Protective monocytes in cerebral ischemia

Stroke is a highly prevalent and devastating disease with limited therapeutic options. Most efforts to develop treatments for stroke have thus far met with limited success in large clinical trials. The brain has an enormous capacity for self-preservation, and elucidating how the brain protects itself may provide novel insights into stroke treatment. Ischemic tolerance (IT), a phenomenon in which application of sub-lethal stress [preconditioning (PC) stimulus; PC] induces a state of tolerance to a subsequent ischemic insult, represents an example of endogenous neuroprotection. IT can be induced in brain by systemic administration of the Toll-like receptor-4 ligand lipopolysaccharide (LPS). Different populations of immune cell could potentially play a role in PC induced by LPS. In particular, monocytes, which are the main target of LPS, can exert a powerful protective effect either directly or by modulating the immune system. C57Bl/6 male mice (8-12 weeks old) were treated with 0.5 mg/kg LPS i.p. (experimental) or saline (control). Twentyfour hours later mice were transcardially perfused with cold PBS and brains were harvested. Brains from 3-4 mice were pooled and enzymatically digested with Liberase DH (Roche Diagnostics) and 50 U/ml DNase I (Worthington Biochemical) at 37°C for 45 min in an orbital shaker at 160 rpm. Mononuclear cells were harvested from digested brains after fractionation over a discontinuous Percoll gradient. Nonspecific binding was blocked by incubation for 10 min at 4 °C with anti–CD16-CD32 antibody (BioLegend, clone 93, 5 ng/μl) and stained with the appropriate antibodies for 15 min at 4 °C. The following antibodies were used for extracellular staining: CD45 (clone 30F-11, 0.5 ng/μl), CD115 (clone AFS98, 0.5 ng/µl), Ly6G (clone 1A8, 2 ng/μl), and Ly6C (clone HK1.4, 0.5 ng/μl). Inflammatory monocytes (CD45highCD115+Ly6ChighLy6G–) were sorted on a FACSVantage cytometer (BD Biosciences) and collected in Trizol LS (Invitrogen).