neuron loss after nerve injury and to test the hypothesis that loss is
not equally distributed across molecular populations.
2. Methods
2.1. Animals
Mice were housed in groups in humidity- and temperature-controlled
rooms with free access to food and water, on a 12-hour light–dark
cycle, and with environmental enrichment. Animal procedures were
performed under a UK Home Office Project Licence and in
accordance with the UK Home Office (Scientific Procedures) Act
(1986). All studies were approved by the Ethical Review Process
Applications Panel of the University of Glasgow or Oxford and conform
to the ARRIVE guidelines. Experiments were performed on adult male
and female mice aged 7 to 16 weeks at the start of the experiments. All
experimental cohorts contained a mix of male and female mice, apart
from the cohort of MrgprdCreERT2;Ai32 mice that underwent SNIcrush
surgery, which was exclusively female. Details of transgenic lines are
provided in Table 1.T a m o x i f e nw a sa d m i n i s t e r e db yi . p .i n j e c t i o no f
20 mg/mL tamoxifen (Sigma-Aldrich) dissolved in wheat germ oil
(doses described inTable 1). There were 2 instances where animals
were excluded from data analysis : One (cyan fluorescent protein)
Thy1-CFP died of unknown causes not related to the procedure and
before the experimental endpoint, and one MrgDCreERT2;Ai32
exhibited no fluorophore expression and was therefore deemed to
have been incorrectly genotyped. Group sizes were based on the
extent of neuronal loss 28d following sciatic nerve transection
identified by Shi et al.50 Givena 5 0.05, power5 0.8, and an effect
size of 4.81, power analysis projects that a group size of 3 mice would
be needed.
2.2. Spared nerve transection and crush surgeries
Spared nerve injury (transection of the common peroneal and
tibial branches of the sciatic nerve; SNI trans) and common
peroneal and tibial crush injury (SNI crush), in which nerve axons
were severed but the epineurium remained intact, were
performed as previously described.12 Anesthesia was induced
with 3% to 5% isoflurane and then maintained at 1.5% to 2% as
required. Analgesia, consisting of carprofen (10 mg/kg) and
buprenorphine (0.05 mg/kg) (Glasgow) or carprofen (5 mg/kg)
and local bupivacaine (2 mg/kg) (Oxford) was provided perioper-
atively. The left hindpaw was secured with tape in hip abduction,
and the operative field (lateral surface of the thigh) was shaved.
Ophthalmic ointment was applied to the eyes, and the shaved
area was swabbed with chlorhexidine solution. A longitudinal
incision was made in the skin at the lateral mid-thigh. Using blunt
dissection, an opening was made through the biceps femoris,
exposing the sciatic nerve and the 3 peripheral branches (sural,
tibial, and common peroneal nerves). For SNI trans, the common
peroneal and tibial nerves were ligated using a 6-0 Vicryl suture
(Ethicon, Raritan, NJ), and a 1- to 2-mm piece distal to the suture
was removed using spring scissors. For SNIcrush, the exposed
tibial and common peroneal nerves were clamped using a pair of
fine hemostats (Fine Science Tools, Heidelberg, Germany) closed
to their second clip, leaving the nerve branches intact but
translucent. The muscle was closed with one 6-0 Vicryl suture
(Ethicon), and the skin incision was closed with one 10 mm
wound clip (Alzet, Cupertino, CA). Animals were monitored daily
for self-mutilation, and no animals required sacrifice due to tissue
damage.
Table 1
Transgenic lines used in the study.
Used name Full name Putative population Ref Source Tamoxifen regime
Atf3CreERT2 Atf3tm1.1(cre/ERT2)Msra Axotomised afferents 13 Gift: Dr Franziska Denk 50 mg/kg on days 0, 3, and 7 after surgery
AvilFlpO Aviltm1(flpo)Ddg Sensory neurons 1 Gift: Prof David Ginty N.A.
MrgDCreERT2 Mrgprdtm1.1(cre/ERT2)Wql Major class of nonpeptidergic
neurons
39 The Jackson Laboratory (RRID:
IMSR_JAX:031286)
General: 1x 50 mg/kg in adulthood, (.1 week
before experiment)
3D volumetric analysis: 5x i.p. (0.5 mg/animal/