cell death and apoptosis with more than 10 genes were
examined. Filtered count data of expressed and nondifferentially
expressed genes were used as a background.
2.8. Dorsal root ganglion culture
Dorsal root ganglia were dissected from MrgD CreERT2;Ai32 and
CalcaCreERT2;Ai32 mice .1 week after dosing with tamoxifen and
enzymatically digested at 37˚˚C for 80 minutes in dispase type II
(4.7 mg/mL) plus collagenase type II (4 mg/mL) (Worthington
Biochemical), as described previously. 63 Mechanically dissoci-
ated cells were plated onto laminin/poly-D-lysine (R&D Systems,
Minneapolis, MN) treated coverslips in complete Neurobasal Plus
medium (Neurobasal Plus media supplemented with 2% (vol/vol)
B27 Plus, 1% N2, 1% Glutamax, and 1% antibiotic–antimycotic
[ThermoFisher Scientific, Waltham, MA]). Mouse nerve growth
factor (GF) (50 ng/mL; nerve growth factor (NGF), PeproTech,
Cranbury, NJ) and 10 ng/mL glial-derived neurotrophic factor
(GDNF, PeproTech) were added to the media under some
conditions. Cytosine b-D-arabinofuranoside (4 mM) was added to
the media for 24 hours the day after plating to reduce the
proliferation of nonneuronal cells. Media was refreshed 3 times
per week thereafter. Cultures were fixed for 10 minutes at room
temperature with 4% paraformaldehyde and subsequently
processed by immunocytochemistry (described earlier).
2.9. Statistical analysis
Data are expressed as mean 6 SEM unless otherwise specified,
and P values of less than 0.05 were considered significant. Power
calculations were performed using G*Power 3.1.9.7. 15 A
quantitative Venn diagram was created using BioVenn. 25 All
other statistical analyses were performed in Prism 10 (GraphPad
Software, Inc, Boston, MA) or R using paired t tests or 1- or 2-way
RM ANOVAs (repeated measures analysis of variance), where
appropriate. Normality was assessed by the Shapiro–Wilk test. If
the main analysis of variance effect was significant,ˇS´ıd ´ak or
Tukey multiple comparisons tests were performed. To compare
population distributions of soma cross-sectional area or volume,
Kolmogorov–Smirnov tests were performed.
3. Results
3.1. Peripheral nerve injury induces a loss of small neurons
from the dorsal root ganglion
To assess the gross loss of neurons from DRG following nerve
injury, we generated the Avil FlpO;Atf3CreERT2;RC::FLTG mouse
line in which na ¨ıve and axotomized sensory neurons were
differentially labelled. In this mouse line, all neurons express
tdTomato (Flp-dependent) in the na ¨ıve state and switch to
expressing green fluorescent protein (GFP) upon axonal damage
and concurrent tamoxifen treatment (Flp- and Cre-dependent)
(Figs. 1A and B ). Following pilot experiments to optimize
tamoxifen dosing regimen, this approach was both highly efficient
and specific (with the caveat that it was necessary to wait for
several days after nerve injury for Cre-induced GFP expression):
14 days after SNI trans surgery, GFP was expressed by 99.1 6
0.6% of Atf3-expressing ipsilateral L4 DRG neurons, while we
observed GFP in only 4.66 0.7% of contralateral DRG neurons
(Figs. S2A–D, http://links.lww.com/PAIN/C84). We then used
a stereological approach to quantify the total number of neurons
in L4 DRG ipsilateral to injury 1, 2, 4, and 8 weeks after SNItrans, as
well as contralateral to injury. One week after SNI trans,w e
observed 7809 6153 neurons per DRG; this was not significantly
different to the number of neurons in the contralateral DRG
(7917 6 349), whereas cell number approximately halved by
8 weeks postinjury to 3963 6 410 neurons per DRG ( Fig. 1C).
Separating analysis into intact vs axotomized afferents revealed
that only axotomized afferents were lost, with no difference
observed in numbers of intact afferents ( Fig. 1D). Between 1 and
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)