Experimental Brain Research
Exp. Brain Res. 32, 459-469 (1978)
@ Springer-Verlag 1978
Histochemical Study of Cobalt-Induced Focal Epilepsy J. Brotchi, A. Dresse, J. Scuvee-Moreau and M.A. Gerebtzoff Departments of Neurosurgery, Anatomy and Pharmacology, University of Liege, B-4020 Liege, Belgium
Summary. A previous study showed a strong relationship between human focal epilepsy and the presence in the cortex of "activated" astrocytes characterized by an intense activity of dehydrogenases (DH) involved in glucose metabolism and of glutamate DH. Using the semi-chronic model of cobalt-induced experimental focal epilepsy in the rat, we investigated a possible correlation between astrocyte modifications and the chronological development of the epileptic manifestations on the E C o G . After a few days the cobalt-implanted rats present spikes, then sharp waves followed by an electrical crisis and ultimately motor seizures. Activated astrocytes were found in each phase of this evolution. Their number increases with the intensity of the manifestations. There is a close relationship between activated astrocytes and focal epileptic phenomena. At this stage of our study it is clear that the presence of activated astrocytes is not a consequence of seizures. However, it is impossible to say whether the activation is secondary to the hyperactivity of the neurons or directly responsib!e for the constitution of the epileptic focus. In any case, activated astrocytes provide a new means of localizing an epileptogenic focus.
Key words: Experimental epilepsy - Astrocytes - Histoenzymology Dehydrogenases.
It is generally accepted that there is no major morphological difference between the "epileptic n e u r o n " and the normal one (Brown, 1973). However, some altered dendrites have been described by the Scheibels (1968), using the technique of Golgi. A mechanical influence on the activity of the neuronal cell has also been postulated (Ward, 1961). In these previous studies the role of neuroglia has often been neglected except when considering the meningocerebral scar (Penfield, 1932; Penfield and Jasper, 1954). Offprint requests to: Prof. M.A. Gerebtzoff, Institut d'Anatomie, rue de Pitteurs, 20, B - 4020 Liege, Belgium 0 0 1 4 - 4 8 1 9 / 7 8 / 0 0 3 2 / 0 4 5 9 / $ 2.20
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While remaining faithful to the present biochemical and physiological concepts on the dynamic role of neuroglia and particularly of the astrocyte in the polarization of the neuron (Tower, 1960, 1965; Kuffler and Nicholls, 1966, 1976), we have attempted to study the astrocyte by a histochemical approach. This method has the advantage of respecting the anatomical relations between the various cellular elements. A direct visualization of the oxidative metabolism of the astrocyte may be obtained by a histochemical evaluation of the activity of certain dehydrogenases (DH). Among the numerous enzymes which catalyze oxidoreduction phenomena, we have chosen five DH which are situated at strategic sites of metabolic pathways. 1. The Glucose-6-Phosphate dehydrogenases (G6PDH) situated in the first stage of the pentose shunt. 2. The Glutamate dehydrogenase (GDH) participating in the transformation of c~-ceto-glutarate into glutamate. 3. The Lactate dehydrogenase (LDH) situated at the end of glycolysis and possessing five isoenzymes characterized by electrophoretic properties. With our method we can establish two groups (Gerebtzoff, 1968, 1970): a) the slow moving group called ISO S. b) the fast moving group called ISO F. It is not yet possible to ascertain if the borderline is situated between the isoenzymes 2 and 3 or 3 and 4. 4. The succinate dehydrogenase (SDH) situated in Kreb's cycle. The normal astrocyte demonstrates very little activity of oxidase enzymes as compared with oligodendroglia, nerve cells or ependyma except for GDH. There is a moderate G6PDH activity, very little ISO S and ISO F and no SDH activity within the astrocytes (Friede, 1965; Dimova and Gerebtzoff, 1966; Gerebtzoff et al., 1974). In some conditions, astrocytes can become morphologically reactive. There is a transformation from the protoplasmic to the fibrous form and a production of gliofibrils (Penfield, 1932). In the rat, an experimental lesion limited to the cortex induces morphologically reactive astrocytes in the neighborhood of the lesion (Dimova, 1966). An extension of this morphological reaction is observed along the pyramidal tract as far as the end of the lumbar medulla when the lesion is in the motor cortex (Demolin and Gerebtzoff, 1975). A histochemical study of these reactive cells has shown that the astrocytes in close proximity to the lesion acquire a high level of DH activity contrary to more caudally placed astrocytes which remain normal. The morphologically reactive astrocytes may thus remain normal or acquire an hyperactivity of DH. Proximal reactive astrocytes after a cortical lesion are revealed by our histoenzymological techniques in the white matter near the lesion, but not in the adjacent cerebral cortex. There, the neuropil has an activity masking that of astrocytes. But cortical astrocytes presenting an intense DH activity do emerge from the neuropil in epileptogenic loci of tumoral or nontumoral human epilepsy (Brotchi, 1972, 1973a, 1973b; Gerebtzoff et al., 1974). The role of these "activated" astrocytes in the genesis of epileptic manifestations has been the object of recent experimental investigations in our laboratories.
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A s e m i - c h r o n i c m o d e l e n a b l i n g us t o f o l l o w t h e s e q u e n c e o f h i s t o c h e m i c a l and
electrocorticographic
changes
appearing
after
the
implantation
of
a
c o b a l t - g e l a t i n e p e l l e t in t h e p a r i e t a l c o r t e x o f t h e r a t h a s b e e n c h o s e n .
Methods Among the numerous experimental models of epilepsy (Purpura et al., 1972), we have chosen the implantation of a cobalt-gelatine rod according to the method of Dow et al. (1961) modified by Fischer et al. (1967). This mixture has the advantage of decreasing the necrotic power of cobalt and gives a stable model of focal epilepsy during 4 to 6 weeks. The pellet is prepared as follows: a suspension of cobalt metallic powder in warm 5 % gelatine is allowed to sediment and is then decanted on to a horizontal slide to form a layer 0.75 mm thick. The solidified film is dehydrated in acetone, fixed in formaldehyde vapors and kept in 90 ~ alcohol. Pellets measuring 0.75 x 0.75 x 1.5 mm are then cut from the film with a razor blade. Control rats are implanted simultaneously with glass powder pellets instead of cobalt. Eighty male Wistar rats of 200-250 g were used in these experiments. The animals were operated under general (Chloral hydrate, 400 mg/kg i.p.) anesthesia. A bone flap was cut in the left parietal side. The dura was gently opened and the pellet inserted perpendicularly into the cortex. The bone was closed to prevent the expulsion of the pellet from the brain. Four stainless steel screws were set as follows: the two anterior ones were situated 2 mm before and 2 mm lateral to the bregma, the two posterior ones 1.5 mm in front of the lambda and 3.2 mm laterally. Electrocorticograms were daily recorded during thirty minutes. The criteria used to follow the different stages of the epileptic manifestations will be described and illustrated in the results. For histochemical analysis, the animals were killed by decapitation. The brain was immediately frozen on dry ice. Slices 7-10 g thick were cut at-18~ and then incubated in appropriated baths for the studied DH. The slices were fixed in 10% formalin, washed in distilled water, dehydrated in alcohols, clarified in toluol and mounted in Caedax. The principle of the method is to reduce a soluble and colourless salt of tetrazolium into an insoluble and colored form detectable with a photonic microscope. To facilitate the solubilization of the tetrazolium salt named "tetranitroblue of tetrazolium" (TNBT) it is dissolved in dimethylsulfoxyde (DMSO). The incubation baths are prepared as follows for the different enzymes: i. G6PDH (D-glucose-6-phosphate: NADP oxidoreductase, E.N.l.1.l.49). Tris-HC1 buffer 0.1 M, pH 7.6 5 ml. D-glucose-6-phosphate 50 mg in 5 ml water. NADP 5 mg in 2.5 ml water. TNBT 6.25 mg in 0.3 ml DMSO and 2.2 ml water. 2. GDH (L-glutamate: NAD oxidoreductase, E.N. 1.4.1.2). Monosodic salt of L-glutamic acid 50 mg and NaC1 50 mg in 5 ml water. NAD 5 mg in 2.5 ml water. TNBT 6.25 mg in 0.3 ml DMSO and 2.2 ml water. 3.1SO S of L D H (L-lactate: NAD oxidoreductase, E.N.l.i. 1.27). Tris-HC1 buffer 5 ml. Sodium lactate 50%. 2.5 ml NAD 5 mg in 5 ml water. TNBT 6.25 mg in 0.3 ml DMSO and 2.2 ml water. 4. ISO F of LDH. Tris-HCI buffer 5 ml. Sodium lactate 50 % 0.1 ml in 2.4 ml water. NAD 5 mg and Urea 1.175 mg in 5 ml water. TNBT 6.25 mg in 0.3 ml DMSO and 2.2 ml water. 5. SDH (succinate oxidoreductase, E.N.1.3.99.1). Tris-HC1 buffer 5 ml. Sodium succinate 1% 5 ml. TNBT 12.5 mg in 0.5 ml DMSO and 4.5 ml water. It has already been shown (Gerebtzoff and Brotchi, 1965) that the implication of the NADH or NADPH oxido-reductase (diaphorase) depends upon an intimate correlation with the DH investigated. Moreover, the use of TNBT, which is chemically reduced when in contact with these coenzymes, realizes a relative by-pass of the diaphorase. In photonic microscopy at least, the localization of the DH is precise enough. Complete information and criticism of the technique may be found in the publications of Gerebtzoff (1966, 1968, 1970).
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Results
Classification of Animals Our experimental animals may be classified into four main groups (at least five animals per group). 1. The control group with gelatine-glass powder pellet. 2. Cobalt-implanted rats with no electrical or motor manifestations. 3. Cobalt-implanted animals presenting various homolateral E C o G signs of epilepsy without motor jerks. They may be divided into the following subgroups based on the presence of: (a) spikes, (b) sharp waves, (c) focus: repetitive discharges, (d) electrical crisis. 4. Cobalt-implanted rats with motor seizures.
Histochemical and ECoG Results Group 1: the animals were sacrificed between the 14th and the 72nd day after the implantation. During this period all the animals had a normal E C o G . As we can see in Figure 1, there are no abnormal astrocytes in the cortex. As known, the D H activity of the normal protoplasmic astrocyte is masked by the high level of enzyme activity of neurons and neuropil. On the other hand, in the white matter, we can see some normal fibrous astrocytes with a detectable activity of G D H only. Some reactive astrocytes with a high activity of all the D H are present in the corpus callosum, near the operative site. Group 2: the animals were sacrificed between the 4th and the 8th day. During this period all these animals had a normal E C o G . Histochemical analysis shows a normal cortex but more reactive and activated astrocytes are present in the corpus callosum as well as in the hippocampus. Group 3: the animals were sacrificed between the 7th and the 16th day. Some animals (subgroup (a)) had spikes on the left side during several days without any other epileptic manifestation. In their cortex some astrocytes are activated (Fig. 2). These cells are hot numerous but their histochemical aspect is strongly modified. Other animals were killed when they had sharp waves on their E C o G (subgroup (b)). At this stage we observe more activated astrocytes in the cortex but they remain localized in the neighborhood of the lesion. In the rats which have an epileptic focus (subgroup (c)) the activated astrocytes are very numerous and infiltrate the cortex far from the lesion. Even SDH, almost negative in normal astrocytes, has here a high activity and cortical astrocytes are revealed by the appropriate histochemical techniques. In the corpus callosum, there are many reactive astrocytes (Cajal gold-sublimate stain) histochemically activated which remain on the side of the lesion. They do not cross the midline. In our experiment we have never observed activated astrocytes controlateral to the lesion. We have been able to analyze animals which were killed when they had a unilateral electrical crisis (subgroup (d)). In these cases the histochemical aspect is quite similar to the one described for subgroup (c). No difference is apparent between the rats with focus and those with electrical crisis. In both groups
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Fig. 1. A ECoG record from a control rat 60 days after implantation of a glass-powder pellet into the left parietal cortex (group 1). Apparatus: Alvar electroencephalograph (8 channels) -Left F.-0: left front-occipital derivation; right F.-0: right fronto-occipital derivation. B Transverse section through the parietal cortex. In all the pictures, the implantation site is situated on the left and the surface of the cortex on the upper side. GDH-Rat 21 - the cortex is normal and contains no activated astrocytes, x 250
a c t i v a t e d a s t r o c y t e s are v e r y n u m e r o u s a n d s p r e a d to t h e c o r t e x at s o m e d i s t a n c e f r o m t h e i m p l a n t a t i o n site (Fig. 3). Group 4: in this g r o u p , the a n i m a l s w e r e sacrificed on t h e d a y o f a m o t o r crisis. W e h a v e r e c o r d e d the e l e c t r i c a l m a n i f e s t a t i o n s o f t h e s e seizures a n d as shown by Figure 4 both hemispheres are concerned. However, the histochemical analysis shows t h a t a c t i v a t e d a s t r o c y t e s are l i m i t e d to the left side. T h e y are v e r y n u m e r o u s a n d e x t e n d far f r o m t h e lesion. N e a r t h e i m p l a n t a t i o n site we o b s e r v e an a n a t o m i c a l a s p e c t o f b r a i n e d e m a .
Discussion E x p e r i m e n t a l e p i l e p s y has b e e n histologically s t u d i e d in t h e a l u m i n a c r e a m m o d e l ( W e s t r u m et al., 1964; S t e r c o v a , 1966; H a r r i s , 1975), as well as in t h e
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Fig. 2. A Spikes appear on the left side 7 days after cobalt implantation (group 3 - subgroup (a)). B Iso F - Rat 26 - Only some activated astrocytes are visible in the neighborhood of the pellet, x 250
cobalt powder model (Fischer et al., 1968a, 1968b, 1968c; L u p p a et al., 1973; Bogolepov and Pushkin, 1975). However, most of these studies have been made in the acute phase and the results are probably in relation with the general n e u r o h o r m o n a l variations described in the status epilepticus (Meldrum and Brierley, 1973; Meldrum and Horton, 1973). The histochemical analysis usually concerns the neuron (Alberici et al., 1969; Meldrum and Brierley, 1972; Cazzullo et al., 1973). The astrocyte appears to have been generally neglected and only some histochemical papers mention it, often in the acute phase (Mison-Crighel and Badiu, 1971; L u p p a et al., 1973; Gussel et al., 1975), exceptionally in a chronic or semi-chronic model (Fischer et al., 1968a, 1968b, 1968c). Fischer et al. (1968a, 1968b, 1968c) have studied the cobalt-gelatine focus and describe three zones: 1. The first zone showing coagulation necrosis. 2. The second one characterized by e d e m a and a glio-mesenchymal or pio-cerebral scar where ganglion cells have disappeared. 3. The transitory zone where the ganglion cells are present but altered (vacuolization, tigrolysis).
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Fig. 3. A Unilateral electrical crisis 13 days after implantation (group 3 - subgroup (d)). B Iso F Rat 20 - Numerous activated astrocytes are seen in the cortex at a distance from the pellet. • 250
They have studied astrocytes in the implantation site but did not describe the cortex at a distance from the lesion. In this work we have investigated the potential link between activated astrocytes (see our definition above) and epileptic manifestations. We have previously postulated this relation on human material (Brotchi, 1972, 1973a, 1973b) and we wanted to confirm it and to define its chronological sequence. This is why we have developed in this work an approach based on the following questions. 1. Is there a close relation between seizures and activated astrocytes in the motor cortex? We have chosen two kinds of rats implanted with cobalt-gelatine rods, a group with m o t o r jerks and another with no motor or electrical epileptic manifestations (groups 4 and 2). We observe that activated astrocytes appear in the motor cortex only in group 4. We thus have strong arguments to postulate the existence of a close relation between seizures and activated astrocytes. 2. Do these activated astrocytes appear in the cortex only after motor jerks or are they already there before?
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Fig. 4. A Motor crisis 9 days after implantation (group 4). B G-6-PDH - Rat 2 - Many activated astrocytes are seen in the swelling cortex. X 300
To reply to this question, we killed animals presenting an electrical crisis without any m o t o r manifestation (group 3, s u b g r o u p (d)). We f o u n d as m a n y activated astrocytes as in g r o u p 4, without brain e d e m a in this case. Se we have dismissed the possibility that activated astrocytes may be the c o n s e q u e n c e of m o t o r seizures. O n e m a y object that we did not examine our rats t w e n t y - f o u r hours a day. Some rats could have m o t o r jerks outside the observation period but we want to stress that the animals o f g r o u p 3, subgroup (d) had electrical crises without any extension to m o t o r manifestations. Nevertheless, we felt the necessity of pursuing our investigations. 3. A r e activated astrocytes already present before electrical crises and, if they are, when do they a p p e a r in relation to early epileptic manifestations in the ECoG? W e killed animals with an electrical focus (group 3, s u b g r o u p (c)) and we f o u n d as m a n y activated astrocytes as in groups 4 and 3, s u b g r o u p (d). The
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histochemical aspect was quite the same in these three groups. Furthermore, when sharp waves (group 3, subgroup (b)) are recorded, activated astrocytes are already present. Then we attempted to detect the early stages of epilepsy and killed animals which had only a few spikes on E C o G (group 3, subgroup (a)). At this stage, we observed some astroeytes with an abnormal metabolism. They were less numerous when compared with the previous groups. It seems that astrocytes are activating when spikes are recorded. So we may say that there is not only a close relation between activated astrocytes and focal epileptic phenomena but also between the histochemical modifications in the astrocytes and the development of an epileptic focus. It is important to remember that all our control animals (group 1) implanted with gelatine-glass powder rods had no activated astrocytes in their cerebral cortex and no epileptic manifestation in their ECoG. Our experimental results confirm the previous research we have done in human focal epilepsy. The role of the astrocyte in epileptic phenomena has been stressed in many biochemical papers (Orkand et al., 1966; Trachtenberg and Pollen, 1970). They attribute to the astrocyte a buffer role in potassium exchanges between the neuron and the extracellular space. Thus, it could play a role in the control of neuron polarization. One of the mechanisms which allows the neuron to become synchronized during epileptic phenomena is the accumulation of potassium in perineuronal extracellular space (Pollen and Trachtenberg, 1970; Adelman and Palti, 1972). Perhaps abnormal astrocytes could not buffer extracellular space and protect neurons against the accumulation of potassium. However, the accumulation of potassium is known to raise the oxidative metabolism of glial cells (O'Connor et al., 1973). This can explain the histochemical aspect of our activated astrocytes. They could also help to raise the level of glutamic acid ( G D H is very high in activated astrocytes) which is known to have an excitatory effect on nervous cells (Wiechert and Gollnitz, 1969; Beart, 1976). So our activated astrocytes can really play a role in some basic mechanisms of focal epilepsy. They are not the consequence of seizures but at this stage of the study it is impossible to say if the activation is secondary to the hyperactivity of the neuron or directly responsible for the constitution of the epileptogenic focus. When we remember the difficulties of accurately localizing a human epileptic focus either with neurophysiological methods (Ajmone Marsan and Goldhammer, 1973; Bancaud et al., 1973), or with anatomic methods (Pope, 1969; Peiffer, 1970) we postulate that the activated astrocytes could represent the anatomic features of some focal seizures. This assertion might be important in view of the difficulties of distinguishing the "epileptic neuron" from the normal one (Brown, 1973).
Acknowledgements. We thank Mr. J.C. Caro for his technical assistance and Mr. F. Letihon for the photographic work. References
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Received August 11, 1977/Accepted April 3, 1978
Exp. Brain Res. 32, 459-469 (1978)
@ Springer-Verlag 1978
Histochemical Study of Cobalt-Induced Focal Epilepsy J. Brotchi, A. Dresse, J. Scuvee-Moreau and M.A. Gerebtzoff Departments of Neurosurgery, Anatomy and Pharmacology, University of Liege, B-4020 Liege, Belgium
Summary. A previous study showed a strong relationship between human focal epilepsy and the presence in the cortex of "activated" astrocytes characterized by an intense activity of dehydrogenases (DH) involved in glucose metabolism and of glutamate DH. Using the semi-chronic model of cobalt-induced experimental focal epilepsy in the rat, we investigated a possible correlation between astrocyte modifications and the chronological development of the epileptic manifestations on the E C o G . After a few days the cobalt-implanted rats present spikes, then sharp waves followed by an electrical crisis and ultimately motor seizures. Activated astrocytes were found in each phase of this evolution. Their number increases with the intensity of the manifestations. There is a close relationship between activated astrocytes and focal epileptic phenomena. At this stage of our study it is clear that the presence of activated astrocytes is not a consequence of seizures. However, it is impossible to say whether the activation is secondary to the hyperactivity of the neurons or directly responsib!e for the constitution of the epileptic focus. In any case, activated astrocytes provide a new means of localizing an epileptogenic focus.
Key words: Experimental epilepsy - Astrocytes - Histoenzymology Dehydrogenases.
It is generally accepted that there is no major morphological difference between the "epileptic n e u r o n " and the normal one (Brown, 1973). However, some altered dendrites have been described by the Scheibels (1968), using the technique of Golgi. A mechanical influence on the activity of the neuronal cell has also been postulated (Ward, 1961). In these previous studies the role of neuroglia has often been neglected except when considering the meningocerebral scar (Penfield, 1932; Penfield and Jasper, 1954). Offprint requests to: Prof. M.A. Gerebtzoff, Institut d'Anatomie, rue de Pitteurs, 20, B - 4020 Liege, Belgium 0 0 1 4 - 4 8 1 9 / 7 8 / 0 0 3 2 / 0 4 5 9 / $ 2.20
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While remaining faithful to the present biochemical and physiological concepts on the dynamic role of neuroglia and particularly of the astrocyte in the polarization of the neuron (Tower, 1960, 1965; Kuffler and Nicholls, 1966, 1976), we have attempted to study the astrocyte by a histochemical approach. This method has the advantage of respecting the anatomical relations between the various cellular elements. A direct visualization of the oxidative metabolism of the astrocyte may be obtained by a histochemical evaluation of the activity of certain dehydrogenases (DH). Among the numerous enzymes which catalyze oxidoreduction phenomena, we have chosen five DH which are situated at strategic sites of metabolic pathways. 1. The Glucose-6-Phosphate dehydrogenases (G6PDH) situated in the first stage of the pentose shunt. 2. The Glutamate dehydrogenase (GDH) participating in the transformation of c~-ceto-glutarate into glutamate. 3. The Lactate dehydrogenase (LDH) situated at the end of glycolysis and possessing five isoenzymes characterized by electrophoretic properties. With our method we can establish two groups (Gerebtzoff, 1968, 1970): a) the slow moving group called ISO S. b) the fast moving group called ISO F. It is not yet possible to ascertain if the borderline is situated between the isoenzymes 2 and 3 or 3 and 4. 4. The succinate dehydrogenase (SDH) situated in Kreb's cycle. The normal astrocyte demonstrates very little activity of oxidase enzymes as compared with oligodendroglia, nerve cells or ependyma except for GDH. There is a moderate G6PDH activity, very little ISO S and ISO F and no SDH activity within the astrocytes (Friede, 1965; Dimova and Gerebtzoff, 1966; Gerebtzoff et al., 1974). In some conditions, astrocytes can become morphologically reactive. There is a transformation from the protoplasmic to the fibrous form and a production of gliofibrils (Penfield, 1932). In the rat, an experimental lesion limited to the cortex induces morphologically reactive astrocytes in the neighborhood of the lesion (Dimova, 1966). An extension of this morphological reaction is observed along the pyramidal tract as far as the end of the lumbar medulla when the lesion is in the motor cortex (Demolin and Gerebtzoff, 1975). A histochemical study of these reactive cells has shown that the astrocytes in close proximity to the lesion acquire a high level of DH activity contrary to more caudally placed astrocytes which remain normal. The morphologically reactive astrocytes may thus remain normal or acquire an hyperactivity of DH. Proximal reactive astrocytes after a cortical lesion are revealed by our histoenzymological techniques in the white matter near the lesion, but not in the adjacent cerebral cortex. There, the neuropil has an activity masking that of astrocytes. But cortical astrocytes presenting an intense DH activity do emerge from the neuropil in epileptogenic loci of tumoral or nontumoral human epilepsy (Brotchi, 1972, 1973a, 1973b; Gerebtzoff et al., 1974). The role of these "activated" astrocytes in the genesis of epileptic manifestations has been the object of recent experimental investigations in our laboratories.
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A s e m i - c h r o n i c m o d e l e n a b l i n g us t o f o l l o w t h e s e q u e n c e o f h i s t o c h e m i c a l and
electrocorticographic
changes
appearing
after
the
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of
a
c o b a l t - g e l a t i n e p e l l e t in t h e p a r i e t a l c o r t e x o f t h e r a t h a s b e e n c h o s e n .
Methods Among the numerous experimental models of epilepsy (Purpura et al., 1972), we have chosen the implantation of a cobalt-gelatine rod according to the method of Dow et al. (1961) modified by Fischer et al. (1967). This mixture has the advantage of decreasing the necrotic power of cobalt and gives a stable model of focal epilepsy during 4 to 6 weeks. The pellet is prepared as follows: a suspension of cobalt metallic powder in warm 5 % gelatine is allowed to sediment and is then decanted on to a horizontal slide to form a layer 0.75 mm thick. The solidified film is dehydrated in acetone, fixed in formaldehyde vapors and kept in 90 ~ alcohol. Pellets measuring 0.75 x 0.75 x 1.5 mm are then cut from the film with a razor blade. Control rats are implanted simultaneously with glass powder pellets instead of cobalt. Eighty male Wistar rats of 200-250 g were used in these experiments. The animals were operated under general (Chloral hydrate, 400 mg/kg i.p.) anesthesia. A bone flap was cut in the left parietal side. The dura was gently opened and the pellet inserted perpendicularly into the cortex. The bone was closed to prevent the expulsion of the pellet from the brain. Four stainless steel screws were set as follows: the two anterior ones were situated 2 mm before and 2 mm lateral to the bregma, the two posterior ones 1.5 mm in front of the lambda and 3.2 mm laterally. Electrocorticograms were daily recorded during thirty minutes. The criteria used to follow the different stages of the epileptic manifestations will be described and illustrated in the results. For histochemical analysis, the animals were killed by decapitation. The brain was immediately frozen on dry ice. Slices 7-10 g thick were cut at-18~ and then incubated in appropriated baths for the studied DH. The slices were fixed in 10% formalin, washed in distilled water, dehydrated in alcohols, clarified in toluol and mounted in Caedax. The principle of the method is to reduce a soluble and colourless salt of tetrazolium into an insoluble and colored form detectable with a photonic microscope. To facilitate the solubilization of the tetrazolium salt named "tetranitroblue of tetrazolium" (TNBT) it is dissolved in dimethylsulfoxyde (DMSO). The incubation baths are prepared as follows for the different enzymes: i. G6PDH (D-glucose-6-phosphate: NADP oxidoreductase, E.N.l.1.l.49). Tris-HC1 buffer 0.1 M, pH 7.6 5 ml. D-glucose-6-phosphate 50 mg in 5 ml water. NADP 5 mg in 2.5 ml water. TNBT 6.25 mg in 0.3 ml DMSO and 2.2 ml water. 2. GDH (L-glutamate: NAD oxidoreductase, E.N. 1.4.1.2). Monosodic salt of L-glutamic acid 50 mg and NaC1 50 mg in 5 ml water. NAD 5 mg in 2.5 ml water. TNBT 6.25 mg in 0.3 ml DMSO and 2.2 ml water. 3.1SO S of L D H (L-lactate: NAD oxidoreductase, E.N.l.i. 1.27). Tris-HC1 buffer 5 ml. Sodium lactate 50%. 2.5 ml NAD 5 mg in 5 ml water. TNBT 6.25 mg in 0.3 ml DMSO and 2.2 ml water. 4. ISO F of LDH. Tris-HCI buffer 5 ml. Sodium lactate 50 % 0.1 ml in 2.4 ml water. NAD 5 mg and Urea 1.175 mg in 5 ml water. TNBT 6.25 mg in 0.3 ml DMSO and 2.2 ml water. 5. SDH (succinate oxidoreductase, E.N.1.3.99.1). Tris-HC1 buffer 5 ml. Sodium succinate 1% 5 ml. TNBT 12.5 mg in 0.5 ml DMSO and 4.5 ml water. It has already been shown (Gerebtzoff and Brotchi, 1965) that the implication of the NADH or NADPH oxido-reductase (diaphorase) depends upon an intimate correlation with the DH investigated. Moreover, the use of TNBT, which is chemically reduced when in contact with these coenzymes, realizes a relative by-pass of the diaphorase. In photonic microscopy at least, the localization of the DH is precise enough. Complete information and criticism of the technique may be found in the publications of Gerebtzoff (1966, 1968, 1970).
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Results
Classification of Animals Our experimental animals may be classified into four main groups (at least five animals per group). 1. The control group with gelatine-glass powder pellet. 2. Cobalt-implanted rats with no electrical or motor manifestations. 3. Cobalt-implanted animals presenting various homolateral E C o G signs of epilepsy without motor jerks. They may be divided into the following subgroups based on the presence of: (a) spikes, (b) sharp waves, (c) focus: repetitive discharges, (d) electrical crisis. 4. Cobalt-implanted rats with motor seizures.
Histochemical and ECoG Results Group 1: the animals were sacrificed between the 14th and the 72nd day after the implantation. During this period all the animals had a normal E C o G . As we can see in Figure 1, there are no abnormal astrocytes in the cortex. As known, the D H activity of the normal protoplasmic astrocyte is masked by the high level of enzyme activity of neurons and neuropil. On the other hand, in the white matter, we can see some normal fibrous astrocytes with a detectable activity of G D H only. Some reactive astrocytes with a high activity of all the D H are present in the corpus callosum, near the operative site. Group 2: the animals were sacrificed between the 4th and the 8th day. During this period all these animals had a normal E C o G . Histochemical analysis shows a normal cortex but more reactive and activated astrocytes are present in the corpus callosum as well as in the hippocampus. Group 3: the animals were sacrificed between the 7th and the 16th day. Some animals (subgroup (a)) had spikes on the left side during several days without any other epileptic manifestation. In their cortex some astrocytes are activated (Fig. 2). These cells are hot numerous but their histochemical aspect is strongly modified. Other animals were killed when they had sharp waves on their E C o G (subgroup (b)). At this stage we observe more activated astrocytes in the cortex but they remain localized in the neighborhood of the lesion. In the rats which have an epileptic focus (subgroup (c)) the activated astrocytes are very numerous and infiltrate the cortex far from the lesion. Even SDH, almost negative in normal astrocytes, has here a high activity and cortical astrocytes are revealed by the appropriate histochemical techniques. In the corpus callosum, there are many reactive astrocytes (Cajal gold-sublimate stain) histochemically activated which remain on the side of the lesion. They do not cross the midline. In our experiment we have never observed activated astrocytes controlateral to the lesion. We have been able to analyze animals which were killed when they had a unilateral electrical crisis (subgroup (d)). In these cases the histochemical aspect is quite similar to the one described for subgroup (c). No difference is apparent between the rats with focus and those with electrical crisis. In both groups
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Fig. 1. A ECoG record from a control rat 60 days after implantation of a glass-powder pellet into the left parietal cortex (group 1). Apparatus: Alvar electroencephalograph (8 channels) -Left F.-0: left front-occipital derivation; right F.-0: right fronto-occipital derivation. B Transverse section through the parietal cortex. In all the pictures, the implantation site is situated on the left and the surface of the cortex on the upper side. GDH-Rat 21 - the cortex is normal and contains no activated astrocytes, x 250
a c t i v a t e d a s t r o c y t e s are v e r y n u m e r o u s a n d s p r e a d to t h e c o r t e x at s o m e d i s t a n c e f r o m t h e i m p l a n t a t i o n site (Fig. 3). Group 4: in this g r o u p , the a n i m a l s w e r e sacrificed on t h e d a y o f a m o t o r crisis. W e h a v e r e c o r d e d the e l e c t r i c a l m a n i f e s t a t i o n s o f t h e s e seizures a n d as shown by Figure 4 both hemispheres are concerned. However, the histochemical analysis shows t h a t a c t i v a t e d a s t r o c y t e s are l i m i t e d to the left side. T h e y are v e r y n u m e r o u s a n d e x t e n d far f r o m t h e lesion. N e a r t h e i m p l a n t a t i o n site we o b s e r v e an a n a t o m i c a l a s p e c t o f b r a i n e d e m a .
Discussion E x p e r i m e n t a l e p i l e p s y has b e e n histologically s t u d i e d in t h e a l u m i n a c r e a m m o d e l ( W e s t r u m et al., 1964; S t e r c o v a , 1966; H a r r i s , 1975), as well as in t h e
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Fig. 2. A Spikes appear on the left side 7 days after cobalt implantation (group 3 - subgroup (a)). B Iso F - Rat 26 - Only some activated astrocytes are visible in the neighborhood of the pellet, x 250
cobalt powder model (Fischer et al., 1968a, 1968b, 1968c; L u p p a et al., 1973; Bogolepov and Pushkin, 1975). However, most of these studies have been made in the acute phase and the results are probably in relation with the general n e u r o h o r m o n a l variations described in the status epilepticus (Meldrum and Brierley, 1973; Meldrum and Horton, 1973). The histochemical analysis usually concerns the neuron (Alberici et al., 1969; Meldrum and Brierley, 1972; Cazzullo et al., 1973). The astrocyte appears to have been generally neglected and only some histochemical papers mention it, often in the acute phase (Mison-Crighel and Badiu, 1971; L u p p a et al., 1973; Gussel et al., 1975), exceptionally in a chronic or semi-chronic model (Fischer et al., 1968a, 1968b, 1968c). Fischer et al. (1968a, 1968b, 1968c) have studied the cobalt-gelatine focus and describe three zones: 1. The first zone showing coagulation necrosis. 2. The second one characterized by e d e m a and a glio-mesenchymal or pio-cerebral scar where ganglion cells have disappeared. 3. The transitory zone where the ganglion cells are present but altered (vacuolization, tigrolysis).
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Fig. 3. A Unilateral electrical crisis 13 days after implantation (group 3 - subgroup (d)). B Iso F Rat 20 - Numerous activated astrocytes are seen in the cortex at a distance from the pellet. • 250
They have studied astrocytes in the implantation site but did not describe the cortex at a distance from the lesion. In this work we have investigated the potential link between activated astrocytes (see our definition above) and epileptic manifestations. We have previously postulated this relation on human material (Brotchi, 1972, 1973a, 1973b) and we wanted to confirm it and to define its chronological sequence. This is why we have developed in this work an approach based on the following questions. 1. Is there a close relation between seizures and activated astrocytes in the motor cortex? We have chosen two kinds of rats implanted with cobalt-gelatine rods, a group with m o t o r jerks and another with no motor or electrical epileptic manifestations (groups 4 and 2). We observe that activated astrocytes appear in the motor cortex only in group 4. We thus have strong arguments to postulate the existence of a close relation between seizures and activated astrocytes. 2. Do these activated astrocytes appear in the cortex only after motor jerks or are they already there before?
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Fig. 4. A Motor crisis 9 days after implantation (group 4). B G-6-PDH - Rat 2 - Many activated astrocytes are seen in the swelling cortex. X 300
To reply to this question, we killed animals presenting an electrical crisis without any m o t o r manifestation (group 3, s u b g r o u p (d)). We f o u n d as m a n y activated astrocytes as in g r o u p 4, without brain e d e m a in this case. Se we have dismissed the possibility that activated astrocytes may be the c o n s e q u e n c e of m o t o r seizures. O n e m a y object that we did not examine our rats t w e n t y - f o u r hours a day. Some rats could have m o t o r jerks outside the observation period but we want to stress that the animals o f g r o u p 3, subgroup (d) had electrical crises without any extension to m o t o r manifestations. Nevertheless, we felt the necessity of pursuing our investigations. 3. A r e activated astrocytes already present before electrical crises and, if they are, when do they a p p e a r in relation to early epileptic manifestations in the ECoG? W e killed animals with an electrical focus (group 3, s u b g r o u p (c)) and we f o u n d as m a n y activated astrocytes as in groups 4 and 3, s u b g r o u p (d). The
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histochemical aspect was quite the same in these three groups. Furthermore, when sharp waves (group 3, subgroup (b)) are recorded, activated astrocytes are already present. Then we attempted to detect the early stages of epilepsy and killed animals which had only a few spikes on E C o G (group 3, subgroup (a)). At this stage, we observed some astroeytes with an abnormal metabolism. They were less numerous when compared with the previous groups. It seems that astrocytes are activating when spikes are recorded. So we may say that there is not only a close relation between activated astrocytes and focal epileptic phenomena but also between the histochemical modifications in the astrocytes and the development of an epileptic focus. It is important to remember that all our control animals (group 1) implanted with gelatine-glass powder rods had no activated astrocytes in their cerebral cortex and no epileptic manifestation in their ECoG. Our experimental results confirm the previous research we have done in human focal epilepsy. The role of the astrocyte in epileptic phenomena has been stressed in many biochemical papers (Orkand et al., 1966; Trachtenberg and Pollen, 1970). They attribute to the astrocyte a buffer role in potassium exchanges between the neuron and the extracellular space. Thus, it could play a role in the control of neuron polarization. One of the mechanisms which allows the neuron to become synchronized during epileptic phenomena is the accumulation of potassium in perineuronal extracellular space (Pollen and Trachtenberg, 1970; Adelman and Palti, 1972). Perhaps abnormal astrocytes could not buffer extracellular space and protect neurons against the accumulation of potassium. However, the accumulation of potassium is known to raise the oxidative metabolism of glial cells (O'Connor et al., 1973). This can explain the histochemical aspect of our activated astrocytes. They could also help to raise the level of glutamic acid ( G D H is very high in activated astrocytes) which is known to have an excitatory effect on nervous cells (Wiechert and Gollnitz, 1969; Beart, 1976). So our activated astrocytes can really play a role in some basic mechanisms of focal epilepsy. They are not the consequence of seizures but at this stage of the study it is impossible to say if the activation is secondary to the hyperactivity of the neuron or directly responsible for the constitution of the epileptogenic focus. When we remember the difficulties of accurately localizing a human epileptic focus either with neurophysiological methods (Ajmone Marsan and Goldhammer, 1973; Bancaud et al., 1973), or with anatomic methods (Pope, 1969; Peiffer, 1970) we postulate that the activated astrocytes could represent the anatomic features of some focal seizures. This assertion might be important in view of the difficulties of distinguishing the "epileptic neuron" from the normal one (Brown, 1973).
Acknowledgements. We thank Mr. J.C. Caro for his technical assistance and Mr. F. Letihon for the photographic work. References
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Received August 11, 1977/Accepted April 3, 1978
