Virus Injection into the Brain
General Protocol to inject viruses into the brain of rats and mice
Placement of animal in stereotaxic frame
- Anaesthetise animal.
- Shave head.
- Place upper front teeth into stereotaxic equipment and position tongue so that the animal won’t choke.
- Ensure nose is inside anaesthetic (in case of gas anaesthesia).
- If using ear bars, introduce one of them gently in the ear of the animal and find the area where you feel that the tip of the ear bar does not move. Hold this bar to the stereotaxic while holding the animal's head so that the ear bar tip does not move. Then, holding the animals head from bellow, introduce the other ear bar on the opposite side using the same approach.
- If using zygoma cups, hold the animal by the ears, position the side holders of the frame and tighten over temporal lobes - if head moves when pushing down, tighten further. There should be a straight line between the top of the frame and the corners of the ear and eye ("cheeks").
- Looking from behind the animal, check front-back alignment (can measure several co-ordinates, e.g. DV (dorsal-ventral) co-ordinates between Bregma and Lambda to check perfect alignment).
- Push down on the animal's head to check everything is secure before beginning surgery.
- Add protective cream to the animal’s eyes using cotton tip.
- Rub surgery area with alcohol and then iodine/betadine swabs to sterilize.
- Make incision (try to make only one cut) between the front of the ears and the beginning of the eyes (back to front) - hold skin tight with one hand while cutting. Insert clamp or skin holder to hold skin open.
- Clean tissue from skull (periostium - shiny part) using hydrogen peroxide. Can also just scrape skull with scalpel if prefer - H2O2 will digest periostium; clean well with cotton tip.
- Attach needle to stereotaxic frame and position over Bregma (if fissures in skull are not exactly in line place in middle). Make note of anterior-posterior (AP) and medial-lateral (ML) co-ordinates.
- Calculate required co-ordinates and mark the position(s) on skull using ink on the end of the needle - use surgery pen to 'paint' tip then fill in better manually.
- Make holes (craniotomy) using drill, pay attention to the size of the drill bits.
- Rinse down the area with saline if need a better view. Use absorbent padding (gel foam) if bleeding occurs. Gel foam can also be used to reduce drying out and swelling of exposed brain regions. Keep hydrated with saline!