Quantification of Prostaglandin E2 Concentration in Interstitial Fluid from the Hypothalamic Region of Free-moving Mice

Abstract

Prostaglandin E2 (PGE2) is a well-established chemical mediator for the generation of the fever at the hypothalamus of the brain. PGE2 mediates fever generation via PGE receptor 3 (i.e., EP3) on neurons in the preoptic area. The role of PGE2 has been analyzed by measuring PGE2 concentration in cerebrospinal fluid (Ccsf); however, local PGE2 concentration at the hypothalamus may not necessarily be consistent with Ccsf. In this protocol, we introduce our method to measure directly the alteration in PGE2 concentration in interstitial fluid in the hypothalamus (Cisf) of awake (free-moving) mice using a microdialysis technique. Male mice (c57BL/6J) were anesthetized and fixed in the stereotaxic instrument, and a microdialysis probe was inserted into the hypothalamus through a guide cannula. On the fifth postoperative day, Cisf was monitored in free-moving mice that were intraperitoneally (i.p.) injected with lipopolysaccharide (LPS). PGE2 and other eicosanoids recovered in Krebs-Ringer phosphate buffer and defused through a microdialysis probe were extracted into ethyl acetate/formic acid and then quantified with LC-MS/MS. Our method is useful to understand the role of key regulators of prostaglandin concentration such as those of transporters, which have been unappreciated in inflammation-based brain diseases.