Traumatic brain injury in young rats leads to progressive behavioral deficits coincident with altered tissue properties in adulthood

Abstract

Traumatic brain injury (TBI) affects many infants and children, and results in enduring motor and cognitive impairments with accompanying changes in white matter tracts, yet few experimental studies in rodent juvenile models of TBI (jTBI) have examined the timeline and nature of these deficits, histologically and functionally. We used a single controlled cortical impact (CCI) injury to the parietal cortex of rats at post-natal day (P) 17 to evaluate behavioral alterations, injury volume, and morphological and molecular changes in gray and white matter, with accompanying measures of electrophysiological function. At 60 days post-injury (dpi), we found that jTBI animals displayed behavioral deficits in foot-fault and rotarod tests, along with a left turn bias throughout their early developmental stages and into adulthood. In addition, anxiety-like behaviors on the zero maze emerged in jTBI animals at 60 dpi. The final lesion constituted only ∼3% of brain volume, and morphological tissue changes were evaluated using MRI, as well as immunohistochemistry for neuronal nuclei (NeuN), myelin basic protein (MBP), neurofilament-200 (NF200), and oligodendrocytes (CNPase). White matter morphological changes were associated with a global increase in MBP immunostaining and reduced compound action potential amplitudes at 60 dpi. These results suggest that brain injury early in life can induce long-term white matter dysfunction, occurring in parallel with the delayed development and persistence of behavioral deficits, thus modeling clinical and longitudinal TBI observations.

FIG. 1.

(A) Sensorimotor functions were tested with the foot-fault test. There was a significant increase in the number of faults in controlled cortical impact (CCI) rats compared to their sham counterparts at 7 and 60 dpi (*p<0.05, **p<0.01). (B) Balance and coordination skills were measured with the beam balance test. The distance covered by CCI animals was significantly decreased compared to sham animals at 1, 3, 7, and 60 dpi. (C) Balance and coordination skills were tested with the rotarod test. Fall latency was significantly lower in CCI animals at 30 dpi (*p<0.05). (D) Turn bias measurements indicated that the percentage of left turns (contralateral to the injury) at 30 and 60 dpi was significantly increased in CCI compared to sham animals (*p<0.05; SEM, standard error of the mean).

FIG. 2.

(A) During open-field testing, the overall distance traveled over 30 min (10 blocks of 3 min each) revealed a significant decrease in activity in the controlled cortical impact (CCI) group compared to sham animals at 30 dpi (*p<0.05). (B) The first 3-min block of activity was evaluated in sham and CCI animals, as this time would normally be used to explore the new environment into which they have been placed. At 60 dpi (but not at 30 dpi) there was a significant difference in the distance traveled in CCI compared to sham animals (*p<0.05), when the CCI animals showed decreased exploratory activity. (C) The zero maze was used to assess anxiety-like behaviors, and in this test more time spent in the dark is thought to correlate with increased anxiety. We observed a significant increase in the time spent in the dark at 60 dpi (*p<0.05). (D) Cumulative distance traveled by the rats to find the platform at 30 and 60 dpi was no different between the CCI and sham groups in the Morris water maze spatial test.

FIG. 3.

(A) Magnetic resonance imaging (MRI) at 3, 30, and 60 dpi identifies the lesion (arrows) as an increase in the signal intensity at 3 and 30 dpi, and as a hole at 60 dpi, corresponding to the formation of the lesion cavity. (B) A three-dimensional reconstruction of the brain (gray) and lesion (red) at 3 dpi extends to include ∼3% of the total brain volume. (C) The lesion volumes were significantly larger in controlled cortical impact (CCI) rats compared to their sham counterparts receiving craniotomy only (**p<0.001) at all time points during our 60-day observation period. Color image is available online at www.liebertonline.com/neu

FIG. 4.

(A–D) Neuronal nuclei (NeuN) immunohistochemistry at 60 dpi in the sham (A and B) and controlled cortical impact (CCI; C and D) groups showed the lesion cavity in the parietal cortex (arrow in C). The number of NeuN-positive neurons (green) was decreased in CCI (D) compared to sham (B) animals in the parietal cortex (Par Cx) at close proximity to the injury site, in tissue adjacent to the cavity. Nuclei of all cell types were stained with DAPI (blue in B and D), and automated counting of the NeuN-positive cells in the parietal cortex (E) showed a significant reduction in CCI compared to sham animals in the ipsilateral and contralateral hemispheres (*p<0.05; scale bars in A and C=200 μm; in B and D=50 μm; DAPI, 4,6-diamino-2-phenylindole). Color image is available online at www.liebertonline.com/neu

FIG. 5.

(A) Slices from magnetic resonance imaging (MRI) and neurofilament-200 (NF200) immunostaining show a decreased corpus callosum (CC) size on MRI (green and yellow outlines), and NF200 immunostaining (arrows). (B) Quantification of the CC area from the MRI at 60 dpi (mm²) showed a significant decrease in CCI animals (*p<0.05). (C) Quantification of the CC area from the NF200 infrared immunostaining (% of sham animals) shows a significant decrease in the CCI group at 7, 30, and 60 dpi (*p<0.05). (D–G) Classical immunostaining with NF200 images at higher magnification in the corpus callosum (CC, red dashed outlines), and overlying cortex (Cx), showed similar morphological staining patterns, and no changes between the sham (D and E) and CCI (F and G) groups. (H) Infrared (IR) quantification of NF200 staining intensity in the CC was unchanged between groups at 60 dpi (scale bar in A=1 mm; in D and F=500 μm; in E and G=50 μm; A.U., arbitrary units). Color image is available online at www.liebertonline.com/neu

FIG. 6.

Myelin basic protein (MBP) immunostaining in the sham (A, B, and G) and controlled cortical impact (CCI; A, C, F, and G) animals from bregma +1.7 mm to −6.0 mm. (A) MBP immunoreactivity using infrared antibodies with mouse anti-MBP (ms-MBP) was increased in the CCI animals compared to sham animals at the anteroposterior bregma level in white matter tracts. This global increase of MBP was also seen distant from the lesion cavity (asterisk in A). (B and C) Higher-magnification images of classical MBP immunostaining show higher intensity in the corpus callosum (CC) compared to the striatum (STR) and cortex (CX), in (B) sham and (C) CCI animals, and increased MBP staining in the CC white matter of CCI animals (insets in B and C). (D) Quantification of infrared MBP staining showed a significant increase in the white matter tracts (CC and anterior commissure) of CCI animals compared to sham animals at 3 and 60 dpi (*p<0.05). (E) CC area measurements of MBP immunostaining showed a decrease in CCI animals compared to sham animals (p<0.05). (F) Double-immunolabeling with infrared MBP antibodies recognizing two different epitopes, mouse anti-MBP (ms-MBP, green) and rabbit anti-MBP (rb-MBP, red), showed co-localization in the merged images. (G) The intensity of the rb-MBP was increased in the CCI compared to the sham group, as indicated by ms-MBP (asterisks in A, F, and G indicate the lesion cavity; scale bars in A, F, and G=1 mm; in B and C=200 μm; in insets in B and C=50 μm). Color image is available online at www.liebertonline.com/neu

FIG. 7.

(A) CNPase immunolabeling along white matter tracts is shown in representative coronal sections near the lesion (bregma −1.8 mm), and anterior to the lesion cavity (bregma +0.5 mm), with bright levels of CNPase staining with infrared (IR) antibodies localized to white matter tracts in the corpus callosum (CC; groups of red arrows). (B–E) Classical CNPase staining at higher magnification shows similar staining patterns in the CC (red dashed outlines), and on individual oligodendrocyte cell bodies (arrows), and processes (arrowheads) of (B and C) sham, and (D and E) controlled cortical impact (CCI) animals. (F) Quantification of infrared staining in the CC across several coronal slices shows no statistically significant differences between the sham and CCI groups for CNPase staining levels (CX, cortex; asterisk in A indicates lesion cavity; scale bar in A=1 mm; in B and D=200 μm; in insets in C and E=20 μm; A.U, arbitrary units). Color image is available online at www.liebertonline.com/neu

FIG. 8.

Electrophysiology in the corpus callosum (CC) at 60 dpi shows N1 amplitudes recorded after compound action potentials (CAP) were evoked. The stimulating electrode (S) was placed on the side of injury (or sham surgery), located approximately 1 mm from the recording electrode (R). Quantification shows that N1 amplitudes were significantly decreased in controlled cortical impact animals compared to sham animals (*p<0.005).