We found that nVNS significantly inhibited the nocifensive response to mechanical stimulation of primary trigeminal neurons in a novel rodent model of migraine pathology. In our model, neck muscle tension/inflammation was used to promote sensitization of trigeminal neurons that become activated on stimulation of nasal/sinus TRPA1 receptors by the pungent stimulus derived from California Bay leaves (CBLs). Neck muscle inflammation was chosen as a means to facilitate sensitization of the trigeminal system because neck/shoulder tension is often cited as a risk factor and a comorbid symptom of migraine.4,14,21,22 The stimulus, complete Freund adjuvant, was used to induce prolonged inflammation by eliciting a sustained immune response for up to 2 weeks.11,26,68 The amount of adjuvant used in our study did not negatively affect the normal feeding and eating behavior of the animals or cause excessive grooming. However, multiple injections of 10 μL CFA resulted in low-grade inflammation and muscle tenderness that mediated sensitization of primary trigeminal nociceptors rather than nociception, which was observed with a single 50 μL injection (data not shown). This sensitizing effect is likely attributable to the fact that afferent processes of the nerves providing sensory innervation of the muscle tissue terminate in the upper spinal cord where there is convergence with the trigeminal system.5,6,50 To cause activation of sensitized trigeminal nociceptive neurons, we used an oil extract containing volatile, stimulatory chemicals that was prepared from the leaves of the California Bay tree. The rationale for choosing this type of stimulus was based on pungent odors being a reported migraine trigger,35 and the seminal work by Nassini et al. that demonstrated application of umbellulone, a chemical isolated from CBL, could elicit a nocifensive trigeminal response on topical application of this compound to the nasal mucosa.53 In addition, umbellulone was shown to cause activation of the TRPA1 receptors expressed on trigeminal neurons and mediate the release of CGRP, a molecule strongly implicated in migraine pathology.7 Our goal was to develop a clinically relevant rodent migraine model that involved inducing or triggering trigeminal activation without having to directly apply or inject the stimulus, and hence why we chose to expose the animals to volatile compounds by inhalation, a more natural and less-invasive process. It should be noted that trigeminal nociceptor activation was induced on activation of V1/V2 neurons that provide sensory innervation of the nasal and sinus mucosa,62,63 which led to an enhanced nocifensive response in V1 (eyebrow) neurons. Taken together, results from our rodent model mimic several key features of episodic migraine pathology including increased mechanical nociception and temporally correlated changes in the expression of several proteins implicated in peripheral and central sensitization.
A major finding from our study was that nVNS was sufficient to significantly inhibit nocifensive head withdrawal response from mechanical stimulation of V1 trigeminal nociceptors. Our finding is similar to the results from other studies in which VNS inhibited trigeminal activation in novel models involving trigeminal allodynia, primary headache, and cortical spreading depression.2,16,55 Interestingly, a single nVNS before trigeminal activation did not block the nocifensive response to the pungent odor in our study. This finding is similar to the reported effect of the antimigraine triptan drugs, which are most effective when administered at the onset of trigeminal activation. In agreement with the change in nociception, nVNS resulted in an inhibitory effect on the expression of proteins associated with peripheral and central sensitization. The elevated level of P-ERK expression in the cytoplasm and nucleus of trigeminal neurons was significantly inhibited by nVNS as were the elevated levels of Iba1 and GFAP. Thus, nVNS seems to function at multiple levels within the trigeminal system to repress the activation of sensitized primary trigeminal neurons and repress the activation of microglia and astrocytes. The exact mechanism by which nVNS modulates nociception and regulates protein expression in our model is not known but may involve increased expression of IL-10 and MAP kinase phosphatases in the TG, activation of the descending inhibitory pathway, and suppression of glia activation. In support of a possible role for IL-10 in mediating the inhibitory effects of nVNS on P-ERK levels, VNS has been shown to cause an increase in peripheral levels of the anti-inflammatory cytokine IL-10.44 Elevated levels of IL-10 could suppress the stimulatory effects of proinflammatory cytokines on P-ERK expression36 in the TG to inhibit trigeminal sensitization and activation.15 Given the key role of MAP kinase phosphatases (MKPs) in regulating activity of the MAP kinases including ERK,34,60 increased levels of the phosphatases MKP-1 and MKP-3 are candidates for dephosphorylation and hence decreasing ERK levels. We have previously reported that increased levels of MKP-1 are associated with the inhibition of stimulated expression of signaling proteins in trigeminal neurons and satellite glial cells.12,13 Enhanced descending modulation mediated by VNS could lead to increased release of the inhibitory neurotransmitter GABA and inhibition of second-order nociceptive neurons, astrocytes, and microglia.41,56 Although evidence for direct activation of this pathway was not demonstrated in our study, enhanced descending modulation provides a possible mechanism to help explain the inhibitory effects of VNS in the STN on glial cell activation.51,57
The authors have no conflicts of interest to declare.
The authors thank Angela Goerndt for her assistance in the care and maintenance of the animals used in our study. Funding for this study, the noninvasive vagus nerve stimulator adapted for rodent use, was provided by electroCore.
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