E that neuroinflammation is involved in PQ-mediated, ROS-induced dopaminergic neurodegeneration. There
E that neuroinflammation is involved in PQ-mediated, ROS-induced dopaminergic neurodegeneration. There were also microglia-like, large cellular structures in SN and hippocampus of PQtreated brain. Therefore, to evaluate involvement of microglial cells in PQ-mediated neurotoxicity, the appearance of microglial cells and expression levels of microglial markers were assessed by immunofluorescence. We observed that Iba1 immunoreactivity and expression levels significantly increased in SN, but significantly decreased in FC with no significant change in hippocampus, of PQ-treated animal compared controls (Figure 13A, B, C and 13D). To assess whether microglial cells appeared activated or not, weevaluated the microglial activation marker Mac1. Western blot analysis revealed that there was a significant increment in Mac 1 expression in SN, but this was decreased significantly in FC and no changes were observed in hippocampus of PQ-treated brain compared controls (Figure 13D). Supplementation with a-tocopherol in PQ-treated animals did not reproduce any change in expression levels of Iba1 or of Mac 1 in any of the three regions of brain examined, compared to PQ-treated mouse brain (data not shown).Histological changes in microglial cellsTo further assess microglia, we used Weil and Davenport’s method for microglia-specific silver staining [23], applied to determine activation of microglial cells orMitra et al. Journal of Neuroinflammation 2011, 8:163 http://www.jneuroinflammation.com/content/8/1/Page 16 ofFigure 11 PQ (10 mg/kg b.w.) treatment increases TNF-a immunoreactivity in brain. TNF-a immunolocalization was assessed in SN, FC and hippocampus (A, B and C respectively). Densitometric analyses of western blots for TNF-a (D) were performed to assess expression levels of TNF-a in SN, FC and hippocampus. (A): PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27484364 TNF-a immunoreactivity was more intense in SN of PQ-treated animals (b) compared to controls (a). TNF-a immunopositivity was found in both neuritic areas and in large, oval-shaped cells (putative microglia-macrophages, black arrows) in SN of PQ-treated animals (b). (B): The FC of PQ-treated animals (b) showed more intense TNF-a immunoreactivity compared to controls (a). order Ornipressin immunoreaction appeared in neurites and in small round cells (putative microglia, black arrows) in FC of PQ-treated animals (b). (C): TNF-a immunoreactivity was found in both nucleated and non-nucleated areas of hippocampus, with more intense immunoreaction in hippocampal regions of PQ-treated animals (dentate gyrus, CA3 and CA1; b, d and f respectively). TNF-a immunopositive small round cells (putative microglia, black arrows) appeared in both nucleated and non-nucleated areas of hippocampus of PQ-treated animals (b, d and f). (D): Densitometric analyses of western blots indicate that TNF-a expression levels increased significantly in all three regions (SN, FC and hippocampus) of PQtreated animals compared to controls. b-Actin was used as a reference control in western blots. Data are presented as mean ?SEM (n = 3). Asterisks (*) represent significant differences at a level of p < 0.05 (Student's t-test) for densitometric analyses. Magnification of IHC images is 40?and the scale bars = 40 .microgliosis, as a priming event of neuroinflammation (Figure 13). Microscopical observation revealed numerous aggregations of microglial cells in SN, compared to controls, and also changes in microglial morphology (Figure 13E). Microglia were less apparent in FC of PQtreated brain compa.