Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas.

BACKGROUND: Several human cancers have been found to contain cancer stem-like cells (CSCs) having cancer-initiating ability. However, only a few reports have shown the existence of CSCs in bone and soft tissue sarcomas. In this study, we identified and characterised side population (SP) cells that showed drug-resistant features in human bone sarcoma cell lines. METHODS: In seven osteosarcoma cell lines (OS2000, KIKU, NY, Huo9, HOS, U2OS and Saos2) and in one bone malignant fibrous histiocytoma (MFH) cell line (MFH2003), the frequency of SP cells was analysed. Tumourigenicity of SP cells was assessed in vitro and in vivo. Gene profiles of SP cells and other populations (main population; MP) of cells were characterised using cDNA microarrays. RESULTS: SP cells were found in NY (0.31%) and MFH2003 (5.28%). SP cells of MFH2003 formed spherical colonies and re-populated into SP and MP cells. In an NOD/SCID mice xenograft model, 1 x 10(3) sorted SP cell-induced tumourigenesis. cDNA microarray analysis showed that 23 genes were upregulated in SP cells. CONCLUSIONS: We showed that SP cells existed in bone sarcoma cell lines. SP cells of MFH2003 had cancer-initiating ability in vitro and in vivo. The gene profiles of SP cells could serve as candidate markers for CSCs in bone sarcomas.

The disruptive effects of ketamine on passive avoidance learning in mice: involvement of dopaminergic mechanism.

The involvement of dopaminergic mechanisms in ketamine-induced disruption of one trial step-through passive avoidance performance was assessed through the coadministration with the dopamine D1 antagonist SCH 23390, the dopamine D2 antagonist YM-091512 and the dopamine autoreceptor agonist at low doses, apomorphine, in mice. Pretraining (10 min before) administration of ketamine (0; saline, 2.5, 5 and 10 mg/kg SC) dose-dependently reduced the latency in the retention trial conducted 24 h after the training. However, ketamine did not affect the retention latency when administered immediately after the training or prior to retention. YM-09151-2 (0.01 and 0.03 mg/kg SC) and apomorphine (0.01 and 0.03 mg/kg SC), but not SCH 23390 (0.01 and 0.03 mg/kg SC), ameliorated the impaired reduction by ketamine (10 mg/kg) in a dose-dependent manner. These results suggest that ketamine obstructs the acquisition of the passive avoidance task, and that this effect is induced by stimulation of dopamine D2 receptors through dopamine release from the presynaptic terminals.

Repeated ketamine administration produces up-regulation of muscarinic acetylcholine receptors in the forebrain, and reduces behavioral sensitivity to scopolamine in mice.

To study the effects of repeated ketamine administration on central muscarinic acetylcholine receptors (mAchRs), ddY male mice were administered subcutaneous doses of 25 mg/kg ketamine every 3 days for a total of five times. Receptor binding assays of mAchR were carried out in the forebrain (FB), cerebellum (CB) and brainstem (BS), using [3H]quinuclidinyl benzilate ([3H]QNB) as a ligand. In addition, we examined whether repeated ketamine (12.5, 25 and 50 mg/kg) or saline (five times) could modify the hyperlocomotion induced by scopolamine (0.5 mg/kg, SC) (a muscarinic antagonist), using a behavior-pharmacological technique. Repeating the ketamine administration resulted in a significant increase in the receptor density value (Bmax) for [3H]QNB only in FB, dependent on the numbers of administrations (1270 +/- 33 fmol/mg protein for a single dose, 1620 +/- 59 for four treatments, 1738 +/- 70 for five treatments without any change in apparent affinity (defined as the reciprocal of the dissociation constant) (Kd). A competitive inhibition study of repeated (5 times) administration of ketamine failed to detect any subtype-specific changes in mAchRs. Repeated ketamine administration reduced the scopolamine-induced hyperlocomotion in a dose-related way, and the changes were significant at 50 mg/kg. Our results suggest that repeated ketamine administration produces an up-regulation of mAchRs, and this change may be associated with altered Ach transmission in the central nervous system.

Effect of dexmedetomidine on the release of [3H]-noradrenaline from rat kidney cortex slices: characterization of alpha2-adrenoceptor.

The presynaptic modulation of [3H]-noradrenaline (NA) release from rat kidney cortex slices, a method used for the first time, was investigated. Rat kidney cortex slices were loaded with [3H]-NA and the release of radioactivity at rest and in response to field stimulation was determined. The alpha(2)-adrenoceptor agonist, dexmedetomidine inhibited the stimulation-evoked release of NA from kidney slices in a concentration-dependent manner, whereas alpha(2)-adrenoceptor antagonist CH-38083 (7,8-methyenedioxy-14-alpha-hydroxyalloberbane HCl), an alpha(2)-adrenoceptor antagonists, enhanced it. When dexmedetomidine and BRL-44408, a selective alpha(2A) antagonist, were added together, the effect of dexmedetomidine was significantly antagonized. In contrast, ARC-239 (2-(2,4-(o-piperazine-1-yl)-ethyl-4,4-dimethyl-1,3-(2H, 4H)disoguinolinedione chloride), a selective alpha(2B)-antagonist, had no effect on the release and failed to prevent the effect of dexmedetomidine. Prazosin, an alpha(1)- and alpha(2B/C)-adrenoceptor antagonist enhanced the release evoked by field stimulation. It is therefore suggested that there is a negative feedback modulation of NA release at the sympathetic innervation of kidney cortex, and dexmedetomidine, a clinically used anesthetic adjunct inhibits the release via activation of alpha(2C)-adrenoceptors.

Excessive release of [3H]noradrenaline and glutamate in response to simulation of ischemic conditions in rat spinal cord slice preparation: effect of NMDA and AMPA receptor antagonists.

In the present study we investigated the effects of NMDA and non-NMDA glutamate receptor antagonists on the ischemia-evoked release of [3H]noradrenaline from rat spinal cord slices. An in vitro ischemia model (oxygen and glucose deprivation) was used to simulate the ischemic conditions known to cause neuronal injury. Spinal cord slices were loaded with [3H]noradrenaline and superfused with Krebs solution in a micro-organ bath. Both axonal stimulation and ischemia increased the release of [3H]noradrenaline, but the release in response to glucose and oxygen deprivation was [Ca2+]o independent. Dizocilpine (MK-801), an NMDA receptor antagonist, suppressed the release of [3H]noradrenaline produced by ischemia, while it enhanced the release of [3H]noradrenaline evoked by electrical field stimulation. In contrast, LY300168 (GYKI-53655) [(+/-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylen e-dioxy-5H-2.3-benzodiazepine] and its (-)isomer LY303070 (GYKI-53784) [(-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene- dioxy-5H-2.3-benzodiazepine] AMPA receptor antagonists, had no effect on the release of [3H]noradrenaline evoked by either electrical stimulation or ischemia. Desipramine, a noradrenaline uptake inhibitor, potentiated the release of [3H]noradrenaline evoked by ischemia, while in the absence of [Ca2+]o but under conditions when [3H]noradrenaline release was further increased, it reduced the release. Dizocilpine also decreased glutamate and aspartate release, measured by high performance liquid chromatography, during ischemia. It is concluded that glutamate release and NMDA receptors, but not AMPA receptors, are involved in the acute effect of oxygen and glucose deprivation on the excessive release of noradrenaline and that this release is not related to physiological axonal conduction.

Na+ channel block prevents the ischemia-induced release of norepinephrine from spinal cord slices.

The principal finding of the present study with rat spinal cord slices was the novel demonstration of the [Ca2+]o-independent effect of ischemia on norepinephrine release and its antagonism by tetrodotoxin and low temperature (10 degrees C). Our finding that tetrodotoxin antagonized the effects of glucose deprivation on norepinephrine release in a [Ca2+]o-independent way suggests that Na+ channel block alone, i.e., the prevention of Na+ accumulation, may account for the protective action. Low temperature completely prevented the effect of ischemia on norepinephrine release but did not change the release associated with axonal activity. This finding is in good agreement with the observation that small changes in brain temperature critically determine the extent of neuronal injury from ischemia and suggests that both [Ca2+]o-independent release and cell injury are associated with the norepinephrine membrane carrier. It is suggested, therefore, that drugs able to attenuate the increase in [Na+]i during ischemia may be useful agents to protect against ischemic damage if given before the insult.

Long-continuous observation of the effects of methamphetamine on wheel-running and drinking in mice.

1. Effects of methamphetamine (MAP) on wheel-running and drinking in mice, housed under 12-hr light-dark schedule (light period; 06:00-18:00), were investigated through long-continuous observation. 2. MAP (1, 2 and 4 mg/kg, s.c.) acutely increased the wheel-running and drinking for 2-4 hr in a dose-dependent manner after the administration at 11:00, midpoint of light period. 3. MAP administered at 11:00 sub-acutely suppressed the spontaneous increment during dark period (18:00-06:00) in both the behaviors. 4. Many factors, such as the time-of-day and interval of the administration as well as the dose administered, affected the behavioral suppression induced by MAP. 5. In addition to these findings, the wheel-running and drinking during the light period increased even on the days without MAP administration. 6. These results suggest that MAP have not only acute stimulant and sub-acute depressant effects, but also long-lasting effects.

Effects of dopamine antagonism on methamphetamine sensitization: evaluation by ambulatory activity in mice.

SCH 23390 (SCH: 0.001-0.03 mg/kg SC) and YM-09151-2 (YM: 0.001-0.03 mg/kg SC), the selective dopamine D1 and D2 antagonists, respectively, reduced dose-dependently the ambulation-increasing effect of methamphetamine (MAP: 2 mg/kg SC) in mice. The sensitization to MAP was inhibited when it was administered in combination with SCH (0.003-0.03 mg/kg) or YM (0.003-0.03 mg/kg) in the repeated administration regimen. The inhibitory action of YM on the MAP sensitization was more prominent than that of SCH. However, the repeated treatment with either SCH or YM could not ameliorate the established MAP sensitization. Rather, the repeated treatment with the highest dose of YM (0.03 mg/kg) increased the MAP sensitivity in both the MAP-sensitized and drug-naive mice. SCH had no such action. The present results suggest that the dopamine D2 receptors are more intimately involved than the dopamine D1 receptors in the increased sensitivity to MAP induced by the repeated treatment with MAP itself, behavioral sensitization, or dopamine antagonists, denervation supersensitivity.

Interactions of opioids with caffeine: evaluation by ambulatory activity in mice.

Morphine (up to 10 mg kg-1), buprenorphine (up to 0.1 mg kg-1), pentazocine (30 mg kg-1) and caffeine (up to 10 mg kg-1), significantly increased mouse ambulation. The combination of morphine, buprenorphine and pentazocine with caffeine generally enhanced the effect. Dopamine D1- and D2-receptor bockade, depletion of stored dopamine, and inhibition of dopamine synthesis could reduce the ambulation increased by single administration of morphine, buprenorphine and caffeine, and by combined administration of morphine and buprenorphine with caffeine. Although naloxone (0.1-3 mg kg-1) itself did not change mouse ambulation, at 3 mg kg-1, it reduced the effect of caffeine. The repeated administration of morphine (10 mg kg-1) induced a sensitization to the ambulation-increasing effect, and was inhibited by the combination of caffeine (10 mg kg-1) in the repeated administration schedule. The repeated administration of caffeine (10 mg kg-1) with buprenorphine (0.3 mg kg-1) resulted in a decrease in the effect to the level of caffeine alone. The development of cross-sensitization to morphine (10 mg kg-1) by the repeated treatment with buprenorphine (0.3 mg kg-1) was inhibited by caffeine (10 mg kg-1). Our results suggest that the dopaminergic systems are involved in the enhanced interaction of opioids having agonistic action on mu- or sigma-receptors with caffeine. However, it is also considered that, following the repeated administration, caffeine acts to reduce the sensitivity to the ambulation-increasing effect of opioids, probably inducing up-regulation of adenosinergic systems.

Dopamine antagonists can inhibit methamphetamine sensitization, but not cocaine sensitization, when assessed by ambulatory activity in mice.

The repeated subcutaneous administration of methamphetamine (2 mg kg-1) and cocaine (10 mg kg-1) at 3-4 day intervals induced sensitization to their ambulation-increasing effects in mice. Subcutaneous administration of SCH 23390 (R-(+)-7-chloro-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; 0.003-0.03 mg kg-1) and YM-09151-2 (cis-N-(1-benzyl-2-methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4- methylaminobenzamide; 0.003-0.03 mg kg-1), the selective dopamine D1 and D2 antagonists, respectively, reduced dose-dependently the acute ambulation-increasing effect of methamphetamine. The development of methamphetamine sensitization was inhibited when it was administered in combination with either SCH 23390 or YM-09151-2 in the repeated administration schedule. Although SCH 23390 (0.01-0.1 mg kg-1) and YM-09151-2 (0.01-0.1 mg kg-1) also reduced the ambulation-increasing effect of cocaine (10 mg kg-1), neither drug inhibited the cocaine sensitization. Mice given cocaine with SCH 23390 (0.03 mg kg-1) or YM-09151-2 (0.03 and 0.1 mg kg-1) showed higher sensitivity than those given cocaine alone. The present results suggest that, although both the dopamine D1 and D2 antagonists reduce the acute stimulant effects of both methamphetamine and cocaine, they are only effective for inhibition of the methamphetamine sensitization. Mechanisms other than the dopaminergic system appear to be involved in the cocaine sensitization.

Long-term clomipramine treatment upregulates forebrain acetylcholine muscarinic receptors, and reduces behavioural sensitivity to scopolamine in mice.

We have investigated the effects of long-term treatment with clomipramine, a tricyclic antidepressant, on central muscarinic acetylcholine receptors (mAChR) in mice. Repeated clomipramine administration resulted in an increase in the forebrain receptor density value (Bmax) for [3H]quinuclidinyl benzilate, a muscarinic ligand (P < 0.05), that was dependent on dose per administration (saline or 5, 10, or 20 mg kg(-1) once a day for 7 days) and number of days treated (20 mg kg(-1) for 1, 3, 5, or 7 days). No change in apparent affinity (defined as the reciprocal of the dissociation constant) (KD) occurred. Seven daily treatments with clomipramine (saline or 5, 10, or 20 mg kg(-1)) reduced hyperlocomotion induced by scopolamine (0.5 mg kg(-1), s.c.) dose-dependently, and the effect of 20 mg kg(-1) clomipramine was significant (P < 0.05). These results suggest that an upregulation of mAChR is produced by repeated clomipramine administration, and such a change is responsible for the decreased sensitivity to the muscarinic antagonist scopolamine.

Delayed effects of ethanol, caffeine and nicotine assessed by wheel-running and drinking in mice.

Effects of ethanol, caffeine and nicotine, pleasurable substances, on wheel-running and drinking in mice that were housed under a 12 : 12-hr light-dark schedule (lighting period ; 6 : 00-18 : 00) were investigated. All drug administrations were carried out at 11: 00, a mid-light period. Although ethanol (0.8-2.4 g/kg p.o.) scarcely changed both the wheel-running and drinking during the light period, it was followed by a strong suppression of both behaviors during the coming dark period (18 : 00-6 : 00). The same treatment with caffeine (1-10 mg/kg s.c.) produced significant increase in the drinking during the light period, but suppression of the wheel-running during the dark period. Nicotine (0.1-1 mg/kg s.c.) significantly suppressed the wheel-running, but not drinking, during the dark period. The coadministration of nicotine (0.1-1 mg/kg) with ethanol (2.4 g/kg) reduced the behavioral suppression during the dark period. Whereas nicotine (0.1-1 mg/kg) reduced the increased drinking during light period by caffeine (10 mg/kg), but enhanced the caffeine-induced behavioral suppression during the dark period. These results indicate that the administration of pleasurable substances in the mid-light period results in a delayed effect which is characterized by a suppression of either and/or both wheel-running and drinking during the coming dark period starting 7 hr after the administration, and that nicotine acts to antagonize the effect of ethanol, but contrally to enhance the effect of caffeine.

Evaluation of the cardiovascular responses to fiberoptic orotracheal intubation with television monitoring: comparison with conventional direct laryngoscopy.

STUDY OBJECTIVE: To evaluate and compare cardiovascular responses to a new method of orotracheal intubation incorporating TV monitoring, with conventional orotracheal intubation via rigid blade laryngoscopy. DESIGN: Prospective single-blind study. SETTING: Operating room of a medical college hospital. PATIENTS: 90 ASA physical status I and II surgical patients requiring general anesthesia and orotracheal intubation. INTERVENTIONS: Patients were randomly allocated to two groups, one for the new intubation method and the other for conventional intubation using a rigid laryngoscope. In the new method, an anesthesiologist inserted an endotracheal tube alone into the trachea via TV monitoring through the bronchoscope, which was inserted by an assistant through the mouth to the middle larynx. The patient's trachea was intubated without extreme stretching of laryngeal tissues or deep insertion of the tip of the bronchoscope. In the conventional method, orotracheal intubation was performed with rigid direct laryngoscopy. MEASUREMENTS: Noninvasive blood pressure (BP) and heart rate (HR) were measured before arrival at the operating room, and before and after orotracheal intubation. MAIN RESULTS: Although this method was expected to be a minimally invasive fiberoptic intubation technique, the patients showed significant increases in BP and HR. No significant differences between the two groups were observed in cardiovascular responses immediately after intubation: the systolic BP, 169.5 +/- 28.3 versus 167.0 +/- 23.1 mmHg, and HR, 100.2 +/- 18.2 versus 98.8 +/- 16.6 bpm. CONCLUSIONS: Insertion of an endotracheal tube may itself be the most invasive stimulus during intubation procedures.

[Effects of systemic lidocaine, mepivacaine and bupivacaine on passive avoidance learning in mice].

The effects of local anesthetics on learning were assessed in mice through one trial step-through passive avoidance performance. Lidocaine HCl (10, 20 or 40 mg.kg-1), mepivacaine HCl (15, 30 or 60 mg.kg-1) or bupivacaine HCl (2.5, 5 or 10 mg.kg-1) were injected subcutaneously 10 min before acquisition training. Lidocaine, but not mepivacaine and bupivacaine, reduced the latency in the retention trial conducted 24hr after the training in a dose-dependent manner. However, lidocaine did not alter the latency when it was given 10 min before the retention trial. These results suggest that lidocaine impairs acquisition, but not retention or retrieval phase in the memory process.

[Urinary retention as a transient neurologic symptom after accidental total spinal anesthesia with mepivacaine hydrochloride].

We report a patient in whom urinary retention as a transient neurologic symptoms (TNS) developed after accidental total spinal anesthesia with mepivacaine hydrochloride. Mepivacaine, an amide local anesthetic, has been used for spinal anesthesia and considered one of the best for spinal anesthesia for its low incidence of TNS. However, we suggest that TNS associated with mepivacaine might not be a rare complication in spinal anesthesia.

[Rhabdomyolysis induced by succinylcholine chloride and sevoflurane in an elderly man].

An 81-year-old man was scheduled for cervical lymph node biopsy. His laboratory data were within normal ranges. After induction of anesthesia with thiopental 175 mg and succinylcholine chloride (SCC) 40 mg, moderate masseter spasm was observed. Anesthesia was maintained with nitrous oxide, oxygen and sevoflurane. After the operation he had severe muscle pain and CK was elevated up to 81,400IU.l-1. The body temperature was not elevated above 37.2 degrees C during and after the operation. The skinned fiber examination, performed one month later, showed his calcium-induced-calcium-release (CICR) to be within normal ranges. We diagnosed him as rhabdomyolysis induced by coadministration of SCC and sevoflurane, especially SCC. We concluded that even in an elderly man, SCC should be administered cautiously.

[Halothane anesthesia decreases the level of interstitial striatal dopamine of awake freely moving rats in an in vivo microdialysis study].

We investigated the effect of halothane on the level of interstitial dopamine of in vivo awake, free moving rats brain striatum using microdialysis techniques. Rats were implanted a microdialysis probe to right striatum of the brain and administered 1.5% of halothane (approximately 1.2 MAC) for 1 or 2 hours, and dialysates from the probe were determined every 20 minutes. Halothane anesthesia reduced the amount of dopamine derived from dialysate, and after discontinuation of halothane and at emergence from anesthesia, the level of dopamine was increased. The levels of metabolites of dopamine during anesthesia were increased lineally in a time dependent manner. We hypothesized that halothane might increase the rate of re-uptake of dopamine at nerve endings and decreased level of interstitial dopamine is compensated by dopamine releases during anesthesia.

[Anesthetic management of a patient with microcephaly associated with cerebral atrophy].

We describe the management of a 35-year-old female with microcephaly, cerebral atrophy especially in frontal lobes, dwarfism and severe mental retardation. The patient underwent open reduction of fracture of the humerus neck suffered from traffic accident. Authors could not find any article on the anesthetic management of microcephaly but could locate some about mentally retarded patients claiming the difficulty in anesthetic induction as well as emergence and frequent incidence of convulsions. We anesthetized the patient with modified NLA with nitrous oxide-oxygen and interestingly very small doses of fentanyl (1.7 micrograms.kg-1) were required. The anesthetic and surgical courses were uneventful. The patient emerged immediately with intravenously administered naloxone and flumazenil with no excitement nor convulsion. We conclude that fentanyl is the drug of choice for mentally retarded patient with microcephaly and smaller doses per kg.weight are sufficient for orthopedic surgery.

[The effect of sevoflurane and isoflurane on striatal dopamine of awake freely moving rats observed in an in vivo microdialysis study].

We investigated the effect of sevoflurane and isoflurane on the level of interstitial dopamine of in vivo awake, free moving rats brain striatum using microdialysis techniques. Rats were implanted with a microdialysis probe to the right striatum of the brain and administered with 1.2 MAC of each volatile anesthetics for 1 hour, and dialysates from the probe were determined every 20 minutes. Both anesthetics reduced the amount of dopamine derived from dialysate, and increased the efflux of dopamine with pretreatment of nomifensine 10mg. kg-1 i.p. The change of metabolites of dopamine during anesthesia was increased. No significant difference was found between sevoflurane and isoflurane. We hypothesized that these anesthetics might have special actions on interactions between metabolism and re-uptake of dopamine in rats striatum during anesthesia.

[Preliminary report: the effect of flumazenil on the hypnotic dose of propofol in ddY mice].

The present investigation dealt with the effect of simultaneous administration of flumazenil on the hypnotic activity of propofol using a behavioral model of ddY mice. The mixed solution of propofol and flumazenil was administered intravenously into the mice tail vein and the achievement of hypnosis was defined as the loss of the righting reflex. Flumazenil 0.2 mg.kg-1 significantly decreased the required dose of propofol for hypnosis (8.43 +/- 0.46 mg.kg-1) compared to the control group (10.55 +/- 0.55 mg.kg-1). The mixture with a pH-3.9 acetate buffer solution did not change the hypnotic dose of propofol (10.88 +/- 0.62 mg.kg-1). The results suggest that flumazenil might potentiate the hypnotic activity of propofol in ddY mice.