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 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

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

Moderate CCI induced hyperactivity and spatial learning deficits.

<p><b>(A)</b> Moderate CCI mice exhibited hyperactivity in the open field, traveling a significantly greater distance at 7 dpi compared to sham (^≠≠≠<i>p</i><0.001), rmCHI (^øøø<i>p</i><0.001), and mCHI (^∂∂<i>p</i><0.01). CCI-induced hyperactivity was not observed at 90+ dpi. <b>(B)</b> At 90+ dpi, CCI mice exhibited severe deficits in the spatial water maze task, with no evidence of learning the escape platform’s location on day 1. These learning deficits persisted when the platform’s location was changed on day 2. (^≠≠<i>p</i><0.01 for CCI compared to sham, ^≠≠≠<i>p</i><0.001 for CCI compared to sham, ^ø<i>p</i><0.05 for CCI compared to rmCHI, ^øø<i>p</i><0.01 for CCI compared to rmCHI, ^∂∂<i>p</i><0.01 for CCI compared to mCHI, ^Ω<i>p</i> < .05 for mCHI compared to sham).</p

Turn bias was most evident in the mice with CCI.

<p><b>(A)</b> At 1 dpi, CCI mice made significantly fewer left turns (contralateral to the injury) on the balance beam than sham (^≠<i>p</i><0.05) and mCHI (^∂∂<i>p</i> < .01) mice. <b>(B)</b> Both rmCHI and CCI mice exhibited more turns to the right throughout balance beam testing than shams, though these results did not reach statistical significance. <b>(C)</b> CCI mice made more left-sided slips on the balance beam than other animals. These deficits were most prominent at 7 dpi compared to shams and other injury groups (^aaa<i>p</i><0.001) and persisted up to 90+ dpi (^≠≠<i>p</i><0.01 vs. sham, ^≠≠≠<i>p</i><0.001 vs. sham, ^ø<i>p</i><0.05 vs. rmCHI, ^øø<i>p</i><0.01 vs. rmCHI, ^∂<i>p</i><0.05 vs. mCHI, ^∂∂<i>p</i><0.01 vs. mCHI, ^∂∂∂<i>p</i><0.001 vs. mCHI). <b>(D)</b> In the water maze, rmCHI and CCI mice tended to swim to the left (contralateral to the injury) compared to sham and mCHI animals (*<i>p</i><0.05, **<i>p</i><0.01; mean +/- 95% CI). 95% confidence intervals show that the left turn biases in rmCHI and CCI mice are statistically significant (<i>p</i><0.05), whereas no significant turn bias is observed in the sham and mCHI mice. (The clock graphic above shows variance markings of the relative angular velocity of each injury group with each mark on the clock representing a separation of 6° in relation to 12 o’clock. Flag symbol marks zero degrees).</p

Motor deficits are evident in the CCI, but not mCHI or rmCHI mice.

<p><b>(A)</b> At 1 dpi, CCI mice spent significantly less active time on the beam than sham (^≠≠<i>p</i><0.01), rmCHI (^ø<i>p</i><0.05), and mCHI (^∂<i>p</i><0.05) mice. They also spent significantly less time being active on the beam at 3 dpi than sham (^≠≠<i>p</i><0.01) and mCHI (^∂<i>p</i><0.05). <b>(B)</b> At 3 dpi, the CCI mice fell off the balance beam significantly more often than the other injury groups (^ΩΩ<i>p</i><0.01). <b>(C)</b> rmCHI and mCHI mice exhibited no deficits on the foot fault task, but CCI mice had more foot faults than shams, rmCHI, and mCHI mice at 3 dpi (^≠≠≠<i>p</i><0.001, ^øøø<i>p</i><0.001, and ^∂∂∂<i>p</i><0.001, respectively), 5 dpi (^≠≠≠<i>p</i><0.001, ^øøø<i>p</i><0.001, and ^∂∂∂<i>p</i><0.001), and 7 dpi (^≠≠<i>p</i><0.01, ^øø<i>p</i><0.01, and ^∂∂∂<i>p</i><0.001). CCI mice still had significant deficits at 90+ dpi versus shams (^≠≠<i>p</i><0.01) and mCHI (^∂<i>p</i><0.05) mice. <b>(D)</b> CCI, but not mCHI or rmCHI, mice performed significantly worse on the accelerating rotarod test (5 RPM every 3 sec) at 1 and 3 dpi (^ΩΩΩ<i>p</i><0.001). These deficits were still present at 90+ dpi compared to shams (^≠≠<i>p</i><0.01) and mCHI mice (^∂∂<i>p</i><0.01).</p

Tests of affective behavior at 90 dpi suggest that TBI induces depression and increased passivity towards other mice.

<p><b>(A)</b> Depression-like behaviors were tested using the tail suspension test, in which both mCHI and rmCHI animals gave up (became immobile) more quickly than shams (**<i>p</i><0.01 and *<i>p</i><0.05, respectively), and rmCHI mice gave up more quickly than CCI animals (*<i>p</i><0.05). Interestingly, CCI mice were not different than shams. <b>(B)</b> When aggressive behavior was assessed in a social recognition test, sham, but not injured, mice engaged in more fighting with the stimulus mouse by trial 3 (*<i>p</i><0.05). rmCHI and CCI, but not mCHI, mice were more passive than shams when a new mouse was introduced on the 4^th (novel) trial, although not significantly.</p

MRI assessment of severity and volumetric analysis (A) T2-weighted MRI at the level of the injury site (*) revealed no overt brain damage in sham, mCHI or rmCHI mice. In contrast, mice subjected to a moderate CCI exhibited a visible lesion within the cortex. (B) 3-D reconstruction of the T2-weighted MRI of the brain revealed no overt cortical damage in sham, mCHI or rmCHI mice. Mice subjected to a moderate CCI revealed a visible lesion (red) within the cortex. (C) MRI analysis revealed significant tissue loss in CCI mice compared to sham, mCHI and rmCHI at 90 dpi (<i>p</i> < .01).

<p>MRI assessment of severity and volumetric analysis (A) T2-weighted MRI at the level of the injury site (*) revealed no overt brain damage in sham, mCHI or rmCHI mice. In contrast, mice subjected to a moderate CCI exhibited a visible lesion within the cortex. (B) 3-D reconstruction of the T2-weighted MRI of the brain revealed no overt cortical damage in sham, mCHI or rmCHI mice. Mice subjected to a moderate CCI revealed a visible lesion (red) within the cortex. (C) MRI analysis revealed significant tissue loss in CCI mice compared to sham, mCHI and rmCHI at 90 dpi (<i>p</i> < .01).</p