This work was supported by National Institutes of Health grant, R01 AG033620 (BG) and by Robert Draper Technology Innovation Funding (DAW).

1. Construction of the HIT Device&#160;
<ol>
	<li>Attach the spring to the board using two clamps and four screws (Figure 1A). Center the clamps relative to the width of the board and butt them up against one another with the outer clamp flush with the edge of the board. Prior to attaching the clamps, bend them using pliers to fit tightly over the spring. 
	NOTE: See Table 1 for descriptions of the materials required for constructing the HIT device.&#160;The clamped end of the spring should be 1/8 inch (3.2 mm) from the edge of the board, and the free end should extend over the board by 3/4 inch (19 mm). Adjust the spring so that it lies parallel with the length of the board.</li>
	<li>Wrap the free end of the spring once around with the adhesive strip of Velcro loops. The outer edge of the Velcro should be flush with the end of the spring. The Velcro is important because it is used to secure the vial to the spring by creating a tight compression fitting. The Velcro also permits easy connection and removal of vials, allowing many vials to be processed in a short period of time.</li>
	<li>Place the ice bucket cover upside down, centered, tight against the wooden board. Orient the raised region of the ice bucket cover such that the long edge is parallel to the width of the board. Note that the raised region of the ice bucket is 1/2 inch (13&#160;mm)&#160;higher than the wooden board, so that when a vial is attached to the spring the spring will not lie flat on the board.</li>
	<li>Slide the whole device against a fixed object such as a wall, so that the ice bucket cover is wedged between the board and the object and does not move.</li>
	<li>Tape the paper protractor to the bottom of a cardboard fly vial tray and stand it on edge against the length of the board so that the 90&#176; mark is aligned with the spring when it is pulled back to a perfectly vertical position.</li>
</ol>
2. Operation of the HIT Device
<ol>
	<li>Place between 1 and 60 CO2-anesthetized flies in an empty vial and stopper the vial using a tight-fitting cotton ball.</li>
	<li>Confine the flies to the bottom 1 inch (2.5 cm) of the vial by pushing the cotton ball into the vial until it is 1 inch (2.5 cm)&#160;from the bottom. It is helpful to draw a line on the vial at the 1&#160;inch (2.5 cm)&#160;mark. Note that confining flies to larger or smaller regions of the vial can affect the severity of phenotypes.</li>
	<li>Wait 5 min for the flies to recover mobility from the CO2. Note that it is not known whether 5 min is sufficient to completely remove the effects of CO2.</li>
	<li>Insert the end of the spring into the vial until the inner edge of the Velcro is flush with the top of the vial (Figure 1B). When the spring is lying flat, 1 inch (2.5 cm)&#160;of the vial should overlap the raised region of the ice bucket cover. Vials can be reused many times.</li>
	<li>While sitting, hold the vial at the Velcro region using the thumb and forefinger of your left hand. Hold the board tight to the benchtop using your right hand. Alternatively, use C-clamps to hold the board tight to the benchtop.</li>
	<li>Pull the spring perfectly straight back to the desired angle. Release the spring. Allow the spring to come to a complete stop.</li>
	<li>Remove the vial from the spring and allow the flies to recover for &#8805;5 min. Subject the flies to another strike or transfer the flies to a vial with fly food. 
	NOTE: A variety of assays can be used to evaluate the phenotypic effects of strikes from the HIT device. For example, effects on longevity can be determined by analyzing the percent of flies that survive at times after injury, effects on brain morphology can be determined by histological analysis of the head, and effects on gene expression can be determined by quantitative analysis of mRNA levels10.</li>
	<li>Determine effects of the procedure that are not due to strikes by identically treating control flies that are not subjected to strikes. Wear hearing protection because the impact of the vial against the ice bucket cover produces a loud noise.</li>
</ol>

<ol>
	<li>Harrison-Felix, C. L., Whiteneck, G. G., Jha, A., DeVivo, M. J., Hammond, F. M., Hart, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+over+four+decades+after+traumatic+brain+injury+rehabilitation:+A+retrospective+cohort+study.">Mortality over four decades after traumatic brain injury rehabilitation: A retrospective cohort study.</a> Arch Phys Med Rehabil. 90, 1506-1513 (2009).</li><li>Coronado, V. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveillance+for+traumatic+brain+injury-related+deaths+&#8211;+United+States.">Surveillance for traumatic brain injury-related deaths &#8211; United States.</a> MMWR Surveill Summ. 60, 1-32 (1997).</li><li>Masel, B., DeWitt, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+brain+injury:+A+disease+process,+not+an+event.">Traumatic brain injury: A disease process, not an event.</a> J. Neurotrauma. 27, 1529-1540 (2010).</li><li>Blennow, K., Hardy, J., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuropathology+and+neurobiology+of+traumatic+brain+injury.">The neuropathology and neurobiology of traumatic brain injury.</a> Neuron. 76, 886-899 (2012).</li><li>Prins, M., Greco, T., Alexander, D., Giza, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+of+traumatic+brain+injury+at+a+glance.">The pathophysiology of traumatic brain injury at a glance.</a> Disease Models Mech. 6, 1307-1315 (2013).</li><li>Menon, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+challenges+in+clinical+trails+in+traumatic+brain+injury.">Unique challenges in clinical trails in traumatic brain injury.</a> Crit Care Med. 37, S129-S135 (2009).</li><li>Xiong, Y., Mahmood, A., Chopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+traumatic+brain+injury.">Animal models of traumatic brain injury.</a> Nature Rev Neurosci. 14, 128-142 (2013).</li><li>Balsiger, Z., Leudkte, J., Mawer, S., Willey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> HIT device high speed analysis. , (2014).</li><li>Davceva, N., Janevska, V., Illevski, B., Petrushevska, G., Popeska, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occurrence+of+acute+subdural+haematoma+and+diffuse+axonal+injury+as+two+typical+acceleration+injuries.">The occurrence of acute subdural haematoma and diffuse axonal injury as two typical acceleration injuries.</a> J Forensic Leg Med. 19, 480-484 (2012).</li><li>Katzenberger, R. J., Loewen, C. A., Wassarman, D. R., Petersen, A. J., Ganetzky, B., Wassarman , D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila.+model+of+closed+head+traumatic+brain+injury.">A Drosophila. model of closed head traumatic brain injury.</a> Proc Natl Acad Sci USA. 110, E4152-E4159 (2013).</li><li>Fang, Y., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+degeneration+and+regeneration:+insights+from+Drosophila+.models+of+nerve+injury.">Axon degeneration and regeneration: insights from Drosophila .models of nerve injury.</a> Annu Rev Cell Biol. 28, 575-597 (2012).</li><li>Babcock, D. T., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+accurate+and+rapid+measurement+of+flight+performance+in+Drosophila.">An improved method for accurate and rapid measurement of flight performance in Drosophila.</a> J Vis Exp. (84), e51223 (2014).</li><li>Tully, T., Preat, T., Boynton, S. C., Vecchio, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+consolidated+memory+in+Drosophila.">Genetic dissection of consolidated memory in Drosophila.</a> Cell. 79, 35-47 (1994).</li><li>Andretic, R., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+sleep+recordings+in+Drosophila.:+moving+beyond+sleep+time.">Essentials of sleep recordings in Drosophila.: moving beyond sleep time.</a> Methods Enzymol. 393, 759-772 (2005).</li></ol>

We have no conflicts of interest to disclose.

The HIT device method is distinguished from other methods that inflict traumatic injury in flies&#160;by the fact that it causes closed head rather than penetrating TBI11. Furthermore, the HIT device method takes less time, effort, and skill to inflict TBI in many flies, so the method is more amenable than other methods to large-scale genetic screens. Lastly, the fact that primary injuries inflicted by the HIT device are not limited to the brain is both a limitation and an advantage. It is a limitation because...

An author's name was corrected in the publication of <a href="http://www.jove.com/video/52905">A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila</a>. The second to last author's was spelled incorrectly. It has been updated from:
Barry Ganetky
to:
Barry Ganetzky

An author's name was corrected in the publication of <a href="http://www.jove.com/video/52905">A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila</a>.

Erratum: A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) is defined as injury to the brain from an external mechanical force. Most commonly, TBI results from closed head forces such as blunt forces and inertial acceleration and deceleration forces that cause the brain to strike the inside of the skull. In the United States, it is estimated that 50,000 individuals die each year from TBI and 2.5-6.5 million individuals are living with the consequences of TBI, including debilitating physical, cognitive, and behavioral problems1,2. The consequences of TBI are not only due to primary mechanical injuries to the brain but also to secondary cellular and molecular injuries to the brain as well as other tissues that occur over time3-5. The development of approaches to diagnose and treat TBI has proven to be difficult because TBI is a complex disease process. The variable nature of primary injuries, human physiology, and environmental factors results in heterogeneous secondary injuries and pathologies. Underlying variable factors include the severity of the primary injury, the time between repetitive primary injuries, and the age and genotype of the individual. Understanding how each variable factor contributes to the consequences of TBI is likely to aid in the development of approaches to diagnose and treat TBI6,7.
Here we describe a method for inflicting closed head TBI in Drosophila melanogaster (fruit flies) that can be used to delineate the contribution of variable factors to the consequences of TBI. The method is based on an initial observation that intensely hitting the side of a fly culture vial against the palm of a hand caused wild-type flies to become temporarily incapacitated, a likely consequence of TBI. Thus, we constructed the High-Impact Trauma (HIT) device to recapitulate the acceleration and deceleration forces from the hand-hitting action. A high-speed movie shows that a single strike from the HIT device causes flies to contact the vial wall several times with their head and body8. To some extent, all contacts are likely to cause the fly brain to ricochet and deform against the head capsule, similar to what happens to humans in falls and car crashes9. Accordingly, flies treated with the HIT device display phenotypes consistent with brain injury, including temporary incapacitation followed by ataxia, gradual recovery of mobility, gene expression changes in the head, and progressive neurodegeneration in the brain10. Thus, the HIT device makes it possible to study TBI using the enormous arsenal of experimental tools and techniques developed for flies.

<table><tbody><tr><td>Zinc plated compression spring</td><td>The Hillman&amp;nbsp; Group</td><td>540189</td><td>9 7/8 inch (length, 2.2 cm), 15/16 inch (outer diameter, 2.4 cm), 0.12 inch (wire size, 0.3 cm)</td></tr><tr><td>Wooden board</td><td></td><td></td><td>9 inch (length, 22.9 cm), 6.5 inch (width, 16.5 cm), 0.75 inch (height, 1.9 cm)</td></tr><tr><td>Clamps</td><td>Sigma Electrical Manufacturing Corporation</td><td>49822</td><td>3.10 inch (length, 7.9 cm), 0.68 inch (width, 1.7 cm), 1.11 inch (height, 2.8 cm), EMT Two Hole Straps, click on type for 1 inch (2.5 cm) steel EMT conduit</td></tr><tr><td>Loop half of self-adhesive velcro</td><td></td><td></td><td>3 inch (length, 7.6 cm), (3/4 inch width, 1.9 cm)</td></tr><tr><td>Polyurethane ice bucket cover</td><td>Fisher Scientific</td><td>02-591-45</td><td>9 1/8 inch (length, 23.2 cm), 9 1/8 inch (width, 23.2 cm), 1 1/4 inch (height, 3.2 cm)</td></tr><tr><td>Plastic fly vials</td><td>Applied Scientific</td><td>AS-510</td><td>3 11/16 inch (height, 9.4 cm), 1 1/16 inch (inner diameter, 2.7 cm), 1 1/8 inch (outer diameter, 2.9 cm)</td></tr><tr><td>Large cotton balls</td><td>Fisher Scientific</td><td>22-456-883</td><td></td></tr><tr><td>Paper protractor</td><td></td><td></td><td>10 inch (diameter, 25.4 cm)</td></tr></tbody></table>

a method to inflict closed head traumatic brain injury in drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

We are interested in understanding why flies die shortly after primary injury. To quantify death, we determined the Mortality Index at 24 hr (MI24), which is the percentage of flies that died within 24 hr of the primary injury. Flies subjected to strikes from the HIT device were incubated at 25 &#176;C in a vial with fly food, and the number of dead flies was counted after 24 hr. We used this approach to identify factors that affect the MI24 and found that the MI24 is not affected by the ...

Watch this Scientific Journal Video about A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila at JoVE.com

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

a-method-to-inflict-closed-head-traumatic-brain-injury-in-drosophila

Research

JoVE Journal

Neuroscience

11.5K Views.  University of Wisconsin-Madison.  Hier wordt een methode beschrijven we gesloten hoofd traumatisch hersenletsel (TBI) toebrengen in Drosophila. Deze methode biedt een toegangspoort tot de cellulaire en moleculaire mechanismen die TBI pathologieën met behulp van de enorme serie van experimentele instrumenten en technieken beschikbaar voor het vliegen ten grondslag liggen te onderzoeken. 

Video: A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Bioengineering

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Medicine

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

Mechanically Injuring Drosophila Larvae Segmental Nerves to Induce Neurodegeneration

Source: Danella, E. B., et al. <a href="https://app.jove.com/v/56128">A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration.</a> J. Vis. Exp. (2017).
The video demonstrates inducing neurodegeneration in Drosophila larva segmental nerves via mechanical injury. The ventral cuticle of anesthetized larvae is pinched using forceps to cause injury to the segmental nerves. The injured larvae are maintained with food, allowing the injury to cause degeneration of the motor neurons in the nerves, causing disruption of their neuromuscular junctions (NMJs).

1. Preparation of Reagents and Equipment 

	Prepare 1x Dissection Buffer (70 mM NaCl, 5 mM KCl, 0.02 mM CaCl2, 20 mM MgCl2, 10 mM NaHCO3, 115 mM sucrose, ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

Drosophila Adult Brain Dissection: A Method in Fly Neurobiology

Source: Kelly, S. M., et al. <a href="https://www.jove.com/video/56174/" target="_blank">Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains</a>. J. Vis. Exp. (2017).
This video describes how to dissect and isolate the adult Drosophila brain&#8211;a procedure necessary for visualizing the organ, which is otherwise obscured by the cuticle, eye, and tracheal tissue. The example protocol shows a detailed demonstration yielding high-quality preparations that can be used for immunostaining and imaging methods in Drosophila neurobiology.

Source: Kelly, S. M., et al. Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains. ...

Biology

Drosophila melanogaster (fruit fly)

Adult

Inducing Targeted Neuronal Injury Using a High-Power Laser in a Drosophila Larva

Source: Li, D., Li, F., et al. <a href="https://app.jove.com/v/57557">A Drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system.</a> J. Vis. Exp. (2018)
This video demonstrates a method for inducing targeted neuronal injury in Drosophila larvae using a high-power laser. After positioning the larva under a microscope, a low-power laser scans for fluorescent neurons, and then the laser power is increased to induce localized damage. The injury is confirmed by increased fluorescence and a crater at the injury site.

Source: Li, D., Li, F., et al. A Drosophila in vivo injury model for studying neuroregeneration in the peripheral and central nervous system. J. Vis. Exp. ...

Trauma

Source: Katzenberger, R. J., et al.,&#160;<a href="https://app.jove.com/v/52905">A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila.</a> J. Vis. Exp. (2015).
The video demonstrates the use of a High-Impact Trauma (HIT) device to induce closed-head traumatic brain injury (TBI) in Drosophila. TBI causes mechanical brain trauma affecting neuronal function, which over time leads to neurodegeneration and mortality.

Source: Katzenberger, R. J., et al.,&#160;A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila. J. Vis. Exp. (2015).
The video ...

A Technique to Inflict Closed-Head Traumatic Brain Injury in Drosophila Flies

Source: Sun, M., et al. <a href="https://app.jove.com/v/55602">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila.</a> J. Vis. Exp. (2017).
This video demonstrates a procedure to inflict a closed-head traumatic injury in anesthetized Drosophila flies using a strike device with a gas-propelled impactor. The injury affects neuronal function and impairs walking ability.

1. Assembly of the Strike Device (Figure 1)

	Remove the plunger from a 1 mL tuberculin syringe. Cut the barrel at the 1 mL mark.
	Remove an aerosol ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

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