Expansion of a fly TBI model to four levels of injury severity reveals synergistic effects of repetitive injury for moderate injury conditions

Abstract

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.

Repetitive mild traumatic brain injury causes synergistic effects on mortality

“…Stocks of w 1118 (BL 5905) and y 1 w 1 (BL 1495) were obtained from the Bloomington Drosophila Stock Center (Bloomington, Indiana, USA). Flies were maintained in a humidified 25°C incubator, on a 12H:12H light:dark cycle, and on a standard glucose-cornmeal-yeast food (Putnam et al 2019).…”

“…Methods for TBI were based on those reported previously (Katzenberger et al 2013; Putnam et al 2019). Briefly, flies were collected using CO2 anesthesia (35 – 60 flies per vial), and subjected to TBI by 5 days after eclosion.…”

“…We previously reported an extended fly (D. melanogaster) model of TBI based on the high-impact trauma (HIT) method (Katzenberger et al 2013; Putnam et al 2019). 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).…”

“…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 HIT number (MI24/HIT) (Katzenberger et al 2013; Putnam et al 2019). If mortality from each HIT is additive then the MI24/HIT values should be equivalent, but, if mortality is synergistic, then the MI24/HIT values should become increasingly large with HIT number and differ.…”

The polarity protein Dlg5 regulates collective cell migration during Drosophila oogenesis

“…To generate the ubiquitous expression construct Ubi-Dlg5 . TagRFP-T , Dlg5 CDS was amplified from LD32687 (DGRC) and subcloned into the vector pUbRPT.attB [42]. After sequencing, the construct was inserted into the ZH-51C attP docking site using established PhiC31-based methods.…”