ischemic neuronal damage specific to monkey hippocampus: histological investigation

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Brain Research Bulletin, Vol. 37, No. 1, 73-87, 1995 pp. Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserwl

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lschemic Neuronal Damage Specific to Monkey

Hippocampus: Histological Investigation

EIICHI TABUCHI,* TAKETOSHI ONO,*’ HISAO NISHIJO,* SHUNRO ENDOt AND SHOUGO KUZES

Departments of *Physiology, tNeurosurgery, and #Anesthesiology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-07, Japan

[Received 29 August 1994; Accepted 21 October 19941

ABSTRACT: We previously reported lesions confined specifically to the hippocampus when produced by occluding eight vessels (the bilateral vertebral, common, internal, and external carotid arteries), which supply blood to the brain. However, his- topathological changes in the primate brain, caused by ischemic injury, have not previously been thoroughly investigated. In the present study, macaque monkeys were subjected to 5-18-min ischemia by occluding the eight vessels. After the brains were perfused and fixed 5 days after the occlusion, all regions were histologically investigated for ischemic cell changes. lschemia for 5 min produced no ischemic cell change. lschemia for IO-15 min produced cell death limited to the deeper portion of the pyramidal cell layer of the CA1 subfield in the hippocampus. In most monkeys, no cell death was observed in any brain region outside of the hippocampus after ischemia for up to 15 min. lschemia for 18 min produced more widespread cell death in the CA1 subfield of the hippocampus, and cell death was no longer confined to the hippocampus. but was observed in layers Ill, V, and VI of the neocotices, the striatum, and some other regions. Brains that were perfused and fixed 1 year after 1 C-min ischemic insult revealed no ischemic cell morphological change in any region, but the number of pyramidal cells in the CA1 subfield was decreased to about half. The results indicate that the CA1 subfield of the monkey hippocampus is the precise region of the brain most susceptible to ischemic insult in the primate forebrain, and after a critical time (15-min ischemia in this procedure) ischemic cell changes occur suddenly and extensively. lschemia due to occlusion of eight arteries for lo-15 min could produce a model of human amnesia caused by transient isch- emit insult.

KEY WORDS: Brain ischemia, Cresyl violet, Luxol fast blue.

INTRODUCTION

Neurons in the central nervous systems of gerbils and rats have been reported to be selectively vulnerable to ischemic injury [8,11,23,40,46]. The hippocampus, the neocortex, and the stria- turn are known to be selectively vulnerable brain regions [8,12,42]. Among these regions, the CA1 subfield of the hippocampus was particularly susceptible to ischemic damage [23,42]. In humans, also, pyramidal neurons in the CA1 subfield are among the regions most vulnerable to ischemia [38]. There have been a few reports of histological changes due to ischemic injury

’ To whom requests for reprints should be addressed.

in the primate brain [7,33], but these results differed somewhat from recent reports for animals and for humans. Profound arterial hypotension caused by combined intravenous injection of hy- potensive drugs, blood withdrawal, and head-up tilt, produced ischemic necrosis in the arterial boundary zones of the cerebral cortices, the cerebellum, the basal ganglia, and the hippocampus of monkeys [7]. It is considered that damage in the arterial boundary zones was a consequence of the anastomosis of small vessels with their counterparts from adjacent arterial fields after loss of autoregulation of blood flow by some combination of reduced perfusion pressure and hypoxemia [8]. Complete monkey brain ischemia for 16 min by a tourniquet wrapped around the neck plus simultaneous drug-induced hypotension produced severe and variable ischemic changes: infarction, neuronal necrosis, and edema [33]. The brain damage was most severe in the brain stem, and slightly less in the cerebral cortices, and relatively still less in the hippocampus. However, the severity of damage is far stronger than, and damaged areas are quite different from, recent reports with the same technique [45,60]. It may relate insuffi- ciency of some postoperative treatments and lack of maintenance of some physiological values.

When studying ischemic damage in human or nonhuman sub- jects, we must consider the elapsed time between the ischemic insult and the histological investigation. Cell damage has been reported to be inversely related to the time elapsed from ischemia to histology [14]. This has been explained by almost complete autolysis or phagocytosis of damaged cells within a certain period after ischemic insult. This disappearance of damaged cells was verified in the present study, by comparing acute histological ischemic change with long-term effects. Therefore, it is difficult to determine in detail which parts of the brain were damaged in most human cases when histology usually followed long after the ischemic insult.

A primate ischemic model with lesions limited exclusively to the hippocampus, especially the CA1 subfield, was recently reported [34,52]. It was produced by lo- 15min occlusion of eight major arteries that supply the brain. Lesions limited mostly to the hippocampus, but with some other minor lesions due to ischemic insult have been reported in primates [4,45,60] and humans [13,30,53,58]. However, there has been no report of histological study of primates to investigate relations between ischemic severity and the extent or grade of damage in different regions. In

73

74 TABUCHI ET AL.

the present report, we present a more detailed and complete ac- count of the effects of transient monkey forebrain ischemia. We describe the results of histological investigation of ischemic cell changes in brain regions both inside and outside of the monkey hippocampus. Correlation between histological severity and duration of ischemia in the CA 1 subfield of the hippocampus, plus the histological change during the time elapsed from ischemic insult until histological investigation are also described.

In addition, amnesic syndromes produced by transient brain ischemia, such as that caused by anesthetic accident, carbon mon- oxide intoxication, and cardiopulmonary arrest have been reported [ 1,26,30]. Clinicopathological investigation has shown that hippocampal damage due to transient global ischemia is re- sponsible for anterograde amnesia [ 13,38,58]. Lesion studies in primates also confirm the critical role of the hippocampal formation in memory formation and temporal storage of memory [48,59]. Our model is produced to be an animal model of human amnesia, and is expected to aid in the assessment of memory disorders that are due to lesions confined to the hippocampus. The amnesic syndrome produced in this model is discussed in another report now being prepared.

METHOD

Materials and Surgery

Sixteen macaque monkeys (Macacafiscata; male 13, female 3) weighing 6.2- 10.1 kg were used. Surgical procedures to oc- clude eight vessels were described in a previous paper [52]. Briefly, on the day of surgery, a monkey was anesthetized with a gas mixture of 0.7- 1.0% halothane, 66% N20, and the balance oxygen. The lungs were manually ventilated through a 6.0 mm inner diameter endotracheal tube by an anesthetist (S.K.). During the operation, the monkeys continuously received Ringer’s so- lution with lactate at 1 ml per min through a peripheral IV cath- eter. Their general condition was continuously monitored for electrocardiogram, heart rate, blood pressure, mean blood pressure, rectal temperature, electroencephalogram, expiratoty Pco, (refer to the second article on data analysis).

Following the latest surgical procedures, the skin around the neck was incised at the midline. The bilateral vertebral, and common carotid arteries, and the internal and external carotid arteries were then exposed by an anterior approach to the cervix by an experienced neurosurgeon (S.E.). After completing their expo- sure, these eight vessels were clamped with vascular clamps in the following order, as simultaneously as possible by two people (T.O. and S.E.) (Fig. 1). The occlusion time was recorded from the beginning of clamping of the bilateral common carotid arteries and ended at the removal of these clamps. The numbers of monkeys and times of ischemia were: 2 for 5 min, 4 for 10 mm, 1 for 13 min, 4 for 15 min, and 2 for 18 min. In addition, three control monkeys were sham operated; they were subjected to the complete procedure except that no arteries were occluded. These three monkeys were returned to their cages without ischemic insult.

In the one monkey subjected to 5-min and one of the four subjected to IO-min ischemia, only four vessels-the bilateral vertebral and common carotid arteries-were clamped. These two monkeys were the first two examined [52]. The eight-vessel occlusion was used mainly to block collateral blood flow from the inferior thyroid artery to the superior thyroid artery (Fig. 1). After removal of the occlusions, blood flow to the brain was resumed and the surgical wound was closed. Anesthesia was discontinued and the monkeys were returned to their cages after recovery of EEG signals.

Rectal temperature was kept at 37.0 2 1 .O”C. The temperature did not significantly change before, during, and after ischemia, both within individuals @ > 0.1, ANOVA) and in a group (p > 0.1, ANOVA). The end tidal Pco2 was maintained within the range of 35-45 mmHg under the anesthetist’s manual control, while the monkeys continued their spontaneous respiration during surgery, or when spontaneous respiration stopped transiently for less than 5 min in a few monkeys during ischemia. During the operations, the loss of blood was less than 5 ml. When the occlusion started, face paleness and mild hemi- or bilateral eye pupil opening appeared transiently in most monkeys. All animals recovered consciousness several minutes after anesthesia was discontinued, and there was no postoperative convulsion. All monkeys were treated in strict compliance with National Insti- tutes of Health and the Society for Neuroscience policy on the humane care and use of laboratory animals.

Histological Investigation

Five days after the surgery, the animals were deeply anesthetized with 60 mg/kg pentobarbital sodium (IM), following 10 mg/kg ketamine (IM). Under artificial respiration, they were sacrificed by transcardial perfusion with 1.0 liter of so- lution composed of 0.8% sodium chloride, 0.8% sucrose, 0.4% glucose, and 10 units/ml heparin in 0.05 M phosphate buffer (pH 7.4), and then fixed with 3.0 liter fixative composed of 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Based on previous reports [ 12,23,38], we assumed that ischemic cell changes and differ- ences would be greatest and clearest in the hippocampus and other brain regions by the fifth day after ischemia. However, one monkey subjected to IO-min ischemia and one subjected to 15min ischemia were sacrificed 4 months and 1 year after the ischemia, respectively, to investigate chronic histological and behavioral consequences.

After removal, the brain was kept in the same fixative at 4.O”C for 7 days. The whole brain divided into three blocks was embedded in paraffin, after dehydration in a series of al- cohol over 4-7 days, dealcoholization by methyl benzoate with 1% celloidine and xylene for 1 day, and replacement of xylene with paraffin for 2 days. Coronal 6 pm sections of the whole brain were then made with a microtome (Yamato, US- 11) with microtome blades (Feather, S35L type). After ex- panding in warm water at 40-5O”C, they were mounted on glass slides coated with a mixture of chicken egg albumen and glycerin. The specimens were ordinarily stained with cresyl violet or by Kltiver-Barrera (cresyl violet and Luxol fast blue) stain, after replacing the paraffin with xylene followed by al- cohol. Some specimens were stained with hematoxylin and eosin. Sections including the hippocampus were examined at 100 pm intervals, and others were examined at 200 ,um intervals. All regions of the brain were investigated for the exis- tence and the degree of ischemic cell changes macroscopically and microscopically in detail (Tables 1 and 2).

Normal cells in the pyramidal layer of the CA1 subfield were counted under a light microscope in a 250 pm X 500 pm strip 1 .O mm lateral to the boundary between the CA 1 and the CA3 subfields in every monkey, by individuals blinded to the treatment. The average pyramidal cell density in the right and left CA1 subfields of monkeys, calculated from eight sections 11 to 13 mm anterior to the interaural line, was assessed by Student’s t-test. The neuropathology was assessed by procedures reported previously [6,8,10,14,57]. Ischemic cell changes seen in the present study were: dilatation of perineural space, shrincage of cell body, cell death, and increase of glial

ISCHEMIC DAMAGE SPECIFIC TO HIPPOCAMPUS 75

Glandula thyreoidea. Vi

A.carotfs

A.carotis interna

A.carotis externa

A.thyreoidea inferior

,A.vertebralls

Truncub brachioceDhalicus

A.subclavia sinistra

A.carotfs communis einistr

Arcus aortae

Aurlcula dextra

runcus pulmonaris Auricula SiniStra

entriculus sinister

FIG. 1. Generalized schema of main arteries supplying the monkey brain. A. Vertebralis, A. carotis communis, intema, and extema were transiently occluded bilaterally by vascular clamps, to produce brain ischemia. A. carotis intema and extema were occluded mainly to interrupt the collateral flow from A. thyreoidea inferior to A. thyreoidea superior.

cells. Infarction, necrosis, and gliosis were not observed in all monkeys. Cell death was defined by nuclear pyknosis, a sen- sitive indicator of anoxia, which start with chromatin aggre- gation, followed by dilation of the nuclear membrane and chromatin condensation [6].

RESULTS

Subdivisions of Brain Regions

Tables 1 and 2 summarize the severity of the neuropatbolog- ical changes in each brain region for the various periods of isch-

76 TABUCHI ET AL.

TABLE 1

HISTOPATHOLOGY IN THE HIPPOCAMPUS, CEREBRAL CORTICES, STRIATUM, AND CEREBELLAR CORTEX

lschemic Period (min) (Number of Monkeys) Brain Region

Hippocampus

CA1 - ++-+++ +++ +++

CA3 - --+ - --•+f

Dentate gyrus

Polymorphic cell layer - --++ _ --++

Granule cell layer - _ _ _

Molecular layer - - _ -

Subiculum _ _ _ _

Entorhinal cortex - + _ +

Cerebral cortices (Layers III, V, VI)

Frontal cortex - + _ +

Temporal cortex - + _ _

Parietal cortex - + _ -

Occipital cortex - + _ +

Striatum

Caudate - _ - _

Putamen - _ - +

Globus pallidus - --+ _ _

Cerebellar cortex

Molecular layer - _ _ -

Purkinje cell layer - --+ _ -

Granule cell layer - _ _ _

t++++ ++++

++++ _

++ ++ +

+-I-

++

++

+

++

++

++

_

_

_

The severity of cell damage was assessed as: -, normal; +, cell shrinkage and/or dilation of perineural space, but less than I % cell death; ++, l-30% cell death; +++, 30-50% cell death; ++++, 50-80% cell death; + + + + +, more than 80% cell death. Ischemic cell changes were bilaterally equal in each region.

* In three of three and two of three animals tested. no cell death was observed in any brain region, except the hippocampus.

emia. Table 1 shows the histopathology in the hippocampus, the cerebral cortices, the striatum, and the cerebellar cortex. Each subdivision was investigated separately: the hippocampus- CA1 subfield, CA3 subfield, dentate gyrus (polymorphic cell layer, granule cell layer, and molecular layer), subiculum (in- clude presubiculum and prosubiculum), and entorhinal cortex; the cerebral cortices-frontal cortex (rostral, dorsal, ventral, medial, lateral, and caudal), temporal cortex (rostral, ventral, medial, lateral, and caudal), parietal cortex (dorsal, medial and lateral), occipital cortex (dorsal, ventral, medial, lateral, and caudal); the striatum-caudate (rostral, ventral, dorsal, medial, and caudal), putamen (rostra& ventral, dorsal, medial, lateral, and caudal), globus pallidus (internal and external); the cerebellar cortex (molecular layer, Purkinje cell layer, and granule cell layer). Other brain regions investigated (Table 2) were: nucleus of diagonal band of Broca (DBB), claustrum, septum (medial and lateral), nucleus accumbens, preoptic area, amygdala (corticomedial and basolat- eral), thalamus (anterior nuclei (A), ventral lateral nuclei (VL), ventral posterior nuclei (VP), intralaminar nuclei (IL), subthalamic nucleus (ST), mediodorsal nucleus (MD), medial and lateral geniculate bodies (MGB and LGB), nucleus reticularis, pulvinar), nucleus ruber, substantia nigra, hypothalamus (anterior (AHA), and lateral hypothalamic areas (LHA), ventromedial (VMH) and posterior hypothalami (PH)), nucleus mammillaris, reticular formation, nucleus pontis, nucleus centralis superior, nuclei raphes, substantia grisea centralis (SGC), nucleus olivaris, nucleus colliculus superior (CS) and inferior (CI), and nucleus parabrachialis. These formations are according to descriptions by Brodal, [9], Jones [21], and Amaral [2].

Macroscopic Changes

The lateral ventricles were enlarged in brains subjected to more than IO-min ischemia. The other macroscopic change was atrophy throughout the cerebral cortex after 1%min ischemia (Fig. 2C). Coronal sections of the monkey brain are shown in Fig. 2. The control monkey brain, sectioned coronally at a level that included the hippocampus (11 mm anterior to the interaural line), was intact and small lateral ventricles could be seen dorsal to the hippocampus (Fig. 2A, arrowheads). The coronal brain section prepared after 15-min ischemia showed equal bilateral enlargements of the lateral ventricle (Fig. 2B, arrowheads); after 18-min ischemia, the lateral ventricles were more enlarged (Fig. 2C, arrowheads), and the cerebral cortex appeared clearly atrophic. In Fig. 2, the darker areas were neural fibers stained with Luxol fast blue, and the lighter areas were cortices and some nuclei stained with cresyl violet. Coronal sections of the hippocampal formation shown in Fig. 3, were subdivided into the dentate gyrus (DG), the CA3 subfield (CA3), the CA1 subfield (CAl), and the subiculum (S). The sections are: control (A), after 15min (B), and after 1%min (C) ischemia. The hippocampal formation after 15min ischemia seemed to be compressed ventrally by the enlarged lateral ventricle (Fig. 3B), and after 1%min ischemia it was compressed more than after 15 min.

Microscopic Changes in the Hippocampus

Microscopic changes observed in the CA1 subfield on the fifth day after ischemic insult for 0,5, 10, 13, 15, and 18 min are shown in Fig. 4. A normal pyramidal cell in the CA1 subfield has a large

ISCHEMIC DAMAGE SPECIFIC TO HIPPGCAMPUS 77

TABLE 2

HISTOPATHOLOGY IN BRAIN REGIONS OTHER THAN THOSE IN TABLE 1

Ischemic Period (min) (Number of Monkeys) Brain Region

5 IO 13 15 18 (2) (3) (1) (3) (2)

DBB

Septum

N. accumbens

Preoptic area

Claustrum

Amygdala

Thalamus

A, VL, VP, IL, ST

MD

MGB, LGB

N. reticularis Pulvinar

N. tuber

Substantia nigra

Hypothalamus

N. mammillaris

Reticular formation

N. pontis

N. centralis superior N. raphes

SGC

N. olivaris

cs, Cl N. parabrachialis

_

_ - - - _ -

- _ _ _ _ _ _

-+ _ - - _ _ _ - _ _

_ _ _ _

+ +

+ _

--+ +

+ -

+ _ _ _

+ _

+ _

+ _ _

_ _

+ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ -

--+ +

_

+ + +

_

+ _ _ - _ _

_ _

+ _ _ _

+ +

++ ++ ++ +

++ +++ ++ _ - _

++ + _ _ _ _ _

+ _ _ _

Abbreviations: DBB; nucleus of diagonal band of Broca. A; anterior nuclei, VL; ventral lateral nuclei, VP; ventral posterior nuclei, IL; intralaminar nuclei, ST; subthalamic nucleus, MD; mediodorsal nucleus of thalamus. MGB and LGB; media1 and lateral geniculate bodies. AHA and LHA; anterior and lateral hypothalamic areas, VMH and PH; ventromedial and posterior hypothalami. SGC; substantia grisea centralis. CS and CI; nucleus colliculus superior and inferior.

rounded nucleus with a nucleolus and its small surrounding peri- karyon, stained with cresyl violet (Fig. 4A). Negligible ischemic cell change was observed after S-min ischemia (Fig. 4B). Darkly stained acidophilic, shrunken cell bodies with dilatation of the perineural space, i.e., pyknotic nuclei, were observed in deeper portions of the pyramidal cell layer after lO-min ischemia (upper part of the photo- micrograph in Fig. 4C). Cell death similar to that after lO-min ischemia was observed in the corresponding region after 13-min ischemia (Fig. 4D). After 15-min ischemia, cell bodies were more darkly stained and shrunken, and had more dilated perineural space than after lO- and 13-min ischemia (Fig. 4E). Extensive cell loss, showing collapsed neural cell bodies and fragmentation, and almost no normal pyramidal cells were observed after 18-min ischemia (Fig. 4F).

The average normal pyramidal cell density (neurons in a 250 pm x 500 pm stop) of the right and left CA1 subfields in each ischemic period was shown in Fig. 5. The mean number of the CA1 pyramidal cells in the control (0-min occlusion) was similar to that after 5-min occlusion. However, the numbers decreased significantly after more than l@min ischemia (p < 0.05), compared with those of control. After 18-min occlusion, few normal cells could be found. The duration of ischemia correlated with the neuronal density (Pearson correlation coefficient = -0.875, p < 0.001).

Microscopic changes in the hippocampus, except in the CA1 subfield are shown in Fig. 6. The granule cell layer and poly-

FIG. 2. The monkey brain sectioned coronally at a level that includes the hippocampus. (A) Intact monkey brain (control). (B) Corona1 brain section 5 days after 15min ischemia. (C) Corona1 brain section 5 days after l8-min ischemia. Lateral ventricle was macroscopically enlarged in 1%min ischemia (B), and more enlarged in Il-min ischemia (C) (arrowheads). Stained by Kltiver-Barrera method. All sections at 11 mm anterior to the interaural line. Bar = 1 cm.

78 TABUCHI ET AL.

BIG. 3. Coronal section of the hippocampal formation. (A) Intact hippocampus (control). (B) Hippocampal formation 5 days after 15min &hernia. The hippocampal formation seems to be compressed ventrally by lateral ventricle enlargement. (C) Hippocampal formation 5 days after 1%min ischemia. The hippocampus is more compressed ventrally than that after 1%min ischemia. DC, dentate gyms; CA 1, CA 1 subfield of the hippocampus; CA3, CA3 subfield of the hippocampus; S, subiculum. Sectioned at 11 mm anterior to the interaural line. Arrowheads, the bor- der between CA3 and CAI subfields. Bar = 1 mm.

morphic cell layer of the dentate gyrus are shown in Fig. 6A-C. Rounded nuclei with nucleoli and relatively dark-stained perikarya can be seen in both cell layers (Fig. 6A). Even after 15 mitt ischemia, no ischemic cell change was observed in most

monkeys (Fig. 6B), although ischemic cell changes were observed occasionally in the polymorphic cell layer of the dentate gyrus of one monkey after IO-min ischemia and another one after 15 min. However, extensive cell death was observed in the polymorphic cell layer and throughout the molecular layer of the dentate gyrus (arrowheads) after 18-min ischemia, but granule cells were well preserved (Fig. 6C). The CA3 subfields are shown in Fig. 6D-F. Normal pyramidal cells in the CA3 subfield have histological characteristics similar to those in the CA1 subfield,

and the cells are more compactly distributed than in the CA1 subfield (Fig. 6D). No ischemic cell change was observed even after 15min ischemia (Fig. 6E). Ischemic cell changes and extensive cell death were observed and cells with well-preserved perikarya were fewer after 18-min ischemia (Fig. 6F).

Microscopic Changes in Other Brain Regions

The temporal cortex in the control, and after 15 and 18-mitt ischemia revealed no ischemic change under low magnification. High magnification photomicrographs of layers V and VI in the temporal cortex are shown in Fig. 7. Polymorphic cells can be identified in layers V and VI in the control (Fig. 7A). Nuclei and perikarya of the cell bodies were still well preserved after 15 min ischemia (Fig. 7B). In the III, V, and VI layers of neocorti- ces, mild dilatation of perineural space and mild shrinkage of cell bodies with slight increment of glial cells were often and diffusely observed. Ischemic cell changes including cell death were observed frequently and diffusely in layers III, V, and VI of the overall cerebral cortices after 18-min ischemia (Fig. 7C). Cell death, collapse, and fragmented cell bodies, can be seen in Fig. 7D, an enlarged section of Fig. 7C. In addition, neural cells with glial cells, morphological astrocytes, were frequently seen around perineural spaces in cerebral cortices (Fig. 7D, arrows) and some other regions after ischemic insult after any occlusion period. Ischemic changes in the putamen and cerebellar cortex, which are highly vulnerable to brain ischemia, are shown in Fig. 8. Figures 8A-C are photomicrographs of the putamen. Small cells with rounded nuclei and nucleoli were diffuse in the control (Fig. 8A). No ischemic cell change was observed after 15-min ischemia (Fig. 8B). Occasionally, mild dilation of perineural space and shrinkage of cell bodies were also observed in the striatum, as well as in the claustrum, globus pallidus, thalamus, substantia nigra, nuclei raphes, etc. Darkly stained areas show perforans fibers stained with Luxol fast blue. Ischemic cell changes and fragmentation of cells were extensively observed after 18-mitt ischemia (Fig. 8C). Figures 8D-F are photomicrographs of the cerebellar cortex. The molecular layer (upper part of each plate), Purkinje cells (arrowheads), and granule cell layer (lower part of each plate) appear normal in the control (Fig. 8D). No ischemic cell change was observed after 15-min ischemia (Fig. 8E). In the cerebellar cortex, no ischemic changes were observed even after 18-min ischemia (Fig. 8F).

Histological Changes at Chronic State Afrer Ischemic Insult

Histological changes 1 year after 15-min ischemic insult are shown in Fig. 9. No ischemic cell change was observed in the CA1 subfield of the hippocampus, but pyramidal cells in the deeper part of the pyramidal cell layer were diminished and the number of cells in the CA1 subfield decreased to about half (19.7 -+ 3.1, n = 8) (Fig. 9A). Histologically, the evidence suggests that damaged cells might have been phagocytosed or au- tolyzed, but the remaining proportion of normal cells was about equal to the proportion (about half) 5 days after an equal occlusion period. Layers V and VI in the temporal cortex (Fig. 9B), putamen (Fig. 9C), and cerebellar cortex (Fig. 9D) appear to be

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TABUCHI ET AL,

Right

cl Left

Control 5 min 10 min 13 min 15 min 18 min

lschemia

FIG. 5. Number of surviving pyramidal cells in the CA1 subfield of the hippocampus as a function of time of occlusion. Normal cells in the pyramidal cell layer of the CA1 subfield were counted under a light microscope in a 250 pm X 500 pm strip 1 .O mm lateral from the boundary between the CA1 and the CA3 subfields. The mean number of the CA1 pyramidal cells in control (0-min occlusion) was similar that after 5-min occlusion. The numbers decreased significantly after 10 min or longer occlusion. After IS-min occlusion, few normal cells were found. There was no significant difference between the number of normal cells in the right CA1 subfield and those in the left CA1 subfield in any ischemic period @ > 0.05, respectively). * and **, p < 0.05 and p < 0.01 between control and each ischemic period. Numbers in parentheses, numbers of monkeys used for each point. Mean 2 SD.

intact. Histological changes due to lo-min ischemia in the chronic state, 4 months after ischemic insult, were similar to those of 15min ischemia, but the number of cells in the CA1 subfield was reduced about l/3 (32.8 + 4.5, n = 8).

Increase of Glial Cells

Various small portions of perineural glial cells (Fig. 7D, arrows), probably astrocytes, were diffuse throughout many regions, mainly in layers III, V, and VI of the cerebral cortices, the striatum, and the claustrum, after ischemia of any duration, but increase of glial cells in the hippocampus was rare. No mi- croglial proliferation was evident in the present study.

Behavioral Changes by Ischemic Insult

Behavior of the monkeys was observed until fixation and perfusion of their brains, ordinarily for 5 days after the occlusion. Behavioral changes were evident in four monkeys. Two monkeys subjected to 5- and 15min ischemia showed transient distur- bance of motor coordination, but recovered completely by the third day after the maneuver. One monkey subjected to 15min &hernia developed transient dyskinesia, and hypalgesia was evident when it was pricked with a pin. Its grasping power and motor coordination were diminished, and escape behavior from noxious stimulation became dull or absent, but recovery from these symptoms was complete within 1 month. Feeding behavior was near normal after 1 week. The monkey subjected to 18-min ischemia had, in addition to the other symptoms, apparent ag- nosia. This monkey did not respond to or attempt to escape from

a gorilla mask, which monkeys ordinarily dislike and show a fear response to. The appetites of most monkeys decreased the first day after the maneuver, but after the second day, they recovered and could eat their regular diet and fruits, and drink water nor- mally with no assistance.

Summary of Histology

Five-minute ischemia produced no ischemic cell change except a slight increment of glial cells that appeared morphologically to be astrocytes, mainly in layers III, V, and Vl of the cerebral cortices and the striatum. After lo- to 15-min ischemia, cell changes were small and relatively mild in every brain region except the hippocampus, and cell death was confined to the hippocampus, especially to the CA1 subfield (Table 1). About l/3 to 1Q of the cells in the CA1 subfield appeared to be damaged (Figs. 4C-E and 5). In addition, no cell death was observed anywhere, other than the hippocampus, after lO- to 15-min occlusion, except in one brain that was occluded for 10 min and one that was occluded for 15 min. Cell death was diffusely and sparsely observed in some parts of layers JII, V, and VI of the frontal and occipital cotices, putamen, entorhinal cortex of the hip pocampus, most thalamic nuclei, and so on, but the damage was mild and was restricted to less than 1% of the total region. Cells of the polymorphic cell layer of the dentate gyms in the hippocampus were damaged in the two monkeys mentioned above. The damage was limited to the boundary area of the polymorphic cell layer. Death of some cells was also observed in the CA3 subfield of one monkey subjected to 15-min ischemia. Ischemia for 18-min pmduced severe cell death in the hippocampus, but cell death was no longer confined to the hippocampus. Damage was observed in layers III, V, and VI

FIG

. 6.

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82 TABUCHI ET AL.

FIG. 7. High magnification photomicrographs of layers V and VI in the temporal cortex. (A) Control. (B) After 15min ischemia, nuclei, and perikarya of cell bodies were well preserved. (C) After 1%min ischemia, ischemic cell changes and cell death were observed frequently and diffusely in layers III, V, and VI throughout the cerebral cortices. (D) After ll-min ischemia, with double the magnification of C, collapsed and fragmented cell bodies were observed. In addition, neural cells with glial cells around the perineural space (arrows) were frequently seen in many brain regions after ischemic insult, not only after 18 min but also after shorter occlusion times. Bar = 100 ,um.

of tbe cerebral cortices, the shiatum, the mediodorsal nucleus of the thalamus, and other regions listed in Tables 1 and 2. In the hippocampus, there was extensive cell loss in the CA1 subfield, and few or no normal pyramidal cells were found (Figs. 4F and 5). The CA3 subfield

(Fig. 6F) and the polymorphic cell layer of the dentate gyms (Fig. 6C) were also severely damaged.

The ischemic cell changes described here were observed bilaterally in all regions. No obvious difference in ischemic damage between the

FIG

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84 TABUCHI ET AL.

PIG. 9. Microscopic changes 1 year after 15-min ischemia. (A) CA1 subfield of the hippocampus. No ischemic cell change was observed, but the number of pyramidal cells was decreased about half. Pyramidal cells in deeper portion of pyramidal layer were fewer. Layers V and VI in the temporal cortex (B), putamen (C) and cerebellar cortex (D) were intact. Bar = 100 pm.

right and the left hemispheres could be determined in any region. Infarction, necrosis, and gliosis were not observed anywhere in all monkeys.

DISCUSSION

Vulnerable Brain Regions to Ischemia

It has been reported that in the gerbil and the rat, the regions most vulnerable to ischemic damage were the CA1 subfield in the hippocampus, layers III, V, and VI of the cerebral cortices and the striatum [43,46]. Even in humans, those regions are com- monly known to be the most susceptible to ischemic injury [10,38]. It is also reported that in the gerbil, bilateral carotid occlusion for 5 min always produced hippocampal lesions [23]. Although lesions in the CA1 subfields of rodents and human were

extensive and irreversible, the development of neuronal death in the CA1 subfield is characteristically slower than in other hippocampal areas or brain regions [12,24,38]. This delayed phe- nomenon was also suggested in human [38]. In relations between severity of ischemia and time of appearance of ischemic damage, it is known that the damage appeared slowly after weak ischemic insult, but rapidly after severe ischemia [20,23]. Therefore, the damage in the CA1 subfield of macaque monkeys in this study was expected to appear slowly, because of the relatively mild ischemia. We, thus, sacrificed the monkeys on the fifth day, by which time the histological evidence of brain ischemia was expected to be maximum, and ischemic cell change could be observed.

Purkinje cells in the cerebellar cortex are considered to be

ISCHEMIC DAMAGE SPECIFIC TO HIPPGCAMPUS 85

among the most vulnerable to ischemic injury [8]. However, the cerebellar cortex was almost completely intact even after 18 min of ischemia in the present study. In a similar model of rats with four vessels occluded, autoradiographic study revealed cerebral blood flow in the cerebellar cortex [41]. This blood flow would have been maintained through the spinal artery and muscle branches [Xl.

Histological Changes Corresponding to Ischemic Period

In the macaque monkeys, lo- to 15min ischemia produced lesions confined exclusively to the CA1 subfield of the hippocampus [52]. However, possibly reversible ischemic cell changes which were dilatation of perineural space, shrinkage of cell bodies, and increase in the number of glial cells, were slightly evident in layers III, V, and VI of the cerebral cortices, the striatum, and some other regions in brains that had received lo- to 15min ischemia. They are indicators for the degenerative process of ischemic cells, but cell death could not be truly identified by such indicators, because they did not accompany nuclear pyknosis. Dead or dying cells were identifiable by shrunken, dark, and often fragmented nuclei in the optic tectum of the developing rat [3].

After 18-min ischemia, cell death appeared in many regions, and was most severe in the CAI subfield of the hippocampus where almost no pyramidal cells were survived. The next most severe damage was in the CA3 subfield and the polymorphic cell layer of the dentate gyrus where about 30% of the cells were normal. Third, was the mediodorsal nucleus of the thalamus. where damage was almost 30%. This nucleus had been reported to be a region particularly vulnerable to ischemic injury [8]. Cells in the cerebral cortices, the striatum, and parts of several other regions suffered about lo-20% damage. Thus, the damage by 18-min ischemia was histologically severe, and morphological and functional recovery was never complete, at least by the fifth day after ischemic insult.

there are many case reports of, among other things, cardiac arrest, hypotension, oxygen deprivation, insulin coma, and so on, producing ischemic cerebral damage [8,13,54].

Our method of brain ischemia can minimize operative inva- sion of the monkeys. Bleeding is slight and less troublesome than that produced by some other methods. One major distinction of the present monkey model is that the damage is exclusively confined to the hippocampus, which is not, of itself, believed to be related to motor function or perception [31,48,56,59]. Therefore, postoperative care is easier because the lesions are restricted. In addition, we anesthetized the monkeys with a mixture of halothane, N20, and O2 to avoid, as much as possible, the protective effects of some induction and anesthetic drugs, such as ketamine and pentobarbital [5,44]. Histological outcome was clearly correlated with the ischemic period, although blood pressure was not controlled and possible protection of anesthetics was probably not completely avoided. It seems that control of blood pressure, at least within normal range, is not effective on histopath- ological results when ischemia is incomplete.

The results in the present study were less severe than those reported for complete brain ischemia in monkeys [17,33]. It is suggested that the occlusion of eight arteries, with no control of blood pressure, produced incomplete ischemia, because blood could be supplied to the brain through the spinal arteries and some collaterals through muscles [55]. It has been ascertained by enhanced angiographs that contrast medium injected into the subclavian artery appeared in cerebral arteries during the occlusion of eight arteries (Ono et al., unpublished data).

Histological Changes at Chronic State After Ischemic Insult

Macroscopically, more or less lateral ventricle enlargement appeared in all monkeys with hippocampal lesion, and the hippocampus seemed to be compressed ventrally. This was probably due, not only to atrophy or loss of pyramidal cells in the CA1 subfield of the hippocampus, but also to transient intracranial hypertension after reperfusion [5 11. The lateral ventricle was larger than the size estimated to be due to atrophy of the CA1 subfield, and the ventricle beyond the hippocampus was also enlarged. Similar macroscopic change has also been revealed by magnetic resonance imaging in amnesic patients with hippocampal lesion [39,49]. The hippocampal formation in non-Korsakoff amnesic patients was markedly reduced in size, and the lateral ventricles were enlarged.

Comparison With Other Models or Human Case Reports of Brain Ischemia

Various methods can be used to produce cerebral ischemia in vertebrates. In primates, complete ischemia by a combination of a tourniquet around the neck and drug-induced hypotension [33,45,50,60], intrathoracic clamping of the brachiocephahc and left subclavian arteries, and ligature of the internal mammary artery [ 181, arterial hypotension [27], and prolonged epileptic seizures induced by injecting alkaloid bicuculline [28] have all produced cerebral lesions. In rodents, occlusion of bilateral carotid and vertebral arteries (four vessels) in rats [37,40,46] and occlusion of bilateral carotid arteries in gerbils 112.22.321 lead

The CA1 subfield of the monkey brain received 15-rnin ischemia, which was perfused and fixed 1 year after, rather than 5 days after, ischemic insult, revealed no ischemic cell change, but the number of pyramidal cells was decreased to half. This proportion was similar to that in the CA1 subfield 5 days after ischemia. It indicates that the extent of damage was maximal at 5 days and did not further mature over a period of 1 year. Damaged cells disappear almost completely if given enough time [6]. We must carefully consider this disappearance of damage evidence after ischemic insult in any brain region.

Giial Cells’ Response to Ischemia

Glial cells that were morphologically identified as astrocytes, were frequently and diffusely observed in the present study. They often appeared around perineural spaces in many brain regions that received ischemic injury, but rarely in the hippocampus. This might be related to the vulnerability of the hippocampus. Astro- cytes are important in the regulation of the neural networks [8,36]. Early proliferative changes in astrocytes, i.e., the imme- diate increases in mitochondria and endoplasmic reticulum, suggest increased metabolic activity in the postischemic brain [36]. Thus, astrocytes might control neural cell recovery or death [43]. It is plausible that after neural cells are exposed to ischemic insult, astrocytes try to deliver substrates for the production of energy to the damaged cells and induce cell recovery if resuscitation is possible, but if this is impossible, the astrocyte will stop the delivery of energy to that cell and try to induce recovery of another cell.

Brain Function and Ischemic Damage _ _

to cerebral ischemia. The four-vessel occlusion was also used in primates and some other vertebrates [ 15,19,55]. Even in humans

Because dead cells never recover morphologically, functions performed by the dead cells ceased temporarily or permanently.

86 TABUCHI ET AL.

Compensation for damaged functions, through plastic change, might be observed in less injured regions, such as the cerebral cortex and the striatum after 18&n brain ischemia. It is reported that memory storage in the nervous system is specifically and causally linked to particular kinds of experience [ 16,471. It is also reported that neurons have different forms of synaptic plasticity that may be involved in different processes of learning [29]. The plasticity of functioning neurons can lead to the recovery of processes, including memory, that have been damaged by ischemic insult. This suggests that small lesions in particular regions, such as those in the cerebral cortex and the striatum after 18-min ischemia, whether produced by ischemia or another cause, might not permanently affect functioning of the brain.

ACKNOWLEDGEMENTS

We thank Dr. A. Simpson, Showa University, for help in preparing the manuscript, and Ms. M. Yamazaki and Ms. A. Tabuchi for typing. This work was supported partly by the Japanese Ministry of Education, Science and Culture Grants-in-Aid for Scientific Research, 06454706, 06680786, and 05267103, by Research Grants of Human Frontier Sci- ence Program for third fiscal year, and by Uehara Memorial Foundation.

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