Fig. 2
Induction of neuroinflammation by chronic HP infection. Using an oral gavage, we inoculated C57BL/6 mice with 300-μl Brucella broth containing 4 × 107 H. pylori daily for 5 days. Three weeks (acute) or 5 months (chronic) after HP infection, we harvested the brain and stomach and conducted immunohistochemistry (IHC) using formalin-fixed and paraffin-embedded sections. A Activated microglia (brown color, arrows) and T cells were visualized by antibodies against Iba1 (GeneTex, Irvine, CA) and CD3 (Biocare medical, Concord, CA), respectively. We determined the level of neuroinflammation by the microglia activation score, using five coronal brain slices per mouse; we quantified Iba1-positive cells (positive cells/field 0.1 mm2: score 0 = 0–2; 1 = 3–5; 2 = 6–10; 3 = 11–15; 4 = 16–20; and 5 = 21 or more) and calculated the average [mean + standard error of the mean (SEM)] of the acute or chronic HP-infected group and the uninfected control group (nine mice per group). We found that the chronic HP-infected group had significantly higher scores than the control and acute groups. *P < 0.05 by the Student’s t test. Bar = 50 μm. We did not detect infiltration of CD3+ T cells in the brain parenchyma in any groups. Bar = 50 μm. ND, not detected. B IHC of HP antigens in the stomach (pylorus) and brain from HP-infected mice. Using anti-HP antibody (Thermo Fisher Scientific, Waltham, MA), we found HP antigen-positive bacteria (brown color, arrows) in the stomach during the acute and chronic phases, but not in the brain. The HP scores in the stomach were determined as follows: HP antigen-positive gastric crypts/total gastric crypts × 100 (%) (score 0 = negative; 1 = 1–20%; 2 = 21–50%; and 3 = 51–100%). The representative stomach image is scored as 3, which was determined as follow: eight HP-antigen positive crypts/11 total crypts × 100 = 73%. Bar = 50 μm. *P < 0.05 by the Student’s t test. ND, not detected