ABSTRACT
Preconditioning with lipopolysaccharide (LPS) induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are resident immune cells of the central nervous system (CNS) that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, - using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins signal transducer and activator of transcription1/2 (STAT1/2).
Subject(s)
Lipopolysaccharides , Microglia , Proteome , Proteomics , Animals , Microglia/metabolism , Microglia/immunology , Mice , Proteomics/methods , Male , Brain Ischemia/metabolism , Brain Ischemia/immunology , Ischemic Preconditioning/methods , Mice, Inbred C57BL , Disease Models, AnimalABSTRACT
Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive.
ABSTRACT
Hematopoietic stem cell performance and identity, crucial for homeostasis of the blood-forming system, is governed by extrinsic factors found in the bone marrow microenvironment. Communication within hematopoietic stem cell niches occurs via soluble factors or cell-to-cell contacts between niche and blood-forming cells - which in turn are influenced by systemic factors distributed by the bone marrow extracellular fluid. Although hematopoietic cell-intrinsic aging contributes to the aging phenotype of the hematopoietic system, the architecture and cellular composition of the bone marrow microenvironment have emerged to be highly dynamic during aging and suggested as a major driver for the functional limitations of the blood system observable in old individuals. Recent attention has been paid to the interface between the peripheral nervous system and blood-forming cells in the bone marrow in several clinical contexts and in aging - the latter is reviewed here.
