Microinjection-Based Delivery of a Therapeutic Agent into an Awake Rodent Brain

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparing the microinjection syringe and microinjector for injection:
   1. Lock each gastight microinjection syringe into the rack of the microinjection pump.
   2. Place the tip of the gastight syringe into the sterile water contained in a small weigh boat and gently flutter the plunger to lubricate the internal wire. Evenly pull the plunger to the backstop on the pump to fill it with water.
   3. Slide the PE20 tubing that is connected to the microinjector onto a 26 G (brown hub) needle attached to a 1 ml syringe that is filled with sterile water. Spray sterile water through the microinjector, and inspect spray pattern and distance.
      NOTE: Water should spray very far and straight out of the microinjector tip. If the stream is not straight, the injection location will not be correct.
   4. Place the sterilized microinjector onto a sterile field.
   5. Slide the tubing off of the needle, keeping the microinjector tubing upright to prevent water from dripping out while also cutting the tubing below the area damaged by the needle.
   6. Push the microinjection syringe plunger slightly forward to create a droplet on the end of the needle and slide the water-filled PE20 tubing (that is connected to the microinjector) onto the needle.
      NOTE: This will create a liquid-to-liquid connection that will allow appropriate backpressure that could dislodge a clog that could form at the tip of the microinjector.
   7. Push the plunger completely forward to prepare for sample loading and ensure that no trace ethanol remains in the microinjector.
   8. Check the setup for leaks by touching the water droplet that has formed at the tip of the microinjector to a clean lab wipe multiple times.
2. NOTE: When touching the lab wipe, only one wet spot should form. If there are more drops, there is a leak in the system.
3. Avoid leaks by controlling super glue application and by trimming the PE20 tubing with sharp scissors or a razor blade any time that the PE20 tubing is removed from any connection.
4. Pull the syringe plunger back 0.2 μl; creating a bubble between the sterile water in the PE20 tubing/ microinjector and the solution to be injected.
5. NOTE: This bubble prevents dilution of the injectate, contamination of the water, and allows for monitoring of the injection process since it will move during the injection. A single, solid bubble should be produced.
6. Place the microinjector into the reagent to be injected and slowly pull the syringe plunger to the backstop.
7. Preparing an animal for injection:
8. Hold the rat’s chest against the experimenter’s chest. Clean the area around the cannulae with a cotton applicator soaked in 70% ethanol (v/v).
9. NOTE: With proper handling, mice can be gently restrained in a cupped hand. Otherwise, scruffing may be required.
10. Remove obturators using small forceps and place them into the weigh boat with 70% ethanol (v/v).
11. Performing injections:
12. Place the filled microinjector into the cannula. Press start on the microinjection pump and monitor bubble movement.
13. NOTE: It may be necessary to apply light pressure to ensure that microinjectors do not lift. The bubble should uniformly advance and fully enter into the microinjector.
14. Remove the microinjectors from the cannula and replace obturators, once the injection is complete and the post-injection diffusion period has passed.
15. NOTE: Typical post-injection diffusion times are 2 – 4 min for pharmacological reagents and 2 – 10 min for viruses. Leaving the microinjector in during the post-infusion period prevents the injectate from refluxing back up the cannulae rather than diffusing into the tissue. Place obturators on the side of the ethanol-filled weigh boat to allow the ethanol to drain before reinserting into the cannula.