Photoreceptor Degeneration in Mice: Adeno-Associated Viral Vector-Mediated Delivery of Erythropoietin - Tissue-Protective Cytokines: Methods and Protocols - page 238

Fig. 3 Schematic representation of eye. Transverse section of the murine eye

5. Introduce the lower blade of the scissors into the cut and
perform a 180° conjunctival peritomy extending superiorly
and inferiorly. You have thus created a conjunctival flap to
which you can grab to by using the tweezers in the following
steps of the procedure.
6. Using the tweezers grab onto the conjunctival flap and pull it
towards the nasal side of the eye to expose its temporal poste-
rior part.
7. Punch through the sclera and choroid at the level of the pars
plana posteriorly to the sclera-corneal margin using the tip of a
sterile 30 gauge needle. Usually a drop of vitreous fluid leaks
from the punctured site.
8. Using the tweezers grab onto the conjunctival flap and pull it
towards the nasal side of the eye to expose its temporal poste-
rior part and the puncture.
9. An assistant has previously loaded the Hamilton syringe with
2 L (see Subheading 3.3.1 , step 3) of the AAV solution. Avoid

making air bubbles when aspirating the vector solution.
10. Insert the tip of the 33 gauge needle (mounted on the Hamilton
syringe) through the puncture into the subretinal space (the
area between the RPE/choroid and the retina). To maximize
the chances of applying the vector-containing solution to the
subretinal space the needle needs to be oriented tangentially to
the globe and with the hub turned towards the operator.
11. Keep the hand holding the syringe steady while the assistant
presses the piston to deliver the injection solution. Notably, in
albino animals the operator should be able to clearly see the
needle through the transparent sclera and to monitor the liquid
delivery. A proper subretinal injection would result in a local

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