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A Drosophila model of closed head traumatic brain injury

Rebeccah J. Katzenbergera, Carin A. Loewenb, Douglas R. Wassarmana, Andrew J. Petersena, Barry Ganetzkyb,1, and David A. Wassarmana,1
a Department of Cell and Regenerative Biology, School of Medicine and Public Health, and bLaboratory of Genetics, University of WisconsinMadison, Madison, WI 53706

Contributed by Barry Ganetzky, September 6, 2013 (sent for review August 6, 2013)

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 ies. Closed head TBI was inicted with a mechanical device that subjects ies to rapid acceleration and deceleration. Similar to humans with TBI, ies 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 ies for providing key insights into human TBI that may ultimately provide unique opportunities for therapeutic intervention.
concussion

| insect | chronic traumatic encephalopathy

raumatic brain injury (TBI) is a leading cause of neurological decits and mortality worldwide (1, 2). TBI outcomes result from primary and secondary injuries that cause cell damage and death in the brain. Primary injuries occur during the initial impact and are triggered by external mechanical forces that deform the brain, whereas secondary injuries are triggered by cellular and molecular responses that occur over time in reaction to the primary injuries. TBI outcomes are heterogeneous in the human population owing to variation in the location and strength of primary injuries as well as genetic and environmental factors that affect the severity of primary and secondary injuries. This heterogeneity is one of the most signicant barriers to the development of therapeutic interventions (35). Therefore, research aimed at determining the genetic and environmental factors that affect the severity of primary and secondary injuries is essential for developing treatments for TBI. To investigate the underlying cellular and molecular basis of TBI, we developed a Drosophila melanogaster model. Key advantages of ies are that (i) large numbers of animals can be rapidly and inexpensively analyzed to establish causality between injuries and outcomes, (ii) many molecular and genetic tools are available to investigate the molecules and pathways that underlie injuries, and (iii) outcomes can readily be evaluated over the entire animal lifespan. Thus, ies provide unique opportunities to understand TBI. It is reasonable to expect that TBI can be modeled in ies because Drosophila has already proved to be an extremely useful model for studying other human neurodegenerative disorders (6). In fact, research in ies has already provided novel insights into neurodegeneration, memory, and sleep, all of which are affected in human TBI (68). Additionally, y and human brains have common architectural, cellular, and molecular features. The y brain is bilaterally symmetrical and is joined to the ventral ganglion that innervates the body, similar to the way that the spinal cord innervates the human body (9). The y brain is organized into
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three regions, the protocerebrum, deutocerebrum, and tritocerebrum, which are homologous to the forebrain, midbrain, and hindbrain, respectively, of humans (10). The y brain is composed of functionally diverse neurons with all of the structural features, neurotransmitters, and ion channels found in human neurons (11). Glial cell types and functions in ies are also similar to those in humans (12). For instance, surface glial cells cover brain neurons to form the bloodbrain barrier, and glial cells are part of the innate immune system (13, 14). The y brain is encapsulated by an exoskeleton, also known as the cuticle (15). Like the human cranium, the cuticle is relatively inelastic. It provides protection from the environment, and it denes the structure of the head. The brain is further separated from the cuticle by uid hemolymph, a blood-like carrier system for macrophages and nutrients (16). The topology of the y brain relative to the cuticle is such that an impact to the head or body most likely causes the brain to ricochet and deform against the cuticle resulting in TBI. Here, we describe the development and initial characterization of a y model of TBI. Using the model, we found that fundamental characteristics of human TBI also occur in ies. We also found that primary injuries exacerbate the normal agerelated decline in ies. This may explain why human TBI is associated with cognitive and neurodegenerative disorders that are typical of older individuals and why TBI outcomes are worse in older individuals. Results
The HIT Device Reproducibly Inicts Closed Head TBI in Flies. To inict TBI in ies, we built the high-impact trauma (HIT) device, which consists of a metal spring clamped at one end to a wooden

Signicance
Traumatic brain injury (TBI) occurs when a strong jolt to the head causes damage to brain cells, resulting in immediate and long-term consequences including physical, behavioral, and cognitive problems. Despite the importance of TBI as a major health issue, our understanding of the underlying cellular and molecular mechanisms is limited. To unravel these mechanisms, we have developed a model of TBI in the fruit y, Drosophila melanogaster, where we can apply many powerful experimental tools. The main features of human TBI also occur in ies, suggesting that the underlying mechanisms are conserved. Our studies demonstrate the value of a y model for understanding the consequences of TBI and may ultimately enable development of therapies for their prevention and treatment.
Author contributions: R.J.K., A.J.P., B.G., and D.A.W. designed research; R.J.K., C.A.L., D.R.W., A.J.P., and D.A.W. performed research; D.A.W. contributed new reagents/analytic tools; R.J.K., C.A.L., B.G., and D.A.W. analyzed data; and R.J.K., C.A.L., B.G., and D.A.W. wrote the paper. The authors declare no conict of interest.
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To whom correspondence may be addressed. E-mail: ganetzky@wisc.edu or dawassarman@ wisc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1316895110/-/DCSupplemental.

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Fig. 1. The HIT device was used to inict TBI in 0- to 4-d-old w1118 ies. Images show the HIT device and ies before and after a strike. Shown are (A) the HIT device deected to 90 before a strike, (B) the HIT device immediately after a strike, (C ) 60 ies in a vial before a strike, and (D) after a strike.

board with the free end positioned over a polyurethane pad (Fig. 1A and Fig. S1). A standard plastic vial containing unanesthetized ies that are conned to the bottom quarter of the vial by a stationary cotton ball is connected to the free end of the spring. When the spring is deected and released, the vial rapidly contacts the polyurethane pad, and a mechanical force is delivered to the ies as they contact the vial wall and rebound (Fig. 1B). Individual ies presumably contact the wall with different regions of their head and/or body and with different forces, so primary injuries will vary among ies in the same vial. The lack of penetrating injuries and the randomness of impact location and strength are features of closed head TBI in the human population. Since closed head TBI is the most common form of TBI occurring in falls, sports collisions, and automobile crashes, a y model is of signicant potential value (17). As shown in Fig. 2, the phenotypic effects on ies subjected to HIT device-induced injury are highly reproducible. The strength of the primary injuries inicted by the HIT device can be adjusted, either by varying the extent of spring deection or by varying the number of strikes. Deection of the spring to 90 resulted in an impact velocity of 3.0 m/s (6.7 miles/h) and an average force of 2.5 N. Some ies subjected to a single strike with the spring deected to 90 became temporarily incapacitated and fell to the bottom of the vial; however, they did not incur obvious external damage to the head, body, or appendages (Fig. 1 C and D). During the rst minute after a strike, 8.8 3.8% of ies were incapacitated, lying on their back or side on the bottom of the vial. Most of the incapacitated ies recovered locomotor activity within 5 min (Fig. S2A). Subsequently, their mobility, as measured by climbing ability, was reduced but gradually returned to normal over a 2-d period (Fig. S2B and Table S1). The immediate loss of motor ability, followed by ataxia and gradual recovery of mobility are reminiscent of concussion in humans and are consistent with the idea that the HIT device inicts brain injury in ies.
Primary Injuries That Exceed a Threshold Cause Death Within 24 h. To determine the effect of subjecting ies to multiple TBI incidents, we varied the number of strikes that ies received and measured
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the percentage of ies that died within 24 h. We dene the percentage of ies that died within 24 h after injury as the mortality index at 24 h (MI24). To minimize any variation in outcome associated with differences in age, genotype, or sex, we used 0- to 3-d-old white (w1118) ies in every experiment, with an approximately equal number of males and females. Flies received 010 strikes with the spring deected to 90 and with 5 min recovery periods between strikes. After a single strike, the MI24 was 4.5 1.2 (Fig. 2A). Additional strikes resulted in an increase in the MI24; however, additional strikes did not signicantly increase the MI24 per strike (Fig. 2B). The fact that not all ies died after a single strike indicates that primary injuries cause death within 24 h only if the injuries exceed a specic threshold, where threshold is presumably a composite measure of impact location and strength. Furthermore, the fact that the number of strikes did not affect the MI24 per strike indicates that ies subjected to primary injuries below the threshold do not have increased susceptibility for mortality from subsequent primary injuries. We also examined whether varying the recovery time between repeated strikes had any effect on the MI24. The expectation was that if the time between primary injuries was increased, then secondary injuries or injury repair mechanisms would have more time to occur, thereby altering the threshold for death caused by subsequent primary injuries. Thus, the MI24 would change as the time between strikes changed. To test this hypothesis, ies received four strikes with time intervals from 1 to 60 min between successive strikes. The temporal spacing of strikes did not signicantly affect the MI24 (Fig. S3A). Therefore, over the times tested, the time between successive strikes neither exacerbates nor ameliorates the consequences of multiple primary injuries. The fact that the time between strikes did not affect the MI24 indicates that secondary injuries and injury repair mechanisms either neutralize one another or do not contribute to the primary injury threshold for death within 24 h. Another parameter that could affect the MI24 is the number of ies per vial. To investigate this possibility, we placed 1060 ies per vial in increments of 10 and subjected the vials to four strikes with 5-min recovery periods between strikes. The number of ies in a vial did not signicantly affect the MI24 (Fig. S3B). Thus, in vials containing 1060 ies, contact among ies does not appear to affect the primary injury threshold for death within 24 h.
TBI Reduces Lifespan. To investigate whether multiple TBI incidents have long-term effects on survival, we determined the lifespan of ies that survived beyond 24 h after receiving 010 strikes with 5 min recovery periods between strikes. We assumed that death from primary injuries was complete by 24 h because the percent survival at 24 h was not substantially different from the percent survival at 48 h. Relative to untreated ies, ies that received one strike had a substantially reduced median lifespan (48.3 1.2 d vs. 38.3 3.5 d) and signicantly reduced maximum lifespan (85.7 3.6 d vs. 73.0 0.6 d) (Fig. 2C and Table S2). Additional strikes caused further reductions in the median and maximum lifespan, but an upper limit for median lifespan was reached at four strikes; additional strikes did not further reduce the median lifespan. These results demonstrate that primary injuries that are below the threshold for death within 24 h nonetheless cause a reduction in lifespan that likely results from the long-term consequences of secondary injuries. Age-Associated Processes Lower the Primary Injury Threshold. For subsequent experiments, we developed a standard TBI protocol consisting of four strikes separated by 5 min intervals. This protocol resulted in an MI24 of 19.0 4.0 for 0- to 3-d-old w1118 ies (Fig. 2A). This moderate MI24 is convenient for identifying genetic and environmental factors that enhance or suppress the effect of primary injuries.
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Fig. 2. The effect of the number of strikes on the MI24 and the lifespan of w1118 ies. (A) The MI24 is graphed vs. the number of strikes. MI24 values were normalized to those of untreated ies. (B) Data from A are graphed as the MI24 per strike vs. the number of strikes. The MI24 per strike was not signicantly affected by the number of strikes (P = 0.82, one-way ANOVA). (C ) The percent survival is graphed vs. age for ies that received the indicated number of strikes. Only ies 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 ies each. Exact values and SD for median and maximum lifespans are provided in Table S2.

We used the standard TBI protocol to determine the effect of sex and age on the MI24. w1118 male and female ies at 04 and 2022 d old were assayed separately. Sex did not have a signicant effect on the MI24 at either age (Fig. 3A). In contrast, age did have a signicant effect; the MI24 was approximately twofold higher for older ies compared with younger ies. To more systematically examine the effect of age on the MI24, we examined ies in 12 age groups ranging from 03 to 2829 d old that were treated with the standard TBI protocol. The data revealed an increase in the MI24 as age increased (Fig. 3B). These results suggest that cellular and molecular changes occur during aging that lower the primary injury threshold for death within 24 h.
Age-Associated Processes Enhance Neurodegeneration Due to Brain Injuries. TBI in rodents and humans increases the occurrence of

chronic traumatic encephalopathy (CTE), a form of neurodegeneration that generally appears long after (years to decades in the case of humans) the primary injury (18). To determine if neurodegeneration is also a long-term outcome of TBI in ies and whether it is affected by age at the time of primary injury, we
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performed histological analysis on brains of younger (0- to 4-dold) and older (20- to 21-d-old) ies 14 d after they were subjected to the standard TBI protocol. Neurodegeneration in ies is commonly manifested by the appearance of vacuolar lesions in the brain neuropil, a region enriched for axons, synaptic terminals, and glial cells (6). In contrast with untreated ies whose brains had a smooth and uniform appearance throughout the neuropil (Fig. 4A), treated ies exhibited neuropathology manifested by the appearance of numerous small vacuolar lesions (1.0 m in diameter) (Fig. 4B). We also observed large vacuolar lesions (5.010.0 m in diameter) in injured ies. The incidence of large vacuolar lesions in the central region of the brain increased with the number of times ies were subjected to the standard TBI protocol (Fig. 4C). In addition, the incidence of large vacuolar lesions was higher in older ies subjected to TBI compared with similarly treated younger ies. These data indicate that brain injuries in ies elicit neurodegeneration as a long-term consequence and that the extent of neurodegeneration is determined by the severity of primary injuries and the age at the time of primary injuries.
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Fig. 3. The MI24 of w1118 ies subjected to the standard TBI protocol is not affected by sex but is affected by age at the time of injury. (A) For ies 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 ies. MI24 values were normalized to those of untreated ies. Sex did not have a signicant effect on the MI24 for either 0- to 4-d-old ies (P = 0.27, one-tailed t test) or 20- to 22-dold ies (P = 0.34, one-tailed t test). Age at the time of injury did have a signicant 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 ies. Error bars indicate the SD for at least three independent trials of 60 ies each.

Primary Injuries Activate the Innate Immune Response. One important cause of secondary injuries in TBI is activation of the innate immune response (19). The innate immune response is triggered by pathogen-derived molecules and by endogenous molecules generated by stressed and injured cells (20). A component of this response is the production of proinammatory cytokines, such as tumor necrosis factor (TNF), by microglial cells and astrocytes. In some studies, elevated levels of TNF in the cerebrospinal uid of TBI patients are correlated with unfavorable clinical outcomes, providing evidence of the injurious role of cytokines (21). Likewise, inhibition of TNF shortly after primary injuries in rodents reduces the severity of some deleterious outcomes, such as tissue loss (22). On the other hand, long-term recovery of neurological damage in TBI is impaired in TNF-decient mice, providing evidence of a benecial role of cytokines (23). Therefore, the innate immune response plays a critical but not yet fully understood role in TBI. Flies provide an opportunity to advance our understanding of this role because innate immune response pathways are highly conserved between ies and humans (24). The Toll pathway in ies is analogous with mammalian Toll-like receptor (TLR) pathways, and the Immune deciency (Imd) pathway is analogous with the mammalian TNF pathway. Among the key functions of both the Toll and Imd pathways is the transcriptional activation of antimicrobial peptide (AMP) genes (24). To assess activation of the innate immune response in ies following primary injuries, we used quantitative real-time reverse transcriptionPCR (qRT-PCR) to determine AMP gene mRNA levels in heads of w1118 ies subjected to the standard TBI protocol. To control for the usual increase in expression of innate immunity genes that occurs as a function of age, treated ies were normalized to age-matched untreated ies (25). Flies that survived the standard TBI protocol exhibited an increase in AMP gene expression within 24 h after the primary injuries (Fig. 5). Similar results were observed in 0- to 4-d-old and 20- to 21-dold ies. Some AMP genes were activated within 1 h after the primary injury and all AMP genes were activated within 24 h. Activation of innate immunity genes following primary injuries is
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at least somewhat specic because the expression of other genes such as TAF1 was not similarly increased. Thus, TBI in ies, as in mammals, elicits activation of an innate immune response pathway.
Genetic Background Affects the Primary Injury Threshold. An increasing body of evidence implicates genetic factors in the variable clinical outcomes of TBI in humans (26). To investigate the effect of genetic background on the primary injury threshold in ies, we determined the MI24 for 42 y lines (aged 07 d) that were subjected to the standard TBI protocol. Five of these lines are commonly used as wild-type controls in various Drosophila experiments; the remaining 37 lines contain mutations in genes that encode components of the Imd or Toll pathways (24). The MI24 showed wide variation among the lines, ranging from 14.9 1.2 to 82.5 5.0 (Fig. 6A). Variability was even observed among wild-type controls, for which the MI24 ranged from 20.3 1.4 to 39.9 3.8. Among the mutant lines there were striking differences for different alleles of the same gene. For example, the MI24 for ImdSDK and Imd10191 was 21.0 3.3 and 40.9 4.8, respectively. Additionally, the MI24 did not correlate with either the Imd or the Toll pathway. For example, mutations in Relish (Rel), which encodes the NF-B transcription factor in the Imd pathway, had a signicantly different effect on the MI24 than mutations in kenny (key), which encodes an activator of Rel (RelE20, 47.2 2.3; RelE38, 41.7 3.3; keyf05097, 8.5 1.2; and keyc02831, 7.9 1.9) (24). The wide range of effects for wild-type lines and the lack of coherent effects for Imd and Toll pathway lines suggests that in young ies many genes in the genetic background affect the primary injury threshold for death within 24 h. We suspect that, in some cases, the genetic background masks the effect of mutations in Imd or Toll pathway genes making it impossible to determine from these data whether the innate immune response specically inuences the MI24. Genetic Background Affects the Age-Dependent Reduction of the Primary Injury Threshold. To investigate the effect of genetic

background on the primary injury threshold in older ies, we

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Fig. 4. Treatment of w1118 ies with the standard TBI protocol causes neurodegeneration. Images of sections of y brains from 14- to 18-d-old (A, Upper and Lower) untreated and (B, Upper and Lower) treated ies are shown. The treated y received the standard TBI protocol at 04 d old. The large vacuole indicated by the arrow in the magnied image (B, Lower) is 7.75 m in diameter. (Scale bars in A, Upper and B, Upper: 100 m. Images in Lower panels are magnied an additional 5.) (C ) The number of large, i.e., 5.0- to 10.0-m-diameter vacuoles in the central region of the brain is graphed vs. the number of times ies were subjected to the standard TBI protocol. Brain sections chosen for analysis were at equivalent depths in the brain, as exemplied by A and B. Multiple treatments with the standard TBI protocol occurred over successive days. All ies were analyzed 14 d after the time of the rst treatment, i.e., ies labeled 1418 d were treated beginning at 04 d old, and ies labeled 3435 d were treated beginning at 2021 d old. Error bars indicate the SD for at least three heads. When treated with one standard TBI protocol, young ies (1418 d old) developed signicantly fewer large vacuoles that older ies (3435 d old) (P = 0.003, one-tailed t test), but the difference between young and older ies was not signicant 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 ies developed signicantly more large vacuoles when treated with three vs. one standard TBI protocol (P = 0.029 for young ies and P = 0.03 for older ies, one-tailed t test).

reexamined the 42 y lines at 2027 d old. The results largely paralleled those observed in young ies in that wide variability was observed in the MI24, and the variability did not correlate with the Imd or the Toll pathway (Fig. 6B). In addition, for every line, the MI24 for 20- to 27-d-old ies was higher than the MI24 for 0- to 7-d-old ies (Fig. S4). MIs24 of younger and older ies had a correlation coefcient (r) of 0.77. Thus, in all genetic backgrounds, age appears to be a critical determinant of the primary injury threshold for death within 24 h. However, the MI24 in younger ies was not entirely predictive of the extent of increase in the MI24 in older ies. For example, 0- to 7-d-old keyf05097 and spatzle (spz3) ies had similar MIs24 (14.9 1.2 and 15.9 3.2, respectively) but 20- to 27-d-old keyf05097 and
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spz3 ies had substantially different MIs24 (22.5 4.2 and 55.1 9.4, respectively) (Fig. S4). Conversely, 0- to 7-d-old necrotic (nec2) and TAK1-associated binding protein 2 (Tab2EY00380) ies had substantially different MIs24 (17.6 4.1 and 44.2 4.6, respectively) but 20- to 27-d-old nec2 and Tab2EY00380 ies had similar MIs24 (62.4 5.9 and 65.1 6.3, respectively). These data indicate that the genes that affect the primary injury threshold for death within 24 h in young ies are different from those that affect the age-dependent reduction in the primary injury threshold.
Variation in Primary Injury Threshold Correlates with Variation in Longevity. Because the primary injury threshold varies with

age, we examined the correlation between MI24 and longevity


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Fig. 5. Treatment of w1118 ies with the standard TBI protocol activates the innate immune response. The histogram shows the fold-increase in mRNA levels in treated vs. untreated ies at the indicated timepoints after treatment of 0- to 4- or 20- to 21-dold ies. 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.

in the absence of TBI. We analyzed 14 y lines, including lines with low, average, or high MI24 after treatment with the standard TBI protocol. We found a negative linear relationship between the MI24 and the median lifespan for both 0- to 7-d-old and 20- to 27-d-old ies (Fig. 7A). The correlation coefcient r

between the MI 24 and the median lifespan was 0.67 for 0to 7d-old ies and 0.84 for 20- to 27-d-old ies. Thus, the longevity of a particular y line and its primary injury threshold for death within 24 h are largely determined by the same genetic factors.

Fig. 6. The MI24 is strongly affected by genetic background. Histograms show the MI24 for 42 different y lines treated with the standard TBI protocol and tested at (A) 07 d old and (B) 2027 d old. The MI24 vs. y genotype is graphed. MI24 values were normalized to those of untreated ies. Genotypes are listed in Table S3. White bars indicate y lines containing mutations in genes implicated in the Imd pathway. Gray bars indicate y lines containing mutations in genes implicated in the Toll pathway. Black bars indicate y lines commonly used as wildtype controls in Drosophila experiments. For a reference, y line number 7 is w1118. Error bars indicate the SD for at least three independent trials of 60 ies each.

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signicantly associated with increasing age, 15% at <18 y, 44% at 1859 y, and 52% at >59 y (29). These similarities between ies in our TBI model and human TBI patients strongly indicate that ies and humans incur TBI through similar cellular and molecular mechanisms.
The Fly Model of TBI Can Provide Unique Insights into Human TBI.

Fig. 7. The susceptibility of different y lines to TBI-induced mortality is inversely correlated with their respective longevity. (A) The MI24 of 20- to 27d-old ies is graphed vs. the median lifespan for 14 of the y lines that were analyzed in Fig. 6. The lines that were analyzed are listed in Table S3. RelE20 and RelE38 ies 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 ies of the indicated age that were raised at either 18 C or 25 C. MI24 values were normalized to those of untreated ies. Temperature had a signicant effect on the MI24 for both 0- to 4-d-old ies (P = 0.012, one-tailed t test) and 21- to 22-d-old ies (P = 0.001, one-tailed t test). Error bars indicate the SD for at least three independent trials of 60 ies each.

To test this proposal, we extended the lifespan of ies and examined the effect on the MI24. To extend the lifespan, we raised ies at 18 C rather than at 25 C, the temperature at which ies were raised for all of the prior experiments. Untreated w1118 ies raised at 18 C had a signicantly longer median lifespan relative to ies raised at 25 C (68.5 0.8 d vs. 48.3 1.2 d, P < 0.0001, one-tailed t test). After treatment with the standard TBI protocol, w1118 ies raised at 18 C had a signicantly lower MI24 than equivalent-age ies raised at 25 C (Fig. 7B). The negative correlation between natural longevity and MI24 was observed for both 0- to 3-d-old and 21- to 22-d-old ies. Thus, ies of the same genotype and chronological age but different median lifespan differed in their primary injury threshold for death within 24 h. This result indicates that environmental factors such as temperature determine both the longevity of a particular line in the absence of injury and its primary injury threshold for death within 24 h. Discussion
Flies Can Model Human TBI. Here, we have described the development and initial characterization of a y model of TBI. We found that inicting mechanical injury on ies by rapid acceleration and deceleration produces outcomes that are similar to outcomes characteristic of closed head TBI in humans (1, 2). These outcomes include temporary incapacitation (Fig. S2A), ataxia (Fig. S2B and Table S1), activation of the innate immune response (Fig. 5), neurodegeneration (Fig. 4), and death (Fig. 2). In addition, we found that risk factors for mortality in ies are shared with humans. Our data indicate that the MI24 following injury does not vary with sex (Fig. 3A). Similarly, a study of 914 women and 916 men by Coimbra et al. concluded that sex is not a risk factor for mortality in TBI patients (27). The MI24, however, is dependent on the age of ies at the time of injury (Figs. 3B and 6). Likewise, in a study of 5,612 TBI patients, Hukkelhoven et al. found that the percent mortality within 6 mo of primary injuries increased with age, 21% at <35 y and 52% at >55 y (28). Also, in a study of 244 TBI patients, Dhandapani et al. found that the percent mortality within 1 mo of primary injuries was
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Because the y TBI model enables us to analyze many animals throughout their lifespan, we have found several properties of TBI that may be of clinical relevance: age at the time of primary injury as well as the number of primary TBI incidents are risk factors for neurodegeneration (Fig. 4C); the number of primary TBI incidents affects longevity (Fig. 2C and Table S2); the MI24 following TBI varies with genetic background (Fig. 6); and nally, the genes that affect the susceptibility to mortality are different at different ages (Fig. 6 and Fig. S4). Although it is difcult to obtain exactly comparable information from human TBI patients, available data are consistent with our conclusions. For example, as in ies, human studies suggest that age and the severity of primary injury are important factors in CTE development (30, 31). In addition, studies in different rodent strains as well as genetic association studies in TBI patients indicate that, as in ies, TBI outcomes depend on genetic background (26, 32). Finally, we found that the genetic and environmental factors that affect longevity in the absence of injury also affect the susceptibility to mortality following TBI (Fig. 7). Two lines of evidence suggest that fractional age rather than chronological age per se is a risk factor for TBI-induced mortality in humans as it is in ies, where fractional age is dened as chronological age relative to the maximum longevity of ies of that genotype. First, drugs such as rapamycin and resveratrol that alleviate TBI outcomes in mammals also extend normal longevity (3336). Second, the gene apolipoprotein E (apoE ), implicated in controlling longevity, is also found to be a risk factor for TBI outcomes in mammals (37, 38). Interestingly, rapamycin, resveratrol, and apoE also appear to affect the severity of neurodegeneration in Alzheimers disease, which is phenotypically similar to CTE (18, 39). Collectively, these ndings suggest that secondary injuries in TBI accelerate some normal aging processes in the brain. This idea may explain why the average age of onset (AAO) for CTE is substantially earlier than for Alzheimers disease (CTE: AAO = 42.8 12.7 y, range = 2576 y; Alzheimers disease: AAO = 72.8 6.8 y, range = 4997 y) (40, 41). In summary, our studies demonstrate that a y TBI model offers considerable potential for understanding the cellular and molecular mechanisms that underlie the pathological consequences of human TBI and may ultimately facilitate development of unique therapeutic strategies. Materials and Methods
Fly Lines and Culturing. Flies were maintained on standard molasses medium at 25 C unless otherwise stated. All y lines were obtained from the Bloomington Stock Center except for ImdSDK and Imd10191, which were obtained from Mimi Shirasu-Hiza (Columbia University, New York). Genotypes of ies used in Figs. 6 and 7A and Fig. S4 are described in Table S3. Assays for TBI Outcomes. Construction and use of the HIT device is described in Fig. S1. The impact velocity of the HIT device was determined by analyzing images from a high-speed camera. MIs24 were determined by analyzing at least 180 ies (three experiments of 60 ies). The percentage of ies that were incapacitated was determined by analyzing movie recordings of ies after a single strike. Flies that did not show obvious locomotor activity were considered incapacitated. Before each timepoint, hitting the benchtop upon which the y vial sat was used to stimulate locomotion. Climbing, lifespan, histology, and qRT-PCR assays were performed as described in Petersen et al. (14, 42).

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ACKNOWLEDGMENTS. We thank Nicole Bertram, Grace Boekhoff-Falk, members of the D.A.W. and B.G. laboratories, and Ron Kalil for their insights that greatly improved this research; Russell Little for help in determining the impact

velocity; and Satoshi Kinoshita for performing the histology. This work was supported by National Institutes of Health Grants R01 NS059001 (to D.A.W.) and R01 AG033620 (to B.G.).

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