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.

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.

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.

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.

A dual treatment blocks alcohol binge-drinking relapse: Microbiota as a new player.

RATIONALE: Gut microbiota communicates information to the brain. Some animals are born with a gut microbiota that predisposes to high alcohol consumption, and transplantation of fecal material from alcoholics to mice increases animal preference for ethanol. Alcohol-use-disorders are chronic conditions where relapse is the hallmark. A predictive animal model of relapse is the "alcohol deprivation effect" where ethanol re-access is allowed following chronic alcohol intake and a long alcohol deprivation. The present study evaluates the effect of gut microbiota modification on relapse, as an adjunct to N-acetylcysteine + Acetylsalicylic acid administration, which inhibits the alcohol-induced hyper-glutamatergic condition. METHODS: Rats bred as heavy alcohol consumers (UChB) were allowed ethanol intake for one month, were deprived of alcohol for two-weeks and subsequently offered re-access to ethanol. Prior to ethanol re-access animals received orally either (i) vehicle-control, (ii) Lactobacillus-rhamnosus-GG after antibiotic treatment (LGG); (iii) N-acetylcysteine+Acetylsalicylic acid (NAC/ASA) or (iv) both treatments: LGG+ (NAC/ASA). RESULTS: Marked binge drinking (1.75 g ethanol/kg in 60 min) and blood alcohol levels exceeding 80 mg/dl were observed in the control group upon ethanol-re-access. Lactobacillus-GG or (NAC+ASA) treatments inhibited alcohol intake by 66-80%. The combination of both treatments virtually suppressed (inhibition of 90%) the re-access binge-like drinking, showing additive effects. Treatment with NAC+ASA increased the levels of glutamate transporters xCT and GLT-1 in nucleus accumbens, while Lactobacillus-GG administration increased those of the dopamine transporter (DAT). CONCLUSIONS: The administration of a well-accepted probiotic may be of value as an adjunct in the treatment of alcohol-use-disorders.

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.

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.

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.

Sustained Energy Deficit Following Perinatal Asphyxia: A Shift towards the Fructose-2,6-bisphosphatase (TIGAR)-Dependent Pentose Phosphate Pathway and Postnatal Development.

Labor and delivery entail a complex and sequential metabolic and physiologic cascade, culminating in most circumstances in successful childbirth, although delivery can be a risky episode if oxygen supply is interrupted, resulting in perinatal asphyxia (PA). PA causes an energy failure, leading to cell dysfunction and death if re-oxygenation is not promptly restored. PA is associated with long-term effects, challenging the ability of the brain to cope with stressors occurring along with life. We review here relevant targets responsible for metabolic cascades linked to neurodevelopmental impairments, that we have identified with a model of global PA in rats. Severe PA induces a sustained effect on redox homeostasis, increasing oxidative stress, decreasing metabolic and tissue antioxidant capacity in vulnerable brain regions, which remains weeks after the insult. Catalase activity is decreased in mesencephalon and hippocampus from PA-exposed (AS), compared to control neonates (CS), in parallel with increased cleaved caspase-3 levels, associated with decreased glutathione reductase and glutathione peroxidase activity, a shift towards the TIGAR-dependent pentose phosphate pathway, and delayed calpain-dependent cell death. The brain damage continues long after the re-oxygenation period, extending for weeks after PA, affecting neurons and glial cells, including myelination in grey and white matter. The resulting vulnerability was investigated with organotypic cultures built from AS and CS rat newborns, showing that substantia nigra TH-dopamine-positive cells from AS were more vulnerable to 1 mM of H2O2 than those from CS animals. Several therapeutic strategies are discussed, including hypothermia; N-acetylcysteine; memantine; nicotinamide, and intranasally administered mesenchymal stem cell secretomes, promising clinical translation.

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.

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.

Oxidative Stress and Neuroinflammation as a Pivot in Drug Abuse. A Focus on the Therapeutic Potential of Antioxidant and Anti-Inflammatory Agents and Biomolecules.

Drug abuse is a major global health and economic problem. However, there are no pharmacological treatments to effectively reduce the compulsive use of most drugs of abuse. Despite exerting different mechanisms of action, all drugs of abuse promote the activation of the brain reward system, with lasting neurobiological consequences that potentiate subsequent consumption. Recent evidence shows that the brain displays marked oxidative stress and neuroinflammation following chronic drug consumption. Brain oxidative stress and neuroinflammation disrupt glutamate homeostasis by impairing synaptic and extra-synaptic glutamate transport, reducing GLT-1, and system Xc- activities respectively, which increases glutamatergic neurotransmission. This effect consolidates the relapse-promoting effect of drug-related cues, thus sustaining drug craving and subsequent drug consumption. Recently, promising results as experimental treatments to reduce drug consumption and relapse have been shown by (i) antioxidant and anti-inflammatory synthetic molecules whose effects reach the brain; (ii) natural biomolecules secreted by mesenchymal stem cells that excel in antioxidant and anti-inflammatory properties, delivered via non-invasive intranasal administration to animal models of drug abuse and (iii) potent anti-inflammatory microRNAs and anti-miRNAs which target the microglia and reduce neuroinflammation and drug craving. In this review, we address the neurobiological consequences of brain oxidative stress and neuroinflammation that follow the chronic consumption of most drugs of abuse, and the current and potential therapeutic effects of antioxidants and anti-inflammatory agents and biomolecules to reduce these drug-induced alterations and to prevent relapse.

N-Acetylcysteine and Acetylsalicylic Acid Inhibit Alcohol Consumption by Different Mechanisms: Combined Protection.

Chronic ethanol intake results in brain oxidative stress and neuroinflammation, which have been postulated to perpetuate alcohol intake and to induce alcohol relapse. The present study assessed the mechanisms involved in the inhibition of: (i) oxidative stress; (ii) neuroinflammation; and (iii) ethanol intake that follow the administration of the antioxidant N-acetylcysteine (NAC) and the anti-inflammatory acetylsalicylic acid (ASA) to animals that had consumed ethanol chronically. At doses used clinically, NAC [40 mg/kg per day orally (p.o.)] and ASA (15 mg/kg per day p.o.) significantly inhibited chronic alcohol intake and relapse intake in alcohol-preferring rats. The coadministration of both drugs reduced ethanol intake by 65% to 70%. N-acetylcysteine administration: (a) induced the Nrf2-ARE system, lowering the hippocampal oxidative stress assessed as the ratio of oxidized glutathione (GSSG)/reduced glutathione (GSH); (b) reduced the neuroinflammation assessed by astrocyte and microglial activation by immunofluorescence; and (c) inhibited chronic and relapse ethanol intake. These effects were blocked by sulfasalazine, an inhibitor of the xCT transporter, which incorporates cystine (precursor of GSH) and extrudes extracellular glutamate, an agonist of the inhibitory mGlu2/3 receptor, which lowers the synaptic glutamatergic tone. The inhibitor of mGlu2/3 receptor (LY341495) blocked the NAC-induced inhibition of both relapse ethanol intake and neuroinflammation without affecting the GSSG/GSH ratio. Unlike N-acetylcysteine, ASA inhibited chronic alcohol intake and relapse via lipoxin A4, a strong anti-inflammatory metabolite of arachidonic acid generated following the ASA acetylation of cyclooxygenases. Accordingly, the lipoxin A4 receptor inhibitor, WRW4, blocked the ASA-induced reduction of ethanol intake. Overall, via different mechanisms, NAC and ASA administered in clinically relevant doses combine their effects inhibiting ethanol intake.

Administration of N-acetylcysteine Plus Acetylsalicylic Acid Markedly Inhibits Nicotine Reinstatement Following Chronic Oral Nicotine Intake in Female Rats.

BACKGROUND: Nicotine is the major addictive component of cigarette smoke and the prime culprit of the failure to quit smoking. Common elements perpetuating the use of addictive drugs are (i) cues associated with the setting in which drug was used and (ii) relapse/reinstatement mediated by an increased glutamatergic tone (iii) associated with drug-induced neuroinflammation and oxidative stress. AIMS: The present study assessed the effect of the coadministration of the antioxidant N-acetylcysteine (NAC) plus the anti-inflammatory acetylsalicylic acid (ASA) on oral nicotine reinstatement intake following a post-deprivation re-access in female rats that had chronically and voluntarily consumed a nicotine solution orally. The nicotine-induced oxidative stress and neuroinflammation in the hippocampus and its effects on the glutamate transporters GLT-1 and XCT mRNA levels in prefrontal cortex were also analyzed. RESULTS: The oral coadministration of NAC (40 mg/kg/day) and ASA (15 mg/kg/day) inhibited by 85% of the oral nicotine reinstatement intake compared to control (vehicle), showing an additive effect of both drugs. Acetylsalicylic acid and N-acetylcysteine normalized hippocampal oxidative stress and blunted the hippocampal neuroinflammation observed upon oral nicotine reinstatement. Nicotine downregulated GLT-1 and xCT gene expression in the prefrontal cortex, an effect reversed by N-acetylcysteine, while acetylsalicylic acid reversed the nicotine-induced downregulation of GLT-1 gene expression. The inhibitory effect of N-acetylcysteine on chronic nicotine intake was blocked by the administration of sulfasalazine, an inhibitor of the xCT transporter. CONCLUSION: Nicotine reinstatement, following post-deprivation of chronic oral nicotine intake, downregulates the mRNA levels of GLT-1 and xCT transporters, an effect reversed by the coadministration of N-acetylcysteine and acetylsalicylic acid, leading to a marked inhibition of nicotine intake. The combination of these drugs may constitute a valuable adjunct in the treatment of nicotine-dependent behaviors.

Intranasal mesenchymal stem cell secretome administration markedly inhibits alcohol and nicotine self-administration and blocks relapse-intake: mechanism and translational options.

BACKGROUND: Chronic consumption of most drugs of abuse leads to brain oxidative stress and neuroinflammation, which inhibit the glutamate transporter GLT-1, proposed to perpetuate drug intake. The present study aimed at inhibiting chronic ethanol and nicotine self-administration and relapse by the non-invasive intranasal administration of antioxidant and anti-inflammatory secretome generated by adipose tissue-derived activated mesenchymal stem cells. The anti-addiction mechanism of stem cell secretome is also addressed. METHODS: Rats bred for their alcohol preference ingested alcohol chronically or were trained to self-administer nicotine. Secretome of human adipose tissue-derived activated mesenchymal stem cells was administered intranasally to animals, both (i) chronically consuming alcohol or nicotine and (ii) during a protracted deprivation before a drug re-access leading to relapse intake. RESULTS: The intranasal administration of secretome derived from activated mesenchymal stem cells inhibited chronic self-administration of ethanol or nicotine by 85% and 75%, respectively. Secretome administration further inhibited by 85-90% the relapse "binge" intake that occurs after a protracted drug deprivation followed by a 60-min drug re-access. Secretome administration fully abolished the oxidative stress induced by chronic ethanol or nicotine self-administration, shown by the normalization of the hippocampal oxidized/reduced glutathione ratio, and the neuroinflammation determined by astrocyte and microglial immunofluorescence. Knockdown of the glutamate transporter GLT-1 by the intracerebral administration of an antisense oligonucleotide fully abolished the inhibitory effect of the secretome on ethanol and nicotine intake. CONCLUSIONS: The non-invasive intranasal administration of secretome generated by human adipose tissue-derived activated mesenchymal stem cells markedly inhibits alcohol and nicotine self-administration, an effect mediated by the glutamate GLT-1 transporter. Translational implications are envisioned.

Activation of mitochondrial aldehyde dehydrogenase (ALDH2) by ALDA-1 reduces both the acquisition and maintenance of ethanol intake in rats: A dual mechanism?

A number of pre-clinical studies have shown that brain-generated acetaldehyde, the first metabolite of ethanol, exerts reinforcing effects that promote the acquisition of ethanol intake, while chronic intake maintenance appears to be mediated by alcohol-induced brain neuroinflammation/oxidative stress. Recently, it was described that N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide (ALDA-1) activates aldehyde dehydrogenase-2 (ALDH2), enzyme that catalyzes the oxidation of ethanol-derived acetaldehyde to acetate. The aim of this study was to determine the effects of ALDA-1 on both the acquisition and the maintenance of alcohol intake in alcohol-preferring UChB rats. For ethanol acquisition studies, naïve UChB rats were treated with five daily doses of ALDA-1 (12.5, 25 or 50 mg/kg, i.p.) from one day before the start of ethanol exposure. For chronic intake studies, UChB rats exposed for 98 days to a free access to 10% ethanol and water were treated daily with ALDA-1 (12.5, 25 or 50 mg/kg, i.p.) for five days. The administration of ALDA-1 reduced by 72-90% (p < 0.001) the acquisition of ethanol consumption in naïve rats. At chronic ethanol consumption, ALDA-1 reduced ethanol intake by 61-82% (p < 0.001). ALDA-1 administration increased by 3- and 2.3-fold the activity of ALDH2 in brain and liver, respectively. ALDA-1 did not affect saccharin consumption, nor it modified the rate of ethanol elimination. The study shows that the activation of ALDH2 by ALDA-1 is effective for inhibiting both the acquisition and the maintenance of chronic ethanol intake by alcohol-preferring rats. Thus, the activation of brain ALDH2 may constitute a novel approach 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.

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.

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.

Intravenous administration of anti-inflammatory mesenchymal stem cell spheroids reduces chronic alcohol intake and abolishes binge-drinking.

Chronic alcohol intake leads to neuroinflammation and astrocyte dysfunction, proposed to perpetuate alcohol consumption and to promote conditioned relapse-like binge drinking. In the present study, human mesenchymal stem cells (MSCs) were cultured in 3D-conditions to generate MSC-spheroids, which greatly increased MSCs anti-inflammatory ability and reduced cell volume by 90% versus conventionally 2D-cultured MSCs, enabling their intravenous administration and access to the brain. It is shown, in an animal model of chronic ethanol intake and relapse-drinking, that both the intravenous and intra-cerebroventricular administration of a single dose of MSC-spheroids inhibited chronic ethanol intake and relapse-like drinking by 80-90%, displaying significant effects over 3-5 weeks. The MSC-spheroid administration fully normalized alcohol-induced neuroinflammation, as shown by a reduced astrocyte activation, and markedly increased the levels of the astrocyte Na-glutamate (GLT-1) transporter. This research suggests that the intravenous administration of MSC-spheroids may constitute an effective new approach for the treatment of alcohol-use disorders.