Biology Reference
In-Depth Information
AAV2-BDNF, we obtained a significant increase in RGC viability after
ON crush, however there was no discernible impact on axonal
regeneration, with few axons crossing the injury site (
Leaver, Cui, et al.,
2006
). Thus, this factor, and most likely NT-4/5, whether delivered by
direct injection of recombinant protein or by vector-mediated methods,
is a potent RGC survival factor after ON injury but appears to induce
local sprouting proximal to the injury site (e.g.,
Cui et al., 2003; Kl
¨
cker,
Jung, Stuermer, & B¨hr, 2001; Sawai et al., 1996
) and is not effective in
promoting long-distance axonal regeneration. In contrast, injection of
AAV2 encoding a secretable form of CNTF increased RGC viability
about fourfold, and in both rats and mice, RGC axons regenerated across
the crush site for several millimeters within distal ON, in mice some
reaching as far as the optic chiasm (
Leaver, Cui, Bernard & Harvey,
2006; Leaver, Cui, et al., 2006
).
The contrasting effects of BDNF and CNTF delivery on RGC axonal
regeneration has recently been confirmed in studies in which we injected
AAV2 vectors prior to grafting an autologous PN onto the cut ON in
adult rats (
Hellstr¨m & Harvey, 2011; Leaver, Cui, Bernard & Harvey,
2006
). All of our AAV2 vectors are bi-
cis
tronic, that is, the therapeutic
transgene is linked via an internal ribosome reentry site to the green
fluorescent protein (GFP) reporter gene. The
GFP
gene is downstream of
the growth-promoting gene and is expressed only when the therapeutic
gene is expressed, allowing direct visualization of the extent of RGC
transduction in injected eyes. The PN grafts are generally about 1.5 cm long
and are blind ended, sutured to fascia on the cranium. Prior to sacrifice, all
PN grafts are injected at their distal end with fluorogold (FG) in order to
retrogradely label all RGCs that have regenerated an axon through the graft.
Immunostaining the retinal whole mounts with an antibody to
b
-III tubulin
(
Cui et al., 2003
) allows quantification of total RGC survival, transduction
efficiency and localization, and the proportion of surviving RGCs that
regenerated an axon (FG positive). In most cases (but see later), to allow
time for activation of transgene expression, the chosen AAV2 vector is
injected into the vitreous 10-14 days before ON injury and surgery.
In PN-grafted rats, AAV-CNTF but not control AAV-GFP promoted
excellent RGC survival, with on average, about 25,000 RGCs viable
7 weeks after ON-PN surgery (
Leaver, Cui, Bernard & Harvey, 2006
).
Remarkably, close to 50% of these surviving RGCs were retrogradely
labeled with FG and thus had successfully regrown an axon at least 1 cm
within a PN graft. Even though the PN grafts are blind ended and not
