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Electron microscopic investigation of the cerebral cortex after cerebral ischemia and reperfusion in the gerbil
Brain Research, 598 (1992) 87-97 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
87
BRES 18326
Electron microscopic investigation of the cerebral cortex after cerebral ischemia and reperfusion in the gerbil Hidekazu Tomimoto and Takehiko Yanagihara Department of Neurology, Mayo Clinic and Mayo Foundation, Rochester, MN 55905 (USA) (Accepted 14 July 1992)
Key words: Cerebral cortex; Cerebral ischemia; Electron microscopy; Gerbil
Prompt dendritic damage has been observed in the hippocampus of the gerbil brain after transient cerebral ischemia. In the present study, we studied the frontoparietal cortex of the gerbil brain electron microscopically after brief bilateral carotid occlusion to assess the vulnerability of dendritic processes. After ischemia for 5 min, there was swelling of the periphery of dendrites accompanied by swelling of mitochondria, cytoplasmic vacuolation and disintegration of microtubules in layer I, which spread to layer III after ischemia for 20 min. After reperfusion for 3-24 h following ischemia for 20 min, swelling in the periphery of dendrites and of mitochondria inside receded but vacuole formation and disintegration of microtubules propagated proximally. In neuronal perikarya, polyribosomal disaggregation was observed after ischemia for 20 min and persisted thereafter, while fragmentation of rough endoplasmic reticulum (ER) and microvaeuolation occurred after reperfusion for 3 h. Electron-dense clumping of neuronal perikarya was observed after reperfusion for 6 h particularly in layers III and Vb, which increased in number for up to 72 h. The observed progressive damage in dendrites may be common to neurons vulnerable to cerebral ischemia and may significantly contribute to development of delayed neuronal death.
INTRODUCTION
the layer V a r e very v u l n e r a b l e to global a n d r e g i o n a l ischemia 3"I°A6'32'34'36. Since n e u r o n s in t h e c e r e b r a l cor-
N e u r o n s e x p o s e d to ischemic insult m a y lose t h e i r s t r u c t u r a l integrity soon a f t e r r e s t o r a t i o n o f b l o o d flow with s h r i n k a g e a n d h y p e r c h r o m a s i a o f n e u r o n a l p e r i k a r y a a n d f o r m a t i o n o f t r i a n g u l a r n u c l e u s 2,4,17. W h i l e t h e s e c h a n g e s c a n o c c u r in any n e u r o n s in the b r a i n a n d soon a f t e r ischemic insults, s o m e a r e a s of t h e brain develop delayed neuronal death after brief i s c h e m i a t3'14'22. T h e m e c h a n i s m for d e l a y e d n e u r o n a l
tex a r e h e t e r o g e n e o u s in t h e i r s t r u c t u r e s including t h e i r d e n d r i t i c p r o c e s s e s as c o m p a r e d to t h o s e in the CA1 r e g i o n o f t h e h i p p o c a m p u s , we s t u d i e d layers I - V I o f the f r o n t o p a r i e t a l cortex o f t h e gerbil b r a i n by using t r a n s m i s s i o n e l e c t r o n microscopy. T h e results o f the p r e s e n t investigation have b e e n r e p o r t e d in abstract form 27.
death remains uncertain, and the structural derangem e n t l e a d i n g to d e l a y e d n e u r o n a l d e a t h has not b e e n well e l u c i d a t e d , b e c a u s e m o s t p r e v i o u s efforts have b e e n c o n c e n t r a t e d on t h e u l t r a s t r u c t u r a l a l t e r a t i o n s o f t h e p y r a m i d a l cell b o d i e s in t h e h i p p o c a m p u s 14'2~. O u r p r e v i o u s investigations in t h e h i p p o c a m p u s o f t h e gerbil b r a i n including t h e CA1 r e g i o n r e v e a l e d p r o m p t swelling o f t h e p e r i p h e r y o f apical a n d b a s a l d e n d r i t e s a n d p r o p a g a t i o n o f s t r u c t u r a l d a m a g e s to the p r o x i m a l part during progressive cerebral ischemia and reperfusion 31'33. In t h e c e r e b r a l cortex, n e u r o n s in layers
MATERIALS AND METHODS
I l l / I V a n d t h e layer just b e n e a t h t h e large n e u r o n s o f
Mongolian gerbils (Meriones unguiculatus) of both sexes weighing 60-80 g were used for the present investigation. Each gerbil was allowed free access to food and water before and after surgery. Under inhalation anesthesia with ether, the common carotid arteries were exposed bilaterally in the neck and occluded by miniature Mayfield aneurysmal clips for 5, 10 and 20 min I°. For investigation of the effect of reperfusion, the clips were released after 3, 6, 12, 24 and 72 h following ischemia for 20 rain. Three gerbils were used for each ischemic and postischemic period. Sham operated gerbils had exposure of both common carotid arteries without occlusion and were allowed to live for 20 min or 72 h. At a predetermined ischemic or postischemic period, each gerbil was reanesthetized with ketamine hydrochloride (60 mg/kg) and perfused through the left ventricle of the heart with 0.02% phosphate buffered saline at pH 7.4 for 20 s
Correspondence: H. Tomimoto, Department of Neurology, Kyoto University Hospital, Kyoto 606, Japan.
88 and then with 2% paraformaldehyde-2% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4 for 10 min. After decapitation, the head was immersed in the same fixative overnight. On the following day, the skull was removed and the brain was cut into blocks and fixed for an additional day. Coronal sections of the frontoparietal cortex including the caudoputamen were further sectioned at 100 # m thickness with a vibratome and kept in 0.1 M PB (pH 7.4) overnight and further fixed for 1 h in 2% osmium tetroxide in 0.1 M PB (pH 7.4). After rinsing in 0.1 M PB for 1 h, each section was dehydrated in 60% ethanol, 70% ethanol containing 2% uranyl acetate and 80-100% ethanol and finally propylene oxide. Each section was flat-embedded in Spurr's embedding medium and narrow strips of the frontoparietal cortex were dissected perpendicularly. Semithin sections (1 ~m) containing each layer were prepared and stained with 1% Toluidine blue to identify layers I-VI. Adjoining ultrathin sections were further sectioned with an ultramicrotome and contrasted with 2% lead citrate and examined under a transmission electron microscope (Phillips 201).
RESULTS
Light microscopic findings There was no vacuole formation or any other abnorm a l i t i e s in s e m i t h i n s e c t i o n s f r o m s h a m o p e r a t e d bils (Fig.
1A,B). After
ischemia
for 5 and
ger-
10 m i n ,
v a c u o l a t i o n o c c u r r e d i n l a y e r I, f o r m i n g a h o n e y c o m b appearance.
It was most notable adjacent to the pia-
arachnoid membrane. more
prominent
The honeycomb
appearance
was
a f t e r i s c h e m i a f o r 20 m i n ( F i g . 1C).
H o w e v e r , n e u r o n a l p e r i k a r y a w e r e n o t a f f e c t e d in a n y layer after ischemia (Fig.
1D). A f t e r
f o r 20 m i n w i t h o u t
reperfusion
reperfusion
f o r 3 h, v a c u o l a t i o n
in
Fig. 1. Light microscopic photographs of layer I (A,C,E) and layer III (B,D,F) in a sham operated gerbil (A,B), after ischemia for 20 min without reperfusion (C,D) and after ischemia for 20 min and reperfusion for 24 h (E,F). The honeycomb appearance was clearly visible in the area facing the suharachnoid space after ischemia for 20 min (C) but only mildly after reperfusion for 24 h (E). Neuronal perikarya in the layer III was essentially normal after ischemia for 20 rain (D) but some of them (arrows) were clumped after reperfusion for 24 h (F). Semithin sections were stained with Toluidine blue. The original photographs were taken at × 400.
89 layer I was less notable but neuronal perikarya were occasionally swollen in layers III and Vb. Vacuolation in layer I markedly receded after reperfusion for 6 h or longer (Fig. 1E), but clumped neuronal perikarya having hyperchromasia with Toluidine blue began to evolve in layers III and Vb after reperfusion for 6 h. The
number and severity of degenerated neurons increased notably after reperfusion for 24 and 72 h (Fig. 1F).
Electron microscopic findings There was no vacuole formation or any other abnormalities in semithin sections from sham operated ger-
Fig. 2. Electron micrographs of layer I in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). After ischemia for 20 min, the periphery of dendrites and mitochondria inside became markedly swollen even without reperfusion (B). Arrowheads indicate the location of postsynaptic densities, the marker for dendrites. Swelling receded already after reperfusion for 3 h (C) and markedly reduced after reperfusion for 24 h (D). Bar = 1 ~tm.
90
Fig. 3. Electron micrographs of layer II in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). Microtubules in some dendrites were partially disintegrated after ischemia for 20 min without reperfusion (B), which became obvious after reperfusion for 3 h (C). Tortuous dendrites with disorganized and disintegrated microtubules were seen after reperfusion for 24 h (D). Bar = 1 /zm.
bils (Fig. 2A). A f t e r ischemia for 5 a n d 10 min, swelling of d e n d r i t i c p r o c e s s e s was o b s e r v e d in layer I. A similar but m o r e p r o n o u n c e d c h a n g e was o b s e r v e d after ischemia for 20 min ( T a b l e I). Swollen d e n d r i t i c p r o cesses as positively i d e n t i f i e d by the p r e s e n c e o f postsyn a p t i c densities, c o n t a i n e d d i s t e n d e d vacuoles filled
with a m o r p h o u s m a t e r i a l s , swollen m i t o c h o n d r i a a n d d i s i n t e g r a t e d m i c r o t u b u l e s . M e m b r a n e limited inclusions, h e r e t e r m e d microvacuoles, w e r e p r e s e n t inside the d e n d r i t e s in layer I. T h e y h a d an e m p t y a p p e a r ance a n d w e r e e n c i r c l e d by a m o n o l a y e r of i r r e g u l a r m e m b r a n e (Fig. 2B). Swelling of m i t o c h o n d r i a and
91
Fig. 4. Electron micrographs of layer III in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). Polyribosomes were disaggregated in some neuronal perikarya even after ischemia without reperfusion (B), which were more complete and widespread after reperfusion for 3 h (C). Rough ERs were fragmented and the density of attached polyribosomes was markedly reduced. After reperfusion for 24 h, some neuronal cell bodies were already degenerated to electron dense cell debris (D). Bar = 1/xm.
d i s i n t e g r a t i o n o f m i c r o t u b u l e s w e r e o b s e r v e d in l a y e r I a f t e r i s c h e m i a for 5 min a n d in layer I a n d less freq u e n t l y in layers II a n d I I I a f t e r i s c h e m i a for 10 a n d 20 m i n ( T a b l e I a n d Fig. 3B). N o a p p a r e n t c h a n g e was o b s e r v e d in the n e u r o p i l o f layers I V - V I (Figs. 5B a n d
7B). H o w e v e r , d i s a g g r e g a t i o n o f p o l y r i b o s o m e s was o b s e r v e d in a small p e r c e n t a g e o f n e u r o n a l p e r i k a r y a in layers I I - V I ( T a b l e I, Figs. 4B a n d 6B). N o glial r e a c t i o n was o b s e r v e d d u r i n g p r o g r e s s i v e i s c h e m i a for 20 min.
92
Fig. 5. Electron micrographs of layer IV in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). While there was no obvious change after ischemia for 20 rain without reperfusion (B), the membrane bound structure with scattered mitochondria indented the trunks of dendrites after reperfusion for 3 h (C). The structure was identified as astrocytic processes by the absence of postsynaptic densities at the contact sites (arrowheads) and the paler cytoplasmic matrix as compared to the swollen dendritic matrix shown in Fig. l. Microtubules were also visible inside the dendritic trunks. Bar = I /sin.
A f t e r r e p e r f u s i o n for 3 h, swelling of the d e n d r i t i e s b e c a m e less notable, b u t d i s i n t e g r a t i o n of m i c r o t u b u l e s a n d m i c r o v a c u o l a t i o n within d e n d r t i t e s persisted in layer I a n d b e c a m e c o n s t a n t in layers II a n d III (Figs. 2C a n d 3C). N e u r o n a l perikarya in layers III a n d Vb
were swollen with a hydropic a p p e a r a n c e where polyrib o s o m e s were disaggregated a n d rough E R was occasionally f r a g m e n t e d with r e d u c e d densities of attached ribosomes (Figs. 4C a n d 6C). E m p t y a p p e a r i n g structures, s u r r o u n d e d by a m o n o l a y e r of m e m b r a n e b u t
93
Fig. 6. Electron micrographs of layer V in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). Polyribosomes in some neuronal perikarya were already disaggregated after ischemia for 20 min without reperfusion (B), which persisted after reperfusion for 3 h along with fragmentation of rough ERs (C) though they were less prominent than those observed in layer III as shown in Fig. 4C. After reperfusion for 24 h, some neuronal perikarya manifested with a hydropic appearance with disaggregated polyribosomes and microvacuoles (D). Bar = 1 #m.
d i f f e r e n t f r o m t h e a f o r e m e n t i o n e d m i c r o v a c u o l e s inside d e n d r i t e s , w e r e f r e q u e n t l y e n c o u n t e r e d in t h e n e u r o p i l o f layers I I I - V I , b u t m o s t p r o m i n e n t l y in layer I V ( T a b l e I, Figs. 5C a n d 7C). T h e a s c e n d i n g d e n d r i t e s b e c a m e t o r t u o u s in s o m e a r e a s b e c a u s e o f
i n d e n t a t i o n c a u s e d by t h e s e e m p t y a p p e a r i n g structures (Fig. 5C). T h e y w e r e d e v o i d o f p o s t s y n a p t i c d e n sities a n d f r e q u e n t l y c o n t a i n e d a few m i t o c h o n d r i a a n d fibrils inside. T h e s e characteristics, along with t h e i r a d d i t i o n a l l o c a t i o n a p p o s e d to n e u r o n a l p e r i k a r y a o r
94 around small blood vessels indicated that they were expanded astrocytic processes. After reperfusion for 6 h, some dendrites in layer I were still swollen. Neuronal perikarya in layers III and Vb continued to show disaggregation of polyribosomes and disintegration of mitochondrial cristae and formation of microvacuoles were also observed. Two of three gerbils showed neuronal perikarya clumped with electron dense autophagosomes and degenerated mitochondria (Table I). Electron-dense cell debris, most likely degenerated neuronal perikarya or proximal dendrites, were occasionally encountered in the same region. They were surrounded by glial processes. After reperfusion for 12 h, swelling of the periphery of ascending dendrites in layer I was reduced considerably but some of them had disintegrated mitochondrial cristae and microtubules as well as microvacuoles. Neuronal perikarya in layers III and Vb continued to show disaggregation of polyribosomes, disintegration of mitochondria, fragmentation of rough ER and microvacuoles. While these degenerating neurons increased in number as compared to those after reperfusion for 6 h, a large number of neuronal perikarya in layers II-VI showed re-aggregation of polyribosomes and normalization of other structures, suggesting that they were surviving neurons in the process of resuming the cellular function. After reperfusion for 24 h, swelling of the periphery of dendrites in layer I was minimal but marked disintegration of microtubules was noted in the more proximal part of dendrites (Table I, Figs. 2D and 3D). The number of degenerated neuronal perikarya increased further (Figs. 4D and 6D).
After reperfusion for 72 h, electron-dense materials were scattered in dendrites in each layer and occasionally even in the extracellular location, suggesting that some dendrites were degenerated from the trunk to the periphery and replaced by these materials. Electron-dense cell debris were also scattered in layers III and Vb. The expansion of astrocytic processes became prominent after reperfusion for 3 h (Table I, Figs. 5C and 7C). However, the number of processes with glial fibrils increased in layers III-VI after reperfusion for 24-72 h. DISCUSSION Our previous immunohistochemical investigation revealed delayed neuronal damage in the cerebral cortex after bilateral carotid occlusion in gerbils ~°, where the extent and rapidity of the evolution of neuronal damage appeared to be dependent on the duration of transient cerebral ischemia. In the present investigation, we employed an ischemic period of 20 min to facilitate the electron microscopic observation of neuronal degeneration during early postischemic periods. This resulted in more rapid visualization of neuronal degeneration with light microscopy than what we have observed in the past 1°. The present investigation clearly demonstrated that the dendrosomatic propagation of ischemic and postischemic neuronal damages in the cerebral cortex for the first time. After ischemia for 5 min, the morphological changes first occurred in layer I in the periphery of dendrites without affecting the proximal part or neu-
TABLE I
Electron microscopic findings during ischemia and reperfusion in the cerebral cortex Numericals indicate the n u m b e r of animals with positive findings. Three animals were used for each ischemic and reperfusion period.
Anatomical location Nerve cell bodies Layer 111 and Vb
Dendrites Layer I Layer II Layer IV Astroglia Layer I I I - V I
EM findings
Progressive ischemia 0 5 10
20 m#~
Reperfusion 3 6
12
24
72 h
0
0
0
2
3
3
3
3
0
0
0
0
0
3
3
3
3
1
0
0
0
0
(I
1
3
3
3
Marked expansion Disintegration of microtubules Disintegration of microtubules
0
3
3
3
2
1
1
I)
0
()
2
3
3
3
3
3
3
3
0
0
0
1
3
3
3
3
3
Cytoplasmic swelling
0
0
0
0
3
3
3
3
3
Disaggregation of polyribosomes Fragmentation of rough E R Electron-dense clumping
95 ronal perikarya (Table I). However, swelling of the periphery of dendrites and of mictochondria inside receded during reperfusion. On the other hand, central propagation of the dendritic damage ensued during
reperfusion with disintegration of mitochondrial cristae and microtubules as well as formation of microvacuoles. The vulnerability of dendrites in cerebral ischemia has been described in light microscopic studies
Fig. 7. Electron micrographs of layer VI in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). While there was no obvious change after ischemia for 20 min without reperfusion (B), a large number of swollen astrocytic processes with pale cytoplasm were observed after reperfusion for 3 h (C). Swelling was more notable than layer IV as shown in Fig. 5C. After reperfusion for 24 h, swelling of astrocytic processes was less notable (D). Some thinly myelinated fibers show degeneration. Bar = 1 ~m.
96 with loss of the immunohistochemical reactions for cytoskeletal proteins 16'32'34'36 and varicose swelling of dendritic terminals t. Electron microscopically, prompt swelling of the periphery of dendrites 31'33, later shrinkage of the dendritic trunk adjacent to neuronal perikarya 21 and dilatation of rough ER and swelling of mitochondria inside the dendrites in the distal stratum radiatum 29 have been observed in the hippocampus during progressive ischemia and after reperfusion as well as in the substantia nigra after hyperglycemic ischemia ~1. While dendritic damages progressed rapidly and irreversibly after hyperglycemic ischemia ~1, swelling of the periphery of dendrites seen in the present study receded during reperfusion, as we have observed in the hippocampus 32. The presence of sporadic electrondense debris in layer I after prolonged reperfusion suggested that only a limited number of neurons and their dendrites were irreversibly damaged and disintegrated in the cerebral cortex under the present experimental condition. In neuronal perikarya, disaggregation of polyribosomes was the earliest manifestation, notable in some neurons even after ischemia for 20 min without reperfusion and in most neurons after reperfusion for 3 h. While the same phenomenon has been observed in earlier studies of the cerebral cortex after unilateral carotid occlusion in gerbils 9'19, disaggregation of polyribosomes in the present study was persistent in some neuronal perikarya and followed by disintegration of mitochondria, fragmentation of rough E R and formation of microvacuoles leading to eventual neuronal disintegration. However, re-aggregation of polyribosomes occurred in many neurons after 12 h, indicating recovery of the neuronal function. Thus, the present study clearly demonstrated the structural alterations leading to neuronal disintegration and those associated with transient dysfunction. Microvacuoles in neuronal perikarya have been reported to be the earliest manifestation of anoxic ischemia but these microvacuoles appear to be mitochondrial in origin because of the presence of the remnant double membrane structure and mitochondrial cristae 4'17. Parallel stacking of rough ER was one of the characteristics observed in the CA1 region of the hippocampus 14'33, but it was not found in the cerebral cortex. The observed difference could be based on the specificity of the neurons in two locations. Swelling of perineuronal and perivascular astroglial foot processes has been described in ischemic and postischemic lesions 3'5. In the present investigation, swelling of astrocytic processes became apparent after reperfusion for 3 h, and caused indentation of dendritic trunks and even invagination into dendritic trunks in some instances. Thus, some microvacuoles in den-
drites may be a part of swollen astrocytic processes invaginated into the dendroplasm. A similar process has been described in dendrites of rat cortical neurons after ethanol consumption 7. Swelling and other changes in the periphery of dendrites without involvement of axons have been observed after sustained electric stimulation of the brain and experimental status epilepticus and these changes are believed to represent excitotoxic neuronal damage 12"2°. N-methyl-D-aspartate, an excitatory amino acid, is considered as the cause of ischemic neuronal damage and delayed neuronal death by many investigators 6'23'24 and its receptor is widely distributed in the stratum radiatum of the CA1 region of the hippocampus and the superficial layers of the cerebral cortex in the rat brain s'as. Therefore, the observed central spreading of ultrastructural damage may represent an excitotoxic dendritic damage with secondary involvement of neuronal perikarya. However, it is also possible that the initial dendritic swelling in layer I could have been caused by an influx of cerebrospinal fluid (CSF) from the subarachnoid space. When the ionic pump failure ensues because of energy depletion in cerebral isehemia, it is also possible that water and calcium ions may enter the brain parenchyma from the CSF space and cause neuronal damage 3°. Although it may not be apparent structurally, the deleterious effect of ischemia and reperfusion can occur promptly in neuronal cell bodies. In parallel with disaggregation of polyribosomes, protein synthesis comes to a standstill 19'25 and a steady state decline of messenger RNA has been observed in neuronal perikarya in the gerbil brain ~5. Within neuronal nuclei, the deleterious effect on chromatin proteins has been observed 26 and immediate-early gene activation has failed to occur in those neurons destined to suffer delayed neuronal death 28. Therefore, ischemia and reperfusion may exert their devastating effect simultaneously to neuronal cell bodies and their dendrites. If this is the case, central propagation of ultrastructural damage seen in the present investigation could be similar to central propagation of axonal damage seen in dying-back neuropathy. While the elucidation of the precise mechanism of delayed neuronal death requires further investigation, the present study demonstrated a prompt morphological event in the periphery of dendrites in cerebral ischemia and after reperfusion, which spread centrally to neuronal cell bodies before light microscopic evidence of delayed neuronal death became apparent. This process in the cerebral cortex appears to be similar to what has been observed in the CA1 region of the hippocampus, suggesting that the observed process
97 may be c o m m o n to neurons vulnerable to cerebral ischemia irrespective of the anatomical location within the central nervous system.
18
Acknowledgements. The present investigation was supported by Grant
19
NS-06663 from the National Institutes of Health, US Public Health Services.
20
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bined light and electron microscope study of early anoxic-ischemic cell change in rat brain, Brain Res., 20 (1970) 193-200. Monaghan, D.T., Holets, V.R., Toy, D.W. and Cotman, C.W., Anatomical distributions of four pharmacologically distinct 3H-Lglutamate binding sites, Nature, 306 (1983) 176-179. Morimoto, K. and Yanagihara, T., Cerebral ischemia in gerbils; polyribosomal function during progression and recovery, Stroke, 12 (1981) 105-110. Olney, J.W., de Gubareff, T. and Sloviter, R.S., 'Epileptic' brain damage in rats induced by sustained electrical stimulation of the perforant path. II. Ultrastructural analysis of acute hippocampal pathology, Brain Res. Bull., 10 (1983) 699-712. Petito, C. and Pulsinelli, W.A., Delayed neuronal recovery and neuronal death in rat hippocampus following severe cerebral ischemia: possible relationship to abnormalities in neuronal processes, J. Cereb. Blood Flow Metab., 4 (1984) 194-205. Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982)491-498. Rothman, S., Synaptic activity mediates death of hypoxic neurons, Science, 220 (1983) 536-537. Simon, R.P., Swan, J.H., Griffiths, T. and Meldrum, B.S., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850-852. Thilman, R., Xie, Y., Kleihues, P. and Kiessling, M., Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus, Acta Neuropathol., 71 (1986) 88-93. Thrall, C.L. and Yanagihara, T., Nuclear binding sites for triiodothyronine in the gerbil brain following ischemia and recirculation, Brain Res., 301 (1984) 179-183. Tomimoto, H. and Yanagihara, T., Electron microscopic study of the cerebral cortex after transient ischemia in gerbils, Soc. Neurosci. Abstr., 17 (1991) 1265. Uemura, Y., Kowall, N.W. and Beal, M.F., Global ischemia induces NMDA receptor-mediated c-fos expression in neurons resistent to injury in gerbil hippocampus, Brain Res., 542 (1991) 343 -347. von Lubitz, D.E. and Diemer, N.H., Complete cerebral ischemia in the rat; an ultrastructural and stereological analysis of the distal stratum radiatum in the hippocampal CA-1 region, NeuropathoL Appl. Neurol., 8 (1982) 197-215 Welsh, F.A., Marcy, V.R., Mckee, A.E. and Sims, R.E., Microchemistry of ischemic damage in rat hippocampus, J. Cereb. Blood Flow. Metab., 7 (1987) $77. (Abstract.) Yamamoto, K., Morimoto, K. and Yanagihara, T., Cerebral ischemia in the gerbil; transmission electron microscopic and immunoelectron microscopic investigation, Brain Res., 384 (1986) 1-10. Yamamoto, K., Akai, F. and Yoshimine, T. and Yanagihara, T., Immunohistochemical investigation of cerebral ischemia after middle cerebral artery occlusion in gerbils, J. Neurosurg., 67 (1987) 414-420. Yamamoto, K., Hayakawa, T., Mogami, H., Akai, F. and Yanagihara, T., Ultrastructural investigation of the CA1 region of the hippocampus after transient cerebral ischemia in gerbils, Acta Neuropathol., 80 (1990) 487-492. Yanagihara, T., Yoshimine, T., Morimoto, K. and Homburger, H.A., Immunohistochemical investigation of cerebral ischemia in gerbils, J. Neuropathol. Exp. Neurol., 44 (1985) 204-215. Yanagihara, T., Brengman, J.M. and Mushynski, W.E., Differential vulnerability of microtubule components in cerebral ischemia, Acta Neuropathol., 80 (1992) 499-505. Yoshimine, T., Morimoto, K., Brengman, J.M., Homburger, H.A., Mogami, H. and Yanagihara, T., Immunohistochemical investigation of cerebral ischemia during recirculation, J. Neurosurg., 63 (1985) 922-928.
87
BRES 18326
Electron microscopic investigation of the cerebral cortex after cerebral ischemia and reperfusion in the gerbil Hidekazu Tomimoto and Takehiko Yanagihara Department of Neurology, Mayo Clinic and Mayo Foundation, Rochester, MN 55905 (USA) (Accepted 14 July 1992)
Key words: Cerebral cortex; Cerebral ischemia; Electron microscopy; Gerbil
Prompt dendritic damage has been observed in the hippocampus of the gerbil brain after transient cerebral ischemia. In the present study, we studied the frontoparietal cortex of the gerbil brain electron microscopically after brief bilateral carotid occlusion to assess the vulnerability of dendritic processes. After ischemia for 5 min, there was swelling of the periphery of dendrites accompanied by swelling of mitochondria, cytoplasmic vacuolation and disintegration of microtubules in layer I, which spread to layer III after ischemia for 20 min. After reperfusion for 3-24 h following ischemia for 20 min, swelling in the periphery of dendrites and of mitochondria inside receded but vacuole formation and disintegration of microtubules propagated proximally. In neuronal perikarya, polyribosomal disaggregation was observed after ischemia for 20 min and persisted thereafter, while fragmentation of rough endoplasmic reticulum (ER) and microvaeuolation occurred after reperfusion for 3 h. Electron-dense clumping of neuronal perikarya was observed after reperfusion for 6 h particularly in layers III and Vb, which increased in number for up to 72 h. The observed progressive damage in dendrites may be common to neurons vulnerable to cerebral ischemia and may significantly contribute to development of delayed neuronal death.
INTRODUCTION
the layer V a r e very v u l n e r a b l e to global a n d r e g i o n a l ischemia 3"I°A6'32'34'36. Since n e u r o n s in t h e c e r e b r a l cor-
N e u r o n s e x p o s e d to ischemic insult m a y lose t h e i r s t r u c t u r a l integrity soon a f t e r r e s t o r a t i o n o f b l o o d flow with s h r i n k a g e a n d h y p e r c h r o m a s i a o f n e u r o n a l p e r i k a r y a a n d f o r m a t i o n o f t r i a n g u l a r n u c l e u s 2,4,17. W h i l e t h e s e c h a n g e s c a n o c c u r in any n e u r o n s in the b r a i n a n d soon a f t e r ischemic insults, s o m e a r e a s of t h e brain develop delayed neuronal death after brief i s c h e m i a t3'14'22. T h e m e c h a n i s m for d e l a y e d n e u r o n a l
tex a r e h e t e r o g e n e o u s in t h e i r s t r u c t u r e s including t h e i r d e n d r i t i c p r o c e s s e s as c o m p a r e d to t h o s e in the CA1 r e g i o n o f t h e h i p p o c a m p u s , we s t u d i e d layers I - V I o f the f r o n t o p a r i e t a l cortex o f t h e gerbil b r a i n by using t r a n s m i s s i o n e l e c t r o n microscopy. T h e results o f the p r e s e n t investigation have b e e n r e p o r t e d in abstract form 27.
death remains uncertain, and the structural derangem e n t l e a d i n g to d e l a y e d n e u r o n a l d e a t h has not b e e n well e l u c i d a t e d , b e c a u s e m o s t p r e v i o u s efforts have b e e n c o n c e n t r a t e d on t h e u l t r a s t r u c t u r a l a l t e r a t i o n s o f t h e p y r a m i d a l cell b o d i e s in t h e h i p p o c a m p u s 14'2~. O u r p r e v i o u s investigations in t h e h i p p o c a m p u s o f t h e gerbil b r a i n including t h e CA1 r e g i o n r e v e a l e d p r o m p t swelling o f t h e p e r i p h e r y o f apical a n d b a s a l d e n d r i t e s a n d p r o p a g a t i o n o f s t r u c t u r a l d a m a g e s to the p r o x i m a l part during progressive cerebral ischemia and reperfusion 31'33. In t h e c e r e b r a l cortex, n e u r o n s in layers
MATERIALS AND METHODS
I l l / I V a n d t h e layer just b e n e a t h t h e large n e u r o n s o f
Mongolian gerbils (Meriones unguiculatus) of both sexes weighing 60-80 g were used for the present investigation. Each gerbil was allowed free access to food and water before and after surgery. Under inhalation anesthesia with ether, the common carotid arteries were exposed bilaterally in the neck and occluded by miniature Mayfield aneurysmal clips for 5, 10 and 20 min I°. For investigation of the effect of reperfusion, the clips were released after 3, 6, 12, 24 and 72 h following ischemia for 20 rain. Three gerbils were used for each ischemic and postischemic period. Sham operated gerbils had exposure of both common carotid arteries without occlusion and were allowed to live for 20 min or 72 h. At a predetermined ischemic or postischemic period, each gerbil was reanesthetized with ketamine hydrochloride (60 mg/kg) and perfused through the left ventricle of the heart with 0.02% phosphate buffered saline at pH 7.4 for 20 s
Correspondence: H. Tomimoto, Department of Neurology, Kyoto University Hospital, Kyoto 606, Japan.
88 and then with 2% paraformaldehyde-2% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4 for 10 min. After decapitation, the head was immersed in the same fixative overnight. On the following day, the skull was removed and the brain was cut into blocks and fixed for an additional day. Coronal sections of the frontoparietal cortex including the caudoputamen were further sectioned at 100 # m thickness with a vibratome and kept in 0.1 M PB (pH 7.4) overnight and further fixed for 1 h in 2% osmium tetroxide in 0.1 M PB (pH 7.4). After rinsing in 0.1 M PB for 1 h, each section was dehydrated in 60% ethanol, 70% ethanol containing 2% uranyl acetate and 80-100% ethanol and finally propylene oxide. Each section was flat-embedded in Spurr's embedding medium and narrow strips of the frontoparietal cortex were dissected perpendicularly. Semithin sections (1 ~m) containing each layer were prepared and stained with 1% Toluidine blue to identify layers I-VI. Adjoining ultrathin sections were further sectioned with an ultramicrotome and contrasted with 2% lead citrate and examined under a transmission electron microscope (Phillips 201).
RESULTS
Light microscopic findings There was no vacuole formation or any other abnorm a l i t i e s in s e m i t h i n s e c t i o n s f r o m s h a m o p e r a t e d bils (Fig.
1A,B). After
ischemia
for 5 and
ger-
10 m i n ,
v a c u o l a t i o n o c c u r r e d i n l a y e r I, f o r m i n g a h o n e y c o m b appearance.
It was most notable adjacent to the pia-
arachnoid membrane. more
prominent
The honeycomb
appearance
was
a f t e r i s c h e m i a f o r 20 m i n ( F i g . 1C).
H o w e v e r , n e u r o n a l p e r i k a r y a w e r e n o t a f f e c t e d in a n y layer after ischemia (Fig.
1D). A f t e r
f o r 20 m i n w i t h o u t
reperfusion
reperfusion
f o r 3 h, v a c u o l a t i o n
in
Fig. 1. Light microscopic photographs of layer I (A,C,E) and layer III (B,D,F) in a sham operated gerbil (A,B), after ischemia for 20 min without reperfusion (C,D) and after ischemia for 20 min and reperfusion for 24 h (E,F). The honeycomb appearance was clearly visible in the area facing the suharachnoid space after ischemia for 20 min (C) but only mildly after reperfusion for 24 h (E). Neuronal perikarya in the layer III was essentially normal after ischemia for 20 rain (D) but some of them (arrows) were clumped after reperfusion for 24 h (F). Semithin sections were stained with Toluidine blue. The original photographs were taken at × 400.
89 layer I was less notable but neuronal perikarya were occasionally swollen in layers III and Vb. Vacuolation in layer I markedly receded after reperfusion for 6 h or longer (Fig. 1E), but clumped neuronal perikarya having hyperchromasia with Toluidine blue began to evolve in layers III and Vb after reperfusion for 6 h. The
number and severity of degenerated neurons increased notably after reperfusion for 24 and 72 h (Fig. 1F).
Electron microscopic findings There was no vacuole formation or any other abnormalities in semithin sections from sham operated ger-
Fig. 2. Electron micrographs of layer I in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). After ischemia for 20 min, the periphery of dendrites and mitochondria inside became markedly swollen even without reperfusion (B). Arrowheads indicate the location of postsynaptic densities, the marker for dendrites. Swelling receded already after reperfusion for 3 h (C) and markedly reduced after reperfusion for 24 h (D). Bar = 1 ~tm.
90
Fig. 3. Electron micrographs of layer II in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). Microtubules in some dendrites were partially disintegrated after ischemia for 20 min without reperfusion (B), which became obvious after reperfusion for 3 h (C). Tortuous dendrites with disorganized and disintegrated microtubules were seen after reperfusion for 24 h (D). Bar = 1 /zm.
bils (Fig. 2A). A f t e r ischemia for 5 a n d 10 min, swelling of d e n d r i t i c p r o c e s s e s was o b s e r v e d in layer I. A similar but m o r e p r o n o u n c e d c h a n g e was o b s e r v e d after ischemia for 20 min ( T a b l e I). Swollen d e n d r i t i c p r o cesses as positively i d e n t i f i e d by the p r e s e n c e o f postsyn a p t i c densities, c o n t a i n e d d i s t e n d e d vacuoles filled
with a m o r p h o u s m a t e r i a l s , swollen m i t o c h o n d r i a a n d d i s i n t e g r a t e d m i c r o t u b u l e s . M e m b r a n e limited inclusions, h e r e t e r m e d microvacuoles, w e r e p r e s e n t inside the d e n d r i t e s in layer I. T h e y h a d an e m p t y a p p e a r ance a n d w e r e e n c i r c l e d by a m o n o l a y e r of i r r e g u l a r m e m b r a n e (Fig. 2B). Swelling of m i t o c h o n d r i a and
91
Fig. 4. Electron micrographs of layer III in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). Polyribosomes were disaggregated in some neuronal perikarya even after ischemia without reperfusion (B), which were more complete and widespread after reperfusion for 3 h (C). Rough ERs were fragmented and the density of attached polyribosomes was markedly reduced. After reperfusion for 24 h, some neuronal cell bodies were already degenerated to electron dense cell debris (D). Bar = 1/xm.
d i s i n t e g r a t i o n o f m i c r o t u b u l e s w e r e o b s e r v e d in l a y e r I a f t e r i s c h e m i a for 5 min a n d in layer I a n d less freq u e n t l y in layers II a n d I I I a f t e r i s c h e m i a for 10 a n d 20 m i n ( T a b l e I a n d Fig. 3B). N o a p p a r e n t c h a n g e was o b s e r v e d in the n e u r o p i l o f layers I V - V I (Figs. 5B a n d
7B). H o w e v e r , d i s a g g r e g a t i o n o f p o l y r i b o s o m e s was o b s e r v e d in a small p e r c e n t a g e o f n e u r o n a l p e r i k a r y a in layers I I - V I ( T a b l e I, Figs. 4B a n d 6B). N o glial r e a c t i o n was o b s e r v e d d u r i n g p r o g r e s s i v e i s c h e m i a for 20 min.
92
Fig. 5. Electron micrographs of layer IV in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). While there was no obvious change after ischemia for 20 rain without reperfusion (B), the membrane bound structure with scattered mitochondria indented the trunks of dendrites after reperfusion for 3 h (C). The structure was identified as astrocytic processes by the absence of postsynaptic densities at the contact sites (arrowheads) and the paler cytoplasmic matrix as compared to the swollen dendritic matrix shown in Fig. l. Microtubules were also visible inside the dendritic trunks. Bar = I /sin.
A f t e r r e p e r f u s i o n for 3 h, swelling of the d e n d r i t i e s b e c a m e less notable, b u t d i s i n t e g r a t i o n of m i c r o t u b u l e s a n d m i c r o v a c u o l a t i o n within d e n d r t i t e s persisted in layer I a n d b e c a m e c o n s t a n t in layers II a n d III (Figs. 2C a n d 3C). N e u r o n a l perikarya in layers III a n d Vb
were swollen with a hydropic a p p e a r a n c e where polyrib o s o m e s were disaggregated a n d rough E R was occasionally f r a g m e n t e d with r e d u c e d densities of attached ribosomes (Figs. 4C a n d 6C). E m p t y a p p e a r i n g structures, s u r r o u n d e d by a m o n o l a y e r of m e m b r a n e b u t
93
Fig. 6. Electron micrographs of layer V in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). Polyribosomes in some neuronal perikarya were already disaggregated after ischemia for 20 min without reperfusion (B), which persisted after reperfusion for 3 h along with fragmentation of rough ERs (C) though they were less prominent than those observed in layer III as shown in Fig. 4C. After reperfusion for 24 h, some neuronal perikarya manifested with a hydropic appearance with disaggregated polyribosomes and microvacuoles (D). Bar = 1 #m.
d i f f e r e n t f r o m t h e a f o r e m e n t i o n e d m i c r o v a c u o l e s inside d e n d r i t e s , w e r e f r e q u e n t l y e n c o u n t e r e d in t h e n e u r o p i l o f layers I I I - V I , b u t m o s t p r o m i n e n t l y in layer I V ( T a b l e I, Figs. 5C a n d 7C). T h e a s c e n d i n g d e n d r i t e s b e c a m e t o r t u o u s in s o m e a r e a s b e c a u s e o f
i n d e n t a t i o n c a u s e d by t h e s e e m p t y a p p e a r i n g structures (Fig. 5C). T h e y w e r e d e v o i d o f p o s t s y n a p t i c d e n sities a n d f r e q u e n t l y c o n t a i n e d a few m i t o c h o n d r i a a n d fibrils inside. T h e s e characteristics, along with t h e i r a d d i t i o n a l l o c a t i o n a p p o s e d to n e u r o n a l p e r i k a r y a o r
94 around small blood vessels indicated that they were expanded astrocytic processes. After reperfusion for 6 h, some dendrites in layer I were still swollen. Neuronal perikarya in layers III and Vb continued to show disaggregation of polyribosomes and disintegration of mitochondrial cristae and formation of microvacuoles were also observed. Two of three gerbils showed neuronal perikarya clumped with electron dense autophagosomes and degenerated mitochondria (Table I). Electron-dense cell debris, most likely degenerated neuronal perikarya or proximal dendrites, were occasionally encountered in the same region. They were surrounded by glial processes. After reperfusion for 12 h, swelling of the periphery of ascending dendrites in layer I was reduced considerably but some of them had disintegrated mitochondrial cristae and microtubules as well as microvacuoles. Neuronal perikarya in layers III and Vb continued to show disaggregation of polyribosomes, disintegration of mitochondria, fragmentation of rough ER and microvacuoles. While these degenerating neurons increased in number as compared to those after reperfusion for 6 h, a large number of neuronal perikarya in layers II-VI showed re-aggregation of polyribosomes and normalization of other structures, suggesting that they were surviving neurons in the process of resuming the cellular function. After reperfusion for 24 h, swelling of the periphery of dendrites in layer I was minimal but marked disintegration of microtubules was noted in the more proximal part of dendrites (Table I, Figs. 2D and 3D). The number of degenerated neuronal perikarya increased further (Figs. 4D and 6D).
After reperfusion for 72 h, electron-dense materials were scattered in dendrites in each layer and occasionally even in the extracellular location, suggesting that some dendrites were degenerated from the trunk to the periphery and replaced by these materials. Electron-dense cell debris were also scattered in layers III and Vb. The expansion of astrocytic processes became prominent after reperfusion for 3 h (Table I, Figs. 5C and 7C). However, the number of processes with glial fibrils increased in layers III-VI after reperfusion for 24-72 h. DISCUSSION Our previous immunohistochemical investigation revealed delayed neuronal damage in the cerebral cortex after bilateral carotid occlusion in gerbils ~°, where the extent and rapidity of the evolution of neuronal damage appeared to be dependent on the duration of transient cerebral ischemia. In the present investigation, we employed an ischemic period of 20 min to facilitate the electron microscopic observation of neuronal degeneration during early postischemic periods. This resulted in more rapid visualization of neuronal degeneration with light microscopy than what we have observed in the past 1°. The present investigation clearly demonstrated that the dendrosomatic propagation of ischemic and postischemic neuronal damages in the cerebral cortex for the first time. After ischemia for 5 min, the morphological changes first occurred in layer I in the periphery of dendrites without affecting the proximal part or neu-
TABLE I
Electron microscopic findings during ischemia and reperfusion in the cerebral cortex Numericals indicate the n u m b e r of animals with positive findings. Three animals were used for each ischemic and reperfusion period.
Anatomical location Nerve cell bodies Layer 111 and Vb
Dendrites Layer I Layer II Layer IV Astroglia Layer I I I - V I
EM findings
Progressive ischemia 0 5 10
20 m#~
Reperfusion 3 6
12
24
72 h
0
0
0
2
3
3
3
3
0
0
0
0
0
3
3
3
3
1
0
0
0
0
(I
1
3
3
3
Marked expansion Disintegration of microtubules Disintegration of microtubules
0
3
3
3
2
1
1
I)
0
()
2
3
3
3
3
3
3
3
0
0
0
1
3
3
3
3
3
Cytoplasmic swelling
0
0
0
0
3
3
3
3
3
Disaggregation of polyribosomes Fragmentation of rough E R Electron-dense clumping
95 ronal perikarya (Table I). However, swelling of the periphery of dendrites and of mictochondria inside receded during reperfusion. On the other hand, central propagation of the dendritic damage ensued during
reperfusion with disintegration of mitochondrial cristae and microtubules as well as formation of microvacuoles. The vulnerability of dendrites in cerebral ischemia has been described in light microscopic studies
Fig. 7. Electron micrographs of layer VI in a sham operated gerbil (A), after ischemia for 20 min without reperfusion (B), after reperfusion for 3 h (C) and after reperfusion for 24 h (D). While there was no obvious change after ischemia for 20 min without reperfusion (B), a large number of swollen astrocytic processes with pale cytoplasm were observed after reperfusion for 3 h (C). Swelling was more notable than layer IV as shown in Fig. 5C. After reperfusion for 24 h, swelling of astrocytic processes was less notable (D). Some thinly myelinated fibers show degeneration. Bar = 1 ~m.
96 with loss of the immunohistochemical reactions for cytoskeletal proteins 16'32'34'36 and varicose swelling of dendritic terminals t. Electron microscopically, prompt swelling of the periphery of dendrites 31'33, later shrinkage of the dendritic trunk adjacent to neuronal perikarya 21 and dilatation of rough ER and swelling of mitochondria inside the dendrites in the distal stratum radiatum 29 have been observed in the hippocampus during progressive ischemia and after reperfusion as well as in the substantia nigra after hyperglycemic ischemia ~1. While dendritic damages progressed rapidly and irreversibly after hyperglycemic ischemia ~1, swelling of the periphery of dendrites seen in the present study receded during reperfusion, as we have observed in the hippocampus 32. The presence of sporadic electrondense debris in layer I after prolonged reperfusion suggested that only a limited number of neurons and their dendrites were irreversibly damaged and disintegrated in the cerebral cortex under the present experimental condition. In neuronal perikarya, disaggregation of polyribosomes was the earliest manifestation, notable in some neurons even after ischemia for 20 min without reperfusion and in most neurons after reperfusion for 3 h. While the same phenomenon has been observed in earlier studies of the cerebral cortex after unilateral carotid occlusion in gerbils 9'19, disaggregation of polyribosomes in the present study was persistent in some neuronal perikarya and followed by disintegration of mitochondria, fragmentation of rough E R and formation of microvacuoles leading to eventual neuronal disintegration. However, re-aggregation of polyribosomes occurred in many neurons after 12 h, indicating recovery of the neuronal function. Thus, the present study clearly demonstrated the structural alterations leading to neuronal disintegration and those associated with transient dysfunction. Microvacuoles in neuronal perikarya have been reported to be the earliest manifestation of anoxic ischemia but these microvacuoles appear to be mitochondrial in origin because of the presence of the remnant double membrane structure and mitochondrial cristae 4'17. Parallel stacking of rough ER was one of the characteristics observed in the CA1 region of the hippocampus 14'33, but it was not found in the cerebral cortex. The observed difference could be based on the specificity of the neurons in two locations. Swelling of perineuronal and perivascular astroglial foot processes has been described in ischemic and postischemic lesions 3'5. In the present investigation, swelling of astrocytic processes became apparent after reperfusion for 3 h, and caused indentation of dendritic trunks and even invagination into dendritic trunks in some instances. Thus, some microvacuoles in den-
drites may be a part of swollen astrocytic processes invaginated into the dendroplasm. A similar process has been described in dendrites of rat cortical neurons after ethanol consumption 7. Swelling and other changes in the periphery of dendrites without involvement of axons have been observed after sustained electric stimulation of the brain and experimental status epilepticus and these changes are believed to represent excitotoxic neuronal damage 12"2°. N-methyl-D-aspartate, an excitatory amino acid, is considered as the cause of ischemic neuronal damage and delayed neuronal death by many investigators 6'23'24 and its receptor is widely distributed in the stratum radiatum of the CA1 region of the hippocampus and the superficial layers of the cerebral cortex in the rat brain s'as. Therefore, the observed central spreading of ultrastructural damage may represent an excitotoxic dendritic damage with secondary involvement of neuronal perikarya. However, it is also possible that the initial dendritic swelling in layer I could have been caused by an influx of cerebrospinal fluid (CSF) from the subarachnoid space. When the ionic pump failure ensues because of energy depletion in cerebral isehemia, it is also possible that water and calcium ions may enter the brain parenchyma from the CSF space and cause neuronal damage 3°. Although it may not be apparent structurally, the deleterious effect of ischemia and reperfusion can occur promptly in neuronal cell bodies. In parallel with disaggregation of polyribosomes, protein synthesis comes to a standstill 19'25 and a steady state decline of messenger RNA has been observed in neuronal perikarya in the gerbil brain ~5. Within neuronal nuclei, the deleterious effect on chromatin proteins has been observed 26 and immediate-early gene activation has failed to occur in those neurons destined to suffer delayed neuronal death 28. Therefore, ischemia and reperfusion may exert their devastating effect simultaneously to neuronal cell bodies and their dendrites. If this is the case, central propagation of ultrastructural damage seen in the present investigation could be similar to central propagation of axonal damage seen in dying-back neuropathy. While the elucidation of the precise mechanism of delayed neuronal death requires further investigation, the present study demonstrated a prompt morphological event in the periphery of dendrites in cerebral ischemia and after reperfusion, which spread centrally to neuronal cell bodies before light microscopic evidence of delayed neuronal death became apparent. This process in the cerebral cortex appears to be similar to what has been observed in the CA1 region of the hippocampus, suggesting that the observed process
97 may be c o m m o n to neurons vulnerable to cerebral ischemia irrespective of the anatomical location within the central nervous system.
18
Acknowledgements. The present investigation was supported by Grant
19
NS-06663 from the National Institutes of Health, US Public Health Services.
20
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