Research in
Res Exp Med (1992) 192 :79-88
ExperimentalMedicine 9 Springer-Verlag 1992
Postischemic alteration of muscarinic acetylcholine and adenosine A1 binding sites in gerbil brain Protective effects of a novel vinca alkaloid derivative, vinconate, and pentobarbitai using an autoradiographic study Tsutomu Araki, Hiroyuki Kato, and Kyuya Kogure Department of Neurology,Institute of Brain Diseases, Tohoku UniversitySchoolof Medicine, 1-1 Seiryo-machi,Sendai, Japan Received June 25, 1991 / accepted December 3, 1991
Summary. We studied the alterations in the binding of muscarinic cholinergic and adenosine A1 receptors following~transient cerebral ischemia in Mongolian gerbils and examined the effects of the novel vinca alkaloid derivative vinconate and pentobarbital against the alteraltions in the binding of these receptors. Animals were allowed to survive for 5 h and 7 days after 10 min of cerebral ischemia induced by bilateral occlusion of common carotid arteries. [3H]Quinuclidinyl benzilate (QNB) and [3H]cyclohexyladenosine (CHA) were used to label muscarinic cholinergic and adenosine A1 receptors, respectively. The [3H]QNB and [3H]CHA bindings showed no significant alteration in the gerbil brain 5 h after ischemia. However, these bindings in the striatium, the hippocampal CA1 sector, and the hippocampal CA3 sector revealed a significant reduction 7 days after ischema. The [3H]CHA binding also showed a significant decline in the dentate molecular layer 7 days after ischemia, lntraperitoneal application of vinconate (100 and 300mg/kg) 10rain and pentobarbital (40mg/kg) 30rain before ischemia showed a mild reduction in the [3H]CHA binding in the brain 5 h after ischemia. Especially, the reduction was found in the hippocampal CA1 sector and the dentate molecular layer. However, the [3H]QNB binding revealed no significant alteration in the brain 5 h after ischemia. Seven days after ischemia, both drugs prevented a marked reduction in the [3H]CHA binding in the striatium, but not in the hippocampal CA1 sector, the hippocampal CA3 sector, and the dentate molecular layer. By contrast, vinconate and pentobarbital failed to prevent the reduction in the [3H]QNB binding in the striatum. Morphological study indicated that vinconate and pentobarbital ameliorated the neuronal damage to the striatum, but not the hippocampal damage 7 days after ischemia. This histological finding was relatively consistent with the alteration in the [3H]CHA binding. These receptor autoradiographic and histological data suggest that vinconate and pentobarbital can protect the brain from both cellular and functional Offprint requests to: T. Araki
80 c o n s e q u e n c e s of ischemia. These findings are of interest in relation to the mechanisms of ischemic brain damage.
Key words: Acetylcholine - A d e n o s i n e A1 - R e c e p t o r a u t o r a d i o g r a p h y - Cerebral ischemia - Vinconate - Pentobarbital - Gerbil
Introduction It is well k n o w n that transient cerebral ischemia produces selectively n e u r o n a l d a m a g e in specific brain areas [1, 3, 8, 20]. The dorsolateral part of striatum and the h i p p o c a m p a l C A 1 sector are most vulnerable to the ischemic insult. We recently r e p o r t e d that the novel vinca alkaloid derivative vinconate and p e n t o b a r bital can offer the n e u r o n a l protection of the striatum after transient ischemia, but these drugs cannot protect the d a m a g e to the h i p p o c a m p a l C A 1 sector [2, 4]. H o w e v e r , the precise m e c h a n i s m for such effects is not fully u n d e r s t o o d . Acetylcholine is a m a j o r n e u r o t r a n s m i t t e r in the central nervous system and is t h o u g h t to contribute to m e m o r y and cognitive processes [7]. In cerebrovascular accident and A l z h e i m e r ' s disease, therefore, the d a m a g e to cholinergic systems is related to m e m o r y and cognitive impairments. A d e n o s i n e plays neurom o d u l a t o r y roles in the central nervous system [24]. Especially, adenosine A1 receptors inhibit adenylate cyclase activity and mediate excitatory neuronal pathways by reducing n e u r o n a l activity [26]. A n a u t o r a d i o g r a p h y study suggests that the acetylcholine and adenosine A~ receptors were very rich in the striatum and the h i p p o c a m p u s [18]. T h e r e f o r e , using r e c e p t o r a u t o r a d i o g r a p h y , the present study was u n d e r t a k e n to elucidate alterations in the binding of the acetylcholine and adenosine A1 receptors after transient cerebral ischemia in the gerbil and to examine the effects of vinconate and pentobarbital on these alterations.
Materials and methods Male adult Mongolian gerbils weighing 65-95 kg were used. They were anesthetized with 2% halothane in a mixture of 70% N20 and 30% 02. Bilateral common carotid arteries were exposed and anesthesia was discontinued to minimize its effect. The bilateral common carotid arteries were clamped by aneurysmal clips for 10min. Vinconate ((_+)-methyl 3-ethyl-2,3,3a,4tetrahydroqH-indolo [3,2,1-de] [1,5]naphtyridine-6-carboxylate monohydrochloride)was given i.p. 10 min before ischemia. Pentobarbital was given i.p. 30 min before ischemia. Vehicle (distilled water) was administered i.p. 10 min before ischemia. Body temperature was maintained at 37-39~ using a heating pad with thermostat throughout the experiment. Sham-operated animals were treated in the same manner except for clipping of the bilateral common carotid arteries and treatment with drug. For receptor autoradiography, the animals were decapitated 5 h and 7 days after ischemia. The brains were removed quickly, frozen in powdered dry-ice, and stored at -80~ Coronal sections 12 ~tm in thickness were cut on a cryostat at -20~ and thaw-mounted onto gelatin-coated slides. Adjacent sections were stained with Cresyl violet and used for histopathology.
Receptor autoradiography Muscarinie cholinergic receptor, Muscarinic cholinergic receptors were quantified using the [3H]quinuclidinyl benzilate ([3H]QNB, spect, act. 41.5 Ci/mmol, Amersham, International plc, Buckingham, UK) according to the method of Onodera et al. [18]. Sections were incubated
81 with lnM [3H]QNB in phosphate buffer (pH 7.4) at room temperature for 90 min. The slides were then washed in the buffer at 4~ for 5 min. Non-specific binding was determined using 1 gM atropine (Sigma, Chemical Company, St. Louis, USA).
AdenosineA1 receptor.Adenosine A1 receptors were visualized using [3H]cyclohexyl-adenosine ([3H]CHA, spect, act. 34.4 Ci/mmol, Dupon NEN Products, Boston, USA) according to the method of Onodera et al. [18]. Sections were incubated with 5 nM [3H]CHA and 2 units/ml adenosine deaminase (Boehringer-Mannheim, Mannheim, FRG) in 50mM Tris-HC1 buffer (pH 7.4) at room temperature for 90 min. The slides were then washed in the buffer at 4~ for 5min. Non-specific binding was determined using 10gM L-phenylisopropyl-adenosine (Boehringer-Mannheim). Slides were dried under a cold stream of air and apposed to HyperfilmJH (Amersham, Sweden AB, Solna, Sweden) for 2-3 weeks in X-ray cassettes with a set of tritium standards ([3H]micro-scales, Amersham, International plc, Buckingham, UK). Optical density of the brain regions was measured with a computer-assisted limage analyzer (Zeiss, IBAS image analyzer system, FRG) without the examiner knowing the experimental protocol. The relationship between optical density and radioactivity was obtained with reference to the [3H]microscales co-exposed with the tissue sections using a third-order polynomial function. The optical density of the brain regions measured in the present study was in the range where optical density and radioactivity of the [3H]micro-scales showed a near linear relationship. A possible drawback of quantitative autoradiography may be a change in quenching level after ischemia. As discussed previously [18], there was no need to make a quench correction due to of increased gliosis. The affinity constant (Kd) and the maximal number of receptor sites (Bmax) in adult gerbils were 1.6 nM and 3 500 fmol/mg protein, respectively, for [3H]QNB, and 1.1 nM and 560 fmol/mg protein, respectively, for [3H]CHA [18]. Each group contained 5-7 animals. Binding assays were performed in duplicate. Values were expressed as means _+_SD. Statistical comparisons were made using Duncan's multiple range test. Results
Receptor autoradiography [3H]QNB binding. Postischemic alteration of [3H]QNB binding sites is summarized in Table 1. Representative autoradiograms are shown in Fig. 1. In sham-operated gerbils, the highest [3H]QNB binding sites was noticed in the striatum and the hippocampus. However, the dentate molecular layer of the hippocampus had relatively low grain density. At 5 h after ischemia, the striatum and the hippocampus in the ischemia-replacing vehicle group showed no significant alteration in the [3H]QNB binding sites. At 7 days after ischemia, the striatum, the hippocampal CA1 sector, and the hippocampal CA3 sector in the ischemia-replacing vehicle group showed a marked reduction in [3H]QNB binding. Administration of vinconate caused no significant alteration in the [3H]QNB binding in various brain regions 5 h and 7 days after ischemia. However, vinconate (100 mg/kg) significantly preventes a decrease in [3H]QNB binding only in the stratum lacunosum-moleculare of the hippocampal CA1 sector 7 days after ischemia. The administration of pentobarbital also showed no signficiant change in [3H]QNB binding in all areas 5 h after ischemia. By contrast, this drug significantly prevented [3H]QNB binding reduction in the stratum radiatum and lacunosum-moleculare of the hippocampal CA1 and CA3 sectors 7 days after ischemia. H o w e v e r , pentobarbital failed to prevent a decrease in [3H]QNB binding in the striatum and stratum oriens of the hippocampal C A I sector.
[SH]CHA binding. Postischemic alteration of [3H]CHA binding sites is summarized in Table 2. Representative autoradiograms are shown in Fig. 2.
82 Table 1. Effect of vinconate and pentobarbital against [3H]QNB bindings in the gerbil brain
after transient cerebral ischemia Sham-ope
Ischemiareplacing vehicle
Vinconate 100mg/kg
300mg/kg
Pentobarbital 40 mg/kg
5 h after ischernia Striatum lateral
518•
84**
485•
63
495•
506•
548•
medial
534•
85**
496•
58
496+77
508•
543+64
CA1 sector stratum oriens stratum radiatum stratum lacunosummoleculare
528_+109"* 586• 56** 479• 62*
496• 541• 432•
41 85 53
487• 577• 461280
478• 549• 436259
492• 584• 476+42
CA3 sector average
410 •
40**
401 •
25
409 _+_24
395 + 29
392 _+48
Dentate molecular layer
598•
46
535•
61
550•
537•
561+51
lateral
372_+ 69
370•
403+58
444_+89
medial
374 + 151
416 • 77
427 • 48
479 + 86
CA1 sector stratum oriens stratum radiatum stratum lacunosummoleculare
400+ 34 499_+ 18 410 + 37
434• 548• 467 _+37*
445+28 512+44 453 + 41
461+38 565+36" 480 +_33*
CA3 sector average
274 _+ 64
336 • 76
343 • 48
404 • 38**
Dentate molecular layer
582_+ 20
596•
565+35
620+35
Hippocampus
7 days after ischemia Striatum
Hippocampus
Optical densities were converted to fmol/mg tissue using [3H] micro-scales. Values are expressed as means _+ SD. Striatum lateral, the dorsolateral part of the striatum; striatum medial, the ventromedial part of the striatum, n = 5-7. * P < 0.05, ** P < 0.01 vs ischemia-replacing vehicle (5h after ischemia), *P
Res Exp Med (1992) 192 :79-88
ExperimentalMedicine 9 Springer-Verlag 1992
Postischemic alteration of muscarinic acetylcholine and adenosine A1 binding sites in gerbil brain Protective effects of a novel vinca alkaloid derivative, vinconate, and pentobarbitai using an autoradiographic study Tsutomu Araki, Hiroyuki Kato, and Kyuya Kogure Department of Neurology,Institute of Brain Diseases, Tohoku UniversitySchoolof Medicine, 1-1 Seiryo-machi,Sendai, Japan Received June 25, 1991 / accepted December 3, 1991
Summary. We studied the alterations in the binding of muscarinic cholinergic and adenosine A1 receptors following~transient cerebral ischemia in Mongolian gerbils and examined the effects of the novel vinca alkaloid derivative vinconate and pentobarbital against the alteraltions in the binding of these receptors. Animals were allowed to survive for 5 h and 7 days after 10 min of cerebral ischemia induced by bilateral occlusion of common carotid arteries. [3H]Quinuclidinyl benzilate (QNB) and [3H]cyclohexyladenosine (CHA) were used to label muscarinic cholinergic and adenosine A1 receptors, respectively. The [3H]QNB and [3H]CHA bindings showed no significant alteration in the gerbil brain 5 h after ischemia. However, these bindings in the striatium, the hippocampal CA1 sector, and the hippocampal CA3 sector revealed a significant reduction 7 days after ischema. The [3H]CHA binding also showed a significant decline in the dentate molecular layer 7 days after ischemia, lntraperitoneal application of vinconate (100 and 300mg/kg) 10rain and pentobarbital (40mg/kg) 30rain before ischemia showed a mild reduction in the [3H]CHA binding in the brain 5 h after ischemia. Especially, the reduction was found in the hippocampal CA1 sector and the dentate molecular layer. However, the [3H]QNB binding revealed no significant alteration in the brain 5 h after ischemia. Seven days after ischemia, both drugs prevented a marked reduction in the [3H]CHA binding in the striatium, but not in the hippocampal CA1 sector, the hippocampal CA3 sector, and the dentate molecular layer. By contrast, vinconate and pentobarbital failed to prevent the reduction in the [3H]QNB binding in the striatum. Morphological study indicated that vinconate and pentobarbital ameliorated the neuronal damage to the striatum, but not the hippocampal damage 7 days after ischemia. This histological finding was relatively consistent with the alteration in the [3H]CHA binding. These receptor autoradiographic and histological data suggest that vinconate and pentobarbital can protect the brain from both cellular and functional Offprint requests to: T. Araki
80 c o n s e q u e n c e s of ischemia. These findings are of interest in relation to the mechanisms of ischemic brain damage.
Key words: Acetylcholine - A d e n o s i n e A1 - R e c e p t o r a u t o r a d i o g r a p h y - Cerebral ischemia - Vinconate - Pentobarbital - Gerbil
Introduction It is well k n o w n that transient cerebral ischemia produces selectively n e u r o n a l d a m a g e in specific brain areas [1, 3, 8, 20]. The dorsolateral part of striatum and the h i p p o c a m p a l C A 1 sector are most vulnerable to the ischemic insult. We recently r e p o r t e d that the novel vinca alkaloid derivative vinconate and p e n t o b a r bital can offer the n e u r o n a l protection of the striatum after transient ischemia, but these drugs cannot protect the d a m a g e to the h i p p o c a m p a l C A 1 sector [2, 4]. H o w e v e r , the precise m e c h a n i s m for such effects is not fully u n d e r s t o o d . Acetylcholine is a m a j o r n e u r o t r a n s m i t t e r in the central nervous system and is t h o u g h t to contribute to m e m o r y and cognitive processes [7]. In cerebrovascular accident and A l z h e i m e r ' s disease, therefore, the d a m a g e to cholinergic systems is related to m e m o r y and cognitive impairments. A d e n o s i n e plays neurom o d u l a t o r y roles in the central nervous system [24]. Especially, adenosine A1 receptors inhibit adenylate cyclase activity and mediate excitatory neuronal pathways by reducing n e u r o n a l activity [26]. A n a u t o r a d i o g r a p h y study suggests that the acetylcholine and adenosine A~ receptors were very rich in the striatum and the h i p p o c a m p u s [18]. T h e r e f o r e , using r e c e p t o r a u t o r a d i o g r a p h y , the present study was u n d e r t a k e n to elucidate alterations in the binding of the acetylcholine and adenosine A1 receptors after transient cerebral ischemia in the gerbil and to examine the effects of vinconate and pentobarbital on these alterations.
Materials and methods Male adult Mongolian gerbils weighing 65-95 kg were used. They were anesthetized with 2% halothane in a mixture of 70% N20 and 30% 02. Bilateral common carotid arteries were exposed and anesthesia was discontinued to minimize its effect. The bilateral common carotid arteries were clamped by aneurysmal clips for 10min. Vinconate ((_+)-methyl 3-ethyl-2,3,3a,4tetrahydroqH-indolo [3,2,1-de] [1,5]naphtyridine-6-carboxylate monohydrochloride)was given i.p. 10 min before ischemia. Pentobarbital was given i.p. 30 min before ischemia. Vehicle (distilled water) was administered i.p. 10 min before ischemia. Body temperature was maintained at 37-39~ using a heating pad with thermostat throughout the experiment. Sham-operated animals were treated in the same manner except for clipping of the bilateral common carotid arteries and treatment with drug. For receptor autoradiography, the animals were decapitated 5 h and 7 days after ischemia. The brains were removed quickly, frozen in powdered dry-ice, and stored at -80~ Coronal sections 12 ~tm in thickness were cut on a cryostat at -20~ and thaw-mounted onto gelatin-coated slides. Adjacent sections were stained with Cresyl violet and used for histopathology.
Receptor autoradiography Muscarinie cholinergic receptor, Muscarinic cholinergic receptors were quantified using the [3H]quinuclidinyl benzilate ([3H]QNB, spect, act. 41.5 Ci/mmol, Amersham, International plc, Buckingham, UK) according to the method of Onodera et al. [18]. Sections were incubated
81 with lnM [3H]QNB in phosphate buffer (pH 7.4) at room temperature for 90 min. The slides were then washed in the buffer at 4~ for 5 min. Non-specific binding was determined using 1 gM atropine (Sigma, Chemical Company, St. Louis, USA).
AdenosineA1 receptor.Adenosine A1 receptors were visualized using [3H]cyclohexyl-adenosine ([3H]CHA, spect, act. 34.4 Ci/mmol, Dupon NEN Products, Boston, USA) according to the method of Onodera et al. [18]. Sections were incubated with 5 nM [3H]CHA and 2 units/ml adenosine deaminase (Boehringer-Mannheim, Mannheim, FRG) in 50mM Tris-HC1 buffer (pH 7.4) at room temperature for 90 min. The slides were then washed in the buffer at 4~ for 5min. Non-specific binding was determined using 10gM L-phenylisopropyl-adenosine (Boehringer-Mannheim). Slides were dried under a cold stream of air and apposed to HyperfilmJH (Amersham, Sweden AB, Solna, Sweden) for 2-3 weeks in X-ray cassettes with a set of tritium standards ([3H]micro-scales, Amersham, International plc, Buckingham, UK). Optical density of the brain regions was measured with a computer-assisted limage analyzer (Zeiss, IBAS image analyzer system, FRG) without the examiner knowing the experimental protocol. The relationship between optical density and radioactivity was obtained with reference to the [3H]microscales co-exposed with the tissue sections using a third-order polynomial function. The optical density of the brain regions measured in the present study was in the range where optical density and radioactivity of the [3H]micro-scales showed a near linear relationship. A possible drawback of quantitative autoradiography may be a change in quenching level after ischemia. As discussed previously [18], there was no need to make a quench correction due to of increased gliosis. The affinity constant (Kd) and the maximal number of receptor sites (Bmax) in adult gerbils were 1.6 nM and 3 500 fmol/mg protein, respectively, for [3H]QNB, and 1.1 nM and 560 fmol/mg protein, respectively, for [3H]CHA [18]. Each group contained 5-7 animals. Binding assays were performed in duplicate. Values were expressed as means _+_SD. Statistical comparisons were made using Duncan's multiple range test. Results
Receptor autoradiography [3H]QNB binding. Postischemic alteration of [3H]QNB binding sites is summarized in Table 1. Representative autoradiograms are shown in Fig. 1. In sham-operated gerbils, the highest [3H]QNB binding sites was noticed in the striatum and the hippocampus. However, the dentate molecular layer of the hippocampus had relatively low grain density. At 5 h after ischemia, the striatum and the hippocampus in the ischemia-replacing vehicle group showed no significant alteration in the [3H]QNB binding sites. At 7 days after ischemia, the striatum, the hippocampal CA1 sector, and the hippocampal CA3 sector in the ischemia-replacing vehicle group showed a marked reduction in [3H]QNB binding. Administration of vinconate caused no significant alteration in the [3H]QNB binding in various brain regions 5 h and 7 days after ischemia. However, vinconate (100 mg/kg) significantly preventes a decrease in [3H]QNB binding only in the stratum lacunosum-moleculare of the hippocampal CA1 sector 7 days after ischemia. The administration of pentobarbital also showed no signficiant change in [3H]QNB binding in all areas 5 h after ischemia. By contrast, this drug significantly prevented [3H]QNB binding reduction in the stratum radiatum and lacunosum-moleculare of the hippocampal CA1 and CA3 sectors 7 days after ischemia. H o w e v e r , pentobarbital failed to prevent a decrease in [3H]QNB binding in the striatum and stratum oriens of the hippocampal C A I sector.
[SH]CHA binding. Postischemic alteration of [3H]CHA binding sites is summarized in Table 2. Representative autoradiograms are shown in Fig. 2.
82 Table 1. Effect of vinconate and pentobarbital against [3H]QNB bindings in the gerbil brain
after transient cerebral ischemia Sham-ope
Ischemiareplacing vehicle
Vinconate 100mg/kg
300mg/kg
Pentobarbital 40 mg/kg
5 h after ischernia Striatum lateral
518•
84**
485•
63
495•
506•
548•
medial
534•
85**
496•
58
496+77
508•
543+64
CA1 sector stratum oriens stratum radiatum stratum lacunosummoleculare
528_+109"* 586• 56** 479• 62*
496• 541• 432•
41 85 53
487• 577• 461280
478• 549• 436259
492• 584• 476+42
CA3 sector average
410 •
40**
401 •
25
409 _+_24
395 + 29
392 _+48
Dentate molecular layer
598•
46
535•
61
550•
537•
561+51
lateral
372_+ 69
370•
403+58
444_+89
medial
374 + 151
416 • 77
427 • 48
479 + 86
CA1 sector stratum oriens stratum radiatum stratum lacunosummoleculare
400+ 34 499_+ 18 410 + 37
434• 548• 467 _+37*
445+28 512+44 453 + 41
461+38 565+36" 480 +_33*
CA3 sector average
274 _+ 64
336 • 76
343 • 48
404 • 38**
Dentate molecular layer
582_+ 20
596•
565+35
620+35
Hippocampus
7 days after ischemia Striatum
Hippocampus
Optical densities were converted to fmol/mg tissue using [3H] micro-scales. Values are expressed as means _+ SD. Striatum lateral, the dorsolateral part of the striatum; striatum medial, the ventromedial part of the striatum, n = 5-7. * P < 0.05, ** P < 0.01 vs ischemia-replacing vehicle (5h after ischemia), *P
