We are currently examining several of the changes in glial cell gene expression by quantitative real time polymerase chain Wortmannin cost reaction to analyze the duration of the effects, and find that some changes persist for as long as 5 days. In two previous papers, we summarized the effects of these cytokine mix tures on immune related molecules and on neural and glial related proteins, including neurotrophins, growth factors and structural proteins. Each of the cytokine mixtures induced an unique and complex pat tern of changes after 6 hours of incubation. In this third paper, we present the effects of the Th1, MM and Th2 cytokine mixtures on early gene expression for molecules involved in metabolism, signaling and regula tory mechanisms in CNS glia.

A number of the changes found are similar to those found in a gene array analysis of changes in rat spinal cord during the course of myelin basic protein induced experimental autoimmune encephalomyelitis. including changes Inhibitors,Modulators,Libraries in ion homeostasis, mitochondrial function, neurotransmitter related enzymes, and a variety of signaling pathways. An unexpected finding was the large number of changes in early gene expression related to lipid metabolism. The culture system we have analyzed is devoid of neurons to enable identification of the responses of the several types of glia to the cytokines in the absence of cross talk with neuronal signaling. For example, although classically thought of as neuron specific, neurotransmitter receptors Inhibitors,Modulators,Libraries on glial cells are now known to play prominent roles in glial differentiation, Inhibitors,Modulators,Libraries axonalneuronal protection, microglial activation and impulse conduc tion along myelinated axons.

We are initiating stud ies on enriched neuronal cultures to identify the direct effects of Inhibitors,Modulators,Libraries the three cytokine mixtures on early gene expres sion in neurons for comparison with the changes found in glia, with the goal of identifying those cytokines most sup portive of preventing damage and promoting normal axonal function. Methods The methodology has been described Inhibitors,Modulators,Libraries in detail in the prior papers. Mixed CNS glial cell cultures Mixed CNS glial cell cultures were obtained from neonatal rat brain using a modification of the so called shake off technique as we described previously. Fol lowing shakeoff of Pazopanib FGFR cells from the astroglial bed layer, the time for partial removal of microglia by adherence to plas tic was 1 hour prior to plating on poly lysine coated flasks. Cells were maintained in defined medium containing 2% fetal bovine serum for 68 days, then treated with the cytokines.