Tau reduction diminishes spatial learning and memory deficits after mild repetitive traumatic brain injury in mice

Abstract

Objective: Because reduction of the microtubule-associated protein Tau has beneficial effects in mouse models of Alzheimer's disease and epilepsy, we wanted to determine whether this strategy can also improve the outcome of mild traumatic brain injury (TBI).

Methods: We adapted a mild frontal impact model of TBI for wildtype C57Bl/6J mice and characterized the behavioral deficits it causes in these animals. The Barnes maze, Y maze, contextual and cued fear conditioning, elevated plus maze, open field, balance beam, and forced swim test were used to assess different behavioral functions. Magnetic resonance imaging (MRI, 7 Tesla) and histological analysis of brain sections were used to look for neuropathological alterations. We also compared the functional effects of this TBI model and of controlled cortical impact in mice with two, one or no Tau alleles.

Results: Repeated (2-hit), but not single (1-hit), mild frontal impact impaired spatial learning and memory in wildtype mice as determined by testing of mice in the Barnes maze one month after the injury. Locomotor activity, anxiety, depression and fear related behaviors did not differ between injured and sham-injured mice. MRI imaging did not reveal focal injury or mass lesions shortly after the injury. Complete ablation or partial reduction of tau prevented deficits in spatial learning and memory after repeated mild frontal impact. Complete tau ablation also showed a trend towards protection after a single controlled cortical impact. Complete or partial reduction of tau also reduced the level of axonopathy in the corpus callosum after repeated mild frontal impact.

Interpretation: Tau promotes or enables the development of learning and memory deficits and of axonopathy after mild TBI, and tau reduction counteracts these adverse effects.

Figure 1. Wildtype mice show learning and memory deficits one month after 2-hit frontal injury.

Wildtype mice (n = 9–10 per group) received a 1-hit or a 2-hit frontal impact injury or sham treatments, and were tested in the Barnes maze one month later. (A, B) Learning curves of the 1-hit (A) and 2-hit (B) groups, reflecting the time it took mice to find the target, averaged from 2 trials per day. Only the 2-hit group differed significantly from sham-treated controls (p = 0.0062 by linear mixed effects model analysis). (C) The 2-hit group and sham-treated controls showed a comparable latency to target during the first trial on the first training day. (D) Probe trial administered 24 h after the last training trial. *p<0.05, **p<0.01 vs corresponding sham group or as indicated by bracket. Sh, Sham; H, Hit. Data are means ± SEM.

Figure 2. Wildtype mice show no impairments in context or cued fear learning and memory after 2-hit injury.

Wildtype mice (n = 8–10 per group) received a 2-hit frontal impact injury or sham treatment, and underwent cued and contextual fear conditioning 1 month later. (A) On day 1, the sham-treated and 2-hit groups showed comparable amounts of freezing at baseline as well as during and between training trials. BL, baseline (3 min); CS, conditional stimulus (auditory stimulus followed by foot shock); IT, interval between CS (2 min); End, period following last CS (2 min). See methods for additional details. (B, C) Both groups also showed a comparable amount of freezing when they were introduced into the same context on day 2 without receiving an auditory stimulus or foot shock (B) or into a novel environment on day 3 after hearing the auditory stimulus without receiving a foot shock (C). AS, auditory stimulus. One-way ANOVA revealed no significant differences between the groups (A–C). Data are means ± SEM.

Figure 3. Wildtype mice show no alterations in exploratory activity or anxiety-like behavior after 2-hit injury.

Wildtype mice (n = 8–10 per group) received a 2-hit frontal impact injury or sham treatment, followed by assessment in different behavioral tests. (A–D) Open field activity 2 weeks post-injury. (E–H) Behavior in elevated plus maze 4 weeks post-injury. Student's t test revealed no significant differences between the 2-hit and sham treated groups for any of the tests and outcome measures. Data are means ± SEM.

Figure 4. Wildtype mice show no alterations in motor performance or depression-like behavior after 2-hit injury.

Wildtype mice (n = 8–10 per group) received a 2-hit frontal impact injury or sham treatment, followed by behavioral assessment. (A–B) Balance beam performance 3 weeks post-injury. (C–E) Y-maze activity 2 weeks post-injury. (F–G) Forced swim test 5 days (F) and 6 months (G) post-injury. Student's t test revealed no significant differences between the 2-hit and sham treated groups for any of the tests and outcome measures. Data are means ± SEM.

Figure 5. Wildtype mice show no focal radiographic abnormalities on MRI after 2-hit injury.

Wildtype mice (n = 6 per group) received a 2-hit frontal impact injury or sham treatment. Their brains were imaged by MRI two days after the second injury. (A–B) Representative T1 weighted coronal images of sham treated (A) and 2-hit injured (B) mice.

Figure 6. Tau reduction diminishes learning and memory deficits caused by 2-hit frontal injury.

Tau ^+/+, Tau ^+/– and Tau ^–/– mice (n = 9–10 per genotype and treatment) received a 2-hit frontal impact injury or sham treatment, and were tested in the Barnes maze one month later. (A) Learning curves of the indicated groups. By linear mixed effects model analysis, injured Tau ^+/+ mice differed from sham treated Tau ^+/+ mice (p = 0.0001), injured Tau ^+/– mice (p = 0.02) and possibly also injured Tau ^–/– mice (p = 0.09). Injured Tau ^+/– and Tau ^–/– mice did not differ from each other or from their respective sham treated controls. (B) Probe trial administered 24 h after the last training trial. Two-way ANOVA revealed a Tau genotype effect (p = 0.016, F(2, 53) = 4.54) and an interaction between Tau genotype and injury (p = 0.011, F(2, 53) = 4.95). *p<0.05, **p<0.01 vs. sham treated group of same genotype or as indicated by brackets (Tukey-Kramer test). ns, not significant; Sh, Sham. Data are means ± SEM.

Figure 7. Tau reduction has less effect on memory deficits caused by controlled cortical impact (CCI) injury.

Tau ^+/+, Tau ^+/– and Tau ^–/– mice (n = 7–10 per genotype and treatment) received a single focal CCI injury to the right frontal cortex or a sham treatment, and were tested in the Barnes maze one month later. (A) Learning curves did not differ significantly among the indicated groups (linear mixed effects model analysis). (B) Probe trial administered 24 h after the last training trial. Two-way ANOVA revealed an injury effect (p<0.0001, F(1, 46) = 29.15) but no Tau genotype effect or interaction between injury and Tau genotype. *p<0.05, **p<0.01 vs. sham treated group of same genotype. ns, not significant. Sh, Sham. Data are means ± SEM.

Figure 8. Tau reduction prevents chronic axonopathy in corpus callosum of 2-hit mice.

Tau ^+/+, Tau ^+/– and Tau ^–/– mice (n = 8–10 per genotype and treatment) received a 2-hit frontal impact injury or sham treatment, underwent behavioral testing 2–6 weeks later, and were analyzed histologically 12 months after the initial injury. (A, B) Coronal brain sections were immunostained for mAPP (22C11). (A) Photomicrographs depicting abnormal mAPP-positive profiles in the corpus callosum of a 2-hit Tau ^+/+ mouse that are not seen in the other mice. (B) Quantitation of mAPP-positive profiles in the corpus callosum. Two-way ANOVA revealed a Tau genotype effect (p = 0.0011, F (2, 45) = 7.94), a 2-hit injury effect (p = 0.027, F (1, 45) = 5.25) and an interaction between Tau genotype and injury (p = 0.008, F (2, 45) = 5.42). ***p<0.001 vs. sham-treated group of same genotype (Tukey-Kramer test). (C,D) Axons in the corpus callosum of 2-hit and 2-sham Tau ^+/+ mice were labeled by silver staining. (C) Representative photomicrographs. (D) The thickness of the corpus callosum was quantitated as described in Methods. Student's t test revealed no significant difference between the groups. Sh, Sham, H, Hit. Scale bar: 80 µm. Data are means ± SEM.

Figure 9. Wildtype mice have no callosal astrocytosis or microgliosis 12 months after 2-hit frontal injury.

Wildtype mice (n = 8–10 per group) received a 2-hit frontal impact injury or sham treatment, underwent behavioral testing 2–6 weeks later, and were analyzed histologically 12 months after the initial injury. Coronal brain sections were immunostained for GFAP or Iba-1(A, B) Representative images (A) and quantitation (B) of GFAP immunoreactivity in the corpus callosum. (C, D) Representative images (C) and quantitation (D) of Iba-1 immunoreactivity in the corpus callosum. Sh, Sham, H, Hit. Scale bar: 80 µm. Data are means ± SEM.