Temporal changes in innate immune signals in a rat model of alcohol withdrawal in emotional and cardiorespiratory homeostatic nuclei

Abstract

BackgroundChronic alcohol use changes the brain’s inflammatory state. However, there is little work examining the progression of the cytokine response during alcohol withdrawal, a period of profound autonomic and emotional upset. This study examines the inflammatory response in the central nucleus of the amygdala (CeA) and dorsal vagal complex (DVC), brain regions neuroanatomically associated with affective and cardiorespiratory regulation in an in vivo rat model of withdrawal following a single chronic exposure.MethodsFor qRT-PCR studies, we measured the expression of TNF-α, NOS-2, Ccl2 (MCP-1), MHC II invariant chain CD74, and the TNF receptor Tnfrsf1a in CeA and DVC samples from adult male rats exposed to a liquid alcohol diet for thirty-five days and in similarly treated animals at four hours and forty-eight hours following alcohol withdrawal. ANOVA was used to identify statistically significant treatment effects. Immunohistochemistry (IHC) and confocal microscopy were performed in a second set of animals during chronic alcohol exposure and subsequent 48-hour withdrawal.ResultsFollowing a chronic alcohol exposure, withdrawal resulted in a statistically significant increase in the expression of mRNAs specific for innate immune markers Ccl2, TNF-α, NOS-2, Tnfrsf1a, and CD74. This response was present in both the CeA and DVC and most prominent at 48 hours. Confocal IHC of samples taken 48 hours into withdrawal demonstrate the presence of TNF-α staining surrounding cells expressing the neural marker NeuN and endothelial cells colabeled with ICAM-1 (CD54) and RECA-1, markers associated with an inflammatory response. Again, findings were consistent in both brain regions.ConclusionsThis study demonstrates the rapid induction of Ccl2, TNF-α, NOS-2, Tnfrsf1a and CD74 expression during alcohol withdrawal in both the CeA and DVC. IHC dual labeling showed an increase in TNF-α surrounding neurons and ICAM-1 on vascular endothelial cells 48 hours into withdrawal, confirming the inflammatory response at the protein level. These findings suggest that an abrupt cessation of alcohol intake leads to an acute central nervous system (CNS) inflammatory response in these regions that regulate autonomic and emotional state.

Transgenic mice with increased astrocyte expression of CCL2 show altered behavioral effects of alcohol

“…In rats, alcohol consumption for 12 months increased mean CCL2 levels (measured by ELISA) in the cortex to 23.8±3.7 pg/mg protein compared to 16.5±1.2 pg/mg protein in the cortex of control animals (Ehrlich et al, 2012). In other studies, increased expression of CCL2 mRNA was observed after withdrawal from a liquid alcohol diet, and the level of CCL2 mRNA expression correlated with blood levels of alcohol observed in the animals prior to withdrawal (Freeman et al, 2012; Harper et al, 2015; Knapp et al, 2016). In mice subjected to repeated alcohol exposure/withdrawal cycles at a high dose of alcohol (5 gm/kg by gavage), the increase in CCL2 mRNA persisted for days after withdrawal, indicating that alcohol can have long-term effects on CCL2 mRNA levels in the CNS (Qin et al, 2008).…”

“…Studies in experimental animals confirmed that increased expression of CCL2 is produced by alcohol (ethanol) exposure/withdrawal. In these studies, significant increases in the levels of CCL2 mRNA and/or protein were observed in several brain regions following either single or repeated alcohol exposure/withdrawal (Qin et al, 2008; Knapp et al, 2011; Ehrlich et al, 2012; Freeman et al, 2012; Kane et al, 2013; Vetreno et al, 2013; Kane et al, 2014; Chang et al, 2015; Drew et al, 2015; Harper et al, 2015; Pascual et al, 2015; Knapp et al, 2016). For example, elevated levels of CCL2 mRNA were observed in the hippocampus of mice one day following a 10 day exposure to alcohol by gavage (BALs ~ 300 mg/dl) (Kane et al, 2013; Kane et al, 2014).…”

Substance use disorders: Psychoneuroimmunological mechanisms and new targets for therapy

“…Over twenty years ago Jerrells et al (1989) suggested that the most prominent immune effects relate to the induction of stress hormones ( e.g ., corticosteroids) following alcohol withdrawal (Jerrells et al, 1989). These initial observations have been confirmed by a number of investigators showing that not only does alcohol withdrawal increase hypothalamic-pituitary-adrenal (HPA) axis reactivity, but it also leads to an acute inflammatory response in the CNS (Buck et al, 2012; Freeman et al, 2012). This is important because increased stress and inflammation can lead to depression, cognitive impairments, and anxiety and can contribute to increased relapse rates, lower treatment retention rates, and reduced daily functioning.…”

The role of neuroimmune signaling in alcoholism

“…Inflammation and cytokine induction are known to contribute to negative affect. Withdrawal from alcohol increases inflammatory cytokines in the brain (Freeman et al, 2012). Also, intracerebroventricular injection of cytokines sensitized anxiety-like behavior during withdrawal from ethanol (Breese et al, 2008).…”