Brains of marmosets in the untreated group were also collected for use as controls

Brains of marmosets in the untreated group were also collected for use as controls. We also generated a mouse model of massive demyelination by continuously feeding male in-house bred C57BL/6J mice aged 8 weeks with a powdered diet containing 0.2% (w/w) cuprizone, as described previously (Chen et al., 2004). differential PBR upregulation: (1) PBR(?) astrogliosis uncoupled with microgliosis or coupled with PBR(+) microgliosis associated with irreversible neuronal insults; and (2) PBR(+) astrogliosis coupled with PBR(? or ) microgliosis associated with minimal or reversible neuronal toxicity. Intracranial transplantation of microglia also indicated that nontoxic microglia drives astroglial PBR expression. Moreover, levels of glial cell line-derived neurotrophic factor (GDNF) in astrocytes were correlated with astroglial PBR, except for increased GDNF in PBR(-) astrocytes in the model of AD-like tau pathology, thereby suggesting that PBR upregulation in astrocytes is an indicator of neurotrophic support. Together, PBR expressions in astrocytes and microglia reflect beneficial and deleterious glial reactions, respectively, in diverse neurodegenerative disorders including AD, pointing to new applications of PBR imaging for monitoring the impact of gliosis on the pathogenesis and treatment MK-8998 of AD. autoradiographic and positron emission tomographic (PET) MK-8998 techniques. For example, [11C]PK11195 was the first to enable PET measurement of PBR in diverse CNS pathologies (Banati et al., 2000; Pappata et al., 2000; Cagnin et al., 2001; Rojas et al., 2007), but other radiolabeled ligands were developed, such as ((HRP-ILB4), cuprizone, GBR12909 and PK11195 from Sigma-Aldrich. We raised a rabbit polyclonal antibody against the C-terminal sequence of PBR using synthetic peptide spanning residues 155C169 of murine PBR (WRDNSGRRGGSRLAE). This antibody (NP155) was affinity-purified and characterized by immunoblotting as well as immunostaining for comparison with commercial anti-PBR antibodies (rabbit polyclonal, R&D Systems; rabbit polyclonal, BioVision; rabbit polyclonal, FL-169, Santa Cruz Biotechnology; goat polyclonal, W-12, Santa Cruz Biotechnology). Other antibodies used in this study are as follows: rabbit polyclonal antibody against ionized calcium binding adapter molecule-1 Rabbit Polyclonal to BRS3 (Iba-1) (Wako Pure Chemicals), mouse monoclonal antibody against rat CD11b (OX42; AbD Serotec), rat monoclonal antibody against mouse CD11b (M1/70; BMA Biomedicals), rabbit polyclonal (Dako) and rat monoclonal (clone 2.2B10; Zymed/Invitrogen) antibodies against glial fibrillary acidic protein (GFAP), rat monoclonal antibody against myelin basic protein (MBP) (Millipore), rabbit polyclonal antibody against glial cell line-derived neurotrophic factor (GDNF) (Santa Cruz Biotechnology) and rabbit polyclonal antibody against tyrosine hydroxylase (TH) (Millipore). Western blot analysis. For evaluation of NP155, the microglial clone termed Ra2 was maintained in Eagle’s minimum amount essential medium (M4655; Sigma-Aldrich) supplemented with 10% fetal bovine serum, 5 g/ml bovine insulin, 0.2% glucose and 1 ng/ml murine granulocyte-macrophage colony-stimulating element (G0282; Sigma-Aldrich), as explained previously (Sawada et al., 1998). For biochemical analyses, the cells (5 106) were scraped and homogenized in 50 mm Tris-HCl, pH 7.4, 4C, containing 0.1% protease inhibitor mixture (5 mm leupeptin, 1 mg pepstatin and 5 mg aprotinin in 1 ml dimethyl sulfoxide) and 0.5 mm phenylmethylsulfonyl fluoride (PMSF). The suspension was centrifuged at 12,000 rpm (10,000 g) for 15 min, and the resultant pellet was resuspended in 50 mm Tris-HCl buffer. The protein amounts in the samples were measured relating to Lowry’s method. For immunocytochemistry, the cells were cultured on cover glasses for 7 d, and washed with PBS, followed by fixation with 4% paraformaldehyde for 20 min. Hippocampal samples from KA-treated and untreated rats were homogenized in 50 mm Tris-HCl, pH 7.4, 4C, containing 0.1% protease inhibitor mixture (as with experiment for Ra2 cells) and 0.5 mm PMSF. The suspended homogenates of cultured microglial (Ra2) cells and rat hippocampal cells, related to 10 and 100 g protein, respectively, were applied to a 15% SDS polyacrylamide gel. After electrophoresis and transfer of proteins to a polyvinylidene fluoride membrane (Immobilon-P; Millipore), the membrane was immersed in Tris-buffered saline (150 mm NaCl, 10 mm Tris-HCl, pH 8.0) containing 0.05% (v/v) Tween 20 and 3% (w/v) bovine serum albumin (BSA), and then reacted for 1 h with anti-PBR antibodies in TBS containing 0.05% (v/v) Tween 20 and 0.1% (w/v) BSA. The primary antibodies were recognized by HRP-conjugated anti-IgG antibodies (GE Healthcare) and enhanced chemiluminescence method (GE Healthcare). Animal models. The mice analyzed here were managed and handled in accordance with the National Research Council’s Guideline for the Care and Use of Laboratory Animals and our institutional recommendations. Protocols for the present animal experiments were approved by the Animal Ethics Committees of the National Institute of Radiological Sciences and Central Institute for Experimental Animals (CIEA). APP Tg mice termed APP23, which overexpress the Swedish doubly mutant APP751 under the control of a neuron-specific Thy-1 promoter element, were developed as described in detail previously (Sturchler-Pierrat et al., 1997), and were maintained on a C57BL/6J background. Tau Tg mice MK-8998 dubbed PS19 were generated by using a cDNA coding a tau isoform comprising 1 N-terminal and 1 C-terminal on the other hand spliced exons and the P301S.