Masahisa Hirakawa

Protective effect of heme oxygenase induction in ischemic acute renal failure

Objective: To examine the role of heme oxygenase‐1 (HO‐1) induction in the recovery of renal function in rats with ischemic acute renal failure. Design: Randomized, masked, controlled animal study. Setting: University‐based animal research facility. Subjects: Sprague‐Dawley male rats, weighing 200‐250 g. Interventions: Anesthetized rats were subjected to bilateral flank incisions, and the right kidney was removed. Renal ischemia was performed by left renal microvascular clamping, followed by reflow of the blood. Measurements and Main Results: Ischemia of the kidney in the uninephrectomized rat significantly induced HO‐1 messenger RNA, protein, and enzyme activity, reaching a maximum at 6 hrs, which was mediated in part through an increase in microsomal heme concentration. Heat shock protein 70 was induced extremely rapidly, reaching a maximum at 1 hr, suggesting that HO‐1 and heat shock protein 70 gene expression are regulated separately. Inhibition of HO activity by tin mesoporphyrin, which resulted in an increase in microsomal heme concentration, significantly exacerbated renal function, as judged by the sustained increase in serum creatinine concentration and extensive tubular epithelial cell injuries. In contrast, animals that did not receive tin mesoporphyrin showed normal creatinine concentration and microsomal heme concentration 24 hrs after reperfusion, as well as restoration of abnormal renal histology. Conclusion: These findings indicate that the expression of HO‐1 in the ischemic kidney may be critical in the recovery of renal cell function in this animal model. These findings also suggest that HO‐1 induction may play an important role in conferring protection on renal cells from oxidative damage caused by heme.

Hyperbaric oxygen reduces infarct volume in rats by increasing oxygen supply to the ischemic periphery.

ObjectiveHyperbaric oxygen (HBO) increases oxygen supply to anoxic areas. To examine the therapeutic effect of HBO on ischemic stroke, we measured infarct volume as well as cerebral blood flow (CBF), oxygen supply, and lipid peroxidation in the ischemic periphery. DesignProspective experimental study in rats. SettingExperimental laboratory in a university teaching hospital. SubjectsThirty-eight adult rats. InterventionThe rats were anesthetized (1% halothane) and intubated. Focal ischemia was induced by ligating the right middle cerebral and right common carotid arteries. Nineteen animals were exposed to 2 hrs of HBO (100% oxygen, 3 atmospheres absolute), initiated 10 mins after the onset of ischemia. The remaining animals were kept at ambient pressure and used as controls. Measurements and Main ResultsAt the initiation of ischemia, CBF measured by a laser-Doppler flow probe placed in the ischemic periphery was reduced to 47% ± 11% and 51% ± 15% of normal levels in animals exposed or not to HBO, respectively. These altered values were not affected further by administration of HBO and remained stable throughout a 2-hr observation period. Arterial oxygen pressure and content were significantly increased to 1571 ± 130 torr (209.41 ± 17.32 kPa;p < .0001) and 1.03 ± 0.04 mmol/dL (p < 0.0001), respectively, in HBO-treated animals compared with nontreated animals (139 ± 14 torr [18.53 ± 1.87 kPa] and 0.86 ± 0.04 mmol/dL, respectively). The calculated increase in the oxygen supply to the ischemic periphery was 20%. The infarct volume of HBO-treated animals measured 24 hrs after the onset of focal cerebral ischemia was significantly reduced by 18% (HBO-treated, 132 ± 13 mm3 vs. nontreated, 161 ± 29 mm3;p = .02). Lipid peroxidation was unchanged after 120 mins of HBO administration in the cerebral cortex where the laser-Doppler flow probe was placed. ConclusionsHBO at 3 atmospheres absolute reduced infarct volume by increasing oxygen supply to the ischemic periphery without aggravating lipid peroxidation, suggesting that HBO can be useful in treating stroke victims.

Hemodynamic effect of the prone position during anesthesia.

We studied 21 patients undergoing lumbar spinal surgery under halothane anesthesia on a convex saddle frame, in order to determine the hemodynamic effect of the prone position. A thermodilution pulmonary arterial catheter was placed in 14 patients (Group PA‐1: n = 8; and Group PA‐2: n = 6), and an inferior vena caval catheter in the remaining seven patients (Group IVC). Group PA‐1 and Group IVC patients were placed in the prone position on a convex saddle frame. In the prone position, the cardiac index (CI) decreased significantly from 3.1 ± 0.5 to 2.5 ± 0.3 (1·min‐1·m‐2 mean ± s.d., P<0.01) without accompanying significant changes in the other hemodynamic variables in Group PA‐1. The postural change in Group IVC did not exert a significant effect on the inferior vena caval pressure. Group PA‐2 were initially placed in the flat prone position on a flat saddle frame, which produced no significant changes in the hemodynamic variables. Then the convex curvature of the frame was adjusted to the grade appropriate for surgery, which produced a significant reduction in CI (from 2.9 ± 0.3 to 2.4 ± 0.4, P<0.05). We conclude that the prone position itself may not interfere with the circulatory function. The prone position using a convex saddle frame causes significant reductions in CI, but little change in the other hemodynamic variables.

Protective role of heme oxygenase-1 in the intestinal tissue injury in an experimental model of sepsis.

ObjectiveThe aim of this study was to examine the role of heme oxygenase-1 induction in the intestinal tissue injury in a rat model of sepsis. DesignRandomized, masked, controlled animal study. SettingUniversity-based animal research facility. SubjectsSprague-Dawley male rats, weighing 220–250 g (n = 126). InterventionsRats were injected with lipopolysaccharide (10 mg/kg) intraperitoneally. Another group of rats was injected with interleukin-6 (10 &mgr;g/kg) intravenously. In some rats, tin mesoporphyrin (1 &mgr;mol/kg) was administered intravenously 1 hr before lipopolysaccharide treatment. Measurements and Main ResultsFollowing lipopolysaccharide treatment, expression of heme oxygenase-1 and nonspecific &dgr;-aminolevulinate synthase (ALAS-N), the rate-limiting enzymes of heme catabolism and biosynthesis, respectively, was examined in various regions of the intestine. Lipopolysaccharide treatment markedly increased heme oxygenase-1 messenger RNA and protein concentrations in the mucosal epithelial cells in the duodenum and the jejunum, whereas its expression in the ileum and the colon was hardly detectable and was not influenced by the treatment. ALAS-N messenger RNA was also more markedly increased in the duodenum, the jejunum, and the ileum than in the colon following lipopolysaccharide treatment. Interleukin-6 administration also induced heme oxygenase-1 and ALAS-N gene expression in a pattern similar to that following lipopolysaccharide treatment. In contrast to the marked heme oxygenase-1 expression in the upper intestine, lipopolysaccharide-induced mucosal injury and inflammation in the upper intestine were far less than observed in the lower intestine as judged both by tumor necrosis factor-&agr; gene expression and by histologic analysis. Of note, inhibition of heme oxygenase activity by tin mesoporphyrin produced a significant tissue injury in the upper intestine of the lipopolysaccharide-treated animals. ConclusionsIntestinal heme oxygenase-1 and ALAS-N gene expression was regulated in a site-specific manner in a rat model of sepsis. Our findings also suggest that heme oxygenase-1 induction may play a fundamental role in protecting mucosal epithelial cells of the intestine from oxidative damages that occur in sepsis.

Dynamic changes of NADH fluorescence images and NADH content during spreading depression in the cerebral cortex of gerbils

To elucidate the changes in the mitochondrial redox state during spreading depression (SD), tissue NADH content was measured in 20 anesthetized gerbils by the enzymatic cycling assay in a small cortical region (0.30+/-0.07 mg) where the direct-current (DC)-potential was measured. Sequential imaging of NADH fluorescence with a CCD camera and continuous monitoring of DC-potential and regional CBF were also performed in another 5 gerbils. Biphasic fluorescence waves propagating at the rate of 3 mm/min were observed using the CCD camera. An initial narrow (1.6+/-0.4 mm) wave, which showed a modest increase in fluorescence (108+/-6.4%), was observed simultaneously with the onset of negative DC-deflection. During depolarization, CBF was unchanged and tissue NADH content increased to 25.3+/-7.9 micromol/kg brain, which was higher than the value in the sham-control (11.0+/-2.5 micromol/kg brain). At 30 s after the deflection, a subsequent wide (7.0+/-2.1 mm) wave, which showed a moderate decrease in fluorescence (87.1+/-5.7%), was observed simultaneously with the increase in CBF and repolarization in DC-potential. Then NADH fluorescence recovered along with normalization of CBF at 152.2+/-38.6 s after the onset of DC-deflection. Tissue NADH concentration sampled at 120 s after the deflection was 11.6+/-4.6 micromol/kg brain. Since NADH fluorescence is absorbed by hemoglobin, the initial increase and subsequent decrease in fluorescence seem to have been induced by increases in NADH content and CBF, respectively. These findings indicate that the mitochondrial redox state transiently inclines to the reduction side synchronous to the onset of DC-deflection and that it normalizes within 120 s after deflection.

Changes in nitric oxide production and cerebral blood flow before development of hyperbaric oxygen-induced seizures in rats

The predictive value of increase in cerebral blood flow (CBF) was examined to detect hyperbaric oxygen (HBO(2))-induced electrical discharge in artificially ventilated rats at three PaCO(2) levels under 5 atmospheric pressures. The possible involvement of NO production in the mechanism of the increase in CBF was also assessed by measurement of major NO metabolites (NO(2)(-) plus NO(3)(-)) using a microdialysis technique at the left parietal cortex during HBO(2) exposure. The onset times of electrical discharge, measured in the right frontal region, were significantly prolonged and shortened in the low PaCO(2) group (79+/-21 min) and high PaCO(2) group (27+/-7 min), respectively, compared to that in the normal PaCO(2) group (37+/-5 min). Increase in CBF (200% of the pre-exposure level) was observed in every animal and was sustained until the appearance of electrical discharge. The onset time of increase in CBF was closely related to that of electrical discharge (R(2)=0.987), and the durations of increase in CBF were almost identical (11-14 min in mean) regardless of the PaCO(2) level. The level of NO(2)(-) plus NO(3)(-) was unaffected by the initiation of HBO(2) exposure and simultaneously increased up to 246+/-59% of control level with the onset of increase in CBF. There was a close relationship between changes in CBF and levels of NO(2)(-) plus NO(3)(-) (R(2)=0.544). These results indicate that monitoring of CBF is useful for the prediction of electrical discharge in artificially ventilated rats regardless of their PaCO(2) levels and that the increase in NO production is related to the mechanism of increase in CBF.

Prevention of halothane-induced hepatotoxicity by hemin pretreatment: Protective role of heme oxygenase-1 induction

Reductive metabolism of halothane in phenobarbital-pretreated rats is known to increase free radical formation that results in hepatotoxicity. It also is associated with a marked induction of microsomal heme oxygenase-1 (HO-1), suggesting that there is an alteration in heme metabolism. In this study, we examined heme metabolism in rats pretreated with phenobarbital, followed by exposure to halothane-hypoxia. In this model, there was a significant decrease in microsomal cytochrome P450 content in the liver, followed by a rapid increase in free heme concentration and a decrease in the level of mRNA for the nonspecific delta-aminolevulinate synthase. A transient but dramatic induction of HO-1 mRNA and a prolonged induction of heat shock protein 70 mRNA also occurred. The HO-1 protein was detected principally in the hepatocytes around the central vein. Serum alanine transaminase (ALT) activity, an indicator of hepatic dysfunction, increased continuously throughout the experiment. Hemin pretreatment induced hepatic HO-1 with abrogation of the halothane-induced hepatotoxicity in this model, as judged by ALT activity and normal histology. Our findings in this study thus indicate that halothane-induced hepatotoxicity is due not only to its reductive metabolite formation, but also to an increase in hepatic free heme concentration, which is a potent prooxidant; HO-1 induction is an important protective response against such changes. This is also the first study to demonstrate that hemin pretreatment, which induces HO-1 prior to exposure to halothane, effectively prevents halothane-induced hepatotoxicity.

Effect of hyperbaric oxygenation on the Na+, K+-ATPase and membrane fluidity of cerebrocortical membranes after experimental subarachnoid hemorrhage

It is reported that CNS hemorrage causes membrane dysfunction and may exacerbate this damage as a result of secondary ischemia or hypoxia. Since hyperbaric oxygenation improves oxygen metabolism, it may reduce this membrane damage. The present study was conducted to reveal whether hyperbaric oxygenation influences membrane alteration after hemorrhage. Thirty minutes after subarachnoid hemorrhage induction, rats were treated with hyperbaric oxygenation 2 ATA for 1 hour. Rats were decapitated 2 hours after subarachnoid hemorrhage induction. Na+, K+-ATPase activity measurement, and spin-label studies were performed on crude synpatosomal membranes. Subarachnoid hemorrhage decreased Na+, K+-ATPase activity. Spin label studies showed that hydrophobic portions of near the membrane surface became more rigid and the mobility of the membrane protein labeled sulfhydryl groups decreased after subarachnoid hemorrhage. Hyperbaric oxygenation significantly ameliorated most of the subarachnoid hemorrhage induced alterations. We conclude that hyperbaric oxygenation may be a beneficial treatment for acute subarachnoid hemorrhage.

Tin chloride pretreatment prevents renal injury in rats with ischemic acute renal failure.

Objective To investigate whether tin chloride pretreatment ameliorates renal injury in rats with ischemic acute renal failure (IARF) by virtue of its kidney-specific heme oxygenase-1 induction. Design Randomized, masked, controlled animal study. Setting University-based animal research facility. Subjects Sprague-Dawley male rats, weighing 200–230 g (n = 359). Interventions Rats were injected with tin chloride subcutaneously, because subcutaneous administration of tin chloride is known to specifically and potently induce renal heme oxygenase activity in the rat. Anesthetized rats were subjected to bilateral flank incisions, and the right kidney was removed. Renal ischemia for 40 mins was performed by left renal microvascular clamping, followed by reflow of the blood. Measurements and Main Results Tin chloride treatment specifically induced heme oxygenase-1 mRNA and protein in the proximal tubular epithelial cells of the kidney without apparent cell injury in the rat. Tin chloride treatment before renal ischemia augmented the induction of heme oxygenase-1 in IARF rats at both transcriptional and protein concentrations in the renal epithelial cells compared with IARF animals. Tin chloride pretreatment, which decreased microsomal heme concentration, ameliorated the ischemic renal injury as judged by the significant decrease in serum creatinine and blood urea nitrogen concentrations and the lesser tubular epithelial cell injuries. In contrast, inhibition of heme oxygenase activity by treatment with tin mesoporphyrin, which increased microsomal heme concentration, abolished the beneficial effect of tin chloride pretreatment. Conclusion These findings indicate that tin chloride pretreatment significantly ameliorates renal injury in rats with IARF by virtue of its specific heme oxygenase-1 induction in renal epithelial cells. These findings also suggest that heme oxygenase-1 induction plays an important role in protecting renal cells from oxidative damage caused by heme.

Tissue-specific gene expression of heme oxygenase-1 (HO-1) and non-specific δ-aminolevulinate synthase (ALAS-N) in a rat model of septic multiple organ dysfunction syndrome

Reactive oxygen species are thought to be involved in the pathogenesis of septic multiple organ dysfunction syndrome (MODS). It has been reported that heme oxygenase-1 (HO-1) (EC 1.14.99.3) is induced in septic animal models and is thought to confer protection against oxidative tissue injury. In this study, we examined changes in gene expression of HO-1 and non-specific delta-aminolevulinate synthase (ALAS-N) (EC 2.3.1.37), the rate-limiting enzymes in heme catabolism and heme synthesis, respectively, after intraperitoneal administration of bacterial lipopolysaccharide (LPS) to rats. LPS treatment caused the elevation of body temperature, increases in white blood cell counts, and marked elevation of serum interleukin-6 levels associated with liver, lung, and kidney injuries, characteristic of septic MODS. LPS administration significantly induced HO-1 mRNA, protein, and enzyme activity in the liver, lung, and kidney. In contrast, ALAS-N mRNA was decreased rapidly in the liver, followed by an oscillating recovery pattern. Induction of hepatic HO-1 mRNA and rapid suppression of ALAS-N mRNA were likely the result of a rapid increase in hepatic free heme concentration as judged by the increase in heme saturation of tryptophan pyrrolase. In contrast to that in the liver, the ALAS-N mRNA level in the lung and kidney was increased significantly after LPS administration, suggesting a novel mechanism of ALAS-N regulation in these tissues. These findings suggest that HO-1 and ALAS-N mRNA are regulated in a tissue-specific manner in a rat model of septic MODS.