Evidence has accumulated to indicate that rodents share with humans almost the same heatstroke syndromes such as hyperpyrexia, hypotension, activated inflammation, and multiorgan dysfunction (in particular, cerebral ischemia, injury, and dysfunction) (1-3). After the onset of heatstroke, both hypotension and intracranial hypertension lead to cerebral ischemia, hypoxia, and neuronal damage. In the central nervous system, overproduction of reactive nitrogen species, reactive oxygen species, cytokines, and cellular ischemia (e.g., glutamate and lactate-pyruvate ratio) and injury (e.g., glycerol) markers in the brain (4, 5) occur during heatstroke.
Another series of evidence has also documented that severe stress and injury (6) and sepsis (7) have all the characteristics of an arginine deficiency state. Heatstroke resembles sepsis in many aspects (2), suggesting the need to replenish l-arginine (6). Earlier work of Poduval et al. (8) have demonstrated that when l-arginine is administered just before heat stress, there is an increased production of nitrite in the blood at approximately 2 h after heat stress and an increased mortality rate. In contrast, in their subsequent work (9), l-arginine at the same dose, administered just after heat stress, rescued the mice from heat-induced death and reduced the hypothermia. These data suggest the potential use of l-arginine, a nonessential amino acid that is used as an external diet supplement, to treat heatstroke-related injury when administered at the appropriate dose and time. However, the effects of l-arginine on cerebrovascular dysfunction and brain inflammation during heatstroke remains unclear.
To deal with the question, the present experiments were performed to compare the temporal profiles of MAP, intracranial pressure, cerebral perfusion pressure, cerebral ischemia and damage markers, and brain cytokines during heatstroke in the rat with or without l-arginine therapy.
MATERIALS AND METHODS
Adult Sprague-Dawley rats (weight, 245 ± 8 g) were obtained from the Animal Resource Center of the Nation Science Council of the Republic of China (Taipei, Taiwan). The animals were housed four in a group at an ambient temperature of 22 ± 1°C with a 12-h light/dark cycle. Pellet rat chow and tap water were available ad libitum. All protocols were approved by the Animal Ethics Committee of the Chi Mei Medical Center (Tainan, Taiwan) in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the guidelines of the Animal Welfare Act. Adequate anesthesia was maintained to abolish the corneal reflex and pain reflexes induced by tail-pinching throughout all experiments (approximately 8 h) by a single i.p. dose of urethane (1.4 g/kg body weight). At the end of the experiments, control rats and any rats that had survived heatstroke were killed with an overdose of urethane.
Induction of heatstroke
Before induction of heatstroke, the core temperature (TCO) of urethane-anesthetized rats was maintained at approximately 36°C with a folded heating pad except during heat stress at a room temperature of 24°C. Heatstroke was induced by increasing the temperature of the folded heating pad to 43°C with circulating hot water. The instant at which MAP dropped to a value of 25 mmHg from the peak level was approximately 70 min after the initiation of heat stress; this time point was arbitrarily taken as the onset of heatstroke (1, 9). The heating pad was then removed, and the animals were allowed to recover at room temperature (24°C). Physiological parameters and survival times (intervals between the initiation of heatstroke and animals death) were then observed to 480 min (or the end of experiments). Physiological data were monitored during the entire course, whereas biochemical parameters were measured at initiation of heat stress (0 min), 70 min after initiation of heat stress, or 85 min after initiation of heat stress.
The animals, under urethane anesthesia, were divided into the following groups. In normothermic groups, animals were treated with normal saline (i.v.; 1 mL/kg body weight; n = 8) In the vehicle-treated heatstroke groups, the animals were treated with a dose of sodium chloride solution (i.v.; 1 mL/kg body weight) 70 min after heat stress. In the drug-treated heatstroke groups, the animals received an i.v. dose of l-arginine (Sigma-Aldrich Chemical Co., St. Louis, Mo; 50 - 250 mg/kg) 70 min after initiation of heat exposure. The volume injected for the drug-treated heatstroke group was the same as for the vehicle-treated groups.
Surgery and physiological parameter monitoring
The right femoral artery and vein of rats were cannulated with polyethylene tubing (PE50) under urethane anesthesia for blood pressure monitoring and drug administration, respectively. The animals were positioned in a stereotaxic apparatus (Kopf 1406; Grass Instrument, Quincy, Mass) to insert probes for measurement of intracranial pressure (ICP). The ICP was monitored with a Statham P23AC transducer via a 20-gauge stainless-steel needle probe (diameter, 0.90 mm; 38 mm), which was introduced into the right lateral cerebral ventricle according to the stereotaxic coordinates of Paxinos and Watson (10): A, interaural, 7.7 mm; L, 2.0 mm from the midline; and H, 3.5 mm from the top of the skull. All recordings were made on a four-channel Gould polygraph. Core temperature was monitored continuously by a thermocouple, and MAP and heart rate were continuously monitored with a pressure transducer. Different groups of animals were used for the different sets of experiments: measurement of survival time; measurement of latency for onset of heatstroke; measurement of TCO, MAP, ICP, cerebral perfusion pressure (CPP = MAP-ICP), brain PO2, and cerebral blood flow (CBF); measurement of extracellular levels of glutamate, glycerol, NO2−, lactate/pyruvate, and dihydroxybenzoic acid (DHBA); and measurement of brain levels of TNF-α, IL-1β, and IL-10. The hypothalamus was chosen as the representative brain region for measurement of CBF, brain PO2, and extracellular levels of glutamate, glycerol, NO2−, lactate/pyruvate, DHBA, IL-1β, IL-10, and TNF-α.
Measurements of extracellular glutamate, glycerol, and lactate-pyruvate ratio in the hypothalamus
Each animal was anesthetized with urethane intraperitoneally. The animal's head was mounted in a stereotaxic apparatus (Kopf 1406; Grass Instrument) with the nose bar positioned 3.3 mm below the horizontal line. After a midline incision, the skull was exposed, and a burr hole was made in the skull for the insertion of a microdialysis probe (4 mm in length, CMA/12: inside diameter, 150 μm; outside diameter, 220 μm; Carnegie Medicine, Stockholm, Sweden). The microdialysis probe was implanted stereotaxically into the hypothalamus according to the atlas and coordinates of Paxinos and Watson (10). The coordinates for the right hypothalamus were P, 1.8 mm; L, 0.8 mm from the midline; and H, 8.8 mm from the top of the skull. According to the methods described previously (4), the microdialysis probe was perfused at 2.0 μL/min, and the dialysates were sampled in microvials. The dialysates were collected every 10 min in a CMA/140 fraction collector (Carnegie Medicine). Aliquots of dialysates (5 μL) were injected onto a CMA600 microdialysis analyzer (Carnegie Medicine) for measurement of lactate, glycerol, pyruvate, and glutamate. Four analytes can be analyzed per sample, and the result is displayed graphically within minutes.
Lactate is enzymatically oxidized by lactate oxidase. The hydrogen peroxide formed reacts with 4-chlorophenol and 4-aminoantipyrine. This reaction is catalyzed by peroxidase (POD) and yields the red violet-colored quinoneimine. The rate of formation is measured photometrically at 546 nm and is proportional to the lactate concentration.
Pyruvate is enzymatically oxidized by pyruvate oxidase. The hydrogen peroxide formed reacts with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine and 4-amino-antipyrine. This reaction is catalyzed by POD and yields the red violet-colored quinoneimine. The rate of formation is proportional to the pyruvate concentration.
Glutamate is enzymatically oxidized by glutamate oxidase. The hydrogen peroxide formed reacts with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine and 4-amino-antipyrine. This reaction is catalyzed by POD and yields the red violet-colored quinoneimine. The rate of formation is measured photometrically at 546 nm and is proportional to the glutamate. After histological verification of the probe's path, all the data obtained were included in our results.
Measurements of CBF and brain PO2
A 100-μm-diameter thermocouple and two 230-μm fibers are attached to the oxygen probe. This combined probe measures oxygen, temperature, and microvascular blood flow. The measurement requires OxyLite and OxyFlo instruments. OxyLite 2000 (Oxford Optronix, Oxford, UK) is a two-channel device (measuring PO2 and temperature at two sites simultaneously), and OxyFlo 2000 is a two-channel laser Doppler perfusion monitoring instrument. The OxyLite has been designed to operate in conjunction with the OxyFlo. The combination of these two instruments provides simultaneous tissue blood flow, oxygenation, and temperature data. After each animal, under urethane anesthesia, was placed in a stereotaxic apparatus, a combined probe was implanted in hypothalamus of rat brain by using the atlas and coordinates of Paxinos and Watson (10). The probe calibration parameters are transferred from the probe packaging to the OxyLite instrument using the bar code wand. For each PO2 input on the OxyLite front panel, there is a corresponding temperature input. A thermocouple sensor may be attached to these temperature inputs using the thermocouple adapters provided. The temperature measurement serves two purposes: (1) to automatically compensate the PO2 measurement and (2) to continuously monitor tissue temperature. The OxyFlo is a laser Doppler flow meter whose primary purpose is to measure real-time microvascular red blood cell perfusion. Laser Doppler signals are recorded in blood perfusion unit, which is a relative unit scale defined using a carefully controlled motility standard. The OxyFlo is calibrated before leaving the factory, using a motility standard solution of carefully selected latex spheres undergoing Brownian motion. The OxyFlo is a stable instrument and should not under normal circumstances require recalibration.
Hypothalamic NO2− and DHBA monitoring
For determination of NO2− or DHBA, extracellular fluids of hypothalamus were taken 0, 70, and 85 min after the start of heat exposure. A microdialysis probe (CMA20; Carnegie Medicine, Stockholm, Sweden) with a 4-mm-long dialysis membrane was vertically implanted into the left hypothalamus. Ringer solution was perfused through the microdialysis probe at a constant flow (2.0 μL/min). After 6 h of stabilization, the dialysates from the hypothalamus were collected at 20-min intervals. The NO2− concentrations in the dialysates were measured with the Eicom ENO-20 NO2− analysis system (Eicom, Kyoto, Japan) (11). In the Eicom ENO-20 NO2− analysis system, after the NO2− and NO3− in the sample have been separated by the column, the NO2- reacts in the acidic solution with the primary aromatic amine to produce an azo compound. After this, the addition of aromatic amines to the azo compound results in a coupling that produces a diazo compound, and the absorbance rate of the red color in this compound is then measured. The system is capable of detecting up to 0.1 pmol. At that time, experiments were performed to determine the effects of heatstroke on the NO2− release in the hypothalamus. After histological verification of the probe's path, all the data obtained were included in our results.
The concentrations of DHBA were measured by a modified procedure based on the hydroxylation of sodium salicylate by hydroxyl radicals, leading to production of 2,3-DHBA and 2,5-DHBA (12).
Determination of TNF-α, IL-1β, and IL-10
For determination of TNF-α, IL-1β, and IL-10, brains (hypothalamic) were taken 0, 70, and 85 min after the start of heat stress. The brain samples were disintegrated in five volumes of ice-cold Ripa buffer. The homogenates were incubated on ice for 30 min and then centrifuged (15,000g, 30 min, 4°C) twice. The supernatants were stored at 70°C until measurement. The concentrations of TNF-α, IL-1β, and IL-10 in tissue lysates were determined using double-antibody sandwich enzyme-linked immunosorbent assay (R & D System, Minneapolis, Mn) according to the manufacturer's instruction. Optical densities were read on a plate reader set at 450 nm for TNF-α, IL-1β, and IL-10. The concentrations of TNF-α, IL-1β, or IL-10 in the samples were calculated from the standard curve multiplied by the dilution factor and were expressed as picograms per gram. Protein concentration was determined by the method of Lowry et al. (13).
All values are expressed as the mean ± SEM and were analyzed by two-way ANOVA. Duncan multiple range test was used for post hoc multiple comparison among means. All results were considered statistically significant at P < 0.05.
l-Arginine improves survival rate during heatstroke
Table 1 summarizes the survival time values for normothermic controls, vehicle-treated heatstroke rats, and l-arginine-treated heatstroke rats. Normothermic controls without treatment (Table 1) were killed approximately 480 min after the initiation of heat stress (or at the end of the experiments). Survival time values during heatstroke for rats treated with vehicle solution were found to be 20 to 26 min. Treatment with L-arginine (50 - 250 mg/kg, i.v.) dose-dependently increased the survival time values to new levels of 54 to 245 min.
l-Arginine attenuates hypotension and cerebrovascular dysfunction during heatstroke
Table 2 summaries the effects of heat exposure (43°C for 70 min) on several physiological parameters in rats treated with vehicle solution, rats treated with L-arginine (150 mg/kg body weight, i.v.), and normothermic controls. In vehicle-treated heatstroke groups, the TCO and ICP were all significantly higher at 70 to 85 min after the start of heat exposure than they were for normothermic controls. In contrast, the values for MAP, CPP, CBF, and brain PO2 at 85 min were significantly lower than those of normothermic controls. Treatment with L-arginine (150 mg/kg, i.v.) 70 min after initiation of heat exposure significantly attenuated the heat stress-induced arterial hypotension, intracranial hypertension, and cerebral hypoperfusion and hypoxia in the hypothalamus. The basal levels of physiological parameters measured in normothermic rats treated with L-arginine (150 mg/kg body weight, i.v.) were indistinguishable from those of normothermic rats without treatment.
l-Arginine attenuates TNF-α and IL-1β overproduction but enhances IL-10 production in the hypothalamus during heatstroke
The hypothalamic levels of TNF-α, IL-1β, and IL-10 for normothermic controls, vehicle-treated heatstroke rats, and L-arginine-treated heatstroke rats are summarized in Table 3. It can be seen from the table that the hypothalamic levels of TNF-α and IL-1β were all significantly higher at 70 to 85 min after the start of heat stress than they were for normothermic controls. Treatment with L-arginine (150 mg/kg body weight, i.v.) 70 min after initiation of heat exposure significantly attenuated the heat stress-induced increased levels of TNF-α and IL-1β in the hypothalamus. In vehicle-treated heatstroke rats, hypothalamic levels of IL-10 were maintained at a negligible level. However, the hypothalamic levels of IL-10 were greatly elevated in heatstroke rats treated with an i.v. dose of l-arginine.
l-Arginine attenuates hypothalamic levels of glycerol, lactate-pyruvate ratio, glutamate, NO2−, and DHBA during heatstroke
Table 4 summarizes the effects of heat exposure (43°C for 70 min) plus 15 min room temperature (24°C) exposure after heatstroke onset on cellular ischemia and injury marker values of the hypothalamus from vehicle-treated or l-arginine-treated rats. In vehicle-treated heatstroke rats, after onset of heat exposure, the values of glycerol, glutamate, lactate-pyruvate ratio, NO2−, and DHBA values were all greater than those in the normothermic controls. However, the heatstroke-induced increase of these substances in the hypothalamus was greatly attenuated by l-arginine therapy.
As mentioned in the " Introduction," l-arginine (120 mg/kg, i.p.), when administered at 1 h before or 1 h after heat stress, led to increased mortality of mice (8, 9). In contrast, i.p. administration of L-arginine (120 mg/kg) 2 to 4 h after the termination of heat stress rescued the mice from heatstroke and profound hypothermia (9, 14). These results suggest that therapeutic administration of L-arginine at appropriate concentration and time leads to the rescue of mice from heatstroke. In the present study, we further demonstrated that i.v. injection of l-arginine (50 - 250 mg/kg) immediately after termination of heat stress in rats significantly improved the survival rate during heatstroke in a dose-dependent way. Therapeutic administration of l-arginine to patients significantly decreased the frequency and severity of stroke-like episodes induced by impaired vasodilation in an intracerebral artery (15). Therefore, the potential use of l-arginine to treat heatstroke related injury is indicated.
In the current studies, all heat-stressed rats displayed hypotension and cerebral dysfunction (evidenced by increased hypothalamic levels of glycerol, glutamate, lactate/pyruvate ratio, DHBA, and NO2−) and inflammation (evidenced by increased IL-1β and TNF-α levels in brain) during heatstroke. Both body temperature and ICP were also increased during heatstroke. In contrast, the values of MAP, CPP, and hypothalamic levels of local blood flow and partial pressure of oxygen were all significantly lower during heatstroke. The present results further showed that L-arginine improved the survival rate during heatstroke by reducing hypotension, cerebral dysfunction, and inflammation. However, the heatstroke-induced hyperthermia was not altered by L-arginine. This demonstrates that even under the absence of whole-body or brain cooling, L-arginine is still able to improve the survival rate during heatstroke by reducing these heatstroke reactions.
In our rat model, overproduction of both IL-1β and TNF-α in the central nervous system (including hypothalamus) occurs during heatstroke. The arterial hypotension occurred during heatstroke can be mimicked by i.v. infusion of IL-1β (16). Pretreatment with IL-1β receptor antagonists (16) is able to improve survival during heatstroke by reducing arterial hypotension and cerebral ischemia and damage. As shown in present results, L-arginine attenuates overproduction of both IL-1β and TNF-α in the central nervous system during heatstroke. In addition, IL-10, an anti-inflammatory cytokine that potently inhibits the production of both IL-1β and TNF-α (17-19), is accelerated by L-arginine during heatstroke in the rat. Thus, it seems that L-arginine may reduce both IL-1β and TNF-α production during heatstroke by increasing IL-10 production.
It has been repeatedly documented that brain NO levels are elevated in heatstroke rats (20-22). Aminoguanidine (an inducible NO synthase inhibitor) (21) and 7-nitroindazole (a neuronal NO synthase inhibitor) (22) improved heat tolerance in rats by reducing intracranial hypertension and cerebral ischemia and injury. Like aminoguanidine or 7-nitroindazole, L-arginine ameliorated the heatstroke-induced cerebral NO2− overproduction and cerebrovascular dysfunction. The increase in extracellular levels of ischemia (e.g., glutamate and lactate-pyruvate ratio) and injury (e.g., glycerol) markers in the hypothalamus (4) that occurred during heatstroke was significantly suppressed by therapeutic doses of l-arginine. These results strongly indicate that L-arginine may attenuate cerebral ischemia and injury during heatstroke by reducing brain NO production.
Studies have been undertaken to elucidate the role of l-arginine in the immunomodulation of the heat-stressed mice (14). Levels of IL-1β, nitrite, TNF-α, iNOS, and corticosterone significantly increased in these heat-stressed mice. The elevated levels of TH1 cytokines, namely, TNF-α, IL-1β, nitrite, and iNOS decreased significantly after L-arginine administration. These results suggest that therapeutic administration of L-arginine at appropriate concentration and time attenuates the acute inflammatory response, leading to the rescue of mice from heatstroke. Because TH2 cytokines and arginase are known to be involved in the regulation of acute inflammation and tissue repair (23-26), it has been hypothesized that the up-regulation of TH2 cytokines and arginase as well as the down-regulation of iNOS by the administration of L-arginine is central to the rescue of the rodents from heatstroke-induced death (14).
Both our previous (5, 12, 27) and present results demonstrated that overproduction of DHBA in the brain (or hypothalamus) occurred during heatstroke. In the present studies, we further showed that the increased production of DHBA in the hypothalamus during heatstroke was significantly reduced by L-arginine therapy. Overproduction of DHBA in tissues has been reported to be directly involved in oxidative damage with cellular macromolecules in ischemic tissues, which lead to cell death (28). Thus, it is likely that L-arginine may attenuate cerebral ischemia and damage occurred during heatstroke by inhibiting oxidative stress in brain.
In summary, our results have demonstrated that cerebral ischemia or hypoxia (resulting from both arterial hypotension and intracranial hypertension) leads to neuronal injury that results in cerebral dysfunction and death. The neurotoxic cascade involves overproduction of glutamate, proinflammatory cytokines, reactive nitrogen species, and reactive oxygen species in the brain. L-Arginine is able to maintain appropriate levels of cerebral blood flow and oxygenation to prevent the secondary neurotoxic cascades and to improve survival during heatstroke.
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Heatstroke; cerebral blood flow; hypotension; cytokines; intracranial pressure; l-arginine