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Cerebral ischemia in the gerbil: Transmission electron microscopic and immunoelectron microscopic investigation
Brain Research, 384 (1986) 1-10
1
Elsevier BRE 12023
Research Reports Cerebral Ischemia in the Gerbil: Transmission Electron Microscopic and Immunoelectron Microscopic Investigation KAZUMI YAMAMOTO, KAZUYOSHI MORIMOTO and TAKEHIKO YANAGIHARA
Department of Neurology, Mayo Clinic and Mayo Medical School, Rochester, MN 55905 (U. S. A.) (Accepted 25 February 1986) Key words: Cerebral ischemia - - Dendrite - - Gerbil - - Immunoelectron microscopy - Microtubule - - Transmission electron microscopy
Progression of cerebral ischemia from 5 min to 3 h after occlusion of a common carotid artery was investigated in the subiculum-CA1 region of the hippocampus of the gerbil by transmission electron microscopic and immunoelectron microscopic technique. The earliest change was found after 5 min in the periphery of the apical dendrites in the stratum moleculare, where mitochondrial swelling and disintegration of microtubules were clearly seen inside swollen dendritic processes. After ischemia for 10 min, similar abnormalities were observed in the more proximal part of the apical dendrites, and the basal dendrites also became similarly affected. After ischemia for 30 min to 1 h, the pyramidal cell bodies showed mitochondrial swelling, distension of endoplasmic reticulum and disaggregation of polyribosomes. The immunoelectron microscopic procedure for tubulin revealed irregularity of reaction products associated with microtubules after ischemia for 5 min in the dendritic terminals in the stratum moleculare and in the stratum radiatum after ischemia for 10 min. Reaction products in the pyramidal cell bodies became sparse after ischemia for 30 min to 1 h. The present investigation revealed early onset of ischemic damage in the dendritic terminals and subsequent proximal extension, with disintegration of microtubules and mitochondrial swelling.
INTRODUCTION A recent investigation from our l a b o r a t o r y revealed p r o m p t d i s a p p e a r a n c e of the light microscopic immunohistochemical reaction for tubulin and creatine kinase BB-isoenzyme from the ischemic areas as early as 5 min after occlusion of a c o m m o n carotid artery in the gerbil 27. A l t h o u g h disintegration of microtubules was p r o p o s e d as one of the possible causes for d i s a p p e a r a n c e of the reaction for tubulin, the underlying mechanism remains uncertain. The immunohistochemical investigation also indicated early involvement of the dendrites, particularly in the hippocampus. In the present investigation, we therefore examined the subiculum-CA1 region of the hippocampus during progression of unilateral cerebral ischemia in the gerbil by transmission electron microscopy ( T E M ) and i m m u n o e l e c t r o n microscopy
( I E M ) for tubulin with special attention to the dendritic structure. The present investigation has been r e p o r t e d in abstract form 24. MATERIALS AND METHODS Mongolian gerbils (Meriones unguiculatus) weighing 60-70 g were used for the present investigation. The details of animal p r e p a r a t i o n have been published previously 14'26. U n d e r anesthesia with ether inhalation, the right c o m m o n carotid artery of each gerbil was occluded in the neck with a miniature Mayfield aneurysm clip. The symptomatic gerbils were identified by torsion of the neck and circling in less than 15 min after surgery. T h r e e gerbils were used for investigation of the ischemic period for 5, 10 and 30 min, respectively, and 2 gerbils were taken for investigation of the ischemic p e r i o d of 1 and 3 h, re-
Correspondence: T. Yanagihara, Department of Neurology, Mayo Clinic, Rochester, MN 55905, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
spectively. After a predetermined ischemic period, each gerbil was re-anesthetized with ether and thoracotomy was carried out. The clip on the common carotid artery was removed promptly and transcardiac perfusion was carried out through the left ventricle of the heart for 20 s with 0.9% saline and then for 15 rain with 2% paraformaldehyde-2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for TEM investigation or 4% paraformaldehyde-0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for IEM investigation. Actual tissue fixation started 20 s after removal of the clip. The brain was promptly removed, sectioned into blocks and further fixed for 4 h at 4 °C in the same aldehyde-based fixative as used for perfusion fixation. Tissue blocks were washed in 0.1 M phosphate buffer (pH 7.4) and further sectioned with a vibratome (Lancer) at 100/~m for TEM and 50/~m for IEM. The sections for TEM were further fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 h, washed in the same buffer solution, contrasted with 2% uranyl acetate in 70% alcohol for 40 min, dehydrated and flat-embedded in Spurr's embedding medium 2°. For IEM, the procedure described by Matus et al. lz was used with slight modification. Each tissue section was incubated with 20% normal rabbit serum at room temperature for 30 min, the anti-tubulin goat serum (1:200) for 1 h, the rabbit anti-goat IgG (1:40) for 1 h and the peroxidase-antiperoxidase complex (1:100) for 1 h. The antiserum for tubulin has been prepared in a goat by administration of purified tubulin from gerbil brains 29. The control specimen was treated with the absorbed antiserum or non-immunized goat serum. Between each incubation, tissue sections were washed 3 times for 1 h each with 0.9% saline in 0.02 M phosphate buffer (pH 7.4) at 4 °C. Each tissue section was incubated with 0.05% 3,3'diaminobenzidine tetrahydrochloride in 0.05 M TrisHC1 buffer (pH 7.6) for 15 min and then with 0.05% 3,Y-diaminobenzidine tetrahydrochloride-0.01% hydrogen peroxide in the same Tris-HC1 buffer for 7 min. Each section was washed with the same TrisHCI buffer and then with 0.1 M phosphate buffer (pH 7.4) prior to fixation with 1% osmium tetroxide in the same buffer for 1 h. The subsequent procedure was the same as for TEM. One adjacent section was examined under a light microscope without fixation
with osmium tetroxide for the presence of ischemic lesions. Under a dissection microscope, the subiculumCA1 region of the hippocampus was dissected from the flat-embedded tissue section and re-embedded in a silicone rubber mold with fresh Spurr's embedding medium 2°. Serial ultrathin sections were prepared with an ultramicrotome (Porter-Blum MT-2B) and examined under a transmission electron microscope (Philips 201). Some sections for TEM were further contrasted with 0.5% lead citrate. Semi-thin sections (1 btm) were stained with 1% toluidine blue and examined under a light microscope. Control specimens were prepared from the sham-operated gerbils and the left (non-occluded) side of the operated gerbils. The specimens were examined by 2 investigators independently. In order to detect subtle abnormalities, 1 investigator carried out evaluation without knowing the identity of specimens, whenever necessary. RESULTS
Transmission electron microscopic investigation Ischemia for 5 min. Light microscopic examination of semi-thin sections after toluidine blue staining revealed honeycomb appearance of the stratum moleculare (Fig. 1). Under electron microscopic examination (Table I), marked swelling was observed in the peripheral part of the apical dendrites which were positively identified by the presence of postsynaptic densities (Fig. 2). Inside, swollen mitochondria with disrupted internal cristae, disintegrated microtubules and vacuoles were found. Disruption of cytoplasmic membranes was occasionally observed. On the other hand, the nerve endings in the same area appeared unaffected. The postsynaptic densities were also unaffected. Swollen mitochondria also existed inside the apical dendrites in the distal part of the stratum radiatum (Fig. 3) and in 1 gerbil in the proximal part of the stratum radiatum, particularly in the small dendritic processes (Fig. 4), and within the basal dendrites in the stratum oriens. No abnormality was found in the pyramidal cell bodies. Swollen mitochondria were also seen within the astrocytic processes in the stratum radiatum and oriens in all gerbils and even in the stratum pyramidale in 1 gerbil. Because of marked dendritic swelling, it was
'A
Fig. 1. Light microscopic appearance of the stratum moleculare during progression of cerebral ischemia. Already after ischemia for 5 min, mild honeycomb appearance became visible at the periphery of the stratum moleculare (B). Vacuolation expanded further after ischemia for 10 min (C) and 30 min (D). The control section (A) was taken from the corresponding area on the non-ischemic side of the gerbil rendered ischemic for 5 min. Semi-thin sections were stained with 1% toluidine blue. The photographs were taken at x400 magnification.
difficult to ascertain swelling of astrocytic mitochondria in the stratum moleculare. No a b n o r m a l i t y was found in any stratum on the left side as c o m p a r e d to the corresponding areas of the control gerbils. Ischernia for 10 min. F u r t h e r expansion of honeycomb a p p e a r a n c e was observed in the stratum moleculare under light microscopic examination (Fig. 1). Electron microscopic examination revealed further distension of the p e r i p h e r a l part of the apical dendrites in the stratum moleculare where irregular m e m b r a n e - b o u n d vacuoles and further swelling of mitochondria with disintegration of internal cristae were seen (Fig. 2). Disintegration of microtubules was observed in all gerbils and disruption of p l a s m a
Fig. 2. Transmission electron microscopic appearance of the stratum moleculare during progression of cerebral ischemia. The peripheral parts of the apical dendrites, which contained mitochondria and microtubules, were intermingled with more osmiophilic presynaptic elements in the control brain (A). After ischemia for 5 min, the distal parts of the apical dendrites which could be identified positively by the presence of postsynaptic densities (arrowhead), were markedly swollen with disintegration of microtubules, swollen mitochondria and formation of vacuoles (B). Disintegration of microtubules and mitochondria further progressed after ischemia for 10 min (C) and 30 min (D). The control section (A) was taken from the corresponding area on the non-ischemic side of the gerbil rendered ischemic for 5 min. The photographs were taken at x17,500 magnification.
m e m b r a n e s existed in 2 gerbils (Table I). The distal part of the stratum r a d i a t u m also showed m o r e extensive alteration including swollen m i t o c h o n d r i a with disruption of internal cristae and disintegration of microtubules (Fig. 3). Mild distension of the dendritic processes with cytoplasmic vacuoles was observed in 1 gerbil (Table I). In the proximal part of the stratum radiatum, swelling of m i t o c h o n d r i a became m o r e obvious and mild to m o d e r a t e disintegration of microtubules was o b s e r v e d inside the apical dendrites (Fig. 4). In the stratum oriens, mitochondrial swelling and disintegration of microtubules were observed to the extent similar to those in the
TABLE I Transmission electron microscopic findings of neuronal perikarya and dendrites (-) Designates the subcellular components not encountered in the area, while (U) designates the uncertain status. Three symptomatic gerbils were used for each ischemic period. Ischemic period (rain) Cytoplasmic swelling
Mitochondrial swelling
Disintegration of mitochondrial cristae
Cytoplasmic vacuoles
Swellingof endoplasmic reticulum
Polyribosomal disaggregation
Disintegration of microtubules
Disruption of plasma membrane
Stratum oriens
Stratum pyramidale
Stratum radiatum (prox. )
Stratum radiatum (dist.)
Stratum moleculare
5
0
0
0
0
3
10
0
0
0
1
3
30
3
3
0
3
3
5 10 30
3 3 3
0 2 3
1 3 3
3 3 3
3 3 3
5
0
0
0
o
3
10
1
0
1
2
3
30
3
3
3
3
3
5 10 30
0 0 3
0 0 0
0 0 3
0 1 3
3 3 3 -
5
-
0
-
-
10
-
0
-
-
30
-
3
-
-
-
-
-
5
-
0
-
10
-
0
-
30
-
3
-
-
-
5 10 30
0 3 3
0 0 U
0 3 3
0 3 3
3 3 3 2
0
0
0
0
i0
5
0
0
0
0
2
30
0
0
0
0
3
distal part of the stratum radiatum. The pyramidal
chondria and microtubules further advanced in the
cell bodies showed slightly swollen mitochondria in 2 gerbils (Table I) but more definite mitochondrial swelling was observed in the adjacent dendrites (Fig.
apical dendrites along with cytoplasmic distension (Fig. 3 and Table I). In the proximal part of the stra-
5). The nerve endings remained morphologically unaffected except for suggestive swelling of mitochondria in the stratum moleculare. Astrocytic mitochondria were swollen in every stratum in all gerbils. Because of marked distension of the dendrites, alteration of astrocytes was not well observed in the stratum moleculare. Ischemia f o r 30 rain. In the peripheral part of the apical dendrites, disintegration of mitochondria and microtubules further advanced and the distended dendrites were often empty or contained only vacuoles in the stratum moleculare (Fig. 2). In the distal part of the stratum radiatum, disintegration of mito-
tum radiatum, some apical dendrites lost microtubules almost completely and swollen mitochondria formed vacuoles because of disintegration of internal cristae. O n the other hand, some apical dendrites still retained microtubules in spite of the presence of markedly swollen mitochondria. The findings in the stratum oriens were similar to those seen in the distal part of the stratum radiatum. Some pyramidal cell bodies showed swollen mitochondria (Fig. 5), mildly enlarged endoplasmic reticulum and disaggregation of polyribosomes (Table I). While swollen mitochondria were clearly seen within the nerve endings in the distal part of the stratum radiatum and the stratum oriens, integrity of the nerve endings with synaptic vesicles was well re-
D
0 Fig. 3. Transmission electron microscopic appearance of the distal part of the stratum radiatum during progression of cerebral ischemia. The apical dendrites, which contained mitochondria and microtubules, were intermingled with more osmiophilic presynaptic elements and could be positively identified by the presence of postsynaptic densities in the control brain (A). After ischemia for 5 min, mitochondria became swollen (B). After ischemia for 10 min, swelling of mitochondria became more notable. In addition, microtubules began to disintegrate and some dendritic cytoplasm appeared swollen (C). After ischemia for 30 min, further disintegration of mitochondria and microtubules ensued along with swelling of dendritic cytoplasm (D). The control section (A) was taken from the corresponding area on the non-ischemic side of the gerbil rendered ischemic for 5 min. The original photographs were taken at × 17,500 magnification.
Fig. 4. Transmission electron microscopic appearance of the proximal part of the stratum radiatum during progression of cerebral ischemia. Abundance of microtubules were identified inside the apical dendrites along with mitochondria in the control brain (A). After ischemia for 5 min, no abnormality of rnicrotubules was found but mitochondria in small dendritic processes were swollen and those in the main trunks of the apical dendrites also appeared mildly swollen (B). After ischemia for 10 min, mitochondria became more swollen inside the apical dendrites but those in the nerve endings were still small. Microtubules began to show irregular contour and density (C). After ischemia for 1 h, further disintegration of microtubules occurred (D). The control section (A) was taken from the corresponding area on the non-ischemic side of the gerbil rendered ischemia for 5 min. The photographs were taken at ×17,500 magnification.
tained. Myelinated axons were still intact. Distension of the astrocytic processes with swollen mitochondria was observed everywhere.
addition, shrunken pyramidal cell bodies with increased nuclear and cytoplasmic electron density were also observed.
Ischemia for 1 h. Disintegration of the peripheral part of the apical dendrites progressed with disruption of plasma m e m b r a n e s . Many apical dendrites in the proximal part of the stratum radiatum lost microtubules extensively or completely (Fig. 4). However, there were still scattered dendrites which retained microtubules relatively well. The pyramidal cell bodies revealed further distension of endoplasmic reticulum and increase in intracytoplasmic vacuoles. In
In the distal part of the stratum radiatum and the stratum oriens, some nerve endings appeared to be distended with swollen mitochondria and aggregated synaptic vesicles. Swelling of the astrocytic processes with swollen mitochondria with disrupted internal cristae was more advanced in each stratum.
Ischemia for 3 h. Morphological alterations observed after ischemia for 1 h advanced further. Various degrees of distension existed in the entire course
Fig. 5. Transmission electron microscopic appearance of the stratum pyramidale during progression of cerebral ischemia. After ischemia for 10 rain, no remarkable change was observed in the neuronal perikarya except for mildly swollen mitochondria but they were more definitely swollen in the dendrites in the surrounding area (B). After ischemia for 30 rain, further swelling of mitochondria occurred in the neuronal perikarya, where swelling of the endoplasmic reticulum and disaggregation of polyribosomes began to ensue (C). After ischemia for 3 h, extensive ischemic damage with swollen endoplasmic reticulure, disaggregated polyribosomes, and vacuolation of mitochondria occurred in the neuronal perikarya (D). The control section (A) was taken from the corresponding area on the nonischemic side of the gerbil rendered ischemic for 5 rain. The photographs were taken at x 11,250 magnification.
of the apical a n d basal d e n d r i t e s with d i s r u p t e d plasm a m e m b r a n e s , v a c u o l a t i o n a n d extensively disintegrated m i t o c h o n d r i a a n d m i c r o t u b u l e s . H o w e v e r . there were s o m e d e n d r i t e s which a p p e a r e d to be c o m p r e s s e d by s u r r o u n d i n g tissue b u t still r e t a i n e d intact m i c r o t u b u l e s inside. T h e p y r a m i d a l cell bodies showed d i s t e n d e d c y t o p l a s m with dispersed intracytoplasmic organelle, d i s i n t e g r a t e d m i t o c h o n d r i a , disaggregated r i b o s o m e s , vacuoles a n d d i s r u p t e d plasm a m e m b r a n e s (Fig. 5). H o w e v e r , there were some which showed f u r t h e r s h r i n k a g e with increased cytoplasmic electron density. M a n y nerve e n d i n g s were swollen with e n l a r g e d
Fig. 6. Immunoelectron microscopic appearance of the trunks of the apical dendrites in the proximal part of the stratum radiaturn during progression of cerebral ischemia. Reaction products after the immunohistochemical procedure for tubulin were observed in an arrangement indicative of microtubules inside the dendritic trunk (A). After ischemia for 10 min, population of reaction products associated with microtubules became sparse and irregular in many dendrites (B). After ischemia for 30 rain, reaction products with contour of microtubules were markedly reduced in many dendrites (C) and reaction products were virtually gone after ischemia for 1 h except those along plasma membranes and degraded mitochondria (D). The control section (A) was taken from the corresponding area on the non-ischemic side of the gerbil rendered ischemic for 5 min. The photographs were taken at x 17,500 magnification.
m i t o c h o n d r i a a n d clustered synaptic vesicles. Disr u p t i o n of synaptic p l a s m a m e m b r a n e s was also observed. T h e postsynaptic densities along the course of the apical a n d basal d e n d r i t e s were relatively intact. Some m y e l i n a t e d axons s h o w e d swollen mitoc h o n d r i a b u t m i c r o t u b u l e s r e m a i n e d intact. A s t r o cytic processes were e x p a n d e d a n d swollen mitoc h o n d r i a with disintegrated i n t e r n a l cristae were seen inside astrocytes everywhere.
Immunoelectron microscopic investigation In the n o r m a l gerbil b r a i n , reaction p r o d u c t s were
found as electron-dense precipitates in the pyramidal nerve cell bodies, dendrites and axons. Inside the dendrites and axons, the electron-dense precipitates were associated with microtubules (Fig. 6). Small precipitates were found widely in the pyramidal cell bodies suggesting their association with endoplasmic reticulum or polyribosomes (Fig. 7). Reaction products were also associated with postsynaptic densities. No reaction was found in the ischemic or control specimens treated with the control serum. Penetration of the antibody to the depth of the specimen was restricted and the treatment to enhance penetration such as freeze-thawing 19, Triton X-100 (ref. 13), or sodium borohydride 6 was either ineffective or detrimental for preservation of ultrastructure. On the other hand, the cutting edge, where reaction products
were seen diffusely, had inherent artifacts. Therefore, the outer 10 ~ m of each tissue block was used for this investigation. Penetration was better after extended ischemia. After ischemia for 5 min, reaction products a s s o ciated with microtubules became sparse and irregular inside the distended apical dendrites in the stratum moleculare, but there was no abnormality in other areas. After ischemia for 10 min, reaction products associated with microtubules were gone from most of the distended apical dendrites in the stratum moleculare. In the distal and proximal part of the stratum radiatum, reaction products associated with microtubules became irregular and sparse (Fig. 6). Mildly irregular reaction products were seen in some dendrites but profound loss of the reaction also occurred
Fig. 7. Immunoelectron microscopic appearance of the neuronal perikarya after ischemia for 1 h. In the normal gerbil brain (A), reaction products after the immunohistochemical procedure for tubulin were widely scattered inside the neuronal perikarya but not associated with mitochondria or inside the endoplasmic reticulum. The appearance suggested the location of reaction products to be polyribosomes. After ischemia for 1 h (B), reaction products were still observed but less densely. Swollen mitochondria and endoplasmic reticulum were devoid of reaction products. In this particular neuron, reaction products associated with microtubules were still visible (double arrows). The photographs were taken at × 11,250 magnification.
in others. In the severely affected dendrites, reaction products could be seen only around swollen mitochondria or along plasma membranes. After ischemia for 31) rain, configuration of reaction products associated with microtubules inside many apical dendrites was almost lost (Fig. 6). However, some apical dendrites showed only moderate loss of reaction products. Reaction products in the pyramidal cell bodies became slightly sparse. After ischemia for 1 h, many apical dendrites had empty cytoplasm with reaction products only along plasma membranes (Fig. 6). In the pyramidal cell bodies, reaction products became more sparse and intermingled with enlarged endoplasmic reticulum, disintegrated mitochondria and intracytoplasmic vacuoles (Fig. 7). After ischemia for 3 h, many pyramidal cell bodies showed only sparse reaction products. DISCUSSION Morphological alterations have been investigated extensively for various types of cerebral ischemia in the past by using electron microscopy. During progressive ischemia following unilateral carotid occlusion in the gerbil, only mild swelling of perivascular astrocytic end-feet has been noted up to ischemia for 10 min and breakdown of ribosomal units and swelling of rough endoplasmic reticulum have been observed in nerve cell bodies after ischemia for 15 min 5. The findings after re-perfusion included disaggregation of polyribosomes and swelling of endoplasmic reticulum and mitochondria soon after re-establishment of cerebral circulation s'14. Delayed neuronal reaction with central accumulation of mitochondria and vesicles has been reported in the H3 sector of the hippocampus 1'2, while proliferation of endoplasmic reticulum cisterns has been observed in the CA1 region of the hippocampus 9. The present study was prompted by our recent observation 27 of prompt loss of the immunohistochemical reaction for tubulin and creatine kinase BB-isoenzyme in the vulnerable areas of the hippocampus. In the present study, electron microscopic abnormalities occurred even after ischemia as short as 5 min and particularly in the dendritic processes, which were more vulnerable than the nerve cell bodies or presynaptic nerve terminals in the same anatomical location. Within the dendritic process, the dendritic
terminal was the most vulnerable, providing the appearance of central propagation of the ischemic damage. It is uncertain whether this is a common phenomenon in cerebral ischemia or specific for this particular part of the brain. The CA1 region of the hippocampus is enriched with receptors for excitatory amino acids such as L-glutamic acid and N-methyl-Daspartate 13. An antagonist for N-methyl-D-asparrate, 2-amino-7-phosphonoheptanoic acid, has been shown to prevent convulsion; and the neural damage caused by cerebral ischemia ~s and hypoglycemia e3. Therefore, it is possible that the observed ultrastructural alterations at the dendritic terminals were mediated by the receptors for excitatory amino acids. The area of the hippocampus we investigated was in contact with cerebrospinal fluid. Since osmolality of brain tissue may increase during ischemia It, the observed expansion of the dendritic terminals could be the osmotic effect. Those possibilities have to be investigated in the future. At the initial stage of the present investigation, the methods for TEM and IEM were optimized with normal gerbil brain tissue. The initial brief perfusion with saline was used to ensure adequate perfusion fixation afterwards. Since the lag time between release of the clip to initiation of perfusion fixation with aldehydes was less than 20 s, it is unlikely that we introduced the effect of further ischemia or the effect of re-perfusion. Since the site of the present investigation was based on our earlier immunohistochemical identification of this area being particularly vulnerable to ischemia after tissue fixation with immersion in alcohol-5% acetic acid 27 or 2% paraformaldehyde-0.2% picric acid m and since perfusion fixation with 2% paraformatdehyde-0.2% picric acid provided the same results (Matsumoto, M. and Yanagihara, T., unpublished data), the observed electron microscopic alterations likely reflect the in vivo changes. There is a possibility, however, that brief perfusion with saline might have exaggerated the findings. While we only dealt with progressive ischemia without recirculation in this investigation, it is quite conceivable that the same phenomenon can occur during evolution of postischemic lesions and it is possible that 'delayed neuronal death' observed by others 9 may reflect the delay in appearance of the morphological damage in the neuronal perikarya. In-
volvement of the proximal part of the apical dendrites adjacent to the nerve cell bodies has been observed 48-72 h after re-perfusion in cerebral ischemia in the rat 15. The present study confirmed prompt disintegration of microtubules inside the dendritic processes. The earliest sign of disintegration was observed in the very peripheral part of the apical dendrites after ischemia for 5 min and later observed more centrally. With the I E M method, the earliest alteration of reaction products associated with microtubules became visible again in the peripheral part of the apical dendrites after ischemia for 5 rain and irregularity and loss of reaction products were later observed more centrally. Because of simultaneous disappearance of microtubules and reaction products associated with microtubules, we conclude that loss of the immunohistochemical reaction for tubulin which we have observed earlier with the light microscopic investigation 27 was caused by disintegration of microtubules inside the dendritic processes. In contrast to the dendrites, reaction products in the neuronal perikarya were better preserved up to an ischemic period of 30 min with the I E M method. This is in agreement with our recent light microscopic investigation using 2% p a r a f o r m a l d e h y d e - 0 . 2 % picric acid for tissue fixation, where delayed disappearance of the immunohistochemical reaction occurred in the neuronal perikarya 1°. Judging from the distribution pattern of reaction products seen by IEM, it is possible that disappearance of reaction products from the neuronal REFERENCES 1 Brown, A.W., Levy, D.E., Kublik, M., Harrow, J., Plum, F. and Brierley, J.B., Selective chromatolysis of neurons in the gerbil brain: a possible consequence of 'epileptic' activity produced by common carotid artery occlusion, Ann. Neurol., 5 (1979) 127-138. 2 Bubis, J.J., Fujimoto, T., Ito, U., Mrgulja, B.J., Spatz, M. and Klatzo, I., Experimental cerebral ischemia in mongolian gerbils. V. Ultrastructural changes in H3 sector of the hippocampus, Acta Neuropathol., 36 (1976) 285-294. 3 Croucher, M.J., Collins, J.F. and Meldrum, B.S., Anticonvulsant action of excitatory amino acid antagonists, Science, 216 (1982) 899-901. 4 Deery, W.J., Means, A.R. and Brinkley, B.R., Calmodulin-microtubule association in cultured mammalian cells, J. Cell Biol., 98 (1984) 904-910. 5 Dodson, R.F., Chu, L.W.F., Welch, K.M.A. and Achar, V.S., Acute tissue response to cerebral ischemia in the gerbil. An ultrastructural study, J. Neurol. Sci., 33 (1977) 161-170.
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ACKNOWLEDGEMENT The present investigation was supported by the Grant NS-06663 from the National Institutes of Health, the U.S. Public Health Services. 6 Eldred, W.D., Zucker, C., Karten, H.J. and Yazulla, S., Comparison of fixation and penetration enhancement techniques for use in ultrastructural immunocytochemistry, J. Histochem. Cytochem., 31 (1983) 285-292. 7 Garcia, J.H., Mitchem, H.L., Briggs, L., Morawetz, R., Hudetz, A.G., Hazelrig, J.B., Halsey, J.H., Jr. and Conger, K.A., Transient focal ischemia in subhuman primates. Neuronal injury as a function of local cerebral blood flow, J. Neuropathol. Exp. Neurol., 42 (1983) 44-60. 8 Hartmann, J.F. and Becker, R.A., Disaggregation of polyribosomes in intact gerbils following ischemia. An ultrastructural study, Stroke, 4 (1973) 964-968. 9 Kirino, T. and Sano, K., Fine structural nature of delayed neuronal death following ischemia in the gerbil hippocampus, Acta Neuropathol., 62 (1984) 209-218. 10 Matsumoto, M., Yamamoto, K. and Yanagihara, T., Cerebral ischemia in gerbils: immunohistochemical study for neuronal and astrocytic proteins, J. Cereb. Blood Flow Metab., 5 (Suppl. 1) (1985) 299-300. 11 Matsuoka, Y. and Hossmann, K.-A., Brain tissue osmolality after middle cerebral artery occlusion in cats, Exp. Neu-
10 rol., 77 (1982) 599-611. 12 Matus, A.I., Waiters, B.B. and Mughal, S., lmmunohistochemical demonstration of tubulin associated with microtubules and synaptic junctions in mammalian brain, J. Neurocytol., 4 (1975) 733-744. 13 Monaghan, D.T. and Cotman, C.W., Distribution of Nmethyl-D-aspartate-sensitive L-[3H]glutamate-binding sites in rat brain, J. Neurosci., 5 (1985) 2909-2919. 14 Morimoto, K. and Yanagihara, T., Cerebral ischemia in gerbils: polyribosomal function during progression and recovery, Stroke. 12 (1981) 105-110. 15 Petito, C.K. 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. 16 Piekut, D.T. and Casey, S.M., Penetration of immunoreagents in vibratome-sectioned brain: a light and electron microscope study, J. Histochem. Cytochem., 31 (1983) 669-674. 17 Simon, R.P., Griffiths, T., Evans, M.C., Swan, J.H. and Meldrum, B.S., Calcium overload in selectively vulnerable neurons of the hippocampus during and after ischemia: an electron microscopy study in the rat, J. Cereb. Blood Flow Metab., 4 (1984) 350-361. 18 Simon, R.P., Swan, J.H., Griffiths, T. and Meldrum, B.S., Blockade of N-methyl-o-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850-852. 19 Somogyi, P. and Takagi, H., A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry, Neuroscience, 7 (1982) 1779-1783. 20 Spurr, A.R., A low-viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res., 26 (1969) 31-43.
21 Walker, P.R. and Whitfield, J.F., Cytoplasmic microtubules are essential for the formation of membrane-bound polyribosomes, J. Biol. Chem.. 260 (1985) 765 770. 22 Weisenberg, R.C. and Decry, W.J., The mechanism of calcium-induced microtubule disassembly, Biochem. Biophys. Res. Commun., 102 (1981) 924-931. 23 Wieloch, T., Hypoglycemia-induced neuronal damage prevented by an N-methyl-D-aspartate antagonist, Science. 230 (1985) 681-683. 24 Yamamoto, K. and Yanagihara, T., Immunoelectronmicroscopic investigation of cerebral ischemia in gerbils (Abstr.), J. Neuropathol. Exp. Neurol., 44 (1985) 337. 25 Yamamoto, K., Yoshimine, T., Homburger, H.A. and Yanagihara, T., Immunohistochemical investigation of regional cerebral ischemia: occlusion of the posterior communicating artery in the gerbil, Brain Research, in press. 26 Yanagihara, T., Experimental stroke in gerbils: correlation of clinical, pathological and electroencephalographic findings and protein synthesis, Stroke, 9 (1978) 155-159. 27 Yanagihara, T., Yoshimine, T., Morimoto, K., Yamamoto, K. and Homburger, H.A., Immunohistochemical investigation of cerebral ischemia in gerbils, J. Neuropathol. Exp. Neurol., 44 (1985) 204-215. 28 Yoshimine, T. and Yanagihara, T., Regional cerebral ischemia by occlusion of the posterior communicating artery and of the middle cerebral artery in gerbils, J. Neurosurg.. 58 (1983) 362-367. 29 Yoshimine, T., Morimoto, K., Homburger, H.A. and Yanagihara, T., Immunohistochemical localization of creatine kinase BB-isoenzyme and tubulin in gerbil brain, Neuroscience, 12 (1984) 959-969. 30 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.
1
Elsevier BRE 12023
Research Reports Cerebral Ischemia in the Gerbil: Transmission Electron Microscopic and Immunoelectron Microscopic Investigation KAZUMI YAMAMOTO, KAZUYOSHI MORIMOTO and TAKEHIKO YANAGIHARA
Department of Neurology, Mayo Clinic and Mayo Medical School, Rochester, MN 55905 (U. S. A.) (Accepted 25 February 1986) Key words: Cerebral ischemia - - Dendrite - - Gerbil - - Immunoelectron microscopy - Microtubule - - Transmission electron microscopy
Progression of cerebral ischemia from 5 min to 3 h after occlusion of a common carotid artery was investigated in the subiculum-CA1 region of the hippocampus of the gerbil by transmission electron microscopic and immunoelectron microscopic technique. The earliest change was found after 5 min in the periphery of the apical dendrites in the stratum moleculare, where mitochondrial swelling and disintegration of microtubules were clearly seen inside swollen dendritic processes. After ischemia for 10 min, similar abnormalities were observed in the more proximal part of the apical dendrites, and the basal dendrites also became similarly affected. After ischemia for 30 min to 1 h, the pyramidal cell bodies showed mitochondrial swelling, distension of endoplasmic reticulum and disaggregation of polyribosomes. The immunoelectron microscopic procedure for tubulin revealed irregularity of reaction products associated with microtubules after ischemia for 5 min in the dendritic terminals in the stratum moleculare and in the stratum radiatum after ischemia for 10 min. Reaction products in the pyramidal cell bodies became sparse after ischemia for 30 min to 1 h. The present investigation revealed early onset of ischemic damage in the dendritic terminals and subsequent proximal extension, with disintegration of microtubules and mitochondrial swelling.
INTRODUCTION A recent investigation from our l a b o r a t o r y revealed p r o m p t d i s a p p e a r a n c e of the light microscopic immunohistochemical reaction for tubulin and creatine kinase BB-isoenzyme from the ischemic areas as early as 5 min after occlusion of a c o m m o n carotid artery in the gerbil 27. A l t h o u g h disintegration of microtubules was p r o p o s e d as one of the possible causes for d i s a p p e a r a n c e of the reaction for tubulin, the underlying mechanism remains uncertain. The immunohistochemical investigation also indicated early involvement of the dendrites, particularly in the hippocampus. In the present investigation, we therefore examined the subiculum-CA1 region of the hippocampus during progression of unilateral cerebral ischemia in the gerbil by transmission electron microscopy ( T E M ) and i m m u n o e l e c t r o n microscopy
( I E M ) for tubulin with special attention to the dendritic structure. The present investigation has been r e p o r t e d in abstract form 24. MATERIALS AND METHODS Mongolian gerbils (Meriones unguiculatus) weighing 60-70 g were used for the present investigation. The details of animal p r e p a r a t i o n have been published previously 14'26. U n d e r anesthesia with ether inhalation, the right c o m m o n carotid artery of each gerbil was occluded in the neck with a miniature Mayfield aneurysm clip. The symptomatic gerbils were identified by torsion of the neck and circling in less than 15 min after surgery. T h r e e gerbils were used for investigation of the ischemic period for 5, 10 and 30 min, respectively, and 2 gerbils were taken for investigation of the ischemic p e r i o d of 1 and 3 h, re-
Correspondence: T. Yanagihara, Department of Neurology, Mayo Clinic, Rochester, MN 55905, U.S.A. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
spectively. After a predetermined ischemic period, each gerbil was re-anesthetized with ether and thoracotomy was carried out. The clip on the common carotid artery was removed promptly and transcardiac perfusion was carried out through the left ventricle of the heart for 20 s with 0.9% saline and then for 15 rain with 2% paraformaldehyde-2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for TEM investigation or 4% paraformaldehyde-0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for IEM investigation. Actual tissue fixation started 20 s after removal of the clip. The brain was promptly removed, sectioned into blocks and further fixed for 4 h at 4 °C in the same aldehyde-based fixative as used for perfusion fixation. Tissue blocks were washed in 0.1 M phosphate buffer (pH 7.4) and further sectioned with a vibratome (Lancer) at 100/~m for TEM and 50/~m for IEM. The sections for TEM were further fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 h, washed in the same buffer solution, contrasted with 2% uranyl acetate in 70% alcohol for 40 min, dehydrated and flat-embedded in Spurr's embedding medium 2°. For IEM, the procedure described by Matus et al. lz was used with slight modification. Each tissue section was incubated with 20% normal rabbit serum at room temperature for 30 min, the anti-tubulin goat serum (1:200) for 1 h, the rabbit anti-goat IgG (1:40) for 1 h and the peroxidase-antiperoxidase complex (1:100) for 1 h. The antiserum for tubulin has been prepared in a goat by administration of purified tubulin from gerbil brains 29. The control specimen was treated with the absorbed antiserum or non-immunized goat serum. Between each incubation, tissue sections were washed 3 times for 1 h each with 0.9% saline in 0.02 M phosphate buffer (pH 7.4) at 4 °C. Each tissue section was incubated with 0.05% 3,3'diaminobenzidine tetrahydrochloride in 0.05 M TrisHC1 buffer (pH 7.6) for 15 min and then with 0.05% 3,Y-diaminobenzidine tetrahydrochloride-0.01% hydrogen peroxide in the same Tris-HC1 buffer for 7 min. Each section was washed with the same TrisHCI buffer and then with 0.1 M phosphate buffer (pH 7.4) prior to fixation with 1% osmium tetroxide in the same buffer for 1 h. The subsequent procedure was the same as for TEM. One adjacent section was examined under a light microscope without fixation
with osmium tetroxide for the presence of ischemic lesions. Under a dissection microscope, the subiculumCA1 region of the hippocampus was dissected from the flat-embedded tissue section and re-embedded in a silicone rubber mold with fresh Spurr's embedding medium 2°. Serial ultrathin sections were prepared with an ultramicrotome (Porter-Blum MT-2B) and examined under a transmission electron microscope (Philips 201). Some sections for TEM were further contrasted with 0.5% lead citrate. Semi-thin sections (1 btm) were stained with 1% toluidine blue and examined under a light microscope. Control specimens were prepared from the sham-operated gerbils and the left (non-occluded) side of the operated gerbils. The specimens were examined by 2 investigators independently. In order to detect subtle abnormalities, 1 investigator carried out evaluation without knowing the identity of specimens, whenever necessary. RESULTS
Transmission electron microscopic investigation Ischemia for 5 min. Light microscopic examination of semi-thin sections after toluidine blue staining revealed honeycomb appearance of the stratum moleculare (Fig. 1). Under electron microscopic examination (Table I), marked swelling was observed in the peripheral part of the apical dendrites which were positively identified by the presence of postsynaptic densities (Fig. 2). Inside, swollen mitochondria with disrupted internal cristae, disintegrated microtubules and vacuoles were found. Disruption of cytoplasmic membranes was occasionally observed. On the other hand, the nerve endings in the same area appeared unaffected. The postsynaptic densities were also unaffected. Swollen mitochondria also existed inside the apical dendrites in the distal part of the stratum radiatum (Fig. 3) and in 1 gerbil in the proximal part of the stratum radiatum, particularly in the small dendritic processes (Fig. 4), and within the basal dendrites in the stratum oriens. No abnormality was found in the pyramidal cell bodies. Swollen mitochondria were also seen within the astrocytic processes in the stratum radiatum and oriens in all gerbils and even in the stratum pyramidale in 1 gerbil. Because of marked dendritic swelling, it was
'A
Fig. 1. Light microscopic appearance of the stratum moleculare during progression of cerebral ischemia. Already after ischemia for 5 min, mild honeycomb appearance became visible at the periphery of the stratum moleculare (B). Vacuolation expanded further after ischemia for 10 min (C) and 30 min (D). The control section (A) was taken from the corresponding area on the non-ischemic side of the gerbil rendered ischemic for 5 min. Semi-thin sections were stained with 1% toluidine blue. The photographs were taken at x400 magnification.
difficult to ascertain swelling of astrocytic mitochondria in the stratum moleculare. No a b n o r m a l i t y was found in any stratum on the left side as c o m p a r e d to the corresponding areas of the control gerbils. Ischernia for 10 min. F u r t h e r expansion of honeycomb a p p e a r a n c e was observed in the stratum moleculare under light microscopic examination (Fig. 1). Electron microscopic examination revealed further distension of the p e r i p h e r a l part of the apical dendrites in the stratum moleculare where irregular m e m b r a n e - b o u n d vacuoles and further swelling of mitochondria with disintegration of internal cristae were seen (Fig. 2). Disintegration of microtubules was observed in all gerbils and disruption of p l a s m a
Fig. 2. Transmission electron microscopic appearance of the stratum moleculare during progression of cerebral ischemia. The peripheral parts of the apical dendrites, which contained mitochondria and microtubules, were intermingled with more osmiophilic presynaptic elements in the control brain (A). After ischemia for 5 min, the distal parts of the apical dendrites which could be identified positively by the presence of postsynaptic densities (arrowhead), were markedly swollen with disintegration of microtubules, swollen mitochondria and formation of vacuoles (B). Disintegration of microtubules and mitochondria further progressed after ischemia for 10 min (C) and 30 min (D). The control section (A) was taken from the corresponding area on the non-ischemic side of the gerbil rendered ischemic for 5 min. The photographs were taken at x17,500 magnification.
m e m b r a n e s existed in 2 gerbils (Table I). The distal part of the stratum r a d i a t u m also showed m o r e extensive alteration including swollen m i t o c h o n d r i a with disruption of internal cristae and disintegration of microtubules (Fig. 3). Mild distension of the dendritic processes with cytoplasmic vacuoles was observed in 1 gerbil (Table I). In the proximal part of the stratum radiatum, swelling of m i t o c h o n d r i a became m o r e obvious and mild to m o d e r a t e disintegration of microtubules was o b s e r v e d inside the apical dendrites (Fig. 4). In the stratum oriens, mitochondrial swelling and disintegration of microtubules were observed to the extent similar to those in the
TABLE I Transmission electron microscopic findings of neuronal perikarya and dendrites (-) Designates the subcellular components not encountered in the area, while (U) designates the uncertain status. Three symptomatic gerbils were used for each ischemic period. Ischemic period (rain) Cytoplasmic swelling
Mitochondrial swelling
Disintegration of mitochondrial cristae
Cytoplasmic vacuoles
Swellingof endoplasmic reticulum
Polyribosomal disaggregation
Disintegration of microtubules
Disruption of plasma membrane
Stratum oriens
Stratum pyramidale
Stratum radiatum (prox. )
Stratum radiatum (dist.)
Stratum moleculare
5
0
0
0
0
3
10
0
0
0
1
3
30
3
3
0
3
3
5 10 30
3 3 3
0 2 3
1 3 3
3 3 3
3 3 3
5
0
0
0
o
3
10
1
0
1
2
3
30
3
3
3
3
3
5 10 30
0 0 3
0 0 0
0 0 3
0 1 3
3 3 3 -
5
-
0
-
-
10
-
0
-
-
30
-
3
-
-
-
-
-
5
-
0
-
10
-
0
-
30
-
3
-
-
-
5 10 30
0 3 3
0 0 U
0 3 3
0 3 3
3 3 3 2
0
0
0
0
i0
5
0
0
0
0
2
30
0
0
0
0
3
distal part of the stratum radiatum. The pyramidal
chondria and microtubules further advanced in the
cell bodies showed slightly swollen mitochondria in 2 gerbils (Table I) but more definite mitochondrial swelling was observed in the adjacent dendrites (Fig.
apical dendrites along with cytoplasmic distension (Fig. 3 and Table I). In the proximal part of the stra-
5). The nerve endings remained morphologically unaffected except for suggestive swelling of mitochondria in the stratum moleculare. Astrocytic mitochondria were swollen in every stratum in all gerbils. Because of marked distension of the dendrites, alteration of astrocytes was not well observed in the stratum moleculare. Ischemia f o r 30 rain. In the peripheral part of the apical dendrites, disintegration of mitochondria and microtubules further advanced and the distended dendrites were often empty or contained only vacuoles in the stratum moleculare (Fig. 2). In the distal part of the stratum radiatum, disintegration of mito-
tum radiatum, some apical dendrites lost microtubules almost completely and swollen mitochondria formed vacuoles because of disintegration of internal cristae. O n the other hand, some apical dendrites still retained microtubules in spite of the presence of markedly swollen mitochondria. The findings in the stratum oriens were similar to those seen in the distal part of the stratum radiatum. Some pyramidal cell bodies showed swollen mitochondria (Fig. 5), mildly enlarged endoplasmic reticulum and disaggregation of polyribosomes (Table I). While swollen mitochondria were clearly seen within the nerve endings in the distal part of the stratum radiatum and the stratum oriens, integrity of the nerve endings with synaptic vesicles was well re-
D
0 Fig. 3. Transmission electron microscopic appearance of the distal part of the stratum radiatum during progression of cerebral ischemia. The apical dendrites, which contained mitochondria and microtubules, were intermingled with more osmiophilic presynaptic elements and could be positively identified by the presence of postsynaptic densities in the control brain (A). After ischemia for 5 min, mitochondria became swollen (B). After ischemia for 10 min, swelling of mitochondria became more notable. In addition, microtubules began to disintegrate and some dendritic cytoplasm appeared swollen (C). After ischemia for 30 min, further disintegration of mitochondria and microtubules ensued along with swelling of dendritic cytoplasm (D). The control section (A) was taken from the corresponding area on the non-ischemic side of the gerbil rendered ischemic for 5 min. The original photographs were taken at × 17,500 magnification.
Fig. 4. Transmission electron microscopic appearance of the proximal part of the stratum radiatum during progression of cerebral ischemia. Abundance of microtubules were identified inside the apical dendrites along with mitochondria in the control brain (A). After ischemia for 5 min, no abnormality of rnicrotubules was found but mitochondria in small dendritic processes were swollen and those in the main trunks of the apical dendrites also appeared mildly swollen (B). After ischemia for 10 min, mitochondria became more swollen inside the apical dendrites but those in the nerve endings were still small. Microtubules began to show irregular contour and density (C). After ischemia for 1 h, further disintegration of microtubules occurred (D). The control section (A) was taken from the corresponding area on the non-ischemic side of the gerbil rendered ischemia for 5 min. The photographs were taken at ×17,500 magnification.
tained. Myelinated axons were still intact. Distension of the astrocytic processes with swollen mitochondria was observed everywhere.
addition, shrunken pyramidal cell bodies with increased nuclear and cytoplasmic electron density were also observed.
Ischemia for 1 h. Disintegration of the peripheral part of the apical dendrites progressed with disruption of plasma m e m b r a n e s . Many apical dendrites in the proximal part of the stratum radiatum lost microtubules extensively or completely (Fig. 4). However, there were still scattered dendrites which retained microtubules relatively well. The pyramidal cell bodies revealed further distension of endoplasmic reticulum and increase in intracytoplasmic vacuoles. In
In the distal part of the stratum radiatum and the stratum oriens, some nerve endings appeared to be distended with swollen mitochondria and aggregated synaptic vesicles. Swelling of the astrocytic processes with swollen mitochondria with disrupted internal cristae was more advanced in each stratum.
Ischemia for 3 h. Morphological alterations observed after ischemia for 1 h advanced further. Various degrees of distension existed in the entire course
Fig. 5. Transmission electron microscopic appearance of the stratum pyramidale during progression of cerebral ischemia. After ischemia for 10 rain, no remarkable change was observed in the neuronal perikarya except for mildly swollen mitochondria but they were more definitely swollen in the dendrites in the surrounding area (B). After ischemia for 30 rain, further swelling of mitochondria occurred in the neuronal perikarya, where swelling of the endoplasmic reticulum and disaggregation of polyribosomes began to ensue (C). After ischemia for 3 h, extensive ischemic damage with swollen endoplasmic reticulure, disaggregated polyribosomes, and vacuolation of mitochondria occurred in the neuronal perikarya (D). The control section (A) was taken from the corresponding area on the nonischemic side of the gerbil rendered ischemic for 5 rain. The photographs were taken at x 11,250 magnification.
of the apical a n d basal d e n d r i t e s with d i s r u p t e d plasm a m e m b r a n e s , v a c u o l a t i o n a n d extensively disintegrated m i t o c h o n d r i a a n d m i c r o t u b u l e s . H o w e v e r . there were s o m e d e n d r i t e s which a p p e a r e d to be c o m p r e s s e d by s u r r o u n d i n g tissue b u t still r e t a i n e d intact m i c r o t u b u l e s inside. T h e p y r a m i d a l cell bodies showed d i s t e n d e d c y t o p l a s m with dispersed intracytoplasmic organelle, d i s i n t e g r a t e d m i t o c h o n d r i a , disaggregated r i b o s o m e s , vacuoles a n d d i s r u p t e d plasm a m e m b r a n e s (Fig. 5). H o w e v e r , there were some which showed f u r t h e r s h r i n k a g e with increased cytoplasmic electron density. M a n y nerve e n d i n g s were swollen with e n l a r g e d
Fig. 6. Immunoelectron microscopic appearance of the trunks of the apical dendrites in the proximal part of the stratum radiaturn during progression of cerebral ischemia. Reaction products after the immunohistochemical procedure for tubulin were observed in an arrangement indicative of microtubules inside the dendritic trunk (A). After ischemia for 10 min, population of reaction products associated with microtubules became sparse and irregular in many dendrites (B). After ischemia for 30 rain, reaction products with contour of microtubules were markedly reduced in many dendrites (C) and reaction products were virtually gone after ischemia for 1 h except those along plasma membranes and degraded mitochondria (D). The control section (A) was taken from the corresponding area on the non-ischemic side of the gerbil rendered ischemic for 5 min. The photographs were taken at x 17,500 magnification.
m i t o c h o n d r i a a n d clustered synaptic vesicles. Disr u p t i o n of synaptic p l a s m a m e m b r a n e s was also observed. T h e postsynaptic densities along the course of the apical a n d basal d e n d r i t e s were relatively intact. Some m y e l i n a t e d axons s h o w e d swollen mitoc h o n d r i a b u t m i c r o t u b u l e s r e m a i n e d intact. A s t r o cytic processes were e x p a n d e d a n d swollen mitoc h o n d r i a with disintegrated i n t e r n a l cristae were seen inside astrocytes everywhere.
Immunoelectron microscopic investigation In the n o r m a l gerbil b r a i n , reaction p r o d u c t s were
found as electron-dense precipitates in the pyramidal nerve cell bodies, dendrites and axons. Inside the dendrites and axons, the electron-dense precipitates were associated with microtubules (Fig. 6). Small precipitates were found widely in the pyramidal cell bodies suggesting their association with endoplasmic reticulum or polyribosomes (Fig. 7). Reaction products were also associated with postsynaptic densities. No reaction was found in the ischemic or control specimens treated with the control serum. Penetration of the antibody to the depth of the specimen was restricted and the treatment to enhance penetration such as freeze-thawing 19, Triton X-100 (ref. 13), or sodium borohydride 6 was either ineffective or detrimental for preservation of ultrastructure. On the other hand, the cutting edge, where reaction products
were seen diffusely, had inherent artifacts. Therefore, the outer 10 ~ m of each tissue block was used for this investigation. Penetration was better after extended ischemia. After ischemia for 5 min, reaction products a s s o ciated with microtubules became sparse and irregular inside the distended apical dendrites in the stratum moleculare, but there was no abnormality in other areas. After ischemia for 10 min, reaction products associated with microtubules were gone from most of the distended apical dendrites in the stratum moleculare. In the distal and proximal part of the stratum radiatum, reaction products associated with microtubules became irregular and sparse (Fig. 6). Mildly irregular reaction products were seen in some dendrites but profound loss of the reaction also occurred
Fig. 7. Immunoelectron microscopic appearance of the neuronal perikarya after ischemia for 1 h. In the normal gerbil brain (A), reaction products after the immunohistochemical procedure for tubulin were widely scattered inside the neuronal perikarya but not associated with mitochondria or inside the endoplasmic reticulum. The appearance suggested the location of reaction products to be polyribosomes. After ischemia for 1 h (B), reaction products were still observed but less densely. Swollen mitochondria and endoplasmic reticulum were devoid of reaction products. In this particular neuron, reaction products associated with microtubules were still visible (double arrows). The photographs were taken at × 11,250 magnification.
in others. In the severely affected dendrites, reaction products could be seen only around swollen mitochondria or along plasma membranes. After ischemia for 31) rain, configuration of reaction products associated with microtubules inside many apical dendrites was almost lost (Fig. 6). However, some apical dendrites showed only moderate loss of reaction products. Reaction products in the pyramidal cell bodies became slightly sparse. After ischemia for 1 h, many apical dendrites had empty cytoplasm with reaction products only along plasma membranes (Fig. 6). In the pyramidal cell bodies, reaction products became more sparse and intermingled with enlarged endoplasmic reticulum, disintegrated mitochondria and intracytoplasmic vacuoles (Fig. 7). After ischemia for 3 h, many pyramidal cell bodies showed only sparse reaction products. DISCUSSION Morphological alterations have been investigated extensively for various types of cerebral ischemia in the past by using electron microscopy. During progressive ischemia following unilateral carotid occlusion in the gerbil, only mild swelling of perivascular astrocytic end-feet has been noted up to ischemia for 10 min and breakdown of ribosomal units and swelling of rough endoplasmic reticulum have been observed in nerve cell bodies after ischemia for 15 min 5. The findings after re-perfusion included disaggregation of polyribosomes and swelling of endoplasmic reticulum and mitochondria soon after re-establishment of cerebral circulation s'14. Delayed neuronal reaction with central accumulation of mitochondria and vesicles has been reported in the H3 sector of the hippocampus 1'2, while proliferation of endoplasmic reticulum cisterns has been observed in the CA1 region of the hippocampus 9. The present study was prompted by our recent observation 27 of prompt loss of the immunohistochemical reaction for tubulin and creatine kinase BB-isoenzyme in the vulnerable areas of the hippocampus. In the present study, electron microscopic abnormalities occurred even after ischemia as short as 5 min and particularly in the dendritic processes, which were more vulnerable than the nerve cell bodies or presynaptic nerve terminals in the same anatomical location. Within the dendritic process, the dendritic
terminal was the most vulnerable, providing the appearance of central propagation of the ischemic damage. It is uncertain whether this is a common phenomenon in cerebral ischemia or specific for this particular part of the brain. The CA1 region of the hippocampus is enriched with receptors for excitatory amino acids such as L-glutamic acid and N-methyl-Daspartate 13. An antagonist for N-methyl-D-asparrate, 2-amino-7-phosphonoheptanoic acid, has been shown to prevent convulsion; and the neural damage caused by cerebral ischemia ~s and hypoglycemia e3. Therefore, it is possible that the observed ultrastructural alterations at the dendritic terminals were mediated by the receptors for excitatory amino acids. The area of the hippocampus we investigated was in contact with cerebrospinal fluid. Since osmolality of brain tissue may increase during ischemia It, the observed expansion of the dendritic terminals could be the osmotic effect. Those possibilities have to be investigated in the future. At the initial stage of the present investigation, the methods for TEM and IEM were optimized with normal gerbil brain tissue. The initial brief perfusion with saline was used to ensure adequate perfusion fixation afterwards. Since the lag time between release of the clip to initiation of perfusion fixation with aldehydes was less than 20 s, it is unlikely that we introduced the effect of further ischemia or the effect of re-perfusion. Since the site of the present investigation was based on our earlier immunohistochemical identification of this area being particularly vulnerable to ischemia after tissue fixation with immersion in alcohol-5% acetic acid 27 or 2% paraformaldehyde-0.2% picric acid m and since perfusion fixation with 2% paraformatdehyde-0.2% picric acid provided the same results (Matsumoto, M. and Yanagihara, T., unpublished data), the observed electron microscopic alterations likely reflect the in vivo changes. There is a possibility, however, that brief perfusion with saline might have exaggerated the findings. While we only dealt with progressive ischemia without recirculation in this investigation, it is quite conceivable that the same phenomenon can occur during evolution of postischemic lesions and it is possible that 'delayed neuronal death' observed by others 9 may reflect the delay in appearance of the morphological damage in the neuronal perikarya. In-
volvement of the proximal part of the apical dendrites adjacent to the nerve cell bodies has been observed 48-72 h after re-perfusion in cerebral ischemia in the rat 15. The present study confirmed prompt disintegration of microtubules inside the dendritic processes. The earliest sign of disintegration was observed in the very peripheral part of the apical dendrites after ischemia for 5 min and later observed more centrally. With the I E M method, the earliest alteration of reaction products associated with microtubules became visible again in the peripheral part of the apical dendrites after ischemia for 5 rain and irregularity and loss of reaction products were later observed more centrally. Because of simultaneous disappearance of microtubules and reaction products associated with microtubules, we conclude that loss of the immunohistochemical reaction for tubulin which we have observed earlier with the light microscopic investigation 27 was caused by disintegration of microtubules inside the dendritic processes. In contrast to the dendrites, reaction products in the neuronal perikarya were better preserved up to an ischemic period of 30 min with the I E M method. This is in agreement with our recent light microscopic investigation using 2% p a r a f o r m a l d e h y d e - 0 . 2 % picric acid for tissue fixation, where delayed disappearance of the immunohistochemical reaction occurred in the neuronal perikarya 1°. Judging from the distribution pattern of reaction products seen by IEM, it is possible that disappearance of reaction products from the neuronal REFERENCES 1 Brown, A.W., Levy, D.E., Kublik, M., Harrow, J., Plum, F. and Brierley, J.B., Selective chromatolysis of neurons in the gerbil brain: a possible consequence of 'epileptic' activity produced by common carotid artery occlusion, Ann. Neurol., 5 (1979) 127-138. 2 Bubis, J.J., Fujimoto, T., Ito, U., Mrgulja, B.J., Spatz, M. and Klatzo, I., Experimental cerebral ischemia in mongolian gerbils. V. Ultrastructural changes in H3 sector of the hippocampus, Acta Neuropathol., 36 (1976) 285-294. 3 Croucher, M.J., Collins, J.F. and Meldrum, B.S., Anticonvulsant action of excitatory amino acid antagonists, Science, 216 (1982) 899-901. 4 Deery, W.J., Means, A.R. and Brinkley, B.R., Calmodulin-microtubule association in cultured mammalian cells, J. Cell Biol., 98 (1984) 904-910. 5 Dodson, R.F., Chu, L.W.F., Welch, K.M.A. and Achar, V.S., Acute tissue response to cerebral ischemia in the gerbil. An ultrastructural study, J. Neurol. Sci., 33 (1977) 161-170.
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