The mechanism by which barbiturates protect neurons against ischemia is unclear, particularly when they are given after ischemia or reperfusion begins. Because an excess release of excitatory neurotransmitters causes postsynaptic membrane depolarization, which triggers neuronal damage in ischemia, the effects of thiopental on histologic outcome, ischemia-induced amino acid release, and anoxic depolarization in gerbils were studied.
The effects of different doses of thiopental administered before or after ischemia were examined morphologically by assessing delayed neuronal death in hippocampal CA1 pyramidal cells produced by forebrain ischemia for 3 min in gerbils. The ischemia-induced changes in output of aspartate, glutamate, glycine, taurine, and gamma-aminobutyric acid were measured using a microdialysis-high-performance liquid chromatography procedure, and the differences among a halothane-anesthetized group, a thiopental-administered group, and a group given thiopental after a period of ischemia were evaluated. The changes induced in the direct-current potential in the hippocampal CA1 area by forebrain ischemia were compared in animals anesthetized with halothane and those given thiopental.
Preischemic administration of thiopental at all doses decreased the risks for delayed neuronal death (P < 0.01). Post-ischemic administration at a dosage of 2 mg.kg-1.min-1 for 60 min protected neurons, but the same dose for 10 min did not ameliorate the cell injury. Forebrain ischemia produced marked increases in all amino acids 3 to 6 min after the start of recirculation in the halothane-anesthetized gerbils, whereas thiopental anesthesia (2 mg.kg-1.min-1) reduced these increases throughout the experimental period, except for glycine (P < 0.01). The initiation of thiopental after reflow did not markedly diminish these increases. Thiopental anesthesia prolonged the onset of anoxic depolarization and reduced its maximal amplitude.
Thiopental helps protect the brain from ischemia, although treatment with this agent after ischemia requires a larger dose than that before ischemia. The effect of preischemic treatment may be related to the suppression of the excitatory amino acid release and the direct-current potential shift.
Key words: Anesthetics, intravenous: thiopental. Animals: gerbils. Brain: anoxic depolarization; hippocampus; ischemia. Measurement techniques: direct-current potential; microdialysis. Neurotransmitters, excitatory: amino acids.
Although the neuroprotective efficacy of barbiturates under some ischemic conditions is unconfirmed, [1,2]these drugs are clearly valuable in other circumstances, particularly to treat temporary focal ischemia. Early studies indicated that the mechanism of barbiturates' action was diminution of the cerebral metabolic rate, which leads to decreasing use of adenosine triphosphate (ATP) during ischemia. However, more recent studies have shown that the protective efficacy of barbiturates is not caused solely by the diminution of the cerebral metabolic rate, because the degree of protection provided by various anesthetics or by hypothermia does not correlate with the magnitude of their depression of the cerebral metabolic rate. Because excitatory amino acid release is believed to play an important part in ischemic disease, [5,6]we studied the effect of barbiturates on the release of excitatory amino acids. We also examined the relations between this effect and the histopathologic outcome and the changes in the direct-current (DC) potential, which is a measure of membrane functional integrity.
Materials and Methods
The study was approved by the Committee on Animal Experimentation at Ehime University School of Medicine, Ehime, Japan. Male Mongolian gerbils weighing 60 to 80 g (Seiwa Experimental Animals, Fukuoka, Japan) were housed in groups in a room controlled at 23 +/- 2 degrees Celsius and maintained in an alternating 12-h light/12-h dark cycle (lights on at 6:00 A.M.). Animals were deprived of food for at least 6 h before ischemia. All experiments were performed under spontaneous ventilation.
In experiment 1, the physiologic variables affecting the ischemia-induced neuronal damage were measured in a group of animals given thiopental and in a group anesthetized with halothane. In experiment 2, delayed neuronal death in the hippocampal CA1 sector provoked by forebrain ischemia was examined, and the differences in the effect of thiopental before and after ischemia were compared. In experiment 3, extracellular concentrations of transmitter amino acids were measured using a microdialysis-high-performance liquid chromatography procedure, and the changes in amino acids produced by the administration of thiopental were assessed in normal and ischemic conditions. In experiment 4, the changes in the DC potential produced by forebrain ischemia in the hippocampal CA1 region were monitored in two groups of animals-those anesthetized with halothane and those anesthetized with thiopental.
Experiment 1: Measurement of Physiologic Variables
Eight gerbils were used: four gerbils were given thiopental and the other four served as controls. Each animal was anesthetized with 2% halothane given by inhalation. With the gerbil in the supine position, the right femoral vein was exposed and a Teflon catheter was inserted to administer the drug.
In the thiopental group, sodium thiopental was injected into the right femoral vein at a rate of 2 mg [centered dot] kg sup -1 min [centered dot] sup -1. Simultaneous with the start of the infusion, the halothane flow was changed to oxygen. In the control animals, saline was administered under halothane anesthesia.
The abdominal aorta was exposed after making an incision along the ventral median line of the abdomen, and a 24-gauge Teflon catheter was inserted into the abdominal aorta to measure blood pressure using a model AP-641G blood pressure amplifier (Nihon Kohden, Tokyo, Japan). With the animal in the prone position, a thermocouple needle probe (TN-800; Unique Medical Corp., Tokyo, Japan) and thermocouple meter assembly (TME-800; Unique Medical Corp.) were used to monitor rectal temperature. Rectal temperature was maintained at 37 to 38 degrees Celsius with a heating lamp. After administering thiopental for 60 min, 10 units of heparin were injected intravenously and 1 ml arterial blood was collected through the abdominal aorta to analyze serum glucose, hematocrit, hemoglobin, and arterial blood gas levels according to routine laboratory procedures (blood glucose testing system by electrode, MPG01; Daikin, Osaka, Japan; ABL505, Radiometer; Copenhagen, Denmark).
Experiment 2: Histologic Analysis
In experiment 2, 62 animals were prepared and then assigned to nine groups. Two groups continued to receive halothane as controls and the other seven groups received varying doses of thiopental before or after ischemia. After anesthesia was induced with 2% halothane, a Teflon catheter was inserted into the right femoral vein of each animal as described in experiment 1. Through a ventral middle cervical incision, both common carotid arteries were exposed and carefully separated from adjacent nerves and tissues. Silk threads (4.0) were looped around them.
After the animal was placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) in the prone position, the skull was exposed and a small burr hole was drilled in the left hemisphere at 2 mm anterior and 2 mm lateral to the bregma to insert a thermocouple needle probe. The dura was carefully cut. The same thermocouple needle probe (0.4 mm diameter) and thermocouple meter assembly used in experiment 1 were used. The thermocouple probe was inserted into the brain with a micromanipulator. The tip was positioned about 2 mm anterior and 2 mm lateral to the bregma and 2 mm below the brain surface. An identical thermocouple needle probe and thermocouple meter assembly were used to monitor rectal temperature. Rectal and brain temperatures were maintained at 37 to 38 degrees Celsius with a heating lamp (Koehler type illumination lamp; Olympus, Tokyo, Japan).
Three minutes of transient forebrain ischemia was achieved by pulling the threads around the bilateral common carotid arteries with 8-g weights. After the 3-min ischemia, the threads were cut to restore blood flow. Rectal and brain temperatures were maintained at 37 to 38 degrees Celsius under halothane anesthesia for 90 min after the reflow. The thermocouple probes were then gently removed. After all surgical incisions were carefully sutured, the animal was removed from the stereotaxic apparatus. The animal was brought to its cage in a room maintained at constant temperature and allowed access to food and water ad libitum.
Sodium thiopental was administered into the right femoral vein as follows: (1) 0.4, 1, and 2 mg [centered dot] kg sup -1 [centered dot] min sup -1 for 10 min immediately before the start of ischemia to six, six, and eight gerbils, respectively; (2) 0.4, 1, and 2 mg [centered dot] kg sup -1 [centered dot] min sup -1 for 10 min immediately after reflow to six gerbils for each, and 2 mg [centered dot] kg sup -1 [centered dot] min sup -1 for 60 min immediately after the reflow to 12 gerbils. In all groups, halothane was continued until the start of thiopental infusion. Thereafter the inspired gas contained only oxygen. Control animals were given saline under halothane anesthesia.
Seven days after the transient forebrain ischemia just described, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbital. The brains were perfused with heparinized saline and fixed with 10% buffered formalin. After dehydration with graded concentrations of alcohol solutions, the brains were embedded in paraffin. Brain slices, 5 micro meter thick, were stained with hematoxylin and eosin. The numbers of pyramidal cells in the hippocampal CA1 region per constant area (1 x 1 mm) were counted in the same level of coronal sections (1.5 mm posterior to bregma). Then the percentages of necrotic cells were determined and compared with the number of pyramidal cells in the corresponding area from the sections of intact animals, and the average of values on both sides was obtained for each animal.
Experiment 3: Brain Microdialysis
In this experiment, extracellular concentrations of amino acids were measured in 18 gerbils. Each animal was anesthetized with 2% halothane, a Teflon catheter was inserted into the right femoral vein, preparations were made for forebrain ischemia, and thermocouple needle probes were inserted as described in experiment 2. The microdialysis probe (Eicom, A-I-8-01, Kyoto, Japan) was I shaped and made of cellulose membrane (1 mm long, 0.22 mm outside diameter, molecular weight cutoff at 50,000). After the animal was fixed to a stereotaxic apparatus, a small burr hole was drilled. The probe was implanted in the right hemisphere and fixed to the skull with dental cement. The coordinates were 1.5 mm posterior and 1.7 mm lateral to the bregma, and 2.3 mm below the brain surface as specified in a brain atlas. Ringer's solution was perfused at a rate of 2 micro liter/min, and brain perfusates were collected into microtubes every 3 min. After a stabilization period of 2 h, transient forebrain ischemia for 3 min was induced according to the method described in experiment 2. At the end of the experiment, the animals were decapitated and the location of the probe was verified.
Sodium thiopental administered at a continuous rate of 2 mg [centered dot] kg sup -1 [centered dot] min sup -1 was started in two groups as follows: group 1, 30 min before ischemia until the end of the experiment in seven gerbils; and group 2, immediately after recirculation of the cerebral blood flow to the end of the experiment in four gerbils. As before, halothane administration was discontinued simultaneously to the start of thiopental infusion. Seven animals served as controls and were given saline under halothane anesthesia.
The collected dialysates were analyzed for five amino acids according to the method of Tossman and Ungerstedt as modified by Ogata and coworkers. The dialysates were reacted with the same volume of reagent containing 37 mM o-phthalaldehyde, 72 mM 2-mercaptoethanol, and 0.36 M potassium borate (pH 10.4) for 2 min, and then injected into the high-performance liquid chromatography system using a model CMA-200 autosampler (BAS, Tokyo, Japan). Chromatography on a reverse-phase column (Cosmosil, 4.0 x 250 mm; Nacalai Tesque, Kyoto, Japan) was performed with a linear gradient of methanol for 20 min: 0.05 M sodium phosphate (pH 6.0) plus 1% tetrahydrofuran from 25:75 to 70:30 (vol:vol) at 1 ml/min and 30 degrees Celsius using a model 2249 pump (Pharmacia LKB, Bromma, Sweden). Fluorescence (measured at 450 nm, excited at 360 nm) was monitored using a 12-micro liter flow cell in a model F2000 fluorescence spectrometer (Hitachi, Tokyo, Japan). The detection limits for aspartate, glutamate, glycine, taurine, and gamma-aminobutyric acid (GABA) were each about 50 fmol using this system, respectively. The reproducibility of the method of analysis was tested by repeated analysis of a standard amino acid solution (1 micro Meter).
An in vitro recovery test was performed by immersing the dialysis probe into 37 degrees Celsius Ringer's solution containing 10 micro Meter of each amino acid. The recovery of aspartate, glutamate, glycine, taurine, and GABA in the perfusate was 7.1 +/- 0.2%, 7.6 +/- 0.2%, 9.7 +/- 0.3%, 9.5 +/- 0.3%, and 8.4 +/- 0.3% (mean +/- SEM; n = 5), respectively, when the probe was perfused at 2 micro liter/min.
Experiment 4: Measurement of the Direct-current Potential
Direct-current potential in the hippocampal CA1 area was measured in 19 gerbils. After halothane anesthesia, each gerbil was prepared for forebrain ischemia according to the procedure described in experiment 2. The electrode consisted of a glass micropipette with a tip diameter of about 6 micro meter, which was filled with 3 M KCl with an Ag/AgCl electrode in the barrel. This local electrode was implanted in the right hippocampal CA1 region (1.5 mm posterior and 1.7 mm lateral to the bregma, and 1.8 mm below the brain surface). The remote electrode (Ag/AgCl) was inserted into the subcutaneous portion of the neck. The DC potential was monitored between these electrodes with a model AB-621G DC amplifier (Nihon Kohden). Transient forebrain ischemia for 3 min was performed after a stabilization period of 60 min while maintaining the brain and rectal temperatures at 37.5 +/- 0.2 degrees Celsius.
Sodium thiopental was administered intravenously 30 min before ischemia at a rate of 2 mg [centered dot] kg sup -1 [centered dot] min sup -1 with oxygen inhalation to the end of the experiment. Control animals were subjected to ischemia under halothane anesthesia.
The difference in anoxic depolarization (AD) between thiopental anesthesia and halothane anesthesia was compared by analyzing its onset latency, amplitude, recovery time of depolarization to half-maximal amplitude, and duration at half-maximal amplitude.
The data obtained from experiment 1 were analyzed by unpaired t tests. The data from experiment 2 were evaluated with the Kruskal-Wallis test for the groups administered before and after ischemia, respectively. Post hoc comparisons between the control group and each thiopental group were performed with the Mann-Whitney test in the groups administered before and after ischemia, respectively. The data from the microdialysis study (experiment 3) under normal conditions were evaluated using paired t tests. The effects of thiopental on changes in amino acids in ischemia were analyzed using repeated two-way analysis of variance to detect differences among groups. When differences were found, Scheffe's test was used post hoc to compare each fractional value with that in the control group. The data obtained from measuring the DC potential were evaluated using unpaired t tests. Probability values less than 5% were considered significant.
Chemicals and Drugs
Sodium thiopental (Ravonal) was obtained from Tanabe Pharmaceutical Co. Ltd. (Osaka, Japan). Other chemicals used were of guaranteed reagent grade.
(Table 1) shows physiologic variables. There were no differences in any of the physiologic variables between the controls and the gerbils given thiopental.
In the histologic experiment (experiment 2), animals in the control group regained consciousness and righting reflex within 30 min after halothane anesthesia was stopped. Similar to the control animals, the animals treated with thiopental at all doses for 10 min recovered from anesthesia within 30 min. However, the effects of anesthesia were prolonged for several hours in animals treated with thiopental (2 mg [centered dot] kg sup -1 [centered dot] min sup -1) for 60 min after ischemia. In this group, 2 of the 12 animals died the next day without waking from anesthesia. No animals in the other groups died.
(Figure 1) shows the percentages of necrotic neurons in the hippocampal CA1 region of individual animals. Nearly all pyramidal cells degenerated after 7 days in the control animals. Preischemic thiopental treatment at all doses significantly reduced the rate of delayed neuronal death. When thiopental was given after ischemia, neither of the 10-min infusions influenced neuronal injury. However, in the animals receiving 2 mg [centered dot] kg sup -1 [centered dot] min sup -1 for 60 min, nearly all pyramidal cells remained intact 7 days after ischemia.
(Table 2) shows the values of aspartate, glutamate, glycine, taurine, and GABA in the dialysate (6 micro liter) 12 to 15 min after the start of thiopental. The initiation of 2 mg [centered dot] kg sup -1 [centered dot] min sup -1 thiopental given intravenously significantly diminished the outputs of glutamate, glycine, and taurine.
(Figure 2) shows the changes in amino acid output before and after forebrain ischemia. In the control group, the outputs of all amino acids began to increase immediately after the reflow. In the fraction 3 to 6 min after the reflow, the values of aspartate, glutamate, glycine, taurine, and GABA reached 950%, 870%, 290%, 670%, and 2,040% of the values immediately before ischemia, respectively. Thiopental anesthesia for the entire period had a significant preventive effect on these increases, except for glycine. The values of amino acids 3 to 6 min after the reflow decreased to 17%, 14%, 36%, 20%, and 7% of each corresponding peak value in the control group, respectively. Postischemic continuous injection of thiopental had no effect on the outputs of amino acids compared with those in the control group within 3 min after reflow. However, in the next fraction (3 to 6 min after the reflow), increased values in this group decreased to basal values before ischemia.
As shown in Figure 3, the forebrain ischemia provoked an AD that was characterized by a sudden shift in the extracellular DC potential. In the control group, the AD was observed at an onset latency of 53.6 +/- 4.3 s, and the amplitude of the DC potential shift was 21.3 +/- 1.0 mV. The recovery time as measured by the duration between the start of the reflow and the point at half-maximal amplitude was 90.4 +/- 17.5 s, and the duration at half-maximal amplitude was 203.9 +/- 24.5 s. Thiopental anesthesia prolonged its onset latency by 62% and reduced the maximal amplitude of the AD by 29%, the effects being significant compared with those of the control group. The recovery time of the AD to half-maximal amplitude and the duration at half-maximal amplitude were not significantly different.
We have shown the protective effects of thiopental against incomplete global ischemia in the gerbil: suppression of excitatory amino acid release and prolongation of the onset of the AD.
In the histologic experiment, transient forebrain ischemia at a brain temperature of 37.5 degrees Celsius produced marked damage in the hippocampal CA1 region in control gerbils. This result is consistent with the previous report that transient forebrain ischemia for 3 min consistently induces severe neuronal damage in the CA1 field in normothermic gerbils. Preischemic administration of thiopental at all the doses we used ameliorated neuronal damage. 
Continuous thiopental administration after ischemia at 2 mg [centered dot] kg sup -1 [centered dot] min sup -1 for 60 min improved neuronal outcome; the same infusion rate for only 10 min did not provide the same protection. Two of the 12 animals in the 60-min group died the next day. However, even if we include the data of these two animals in this group as 100% necrosis, we still observed the protective effect of postischemic treatment. In groups treated after ischemia, the animals in the control group regained consciousness within 30 min after halothane anesthesia was stopped, whereas the effects of anesthesia were prolonged for several hours in the animals treated with thiopental for 60 min. This retardation shows that anesthesia-induced hypothermia with thiopental might contribute to brain protection, because experimental protection with barbiturates in previous studies may have been a consequence of reduced brain temperature. Furthermore, hypothermia for 12 h initiated 1 h after ischemia recently was shown to attenuate hippocampal CA1 necrosis produced by 3 min of forebrain ischemia in gerbils. However, this treatment did not completely eliminate damage, whereas thiopental as used in the current experiments almost completely ameliorated injury despite postischemic temperature control for 90 min after the start of reflow. Thus another element specific to barbiturates besides hypothermia seems to have provided the protection.
In this study, we did not use mechanical ventilation because of the difficulty in applying it to such small animals. However, we observed no differences in physiologic variables between the two groups treated with different anesthetic agents. Therefore, it is not likely that differences in physiologic variables affected histologic outcome, although those values represent a preischemic state. We inserted a thermocouple needle probe to measure brain temperature, which may have triggered a wave of spreading depression, which is thought to be protective against subsequent ischemic events. However, spreading depression in the left hemisphere, where the probe was placed, probably did not protect the ipsilateral hemisphere, and we observed no differences between hemispheres in terms of the number of necrotic neurons.
In the nonischemic condition, we observed a reduction in the extracellular concentrations of some transmitter amino acids by thiopental in our microdialysis experiment. In nerve endings, subsequent to depolarization, Calcium2+ flows into neurons, which causes fusion of the vesicle membrane with the cell membrane, resulting in neurotransmitter release by exocytosis. Barbiturates impair depolarization-induced Calcium2+ influx into nerve terminals and depress subsequent neurotransmitter release with sedative concentrations. [15,16]The lower concentrations of amino acids seen in normal conditions may be caused by this inhibition.
In these experiments with forebrain ischemia, we confirmed the earlier findings that concentrations of both excitatory and inhibitory amino acids increase in the extracellular space during the acute stage of ischemia. [17,18]Because an imbalance between excitatory and inhibitory neurotransmission, with an excess of the former, is thought to play an important role in postsynaptic injury, the inhibition of ischemia-induced increases in excitatory amino acids by thiopental may be the mechanism by which the drug attenuates brain damage. Barbiturates have been reported to prevent axonal conduction by reducing the Sodium sup + conductance at rather high concentrations. In our previous study, we showed that the blockade of Sodium sup + channels with lidocaine helps protect the brain by reducing increases in excitatory amino acids. In another study, researchers have shown that blockade of Sodium sup + channels by tetrodotoxin reduces Calcium2+ -independent excitatory amino acid release. Therefore, the action of Sodium sup + channel blockade with thiopental may have partly caused the improvement in histologic outcome by limiting cytosolic glutamate release. In contrast to our findings, extracellular glutamate and glycine concentrations are reportedly greater with thiopental anesthesia than with halothane anesthesia in rats subjected to forebrain ischemia for 10 min. Although the explanation of these contradictory results is not clear, the differences in the magnitude of ischemia among animal species might produce these results. In other reports in gerbils, the extracellular concentration of glutamate increased to about 20 times as much as that of values before ischemia by transient forebrain ischemia for 3 to 5 min with halothane anesthesia. [18,22]
With respect to the effect of postischemic administration, the output of amino acids within 3 min after reflow revealed no differences compared with those of the control group. In this period, thiopental might not act completely to exert an influence on amino acid outputs, because of its insufficient dose compared with the group given thiopental during the experiment. However, in the next fraction (3 to 6 min after the reflow), concentrations of amino acids except taurine decreased rapidly, whereas those in control animals attained peak values. In another preliminary experiment, postischemic administration for 10 min did not reduce the increase in amino acids that was markedly similar to that for 60 min, and an increase after the cessation of the administration was not found: The values of fractional glutamate output immediately before ischemia to the end of the experiment were 1.3, 2.4, 7.1, 3.3, 2.4, 1.9, 1.2, 1.2, 1.3, 1.3, and 1.2 nmol/3 min, respectively. In contrast to pretreatment, postischemic administration for 10 min did not improve injury in this histologic experiment. This failure may be due to the insufficient reduction of extracellular excitatory amino acids. Because the same dose for 60 min protected neurons, in the histologic experiment, inhibition of the release of excitatory amino acids does not seem to be a mechanism for protection by postischemic administration for 60 min.
The DC potential displays distinct changes closely related to Sodium sup +, Potassium sup +, Chlorine sup -, and Calcium2+. Cerebral ischemia produces a slow change in this potential in the early phase, which is explained by insufficient pumping due to the reduced activity of Sodium sup + -Potassium sup + -ATPase. Subsequently, a rapid and marked change is observed, which represents a sudden increase in overall ionic permeability. This rapid phase reflects the severe limitation in ATP for the maintenance of the membrane potential by Sodium sup + -Potassium sup + -ATPase. We found a latency of initiation of this abrupt phase and reduced AD amplitude in animals given thiopental. These findings suggest the preservation of ATP by thiopental anesthesia, which might account for the reduced cerebral metabolic rate by barbiturates in earlier studies. In ischemia, most of the efflux of glutamate from the metabolic pool is provoked by reversing the Sodium sup + -cotransport system. This glutamate efflux and the Calcium2+ entry into neurons occur after the onset of the AD. The preischemic administration of thiopental may have reduced the glutamate toxicity simply by delaying the onset of the AD, and thus may not be a specific effect of thiopental.
However, the postischemic administration of thiopental does not seem to have exerted an effect on the AD, because thiopental anesthesia failed to reduce the recovery time of the AD, which represents the recovery of the membrane potential by the activity of the Sodium sup + -Potassium sup + -ATPase during reflow. Therefore another component, not the inhibition of the transmitter release nor the latency of the AD, seems to take part in the protection. Enhanced GABAergic activity, scavenging of free radicals, and improvement of protein synthesis after ischemia are conceivable. In addition, the anticonvulsant effect of thiopental against seizures after ischemia may have contributed to the protection. The administration of thiopental for a long duration after ischemia might exert an influence on these postischemic processes.
Our findings show that thiopental contributes to brain protection against ischemia, although a longer administration period for this agent is required after ischemia than when it is given before ischemia. We did not determine the mechanism of neuronal protection of thiopental given after ischemia. However, the effect of preischemic administration may be related to suppression of the excitatory amino acid release and a shift in DC potential.