Intranasal mesenchymal stem cell secretome administration markedly inhibits alcohol and nicotine self-administration and blocks relapse-intake: mechanism and translational options
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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.
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.
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.
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.
KeywordsROS Neuroinflammation Knockdown GLT-1 antisense Intranasal Mesenchymal stem cells
Blood alcohol concentration
Cytochrome P450 family 2 subfamily E member 1
Glial fibrillary acidic protein
Glutamate transporter 1
Ionized calcium-binding adaptor molecule 1
Inhibitor of kappa B
Mesenchymal stem cells
Nuclear factor kappa-light-chain-enhancer of activated B cells
Reactive oxygen species
Tumor necrosis factor alpha
University of Chile Bibulous
Alpha minimal essential medium
Numerous preclinical and clinical studies have shown that oxidative stress and neuroinflammation are generated following chronic alcohol consumption or its administration [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11]. It has been reported that ethanol intake, via the generation of acetaldehyde, weakens the tight junction intestinal barrier and promotes lipopolysaccharide influx from the gut into the portal circulation , leading to the increases in systemic TNF-α and the induction of neuroinflammation via the brain TNF-α receptor .
Heavy smoking often accompanies alcohol use disorders [14, 15]. Nicotine is the major addictive component of cigarette smoke and the prime culprit of the failure to quit smoking . Chronic nicotine administration also leads to brain oxidative stress and activates NF-κB transcription, inducing neuroinflammation [17, 18, 19, 20].
Recent studies  showed that rats that had ingested a nicotine solution (40 mg/l) for 2 months, under a free-choice paradigm versus water, display marked increases in brain oxidative stress, as seen by an increased oxidized/reduced glutathione (GSSG/GSH) hippocampal ratio. Nicotine is known to release dopamine into the nucleus accumbens, where high dopamine levels induce oxidative stress via DA auto-oxidation . Further, the monoamine oxidase metabolism of dopamine generates hydrogen peroxide, a precursor to hydroxyl radicals [21, 22]. Nicotine also elevates both cytochrome P450 (CYP) 2A6 and CYP2E1, potent generators of oxygen radicals .
It has been shown that oxidative stress and neuroinflammation are tightly linked; TNF-α increases the generation of reactive oxygen species (ROS) via cytosolic and mitochondrial mechanisms [24, 25, 26]. In turn, ROS activates the NF-κB system partly by inactivating the NF-κB inhibitor IκB  allowing the NF-κB-mediated synthesis of TNF-α, thus creating a self-perpetuating neuroinflammation-oxidative stress vicious-like cycle.
Beyond the initial receptor-specific reinforcing effects of different drugs, the perpetuation of intake and the relapse of most drugs of abuse occur mainly by a general drug-induced cued-recall activation of the nucleus accumbens by glutamate released via hippocampal connection [28, 29]. In operant studies, chronic ethanol self-administration leads to a cued-induced elevation of extracellular glutamate in the nucleus accumbens , an elevation that is greatly increased if glutamate reuptake by the glial glutamate transporter GLT-1 is inefficient; this is also expected for nicotine . Reactive oxygen species directly inhibit the glutamate GLT-1 transporter ; additionally, the ROS-generated lipoperoxidation product 4-hydroxynonenal (4HNE) forms adducts with the GLT-1 transporter, further inhibiting glutamate uptake by astrocytes [32, 33].
Mesenchymal stem cells (MSCs) are referred to as “guardians of inflammation” , being activated by pro-inflammatory signals to release anti-inflammatory cytokines and antioxidant molecules [8, 34]. Thus, MSCs have been proposed as potent therapeutic agents in the treatment of several conditions associated with neuroinflammation and oxidative stress . Recent studies showed that the intranasal administration of MSC-derived exosomes (nanovesicles separated by ultracentrifugation from the secretome released from MSCs) to animals chronically ingesting ethanol temporally reverses neuroinflammation, lowers brain ROS levels, and inhibits alcohol intake . However, the inhibitory effect of MSC exosomes on ethanol intake was considerably less effective and shorter acting than that reported  to follow the intravenous administration of intact MSCs, which after been activated release both exosomes plus soluble molecules, likely acting via a paracrine effect. Since whole secretome is expected to display effects more akin to MSC “pumps,” in the present study, we tested whether the non-invasive intranasal administration of the complete secretome derived from adipose tissue MSCs activated in vitro in pro-inflammatory conditions results in a strong therapeutic effect in inhibiting chronic ethanol intake and a longer action in preventing alcohol relapse binge drinking than that observed for MSC-derived exosomes .
Present studies also allowed us to determine the secretome effects on the self-administration of another drug of abuse, namely nicotine, thus testing the hypothesis that intake perpetuation of drugs of abuse (alcohol and nicotine) shares a common mechanism.
Studies presented determine if intranasally administered human adipose tissue-derived MSC secretome (i) inhibits chronic alcohol and nicotine intake and their relapse, (ii) normalizes chronic ethanol- and nicotine-induced hippocampal oxidative stress, and (iii) reverses the neuroinflammation induced by both drugs of abuse. (iv) Knockdown studies tested the hypothesis that the glutamate transporter GLT-1 mediates the inhibitory effect of MSC secretome on chronic alcohol and nicotine intake.
Two-month-old female Wistar-derived rats selectively bred as alcohol consumers (University of Chile Bibulous (UChB)) [11, 36] were used in the experiments. All animal procedures were approved by the Committee for Experiments with Laboratory Animals at the Medical Faculty of the University of Chile (Protocol CBA# 0994 FMUCH).
Isolation, expansion, and characterization of human adipose tissue-derived MSCs
Human adipose tissue-derived MSCs were isolated from subcutaneous adipose tissue (abdominal region), obtained from liposuction aspirates of patients undergoing cosmetic liposuction at Clínica Alemana, Santiago, Chile, as previously described . For all samples, a written informed consent was obtained. All protocols were approved by the Ethics Committee of Facultad de Medicina, Clínica Alemana-Universidad del Desarrollo. After two subcultures, cells were characterized according to their adipogenic and osteogenic differentiation potential, by the presence of putative hMSC markers (CD29, CD13, CD105, CD73, and CD90), and the absence of marker characteristics of other cell lineages (CD235a, CD31, and CD45) as previously described .
Activation of human adipose tissue-derived MSCs and secretome generation
Human adipose tissue-derived MSCs (passage 3) at 70% of confluence were activated by incubation in minimal essential medium (α-MEM Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT) plus 10 ng/ml TNF-α and 15 ng/ml IFN-γ (R&D System, Minneapolis, MN) for 40 h. We and others [8, 38] have previously described that this activation strategy greatly improves the production of anti-inflammatory factors compared to non-activated MSCs. After the activation, the cells were washed three times with phosphate-buffered saline (PBS) and incubated for 48 h with α-MEM. After that, the culture media (secretome) were collected and centrifuged at 400g for 10 min to remove the whole cells. The supernatant was centrifuged again at 5000g for 10 min to remove cell debris. This process reduces the contamination of the secretome with proteins release by the rupture of the cells. Finally, the secretome was filtered in 0.22-μm filters.
Secretomes were concentrated 30 times (v/v) by 3 kDa cutoff filters (Millipore, Carrigtwohill CO). The concentrates were washed with 15 ml of PBS and re-concentrated again with the same filters. The protein concentration was determined by the BCA protein assay kit (Thermo Scientific, Waltham, MA), and the secretome was frozen at − 80 °C until use. As a control, secretome obtained from the same number of non-activated MSCs was also generated.
Determination of antioxidant capacity of MSC secretome
The total antioxidant capacity of 2 μg of secretome protein obtained from activated adipose tissue-derived MSCs was determined by the Antioxidant Assay Kit (Cayman Chemical). The antioxidant system includes superoxide dismutase, catalase, glutathione peroxidase, macromolecules such as ferritin, and an array of small molecules including ascorbic acid, α-tocopherol, β-carotene, reduced glutathione, uric acid, and bilirubin. The assay relies on the ability of antioxidants in the sample to inhibit the oxidation of 2,2′-azino-di-3-ethylbenzthiazoline sulphonate. Thus, the combined antioxidant activities of all the constituents of the MSC secretome were assessed. Data were compared against the antioxidant capacity of 2 μg of secretome protein obtained from non-activated MSCs and 2 μg of human albumin.
Quantification of IL-10 and TGF-B1 levels in MSC secretome
IL-10 and TGF-B1 were used as markers of anti-inflammatory activity. IL-10 and TGF-B1 abundance were evaluated in the secretome obtained from 1 × 106 activated and 1 × 106 non-activated MSCs using the human IL-10 High sensitivity ELISA Kit (Invitrogen) and TGF-B1 ELISA Kit (Life Technologies), respectively.
Voluntary alcohol consumption test: free choice drinking paradigm
Twenty-six female UChB rats were used. Only female rats were used because (unlike males) females do not increase their body weight during the long-term (3–4 months) voluntary alcohol or nicotine consumption, allowing a more stable alcohol or nicotine intake over time, when ethanol or nicotine intake are expressed as grams of ethanol/nicotine consumed per kilogram body weight. Additionally, for rats selectively bred for their high-ethanol intake, the Indianapolis female rats have a higher (HAD-2) or equal (HAD-1) ethanol intake than males . For Sardinian high-ethanol intake bred rats, females show higher levels of ethanol intake than males . Females of the UChB rat line show a 20% higher ethanol intake than males (unpublished). Oral alcohol intake (10% ethanol v/v prepared from absolute ethanol diluted in tap water) was allowed for 10–12 weeks in a free-choice drinking paradigm versus tap water. On the last week, animals were offered 10% ethanol, 20% ethanol, and water. Ethanol intake was expressed as grams of ethanol consumed per kilogram body weight per day.
Relapse-like alcohol drinking
Following over 13 weeks of continuous ethanol access, animals were deprived of ethanol solutions, but not water, for 14 days after which the animals were allowed re-access to 10% and 20% ethanol solutions for only 60 min as previously described . Ethanol intake was expressed as grams of ethanol consumed per kilogram body weight per 60 min.
Voluntary nicotine consumption and relapse-like nicotine self-administration
Twenty-six female UChB rats were allowed to self-administer nicotine solutions as described by Quintanilla et al. . Essentially, rats were pre-treated for 14 days with a daily intraperitoneal dose (0.5 mg/kg) of nicotine hydrogen tartrate (Sigma-Aldrich, St. Louis, MO) dissolved in saline . On day 15, nicotine intraperitoneal injections were discontinued, and rats were offered a free choice between ingesting increasing concentration of nicotine solutions (5 to 40 mg/l nicotine dissolved in water) or water. The nicotine solution was kept constant (24 h/day) at 40 mg/l for the last 15 weeks. Nicotine intake was expressed as milligrams of nicotine consumed per kilogram body weight per day. The development of the nicotine self-administration model is shown in Additional file 1: Figure S1. Following 22 weeks of continuous access to nicotine and water, animals were deprived of nicotine for 13 days and were thereafter allowed re-access to a 40-mg/l nicotine solution and water for only 60 min . Nicotine intake upon re-access was expressed as milligrams of nicotine consumed per kilogram body weight per 60 min.
Intranasal administration of MSC-derived secretome
Rats were anesthetized with chloral hydrate (280 mg/kg, i.p.) and placed in the supine position. Twenty microliters of secretome solution was administered intranasally as drops delivered from a small pipette tip every 5 min (four times in each nostril) into alternative sides of the nasal cavity for a total of 20 min. A total volume of 160 μl of secretome containing 25 μg of total proteins derived from 1 × 106 adipose tissue-derived activated MSCs was delivered into the nasal cavity. Control animals received 160 μl of saline by the same administration scheme .
Determination of chronic ethanol and nicotine intake and relapse following the intranasal MSC secretome administration
Two groups of rats were used. The first group of rats (n = 10) had ingested ethanol under the free-choice condition for 74 days, while the second group (n = 10) had ingested nicotine under the free-choice condition for 138 days. Each group was divided into two subgroups that received a weekly intranasal dose of secretome or vehicle (saline) for 5 weeks. The first three doses were administered while the rats were under the free choice of chronic ethanol or chronic nicotine intake, while the last two doses were administered while the animals were under a 13- to 14-day period of nicotine or ethanol deprivation. Three days after the last intranasal secretome or vehicle administration, re-access to 10% and 20% ethanol or to 40 mg/l nicotine and water for a 60-min period was allowed. Immediately after re-access to the ethanol or nicotine solutions, the animals were anesthetized with chloral hydrate (280 mg/kg, i.p.) and blood samples were obtained for the assessment of the blood ethanol or plasma cotinine, the major metabolite of nicotine. Then, the animals were perfused intracardially with 100 ml of PBS (pH 7.4) and euthanized to obtain brain samples for the determination of hippocampus GSSG/GSH ratio and glial reactivity.
Blood ethanol determination
Blood samples (0.1 ml) were collected without anticoagulant from the tip of the tail under moderate sedation with acepromazine (1 mg/kg, i.p.). The samples were analyzed by headspace gas chromatography (Perkin Elmer SRI 8610) as previously described .
Plasma cotinine determination
Immediately following a 60-min re-access to nicotine (40 mg/l) after the 13-day nicotine deprivation period, rats were anesthetized with chloral hydrate (280 mg/kg, i.p.) and blood (1.5 ml) samples were collected by cardiac puncture. Cotinine level was determined in the plasma by ELISA using the Cotinine ELISA Kit (MyBiosource, San Diego, CA).
GSSG/GSH ratio determination
The hippocampi were extracted and mixed with three volumes of ice-cold potassium buffer (0.1 M) containing 5 mM EDTA, pH 7.4. Reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG) content were determined as described previously .
Tissue preparation and glial immunohistochemistry
Preparation of brain tissue for immunohistochemistry against glial fibrillary acidic protein (GFAP)-positive astrocytes and ionized calcium-binding adaptor molecule 1 (Iba-1) for microglial cells was performed according to Morales et al. , Perez-Lobos et al. , and Ezquer et al. . The samples were examined by confocal microscopy (Olympus-fv10i). An unbiased stereological approach was used to analyze GFAP-positive astrocytes, quantifying the total length and thickness of primary processes and density of Iba-1-positive microglial cells in the hippocampus, since this glia has a significant role in the development and maintenance of drug addiction [1, 43, 44].
Effect of glutamate transporter GLT-1 knockdown on chronic ethanol or nicotine intake following the intranasal administration of MSC-derived secretome
Two groups of rats were used: rats of the alcohol group (n = 16) that had ingested ethanol for 88 days and rats of the nicotine group (n = 16) that had ingested nicotine for 153 days. All animals were pre-anesthetized with chloral hydrate (280 mg/kg, i.p.), maintained under anesthesia with a mixture of air and isofluorane, and mounted on a stereotaxic frame to be injected with the oligonucleotides into the left lateral ventricle (AP 0, − 1.2, − 3.8 mm), according to Paxinos and Watson . The alcohol and nicotine groups were divided into four subgroups (n = 4) that were injected intracerebro-ventricularly (icv) with: group #1, 10 μl of GLT-1 oligo antisense (17 nmol); group #2, 10 μl of control oligo antisense (17 nmol); group #3, 10 μl of vehicle (saline); or group #4, 10 μl of vehicle (saline). The sequences of antisense oligonucleotide anti-GLT-1 and the control oligonucleotide (Integrated DNA Technologies, NC) were those described by Reissner et al. . However, phosphorothioate rather than morpholino moieties constituted the backbone of the molecule administered. Thereafter, the animals were returned to their cages and allowed access to ethanol or nicotine solutions and water under free choice. Twenty-four hours after the intracerebral administration rats of group #1, group #2, and group #3 received intranasally 160 μl of secretome (25 μg of secretome proteins, derived from 1 × 106 activated MSCs), while rats of group #4 received 160 μl of vehicle, all under chloral hydrate (280 mg/kg, i.p.) anesthesia. After the intranasal administration, the animals were allowed to continue drinking alcohol or nicotine under free choice, for 48 additional hours.
Statistics were performed using GraphPad Prism software (San Diego, CA). For behavioral experiments, two-way ANOVA (treatment × day) with or without repeated measures followed by Bonferroni’s or Tukey’s post-hoc tests were used. For GSSG/GSH ratio, the total length and thickness of primary astrocytic processes and microglia density, one-way ANOVA followed by Tukey or Bonferroni post-hoc test were used for testing the differences among the groups. When only two groups were compared, statistical significance was determined by Student’s t test. Significance was set at p < 0.05, and all data are presented as mean ± SEM. Full statistical analyses of each figure are presented in the legend of the figure.
Intranasal administration of secretome derived from adipose tissue-derived activated MSCs inhibits chronic alcohol and chronic nicotine intake and blocks post-deprivation relapse
It has been previously reported that human MSCs can produce a broad range of anti-inflammatory and antioxidant factors, and this paracrine potential can be boosted by activating the cells by incubation with pro-inflammatory cytokines, enhancing their therapeutic efficacy . Initially, as a proof of concept, we evaluated if the intranasal administration of secretome derived from non-activated MSCs could inhibit chronic ethanol intake. Additional file 2: Figure S2A shows that secretome obtained from 1 × 106 adipose tissue-derived MSCs that were not activated only marginally and transiently inhibited chronic alcohol intake and did not prevent a relapse binge drinking (i.e., blood alcohol concentration BAC > 80 mg/dl) (Additional file 2: Figure S2B), suggesting (vide infra) that the activation of MSCs is needed to improve the anti-addictive effect of the secretome.
Intranasal administration of secretome derived from adipose tissue-derived activated MSCs reduced hippocampus oxidative stress induced by chronic ethanol and chronic nicotine intake
Intranasal administration of secretome derived from adipose tissue-derived activated MSCs normalized both the increased astrocyte reactivity and the increased microglial density induced by chronic ethanol and chronic nicotine intake
A single intranasal dose of secretome derived from adipose tissue-derived activated MSCs inhibited ethanol and nicotine intake and reduced the hippocampal oxidative stress induced by chronic ethanol or nicotine intake
Knockdown of GLT-1 prevented the inhibition of ethanol and nicotine intake induced by intranasal administration of secretome derived from adipose tissue-derived activated MSCs
The present study shows that, as for other drugs of abuse [16, 17, 18, 19, 20, 47, 48, 49], chronic ethanol and chronic nicotine intakes lead to brain oxidative stress. The intranasal administration of antioxidant secretome derived from adipose tissue-derived activated MSCs inhibited chronic ethanol self-administration by 80% to 85% and virtually abolished the relapse binge drinking that followed a prolonged ethanol deprivation and a 60-min re-access to ethanol. Nearly identical results were obtained for nicotine intake following the intranasal administration of MSC-derived secretome. Thus, both chronic intake and relapse of two drugs of abuse were markedly inhibited by the intranasal administration of MSC-derived secretome.
The study shows that the brain oxidative stress (elevated GSSG/GSH ratio) induced by the chronic intake of both ethanol and nicotine was fully suppressed by the administration of MSC-derived secretome. A similar inhibition applied for neuroinflammation (astrocyte GFAP and microglia Iba-1 profiles). The question can be addressed as to which of these changes—oxidative stress or neuroinflammation—is directly related to the perpetuation of chronic ethanol or nicotine intake and is responsible for their relapse. Considering the existence of an oxidative stress/neuroinflammation self-perpetuating cycle [24, 25, 26, 27], both the antioxidant and the anti-inflammatory activities of MSC-derived secretome likely contribute to reducing drug intake. Accordingly, the administration of the anti-inflammatory drug ibudilast was shown to inhibit chronic ethanol intake and relapse by 50%, both in rats and mice , while the oral administration of the potent antioxidant N-acetyl-cysteine reduced ethanol intake by 70 to 75% .
What is clear from the knockdown study is that the astrocyte GLT-1 transporter mediates the inhibitory effect of MSC secretome on chronic ethanol and nicotine self-administration, since the reduction in GLT-1 expression prevents the anti-addictive effects of the secretome. An unbiased brain transcriptome analysis of 6 strains/lines of rats (including the UChB rat used in these experiments) and mice bred for either high or low alcohol preference  showed that the differentially expressed genes between high and low drinking lines were primarily associated with glial (astrocytes and microglia) function, glial-neuronal communication, and energy metabolism (the main generator of reactive oxygen species), thus consistent with the present studies.
Glutamate homeostasis by astrocytes appears to play a major role in alcohol and other drug use disorders [28, 29, 43]. Several reports show that the astrocyte glutamate transporter GLT-1 activity is inhibited under oxidative stress conditions, both by oxidation of its sulfhydryl moieties  and by inactivation mediated by the lipid peroxidation product 4-HNE [32, 33]. As shown for chronic ethanol and nicotine studies, marked oxidative stress has also been reported for chronic cocaine administration . Noteworthy, Reissner et al.  showed that the inhibition of cocaine relapse induced by the antioxidant N-acetyl cysteine was also prevented by GLT-1 transporter knockdown. Thus, the present study supports the view that glutamate overflow likely at the tripartite synapse in the nucleus accumbens  mediates the relapse of drugs of abuse.
Overall, studies conducted show that the non-invasive intranasal administration of adipose tissue-derived activated mesenchymal stem cell secretome (i) markedly inhibited chronic intake and relapse of ethanol and nicotine, (ii) suppressed both the increased oxidative stress and neuroinflammation induced by chronic ethanol or nicotine intake, and (iii) demonstrate that the glutamate transporter GLT-1 mediates the inhibitory effect of the secretome on chronic ethanol and nicotine intake, possibly via a reduction of oxidative stress and neuroinflammation. Given the marked inhibitory effects and the duration of the effects of intranasally administered secretome on the self-administration and relapse of two drugs of abuse, possible translational implications are envisioned.
The technical assistance of Mr. Juan Santibáñez and Ms. Carmen Almeyda is very much appreciated.
MEQ was responsible for the conception and design, provision of the study material, collection of data, data analysis, and manuscript writing. FE was responsible for the conception and design, provision of the study material, collection of data, data analysis, financial support, and manuscript writing. PM, DS, PB, and ME were responsible for the collection of data and data analysis. MHM was responsible for the conception and design and data analysis. YI was responsible for the conception and design, financial support, data analysis, and manuscript writing. All authors read and approved the final manuscript.
This work was supported by FONDECYT #1180042 to Yedy Israel.
Ethics approval and consent to participate
Human MSCs were isolated from subcutaneous adipose tissue (abdominal liposuction), after written informed consent. All protocols were approved by the Ethics Committee of Facultad de Medicina, Clínica Alemana-Universidad del Desarrollo (Protocol 2015-40).
All animal procedures were approved by the Committee for Experiments with Laboratory Animals at the Medical Faculty of the University of Chile (Protocol CBA# 0994 FMUCH).
Consent for publication
The authors declare that they have no competing interests.
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