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Page 12 of 32 Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18

material and less encapsulating sheath compared to SCNs treated with 4-0 CCI [Figure 1G and H]. Upon
further dissection of the sheath and sutures away from the nerve in 4-0 and 5-0 CCI, marked indentations
beneath the ligature in both conditions were observed. These observations suggest that reversal of
allodynia from CCI in mice involves processes that are independent of the presence of the sutures. That is,
the physiological response to peri-sciatic CCI is critical in the resolution of allodynia, and is not dependent
on the presence of factors from the suture material itself.

The LFA-1 antagonist BIRT377 reverses allodynia in male and female mice
Given the onset, intensity, and duration of allodynia was similar following either 4-0 or 5-0 peri-sciatic
CCI in both males and females, subsequent experiments applied 5-0 chromic gut suture for CCI. Mice
were assessed using the von Frey fiber test at BL, and no significant differences were observed. Mice with
CCI developed maximal bilateral allodynia by Day 8 post-CCI [Figure 2A]. On Day 10 post-CCI, when all
animals revealed stable and maximal allodynia, an i.v. injection of BIRT377 or vehicle was given followed
by hindpaw re-assessment. Compared to mice given vehicle, complete reversal from allodynia was observed
in both male and female animals following BIRT377 injection. Interestingly, a slight delay and duration
of reversal of ipsilateral hindpaw sensitivity was observed in females compared to males. Specifically,
BIRT377-mediated reversal of allodynia was delayed by 24 h in female mice, with allodynia returning 24 h
earlier than their male counterparts. Contralateral hindpaw sensitivity was reduced by BIRT377 treatment
to a similar degree and magnitude between males and females, as no statistical differences were observed.
While it is clear that both male and female mice develop allodynia to the same degree with a similar
duration, the difference in their response to i.v. BIRT377 suggests that the underlying processes leading to
allodynia may not simply overlap, but instead may include distinct mechanisms between male and female
mice.

In an effort to expand on characterizing potential sex-dependent differences in expression levels of
peripheral immune signaling molecules (pro- and anti-inflammatory cytokines) during established
peripheral neuropathy or BIRT377-induced reversal from neuropathy, a separate experiment was conducted
to replicate the effect of BIRT377 on allodynia which was terminated at the peak of BIRT377 mediated
pain reversal [Figure 2B] and tissues were collected for protein or mRNA (represented in subsequent
figures) analysis. In this replication study, while differences in ipsilateral, but not the contralateral hindpaw
threshold responses were observed at BL (F = 2.27, P = 0.048), these differences may simply be due to an
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exceptionally small variance in the threshold responses of female mice compared to males. However, these
differences are not considered physiologically meaningful, as such variance was not observed previously
or routinely in either the ipsilateral or contralateral hindpaws. Compared to sham-operated animals, male
and female mice with CCI developed clear allodynia through Day 10 [Figure 2B]. A main effect of time
(ipsilateral: F 2.22,88.85 = 265.36, P < 0.001; contralateral: F 2.11,84.20 = 213.38, P < 0.001) and surgery (ipsilateral:
F = 1612.46, P < 0.001; contralateral: F = 978.01, P < 0.001), and an interaction between time and
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surgery (ipsilateral: F 2.22,88.85 = 240.45, P < 0.001; contralateral: F 2.11,84.20 = 200.82, P < 0.001) was observed.
Following BIRT377 treatment, while sham animals remained stably responsive and close to BL thresholds
throughout the timecourse, partial bilateral reversal from allodynia was observed by 24 h in males, but
not females. Additionally, maximal effects of BIRT377 on allodynia were observed a full day sooner in
males than in females [Figure 2B]. Main effects of time (ipsilateral: F 3,120 = 65.14, P < 0.001; contralateral:
F 3,120 = 71.58, P < 0.001), injection (ipsilateral: F = 218.80, P < 0.001; contralateral: F = 306.81, P < 0.001),
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and surgery (ipsilateral: F = 1818.98, P < 0.001; contralateral: F = 1816.36, P < 0.001), and interactions
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between time and sex (ipsilateral: F 3,120 = 5.31, P = 0.002; contralateral: F 3,120 = 2.86, P = 0.040), time and
injection (ipsilateral: F 3,120 = 62.77, P < 0.001; contralateral: F 3,120 = 69.52, P < 0.001), and sex and injection
(ipsilateral: F = 16.08, P < 0.001; contralateral: F = 10.70, P = 0.002) were observed. However, by Day 3
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post-injection, both males and females achieved similar levels of reversal from allodynia.