Involvement of calcitonin gene-related peptide and CCL2 production in CD40-mediated behavioral hypersensitivity in a model of neuropathic pain

Abstract

The neuropeptide calcitonin gene-related peptide (CGRP) is known to play a pro-nociceptive role after peripheral nerve injury upon its release from primary afferent neurons in preclinical models of neuropathic pain. We previously demonstrated a critical role for spinal cord microglial CD40 in the development of spinal nerve L5 transection (L5Tx)-induced mechanical hypersensitivity. Herein, we investigated whether CGRP is involved in the CD40-mediated behavioral hypersensitivity. First, L5Tx was found to significantly induce CGRP expression in wild-type (WT) mice up to 14 days post-L5Tx. This increase in CGRP expression was reduced in CD40 knockout (KO) mice at day 14 post-L5Tx. Intrathecal injection of the CGRP antagonist CGRP8–37 significantly blocked L5Tx-induced mechanical hypersensitivity. In vitro, CGRP induced glial IL-6 and CCL2 production, and CD40 stimulation added to the effects of CGRP in neonatal glia. Further, there was decreased CCL2 production in CD40 KO mice compared to WT mice 21 days post-L5Tx. However, CGRP8–37 did not significantly affect spinal cord CCL2 production following L5Tx in WT mice. Altogether, these data suggest that CD40 contributes to the maintenance of behavioral hypersensitivity following peripheral nerve injury in part through two distinct pathways, the enhancement of CGRP expression and spinal cord CCL2 production.

Whole-body Vibration at Thoracic Resonance Induces Sustained Pain and Widespread Cervical Neuroinflammation in the Rat

“…The sensitivity to pain and injury severity is also evident in spinal CGRP expression (Fig. 5), which also increases after a host of painful peripheral traumas and inflammatory stimuli [18,50,56,69,92]. In contrast, it decreases after a painful root compression [37,92], most likely from axonal degeneration in the root decreasing trafficking to the dorsal horn.…”

Calcitonin gene-related peptide contributes to peripheral nerve injury-induced mechanical hypersensitivity through CCL5 and p38 pathways

“…Mice were randomly assigned to different treatment groups, and then received intrathecal injections (5 μl/mouse) (see Table 1) daily from days 6 to 13 post-surgery. This time period was chosen based on the spinal cord level of CGRP expression (peaked at day 7 and day 14 post-L5Tx) (Malon et al, 2011) and previous report (Yu et al, 1996a; Yu et al, 1996b). Intrathecal injections were performed as described previously (Malon et al, 2011).…”

“…This time period was chosen based on the spinal cord level of CGRP expression (peaked at day 7 and day 14 post-L5Tx) (Malon et al, 2011) and previous report (Yu et al, 1996a; Yu et al, 1996b). Intrathecal injections were performed as described previously (Malon et al, 2011). Besides vehicle-injected mice, animals that underwent L5Tx but received no i.t.…”

“…Primary adult mixed glia cultures were established using 8-week-old BABL/c NCI mice and maintained in complete Dulbecco’s Modified Eagle Media (cDMEM) (Lonza, Walkersville, MD) containing 50 μM 2-mercaptoethanol (2-ME, Sigma-Aldrich) in 24-well plates as we have previously described (Malon et al, 2011). Media was replaced on days 2, 4, 8, and 12 after the establishment of each culture.…”

“…All cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 . Mixed glia were treated at day 14 following the establishment of each culture with CGRP at 25, 100, 1000, and 4000 ng/ml as determined from previous experiments (Malon et al, 2011). Supernatants and cell lysate were collected 24 and 48 hours after treatment for CCL5 ELISAs and CBA respectively (see above).…”