Eiichi Sekizuka

Carbon Monoxide From Heme Oxygenase-2 Is a Tonic Regulator Against NO-Dependent Vasodilatation in the Adult Rat Cerebral Microcirculation

Although the brain generates NO and carbon monoxide (CO), it is unknown how these gases and their enzyme systems interact with each other to regulate cerebrovascular function. We examined whether CO produced by heme oxygenase (HO) modulates generation and action of constitutive NO in the rat pial microcirculation. Immunohistochemical analyses indicated that HO-2 occurred in neurons and arachnoid trabecular cells, where NO synthase 1 (NOS1) was detectable, and also in vascular endothelium–expressing NOS3, suggesting colocalization of CO- and NO-generating sites. Intravital microscopy using a closed cranial window preparation revealed that blockade of the HO activity by zinc protoporphyrin IX significantly dilates arterioles. This vasodilatation depended on local NOS activities and was abolished by CO supplementation, suggesting that the gas derived from HO-2 tonically regulates NO-mediated vasodilatory response. Bioimaging of NO by laser-confocal microfluorography of diaminofluorescein indicated detectable amounts of NO at the microvascular wall, the subdural mesothelial cells, and arachnoid trabecular cells, which express NOS in and around the pial microvasculature. On CO inhibition by the HO inhibitor, regional NO formation was augmented in these cells. Such a pattern of accelerated NO formation depended on NOS activities and was again attenuated by the local CO supplementation. Studies using cultured porcine aortic endothelial cells suggested that the inhibitory action of CO on NOS could result from the photo-reversible gas binding to the prosthetic heme. Collectively, CO derived from HO-2 appears to serve as a tonic vasoregulator antagonizing NO-mediated vasodilatation in the rat cerebral microcirculation.

Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis.

Abstract Background: Overproduction of nitric oxide by the inducible form of nitric oxide synthase (iNOS) has been implicated in colitis. Different authors have postulated both toxic and protective effects of nitric oxide (NO) in the pathophysiology of active inflammation. The objective of this study was to examine the role of iNOS in experimental chronic colitis using iNOS-deficient mice. Methods: For induction of colitis, mice received three cycles of 2% of dextran sodium sulfate (DSS) (M.W. 40,000) treatment in drinking water. The degree of colonic inflammation, leukocyte infiltration, and the expression of cell adhesion molecules were determined. INOS expression and nitrotyrosine were also determined by immunohistochemistry. Results: After DSS treatment, a moderate colitis with marked cell infiltration was observed. Intense expression of iNOS was observed on infiltrating cells as well as on the colonic mucosal epithelium in these animals. In the iNOS-deficient mice, tissue damage was significantly diminished. No iNOS or nitrotyrosine staining was found in iNOS-deficient mice. The number of infiltrating cells and the expression of mucosal adressin cell adhesion molecule-1 were significantly attenuated in the DSS-treated colon of iNOS-deficient mice. Conclusion: Induction of iNOS seems to act as a critical toxic effector molecule in the pathogenesis of chronic colonic inflammation.

Platelet and leukocyte adhesion in the microvasculature at the cerebral surface immediately after subarachnoid hemorrhage.

OBJECTIVEPathophysiology after subarachnoid hemorrhage (SAH) caused by aneurysmal rupture has not been well examined. The purpose of this study was to observe platelet-leukocyte-endothelial cell interactions as indexes of inflammatory and prothrombogenic responses in the acute phase of SAH, using an in vivo cranial window method. METHODSSubarachnoid hemorrhage was induced in C57Bl/6J mice by using the endovascular perforation method. Intravital microscopy was used to monitor the rolling and adhesion of platelets and leukocytes that were labeled with different fluorochromes. Regional cerebral blood flow was measured with laser Doppler flowmetry. The platelet-leukocyte-endothelial cell interactions were observed 30 minutes, 2 hours, and 8 hours after SAH. The effect of P-selectin antibody and apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, on these responses was examined at 2 hours after SAH, and compared with a different SAH model in which autologous blood was injected into the foramen magna. RESULTSSAH was accompanied by a 60% decrease in regional cerebral blood flow, whereas no changes in regional cerebral blood flow were observed on the contralateral side. SAH elicited time- and size-dependent increases in rolling and adherent platelets and leukocytes in cerebral venules. All of these interactions were attenuated by treatment with a P-selectin antibody or apocynin. There was no significant blood cell recruitment observed in the blood-injected SAH model. CONCLUSIONSAH at the skull base induced P-selectin- and oxygen radical-mediated platelet-leukocyte-endothelial cell interactions in venules at the cerebral surface. These early inflammatory and prothrombogenic responses may cause a whole-brain injury immediately after SAH.

Ischemic bowel necrosis induced by endothelin-1: an experimental model in rats.

Local intra-arterial administration of endothelin-1 induced small intestinal mucosal damage in rats in a dose-dependent manner. A remarkable decrease in mucosal blood flow (15% of control values) was observed by a laser Doppler flowmetry 10 min after injection of endothelin-1 (1 nmol/kg). Endothelin-1 at this dose induced significant hemorrhagic and necrotic lesions in the small intestinal mucosa 30 min after the injection. Decreased mucosal blood flow was attenuated to some extent by the pretreatment with platelet-activating factor (PAF) inhibitor, CV-6209, superoxide dismutase plus catalase or the calcium antagonist, nicorandil. All these inhibitors significantly prevented the endothelin-1-induced ischemic necrotic damage in the small intestine. These results suggest a potential role of endothelin-1 in the pathogenesis of ischemic bowel diseases in clinical situations and also the possibility that PAF and oxygen-derived free radicals may be involved as secondary mediators in endothelin-induced intestinal tissue damage.

Role of platelet activating factor on the fibrinolytic activation in the pathogenesis of gastric mucosal damage induced by endothelin-1.

We have examined the hypothesis that the release of tissue type plasminogen activator may play a prominent role in endothelin induced gastric mucosal injury. We determined tissue type plasminogen activator activity in the regional blood sample and the concentration of platelet activating factor in the gastric mucosa after the administration of endothelin-1 in a range of 50-500 pmol/kg into the left gastric artery of male Wistar rats. Endothelin-1 increased the tissue type plasminogen activator release and platelet activating factor formation, and induced subsequent gastric mucosal haemorrhagic change in a dose dependent manner. In addition CV-6209, a selective platelet activating factor blocker, attenuated the activation of regional tissue type plasminogen activator and the development of mucosal damage induced by endothelin-1. The results of this study showed that tissue type plasminogen activator activation may play an important role in the pathogenesis of endothelin induced mucosal injury of rat stomach, and suggest that the platelet activating factor may be involved in the process of regional fibrinolytic activation induced by endothelin-1.

Continuous measurement of blood oxygen pressure using a fiber optic sensor based on phosphorescence quenching

Measuring oxygen pressure within an artery is essential for providing information on blood perfusion. Optic-sensor is particularly well suited for oxygen pressure measurement because electrolytic or magnetic changes of the environment do not affect the measurement. We developed a fiber optical sensor based on phosphorescence quenching method. The probe, Pd-meso-tetra-(4-carboxyphenyl)porphyrin, was immobilized to the silicone resin membrane, which has high oxygen permeability, and attached to the tip of POF (outer diameter: 500 /spl mu/m). The light source used for exciting the probe was a Nd:YAG-SH pulse laser (532 nm) and the phosphorescent emission was detected by a photomultiplier tube. The signal was fitted to a single exponential decay curve to acquire the time constant (life time), thereafter the oxygen pressure was calculated from Stern-Volmer relationship. The effectiveness of the sensor was obtained to measure the dissolved oxygen with high precision (correlation coefficient 0.999) and high response time (about 7 sec). In this study, measurement of oxygen pressure was also made in the femoral artery of the rat by using the developed sensor and it indicated that it was very useful to measure arterial oxygen pressure continuously.

In vivo visualization of lymphatic microvessels and lymphocyte migration through rat Peyer's patches

BACKGROUND/AIMS In the small intestine, lymphocytes migrate through Peyers patches. The distribution of lymphatic microvessels in rat Peyers patches and lymphocyte traffic through them were studied. METHODS Vital dyes were injected via a micropipette into the Peyers patches tissue to fill lymphatic microvessels and to stain lymphocytes in lymphatic microvessels. RESULTS Direct microscopic observation revealed a dense plexus of lymphatic microvessels in the perifollicular and interfollicular areas. Injection of the dyes into the germinal center failed to delineate lymphatic microvessels. The lymphatic microvessels in the perifollicular area were filled with lymphocytes. Most lymphocytes in the perifollicular lymphatics stayed in the lymphatic microvessels. Some lymphocytes became detached and drained into lymphatic microvessels in the interfollicular areas. Lymphocytes then moved toward the submucosal lymphatics beneath the villi around the Peyers patches. The interfollicular lymphatics did not display contractile activity but had valves. Opening and closing of valves was synchronized with the respiration and the back and forth flow of lymphocytes. CONCLUSIONS There are numerous lymphocytes in a dense lymphatic network in the perifollicular and interfollicular areas of Peyers patches. This well-developed lymphatic network has the potential capacity for storage of lymphocytes and modulation of lymphocyte migration.

Platelet adhesion and arteriolar dilation in the photothrombosis: observation with the rat closed cranial and spinal windows.

The mechanism of cerebral infarction, in which thrombus formation and platelet-endothelium interaction play an important part, have not yet been clearly elucidated in vivo. The aim of this study was to observe rolling and adherent platelets and to analyze adherent leukocytes and vessel diameter change in vivo using a photothrombotic vessel occlusion model.A photothrombosis, which is mediated by free radicals, was induced in male Wistar rats in the presence of a photosensitizing dye (Photofrin II) and exposure to a filtered light. Rhodamine 6G-labeled platelets and leukocytes were visualized with intravital fluorescence videomicroscopy through a closed cranial or spinal window. The vessel diameter, photothrombosis and leukocyte adhesion were analyzed. Rolling and adherent platelets were observed during irradiation through the cerebral and spinal window. Before the platelets were recognized, the irradiated arteriole dilated significantly. After the photochemical occlusion of an arteriole, other arterioles also dilated and the adherent leukocytes increased in the venules. The photothrombosis were almost completely composed of platelets according to electron microscopic analysis. The arteriolar dilation rate and the number of adherent leukocytes in the cerebrum were greater than those in the spinal cord. By combining the photochemical thrombus formation and the fluorescence microscope techniques, we were able for the first time to observe rolling and adherent platelets and microvascular responses during photothrombosis in the cerebral and spinal microvasculature. It is suggested that free radicals, which can lead to platelet aggregation, play an important role as a cerebral vasodilator. This model is useful for cerebral and spinal microcirculatory analysis to investigate the platelet-endothelium interaction, the platelet aggregation and the effect of free radicals on cerebral and spinal microcirculation.

Involvement of superoxide anion and platelet-activating factor in increased tissue-type plasminogen activator during rat gastric microvascular damages.

The role of tissue-type plasminogen activator (t-PA) was investigated in the gastric ulcer formation induced by microvascular derangement. The rat stomach was exposed and repeated electrical stimuli (irritation) were applied on the small arterial wall close to the lesser curvature to induce mucosal ischemia followed by hyperemia. The t-PA activity in the regional blood of the stomach was significantly elevated as early as 5 min after the irritation. Immunohistochemical study using anti-t-PA monoclonal antibody revealed that t-PA was detectable in the endothelial cells of capillaries and collecting venules, suggesting the involvement of endothelium-mediated fibrinolytic activity in the irritation-induced ulcer formation. Pretreatment of SOD or allopurinol significantly attenuated the irritation-induced t-PA activation, suggesting that the t-PA activity was modulated by xanthine oxidase-associated superoxide anions. CV-6209, a selective antagonist of platelet-activating factor (PAF), also prevented the activation of t-PA as well as ulcer formation, providing a concept that PAF may be associated with the local fibrinolytic activation which may cause hemorrhagic changes in the gastric mucosal microvasculature. The present study supports the hypothesis that increased t-PA activity may reflect the microvascular endothelial damages caused by vasomotor derangement and suggests that oxygen-derived free radicals may participate in the regulation of endothelium-derived fibrinolytic activities in the mucosal microvasculature.

Fluorographic study on the oxidative stress in the process of gastric mucosal injury: Attenuating effect of Vitamin E

Abstract In vivo oxidative change was visualized in the gastric mucosa of rats and the alteration was analysed by using a fluorescence microscope equipped with a digital imaging processor during the development of mucosal damage. Dichlorofluorescein (DCF)‐associated fluorescence increased after the repeated electrical stimulation on the gastric artery (irritation), suggesting the occurrence of lipid peroxidation. The increase was enhanced in the mid‐zone of two adjacent collecting venules. Allopurinol attenuated the oxidative stress in mucosa, showing the involvement of xanthine oxidase. Luminol‐dependent chemiluminescence value in the blood taken from gastric vein was elevated by the irritation, suggesting that leucocyte‐generated oxygen radicals also participate in this oxidative process. α‐Tocopherol attenuated both the DCF activation and the increase in chemiluminescence value and prevented gastric mucosal injury. The present results suggest that α‐tocopherol may be useful for the prevention of oxidative alteration in gastric mucosa.