Cytokine and chemokine responses in serum and brain after single and repeated injections of lipopolysaccharide: multiplex quantification with path analysis

Administration of the proinflammatory molecule lipopolysaccharide (LPS) alters transport rates for many peptides across the blood–brain barrier (BBB). We and others have previously shown that effects of LPS on BBB transport are highly dependent on the injection paradigm used, and timing of the study. Cytokine expression in both brain and serum compartments influences the BBB response to an inflammatory stimulus, and mediates changes in BBB transport. Here, we used multianalyte technology to simultaneously determine the responses of 13 cytokines and chemokines (G-CSF, GM-CSF, IL-1α, IL-1β, IL-6, IL-10, IL-13, IP-10, KC, MCP-1, MIP-1α, RANTES, and TNF-α) in brain and blood to single and repeated injections of LPS and path analysis to determine the major relations among these analytes. Major findings are: (1) in comparison to measurements taken from a time course after a single injection of LPS, the three injection regimen of LPS produced significantly higher levels in brain for G-CSF, IL-1α, IL-6, MCP-1, MIP-1α, and TNF and in serum for G-CSF, IL-6, and GM-CSF and (2) path analysis distinguished direct from indirect correlations between analyte pairs, with MCP-1, IL-6, G-CSF, and KC mediating relations among these cytokines both within and between serum and brain compartments. These results suggest that potentiation of cytokine levels in brain and serum compartments could play important roles in the regulation of BBB transport, and that our novel application of an established statistical method can be used to assess direct correlations within multiplexed datasets.