Yoshimasa Takeda

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.

The effects of continuous epidural anesthesia and analgesia on stress response and immune function in patients undergoing radical esophagectomy

We investigated whether perioperative extensive epidural block (C3-L) affects postoperative immune response in patients undergoing radical esophagectomy. Patients undergoing radical esophagectomy were randomly assigned to either general anesthesia with continuous epidural infusion via 2 epidural catheters that was continued for postoperative analgesia (group E, n = 15) or intraoperative general anesthesia and postoperative IV morphine analgesia (group G, n = 15). Plasma levels of stress hormones, cytokines, C-reactive protein (CRP), leukocyte counts, and distribution of lymphocyte subsets were assessed before and after surgery and on postoperative days (PODs) 1 and 3. In comparison with group E, significant increases in plasma epinephrine level at the end of surgery (P < 0.05) and norepinephrine level at the end of surgery (P < 0.01) and on POD1 (P < 0.01) and POD3 (P < 0.01) and significant decrease in cluster of differentiation (CD4/CD8 ratio) at the end of surgery (P < 0.05) were observed in group G. However, there were no significant differences in other variables between groups. In both groups, plasma cortisol, adrenocorticotropic hormone, interleukin (IL)-1β, IL-6, IL-10, and CRP levels were increased after surgery (each group P < 0.01) and IL-1β, IL-6, IL-10, and CRP were still increased on POD1 and POD3 (each change, each group P < 0.01). Leukocyte counts were increased on POD1 (each group P < 0.05) and POD3 (each group P < 0.01). The proportion of lymphocytes decreased from the end of surgery to POD3 (each group P < 0.01). The proportion of B cells was increased on POD1 (each group P < 0.01); that of natural killer cells was decreased at POD1 and POD3 (each group P < 0.01). We conclude that tissue damage and inflammation apparently overcome the effects of extensive epidural block on stress response and immune function in radical esophagectomy.

Effects of hypothermia for a short period on histologic outcome and extracellular glutamate concentration during and after cardiac arrest in rats.

Objective:To evaluate the therapeutic effects of hypothermia for a short period (20 mins, 31°C) using a cardiac arrest model (5 mins) in rats. Design:Prospective animal study. Setting:Experimental laboratory in a university hospital. Subjects:Male Wistar rats (n = 42). Intervention:Direct current (DC) potential and extracellular glutamate concentrations (microdialysis) were monitored in the hippocampal region. Histologic observation was performed 7 days later. Measurements and Main Results:No animal died or showed severe complications as a result of hypothermia for a short period. In nontreated animals (group F), extracellular glutamate concentration simultaneously increased with the onset of membrane depolarization and continued to increase during the reperfusion period (maximum, 212% ± 40% of the pre-ischemia level) until the onset of DC recovery. In animals in which hypothermia was initiated before the onset of ischemia (group A), extracellular glutamate concentration did not increase during the ischemia period. When hypothermia was initiated at the onset of resuscitation (group B), the glutamate concentration immediately decreased. In animals in which hypothermia was initiated at 4.9 ± 1.3 mins (immediately after DC recovery, group C), 10 mins (group D), and 20 mins (group E) after the onset of resuscitation, changes in extracellular glutamate concentration were the same as those in nontreated animals. The percentage of injured neurons was significantly attenuated (compared with group F, 82% ± 10%) when hypothermia was initiated before DC recovery (group A, 5% ± 3%; group B, 29% ± 22%) or immediately after DC recovery (group C, 58% ± 18%, 9.9 ± 1.3 mins after the onset of ischemia). Conclusions:Hypothermia for a short period decreased glutamate concentration when it was initiated before DC recovery and attenuated neuronal damage when it was initiated before or immediately after DC recovery. The therapeutic time window for hypothermia for a short period is about 10 mins after the onset of ischemia.

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.

Dynamic Changes in Cortical NADH Fluorescence and Direct Current Potential in Rat Focal Ischemia: Relationship between Propagation of Recurrent Depolarization and Growth of the Ischemic Core

Forty rats were subjected to 3 hours of focal ischemia by occluding the left middle cerebral and left common carotid arteries. The propagation of recurrent depolarization around the ischemic core was analyzed using direct-current potential and NADH (reduced nicotinamide adenine dinucleotide) fluorescence images by irradiating the parietal-temporal cortex with ultraviolet light. Based on histological evaluation at direct-current recording sites, the total time of depolarization causing 50% neuronal injury was estimated to be 18.2 minutes. The sites showing recurrent depolarizations resulted in 23 ± 29% neuronal injury due to the short depolarization time, whereas the sites showing recurrent depolarizations and eventually persistent depolarization resulted in infarction. The NADH fluorescence images showed that recurrent depolarizations propagated along the margin of the ischemic core. In 85.9% of the recurrent depolarizations, the fluorescence disappeared without leaving any traces and did not affect the area of the ischemic core. However, in 47.5% of the animals, 14.1% of recurrent depolarizations merged with the ischemic core and increased the area by 6 ± 4 mm2. These findings suggest that recurrent depolarization increases the severity of neuronal injury but does not cause infarction by itself if persistent depolarization does not follow, and that the area of persistent depolarization is enlarged with 14.1% of recurrent depolarizations.

Effects of 7-nitroindazole and N-nitro-l-arginine methyl ester on changes in cerebral blood flow and nitric oxide production preceding development of hyperbaric oxygen-induced seizures in rats

Hyperbaric oxygen (HBO(2)) exposure induces increases in cerebral blood flow (CBF) and extracellular concentrations of nitric oxide (NO) that precede the appearance of central nervous system toxicity, which may manifest as convulsions. To elucidate the origins of NO production during HBO(2) exposure, we examined the effects of the selective neuronal NO synthase (NOS) inhibitor, 7-nitroindazole (7-NI), and the non-selective NOS inhibitor, N-nitro-l-arginine methyl ester (l-NAME), on changes in CBF and NO metabolites (NO(x), nitrite and nitrate) using a laser Doppler flow probe and in vivo microdialysis techniques, respectively. Rats were anesthetized, artificially ventilated, and pressurized to 5 atmosphere absolute (ATA) with pure oxygen for 60 min. In rats treated with vehicle, CBF and NO(x) levels in the cortex increased to 201% and 239% of basal levels, respectively, before the onset of electrical discharges, measured by electroencephalogram. The increase in CBF and NO(x) was completely inhibited by 7-NI and l-NAME. Both drugs also inhibited the appearance of electrical discharges for 60 min. Dynamic changes in CBF and NO(x) were not significantly different between 7-NI and l-NAME. These findings suggest that neuronal NOS is the main mediator of NO production associated with increase in CBF leading to the appearance of electrical discharge during HBO(2) exposure.

Nasopharyngeal cooling selectively and rapidly decreases brain temperature and attenuates neuronal damage even if initiated at the onset of cardiopulmonary resuscitation in rats

ObjectiveTo determine the effectiveness of nasopharyngeal cooling for selective brain cooling and neuroprotection from ischemia. DesignProspective animal study. SettingExperimental laboratory in a university hospital. SubjectsMale Wistar rats (n = 28). InterventionsIn study 1, hippocampal temperature was decreased to 31°C under spontaneous circulation. In the nasopharyngeal cooling group, physiologic saline (5°C) was infused to the bilateral nasal cavities at the rate of 100 mL·min−1·kg weight−1. In the whole body cooling group, a fan and a water blanket (5°C) were used. In study 2, ischemia and resuscitation were performed in normothermic and nasopharyngeal cooling (initiated with resuscitation after 5 mins of ischemia and continued for 20 mins) groups. Measurements and Main ResultsThe hippocampal temperature was decreased to 31°C in 7 ± 2 mins without any change in the rectal temperature in the nasopharyngeal cooling group, whereas a decrease in hippocampal temperature to 31°C took 33 ± 1 mins in the whole body cooling group. Although skull base region was cooled by nasopharyngeal cooling, the epidural temperature of the parietal region was lower than the hippocampal temperature, indicating that brain temperature was hematogeneously lowered. There was no difference between changes in cerebral blood flow or between the ratios of oxygen extraction from arterial blood in the head region in the nasopharyngeal cooling and whole body cooling groups. In the second study, all animals were successfully resuscitated, and the times required for recovery of mean arterial blood pressure (60 mm Hg) after resuscitation in the nasopharyngeal cooling and normothermic groups were the same. The histologic damage was significantly attenuated in the nasopharyngeal cooling group (33 ± 21% cell death in the hippocampus) compared with that in the normothermic group (73 ± 11%). ConclusionsNasopharyngeal cooling enables rapid and selective reductions in cortical and subcortical temperatures without disturbing the recovery of systemic circulation after resuscitation.

Temporal profiles of the levels of endogenous antioxidants after four-vessel occlusion in rats.

Although it is known that development of lipid peroxidation after ischemia occurs predominantly in vulnerable regions, temporal profiles of antioxidants after ischemia have not been regionally elucidated. After reperfusion periods of 0, 3, 24, and 72 hours following 20 minutes of four-vessel occlusion (n = 6 in each group), the concentration of total glutathione (GSH) and the activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) were assayed in the hippocampus, parietal cortex, striatum, thalamus, and brain stem. The levels of all antioxidants were unchanged in all regions without reperfusion; however, the concentration of total GSH significantly decreased in the hippocampus at 3 hours after the onset of reperfusion, and showed a maximum decrease in the hippocampus (68% of the sham-control level), parietal cortex (78% of the sham-control level), and striatum (76% of the sham-control level) after 24 hours of reperfusion. After 72 hours of reperfusion, these regions and the thalamus showed restoration and an increase in the total GSH concentration, respectively. The activities of SOD, GSH-Px, and catalase were stable during the reperfusion period, but the hippocampus showed significant increases in these enzyme activities and the parietal cortex and striatum showed significant increases in SOD activities at 72 hours after the onset of reperfusion. These results indicate that endogenous antioxidants take 72 hours for restoration in vulnerable regions after 20 minutes of four-vessel occlusion in rats.

Does diffusion-weighted magnetic resonance imaging enable detection of early ischemic change following transient cerebral ischemia?

To examine the usefulness of diffusion-weighted imaging for detecting neuronal damage following ischemia, dynamic changes in diffusion-, T1- and T2-weighted images of rats subjected to 10 min of 4-vessel occlusion and of humans who had suffered 10-20 min of cardiac arrest were observed. In rats, no remarkable alteration was observed on day 1. On day 3, however, diffusion-weighted images showed high signal intensity in the hippocampal area, in which the apparent diffusion coefficient was significantly lower than that of the control (760+/-28x10(-6) mm(2)/s in control vs. 480+/-29x10(-6) mm(2)/s on day 3, P<0.0001). Histological observation revealed microvacuolation in 92+/-4% of pyramidal neurons in the CA1 region. On day 7, the hyperintensity in diffusion-weighted images had disappeared and microvacuolation had also disappeared in the CA1 region, but severely disrupted pyramidal neurons containing pyknotic nuclei had appeared in the CA1 region instead. In humans, diffusion-weighted images did not show any apparent abnormality in the cerebral cortex on the day of resuscitation. On day 3, however, diffusion-weighted images consistently showed hyperintensities in the temporal or occipital cortex, and these hyperintensities had disappeared in images obtained on days 7 and 14. From day 14, T1-weighted images showed laminar hyperintensity, suggesting laminar necrosis, along the cortex, where diffusion-weighted images showed high signal intensity on day 3. These results suggested that diffusion-weighted imaging has a potential for detection of the occurrence of microvacuolation and is useful for detecting the progression of ischemic changes in humans following global ischemia.