Urokinase-Type Plasminogen Activator Promotes Dendritic Spine Recovery and Improves Neurological Outcome Following Ischemic Stroke

uPA promotes neurological recovery after ischemic stroke. A, B, Mean LI (A) and forelimb strength (B) in Wt (black bars in A and black squares in B), uPA^−/− (dark gray bars in A and white diamonds in B), and uPAR^−/− (light gray bars in A and black triangles in B) mice, between 6 h and 7 d after tMCAO. White bars in A and white circles in B correspond to sham-operated controls. ^†p < 0.0001 compared with sham-operated animals. *p = 0.01, **p < 0.0001, ^§p < 0.0001, and ^̂p < 0.0001 in A compared with Wt mice 6 h after tMCAO, and with uPA^−/− and uPAR^−/− mice at each time point. *p = 0.02, **p < 0.0001, ^̂p < 0.0001, and ***p < 0.0001 in B compared with sham-operated, and uPA^−/− and uPAR^−/− mice at each time point; n = 20 per strain. Lines denote SD. Statistical analysis was performed with one-way ANOVA. C–H, Mean LI (C, E, G) and forelimb strength (D, F, H), in Wt (C, D), uPA^−/− (E, F), and uPAR^−/− (G, H) mice, between 24 h and 7 d after tMCAO. Mice were treated immediately after tMCAO with either intravenous saline solution (black bars in C, E, G; black circles in D, F, H) or with 0.1/mg/kg per IA of ruPA (gray bars in C, E, G; gray squares in D, F, H); n = 15 observations per time point. Lines denote SD. Compared with mice treated with saline solution at each time point: C, *p = 0.01, **p = 0.04, ^§p = 0.01, and ^̂p = 0.03. D, *p = 0.001, **p = 0.02, ^̂p = 0.04, and ^§p = 0.02. E, *p = 0.01,**p = 0.03, ^§p = 0.02, and ^̂p = 0.001. F, *p = 0.001, **p < 0.0001, ^̂p = 0.02, and ^§p = 0.04. Ns in G and H: nonsignificant compared with saline solution-treated mice (G) and controls (H) at each time point. Statistical analysis was performed with one-way ANOVA.

Effect of uPA deficiency on the anatomical structure of the peri-infarct cortex. A, Diagram (top) and representative TTC staining (bottom) of a coronal cut through bregma + 0.50 24 h after MCAO. The asterisk denotes the area of the brain where quantifications and observations presented in E and F were performed. M1 and M2 denote primary and secondary motor cortices, respectively. B, Representative photographs of combined DTIs and Fa maps in Wt and uPA ^−/− mice 24 h after MCAO. Colors represent fiber orientation: red, mediolateral; green, dorsoventral; blue, rostrocaudal. C, D, MD of water (C) and Fa (D) in the area denoted by the asterisk in A and B in the nonischemic (NI; white bars) and ischemic (I; black bars) cortex of Wt and uPA^−/− mice 24 h after MCAO. C,*p = 0.001 compared with the nonischemic hemisphere in uPA^−/− mice. D, *p = 0.02 compared with the nonischemic hemisphere in Wt mice. **p < 0.0001 compared with the nonischemic hemisphere in uPA^−/− mice; n = 6 mice per experimental group. Lines denote SD. Statistical analysis was performed with two-sample t test. E, Mean length of distal neurites located within 0.6 mm of the border of the necrotic core in Wt (white bar) and uPA^−/− (black bar) mice 24 h after MCAO; n = 6 mice per experimental group. Bars represent mean values obtained from the analysis of 2000 extensions per mouse; *p < 0.0001. Lines indicate SD. F, Representative micrographs of a tridimensional reconstruction of DiI-stained distal dendrites from neurons originated in cortical layer V from Wt and uPA^−/− mice either under nonischemic conditions (controls) or 24 h after tMCAO. Red is the dendritic shaft and blue corresponds to dendritic protrusions.

uPA mediates dendritic spine recovery in the peri-infarct cortex. A, Representative micrograph of a Golgi-stained coronal section at bregma: +0.5 mm from a Wt brain 24 h after tMCAO. M1 and M2 denote primary and secondary motor cortices, respectively. The square denotes the interface between the necrotic core (Nc) and peri-infarct cortex where observations presented in C–F were performed. Magnification: 4×. B, Representative micrographs taken from the interphase between the Nc and peri-infarct cortex (a). b, Shows the area located within 0.6 mm from the border of the Nc where dendritic spines (arrowheads) are intermixed with dendritic varicosities (arrows). Magnification: 40× in a, and 100× in b. C, Representative micrographs of Golgi-stained dendrites located within 0.6 mm from the border of the Nc from Wt and uPA^−/− mice 6, 24, and 168 h (7 d) after tMCAO. NI, nonischemic brains (controls). Arrows in c and d denote dendritic varicosities. Arrows and arrowheads in e denote filopodia and varicosities, respectively. Arrows and arrowheads in f denote dendritic varicosities and small protrusions, respectively. Arrowhead and arrows in g denote dendritic spines and long thin protrusions, respectively. D, E, Mean number of dendritic protrusions per 10 μm (D) and percentage of neurons with dendritic blebbing (E) 0.6 mm from the border of the Nc of Wt (white bars), uPA^−/− (black bars), uPAR^−/− (gray bars), and Plau^GFDhu/GFDhu (silver bars) mice 0–168 h after tMCAO. C, controls, nonischemic brains. Lines denote SD; n = 12 mice per strain at each time point. D, *p < 0.0001 and **p < 0.0001 compared with Wt at 6 h and uPA^−/−, uPAR^−/−, and Plau^GFDhu/GFDhu brains 6, 24, and 168 h after tMCAO. E, *p < 0.0001 compared with Wt at 6 h and with uPA^−/−, uPAR^−/−, and Plau^GFDhu/GFDhu brains and 6, 24, and 168 h after tMCAO. Statistical analysis was performed with two-way ANOVA test. F, Cumulative frequency plot of spine length in the distal 10 μm of 600 dendrites examined from Wt mice either under nonischemic conditions (white squares) or 24 and 168 h after 60 min of tMCAO (black circles and gray triangles, respectively).

uPAR expression in cerebral cortical neurons. A, Representative micrograph of uPAR expression in cerebral cortical neurons. Green, uPAR; blue, DAPI. Magnification: 20×. B, C, Representative micrographs of wild-type cerebral cortical neurons stained with antibodies against uPAR (white and green in B and C) and MAP-2 (red in B) or synaptophysin (red in C). Arrows in C denote examples where presynaptic synaptophysin-containing vesicles face uPAR-expressing postsynaptic dendrites. Magnification: 60×. D, E, Representative micrograph of uPAR immunostaining (green in D and white and green in E) and phalloidin (red in D and E) in a distal dendrite of a Wt cerebral cortical neuron. The dashed and continuous squares correspond to the areas magnified in E a–c and d–f, respectively. Arrows in c indicate colocalization of uPAR and phalloidin. Arrows in f denote an example where uPAR is localized in the tip of dendritic protrusions. Magnification: 20× in D and 60× in E.

uPA promotes dendritic protrusion recovery following an excitotoxic injury. A, Diagram of the experimental paradigm used to study the effect of uPA on dendritic spine recovery following an excitotoxic injury. a–c, Correspond to examples of DiI-stained distal dendrites either at baseline (a), or 10 min after exposure to NMDA (b; acute injury), or 180 min after withdrawing NMDA (c; recovery). Arrowheads denote dendritic spines in a and varicosities in b. Arrows in c show dendritic spines and filopodia re-emerging from varicosities. B, Representative micrograph of MAP-2 (red) and uPAR (white in b and yellow in c) staining in the distal dendrite of a Wt neuron 3 h after 10 min of incubation with NMDA. Arrows in c denote uPAR clustered within dendritic varicosities. Magnification: 40×. C, Mean percentage of uPAR-negative (white bar) and uPAR-positive (black bar) varicosities in the distal 10 μm of Wt dendrites following 10 min of treatment with NMDA (n = 200 neurons from three different cultures; p < 0.001; two-sample t test). Lines denote SD. D, Magnification of the area denoted by the square in B. Arrows denote uPAR in filopodia re-emerging from a uPAR-enriched varicosity. Red is MAP-2, white (b) and green (c) are uPAR. Magnification: 100×. E, Mean number of dendritic protrusions per10 μm in the distal dendrites of Wt (white bars), uPA^−/− (black bars), and uPAR^−/− (gray bars) cerebral cortical neurons either at baseline (time 0), or immediately after 10 min of incubation with NMDA, or 3 h after recovery from 10 min of incubation with NMDA. A subset of Wt, uPA^−/−, and uPAR^−/− neurons were treated with 10 nm uPA immediately after the end of the excitotoxic injury (ruPA). *p < 0.0001 compared with Wt neurons immediately after NMDA exposure; **p = 0.001 compared with untreated Wt neurons 3 h after NMDA exposure; ^§p < 0.0001 compared with uPA^−/− neurons either immediately after exposure to NMDA or 3 h after NMDA exposure but no treatment with ruPA; n = 60 per time point. Each observation was repeated in cultures from three different animals. Statistical analysis was performed with one-way ANOVA.