Premium
Microglial activation state and lysophospholipid acid receptor expression
Author(s) -
Tham ChuiSe,
Lin FenFen,
Rao Tadimeti S.,
Yu Naichen,
Webb Michael
Publication year - 2003
Publication title -
international journal of developmental neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.761
H-Index - 88
eISSN - 1873-474X
pISSN - 0736-5748
DOI - 10.1016/j.ijdevneu.2003.09.003
Subject(s) - lysophosphatidic acid , microglia , receptor , messenger rna , lipopolysaccharide , microbiology and biotechnology , sphingosine , biology , sphingosine 1 phosphate , cell culture , stimulation , real time polymerase chain reaction , gene expression , chemistry , biochemistry , immunology , gene , endocrinology , inflammation , genetics
We used a simple commercial magnetic immunobead method for the preparation of acutely isolated microglial cells from postnatal days 1–3 rat brain. With the exception of a 15 min enzyme incubation, all stages are carried out at 4 °C, minimizing the opportunity for changes in gene expression during the isolation to be reflected in changes in accumulated mRNA. The composition of the isolated cells was compared with that of microglial cultures prepared by conventional tissue culture methods, and the purity of microglia was comparable between the two preparations. RT‐PCR analysis of several genes related to inflammatory products indicated that the acutely prepared cells were in a less activated condition than the conventionally tissue cultured cells. We examined the pattern of expression of receptors for lysophosphatidic acid (lpa) and sphingosine‐1‐phosphate (S1P) using quantitative real‐time PCR (TaqMan PCR) techniques. mRNA for LPA 1 , S1P 1 , S1P 2 , S1P 3 and S1P 5 was detected in these preparations, but the levels of the different receptor mRNAs varied according to the state of activation of the cells. mRNA for LPA 3 was only detected significantly in cultured cell after lipopolysaccharide (LPS) stimulation, being almost absent in cultured microglia and undetectable in the acutely isolated preparations. The levels of mRNA of LPA 1 and S1P receptors was reduced by overnight exposure to S1P, while the same treatment significantly up‐regulated the level of LPA 3 mRNA.