Fig 1.
Schematic representation of the 4-day drinking in the dark (DID) procedure for 4 weeks.
Table 1.
Biochemical parameters in the plasma and liver of rats exposed to alcohol during adolescence.
Fig 2.
Plasma concentrations of chemokines in rats exposed to alcohol during adolescence and control rats grouped by sex.
(A) CCL2 (pg/mL); (B) CX3CL1 (pg/mL); (C) TNF-α (pg/mL); (D) IL-1β (pg/mL); (E) IL-6 (pg/mL); and (F) IL-10 (pg/mL). The bars represent means ± SEM. The bars represent means ± SEM. The data were analyzed by two-way ANOVA and significant main effects or interactions between factors were indicated as follows: (&&&) denotes significant main effect of sex; (##) p<0.01 and (###) p<0.001 denote significant interaction between adolescent alcohol exposure and sex; (aaa) p<0.001 denotes significant differences compared to female control rats after post hoc tests.
Fig 3.
Representative images after histological analysis of spleen sections using haematoxylin and eosin stain.
Spleen sections are shown at 4x and 40x magnification. Abbreviations: A = Central artery; F = Follicle; MS = Marginal sinus region; MZ = Marginal zone; P = Periarteriolar lymphoid sheath; RP = Red pulp; T = Trabeculus; VS = Venous sinus; WP = White pulp.
Fig 4.
Relative mRNA expression of endocannabinoid signaling-related proteins in the spleen of rats exposed to alcohol during adolescence and control rats grouped by sex.
(A) CB1 mRNA levels (fold change); (B) CB2 mRNA levels (fold change); (C) PPARα mRNA levels (fold change); (D) NAPE-PLD mRNA levels (fold change); (E) DGLα mRNA levels (fold change); (F) DGLβ mRNA levels (fold change); (G) FAAH mRNA levels (fold change); (H) MGL mRNA levels (fold change); and (I) FAT/CD36 mRNA levels (fold change). The bars represent means ± SEM. The data were analyzed by two-way ANOVA and significant main effects or interaction between factors was indicated as follows: (*) p<0.05 and (**) p<0.01 denote significant main effect of adolescent alcohol exposure; (###) p<0.001 denotes significant interaction between adolescent alcohol exposure and sex. (aaa) p<0.001 denotes significant differences compared to female control rats after post hoc tests; (bbb) p<0.001 denotes significant differences compared to female alcohol-exposed rats after post hoc tests.
Fig 5.
Expression of CB1, CB2, PPARα and NAPE-PLD proteins in the spleen of rats exposed to alcohol during adolescence and control rats grouped by sex.
Relative protein expression and representative immunoblots of: (A) CB1 protein (fold change); (B) CB2 protein (fold change); (C) PPARα protein (fold change); and (D) NAPE-PLD protein (fold change). Representative immunohistochemical images at 4x magnification of: (E) CB1 protein; (F) CB2 protein; (G) PPARα protein; and (H) NAPE-PLD protein. The bars represent means ± SEM. The data were analyzed by two-way ANOVA and significant main effects or interaction between factors was indicated as follows: (*) p<0.05 denotes significant main effect of adolescent alcohol exposure; (#) p<0.05 denotes significant interaction between adolescent alcohol exposure and sex. (a) p<0.05 and (aa) p<0.01 denote significant differences compared to the respective control rats after post hoc tests; (b) p<0.05 denotes significant differences compared to female alcohol-exposed rats after post hoc tests. The black arrows indicate positive cells. Abbreviations: RP = Red pulp; VS = Venous sinus; WP = White pulp.
Fig 6.
Representative immunohistochemical images of spleen sections at 10x and 40x magnification of CB1, CB2, PPARα and NAPE-PLD from female control rats.
The squares indicate the areas of 40x magnification. Abbreviations: A = Central artery; F = Follicle; MZ = Marginal zone; P = Periarteriolar lymphoid sheath; RP = Red pulp; VS = Venous sinus; WP = White pulp.