Satoshi Shibuta

A comparative study of the antinociceptive action of xenon and nitrous oxide in rats

We attempted to clarify the mechanism of antinociceptive action induced by xenon and nitrous oxide.Eighty percent of nitrous oxide or 80% xenon was applied to rats inside enclosed clear plastic glass cylinders with their tails protruding for assessment of the tail-flick response to radiant heat. With repeated testing, there was a rapid reduction to nitrous oxide antinociception within 90 min, which was interpreted as development of tolerance, but not to xenon antinociception. Nitrous oxide antinociception was blocked by the intraperitoneal administration of 0.1 or 1.0 mg/kg yohimbine, but not by 1.0 or 5.0 mg/kg L659-066 or by 5.0 or 10 mg/kg naloxone. Xenon antinociception was not affected by any of these drugs. Yohimbine and L659-066 are characterized as alpha2-adrenoceptor antagonists. Although yohimbine penetrates the blood-brain barrier after systemic administration, L659-066 does not penetrate it and act peripherally. Therefore, the results indicate that alpha2-adrenoceptors, but not opioid receptors, may play a key role in antinociception induced by nitrous oxide in the central nervous system. Furthermore, the mechanism of xenon antinociception differs from that of nitrous oxide because it does not involve either alpha2 or opioid receptors. Implications: The precise mechanism of antinociceptive action of nitrous oxide and xenon remains unknown. It is still controversial whether an opioid system plays a role in antinociception induced by nitrous oxide. The results of the study showed that antagonism of central alpha2-adrenoceptors, but not opioid receptors, reverses the antinociception induced by nitrous oxide but not by xenon, which indicates that alpha2-adrenoceptors may play a key role in nitrous oxide antinociception. (Anesth Analg 1997;85:931-6)

Intrathecal administration of a new nitric oxide donor, NOC-18, produces acute thermal hyperalgesia in the rat.

A nitric oxide releasing compound, NOC-18, was injected intrathecally in order to determine the role of NO in spinal nociceptive mechanisms in rats. The nociceptive threshold was evaluated by the radiant heat tail-flick test. The effects of intrathecal injection of N-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor; methylene blue (MB), a soluble guanylate cyclase inhibitor and hemoglobin (Hb), an NO scavenger, on the nociceptive threshold were measured in the presence and absence of 0.1, 1 and 10 microg of NOC-18. The results were compared with a control group of rats which were injected with the same volume of normal saline. NOC-18 caused a dose-dependent curtailment of the tail-flick latency during the period from 15 to 150 min. L-NAME, MB and Hb all produced prolongation of the tail-flick latency during the same time period. The hyperalgesia induced by this concentration range of NOC-18 was completely blocked by Hb, but was not affected by either L-NAME or MB. These findings indicate that NO plays a direct role in thermal hyperalgesia in the spinal cord, and that an another pathway in addition to the NO-cGMP pathway may be involved.

Nitric oxide-induced cytotoxicity attenuation by thiopentone sodium but not pentobarbitone sodium in primary brain cultures

We describe the effects of barbiturates on the neurotoxicity induced by nitric oxide (NO) on foetal rat cultured cortical and hippocampal neurones. Cessation of cerebral blood flow leads to an initiation of a neurotoxic cascade including NO and peroxynitrite. Barbiturates are often used to protect neurones against cerebrovascular disorders clinically. However, its neuroprotective mechanism remains unclear. In the present experiment, we established a new in vitro model of brain injury mediated by NO with an NO‐donor, 1‐hydroxy‐2‐oxo‐3‐(3‐aminopropyl)‐3‐isopropyl‐1‐triazene (NOC‐5) on grid tissue culture wells. We also investigated the mechanisms of protection of CNS neurones from NO‐induced neurotoxicity by thiopentone sodium, which contains a sulphhydryl group (SH‐) in the medium, and pentobarbitone sodium, which does not contain SH‐. Primary cultures of cortical and hippocampal neurones (prepared from 16‐day gestational rat foetuses) were used after 13–14 days in culture. The cells were exposed to NOC‐5 at the various concentrations for 24 h in the culture to evaluate a dose‐dependent effect of NOC‐5. To evaluate the role of the barbiturates, neurones were exposed to 4, 40 and 400 μM of thiopentone sodium or pentobarbitone sodium with or without 30 μM NOC‐5. In addition, superoxide dismutase (SOD) at 1000 u ml−1 and 30 μM NOC‐5 were co‐administered for 24 h to evaluate the role of SOD. Exposure to NOC‐5 induced neural cell death in a dose‐dependent manner in both cortical and hippocampal cultured neurones. Approximately 90% of the cultured neurones were killed by 100 μM NOC‐5. This NOC‐5‐induced neurotoxicity was significantly attenuated by high concentrations of thiopentone sodium (40 and 400 μM) as well as SOD, but not by pentobarbitone sodium. The survival rates of the cortical neurones and hippocampal neurones that were exposed to 30 μM NOC‐5 were 11.2±4.2% and 37.2±3.0%, respectively, and in the presence of 400 μM thiopentone sodium, the survival rate increased to 65.3±3.5% in the cortical neurones and 74.6±2.2% in the hippocampal neurones. These findings demonstrate that thiopentone sodium, which acts as a free radical scavenger, protects the CNS neurones against NO‐mediated cytotoxicity in vitro. In conclusion, thiopentone sodium is one of the best of the currently available pharmacological agents for protection of neurones against intraoperative cerebral ischaemia.

Intracerebroventricular administration of a nitric oxide-releasing compound, NOC-18, produces thermal hyperalgesia in rats

This study was undertaken to define the role of nitric oxide (NO) in central nociceptive mechanisms by intracerebroventricular injection of an NO-releasing compound, NOC-18, in rats. The nociceptive threshold was evaluated by the radiant heat tail-flick test. Sixty-nine rats were divided into the seven groups, and the following drugs were injected intracerebroventricularly in 5 microliters of saline: no drug (control) (n = 13), 15 micrograms of NOC-18 (n = 15); 150 micrograms of NOC-18 (n = 9); 100 micrograms of N-nitro-L-arginine methyl ester (L-NAME) (n = 8); 15 micrograms of NOC-18 + 100 micrograms of L-NAME (n = 8); 10 micrograms of methylene blue (MB) (n = 8); 15 micrograms of NOC-18 + 10 micrograms of MB (n = 8). NOC-18 caused a dose-dependent curtailment (7% and 23% decreases for 15 micrograms and 150 micrograms of NOC-18, respectively) of the tail-flick latency during the period from 15 to 120 min. L-NAME caused prolongation (15% maximum) of the tail-flick latency during the period from 15 to 150 min. However, NOC-18-induced hyperalgesia was not influenced by L-NAME. MB also caused prolongation (9% maximum) of the tail-flick latency during the period from 15 to 150 min, and completely blocked the hyperalgesia induced by 15 micrograms of NOC-18. These findings indicate that the NO-cGMP pathway is directly involved in thermal hyperalgesia in the brain.

Cardiovascular Effects of an Ultra–Short-Acting Nitric Oxide–Releasing Compound, Zwitterionic Diamine/NO Adduct, in Dogs

BACKGROUND This study was conducted to clarify the cardiovascular effects of a new NO-releasing compound, NOC-7, and to compare it with other nitrovasodilators, sodium nitroprusside (SNP) and nitroglycerin, in dogs anesthetized with pentobarbital. METHODS AND RESULTS A bolus injection of NOC-7 decreased mean aortic blood pressure in a dose-dependent manner. The onset was rapid and the recovery quick. Continuous infusion of NOC-7 decreased mean aortic pressure from 115 +/- 3.9 to 84 +/- 2.9 mm Hg and infusion of SNP, from 118 +/- 3.8 to 87 +/- 3.1 mm Hg. The optimum doses of NOC-7 and SNP were determined to be 2.73 +/- 0.77 and 11.5 +/- 6.1 micrograms.kg-1.min-1, respectively. During infusion of NOC-7, heart rate and cardiac output were increased (P < .05), pulmonary artery pressure was not changed, and systemic and pulmonary vascular resistances were decreased (P < .05). Electromagnetic flowmetry showed that portal venous and internal carotid arterial blood flow were increased (P < .05) and that hepatic and renal arterial blood flows were not changed. These hemodynamic changes during NOC-7 infusion were similar to those with SNP. The plasma level of NO2-/NO3 did not change, but methemoglobin increased slightly (P < .05). Comparison between hypotensive responses before and after a 3.5-hour infusion of NOC-7 or nitroglycerin showed that acute tolerance developed to nitroglycerin but not to NOC-7. CONCLUSIONS The results indicate that NOC-7 may be useful as an ultra-short-acting nitrovasodilator that has no major adverse effect or tolerance.

The effects of propofol on NMDA- or nitric oxide-mediated neurotoxicity in vitro

Acute brain ischemia causes neurotoxic cascades including NMDA receptors and NO. Propofol, an i.v. anesthetic, is thought to have a neuroprotective effect. We investigated the influence of propofol on NMDA/NO neurotoxicity using Shibutas established model of primary brain cultures. Cortical neurons prepared from E16 were used after 13–14 days in culture. The neurons were exposed to various concentrations of propofol with NMDA or NO-donor. The survival rates of neurons exposed to 30 μM NMDA with or without 300 μM propofol were 12.1 ± 2.2% and 11.9 ± 2.2%, respectively. The survival rates exposed to 30 μM NO-donor with or without 300 μM propofol were 11.2 ± 4.2% and 14.0 ± 3.9%, respectively. These results suggest that neuroprotective effect of propofol is limited and propofol does not offer advantages over thiopental against NMDA/NO-induced cytotoxicity.

A new nitric oxide donor, NOC-18, exhibits a nociceptive effect in the rat formalin model

The utility of a new nitric oxide (NO) donor, NOC-18, and the contribution of the neurotransmitter NO to nociception in response to tissue injury in rats, were examined following the subcutaneous injection of formalin into the hindpaw. This model induces biphasic responses in pain-related behavior, such that C-fiber activation during the first phase triggers a state of central sensitization characterized by the second phase. Formalin-induced nociceptive behavior was facilitated by intracerebroventricular administration of NOC-18 in the second phase, but not the first phase. This enhancement was completely abolished by the soluble guanylate cyclase inhibitor, methylene blue. These findings indicate that NO causes nociception via the NO-cGMP pathway in the central nervous system and NOC-18 proved to be a convenient and useful tool for the investigation of nociception-related NO.

Hypothermia and thiopentone sodium: Individual and combined neuroprotective effects on cortical cultures exposed to prolonged hypoxic episodes

Because there are many conflicting reports on cerebroprotective effects of hypothermia and barbiturates, we examined the degree of neuroprotection at defined temperatures (normothermia, 37°C; mild hypothermia, 32°C; deep hypothermia, 22°C; and profound hypothermia, 17°C) and various concentrations (low, 4 μM; moderate, 40 μM; and high, 400 μM) of thiopentone sodium (TPS), alone and in combination in cortical cultures exposed to prolonged hypoxia (24–48 hr). The survival rate of embryonic day (E)16 Wistar rat cortical neurons was evaluated on photomicrographs before and after experiments. During the 24‐hr hypoxic period, the survival rate of neurons was maximal with combinations of mild hypothermia with 40 μM (91.6 ± 0.7%) and 400 μM TPS (90.8 ± 0.7%) or deep hypothermia combined with all concentrations of TPS (4 μM, 90.6 ± 1.0%; 40 μM, 91.4 ± 0.8%; 400 μM, 91.8 ± 1.2%). During 48 hr hypoxia, the highest survival rate was seen with the combination of deep hypothermia and either 40 μM (90.9 ± 0.6%) or 400 μM (91.1 ± 1.4%) TPS. In the presence of profound hypothermia in combination with all concentrations of TPS, the survival rate was significantly reduced (P< 0.01) compared to combined application of either mild or deep hypothermia with TPS. In summary, maximal neuroprotection was attained with hypothermia and TPS in combination rather than applied individually, during prolonged hypoxic episodes (24– 48 hr). During a 24‐hr hypoxic period, both mild and deep hypothermia combined with a clinically relevant concentration of TPS (40 μM) offered the highest neuroprotection. Only deep hypothermia provided maximal neuroprotection when combined with 40 μM TPS, during 48‐hr hypoxia. Combination of profound hypothermia and TPS did not confer considerable neuroprotection during long lasting hypoxia.

Neuroprotective effect of hypothermia at defined intraischemic time courses in cortical cultures

Many experimental and clinical studies have shown that hypothermia confers cerebroprotective benefits against ischemic insults. Because of the many conflicting reports on hypothermic neuroprotection, we undertook this cellular study to identify the optimal temperature or a range of temperatures for maximal neuroprotection at different times (6–24 hr) during ischemic insults. Cultured Wistar rat cortical neurons were exposed to oxygen deprivation at defined times and temperatures (37°C normothermia, 32°C mild hypothermia, 27°C moderate hypothermia, 22°C deep hypothermia, and 17°C profound hypothermia). The survival rate of neurons was evaluated by assessing viable neurons on photomicrographs. The normothermic group demonstrated a significantly lower survival rate of cultured neurons (6 hr, 80.3% ± 2.7%; 12 hr, 56.1% ± 2.1%; 18 hr, 34.2% ± 1%; 24 hr, 18.1% ± 2.2%) compared to hypothermic groups (P < 0.001). The survival rate for the profound hypothermic group was significantly reduced (P < 0.01) compared to other hypothermic groups (at 17°C: 12 hr, 85.9% ± 2.5%, 18 hr, 74.7% ± 3.7%, 24 hr, 58.7% ± 2.7%). Almost equal survival rates were observed among mild, moderate, and deep hypothermic groups following <18 hr exposure to hypoxia, but the deep hypothermic group showed a significantly higher survival rate (84.1% ± 1.6%; P < 0.001) when subjected to hypoxia for 24 hr. In conclusion, hypothermia offers marked neuroprotection against hypoxia, but attenuation of neuronal cell death was less with profound hypothermia compared to mild, moderate, and deep hypothermia. Deep hypothermia affords maximal protection of neurons compared to mild and moderate hypothermia during long‐lasting hypoxia (>18 hr). J. Neurosci. Res. 65:583–590, 2001.

The neuroprotective effect of ONO-1714 on NMDA-mediated cytotoxicity in vitro

We report the effects of a newly developed NOS inhibitor on the neurotoxicity induced by NMDA on cultured fetal rat cortical neurons. To date, three different isoforms of NOS have been characterized. It has been considered that both neuronal NOS and inducible NOS activities are detrimental to the ischemic brain, whereas endothelial NOS plays a prominent role in maintaining cerebral blood flow and prevents neuronal injury during ischemia. ONO-1714 is a newly developed competitive NOS inhibitor that has selective inhibitory potency for iNOS than eNOS. However, its effect on nNOS has not been investigated yet. In this study, we investigated the neuroprotective effect of ONO-1714 on NMDA-induced neurotoxicity in our established model of primary cultured cortical neurons of rat foetus. Cortical neurons (prepared from E16 rat foetuses) were used after 13-14 days in culture. The cells were exposed to 30 muM NMDA for 24 h in the culture. To evaluate the neuroprotective effects of NOS inhibitors, ONO-1714 and L-NAME, neurons were exposed to various concentrations of an NOS inhibitor with 30 muM NMDA. The NMDA induced neurotoxicity was significantly attenuated by ONO-1714 in all concentrations, but not in low to moderate concentrations of L-NAME. These findings demonstrate that the neuroprotective effect of ONO-1714 was more potent than L-NAME. Moreover, ONO-1714 has a strong inhibitory effect on nNOS and would be a powerful tool for the protection of neurons against cerebral ischemia.