Chronic Voluntary Morphine Intake Is Associated with Changes in Brain Structures Involved in Drug Dependence in a Rat Model of Polydrug Use.

Chronic opioid intake leads to several brain changes involved in the development of dependence, whereby an early hedonistic effect (liking) extends to the need to self-administer the drug (wanting), the latter being mostly a prefrontal-striatal function. The development of animal models for voluntary oral opioid intake represents an important tool for identifying the cellular and molecular alterations induced by chronic opioid use. Studies mainly in humans have shown that polydrug use and drug dependence are shared across various substances. We hypothesize that an animal bred for its alcohol preference would develop opioid dependence and further that this would be associated with the overt cortical abnormalities clinically described for opioid addicts. We show that Wistar-derived outbred UChB rats selected for their high alcohol preference additionally develop: (i) a preference for oral ingestion of morphine over water, resulting in morphine intake of 15 mg/kg/day; (ii) marked opioid dependence, as evidenced by the generation of strong withdrawal signs upon naloxone administration; (iii) prefrontal cortex alterations known to be associated with the loss of control over drug intake, namely, demyelination, axonal degeneration, and a reduction in glutamate transporter GLT-1 levels; and (iv) glial striatal neuroinflammation and brain oxidative stress, as previously reported for chronic alcohol and chronic nicotine use. These findings underline the relevance of polydrug animal models and their potential in the study of the wide spectrum of brain alterations induced by chronic morphine intake. This study should be valuable for future evaluations of therapeutic approaches for this devastating condition.

A dual mechanism fully blocks ethanol relapse: Role of vagal innervation.

Previous studies showed that vagotomy markedly inhibits alcohol self-administration. Present studies hypothesised that vagotomy significantly adds to the inhibition of alcohol relapse induced by drugs that reduce the alcohol-induced hyperglutamatergic state (e.g., N-acetylcysteine + acetylsalicylic acid). The alcohol relapse paradigm tested gauges the elevated alcohol intake observed in animals that had consumed ethanol chronically, were subjected to a prolonged alcohol deprivation and are subsequently allowed ethanol re-access. Ethanol-drinker rats (UChB) were exposed to 10% and 20% ethanol and water concurrently for 4 months, were alcohol deprived for 14 days and were thereafter allowed re-access to the ethanol solutions. An initial binge-like drinking episode is observed upon ethanol re-access, followed by a protracted elevated ethanol intake that exceeds the predeprivation intake baseline. Prior to ethanol re-access, animals were (i) administered N-acetylcysteine (40 mg/kg/day) + acetylsalicylic acid (15 mg/kg/day), (ii) were bilaterally vagotomised, (iii) were exposed to both treatments or (iv) received no treatments. The initial binge-like relapse intake and a protracted elevated ethanol intake observed after repeated ethanol deprivations/re-access cycles were inhibited by 50%-70% by the administration of N-acetylcysteine + acetylsalicylic acid and by 40%-70% by vagotomy, while the combined vagotomy plus N-acetylcysteine + acetylsalicylic acid treatment inhibited both the initial binge-like intake and the protracted ethanol intake by >95% (p < 0.001), disclosing a dual mechanism of ethanol relapse and subsequent inhibition beyond that induced by either treatment alone. Future exploration into the mechanism by which vagal activity contributes to ethanol relapse may have translational promise.

A Novel Morphine Drinking Model of Opioid Dependence in Rats.

An animal model of voluntary oral morphine consumption would allow for a pre-clinical evaluation of new treatments aimed at reducing opioid intake in humans. However, the main limitation of oral morphine consumption in rodents is its bitter taste, which is strongly aversive. Taste aversion is often overcome by the use of adulterants, such as sweeteners, to conceal morphine taste or bitterants in the alternative bottle to equalize aversion. However, the adulterants' presence is the cause for consumption choice and, upon removal, the preference for morphine is not preserved. Thus, current animal models are not suitable to study treatments aimed at reducing consumption elicited by morphine itself. Since taste preference is a learned behavior, just-weaned rats were trained to accept a bitter taste, adding the bitterant quinine to their drinking water for one week. The latter was followed by allowing the choice of quinine or morphine (0.15 mg/mL) solutions for two weeks. Then, quinine was removed, and the preference for morphine against water was evaluated. Using this paradigm, we show that rats highly preferred the consumption of morphine over water, reaching a voluntary morphine intake of 15 mg/kg/day. Morphine consumption led to significant analgesia and hyperlocomotion, and to a marked deprivation syndrome following the administration of the opioid antagonist naloxone. Voluntary morphine consumption was also shown to generate brain oxidative stress and neuroinflammation, signs associated with opioid dependence development. We present a robust two-bottle choice animal model of oral morphine self-administration for the evaluation of therapeutic interventions for the treatment of morphine dependence.

Aspirin and N-acetylcysteine co-administration markedly inhibit chronic ethanol intake and block relapse binge drinking: Role of neuroinflammation-oxidative stress self-perpetuation.

Chronic alcohol intake leads to neuroinflammation and cell injury, proposed to result in alterations that perpetuate alcohol intake and cued relapse. Studies show that brain oxidative stress is consistently associated with alcohol-induced neuroinflammation, and literature implies that oxidative stress and neuroinflammation perpetuate each other. In line with a self-perpetuating mechanism, it is hypothesized that inhibition of either oxidative stress or neuroinflammation could reduce chronic alcohol intake and relapse. The present study conducted on alcohol-preferring rats shows that chronic ethanol intake was inhibited by 50% to 55% by the oral administration of low doses of either the antioxidant N-acetylcysteine (40 mg/kg/d) or the anti-inflammatory aspirin (ASA; 15 mg/kg/d), while the co-administration of both dugs led to a 70% to 75% (P < .001) inhibition of chronic alcohol intake. Following chronic alcohol intake, a prolonged alcohol deprivation, and subsequent alcohol re-access, relapse drinking resulted in blood alcohol levels of 95 to 100 mg/dL in 60 minutes, which were reduced by 60% by either N-acetylcysteine or aspirin and by 85% by the co-administration of both drugs (blood alcohol: 10 to 15 mg/dL; P < .001). Alcohol intake either on the chronic phase or following deprivation and re-access led to a 50% reduction of cortical glutamate transporter GLT-1 levels, while aspirin administration fully returned GLT-1 to normal levels. N-acetylcysteine administration did not alter GLT-1 levels, while N-acetylcysteine may activate the cystine/glutamate transport xCT, presynaptically inhibiting relapse. Overall, the study suggests that a neuroinflammation/oxidative stress self-perpetuation cycle maintains chronic alcohol intake and relapse drinking. The co-administration of anti-inflammatory and antioxidant agents may have translational value in alcohol-use disorders.

Innate gut microbiota predisposes to high alcohol consumption.

Gut microbiota is known to be transferred from the mother to their offspring. This study determines whether the innate microbiota of rats selectively bred for generations as high alcohol drinkers play a role in their alcohol intake. Wistar-derived high-drinker UChB rats (intake 10-g ethanol/kg/day) administered nonabsorbable oral antibiotics before allowing access to alcohol, reducing their voluntary ethanol intake by 70%, an inhibition that remained after the antibiotic administration was discontinued. Oral administration of Lactobacillus rhamnosus Gorbach-Goldin (GG) induced the synthesis of FGF21, a vagal ß-Klotho receptor agonist, and partially re-invoked a mechanism that reduces alcohol intake. The vagus nerve constitutes the main axis transferring gut microbiota information to the brain ("microbiota-gut-brain" axis). Bilateral vagotomy inhibited rat alcohol intake by 75%. Neither antibiotic treatment nor vagotomy affected total fluid intake. A microbiota-mediated marked inflammatory environment was observed in the gut of ethanol-naïve high-drinker rats, as gene expression of proinflammatory cytokines (TNF-α; IL-6; IL-1ß) was significantly reduced by nonabsorbable antibiotic administration. Gut cytokines are known to activate the vagus nerve, while vagal activation induces pro-rewarding effects in nucleus accumbens. Both alcoholics and alcohol-preferring rats share a marked preference for sweet tastes-likely an evolutionary trait to seek sweet fermented fruits. Saccharin intake by UChB rats was inhibited by 75%-85% by vagotomy or oral antibiotic administration, despite saccharin-induced polydipsia. Overall, data indicate that the mechanisms that normally curtail heavy drinking are inhibited in alcohol-preferring animals and inform a gut microbiota origin. Whether it applies to other mammals and humans merits further investigation.

Intranasal Administration of Mesenchymal Stem Cell Secretome Reduces Hippocampal Oxidative Stress, Neuroinflammation and Cell Death, Improving the Behavioral Outcome Following Perinatal Asphyxia.

Perinatal Asphyxia (PA) is a leading cause of motor and neuropsychiatric disability associated with sustained oxidative stress, neuroinflammation, and cell death, affecting brain development. Based on a rat model of global PA, we investigated the neuroprotective effect of intranasally administered secretome, derived from human adipose mesenchymal stem cells (MSC-S), preconditioned with either deferoxamine (an hypoxia-mimetic) or TNF-α+IFN-γ (pro-inflammatory cytokines). PA was generated by immersing fetus-containing uterine horns in a water bath at 37 °C for 21 min. Thereafter, 16 µL of MSC-S (containing 6 µg of protein derived from 2 × 105 preconditioned-MSC), or vehicle, were intranasally administered 2 h after birth to asphyxia-exposed and control rats, evaluated at postnatal day (P) 7. Alternatively, pups received a dose of either preconditioned MSC-S or vehicle, both at 2 h and P7, and were evaluated at P14, P30, and P60. The preconditioned MSC-S treatment (i) reversed asphyxia-induced oxidative stress in the hippocampus (oxidized/reduced glutathione); (ii) increased antioxidative Nuclear Erythroid 2-Related Factor 2 (NRF2) translocation; (iii) increased NQO1 antioxidant protein; (iv) reduced neuroinflammation (decreasing nuclearNF-κB/p65 levels and microglial reactivity); (v) decreased cleaved-caspase-3 cell-death; (vi) improved righting reflex, negative geotaxis, cliff aversion, locomotor activity, anxiety, motor coordination, and recognition memory. Overall, the study demonstrates that intranasal administration of preconditioned MSC-S is a novel therapeutic strategy that prevents the long-term effects of perinatal asphyxia.

Activated mesenchymal stem cell administration inhibits chronic alcohol drinking and suppresses relapse-like drinking in high-alcohol drinker rats.

Neuroinflammation has been reported to follow chronic ethanol intake and may perpetuate alcohol consumption. Present studies determined the effect of human mesenchymal stem cells (hMSCs), known for their anti-inflammatory action, on chronic ethanol intake and relapse-like ethanol intake in a post-deprivation condition. Rats were allowed 12-17 weeks of chronic voluntary ethanol (10% and 20% v/v) intake, after which a single dose of activated hMSCs (5 × 105 ) was injected into a brain lateral ventricle. Control animals were administered vehicle. After assessing the effect of hMSCs on chronic ethanol intake for 1 week, animals were deprived of ethanol for 2 weeks and thereafter an ethanol re-access of 60 min was allowed to determine relapse-like intake. A single administration of activated hMSCs inhibited chronic alcohol consumption by 70% (P < 0.001), an effect seen within the first 24 hours of hMSCs administration, and reduced relapse-like drinking by 80% (P < 0.001). In the relapse-like condition, control animals attain blood ethanol ('binge-like') levels >80 mg/dl. The single hMSC administration reduced relapse-like blood ethanol levels to 20 mg/dl. Chronic ethanol intake increased by 250% (P < 0.001) the levels of reactive oxygen species in hippocampus, which were markedly reduced by hMSC administration. Astrocyte glial acidic fibrillary protein immunoreactivity, a hallmark of neuroinflammation, was increased by 60-80% (P < 0.001) by chronic ethanol intake, an effect that was fully abolished by the administration of hMSCs. This study supports the neuroinflammation-chronic ethanol intake hypothesis and suggest that mesenchymal stem cell administration may be considered in the treatment of alcohol use disorders.

Intranasal delivery of mesenchymal stem cell-derived exosomes reduces oxidative stress and markedly inhibits ethanol consumption and post-deprivation relapse drinking.

Chronic ethanol consumption leads to brain oxidative stress and neuroinflammation, conditions known to potentiate and perpetuate each other. Several studies have shown that neuroinflammation results in increases in chronic ethanol consumption. Recent reports showed that the intra-cerebroventricular administration of mesenchymal stem cells to rats consuming alcohol chronically markedly inhibited oxidative-stress, abolished neuroinflammation and greatly reduced chronic alcohol intake and post deprivation relapse-like alcohol intake. However, the intra-cerebroventricular administration of living cells is not suitable as a treatment of a chronic condition. The present study aimed at inhibiting ethanol intake by the non-invasive intranasal administration of human mesenchymal stem cell products: exosomes, microvesicles (40 to 150 nm) with marked antioxidant activity extruded from mesenchymal stem cells. The exosome membrane can fuse with the plasma membrane of cells in different tissues, thus delivering their content intracellularly. The study showed that the weekly intranasal administration of mesenchymal stem cell-derived exosomes to rats consuming alcohol chronically (1) inhibited their ethanol intake by 84 percent and blunted the relapse-like 'binge' drinking that follows an alcohol deprivation period and ethanol re-access. (2) Intranasally administered exosomes were found in the brain within 24 hours; (3) fully reversed both alcohol-induced hippocampal oxidative-stress, evidenced by a lower ratio of oxidized to reduced glutathione, and neuroinflammation, shown by a reduced astrocyte activation and microglial density; and (4) increased glutamate transporter GLT1 expression in nucleus accumbens, counteracting the inhibition of glutamate transporter activity, reportedly depressed under oxidative-stress conditions. Possible translational implications are envisaged.

Commonality of Ethanol and Nicotine Reinforcement and Relapse in Wistar-Derived UChB Rats: Inhibition by N-Acetylcysteine.

BACKGROUND: Life expectancy is greatly reduced in individuals presenting alcohol use disorders and chronic smoking. Literature studies suggest that common mechanisms may apply to the chronic use and relapse of both alcohol and nicotine. It is hypothesized that an increased brain oxidative stress and neuroinflammation are involved in perpetuating these conditions and that a common treatment may be considered for both. METHODS: Rats bred as high ethanol (EtOH) drinkers (UChB) were allowed chronic access to EtOH solutions and water and were thereafter deprived of EtOH for a prolonged period, subsequently allowing reaccess to EtOH, which leads to marked relapse binge-like drinking. Separately, EtOH-naïve animals were chronically administered nicotine intraperitoneally and tested under either a conditioned place preference (CPP) reinstatement condition or allowed a free-choice drinking of nicotine solutions and water. Oral N-acetylcysteine (NAC) (100 mg/kg) was administered daily to the animals to determine its effect on both chronic voluntary EtOH and nicotine intake, on EtOH relapse and nicotine-CPP reinstatement. Oxidative stress was evaluated in hippocampus as the oxidized/reduced glutathione ratio (GSSG/GSH), and neuroinflammation by glial fibrillary acidic protein (GFAP) immunohistochemistry. RESULTS: Marked increases in hippocampal oxidative stress (GSSG/GSH) and neuroinflammation (astrocyte reactivity, GFAP) were observed after both chronic EtOH and chronic nicotine treatment. Oral NAC administration (i) fully abolished the increased oxidative stress and the neuroinflammation induced by both drugs, (ii) greatly inhibited EtOH intake (70%) and EtOH relapse binge-like drinking (76%), and (iii) markedly inhibited (90%) voluntary nicotine intake and fully suppressed nicotine-CPP reinstatement. CONCLUSIONS: Data indicate that (i) oxidative stress and neuroinflammation are tightly associated with chronic EtOH and nicotine intake and drug relapse and (ii) NAC inhibits the relapse for both drugs, suggesting that the oral chronic administration of NAC may be of value in the concomitant treatment of alcohol and nicotine use disorders.

Intracerebral Stem Cell Administration Inhibits Relapse-like Alcohol Drinking in Rats.

Study describes the blockade of relapse-like alcohol drinking by mesenchymal stem cells (MSCs). High alcohol-intake bred rats consumed alcohol for 3 months and were subjected to repeated alcohol deprivations for 7-14 days, followed by alcohol reaccess. Upon reaccess, animals consumed 2.2 g alcohol/kg in 60 minutes. A single intra-cerebroventricular MSC administration inhibited relapse-like drinking up to 80-85% for 40 days (P < 0.001). An alcohol-use-disorder was prevented.

Beyond the "First Hit": Marked Inhibition by N-Acetyl Cysteine of Chronic Ethanol Intake But Not of Early Ethanol Intake. Parallel Effects on Ethanol-Induced Saccharin Motivation.

BACKGROUND: A number of studies have shown that acetaldehyde synthesized in the brain is necessary to induce ethanol (EtOH) reinforcement in naïve animals (acquisition phase). However, after chronic intake is achieved (maintenance phase), EtOH intake becomes independent of acetaldehyde generation or its levels. Glutamate has been reported to be associated with the maintenance of chronic EtOH intake. The levels of brain extracellular glutamate are modulated by 2 glial processes: glutamate reabsorption via an Na(+) -glutamate transporter (GLT1) and a cystine-glutamate exchanger. Chronic EtOH intake lowers GLT1 levels and increases extracellular glutamate. The administration of N-acetyl cysteine (NAC), a precursor of cystine, has been shown to reduce the relapse of several drugs of abuse, while NAC has not been tested on chronic EtOH intake or on EtOH's influence on the motivation for another drug. These were investigated in the present study. METHODS: (i) Rats bred for their high EtOH intake were allowed access to 10% EtOH and water up to 87 days. NAC was administered (30 and 60 mg/kg daily, intraperitoneally) for 14 consecutive days, either during the acquisition phase or the maintenance phase of EtOH drinking. (ii) In additional experiments, rats were allowed EtOH (10%) and water access for 61 days, after which EtOH was replaced by saccharin (0.3%) to determine both if chronic EtOH consumption influences saccharin intake and whether NAC modifies the post chronic EtOH saccharin intake. RESULTS: NAC did not influence the acquisition ("first hit") of chronic EtOH intake, but greatly inhibited (60 to 70%; p < 0.0001) EtOH intake when NAC was administered to animals that were consuming EtOH chronically. NAC did not influence saccharin intake in naïve animals. In animals that had consumed EtOH chronically and were thereafter offered a saccharin solution (0.3%), saccharin intake increased over 100% versus that of EtOH-untreated animals, an effect that was fully suppressed by NAC. CONCLUSIONS: N-acetyl cysteine, a drug approved for use in humans, markedly reduces chronic EtOH intake and abolishes the increased intake of saccharin stimulated by chronic EtOH drinking.

(R)-Salsolinol, a product of ethanol metabolism, stereospecifically induces behavioral sensitization and leads to excessive alcohol intake.

Ethanol is oxidized in the brain to acetaldehyde, which can condense with dopamine to generate (R/S)-salsolinol [(RS)-SAL]. Racemic salsolinol [(RS)-SAL] is self-infused by rats into the posterior ventral tegmental area (VTA) at significantly lower concentrations than those of acetaldehyde, suggesting that (RS)-SAL is a most active product of ethanol metabolism. Early studies showed that repeated intraperitoneal or intra-VTA administration of (RS)-SAL (10 mg/kg) induced conditioned place preference, led to locomotor sensitization and increased voluntary ethanol consumption. In the present study, we separated the (R)- and (S)-enantiomers from a commercial (RS)-SAL using a high-performance liquid chromatography with electrochemical detection system fitted with a ß-cyclodextrin-modified column. We injected (R)-SAL or (S)-SAL (30 pmol/1.0 µl) into the VTA of naïve UChB rats bred as alcohol drinkers to study whether one or both SAL enantiomers are responsible for the motivated behavioral effects, sensitization and increase in voluntary ethanol intake. The present results show that repeated administration of (R)-SAL leads to (1) conditioned place preference; (2) locomotor sensitization; and (3) marked increases in binge-like ethanol intake. Conversely, (S)-SAL did not influence any of these parameters. Overall, data indicate that (R)-SAL stereospecifically induces motivational effects, behavioral sensitization and increases ethanol intake.

The "first hit" toward alcohol reinforcement: role of ethanol metabolites.

This review analyzes literature that describes the behavioral effects of 2 metabolites of ethanol (EtOH): acetaldehyde and salsolinol (a condensation product of acetaldehyde and dopamine) generated in the brain. These metabolites are self-administered into specific brain areas by animals, showing strong reinforcing effects. A wealth of evidence shows that EtOH, a drug consumed to attain millimolar concentrations, generates brain metabolites that are reinforcing at micromolar and nanomolar concentrations. Salsolinol administration leads to marked increases in voluntary EtOH intake, an effect inhibited by mu-opioid receptor blockers. In animals that have ingested EtOH chronically, the maintenance of alcohol intake is no longer influenced by EtOH metabolites, as intake is taken over by other brain systems. However, after EtOH withdrawal brain acetaldehyde has a major role in promoting binge-like drinking in the condition known as the "alcohol deprivation effect"; a condition seen in animals that have ingested alcohol chronically, are deprived of EtOH for extended periods, and are allowed EtOH re-access. The review also analyzes the behavioral effects of acetate, a metabolite that enters the brain and is responsible for motor incoordination at low doses of EtOH. Also discussed are the paradoxical effects of systemic acetaldehyde. Overall, evidence strongly suggests that brain-generated EtOH metabolites play a major role in the early ("first-hit") development of alcohol reinforcement and in the generation of relapse-like drinking.

Long-term inhibition of ethanol intake by the administration of an aldehyde dehydrogenase-2 (ALDH2)-coding lentiviral vector into the ventral tegmental area of rats.

Previous studies suggest that acetaldehyde generated from ethanol in the brain is reinforcing. The present studies tested the feasibility of achieving a long-term reduction of chronic and post-deprivation binge ethanol drinking by a single administration into the brain ventral tegmental area (VTA) of a lentiviral vector that codes for aldehyde dehydrogenase-2 (ALDH2), which degrades acetaldehyde. The ALDH2 gene coding vector or a control lentiviral vector were microinjected into the VTA of rats bred for their alcohol preference. In the chronic alcohol administration model, naïve animals administered the control vector and subsequently offered 10% ethanol and water ingested 8-9 g ethanol/kg body weight/day. The single administration of the ALDH2-coding vector prior to allowing ethanol availability reduced ethanol drinking by 85-90% (P < 0.001) for the 45 days tested. In the post-deprivation binge-drinking model, animals that had previously consumed ethanol chronically for 81 days were administered the lentiviral vector and were thereafter deprived of ethanol for three 7-day periods, each interrupted by a single 60-minute ethanol re-access after the last day of each deprivation period. Upon ethanol re-access, control vector-treated animals consumed intoxicating 'binge' amounts of ethanol, reaching intakes of 2.7 g ethanol/kg body weight in 60 minutes. The administration of the ALDH2-coding vector reduced re-access binge drinking by 75-80% (P < 0.001). This study shows that endowing the ventral tegmental with an increased ability to degrade acetaldehyde greatly reduces chronic alcohol consumption and post-deprivation binge drinking for prolonged periods and supports the hypothesis that brain-generated acetaldehyde promotes alcohol drinking.

Morphine self-administration is inhibited by the antioxidant N-acetylcysteine and the anti-inflammatory ibudilast; an effect enhanced by their co-administration.

BACKGROUND: The treatment of opioid addiction mainly involves the medical administration of methadone or other opioids, aimed at gradually reducing dependence and, consequently, the need for illicit opioid procurement. Thus, initiating opioid maintenance therapy with a lower level of dependence would be advantageous. There is compelling evidence indicating that opioids induce brain oxidative stress and associated glial activation, resulting in the dysregulation of glutamatergic homeostasis, which perpetuates drug intake. The present study aimed to determine whether inhibiting oxidative stress and/or neuroinflammation reduces morphine self-administration in an animal model of opioid dependence. METHODS: Morphine dependence, assessed as voluntary morphine self-administration, was evaluated in Wistar-derived UChB rats. Following an extended period of morphine self-administration, animals were administered either the antioxidant N-acetylcysteine (NAC; 40 mg/kg/day), the anti-inflammatory ibudilast (7.5 mg/kg/day) or the combination of both agents. Oxidative stress and neuroinflammation were evaluated in the hippocampus, a region involved in drug recall that feeds into the nucleus accumbens, where the levels of the glutamate transporters GLT-1 and xCT were further assessed. RESULTS: Daily administration of either NAC or ibudilast led to a mild reduction in voluntary morphine intake, while the co-administration of both therapeutic agents resulted in a marked inhibition (-57%) of morphine self-administration. The administration of NAC or ibudilast markedly reduced both the oxidative stress induced by chronic morphine intake and the activation of microglia and astrocytes in the hippocampus. However, only the combined administration of NAC + ibudilast was able to restore the normal levels of the glutamate transporter GLT-1 in the nucleus accumbens. CONCLUSION: Separate or joint administration of an antioxidant and anti-inflammatory agent reduced voluntary opioid intake, which could have translational value for the treatment of opioid use disorders, particularly in settings where the continued maintenance of oral opioids is a therapeutic option.

Amelioration of morphine withdrawal syndrome by systemic and intranasal administration of mesenchymal stem cell-derived secretome in preclinical models of morphine dependence.

BACKGROUND: Morphine is an opiate commonly used in the treatment of moderate to severe pain. However, prolonged administration can lead to physical dependence and strong withdrawal symptoms upon cessation of morphine use. These symptoms can include anxiety, irritability, increased heart rate, and muscle cramps, which strongly promote morphine use relapse. The morphine-induced increases in neuroinflammation, brain oxidative stress, and alteration of glutamate levels in the hippocampus and nucleus accumbens have been associated with morphine dependence and a higher severity of withdrawal symptoms. Due to its rich content in potent anti-inflammatory and antioxidant factors, secretome derived from human mesenchymal stem cells (hMSCs) is proposed as a preclinical therapeutic tool for the treatment of this complex neurological condition associated with neuroinflammation and brain oxidative stress. METHODS: Two animal models of morphine dependence were used to evaluate the therapeutic efficacy of hMSC-derived secretome in reducing morphine withdrawal signs. In the first model, rats were implanted subcutaneously with mini-pumps which released morphine at a concentration of 10 mg/kg/day for seven days. Three days after pump implantation, animals were treated with a simultaneous intravenous and intranasal administration of hMSC-derived secretome or vehicle, and withdrawal signs were precipitated on day seven by i.p. naloxone administration. In this model, brain alterations associated with withdrawal were also analyzed before withdrawal precipitation. In the second animal model, rats voluntarily consuming morphine for three weeks were intravenously and intranasally treated with hMSC-derived secretome or vehicle, and withdrawal signs were induced by morphine deprivation. RESULTS: In both animal models secretome administration induced a significant reduction of withdrawal signs, as shown by a reduction in a combined withdrawal score. Secretome administration also promoted a reduction in morphine-induced neuroinflammation in the hippocampus and nucleus accumbens, while no changes were observed in extracellular glutamate levels in the nucleus accumbens. CONCLUSION: Data presented from two animal models of morphine dependence suggest that administration of secretome derived from hMSCs reduces the development of opioid withdrawal signs, which correlates with a reduction in neuroinflammation in the hippocampus and nucleus accumbens.

The alcohol deprivation effect: marked inhibition by anticatalase gene administration into the ventral tegmental area in rats.

BACKGROUND: Animals that have chronically consumed alcohol and are subsequently deprived of it markedly increase their intake above basal levels when access to alcohol is reinstated. Such an effect, termed the alcohol deprivation effect (ADE), has been proposed to reflect (i) an obsessive-compulsive behavior, (ii) craving, or (iii) an increased reinforcing value of ethanol (EtOH). It has been reported that acetaldehyde, a highly reinforcing metabolite of EtOH, is generated in the brain by the action of catalase. Recent studies show that the administration of an anticatalase (shRNA)-encoding lentiviral vector into the brain ventral tegmental area (VTA) of naïve rats virtually abolishes (85 to 95%) their EtOH intake. It is hypothesized that the antireinforcing effect of the anticatalase vector will also inhibit the ADE. METHODS: Two-month-old Wistar-derived UChB alcohol drinker rats were offered free access to water and 10 and 20% EtOH for 67 days. Thereafter, the animals were deprived of EtOH for 15 days and were subsequently offered access to the EtOH solutions. At the start of the deprivation period, animals were microinjected a single dose of an anticatalase (or control) vector into the VTA. EtOH intake was measured on the first hour of EtOH re-exposure as well as on a 24-hour basis for 7 days. RESULTS: A marked ADE was observed when EtOH intake was measured on the first hour or 24 hours following EtOH re-exposure, compared to the corresponding controls. The administration of the anticatalase vector reduced ADE by 60 to 80% (p < 0.001) on the first hour and by 63 to 80% (p < 0.001) on the initial 24 hours of EtOH re-exposure (first and second ADE, respectively) without changing the total fluid intake, indicating a specific effect on EtOH drinking. CONCLUSIONS: Ethanol intake associated with ADE--a binge-like drinking behavior--is markedly inhibited by the administration of an anticatalase vector into the VTA, which blocks the conversion of EtOH into acetaldehyde, strongly suggesting that the marked increased EtOH intake that follows an alcohol deprivation period is mediated by acetaldehyde and its reinforcing metabolite.

Role of Metabolism on Alcohol Preference, Addiction, and Treatment.

Studies presented in this chapter show that: (1) in the brain, ethanol is metabolized by catalase to acetaldehyde, which condenses with dopamine forming salsolinol; (2) acetaldehyde-derived salsolinol increases the release of dopamine mediating, via opioid receptors, the reinforcing effects of ethanol during the acquisition of ethanol consumption, while (3) brain acetaldehyde does not influence the maintenance of chronic ethanol intake, it is suggested that a learned cue-induced hyperglutamatergic system takes precedence over the dopaminergic system. However, (4) following a prolonged ethanol deprivation, the generation of acetaldehyde in the brain again plays a role, contributing to the increase in ethanol intake observed during ethanol re-access, called the alcohol deprivation effect (ADE), a model of relapse behavior; (5) naltrexone inhibits the high ethanol intake seen in the ADE condition, suggesting that acetaldehyde-derived salsolinol via opioid receptors also contributes to the relapse-like drinking behavior. The reader is referred to glutamate-mediated mechanisms that trigger the cue-associated alcohol-seeking and that also contribute to triggering relapse.

Reward and relapse: complete gene-induced dissociation in an animal model of alcohol dependence.

BACKGROUND: In animal models of continuous alcohol self-administration, in which physical dependence does not constitute the major factor of ethanol intake, 2 factors likely contribute to the perpetuation of alcohol self-administration: (i) the rewarding effects of ethanol and (ii) the contextual conditioning cues that exist along with the process of self-administration. Present studies are aimed at understanding the relative contribution of these factors on the perpetuation of heavy alcohol self-administration, as an indication of relapse. METHODS: Wistar-derived UChB high ethanol drinker rats were allowed access to 10% ethanol and water on a 24-hour basis. In initial studies, an anticatalase shRNA gene-coding lentiviral vector aimed at inhibiting acetaldehyde generation was administered into the ventral tegmental area (VTA) of the animals prior to ethanol access. In subsequent studies, the lentiviral vector was administered to animals, which had consumed ethanol on a 24-hour basis, or a 1-hour basis, after the animals had reached high levels of ethanol intake for 60 to 80 days. In final studies, quinine (0.01%) was added to the ethanol solution to alter the conditioning taste/smell cues of alcohol that animals had chronically ingested. RESULTS: Data indicate that the administration of an anticatalase vector into the VTA of naïve animals blocked reward and alcohol self-administration, while it was, nevertheless, inactive in inhibiting alcohol self-administration in rats that had been conditioned to ingest ethanol for over 2 months. The lack of inhibitory effect of the anticatalase vector on ethanol intake in animals that had chronically self-administered ethanol was fully reversed when the contextual conditioning cues of the alcohol solution were changed. CONCLUSIONS: Data highlight the importance of conditioning factors in relapse and suggest that only abolishing or blunting it, along with long-lasting pharmacological treatment to reduce ethanol reward, may have protracted effects in reducing alcohol self-administration.

Effect of human mesenchymal stem cell secretome administration on morphine self-administration and relapse in two animal models of opioid dependence.

The present study investigates the possible therapeutic effects of human mesenchymal stem cell-derived secretome on morphine dependence and relapse. This was studied in a new model of chronic voluntary morphine intake in Wistar rats which shows classic signs of morphine intoxication and a severe naloxone-induced withdrawal syndrome. A single intranasal-systemic administration of MSCs secretome fully inhibited (>95%; p < 0.001) voluntary morphine intake and reduced the post-deprivation relapse intake by 50% (p < 0.02). Since several studies suggest a significant genetic contribution to the chronic use of many addictive drugs, the effect of MSCs secretome on morphine self-administration was further studied in rats bred as high alcohol consumers (UChB rats). Sub-chronic intraperitoneal administration of morphine before access to increasing concentrations of morphine solutions and water were available to the animals, led UChB rats to prefer ingesting morphine solutions over water, attaining levels of oral morphine intake in the range of those in the Wistar model. Intranasally administered MSCs secretome to UChB rats dose-dependently inhibited morphine self-administration by 72% (p < 0.001); while a single intranasal dose of MSC-secretome administered during a morphine deprivation period imposed on chronic morphine consumer UChB rats inhibited re-access morphine relapse intake by 80 to 85% (p < 0.0001). Both in the Wistar and the UChB rat models, MSCs-secretome administration reversed the morphine-induced increases in brain oxidative stress and neuroinflammation, considered as key engines perpetuating drug relapse. Overall, present preclinical studies suggest that products secreted by human mesenchymal stem cells may be of value in the treatment of opioid addiction.