期刊
BIO-PROTOCOL
卷 9, 期 15, 页码 -出版社
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.3324
关键词
Prostaglandin E-2 (PGE(2)); Microdialysis; Hypothalamus; Cerebrospinal fluid; Interstitial fluid; Mice; Free-moving; Transporters
类别
资金
- (KAKENHI) from Japan Society for the Promotion of Science [15H04755, 15K15181, 055]
- Japan Society for the Promotion of Science [055]
- Smoking Research Foundation [055]
- Grants-in-Aid for Scientific Research [15K15181] Funding Source: KAKEN
Prostaglandin E-2 (PGE(2)) is a well-established chemical mediator for the generation of the fever at the hypothalamus of the brain. PGE(2) mediates fever generation via PGE receptor 3 (i.e., EP3) on neurons in the preoptic area. The role of PGE(2) has been analyzed by measuring PGE(2) concentration in cerebrospinal fluid (C-csf); however, local PGE(2) 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 PGE(2) 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). PGE(2) 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.
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