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Page 6 of 32 Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18

Sciatic nerve biopsy
Following characterization of the timecourse of hindpaw sensory thresholds from sham or CCI treated
mice using either 4-0 or 5-0 chromic gut, the presence of suture material and the condition of the SCN
were carefully examined. Following complete return of sensory thresholds similar to BL levels, the
ipsilateral SCN was dissected, and the degree of both suture absorption and nerve perturbation was noted.
The appearance of each SCN at the time of dissection was documented by photograph.

To accomplish biopsies, animals were euthanized just prior to biopsy using 2% CO in a closed container
2
followed by cervical dislocation. An incision was then made on the ipsilateral skin overlaying the CCI
manipulation, and the skin was retracted to expose the underlying muscle and surrounding area,
which appeared to be healthy. Blunt dissection scissors were used to re-expose the underlying SCN.
The surrounding muscle and remaining sutures and encapsulating sheath were removed, followed by
dissection of an approximately 1 cm length of SCN. The nerve segment was then placed next to a ruler (in
centimeters), on a clean black surface for documentation.

Tissue collection for RNA and protein analysis
Tissue collection was conducted in six cohorts of mice (8 mice in each cohort, N of 1 from each
experimental condition) as previously described [47,51] and modified as described here. Immediately following
behavioral analysis on Day 13 post-surgery (Day 3 post-injection), mice were deeply anesthetized under
isoflurane (10 min in 5% isoflurane and in oxygen at 2 L/min), followed by rapid transcardial perfusion
with ice cold 0.1 M phosphate buffered saline (PBS; pH = 7.4; flow rate 10 mL/min). Following collection
of the spleen, mice were placed on a frozen gel refrigerant pack (Glacier Ice, Pelton Shepherd Industries),
and the LSC (L3-L6) was dissected, with the dorsal spinal cord ipsilateral and contralateral to the sciatic
ligation stored separately. Additionally, lumbar DRG (L4-L6) ipsilateral to the sciatic ligation, and the SCN
were dissected. All tissues were immediately placed in DNase/RNase/Protease-free 1.5 mL disposable pellet
mixer microtubes (VWR International; Cat#47747-358), briefly spun down, frozen on dry ice, and stored at
-80 °C for future analysis.

Total RNA isolation
[47]
Total RNA was extracted as described previously , with minor modifications as briefly described here.
Extraction was performed using the miRNeasy Micro Kit (Qiagen; Cat#217084) per manufacturer’s
instructions except where noted. Homogenization was performed using a motormlized VWR disposable
pellet mixer and cordless motor pestle system (VWR; cordless pestle motor: Cat#47747-370; 1.5 mL
microtubes: Cat#47747-362; 1.5 mL pestle: Cat#47747-358; and 1.5 pestle and microtube combo: Cat#47747-
366: DRGs only) followed by addition of Qiazol Lysis Reagent (Qiazol; Qiagen; Cat#79306). DRGs were
then transferred into microtubes prior to homogenization. Samples were homogenized in Qiazol with the
motorized pestle for 60 s, and used for RNA extraction (Qiagen; miRNeasy Micro Kit). SCN and LSC were
homogenized prior to aliquoting the tissue into two microtubes for protein or RNA extraction. 100 µL of
chilled 1 × phosphate buffered saline (PBS; 10 × PBS diluted to 1 × with DNase/RNase free water; Sigma-
Aldrich; Cat#P7059; Cat#W4502 respectively) was added to the tube containing the tissue. The SCN was
chopped quickly using scissors for 30 s. Both SCN and LSC were then homogenized with the motorized
pestle for 15 s. After initial homogenization, 40 µL of the homogenized solution was removed and placed
into a separate 1.5 microtube containing 150 µL of chilled Qiazol and homogenized for an additional 15 s
for LSC, or 30 s for SCN, prior to using the miRNeasy Micro Kit for RNA extraction.

Minor changes were incorporated for mRNA extraction using the miRNeasy kits as follows. An initial
homogenization in 150-200 µL of Qiazol occurred, with the final volume increased to 700 µL following
homogenization. Samples were vortexed and incubated at room temperature (RT) for 7 min, followed by
the addition of 140 µL of chloroform (Sigma-Aldrich; Cat#C2432). The samples were then hand-shaken