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.

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.

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.

Evidence of a role for spinal HMGB1 in ischemic stress-induced mechanical allodynia in mice.

We have previously showed that spinal high-mobility group box-1 (HMGB1) plays an important role in the induction of central post-stroke pain (CPSP). It has been reported that HMGB1 exacerbates inflammation and pain via TLR4 or RAGE. Furthermore, the relationship between glial cells, such as microglia and astrocytes, involved in pain exacerbation and HMGB1 has also attracted attention. In this study, we investigated whether the interaction between spinal glial cells and HMGB1 signaling, including its receptors TLR4 or RAGE, is directly involved in the induction of CPSP. Spinal HMGB1 expression increased on day 3 after bilateral carotid artery occlusion (BCAO), and spinal microglia and astrocytes were clearly activated. HMGB1 colocalized with neurons, but not with microglia and astrocytes after BCAO. Intrathecal (i.t.) injection of lipopolysaccharides from Rhodobacter sphaeroides (LPS-RS, a TLR4 antagonist) and low-molecular-weight heparin (LMWH, a RAGE antagonist) significantly blocked mechanical allodynia on day 3 after BCAO. BCAO-induced activation of spinal microglia and astrocyte were suppressed by i.t. anti-HMGB1 monoclonal antibody (mAb) and LPS-RS administration. In addition, i.t. injection of NG-nitro-l-arginine methyl ester [a nonselective nitric oxide synthetase (NOS) inhibitor] significantly blocked mechanical allodynia on day 3 after BCAO and i.t. administration of anti-HMGB1 mAb, LPS-RS, and LMWH significantly inhibited the increase of NOS activity in the spinal cord on day 3 after BCAO. These results showed that the interaction between spinal glial cells and HMGB1/TLR4/NOS or HMGB1/RAGE/NOS is directly involved in the induction of CPSP.

RETRACTED: Cerebral ischemia-induced elevation of hepatic inflammatory factors accompanied by glucose intolerance suppresses hypothalamic orexin-A-mediated vagus nerve activation.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the authors following an investigation into data manipulation (Fig.3A-D and Fig.4A-F) by an Investigation Committee at Kobe Gakuin University. Namely: Fig.3A-D and Fig.4A-F ­ numerical disagreement (numbers removed) was found in some parts between the raw data and the article data, hence the significant difference illustrated in the published article was not obtained.

RETRACTED: Possible involvement of the HMGB1/RAGE signaling mechanism in the induction of central post-stroke pain induced by acute global cerebral ischemia.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Authors following an investigation into data manipulation (Figures 2A,B and 5) by an Investigation Committee at Kobe Gakuin University. Namely: Figures 2A and 2B - numerical disagreement (numbers removed) was found in some parts between the raw data and the article data, hence the significant difference illustrated in the published article was not obtained. Figure 5 - multiple numbers of the original raw data were changed, hence significant difference the significant difference illustrated in the published article was not obtained.

Unique action mechanisms of tramadol in global cerebral ischemia-induced mechanical allodynia.

Central poststroke pain is associated with specific somatosensory abnormalities, such as neuropathic pain syndrome. Although central poststroke pain is a serious condition, details pertaining to underlying mechanisms are not well established, making current standard treatments only partially effective. Here, we assessed the effects of tramadol, an analgesic drug mediated by opioid receptors, using a mouse model of global cerebral ischemia. Ischemia was induced by bilateral carotid artery occlusion (30 min) in male ddY mice. Development of hind-paw mechanical allodynia was measured 3 days after bilateral carotid artery occlusion using the von Frey test. Mechanical allodynia was significantly and dose dependently suppressed by intraperitoneal tramadol (10 or 20 mg/kg). These effects, which peaked at 10 min and continued for at least 60 min, were inhibited by naloxone (nonselective opioid receptor antagonist, 1 mg/kg, intraperitoneal). Tramadol antinociception was significantly negated by ß-funaltrexamine (selective µ-opioid receptor antagonist, 20 mg/kg, intraperitoneal), but not naltrindole (selective δ-opioid receptor antagonist, 5 mg/kg, intraperitoneal) or nor-binaltorphimine (selective κ-opioid receptor antagonist, 10 mg/kg, intraperitoneal) after 5 min, by ß-funaltrexamine and nor-binaltorphimine but not naltrindole after 10 min, and by all selective opioid receptor antagonists at 15 and 30 min after tramadol treatment. These results suggested that antinociception induced by tramadol through various opioid receptors was time dependent. Furthermore, it is possible that the opioid receptors involved in tramadol-induced antinociception change over time with the metabolism of this drug.

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.

Involvement of GPR40, a long-chain free fatty acid receptor, in the production of central post-stroke pain after global cerebral ischemia.

Central post-stroke pain (CPSP), one of the complications of cerebral ischemia and neuropathic pain syndrome, is associated with specific somatosensory abnormalities. Although CPSP is a serious problem, detailed underlying mechanisms and standard treatments for CPSP are not well established. In this study, we assessed the role of GPR40, a long-chain fatty acid receptor, showing anti-nociceptive effects, in CPSP. We also examined the role of astrocytes in CPSP due to their effects in mediating the release of polyunsaturated fatty acids, which act as potential GPR40 ligands. The aim of this study was to determine the interactions between CPSP and astrocyte/GPR40 signaling. Male ddY mice were subjected to 30 min of bilateral carotid artery occlusion (BCAO). The development of hind paw mechanical hyperalgesia was measured after BCAO using the von Frey test. Neuronal damage was estimated by histological analysis on day 3 after BCAO. The thresholds for hind paw mechanical hyperalgesia were significantly decreased on days 1-28 after BCAO when compared with those of pre-BCAO assessments. BCAO-induced mechanical hyperalgesia was significantly decreased by intracerebroventricular injection of docosahexaenoic acid or GW9508, a GPR40 agonist; furthermore, these effects were reversed by GW1100, a GPR40 antagonist. The expression levels of glial fibrillary acidic protein, an astrocytic marker, and some free fatty acids were significantly decreased 5h after BCAO, although no effects of BCAO were noted on hypothalamic GPR40 protein expression. Our data show that BCAO-induced mechanical hyperalgesia is possible to be regulated by astrocyte activation and stimulation of GPR40 signaling.

SLC39A9 (ZIP9) regulates zinc homeostasis in the secretory pathway: characterization of the ZIP subfamily I protein in vertebrate cells.

The SLC39A family of zinc transporters can be divided into four subfamilies (I, II, LIV-1, and gufA) in vertebrates, but studies of their functions have been restricted exclusively to members of subfamilies II and LIV-1. In this study, we characterized SLC39A9 (ZIP9), the only member of subfamily I in vertebrates. Confocal microscopy demonstrated that transiently expressed, HA-tagged human ZIP9 (hZIP9-HA) was localized to the trans-Golgi network regardless of zinc status. Disruption of the ZIP9 gene in DT40 cells did not change the growth rate, sensitivity to high zinc and manganese concentrations during long-term culture, or cellular zinc status after short-term incubation with zinc. The alkaline phosphatase activity of ZIP9(-/-) cells did not change in cells cultured in medium containing normal zinc levels. In contrast, the activity of this enzyme decreased in wild-type cells cultured in zinc deficient medium but less so in ZIP9(-/-) cells under these conditions. Stable over-expression of hZIP9-HA moderately decreased alkaline phophatase activity. These results suggest that ZIP9 functions to regulate zinc homeostasis in the secretory pathway without significantly altering cytosolic zinc homeostasis.

Pulmonary endarteritis and subsequent pulmonary embolism associated with clinically silent patent ductus arteriosus.

A 49-year-old man without heart murmur was admitted with fever because of bacteremia following a tooth extraction. Antibiotics rapidly alleviated the fever; however, a small nodule in the pulmonary artery was identified on computed tomography (CT). When the patient experienced chest discomfort with fever, CT demonstrated the absence of the nodule and the appearance of an abnormal lung opacity, and echocardiography showed turbulent retrograde flow in the pulmonary artery. We had the rare opportunity to follow a case of pulmonary bacterial endarteritis and subsequent pulmonary embolism with clinically silent patent ductus arteriosus (PDA) that was confirmed by 3-dimensional CT.

Infective endocarditis and acute purulent pericarditis in a patient with hyperglycemia.

A diabetic patient was admitted to our hospital for infective endocarditis with acute purulent pericarditis and diabetic ketoacidosis. Echocardiography revealed attachment of vegetation to the chordae tendineae in the left ventricle and pericaridial effusion. The vegetation was enlarged and pendulated for a few days despite maximal antimicrobial therapy. Surgical resection was desirable to decrease the risk of embolic complications and cardiovascular collapse. We could not open the heart because of accumulation of purulent pericardial fluid, and right renal infarction was complicated. We believe that the immunocompromised and hypercoagulable state due to diabetes caused these conditions.

Zinc transporters, ZnT5 and ZnT7, are required for the activation of alkaline phosphatases, zinc-requiring enzymes that are glycosylphosphatidylinositol-anchored to the cytoplasmic membrane.

Numerous proteins are properly folded by binding with zinc during their itinerary in the biosynthetic-secretory pathway. Several transporters have been implicated in the zinc entry into secretory compartments from cytosol, but their precise roles are poorly understood. We report here that two zinc transporters (ZnT5 and ZnT7) localized in the secretory apparatus are responsible for loading zinc to alkaline phosphatases (ALPs) that are glycosylphosphatidylinositol-anchored membrane proteins exposed to the extracellular site. Disruption of the ZnT5 gene in DT40 cells decreased the ALP activity to 45% of that in the wild-type cells. Disruption of the ZnT7 gene lowered the ALP activity only by 20%. Disruption of both genes markedly decreased the ALP activity to <5%. Overexpression of human ZnT5 or ZnT7 in DT40 cells deficient in both ZnT5 and ZnT7 genes recovered the ALP activity to the level comparable to that in the wild-type cells. The inactive ALP protein in DT40 cells deficient in both ZnT5 and ZnT7 genes was transported to cytoplasmic membrane like the active ALP protein in the wild-type cells. Thus both ZnT5 and ZnT7 contribute to the conversion of apo-ALP to holo-ALP.

Long-term effects of quinapril with high affinity for tissue angiotensin-converting enzyme after coronary intervention in Japanese.

BACKGROUND: Angiotensin-converting enzyme inhibitors have been shown experimentally to prevent restenosis after balloon injury. We previously reported that quinapril reduced the 6-month restenosis (percent diameter stenosis >or=50%) rate after percutaneous coronary intervention (PCI). However, it was not established whether this favorable outcome was maintained for longer periods. METHODS: This study was a prospective, randomized, open, and non-placebo controlled trial. Patients with coronary artery disease were enrolled after successful coronary balloon angioplasty or stenting. Two hundred and fifty-three patients were randomly assigned to the quinapril (10-20 mg per day) or control groups. The major clinical end points included death, myocardial infarction, cerebrovascular accident, or revascularization (either coronary artery bypass grafting or repeat PCI). These were tabulated according to the intention-to-treat principle. RESULTS: Long-term follow-up was available with a median of 4.8 (interquartile range 4.2-5.1) years after the procedure. The incidence of combined end points of mortality and morbidity (myocardial infarction and cerebrovascular accident) in the quinapril group was lower than that in the control group (6.1% vs 14.8%; relative risk [RR] 0.42, 95% CI 0.18-0.96, P =.033). The overall incidence of end-point events in patients with quinapril also occurred less frequently (29.8% vs 46.7%; RR 0.58, 95% CI 0.38-0.86, P =.007). CONCLUSIONS: These clinical outcomes show that the benefit of quinapril in patients following PCI is maintained for 4 years.