Introduction 3. Opioid receptor signaling 3.1. Receptor phosphorylation by protein kinase C 3.2. ... more Introduction 3. Opioid receptor signaling 3.1. Receptor phosphorylation by protein kinase C 3.2. Modulation of receptor internalization by protein kinase C 3.3. PKC hypothesis for morphine tolerance/ desensitization 3.4. Heterologous regulation of opioid receptor endocytosis 4. Plasticity in neuronal networks 4.1. Anti-opioid glutamate-NMDA receptor system 4.2. BDNF system that supports the anti-opioid NMDA receptor system 4.3. Other systems that support the anti-opioid NMDA receptor system 4.4. Anti-opioid neuropeptide systems 4.5 Opioid-induced hyperalgesia by neuropeptides 4.6 Glial cell involvements in the anti-opioid system 5. Conclusion 6. Acknowledgments 7. References
Journal of Pharmacology and Experimental Therapeutics, 2004
The retinal ischemic-reperfusion stress (130 mmHg, 45 min) caused neuronal damages throughout all... more The retinal ischemic-reperfusion stress (130 mmHg, 45 min) caused neuronal damages throughout all cell layers and reduced the thickness of retinal layer by 30% at 7days after the stress of mouse retina. The intravitreous injection of 100 pmol of nefiracetam, a cognition-enhancer, completely prevented the damages when it was given 30 min prior to and 3 h after the stress. Partial prevention was observed when it was given 24 h after the stress, or low dose (10 pmol) nefiracetam was given 30 min prior to the stress. However, aniracetam had no effect. In the retinal cell line N18-RE-105, the ischemic-reperfusion stress by 2 h culture under the serum-free condition with low oxygen (less of 0.4% O 2) and low glucose (1 mM), caused necrosis or apoptosis in the low-density (0.5x10 4 cell/cm 2) or high-density (5x10 4 cell/cm 2) culture, respectively. The necrosis showed membrane disruption, loss of electron density, and mitochondrial swelling, while apoptosis showed nuclear fragmentation and condensation in transmission electron microscopical analyses and in experiments using specific cell death markers. Nefiracetam inhibited both necrosis and apoptosis, while brain-derived neurotrophic factor (BDNF) inhibited only apoptosis. The cell-protective actions of nefiracetam were abolished by nifedipine and ω-conotoxin GVIA, L-type and N-type calcium channel blocker, but not by PD98059 or wortmannin, ERK1/2 or PI 3-kinase inhibitor, respectively, while those of BDNF were abolished by PD98059 and wortmannin, but not by nifedipine and ω-conotoxin GVIA. All these findings suggest that nefiracetam inhibit necrosis and apoptosis occurred in the ischemic/hypoxic neuronal injury through an increase in Ca 2+-influx.
Cortical neurons rapidly die in necrosis due to poor glucose uptake in the low-density (LD) cultu... more Cortical neurons rapidly die in necrosis due to poor glucose uptake in the low-density (LD) culture under serum-free condition without any supplements. The scanning and transmission electron microscopical analyses characterized the necrosis by membrane disruption, mitochondrial swelling and loss of cytoplasmic electron density. High-glucose treatment delayed the neuronal death by suppressing necrosis, but induced apoptosis through increase in Bax levels, cytochrome c release, caspase-3 activation and DNA ladder formation. Although pyruvate as well as high glucose inhibited necrotic cell death and rapid decrease in cellular ATP levels, possibly related to decreased [ 3 H]-2-deoxy glucose uptake under the serum-free condition, it did not induce apoptosis. Protein kinase C inhibitors blocked these changes related to the cell death mode switch. Several neurotrophic factors did not affect the necrosis, but potentiated high-glucose-induced survival activity, while inhibiting cytochrome c release. All these results suggest that high-glucose treatment causes neuronal cell death mode switch by inhibiting necrosis, while inducing apoptosis, which is prevented by neurotrophic factors.
Morphine is now said to have no problematic side effects such as analgesic tolerance and physical... more Morphine is now said to have no problematic side effects such as analgesic tolerance and physical dependence for cancer pain patients in clinic, as far as it is appropriately used. However, sub-sensitivity to morphine might be developed when higher doses of morphine are used for terminal cancer pain patients. Along with the severity of cancer, the nature of pain becomes changed to neuropathic pain, which is resistant to morphine or NSAIDS. In order to safely use morphine in the clinic, we need to know how morphine tolerance and neuropathic pain are developed and what adjuvants could be used to completely suppress the pain. Here I overview the proposed mechanisms for morphine tolerance and neuropathic pain in relation to the availability of analgesic adjuvants.
Journal of Pharmacology and Experimental Therapeutics, 2020
Treatment for fibromyalgia is an unmet medical need; however, its pathogenesis is still poorly un... more Treatment for fibromyalgia is an unmet medical need; however, its pathogenesis is still poorly understood. In a series of studies, we have demonstrated that some pharmacological treatments reverse generalized chronic pain, but do not affect the lack of morphine analgesia in the intermittent cold stress (ICS)-induced fibromyalgia-like pain model in mice. Here we report that repeated intraperitoneal treatments with mirtazapine, which is presumed to disinhibit 5-HT release and activate 5-HT1 receptor through mechanisms of blocking presynaptic adrenergic α2, postsynaptic 5-HT2 and 5-HT3 receptors, completely reversed the chronic pain for more than 4-5 days after the cessation of treatments. The repeated mirtazapine-treatments also recovered the morphine analgesia after the return of nociceptive threshold to the normal level. The microinjection of siRNA adrenergic α2a receptor (ADRA2A) into the habenula, which showed a selective upregulation of α2 receptor gene expression after ICS, reversed the hyperalgesia, but did not recover the morphine analgesia. However, both reversal of hyperalgesia and recovery of morphine analgesia were observed when siRNA ADRA2A was administered intracerebroventricularly. As the habenular is reported to be involved in the emotion/reward-related pain and hypoalgesia, these results suggest that mirtazapine could attenuate pain and/or augment hypoalgesia by blocking the habenular α2 receptor after ICS. The recovery of morphine analgesia in the ICS model, on the other hand, seems to be mediated through a blockade of α2 receptor in unidentified brain regions.
In the present study, we first report an in vivo characterization of flexor responses induced by ... more In the present study, we first report an in vivo characterization of flexor responses induced by three distinct sine-wave stimuli in the electrical stimulation-induced paw flexion (EPF) test in mice. The fixed sine-wave electric stimulations of 5 Hz (C-fiber), 250 Hz (Adelta-fiber) and 2000 Hz (Abeta-fiber) to the hind paw of mice induced a paw-flexion response and vocalization. The average threshold for paw flexor responses by sine-wave stimulations was much lower than that for vocalization. Neonatally (P3) pretreatment with capsaicin to degenerate polymodal substance P-ergic C-fiber neurons increased the threshold to 5 Hz (C-fiber) stimuli, but not to 250 Hz (Adelta-fiber) and 2000 Hz (Abeta-fiber). The flexor responses to 5 Hz stimuli were significantly blocked by intrathecal (i.t.) pretreatment with both CP-99994 and MK-801, an NK1 and NMDA receptor antagonist, respectively, but not by CNQX, an AMPA/kainate receptor antagonist. On the other hand, the flexor responses induced by ...
Peripheral nerve injury causes neuropathic pain, which is characterized by the paradoxical sensat... more Peripheral nerve injury causes neuropathic pain, which is characterized by the paradoxical sensations of positive and negative symptoms. Clinically, negative signs are frequently observed; however, their underlying molecular mechanisms are largely unknown. Dysfunction of C-fibers is assumed to underlie negative symptoms and is accompanied by long-lasting downregulation of Nav1.8 sodium channel and μ-opioid receptor (MOP) in the dorsal root ganglion (DRG). In the present study, we found that nerve injury upregulates neuron-restrictive silencer factor (NRSF) expression in the DRG neurons mediated through epigenetic mechanisms. In addition, chromatin immunoprecipitation analysis revealed that nerve injury promotes NRSF binding to the neuron-restrictive silencer element within MOP and Nav1.8 genes, thereby causing epigenetic silencing. Furthermore, NRSF knockdown significantly blocked nerve injury-induced downregulations of MOP and Nav1.8 gene expressions, C-fiber hypoesthesia, and the ...
Proceedings of the National Academy of Sciences, 1998
We have studied the in vivo signaling mechanisms involved in nociceptin/orphanin FQ (Noci)-induce... more We have studied the in vivo signaling mechanisms involved in nociceptin/orphanin FQ (Noci)-induced pain responses by using a flexor-reflex paradigm. Noci was 10,000 times more potent than substance P (SP) in eliciting flexor responses after intraplantar injection into the hind limb of mice, but the action of Noci seems to be mediated by SP. Mice pretreated with an NK1 tachykinin receptor antagonist or capsaicin, or mice with a targeted disruption of the tachykinin 1 gene no longer respond to Noci. The action of Noci appears to be mediated by the Noci receptor, a pertussis toxin-sensitive G protein–coupled receptor that stimulates inositol trisphosphate receptor and Ca 2+ influx. These findings suggest that Noci indirectly stimulates nerve endings of nociceptive primary afferent neurons through a local SP release.
Lysophosphatidic acid receptor (LPA 1) signaling initiates neuropathic pain and several pathologi... more Lysophosphatidic acid receptor (LPA 1) signaling initiates neuropathic pain and several pathological events in a partial sciatic nerve injury model. Recently, we reported that lysophosphatidic acid (LPA) induces neuropathic pain as well as demyelination and pain-related protein expression changes via LPA 1 receptor signaling. Lysophosphatidylcholine (LPC), also known as lysolecithin, which is hydrolyzed by autotaxin/ATX into LPA, induces similar plastic changes. Here, we attempted to clarify whether ATX and LPA 1 receptor signaling is involved in the LPC-induced neuropathic pain. In wild-type mice, a single intrathecal (i.t.) injection of LPC induced mechanical allodynia and thermal hyperalgesia 2 days after injection; this persisted for 7 days at least. On the other hand, LPC-induced mechanical allodynia and thermal hyperalgesia were completely abolished in mice lacking an LPA 1 receptor gene. Furthermore, the LPC-induced response was also significantly, but partially reduced in heterozygous mutant mice for the ATX gene. These findings suggest that intrathecally-injected LPC is converted to LPA by ATX, and this LPA activates the LPA 1 receptor to initiate neuropathic pain.
Background: We have previously demonstrated that different spinal transmissions are involved in t... more Background: We have previously demonstrated that different spinal transmissions are involved in the nociceptive behavior caused by electrical stimulation of Aβ-, Aδ- or C-fibers using a Neurometer® in naïve mice. In this study, we attempted to pharmacologically characterize the alteration in spinal transmission induced by partial sciatic nerve injury in terms of nociceptive behavior and phosphorylation of extracellular signal-regulated kinase (pERK) in the spinal dorsal horn. Results: Aβ-fiber responses (2000-Hz), which were selectively blocked by the AMPA/kainate antagonist CNQX in naïve mice, were hypersensitized but blocked by the NMDA receptor antagonists MK-801 and AP-5 in injured mice in an electrical stimulation-induced paw withdrawal (EPW) test. Although Aδ-fiber responses (250-Hz) were also hypersensitized by nerve injury, there was no change in the pharmacological characteristics of Aδ-fiber responses through NMDA receptors. On the contrary, C-fiber responses (5-Hz) were h...
Background: We have proposed that nerve injury-specific loss of spinal tonic cholinergic inhibiti... more Background: We have proposed that nerve injury-specific loss of spinal tonic cholinergic inhibition may play a role in the analgesic effects of nicotinic acetylcholine receptor (nAChR) agonists on neuropathic pain. However, the tonic cholinergic inhibition of pain remains to be well characterized. Results: Here, we show that choline acetyltransferase (ChAT) signals were localized not only in outer dorsal horn fibers (lamina I–III) and motor neurons in the spinal cord, but also in the vast majority of neurons in the dorsal root ganglion (DRG). When mice were treated with an antisense oligodeoxynucleotide (AS-ODN) against ChAT, which decreased ChAT signals in the dorsal horn and DRG, but not in motor neurons, they showed a significant decrease in nociceptive thresholds in paw pressure and thermal paw withdrawal tests. Furthermore, in a novel electrical stimulation-induced paw withdrawal (EPW) test, the thresholds for stimulation through C-, Aδ- and Aβ-fibers were all decreased by AS-O...
Background: Cyclic phosphatidic acid (cPA) is a structural analog of lysophosphatidic acid (LPA),... more Background: Cyclic phosphatidic acid (cPA) is a structural analog of lysophosphatidic acid (LPA), but possesses different biological functions, such as the inhibition of autotaxin (ATX), an LPA-synthesizing enzyme. As LPA is a signaling molecule involved in nociception in the peripheral and central systems, cPA is expected to possess analgesic activity. We characterized the effects of cPA and 2-carba-cPA (2ccPA), a chemically stable cPA analog, on acute and chronic pain. Results: (1) The systemic injection of 2ccPA significantly inhibited somato-cardiac and somato-somatic C-reflexes but not the corresponding A-reflexes in anesthetized rats. (2) 2ccPA reduced sensitivity measured as the paw withdrawal response to electrical stimulation applied to the hind paws of mice through the C-fiber, but not Aδ or Aβ. (3) In mice, pretreatment with 2ccPA dose-dependently inhibited the second phase of formalin-induced licking and biting responses. (4) In mice, pretreatment and repeated post-treat...
Lysophosphatidic acid receptor subtype LPA1 is crucial for the initiation of neuropathic pain and... more Lysophosphatidic acid receptor subtype LPA1 is crucial for the initiation of neuropathic pain and underlying changes, such as up-regulation of Ca2+ channel α2δ-1 subunit in dorsal root ganglia (DRG), up-regulation of PKCγ in the spinal dorsal horn, and demyelination of dorsal root fibers. In the present study, we further examined the involvement of LPA1 signaling in the reorganization of Aβ-fiber-mediated spinal transmission, which is presumed to underlie neuropathic allodynia. Following nerve injury, the phosphorylation of extracellular-signal regulated kinase (pERK) by Aβ-fiber stimulation was observed in the superficial layer of spinal dorsal horn, where nociceptive C-or Aδ-fibers are innervated, but not in sham-operated wild-type mice. However, the pERK signals were largely abolished in LPA1 receptor knock-out ( Lpar1-/-) mice, further supported by quantitative analyses of pERK-positive cells. These results suggest that LPA1 receptor-mediated signaling mechanisms also participat...
Journal of Pharmacology and Experimental Therapeutics, 2003
Topical capsaicin is believed to alleviate pain by desensitizing the vanilloid receptor 1 (VR1) a... more Topical capsaicin is believed to alleviate pain by desensitizing the vanilloid receptor 1 (VR1) at the peripheral nerve endings. Here, we report that an upregulation of VR1 expression on myelinated fibers contributes to the antihyperalgesic effect of capsaicin cream in streptozotocin (STZ)-induced diabetic neuropathic pain. Intravenous injection of STZ (200 mg/kg) in mice caused rapid onset of diabetes within 24 h. Thermal and mechanical hyperalgesia developed by 3 days after STZ injection and persisted at all time points tested until 28 days. There was also hyperalgesic response to intraplantar (i.pl.) prostaglandin I 2 (PGI 2) agonist-induced nociception in such mice. Application of capsaicin cream dose-dependently reversed the thermal, mechanical and PGI 2 agonist-induced hyperalgesia observed in the diabetic mice. The i.pl. injection of capsaicin solution (0.4 µg/20µl) produced nociceptive biting-licking responses in control mice, and these responses were significantly increased in STZ-induced diabetic mice. After neonatal capsaicin-treatment, which destroys most unmyelinated C-fibers, the i.pl. capsaicin-induced biting-licking responses were almost abolished. However, in neonatal capsaicin-treated diabetic mice, the i.pl. capsaicin-induced biting-licking responses reappeared. The i.pl. capsaicin-induced biting-licking responses were blocked by the competitive VR1 antagonist capsazepine. All these results suggest an increase in capsaicin receptor on myelinated fibers due to diabetes. Finally, we confirmed the upregulation of VR1 expression on myelinated primary afferent neurons of diabetic mice by immunohistochemistry. Altogether our results suggest that increased expression of VR1 on myelinated fibers might contribute to the antihyperalgesic effect of topical capsaicin in diabetic neuropathic pain.
Journal of Pharmacology and Experimental Therapeutics, 2007
Using novel apparatus, KUROBOX, learning and memory behaviors as well as various parameters of mo... more Using novel apparatus, KUROBOX, learning and memory behaviors as well as various parameters of movement activity were re-evaluated in mice deficient for nociceptin/orphanin FQ receptor (NOP-/mice) or µ-opioid receptor (MOP-/mice). This method has the advantages that no handling procedures are required throughout the experiments performed over 3 days; positive cue paradigms are used without water or shock stress; and the method does not disturb the nocturnal habit of mice. NOP-/mice displayed a significant enhancement of learning and memory under stress-free conditions, but there were no changes in the various physical and psychological parameters of movement activity (nest stay ratio, distance moved, speed and angle in the movement) and biological rhythm that were measured. Enhancement of nocturnal learning was observed during the first 12 h dark cycle, and enhancement of memory was observed at the beginning of the second dark cycle, in NOP-/mice. By contrast, MOP-/mice showed no significant change in learning and memory behaviors or in physical and psychological parameters of movement activity, except for speed: MOP-/mice showed a significant decrease in speed of movement. Thus, the KUROBOX apparatus provides a useful alternative method to evaluate learning and memory activity under the more physiological conditions. In addition, this apparatus has an advantage that various physical and psychological parameters of movement activity affecting learning and memory behavior are also evaluated at the same time.
Fibromyalgia (FM) is a generalized chronic pain condition whose pathophysiology is poorly underst... more Fibromyalgia (FM) is a generalized chronic pain condition whose pathophysiology is poorly understood, and both basic and translational research are needed to advance the field. Here we used the Sluka model to test whether FM-like pain in mice would produce detectable brain modifications using resting-state (rs) functional Magnetic Resonance Imaging (fMRI). Mice received intramuscular acid saline treatment, images were acquired at 7T 5 days post-treatment, and pain thresholds tested 3 weeks post-scanning. Data-driven Independent Component Analysis revealed significant reduction of functional connectivity (FC) across several component pairs, with major changes for the Retrosplenial cortex (RSP) central to the default mode network, and to a lesser extent the Periaqueductal gray (PAG), a key pain processing area. Seed-to-seed analysis focused on 14 pain-related areas showed strongest FC reduction for RSP with several cortical areas (somatosensory, prefrontal and insular), and for PAG with both cortical (somatosensory) and subcortical (habenula, thalamus, parabrachial nucleus) areas. RSP-PAG FC was also reduced, and this decreased FC tended to be positively correlated with pain levels at individual subject level. Finally, seed-voxelwise analysis focused on PAG confirmed seed-to-seed findings and, also detected reduced PAG FC with the anterior cingulate cortex, increasingly studied in aversive pain effects. In conclusion, FM-like pain triggers FC alterations in the mouse, which are detected by rs-fMRI and are reminiscent of some human findings. The study reveals the causal fingerprint
Lysophosphatidic acid (LPA, 1-acyl-sn-glycerol-3-phosphate) is a well-known lipid growth factor t... more Lysophosphatidic acid (LPA, 1-acyl-sn-glycerol-3-phosphate) is a well-known lipid growth factor that is found widely in various tissues including brain and is reported to drive different intracellular signaling pathways. In the nervous system, LPA studies have drawn many neuroscientists’ attention because it has some actions related to neurogenesis such as cell rounding and proliferation. Remarkable advances in this field have been obtained along with the discovery of the cDNA clone for its receptor, vzg1 /edg2, a member of the seven transmembrane-type edg family. Successive studies have revealed that edg2 activation by LPA mediates several neurobiological actions related to neurogenesis, neuronal excitability and survival activity on developing and postnatal neurons. Here we focused their molecular basis of signaling through G proteins and in vivo roles of edg2 in such neurobiological events.
Lysophosphatidic acid (LPA) signaling is known to play key roles in the initiation and maintenanc... more Lysophosphatidic acid (LPA) signaling is known to play key roles in the initiation and maintenance of various chronic pain models. Here we examined whether LPA signaling is also involved in diabetes-induced abnormal pain behaviors. The high-fat diet (HFD) showing elevation of blood glucose levels and body weight caused thermal, mechanical hyperalgesia, hypersensitivity to 2000 or 250 Hz electrical-stimulation and hyposensitivity to 5 Hz stimulation to the paw in wild-type (WT) mice. These HFD-induced abnormal pain behaviors and body weight increase, but not elevated glucose levels were abolished in LPA1−/− and LPA3−/− mice. Repeated daily intrathecal (i.t.) treatments with LPA1/3 antagonist AM966 reversed these abnormal pain behaviors. Similar abnormal pain behaviors and their blockade by daily AM966 (i.t.) or twice daily Ki16425, another LPA1/3 antagonist was also observed in db/db mice which show high glucose levels and body weight. Furthermore, streptozotocin-induced similar abno...
Journal of Pharmacology and Experimental Therapeutics, 2020
We have developed an experimental fibromyalgia (FM)-like mouse model using intermittent cold stre... more We have developed an experimental fibromyalgia (FM)-like mouse model using intermittent cold stress (ICS), where chronic pain is generalized, female-predominant and abolished in type 1 lysophosphatidic acid receptor-KO (LPA1-/-) mice, but is not reversed by systemic or brain treatment with morphine. We investigated two issues in the present study; first whether chronic pain mechanisms and lack of brain morphine analgesia are associated in the ICS model; and second what mechanisms are involved in the lack of morphine analgesia. ICS-induced hyperalgesia was not affected in μ-opioid receptor-KO (MOPr-/-) mice, while the lack of brain morphine analgesia remained unchanged in LPA1-/mice, which completely abolish the hyperalgesia in the ICS model. In contrast, the lack of morphine analgesia was abolished in NR2A-NMDA receptor-KO (NR2A-/-) mice, and blocked by intracerebroventricular (i.c,v,) injection of (R)-CPP, an NR2A antagonist, or by microinjection of siRNA NR2A into the PAG region, while no change was observed with Ro 04-5595, an NR2B antagonist (i.c.v.). The lack of morphine analgesia was also reversed by concomitant treatment with 1 mg/kg (i.p.) of dextromethorphan, which possesses NMDA receptor antagonist activity but no analgesic activity. Finally, the hyperalgesia was completely reversed by methadone, which possesses both MOPr agonist and NMDA receptor antagonist activity. Indeed, methadone analgesia was abolished in MOPr-/mice. These results suggest that chronic pain status and lack of morphine analgesia are independent of each other, and that lack of morphine analgesia is mediated by activation of the NR2A-NMDA receptor system. Significance Statement This study reports that a type of intermittently repeated stress (ICS) causes widespread pain that does not respond to morphine. As this lack of morphine analgesia is not affected in LPA1-KO (LPA1-/-) mice, in which chronic pain is abolished, the mechanisms underlying chronic pain and lack of morphine analgesia are independent of each other. Through speculation that a lack of morphine analgesia may be a secondary event to endogenous opioid analgesic tolerance, the authors demonstrate that an anti-opioid NMDAr system counterbalances the MOPr-mediated analgesic mechanisms in the ICS model.
Introduction 3. Opioid receptor signaling 3.1. Receptor phosphorylation by protein kinase C 3.2. ... more Introduction 3. Opioid receptor signaling 3.1. Receptor phosphorylation by protein kinase C 3.2. Modulation of receptor internalization by protein kinase C 3.3. PKC hypothesis for morphine tolerance/ desensitization 3.4. Heterologous regulation of opioid receptor endocytosis 4. Plasticity in neuronal networks 4.1. Anti-opioid glutamate-NMDA receptor system 4.2. BDNF system that supports the anti-opioid NMDA receptor system 4.3. Other systems that support the anti-opioid NMDA receptor system 4.4. Anti-opioid neuropeptide systems 4.5 Opioid-induced hyperalgesia by neuropeptides 4.6 Glial cell involvements in the anti-opioid system 5. Conclusion 6. Acknowledgments 7. References
Journal of Pharmacology and Experimental Therapeutics, 2004
The retinal ischemic-reperfusion stress (130 mmHg, 45 min) caused neuronal damages throughout all... more The retinal ischemic-reperfusion stress (130 mmHg, 45 min) caused neuronal damages throughout all cell layers and reduced the thickness of retinal layer by 30% at 7days after the stress of mouse retina. The intravitreous injection of 100 pmol of nefiracetam, a cognition-enhancer, completely prevented the damages when it was given 30 min prior to and 3 h after the stress. Partial prevention was observed when it was given 24 h after the stress, or low dose (10 pmol) nefiracetam was given 30 min prior to the stress. However, aniracetam had no effect. In the retinal cell line N18-RE-105, the ischemic-reperfusion stress by 2 h culture under the serum-free condition with low oxygen (less of 0.4% O 2) and low glucose (1 mM), caused necrosis or apoptosis in the low-density (0.5x10 4 cell/cm 2) or high-density (5x10 4 cell/cm 2) culture, respectively. The necrosis showed membrane disruption, loss of electron density, and mitochondrial swelling, while apoptosis showed nuclear fragmentation and condensation in transmission electron microscopical analyses and in experiments using specific cell death markers. Nefiracetam inhibited both necrosis and apoptosis, while brain-derived neurotrophic factor (BDNF) inhibited only apoptosis. The cell-protective actions of nefiracetam were abolished by nifedipine and ω-conotoxin GVIA, L-type and N-type calcium channel blocker, but not by PD98059 or wortmannin, ERK1/2 or PI 3-kinase inhibitor, respectively, while those of BDNF were abolished by PD98059 and wortmannin, but not by nifedipine and ω-conotoxin GVIA. All these findings suggest that nefiracetam inhibit necrosis and apoptosis occurred in the ischemic/hypoxic neuronal injury through an increase in Ca 2+-influx.
Cortical neurons rapidly die in necrosis due to poor glucose uptake in the low-density (LD) cultu... more Cortical neurons rapidly die in necrosis due to poor glucose uptake in the low-density (LD) culture under serum-free condition without any supplements. The scanning and transmission electron microscopical analyses characterized the necrosis by membrane disruption, mitochondrial swelling and loss of cytoplasmic electron density. High-glucose treatment delayed the neuronal death by suppressing necrosis, but induced apoptosis through increase in Bax levels, cytochrome c release, caspase-3 activation and DNA ladder formation. Although pyruvate as well as high glucose inhibited necrotic cell death and rapid decrease in cellular ATP levels, possibly related to decreased [ 3 H]-2-deoxy glucose uptake under the serum-free condition, it did not induce apoptosis. Protein kinase C inhibitors blocked these changes related to the cell death mode switch. Several neurotrophic factors did not affect the necrosis, but potentiated high-glucose-induced survival activity, while inhibiting cytochrome c release. All these results suggest that high-glucose treatment causes neuronal cell death mode switch by inhibiting necrosis, while inducing apoptosis, which is prevented by neurotrophic factors.
Morphine is now said to have no problematic side effects such as analgesic tolerance and physical... more Morphine is now said to have no problematic side effects such as analgesic tolerance and physical dependence for cancer pain patients in clinic, as far as it is appropriately used. However, sub-sensitivity to morphine might be developed when higher doses of morphine are used for terminal cancer pain patients. Along with the severity of cancer, the nature of pain becomes changed to neuropathic pain, which is resistant to morphine or NSAIDS. In order to safely use morphine in the clinic, we need to know how morphine tolerance and neuropathic pain are developed and what adjuvants could be used to completely suppress the pain. Here I overview the proposed mechanisms for morphine tolerance and neuropathic pain in relation to the availability of analgesic adjuvants.
Journal of Pharmacology and Experimental Therapeutics, 2020
Treatment for fibromyalgia is an unmet medical need; however, its pathogenesis is still poorly un... more Treatment for fibromyalgia is an unmet medical need; however, its pathogenesis is still poorly understood. In a series of studies, we have demonstrated that some pharmacological treatments reverse generalized chronic pain, but do not affect the lack of morphine analgesia in the intermittent cold stress (ICS)-induced fibromyalgia-like pain model in mice. Here we report that repeated intraperitoneal treatments with mirtazapine, which is presumed to disinhibit 5-HT release and activate 5-HT1 receptor through mechanisms of blocking presynaptic adrenergic α2, postsynaptic 5-HT2 and 5-HT3 receptors, completely reversed the chronic pain for more than 4-5 days after the cessation of treatments. The repeated mirtazapine-treatments also recovered the morphine analgesia after the return of nociceptive threshold to the normal level. The microinjection of siRNA adrenergic α2a receptor (ADRA2A) into the habenula, which showed a selective upregulation of α2 receptor gene expression after ICS, reversed the hyperalgesia, but did not recover the morphine analgesia. However, both reversal of hyperalgesia and recovery of morphine analgesia were observed when siRNA ADRA2A was administered intracerebroventricularly. As the habenular is reported to be involved in the emotion/reward-related pain and hypoalgesia, these results suggest that mirtazapine could attenuate pain and/or augment hypoalgesia by blocking the habenular α2 receptor after ICS. The recovery of morphine analgesia in the ICS model, on the other hand, seems to be mediated through a blockade of α2 receptor in unidentified brain regions.
In the present study, we first report an in vivo characterization of flexor responses induced by ... more In the present study, we first report an in vivo characterization of flexor responses induced by three distinct sine-wave stimuli in the electrical stimulation-induced paw flexion (EPF) test in mice. The fixed sine-wave electric stimulations of 5 Hz (C-fiber), 250 Hz (Adelta-fiber) and 2000 Hz (Abeta-fiber) to the hind paw of mice induced a paw-flexion response and vocalization. The average threshold for paw flexor responses by sine-wave stimulations was much lower than that for vocalization. Neonatally (P3) pretreatment with capsaicin to degenerate polymodal substance P-ergic C-fiber neurons increased the threshold to 5 Hz (C-fiber) stimuli, but not to 250 Hz (Adelta-fiber) and 2000 Hz (Abeta-fiber). The flexor responses to 5 Hz stimuli were significantly blocked by intrathecal (i.t.) pretreatment with both CP-99994 and MK-801, an NK1 and NMDA receptor antagonist, respectively, but not by CNQX, an AMPA/kainate receptor antagonist. On the other hand, the flexor responses induced by ...
Peripheral nerve injury causes neuropathic pain, which is characterized by the paradoxical sensat... more Peripheral nerve injury causes neuropathic pain, which is characterized by the paradoxical sensations of positive and negative symptoms. Clinically, negative signs are frequently observed; however, their underlying molecular mechanisms are largely unknown. Dysfunction of C-fibers is assumed to underlie negative symptoms and is accompanied by long-lasting downregulation of Nav1.8 sodium channel and μ-opioid receptor (MOP) in the dorsal root ganglion (DRG). In the present study, we found that nerve injury upregulates neuron-restrictive silencer factor (NRSF) expression in the DRG neurons mediated through epigenetic mechanisms. In addition, chromatin immunoprecipitation analysis revealed that nerve injury promotes NRSF binding to the neuron-restrictive silencer element within MOP and Nav1.8 genes, thereby causing epigenetic silencing. Furthermore, NRSF knockdown significantly blocked nerve injury-induced downregulations of MOP and Nav1.8 gene expressions, C-fiber hypoesthesia, and the ...
Proceedings of the National Academy of Sciences, 1998
We have studied the in vivo signaling mechanisms involved in nociceptin/orphanin FQ (Noci)-induce... more We have studied the in vivo signaling mechanisms involved in nociceptin/orphanin FQ (Noci)-induced pain responses by using a flexor-reflex paradigm. Noci was 10,000 times more potent than substance P (SP) in eliciting flexor responses after intraplantar injection into the hind limb of mice, but the action of Noci seems to be mediated by SP. Mice pretreated with an NK1 tachykinin receptor antagonist or capsaicin, or mice with a targeted disruption of the tachykinin 1 gene no longer respond to Noci. The action of Noci appears to be mediated by the Noci receptor, a pertussis toxin-sensitive G protein–coupled receptor that stimulates inositol trisphosphate receptor and Ca 2+ influx. These findings suggest that Noci indirectly stimulates nerve endings of nociceptive primary afferent neurons through a local SP release.
Lysophosphatidic acid receptor (LPA 1) signaling initiates neuropathic pain and several pathologi... more Lysophosphatidic acid receptor (LPA 1) signaling initiates neuropathic pain and several pathological events in a partial sciatic nerve injury model. Recently, we reported that lysophosphatidic acid (LPA) induces neuropathic pain as well as demyelination and pain-related protein expression changes via LPA 1 receptor signaling. Lysophosphatidylcholine (LPC), also known as lysolecithin, which is hydrolyzed by autotaxin/ATX into LPA, induces similar plastic changes. Here, we attempted to clarify whether ATX and LPA 1 receptor signaling is involved in the LPC-induced neuropathic pain. In wild-type mice, a single intrathecal (i.t.) injection of LPC induced mechanical allodynia and thermal hyperalgesia 2 days after injection; this persisted for 7 days at least. On the other hand, LPC-induced mechanical allodynia and thermal hyperalgesia were completely abolished in mice lacking an LPA 1 receptor gene. Furthermore, the LPC-induced response was also significantly, but partially reduced in heterozygous mutant mice for the ATX gene. These findings suggest that intrathecally-injected LPC is converted to LPA by ATX, and this LPA activates the LPA 1 receptor to initiate neuropathic pain.
Background: We have previously demonstrated that different spinal transmissions are involved in t... more Background: We have previously demonstrated that different spinal transmissions are involved in the nociceptive behavior caused by electrical stimulation of Aβ-, Aδ- or C-fibers using a Neurometer® in naïve mice. In this study, we attempted to pharmacologically characterize the alteration in spinal transmission induced by partial sciatic nerve injury in terms of nociceptive behavior and phosphorylation of extracellular signal-regulated kinase (pERK) in the spinal dorsal horn. Results: Aβ-fiber responses (2000-Hz), which were selectively blocked by the AMPA/kainate antagonist CNQX in naïve mice, were hypersensitized but blocked by the NMDA receptor antagonists MK-801 and AP-5 in injured mice in an electrical stimulation-induced paw withdrawal (EPW) test. Although Aδ-fiber responses (250-Hz) were also hypersensitized by nerve injury, there was no change in the pharmacological characteristics of Aδ-fiber responses through NMDA receptors. On the contrary, C-fiber responses (5-Hz) were h...
Background: We have proposed that nerve injury-specific loss of spinal tonic cholinergic inhibiti... more Background: We have proposed that nerve injury-specific loss of spinal tonic cholinergic inhibition may play a role in the analgesic effects of nicotinic acetylcholine receptor (nAChR) agonists on neuropathic pain. However, the tonic cholinergic inhibition of pain remains to be well characterized. Results: Here, we show that choline acetyltransferase (ChAT) signals were localized not only in outer dorsal horn fibers (lamina I–III) and motor neurons in the spinal cord, but also in the vast majority of neurons in the dorsal root ganglion (DRG). When mice were treated with an antisense oligodeoxynucleotide (AS-ODN) against ChAT, which decreased ChAT signals in the dorsal horn and DRG, but not in motor neurons, they showed a significant decrease in nociceptive thresholds in paw pressure and thermal paw withdrawal tests. Furthermore, in a novel electrical stimulation-induced paw withdrawal (EPW) test, the thresholds for stimulation through C-, Aδ- and Aβ-fibers were all decreased by AS-O...
Background: Cyclic phosphatidic acid (cPA) is a structural analog of lysophosphatidic acid (LPA),... more Background: Cyclic phosphatidic acid (cPA) is a structural analog of lysophosphatidic acid (LPA), but possesses different biological functions, such as the inhibition of autotaxin (ATX), an LPA-synthesizing enzyme. As LPA is a signaling molecule involved in nociception in the peripheral and central systems, cPA is expected to possess analgesic activity. We characterized the effects of cPA and 2-carba-cPA (2ccPA), a chemically stable cPA analog, on acute and chronic pain. Results: (1) The systemic injection of 2ccPA significantly inhibited somato-cardiac and somato-somatic C-reflexes but not the corresponding A-reflexes in anesthetized rats. (2) 2ccPA reduced sensitivity measured as the paw withdrawal response to electrical stimulation applied to the hind paws of mice through the C-fiber, but not Aδ or Aβ. (3) In mice, pretreatment with 2ccPA dose-dependently inhibited the second phase of formalin-induced licking and biting responses. (4) In mice, pretreatment and repeated post-treat...
Lysophosphatidic acid receptor subtype LPA1 is crucial for the initiation of neuropathic pain and... more Lysophosphatidic acid receptor subtype LPA1 is crucial for the initiation of neuropathic pain and underlying changes, such as up-regulation of Ca2+ channel α2δ-1 subunit in dorsal root ganglia (DRG), up-regulation of PKCγ in the spinal dorsal horn, and demyelination of dorsal root fibers. In the present study, we further examined the involvement of LPA1 signaling in the reorganization of Aβ-fiber-mediated spinal transmission, which is presumed to underlie neuropathic allodynia. Following nerve injury, the phosphorylation of extracellular-signal regulated kinase (pERK) by Aβ-fiber stimulation was observed in the superficial layer of spinal dorsal horn, where nociceptive C-or Aδ-fibers are innervated, but not in sham-operated wild-type mice. However, the pERK signals were largely abolished in LPA1 receptor knock-out ( Lpar1-/-) mice, further supported by quantitative analyses of pERK-positive cells. These results suggest that LPA1 receptor-mediated signaling mechanisms also participat...
Journal of Pharmacology and Experimental Therapeutics, 2003
Topical capsaicin is believed to alleviate pain by desensitizing the vanilloid receptor 1 (VR1) a... more Topical capsaicin is believed to alleviate pain by desensitizing the vanilloid receptor 1 (VR1) at the peripheral nerve endings. Here, we report that an upregulation of VR1 expression on myelinated fibers contributes to the antihyperalgesic effect of capsaicin cream in streptozotocin (STZ)-induced diabetic neuropathic pain. Intravenous injection of STZ (200 mg/kg) in mice caused rapid onset of diabetes within 24 h. Thermal and mechanical hyperalgesia developed by 3 days after STZ injection and persisted at all time points tested until 28 days. There was also hyperalgesic response to intraplantar (i.pl.) prostaglandin I 2 (PGI 2) agonist-induced nociception in such mice. Application of capsaicin cream dose-dependently reversed the thermal, mechanical and PGI 2 agonist-induced hyperalgesia observed in the diabetic mice. The i.pl. injection of capsaicin solution (0.4 µg/20µl) produced nociceptive biting-licking responses in control mice, and these responses were significantly increased in STZ-induced diabetic mice. After neonatal capsaicin-treatment, which destroys most unmyelinated C-fibers, the i.pl. capsaicin-induced biting-licking responses were almost abolished. However, in neonatal capsaicin-treated diabetic mice, the i.pl. capsaicin-induced biting-licking responses reappeared. The i.pl. capsaicin-induced biting-licking responses were blocked by the competitive VR1 antagonist capsazepine. All these results suggest an increase in capsaicin receptor on myelinated fibers due to diabetes. Finally, we confirmed the upregulation of VR1 expression on myelinated primary afferent neurons of diabetic mice by immunohistochemistry. Altogether our results suggest that increased expression of VR1 on myelinated fibers might contribute to the antihyperalgesic effect of topical capsaicin in diabetic neuropathic pain.
Journal of Pharmacology and Experimental Therapeutics, 2007
Using novel apparatus, KUROBOX, learning and memory behaviors as well as various parameters of mo... more Using novel apparatus, KUROBOX, learning and memory behaviors as well as various parameters of movement activity were re-evaluated in mice deficient for nociceptin/orphanin FQ receptor (NOP-/mice) or µ-opioid receptor (MOP-/mice). This method has the advantages that no handling procedures are required throughout the experiments performed over 3 days; positive cue paradigms are used without water or shock stress; and the method does not disturb the nocturnal habit of mice. NOP-/mice displayed a significant enhancement of learning and memory under stress-free conditions, but there were no changes in the various physical and psychological parameters of movement activity (nest stay ratio, distance moved, speed and angle in the movement) and biological rhythm that were measured. Enhancement of nocturnal learning was observed during the first 12 h dark cycle, and enhancement of memory was observed at the beginning of the second dark cycle, in NOP-/mice. By contrast, MOP-/mice showed no significant change in learning and memory behaviors or in physical and psychological parameters of movement activity, except for speed: MOP-/mice showed a significant decrease in speed of movement. Thus, the KUROBOX apparatus provides a useful alternative method to evaluate learning and memory activity under the more physiological conditions. In addition, this apparatus has an advantage that various physical and psychological parameters of movement activity affecting learning and memory behavior are also evaluated at the same time.
Fibromyalgia (FM) is a generalized chronic pain condition whose pathophysiology is poorly underst... more Fibromyalgia (FM) is a generalized chronic pain condition whose pathophysiology is poorly understood, and both basic and translational research are needed to advance the field. Here we used the Sluka model to test whether FM-like pain in mice would produce detectable brain modifications using resting-state (rs) functional Magnetic Resonance Imaging (fMRI). Mice received intramuscular acid saline treatment, images were acquired at 7T 5 days post-treatment, and pain thresholds tested 3 weeks post-scanning. Data-driven Independent Component Analysis revealed significant reduction of functional connectivity (FC) across several component pairs, with major changes for the Retrosplenial cortex (RSP) central to the default mode network, and to a lesser extent the Periaqueductal gray (PAG), a key pain processing area. Seed-to-seed analysis focused on 14 pain-related areas showed strongest FC reduction for RSP with several cortical areas (somatosensory, prefrontal and insular), and for PAG with both cortical (somatosensory) and subcortical (habenula, thalamus, parabrachial nucleus) areas. RSP-PAG FC was also reduced, and this decreased FC tended to be positively correlated with pain levels at individual subject level. Finally, seed-voxelwise analysis focused on PAG confirmed seed-to-seed findings and, also detected reduced PAG FC with the anterior cingulate cortex, increasingly studied in aversive pain effects. In conclusion, FM-like pain triggers FC alterations in the mouse, which are detected by rs-fMRI and are reminiscent of some human findings. The study reveals the causal fingerprint
Lysophosphatidic acid (LPA, 1-acyl-sn-glycerol-3-phosphate) is a well-known lipid growth factor t... more Lysophosphatidic acid (LPA, 1-acyl-sn-glycerol-3-phosphate) is a well-known lipid growth factor that is found widely in various tissues including brain and is reported to drive different intracellular signaling pathways. In the nervous system, LPA studies have drawn many neuroscientists’ attention because it has some actions related to neurogenesis such as cell rounding and proliferation. Remarkable advances in this field have been obtained along with the discovery of the cDNA clone for its receptor, vzg1 /edg2, a member of the seven transmembrane-type edg family. Successive studies have revealed that edg2 activation by LPA mediates several neurobiological actions related to neurogenesis, neuronal excitability and survival activity on developing and postnatal neurons. Here we focused their molecular basis of signaling through G proteins and in vivo roles of edg2 in such neurobiological events.
Lysophosphatidic acid (LPA) signaling is known to play key roles in the initiation and maintenanc... more Lysophosphatidic acid (LPA) signaling is known to play key roles in the initiation and maintenance of various chronic pain models. Here we examined whether LPA signaling is also involved in diabetes-induced abnormal pain behaviors. The high-fat diet (HFD) showing elevation of blood glucose levels and body weight caused thermal, mechanical hyperalgesia, hypersensitivity to 2000 or 250 Hz electrical-stimulation and hyposensitivity to 5 Hz stimulation to the paw in wild-type (WT) mice. These HFD-induced abnormal pain behaviors and body weight increase, but not elevated glucose levels were abolished in LPA1−/− and LPA3−/− mice. Repeated daily intrathecal (i.t.) treatments with LPA1/3 antagonist AM966 reversed these abnormal pain behaviors. Similar abnormal pain behaviors and their blockade by daily AM966 (i.t.) or twice daily Ki16425, another LPA1/3 antagonist was also observed in db/db mice which show high glucose levels and body weight. Furthermore, streptozotocin-induced similar abno...
Journal of Pharmacology and Experimental Therapeutics, 2020
We have developed an experimental fibromyalgia (FM)-like mouse model using intermittent cold stre... more We have developed an experimental fibromyalgia (FM)-like mouse model using intermittent cold stress (ICS), where chronic pain is generalized, female-predominant and abolished in type 1 lysophosphatidic acid receptor-KO (LPA1-/-) mice, but is not reversed by systemic or brain treatment with morphine. We investigated two issues in the present study; first whether chronic pain mechanisms and lack of brain morphine analgesia are associated in the ICS model; and second what mechanisms are involved in the lack of morphine analgesia. ICS-induced hyperalgesia was not affected in μ-opioid receptor-KO (MOPr-/-) mice, while the lack of brain morphine analgesia remained unchanged in LPA1-/mice, which completely abolish the hyperalgesia in the ICS model. In contrast, the lack of morphine analgesia was abolished in NR2A-NMDA receptor-KO (NR2A-/-) mice, and blocked by intracerebroventricular (i.c,v,) injection of (R)-CPP, an NR2A antagonist, or by microinjection of siRNA NR2A into the PAG region, while no change was observed with Ro 04-5595, an NR2B antagonist (i.c.v.). The lack of morphine analgesia was also reversed by concomitant treatment with 1 mg/kg (i.p.) of dextromethorphan, which possesses NMDA receptor antagonist activity but no analgesic activity. Finally, the hyperalgesia was completely reversed by methadone, which possesses both MOPr agonist and NMDA receptor antagonist activity. Indeed, methadone analgesia was abolished in MOPr-/mice. These results suggest that chronic pain status and lack of morphine analgesia are independent of each other, and that lack of morphine analgesia is mediated by activation of the NR2A-NMDA receptor system. Significance Statement This study reports that a type of intermittently repeated stress (ICS) causes widespread pain that does not respond to morphine. As this lack of morphine analgesia is not affected in LPA1-KO (LPA1-/-) mice, in which chronic pain is abolished, the mechanisms underlying chronic pain and lack of morphine analgesia are independent of each other. Through speculation that a lack of morphine analgesia may be a secondary event to endogenous opioid analgesic tolerance, the authors demonstrate that an anti-opioid NMDAr system counterbalances the MOPr-mediated analgesic mechanisms in the ICS model.
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Papers by Hiroshi Ueda