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

Abstract

Background: Chronic 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.

Methods: For 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.

Results: Following 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.

Conclusions: This 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.

Figure 1

Experimental Design.(A) For the gene expression qRT-PCR study, triplet rats were assigned to control, chronic exposure, and four- or forty-eight-hour withdrawal. Following a 35-day liquid alcohol exposure, animals were sacrificed and the RNA extracted from their CeA and DVC. (B) For IHC study, four pairs of animals were assigned to either the chronic exposure or 48 hours of alcohol withdrawal following eight months on the alcohol diet. Forty-eight hours prior to the time of sacrifice, the withdrawal animals had their diets removed to initiate withdrawal. Following intracardiac perfusion, both rats were sacrificed and their CeA and DVC collected for immunohistochemistry and confocal microscopy. CeA, central nucleus of the amygdala; DVC, dorsal vagal complex; qRT-PCR, quantitative reverse transcription polymerase chain reaction; IHC, immunohistochemistry.

Figure 2

qRT-PCR analysis of expression ofCcl2,NOS-2,TNF-α,Tnfrsf1aandCD74during the first 48 hours of alcohol withdrawal. Alcohol-treated rats were fed the Lieber-DeCarli liquid alcohol diet for 35 days prior to forced withdrawal. qRT-PCR was performed on CeA and DVC samples. All transcripts were found to have a significant treatment effect by two-factor ANOVA, and to change significantly as a result of alcohol treatment and withdrawal. (A) CeA mean ∆∆C_T values for the transcripts Ccl2, NOS-2, TNF-α and Tnfrsf1a and CD74 in control (C; N = 10), chronically alcohol exposed (E; N = 5), and 4 hours (4 h W; N = 5) and 48 hours (48 h W, N = 3) withdrawn rats. (B) DVC mean ∆∆ C_T values (C N = 11; E = 5, 4 h W n = 7, 48 h N = 5). A ∆∆C_T = 1 is a doubling of control mRNA levels. Error bars represent the +/−SEM. *Significant treatment effect identified via two-way ANOVA (P ≤ 0.05); **Significant treatment effect identified via two-way ANOVA (P ≤ 0.01); ***Significant treatment effect identified via two-way ANOVA (P ≤ 0.005); † Significant post hoc Tukey’s test versus control, (P ≤ 0.01.) ANOVA, two-way analysis of variance; Ccl2, chemokine (C-C motif) ligand 12; CeA, central nucleus of the amygdala; DVC, dorsal vagal complex; NOS-2, inducible nitric oxide synthase; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Figure 3

Immunohistochemical evaluation of the alcohol-induced inflammatory response. (A to F). TNF-α reactivity in the neural compartment in the CeA (A to C) and DVC (D to F) in control animals (A, D; N = 4) and following chronic alcohol exposure (B, E; N = 2) and 48 hours into withdrawal (C, F; N = 2). Neurons are stained red with neuronal nuclear antigen (NeuN) and nuclei with DAPI shown in blue. The arrows in panels C., E. and F. show the expression of TNF-α (green) surrounding cells expressing the neural marker NeuN. Dual labeling of a cell for both NeuN and TNF is shown in yellow. (G to L). ICAM-1 reactivity (green) in the endothelial compartment in the CeA (G to I) and DVC (J to L), following chronic alcohol exposure (H,K) and 48 hours into withdrawal (I,L). Endothelia are stained red with RECA-1 and nuclei with DAPI shown in blue. The arrow in panel H shows the expression of ICAM-1 by cells also expressing the endothelial cell marker RECA-1. Panels I and L have arrows showing small cells with a high degree of ICAM-1 staining found commonly in the chronic and withdrawal tissue that are absent in the control condition. Dual labeling of a cell for both RECA-1 and ICAM-1 is shown in yellow. CeA, central nucleus of the amygdale; DAPI, 5 μg/ml 4′-6-diamidino-2-phenylindole; DVC, dorsal vagal complex; ICAM‐1, intercellular adhesion molecule 1;NeuN, neuronal nuclear antigen; RECA‐1, rat endothelial cell antigen‐1.