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Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18 Page 13 of 32

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Figure 2. The LFA-1 antagonist, BIRT377, similarly reverses allodynia in males and females. Mice were either sham or treated with
perisciatic 5-0 suture. (A) All groups of mice show similar BL threshold hindpaw sensitivity. Following 5-0 CCI, all animals develop
clear allodynia during an 8-day timecourse, showing stable allodynia on Day 10, with a main effect of time (ipsilateral: F 3.02,60.48 =
1003.02, P < 0.001; contralateral: F 3.56,71.15 = 528.60, P < 0.001). Additionally, a main effect of sex during the development of allodynia
is observed (ipsilateral: F 1,20 = 21.27, P < 0.001; contralateral: F 1,20 = 13.06, P = 0.002), with a significant interaction between time
and sex (ipsilateral: F 3.02,60.48 = 10.899, P < 0.001; contralateral: F 3.56,71.15 = 3.23, P = 0.021). Following injections on Day 10 post-
surgery, clear reversal from allodynia resulting from BIRT377 injection is observed compared to vehicle treated mice, supported by
a main effect of injection (ipsilateral: F 1,20 = 328.97, P < 0.001; contralateral: F 1,20 = 74.47, P < 0.001). In addition, male mice treated
with BIRT377 appeared to reverse from allodynia 1 day sooner than female BIRT377-treated mice, as observed in hindpaw responses
ipsilateral (F 1,20 = 12.12, P = 0.002) but not contralateral to the CCI, with an interaction between time and sex (ipsilateral: F 4.33,86.61 =
9.33, P < 0.001; contralateral: F 4.92,98.34 = 3.15, P = 0.012), and time and injection (ipsilateral: F 4.33,86.61 = 70.29, P < 0.001; contralateral:
F 4.92,98.34 = 32.77, P < 0.001). (B) Experimental replication of BIRT377 reversal in males and females following CCI, with the onset and
full development of bilateral allodynia occuring during a 10-day timecourse. Female mice reveal delayed onset of allodynia but no
sex differences are observed by Day 10 post-surgery, when maximal allodynia is observed in both males and females. As previously
observed, female 5-0 CCI mice treated with BIRT377 displayed slightly slower reversal from allodynia compared to males, with
maximal bilateral reversal observed by Day 3 post-injection. n = 6 for all groups

Characterization of sciatic nerve anti- and proinflammatory cytokine/chemokine mRNA levels in
males and females following BIRT377 treatment
Prior studies suggest contralateral allodynia referred to as “mirror pain” corresponds to pathological events
at the spinal cord [22,72,73,76-81] . In the current study, inflammatory cytokine changes were examined in the
ipsilateral SCN and DRGs, as well as in both the ipsilateral and contralateral LSC dorsal horn to complement
prior reports. In the ipsilateral SCN, mRNA levels of the proinflammatory cytokines, CCL2, IL-1β and TNF,
were robustly elevated in both males (CCL2, IL-1β and TNF: P < 0.0001) and females (CCL2: P = 0.039; IL-1β:
P = 0.0007; TNF: P = 0.0006), compared to the corresponding sham-treated controls [Figure 3A-C]. A greater
magnitude of CCL2 (P = 0.015) and IL-1β (P < 0.0002) increase was observed in CCI + Veh males when
compared to CCI + Veh females. Treatment with BIRT377 in CCI-operated mice (CCI + BIRT) revealed a
reduction in CCL2 in males (P = 0.006), and in both males and females, a reduction in IL-1β (male: P < 0.0001;
female: P = 0.049) and TNF (male: P = 0.0001; female: P = 0.022) mRNA levels, with the largest magnitude