Several million traumatic brain injury (TBI) events are reported in the United States annually. However, mild TBI events often go unreported, and mild and repetitive mild TBI conditions are challenging to model. Fruit flies (Drosophila melanogaster) have gained traction for the study of TBI. The best-characterized fly TBI model is the high-impact trauma (HIT) method. We replicated the HIT method and confirmed several previous findings at the standard level of injury severity. We then expanded upon the HIT model by characterizing mortality across three reduced levels of injury severity. Importantly, we found reduced mortality with reduced injury severity and synergistic effects on mortality in response to repetitive TBI by our moderate injury conditions. Last, we compared moderate, repetitive TBI to a single severe TBI via assessment of the pattern of mortality and geotaxis performance in the 24 h following TBI. We found the number and severity of injuries could result in different patterns of death, while all TBI conditions led to impaired geotaxis compared to uninjured flies at 0.5 h and 6 h post-TBI. Thus, we have extended a wellcharacterized model of TBI in flies, and shown the utility of this model for making unique insights into TBI across various severities, injury numbers, and time-points post-injury.

Running Title: Expansion of a fly TBI model and synergistic effects 10 11 Expansion of a fly TBI model and synergistic effects 2 ABSTRACT 12Several million traumatic brain injury (TBI) events are reported in the United States annually. 13 However, mild TBI events often go unreported, and mild and repetitive mild TBI conditions are 14 challenging to model. Fruit flies (Drosophila melanogaster) have gained traction for the study of 15 TBI. The best-characterized fly TBI model is the high-impact trauma (HIT) method. We 16 replicated the HIT method and confirmed several previous findings at the standard level of injury 17 severity. We then expanded upon the HIT model by characterizing mortality across three 18 reduced levels of injury severity. Importantly, we found reduced mortality with reduced injury 19 severity and synergistic effects on mortality in response to repetitive TBI by our moderate injury 20 conditions. Last, we compared moderate, repetitive TBI to a single severe TBI via assessment 21 of the pattern of mortality and geotaxis performance in the 24 h following TBI. We found the 22 level of α = 0.05. Only vials containing at least 30 flies were used in median MI24/HIT 130 comparisons. Full count data were used for comparisons of trends across MI24/HIT data; overall 131 MI24 values were divided by their respective HIT number, plotted across 1-4HITs, and fitted 132 using the linear fit mode within the nonlinear regression analysis toolkit (GraphPad Prism 7). 133 Lines were fitted using the least squares fit mode, compared to a hypothetical slope of zero via 134 the extra sum-of-squares F test at a level of α = 0.05, and the 95% confidence interval (CI) 135 determined asymmetrically. Dead fly counts from determination of death across time-points 136 were compared by Fisher's Exact Tests of 4x2 matrices in R (version 3.5.1) with post-hoc, 137 pairwise comparisons (dead within window:dead outside of window) via 2x2 Fisher's Exact 368 University of Wisconsin -Green Bay for careful reading of the manuscript and suggested 369 revisions.