Dietary supplementation of gingerols- and shogaols-enriched ginger root extract attenuate pain-associated behaviors while modulating gut microbiota and metabolites in rats with spinal nerve ligation.

Neuroinflammation is a central factor in neuropathic pain (NP). Ginger is a promising bioactive compound in NP management due to its anti-inflammatory property. Emerging evidence suggests that gut microbiome and gut-derived metabolites play a key role in NP. We evaluated the effects of two ginger root extracts rich in gingerols (GEG) and shogaols (SEG) on pain sensitivity, anxiety-like behaviors, circulating cell-free mitochondrial DNA (ccf-mtDNA), gut microbiome composition, and fecal metabolites in rats with NP. Sixteen male rats were divided into four groups: sham, spinal nerve ligation (SNL), SNL+0.75%GEG in diet, and SNL+0.75%SEG in diet groups for 30 days. Compared to SNL group, both SNL+GEG and SNL+SEG groups showed a significant reduction in pain- and anxiety-like behaviors, and ccf-mtDNA level. Relative to the SNL group, both SNL+GEG and SNL+SEG groups increased the relative abundance of Lactococcus, Sellimonas, Blautia, Erysipelatoclostridiaceae, and Anaerovoracaceae, but decreased that of Prevotellaceae UCG-001, Rikenellaceae RC9 gut group, Mucispirillum and Desulfovibrio, Desulfovibrio, Anaerofilum, Eubacterium siraeum group, RF39, UCG-005, Lachnospiraceae NK4A136 group, Acetatifactor, Eubacterium ruminantium group, Clostridia UCG-014, and an uncultured Anaerovoracaceae. GEG and SEG had differential effects on gut-derived metabolites. Compared to SNL group, SNL+GEG group had higher level of 1'-acetoxychavicol acetate, (4E)-1,7-Bis(4-hydroxyphenyl)-4-hepten-3-one, NP-000629, 7,8-Dimethoxy-3-(2-methyl-3-buten-2-yl)-2H-chromen-2-one, 3-{[4-(2-Pyrimidinyl)piperazino]carbonyl}-2-pyrazinecarboxylic acid, 920863, and (1R,3R,7R,13S)-13-Methyl-6-methylene-4,14,16-trioxatetracyclo[11.2.1.0∼1,10∼.0∼3,7∼]hexadec-9-en-5-one, while SNL+SEG group had higher level for (±)-5-[(tert-Butylamino)-2'-hydroxypropoxy]-1_2_3_4-tetrahydro-1-naphthol and dehydroepiandrosteronesulfate. In conclusion, ginger is a promising functional food in the management of NP, and further investigations are necessary to assess the role of ginger on gut-brain axis in pain management.

Fear extinction learning ability predicts neuropathic pain behaviors and amygdala activity in male rats.

Background The amygdala plays a key role in fear learning and extinction and has emerged as an important node of emotional-affective aspects of pain and pain modulation. Impaired fear extinction learning, which involves prefrontal cortical control of amygdala processing, has been linked to neuropsychiatric disorders. Here, we tested the hypothesis that fear extinction learning ability can predict the magnitude of neuropathic pain. Results We correlated fear extinction learning in naive adult male rats with sensory and affective behavioral outcome measures (mechanical thresholds, vocalizations, and anxiety- and depression-like behaviors) before and after the induction of the spinal nerve ligation model of neuropathic pain compared to sham controls. Auditory fear conditioning, extinction learning, and extinction retention tests were conducted after baseline testing. All rats showed increased freezing responses after fear conditioning. During extinction training, the majority (75%) of rats showed a decline in freezing level to 50% in 5 min (fear extinction+), whereas 25% of the rats maintained a high freezing level (>50%, fear extinction-). Fear extinction- rats showed decreased open-arm preference in the elevated plus maze, reflecting anxiety-like behavior, but there were no significant differences in sensory thresholds, vocalizations, or depression-like behavior (forced swim test) between fear extinction+ and fear extinction- types. In the neuropathic pain model (four weeks after spinal nerve ligation), fear extinction- rats showed a greater increase in vocalizations and anxiety-like behavior than fear extinction+ rats. Fear extinction- rats, but not fear extinction+ rats, also developed depression-like behavior. Extracellular single unit recordings of amygdala (central nucleus) neurons in behaviorally tested rats (anesthetized with isoflurane) found greater increases in background activity, bursting, and evoked activity in fear extinction- rats than fear extinction+ rats in the spinal nerve ligation model compared to sham controls. Conclusion The data may suggest that fear extinction learning ability predicts the magnitude of neuropathic pain-related affective rather than sensory behaviors, which correlates with differences in amygdala activity changes.

Small-conductance calcium-activated potassium (SK) channels in the amygdala mediate pain-inhibiting effects of clinically available riluzole in a rat model of arthritis pain.

BACKGROUND: Arthritis pain is an important healthcare issue with significant emotional and affective consequences. Here we focus on potentially beneficial effects of activating small-conductance calcium-activated potassium (SK) channels in the amygdala, a brain center of emotions that plays an important role in central pain modulation and processing. SK channels have been reported to regulate neuronal activity in the central amygdala (CeA, output nucleus). We tested the effects of riluzole, a clinically available drug for the treatment of amyotrophic lateral sclerosis, for the following reasons. Actions of riluzole include activation of SK channels. Evidence in the literature suggests that riluzole may have antinociceptive effects through an action in the brain but not the spinal cord. Mechanism and site of action of riluzole remain to be determined. Here we tested the hypothesis that riluzole inhibits pain behaviors by acting on SK channels in the CeA in an arthritis pain model. RESULTS: Systemic (intraperitoneal) application of riluzole (8 mg/kg) inhibited audible (nocifensive response) and ultrasonic (averse affective response) vocalizations of adult rats with arthritis (5 h postinduction of a kaolin-carrageenan monoarthritis in the knee) but did not affect spinal withdrawal thresholds, which is consistent with a supraspinal action. Stereotaxic administration of riluzole into the CeA by microdialysis (1 mM, concentration in the microdialysis fiber, 15 min) also inhibited vocalizations, confirming the CeA as a site of action of riluzole. Stereotaxic administration of a selective SK channel blocker (apamin, 1 µM, concentration in the microdialysis fiber, 15 min) into the CeA had no effect by itself but inhibited the effect of systemic riluzole on vocalizations. Off-site administration of apamin into the basolateral amygdala (BLA) as a placement control or stereotaxic application of a selective blocker of large-conductance calcium-activated potassium (BK) channels (charybdotoxin, 1 µM, concentration in the microdialysis fiber, 15 min) into the CeA did not affect the inhibitory effects of systemically applied riluzole. CONCLUSIONS: The results suggest that riluzole can inhibit supraspinally organized pain behaviors in an arthritis model by activating SK, but not BK, channels in the amygdala (CeA but not BLA).

CB1 augments mGluR5 function in medial prefrontal cortical neurons to inhibit amygdala hyperactivity in an arthritis pain model.

The medial prefrontal cortex (mPFC) serves executive control functions and forms direct connections with subcortical areas such as the amygdala. Our previous work showed abnormal inhibition of mPFC pyramidal cells and hyperactivity of amygdala output neurons in an arthritis pain model. To restore mPFC activity and hence control pain-related amygdala hyperactivity this study focused on CB1 and mGluR5 receptors, which are important modulators of cortical functions. Extracellular single-unit recordings of infralimbic mPFC pyramidal cells and of amygdala output neurons in the laterocapsular division of the central nucleus (CeLC) were made in anesthetised adult male rats. mPFC neurons were classified as 'excited' or 'inhibited' based on their response to brief innocuous and noxious test stimuli. After arthritis pain induction, background activity and evoked responses of excited neurons and background activity and inhibition of inhibited neurons decreased. Stereotaxic application of an mGluR5-positive allosteric modulator (N-cyclobutyl-6-((3-fluorophenyl)ethynyl) nicotinamide hydrochloride, VU0360172) into the mPFC increased background and evoked activity of excited, but not inhibited, mPFC neurons under normal conditions but not in arthritis. A selective CB1 receptor agonist (arachidonyl-2-chloroethylamide) alone had no effect but restored the facilitatory effects of VU0360172 in the pain model. Coactivation of CB1 and mGluR5 in the mPFC inhibited the pain-related activity increase of CeLC neurons but had no effect under normal conditions. The data suggest that excited mPFC neurons are inversely linked to amygdala output (CeLC) and that CB1 can increase mGluR5 function in this subset of mPFC neurons to engage cortical control of abnormally enhanced amygdala output in pain.

Pain-related deactivation of medial prefrontal cortical neurons involves mGluR1 and GABA(A) receptors.

Pain-related hyperactivity in the amygdala leads to deactivation of the medial prefrontal cortex (mPFC) and decision-making deficits. The mechanisms of pain-related inhibition of the mPFC are not yet known. Here, we used extracellular single-unit recordings of prelimbic mPFC neurons to determine the role of GABA(A) receptors and metabotropic glutamate receptor (mGluR) subtypes, mGluR1 and mGluR5, in pain-related activity changes of mPFC neurons. Background and evoked activity of mPFC neurons decreased after arthritis induction. To determine pain-related changes, the same neuron was recorded continuously before and after induction of arthritis in one knee joint by intra-articular injection of kaolin/carrageenan. Stereotaxic administration of a GABA(A) receptor antagonist {[R-(R*,S*)]-5-(6,8-dihydro-8-oxofuro[3,4-e]-1,3-benzodioxol-6-yl)-5,6,7,8-tetrahydro-6,6-dimethyl-1,3-dioxolo[4,5-g]isoquinolinium iodide (bicuculline)} into the mPFC by microdialysis reversed pain-related inhibition, whereas offsite injections into the adjacent anterior cingulate cortex had no or opposite effects on prelimbic mPFC neurons. A selective mGluR1/5 agonist [(S)-3,5-dihydroxyphenylglycine (DHPG)] inhibited background and evoked activity under normal conditions through a GABAergic mechanism, because the inhibitory effect was blocked with bicuculline. In the arthritis pain state, DHPG, alone or in the presence of bicuculline, had no effect. Consistent with the involvement of mGluR1 in pain-related inhibition of the mPFC, a selective mGluR1 antagonist [(S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid] reversed the pain-related decrease of background and evoked activity of mPFC neurons in arthritis, whereas a selective mGluR5 antagonist [2-methyl-6-(phenylethynyl)pyridine hydrochloride] had no effect. The mGluR antagonists had no effect under normal conditions. We interpret our data to suggest that pain-related inhibition of mPFC neurons in the arthritis model depends on mGluR1-mediated endogenous activation of GABA(A) receptors. Exogenous activation of mGluR1/5 produces GABAergic inhibition under normal conditions. Restoring normal activity in the mPFC may be a therapeutic strategy to improve cognitive deficits associated with persistent pain.

Kynurenic acid enhances electroacupuncture analgesia in normal and carrageenan-injected rats.

The interaction between electroacupuncture (EA) and an intrathecally administered wide-spectrum excitatory amino acid (EAA) receptor(s) antagonist, kynurenic acid (KYNA) on carrageenan-induced thermal hyperalgesia and spinal Fos expression was investigated. Intrathecal (i.t.) injection of 0.1, 1, 10, and 100 nmol KYNA markedly and dose-dependently increased the latency of paw withdrawal (PWL) of the carrageenan-injected paw. While the PWLs of the non-injected and normal saline (NS)-injected paws were not obviously affected by application of KYNA at the doses tested. Intrathecal injection of 0.1 nmol KYNA significantly potentiated the anti-nociception induced by EA stimulation of contralateral 'Zu-San-Li' and 'Kun-Lun' acupoints either in the carrageenan- or NS-injected rats. Three hours after intraplantar (i.pl.) injection of carrageenan, the number of Fos-like immunoreactive (Fos-LI) neurons was significantly increased in all layers of ipsilateral spinal cord at L(4)-L(5) with the higher density in laminae I-II and V-VI. Intrathecally pre-administered KYNA (10 nmol) significantly reduced the total number of carrageenan-induced Fos-LI neurons with more apparent reduction in laminae I-II and IV-V. Pre-coapplication of 10 nmol KYNA and EA of bilateral 'Zu-San-Li' and 'Kun-Lun' acupoints, the numbers of carrageenan-induced Fos-LI neurons in laminae I-II and V-VI further reduced. The level of Fos expression in the spinal cord induced by carrageenan was significantly lower compared with that of i.t. injection of KYNA or EA alone. These results demonstrated that EAA receptor(s) antagonist could enhance EA-induced anti-nociception and anti-hyperalgesia.

Excitatory amino acid receptor antagonists and electroacupuncture synergetically inhibit carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats.

The interaction between electroacupuncture and an N-methyl-D-aspartic acid (NMDA) receptor antagonist, (DL-2-amino-5-phosphonopentanoic acid; AP5), or an (+/-)-alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainite (AMPA/KA) receptor antagonist, (6,7-dinitroquinoxaline-2,3 (1H,4H); DNQX) administered intrathecally on carrageenan-induced thermal hyperalgesia and spinal c-Fos expression was investigated. The latency of paw withdrawal (PWL) from a thermal stimulus was used as a measure of hyperalgesia in awake rats. Intrathecal (i.t.) injection of 1 and 10 nmol AP5, but not DNQX, markedly increased the PWL of the carrageenan-injected paw. At a dose of 100 nmol, either AP5 or DNQX significantly increased the PWL of carrageenan-injected paw, with AP5 being more potent. The PWLs of the non-injected and normal saline (NS)-injected paws were not detectably affected by the administration of NMDA or AMPA/KA receptor antagonists at the doses tested. Unilateral electroacupuncture stimulation of the 'Zu-San-Li' (St 36) and 'Kun-Lun' (UB 60) acupuncture points (60 and 2 Hz alternately, 1-2-3 mA) contralateral to the carrageenan-injected paw significantly elevated the PWLs of carrageenan- and NS-injected paws. Although neither i.t. injection of 0.1 nmol AP5 nor 1 nmol DNQX alone had an effect on the PWL of the carrageenan- and NS-injected paws, both significantly potentiated electroacupuncture-induced analgesia in carrageenan-injected rats, especially 0.1 nmol AP5. Fos expression evoked by intraplantar (i.pl.) injection of carrageenan was examined in the spinal cord with immunohistochemical methods. Three hours after i.pl. injection of carrageenan, the number of Fos-like immunoreactive (Fos-LI) neurons was significantly increased in all the layers of the ipsilateral spinal cord at L(4-5), with the highest density in laminae I-II and V-VI. Intrathecally pre-administered AP5 (10 nmol) or DNQX (100 nmol) significantly reduced the total number of carrageenan-induced Fos-LI neurons. The reduction was most apparent in laminae I-II and IV-V. Similarly, following bilateral electroacupuncture stimulation of the 'Zu-San-Li' and 'Kun-Lun' acupuncture points, the numbers of carrageenan-induced Fos-LI neurons in laminae I-II and V-VI were also markedly reduced. When a combination of electroacupuncture with 10 nmol AP5 or 100 nmol DNQX was used, the level of Fos expression in the spinal cord induced by carrageenan was significantly lower than electroacupuncture or i.t. injection of AP5 or DNQX alone. These results demonstrate that electroacupuncture and NMDA or AMPA/KA receptor antagonists have a synergetic anti-nociceptive action against inflammatory pain. Furthermore, this study supports the idea that both NMDA and AMPA/KA receptors are involved in spinal nociceptive transmission in carrageenan-inflamed rats, with the former more preferentially mediating transmission of nociceptive information from cutaneous tissue.