3.3. Spared nerve injury induces a loss of Trpm81 and
calcitonin gene-related peptide1 but not myelinated dorsal
root ganglion neurons
Loss restricted to nonpeptidergic nociceptors would not fully
account for the degree of total neuron loss that we observed.
Therefore, we studied a range of other subpopulations, both
small and large in diameter, for their vulnerability to injury-
induced loss. To investigate potential loss of Trpm8 1 (cold-
sensitive), calcitonin gene-related peptide 1 (CGRP) (peptider-
gic), and myelinated subpopulations of DRG neurons following
nerve injury, we applied our FB-labelling approach in Trpm8 FlpO;
RC::FLTG (FlpO-dependent tdTom expression), Calca CreERT2;
Ai32 (Cre-dependent ChR2-YFP expression) and Thy1-CFP
mice, respectively ( Figs. 4A–D). Trpm8-tdTom was expressed
Figure 2.Spared nerve crush and transection lead to a loss of small DRG neurons. (A) Approach to restrict analysis to damaged afferents: a subcutaneous
injection of the tracer FB into both hindpaws labelled tibial afferents, before unilateral SNI trans or SNIcrush surgery. (B) Representative image of FB labelling and NeuN
immunostaining in the L4 DRG. The image is a projection of optical sections at 3- mm intervals through the entirety of a 30- mm-thick tissue section. Scale bar 5
100 mm. (C and D) Quantification of the cross-sectional area of FastBlue labelled DRG neurons ipsilateral and contralateral to SNI trans (C) or SNI crush injury (D)
reveals a loss of small afferents and subsequent shift in population distribution. Kolmogorov–Smirnov tests of cumulative distributions; SNI trans:D 5 0.25, P ,
0.001; n 5 183 or 191 neurons from 3 mice; SNI crush:D 5 0.22, P , 0.001, n 5 319 or 325 neurons from 3 mice. (E) Experimental approach for whole DRG
volumetric analyses after SNI trans. (F) Representative 3D rendering of TDP-43 profiles and corresponding nuclear spot profiles following Imaris-based spot
detection feature. Scale bar 5 100 mm. (G) Quantification of DRG nuclear spot volume ipsilateral and contralateral to SNI trans. Kolmogorov–Smirnov tests of
cumulative distribution: D 5 0.06, P , 0.001, n 5 30,206 (contra) or 32,544 (ipsi) nuclei from 4 (contra) or 5 (ipsi) mice. (H) Total number of nuclear spots, by size,
per DRG. Two-way RM ANOVA; size bin 3 injury interaction: F2,145 8.26, P 5 0.004; n 5 4 to 5 mice; ˇS´ıd ´ak multiple comparisons tests: ** P , 0.01. ANOVA,
analysis of variance; DRG, dorsal root ganglion; FB, FastBlue; RM, repeated measures.
December 2024 ·Volume 165 ·Number 12 www.painjournalonline.com 2869