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Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18 Page 9 of 32
[51]
surface antigens, and intracellular proteins, as described previously, with minor modifications . Briefly,
cells were pelleted into FACS staining tubes (BD Falcon) and incubated with Fc-block (blocking buffer)
for 10 min on ice, stained with viability dye (25 min, on ice, dark) and then incubated with CD4 antibody
(25 min, on ice, dark). To stain for RORɣt and intracellular cytokines (IL-17, TNF, IL-10 and TGF-β1), an
intracellular cytokine staining kit (Cat#00-5523-00, eBioScience, Thermofisher Scientific, MA, USA) was
used. With this protocol, cells were fixed and permeabilized for 60 min at RT and protected from light.
Then cells were washed 2× with permeabilization buffer (2 mL/tube) for 5 min at RT. To prevent non-
specific binding of the antibodies, cells were incubated with blocking buffer (containing 2.5 µg anti-mouse
CD32 purified antibody) for 15 min on ice. Without washing, fluorochrome conjugated antibodies against
RORɣt, IL-17A, and TNF (cells from Th17 differentiation wells), or against IL-10 and TGF-β1 (cells from
Treg differentiation wells), were added, and cells were incubated for 45 min at RT in the dark. Cells were
then washed 2 × again with 2mL of permeabilization buffer at 300 × g for 5 min, at 4 °C, resuspended in
300 μL FACs buffer (1 × PBS containing 1% bovine serum albumin and 1mM EDTA), and kept on ice
protected from light until data acquisition. Blocking buffer was purchased from BD Biosciences (Fc block,
Cat#553141). All the fluorochrome conjugated antibodies were purchased from eBioscience (Thermofisher
6
Scientific, MA, USA) and used at 0.125-1 μg/per 10 cells per tube, as recommended by the manufacturer.
T cell events (50,000) were collected using a BD LSR Fortessa Cell Analyzer, and later analyzed via FlowJo
software v.8.7.4. Only viable (based on light scatter properties and viability dye) CD4 T cells (based
on positive staining of surface CD4 antigen) were included for further analysis. Positive staining for
transcription factor and/or cytokines were determined based on staining with fluorochrome conjugated
isotype controls (IgG2b and IgGa).

Experimental design and statistical analysis
Three independent behavioral experiments were conducted with an n = 6 mice in each group in each
experiment. Prior work demonstrates n = 4-6 mice/experimental condition is sufficient to yield reliable
group differences when examining similar endpoints [3,4,13,33-35,47] . The initial experiment was an examination
of differences in hindpaw sensitivity between sexes following peri-sciatic manipulations (sham vs. 4-0 vs.
5-0 CCI) during a 56-days timecourse. Thus, a 2 × 3 repeated measures analysis of variance (ANOVA)
was conducted, with hindpaw thresholds assessed at BL and re-assessed every 1, 2, 3, or 5 days after
surgical manipulation until complete resolution of allodynia was observed. The behavioral profile of the
mice presented in Figure 1 was predicted to inform parameters of Experiment 2. That is, to characterize
the earliest maximal onset, stable maintenance and duration of allodynia in both male and female mice.
For experiment 2, injection of BIRT377 was administered on Day 10 after surgery, and the efficacy and
duration of reversal from allodynia by BIRT377 was assessed. Experiment 2 design was a 2 (male vs.
female) × 2 (vehicle vs. BIRT377) and analyzed by a 2-way repeated measures ANOVA where hindpaw
assessment occurred prior to and following BIRT377 treatment. The goal of Experiment 3 was to examine
the biochemical profile (protein and mRNA) in discrete tissue systems at a time when BIRT377 exerts
maximal efficacy on stable allodynia as determined by Experiment 2. Therefore, Experiment 3 design was a
2 (male vs. female) × 2 (sham vs. CCI) × 2 (vehicle vs. BIRT377) and analyzed by a 3-way repeated measures
ANOVA, with re-assessment of hindpaw thresholds terminating at peak BIRT377 efficacy. At this time,
spleen, SCN, DRG, ipsilateral and contralateral LSC tissues were dissected and processed.

All behavioral data was graphed in GraphPad Prism version 7.02 (GraphPad Software Inc.;
RRID:SCR_002798). All statistics were run using IBM SPSS Statistics version 24 (IBM; RRID:SCR_002865).
ANOVAs were performed for data collected at BL and on injection day. For all other behavioral timepoints,
repeated measures ANOVA were performed to assess differences of group and timecourse between
treatments. The assumption of sphericity was assessed using Mauchly’s Test of Spericity (α = 0.05) and, if
the assumption of sphericity was violated (P > 0.05), the reported degrees of freedom and p-values were
adjusted using the Greenhouse-Geisser correction to protect against Type I errors [47,51] . Fisher’s LSD test was