The online version of this article (doi:10.1186/s12974-017-0856-z) contains supplementary material, which is available to authorized users.
Recent studies have implicated microglia—the resident immune cells of the brain—in the pathophysiology of alcoholism. Indeed, post-mortem alcoholic brains show increased microglial markers and increased immune gene expression; however, the effects of ethanol on microglial functioning and how this impacts the brain remain unclear. In this present study, we investigate the effects of acute binge ethanol on microglia and how microglial depletion changes the brain neuroimmune response to acute binge ethanol withdrawal.
C57BL/6J mice were treated intragastrically with acute binge ethanol for time course and dose-response studies. Cultured mouse BV2 microglia-like cells were treated with ethanol in vitro for time course studies. Mice were also administered the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia from the brain. These mice were subsequently treated with acute binge ethanol and sacrificed during withdrawal. Brain and BV2 mRNA were isolated and assessed using RT-PCR to examine expression of microglial and neuroimmune genes.
Acute binge ethanol biphasically changed microglial (e.g., Iba1, CD68) gene expression, with initial decreases during intoxication and subsequent increases during withdrawal. Acute ethanol withdrawal dose dependently increased neuroimmune gene (e.g., TNFα, Ccl2, IL-1ra, IL-4) expression beginning at high doses. BV2 cells showed biphasic changes in pro-inflammatory (e.g., TNFα, Ccl2) gene expression following ethanol treatment in vitro. Administration of PLX5622 depleted microglia from the brains of mice. Although some neuroimmune genes were reduced by microglial depletion, many others were unchanged. Microglial depletion blunted pro-inflammatory (e.g., TNFα, Ccl2) gene expression and enhanced anti-inflammatory (e.g., IL-1ra, IL-4) gene expression during acute binge ethanol withdrawal.
These studies find acute binge ethanol withdrawal increases microglial and neuroimmune gene expression. Ethanol exposure also increases microglial pro-inflammatory gene expression in vitro. Furthermore, microglial depletion decreases expression of microglia-specific genes but has little effect on expression of many other neuroimmune signaling genes. Microglial depletion blunted the acute binge ethanol withdrawal induction of pro-inflammatory genes and enhanced induction of anti-inflammatory genes. These findings indicate microglia impact the brain response to acute binge ethanol withdrawal.
Additional file 1: Figure S1. Schematic of experimental designs. a) For the Dose-Response Experiment, mice were gavaged with 3.0, 4.5 or 6.0 g/kg ethanol (25% v/v) and pain response and movement were qualitatively assessed each following hour. For each group, BACs were collected 1 hour post-gavage. Mice in these groups were sacrificed at 18 hours and brain mRNA was collected for RT-PCR. Note that mice in the group receiving 3.0 g/kg ethanol were not qualitatively assessed, as they exhibited only minor behavioral changes. b) For the Time Course Experiment, mice were gavaged with 6.0 g/kg ethanol (25% v/v) and sacrificed at 6, 12, 18 or 24 hours. A non-gavaged “0 hour” control group was also included. Brain mRNA was collected for RT-PCR. c) A separate group of mice was gavaged with 6.0 g/kg ethanol (25% v/v) and tail blood was collected at 1, 6, 12 and 18 hours for assessment of BACs. d) Mice were given either Control chow or PLX5622 chow. One week after starting PLX5622 chow to deplete microglia, mice were injected intraperitoneally with 2.0 g/kg ethanol (20% v/v) and tested on the rotarod. After two more weeks of treatment with PLX5622 chow, mice were gavaged with 6.0 g/kg ethanol (25% v/v) and brain mRNA was collected 18 hours later for RT-PCR. (TIF 108 kb)12974_2017_856_MOESM1_ESM.tif
Additional file 2: Figure S2. Behavioral characterization of the acute binge ethanol model. Mice were gavaged with ethanol (4.5 or 6 g/kg, 25% v/v) and pain response and movement were qualitatively assessed each following hour. Note that there was 0% mortality, as indicated by behavior recorded for 100% of mice in each group at each time point. A, C) For movement, a complete absence of movement other than breathing was recorded as “No Activity.” Head movement, limb movement, or impaired ambulation was recorded as “Impaired Activity.” Movement that was indistinguishable from a control mouse was recorded as “Full Activity.” B, D) For pain assessment, each hindpaw was pinched. A complete absence of a response was recorded as “No response.” Slight flinching or movement following any pinch was recorded as “Weak Response.” Full-paw withdrawal following any pinch was recorded as “Full Response.” n = 8/group (TIF 175 kb)12974_2017_856_MOESM2_ESM.tif
Additional file 3: Figure S3. Acute binge ethanol causes biphasic changes in brain Iba1 and CD68 mRNA compared to controls. Mice were gavaged with acute binge ethanol (6 g/kg, 25% v/v) or water and sacrificed various times post-treatment. A) Brain Iba1 was measured over time by RT-PCR in both water- and ethanol-treated mice. B) Brain CD68 was measured over time by RT-PCR in both water- and ethanol-treated mice. Note there is no change in brain Iba1 or CD68 mRNA in water-treated mice over time. Data are represented as mean ± SEM. *p < 0.05 compared to controls. n = 5–6/group (TIF 1013 kb)12974_2017_856_MOESM3_ESM.tif
Additional file 4: Figure S4. Acute binge ethanol withdrawal increases brain TNFα, Ccl2, and IL-4 mRNA compared to controls. Mice were gavaged with acute binge ethanol (6 g/kg, 25% v/v) or water and sacrificed various times post-treatment. A) Brain TNFα, B) Ccl2, C) IL-1ra, and D) IL-4 were measured over time by RT-PCR in both water- and ethanol-treated mice. Data are represented as mean ± SEM. *p < 0.05 compared to controls. n = 5–7/group (TIF 136 kb)12974_2017_856_MOESM4_ESM.tif
Additional file 5: Figure S5. Acute binge ethanol increases brain TNFα, Ccl2, and IL-4 protein during withdrawal. Mice were treated with ethanol (6 g/kg, 25% v/v) or water and sacrificed 18 h post-treatment. Brain protein was collected and A) TNFα, B) Ccl2, C) IL-1ra, and D) IL-4 protein levels were determined via ELISA. Data are represented as mean ± SEM. *p < 0.05, Student’s t test, n = 6/group (TIF 97 kb)12974_2017_856_MOESM5_ESM.tif
Additional file 6: Figure S6. Acute ethanol treatment and evaporation changes BV2 TNFα, Ccl2, IL-1ra, and IL-4 mRNA compared to controls. Time course of cytokine expression in ethanol-treated and PBS-treated control BV2 cells: Microglia-like BV2 cells were treated with either ethanol (85 mM) or PBS and the ethanol was allowed to evaporate away over time. Transcript levels of A) TNFα B) Ccl2 C) IL-1ra, and D) IL-4 were assessed in ethanol-treated cells (black dots) and PBS-treated control cells (white dots). Baseline changes in gene expression likely occur because the BV2 cells are proliferating and engaging in autocrine-paracrine signaling in a closed system. Data are represented as mean ± SEM. *p < 0.05, Student’s t test, n = 4–6/group (TIF 144 kb)12974_2017_856_MOESM6_ESM.tif
Additional file 7: Figure S7. BV2 viability following ethanol treatment. Microglia-like BV2 cells were treated with PBS or ethanol (85 mM) and the ethanol was allowed to evaporate away over time. At 0, 1.5, 12, and 24 h, cell viability was determined with the vital stain, Trypan blue. The number of live and dead cells was counted, and the number of live cells was divided by the number of total cells to calculate percent viability. Note that ethanol treatment did not affect cell viability at any time point. Data are represented as mean ± SEM. n = 3/group (TIF 514 kb)12974_2017_856_MOESM7_ESM.tif
Additional file 8: Figure S8. Continuous ethanol treatment does not increase BV2 TNFα or Ccl2 expression. Microglia-like BV2 cells were treated with either ethanol (85 mM) or PBS. A) For ethanol-treated cells, ethanol was vaporized into the incubator to keep media ethanol concentrations constant. After 24 h of continuous ethanol exposure, BV2 mRNA was isolated and B) TNFα and C) Ccl2 gene expression were measured. Note that continuous ethanol exposure does not increase BV2 pro-inflammatory cytokine expression at 24 h. Data are represented as mean ± SEM. *p < 0.05, Student’s t test, n = 6/group (TIF 94 kb)12974_2017_856_MOESM8_ESM.tif
Additional file 9: Figure S9. The role of microglia in ethanol-induced motor impairment. Mice were treated with compounds that impact microglia—either the CSF1R inhibitor PLX5622 or recombinant IL-1ra (Kineret). A) Mice were fed PLX5622 chow for 1 week to deplete microglia and injected i.p. with ethanol (2.0 g/kg, 20% v/v). Mice were then tested on the rotarod at 2, 5, 8, 14, 20 min and every subsequent 10 min post-injection until 110 min had passed. The time the mice remained on the rotarod was recorded. Note that microglial depletion with 1 week on PLX5622 chow did not alter the ethanol-induced motor impairments. B, C) Mice received an intraperitoneal injection of B) 100 or C) 300 mg/kg IL-1ra 30 min prior to an i.p. injection of ethanol (2.0 g/kg, 20% v/v). Mice were then tested on the rotarod at 2, 5, 8, 14, 20 min and every subsequent 10 min after ethanol injection until 110 min had passed. The time the mice remained on the rotarod was recorded. Note that IL-1ra did not alter the ethanol-induced motor impairments. n = 8/group (TIF 147 kb)12974_2017_856_MOESM9_ESM.tif
Additional file 10: Table S1. Effects of acute binge ethanol on brain gene expression over time (DOC 53 kb)12974_2017_856_MOESM10_ESM.doc
Additional file 11: Table S2. Effects of acute binge ethanol dose on brain gene expression during withdrawal (DOC 36 kb)12974_2017_856_MOESM11_ESM.doc
Additional file 12: Table S3. Effects of ethanol treatment on BV2 gene expression over time (DOC 47 kb)12974_2017_856_MOESM12_ESM.doc
NIAAA. National Institute of Alcohol Abuse and Alcoholism Council approves definition of binge drinking. 2004.
Crews FT. Alcohol and neurodegeneration. CNS Drug Rev. 1999;5:379–94. CrossRef
Karlsson C, Schank JR, Rehman F, Stojakovic A, Bjork K, Barbier E, Solomon M, Tapocik J, Engblom D, Thorsell A, Heilig M. Proinflammatory signaling regulates voluntary alcohol intake and stress-induced consumption after exposure to social defeat stress in mice. Addict Biol. 2016
Alfonso-Loeches S, Pascual-Lucas M, Blanco AM, Sanchez-Vera I, Guerri C. Pivotal role of TLR4 receptors in alcohol-induced neuroinflammation and brain damage. J Neurosci Off J Soc Neurosci. 2010;30:8285–95. CrossRef
Kane CJ, Phelan KD, Han L, Smith RR, Xie J, Douglas JC, Drew PD. Protection of neurons and microglia against ethanol in a mouse model of fetal alcohol spectrum disorders by peroxisome proliferator-activated receptor-gamma agonists. Brain Behav Immun. 2011;25 Suppl 1:S137–145. CrossRefPubMedPubMedCentral
Rubio-Araiz A, Porcu F, Perez-Hernandez M, Garcia-Gutierrez MS, Aracil-Fernandez MA, Gutierrez-Lopez MD, Guerri C, Manzanares J, O'Shea E, Colado MI. Disruption of blood-brain barrier integrity in postmortem alcoholic brain: preclinical evidence of TLR4 involvement from a binge-like drinking model. Addict Biol. 2016
Elmore MR, Najafi AR, Koike MA, Dagher NN, Spangenberg EE, Rice RA, Kitazawa M, Matusow B, Nguyen H, West BL, Green KN. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron. 2014;82:380–97. CrossRefPubMedPubMedCentral
Babcock AA, Kuziel WA, Rivest S, Owens T. Chemokine expression by glial cells directs leukocytes to sites of axonal injury in the CNS. J Neurosci. 2003;23:7922–30. PubMed
Lewis KE, Rasmussen AL, Bennett W, King A, West AK, Chung RS, Chuah MI. Microglia and motor neurons during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis: changes in arginase1 and inducible nitric oxide synthase. J Neuroinflammation. 2014;11:55. CrossRefPubMedPubMedCentral
Marshall SA, Geil CR, Nixon K. Prior binge ethanol exposure potentiates the microglial response in a model of alcohol-induced neurodegeneration. Brain Sci. 2016;6:2076-2095
- Microglial depletion alters the brain neuroimmune response to acute binge ethanol withdrawal
T. Jordan Walter
Fulton T. Crews
- BioMed Central
Neu in den Fachgebieten Neurologie und Psychiatrie
Meistgelesene Bücher in der Neurologie & Psychiatrie