Amelioration of Cognitive and Behavioral Deficits after Traumatic Brain Injury in Coagulation Factor XII Deficient Mice

Figures

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  Figure 1. Flow diagram of experimental design. TBI–traumatic brain injury, NSS neuroscore, NOR novel object recognition test.

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  Figure 2. Functional outcome was measured using the Neurological Severity Score (NSS) during the first 7 days following TBI in sham, wildtype (WT) and FXII−/− mice. Neurological deficits at 1 h after TBI and on days 1 and 3 after trauma were similar in FXII−/− and WT mice whereas FXII−/− mice had a significantly better outcome than wild type mice (WT) on day 7 after weight-drop TBI. Number of animals n = 25 in sham, n = 23 in WT and n = 16 in FXII−/− in panel (A), in panel (B) (females) n = 13 in sham, n = 9 in WT and n = 7 in FXII−/−, and in (C) (males) n = 12 in sham, n = 14 in WT and n = 9 in FXII−/−. Box plots represent median ± 25th and 75th percentiles, whiskers minimum and maximum values, dots indicate individual data points with each boxplot, *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant 2-way ANOVA with Bonferroni test.

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  Figure 3. Glycoprotein Ib (GPIb)-positive platelet aggregates in CD31-positive brain microvasculature 3 months after TBI. (A). Quantitative analysis of the GPIb immunofluorescence reveals significantly more intravascular platelet depositions in brain sections of wild type mice (n = 3) as compared to sham-operated (n = 4) or FXII−/− mice (n = 5). Box plots represent median ± 25th and 75th per-centiles, whiskers minimum and maximum values, dots indicate individual data points with each boxplot * p < 0.05, ns = not significant one-way ANOVA with Bonferroni test. In panels (B) to (D) images of the frontoparietal cortex showing yellow-green GPIb-positive platelet aggregates (white arrows) in CD31-positive brain microvessels (red) in (B) sham, (C) wild type (WT) and (D) FXII−/− mouse; scale bar = 50 µm.

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  Figure 4. Cognitive recovery in novel object recognition test in sham (n = 22), wild type (WT, n = 22) and FXII−/− mice (n = 15). (A) Setup for the novel object test on days d1/d2 and d3. (B) Black wooden objects of 6 × 6 × 6 cm size were used in the test trials. The novel object used on day 3 is marked with a red arrow. (C–E) Retention memory for novel object 6 weeks after TBI was significantly reduced in wild type mice as compared to sham-operated or FXII−/− mice. Number of animals n = 22 in sham, n = 22 in WT and n = 15 in FXII−/− in panel (C), n = 10 in sham, n = 9 in WT and n = 5 in FXII−/− in panel (D) (females), and n = 12 in sham, n = 13 in WT and n = 10 in FXII−/− in panel (E) (males), *** p < 0.001,** p < 0.01, * p < 0.05, ns = not significant one-way ANOVA with Bonferroni test.

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  Figure 5. Behavioral phenotyping after TBI in sham, wildtype (WT) and FXII−/− mice. Investigative activity reflected by visits (B–D) and nosepokes (E–G) in the IntelliCage corners during the first day of testing is significantly lower in wild type mice than in sham-operated or FXII−/− mice 3 weeks after TBI. All mice made more visits and nosepokes during dark night period (7 pm to 7 am) than during daytime (7 am to 7 pm) when light was on. Number of animals n = 17 in sham, n = 14 in WT and n = 13 in FXII−/− in panels (A–D), in panels (E) and (G) (females) n = 11 in sham, n = 5 in WT and n = 6 in FXII−/−, and in (F) and (H) (males) n = 6 in sham, n = 9 in WT and n = 7 in FXII−/−. Box plots represent median ± 25th and 75th per-centiles, whiskers minimum and maximum values, dots indicate individual data points with each boxplot, *** p < 0.001, ** p < 0.01, * p < 0.05, ns not significant one-way ANOVA with Bonferroni test).

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  Figure 6. Behavioral phenotyping after TBI in sham, wild type (WT) and FXII−/− mice. (A) Setup for appetitive reward and extinction in IntelliCages. During the trial water or 1% (w/v) sucrose water was available at designated corners from 10 p.m. to 12 p.m. Blue droplets depict corners with access to water, orange droplets access to 1% (w/v) sucrose water, and red crosses closed corners with no drinking access. (B) Percent of nosepokes in the rewarded corner during the reward/extinction testing. (C–E) Negative emotional response to appetitive reward extinction 4 weeks after TBI: Wild type mice in contrast to FXII−/− or sham-treated mice avoid using the previously rewarded corner when the drinking supply is shifted from 1% sucrose to water on day 5 of testing. ** p < 0.01, * p < 0.05, ns no significant difference, one-way ANOVA with Bonferroni test, Number of animals n = 17 in sham, n = 14 in WT and n = 13 in FXII−/− in panel (C), n = 11 in sham, n = 5 in WT and n = 6 in FXII−/− in panel (D) (females) and n = 6 in sham, n = 9 in WT and n = 7 in FXII−/− in panel (E) (males). Line graphs represent mean ± SD, box plots median ± 25th and 75th percentiles, whiskers minimum and maximum values,…

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  Figure 7. Plasma corticosterone level 3 months after TBI in sham, wild type (WT) and FXII−/− mice. Peripheral corticosterone levels are decreased in wild type mice, but not in FXII−/− mice when compared to sham-operated mice. Number of animals n = 25 in sham, n = 23 in WT and n = 16 in FXII−/− in (A), n = 13 in sham, n = 9 in WT and n = 7 in FXII−/− in (B) (females) and n = 12 in sham, n = 14 in WT and n = 9 in FXII−/− in (C) (males). Box plots represent median ± 25th and 75th percentiles, whiskers minimum and maximum values, dots indicate individual data points with each boxplot, ** p < 0.01, * p < 0.05, ns = not significant one-way ANOVA with Bonferroni test.