The cardioprotective effects of glucocorticoids in the acute setting of ischemia/reperfusion have been experimentally demonstrated in animals1–7 and humans.8 Although the mechanisms underlying glucocorticoid cardioprotection are not known with certainty, glucocorticoids are reported to preserve cellular function in ischemia thereby prolonging the period of myocardial viability. In previous studies, glucocorticoids were reported to improve lactate imbalance and prevent the leakage of intracellular enzymes caused by myocardial ischemia.6,7
At the same time, deleterious effects of glucocorticoids on ischemia-induced neuronal damage have been shown in various animal models, despite the ability of glucocorticoids to alleviate cerebral edema caused by traumatic brain injury and hemorrhage.9–13 Further, steroids are not indicated for traumatic brain injury in humans, as they do not improve clinical outcomes.14
Because adenosine 5′-triphosphate (ATP) provides energy to maintain membrane functional integrity in both the heart and brain, and ATP depletion precedes loss of cellular viability, the different effects of the agents in ATP utilization in the heart and brain during ischemia could well explain differences in ischemia-induced organ injury. In the present study, therefore, we investigated the effect of dexamethasone, a glucocorticoid without mineralocorticoid activity, on ischemia-induced reduction of ATP in the mouse heart and brain. In addition, we examined the effect of metyrapone, a potent inhibitor of the synthesis of glucocorticoids, to assess the role of endogenous glucocorticoids. We hypothesized that ATP levels would be diminished in neuronal tissue subjected to dexamethasone and improved in cardiac tissue subjected to dexamethasone. In contrast, ATP levels would be improved in neuronal tissue subjected to metyrapone and diminished in cardiac tissue subjected to metyrapone.
MATERIALS AND METHODS
This study was approved by the Committee on Animal Experimentation at Ehime University Graduate School of Medicine, Ehime, Japan. All animals were cared for in compliance with the Principles of Laboratory Animal Care formulated by Ehime University Graduate School of Medicine. Male ddY mice at 10 weeks of age, weighing about 40 g (Japan SLC, Shizuoka, Japan), were housed in an acrylic cage (5 mice in each cage) (CLEA Japan Inc., Tokyo, Japan) with paper bedding (Japan SLC) at 23±1°C, and maintained in an alternating 12-hour light/12-hour dark cycle (lights on at 06:00 h). A certified diet (MF; Oriental Yeast Co. Ltd., Tokyo, Japan) and water filtrated with activated carbon were provided ad libitum. The mice were deprived of food for at least 6 hours preceding the start of experiments to eliminate the influence of glucocorticoids on the plasma concentration of glucose.
Seventy-two mice were prepared and then randomly assigned to 4 saline groups, 4 dexamethasone groups, and 4 metyrapone groups (6 animals in each). Saline, dexamethasone (5 mg/kg), or metyrapone (100 mg/kg) was administered intraperitoneally, and mice were decapitated after 3 hours. Previous studies reported that these agents did not affect plasma concentrations of glucose and electrolytes 3 hours after treatments.11,13,15
The heart was removed and incubated in a hypoxic phosphate buffer solution equilibrated with nitrogen (pH 7.4, 37°C). The partial pressure of oxygen in the hypoxic phosphate buffer was kept between 29.9 and 41.5 mm Hg. Then, the heart was frozen in liquid nitrogen after 5, 10, or 20 minutes of incubation. In the nonischemic (0 min) groups, the heart was frozen immediately after its removal, and body temperature was quickly measured in the sternocleidomastoid muscle on the side to the sternal origin of the body by a thermocouple needle probe (DTE-10 N; Inter Medical, Aichi, Japan).
After decapitation, the head was frozen in liquid nitrogen after 0, 0.5, 1, or 2 minutes. The thermocouple needle probe was inserted into the sternocleidomastoid muscle on the side to its insertion of the skull, and head temperature was kept approximately 37.5°C with a heating lamp during decapitation ischemia in the 0.5-, 1-, and 2-minute groups. The temperature of the sternocleidomastoid muscle was recorded before freezing the head after 0.5, 1, and 2 minutes. The brain was removed from the frozen skull on dry ice to prevent the degeneration of adenylates.
We monitored temperature in the sternocleidomastoid muscle as a surrogate for temporal muscle temperature, as hypothermia is well known to provide benefits on ischemia-induced organ injury, and temporal muscle temperature well reflects brain temperature.16,17 However, we could measure neither brain nor temporal muscle temperature in the 0-minute groups, because the head had been frozen immediately after decapitation. We measured temperature in the sternocleidomastoid muscle near its insertion on the skull and its sternal origin as a surrogate for brain and body temperatures, respectively, immediately after decapitation in the 0-minute groups, although there are no reports that showed the relationship between sternocleidomastoid muscle temperature and temporal muscle and body temperatures.
The frozen heart and brain were weighed and quickly homogenized with ice-cold perchloric acid (3 mL of 0.4 mol/L). After centrifugation at 20,000g for 30 minutes, the supernatant was injected into a high-performance liquid chromatography (HPLC) system to determine the tissue concentrations of ATP, adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP).12 The HPLC system consisted of a pump (L-7100; Hitachi, Tokyo, Japan) used to deliver the mobile phase, a model L-7250 sample injector (Hitachi) with a 100 μL sampling loop, a separation column (GL-W510-S, 7.8×300 mm inside diameter; Hitachi), and an ultraviolet detector (L-7400; Hitachi). The mobile phase was 0.2 mol/L NaH2PO4, with an adjusted pH of 3.5 with 0.2 mol/L H3PO4, and the flow rate was 0.5 mL/min. The absorption intensity (peak height) was measured at a wavelength of 270 nm.
The concentrations of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-hydroxytryptamine, 5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in the brain homogenates were determined using an HPLC system with electrochemical detection (Eicom, Kyoto, Japan).18 The HPLC system consisted of a pump equipped with a damper (EP-300; Eicom), an electrochemical detector (ECD-300; Eicom) with a graphite-working electrode operated at 750 mV versus a silver-silver chloride reference electrode (RE-100; Eicom), and a reverse-phase column (MA-5ODS, 2.1×150 mm inside diameter; Eicom). The mobile phase consisted of 0.1 mol/L citrate and 0.1 mol/L sodium acetate buffer (pH 3.3) containing 15% methanol, 1 mmol/L sodium 1-octanesulfonate, and 10 μmol/L disodium ethylenediaminetetraacetic acid.
Although agents were not injected in a blinded manner, the sampling of organs and the measurements of adenylates and monoamines were performed by investigators who were unaware of the particular treatment group.
The data were analyzed using 2-way analysis of variance to evaluate interactions between treatments and time points. Then, the data on the 3 treatment groups at each time point were analyzed by 1-way analysis of variance, and post hoc comparisons were performed with the Scheffé test (StatView for Windows, ver. 5.0; SAS Institute Inc., Cary, NC). P<0.05 was considered statistically significant. Confidence intervals were calculated with SPSS Statistics, ver. 11.0.1 J (SPSS Inc., Chicago, IL).
Body temperature measured in the sternocleidomastoid muscle, on the side of the sternal origin of the body, immediately after decapitation in the saline, dexamethasone, and metyrapone groups was 37.8±0.4, 37.6±0.4, and 36.9±0.6°C (mean±SD, n=6), respectively. Body temperature in the metyrapone group was significantly lower than that in the saline group (P<0.05). No significant differences were observed between the dexamethasone and saline groups (P=0.76), and between the dexamethasone and metyrapone groups (P=0.11). Consistent with our attempt to maintain head temperature at 37.5°C with a heating lamp after decapitation, no statistical differences were observed in temperatures measured in the sternocleidomastoid muscle, on the side to its insertion of the skull, among the 3 groups at each time point between 0.5 and 2 minutes of ischemia, although the results were not sufficiently powered with an n=6 per experimental group (Table 1).
There were no differences in the ATP concentration in the heart among the 3 groups when the hearts were frozen immediately after removal (Fig. 1, Table 2). However, the power value was not sufficient. In the saline group, ischemia for 5 minutes decreased the ATP content to 21% of the basal level. However, the extent of the ATP decrease caused by 5 minutes of ischemia was significantly suppressed in the dexamethasone and metyrapone groups. The ATP levels were 63% and 64% of each basal level, respectively. Longer duration of ischemia further lowered the ATP content in each group and no differences were found among the 3 groups after 10 or 20 minutes of ischemia.
Similar to the decrease in ATP levels, the heart ADP content decreased during ischemia. Pretreatment with dexamethasone suppressed the extent of the ADP decrease, and the effect was significant after 5 minutes of ischemia. In the metyrapone group, the ADP level was significantly higher at any duration of ischemia than that in the saline and dexamethasone groups. In the dexamethasone and metyrapone groups, the heart AMP content at 5 minutes of ischemia was significantly lower than that in the saline group (P<0.01).
Concerning the brain concentrations of adenylates, the ATP levels in the saline and dexamethasone groups at 0.5 minute of ischemia were 34% and 37% of those at the beginning of ischemia, respectively (Fig. 2, Table 3). Suppression of the ATP decrease by dexamethasone was not significant in the brain. The ATP concentration in the metyrapone group was lower than that in the other groups at the start of cerebral ischemia with 62% of the concentration of the saline group. The ADP level in the dexamethasone group was higher than that in the saline group at both 0 and 1 minute of ischemia. The ADP concentration in the metyrapone group was lower than that in the saline and dexamethasone groups at 0.5 minute of ischemia. Conversely, the AMP concentration in the metyrapone group was 276% of that in the saline group at 0 minute and was still marked at 0.5 minute of ischemia. There was no significant difference in the AMP level between the saline and dexamethasone groups.
The dopamine concentration in the brain was almost constant in the saline and metyrapone groups during cerebral ischemia (Fig. 3, Table 4). However, the dopamine level in the dexamethasone group increased at 0.5 minute and returned to the basal level at 1 minute of ischemia. Although the concentration of DOPAC, a metabolite of dopamine, in the saline group was almost constant during ischemia, its level in the dexamethasone group was higher than that in the saline group at 0.5 minute of ischemia. The DOPAC level in the metyrapone group was lower than that in the other 2 groups during the ischemic period. In the saline group, the concentration of HVA, a metabolite of dopamine, increased at 0.5 minute of ischemia and returned to the basal level at 1 minute of ischemia. In the dexamethasone group, the HVA concentration remained high for 1 minute, and its level in the metyrapone group did not change during ischemia.
The 5-HT level did not differ among groups during ischemia (Fig. 4, Table 5). Changes in the concentration of its metabolite, 5-HIAA, were similar to those in the HVA level. The 5-HIAA level at 0 minute was higher in the dexamethasone group than in the other groups, and its level in the metyrapone group remained low during ischemia.
The findings in the present study are: (1) in the heart, metyrapone increased ADP at preischemia and during ischemia, and both metyrapone and dexamethasone increased ATP and decreased AMP in the early ischemic period; (2) in the brain, metyrapone decreased ATP and increased AMP at preischemia, and decreased ADP and increased AMP in the early ischemic period; and (3) in the brain, dexamethasone increased metabolites of dopamine and 5-HT before and during ischemia.
There are many studies reporting that glucocorticoids alleviate ischemia-induced myocardial injury. In this study, dexamethasone attenuated an ischemia-induced decrease in the heart ATP concentration. This may be caused by an enhancement of ATP synthesis in the heart or a suppression of ATP consumption in an anaerobic state. In the present study, however, the ATP level at the beginning of ischemia did not differ among the groups, and generation of ATP under ischemic states is unlikely in any of the groups. Therefore, it is highly probable that dexamethasone reduced ATP consumption due to a decrease in the energy requirement in the heart. In a canine study on myocardial ischemia, a pretreatment with methylprednisolone improved the segmental myocardial function and lactate imbalance, indicating the improvement of energy metabolism in an anaerobic state.7 These findings, taken together with our present results, indicate that glucocorticoids provide benefits in the heart by reducing energy requirements during ischemia, although the power value was not sufficient.
Preservation of ATP in the heart during ischemia was also reported in studies on ischemic preconditioning.19–21 Preconditioning myocardium with 4 cycles of 5-minute ischemia and reperfusion slowed the rate of ATP depletion during sustained ischemia, and inhibited irreversible injury in the canine heart.19 Considering preconditioning effects of preischemic administration of glucocorticoids, reduction of energy demand may be a contributing factor in alleviation of ischemia-induced myocardial damage.
There are some controversial reports that glucocorticoids do not protect against ischemia-induced myocardial changes.22,23 There are differences in the methodology of positive and negative reports, such as timing of drug administration, dosage, duration of ischemia, and animal species. In most studies that failed to show benefits, glucocorticoids were administered after ischemic events.23 Because postischemic treatments cannot exert an influence on energy metabolism during ischemia, preischemic administration seems to be considered. An acute cardioprotective effect of a high-dose methylprednisolone before cardiopulmonary bypass on ischemia-reperfusion injury has also been reported in a clinical study.24
In the present study, the brain concentration of dopamine in the dexamethasone group was higher than that in the other 2 groups. Furthermore, the concentrations of its metabolites, DOPAC and HVA, increased during ischemia. Particularly, the HVA concentration in the dexamethasone group was higher at the start of ischemia than that in the control group, suggesting facilitated activity of the central dopaminergic system by dexamethasone before induction of ischemia. Despite the enhancement of neuronal activity, ATP consumption was not facilitated. In our previous studies on ATPase activity, identical doses of dexamethasone suppressed enzyme activity of the sodium pump in acidic conditions and suppressed ATP hydrolysis.25 Both decreased supply and increased demand of energy may make neurons relatively energy depleted, and thus vulnerable.
Besides energy depletion, an increase in dopamine metabolism is considered to be an important mechanism for dopamine toxicity, because oxidation of dopamine by monoamine oxidase generates reactive oxygen species as well as DOPAC.26 Furthermore, spontaneous and enzymatic oxidation of the catechol ring forms hydrogen peroxide.27 Dissimilar to neurotoxicity of the dopaminergic system, the serotonergic system has been reported to provide benefits on ischemia-induced neuronal injury.28,29 In the present study, the 5-HIAA concentrations in the dexamethasone group were higher than those in the saline group. As glucocorticoids aggravate ischemia-induced neuronal injury, the protective effects of the serotonergic system may be insufficient.
Glucocorticoids have been shown to increase the plasma glucose level, and hyperglycemia is known to exacerbate ischemia-induced neuronal damage due to the intracellular lactic acidosis associated with enhanced anaerobic metabolism. In the current study, agents were administered 3 hours before induction of ischemia, and the treatments did not affect physiological variables, plasma levels of glucose, electrolytes, and arterial blood gas tensions in our previous studies.11,13,15 Furthermore, effects of dexamethasone were evaluated by isolated hearts, using a glucose-free medium, and isolated brains. Therefore, it is unlikely that the glucose level affected the current results. Likewise, genomic effects by modifying nucleic acids through steroid receptors in the nucleus may not be relevant, as agents were administered 3 hours before induction of ischemia. Rapidly induced enzymes by glucocorticoids that detoxify mediators released during stress are conceivable as mechanisms of acute effects of glucocorticoids.30,31
Metyrapone is a potent and rapid inhibitor of the synthesis of glucocorticoids. It blocks the 11β-hydroxylation step by inhibiting cytochrome P450 and reduces the plasma concentration of endogenous glucocorticoids.32,33 In the present study, the agent attenuated a ischemic decrease in the ATP concentration, as did the dexamethasone treatment. Furthermore, it increased the heart ADP content for the entire duration of ischemia. Metyrapone is reported to reduce oxygen consumption and decrease body temperature as observed in the present study.33,34 Although the precise mechanism underlying hypothermia is obscure, hypothermia may have resulted in preservation of ATP during ischemia and accumulation of ADP by depressing hydrolysis of ATP and phosphorylation of ADP. For these reasons, the preservation of ATP in metyrapone-treated animals may be caused by another mechanism besides depletion of endogenous glucocorticoids.
Although there are no reports of metyrapone aggravating ischemia-induced myocardial injury, the agent has been shown to reduce brain damage induced by focal and global ischemia and seizure.35 We also confirmed beneficial effects of metyrapone on ischemia-induced neuronal damage.10,15 It reduced glutamate toxicity and ameliorated delayed neuronal death caused by transient ischemia. In the present study, however, the ATP concentration in the brain in the metyrapone group was markedly low, whereas the AMP concentration was high at the beginning of ischemia, showing suppression of the energy charge in the brain by metyrapone. Hence, changes in the energy level cannot explain beneficial effects of the agent in the brain.
There is a study in which protective effects of metyrapone independent of plasma corticosterone levels was demonstrated.36 In the study, supplemental administration of corticosterone in metyrapone-treated rats did not result in a subsequent increase in brain damage and seizures when compared with metyrapone-treated animals.36 In another study, the hippocampal levels of glutamate and γ-aminobutyric acid, neurotransmitter amino acids, have been shown to be suppressed by metyrapone.37 These are consistent with our present results on the dopaminergic and serotonergic activity. The neuroprotective properties of metyrapone may be partly attributed to the metabolic modifications on the nervous system.
In conclusion, exogenous administration of dexamethasone and blockade of endogenous glucocorticoids by metyrapone did not act oppositely on energy levels in the present study. Further, we could not clearly elucidate the relationship between changes in energy levels by glucocorticoids and cellular viability, as morphologic and functional changes by the agents were not evaluated in the present animal model. Studies that can correlate functional outcomes to energy levels are required, at least, and the relevance to clinical practice of glucocorticoids remains to be clarified in future studies.
The authors thank Dr. Keizo Ikemune for his assistance on statistical data analysis.
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