Abstract: BACKGROUND: Oxidants including reactive oxygen species have been indicated to play an important role in the pathogenesis of asthma. Objective: We investigated oxidative status in patients with acute exacerbations of asthma and evaluated the therapeutic response using the D-ROM test which is simple to use and quick. METHODS: We measured reactive oxygen metabolite (ROM) levels in the serum of 42 outpatients with acute exacerbations of asthma, 11 outpatients with stable asthma and 40 healthy subjects using the D-ROM test. Seven inpatients admitted due to acute exacerbations of asthma were also enrolled to evaluate the effects of treatment. Serum eosinophil cationic protein and plasma polymorphonuclear elastase were also measured by EIA or ELISA to evaluate the correlation between inflammation and oxidative status. RESULTS: Serum ROM levels were significantly higher in patients with acute exacerbation of asthma than in patients with stable asthma or healthy subjects. Levels of serum eosinophil cationic protein and plasma polymorphonuclear elastase were increased in acute exacerbation and moderately correlated to ROM levels. Levels of ROM were significantly decreased after treatment with systemic steroids and bronchodilators. CONCLUSION: These findings suggest that acute exacerbation of asthma is associated with increased oxidative stress. Serum ROM levels would partly reflect the inflammation with eosinophils and neutrophils and may be useful as biomarkers of asthma.
Abstract: Orexins/hypocretins are neuropeptides that have various physiological effects, including the regulation of both the feeding behavior neuroendocrine functions and sleep-wakefulness cycle. Recent studies have suggested that the orexin system may also be involved in neuronal damage in the clinical setting and animal experiments. The aim of this study was to examine the role of the hypothalamic orexin-A/hypocretin-1 system in patients with intracerebral hemorrhage (ICH). The CSF orexin-A/hypocretin-1 levels were measured in 11 ICH patients. CSF orexin-A/hypocretin-1 levels were low in ICH patients during the 13 days following the ICH event. The mean CSF orexin-A/hypocretin-1 levels were 61.1+/-22.3 (S.D.) pg/ml (range 27.5-106.9 pg/ml).The decreasing in the CSF orexin-A/hypocretin-1 levels was not related to the severity of ICH. The CSF orexin-A/hypocretin-1 levels were lower in the thalamic hemorrhage patients than those in other patients (48.5+/-23.3 pg/ml vs. 65.2+/-21.2 pg/ml; p=0.03.) These data indicate that orexin-A/hypocretin-1 may therefore play an important role in the various physiological responses including sleep, feeding, and the overall metabolism in ICH patients.
Abstract: Activity-dependent neurotrophic protein (ADNP) was discovered as a novel response gene for vasoactive intestinal polypeptide. We have reported that PACAP strongly stimulated ADNP mRNA expression in a mouse neuron/glial cell culture; however, the distribution of ADNP in the brain and its possible co-expression with the PACAP receptor (PAC1R) are unknown. In this study, the specificity of the ADNP antibody used in subsequent immunohistochemistry experiments was first characterized. Mouse brain lysates were analysed by Western blot, with an ADNP-immunopositive signal corresponding to the expected molecular weight of ADNP detected as a 124 kDa band. Immunohistochemical staining to identify ADNP and PAC1R immunoreactivity in mouse brain was then performed. ADNP immunoreactive cells were observed in the cerebral cortex, cerebellum, hippocampus, and medial septum of brain slices. ADNP-immunoreactive cells in the cerebral cortex were multi-polar-shaped and co-immunostained with the astrocyte marker, glial fibrillary acidic protein (GFAP). ADNP-immunoreactive cells in the cerebellum were found to surround Purkinje cells and showed GFAP immunoreactivity. On the other hand, ADNP-immunoreactive cells in the hippocampus and septum were round in shape and co-immunostained with the neuron marker, neuron-specific enorase. All of the ADNP-immunopositive cells co-localized with PAC1R immunoreactivity. These observations suggest that ADNP is expressed in both astrocytes and neurons, and that ADNP expression may be regulated by PACAP.
Abstract: Edaravone (MCI-186) is a novel synthetic free radical scavenger intended to have neuroprotective effect against ischemic insult. It is currently used on patients with cerebral infarction. Here, we note beneficial pharmaceutical effects of edaravone in rat experimental traumatic brain injury. Under specific experimental conditions, edaravone minimized traumatic brain injury by functioning as a synthetic antioxidant. Clinical trials testing the efficacy of edaravone are warranted.
Abstract: Oxidative stress and DNA oxidation play important roles in the induction of ischemic neuronal cell death. However, the subcellular source of oxidized DNA detected by 8-hydroxy-2'-deoxyguanosine (8-OHdG) after ischemia has not been clarified although it is known to increase in the brain after ischemia. One-hour transient ischemia of the middle cerebral artery was induced in mice utilizing an intraluminal filament. The occurrence of superoxide anion as an ethidium (Et) signal, 8-OHdG, cytochrome c release and neuronal cell death were examined using immunohistological and biochemical techniques in sham-operated control (0h) and 1, 3, 6, 24, or 96h after reperfusion. Et signals were prominent in the cortical neurons of ipsilateral hemisphere 3h after reperfusion. Strong 8-OHdG immunoreactivity was observed 3-6h after reperfusion. Immunoassays after cell fractionation revealed a significant increase of 8-OHdG in mitochondria 6h after reperfusion. Immunohistochemistry revealed that the 8-OHdG immunoreactivity colocalized with a neuronal marker, microfilament 200 and a mitochondrial marker, cytochrome oxidase subunit I. Cytochrome c rose in cytoplasm at 6h and TUNEL-positive neurons noted 6-24h after ischemia. The present results suggest the possibility that the mitochondrial damage including mitochondrial DNA oxidation might be responsible for the induction of ischemic neuronal cell death.
Abstract: We evaluated the effects of a novel pharmacological brain cooling (PBC) method with indomethacin (IND), a nonselective cyclooxygenase inhibitor, without the use of cooling blankets in patients with hemorrhagic stroke. Forty-six patients with hemorrhagic stroke (subarachnoid hemorrhage; n = 35, intracerebral hemorrhage; n = 11) were enrolled in this study. Brain temperature was measured directly with a temperature sensor. Patients were cooled by administering transrectal IND (100 mg) and a modified nasopharyngeal cooling method (positive selective brain cooling) initially. Brain temperature was controlled with IND 6 mg/kg/day for 14 days. Cerebrospinal fluid concentrations of interleukin-1beta (CSF IL-1beta) and serum bilirubin levels were measured at 1, 2, 4, and 7 days. The incidence of complicating symptomatic vasospasm after subarachnoid hemorrhage was lower than in non-PBC patients. CSF IL-1beta and serum bilirubin levels were suppressed in treated patients. IND has several beneficial effects on damaged brain tissues (anticytokine, free radical scavenger, antiprostaglandin effects, etc.) and prevents initial and secondary brain damage. PBC treatment for hemorrhagic stroke in patients appears to yield favorable results by acting as an antiinflammatory cytokine and reducing oxidative stress.
Abstract: A stable model of neuronal damage after ischemia is needed in mice to enable progression of transgenic strategies. We performed transient global ischemia induced by common carotid artery occlusions with and without maintaining normal rectal temperature (Trec) in order to determine the importance of body temperature control during ischemia. We measured brain temperature (Tb) during ischemia/reperfusion. Mice with normothermia (Trec within +/- 1 degrees C) had increased mortality and neuronal cell death in the CA1 region of hippocampus, which did not occur in hypothermic animals. If the Trec was kept within +/- 1 degrees C, the Tb decreased during ischemia. After reperfusion, Tb in the normothermia group developed hyperthermia, which reached > 40 degrees C and was > 2 degrees C higher than Trec. We suggest that tightly controlled normothermia and prevention of hypothermia (Trec) during ischemia are important factors in the development of a stable neuronal damage model in mice.
Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to decrease ischemic neuronal damage and increase IL-6 secretion in rats. However, the mechanisms underlying neuroprotection are still to be fully elucidated. The present study was designed to investigate the role played by PACAP and IL-6 in mediating neuroprotection after ischemia in a null mouse. Infarct volume, neurological deficits, and cytochrome c in cytoplasm were higher in PACAP(+/-) and PACAP(-/-) mice than in PACAP(+/+) animals after focal ischemia, although the severity of response was ameliorated by the injection of PACAP38. A decrease in mitochondrial bcl-2 was also accentuated in PACAP(+/-) and PACAP(-/-) mice, but the decrease could be prevented by PACAP38 injection. PACAP receptor 1 (PAC1R) immunoreactivity was colocalized with IL-6 immunoreactivity in neurons, although the intensity of IL-6 immunoreactivity in PACAP(+/-) mice was less than that in PACAP(+/+) animals. IL-6 levels increased in response to PACAP38 injection, an effect that was canceled by cotreatment with the PAC1R antagonist. However, unlike in wild-type controls, PACAP38 treatment did not reduce the infarction in IL-6 null mice. To clarify the signaling pathway associated with the activity of PACAP and IL-6, phosphorylated STAT (signal transducer and activator of transcription) 3, ERK (extracellular signal-regulated kinase), and AKT levels were examined in PACAP(+/-) and IL-6 null mice after ischemia. Lower levels of pSTAT3 and pERK were observed in the PACAP(+/-) mice, whereas a reduction in pSTAT3 was recorded in the IL-6 null mice. These results suggest that PACAP prevents neuronal cell death after ischemia via a signaling mechanism involving IL-6.
Abstract: OBJECTIVE: Severe global ischemia often results in severe damage to the central nervous system of survivors. Hind-limb paralysis is a common deficit caused by global ischemia. Until recently, most studies of global ischemia of the central nervous system have examined either the brain or spinal cord, but not both. Spinal cord damage specifically after global ischemia has not been studied in detail. Because the exact nature of the neuronal damage to the spinal cord and the differences in neuronal damage between the brain and spinal cord after global ischemia are poorly understood, we developed a new global ischemia model in the rat and specifically studied spinal cord damage after global ischemia. Further, we compared the different forms of neuronal damage between the brain and spinal cord after global ischemia. DESIGN: Randomized, controlled study using three different global ischemia models in the rat. SETTING: University research laboratory. SUBJECTS: Male, adult Sprague-Dawley rats (300 g). INTERVENTIONS: Animals were divided into three experimental groups, group A (n = 6, survived for 7 days), 12 mins of hemorrhagic shock; group B (n = 6, survived for 7 days), 5 mins of cardiac arrest; or group C (n = 6, each for 6 hrs, 12 hrs, 1 day, 3 days, and 7 days), 7 mins of hemorrhagic shock and 5 mins of cardiac arrest. Motor deficit of the hind limbs was studied 6 hrs to 7 days after resuscitation. Also, nonoperated animals (n = 6) were used as the control. Histologic analysis (hematoxylin and eosin, Fluoro-Jade B, terminal deoxynucleotidyl transferase- mediated dUTP end-labeling [TUNEL], Klüver-Barrera) and ultrastructural analysis using electron microscopy were performed on samples from the CA1 region of the hippocampus and lumbar spinal cord. Demyelination of the white matter of the lumbar spinal cord was analyzed semiquantitatively using Scion Image software. MAIN RESULTS: No paraplegic animals were observed in either group A or B. All group C animals showed severe hind-limb paralysis. Severe neuronal damage was found in the CA1 region of the hippocampus in all groups, and the state of delayed neuronal cell death was similar among the three groups. Neuronal damage in the lumbar spinal cord was detected only in group C animals, mainly in the dorsal horn and intermediate gray matter. Demyelination was prominent in the ventral and ventrolateral white matter in group C. A significant difference was observed between control and group C rats with Scion Image software. Ultrastructural analysis revealed extensive necrotic cell death in the intermediate gray matter in the lumbar spinal cord in group C rats. CONCLUSION: The combination in the global ischemia model (i.e., hemorrhagic shock followed by cardiac arrest) caused severe neuronal damage in the central nervous system. Thereby, hind-limb paralysis after global ischemia might result from spinal cord damage. These results suggest that therapeutic strategies for preventing spinal cord injury are necessary when treating patients with severe global ischemia.
Abstract: Lipid peroxidation is caused by reactive oxygen species (ROS) and is involved in traumatic brain injury (TBI). Consequently, a therapeutic strategy for TBI may be to control lipid peroxidation. The only drug approved to date for blocking lipid peroxidation is edaravone (MCI-186), a novel free-radical scavenger shown to exert neuroprotective effects in acute ischemic stroke. Although edaravone scavenges hydroxyl and nitric oxide radicals, its effect on alkoxyl radicals (OR-), which also contribute to lipid peroxidation, is unknown. To date, the study of free radicals in blood has been severely hampered by technical difficulties in their detection. We used an in vitro and ex vivo electron spin resonance (ESR) method employing 5,5-dimethyl-1-pyrroline-N-oxide as a spin trap to investigate whether edaravone can scavenge OR-. By mixing either methemoglobin or human blood with tert-butyl hydroperoxide, we found that this technique can detect OR- generated in vitro. We also found that generated OR- can be completely absorbed by administration of edaravone in vitro (400 microM). Analysis of jugular venous blood collected from 17 TBI patients immediately before and 20 minutes after the administration of edaravone (30 mg, i.v.) revealed higher OR- levels in the untreated patients blood than in normal control blood samples. However, treatment with edaravone suppressed these OR- levels by 24.6% (radical intensity = 71.1 +/- 5.2-53.6 +/- 5.2; p < 0.01). Thus, edaravone can scavenge OR- and significantly reduce levels of these radicals in TBI patients. The novel ex vivo ESR method described here provides a valuable clinical measure of oxidative stress.
Abstract: Orexins/hypocretins are neuropeptides that have various physiological effects, including the regulation of feeding behavior, neuroendocrine functions and sleep-wake cycles. Recent studies have suggested that the orexin system may also be involved in brain ischemic reactions. It is also known that changes in sleep patterns, energy homeostasis and neuroendocrine functions are often occur in neurological conditions associated brain ischemia. In the current study, we investigated the time-dependent changes in cerebrospinal fluid (CSF) orexin-A concentration and the expression of the orexin-1 receptor (OX1R) in the rat hippocampus after global ischemia-reperfusion (5 min cardiopulmonary arrest), which is known to induce delayed cell death in the CA1 region of the hippocampus. The CSF orexin-A concentration was elevated transiently at 24 h after ischemia. On days 2 and 4 after ischemia, CSF orexin concentrations were significantly reduced relative to the baseline, and returned to the baseline level by day 7. These changes were correlated with increased expression of OX1R in the CA1 on days 1 and 2 post-ischemia. These results suggest that dynamics of orexin signaling observed may have functional roles for neuronal damage associated with transient ischemia.
Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. PACAP prevents ischemic delayed neuronal cell death (apoptosis) in the hippocampus. PACAP inhibits the activity of the mitogen-activated protein kinase (MAPK) family, especially JNK/SAPK and p38, thereby protecting against apoptotic cell death. After the ischemia-reperfusion, both pyramidal cells and astrocytes increased their expression of the PACAP receptor (PAC1-R). Reactive astrocytes increased their expression of PAC1-R, released interleukin-6 (IL-6) that is a proinflammatory cytokine with both differentiation and growth-promoting effects for a variety of target cell types, and thereby protected neurons from apoptosis. These results suggest that PACAP itself and PACAP-stimulated secretion of IL-6 synergistically inhibit apoptotic cell death in the hippocampus. The PAC1-R is expressed in the neuroepithelial cells from early developmental stages and in various brain regions during development. We have recently found that PACAP, at physiological concentrations, induces differentiation of mouse neural stem cells into astrocytes. Neural stem cells were prepared from the telencephalon of mouse embryos and cultured with basic fibroblast growth factor. The PAC1-R immunoreactivity was demonstrated in the neural stem cells. When neural stem cells were exposed to PACAP, about half of these cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. This phenomenon was significantly antagonized by a PAC1-R antagonist (PACAP6-38), indicating that PACAP induces differentiation of neural stem cell into astrocytes. Other our physiological studies have demonstrated that PACAP acts on PAC1-R in mouse neural stem cells and its signal is transmitted to the PAC1-R-coupled G protein Gq but not to Gs. These findings strongly suggest that PACAP plays very important roles in neuroprotection in adult brain as well as astrocyte differentiation during development.
Abstract: Brain damage is worsened by hyperthermia and prevented by hypothermia. Conventional hypothermia is a non-selective brain cooling method that employs cooling blankets to achieve surface cooling. This complicated method sometimes induces unfavorable systemic complications. We have developed a positive selective brain cooling (PSBC) method to control brain temperature quickly and safely following brain injury. Brain temperature was measured in patients with a ventriculostomy CAMINO catheter. A Foley balloon catheter was inserted to direct chilled air (8 to 12 L/min) into each side of the nasal cavity. The chilled air was exhaled through the oral cavity. In most patients, PSBC maintained normal brain temperature. This new technique provides quick induction of brain temperature control and does not require special facilities.
Abstract: The aim of this study was to examine the role of the hypothalamic hypocretin/orexin system in complications of delayed ischemic neuronal deficit (DIND) resulting from symptomatic vasospasm in patients with aneurysmal subarachnoid hemorrhage (SAH). CSF hypocretin-1/orexin-A levels were measured in 15 SAH patients. DIND complications occurred in seven patients with symptomatic vasospasm. Hypocretin-1/orexin-A levels were low in SAH patients during the 10 days following the SAH event. CSF hypocretin-1/orexin-A levels were lower in patients with DIND complications than in those who did not develop DIND. A significant transient decline in CSF hypocretin-1/orexin-A levels was also observed at the onset of DIND in all patients with symptomatic vasospasm. The reduced hypocretin/orexin production observed in SAH patients may reflect reduced brain function due to the decrease in cerebral blood flow. These results, taken together with recent experimental findings in rats that indicate hypocretin receptor 1 (orexin 1 receptor) mRNA and protein are elevated following middle cerebral artery occlusion, suggest that a reduction in hypocretin/orexin production in SAH and DIND patients is associated with alterations in brain hypocretin/orexin signaling in response to ischemia.
Abstract: Bilirubin, a powerful endogenous antioxidant, is one of the catabolites of heme oxygenases (HOs). In this study, the plasma bilirubin concentration was measured to establish bilirubin kinesis after traumatic brain injury (TBI). Furthermore, in in vitro studies, the free radical scavenging activity and antioxidant potency of bilirubin was also investigated at various concentrations, including physiological ones. Indirect plasma bilirubin was measured in 25 patients on days 1, 2, 3 and 4 after presentation with TBI. The ability of bilirubin to scavenge the hydroxyl (OH) and 1,1-diphenyl-2-picrylhyrazyl (DPPH) radicals, and its antioxidant potency, were also analyzed using electron spin resonance (ESR) and the bioantioxidant power (BAP) methods, respectively. Plasma bilirubin levels were significantly higher on days 2, 3 and 4 than on patient admission (day 1; p < 0.05). ESR and BAP results revealed that bilirubin has direct OH and DPPH radical scavenging activities and potent antioxidant effects in vitro at physiological concentrations. These data indicate that physiological concentrations of bilirubin have antioxidant properties and that it constitutes one of the biological defense mechanisms in neurotrauma patients.
Abstract: The expression of tumor necrosis factor alpha (TNFalpha) increases and participates in several central nervous system (CNS) disorders. However, its expression after transient middle cerebral artery occlusion (tMCAO) in mice is not fully discussed yet. Therefore, we examined gene expression and protein localization of TNFalpha in brain using real-time polymerase chain reaction (PCR) and immunostaining after 1 h tMCAO in mice. After 1 h of ischemic conditions, we observed an increase in the expression of TNFalpha mRNA from basal level. While the expression decreased immediately to control level after reperfusion, it increased again significantly at 24 and 48 h after tMCAO. TNFalpha-like immunoreactivity (TNFalpha-LI) was slightly detected in fibrous structures of the neurons before ischemia. After ischemia, TNFalpha-LI spread widely to the soma of neurons and became more abundant in the nerve fibers, including axonal and dendritic processes. Moreover, TNFalpha-LI was also expressed in the oligodendrocytes and, occasionally, in microglia/macrophages, but not in astrocytes 24 h after tMCAO. These results suggest that TNFalpha shows biphasic expression that corresponds with ischemia and reperfusion, and might play a role in various cells to regulate CNS disorders such as neuronal and oligodendritic cell death after transient ischemia.
Abstract: It has been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in preventing neuronal cell death and is also a potent vasodilator. Cerebral hypotension and hypoperfusion during cerebral ischemia and neurodegenerative diseases are well known as some of the negative factors which aggravate neuronal cell death. Nevertheless, the effect of PACAP on the cerebral circulation was not understood well. Therefore, in the present study, we determined the mean arterial blood pressure (MBP), regional cerebral blood flow (rCBF) and cerebral oxygen content (pO2) in mice, and estimated the therapeutically useful doses of PACAP. Under barbiturate anesthesia, polyethylene tubes were inserted into mice to monitor MBP and to administer PACAP (5 x 10(-13)-5 x 10(-8) mol/kg) or vasoactive intestinal peptide (VIP; 5 x 10(-12) and 5 x 10(-9) mol/kg). Then, MBP, rCBF and cerebral pO2 were simultaneously measured in the mice. PACAP (5 x 10(-10)-5 x 10(-9) mol/kg) injections transiently decreased MBP, and cerebral pO2. PACAP (5 x 10(-8) mol/kg) injections produced a long-lasting potent decline of MBP, rCBF and cerebral pO2. Therefore, PACAP should be applied at low doses which do not influence the MBP and cerebral circulation to determine the therapeutically useful doses of PACAP for neuroprotection.
Abstract: Ischemic delayed neuronal cell death (apoptosis) in the hippocampus is prevented by PACAP. PACAP inhibits the increasing activity of the MAP kinase family, especially on JNK/SAPK and p38, thereby protecting against apoptotic cell death. After the ischemia-reperfusion, both pyramidal cells and astrocytes increased their expression of PACAP receptors (PAC1-Rs). The pyramidal cells degenerated but reactive astrocytes increased their expression of PAC1-R. PACAP does not only inhibit apoptotic cell death directly, but also affects astrocytes through PAC1-Rs. Interleukin-6 (IL-6), produced in astrocytes, has several effects on the prevention of brain ischemia and trauma and stimulating neuronal growth. IL-6 secretion into the CSF was markedly stimulated after PACAP infusion, suggesting that PACAP stimulates IL-6 secretion from astrocytes. We studied the effects of PACAP on the wild type and IL-6 KO mice after brain ischemia. In wild-type animals, PACAP significantly inhibited cell death, but in IL-6 KO animals, no cytoprotective effect of PACAP was seen. These results suggest that PACAP inhibits apoptotic cell death partly through IL-6. It is considered that PACAP itself and IL-6, stimulated secretion by PACAP, both synergistically inhibit the JNK/SAPK and p38 signaling pathway, thereby protecting against neuronal cell death.
Abstract: NO is a putative neurotransmitter and neuromodulator in the brain. NO is not functioning as a direct neurotoxin. NO with the superoxide radical product peroxynitrite (ONOO-) is much more cytotoxic under tissue impairment conditions. Caspase-3, a potent effector of apoptosis that is triggered via several different signaling pathways, may play a very important role in neuronal cell death caused by various brain injuries. The relationship between mouse caspase-3 and peroxynitrite remains unclear. In the present study, we examined the in vivo expression of 3-nitrotyrosine (a metabolite of peroxinitrite) and caspase-3 after cerebral ischemia produced in a global ischemia model using mice (i.e., a cardiac arrest model). 3-nitrotyrosine immunoreactivity was detected in neuronal cells in the hippocampal dentate nucleus, and cortical regions starting at 12 hrs after ischemia. In particular, numerous neuronal cells were highly immunoreactive for 3-nitrotyrosine in the cortical regions. In hippocampal CA1 pyramidal neurons, 3-nitrotyrosine immunoreactivity was detected from 24 hrs. Caspase-3 immunopositive cells were observed in approximately the same area in which the positive reaction to the anti-nitrotyrosine antibody was observed. These results provide direct evidence for the induction of 3-nitrotyrosine and caspase-3 expression in vivo in an ischemia model using mice. The present findings suggest that peroxynitrite generated by cerebral ischemia/ reperfusion was strongly cytotoxic and induced neuronal cell death (apoptosis) mediated by caspase-3.
Abstract: Three hot water extracts of black tea, green tea and powdered green tea and five Chinese medicines (Shosaiko-tou, Orengedoku-tou, Goshuyu-tou, Choto-san, Keishininjinn-tou) were investigated for their ability to modify nitric oxide (NO) production by lipopolysaccharide (LPS)-stimulated mouse macrophage-like Raw 264.7 cells, and for their cytotoxicity, radical intensity and scavenging activity. All eight materials significantly reduced the extracellular concentration of NO in the LPS-stimulated Raw 264.7 cells. ESR spectroscopy shows that tea extracts, which had higher cytotoxicity, generated higher amounts of radicals, and more efficiently scavenged O2- (generated by hypoxanthine-xanthine oxidase reaction), hydroxyl radical (generated by Fenton reaction) and NO (generated by 1-hydroxyl-2-oxo-3-(N-3-methyl-3-aminopropyl)-3-methyl-1-triazene) than Chinese medicines. Close association between the radical intensity and radical scavenging activity suggests their bimodal (anti-oxidant and pro-oxidant) action. Pretreatment of mice with tea extracts significantly reduced the lethality of Escherichia coli-infection. All tea extracts showed no apparent anti-HIV activity. The present study demonstrates, for the first time, several attractive features of tea extracts in comparison with Chinese medicines, suggesting the possible application of the tea extracts for radical-mediated diseases.
Abstract: Choto-san is a kampo medicine that is widely used in patients with cerebral infarction, but the details of its mechanism of action remain unclear. We examined the neuroprotective effects of Choto-san using an experimental cerebral ischemia model (i.e., a rat cardiac arrest model). We also investigated the ability of Choto-san to eliminate or inhibit the activity of free radicals. It was found that Choto-san significantly prevents delayed neuronal cell death after ischemic reperfusion. Electron spin resonance demonstrated that the formation of hydroxyl- and superoxide-DMPO spin adducts were inhibited by Choto-san. The results of this study indicated that Choto-san prevents delayed neuronal cell death in the hippocampal CA1 region after ischemia. Direct free radical scavenging activity is among the pharmacological effects of Choto-san.
Abstract: Interleukin-1 (IL-1) contributes to ischemic neurodegeneration. However, the mechanisms regulating action of IL-1 are still poorly understood. In order to clarify this central issue, mice that were gene deficient both IL-1alpha and beta (IL-1 KO) and wild-type mice were subjected to 1 hour transient middle cerebral artery occlusion (tMCAO). The concentration of 8-hydroxy deoxyguanosine (8OHdG) which is considered to be a reliable oxidative DNA damage by superoxide anion, in brain and of total nitric oxide (NO) in plasma were determined by use of HPLC. Twenty-four hours after tMCAO, the ratio of 8OHdG to dG in the ipsilateral hemisphere of wild-type mice were 2.24 x 10(-3) and 4.41 x 10(-3) in the neocortex and striatum, respectively. The concentration of 8OHdG in the ipsilateral hemisphere of the wild-type mice was higher than that of the IL-1 KO mice. The concentration of total NO in the plasma of IL-1 KO mice was also lower than that of the wild-type 24 hours after tMCAO. These results strongly suggest that IL-1 is participated in generating reactive oxygen spices and it aggravates and induces the ischemic neuronal cell death.(183 words).
Abstract: Bilirubin (Bil) is the end product of heme catabolism. The production of Bil reflects heme oxygenase-1 expression in response to oxidative stress in various diseases. To assess the role of Bil as a marker of oxidative stress in cases of brain damage, we measured serum Bil concentrations in patients with hemorrhagic stroke. Serum levels of total Bil were measured in 20 subarachnoid hemorrhage patients with symptomatic vasospasms and in 23 patients with intracerebral hemorrhage; concentrations were measured every day for 14 consecutive days. Serum Bil levels were significantly elevated in the early phases in both groups. Moreover, transient elevation was observed on the day prior to the observation of clinical manifestations of symptomatic vasospasm after SAH. Bil, known to be a powerful antioxidant, was induced after hemorrhagic stroke, reflecting the intensity of oxidative stress. Plasma Bil concentrations might serve as a useful marker of oxidative stress in hemorrhagic stroke patients.
Abstract: We have demonstrated that ischemic neuronal death (apoptosis) of rat CA1 region of the hippocampus was prevented by infusing pituitary adenylate cyclase-activating polypeptide (PACAP) either intracerebroventricularly or intravenously. We have also demonstrated that the activity of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38, was increased in the hippocampus within 1-6 h after brain ischemia. The molecular mechanisms underlying the PACAP anti-apoptotic effect were demonstrated in this study. Ischemic stress had a strong influence on MAP kinase family, especially on JNK/SAPK and p38. PACAP inhibited the activation of JNK/SAPK and p38 after ischemic stress, while ERK is not suppressed. These findings suggest that PACAP inhibits the JNK/SAPK and p38 signaling pathways, thereby protecting neurons against apoptosis.
Abstract: Nonsteroidal anti-inflammatory drugs (NSAIDs), which are used widely to manage pain, are known to inhibit cyclooxygenase, but details of the mechanisms of NSAID action remain unclear. We investigated the ability of three NSAIDs (indomethacin, loxoprofen, and etodolac) to eliminate and inhibit free radicals. Superoxide scavenging activity of these NSAIDs was measured in vitro by electron spin resonance spectrometry using 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) as a spin trap. Electron spin resonance demonstrated that formation of superoxide-DMPO spin adduct was completely inhibited by two nonselective cyclooxygenase inhibitors, indomethacin (3 mmol) and loxoprofen (3 mmol). The electron spin resonance study also demonstrated that the formation of superoxide-DMPO spin adduct was strongly inhibited by a selective cyclooxygenase-2 inhibitor, etodolac, in a concentration-dependent manner. These results indicate that NSAIDs, including indomethacin, loxoprofen, and etodolac, have direct superoxide scavenging activity.
Abstract: Impotence commonly occurs after events such as acute myocardial infarction, coronary bypass, head trauma, and cerebral bleeding, including subarachnoid hemorrhage. We hypothesize that the hypoxia accompanying these events could damage the blood-testis barrier (BTB) and so cause testicular dysfunction, a possible cause of impotence. We examined the effect of cardiac arrest in mice on testis weight and various aspects of BTB function. Testis weight was decreased by about 24% 12 hours after cardiac arrest but had recovered fully by day 7. The testis/serum ratio for albumin was increased 12 hours after arrest, showing a disruption in the vascular BTB with recovery by 24 hours. The testis/serum ratio for sucrose was not consistently elevated, showing that the Sertoli cell BTB remained intact. The testis/serum ratio for verapamil was increased on day 3 of cardiac arrest, suggesting impaired function of the BTB's p-glycoprotein efflux transporter. Transporters for pituitary adenylate cyclase activating polypeptide and tumor necrosis factor-alpha were not affected by cardiac arrest. These results show that cardiac arrest affects testis weight and some aspects of BTB function. Such changes might have long-term effects on testicular function.
Abstract: Oxygen free radicals have been proposed to be one of the major mechanisms of secondary brain damage in traumatic brain injury. Protective effect by vitamin E against oxidative damage has attracted much attention. Recent studies have demonstrated a novel vitamin E derivative, 2-(alpha-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol (TMG), has excellent water-solubility and antioxidant activity. The purpose of this study was to investigate protective effects of TMG on experimental traumatic brain edema (BE). Male Wistar rats were anaesthetized with chloral hydrate. Traumatic BE was produced by a cortical freezing lesion. Animals were separated into three groups: saline-treated rats (n = 4), TMG-treated (4 mg/kg) rats (n = 7) and TMG-treated (40 mg/kg) rats (n = 8). Saline or TMG was administered intravenously before lesion production. Animals were sacrificed at 6 hours after lesion production and the brain water content was determined by the dry-wet weight method. Half-life of TMG after intravenous administration of TMG was also investigated. The half life of TMG was approximately 5 minutes. TMG (40 mg/kg) significantly attenuated BE following cryogenic brain injury (p < 0.01). In conclusion, this preliminary study has demonstrated that a novel vitamin E derivative might be promising in the treatment of traumatic BE.
Abstract: Oxygen free radicals have been implicated in the genesis of traumatic brain injury and brain edema (BE). Recent studies have suggested that hydroxyl radical can initiate lipid peroxidation, thus producing lipid-free radicals that may become important sources of singlet oxygen. L-histidine, a singlet oxygen scavenger, potentially can be used to treat BE. In this study we investigated the effects of L-histidine and D-histidine on BE following cryogenic injury in rats. Male Wistar rats were anaesthetized with chloral hydrate. Vasogenic BE was produced by a cortical freezing lesion. Generation of singlet oxygen from photoactivation of rose bengal was studied by electron spin resonance (ESR). Animals were separated into four groups: sham rats (n = 5), saline-treated rats (n = 10), L-histidine treated rats (n = 6) and D-histidine treated rats (n = 7). Each agent (100 mg/kg) was administered intravenously at 30 minutes before lesion production. Animals were sacrificed at 24 hours after lesion production and the brain water content was determined by the dry-wet weight method. L-histidine had no effect on rectal and brain temperature. Election Spin Resonance studies demonstrated that L-histidine is a singlet oxygen scavenger. L-histidine but not D-histidine significantly attenuated BE following cryogenic injury (p < 0.05). In conclusion, L-histidine is useful in the treatment of traumatic BE.
Abstract: We report a case of a patient with traumatic carotid cavernous fistula (CCF) caused by transnasal-transsphenoidal surgery, who was successfully treated using detachable coils. A 47-year-old man was admitted to our hospital because of severe headache. He was confirmed to have a nonfunctioning pituitary adenoma with presellar-type sphenoid sinus. Cerebral angiography initially disclosed no vascular lesions. A transnasal-transsphenoidal adenomectomy was performed. When the anterior wall of the sphenoid sinus was dissected with a chisel, the chisel deeply stuck into the posterolateral part of the sinus. Profuse arterial bleeding was observed through the sphenoid sinus. The bleeding was stopped easily by compression and packing with bone wax. The operation was continued, the sellar floor was opened widely and the tumor was removed subtotally. The medial wall of the cavernous sinus was intact. Histological examination revealed a pituitary adenoma. Immediately after surgery, the patient noticed a bruit. He developed chemosis and abducent palsy on the right side. Cerebral angiography displayed a high-flow CCF, which was attributed to the carotid artery injury caused by the transnasal-transsphenoidal surgery. The CCF disappeared after two-staged embolization using detachable coils, 1st transvenous and 2nd transarterial. Ten months later, cerebral angiography showed persistent occlusion of the fistula, and the patient experienced no tumor recurrence. It is suggested that drilling is a safer procedure than using a chisel for dissection of a sphenoid sinus with incomplete pneumatization. Endovascular treatment using detachable coils proved useful to manage the CCF, an unusual complication of transsphenoidal surgery.
Abstract: Neuropsychological disturbances following surgery for anterior communicating artery aneurysms were analyzed in 26 patients (11 males, 15 females) using the Hasegawa dementia scale-revised (HDS-R) over a 3-year period. The patients were aged from 34 to 76 years (mean 54.1 years). Lesions in the frontal lobe were evaluated using computed tomography (CT). Twenty-three patients had symptoms over the course. Four patients had basal forebrain lesion, five had ventral frontal lesion, and 12 had no lesion. Patients with basal forebrain lesion and no lesion tended to show disorientation. The mean HDS-R score was 10.2 points in the patients with ventral frontal lesion, and 13.5 points in the patients with no lesion. These scores are within the range for dementia. The mean HDS-R score in patients with basal forebrain and striate lesions was over 25 points and beyond the range for dementia. Significant differences were observed in the HDS-R score between patients with ventral frontal lesion and basal forebrain lesion, and between patients with no lesion and basal forebrain lesion (p < 0.05). Recovery from neuropsychological disturbances was poorer in patients with ventral frontal lesion and no lesion compared to those with basal forebrain and striate lesions, and their symptoms tended to persist.
Abstract: A 37-year-old female died of cerebral vasospasm as a complication of rewarming following hypothermia therapy for severe head injury. She presented with severe consciousness disturbance and anisocoria after falling down a flight of stairs. Computed tomography (CT) revealed a right acute subdural hematoma and temporal contusion. Following surgery, mild hypothermia was started and rewarming was completed by the 11th day. Neurological examination showed no abnormalities, but intracranial pressure (ICP) suddenly increased and she manifested anisocoria on the 13th day. Repeat CT revealed a low density area in the right middle cerebral artery region and cerebral angiography showed diffuse narrowing of the main arterial trunks. A cerebrospinal fluid (CSF) sample was collected using an intraventricular ICP monitoring catheter. The CSF level of 8-hydroxy-2'-deoxyguanosine was elevated during the rewarming period, indicating substantial deoxyribonucleic acid (DNA) oxidation. She died on the 15th day due to uncontrollable ICP. Histological examination at autopsy of the narrowed artery found the waving phenomenon in the internal elastic lamina and invasion of inflammatory cells into the adventitia. These findings constitute the possible evidence that free-radical-mediated oxidative DNA damage may be important in the genesis of severe vasospasm due to rewarming following hypothermia.
Abstract: Micro-pressure-suction-irrigation system (MPSIS), introduced by Luedecke et al, is an instrumentation for the direct transnasal pituitary procedure. We improved this system for use in Japan. The irrigation system can effectively clean the operating field by one-hand manipulation and dissect tumor tissue by its rapid flow. The pressure of suction and irrigation can be adjusted respectively by a device in the handpiece. The MPSIS is applicable to different stages of intervention because it is equipped with separate tips of various diameters, lengths and angles. This system is especially useful in combination with a micromirror or an endoscopy for direct inspection of the eccentric tumor sites such as the cavernous sinus, the upper part of the planum sphenoidale, or the posterior suprasellar regions. The use of the MPSIS helps to avoid injury to normal tissue structures, and prevents tiny soft microadenoma from being lost during preparation. We have proved the suitability and usefulness of the MPSIS in 23 surgical interventions for transnasal microsurgery of pituitary adenomas.
Abstract: Transient global ischemia caused by 5 min of cardiac arrest induced delayed neuronal cell death (apoptosis) in the CA1 region of the rat hippocampus. To characterize the molecular mechanisms that regulate apoptosis in vivo, the contributions to cell death of mitogen-activated protein kinase family members were examined in the hippocampal region after brain ischemia-reperfusion. Ischemia-reperfusion led to a strong activation of the JNK/SAPK (c-Jun NH2-terminal protein kinase/stress activated protein kinase), ERK (extracellular signal-regulated kinase), and p38 enzymes. These results with other previous studies suggest that the activation of JNK/SAPK in accordance with p38 contributes to the induction of apoptosis in CA1 neurons.
Abstract: The incidence of metastatic brain tumors is increasing because of the recent progress in the detection and management of primary cancer. However, metastatic skull tumors from cancers associated with giant subcutaneous mass lesions are rare. We present four patients with metastatic skull tumors: two from hepatic cancer, one from lung cancer, and one from mamma cancer. In these patients, plain skull X-ray and bone CT showed osteolytic lesions. Angiograms revealed a tumor stain fed by abnormal vessels from the external carotid artery. MRI demonstrated masses with marked homogeneous enhancement with the "dural tail sign" in the dura adjacent to the tumors in three skull tumors from hepatic and mamma cancers, and a mass with slightly enhancement without the "dural tail sign" in a skull tumor from lung cancer. At surgery, hemorrhagic well-demarcated tumors were totally removed. The histological diagnosis was skull metastases from cancers in all cases. In cases with the "dural tail sign" on MRI, no tumor cells were seen in the inner layer of the dura and the dura adjacent to the tumors. It is possible that the "dural tail" is due to increased vascular permeability of the dural vessels. The recurrence of these skull tumors was not observed during the follow-up period. Surgical treatment for the metastatic skull tumors from cancers may be indicated to prevent deteriorating neurological symptoms affecting the quality of life.
Abstract: We have demonstrated that the ischemia-induced apoptosis of neurons in the CA1 region of the rat hippocampus was prevented by either intracerebroventricular or intravenous infusion of pituitary adenylate cyclase-activating polypeptide (PACAP). However, the molecular mechanisms underlying the anti-apoptotic effect of PACAP remain to be determined. Within 3-6 h after ischemia, the activities of members of the mitogen-activated protein (MAP) kinase family, including extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), and p38 were increased in the hippocampus. The ischemic stress had a potent influence on the MAP kinase family, especially on JNK/SAPK. PACAP inhibited the activation of JNK/SAPK after ischemic stress. Secretion of interleukin-6 (IL-6) into the cerebrospinal fluid was intensely stimulated after PACAP infusion. IL-6 inhibited the activation of JNK/SAPK, while it activated ERK. These observations suggest that PACAP and IL-6 act to inhibit the JNK/SAPK signaling pathway, thereby protecting neurons against apoptosis.
