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Page 4 of 32 Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18

Chronic constriction injury
[46]
A modification of Bennett and Xie’s sciatic nerve (SCN) Chronic constriction injury (CCI) was used
[47]
after BL hindpaw threshold assessment, as detailed previously . Briefly, following isoflurane anesthesia
(induction at 3.0 vol.% followed by 1.5 vol.%-2.0 vol.% in oxygen, 2.0 L/min), the dorsal left thigh was shaved
and cleaned using 70% Ethanol (EtOH) that was air dried prior to surgery. Using aseptic procedures, the
SCN was exposed using blunt dissection scissors through the muscular fascia. Sterile plastic probes were
used to locate and lift the SCN from its position between the muscles. Three, 2 cm-length pieces of chromic
gut suture (Ethicon: 4-0 or 5-0, Cat#635H and Cat#634G, respectively) were then snugly tied around the
SCN proximal to the trifurcation with care to avoid pinching the nerve, with ~1.5 mm spacing between
sutures. Throughout this process, the nerve was kept thoroughly irrigated using isotonic sterile saline
(Hospira; Cat#NDC 0409-4888-03). Sham surgeries were performed identically, but without the chromic
gut ligation. The nerve was then placed back into its position using the plastic probes, and the muscles were
TM
then closed using one 4-0 silk suture (Ethicon; Cat#83G). Skin was closed using two Reflex wound clips
(Kent Scientific Corp; Cat#INS750344). Total time for the surgical procedure was ~20 min, followed by a ~10
min recovery from anesthesia. Body weight was monitored prior to and after surgery to confirm healthy
recovery [Supplementary Figure]. Following surgery, wound condition, hindpaw autotomia, activity levels
and grooming appearance were checked routinely (each 1-2 days). Less than 1.0% of animals revealed
abnormal recovery and were immediately euthanized when identified.

Behavioral assessment of hindpaw mechanical allodynia
Mechanical allodynia was chosen for investigation because pathological pain intensity (touch sensitivity)
occurs clinically at much lower ranges of stimulus intensity compared to that observed when examining
[48]
mechanical or thermal hyperalgesia. Thus, the impact of clinical allodynia is thought to be much greater .
Mice were habituated to the testing environment for ~45 min within the first 4 h of the light cycle, for four
periods over the course of one week prior to BL hindpaw assessment. Hindpaw threshold responses to light
mechanical stimuli were assessed by adopting principles of the von Frey fiber test originally developed for
[49]
[47]
the rat , and modified for the mouse, as recently described in detail . Hindpaw assessment occurred
within the first 2 h of the light cycle in testing groups of 4-6, with testers blind to experimental conditions.
Each group comprised a minimum of 1-4 mouse/mice per condition to ultimately reach n = 6 mice/
experimental condition. Time points for behavior assessments were chosen based on pilot studies and prior
reports to capture potential subtle differences during the development of allodynia and BIRT377-mediated
pain reversal.

The von Frey test was applied using nine calibrated monofilaments (touch-test sensory evaluator: North
Coast Medical; Cat#NC12775) applied for a maximum of 3.0s to the plantar surface of both the left
and right hindpaws, with laterality of hindpaw testing occurring randomly, with repeated stimulus
presentations to a single animal using a minimum inter-trial stimulus period of 30s. The log intensity
of the nine monofilaments used is defined as log (grams × 10,000) with the range of intensity being as
10
follows, reported in log (grams): 2.36 (0.022 g), 2.44 (0.028 g), 2.83 (0.068 g), 3.22 (0.166 g), 3.61 (0.407 g),
3.84 (0.692 g), 4.08 (1.202 g), 4.17 (1.479 g), and 4.31 (2.042 g). Testing began using the fiber marked 3.22
with subsequent monofilaments used based on the response/non-response of the mouse to the previous
monofilament tested: if no response was elicited by the monofilament stimulus presented (e.g., 3.22),
the next “heavier” monofilament was tested (e.g., 3.61); if a response was elicited by the monofilament
stimulus presented (e.g., 3.22), the next “lighter” monofilament was tested (e.g., 2.83). A maximum total of
six stimulus presentations were applied to each paw. The total number of positive and negative responses
were then entered into the computer software program, PsychoFit (http://psych.colorado.edu/~lharvey:
RRID: SCR_015381) to determine the absolute withdrawal threshold (50% paw withdrawal threshold),
[50]
as previously described . The PsychoFit program fits a Gaussian integral psychometric function to the