To compare the long-term recovery of gene
expression in dorsal root ganglion (DRG)
neurons under conditions of regeneration vs.
non-regeneration, Northern blotting and in situ
hybridization were used to assess steady-state
neurofilament (NF) and beta tubulin mRNA
levels 12 weeks following axonal injury. Adult
male rats sustained either a crush lesion of the
mid-sciatic nerve (regeneration occurs), or a cut
lesion of the sciatic nerve combined with ligation
of the proximal nerve stump and removal of a
large segment of the distal nerve (regeneration
does not occur). In the latter case, neuroma
formation physically prevented axonal regeneration.
Results of Northern blotting of total RNA
obtained from the DRG indicated that NF-L and NF-Μ
mRNA levels had largely returned to
control levels at 12 weeks following crush
axotomy but were still substantially depressed
following cut/ligation injury of the sciatic nerve
at that time. in situ hybridization studies
indicated that both crush and cut/ligation
axotomy resulted in significantly lower NF-L
mRNA levels in large-sized (>
1000 μm2) DRG
neurons at 12 weeks post-axotomy. Discrepancies
in the conclusions from Northern
blotting and in situ hybridization experiments
were also noted in the case of tubulin
mRNA changes at long intervals after axotomy. in situ
hybridization data derived from the large-sized
DRG neurons using a coding region β-tubulin
cDNA (which recognizes both βΙΙ
and βΙΙΙ
mRNAs) showed complete recovery of β-tubulin
mRNA levels in surviving, large-sized DRG
neurons after crush axotomy, but significantly
elevated tubulin mRNA levels in surviving large
DRG cells at 12 weeks after cut/ligation
axotomy. In contrast, Northern blotting results
indicated that βΙΙ-tubulin mRNA levels in the
crush axotomy condition remained elevated
relative to control while they were substantially
lower than control in cut/ligation axotomy
samples. Results from analysis of βΙΙΙ-tubulin
mRNA changes were not conclusive. The lack of
complete correspondence in the results from the
two different methods of analysis of mRNA
changes (blotting vs. in situ) is likely to be due to
selective loss of large-sized DRG neurons in the
long-standing cut/ligation injury condition. This
would influence results from blotting data,
where RNA is derived from the DRG as a whole,
more so than in situ hybridization experiments
which specifically focus on the surviving largesized
neurons. Overall, data from these
experiments indicate that altered patterns of
gene expression remain in the DRG for long
intervals after axonal injury, whether or not
axonal regeneration has been successful.
However, recovery of “normal8221; patterns of
cytoskeletal gene expression in the DRG is
considerably more complete after crush injury
than after cut/ligation injury.
