33
Brain Research, 553 (1991) 33-38 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391167383 BRES 16738
Sequential changes in muscarinic acetylcholine, adenosine A 1 and calcium antagonist binding sites in the gerbil hippocampus following repeated brief ischemia Hiroyuki Kato, Tsutomo Araki, Hideaki Hara and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan) (Accepted 22 January 1991) Key words: Cerebral ischemia; Hippocampus; Gerbil; Autoradiography; Acetylcholine; Adenosine; Calcium antagonist
We performed quantitative autoradiography to determine sequential alterations in the binding of muscarinic cholinerglc and adenosine A 1 receptors and of an L-type calcium channel blocker in the gerbil hippocampus following repeated brief ischemic insults. [3H]Quinucfidinyl benzilate (QNB), [3H]cyclohexyladenosine (CHA) and [3H]PN200-110were used to label muscarinic and adnosine A 1 receptors and L-type calcium channels, respectively. Changes at 1 h, 6 h, 1 day, 4 days and 1 month after three 2-min ischemic insults were compared with changes after single 2- or 6-rain ischemia. Two-minute ischemia, which causes no histopathological neuronal damage, produced no persistent alterations in binding sites. We observed a transient and mild increase in binding activities, especially in [3H]CHA binding, at 1 h of recireulation. Following 6-rain ischemia and three 2-rain ischemic insults, [3H]QNB and [3H]PN200-110binding decreased by more than 50% in the CA1 subfield by 1 month, but [3H]CHA binding decreased transiently by 20-30% at 4 days when delayed neuronal death of hippocampal CA1 pyramidal cells took place. Reductions in binding, especially in [3H]QNB binding, following three 2-min ischemic insults were greater and appeared earlier than those after 6-rain ischemia. Furthermore, alterations extended to the CA3 subfield and the dentate gyms following repeated insults. Thus, alterations in receptor binding after repeated ischemic insults were greater than those after equivalent single period of ischemia.
INTRODUCTION We have recently demonstrated that brief and nonlethal cerebral ischemia can injure selectively vulnerable neurons when such ischemia is induced repeatedly at certain intervals 3'8,9. Two-minute bilateral c o m m o n carotid artery occlusion in the gerbil produces no histopathological neuronal damage in the brain, whereas 3 such occlusions at 1-h intervals consistently lead to severe destruction of hippocampal CA1 pyramidal neurons. Of interest is that the neuronal damage is most severe when ischemia is repeated at 1-h intervals and is relatively mild when repeated at shorter or longer intervals 9. This p h e n o m e n o n is consistent with other reports using different ischemia models 15"2°. However, the underlying mechanisms remain to be elucidated, although postischemic circulatory and metabolic disturbances, such as postischemic hypoperfusion which is maximal at 1 h after 2-min ischemia 11 and impairment of protein synthesis which is depressed for several hours after 2-min ischemia 2, as well as excitotoxic mechanisms 1°, may explain the cumulative damage.
Recent experimental evidence from our laboratory indicates that a decrease in binding of certain kinds of neurotransmitter receptors and calcium channel blockers precedes delayed neuronal death of hippocampal CA1 pyramidal cells following transient cerebral ischemia 17'19. Furthermore, it is possible that alterations in the receptor sensitivity and in calcium conductance take place when ischemia is repeated, resulting in the cumulative damage. The purpose of this study was, therefore, to reveal the sequential alterations in the binding of neurotransmitters, muscaxinic acetylcholine and adenosine A1, and of an L-type calcium channel blocker, PN200-110, in the gerbil hippocampus following three 2-rain ischemic insults in comparison with those following an equivalent single period of 6-min ischemia and following single 2-min ischemia. MATERIALS AND METHODS Induction of ischemia A total of 75 male Mongolian gerbils (Seiwa Experimental Animals, Fukuoka, Japan), aged 12-15 weeks and weighing 68-92 g, were used. They were allowed free access to food and water before and after surgery. Anesthesia was induced with 2% halo-
Correspondence: H. Kato, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendal 980, Japan.
34 thane 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. The arteries were then occluded with aneurysm clips. Anesthesia was discontinued when the clips were in place. Occlusion and reperfusion of the carotid arteries were verified by visual observation. Body temperature during surgery and ischemia was maintained at close to 37 °C except for second and third ischemic insults during which body temperatures were 0.60.8 *C higher than normal as reported previouslyTM. Animals were subjected to a 2-min occlusion, a 6-min occlusion or three 2-min occlusions at 1-h intervals. Animals subjected to 2- or 6-min ischemia were decapitated at 1 h, 6 h, 1 day, 4 days and 1 month after ischemia. Animals subjected to three 2-min ischemic insults were decapitated at 6 h, 1 day, 4 days and 1 month. Five control animals were also included. The brains were quickly removed and frozen in powdered dry ice. Coronal frozen sections, 15 /~m in thickness, were cut in a cryostat and thaw-mounted onto getatincoated cover-slips, and stored at -80 °C until assay. Adjacent sections were stained with Cresyl violet and used for histopathology.
different, left and right hemispheres were analyzed separately. Values were expressed as means + S.D. Statistical significance was analyzed using two-tailed Mann-Whitney U-test.
RESULTS
Histopathology CA1 p y r a m i d a l cells exhibited d e l a y e d neuronal death following 6-min ischemia and t h r e e 2-min ischemic insults. The CA1 neurons a p p e a r e d intact by light microscopy at 1 and 6 h and 1 day, but almost all p y r a m i d a l cells had been d e s t r o y e d 4 days and 1 month after ischemia. C A 3 p y r a m i d a l cells and d e n t a t e granule cells showed no visible d a m a g e at any time. Two-minute ischemia caused no d a m a g e in the hippocampus.
[3H]QNB binding Muscarinic acetylcholine receptors were quantified using the radiolabeled antagonist [3H]quinuclidinyl benzilate (QNB; 41.5 Ci/mmol, Amersham) as reported previously17a9. Sections were incubated with 1 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 rain at 4 °C. Non-specific bindings were determined using 1 gM atropine (Sigma).
[~H]CHA binding Adenosine A 1 receptors were measured using [3H]cyciohexyladenosine (CHA; 34.4 Ci/mmol, New England Nuclear) as described previously17'~9. Sections were incubated with 5 nM [3H]CHA and 2 units/ml adenosine deaminase (Boehringer-Mannheim) in 50 mM Tris-buffer (pH 7.4) for 90 min at room temperature. The sections were then washed in the buffer for 1 rain at 4 °C. Non-specific binding was determined using 10 /JM Lphenylisopropyladenosine (Boehringer-Mannheim).
[3H]PN200-110binding The method for the autoradiographic visualization of L-type calcium channel blocker binding using [3H]PN200-110, a 1,4dihydropyridine calcium channel blocker, has been described previously19. Sections were incubated with 0.1 nM [3H]PN200-110 (71.5 Ci/mmol, New England Nuclear) in 170 mM Tris-HCl buffer (pH 7.7) for 1 h at room temperature under subdued lighting. The sections were then washed in the buffer for 20 min at 4 °C. Non-specific bindings were determined using 1 gM nitrendipine (Sigma).
A utoradiography 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 computer-assisted image analyzer system (IBAS image analyzer system, 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. A possible drawback of quantitative autoradiography may be a change in quenching level after ischemia. As discussed previously17, there was no need to make a quench correction because of increased ghosis. The affinity constant (Kd) and the maximal number of receptor sites (B,~) in adult gerbils were 1.6 nM and 3500 fmoi/mg protein, respectively, for [3H]QNB, and 1.1 nM and 560 fmol/mg protein, respectively, for [3H]CHA17.
Statistics Each group contained 5 animals. Binding assays were performed in duplicate. Because many values of both hippocampi were
[3H] Q N B binding The CA1 subfield and the stratum moleculare of the d e n t a t e gyrus had the highest density of [3H]QNB receptors in control gerbils (Fig. 1 and Table I). T h e C A 3 subfield had the m o d e r a t e n u m b e r of the binding sites. Following 2-min ischemia, [3H]QNB binding sites in the hippocampus r e m a i n e d unchanged except for a slight reduction in the binding at 6 h and 1 d a y of recirculation and a slight increase in the C A 3 subfield at 1 m o n t h (Table I). Six minutes of ischemia caused a slight but significant increase in [3H]QNB binding in the C A 3 subfield and the d e n t a t e gyrus at 1 h. The density of [3H]QNB binding sites was u n a l t e r e d at 6 h and 1 day, but slightly (by 12%) d e c r e a s e d in the stratum oriens of CA1 at 4 days. [3H]QNB binding d e c r e a s e d to 45% of control in the C A 1 subfield by 1 m o n t h (Fig. 1, Table I). T h r e e 2-min ischemic insults caused a significant reduction in [3H]QNB binding in the stratum oriens of the CA1 subfield, the C A 3 subfield and in the d e n t a t e gyrus at as early as 6 h of recirculation. By 1 m o n t h , [3H]QNB binding decreased to 45% of control in the C A 1 subfield, and increased by 10% in the C A 3 subfield and the dentate gyrus (Fig. 1 and Table I).
[3H]CHA binding The dendritic fields of the CA1 and C A 3 subfields and the stratum moleculare of the d e n t a t e gyrus had high densities of [3H]CHA binding sites in control gerbils (Fig. 1, Table II). In contrast, the stratum p y r a m i d a l e had few binding sites. We found a transient increase by 1 5 - 2 5 % in [3H]CHA binding in the whole h i p p o c a m p u s 1 h after 2 and 6 min of ischemia (Table II). [3H]CHA binding again slightly (by 15%) increased in the CA1 at 4 days after 2-min ischemia, but had r e t u r n e d to control levels by 1 month. [3H]CHA binding in the CA1 subfield decreased by 30% 4 days after 6-rain ischemia, but h a d returned to control levels by 1 m o n t h (Table II). By 4
35
QNB
CHA
PN200-110
Fig. 1. Representative autoradiographs of [3H]QNB (a,d,g), [3H]CHA (b,e,h) and [3H]PN200-110 (c,f,i) binding in the gerbil hippocampus of control ( a - c ) , and 4 days ( d - 0 and 1 m o n t h (g-i) after three 2-min bilateral carotid artery occlusions. Note a conspicuous reduction in [3H]QNB and [3H]PN200-110 binding in the CA1 subfield at 1 m o n t h and relative preservation of [3H]CHA binding.
TABLE I
Sequential alterations in [JH]QNB binding (fraol/mg tissue) in the hippocampus following 2- and 6-rain ischemia and foUowing three 2-min ischemic insults Values are m e a n s + S.D., n = 9-10 hemispheres.
Control
1h
6h
1 day
4 days
1 month
515 + 33.2 566 + 41.3 450 + 34.5
495 + 60.3 544 + 68.1 446 + 63.2
418 + 51.3"* 546 + 55.0 458 + 54.2
446 ___57.3** 540 + 54.1 468 + 54.3
515 + 33.2 590 + 46.0 463 + 40.1
518 + 66.6 546 + 100.2 443 + 88.5
350 + 28.7
384 + 43.8
311 + 32.8*
312 ___44.2*
366 + 24.7
398 + 36.5**
542 + 24.8
552+57.6
487+36.2**
494+54.8
540+31.6
577+44.8
516 + 33.2 566 + 41.3 450 + 34.5
513+54.0 590 + 64.9 493 + 60.0
476+51.9 567 + 36.0 460 + 34.7
496+55.8 555 _+_68.8 453 + 64.7
454+54.0* 550 _ 45.4 464 + 44.3
232+30.1"* 254 + 30.1"* 209 + 16.7"*
350 + 28.7
429+43.2**
350+26.7
332+39.2
298+61.0
340+55.8
542 + 24.8
603+26.9**
516+32.7
530+45.8
546+44.5
572+63.6
515 _+ 33.2 566 + 41.3 450 + 34.5
428 + 49.5** 513 + 81.1 428 + 73.7
423 + 24.5** 511 + 50.7* 419 + 51.2
450 +_ 44.2** 536 + 42.4 444 + 35.8
224 + 31:3"* 254 + 35.5** 202 + 21.6"*
350 + 28.7
292 + 49.1"*
271 ___40.1"*
321 + 35.9
386 + 54.1"
542 + 24.8
458 + 68.8*
464 + 44.4**
543 + 42.5
591 + 65.2*
2-min ischemia C A 1 subfield Stratum oriens Stratum radiatum Stratum l a c u n o s u m moleculare C A 3 subfield Average D e n t a t e gyrus Stratum moleculare
6-min ischemia CA1 subfield Stratum oriens Stratum radiatum Stratum l a c u n o s u m moleculare C A 3 subfield Average D e n t a t e gyrus Stratum moleculare
3 x 2-rain ischemia CA1 subfield Stratum oriens Stratum radiatum Stratum l a c u n o s u m moleculare C A 3 subfield Average D e n t a t e gyrus Stratum moleculare
*P < 0.05, **P < 0.01 compared to control (two-tailed M a n n - W h i t n e y U-test).
36 TABLE II
Sequential alterations in [3H]CHA binding (fmol/mg tissue) in the hippocampus following 2- and 6-rain ischemia and following three 2-min ischemic insults Values are means + S.D., n = 8-10 hemispheres.
Control
Ih
6h
I day
4 days
I month
285 + 40.9 331 + 40.7 236 + 24.9
355 + 39.8** 419 + 51.7"* 269 + 33.8*
303 + 46.0 351 + 47.9 249 + 24.8
300 + 57.3 328 + 72.5 248 + 67.3
325 + 33.1" 383 - 50.8* 242 + 39.4
323 + 25.9 371 + 50.7 258 + 38.0
225 + 31.6 274 + 38.4
257 + 28.8 320 + 27.9*
239 + 16.0 279 + 27.4
225 + 35.6 264 + 58.2
247 + 18.4 302 + 40.9
231 + 27.5 285 + 46.3
227 _+ 17.1
257 + 21.6"*
246 + 17.8"
231 + 44.0
236 + 35,4
232 + 27.8
285 + 40.9 331 _+40.7 236 _+ 24.9
355 + 55.8* 416 _+ 81.7"* 265 -+ 44.2
298 + 46.4 343 + 51.2 255 + 51.3
289 _+ 64.7 341 _+ 83.5 238 + 46.8
203 + 47.3** 229 + 54,8** 191 + 46.8*
283 _+ 96.3 305 _+ 125.5 210 + 65.0*
225 _+ 31.6 274 + 38.4
262 -+ 41.9" 319 -+ 54.8
241 -+ 38.7 277 + 38.0
229 _+ 40.7 273 _+ 55.7
229 + 42.6 282 + 62.9
225 _+ 64.5 298 _+ 71.3
227 _+ 17.7
263 -+ 45.3*
248 -+ 42.7
241 + 49.6
223 +_ 47.8
258 + 43.0
285 _+ 40.9 331 __-40.7 236 _ 24.9
268 +_ 43.4 325 -- 21.7 232 _+ 24.9
280 -+ 68.4 340 _+ 66.8 239 _+ 36.2
216 + 32.8** 256 + 37.9** 185 _+ 17.1 **
355 + 52.1 ** 400 + 75.8 210 _+ 29.6*
225 + 31.7 274 __-38.4
232 + 34.2 273 + 25.1
235 _+ 30.3 295 + 54.2
217 + 30.8 280 + 43.4
270 + 37.0* 344 + 17.4"*
227 _+ 17.7
226 + 24.7
243 + 34.4
215 _+ 27.2
276 + 27.2**
2-min ischemia CA1 subfield Stratum oriens Stratum radiatum Stratum laeunosum moleculare CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum moleeulare
6-min ischemia CA1 subfield Stratum oriens Stratum radiatum Stratum lacunosum moleculare CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum moleculare
3 x 2-min ischemia CA1 subfield Stratum oriens Stratum radiatum Stratum lacunosum moleculare CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum moleculare
*P < 0.05, **P < 0.01 compared to control (two-tailed Mann-Whitney U-test).
TABLE III
Sequential alterations in f H] PN200-110 binding (fmol/mg tissue) in the hippocampus following 2- and 6-rain ischemia and following three 2-min iscemic insults Values are means + S.D., n = 9-10 hemispheres.
Control
1h
6h
1 day
4 days
1 month
10 + 2.5 16 + 2.5 20 + 5.4
12 + 2.5 18 + 4.4 23 + 4.0
12 + 1.4 17 + 3.4 24 + 3.1"
10 + 2.5 15 + 2.9 20 + 4.3
11 + 2.3 16 + 2.9 22 + 4.3
11 + 2.8 18 + 5.8 23 + 3.5
10 + 2.5 16 + 2.5 20 + 5.4
12 + 1.6 20 _+_4.6 26 + 3.9*
9 +- 1.9 16 + 3.4 20 + 4.1
11 --- 2.1 16 + 2.4 22 + 4.3
10 + 2.5 14 + 2.5 19 + 5.0
4 ___1.6"* 17 + 5.9 28 + 8.4*
11 + 1.4 18 + 3.6 22 _+ 2.5
12 -+ 1.6 17 -+ 2.9 23 + 3.9
10 +_ 1.6 16 _+ 2.0 22 _+ 2.9*
4 _+ 1.5"* 21 + 6.0* 25 _+ 4.2*
2-min ischemia CA1 subfield CA3 subfield Dentate gyrus
6-rain ischemia CA1 subfield CA3 subfield Dentate gyrus
3 x 2-rain ischemia CA1 subfield CA3 subfield Dentate gyrus
10 +_ 2.5 16 _+ 2.5 20 _+ 5.4
*P < 0.05, **P < 0.01 compared to control (two-tailed Mann-Whitney U-test).
37 days after three 2-min ischemic insults, the density of [3H]CHA binding sites in the CA1 subfield decreased to 76% of control, but the binding was slightly increased at 1 month. [3H]CHA binding in the CA3 subfield and the dentate gyrus also increased at 1 month by more than 20% (Table II).
[3H]PN200-110binding There was a high density of [3H]PN200-110 binding sites in the stratum moleculare of the dentate gyrus in control gerbils (Fig. 1, Table III). The CA3 subfield also had a high density of the binding sites, but the CA1 subfield had a relatively low density. No significant alterations in [3H]PN200-110 binding were observed after 2-rain ischemia except for a transient increase at 6 h in the dentate gyrus (Table III). Six-minute ischemia caused a 30% increase in [3H]PN200-110 binding in the dentate gyrus at 1 h of recirculation. We also found a 60% reduction in the CA1 subfield and a 40% increase in the dentate gyrus at 1 month, but otherwise unaltered (Table III). Following three 2-min ischemic insults, [3H]PN200110 binding decreased to 40% of control in the CA1 subfield by 1 month (Fig. 1). We noted a 25% increase in [3H]PN200-110 binding in the CA3 subfield and the dentate gyrus at 1 month (Table III). DISCUSSION Previous reports have shown the temporal profile of histopathological changes in the gerbil hippocampus following transient ischemia and repeated ischemic insults 1'8'9A2'13. Two minutes of ischemia produces no neuronal damage in the hippocampus. However, CA1 pyramidal cells are selectively destroyed at 2--4 days after three 2-min ischemic insults and after 5-10 min of ischemia (delayed neuronal death12), whereas neurons in the CA3 subfield and the dentate gyrus are not damaged. Thus, histopathological observations in this study confirmed the previous results. [3H]QNB and [3H]PN200-110 binding in the CA1 subfield decreased to 45 and 40% of control, respectively, 1 month after 6-min ischemia and three 2-min ischemic insults. However, at 4 days when CA1 pyramidal cells had been destroyed, a significant decrease in [3H]QNB binding was observed only in the stratum oriens (by 13%), and a reduction in [3H]PN200-110 binding was not observed. Therefore, recognition sites labeled by [3H]QNB and [3H]PN200-110 may be resistant to degradation processes. Furthermore, because almost all CA1 pyramidal cells are destroyed following 6-min ischemia and following three 2-min ischemic insults, [3H]QNB and [3H]PN200-110 binding sites in the CA1 subfield for up to 40--45% of control may be localized on
interneurons and presynaptic sites which are resistant to ischemic insult6'7'14. In contrast, [3H]CHA binding in the CA1 subfield decreased to 70-80% of control at 4 days after 6-min ischemia and after three 2-min ischemic insults when CA1 pyramidal cells were destroyed, but the binding had returned to control level 1 month after 6-min ischemia and even increased after three 2-min ischemic insults. The absence of reductions in [3H]CHA binding in the CA1 subfield at 1 month is incompatible with previous reports which showed a significant loss of [3H]CHA binding sites in the CA1 subfield following 15 min ischemia in gerbils 16'17. However, 15-rain ischemia is severe enough to cause damage to CA3 pyramidal cells which terminate in the CA1 region (Schaffer collaterals) as presynaptic sites TM. Taken together, many [3H]CHA binding sites may be located on cells other than CA1 pyramidal neurons such as presynaptic sites, interneurons, blood vessels and glial cells. [3H]QNB, [3H]CHA and [3H]PN200-110 binding increased in the CA3 subfield and the dentate gyrus at 1 month after three 2-min ischemic insults. Such an increase was not observed after 6-min ischemia except for an increase in [3H]PN200-110 binding in the dentate gyrus. The physiological meaning of the increase at 1 month is difficult to explain in the present study. Elimination of CA1 pyramidal cells may play a role to induce synaptic modification of the neurotransmitter system resulting in an increase in receptor density on surviving neurons or glial cells. An increase in receptor density can take place after lesioning of neuronal circuits5A8. Increase in [3H]CHA binding in the CA1 subfield which was observed 4 days after 2-min ischemia and 1 month after three 2-min ischemic insults may also be explained by the synaptic modification of this inhibitory neurotransmitter system. Among 3 ligands used, [3H]QNB binding was most susceptible to ischemic insult. The binding decreased as early as 6 h after three 2-min ischemic insults. Thus, the decrease in [3H]QNB binding preceded delayed neuronal death of CA1 pyramidal cells following repeated insults as reported previously in transient ischemia 17'19. Furthermore, [3H]QNB binding transiently decreased after 2-min ischemia which causes no morphological neuronal damage. One of the unique features of neuronal damage following repeated ischemic insults is that the damage is most severe when ischemia is repeated at 1-h intervals9' 15.20. This phenomenon may be explained by the alterations in cerebral blood flow and metabolism which is maximally disturbed 1 h after ischemiaTM. In this study, we found a transient increase in [3H]QNB, [3H]PN200110, and especially [3H]CHA binding in the hippoeampus
38 1 h after 2- or 6-min ischemia. The increase may reflect the possible alterations in receptor sensitivity and calcium conduction. Therefore, these changes in receptor binding might explain the mechanism of the cumulative damage following repeated ischemic insults which is most severe at 1-h intervals. Patterns of alterations in the binding activities after three 2-min ischemic insults were different from that after 6-min ischemia. Reductions in [3H]QNB binding after three 2-min insults were greater and earlier that those after 6-min ischemia. Furthermore, changes were extended to the CA3 subfield and the dentate gyrus after repeated ischemic insults. These observations may further demonstrate the cumulative effect of neuronal damage after three 2-min ischemic insults which is greater than that after 6-rain ischemia 9. In conclusion, the present study showed the sequential alterations in binding sites of two major neurotransmitters, muscarinic acetylcholine and adenosine ml, and of a dihydropyridine calcium antagonist following three 2-min ischemic insults in comparison with those following 2 or 6 min ischemia. Findings observed were: (1) 2-min
REFERENCES 1 Araki, T., Kato, H. and Kogure, K., Selective neuronal vulnerability following transient cerebral ischemia in the gerbil: distribution and time course, Acta Neurol. Scand., 80 (1989) 548-553. 2 Araki, T., Kato, H., Inoue, T. and Kogure, K., Regional impairment of protein synthesis following brief cerebral ischemia in the gerbil, Acta Neuropathol., 79 (1990) 501-505. 3 Araki, T., Kato, H. and Kogure, K., Neuronal damage and calcium accumulation following brief cerebral ischemia in the gerbil, Brain Research, 588 (1990) 114-122. 4 Araki, T., Kato, H., Inoue, T. and Kogure, K., Long-term observations on calcium accumulation in postischemic gerbil brain, Acta Neurol. Scand., in press. 5 Block, G.A., Buchan, A.M. and Pulsinelli, W.A., Alteration of A1 adenosine receptor density following transient forebrain ischemia and fimbria/fornix lesions, Soc. Neurosci. Abstr., 13 (1987) 1080. 6 Francis, A. and Pulsinelli, W.A., Response of GABAergic and cholinergic neurons to transient cerebral ischemia, Brain Research, 243 (1982) 271-278. 7 Johansen, EE, Jorgensen, M.B. and Diemer, N.H., Resistance of hippocampal CA-1 interneurons to 20 min of transient cerebral ischemia in the rat, Acta Neuropathol., 61 (1983) 135-140. 8 Kato, H., Kogure, K. and Nakano, S., Neuronal damage following repeated brief ischemia in the gerbil, Brain Research, 479 (1989) 366-370. 9 Kato, H. and Kogure, K., Neuronal damage following nonlethal but repeated cerebral isehemia in the gerbil, Acta Neuropathol., 79 (1990) 494-500. 10 Kato, H., Araki, T. and Kogure, K., Role of the excitotoxic mechanisms in the development of neuronal damage following repeated cerebral ischemia in the gerbil: protective effects of
ischemia, which is brief enough to permit recovery without any morphological damage, produced no persistent alterations in binding activities; (2) [3H]QNB and [3H]PN200-110 binding decreased by more than 50% in the CA1 subfield by 1 month after elimination of CA1 pyramidals cells by ischemia. In contrast, [3H]CHA binding in the CA1 subfield decreased by 20-30% transiently at 4 days although CA1 pyramidal neurons were destroyed; (3) there was a transient and mild increase in binding activities, especially in [3H]CHA binding, at 1 h of recirculation, which may be one of the reasons why neuronal damage following repeated brief ischemic insults is most severe at 1-h intervals; and (4) alterations in binding activities, especially in [3H]QNB binding, following three 2-min ischemic insults were greater and appeared earlier than those after 6-min ischemia. Furthermore, alterations extended to the CA3 subfield and the dentate gyrus following repeated insults. Thus, alterations in receptor binding after repeated ischemic insults were greater than those after equivalent single period of ischemia.
MK-801 and pentobarbital, Brain Research, 516 (1990) 175-179. 11 Kato, H., Araki, T., Kogure, K., Murakami, M. and Uemura, K., Sequential cerebral blood flow changes in short-term cerebral ischemia in gerbils, Stroke, 21 (1990) 1346-1349. 12 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Research, 239 (1982) 57-69. 13 Kirino,T. and Sano, K., Selective vulnerability in the gerbil hippocampus following transient ischemia, Acta Neuropathol., 60 (1984) 207-216. 14 Kirino, T., Tamura, A. and Sano, K., Chronic maintenance of presynaptic terminals in gliotic hippocampus following ischemia, Brain Research, 510 (1990) 17-25. 15 Nakano, S., Kato, H., and Kogure, K., Neuronal damage in the rat hippocampus in a new model of repeated reversible transient cerebral ischemia, Brain Research, 490 (1989) 178-180. 16 Onodera, H. and Kogure, K., Autoradiographic visualization of adenosine A1 receptors in the gerbil hippocampus: changes in the receptor density after transient ischemia, Brain Research, 345 (1985) 406-408. 17 Onodera, H., Sato, G. and Kogure, K., Quantitative autoradiographic analysis of muscarinic cholinergic and adenosine A1 binding sites after transient forebrain ischemia in the gerbil, Brain Research, 415 (1987) 309-322. 18 Onodera, H., Sato, G. and Kogure, K., G A B A and benzodiazepine receptors in the gerbil brain after transient ischemia: demonstration by quantitative receptor autoradiography, J. Cereb. Blood Flow Metabol., 7 (1987) 82-88. 19 Onodera, H. and Kogure, K., Calcium antagonist, adenosine A1, and muscarinic binding in rat hippocampus after transient ischemia, Stroke, 21 (1990) 771-776. 20 Tomida, S., Nowak, Jr., T.S., Vass, K., Lohr, J.M. and Klatzo, I., Experimental model for repetitive ischemic attacks in the gerbil: the cumulative effect of repeated ischemic insults, J. Cereb. Blood Flow Metabol., 7 (1987) 773-782.
Brain Research, 553 (1991) 33-38 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391167383 BRES 16738
Sequential changes in muscarinic acetylcholine, adenosine A 1 and calcium antagonist binding sites in the gerbil hippocampus following repeated brief ischemia Hiroyuki Kato, Tsutomo Araki, Hideaki Hara and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan) (Accepted 22 January 1991) Key words: Cerebral ischemia; Hippocampus; Gerbil; Autoradiography; Acetylcholine; Adenosine; Calcium antagonist
We performed quantitative autoradiography to determine sequential alterations in the binding of muscarinic cholinerglc and adenosine A 1 receptors and of an L-type calcium channel blocker in the gerbil hippocampus following repeated brief ischemic insults. [3H]Quinucfidinyl benzilate (QNB), [3H]cyclohexyladenosine (CHA) and [3H]PN200-110were used to label muscarinic and adnosine A 1 receptors and L-type calcium channels, respectively. Changes at 1 h, 6 h, 1 day, 4 days and 1 month after three 2-min ischemic insults were compared with changes after single 2- or 6-rain ischemia. Two-minute ischemia, which causes no histopathological neuronal damage, produced no persistent alterations in binding sites. We observed a transient and mild increase in binding activities, especially in [3H]CHA binding, at 1 h of recireulation. Following 6-rain ischemia and three 2-rain ischemic insults, [3H]QNB and [3H]PN200-110binding decreased by more than 50% in the CA1 subfield by 1 month, but [3H]CHA binding decreased transiently by 20-30% at 4 days when delayed neuronal death of hippocampal CA1 pyramidal cells took place. Reductions in binding, especially in [3H]QNB binding, following three 2-min ischemic insults were greater and appeared earlier than those after 6-rain ischemia. Furthermore, alterations extended to the CA3 subfield and the dentate gyms following repeated insults. Thus, alterations in receptor binding after repeated ischemic insults were greater than those after equivalent single period of ischemia.
INTRODUCTION We have recently demonstrated that brief and nonlethal cerebral ischemia can injure selectively vulnerable neurons when such ischemia is induced repeatedly at certain intervals 3'8,9. Two-minute bilateral c o m m o n carotid artery occlusion in the gerbil produces no histopathological neuronal damage in the brain, whereas 3 such occlusions at 1-h intervals consistently lead to severe destruction of hippocampal CA1 pyramidal neurons. Of interest is that the neuronal damage is most severe when ischemia is repeated at 1-h intervals and is relatively mild when repeated at shorter or longer intervals 9. This p h e n o m e n o n is consistent with other reports using different ischemia models 15"2°. However, the underlying mechanisms remain to be elucidated, although postischemic circulatory and metabolic disturbances, such as postischemic hypoperfusion which is maximal at 1 h after 2-min ischemia 11 and impairment of protein synthesis which is depressed for several hours after 2-min ischemia 2, as well as excitotoxic mechanisms 1°, may explain the cumulative damage.
Recent experimental evidence from our laboratory indicates that a decrease in binding of certain kinds of neurotransmitter receptors and calcium channel blockers precedes delayed neuronal death of hippocampal CA1 pyramidal cells following transient cerebral ischemia 17'19. Furthermore, it is possible that alterations in the receptor sensitivity and in calcium conductance take place when ischemia is repeated, resulting in the cumulative damage. The purpose of this study was, therefore, to reveal the sequential alterations in the binding of neurotransmitters, muscaxinic acetylcholine and adenosine A1, and of an L-type calcium channel blocker, PN200-110, in the gerbil hippocampus following three 2-rain ischemic insults in comparison with those following an equivalent single period of 6-min ischemia and following single 2-min ischemia. MATERIALS AND METHODS Induction of ischemia A total of 75 male Mongolian gerbils (Seiwa Experimental Animals, Fukuoka, Japan), aged 12-15 weeks and weighing 68-92 g, were used. They were allowed free access to food and water before and after surgery. Anesthesia was induced with 2% halo-
Correspondence: H. Kato, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendal 980, Japan.
34 thane 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. The arteries were then occluded with aneurysm clips. Anesthesia was discontinued when the clips were in place. Occlusion and reperfusion of the carotid arteries were verified by visual observation. Body temperature during surgery and ischemia was maintained at close to 37 °C except for second and third ischemic insults during which body temperatures were 0.60.8 *C higher than normal as reported previouslyTM. Animals were subjected to a 2-min occlusion, a 6-min occlusion or three 2-min occlusions at 1-h intervals. Animals subjected to 2- or 6-min ischemia were decapitated at 1 h, 6 h, 1 day, 4 days and 1 month after ischemia. Animals subjected to three 2-min ischemic insults were decapitated at 6 h, 1 day, 4 days and 1 month. Five control animals were also included. The brains were quickly removed and frozen in powdered dry ice. Coronal frozen sections, 15 /~m in thickness, were cut in a cryostat and thaw-mounted onto getatincoated cover-slips, and stored at -80 °C until assay. Adjacent sections were stained with Cresyl violet and used for histopathology.
different, left and right hemispheres were analyzed separately. Values were expressed as means + S.D. Statistical significance was analyzed using two-tailed Mann-Whitney U-test.
RESULTS
Histopathology CA1 p y r a m i d a l cells exhibited d e l a y e d neuronal death following 6-min ischemia and t h r e e 2-min ischemic insults. The CA1 neurons a p p e a r e d intact by light microscopy at 1 and 6 h and 1 day, but almost all p y r a m i d a l cells had been d e s t r o y e d 4 days and 1 month after ischemia. C A 3 p y r a m i d a l cells and d e n t a t e granule cells showed no visible d a m a g e at any time. Two-minute ischemia caused no d a m a g e in the hippocampus.
[3H]QNB binding Muscarinic acetylcholine receptors were quantified using the radiolabeled antagonist [3H]quinuclidinyl benzilate (QNB; 41.5 Ci/mmol, Amersham) as reported previously17a9. Sections were incubated with 1 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 rain at 4 °C. Non-specific bindings were determined using 1 gM atropine (Sigma).
[~H]CHA binding Adenosine A 1 receptors were measured using [3H]cyciohexyladenosine (CHA; 34.4 Ci/mmol, New England Nuclear) as described previously17'~9. Sections were incubated with 5 nM [3H]CHA and 2 units/ml adenosine deaminase (Boehringer-Mannheim) in 50 mM Tris-buffer (pH 7.4) for 90 min at room temperature. The sections were then washed in the buffer for 1 rain at 4 °C. Non-specific binding was determined using 10 /JM Lphenylisopropyladenosine (Boehringer-Mannheim).
[3H]PN200-110binding The method for the autoradiographic visualization of L-type calcium channel blocker binding using [3H]PN200-110, a 1,4dihydropyridine calcium channel blocker, has been described previously19. Sections were incubated with 0.1 nM [3H]PN200-110 (71.5 Ci/mmol, New England Nuclear) in 170 mM Tris-HCl buffer (pH 7.7) for 1 h at room temperature under subdued lighting. The sections were then washed in the buffer for 20 min at 4 °C. Non-specific bindings were determined using 1 gM nitrendipine (Sigma).
A utoradiography 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 computer-assisted image analyzer system (IBAS image analyzer system, 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. A possible drawback of quantitative autoradiography may be a change in quenching level after ischemia. As discussed previously17, there was no need to make a quench correction because of increased ghosis. The affinity constant (Kd) and the maximal number of receptor sites (B,~) in adult gerbils were 1.6 nM and 3500 fmoi/mg protein, respectively, for [3H]QNB, and 1.1 nM and 560 fmol/mg protein, respectively, for [3H]CHA17.
Statistics Each group contained 5 animals. Binding assays were performed in duplicate. Because many values of both hippocampi were
[3H] Q N B binding The CA1 subfield and the stratum moleculare of the d e n t a t e gyrus had the highest density of [3H]QNB receptors in control gerbils (Fig. 1 and Table I). T h e C A 3 subfield had the m o d e r a t e n u m b e r of the binding sites. Following 2-min ischemia, [3H]QNB binding sites in the hippocampus r e m a i n e d unchanged except for a slight reduction in the binding at 6 h and 1 d a y of recirculation and a slight increase in the C A 3 subfield at 1 m o n t h (Table I). Six minutes of ischemia caused a slight but significant increase in [3H]QNB binding in the C A 3 subfield and the d e n t a t e gyrus at 1 h. The density of [3H]QNB binding sites was u n a l t e r e d at 6 h and 1 day, but slightly (by 12%) d e c r e a s e d in the stratum oriens of CA1 at 4 days. [3H]QNB binding d e c r e a s e d to 45% of control in the C A 1 subfield by 1 m o n t h (Fig. 1, Table I). T h r e e 2-min ischemic insults caused a significant reduction in [3H]QNB binding in the stratum oriens of the CA1 subfield, the C A 3 subfield and in the d e n t a t e gyrus at as early as 6 h of recirculation. By 1 m o n t h , [3H]QNB binding decreased to 45% of control in the C A 1 subfield, and increased by 10% in the C A 3 subfield and the dentate gyrus (Fig. 1 and Table I).
[3H]CHA binding The dendritic fields of the CA1 and C A 3 subfields and the stratum moleculare of the d e n t a t e gyrus had high densities of [3H]CHA binding sites in control gerbils (Fig. 1, Table II). In contrast, the stratum p y r a m i d a l e had few binding sites. We found a transient increase by 1 5 - 2 5 % in [3H]CHA binding in the whole h i p p o c a m p u s 1 h after 2 and 6 min of ischemia (Table II). [3H]CHA binding again slightly (by 15%) increased in the CA1 at 4 days after 2-min ischemia, but had r e t u r n e d to control levels by 1 month. [3H]CHA binding in the CA1 subfield decreased by 30% 4 days after 6-rain ischemia, but h a d returned to control levels by 1 m o n t h (Table II). By 4
35
QNB
CHA
PN200-110
Fig. 1. Representative autoradiographs of [3H]QNB (a,d,g), [3H]CHA (b,e,h) and [3H]PN200-110 (c,f,i) binding in the gerbil hippocampus of control ( a - c ) , and 4 days ( d - 0 and 1 m o n t h (g-i) after three 2-min bilateral carotid artery occlusions. Note a conspicuous reduction in [3H]QNB and [3H]PN200-110 binding in the CA1 subfield at 1 m o n t h and relative preservation of [3H]CHA binding.
TABLE I
Sequential alterations in [JH]QNB binding (fraol/mg tissue) in the hippocampus following 2- and 6-rain ischemia and foUowing three 2-min ischemic insults Values are m e a n s + S.D., n = 9-10 hemispheres.
Control
1h
6h
1 day
4 days
1 month
515 + 33.2 566 + 41.3 450 + 34.5
495 + 60.3 544 + 68.1 446 + 63.2
418 + 51.3"* 546 + 55.0 458 + 54.2
446 ___57.3** 540 + 54.1 468 + 54.3
515 + 33.2 590 + 46.0 463 + 40.1
518 + 66.6 546 + 100.2 443 + 88.5
350 + 28.7
384 + 43.8
311 + 32.8*
312 ___44.2*
366 + 24.7
398 + 36.5**
542 + 24.8
552+57.6
487+36.2**
494+54.8
540+31.6
577+44.8
516 + 33.2 566 + 41.3 450 + 34.5
513+54.0 590 + 64.9 493 + 60.0
476+51.9 567 + 36.0 460 + 34.7
496+55.8 555 _+_68.8 453 + 64.7
454+54.0* 550 _ 45.4 464 + 44.3
232+30.1"* 254 + 30.1"* 209 + 16.7"*
350 + 28.7
429+43.2**
350+26.7
332+39.2
298+61.0
340+55.8
542 + 24.8
603+26.9**
516+32.7
530+45.8
546+44.5
572+63.6
515 _+ 33.2 566 + 41.3 450 + 34.5
428 + 49.5** 513 + 81.1 428 + 73.7
423 + 24.5** 511 + 50.7* 419 + 51.2
450 +_ 44.2** 536 + 42.4 444 + 35.8
224 + 31:3"* 254 + 35.5** 202 + 21.6"*
350 + 28.7
292 + 49.1"*
271 ___40.1"*
321 + 35.9
386 + 54.1"
542 + 24.8
458 + 68.8*
464 + 44.4**
543 + 42.5
591 + 65.2*
2-min ischemia C A 1 subfield Stratum oriens Stratum radiatum Stratum l a c u n o s u m moleculare C A 3 subfield Average D e n t a t e gyrus Stratum moleculare
6-min ischemia CA1 subfield Stratum oriens Stratum radiatum Stratum l a c u n o s u m moleculare C A 3 subfield Average D e n t a t e gyrus Stratum moleculare
3 x 2-rain ischemia CA1 subfield Stratum oriens Stratum radiatum Stratum l a c u n o s u m moleculare C A 3 subfield Average D e n t a t e gyrus Stratum moleculare
*P < 0.05, **P < 0.01 compared to control (two-tailed M a n n - W h i t n e y U-test).
36 TABLE II
Sequential alterations in [3H]CHA binding (fmol/mg tissue) in the hippocampus following 2- and 6-rain ischemia and following three 2-min ischemic insults Values are means + S.D., n = 8-10 hemispheres.
Control
Ih
6h
I day
4 days
I month
285 + 40.9 331 + 40.7 236 + 24.9
355 + 39.8** 419 + 51.7"* 269 + 33.8*
303 + 46.0 351 + 47.9 249 + 24.8
300 + 57.3 328 + 72.5 248 + 67.3
325 + 33.1" 383 - 50.8* 242 + 39.4
323 + 25.9 371 + 50.7 258 + 38.0
225 + 31.6 274 + 38.4
257 + 28.8 320 + 27.9*
239 + 16.0 279 + 27.4
225 + 35.6 264 + 58.2
247 + 18.4 302 + 40.9
231 + 27.5 285 + 46.3
227 _+ 17.1
257 + 21.6"*
246 + 17.8"
231 + 44.0
236 + 35,4
232 + 27.8
285 + 40.9 331 _+40.7 236 _+ 24.9
355 + 55.8* 416 _+ 81.7"* 265 -+ 44.2
298 + 46.4 343 + 51.2 255 + 51.3
289 _+ 64.7 341 _+ 83.5 238 + 46.8
203 + 47.3** 229 + 54,8** 191 + 46.8*
283 _+ 96.3 305 _+ 125.5 210 + 65.0*
225 _+ 31.6 274 + 38.4
262 -+ 41.9" 319 -+ 54.8
241 -+ 38.7 277 + 38.0
229 _+ 40.7 273 _+ 55.7
229 + 42.6 282 + 62.9
225 _+ 64.5 298 _+ 71.3
227 _+ 17.7
263 -+ 45.3*
248 -+ 42.7
241 + 49.6
223 +_ 47.8
258 + 43.0
285 _+ 40.9 331 __-40.7 236 _ 24.9
268 +_ 43.4 325 -- 21.7 232 _+ 24.9
280 -+ 68.4 340 _+ 66.8 239 _+ 36.2
216 + 32.8** 256 + 37.9** 185 _+ 17.1 **
355 + 52.1 ** 400 + 75.8 210 _+ 29.6*
225 + 31.7 274 __-38.4
232 + 34.2 273 + 25.1
235 _+ 30.3 295 + 54.2
217 + 30.8 280 + 43.4
270 + 37.0* 344 + 17.4"*
227 _+ 17.7
226 + 24.7
243 + 34.4
215 _+ 27.2
276 + 27.2**
2-min ischemia CA1 subfield Stratum oriens Stratum radiatum Stratum laeunosum moleculare CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum moleeulare
6-min ischemia CA1 subfield Stratum oriens Stratum radiatum Stratum lacunosum moleculare CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum moleculare
3 x 2-min ischemia CA1 subfield Stratum oriens Stratum radiatum Stratum lacunosum moleculare CA3 subfield Stratum oriens Stratum radiatum Dentate gyrus Stratum moleculare
*P < 0.05, **P < 0.01 compared to control (two-tailed Mann-Whitney U-test).
TABLE III
Sequential alterations in f H] PN200-110 binding (fmol/mg tissue) in the hippocampus following 2- and 6-rain ischemia and following three 2-min iscemic insults Values are means + S.D., n = 9-10 hemispheres.
Control
1h
6h
1 day
4 days
1 month
10 + 2.5 16 + 2.5 20 + 5.4
12 + 2.5 18 + 4.4 23 + 4.0
12 + 1.4 17 + 3.4 24 + 3.1"
10 + 2.5 15 + 2.9 20 + 4.3
11 + 2.3 16 + 2.9 22 + 4.3
11 + 2.8 18 + 5.8 23 + 3.5
10 + 2.5 16 + 2.5 20 + 5.4
12 + 1.6 20 _+_4.6 26 + 3.9*
9 +- 1.9 16 + 3.4 20 + 4.1
11 --- 2.1 16 + 2.4 22 + 4.3
10 + 2.5 14 + 2.5 19 + 5.0
4 ___1.6"* 17 + 5.9 28 + 8.4*
11 + 1.4 18 + 3.6 22 _+ 2.5
12 -+ 1.6 17 -+ 2.9 23 + 3.9
10 +_ 1.6 16 _+ 2.0 22 _+ 2.9*
4 _+ 1.5"* 21 + 6.0* 25 _+ 4.2*
2-min ischemia CA1 subfield CA3 subfield Dentate gyrus
6-rain ischemia CA1 subfield CA3 subfield Dentate gyrus
3 x 2-rain ischemia CA1 subfield CA3 subfield Dentate gyrus
10 +_ 2.5 16 _+ 2.5 20 _+ 5.4
*P < 0.05, **P < 0.01 compared to control (two-tailed Mann-Whitney U-test).
37 days after three 2-min ischemic insults, the density of [3H]CHA binding sites in the CA1 subfield decreased to 76% of control, but the binding was slightly increased at 1 month. [3H]CHA binding in the CA3 subfield and the dentate gyrus also increased at 1 month by more than 20% (Table II).
[3H]PN200-110binding There was a high density of [3H]PN200-110 binding sites in the stratum moleculare of the dentate gyrus in control gerbils (Fig. 1, Table III). The CA3 subfield also had a high density of the binding sites, but the CA1 subfield had a relatively low density. No significant alterations in [3H]PN200-110 binding were observed after 2-rain ischemia except for a transient increase at 6 h in the dentate gyrus (Table III). Six-minute ischemia caused a 30% increase in [3H]PN200-110 binding in the dentate gyrus at 1 h of recirculation. We also found a 60% reduction in the CA1 subfield and a 40% increase in the dentate gyrus at 1 month, but otherwise unaltered (Table III). Following three 2-min ischemic insults, [3H]PN200110 binding decreased to 40% of control in the CA1 subfield by 1 month (Fig. 1). We noted a 25% increase in [3H]PN200-110 binding in the CA3 subfield and the dentate gyrus at 1 month (Table III). DISCUSSION Previous reports have shown the temporal profile of histopathological changes in the gerbil hippocampus following transient ischemia and repeated ischemic insults 1'8'9A2'13. Two minutes of ischemia produces no neuronal damage in the hippocampus. However, CA1 pyramidal cells are selectively destroyed at 2--4 days after three 2-min ischemic insults and after 5-10 min of ischemia (delayed neuronal death12), whereas neurons in the CA3 subfield and the dentate gyrus are not damaged. Thus, histopathological observations in this study confirmed the previous results. [3H]QNB and [3H]PN200-110 binding in the CA1 subfield decreased to 45 and 40% of control, respectively, 1 month after 6-min ischemia and three 2-min ischemic insults. However, at 4 days when CA1 pyramidal cells had been destroyed, a significant decrease in [3H]QNB binding was observed only in the stratum oriens (by 13%), and a reduction in [3H]PN200-110 binding was not observed. Therefore, recognition sites labeled by [3H]QNB and [3H]PN200-110 may be resistant to degradation processes. Furthermore, because almost all CA1 pyramidal cells are destroyed following 6-min ischemia and following three 2-min ischemic insults, [3H]QNB and [3H]PN200-110 binding sites in the CA1 subfield for up to 40--45% of control may be localized on
interneurons and presynaptic sites which are resistant to ischemic insult6'7'14. In contrast, [3H]CHA binding in the CA1 subfield decreased to 70-80% of control at 4 days after 6-min ischemia and after three 2-min ischemic insults when CA1 pyramidal cells were destroyed, but the binding had returned to control level 1 month after 6-min ischemia and even increased after three 2-min ischemic insults. The absence of reductions in [3H]CHA binding in the CA1 subfield at 1 month is incompatible with previous reports which showed a significant loss of [3H]CHA binding sites in the CA1 subfield following 15 min ischemia in gerbils 16'17. However, 15-rain ischemia is severe enough to cause damage to CA3 pyramidal cells which terminate in the CA1 region (Schaffer collaterals) as presynaptic sites TM. Taken together, many [3H]CHA binding sites may be located on cells other than CA1 pyramidal neurons such as presynaptic sites, interneurons, blood vessels and glial cells. [3H]QNB, [3H]CHA and [3H]PN200-110 binding increased in the CA3 subfield and the dentate gyrus at 1 month after three 2-min ischemic insults. Such an increase was not observed after 6-min ischemia except for an increase in [3H]PN200-110 binding in the dentate gyrus. The physiological meaning of the increase at 1 month is difficult to explain in the present study. Elimination of CA1 pyramidal cells may play a role to induce synaptic modification of the neurotransmitter system resulting in an increase in receptor density on surviving neurons or glial cells. An increase in receptor density can take place after lesioning of neuronal circuits5A8. Increase in [3H]CHA binding in the CA1 subfield which was observed 4 days after 2-min ischemia and 1 month after three 2-min ischemic insults may also be explained by the synaptic modification of this inhibitory neurotransmitter system. Among 3 ligands used, [3H]QNB binding was most susceptible to ischemic insult. The binding decreased as early as 6 h after three 2-min ischemic insults. Thus, the decrease in [3H]QNB binding preceded delayed neuronal death of CA1 pyramidal cells following repeated insults as reported previously in transient ischemia 17'19. Furthermore, [3H]QNB binding transiently decreased after 2-min ischemia which causes no morphological neuronal damage. One of the unique features of neuronal damage following repeated ischemic insults is that the damage is most severe when ischemia is repeated at 1-h intervals9' 15.20. This phenomenon may be explained by the alterations in cerebral blood flow and metabolism which is maximally disturbed 1 h after ischemiaTM. In this study, we found a transient increase in [3H]QNB, [3H]PN200110, and especially [3H]CHA binding in the hippoeampus
38 1 h after 2- or 6-min ischemia. The increase may reflect the possible alterations in receptor sensitivity and calcium conduction. Therefore, these changes in receptor binding might explain the mechanism of the cumulative damage following repeated ischemic insults which is most severe at 1-h intervals. Patterns of alterations in the binding activities after three 2-min ischemic insults were different from that after 6-min ischemia. Reductions in [3H]QNB binding after three 2-min insults were greater and earlier that those after 6-min ischemia. Furthermore, changes were extended to the CA3 subfield and the dentate gyrus after repeated ischemic insults. These observations may further demonstrate the cumulative effect of neuronal damage after three 2-min ischemic insults which is greater than that after 6-rain ischemia 9. In conclusion, the present study showed the sequential alterations in binding sites of two major neurotransmitters, muscarinic acetylcholine and adenosine ml, and of a dihydropyridine calcium antagonist following three 2-min ischemic insults in comparison with those following 2 or 6 min ischemia. Findings observed were: (1) 2-min
REFERENCES 1 Araki, T., Kato, H. and Kogure, K., Selective neuronal vulnerability following transient cerebral ischemia in the gerbil: distribution and time course, Acta Neurol. Scand., 80 (1989) 548-553. 2 Araki, T., Kato, H., Inoue, T. and Kogure, K., Regional impairment of protein synthesis following brief cerebral ischemia in the gerbil, Acta Neuropathol., 79 (1990) 501-505. 3 Araki, T., Kato, H. and Kogure, K., Neuronal damage and calcium accumulation following brief cerebral ischemia in the gerbil, Brain Research, 588 (1990) 114-122. 4 Araki, T., Kato, H., Inoue, T. and Kogure, K., Long-term observations on calcium accumulation in postischemic gerbil brain, Acta Neurol. Scand., in press. 5 Block, G.A., Buchan, A.M. and Pulsinelli, W.A., Alteration of A1 adenosine receptor density following transient forebrain ischemia and fimbria/fornix lesions, Soc. Neurosci. Abstr., 13 (1987) 1080. 6 Francis, A. and Pulsinelli, W.A., Response of GABAergic and cholinergic neurons to transient cerebral ischemia, Brain Research, 243 (1982) 271-278. 7 Johansen, EE, Jorgensen, M.B. and Diemer, N.H., Resistance of hippocampal CA-1 interneurons to 20 min of transient cerebral ischemia in the rat, Acta Neuropathol., 61 (1983) 135-140. 8 Kato, H., Kogure, K. and Nakano, S., Neuronal damage following repeated brief ischemia in the gerbil, Brain Research, 479 (1989) 366-370. 9 Kato, H. and Kogure, K., Neuronal damage following nonlethal but repeated cerebral isehemia in the gerbil, Acta Neuropathol., 79 (1990) 494-500. 10 Kato, H., Araki, T. and Kogure, K., Role of the excitotoxic mechanisms in the development of neuronal damage following repeated cerebral ischemia in the gerbil: protective effects of
ischemia, which is brief enough to permit recovery without any morphological damage, produced no persistent alterations in binding activities; (2) [3H]QNB and [3H]PN200-110 binding decreased by more than 50% in the CA1 subfield by 1 month after elimination of CA1 pyramidals cells by ischemia. In contrast, [3H]CHA binding in the CA1 subfield decreased by 20-30% transiently at 4 days although CA1 pyramidal neurons were destroyed; (3) there was a transient and mild increase in binding activities, especially in [3H]CHA binding, at 1 h of recirculation, which may be one of the reasons why neuronal damage following repeated brief ischemic insults is most severe at 1-h intervals; and (4) alterations in binding activities, especially in [3H]QNB binding, following three 2-min ischemic insults were greater and appeared earlier than those after 6-min ischemia. Furthermore, alterations extended to the CA3 subfield and the dentate gyrus following repeated insults. Thus, alterations in receptor binding after repeated ischemic insults were greater than those after equivalent single period of ischemia.
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