Figure 1.
Ontogeny of gelatinase activity.
Gelatin zymograms of mouse cortical homogenates prepared at postnatal days 1 (P1), to P60, in WT, t-PA−/− or PAI-1−/− animals. MMP-9 and MMP-2 activities are detected at 70 and 90 KDa respectively.
Figure 2.
Quantification of gelatinase activities in cortical extracts 6 hours after insults in P5 mice.
MMP-2 (A,C,E) and MMP-9 (B,D,F) gelatinolytic activities measured in hypoxia-ischemia (A,B), ibotenate excitotoxic paradigm (C,D) and PBS injection trauma (E,F). Data are expressed relative to the average densitometry in WT mice. * indicate significant difference with activity in respective controls, i.e. WT sham operated (A,B), PBS injected (C,D) or non-treated animals (E,F). *p<0.05; **p<0.01; ***p<0.001 vs WT controls, ##p<0.001 and ###p<0.001 vs PAI-1−/− controls. Numbers in parentheses indicate the number of animals used. ND; non detected upon standard incubation.
Figure 3.
In situ gelatinolytic activity labeling 6 h after hypoxic-ischemic procedure.
Gelatinolytic activity (green) and vessel labeling by IB4 (red) were obtained in WT (A–D), t-PA−/− (E–H) and PAI-1−/− (I–L) mice. Low magnification observation of gelatinolytic activity (A,E,I). Note gross tissue alteration in PAI-1−/− and low level of fluorescence in t-PA−/− mice sections. Higher magnification allows to visualizing micro-vascularization. Gelatinolytic activity is hardly detectable on vessels in WT (B–D), while it is undetectable in t-PA−/− (F–H) and high in PAI-1−/− (J–L) mice. Arrowheads point to spots of high activity; arrows point to microvessels. ISZ; in situ zymographic activity; Ib4; isolectin B4 vessels labeling.
Figure 4.
In situ gelatinolytic activity labeling 6 h after ibotenate injection.
Ibotenate was injected in WT (A–C) and in t-PA−/− (D–F) mice and PBS (control) was injected in WT (G–I) and PAI-1−/− (J–L) mice. Ibotenate induced high gelatinolytic activity spots in WT but not in t-PA−/− mice (A,D), but did not induce activity in microvessels (C,F). PBS controls in WT did not exhibit enhanced gelatinolytic ativity (G–I) but a strong intracellular activity together with vessel activity in PAI-1−/− (J–L) mice. Arrowheads point to spots of high activity; arrows point to microvessels. ISZ; in situ zymographic activity; Ib4; isolectin B4 vessels labeling.
Figure 5.
In situ and in vitro gelatinolytic activity 5 days after PBS injection in WT and PAI-1−/− mice.
Effect of PBS intracortical injection in WT (A–C) or PAI-1−/− (D–F) 5 days post-insult. Gelatinolytic activity remained elevated in PAI-1−/− mice in cell nuclei, in extracellular spots and in vessels. (G) Gel zymogram of gelatinase activity in PAI-1−/− mouse cortex 5 days after PBS injection at P5 and non-injected control. (H) Quantification of MMP-2 and MMP-9 activities in gels. Arrowheads point to spots of high activity; arrows point to microvessels. Numbers in parentheses indicate the number of animals used. *p<0.05 compared to corresponding controls, according to Student’t test.
Figure 6.
Vascular gelatinolytic activity labeling in 5 day-old mice brain sections exposed to glutamate and hrt-PA.
Gelatinolytic activity (green) and vessel labeling by IB4 (red) were obtained in WT (A–F) or t-PA−/− (G–L) mouse brain sections (250 µm thick) after 3 hours ex vivo exposure to control medium (A–C), glutamate 200 µM alone (D–I) or in association with 20 µg/mL hrt-PA (J–L). Arrows point out gelatinolytic activity in vessels. Bar = 200 µm.