A Drosophila model of closed head traumatic brain injury

Abstract

Traumatic brain injury (TBI) is a substantial health issue worldwide, yet the mechanisms responsible for its complex spectrum of pathologies remains largely unknown. To investigate the mechanisms underlying TBI pathologies, we developed a model of TBI in Drosophila melanogaster. The model allows us to take advantage of the wealth of experimental tools available in flies. Closed head TBI was inflicted with a mechanical device that subjects flies to rapid acceleration and deceleration. Similar to humans with TBI, flies with TBI exhibited temporary incapacitation, ataxia, activation of the innate immune response, neurodegeneration, and death. Our data indicate that TBI results in death shortly after a primary injury only if the injury exceeds a certain threshold and that age and genetic background, but not sex, substantially affect this threshold. Furthermore, this threshold also appears to be dependent on the same cellular and molecular mechanisms that control normal longevity. This study demonstrates the potential of flies for providing key insights into human TBI that may ultimately provide unique opportunities for therapeutic intervention.

Keywords: chronic traumatic encephalopathy; concussion; insect.

Fig. 1.

The HIT device was used to inflict TBI in 0- to 4-d-old w¹¹¹⁸ flies. Images show the HIT device and flies before and after a strike. Shown are (A) the HIT device deflected to 90° before a strike, (B) the HIT device immediately after a strike, (C) 60 flies in a vial before a strike, and (D) after a strike.

Fig. 2.

The effect of the number of strikes on the MI₂₄ and the lifespan of w¹¹¹⁸ flies. (A) The MI₂₄ is graphed vs. the number of strikes. MI₂₄ values were normalized to those of untreated flies. (B) Data from A are graphed as the MI₂₄ per strike vs. the number of strikes. The MI₂₄ per strike was not significantly affected by the number of strikes (P = 0.82, one-way ANOVA). (C) The percent survival is graphed vs. age for flies that received the indicated number of strikes. Only flies that survived 24 h after treatment were included in the analysis. The line at 50% indicates the median lifespan. Error bars indicate the SD for at least three independent trials of 60 flies each. Exact values and SD for median and maximum lifespans are provided in Table S2.

Fig. 3.

The MI₂₄ of w¹¹¹⁸ flies subjected to the standard TBI protocol is not affected by sex but is affected by age at the time of injury. (A) For flies treated with the standard TBI protocol, the MI₂₄ is graphed vs. sex. The experiment was performed with 0- to 4-d-old and 20- to 22-d-old flies. MI₂₄ values were normalized to those of untreated flies. Sex did not have a significant effect on the MI₂₄ for either 0- to 4-d-old flies (P = 0.27, one-tailed t test) or 20- to 22-d-old flies (P = 0.34, one-tailed t test). Age at the time of injury did have a significant effect on the MI₂₄ for both males (P = 0.004, one-tailed t test) and females (P = 0.002, one-tailed t test). (B) The MI₂₄ is graphed vs. age at the time of treatment with the standard TBI protocol. MI₂₄ values were normalized to those of untreated flies. Error bars indicate the SD for at least three independent trials of 60 flies each.

Fig. 4.

Treatment of w¹¹¹⁸ flies with the standard TBI protocol causes neurodegeneration. Images of sections of fly brains from 14- to 18-d-old (A, Upper and Lower) untreated and (B, Upper and Lower) treated flies are shown. The treated fly received the standard TBI protocol at 0–4 d old. The large vacuole indicated by the arrow in the magnified image (B, Lower) is 7.75 μm in diameter. (Scale bars in A, Upper and B, Upper: 100 μm. Images in Lower panels are magnified an additional 5×.) (C) The number of large, i.e., 5.0- to 10.0-μm-diameter vacuoles in the central region of the brain is graphed vs. the number of times flies were subjected to the standard TBI protocol. Brain sections chosen for analysis were at equivalent depths in the brain, as exemplified by A and B. Multiple treatments with the standard TBI protocol occurred over successive days. All flies were analyzed 14 d after the time of the first treatment, i.e., flies labeled 14–18 d were treated beginning at 0–4 d old, and flies labeled 34–35 d were treated beginning at 20–21 d old. Error bars indicate the SD for at least three heads. When treated with one standard TBI protocol, young flies (14–18 d old) developed significantly fewer large vacuoles that older flies (34–35 d old) (P = 0.003, one-tailed t test), but the difference between young and older flies was not significant for treatment with two or three standard TBI protocols (P = 0.07 and P = 0.07, one-tailed t test, respectively). Both young and older flies developed significantly more large vacuoles when treated with three vs. one standard TBI protocol (P = 0.029 for young flies and P = 0.03 for older flies, one-tailed t test).

Fig. 5.

Treatment of w¹¹¹⁸ flies with the standard TBI protocol activates the innate immune response. The histogram shows the fold-increase in mRNA levels in treated vs. untreated flies at the indicated timepoints after treatment of 0- to 4- or 20- to 21-d-old flies. The AMP genes examined were Attacin-C (AttC), Diptericin B (DiptB), and Metchnikowin (Mtk), and the control gene examined was TBP-associated factor 1 (TAF1). Error bars indicate the SEM for at least three independent trials.

Fig. 6.

The MI₂₄ is strongly affected by genetic background. Histograms show the MI₂₄ for 42 different fly lines treated with the standard TBI protocol and tested at (A) 0–7 d old and (B) 20–27 d old. The MI₂₄ vs. fly genotype is graphed. MI₂₄ values were normalized to those of untreated flies. Genotypes are listed in Table S3. White bars indicate fly lines containing mutations in genes implicated in the Imd pathway. Gray bars indicate fly lines containing mutations in genes implicated in the Toll pathway. Black bars indicate fly lines commonly used as wild-type controls in Drosophila experiments. For a reference, fly line number 7 is w¹¹¹⁸. Error bars indicate the SD for at least three independent trials of 60 flies each.

Fig. 7.

The susceptibility of different fly lines to TBI-induced mortality is inversely correlated with their respective longevity. (A) The MI₂₄ of 20- to 27-d-old flies is graphed vs. the median lifespan for 14 of the fly lines that were analyzed in Fig. 6. The lines that were analyzed are listed in Table S3. Rel^E20 and Rel^E38 flies had the same MI₂₄ and median lifespan, so they appear as a single open box on the graph. (B) The MI₂₄ is graphed for w¹¹¹⁸ flies of the indicated age that were raised at either 18 °C or 25 °C. MI₂₄ values were normalized to those of untreated flies. Temperature had a significant effect on the MI₂₄ for both 0- to 4-d-old flies (P = 0.012, one-tailed t test) and 21- to 22-d-old flies (P = 0.001, one-tailed t test). Error bars indicate the SD for at least three independent trials of 60 flies each.