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

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 (C_csf); however, local PGE₂ concentration at the hypothalamus may not necessarily be consistent with C_csf. In this protocol, we introduce our method to measure directly the alteration in PGE₂ concentration in interstitial fluid in the hypothalamus (C_isf) 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, C_isf 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.