Camila S. Dale

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.

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.

Anxiogenic-like effects induced by hemopressin in rats.

Hemopressin (PVNFKFLSH; HP) is an orally active peptide derived from rat hemoglobin α-chain that could act as an inverse agonist at cannabinoid type 1 receptors (CB1). Here, we aim to investigate possible behavioral effects of HP in male Wistar rats tested in the elevated plus maze (EPM), following HP intraperitoneal (i.p., 0.05 mg/kg), oral (P.O., 0.05 and 0.5 mg/kg) or intracerebroventricular (I.C.V., 3 and 10 nmol) administration. HP induced a decrease in EPM open arm exploration, indicating an anxiogenic-like effect. However, i.p. administration of HP (1 mg/kg) followed by mass spectrometry analysis of brain-peptide extracts suggested that the intact HP does not cross the blood brain barrier. I.C.V. administrated HP produced anxiogenic-like effects that were prevented by Transient Receptor Potential Vanilloid Type 1 (TRPV1) antagonists, 6-iodonordihydrocapsaicin (1 nmol) or SB366791 (1 nmol), but not by the CB1 receptor antagonist AM251 (0.1 and 1 nmol). Altogether, these data suggest that I.C.V. administrated HP induces anxiogenic-like effects by activating TRPV1 receptors. The similar anxiogenic effects observed after i.p. or P.O. administration could be due to HP fragment(s) crossing the blood brain barrier. The present results advance our knowledge about HP pharmacology and suggest concerns in future clinical studies.

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 cm2) was irradiated by LEDs at 30 min and 3 h after venom inoculation. Both 635 nm (110 mW, fluence of 3.76 J/cm2 and 41 s of irradiation time) and 945 nm (120 mW, fluence of 3.8 J/cm2 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.

Analgesic Effect of Photobiomodulation on Bothrops Moojeni Venom-Induced Hyperalgesia: A Mechanism Dependent on Neuronal Inhibition, Cytokines and Kinin Receptors Modulation

Background Envenoming induced by Bothrops snakebites is characterized by drastic local tissue damage that involves an intense inflammatory reaction and local hyperalgesia which are not neutralized by conventional antivenom treatment. Herein, the effectiveness of photobiomodulation to reduce inflammatory hyperalgesia induced by Bothrops moojeni venom (Bmv), as well as the mechanisms involved was investigated. Methodology/Principal Findings Bmv (1 μg) was injected through the intraplantar route in the right hind paw of mice. Mechanical hyperalgesia and allodynia were evaluated by von Frey filaments at different time points after venom injection. Low level laser therapy (LLLT) was applied at the site of Bmv injection at wavelength of red 685 nm with energy density of 2.2 J/cm2 at 30 min and 3 h after venom inoculation. Neuronal activation in the dorsal horn spinal cord was determined by immunohistochemistry of Fos protein and the mRNA expression of IL-6, TNF-α, IL-10, B1 and B2 kinin receptors were evaluated by Real time-PCR 6 h after venom injection. Photobiomodulation reversed Bmv-induced mechanical hyperalgesia and allodynia and decreased Fos expression, induced by Bmv as well as the mRNA levels of IL-6, TNF-α and B1 and B2 kinin receptors. Finally, an increase on IL-10, was observed following LLLT. Conclusion/Significance These data demonstrate that LLLT interferes with mechanisms involved in nociception and hyperalgesia and modulates Bmv-induced nociceptive signal. The use of photobiomodulation in reducing local pain induced by Bothropic venoms should be considered as a novel therapeutic tool for the treatment of local symptoms induced after bothropic snakebites.

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.

Epigenetics insights into chronic pain: DNA hypomethylation in fibromyalgia - A controlled pilot-study

Abstract To evaluate changes in DNA methylation profiles in patients with fibromyalgia (FM) compared to matched healthy controls (HCs). All individuals underwent full clinical and neurophysiological assessment by cortical excitability (CE) parameters measured by transcranial magnetic stimulation. DNA from the peripheral blood of patients with FM (n = 24) and HC (n = 24) were assessed using the Illumina-HumanMethylation450 BeadChips. We identified 1610 differentially methylated positions (DMPs) in patients with FM displaying a nonrandom distribution in regions of the genome. Sixty-nine percent of DMP in FM were hypomethylated compared to HC. Differentially methylated positions were enriched in 5 genomic regions (1p34; 6p21; 10q26; 17q25; 19q13). The functional characterization of 960 genes related to DMPs revealed an enrichment for MAPK signaling pathway (n = 18 genes), regulation of actin cytoskeleton (n = 15 genes), and focal adhesion (n = 13 genes). A gene–gene interaction network enrichment analysis revealed the participation of DNA repair pathways, mitochondria-related processes, and synaptic signaling. Even though DNA was extracted from peripheral blood, this set of genes was enriched for disorders such as schizophrenia, mood disorders, bulimia, hyperphagia, and obesity. Remarkably, the hierarchical clusterization based on the methylation levels of the 1610 DMPs showed an association with neurophysiological measurements of CE in FM and HC. Fibromyalgia has a hypomethylation DNA pattern, which is enriched in genes implicated in stress response and DNA repair/free radical clearance. These changes occurred parallel to changes in CE parameters. New epigenetic insights into the pathophysiology of FM may provide the basis for the development of biomarkers of this disorder.

Pregabalin for the Prevention of Oxaliplatin‐Induced Painful Neuropathy: A Randomized, Double‐Blind Trial

Abstract Lessons Learned. Pregabalin is a medication that can decrease neuronal hyperexcitability, relieve neuropathic pain, and reach stable plasma levels after a titration period of only a few days. Its use during oxaliplatin infusions was not able to decrease the incidence of chronic, oxalipaltin‐related neuropathic pain, compared with placebo. Background. Patients with colorectal cancer (CRC) receiving oxaliplatin (OXA) develop acute and chronic painful oxaliplatin‐induced peripheral neuropathy (OXAIPN). Acute and chronic OXA‐related neuropathies have different pathophysiological bases, but both lead to a common phenomenon: central sensitization (CS) of nociceptive neuronal networks, leading to increased sensitivity (hyperlgesia, allodynia) in the somatosensory system, the common ground of chronic neuropathic pain. Because CS is related to increased risk of painful OXAIPN, we hypothesized that preemptive use of the anti‐hyperalgesic drug pregabaline (known to decrease CS) during OXA infusions would decrease the incidence of chronic OXAIPN. Methods. Pain‐free, chemotherapy‐naïve CRC patients receiving at least one cycle of modified‐FLOX [5‐FU(500 mg/m2)+leucovorin(20 mg/m2)/week for] 6 weeks+oxaliplatin(85 mg/m2) at weeks 1‐3‐5 every 8 weeks] were randomized (1:1) into the study. Patients received either pregabalin or placebo for 3 days before and 3 days after each OXA infusion and were followed for up to 6 months. Clinical assessments were performed at baseline, at the end of chemotherapy, and after the follow‐up period. The main outcome was average pain at the last visit assessed by the visual analogic scale (0–10) item of the Brief Pain Inventory (BPI). Secondary endpoints were presence of neuropathic pain according to the Douleur Neuropathique‐4 (DN‐4), pain dimensions (short‐ form McGill Pain Questionnaire [MPQ]), Neuropathic Pain Symptom Inventory (NPSI), and changes in nerve conduction studies (NCS) and side effect profile. Results. One hundred ninety‐nine patients (57.0 ± 10.7 years old, 98 female, 101 male) were randomized. Data from 56 patients were not included in the analyses (as they did not receive at least one full cycle of modified FLOX). Data from 78 patients in the pregabalin group and 65 patients in the placebo group were retained for analyses. At the last visit, pain intensity in the pregabalin group was 1.03 (95% confidence interval [CI] = 0.79–1.26), and 0.85 (95% CI = 0.64–1.06) in the placebo group, which did not reach significance. Scores from the BPI, MPQ, DN‐4, NPSI, and NCS and side‐effect profiles and incidence of death did not differ between groups. Quality of life (QoL) score did not differ between groups (placebo = 76.9 ± 23.1, pregabalin group 79.4 ± 20.6). Mood scores were not significantly different between groups (placebo 9.7 [8.1–11.2]; pregabalin 6.8 [5.6–8.0]). Conclusion. The preemptive use of pregabalin during OXA infusions was safe, but did not decrease the incidence of chronic pain related to OXAIPN.

Light emitting diode (LED) therapy reduces local pathological changes induced by Bothrops asper snake venom

ABSTRACT The therapeutic effect of the Light Emitting Diode (LED) treatment in two wavelengths (635 or 945 nm) was evaluated in the local pathological alterations induced by Bothrops asper snake venom. Mice received irradiation of infrared LED (120 mW, 945 nm) or red LED (110 mW, 635 nm) applied immediately, 1 and 2 h after venom injection. LED treatment reduced edema formation in the plantar region and gastrocnemius muscle and significantly reduced neutrophil migration and hyperalgesia after the venom injection. Also, both infrared LED and red LED treatment significantly reduced myonecrosis, as revealed by muscle CK and plasma CK levels. Histological analysis corroborated the reduction in the extent of venom‐induced myonecrosis. In conclusion, our data demonstrates that PBM with LED light in both red and infrared wavelengths, when applied after envenomation in mice, reduces the extent of myotoxicity, edema, inflammatory infiltrate and hyperalgesia, suggesting that photobiomodulation is a potential therapeutic approach that should be further investigated for the treatment of local effects of Bothrops snakebite. HighlightsBothrops asper venom causes prominent local pathological and inflammatory effects.Antivenom therapy does not neutralize the local effects caused by Bothrops asper venom.LED therapy reduces local effects caused by Bothrops asper venom.LED is a promising tool in the treatment of local envenomation in snakebites.

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.