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Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18 Page 3 of 32
[30]
implicated a T cell role by examining cell migration to the DRGs [27,36] or the spinal cord in neuropathic
females. However, it is critical to identify T cell subtypes present in these key anatomical regions because
discrete subtypes exert a distinctly different impact on surrounding tissue during chronic pain. The critical
roles of subtypes of T cells within discrete anatomical pain-related regions (peripheral or central) remain
unclear.

We hypothesized that peripheral immune and glial responses following peripheral nerve damage are
quantitatively and qualitatively (specific immune cells) different between sexes. If true, pain therapy that
targets specific immune actions may require distinctly different mechanisms to exert efficacy. Lymphocyte
function-associated antigen-1 (LFA-1) is an adhesion molecule expressed on myeloid and T cells and
[37]
possibly spinal microglia and is critical for immune cell adhesion and migration . In addition to the
widely characterized role of LFA-1, emerging evidence suggests that LFA-1 regulates various macrophage
proinflammatory functions as well as T cell activation and differentiation [38-43] . Based on existing gaps in
understanding sex differences in glial, innate or adaptive immune cell function driving neuropathic pain
and the related cytokine/chemokine repertoire, the current study examined whether: (1) the development,
magnitude, and duration of mechanical allodynia were different between male and female mice subjected
to a well-established peripheral nerve injury (CCI) model; (2) blocking immune cell migration and/or
altering the proinflammatory phenotype by preventing peripheral LFA-1 actions using a blood-spinal
barrier impermeable small molecule antagonist, BIRT377 [42,44,45] , reduces allodynia in males and females;
and (3) BIRT377 directly modulates myeloid/glial-derived and T cell-related pro- and anti-inflammatory
cytokine expression. The results identified differences in the magnitude of immune factor expression
contributing to neuropathy between males and females, and that BIRT337 reversed allodynia similarly
between males and females, and altered the corresponding expression of sex-specific immune factors.

METHODS
Animals
Experiments were performed using 10-14 week-old C57BL/6 mice (wildtype; FFID: IMSR JAX:000664)
purchased from Jackson Laboratories or were bred in-house with parent mice purchased from Jackson
Laboratories (Bar Harbor, ME, USA). Age at the time of surgery ranged from 11-12 weeks for males and
10-12 weeks for females. Mice were housed with their cage-mates in groups of 2-5, in temperature (23 °C ±
2 °C) and light (12:12 light:dark; lights on at 6:00 am) controlled rooms, fed standard rodent chow and
water ad libitum, and acclimated for 1-2 weeks prior to handling. All mice were routinely monitored by
the animal care staff under the direction of the institutional veterinarian, with cages and bedding changed
every 7 days. Mice were maintained in separate male or female mouse colonies and were behaviorally
assessed in separate testing rooms at the University of New Mexico (UNM) Health Sciences Center (HSC)
Animal Facility. Pilot studies of behavioral hindpaw threshold responses were conducted to determine
whether different phases of the estrous cycle altered behavioral outcomes at baseline (BL) and after surgical
manipulation. Despite female mice entering experiments at different phases of the estrous cycle, hindpaw
responses remained stable and predictable. Consequently, the stage of the estrous cycle varied and was
not considered a key factor influencing hindpaw sensory responses throughout the chronic neuropathy
paradigm.

All procedures were approved by the Institutional Animal Care and Use Committee of the UNM HSC,
conducted in accordance to the NIH Guidelines for the Care and Use of Laboratory Animals, and closely
adhered to recommendations from the International Association for the Study of Pain for the use of
animals in research (Foundation for Biomedical Research, The Biomedical Investigator’s Handbook for
Researchers Using Animal Models. Washington, D.C.: FBR, 1987. WWW: http://www.fbresearch.org/).