Neuroscience Vol. 42, No. 3, pp. 661~70, 1991 Printed in Great Britain
0306-4522/91 $3.00 + 0.00 Pergamon Press plc © 1991 IBRO
CRITICAL LEVELS OF EXTRACELLULAR GLUTAMATE MEDIATING GERBIL HIPPOCAMPAL DELAYED NEURONAL DEATH DURING HYPOTHERMIA: BRAIN MICRODIALYSIS STUDY A. MITANI* a n d K. KATAOKA Department of Physiology, The University of Ehime, School of Medicine, Shigenobu, Onsen-Gun, Ehime 791-02, Japan Abstract--When the brain temperature was lowered by 2°C from normothermic temperature, a protective effect on postischemic neuronal death was exhibited and levels of extracellular glutamate were attenuated to about half of those at normothermic brain temperature in the gerbil hippocampus. Hypothermia has been reported to confer a protective effect on ischemia-induced delayed neuronal death. The present study was carried out to quantify this protective effect of hypothermia on the degree of alteration in extracellular release of glutamate during ischemia and the final histopathological outcome in the hippocampus. Extracellular glutamate levels were measured by microdialysis. In gerbils whose brain temperature was maintained at normothermia (37°C), glutamate increased during ischemia and the early period of recirculation (by 15-fold), and CA1 neurons were consistently damaged. In animals whose brain temperature was maintained at 35 or 33°C during ischemia, the release of glutamate was significantly attenuated to half or a quarter, respectively, at 37°C. In animals whose brain temperature was maintained at 31°C during ischemia, the release of glutamate was slightly lower than that at 33°C. No CA1 isehemic neuronal damage was seen in 60% of gerbils at 35°C and none was seen in any gerbils at 33 and 31°C. In animals whose brain temperature was maintained at 39°C during ischemia, the release of glutamate was slightly higher than that at 37°C, and a high mortality rate of animals (75%) was observed. Our results reinforce other recent evidence suggesting that one of the mechanisms by which lowering of the brain temperature by only a few degrees during ischemia exerts a protective effect in the hippocampus, involves the reduction of ischemia-induced glutamate release.
P y r a m i d a l n e u r o n s in the h i p p o c a m p a l CA1 region are particularly vulnerable to transient ischemia.13,2L23 T h e C A I n e u r o n a l d a m a g e is t h o u g h t to be mediated by e n d o g e n o u s excitotoxins, 24 such as g l u t a m a t e which has the potential to destroy central n e u r o n s by a n excitatory mechanism. ~s,~9 Recent studies have described elevations in the extracellular c o n c e n t r a t i o n o f g l u t a m a t e in the h i p p o c a m p u s d u r i n g a n d shortly after transient ischemia, ~,6,14 a n d others have d e m o n strated t h a t ischemia-induced delayed n e u r o n a l d e a t h in the C A I region o f the h i p p o c a m p u s is d e p e n d e n t o n the integrity o f its glutamatergic input. 9,1°,2°,22,27 These results implicate the release o f g l u t a m a t e in the d e v e l o p m e n t o f ischemic n e u r o n a l d e a t h o f C A I n e u r o n s in the h i p p o c a m p u s . M o r e recent studies have d e m o n s t r a t e d t h a t a m o d e r a t e reduction in b r a i n t e m p e r a t u r e protects C A I n e u r o n s f r o m injury following transient ischemia 2,3,27 a n d inhibits the release o f n e u r o t r a n s m i t t e r s in the striatum. 4 These results suggest t h a t a m o d e r a t e reduction in b r a i n t e m p e r a t u r e inhibits g l u t a m a t e release d u r i n g a n d shortly after ischemia a n d improves the h i s t o p a t h o logical o u t c o m e o f the ischemic insult in the hippocampus. In the present study, we quantified the effect o f h y p o t h e r m i a o n the release o f extracellular g l u t a m a t e d u r i n g ischemia a n d a n i m p r o v e m e n t in *To whom correspondence should be addressed. 661
the final histopathological o u t c o m e in the hippocampus. EXPERIMENTAL PROCEDURES
Monitoring normothermic brain temperature
Normothermic brain temperature was measured directly in the awake and undrugged Mongolian gerbils. The procedure used in this study was referred to that of sampling brain dialysate, 7,~ being dependent on a monitoring probe inserted down a previously implanted guide cannula. Six gerbils were anesthetized and maintained with 2.5% halothane: 70% N20/30% 02 mixture. Surgical procedures were performed as previously described. 15 A stainless-steel guide cannula (0.6 mm i.d.) for the subsequent insertion of a thermocouple probe was perpendicularly implanted at 2.0 mm posterior and 2.0 mm lateral to bregma and 1.2 mm ventral to the cortical surface, and glued to the surface of the skull with dental cement. The wound was sutured and the animal was treated with antibiotics and allowed to recover for seven to eight days before it was used. The thermocouple needle-probe with 0.4 mm diameter (TN-800, Unique Medical Corporation, Japan) and a thermocouple meter (TME-300, Unique Medical Corporation, Japan) were employed, having a resolution of 0.2°C (time constant: 0.1 s). Seven to eight days after surgery, brain temperature monitoring was performed between 13:00 and 17:00, which corresponded to the period of the microdialysis experiments. The gerbil was placed in a Perspex cage (30 x 30 x 20 cm high) with water freely available, and the thermocouple probe was inserted into the hippocampal guide cannula (2.5 mm ventral to the cortical surface) using the same procedure as previous studies. TM Normothermic brain tem-
662
A. MITANI and K. KATAOKA
perature was monitored for the first 20 min after insertion in freely moving gerbils. After the brain temperature monitoring, rectal temperature was monitored using the same probe and meter. After the monitor, the animals were anesthetized with pentobarbital and perfused with 10% formalin. The 40-#m-thick brain sections were made for examination of the position of the thermocouple probe.
Dialysis experiments Animal and surgery. Male Mongolian gerbils, weighing 60-80 g, were used. The animals were anesthetized and maintained with a mixture of 2.5% halothane and nitrous oxide-oxygen (7:3). Through a ventral midline cervical incision, both common carotid arteries were exposed and dissected free of surrounding tissues, and a 4-0 silk suture was looped around each artery. Body and brain temperature was maintained at 37°C with a heating blanket and a heating lamp during operative procedures. The head of the animal was fixed to a stereotaxic apparatus (David Kopf). A small burr hole for insertion of a microdialysis probe was drilled at 1.5-2.5 mm posterior and 1.5-2.5 mm lateral to bregma. Another small burr hole for insertion of a thermocouple needle-probe was drilled at 1.5-2.5 mm anterior and 1.5-2.5mm lateral to bregma. The dura was carefully incised. Thermo-monitor. The thermocouple needle-probe of 0.4 mm diameter (TN-800) and thermocouple meter (TME300) were employed. The thermocouple probe was inserted in the brain at an angle of 400 rostral to the vertical plane: the tip of the thermocouple probe was positioned about 2 mm lateral to bregma and 2.2 mm ventral to the cortical surface. Another set of same thermocouple needle-probe and thermocouple meter was employed to monitor rectal temperature.
Brain dialysis and hypo- and hyperthermia procedures. A microdialysis probe ( l m m long dialysis membrane, 0.22mm o.d.; MW cut-off= 50,000; Eicom, Japan) was attached to the micromanipulator (Narishige, Japan) and positioned perpendicularly into the hippocampus (2.2 mm ventral to the cortical surface, 2.0 mm posterior and 2.0 mm lateral to bregma). The 1-mm-long dialysis membrane was situated in the CA 1 sector and dentate gyrus (approximately 70% of the dialysis membrane was in the CA1 region, and the remaining 30% was in the dentate gyrus). Subsequently, it was perfused with Ringer solution at a flow rate of 0.6pl/min by means of a microinfusion pump (BRC, Japan). The solution was heated, to approximately the same temperature as that of the animal's brain, with a lamp. Halothane administration was decreased, and the animals were maintained on halothane (1%) and a mixture of nitrous oxide (70%) and oxygen (30%). Following a 2-h stabilization period, brain temperature was altered from 37 to 31°C [ischemic group (i.g.): n = 8; control group (c.g.: n = 6), 33°C (i.g.: n = 8, c.g.: n = 6), 35°C (i.g.: n = 10, c.g.: n = 6), 37°C (i.g.: n = 8, c.g.: n = 6) or 39°C (i.g.: n = 8, c.g.: n = 6) and was maintained until 20 min after the onset of recirculation by manipulating a heating lamp (Koehler's type illumination lamp; Olympus, Japan) and a cooling, small, high-speed fan (Panasonic, Japan) which were placed near the head. Bilateral transient forebrain ischemia was performed while maintaining brain temperature at 31, 33, 35, 37 or 39°C. Twenty minutes after the onset of recirculation, the brain temperature was re-manipulated to become 37°C. One hundred-second samples (1 #1) of the dialysate, representing hippocampal extracellular fluid, were collected in paraffin oil in polyethylene sampling tubes in an ice bath; the end of the small outlet Teflon tube was dipped into the paraffin oil. Five 100-s samples were collected during a period of altering temperature from 37°C to 31, 33, 35, 37 or 39°C in order to assess whether basal levels of extracellular glutamate were changed by a difference in brain temperature. Twenty consecutive 100-s samples were collected
during maintaining brain temperature at 31, 33, 35, 37 or 39°C: five samples were collected prior to iscbemia, three samples were collected during ischemia, and 12 samples were collected after the onset of recirculation. Then, five 100-s samples were collected during a period of returning temperature from 31, 33, 35, 37 or 39°C to 37°C. Transient ischemia. The bilateral ischemic insult was initiated after collecting five consecutive 100-s samples during maintaining brain temperature at 31, 33, 35, 37 or 39°C. The sutures around the two common carotid arteries were pulled by 12-g weights to occlude the circulation. 15,25 Following 5 min of ischemia, the sutures were cut and removed to restore the blood flow. Brain temperature tended to fall spontaneously by 2-3°C during the ischemic insult, and then adjustments of the heating lamp were needed to keep the brain temperature stable. After the collection of samples, the microdialysis and thermocouple probes were gently pulled out. All surgical incisions were carefully sutured. Animals were treated with antibiotics, removed from the stereotaxic apparatus and brought into a comfortable position on a warming blanket. After awakening, the gerbils were returned to individual cages in a room maintained at constant temperature (30°C) and allowed access to food and water ad libitum. Glutamate assay. The method of glutamate assay is in principle the same as described in our previous study; ~4this method has a sensitivity of 0.24).5 pmol/sample. Immediately after the collection, all sampling tubes were centrifuged to make a bolus of dialysate at the bottom of the tubes. Glutamate was analysed by the following serial enzymatic reactions. In brief, the dialysate in the paraffin oil was reacted first with 20/zl of enzymatic reagent for 30 min to form NADH. The reaction was stopped by the addition of 5 #l of I M NaOH followed by heating at 60°C for 20 min. Subsequently, for triplicate determinations, three 5-#1 aliquots were transferred into fluorometer tubes and used for NAD +-NADH cycling by means of the enzymatic cycling reactions described by previous studies.t2'17The fluorescence of NADH was measured with a Farrand's fluorometer. L-Glutamate standards of zero and 0.5-50 x 10-J2mol/l were quantified in parallel with the samples throughout the assay in each experiment, and glutamate concentrations of samples were read from the standard curve; mean values and standard errors were calculated for all animals studied. Histological analysis. Seven days after the ischemic insult, the animals were anesthetized with pentobarbital and perfused transcardially with heparinized saline and then with 10% formalin in 0.I M phosphate buffer (pH 7.4). The brains were removed and saturated with a cold solution of 30% sucrose in 10% formalin. Serial coronal sections were cut at 40pro and 4/~m alternately with a cryostat and mounted on gelatine-coated slides: the 40-#m-thick sections were stained with Cresyl Violet to examine the positions of the microdialysis probe, and the 4-/~m-thick sections were stained with hematoxylin and eosin to count the numbers of normal-appearing CA1 neurons. Cell counts were performed using a microscope (Nikon Biophoto, Japan) with x 40 objective and x 10 ocular by placing a ocular grid with a length of 500 #m along the CA1 pyramidal layer and with a width of 125 #m. In three frontal sections taken 2.5 mm behind bregma, all normal-appearing neurons with intact morphology within three grids were counted: i.e. no eosinophilic changes and no Karyorrhexis. In each animal the mean surviving neuron number was calculated. Data analysis. The statistical significance of differences in glutamate levels and cell counts between control and ischemic animals with same brain temperature was assessed by two-tailed Student's t-test. The statistical significance of differences in glutamate levels and cell counts between the separate temperature groups was analysed by two-way ANOVA; this analysis included Tukey's or Dunnett's multiple-comparison procedure.
Glutamate during hypothermia and isehemic damage Table 1. Normothermic brain temperature of gerbil Animal
Brain temperature (°C)
1 2 3 4 5 6 Mean + S.E.M.
36.4 37.2 37.5 37.6 37.0 37.0 37.1 + 0.2
Brain temperature monitoring was performed for 20 min at the time between 13:00 and 17:00 in each animal.
RESULTS
Normothermic brain temperature of gerbil Normothermic brain temperature was monitored from six freely moving gerbils. Table 1 displays the brain temperature of each animal. The brain temperature of each animal was constant during the period of monitoring. Mean brain temperature of six animals was 37.1°C (mean rectal temperature was 37.7°C). Therefore, the brain temperature at 37°C was set as normotbermic brain temperature in the present study.
Effect of hypo- or hyperthermia on extracellular glutamate In order to confirm that there is no influence of variations in temperature on the recovery rate of dialysis membrane for glutamate, in vitro experiments were performed. Six similar microdialysis probes were used for this test. Each probe was dipped in Ringer solution which contained 50 #M glutamate. Dialysates were collected from the solution at 31, 33, 35, 37 and 39°C as described above. As shown in Table 2, no significant differences in glutamate values were detected among dialysates collected at different temperatures. The time-course of change in glutamate release during ischemia and recirculation in ischemic (n = 8) and control (n = 6) animals whose brain temperature was maintained at 37°C is illustrated in Fig. 1. Stable basal concentrations of extracellular glutamate were detected in the 10 consecutive samples collected before ischemia. In ischemic gerbils, a significant increase in hippocampal glutamate was detected in Table 2. Recovery test of dialysis membrane at different solution temperature Solution temperature (°C) 31(n = 6) 33(n = 6) 35(n = 6) 37(n = 6) 39(n = 6)
Concentration of glutamate in dialysate (#M) 6.7 + 0.6 6.9 + 0.5 7.1 + 0.5 6.9 + 0.4 6.5 + 0.6
Mean values + S.E.M. are represented. Six microdiaiysis probes were used; each probe was used to collect dialysate from Ringer solution (containing 50 #M glutamate) at 31, 33, 35, 37 and 39°C.
663
the dialysate during ischemia and the early period of recirculation. The increase was acute and massive; maximal levels were attained at the end of 5 min of ischemia (---15-fold increase). Glutamate extracellular levels returned to baseline within 6 - 7 m in of recirculation. In control gerbils, stable concentrations of extracellular glutamate were detected in all consecutive samples. The time-course of change in glutamate release during ischemia and recirculation in iscbemic (n = 10) and control (n = 6) animals whose brain temperature was maintained at 35°C is illustrated in Fig. 2. Stable basal concentrations of extracellular glutamate were detected in the five consecutive samples collected before isebemia and also in five other samples collected during a period of altering brain temperature from 37 to 35°C. Variations in brain temperature between 37 and 35°C had no significant influence on basal glutamate levels. In ischemic gerbils, a significant increase in hippocampal glutamate was detected in the dialysate during ischemia and the early period of recirculation. However, the increase was more gradual and of a significantly lesser extent compared with the acute and massive changes detected in animals whose brain temperature was maintained at 37°C (Table 3). The maximal levels were attained at the end of 5 rain of iscbemia (--7.6-fold increase); the levels of glutamate were attenuated to about half of those at 37°C. Glutamate extracellular levels returned to baseline within 5 min of recirculation. In control gerbils, stable concentrations of extracellular glutamate were detected in all samples. The time-course of changes in glutamate release during ischemia and recirculation in ischemic (n = 8) and control (n = 6) animals whose brain temperature was maintained at 33°C is illustrated in Fig. 3. Stable basal concentrations of extracellular glutamate were detected in the five consecutive samples collected prior to ischemia and also in five other samples collected during a period of altering brain temperature from 37 to 33°C. Variations in brain temperature between 37 and 33°C had no significant influence on basal glutamate levels. In ischemic gerbils, glutamate levels were still significantly increased during ischemia and the early period of recirculation. However, the increases were more gradual and of a lesser extent compared with changes detected in animals whose brain temperature was maintained at 35°C. The increase almost reached a plateau during the second 100 s of 5-min iscbemia. The maximal levels were attained at the end of 5 min of ischemia (--~4.2fold increase); the levels of glutamate were attenuated to about a quarter of those at 37°C (Table 3) and were significantly lower than those at 35°C (P < 0.01; Tukey's multiple-comparison procedure). Extracellular glutamate levels returned to baseline by 5 min of recirculation. In control gerbils, stable concentrations of extracellular glutamate were detected in all samples.
664
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0306-4522/91 $3.00 + 0.00 Pergamon Press plc © 1991 IBRO
CRITICAL LEVELS OF EXTRACELLULAR GLUTAMATE MEDIATING GERBIL HIPPOCAMPAL DELAYED NEURONAL DEATH DURING HYPOTHERMIA: BRAIN MICRODIALYSIS STUDY A. MITANI* a n d K. KATAOKA Department of Physiology, The University of Ehime, School of Medicine, Shigenobu, Onsen-Gun, Ehime 791-02, Japan Abstract--When the brain temperature was lowered by 2°C from normothermic temperature, a protective effect on postischemic neuronal death was exhibited and levels of extracellular glutamate were attenuated to about half of those at normothermic brain temperature in the gerbil hippocampus. Hypothermia has been reported to confer a protective effect on ischemia-induced delayed neuronal death. The present study was carried out to quantify this protective effect of hypothermia on the degree of alteration in extracellular release of glutamate during ischemia and the final histopathological outcome in the hippocampus. Extracellular glutamate levels were measured by microdialysis. In gerbils whose brain temperature was maintained at normothermia (37°C), glutamate increased during ischemia and the early period of recirculation (by 15-fold), and CA1 neurons were consistently damaged. In animals whose brain temperature was maintained at 35 or 33°C during ischemia, the release of glutamate was significantly attenuated to half or a quarter, respectively, at 37°C. In animals whose brain temperature was maintained at 31°C during ischemia, the release of glutamate was slightly lower than that at 33°C. No CA1 isehemic neuronal damage was seen in 60% of gerbils at 35°C and none was seen in any gerbils at 33 and 31°C. In animals whose brain temperature was maintained at 39°C during ischemia, the release of glutamate was slightly higher than that at 37°C, and a high mortality rate of animals (75%) was observed. Our results reinforce other recent evidence suggesting that one of the mechanisms by which lowering of the brain temperature by only a few degrees during ischemia exerts a protective effect in the hippocampus, involves the reduction of ischemia-induced glutamate release.
P y r a m i d a l n e u r o n s in the h i p p o c a m p a l CA1 region are particularly vulnerable to transient ischemia.13,2L23 T h e C A I n e u r o n a l d a m a g e is t h o u g h t to be mediated by e n d o g e n o u s excitotoxins, 24 such as g l u t a m a t e which has the potential to destroy central n e u r o n s by a n excitatory mechanism. ~s,~9 Recent studies have described elevations in the extracellular c o n c e n t r a t i o n o f g l u t a m a t e in the h i p p o c a m p u s d u r i n g a n d shortly after transient ischemia, ~,6,14 a n d others have d e m o n strated t h a t ischemia-induced delayed n e u r o n a l d e a t h in the C A I region o f the h i p p o c a m p u s is d e p e n d e n t o n the integrity o f its glutamatergic input. 9,1°,2°,22,27 These results implicate the release o f g l u t a m a t e in the d e v e l o p m e n t o f ischemic n e u r o n a l d e a t h o f C A I n e u r o n s in the h i p p o c a m p u s . M o r e recent studies have d e m o n s t r a t e d t h a t a m o d e r a t e reduction in b r a i n t e m p e r a t u r e protects C A I n e u r o n s f r o m injury following transient ischemia 2,3,27 a n d inhibits the release o f n e u r o t r a n s m i t t e r s in the striatum. 4 These results suggest t h a t a m o d e r a t e reduction in b r a i n t e m p e r a t u r e inhibits g l u t a m a t e release d u r i n g a n d shortly after ischemia a n d improves the h i s t o p a t h o logical o u t c o m e o f the ischemic insult in the hippocampus. In the present study, we quantified the effect o f h y p o t h e r m i a o n the release o f extracellular g l u t a m a t e d u r i n g ischemia a n d a n i m p r o v e m e n t in *To whom correspondence should be addressed. 661
the final histopathological o u t c o m e in the hippocampus. EXPERIMENTAL PROCEDURES
Monitoring normothermic brain temperature
Normothermic brain temperature was measured directly in the awake and undrugged Mongolian gerbils. The procedure used in this study was referred to that of sampling brain dialysate, 7,~ being dependent on a monitoring probe inserted down a previously implanted guide cannula. Six gerbils were anesthetized and maintained with 2.5% halothane: 70% N20/30% 02 mixture. Surgical procedures were performed as previously described. 15 A stainless-steel guide cannula (0.6 mm i.d.) for the subsequent insertion of a thermocouple probe was perpendicularly implanted at 2.0 mm posterior and 2.0 mm lateral to bregma and 1.2 mm ventral to the cortical surface, and glued to the surface of the skull with dental cement. The wound was sutured and the animal was treated with antibiotics and allowed to recover for seven to eight days before it was used. The thermocouple needle-probe with 0.4 mm diameter (TN-800, Unique Medical Corporation, Japan) and a thermocouple meter (TME-300, Unique Medical Corporation, Japan) were employed, having a resolution of 0.2°C (time constant: 0.1 s). Seven to eight days after surgery, brain temperature monitoring was performed between 13:00 and 17:00, which corresponded to the period of the microdialysis experiments. The gerbil was placed in a Perspex cage (30 x 30 x 20 cm high) with water freely available, and the thermocouple probe was inserted into the hippocampal guide cannula (2.5 mm ventral to the cortical surface) using the same procedure as previous studies. TM Normothermic brain tem-
662
A. MITANI and K. KATAOKA
perature was monitored for the first 20 min after insertion in freely moving gerbils. After the brain temperature monitoring, rectal temperature was monitored using the same probe and meter. After the monitor, the animals were anesthetized with pentobarbital and perfused with 10% formalin. The 40-#m-thick brain sections were made for examination of the position of the thermocouple probe.
Dialysis experiments Animal and surgery. Male Mongolian gerbils, weighing 60-80 g, were used. The animals were anesthetized and maintained with a mixture of 2.5% halothane and nitrous oxide-oxygen (7:3). Through a ventral midline cervical incision, both common carotid arteries were exposed and dissected free of surrounding tissues, and a 4-0 silk suture was looped around each artery. Body and brain temperature was maintained at 37°C with a heating blanket and a heating lamp during operative procedures. The head of the animal was fixed to a stereotaxic apparatus (David Kopf). A small burr hole for insertion of a microdialysis probe was drilled at 1.5-2.5 mm posterior and 1.5-2.5 mm lateral to bregma. Another small burr hole for insertion of a thermocouple needle-probe was drilled at 1.5-2.5 mm anterior and 1.5-2.5mm lateral to bregma. The dura was carefully incised. Thermo-monitor. The thermocouple needle-probe of 0.4 mm diameter (TN-800) and thermocouple meter (TME300) were employed. The thermocouple probe was inserted in the brain at an angle of 400 rostral to the vertical plane: the tip of the thermocouple probe was positioned about 2 mm lateral to bregma and 2.2 mm ventral to the cortical surface. Another set of same thermocouple needle-probe and thermocouple meter was employed to monitor rectal temperature.
Brain dialysis and hypo- and hyperthermia procedures. A microdialysis probe ( l m m long dialysis membrane, 0.22mm o.d.; MW cut-off= 50,000; Eicom, Japan) was attached to the micromanipulator (Narishige, Japan) and positioned perpendicularly into the hippocampus (2.2 mm ventral to the cortical surface, 2.0 mm posterior and 2.0 mm lateral to bregma). The 1-mm-long dialysis membrane was situated in the CA 1 sector and dentate gyrus (approximately 70% of the dialysis membrane was in the CA1 region, and the remaining 30% was in the dentate gyrus). Subsequently, it was perfused with Ringer solution at a flow rate of 0.6pl/min by means of a microinfusion pump (BRC, Japan). The solution was heated, to approximately the same temperature as that of the animal's brain, with a lamp. Halothane administration was decreased, and the animals were maintained on halothane (1%) and a mixture of nitrous oxide (70%) and oxygen (30%). Following a 2-h stabilization period, brain temperature was altered from 37 to 31°C [ischemic group (i.g.): n = 8; control group (c.g.: n = 6), 33°C (i.g.: n = 8, c.g.: n = 6), 35°C (i.g.: n = 10, c.g.: n = 6), 37°C (i.g.: n = 8, c.g.: n = 6) or 39°C (i.g.: n = 8, c.g.: n = 6) and was maintained until 20 min after the onset of recirculation by manipulating a heating lamp (Koehler's type illumination lamp; Olympus, Japan) and a cooling, small, high-speed fan (Panasonic, Japan) which were placed near the head. Bilateral transient forebrain ischemia was performed while maintaining brain temperature at 31, 33, 35, 37 or 39°C. Twenty minutes after the onset of recirculation, the brain temperature was re-manipulated to become 37°C. One hundred-second samples (1 #1) of the dialysate, representing hippocampal extracellular fluid, were collected in paraffin oil in polyethylene sampling tubes in an ice bath; the end of the small outlet Teflon tube was dipped into the paraffin oil. Five 100-s samples were collected during a period of altering temperature from 37°C to 31, 33, 35, 37 or 39°C in order to assess whether basal levels of extracellular glutamate were changed by a difference in brain temperature. Twenty consecutive 100-s samples were collected
during maintaining brain temperature at 31, 33, 35, 37 or 39°C: five samples were collected prior to iscbemia, three samples were collected during ischemia, and 12 samples were collected after the onset of recirculation. Then, five 100-s samples were collected during a period of returning temperature from 31, 33, 35, 37 or 39°C to 37°C. Transient ischemia. The bilateral ischemic insult was initiated after collecting five consecutive 100-s samples during maintaining brain temperature at 31, 33, 35, 37 or 39°C. The sutures around the two common carotid arteries were pulled by 12-g weights to occlude the circulation. 15,25 Following 5 min of ischemia, the sutures were cut and removed to restore the blood flow. Brain temperature tended to fall spontaneously by 2-3°C during the ischemic insult, and then adjustments of the heating lamp were needed to keep the brain temperature stable. After the collection of samples, the microdialysis and thermocouple probes were gently pulled out. All surgical incisions were carefully sutured. Animals were treated with antibiotics, removed from the stereotaxic apparatus and brought into a comfortable position on a warming blanket. After awakening, the gerbils were returned to individual cages in a room maintained at constant temperature (30°C) and allowed access to food and water ad libitum. Glutamate assay. The method of glutamate assay is in principle the same as described in our previous study; ~4this method has a sensitivity of 0.24).5 pmol/sample. Immediately after the collection, all sampling tubes were centrifuged to make a bolus of dialysate at the bottom of the tubes. Glutamate was analysed by the following serial enzymatic reactions. In brief, the dialysate in the paraffin oil was reacted first with 20/zl of enzymatic reagent for 30 min to form NADH. The reaction was stopped by the addition of 5 #l of I M NaOH followed by heating at 60°C for 20 min. Subsequently, for triplicate determinations, three 5-#1 aliquots were transferred into fluorometer tubes and used for NAD +-NADH cycling by means of the enzymatic cycling reactions described by previous studies.t2'17The fluorescence of NADH was measured with a Farrand's fluorometer. L-Glutamate standards of zero and 0.5-50 x 10-J2mol/l were quantified in parallel with the samples throughout the assay in each experiment, and glutamate concentrations of samples were read from the standard curve; mean values and standard errors were calculated for all animals studied. Histological analysis. Seven days after the ischemic insult, the animals were anesthetized with pentobarbital and perfused transcardially with heparinized saline and then with 10% formalin in 0.I M phosphate buffer (pH 7.4). The brains were removed and saturated with a cold solution of 30% sucrose in 10% formalin. Serial coronal sections were cut at 40pro and 4/~m alternately with a cryostat and mounted on gelatine-coated slides: the 40-#m-thick sections were stained with Cresyl Violet to examine the positions of the microdialysis probe, and the 4-/~m-thick sections were stained with hematoxylin and eosin to count the numbers of normal-appearing CA1 neurons. Cell counts were performed using a microscope (Nikon Biophoto, Japan) with x 40 objective and x 10 ocular by placing a ocular grid with a length of 500 #m along the CA1 pyramidal layer and with a width of 125 #m. In three frontal sections taken 2.5 mm behind bregma, all normal-appearing neurons with intact morphology within three grids were counted: i.e. no eosinophilic changes and no Karyorrhexis. In each animal the mean surviving neuron number was calculated. Data analysis. The statistical significance of differences in glutamate levels and cell counts between control and ischemic animals with same brain temperature was assessed by two-tailed Student's t-test. The statistical significance of differences in glutamate levels and cell counts between the separate temperature groups was analysed by two-way ANOVA; this analysis included Tukey's or Dunnett's multiple-comparison procedure.
Glutamate during hypothermia and isehemic damage Table 1. Normothermic brain temperature of gerbil Animal
Brain temperature (°C)
1 2 3 4 5 6 Mean + S.E.M.
36.4 37.2 37.5 37.6 37.0 37.0 37.1 + 0.2
Brain temperature monitoring was performed for 20 min at the time between 13:00 and 17:00 in each animal.
RESULTS
Normothermic brain temperature of gerbil Normothermic brain temperature was monitored from six freely moving gerbils. Table 1 displays the brain temperature of each animal. The brain temperature of each animal was constant during the period of monitoring. Mean brain temperature of six animals was 37.1°C (mean rectal temperature was 37.7°C). Therefore, the brain temperature at 37°C was set as normotbermic brain temperature in the present study.
Effect of hypo- or hyperthermia on extracellular glutamate In order to confirm that there is no influence of variations in temperature on the recovery rate of dialysis membrane for glutamate, in vitro experiments were performed. Six similar microdialysis probes were used for this test. Each probe was dipped in Ringer solution which contained 50 #M glutamate. Dialysates were collected from the solution at 31, 33, 35, 37 and 39°C as described above. As shown in Table 2, no significant differences in glutamate values were detected among dialysates collected at different temperatures. The time-course of change in glutamate release during ischemia and recirculation in ischemic (n = 8) and control (n = 6) animals whose brain temperature was maintained at 37°C is illustrated in Fig. 1. Stable basal concentrations of extracellular glutamate were detected in the 10 consecutive samples collected before ischemia. In ischemic gerbils, a significant increase in hippocampal glutamate was detected in Table 2. Recovery test of dialysis membrane at different solution temperature Solution temperature (°C) 31(n = 6) 33(n = 6) 35(n = 6) 37(n = 6) 39(n = 6)
Concentration of glutamate in dialysate (#M) 6.7 + 0.6 6.9 + 0.5 7.1 + 0.5 6.9 + 0.4 6.5 + 0.6
Mean values + S.E.M. are represented. Six microdiaiysis probes were used; each probe was used to collect dialysate from Ringer solution (containing 50 #M glutamate) at 31, 33, 35, 37 and 39°C.
663
the dialysate during ischemia and the early period of recirculation. The increase was acute and massive; maximal levels were attained at the end of 5 min of ischemia (---15-fold increase). Glutamate extracellular levels returned to baseline within 6 - 7 m in of recirculation. In control gerbils, stable concentrations of extracellular glutamate were detected in all consecutive samples. The time-course of change in glutamate release during ischemia and recirculation in iscbemic (n = 10) and control (n = 6) animals whose brain temperature was maintained at 35°C is illustrated in Fig. 2. Stable basal concentrations of extracellular glutamate were detected in the five consecutive samples collected before isebemia and also in five other samples collected during a period of altering brain temperature from 37 to 35°C. Variations in brain temperature between 37 and 35°C had no significant influence on basal glutamate levels. In ischemic gerbils, a significant increase in hippocampal glutamate was detected in the dialysate during ischemia and the early period of recirculation. However, the increase was more gradual and of a significantly lesser extent compared with the acute and massive changes detected in animals whose brain temperature was maintained at 37°C (Table 3). The maximal levels were attained at the end of 5 rain of iscbemia (--7.6-fold increase); the levels of glutamate were attenuated to about half of those at 37°C. Glutamate extracellular levels returned to baseline within 5 min of recirculation. In control gerbils, stable concentrations of extracellular glutamate were detected in all samples. The time-course of changes in glutamate release during ischemia and recirculation in ischemic (n = 8) and control (n = 6) animals whose brain temperature was maintained at 33°C is illustrated in Fig. 3. Stable basal concentrations of extracellular glutamate were detected in the five consecutive samples collected prior to ischemia and also in five other samples collected during a period of altering brain temperature from 37 to 33°C. Variations in brain temperature between 37 and 33°C had no significant influence on basal glutamate levels. In ischemic gerbils, glutamate levels were still significantly increased during ischemia and the early period of recirculation. However, the increases were more gradual and of a lesser extent compared with changes detected in animals whose brain temperature was maintained at 35°C. The increase almost reached a plateau during the second 100 s of 5-min iscbemia. The maximal levels were attained at the end of 5 min of ischemia (--~4.2fold increase); the levels of glutamate were attenuated to about a quarter of those at 37°C (Table 3) and were significantly lower than those at 35°C (P < 0.01; Tukey's multiple-comparison procedure). Extracellular glutamate levels returned to baseline by 5 min of recirculation. In control gerbils, stable concentrations of extracellular glutamate were detected in all samples.
664
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