0361-9230/92 $5.00 + .OO Copyright 0 1992 Pergamon Press Ltd.
Brain Research Bullelin, Vol. 29, pp. 395-400, 1992 Printed in the USA. All rights reserved.
Preserved Neurotransmitter Receptor Binding Following Ischemia in Preconditioned Gerbil Brain HIROYUKI
KATO,’
TSUTOMU
ARAKI
AND
KYUYA
KOGURE
Department yf’Newology, Institute QfBrain Diseases, Tohokzl University School Received
2 December
19? 1; Accepted
3 1 January
qfMedicine, Sendai, Japan
1992
KATO, H., T. ARAKI AND K. KOGURE. Preserved neurolransmitler receptor binding following ix-hernia in preconditioned gerbil brain. BRAIN RES BULL 29(3/4)395-400, 1992.-Preconditioning the brain with sublethal ischemia induces tolerance to subsequent ischemic insult. Using [‘Hlquinuclidinyl benzilate (QNB), [)H]MK 801, [‘H]cyclohexyladenosine, [‘H]muscimol, and [3H]PN200-I 10, we investigated the alterations in neurotransmitter receptor and calcium channel binding in the gerbil hippocampus following ischemia with or without preconditioning. Two-minute forebrain ischemia, which produced no neuronal damage, resulted in no alterations in binding except for a slight reduction in [‘HIQNB binding in the CA I subfield. Three-minute ischemia destroyed the majority of CA 1 pyramidal cells and caused, in CA I, reductions in binding of all ligands used. Preconditioning with 2-min ischemia followed by 4 days of reperfusion protected against CA I neuronal damage and prevented the reductions in binding although [3H]QNB and [3H]PN200-I 10 binding transiently decreased in the early reperfusion period, suggesting downregulation. Thus, preconditioning protects against damage to the neurotransmission system as well as histopathological neuronal death. Preconditioning Cerebral ischemia Hippocampus Gerbil
Tolerance
Neurotransmitter systems
RECENT experiments have shown that selective neuronal vulnerability is altered by preconditioning the brain with sublethal ischemia. Vulnerability is increased at around 1 h after preconditioning, and extensive neuronal damage results following subsequent ischemic insult (7,8,9,14). By contrast, such sublethal ischemia induces tolerance to ischemic insult rendered l-7 days later, and neuronal damage from secondary insult is prevented (9, IO. 1I). However, the mechanism of the induction of tolerance is not known at present. Ischemia-induced neuronal damage is triggered by a massive release of neurotransmitters, especially excitatory amino acids, during &hernia. followed by receptor activation and intracellular calcium influx ( 1,13,18). Reductions in neurotransmitter receptor and calcium channel binding take place early after cerebral ischemia and precede delayed neuronal death of hippocampal CAI pyramidal cells (15). Therefore, we performed quantitative autoradiography to determine sequential alterations in neurotransmitter receptor and calcium channel binding following ischemia in preconditioned gerbil brain. For this purpose, we used [3H]quinuclidinyl benzilate (QNB), [3H]MK 801, [3H]cyclohexyladenosine (CHA), [3H]muscimol, and [‘H]PN200-I 10 to label muscarinic acetylcholine. NMDA,
Receptor
adenosine Al. and GABA-A channels, respectively.
Autoradiography
receptors
and
L-type
calcium
METHOD
A total of 40 male Mongolian gerbils (Seiwa Experimental Animals, Fukuoka. Japan), aged 12-14 weeks and weighing 6584 g, were used. They were allowed free access to food and water before and after surgery. Anesthesia was induced with 2% halothane in a mixture of 30% oxygen and 70% nitrous oxide. A midline cervical skin incision was made and bilateral common carotid arteries were gently exposed. Anesthesia was then discontinued, and I min later the arteries were occluded with aneurysm clips. Occlusion and reperfusion of the carotid arteries were verified by visual observation. Severe depression of electroencephalograph activity was confirmed in each animal using a pair of needle electrodes placed under the scalp. Body temperature during surgery and ischemia was maintained at close to 37°C. Postischemic body temperature was also monitored for 2 h to confirm the presence of postischemic hyperthermia and absence of hypothermia (9). Animals were subjected to a 2-min occlusion,
’ Requests for reprints should be addressed to Hiroyuki Kato, M.D., Department of Neurology. Institute of Brain Diseases, Tohoku University School of Medicine, I-I Seiryo-machi, Aoba-ku, Sendai 980, Japan.
395
KATO.
396
TABLE [‘H]QNB
BINDING (fmoI/mg AND FOLLOWING
AND
KOGURE
I
TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3.mm ISCHEMIA 3-min ISCHEMIA INDUCED 4 DAYS AFTER 2-min ISCHEMIA
Control
CA
ARAKI
2-min Ischemia (7 days)
2.
3-mln Ischemia (7 days)
t
3.mln lschemia
Ih
I day
7 days
I subfield
Stratum oriens Stratum radiatum Stratum lacunosum-moleculare CA3 subfield average Dentate gyrus Stratum
531 618 519 396
k k * +
13.5 11.4 14.1 13.1
560 i 12.6
moleculare
Mean t SD, n = 6-7. * p < 0.01, tp < 0.05 compared
498 588 493 397
zk 16.8 If- 25.2 t + 33.5 + I I .6
541 +- 21.7
Muscarinic acetylcholine receptors were quantified using the radiolabeled antagonist [3H]QNB (4 1.5 Ci/mmol, Amersham Corp., Arlington Heights, IL) as reported previously (5.15,16). Sections were incubated with I nM [3H]QNB in phosphate buffer (pH 7.4) for 90 min at room temperature. The sections were then washed in the buffer for 5 min at 4°C. Nonspecific binding was determined using 1 PM atropine (Sigma Chemical Co.. St. Louis, MO).
TABLE
CA I subfield Stratum oriens Stratum radiatum Stratum lacunosum-moleculare
CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum moleculare Mean & SD, n = 6-7. *p < 0.05.
t + k t
24.8* 20.9* 24.0 17.8
545 + 19.7
465 562 482 365
2 k f i
3h.4* 33.3* 53.9 13.9*
536 2 19.5
358 573 506 364
of 30.6* f 18.6* -+ 28. I t 20.4*
561 I 36.2
490 585 482 380
i 23.6t Y!?19.9t k 19.8 i 5.7
543 t 19.5
to control.
a 3-min occlusion, or a 2-min occlusion followed by a 3-min occlusion 4 days later. Animals subjected to single ischemia were decapitated at 7 days of survival and animals subjected to double ischemia were killed at I h, 1 day, and 7 days of survival. Six sham-operated animals were treated in the same manner except for the occlusion of the carotid arteries. Brains were quickly removed and frozen in powdered dry ice. Coronal frozen sections, 15 pm in thickness, were cut in a cryostat, thaw mounted onto gelatin-coated cover-slips, and stored at -80°C until assay. Adjacent sections were stained with cresyl violet and hematoxylin & eosin and used for histopathology. Neuronal damage in the hippocampus was semiquantitatively graded using a O-3 rating system with 0 = no damage, 1 = a few neurons damaged, 2 = many neurons damaged, and 3 = majority of neurons damaged.
[‘H]MK
465 547 487 378
NMDA receptors were quantified using the radiolabeled noncompetitive antagonist [‘H]MK 801 (28.8 Ci/mmol, New England Nuclear Corp.. Newton. MA) as reported previously (2). Sections were rinsed in 50 mM Tris-HCI buffer (pH 7.4) with 190 mM sucrose. air-dried, and then incubated with 30 nM [‘H]MK 801 in the same buffer for 20 min at room temperature. The sections were then washed twice in the buffer for 20 s. Nonspecific binding was determined using 100 PM MK 801 (Research Biochemicals. Inc., Natick. MA). [3tfjC’If,4
Binding
Adenosine A 1 receptors were measured using [3H]CHA (34.4 Ci/mmoI. New England Nuclear) as described previously (5,15,16). Sections were incubated with 5 nM [3H]CHA and 2 U/ml adenosine deaminase (Boehringer Mannheim. Indianapolis, IN) in 50 mM Tris-buffer (pH 7.4) for 90 min at room temperature. The sections were then washed in the buffer for 1 min at 4°C. Nonspecific binding was determined using IO PM L-phenylisopropyladenosine (Boehringer Mannheim).
GABA A receptors were measured using [‘Hlmuscimol (20 Ci/mmol, New England Nuclear) as described previously ( 17).
7
801 BINDING (fmol/mg TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3.mm AND FOLLOWING 3-min ISCHEMIA INDUCED 4 DAYS AFTER 2-min ISCHEMIA
ISCHEMIA
2. + 3.min Ischemla
Control
2.min Ischemia (7 days)
3.mm lschemia (7 days)
Ih
I day
7 days
192 _t 64.6 213 _t 71.0 164 k 75.7
2 17 t 66.4 232 + 56.6 I56 f 32.8
132 ? 33.9* 146 +- 36.0
181 lr 32.7 200 k 38.2
200 +- 39.7 21 I * 31.2
171 + 41.5 I84 f 43.0
IO1 t 28.5*
143 + 25.0
I65 ? 22.3
133 -t 32.7
146 ir 32.5 I74 * 50. I
173 & 53.7 194 * 44.7
I37 * 40.8 I65 i 35.6
I51 f 28.6 I74 + 26.8
I52 + 29.9 I75 -t 22.6
I50 f 23.9 174 t 34.1
I91 f 61.4
21 I i- 50.9
I55 t 26.8
183 i 23.0
190 i 28.9
176 + 41.8
ISCHEMIC TOLERANCE
AND RECEPTOR
BINDING
397
TABLE 3 [3H]CHA
BINDING (fmol/mg AND FOLLOWING
TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3-min ISCHEMIA 3-min ISCHEMIA INDUCED 4 DAYS AFfER 2-min ISCHEMIA
Control
CA 1 subfield Stratum oriens Stratum radiatum Stratum lacunosum-moleculare CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum moleculare
2-min Ischemia (7 days)
2- + 3-min Ischemia
3-min Ischemia (7 days)
Ih
I day
7 days
309 f 40.9 347 f 30.6 218 + 25.1
280 z!z32.8 314 + 24.9 208 f 18.4
278 f 42.0 297 k 40.4* 183 t 20.5*
305 f 26.5 352 + 36.5 235 k 30.9
280 + 34.7 322 f 32.8 207 f 26.4
317 + 33.1 364 + 25.8 230 rl: 26.3
228 + 24.7 253 t 27.4
215 f 22.9 244 f 28.9
229 i 22.7 268 t 28.7
235 k 24.7 266 + 34.7
213 f 26.1 249 f 28.5
235 k 21.1 266 + 22.7
205 + 24.6
192 + 13.5
21 I f 27.8
212 f 20.7
194 f 24.7
216 + 17.9
Mean + SD, n = 6-7. * p < 0.05.
Sections were preincubated in 50 mM T&citrate buffer (pH7.1) for 20 min at 4°C and then incubated with 30 nM [3H]muscimol in the same buffer for 40 min at 4°C. The sections were then washed in the buffer for I min and in distilled water rapidly at 4°C. Nonspecific binding was determined using 10 rM GABA (Wako Chemicals).
Zeiss). The relation between optical density and radioactivity was determined using a third-order polynomial function with reference to tritium standards ( [3H]microscale, Amersham) exposed along with the tissue sections. Optical densities of the brain regions measured were in the range in which the radioactivity of the [3H]microscale showed a near-linear relation.
[‘H]PNZOO-I 10 Binding
Statistics
The method for the autoradiographic visualization of L-type calcium channel blocker binding using [‘H]PN200-110, a I ,4dihydropyridine calcium channel blocker, has been described previously (5,lS). Sections were incubated with 0. I nM [3H]PN200-I IO (71.5 Ci/mmol, New England Nuclear) in 170 mM Tris-HCI buffer (pH 7.7) for I h at room temperature under subdued lighting. The sections were then washed in the buffer for 20 min at 4°C. Nonspecific binding was determined using 1 MM nitrendipine (Sigma).
Each group consisted of six to seven animals. Binding assays were performed in duplicate. Average values of left and right hippocampi were used for analysis. Values were expressed as means f SD. Statistical significance was analyzed using the Kruskal-Wallis nonparametric analysis of variance (ANOVA) followed by the Williams-Wilcoxon rank sum test. RESULTS
Histoputhology
The sections were dried under a stream of cold air and apposed to Hyperfilm-3H (Amersham) for 2-4 weeks. The optical density of the regions of interest was measured using a computerassisted image analyzer system (IBAS image analyzer system,
No neuronal damage was observed following 2-min ischemia, but the majority of CA I pyramidal cells were destroyed following 3 min of ischemia with the damage score of 2.3 + 0.8 SD (p < 0.0 I vs. sham operation). Preconditioning with 2-min ischemia followed by 4 days of reperfusion prevented the CA 1 neuronal
TABLE 4 [‘HIMUSCIMOL
BINDING (fmol/mg TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3-min AND FOLLOWING 3-min ISCHEMIA INDUCED 4 DAYS AFTER 2-min ISCHEMIA
Control
CA I subfield Stratum oriens Stratum radiatum Stratum lacunosum-moleculare CA3 subfield average Dentate gyrus Stratum moleculare Mean f SD, n = 6-7 * p < 0.05.
183 + 200 f 172 f 95 f
14.0 15.4 17.5 8.8
234 f 24.6
2-min Ischemia (7 days)
191 f 209 f 185 + 98 f
10.5 12.7 16.7 6.7
236 t 20.3
3-min Ischemia (7 days)
152 f 174 f 158 + 92 +
l6.6* 21.3: 18.9 8.3
217 It 19.8
ISCHEMIA
2- + 3-min khemia Ih
190 f 209 f 178 f 96 f
I day
25.9 28.0 22.3 12.4
234 + 40.2
187 + 210 f 181 + 97 ?
35.0 37.5 29.8 12.6
231 k 31.0
7 days
184 + 212 f 178 f 98 f
15.9 19.8 20.5 10.8
218 f 19.1
398
KATQ.
TABLE
ARAKl
AND
KQGURE
5
[“H]PNZOO-I 10 BINDING (fmol/mg TISSUE) IN THE HIPPOCAMPUS FOLLOWING 7. ;\ND 3.mm ISCHFMIA AND FOLLOWING 3-min ISCHEMIA INDUCED 3 DAYS AFTER 2.min ISCfiEMIA
CA I subfield CA3 subfield Dentate gyrus Mean + SD. II r 6-7. * 11 < 0.01. T/J < 0.05 compared to control
damage following 3 min of ischemia. and no neuronal damage was observed. CA3 pyramidal cells and dentate granule cells showed no visible damage in any group.
Following 2-min ischemia. we found no alterations in the binding in the hippocampus except for a slight (5a) but significant reduction in [3H]QNB binding in the CA I subfield (Tables i-5). Three-minute ischemia caused significant reductions in the binding of all ligands in the CA1 subfield (Fig. I, Tables l-5).
The reductions wcrc greater in [‘H]MK 801 and [3H]PN200I IO binding. in which more than 30% reductions were observed, compared to [jH]QNB. [‘H]CHA, and [3H]muscimol binding. in which the reductions were by IO- 15%). There were no reductions in the binding in the CA3 subheld and dentate g:rus. Changes following double ischcmia are shown in Frg. I and fables l-5. [‘H]MK 801. [‘H]CHA. and [3H]muscimol binding in the CA1 subheld was comparable to control levels throughout the observation period following secondary ischemia. [3H]PN200-I IO binding in CAI decreased (by 25%) I h after secondary ischemia but recovered to control levels thereafter.
contr 01 FIG. I, Representative autoradiographs of [jH]QNB. [‘H]MK 801 (MK), [‘H]CHA. [3H]muscimol (MUS). and [‘HIPNZOOI IO (PN) binding in the gerbil hippocampus ofcontrol (left column). 7 days after 3-min forebrain ischemia (middle column). and 7 days after 3-min ischemia induced 4 days after preconditioning with ?-min ischemia (right column) groups. The binding. especially [‘H]MK and [‘H]PN, is reduced following 3-min ischemia but preserved by preconditioning.
ISCHEMIC
TOLERANCE
AND
RECEPTOR
399
BINDING
[3H]QNB binding in CA I decreased by 30% 1 h and I day after secondary ischemia but recovered to the slightly decreased level comparable to that after 2-min ischemia. [3H]QNB binding in the CA3 subfield also decreased at 1 h and 1 day after secondary ischemia. Otherwise, there were no alterations in the binding in CA3 and the dentate gyrus. DISCUSSION
The present study demonstrated that the binding of neurotransmitter receptors and calcium channels significantly decreased in the CA1 subfield by 7 days after 3 min of ischemia, where selective pyramidal cell death was observed by histopathology. but not after 2 min of ischemia, which caused no neuronal damage. The reduction in binding after elimination of CA 1 pyramidal cells is consistent with previous reports although the rate of reduction is smaller compared to that after longer periods of ischemia (5,16). Interestingly, the reductions in [3H]MK 80 I and [3H]PN200-I 10 binding were the greatest among the five ligands used. This suggests the predominant localization of NMDA receptors and L-type calcium channels on pyramidal cells in the CA I subfield, and may be related to the role of NMDA receptors and the calcium channels in selective CA I neuronal damage as has been emphasized (13,I8). Preconditioning the brain with sublethal ischemia prevented reductions in the receptor and calcium channel binding, as well as histopathological neuronal destruction. By 7 days after secondary ischemia, the binding activity recovered to preischemia levels. Interestingly, we observed a transient reduction in [3H]QNB and [3H]PN200-1 IO binding in the early reperfusion period after secondary ischemia, which may be important because the down regulation of an excitatory neurotransmitter system and the voltage-dependent calcium channels is likely to ameliorate neuronal damage. The mechanism of the induction of tolerance to subsequent ischemic insult after preconditioning with sublethal ischemia remains elusive. However, a role of stress response is suggested
because the stage of tolerance accompanies the synthesis of heat shock/stress proteins (10) and because pretreatment with high temperature (hyperthermia) also induces tolerance to subsequent ischemic insult (3.12). But, as Kirino et al. (10) reported, tolerance may not simply be induced by stress proteins. Tolerance is induced by I day after preconditioning but immunohistochemical observations revealed only minimum heat shock protein staining at that time. Important may be the alterations in the intracellular second messenger systems. In a separate study, we observed that [3H]inositol 1.45triphosphate (IP,) binding in CA 1 was reduced by 30% 4 days after 2 min of ischemia in gerbils (6). IP3 is massively released during ischemia and the stimulation of IP3 receptors mobilizes calcium from intracellular stores (4,19). Because increased intracellular calcium concentration is believed to lead to cellular damage ( I@, the downregulation of IP, binding may play a role in protection against ischemic neuronal damage. Because preconditioning protects the neurotransmitter receptor system as shown in this study, the neurons are expected to resume normal or near-normal activity. However, a behavioral study demonstrated that gerbils subjected to 5 min of ischemia 2 days after preconditioning show poor passive avoidance test performance 4 days after the secondary insult as compared to control animals despite the fact that animals in both groups had normal density of CA 1 neurons (20). The present results, which showed disturbed binding activity in the early reperfusion period after secondary ischemia. may be related to the disturbed learning ability. But, long-term observations may be necessary. In conclusion. the present results show that preconditioning prevents both histopathologic neuronal death and reductions in neurotransmitter receptor and calcium channel binding. Transient downregulation of [‘H]QNB and [3H]PN200-l 10 binding, observed in the early reperfusion period, may play a role in protection against neuronal damage. Further studies should be conducted to reveal the mechanisms of the induction of tolerance.
REFERENCES 1.
2.
3.
4. 5.
6. 7. 8.
Benveniste, H.: Drejer. J.; Schousboue, A.; Diemer, H. N. Elevation of extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43: 1369- 1373; 1984. Bowerv. N. G.: Wona. E. H. F.: Hudson. A. L. Ouantitative autoradiography of [‘HI-MK-801 binding sites in mammalian brain. Br. J. Pharmacol. 93:944-954; 1988. Chopp, M.: Chen. H.; Ho, K.-L.; Dreski, M. 0.; Brown, E.: Hetzel. F. W.: Welch, K. M. A. Transient hyperthermia protects against subsequent forebrain ischemia cell damage in the rat. Neurology 39:1396-1398; 1989. Chueh, S. H.; Gill, D. L. lnositol 1,4,5_triphosphate and guanine nucleotides activate calcium release from kndoplasmic reiiculum via distinct mechanisms. J. Biol. Chem. 26 I : I3883- 13886: 1986. Kato, H.; Araki, T.; Hara, H.; Kogure, K. Sequential changes in muscarinic acetylcholine, adenosine A 1 and calcium antagonist binding sites in the gerbil hipuocampus followina_ repeated brief . ischemya. Brain Res. 553:33-j& 1991: Kato, H.; Araki, T.; Hara, H.; Kogure, K. Autoradiographic analysis of second-messenger systems in the gerbil hippocampus following reoeated brief ischemic insults. Brain Res. Bull. 27:759-765; 199 I. K&o, H.; Kogure, K. Neuronal damage following non-lethal but repeated cerebral ischemia in the gerbil. Acta Neuropathol. 79:494500: 1990. Kato, H.: Kogure, K.; Nakano, S. Neuronal damage following repeated brief ischemia in the gerbil. Brain Res. 479:366-370; 1989.
9. Kato, H.: Liu. Y.; Araki. T.: Kogure, K. Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: Cumulative damaae and orotective effects. Brain Res. 553:238-242: 1991. IO. Kit-&, T.; T’sujita. Y.; Tamura, A. Induced tolerance to ischemia in gerbil hippocampul neurons. J. Cereb. Blood Flow Metab. 11: 299-307; 1991. I I. Kitagawa, K.; Matsumoto, M.; Tagata, M.; Hata, R.; Ueda. H.; Niinobe, M.: Handa. N.; Hukunaga, R.; Kimura, K.; Mikoshiba, K.; Kamada, T. “lschemic tolerance” phenomenon found in the brain. Brain Res. 528:2 l-24; 1990. 12. Kitagawa, K.; Matsumoto, M.; Tagaya, M.; Kuwabara, K.; Hata. R.: Handa, N.: Fukunaga. R.; Kimura. K.; Kamada. T. Hyperthermia-induced neuronal protection against ischemic injury in gerbils. J. Cereb. Blood Flow Metab. 1 1:449-452; I99 I. 13. Kogure. K.; Tanaka, J.: Araki. T. The mechanism of ischemia-induced brain cell injury. The membrane theory. Neurochem. Pathol. 9:145-170; 1988. 14. Nakano, S.: Kato, H.; Kogure, K. Neuronal damage in the rat hippocampus ischemia. 15. Onodera.
in a new model of repeated reversible Brain Res. 490: 178- 180; 1989.
transient
cerebral
H.; Kogure. K. Calcium antagonist, adenosine Al, and muscarinic binding in rat hippocampus after transient ischemia. Stroke 21:771-776:
1989.
16. Onodera, H.: Sato, G.; Kogure, K. Quantitative autoradiographic analysis of muscarinic cholinergic and adenosine Al binding sites
400
after transient forebrain ischemia in the gerbil. Brain Res. 4 15:309322; 1989. 17. Onodera. H.; Sato. G.; Kogure, K. GABA and benzodiazepine receptors in the gerbil brain after transient ischemia: Demonstration by quantitative receptor autoradiography. J. Cereb. Blood Flow Metab. 7:82-88; 1987. 18. Siesjo. B. K.: Bengtsson. F. Calcium fluxes, calcium antagonists. and calcium-related pathology in brain ischemia. hypoglycemia, and spreading depression: A unifying hypothesis. J. Cereb. Blood Flow Metab. 9:127-140: 1989.
KATO.
ARAKI
AND
KOGURE
19. Streb. H.: Invine, R. F.; Berridge. M. J.: Shulz, 1. Release of Ca*’ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-l.4,5-triphosphate. Nature 306:67-68; 1983. 20. Tanaka. J.; Yamada. F.: Yamauchi. H.; Kogure, K. Preliminary exposure to brief cerebral ischemia prevents delayed neuronal death but does not ameliorate functional brain damage in Mongolian gerbils. J. Cereb. Blood Flow Metab. I I(suppl. 2):S325: 199 I.
Brain Research Bullelin, Vol. 29, pp. 395-400, 1992 Printed in the USA. All rights reserved.
Preserved Neurotransmitter Receptor Binding Following Ischemia in Preconditioned Gerbil Brain HIROYUKI
KATO,’
TSUTOMU
ARAKI
AND
KYUYA
KOGURE
Department yf’Newology, Institute QfBrain Diseases, Tohokzl University School Received
2 December
19? 1; Accepted
3 1 January
qfMedicine, Sendai, Japan
1992
KATO, H., T. ARAKI AND K. KOGURE. Preserved neurolransmitler receptor binding following ix-hernia in preconditioned gerbil brain. BRAIN RES BULL 29(3/4)395-400, 1992.-Preconditioning the brain with sublethal ischemia induces tolerance to subsequent ischemic insult. Using [‘Hlquinuclidinyl benzilate (QNB), [)H]MK 801, [‘H]cyclohexyladenosine, [‘H]muscimol, and [3H]PN200-I 10, we investigated the alterations in neurotransmitter receptor and calcium channel binding in the gerbil hippocampus following ischemia with or without preconditioning. Two-minute forebrain ischemia, which produced no neuronal damage, resulted in no alterations in binding except for a slight reduction in [‘HIQNB binding in the CA I subfield. Three-minute ischemia destroyed the majority of CA 1 pyramidal cells and caused, in CA I, reductions in binding of all ligands used. Preconditioning with 2-min ischemia followed by 4 days of reperfusion protected against CA I neuronal damage and prevented the reductions in binding although [3H]QNB and [3H]PN200-I 10 binding transiently decreased in the early reperfusion period, suggesting downregulation. Thus, preconditioning protects against damage to the neurotransmission system as well as histopathological neuronal death. Preconditioning Cerebral ischemia Hippocampus Gerbil
Tolerance
Neurotransmitter systems
RECENT experiments have shown that selective neuronal vulnerability is altered by preconditioning the brain with sublethal ischemia. Vulnerability is increased at around 1 h after preconditioning, and extensive neuronal damage results following subsequent ischemic insult (7,8,9,14). By contrast, such sublethal ischemia induces tolerance to ischemic insult rendered l-7 days later, and neuronal damage from secondary insult is prevented (9, IO. 1I). However, the mechanism of the induction of tolerance is not known at present. Ischemia-induced neuronal damage is triggered by a massive release of neurotransmitters, especially excitatory amino acids, during &hernia. followed by receptor activation and intracellular calcium influx ( 1,13,18). Reductions in neurotransmitter receptor and calcium channel binding take place early after cerebral ischemia and precede delayed neuronal death of hippocampal CAI pyramidal cells (15). Therefore, we performed quantitative autoradiography to determine sequential alterations in neurotransmitter receptor and calcium channel binding following ischemia in preconditioned gerbil brain. For this purpose, we used [3H]quinuclidinyl benzilate (QNB), [3H]MK 801, [3H]cyclohexyladenosine (CHA), [3H]muscimol, and [‘H]PN200-I 10 to label muscarinic acetylcholine. NMDA,
Receptor
adenosine Al. and GABA-A channels, respectively.
Autoradiography
receptors
and
L-type
calcium
METHOD
A total of 40 male Mongolian gerbils (Seiwa Experimental Animals, Fukuoka. Japan), aged 12-14 weeks and weighing 6584 g, were used. They were allowed free access to food and water before and after surgery. Anesthesia was induced with 2% halothane in a mixture of 30% oxygen and 70% nitrous oxide. A midline cervical skin incision was made and bilateral common carotid arteries were gently exposed. Anesthesia was then discontinued, and I min later the arteries were occluded with aneurysm clips. Occlusion and reperfusion of the carotid arteries were verified by visual observation. Severe depression of electroencephalograph activity was confirmed in each animal using a pair of needle electrodes placed under the scalp. Body temperature during surgery and ischemia was maintained at close to 37°C. Postischemic body temperature was also monitored for 2 h to confirm the presence of postischemic hyperthermia and absence of hypothermia (9). Animals were subjected to a 2-min occlusion,
’ Requests for reprints should be addressed to Hiroyuki Kato, M.D., Department of Neurology. Institute of Brain Diseases, Tohoku University School of Medicine, I-I Seiryo-machi, Aoba-ku, Sendai 980, Japan.
395
KATO.
396
TABLE [‘H]QNB
BINDING (fmoI/mg AND FOLLOWING
AND
KOGURE
I
TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3.mm ISCHEMIA 3-min ISCHEMIA INDUCED 4 DAYS AFTER 2-min ISCHEMIA
Control
CA
ARAKI
2-min Ischemia (7 days)
2.
3-mln Ischemia (7 days)
t
3.mln lschemia
Ih
I day
7 days
I subfield
Stratum oriens Stratum radiatum Stratum lacunosum-moleculare CA3 subfield average Dentate gyrus Stratum
531 618 519 396
k k * +
13.5 11.4 14.1 13.1
560 i 12.6
moleculare
Mean t SD, n = 6-7. * p < 0.01, tp < 0.05 compared
498 588 493 397
zk 16.8 If- 25.2 t + 33.5 + I I .6
541 +- 21.7
Muscarinic acetylcholine receptors were quantified using the radiolabeled antagonist [3H]QNB (4 1.5 Ci/mmol, Amersham Corp., Arlington Heights, IL) as reported previously (5.15,16). Sections were incubated with I nM [3H]QNB in phosphate buffer (pH 7.4) for 90 min at room temperature. The sections were then washed in the buffer for 5 min at 4°C. Nonspecific binding was determined using 1 PM atropine (Sigma Chemical Co.. St. Louis, MO).
TABLE
CA I subfield Stratum oriens Stratum radiatum Stratum lacunosum-moleculare
CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum moleculare Mean & SD, n = 6-7. *p < 0.05.
t + k t
24.8* 20.9* 24.0 17.8
545 + 19.7
465 562 482 365
2 k f i
3h.4* 33.3* 53.9 13.9*
536 2 19.5
358 573 506 364
of 30.6* f 18.6* -+ 28. I t 20.4*
561 I 36.2
490 585 482 380
i 23.6t Y!?19.9t k 19.8 i 5.7
543 t 19.5
to control.
a 3-min occlusion, or a 2-min occlusion followed by a 3-min occlusion 4 days later. Animals subjected to single ischemia were decapitated at 7 days of survival and animals subjected to double ischemia were killed at I h, 1 day, and 7 days of survival. Six sham-operated animals were treated in the same manner except for the occlusion of the carotid arteries. Brains were quickly removed and frozen in powdered dry ice. Coronal frozen sections, 15 pm in thickness, were cut in a cryostat, thaw mounted onto gelatin-coated cover-slips, and stored at -80°C until assay. Adjacent sections were stained with cresyl violet and hematoxylin & eosin and used for histopathology. Neuronal damage in the hippocampus was semiquantitatively graded using a O-3 rating system with 0 = no damage, 1 = a few neurons damaged, 2 = many neurons damaged, and 3 = majority of neurons damaged.
[‘H]MK
465 547 487 378
NMDA receptors were quantified using the radiolabeled noncompetitive antagonist [‘H]MK 801 (28.8 Ci/mmol, New England Nuclear Corp.. Newton. MA) as reported previously (2). Sections were rinsed in 50 mM Tris-HCI buffer (pH 7.4) with 190 mM sucrose. air-dried, and then incubated with 30 nM [‘H]MK 801 in the same buffer for 20 min at room temperature. The sections were then washed twice in the buffer for 20 s. Nonspecific binding was determined using 100 PM MK 801 (Research Biochemicals. Inc., Natick. MA). [3tfjC’If,4
Binding
Adenosine A 1 receptors were measured using [3H]CHA (34.4 Ci/mmoI. New England Nuclear) as described previously (5,15,16). Sections were incubated with 5 nM [3H]CHA and 2 U/ml adenosine deaminase (Boehringer Mannheim. Indianapolis, IN) in 50 mM Tris-buffer (pH 7.4) for 90 min at room temperature. The sections were then washed in the buffer for 1 min at 4°C. Nonspecific binding was determined using IO PM L-phenylisopropyladenosine (Boehringer Mannheim).
GABA A receptors were measured using [‘Hlmuscimol (20 Ci/mmol, New England Nuclear) as described previously ( 17).
7
801 BINDING (fmol/mg TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3.mm AND FOLLOWING 3-min ISCHEMIA INDUCED 4 DAYS AFTER 2-min ISCHEMIA
ISCHEMIA
2. + 3.min Ischemla
Control
2.min Ischemia (7 days)
3.mm lschemia (7 days)
Ih
I day
7 days
192 _t 64.6 213 _t 71.0 164 k 75.7
2 17 t 66.4 232 + 56.6 I56 f 32.8
132 ? 33.9* 146 +- 36.0
181 lr 32.7 200 k 38.2
200 +- 39.7 21 I * 31.2
171 + 41.5 I84 f 43.0
IO1 t 28.5*
143 + 25.0
I65 ? 22.3
133 -t 32.7
146 ir 32.5 I74 * 50. I
173 & 53.7 194 * 44.7
I37 * 40.8 I65 i 35.6
I51 f 28.6 I74 + 26.8
I52 + 29.9 I75 -t 22.6
I50 f 23.9 174 t 34.1
I91 f 61.4
21 I i- 50.9
I55 t 26.8
183 i 23.0
190 i 28.9
176 + 41.8
ISCHEMIC TOLERANCE
AND RECEPTOR
BINDING
397
TABLE 3 [3H]CHA
BINDING (fmol/mg AND FOLLOWING
TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3-min ISCHEMIA 3-min ISCHEMIA INDUCED 4 DAYS AFfER 2-min ISCHEMIA
Control
CA 1 subfield Stratum oriens Stratum radiatum Stratum lacunosum-moleculare CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum moleculare
2-min Ischemia (7 days)
2- + 3-min Ischemia
3-min Ischemia (7 days)
Ih
I day
7 days
309 f 40.9 347 f 30.6 218 + 25.1
280 z!z32.8 314 + 24.9 208 f 18.4
278 f 42.0 297 k 40.4* 183 t 20.5*
305 f 26.5 352 + 36.5 235 k 30.9
280 + 34.7 322 f 32.8 207 f 26.4
317 + 33.1 364 + 25.8 230 rl: 26.3
228 + 24.7 253 t 27.4
215 f 22.9 244 f 28.9
229 i 22.7 268 t 28.7
235 k 24.7 266 + 34.7
213 f 26.1 249 f 28.5
235 k 21.1 266 + 22.7
205 + 24.6
192 + 13.5
21 I f 27.8
212 f 20.7
194 f 24.7
216 + 17.9
Mean + SD, n = 6-7. * p < 0.05.
Sections were preincubated in 50 mM T&citrate buffer (pH7.1) for 20 min at 4°C and then incubated with 30 nM [3H]muscimol in the same buffer for 40 min at 4°C. The sections were then washed in the buffer for I min and in distilled water rapidly at 4°C. Nonspecific binding was determined using 10 rM GABA (Wako Chemicals).
Zeiss). The relation between optical density and radioactivity was determined using a third-order polynomial function with reference to tritium standards ( [3H]microscale, Amersham) exposed along with the tissue sections. Optical densities of the brain regions measured were in the range in which the radioactivity of the [3H]microscale showed a near-linear relation.
[‘H]PNZOO-I 10 Binding
Statistics
The method for the autoradiographic visualization of L-type calcium channel blocker binding using [‘H]PN200-110, a I ,4dihydropyridine calcium channel blocker, has been described previously (5,lS). Sections were incubated with 0. I nM [3H]PN200-I IO (71.5 Ci/mmol, New England Nuclear) in 170 mM Tris-HCI buffer (pH 7.7) for I h at room temperature under subdued lighting. The sections were then washed in the buffer for 20 min at 4°C. Nonspecific binding was determined using 1 MM nitrendipine (Sigma).
Each group consisted of six to seven animals. Binding assays were performed in duplicate. Average values of left and right hippocampi were used for analysis. Values were expressed as means f SD. Statistical significance was analyzed using the Kruskal-Wallis nonparametric analysis of variance (ANOVA) followed by the Williams-Wilcoxon rank sum test. RESULTS
Histoputhology
The sections were dried under a stream of cold air and apposed to Hyperfilm-3H (Amersham) for 2-4 weeks. The optical density of the regions of interest was measured using a computerassisted image analyzer system (IBAS image analyzer system,
No neuronal damage was observed following 2-min ischemia, but the majority of CA I pyramidal cells were destroyed following 3 min of ischemia with the damage score of 2.3 + 0.8 SD (p < 0.0 I vs. sham operation). Preconditioning with 2-min ischemia followed by 4 days of reperfusion prevented the CA 1 neuronal
TABLE 4 [‘HIMUSCIMOL
BINDING (fmol/mg TISSUE) IN THE HIPPOCAMPUS FOLLOWING 2- AND 3-min AND FOLLOWING 3-min ISCHEMIA INDUCED 4 DAYS AFTER 2-min ISCHEMIA
Control
CA I subfield Stratum oriens Stratum radiatum Stratum lacunosum-moleculare CA3 subfield average Dentate gyrus Stratum moleculare Mean f SD, n = 6-7 * p < 0.05.
183 + 200 f 172 f 95 f
14.0 15.4 17.5 8.8
234 f 24.6
2-min Ischemia (7 days)
191 f 209 f 185 + 98 f
10.5 12.7 16.7 6.7
236 t 20.3
3-min Ischemia (7 days)
152 f 174 f 158 + 92 +
l6.6* 21.3: 18.9 8.3
217 It 19.8
ISCHEMIA
2- + 3-min khemia Ih
190 f 209 f 178 f 96 f
I day
25.9 28.0 22.3 12.4
234 + 40.2
187 + 210 f 181 + 97 ?
35.0 37.5 29.8 12.6
231 k 31.0
7 days
184 + 212 f 178 f 98 f
15.9 19.8 20.5 10.8
218 f 19.1
398
KATQ.
TABLE
ARAKl
AND
KQGURE
5
[“H]PNZOO-I 10 BINDING (fmol/mg TISSUE) IN THE HIPPOCAMPUS FOLLOWING 7. ;\ND 3.mm ISCHFMIA AND FOLLOWING 3-min ISCHEMIA INDUCED 3 DAYS AFTER 2.min ISCfiEMIA
CA I subfield CA3 subfield Dentate gyrus Mean + SD. II r 6-7. * 11 < 0.01. T/J < 0.05 compared to control
damage following 3 min of ischemia. and no neuronal damage was observed. CA3 pyramidal cells and dentate granule cells showed no visible damage in any group.
Following 2-min ischemia. we found no alterations in the binding in the hippocampus except for a slight (5a) but significant reduction in [3H]QNB binding in the CA I subfield (Tables i-5). Three-minute ischemia caused significant reductions in the binding of all ligands in the CA1 subfield (Fig. I, Tables l-5).
The reductions wcrc greater in [‘H]MK 801 and [3H]PN200I IO binding. in which more than 30% reductions were observed, compared to [jH]QNB. [‘H]CHA, and [3H]muscimol binding. in which the reductions were by IO- 15%). There were no reductions in the binding in the CA3 subheld and dentate g:rus. Changes following double ischcmia are shown in Frg. I and fables l-5. [‘H]MK 801. [‘H]CHA. and [3H]muscimol binding in the CA1 subheld was comparable to control levels throughout the observation period following secondary ischemia. [3H]PN200-I IO binding in CAI decreased (by 25%) I h after secondary ischemia but recovered to control levels thereafter.
contr 01 FIG. I, Representative autoradiographs of [jH]QNB. [‘H]MK 801 (MK), [‘H]CHA. [3H]muscimol (MUS). and [‘HIPNZOOI IO (PN) binding in the gerbil hippocampus ofcontrol (left column). 7 days after 3-min forebrain ischemia (middle column). and 7 days after 3-min ischemia induced 4 days after preconditioning with ?-min ischemia (right column) groups. The binding. especially [‘H]MK and [‘H]PN, is reduced following 3-min ischemia but preserved by preconditioning.
ISCHEMIC
TOLERANCE
AND
RECEPTOR
399
BINDING
[3H]QNB binding in CA I decreased by 30% 1 h and I day after secondary ischemia but recovered to the slightly decreased level comparable to that after 2-min ischemia. [3H]QNB binding in the CA3 subfield also decreased at 1 h and 1 day after secondary ischemia. Otherwise, there were no alterations in the binding in CA3 and the dentate gyrus. DISCUSSION
The present study demonstrated that the binding of neurotransmitter receptors and calcium channels significantly decreased in the CA1 subfield by 7 days after 3 min of ischemia, where selective pyramidal cell death was observed by histopathology. but not after 2 min of ischemia, which caused no neuronal damage. The reduction in binding after elimination of CA 1 pyramidal cells is consistent with previous reports although the rate of reduction is smaller compared to that after longer periods of ischemia (5,16). Interestingly, the reductions in [3H]MK 80 I and [3H]PN200-I 10 binding were the greatest among the five ligands used. This suggests the predominant localization of NMDA receptors and L-type calcium channels on pyramidal cells in the CA I subfield, and may be related to the role of NMDA receptors and the calcium channels in selective CA I neuronal damage as has been emphasized (13,I8). Preconditioning the brain with sublethal ischemia prevented reductions in the receptor and calcium channel binding, as well as histopathological neuronal destruction. By 7 days after secondary ischemia, the binding activity recovered to preischemia levels. Interestingly, we observed a transient reduction in [3H]QNB and [3H]PN200-1 IO binding in the early reperfusion period after secondary ischemia, which may be important because the down regulation of an excitatory neurotransmitter system and the voltage-dependent calcium channels is likely to ameliorate neuronal damage. The mechanism of the induction of tolerance to subsequent ischemic insult after preconditioning with sublethal ischemia remains elusive. However, a role of stress response is suggested
because the stage of tolerance accompanies the synthesis of heat shock/stress proteins (10) and because pretreatment with high temperature (hyperthermia) also induces tolerance to subsequent ischemic insult (3.12). But, as Kirino et al. (10) reported, tolerance may not simply be induced by stress proteins. Tolerance is induced by I day after preconditioning but immunohistochemical observations revealed only minimum heat shock protein staining at that time. Important may be the alterations in the intracellular second messenger systems. In a separate study, we observed that [3H]inositol 1.45triphosphate (IP,) binding in CA 1 was reduced by 30% 4 days after 2 min of ischemia in gerbils (6). IP3 is massively released during ischemia and the stimulation of IP3 receptors mobilizes calcium from intracellular stores (4,19). Because increased intracellular calcium concentration is believed to lead to cellular damage ( I@, the downregulation of IP, binding may play a role in protection against ischemic neuronal damage. Because preconditioning protects the neurotransmitter receptor system as shown in this study, the neurons are expected to resume normal or near-normal activity. However, a behavioral study demonstrated that gerbils subjected to 5 min of ischemia 2 days after preconditioning show poor passive avoidance test performance 4 days after the secondary insult as compared to control animals despite the fact that animals in both groups had normal density of CA 1 neurons (20). The present results, which showed disturbed binding activity in the early reperfusion period after secondary ischemia. may be related to the disturbed learning ability. But, long-term observations may be necessary. In conclusion. the present results show that preconditioning prevents both histopathologic neuronal death and reductions in neurotransmitter receptor and calcium channel binding. Transient downregulation of [‘H]QNB and [3H]PN200-l 10 binding, observed in the early reperfusion period, may play a role in protection against neuronal damage. Further studies should be conducted to reveal the mechanisms of the induction of tolerance.
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