Neurochemical Research (1) 313-327 (1976)
A SPECIFIC PROTEIN A B N O R M A L I T Y ASSOCIATED WITH C O B A L T - I N D U C E D E P I L E P S Y IN MICE M. GREAVES, 1 M. BELANGER, AND N . M. VAN GELDER Neurological Sciences Group of the Medical Research Council of Canada Ddpartement de physiologic, Facultd de mOdecine UniversitO de Montrdal
Accepted March 22, 1976
Density profiles of protein patterns from cortical tissue exhibit an increase in only one peak when mice are rendered epileptic by application of cobalt to the cortex. The increase and diminution in peak height, attributed to a change in the concentration o f a single protein (protein 3), coincides with the severity of seizure activity; with the degree of abnormality o f the cortex region affected; and with the time of onset, duration, and disappearance of the epileptic condition. Thus, the concentration of protein 3 is highest in tissue from the site of cobalt application (up to 10x normal), is increased less in the focus (up to 5 x normal), while in the mirror focus (contralateral, not exposed surgically), the increase in the concentration o f protein 3 is still detectable, but not as pronounced. The concentrations in these cortex regions decrease to normal in reverse order to their elevation when the epileptic signs begin to diminish. Furthermore, the increase of protein 3 in all three areas is proportional to the severity of epilepsy. The concentration of protein 3 also becomes enhanced when the cortex is injured, but no progressive increase in the concentration occurs with time, nor does the concentration reach that observed in the site o f cobalt application or the focal region. These mice do not exhibit spontaneous seizures, but injection of pentelynetetrazol confirms that animals with brain injury only are more susceptible to seizures. The results of this study suggest that both the area of cortex affected and the intensity of metabolic alterations may be precipitating factors in establishing an epileptic condition. This view is in agreement with clinical observations on epilepsy.
INTRODUCTION Several lines of evidence suggest that protein metabolism may become altered during experimentally induced seizure states. Dunn and collabo1This work is in partial fulfillment of M.Sc. requirements.
313 9 1976 Plenum PublishingCorporation,227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying,microfilming,recording,or otherwise,without written permissionof the publisher,
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rators (7,8) reported a transient inhibition of protein synthesis following electroconvulsive seizures, while Mclnnes et al. (20), Wasterlain (33), and Vesco and Giuditta (30) have shown that electroconvulsive treatment causes disaggregation of brain polysomes. Other data are available that further support a conclusion that seizure states are accompanied by changes in protein metabolism (4,14,21,23). Enzyme activities have also been shown to fluctuate during convulsive conditions (9,25), and a decrease of glutamic acid in regions of maximal epileptogenic activity is by now well established (17,26,28). This amino acid is a normal and often prevalent constituent of proteins (e.g., 35). The work described here represents the first study in a series of planned investigations directed to answering several important questions. First, are the reported changes in protein metabolism translated into altered protein patterns in the focal region, and are they unique to that region? Second, are such alterations a permanent characteristic of the focus? Third, is the (possible) abnormality a prerequisite for the establishment of an epileptogenic focus? Questions of this type can be best answered by using experimental models of epilepsy that resemble, at least in some major aspects, the conditions encountered in patients with chronic, focal epilepsy. In these investigations, mice with cobaltinduced epilepsy were used (15). This epilepsy model, while not necessarily ideal, has been shown to be associated with an altered amino acid content of the CNS that parallels the development, and also the reversal, of the epileptic condition (16,24,27). The biochemical abnormalities, moreover, appear to resemble closely those found in the epileptic focus of patients (28). Finally, metabolic changes not only occur in the primary focus, but also extend to the secondary, contralateral mirror focus (27,34). The results of this study will show that an abnormal protein pattern is indeed characteristic of seizure-susceptible cortex. Furthermore, the time course of the appearance of a protein abnormality may be associated with the development of an epileptic condition.
EXPERIMENTAL PROCEDURE Cobalt-Epilepsy (27). Adult (30 g) female mice (Charles River CD-1, strain COBS) were anesthetized with Nembutal (80 mg/kg) and a 2-mm2 area of cortex was exposed just behind the sutura coronalis with the aid of a small dental "diamond" wheel. Approximately 3 mg cobalt powder (300 mesh) was placed on the cortex. The region was covered by dental cement to retain the cobalt powder, and the skin incision was closed by two stainless steel wound clips. Over 95% of the animals usually recover in good condition. Postoperative infections are extremely rare with this procedure.
P R O T E I N A B N O R M A L I T Y IN E P I L E P S Y
315
During the next 24 h, the animals were classified according to the severity of epileptic signs into three groups: severe, very severe, and status epilepticus (24). Removal of Tissue. Animals were killed by decapitation, and the appropriate area of cortex was removed with a small scalpel. The tissue was immediately dropped into a beaker of petroleum ether cooled to -60~ by a dry ice-alcohol bath. The total time between decapitation and freezing did not exceed 30 s, and was often as short as 20 s. Tissues removed faster or slower were rejected in order to preserve uniformity in metabolic postmortem changes. Classification of Tissues. A total of 10 groups of tissues were used in these experiments: 1. Normal 2. Sham-Operated: 3. Damaged: 4. Sham Focus:
5. Cobalt: 6. Cobalt S.E.: 7. Focus: 8. Focus S.E.: 9. Mirror Focus: 10. Sham Mirror:
Normal cortex from nonoperated animals. Bone removed and replaced. No deliberate damage to cortex. Cortex was scratched or crosshatched with a scalpel and region covered by dental cement. Area of cortex between (behind) lesion and occipital pole of the cortex. This area was not exposed during surgery, and no overt damage was noticed. Area to which cobalt powder was applied. Animals very severely epileptic. As #5, but animals in status epilepticus. Area of cortex behind (excluding) region of cobalt application. Area not exposed during surgery. As #7, but animals in status epilepticus. The same area as #7 or #8, but from the contralateral cortex. This area was also not exposed during surgery. Same area as #9, but from sham-operated animals (#2).
Preparation of Tissue. The frozen cortex was lightly blotted, weighed (
A SPECIFIC PROTEIN A B N O R M A L I T Y ASSOCIATED WITH C O B A L T - I N D U C E D E P I L E P S Y IN MICE M. GREAVES, 1 M. BELANGER, AND N . M. VAN GELDER Neurological Sciences Group of the Medical Research Council of Canada Ddpartement de physiologic, Facultd de mOdecine UniversitO de Montrdal
Accepted March 22, 1976
Density profiles of protein patterns from cortical tissue exhibit an increase in only one peak when mice are rendered epileptic by application of cobalt to the cortex. The increase and diminution in peak height, attributed to a change in the concentration o f a single protein (protein 3), coincides with the severity of seizure activity; with the degree of abnormality o f the cortex region affected; and with the time of onset, duration, and disappearance of the epileptic condition. Thus, the concentration of protein 3 is highest in tissue from the site of cobalt application (up to 10x normal), is increased less in the focus (up to 5 x normal), while in the mirror focus (contralateral, not exposed surgically), the increase in the concentration o f protein 3 is still detectable, but not as pronounced. The concentrations in these cortex regions decrease to normal in reverse order to their elevation when the epileptic signs begin to diminish. Furthermore, the increase of protein 3 in all three areas is proportional to the severity of epilepsy. The concentration of protein 3 also becomes enhanced when the cortex is injured, but no progressive increase in the concentration occurs with time, nor does the concentration reach that observed in the site o f cobalt application or the focal region. These mice do not exhibit spontaneous seizures, but injection of pentelynetetrazol confirms that animals with brain injury only are more susceptible to seizures. The results of this study suggest that both the area of cortex affected and the intensity of metabolic alterations may be precipitating factors in establishing an epileptic condition. This view is in agreement with clinical observations on epilepsy.
INTRODUCTION Several lines of evidence suggest that protein metabolism may become altered during experimentally induced seizure states. Dunn and collabo1This work is in partial fulfillment of M.Sc. requirements.
313 9 1976 Plenum PublishingCorporation,227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying,microfilming,recording,or otherwise,without written permissionof the publisher,
314
GREAVES ET AL.
rators (7,8) reported a transient inhibition of protein synthesis following electroconvulsive seizures, while Mclnnes et al. (20), Wasterlain (33), and Vesco and Giuditta (30) have shown that electroconvulsive treatment causes disaggregation of brain polysomes. Other data are available that further support a conclusion that seizure states are accompanied by changes in protein metabolism (4,14,21,23). Enzyme activities have also been shown to fluctuate during convulsive conditions (9,25), and a decrease of glutamic acid in regions of maximal epileptogenic activity is by now well established (17,26,28). This amino acid is a normal and often prevalent constituent of proteins (e.g., 35). The work described here represents the first study in a series of planned investigations directed to answering several important questions. First, are the reported changes in protein metabolism translated into altered protein patterns in the focal region, and are they unique to that region? Second, are such alterations a permanent characteristic of the focus? Third, is the (possible) abnormality a prerequisite for the establishment of an epileptogenic focus? Questions of this type can be best answered by using experimental models of epilepsy that resemble, at least in some major aspects, the conditions encountered in patients with chronic, focal epilepsy. In these investigations, mice with cobaltinduced epilepsy were used (15). This epilepsy model, while not necessarily ideal, has been shown to be associated with an altered amino acid content of the CNS that parallels the development, and also the reversal, of the epileptic condition (16,24,27). The biochemical abnormalities, moreover, appear to resemble closely those found in the epileptic focus of patients (28). Finally, metabolic changes not only occur in the primary focus, but also extend to the secondary, contralateral mirror focus (27,34). The results of this study will show that an abnormal protein pattern is indeed characteristic of seizure-susceptible cortex. Furthermore, the time course of the appearance of a protein abnormality may be associated with the development of an epileptic condition.
EXPERIMENTAL PROCEDURE Cobalt-Epilepsy (27). Adult (30 g) female mice (Charles River CD-1, strain COBS) were anesthetized with Nembutal (80 mg/kg) and a 2-mm2 area of cortex was exposed just behind the sutura coronalis with the aid of a small dental "diamond" wheel. Approximately 3 mg cobalt powder (300 mesh) was placed on the cortex. The region was covered by dental cement to retain the cobalt powder, and the skin incision was closed by two stainless steel wound clips. Over 95% of the animals usually recover in good condition. Postoperative infections are extremely rare with this procedure.
P R O T E I N A B N O R M A L I T Y IN E P I L E P S Y
315
During the next 24 h, the animals were classified according to the severity of epileptic signs into three groups: severe, very severe, and status epilepticus (24). Removal of Tissue. Animals were killed by decapitation, and the appropriate area of cortex was removed with a small scalpel. The tissue was immediately dropped into a beaker of petroleum ether cooled to -60~ by a dry ice-alcohol bath. The total time between decapitation and freezing did not exceed 30 s, and was often as short as 20 s. Tissues removed faster or slower were rejected in order to preserve uniformity in metabolic postmortem changes. Classification of Tissues. A total of 10 groups of tissues were used in these experiments: 1. Normal 2. Sham-Operated: 3. Damaged: 4. Sham Focus:
5. Cobalt: 6. Cobalt S.E.: 7. Focus: 8. Focus S.E.: 9. Mirror Focus: 10. Sham Mirror:
Normal cortex from nonoperated animals. Bone removed and replaced. No deliberate damage to cortex. Cortex was scratched or crosshatched with a scalpel and region covered by dental cement. Area of cortex between (behind) lesion and occipital pole of the cortex. This area was not exposed during surgery, and no overt damage was noticed. Area to which cobalt powder was applied. Animals very severely epileptic. As #5, but animals in status epilepticus. Area of cortex behind (excluding) region of cobalt application. Area not exposed during surgery. As #7, but animals in status epilepticus. The same area as #7 or #8, but from the contralateral cortex. This area was also not exposed during surgery. Same area as #9, but from sham-operated animals (#2).
Preparation of Tissue. The frozen cortex was lightly blotted, weighed (
