Biomedical Engineering Reference
In-Depth Information
the intrathecal space with considerable success. Storek and colleagues
investigated intrathecal injections of conventional and self comple-
mentary (sc) AAV vectors expressing eGFP packaged with capsids
of the serotypes 1, 2, 3, and 5 in rats [
20
]. No expression was seen
with conventional AAV serotypes 1 and 5 and with sc-AAV serotypes
2 and 3. Long term strong eGFP expression was observed for
sc-AAV serotype 1 and weak expression for sc-AAV serotype 5, as
measured by Western Blots of the lumbar segments of the spinal
cord and cauda equina. Additionally they achieved successful long-
term transduction of DRG neurons using sc-AAV serotype 8
expressing eGFP in rats [
21
] and demonstrated functional effects of
rIL-10 and prepro-
-endorphin delivered with the same sc-AAV
serotype 8 in a chronic neuropathic pain model. Towne and col-
leagues have seen similar transduction effi ciency of DRG neurons in
mice using AAV serotype 6 through different delivery methods
[
22
]. They concluded that intrathecal delivery of AAV6 expressing
eGFP is an effi cient method to transduce DRG neurons. Xu and
colleagues showed successful transduction of lumbar DRG using
sc-AAV serotype 5 expressing eGFP and shRNA targeting mTor in
rats, demonstrating the feasibility of gene knockdown in DRG neu-
rons using AAV injected into the intrathecal space [
23
]. Other
groups have used direct lumbar punctures in rodents [
26
,
27
].
However, most of these injections lead to inferior transduction effi -
ciency of DRG neurons and a laminectomy is needed in some cases
[
27
]. Furthermore, considerable experience is needed to perform
these direct lumbar punctures in a reproducible fashion.
ʲ
2.5 Method
B. Intrathecal AAV
Delivery Targeting
the Lumbar DRG Using
a Catheter
The catheter is made from approximately 30 cm of stretched PE-10
tubing (800/100/100; 0.28 mm I.D., 0.61 mm O.D.; Hythe,
UK). The stretching of tubing is done manually by warming it up
between two hands after which the tips are grasped between two
haemostatic forceps and pulled away from each other until an outer
diameter of 0.4 mm is reached. This can best be done parallel to a
fl at surface which helps to avoid fl exing of the tubing. Cut of both
ends of the tubing and make sure that the tip on the side that will
be introduced into the intrathecal space has no sharp edges that
could damage the spinal cord while inserting. The tubing is then
disinfected using 70 % ethanol and marked at 8.5 cm from the tip.
We have determined that with this placement distance the tip of
the tubing ends up around L2 level in Fischer 344 rats that weigh
180-250 g, which should result in bilateral transduction of the L2
to L6 DRG with maximum transduction rates at the L4/L5 level.
The tubing is then connected to a 50
l Hamilton syringe (549-
1154; Hamilton) which should be properly sealed without need
for adhesives. The syringe and tubing are fi lled with sterile water by
removing the plunger, as described above for direct injections.
After replacement of the Hamilton plunger, 2
ʼ
l of air should be
taken up prior to loading with virus to prevent mixing.
ʼ
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