Deferoxamine protects cochlear hair cells and hair cell-like HEI-OC1 cells against tert-butyl hydroperoxide-induced ototoxicity.

Oxidative stress is the common mechanism of sensorineural hearing loss (SNHL) caused by many factors, such as noise, drugs and ageing. Here, we used tert-butyl hydroperoxide (t-BHP) to cause oxidative stress damage in HEI-OC1 cells and in an in vitro cochlear explant model. We observed lipid peroxidation, iron accumulation, mitochondrial shrinkage and vanishing of mitochondrial cristae, which caused hair cell ferroptosis, after t-BHP exposure. Moreover, the number of TUNEL-positive cells in cochlear explants and HEI-OC1 cells increased significantly, suggesting that t-BHP caused the apoptosis of hair cells. Administration of deferoxamine (DFOM) significantly attenuated t-BHP-induced hair cell loss and disordered hair cell arrangement in cochlear explants as well as HEI-OC1 cell death, including via apoptosis and ferroptosis. Mechanistically, we found that DFOM treatment reduced t-BHP-induced lipid peroxidation, iron accumulation and mitochondrial pathological changes in hair cells, consequently mitigating apoptosis and ferroptosis. Moreover, DFOM treatment alleviated GSH depletion caused by t-BHP and activated the Nrf2 signalling pathway to exert a protective effect. Furthermore, we confirmed that the protective effect of DFOM mainly depended on its ability to chelate iron by constructing Fth1 knockout (KO), TfR1 KO and Nrf2 KO HEI-OC1 cell lines using CRISPR/Cas9 technology and a Flag-Fth1 (overexpression) HEI-OC1 cell line using the FlpIn™ System. Our findings suggest that DFOM is a potential drug for SNHL treatment due to its ability to inhibit apoptosis and ferroptosis by chelating iron and scavenging reactive oxygen species (ROS).

Functional to structural plasticity in unilateral sudden sensorineural hearing loss: neuroimaging evidence.

A cortical plasticity after long-duration single side deafness (SSD) is advocated with neuroimaging evidence while little is known about the short-duration SSDs. In this case-cohort study, we recruited unilateral sudden sensorineural hearing loss (SSNHL) patients and age-, gender-matched health controls (HC), followed by comprehensive neuroimaging analyses. The primary outcome measures were temporal alterations of varied dynamic functional network connectivity (dFNC) states, neurovascular coupling (NVC) and brain region volume at different stages of SSNHL. The secondary outcome measures were pure-tone audiograms of SSNHL patients before and after treatment. A total of 38 SSNHL patients (21 [55%] male; mean [standard deviation] age, 45.05 [15.83] years) and 44 HC (28 [64%] male; mean [standard deviation] age, 43.55 [12.80] years) were enrolled. SSNHL patients were categorized into subgroups based on the time from disease onset to the initial magnetic resonance imaging scan: early- (n = 16; 1-6 days), intermediate- (n = 9; 7-13 days), and late- stage (n = 13; 14-30 days) groups. We first identified slow state transitions between varied dFNC states at early-stage SSNHL, then revealed the decreased NVC restricted to the auditory cortex at the intermediate- and late-stage SSNHL. Finally, a significantly decreased volume of the left medial superior frontal gyrus (SFGmed) was observed only in the late-stage SSNHL cohort. Furthermore, the volume of the left SFGmed is robustly correlated with both disease duration and patient prognosis. Our study offered neuroimaging evidence for the evolvement from functional to structural brain alterations of SSNHL patients with disease duration less than 1 month, which may explain, from a neuroimaging perspective, why early-stage SSNHL patients have better therapeutic responses and hearing recovery.

Comparative transcriptome profiling reveals RNA splicing alterations and biological function in patients exposed to occupational noise.

Genetic factors play an important role in susceptibility to noise-induced hearing loss (NIHL). Alternative splicing (AS) is an essential mechanism affecting gene expression associated with disease pathogenesis at the post-transcriptional level, but has rarely been studied in NIHL. To explore the role of AS in the development of NIHL, we performed a comprehensive analysis of RNA splicing alterations by comparing the RNA-seq data from blood samples from NIHL patients and subjects with normal hearing who were exposed to the same noise environment. A total of 356 differentially expressed genes, including 23 transcription factors, were identified between the two groups. Of particular note was the identification of 56 aberrant alternative splicing events generated by 41 differentially expressed genes between the two groups, with exon skipping events accounting for 54% of all the differentially alternative splicing (DAS) events. The results of functional enrichment analysis showed that these intersecting DAS genes and differentially expressed genes were significantly enriched in autophagy and mitochondria-related pathways. Together, our findings provide insights into the role of AS events in susceptibility and pathogenesis of NIHL.

Development of an audiological assessment and diagnostic model for high occupational noise exposure.

PURPOSE: To explore the diagnostic auditory indicators of high noise exposure and combine them into a diagnostic model of high noise exposure and possible development of hidden hearing loss (HHL). METHODS: We recruited 101 young adult subjects and divided them according to noise exposure history into high-risk and low-risk groups. All subjects completed demographic characteristic collection (including age, noise exposure, self-reported hearing status, and headset use) and related hearing examination. RESULTS: The 8 kHz (P = 0.039) and 10 kHz (P = 0.005) distortion product otoacoustic emission amplitudes (DPOAE) (DPs) in the high-risk group were lower than those in the low-risk group. The amplitudes of the summating potential (SP) (P = 0.017) and action potential (AP) (P = 0.012) of the electrocochleography (ECochG) in the high-risk group were smaller than those in the low-risk group. The auditory brainstem response (ABR) wave III amplitude in the high-risk group was higher than that in the low-risk group. When SNR = - 7.5 dB (P = 0.030) and - 5 dB (P = 0.000), the high-risk group had a lower speech discrimination score than that of the low-risk group. The 10 kHz DPOAE DP, ABR wave III amplitude and speech discrimination score under noise with SNR = - 5 dB were combined to construct a combination diagnostic indicator. The area under the ROC curve was 0.804 (95% CI 0.713-0.876), the sensitivity was 80.39%, and the specificity was 68.00%. CONCLUSIONS: We expect that high noise exposure can be detected early with this combined diagnostic indicator to prevent HHL or sensorineural hearing loss (SNHL). TRIAL REGISTRATION NUMBER/DATE OF REGISTRATION: ChiCTR2200057989, 2022/3/25.

Correlation between clinical characteristics and tinnitus severity in tinnitus patients of different sexes: an analytic retrospective study.

PURPOSE: This study aimed to explore whether sex is influences tinnitus severity and whether the risk factors for tinnitus severity are the same in tinnitus patients of different sexes. METHODS: This was a retrospective study of data from 1427 patients complaining of tinnitus in a local hospital otolaryngology clinic from November 2019 to January 2022. All patients were interviewed and assessed by otoscopy, pure-tone audiometry, tinnitus handicap inventory (THI), visual analogue scale (VAS), and tinnitus refinement test. RESULTS: THI values were higher in females than in males (P = 0.00). Types of tinnitus sounds (OR 0.667, P = 0.000) and degree of hearing loss (OR 1.318, P = 0.000) were risk factors for tinnitus severity in males. Types of tinnitus sounds (OR 0.789, P = 0.005), sensation level (OR 1.023, P = 0.037), tinnitus types (OR 1.163, P = 0.041), tinnitus location (OR 1.198, P = 0.026), and the degree of hearing loss (OR 1.303, P = 0.000) were risk factors for tinnitus severity in females. Sex was an influencing factor for tinnitus severity. There were different risk factors for the tinnitus severity in different sexes. CONCLUSION: The risk factors for tinnitus severity differed according to sex in tinnitus patients, and the risk factors for tinnitus severity were greater in women than in men. These findings add to the literature on sex differences in tinnitus and suggest that medical and psychological screening of affected individuals and customized tinnitus treatment for each individual with tinnitus are needed. TRIAL REGISTRATION NUMBER/DATE OF REGISTRATION: ChiCTR2200057958, 2022/3/24 (retrospectively registered trials).

Treatment with the Ferroptosis Inhibitor Ferrostatin-1 Attenuates Noise-Induced Hearing Loss by Suppressing Ferroptosis and Apoptosis.

Hair cell death induced by excessive reactive oxygen species (ROS) has been identified as the major pathogenesis of noise-induced hearing loss (NIHL). Recent studies have demonstrated that cisplatin- and neomycin-induced ototoxicity can be alleviated by ferroptosis inhibitors. However, whether ferroptosis inhibitors have a protective effect against NIHL remains unknown. We investigated the protective effect of the ferroptosis inhibitor ferrostatin-1 (Fer-1) on NIHL in vivo in CBA/J mice and investigated the protective effect of Fer-1 on tert-butyl hydroperoxide (TBHP)-induced hair cell damage in vitro in cochlear explants and HEI-OC1 cells. We observed ROS overload and lipid peroxidation, which led to outer hair cell (OHC) apoptosis and ferroptosis, in the mouse cochlea after noise exposure. The expression level of apoptosis-inducing factor mitochondria-associated 2 (AIFM2) was substantially increased following elevation of the expression of its upstream protein P53 after noise exposure. The ferroptosis inhibitor Fer-1was demonstrated to enter the inner ear after the systemic administration. Administration of Fer-1 significantly alleviated noise-induced auditory threshold elevation and reduced the loss of OHCs, inner hair cell (IHC) ribbon synapses, and auditory nerve fibers (ANFs) caused by noise. Mechanistically, Fer-1 significantly reduced noise- and TBHP-induced lipid peroxidation and iron accumulation in hair cells, alleviating ferroptosis in cochlear cells consequently. Furthermore, Fer-1 treatment decreased the levels of TfR1, P53, and AIFM2. These results suggest that Fer-1 exerted its protective effects by scavenging of ROS and inhibition of TfR1-mediated ferroptosis and P53-AIFM2 signaling pathway-mediated apoptosis. Our findings suggest that Fer-1 is a promising drug for treating NIHL because of its ability to inhibit noise-induced hair cell apoptosis and ferroptosis, opening new avenues for the treatment of NIHL.

Systemic steroid administration combined with intratympanic steroid injection in the treatment of a unilateral sudden hearing loss prognosis prediction model: A retrospective observational study.

Idiopathic sudden sensorineural hearing loss (ISSNHL) is an emergency ear disease that is referred to as a sensorineural hearing loss of at least 30 dB in three sequential frequencies and occurs over a period of < 72 h. Because of its etiology, pathogenesis, and prognostic factors, the current treatment methods are not ideal. Previous studies have developed prognostic models to predict hearing recovery from ISSNHL, but few studies have incorporated serum biochemical indicators into previous models. The aim of this study was to explore the factors influencing the ISSNHL prognosis of combination therapy (combined intratympanic and systemic use of steroids, CT), among the patient population data, the serum biochemical indicators before the treatment, and the clinical features of ISSNHL. The new prediction model was developed through these factors. From November 2015 to April 2022, 430 patients who underwent CT at the Department of Otorhinolaryngology Head and Neck Surgery, Tangdu Hospital, Air Force Medical University for ISSNHL, were reviewed retrospectively. We found significant differences in age (P = 0.018), glucose (P = 0.035), white blood cell (WBC) (P = 0.021), vertigo (P = 0.000) and type (P = 0.000) with different therapeutic efficacies. Multivariate logistic regression analysis showed that age (OR = 0.715, P = 0.023), WBC (OR = 0.527, P = 0.01), platelet to lymphocyte ratio (PLR) (OR = 0.995, P = 0.038), vertigo (OR = 0.48, P = 0.004), course (time from onset to treatment) (OR = 0.681, P = 0.016) and type (OR = 0.409, P = 0.000) were independent risk factors for ISSNHL prognosis. Based on independent risk factors, a predictive model and nomogram were developed to predict hearing outcomes in ISSNHL patients. The area under the curve (AUC) value of the model developed in this study was 0.773 (95% CI = 0.730-0.812), which has a certain predictive ability. The calibration curve indicated good consistency between the actual diagnosed therapeutic effectiveness and the predicted probability. The model and nomogram can predict the hearing prognosis of ISSNHL patients treated with CT and can provide help for medical staff to make the best clinical decision. This study has been registered with the registration number ChiCTR2200061379.

mito-TEMPO Attenuates Oxidative Stress and Mitochondrial Dysfunction in Noise-Induced Hearing Loss via Maintaining TFAM-mtDNA Interaction and Mitochondrial Biogenesis.

The excessive generation of reactive oxygen species (ROS) and mitochondrial damage have been widely reported in noise-induced hearing loss (NIHL). However, the specific mechanism of noise-induced mitochondrial damage remains largely unclear. In this study, we showed that acoustic trauma caused oxidative damage to mitochondrial DNA (mtDNA), leading to the reduction of mtDNA content, mitochondrial gene expression and ATP level in rat cochleae. The expression level and mtDNA-binding function of mitochondrial transcription factor A (TFAM) were impaired following acoustic trauma without affecting the upstream PGC-1α and NRF-1. The mitochondria-target antioxidant mito-TEMPO (MT) was demonstrated to enter the inner ear after the systemic administration. MT treatment significantly alleviated noise-induced auditory threshold shifts 3d and 14d after noise exposure. Furthermore, MT significantly reduced outer hair cell (OHC) loss, cochlear ribbon synapse loss, and auditory nerve fiber (ANF) degeneration after the noise exposure. In addition, we found that MT treatment effectively attenuated noise-induced cochlear oxidative stress and mtDNA damage, as indicated by DHE, 4-HNE, and 8-OHdG. MT treatment also improved mitochondrial biogenesis, ATP generation, and TFAM-mtDNA interaction in the cochlea. These findings suggest that MT has protective effects against NIHL via maintaining TFAM-mtDNA interaction and mitochondrial biogenesis based on its ROS scavenging capacity.

Dynamic alterations of functional connectivity and amplitude of low-frequency fluctuations in patients with unilateral sudden sensorineural hearing loss.

Unilateral sudden sensorineural hearing loss (SSNHL) adversely affects the quality of life, leading to increased risk of depression and cognitive decline. Our previous studies have mainly focused on the static brain function abnormalities in SSNHL patients. However, the dynamic features of brain activity in SSNHL patients are not elucidated. To explore the dynamic brain functional alterations in SSNHL patients, age- and sex- matched SSNHL patients (n = 38) and healthy controls (HC, n = 44) were enrolled. The dynamic functional connectivity (dFC) and dynamic amplitude of low-frequency fluctuation (dALFF) methods were used to compare the temporal features and dynamic neural activity between the two groups. In dFC analyses, the multiple functional connectivities (FCs) were clustered into 2 different states; a greater proportion of FCs in SSNHL patients showed sparse state compared with HC. In dALFF analyses, SSNHL individuals exhibited decreased dALFF variability in bilateral inferior occipital gyrus, middle occipital gyrus, calcarine, right lingual gyrus, and right fusiform gyrus. dALFF variability showed a negative correlation with activated partial thromboplatin time. The dynamic characteristics of SSNHL patients were different from static functional connectivity and static amplitude of low-frequency fluctuation, especially within the visual cortices. These findings suggest that SSNHL patients experience cross-modal plasticity and visual compensation, which may be closely related to the pathophysiology of SSNHL.

Treating ischaemic stroke with intravenous tPA beyond 4.5 hours under the guidance of a MRI DWI/T2WI mismatch was safe and effective.

Purpose: Clinical trials have provided evidence that treating patients with acute ischaemic stroke (AIS) beyond 4.5 hours was feasible. Among them using MRI diffusion-weighted imaging/fluid attenuation inversion response (DWI/FLAIR) mismatch to guide intravenous tissue plasminogen activator (tPA) was successful. Our study explored the outcome and safety of using DWI/T2-weighted imaging (T2WI) mismatch to guide intravenous tPA therapy for patients with AIS between 4.5 hours and 12 hours of onset. Method: This was a retrospective study. Records of 1462 AIS patients with the time of onset of <12 hours were reviewed. Those had MRI rapid sequence study and had hyperintense signal on DWI but normal T2WI and received intravenous tPA up to 12 hours of onset were included in the analysis. Their demographics, risk factors, post-tPA complications, National Institutes of Health Stroke Scale (NIHSS) scores and outcome were recorded and analyse. χ2 was used to compare the intergroup variables. SAS was used to perform statistical calculation. A p<0.05 was considered statistically significant. Results: Of 1462 identified, 601 (41%) patients were entered into the final analysis. Among them, 327 (54%) had intravenous tPA within 4.5 hours of onset and 274 (46%) were treated between 4.5-12 hours. After intravenous tPA, 426 cases (71%) had >4 pints of improvement on NIHSS score within 24 hours. Postintravenous tPA, 32 (5.32%) cases had haemorrhagic transformation. 26 (4.33%) were asymptomatic ICH and 4 (0.67%) died. At 90 days, 523 (87%) achieved a modified Rankin scale of 0-2. Conclusion: Using MRI DWI/T2WI mismatch to identify patients with AIS for intravenous tPA between 4.5 hours and 12 hours was safe and effective. The outcome was similar to those used DWI/PWI or DWI/FLAIR mismatch as the screening tool. However, obtaining DWI/T2WI was faster and avoided the need of contrast material.

Exposure to acoustic stimuli promotes the development and differentiation of neural stem cells from the cochlear nuclei through the clusterin pathway.

Stem cell therapy has attracted widespread attention for a number of diseases. Recently, neural stem cells (NSCs) from the cochlear nuclei have been identified, indicating a potential direction for the treatment of sensorineural hearing loss. Acoustic stimuli play an important role in the development of the auditory system. In this study, we aimed to determine whether acoustic stimuli induce NSC development and differentiation through the upregulation of clusterin (CLU) in NSCs isolated from the cochlear nuclei. To further clarify the underlying mechanisms involved in the development and differentiation of NSCs exposed to acoustic stimuli, we successfully constructed animal models in which was CLU silenced by an intraperitoneal injection of shRNA targeting CLI. As expected, the NSCs from rats treated with LV-CLU shRNA exhibited a lower proliferation ratio when exposed to an augmented acoustic environment (AAE). Furthermore, the inhibition of cell apoptosis induced by exposure to AAE was abrogated after silencing the expression of the CLU gene. During the differentiation of acoustic stimuli-exposed stem cells into neurons, the number of astrocytes was significantly reduced, as evidenced by the expression of the cell markers, microtubule associated protein­2 (MAP-2) and glial fibrillary acidic protein (GFAP), which was markedly inhibited when the CLU gene was silenced. Our results indicate that acoustic stimuli may induce the development and differentiation of NSCs from the cochlear nucleus mainly through the CLU pathway. Our study suggests that CLU may be a novel target for the treatment of sensorineural hearing loss.

Noise alters guinea pig's blood-labyrinth barrier ultrastructure and permeability along with a decrease of cochlear Claudin-5 and Occludin.

BACKGROUND: Noise exposure (NE) is a severe modern health hazard that induces hearing impairment. However, the noise-induced ultrastructural changes of blood-labyrinth barrier (BLB) and the potential involvements of tight junction proteins (TJP) remain inconclusive. We investigated the effects of NE on not only the ultrastructure of cochlea and permeability of BLB but also the expression of TJP within the guinea pig cochlea. RESULTS: Male albino guinea pigs were exposed to white noise for 4 h or 2 consecutive days (115 dB sound pressure level, 6 hours per day) and the hearing impairments and light microscopic change of BLB were evaluated with auditory brainstem responses (ABR) and the cochlear sensory epithelia surface preparation, respectively. The cochlear ultrastructure and BLB permeability after NE 2d were revealed with transmission electron microscope (TEM) and lanthanum nitrate-tracing techniques, respectively. The potential alterations of TJPs Claudin-5 and Occludin were quantified with immunohistochemistry and western blot. NE induced significant hearing impairment and NE 2d contributed to significant outer hair cell (OHC) loss that is most severe in the first row of outer hair cells. Furthermore, the loosen TJ and an obvious leakage of lanthanum nitrate particles beneath the basal lamina were revealed with TEM. Moreover, a dose-dependent decrease of Claudin-5 and Occludin was observed in the cochlea after NE. CONCLUSIONS: All these findings suggest that both decrease of Claudin-5 and Occludin and increased BLB permeability are involved in the pathologic process of noise-induced hearing impairment; however, the causal relationship and underlying mechanisms should be further investigated.

Favorable proliferation and differentiation capabilities of neural precursor cells derived from rat cochlear nucleus.

NSCs/NPCs could be used for Sensorineural hearing loss treatment, because of the extensive capacity for self-renewal and pluripotency. In order to isolate and identify neural precursor cells (NPCs), we established a strategy to isolate and cultivate NPCs. Immunohistochemistry, immunofluorescence, Western blotting, and electron microscopy were used to characterize the cells and compare their differentiation patterns with those of olfactory bulb and olfactory epithelium NPCs. Furthermore, NPCs from the cochlear nucleus were sustained good cell viability and cloning efficiency after cryopreservation and thawing. Finally, high capacity to differentiate into astrocytes, oligodendrocytes, and neurons of NPCs was found. In conclusion, NPCs isolated from the cochlear nucleus can proliferate and differentiate into functional neurons, which offers a potential strategy for sensorineural hearing loss treatment. In addition, the storage method developed here will benefit further exploration of NPCs.

Publications about hearing in otorhinolaryngology journals from chinese authors: a 11-year survey of the literature.

Hearing loss is a leading cause of disability in China. However, the research status in the field of hearing among Chinese individuals in the three major regions of China: Mainland (ML), Hong Kong (HK) and Taiwan (TW), are unknown. The output of hearing articles published in international otorhinolaryngology journals from these three regions were compared in this study. Articles published in 31 international otorhinolaryngology journals related to hearing originating from the ML, TW and HK from 2000 to 2011 were retrieved from the PubMed database search. The number of total articles, clinical trials, randomized controlled trials, case reports, and articles published in the top 5 international otorhinolaryngology journals were assessed in terms of quantity and quality comparisons. The total number of articles from the three regions increased significantly from 2000 to 2011. There were 379 articles from ML (143), TW (180) and HK (56) in the past 10 years. The number of articles published per year from the ML has exceeded those from TW in 2009 and HK in 2003. TW had the most articles (46) published in the top 5 international otorhinolaryngology journals among the three regions. The total number of articles from the three major regions of China increased significantly from 2000 to 2011. The numbers of articles published per year from the ML have exceeded those from TW and HK. However, the quality of articles from TW is better than that from ML.

Can global variation of nasopharynx cancer be retrieved from the combined analyses of IARC Cancer Information (CIN) databases?

BACKGROUND: The international nasopharynx cancer (NPC) burdens are masked due to the lack of integrated studies that examine epidemiological data based on up-to-date international disease databases such as the Cancer Information (CIN) databases provided by the International Agency for Research on Cancer (IARC). METHODS: By analyzing the most recently updated NPC epidemiological data available from IARC, we tried to retrieve the worldwide NPC burden and patterns from combined analysis with GLOBOCAN2008 and the Cancer Incidence in Five Continents (CI5) databases. We provide age-standardized rates (ASR) for NPC mortality in 20 highest cancer registries from GLOBOCAN2008 and the World Health Organization (WHO) mortality databases, respectively. However, NPC incidence data can not be retrieved since it is not individually listed in CI5 database. The trend of NPC mortality was investigated with Joinpoint analysis in the selected countries/regions with high ASR. RESULTS: GLOBOCAN 2008 revealed that the highest NPC incidence rates in 2008 were in registries from South-Eastern Asia, Micronesia and Southern Africa with Malaysia, Indonesia and Singapore ranking the top 3. WHO mortality database analysis revealed that China Hong Kong, Singapore and Malta ranks the top 3 regions with the highest 5-year mortality rates. CONCLUSIONS: NPC mortality rate is about 2-3 times higher in male than that in female, and shows decrease tendency in those selected countries/regions during the analyzed periods. However, the integrated analyses of the current IARC CIN databases may not be suitable to retrieve epidemiological data of NPC. Much effort is required to improve the local cancer entry and regional death-reporting systems so as to aid similar studies.

[Inhibitory effect of 1,25(OH)2D3 on proliferation of human laryngeal carcinoma cells and potential mechanisms].

BACKGROUND AND OBJECTIVE: 1Alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], the biologically active form of vitamin D3, has antiproliferative activity against various tumor cells. This study was to explore the inhibitory effect of 1,25(OH)2D3 on human laryngeal carcinoma Hep-2 cells and potential mechanisms. METHODS: Hep-2 cells were treated by 1,25(OH)2D3 (0, 1, 10 and 100 nmol/L) for 24, 48, 72 and 96 h, respectively. Cell proliferation was measured by MTT assay. Cell apoptosis was measured by flow cytometry (FCM). The expression and phosphorylation of ERK, p38MAPK, and JNK proteins were detected by Western blot. RESULTS: 1,25(OH)2D3 significantly inhibited Hep-2 cell proliferation and induced cell apoptosis. 1,25(OH)2D3 increased p38MPAK phosphorylation but not ERK and JNK phosphorylation. The 1,25(OH)2D3-induced apoptosis of Hep-2 cells was partly blocked by p38 inhibitor SB2035080. CONCLUSION: 1,25(OH)2 D3 could induce apoptosis of Hep-2 cells and p38MAPK plays an important role in this process.

Effect of cryopreservation on proliferative features of neural progenitor cells derived from olfactory bulb of embryonic rat.

OBJECTIVE: Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. The use of olfactory bulb neural progenitor cells for transplantation requires efficient recovery methods and cryopreservation procedures. The purpose of this study was to determine cryopreservation techniques for neural progenitor cells derived from olfactory bulb (OB NPCs) of embryonic rat. METHODS: Initially, we compared the survival rates of cryopreserved OB NPCs using three cryoprotectants: dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol with or without 10% FBS. Cells were held at liquid nitrogen (-186 degrees C) for 7 days ("short-term storage") or 6 months ("long-term storage"). We assessed OB NPCs recovery efficiency after freezing and thawing by viability testing, colony-forming ability and immunocytochemistry under different conditions. RESULTS: The survival rate of cryopreserved-thawed OB NPCs was estimated by counting colony numbers under a stereomicroscope. No significant difference in survival rate was observed between cryoprotectants. CONCLUSIONS: These observations indicate that cryopreservation of OB NPCs is possible for up to 6 months in optimal conditions without losing proliferation activity.

Polyclonal antibodies to LIM proteins CRP2 and CRIP2 reveal their subcellular localizations in olfactory precursor cells.

In this study, we describe the presence of CRP2 (cysteine- and glycine-rich protein 2) and CRIP2 (cysteine-rich intestinal protein 2), which are members of group 2 LIM proteins, in rat olfactory precursor cells by reverse transcription polymerase chain reaction. We have developed polyclonal antibodies against CRP2 and CRIP2 individually. Specificity of the antibodies was demonstrated by Western blot analysis, using CRP2 and CRIP2 transfected cells. No cross-reactivity was observed between the antibodies. Furthermore, we used the antibodies to determine the expression and localization of CRP2 and CRIP2 in olfactory precursor cells by Western blot analysis and immunofluorescence staining. Our results demonstrated that in undifferentiated olfactory precursor cells CRP2 was distributed both in the nucleus and the cytoplasm, whereas CRIP2 was predominantly localized in the cytoplasm. While the olfactory precursor cells differentiated into end cells, only the expression of CRIP2 would be detected. The function of these LIM proteins in olfactory precursor cells warrants further study.