The brain mapping of the retrieval of conditioned taste aversion memory using manganese-enhanced magnetic resonance imaging in rats.

Manganese-enhanced MRI (MEMRI) is a newly developed noninvasive imaging technique of brain activities. The signal intensity of MEMRI reflects cumulative activities of the neurons. To validate the use of MEMRI technique to investigate the neural mechanisms of learning and memory, we tried to map brain areas involved in the retrieval of conditioned taste aversion (CTA) memory. CTAs were established to saccharin (conditioned stimulus: CS) by pairing its ingestion with an i.p. injection of LiCl (unconditioned stimulus: US). LiCl solutions (as a robust aversion chemical) of 0.15 M were injected i.p. 15 min after drinking the saccharine solution (CS). After the two times conditionings, these rats showed a robust aversion to the saccharine solution (CS). Rats of the control group were injected saline i.p. instead of LiCl solutions. The MRI signal intensities at the gustatory cortex (GC), the core subregion of the nucleus accumbens (NAcC), the shell subregion of the nucleus accumbens (NAcSh), the ventral pallidum (VP), the central nucleus of amygdala (CeA), the lateral hypothalamus (LH), and the basolateral nucleus of amygdala (BLA) of the conditioned group were higher than those of the control group. There were no significant differences between the conditioned and the control groups in the intensities for other regions, such as the striatum area, motor cortex, cingulate cortex, interstitial nucleus of the posterior limb of the anterior commissure and hippocampus. These indicate that the GC, NAcC, NAcSh, VP, CeA, LH and BLA have important roles in the memory retrieval of CTA.

Effect of glycolysis on pancreatic microcirculation and cellular functions in anesthetized rats.

It has been reported that aerobic glycolysis in the pancreas contributes less than 10% to oxidative phosphorylation based on in vitro experiments using pancreatic tissue segments. However, its contribution to aerobic glycolysis in vivo remains uncertain. We investigated the effect of inhibiting glycolysis on O2 metabolism in microvessels, exocrine enzyme secretion, and the blood glucose level in the pancreas of anesthetized rats in vivo. Inhibition of glycolysis, by superfusing the pancreas of anesthetized rats with 2-deoxyglucose (10 mM) or sodium fluoride (2 mM), significantly decreased O2 release from erythrocytes flowing in the microvessels by 30-40%. Inhibiting glycolysis did not affect the exocrine secretion of pancreatic juice but decreased the secretion of total protein by approximately or = to 40%. Inhibiting glycolysis decreased blood glucose levels by approximately or = to 40% and increased glucagon release twofold. Aerobic glycolysis may play more important roles in the regulation of O2, metabolism, pancreatic exocrine enzyme secretion and the blood glucose level in rat pancreas.

Vagus nerve is involved in lack of blood reflow into sinusoids after rat hepatic ischemia.

Although recovery of microcirculation is an important determinant for ischemia-reperfusion injury, little information is available about hepatic blood flow after ischemia. To examine regulatory mechanisms of postischemic hepatic microcirculation, we studied the sinusoidal blood flow after portal triad clamping of rat livers for 5, 15, or 30 min. Hepatic tissue blood flow and erythrocyte blood flow in sinusoids were measured using a laser-Doppler flowmeter and an intravital microspectroscope, respectively. There was a time of no blood flow (lag time) in sinusoids after declamping, dependent on the ischemic time. Cholinergic blockade agents eliminated the lag time, whereas nerve stimulation at the hiatus esophagus or on the hepatoduodenal ligament during reperfusion prolonged it. Chemical denervation with 10% phenol or surgical denervation on the hepatoduodenal ligament eliminated the lag time. The prolongation of lag time by nerve stimulation was completely abrogated by truncal vagotomy. These results suggest that the cholinergic vagus nerve is involved in causing the lag time of sinusoidal blood flow in hepatic ischemia-reperfusion.

Recovery of blood flow and oxygen transport after temporary ischemia of rat liver.

Hepatic tissue perfusion and O2 supply after ischemia are indispensable for recovery of cellular functions, but few studies have been performed regarding the recovery of tissue blood flow and O2 transport. After 5, 15, and 30 min of ischemia of rat livers, hepatic tissue perfusion, hepatic arterial and portal blood flow, plasma PO2, and O2 transport parameters were measured. Hepatic tissue blood flow and erythrocyte velocity in the sinusoids showed biphasic recoveries after temporal ischemia for 5, 15, and 30 min. The first peak in the flow appeared at 3-4 min after the initiation of tissue perfusion, and the second peak appeared at approximately 20 min, irrespective of the ischemic period. Hepatic blood flow during the initial increase contained relatively low O2-saturated blood compared with that in the second increase. Livers that had been subjected to a prior hepatic artery ligation only showed the first peak at approximately 4 min. The first increase in hepatic blood flow corresponded to the peak in the portal venous flow, and the second increase corresponded to that of the hepatic artery. These results suggested that hepatic microcirculation after temporary hepatic ischemia showed biphasic recoveries because of different restoration patterns of the portal vein and hepatic artery.

Endothelin A-receptor blockade worsens endotoxin-induced hepatic microcirculatory changes and necrosis.

BACKGROUND & AIMS: Endothelin 1 is considered to be an important regulator of sinusoidal blood flow and increases during endotoxemia. The purpose of this study was to investigate the role of endothelin 1 in hepatic microcirculation, oxygen transport, and liver injury during endotoxemia. METHODS: Male Sprague-Dawley rats were continuously infused with 2.5 mL/h of saline, 0.8 mg . kg-1 . h-1 of lipopolysaccharide (LPS), 3 mg . kg-1 . h-1 of BQ-485, an endothelin A-receptor antagonist, or LPS plus BQ-485 for 7 hours. RESULTS: BQ-485 infusion had no significant effect on hepatic microcirculation and liver injury. LPS increased the plasma levels of aspartate aminotransferase (AST) and total bilirubin and decreased the hepatic adenosine triphosphate (ATP) level and bile flow rate. LPS + BQ-485 infusion further increased the plasma levels of AST and total bilirubin and decreased the bile flow rate and the hepatic ATP level. Dual-spot microspectroscopy revealed mild decreases in sinusoidal erythrocyte velocity and oxygen transport in the LPS group and profound decreases in these parameters in the LPS + BQ-485 group. Histological examinations revealed massive necrotic changes in the pericentral regions of the LPS + BQ-485 group. CONCLUSIONS: These results suggest that blockade of endothelin A receptors disturbs hepatic microcirculation and oxygen transport and aggravates the necrotic injury induced by endotoxin.

Pentoxifylline attenuates reperfusion injury in skeletal muscle after partial ischemia.

Leukocytes have been shown to contribute to ischemia-reperfusion injury in skeletal muscle. Pentoxifylline (PTXF), a xanthine-derived phosphodiesterase inhibitor, has received recent attention because of its action on leukocytes. To clarify the effects of PTXF in reperfusion injury, we measured the resting transmembrane potential difference (Em) and evaluated postcapillary venule microcirculation using intravital microscopy in rat skeletal muscle during ischemia and reperfusion. The infrarenal aorta was clamped for 90 min and then reperfused for 60 min. Persistent depolarization of the resting Em was observed in an ischemia-reperfusion (IR) group and was significantly repolarized in a PTXF group during the reperfusion period. The tissue water content was significantly reduced in the PTXF group, although no difference was noted in the tissue lactate content. Flowing erythrocyte velocity and wall shear rate in the PTXF group were significantly higher than in the IR group during the reperfusion period but without significant differences in vessel diameter and hemoglobin oxygenation. Blood flow measured by laser-Doppler flowmeter was also significantly improved in the PTXF group. Furthermore, the adherent leukocyte count was significantly reduced in the PTXF group during this same period. These results indicate that PTXF attenuated reperfusion-associated membrane injury and tissue edema and that PTXF suppressed leukocyte adhesion and improved hindlimb blood flow during the reperfusion period.

Increase in experimental pulmonary metastasis in mice by L-arginine under inhibition of nitric oxide production by NG-nitro-L-arginine methyl ester.

As we have previously reported, intraperitoneal injections of NG-nitro-L-arginine methyl ester [L-NAME; a competitive inhibitor of nitric oxide (NO) synthase] before and after the injection of B16 melanoma cells through a tail vein increased experimental pulmonary metastasis, while simultaneous injections of L-arginine (a substrate of NO synthase) at a 20-fold higher dose synergistically increased pulmonary metastasis. Our present study was intended to elucidate the mechanisms by which L-NAME alone or together with L-arginine increases metastasis. Injections of L-NAME decreased the serum concentration of nitrite plus nitrate (metabolites of NO) by about 50%, which was not reversed by simultaneous injections of L-arginine. Injections of L-NAME also decreased the diameter of arterioles and venules by 20-30%, while simultaneous injections of L-arginine did not show any significant effect. When collagen- or ADP-induced platelet aggregation was examined using platelet-rich plasma, injections of L-NAME showed little effects on platelet aggregation, while simultaneous injections of L-arginine rather suppressed platelet aggregation. B16 melanoma cells produced NO in culture, and L-NAME (0.2 mM) decreased NO production without effects on viability. Our results suggest that the increased experimental pulmonary metastasis induced by L-NAME can be ascribed partly to the contraction of arterioles and venules, which is induced by the inhibition of endogenous NO production by L-NAME, and that the synergistic effect of L-arginine on metastasis is related to the inhibition of endogenous NO production through unknown mechanisms.

Role of nitric oxide in oxygen transport in rat liver sinusoids during endotoxemia.

To evaluate the role of nitric oxide (NO) in hepatic microcirculation and liver injury during endotoxemia, we studied O2 transport in the hepatic microcirculation of endotoxin-infused rats. Rats were continuously infused with Escherichia coli lipopolysaccharide (LPS) (0.8 mg/kg/h) for 7 hours. LPS increased the plasma levels of NO2- + NO3- and aspartate transaminase (AST), and decreased the bile flow rate and hepatic adenosine triphosphate (ATP) level. Hepatic microcirculation was evaluated by two methods: reflectance spectrophotometry showed a decrease in the oxygenation of hemoglobin (Hb) in the liver, and dual-spot microspectroscopy indicated that LPS administration decreased blood velocity, the oxygenation of Hb, and O2 release from sinusoids to hepatocytes. The observed decreases in the O2 transport parameters were prominent in pericentral sinusoids. All of these phenomena were further aggravated by the administration of N(w)-nitro-L-arginine methyl ester (L-NAME) (5 mg/kg/h) plus LPS, and by aminoguanidine (AMG) (5 mg/kg/h) plus LPS, and these could be reversed by the concomitant administration of L-arginine (L-Arg) (100 mg/kg/h). These results suggest that deterioration of hepatic oxygen transport and liver function induced by endotoxin can be ameliorated by NO.

A near-infrared spectroscopic study of cerebral ischemia and ischemic tolerance in gerbils.

BACKGROUND AND PURPOSE: To explore the physiological mechanism of ischemic tolerance, we studied intracerebral oxygenation states noninvasively using near-infrared spectroscopy after bilateral common carotid artery occlusion (BCO) in gerbils with and without ischemic pretreatment. METHODS: Under ether anesthesia, gerbils with sham operation (S group, n = 8) and those with pretreatment consisting of BCO for 2 minutes, twice at 3 days and 2 days earlier (T group, n = 8), were again subjected to BCO for 5 minutes. Changes in oxyhemoglobin (HbO2), deoxyhemoglobin (Hb), and total hemoglobin (HbT) as well as reduction in cytochrome oxidase (cyt.aa3) were calculated from the absorbance changes of the light transmitted through the brain. Seven days after the ischemic study, immunohistochemical examination was performed with an antiserum against microtubule-associated proteins. RESULTS: In both groups, the increase of Hb and decrease of HbO2 and HbT proceeded rapidly after BCO, and the maximal deoxygenation of hemoglobin occurred within 2.5 minutes. Reduction of cyt.aa3 also ensued rapidly and reached the maximal reduction within 3 minutes in both groups. In the T group, however, both deoxygenation of hemoglobin and reduction of cyt.aa3 progressed more slowly than in the S group. The time (seconds) necessary for a maximal change for cyt.aa3 was significantly longer in the T group (203.8 +/- 34.0 [mean +/- SD]; P < .01) than in the S group (68.0 +/- 14.7). The time necessary for a half-maximal change was also significantly longer in the T group than in the S group for both Hb (22.0 +/- 7.5 and 13.5 +/- 4.0, respectively; P < .05) and cyt.aa3 (23.9 +/- 5.7 and 11.6 +/- 4.3; P < .01). After recirculation for 7 days, all gerbils in the S group were found to have neuronal death in the hippocampus, while those in the T group did not. CONCLUSIONS: The present study indicated that mild ischemic stress can induce improvement in oxygen metabolism during subsequent ischemia, which might be causally related to the phenomenon known as "ischemic tolerance," in which a protective effect toward ischemic/postischemic injury is induced by earlier mild ischemic pretreatment.

Elevation of oxygen release by nitroglycerin without an increase in blood flow in the hepatic microcirculation.

The effect of nitroglycerin on oxygen (O2) release in the microcirculation was investigated by examining single, unbranched hepatic sinusoids of rats using dual-spot microspectroscopy. Nitroglycerin significantly increased O2 release from erythrocytes flowing in the sinusoids. Differences in O2 saturation of hemoglobin per unit length of the sinusoid were significantly enhanced, while there were no significant changes in erythrocyte velocity, hemoglobin concentration or oxyhemoglobin flow into the sinusoids, or in regional hepatic blood flow measured with a laser tissue blood flow meter. No change was noted for hepatic O2 consumption measured in isolated liver perfused with hemoglobin-free oxygenated buffer. Isosorbide dinitrate showed a similar but slower effect. These findings suggest that nitroglycerin and isosorbide dinitrate enhance O2 release from erythrocytes without significantly increasing tissue blood flow.

Absence of mast cell involvement in leukocyte adhesion and emigration induced by inhibition of nitric oxide synthase.

Leukocyte adhesion on venules and their emigration to extravascular connective tissue are induced by administration of a nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester. In the present study, the involvement of mast cells in the process was examined in genetically mast cell-deficient and normal rats by intravital microscopy. Superfusion of the NOS inhibitor into the mesentery induced partial degranulation of mast cells in normal rats. However, leukocyte adhesion on mesenteric venules and emigration to extravascular connective tissue occurred even in mast cell-deficient rats, with no significant difference from the normal rats. When the reverse-passive Arthus reaction, characterized by generalized antigen-antibody complex formation, was induced in the rat mesentery, the immune complex increased both the adhesion and emigration in normal rats but not in mast cell-deficient rats. These results show that mast cells are not involved in the leukocyte adhesion and emigration induced by NOS inhibition, but are in the reverse-passive Arthus reaction.

Effect of ACh and calmodulin inhibitor on O2 transfer from exocrine pancreatic microvessels of rats.

Effects of acetylcholine (ACh) and calmodulin (CaM) inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), on the rate of O2 release (Ro2) from single exocrine pancreatic microvessels of anesthetized rats were investigated with dual-spot microspectroscopy. The surface of the pancreas was superfused with Krebs-Henseleit buffer containing various concentrations of ACh and/or W-7. Superfusion of ACh (> or = 20 microM) elevated Ro2 as well as pancreatic secretion approximately 2.5 times higher than that of control level, whereas superfusion of W-7 (> or = 100 microM) reduced approximately 50%. In both cases, O2 inflow in single microvessels, as quantified by oxyhemoglobin inflow into the microvessels, was maintained at control level. On the other hand, superfusion of both ACh and W-7 did not modify Ro2 and pancreatic secretion, despite significant reduction in O2 inflow. These results indicate that 1) the ACh-induced elevation of O2 release from single microvessels is accomplished by increased O2 extraction instead of increased O2 inflow in the microvessels, and 2) the activity of a W-7-sensitive Ca2+ binding protein, most likely CaM, is responsible for half of the microvascular O2 transfer and of the pancreatic exocrine secretion.

Effect of hypothermia on skeletal muscle metabolism in perfused rat hindlimb.

Temperature-induced metabolic change was studied with isolated rat hindlimb muscle to elucidate how tissue viability is maintained under hypothermia. The hindlimb was perfused with Krebs-bicarbonate buffer containing 4% (w/v) polyvinylpyrrolidone (PVP-40T) in a flowthrough mode at 35-8 degrees C. When the temperature was lowered, the following results were observed: (i) Vascular resistance (defined as perfusion pressure divided by flow rate) increased proportionally with elevation of the viscosity of the perfused medium, suggesting that the capillary bed in the perfused muscle is maintained under a similar condition under these temperatures; (ii) the Arrhenius plot of the O2 uptake rate showed a break at ca. 20 degrees C; (iii) the rates of O2 uptake and lactate release decreased, but the lactate/pyruvate ratio increased even under aerobic conditions; (iv) oxygenation of myoglobin and oxidation of cytochromes increased, suggesting a reduced electron-transfer rate in spite of improved or sufficient oxygenation of the tissue. Based on these results, we concluded that oxidative phosphorylation is more affected by temperature than glycolysis, and thus under hypothermia, the role of glycolysis in energy production increases in rat skeletal muscle, especially below 20 degrees C.

O2 transfer from single microvessels to acinar cells in secretin-stimulated pancreas of rat.

O2 transfer from the inside to the outside of single microvessels in the resting and secretin-stimulated exocrine pancreas of rats was investigated by dual-spot microspectroscopy. Measurements of intravascular hemoglobin (Hb) concentration, O2 saturation of Hb, and velocity of flowing red blood cells were carried out in single microvessels at the edge of the exocrine pancreas of anesthetized rats. The rate of O2 release (Ro2) from a single microvessel wall was constant [approximately 2 nmol.cm-2.s-1] over a wide range of oxyhemoglobin inflow ([HbO2] inflow; 200-700 fmol/s) but decreased almost linearly with an [HbO2] inflow < 200 fmol/s, where [HbO2] inflow is defined as the product of inflowing oxyhemoglobin concentration ([HbO2]) and blood flow rate. When the exocrine pancreas was stimulated with secretin either by superfusion (> or = 0.3 nM) or by intravenous infusion (> or = 0.5 microgram.kg-1.h-1), the Ro2 as well as the pancreatic secretion increased about two times higher than the basal values. With secretin administration, it was found that 1) an inverse relationship between red blood cell velocity and intravascular Hb concentration held and thus 2) [HbO2] inflow was maintained within the basal level (i.e., 200-700 fmol/s). Furthermore, 3) the elevation of Ro2 from single microvessels was accomplished by the increased O2 extraction instead of the increased O2 supply in the microvessels.

Physiological role of nitric oxide as an enhancer of oxygen transfer from erythrocytes to tissues.

The present study revealed the physiological role of NO as an enhancer of oxygen release from erythrocytes to peripheral tissue by breaking or stretching the heme iron-proximal histidine bond in the alpha subunit of hemoglobin (Hb), while maintaining oxygen binding capacity by restoring the bond in the lungs. Oxygen affinity was significantly decreased in blood containing NO-bound Hb alpha. The results may partly explain nitrosothiols in blood as an NO reservoir for an emergency and why cytokines induce NO synthase in the vascular system during such events as inflammation.

Ethanol induces heterogeneous reduction of cytochrome aa3 within perfused rat liver lobule assessed using microspectroscopy.

The redox state of cytochrome aa3 was measured at microspots (20 microns diameter) within the lobule of perfused rat livers, using reflectance microspectroscopy, and the effect of ethanol infusion on sublobular distribution of the redox states was evaluated. A sigmoidal relationship was observed between oxygen delivery and the reduction of cytochrome aa3 in both the periportal and pericentral regions of the liver lobule when the influent O2 concentration was decreased in a graduated manner. This sigmoidal curve was shifted to the more reduced state by ethanol infusion, with ethanol (25-100 mM) increasing the degree of cytochrome aa3 reduction in a dose-dependent manner according to the distance from the periportal region along a sinusoid. This increase was spatially heterogeneous within a liver lobule. These data indicate that ethanol accelerates cytochrome aa3 reduction, with a distinct gradient of reduction along sinusoids but a heterogeneous distribution within the liver lobule.

Direct proof of nitric oxide formation from a nitrovasodilator metabolised by erythrocytes.

When 15N-isosorbide dinitrate was injected into rats, the venous blood showed a 2-line hyperfine structure in the electron spin resonance (ESR) spectra of NO hemoglobin (HbNO), proving generation of 15N-NO. When human blood was mixed with 15N-isosorbide dinitrate anaerobically, ESR spectra showed initially 14N-HbNO, which was then overwhelmed with the increased 15N-HbNO. With deoxygenation of the blood of another 4 male donors without addition of isosorbide dinitrate, two showed 14N-HbNO, but others did not, suggesting varied amount of NO-precursers, nitrosothiols, in human blood. Neither N-ethylmaleimide nor S-hexylglutathione, a glutathione-S-transferase inhibitor, inhibited the formation of UbNO in the hemolysate mixed anaerobically with isosorbide dinitrite.

Effects of NG-nitro-L-arginine and/or L-arginine on experimental pulmonary metastasis in mice.

Effects of NG-nitro-L-arginine methyl ester (L-NAME; an inhibitor of nitric oxide (NO) synthase) and/or L-arginine (substrate of NO synthase) on pulmonary metastasis of murine melanoma and Lewis lung carcinoma cells were investigated. L-NAME, L-arginine or both L-NAME and L-arginine was injected i.p. into mice 5, 3, and 1 h before and 1, 3, 5, and 7 h after the injection of tumor cells into mice via a tail vein. The administration of L-NAME (9.3 mumol/mouse) alone or L-arginine alone (46.5 or 186 mumol/mouse) potentiated pulmonary metastasis of highly and poorly metastatic B16 melanoma cells. L-NAME alone also increased the number of pulmonary metastasis of Lewis lung carcinoma cells, but L-arginine (185 mumol/mouse) did not. However, the combination of L-NAME and L-arginine increased the number of pulmonary metastasis of both the melanoma and Lewis lung carcinoma cells synergistically. L-NAME or L-arginine administration enhanced the retention of B16 melanoma cells in the lungs examined 24 h after the tumor cell injection. Synergistic effect of L-NAME and L-arginine was also seen in the tumor cell retention. The present results suggest that the metastatic potentials of the tumor cells do not simply correlate to NO production in vivo.