Effect of pentobarbital,chloralose, and urethane on inhibitory postsynaptic potentials of cortical neurons

The effect of pentobarbital, chloralose, and urethane on IPSPs arising in auditory cortical neurons in response to electrical stimulation of geniculocortical fibers was studied in experiments on cats immobilized with D-tubocurarine. Pentobarbital (60–80 mg/kg body weight, intraperitoneally) sharply reduced the number of neurons responding by spikes to geniculocortical stimulation. Only short-latency responses remained. The number of neurons responding with IPSPs was unchanged. Pentobarbital increased the duration of the IPSPs by 1.5–2 times and shortened their latent periods. Under the influence of chloralose (60 mg/kg, intraperitoneally) the number of responses of EPSP—spike—IPSP type was increased and the duration of the IPSPs also was increased by 3–4 times. The latent period of the primary IPSPs was shortened. Unlike pentobarbital and chloralose, urethane (1000 mg/kg, intravenously) reduced the duration of the IPSPs to 30 msec. About 2% of IPSPs recorded before anesthesia had a latent period of 1.0–1.5 msec. Under the influence of anesthesia the relative number of these IPSPs increased to 5.7%. It it postulated that they are monosynaptic. The mechanism of action of general anesthetics on the cortical inhibitory system is discussed.     

microRNA-124 attenuates isoflurane-induced neurological deficits in neonatal rats via binding to EGR1

Isoflurane anesthesia induces neuroapoptosis in the development of the brain. In this study, neonatal rats and hippocampal neurons were subjected to isoflurane exposure, in which the effect of miR-124 on the neurological deficits induced by isoflurane was evaluated. Isoflurane anesthesia models were induced in neonatal SD rats aged 7 days and then treated with miR-124 agomir, miR-124 antagomir, or LV-CMV-early growth response 1 (EGR1) plasmids. Then, the spatial learning and memory ability of rats were evaluated by Morris water maze. Furthermore, primary hippocampal neurons cultured 7 days were also exposed to isoflurane and transfected with miR-124 agomir, miR-124 antagomir, or LV-CMV-EGR1 plasmids. The targeting relationship of miR-124 and EGR1 was verified by the dual-luciferase reporter gene assay. To identify the effect of miR-124 on neuron activities, the viability and apoptosis of hippocampal neurons were assessed. In response to isoflurane exposure, miR-124 expression was reduced and EGR1 expression was increased in the hippocampal tissues and neurons. The isoflurane anesthesia damaged rats' spatial learning and memory ability, and reduced viability, and promoted apoptosis of hippocampal neurons. EGR1 was targeted and negatively regulated by miR-124. The treatment of miR-124 agomir improved rats' spatial learning and memory ability and notably increased hippocampal neuron viability and resistance to apoptosis, corresponding to an increased brain-derived neurotrophic factor (BDNF) expression, inhibited expression of proapoptotic factors (cleaved-Caspase-3 and Bax), and enhanced the expression of antiapoptotic factor (Bcl-2). Upregulated miR-124 inhibited the expression of EGR1, by which mechanism miR-124 reduced the neurological deficits induced by isoflurane in neonatal rats through inhibiting apoptosis of hippocampal neurons.     

Effect of mutagenic replacement of the carboxyl terminal amino acid, val124, on the properties and regeneration of bovine pancreatic ribonuclease A

An important role of C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) in the formation of the three-dimensional structure was previously implied. In this study, we replaced the C-terminal amino acid, Val124, with amino acid residues with different properties by site-directed mutagenesis. The recombinant mutant enzymes were purified and subjected to a refolding study after being converted to a fully reduced and denatured state. There was a significant difference among the mutant enzymes in the rate of recovery of the activity when air oxidation was performed: the rate decreased in the order of V124E, V124L, V124G, V124K, V124A, and V124W. On the other hand, the recovery rates for all the mutant RNase A in the presence of GSH and GSSG were almost the same. The recovered activity of V124E after 24 h incubation reached approximately 90% of that of the wild type enzyme, followed by V124L 80%, V124A and V124W 65%, and V124K and V124G 50%. The duration of the initial lag phase became shorter in the order of V124W, V124A, V124K or V124G, V124E, or V124L. The results imply that the C-terminal amino acid significantly influences the formation of correct disulfide bonds during the refolding process and that the hydrophobic interaction of Val124 is important for efficient packing of the RNase A molecule.     

Effects of a high concentration of CO2 on electrocardiograms in the carp, Cyprinus carpio

1. The effects of a high concentration of CO2 (PCO2 = 250 mmHg and PO2 = 360 mmHg in water) and MS222 (tricaine methanesulfonate, 1/8000 or 1/5000) on the electrocardiogram (ECG) in carp were examined using five kinds of bipolar leads from the body surface. 2. In the carp anesthetized with the high concentration of CO2 for 30 min, the QRS duration, PQ interval and and direction of the QRS axis on the frontal plane significantly changed. Even after recovery from anesthesia, delay in the QRS duration was still recognized. 3. The concentration of CO2 used in this study had an anesthetic action to the same degree as 1/8000 of MS222 and had a much more severe effect on the ECG of the carp than 1/5000 of MS222.     

A Lack of Premature Termination Codon Read-Through Efficacy of PTC124 (Ataluren) in a Diverse Array of Reporter Assays

The drug molecule PTC124 (Ataluren) has been described as a read-through agent, capable of suppressing premature termination codons (PTCs) and restoring functional protein production from genes disrupted by nonsense mutations. Following the discovery of PTC124 there was some controversy regarding its mechanism of action with two reports attributing its activity to an off-target effect on the Firefly luciferase (FLuc) reporter used in the development of the molecule. Despite questions remaining as to its mechanism of action, development of PTC124 continued into the clinic and it is being actively pursued as a potential nonsense mutation therapy. To thoroughly test the ability of PTC124 to read through nonsense mutations, we conducted a detailed assessment comparing the efficacy of PTC124 with the classical aminoglycoside antibiotic read-through agent geneticin (G418) across a diverse range of in vitro reporter assays. We can confirm the off-target FLuc activity of PTC124 but found that, while G418 exhibits varying activity in every read-through assay, there is no evidence of activity for PTC124.     

Formation of experimental alcoholism in a population of crossbred rats

Experiments on random-bred rats were made to study the relationship between the duration of ethanol anesthesia and the time course of alcohol consumption for 8 months. Short-sleeping rats with ethanol anesthesia lasting less than 90 minutes exhibited initially high and steady alcoholic motivation. Meanwhile long-sleeping rats with ethanol anesthesia lasting over 175 minutes demonstrated high level of alcoholic motivation only under stressful conditions. After 8 months of keeping in isolated cages long-sleeping rats demonstrated the same intensity and steadiness of alcoholic motivation as was the case in short-sleeping animals. Long-sleeping rats differed from short-sleeping ones in hypersensitivity to the toxic action of ethanol. It is inferred that in random-bred rats, there are at least two forms of experimental alcoholism.     

Evaluation of two combinations of Domitor, Zoletil 100, and Euthatal to obtain long-term nonrecovery anesthesia in Sprague-Dawley rats

We sought to evaluate a new protocol designed to maintain long-term, nonrecovery, surgical anesthesia in Sprague-Dawley rats. In the first phase, two groups of rats were anesthetized with two different dose combinations of Domitor (medetomidine) and Zoletil 100 (tiletamine-zolazepam) to investigate their efficacy in induction of anesthesia. One combination comprised Domitor at 35 microg/kg and Zoletil 100 at 40 mg/kg, whereas the other comprised Domitor at 50 microg/kg and Zoletil 100 at 20 mg/kg. Both combinations effectively induced deep anesthesia and caused no mortality, but the duration of anesthesia differed statistically. In the second phase, we induced anesthesia with both Domitor-Zoletil 100 dose combinations then investigated the possibility of maintaining anesthesia for 5 h by administering Euthatal (pentobarbitone) intra-arterially at 10 mg/kg hourly. Depth of anesthesia, mortality, physiological parameters, blood gas analysis, hematology, clinical chemistry, and postmortem histopathology were recorded. Euthatal provided stable long-term anesthesia with both dose combinations of Domitor-Zoletil 100. Seven of 8 (88%) animals anesthetized with Domitor at 50 microg/kg and Zoletil 100 at 20 mg/kg successfully were maintained under deep anesthesia for 5 h. Higher mortality (36% versus 12%) occurred in group of animals treated with Domitor at 35 microg/kg and Zoletil 100 at 40 mg/kg. This difference may be linked to dose-related respiratory depression, as alterations of arterial gas levels were noted. Our findings suggest that, when long-term nonrecovery anesthesia is required, doses of 50 microg/kg Domitor and 20 mg/kg Zoletil 100 are preferable when given with Euthatal to maintain physiological conditions in animals.     

Novel gonadotropin-releasing hormone antagonists with substitutions at position 5

Gonadotropin-releasing hormone (GnRH) antagonists with high potency and improved duration of action are needed for potential clinical applications. We synthesized four new antagonists (2-5) of GnRH homologues to Azaline B (1), with a common core sequence of [Aph(X)5, D-Aph(Cbm)6]Azaline B. In these analogs, (X) contains hydrophobic aromatic moieties (like homoveratoyl in 2, homovanillyl in 3, 2,5-dimethoxyphenylacetyl in 4, and 3,5-dimethoxyphenylacetyl in 5) designed to improve the duration of action over that of Azaline B. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analogs 2, 4, and 5 were potent in vitro, but were found to be short acting in vivo. However, analog 3 [Aph(Hvn)5,D-Aph(Cbm)6]Azaline B is a potent human GnRH receptor antagonist in vitro (IC50 1.47 nM) and exhibits a longer duration of action than azaline B.     

Long-acting oxytocin antagonists: effects of 2-D-stereoisomer substitution on antagonistic potency and duration of action

Recently we reported the discovery of a series of 2-O-alkyltyrosine- (or 2-p-alkylphenylalanine), 4-threonine-, and 8-ornithine-substituted analogs of [1-penicillamine]oxytocin [( Pen1]OT) which possess prolonged anti-OT activity. In this study, we attempt to improve the potency and the duration of action of this series of OT antagonists by exploring the effects of D-stereoisomer substitution in the 2 position. We compare the in vitro anti-OT potency, expressed in pA2 values, and the duration of in vivo inhibitory action, expressed in recovery t1/2, of [Pen1]OT, [Pen1,Orn8]OT, [Pen1,Thr4]OT, [Pen1,Tyr(OMe)2,Thr4, Orn8]OT, [Pen1, Tyr(OEt)2,Thr4,Orn8]OT, [Pen1,D-Tyr(OEt)2,Thr4,Orn8]OT, [Pen1,Phe2,Thr4]OT, [Pen1,Phe(Me)2,Thr4,Orn8]OT, [Pen1,D-Phe(Me)2,Thr4,Orn8]OT, [Pen1,Phe(Et)2,Thr4,Orn8]OT, and [Pen1,D-Phe(Et)2,Thr4,Orn8]OT. The results show that modifications of the amino acid in position 2 by alkylation of the aromatic ring and use of D-stereoisomerism produce nonparallel effects on the in vitro potency and duration of action of OT antagonists. Time-action curve determinations show that long-acting OT antagonists exhibit delayed peak inhibitory action. Long action is not coupled with high potency in all cases. This dissociation between potency and duration of action gives support to our hypothesis that the potency and duration of action of these peptides may each have different conformational structure requirements.     

Design and synthesis of long-acting inhibitors of dipeptidyl peptidase IV

A series of (4-substituted prolyl)prolinenitriles were synthesized and evaluated as inhibitors of dipeptidylpeptidase IV (DPP-IV). Among those tested, the 4beta-[4-(hydroxyphenyl)prolyl]prolinenitriles showed a potent inhibitory activity with a long duration of action. Metabolic formation of the corresponding phenol glucuronates was found to contribute to their long duration of action. The activity profiles of the synthesized compounds are reported and structure-activity relationships are also presented.     

General Anesthetics Effects on Circadian Temporal Structure: An Update

Disruptions of circadian and biological rhythms as well as general anesthesia can induce sleep disorders, resulting in an increase in sleepiness and drowsiness and a decrease in vigilance. It has been previously shown that circadian time can influence the pharmacologic sensitivity and the duration of action of general anesthetics. Studies on interactions between general anesthesia and circadian rhythms are few, but all of them suggest an important role of general anesthetics on circadian rhythms. General anesthesia is a particular wake‐sleep state that could potentially alter circadian rhythms on the days following anesthesia. The aim of this review is to discuss the various effects of general anesthesia on animal and human circadian time structure. This topic is highly relevant to clinicians, especially those involved in that field of ambulatory practice responsible for post‐operative patient care, including patient recovery and fatigue.     

Organophosphorus pesticide‐induced butyrylcholinesterase inhibition and potentiation of succinylcholine toxicity in mice

Succinylcholine is the most important rapid‐acting depolarizing muscle relaxant during anesthesia. Its desirable short duration of action is controlled by butyrylcholinesterase, the detoxifying enzyme. There are two reported cases of prolonged paralysis from succinylcholine in patients poisoned with the organophosphorus insecticides parathion and chlorpyrifos. The present study examines the possibility that other organophosphorus and methylcarbamate pesticides might also prolong succinylcholine action by inhibiting butyrylcholinesterase using mice treated intraperitoneally as a model and relating inhibition of blood serum hydrolysis of butyrylthiocholine to potentiated toxicity (mouse mortality). The organophosphorus plant defoliant tribufos (4 h pretreatment, 160 mg/kg) and organophosphorus plant growth regulator ethephon (1 h pretreatment, 200 mg/kg) potentiate the toxicity of succinylcholine by seven‐ and fourfold, respectively. Some other pesticides or analogs are more potent sensitizers for succinylcholine toxicity with threshold levels of 0.5, 1.0, 1.7, 8, 10, and 67 mg/kg for phenyl saligenin cyclic phosphonate, profenofos, methamidophos, tribufos, chlorpyrifos, and ethephon, respectively. Enhanced mortality from succinylcholine is generally observed when serum butyrylcholinesterase is inhibited 55–94%. Mivacurium, a related nondepolarizing muscle relaxant also detoxified by butyrylcholinesterase, is likewise potentiated by at least threefold on 4 hour pretreatment with tribufos (25 mg/kg) or profenofos (10 mg/kg). © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 113–118, 1999     

A comparison of pentobarbital, fentanyl-droperidol, ketamine-xylazine and ketamine-diazepam anesthesia in adult male rats

Proper use of anesthetics is of paramount importance for humane animal care. Current research trends show a greater reliance on rats for laboratory investigations. This study compared several dosages for four different drugs, (pentobarbital, fentanyl-droperidol, ketamine-xylazine and ketamine-diazepam) for use in the laboratory rat. Each drug was evaluated in respect to its onset, duration of effect, recovery, adverse effects and mortality. A quantitative assessment of the depth of anesthesia also was obtained for all dosages of each drug. Results showed that all tested dosages of pentobarbital, ketamine-xylazine and ketamine-diazepam were suitable anesthetics for use in the laboratory rat. Low dosages of fentanyl-droperidol (Innovar-Vet), however, appeared to produce a state known as neuroleptanalgesia as opposed to anesthesia seen with the other agents.     

Isoflurane and Sevoflurane Induce Severe Hepatic Insulin Resistance in a Canine Model

IntroductionAnesthesia induces insulin resistance, which may contribute to elevated blood glucose and adverse post-operative outcomes in critically ill patients, and impair glycemic control in surgical patients with diabetes. However, little is known about the mechanisms by which anesthesia impairs insulin sensitivity. Here we investigate the effects of anesthesia on insulin sensitivity in metabolic tissues.MethodsHyperinsulinemic-euglycemic clamps were performed in 32 lean (control diet; n = 16 conscious versus n = 16 anesthetized) and 24 fat-fed (6 weeks fat-feeding; n = 16 conscious versus n = 8 anesthetized) adult male mongrel dogs in conjunction with tracer methodology to differentiate hepatic versus peripheral insulin sensitivity. Propofol was administered as an intravenous bolus (3mg/kg) to initiate anesthesia, which was then maintained with inhaled sevoflurane or isoflurane (2–3%) for the duration of the procedure.ResultsAnesthesia reduced peripheral insulin sensitivity by approximately 50% in both lean and fat-fed animals as compared to conscious animals, and insulin action at the liver was almost completely suppressed during anesthesia such that hepatic insulin sensitivity was decreased by 75.5% and; 116.2% in lean and fat-fed groups, respectively.ConclusionInhaled anesthesia induces severe hepatic insulin resistance in a canine model. Countermeasures that preserve hepatic insulin sensitivity may represent a therapeutic target that could improve surgical outcomes in both diabetic and healthy patients.     

Sleep-time variation for ethanol and the hypnotic drugs tribromoethanol, urethane, pentobarbital, and propofol within outbred ICR mice.

To evaluate the phenotypic variation within a commercial outbred mouse stock, we examined sleep-time (or duration of loss of righting reflex) of outbred ICR mice after i.p. injection of ethanol (4.0 g/kg of body weight), urethane (1.3 g), tribromoethanol (250 mg), and pentobarbital (60 mg), and after i.v. injection of propofol (30 mg). We observed high-grade individual differences in sleep-time that ranged from 0 to 179 min, 83.1 +/- 4.3 (mean and SEM of 100 mice) for ethanol; 0 to 169 min, 64.5 +/- 3.1 for pentobarbital; 0 to 160 min, 36.6 +/- 3.6 for urethane; 0 to 120 min, 21.5 +/- 2.2 for tribromoethanol; and 3 to 20.5 min, 7.1 +/- 0.3 for propofol. This extensive phenotypic variance within the outbred stock was as great as the variation reported among inbred strains or selected lines, and the varied susceptibility within the colony was inherited by Jcl:ICR-derived inbred strains IAI, ICT, IPI, and IQI. The range of sleep-time variance for ethanol, pentobarbital, urethane, tribromoethanol, and propofol within four-way cross hybrid Jcl:MCH(ICR) mice was 86.6%, 63.3%, 124%, 61.0%, and 53.1% that of outbred Jcl:ICR mice, respectively. The present study indicates that phenotypic variance within an outbred Jcl:ICR stock was at high risk for susceptibility to the drugs that depress the central nervous system and that Jcl:ICR-derived inbreds may be an excellent source of animal models for studying the anesthesia gene.     

The mechanism of the rate-dependent changes of the conducted action potential in rabbit ventricle

Blockers of the transient outward current (4-aminopyridine) and the Ca current (Co2+) as well as injection of polarizing current during the plateau were used to assess the role of these current systems as determinants of action potential duration at different pacing rates. Papillary muscles and ventricular trabecula were superfused with oxygenated Krebs solution at 33 degrees C and driven at a basic rate of 1 Hz. The effects of varying the frequency of stimulation between 0.1 and 4 Hz on action potential parameters were determined under control conditions and during exposure to 2 mM 4-aminopyridine, 1-3 mM CoCl2, or a mixture of 4-aminopyridine and CoCl2. The control relationship between action potential duration and pacing rate showed a maximum between 1 and 2 Hz. Under 4-aminopyridine, the plateau height and the action potential duration increased. The rate-dependent shortening of the action potential at frequencies below 1 Hz was reduced or abolished, and enhanced shortening was observed at rates above 1 Hz. Exposure to Co2+ reduced the action potential shortening at rates higher than 1 Hz. Both blockers, 4-aminopyridine and Co2+ were necessary to eliminate the rate-dependent changes of the action potential duration. Our results indicated that both the transient outward current and the inward calcium current determine the plateau height and duration for frequencies less than or equal to 2 Hz, whereas at higher rates, the Ca current plays a dominant role.     

Effects of an anesthetic mixture of medetomidine,midazolam, and butorphanol in rats—strain difference and antagonism by atipamezole

An anesthetic mixture of medetomidine (MED), midazolam (MID), and butorphanol (BUT) has been used in laboratory animals. We previously reported that this anesthetic mixture produced closely similar anesthetic effects in BALB/c and C57BL/6J strains. We also demonstrated the efficacy of atipamezole (ATI), an antagonist of MED that produced quick recovery from anesthesia in mice. Anesthetics have various anesthetic effects among animal strains. However, the differences in the effects of anesthetic mixtures in rats are unclear. In the present study, we first examined effects of the abovementioned anesthetic mixture using three different rat strains: Wistar (WST), Sprague-Dawley (SD), and Fischer 344 (F344). Second, we examined how different dosages and optimum injection timing of ATI affected recovery from anesthesia in rats. We used the anesthetic score to measure anesthetic duration and a pulse oximeter to monitor vital signs. We found no significant differences in anesthetic duration among the three different strains. However, recovery from anesthesia in the SD strain took significantly longer than in the other strains. The antagonistic effects of ATI (0.15 mg/kg and 0.75 mg/kg) were equivalent when administered at 30 min after anesthetic mixture administration. The antagonistic effects of ATI 0.75 mg/kg were stronger than those of ATI 0.15 mg/kg at 10 min after anesthetic mixture administration. This anesthetic mixture is a useful drug that can induce similar anesthetic effects in three different strains and has an antagonist, ATI, that makes rats quickly recover from anesthesia. These results may contribute to the welfare of laboratory animals.     

Effects of hirsutine and dihydrocorynantheine on the action potentials of sino-atrial node, atrium and ventricle

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla MIQ. JACKSON with antihypertensive, negative chronotropic and antiarrhythmic activity, and its C3 structural epimer, dihydrocorynantheine, on membrane potentials of rabbit sino-atrial node and guinea-pig right ventricle and left atrium were studied with microelectrode techniques. In sino-atrial node preparations, hirsutine and dihydrocorynantheine (0.1 microM to 10 microM) concentration-dependently increased cycle length, decreased slope of the pacemaker depolarization (phase 4 depolarization), decreased maximum rate of rise and prolonged action potential duration. In atrial and ventricular preparations, both compounds (0.1 microM to 30 microM) concentration-dependently decreased maximum rate of rise and prolonged action potential duration. These results indicate that hirsutine and dihydrocorynantheine have direct effects on the action potential of cardiac muscle through inhibition of multiple ion channels, which may explain their negative chronotropic and antiarrhythmic activity.     

Effects of timolol, indomethacin, and MK-571 on bronchoconstriction to infused leukotriene D4 in guinea pigs

Continuous intravenous infusions of leukotriene D4 produced a prolonged but variable bronchoconstriction (approximately a 200% increase in lung resistance (RL) and a 50% decrease in dynamic compliance (Cdyn] in anesthetized and paralysed guinea pigs that peaked within 1-1.5 min and was followed by a somewhat smaller secondary plateau response. The overall response was delayed (time to peaks) but not significantly reduced by pretreatment with the cyclooxygenase inhibitor indomethacin (1 mg/kg), was markedly potentiated by the beta-adrenoceptor antagonist timolol (5 micrograms/kg), and was partially and completely blocked by pretreatment with 0.1 and 1.0 mg/kg, respectively, of the leukotriene D4 receptor antagonist MK-571. MK-571 prevented the response in indomethacin-treated guinea pigs but was considerably more active at preventing and reversing the potentiated responses (lower dose of leukotriene D4) in animals treated with indomethacin and timolol. Additional studies in indomethacin- and timolol-treated animals demonstrated that MK-571 was active with good duration of action by the aerosol route of administration (30 min and 4 h pretreatment). The technique of infusing leukotrienes into untreated, indomethacin-treated, and indomethacin- and timolol-treated guinea pigs is a useful method to study the action and interaction of leukotriene receptor antagonists.