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

AbstractBackgroundChronic 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 anin vivorat model of withdrawal following a single chronic exposure.MethodsFor qRT-PCR studies, we measured the expression ofTNF-α,NOS-2,Ccl2 (MCP-1), MHC II invariant chainCD74, and the TNF receptorTnfrsf1ain 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 markersCcl2,TNF-α,NOS-2,Tnfrsf1a, andCD74. 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 ofCcl2,TNF-α,NOS-2,Tnfrsf1aandCD74expression 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.