Duration Of Loss Of The Righting Reflex Research Articles

C57BL/6J and C57BL/6N mice exhibit different neuro-behaviors and sensitivity to midazolam- and propofol-induced anesthesia

Phenotypes of inbred mice are strain-dependent, indicating the important influence of genetic background in biomedical research. C57BL/6 is one of the most commonly used inbred mouse strains, and its two closely related substrains, C57BL/6J and C57BL/6N, have been separated for only about 70 years. These two substrains have accumulated genetic variations and exhibit different phenotypes, but it remains unclear whether they respond to anesthetics differently. In this study, commercially acquired wildtype C57BL/6J or C57BL/6N mice from two different sources were analyzed and compared for their response to a spectrum of anesthetics (midazolam, propofol, esketamine or isoflurane anesthesia) and their performance in a series of behavioral tests associated with neurological functions including open field test (OFT), elevated plus maze (EPM), Y maze, prepulse inhibition (PPI), tail strain test (TST) and forced swimming test (FST). Loss of the righting reflex (LORR) is used to measure the anesthetic effects. Our results suggested that the anesthesia induction time induced by either of the four anesthetics were comparable for the C57BL/6J and C57BL/6N mice. However, C57BL/6J or C57BL/6N mice do exhibit different sensitivity to midazolam and propofol. The anesthesia duration of midazolam of C57BL/6J mice was about 60% shorter than that of the C57BL/6N mice, while the LORR duration induced by propofol in C57BL/6J mice was 51% longer than that of the C57BL/6N. In comparison, the two substrains were anesthetized by esketamine or isoflurane similarly. In the behavioral analysis, the C57BL/6J mice exhibited a lower level of anxiety- and depression-like behaviors in OFT, EPM, FST and TST than the C57BL/6N mice. Locomotor activity and sensorimotor gating of these two substrains remained comparable. Our results stress the point that when selecting inbred mice for allele mutation or behavioral testing, the influence of even subtle differences in genetic background should be fully considered.

The preclinical pharmacological study on HX0969W, a novel water-soluble pro-drug of propofol, in rats.

BackgroundPropofol is the most widely used intravenous sedative-hypnotic anesthetic in clinical practice. However, many serious side effects have been related to its lipid emulsion formulation. The pro-drug design approach was used to develop the water-soluble propofol, which could effectively resolve the limitations associated with the lipid emulsion formulation. Thus, the new water-soluble pro-drug of propofol, HX0969W, was designed and synthesized. The objective of this study was to conduct preclinical pharmacological studies on this novel water-soluble pro-drug of propofol.MethodsThe assessment of the loss of the righting reflex (LoRR) was used for the pharmacodynamic study, and liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography- fluorescence were used for the pharmacokinetic study.ResultsThe potency of HX0969W (ED50 [95% CI], 46.49 [43.89–49.29] mg/kg) was similar to that of fospropofol disodium (43.66 [43.57–43.75] mg/kg), but was lower than that of propofol (4.82 [4.8–14.82] mg/kg). Administered with a dose of 2-fold ED50, propofol required a shorter time to cause LoRR than that of HX0969W and fospropofol. However, the LoRR duration was significantly longer in response to the administration of HX0969W and fospropofol disodium than that caused by propofol. In the pharmacokinetic study, the Cmax of fospropofol was higher than that of HX0969W. HX0969W had a shorter mean residual time and a rapid clearance rate than that of fospropofol disodium. There was no significant difference between the Tmax of the propofol whether it was released by HX0969W or fospropofol disodium; the Cmax of propofol released by HX0969W was similar to that of propofol, which was higher than the propofol released by fospropofol disodium.

Aquaporin-4 knockout mice exhibit increased hypnotic susceptibility to ketamine.

PurposeThis study examines anesthetic/hypnotic effects of ketamine in AQP4 knockout (KO) and wild‐type (WT) mice with the particular focus on neurotransmission.Materials and MethodsKetamine (100 mg/kg) was intraperitoneally injected in 16 WT and 16 KO mice. The hypnotic potencies were evaluated by the loss of the righting reflex (LORR). The amino acids neurotransmitter levels in prefrontal cortex were measured by microdialysis.ResultsThis study demonstrated that AQP4 knockout significantly shortened the latency compared with WT mice (98.0 ± 4.2 vs. 138.1 ± 15.0 s, p < .05) and prolonged duration of LORR (884.7 ± 58.6 vs. 562.0 ± 51.7 s, p < .05) compared with WT mice in LORR induced by ketamine. Microdialysis showed that lack of AQP4 markedly decreased glutamate level within 20 min (p < .05) and increased γ‐aminobutyric acid (GABA) level within 30–40 min (p < .05) after use of ketamine. Moreover, the levels of taurine were remarkably higher in KO mice than in WT mice, but no obvious differences in aspartate were observed between two genotypes.Conclusion AQP4 deficiency led to more susceptibility of mice to ketamine, which is probably due to the modulation of specific neurotransmitters, hinting an essential maintenance of synaptic activity mediated by AQP4 in the action of ketamine.

Establishment of a BALB/c Mouse Model for Assessing the Degree of Inebriation Induced by Luzhou-Flavor Liquors.

This investigation was carried out to determine the differences in the degree of inebriation induced by Luzhou-flavor liquors having the same ethanol content. A BALB/c mouse model was used to test the effects of two liquors on the loss of the righting reflex (LORR) and the duration (DUR) of the LORR, as indices of the degree of inebriation. The blood ethanol concentration, blood acetaldehyde concentration, acetylcholinesterase activity in the hippocampus, and concentrations of dopamine and serotonin in the striatum were also determined. The degrees of inebriation induced by the two liquors were 0.694 and 0.404, as quantified by LORR and LORR DUR. The liquor that induced the lower degree of inebriation also induced lower blood ethanol and blood acetaldehyde concentrations. Moreover, it had no significant effects on acetylcholinesterase activity in the hippocampus or on the concentrations of dopamine or serotonin in the striatum. Chinese liquors with the same ethanol content can be distinguished by the degree of inebriation they induce. A relationship was found between the internal composition of the liquor and the degree of inebriation it induced. Our data support choosing liquors with low degrees of inebriation to reduce their harmful effects.

Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice

AimsAldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is highly expressed in the liver and is involved in both the detoxification of carbonyl compounds and ascorbic acid biosynthesis. By comparison with wild-type mice, Akr1a-knockout (Akr1a−/−) mice and human Akrla-transgenic (Akr1atg/+) mice experience different anesthetic actions from pentobarbital—prolonged in Akr1a-knockout (Akr1a−/−) mice and shortened in human Akrla-transgenic (Akr1atg/+) mice. Main methodsWe investigated this alteration in the anesthetic efficacy of pentobarbital in Akr1a genetically modified mice. Key findingsNeither the cytosolic protein of wild-type mouse liver nor purified rat AKR1A directly reduced pentobarbital. Ascorbic acid administration neutralized the prolonged duration of the loss of the righting reflex (LORR) in Akr1a−/− mice, but preincubation of pentobarbital with ascorbic acid prior to administration did not change the anesthetic effect. Those results indicated that ascorbic acid does not directly reduce pentobarbital. Enzymatic activities and levels of the proteins of some cytochrome P450s that make up a potent detoxification system for pentobarbital showed no changes in the genetically modified mice examined. Thus, ascorbic acid also had no effect on the detoxification system in the liver. The prolonged duration of LORR in the Akr1a−/− mice caused by pentobarbital and the neutralization of the anesthetic effect by ascorbic acid together with other results imply that ascorbic acid alters the responses of the neuronal system to anesthetics. SignificancePentobarbital action is increased under conditions of ascorbic acid deficiency, and this may have to be taken into account when anesthetizing malnourished patients.

Dexmedetomidine and hydroxyzine synergistically potentiate the hypnotic activity of propofol in mice

Investigation into the characteristics of anesthetic interactions may provide clues to anesthesia mechanisms. Dexmedetomidine, an α(2)-adrenergic receptor agonist, has become a popular sedative in intensive care, and hydroxyzine, a histamine receptor antagonist, is well known as a tranquilizing premedication for anesthesia. However, no experimental or pharmacological evaluation has been reported concerning their combination with propofol. Thus, we studied their combined effect with a hypnotic dose of propofol in ddY mice. Male adult mice were intravenously administered either dexmedetomidine (30μg/kg) or hydroxyzine (5mg/kg) with propofol (3.75-10mg/kg) to induce hypnosis, defined as a loss of the righting reflex (LRR). Other mice were intravenously administered propofol, dexmedetomidine (300μg/kg), or hydroxyzine (50mg/kg) alone, and subsequent behavioral changes were observed. The 50% effective dose (ED(50)) for LRR was calculated, and the duration of LRR was determined. The hypnotic dose of propofol was 9.95±1.04mg/kg (ED(50)±SEM) without combination. Dexmedetomidine and hydroxyzine reduced the ED(50) of propofol to 5.32±0.57 and 5.63±0.57mg/kg, respectively. Coadministration of dexmedetomidine significantly extended LRR duration compared with propofol alone, whereas hydroxyzine significantly shortened LRR duration. A maximal dose of dexmedetomidine or hydroxyzine alone did not induce hypnosis. Dexmedetomidine and hydroxyzine demonstrated no hypnotic action alone; however, their coadministration potentiated the hypnotic activity of propofol. Although reduction in the dose of propofol was similar, only dexmedetomidine prolonged the duration of hypnosis.

Systemic Administration of Arecoline Reduces Ethanol‐Induced Sleeping Through Activation of Central Muscarinic Receptor in Mice

Epidemiological evidence of co-use of alcohol and areca nuts suggests a potential central interaction between arecoline, a major alkaloid of areca and a muscarinic receptor agonist, and ethanol. Moreover, the central cholinergic system plays an important role in the depressant action of ethanol and barbiturates. The purpose of this study was to investigate the effects of arecoline on pentobarbital- and ethanol-induced hypnosis in mice. Male ICR mice were tested for locomotor activity following acute systemic administration of ethanol alone, arecoline alone, or ethanol plus arecoline. For the loss of the righting reflex (LORR) induced by pentobarbital and ethanol, sleep latency and sleeping duration were evaluated in mice treated with arecoline alone or the combination of arecoline and scopolamine or methscopolamine. Ethanol (1.0 to 3.0 g/kg, i.p.) reduced locomotor activity significantly and a declining trend was observed after treatment with arecoline (0.25 to 1.0 mg/kg, i.p.), but there were no synergistic effects of ethanol and arecoline on locomotor activity. The experiments on LORR demonstrated that arecoline (0.125 to 1.0 mg/kg, s.c.) shortened the duration of sleeping induced by ethanol (4.0 g/kg, i.p.), but not pentobarbital (45 mg/kg, i.p.). In addition, alterations of sleep latency were not obvious in both pentobarbital- and ethanol-induced LORR. Statistical analyses revealed that scopolamine (centrally acting), but not methscopolamine (peripherally acting), could antagonize the effect of arecoline on the duration of ethanol-induced LORR in mice. These results suggest that central muscarinic receptor is a pharmacological target for the action of arecoline to modulate ethanol-induced hypnosis.

Restraint stress and exogenous corticosterone differentially alter sensitivity to the sedative-hypnotic effects of ethanol in inbred long-sleep and inbred short-sleep mice

Decreased sensitivity to ethanol is a genetically mediated trait implicated in susceptibility to developing alcoholism. Here, we explore genotype by environment differences in ethanol sensitivity. The relationship between acute- and repeated-restraint stress, corticosterone (CORT) levels, and sensitivity to sedative-hypnotic properties of ethanol was explored using inbred long-sleep (ILS) and inbred short-sleep (ISS) mice. In ILS mice, acute restraint decreased ethanol sensitivity at a 4.1 g/kg dose, as measured by a decrease in the duration of loss of the righting reflex (LORE) and an increase in blood ethanol concentration at regain of the righting response (BECRR). Repeated restraint also decreased LORE duration, but had no effect on BECRR. In the ISS mice, there was no effect of acute restraint on either LORE duration or BECRR. However, repeated restraint increased ethanol sensitivity at a 4.1 g/kg dose; with an increase in LORE duration, but a decrease in BECRR. Differences in hypothalamic-pituitary-adrenal (HPA) axis responsiveness to restraint stress (as measured by plasma CORT) were also examined between genotypes. ILS mice displayed habituation to repeated restraint, whereas ISS mice did not. Lastly, the effect of enhanced CORT levels independent of psychological stress was examined for its effects on the sedative-hypnotic effects of ethanol. There were no effects of CORT pretreatment on LORE duration or BECRR in ILS mice compared to saline- or noninjected littermates. In contrast, ISS mice injected with CORT showed a decreased duration of LORE, but no effects on BECRR. These findings suggest that in addition to genetic susceptibility, environmental factors (e.g., restraint stress, exogenous CORT administration) also influence sensitivity to the sedative effects of ethanol through alteration of central nervous system sensitivity and pharmacokinetic parameters, and do so in a genotype-dependent manner.

Short-term selection for acute ethanol tolerance and sensitization from an F 2 population derived from the high and low alcohol-sensitive selectively bred rat lines

Previous studies have identified quantitative trait loci (QTL) in the inbred high and low alcohol-sensitive rat (IHAS1 and ILAS1) strains. The original development of the strains involved selection for ethanol sensitivity based on duration of the loss of the righting reflex (LORR) after a standard dose of ethanol. This paper confirms some of these QTL using a short-term selection procedure based on the difference between the blood ethanol level at LORR and regain of the righting response. An F 2 population of rats was developed by a reciprocal cross of IHAS1 and ILAS1 rats. Selection for five generations was carried out using delta-blood ethanol concentration (dBEC) as the selection trait, where dBEC = BECLR (BEC at loss of righting reflex) − BECRR (BEC at regain of righting reflex). The lines were labeled tolerant (TOL) or sensitive (SENS). Approximately one-third of the offspring for each generation in each line were genotyped using DNA markers that had been previously found to be linked to QTL on chromosomes 1, 2, 5, 12, and 13. By the fifth generation of selection, the lines showed a very large difference in dBEC, BECRR, and duration of LORR; BECLR showed little segregation during the selection, and latency to lose the righting reflex showed none. IHAS allele frequency increased in the SENS line for markers on chromosomes 1, 5, 12, and 13 while ILAS allele frequency increased in the TOL line. These results were in good agreement with the two previous QTL studies. On chromosome 2, the selection resulted in an accumulation of ILAS alleles in both lines. This study provides independent confirmation of the location of QTL on chromosomes 1, 5, 12, and 13 for ethanol sensitivity. It also suggests that genetic differences in duration of LORR are mediated primarily by the dBEC phenotype.

Effects of First- and Second-Generation Histamine-H1-Receptor Antagonists on the Pentobarbital-Induced Loss of the Righting Reflex in Streptozotocin-Induced Diabetic Mice

The second-generation histamine-H1-receptor antagonists, such as epinastine and cetirizine, are used as non-sedating antihistamines for treating allergic symptoms due to their poor ability to penetrate blood-brain barrier. Because it has been reported that the blood-brain barrier system is disturbed in diabetes, it is possible that second-generation histamine-H1-receptor antagonists may easily penetrate the blood-brain barrier and cause potent sedation in diabetics. In the present study, we investigated the effects of first-generation (diphenhydramine) and second-generation (epinastine and cetirizine) histamine-H1-receptor antagonists on the duration of pentobarbital-induced loss of the righting reflex (LORR) in non-diabetic and diabetic mice. Systemic treatment with diphenhydramine (3 – 30 mg/kg, s.c.), and intracerebroventricular treatment with epinastine (0.03 – 0.3 μg/mouse) and cetirizine (0.03 – 0.3 μg/mouse) dose-dependently and significantly increased the duration of pentobarbital-induced LORR in both nondiabetic and diabetic mice. Although systemic treatment with epinastine (3 – 30 mg/kg, s.c.) and cetirizine (3 – 30 mg/kg, s.c.) did not affect the duration of pentobarbital-induced LORR in nondiabetic mice, these treatments significantly prolonged it in diabetic mice. Our results suggest that the systemic administration of second-generation histamine-H1-receptor antagonists may produce a central nervous system depressant effect in diabetes.

Corticotropin-releasing factor overexpression decreases ethanol drinking and increases sensitivity to the sedative effects of ethanol.

Corticotropin-releasing factor (CRF) may play a significant role in drug and alcohol abuse. To evaluate the role of CRF in these processes, we examined several ethanol (EtOH) related behaviors in mice that carry a transgene that causes overexpression of CRF. We examined voluntary EtOH drinking, loss of the righting reflex (LORR), EtOH-induced conditioned taste aversion (CTA), and EtOH clearance in littermate transgenic (TG) and non-transgenic (non-TG) mice. In addition, because preliminary results indicated that age exacerbated differences in EtOH consumption between the two genotypes, we performed a cross-sectional and longitudinal evaluation of this trait at two ages ( approximately 100 and 200 days old). We found that TG mice consumed significantly less EtOH and had a lower preference for EtOH-containing solutions compared with their non-TG littermates. We also found that the older drug-naive TG mice drank less EtOH as compared with the younger mice of the same genotype; however, the same relationship did not exist for drug-naive non-TG mice. Prior experience in drinking EtOH when 100 days old led to decreased EtOH drinking when 200 days old in both genotypes. Duration of LORR was longer in the TG mice, EtOH-induced CTA was marginally greater in non-TG mice at the highest dose tested, and there were significant but small differences in EtOH clearance parameters. These data show that CRF overexpressing mice voluntarily consume less EtOH. This difference is associated with greater sensitivity to the sedative-hypnotic effects of EtOH, but not with increased sensitivity to the aversive effects of EtOH.

Potentiation of Ethanol-Induced Loss of the Righting Reflex by Ascorbic Acid in Mice: Interaction With Dopamine Antagonists

WU, C. F., H. L. ZHANG AND W. LIU. Potentiation of ethanol-induced loss of the righting reflex by ascorbic acid in mice: Interaction with dopamine antagonists. PHARMACOL BIOCHEM BEHAV 66(2) 413–418, 2000.—The present investigation was carried out to determine the effect of ascorbic acid on ethanol-induced loss of the righting reflex (LORR) and the interactions between ascorbic acid and dopamine receptor antagonists in affecting this action of ethanol in mice. To test the effect of each drug on ethanol-induced LORR, ascorbic acid (31.25, 62.5, 125, 250, 500, 1000 mg/kg intraperitoneally [IP]) and dopamine receptor antagonists (haloperidol 0.5, 1.0 mg/kg; L-sulpiride 20, 40, 80 mg/kg; clozapine 0.625, 1.25, 2.5 mg/kg; SCH 23390 0.5, 1.0, 2.0 mg/kg subcutaneously [SC]) were administered, respectively, 30 min before ethanol (4.0 g/kg IP) administration. Ascorbic acid, at the dose of 1000 mg/kg, significantly potentiated ethanol-induced LORR in mice. Dopamine D 2 antagonists haloperidol (0.5, 1.0 mg/kg SC), and L-sulpiride (80 mg/kg SC) also significantly prolonged the duration of LORR induced by ethanol. Clozapine and SCH 23390, at the doses used, did not affect ethanol-induced LORR. In the interaction study, the synergistic effect of ascorbic acid (1000 mg/kg IP) on ethanol-induced LORR was significantly enhanced by dopamine D 2 antagonists haloperidol, L-sulpiride, and clozapine, and the highest dose of dopamine D 1 antagonist SCH 23390. These results suggest that ascorbic acid may potentiate ethanol-induced LORR partially via a mechanism mainly linked to blockade of dopamine D 2 receptors.

Effects of Nitric Oxide‐Related Agents on Alcohol Narcosis

To examine whether nitric oxide (NO) affects alcohol (ethanol) narcosis, adult male rats were pretreated with a NO synthase (NOS) inhibitor, NG-nitro-l-arginine methyl ester (NAME); a NOS substrate, l-arginine methyl ester (AME); or a NO donor, isosorbide dinitrate (ISDN); then treated with anesthetic doses (3-5 g/kg, ip) of alcohol. Pretreatment with NAME (30-100 mg/kg, sc) 40 min before alcohol treatment delayed the onset of alcohol-induced loss of the righting reflex (LORR) and increased the duration of the LORR. NAME (100 mg/kg) pretreatment combined with high doses of alcohol (4-5 g/kg) exerted significant lethal effects, even though treatment with either agent alone or NAME combined with lower doses of alcohol (3-3.5 g/kg) was not lethal. Simultaneous treatment with the NOS substrate AME (100 mg/kg, subcutaneous) blocked the effects of NAME on LORR duration times, but did not alter LORR onset times. The NO donor ISDN (30 mg/kg) given by oral gavage 2 hr before alcohol decreased LORR duration times without affecting the onset of LORR. In addition, ISDN dose-dependently inhibited NAME-induced LORR duration increases during alcohol narcosis without significantly altering LORR onset times. Neither ISDN nor NAME significantly altered blood alcohol concentrations. These results suggest that NOS inhibition and subsequent decreases in NO production enhance alcohol-induced narcosis and that increases in NO concentrations inhibit alcohol narcosis, supporting the hypothesis that inhibition of arginine-NOS-NO systems mediates part of the sedative-hypnotic effect of alcohol.