Recruitment of GFP-expressing M2 macrophages to <i>SNpc</i> in 6-OHDA-intoxicated mice.

<p>Macrophages were transfected with GFP-encoding <i>p</i>DNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106867#s2" target="_blank">Materials and Methods</a> section and stained with primary antibodies to CD206, a marker for M2 macrophages, and secondary fluorescently-labeled anti-Mouse-IgG-atto 647N (red). BALB/c mice were <i>i.c.</i> intoxicated with 6-OHDA into <i>SNpc</i>. Twenty one day later, the animals were <i>i.v.</i> injected with GFP-expressing RAW 264.7 macrophages (green, 5×10⁶ cells/ mouse in 100 µl). Twenty four hours later mice were sacrificed, and perfused with PBS and 4% PFA. Brains were frozen, sectioned with a cryostat (10 µm thick), and examined by confocal microscopy (60×magnification) (<b>A, B</b>). Healthy mice without brain inflammation (with PBS <i>i.c.</i> injections) were used as a control group (<b>C</b>). Slides were stained with for expression of mannose receptor (CD206 antibodies). Co-localization of GFP-expressing macrophages and CD206 antibodies to mannose receptor manifested in yellow staining (arrows) confirmed presence of significant amounts of the M2 genetically-modified cells in the intoxicated brain endothelial microvesseles (<b>A</b>), and parenchyma (<b>B</b>). No fluorescence in the healthy brain was found (<b>C</b>) indicating that systemically administered Raw 264.7 macrophages did not cross the BBB in the absence of brain inflammation. The bar: 20 µm.