Reduced infiltration of neutrophils and disruption of vascular barrier in the injured spinal cord in PAR-1 null mice.

<p>Aggregation of immunolabled GR1-positive neutrophils is evident in the lesion epicenter in both wild-type (A) and PAR-1 null mice (B) 24 hours after injury. Quantitative analysis reveals that the number of infiltrated neutrophils within the lesion epicenter is significantly lower in the PAR-1 null mice than in the wild-type mice (C). Such a difference in number between the 2 groups of mice is not apparent in segments rostral or caudal to the epicenter. The luciferase luminescence, which inversely represents the integrity of the blood-spinal cord barrier, is significantly decreased at the lesion epicenter in PAR-1 null mice as compared to that of the wild-type mice. (n = 7/genotype, means ± SEM, unpaired Student's <i>t</i>-test, *p < 0.05, **p < 0.01).</p

Improved motor function recovery in PAR-1 null mice after spinal cord injury.

<p>Representative pawprints show the walking patterns of the injured wild-type (A) and PAR-1 null mice (B) 42 days post injury as well as that of the uninjured PAR-1 null mice (C). After spinal cord injury, dragging of the hindlimbs with poor coordination is evident in the wild-type mice, whereas considerable improvement with weight-bearing stepping and slight external rotation of the hindpaws is consistent in the PAR-1 null mice. Injured PAR-1 null mice also show significant locomotor recovery, assessed by the Basso Mouse Scale, as compared to the wild-type mice (D). This result parallels the better performance of PAR-1 null mice on the inclined grid (E). (n = 10/genotype, means ± SEM, 2-way ANOVA for locomotor assessment, unpaired Student's <i>t</i>-test for inclined grid, *p < 0.05, **p < 0.01).</p

Enhanced white matter sparing and reduced glial scar formation at the lesion epicenter in PAR-1 null mice 42 days post injury.

<p>Residual white matter is visualized by luxol fast blue staining using dark-field microscopy. Wild-type mice (A) have less residual white matter, mainly located at the ventral-most part of the spinal cord cross section, than the PAR-1 null mice (B). Such a difference in the size of spared white matter is statistically significant (C). The glial scar, characterized by intense GFAP immunoreactivity, is more widespread at the lesion epicenter in the wild-type mice (D) relative to the PAR-1 null mice (E). Boxed areas enclose part of the glial limitans, an interface separating the GFAP-quiescent areas (asterisks) in the lesion epicenter from the residual cord tissue. At higher magnification, more densely entangled astrocytic processes are apparent in the wild-type mice than in the PAR-1 null mice as demonstrated in the insets. As described in Materials and Methods, the quantitative analysis demonstrates that the total score of the glial scarring, which represents the severity of glial scar formation, is significantly lower in the PAR-1 null mice than in the wild-type controls (F). Scale bar = 500 μm. (n = 7 and 5/genotype for the measurement of spared white matter and glial scarring, respectively, means ± SEM, unpaired Student's <i>t</i>-test, *p < 0.05, **p < 0.01).</p

Improved locomotor recovery after the treatment with rAPC in spinal cord-injured wild-type mice.

<p>Administration of rAPC 20 minutes after the injury significantly improves locomotor performance of wild-type mice (A), starting from 14 days post injury, to an extent comparable to that of the injured PAR-1 null mice receiving no APC treatment. However, APC shows no additional benefit of functional improvements in injured PAR-1 null mice (B), suggesting that the effectiveness of APC is associated with PAR-1. (n = 10/genotype, means ± SEM, 2-way ANOVA,*p < 0.05, **p < 0.01).</p

Immunolocalization of PAR-1 in the spinal cord of the wild-type mice.

<p>PAR-1 is expressed by neurons in the ventral horn in the uninjured spinal cord (A). At higher magnification, the PAR-1-positive neuron exhibits typical multipolar morphology of the spinal motor neurons (B). PAR-1 (arrow, C) also co-localizes with PECAM-1-positive capillaries (arrow, D) in the uninjured cord. After spinal cord injury, PAR-1 is expressed by reactive astrocytes 24 hours post-injury (E). These reactive astrocytes in the lesion show increased expression of GFAP and hypertrophic morphology (F) as demonstrated in the digitally merged image (G). Scale bars = 100 μm for A, C, D; 50 μm for B, E, F, G.</p