A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Abstract

Source: Katzenberger, R. J., et al., A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila. 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.

Protocol

1. Construction of the HIT Device

1. 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. 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.
2. 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.
3. 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 mm) higher than the wooden board, so that when a vial is attached to the spring the spring will not lie flat on the board.
4. 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.
5. 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° mark is aligned with the spring when it is pulled back to a perfectly vertical position.

2. Operation of the HIT Device

1. Place between 1 and 60 CO₂-anesthetized flies in an empty vial and stopper the vial using a tight-fitting cotton ball.
2. 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) from the bottom. It is helpful to draw a line on the vial at the 1-inch (2.5 cm) mark. Note that confining flies to larger or smaller regions of the vial can affect the severity of phenotypes.
3. 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.
4. 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) of the vial should overlap the raised region of the ice bucket cover. Vials can be reused many times.
5. 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.
6. Pull the spring perfectly straight back to the desired angle. Release the spring. Allow the spring to come to a complete stop.
7. Remove the vial from the spring and allow the flies to recover for ≥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 percentage 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 levels.
8. 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.

Results

Figure 1: Diagram of the HIT device. (A) Illustration of the top view of the HIT device in the resting position. (B) Illustration of the side view of the HIT device. The spring is shown in the resting position (dark red) and...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Zinc plated compression spring	The Hillman Group	540189	9 7/8 in (length), 15/16 in (outer diameter), 0.12 in (wire size)
Wooden board			9 in (length), 6.5 in (width), 0.75 in (height)
Clamps	Sigma Electrical Manufacturing Corporation	49822	3.10 in (length), 0.68 in (width), 1.11 in (height), EMT Two Hole Straps, click on type for 1 inch steel EMT conduit
Loop half of self-adhesive velcro			3 in (length), (3/4 in width)
Polyurethane ice bucket cover	Fisher Scientific	02-591-45	9 1/8 in (length), 9 1/8 in (width), 1 1/4 in (height)
Plastic fly vials	Applied Scientific	AS-510	3 11/16 in (height), 1 1/16 in (inner diameter), 1 1/8 in (outer diameter)
Large cotton balls	Fisher Scientific	22-456-883
Paper protractor			10 in (diameter)