Repetitive Concussive and Subconcussive Injury in a Human Tau Mouse Model Results in Chronic Cognitive Dysfunction and Disruption of White Matter Tracts,But Not Tau Pathology

Animals

Fifty hTau mice (C57BL/6 Cg-Mapt<tm1(EGFP)Klt>(MAPT), Cat #00549, Jackson Laboratory, Bar Harbor, ME) aged 4–8 weeks were purchased as two cohorts of 25 animals each. All mice were allowed to acclimate to 12-h light-dark cycles. Food and water were supplied ad libitum.

CHIMERA injury and anesthesia

Injuries were performed using the CHIMERA injury method, which involves a midline closed-head impact followed by rotational acceleration. Each animal was anesthetized with 5% isoflurane for 2.5 min followed by maintenance at 2.5% isoflurane during positioning on the platform. Total isoflurane exposure did not exceed 5 min and delivery was stopped immediately before impact. Sham animals underwent the same anesthesia protocol and head positioning on the CHIMERA device but no impact by the piston.

Determination of injury thresholds

Because animals were exposed to 20 daily impacts, the energy levels for subconcussive and concussive impacts were determined empirically (Supplementary Fig. 1A; see online supplementary material at http://www.liebertpub.com). Latency to right reflex (LRR), defined as the time from impact to regaining consciousness and becoming upright, was used as the acute metric of injury. In an initial study, wild-type mice (C57Bl6/J, Jackson Laboratory) were randomly assigned to receive a single impact with piston pressure set to range between 0 and 2.4 psi. Injuries were performed using the same anesthesia and head placement protocol described above, and righting time was recorded and used to determine the subconcussive threshold. Mice injured with piston pressures greater than 1.9 psi (0.13J energy intensity) all had mean righting times greater than the sham average.

Previous work using the CHIMERA device has determined that a single impact below 0.5J does not result in phenotypic deficits, including any increases in LRR time. ²⁸ However, our pilot study with a single impact showed that animals receiving impacts between energy intensities of 0.13J and 0.24J (3.1 psi piston pressure) had increased LRR times (Supplementary Fig. 1A). Further, because we were interested in the cumulative effect of repeated injuries rather than a single impact, a second study was performed using wild-type mice. These mice were randomly assigned to receive 20 × sham, 20 × 0.13J or 20 × 0.24J injuries spaced 24 ± 1 h apart. Righting times were recorded daily. The average righting time over the course of the 20 injuries was then calculated for each group and analyzed using a one-way analysis of variance (ANOVA) followed by a post hoc Tukey's test (Supplementary Fig. 1B). There was a significant effect of injury in this study [F(2,31) = 28.99, η² _p = 0.652, p < 0.00001]. Animals in the 0.24J group had increased righting times relative to shams and 20 × 0.13J mice (20 × sham vs. 20 × 0.24J, p = 0.000126, 20 × 0.13J vs. 0.24J, p = 0.000125). Based on these data, we determined a subconcussive energy threshold of 0.13J and a concussive energy threshold of 0.24J.

Injury timeline

At 4 months of age, hTau mice were assigned to 20 × concussive injury, 20 × subconcussive injury, or 20 × sham groups using a random number generator. Following impact or sham, each animal was placed supine in a warmed chamber (37°C) and monitored by an investigator blinded to injury status in a quiet, separate room until fully upright and ambulatory. LRR was recorded by this investigator. For sham animals, LRR was defined as the time from when isoflurane delivery was stopped to regaining consciousness. After regaining consciousness, each animal was returned to its home cage. This procedure was repeated for 20 consecutive days with injuries spaced 24 ± 1 h apart, such that each animal received 20 head impacts or sham procedures (Fig. 1A). A Cox regression for survival analysis was performed to determine the effect of injury on survival post-injury (Fig. 1B), and showed that there was a significant effect of injury status on survival (p = 0.0304), with a trend towards more deaths in the 20 × concussive injury group.

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FIG. 1.

Injury timeline and survival post-injury. (A) Fifty male hTau mice were purchased in two separate cohorts. Animals were randomly assigned to sham, 20 × subconcussive (0.13J) or 20 × concussive (0.24J) injury groups. Following 20 days of injuries, behavioral outcomes including social interaction (SI), elevated plus maze (EPM), open field maze (OFM), Morris water maze (MWM) and tail suspension (TS) were assessed acutely, at 3 months, and 1 year post-injury. Following the 12-month behavioral test session, all animals were perfused. (B) Survival and mortality risk were assessed using a Cox survival and hazard regression analysis. The data showed a modest but significant (p = 0.03) effect of subconcussive or concussive injury on mortality rate. Color image is available online.

Behavioral testing

All neurobehavioral testing was performed in a specified behavior room isolated from external noise and with light and sound controls. We controlled for 60 dB white noise (Marpac Dohm-DS, Wilmington, NC) and lighting (40 lux for Morris water maze, 20 lux for all other tests) for all testing. Neurobehavioral tests were performed during the work day (6 am-6 pm). All equipment was cleaned with 70% ethanol prior to each test to remove scents. Animals were allowed to acclimate in the room for at least 1 h before testing. Following injuries, cages were renumbered such that the investigator remained blinded to injury status during the following behavioral testing. Behavioral testing was performed at three time-points: acutely, 3 months and one year following injuries. Acute behavioral testing began 22 days after the first injury (Fig. 1A). Social interaction testing was performed on Days 22–24, to provide sufficient testing time for each animal (25 min per animal with 5 min between animals). Acute elevated plus maze testing was performed 25 days after the first injury and acute open field testing was performed 28 days after the first injury. Acute Morris water maze testing began with the first day of the visible platform phase 29 days after the first injury and ending with the probe trial test 38 days after the first injury. Finally, acute tail suspension was performed 40 days after the first injury. Animals were allowed one day of rest between each test to minimize effects of stress induced by each behavioral assay. With the exception of tail suspension, all tests were recorded on video and subsequently analyzed using Panlab SMART (Harvard Bioscience, Holliston, MA).

CSF, blood, and tissue collection

Following the 1-year time-point of behavioral testing, mice were sacrificed. Animals were randomly assigned a new identification number to assure blinding during histological analysis. Mice were anesthetized with a 75 mg/kg dose of pentobarbital administered IP. When mice remained unresponsive to tail or toe pinch, they were placed on a pad heated to 37°C, and CSF was extracted from the cisterna magna. ³⁰ Mice were then transcardially perfused using ice cold 0.3% phosphate-buffered saline (PBS)-heparin solution and approximately 800 μL of blood was collected using the cardiac bleeding method. Following tissue extraction, the brain was separated into two hemispheres. The left hemisphere was drop fixed overnight in 4% paraformaldehyde and then switched to 30% sucrose solution, stored at 4°C. The right hemisphere was dissected on a chilled aluminum foil plate into cortical and hippocampal regions and stored at −80°C. Blood samples were allowed to clot for a minimum of 1 h, and then blood and CSF were centrifuged at 2000 × g to extract serum samples, which were stored at −80°C. Results from CSF, blood and frozen brain tissue analyses will be reported separately.

Immunohistochemistry

Fixed brain tissue was sectioned coronally into 50-μm thick sections using a freezing sliding microtome (Microm HM 430, ThermoFisher Scientific, Waltham, MA) in a 1:6 series and stored in cryoprotectant (sucrose dissolved in sodium phosphate buffer, pH 7.4 and ethylene glycol) at 4°C. Total human tau was detected using monoclonal HT7 (Cat# MN1000, ThermoFisher Scientific). Primary antibodies used to detect phosphorylated tau included monoclonal AT8 (Cat# MN1020, ThermoFisher Scientific), CP13 (courtesy of Peter Davies, Albert Einstein College of Medicine, NY) and RZ3 (courtesy of Peter Davies). Astrocytes and activated microglia were detected with polyclonal GFAP (Cat# AB5541, Millipore Sigma, St Louis, MO) and polyclonal Iba1 (Cat# 019-19741, Wako Chemicals USA Inc., Richmond, VA) respectively. APP immunoreactivity was detected using polyclonal βAPP (Cat# 51-2700 Invitrogen, Camarillo, CA). For tau staining, antigen retrieval was performed using a 5-min incubation in 70% formic acid. Sections were then incubated in 0.3% hydrogen peroxide solution to block endogenous peroxidase and then blocked for 30 min in 3% serum diluted in TBS-X. Normal goat serum (Cat# S-1000, Vector Laboratories, Burlingame, CA) was used for tau, Iba1 and APP staining, while normal donkey serum (Cat# 017-000-121, Jackson Immunoresearch, West Grove, PA) was used for GFAP staining. Sections were then incubated overnight at 4°C in primary antibody diluted 1:1000 in 3% serum. Tissue from an uninjured 22-month-old 3xTG mouse was used as a positive control for tau pathology, while tissue from a mouse injured using the controlled cortical impact injury model was used as a positive control for GFAP, Iba1 and APP staining. ^31,32 Negative controls with the primary antibody excluded were performed for all stains. The next day, sections were incubated in secondary antibody diluted 1:1000 in TBS-X for 1 h. For tau staining, biotinylated goat anti-mouse secondary antibody (Cat# BA-9200, Vector Laboratories) was used while biotinylated donkey anti-chicken secondary antibody (Cat# 703-065-115, Jackson Immunoresearch) was used for GFAP staining. Biotinylated goat anti-rabbit secondary antibody (Cat# BA-1000, Vector Laboratories) was used for Iba1 and APP staining Sections were incubated for one 1 h in ABC solution (Cat# PK-6100, Vector Laboratories) diluted 1:400 A and B in 1xTBS, followed by visualization using 3′3′ diaminobenzidine (Cat# D5905-100TAB, Sigma Aldrich, St. Louis, MO). Sections were washed three times in 1 × TBS between each step.

Myelin Black Gold II staining

Tissue sections were first washed in TBS to remove residual cryoprotectant. Black Gold II solution was prepared by resuspending 150 mg dry Black Gold II powder (Cat# AG400, Millipore Sigma) in 0.9% saline solution. The Black Gold II solution was then heated to 60°C. Free-floating tissue sections were incubated in the solution for 8–12 min until fibers were completely stained (Supplementary Fig. 2; see online supplementary material at http://www.liebertpub.com). Following two washes in Milli-Q water, sections were incubated in 0.1% sodium thiosulfate solution heated to 60°C for 3 min. The tissue sections were then washed three times in TBS to remove trace sodium thiosulfate.

Quantification of histology

Following staining, tissue sections were mounted onto positively charged slides, dehydrated in a series of graded ethanols (50%-70%-95%-100%-100%), and cleared in three changes of xylenes before being cover-slipped (No. 1.5, Cat # 48393-241; VWR). Images of stained sections were acquired on a Zeiss Axioscan (Carl Zeiss AG, Oberkochen, Germany) using a brightfield 10 × objective lens and then exported at their original resolution into tiff format for quantification. Regions of interest (ROIs) in the gray matter included cortical gray matter (4–5 sections/animal), lateral septal nucleus (1 section/animal), and the hippocampus (3–4 sections/animal). White matter intact (> 50% of region without folds or tears) ROIs included the corpus callosum (4–5 sections/animal) anterior commissure (one section/animal), hippocampal commissure (1 section/animal) and fimbria (2 sections/animal).

To quantify GFAP staining, the Renyi entropy thresholding method was applied to all images (Supplementary Fig. 3; see online supplementary material at http://www.liebertpub.com). The Sauvola local thresholding method (radius of 15) was used to quantify microglial cell bodies stained using Iba1. Similar to Myelin Black Gold II analysis, ROIs were included and considered intact only if >50% of the region was without folds or tears. To exclude remaining edge and fold artifacts, only particles with an area of 10–500 pixels were included in analysis.

Because there were no signs of severe demyelination in any of the tissue sections, we used power coherence as a measure of white matter integrity as previously described. ³³ Briefly, each ROI was subdivided into square regions with an area of 10 pixels ² . This area was used to sustain sufficient spatial resolution and prevent artifacts caused by multiple crossing fibers, while also removing noise related artifacts. A two-tensor model was then fit to the power spectrum of each subdivided region. Power coherence, a metric ranging from 0 to 1, was calculated by subtracting the ratio of the major and minor axes of the tensors from one.

PCR confirmation of genotype

PCR was used to confirm the hTau genotype from one animal selected at random from each cohort of hTau mice. DNA from the leftover regions of the fresh-frozen brain tissue was extracted using the EZ High MW Mouse Tail DNA Isolation Kit (Cat# M1007-100, EZ Bioresearch, St. Louis, MO). Nuclei lysis and Proteinase K solution were added to the tissue sample and incubated overnight at 55°C with gentle shaking until the tissue was completely digested. The next day, protein precipitation solution was added to the mixture. Following a 5-min incubation on ice, the solution was centrifuged at 16,000 × g for 3 min at room temperature, separating the protein pellet from supernatant. Isopropanol was added to the supernatant and inverted until DNA was visible as threads. The supernatant and isopropanol solution was then centrifuged 16,000 × g for 1 min at room temperature. The supernatant was then removed from the microcentrifuge tube and the remaining DNA pellet was washed with 70% ethanol. The DNA pellet was centrifuged and washed again with 70% ethanol. Any residual supernatant was then removed and the DNA pellet was allowed to air dry for 10 min. DNA Hydration Solution was added to the pellet and incubated at 65°C for 1 h with gentle shaking. The extracted DNA was then stored at 4°C until PCR genotyping. Tail DNA from an hTau mouse was obtained from an independent investigator to use as an external positive control.

Control (hTau ctrl R : 5′- GTA GGT GGA AAT TCT AGC ATC ATC C -3′, hTau ctrl F : 5′- CTA GGC CAC AGA ATT GAA AGA TCT -3′) and hTau specific oligonucleotides (hTau R : 5′- CTC TGC ATG GCT GTC CAC TAA CCT T -3′, hTau F : 5′- ACT TTG AAC CAG GAT GGC TGA GCC C -3′) were resuspended to 100 μM concentration in molecular grade water. The extracted DNA was thawed on ice and diluted to a final concentration of 100 ng/μL. A solution of DNA, control or hTau oligonucleotides (0.2 μM), GoTaq Flex (Promega, Madison, WI), deoxyribonucleotide triphosphates, reaction buffer, and enhancer was then prepared in molecular grade water and heated using a thermocycler. Thermocycler conditions were 94°C for 30 sec, 58°C for 1 min, and 72°C for 1 min for 35 cycles each, with a final extension phase of 72°C for 2 min. PCR was visualized on a 1% agarose gel.

Western blotting

Western blotting for total human tau and phosphorylated tau was performed as previously described. ^17,34 Briefly, frozen hippocampi from 5–6 randomly selected animals from each injury group were homogenized in RIPA buffer with added protease and phosphatase inhibitors. Hippocampal tissue from an uninjured 22-month-old 3xTG animal was used as a positive control for total and phosphorylated tau. Homogenates were centrifuged (13,000 rpm, 20 min at 4°C) and protein concentrations were determined using the bicinchoninic acid assay methods in order to obtain equal amounts of protein sample. Samples were then heated with mercapto-ethanol. Dot blot analysis was performed by pipetting 100 μg of protein onto 0.2 μm nitrocellulose membranes which were then allowed to dry completely. In parallel, 20 μg of protein with loading buffer was loaded per well and then electrophoresed on 10% BisTris NUPAGE gels (Cat#NP0301, ThermoFisher Scientific, Carlsbad, CA), followed by transfer to 0.2 μm nitrocellulose membranes. These membranes were blocked with 5% milk in PBS with Tween (PBS-T) for 1 h, followed by incubation in primary antibody (HT7, total human tau or AT8, ptau) diluted 1:1000 in 5% milk in TBS-T overnight at 4°C. Antibodies were detected using sheep anti-mouse horseradish peroxidase (GE Healthcare, Cat#NXA931, Buckinghamshire, UK; 1:10,000 dilution in 5% milk and PBS-T solution) and ECL Western blotting kit (GE Healthcare, Cat#RPN2235, Buckinghamshire, UK). Membranes were then stripped and reprobed for tubulin (1:1000 dilution).

Statistical analysis

All analysis was performed using Statistica (Tibco Software Inc., Palo Alto, CA). All data was tested for normality using the Shapiro-Wilk normality test. Any datasets that violated the assumption of normality were transformed so that parametric analyses could be performed (Supplementary Table 2; see online supplementary material at http://www.liebertpub.com). A two-way, repeated measures ANOVA was performed for each neurobehavioral metric to test for the effects of injury and cohort, with test session (acute, 3-month, one1data and histological data. We pre-specified the statistical plan to collapse across the two cohorts of mice for analyses for which there were no significant main effects or cohort of interactions with of other factors with cohort. Statistical power calculations for main effects of injury was 80% at p < 0.05 with an effect size of 0.50. Animals that died before the end of the experiment were excluded from behavioral and histological analysis. Post hoc Tukey's tests were then used to determine significance for specific pairwise comparisons (p < 0.05). Because the residuals were not normally distributed, all correlational analyses were performed using Spearman's rank correlations, followed by Bonferroni correction for multiple comparisons. The relationship between power coherence and microgliosis in white matter tracts was assessed using Spearman's correlations, followed by Bonferroni correction for multiple comparisons. When determining the relationship between white matter power coherence and Morris water maze measures (four ROIs × five behavioral measures = 20 correlations), the effective number of tests was adjusted to account for potential correlation between measures. ³⁵ Without this adjustment, the Bonferroni correction at p < 0.05/20 = 0.0025; however, with the variance of the eigenvalues of the correlation matrix (vλobs = 1.265), the adjusted Bonferroni at p < 0.05/18.79 = 0.0027.

Correlations between microgliosis in both gray and white matter with Morris water maze measures (seven ROIs × five behavioral measures = 35 correlations) were similarly adjusted. After adjustment using the variance of the eigenvalues of the correlation matrix (vλobs = 1.041), the adjusted Bonferroni at p < 0.05/33.99 = 0.0015. Effect sizes were calculated for each test that was performed (Supplementary Table 3; see online supplementary material at http://www.liebertpub.com). Graphs were plotted using Prism v7.0 (Graph Pad, La Jolla, CA). All error bars represent standard error of mean (SEM).

hTau mice show injury dependent differences in righting time

Loss of consciousness, measured by righting time (LRR) was used to measure the immediate effects of injury during the 20 days of impacts (Fig. 2). LRR was calculated by measuring the righting time each day of injury, and averaging across days. A two-way ANOVA showed that there was a significant effect of injury [F(2,44) = 11.8092, η² _p = 0.349, p < 0.0001]. Animals in the 20 × concussive group had elevated righting times compared with 20 × shams (p = 0.00059); as well as animals in the 20 × subconcussive group (p = 0.00046) after post hoc Tukey's testing. There was no difference in righting times between 20 × shams and animals in the 20 × subconcussive group (p = 0.985). There was no effect of cohort or interaction between injury and cohort. These data show that the higher-energy concussive impacts caused a significantly more substantial acute injury than the lower-energy subconcussive impacts.

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FIG. 2.

Loss of consciousness following injury. Latency righting reflex was used as a measure loss of consciousness. Righting times were measured by an observer blinded to injury status. Animals in the concussive injury group had increased righting times relative to the subconcussive and sham groups, which were indistinguishable. There was a significant effect of concussive injury on righting time, with animals in the 20 × concussive group having increased righting times relative to 20 × shams (p = 0.00059) and 20 × subconcussive mice (p = 0.00046). Color image is available online.

hTau mice subjected to repeated concussive injuries show persistent chronic cognitive deficits

At acute, 3-month and 12-month time-points, animals were tested for learning deficits using the Morris water maze task. During the 3 days of visible platform testing (Fig. 3; Supplementary Fig. 4; see online supplementary material at http://www.liebertpub.com), the two-way, repeated measures ANOVA, showed a main effect of injury on swim speed [F(2,36) = 29.9335, η² _p = 0.624, p < 0.00001]. Animals in the 20 × concussive group had reduced swim speeds relative to both shams (p = 0.000127) and mice in the 20 × subconcussive injury group (p = 0.000128). There was no interaction between test session (acute, 3-month, 12 month) and injury. There was also a main effect of injury on distance to the target [F(2,36) = 25.642, η² _p = 0.588, p < 0.00001]. Animals in the 20 × concussive group had increased swimming distances relative to shams (p = 0.000127) and mice in the 20 × subconcussive injury group (p = 0.000127). There were no differences between subconcussive injury and sham groups (p = 0.923). There was no effect of test session or interaction between test session and injury on swim speed or distance swum.

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FIG. 3.

Effects of injury on visible platform phase of the Morris water maze. (A) Average swimming speeds for each day of the visible phase of the Morris water maze during each of the three test sessions. There was a significant effect of injury on swim speed (0.24J vs. sham: p = 0.000127, 20 × concussive vs. 20 × subconcussive; p = 0.000128). There was no interaction between test session and injury. (B) Total distance to the target platform during 3 days of visible platform testing. There was a significant effect of repetitive concussive injury (20 × concussive vs. sham: p = 0.000127, 20 × concussive vs. 20 × subconcussive: p = 0.000127). Color image is available online.

At each test session, 2 days following the final day of visible platform testing, animals underwent hidden platform testing of the Morris water maze task for 4 consecutive days (Fig. 4; Supplementary Fig. 5; see online supplementary material at http://www.liebertpub.com). Similar to the visible platform phase, the two-way repeated measures ANOVA showed an effect of injury on swim speed [F(2,36) = 36.5009, η² _p = 0.663, p < 0.00001] that remained significant after post hoc testing. Mice in the 20 × concussive group had reduced swim speeds relative to 20 × shams (p = 0.000125) and mice in the 20 × subconcussive group (p =0.000128). Animals in the 20 × subconcussive group also had reduced swimming speeds relative to 20 × shams (p = 0.028). There also was an effect of injury on swimming distance during the hidden platform phase [F(2,36) = 6.272, η² _p = 0.564, p < 0.01] that remained significant following post hoc Tukey's testing. Animals in the 20 × concussive group had increased swimming distances relative to 20 × shams (p = 0.044) and animals in the 20 × subconcussive group (p = 0.009). There was no difference in swimming distance between subconcussive injury and sham groups (p = 0.867). There no effect of test session on either swim speed or swimming distance. There was also was no interaction between test session and injury on either of these measures.

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FIG. 4.

Effects of injury on hidden platform phase of the Morris water maze. (A) Average swimming speeds for each of the 4 days of the hidden platform task during each of the three test sessions. There was a significant effect of injury (20 × concussive vs. sham, p = 0.000125, 20 × concussive vs. subconcussive, p = 0.000128). Mice in the 20 × subconcussive group also had reduced swimming speeds relative to shams (p = 0.028). (B) Total distance to the target platform during four days of hidden platform testing. There was a significant effect of concussive injury (20 × concussive vs. sham: p = 0.044, 20 × concussive vs. 20 × subconcussive: p = 0.009). There was no interaction of test session and injury. Color image is available online.

Twenty-four hours after the final day of hidden platform testing, hTau mice were tested for memory retention using the probe trial of the Morris water maze task (Fig. 5; Supplementary Fig. 6; see online supplementary material at http://www.liebertpub.com). Mean proximity to platform and percent time spent in the target quadrant were used as metrics of memory retention for each test session. For the measure of mean proximity to platform, there was an effect of injury [F(2,36) = 23.173, η² _p = 0.570, p < 0.00001] and time [F(2,70) = 6.9935, η² _p = 0.167, p < 0.01] on mean proximity to platform that remained significant after post hoc testing. Animals in the 20 × concussive injury group were further away from where the hidden platform had previously been placed relative to both shams (p = 0.000124) and 20 × subconcussive animals (p = 0.000126). Animals also had increased proximity to the target platform during the 3-month test session (p = 0.000280) relative to the acute test session. There was also an effect of injury on percent time in the target quadrant [F(2,36) = 22.851, η² _p = 0.566, p < 0.00001] that remained significant after post hoc testing. These same animals also spent significantly less time in the target quadrant relative to shams (p = 0.000124) and 20 × subconcussive animals (p = 0.000123). There were no significant differences between animals in the 20 × subconcussive group or 20 × sham group for either proximity to platform or percent time spent in the target quadrant. There was no interaction between time and injury for either parameter.

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FIG. 5.

Effects of injury on Morris water maze probe trial performance. (A) There was a significant effect of concussive injury on average proximity to platform during probe trial; 20 × concussive vs. 20 × sham: p = 0.000124, 20 × concussive vs. 20 × subconcussive: p = 0.000126). There was also an effect of time, where animals were significantly further away from the platform at the 3-month test session (p = 0.000280) relative to the acute session (###). Animals in the repetitive concussive injury group were on average further away from the location of the target platform. (B) There was a significant effect of concussive injury on the percent time spent in the quadrant where the platform had previously been placed (20 × concussive vs. sham: p = 0.000124, 20 × concussive vs. 20 × subconcussive: p = 0.000123). Animals in the repetitive concussive injury group tended to spend less than 25% of their time (chance performance, dotted line) in the target quadrant. There was no interaction between test session and injury. Color image is available online.

In summary, repetitive concussive injury resulted in reduced swim speed during both visible and hidden platform phases of Morris water maze, a possible indication of reduced motor function or motivation following injury. Further, repetitive concussive injury also caused an increase in distance swum to the target. This effect indicates an impairment in ability to learn the platform location. Moreover, repetitive concussive injury resulted in impaired memory, demonstrated by increased proximity to platform and reduced percent time in target quadrant during the probe trial. These findings, combined with the effect of time on proximity to the platform during only intermediate session of probe trial indicates that the 20 × concussive injury method implemented in this study causes acute cognitive deficits that neither progressively worsen nor resolve at 1 year post-injury. Instead, these changes in learning and memory appear to remain sustained at a reduced level relative to shams and animals in the 20 × subconcussive group.

No social interaction deficits, anxiety or depressive-like behaviors detected after repeated injury in hTau mice

The sociability and social novelty phases of Crawley's three-chamber social interaction test were used to measure both acute and chronic social behaviors (Fig. 6; Supplementary Fig. 7; see online supplementary material at http://www.liebertpub.com). For the sociability test, the ratio of time spent interacting with the stimulus mouse to time spent interacting with the novel object was used as an index of sociability. For the social novelty test, the ratio of time spent interacting with the novel mouse to time spent interacting with the previous stimulus mouse was used as an index of social novelty. The two-way repeated measures ANOVA showed no effect of injury on either sociability index [F(2,37) = 0.300, p = 0.74] or social novelty testing index [F(2,37) = 2.729, p = 0.78]. There also was no effect of test session on either index.

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FIG. 6.

No change in social interaction following injury. (A) Injured animals did not show any significant changes in sociability compared with shams. Dotted line indicates a sociability index of 1. Points above this line indicate that the animal showed preference for the stimulus mouse rather than the novel inanimate object. (B) Injured animals did not show any significant changes in social novelty compared with shams. Dotted line indicates a social novelty index of 1. Points above this line indicate that the animal showed preference for the novel mouse rather than the stimulus mouse from the previous sociability session. Color image is available online.

Anxiety-like behavior was measured using two behavioral measures: open arm time during the elevated plus maze task, and thigmotaxis during the open field maze task (Fig. 7; Supplementary Fig. 8; see online supplementary material at http://www.liebertpub.com). Animals displaying increased risk taking and anxious behavior would be expected to have increased open arm time and elevated thigmotaxis scores. There was no effect of injury on open arm time [F(2,37) = 0.195, p = 0.98]. While the two-way repeated measures ANOVA did show an effect of injury on open field thigmotaxis [F(2,37) = 3.610, p = 0.0367] there were no significant differences between groups following post hoc testing. There was no effect of test session on open arm time or thigmotaxis.

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FIG. 7.

No changes in anxiety related behavior following injury. (A) Animals in the 20 × concussive and 20 × subconcussive did not show any changes in percent time spent in open arms of the elevated plus maze relative to shams. (B) Injured animals did not show any changes in open field thigmotaxis (time weighted distance from the wall) relative to sham animals. Color image is available online.

Time immobile during the tail suspension test was used as a metric of depressive-like behavior (Fig. 8; Supplementary Fig. 9; see online supplementary material at http://www.liebertpub.com). The two-way repeated measures ANOVA showed an effect of injury on immobility time [F(2,37) = 12.954, p < 0.0001] that remained significant after post hoc testing. hTau mice in the 20 × concussion group showed reduced immobility times rather than the expected increased immobility times relative to shams (p = 0.00427) and relative to mice in the subconcussive group (p = 0.000212). The interpretation of this result is not clear (see Discussion). There was no significant difference between animals in the 20 × subconcussive group and 20 × shams (p = 0.546). There also was no significant effect of test session on time spent immobile, though it appeared that the effect was less apparent at the 3-month test session. These findings show that repetitive concussive or subconcussive injury does not result in acute social, anxiety related, or depressive-like deficits. Further, based on the lack of an effect of test session, deficits in these domains do not manifest during the chronic stages following injury.

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FIG. 8.

Effects of injury on depressive-type behavior. The tail suspension test was used to assess depressive-type behavior in animals. There was a significant effect of injury (20 × concussive vs. sham: p = 0.00427, 20 × concussive vs. 20 × subconcussive: p = 0.000212) with less immobility in the 20 × concussive group. Color image is available online.

Repetitive brain injury does not result in chronic astrogliosis

GFAP staining was used to assess the effect of repetitive head injury on chronic astrogliosis. Regions of interest were drawn in the gray matter (cortex, lateral septal nucleus and hippocampus) and white matter (corpus callosum, anterior commissure, hippocampal commissure and fimbria). Percent area of positive staining was used as a measure of astrogliosis. The two-way ANOVA showed no effect of injury (p > 0.05 all regions; details in Supplementary Table 3) on percent area of GFAP staining in any of the regions of interest (Fig. 9). In summary, our analysis of activated astrocytes shows that our 20 × concussion model does not result in chronic astrogliosis.

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FIG. 9.

No effects of injury on chronic astrogliosis. (A) Percent area of glial fibrillary acidic protein (GFAP) staining was measured in cortical gray matter. There were no differences in staining between injury groups. (B) There was no difference in GFAP staining between injury groups in the lateral septal nucleus. (C) No differences in GFAP staining were detected in the hippocampus (D). No differences in GFAP staining were detected in the corpus callosum (E). There were no differences between injury groups detected in the anterior commissure. (F). No differences in GFAP staining were detected in the hippocampal commissure. (G). No differences in GFAP staining were detected in the fimbria. (H). GFAP staining in a positive control wild-type mouse injured using controlled cortical impact shows extensive astrogliosis in the hippocampus contralateral to the injury site. Scale bar = 100 microns. Color image is available online.

Repetitive brain injury results in chronic microgliosis in the corpus callosum

Iba1 staining was used to determine whether there was an effect of repetitive head injury on chronic microgliosis. Regions of interest were drawn in the gray matter (cortex, lateral septal nucleus and hippocampus) and white matter (corpus callosum, anterior commissure, hippocampal commissure and fimbria). Percent area of positive thresholded staining was used to quantify the extent of microgliosis in each region. For all regions except for the corpus callosum, the two-way ANOVA showed no effect of injury (Supplementary Fig. 10, p > 0.05; see online supplementary material at http://www.liebertpub.com; details in Supplementary Table 3). In the corpus callosum, the two-way ANOVA revealed that there was an effect of injury on microgliosis [F(2,34) = 7.984, η² _p = 0.320, p < 0.01]. Post hoc Tukey's testing showed that animals in the repetitive concussive injury group had modest increases in microgliosis in the corpus callosum (Fig. 10; p = 0.0327). These findings show that repetitive concussive injury results in white matter microgliosis in the chronic phase of injury.

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FIG. 10.

Repetitive head injuries results in modest increases of activated microglia in the corpus callosum. (A). Percent area of thresholded Iba1 staining was significantly increased in animals sustaining 20 × concussive injuries at the chronic time-point relative to 20 × shams (p = 0.0327). (B) Staining was thresholded using the Sauvola thresholding method in order to quantify Iba1 staining. The automated thresholding method identified increased microglial cell bodies in the 20 × concussive animals relative to shams. Scale bar = 200 microns. Color image is available online.

No evidence of phosphorylated tau pathology in hTau mice

PCR genotyping confirmed that mice from both cohorts were positive for human tau (Supplementary Fig. 11A; see online supplementary material at http://www.liebertpub.com). Total human tau (HT7) was detected using Western blotting for animals from all injury groups and in both cohorts (Supplementary Fig. 11B). A 22-month-old 3xTG mouse was used as a positive control for total and phosphorylated tau. Phosphorylated tau could not be detected in either hTau mice or 3xTG mice using Western blotting for AT8, (Supplementary Fig. 11B), CP13 or AT180. Dot blot analysis with high levels of protein showed that all three antibodies for phosphorylated tau were successfully capable of detecting phosphorylated tau in aged 3xTG but not hTau mice (Supplementary Fig. 11C). Further, hTau mice did not show any substantive staining for HT7, AT8 (ptau, ser202/thr205), CP13 (ptau, ser202) or RZ3 (ptau, thr231; Fig. 11A). However, the aged 3xTG positive control tissue showed extensive positive staining for total tau and phosphorylated tau epitopes (Fig. 11B).

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FIG. 11.

No detection of tau pathology using immunohistochemistry in any of the hTau mice. (A) Monoclonal AT8 (ptau, ser202/thr205), CP13 (ptau, ser202), RZ3 (ptau, Thr231) and HT7 (total tau) were tested to detect phosphorylated and total tau in the hTau mice line. No positive staining was observed for any of the antibodies in any of the groups. (B) Tissue from a 22-month-old 3xTG mouse was used as a positive control. When stained with AT8, CP13, RZ3 and HT7 there is extensive positive staining in the hippocampal neurons. Scale bar = 100 microns. Color image is available online.

Repetitive injury results in chronic white matter disruption

βAPP staining in hTau animals revealed no abnormalities of axonal profiles at 1 year post-injury, particularly when compared with the axonal swellings clearly visible in the ipsilesional regions of animals injured using a controlled cortical impact injury as a positive control (Supplementary Fig. 12; see online supplementary material at http://www.liebertpub.com). We therefore assessed white matter integrity using power coherence as previously described. ³³ Power coherence analysis of Myelin Black Gold II stained images was used to measure chronic white matter disruption (Fig. 12). White matter regions of interest included the corpus callosum, anterior commissure, hippocampal commissure and fimbria. The two-way ANOVA showed an effect of injury on power coherence in the corpus callosum [F(2,37) = 28.048, η² _p = 0.644, p < 0.00001], hippocampal commissure [F(2,37) = 34.015, η² _p = 0.687, p < 0.00001] and fimbria [F(2,37) = 17.262, η² _p = 0.527, p < 0.00001] that remained significant following post hoc Tukey's testing (Fig. 13). hTau animals in the 20 × concussive group had reduced power coherence in the corpus callosum (p = 0.000125), hippocampal commissure (p = 0.000125) and fimbria (p = 0.000136) relative to shams. Animals in the 20 × subconcussive group also showed more modestly reduced power coherence only in the corpus callosum (p = 0.0091) and hippocampal commissure (p = 0.027) relative to shams. Power coherence was also significantly reduced in the corpus callosum (p = 0.000367), hippocampal commissure (p = 0.000150), and fimbria (p = 0.000189) in the 20 × concussive group relative to animals in the 20 × subconcussive group.

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FIG. 12.

Effects of injury on chronic white matter disruption, assessed using power coherence. (A) Power coherence was measured in the corpus callosum of tissue sections stained with Myelin Black Gold II. Fibers in sham animals have high coherence, indicating that fibers are aligned and intact. However, white matter fibers in injured animals show reduced power coherence, indicating white matter disruption. (B) Fibers in the anterior commissure do not show any differences in power coherence, indicating intact white matter. (C) Similar to the corpus callosum, white matter in the hippocampal commissure has reduced power coherence, indicating fiber disruption. (D) White matter in the fimbria of sham and 20 × subconcussive animals shows high power coherence, while the fimbria of 20 × concussive animals shows reduced power coherence. Because the fimbria tended to stain more heavily relative to other regions, brightness and contrast levels were uniformly adjusted adjusted for the exemplars so fibers could be better visualized. Scale bars = 50 microns. Color image is available online.

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FIG. 13.

Reduced white matter integrity following repetitive injury. (A) Power coherence in the corpus callosum was significantly reduced in injured animals (20 × concussive vs. sham, p = 0.000125, 20 × subconcussive vs. sham, p = 0.0091). There was also a significant difference in power coherence between animals in the 20 × concussive and 20 × subconcussive groups (p = 0.000367). (B) Power coherence in the anterior commissure showed no effect of injury. (C) There was a significant effect of injury on power coherence in the hippocampal commissure of hTau animals (20 × concussive vs. sham, p = 0.000125, 20 × subconcussive vs. sham, p = 0.027). There was also a significant difference between animals in the 20 × concussive and 20 × subconcussive groups (p = 0.000150). (D) Power coherence in the fimbria was significantly reduced in animals in the 20 × concussive group (20 × concussive vs. sham, p = 0.000136, 20 × concussive vs. 20 × subconcussive, p = 0.000189). Color image is available online.

Because we found evidence of chronic white matter microgliosis following repetitive head injury, we also asked whether there was a relationship between power coherence and microgliosis in white matter tracts (Fig. 14). Interestingly, we found a weak-modest negative correlation between microgliosis and power coherence in the corpus callosum that remained significant after correction for multiple comparisons (r = -0.441, p = 0.0081). This relationship was weak and insignificant when analyzing the 20 × concussive group alone. None of the other white matter tracts showed significant relationships when considering the full sample of animals or injury groups separately. The injury dependent reduction in power coherence suggests that repetitive brain injuries cause acute white matter disruption that remains unresolved in the chronic phase of injury. The difference in effect sizes when comparing coherence in white matter indicates that the corpus callosum and hippocampal commissure may be especially useful regions to assess chronic histological outcomes. Further, white matter disruption in these tracts white matter disruption is concomitant, not correlated with microgliosis.

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FIG. 14.

Relationship between microgliosis and white matter disruption. (A) Microgliosis, assessed by percent area of Iba1 staining, showed modest, negative correlation with power coherence in the corpus callosum when considering the full sample of hTau animals. (B) In the anterior commissure, microgliosis had a weak and insignificant relationship with power coherence. (C) Power coherence in the hippocampal commissure was weakly correlated with microgliosis, a relationship that was not significant after correction for multiple comparisons. (D) Spearman's correlation of microgliosis and power coherence in the fimbria showed no significant relationship. Color image is available online.

Correlation between white matter disruption and behavioral performance deficits

To address the question of whether there was a relationship between white matter disruption and the cognitive and motor deficits observed during the Morris water maze task, we performed Spearman's rank correlations between white matter power coherence data collected from the chronic test session of Morris water maze (Fig. 15; Table 1). This was followed by Bonferroni correction for multiple comparisons where the effective number of tests was adjusted by the variance of the eigenvalues of the correlation matrix. ³⁵ We found a modest but significant relationship between power coherence in the corpus callosum and swimming speed during both visible and hidden phases. When analyzing the 20 × concussive group in isolation, this correlation remained moderate and significant. There also was a moderate correlation between white matter power coherence in the hippocampal commissure and swimming speed during both visible and hidden phases. However, when considering the 20 × concussive injury group in isolation, this relationship was weak and not significant. There were no significant correlations between white matter damage and distance to platform measures. Finally, we found a modest but significant relationship between power coherence in the fimbria and proximity to platform during probe trial. This relationship weakened and was not significant when considering the 20 × concussive group in isolation.

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FIG. 15.

Relationship between white matter disruption and Morris water maze deficits. (A) There was a modest, significant relationship between power coherence in the corpus callosum and swimming speed during the visible platform phase. When considering the 20 × concussive group alone, this relationship remained consistent. In the hippocampal commissure, animals in the 20 × concussive groups had weak, insignificant correlation of power coherence and swim speed. (B) A similar trend was observed in both the corpus callosum and hippocampal commissure during the hidden platform phase. (C) Animals that were further away from the platform during probe trial tended to sustain more severe chronic white matter damage in the fimbria. There was a modest but significant relationship between power coherence in this region and proximity to platform. However, in the 20 × concussive group, this relationship was weak and no significant relationship between power coherence in the fimbria and proximity to platform during probe trial. Color image is available online.

We next asked whether there was an association of microgliosis in gray and white matter regions with cognitive deficits observed during the chronic test session of Morris water maze. Spearman's rank analyses showed modest and positive relationships between percent area of Iba1 staining in the corpus callosum, distance to target during the hidden phase, and proximity to platform during probe trial that remained significant after adjusted Bonferroni correction for multiple comparisons (Fig. 16; Table 2). When analyzing the 20 × concussive group alone, these relationships weakened and were not significant. The modest correlations for total hTau mice appeared to be driven by animals in the 20 × sham group, where the relationships remained moderate but insignificant. Interestingly, we also found that while the relationship between microgliosis in the anterior commissure and distance to target during hidden platform phase was weak and insignificant for the full sample of hTau mice, this correlation was strong and significant when considering animals in the 20 × subconcussive group in isolation. These results indicate that microgliosis and white matter disruption in the corpus callosum, along with damage to tracts such as the hippocampal commissure and fimbria is related to behavioral deficits exhibited during the Morris water maze task.

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FIG. 16.

Relationship between white matter microgliosis and Morris water maze deficits. (A) There was a modest, positive relationship between microgliosis in the corpus callosum and distance to target during the hidden platform phase. When considering the 20 × concussive group separately, this relationship was weak and not significant. (B) Microgliosis in the corpus callosum was also modestly and significantly correlated with proximity to platform during probe trial. In the 20 × concussive group, this relationship was weak and insignificant. (C) While microgliosis in the anterior commissure did not show a strong relationship with any Morris water maze measures when considering the full sample of hTau animals, analysis of the 20 × subconcussive group showed strong and significant correlation of anterior commissure microgliosis and distance to target during hidden platform. Color image is available online.