Assessment of Extracellular Cytokines in the Hippocampus of the Awake Behaving Rat Using Large‐Molecule Microdialysis Combined with Multiplex Arrays After Acute and Chronic Ethanol Exposure

Abstract

Background: Studies have demonstrated persistent changes in central nervous system (CNS) cytokine gene expression following ethanol (EtOH) exposure. However, the low endogenous expression and short half-lives of cytokines in the CNS have made cytokine protein detection challenging. The goal of these studies was to establish parameters for use of large-molecule microdialysis and sensitive multiplexing technology for the simultaneous detection of brain cytokines, corticosterone (CORT), and EtOH concentrations in the awake behaving rat.Methods: Adult (P75+) male Sprague Dawley rats that were either na€ ıve to EtOH (Experiment 1) or had a history of adolescent chronic intermittent EtOH (CIE; Experiment 2) were given an acute EtOH challenge during microdialysis. Experiment 1 examined brain EtOH concentrations, CORT and a panel of neuroimmune analytes, including cytokines associated with innate and adaptive immunity. The natural time course of changes in these cytokines was compared to the effects of an acute 1.5 or 3.0 g/kg intraperitoneal (i.p.) EtOH challenge. In Experiment 2, rats with a history of adolescent CIE or controls exposed to vehicle were challenged with 3.0 g/kg i.p. EtOH during microdialysis in adulthood, and a panel of cytokines was examined in parallel with brain EtOH concentrations and CORT.Results: The microdialysis procedure itself induced a cytokine-specific response that replicated across studies, specifically a sequential elevation of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-a), and IL-10. Surprisingly, acute EtOH did not significantly alter this course of cytokine fluctuations in the hippocampus. However, a history of adolescent CIE showed drastic effects on multiple neuroimmune analytes when rechallenged with EtOH as adults. Rats with a history of adolescent EtOH displayed a severely blunted neuroimmune response in adulthood, evinced by suppressed IL-1b, IL-10, and TNF-a.Conclusions: Together, these findings provide a methodological framework for assessment of cytokine release patterns, their modulation by EtOH, and the long-lasting changes to neuroimmune reactivity evoked by a history of adolescent CIE.

Progenitor Cells Play a Role in Reinstatement of Ethanol Seeking in Adult Male and Female Ethanol Dependent Rats

“…In support of these immune changes, the postmortem brain tissue of individuals with moderate to severe AUD shows increased levels of chemokines and pro-inflammatory cytokines [82,83]. In support of the clinical findings, preclinical studies using a variety of ethanol exposure paradigms indicate immune responses in the brain and serum and that the pro-inflammatory cytokines IL-6, IL-1β, IL-10, and TNF-α, are amongst the most well-described targets of ethanol's action on immune signaling in the brain [84][85][86][87][88][89][90][91][92][93]. However, the literature concerning the functional effects of cytokines on ethanol-related behaviors is comparatively scant, though the behavioral validation of the changes in neuroimmune-associated genes has emerged.…”

Neuroimmune Activation and Microglia Reactivity in Female Rats Following Alcohol Dependence

“…Examinations of microglia specifically, through proteomic approaches with pathway analyses or flow cytometry, do not show alcohol-induced neuroinflammatory signatures in either males or females [43,58,59,90]. Alternatively, in studies where broad panels of neuroimmune mediators have been assessed, it is clear that rats do not show classic inflammatory effects from alcohol and appear to have microglia that are pro-repair, support resolution of damage, or even exhibit a blunted response [43,[90][91][92][93][94] (for a review, see [37]). Another critical function of microglia is synaptic pruning [95], which may be impacted by binge alcohol exposure [96,97].…”

Adolescent Intermittent Ethanol Drives Modest Neuroinflammation but Does Not Escalate Drinking in Male Rats

“…Indeed, while C57BL/6J mice will escalate their alcohol drinking in adulthood after adolescent exposure, DBA/2J mice, without the mutated nnt gene, do not [85]. Rats also do not show the dramatic inflammatory effects of alcohol but instead may have more reparative types of microglia that help to resolve damage or even a blunted response [28,[105][106][107]; for review see [23]. Here, our histological analyses support that AIE with 2 g/kg ethanol does not cause a proinflammatory microglia reaction (Figure 5) or much of an astroglial reaction (Figure 6).…”