data_sheet_1_Mild Traumatic Brain Injury in Adolescent Mice Alters Skull Bone Properties to Influence a Subsequent Brain Impact at Adulthood: A Pilot Study.docx

<p>Mild traumatic brain injuries (mTBI) are common during adolescence, and limited clinical evidence suggests that a younger age at first exposure to a mTBI may lead to worse long-term outcomes. In this study, we hypothesized that a mTBI during adolescence would predispose toward poorer neurobehavioral and neuropathological outcomes after a subsequent injury at adulthood. Mice received a mild weight drop injury (mTBI) at adolescence (postnatal day 35; P35) and/or at adulthood (P70). Mice were randomized to 6 groups: ‘sham’ (sham-surgery at P35 only); ‘P35’ (mTBI at P35 only); ‘P35 + sham’ (mTBI at P35 + sham at P70); ‘sham + P70’ (sham at P35 + mTBI at P70); ‘sham + sham’ (sham at both P35 and P70); or ‘P35 + P70’ (mTBI at both P35 and P70). Acute apnea and an extended righting reflex time confirmed a mTBI injury at P35 and/or P70. Cognitive, psychosocial, and sensorimotor function was assessed over 1-week post-injury. Injured groups performed similarly to sham controls across all tasks. Immunofluorescence staining at 1 week detected an increase in glial activation markers in Sham + P70 brains only. Strikingly, 63% of Sham + P70 mice exhibited a skull fracture at impact, compared to 13% of P35 + P70 mice. Micro computed tomography of parietal skull bones found that a mTBI at P35 resulted in increased bone volume and strength, which may account for the difference in fracture incidence. In summary, a single mTBI to the adolescent mouse brain did not exacerbate the cerebral effects of a subsequent mTBI in adulthood. However, the head impact at P35 induced significant changes in skull bone structure and integrity. These novel findings support future investigation into the consequences of mTBI on skull bone.</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

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

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

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

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

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

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

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