Ted H. Chiu

A low-noise flexible integrated system for recording and analysis of multiple electrical signals during sleep-wake states in rats.

A low-noise flexible system for the simultaneous recording and analysis of several electrical signals (EEG, ECG, EMG, and diaphragm EMG) from the same rat was constructed for studying changes in physiological functions during the sleep-wake cycle. The hardware in the system includes a multichannel amplifier, a video camera, a timer code generator, and a PC. A miniature buffer headstage with high-input impedance connected to a 6-channel amplifier was developed. All electrical activities devoid of 60 Hz interference could be consistently recorded by our low-cost amplifier with no shielding treatment. The analytical software was established in the LabVIEW environment and consisted of three major frames: temporal, spectral, and nonlinear analyses. These analytical tools demonstrated several distinct utilities. For example, the sleep-wake states could be successfully distinguished by combining temporal and spectral analyses. An obvious theta rhythm during rapid-eye-movement sleep (REMS) was recorded from parietal to occipital cortical areas but not from the frontal area. In addition, two types of sleep apnea with/without cardiac arrhythmias were observed under REMS condition. Moreover, the evoked potentials of the primary somatosensory cortex elicited by innocuous electrical pulses were modulated by vigilant states, especially under a slow-wave sleep state. These results show that our system delivers high-quality signals and is suitable for sleep investigations. The system can be easily expanded by combining other recording devices, like a plethysmograph. This compact system can also be easily modified and applied to other related physiological or pharmacological studies.

Caffeine and a selective adenosine A2A receptor antagonist induce sensitization and cross-sensitization behavior associated with increased striatal dopamine in mice

BackgroundCaffeine, a nonselective adenosine A1 and A2A receptor antagonist, is the most widely used psychoactive substance in the world. Evidence demonstrates that caffeine and selective adenosine A2A antagonists interact with the neuronal systems involved in drug reinforcement, locomotor sensitization, and therapeutic effect in Parkinsons disease (PD). Evidence also indicates that low doses of caffeine and a selective adenosine A2A antagonist SCH58261 elicit locomotor stimulation whereas high doses of these drugs exert locomotor inhibition. Since these behavioral and therapeutic effects are mediated by the mesolimbic and nigrostriatal dopaminergic pathways which project to the striatum, we hypothesize that low doses of caffeine and SCH58261 may modulate the functions of dopaminergic neurons in the striatum.MethodsIn this study, we evaluated the neuroadaptations in the striatum by using reverse-phase high performance liquid chromatography (HPLC) to quantitate the concentrations of striatal dopamine and its metabolites, dihydroxylphenylacetic acid (DOPAC) and homovanilic acid (HVA), and using immunoblotting to measure the level of phosphorylation of tyrosine hydroxylase (TH) at Ser31, following chronic caffeine and SCH58261 sensitization in mice. Moreover, to validate further that the behavior sensitization of caffeine is through antagonism at the adenosine A2A receptor, we also evaluate whether chronic pretreatment with a selective adenosine A2A antagonist SCH58261 or a selective adenosine A1 antagonist DPCPX can sensitize the locomotor stimulating effects of caffeine.ResultsChronic treatments with low dose caffeine (10 mg/kg) or SCH58261 (2 mg/kg) increased the concentrations of dopamine, DOPAC and HVA, concomitant with increased TH phosphorylation at Ser31 and consequently enhanced TH activity in the striatal tissues in both caffeine- and SCH58261-sensitized mice. In addition, chronic caffeine or SCH58261 administration induced locomotor sensitization, and locomotor cross-sensitization to caffeine was observed following chronic treatment of mice with SCH58261 but not with DPCPX.ConclusionsOur study demonstrated that low dosages of caffeine and a selective adenosine A2A antagonist SCH58261 elicited locomotor sensitization and cross-sensitization, which were associated with elevated dopamine concentration and TH phosphorylation at Ser31 in the striatum. Blockade of adenosine A2A receptor may play an important role in the striatal neuroadaptations observed in the caffeine-sensitized and SCH58261-sensitized mice.

Hydrogen peroxide increases the activity of rat sympathetic preganglionic neurons in vivo and in vitro

Reactive oxygen species (ROS) have been shown to modulate neuronal synaptic transmission and have also been implicated in cardiovascular diseases such as hypertension. The hypothesis that H(2)O(2) acting on sympathetic preganglionic neurons (SPNs) affects spinal sympathetic outflow was tested in the present study. H(2)O(2) was applied intrathecally via an implanted cannula to the T7-T9 segments of urethane-anesthetized rats. Blood pressure and heart rate were used as indices to evaluate the spinal sympathetic effects of H(2)O(2) in vivo. Intrathecal H(2)O(2) (100-1000 nmol) dose-dependently increased both the mean arterial pressure and heart rate. Reproducible pressor effects of H(2)O(2) (1000 nmol) applied consecutively at intervals of 30 min were observed. The pressor effects of intrathecal H(2)O(2) (1000 nmol) were attenuated by pretreatment with intrathecal administration of catalase (500 units), or N-acetyl-cysteine (1000 nmol). The pressor effects of intrathecal H(2)O(2) (1000 nmol) were also antagonized dose-dependently by prior intrathecal injection of AP-5 (DL-2-amino-5- phosphonovaleric acid, 10 and 30 nmol), or 6-cyano-7- nitroquinoxaline-2,3-dione, 10 and 30 nmol. In vitro electrophysiological study in spinal cord slices showed that superfusion of 1 mM H(2)O(2) for 3 min, which had no effect on membrane potential, caused an increase in amplitude of excitatory postsynaptic potentials in SPNs, but had little effect on that of inhibitory postsynaptic potentials. Taken together, these results demonstrated that oxidative stress in spinal cord may cause an increase in spinal sympathetic tone by acting on SPNs, which may contribute to ROS-induced cardiovascular dysfunction.

Peroxisomal proliferator-activated receptor-α protects renal tubular cells from doxorubicin-induced apoptosis

Peroxisome proliferator-activated receptor-α (PPAR-α) is a transcription factor and has been reported to inhibit cisplatin-mediated proximal tubule cell death. In addition, doxorubicin (Adriamycin)-induced nephrosis in rats is a commonly used experimental model for pharmacological studies of human chronic renal diseases. In this study, we investigated the protective effect of PPAR-α on doxorubicin-induced apoptosis and its detailed mechanism in NRK-52E cells and animal models. The mRNA level of PPAR-α was found to be reduced by doxorubicin treatment in NRK-52E cells. PPAR-α overexpression in NRK-52E cells significantly inhibited doxorubicin-induced apoptosis and the quantity of cleaved caspase-3. Endogenous prostacyclin (PGI2) augmentation, which has been reported to protect NRK-52E cells from doxorubicin-induced apoptosis, induced the translocation and activation of PPAR-α. The transformation of PPAR-α short interfering RNA was applied to silence the PPAR-α gene, which abolished the protective effect of PGI2 augmentation in doxorubicin-treated cells. To confirm the protective role of PPAR-α in vivo, PPAR-α activator docosahexaenoic acid (DHA) was administered to doxorubicin-treated mice, and it has been shown to significantly reduce the doxorubicin-induced apoptotic cells in renal cortex. However, this protective effect of DHA did not exist in PPAR-α-deficient mice. In NRK-52E cells, the overexpression of PPAR-α elevated the activity of catalase and superoxide dismutase and inhibited doxorubicin-induced reactive oxygen species (ROS). PPAR-α overexpression also inhibited the doxorubicin-induced activity of nuclear factor-κB (NF-κB), which was associated with the interaction between PPAR-α and NF-κB p65 subunit as revealed in immunoprecipitation assays. Therefore, PPAR-α is capable of inhibiting doxorubicin-induced ROS and NF-κB activity and protecting NRK-52E cells from doxorubicin-induced apoptosis.

Ceftriaxone attenuates hypoxic-ischemic brain injury in neonatal rats.

BackgroundPerinatal brain injury is the leading cause of subsequent neurological disability in both term and preterm baby. Glutamate excitotoxicity is one of the major factors involved in perinatal hypoxic-ischemic encephalopathy (HIE). Glutamate transporter GLT1, expressed mainly in mature astrocytes, is the major glutamate transporter in the brain. HIE induced excessive glutamate release which is not reuptaked by immature astrocytes may induce neuronal damage. Compounds, such as ceftriaxone, that enhance the expression of GLT1 may exert neuroprotective effect in HIE.MethodsWe used a neonatal rat model of HIE by unilateral ligation of carotid artery and subsequent exposure to 8% oxygen for 2 hrs on postnatal day 7 (P7) rats. Neonatal rats were administered three dosages of an antibiotic, ceftriaxone, 48 hrs prior to experimental HIE. Neurobehavioral tests of treated rats were assessed. Brain sections from P14 rats were examined with Nissl and immunohistochemical stain, and TUNEL assay. GLT1 protein expression was evaluated by Western blot and immunohistochemistry.ResultsPre-treatment with 200 mg/kg ceftriaxone significantly reduced the brain injury scores and apoptotic cells in the hippocampus, restored myelination in the external capsule of P14 rats, and improved the hypoxia-ischemia induced learning and memory deficit of P23-24 rats. GLT1 expression was observed in the cortical neurons of ceftriaxone treated rats.ConclusionThese results suggest that pre-treatment of infants at risk for HIE with ceftriaxone may reduce subsequent brain injury.

Excitatory action of lead on rat sympathetic preganglionic neurons in vitro and in vivo

Lead exposure elicited an increase in blood pressure and was considered to be a cardiovascular risk factor. The involvements of sympathetic nervous system and circulating catecholamines have been implicated in lead-induced hypertension. This study examined the effects of PbCl(2) on sympathetic preganglionic neurons (SPNs) in vitro and in vivo. In vitro electrophysiological study showed that superfusion of a low concentration (5 microM) of PbCl(2), which had no effects on membrane potential and spontaneous discharge rate, enhanced excitatory postsynaptic potentials (EPSPs) in some of the SPNs examined but inhibited inhibitory postsynaptic potentials (IPSPs) in other SPNs tested. A higher concentration (50 microM) of PbCl(2) inhibited both EPSPs and IPSPs in all SPNs examined. In vivo study showed that intrathecal injection of PbCl(2) (10 and 100 nmol) via an implanted cannula to the T7-T9 segments of urethane-anesthetized rats increased both the heart rate and mean arterial pressure. The pressor and tachycardic responses of intrathecal PbCl(2) (100 nmol) were attenuated by pretreatment with intravenous administration of hexamethonium (10 mg/kg) or intrathecal AP-5 (DL-2-amino-5-phosphonovaleric acid, 100 nmol), but were not significantly antagonized by prior intrathecal administration of CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, 100 nmol). Taken together, these results demonstrated that lead may exert a stimulatory effect on SPNs, which may result from the enhancement of EPSPs and inhibition of IPSPs by low concentrations of lead.

Novel Survivin Inhibitor YM155 elicits Cytotoxicity in Glioblastoma Cell Lines with Normal or Deficiency DNA-Dependent Protein Kinase Activity

BACKGROUND Pediatric glioblastoma is a malignant disease with an extremely poor clinical outcome. Patients usually suffer from resistance to radiation therapy, so targeted drug treatment may be a new possibility for glioblastoma therapy. Survivin is also overexpressed in glioblastoma. YM155, a novel small-molecule survivin inhibitor, has not been examined for its use in glioblastoma therapy. METHODS The human glioblastoma cell line M059K, which expresses normal DNA-dependent protein kinase (DNA-PK) activity and is radiation-resistant, and M059J, which is deficient in DNA-PK activity and radiation-sensitive, were used in the study. Cell viability, DNA fragmentation, and the expression of survivin and securin following YM155 treatment were examined using MTT (methylthiazolyldiphenyl-tetrazolium) assay, ELISA assay, and Western blot analysis, respectively. RESULTS YM155 caused a concentration-dependent cytotoxic effect, inhibiting the cell viability of both M059K and M059J cells by 70% after 48 hours of treatment with 50 nM YM155. The half-maximal inhibitory concentration (IC50) was around 30-35 nM for both cell lines. Apoptosis was determined to have occurred in both cell lines because immunoreactive signals from the DNA fragments in the cytoplasm were increased 24 hours after treatment with 30 nM YM155. The expression of survivin and securin in the M059K cells was greater than that measured in the M059J cells. Treatment with 30 nM YM155, for both 24 and 48 hours, significantly suppressed the expression of survivin and securin in both cell lines. CONCLUSION The novel survivin inhibitor YM155 elicits potent cytotoxicity in glioblastoma cells in vitro via DNA-PK-independent mechanisms. YM155 could be used as a new therapeutic agent for the treatment of human glioblastomas.

Inhibition by ethanol of NMDA‐induced responses and acute tolerance to the inhibition in rat sympathetic preganglionic neurons in vitro and in vivo

N‐methyl‐D‐aspartate (NMDA) receptors have been demonstrated to be a pivotal target for ethanol action. The present study examined the actions of acute ethanol exposure on NMDA‐induced responses and the acute tolerance to ethanol actions in rat sympathetic preganglionic neurons (SPNs) in vitro and in vivo. NMDA (50 μM) applied every 5 min induced reproducible membrane depolarizations of SPNs in neonatal spinal cord slice preparations. Ethanol (50 – 100 mM) applied by superfusion for 15 min caused a sustained decrease in NMDA‐induced depolarizations in a dose‐dependent and reversible manner. When the superfusion time of ethanol (100 mM) was increased to 50 min, NMDA‐induced depolarizations were attenuated initially but a gradual recovery was seen in ∼40% of SPNs tested. Repeated injections of NMDA (2 nmol) intrathecally at 30 min interval caused reproducible increases in mean arterial pressure (MAP) in urethane‐anesthetized rats. Intravenous injections of ethanol (0.16 or 0.32 g, 1 ml) inhibited NMDA‐induced pressor effects in a blood concentration‐dependent manner. The inhibition by ethanol of NMDA‐induced pressor effects was reduced over time during continuous infusion of ethanol or on the second injection 3.5 h after prior injection of a higher dose of ethanol. Ethanol, at concentrations significantly inhibited NMDA‐induced responses, had no significant effects on α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid‐induced responses. The study demonstrated the selective inhibition by ethanol of NMDA‐induced responses and the development of acute tolerance to the inhibitory effects in SPNs both in vitro and in vivo. These effects may play important roles in the ethanol regulation of cardiovascular function.

Acute exposure to trichloroethylene differentially alters the susceptibility to chemoconvulsants in mice

The effects of a common industrial solvent, trichloroethylene (TCE), which was once used as an anesthetic agent but its in vivo mechanism is still unknown, on convulsant-induced seizures in mice were examined. Pretreatment with TCE (250-2000 mg/kg, i.p.) significantly increased pentylenetetrazol (PTZ)-, picrotoxin (PIC)-, bicuculline (BIC)-, strychnine (STY)-, 4-aminopyridine (4-AP)- and N-methyl-D-aspartate (NMDA)-induced convulsion thresholds and lethal doses. However, the increase in convulsion thresholds and lethal doses was much greater for GABAergic antagonists (PIC, BIC, and PTZ) than non-GABAergic convulsants (STY, 4AP, and NMDA) following 2000 mg/kg TCE administration. Pre-treatment of mice with disulfiram (an inhibitor of CYP 4502E1) but not 4-methyl pyrazole (an inhibitor of alcohol dehydrogenase) significantly prolonged the time required for TCE (5000 mg/kg, i.p.) to induce the loss of righting reflex. These results suggest that acute exposure to TCE differentially alters the susceptibility to chemically induced convulsions in mice. The anticonvulsive effect of TCE may be predominantly mediated by GABA(A) receptors. In addition, TCE appears to exert a direct anesthetic effect.

Therapeutic potential of sepantronium bromide YM155 in gemcitabine-resistant human urothelial carcinoma cells

Survivin is overexpressed in transitional cell carcinoma (TCC), the most common type of bladder cancer. Previous reports demonstrated that knockdown of survivin by siRNA induced apoptosis of TCC cells. The present study evaluated the therapeutic effects of sepantronium bromide (YM155), a novel small molecule survivin inhibitor under clinical trials, on TCC cells in vitro. BFTC905, a grade III TCC cell line derived from a patient of blackfoot disease in Taiwan, was the most gemcitabine-resistant cell line when compared to BFTC909, TSGH8301 and T24 in cytotoxicity assay, resulting from upregulation of securin and bcl-2 after gemcitabine treatment. YM155 caused potent concentration‑dependent cytotoxicity in 4 TCC cell lines (IC50s ≤20 nM), but exhibited no cytotoxicity in survivin-null primary human urothelial cells. For BFTC905 cells, addition of gemcitabine and/or cisplatin, the standard TCC chemotherapy regimen, to YM155 did not exert additive cytotoxic effects. Molecular analyses indicated that YM155 inhibited the proliferation of BFTC905 cells by increasing p27kip1, suppressing Ki-67, and inducing quiescence. In addition, YM155 elicited apoptosis manifested with DNA fragmentation through suppressing the expression of survivin, securin and bcl-2. Furthermore, YM155 induced autophagy in BFTC905 cells as autophagic inhibitor, 3-methyladenine, attenuated YM155-induced LC3B-II levels and reversed the cytotoxicity of YM155. mTOR inhibitors sirolimus and everolimus did not increase YM155-induced expression of LC3B-II nor augment YM155-induced cytotoxicity. These results indicate that YM155 exerts its lethal effect on BFTC905 cells via apoptotic and autophagic death pathways and suggest that YM155 may be a potential drug for the therapy of gemcitabine-resistant bladder cancer.