Clinical correlates in an experimental model of repetitive mild brain injury

Abstract

Objective: Although there is growing awareness of the long-term cognitive effects of repetitive mild traumatic brain injury (rmTBI; eg, sports concussions), whether repeated concussions cause long-term cognitive deficits remains controversial. Moreover, whether cognitive deficits depend on increased amyloid β deposition and tau phosphorylation or are worsened by the apolipoprotein E4 allele remains unknown. Here, we use an experimental model of rmTBI to address these clinical controversies.

Methods: A weight drop rmTBI model was used that results in cognitive deficits without loss of consciousness, seizures, or gross or microscopic evidence of brain damage. Cognitive function was assessed using a Morris water maze (MWM) paradigm. Immunostaining and enzyme-linked immunosorbent assay (ELISA) were used to assess amyloid β deposition and tau hyperphosphorylation. Brain volume and white matter integrity were assessed by magnetic resonance imaging (MRI).

Results: Mice subjected to rmTBI daily or weekly but not biweekly or monthly had persistent cognitive deficits as long as 1 year after injuries. Long-term cognitive deficits were associated with increased astrocytosis but not tau phosphorylation or amyloid β (by ELISA); plaques or tangles (by immunohistochemistry); or brain volume loss or changes in white matter integrity (by MRI). APOE4 was not associated with worse MWM performance after rmTBI.

Interpretation: Within the vulnerable time period between injuries, rmTBI produces long-term cognitive deficits independent of increased amyloid β or tau phosphorylation. In this model, cognitive outcome is not influenced by APOE4 status. The data have implications for the long-term mental health of athletes who suffer multiple concussions.

FIGURE 1

Study flow diagram. AB = amyloid β; ELISA = enzyme-linked immunosorbent assay; IHC = immunohistochemistry; MWM = Morris water maze; rmTBI = repetitive mild traumatic brain injury.

FIGURE 2

Morris water maze performance in wild-type (WT) mice that underwent 5 repetitive mild traumatic brain injuries (rmTBIs) in 5 days. (A) Three days after the last injury, injured mice had worse performance on hidden platform testing than sham-injured mice (n = 8/group, p = 0.01), but there was no difference in injured versus sham-injured mice on visible platform testing. (B) Two months after the last injury, injured mice persisted with deficits in hidden platform performance compared to sham-injured mice (n = 8/group, p = 0.01), with no differences in visible platform testing. (C) One year after injury, injured mice demonstrated worse hidden platform performance compared to sham-injured mice (n = 7–8/group, p = 0.02), with no differences in visible platform performance.

FIGURE 3

The effect of time interval between repetitive mild traumatic brain injuries (rmTBIs) on Morris water maze performance 6 months after the last injury. All groups demonstrated time-dependent improvement in hidden platform performance. There were no group differences in visual platform performance. Mice that underwent 5 concussive injuries in 5 days (A) and weekly concussive injuries for 5 weeks (B) had deficits in hidden platform performance when compared to sham-injured mice (n = 5–8/group, p = 0.001 and n = 11–16/group, p = 0.002, respectively). There were no differences in hidden platform performance between injured and sham-injured mice that underwent biweekly injuries for 10 weeks (n = 11–14, p = 0.1; C), 5 monthly injuries (n = 12–16/group, p = 0.2; D), or 1 injury (n = 11–15/group, p = 0.4; E). WT = wild type.

FIGURE 4

Comparisons of group-averaged brain data between sham-injured (top) and repetitive mild traumatic brain injury (rmTBI)-injured (bottom) mice (n = 4/group) using fast spin-echo (FSE) images, fractional anisotropy (FA) computed from diffusion tensor imaging, and magnetization transfer ratio (MTR). Group averages were obtained at a coronal slice 2mm posterior to bregma and an axial level 1.75mm ventral to bregma. WT = wild type.

FIGURE 5

There were no genotype-specific differences in Morris water maze (MWM) performance after repetitive mild traumatic brain injury (rmTBI). All sham-injured and injured groups demonstrated time-dependent improvements in latency to the hidden platform, indicating the ability to learn the MWM paradigm before and after rmTBI (p < 0.05 for time for all groups). Injured mice performed worse than their sham-injured controls (p < 0.05 for all groups, data not shown). (A) MWM performance in APOE4 versus wild-type (WT) mice immediately after last injury in mice that underwent 5 rmTBIs in 5 days. Hidden platform trial performance did not differ between injured APOE4 and injured WT animals (p = 0.9) or between sham-injured APOE4 and sham-injured WT animals (p = 0.3, data not shown). Injured WT animals had worse performance on visual platform testing compared to injured APOE4 mice (p = 0.005). (B) MWM performance immediately after last injury in mice that underwent 7 rmTBIs in 9 days. Hidden platform trial performance did not differ between injured APOE4 and injured WT animals (p = 0.1), but was better in sham-injured APOE4 compared to sham-injured WT animals (p = 0.003, data not shown).

FIGURE 6

APOE4 does not worsen long-term Morris water maze (MWM) performance after repetitive mild traumatic brain injury (rmTBI). There were no baseline differences in MWM performance in sham-injured APOE4 versus wild-type (WT) mice (data not shown). (A) MWM performance 2 months after last injury in mice that underwent 5 rmTBIs in 5 days. Injured WT mice performed worse than injured APOE4 mice (p = 0.003) and WT sham-injured controls (p = 0.001, data not shown). Injured WT animals had worse performance on visible platform testing compared to injured APOE4 animals (p = 0.04). (B) MWM performance 2 months after last injury in mice that underwent 7 rmTBIs in 9 days. There were no differences in hidden platform performance between injured APOE4 mice and injured WT mice (p = 0.8). There were no differences in visual platform performance in injured APOE4 versus injured WT mice (p = 0.06). (C) MWM performance 6 months after last injury in mice that underwent daily 7 concussions in 9 days. There were no differences between injured APOE4 animals and injured WT animals (p = 0.2) on hidden platform performance. On visual platform testing, there were no differences in performance in injured APOE4 versus injured WT mice (p = 0.1). (D) Mice that were naive to the MWM when tested 6 months after their final injury showed no differences in hidden platform performance between WT and APOE4 mice (p = 0.1).

FIGURE 7

Enzyme-linked immunosorbent assay testing for soluble amyloid β (Aβ) 40, total tau, and phosphorylated tau in cortical and hippocampal samples 6 months after final injury. (A) There was no difference in cortical soluble Aβ40 in injured APOE4 versus injured wild-type (WT) animals (16 ± 3.0pmol/g vs 12 ± 3.0pmol/g, p = 0.4), injured APOE4 versus sham-injured APOE4 mice (16 ± 3.0pmol/g vs 10 ± 0.1pmol/g, p = 0.4), or injured WT versus sham-injured WT animals (12 ± 3.0pmol/g vs 8.0 ± 0.9pmol/g, p = 0.3). (B) There was no difference in hippocampal soluble Aβ40 in injured APOE4 versus injured WT animals (1.3 ± 0.1pmol/g vs 1.9 ± 0.3pmol/g, p = 0.1), injured APOE4 versus sham-injured APOE4 mice (1.3 ± 0.1pmol/g vs 1.2 ± 0.6pmol/g, p = 0.8), or injured WT versus sham-injured WT animals (12 ± 3.0pmol/g vs 8.0 ± 0.9pmol/g, p = 0.3). (C) Total tau was not elevated in injured APOE4 versus sham-injured APOE4 mice (92,245 ± 5,468pg/mg vs 60,911 ± 15,606pg/mg, p = 0.05), injured APOE4 versus injured WT animals (92,445 ± 5,468pg/mg vs 93,042 ± 5,587pg/mg, p = 0.9), or injured WT versus sham-injured WT mice (93,402 ± 5,587pg/mg vs 102,904 ± 4,056pg/mg, p = 0.2). (D) No differences were seen in cortical soluble phosphorylated tau injured APOE4 versus sham-injured APOE4 mice (33,019 ± 2,267pg/mg vs 28,785 ± 4,398pg/mg, p = 0.4), injured APOE4 versus injured WT animals (33,019 ± 2,267pg/mg vs 33,767 ± 1,760pg/mg, p = 0.8), or injured WT versus sham-injured WT mice (33,767 ± 1,760pg/mg vs 28,785 ± 4,398pg/mg, p = 0.2). (E) Total tau was not elevated in hippocampal samples from injured APOE4 versus sham-injured APOE4 mice (96,792 ± 8,683pg/mg vs 97,125 ± 2,510pg/mg, p = 1), injured APOE4 versus injured WT animals (96,792 ± 8,683pg/mg vs 120,074 ± 11,200pg/mg, p = 0.4), or injured WT versus sham-injured WT mice (120,074 ± 11,200pg/mg vs 128,117 ± 8,031pg/mg, p = 0.6). (F) No differences were seen in hippocampal soluble phosphorylated tau in injured APOE4 versus sham-injured APOE4 mice (37,784 ± 3,543pg/mg vs 35,032 ± 1426pg/mg, p = 0.7), injured APOE4 versus injured WT animals (37,784 ± 3,543pg/mg vs 39,189 ± 3,086pg/mg, p = 0.5), or injured WT versus sham-injured WT mice (39,189 ± 3,086pg/mg vs 36,903 ± 2,190pg/mg, p = 0.6).

FIGURE 8

No focal amyloid β (Aβ) deposition is seen 6 months after repetitive mild traumatic brain injury. Representative images show no focal deposition of Aβ in injured wild-type (A) or APOE4 (B) mice compared to an Alzheimer disease transgenic naive positive control (C). [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]

FIGURE 9

Increased astrocytosis after repetitive mild traumatic brain injury (rmTBI; 5 rmTBIs in 5 days) versus sham-injured mice. Representative photomicrographs show immunoreactivity of glial fibrillary acidic protein (GFAP; red) and Hoechst staining (blue) in injured versus sham-injured mice. Increased GFAP immunoreactivity is demonstrated in hippocampus of injured (A) versus sham-injured mice (B). No qualitative differences in neuronal density by Hoechst staining in hippocampus (dentate gyrus is shown) was observed between injured (C) and sham-injured (D) mice. (E) Quantitative analysis of GFAP-positive cells counted in cortex (CX), corpus callosum (CC), and hippocampus (HIP) of injured (5 rmTBIs in 5 days, n = 5) and sham-injured mice (n = 5) 6 months after injury (mean ± standard deviation); *p = 0.009 for CX, *p = 0.02 for CC, and *p = 0.04 for HIP. HPF = high-power magnification field. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]