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

Samenvatting

Source: Sun, M., et al. A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila. 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.

Protocol

1. Assembly of the Strike Device (Figure 1)

1. Remove the plunger from a 1 mL tuberculin syringe. Cut the barrel at the 1 mL mark.
2. Remove an aerosol barrier (3 mm height x 4 mm diameter) from a 200 µL pipette tip and use it as an impactor. Place the impactor inside the syringe barrel. Gently tap the barrel to move the impactor to the tip end, with the flat side covering the nozzle opening.
3. Attach the barrel tip end to plastic tubing that is connected to the carbon dioxide (CO₂) flow regulator of a Drosophila anesthesia station.
4. Hold the barrel vertically and clamp it to a standard clamp holder stand so that the impactor stays at the bottom of the barrel.
5. Modify a 200 µL pipette tip to make the fly holder.
   1. Cut 4 mm from the tip to make a 0.8 mm-diameter opening, allowing only the fly head to be exposed.
      ​NOTE: The thorax and all the other parts of the fly body will stay inside the pipette tip.
6. Modify a 1,000 µL pipette tip and a 1 mL syringe needle cap to make the connector.
   1. Cut off 44 mm from the opening of the tip. Take a 6 mm length of a 1 mL syringe needle cap and push it tightly into the remaining segment of the 1,000 µL pipette tip.

2. Operation of the Strike Device

1. Anesthetize a single 2-day-old adult female fly using CO₂ on a fly pad.
2. Gently transfer it to the fly holder using a fine brush. Tap the holder gently so that the fly head is seen outside of the tip end. If the fly proboscis is exposed outside the tip, gently tuck it back inside the tip with a blunted 1 mL syringe needle.
   NOTE: Make sure to keep the fly proboscis inside the holder. Otherwise, the fly may die from a sucking proboscis injury.
3. Tighten the fly holder to the syringe barrel with the connector so that the fly head is facing downwards.
4. Set the gas pressure to 100 kPa. Adjust the flow rate according to the experiment design.
5. Quickly turn the flow regulator toggle switch on and off so that the impactor strikes the fly head once.
6. Lift the fly holder and move it over a fly pad. Reverse the fly holder and gently tap the side to let the fly out. Leave the fly in an empty vial to recover.

Resultaten

Figure 1. Diagram of the Strike Device. The device used materials that are readily available in a Drosophila lab: 1) 200 µL pipette tip, 2) 1 mL tuberculin syringe, 3) impactor, 4) connecting tube hooked to a Drosophila anesthesia s...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Aerosol Barrier	USA Scientific	1120-8810	Used as an impactor
200 ul Pipette Tip	USA Scientific	1111-0706	Used as a fly head holder
1000 ul Pipette Tip	USA Scientific	1122-1830	Used as a connector
1 ml Tuberculin Syringe	Becton Dickinson	309625
60 mm Petri Dishes	Fisher Scientific	FB0875713A	Used as a tracking arenas
Flow Regulator	Genesee Scientific	59-122WC
Standard Clamp Holder/stand	EISCO Scientific	CH0688
Fine Brush	Genesee Scientific	59-204
Flypad	Genesee Scientific	59-114