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Changes in Cholinergic Neurons and Failure in Learning Function After Microsphere Embolism-Induced Cerebral Ischemia
Brain Research Bulletin, Vol. 43, No. 1, pp. 87–92, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
PII S0361-9230(96)00350-4
Changes in Cholinergic Neurons and Failure in Learning Function After Microsphere Embolism-Induced Cerebral Ischemia NORIO TAKAGI, KEIKO MIYAKE, TAKU TAGUCHI, NAOKO SUGITA, KAORI TAKAGI, HIROAKI TAMADA, AND SATOSHI TAKEO* Department of Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, 192-03, Japan
ABSTRACT: Central cholinergic neurons play an important role in learning and memory functions. The present study was undertaken to elucidate the pathological changes in learning function and acetylcholine metabolism of the cerebral cortex and hippocampus, following microsphere embolism in rats. Microspheres (48 mm) were injected into the right internal carotid artery of the rats. Learning function was determined using a passive avoidance task on the seventh day after the embolism. In the biochemical study, acetylcholine and choline contents, and choline acetyltransferase activity were measured in the cerebral cortex and hippocampus. Cortical acetylcholinesterase-containing fibers were quantitatively estimated in the embolized rat. Passive avoidance was impaired in the microsphere-embolized rat. Microsphere embolism decreased the acetylcholine concentration and choline acetyltransferase activity in the cerebral cortex and hippocampus. In the histochemical study, the length of cortical acetylcholinesterase-containing fibers was decreased, but cell density was unchanged in the ipsilateral hemisphere of the microsphere-embolized rat. The results suggest that microsphere embolism induces severe damage to cholinergic neurons, which may be related to the impairment of learning function in the ischemic brain. Q 1997 Elsevier Science Inc.
information is available, however, concerning the relationship between learning deficits and pathological changes in cholinergic neuron in cerebral embolism, which mimics sustained brain ischemia and eventually leads to multiinfarct pathology. In previous studies, we have shown that microsphere embolism induced a marked decrease in cerebral blood flow and a pronounced disturbance of energy metabolism in the brain regions, demonstrating that microsphere embolism was capable of inducing progressive and sustained cerebral ischemia and/or oligemia [22,33]. It is suggested that microsphere-induced multiple small infarction resembles clinical vascular dementia more closely than the infarction induced by middle cerebral artery occlusion in terms of pathophysiological alterations induced [23]. Studies on the learning function and cholinergic system following microsphere embolism may, therefore, provide a better understanding of neuronal disturbance in multiinfarction. The purpose of the present study was to elucidate the pathological changes in learning function and biochemical profile of cholinergic neurons in the cerebral cortex and hippocampus, which are vulnerable to ischemia [15,24,29]. The morphological profile of cortical cholinergic neurons of the microsphere-embolized rat was also examined.
KEY WORDS: Cerebral ischemia, Microsphere embolism, Cholinergic neuron, Learning function, Rats.
MATERIALS AND METHODS Operation for Microsphere Embolism
INTRODUCTION
One hundred and fifty-three male Wistar rats weighing 180 to 220 g (Charles River Japan, Inc., Atsugi, Japan) were used in the present study. The animals were maintained under artificial conditions at 23 { 17C with a constant humidity of 55 { 5% with a cycle of 12-h light and 12-h dark, and had free access to food and tap water according to the Guideline of Experimental Animal Care issued by the Prime Minister’s Office of Japan. Microsphere-induced cerebral embolism was performed by the method previously described [32]. Briefly, 88 rats were anesthetized with sodium pentobarbital (35 mg/kg, IP) and fixed in the supine position on an operation plate. After cervical incision, the right common carotid artery was isolated. The right
It is well recognized that cortical and hippocampal cholinergic input originates in the nucleus basalis of Meynert and in the medial septum in primates, respectively [4], and that the cholinergic system in the central nervous system plays an important role in learning and memory functions [26]. A failure in cortical cholinergic activity has been shown to underlie the memory loss associated with aging and Alzheimer’s disease [3,31]. In rodents, lesions of cholinergic neuron and pharmacological manipulations of the cholinergic system have been correlated with altered cognitive performance [1,9,25]. It has also been reported that hypoxia induces a reduction of memory and judgment that is associated with a decrease in acetylcholine synthesis [8]. Little * To whom requests for reprints should be addressed.
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external carotid and right pterygopalatine arteries were ligated with string. A polyethylene catheter (3F size, 1.0 mm in diameter, Atom Co., Tokyo) was inserted into the right common carotid artery. Nine hundred microspheres (47.5 { 0.5 mm in diameter; NEN-005, New England Nuclear Inc., Boston, MA) suspended in 20% dextran solution were injected into the right internal carotid artery through this cannula. Fifty-one rats that had undergone a sham operation were injected with same volume of vehicle without microspheres. The control group comprised 14 nonoperated rats. Neurological Deficits Sixteen hours after the operation, the behavior of the rats was scored on the basis of paucity of movement, truncal curvature, and forced circling during locomotion, which are considered to be typical symptoms of stroke in rats [7,21]. The score of each item was ranked from 3 to 0 (3, very severe; 2, severe; 1, moderate). The rats which had more than seven points were considered to be type A, six to four type B and less than four type C. We used only type A animals in the following study. Passive Avoidance Response Rats were tested in a step-through type passive avoidance task [12]. The experimental apparatus consists of two compartments (illuminated one: 20 1 20 1 20 cm, dark one: 20 1 20 1 20 cm equipped with a grid floor), divided by a guillotine door (8 1 8 cm). In the acquisition trial, the rat was placed in the illuminated compartment and allowed to enter the dark compartment after opening of the guillotine door. As soon as the rat entered the dark compartment, the guillotine door was closed and an inescapable footshock (2.0 mA, 5 s) was delivered through the grid floor. In the retention test, the rats were placed in the illuminated compartment and the time taken to enter the dark compartment was measured. Acetylcholinesterase-Containing Fibers Morphological changes in the cholinergic pathway of the microsphere-embolized rat were measured by acetylcholinesterase histochemistry [10]. After passive avoidance task, 11 microsphere-embolized rats were perfused via the heart with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After immersion in cold fixative for 1 day, the brain was transferred through several changes of 30% sucrose. Frozen sections were cut on a cryostat (20 mm). Sections were rinsed in 0.1 M acetate buffer (pH 6.0), and incubation was performed for 40 min in the following medium: 25 mg acetylthiocholine iodide, 32.5 ml of 0.1 M sodium acetate buffer (pH 6.0), 2 ml of 0.1 M sodium citrate, 5 ml of 0.03 M cupric sulfate, 9.5 ml of distilled H2O, and 1 ml of 5 mM potassium ferricyanide. After five changes of acetate buffer, sections were treated with 1% ammonium sulfide solution for 1 min followed by five changes of 0.1 M sodium nitrate. Sections were then exposed to 0.1% silver nitrate followed by five changes of 0.1 M sodium nitrate. Free-floating sections were rinsed and mounted. Sections were viewed with light microscope (OLYMPUS BH-2) and a photograph of each section was taken. Quantitative analysis of the acetylcholinesterase histochemistry was carried out with an aid of the Image 1.56 software program running on a Macintosh Centris 660AV. In brief, the two-tone gray image of the acetylcholinesterase containing axons was extracted from the original 256 gray-tone image, which was taken by scanner (SCANTOUCH, Nikon), by binarization. Then, binarized image was converted to the twotone gray image of axons and then to a binary line image with
1-pixel width. Measurement was made on the basis of length of the binary line image with 1-pixel width in a selected field. The sections were counterstained by cresyl violet and cells were counted in the same field as above. Measurement of Acetylcholine and Choline Contents The animals were sacrificed with a focal irradiation of microwave to the head with a strength of 5 kW for 0.85 s by a microwave applicator (TMW-6402C, Muromachikikai Co., Tokyo). After decapitation, the head of the animals was immersed in liquid nitrogen and left for 10 s (near freezing). The cerebral hemisphere was isolated, and the cerebral cortex and hippocampus of both hemisphere were separated. The tissues were homogenized in 0.2 M HClO4 and 0.01% disodium EDTA with a Polytron homogenizer (PT-10, Kinematica, Switzerland). The homogenate was centrifuged at 10,000 1 g for 15 min at 47C. The supernatant fluid was filtered through a membrane filter (0.45 mm). A 5 ml aliquot of the supernatant fluid was applied to a HPLCECD to determine the concentrations of acetylcholine and choline. The HPLC-ECD system was composed of enzymatic column (Eicompak AC-gel / AC-enzymepak; Eicom, Kyoto), a model L-6000 pump (Hitachi, Japan) and an electrochemical detector (ECD-100, Eicom). The mobile phase contained 0.1 M phosphate buffer at pH 8.5, containing 1.2 mM sodium 1-decanesulfonate (Tokyo Kasei, Tokyo) and 0.6 mM tetramethylammonium chloride (Nacalai Tesque, Kyoto). Measurement of Choline Acetyltransferase Activity Choline acetyltransferase (ChAT) activity was determined according to the method of Kaneda and Nagatsu [13]. The cortex and hippocampus of both hemispheres were homogenized by Teflon homogenizer (20 updown) in 12.5 ml of cold 25 mM phosphate buffer, pH 7.4, containing 0.5% Triton X-100, per gram of wet weight. The homogenate was centrifuged at 20,000 1 g for 60 min. The ChAT activity in the supernatant fluid was assayed. For the assay, 100 ml substrate solution containing 10 mM choline chloride, 0.4 mM acetyl CoA, 0.2 mM eserine sulfate, 0.3 mM sodium chloride, and 20 mM EDTA-2Na in 0.1 M sodium phosphate buffer, pH 7.4 was added to 100 ml of enzyme solution in 25 mM sodium phosphate buffer, pH 7.4, and incubated at 377C for 20 min. The enzyme reaction was stopped using 50 ml of 1 M perchloric acid in ice. After 10 min, 6 ml of 1.0 mM isopropylhomocholine, as an internal standard, was added and the mixture was centrifuged at 1,600 1 g for 10 min at 47C. The supernatant fluid was filtered through a membrane filter (0.45 mm). A 5-ml aliquot of the supernatant fluid was applied to a HPLC-ECD as described above to determine the concentration of acetylcholine. Protein concentration of the supernatant fluid was determined according to the method of Lowry et al. [19]. Statistical Analysis The results are expressed as mean { SEM, except for the stepthrough latency, which is expressed as mean { SD. Statistical significance between different groups was evaluated by Student’s t-test and Mann–Whitney U-test (two tailed). Analysis of variance followed by Dunnett’s multiple comparison was employed for evaluation of significance of the changes in time course of variables. p õ 0.05 was considered to be statistically significant. RESULTS Microsphere Embolism Fifteen (17%) rats out of 88 rats that were injected with microspheres died before their symptoms were assessed (within
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Choline Acetyltransferase Activity, Acetylcholine, and Choline Contents
FIG. 1. The step-through latency of the passive avoidance response of the sham-operated and microsphere-embolized rat on the seventh day after the operation. Each value represents the mean { SD of 11 experiments. *Significantly different from sham-operated group (p õ 0.05).
16 h of the embolism). Microsphere injection induced type A strokelike symptoms in 61 (69%) of the operated rats. Among these rats, 10 (16%) of type A rats died by the third day after the operation. Eight (9%) rats showed type B symptoms, and four (5%) rats showed type C symptoms. The sham-operated rats showed no strokelike symptoms and all survived. Passive Avoidance Task We confirmed that the response of the microsphere-embolized rat to electric shock, such as startle or crouch, on the seventh day was comparable with that of the sham-operated rat. The behavioral score of microsphere-embolized rats on the seventh day after the operation was 2.1 { 0.5. Analysis of the passive avoidance acquisition time of shamoperated (18.5 { 11.5 s; mean { SD) and of microsphere-embolized (21.1 { 14.4 s; mean { SD), which were carried out 7 days after the operation, revealed no group differences. The latency period of the step-through, which was carried out 24 h after the acquisition test, in the passive avoidance task of microsphereembolized and sham-operated rats are shown in Fig. 1. The latency of step-through in microsphere-embolized rats was significantly shortened compared to sham-operated rats. Acetylcholinesterase-Containing Fiber Density and Cell Density of the Microsphere-Embolized Rat On the seventh day after the microsphere embolism, a significant loss of acetylcholinesterase-containing fibers in the right frontal cortex was seen compared with the left frontal cortex (Table 1). In contrast, there were no differences in the cell density of both frontal cortices of the microsphere-embolized rat (Table 1).
Figure 2 shows the time courses of changes in ChAT activities of the cerebral cortex and hippocampus of both hemispheres in microsphere-embolized and sham-operated rats. ChAT activity in the brain regions of the right hemisphere was unchanged on the first day after the microsphere embolism. Thereafter, microsphere embolism induced a significant decrease in ChAT activity of the right hemisphere throughout the experiment. The time course of the changes in acetylcholine and choline contents of the cerebral cortex and hippocampus of the both hemispheres in microsphere-embolized and sham-operated rats are shown in Figs. 3 and 4, respectively. In the sham-operated rats, there were no appreciable changes in acetylcholine and choline contents of either hemisphere throughout the experiment. In the microsphere-embolized rat, a marked reduction in acetylcholine content in the brain regions of the right hemisphere was seen on the first day after the operation. Thereafter, the acetylcholine content remained at a low level. The time course of changes in acetylcholine content of the left hemisphere in the microsphereembolized rats revealed a similar trend to that of the right hemisphere, but the changes were of a smaller magnitude. In microsphere-embolized rats, a marked increase in choline content of the right cerebral cortex and hippocampus was seen on the first day after the operation. Thereafter, the choline content of these regions returned toward the control level. DISCUSSION The protocol of the passive avoidance task used in the present study eliminates disability of behavior caused by neurological deficits in the microsphere-embolized rat, because neurological deficits disappeared almost completely 7 or more days after the operation [33]. We also confirmed that the response of the microsphere-embolized rat to electric shock 7 or more days after the operation did not differ from that of the control or shamoperated rat in the present study. It has been reported that the sensitivity to electric shock of the microsphere-embolized rat is normal 1 or 2 weeks after the operation [16]. Thus, it appears that the impairment in the passive avoidance response using electric shock is not due to a decrease in sensitivity to the electric shock and/or disability of behavior caused by neurological deficits, but due to a failure in learning function. Microsphere-embolized rats showed an impairment of passive avoidance response in the present study. Impairments of passive avoidance task have been reported in several ischemic models [11,17,35]. In transiently ischemic rats, an impairment of passive avoidance learning, which disappears with time after the ischemia [2], matches the period of disturbance of brain energy metabolism. Therefore, an impairment of the passive avoidance task may be, in part, attributed to transient dysfunction of the central TABLE 1 ACETYLCHOLINESTERASE-CONTAINING FIBER DENSITY AND CELL DENSITY OF THE MICROSPHERE-EMBOLIZED RAT
Right hemisphere Left hemisphere
AChE-Positive Fiber Density (mm/mm2 Frontal Cortex)
Cell Density (1103/mm2 Frontal Cortex)
60.7 { 7.0* 102.9 { 8.8
1.408 { 0.069 1.257 { 0.039
Each value represent the mean { SEM of 11 experiments. * Significantly different from the left hemisphere ( p õ 0.05).
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TAKAGI ET AL. examined 12 weeks after ischemia, despite the presence of appreciable postischemic hippocampal damage [17]. It cannot be determined, however, whether damage to the hippocampal cholinergic neurons is responsible for the impairment of passive avoidance response in transiently ischemic rats. In the present study, microsphere embolism resulted in a sustained decrease in acetylcholine content and ChAT activity in the hippocampus, suggesting that the hippocampal cholinergic system and/or structure may be severely impaired. The disturbance of learning function in the microsphere-embolized rat may, at least in part, relate to dysfunction of hippocampus and/or hippocampal cholinergic system. It is recognized that cortical cholinergic innervation plays an important role in cognitive function in humans [3]. Cortical cholinergic neurons [5] that receive large afferent neuron from the nucleus basalis magnocellularis located near the globus pallidus
FIG. 2. The time course of changes in choline acetyltransferase activity in the cerebral cortex and hippocampus of the right and left hemispheres of microsphere-embolized ( l and j ), sham-operated ( s and h ), and control ( s and h at day 0) rats, respectively. Each value represents the mean { SEM of five to seven experiments. *Significantly different from control group (p õ 0.05).
nervous system due to disturbance of energy metabolism in the ischemic brain [17]. Because microsphere embolism induces a sustained impairment of brain energy metabolism [33], an impairment of energy metabolism in the microsphere-embolized rat may result in a deterioration of memory function. In contrast to transiently ischemic models, the step-through latency of the middle cerebral artery-occluded rat is shortened for a period of 16 weeks [35]. This suggests that differences in ischemic conditions or ischemic models results in differences in the avoidance response changes. From the present result on avoidance task, it appears that microsphere embolism induces an impairment of learning function. It is generally considered that the hippocampus plays an important role in the formation and preservation of memory [30]. Furthermore, the septohippocampal cholinergic system is considered to play a pivotal role in the memory processes [11]. It has been reported, however, in rats that exposure to transient forebrain ischemia did not alter the passive avoidance task when
FIG. 3. The time course of changes in acetylcholine content in the cerebral cortex and hippocampus of the right and left hemispheres of microsphere-embolized ( l and j ), sham-operated ( s and h ) and control ( s and h at day 0) rats, respectively. Each value represents the mean { SEM of five to seven experiments. *Significantly different from control group (p õ 0.05).
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FIG. 4. The time course of changes in choline content in the cerebral cortex and hippocampus of the right and left hemispheres of microsphere-embolized (l and j), sham-operated (s and h), and control ( s and h at day 0) rats, respectively. Each value represents the mean { SEM of five to seven experiments. *Significantly different from control group (p õ 0.05).
[20] are believed to play a role in learning function in animals. It is also suggested that the impairment of passive avoidance performance defect in nucleus basalis-lesioned rats is caused by cholinergic neuron loss [6]. It is hypothesized, therefore, that the cortical cholinergic system plays an important role in learning function in animals. The role of morphological changes in cortical cholinergic neurons in microsphere-embolized rats is, however, little understood. In the present study, a loss of acetylcholinesterase-containing fibers was detected in the ipsilateral frontal cortex of the microsphere-embolized rat, but cell density remained unchanged. It has been shown that acetylcholinesterasecontaining fibers in the frontal cortex on the ipsilateral hemisphere is significantly reduced in the middle cerebral artery-occluded rat, suggesting that there is a disruption of the cholinergic pathway between the frontal cortex and the nucleus basalis [14]. Some noncholinergic neurons may also be stained by the method used in the present study, because acetylcholinesterase is not a specific marker for determination of the cholinergic pathway [18]. It is conceivable, however, that the loss of acetylcholinesterase-containing fibers in the frontal cortex of the microsphere-embolized rat represents a disruption of the neu-
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ronal innervation in the frontal cortex and, in part, results in an alteration of learning function. Furthermore, we observed that microsphere embolism decreased the acetylcholine content and ChAT activity of the right cerebral cortex during most periods of the experiment, although the ChAT activity was unchanged on the first day after the embolism. Acetyl-CoA, required for ACh synthesis, is normally generated by oxidation of pyruvate by the pyruvate dehydrogenase complex [34], and pyruvate availability is known to be decreased in ischemic conditions [28]. In our previous study, microsphere embolism induced a sustained impairment of cerebral energy metabolism [33]. The decrease in acetylcholine concentration of the ipsilateral hemisphere on the first day may, therefore, be attributed to low levels of acetyl-CoA concentration due to impaired energy metabolism rather than altered ChAT activity. After the third day, it is likely that the decrease in acetylcholine concentration is related to both low level of acetylCoA and low activity of ACh synthesis regulated by ChAT. In the present study, microsphere embolism induced an increase in the choline content of the right hemisphere throughout the experiment. It is possible that an increase in choline content of the microsphere-embolized rat is not attributable to breakdown of acetylcholine, but is a result of hydrolysis of phospholipids [27]. In the present study, the ACh contents in the contralateral hemisphere were decreased, although the activity of ChAT and choline contents did not change. This result may indicate that cerebral ACh is the most sensitive biochemical parameter among cholinergic variables under ischemic and/or oligemic conditions. Furthermore, we showed in a previous study [33] that microsphere embolizm induced sustained damage to cerebral energy metabolism in the contralateral hemisphere, but to a lesser degree than the ipsilateral hemisphere. Thus, it is likely that the decrease in ACh contents of contralateral hemisphere contributes to low levels of acetyl-CoA concentration due to impairment of cerebral energy metabolism in the contralateral hemisphere of the microsphere-embolized rat. The results in the present study suggest that sustained and severe impairment of acetylcholine metabolism occurs in the microsphere-embolized rat. Pharmacological studies support the cholinergic specificity of the passive avoidance defect caused by lesions in the nucleus basalis [1,9,25]. Thus, it is suggested that the disturbance of cortical acetylcholine metabolism and/or the loss of cholinergic fibers in the microsphere-embolized rat lead to a deterioration of cortical cholinergic neurotransmission, and may contribute to an impairment of learning function. In conclusion, we demonstrate in the present study that microsphere embolism induce an impairment of passive avoidance response and the loss of acetylcholinesterase-containing fibers in the ipsilateral frontal cortex. Furthermore, we showed a decrease in acetylcholine content and ChAT activity in the ipsilateral hemisphere of the microsphere-embolized rat. These results suggest that a disturbance of cholinergic neurons by microsphere embolism is comparable to the pathophysiological alterations of multiinfarction. ACKNOWLEDGEMENTS
This work was in part supported by grant-in-aid for general scientific research C from the Japanese Ministry of Education, Science and Culture.
REFERENCES 1. Aaltonen, M.; Riekkinen, P.; Sirvio¨, J.; Riekkinen, P. Jr. Effects of THA on passive avoidance and spatial performance in quisqualic
/ 2a37 2303 Mp 91 Friday May 30 08:17 AM EL–BRB (v. 42, no. 5) 2303
92
2.
3. 4. 5.
6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
TAKAGI ET AL. acid nucleus basalis-lesioned rats. Pharmacol. Biochem. Behav. 39:563–567; 1991. Bothe, H. W.; Bosma, H. J.; Hofer, H.; Hossmann, K.-A.; Angermeier, W. F. Operant learning, cerebral blood flow and neurohistology in Meriones unguiculatus after unilateral carotid artery ligation. In: Meyer, J. S.; Lechner, H.; Reivich, M.; Ott, E. O., eds. Cerebral vascular disease, vol. 4, Amsterdam: Excerpta Medica; 1983:250– 255. Coyle, J. T.; Price, D. L.; Delong, M. R. Alzheimer’s disease: A disorder of cortical cholinergic innervation. Science 219:1184– 1190; 1983. Divac, I. Magnocellular nuclei of the basal forebrain project to neocortex, brain stem, and olfactory bulb. Review of some functional correlates. Brain Res. 93:385–398; 1975. Dubois, B.; Mayo, W.; Agid, Y.; Le Moal, M.; Simon, H. Profound disturbances of spontaneous and learned behaviors following lesions of the nucleus basalis magnocellularis in the rat. Brain Res. 338:249–258; 1985. Dunnett, S. B.; Whishaw, I. Q.; Jones, G. H.; Bunch, S. T. Behavioural biochemical and histochemical effects of different neurotoxic amino acids injected into nucleus basalis magnocellularis of rats. Neuroscience 20:653–669; 1987. Furlow, T.; Bass, N. H. Arachidonate-induced cerebrovascular occlusion in the rat. Neurology 26:297–304; 1976. Gibson, G. E.; Duffy, T. E. Impaired synthesis of acetylcholine by mild hypoxic hypoxia or nitrous oxide. J. Neurochem. 36:28–33; 1981. Haroutunian, V.; Kanof, P.; Davis, K. L. Pharmacological alleviation of cholinergic lesion induced memory deficits in rats. Life Sci. 37:945–952; 1985. Hedreen, J. C.; Bacon, S. J.; Price, D. L. A modified histochemical technique to visualize acetylcholinesterase-containing axons. J. Histochem. Cytochem. 33:134–140; 1985. Hirakawa, M.; Tamura, A.; Nagashima, H.; Nakayama, H.; Sano, K. Disturbance of retention of memory after focal cerebral ischemia in rats. Stroke 25:2471–2475; 1994. Itoh, J.; Ukai, M.; Kameyama, T. Dynorphin A-(1–13) potently prevents memory dysfunctions induced by transient cerebral ischemia in mice. Eur. J. Pharmacol. 234:9–15; 1993. Kaneda, N.; Nagatsu, T. Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection. J. Chromatogr. 341:23–30; 1985. Kataoka, K.; Hayakawa, T.; Kuroda, R.; Yuguchi, T.; Yamada, K. Cholinergic deafferentation after focal cerebral infarct in rats. Stroke 22:1291–1296; 1991. Kirino, T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 39:57–69; 1982. Kiyota, Y.; Miyamoto, M.; Nagaoka, A.; Nagawa, Y. Cerebral embolization leads to memory impairment of several learning tasks in rats. Pharmacol. Biochem. Behav. 24:687–692; 1986. Kiyota, Y.; Miyamoto, M.; Nagaoka, A. Relationship between brain damage and memory impairment in rats exposed to transient forebrain ischemia. Brain Res. 538:295–302; 1991.
18. Kristt, D. A. Acetylcholinesterase in the cortex. In: Jones, E. G.; Peters, A., eds. Cerebral cortex, vol. 6. New York: Plenum Publishing Corp.; 1987:187–236. 19. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randal, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951. 20. Luiten, P. G. M.; Gaykema, R. P. A.; Traber, J.; Spencer, D. G. Jr. Cortical projection patterns of magnocellular basal nucleus subdivisions as revealed by anterogradely transported Phaseolus vulgaris leucoagglutinin. Brain Res. 413:229–250; 1987. 21. McGraw, C. P. Experimental cerebral infarction: effect of pentobarbital in mongolian gerbils. Arch. Neurol. 34:334–336; 1977. 22. Miyake, K.; Takeo, S.; Kajihara, H. Sustained decrease in brain regional blood flow after microsphere embolism in rats. Stroke 24:415–420; 1993. 23. Naritomi, H. Experimental basis of multi-infarct dementia: memory impairments in rodent models of ischemia. Alzheimer Dis. Assoc. Disord. 5:103–111; 1991. 24. Pulsinelli, W. A.; Brierley, J. B.; Plum, F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11:491–498; 1982. 25. Riekkinen, P., Jr.; Sirvio¨, J.; Riekkinen, M.; Riekkinen, P. Effects of THA on passive avoidance retention performance of intact, nucleus basalis, frontal cortex and nucleus basalis / frontal cortexlesioned rats. Pharmacol. Biochem. Behav. 39:841–846; 1991. 26. Sarter, M. F.; Bruno, J. P. Cognitive functions of cortical ACh: Lessons from studies on trans-synaptic modulation of activated efflux. Trends Neurosci. 17:217–221; 1994. 27. Scremin, O. U.; Jenden, D. J. Focal ischemia enhances choline output and decreases acetylcholine output from rat cerebral cortex. Stroke 20:92–95; 1989. 28. Siesjo¨, B. K. Brain energy metabolism. New York: John Wiley & Sons; 1978. 29. Smith, M. L.; Auer, R. N.; Siesjo¨, B. K. The density and distribution of ischemic brain injury in the rat following 2–10 min of forebrain ischemia. Acta Neuropathol. 64:319–332; 1984. 30. Squire, L. R. Memory and brain. Oxford, England: Oxford University Press; 1987:56–74, 109–123. 31. Summers, W. K.; Majovski, L. V.; Marsh, G. M.; Tachiki, K.; Kling, A. Oral tetrahydroaminoacridine in long-term treatment of senile dementia. N. Engl. J. Med. 315:1241–1245; 1986. 32. Takagi, N.; Tsuru, H.; Yamamura, M.; Takeo, S. Changes in striatal dopamine metabolism after microsphere embolism in rats. Stroke 26:1101–1106; 1995. 33. Takeo, S.; Taguchi, T.; Tanonaka, K.; Miyake, K.; Horiguchi, T.; Takagi, N.; Fujimori, K. Sustained damage to energy metabolism of brain regions after microsphere embolism in rats. Stroke 23:62–68; 1992. 34. Tucek, S. Problems in the organization and control of acetylcholine synthesis in brain neurons. Prog. Biophys. Mol. Biol. 44:1–46; 1984. 35. Yamamoto, M.; Tamura, A.; Kirino, T.; Shimizu, M.; Sano, K. Behavioral changes after focal cerebral ischemia by left middle cerebral artery occlusion in rats. Brain Res. 452:323–328; 1988.
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Changes in Cholinergic Neurons and Failure in Learning Function After Microsphere Embolism-Induced Cerebral Ischemia NORIO TAKAGI, KEIKO MIYAKE, TAKU TAGUCHI, NAOKO SUGITA, KAORI TAKAGI, HIROAKI TAMADA, AND SATOSHI TAKEO* Department of Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, 192-03, Japan
ABSTRACT: Central cholinergic neurons play an important role in learning and memory functions. The present study was undertaken to elucidate the pathological changes in learning function and acetylcholine metabolism of the cerebral cortex and hippocampus, following microsphere embolism in rats. Microspheres (48 mm) were injected into the right internal carotid artery of the rats. Learning function was determined using a passive avoidance task on the seventh day after the embolism. In the biochemical study, acetylcholine and choline contents, and choline acetyltransferase activity were measured in the cerebral cortex and hippocampus. Cortical acetylcholinesterase-containing fibers were quantitatively estimated in the embolized rat. Passive avoidance was impaired in the microsphere-embolized rat. Microsphere embolism decreased the acetylcholine concentration and choline acetyltransferase activity in the cerebral cortex and hippocampus. In the histochemical study, the length of cortical acetylcholinesterase-containing fibers was decreased, but cell density was unchanged in the ipsilateral hemisphere of the microsphere-embolized rat. The results suggest that microsphere embolism induces severe damage to cholinergic neurons, which may be related to the impairment of learning function in the ischemic brain. Q 1997 Elsevier Science Inc.
information is available, however, concerning the relationship between learning deficits and pathological changes in cholinergic neuron in cerebral embolism, which mimics sustained brain ischemia and eventually leads to multiinfarct pathology. In previous studies, we have shown that microsphere embolism induced a marked decrease in cerebral blood flow and a pronounced disturbance of energy metabolism in the brain regions, demonstrating that microsphere embolism was capable of inducing progressive and sustained cerebral ischemia and/or oligemia [22,33]. It is suggested that microsphere-induced multiple small infarction resembles clinical vascular dementia more closely than the infarction induced by middle cerebral artery occlusion in terms of pathophysiological alterations induced [23]. Studies on the learning function and cholinergic system following microsphere embolism may, therefore, provide a better understanding of neuronal disturbance in multiinfarction. The purpose of the present study was to elucidate the pathological changes in learning function and biochemical profile of cholinergic neurons in the cerebral cortex and hippocampus, which are vulnerable to ischemia [15,24,29]. The morphological profile of cortical cholinergic neurons of the microsphere-embolized rat was also examined.
KEY WORDS: Cerebral ischemia, Microsphere embolism, Cholinergic neuron, Learning function, Rats.
MATERIALS AND METHODS Operation for Microsphere Embolism
INTRODUCTION
One hundred and fifty-three male Wistar rats weighing 180 to 220 g (Charles River Japan, Inc., Atsugi, Japan) were used in the present study. The animals were maintained under artificial conditions at 23 { 17C with a constant humidity of 55 { 5% with a cycle of 12-h light and 12-h dark, and had free access to food and tap water according to the Guideline of Experimental Animal Care issued by the Prime Minister’s Office of Japan. Microsphere-induced cerebral embolism was performed by the method previously described [32]. Briefly, 88 rats were anesthetized with sodium pentobarbital (35 mg/kg, IP) and fixed in the supine position on an operation plate. After cervical incision, the right common carotid artery was isolated. The right
It is well recognized that cortical and hippocampal cholinergic input originates in the nucleus basalis of Meynert and in the medial septum in primates, respectively [4], and that the cholinergic system in the central nervous system plays an important role in learning and memory functions [26]. A failure in cortical cholinergic activity has been shown to underlie the memory loss associated with aging and Alzheimer’s disease [3,31]. In rodents, lesions of cholinergic neuron and pharmacological manipulations of the cholinergic system have been correlated with altered cognitive performance [1,9,25]. It has also been reported that hypoxia induces a reduction of memory and judgment that is associated with a decrease in acetylcholine synthesis [8]. Little * To whom requests for reprints should be addressed.
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external carotid and right pterygopalatine arteries were ligated with string. A polyethylene catheter (3F size, 1.0 mm in diameter, Atom Co., Tokyo) was inserted into the right common carotid artery. Nine hundred microspheres (47.5 { 0.5 mm in diameter; NEN-005, New England Nuclear Inc., Boston, MA) suspended in 20% dextran solution were injected into the right internal carotid artery through this cannula. Fifty-one rats that had undergone a sham operation were injected with same volume of vehicle without microspheres. The control group comprised 14 nonoperated rats. Neurological Deficits Sixteen hours after the operation, the behavior of the rats was scored on the basis of paucity of movement, truncal curvature, and forced circling during locomotion, which are considered to be typical symptoms of stroke in rats [7,21]. The score of each item was ranked from 3 to 0 (3, very severe; 2, severe; 1, moderate). The rats which had more than seven points were considered to be type A, six to four type B and less than four type C. We used only type A animals in the following study. Passive Avoidance Response Rats were tested in a step-through type passive avoidance task [12]. The experimental apparatus consists of two compartments (illuminated one: 20 1 20 1 20 cm, dark one: 20 1 20 1 20 cm equipped with a grid floor), divided by a guillotine door (8 1 8 cm). In the acquisition trial, the rat was placed in the illuminated compartment and allowed to enter the dark compartment after opening of the guillotine door. As soon as the rat entered the dark compartment, the guillotine door was closed and an inescapable footshock (2.0 mA, 5 s) was delivered through the grid floor. In the retention test, the rats were placed in the illuminated compartment and the time taken to enter the dark compartment was measured. Acetylcholinesterase-Containing Fibers Morphological changes in the cholinergic pathway of the microsphere-embolized rat were measured by acetylcholinesterase histochemistry [10]. After passive avoidance task, 11 microsphere-embolized rats were perfused via the heart with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After immersion in cold fixative for 1 day, the brain was transferred through several changes of 30% sucrose. Frozen sections were cut on a cryostat (20 mm). Sections were rinsed in 0.1 M acetate buffer (pH 6.0), and incubation was performed for 40 min in the following medium: 25 mg acetylthiocholine iodide, 32.5 ml of 0.1 M sodium acetate buffer (pH 6.0), 2 ml of 0.1 M sodium citrate, 5 ml of 0.03 M cupric sulfate, 9.5 ml of distilled H2O, and 1 ml of 5 mM potassium ferricyanide. After five changes of acetate buffer, sections were treated with 1% ammonium sulfide solution for 1 min followed by five changes of 0.1 M sodium nitrate. Sections were then exposed to 0.1% silver nitrate followed by five changes of 0.1 M sodium nitrate. Free-floating sections were rinsed and mounted. Sections were viewed with light microscope (OLYMPUS BH-2) and a photograph of each section was taken. Quantitative analysis of the acetylcholinesterase histochemistry was carried out with an aid of the Image 1.56 software program running on a Macintosh Centris 660AV. In brief, the two-tone gray image of the acetylcholinesterase containing axons was extracted from the original 256 gray-tone image, which was taken by scanner (SCANTOUCH, Nikon), by binarization. Then, binarized image was converted to the twotone gray image of axons and then to a binary line image with
1-pixel width. Measurement was made on the basis of length of the binary line image with 1-pixel width in a selected field. The sections were counterstained by cresyl violet and cells were counted in the same field as above. Measurement of Acetylcholine and Choline Contents The animals were sacrificed with a focal irradiation of microwave to the head with a strength of 5 kW for 0.85 s by a microwave applicator (TMW-6402C, Muromachikikai Co., Tokyo). After decapitation, the head of the animals was immersed in liquid nitrogen and left for 10 s (near freezing). The cerebral hemisphere was isolated, and the cerebral cortex and hippocampus of both hemisphere were separated. The tissues were homogenized in 0.2 M HClO4 and 0.01% disodium EDTA with a Polytron homogenizer (PT-10, Kinematica, Switzerland). The homogenate was centrifuged at 10,000 1 g for 15 min at 47C. The supernatant fluid was filtered through a membrane filter (0.45 mm). A 5 ml aliquot of the supernatant fluid was applied to a HPLCECD to determine the concentrations of acetylcholine and choline. The HPLC-ECD system was composed of enzymatic column (Eicompak AC-gel / AC-enzymepak; Eicom, Kyoto), a model L-6000 pump (Hitachi, Japan) and an electrochemical detector (ECD-100, Eicom). The mobile phase contained 0.1 M phosphate buffer at pH 8.5, containing 1.2 mM sodium 1-decanesulfonate (Tokyo Kasei, Tokyo) and 0.6 mM tetramethylammonium chloride (Nacalai Tesque, Kyoto). Measurement of Choline Acetyltransferase Activity Choline acetyltransferase (ChAT) activity was determined according to the method of Kaneda and Nagatsu [13]. The cortex and hippocampus of both hemispheres were homogenized by Teflon homogenizer (20 updown) in 12.5 ml of cold 25 mM phosphate buffer, pH 7.4, containing 0.5% Triton X-100, per gram of wet weight. The homogenate was centrifuged at 20,000 1 g for 60 min. The ChAT activity in the supernatant fluid was assayed. For the assay, 100 ml substrate solution containing 10 mM choline chloride, 0.4 mM acetyl CoA, 0.2 mM eserine sulfate, 0.3 mM sodium chloride, and 20 mM EDTA-2Na in 0.1 M sodium phosphate buffer, pH 7.4 was added to 100 ml of enzyme solution in 25 mM sodium phosphate buffer, pH 7.4, and incubated at 377C for 20 min. The enzyme reaction was stopped using 50 ml of 1 M perchloric acid in ice. After 10 min, 6 ml of 1.0 mM isopropylhomocholine, as an internal standard, was added and the mixture was centrifuged at 1,600 1 g for 10 min at 47C. The supernatant fluid was filtered through a membrane filter (0.45 mm). A 5-ml aliquot of the supernatant fluid was applied to a HPLC-ECD as described above to determine the concentration of acetylcholine. Protein concentration of the supernatant fluid was determined according to the method of Lowry et al. [19]. Statistical Analysis The results are expressed as mean { SEM, except for the stepthrough latency, which is expressed as mean { SD. Statistical significance between different groups was evaluated by Student’s t-test and Mann–Whitney U-test (two tailed). Analysis of variance followed by Dunnett’s multiple comparison was employed for evaluation of significance of the changes in time course of variables. p õ 0.05 was considered to be statistically significant. RESULTS Microsphere Embolism Fifteen (17%) rats out of 88 rats that were injected with microspheres died before their symptoms were assessed (within
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Choline Acetyltransferase Activity, Acetylcholine, and Choline Contents
FIG. 1. The step-through latency of the passive avoidance response of the sham-operated and microsphere-embolized rat on the seventh day after the operation. Each value represents the mean { SD of 11 experiments. *Significantly different from sham-operated group (p õ 0.05).
16 h of the embolism). Microsphere injection induced type A strokelike symptoms in 61 (69%) of the operated rats. Among these rats, 10 (16%) of type A rats died by the third day after the operation. Eight (9%) rats showed type B symptoms, and four (5%) rats showed type C symptoms. The sham-operated rats showed no strokelike symptoms and all survived. Passive Avoidance Task We confirmed that the response of the microsphere-embolized rat to electric shock, such as startle or crouch, on the seventh day was comparable with that of the sham-operated rat. The behavioral score of microsphere-embolized rats on the seventh day after the operation was 2.1 { 0.5. Analysis of the passive avoidance acquisition time of shamoperated (18.5 { 11.5 s; mean { SD) and of microsphere-embolized (21.1 { 14.4 s; mean { SD), which were carried out 7 days after the operation, revealed no group differences. The latency period of the step-through, which was carried out 24 h after the acquisition test, in the passive avoidance task of microsphereembolized and sham-operated rats are shown in Fig. 1. The latency of step-through in microsphere-embolized rats was significantly shortened compared to sham-operated rats. Acetylcholinesterase-Containing Fiber Density and Cell Density of the Microsphere-Embolized Rat On the seventh day after the microsphere embolism, a significant loss of acetylcholinesterase-containing fibers in the right frontal cortex was seen compared with the left frontal cortex (Table 1). In contrast, there were no differences in the cell density of both frontal cortices of the microsphere-embolized rat (Table 1).
Figure 2 shows the time courses of changes in ChAT activities of the cerebral cortex and hippocampus of both hemispheres in microsphere-embolized and sham-operated rats. ChAT activity in the brain regions of the right hemisphere was unchanged on the first day after the microsphere embolism. Thereafter, microsphere embolism induced a significant decrease in ChAT activity of the right hemisphere throughout the experiment. The time course of the changes in acetylcholine and choline contents of the cerebral cortex and hippocampus of the both hemispheres in microsphere-embolized and sham-operated rats are shown in Figs. 3 and 4, respectively. In the sham-operated rats, there were no appreciable changes in acetylcholine and choline contents of either hemisphere throughout the experiment. In the microsphere-embolized rat, a marked reduction in acetylcholine content in the brain regions of the right hemisphere was seen on the first day after the operation. Thereafter, the acetylcholine content remained at a low level. The time course of changes in acetylcholine content of the left hemisphere in the microsphereembolized rats revealed a similar trend to that of the right hemisphere, but the changes were of a smaller magnitude. In microsphere-embolized rats, a marked increase in choline content of the right cerebral cortex and hippocampus was seen on the first day after the operation. Thereafter, the choline content of these regions returned toward the control level. DISCUSSION The protocol of the passive avoidance task used in the present study eliminates disability of behavior caused by neurological deficits in the microsphere-embolized rat, because neurological deficits disappeared almost completely 7 or more days after the operation [33]. We also confirmed that the response of the microsphere-embolized rat to electric shock 7 or more days after the operation did not differ from that of the control or shamoperated rat in the present study. It has been reported that the sensitivity to electric shock of the microsphere-embolized rat is normal 1 or 2 weeks after the operation [16]. Thus, it appears that the impairment in the passive avoidance response using electric shock is not due to a decrease in sensitivity to the electric shock and/or disability of behavior caused by neurological deficits, but due to a failure in learning function. Microsphere-embolized rats showed an impairment of passive avoidance response in the present study. Impairments of passive avoidance task have been reported in several ischemic models [11,17,35]. In transiently ischemic rats, an impairment of passive avoidance learning, which disappears with time after the ischemia [2], matches the period of disturbance of brain energy metabolism. Therefore, an impairment of the passive avoidance task may be, in part, attributed to transient dysfunction of the central TABLE 1 ACETYLCHOLINESTERASE-CONTAINING FIBER DENSITY AND CELL DENSITY OF THE MICROSPHERE-EMBOLIZED RAT
Right hemisphere Left hemisphere
AChE-Positive Fiber Density (mm/mm2 Frontal Cortex)
Cell Density (1103/mm2 Frontal Cortex)
60.7 { 7.0* 102.9 { 8.8
1.408 { 0.069 1.257 { 0.039
Each value represent the mean { SEM of 11 experiments. * Significantly different from the left hemisphere ( p õ 0.05).
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TAKAGI ET AL. examined 12 weeks after ischemia, despite the presence of appreciable postischemic hippocampal damage [17]. It cannot be determined, however, whether damage to the hippocampal cholinergic neurons is responsible for the impairment of passive avoidance response in transiently ischemic rats. In the present study, microsphere embolism resulted in a sustained decrease in acetylcholine content and ChAT activity in the hippocampus, suggesting that the hippocampal cholinergic system and/or structure may be severely impaired. The disturbance of learning function in the microsphere-embolized rat may, at least in part, relate to dysfunction of hippocampus and/or hippocampal cholinergic system. It is recognized that cortical cholinergic innervation plays an important role in cognitive function in humans [3]. Cortical cholinergic neurons [5] that receive large afferent neuron from the nucleus basalis magnocellularis located near the globus pallidus
FIG. 2. The time course of changes in choline acetyltransferase activity in the cerebral cortex and hippocampus of the right and left hemispheres of microsphere-embolized ( l and j ), sham-operated ( s and h ), and control ( s and h at day 0) rats, respectively. Each value represents the mean { SEM of five to seven experiments. *Significantly different from control group (p õ 0.05).
nervous system due to disturbance of energy metabolism in the ischemic brain [17]. Because microsphere embolism induces a sustained impairment of brain energy metabolism [33], an impairment of energy metabolism in the microsphere-embolized rat may result in a deterioration of memory function. In contrast to transiently ischemic models, the step-through latency of the middle cerebral artery-occluded rat is shortened for a period of 16 weeks [35]. This suggests that differences in ischemic conditions or ischemic models results in differences in the avoidance response changes. From the present result on avoidance task, it appears that microsphere embolism induces an impairment of learning function. It is generally considered that the hippocampus plays an important role in the formation and preservation of memory [30]. Furthermore, the septohippocampal cholinergic system is considered to play a pivotal role in the memory processes [11]. It has been reported, however, in rats that exposure to transient forebrain ischemia did not alter the passive avoidance task when
FIG. 3. The time course of changes in acetylcholine content in the cerebral cortex and hippocampus of the right and left hemispheres of microsphere-embolized ( l and j ), sham-operated ( s and h ) and control ( s and h at day 0) rats, respectively. Each value represents the mean { SEM of five to seven experiments. *Significantly different from control group (p õ 0.05).
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FIG. 4. The time course of changes in choline content in the cerebral cortex and hippocampus of the right and left hemispheres of microsphere-embolized (l and j), sham-operated (s and h), and control ( s and h at day 0) rats, respectively. Each value represents the mean { SEM of five to seven experiments. *Significantly different from control group (p õ 0.05).
[20] are believed to play a role in learning function in animals. It is also suggested that the impairment of passive avoidance performance defect in nucleus basalis-lesioned rats is caused by cholinergic neuron loss [6]. It is hypothesized, therefore, that the cortical cholinergic system plays an important role in learning function in animals. The role of morphological changes in cortical cholinergic neurons in microsphere-embolized rats is, however, little understood. In the present study, a loss of acetylcholinesterase-containing fibers was detected in the ipsilateral frontal cortex of the microsphere-embolized rat, but cell density remained unchanged. It has been shown that acetylcholinesterasecontaining fibers in the frontal cortex on the ipsilateral hemisphere is significantly reduced in the middle cerebral artery-occluded rat, suggesting that there is a disruption of the cholinergic pathway between the frontal cortex and the nucleus basalis [14]. Some noncholinergic neurons may also be stained by the method used in the present study, because acetylcholinesterase is not a specific marker for determination of the cholinergic pathway [18]. It is conceivable, however, that the loss of acetylcholinesterase-containing fibers in the frontal cortex of the microsphere-embolized rat represents a disruption of the neu-
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ronal innervation in the frontal cortex and, in part, results in an alteration of learning function. Furthermore, we observed that microsphere embolism decreased the acetylcholine content and ChAT activity of the right cerebral cortex during most periods of the experiment, although the ChAT activity was unchanged on the first day after the embolism. Acetyl-CoA, required for ACh synthesis, is normally generated by oxidation of pyruvate by the pyruvate dehydrogenase complex [34], and pyruvate availability is known to be decreased in ischemic conditions [28]. In our previous study, microsphere embolism induced a sustained impairment of cerebral energy metabolism [33]. The decrease in acetylcholine concentration of the ipsilateral hemisphere on the first day may, therefore, be attributed to low levels of acetyl-CoA concentration due to impaired energy metabolism rather than altered ChAT activity. After the third day, it is likely that the decrease in acetylcholine concentration is related to both low level of acetylCoA and low activity of ACh synthesis regulated by ChAT. In the present study, microsphere embolism induced an increase in the choline content of the right hemisphere throughout the experiment. It is possible that an increase in choline content of the microsphere-embolized rat is not attributable to breakdown of acetylcholine, but is a result of hydrolysis of phospholipids [27]. In the present study, the ACh contents in the contralateral hemisphere were decreased, although the activity of ChAT and choline contents did not change. This result may indicate that cerebral ACh is the most sensitive biochemical parameter among cholinergic variables under ischemic and/or oligemic conditions. Furthermore, we showed in a previous study [33] that microsphere embolizm induced sustained damage to cerebral energy metabolism in the contralateral hemisphere, but to a lesser degree than the ipsilateral hemisphere. Thus, it is likely that the decrease in ACh contents of contralateral hemisphere contributes to low levels of acetyl-CoA concentration due to impairment of cerebral energy metabolism in the contralateral hemisphere of the microsphere-embolized rat. The results in the present study suggest that sustained and severe impairment of acetylcholine metabolism occurs in the microsphere-embolized rat. Pharmacological studies support the cholinergic specificity of the passive avoidance defect caused by lesions in the nucleus basalis [1,9,25]. Thus, it is suggested that the disturbance of cortical acetylcholine metabolism and/or the loss of cholinergic fibers in the microsphere-embolized rat lead to a deterioration of cortical cholinergic neurotransmission, and may contribute to an impairment of learning function. In conclusion, we demonstrate in the present study that microsphere embolism induce an impairment of passive avoidance response and the loss of acetylcholinesterase-containing fibers in the ipsilateral frontal cortex. Furthermore, we showed a decrease in acetylcholine content and ChAT activity in the ipsilateral hemisphere of the microsphere-embolized rat. These results suggest that a disturbance of cholinergic neurons by microsphere embolism is comparable to the pathophysiological alterations of multiinfarction. ACKNOWLEDGEMENTS
This work was in part supported by grant-in-aid for general scientific research C from the Japanese Ministry of Education, Science and Culture.
REFERENCES 1. Aaltonen, M.; Riekkinen, P.; Sirvio¨, J.; Riekkinen, P. Jr. Effects of THA on passive avoidance and spatial performance in quisqualic
/ 2a37 2303 Mp 91 Friday May 30 08:17 AM EL–BRB (v. 42, no. 5) 2303
92
2.
3. 4. 5.
6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
TAKAGI ET AL. acid nucleus basalis-lesioned rats. Pharmacol. Biochem. Behav. 39:563–567; 1991. Bothe, H. W.; Bosma, H. J.; Hofer, H.; Hossmann, K.-A.; Angermeier, W. F. Operant learning, cerebral blood flow and neurohistology in Meriones unguiculatus after unilateral carotid artery ligation. In: Meyer, J. S.; Lechner, H.; Reivich, M.; Ott, E. O., eds. Cerebral vascular disease, vol. 4, Amsterdam: Excerpta Medica; 1983:250– 255. Coyle, J. T.; Price, D. L.; Delong, M. R. Alzheimer’s disease: A disorder of cortical cholinergic innervation. Science 219:1184– 1190; 1983. Divac, I. Magnocellular nuclei of the basal forebrain project to neocortex, brain stem, and olfactory bulb. Review of some functional correlates. Brain Res. 93:385–398; 1975. Dubois, B.; Mayo, W.; Agid, Y.; Le Moal, M.; Simon, H. Profound disturbances of spontaneous and learned behaviors following lesions of the nucleus basalis magnocellularis in the rat. Brain Res. 338:249–258; 1985. Dunnett, S. B.; Whishaw, I. Q.; Jones, G. H.; Bunch, S. T. Behavioural biochemical and histochemical effects of different neurotoxic amino acids injected into nucleus basalis magnocellularis of rats. Neuroscience 20:653–669; 1987. Furlow, T.; Bass, N. H. Arachidonate-induced cerebrovascular occlusion in the rat. Neurology 26:297–304; 1976. Gibson, G. E.; Duffy, T. E. Impaired synthesis of acetylcholine by mild hypoxic hypoxia or nitrous oxide. J. Neurochem. 36:28–33; 1981. Haroutunian, V.; Kanof, P.; Davis, K. L. Pharmacological alleviation of cholinergic lesion induced memory deficits in rats. Life Sci. 37:945–952; 1985. Hedreen, J. C.; Bacon, S. J.; Price, D. L. A modified histochemical technique to visualize acetylcholinesterase-containing axons. J. Histochem. Cytochem. 33:134–140; 1985. Hirakawa, M.; Tamura, A.; Nagashima, H.; Nakayama, H.; Sano, K. Disturbance of retention of memory after focal cerebral ischemia in rats. Stroke 25:2471–2475; 1994. Itoh, J.; Ukai, M.; Kameyama, T. Dynorphin A-(1–13) potently prevents memory dysfunctions induced by transient cerebral ischemia in mice. Eur. J. Pharmacol. 234:9–15; 1993. Kaneda, N.; Nagatsu, T. Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection. J. Chromatogr. 341:23–30; 1985. Kataoka, K.; Hayakawa, T.; Kuroda, R.; Yuguchi, T.; Yamada, K. Cholinergic deafferentation after focal cerebral infarct in rats. Stroke 22:1291–1296; 1991. Kirino, T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 39:57–69; 1982. Kiyota, Y.; Miyamoto, M.; Nagaoka, A.; Nagawa, Y. Cerebral embolization leads to memory impairment of several learning tasks in rats. Pharmacol. Biochem. Behav. 24:687–692; 1986. Kiyota, Y.; Miyamoto, M.; Nagaoka, A. Relationship between brain damage and memory impairment in rats exposed to transient forebrain ischemia. Brain Res. 538:295–302; 1991.
18. Kristt, D. A. Acetylcholinesterase in the cortex. In: Jones, E. G.; Peters, A., eds. Cerebral cortex, vol. 6. New York: Plenum Publishing Corp.; 1987:187–236. 19. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randal, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951. 20. Luiten, P. G. M.; Gaykema, R. P. A.; Traber, J.; Spencer, D. G. Jr. Cortical projection patterns of magnocellular basal nucleus subdivisions as revealed by anterogradely transported Phaseolus vulgaris leucoagglutinin. Brain Res. 413:229–250; 1987. 21. McGraw, C. P. Experimental cerebral infarction: effect of pentobarbital in mongolian gerbils. Arch. Neurol. 34:334–336; 1977. 22. Miyake, K.; Takeo, S.; Kajihara, H. Sustained decrease in brain regional blood flow after microsphere embolism in rats. Stroke 24:415–420; 1993. 23. Naritomi, H. Experimental basis of multi-infarct dementia: memory impairments in rodent models of ischemia. Alzheimer Dis. Assoc. Disord. 5:103–111; 1991. 24. Pulsinelli, W. A.; Brierley, J. B.; Plum, F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11:491–498; 1982. 25. Riekkinen, P., Jr.; Sirvio¨, J.; Riekkinen, M.; Riekkinen, P. Effects of THA on passive avoidance retention performance of intact, nucleus basalis, frontal cortex and nucleus basalis / frontal cortexlesioned rats. Pharmacol. Biochem. Behav. 39:841–846; 1991. 26. Sarter, M. F.; Bruno, J. P. Cognitive functions of cortical ACh: Lessons from studies on trans-synaptic modulation of activated efflux. Trends Neurosci. 17:217–221; 1994. 27. Scremin, O. U.; Jenden, D. J. Focal ischemia enhances choline output and decreases acetylcholine output from rat cerebral cortex. Stroke 20:92–95; 1989. 28. Siesjo¨, B. K. Brain energy metabolism. New York: John Wiley & Sons; 1978. 29. Smith, M. L.; Auer, R. N.; Siesjo¨, B. K. The density and distribution of ischemic brain injury in the rat following 2–10 min of forebrain ischemia. Acta Neuropathol. 64:319–332; 1984. 30. Squire, L. R. Memory and brain. Oxford, England: Oxford University Press; 1987:56–74, 109–123. 31. Summers, W. K.; Majovski, L. V.; Marsh, G. M.; Tachiki, K.; Kling, A. Oral tetrahydroaminoacridine in long-term treatment of senile dementia. N. Engl. J. Med. 315:1241–1245; 1986. 32. Takagi, N.; Tsuru, H.; Yamamura, M.; Takeo, S. Changes in striatal dopamine metabolism after microsphere embolism in rats. Stroke 26:1101–1106; 1995. 33. Takeo, S.; Taguchi, T.; Tanonaka, K.; Miyake, K.; Horiguchi, T.; Takagi, N.; Fujimori, K. Sustained damage to energy metabolism of brain regions after microsphere embolism in rats. Stroke 23:62–68; 1992. 34. Tucek, S. Problems in the organization and control of acetylcholine synthesis in brain neurons. Prog. Biophys. Mol. Biol. 44:1–46; 1984. 35. Yamamoto, M.; Tamura, A.; Kirino, T.; Shimizu, M.; Sano, K. Behavioral changes after focal cerebral ischemia by left middle cerebral artery occlusion in rats. Brain Res. 452:323–328; 1988.
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