Biology Reference
In-Depth Information
19. To properly perform subretinal injection, it is necessary that
the novice observes an experienced operator and that he/she
performs the injection on his/her own several times to get
acquainted before attempting the injections in actual experi-
ments. A good practice is important to efficiently transduce the
retina and/or the RPE and to avoid damages to the retina and
the lens.
20. The efficiency of retinal and RPE transduction following sub-
retinal injection may vary from one injection to another due to
the intrinsic variability of the procedure.
21. The animals that are treated with AAV vectors should be
housed in a separate room of the animal facility.
22. The surgical instruments must be cleaned with disinfectant and
sterilized under UV light before and after their use.
23. To clean the Hamilton syringes and needles after the injections
wash them five times with 70% Ethanol and five times with
H
2
O, then put the syringes under UV light overnight.
24. When injecting different AAV vectors during the same surgical
session (e.g., an AAV vector encoding for EPO, another one
encoding for S100E or EGFP) clean the Hamilton syringes as
described in Note 11 when switching from one vector to
another.
25. If experienced, operators can perform intramuscular injections
directly into the gastrocnemius of 4-week old rats without
making a skin incision to expose the muscle.
26. The custom device for light damage in rats is made of a wired
support for the cages (that allows the cage insertion and
removal as a drawer). The wired support we use can accom-
modate a total of four cages (therefore a total of four rats per
light damage cycle, as each rat has to be housed in a single cage
to avoid that more animals in the same cage shield each other
from the light). A white Plexiglas sheet is located above and
below the cages (at a distance of 30 cm). White light bulbs are
placed above and below (four bulbs per side) the superior and
inferior Plexiglas sheets, respectively. The lights can be turned
on and off at will. The bottom of each cage is grated to allow
the excrements to pass through the cage in order to be removed
by the animal house assistants.
27. The average luminance generated by the lights should be around
2,000 lx as measured using a luxometer before the rats are sub-
mitted to light damage. After many hours of use the light bulbs
may wear out, therefore if the luminance measured is significantly
lower than 2,000 lx the light bulbs need to be replaced.
28. The retinal damage induced by light can be modulated by
varying the light intensity as well as the onset and duration of
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