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.

The overall goal of this procedure is to inflict closed head traumatic brain injury. In flies. This is accomplished by first placing flies in a vial one inch from the bottom.

The second step is to attach the vial to the HIIT device. Next, the spring is deflected to a particular angle and the vial is released and allowed to come to a complete rest. Ultimately, a variety of behavioral morphological and molecular assays can be used to characterize the consequences of the resulting traumatic brain injury.

The main advantage of this method over existing methods, such as the rodent weight drop method, is that many animals can be studied over a short period of time. This method can help answer key questions in a traumatic brain injury field, such as why the consequences of traumatic brain injury vary in different individuals. To begin, assemble the raw materials listed in table one of the accompanying text protocol.

Bend the clamps using a pair of pliers so that they fit tightly over the spring. Then 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. Adjust the spring so that it lies parallel with the length of the board, and then attach the clamps over the spring and onto the board using four screws.

Next, wrap a strip of Velcro adhesive tape. Once around the free end of the spring, the outer edge of the Velcro should be flush with the end of the spring. Place the ice bucket cover upside down, centered and 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. 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. Then 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 degree mark is aligned with the spring.

When it is pulled back to a perfectly vertical position, place between one and 60 carbon dioxide anesthetized flies in an empty vial and stopper the vial using a tight fitting cotton ball. Then draw a line on the vial one inch from its base, knock the flies down from the sides of the vial, and push in the cotton ball until it matches up with a newly drawn line. Wait five minutes for the flies to recover mobility from the carbon dioxide.

Next, insert the end of the spring into the vial until the inner edge of the Velcro is flush with the top of the vial. This secures the vial to the spring by creating a tight compression fitting with the spring lying flat. Ensure that one inch of the vial overlaps the raised region of the ice bucket cover at this time.

Place on hearing protection because the impact of the vial against the ice bucket cover produces a loud noise. Next, hold the vial at the Velcro region using the thumb and forefinger of the left hand with the right hand, hold the board tight to the bench top. Next, pull the spring perfectly straight back to the desired angle, and then release the spring.

Wait for the spring to come to a complete stop, and then remove the vial from the spring. Set the vial on the bench top and allow the flies to recover for at least five minutes. During this time, the flies will slowly regain their normal motion.

Next, transfer the flies to a vial with fly food for later analysis or subject the flies to another strike, the vials can be reused many times. The purpose of the experiment was to determine the effect of injury severity on the mortality index at 24 hours. The severity of trauma was determined by the angle of deflection of the spring from zero to 90 degrees prior to release.

The experiment was performed on flies of two different ages, zero to seven days old flies, and 20 to 27 days old flies. The results show that more severe injuries cause a higher mortality index. Older flies had an increased mortality index compared to younger flies when spring deflection was 50 degrees or higher.

After watching this video, you should have a good understanding of how to inflict traumatic brain injury using flies.