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.

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.

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.

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.

Activation of c-Jun N-terminal kinase and p38 after cerebral ischemia upregulates cerebral sodium-glucose transporter type 1.

Cerebral ischemic stress increases cerebral sodium-glucose transporter type 1 (SGLT-1). However, the mechanism by which cerebral ischemia leads to the up-regulation of SGLT-1 remains unclear. In peripheral tissue, the activation of mitogen-activated protein kinases (MAPKs) increases SGLT-1. MAPK pathways [c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated protein kinase (ERK)] are activated by cerebral ischemic stress. Therefore, we confirmed the involvement of MAPKs in the up-regulation of cerebral SGLT-1 after cerebral ischemia. Male ddY mice were subjected to middle cerebral artery occlusion (MCAO). Protein expression was assessed by western blotting. Mice received an intracerebroventricular (i.c.v.) injection of SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), and PD98059 (MEK inhibitor) immediately after reperfusion. The infarction and behavioral abnormalities were assessed on days 1 and 3 after MCAO. The MAPK inhibitors suppressed the activation of JNK, p38, and ERK 3 h after MCAO. SP600125 and SB203580 administration ameliorated cerebral ischemic neuronal damage, whereas PD98059 administration exacerbated cerebral ischemic neuronal damage. SP600125 and SB203580 significantly suppressed the increase in SGLT-1 12 h after MCAO. PD98059 had no effect on SGLT-1 expression after MCAO. Our results indicate that the activation of JNK and p38 participate in the up-regulation of cerebral SGLT-1 after MCAO.

Dexamethasone Downregulates Endothelin Receptors and Reduces Endothelin-Induced Production of Matrix Metalloproteinases in Cultured Rat Astrocytes.

In brain disorders, astrocytes change phenotype to reactive astrocytes and are involved in the induction of neuroinflammation and brain edema. The administration of glucocorticoids (GCs), such as dexamethasone (Dex), reduces astrocytic activation, but the mechanisms underlying this inhibitory action are not well understood. Endothelins (ETs) promote astrocytic activation. Therefore, the effects of Dex on ET receptor expressions were examined in cultured rat astrocytes. Treatment with 300 nM Dex for 6-48 hours reduced the mRNA expression of astrocytic ETA and ETB receptors to 30-40% of nontreated cells. Levels of ETA and ETB receptor proteins became about 50% of nontreated cells after Dex treatment. Astrocytic ETA and ETB receptor mRNAs were decreased by 300 nM hydrocortisone. The effects of Dex and hydrocortisone on astrocytic ET receptors were abolished in the presence of mifepristone, a GC receptor antagonist. Although Dex did not decrease the basal levels of matrix metalloproteinase (MMP) 3 and MMP9 mRNAs, pretreatment with Dex reduced ET-induced increases in MMP mRNAs. The effects of ET-1 on the release of MMP3 and MMP9 proteins were attenuated by pretreatment with Dex. ET-1 stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in cultured astrocytes. Pretreatment with Dex reduced the ET-induced increases in ERK1/2 phosphorylation. In contrast, pretreatment with Dex did not affect MMP production or ERK1/2 phosphorylation induced by phorbol myristate acetate, a protein kinase C activator. These results indicate that Dex downregulates astrocytic ET receptors and reduces ET-induced MMP production.

The Deletion of GPR40/FFAR1 Signaling Damages Maternal Care and Emotional Function in Female Mice.

The free fatty acid receptor 1 (GPR40/FFAR1) is activated by polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acids (DHA). This receptor has been the focus of many studies regarding physiological functions of the central nervous system. PUFAs are essential for neuronal development and maintenance of neuronal function; thus, the decrease of PUFAs in the brain is closely related to the induction of psychiatric diseases associated with emotional disorder, such as anxiety, depression, and schizophrenia. However, details of the mechanisms remain unclear. In this study, we investigated changes of maternal and/or emotional behavior caused by a deficiency of GPR40/FFAR1 signaling. GPR40/FFAR1 deficient (FFAR1-/-) female mice exhibited impaired maternal care such as retrieving behaviors and an increased rate of neglect and infanticide when compared to wild type (WT) female mice. Furthermore, FFAR1-/- female mice showed increased time spent in the open arms in an elevated plus maze test, reduction of locomotor activity and social interaction behavior, and decreased sucrose intake, when compared to WT female mice. In conclusion, these findings suggest that PUFAs-GPR40/FFAR1 signaling might function, at least in part, as a regulatory factor of emotional and maternal behavior in mice.

GPR40/FFAR1 deficient mice increase noradrenaline levels in the brain and exhibit abnormal behavior.

The free fatty acid receptor 1 (GPR40/FFAR1) is a G protein-coupled receptor, which is activated by long chain fatty acids. We have previously demonstrated that activation of brain GPR40/FFAR1 exerts an antinociceptive effect that is mediated by the modulation of the descending pain control system. However, it is unclear whether brain GPR40/FFAR1 contributes to emotional function. In this study, we investigated the involvement of GPR40/FFAR1 in emotional behavior using GPR40/FFAR1 deficient (knockout, KO) mice. The emotional behavior in wild and KO male mice was evaluated at 9-10 weeks of age by the elevated plus-maze test, open field test, social interaction test, and sucrose preference test. Brain monoamines levels were measured using LC-MS/MS. The elevated plus-maze test and open field tests revealed that the KO mice reduced anxiety-like behavior. There were no differences in locomotor activity or social behavior between the wild and KO mice. In the sucrose preference test, the KO mice showed reduction in sucrose preference and intake. The level of noradrenaline was higher in the hippocampus, medulla oblongata, hypothalamus and midbrain of KO mice. Therefore, these results suggest that brain GPR40/FFAR1 is associated with anxiety- and depression-related behavior regulated by the increment of noradrenaline in the brain.

Effects of Adjuvant Analgesics on Cerebral Ischemia-Induced Mechanical Allodynia.

Central post-stroke pain (CPSP), a potential sequela of stroke, is classified as neuropathic pain. Although we recently established a CPSP-like model in mice, the effects of adjuvant analgesics as therapeutic drugs for neuropathic pain in this model are unknown. Hence, the aim of the present study was to assess the usefulness of our model by evaluating the effects of adjuvant analgesics used for treating neuropathic pain in this mouse model of CPSP. Male ddY mice were subjected to 30 min of bilateral carotid artery occlusion (BCAO). The development of hind paw mechanical allodynia was measured after BCAO using the von Frey test. The mechanical allodynia was significantly increased on day 3 after BCAO compared with that during the pre-BCAO assessment. BCAO-induced mechanical allodynia was significantly decreased by intraperitoneal injections of imipramine (a tricyclic antidepressant), mexiletine (an antiarrhythmic), gabapentin (an antiepileptic), or a subcutaneous injection of morphine (an opioid receptor agonist) compared with that following vehicle treatment in BCAO-mice. By contrast, milnacipran (a serotonin and norepinephrine reuptake inhibitor), paroxetine (selective serotonin reuptake inhibitor), carbamazepine (antiepileptic), and indomethacin (nonsteroidal anti-inflammatory drug) did not affect the BCAO-induced mechanical allodynia. Our results show that BCAO in mice may be useful as an animal model of CPSP. In addition, BCAO-induced mechanical allodynia may be suppressed by some adjuvant analgesics used to treat neuropathic pain.

Proteomic Profiling in the Spinal Cord and Sciatic Nerve in a Global Cerebral Ischemia-Induced Mechanical Allodynia Mouse Model.

Central post-stroke pain (CPSP) is one of the complications of cerebral ischemia and neuropathic pain syndrome. At present, there are few studies of pain in regions such as the spinal cord or sciatic nerve in cerebral ischemic animal models. To identify proteomic changes in the spinal cord and sciatic nerve in global cerebral ischemic model mice, in the present study we performed an investigation using proteomic methods. In a comparison between the intensity of protein spots obtained from a sham and that from a bilateral carotid artery occulusion (BCAO) in spinal cord and sciatic nerve, the levels of 10 (spinal cord) and 7 (sciatic nerve) protein spots were altered. The protein levels in the spinal cord were significantly increased in N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 (DDAH1), 6-phosphogluconolactonase isoform 1, and precursor apoprotein A-I and decreased in dihydropyrimidinase-related protein 2 (CRMP-2), enolase 1B, rab guanosine 5'-diphosphate (GDP) dissociation inhibitor beta, septin-2 isoform a, isocitrate dehydrogenase subunit alpha, cytosolic malate dehydrogenase, and adenosine triphosphate synthase. The protein levels in the sciatic nerve were significantly increased in a mimecan precursor, myosin light chain 1/3, and myosin regulatory light chain 2 (MLC2), and decreased in dihydropyrimidinase-related protein 3 (CRMP-4), protein disulfide-isomerase A3, 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, and B-type creatine kinase. In addition, CRMP-2 and CRMP-4 protein levels were decreased, and DDAH1 and MLC2 protein levels were increased on day 1 after BCAO using Western blotting. These results suggested that changes in these proteins may be involved in the regulation of CPSP.

Astrocytes Release Polyunsaturated Fatty Acids by Lipopolysaccharide Stimuli.

We previously reported that levels of long-chain fatty acids (FAs) including docosahexaenoic acids (DHA) increase in the hypothalamus of inflammatory pain model mice. However, the precise mechanisms underlying the increment of free fatty acids (FFAs) in the brain during inflammation remains unknown. In this study, we characterized FFAs released by inflammatory stimulation in rat primary cultured astrocytes, and tested the involvement of phospholipase A2 (PLA2) on these mechanisms. Lipopolysaccharide (LPS) stimulation significantly increased the levels of several FAs in the astrocytes. Under these conditions, mRNA expression of cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2) in LPS-treated group increased compared with the control group. Furthermore, in the culture media, the levels of DHA and arachidonic acid (ARA) significantly increased by LPS stimuli compared with those of a vehicle-treated control group whereas the levels of saturated FAs (SFAs), namely palmitic acid (PAM) and stearic acid (STA), did not change. In summary, our findings suggest that astrocytes specifically release DHA and ARA by inflammatory conditions. Therefore astrocytes might function as a regulatory factor of DHA and ARA in the brain.

Pancreatic Changes in Nerve Growth Factor/TrkA Associated with Insulin Secretion in Cerebral Ischemia.

Regulation of blood glucose levels as a therapeutic strategy for cerebral ischemia plays an important role in suppressing neuronal damage. In particular, suppression of post-ischemic glucose intolerance improves cerebral ischemia. We have reported that cerebral ischemia induces glucose intolerance and an increase in plasma insulin levels. However, the mechanism of insulin secretion after cerebral ischemia is unclear. Nerve growth factor (NGF), a member of the neurotrophin family, has high affinity for tropomyosin-related kinase A (TrkA). NGF/TrkA signaling is associated with neuronal survival, differentiation, and function. Recently, NGF/TrkA signaling has been reported to be associated with insulin synthesis and secretion. In the present study, we evaluated the insulin content and expression of NGF/TrkA by immunofluorescence and Western blotting after middle cerebral artery occlusion (MCAO) as a cerebral ischemia model. At 6, 12, and 24 h after MCAO, insulin contents were increased in MCAO mice. The expression of NGF was increased at 6, 12, and 24 h, whereas the expression of TrkA tended to decrease in pancreas after MCAO. These results suggest that NGF/TrkA signaling is an important factor in cerebral ischemia-induced insulin synthesis and secretion in the pancreas.

Endothelin-1 increases the expression of VEGF-R1/Flt-1 receptors in rat cultured astrocytes through ETB receptors.

Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin-1 (ET-1) were examined in rat cultured astrocytes. Expressions of VEGF-R1 and -R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF-R3 and Tie-2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF-R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF-E, a selective VEGF-R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF165 were reduced by selective antagonists for VEGF-R1 and -R2. Treatment with ET-1 increased VEGF-R1 mRNA and protein levels. The effects of ET-1 on VEGF-R1 mRNA were mimicked by Ala(1,3,11,15) -ET-1, a selective agonist for ETB receptors, and inhibited by BQ788, an ETB antagonist. ET-1 did not affect the mRNA levels of VEGF-R2, -R3, and Tie-2. Pre-treatment with ET-1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET-1 induces up-regulation of VEGF-R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues. To clarify the regulatory mechanisms of vascular endothelial growth factor (VEGF) receptors, the effects of endothelin-1 (ET-1) were examined in rat cultured astrocytes. Effects of selective VEGF-R1 and R2 agonist showed that these receptors were linked to focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Treatment with ET-1 increased expression of VEGF-R1, which was mediated by ETB receptors. Pre-treatment with ET-1 potentiated the VEGF-R1-mediated activations of FAK and ERK1/2. These findings suggest that ET-1 induces up-regulation of VEGF-R1 receptors in astrocytes.

Mechanisms of P-glycoprotein alteration during anticancer treatment: role in the pharmacokinetic and pharmacological effects of various substrate drugs.

In clinical pharmacotherapy, therapeutic benefits and adverse effects of medicines differ substantially between individuals and are often determined by their blood levels. Critical regulators influencing the pharmacokinetics and pharmacodynamics of drugs include drug transporters and drug-metabolizing enzymes. Among these, we have focused on P-glycoprotein (P-gp), a drug efflux transporter. A growing body of evidence indicates that the expression and functional activity of P-gp are altered under several pathological conditions, by exposure to substrate drugs of P-gp, and by ingestion of certain foods. In this critical review, we discuss the mechanisms by which anticancer drugs, most of which are P-gp substrates, alter the expression and functional activity of P-gp in tumors and normal tissues after chronic treatment. Accumulating evidence shows that various transcription factors, in addition to epigenetic and post-translational factors, modulate P-gp expression, which alters the pharmacokinetics and pharmacological effects of drugs. Therefore, it is important to consider individual patients with regard to drug-taking history, as well as levels of P-gp expression and function, when providing clinical pharmacotherapy.

Involvement of the long-chain fatty acid receptor GPR40 in depression-related behavior.

The functional role of brain G protein-coupled receptor 40 (GPR40) remains unclear. We investigated GPR40 signaling in depression-related behavior in mice via the forced swim test. A repeated but not a single intracerebroventricular administration of the GPR40 agonist, GW9508, reduced the duration of immobility behavior. Moreover, the levels of hippocampal non-esterified docosahexaenoic acid and arachidonic acid were decreased immediately after the forced swimming. These results suggested that brain GPR40 signaling may regulate depression-related behavior.

Pharmacological characterization of standard analgesics on oxaliplatin-induced acute cold hypersensitivity in mice.

Oxaliplatin, a platinum-based chemotherapeutic agent, causes an acute peripheral neuropathy triggered by cold in almost all patients during or within hours after its infusion. We recently reported that a single administration of oxaliplatin induced cold hypersensitivity 2 h after the administration in mice. In this study, we examined whether standard analgesics relieve the oxaliplatin-induced acute cold hypersensitivity. Gabapentin, tramadol, mexiletine, and calcium gluconate significantly inhibited and morphine and milnacipran decreased the acute cold hypersensitivity, while diclofenac and amitriptyline had no effects. These results suggest that gabapentin, tramadol, mexiletine, and calcium gluconate are effective against oxaliplatin-induced acute peripheral neuropathy.

Changes in PtdIns(4,5)P2 induced by etoposide treatment modulates small intestinal P-glycoprotein via radixin.

Previously, we reported that repeated oral administration of etoposide (ETP) increases P-glycoprotein (P-gp) expression in association with activation of ezrin/radixin/moesin (ERM) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK) signaling in the small intestine. However, the detailed mechanisms of this pathway have yet to be fully elucidated. Recently, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], one of the most abundant phosphoinositides in the plasma membrane, has attracted attention regarding its involvement in the plasma membrane localization of various membrane proteins. PtdIns(4,5)P2 is an essential factor in the dissociation and subsequent membrane translocation (activation) of ERM, and its synthetic pathway is known to be highly regulated by RhoA/ROCK signaling. Here, we examined the involvement of PtdIns(4,5)P2 in the mechanism by which ETP treatment increases small intestinal P-gp levels, and we determined which protein within ERM contributes to this phenomenon. Repeated oral treatment with ETP (10 mg/kg/d) over 5 d significantly increased PtdIns(4,5)P2 expression in the ileal membrane as measured by dot blot. Furthermore, this increase was suppressed by co-administration of a RhoA inhibitor, rosuvastatin (5 mg/kg/d, per os (p.o.)), or a ROCK inhibitor, fasudil (5 mg/kg/d, p.o.). In immunoprecipitation assays, radixin (but not ezrin or moesin) binding to PtdIns(4,5)P2 was observed to increase in association with the up-regulation of P-gp in the same fraction, and immunofluorescence studies indicated that radixin co-localized with PtdIns(4,5)P2 in the ileal tissue. In conclusion, ETP treatment appears to up-regulate PtdIns(4,5)P2 expression via RhoA/ROCK signaling, leading to the activation of ERM, presumably through the physical interaction of radixin with PtdIns(4,5)P2. This in turn increases the expression of ileal P-gp.

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.

Orexin-A suppresses postischemic glucose intolerance and neuronal damage through hypothalamic brain-derived neurotrophic factor.

Orexin-A (a glucose-sensing neuropeptide in the hypothalamus) and brain-derived neurotrophic factor (BDNF; a member of the neurotrophin family) play roles in many physiologic functions, including regulation of glucose metabolism. We previously showed that the development of postischemic glucose intolerance is one of the triggers of ischemic neuronal damage. The aim of this study was to determine whether there was an interaction between orexin-A and BDNF functions in the hypothalamus after cerebral ischemic stress. Male ddY mice were subjected to 2 hours of middle cerebral artery occlusion (MCAO). Neuronal damage was estimated by histologic and behavioral analyses. Expression of protein levels was analyzed by Western blot. Small interfering RNA directed BDNF, orexin-A, and SB334867 [N-(2-methyl-6-benzoxazolyl)-N'-1,5-naphthyridin-4-yl urea; a specific orexin-1 receptor antagonist] were administered directly into the hypothalamus. The level of hypothalamic orexin-A, detected by immunohistochemistry, was decreased on day 1 after MCAO. Intrahypothalamic administration of orexin-A (1 or 5 pmol/mouse) significantly and dose-dependently suppressed the development of postischemic glucose intolerance on day 1 and development of neuronal damage on day 3. The MCAO-induced decrease in insulin receptor levels in the liver and skeletal muscle on day 1 was recovered to control levels by orexin-A, and this effect of orexin-A was reversed by the administration of SB334867 as well as by hypothalamic BDNF knockdown. These results suggest that suppression of postischemic glucose intolerance by orexin-A assists in the prevention of cerebral ischemic neuronal damage. In addition, hypothalamic BDNF may play an important role in this effect of orexin-A.

Functional alterations of intestinal P-glycoprotein under diabetic conditions.

The maintenance of an appropriate serum concentration of a drug is known to be important for its pharmacological effects and the prevention of unexpected adverse effects. Functional alterations of drug transporters and drug-metabolizing enzymes may influence the serum concentration of drugs through changes in its pharmacokinetics and pharmacodynamics (PK/PD). There are many drug transporters expressed in the brain, liver, kidneys, and intestine including ATP-binding cassette (ABC) transporters and solute carriers (SLCs), which contribute to the systemic distribution of various drugs. Furthermore, the expression and function of P-glycoprotein (P-gp), one of the ABC transporters, is altered by environmental factors such as lifestyle and disease. In this review, we have focused on the influence of functional alterations in the intestinal P-gp on the PK/PD of drugs administered via the oral route under diabetic conditions. Altered expression patterns of intestinal P-gp observed under diabetic conditions exhibit pathological stage-dependency. Furthermore, many factors, such as serum glucose, insulin, nitric oxide, and cytokines, influence expression of intestinal P-gp. Finally, to design appropriate and individually targeted pharmacotherapy, it is necessary to consider the influence of alterations in the intestinal P-gp as well as drug metabolizing enzymes under diabetic conditions based on experimental results obtained from fundamental animal research.