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Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18 Page 5 of 32

observed withdrawal rates for each monofilament using a maximum-likelihood fitting method [47,51] . The
interpolated 50% withdrawal thresholds were then used for statistical analysis.

BIRT377 preparation
(R)-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione (BIRT377) was
[45]
first reported and characterized by Kelly et al. . BIRT377 is a small molecule that blocks the active
conformational change of the transmembrane β -integrin adhesion molecule, leukocyte function-associated
2
antigen-1 (LFA-1), that is expressed on leukocytes (e.g., T cell and myeloid cells) . Upon activation from
[37]
chemotactic signaling, LFA-1 undergoes a series of conformational changes from a bent inactive position to
a progressively straightened and active position, thus allowing binding of LFA-1 with the surface receptor,
[52]
intercellular adhesion molecule-1 (ICAM-1) expressed on endothelial cells . Upon LFA-1/ICAM-1 interaction,
cells expressing LFA-1 (leukocytes) are capable of undergoing transendothelial migration, and subsequently
traffic to regions where damage- or pathogen-associated tissue signals arise. Therefore, BIRT377 binding to
LFA-1 inhibits LFA-1/ICAM-1 molecular interactions, and prevents circulating leukocyte cell adhesion and
migration [44,45,53] to sites of inflammation. BIRT377 abolishes T cell and antigen presenting cell interactions
[54]
(referred as immune synapse), which is crucial for T cell activation . Moreover, BIRT377 is bioavailable and
easier to formulate for oral administration than antibodies against LFA-1 [44,45] and BIRT377 is impermeable
[42]
to blood-spinal cord barrier . Therefore, i.v. injection of BIFT377 is expected to impact: (1) leukocyte
migration to peripheral sites and across spinal endothelial cells; (2) macrophage proinflammatory function
and possibly T cell differentiation in the periphery; and (3) neuron-to-glial and immune communication in
the LSC due to BIRT377-mediated modulation in the periphery during neuropathy.

In an initial experiment, BIRT377 was gifted by HTW and CRW (University of Minnesota, College of
Pharmacy, MN, USA), with later experiments where BIRT377 was made commercially available (Tocris;
Cat#4776). BIRT377 was initially reconstituted in 200 proof ethyl alcohol EtOH (Sigma-Aldrich; Cat#7023)
as a stock solution (22.156 mg/mL), followed by creating aliquots (221.56 μg of BIRT377 in 10 μL), which
were stored in a clean sealed container at 4 °C for later use. On the day of intravenous (i.v.) injection,
one aliquot was diluted using sterile water (Hospira; Cat# NDC 0409-4887-10), such that each 50 µL i.v.
injection contained 2.5 µg BIRT377 (113.089 μM), and vortexed for 2 min. This dose was chosen based on
a pilot study of various doses (ranging from 100 ng to 5 µg) that 2.5 µg was the lowest reliably efficacious
[42]
dose in rats (unpublished data and ). Vehicle contained 0.226% EtOH in sterile water. Animals were
injected within the hour following BIRT377 dilution.

Intravenous BIRT377 injection
For all experiments characterizing BIRT377 efficacy, i.v. BIRT377 or equivolume vehicle injection into tail
veins of unanesthetized mice occurred on Day 10 post-surgery within 2.5 h of the initiation of the light
cycle. Using aseptic procedures, 50 µL of BIRT377 or vehicle was collected into individual 1 cc, 27-G 5/8
insulin syringes (Becton Dickinson; Cat#329412). The weight of each mouse was recorded followed by
placement for 30 s under a heat lamp with the mouse held in place by the tail and a soft, clean cloth placed
over the body to avoid excessive heat to the body while leaving the tail exposed. Heating the tail facilitates
tail vein dilation for ease of injection. Each mouse was moved immediately into a plastic restraint with
a slit through the top and back so as to allow easy placement of the mouse into the restraint, and proper
positioning of the tail. Held firmly in place, a 27-G sterile needle attached to a sterile 1 cc syringe was
inserted into the lateral tail vein, followed by a small amount of blood efflux into the syringe hub, with a
subsequent 5 s injection. Success of achieving accurate needle placement upon the first attempt was greater
than 99%. Following injection, a small piece of sterile gauze was placed over the injection site to stem
bleeding, and the mouse was removed from the restraint and placed back into its home cage. All mice
appeared normal (e.g., moving, grooming, interacting) following injections. The total time required for
handling and injection was less than 2 min without anesthesia.