Modeling spontaneous opioid withdrawal in male and female outbred mice using traditional endpoints and hyperalgesia.

Opioid withdrawal significantly impacts drug dependence cycles as hyperalgesia associated with withdrawal is often a reason for continued drug use. Animal models of addiction are important tools for studying how drug dependence and withdrawal impact not only normal neurocircuitry but also the effectiveness of potential treatments for dependence and withdrawal. We conducted a study of the time course of spontaneous morphine withdrawal in outbred male and female mice that can be used to examine sex differences in male and female mice using both traditional somatic endpoints and mechanical hyperalgesia as an endpoint of withdrawal. Male and female national institute of health (NIH) Swiss mice were made dependent upon morphine using an escalating dosing schedule. Injections were stopped after 5 days. Withdrawal behavior was assessed at time intervals up to 106 h after the final injection. Numbers of forepaw tremors, wet-dog shakes, jumps and other behaviors were scored to create a global score. Paw pressure readings were then also taken to track changes in sensitivity to a painful stimulus over time. Male and female mice had approximately similar withdrawal severity peaking at 24 h after the final injection as measured by composite global scores. Females did exhibit an earlier and greater frequency of tremors than males. Although males and females showed similar hyperalgesia during withdrawal, females recovered faster. Spontaneous opioid withdrawal peaking at 24 h was demonstrated in male and female NIH Swiss mice. We also successfully demonstrated that hyperalgesia is an endpoint that varies over the course of withdrawal.

Hyperbaric Oxygen Therapy for Pain, Opioid Withdrawal, and Related Symptoms: A Pilot Randomized Controlled Trial.

BACKGROUND: Pain, drug cravings, and opioid withdrawal symptoms can interfere with substance use disorder or opioid tapering treatment goals. AIM: This pilot study investigated the feasibility of a protocol designed to test opioid withdrawal symptom relief relative to a sham condition after two consecutive days of hyperbaric oxygen therapy (HBOT) for adults prescribed daily methadone for opioid use disorder. METHOD: Using a double-blind protocol, eight adults were randomized to receive either a full 90-minute HBOT dose in a pressurized chamber with 100% oxygen at 2.0 atmospheres absolute (ATA) or a sham condition receiving 21% oxygen (equivalent to room air within the chamber) at a minimal pressure of ≤1.3 ATA. Measures included study retention, treatment satisfaction, and pre- and post-intervention effects for opioid withdrawal symptoms, drug cravings, pain intensity and interference, sleep quality, and mood. RESULTS: Study retention and treatment satisfaction was high. All measurements improved more, on average, for participants receiving full-dose HBOT treatment than among participants receiving sham treatments except for clinically observed withdrawal symptoms. The largest positive effects were observed in measurements of pain intensity and drug craving. CONCLUSIONS: These pilot results provide evidence to support a fully powered study of HBOT as a potential treatment adjunct for adults receiving methadone for opioid use disorder. Trends towards symptom improvements were detected from pre- to post-HBOT in the full treatment arm versus sham condition. More research into novel non-pharmacologic options to relieve distressing symptoms related to pain and opioid use disorder is essential to improve clinical outcomes.

Effect of hyperbaric oxygen on chemotherapy-induced neuropathy in male and female rats.

Chemotherapeutic agents can cause peripheral neuropathy, a deleterious side effect of cancer treatment. Hyperbaric oxygen (HBO2) treatment has shown great potential for decreasing pain in numerous clinical pain conditions and in preclinical studies. This study was designed to test whether HBO2 might also be useful for treating chemotherapy-induced peripheral neuropathy. Male and female Sprague-Dawley rats were injected with 1 mg/kg paclitaxel or vehicle every other day for 7 days to induce allodynia, followed by either one single, or four daily 60-min exposures to HBO2 or room air. Mechanical and cold allodynia as well as locomotor behavior and body weight were assessed intermittently for several weeks. Estrous cycling was also tracked in female rats. Paclitaxel caused pronounced mechanical allodynia in both sexes that was completely reversed by either one or four treatments of HBO2. Females in all treatment groups showed greater cold acetone scores than males, and acetone scores were not reliably reduced by HBO2 treatment. Neither paclitaxel nor HBO2 treatment altered locomotor behavior or estrous cycling. We conclude that HBO2 treatment was highly effective at reducing mechanical allodynia in paclitaxel-treated rats without affecting weight gain, locomotion, or estrous cycling, suggesting that HBO2 may be effective for treating chemotherapy-induced neuropathic pain without producing significant side effects.

Hyperbaric Oxygen to Assist Adults With Opioid Use Disorder in Reducing Methadone Dose.

ABSTRACT: Opioid withdrawal symptoms can interfere with substance use disorder treatment goals. This study investigated the acceptability, feasibility, and treatment effects of hyperbaric oxygen therapy (HBOT) as an adjunct to reduce withdrawal symptoms for adults initiating a medically supervised methadone dose reduction. Adults prescribed methadone for opioid use disorder were randomized into either a hyperbaric oxygen group (n = 17) or an attention control group (n = 14). The study site was an outpatient opioid treatment program in the northwestern United States. Participants were asked to attend five consecutive daily 90-minute HBOT sessions offered at 2.0 atmospheres absolute with 100% oxygen in a pressurized chamber. Treatment attendance and reported satisfaction were measures of acceptability and feasibility. Medication doses were tracked posttreatment at 1 week, 1 month, and 3 months. Withdrawal symptoms were assessed at baseline and daily during the 5-day intervention period. After randomization, 13 (76.5%) followed through with medical screening and HBOT sessions, and of those, nine (69.2%) completed all five 90-minute HBOT sessions. At 3 months, the treatment group maintained, on average, a 4.3-mg methadone dose reduction compared with an average reduction of 0.25 mg for control group participants. Opioid withdrawal symptoms were reduced after Day 1 of HBOT by twice as much, on average, compared with the control condition. Satisfaction surveys found participants were generally satisfied with ease and comfort of the treatment. The evidence that HBOT is an acceptable, feasible adjunct warrants future trials to determine more conclusively effects on withdrawal symptoms associated with methadone dose taper.

Genome-to-phenome research in rats: progress and perspectives.

Because of their relatively short lifespan (<4 years), rats have become the second most used model organism to study health and diseases in humans who may live for up to 120 years. First-, second- and third-generation sequencing technologies and platforms have produced increasingly greater sequencing depth and accurate reads, leading to significant advancements in the rat genome assembly during the last 20 years. In fact, whole genome sequencing (WGS) of 47 strains have been completed. This has led to the discovery of genome variants in rats, which have been widely used to detect quantitative trait loci underlying complex phenotypes based on gene, haplotype, and sweep association analyses. DNA variants can also reveal strain, chromosome and gene functional evolutions. In parallel, phenome programs have advanced significantly in rats during the last 15 years and more than 10 databases host genome and/or phenome information. In order to discover the bridges between genome and phenome, systems genetics and integrative genomics approaches have been developed. On the other hand, multiple level information transfers from genome to phenome are executed by differential usage of alternative transcriptional start (ATS) and polyadenylation (APA) sites per gene. We used our own experiments to demonstrate how alternative transcriptome analysis can lead to enrichment of phenome-related causal pathways in rats. Development of advanced genome-to-phenome assays will certainly enhance rats as models for human biomedical research.

Homeostatic synapse-driven membrane plasticity in nucleus accumbens neurons.

Stable brain function relies on homeostatic maintenance of the functional output of individual neurons. In general, neurons function by converting synaptic input to output as action potential firing. To determine homeostatic mechanisms that balance this input-output/synapse-membrane interaction, we focused on nucleus accumbens (NAc) neurons and demonstrated a novel form of synapse-to-membrane homeostatic regulation, homeostatic synapse-driven membrane plasticity (hSMP). Through hSMP, NAc neurons adjusted their membrane excitability to functionally compensate for basal shifts in excitatory synaptic input. Furthermore, hSMP was triggered by synaptic NMDA receptors (NMDARs) and expressed by the modification of SK-type Ca(2+)-activated potassium channels. Moreover, hSMP in NAc neurons was abolished in rats during a short- (2 d) or long- (21 d) term withdrawal from repeated intraperitoneal injections of cocaine (15 mg/kg/d, 5 d). These results suggest that hSMP is a novel form of synapse-to-membrane homeostatic plasticity and dysregulation of hSMP may contribute to cocaine-induced cellular alterations in the NAc.

Hyperbaric oxygen produces a nitric oxide synthase-regulated anti-allodynic effect in rats with paclitaxel-induced neuropathic pain.

Research has demonstrated that hyperbaric oxygen (HBO2) treatment produced relief of both acute and chronic pain in patients and animal models. However, the mechanism of HBO2 antinociceptive effect is still elusive. Based on our earlier findings that implicate NO in the acute antinociceptive effect of HBO2, the purpose of this study was to ascertain whether HBO2-induced antinociception in a chronic neuropathic pain model is likewise dependent on NO. Neuropathic pain was induced in male Sprague Dawley rats by four injections of paclitaxel (1.0 mg/kg, i.p.). Twenty-four hours after the last paclitaxel injection, rats were treated for one day or four consecutive days with 60-min HBO2 at 3.5 atmospheres absolute (ATA). Two days before HBO2 treatment, some groups of rats were implanted with Alzet® osmotic minipumps that continuously infused a selective inhibitor of neuronal NO synthase (nNOS) into the lateral cerebral ventricle for 7 days. Mechanical and cold allodynia were assessed every other day, using electronic von Frey and acetone assays, respectively. Rats in the paclitaxel control group exhibited a mechanical or cold allodynia that was significantly reversed by one HBO2 treatment for mechanical allodynia and four HBO2 treatments for cold allodynic. In rats treated with the nNOS inhibitor, the effects of HBO2 were nullified in the mechanical allodynia test but unaffected in the cold allodynia test. In summary, these results demonstrate that the antiallodynic effect of HBO2 in two different pain tests is dependent on NO in the CNS.

Nitrous oxide-antinociception is mediated by opioid receptors and nitric oxide in the periaqueductal gray region of the midbrain.

Previous studies have shown that nitrous oxide (N(2)O)-induced antinociception is sensitive to antagonism by blockade of opioid receptors and also by inhibition of nitric oxide (NO) production. The present study was conducted to determine whether these occur within the same brain site. Mice were stereotaxically implanted with microinjection cannulae in the periaqueductal gray (PAG) area of the midbrain. In saline-pretreated mice, exposure to 70% N(2)O resulted in a concentration-dependent antinociceptive effect in the mouse abdominal constriction test. Pretreatment with an opioid antagonist in the PAG significantly antagonized the antinociceptive effect. Pretreatment with an inhibitor of NO production in the PAG also significantly antagonized the antinociceptive effect. These findings suggest that N(2)O acts in the PAG via an NO-dependent, opioid receptor-mediated mechanism to induce antinociception.

The effect of hyperbaric oxygen on regional brain and spinal cord levels of nitric oxide metabolites in rat.

Hyperbaric oxygen (HBO(2)) therapy is reported to be beneficial in transient brain ischemia. The present study was conducted to determine the influence of HBO(2) on metabolites of nitric oxide (NO) in brain and spinal cord of rats. Rats were exposed to room air (RA), normobaric air (NBA), normobaric oxygen (NBO(2)), hyperbaric air (HBA) or HBO(2), the last two conditions at 2.5ATA (atmosphere absolute) for 60 min. The results demonstrate that, compared to the NBA control, oxygen alone generally reduced tissue levels of NO(x)(-) (nitrite plus nitrate). On the other hand, 2.5ATA alone tended to have a slight, if any, effect on tissue levels of NO(x)(-). The combination of oxygen and pressure (i.e., HBO(2)) generally led to an increase in tissue levels of NO(x)(-). Based on these findings, it is concluded that HBO(2) appears to markedly increase NO function most notably in the corpus striatum, brainstem, cerebellum and spinal cord.

nNOS immunoreactivity co-localizes with GABAergic and cholinergic neurons, and associates with ß-endorphinergic and met-enkephalinergic opioidergic fibers in rostral ventromedial medulla and A5 of the mouse.

The present study examined the co-expression of neuronal nitric oxide synthase (nNOS) in the rostral ventromedial medulla (RVM) and A5 regions of the mouse brainstem within several neurochemical populations involved in nociceptive modulation. Double immunohistochemical methods showed that nNOS+ neurons do not co-localize with serotonergic neurons within any of these regions. Within the RVM, the nuclei raphe magnus and gigantocellularis contain a population of nNOS+/GAD67+ neurons, and within the paragigantocellularis lateralis, there is a smaller population of nNOS+/CHAT+ neurons. Further, nNOS+ neurons overlap the region of expression of ß-endorphinergic and met-enkephalinergic fibers within the RVM. No co-labeling was found within the A5 for any of these populations. These findings suggest that pain-modulatory serotonergic neurons within the brainstem do not directly produce nitric oxide (NO). Rather, NO-producing neurons within the RVM belong to GABAergic and cholinergic cell populations, and are in a position to modulate or be modulated by local opioidergic neurons.

Role of spinal GABA receptors in the acute antinociceptive response of mice to hyperbaric oxygen.

New pain treatments are in demand due to the pervasive nature of pain conditions. Hyperbaric oxygen (HBO2) has shown potential in treating pain in both clinical and preclinical settings, although the mechanism of this effect is still unknown. The aim of this study was to investigate whether the major inhibitory neurotransmitter γ-aminobutyric acid (GABA) is involved in HBO2-induced antinociception in the central nervous system (CNS). To accomplish this goal, pharmacological interactions between GABA drugs and HBO2 were investigated using the behavioral acetic acid abdominal constriction test. Western blotting was used to quantify protein changes that might occur as a result of the interactions. GABAA but not GABAB receptor antagonists dose-dependently reduced HBO2 antinociception, while antagonism of the GABA reuptake transporter enhanced this effect. Western blot results showed an interaction between the pain stimulus and HBO2 on expression of the phosphorylated ß3 subunit of the GABAA receptor at S408/409 in homogenates of the lumbar but not thoracic spinal cord. A significant interaction was also found in neuronal nitric oxide synthase (nNOS) expression in the lumbar but not thoracic spinal cord. These findings support the notion that GABA may be involved in HBO2-induced antinociception at the GABAA receptor but indicate that more study will be needed to understand the intricacies of this interaction.

Antagonism of phosphoramidon-induced antinociception in mice by mu- but not kappa-opioid receptor blockers.

Intracerebroventricular (i.c.v.) administration of the neutral endopeptidase 24.11-inhibitor phosphoramidon evoked a dose-dependent antinociceptive effect in the mouse acetic acid abdominal constriction test. The present study was conducted to identify the opioid receptor subtype(s) that mediate phosphoramidon antinociception in this paradigm. Mice were pretreated with different opioid antagonists prior to being challenged with phosphoramidon, i.c.v., the mu-opioid agonist sufentanil, s.c., or the kappa-opioid agonist U-50,488H, s.c. Naltrexone significantly attenuated phosphoramidon-induced antinociception at an i.c.v. dose that also blocked both sufentanil and U-50,488H. The mu-opioid antagonist beta-funaltrexamine (beta-FNA) blocked phosphoramidon and sufentanil at an i.c.v. dose that did not block U-50,488H. The kappa-opioid antagonist nor-binaltorphimine (nor-BNI) produced dose-related effects. A low dose (10 microg) of nor-BNI had no effect on either phosphoramidon or sufentanil but did reduce U-50,488H antinociception. A higher dose (30 microg) of nor-BNI blocked phosphoramidon, sufentanil, and U-50,488H, suggesting a loss of kappa-opioid receptor selectivity at this dose. These findings suggest that mu- but not kappa-opioid receptors mediate phosphoramidon-induced antinociception in the abdominal constriction test.

Advances in understanding the actions of nitrous oxide.

Nitrous oxide (N(2)O) has been used for well over 150 years in clinical dentistry for its analgesic and anxiolytic properties. This small and simple inorganic chemical molecule has indisputable effects of analgesia, anxiolysis, and anesthesia that are of great clinical interest. Recent studies have helped to clarify the analgesic mechanisms of N(2)O, but the mechanisms involved in its anxiolytic and anesthetic actions remain less clear. Findings to date indicate that the analgesic effect of N(2)O is opioid in nature, and, like morphine, may involve a myriad of neuromodulators in the spinal cord. The anxiolytic effect of N(2)O, on the other hand, resembles that of benzodiazepines and may be initiated at selected subunits of the gamma-aminobutyric acid type A (GABA(A)) receptor. Similarly, the anesthetic effect of N(2)O may involve actions at GABA(A) receptors and possibly at N-methyl-D-aspartate receptors as well. This article reviews the latest information on the proposed modes of action for these clinical effects of N(2)O.

Dynorphin-mediated antinociceptive effects of L-arginine and SIN-1 (an NO donor) in mice.

The antinociceptive response of mice to the amino acid L-arginine (L-ARG) has been attributed to either an opioid mechanism or a non-opioid but nitric oxide (NO)-dependent mechanism. Earlier it was reported that the mechanism of nitrous oxide-induced antinociception involved opioid components and was also dependent on brain NO. This study was designed to determine whether the antinociceptive effects of L-ARG and the NO donor 3-morpholinosydnoimine (SIN-1) might be mediated by brain mechanisms similar to those that are responsible for nitrous oxide (N(2)O) antinociception. L-ARG and SIN-1 were administered to mice intracerebroventricularly (i.c.v.), and antinociception was assessed using the acetic acid abdominal constriction test. Both L-ARG and SIN-1 caused dose-related antinociceptive effects that were blocked by naloxone and norbinaltorphimine. The antinociceptive effects of both SIN-1 and L-ARG were also blocked to a greater extent by i.c.v. administration of a rabbit antiserum against rat dynorphin 1-13 than an antiserum against methionine-enkephalin, suggesting that the SIN-1 and L-ARG effects may be related to stimulated release of dynorphin. The antinociceptive effect of L-ARG was antagonized by an inhibitor of neuoronal NO synthase enzyme, indicating that L-ARG had to be converted to NO for its antinociceptive action. These findings indicate that the mechanisms of antinociceptive action of L-ARG and SIN-1 are both mediated by dynorphin and dependent on NO.

A study of the role of serotonin in the anxiolytic effect of nitrous oxide in rodents.

RATIONALE: In earlier studies, we have shown that nitrous oxide (N2O)-induced behavioral effects in rats and mice are mediated by benzodiazepine receptors. OBJECTIVES: This two-part study was conducted in order to investigate the possible role of serotonin (5-HT) in the behavioral effects of N2O by clarifying its effects on regional brain concentrations of 5-HT and assessing the influence of 5-HT antagonist and reuptake inhibiting drugs on the anxiolytic-like behavioral effect of N2O. METHODS: In experiment A, male, 150-200 g Sprague-Dawley rats were killed following a 15-min exposure to room air or 70% N2O. The frontal cortex, hippocampus, corpus striatum and hypothalamus were dissected out and analyzed by HPLC with electrochemical detection for content of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA); dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were also measured. In experiment B, male 18-22 g NIH Swiss mice were pretreated with the 5-HT2 antagonist cinanserin, the 5-HT3 antagonist LY-278,584, the 5-HT reuptake inhibitor fluoxetine or saline and tested in the light/dark exploration test under 70% N2O 30 min after pretreatment. RESULTS: In experiment A, N2O produced differential effects on 5-HT neurons in distinct brain areas. There was increased 5-HT turnover in the hypothalamus, decreased turnover in the frontal cortex but no changes in either hippocampus or corpus striatum. By comparison, dopamine turnover in these brain regions was unaltered by N2O exposure. In experiment B, pretreatment with neither cinanserin, LY-278,584 nor fluoxetine had any appreciable effect on the N2O-induced increase in time spent in the light compartment. Only cinanserin significantly reduced the N2O-induced increase in transitions. CONCLUSIONS: While neurochemical results suggest an effect of N2O on brain 5-HT function, there was no effect of 5-HT2 or 5-HT3 antagonists or 5-HT reuptake inhibitor on N2O-induced anxiolytic-like behavior.

Increasing the availability of l-arginine and nitric oxide increases sensitivity of nitrous oxide (N2O)-insensitive inbred mice to N2O-induced antinociception.

Nitrous oxide (N2O)-induced antinociception in mice is dependent on the neuromodulator nitric oxide (NO). In contrast to C57BL/6J (B6) mice, DBA/2J (D2) mice fail to respond to N2O with a robust antinociceptive response or with an increase in brain nitric oxide synthase (NOS) enzyme activity, suggesting that failure of D2 mice to respond to N2O might result from a deficit of NO function. Therefore, it was of interest to determine whether increasing the availability of NO might increase sensitivity of D2 mice to N2O. Male D2 mice were pretreated with sub-antinociceptive intracerebroventricular doses of the NO donor 3-morpholinosydnoimine or the NO precursor l-arginine then assessed for responsiveness to N2O-induced antinociception using the acetic acid abdominal constriction test. Both pretreatments increased the antinociceptive responsiveness of D2 mice to N2O. These results indicate that the NOS enzyme in D2 mice is functional and that the deficit in NO function that obstructs sensitivity to N2O-induced antinociception may lie in availability or utilization of l-arginine.

Hyperbaric oxygen treatment suppresses withdrawal signs in morphine-dependent mice.

Hyperbaric oxygen (HBO2) therapy reportedly reduces opiate withdrawal in human subjects. The purpose of this research was to determine whether HBO2 treatment could suppress physical signs of withdrawal in opiate-dependent mice. Male NIH Swiss mice were injected s.c. with morphine sulfate twice a day for 4 days, the daily dose gradually increasing from 50mg/kg on day 1 to 125mg/kg on day 4. On day 5, withdrawal was precipitated by i.p. injection of 5.0mg/kg naloxone. Mice were observed for physical withdrawal signs, including jumping, forepaw tremor, wet-dog shakes, rearing and defecation for 30min. Sixty min prior to the naloxone injection, different groups of mice received either a 30-min or 60-min HBO2 treatment at 3.5atm absolute. HBO2 treatment significantly reduced naloxone-precipitated jumping, forepaw tremor, wet-dog shakes, rearing and defecation. Based on these experimental findings, we concluded that treatment with HBO2 can suppress physical signs of withdrawal syndrome in morphine-dependent mice.

Involvement of cyclic GMP-dependent protein kinase in nitrous oxide-induced anxiolytic-like behavior in the mouse light/dark exploration test.

The second messenger cyclic GMP (cGMP) plays a role in the anxiolytic-like behavioral response of mice to nitrous oxide (N2O). This study was conducted to determine whether this behavioral effect of N2O is affected by inhibition of cGMP-dependent protein kinase (PKG). N2O-induced behavior in the light/dark exploration test was significantly attenuated by the PKG inhibitors H-8 and Rp-8-pCPT-cGMPS but not Rp-8-pCPT-cAMPS, an inhibitor of cAMP-dependent protein kinase. These findings implicate PKG in the mediation or modulation of the anxiolytic-like behavioral response to N2O.

Do inhalation general anesthetic drugs induce the neuronal release of endogenous opioid peptides?

The antagonism of some effects of inhalation general anesthetic agents by naloxone suggests that there may be an opioid component to anesthetic action. There is evidence that this opioid action component is due to neuronal release of endogenous opioid peptides. The strongest evidence is provided by studies that monitor changes in the concentration of opioid peptides in the perfused brain following inhalation of the anesthetic. Indirect or circumstantial evidence also comes from studies of anesthetic effects on regional brain levels of opioid peptides, antagonism of selected anesthetic effects by antisera to opioid peptides and anesthetic-induced changes radioligand binding to opioid receptors. It is likely that some inhalation general anesthetics (e.g., nitrous oxide) can induce neuronal release of opioid peptides and that this may contribute to certain components of general anesthesia (e.g., analgesia). More definitive studies utilizing in vivo microdialysis or autoradiography in selected areas of the brain during induction and successive states of general anesthesia have yet to be conducted.

Correlation of inbred mouse sensitivity to nitrous oxide antinociception with brain nitric oxide synthase activity following exposure to nitrous oxide.

Inhibition of nitric oxide synthase (NOS) antagonizes nitrous oxide (N2O)-induced antinociception in mice. This study was conducted to compare brain NOS activity in high responding C57BL/6 mice, low responding DBA/2 mice and S5 mice selectively bred for low responsiveness to N2O. Exposure to 70% N2O suppressed acetic acid-induced abdominal constrictions in C57BL/6 mice but not DBA/2 or S5 mice. N2O exposure also elevated NOS activity in brains of C57BL/6 mice but not DBA/2 or S5 mice. The absence of these effects in DBA/2 or S5 mice is further support for the hypothesis that nitric oxide (NO) may play a critical role in N2O-induced antinociception in mice.