![]() In this protocol, we describe the assembly of the dTBI circuit (steps 1-24), construction (steps 25-31) and calibration of the device (steps 32-47), the technique of collaring flies (steps 48-54) and administering dTBI (steps 55-65), and discuss various factors that affect the reproducibility of the technique. A full list of the features observed with dTBI along with the timeframes of their occurrence is presented in Fig 1. The injured head also acutely upregulates proteasomal activity, markers of oxidative stress, and molecular chaperones. By contrast, cellular necrosis and blood brain barrier dysfunction occur early after injury and subside with time. The brain undergoes injury severity-dependent vacuolization within a day of dTBI, which progresses with time. Head-injured flies display a remarkable similarity to mammalian TBI, and several phenotypes are observed in an injury severity dose-dependent manner 22 Notably, neurological deficits such as immediate loss of righting reflex, locomotor deficits, spontaneous seizures, age-onset learning deficits, and a reduction in lifespan are observed as a consequence of dTBI. We define 3 thresholds of injury – mild, moderate and severe – caused by compressing the fly head by 35%, 40% and 45% respectively. The protocol uses a Heisenberg collar to immobilize the flies and a piezoelectric actuator to deliver a compressive injury to the fly head within 250ms. We have recently developed a head-specific model of Drosophila TBI, which we term dTBI, that is suitable for large-scale genetic or pharmacological screens 22. Since the experimental setup involves loading and injuring flies individually, it could be challenging to perform high-throughput screens. A head-specific model of TBI was described, which uses a pipette tip to immobilize flies and a ballistic impactor to strike the head 11. Although these models demonstrate that a portion of body-injured flies have neurodegenerative deficits 9, 10, 21, the injury is neither tissue-specific nor uniform between individual flies, thus increasing the heterogeneity in the population response. Another model employs a bead-mill homogenizer to deliver the force and similarly traumatize the flies 10. The first model of non-penetrating, closed head TBI in Drosophila uses a spring to deliver a mechanical force to a cohort of flies in a plastic vial, causing whole body injury 9. However, these models do not reflect the diffuse, brain-wide injury that occurs during a closed head trauma. These include models of axon injury through selectively severing particular neurons like the olfactory nerve 17 and wing axons 18, 19, and penetrative brain injury by piercing a needle through the head to cause damage to brain tissue 20. With sophisticated genetic toolkits that enable researchers to activate or inhibit specific genes in defined tissues with temporal control, Drosophila has provided mechanistic insight into many neurodegenerative diseases. Importantly, many central nervous system (CNS) genes and pathways are conserved between flies and humans, with an estimated 75% of human disease-causing genes also having functional fly orthologs 15, 16. The dTBI device can be used to harness the power of Drosophila genetics and perform large-scale genetic or pharmacological screens, using a 7-day post-injury survival curve to identify modifiers of injury.ĭrosophila has been instrumental in elucidating the mechanisms of nervous system development and complex behaviors such as learning, memory, circadian cycles and courtship 13, 14. The device components and the necessary electrical tools are readily available and cost ~$800. The entire device can be assembled and calibrated in under a week. The extent of head compression can be controlled through an electrical circuit, allowing the operator to set different levels of injury. This protocol describes the construction, calibration and use of the Drosophila TBI (dTBI) device, a platform that employs a piezoelectric actuator to reproducibly deliver a force in order to briefly compress the fly head against a metal surface. We have recently developed a device that can be used to inflict controlled head injury to flies, resulting in physiological responses that are remarkably similar to what is observed in humans with TBI. However, the injuries produced by these models are not specific in location and are inconsistent between individual animals. The brain degeneration associated with traumatic brain injury (TBI) has been modeled in Drosophila using devices that inflict trauma on multiple parts of the fly body, including the head. Drosophila models have been instrumental in providing insights into molecular mechanisms of neurodegeneration, with wide applications to human disease.
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