8 weeks after injury, we observed a 61.0 6 7.0% decrease in the
number of GFP 1 neurons. This loss of injured afferents resulted
in a loss of neuron-containing (ie, excluding white matter regions)
DRG volume (Fig. 1E), but not neuron density ( Fig. 1F). Cell loss
predominantly occurred between 1 and 2 weeks postinjury and
stabilized after this timepoint. Population distributions of the
cross-sectional area of nucleated, tdTomato-expressing cell
profiles were not significantly different at 1 vs 8 weeks post-
SNItrans, in contrast to GFP-expressing/injured afferents, in which
a loss of a population of small afferents at 8 weeks postinjury was
observed (Fig. 1G).
SNItrans resulted in a mixed population of axotomized and intact
afferents within the L4 DRG. Therefore, we developed an approach
to restrict our analysis to axotomized afferents, without relying on
transgenic labelling, and used this as a complementary approach to
confirm our findings. We injected the neuronal tracer FB into the
glabrous, tibial innervation territory of both hindpaws 1 week before
common peroneal and tibial transection (SNItrans)o rc r u s h( S N Icrush)
surgeries (Figs. 2A and B). FastBlue-uptake was complete across
neurons of all sizes by 1 week (Fig. S3, http://links.lww.com/PAIN/
C84), so this approach allowed us to profile a sample of the
axotomized afferents. Both SNItrans (Fig. 2C)a n dS N Icrush (Fig. 2D)
injuries resulted in a rightward shift in population distributions of the
cross-sectional area of nucleated, FB-labelled DRG neurons when
compared with contralateral DRG, consistent with a loss of small
afferents post–nerve injury.
As a third complementary approach, we applied semiauto-
mated volumetric analyses of nuclei size following tissue clearing.
In this study, whole DRGs were cleared 4 weeks after SNI trans for
nuclei counting in “complete” tissue ( Figs. 2E–H). Nuclei were
labelled by TDP-43, in line with the study by West et al., 67 and
were quantified using Imaris software ( Fig. 2F, Video 1). We
observed a slight but significant rightward shift in nuclear spot
volume population distribution 4 weeks after SNItrans (Fig. 2G). In
addition, there was a significant reduction in the number of small
but not medium or large nuclear spots, in support of a loss of
small-diameter neuron populations (Fig. 2H).
Together, our data derived from several different experimental
approaches show that a population of small-diameter afferents
are lost following peripheral nerve injury.
3.2. Spared nerve crush or transection results in death of
Mrgprd-expressing neurons
To date, determining cell loss among specific populations of
afferent neurons has proved challenging due to the down-
regulation of subpopulation-specific marker genes following
axonal transection. 37,44 To overcome this issue, we took
advantage of transgenic strategies to label populations in
a manner that persisted after injury. Owing to the bias for the
loss of small neurons and the known loss of IB4-binding central
terminals postinjury, 36 we initially focused on nonpeptidergic
nociceptive neurons. We used MrgD ChR2-YFP mice to identify
neurons belonging to the largest of the 3 classes of non-
peptidergic nociceptors, NP1. 55,59 To determine whether these
neurons are lost following nerve injury, we used a stereological
method to quantify L4 DRG MrgD-YFP 1 (yellow fluorescent
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