells was stained with fluorescent phalloidin, and its nucleus was stained with to pro-3 DNA probe. The vestibular hair cells were quantitatively counted and photographed under confocal fluorescent microscope., Results: Morphological feature of vestibular hair cells were good in normal control cultures. However, the density of hair cells was decreased in evidence with increase of streptomycin sulfate concentrations. Twenty-four hours after streptomycin cultures, 0.25 mmol/L streptomycin caused a 10% hair cell missing, 50% hair cell loss from 1 mmol/L streptomycin treatment, and more than 75% hair cells gone post-3 mmol/L streptomycin cultures. After streptomycin treatment, the nuclear shrinkage and fragmentation were found in vestibular hair cells, whereas the vestibular supporting cells were normal., Conclusion: Streptomycin induced-vestibular hair cells lesion was in a dose dependent manner with nuclear shrinkage and fragmentation. This may suggest that streptomycin leads vestibular hair cell die through apoptosis.

Gonadectomized and intact adult C57BL/6J (B6) mice of both sexes were exposed for 12h nightly to an augmented acoustic environment (AAE): repetitive bursts of a 70dB SPL noise band. The high-frequency AAE (HAAE) was a half-octave band centered at 20kHz; the low-frequency AAE (LAAE) was a 2-8kHz band. The effects of sex, gonadectomy, and AAE treatment on genetic progressive hearing loss (a trait of B6 mice) were evaluated by obtaining auditory brainstem response (ABR) thresholds at ages 3-, 6-, and 9-months. At 9-months of age, hair cell counts (cytocochleograms) were obtained, and morphometric measures of the anteroventral cochlear nucleus (AVCN) were obtained. LAAE treatment caused elevation in ABR thresholds (8-24kHz), with the highest thresholds occurring in intact females. LAAE treatment caused some loss of outer hair cells in the basal half of the cochlea (in addition to losses normally occurring in B6 mice), with intact females losing more cells than intact males. The loss of AVCN neurons and shrinkage of tissue volume that typically occur in 9-month-old B6 mice was lessened by LAAE treatment in intact (but not gonadectomized) male mice, whereas the degenerative changes were exacerbated in intact (but not gonadectomized) females. These LAAE effects were prominent in, but not restricted to, the tonotopic low-frequency (ventral) AVCN. HAAE treatment resulted in some loss of neurons in the high-frequency (dorsal) AVCN. In general, LAAE treatment plus male gonadal hormones (intact males) had an ameliorative effect whereas HAAE or LAAE treatment plus ovarian hormones (intact females) had a negative effect on age-related changes in the B6 auditory system.

The goals were to study the physiological effects of auditory nerve myelinopathy in chinchillas and to test the hypothesis that myelin abnormalities could account for auditory neuropathy, a hearing disorder characterized by absent auditory brainstem responses (ABRs) with preserved outer hair cell function. Doxorubicin, a cytotoxic drug used as an experimental demyelinating agent, was injected into the auditory nerve bundle of 18 chinchillas; six other chinchillas were injected with vehicle alone. Cochlear microphonics, compound action potentials (CAPs), inferior colliculus evoked potentials (IC-EVPs), cubic distortion product otoacoustic emissions and ABRs were recorded before and up to 2 months after injection. Cochleograms showed no hair cell loss in any of the animals and measures of outer hair cell function were normal (cubic distortion product otoacoustic emissions) or enhanced (cochlear microphonics) after injection. ABR was present in animals with mild myelin damage (n = 10) and absent in animals with severe myelin damage that included the myelin surrounding spiral ganglion cell bodies and fibers in Rosenthal's canal (n = 8). Animals with mild damage had reduced response amplitudes at 1 day, followed by recovery of CAP and enhancement of the IC-EVP. In animals with severe damage, CAP and IC-EVP thresholds were elevated, amplitudes were reduced, and latencies were prolonged at 1 day and thereafter. CAPs deteriorated over time, whereas IC-EVPs partially recovered; latencies remained consistently prolonged despite changes in amplitudes. The results support auditory nerve myelinopathy as a possible pathomechanism of auditory neuropathy but indicate that myelinopathy must be severe before physiological measures are affected.

Gonadectomized and surgically intact adult C57BL/6J (B6) mice of both sexes were exposed for 12h nightly to a high-frequency augmented acoustic environment (AAE): repetitive bursts of a half-octave noise band centered at 20 kHz, 70 dB SPL. The effects of sex, gonadectomy, and AAE treatment on genetic progressive hearing loss (exhibited by B6 mice) were evaluated by obtaining auditory brainstem response thresholds at ages 3-, 6-, and 9-months; hair cell counts (cytocochleograms) were obtained at 9 months. A sex difference in the rate of genetic progressive hearing loss in B6 mice (observed by earlier studies) was confirmed, with females exhibiting a faster rate of threshold elevations and more severe loss of hair cells at age 9 months. Gonadectomy had no consistent effects on the rate or severity of hearing loss in non-exposed mice of either sex. An unexpected finding was that the high-frequency AAE treatment caused additional ABR threshold elevations and hair cell loss. In an earlier study, the same high-frequency AAE treatment on DBA/2J mice ameliorated hearing loss. The most severe AAE-induced losses occurred in surgically intact females, suggesting a potentiating effect of ovarian hormone(s).

DBA/2J (D2) mice, which exhibit very early progressive sensorineural hearing loss, were treated for 12h nightly with an augmented acoustic environment (AAE) initiated before the onset of hearing. The AAE consisted of repetitive bursts of a 70 dB sound pressure level, half-octave noise band centered at 20 kHz (i.e. low frequencies were excluded). At 55 days of age, AAE-treated mice, compared to control mice, exhibited less elevation of auditory brainstem response thresholds for tone frequencies from 16 to 32 kHz and fewer missing outer hair cells in the high-frequency tonotopic region of the cochlea. The dorsal region of their anteroventral cochlear nucleus (most strongly stimulated by the AAE) was larger, had more surviving neurons, and larger neurons than those of untreated control mice. These and previous findings using an AAE band containing lower frequencies indicate that AAE treatment effects are frequency-related. The findings provide support for the hypothesis that the beneficial effects of AAE treatment on the cochlea are associated with increased physiological activity evoked by the AAE, and the central AAE effects result from increased AAE-evoked neural activity and a healthier cochlea providing the auditory input.

Amino acid concentrations were measured in the cochlear nucleus for a group of 20 chinchillas: four each of control and 4, 8, 29, and 85 days after treatment with the ototoxic anti-tumor drug carboplatin (100 mg/kg, i.p.). The treated chinchillas showed various extents of inner hair cell loss, generally more complete at longer survival times, but little loss of outer hair cells. Aspartate concentration in rostral anteroventral cochlear nucleus (AVCN) showed a decline to 28% less than the control value at 29 and 85 days after treatment, whereas glutamate concentration showed little change through 29 days, then dropped by 22% at 85 days after treatment. In caudal posteroventral cochlear nucleus (PVCN), the aspartate concentration decreased by 32% at 29 days, in animals with significant inner hair cell loss, and 48% at 85 days after treatment, while the glutamate concentration showed no decrease through 29 days and 40% decrease at 85 days. The concentration of gamma-aminobutyrate (GABA) was about 18% lower than control in caudal PVCN at all survival times. Significant correlations were found between the proportion of inner hair cells remaining and glutamate and aspartate concentrations in PVCN and AVCN, but not GABA or other amino acids.

Substance P (SP), a member of the tachykinin family of neurotransmitters and neuromodulators, has been identified on spiral ganglion neurons (SGNs) in the inner ear; however, its high affinity receptor, neurokinin-1 (NK1), has not been identified and the physiological effects of SP on SGNs are not well understood. To address these issues, immunolabeling, RT-PCR, Western blots and whole-cell patch-clamp recordings were made from SGNs in P0-P5 mouse cochlear organotypic cultures. The NK1 receptor was detected on SGNs by immunocytochemistry, the protein was detected in cochlear tissues by Western blots, and the mRNA for the NK1 receptor was also found in cochlear tissues of postnatal mice (P2) by RT-PCR. Application of SP (1 to 25 microM) significantly increased the latency of SGN action potentials (APs) (mean increase 7.8 +/- 4 ms; 25 microM of SP), prolonged the duration of the action potential and made the resting potential (RP) more positive (mean 9.0 +/- 7 mV) relative to normal values (-54 +/- 6 mV). SP (1 to 25 microM) also suppressed voltage-activated potassium currents (IK+) and calcium currents (ICa2+). Puffing 25 microM of SP onto SGNs suppressed IK+ by 43 +/- 9% (n = 7) and ICa2+ by 40.6 +/- 5.6% (n = 7); both currents recovered when SP was washed out. A SP antagonist blocked the SP-induced suppression of IK+ and ICa2+. These results indicate that SP acting through NK1 receptors can have direct neuromodulatory effects on SGNs., (Copyright 2004 Elsevier B.V.)

It is generally believed that mechanical amplification by cochlear hair cells is necessary to enhance the sensitivity and frequency selectivity of hearing. In the mammalian ear, the basis of cochlear amplification is believed to be the voltage-dependent electromotility of outer hair cells (OHCs). The avian basilar papilla contains tall and short hair cells, with the former being comparable to inner hair cells, and the latter comparable to OHCs, based on their innervation patterns. In this study, we sought evidence for somatic electromotility by direct measurements of voltage-dependent length changes in both tall and short hair cells at nanometre resolution. Microchamber and whole-cell voltage-clamp techniques were used. Motility was measured with a photodiode-based measurement system. Non-linear capacitance, an electrical signature of somatic motility, was also measured to complement motility measurement. Significantly, chick hair cells did not exhibit somatic motility nor express non-linear capacitance. The lack of somatic motility suggests that in avian hair cells the active process resides elsewhere, most likely in the hair cell stereocilia.

Carboplatin, a drug widely used against solid head and neck tumors, selectively destroys cochlear inner hair cells and type I auditory nerve fibers in chinchilla. This should affect neurotransmitter chemistry, involving amino acids, where the type I auditory nerve fibers terminate in the cochlear nucleus. Using microdissection combined with high-performance liquid chromatography, amino acid concentrations were mapped in the cochlear nuclei of chinchillas injected intraperitoneally 6-8 weeks earlier with 100 mg/kg carboplatin and in those of control animals. Glutamate concentrations were 23% lower in the anteroventral cochlear nucleus (AVCN) and 40% lower in the posteroventral cochlear nucleus (PVCN) of carboplatin-injected chinchillas as compared to controls, while aspartate concentrations were 18% lower in AVCN and 27% lower in PVCN. Using a fluorometric assay, activities of glutaminase, an enzyme which catalyzes glutamate synthesis, were 30% lower in AVCN and 38% lower in PVCN of carboplatin-injected chinchillas. Concentrations of glutamine, gamma-aminobutyrate, and glycine were also lower in some ventral and dorsal cochlear nucleus regions of treated animals. These changes probably result mainly from both primary and later effects of reduced type I auditory nerve fiber input to the cochlear nucleus.

Current procedures for treating hemifacial spasm control its symptoms but the condition is often complicated by facial weakness. We undertook this study using a rat facial nerve model to determine whether peripheral glycerol injection could be recommended as a feasible and safe technique in the management of hemifacial spasm. The animals were given a perineural application of glycerol on the facial nerve and killed at 3 days or 2 or 4 weeks after operation. The facial nerves were then examined with light and electron microscopy. Our results show that myelin disintegration and axolysis occurred after glycerol application and both the myelinated and unmyelinated fibers were affected at random, although the most striking histological changes were seen in the myelinated fibers. No evidence of persistent facial weakness in the animals was associated with this method.

Kainic acid (KA) selectively damages afferent synapses that innervate, in chickens, mainly tall hair cells. To better understand the nature of KA-induced excitotoxic damage to the cochlear afferent neurons, KA, at two different concentrations (0.3 or 5 mM), was injected directly into the inner ear of adult chickens. Pathologic changes in the afferent nerve ending and cell body were evaluated with light and transmission electron microscopy at various time points after KA application. The compound action potential (CAP) and cochlear microphonic (CM) potential were recorded to monitor the physiologic status of the afferent neurons and hair cells, respectively. Hair cell morphology and function were essentially normal after KA treatment. However, afferent synapses beneath tall hair cells were swollen within 30 minutes after KA at both low (KA-L) and high (KA-H) doses. In the KA-L group, the swelling disappeared within 1 day and the morphology of the postsynaptic region returned to near normal condition. In the KA-H group, by contrast, the vacant region beneath tall hair cells remained evident even 20 weeks after KA. The number of cochlear ganglion neurons in the KA-H group decreased progressively from 1 to 8-20 weeks, whereas hair cells in the basilar papilla remained morphologically intact out to 20 weeks after KA. There was no significant change in neuron number in the KA-L group. Temporal changes in the CAP amplitude paralleled the anatomic changes, although the CAP only partially recovered. These results suggest that KA induces partially reversible damage to cochlear afferent neurons with low KA concentration; above this level, KA triggers irreversible, progressive neurodegeneration., (Copyright 2001 Wiley-Liss, Inc.)

Acoustic overstimulation produces many anatomical, biochemical and physiological changes in the inner ear. However, the changes in gene expression that underlie these biological changes are poorly understood. Our approach to investigating this problem is to use gene microarrays to measure the changes in gene expression in the chinchilla inner ear following a 3 h or 6 h noise exposure (95 dB SPL, 707-1414 Hz). This noise exposure causes a temporary threshold shift (~40 dB) and a temporary reduction in distortion product otoacoustic emissions (DPOAE), but no permanent hearing loss or hair cell loss. Here, we present data showing (1) the suitability of mouse and human complementary DNA (cDNA) clones for detecting chinchilla cochlear gene transcripts, and (2) the change in cochlear gene transcripts in noise exposed chinchillas. Chinchilla cochlear transcript probes exhibited strong and discrete signals on both mouse and human cDNA filter arrays. Since the strongest hybridization occurred with mouse clones, mouse cDNA microarrays were used to study noise-induced changes in gene expression. Chinchilla cDNA probes were differentially labelled with Cy3 (control) or Cy5 (noise exposed) by random primed synthesis, hybridized to 8750 mouse cDNAs arrayed on microscope slides and analysed by laser fluorescent microscopy. Several classes of genes exhibited time-dependent up regulation of transcription, including those involved in protein synthesis, metabolism, cytoskeletal proteins, and calcium binding proteins. The results are discussed in relationship to previous studies showing noise-induced changes in structural proteins, calcium binding proteins, metabolic enzymes and membrane bound vesicles.

Reactive oxygen species (ROS) and oxidative stress have been implicated in cochlear injury following loud noise and ototoxins. Genetic mutations that impair antioxidant defenses would be expected to increase cochlear injury following acute insults and to contribute to cumulative injury that presents as age-related hearing loss. We examined whether genetically based deficiency of cellular glutathione peroxidase, a major antioxidant enzyme, increases noise-induced hearing loss in mice. Two-month-old "knockout" mice with a targeted inactivating mutation of the gene coding for glutathione peroxidase (Gpx1) and wild type controls were exposed to broadband noise for one hour at 110 dB SPL. Auditory brainstem response (ABR) thresholds at test frequencies ranging from 5 to 40 kHz were obtained two and four weeks after exposure to determine the stable permanent component of the hearing loss. Depending on test frequency, (compared with controls) Gpx1 knockout mice showed up to 16 dB higher ABR thresholds prior to noise exposure, and up to 15 dB greater noise-induced hearing loss, compared with normal control. Within the cochlear base, there was also a significant contribution of the knockout to inner and outer hair cell loss, as well as nerve fiber loss. Our results support a link between genetic impairment of antioxidant defenses, vulnerability of the cochlea injury, and cochlear degeneration. Such impairment produces characteristics expected of some mutations associated with age-related hearing loss and offers one possible mechanism for their action.

The effects of a combination of two antioxidant compounds were studied in a chinchilla model of noise-induced hearing loss. After obtaining baseline hearing thresholds using inferior colliculus evoked potentials, chinchillas were exposed for 6 h to octave band noise centered at 4 kHz (105 dB SPL). Post-noise thresholds were obtained 1 h after the noise exposure, and then animals received either saline or salicylate and N-L-acetylcysteine combination. Another group received antioxidant treatment 1 h prior to noise. Hearing was tested at 1, 2 and 3 weeks post-noise. Subsequently, the cochleae were harvested, and cytocochleograms were prepared. There was a 20-40 dB SPL threshold shift at 3 weeks for tested controls. Permanent threshold shifts (PTS) were significantly reduced (P<0.05) to approximately 10 dB for the pre-treatment group at week 3. The PTS for the post-treatment group at week 3 was similar to the pre-treatment group at 1 and 2 kHz (0-10 dB) but was intermediate between the control and pre-treatment groups at 4 and 8 kHz (23 dB). Animals pre-treated with antioxidant had a significant reduction in hair cell loss but those post-treated with antioxidant had no protection from hair cell loss. These findings demonstrate the feasibility of reduction of noise-induced hearing loss using clinically available antioxidant compounds.

Carboplatin, a second-generation platinum-based antineoplastic drug, preferentially destroys inner hair cells (IHCs) in the chinchilla while sparing outer hair cells (OHCs). D-Methionine (D-Met), a sulfur-containing amino acid, has been shown to protect hair cells from cisplatin damage in rats, but its ability to protect IHCs from carboplatin damage has not yet been evaluated in the chinchilla. We tested whether D-Met would protect the hair cells in the chinchilla from carboplatin. Animals were divided into two groups: a control group that only received carboplatin (100 mg/kg, i.p.) and an experimental group that received 300 mg/kg D-Met (i.p.) 30 min before carboplatin treatment. Ototoxicity was assessed by measuring the amount of IHC and OHC loss. Average IHC loss in the group treated with D-Met was 62% compared with 84% in the untreated control group. Thus, D-Met causes a statistically significant reduction in IHC loss induced by carboplatin., (Copyright 2000 S. Karger AG, Basel)

The olivocochlear bundle (OCB) has been shown to protect the ear from acoustic trauma induced by continuous noise or tones. The present study examines the OCB's role in the ear's response to impulse noise (150 dB pSPL, 100 impulses, 50 s total exposure duration). Successful section of the OCB was achieved through a posterior parafloccular fossa approach for the right ears of six out of 15 adult chinchillas. The left ears from the same animals served as efferent-innervated controls. Measurements of inferior colliculus evoked potentials (ICPs) showed that the de-efferented ears incurred similar temporary and permanent threshold shifts as the control ears. Twenty days after noise exposure, depressed ICP amplitudes had virtually recovered to pre-values in the control ears whereas those in the de-efferented ears remained significantly depressed. Greater loss of inner hair cells was seen in the de-efferented ears than in the control ears. Both control and de-efferented ears incurred large loss of outer hair cells, with no statistically significant differences between groups. The current data are intriguing, yielding tentative evidence to suggest that inner hair cells of de-efferented ears are more susceptible to impulse noise than those in efferented control ears. In contrast, outer hair cell vulnerability to impulse noise appears to be unaffected by de-efferentation.

The effects of eliminating the olivocochlear bundle (OCB) on cochlear electromechanical properties were examined by measuring cochlear microphonics (CM) and distortion product otoacoustic emissions (DPOAEs) in chronically de-efferented chinchillas. The OCB fibers to the right ears were successfully sectioned in six out of 15 adult chinchillas via a posterior paraflocular fossa approach. At the end of the experiment, these ears were histologically verified as being deprived of both lateral and medial OCB fibers. The opposite (left) ears from the animals served as controls. Following de-efferentation, changes of the inter-modulation distortion components (2f(1)-f(2), f(2)-f(1), 3f(1)-2f(2), 3f(2)-2f(1)) varied, depending on the frequencies and levels of the stimuli. DPOAE amplitudes to low-level stimuli were within the 95% confidence intervals around mean DPOAE amplitudes of the control ears at all the frequencies (1-8 kHz). At high stimulus levels, DPOAE amplitudes increased by 5-20 dB at 1 and 2 kHz while remaining in the normal range at 4 and 8 kHz. In contrast, the CM input/output functions to stimuli from 1 to 8 kHz were significantly reduced by approximately 40-50% at all input levels. The results suggest that the OCB may play a role in modulating electrical properties of the outer hair cells and in reducing the magnitude of cochlear distortion to high-level stimuli.

Kainic acid (KA) is a potent glutamate analog that can temporarily or permanently damage glutamatergic neurons. The purpose of the present study was to determine the short- and long-term effects of KA on chicken otoacoustic emissions and cochlear potentials. A chronic electrode was used to record the compound action potential (CAP), cochlear microphonic (CM), and the slow, positive neural potential (SPNP), a predominantly dc response. The CM, CAP, SPNP, and distortion product otoacoustic emissions (DPOAEs) were recorded before and after infusing 10 microl of a low dose (KA-L, 0.3 mM) or high dose (KA-H, 5 mM) of KA into scala tympani. KA caused a rapid and large reduction in CAP and SPNP amplitude in both the KA-H and KA-L groups; however, the CM and DPOAEs were largely unchanged. The amplitude of the CAP and SPNP in the KA-L group began to recover around 1 week post-KA, but was approximately 50% below normal at 4 weeks post-KA. In contrast, the CAP and SPNP showed no signs of recovery in the KA-H group. The results suggest that KA has no effect on the CM and DPOAEs generated by the hair cells, but selectively damages the CAP generated by the cochlear ganglion neurons. The reduction in the avian SPNP suggests that the response originates in the cochlear afferent neurons, unlike the summating potential (SP) in mammals that is generated in hair cells.

Sound conditioning (pre-exposure to a moderate-level acoustic stimulus) can induce resistance to hearing loss from a subsequent traumatic exposure. Most sound conditioning experiments have utilized long-duration tones and noise at levels below 110 dB SPL as traumatic stimuli. It is important to know if sound conditioning can also provide protection from brief, high-level stimuli such as impulses produced by gunfire, and whether there are differences between females and males in the response of the ear to noise. In the present study, chinchillas were exposed to 95 dB SPL octave band noise centered at 0.5 kHz for 6 h/day for 5 days. After 5 days of recovery, they were exposed to simulated M16 rifle fire at a level of 150 dB peak SPL. Animals that were sound conditioned showed less hearing loss and smaller hair cell lesions than controls. Females showed significantly less hearing loss than males at low frequencies, but more hearing loss at 16 kHz. Cochleograms showed slightly less hair cell loss in females than in males. The results show that significant protection from impulse noise can be achieved with a 5-day conditioning regimen, and that there are consistent differences between female and male chinchillas in the response of the cochlea to impulse noise.

Carboplatin preferentially destroys inner hair cells (IHCs) and type-I spiral ganglion neurons while sparing outer hair cells (OHCs). Loss of IHCs and type-I ganglion cells is associated with a significant reduction of the compound action potential (CAP). However, the cochlear microphonic (CM) potential and distortion product otoacoustic emissions (DPOAEs) remain normal, indicating that the OHCs are functionally intact. In the vestibular system, carboplatin selectively destroys type-I hair cells and their afferent neurons. Damage of type-I vestibular hair cells and their afferent terminals is associated with significant depression of nystagmus induced by cold, caloric stimulation. Histochemical studies revealed a rapid decrease in succinate dehydrogenase (SDH) staining in IHCs soon after carboplatin treatment, and staining intensity remained depressed in surviving IHCs for at least 1 month after carboplatin treatment. These results suggest that carboplatin depresses the metabolic function in surviving IHCs. Several lines of evidence suggest that free radicals may contribute to carboplatin-induced sensory cell damage. Intracochlear infusion of L-buthionine-[S,R]-sulfoximine (BSO), which depletes intracellular glutathione (GSH), increases IHC and OHC loss. Previous in vitro studies have shown that neurotrophin 4/5 (NT-4/5) promotes the survival of spiral ganglion neurons from cisplatin ototoxicity. In vivo perfusion of NT-4/5 promoted the survival of spiral ganglion neurons, but did not protect the hair cells.

The present study examines the recovery of the inner hair cell (IHC)/auditory nerve synapse following cochlear excitotoxicity induced by kainic acid (KA). Three hours after KA treatment, there was massive swelling of type I afferent endings under the IHCs. Five to ten days later, the pattern of IHC innervation appeared to be normal. Distortion-product otoacoustic emissions were normal during the whole experiment. The amplitude of the auditory nerve compound action potential (CAP) was significantly reduced immediately after KA treatment and then recovered over a 30-day period. However, it only took five days for the evoked response from the inferior colliculus (IC) to recover from a substantial depression. In contrast to amplitudes, thresholds for the CAP and IC recovered at the same rate and returned to normal within 5 days after KA. Single auditory nerve fibers were also assessed at various times after the KA treatment. Ten days after KA, these fibers had almost normal thresholds, tuning, spontaneous, and driven discharge rates. The results indicate that (1) excitotoxically damaged cochlear afferent neurons can rapidly regenerate and establish viable synapses with the IHCs, and (2) the central auditory system recovers more rapidly than the periphery.

Reactive oxygen species (ROS) such as superoxide, peroxide and hydroxyl radicals are generated during normal cellular metabolism and are increased in acute injury and in many chronic disease states. When their production is inadequately regulated, ROS accumulate and irreversibly damage cell components, causing impaired cellular function and death. Antioxidant enzymes such as superoxide dismutase (SOD) play a vital role in minimizing ROS levels and ROS-mediated damage. The cytosolic form of Cu/Zn-SOD appears specialized to remove superoxide produced as a result of injury. 'Knockout' mice with targeted deletion of Sod1, the gene that codes for Cu/Zn-SOD, develop normally but show enhanced susceptibility to central nervous system injury. Since loud noise is injurious to the cochlea and is associated with elevated cochlear ROS, we hypothesized that Sod1 knockout mice would be more susceptible to noise-induced permanent threshold shifts (PTS) than wild-type and heterozygous control mice. Fifty-nine mice (15 knockout, 29 heterozygous and 15 wild type for Sod1) were exposed to broad-band noise (4.0-45.0 kHz) at 110 dB SPL for 1 h. Hearing sensitivity was evaluated at 5, 10, 20 and 40 kHz using auditory brainstem responses before exposure and 1, 14 and 28 days afterward. Cu/Zn-SOD deficiency led to minor (0-7 dB) threshold elevations prior to noise exposure, and about 10 dB of additional noise-induced PTS at all test frequencies, compared to controls. The distribution of thresholds at 10 and 20 kHz at 28 days following exposure contained three modes, each showing an effect of Cu/Zn-SOD deficiency. Thus another factor, possibly an additional unlinked gene, may account for the majority of the observed PTS. Our results indicate that genes involved in ROS regulation can impact the vulnerability of the cochlea to noise-induced hearing loss.

The aim of the present study was to examine the role of the olivocochlear system in auditory processing by examining the long-term effects of cochlear de-efferentation on auditory nerve response properties in adult chinchillas. Spontaneous rates, response thresholds, tuning curves, discharge rate-level functions, and adaptation of single auditory nerve fibers were measured in chinchillas with complete cochlear de-efferentation produced by sectioning the olivocochlear bundle in the internal auditory meatus. De-efferentation was verified as successful on the basis of acetylcholinesterase staining of surface preparations of the organ of Corti. Following chronic de-efferentation, there was a striking decrease in spontaneous rate, consistent with earlier observations in cats. In addition, the present study shows that complete de-efferentation results in: (1) increased driven discharge rates and decreased dynamic range of discharge rate-level functions, (2) larger onset-to-steady state ratio of discharge rate at moderate intensities, and (3) a hypersensitive tail of the tuning curve. These effects, largely confined to neurons that were most sensitive to frequencies between 2-8 kHz, indicate that the cochlear efferent system is important in maintaining normal function (e.g., frequency and intensity selectivity) of the auditory periphery by modulating auditory nerve fiber response properties.

In the C57 mouse strain, loss of sensory hair cells (HCs) begins during early adulthood, starting in the base of the cochlea and progressing toward the apex as aging continues. In contrast, the CBA mouse strain exhibits no significant cochlear histopathology until relatively late in life. These strain and age differences may be related to differences in cochlear energy metabolism. To examine this possibility, we used dehydrogenase and glycogen histochemistry to evaluate the metabolic capacities of HCs and stria vascularis (SV) in cochleas of C57 and CBA mice. Reaction product density was quantified and compared as a function of strain (1.5-month-old C57 mice vs. CBA mice) and age (CBA mice, 1.5, 18 and 36 months). Young C57 mice had significantly less HC dehydrogenase activity than CBA mice of any age, lower HC glycogen levels than 18-month-old CBA mice and lower SV glycogen levels than 18- or 36-month-old CBA animals. Within the CBA strain, HC dehydrogenase activity decreased significantly between 1.5 and 18 months of age, while glycogen levels in both HCs and SV increased over the same time period. Between 18 and 36 months, HC dehydrogenase activity and SV glycogen levels remained stable. The results show that there are significant age-related changes in energy metabolism in the inner ear of CBA mice that are correlated with age-related hearing loss. Genetically determined deficits in cochlear metabolic capacity in C57 mice could be linked to the early onset of hearing loss in this strain.

The relative contribution of inner hair cells (IHCs) and outer hair cells (OHCs) to the production of the summating potential (SP) is unresolved in the literature. Since OHCs in the base of the cochlea have been reported to produce little dc receptor potential except at very high sound pressure levels [I. J. Russell and P. M. Sellick, J. Physiol (London) 284, 261-290 (1983)], the IHCs appear to be the dominant source of the SP. However, results of intracochlear recordings are conflicting, although deriving from measurements in different turns of the cochlea [e.g., I. J. Russell and P. M. Sellick, J. Physiol. (London) 284, 261-290 (1983) versus P. Dallos and M. A. Cheatham, Sensory Transduction (1992)]. To determine which type of hair cells is the dominant source of the SP recorded at the round window, we used carboplatin to selectively destroy IHCs or a combination of IHCs and OHCs in the chinchilla. Related work, using measurements of distortion product otoacoustic emissions and cochlear potentials to assess the functional status of the OHCs served to validate this animal model [Trautwein et al., Hearing Res. 96(1-2), 71-82 (1996)]. The SP, cochlear microphonic (CM), and compound action potential (CAP) were recorded from the round window, and cochleograms were determined using well-established histological methods. The results were reasonably distinctive among three groups of ears--control (from untreated normal chinchillas), IHC-loss (extensive IHC loss with minor or no loss of OHCs), and IHC-OHC loss (total IHC loss plus extensive loss of OHCs over the basal half of the cochlea). Ears of chinchillas in the IHC loss group had a decrease of over 50% in SP output compared to control ears with the exact reduction depending somewhat upon the stimulus conditions. Ears in the IHC + OHC loss group, nevertheless, showed even further reduction in SP output which was clearly attributable to destruction of OHCs in the cochlear base. It was concluded that, although the IHCs are responsible for a greater contribution of dc-receptor potential to the SP recorded at the round window, a significant contribution is made by the OHCs, as well. The results suggest, specifically, that the round window "sees" SP output roughly in inverse proportion to the IHC:OHC. Lastly, the complexity of SP production, as recorded from the round window, precludes a completely straightforward interpretation of the SP:CAP in clinical ECochG.

The role of the inner hair cells (IHCs) in generating the cochlear summating potentials (SP) was assessed by measuring SP, cochlear nerve action potentials (CAP), cochlear microphonics (CM) and 2f1-f2 distortion product otoacoustic emissions (DPOAEs) in 15 chinchillas with either acute chemical de-afferentation, accomplished by applying kainic acid to the round window, or surgical de-afferentation and basal IHC loss, which developed within two months after sectioning the auditory nerve. In the auditory nerve sectioned ears, type I ganglion cells disappeared whereas most, if not all, type II ganglion cells were still present. Histological analysis of surface preparations and sections through the modiolus verified the de-afferentation in both models and showed a large IHC loss at the base of the cochlea in the ears with the auditory nerve sectioned while other structures of the cochlea remained intact. Unoperated (left) ears of 9 animals served as controls. CM and DPOAEs were normal in all ears whereas the CAP was substantially depressed in de-afferented ears. Comparisons among the SP input-output functions suggest that (1) the IHCs are the major generator of SP recorded from the round window in chinchilla, in particular at low to moderate stimulus levels, (2) the SP recorded from the round window largely reflects the responses from hair cells at the base of the cochlea, and (3) kainic acid results in an increase of SP amplitude to high-level stimuli whereas the SP to low- to moderate-level stimuli remains in the normal range.

The role of the olivocochlear bundle (OCB) in modulating noise-induced permanent injury to the auditory periphery was studied by completely sectioning the OCB fibers in chinchillas and exposing the animals while awake to a broad-band noise at 105 dB SPL for 6 h. Outer hair cell (OHC) function was assessed by measuring 2f1-f2 distortion product otoacoustic emissions (DPOAE) at frequencies from 1.2 to 9.6 kHz and cochlear microphonics (CM) at frequencies from 1 to 8 kHz. As a result of de-efferentation, the CM was decreased but the DPOAEs were unchanged in de-efferented ears as compared with efferented control and sham-operated ears. Following noise exposure, the ears that were de-efferented showed significantly more depression of DPOAE input/output functions and greater decrement of CM amplitude. The differences between de-efferented and efferent-innervated ears were evident across all the frequencies. The cochlear lesions of the OHCs reflected by traditional cytocochleograms, however, were minimal in both efferented and de-efferented ears. The results indicate that cochlear de-efferentation decreases the CM in chinchilla and increases the ear's susceptibility to noise-induced permanent hearing damage. More importantly, de-efferentation increases susceptibility at low frequencies as well as high frequencies.

The influence to endocochlear potential (EP), cochlear microphonics (CM) and summating potential (SP), the ultrastructural changes of the cochlea and the survival time of hair cells in guinea pigs during anoxia and suffocation were investigated. We found that: 1) The stria vascularis was much more sensitive to anoxia than hair cells. 2) 85% CM was highly sensitive to anoxia, and 15% kept stable until the destruction of outer hair cells. 3) -SP turned into +SP after anoxia. 4) EP was related to the destruction of outer hair cells and the disappearance of CM.

A comparison of stereociliary changes at different post-exposure intervals in ears with temporary and permanent hearing losses has been made. Twenty guinea pigs were exposed to either 110 dB SPL broadband white noise for 30 min (N = 10) or 120 dB SPL white noise for 150 min (N = 10). The recovery patterns for threshold shifts for both groups were systematically assessed at regular post-exposure intervals for 80 days, using the auditory cortex evoked response to tone bursts between 0.5 and 8kHz. Thirty-two animals that had been exposed to the same noise at either 110 dB for 30 min (N = 16) or 120 dB for 150 min (N = 16) were decapitated for scanning electron microscopic examination at the same post-exposure intervals. The threshold shifts induced by 110 dB noise were reversible while those induced by 120 dB were generally irreversible, although extreme variabilities existed among the animals. In the acute TTS ears, damage was confined to the third row of OHCs, where only the tips of the stereocilia were affected. Neither discontinuity of cuticular plate nor expelled cytoplasm was found in these cells. In the lesions of PTS, either all the three rows of OHCs or the IHCs and the first row of OHCs were involved. The entire length of the stereocilia, more severe in the lower part was always damaged. Expelled cytoplasm and fusion between stereocilia were frequently seen. In the chronic TTS ears, no abnormalities of stereocilia were found while in the PTS ears, a complete absence of the organ of Corti was noticed. The results of the present study clearly suggest that the status of the lower part of the stereocilia and the continuity of the cuticular plate play an important role in determining the reversibility of threshold shifts.