Blockage of mechanosensitive Piezo1 channel alleviates the severity of experimental malaria-associated acute lung injury.

BACKGROUND: Malaria-associated acute lung injury (MA-ALI) is a well-recognized clinical complication of severe, complicated malaria that is partly driven by sequestrations of infected red blood cells (iRBCs) on lung postcapillary induced impaired blood flow. In earlier studies the mechanosensitive Piezo1 channel emerged as a regulator of mechanical stimuli, but the function and underlying mechanism of Piezo1 impacting MA-ALI severity via sensing the impaired pulmonary blood flow are still not fully elucidated. Thus, the present study aimed to explore the role of Piezo1 in the severity of murine MA-ALI. METHODS: Here, we utilized a widely accepted murine model of MA-ALI using C57BL/6 mice with Plasmodium berghei ANKA infection and then added a Piezo1 inhibitor (GsMTx4) to the model. The iRBC-stimulated Raw264.7 macrophages in vitro were also targeted with GsMTx4 to further explore the potential mechanism. RESULTS: Our data showed an elevation in the expression of Piezo1 and number of Piezo1+-CD68+ macrophages in lung tissues of the experimental MA-ALI mice. Compared to the infected control mice, the blockage of Piezo1 with GsMTx4 dramatically improved the survival rate but decreased body weight loss, peripheral blood parasitemia/lung parasite burden, experimental cerebral malaria incidence, total protein concentrations in bronchoalveolar lavage fluid, lung wet/dry weight ratio, vascular leakage, pathological damage, apoptosis and number of CD68+ and CD86+ macrophages in lung tissues. This was accompanied by a dramatic increase in the number of CD206+ macrophages (M2-like subtype), upregulation of anti-inflammatory cytokines (e.g. IL-4 and IL-10) and downregulation of pro-inflammatory cytokines (e.g. TNF-α and IL-1ß). In addition, GsMTx4 treatment remarkably decreased pulmonary intracellular iron accumulation, protein level of 4-HNE (an activator of ferroptosis) and the number of CD68+-Piezo1+ and CD68+-4-HNE+ macrophages but significantly increased protein levels of GPX4 (an inhibitor of ferroptosis) in experimental MA-ALI mice. Similarly, in vitro study showed that the administration of GsMTx4 led to a remarkable elevation in the mRNA levels of CD206, IL-4, IL-10 and GPX-4 but to a substantial decline in CD86, TNF-α, IL-1ß and 4-HNE in the iRBC-stimulated Raw264.7 cells. CONCLUSIONS: Our findings indicated that blockage of Piezo1 with GsMTx4 alleviated the severity of experimental MA-ALI in mice partly by triggering pulmonary macrophage M2 polarization and subsequent anti-inflammatory responses but inhibited apoptosis and ferroptosis in lung tissue. Our data suggested that targeting Piezo1 in macrophages could be a promising therapeutic strategy for treating MA-ALI.

PnPP-15, a Synthetic Peptide Derived from a Toxin from Phoneutria nigriventer Spider Venom, Alleviates Diabetic Neuropathic Pain and Acts Synergistically with Pregabalin in Mice.

Diabetic neuropathic pain is one of the complications that affect a wide variety of the diabetic population and is often difficult to treat. Only a small number of patients experience pain relief, which usually comes with onerous side effects and low levels of satisfaction. The search for new analgesic drugs is necessary, given the limitations that current drugs present. Combining drugs to treat neuropathic pain has been attracting interest to improve their efficacy compared to single-drug monotherapies while also reducing dose sizes to minimize side effects. The aim of our study was to verify the antinociceptive effect of a synthetic peptide, PnPP-15, alone and combined with pregabalin, in male Swiss diabetic mice using the von Frey method. PnPP-15 is a synthetic peptide derived from PnPP19, a peptide representing a discontinuous epitope of the primary structure of the toxin PnTx2-6 from the venom of the spider Phoneutria nigriventer. The antinociceptive activity of both compounds was dose-dependent and showed synergism, which was verified by isobolographic analysis. Treatment with PnPP-15 did not cause spontaneous or forced motor changes and did not cause any damage or signs of toxicity in the analyzed organs (pancreas, lung, heart, kidney, brain, or liver). In conclusion, PnPP-15 is a great candidate for an analgesic drug against neuropathic pain caused by diabetes and exerts a synergistic effect when combined with pregabalin, allowing for even more efficient treatment.

Evolution in the choice of therapies used to treat latrodectism: Redback spider antivenom or standard analgesic medications. Nothing to rave about.

OBJECTIVES: Redback spider (RBS) antivenom (RBSAV) use appears to have decreased since the results of the RAVE-2 antivenom efficacy study were released. The aims of this study were to assess change in RBSAV use over time and compare responses to treatment for antivenom and other analgesics. METHODS: Retrospective audit of RBS bite referrals to a toxicology unit, from January 2010 to January 2022. Data included demographics, pain severity, treatment (analgesia or RBSAV), response to treatment, re-presentation rate, adverse events, change in antivenom use over time. RESULTS: Of 270 presentations, 157 with moderate or severe pain were included (RBSAV n = 51, analgesia n = 106). Median age was 39 years, n = 81 (51%) female. Those receiving antivenom were more likely to report severe pain n = 46/51 (84%) versus n = 68/106 (58%) (P = 0.006). Eighty-three percent of antivenom doses were administered between 2010 and 2013. Analgesia-only group received various combinations of paracetamol, NSAIDs, and opioids. In those receiving RBSAV, 17/48 (35%), 26/48 (54%), 5/48 (10%) reported a partial, complete or no reduction in pain, respectively, versus 30/77 (39%), 43/77 (58%) and 4/77 (5%), for analgesia-only group. Post-treatment pain was not recorded in three RBSAV and 28 analgesia-only patients. Pain reduction was no different for intravenous and intramuscular antivenom. Re-presentation for ongoing pain was more common in the analgesia-only group, 16/106 (15%) versus 1/51 (2%) for antivenom (P = 0.013). CONCLUSION: Antivenom use fell over the study period. There was no difference in pain relief between RBSAV and analgesia-only groups. RBSAV, regardless of route of administration, was no better than standard analgesics in pain reduction in the present study.

Gsmtx4 Alleviated Osteoarthritis through Piezo1/Calcineurin/NFAT1 Signaling Axis under Excessive Mechanical Strain.

Excessive mechanical strain is the prominent risk factor for osteoarthritis (OA), causing cartilage destruction and degeneration. However, the underlying molecular mechanism contributing to mechanical signaling transduction remains unclear in OA. Piezo type mechanosensitive ion channel component 1 (Piezo1) is a calcium-permeable mechanosensitive ion channel and provides mechanosensitivity to cells, but its role in OA development has not been determined. Herein, we found up-regulated expression of Piezo1 in OA cartilage, and that its activation contributes to chondrocyte apoptosis. The knockdown of Piezo1 could protect chondrocytes from apoptosis and maintain the catabolic and anabolic balance under mechanical strain. In vivo, Gsmtx4, a Piezo1 inhibitor, markedly ameliorated the progression of OA, inhibited the chondrocyte apoptosis, and accelerated the production of the cartilage matrix. Mechanistically, we observed the elevated activity of calcineurin (CaN) and the nuclear transfection of nuclear factor of activated T cells 1 (NFAT1) under mechanical strain in chondrocytes. Inhibitors of CaN or NFAT1 rescued the pathologic changes induced by mechanical strain in chondrocytes. Overall, our findings revealed that Piezo1 was the essential molecule response to mechanical signals and regulated apoptosis and cartilage matrix metabolism via the CaN/NFAT1 signaling axis in chondrocytes, and that Gsmtx4 could be an attractive therapeutic drug for OA treatment.

Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate.

Atrial fibrillation is the most common sustained cardiac arrhythmia in humans. Current atrial fibrillation antiarrhythmic drugs have limited efficacy and carry the risk of ventricular proarrhythmia. GsMTx4, a mechanosensitive channel-selective inhibitor, has been shown to suppress arrhythmias through the inhibition of stretch-activated channels (SACs) in the heart. The cost of synthesizing this peptide is a major obstacle to clinical use. Here, we studied two types of short peptides derived from GsMTx4 for their effects on a stretch-activated big potassium channel (SAKcaC) from the heart. Type I, a 17-residue peptide (referred to as Pept 01), showed comparable efficacy, whereas type II (i.e., Pept 02), a 10-residue peptide, exerted even more potent inhibitory efficacy on SAKcaC compared with GsMTx4. We identified through mutagenesis important sequences required for peptide functions. In addition, molecular dynamics simulations revealed common structural features with a hydrophobic head followed by a positively charged protrusion that may be involved in peptide channel-lipid interactions. Furthermore, we suggest that these short peptides may inhibit SAKcaC through a specific modification to the mechanogate, as the inhibitory effects for both types of peptides were mostly abolished when tested with a mechano-insensitive channel variant (STREX-del) and a nonmechanosensitive big potassium (mouse Slo1) channel. These findings may offer an opportunity for the development of a new class of drugs in the treatment of cardiac arrhythmia generated by excitatory SACs in the heart.

Systemic, Intrathecal, and Intracerebroventricular Antihyperalgesic Effects of the Calcium Channel Blocker CTK 01512-2 Toxin in Persistent Pain Models.

CTK 01512-2 toxin is a recombinant peptide of the Phα1ß version derived from the venom of the Phoneutria nigriventer spider. It acts as an N-type voltage-gated calcium channel (VGCC) blocker and shows a prolonged effect on preventing and reducing nociception. Herein, CTK 01512-2 was tested on two models of persistent pain, the chronic post-ischemia pain (CPIP) and the paclitaxel-induced peripheral neuropathy, to evaluate its systemic, intrathecal, and intracerebroventricular effects on mechanical hypersensitivity and thermal allodynia. Glial cell viability was also investigated using the MTT test. The results showed that CTK 01512-2 intrathecal and systemic treatments reduced the mechanical hypersensitivity induced by CPIP, mainly between 1-4 h after its administration. Additionally, intrathecal treatment reduced the CPIP-induced thermal allodynia. In its turn, the intracerebroventricular treatment showed mechanical antihyperalgesic and thermal antiallodynic effects in the paclitaxel-induced peripheral neuropathy. These data reinforce the therapeutic potential of CTK 01512-2 to treat persistent pain conditions and offer a perspective to use the systemic route. Moreover, CTK 01512-2 increased the glial cell viability in the MTT reduction assay, and it may indicate a new approach to managing chronic pain. The results found in this study help to pave new perspectives of pain relief treatments to patients affected by chronic pain.

Different effects of GsMTx4 on nocturia associated with the circadian clock and Piezo1 expression in mice.

OBJECTIVES: Nocturia is a major problem in geriatric patients. Clock genes regulate circadian bladder function and Piezo type mechanosensitive ion channel component 1 (Piezo1) that senses bladder fullness. We utilized WT and Clock mutant (ClockΔ19/Δ19: nocturia phenotype) mice to determine if the effects of GsMTx4, a Piezo1 inhibitor, is dependent on circadian Piezo1 expression in the bladder. METHODS: We compared voiding behavior in mice after the administration of vehicle, low dose, or high dose of GsMTx4. Intraperitoneal injections (IP) were performed at Zeitgeber time (ZT) 0, lower Piezo1 expression phase (ZT0-IP) and ZT12, higher Piezo1 expression phase (ZT12-IP). Urine volume (Uvol), voiding frequency (VF), and urine volume per void (Uvol/v) were measured using metabolic cages. RESULTS: VF decreased at ZT12-IP in WT mice only with high dose of GsMTx4 but showed no effects in ClockΔ19/Δ19 mice. VF decreased significantly at ZT0-IP in WT mice after both doses, but only decreased after high dose in ClockΔ19/Δ19 mice. Uvol/v increased in WT mice at ZT0-IP after both doses and at ZT12-IP after high dose. Uvol/v increased in ClockΔ19/Δ19 mice only at ZT0-IP after high dose. GsMTx4 did not affect Uvol in both mice at ZT12-IP. A decrease in Uvol was observed in both mice at ZT0-IP; however, it was unrelated to GsMTx4-IP. CONCLUSIONS: The effects of GsMTx4 changed associated with the circadian clock and Piezo1 expression level. The maximum effect occurred during sleep phase in WT. These results may lead to new therapeutic strategies against nocturia.

A spider-venom peptide with multitarget activity on sodium and calcium channels alleviates chronic visceral pain in a model of irritable bowel syndrome.

ABSTRACT: Chronic pain is a serious debilitating condition that affects ∼20% of the world's population. Currently available drugs fail to produce effective pain relief in many patients and have dose-limiting side effects. Several voltage-gated sodium (NaV) and calcium (CaV) channels are implicated in the etiology of chronic pain, particularly NaV1.1, NaV1.3, NaV1.7-NaV1.9, CaV2.2, and CaV3.2. Numerous NaV and CaV modulators have been described, but with few exceptions, they display poor potency and/or selectivity for pain-related channel subtypes. Here, we report the discovery and characterization of 2 novel tarantula-venom peptides (Tap1a and Tap2a) isolated from Theraphosa apophysis venom that modulate the activity of both NaV and CaV3 channels. Tap1a and Tap2a inhibited on-target NaV and CaV3 channels at nanomolar to micromolar concentrations and displayed moderate off-target selectivity for NaV1.6 and weak affinity for NaV1.4 and NaV1.5. The most potent inhibitor, Tap1a, nearly ablated neuronal mechanosensitivity in afferent fibers innervating the colon and the bladder, with in vivo intracolonic administration reversing colonic mechanical hypersensitivity in a mouse model of irritable bowel syndrome. These findings suggest that targeting a specific combination of NaV and CaV3 subtypes provides a novel route for treatment of chronic visceral pain.

Phα1ß, a dual blocker of TRPA1 and Cav2.2, as an adjuvant drug in opioid therapy for postoperative pain.

Opioids are the "gold standard" treatment for postoperative pain, but these drugs also have limiting adverse effects. Thus, adjuvant drugs might be useful in opioid therapy for postoperative pain. The aim of the present study was to evaluate the effect of Phα1ß, a dual blocker of Cav2 and TRPA1 channels, on antinociceptive and adverse actions of morphine in a model of postoperative pain. Phα1ß (100-300 pmol/site) or morphine (3-10 mg/kg), alone, largely reduced postoperative nociception. However, Phα1ß (100 pmol/site) or morphine (10 mg/kg) also produced motor impairment. Lower doses of Phα1ß (30 pmol/site) or morphine (1 mg/kg), that did not have an effect alone, showed antinociceptive effect when concomitantly administrated. Moreover, co-administration of Phα1ß (30 pmol/site) with morphine (1 or 10 mg/kg) was unable to cause motor impairment. Preoperative repeated treatment with morphine increased the expression of Cav2 and TRPA1 channels in spinal cord, and caused tolerance and withdrawal syndrome, which were reversed with a single injection of Phα1ß (30 pmol/site). When injected postoperatively, escalating doses of morphine worsened postoperative hyperalgesia, induced tolerance, and withdrawal syndrome. Similarly, Phα1ß (30 pmol/site) reversed these adverse effects. Single or repeated morphine caused constipation, which was not altered by Phα1ß. Thus, a low dose of Phα1ß potentiated the analgesia, and reversed some adverse effects of morphine on operated mice, indicating the potential use of this agent as an adjuvant drug in opioid therapy for postoperative pain.

Spider venom administration impairs glioblastoma growth and modulates immune response in a non-clinical model.

Molecules from animal venoms are promising candidates for the development of new drugs. Previous in vitro studies have shown that the venom of the spider Phoneutria nigriventer (PnV) is a potential source of antineoplastic components with activity in glioblastoma (GB) cell lines. In the present work, the effects of PnV on tumor development were established in vivo using a xenogeneic model. Human GB (NG97, the most responsive line in the previous study) cells were inoculated (s.c.) on the back of RAG-/- mice. PnV (100 µg/Kg) was administrated every 48 h (i.p.) for 14 days and several endpoints were evaluated: tumor growth and metabolism (by microPET/CT, using 18F-FDG), tumor weight and volume, histopathology, blood analysis, percentage and profile of macrophages, neutrophils and NK cells isolated from the spleen (by flow cytometry) and the presence of macrophages (Iba-1 positive) within/surrounding the tumor. The effect of venom was also evaluated on macrophages in vitro. Tumors from PnV-treated animals were smaller and did not uptake detectable amounts of 18F-FDG, compared to control (untreated). PnV-tumor was necrotic, lacking the histopathological characteristics typical of GB. Since in classic chemotherapies it is observed a decrease in immune response, methotrexate (MTX) was used only to compare the PnV effects on innate immune cells with a highly immunosuppressive antineoplastic drug. The venom increased monocytes, neutrophils and NK cells, and this effect was the opposite of that observed in the animals treated with MTX. PnV increased the number of macrophages in the tumor, while did not increase in the spleen, suggesting that PnV-activated macrophages were led preferentially to the tumor. Macrophages were activated in vitro by the venom, becoming more phagocytic; these results confirm that this cell is a target of PnV components. Spleen and in vitro PnV-activated macrophages were different of M1, since they did not produce pro- and anti-inflammatory cytokines. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. The identification, optimization and synthesis of antineoplastic drugs from PnV molecules may lead to a new multitarget chemotherapy. Glioblastoma is associated with high morbidity and mortality; therefore, research to develop new treatments has great social relevance. Natural products and their derivatives represent over one-third of all new molecular entities approved by FDA. However, arthropod venoms are underexploited, although they are a rich source of new molecules. A recent in vitro screening of the Phoneutria nigriventer spider venom (PnV) antitumor effects by our group has shown that the venom significantly affected glioblastoma cell lines. Therefore, it would be relevant to establish the effects of PnV on tumor development in vivo, considering the complex neoplastic microenvironment. The venom was effective at impairing tumor development in murine xenogeneic model, activating the innate immune response and increasing tumor infiltrating macrophages. In addition, PnV activated macrophages in vitro for a different profile of M1. These activated PnV-macrophages have potential to fight the tumor without promoting tumorigenesis. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. We aim to synthesize and carry out a formulation with these antineoplastic molecules for clinical trials. Spider venom biomolecules induced smaller and necrotic xenogeneic GB; spider venom activated the innate immune system; venom increased blood monocytes and the migration of macrophages to the tumor; activated PnV-macrophages have a profile different of M1 and have a potential to fight the tumor without promote tumorigenesis.

Spider Knottin Pharmacology at Voltage-Gated Sodium Channels and Their Potential to Modulate Pain Pathways.

Voltage-gated sodium channels (NaVs) are a key determinant of neuronal signalling. Neurotoxins from diverse taxa that selectively activate or inhibit NaV channels have helped unravel the role of NaV channels in diseases, including chronic pain. Spider venoms contain the most diverse array of inhibitor cystine knot (ICK) toxins (knottins). This review provides an overview on how spider knottins modulate NaV channels and describes the structural features and molecular determinants that influence their affinity and subtype selectivity. Genetic and functional evidence support a major involvement of NaV subtypes in various chronic pain conditions. The exquisite inhibitory properties of spider knottins over key NaV subtypes make them the best lead molecules for the development of novel analgesics to treat chronic pain.

Blockade of Acid-Sensing Ion Channels Attenuates Recurrent Hypoglycemia-Induced Potentiation of Ischemic Brain Damage in Treated Diabetic Rats.

Diabetes is a chronic metabolic disease and cerebral ischemia is a serious complication of diabetes. Anti-diabetic therapy mitigates this complication but increases the risk of exposure to recurrent hypoglycemia (RH). We showed previously that RH exposure increases ischemic brain damage in insulin-treated diabetic (ITD) rats. The present study evaluated the hypothesis that increased intra-ischemic acidosis in RH-exposed ITD rats leads to pronounced post-ischemic hypoperfusion via activation of acid-sensing (proton-gated) ion channels (ASICs). Streptozotocin-diabetic rats treated with insulin were considered ITD rats. ITD rats were exposed to RH for 5 days and were randomized into Psalmotoxin1 (PcTx1, ASIC1a inhibitor), APETx2 (ASIC3 inhibitor), or vehicle groups. Transient global cerebral ischemia was induced overnight after RH. Cerebral blood flow was measured using laser Doppler flowmetry. Ischemic brain injury in hippocampus was evaluated using histopathology. Post-ischemic hypoperfusion in RH-exposed rats was of greater extent than that in control rats. Inhibition of ASICs prevented RH-induced increase in the extent of post-ischemic hypoperfusion and ischemic brain injury. Since ASIC activation-induced store-operated calcium entry (SOCE) plays a role in vascular tone, next we tested if acidosis activates SOCE via activating ASICs in vascular smooth muscle cells (VSMCs). We observed that SOCE in VSMCs at lower pH is ASIC3 dependent. The results show the role of ASIC in post-ischemic hypoperfusion and increased ischemic damage in RH-exposed ITD rats. Understanding the pathways mediating exacerbated ischemic brain injury in RH-exposed ITD rats may help lower diabetic aggravation of ischemic brain damage.

Parawixin2 Protects Hippocampal Cells in Experimental Temporal Lobe Epilepsy.

Epilepsy is considered as one of the major disabling neuropathologies. Almost one third of adult patients with temporal lobe epilepsy (TLE) do not respond to current antiepileptic drugs (AEDs). Additionally, most AEDs do not have neuroprotective effects against the inherent neurodegenerative process underlying the hippocampal sclerosis on TLE. Dysfunctions in the GABAergic neurotransmission may contribute not only to the onset of epileptic activity but also constitute an important system for therapeutic approaches. Therefore, molecules that enhance GABA inhibitory effects could open novel avenues for the understanding of epileptic plasticity and for drug development. Parawixin2, a compound isolated from Parawixia bistriata spider venom, inhibits both GABA and glycine uptake and has an anticonvulsant effect against a wide range of chemoconvulsants. The neuroprotective potential of Parawixin2 was analyzed in a model of TLE induced by a long-lasting Status Epilepticus (SE), and its efficiency was compared to well-known neuroprotective drugs, such as riluzole and nipecotic acid. Neuroprotection was assessed through histological markers for cell density (Nissl), astrocytic reactivity (GFAP) and cell death labeling (TUNEL), which were performed 24 h and 72 h after SE. Parawixin2 treatment resulted in neuroprotective effects in a dose dependent manner at 24 h and 72 h after SE, as well as reduced reactive astrocytes and apoptotic cell death. Based on these findings, Parawixin2 has a great potential to be used as a tool for neuroscience research and as a probe to the development of novel GABAergic neuroprotective agents.

GpTx-1 and [Ala5 , Phe6 , Leu26 , Arg28 ]GpTx-1, two peptide NaV 1.7 inhibitors: analgesic and tolerance properties at the spinal level.

BACKGROUND AND PURPOSE: The voltage-gated sodium channel NaV 1.7 is considered a therapeutic target for pain treatment based on human genetic evidence. GpTx-1 and its potent analogue [Ala5 , Phe6 , Leu26 , Arg28 ]GpTx-1 (GpTx-1-71) were recently characterized as NaV 1.7 inhibitors in vitro. Furthermore, the present work was conducted to investigate the analgesic properties of these two peptides in different pain models after spinal administration. EXPERIMENTAL APPROACH: The antinociceptive activities of both GpTx-1 and GpTx-1-71 were investigated in mouse models of acute, visceral, inflammatory and neuropathic pain. Furthermore, the side effects of GpTx-1 and GpTx-1-71 were evaluated in rotarod, antinociceptive tolerance, acute hyperlocomotion and gastrointestinal transit tests. KEY RESULTS: The i.t. administration of both GpTx-1 and GpTx-1-71 dose-dependently produced powerful antinociception in the different pain models. This effect was attenuated by the opioid receptor antagonist naloxone, suggesting the involvement of the opioid system. In this study, repeated administration of these two_peptides produced spinal analgesia without a loss of potency over 8 days in mouse models of acute, inflammatory and neuropathic pain. Moreover, spinal administration of GpTx-1 and GpTx-1-71 did not induce significant effects on motor coordination, evoke acute hyperlocomotion or increase gastrointestinal transit time. CONCLUSIONS AND IMPLICATIONS: Our data indicate that the NaV 1.7 peptide inhibitors GpTx-1 and GpTx-1-71 produce powerful, nontolerance-forming analgesia in preclinical pain models, which might be dependent on the endogenous opioid system. In addition, at the spinal level, the limited side effects imply that these NaV 1.7 peptide inhibitors could be potentially developed as GpTx-1-based drugs for pain relief.

Acid Sensing Ion Channel 1a (ASIC1a) Mediates Activity-induced Pain by Modulation of Heteromeric ASIC Channel Kinetics.

Chronic muscle pain is acutely worsened by exercise. Acid sensing ion channels (ASIC) are heteromeric channels expressed in muscle sensory neurons that detect decreases in pH. We have previously shown ASIC3 is important in activity-induced hyperalgesia. However, ASICs form heteromers with ASIC1a being a key component in sensory neurons. Therefore, we studied the role of ASIC1a in mice using behavioral pharmacology and genetic deletion in a model of activity-induced hyperalgesia. We found ASIC1a-/- mice developed mechanical hyperalgesia similar to wild-type mice, but antagonism of ASIC1a, with psalmotoxin, prevented development of mechanical hyperalgesia in wild-type mice, but not in ASIC1a-/- mice. To explain this discrepancy, we then performed electrophysiology studies of ASICs and examined the effects of psalmotoxin on ASIC heteromers. We expressed ASIC1a, 2 and 3 heteromers or ASIC1 and 3 heteromers in CHO cells, and examined the effects of psalmotoxin on pH sensitivity. Psalmotoxin significantly altered the properties of ASIC hetomeric channels. Specifically, in ASIC1a/2/3 heteromers, psalmotoxin slowed the kinetics of desensitization, slowed the recovery from desensitization, and inhibited pH-dependent steady-state desensitization, but had no effect on pH-evoked current amplitudes. We found a different pattern in ASIC1a/3 heteromers. There was a significant leftward shift in the pH dose response of steady-state desensitization and decrease in pH-evoked current amplitudes. These results suggest that blockade of ASIC1a modulates the kinetics of heteromeric ASICs to prevent development of activity-induced hyperalgesia. These data suggest ASIC1a is a key subunit in heteromeric ASICs and may be a pharmacological target for treatment of musculoskeletal pain.

The NAv1.7 blocker protoxin II reduces burn injury-induced spinal nociceptive processing.

Controlling pain in burn-injured patients poses a major clinical challenge. Recent findings suggest that reducing the activity of the voltage-gated sodium channel Nav1.7 in primary sensory neurons could provide improved pain control in burn-injured patients. Here, we report that partial thickness scalding-type burn injury on the rat paw upregulates Nav1.7 expression in primary sensory neurons 3 h following injury. The injury also induces upregulation in phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB), a marker for nociceptive activation in primary sensory neurons. The upregulation in p-CREB occurs mainly in Nav1.7-immunopositive neurons and exhibits a peak at 5 min and, following a decline at 30 min, a gradual increase from 1 h post-injury. The Nav1.7 blocker protoxin II (ProTxII) or morphine injected intraperitoneally 15 min before or after the injury significantly reduces burn injury-induced spinal upregulation in phosphorylated serine 10 in histone H3 and phosphorylated extracellular signal-regulated kinase 1/2, which are both markers for spinal nociceptive processing. Further, ProTxII significantly reduces the frequency of spontaneous excitatory post-synaptic currents in spinal dorsal horn neurons following burn injury. Together, these findings indicate that using Nav1.7 blockers should be considered to control pain in burn injury. KEY MESSAGES: • Burn injury upregulates Nav1.7 expression in primary sensory neurons. • Burn injury results in increased activity of Nav1.7-expressing primary sensory neurons. • Inhibiting Nav1.7 by protoxin II reduces spinal nociceptive processing. • Nav1.7 represents a potential target to reduce pain in burn injury.

Disease Modifying Effects of the Spider Toxin Parawixin2 in the Experimental Epilepsy Model.

(1) Background: Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults. It is also the one with the highest percentage of drug-resistance to the current available anti-epileptic drugs (AED). Additionaly, most antiepileptic drugs are only able to control seizures in epileptogenesis, but do not decrease the hippocampal neurodegenerative process. TLE patients have a reduced population of interneuronal cells, which express Parvalbumin (PV) proteins. This reduction is directly linked to seizure frequency and severity in the chronic period of epilepsy. There is therefore a need to seek new therapies with a disease-modifying profile, and with efficient antiepileptic and neuroprotective properties. Parawixin2, a compound isolated from the venom of the spider Parawixia bistriata, has been shown to inhibit GABA transporters (GAT) and to have acute anticonvulsant effects in rats. (2) Methods: In this work, we studied the effects of Parawixin2 and Tiagabine (an FDA- approved GAT inhibitor), and compared these effects in a TLE model. Rats were subjected to lithium-pilocarpine TLE model and the main features were evaluated over a chronic period including: (a) spontaneous recurrent seizures (SRS), (b) neuronal loss, and (c) PV cell density in different regions of the hippocampus (CA1, CA3, DG and Hilus). (3) Results: Parawixin2 treatment reduced SRS frequency whereas Tiagabine did not. We also found a significant reduction in neuronal loss in CA3 and in the hilus regions of the hippocampus, in animals treated with Parawixin2. Noteworthy, Parawixin2 significantly reversed PV cell loss observed particularly in DG layers. (4) Conclusions: Parawixin2 exerts a promising neuroprotective and anti-epileptic effect and has potential as a novel agent in drug design.

The spider toxin Phα1ß recombinant possesses strong analgesic activity.

The native Phα1ß - a Voltage-Gated Calcium Channel (VGCC) blocker - and its Recombinant Version - were both tested in rodent pain models with an intraplantar injections of capsaicin or formalin, a chronic constriction injury, and melanoma cancer related pain. The formalin nociceptive behaviour in the neurogenic phase was not affected by the toxin pre-treatments, while in the inflammatory phase, Phα1ß and the Recombinant form caused a significant reduction. The nociception that was triggered by capsaicin, an agonist of the TRPV1 vanilloid receptor, was totally blocked by 100 pmol/site, i.t. of Phα1ß or the recombinant version. For the neuropathic pain that was induced by a chronic constriction injury of the sciatic nerve, Phα1ß and its Recombinant reduced the allodynia that was induced by the CCI procedure in the rats and the hypersensitivity lasted for 4 h. Fourteen days after the inoculation of the B16-F10 melanoma cells in the mice, a marked hyperalgesia was induced in the melanoma cancer pain model. Phα1ß and the Recombinant form reduced the hyperalgesia with a full reversion at 100 pmol/site i.t. The inhibitory effects of the nociception that was induced by native Phα1ß and the Recombinant in the studied pain models were not statistically different and they developed with no side effects.

Manifestaciones de neurotoxicidad en el envenenamiento por mordedura de araña «viuda negra» en edades pediátricas / Neurotoxic manifestations of black widow spider envenomation in paediatric patients

Introducción: El envenenamiento por mordedura de araña «viuda negra» (Latrodectus mactans) en niños se expresa clínicamente con neurotoxicidad. Objetivo: Identificar los signos y síntomas neurológicos característicos, en las diferentes edades pediátricas, la evolución y el tratamiento en pacientes atendidos por esta mordedura de en un hospital pediátrico de noroeste México. Material y métodos: Se revisaron 70 expedientes de niños hospitalizados entre 1978-2014; estableciéndose 2 grupos: grupo 1, de 33 lactantes y preescolares, y grupo 2, con 37 escolares y adolescentes. Las variables consideradas fueron: edad, género, lugar de procedencia, sitio del accidente, área corporal afectada, grado de envenenamiento, tratamiento, evolución clínica, diferencias estadísticas. Resultados: Predominó el género masculino, 61,4%; los lactantes menores de un año fueron un 14,2%. El 70% de los pacientes tuvieron el contacto con el arácnido dentro del domicilio; las áreas anatómicas más afectadas fueron miembros inferiores, cuello, tronco y abdomen; los signos y síntomas neurológicos más notables en el grupo 1 fueron: irritabilidad, llanto constante, naáseas, sialorrea, agitación, taquicardia, arritmias, incapacidad para caminar, espasmos musculares, parestesias, tetania, convulsiones, nistagmo. En el grupo 2 fueron: dolor local, cefalea, sialorrea, parestesias, sudoración profusa, ansiedad, debilidad muscular, espasmos musculares y temblor fino. La manifestación clínica autonómicas predominante en el grupo 1 fue sialorrea, p < 0,0001, y en el grupo 2, parestesias, p < 0,0001. El uso de faboterápicos en el tratamiento permitió mejor evolución, menor tiempo de hospitalización, no hubo mortalidad. Conclusiones: Los signos y los síntomas de la mordedura por araña «viuda negra» son predominantemente autonómicos; identificarlos permite el diagnóstico oportuno y tratamiento eficaz

Introduction: Envenomation by black widow spiders manifests clinically with signs of neurotoxicity in paediatric patients. Objective: Identify typical neurological signs and symptoms in paediatric patients of different ages, and describe treatment and outcomes in a paediatric hospital in northwest Mexico. Material and methods: We reviewed 70 clinical records of patients hospitalised due to black widow spider bite between 1978 and 2014. We divided the total into 2 groups: Group 1, infants and preschool children; and Group 2, school-age children and adolescents. The demographic variables were age, sex, birthplace, place where envenomation occurred, body part(s) affected, degree of envenomation according to signs and symptoms, treatment, clinical outcome, and statistical differences. Results: Boys accounted for 61.4% of all cases, and infants younger than one year old made up 14.2%. Most patients (70%) were bitten by the spider at home; the anatomical areas most frequently affected were the legs, neck, thorax, and abdomen. The neurological signs and symptoms displayed by Group 1 were irritability, constant crying, sialorrhoea, nausea, tachycardia, arrhythmias, fatigue when walking, agitation, muscle spasms paraesthesia, tetany, seizures, and nystagmus. Signs in Group 2 included localized pain, headache, sialorrhoea, paraesthesia, profuse sweating, anxiety, muscle weakness, muscle spasms, and fine tremor. The predominant autonomic sign in Group 1 was sialorrhoea (P<.0001) and in Group 2, paraesthesia (P<.0001). Patients who received Fab antivenom treatment displayed better outcomes and shorter hospital stays than those who did not. No deaths were reported. Conclusions: The neurological signs and symptoms caused by black widow spider bite are predominantly autonomic, and identifying them permits early diagnosis and more effective treatment

Analgesic Effects of GpTx-1, PF-04856264 and CNV1014802 in a Mouse Model of NaV1.7-Mediated Pain.

Loss-of-function mutations of Na(V)1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of Na(V)1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of Na(V)1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of Na(V)1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with Na(V)1.7 inhibitors and significantly reduced in Na(V)1.7(-/-) mice. To validate the use of the model for profiling Na(V)1.7 inhibitors, we determined the Na(V) selectivity and tested the efficacy of the reported Na(V)1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited Na(V)1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited Na(V)1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited Na(V) channels and was only effective in the OD1 model when delivered systemically. Our novel model of Na(V)1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of Na(V)1.7 inhibitors.