Structural analysis of TrkA mutations in patients with congenital insensitivity to pain reveals PLCγ as an analgesic drug target.

Chronic pain is a major health issue, and the search for new analgesics has become increasingly important because of the addictive properties and unwanted side effects of opioids. To explore potentially new drug targets, we investigated mutations in the NTRK1 gene found in individuals with congenital insensitivity to pain with anhidrosis (CIPA). NTRK1 encodes tropomyosin receptor kinase A (TrkA), the receptor for nerve growth factor (NGF) and that contributes to nociception. Molecular modeling and biochemical analysis identified mutations that decreased the interaction between TrkA and one of its substrates and signaling effectors, phospholipase Cγ (PLCγ). We developed a cell-permeable phosphopeptide derived from TrkA (TAT-pQYP) that bound the Src homology domain 2 (SH2) of PLCγ. In HEK-293T cells, TAT-pQYP inhibited the binding of heterologously expressed TrkA to PLCγ and decreased NGF-induced, TrkA-mediated PLCγ activation and signaling. In mice, intraplantar administration of TAT-pQYP decreased mechanical sensitivity in an inflammatory pain model, suggesting that targeting this interaction may be analgesic. The findings demonstrate a strategy to identify new targets for pain relief by analyzing the signaling pathways that are perturbed in CIPA.

Interactions between cannabinoid and opioid receptors in a mouse model of diabetic neuropathy.

ABSTRACT: Diabetic neuropathy, often associated with diabetes mellitus, is a painful condition with no known effective treatment except glycemic control. Studies with neuropathic pain models report alterations in cannabinoid and opioid receptor expression levels; receptors whose activation induces analgesia. We examined whether interactions between CB1R and opioid receptors could be targeted for the treatment of diabetic neuropathy. For this, we generated antibodies that selectively recognize native CB1R-MOR and CB1R-DOR heteromers using a subtractive immunization strategy. We assessed the levels of CB1R, MOR, DOR, and interacting complexes using a model of streptozotocin-induced diabetic neuropathy and detected increased levels of CB1R, MOR, DOR, and CB1R-MOR complexes compared with those in controls. An examination of G-protein signaling revealed that activity induced by the MOR, but not the DOR agonist, was potentiated by low nanomolar doses of CB1R ligands, including antagonists, suggesting an allosteric modulation of MOR signaling by CB1R ligands within CB1R-MOR complexes. Because the peptide endocannabinoid, hemopressin, caused a significant potentiation of MOR activity, we examined its effect on mechanical allodynia and found that it blocked allodynia in wild-type mice and mice with diabetic neuropathy lacking DOR (but have CB1R-MOR complexes). However, hemopressin does not alter the levels of CB1R-MOR complexes in diabetic mice lacking DOR but increases the levels of CB1R-DOR complexes in diabetic mice lacking MOR. Together, these results suggest the involvement of CB1R-MOR and CB1R-DOR complexes in diabetic neuropathy and that hemopressin could be developed as a potential therapeutic for the treatment of this painful condition.

Electrical stimulation of the posterior insula induces mechanical analgesia in a rodent model of neuropathic pain by modulating GABAergic signaling and activity in the pain circuitry.

The insula has emerged as a critical target for electrical stimulation since it influences pathological pain states. We investigated the effects of repetitive electrical stimulation of the insular cortex (ESI) on mechanical nociception, and general locomotor activity in rats subjected to chronic constriction injury (CCI) of the sciatic nerve. We also studied neuroplastic changes in central pain areas and the involvement of GABAergic signaling on ESI effects. CCI rats had electrodes implanted in the left agranular posterior insular cortex (pIC), and mechanical sensitivity was evaluated before and after one or five daily consecutive ESIs (15 min each, 60 Hz, 210 µs, 1 V). Five ESIs (repetitive ESI) induced sustained mechanical antinociception from the first to the last behavioral assessment without interfering with locomotor activity. A marked increase in Fos immunoreactivity in pIC and a decrease in the anterior and mid-cingulate cortex, periaqueductal gray and hippocampus were noticed after five ESIs. The intrathecal administration of the GABAA receptor antagonist bicuculline methiodide reversed the stimulation-induced antinociception after five ESIs. ESI increased GAD65 levels in pIC but did not interfere with GABA, glutamate or glycine levels. No changes in GFAP immunoreactivity were found in this work. Altogether, the results indicate the efficacy of repetitive ESI for the treatment of experimental neuropathic pain and suggest a potential influence of pIC in regulating pain pathways partially through modulating GABAergic signaling.

Photobiomodulation induces antinociception, recovers structural aspects and regulates mitochondrial homeostasis in peripheral nerve of diabetic mice.

Diabetic peripheral neuropathy (DPN) is a nervous disorder caused by diabetes mellitus, affecting about 50% of patients in clinical medicine. Chronic pain is one of the major and most unpleasant symptoms developed by those patients, and conventional available treatments for the neuropathy, including the associated pain, are still unsatisfactory and benefit only a small number of patients. Photobiomodulation (PBM) has been gaining clinical acceptance once it is able to promote early nerve regeneration resulting in significant improvement in peripheral nerves disabilities. In this work, the effects of PBM (660 nm, 30 mW, 1.6 J/cm2 , 0.28 cm2 , 15 s in a continuous frequency) on treating DPN-induced pain and nerve damage were evaluated in an experimental model of diabetic-neuropathy induced by streptozotocin in mice. PBM-induced antinociception in neuropathic-pain mice was dependent on central opioids release. After 21 consecutive applications, PBM increased nerve growth factor levels and induced structural recovery increasing mitochondrial content and regulating Parkin in the sciatic nerve of DPN-mice. Taking together, these data provide new insights into the mechanisms involved in the effects of PBM-therapy emphasizing its therapeutic potential in the treatment of DPN.

Hemopressin, an inverse agonist of cannabinoid receptors, inhibits neuropathic pain in rats.

Direct-acting cannabinoid receptor ligands are well known to reduce hyperalgesic responses after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Hemopressin (Hp) is a nonapeptide that selectively binds CB1 cannabinoid receptors (CB1 receptors) and exerts antinociceptive action in inflammatory pain models. We investigated the effect of Hp on neuropathic pain in rats subjected to chronic constriction injury (CCI) of the sciatic nerve, and explored the mechanisms involved. Oral administration of Hp inhibits mechanical hyperalgesia of CCI-rats up to 6h. Hp treatment also decreases Egr-1 immunoreactivity (Egr-1Ir) in the superficial layer of the dorsal horn of the spinal cord of CCI rats. The antinociceptive effect of Hp seems to be independent of inhibitory descending pain pathway since methysergide (5HT1A receptor antagonist) and yohimbine (α-2 adrenergic receptor antagonist) were unable to prevent Hp antinociceptive effect. Hp decreased calcium flux on DRG neurons from CCI rats, similarly to that observed for AM251, a CB1 receptor antagonist. We also investigated the effect of Hp on potassium channels of CCI rats using UCL 1684 (a blocker of Ca(2+)-activated K(+) channels) which reversed Hp-induced antinociception. Furthermore, concomitant administration of URB-584 (FAAH inhibitor) but not JZL-184 (MAGL inhibitor) potentiates antinociceptive effect of Hp in CCI rats indicating an involvement of anadamide on HP-induced antinociception. Together, these data demonstrate that Hp displays antinociception in pain from neuropathic etiology through local effects. The release of anandamide and the opening of peripheral K(+) channels are involved in the antinociceptive effect.

Analgesic Effect of Light-Emitting Diode (LED) Therapy at Wavelengths of 635 and 945 nm on Bothrops moojeni Venom-Induced Hyperalgesia.

Envenoming induced by Bothrops snakes is characterized by drastic local tissue damage involving hemorrhage, myonecrosis and proeminent inflammatory and hyperalgesic response. The most effective treatment is antivenom therapy, which is ineffective in neutralizing the local response. Herein, it was evaluated the effectiveness of light-emitting diode (LED) at wavelengths of 635 and 945 nm in reducing inflammatory hyperalgesia induced by Bothrops moojeni venom (BmV) in mice, produced by an subplantar injection of BmV (1 µg). Mechanical hyperalgesia and allodynia were assessed by von Frey filaments at 1, 3, 6 and 24 h after venom injection. The site of BmV injection (1.2 cm(2) ) was irradiated by LEDs at 30 min and 3 h after venom inoculation. Both 635 nm (110 mW, fluence of 3.76 J/cm(2) and 41 s of irradiation time) and 945 nm (120 mW, fluence of 3.8 J/cm(2) and 38 s of irradiation time) LED inhibited mechanical allodynia and hyperalgesia of mice alone or in combination with antivenom treatment, even when the symptoms were already present. The effect of phototherapy in reducing local pain induced by BmV should be considered as a novel therapeutic tool for the treatment of local symptoms induced after bothropic snake bites.

AGH is a new hemoglobin alpha-chain fragment with antinociceptive activity.

Limited proteolysis of certain proteins leads to the release of endogenous bioactive peptides. Hemoglobin-derived peptides such as hemorphins and hemopressins are examples of intracellular protein-derived peptides that have antinociceptive effects by modulating G-protein coupled receptors activities. In the present study, a previously characterized substrate capture assay that uses a catalytically inactive form of the thimet oligopeptidase was combined with isotopic labeling and mass spectrometry in order to identify new bioactive peptides. Indeed, we have identified the peptide AGHLDDLPGALSAL (AGH), a fragment of the hemoglobin alpha-chain, which specifically bind to the inactive thimet oligopeptidase in the substrate capture assay. Previous peptidomics studies have identified the AGH as well as many other natural peptides derived from hemoglobin alpha-chain containing this sequence, further suggesting that AGH is a natural endogenous peptide. Pharmacological assays suggest that AGH inhibits peripheral inflammatory hyperalgesic responses through indirect activation of mu opioid receptors, without having a central nervous system activity. Therefore, we have successfully used the substrate capture assay to identify a new endogenous bioactive peptide derived from hemoglobin alpha-chain.

Antinociception induced by motor cortex stimulation: somatotopy of behavioral response and profile of neuronal activation.

Motor cortex stimulation (MCS) is used as a therapy for patients with refractory neuropathic pain. Experimental evidence suggests that the motor cortex (MC) is involved in the modulation of normal nociceptive response, but the underlying mechanisms have not been clarified yet. In previous studies, we demonstrated that MCS increases the nociceptive threshold of naive conscious rats by inhibiting thalamic sensory neurons and disinhibiting the neurons in periaqueductal gray (PAG), with the involvement of the opioid system. The aim of this study was to investigate the possible somatotopy of the motor cortex on MCS-induced antinociception and the pattern of neuronal activation evaluated by Fos and Egr-1 immunolabel in an attempt to better understand the relation between MC and analgesia. Rats received epidural electrode implants placed over the MC, in three distinct areas (forelimb, hindlimb or tail), according to a functional mapping established in previous studies. Nociceptive threshold was evaluated under 15-min electrical stimulating sessions. MCS induced selective antinociception in the limb related to the stimulated cortex, with no changes in other evaluated areas. MCS decreased Fos immunoreactivity (Fos-IR) in the superficial layers of the dorsal horn of the spinal cord for all evaluated groups and increased Fos-IR in the PAG, although no changes were observed in the PAG for the tail group. Egr-1 results were similar to those obtained for Fos. Data shown herein demonstrate that MCS elicits a substantial and selective antinociceptive effect, which is mediated, at least in part, by the activation of descendent inhibitory pain pathway.