Ethanol increases TLR3 and HMGB1 expression. Chronic ethanol treatment of C57BL/6 mice (5 g/kg, i.g., daily for 10 days) increased mRNA and protein expression (+IR) of brain TLR3 and HMGB1. (A) Quantitation of TLR3 mRNA and TLR3 + IR. (A-a) Level of brain TLR3 mRNA 27 hours following the last dose of ethanol treatment was measured using real-time PCR as described in the methods. Ethanol exposure significantly increased brain TLR3 mRNA. (A-b) TLR3 + IR cells were counted in mouse cortex after TLR3 immunostaining. Ethanol significantly increased the number of TLR3 + IR cells. (A-c) Representative images of immunohistochemical staining for TLR3 in the cortex of control and ethanol-treated mice. (B) Quantitation of HMGB1 mRNA and HMGB1 + IR. (B-a) HMGB1 mRNA was measured by real-time PCR in which ethanol increased by about 2 fold. (B-b) Quantitative evaluation of HMGB1 + IR. The number of HMGB1 + IR cells was increased about 2 fold. (B-c) The representative images of immunohistochemical staining for HMGB1 in the cortex of control and ethanol-treated mice. *P <0.05, **P <0.01, compared with water control group. Scale bar, 50 μm.