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10 septembre 19381938/09/10 (A67).Appartient à l’ensemble documentaire : PoitouCh

Primers used for quantitative real-time polymerase chain reaction analysis of SDF-1α, BDNF, IGF-1, VEGF-A, TGF-β1, β-actin transcript levels.

a<p>forward</p>b<p>reverse</p

Confocal analysis of immunoreactivity of NC(4 h) - green - and nestin, GFAP or NG-2 - red - at day 1.

<p>Colocalization (arrows) can be observed between NC(4 h) and nestin, NC(4 h) and GFAP, NC(4 h) and NG-2. Images are taken in striatum, in proximity of the ventricular wall where most cells can be found at this time point. Bar: 25 µm.</p

Confocal analysis of immunoreactivity of NC (green) and microglia/macrophages (red).

<p>CD11b immunoreactivity signal, staining for microglia/macrophages, colocalizes with that of NC(4 h) in striatum at day 1 (A–C) and 7 (D–F). Z- stacks are shown. Bar: 25 µm.</p

Morphology of microglia/macrophages (red).

<p>CD11b immunopositive cells at day 1 after ischemia and NC(4 h) infusion display a round, phagocytic morphology in the injected (A) but not in the contralateral side were no NC are present (B). Microglia/macrophages appear to surround NC as evidenced in C were NC are marked with Hoechst (blue). A further detail of the interaction between these cells is provided in D where one microglia/macrophage appears to cap a NC (marked with 5-CFDA, green). Bar (A–C): 20 µm; bar (D): 7 µm.</p

Box-plot of neuronal count performed in ischemic mice 7 and 14 days after ischemia with the optical dissector method.

<p>Differences of neuronal density between contralateral and ipsilateral striata are sorted by treatment and time. The box represents the median and 25^th–75^th percentiles, whiskers represent the range, + represents the mean of the group. The hierarchical ANOVA showed a significant effect for the treatment (P = 0.02). The Bonferroni post-hoc test showed significant differences (P<0.05) among mean density differences of the isch/NC(4 h) group and the isch/PBS, isch/fibr and isch/NC(7 d) groups. Data plotted have been multiplied by 1×10⁴.</p

Experimental protocols.

<p>Ischemia was induced for 30 minutes, 2 hours after the <i>in vitro</i> placement of the isolated brain. NC were perfused for 1 h immediately either after the reopening of the vessel or 1 our later (protocol 2). The perfusion was followed by a wash-out period with a solution without NCs. At 5 hours <i>in vitro</i> the brains were fixed for immunohistochemistry. The bottom of the panel shows an example of simultaneous DC recordings from 4 different sites in an isolated guinea pig brain. Hypoxic depressions (HD) were recorded in the electrodes located in the regions vascularized by the occluded MCA. Potentials evoked by LOT stimulation before and during the first part of ischemia (arrowhead) disappeared when HD occurred, and recovered during MCA reperfusion. Evoked potentials in the hemisphere contralateral to MCA occlusion were not altered.</p

Distribution of NC(4 h) in ischemic and sham-operated mice at days 1, 7 and 14.

<p>NC were infused icv in the lesioned side. Drawings represent typical results and are based on n = 5 brains for each experimental condition.</p

Figure 4

<p>(A) Changes in extracellular pH <u>(pH_e)</u> in the PC and in mOT induced by ipsilateral MCA occlusion and reperfusion. Occlusion induced a rapid metabolic acidification of the extracellular microenviroment in PC, interrupted by a transient and mild basification (arrow) associated to the hypoxic spreading depression (HD, asterisk). No changes in extracellular [H⁺] were recorded in the mOT, that is not served by the MCA. (B) Simultaneous changes in extracellular potassium concentration ([K⁺]_o)and extracellular pH in the PC after MCA occlusion and reperfusion. An initial enhancement in [K⁺] was followed by a fast and large increase in [K⁺]_o. associated to the HD. The schematic drawing on the right illustrates the position of the two-barrel recording electrodes. The field responses (FP) recorded with the conventional extracellular barrel are also shown. The period of MCA occlusion is marked by the shaded area.</p

Quantification of microglia/macrophage activation.

<p>The area occupied by CD11b immunopositive cells was measured in striatum of sham-operated or ischemic mice receiving PBS, NC(4 h) or NC(7 d) at different time points (see Experimental design, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000373#pone-0000373-g001" target="_blank">Fig. 1A and B</a>). Data are expressed as mean±SEM (n = 4–5). Two-way ANOVA: (A) F: 58.05, p<0.0001; (B) F: 10.19, p = 0.0002. <i>Posthoc</i> Bonferroni test: *p<0.05, **p<0.01, ***p<0.001 vs sham/PBS; °°p<0.01, °°°p<0.001 vs isch/PBS, ##p<0.01, ###p<0.001 vs sham/NC.</p