protein) neurons 28 days after sham surgery or SNI trans (Figs. 3A
and B). SNI trans, but not sham, resulted in a significant decrease
(54.0 6 6.6%) in the total number of MrgD-YFP 1 neurons in L4
DRG ( Fig. 3C).
Yellow fluorescent protein expression in MrgD ChR2-YFP mice is
driven by the endogenous Mrgprd promotor, which has been
reported to be upregulated or downregulated following axonal
damage.44,58 Such changes in promoter activity could affect the
proportion of nonpeptidergic nociceptors identified by YFP
expression. Therefore, to verify these findings, we used
MrgDCreERT2;Ai32 mice and tamoxifen administration before
injury, to permanently label Mrgprd-expressing afferents with
ChR2-YFP ( Figs. 3D–F). We then tested whether the proportion
of cutaneous tibial afferents that were YFP 1 was altered following
nerve injury. Following hindpaw FB injection, ;15% of contralat-
eral, FB-labelled DRG neurons expressed YFP. This was reduced
to 6.0 61.2% 28 days after SNI crush injury and to only 1.7 60.9%
28 days after SNI trans (Fig. 3G). Uptake by uninjured YFP 1
neurons was equivalent 7 and 35 days after FB injection,
demonstrating that this reduction was not because 7 days were
insufficient for YFP 1 neurons to fully uptake FB (Fig. S3C, http://
links.lww.com/PAIN/C84). No significant difference in the per-
centage of FB-labelled YFP 1 DRG neurons between ipsilateral
and contralateral DRG was observed at 7 days following SNI trans
(Figs. S4A and B, http://links.lww.com/PAIN/C84), demonstrat-
ing that loss occurred after this timepoint. Analysis of the cross-
sectional soma area of FB-labelled, YFP 1 neurons in uninjured
DRG revealed an area of 361 6 138 mm2 (mean 6 SD) (Fig. S4C,
http://links.lww.com/PAIN/C84), which is a distribution profile
matching those neurons presumed lost. Collectively, these data
show that peripheral nerve injury results in a substantial loss of
nonpeptidergic,Mrgprd-expressing neurons, with SNI trans (ie, an
unrepaired axonal transection) resulting in an almost complete
loss of this population.
Figure 1. SNItrans induces death of small primary afferent neurons, accompanied by a reduction in volume, not cell density, of the dorsal root ganglion. (A)
Approach to differentially labelled intact afferents with tdTomato and damaged afferents with GFP after peripheral nerve injury using the Avil FlpO;Atf3CreERT2;RC::
FLTG mouse line and schematic of experimental timeline. (B) Representative image of GFP, tdTomato, and NeuN expression in an L4 DRG, 2 weeks after SNI trans.
Scale bars 5100 mm. (C and D) Stereological quantification of the total number of DRG neurons (C) or number of axotomized and intact neurons (D) in the L4 DRG
1, 2, 4, and 8 weeks after SNI trans or contralateral (contra) to injury. (C) One-way ANOVA with Tukey posttests; F4,10 537.98, P ,0.001. (D) Two-way RM ANOVA;
Timepoint 3 Color interaction F4,10 5 39.04, P , 0.001, n 5 3 mice; Tukey posttests (between injured groups): † P , 0.05 vs contra, ‡ P , 0.05 vs 1-week. (E)
Volume of DRG-containing cells (ie, excluding white matter tracts) following SNI trans. One-way ANOVA with Tukey posttests; F4,10 5 21.25, P , 0.001, n 5 3. (F)
Neuronal density within the DRG following SNI trans. One-way ANOVA; F4,10 52.77, P 50.09, n 53. (G) Population distribution of uninjured and injured afferents by
cross-sectional area, 1 and 8 weeks post-SNI trans. Kolmogorov–Smirnov tests of cumulative distributions; Uninjured: D 5 0.08, P 5 0.18; Injured: D 5 0.32, P ,
0.001; n 5 310 to 427 neurons from 3 mice. * P , 0.05, **P , 0.01, ***P , 0.001 vs contra. ANOVA, analysis of variance; DRG, dorsal root ganglion; GFP, green
fluorescent protein.
2868 A.H. Cooper et al. ·165 (2024) 2863–2876 PAIN®