injury (Fig. S6A–C, http://links.lww.com/PAIN/C84), indicating
that any loss of neurons within specific neuronal subpopulations
was not biased towards soma size. Collectively, these data show
that unrepaired axonal damage to peripheral sensory neurons
induces a partial loss of Trpm81 and CGRP 1 subpopulations,
but no major loss of myelinated afferents.
Based on our findings of preferential loss of nonpeptidergic
nociceptors, we re-analyzed a previous population-specific
transcriptomic dataset of mouse DRG neurons following nerve
injury for potential upregulation of cell death pathways (Fig. S7,
http://links.lww.com/PAIN/C84).3 We found that early after injury
(3 days post-SNI trans), nonpeptidergic (MrgD CreERT2-expressing)
neurons showed enhanced enrichment of GO terms associated
with apoptosis, in contrast to a broad population of nociceptors
(labelled with Scn10a CreERT2), peptidergic nociceptors (Calca-
CreERT2), C-LTMRs (Th CreERT2), and A b-RA (rapidly adapting) and
Ad-LTMRs (A d/Ab-LTMR, Ntrk2 CreERT2;AdvillinFlpO), in which
there was less or no enrichment of cell death pathways. By
4 weeks, only C-LTMR and A d/Ab-LTMR subtypes show any
overrepresentation of cell death pathways (in the populations
studied). Both injury-specific and apoptotic signatures in non-
peptidergic neurons were no longer significantly enriched,
consistent with a loss of axotomized nonpeptidergic afferents
by this late timepoint postinjury. These data suggest that
apoptotic pathways are upregulated acutely after injury in a cell-
type-specific manner.
3.4. Mrgprd dorsal root ganglion neurons are sensitive to
loss in vitro
Earlier studies postulated that a lack of neurotrophic support
underlies neuronal loss, which is supported by the observation
that exogenous GDNF treatment at the time of injury, or shortly
after, rescues the loss of IB4- binding central terminals
posttransection. 5 We sought to use the DRG neurons from
MrgDCreERT2 ;Ai32 mice to test this postulate and establish an
in vitro platform capable of probing the molecular basis of loss,
with axonal transection during isolation providing a correlate
for in vivo nerve injury ( Figs. 5A–E). Twenty-four hours after
plating, YFP was expressed by 16.3 6 1.3% of DRG neurons,
which was reduced to 11.8 6 1.7% after 28 days of culture in
the presence of exogenous GFs, NGF and GDNF ( Fig. 5F).
However, in the absence of GFs, YFP 1 neurons only
accounted for 1.7 6 0.6% of neurons after 28 days,
accompanied by an apparent reduction in the overall number
of neurons within the culture, despite all conditions being
seeded at the same initial density ( Figs. 5C and F). YFP 1 cell
loss was partially rescued by t he presence of GDNF, but not
NGF alone, in the culture media ( Figs. 5D–F). These results
contrasted with experiment s using neurons derived from
CalcaCreERT2 ;Ai32 mice, in which we observed no change in
the proportion of neurons that were Calca-YFP 1 after 28 days
in culture, regardless of exogenous GF addition ( Figs. 5G–L).
Collectively, these data support the use of DRG cultures to
probe the mechanisms underlying selective loss of sensory
neurons following nerve injury a nd suggest a role for trophic
support, particularly by GDNF sig naling, in preventing the loss
of nonpeptidergic nociceptors.
4. Discussion
We present data herein to support the hypothesis that
traumatic nerve injury in rodents leads to a profound loss of
small-diameter DRG neurons. Taking advantage of newly
developed transgenic recombinase driver lines, we have
shown that loss is biased across molecularly defined
subpopulations. Nonpeptidergic nociceptive neurons are
particularly susceptible to loss, with almost all Mrgprd 1
axotomized afferents lost following an unrepaired transection
injury (SNI trans ) and roughly half lost following a model which
contrastingly allows for n erve regenerations (SNI crush ).
Finally, we have observed that the vulnerability of Mrgprd 1