Previous studies by our and other groups on an acute experimental model of neurogenic inflammation evoked by intradermal injection of CAP have physiologically and pharmacologically demonstrated that cutaneous inflammatory reactions characterized by local vasodilation (flare) and edema (increased paw-thickness) are predominantly mediated by triggering DRRs [17, 19, 20]. DRR activity has been recorded electrophysiologically from the central end of individual Aδ- and C-primary afferents and shown to be enhanced after CAP injection [18, 21, 29]. In the present study, we have further extended our ongoing project in the following respects. 1) New evidence has been provided to confirm the view that DRRs are triggered and then enhanced by activation of TRPV1 receptors to evoke neurogenic inflammation by driving the release of neuropeptides (CGRP and/or SP). 2) pharmacological studies using dose-response analyses of antagonism of TRPV1 and neuropeptide receptors reveal that the released CGRP and SP participate critically in the neurogenic inflammation; 3) activation of TRPV1 receptors in primary afferent nociceptors following CAP injection initiates this process, including triggering of DRRs.
Many primary nociceptive afferent neurons and their axons (Aδ- and C-fibers) are peptidergic with the capacity to release inflammatory peptides [30–35]. CGRP and SP are major inflammatory mediators that contribute a neurogenic component to inflammation [36, 37]. When released from primary afferent neurons, CGRP and SP produce neurogenic inflammation by interacting with endothelial cells, mast cells, immune cells and arterioles. For instance, CGRP is potent vasodilator that produces a strong and long-lasting vasodilation , and SP results preferentially in stronger plasma extravasation .
A critical concern addressed in the present study is the mechanism by which inflammatory mediators are released in the periphery to induce neurogenic inflammation. It has been suggested that intradermal injection of CAP results in a local vasodilation, increased plasma extravasation, and hyperalgesia through release of neuropeptides from peripheral primary afferent terminals [11, 40–42]. These afferent fibers can be sensitized by CAP due to activation of TRPV1 receptors, a key nociceptive molecule expressed in these fibers, to contribute to nociceptive transmission and neurogenic inflammation [5, 6, 43–45]. Thus, CAP plays not only a sensory role by activating nociceptors, but it also has an efferent function by initiating neurogenic inflammation. The latter results from CAP-induced Ca2+ influx into nerve terminals through TRPV1 receptors and voltage-dependent Ca2+ channels, causing the exocytosis of inflammatory mediators [46–49] and their release into the periphery to produce sensitization of primary afferent nociceptors and neurogenic inflammation [50–53]. The above process can be modulated by antidromic activation of afferent fibers, which would drive and trigger the release of inflammatory mediators that initiates neurogenic inflammation, because experimentally antidromic activation of the cut dorsal roots can evoke obvious vasodilation and plasma extravasation when the electrical stimulus strength is strong enough to active C-fibers [54–56]. In the present study, experiments were designed to determine whether there was a release of CGRP and SP from sensory afferent terminals (nociceptors) and whether this release was antidromically driven by DRRs in the CAP-evoked neurogenic inflammation. We proposed that removal of DRRs would interrupt this pathway to alleviate the neurogenic inflammation induced by CAP injection. The data have shown that local vasodilation and increased paw-thickness evoked by CAP injection were greatly reduced after dorsal rhizotomy or intrathecal bicuculline administration that removed DRRs. In contrast, inflammatory reactions evoked by direct application of CGRP or SP in the periphery that would mimic the DRR-mediated inflammation induced by CAP injection were unchanged under the same conditions when DRRs were removed. Thus, there should be a close relationship between DRRs and the release of these neuropeptides based on the observations of differential effects of DRR removal on CAP- and neuropeptide-evoked inflammation, which suggests that the release of CGRP and/or SP is driven by DRRs to participate critically in the CAP-evoked inflammation. In this process, activation of TRPV1 receptors appears to be an initial step. Therefore, we wanted to analyze further how neurogenic inflammation was initiated and developed via DRRs by differentiating the roles of TRPV1, CGRP and NK1 receptors.
Dose-response analysis of the antagonistic effect of the TRPV1 receptor antagonist, capzasepine, on the CAP-evoked inflammation indicates that vasodilation and edema evoked by CAP injection are inhibited in a dose-dependent manner by capsazepine pretreatment. When the dose of capsazepine was in the range of 30–150 μg, the inhibition seemed to reach a maximum. This result is consistent with studies on other pain models that a blockade of TRPV1 receptors by similar doses of capsazepine antagonized selectively the CAP-evoked hyperalgesia and alleviated other inflammogen-evoked pain behaviors in a dose-dependent manner [57–59]. Importantly, CAP-evoked inflammation was nearly completed blocked with these doses. This suggests that neurogenic inflammation after CAP injection is initiated by activation of TRPV1 receptors that in turn trigger and then enhance DRRs, which release inflammatory neuropeptides.
Since the mechanism underlying neurogenic inflammation evoked by CAP injection and driven by DRRs seems to be the result of CGRP and/or SP release, we assumed that a blockade of either CGRP or NK1 receptors in the periphery should alleviate the inflammation. The analysis of antagonistic effects of blockade of CGRP or NK1 receptors by examining the dose-response relationships when CGRP8–37 or spantide I was given as a pretreatment shows that each antagonist when given individually reduced the CAP-evoked inflammation in a dose-dependent manner, but the inflammation was not completely abolished when the effect of each antagonist was maximal. Thus, each neuropeptide released contributes partially to neurogenic inflammation initiated by CAP injection via activation of TRPV1 receptors. A further analysis of blockade of both CGRP and NK1 receptors revealed that the CAP-evoked inflammation (prominently vasodilation) was more effectively alleviated by co-administration of CGRP8–37 and spantide I compared to the effect of a single antagonist. This suggests that CGRP and SP are two major inflammatory mediators in the neurogenic inflammation initiated by activation of TRPV1 receptors and driven by triggering of DRRs.
In summary, the present results update the role of DRRs in neurogenic inflammation by providing new evidence to suggest that the release of CGRP and SP in the periphery is driven by the generation of DRRs, which participate critically in neurogenic inflammation with that pain perception is exacerbated. Further, this process is initiated by activation of TRPV1 receptors after CAP injection.