Photoreceptor Degeneration in Mice: Adeno-Associated Viral Vector-Mediated Delivery of Erythropoietin - Tissue-Protective Cytokines: Methods and Protocols - page 239

retinal detachment that can be visualized by the operator as a
bleb.
12. Keep maintaining the conjunctival flap using tweezers while
slowly and carefully removing the needle.
13. Let the conjunctival flap go.
14. Gently apply a thin layer of ointment containing antibiotics
and steroids to the operated area.
15. Starting from step 1, repeat the procedure to inject the con-
tralateral eye of the same rat.

Put the cage containing the anesthetized animal on a hot plate
set at 37°C to limit the anesthesia-induced hypothermia and to
speed up the recovery from anesthesia (the complete recovery
usually takes 2-4 h) (see Notes 21-24).

1. Anesthetize the 4 week-old rat (see Subheading 3.4 , step 1).
2. Gently lay the rat flat on its stomach.
3. Gently secure the rat's legs to the support using an adhesive
gauze and sterilize the area of the leg to be operated using a
disinfectant.
4. Carefully shave the area to be injected.
5. Using tweezers grip the skin and carefully make a cut to expose
the underlying gastrocnemius muscle (see Note 25).
6. Keep the skin layer steady with the tweezers.
7. An assistant has previously loaded the Hamilton syringe (armed
with a 30 Gauge needle) with 100

3.3.3 Intramuscular
Injections

L (see Subheading 3.3.1 ,
step 4) of the AAV vector solution. Avoid making air bubbles
when aspirating the vector solution.
8. Puncture the gastrocnemius allowing only the tip of the needle
to enter the muscle.
9. Hold the syringe steadily while the assistant presses the piston
to deliver the injection volume (up to 40

μ

L). A proper intra-
muscular injection results in a local muscular lump.
10. Inject 30

μ

L in two additional locations of the gastrocnemius
to evenly distribute the dose of vector in the muscle.
11. Slowly and carefully remove the needle.
12. Suture the skin using the catgut.
13. Gently apply a thin layer of antibiotic ointment to the operated
area.
14. Relocate the rat into its cage.
15. Put the cage containing the anesthetized animal on a hot plate
set at 37°C to limit the anesthesia-induced hypothermia and to
speed up the recovery from anesthesia (see Notes 21-24).

μ