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Submicroscopic changes of cortex nerve cells in chronic mirror epileptic focus in rat
Brahl Research, 94 (1975) 173-184 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
173
Research Reports
SUBMICROSCOPIC C H A N G E S OF CORTEX NERVE CELLS IN C H R O N I C M I R R O R EPILEPTIC FOCUS IN RAT
N. N. BOGOLEPOV AND A. S. P U S H K I N
Electron Microscopy Laboratory, Brain Research hlstitute, Academy of Medical Sciences, Moscow
(u.s.s.R.) (Accepted March 1 lth, 1975)
SUMMARY
Submicroscopic changes of neurons, synapses, glial cells and some elements of capillaries were established by studying chronic mirror foci which appeared in the rat cerebral cortex after implantation of cobalt-gelatinous rods into the contralateral hemisphere. The results were compared with normal control cortex and with changes after thermocoagulation of the symmetrical contra[ateral cortex. it is shown that epileptogenic lesions produce a significant reduction of RNAcomponents, edema and vacuolization of neuronal cytoplasm, hypertrophy and proliferation of glial cells. The revealed pathological changes of the synapses were manifested by dilatation of some vesicles and the appearance ofheterogenic inclusions. The characteristic features of Wallerian degeneration of callosal synapses and the reversible retrograde changes of the neurons which send their axons into the traumatized zone of the contralateral hemisphere were observed to be similar after both experimental lesions.
INTRODUCTION
The study of the peculiarities of submicroscopic changes in secondary epileptic foci is of interest not only in connection with the analysis of the ultrastructural foundations of the epileptic processZ,V,s,la,19, ~4, but also as a convenient method for the elucidation of the plastic modifications which convey epileptic properties to an initially intact brain zone14, 2~. Previous electrophysiological investigations of the so-called 'mirror' focus have
174 shown that its origin depends on a stable reorganization of functional properties and metabolism, as well as on the structure of the secondary epileptic cortex zone 15,1s:'e. Up to the present time, however, morphological characterization of the mirror foci has been limited by light microscopic data which do not show the intimate damage of the cell structures or the reorganization of the interneuronal mutual relationships. This paper deals with the problem of the first stage of submicroscopic analysis of secondary epileptogenesis, with the aim of showing the qualitative peculiarities of changes in basal cell elements in a rat brain cortex mirror focus, emerging after a chronic epileptogenic lesion of a symmetrical contralateral cortex zone.
MATERIAL AND METHODS
Thirty-eight adult white male rats, weighing 170-250 g, were used. The anterior parietal (PA) region of the cortex was studied 23. Six rats without experimental lesions were used as controls to study the normal ultrastructure. Twenty-two rats were subjected to an implantation of cobalt-gelatinous rods into the above-mentioned zone of the left hemisphere cortex 9, which created a lesion nidus of 8-10 sq.mm with a depth of 1.0-1.2 mm. The animals were sacrificed on days 3, 6, 8, 10, 12, 14, 24 or 30 after the operation. The picture of the submicroscopic changes in the mirror focus was compared with modifications in the cerebral cortex which, of the contralateral hemisphere, inevitably occur when the cortex is subjected to lesions and which are due to the development of a retrograde and Wallerian degeneration in the neurons and fibers comprising the transcaUosal connections. Accordingly 10 rats were subjected to a thermocoagulation of an analogous section of the left hemisphere cortex. The dimensions of the necrotic focus were identical to those of the cobalt-gelatinous lesion. These animals were sacrificed on days 3, 5, 7, 14, and 21 after the operation. For the identification of the epileptic process we used electroencephalograms registered on a 'Galileo' oscilloscope using intracranially implanted electrodes. Under Nembutal anesthesia (4 mg/100 g body weight) the animals were perfused with a 2.5 ~ glutaraldehyde solution in a phosphate buffer (pH 7.4). After previous fixation of the brain a specimen containing the focus of the primary lesion was excised as well as the symmetrical part of the contralateral hemisphere. This sample was immersed in a 5 ~o glutaraldehyde solution for one hour. Then, from the zone symmetrical to the focus of the lesion, samples were taken for electron microscopy. After rinsing in the buffer solution the samples were immersed for 1.5-2.0 h in a 2 ~ OsO4 solution, dehydrated in increasing alcohol concentration gradients and embedded in Epon-812. Ultrathin and semithin (1.0-2.5/~m) slices were then obtained, using an LKB- 111 ultramicrotome. The slices were stained with uranyl acetate and lead citrate after which they were studied and photographed with UEMV-100A, HU-11-E and AEI-EM801 electron microscopes.
175
Fig. 1. Chromatolysis of cortical pyramid cells from layer III. Day 24 after operation. Mirror focus. x 5250.
Fig. 2. The reduction of granular endoplasmic reticulum in the cytoplasm of small neuron. Day 14 after operation. Mirror focus. Layer III. x 18,000.
176
Fig. 3. Small neuron from layer 1V of intacl cortex.
21,000.
Light microscopic investigations and the identification of the cortical layers were conducted on semithin slices stained with methylene blue or p-phenylenediamine. The remaining part of the brain sample taken after the perfusion was embedded in paraffin, cut into 10-15 #m slices, stained with cresyl violet and studied by light microscopy to determine the depth of the primary lesions caused by cobalt-gelatinous rods or by thermocoagulation. RESULTS
The EEGs of rats with cobalt-gelatinous rod implantations showed sporadic
177
Fig. 4. The part of the neuropile from layer IV. Day 14 after operation. Mirror focus. Edema of dendrites is shown. × 20,000.
Fig. 5. Degenerating presynaptic terminal in neuropile of layer VI. Mirror focus. Day 10 after operation, x 55,000.
178 outbursts of high amplitude sharp waves and bursts of discharges of the 'spike-wave' type, which were described as definite equivalents of the epileptic process (Penfield and Jasper1'5,18,21). Convulsive movements, however, were not observed. Light microscopic investigations revealed that in the cortical mirror tbcus the main reaction of the cells consisted of a peripheral chromatolysis which included a vast majority of neurons. These neurons were distributed throughout the cortex but were more frequently found in layers V and VI. In the thermocoagulation samples only a restricted number of cells showed chromatolysis, mainly the pyramidal middlesize neurons and rarely the large pyramidal neurons localized, preferably, in layers III-V. Accordingly, the sattelitosis and hyperplasia of different forms of neuroglial cells is less marked after thermocoagulation than in mirror focus. The p-phenylenediamine stained preparations clearly showed degenerating myelin fibers. Their distribution was similar in the mirror focus and the thermocoagulation samples. Most of the degenerating fibers were concentrated in layers VI and V. The electron microscopic studies of the mirror focus showed that the most noticeable modifications of the nerve cells, interneuronal connections and glial cells were evident during the period of maximal electrophysiological changes - - from the 10th to the 24th day after the implantation of the cobalt-gelatinous rod. The modified nerve cells were characterized by a decreased number of cytoplasmic ribosomes and polysomes in combination with an increase of the depth and length of the nuclear membrane folds and a tendency for the nucleolus to be situated near the membrane. A redistribution of granular and filamentous components was evident. The cytoplasmic ribosomes concentrated around the nucleus and in the zone of the nuclear membrane folds. The cytoplasmic periphery was markedly deprived of these structures (Figs. I and 2), which differentiates the lesioned cells from normal ones (Fig. 3). In many cells these signs were accompanied by an edema of the endoplasmic reticulum cisternae. The modified dendrites were characterized by the appearance of vacuole-like cavities (Fig. 4). Chromatolysis and the disintegration of organelles are mainly characteristic of small neurons (Fig. 2), the pathological changes of which increased up to a disintegration of the cell membrane, and probably to cell death. As opposed to the above mentioned results the ultrastructure of a substantial number of larger neurons was like normal pictures. Two kinds of changes of nerve fibers and synapses were registered in the course of development of the mirror focus. From the 3rd to the 14th day after the operation in some of the axon terminals the well-known signs of Wallerian degeneration of the 'dark' and 'light' type were observed. These presynaptic processes were mostly localized on the spines of the dendrites and on their thin branchings, and sometimes on the bodies of the small neurons. The degenerating synapses (Fig. 5) are distributed throughout the whole cortex, mostly in layers V and VI and slightly less in layers II and III. The appearance of glycogen granules, lysosome-like structures and the increase
179
Fig. 6. Enlarged vesicles in presynaptic bag terminated on demh itic branch. Day 24 after operation. Mirror focus, x 75,000.
Fig. 7. Numerous glycogen inclusions in presynaptie terminals. Day 14 after operation. Mirror focus, x 35,000.
of some vesicles' size without changes in the axoplasmic osmiophilicity were noted in some contacts on day 12-14 after operation (Figs. 6 and 7). The modifications of the bodies and processes of nerve cells were accompanied by an intensification of the glial reaction. The number of perineuronal gliocytes was increased, consisting mostly of oligodendrocytes and rarely of astrocytes and cells of a nondescript type. A marked hypertrophy and hyperplasia of cytoplasmic organelles was observed in these cells (Fig. 8). The same reaction was seen in the processes of
180
Fig. 8. The activation of glial satellite of chromatolytic neuron. Day 14 after operation. Mirror focus. ~. 30,000. astrocytes, which showed a swollen cytoplasm and often contained numerous lipid inclusions and glycogen-like granules. The increases in size and the number of processes ofastrocytes often gave a picture of gliosis loci and changed the neuropil architectonics. The cytoplasm of pericytes, and occasionally of endothelial cells, was characterized by an hypertrophy of the ultrastructures and by the appearance of a large number of heterogenous lysosome-like bodies. To test the specificity of the epiteptogenically induced changes we conducted studies of the cortex after a thermocoagulation of a symmetrical zone of the contralateral hemisphere. It was shown that, in contrast to the phenomena occurring in the mirror focus, the small neurons remained normal. The ultrastructure of large cells, which showed signs of chromatolysis on the semithin slices, was characterized by a substantial decrease of the number of ribosomes of the cytoplasm periphery (Fig. 9). Occasionally such neurons showed a swelling and destruction of the crists of separate mitochondria, an increase of the number of lysosomes and the presence of small vacuoles. On the 21st postoperative day the ultrastructure of most of these neurons had normalized. The thermocoagulation experiments showed submicroscopic signs of Watterian degeneration of some fibers and their synaptic contacts (Fig. 10). An analysis of the mode of termination and of the laminar distribution of the degenerating terminals
181
Fig, 9. Large pyramidal neuron from the layer V. Day 14 after thermocoagulative lesion of contralateral hemisphere. × 4500.
did not disclose any fundamental distinctions between the two forms of experimental lesion. The nerve cell changes were accompanied by an hypertrophy of the organelles of the perineuronal oligodendrocytes' cytoplasm. It was also noted that the astrocyte processes, which engulfed degenerating synapses, were increased in size. In comparison with the mirror focus, however, the activation of the ultrastructures of postthermocoagulation glial elements was considerably less marked. DISCUSSION
The comparison of the changes which were observed in the cortex after both forms of experimental lesion of a symmetrical zone of the contralateral hemisphere disclosed some of the peculiarities of the mirror focus. They consisted of a substantial decrease of the number of ribosomes, a reduction of the endoplasmic reticulum, a disintegration of mitochondria, a vacuolization of neurons (especially of small neurons), a swelling and edema of separate dendrites and polymorphous dystrophic lesions of synapses. These changes were accompanied by an activation of the glial cells. The above mentioned findings are in direct correlation with the biochemical
182
Fig. 10. Degenerating contact with the soma of small neuron from layer VI. Day 5 after thermocoagulation of contralateral hemisphere. ,~,"60,000, data of a decrease of the R N A and protein content of chronic mirror focus neurons and with the changes of activity of certain dehydrogenases, which are oppositely directed in neurons and glial cells 10,1~,~.~. Similar changes of the ultrastructure and of the biochemistry of nerve cells were observed in experiments reproducing acute epileptiform convulsions 13,16,z° as well as in experiments reproducing an increased adequate activity of neurons 6,17. This is an indication of a stable and substantial prevalence of the processes of disintegration of cellular proteins over their synthesis. We have noted an increase of the area of the glial profiles in the neuropile, which can be regarded as one of the stages of gliosis development characteristic of chronic foci4,ag, 24. Swelling of the glial processes, similar to that presently observed, was described by Okada e t al. 16 in acute primary foci. Westmoreland e t al. ~ adhere to the opinion that in a mirror focus there is also developing a proliferation of the glial cells determined by an increase of DNA, probably connected with a degeneration of the interhemispheric bonds. Of great interest is the presence in the mirror focus of disrupted synaptic connections. The typical features of a Wallerian degeneration and particularly of a disintegration of the 'dark'3, 26 type are similar in both models of experiments. The
183
manifested distribution of the degenerating terminals bears a resemb!ance to the data on the localization of synapses formed by callosal fibers~,aL Changes appeared at the region of the mirror focus in the late stages, when a typical Wallerian degeneration was already concluded by a disruption and phagocytosis of terminals. Possibly such symptoms of early stages of degeneration as an enlargement of synaptic vesicles a, an accumulation of glycogen, or the appearance of lysosome-like bodies in the terminals, point to the development of a transneuronal degeneration. Thus in this work are shown the changes of neurons, of interneuronal connections and of glial cells observed by electron microscopy interconnected with the development of a pathological activity in an epileptic mirror focus.
REFERENCES I AKERT, K., CUI~NOD, M., AND MOOR, H., Further observations on the enlargement of synaptic vesicles in degenerating optic nerve terminals of the avian tectum, Brain Research, 25 (1971) 255-263. 2 BALEYDIER, C., Etude ultrastructurale des modifications du cortex c6r6brale en voisinage d'un foyer 6pileptog6ne b. la cr~me d'alumine, Aeta neuropath. (Berl.), 20 (1972) 11-21. 3 BOGOLEPOV, N. N., Questions of degenerative changes of cortical synapses, J. Neuropath. Psyekiat., 12 (1965) 1789-1792. 4 BOGOLEPOV, N. N., MATVEEV, A. S., PUSHKIN, A. S., SAVCHENKO, Ju. N., AND TRISTAN, V. G., Pathological changes of nerve cells in temporal lobe epileptogenic foci in man and in chronic experiments, J. Neuropath. Psychiat., 9 (1973) 1340-1350. 5 BOGOLEPOV,N. N., AND PUSHKIN, A. S., Distribution of synapses of interhemispheric fibres in the sincipital area of the rat cerebral corrtex, Arch. Anat. Histol. and Embriol. (Leningrad), 64 (1974)28-36. 6 DJACHKOVA,L. N., HUMORI, J., AND FEDINA, L., Ultrastructure of ganglion ciliarae synapses in birds under orthodromal and antidromal electrical stimulation, Dokl. Akad. Nauk SSSR, Otd. Biol., 172 (1967) 957-959. 7 FISCHER, J., Electron-microscopic changes in the perikarya and in the processes of ganglion cells in the cobalt-gelatine epileptogenic focus. (Their correlation with electrophysiological data.), Physiol. bohemoslov., 18 (1969) 387-393. 8 FISCHER, J., The cobalt-gelatine necrosis of the cerebral cortex of the rat. A contribution to the ultrastructure of the coagulation necrosis of the central nervous system, Csech. Patol., 3 (1969) 146-152. 9 FISCHER, J., HOLUBAR, J., AND MALIK, V., A new method of producing chronic epileptogenic cortical foci in rats, Physiol. bohemoslov., 16 (1967) 272-277. 10 FISCHER, J., HOLUBAR, J., AND MALIK, V., Some dehydrogenases in the epileptic rat cerebral cortex. A correlation of histochemical and electrophysiological findings, Acta Histoehem. (Jena), 31 (1968) 305-318. 11 HUDOERKOV, R. M., Personal communication, 1972. 12 LUND, J. S., AND LyNn, R. D., The termination of callosal fibers in the paravisual cortex of the rat, Brain Research, 17 (1970) 25-47. 13 MOLBERT, E., BAUMGARTNER,G., UND KETELSEN, W. P., Elektronenmikroscopische Untersuchungen an der Grosshirnrinde der Katze nach Elektrokrfimpfen, Dtsch. Z. Nervenheilk., 190 (1967) 295-315. 14 MORRELL, F., Modifications of R N A as a result of neural activity. In M. A. B. BRAZIER (Ed.), Brain Function I1: R N A and Brain Function, Memory and Learning, Univ. of California Press, Los Angeles, Calif., 1964, pp. 183-202. 15 OCtJDJAVA, V. M., Basic" Neurophysiologieal Mechanisms of Epileptic Activity, Ganatleba, Tbilisi, 1969.
184 16 OKADA, K., AYALA, G. F., AND SUN(;, J. H., Ultrastructure of penicillin induced epileptogenic lesion of the cerebral cortex in cats, J. Nearopath. exp. Neurol., 30 (197l) 337 353. 17 PALLADIN, A. V., BELIK, JA. V., AND POt,JAKOVA, 1. M., Proteins of the Brail~, Naukova 1)umka, Kiev, 1972. I 8 PENFIELD, W., AND JASPER, H., Epilepsy and Functional Anatomy of Human Brain (Translated into Russian), lzdatelstvo lnostrannoj Literaturj, Moscow, 1958. 19 PETROV, V. S., Ultrastructural features of nuclear and cytoplasmatic elements of cerebral cortex cells in patients with convulsions and without them, J. Neuropath. Psychiat., 70 (1970) 106 I l L 20 POGODAEV, K. L., Energy supply of epileptic discharge in the time of generalizated paroxysms, J. Neuropath. Psychiat., 73 (1973) 1604-1607. 21 SHARONOVA,1. N., Functh~nal Characteristics of Visual Cortex Neurons in Norm and Experimetltal Epileptic" Foci, Master's degree thesis, Moscow, 1973. 22 SMmNOV, G. D., Functional and structural aspects of brain's plasticity. In E. N. PoeovA AND L.A. PRON~N (Eds.), Materials of All-Union Plenum on the Problem of Functional Structural Bases of Systemic Activities and Mechanisms of Brain Plasticity, Brain Research Institute USSR Acad. of Med. Sci., Moscow, 1972, pp. 102 104. 23 SVETUCH~NA,V. M., Cytoarchitecture of neocortical regions and areas of the brain in white rats, Arch. amtt. Histol. and Embrh~l. (Leningrad), 42 (19,52) 31-45. 24 UGRJUMOV, B.M., ZEMSKAJA, A . G . , AND PETROV, V.S., Ultrastructural features of cellular elements in cortical epileptogenic foci, J. Neuropath. Psychiat., 71 (1971) 92-100. 25 WESTMOaELAND,B. F., HANNA, G. R., AND BASS, N. H., Cortical alterations in zones of secondary epileptogenesis: a neurophysiologic, morphologic and microchemical correlation study in lhe albino rat, Brain Research, 43 (1972) 485-499. 26 WESTRU~, L. E., Electron microscopy of degeneration in the lateral olfactory tract and plexiform layer of the prepyriform cortex of the rat, Z. Zellt"orsch., 98 (1969) 157-187.
173
Research Reports
SUBMICROSCOPIC C H A N G E S OF CORTEX NERVE CELLS IN C H R O N I C M I R R O R EPILEPTIC FOCUS IN RAT
N. N. BOGOLEPOV AND A. S. P U S H K I N
Electron Microscopy Laboratory, Brain Research hlstitute, Academy of Medical Sciences, Moscow
(u.s.s.R.) (Accepted March 1 lth, 1975)
SUMMARY
Submicroscopic changes of neurons, synapses, glial cells and some elements of capillaries were established by studying chronic mirror foci which appeared in the rat cerebral cortex after implantation of cobalt-gelatinous rods into the contralateral hemisphere. The results were compared with normal control cortex and with changes after thermocoagulation of the symmetrical contra[ateral cortex. it is shown that epileptogenic lesions produce a significant reduction of RNAcomponents, edema and vacuolization of neuronal cytoplasm, hypertrophy and proliferation of glial cells. The revealed pathological changes of the synapses were manifested by dilatation of some vesicles and the appearance ofheterogenic inclusions. The characteristic features of Wallerian degeneration of callosal synapses and the reversible retrograde changes of the neurons which send their axons into the traumatized zone of the contralateral hemisphere were observed to be similar after both experimental lesions.
INTRODUCTION
The study of the peculiarities of submicroscopic changes in secondary epileptic foci is of interest not only in connection with the analysis of the ultrastructural foundations of the epileptic processZ,V,s,la,19, ~4, but also as a convenient method for the elucidation of the plastic modifications which convey epileptic properties to an initially intact brain zone14, 2~. Previous electrophysiological investigations of the so-called 'mirror' focus have
174 shown that its origin depends on a stable reorganization of functional properties and metabolism, as well as on the structure of the secondary epileptic cortex zone 15,1s:'e. Up to the present time, however, morphological characterization of the mirror foci has been limited by light microscopic data which do not show the intimate damage of the cell structures or the reorganization of the interneuronal mutual relationships. This paper deals with the problem of the first stage of submicroscopic analysis of secondary epileptogenesis, with the aim of showing the qualitative peculiarities of changes in basal cell elements in a rat brain cortex mirror focus, emerging after a chronic epileptogenic lesion of a symmetrical contralateral cortex zone.
MATERIAL AND METHODS
Thirty-eight adult white male rats, weighing 170-250 g, were used. The anterior parietal (PA) region of the cortex was studied 23. Six rats without experimental lesions were used as controls to study the normal ultrastructure. Twenty-two rats were subjected to an implantation of cobalt-gelatinous rods into the above-mentioned zone of the left hemisphere cortex 9, which created a lesion nidus of 8-10 sq.mm with a depth of 1.0-1.2 mm. The animals were sacrificed on days 3, 6, 8, 10, 12, 14, 24 or 30 after the operation. The picture of the submicroscopic changes in the mirror focus was compared with modifications in the cerebral cortex which, of the contralateral hemisphere, inevitably occur when the cortex is subjected to lesions and which are due to the development of a retrograde and Wallerian degeneration in the neurons and fibers comprising the transcaUosal connections. Accordingly 10 rats were subjected to a thermocoagulation of an analogous section of the left hemisphere cortex. The dimensions of the necrotic focus were identical to those of the cobalt-gelatinous lesion. These animals were sacrificed on days 3, 5, 7, 14, and 21 after the operation. For the identification of the epileptic process we used electroencephalograms registered on a 'Galileo' oscilloscope using intracranially implanted electrodes. Under Nembutal anesthesia (4 mg/100 g body weight) the animals were perfused with a 2.5 ~ glutaraldehyde solution in a phosphate buffer (pH 7.4). After previous fixation of the brain a specimen containing the focus of the primary lesion was excised as well as the symmetrical part of the contralateral hemisphere. This sample was immersed in a 5 ~o glutaraldehyde solution for one hour. Then, from the zone symmetrical to the focus of the lesion, samples were taken for electron microscopy. After rinsing in the buffer solution the samples were immersed for 1.5-2.0 h in a 2 ~ OsO4 solution, dehydrated in increasing alcohol concentration gradients and embedded in Epon-812. Ultrathin and semithin (1.0-2.5/~m) slices were then obtained, using an LKB- 111 ultramicrotome. The slices were stained with uranyl acetate and lead citrate after which they were studied and photographed with UEMV-100A, HU-11-E and AEI-EM801 electron microscopes.
175
Fig. 1. Chromatolysis of cortical pyramid cells from layer III. Day 24 after operation. Mirror focus. x 5250.
Fig. 2. The reduction of granular endoplasmic reticulum in the cytoplasm of small neuron. Day 14 after operation. Mirror focus. Layer III. x 18,000.
176
Fig. 3. Small neuron from layer 1V of intacl cortex.
21,000.
Light microscopic investigations and the identification of the cortical layers were conducted on semithin slices stained with methylene blue or p-phenylenediamine. The remaining part of the brain sample taken after the perfusion was embedded in paraffin, cut into 10-15 #m slices, stained with cresyl violet and studied by light microscopy to determine the depth of the primary lesions caused by cobalt-gelatinous rods or by thermocoagulation. RESULTS
The EEGs of rats with cobalt-gelatinous rod implantations showed sporadic
177
Fig. 4. The part of the neuropile from layer IV. Day 14 after operation. Mirror focus. Edema of dendrites is shown. × 20,000.
Fig. 5. Degenerating presynaptic terminal in neuropile of layer VI. Mirror focus. Day 10 after operation, x 55,000.
178 outbursts of high amplitude sharp waves and bursts of discharges of the 'spike-wave' type, which were described as definite equivalents of the epileptic process (Penfield and Jasper1'5,18,21). Convulsive movements, however, were not observed. Light microscopic investigations revealed that in the cortical mirror tbcus the main reaction of the cells consisted of a peripheral chromatolysis which included a vast majority of neurons. These neurons were distributed throughout the cortex but were more frequently found in layers V and VI. In the thermocoagulation samples only a restricted number of cells showed chromatolysis, mainly the pyramidal middlesize neurons and rarely the large pyramidal neurons localized, preferably, in layers III-V. Accordingly, the sattelitosis and hyperplasia of different forms of neuroglial cells is less marked after thermocoagulation than in mirror focus. The p-phenylenediamine stained preparations clearly showed degenerating myelin fibers. Their distribution was similar in the mirror focus and the thermocoagulation samples. Most of the degenerating fibers were concentrated in layers VI and V. The electron microscopic studies of the mirror focus showed that the most noticeable modifications of the nerve cells, interneuronal connections and glial cells were evident during the period of maximal electrophysiological changes - - from the 10th to the 24th day after the implantation of the cobalt-gelatinous rod. The modified nerve cells were characterized by a decreased number of cytoplasmic ribosomes and polysomes in combination with an increase of the depth and length of the nuclear membrane folds and a tendency for the nucleolus to be situated near the membrane. A redistribution of granular and filamentous components was evident. The cytoplasmic ribosomes concentrated around the nucleus and in the zone of the nuclear membrane folds. The cytoplasmic periphery was markedly deprived of these structures (Figs. I and 2), which differentiates the lesioned cells from normal ones (Fig. 3). In many cells these signs were accompanied by an edema of the endoplasmic reticulum cisternae. The modified dendrites were characterized by the appearance of vacuole-like cavities (Fig. 4). Chromatolysis and the disintegration of organelles are mainly characteristic of small neurons (Fig. 2), the pathological changes of which increased up to a disintegration of the cell membrane, and probably to cell death. As opposed to the above mentioned results the ultrastructure of a substantial number of larger neurons was like normal pictures. Two kinds of changes of nerve fibers and synapses were registered in the course of development of the mirror focus. From the 3rd to the 14th day after the operation in some of the axon terminals the well-known signs of Wallerian degeneration of the 'dark' and 'light' type were observed. These presynaptic processes were mostly localized on the spines of the dendrites and on their thin branchings, and sometimes on the bodies of the small neurons. The degenerating synapses (Fig. 5) are distributed throughout the whole cortex, mostly in layers V and VI and slightly less in layers II and III. The appearance of glycogen granules, lysosome-like structures and the increase
179
Fig. 6. Enlarged vesicles in presynaptic bag terminated on demh itic branch. Day 24 after operation. Mirror focus, x 75,000.
Fig. 7. Numerous glycogen inclusions in presynaptie terminals. Day 14 after operation. Mirror focus, x 35,000.
of some vesicles' size without changes in the axoplasmic osmiophilicity were noted in some contacts on day 12-14 after operation (Figs. 6 and 7). The modifications of the bodies and processes of nerve cells were accompanied by an intensification of the glial reaction. The number of perineuronal gliocytes was increased, consisting mostly of oligodendrocytes and rarely of astrocytes and cells of a nondescript type. A marked hypertrophy and hyperplasia of cytoplasmic organelles was observed in these cells (Fig. 8). The same reaction was seen in the processes of
180
Fig. 8. The activation of glial satellite of chromatolytic neuron. Day 14 after operation. Mirror focus. ~. 30,000. astrocytes, which showed a swollen cytoplasm and often contained numerous lipid inclusions and glycogen-like granules. The increases in size and the number of processes ofastrocytes often gave a picture of gliosis loci and changed the neuropil architectonics. The cytoplasm of pericytes, and occasionally of endothelial cells, was characterized by an hypertrophy of the ultrastructures and by the appearance of a large number of heterogenous lysosome-like bodies. To test the specificity of the epiteptogenically induced changes we conducted studies of the cortex after a thermocoagulation of a symmetrical zone of the contralateral hemisphere. It was shown that, in contrast to the phenomena occurring in the mirror focus, the small neurons remained normal. The ultrastructure of large cells, which showed signs of chromatolysis on the semithin slices, was characterized by a substantial decrease of the number of ribosomes of the cytoplasm periphery (Fig. 9). Occasionally such neurons showed a swelling and destruction of the crists of separate mitochondria, an increase of the number of lysosomes and the presence of small vacuoles. On the 21st postoperative day the ultrastructure of most of these neurons had normalized. The thermocoagulation experiments showed submicroscopic signs of Watterian degeneration of some fibers and their synaptic contacts (Fig. 10). An analysis of the mode of termination and of the laminar distribution of the degenerating terminals
181
Fig, 9. Large pyramidal neuron from the layer V. Day 14 after thermocoagulative lesion of contralateral hemisphere. × 4500.
did not disclose any fundamental distinctions between the two forms of experimental lesion. The nerve cell changes were accompanied by an hypertrophy of the organelles of the perineuronal oligodendrocytes' cytoplasm. It was also noted that the astrocyte processes, which engulfed degenerating synapses, were increased in size. In comparison with the mirror focus, however, the activation of the ultrastructures of postthermocoagulation glial elements was considerably less marked. DISCUSSION
The comparison of the changes which were observed in the cortex after both forms of experimental lesion of a symmetrical zone of the contralateral hemisphere disclosed some of the peculiarities of the mirror focus. They consisted of a substantial decrease of the number of ribosomes, a reduction of the endoplasmic reticulum, a disintegration of mitochondria, a vacuolization of neurons (especially of small neurons), a swelling and edema of separate dendrites and polymorphous dystrophic lesions of synapses. These changes were accompanied by an activation of the glial cells. The above mentioned findings are in direct correlation with the biochemical
182
Fig. 10. Degenerating contact with the soma of small neuron from layer VI. Day 5 after thermocoagulation of contralateral hemisphere. ,~,"60,000, data of a decrease of the R N A and protein content of chronic mirror focus neurons and with the changes of activity of certain dehydrogenases, which are oppositely directed in neurons and glial cells 10,1~,~.~. Similar changes of the ultrastructure and of the biochemistry of nerve cells were observed in experiments reproducing acute epileptiform convulsions 13,16,z° as well as in experiments reproducing an increased adequate activity of neurons 6,17. This is an indication of a stable and substantial prevalence of the processes of disintegration of cellular proteins over their synthesis. We have noted an increase of the area of the glial profiles in the neuropile, which can be regarded as one of the stages of gliosis development characteristic of chronic foci4,ag, 24. Swelling of the glial processes, similar to that presently observed, was described by Okada e t al. 16 in acute primary foci. Westmoreland e t al. ~ adhere to the opinion that in a mirror focus there is also developing a proliferation of the glial cells determined by an increase of DNA, probably connected with a degeneration of the interhemispheric bonds. Of great interest is the presence in the mirror focus of disrupted synaptic connections. The typical features of a Wallerian degeneration and particularly of a disintegration of the 'dark'3, 26 type are similar in both models of experiments. The
183
manifested distribution of the degenerating terminals bears a resemb!ance to the data on the localization of synapses formed by callosal fibers~,aL Changes appeared at the region of the mirror focus in the late stages, when a typical Wallerian degeneration was already concluded by a disruption and phagocytosis of terminals. Possibly such symptoms of early stages of degeneration as an enlargement of synaptic vesicles a, an accumulation of glycogen, or the appearance of lysosome-like bodies in the terminals, point to the development of a transneuronal degeneration. Thus in this work are shown the changes of neurons, of interneuronal connections and of glial cells observed by electron microscopy interconnected with the development of a pathological activity in an epileptic mirror focus.
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