Figure 1 Effects of subcutaneous injection of the cytokine mixture on 6-OHDA-induced Parkinsonian rats. 6-OHDA was bilaterally administered into the striatum. +Saline or +Cytokine indicate 6-OHDA-administred rats with subcutaneous injection of saline or cytokine, … DA levels were measured by HPLC in the striatum. Approximately 15 μg/mg tissue

weight of DA was detected in the striatum of the sham group, but less than 3 μg/mg DA was present in Inhibitors,research,lifescience,medical the 6-OHDA-treated rats (Fig. 1C). This marked decrease in DA content may selleck screening library underlie the motor dysfunction of the 6-OHDA-treated rats. However, DA contents of the cytokine group increased to 8 μg/mg or more, at 30 days or later. Total RNA was prepared from the ventral midbrain containing the SNpc and then

Inhibitors,research,lifescience,medical reverse transcribed into cDNA for qRT-PCR (Fig. 1D). Although 6-OHDA administration decreased the amount of mRNA encoding the rate-limiting enzyme for DA synthesis TH, the mRNA level was higher in the cytokine group than in the saline-treated group (Fig. 1Da). mRNAs for the microglia marker Iba1 and the oligodendrocyte progenitor cell marker NG2 chondroitin sulphate proteoglycan (NG2) increased in the cytokine group. Protein samples were prepared at 7 and 30 days after 6-OHDA treatment and Inhibitors,research,lifescience,medical used for immunoblotting to estimate the amount of TH, Iba1, and NG2 at the protein level in the SNpc. Representative results from four separate samples are shown in Fig. 1E. β-actin was used as an internal standard. The protein bands from seven separate samples were analyzed by scanning densitometry. Inhibitors,research,lifescience,medical Fig. 1F shows that the TH protein decreased in the saline-treated group compared with the sham group (Fig. 1Fa). Iba1 protein tended to increase in the cytokine group at 7 days, but the level returned to the sham level at 30 days (Fig. 1Fb). NG2 protein was significantly reduced at 30 days in the cytokine

group (Fig. 1Fc). Expression of GM-CSF and IL-3 receptors in neurons and microglia Antibodies to GM-CSF receptor α (GM-CSFRα) and IL-3 receptor α (IL-3Rα) were used in combination with antibodies for TH, and a marker Inhibitors,research,lifescience,medical for microglia, CD11b, to investigate localization of these receptors in the SNpc (Fig. 2). GM-CSFRα-immunoreactivity was localized both in CD11b+ microglia and the TH+ DArgic neurons (Fig. 2A). However, some microglia appeared to express isothipendyl the receptor more strongly than neurons. IL-3Rα immunoreactivity was also localized in both microglia and DArgic neurons (Fig. 2B), but this immunoreactivity was stronger in the neurons than in microglia. Primary cultured microglial cells expressed both receptors (Fig. 2C, D). mRNAs encoding these receptors were evaluated by qRT-PCR. Both mRNAs were detected in the ventral midbrain (Fig. 2E). Only GM-CSFRα-mRNA significantly increased in response to cytokine injection in the 6-OHDA-treated rats. This increase may imply that GM-CSFR expression is regulated in a positive-feedback manner, while IL-3R expression may likely be in a negative-feedback manner.