Traumatic injury to the immature frontal lobe: a new murine model of long-term motor impairment in the absence of psychosocial or cognitive deficits

Abstract

Traumatic brain injury in children commonly involves the frontal lobes and is associated with distinct structural and behavioral changes. Despite the clinical significance of injuries localized to this region during brain development, the mechanisms underlying secondary damage and long-term recovery are poorly understood. Here, we have characterized the first model of unilateral focal traumatic injury to the developing frontal lobe. Male C57Bl/6J mice at postnatal day (p)21, an age approximating a toddler-aged child, received a controlled cortical impact or sham surgery to the left frontal lobe and were euthanized 1 or 7 days later. A necrotic cavity and local inflammatory response were largely confined to the unilateral frontal lobe, dorsal corpus callosum and striatum anterior to the bregma. While cell death and accumulated β-amyloid precursor protein were characteristic features of the pericontusional motor cortex, corpus callosum, cingulum and dorsal striatum, underlying structures including the hippocampus showed no overt pathology. To determine the long-term functional consequences of injury at p21, two additional cohorts were subjected to a battery of behavioral tests in adolescence (p35-45) or adulthood (p70-80). In both cohorts, brain-injured mice showed normal levels of anxiety, sociability, spatial learning and memory. The signature phenotypic features were deficits in motor function and motor learning, coincident with a reduction in ipsilateral cortical brain volumes. Together, these findings demonstrate classic morphological features of a focal traumatic injury, including early cell death and axonal injury, and long-term volumetric loss of cortical volumes. The presence of deficits in sensorimotor function and coordination in the absence of abnormal findings related to anxiety, sociability and memory likely reflects several variables, including the unique location of the injury and the emergence of favorable compensatory mechanisms during subsequent brain development.

Figure 1

Frontal TBI induces a distinct unilateral lesion. Panel (a) depicts representative photographs of whole perfused brains collected at 24 h and 7 d post-injury (scale bar = 3 mm). Panel (b) illustrates representative coronal sections stained with cresyl violet to delineate tissue damage at 24 h and 7 d. Images were captured at ~Bregma +1.70, +0.50, -0.10 and -0.15 mm respectively, from top to bottom.

Figure 2

Local cell death in the dorsal cortex and subcortical structures after frontal TBI. Degenerating neurons were labeled with Fluoro-Jade C at 24 h post-injury (a), revealing abundant neuronal damage in the peri-contusional cortex and dorsal striatum (scale bar = 200 μm). Cell death was confirmed by TUNEL staining (b), which was widespread in the peri-contusional cortex as well as in the underlying corpus callosum and striatum (scale bar = 250 μm). TUNEL-positive cells were summed across 5 sections per brain at 24 h post-injury (c), highlighting the regional localization of acute cell death after frontal TBI (one-way ANOVA, F_3,28=11.07, p<0.0001; n=8).

Figure 3

Immuno-reactivity for astrocytes (GFAP) and reactive microglia/macrophages (Iba-1) is evident in the ipsilateral dorsal cortex (c, e, g, k, m, o) and corpus callosum (d, f, h, j, l, n, p) after frontal TBI. Sham operation did not induce gliosis at 24 h (1^st row). At 24 h post-injury (2^nd row), only few reactive astrocytes are noted, although Iba-1 reactivity was increased above sham levels. By 7 d (3^rd row), both GFAP+ astrocytes and Iba-1+ reactive microglia were visibly accumulating in the peri-contusional parenchyma. By 3 months post-injury, GFAP+ astrocytes formed a glial scar at the cortical lesion edge. The lesion core (o) also stains strongly for Iba-1 at these times. Scale bar =100 μm. Arrows indicate a glial scar; asterisks highlight the enlarged lateral ventricle.

Figure 4

Axonal degeneration was detected by immuno-staining for β-APP accumulation after frontal TBI. Immuno-positive cells were abundant in the cortex at 24 h post-injury (a, b), particularly in the peri-contusional cortex (arrows). Filamentous staining was evident in the corpus callosum ipsilateral to the impact site at 24 h post-injury (a, c), indicating swollen or damaged axons in which β-APP was accumulating. This was most apparent directly ventral to the lesion site, however, immuno-reactive axons were also evident crossing the midline into the contralateral corpus callosum (a). At 7 d (d, e), much of the β-APP staining was granular or globular in appearance in the injured cortex and corpus callosum. Asterisks indicate ipsilateral ventricle. Scale bar =100 μm.

Figure 5

Frontal TBI results in cavity formation and a volumetric reduction in the injured cortex. At 1 and 3 months post-injury, a pronounced unilateral cortical cavity was evident in whole perfused brains (arrows in panel a, scale bar = 3 mm) and representative dorsal cortex sections stained with cresyl violet (b, scale bar = 500 μm). Images were captured at ~Bregma +1.70, +0.50, -0.10 and -0.15 mm respectively, from top to bottom for each time point shown. Note the enlarged lateral ventricle ipsilateral to the injury site at 1 and 3 months (asterisks). Remaining dorsal cortex was quantified at both 1 and 3 months post-injury (c), demonstrating significant volumetric loss in the ipsilateral dorsal cortex at both time points (2-way ANOVA post-hoc, p<0.0001; n=10/group).

Figure 6

Motor learning and rearing deficits were evident after frontal TBI. Mice were tested on the rotarod across three consecutive days (a). At adolescence, TBI mice showed a significant reduction in the mean latency to fall at days 2 and 3 (2-way RM ANOVA, post-hoc *p<0.05, ***p<0.001) compared to sham-operated mice. Although less pronounced, this injury-dependent impairment was also evident at adulthood (post-hoc, *p<0.05). Rearing behavior measured in the open field task was also used to detect forelimb motor function (b). While adolescent sham and TBI mice did not differ, adult TBI mice showed a significant deficit in rearing behavior compared to their respective shams, with a reduction in the time spent rearing (t_1,17=3.560, p=0.0024; n=10/group).

Figure 7

Mice showed normal social function after frontal TBI. The total investigative time in the partition task (a) was similar in sham and TBI mice in both cohorts. Several investigative behaviors in the resident-intruder test were quantified separately (b), including the time spent sniffing the head/torso or ano/genital regions of the stimulus mouse, and time engaged in grooming, following or circling behaviors. Sham and TBI mice showed equivalent investigative behaviors in all measures. Performance in the tube-dominance task, quantified as the chance of a ‘win’ by the test (sham or TBI) mouse (c), was also unaffected by injury. The three-chamber task (d) was used to delineate a preference for sociability (stage 2: choice between stimulus 1 and empty chambers) and social novelty (stage 3: choice between stimulus 1 and 2). At both adolescence and adulthood, sham and TBI mice showed a similar preference for sociability and social novelty in these tasks (n=10/group).

Figure 8

Spatial learning and memory were assessed in the Morris water maze after frontal TBI. The swimming speed of TBI mice at both adolescence and adulthood was reduced compared to sham animals (a; 2-way RM ANOVA, effect of injury). Despite this, brain-injured mice at adolescence were able to locate both the visible and hidden platforms similarly to sham-operated mice (b), with all mice showing learning over time. At adulthood, TBI mice showed a reduction in distance to reach the visible platform during sessions 2 and 3 compared to sham mice (2-way RM ANOVA, post-hoc *p<0.05). However, sham and TBI mice showed equivalent performance during the hidden platform sessions. Probe trials, in which the target platform is removed, were quantified as time spent in the target (black bar), opposite (white bar) or adjacent quadrants (striped bar) were compared within each group (1-way ANOVA). Probe trials 1 and 2 are shown in Suppl. Fig. 2. In probe trial 3 (c), all groups independent of injury or age showed memory retention by a preference for the target quadrant. Probe trial 4 was performed one week following probe trial 3 (d). Regardless of injury, adolescent mice do not show a preference for the target quadrant, indicating a lack of long-term spatial memory. In the adult cohort, both sham and TBI mice show long-term memory retention, with a strong preference for the target quadrant (post-hoc; *p<0.05, **p<0.01, ***p<0.0001; n=10/group).