P-69TLR4 ELIMINATION PREVENTS LONG-LASTING ETHANOL EFFECTS ON COCAINE-INDUCED CONDITIONED PLACE PREFERENCE IN ADOLESCENT MICE
Our previous studies indicated that binge-like ethanol treatment in adolescent rats induces an increase in the conditioned rewarding effects of cocaine. Ethanol induces the production of cytokines and inflammatory mediators, that cause brain damage by activating the toll-like receptor 4 (TLR4) signaling response. To test if these receptor mediated the observed increased in cocaine-induced conditioned …
TLR4 elimination prevents synaptic and myelin alterations and long-term cognitive dysfunctions in adolescent mice with intermittent ethanol treatment.
The adolescent brain undergoes important dynamic and plastic cell changes, including overproduction of axons and synapses, followed by rapid pruning along with ongoing axon myelination. These developmental changes make the adolescent brain particularly vulnerable to neurotoxic and behavioral effects of alcohol. Although the mechanisms of these effects are largely unknown, we demonstrated that ethanol by activating innate immune receptors toll-like receptor 4 (TLR4), induces neuroinflammation and brain damage in adult mice. The present study aims to evaluate whether intermittent ethanol treatment in adolescence promotes TLR4-dependent pro-inflammatory processes, leading to myelin and synapti…
Ketogenic Diet Decreases Alcohol Intake in Adult Male Mice
The classic ketogenic diet is a diet high in fat, low in carbohydrates, and well-adjusted proteins. The reduction in glucose levels induces changes in the body’s metabolism, since the main energy source happens to be ketone bodies. Recent studies have suggested that nutritional interventions may modulate drug addiction. The present work aimed to study the potential effects of a classic ketogenic diet in modulating alcohol consumption and its rewarding effects. Two groups of adult male mice were employed in this study, one exposed to a standard diet (SD, n = 15) and the other to a ketogenic diet (KD, n = 16). When a ketotic state was stable for 7 days, animals were exposed to the oral self-a…
Neuronal polarization is impaired in mice lacking RhoE expression
J. Neurochem. (2012) 121, 903–914. Abstract Proper development of neuronal networks relies on the polarization of the neurons, thus the establishment of two compartments, axons and dendrites, whose formation depends on cytoskeletal rearrangements. Rnd proteins are regulators of actin organization and they are important players in several aspects of brain development as neurite formation, axon guidance and neuron migration. We have recently demonstrated that mice lacking RhoE/Rnd3 expression die shortly after birth and have neuromotor impairment and neuromuscular alterations, indicating an abnormal development of the nervous system. In this study, we have further investigated the specific ro…
Study of surface carbohydrates on isolated Golgi subfractions by fluorescent-lectin binding and flow cytometry
The Golgi complex is a functionally heterogeneous subcellular structure that plays a key role in the synthesis, maturation, and sorting of newly synthesized glycoproteins. Fluorescent lectins have been used extensively to analyze surface glycoproteins by flow cytometry in whole cells and more recently in isolated subcellular organelles, such as mitochondria and chloroplasts. We report here the use of several fluorescein-isothiocyanate-conjugated lectins to detect and quantify specific surface sugars by flow cytometry on isolated elements from purified cis and trans-Golgi fractions from rat liver. Our results show that this approach may be useful to study Golgi composition and function, sinc…
Role of dopamine neurotransmission in the long-term effects of repeated social defeat on the conditioned rewarding effects of cocaine
Numerous studies report that social defeat stress alters dopamine (DA) neurotransmission in several areas of the brain. Alterations of the mesolimbic dopaminergic pathway are believed to be responsible for the increased vulnerability to drug use observed as a result of social stress. In the present study, we evaluated the influence of DA receptors on the long-term effect of repeated social defeat (RSD) on the conditioned rewarding and reinstating effects of cocaine. For this purpose, the D1R antagonist SCH 23390 and the D1R antagonist raclopride were administered 30 min before each social defeat and a cocaine-induced CPP procedure was initiated three weeks later. The expression of the D1R a…
Intermittent ethanol exposure induces inflammatory brain damage and causes long-term behavioural alterations in adolescent rats
Adolescent brain development seems to be important for the maturation of brain structures and behaviour. Intermittent binge ethanol drinking is common among adolescents, and this type of drinking can induce brain damage. Because we have demonstrated that chronic ethanol treatment induces inflammatory processes in the brain, we investigate whether intermittent ethanol intoxication enhances cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in adolescent rats, and whether these mediators induce brain damage and cause permanent cognitive dysfunctions. Adolescent rats were exposed to ethanol (3.0 g/kg) for two consecutive days at 48-h intervals over 14 days. Levels of COX-2, iN…
Additional file 2: of TLR4 response mediates ethanol-induced neurodevelopment alterations in a model of fetal alcohol spectrum disorders
Table S2. Summary table of the two-way ANOVA of biochemical data (only statistically significant data is shown). Table S3. Summary table of the three-way ANOVA to study gender differences in ethanol-treated and non-treated WT and TLR4-KO pups at PND 0 and 20 (only statistically significant data is shown). Table S4. Summary table of the two-way ANOVA of western blot, immunohistochemistry, and electron microscopy data (only statistically significant data is shown). (DOC 74 kb)
Role of Microbiota-Derived Extracellular Vesicles in Gut-Brain Communication
Human intestinal microbiota comprise of a dynamic population of bacterial species and other microorganisms with the capacity to interact with the rest of the organism and strongly influence the host during homeostasis and disease. Commensal and pathogenic bacteria coexist in homeostasis with the intestinal epithelium and the gastrointestinal tract’s immune system, or GALT (gut-associated lymphoid tissue), of the host. However, a disruption to this homeostasis or dysbiosis by different factors (e.g., stress, diet, use of antibiotics, age, inflammatory processes) can cause brain dysfunction given the communication between the gut and brain. Recently, extracellular vesicles (EVs) derived from …
'Up-regulation of histone acetylation induced by social defeat mediates the conditioned rewarding effects of cocaine
Social defeat (SD) induces a long-lasting increase in the rewarding effects of psychostimulants measured using the self-administration and conditioned place procedures (CPP). However, little is known about the epigenetic changes induced by social stress and about their role in the increased response to the rewarding effects of psychostimulants. Considering that histone acetylation regulates transcriptional activity and contributes to drug-induced behavioral changes, we addressed the hypothesis that SD induces transcriptional changes by histone modifications associated with the acquisition of place conditioning. After a fourth defeat, H3(K9) acetylation was decreased in the hippocampus, whil…
TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles
Background Current evidence indicates that extracellular vesicles (EVs) participate in intercellular signaling, and in the regulation and amplification of neuroinflammation. We have previously shown that ethanol activates glial cells through Toll-like receptor 4 (TLR4) by triggering neuroinflammation. Here, we evaluate if ethanol and the TLR4 response change the release and inflammatory content of astrocyte-derived EVs, and whether these vesicles are capable of communicating with neurons by spreading neuroinflammation. Methods Cortical neurons and astrocytes in culture were used. EVs were isolated from the extracellular medium of the primary culture of the WT and TLR4-KO astrocytes treated …
Involvement of TLR4 in the long-term epigenetic changes, rewarding and anxiety effects induced by intermittent ethanol treatment in adolescence
Studies in humans and experimental animals have demonstrated the vulnerability of the adolescent brain to actions of ethanol and the long-term consequences of binge drinking, including the behavioral and cognitive deficits that result from alcohol neurotoxicity, and increased risk to alcohol abuse and dependence. Although the mechanisms that participate in these effects are largely unknown, we have shown that ethanol by activating innate immune receptors, toll-like receptor 4 (TLR4), induces neuroinflammation, impairs myelin proteins and causes cognitive dysfunctions in adolescent mice. Since neuroimmune signaling is also involved in alcohol abuse, the aim of this study was to assess whethe…
Disposition of acamprosate in the rat: Influence of probenecid
The purpose of the present study was to investigate the disposition of acamprosate (calcium bis acetyl-homotaurine) in the rat. Initially, we studied the linearity of acamprosate disposition and the fraction of acamprosate excreted unchanged in the urine of the animals. Rats received 9.3, 36.6 or 73.3 mg/kg of the drug as an intravenous bolus. The statistical analysis of the pharmacokinetic parameters did not reveal any significant difference, indicating that acamprosate disposition was linear within the range of the doses assayed. On average, 95% of the administered dose was excreted unchanged in the urine of the animals in the 0-6 h post-administration period indicating that renal excreti…
Distribution and differential induction of CYP2E1 by ethanol and acetone in the mesocorticolimbic system of rat
Aims: The expression of cytochrome P4502E1 (CYP2E1) in the brain has been demonstrated in several regions, nevertheless there is a lack of specific studies on the constitutive expression and induction at the mesocorticolimbic system, the most relevant brain pathway in the context of drug addiction and alcoholism. Hence, we have performed a detailed study of the CYP2E1 expression and induction in three key areas of the mesocorticolimbic system of the rat brain: prefrontal cortex (PFC), nucleus accumbens (NAc), and ventral tegmental area (VTA). Methods: Expression levels of CYP2E1 were analyzed by Western blot. The induction of the enzyme in the selected brain areas by chronic acetone (1% v/v…
Social defeat-induced increase in the conditioned rewarding effects of cocaine: Role of CX3CL1
Abstract Social stress is associated with higher vulnerability to drug use, as it enhances the reinforcing effects of psychostimulants in rodents. Furthermore, continued or severe stress induces a proinflammatory state of microglial activation and augmented cytokine production. The aim of the present work was to evaluate the role of fractalkine [C-X3-C motif ligand 1 (CX3CL1)], an inflammatory chemokine, in the increased conditioned rewarding effects of cocaine in animals exposed to social defeat stress. In addition, we measured the signaling cascade pathway of CX3CL1 in the hippocampus (HPC) (including p-ERK/ERK, p-p38/p38 MAPK, p-p65/p65 NFκB and p-CREB/CREB ratios). The glutamate recepto…
Astrocytes in culture express the full-length Trk-B receptor and respond to brain derived neurotrophic factor by changing intracellular calcium levels: effect of ethanol exposure in rats.
Abstract Although cultured astroglial cells were reported to express exclusively the truncated non-catalytic Trk B receptor for brain-derived neurotrophic factor (BDNF), we detect here, using a sensitive ribonuclease protection assay, mRNAs for both truncated (TrkB–T) and the full length catalytic (TrkB–fl) form of BDNF receptor in developing cortical astrocytes and neurons in culture. Cortical neurons and immature astroglia, such as radial glia and proliferating astrocytes, express both the protein and mRNAs for TrkB-fl and TrkB-T, whereas the differentiation of astrocytes leads to a decrease in the trkB-fl mRNA, being the truncated TrkB the predominant receptor in differentiating and conf…
Role of mTOR-regulated autophagy in spine pruning defects and memory impairments induced by binge-like ethanol treatment in adolescent mice.
Abstract Adolescence is a brain maturation developmental period during which remodeling and changes in synaptic plasticity and neural connectivity take place in some brain regions. Different mechanism participates in adolescent brain maturation, including autophagy that plays a role in synaptic development and plasticity. Alcohol is a neurotoxic compound and its abuse in adolescence induces neuroinflammation, synaptic and myelin alterations, neural damage and behavioral impairments. Changes in synaptic plasticity and its regulation by mTOR have also been suggested to play a role in the behavioral dysfunction of binge ethanol drinking in adolescence. Therefore, by considering the critical ro…
TLR4 response mediates ethanol-induced neurodevelopment alterations in a model of fetal alcohol spectrum disorders
Background Inflammation during brain development participates in the pathogenesis of early brain injury and cognitive dysfunctions. Prenatal ethanol exposure affects the developing brain and causes neural impairment, cognitive and behavioral effects, collectively known as fetal alcohol spectrum disorders (FASD). Our previous studies demonstrate that ethanol activates the innate immune response and TLR4 receptor and causes neuroinflammation, brain damage, and cognitive defects in the developmental brain stage of adolescents. We hypothesize that by activating the TLR4 response, maternal alcohol consumption during pregnancy triggers the release of cytokines and chemokines in both the maternal …
Acute behavioural and neurotoxic effects of MDMA plus cocaine in adolescent mice.
The poly-drug pattern is the most common among those observed in MDMA users, with cocaine being a frequently associated drug. This study evaluates the acute effects of MDMA (5, 10 and 20 mg/kg), alone or in combination with cocaine (25 mg/kg), on motor activity, anxiety (elevated plus maze and social interaction test), memory and brain monoamines in adolescent mice, Both drugs, administered alone or concurrently, produced hyperactivity and a decrease in social contacts. However, an anxiolytic effect, studied by means of the elevated plus maze and expressed as an increase in the time spent on the open arms, was observed only in those animals treated with cocaine and MDMA. The passive avoidan…
Binge-like ethanol treatment in adolescence impairs autophagy and hinders synaptic maturation: Role of TLR4.
Abstract Adolescence is a developmental period of brain maturation in which remodeling and changes in synaptic plasticity and neural connectivity take place in some brain regions. A different mechanism participates in adolescent brain maturation, including autophagy processes that play a role in synaptic development and plasticity. Alcohol is a neurotoxic compound whose abuse in adolescence causes TLR4 response activation by triggering neuroinflammation, neural damage and behavioral alterations. However, the potential participation of autophagy in long-term neurochemical and cognitive dysfunctions induced by binge ethanol drinking in adolescence is uncertain. We therefore evaluated whether …
Critical role of TLR4 in uncovering the increased rewarding effects of cocaine and ethanol induced by social defeat in male mice
Abstract Background Substance use disorders and social stress are currently associated with changes in the immune system response by which they induce a proinflammatory state in neurons and glial cells that eventually modulates the reward system. Aims The aim of the present work was to assess the role of the immune TLR4 (Toll-like receptors 4) and its signaling response in the increased contextual reinforcing effects of cocaine and reinforcing effects of ethanol (EtOH) induced by social defeat (SD) stress. Methods Adult male C57BL/6 J wild-type (WT) mice and mice deficient in TLR4 (TLR4-KO) were assigned to experimental groups according to stress condition (exploration or SD). Three weeks a…
Assessment and modulation of acamprosate intestinal absorption: comparative studies using in situ, in vitro (CACO-2 cell monolayers) and in vivo models.
The purpose of this study was to explore the intestinal absorption mechanism of acamprosate and to attempt to improve the bioavailability (BA) of the drug through modulation of its intestinal absorption using two enhancers (polysorbate 80 and sodium caprate) based on in situ, in vitro and in vivo models and comparing the results obtained. Intestinal transport of the drug, in the absence and in presence of polysorbate 80 (0.06, 0.28 and 9.6 mM) or sodium caprate (13 and 16 mM) was measured by using an in situ rat gut technique and Caco-2 cell monolayers. Additionally, the effect of sodium caprate on drug oral bioavailability, measured as urinary recovery, was quantified by performing in vivo…
Circulating MicroRNAs in Extracellular Vesicles as Potential Biomarkers of Alcohol-Induced Neuroinflammation in Adolescence: Gender Differences
Current studies evidence the role of miRNAs in extracellular vesicles (EVs) as key regulators of pathological processes, including neuroinflammation and neurodegeneration. As EVs can cross the blood&ndash
Changes in histone acetylation in the prefrontal cortex of ethanol-exposed adolescent rats are associated with ethanol-induced place conditioning
Alcohol drinking during adolescence can induce long-lasting effects on the motivation to consume alcohol. Abnormal plasticity in reward-related processes might contribute to the vulnerability of adolescents to drug addiction. We have shown that binge-like ethanol treatment in adolescent rats induces alterations in the dopaminergic system and causes histone modifications in brain reward regions. Considering that histone acetylation regulates transcriptional activity and contributes to drug-induced alterations in gene expression and behavior, we addressed the hypothesis that ethanol is capable of inducing transcriptional changes by histone modifications in specific gene promoters in adolescen…
Neuroimmune Activation and Myelin Changes in Adolescent Rats Exposed to High-Dose Alcohol and Associated Cognitive Dysfunction: A Review with Reference to Human Adolescent Drinking
Aims: The aim of the study was to assess whether intermittent ethanol administration to adolescent rats activates innate immune response and TLRs signalling causing myelin disruption and long-term cognitive and behavioural deficits. Methods: We used a rat model of intermittent binge-like ethanol exposure during adolescence. Results: Binge-like ethanol administration to adolescent rats increased the gene expression of TLR4 and TLR2 in the prefrontal cortex (PFC), as well as inflammatory cytokines TNF alpha and IL-1 beta. Up-regulation of TLRs and inflammatory mediators were linked with alterations in the levels of several myelin proteins in the PFC of adolescent rats. These events were assoc…
Toll-like receptors in neuroinflammation, neurodegeneration, and alcohol-induced brain damage
Toll-like receptors (TLRs) or pattern recognition receptors respond to pathogen-associated molecular patterns (PAMPs) or internal damage-associated molecular patterns (DAMPs). TLRs are integral membrane proteins with both extracellular leucine-rich and cytoplasmic domains that initiate downstream signaling through kinases by activating transcription factors like AP-1 and NF-κB, which lead to the release of various inflammatory cytokines and immune modulators. In the central nervous system, different TLRs are expressed mainly in microglia and astroglial cells, although some TLRs are also expressed in oligodendroglia and neurons. Activation of TLRs triggers signaling cascades by the host as a…
Unveiling Sex-Based Differences in the Effects of Alcohol Abuse: A Comprehensive Functional Meta-Analysis of Transcriptomic Studies
AbstractThe abuse of alcohol, one of the most popular psychoactive substances, can cause several pathological and psychological consequences, including alcohol use disorder (AUD). An impaired ability to stop or control alcohol intake despite adverse health or social consequences characterize AUD. While AUDs predominantly occur in men, growing evidence suggests the existence of distinct cognitive and biological consequences of alcohol dependence in women. The molecular and physiological mechanisms participating in these differential effects remain unknown. Transcriptomic technology permits the detection of the biological mechanisms responsible for such sex-based differences, which supports t…
Plasma profile of pro-inflammatory cytokines and chemokines in cocaine users under outpatient treatment: influence of cocaine symptom severity and psychiatric co-morbidity
The treatment for cocaine use constitutes a clinical challenge because of the lack of appropriate therapies and the high rate of relapse. Recent evidence indicates that the immune system might be involved in the pathogenesis of cocaine addiction and its co-morbid psychiatric disorders. This work examined the plasma pro-inflammatory cytokine and chemokine profile in abstinent cocaine users (n = 82) who sought outpatient cocaine treatment and age/sex/body mass-matched controls (n = 65). Participants were assessed with the diagnostic interview Psychiatric Research Interview for Substance and Mental Diseases according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition,…
Cocaine-induced changes in CX3CL1 and inflammatory signaling pathways in the hippocampus: Association with IL1β
Cocaine induces neuroinflammatory response and interleukin-1 beta (IL1β) is suggested a final effector for many cocaine-induced inflammatory signals. Recently, the chemokine fractalkine (CX3CL1) has been reported to regulate hippocampus-dependent neuroinflammation and synaptic plasticity via CX3C-receptor 1 (CX3CR1), but little is known about the impact of cocaine. This study is mainly focused on the characterization of CX3CL1, IL1β and relevant inflammatory signal transduction pathways in the hippocampus in acute and repeated cocaine-treated male mice. Complementarily, the rewarding properties of cocaine were also assessed in Cx3cr1-knockout (KO) mice using a conditioned place preference (…
Exosomes as mediators of neuron-glia communication in neuroinflammation
In recent years, a type of extracellular vesicles named exosomes has emerged that play an important role in intercellular communication under physiological and pathological conditions. These nanovesicles (30–150 nm) contain proteins, RNAs and lipids, and their internalization by bystander cells could alter their normal functions. This review focuses on recent knowledge about exosomes as messengers of neuron-glia communication and their participation in the physiological and pathological functions in the central nervous system. Special emphasis is placed on the role of exosomes under toxic or pathological stimuli within the brain, in which the glial exosomes containing inflammatory molecules…
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) 1
Contains fulltext : 232759.pdf (Publisher’s version ) (Closed access) In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to…
Role of neuroinflammation in ethanol neurotoxicity
Abstract Alcohol is a neurotoxic compound whose abuse can cause alterations in brain structure and functions, although these effects are more dramatic while the brain develops. The molecular mechanisms of neurotoxic effects of alcohol are complex and remain largely unknown. Current evidence from human and animal studies supports the role of the neuroimmune system in many actions of ethanol on the central nervous system, including neurotoxicity. Different studies have shown that, by activating innate immune receptors TLRs (Toll-like receptors) and (NOD)-like receptors (inflammasome NLRs) in glial cells, alcohol triggers signaling pathways which culminate in the production of pro-inflammatory…
Additional file 4: of TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles
Table S1. Nucleotide sequences of the primers used for the TaqMan RT-qPCR of miRNAs. Table S2. Nucleotide sequences of the primers used for the RT-PCR of genes. Table S3. Targets for mmu-mir-146a, mmu-mir-182 and mmu-mir-200b obtained by the mirnet.es webserver. Table S4. The KEGG pathways obtained by the DIANA tool webserver. Table S5. The KEGG pathways that derived from the String protein-protrin interaction analysis between the target genes modulated by mmu-miR-146a and mmu-mir-182. (DOCX 57 kb)
Autophagy
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide…
P-01MicroRNA PROFILING IDETIFY NEW REGULATORY GENES IN ETHANOL-INDUCED NEUROINFLAMMATION AND BRAIN DAMAGE THROUGH TLR4 RECEPTORS
MicroRNAs (miRNAs) are small non-protein coding RNA regulating the transcription and translation of other RNA and are involved in several diseases and disorders, including neurodegeneration. We aim to evaluate the impact of miRNAs in the regulation of ethanol-induced neuro-inflammation and brain damage and to assess the potential role of miRNAs …
Adolescent pre-exposure to ethanol and 3,4-methylenedioxymethylamphetamine (MDMA) increases conditioned rewarding effects of MDMA and drug-induced reinstatement
Many adolescents often take ethanol (EtOH) in combination with 3,4-methylenedioxymethylamphetamine (MDMA). In the present work, we used a mouse model to study the effect of repeated pre-exposure during adolescence to EtOH (2 g/kg), MDMA (10 or 20 mg/kg) or EtOH + MDMA on the rewarding and reinstating effects of MDMA in the conditioned place preference (CPP) paradigm. Pre-exposure to EtOH, MDMA or both increased the rewarding effects of a low dose of MDMA (1.25 mg/kg). These pre-treatments did not affect the acquisition of the CPP induced by 5 mg/kg of MDMA. However, the CPP was more persistent in mice pre-exposed to both doses of MDMA or to EtOH + MDMA20. After extinction of the CPP induced…
Additional file 3: of TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles
Figure S3. Analysis of the RNA population isolated from the WT and TLR4-KO, ethanol-treated or not astrocyte-derived EVs by a 2100 Agilent Bioanalyzer. X axis shows the nucleotide length of the RNA population and the Y axis its fluorescence intensity. (TIF 366 kb)
Additional file 1: of TLR4 response mediates ethanol-induced neurodevelopment alterations in a model of fetal alcohol spectrum disorders
Figure S1. Role of TLR4 in the expression of cytokines (IL-1β, IL-17) and chemokines (fractalkine, MCP-1, MIP-1α) in the cerebral cortices of the WT and TLR4-KO male pups on PND 0 and 20 exposed, or not, to ethanol during the embryonic and postnatal periods. PPEE: prenatal and postnatal ethanol exposure. Data represent mean ± SEM, n = 4 mice/group. *p
Additional file 2: of TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles
Figure S2. A) Flow cytometry graph of a mixture of FITC fluorescent beads with different diameters of 100 nm, 300 nm, 500 nm and 900 nm (Megamix-Plus FSC beads), which was used to detect the EVs obtained from the WT and TLR4-KO astrocytes. B) Example of the graph obtained in the nanoparticles tracking analysis using size distribution and the concentration of microvesicles. (TIF 924 kb)
Additional file 1: of TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles
Figure S1. Immunoblot analysis of the calnexin levels present in the EVs from the untreated and ethanol-treated WT and TLR4-KO astrocytes. The absence of the calnexin expression in the exosome samples confirmed the absence of cytosolic protein contamination. A sample of astrocyte lysate was used as positive control of the calnexin expression. (TIF 489 kb)