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Focal epileptogenesis after intracortical hemoglobin injection
SXPERIMENTAL
NEUROLOGY
66,277-284
(1979)
Focal Epileptogenesis Hemoglobin
after lntracortical Injection
A. D. ROSEN AND N. V. FRUMIN’ Depurtment
of Neurology, Received
State March
University
of Neu*
30. 1979; revision
York,
Stony
received
Muy
Brook,
New
York
I1794
29, 1979
Intracortical injection of purified bovine hemoglobin produced chronic focal spike activity in 89% of rats tested; 50% of the animals evidenced spike activity within 48 h. The lesions produced were pathologically similar to those seen in post-traumatic epilepsy. Evidence is presented suggesting that the neural effect of iron released from hemoglobin may be causally related to the development of a trauma-induced epileptiform focus in humans.
INTRODUCTION Intracerebral hemorrhage has been shown to be associated with a significant incidence of both early and late seizures (17). Seizures are a well-known sequela to head trauma and though many factors may be important in the probability of seizure development (4, 6), pathological studies have demonstrated gliosis, neuronal loss, and hemosiderosis in the involved cerebral tissue (13). Blood, especially when first hemolyzed, was effective in producing electrocorticographic spikes in 40% of cats when injected into the subarachnoid space (11). This suggests that some component of blood had epileptogenic properties when in direct contact with the cerebral cortex. Recent studies suggested that this epileptogenic agent may be ionic iron released from hemoglobin. When either the di- or trivalent form of iron was iontophoresed onto the pial surface (21) or injected directly into the cortex of rats (22), electrical seizure activity could be recorded within several minutes and persisted for several weeks. Abbreviation: EEG-electroencephalogram. ’ This work was supported in part by the Medical Research Service of the Veterans Administration. The authors extend their thanks to Mrs. Barbara Fischer for her secretarial assistance. Address A. D. Rosen, Department of Neurology, Health Sciences Center. State University of New York. Stony Brook, NY 11794. 277 0014-4886/79/110277-08$02.00/O Copyrigh: 0 1979 by Academic Press. Inc. All nghts of reproduction in any form reserved.
278
ROSEN
AND
FRUMIN
The present study was undertaken to demonstrate that purified hemoglobin itself is an effective epileptogenic agent and to determine the time course of that activity. METHODS Twenty-seven adult Sprague-Dawley rats (150 to 324 g) of either sex were used. After anesthesia with sodium pentobarbital(5 mg/lOO mg, i.p.) the animal was secured in a stereotaxic apparatus. Under aseptic conditions, a 2-mm hole was drilled through the skull over the left occipital cortex with particular care not to damage the dura. A solution of purified bovine hemoglobin in saline (13 mg/lOO ml) was prepared and placed in a microinjection syringe fitted with a 30-gauge needle. With the aid of a dissecting microscope the needle was placed over a relatively avascular area of cortex and inserted 1.5 mm below the dura. Hemoglobin solution (10 ,ul) was injected during 2 min and the needle removed. Some leakage of the injected material was frequently seen after withdrawal of the needle. The scalp was closed with wound clips and postoperative electroencephalogram (EEG) recordings were made via needle scalp electrodes placed 1 cm on either side of the midline in a plane defined by the interaural line. Both electrodes were referenced to a midline electrode at the level of the interoccular line. Recordings were continued until the animal showed signs of awakening from anesthesia. EEGs were obtained from the same recording sites daily for the next 5 days and twice weekly thereafter. Animals were maintained for as long as 2 months. All EEGs were recorded under pentobarbital anesthesia and continued until the animal showed signs of awakening. During all recording sessions, the animal’s temperature was monitored via a rectal probe and maintained at 38.0 to 38.5”C by a heating pad. At various intervals after the hemoglobin injection, animals were anesthetized with pentobarbital and killed by transcardiac perfusion with saline followed by 10% neutral buffered Formalin. Histological verification of the extent of the lesions was made in IO-$ paraffin sections stained with Highman’s method for iron (7) and &rain-O. Selected sections were processed with a Nissl stain. An additional four rats were injected with saline alone and followed in the same manner as the hemoglobin-injected animals. RESULTS High-amplitude focal spike activity was recorded from 24 of the 27 animals subjected to intracortical hemoglobin injection (89%). This is illustrated in Fig. 1. Although single spikes were most commonly seen, brief runs were occasionally noted. The development of spike activity was
HEMOGLOBIN
EPILEPTOGENESIS
279
FIG. 1. Serial electroencephalograms from left posterior cerebrum of rat before and after intracortical injection of hemoglobin. Calibration: 1 s. 100 FV.
associated with desynchronized background activity. Fifty percent of the animals developed spike activity within 48 h and all that were to develop spikes, did so within 8 days (Fig. 2). The seizure focus, when present, became relatively stable within 2 days after this activity was first noted and, in all cases, persisted until the animal was killed. No obvious behavioral manifestations of seizures were seen although many of the animals showed diminished cage exploration postoperatively. No EEG evidence of seizure activity was seen in those animals subjected to intracortical saline injection. Microscopic examination of the injection sites revealed discrete intracortical lesions (Fig. 3) consisting of a cavity with scattered hemosiderin-laden macrophages in the wall as well as a variable degree of associated gliosis. Surrounding neurons were normal in number and morphology. The cavity usually was in communication with the subarachnoid space via the original needle tract. Tissue stained for ionic iron showed the material within macrophages but iron was not found in neurons (Fig. 4).
280
ROSEN AND FRUMIN
. .
.
.
.
FIG. 2. Cumulative percentage of animals exhibiting epileptiform animals.
activity. Data from 27
DISCUSSION A variety of techniques has been used in the past to produce a model which simulates the epileptogenic focus seen in humans. Cortical freezing (12, 18, 19), topical penicillin (14, 16), topical alumina cream (10, 20), and intracortical cobalt (3, 9) were the most widely used methods but none is a pathophysiological correlate of the clinical disorder. The recent demonstration that intracortical ionic iron, the major metalic cation of blood, is an effective epileptogenic agent (22, 23) suggests a more physiological model for posttraumatic epilepsy. In the present study, intracortical hemoglobin was demonstrated to be epileptogenic, with the time course of epileptogenic activity corresponding to the expected breakdown of hemoglobin and release of iron. The lesions produced in this study were histopathologically similar to those seen in posttraumatic epilepsy (13). Furthermore, intracortical hemoglobin did not produce significant neuronal loss in the tissue surrounding the injection site as was seen with intracortical injection of solutions of ionic iron [Fig. 4 of (23)]. It should be pointed out that although bovine hemoglobin was used in this study, the structure of heme is identical in all mammals (24); species differences are reflected in the amino acid sequences in globin. During hemoglobin catabolism, globin is split from the molecule, degraded, and returned to the body pool of amino acids. Iron is released from the heme portion of the molecule and the latter is broken down to bilirubin. The released iron may link with the transport protein, transfer-tin, or remain in the tissues as an iron-protein complex, ferritan, or hemosiderin.
HEMOGLOBINEPILEPTOGENESIS
282
ROSEN
AND
FRUMIN
HEMOGLOBIN
EPILEPTOGENESIS
283
Although the precise mechanism of action of ionic iron in the central nervous system is unknown, there is some evidence that it exerts a facilitatory effect on excitable membranes. Deposition of iron was shown to increase the neuronal discharge rate when applied in the vicinity of the cell body but not when applied to nerve fibers (2). In addition, deposition of iron ions from steel anodal electrodes was successfully used for exciting nerve cells in the preoptic area (5)) hypothalamus (8)) and amygdala ( 1). It seems probable that in order to exert its effect on the excitable membrane, ionic iron binds in some manner to that membrane. Although no membrane-bound iron was seen with the histochemical techniques used in the present study, the amount present may have been too small to be seen or the binding constant too high to permit histochemical reaction. It is interesting to note that iron has been used to specifically stain nodes of Ranvier in rat sciatic nerves (15). This phenomenon must be cautiously interpreted for physiological significance, as the stain was used in fixed tissue. This observation suggests a specific affinity of the cytoplasmic surface of the nodal axon membrane for iron. Further studies to ascess the degree ofin vivo membrane binding of iron are presently being carried out. REFERENCES 1. BELTRAMINO, C., AND S. TALEISNIK. 1978. Facilitatory and inhibitary effects of electrochemical stimulation of the amygdala on the release of luteinising hormone. Bruin Res. 144: 95-107. 2. COLOMBO, J. A., D. I. WHITMOYER, AND C. H. SAWYER. 1975. Local effects of iron deposition on multiple unit activity in the female rat brain. Bruin Res. 96: 88-92. 3. Dow, R. S., A. FERNANDEZ~JARDIOLA, AND E. MANNI. 1962. The production of experimental cobalt epilepsy in the rat. Electroenceph. Clin. Neurophysioi. 14: 399-407. 4. EVANS, J. H. 1962. Post-traumatic epilepsy. Neurology (Minneupolis) 12: 665-674. 5. EVERTT, J. W.. AND H. M. REDFORD. 1961. Irritative deposits from stainless-steel electrodes in the preoptic rat brain causing release of pituitary gonadotrophin. Proc. Sot. Exp. Biol. Med. 108: 604-609. 6. FEENEY, D. M., AND A. E. WALKER. 1979. The prediction of the posttraumatic epilepsy. Arch. Neural. 36: 8-12. 7. HIGHMAN. B. 1942. A new modification of Perl’s reaction for hemosiderin in tissues. Arch. Patz. 33: 937-938. 8. KALRA, S. P., K. AJIKA, L. KRULICH, C. P. FAWCETT, M. QUIJADA, AND S. M. MCCANN. 1971. Effects of hypothalamic and preoptic electrochemical stimulation on gonadotropin and prolactin release in proestrus rats. Endocrinology 88: 1150- 1158. 9. KOPELOFF. L. M. 1960. Experimental epilepsy in the mouse. Proc. Sot. Exp. Biol. Med. 104: 500-504. 10. KOPELOFF. L. M., S. E. BARRERA, AND N. KOPELOFF. 1942. Recurrent convulsive seizures in animals produced by immunologic and chemical means. Am. J. Psych. 98: 881-902. 11. LEVITT, P., W. P. WILSON, AND R. H. WILKINS. 1971. The effects of subarachnoid blood on the electrocorticogram of the cat. J. Neurosurg. 35: 185- 191,
284
ROSEN AND FRUMIN
17.
F., AND A. FLORENZ. 1958. Modification of the freezing technique for producing experimental epileptogenic lesions. Electroenceph. Clin. Neurophysiol. 10: 187-188. PAYAN, H., M. TOGA, AND M. B~RARD-BADIER. 1970. The pathology of post-traumatic epilepsies. Epilepsia 11: 81-94. PRINCE, D. A. 1966. Modification of focal cortical epileptogenic discharge by afferent influences. Epilepsia 7: 181-201. QUICK,D. C.,ANDS. G. WAXMAN. 1977. Speciticstainingoftheaxonmembraneatnodes of Ravnier with ferric ion and ferrocyanide. J. Nearof. Sci. 31: l-l 1. RALSTON, B. L. 1958. The mechanism of transition of interictal spiking foci into ictal seizure discharge. Electroenceph. Clin. Neurophysiol. 10: 217-232. RICHARDSON, E. P., JR, AND P. R. DODGE. 1954. Epilepsy in cerebrovascular disease.
18.
SCHNEIDER,
12.
13. 14. 15. 16.
MORRELL,
Epilepsia
19. 20. 21. 22.
23.
24.
3: 49-74.
A., AND B. EPSTEIN. 1931. The effects of local freezing of the central nervous system of the cat. Arch. Neurol. Psych. 25: 1263-1270. SMITH, T. G., JR., AND D. P. PURPURA. 1960. Electrophysiological studies on epileptogenic lesions of cat cortex. Electroenceph. C/in. Neurophysiol. 12: 59-82. WARD, A. A., JR., 1969. The epileptic neuron: chronic foci in animals and man. Pages 263-288 in H. A. JASPER, A. A. WARD, JR., AND A. POPE, Eds.,Basic Mechanisms of the Epiiepsies. Little, Brown, Boston. WILLMORE, L. J., R. W. HURD, AND G. W. SYPERT. 1978. Epileptiform activity initiated by pial iontophoresis of ferrous and ferric chloride on rat cerebral cortex. Brain Res. 52: 406-410. WILLMORE, L. J., G. W. SYPERT, J. B. MUNSON, AND R. W. HURD. 1978. Chronic focal epileptiform discharges induced by injection of iron into rat and cat cortex. Science 200: 1501-1503. WILLMORE, L. J., G. W. SYPERT, AND J. B. MUNSON. 1978. Recurrent seizures induced by cortical iron injection: A model of post-traumatic epilepsy. Ann. NeuroL 4: 309-336. WINTROBE, M. W. 1974. Clinical Hematology, 7th ed. Lea & Febiger, Philadelphia.
NEUROLOGY
66,277-284
(1979)
Focal Epileptogenesis Hemoglobin
after lntracortical Injection
A. D. ROSEN AND N. V. FRUMIN’ Depurtment
of Neurology, Received
State March
University
of Neu*
30. 1979; revision
York,
Stony
received
Muy
Brook,
New
York
I1794
29, 1979
Intracortical injection of purified bovine hemoglobin produced chronic focal spike activity in 89% of rats tested; 50% of the animals evidenced spike activity within 48 h. The lesions produced were pathologically similar to those seen in post-traumatic epilepsy. Evidence is presented suggesting that the neural effect of iron released from hemoglobin may be causally related to the development of a trauma-induced epileptiform focus in humans.
INTRODUCTION Intracerebral hemorrhage has been shown to be associated with a significant incidence of both early and late seizures (17). Seizures are a well-known sequela to head trauma and though many factors may be important in the probability of seizure development (4, 6), pathological studies have demonstrated gliosis, neuronal loss, and hemosiderosis in the involved cerebral tissue (13). Blood, especially when first hemolyzed, was effective in producing electrocorticographic spikes in 40% of cats when injected into the subarachnoid space (11). This suggests that some component of blood had epileptogenic properties when in direct contact with the cerebral cortex. Recent studies suggested that this epileptogenic agent may be ionic iron released from hemoglobin. When either the di- or trivalent form of iron was iontophoresed onto the pial surface (21) or injected directly into the cortex of rats (22), electrical seizure activity could be recorded within several minutes and persisted for several weeks. Abbreviation: EEG-electroencephalogram. ’ This work was supported in part by the Medical Research Service of the Veterans Administration. The authors extend their thanks to Mrs. Barbara Fischer for her secretarial assistance. Address A. D. Rosen, Department of Neurology, Health Sciences Center. State University of New York. Stony Brook, NY 11794. 277 0014-4886/79/110277-08$02.00/O Copyrigh: 0 1979 by Academic Press. Inc. All nghts of reproduction in any form reserved.
278
ROSEN
AND
FRUMIN
The present study was undertaken to demonstrate that purified hemoglobin itself is an effective epileptogenic agent and to determine the time course of that activity. METHODS Twenty-seven adult Sprague-Dawley rats (150 to 324 g) of either sex were used. After anesthesia with sodium pentobarbital(5 mg/lOO mg, i.p.) the animal was secured in a stereotaxic apparatus. Under aseptic conditions, a 2-mm hole was drilled through the skull over the left occipital cortex with particular care not to damage the dura. A solution of purified bovine hemoglobin in saline (13 mg/lOO ml) was prepared and placed in a microinjection syringe fitted with a 30-gauge needle. With the aid of a dissecting microscope the needle was placed over a relatively avascular area of cortex and inserted 1.5 mm below the dura. Hemoglobin solution (10 ,ul) was injected during 2 min and the needle removed. Some leakage of the injected material was frequently seen after withdrawal of the needle. The scalp was closed with wound clips and postoperative electroencephalogram (EEG) recordings were made via needle scalp electrodes placed 1 cm on either side of the midline in a plane defined by the interaural line. Both electrodes were referenced to a midline electrode at the level of the interoccular line. Recordings were continued until the animal showed signs of awakening from anesthesia. EEGs were obtained from the same recording sites daily for the next 5 days and twice weekly thereafter. Animals were maintained for as long as 2 months. All EEGs were recorded under pentobarbital anesthesia and continued until the animal showed signs of awakening. During all recording sessions, the animal’s temperature was monitored via a rectal probe and maintained at 38.0 to 38.5”C by a heating pad. At various intervals after the hemoglobin injection, animals were anesthetized with pentobarbital and killed by transcardiac perfusion with saline followed by 10% neutral buffered Formalin. Histological verification of the extent of the lesions was made in IO-$ paraffin sections stained with Highman’s method for iron (7) and &rain-O. Selected sections were processed with a Nissl stain. An additional four rats were injected with saline alone and followed in the same manner as the hemoglobin-injected animals. RESULTS High-amplitude focal spike activity was recorded from 24 of the 27 animals subjected to intracortical hemoglobin injection (89%). This is illustrated in Fig. 1. Although single spikes were most commonly seen, brief runs were occasionally noted. The development of spike activity was
HEMOGLOBIN
EPILEPTOGENESIS
279
FIG. 1. Serial electroencephalograms from left posterior cerebrum of rat before and after intracortical injection of hemoglobin. Calibration: 1 s. 100 FV.
associated with desynchronized background activity. Fifty percent of the animals developed spike activity within 48 h and all that were to develop spikes, did so within 8 days (Fig. 2). The seizure focus, when present, became relatively stable within 2 days after this activity was first noted and, in all cases, persisted until the animal was killed. No obvious behavioral manifestations of seizures were seen although many of the animals showed diminished cage exploration postoperatively. No EEG evidence of seizure activity was seen in those animals subjected to intracortical saline injection. Microscopic examination of the injection sites revealed discrete intracortical lesions (Fig. 3) consisting of a cavity with scattered hemosiderin-laden macrophages in the wall as well as a variable degree of associated gliosis. Surrounding neurons were normal in number and morphology. The cavity usually was in communication with the subarachnoid space via the original needle tract. Tissue stained for ionic iron showed the material within macrophages but iron was not found in neurons (Fig. 4).
280
ROSEN AND FRUMIN
. .
.
.
.
FIG. 2. Cumulative percentage of animals exhibiting epileptiform animals.
activity. Data from 27
DISCUSSION A variety of techniques has been used in the past to produce a model which simulates the epileptogenic focus seen in humans. Cortical freezing (12, 18, 19), topical penicillin (14, 16), topical alumina cream (10, 20), and intracortical cobalt (3, 9) were the most widely used methods but none is a pathophysiological correlate of the clinical disorder. The recent demonstration that intracortical ionic iron, the major metalic cation of blood, is an effective epileptogenic agent (22, 23) suggests a more physiological model for posttraumatic epilepsy. In the present study, intracortical hemoglobin was demonstrated to be epileptogenic, with the time course of epileptogenic activity corresponding to the expected breakdown of hemoglobin and release of iron. The lesions produced in this study were histopathologically similar to those seen in posttraumatic epilepsy (13). Furthermore, intracortical hemoglobin did not produce significant neuronal loss in the tissue surrounding the injection site as was seen with intracortical injection of solutions of ionic iron [Fig. 4 of (23)]. It should be pointed out that although bovine hemoglobin was used in this study, the structure of heme is identical in all mammals (24); species differences are reflected in the amino acid sequences in globin. During hemoglobin catabolism, globin is split from the molecule, degraded, and returned to the body pool of amino acids. Iron is released from the heme portion of the molecule and the latter is broken down to bilirubin. The released iron may link with the transport protein, transfer-tin, or remain in the tissues as an iron-protein complex, ferritan, or hemosiderin.
HEMOGLOBINEPILEPTOGENESIS
282
ROSEN
AND
FRUMIN
HEMOGLOBIN
EPILEPTOGENESIS
283
Although the precise mechanism of action of ionic iron in the central nervous system is unknown, there is some evidence that it exerts a facilitatory effect on excitable membranes. Deposition of iron was shown to increase the neuronal discharge rate when applied in the vicinity of the cell body but not when applied to nerve fibers (2). In addition, deposition of iron ions from steel anodal electrodes was successfully used for exciting nerve cells in the preoptic area (5)) hypothalamus (8)) and amygdala ( 1). It seems probable that in order to exert its effect on the excitable membrane, ionic iron binds in some manner to that membrane. Although no membrane-bound iron was seen with the histochemical techniques used in the present study, the amount present may have been too small to be seen or the binding constant too high to permit histochemical reaction. It is interesting to note that iron has been used to specifically stain nodes of Ranvier in rat sciatic nerves (15). This phenomenon must be cautiously interpreted for physiological significance, as the stain was used in fixed tissue. This observation suggests a specific affinity of the cytoplasmic surface of the nodal axon membrane for iron. Further studies to ascess the degree ofin vivo membrane binding of iron are presently being carried out. REFERENCES 1. BELTRAMINO, C., AND S. TALEISNIK. 1978. Facilitatory and inhibitary effects of electrochemical stimulation of the amygdala on the release of luteinising hormone. Bruin Res. 144: 95-107. 2. COLOMBO, J. A., D. I. WHITMOYER, AND C. H. SAWYER. 1975. Local effects of iron deposition on multiple unit activity in the female rat brain. Bruin Res. 96: 88-92. 3. Dow, R. S., A. FERNANDEZ~JARDIOLA, AND E. MANNI. 1962. The production of experimental cobalt epilepsy in the rat. Electroenceph. Clin. Neurophysioi. 14: 399-407. 4. EVANS, J. H. 1962. Post-traumatic epilepsy. Neurology (Minneupolis) 12: 665-674. 5. EVERTT, J. W.. AND H. M. REDFORD. 1961. Irritative deposits from stainless-steel electrodes in the preoptic rat brain causing release of pituitary gonadotrophin. Proc. Sot. Exp. Biol. Med. 108: 604-609. 6. FEENEY, D. M., AND A. E. WALKER. 1979. The prediction of the posttraumatic epilepsy. Arch. Neural. 36: 8-12. 7. HIGHMAN. B. 1942. A new modification of Perl’s reaction for hemosiderin in tissues. Arch. Patz. 33: 937-938. 8. KALRA, S. P., K. AJIKA, L. KRULICH, C. P. FAWCETT, M. QUIJADA, AND S. M. MCCANN. 1971. Effects of hypothalamic and preoptic electrochemical stimulation on gonadotropin and prolactin release in proestrus rats. Endocrinology 88: 1150- 1158. 9. KOPELOFF. L. M. 1960. Experimental epilepsy in the mouse. Proc. Sot. Exp. Biol. Med. 104: 500-504. 10. KOPELOFF. L. M., S. E. BARRERA, AND N. KOPELOFF. 1942. Recurrent convulsive seizures in animals produced by immunologic and chemical means. Am. J. Psych. 98: 881-902. 11. LEVITT, P., W. P. WILSON, AND R. H. WILKINS. 1971. The effects of subarachnoid blood on the electrocorticogram of the cat. J. Neurosurg. 35: 185- 191,
284
ROSEN AND FRUMIN
17.
F., AND A. FLORENZ. 1958. Modification of the freezing technique for producing experimental epileptogenic lesions. Electroenceph. Clin. Neurophysiol. 10: 187-188. PAYAN, H., M. TOGA, AND M. B~RARD-BADIER. 1970. The pathology of post-traumatic epilepsies. Epilepsia 11: 81-94. PRINCE, D. A. 1966. Modification of focal cortical epileptogenic discharge by afferent influences. Epilepsia 7: 181-201. QUICK,D. C.,ANDS. G. WAXMAN. 1977. Speciticstainingoftheaxonmembraneatnodes of Ravnier with ferric ion and ferrocyanide. J. Nearof. Sci. 31: l-l 1. RALSTON, B. L. 1958. The mechanism of transition of interictal spiking foci into ictal seizure discharge. Electroenceph. Clin. Neurophysiol. 10: 217-232. RICHARDSON, E. P., JR, AND P. R. DODGE. 1954. Epilepsy in cerebrovascular disease.
18.
SCHNEIDER,
12.
13. 14. 15. 16.
MORRELL,
Epilepsia
19. 20. 21. 22.
23.
24.
3: 49-74.
A., AND B. EPSTEIN. 1931. The effects of local freezing of the central nervous system of the cat. Arch. Neurol. Psych. 25: 1263-1270. SMITH, T. G., JR., AND D. P. PURPURA. 1960. Electrophysiological studies on epileptogenic lesions of cat cortex. Electroenceph. C/in. Neurophysiol. 12: 59-82. WARD, A. A., JR., 1969. The epileptic neuron: chronic foci in animals and man. Pages 263-288 in H. A. JASPER, A. A. WARD, JR., AND A. POPE, Eds.,Basic Mechanisms of the Epiiepsies. Little, Brown, Boston. WILLMORE, L. J., R. W. HURD, AND G. W. SYPERT. 1978. Epileptiform activity initiated by pial iontophoresis of ferrous and ferric chloride on rat cerebral cortex. Brain Res. 52: 406-410. WILLMORE, L. J., G. W. SYPERT, J. B. MUNSON, AND R. W. HURD. 1978. Chronic focal epileptiform discharges induced by injection of iron into rat and cat cortex. Science 200: 1501-1503. WILLMORE, L. J., G. W. SYPERT, AND J. B. MUNSON. 1978. Recurrent seizures induced by cortical iron injection: A model of post-traumatic epilepsy. Ann. NeuroL 4: 309-336. WINTROBE, M. W. 1974. Clinical Hematology, 7th ed. Lea & Febiger, Philadelphia.