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Glial immune-related pathways mediate effects of closed head traumatic brain injury on behavior and lethality in Drosophila

['Bart Van Alphen', 'Department Of Neurobiology', 'Northwestern University', 'Evanston', 'Illinois', 'United States Of America', 'Samuel Stewart', 'Marta Iwanaszko', 'Department Of Preventive Medicine Biostatistics', 'Feinberg School Of Medicine']

Date: 2022-02 

In traumatic brain injury (TBI), the initial injury phase is followed by a secondary phase that contributes to neurodegeneration, yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI termed Drosophila Closed Head Injury (dCHI), where well-controlled, nonpenetrating strikes are delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. TBI results in significant changes in the transcriptome, including up-regulation of genes encoding antimicrobial peptides (AMPs). To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB, important for AMP induction, and found that while sleep and motor function effects were reduced, lethality effects were enhanced. Similarly, loss of most AMP classes also renders flies susceptible to lethal TBI effects. These studies validate a new Drosophila TBI model and identify immune pathways as in vivo mediators of TBI effects.

Funding: This study was funded by the Department of Defense (W81XWH-20-1-0211 and W81XWH-16-1-0166 to RA) and the Defense Advanced Research Projects Agency (D12AP00023). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The code used to generate the results that are reported in this study are available from allada-lab@northwestern.edu upon reasonable request. Data supporting the findings are available in the respective supplemental data files, while the NGS data files were deposited in the Gene Expression Omnibus (GEO) under the accession code GSE164377.

Copyright: © 2022 van Alphen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We have chosen to publish the science that includes Dr. Van Alphen’s work, as well as significant contributions of several authors, since it addresses an important scientific and societal problem, and since it was conducted with grant funding that carries the responsibility to communicate scientific discoveries with the broader community. The science presented here is independent of the personal views of any of the investigators.

The research presented here was conducted over several years by a large diverse and multidisciplinary group of scientists. We (the co-authors) became aware of the offensive online posts made by Dr. Van Alphen only shortly before his death, while the manuscript was already in revision. We were shocked by these comments and condemn them as anathema to our core values.

PLOS Biology has tried to ensure that this article’s evaluation was not affected by competing interests and adhered to the journal’s high standards for fair, rigorous, and objective peer review. Given the details of this case we have honored the positions of editors when they requested anonymity. The PLOS Biology Staff Editors are listed as handling editors on this article for this reason. The peer review process involved the PLOS Biology Staff Editors and six external subject matter experts, including two members of our Editorial Board.

After becoming aware of this issue, we took into consideration that the other authors of the article may have been unaware of the first author’s activities, that the research reported in this article was likely unaffected by them, and that the issue had come to light after the authors had invested significant effort and resources in conducting this study and revising the manuscript to address issues raised by PLOS Biology reviewers. Given these factors, we decided to continue considering the article for publication.

During the second round of review of this article, it came to light that the first author, under a pseudonym, had engaged in activities which do not align with PLOS’ values. Please note that PLOS strongly condemns all discriminatory behaviors, attitudes, and actions.

Most Drosophila TBI models [ 40 , 41 ] deliver impacts to the entire body, not just the head, and thus, one cannot definitively attribute ensuing phenotypes to TBI. More recently, a Drosophila TBI assay was published that uses head compression in flies just recovered from anesthesia to induce TBI [ 42 ]. To remove the confounds of bodily injury and anesthesia, we have developed a head-specific Drosophila model for TBI, Drosophila Closed Head Injury (dCHI). Here, we show that by delivering precisely controlled, nonpenetrating strikes to an unanesthetized fly’s head, we can induce cell death and increased mortality in a dose-dependent manner. In addition, TBI results in impaired motor control and decreased, fragmented sleep in flies that survive the injury. Impaired motor control persists for many days after TBI, while the sleep phenotype disappears after 3 days. In wild-type flies, TBI results in changes in glial gene expression, where many immune-related genes, including most antimicrobial peptides (AMPs) are up-regulated 24 to 72 hours after injury. TBI-induced behavioral phenotypes do not occur in mutants lacking the master immune regulator nuclear factor kappa B (NF-κB) Relish (Rel), even though TBI-induced mortality is greatly induced in these mutants, suggesting that these impairments are due to immune activation rather than the injury itself. CRISPR deletions of most AMP classes increase TBI-induced mortality, but survival is increased in flies lacking Defensin, suggesting that the innate immune response to TBI in Drosophila can have both beneficial and detrimental effects. Together, these results establish a platform where powerful Drosophila genetics can be utilized to study the complex cascade of secondary injury mechanisms that occur after TBI in order to genetically disentangle its beneficial and detrimental effects.

Trauma-induced changes in glial gene expression are a highly conserved feature of both mammalian [ 27 , 28 ] and Drosophila glia [ 29 – 32 ] (reviewed in [ 33 ]). In Drosophila, glia are able to perform immune-related functions [ 32 , 34 ]. Ensheathing glia can act as phagocytes and contribute to the clearance of degenerating axons from the fly brain [ 29 , 31 , 35 ]. The Drosophila innate immune system is highly conserved with that of mammals and consists primarily of the Toll, Immunodeficiency (Imd), and Janus Kinase protein and the Signal Transducer and Activator of Transcription (JAK–STAT) pathways, which, together, combat fungal and bacterial infections [ 36 , 37 ]. Dysregulation of cerebral innate immune signaling in Drosophila glial cells can lead to neuronal dysfunction and degeneration [ 38 , 39 ], suggesting that changes in glia cells could underlie secondary injury mechanisms in our Drosophila model of TBI.

To study the mechanisms that mediate TBI pathology in vivo over time, we employ the fruit fly Drosophila melanogaster, a model organism well suited to understanding the in vivo genetics of brain injury. Despite considerable morphological differences between flies and mammals, the fly brain operates on similar principles through a highly conserved repertoire of neuronal signaling proteins, including a large number of neuronal cell adhesion receptors, synapse-organizing proteins, ion channels and neurotransmitter receptors, and synaptic vesicle-trafficking proteins [ 21 ]. This homology makes Drosophila a fruitful model to study neurodegenerative disorders [ 22 ], including amyotrophic lateral sclerosis (ALS) [ 23 ], Alzheimer disease [ 24 ], Huntington disease [ 25 ], and Parkinson disease [ 26 ].

Unlike in most forms of trauma, a large percentage of people killed by TBIs do not die immediately but rather days or weeks after the insult [ 4 ]. The primary brain injury is the result of an external mechanical force, resulting in damaged blood vessels, axonal shearing [ 5 ], cell death, disruption of the blood–brain barrier, edema, and the release of damage-associated molecular patterns (DAMPs) and excitotoxic agents [ 6 ]. In response, local glia and infiltrating immune cells up-regulate cytokines (tumor necrosis factor α) and interleukins (IL-6 and IL-1β) that drive posttraumatic neuroinflammation [ 7 – 10 ]. This secondary injury develops over a much longer time course, ranging from hours to months after the initial injury and is the result of a complex cascade of metabolic, cellular, and molecular processes [ 11 – 13 ]. Neuroinflammation is beneficial when it is promoting clearance of debris and regeneration [ 14 ] but can become harmful, mediating neuronal death, progressive neurodegeneration, and neurodegenerative disorders [ 15 – 18 ]. The mechanisms underlying these opposing outcomes are largely unknown but are thought to depend of the location and timing of the neuroinflammatory response [ 19 , 20 ]. It remains to be determined what the relative roles of TBI-induced neuroinflammation and other TBI-induced changes are in mediating short- and long-term impairments in brain function in vivo.

Traumatic brain injury (TBI) is one of the major causes of death and disability in the developed world [ 1 – 3 ]. Yet, the underlying mechanisms that lead to long-term physical, emotional, and cognitive impairment remain unclear.

Supporting figures for post-TBI days 1, 3, and 7 ( S5 Fig ) show sample comparison of relative log expression in untreated and successfully corrected data (panels A, C, E and B, D, F, respectively). Small deviations, arising from the technical differences, can be observed in D01 and D03; these were removed with UQ between lane correction [ 70 ]. For D07 ( S5E Fig ), we have observed that the replicates are lower quality, and there is a significant deviation in values between replicates within the TBI group, with replicate R3 assumed to be corrupted (see S5 Fig ). For consistency, we applied the same correction method to remove technical differences from post-TBI day 7, but as expected, replicate R3 did not improve. Taking this into consideration, we decided to remove this replicate from further analysis.

Sequencing was done with Illumina HiSeq 2000. All samples are done with single-end reads of 50 base pairs in length. At least approximately 5,000,000 mappable reads were obtained and used for quantification for each sample. Reads were quantified against transcript assembly release 6.10 from Flybase with Kallisto. Results of each gene were calculated by adding up all the transcripts for the gene. RNA-seq data were quantified at transcript level using Kallisto [ 51 ], using FlyBase_r6.14 as a reference transcriptome [ 52 ]. Quantified transcripts were summed up to the gene level using tximport library [ 53 ]. A minimal prefiltering, keeping only rows with more than 2 reads, was applied to gene level data before differential expression (DE) analysis. Differential gene expression analysis was performed on TBI versus control data with DESeq2 [ 54 ], using the likelihood ratio test to correct for batch effect among the biological replicates. Genes with the absolute log2 fold change higher than 0.6, and false discovery rate adjusted p-values ≤ 0.1 were identified as differentially expressed consistent with previous studies [ 55 – 60 ]. Day 7 replicates were corrected for sequencing depth and possibly other distributional differences between lanes, using upper-quartile (UQ) normalization, available through RUVSeq library [ 61 ], before proceeding to DE analysis. One replicate was removed from further analysis, due to extremely low expression across the sample, which was not comparable to the levels observed in the other Day 7 replicates. DEseq2 can assess DE with 2 replicate samples as input [ 62 – 67 ]. Functional annotation of DE genes was performed using the DAVID database (release 6.8 [ 68 , 69 ]) with a focus on gene ontology (GO) terms and Reactome pathways. Fastq data have been uploaded to the GEO repository (Series record GSE164377).

After receiving TBI, flies were collected at one of 3 time points, namely, 1 day postinjury, 3 days postinjury, and 7 days postinjury at ZT0 (lights-on in 12-hour light:12-hour dark). Flies were collected in 15 ml conical tubes and flash frozen in liquid nitrogen. Their heads were collected by vigorously shaking frozen flies and passing them through geological sieves. Approximately 100 heads were used for each experiment. Heads were homogenized for 3 minutes by Pellet Pestle Cordless Motor. Translating Ribosome Affinity Purification and Sequencing (TRAP-Seq) was performed as described [ 43 , 49 ]. Sepharose beads were prepared by rinsing 25 μL of resin per reaction with 1 mL of extraction buffer. Protein A Plus UltraLink (PAS) resin was incubated with 1 mL of extraction buffer and 2.5 uG of HTZ 19C8 antibody and rotated for 2 to 3 hours at room temperature. Beads were then spun at 2,500g for 30 seconds at room temperature and rinsed another 3 times with extraction buffer. The conjugated beads were then incubated with 1 mL of blocking buffer for 15 minutes at 4°C. The beads were then spun again at 2,500g for 30 seconds, and the supernatant was discarded. The beads were washed with 1 ml cold extraction buffer. This process was repeated another 2 times. Beads were incubated with 260 μL of head extract for 1 hour at 4°C and then spun at 2,500g for 30 seconds at 4°C. The beads were rinsed with 1 mL of cold wash buffer at 4°C. This process was repeated 3 times. After the final wash, 1 mL of Trizol was added. The beads were rotated at room temperature for 15 minutes. Chloroform was added, and the beads were subsequently shaken by hand for 30 seconds and incubated for 3 minutes at room temperature. The beads were then centrifuged at 15,000 rpm for 15 minutes at 4°C. The resulting upper aqueous phase was extracted and transferred to a new tube with 70% ethanol. RNA was extracted following the RNeasy Micro Kit protocol (Qiagen, Venlo, the Netherlands). RNA purified from the GFP tagged RpL10 was then reverse transcribed to cDNA. The cDNA was used as template for T7 transcriptase to amplify the original RNA. We synthesized first and second strand cDNA from RNA first with Superscript III and DNA polymerase. Then, we amplified the RNA by synthesizing more RNA from the cDNA template with T7 RNA polymerase. Amplified RNA was purified with RNeasy Mini Kit (Qiagen). A detailed procedure for amplification can be in found in [ 50 ]. After the second round of cDNA synthesis from amplified RNA, the cDNA was submitted to HGAC at University of Chicago for library preparation and sequencing.

A TUNEL assay was performed in whole brain as per manufacturer’s protocol (In situ cell death detection kit, Fluorescein, Sigma Aldrich, St. Louis, MO). The brains were carefully dissected out at different time points and fixed in 4% paraformaldehyde for 20 minutes followed by 3× 15-minute wash in PBST (PBS with 0.5% Triton-X 100). The brains were incubated in TUNEL mixture (prepared as per manufacturer’s instruction) for 60 minutes at 37°C followed by 3× 15-minute wash in PBST. The brains were then mounted in Vectashield mounting medium. TUNEL-positive values were determined for the entire central brain.

All statistical analysis for behavioral experiments was performed using Matlab 2011a for PC. For TRAP-seq analysis, see below. Mortality assay: Survival curves were plotted using the Kaplan–Meier estimator as described [ 47 ]. The statistical significance was calculated using the log-rank test. Plots and log-rank tests were performed in Matlab, using scripts developed by [ 48 ].

Three- to 7-day-old flies were placed into individual 65-mm glass tubes in the Drosophila activity monitoring (DAM) system (Trikinetics, Waltham, MA), which were placed in incubators running a 12-hour light/12-hour dark cycle. All experiments were carried out at 25°C. Sleep data were collected by the DAM system in 1-minute bins and analyzed offline using custom-made Matlab scripts (Matlab 2011a, Mathworks, Natick, MA). Briefly, sleep was defined as any period of inactivity of 5 minutes or more [ 45 , 46 ]. For each fly, total amount of sleep per day, average bout length, number of sleep bouts, number of brief awakenings, and average daily activity were derived from its activity trace (number of infrared beam crossings per minute).

After TBI induction, flies are housed in plastic vials with standard corn meal medium and housed in a 12-hour light/12-hour dark cycle at 25°C and approximately 65% relative humidity. Flies are gently transferred to fresh vials every 3 days. Deceased flies remaining in the old vial are counted.

A climbing assay is used to measure locomotor deficits after TBI in a manner similar to the RING assay [ 44 ]. Flies were individually stored in food vials and kept under the conditions discussed above. Vials were vertically divided into six 1-cm tall segments, labeled in order of ascending height (0 cm, 1 cm, etc.). Vials were tapped on a lab bench as a startle stimulus. Flies were then allowed to climb freely for 4 seconds, after which the highest point reached by the flies was observed and recorded. Three trials were observed for each individual fly; flies were allowed a period of at least 1 minute of recovery in between trials. Measurements from individual flies’ trials were then averaged to calculate a fly’s mean performance.

Immediately after TBI induction, individual flies were placed in 35 mm petri dishes, along with some fly food. Fly positions were recorded at 5 frames per second for 4 consecutive hours using a Blackfly CCD camera (FLIR Systems, Wilsonville, OR). Video data were analyzed using a custom Matlab script, using background subtraction to find fly positions in each frame, from which we derived velocity, latency to move, and the percentage of time each fly is active.

(A, B) To induce head-specific TBI, individual flies are gently aspirated into a modified 200-μL pipette tip that acts as a restraint. Immobilized flies are placed in front of a solenoid, using a set of micromanipulators with 5 degrees of freedom (x,y,z, pitch, roll) and a high-magnification video system to ensure highly replicable positioning. TBI is induced by running a current through the magnetic coil of the solenoid, which retracts a brass trapezoid-shaped block. ( C) By releasing current, a spring drives the brass block forward, hitting the fly on the top of the head. TBI, traumatic brain injury.

Flies were removed from their home vials without the use of anesthetic, using an aspirator and gently transferred to a prepared P200 pipette (see above). By applying some air pressure on the aspirator, the fly is pushed into the P200 pipette in such a way that the fly gets stuck at the end, with only its head sticking out. The restrained fly is then placed in a micromanipulator allowing for movement in 3 dimensions, which was subsequently used to move the fly into the appropriate position, with the back of the fly’s head making contact with the pin of a pull-type solenoid (uxcell DC 12V), which delivers 8.34 Newtons of force. Flies were observed using a high-powered camera lens (Navitar Zoom 6000, Rochester, NY) to ensure that they were in the proper position. A variable-voltage power supply (Tenma Corporation, Tokyo, Japan) was set to 12 V and used to power the solenoid, which then delivered a blow to the fly’s head ( Fig 1 ). Flies were hit 1 time, 5 times, and 10 times when observing effect of number of blows on response to TBI. Flies were hit 5 times for all other experiments. To minimize confounding effects of anesthesia, flies were collected under CO 2 anesthesia at least 24 hours before each experiment. All experiments are carried out in awake, unanesthetized flies.

Aspirators were constructed by wrapping a small square of cheesecloth around one end of aquarium tubing. A P1000 pipette tip was securely attached to covered end of the tubing, and the tip of the pipette tip was cut off to leave an aperture large enough for an individual fly to pass through without difficulty. The aspirator is used to transport individual flies via mouth pipetting. This allows flies to be transferred from their home vials to the experimental setup without using anesthesia. Fly restraints were created by cutting off the last 3 to 4 millimeters of P200 pipette tips to create an aperture large enough to let an individual fly’s head through without letting the entire body through. Multiple sizes of fly restraints were produced to accommodate small variations in size among flies.

Fly stocks were raised on standard cornmeal food under a 12-hour light/12-hour dark cycle at 25°C and approximately 65% relative humidity. TBI inductions and climbing assays were carried out in the lab at room temperatures (approximately 21 to 23°C). For sleep and life span experiments, flies were kept on standard cornmeal food under a 12-hour light/12-hour dark cycle at 25°C and approximately 65% relative humidity. All experiments were carried out in young adult (3 to 7 days old) male iso31 flies, an isogenic w 1118 control strain commonly used for sleep research. NF-κB Relish null mutants (Relish[E20]) were obtained from Bloomington (w 1118 ; Rel [E20] e [s] ; #9457). Repo-Gal4 was obtained from Bloomington (w[1118]; P{w[+m*] = GAL4repo/TM3, Sb [ 1 ] #7415). UAS-GFP::RpL10A was obtained from the Jackson lab [ 43 ]. CRISPR deletions of different classes of AMPs were obtained from Bruno LeMaitre and compared to their iso31 control strain {Hanson, 2019}. For the glial RNAi screen, RNAi lines obtained from Bloomington and VDRC were crossed to repo-Gal4 (BDRC# 7415). Controls consist of the appropriate RNAi control line (y [ 1 ] v [ 1 ]; P{y[+t7.7] = CaryP}attP2 (BDRC# 36303) for attP2 TRiP lines; y [ 1 ] v [ 1 ]; P{y[+t7.7] = CaryP}attP40 (BDRC# 36304 for attP40 TRiP lines; isogenic host strain w1118, for GD lines (VDRC ID 60000), empty insertion line y,w[1118];P{attP,y[+],w[3`]} (VDRC ID 60100) for KK lines). Attacin-A (BDRC 56904, VDRC 50320GD), Attacin-B (VDRC 57392, VDRC 33194GD), Attacin-C (VDRC 101213KK, VDRC 42860GD), Cecropin-A1 (BDRC 64855), Cecropin-A2, (BDRC 65160), Cecropin-B (BDRC 61932), Cecropin-C (BDRC 50602), Diptericin-A (BDRC 53923, VDRC 41285GD), Diptericin-B (BDRC 28975), Drosocin (BDRC 67223, VDRC 42503), Drosomycin (BDRC 55391, 63631), Listericin (VDRC 102769 KK), Metchnikowin (BDRC 28546, VDRC 109740 KK), virus-induced RNA-1 (vir-1) (BDRC 58209). All flies were collected under CO 2 anesthesia at least 24 hours before TBI induction and placed on regular food.

Results

dCHI: A controlled head impact model for TBI in Drosophila To study TBI in flies, we developed a head-specific TBI model where brain injury is inflicted in unanesthetized, individually restrained flies using a solenoid to deliver well-controlled, nonpenetrating strikes to the fly head (Fig 1). For TBI induction, individual flies are transferred from their home vial to a prepared P200 pipette tip, using an aspirator. Flies are gently blown upward until the head emerges from the tip of the pipette (Fig 1B). The pipette is then placed in a micromanipulator platform with 5 degrees of freedom (pitch, roll as well as movement along the XYZ axes). The top of the fly head is pressed against the tip of the solenoid that consists of a metal pin running through a copper coil attached to a spring. By running a current through the coil, it acts as a magnet, drawing the pin back and arming the spring. When the current is halted, the spring causes the pin to shoot out, thus allowing us to deliver one or more blows to the fly’s head (Fig 1C, S1 Movie). After TBI induction, flies are aspirated out of the pipette tip and returned to an empty vial containing regular fly food.

dCHI results in immediate locomotor defects Immediately after TBI induction, flies are often able to stand but only barely respond to tactile stimuli (S2 Movie). However, mobility returns in a manner of minutes (S3 Movie). To quantify locomotor impairments after TBI, we placed flies in 35 mm petri dishes immediately after TBI, along with some fly food, and recorded fly positions using a CCD camera. Sample traces for 3 intensities (1, 5, and 10 strikes; TBIx1, TBIx5, and TBIx10) as well as sham-treated controls are shown in S1A Fig. After TBI, approximately 25% flies in the TBIx1 condition are immobile versus approximately 55% in the TBIx5 and TBIx10 conditions (S1B Fig). Flies in the TBIx1 condition started moving within seconds, while flies in the TBIx5 and TBIx10 conditions started moving after minutes (3.3 and 10 minutes, respectively; S2C Fig). We also observed some locomotor defects (circling, slow walking, sideways walking, backwards walking, and jumping) shortly after TBI onset, in a dose-dependent manner (25%, 45%, and 50% in the TBIx1, TBIx5, and TBIx10 groups, respectively) (S2D Fig). These movement disorders only occurred in flies that were immobile immediately after TBI and were not observed in flies that immediately started walking. Walking speed was reduced in all 3 groups during the first hour post-TBI, but the TBIx1 and TBIx5 groups had recovered by the second hour. Walking speed remained impaired for all 4 hours in the TBIx10 group (S2E Fig). Overall activity (% of time active) was significantly reduced in the TBIx5 and TBIx10 groups for the first hour after TBI but unaffected in the TBIx1 group (S2F Fig).

dCHI increases mortality and impairs negative geotaxis in a dose-dependent manner within 24 hours We next examined the pathological and behavioral effects within the first 24 hours post-dCHI. TBI phenotypes become more severe with consecutive strikes in mammals [71] and Drosophila [40,41]. We subjected male flies to 1, 5, or 10 consecutive solenoid strikes, delivered at 1 strike per second. After TBI induction, treated and sham-treated cohorts were individually housed in vials containing standard food. Twenty-four hours after TBI exposure, surviving flies were counted in each of the 4 groups. We observed a dose-dependent increase in 24-hour mortality (Fig 2A). At 1 strike (TBIx1), there is no effect on 24-hour mortality (p = 0.68). Mortality is increased in a dose-dependent manner (control versus TBIx5, p = 0.03; control versus TBIx10, p = 0.004; ANOVA with Dunnett post hoc test, F(3,8) = 8.41; n = 3 replicates of 10 flies/group). PPT PowerPoint slide

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TIFF original image Download: Fig 2. TBI causes cell death, mortality, and impaired climbing in a dose-dependent manner. Male w1118 flies were exposed to either 1, 5, or 10 strikes to the head, delivered at 1 strike per second (n = 32 per group). (A) 24-hour survival rate decreased with increased number of strikes. (B) In surviving flies, climbing behavior was quantified and compared to sham-treated controls 24 hours after TBI. Climbing behavior became more impaired with increased TBI severity (n.s = not significant, *** p < 0.001, one way ANOVA with Dunnett post hoc test, n = 30/group). Cell death following TBI was quantified with a TUNEL assay. (C) Representative images of TUNEL staining at different time points in control and post-TBI flies. (D) Histogram showing significantly increased TUNEL-positive cells post-TBI in a dose-dependent manner (n = 10, 8, 8 for controls, n = 8, 8, 9 for TBIx5, and n = 7, 7, 8 for TBIx10 at 4 hours, 8 hours, and 24 hours, respectively). * p < 0.05, ** p < 0.01, *** p < 0.001 ANOVA with Dunnett post hoc test. Error bars indicate SEM. All figure-related data are located in S2 Data. TBI, traumatic brain injury. https://doi.org/10.1371/journal.pbio.3001456.g002 Loss of balance and poor motor coordination are symptoms associated with TBI [72–74]. Impairments in motor control, balance, and sensorimotor integration are also a well-studied endophenotype in rodent models of TBI (as quantified by beam balance, beam walk, and rotarod assays; reviewed in [75]). In Drosophila, impairments in sensorimotor integration are quantified by measuring the negative geotaxis response, a reflexive behavior where a fly moves away from gravity’s pull when agitated [76]. Impaired negative geotaxis has been observed in aging and in Drosophila models of neurodegeneration [77–79]. To assess sensorimotor function after TBI, we used a variation of the negative geotaxis assay [44], where the average height climbed in a defined time period is quantified, rather than a pass/fail number for absolute height as more subtle deficits can be observed using this approach. Typically, young adult wild-type flies reach an average climbing height of approximately 4 to 5 cm in a 3-second time period [44]. In our assay, sham-treated w1118 flies (3- to 7-day-old males) reached an average height of approximately 3.4 cm in 4 seconds (Fig 2B; 0 days post-TBI). Climbing behavior, driven by negative geotaxis becomes impaired after TBI. After a single hit, there is no detectable difference in climbing, 24 hours after TBI induction (control versus TBIx1, p = 0.1876; Fig 2B). However, after 5 or 10 consecutive hits, climbing behavior becomes impaired in a dose-dependent manner (Fig 2B; control versus TBIx5, p = 2.67 × 10−6; control versus TBIx10, p = 2.67 ×10−6; ANOVA with Dunnett post hoc test, F(3,99) = 57.54; n = 30 flies/group).

TBI increases apoptotic cell death in a dose- and time-dependent manner To test whether our TBI assay causes neuronal death, apoptosis was quantified using a TUNEL assay [80] after inducing TBI by striking flies either 5 or 10 times and comparing the number of TUNEL-positive cells at 3 different time points (4, 8, and 24 hours) between TBI-treated flies and sham-treated controls. Controls showed, on average, 2 to 4 TUNEL-positive cells, which may be spontaneous apoptotic cells (Fig 2C and 2D). Four hours after TBI induction, we saw an increase in TUNEL-positive cells in the TBIx10 condition (p = 2.56 × 10−6) but not in the TBIx5 condition (p = 0.1027; F(2,23) = 68.29) at this time point (Fig 2D). Eight hours after TBI induction, we also saw an increase in TUNEL-positive cells in the TBIx10 condition (p = 2.93 × 10−6) but not in the TBIx5 condition (p = 0.5623; F(2,22) = 33.41) at this time point (Fig 2D). Twenty-four hours after TBI induction, we saw an increase in TUNEL-positive cells in both the TBIx5 (p = 2.57 × 10−6) and the TBIx10 condition (p = 2.53 × 10−6; F(2,19) = 111.23) at this time point (Fig 2D). ANOVA with Dunnett post hoc test. Taken together, dCHI induces advanced mortality, motor deficits, and cell death within the first 24 hours.

dCHI reduces life span Given the slowly evolving nature of TBI pathology, we next examined the chronic effects of dCHI over time. We first examined life span. Unlike other forms of trauma, death after TBI rarely occurs immediately. To test how our TBI assay affects overall life span, we delivered 5 consecutive strikes to the top of a fly’s head (S1 Movie). After this, flies were housed individually, and survivors were counted every day. dCHI significantly reduces life span (log-rank test on Kaplan–Meier survival curves, p < 0.001). Around 50% of the TBI group had died 14 days after TBI induction, while 50% of the sham-treated controls had died 32 days after the start of the survival assay (Fig 3A). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Long-term effects of TBI on mortality, climbing, and sleep architecture. (A) Kaplan–Meier estimates of survival functions in TBI-treated flies and sham-treated controls. TBI (5 strikes) was induced in male w1118 flies (n = 59), and post-TBI survival was compared to survival in sham-treated controls (n = 57) using a log-rank test. TBI results in a significant decrease in survival rate (p < 0.001). Around 50% of the TBI group was deceased 14 days after TBI induction, while 50% of the sham-treated controls had died 34 days after the start of the survival assay. (B) Climbing behavior was tested in male w1118 flies, after which TBI was induced (n = 30). Climbing behavior was subsequently tested for 7 days after TBI and compared to sham-treated controls (n = 30). Climbing impairments recover on post-TBI days 2 and 3, followed by a relapse on days 4–7. (C) Sleep architecture was quantified in male flies up to 10 days after TBI induction (n = 96) and sham-treated controls (n = 84). TBI induction resulted in (C) decreased total sleep for up to 3 days post-TBI. (D, E) More fragmented sleep (decreased bout length, increased bout number) and (E) increased brief awakenings, suggesting lighter sleep. *** p < 0.001, ** p < 0.01 by t tests with Bonferroni correction. Error bars indicate SEM. All figure-related data are located in S3 Data. TBI, traumatic brain injury. https://doi.org/10.1371/journal.pbio.3001456.g003 To test whether this increase in mortality is mainly due to flies dying during the first 14 days after TBI, we removed flies that died during this peropd from both controls and TBI flies cumulatively, for up to 2 weeks after TBI, and performed log-rank test on the remaining flies. In all cases, survival rate is still significantly decreased in the TBI group, suggesting that the increased mortality is not due to flies that die early (S2 Fig).

[END]

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