Study Goals: Obstructive sleep apnea (OSA) induces cognitive impairment that involves intermittent hypoxia (IH). tended to be lately affected. Seven proinflammatory and anti-inflammatory cytokine messenger RNA (mRNA) were assessed at all time points using semiquantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Comparable mRNA analysis was performed after 6 w IH or normoxia associated for the past 3 w with repeated intraperitoneal low-dose lipopolysaccharide or saline. Measurements and Results: Chronic (6, 24 w) but not acute IH induced significant microglial changes in dH only, including increased density and morphological features of microglia priming. In dH, acute but not chronic IH increased IL-1 and RANTES/CCL5 mRNA, whereas the other cytokines remained unchanged. In contrast, chronic IH plus lipopolysaccharide increased interleukin (IL)-6 and IL10 mRNA whereas lipopolysaccharide alone did not affect these cytokines. Conclusion: The obstructive sleep apnea component intermittent hypoxia (IH) causes low-grade neuroinflammation in the dorsal hippocampus of mice, including early but transient cytokine elevations, delayed but long-term microglial changes, and cytokine response alterations to lipopolysaccharide inflammatory challenge. These changes may contribute to IH-induced cognitive impairment and pathological brain aging. Citation: Sapin E, Peyron C, Roche F, Gay N, Carcenac C, Savasta M, Levy P, Dematteis M. Chronic intermittent hypoxia induces chronic low-grade neuroinflammation in the dorsal hippocampus of mice. 2015;38(10):1537C1546. serotype 055:B5; Sigma-Aldrich, St Louis, MO, USA) or saline 0.9% was administered intraperitoneally (10 mL/kg) at 15:00, twice a week (n = 5 in each group) as described by Sy et al.31 Mice were quickly euthanized at 11:00, 20C22 h after the last LPS injection. Brains were removed, rapidly frozen on dry ice, and kept at ?80C until semiquantitative polymerase string reaction (qPCR) evaluation. Immunohistochemical Evaluation of Microglial Cells Human brain sections were incubated at room temperature in a remedy containing anti-Iba1 antibody successively. In human brain, Iba1 (ionized calcium-binding adaptor molecule 1) is certainly a particular marker of microglial cells. The antibody (1:2500; Wako Chemical substances GmbH, Neuss, Germany) was diluted within a 10 mM PB option also formulated with 0.9% NaCl and 0.3% Triton-100X (PBST) for 18 h, accompanied by a biotinylated donkey antirabbit immunoglobulin G (IgG) option (1:1000; Rockland Immunohistochemicals Inc., Gilbertville, PA, USA) and an avidinbiotin complicated proclaimed by horse-radish peroxidase option (1:1000; Elite package, Vector Laboratories, Burlingame, CA, USA) both for 90 min. Finally, the areas had been immersed within a 0.05 M Tris-HCl buffer (pH 7.6) containing 0.025% 3.3-diaminobenzidine-4 HCl (Sigma-Aldrich, St. Louis, MO, USA), 0.5% nickel ammonium sulfate and 0.003% H2O2. Three washes of 10 min in PBST had been performed between each stage. The sections had been then rinsed 2 times in PBST for 10 min and mounted on glass slides. Sections were counterstained with neutral red, dehydrated and coverslipped with Depex (Vector Laboratories). Controls were run in the absence of primary antibodies to ensure the absence of nonspecific labeling. The labeled sections were digitized at 40 magnification through an Axioscope microscope (Zeiss, Germany) equipped with a motorized XCY-sensitive stage and a video camera connected to a computerized image analysis system (ICS Framework; TRIBVN, Chatillon, France). Iba1 immunoreactive (Iba1+) cells were manually plotted from hemisections of the hippocampus taken at 400 m intervals (7 sections between ?1.06 and ?3.28 anteroposterior from Bregma). Hippo-campus surfaces were automatically calculated by tracing their contours with ICS Framework software and the 443797-96-4 atlas of Paxinos and Franklin.32 The microglial cell density was calculated as the number of Iba1+ cells over the surface area of the counted region. The dorsal hippocampus 443797-96-4 was present in sections 1 to 4 443797-96-4 whereas the ventral hippocampus was in sections 5 to 7. Semiquantitative Real-Time RT-PCR Among the inflammatory mediators, we focused on the key proinflammatory chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted) also known as CCL5 (Chemokine (C-C motif) ligand 5), MCP-1 (monocyte SH3RF1 chemoattractant protein-1)/CCL2 (Chemokine (C-C motif) ligand 2), and on the intercellular adhesion molecule-1 (ICAM-1), as well as around the proinflammatory and anti-inflammatory cytokines (TNF-; IL-1, IL-6, and IL-10 interleukins) that are important mediators in neuroinflammation with linked activities. Brains were sliced in 400-m-thick frontal sections at ?12C on a cryostat. The dorsal hippocampus was dissected bilaterally using a stereomicroscope and a scalpel knife (between ?1.34 and ?2.46 anteroposterior from Bregma). Tissue samples were kept at ?80C until.