Histopathology of the ferric-induced chronic epileptic focus in cat: A Golgi study

EXPERIMENTAL

NEUROLOGY

Histopathology

STEVEN

Veterans Neurology.

66, 205-219 (1979)

of the Ferric-induced Focus in Cat: A Golgi

Chronic Study

A. REID, GEORGE W. SYPERT, WILLIAM AND L. JAMES WILLMORE'

Adntinisrrctrion Uttiversif.v Received

Hospital of Florida Febrtrury

uttd

Depurlmenrs

7. 1979;

revision

M. BOGGS,

of Neuroscience,

of Medicine,

College

Epileptic

received

Gainesville. May

Surgery, Florida

und 32610

7. 1979

Five cats were rendered chronically epileptic via subpial injection of saturated FeCI,, solution. Six weeks postinjection, electrocorticographic recording demonstrated focal epileptiform spiking in the region of the injection. This finding was not observed in saline-injected controls. Histopathological analysis of the epileptic focus using Nissl and Golgi-Cox techniques revealed (i) depopulation of Golgi-impregnated neurons, (ii) astocytic gliosis, (iii) loss of dendritic spines, (iv) decreased dendritic branching, and (v) dendritic varicosities. These are similar to the pathological findings which have been described for human epileptogenic foci. These results. in combination with the frequent observation of hemosiderosis in regions of human epileptogenic foci, implicate the release of iron from extravasated blood elements as a possible etiologic mechanism. Therefore, we believe the FeCI, experimental epileptogenic focus accurately models the human clinical entity (posttraumatic epilepsy) with respect to both electrophysiology and histopathology.

INTRODUCTION Numerous investigators have examined the morphologic and metabolic aspects of epileptogenic cortex. Their studies included histologic observations of experimentally induced foci in laboratory animals (3,4, 10, 12, 13, 25, 26) and of human neuropathological specimens obtained at neurosurgery or autopsy (2,6,8,9, 14, 16, 18,20,23,24). Tissue evidence of microvascular hemorrhage is often present in the region of the epileptic ’ This work was supported by the Medical Research Service of the Veterans Administration. The authors gratefully acknowledge the invaluable technical, histological, and photographic assistance of Carolyn J. Thomas, Barbara J. McGuire, and David W. Gratz, respectively. 205 0014-4886/79/110205-15$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.

206

REID

ET AL.

focus. Hamada et al. (9) reported vascular changes in 71% of surgically resected temporal lobe foci. Hemosiderosis, the hallmark of prior extravasation of red blood cells, is a frequent finding. Such erythrocytes are phagocytized by macrophages and their heme pigments processed to hemosiderin. Payan et al. (18) listed four main lesions which were “always” seen in posttraumatic epilepsy. These were “leptomeningeal fibrosis, hemosiderosis [italics ours], neuronal changes, and astrocytic gliosis.” That gliosis probably does not contribute primarily to the etiology of epileptogenesis was demonstrated convincingly by Hoover et al. (13). Mathieson reported (16) that the presence of hemosiderin-laden macrophages is not an uncommon finding in human temporal lobe foci. It was reported (22) that hemorrhagic cortical infarction and intracerebral hemorrhage are associated with an increased incidence of early and late seizures, especially if blood is within or adjacent to the cerebral cortex. Furthermore, injection of erythrocyte contents into the subarachnoid space of the cat will cause epileptiform discharges (15). These observations suggest a strong association between substances within extravasated blood and the subsequent development of seizure activity. Willmore et al, (26,27) recently reported that cortical application of iron salts resulted in the development of chronic focal seizures. Those investigators suggested that a likely etiologic agent of posttraumatic epilepsy is iron released from hemoglobin into brain substance after local hemorrhage. Of the techniques available for the light microscopic study of epileptogenic cortical foci, the Golgi approach is doubtless among the most useful. Scheibel et al. (23, 24) in a Golgi study of the human hippocampal-dentate complex in chronic temporal lobe epilepsy, reported several characteristic abnormalities. Westrum et al. (25), investigating the chronic, alumina gel-induced epileptogenic focus in monkey cortex, found very similar histopathological changes. Therefore, in an effort to determine if these changes are characteristic of chronic epileptogenic foci in general, we applied the Golgi technique to the chronic FeCl, focus in cat. METHODS Seven cats were used in this study- five in the experimental group and two in the control group. Each cat was premeditated with subcutaneous atropine (0.05 mg/kg) and anesthetized with intraperitoneal pentobarbital (40 mg/kg). Using sterile technique, the scalp was incised and a small burr hole made over the left marginal gyrus. Cortex was exposed via a small

GOLGI

ANALYSIS:

FERRIC

EPILEPTOGENIC

FOCI

207

cruciate incision in the dura. A 30-gauge cannula was mechanically advanced to a depth of 500 pm below the pial surface. Five microliters saturated FeCl, solution was delivered through this cannula to cats in the experimental group; a similar volume of 0.9% NaCl solution was administered to cats in the control group. To prevent leakage of injected solution along the needle track, the rate of administration was limited to 1 pl/min. For the same reason, the cannula was left in place for at least 5 min after delivery of electrolyte, thereby allowing the solution to diffuse into the brain substance. After withdrawal of the cannula, the skull defect was plugged with bone wax, the skin sutured, and the animal returned to its home cage for a 5- to &week recovery period. To obtain electrocorticographic verification of focal seizure activity, the following procedure was carried out for each cat. Premeditation was again subcutaneous atropine (0.05 mg/kg). Inhalation anesthesia with ethyl ether was used during initial surgical preparation. Cats were intubated via tracheotomy, and respirations maintained with a Harvard Apparatus small animal volume-cycle respirator. End-expiratory COZ was monitored with a Beckman LB-l gas analyzer and maintained at 3.5%. To eliminate the possibility of electromyographic and movement artifacts, the animals were paralyzed with intravenous gallamine triethiodide (40 mg). Each cat was mounted in a Kopf sterotaxic frame, and cortical recording sites were exposed as described above for the electrolyte injection. These recording sites were located in the following montage: electrode A- 1 cm posterior to the electrolyte injection site; electrode B-l cm anterior to the electrolyte injection site, electrode C- homotopic cortex contralateral to electrode B, and electrode D- homotopic cortex contralateral to electrode A. Electrocorticographic recordings were taken through silver ball cortical electrodes positioned at each recording site. Signals were appropriately amplified, filtered, and displayed on oscilloscopes for immediate analysis. In addition, records were stored on magnetic tape for later, off-line examination. At the time of recording, the initial ether anesthesia was no longer effective; therefore these records reflected the cerebral electrical activity of the awake animal. During the experiment, all incised tissues and pressure points were repeatedly infiltrated with lidocaine. Records were taken for 20 min; then each cat was deeply anesthetized with a bolus of intravenous pentobarbital(60 mg/kg). These animals were exsanguinated via transcardial saline perfusion. Six brains were removed and placed immediately in freshly prepared fixative for histological processing using the tungstate modification of the Colgi-Cox technique, as described by Ramon-Moliner (21). After impregnation, the brains were embedded in celloidin. Serial 150-pm coronal sections were taken through

208

REID ET AL.

the entire telencephalon. One brain from the experimental group was processed using Einarson’s method for Nissl substance (7). This tissue was fixed in 10% buffered neutral formaldehyde, and subsequently embedded in paraffin. Coronal sections were cut at 6 pm. The electrolyte injection site was clearly identified in each brain by the presence of the cannula track. Sections were examined with light microscopy, comparing the region of the injection site to the homotopic contralateral cortex and to other neocortical loci which were at least 1.5 cm lateral to either of the above sites. RESULTS Each of the five cats in the experimental group demonstrated electrocorticographic epileptic spike activity 6 weeks after FeCI, administration. These focal, paroxysmal discharges, characteristic of

FIG. 1. Electrocorticographic demonstration of paroxysmal epileptiform discharges. Prior to killing, cortical electrical activity was recovered with silver ball electrodes positioned in the referential montage illustrated above. Electrically positive excursions are represented by upward pen deflections in channels B, C, and D, and by downward pen deflections in channel A. Each cat previously injected with FeCla solution demonstrated spontaneous, epileptiform spikes, as shown in this record. This finding was not observed in control cats injected with saline. The asterisk marks the injection site.

GOLGI

ANALYSIS:

FERRIC

EPILEPTOGENIC

FOCI

209

active, chronic epileptogenic foci, were not observed in control animals. Figure 1 shows a typical referential electrocorticographic record from a cat in the experimental group. At the time of brain removal, the injection sites were examined for gross pathology. The region of the FeCl, injection presented as a l-mm-diameter, rust-colored cortical dimple. Except for differences in color, the FeCl, and NaCl injection sites appeared identical. The cortex surrounding each site was normal in consistency, whereas the injection sites themselves were somewhat firmer to palpation. The relatively benign gross appearance of the FeCl, focus belied the tissue destruction that was revealed on microscopic examination. Figure 2 shows a low-power photomicrograph of Golgi-stained tissue in the region of the FeCI,, injection. The striking findings here included a marked depopulation of Golgi-impregnated neurons and an astrocytic gliosis. Although astrocytosis was also prominent near NaCl injection sites, these regions contained normal complements of neuronal forms. Near the FeCl, injection site, however, Golgi-stained neurons were sparse. Figure 3 compares cellular morphology in the region of the FeCI, injection to that of the homotopic cortex, contralateral to the injection site. Again, depopulation of impregnated neurons near the injection was a prominent feature. Also apparent was a dramatic loss of dendritic spines from the cell within the epileptogenic cortex. This pyramidal neuron was characteristic of those near the FeCl, injection site and demonstrated mild nodular dendritic varicosites, as well as reduction of dendritic branching. Figure 4 compares dendritic morphology in the region of the FeCI, injection to that in a distant neocortical locus, at a position 2 cm lateral to the site of electrolyte administration. The dendrites in the distant cortex appeared to be normal and each bore the usual number of spines. In contrast, marked “string of beads” nodular deformities and dendrites devoid of spines were found in the region of the FeCl, injection. This is not to imply that all neurons within the ferric-induced epileptogenic focus demonstrated these changes. In fact, involved cells were intermixed with normal appearing neurons. Furthermore, this mosaic&m extended to occasional neurons in which only a few dendrites, or segments of dendrites, exhibited these alterations. In these latter cells, the remainder of the dendritic arborization appeared unaffected. Many of the histologic changes observed in the Golgi-stained specimens were also detectable through the use of the Nissl stain. Figure 5, taken from Nissl-stained tissue, compares the cortex immediately adjacent to the FeCl, injection site to the contralateral, homotopic cortex. Neuronal

210

REID

ET AL.

GOLGI

ANALYSIS:

FERRIC

EPILEITOGENIC

FOCI

depopulation was a prominent feature of the Nissl-stained focus. Also apparent were thickening of the pia-arachnoid, laden macrophages, and spongiosis.

211 epileptogenic hemosiderin-

DISCUSSION The high degree of localization of gross pathologic changes to the FeCl, injection site stands in sharp contrast to the characteristic appearance of alumina gel-induced epileptogenic foci. Typically, the latter focus presents as a relatively large cortical defect (lo), as in contrast to the l-mm cortical dimple we observed with FeCl,. Very interesting findings, suggestive of microvascular hemorrhage, were observed as prominent features of the alumina gel focus. Harris (10) reported that, in chronic foci, “brown pigment is prominent in macrophages for 2 micra to 300 micra distant from the (alumina gel-induced) granuloma border.” In nonepileptic saline controls, however, there were but “a few macrophages filled with brownish pigment” (10). This description conforms to the characteristic appearance of hemosiderin in Nissl-stained specimens i.e., as collections of brown pigment within macrophages. Positive identification of this material awaits histochemical analysis using the Prussian blue technique (26). The usual time course for the establishment of chronic epileptiform activity in the alumina model is 3 to 8 weeks, prior to which time the electroencephalogram is within normal limits (10, 17). This latency period is compatible with the following hypothesis regarding the development of alumina gel-induced chronic epileptogenic foci. Aluminum hydroxide may effect extravasation of blood elements by toxic disruption of the vascular endothelium. The subsequent degradation of extravasated red blood cells, involving the oxidation of hemoglobin iron from the ferrous to the ferric state, could result in relatively high local concentrations of ferric ions. These latter cations have been shown to produce focal epileptiform discharges both acutely and chronically (26, 27). Further support for this hypothesis is given by Harris’s investigation of the ultrastructure and histochemistry of alumina in cortex. He reported (11) that “no alumina crystals could be identified in any process resembling that originating from

FIG. 2. The ferric injection site. photomicrograph. Demonstrated depopulation, reduction of dendritic morphologic disruption induced by

The injection site is seen as the upper border of this here are large astrocytic forms, profound neuronal branching, and segments of decreased impregnation. The FeCI, is striking. x25.

212

REID ET AL.

GOLGI

ANALYSIS:

FERRIC

EPILEPTOGENIC

FOCI

213

neurons and no neurons were found in any of the animals to contain alumina crystals.” However, in the same animals, “blood vessel walls become encrusted with alumina in areas adjoining the granuloma” (11). In contrast, Willmore et al. (28) reported for the FeCI, focus that ferric ion deposits were seen concentrated within neurons. Therefore, the possibility exists that the delayed epileptogenic action of alumina is mediated through iron originally contained within erythrocytes. The cellular alterations we observed in the FeCl,-induced chronic epileptogenic focus are similar to those which were reported for alumina gel-induced foci. Westrum et al. (25) reported that the chronic alumina gel-induced epileptogenic focus is characterized by “neuronal depopulation and a relative gliosis with marked alterations of the dendritic arborizations including: (i) reduction in branching, (ii) bizarre angulations or distortions, (iii) varicose-like swellings (iv) unexplained segments of poor impregnation, and (v) absence or severe reduction in the number of dendritic spines.” The morphologic alterations induced by FeCl, are also very similar to those in human epileptic foci. Scheibel et al. (23,24) reported on the Golgi histopathology of the human hippocampal-dentate complex in temporal lobe epilepsy. A spectrum of cellular morphologic alterations was observed which was interpreted as representing an ongoing process of destruction. Among the changes noted were (i) a decreased number of dendritic spines, (ii) development of terminal nodules and knobs on the apical shafts of hippocampal pyramids, (ii) a “string of beads”-type of nodular dendritic deformity, and (iv) changes involving long segments of the dendritic shaft, including swelling, irregular nodulation, leaf-like excrescences, distortion, and shrinkage (23). Brown (2), in a combined optical microscopic and ultrastructural study of human temporal lobe foci, also noted neuronal depopulation, loss of dendritic spines, and gliosis. Spongiosis, as demonstrated by our Nissl-stained specimens, was reported (18) in human posttraumatic epileptic foci. The mosaicism of neuronal and dendritic involvement we observed in the FeCl,-induced epileptogenic focus was another similarity to the findings described for human epileptogenic foci (23).

FIG. 3. Comparison of cellular morphology in the FeCI,, injection site to that in the homotopic contralateral cortex. A-this field reveals normal neuronal distribution and morphology in homotopic cortex. located contralateral to the FeCIR injection site. x63. B-this photomicrograph of cortex in the region of FeCI,, administration shows a marked paucity of neurons. The pyramidal cell in this field is completely devoid of dendritic spines: it also demonstrates reduction of dendritic branching and mild nodular dendritic swellings. x63.

214

REID

ET AL.

GOLGI

ANALYSIS:

FERRIC

EPILEPTOGENIC

FOCI

21.5

The combination of loss of gemmules and appearance of nodular dendritic swellings deserves a special comment. These changes were probably first noted in Golgi-stained epileptic cortex by Demoor in 1898 (6). In certain spineless cortical neurons, such as stellate cells, dendritic varicosities may be regularly observed, unassociated with pathological processes (23). Peters (19), in an ultrastructural study, showed that these nodules contained enlarged cisternae within the endoplasmic reticulum. However, such findings in cortical pyramidal cells were almost certainly reflective of disease. Deafferentation has been shown to result in loss of spines and development of moniliform dendritic varicosities (1, 5). Ultrastructural examination of dendritic swellings in spinal cord, deafferented by hemisection, revealed numerous findings (1) of interest. The varicosities contained neurofilaments, neurotubules, and mitochondria-all normal components of dendrites. However, there were also increased numbers of vacuoles and areas of expanded endoplasmic reticulum. These changes suggested a challenge to the cell’s protein manufacturing machinery. Synaptic complexes with normal boutons were observed in the indentations of the varicosities. These features of deafferentation, very similar to those observed with the light microscope in epileptogenic cortex, support those theories which invoke “denervation supersensitivity” or loss of inhibitory inputs as etiologic factors promoting epileptogenesis (25). Neuronal depopulation near the FeCl,-induced epileptogenic focus was demonstrated in both the Golgi- and the Nissl-stained specimens. The Nissl stain confirmed that the loss of Golgi-impregnated neuronal forms was not due to a quirk in the impregnation process. Rather, fewer neurons were available near the ferric ion injection site for impregnation. Our findings of mild astocytic gliosis near NaCl injection sites in control cats, none of which developed epileptiform activity, support the conclusion of Hoover et al. (13) that gliosis probably is not the primary etiologic agent of focal epilepsy. The histopathologic alterations induced by subpial administration of FeCl, clearly have much in common with those seen in other chronic,

FIG. 4. Comparison of dendritic morphology in the FeCl, injection site to that in a distant cortical locus. A-this photomicrograph reveals the normal appearance of dendrites which have not been exposed to FeCI,. x400. B-these dendrites were observed near the site of FeCI, administration. Note that the magnification here is half that of A. Loss of dendritic spines and development of nodular dendritic varicosities are prominent features of chronic epileptogenic foci. x200.

216

REID

ET AL.

GOLGI

ANALYSIS:

FERRIC

EPILEPTOGENIC

FOCI

217

218

REID

ET AL.

epileptogenic foci. Changes associated with deafferentation, in combination with neuronal depopulation and astrocytic gliosis, may indeed be the hallmark of the chronic epileptogenic focus. REFERENCES 1. BERNSTEIN, J. J., M. R. WELLS, AND M. E. BERNSTEIN. 1975. Dendrites and neuroglia following hemisection of rat spinal cord: effects of puromycin. Adv. Neural. 12: 439-451. 2. BROWN, W. J. 1973. Structural substrates of seizure foci in the human temporal lobe. Pages 339-374in M. BRAZIER, Ed.,Epilepsy: Its Phenomena in Man. Academic Press, New York. 3. COLLINS, R. C. 1978. Use of cortical circuits during focal penicillin seizures: an autoradiographic study with IT deoxyglucose. Brain Res. 150: 487-501. 4. COLLINS, R. C. 1978. Kindling of neuroanatomic pathways during recurrent focal penicillin seizures. Brain Res. 150: 503-517. 5. COLONNIER, M. 1964. Experimental degeneration in the cerebral cortex. J. Anat. 98: 47-53. 6.

DEMOOR, J. 1898. La mecanisme et la signification de J’etat moniliforme des neurones. Ann.

Sot.

R. Sci. Med.

Nat.

Brux.

7: 205-250.

7. EINARSON, L. 1968. Einarson’s method for Nissl substance. Page 212in L. G. LUNA, Ed., Manual

of Histologic

Staining

Methods

of the Armed

Forces

Institute

of Pathology,

3rd ed. Mcgraw-Hill, New York. 8. FALCONER, M. A. 1970. The pathological substrate of temporal lobe epilepsy. Guy’s Hosp. Rep. 119: 47-60. 9. HAMADA, H., E. KOBAYASHI, T. MIHARA, T. ASAKURA, AND K. KITAMURA. 1976. A morphological study on vascular changes in the surgically resected tissues of temporal lobe epilepsy-with a special reference to the etiology. Folia Psychiatr. Neural. Jap. 30: 434-453.

10. HARRIS, A. B. 1972. Degeneration

in experimental

epileptic foci. Arch.

Neural.

26:

434-449.

11. HARRIS, A. B. 1973. Ultrastructure

and histochemistry ofaluminain

cortex. Exp. Neural.

38: 33-63. 12.

13. 14. IS. 16. 17.

HARRIS, A. B. 1975. Cortical neurologiia in experimental epilepsy. Exp. Neural. 49: 691-715. HOOVER, D. B., J. L. CULBERSON, AND C. R. CRAIG. 1977. Structural changes in cerebral cortex during cobalt-induced epilepsy in the rat. Neurosci. Lett. 4: 275-280. JENSEN, I., AND L. KLINKEN. 1976. Temporal lobe epilepsy and neuropathology. Acta Neural. Stand. 54: 391-414. 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. MATH~ESON, G. 1975. Pathology of temporal lobe foci. Adv. Neurof. 11: 163-185. MAYMAN, C. I., J. S. MANLAPAZ, H. T. BALLANTINE, JR., AND E. P. RICHARDSON, JR. 1965. A neuropathological study of experimental epileptogenic lesions in the cat. J. Neuropathol.

Exp.

Neural.

24: 502-511.

18. PAYAN, H., M. TOGA, M. BERARD-BADIER. 1970. The pathology of posttraumatic epilepsies. Epilepsia 11: 81-94.

GOLGI ANALYSIS:

FERRIC EPILEPTOGENIC

FOCI

219

19. PETERS, A. 1971. Stellate cells of the rat parietal c0rtex.l. Camp. Neural. 141: 345-373. 20. POTTER, J. M. 1978. The personal factor in the maturation of epileptogenic brain scars: review and hypothesis. J. Neurol. Neurosurg. Psychol. 41: 26.5-271. 21. RAMON-MOLIN!&, E. 1970. The Golgi-Cox technique. Pages 32-55 in W. J. H. NAUTA. AND S. 0. E. EBBESON, Eds., Contemporary Research Methods in Neuroanatomy. Springer-Verlag, New York. 22. RICHARDSON, E. P., AND P. R. DODGE. 1954. Epilespy in cerebrovascular disease. Epilepsia

(Amsterdam)

49-74.

23. SCHEIBEL, M. E., P. H. CRANDALL, AND A. B. SCHEIBEL. 1974. The hippocampal-dentate complex in temporal lobe epilepsy-a Golgi study. Epikpsia 15: 55-80. 24. SCHEIBEL, M. E., AND A. B. SCHEIBEL. 1973. Hippocampal pathology in temporal lobe epilepsy-a Golgi survey. Pages 311-337 in M. BRAZIER, Ed.. Epilepsy: Its Phenomena in Man. Academic Press, New York. 25. WESTRUM, L. E., L. E. WHITE, AND A. A. WARD, JR. 1964. Morphology of the experimental epileptic focus. J. Neurosurg. 21: 1033-1046. 26. 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. 152: 406-410. 27. 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. 28. 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. Neural. 4: 329-336.