Prior Binge Ethanol Exposure Potentiates the Microglial Response in a Model of Alcohol-Induced Neurodegeneration

Abstract

Excessive alcohol consumption results in neurodegeneration which some hypothesize is caused by neuroinflammation. One characteristic of neuroinflammation is microglial activation, but it is now well accepted that microglial activation may be pro- or anti-inflammatory. Recent work indicates that the Majchrowicz model of alcohol-induced neurodegeneration results in anti-inflammatory microglia, while intermittent exposure models with lower doses and blood alcohol levels produce microglia with a pro-inflammatory phenotype. To determine the effect of a repeated binge alcohol exposure, rats received two cycles of the four-day Majchrowicz model. One hemisphere was then used to assess microglia via immunohistochemistry and while the other was used for ELISAs of cytokines and growth factors. A single binge ethanol exposure resulted in low-level of microglial activation; however, a second binge potentiated the microglial response. Specifically, double binge rats had greater OX-42 immunoreactivity, increased ionized calcium-binding adapter molecule 1 (Iba-1+) cells, and upregulated tumor necrosis factor-α (TNF-α) compared with the single binge ethanol group. These data indicate that prior ethanol exposure potentiates a subsequent microglia response, which suggests that the initial exposure to alcohol primes microglia. In summary, repeated ethanol exposure, independent of other immune modulatory events, potentiates microglial activity.

Keywords: TNF-alpha; alcohol; alcoholism; cytokines; ethanol; microglia; microglial priming; neurodegeneration.

Figure 1

Potentiated Microglial Activation in the Hippocampus by Repeated Ethanol Exposure. OX-42 (CD11b/c) is upregulated in the hippocampus of ethanol-exposed rats as shown in representative photomicrographs of the (A–C) hippocampal dentate gyrus for (B) Con/EtOH and (C) EtOH/EtOH groups compared to (A) controls. Analysis of OX-42 immunoreactivity indicated that the EtOH/EtOH group had significantly more staining than the Con/EtOH group in the: (D) cornu amonis 1 (CA1) and (E) cornu amonis 2/3 (CA2/3) regions but not the (F) dentate gyrus (DG). Data expressed as a percentage of ad libitum control (not shown). Images were taken at 50× magnification with insets at 600× magnification. Scale bar = 200 µm; inset 30 µm. * p < 0.05 compared to ad libitum and Con/Con groups; # p < 0.05 compared to Con/EtOH.

Figure 2

Potentiated Microglial Activation in the Entorhinal Cortex by Repeated Ethanol Exposure. OX-42 (CD11b) is upregulated in the entorhinal cortex of ethanol-exposed rats as shown in representative photomicrographs of the (A–C) entorhinal cortex for (B) Con/EtOH and (C) EtOH/EtOH groups compared to (A) controls. Analysis of OX-42 immunoreactivity indicated that the EtOH/EtOH group had significantly more positive pixels than the Con/EtOH group in the (D) entorhinal cortex. Data expressed as a percentage of ad libitum control (not shown). Images were taken at 200× magnification with insets at 600× magnification. Scale bar = 100 µm; inset 30 µm. * p < 0.05 compared to ad libitum and Con/Con groups; # p < 0.05 compared to Con/EtOH.

Figure 3

Lack of ED-1 Positive Cells. ED-1 was not visible in the parenchyma of the (A–C) hippocampus or (D–F) entorhinal cortex as seen in representative photomicrographs in (A,D) controls, (B,E) Con/EtOH (C,F) or EtOH/EtOH groups. ED-1 positive cells could be seen along the hippocampal fissure and blood vessels as shown in the inset of B. Scale bars = 200 µm.

Figure 4

Lack of OX-6 Positive Cells. No OX-6 positive cells were observed regardless of treatment, except in one EtOH/EtOH rat as shown in representative photomicrographs of the (A–C) hippocampus or (E–H) entorhinal cortex in (A,E) controls, (B,F) Con/EtOH (C,G) or EtOH/EtOH groups. OX-6 positive cells could be seen along blood vessels as shown in the inset of B. One EtOH/EtOH animal showed upregulation of OX-6 in both the (D) hippocampus and (H) entorhinal cortex. Scale bars = 200 µm.

Figure 5

Microglial Cell Counts Differentially Altered by Ethanol Experience. Stereological estimates indicate an increase in the number of microglia in the EtOH/EtOH group in the (A) cornu amonis 1 (CA1), (B) cornu amonis 2/3 (CA2/3), and (C) dentate gyrus (DG) compared with all other groups. However, the number of microglia in the Con/EtOH group was decreased throughout the hippocampus. In the (D) entorhinal cortex, microglia were decreased in both the Con/EtOH and EtOH/EtOH groups compared to both the ad libitum and Con/Con groups. * p < 0.05 compared to ad libitum and Con/Con group; # p < 0.05 versus Con/EtOH.

Figure 6

Increased TNF-α in EtOH/EtOH Group. Concentrations of (A,B) interleukin-10 (IL-10) and (C,D) tumor necrosis factor-α (TNF-α) were determined by ELISA in both the hippocampus (A,C) and entorhinal cortex (B,D). No change in IL-10 was measured in either the hippocampus or the entorhinal cortex, but at least a 2.7-fold increase in TNF-α was measured in the (C) hippocampus in the EtOH/EtOH group compared with all other groups. However, no change in TNF-α was seen in the (D) entorhinal cortex. * p < 0.05 compared to ad libitum and Con/Con groups; # p < 0.05 compared to Con/EtOH.

Figure 7

Correlations of Cytokines: (A) A positive correlation between blood ethanol concentration (BEC) and tumor necrosis factor-α (TNF-α) concentration. Animals with BECs over 400 mg/dL appear to have an increase in TNF-α. (B) A positive correlation between hippocampal estimates of microglia number and brain derived neurotrophic factor (BDNF) concentrations in the Con/EtOH group. A decline in the number of microglia cells correlated with decreases in BDNF concentrations.

Figure 8

Ethanol Experience-Contingent Effects on BDNF. Concentrations of brain derived neurotrophic factor (BDNF) were determined by ELISA in the hippocampus. BDNF was decreased by approximately 15% in Con/EtOH treated animals compared with Con/Con or ad libitum groups but increased by 20% in the EtOH/EtOH group. * p < 0.05 vs. ad libitum and Con/Con groups; # p < 0.05 vs. to Con/EtOH.