Description

Millions of Americans suffer traumatic brain injury (TBI) each year (Taylor et al. 2017). The vast majority (~90%) of TBI events in the U.S. are mild TBIs, which includes concussions (Cassidy et al. 2004). Mild TBI commonly causes temporary symptoms, but repetitive mild TBI (rmTBI) is associated with long-term consequences, which may take years to manifest (Bailes et al. 2013). Current animal models of rmTBI have drawbacks which contribute to the scarcity of evidence for mechanistic bases of dysfunction and disease.

We previously reported an extended fly (D. melanogaster) model of TBI based on the spring-based, high-impact trauma (HIT) method (Katzenberger et al. 2013; Putnam et al. 2019). We showed that deflection of the spring and attached vial of flies to 90° from horizontal resulted in severe injuries as evidenced by rates of mortality, but that reduced deflections (60-80°) had lesser effects (Putnam et al. 2019). Moreover, we showed that repetitive injuries at the relatively moderate deflections of 70° and 80°, but not severe injuries at 90° nor mild injuries at 60°, resulted in a synergistic effect on mortality (Putnam et al. 2019). One potentially confounding issue preventing identification of synergistic effects at 60° was the low mortality rate after the 1-4 injuries administered (Putnam et al. 2019). We chose to further investigate the relationship between mortality and rmTBI using the 60° deflection by extending the injury number to 5-12 strikes.

The main outcome measured using the HIT model of TBI in flies is the mortality index at 24 hours (MI24) (Katzenberger et al. 2013). The pattern of mortality across varied HIT numbers can be assessed by dividing each MI24 value by its respective strike number (MI24/strike) (Katzenberger et al. 2013; Putnam et al. 2019). If mortality from each strike is additive then the MI24/ strike values should be equivalent, but, if mortality is synergistic, then the MI24/strike values should become increasingly large with strike number. Neither, w¹¹¹⁸ nor y¹w¹ flies showed any differences in MI24/ strike values across 5-8 strikes (Fig. 1A, 1B respectively). However, by 10 strikes both genotypes showed larger MI24/strike values than for 5 or 6 strikes, and we found a more pronounced effect at 12 strikes (Fig. 1A, 1B). To more fully assess the pattern of mortality across injury number, we checked the MI24/strike data using a line fit. When we used data across only 5-8 strikes neither w¹¹¹⁸ nor y¹w¹ flies deviated from fit to a zero-slope line (w¹¹¹⁸: p = 0.34; y¹w¹: p = 0.45), consistent with additive effects as seen previously. However, inclusion of data from 5-12 strikes for each of w¹¹¹⁸ and y¹w¹ resulted in positive slope lines that significantly deviated from zero-slope lines (Fig. 1C, 1D respectively; w¹¹¹⁸: p = 0.004; y¹w¹: p = 0.006), consistent with a synergistic relationship.

Our data show that mild TBI causes synergistic effects on mortality after a requisite number of injuries, possibly because cumulative cellular stress surpasses a critical threshold. We expect that many of the secondary injury pathways active in animals following our mild TBI (60°) methodology overlap with pathways previously reported (Katzenberger et al. 2015; Katzenberger et al. 2016; Anderson et al. 2018; Saikumar et al. 2020; Swanson, Rimkus, et al. 2020; Swanson, Trujillo, et al. 2020). However, the identity of any specific factor(s) which set or scale the sensitivity to rmTBI remain unknown. Identification of such factor(s) may allow us to develop diagnostic tools more sensitive to tracking rmTBI outcomes and lead to identification of genetic risk factors which contribute to disease following rmTBI.