Greater neurobehavioral deficits occur in adult mice after repeated, as compared to single, mild traumatic brain injury (mTBI)

Abstract

Mild traumatic brain injury (mTBI) accounts for the majority of all brain injuries and affected individuals typically experience some extent of cognitive and/or neuropsychiatric deficits. Given that repeated mTBIs often result in worsened prognosis, the cumulative effect of repeated mTBIs is an area of clinical concern and on-going pre-clinical research. Animal models are critical in elucidating the underlying mechanisms of single and repeated mTBI-associated deficits, but the neurobehavioral sequelae produced by these models have not been well characterized. Thus, we sought to evaluate the behavioral changes incurred after single and repeated mTBIs in mice utilizing a modified impact-acceleration model. Mice in the mTBI group received 1 impact while the repeated mTBI group received 3 impacts with an inter-injury interval of 24h. Classic behavior evaluations included the Morris water maze (MWM) to assess learning and memory, elevated plus maze (EPM) for anxiety, and forced swim test (FST) for depression/helplessness. Additionally, species-typical behaviors were evaluated with the marble-burying and nestlet shredding tests to determine motivation and apathy. Non-invasive vibration platforms were used to examine sleep patterns post-mTBI. We found that the repeated mTBI mice demonstrated deficits in MWM testing and poorer performance on species-typical behaviors. While neither single nor repeated mTBI affected behavior in the EPM or FST, sleep disturbances were observed after both single and repeated mTBI. Here, we conclude that behavioral alterations shown after repeated mTBI resemble several of the deficits or disturbances reported by patients, thus demonstrating the relevance of this murine model to study repeated mTBIs.

Keywords: Behavior; Learning and memory; Mild; Repetitive; Sleep; Traumatic brain injury.

Fig. 1

Timeline of experimental procedures. Two separate behavioral paradigms are depicted. (A) Mice were assessed using the Morris water maze (MWM), marble burying test, and nestlet shredding test. (B) Following acclimation to LABORAS cages, mice were evaluated using LABORAS, elevated-plus maze (EPM), and forced swim test (FST).

Fig. 2

Weight drop device injury parameters. (A) Increasing impact height resulted in a significantly longer duration of unconsciousness as determined by return of righting reflex time. (B) Representative pressure sensing film and associated impact force delivered by the weight drop device from a 70 cm drop height. (C) All mTBI groups exposed to the 70 cm impact height experienced a significant loss of consciousness compared to shams. (* indicates p <0.05 to sham; $\widehat{\mathrm{i}}\text{ndicates}$ p <0.05 to 60 cm; # indicates p <0.05 to 70 cm; + indicates p <0.05 to rmTBI-1).

Fig. 3

Repeated mild traumatic brain injury (rmTBI) induces impairments in spatial learning and short-term recall. A 3 week reversal/ double-reversal paradigm of the Morris water maze was used to assess spatial learning (acquisition) (A–D) and short-term spatial recall (probe) (E–H) on post-injury days 7–11, 14–18, and 21–25. During acquisition, mice receiving rmTBIs took longerto find the hidden platform (A) and travelled a greater distance before reaching the platform (B) throughout the 3 weeks. (C) Compared to shams, the rmTBI group spent significantly more time in the former target quadrant during week 3 (double-reversal) acquisition testing. (D) There were no differences in the average swim speed between groups. Both mTBI and rmTBI groups spent significantly less time in the current target quadrant (E) and significantly more time in the formertarget quadrant (G) compared to shams during the week 3 (double-reversal) probe. There were no differences between groups in the distance travelled (F) or average swim speed (H) during probe tests. (* indicates p <0.05 to sham; $\widehat{\mathrm{i}}\text{ndicates}$ p <0.05 to mTBI).

Fig. 4

Repeated mild traumatic brain injury (rmTBI) causes reduced performance in tasks measuring species-typical behaviors. Compared to shams, mice receiving rmTBIs buried significantly less marbles in the marble burying test (A) and built poorer nests in the nestlet shredding test (B). Tests were performed on post-injury days 10 and 11, respectively. Data are presented are percent change from baseline. (* indicates p <0.05 to sham).

Fig. 5

Single and repeated mild traumatic brain injury (mTBI) results in sleep alterations. (A) Sleep was determined by measuring immobility time in the LABORAS cages for 7 consecutive days post-injury (PIDs 2–9). Data was further separated by light/dark cycle. (B) Mice receiving rmTBIs experienced an acute increase in sleep in the dark cycle compared to shams. (C) Single mTBI resulted in increased sleep in the light cycle compared to shams and rmTBIs. (* indicates p <0.05 to sham; $\widehat{\mathrm{i}}\text{ndicates}$ p <0.05 to rmTBI).

Fig. 6

Mild traumatic brain injury (mTBI) does not alter anxiety or depression-like behaviors. The elevated plus maze was performed on post-injury day 9 (PID 9) to determine anxiolytic behavior by quantifying open arm duration (A) and entries (B). (C) The forced swim test, performed on PID 10, was used to assess depression by calculating the amount of time spent immobile. No significant differences were observed.