GV16 acupoint stimulation with bee venom reduces peripheral hypersensitivity via activation of α2 adrenoceptors in a nitroglycerin-induced migraine mouse model.

Background: Peripheral hypersensitivities develop in the face and hindpaws of mice with nitroglycerin (NTG)-induced migraine. We evaluated whether diluted bee venom (DBV) injections at acupoints prevented these peripheral hypersensitivities and c-Fos expression in the trigeminal nucleus caudalis (TNC). Methods: NTG (10 mg/kg, intraperitoneal, i.p.) was administered every other day for nine days. DBV (0.1 mg/kg) was subcutaneously injected into the ST36 (Zusanli), LI4 (Hegu), or GV16 (Fengfu) acupoints 75 min after each NTG injection. Mice were pretreated with naloxone (5 mg/kg, i.p.) or yohimbine (5 mg/kg, i.p.) 30 min before the DBV injections. Results: NTG injection caused facial cold allodynia, hindpaw mechanical allodynia, and increased c-Fos-immunoreactive (ir) cells in the TNC. Repetitive DBV injections at GV16, but not the ST36, or LI4 acupoints, suppressed NTG-induced hindpaw mechanical allodynia and facial cold allodynia. The number of c-Fos-ir cells also decreased in response to DBV injections at the GV16 acupoint. Remarkably, pretreatment with yohimbine reversed the anti-allodynic effects of DBV injections and attenuated the decreased c-Fos expression in response to GV16 DBV treatment. Naloxone did not block the effects of GV16 DBV stimulation. Conclusion: These findings demonstrate that repetitive DBV treatment at the GV16 acupoint relieves NTG-induced facial and hindpaw hypersensitivities and decreases in c-Fos expression in the TNC via activation of the alpha-2 adrenoceptors, but not the opioid receptors.

The mTOR inhibitor rapamycin suppresses trigeminal neuropathic pain and p-MKK4/p-p38 mitogen-activated protein kinase-mediated microglial activation in the trigeminal nucleus caudalis of mice with infraorbital nerve injury.

Neuropathic pain caused by trigeminal nerve injury is a typical refractory orofacial chronic pain accompanied by the development of hyperalgesia and allodynia. We previously demonstrated that the mammalian target of rapamycin (mTOR) inhibitor rapamycin suppressed orofacial formalin injection-induced nociception; however, the underlying mechanism is unclear, and it is unknown whether it can reduce trigeminal neuropathic pain. In mice, left infraorbital nerve and partial nerve ligation (ION-pNL) was performed using a silk suture (8-0). Fourteen days after surgery, neuropathic pain behavior was examined on a whisker pad and rapamycin (0.1, 0.3, and 1.0 mg/kg) was administered intraperitoneally. Mechanical and cold sensitivities in the orofacial region were quantified using von Frey filaments and acetone solution, respectively. Changes in mTOR and related proteins, such as p-MKK3/6, p-MKK4, p-JNK, p-ERK, p-p38 MAPK, GFAP, and Iba-1, in the trigeminal nucleus caudalis (TNC) or the trigeminal ganglia (TG) tissues were examined via western blot analysis or immunohistochemistry. Mice demonstrated significant mechanical and cold allodynia 2 weeks following ION-pNL injury, both of which were significantly reduced 1 h after the administration of high-dose rapamycin (1.0 mg/kg). In the TG tissue, ION-pNL surgery or rapamycin treatment did not change p-mTOR and p-4EBP1, but rapamycin reduced the increase of p-S6 and S6 induced by ION-pNL. In the TNC tissue, neither ION-pNL surgery nor rapamycin treatment altered p-mTOR, p-S6, and p-4EBP1 expressions, whereas rapamycin significantly decreased the ION-pNL-induced increase in Iba-1 expression. In addition, rapamycin suppressed the increase in p-p38 MAPK and p-MKK4 expressions but not p-MKK3/6 expression. Moreover, p-p38 MAPK-positive cells were colocalized with increased Iba-1 in the TNC. Our findings indicate that rapamycin treatment reduces both mechanical and cold orofacial allodynia in mice with trigeminal neuropathic pain, which is closely associated with the modulation of p-MKK4/p-p38 MAPK-mediated microglial activation in the TNC.

Different Involvement of ASIC and TRPA1 in Facial and Hindpaw Allodynia in Nitroglycerin-Induced Peripheral Hypersensitivities in Mice.

The pathophysiological mechanism underlying migraine-associated peripheral hypersensitivity remains unclear. Acid-sensing ion channels (ASICs) and transient receptor potential ankyrin 1 (TRPA1) are known to be causative pathogenic factors of mechanical and cold allodynia, respectively. Here, we sought to investigate their involvement in cold and mechanical allodynia of the face and hindpaws, respectively, in a mouse model of repetitive nitroglycerin (NTG)-induced migraine. NTG (10 mg/kg) was administered to the mice every other day for 9 days, followed 90 min later by HC-030031 (a TRPA1 blocker) or amiloride (a non-selective ASIC blocker). Mechanical or cold sensitivity of the hindpaw and facial regions was quantified using von-Frey filaments or acetone solution, respectively. Immunohistochemistry revealed that c-Fos expression was significantly increased in the trigeminal nucleus caudalis region but not in the spinal cord. Amiloride treatment only reduced NTG-induced hindpaw mechanical allodynia, whereas HC-030031 treatment only improved facial cold allodynia. Interestingly, the number of c-Fos positive cells decreased to a similar level in each drug treatment group. These findings demonstrate that facial cold allodynia and hindpaw mechanical allodynia are differentially mediated by activation of TRPA1 and ASIC, respectively, in mice with repetitive NTG-induced hypersensitivity.

Dexmedetomidine Co-Administered with Lidocaine Decreases Nociceptive Responses and Trigeminal Fos Expression without Motor Dysfunction and Hypotension in a Murine Orofacial Formalin Model.

Administration of dexmedetomidine significantly induces sedation and anti-nociception in several nociceptive models, but clinical trials are restricted due to adverse side effects, including lethargy, hypotension, and bradycardia. Herein, we investigated whether intraperitoneal inoculation of dexmedetomidine reduced the orofacial nociceptive response and affected motor coordination and blood pressure and examined whether a lower dose of dexmedetomidine in combination with 0.5% lidocaine produced an antinociceptive effect without any adverse side events in a murine model. To perform the experiment, 5% formalin (10 µL) was subcutaneously inoculated into the right upper lip, and the rubbing responses were counted for 45 min. Different doses of dexmedetomidine combined with 0.5% lidocaine were administered 10 and 30 min before formalin injection, respectively. Dexmedetomidine (10 µg/kg) significantly reduced orofacial nociceptive responses during the second phase of the formalin test and decreased the expression of Fos in trigeminal nucleus caudalis (TNC). Besides, a high dose of dexmedetomidine (30 µg/kg) induced lessening physical ability and significantly reduced systolic pressure and heart rate. When 0.5% lidocaine was injected subcutaneously, nociceptive responses were reduced only in the first phase. Interestingly, although a low dose of dexmedetomidine (3 µg/kg) alone did not show an antinociceptive effect, its co-administration with lidocaine significantly reduced the nociceptive response in both phases and decreased TNC Fos expression without motor dysfunction and hypotension. This finding suggests that the combination of a low-dose of systemic dexmedetomidine with lidocaine may be a safe medicinal approach for acute inflammatory pain management in the orofacial region, particularly mucogingival pain.

Rapamycin reduces orofacial nociceptive responses and microglial p38 mitogen-activated protein kinase phosphorylation in trigeminal nucleus caudalis in mouse orofacial formalin model.

The mammalian target of rapamycin (mTOR) plays a role in various cellular phenomena, including autophagy, cell proliferation, and differentiation. Although recent studies have reported its involvement in nociceptive responses in several pain models, whether mTOR is involved in orofacial pain processing is currently unexplored. This study determined whether rapamycin, an mTOR inhibitor, reduces nociceptive responses and the number of Fos-immunoreactive (Fos-ir) cells in the trigeminal nucleus caudalis (TNC) in a mouse orofacial formalin model. We also examined whether the glial cell expression and phosphorylated p38 (p-p38) mitogen-activated protein kinases (MAPKs) in the TNC are affected by rapamycin. Mice were intraperitoneally given rapamycin (0.1, 0.3, or 1.0 mg/kg); then, 30 min after, 5% formalin (10 µl) was subcutaneously injected into the right upper lip. The rubbing responses with the ipsilateral forepaw or hindpaw were counted for 45 min. High-dose rapamycin (1.0 mg/kg) produced significant antinociceptive effects in both the first and second phases of formalin test. The number of Fos-ir cells in the ipsilateral TNC was also reduced by high-dose rapamycin compared with vehicle-treated animals. Furthermore, the number of p-p38-ir cells the in ipsilateral TNC was significantly decreased in animals treated with high-dose rapamycin; p-p38 expression was co-localized in microglia, but not neurons and astrocytes. Therefore, the mTOR inhibitor, rapamycin, reduces orofacial nociception and Fos expression in the TNC, and its antinociceptive action on orofacial pain may be associated with the inhibition of p-p38 MAPK in the microglia.

Analgesic Efficacy of α2 Adrenergic Receptor Agonists Depends on the Chronic State of Neuropathic Pain: Role of Regulator of G Protein Signaling 4.

The analgesic effect of alpha-2 adrenergic receptor (α2AR) agonists, which relieve chronic neuropathic pain, is highly variable among individuals. Here, we used a mouse model of spared nerve injury (SNI) to show that treatment time after the establishment of neuropathic pain was important for the variability in the analgesic efficacy of α2AR agonists, which was related to the activity of regulator of G-protein signaling protein 4 (RGS4). Intrathecal treatment with α2AR agonists, clonidine (0.1-1 nmol) or dexmedetomidine (0.3-1 nmol), relieved mechanical allodynia and thermal hyperalgesia on postoperative day (POD) 14, but their efficacy was weaker on POD28 and absent on POD56. The RGS4 level of plasma membrane was increased on POD56 compared to that on POD14. Moreover, in RGS4-deficient or RGS4 inhibitor (CCG50014)-treated mice, the analgesic effect of the α2AR agonists was conserved even on POD56. The increased plasma membrane RGS4 expression and the reduced level of active Gαi after clonidine injection on POD56 were completely restored by CCG50014. Higher doses of clonidine (10 nmol) and dexmedetomidine (3 nmol) relieved neuropathic pain on POD56 but were accompanied with serious side effects. Whereas, the coadministration of CCG50014 with clonidine (1 nmol) or dexmedetomidine (1 nmol) did not cause side effects. These findings demonstrated that SNI-induced increase in plasma membrane RGS4 expression was associated with low efficacy of α2AR agonists in a model of persistent, chronic neuropathic pain. Furthermore, α2AR agonist administration together with RGS4-targeted intervention represents a novel strategy for the treatment of neuropathic pain to overcome dose-limiting side effects.

Peripheral GABAA receptor-mediated signaling facilitates persistent inflammatory hypersensitivity.

Unlike in the central nervous system (CNS), in the adult peripheral nervous system (PNS), activation of GABAA receptors (GABAAR) is excitatory because of the relatively high concentration of intracellular chloride in these neurons. Indeed, exogenous GABA and muscimol, a GABAAR agonist, exacerbate acute inflammatory hypersensitivity in rodents. However, it remains unclear whether peripheral GABAAR and the endogenous GABA play an important role in persistent inflammatory hypersensitivity. In this study, we thus investigated how peripheral GABAAR affects pain hypersensitivity by using the complete Freund's adjuvant (CFA)-induced persistent inflammatory pain mouse model. We found that intraplantar (i.pl.) administration of GABAAR antagonists, picrotoxin, and 1(S),9(R)-(-)-bicuculline methiodide significantly inhibited both spontaneous nociceptive (paw licking and flinching) behavior and mechanical hypersensitivity in CFA-injected mice at day 3 (D3), but not in naïve mice. Interestingly, CFA-induced mechanical hypersensitivity was significantly reversed by anti-GABA antibody (anti-GABA, i.pl.). In addition, RT-qPCR revealed that glutamate decarboxylase Gad1 (GAD 67) and Gad2 (GAD 65) mRNA expression was also upregulated in the ipsilateral hind paw of CFA-injected mice at D3. Finally, 5α-pregnan-3α-ol-20-one (3α,5α-THP), a selective positive allosteric modulator of GABAAR, produced mechanical hypersensitivity in naïve mice in a dose-dependent manner. Taken together, our results indicate that peripheral GABAAR and endogenous GABA, possibly produced by the inflamed tissue, potentiate CFA-induced persistent inflammatory hypersensitivity, suggesting that they can be used as a therapeutic target for alleviating inflammatory pain.

Differential Development of Facial and Hind Paw Allodynia in a Nitroglycerin-Induced Mouse Model of Chronic Migraine: Role of Capsaicin Sensitive Primary Afferents.

Despite the relatively high prevalence of migraine or headache, the pathophysiological mechanisms triggering headache-associated peripheral hypersensitivities, are unknown. Since nitric oxide (NO) is well known as a causative factor in the pathogenesis of migraine or migraine-associated hypersensitivities, a mouse model has been established using systemic administration of the NO donor, nitroglycerin (NTG). Here we tried to investigate the time course development of facial or hindpaw hypersensitivity after repetitive NTG injection. NTG (10 mg/kg) was administrated to mice every other day for nine days. Two hours post-injection, NTG produced acute mechanical and heat hypersensitivity in the hind paws. By contrast, cold allodynia, but not mechanical hypersensitivity, occurred in the facial region. Moreover, this hindpaws mechanical hypersensitivity and the facial cold allodynia was progressive and long-lasting. We subsequently examined whether the depletion of capsaicin-sensitive primary afferents (CSPAs) with resiniferatoxin (RTX, 0.02 mg/kg) altered these peripheral hypersensitivities in NTG-treated mice. RTX pretreatment did not affect the NTG-induced mechanical allodynia in the hind paws nor the cold allodynia in the facial region, but it did inhibit the development of hind paw heat hyperalgesia. Similarly, NTG injection produced significant hindpaw mechanical allodynia or facial cold allodynia, but not heat hyperalgesia in transient receptor potential type V1 (TRPV1) knockout mice. These findings demonstrate that different peripheral hypersensitivities develop in the face versus hindpaw regions in a mouse model of repetitive NTG-induced migraine, and that these hindpaw mechanical hypersensitivity and facial cold allodynia are not mediated by the activation of CSPAs.

A critical role of spinal Shank2 proteins in NMDA-induced pain hypersensitivity.

Background Self-injurious behaviors (SIBs) are devastating traits in autism spectrum disorder (ASD). Although deficits in pain sensation might be one of the contributing factors underlying the development of SIBs, the mechanisms have yet to be addressed. Recently, the Shank2 synaptic protein has been considered to be a key component in ASD, and mutations of SHANK2 gene induce the dysfunction of N-methyl-D-aspartate (NMDA) receptors, suggesting a link between Shank2 and NMDA receptors in ASD. Given that spinal NMDA receptors play a pivotal role in pain hypersensitivity, we investigated the possible role of Shank2 in nociceptive hypersensitivity by examining changes in spontaneous pain following intrathecal NMDA injection in S hank2-/- ( Shank2 knock-out, KO) mice. Results Intrathecal NMDA injection evoked spontaneous nociceptive behaviors. These NMDA-induced nociceptive responses were significantly reduced in Shank2 KO mice. We also observed a significant decrease of NMDA currents in the spinal dorsal horn of Shank2 KO mice. Subsequently, we examined whether mitogen-activated protein kinase or AKT signaling is involved in this reduced pain behavior in Shank2 KO mice because the NMDA receptor is closely related to these signaling molecules. Western blotting and immunohistochemistry revealed that spinally administered NMDA increased the expression of a phosphorylated form of extracellular signal-regulated kinase (p-ERK) which was significantly reduced in Shank2 KO mice. However, p38, JNK, or AKT were not changed by NMDA administration. The ERK inhibitor, PD98059, decreased NMDA-induced spontaneous pain behaviors in a dose-dependent manner in wild-type mice. Moreover, it was found that the NMDA-induced increase in p-ERK was primarily colocalized with Shank2 proteins in the spinal cord dorsal horn. Conclusion Shank2 protein is involved in spinal NMDA receptor-mediated pain, and mutations of Shank2 may suppress NMDA-ERK signaling in spinal pain transmission. This study provides new clues into the mechanisms underlying pain deficits associated with SIB and deserves further study in patients with ASD.

Repetitive Acupuncture Point Treatment with Diluted Bee Venom Relieves Mechanical Allodynia and Restores Intraepidermal Nerve Fiber Loss in Oxaliplatin-Induced Neuropathic Mice.

UNLABELLED: The chemotherapeutic agent, oxaliplatin, produces a robust painful neuropathy that results in the loss of intraepidermal nerve fibers (IENFs). We have previously reported that an acupuncture point (acupoint) injection of diluted bee venom (DBV) produces a temporary antiallodynic effect in oxaliplatin-induced neuropathic mice. Herein we show a significant long-lasting antinociceptive effect of repetitive DBV acupoint treatment on oxaliplatin-induced mechanical allodynia and a significant reduction in the loss of IENFs. DBV (0.1 mg/kg, subcutaneous) was administered once a day for 18 days beginning on day 15 after oxaliplatin injection. Immunohistochemistry for IENF was performed on the glabrous skin of the hind paw footpad using the pan-neuronal marker, protein gene product 9.5. A temporary increase in mechanical threshold was observed 60 minutes after a single DBV injection into the Zusanli acupoint, and this effect was enhanced over time with repetitive DBV treatments. The basal mechanical threshold before daily DBV injection also increased from day 7 after DBV injections, and peaked at day 14 after DBV treatment. Moreover, the oxaliplatin-induced loss of IENFs was significantly reduced in mice treated repetitively with DBV. Repetitive pretreatment with the α-2 adrenoceptor antagonist, yohimbine, (5 mg/kg, subcutaneous) completely prevented the antiallodynic effects and the increase in IENFs observed in mice treated repetitively with DBV. PERSPECTIVE: We showed that repetitive acupoint stimulation with DBV gradually and significantly reduced oxaliplatin-induced mechanical allodynia and restored the loss of IENFs in neuropathic mice via an α-2 adrenoceptor mechanism. Collectively, results of this study suggest that repetitive acupoint treatment with DBV can be a potential strategy for the management of chemotherapy-induced neuropathy.

Clonidine, an alpha-2 adrenoceptor agonist relieves mechanical allodynia in oxaliplatin-induced neuropathic mice; potentiation by spinal p38 MAPK inhibition without motor dysfunction and hypotension.

Cancer chemotherapy with platinum-based antineoplastic agents including oxaliplatin frequently results in a debilitating and painful peripheral neuropathy. We evaluated the antinociceptive effects of the alpha-2 adrenoceptor agonist, clonidine on oxaliplatin-induced neuropathic pain. Specifically, we determined if (i) the intraperitoneal (i.p.) injection of clonidine reduces mechanical allodynia in mice with an oxaliplatin-induced neuropathy and (ii) concurrent inhibition of p38 mitogen-activated protein kinase (MAPK) activity by the p38 MAPK inhibitor SB203580 enhances clonidine's antiallodynic effect. Clonidine (0.01-0.1 mg kg(-1), i.p.), with or without SB203580(1-10 nmol, intrathecal) was administered two weeks after oxaliplatin injection(10 mg kg(-1), i.p.) to mice. Mechanical withdrawal threshold, motor coordination and blood pressure were measured. Postmortem expression of p38 MAPK and ERK as well as their phosphorylated forms(p-p38 and p-ERK) were quantified 30 min or 4 hr after drug injection in the spinal cord dorsal horn of treated and control mice. Clonidine dose-dependently reduced oxaliplatin-induced mechanical allodynia and spinal p-p38 MAPK expression, but not p-ERK. At 0.1 mg kg(-1), clonidine also impaired motor coordination and decreased blood pressure. A 10 nmol dose of SB203580 alone significantly reduced mechanical allodynia and p-p38 MAPK expression, while a subeffective dose(3 nmol) potentiated the antiallodynic effect of 0.03 mg kg(-1) clonidine and reduced the increased p-p38 MAPK. Coadministration of SB203580 and 0.03 mg kg(-1) clonidine decreased allodynia similar to that of 0.10 mg kg(-1) clonidine, but without significant motor or vascular effects. These findings demonstrate that clonidine treatment reduces oxaliplatin-induced mechanical allodynia. The concurrent administration of SB203580 reduces the dosage requirements for clonidine, thereby alleviating allodynia without producing undesirable motor or cardiovascular effects.

Diluted bee venom injection reduces ipsilateral mechanical allodynia in oxaliplatin-induced neuropathic mice.

Oxaliplatin, which is used as one of anti-cancer drugs, commonly induces peripheral neuropathic pain. We have previously reported that an injection of diluted bee venom (DBV) produced a significant anti-nociceptive effects in several pain models of mice or rats. In this study, we evaluated time- and dose-dependent development of oxaliplatin-induced mechanical allodynia in bilateral hind paws of mice, and investigated the effect of DBV injection on this mechanical allodynia. DBV (0.1 mg/kg) was subcutaneously injected into the Zusanli acupoint 2 weeks after oxaliplatin (10 mg/kg) injection. One hour after DBV injection, we observed a significant reduction of mechanical allodynia in the ipsilateral hind paw, but not in the contralateral hind paw to DBV injection site. We subsequently examined whether this effect of DBV was related to the activation of peripheral nerves in DBV injected site, and then whether it was mediated by the activation of spinal cord alpha-2 adrenoceptors or opioid receptors. Subcutaneous pre-injection of 2% lidocaine (40 mg/kg) into the Zusanli acupoint completely blocked the anti-allodynic effect of DBV. Intrathecal pretreatment with yohimbine (25 µg/mouse), an alpha-2 adrenoceptor antagonist, also prevented the anti-allodynic effect of DBV, whereas pretreatment with naloxone (20 µg/mouse), an opioid receptor antagonist, did not block the effect of DBV. Taken together, these findings demonstrate that DBV injection into the Zusanli acupoint significantly reduces ipsilateral mechanical allodynia generated by oxaliplatin in mice, and also suggest that this anti-allodynic effect is dependent on the peripheral nerve activation in injected site and spinal cord alpha-2 adrenoceptors.