[A long chain fatty acid receptor signaling as a new therapeutic target for stress-induced chronic pain].

Psychological and social stresses are known to be risk factors for psychiatric disorders, including depression and anxiety. On the other hand, exposure to these stresses can also cause prolonged and severe pain. However, the pathological mechanism for stress-induced chronic pain is complex, and there are many unresolved aspects, and no effective therapeutic drugs have been established. Since the discovery of the long-chain fatty acid receptor GPR40/FFAR1 about 20 years ago, research on the mechanism that promotes insulin secretion in the pancreas has progressed. Previously, we have worked to elucidate the physiological effects of GPR40/FFAR1 in the central nervous system and has found that it is involved in the regulation of pain and emotion. Based on these findings, they are now investigating the involvement of fatty acid receptors signaling in the development of stress-related chronic pain. In this review, we discuss the status of psychological stress-related chronic pain and the GPR40/FFAR1-mediated and -striking regulatory mechanisms of stress-induced chronic pain, based on our findings using a mouse model of chronic pain created by loading postoperative pain to a social defeat stress model mouse that mimics psychosocial stress. We summarized about the involvement of fatty acid receptor signaling as a new therapeutic candidate for chronic pain in this review.

Spinal lipocalin 2 as a factor in the development of central post-stroke pain.

Central poststroke pain (CPSP) is a type of central neuropathic pain whose mechanisms remain unknown. Recently, we showed that activated astrocytes and microglial cells are present in the spinal cord of CPSP model mice. Activated glial cells exacerbate cerebral ischemic pathology by increasing the expression of inflammatory factors. However, the involvement of spinal glial cells in CPSP remains unknown. We hypothesized that spinal glial cell-derived molecules cause hyperexcitability or promoted the development of CPSP. In this study, we identified glial cell-derived factors involved in the development of CPSP using a bilateral common carotid occlusion (BCAO)-induced CPSP mouse model. Male ddY mice were subjected to BCAO for 30 min. The von Frey test assessed mechanical hypersensitivity in the right hind paw of mice. BCAO mice showed hypersensitivity to mechanical stimuli and astrocyte activation in the spinal cord 3 days after treatment. DNA microarray analysis revealed a significant increase in lipocalin 2 (LCN2), is known as neutrophil gelatinase-associated lipocalin, in the superficial dorsal horns of BCAO-induced CPSP model mice. LCN2 colocalized with GFAP, an astrocyte marker. Spinal GFAP-positive cells in BCAO mice co-expressed signal transducer and activator of transcription 3 (STAT3). The increase in the fluorescence intensity of LCN2 and GFAP in BCAO mice was suppressed by intrathecal injection of AG490, an inhibitor of JAK2 and downstream STAT3 activation, or anti-LCN2 antibody. Our findings indicated that LCN2 in spinal astrocytes may be a key molecule and may be partly involved in the development of CPSP.

[The involvement of brain fatty acid-GPR40/FFAR1 signaling in the stress-induced chronic pain].

Psychosocial stress is a risk factor for psychiatric disorders, including depression and anxiety, and chronic pain, whereas chronic pain is also closely related to the development of psychiatric disorders. However, the pathological mechanisms of stress-triggered psychiatric disorders and chronic pain are unknown, and their effective treatments have not been established. Recently, the advances in analytical techniques for fatty acids and their metabolites have made it possible to comprehensively measure changes in fatty acid composition in various cells and organs using the lipidomics approach, and to visualize the localization of phospholipids, fatty acids, and other lipid mediators using imaging mass spectrometry. Many researchers have focused on understanding the differences in the distribution of phospholipids, fatty acids, their lipid metabolites in cells and organs, and the changes of fatty acid composition in various diseases. More recently, changes in the fatty acid composition and its distribution during chronic pain and stress have also been reported. We also proposed that modulation of brain fatty acid signaling could be a new therapeutic target for stress-induced chronic pain. In this review, we summarize the latest basic and clinical findings on the role of fatty acid signaling in stress-induced psychiatric disorders and chronic pain.

Stress-Induced Changes in the Endogenous Opioid System Cause Dysfunction of Pain and Emotion Regulation.

Early life stress, such as child abuse and neglect, and psychosocial stress in adulthood are risk factors for psychiatric disorders, including depression and anxiety. Furthermore, exposure to these stresses affects the sensitivity to pain stimuli and is associated with the development of chronic pain. However, the mechanisms underlying the pathogenesis of stress-induced depression, anxiety, and pain control remain unclear. Endogenous opioid signaling is reportedly associated with analgesia, reward, addiction, and the regulation of stress responses and anxiety. Stress alters the expression of various opioid receptors in the central nervous system and sensitivity to opioid receptor agonists and antagonists. µ-opioid receptor-deficient mice exhibit attachment disorders and autism-like behavioral expression patterns, while those with δ-opioid receptor deficiency exhibit anxiety-like behavior. In contrast, deficiency and antagonists of the κ-opioid receptor suppress the stress response. These findings strongly suggest that the expression and dysfunction of the endogenous opioid signaling pathways are involved in the pathogenesis of stress-induced psychiatric disorders and chronic pain. In this review, we summarize the latest basic and clinical research studies on the effects of endogenous opioid signaling on early-life stress, psychosocial stress-induced psychiatric disorders, and chronic pain.

Nicotine suppresses central post-stroke pain via facilitation of descending noradrenergic neuron through activation of orexinergic neuron.

Central post-stroke pain (CPSP) is a type of central neuropathic pain, whose underlying mechanisms remain unknown. We previously reported that bilateral carotid artery occlusion (BCAO)-induced CPSP model mice showed mechanical hypersensitivity and decreased mRNA levels of preproorexin, an orexin precursor, in the hypothalamus. Recently, nicotine was shown to regulate the neuronal activity of orexin in the lateral hypothalamus (LH) and suppress inflammatory and neuropathic pain. In this study, we evaluated whether nicotine could suppress BCAO-induced mechanical allodynia through the activation of orexinergic neurons. Mice were subjected to BCAO for 30 min. Mechanical hypersensitivity was assessed by the von Frey test. BCAO mice showed hypersensitivity to mechanical stimuli three days after BCAO surgery. The intracerebroventricular injection of nicotine suppressed BCAO-induced mechanical hypersensitivity in a dose-dependent manner. These effects were inhibited by α7 or α4ß2-nicotinic receptor antagonists. After nicotine injection, the level of c-fos, a neuronal activity marker, increased in the LH and locus coeruleus (LC) of Sham and BCAO mice. Increased number of c-Fos-positive cells partly colocalized with orexin A-positive cells in the LH, as well as tyrosine hydroxylase-positive cells in the LC. Orexinergic neurons project to the LC area. Nicotine-induced antinociception tended to cancel by the pretreatment of SB334867, an orexin receptor1 antagonist into the LC. Intra-LH microinjection of nicotine attenuated BCAO-induced mechanical hypersensitivity. Nicotine-induced antinociception was inhibited by intrathecal pre-treatment with yohimbine, an α2 adrenergic receptor antagonist. These results indicated that nicotine may suppress BCAO-induced mechanical hypersensitivity through the activation of the descending pain control system via orexin neurons.

Docosahexaenoic Acid Attenuates the Progression of Nonalcoholic Steatohepatitis by Suppressing the Adipocyte Inflammation via the G Protein-Coupled Receptor 120/Free Fatty Acid Receptor 4 Pathway.

INTRODUCTION: Our previous study demonstrated that docosahexaenoic acid (DHA), an endogenous G protein-coupled receptor 120 (GPR120)/free fatty acid receptor (FFAR) 4 agonist, attenuated the liver inflammation in nonalcoholic steatohepatitis (NASH), while exacerbated liver inflammation was observed in the GPR120/FFAR4 knockout (GPR120/FFAR4KO) mice. Recently, abdominal adiposity has been reported to correlate with the severity of inflammation and fibrosis in patients with NASH. In this study, we investigated whether the activation of GPR120/FFAR4 suppressed the inflammation of the adipose tissue and whether these suppressive effects attenuated the development of NASH. METHODS: A choline-deficient and 0.1% methionine-containing high-fat (CDAHF) diet was used to create a mouse model of NASH. DHA was orally administered to the mice for 1 week. Epididymal fat pads which collected from the control-fed wild-type (WT) or GPR120/FFAR4KO mice were used as ex vivo white adipose tissue (WAT) culture systems. RESULTS: The mice fed a CDAHF diet for 2 weeks showed NASH-like liver diseases. In the WAT of mice fed with the CDAHF diet, inflammation and fibrosis were significantly increased, and the administration of DHA suppressed these phenomena. In an ex vivo adipocyte culture study, DHA dose-dependently suppressed the lipopolysaccharide-induced inflammation in the adipocyte tissue of WT mice, which was reversed by pretreatment with AH7614, a GPR120/FFAR4 antagonist, but not GPR40 or peroxisome proliferator-activated receptor γ antagonist. CONCLUSIONS: These findings suggest that the activation of GPR120/FFAR4 may suppress the inflammation of adipocytes, which could be a key pathway to prevent the development of NASH.

Changes in median eminence of fatty acid-binding protein 3 in a mouse model of pain.

AIMS: Fatty acid-binding protein (FABP) regulates polyunsaturated fatty acid (PUFA) intracellular trafficking and signal transduction. Our previous studies demonstrated that the alteration of PUFA in the hypothalamus is involved in pain process. However, how FABP subtypes change during pain remain unclear. Here, we examined the expression changes and localization in the hypothalamic FABP subtype in postoperative pain model mice. METHODS: Paw incision-induced postoperative methods were adopted as a pain model in male ddY mice. Mechanical allodynia was examined using the von Frey test. The analysis of several FABPs mRNA was measured by real-time PCR, and cellular localization of its protein level was measured by immunofluorescent study. RESULTS: Postoperative pain mouse elicited mechanical allodynia on Day 2 after paw incision, and mRNA expression of FABP3 increased significantly in the hypothalamus in the postoperative pain mouse model compared to that in control mice. FABP3 protein expressed in the median eminence and the arcuate nucleus, and colocalized with Iba-1, which is a microglial cell marker. Its protein level significantly increased in the median eminence on Day 2 after incision and returned to the control level on Day 4 after incision. CONCLUSIONS: Our findings indicate that FABP3 in the median eminence may change in pain stimuli and may represent a molecular link controlling pain.

FFAR1/GPR40 Contributes to the Regulation of Striatal Monoamine Releases and Facilitation of Cocaine-Induced Locomotor Activity in Mice.

The free fatty acid receptor 1 (FFAR1) is suggested to function as a G protein-coupled receptor (GPR40) for medium-to-long-chain free fatty acids. Previous studies on the expression of FFAR1 revealed that the nigrostriatal region is one of the areas which express abundant FFAR1 mRNA/protein in the central nervous system (CNS). However, the role of FFAR1 in the CNS has been still largely unclarified. Here, we examined a possible functional role of FFAR1 in the control of extracellular concentrations of striatal monoamines and cocaine-induced locomotor activity. Microdialysis analysis revealed that the basal level of extracellular dopamine (DA) was significantly elevated, while the basal serotonin (5-HT) level tended to be reduced in the striatum of FFAR1 knockout (-/-) mice. Interestingly, local application of a FFAR1 agonist, GW9508, markedly augmented the striatal 5-HT release in FFAR1 wild-type (+/+) mice, whereas topical application of a FFAR1 antagonist, GW1100, significantly reduced the 5-HT release. However, the enhanced 5-HT release was completely lost in -/- mice. Although acute administration of cocaine enhanced the locomotor activity in both +/+ and -/- mice, the magnitude of the enhancement was significantly reduced in -/- mice. In addition, intraperitoneal injection of GW1100 significantly decreased the cocaine-induced locomotor enhancement. These results suggest that FFAR1 has a facilitatory role in striatal 5-HT release, and the evoked 5-HT release might contribute to enhance cocaine-induced locomotor activity.

Changes in opioid receptors, opioid peptides and morphine antinociception in mice subjected to early life stress.

Recent studies have shown that the endogenous opioid system is considerably affected by early life stress such as child abuse. Here, we investigated whether early life stress changes the endogenous opioid receptors and their peptides, and if such stress impacts morphine antinociception. We used mice affected by maternal separation and social isolation (MSSI) as an early life stress model. In the tail-flick test, 10-week-old MSSI mice showed a significant decrease in morphine antinociception compared to age-matched control mice. The number of c-Fos-positive cells increased in the periaqueductal gray (PAG), nucleus accumbens, and thalamus of control mice after the morphine injections, whereas hardly any positive cells were detected in the same areas of MSSI mice. The expression of µ- and κ-opioid receptor (MOR and KOR, respectively) messenger RNA (mRNA) was significantly decreased in the PAG of MSSI mice, whereas KOR expression was significantly increased in the amygdala of MSSI mice. The expression of δ-opioid receptor (DOR) mRNA was significantly reduced in the PAG and rostral ventromedial medulla of MSSI mice compared to control mice. Moreover, the lack of morphine antinociception was observed in 18-week-old MSSI mice. Our findings suggest that the supraspinal opioid system may be affected by early life stress exposure, and that this exposure may impact morphine antinociception.

Retraction Note to: Honokiol suppresses the development of post-ischemic glucose intolerance and neuronal damage in mice.

This article [1] has been retracted at the request of the corresponding author because an Investigation Committee established by Kobe Gakuin University (Kobe, Japan) has found numerous discrepancies between the raw data and the data presented in Figs. 6b, d. Statistical analysis of the raw data showed no significant difference between conditions. Authors S. Harada, K. Nakamoto, W. Fujita-Hamabe, H.-H. Chen, M.-H. Chan, and S. Tokuyama agree with this retraction. Authors M. Kishimoto and M. Kobayashi could not be reached for comment about this retraction.

Involvement of descending pain control system regulated by orexin receptor signaling in the induction of central post-stroke pain in mice.

Central post-stroke pain (CPSP) is a type of neuropathic pain for which the mechanism and relevant drug pathways remain unknown. Recently, it was reported that intracerebroventricular (ICV) administration of orexin-A suppresses pain and ischemia. In this study, we tested the role of orexin-A in CPSP induction in mice. Male ddY mice were subjected to 30 min of bilateral carotid artery occlusion (BCAO). CPSP was assessed by von Frey test. Colocalization of orexin 1 receptor (OX1R) with various neuron markers were determined by double-immunofluorescence. The hindpaw withdrawal responses to mechanical stimuli were significantly increased 3 days post-BCAO compared with those of sham groups. ICV injection of orexin-A dose-dependently suppressed BCAO-induced mechanical allodynia. These effects were inhibited by pre-treatment with SB334867 (an OX1R antagonist; ICV injection), yohimbine (a noradrenaline α2 receptor antagonist; intrathecal (IT) injection), and WAY100635 (a serotonin 5-HT1A receptor antagonist; IT injection), but not TCS OX2 29 (an OX2R antagonist; ICV injection). OX1R colocalized with TH (a noradrenergic neuron marker) and TPH (a serotonergic neuron marker) in the locus ceruleus (LC) and the rostral ventromedial medulla (RVM), respectively. The number of c-Fos positive cells in the LC and the RVM of BCAO mice was increased at 90 min after ICV injection of orexin-A compared to saline group. These results indicate that orexin-A/OX1R signaling plays an important role through activation of the descending pain control system in the induction of CPSP in mice.

[Involvement of the Free Fatty Acid Receptor GPR120/FFAR4 in the Development of Nonalcoholic Steatohepatitis].

Nonalcoholic fatty liver disease (NAFLD) is characterized by the pathological accumulation of fat in the liver in the absence of any other disease related to liver steatosis, which includes a wide spectrum ranging from mild asymptomatic fatty liver to nonalcoholic steatohepatitis (NASH) and cirrhosis. However, the pathogenesis of NASH has not been established. In this study, we investigated the involvement of the G-protein-coupled receptor 120/free fatty acid receptor 4 (GPR120/FFAR4) in the pathogenesis of NASH. Mice fed a 0.1% methionine- and choline-deficient l-amino acid-defined, high-fat (CDAHF) diet showed a significant increase in plasma aspartate aminotransferase and alanine aminotransferase levels, fatty deposition, inflammatory cell infiltration, and slight fibrosis. Docosahexanoic acid (DHA, a GPR120/FFAR4 agonist) suppressed the inflammatory cytokines in hepatic tissues and prevented liver fibrosis. On the other hand, GPR120/FFAR4-deficient CDAHF-fed mice showed increments in the number of hepatic crown-like structures and immunoreactivity to F4/80-positive cells compared with wild-type mice. Furthermore, the levels of hepatic TNF-α mRNA expression increased in GPR120-deficient mice. These findings suggest that the GPR120/FFAR4-mediating system could be a key signaling pathway to prevent the development of NASH. In this review, we describe our recent data showing that GPR120/FFAR4 could be a therapeutic target in NASH/NAFLD.

The Involvement of DDAH1 in the Activation of Spinal NOS Signaling in Early Stage of Mechanical Allodynia Induced by Exposure to Ischemic Stress in Mice.

The pathophysiological mechanism of central post-stroke pain (CPSP) is complicated and not well understood. Recently, it has been reported that an increase in the levels of spinal nitric oxide synthetase (NOS) occurs in cerebral ischemia, and spinal NOS is involved in the development of neuropathic pain. The aim of this study was to elucidate the mechanism of spinal NOS signaling in the development of CPSP. Male ddY mice were subjected to 30-min long bilateral carotid artery occlusion (BCAO). The withdrawal responses to mechanical stimuli were significantly increased as determined with von Frey test on days 1 and 3 after BCAO. Protein expression of spinal N(G),N(G)-dimethylarginine dimethylaminohydralase 1 (DDAH1), a key enzyme involved in the metabolism of the endogenous NOS, increased on day 1 after BCAO, but not on day 3. Intrathecal (i.t.) injection of PD404182, a DDAH1 inhibitor, significantly suppressed mechanical allodynia on day 1, but not on day 3 after BCAO. In addition, i.t. administration of NG-nitro-L-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, significantly blocked mechanical allodynia on days 1 and 3 after BCAO. Furthermore, BCAO-induced increment of spinal NOS activity was inhibited by the pretreatment with PD404182. These results suggest that mechanical allodynia in the early stage of CPSP is caused by increment of NOS activity through upregulated DDAH1 in the spinal cord.

NNC 55-0396, a T-type calcium channel blocker, protects against the brain injury induced by middle cerebral artery occlusion and reperfusion in mice.

We tested whether NNC 55-0396 (NNC), a T-type calcium channel (T-channel) blocker, reduces the brain injury caused by middle cerebral artery occlusion and reperfusion (MCAO/R) in mice. NNC, administered i.c.v. before the occlusion, greatly reduced the MCAO/R-induced brain infarct and neurological dysfunctions, although it, given toward the end of occlusion, was less effective. Systemic administration of NNC before the occlusion also attenuated the infarct and neurological dysfunctions. Our data imply that blood-brain-barrier-permeable T-channel blockers such as NNC are capable of reducing MCAO/R-induced brain damage, and that T-channels are involved in neuronal damage induced by ischemia rather than reperfusion.