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Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18 Page 19 of 32

(male: P = 0.029; female: P < 0.0001) and TNF (males: P = 0.001) mRNA levels were measured in allodynic-
reversed mice given BIRT377. Unexpectedly, no further increases in IL-10 mRNA levels were observed in
BIRT377-treated allodynic-reversed mice. It is notable that the magnitude of IL-1β increase was greater
in female CCI + Veh mice (P = 0.0002) than males. Correspondingly, the magnitude of BIRT377-induced
decreases in IL-1β was greatest in female CCI + BIRT mice [Figure 6B]. In general, the effects of BIRT377 on
these pro- and anti-inflammatory cytokines revealed similar trends in both male and female DRGs. Together,
these data support that BIRT377 not only affects immune cells at the nerve injury, but also is able to modulate
immune cells locally in the DRGs thereby dampening the proinflammatory environment contributing to pain
reversal.

BIRT377 predominantly restores IL-10 levels in the dorsal spinal cord
It is possible that BIRT377-mediated changes in cytokine mRNA levels at the damaged SCN and the
DRG together influence the inflammatory signals ultimately relayed to the spinal cord dorsal horn
where critical pain relays can be facilitated by spinal glial and resident immune cells. Moreover, it is
reasonably possible that BIRT377 additionally modulates leukocyte adhesion and spinal trafficking, thereby
controlling the peripheral leukocyte milieu recruited to the spinal cord as a consequence of nerve injury.
Therefore, spinal mRNA levels of CCL2 was assessed, as CCL2 is a well-established chemokine released
from damaged neurons that signals to circulating leukocytes (macrophages as well as subsets of T cells)
facilitating immune cell migration to the spinal cord. As predicted, a significant induction of CCL2 mRNA
was observed in the dorsal horn of the LSC ipsilateral to the SCN lesion both in males (P = 0.014) and
females (P < 0.0001), with a trend toward increased CCL2 in LSC contralateral to the SCN lesion in CCI
females compared to Sham conditions [Figure 7A and B]. Interestingly, CCL2 mRNA levels were similar in
neuropathic males given vehicle or BIRT377, whereas a significant bilateral reduction of CCL2 was observed
in females given BIRT377 (LSC ipsilateral: P = 0.0006; LSC contralateral: P = 0.04) [Figure 7A and B]. In
support of prior reports documenting the crucial role of IL-1β actions in mediating allodynia, a small but
significant increase in the levels of IL-1β mRNA were observed in female CCI + Veh (P < 0.0001), with a
similar trend observed from the contralateral side. Unexpectedly, BIRT377 treatment did not change IL-1β
mRNA levels in the spinal cord [Figure 7C and D].

Anti-inflammatory cytokines TGF-β1 and IL-10 were analyzed in the LSC both ipsilateral and contralateral
to the SCN lesion [Figure 7E-H]. Compared to Sham + Veh, a significant induction in ipsilateral TGF-β1 (male:
P = 0.0004; female: P = 0.013) and reduction of IL-10 (male: P = 0.0001; female: P < 0.0001) mRNA levels
were measured in all CCI + Veh mice [Figure 7E and G]. However, following BIRT377 treatment, ipsilateral
LSC IL-10 mRNA levels were elevated in both neuropathic males (P = 0.005) and females (P = 0.02), with
similar observations made in female ipsilateral LSC TGF-β1 mRNA levels [Figure 7E and G]. However, the
magnitude of IL-10 increases in ipsilateral LSC was greater in CCI + BIRT males than females, and CCI +
BIRT females displayed lower IL-10 levels than males (P = 0.019). Contralateral IL-10 mRNA levels displayed
the same pattern as ipsilateral dorsal horn following CCI: IL-10 mRNA levels were significantly decreased
in neuropathic females (P = 0.004), along with a similar trend in males (P = 0.07), compared to their
corresponding Sham + Veh groups. BIRT377 treatment increased contralateral IL-10 significantly from
CCI + Veh in males (P = 0.04), with a similar trend observed in females (P = 0.06) [Figure 7H]. These data
indicate that BIRT377-mediated pain reversal corresponds to increased bilateral spinal IL-10 mRNA levels
in neuropathic animals of both sexes.

BIRT377 reduced astrocyte activation in the spinal cord
The data above show that i.v. BIRT377 corresponds to reduced proinflammatory cytokines in both the SCN
and DRGs, and reduced CCL2 in the ipsilateral spinal cord, while elevating anti-inflammatory cytokines in
the SCN, DRGs, and LSC. Persistent microglial and astrocyte activation in the spinal cord is critical for the
chronicity of sciatic neuropathy. Reducing the pro-inflammatory cytokine milieu at peripheral anatomical