Therapeutic role of mesenchymal stem cell‐derived extracellular vesicles in neuroinflammation and cognitive dysfunctions induced by binge‐like ethanol treatment in adolescent mice

Abstract Background Extracellular vesicles (EVs) are heterogeneous membrane vesicles secreted by cells in extracellular spaces that play an important role in intercellular communication under both normal and pathological conditions. Mesenchymal stem cells (MSC) are anti‐inflammatory and immunoregulatory cells capable of secreting EVs, which are considered promising molecules for treating immune, inflammatory, and degenerative diseases. Our previous studies demonstrate that, by activating innate immune receptors TLR4 (Toll‐like receptor 4), binge‐like ethanol exposure in adolescence causes neuroinflammation and neural damage. Aims To evaluate whether the intravenous administration of MSC‐derived EVs is capable of reducing neuroinflammation, myelin and synaptic alterations, and the cognitive dysfunction induced by binge‐like ethanol treatment in adolescent mice. Materials & Methods MSC–derived EVs obtained from adipose tissue were administered in the tail vein (50 microg/dose, one weekly dose) to female WT adolescent mice treated intermittently with ethanol (3.0 g/kg) during two weeks. Results MSC‐derived EVs from adipose tissue ameliorate ethanol‐induced up‐regulation of inflammatory genes (e.g., COX‐2, iNOS, MIP‐1α, NF‐κB, CX3CL1, and MCP‐1) in the prefrontal cortex of adolescent mice. Notably, MSC‐derived EVs also restore the myelin and synaptic derangements, and the memory and learning impairments, induced by ethanol treatment. Using cortical astroglial cells in culture, our results further confirm that MSC‐derived EVs decrease inflammatory genes in ethanol‐treated astroglial cells. This, in turn, confirms in vivo findings. Conclusion Taken together, these results provide the first evidence for the therapeutic potential of the MSC‐derived EVs in the neuroimmune response and cognitive dysfunction induced by binge alcohol drinking in adolescence.


| INTRODUC TI ON
Mesenchymal stem cells (MSC) are adult stem cells capable of stimulating the maintenance, growth, and survival of other cells. 1 Their therapeutic potential seems to be mediated by the paracrine factors contained in microvesicles. 2 Indeed the extracellular vesicles (EVs) that derive from MSC (MSC-EVs) display a different genetic cargo and protein content, which play a significant role in biological processes, including regulation of inflammation, apoptosis, angiogenesis, adipogenesis, blood coagulation, and extracellular matrix remodeling. 3However, MSC-EVs have emerged as therapeutic agents to reduce tissue injury and enhance tissue repair in cerebral injury, lung injury, myocardial injury, and kidney injury cases. 4,5tracellular vesicles are microvesicles that originate from multivesicular bodies and can be isolated from diverse body fluids and multiple cell cultures supernatants. 68][9] Analyses of MSC-EVs have demonstrated that their content is enriched in miRNAs and proteins. 3In the central nervous system, neuron-and astrocyte-derived EVs play critical roles in several processes, such as promoting neurite outgrowth, synaptic plasticity, neuronal survival, and neuroprotection, 7 MSC-EVs can recover the neuronal and astroglial damage induced by several injuries. 10e adolescent brain shows important changes in its structure and function, 11 including synaptic plasticity and neural connectivity, which especially occur in the prefrontal cortex (PFC) and other subcortical areas. 12,13These extensive developing changes in brain maturation might explain the adolescent brain's vulnerability to the deleterious effects of ethanol. 14,15Our previous studies have demonstrated that by activating innate immune receptors TLR4 (Toll-like receptor 4) in glial cells, binge-like ethanol exposure in adolescence leads to the release of cytokines and chemokines, and causes neuroinflammation and neural damage. 16,17Activation of the TLR4 response has also been demonstrated in myelin and synaptic alterations, as well as cognitive and behavioral impairments, induced by binge drinking in adolescent mice. 18This scenario suggests the involvement of the neuroinflammatory immune response in behavioral deficits.Indeed human studies have shown that the one brain region most affected by ethanol drinking in adolescence is the PFC, a region associated with cognitive control and executive function. 19kewise, myelin dysfunctions have been related to attention and spatial working memory deficits in human adolescents with heavy alcohol abuse. 20 agreement with the therapeutic beneficial effects of MSC-EVs to treat neurological and neurodegenerative diseases, 21 the present study provides evidence that the intravenous administration of MSC-EVs from adipose tissue ameliorates neuroinflammation, as well as myelin and synaptic alterations, which lead to the cognitive impairments induced by binge-like ethanol treatment in adolescent mice.

| MSC isolation, culture, and isolation of MSC-EVs
Human adipose tissue was obtained from surplus fat tissue during knee prosthesis operation performed on four patients under sterile conditions.Human samples were anonymized.The experimental procedure was previously evaluated and accepted by the Regional Ethics Committee for Clinical Research with Medicines and Health Products following Code of Practice 2014/01.As the exclusion criteria, no samples were collected from patients with a history of cancer or infectious diseases at the time of (viral or bacterial) surgery.
All the human patients voluntarily signed an informed consent document to use the adipose samples.Cells were expanded and grown in the growth medium [GM: High glucose DMEM basal medium supplemented with 20% FBS (previously centrifuged at 100 000×g for 1 h for EV depletion and then filtered by a 0.2 μm filter), 100 units/ mL penicillin, 100 μg/mL streptomycin and 2 mM l-glutamine in].MSC have been characterized and previously described. 22,23bconfluent cells were incubated in GM for 48 h.Then, media were collected and cleared from detached cells and cell fragments by centrifugation at 300×g for 10 min, and by the supernatant at 2000×g for 10 min, respectively.Subsequently, apoptotic bodies and other cellular debris were pelleted by centrifuging the resulting supernatant at 10 000×g for 30 min.EVs were then pelleted from the previous resulting supernatant at 100 000×g for 1 h.The EVs pellet was washed with PBS and centrifuged at 100 000×g for 1 h.EVs were finally suspended in 100 mL PBS and stored at −80°C.The intermittent ethanol treatment was initiated early in adolescence or during the prepubescent period on postnatal day (PND) 30. 24Morning doses of either saline or 25% (v/v) ethanol (3 g/kg) in isotonic saline were administered intraperitoneally to 30-day-old mice on two consecutive days with 2-day gaps without injections for 2 weeks (PND30-PND43), as previously described. 18,25No signs of peritoneal cavity irritation, pain or distress, or peripheral inflammation induced by intraperitoneal ethanol concentration were noted, which agrees with other studies that have used intraperitoneal ethanol administration. 26After a single ethanol dose, blood alcohol levels peaked at 30 min (~340 mg/ dL) and then progressively lowered until 5 h post-injection.Three hours before ethanol administration, animals were also treated with MSC-EVs (50 μg/dose) or saline (sodium chloride, 0.9%) in the tail vein once a week (with the third and seventh ethanol dose).No changes in either animals' body weight or brain weight were observed during the intermittent treatment (Figure S1).Animals were anesthetized 24 h after the last (8th) ethanol or saline administration (PND 44).Brains were removed and transferred to a plate placed on ice.Olfactory bulbs, cerebellum, and pons were removed.Brains were placed with the ventral side facing the plate.

| Animals and treatments
We used curved forceps to remove the cortical hemispheres from the rest of the brain.Then the PFC dissection was performed based on visual information and using brain atlas coordinates. 27,28Cs (n = 9-10 mice/group) were immediately snap-frozen in liquid nitrogen and stored at −80°C until used.In addition, some animals were anesthetized, perfused with paraformaldehyde/glutaraldehyde, and used for the electron microscopy analysis (n = 6 mice/ group).Other animals (n = 10-12 mice/group) were employed to perform behavioral studies after ethanol treatment on PND 50 in this test daily order: novel object recognition, passive avoidance, and Hebb-Williams maze.
After 24 h of ethanol stimulation, cells were harvested, frozen, and stored at −80°C until further use.

| Extracellular vesicles characterization by transmission electron microscopy and nanoparticles tracking analysis
The freshly isolated EVs were fixed with 2% paraformaldehyde and prepared as previously described. 30Preparations were examined under a transmission FEI Tecnai G2 Spirit electron microscope (FEI Europe) with a Morada digital camera (Olympus Soft Image Solutions GmbH).In addition, an analysis of the absolute size range and concentration of microvesicles was performed using NanoSight NS300 Malvern (NanoSight Ltd.), as previously described. 30

| Western blot analysis
The Western blot technique was performed in MSC-EVs for their characterization, as described elsewhere. 18The employed primary antibodies were: anti-CD9, anti-CD63, anti-CD81, and anti-calnexin (Santa Cruz Biotechnology).Membranes were washed, incubated with the corresponding HRP-conjugated secondary antibodies, and developed by the ECL system (ECL Plus; Thermo Scientific).The cell lysate from the astrocyte primary cell culture was used as the negative control for CD9, CD63, and CD81, and as the positive control for calnexin.The full unedited blots are included in the Supplementary Material.

| RNA isolation, reverse transcription, and quantitative RT-PCR
The frozen PFC samples (10-20 mg) and astroglial cells were used for total RNA extraction.Tissue and cells were disrupted using 1 mL of TRIzol (Sigma-Aldrich), and the total RNA fraction was extracted following the manufacturer's instructions.S1).

| Brain tissue preparation and electron microscopy
Mice were anesthetized by an intraperitoneal injection of sodium penthobarbital (60 mg/kg) and fentanyl (0.05 mg/kg) for analgesia.
Animals were then perfused transcardially with 0.9% saline containing heparin, followed immediately by 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for tissue fixation.The fixed brains were removed, postfixed overnight at 4°C with the same fixative solution, and then stored at 4°C in PBS.After removing the olfactory bulbs, the anterior coronal section, of approximately 1 mm was discarded.The following section of approximately 1 mm from 2.5 to 1.5 mm anterior to Bregma was used to cut PFCs in sections of 200 μm on a Leica VT-1000 vibratome (Leica).

| Novel object recognition test
Mice performed this test in a black open box (24 cm × 24 cm × 15 cm) using small nontoxic objects: two plastic boxes and a plastic toy.The task procedure is described elsewhere 25  one object was identical to the sample and the other was novel.
Object exploration was defined as the orientation of the animal's snout toward the object within a range of ≤2 cm from the object.

| Passive avoidance test
Step-through inhibitory avoidance apparatus for mice (Ugo Basile) was employed for the passive avoidance test.The cage was made of Perspex sheets and was divided into two compartments (15 × 9.5 × 16.5 cm each).The safe compartment was white and illuminated by a light fixture (10 W) fastened to the cage lid.The "shock" compartment was dark and made of black Perspex panels.Both compartments were divided by a door that automatically operated by sliding on the floor.The floor was made of 48 stainless steel bars (0.7 mm in diameter) placed 8 mm apart.Passive avoidance tests were run following the previously described procedure. 32On the training day, each mouse (n = 10-12 mice/group) was placed inside the illuminated compartment facing away from the dark compartment.The door leading to the dark compartment was opened after a 60-s habituation period.When the animal had placed all four paws in the dark compartment, a footshock (0.5 mA, 3 s) was delivered and the animal was immediately removed from the apparatus and returned to its home cage.The time taken to enter the dark compartment (step-through latency) was recorded.
Retention was tested 24 h and 7 days later following the same procedure, but with no footshock.The maximum step-through latency lasted 300 s.

| Hebb-Williams maze
This task was used for the advantages that it offers over other tests because not only can problem-solving, visuo-spatial abilities, and cognitive performance in rodents 33 be assessed, but so can easy and difficult learnings and, consequently, minor cognitive deficits can be differentiated.Motivation to perform this maze is not based on reinforcement (i.e., food), but on escaping from a stressful situation (cold water), which can influence learning and memory. 34,35e maze used in our experiments was made of black plastic and measured 60 cm wide × 60 cm long × 10 cm high.It contained a start box and a goal box (both 14 cm wide × 9 cm long), which were positioned in diagonally opposite corners.The maze contained cold water at a wading depth (15°C, 3.5 cm high), while the goal box was stocked with fresh dry tissue.Several maze designs were produced by fixing different arrangements of barriers to a clear plastic ceiling.This apparatus allows the cognitive process of routed learning and water-escape motivation to be measured.The following procedure was based on that employed by, 36 in which mice must navigate the maze and cross over from the wet start box to the dry goal box to escape cold water.Animals (n = 10-12 mice/group) underwent a 5-min habituation period (dry sand, no barriers) on day 1, and faced problem A on day 2 and problem D on day 3 (4 trials/day) (practice mazes).Mice were subsequently submitted to mazes 1, 5, 3, 4, and 8 on separate days on which eight trials took place.The time limit for reaching the goal box was 5 min, after which time the mouse was guided to the box.The following measurements were recorded: total latency score (the sum of the latencies in all the problem trials in each maze); latency to reach the goal during the eighth trial; error scores, for which a similar total was used (where "error" was considered the act of entering the error zone as previously described).Following, 37 mazes were defined as easy (1, 3, and 4) or difficult (5 and 8).

| Statistical analysis
The results are reported as the mean ± SEM.All the employed statis-

| MSC-EVs ameliorate the neuroinflammatory immune response induced by binge-like ethanol treatment in adolescent mice
We first characterized MSC-EVs by electron microscopy, nanoparticle tracking analysis, and EVs markers (Figure 1).The size distribution and concentration of the MSC-secreted nanoparticles using the NanoSight resulted in a high peak that ranged between 100 and 200 nm, which included the size range of EVs as demonstrated by electron microscopy.In addition, these EVs expressed the exosome markers, named tetraspanin proteins (CD63, CD9, and CD81), whereas no signs of cytosolic protein contamination were observed by the calnexin protein.
Then we analyzed if MSC-EVs administration can restore the up-regulation in the mRNA levels of inflammatory genes induced by ethanol treatment.For these experiments, we used adipose MSC-EVs intravenously administered 3 h before ethanol injection in adolescent mice (see 2. Materials and Methods).We measured the COX-2, NF-κB, iNOS, CX3CL1, MIP-1α, and MCP-1 levels in the PFC under different experimental conditions.Figure 2

| MSC-EVs reduce the myelin and synaptic alterations in the PFC of ethanol-treated adolescent mice
Considering that intermittent binge-like ethanol drinking alters the myelin structure in the PFC of adolescent mice, 18 we herein analyzed the potential beneficial role of MSC-EVs in ethanol-induced myelin structure alterations.Figure 3A shows that while ethanol However, no changes were found in the expression of these genes between the saline-treated and EVs-treated animals.
We also assessed the potential role of MSC-EVs in the ultrastructural alterations in the synaptic elements induced by intermittent F I G U R E 2 MSC-EVs restore the levels of inflammatory genes induced by binge-like ethanol treatment in adolescent mice.The mRNA levels of COX-2 (A), NF-κB (B), iNOS (C), CX3CL1 (D), MIP-1α (E), and MCP-1 (F) were analyzed in the PFC samples of adolescent mice.Data represent mean ± SEM, n = 9-10 mice/ group.**p < 0.01 and ***p < 0.001, compared to their respective salinetreated group; # p < 0.05, ## p < 0.01, and ### p < 0.001, compared to their respective ethanol-treated group.ethanol treatment in adolescent mice (Figure 5A), as previously demonstrated. 18To accomplish this, we analyzed vesicle number, synaptic cleft width, and postsynaptic density thickness in the PFC of adolescent mice.Figure 5

| MSC-EVs restore the cognitive dysfunctions induced by binge-like ethanol treatment in adolescent mice
We next assessed whether the amelioration of the neuroinflammation, myelin and synaptic alterations induced by MSC-EVs in the ethanol-treated adolescent mice would also be capable of restoring cognitive dysfunction.We performed several memory and learning tasks, such as the passive avoidance test, the novel object recognition test, and Hebb-Williams maze.[40][41] In the novel object recognition test (Figure 6A 6C) and the data from Supplementary Material (Figures S2 and S3) also confirm that MSC-EVs treatment was able to revert the impairment in the spatial learning induced by ethanol in adolescent mice.Furthermore, the ANOVA for the total number of errors in the Hebb-Williams maze (Figure 6D) revealed an effect of the variable Ethanol in the easy [F(1, 39) = 5.785, p < 0.05] and difficult [F(1, 39) = 47.493,p < 0.001] mazes.The animals treated with ethanol and ethanol plus MSC-EVs made more errors while learning mazes versus the saline-treated mice (p < 0.01 and p < 0.001 for the easy and difficult mazes, respectively; Figure 6D and Figure S4).
F I G U R E 4 MSC-EVs restore the levels of the myelin-related genes induced by binge-like ethanol treatment in adolescent mice.The mRNA levels of CNPase (A), MAG (B), MBP (C), and NG2 (D) were analyzed in the PFC samples of adolescent mice.Data represent mean ± SEM, n = 9-10 mice/group.*p < 0.05, **p < 0.01 and ***p < 0.001, compared to their respective saline-treated group; # p < 0.05, ## p < 0.01 and ### p < 0.001, compared to their respective ethanol-treated group.

| Role of MSC-EVs in the ethanol-induced inflammatory response in astroglial cells in primary culture
We have previously demonstrated that ethanol induces neuroinflammation by activating the TLR4 inflammatory response in glial cells. 17,42Therefore, to extend the in vivo results on the protective role of MSC-EVs in ethanol-induced neuroinflammation, we also used the primary culture of the astroglial cells exposed to ethanol and treated with and without MSC-EVs.The results demonstrated that ethanol treatment increased the levels of several proinflammatory genes, such as TLR4, NF-κB, iNOS, CX3CL1, and MIP-1α.

| DISCUSS ION
We have previously demonstrated the critical role of the innate immune response in the ethanol-induced PFC damage and cognitive dysfunctions induced by binge-like ethanol exposure in adolescence. 18,25Considering the regenerative potential of MSC-EVs in both brain damage and neurodegenerative disorders, [43][44][45][46] MSC-EVs were herein used to ameliorate the neuroinflammation induced by binge ethanol drinking.The present findings provide evidence that MSC-EVs administration mostly restores the neuroinflammatory response, along with myelin and synaptic structural alterations, as well as cognitive and memory dysfunctions induced by binge-like ethanol treatment in adolescent mice (Figure 8).
Our previous studies revealed the role of EVs as amplifiers of the TLR4 response and the neuroinflammation induced by ethanol treatment in astroglial cells in culture 30 and in in vivo brain tissue. 47wever, different recent studies also report the potential role of EVs to show that EVs emerge as therapeutical agents of different diseases, such as neurodegenerative disorders and brain damage.Experimental studies have specifically evidenced the anti-inflammatory and antioxidative properties of MSC-EVs in the TLR4 activation induced by its specific ligand, lipopolysaccharide (LPS).For instance, MSC-EVs prevent LPS-induced microglia activation and the production of inflammatory mediators in rats 44 and microglial cells. 48Han et al. 49 have shown that MSC-EVs alleviate the brain expression of inflammatory cytokines in rats with subarachnoid hemorrhage by inhibiting the activation of nuclear transcription factor NF-κB.In line with these findings, we show that MSC-EVs can prevent the up-regulation of the expression of inflammatory genes (e.g., iNOS, MIP-1α, NF-κB, and CX3CL1) induced by binge-like ethanol treatment in both the adolescent PFC and ethanol-treated astroglial cells.Similarly, Ezquer et al. 50ve demonstrated that MSC-EVs inhibit the neuroinflammation and glial activation induced by chronic alcohol consumption.Protective functions of these microvesicles have been shown by the miRNAs contained in MSC-EVs, which modulate various cell signaling processes and regulate the expression of multiple target genes with antioxidative and anti-inflammatory properties. 51Other studies have also demonstrated that MSC-EVs perform immunosuppressive functions by decreasing T-and B-cell proliferation, by releasing immunomodulating factors (e.g., iNOS, PGE2, TSG-6, and HLA-G) packed in EVs. 52nversely, the EVs derived by ethanol-treated astrocytes could be internalized by neurons to increase the levels of proinflammatory molecules, by compromising their survival. 30However, the present study confirms that the systemic administration of MSC-EVs exerts antiinflammatory properties in ethanol-treated adolescent mice.Indeed, MSC-EVs incorporated into glial cells and neurons could promote F I G U R E 7 MSC-EVs lower the levels of the inflammatory genes induced by ethanol treatment in astroglial cells.The astrocytes in primary culture treated or not with ethanol in the presence or absence of MSC-EVs were used to analyze the mRNA levels of TLR4 (A), NF-κB (B), iNOS (C), CX3CL1 (D) and MIP-1α (E).Data represent mean ± SEM (n = 6 independent experiments).*p < 0.05, **p < 0.01 and ***p < 0.001, compared to their respective untreated cells; # p < 0.05, ## p < 0.01 and ### p < 0.001, compared to their respective ethanol-treated cells.neuroprotection against the neuroinflammatory response and brain damage. 49,53,54olescence is a critical brain maturation period when neurotoxic ethanol effects can induce structural cortical alterations in humans 55,56 and experimental models, 18 which could be associated with persistent neurobehavioral deficits, including memory and cognitive dysfunction. 18,57,58The present findings show that the intravenous MSC-EVs injection into binge ethanol-treated adolescent mice is able to reduce both alterations in white matter (e.g., irregular myelin fiber shapes, interlaminar splitting of myelin sheaths) and ultrastructural changes in synapses, such as a reduction in both postsynaptic thickness and synaptic vesicle number.In line with our results, a study in rhesus monkeys with cortical injury has reported that MSC-EVs administration can promote therapeutic actions through the recovery of the structure of premotor pyramidal neurons and dendritic plasticity function. 59Treating brain injury with MSC-EVs can also promote the restoration of long-term perinatal microstructural abnormalities of white matter, 44 and remyelination by acting directly on oligodendrocyte progenitor cells and indirectly on microglia activation. 46Therefore, these protective and supportive effects of MSC-EVs can be attributed not only to their anti-inflammatory properties, 44 but also to the up-regulation of myelin-related genes 60 and neural growth factors (i.e., BDNF, VEGF, and EGF) 61 in brain injury.
We have previously demonstrated that alterations in myelin and synaptic structures induced by binge drinking in adolescence can lead to poor synaptic transmission efficacy, which causes cognitive impairments in adolescent mice. 18Recent reports show the involvement of MSC-EVs in the protection of LPS-induced spatio-temporal memory deficits and learning impairments as assessed by a novel object recognition test and Barnes maze. 44gnitive function restoration using MSC-EVs has also been demonstrated in animal models of neurodegenerative diseases, such as Alzheimer's disease, brain injury, Parkinson's disease, schizophrenia, among others. 43,45,46Notably, the present study demonstrates that MSC-EVs ameliorate ethanol-induced spatiotemporal memory dysfunction, as well as learning and recognition memory deficits as evaluated by the novel object recognition test, the passive avoidance task, and Hebb-Williams maze.Indeed, we show that MSC-EVs administration is capable of normalizing the disability to recognize the novel object, the shorter latency time during the passive avoidance test, and the longer times to complete the Hebb-Williams mazes in adolescent mice treated with ethanol.Therefore, we hypothesize that an intravenous MSC-EVs injection may rescue cognitive deficits by regulating inflammatory responses and structural cortical alterations.Several studies suggest that the protective role of MSC-EVs in cognitive deficits could be attributed to the amelioration of white matter disturbances, 44 F I G U R E 8 Schematic representation of the protective effects of MSC-EVs administration on ethanol-induced neuroinflammation.Adipose tissuederived EVs were administered by intravenous injection (iv) prior to ethanol treatment (ip, intraperitoneal injection) in adolescent mice.MSC-EVs administration ameliorates ethanol-induced up-regulation in inflammatory genes in the prefrontal cortex of adolescent mice.Notably, MSC-EVs treatment also restored the myelin and synaptic derangements, as well as the memory and learning impairments, induced by ethanol administration.
Ninety-two female C57BL/6 WT (wild-type) mice (Harlan Ibérica) were used.Mice were housed (3-4 animals/cage) and maintained on a water and solid diet ad libitum.Environmental conditions, such as light and dark (12/12 h), temperature (23°C), and humidity (60%), were controlled for all the animals.All the animal experimental procedures were approved by the Ethical Committee of Animal Experimentation of the Principe Felipe Research Center (Valencia, Spain), following the guidelines approved by European Communities K E Y W O R D S adolescence, binge-like ethanol treatment, cognitive dysfunction, extracellular vesicles, mesenchymal stem cells, neuroinflammation Council Directive (86/609/ECC) and Spanish Royal Decree 53/2013 modified by Spanish Royal Decree 1386/2018.
Total mRNA was reversetranscribed by the NZY First-Strand cDNA Synthesis Kit (NZYTech, Lda.Genes and Enzymes).Quantitative two-step RT-PCR (real-time reverse transcription) was performed with the Light-Cycler 480 detection System (Roche Diagnostics).Genes were amplified employing the AceQ® qPCR SYBR Green Master Mix (NeoBiotech) following the manufacturer's instructions.The mRNA level of housekeeping gene cyclophilin A was used as an internal control for the normalization of the analyzed genes.All the RT-qPCR runs included non-template controls (NTCs).Experiments were performed in triplicates.Quantification of expression (fold change) from the Cq data was calculated by the ΔΔCq method 31 by the LightCycler 480 relative quantification software (Roche Diagnostics).Details of the nucleotide sequences of the used primers are found in the Supplementary Material (Table embedded in Durcupan resin (Fluka, Sigma-Aldrich).Semithin sections (1.5 μm) were cut with an Ultracut UC-6 (Leica) and stained lightly with 1% toluidine blue.Finally, ultrathin sections (0.08 μm) were cut with a diamond knife, stained with lead citrate (Reynolds solution), and examined under a transmission FEI Tecnai G2 Spirit electron microscope (FEI Europe) using a Morada digital camera (Olympus Soft Image Solutions GmbH).Images were analyzed by the ImageJ software (version 1.53a).Synaptic and myelin quantifications were carried out on 4-5 sections per PFC.For each group, 150-175 postsynaptic density thicknesses and 150-175 synaptic cleft width were analyzed.In addition, the number of synaptic vesicles was quantified in 75-100 presynaptic terminals.Damage in the total myelin sheaths was analyzed by measuring the alterations of the total multiple layers of myelin membrane around an axon and was represented as a percentage.Myelin quantification was measured in at least 40-45 axons chosen randomly from each group.
and consists of three phases: habituation, training session (T1), and test session (T2).During the habituation session, mice spent 5 min exploring the openfield arena where T1 and T2 were performed.During the training session, one mouse was placed in the open-field arena containing two identical sample objects placed in the middle of the testing box for 3 min.After a 1-min retention interval, the animal was returned to the open-field arena with two objects during the test session (3 min): tical parameters were calculated with SPSS v28.The Shapiro-Wilk test was used to test for data distribution normality.A one-way ANOVA or a two-way ANOVA was used as parametric tests, and the Mann-Whitney U test or the Kruskal-Wallis test was used as non-parametric alternatives.Values of p < 0.05 were considered statistically significant.Electron microscopy imaging was analyzed with a one-way ANOVA.Quantitative RT-PCR and behavioral data from the novel object recognition were analyzed by a two-way ANOVA with two between-subject variables: ethanol (saline and ethanol); MSC-EVs (with and without EVs).Bonferroni adjustment was employed for the post hoc comparisons in the ANOVA.The number of errors in the Hebb-Williams maze data was analyzed by a two-way ANOVA with the same two between-subject variables in the easy and difficult mazes.As normal distribution was lacking, the time to reach the goal and the particular measures of each trial in the easy and difficult mazes of the Hebb-Williams maze, and the passive avoidance test data, were analyzed by the Kruskal-Wallis test and pairwise comparisons by the Mann-Whitney U test.
), the two-way ANOVA revealed a significant effect of the variables Ethanol [F(1, 43) = 4.487, p < 0.05], MSC-EVs [F(1, 43) = 4.482, p < 0.05], and of the Ethanol × MSC-EVs interaction [F(1, 43) = 6.006, p = 0.01].The ethanol-treated mice failed to recognize the novel object because their discrimination index was significantly lower than in the salinetreated mice and the mice treated with MSC-EVs plus ethanol (p < 0.01 in both cases).The Kruskal-Wallis test for the passive avoidance data (Figure 6B) showed an effect on the training day [χ 2 (2) = 12.440, p < 0.01] and 7 days after the training day [χ 2 (2) = 17.359, p < 0.001].On the training day, the Mann-Whitney U test revealed longer latency to cross to the dark compartment in the animals treated F I G U R E 3 MSC-EVs diminish the myelin sheath disarrangements induced by binge-like ethanol treatment in adolescent mice.(A) The representative transmission electron micrographs of the PFC of the adolescent mice treated with saline, ethanol, and ethanol plus EVs are shown.Arrows indicate the interlaminar splitting of myelin sheaths.(B) Bars represent the percentage of myelin sheath damage.Data denote mean ± SEM, n = 6 mice/group.***p < 0.001, compared to the salinetreated group; ### p < 0.001, compared to the ethanol-treated group.with ethanol compared to all the other experimental groups (saline: U = 23, p = 0.01; ethanol plus MSC-EVs: U = 17, p < 0.01; saline plus MSC-EVs U = 10, p < 0.001).Conversely, 7 days after the training day, the ethanol-treated mice crossed to the dark compartment more quickly than all other groups (saline: U = 10.5, p < 0.001; ethanol plus MSC-EVs: U = 7.5, p < 0.001; saline plus MSC-EVs: U = 23, p < 0.05).These findings suggest that ethanol causes poor retention in memory tasks, while MSC-EVs administration can preserve cognition when harmful ethanol effects come into play.The Kruskal-Wallis test revealed an effect on the time to reach the goal in the easy and difficult mazes [χ 2 (2) = 24.500,p < 0.01; χ 2 (2) = 13.500,p = 0.001, respectively; Figure 6C].Pairwise comparisons showed that the ethanol-treated mice took longer to reach the goal in the easy and difficult mazes compared to the other experimental groups (saline: U = 24.5, p < 0.01; ethanol plus MSC-EVs: U = 35.3,p < 0.05; saline plus MSC-EVs: U = 27, p < 0.05, in the easy mazes; and saline: U = 13.5, p < 0.001; ethanol plus MSC-EVs: U = 13, p < 0.01; saline plus MSC-EVs: U = 11, p = 0.001, in the difficult mazes).These data (Figure

F I G U R E 5
The electron microscopy analysis shows the role of MSC-EVs in the synaptic structure induced by bingelike ethanol treatment in adolescent mice.(A) Representative transmission electron micrographs from the PFC of the adolescent mice treated with saline, ethanol, and ethanol plus EVs.Arrows mark vesicles.Bars represent synaptic vesicle number (B), postsynaptic density thickness (C), and synaptic cleft width (D).Data represent mean ± SEM, n = 6 mice/group.**p < 0.01 and ***p < 0.001, compared to their respective salinetreated group; # p < 0.05 and ### p < 0.001, compared to their respective ethanoltreated group.

F
I G U R E 6 MSC-EVs restore ethanol-induced cognitive dysfunction in adolescent mice.(A) Bars represent the mean (±SEM, n = 10-12 mice/group) of the discrimination index during the novel object recognition task.**p < 0.01 compared to their respective saline-treated group.## p < 0.01 compared to the ethanol-treated group.(B) Bars represent the time taken to enter the dark compartment of the passive avoidance test during the training and test sessions (24 h and 7 days after training).Data are presented as mean (±SEM), n = 10-12 mice/ group.*p < 0.05 and **p < 0.01, compared to all the other experimental groups.(C) Bars represent the mean (±SEM, n = 10-12 mice/group) of the time to reach the goal in the difficult and easy mazes in Hebb-Williams mazes.*p < 0.05 and **p < 0.01, compared to all the other experimental groups.(D) Bars denote the mean (±SEM, n = 10-12 mice/group) of the number of errors to reach the goal in the difficult and easy mazes in Hebb-Williams mazes.**p < 0.01 and ***p < 0.001, compared to their respective saline-treated group.