The forebrain medial septal region and nociception.

The forebrain medial septum, which is an integral part of the septo-hippocampal network, is implicated in sensorimotor integration, fear and anxiety, and spatial learning and memory. A body of evidence also suggests that the septal region affects experimental pain. Indeed, some explorations in humans have raised the possibility that the region may modulate clinical pain as well. This review explores the evidence that implicates the medial septum in nociception and suggests that non-overlapping circuits in the region facilitate acute nociceptive behaviors and defensive behaviors that reflect affect and cognitive appraisal, especially in relation to persistent nociception. In line with a role in nociception, the region modulates nociceptive responses in the neuraxis, including the hippocampus and the anterior cingulate cortex. The aforementioned forebrain regions have also been implicated in persistent/long-lasting nociception. The review also weighs the effects of the medial septum on nociception vis-à-vis the known roles of the region and emphasizes the fact that the region is a part of network of forebrain structures which have been long associated with reward, cognition and affect-motivation and are now implicated in persistent/long-lasting nociception.

Metabolic brain activity suggestive of persistent pain in a rat model of neuropathic pain.

Persistent pain is a central characteristic of neuropathic pain conditions in humans. Knowing whether rodent models of neuropathic pain produce persistent pain is therefore crucial to their translational applicability. We investigated the spared nerve injury (SNI) model of neuropathic pain and the formalin pain model in rats using positron emission tomography (PET) with the metabolic tracer [18F]fluorodeoxyglucose (FDG) to determine if there is ongoing brain activity suggestive of persistent pain. For the formalin model, under brief anesthesia we injected one hindpaw with 5% formalin and the FDG tracer into a tail vein. We then allowed the animals to awaken and observed pain behavior for 30min during the FDG uptake period. The rat was then anesthetized and placed in the scanner for static image acquisition, which took place between minutes 45 and 75 post-tracer injection. A single reference rat brain magnetic resonance image (MRI) was used to align the PET images with the Paxinos and Watson rat brain atlas. Increased glucose metabolism was observed in the somatosensory region associated with the injection site (S1 hindlimb contralateral), S1 jaw/upper lip and cingulate cortex. Decreases were observed in the prelimbic cortex and hippocampus. Second, SNI rats were scanned 3weeks post-surgery using the same scanning paradigm, and region-of-interest analyses revealed increased metabolic activity in the contralateral S1 hindlimb. Finally, a second cohort of SNI rats was scanned while anesthetized during the tracer uptake period, and the S1 hindlimb increase was not observed. Increased brain activity in the somatosensory cortex of SNI rats resembled the activity produced with the injection of formalin, suggesting that the SNI model may produce persistent pain. The lack of increased activity in S1 hindlimb with general anesthetic demonstrates that this effect can be blocked, as well as highlights the importance of investigating brain activity in awake and behaving rodents.

Betulinic acid analogs inhibit N- and T-type voltage-gated calcium channels to attenuate nerve-injury associated neuropathic and formalin models of pain.

Over the past three decades, there has been a significant growth in the use of natural products, with approximately 80% of individuals using them for some aspect of primary healthcare. Our laboratories have identified and studied natural compounds with analgesic effects from dry land plants or their associated fungus during the past ten years. Here, we isolated and characterized thirteen betulin analogs and fifteen betulinic acid analogs for their capacity to prevent calcium influx brought on by depolarization in sensory neurons. The in vitro inhibition of voltage-gated calcium channels by the top drugs was then assessed using whole cell patch clamp electrophysiology. In vivo experiments, conducted at two sites, evaluated the best compound in acute and tonic, neuropathic, inflammatory, post-operative and visceral models of pain. We found that the betulinic acid analog 8 inhibited calcium influx in rat dorsal root ganglion neurons by inhibiting N- (CaV2.2) and T- (CaV3) type voltage-gated calcium channels. Moreover, intrathecal delivery of analog 8 had analgesic activity in both spared nerve injury model of neuropathic pain and acute and tonic pain induced by formalin. The results presented herein highlight the potential antinociceptive properties of betulinic acid analog 8 and set the stage for the development of novel non-opioid pain therapeutics based on the triterpenoid scaffold of betulinic acid.

Activation of Peripheral Cannabinoid Receptors Synergizes the Effect of Systemic Ibuprofen in a Pain Model in Rat.

Pharmacological synergism is a current strategy for the treatment of pain. However, few studies have been explored to provide evidence of the possible synergism between a non-steroidal anti-inflammatory drug (NSAID) and a cannabinoid agonist, in order to establish which combinations might be effective to manage pain. The aim of this study was to explore the synergism between ibuprofen (IBU) and the synthetic cannabinoid WIN 55,212-2 (WIN) to improve pain relief by analyzing the degree of participation of the CB1 and CB2 cannabinoid receptors in the possible antinociceptive synergism using an experimental model of pain in Wistar rats. First, the effective dose thirty (ED30) of IBU (10, 40, 80, and 160 mg/kg, subcutaneous) and WIN (3, 10, and 30 µg/p, intraplantar) were evaluated in the formalin test. Then, the constant ratio method was used to calculate the doses of IBU and WIN to be administered in combination (COMB) to determine the possible synergism using the isobolographic method. The participation of the CB1 and CB2 receptors was explored in the presence of the antagonists AM281 and AM630, respectively. The combination of these drugs produced a supra-additive response with an interaction index of 0.13. In addition, AM281 and AM630 antagonists reversed the synergistic effect in 45% and 76%, respectively, suggesting that both cannabinoid receptors are involved in this synergism, with peripheral receptors playing a relevant role. In conclusion, the combination of IBU + WIN synergism is mainly mediated by the participation of the CB2 receptor, which can be a good option for the better management of pain relief.

Input-dependent synaptic suppression by pregabalin in the central amygdala in male mice with inflammatory pain.

Pregabalin (PGB) is a synthetic amino acid compound most widely prescribed for chronic peripheral and central neuropathic pain. PGB is a ligand for the α2δ1 subunit of voltage-dependent calcium channels, and its binding reduces neurotransmitter release and thus inhibits synaptic transmission. The central nucleus of the amygdala (CeA) is a kernel site for the enhanced nociception-emotion link in chronic pain. The nociceptive information is conveyed to the CeA via the following two pathways: 1) the pathway arising from the basolateral amygdala (BLA), which carries nociceptive information mediated by the thalamocortical system, and 2) that arising from the external part of the pontine lateral parabrachial nucleus (LPB), that forms the final route of the spino-parabrachio-amygdaloid pathway that conveys nociceptive information directly from the superficial layer of the spinal dorsal horn. We compared the effects of PGB on the excitatory postsynaptic currents of neurons in the right CeA in response to electrical stimulation of BLA and LPB pathways using the whole-cell patch-clamp technique. Inflammatory pain was induced by intraplantar injection of formalin solution at the left hind paw. At eight hours post-formalin, PGB reduced EPSCs amplitude of the BLA-to-CeA synaptic transmission, accompanied by a significant increase in the PPR, suggesting a decreased release probability from the presynaptic terminals. In addition, these effects of PGB were only seen in inflammatory conditions. PGB did not affect the synaptic transmission at the LPB-to-CeA pathway, even in formalin-treated mice. These results suggest PGB improves not simply the aberrantly enhanced nociception but also various pain-associated cognitive and affective consequences in patients with chronic nociplastic pain.

C57BL/6 substrain differences in formalin-induced pain-like behavioral responses.

Substantial evidence from preclinical models of pain suggests that basal and noxious nociceptive sensitivity, as well as antinociceptive responses to drugs, show significant heritability. Individual differences to these responses have been observed across species from rodents to humans. The use of closely related C57BL/6 inbred mouse substrains can facilitate gene mapping of acute nociceptive behaviors in preclinical pain models. In this study, we investigated behavioral differences between C57BL/6 J (B6 J) and C57BL/6 N (B6 N) substrains in the formalin test, a widely used tonic inflammatory pain model, using a battery of pain-related phenotypes, including reflexive tests, nesting, voluntary wheel running, sucrose preference and anxiety-like behavior in the light/dark test at two different time points (1-h and 24-h). Our results show that these substrains did not differ in reflexive thermal and mechanical responses at the 1-h time point. However, B6 N substrain mice showed increased sensitivity to spontaneous pain-like behaviors. In addition, B6 N substrain continued to show higher levels of mechanical hypersensitivity compared to controls at 24-h. indicating that mechanical hypersensitivity is a more persistent pain-related phenotype induced by formalin. Finally, no sex differences were observed in our outcome measures. Our results provide a comprehensive behavioral testing paradigm in response to an inflammatory agent for future mouse genetic studies in pain.

Dual-Acting Peripherally Restricted Delta/Kappa Opioid (CAV1001) Produces Antinociception in Animal Models of Sub-Acute and Chronic Pain.

BACKGROUND: The development of highly efficacious alternatives to mu-opioid analgesics represents an urgent unmet medical and public health need. In the presence of inflammation both delta- and kappa-opioid agonists, acting on peripheral sensory neurons, mediate analgesia. The dual-acting, peripherally restricted kappa/delta-opioid agonist, CAV1001, was tested in four rodent pain models. METHODS: Experiment 1 - Formalin testing in mice. Three doses (1-10 mg/kg) of CAV1001 or ICI204448 at 30 minutes were tested after formalin injection. Spontaneous nocifensive responses were video recorded. Experiment 2 - Complete Freund's Adjuvant (CFA)-induced arthritis. CFA was injected into the ankle joint of rats. Joint compression thresholds (JCT) were measured. CAV1001 was compared to celecoxib. Experiment 3 - Spinal nerve ligation (SNL) in rats. Paw compression thresholds (PCT) were measured. CAV1001 was compared to gabapentin. Experiment 4 - MMRT-1 bone cancer implantation into the rat tibia. Weight-bearing was assessed. CAV1001 was compared to morphine. RESULTS: In Phase 2 of the formalin model, CAV1001 (1 mg/kg) significantly reduced pain behaviors to a degree comparable to the peripherally restricted kappa-opioid agonist, ICI204448 (10 mg/kg). CAV1001 (10 mg/kg) effectively eliminated pain behaviors associated with phase 2. In the CFA-induced arthritis model, a significant increase in JCTs, similar to the comparator celecoxib, was observed with CAV1001 at 1 mg/kg at 2 hours; CAV1001 (10 mg/kg) was effective at 1 hour. In the SNL model, both the comparator gabapentin and CAV1001 (5 mg/kg) significantly reduced PCT at 2 hours, but at 4 hours, the CAV1001 thresholds improved to baseline. CAV1001 10 mg/kg significantly improved weight bearing at 4-hour post-dosing compared to baseline following MMRT-1 implantation. CONCLUSION: CAV1001 demonstrated efficacy in several different preclinical pain models. Time- and dose-dependent differences in the efficacy of CAV1001 amongst these rodent pain models parallel the degree of underlying inflammation.

Analgesic Effects of Duloxetine on Formalin-Induced Hyperalgesia and Its Underlying Mechanisms in the CeA.

In rodents, the amygdala has been proposed to serve as a key center for the nociceptive perception. Previous studies have shown that extracellular signal-regulated kinase (ERK) signaling cascade in the central nucleus of amygdala (CeA) played a functional role in inflammation-induced peripheral hypersensitivity. Duloxetine (DUL), a serotonin and noradrenaline reuptake inhibitor, produced analgesia on formalin-induced spontaneous pain behaviors. However, it is still unclear whether single DUL pretreatment influences formalin-induced hypersensitivity and what is the underlying mechanism. In the current study, we revealed that systemic pretreatment with DUL not only dose-dependently suppressed the spontaneous pain behaviors, but also relieved mechanical and thermal hypersensitivity induced by formalin hindpaw injection. Consistent with the analgesic effects of DUL on the pain behaviors, the expressions of Fos and pERK that were used to check the neuronal activities in the spinal cord and CeA were also dose-dependently reduced following DUL pretreatment. Meanwhile, no emotional aversive behaviors were observed at 24 h after formalin injection. The concentration of 5-HT in the CeA was correlated with the dose of DUL in a positive manner at 24 h after formalin injection. Direct injecting 5-HT into the CeA suppressed both the spontaneous pain behaviors and hyperalgesia induced by formalin injection. However, DUL did not have protective effects on the formalin-induced edema of hindpaw. In sum, the activation of CeA neurons may account for the transition from acute pain to long-term hyperalgesia after formalin injection. DUL may produce potent analgesic effects on the hyperalgesia and decrease the expressions of p-ERK through increasing the concentration of serotonin in the CeA.

Analgesic activities of the mixed opioid and NPFF receptors agonist DN-9 in a mouse model of formalin-induced orofacial inflammatory pain.

Orofacial pain is one of the most common pain conditions and compromises the quality of life of the sufferer. Several studies have shown that opioid agonists produced significant analgesia in the orofacial pain, and combination of opioids with drugs belonging to other classes induced synergism in the orofacial pain. However, combination therapy of different analgesic drugs improves the risk of drug-drug interactions. Against this background, we sought to investigate the analgesic effects of the multi-functional opioid peptide DN-9, a mixed opioid and NPFF receptors agonist that produced robust analgesia in acute and inflammatory pain models, on formalin-induced orofacial pain. Our results showed that formalin injection caused significant spontaneous pain behaviors and increased the expressions of the mu-opioid receptor, c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) in the ipsilateral trigeminal ganglion (TG). In mice pretreated with DN-9, there was a significant reduction in nociceptive behaviors, which was selectively mediated by the mu- and kappa-opioid receptors, independently of the NPFF system. Four hours after formalin injection, the level of c-Fos immunoreactivity in the ipsilateral TG neurons was much lower in mice pretreated with DN-9 or morphine. In addition, DN-9 exhibited a significant inhibition in the expression of p-ERK1/2, which was reversed by the selective antagonists of the mu- and kappa-opioid receptors. In conclusion, our present results demonstrate that central administration of DN-9 produces potential antinociceptive effects via the mu- and kappa-opioid receptors, independently of the NPFF system, and this antinociceptive action is tightly linked with the intracellular ERK activation in TG neurons.

Chemical Composition and Antinociceptive Potential of Campomanesia reitziana Fruits.

The methanolic extract from Campomanesia reitziana fruits and the main active principle, identified as 4',6'-dihydroxy-3',5'-dimethyl-2'-methoxy chalcone or dimethyl cardamonin (1), exhibited pronounced antinociceptive effects against two models of pain in mice. Compound 1 caused dose-dependent inhibition of abdominal constrictions, with a calculated ID50 value of 8.1 (6.5-10.1) µmol/kg (i.p.), being about 16-fold more potent than two reference analgesic drugs. Methanolic extract and 1 were also effective against the formalin model, inhibiting both phases of pain, causing reductions of 39.9% and 26.8% (extract, 10 mg/kg) and 52.9% and 57.6% (compound 1, 5 mg/kg) for the first and second phases, respectively.

Discovery of Aryl Sulfonamides as Isoform-Selective Inhibitors of NaV1.7 with Efficacy in Rodent Pain Models.

We report on a novel series of aryl sulfonamides that act as nanomolar potent, isoform-selective inhibitors of the human sodium channel hNaV1.7. The optimization of these inhibitors is described. We aimed to improve potency against hNaV1.7 while minimizing off-target safety concerns and generated compound 3. This agent displayed significant analgesic effects in rodent models of acute and inflammatory pain and demonstrated that binding to the voltage sensor domain 4 site of NaV1.7 leads to an analgesic effect in vivo. Our findings corroborate the importance of hNaV1.7 as a drug target for the treatment of pain.

Antinociceptive properties of N-Mannich bases derived from 3-substituted pyrrolidine-2,5-dione in the formalin model of persistent pain in mice.

BACKGROUND: Accumulated data indicate that anticonvulsants possess antinociceptive properties in rodent pain models. In view of the anticonvulsant activity demonstrated previously among N-Mannich bases derived from 3-mono- (1-6) and 3,3-disubstituted pyrrolidine-2,5-diones (7-14) their analgesic activity has been investigated in the formalin model of tonic pain in mice. METHODS: The compounds 1-14 were tested at doses equal to the respective ED50 values obtained earlier in the MES test. 0.5% formalin solution was given as intraplantar injections into the hind paw of the mouse and the duration of the nocifensive response was counted in drug-treated and vehicle-treated animals in the acute and the late phases of the test. RESULTS: A significant antinociceptive activity was observed for majority of the compounds. In the first phase of the test all the active compounds, except for 9-11, reduced the duration of the licking response up to 88% (compounds 2 and 6; p<0.001). In the late phase the 1-3, 5, 6, 9 and 14 were the most effective agents and their analgesic activities ranged from 92 to 100%. CONCLUSIONS: The results of the research indicate that some of the investigated compounds reduced effectively either both phases of the test or were able to attenuate pain during only the acute or late phase of the formalin test. These properties, which are particularly strong in case of the compounds 1-3, 5, 6, 9 and 14, might be relevant for the development of novel analgesic-active compounds and their possible use in neuropathic pain syndromes.

TRPV4 is necessary for trigeminal irritant pain and functions as a cellular formalin receptor.

Detection of external irritants by head nociceptor neurons has deep evolutionary roots. Irritant-induced aversive behavior is a popular pain model in laboratory animals. It is used widely in the formalin model, where formaldehyde is injected into the rodent paw, eliciting quantifiable nocifensive behavior that has a direct, tissue-injury-evoked phase, and a subsequent tonic phase caused by neural maladaptation. The formalin model has elucidated many antipain compounds and pain-modulating signaling pathways. We have adopted this model to trigeminally innervated territories in mice. In addition, we examined the involvement of TRPV4 channels in formalin-evoked trigeminal pain behavior because TRPV4 is abundantly expressed in trigeminal ganglion (TG) sensory neurons, and because we have recently defined TRPV4's role in response to airborne irritants and in a model for temporomandibular joint pain. We found TRPV4 to be important for trigeminal nocifensive behavior evoked by formalin whisker pad injections. This conclusion is supported by studies with Trpv4(-/-) mice and TRPV4-specific antagonists. Our results imply TRPV4 in MEK-ERK activation in TG sensory neurons. Furthermore, cellular studies in primary TG neurons and in heterologous TRPV4-expressing cells suggest that TRPV4 can be activated directly by formalin to gate Ca(2+). Using TRPA1-blocker and Trpa1(-/-) mice, we found that both TRP channels co-contribute to the formalin trigeminal pain response. These results imply TRPV4 as an important signaling molecule in irritation-evoked trigeminal pain. TRPV4-antagonistic therapies can therefore be envisioned as novel analgesics, possibly for specific targeting of trigeminal pain disorders, such as migraine, headaches, temporomandibular joint, facial, and dental pain, and irritation of trigeminally innervated surface epithelia.