Administration of lutein attenuates trigeminal inflammatory hyperalgesia
The present study produced the following main findings:
(i) the threshold of escape from mechanical stimulation applied to the orofacial area in inflamed rats was significantly lower than that in naïve rats, as described previously [
8,
17];
(ii) from one to three days after inflammation, the lowered mechanical threshold in the inflamed rats tended toward control levels following daily administration of lutein (10 mg/kg, i.p);
(iii) the reduced escape threshold from mechanical stimulation in inflamed rats increased to control levels with lutein at day 3 of inflammation;
(iv) inflammation-induced edema was significantly decreased to control levels with lutein at 2 days inflammation.
Matsumoto et al. [
21] recently reported that pretreatment with resveratrol significantly decreased the mean thickness of inflammation-induced edema in whisker pads compared to those of untreated, inflamed rats and significantly decreased that number of
c-fos-immunoreactive SpVc/C1 neurons in inflamed rats compared to naïve rats. Recently, we also reported that administration of dietary resveratrol attenuates inflammation-induced mechanical inflammatory hyperalgesia and that this effect was mainly due to the suppression of hyperexcitability of SpVc WDR neurons via the inhibition of both peripheral and central Cox-2 cascade signaling pathways [
17]. In accordance with these observations, the present study revealed that
(i) following injection of CFA into the whisker pads, the mean number of Cox-2
-immunoreactive cells in the whisker pad was significantly increased in the inflamed rats compared to those in the naïve rats;
(ii) increased Cox-2 immunoreactivity of whisker pad in inflamed rats was significantly returned to control level by administration of lutein day 3. Taken together, these findings support the idea that daily administration of lutein suppresses inflammation-induced edema and hyperalgesia via the inhibition of prostaglandin E
2 (PGE
2) production by suppression of Cox-2 signaling in the whisker pad.
Suppressive effect of lutein on the hyperexcitability of WDR SpVc neuronal activity associated with hyperalgesia following inflammation
Scholz and Woolf [
4] indicated that peripheral tissue injury/inflammation innervating trigeminal nerves can alter the properties of trigeminal somatic sensory pathways, causing behavioral hypersensitivity and resulting in increased responses to pain caused by noxious stimulation, such as hyperalgesia. Following peripheral inflammation and/or nerve injury, inflammatory mediators such as PGE
2 bind to G-protein-coupled E-type prostanoid receptors and induce activation of protein kinases A and C (PKA and PKC, respectively) in nociceptive peripheral terminals, leading to phosphorylation of mechanosensitive sodium and potassium ion channels and receptors [
26,
27]. As a result, the activation threshold for transducer channels such as TRPA1 is reduced and the membrane excitability of the peripheral terminals increases, resulting in a high frequency of action potentials being conducted to presynaptic central terminals of the SpVc [
2,
26]. This induces the release of large amounts of glutamate into the synaptic cleft, which binds to upregulated post-synaptic glutamate receptors, augmenting excitatory post synaptic potentials (EPSPs), causing a barrage of action potentials to be conducted to higher centers of pain pathways, and creating a state of heightened sensitivity termed peripheral sensitization [
2,
26].
In this study, we found that the decreased mean mechanical stimulation threshold in the inflamed rats was also significantly returned to control levels following daily systemic administration of lutein. In accordance with these findings, both non-noxious and noxious mechanical stimuli-evoked mean discharge frequencies of the SpVc WDR neurons in inflamed rats were significantly returned to control levels after chronic lutein treatment, suggesting that systemic administration of lutein can alter the inflammation-induced hypersensitivity of SpVc WDR neuronal activity, possibly via the suppression of peripheral sensitization. The present study also revealed that lutein administration returned the increased Cox-2
-immunoreactivity of whisker pad in inflamed rats to control levels. To this end, PGE
2 appears to facilitate the activation of TRPA1 and tetrodotoxin-TTX-resistant (TTX-R) Na
+ channels [
4,
28,
29], with the latter channels (eg., Nav1.8 and Nav1.9) selectively expressed in small- and medium-sized DRG neurons [
30]. Based on findings of increased excitability of small-diameter trigeminal ganglion neurons after PGE
2 application and an increase in the TTX-R Na
+ currents [
31], it can be assumed that lutein inhibits the excitability of small-diameter trigeminal ganglion neurons via suppression of Cox-2-related PGE
2 production leading to induced generator potential and TTX-R Na
+ currents.
Previous studies reported after-discharges followed by noxious mechanical stimulation in SpVc WDR neurons tested in a chronic inflammation model, in association with neuronal sensitization during persistent pain [
32,
33]. Interestingly, in this study, we observed that after-discharges following the noxious pinch stimulation in inflamed rats were abolished following daily administration of lutein. Although the precise mechanism underlying this suppressive effect was not elucidated, two possible mechanisms can be postulated. First, previous administration of the substance P receptor, neurokin-1 (NK
1) receptor antagonist inhibited pinch-evoked after-discharges of WDR neurons in the spinal cord [
34]. We also have previously observed the evidence that CFA-induced pinch-evoked after-discharges of SpVc WDR neurons was abolished by resveratrol administration [
17]. Thus, although there are no reports for lutein acting as a NK1 receptor antagonist, it can be assumed that chronic administration of lutein could similarly attenuate NK1 receptor-mediated after-discharges of WDR neurons in the SpVc under inflammatory conditions. In a second series of data, microiontophoretic application of GABA
A receptor agonists/antagonists into the SpVc region using multibarrel electrodes suggested that a local GABAergic mechanism could control nociceptive transmission in the SpVc neurons, contributing to the overall mechanical receptive field properties [
24]. Since Higashima et al. [
35] also reported that GABA
A receptor antagonist, bicuculline, inhibits generation of after-discharges from hippocampal neurons, while GABA
B receptor antagonist, phaclofen, enhances after-discharges in slice preparations, it can be assumed that chronic administration of lutein attenuates GABA
A receptor-mediated after-discharges of WDR neuron in the SpVc. In this study, we also found that the mean receptive field size of inflamed rats was returned to control levels following lutein. Although the precise mechanism by which chronic administration of lutein could suppress the expanded receptive field remains to be understood, it can be postulated that lutein modulates a local GABAergic mechanism tonic control and that nociceptive mechanoreceptive transmission predominantly inhibits central mechanisms through excitatory synaptic transmission. It is clear that further studies are warranted to explore this intriguing possibility, but it is reasonable to speculate that at least part of the peripheral anitinociceptive action of lutein is due to prevention of peripheral sensitization, as well as antipyretic analgesics.
Functional significance of suppression effect of lutein on the hyperexcitability of SpVc neuron associated with hyperalgesia
In this study, spontaneous discharges were observed in 20% of naïve rats, while most neurons fired at a low frequency and all SpVc WDR neurons were spontaneously active in inflamed rats. Previous studies reported that CFA inflammation induced hyperexcitability of SpVc WDR neurons to mechanical stimuli [
3,
17], and there are reports that SpVc WDR neurons contribute to the mechanism of hyperalgesia and/or referred pain associated with dental pain [
3,
20], while ongoing activities were observed in the SpVc associated with ongoing headache and spontaneous pain [
36]. Indeed, the origin of ongoing activity in the central neurons that relay sensory information has become a topic of considerable clinical interest because of findings suggesting such activity as a quantitative determinant of post-traumatic injury and chronic pain [
36,
37]. To this end, Roch et al. [
37] demonstrated that ongoing activity of SpVc WDR neurons is driven from the periphery, since microinjection of lidocaine into the trigeminal ganglia caused a significant decrease in ongoing activity. Taken together with these results, our data suggest that lutein could suppress spontaneous discharge activity of SpVc WDR neurons innervating the facial skin due to peripheral and/or trigeminal ganglion sensitization [
9,
37], with probable contributing effects on spontaneous pain, including clinical headache.
The toxic side effects associated with most commonly prescribed analgesic drugs, such as NSAIDs, cox-inhibitors, and opioids, have increased interest in CAM agents for the treatment of persistent chronic pain [
38,
39]. Indeed, patients frequently turn to CAM therapies including herbal medicines and acupuncture for pain control when other medical treatments become ineffective [
18,
39], and the potential influence of diet and dietary supplementation on conditions associated with pain are increasingly the focus of research [
40‐
42]. In the present study, we found that systemic administration of lutein attenuated inflammation-induced hyperexcitability of trigeminal SpVc neurons associated with hyperalgesia in rats. Sekiguchi et al. [
17] also reported that chronic administration of resveratrol attenuates inflammation-induced mechanical inflammatory hyperalgesia, mainly via suppression of SpVc WDR neurons and inhibition of both peripheral and central Cox-2 signaling pathways. Therefore, our results combined with this finding suggest that dietary constituents such as lutein and resveratrol might additively contribute to the development of analgesic drugs with fewer and less toxic side effects for treating pathological pain, including orofacial pain. In particular, our findings support that lutein is a potential therapeutic alternative for clinical use in preventing trigeminal inflammatory hyperalgesia.