Long-lasting increases in GABAB receptor subunit levels in hippocampal dentate gyrus of mice with a single systemic injection of trimethyltin.

We have recently shown delayed increases in GABAB receptor (GABABR) subunit protein levels in the hippocampal dentate gyrus (DG), but not in the pyramidal CA1 and CA3 regions, at 15-30 days after the systemic single administration of trimethyltin (TMT) in mice. An attempt was thus made to determine whether the delayed increases return to the control levels found in naive mice afterward. In the DG on hippocampal slices obtained at 90 days after the administration, however, marked increases were still seen in protein levels of both GABABR1 and GABABR2 subunits without significant changes in calbindin and glial fibrillary acidic protein (GFAP) levels on immunoblotting analysis. Fluoro-Jade B staining clearly revealed the absence of degenerated neurons from the DG at 90 days after the administration. Although co-localization was invariably detected between GABABR2 subunit and GFAP in the DG at 30 days on immunohistochemical analysis, GABABR2-positive cells did not merge well with GFAP-positive cells in the DG at 90 days. These results suggest that both GABABR1 and GABABR2 subunits would be tardily and sustainably up-regulated by cells other than neurons and astrocytes in the murine DG at 90 days after a systemic single injection of TMT.

A novel model of sensorineural hearing loss induced by repeated exposure to moderate noise in mice: the preventive effect of resveratrol.

Sensorineural hearing loss (SNHL) induced by noise has increased in recent years due to personal headphone use and noisy urban environments. The study shows a novel model of gradually progressive SNHL induced by repeated exposure to moderate noise (8-kHz octave band noise, 90-dB sound pressure level) for 1 hr exposure per day in BALB/cCr mice. The results showed that the repeated exposure led to gradually progressive SNHL, which was dependent on the number of exposures, and resulted in permanent hearing loss after 5 exposures. Repeated exposure to noise causes a loss of synapses between the inner hair cells and the peripheral terminals of the auditory nerve fibers. Additionally, there is a reduction in the expression levels of c-fos and Arc, both of which are indicators of cochlear nerve responses to noise exposure. Oral administration of resveratrol (RSV, 50 mg/kg/day) during the noise exposure period significantly prevented the noise exposure-induced synapse loss and SNHL. Furthermore, the study found that RSV treatment prevented the noise-induced increase in the gene expression levels of the proinflammatory cytokine interleukin-1ß in the cochlea. These results demonstrated the potential usefulness of RSV in preventing noise-induced SNHL in the animal model established as gradually progressive SNHL.

Delayed Expression of Both GABABR1 and GABABR2 Subunits in Murine Hippocampal Dentate Gyrus After a Single Systemic Injection of Trimethyltin.

Trimethyltin (TMT) has been used as a cytotoxin to neurons rather than glial cells in the mammalian hippocampus. The systemic administration of TMT led to declined fluorescence of ZnAF-2 DA staining as a marker of intact mossy fibers and increased fluorescence of Fluoro-Jade B staining as a marker of degenerated neurons during the initial 2 to 5 days after the administration with later ameliorations within 30 days in the hippocampal dentate gyrus (DG) and CA3 region in mice. On immunoblotting analysis, both GABABR1 and GABABR2 subunit levels increased during 15 to 30 days after TMT along with significant decreases in glutamatergic GluA1 and GluA2/3 receptor subunit levels during 2 to 7 days in the DG, but not in other hippocampal regions such as CA1 and CA3 regions. Immunohistochemical analysis revealed the constitutive and inducible expression of GABABR2 subunit in cells immunoreactive to an astrocytic marker as well as neuronal markers in the DG with the absence of neither GABABR1a nor GABABR1b subunit from cells positive to an astrocytic marker. These results suggest that both GABABR1 and GABABR2 subunits may be up-regulated in cells other than neurons and astroglia in the DG at a late stage of TMT intoxication in mice.

Bacopa monnieri (L.) Wettst. Extract Improves Memory Performance via Promotion of Neurogenesis in the Hippocampal Dentate Gyrus of Adolescent Mice.

Bacopa monnieri L. Wettst. (BM) is a botanical component of Ayurvedic medicines and of dietary supplements used worldwide for cognitive health and function. We previously reported that administration of BM alcoholic extract (BME) prevents trimethyltin (TMT)-induced cognitive deficits and hippocampal cell damage and promotes TMT-induced hippocampal neurogenesis. In this study, we demonstrate that administration of BME improves spatial working memory in adolescent (5-week- old) healthy mice but not adult (8-week-old) mice. Moreover, improved spatial working memory was retained even at 4 weeks after terminating 1-week treatment of adolescent mice. One-week BME treatment of adolescent mice significantly enhanced hippocampal BrdU incorporation and expression of genes involved in neurogenesis determined by RNAseq analysis. Cell death, as detected by histochemistry, appeared not to be significant. A significant increase in neurogenesis was observed in the dentate gyrus region 4 weeks after terminating 1-week treatment of adolescent mice with BME. Bacopaside I, an active component of BME, promoted the proliferation of neural progenitor cells in vitro in a concentration-dependent manner via the facilitation of the Akt and ERK1/2 signaling. These results suggest that BME enhances spatial working memory in healthy adolescent mice by promoting hippocampal neurogenesis and that the effects of BME are due, in significant amounts, to bacopaside I.

Bacopa monnieri (L.) Ameliorates Cognitive Deficits Caused in a Trimethyltin-Induced Neurotoxicity Model Mice.

We previously demonstrated that Bacopa monnier (L.) WETTST. extract (BME) ameliorated cognitive dysfunction in animal models of dementia by enhancing synaptic plasticity-related signaling in the hippocampus and protecting cholinergic neurons in the medial septum. To further clarify the pharmacological features and availability of BME as a novel anti-dementia agent, we investigated whether BME affects neuronal repair using a mouse model of trimethyltin (TMT)-induced neuronal loss/self-repair in the hippocampus. Mice pretreated with TMT (2.8 mg/kg, intraperitoneally (i.p.)) on day 0 were given BME (50 mg/kg, per os (p.o.)) once daily for 15-30 d. Cognitive performance of the animals was elucidated twice by the object location test and modified Y maze test on days 17-20 (Phase I) and days 32-35 (Phase II) or by the passive avoidance test on Phase II. TMT impaired hippocampus-dependent spatial working memory and amygdala-dependent fear-motivated memory. The administration of BME significantly prevented TMT-induced cognitive deficits. The protective effects of BME on the spatial memory deficits were confirmed by Nissl staining of hippocampal tissues and propidium iodide staining of organotypic hippocampal slice cultures. Immunohistochemical studies conducted on days 17 and 32 revealed that thirty days of treatment with BME increased the number of 5-bromo-2'-deoxyuridine (BrdU)-immunopositive cells in the dentate gyrus region of TMT-treated mice, whereas fifteen days of treatment with BME had no effect. These results suggest that BME ameliorates TMT-induced cognition dysfunction mainly via protecting the hippocampal neurons from TMT-induced hippocampal lesions and partly via promoting neuroregeneration in the dentate gyrus regions.

[Adult Neurogenesis-activating Signals as Therapeutic Targets for Neurodegenerative Disorders].

In most mammalian species, adult neurogenesis appears to occur only in the olfactory bulb and hippocampal dentate gyrus, where neural stem/progenitor cells exist to create new neurons. The discovery of multi-potential neural stem/progenitor cells (NPCs) in the adult brain has precipitated a novel therapeutic strategy for harnessing these endogenous cells to aid in recovery from neurodegenerative disorders. During neurodegeneration, a plethora of endogenous factors, including cytokines, chemokines, neurotransmitters, blood-derived factors, and reactive oxygen species, are released by the activation of resident microglia, astrocytes, and infiltrating peripheral macrophages. It is interesting that these endogenous factors affect the proliferation, migration, differentiation, and survival of newly generated cells involved in the incorporation of newly generated neurons into the brain's circuitry. The unique profile of these endogenous factors can vary the degree of neuroregeneration after neurodegeneration. We show that adult neurogenesis-activating signals are regulated by endogenous factors produced during neurodegeneration.

Disruption of Gap Junction-Mediated Intercellular Communication in the Spiral Ligament Causes Hearing and Outer Hair Cell Loss in the Cochlea of Mice.

It is well-known that outer hair cell (OHC) loss occurs in the cochlea of animal models of permanent hearing loss induced by intense noise exposure. Our earlier studies demonstrated the production of hydroxynonenal and peroxynitrite, as well as the disruption of gap junction-mediated intercellular communication (GJIC), in the cochlear spiral ligament prior to noise-induced sudden hearing loss. The goal of the present study was to evaluate the mechanism underlying cochlear OHC loss after sudden hearing loss induced by intense noise exposure. In organ of Corti explant cultures from mice, no significant OHC loss was observed after in vitro exposure to 4-hydroxynonenal (a product of lipid peroxidation), H2O2, SIN-1 (peroxynitrite generator), and carbenoxolone (a gap junction inhibitor). Interestingly, in vivo intracochlear carbenoxolone injection through the posterior semicircular canal caused marked OHC and hearing loss, as well as the disruption of gap junction-mediated intercellular communication in the cochlear spiral ligament. However, no significant OHC loss was observed in vivo in animals treated with 4-hydroxynonenal and SIN-1. Taken together, our data suggest that disruption of GJIC in the cochlear lateral wall structures is an important cause of cochlear OHC loss in models of hearing loss, including those induced by noise.

Hearing vulnerability after noise exposure in a mouse model of reactive oxygen species overproduction.

Previous studies have convincingly argued that reactive oxygen species (ROS) contribute to the development of several major types of sensorineural hearing loss, such as noise-induced hearing loss (NIHL), drug-induced hearing loss, and age-related hearing loss. However, the underlying molecular mechanisms induced by ROS in these pathologies remain unclear. To resolve this issue, we established an in vivo model of ROS overproduction by generating a transgenic (TG) mouse line expressing the human NADPH oxidase 4 (NOX4, NOX4-TG mice), which is a constitutively active ROS-producing enzyme that does not require stimulation or an activator. Overproduction of ROS was detected at the cochlea of the inner ear in NOX4-TG mice, but they showed normal hearing function under baseline conditions. However, they demonstrated hearing function vulnerability, especially at high-frequency sounds, upon exposure to intense noise, which was accompanied by loss of cochlear outer hair cells (OHCs). The vulnerability to loss of hearing function and OHCs was rescued by treatment with the antioxidant Tempol. Additionally, we found increased protein levels of the heat-shock protein 47 (HSP47) in models using HEK293 cells, including H2 O2 treatment and cells with stable and transient expression of NOX4. Furthermore, the up-regulated levels of Hsp47 were observed in both the cochlea and heart of NOX4-TG mice. Thus, antioxidant therapy is a promising approach for the treatment of NIHL. Hsp47 may be an endogenous antioxidant factor, compensating for the chronic ROS overexposure in vivo, and counteracting ROS-related hearing loss.

Alleviation by GABAB Receptors of Neurotoxicity Mediated by Mitochondrial Permeability Transition Pore in Cultured Murine Cortical Neurons Exposed to N-Methyl-D-aspartate.

Mitochondrial permeability transition pore (PTP) is supposed to at least in part participate in molecular mechanisms underlying the neurotoxicity seen after overactivation of N-methyl-D-aspartate (NMDA) receptor (NMDAR) in neurons. In this study, we have evaluated whether activation of GABAB receptor (GABABR), which is linked to membrane G protein-coupled inwardly-rectifying K+ ion channels (GIRKs), leads to protection of the NMDA-induced neurotoxicity in a manner relevant to mitochondrial membrane depolarization in cultured embryonic mouse cortical neurons. The cationic fluorescent dye 3,3'-dipropylthiacarbocyanine was used for determination of mitochondrial membrane potential. The PTP opener salicylic acid induced a fluorescence increase with a vitality decrease in a manner sensitive to the PTP inhibitor ciclosporin, while ciclosporin alone was effective in significantly preventing both fluorescence increase and viability decrease by NMDA as seen with an NMDAR antagonist. The NMDA-induced fluorescence increase and viability decrease were similarly prevented by pretreatment with the GABABR agonist baclofen, but not by the GABAAR agonist muscimol, in a fashion sensitive to a GABABR antagonist. Moreover, the GIRK inhibitor tertiapin canceled the inhibition by baclofen of the NMDA-induced fluorescence increase. These results suggest that GABABR rather than GABAAR is protective against the NMDA-induced neurotoxicity mediated by mitochondrial PTP through a mechanism relevant to opening of membrane GIRKs in neurons.

Preventive effect of curcumin and its highly bioavailable preparation on hearing loss induced by single or repeated exposure to noise: A comparative and mechanistic study.

We sought to determine the preventive effects of curcumin and its highly bioavailable preparation on noise-induced hearing loss in a novel murine model of permanent hearing loss developed by repeated exposure to noise. Upon exposure to noise (8-kHz octave band noise, 90 dB sound pressure level, 1 h), hearing ability was impaired in a temporary and reversible manner. During repeated noise exposure (1-h exposure per day, 5 days), there was a progressive increase in the auditory threshold shift at 12 and 20 kHz. The threshold shift persisted for at least 6 days after noise exposure. Oral administration of curcumin for 3 days before and each day during noise exposure significantly alleviated the hearing loss induced by repeated noise exposure. Curcumin abolished intranuclear translocation of nuclear factor-κB-p65 and generation of 4-hydroxynonenal-adducted proteins found in the cochlea after noise exposure. Theracurmin®, a highly absorbable and bioavailable preparation of curcumin, had strong preventive effects on hearing loss induced by repeated noise exposure. Together, these data suggest that curcumin exerts a preventive effect on noise-induced hearing loss and is therefore a good therapeutic candidate for preventing sensorineural hearing loss.

Calpain inhibitor alleviates permanent hearing loss induced by intense noise by preventing disruption of gap junction-mediated intercellular communication in the cochlear spiral ligament.

Our previous studies demonstrated that intense noise-induced hearing loss might be at least in part due to an oxidative stress-induced decrease in the level of gap junction-composing protein connexins in the spiral ligament (SL) of the cochlear lateral wall structures in mice. Further, an in vivo exposure of mice to intense noise activates calpain in the cochlear SL. Based on these studies, we sought to determine whether a calpain inhibitor would prevent an intense noise exposure from causing hearing loss, disruption of gap junction-mediated intercellular communication (GJIC) in the SL. An exposure of mice to intense noise (8-Hz octave band noise, 110-dB sound pressure level, 1h) produced permanent hearing loss and cochlear hair cell death. The results of an ex vivo assay using gap-fluorescence recovery after photobleaching of dissected lateral wall structures revealed that the intense noise disrupted GJIC in the cochlear SL at day-7 post exposure. A prior intracochlear injection of the calpain inhibitor PD150606 significantly abolished this noise-induced hearing loss on days 5 and 7 post exposure. Similarly, PD150606 prevented noise-induced hair cell death and the GJIC disruption on day-7 post exposure. The intense noise temporarily enhanced the gene expression of calpain subtypes Capn1 and Capn2 immediately after exposure. Taken together, our data suggest that calpain inhibitor alleviated the noise-induced hearing loss, at least in part, by preventing disruption of GJIC in the cochlear SL. It possible that calpain inhibitors would be useful as a candidate of therapeutic drugs for sudden sensorineural hearing loss.

Quantitative Analysis of Aquaporin Expression Levels during the Development and Maturation of the Inner Ear.

Aquaporins (AQPs) are a family of small membrane proteins that transport water molecules across the plasma membrane along the osmotic gradient. Mammals express 13 subtypes of AQPs, including the recently reported "subcellular AQPs", AQP11 and 12. Each organ expresses specific subsets of AQP subtypes, and in the inner ear, AQPs are essential for the establishment and maintenance of two distinct fluids, endolymph and perilymph. To evaluate the contribution of AQPs during the establishment of inner ear function, we used quantitative reverse transcription polymerase chain reaction to quantify the expression levels of all known AQPs during the entire development and maturation of the inner ear. Using systematic and longitudinal quantification, we found that AQP11 was majorly and constantly expressed in the inner ear, and that the expression levels of several AQPs follow characteristic longitudinal patterns: increasing (Aqp0, 1, and 9), decreasing (Aqp6, 8, and 12), and peak of expression on E18 (Aqp2, 5, and 7). In particular, the expression level of Aqp9 increased by 70-fold during P3-P21. We also performed in situ hybridization of Aqp11, and determined the unique localization of Aqp11 in the outer hair cells. Immunohistochemistry of AQP9 revealed its localization in the supporting cells inside the organ of Corti, and in the root cells. The emergence of AQP9 expression in these cells was during P3-P21, which was coincident with the marked increase of its expression level. Combining these quantification and localization data, we discuss the possible contributions of these AQPs to inner ear function.

Proof of Learning Outcome by the Advanced Clinical Competency Examination Trial after the Long-term Student's Practice in Pharmaceutical Education.

At Setsunan University, a debrief session (a poster session) is commonly performed by the students who have completed the long-term students' practice. Since the valuable changes in practical competency of the students cannot be evaluated through this session, we specified items that can help evaluate and methods that can help estimate the students' competency as clinical pharmacists. We subsequently carried out a trial called the "Advanced Clinical Competency Examination". We evaluated 103 students who had concluded the students' practice for the second period (Sep 1, 2014, to Nov 16, 2014): 70 students (called "All finish students") who had completed the practice in a hospital and pharmacy, and 33 students (called "Hospital finish students") who had finished the practice at a hospital only. The trial was executed in four stages. In the first stage, students drew pictures of something impressive they had learned during the practice. In the second stage, students were given patient cases and were asked, "What is this patient's problem?" and "How would you solve this problem?". In the third stage, the students discussed their answers in a group. In the fourth stage, each group made a poster presentation in separate rooms. By using a rubric, the teachers evaluated each student individually, the results of which showed that the "All finish students" could identify more problems than the "Hospital finish students".

Protease-activated receptor-1 negatively regulates proliferation of neural stem/progenitor cells derived from the hippocampal dentate gyrus of the adult mouse.

Thrombin-activated protease-activated receptor (PAR)-1 regulates the proliferation of neural cells following brain injury. To elucidate the involvement of PAR-1 in the neurogenesis that occurs in the adult hippocampus, we examined whether PAR-1 regulated the proliferation of neural stem/progenitor cells (NPCs) derived from the murine hippocampal dentate gyrus. NPC cultures expressed PAR-1 protein and mRNA encoding all subtypes of PAR. Direct exposure of the cells to thrombin dramatically attenuated the cell proliferation without causing cell damage. This thrombin-induced attenuation was almost completely abolished by the PAR antagonist RWJ 56110, as well as by dabigatran and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), which are selective and non-selective thrombin inhibitors, respectively. Expectedly, the PAR-1 agonist peptide (AP) SFLLR-NH2 also attenuated the cell proliferation. The cell proliferation was not affected by the PAR-1 negative control peptide RLLFT-NH2, which is an inactive peptide for PAR-1. Independently, we determined the effect of in vivo treatment with AEBSF or AP on hippocampal neurogenesis in the adult mouse. The administration of AEBSF, but not that of AP, significantly increased the number of newly-generated cells in the hippocampal subgranular zone. These data suggest that PAR-1 negatively regulated adult neurogenesis in the hippocampus by inhibiting the proliferative activity of the NPCs.

Involvement of calpain in 4-hydroxynonenal-induced disruption of gap junction-mediated intercellular communication among fibrocytes in primary cultures derived from the cochlear spiral ligament.

The endocochlear potential in the inner ear is essential for hearing ability, and maintained by various K(+) transport apparatuses including Na(+), K(+)-ATPase and gap junction-mediated intercellular communication (GJ-IC) in the lateral wall structures of the cochlea. Noise-induced hearing loss is known at least in part due to disruption of GJ-IC resulting from an oxidative stress-induced decrease in connexins (Cxs) level in the lateral wall structures. The purpose of this study was to investigate, using primary cultures of fibrocytes from the cochlear spiral ligament of mice, the mechanism underlying GJ-IC disruption induced by 4-hydroxynonenal (4-HNE), which is formed as a mediator of oxidative stress. An exposure to 4-HNE produced the following events: i.e., an increase in 4-HNE-adducted proteins; a decrease in the protein levels of Cx43, ß-catenin, and Cx43/ß-catenin complex along with intracellular translocation of this complex from the cell membrane to the cytoplasm; enhanced calpain-dependent degradation of endogenous α-fodrin; and disruption of GJ-IC. The 4-HNE-induced decrease in these protein levels and disruption of GJ-IC were most completely abolished by the calpain inhibitor PD150606. Taken together, our data suggest that 4-HNE disrupted GJ-IC through calpain-mediated degradation of Cx43 and ß-catenin in primary cultures of fibrocytes derived from the cochlear spiral ligament.

[Concept extraction of graduate research by modified grounded theory approach and creating of rubric oriented to performance evaluation].

  A revised core curriculum model for pharmaceutical education, developed on the basis of the principles of outcome-based education, will be introduced in 2015. Inevitably, appropriate assessments of students' academic achievements will be required. Although evaluations of the cognitive domain can be carried out by paper tests, evaluation methods for the attitude domain and problem-solving abilities need to be established. From the viewpoint of quality assurance for graduates, pharmaceutical education reforms have become vital to evaluation as well as learning strategies. To evaluate student academic achievements on problem-solving abilities, authentic assessment is required. Authentic assessment is the evaluation that mimics the context tried in work and life. Specifically, direct evaluation of performances, demonstration or the learners' own work with integrated variety knowledge and skills, is required. To clarify the process of graduate research, we obtained qualitative data through focus group interviews with six teachers and analyzed the data using the modified grounded theory approach. Based on the results, we clarify the performance students should show in graduate research and create a rubric for evaluation of performance in graduate research.

Beneficial effect of cilostazol-mediated neuronal repair following trimethyltin-induced neuronal loss in the dentate gyrus.

Cilostazol acts as an antiplatelet agent and has other pleiotropic effects based on phosphodiesterase-3-dependent mechanisms. We evaluated whether cilostazol would have a beneficial effect on neuronal repair following hippocampal neuronal damage by using a mouse model of trimethyltin (TMT)-induced neuronal loss/self-repair in the hippocampal dentate gyrus [Ogita et al. (2005) J Neurosci Res 82:609-621]; these mice will hereafter be referred to as impaired animals. A single treatment with cilostazol (10 mg/kg, i.p.) produced no significant change in the number of 5-bromo-2'-deoxyuridine (BrdU)-incorporating cells in the dentate granule cell layer (GCL) or subgranular zone on day 3 after TMT treatment. However, chronic treatment with cilostazol on days 3-15 posttreatment resulted in an increase in the number of BrdU-incorporating cells in the dentate GCL of the impaired animals, and these cells were positive for neuronal nuclear antigen or doublecortin. Cilostazol was effective in elevating the level of phosphorylated cyclic adrenosine monophosphate response element-binding protein (pCREB) in the dentate gyrus of impaired animals. The results of a forced swimming test revealed that the chronic treatment with cilostazol improved the depression-like behavior seen in the impaired animals. In the cultures of hippocampal neural stem/progenitor cells, exposure to cilostazol produced not only enhancement of proliferation activity but also elevation of pCREB levels. Taken together, our data suggest that cilostazol has a beneficial effect on neuronal repair following neuronal loss in the dentate gyrus through promotion of proliferation and/or neuronal differentiation of neural progenitor cells in the subgranular zone.

[A self-improvement and participatory career development education program involving internships and volunteer training experience for pharmacy students: results verified in a follow-up survey three years after participation].

The Faculty of Pharmaceutical Sciences, Setsunan University, offers the Self-improvement and Participatory Career Development Education Program: Internship and Volunteer Training Experience for Pharmacy Students to third-year students. We previously reported that the training experience was effective in cultivating important attributes among students, such as a willingness to learn the aims of pharmacists, an awareness of their own role as healthcare workers, and a desire to reflect on their future careers and lives. A follow-up survey of the participants was carried out three years after the training experience. The questionnaire verified that the training experience affected attendance at subsequent lectures and course determination after graduation. We confirmed the relationship between the participants' degree of satisfaction with the training experience and increased motivation for attending subsequent lectures. Through the training experience, participants discovered future targets and subjects of study. In addition, they became more interested in subsequent classroom lessons and their future. The greater the participants' degree of satisfaction with their training experience, the more interest they took in practical training and future courses. The present study clarified that the training experience was effective in cultivating important attributes such as a willingness to learn and an interest in future courses. Moreover, the training positively affected the course determination after graduation.

Possible involvement of caspases in proliferation of neocortical neural stem/progenitor cells in the developing mouse brain.

Caspases are well-known enzymes that work as initiators and effectors of apoptosis. To elucidate the role of caspases in neurodevelopment, we sought to determine if caspases are involved in the proliferation of neural stem/progenitor cells (NPCs) in the developing mouse brain. Labeling with 5-bromo-2'-deoxyuridine (BrdU) from days 14 to 18 of pregnant mice revealed that the 18-d old fetus had many BudU-positive cells in its brain. Double-labeling revealed that active caspase-3 was co-localized with these BrdU-positive cells in the neocortex, hippocampus, and subventricular zone of the fetal brain. Active caspase-3 was detected in cultures of NPCs derived from the neocortex of 15-d old fetuses during culture periods. Importantly, the pan-caspase inhibitor z-VAD-FMK was effective at completely inhibiting neurosphere formation by the NPCs. These results suggest the possibility that the caspase cascade is essential for the proliferation of neocortical NPCs in the developing mouse brain.

Disruption of ion-trafficking system in the cochlear spiral ligament prior to permanent hearing loss induced by exposure to intense noise: possible involvement of 4-hydroxy-2-nonenal as a mediator of oxidative stress.

Noise-induced hearing loss is at least in part due to disruption of endocochlear potential, which is maintained by various K(+) transport apparatuses including Na(+), K(+)-ATPase and gap junction-mediated intercellular communication in the lateral wall structures. In this study, we examined the changes in the ion-trafficking-related proteins in the spiral ligament fibrocytes (SLFs) following in vivo acoustic overstimulation or in vitro exposure of cultured SLFs to 4-hydroxy-2-nonenal, which is a mediator of oxidative stress. Connexin (Cx)26 and Cx30 were ubiquitously expressed throughout the spiral ligament, whereas Na(+), K(+)-ATPase α1 was predominantly detected in the stria vascularis and spiral prominence (type 2 SLFs). One-hour exposure of mice to 8 kHz octave band noise at a 110 dB sound pressure level produced an immediate and prolonged decrease in the Cx26 expression level and in Na+, K(+)-ATPase activity, as well as a delayed decrease in Cx30 expression in the SLFs. The noise-induced hearing loss and decrease in the Cx26 protein level and Na(+), K(+)-ATPase activity were abolished by a systemic treatment with a free radical-scavenging agent, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, or with a nitric oxide synthase inhibitor, N(ω)-nitro-L-arginine methyl ester hydrochloride. In vitro exposure of SLFs in primary culture to 4-hydroxy-2-nonenal produced a decrease in the protein levels of Cx26 and Na(+), K(+)-ATPase α1, as well as Na(+), K(+)-ATPase activity, and also resulted in dysfunction of the intercellular communication between the SLFs. Taken together, our data suggest that disruption of the ion-trafficking system in the cochlear SLFs is caused by the decrease in Cxs level and Na(+), K(+)-ATPase activity, and at least in part involved in permanent hearing loss induced by intense noise. Oxidative stress-mediated products might contribute to the decrease in Cxs content and Na(+), K(+)-ATPase activity in the cochlear lateral wall structures.