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

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

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

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

Acute gelatinase inhibitor does not attenuate deficits in social behavior at adulthood after pediatric TBI.

<p>(A) Social investigation was quantified by the resident-intruder paradigm, revealing that TBI mice as compared to sham controls spent less time investigating a naïve intruder mouse (2-way ANOVA overall effect of TBI, **p<0.01). (B) In the three-chamber social approach task (stage 2), all mice showed an overall preference for sociability with stimulus mouse 1 compared to the empty chamber (2-way RM ANOVA overall effect of chamber, p = 0.0003). (C) Stage 3 of the three-chamber task tested social novelty. Here, sham-operated mice revealed a preference for a novel stimulus mouse compared to the now-familiar mouse (2-way RM ANOVA interaction, p = 0.0055; subsequent Sidak’s post-hoc tests, ***p<0.001, ****p<0.0001 as indicated graphically). In contrast, TBI mice showed a lack of of social memory (n.s. by Sidak's post-hoc) (n = 15/group). Bars represent mean + sem.</p

Cognitive deficits detected in the Morris water maze (MWM) at adulthood after pediatric TBI, are unaffected by gelatinase inhibition.

<p>(A) During the visible sessions, quantification of latency to reach the platform revealed an impairment in task learning by TBI mice compared to sham controls (multivariate ANOVA overall effect of TBI, **p<0.01). (B) During hidden platform sessions, injured mice also showed a greater latency to reach the platform as compared to sham controls (overall effect of TBI, ***p<0.001), indicating an impairment in spatial memory. Cumulative distance to the target was also quantified as an alternative outcome measure (C-D), which similarly detected impairments in task performance and spatial memory in TBI mice compared to sham controls (overall effect of TBI, **p<0.01). (E) Probe trial performance was quantified as cumulative distance to the target. Injured mice traveled a greater distance to reach the target quadrant compared to sham controls (RM ANOVA, overall effect of TBI, **p<0.01) (n = 15/group). Bars represent mean + sem and values represent mean ± sem.</p

Gelatinase inhibition with <i>p</i>-OH SB-3CT does not attenuate extensive injury-induced loss of cortical and hippocampal structures.

<p>Volumetric estimates spanning Bregma 1.5 to -3.8mm in the cortex (Ctx; A), hippocampus (Hpc; B) and dentate gyrus (DG; C) revealed injury-induced reductions (unpaired t-tests *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 as indicated graphically; n = 11–15/group). (D) Unbiased cell counts performed in the ipsilateral DG found similar numbers of surviving neurons in the upper and lower blades of injured mice independent of drug treatment (n = 8–9/group). Bars represent mean + sem.</p

Concentrations of <i>p</i>-OH SB-3CT after multiple-dose s.c. administration.

<p>^<i>a</i> Concentrations in pmol/mg tissue</p><p>^<i>b</i> Concentrations in μM</p><p>^<i>c</i> NQ = not quantifiable</p><p>AUC = area under the curve</p><p>^<i>d</i><i>AUC</i> in pmol·min/mg for brain and in μM·min for plasma</p><p>Concentrations of <i>p</i>-OH SB-3CT after multiple-dose s.c. administration.</p

Enhanced gelatinases detected at 48 h after TBI.

<p>(A) Gelatin zymography from representative brain lysates indicates increased expression of MMP-9 and MMP-2 at 48 h post-injury as compared to sham controls. The pro-forms of MMP-9 and MMP-2 were detected at ~105 and 72 kDa, respectively. Purified human MMP-2 and MMP-9 were used as standards. (B) Quantification of band intensity revealed a robust increase in the pro-enzyme forms of MMP-2 and (C) MMP-9, as well as increases in the active enzyme forms after TBI compared to sham (n = 6 per group; unpaired t-tests, *p<0.05, **p<0.01 and ***p<0.001, TBI compared to sham). Bars represent mean + sem.</p