Biomedical Engineering Reference
In-Depth Information
Before beginning with injections the L4 DRG should be
exposed in the same way.
To load viral vector into the glass needle, it can be fi rst pipetted
onto a hydrophobic surface such as a piece of Nesco fi lm or
Parafi lm, and then withdrawn into the needle while it is mounted
ready for injection, with the aid of the microscope. Manual with-
drawal of the pusher block holding the syringe plunger can be
used, or the pump controls. We routinely add 0.1 % Fast Green
FCF, to visualize the injected fl uid during the injection procedure.
This is useful to monitor the success of the injection and the degree
of leakage into surrounding tissue.
2.3.3 Loading
the Viral Vector
An important factor in the success of this technique is in the posi-
tioning of the needle. This should be done so that the needle tip is
as close to the center of the ganglion as possible when inserted. It
is helpful therefore to make sure the edges of the ganglion are vis-
ible. Best results are achieved by inserting the needle into the
thickest part of the ganglion at a depth of 400-500
2.3.4 Injecting
the Viral Vector
m. This can
be measured using the Vernier gauge of the stereotactic frame. The
needle should be rested on the surface of the ganglion so it is visi-
bly in contact and the vertical position of the holding arm read.
Move the needle down quickly until it penetrates the ganglion,
while observing through the microscope. Then withdraw slightly
and adjust the depth. After the needle is inserted, wait approxi-
mately 2 min before beginning to infuse liquid. This will allow the
tissue to seal around the needle and help prevent leakage. Likewise,
following injection, it is advisable to wait 2 min before removing
the needle to allow the injected liquid to be absorbed into the
endoneurium and avoid leakage from refl ux.
Up to 1.2
ʼ
ʼ
l virus can be injected in one DRG, with a fl ow
speed of 0.2
l/min.
After both DRG have been injected the overlying muscles are
closed with a 5-0 suture and the wound closed with clips. The ani-
mal is allowed to recover in a heated box.
ʼ
Breathing
. Movement of the DRG due to the animal's breathing
can cause problems such as increased leakage. Lifting the animal
slightly by placement of a roll of tissue under the belly, either
directly below the DRG or slightly more rostral, will minimize this
movement.
2.3.5 Notes
Bleeding
. Performing the laminectomy can lead to persistent bleed-
ing. This is particularly the case if the laminectomy is extended too
far rostro-medially and caudo-medially. Bleeding can usually be
stopped by inserting a small piece of gelfoam (spongostan,
MS0002; Johnson & Johnson Medical) between the lamina and
the spinal cord.
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