The effects of mild closed head injuries on tauopathy and cognitive deficits in rodents: Primary results in wild type and rTg4510 mice, and a systematic review

Abstract

In humans, the majority of sustained traumatic brain injuries (TBIs) are classified as 'mild' and most often a result of a closed head injury (CHI). The effects of a non-penetrating CHI are not benign and may lead to chronic pathology and behavioral dysfunction, which could be worsened by repeated head injury. Clinical-neuropathological correlation studies provide evidence that conversion of tau into abnormally phosphorylated proteotoxic intermediates (p-tau) could be part of the pathophysiology triggered by a single TBI and enhanced by repeated TBIs. However, the link between p-tau and CHI in rodents remains controversial. To address this question experimentally, we induced a single CHI or two CHIs to WT or rTg4510 mice. We found that 2× CHI increased tau phosphorylation in WT mice and rTg4510 mice. Behavioral characterization in WT mice found chronic deficits in the radial arm water maze in 2× CHI mice that had partially resolved in the 1× CHI mice. Moreover, using Manganese-Enhanced Magnetic Resonance Imaging with R1 mapping - a novel functional neuroimaging technique - we found greater deficits in the rTg4510 mice following 2× CHI compared to 1× CHI. To integrate our findings with prior work in the field, we conducted a systematic review of rodent mild repetitive CHI studies. Following Prisma guidelines, we identified 25 original peer-reviewed papers. Results from our experiments, as well as our systematic review, provide compelling evidence that tau phosphorylation is modified by experimental mild TBI studies; however, changes in p-tau levels are not universally reported. Together, our results provide evidence that repetitive TBIs can result in worse and more persistent neurological deficits compared to a single TBI, but the direct link between the worsened outcome and elevated p-tau could not be established.

Keywords: CTE; Concussion; Neurodegeneration; Rodent behavior; TBI; Tau.

Figure 1:. Characterization of 2-hit CHI model in WT mice

(A) Graphical summary of the overall experimental design is shown. The white boxes indicating a sham surgery, with the red boxes indicating a CHI surgery. (B) Mice were tested in the voluntary running wheel assay and the total distance traveled per day is shown in the bar graph. At 1d post injury (p.i.) the mice 2x CHI (1d) ran significantly less than the sham injured mice. *sham vs. 2x CHI (1d) p=0.032. A Mesoscale discovery Elisa was used to measure p-tau and total tau in a PBS soluble and detergent (TPER) soluble fraction of the cortex brain homogenate at 7d post injury (C) An increase in detergent soluble p-tau (*sham vs. 2x CHI (7d) p=0.017), (D) and total tau (**sham vs. 2x CHI (1d) p=0.0027. *sham vs. 2x CHI (7d) p=0.0257) was seen in the 2x CHI groups. (E) A shift in the ratio of detergent soluble to PBS was found in the 2x CHI groups. (*sham vs. 2x CHI (1d) p=0.045). (F) Ratio of p-tau to total tau is increased in the PBS soluble fraction (*sham vs. 2x CHI (7d) p=0.033). Sham n=11, 1x CHI n=12, 2x CHI (1d) n=4, 2x CHI (7d) n=8.

Figure 2:. Cognitive deficit associated with 2-hit CHI model in WT mice

(A) Graphical summary of the overall experimental design is shown. The white boxes indicate a sham surgery, and the red boxes indicate a CHI surgery. (B) Nesting behavior was measured at 1d after the second surgery (p.i.). ** p<0.005, compared to sham (Dunn’s multiple comparisons test). (n=10 per group) (C) The 6-arm radial arm water maze (RAWM) was run over 2 consecutive days starting at day 14 after the second injury. The number of entries into arms, without finding the escape platform, was recorded as an error. ** p<0.005, *** p<0.0001 compared to sham (Tukey test). Sham n=15, 1x CHI n=14, 2x CHI n=14. (D) Graphical summary of the overall experimental design is shown. (E) The 6-arm RAWM was run over 3 consecutive days starting on day 28 after the second surgery. * p<0.05, ** p<0.005, *** p<0.0001 compared to sham (Tukey test). _‡ p<0.05, _‡‡ p<0.005 compared to 1x CHI (Tukey test). Sham n=14, 1x CHI n=13, 2x CHI n=9.

Figure 3:. Neuroimaging deficit associated with 2-hit CHI model in Tg4510 mice.

(A) Graphical summary of the overall experimental design is shown. The white boxes indicate a sham surgery, and the red boxes indicate a CHI surgery. (B) representative example of T2 MRI, and (C) quantification of the volume of the cortex in the mice (n= 4 per group). (D) representative example of the MEMRI, and (E) quantification of the ΔR1 relaxation rate in the cortex (n=3 per group).

Figure 4:. Effects of 2-hit CHI on p-tau in the Tg4510 mice.

(A) Western blot for PHF1 (pS396/pS404 tau), and total tau in the PBS fraction of cortex homogenate. (B) Quantification of the PHF1 tau normalized to the actin loading control in the cortex of the Tg4510 mice. * p<0.05 compared to sham (Tukey test).

Figure 5:. Methods flow chart.

Identification through searches on two separate web-based platforms yielded 2,301 articles. Abstracts were screened with 2,169 articles excluded. The full-text examination of the remaining 132, resulted in 107 excluded for not tau protein measurements. A total of 25 repetitive mTBI articles that measured tau changes were included in our review.