Increased MCP-1 and microglia in various regions of the human alcoholic brain

Abstract

Cytokines and microglia have been implicated in anxiety, depression, neurodegeneration as well as the regulation of alcohol drinking and other consumatory behaviors, all of which are associated with alcoholism. Studies using animal models of alcoholism suggest that microglia and proinflammatory cytokines contribute to alcoholic pathologies [Crews, F.T., Bechara, R., Brown, L.A., Guidot, D.M., Mandrekar, P., Oak, S., Qin, L., Szabo, G., Wheeler, M., Zou, J., (2006) Cytokines and alcohol. Alcohol., Clin. Exp. Res. 30:720-730]. In the current study, human postmortem brains from moderate drinking controls and alcoholics obtained from the New South Wales Tissue Resource Center were used to study the cytokine, monocyte chemoattractant protein 1 (MCP-1,CCL2) and microglia markers in various brain regions. Since MCP-1 is a key proinflammatory cytokine induced by chronic alcohol treatment of mice, and known to regulate drinking behavior in mice, MCP-1 protein levels from human brain homogenate were measured using ELISA, and indicated increased MCP-1 concentration in ventral tegmental area (VTA), substantia nigra (SN), hippocampus and amygdala of alcoholic brains as compared with controls. Immunohistochemistry was further performed to visualize human microglia using ionized calcium binding adaptor protein-1 (Iba-1), and Glucose transporter-5 (GluT5). Alcoholics were found to have brain region-specific increases in microglial markers. In cingulate cortex, both Iba-1 and GluT5 were increased in alcoholic brains relative to controls. Alternatively, no detectable change was found in amygdala nuclei. In VTA and midbrain, only GluT5, but not Iba-1 was increased in alcoholic brains. These data suggest that the enhanced expression of MCP-1 and microglia activities in alcoholic brains could contribute to ethanol-induced pathogenesis.

Fig. 1

Increased MCP-1 protein concentrations in alcoholic brains. MCP-1 protein concentrations (pg/mg of total protein) from brain homogenate were measured using ELISA represented as mean ± SEM. Using ANOVA, significantly increased MCP-1 expression was detected in alcoholics as compared to control of in VTA (ventral tegmental area) of alcoholics as compared with controls (*, p<0.05, N=5 controls, N=7 alcoholics), in substantia nigra (SN) (* p<0.05, N=5 controls, N=6 alcoholics), in hippocampus (*, p<0.05, N=6 controls, N=8 alcoholics), and in amygdala (*, p<0.05, N=6 controls, N=8 alcoholics).

Fig.2

Representative pictures of different stages of microglia activation. Ramified microglial cells are believed to be in the resting stage; the bushy-looking microglia indicates early activation; ameboid microglia represent fully activated brain macrophages.

Fig. 3

In cingulate cortex, the expression of both Iba-1 and GluT₅ was significantly increased in the alcoholic brains (N=5) as compared to the controls (N=4). The level of immunoreactive density was quantified by BioQuant Nova analysis system as described in the Methods and presented as mean ± SEM in pixel/mm². ANOVA indicated significant differences in immunoreactive density of both Iba-1 and GluT₅ between control and alcoholic groups (*, p<0.05).

Fig. 4

In VTA, the expression of GluT₅, but not Iba-1 was significantly higher in the alcoholic brains (N=8) than controls (N=8). The immunoreacitve density was measured by BioQuant Nova analysis system as described in the Methods and presented as mean ± SEM in pixel/mm². ANOVA indicated a significant increase in GluT₅ immunoreactivity, but not Iba-1, in alcoholics as compared to controls (*, p<0.05).

Fig. 5

In Midbrain, the expression of GluT₅, but not Iba-1 was significantly higher in the alcoholic brains (N=6) as compared to controls (N=5). The immunoreactive density was measured by BioQuant Nova analysis system as described in the Methods and presented as mean ± SEM in pixel/mm². ANOVA indicated a significant increase in GluT₅ immunoreactivity, but not Iba-1, in alcoholics as compared to controls (*, p<0.05).

Fig. 6

In Amygdala, no significant difference was detected in either Iba-1 or GluT₅ expression between alcoholic (N=8) and control brains (N=8). The immunoreactive density measured by BioQuant Nova systems as described in the Methods and presented as mean ± SEM in pixel/mm². ANOVA indicated no significant differences in either marker between alcoholic and control groups.