Molecular Aspects Involved in the Mechanisms of Bothrops jararaca Venom-Induced Hyperalgesia: Participation of NK1 Receptor and Glial Cells.

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

Environmental factors and their impact on chronic pain development and maintenance.

It is more than recognized and accepted that the environment affects the physiological responses of all living things, from bacteria to superior vertebrates, constituting an important factor in the evolution of all species. Environmental influences range from natural processes such as sunlight, seasons of the year, and rest to complex processes like stress and other mood disorders, infections, and air pollution, being all of them influenced by how each creature deals with them. In this chapter, it will be discussed how some of the environmental elements affect directly or indirectly neuropathic pain, a type of chronic pain caused by a lesion or disease of the somatosensory nervous system. For that, it was considered the edge of knowledge in translational research, thus including data from human and experimental animals as well as the applicability of such findings.

Molecular aspects involved in the mechanisms of Bothrops jararaca venom-induced Hyperalgesia: participation of NK1 Receptor and glial cells

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

Environmental factors and their impact on chronic pain development and maintenance

It is more than recognized and accepted that the environment affects the physiological responses of all living things, from bacteria to superior vertebrates, constituting an important factor in the evolution of all species. Environmental influences range from natural processes such as sunlight, seasons of the year, and rest to complex processes like stress and other mood disorders, infections, and air pollution, being all of them influenced by how each creature deals with them. In this chapter, it will be discussed how some of the environmental elements affect directly or indirectly neuropathic pain, a type of chronic pain caused by a lesion or disease of the somatosensory nervous system. For that, it was considered the edge of knowledge in translational research, thus including data from human and experimental animals as well as the applicability of such findings.

Formyl peptide receptors are involved in CTX-induced impairment of lymphocyte functions.

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA4. These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA4 is a key mediator of the immunosuppressive effects of CTX.

Wnt signaling is involved in crotalphine-induced analgesia in a rat model of neuropathic pain

The aberrant activation of Wnt/β-catenin and atypical Wnt/Ryk signaling pathways in the spinal cord is critical for the development and maintenance of neuropathic pain. Crotalphine is a structural analog to a peptide first identified in Crotalus durissus terrificus snake venom, which induces antinociception by activating kappa-opioid and CB2 cannabinoid receptors. Consistent with previous data, we showed that the protein levels of the canonical Wnt/β-catenin and the atypical Wnt/Ryk signaling pathways are increased in neuropathic rats. Importantly, the administration of crotalphine downregulates these protein levels, including its downstream cascades, such as TCF4 from the canonical pathway and NR2B glutamatergic receptor and Ca2+-dependent signals, via the Ryk receptor. The CB2 receptor antagonist, AM630, abolished the crotalphine-induced atypical Wnt/Ryk signaling pathway activation. However, the selective CB2 agonist affects both canonical and non-canonical Wnt signaling in the spinal cord. Next, we showed that crotalphine blocked hypersensitivity and significantly decreased the concentration of IL-1ɑ, IL-1β, IL-6, IL-10, IL-18, TNF-ɑ, MIP-1ɑ and MIP-2 induced by intrathecal injection of exogenous Wnt-3a agonist. Taken together, our findings show that crotalphine induces analgesia in a neuropathic pain model by down-regulating the canonical Wnt/β-catenin and the atypical Wnt/Ryk signaling pathways and, consequently controlling neuroinflammation. This effect is, at least in part, mediated by CB2 receptor activation. These results open a perspective for new approaches that can be used to target Wnt signaling in the context of chronic pain.

Formyl peptide receptors are involved in CTX-induced impairment of lymphocyte functions

Crotoxin (CTX) is a neurotoxin that is isolated from the venom of Crotalus durissus terrificus, which displays immunomodulatory, anti-inflammatory, and anti-tumoral effects. Previous research has demonstrated that CTX promotes the adherence of leukocytes to the endothelial cells in blood microcirculation and the high endothelial venules of lymph nodes, which reduces the number of blood cells and lymphocytes. Studies have also shown that these effects are mediated by lipoxygenase-derived mediators. However, the exact lipoxygenase-derived eicosanoid involved in the CTX effect on lymphocytes is yet to be characterized. As CTX stimulates lipoxin-derived mediators from macrophages and lymphocyte effector functions could be modulated by activating formyl peptide receptors, we aimed to investigate whether these receptors were involved in CTX-induced redistribution and functions of lymphocytes in rats. We used male Wistar rats treated with CTX to demonstrate that Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe), an antagonist of formyl peptide receptors, prevented CTX-induced decrease in the number of circulating lymphocytes and increased the expression of the lymphocyte adhesion molecule LFA1. CTX reduced the T and B lymphocyte functions, such as lymphocyte proliferation in response to the mitogen Concanavalin A and antibody production in response to BSA immunization, respectively, which was prevented by the administration of Boc2. Importantly, mesenteric lymph node lymphocytes from CTX-treated rats showed an increased release of 15-epi-LXA4. These results indicate that formyl peptide receptors mediate CTX-induced redistribution of lymphocytes and that 15-epi-LXA4 is a key mediator of the immunosuppressive effects of CTX.

PKCζ-Mitogen-activated protein kinase signaling mediates crotalphine-Induced antinociception / A sinalização da proteína quinase ativada por PKCζ-mitógeno medeia a antinocicepção induzida por crotalfina

Crotalphine (CRP) is a structural analogue to a peptide that was first identified in the crude venom from the South American rattlesnake Crotalus durissus terrificus. This peptide induces a potent and long-lasting antinociceptive effect that is mediated by the activation of peripheral opioid receptors. The opioid receptor activation regulates a variety of intracellular signaling, including the mitogen-activated protein kinase (MAPK) pathway. Using primary cultures of sensory neurons, it was demonstrated that crotalphine increases the level of activated ERK1/2 and JNK-MAPKs and this increase is dependent on the activation of protein kinase Cζ (PKCζ). However, whether PKCζ-MAPK signaling is critical for crotalphine-induced antinociception is unknown. Here, we biochemically demonstrated that the systemic crotalphine activates ERK1/2 and JNK and decreases the phosphorylation of p38 in the lumbar spinal cord. The in vivo pharmacological inhibition of spinal ERK1/2 and JNK, but not of p38, blocks the antinociceptive effect of crotalphine. Of interest, the administration of a PKCζ pseudosubstrate (PKCζ inhibitor) prevents crotalphine-induced ERK activation in the spinal cord, followed by the abolishment of crotalphine-induced analgesia. Together, our results demonstrate that the PKCζ-ERK signaling pathway is involved in crotalphine-induced analgesia. Our study opens a perspective for the PKCζ-MAPK axis as a target for pain control.

Activation of PKCε-ALDH2 axis prevents 4-HNE-induced pain in mice / A ativação do eixo PKCε-ALDH2 previne a dor induzida por 4-HNE em camundongos

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

Activation of PKCε-ALDH2 axis prevents 4-HNE-induced pain in mice

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

Mitogen-Activated Protein Kinase Signaling Mediates Morphine Induced-Delayed Hyperalgesia.

The use of morphine, the standard opioid drug, is limited by its undesirable effects, such as tolerance, physical dependence, and hyperalgesia (increased pain sensitivity). Clinical and preclinical studies have reported development of hyperalgesia after prolonged opioid administration or after a single dose of intrathecal (i.t.) morphine in uninjured rats. However, whether a single standard systemic morphine dose is sufficient to decrease the nociceptive threshold in rats is unknown. Here, we showed that a single morphine subcutaneous injection induces analgesia followed by a long-lasting delayed hyperalgesia in uninjured and PGE2 sensitized rats. The i.t injection of extracellular signal-regulated kinase (ERK) inhibitor blocked morphine-induced analgesia, without interfering with the morphine-induced hyperalgesia. However, i.t. injection of SB20358, a p38 inhibitor and SP660125, a JNK inhibitor, decreased the morphine-induced hyperalgesia. Consistently with the behavioral data, Western Blot analysis showed that ERK is more phosphorylated 1 h after morphine, i.e., when the analgesia is detected. Moreover, phospho-p38 and phospho-JNK levels are upregulated 96 h after morphine injection, time that coincides with the hyperalgesic effect. Intrathecal (i.t.) oligodeoxynucleotide (ODN) antisense to cAMP-responsive element binding protein (CREB) attenuated morphine-induced hyperalgesia. Real-time polymerase chain reaction (RT-PCR) analysis showed that CREB downstream genes expressions were significantly up-regulated 96 h after morphine injection in spinal cord. Together, our data suggest that central ERK is involved in the analgesic and hyperalgesic effects of morphine while JNK, p38, and CREB are involved in the morphine-induced delayed hyperalgesia.

Mitogen-activated protein kinase signaling mediates morphine induced-delayed hyperalgesia

The use of morphine, the standard opioid drug, is limited by its undesirable effects, such as tolerance, physical dependence, and hyperalgesia (increased pain sensitivity). Clinical and preclinical studies have reported development of hyperalgesia after prolonged opioid administration or after a single dose of intrathecal (i.t.) morphine in uninjured rats. However, whether a single standard systemic morphine dose is sufficient to decrease the nociceptive threshold in rats is unknown. Here, we showed that a single morphine subcutaneous injection induces analgesia followed by a long-lasting delayed hyperalgesia in uninjured and PGE2 sensitized rats. The i.t injection of extracellular signal-regulated kinase (ERK) inhibitor blocked morphine-induced analgesia, without interfering with the morphine-induced hyperalgesia. However, i.t. injection of SB20358, a p38 inhibitor and SP660125, a JNK inhibitor, decreased the morphine-induced hyperalgesia. Consistently with the behavioral data, Western Blot analysis showed that ERK is more phosphorylated 1 h after morphine, i.e., when the analgesia is detected. Moreover, phospho-p38 and phospho-JNK levels are upregulated 96 h after morphine injection, time that coincides with the hyperalgesic effect. Intrathecal (i.t.) oligodeoxynucleotide (ODN) antisense to cAMP-responsive element binding protein (CREB) attenuated morphine-induced hyperalgesia. Real-time polymerase chain reaction (RT-PCR) analysis showed that CREB downstream genes expressions were significantly up-regulated 96 h after morphine injection in spinal cord. Together, our data suggest that central ERK is involved in the analgesic and hyperalgesic effects of morphine while JNK, p38, and CREB are involved in the morphine-induced delayed hyperalgesia.

Mitogen-activated protein kinase signaling mediates morphine induced-delayed hyperalgesia

The use of morphine, the standard opioid drug, is limited by its undesirable effects, such as tolerance, physical dependence, and hyperalgesia (increased pain sensitivity). Clinical and preclinical studies have reported development of hyperalgesia after prolonged opioid administration or after a single dose of intrathecal (i.t.) morphine in uninjured rats. However, whether a single standard systemic morphine dose is sufficient to decrease the nociceptive threshold in rats is unknown. Here, we showed that a single morphine subcutaneous injection induces analgesia followed by a long-lasting delayed hyperalgesia in uninjured and PGE2 sensitized rats. The i.t injection of extracellular signal-regulated kinase (ERK) inhibitor blocked morphine-induced analgesia, without interfering with the morphine-induced hyperalgesia. However, i.t. injection of SB20358, a p38 inhibitor and SP660125, a JNK inhibitor, decreased the morphine-induced hyperalgesia. Consistently with the behavioral data, Western Blot analysis showed that ERK is more phosphorylated 1 h after morphine, i.e., when the analgesia is detected. Moreover, phospho-p38 and phospho-JNK levels are upregulated 96 h after morphine injection, time that coincides with the hyperalgesic effect. Intrathecal (i.t.) oligodeoxynucleotide (ODN) antisense to cAMP-responsive element binding protein (CREB) attenuated morphine-induced hyperalgesia. Real-time polymerase chain reaction (RT-PCR) analysis showed that CREB downstream genes expressions were significantly up-regulated 96 h after morphine injection in spinal cord. Together, our data suggest that central ERK is involved in the analgesic and hyperalgesic effects of morphine while JNK, p38, and CREB are involved in the morphine-induced delayed hyperalgesia.