Maturation of kainic acid seizure-brain damage syndrome in the rat. II. Histopathological sequelae

03064522:84

~rrtro~cience Vol. 13, No 4. pp 1073-1094, 1984 Printed in Great Rrltaln

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MATURATION OF KAINIC ACID SEIZURE-BRAIN DAMAGE SYNDROME IN THE RAT. II. HISTOPATHOLOGICAL SEQUELAE I_.NITECKA*,

E. TREMBLAY, G. CHARTON,

J. P. BOLJ~LLOT,

M. L. BERGER and Y. BEN-ARIt Laboratoire

de Physiologie

Nerveuse,

DCpartement de Neurophysiologie Gif-sur-Yvette, France

AppliquCe,

C.N.R.S..

9 I190

Abstract-The histopathological sequelae of parenteral administration of kainic acid were investigated in immature rats (3-35 days of age). The brains were fixed l-14 days after the administration of kainate and the damage evaluated by means of argyrophylic (Fink-Heimer. Gallyas or Nauta-Gygax) and Nissl stains. In animals of less than 18 days of age there was no sign of damage even after 1-2 h of severe tonico-clonic convulsions. Between 18 and 35 days after birth, there was a progressive increase in the severity of the damage, the adult pattern being reached at the latter age. As in adult animals, brain damage was most severe in structures which are part of the limbic system, i.e. the hippocampal formation, lateral septum, amygdaloid complex, claustrum, piriform cortex, etc. In addition to neuronal abnormalities. the following reactions were observed: hypertrophy and swelling of satellite oligodendroglia, proliferation of hypertrophic microglia, proliferation of astroglia and hypertrophy of endothelial cells in the capillary wall. The latter type of change, together with local coagulative necrosis. was almost exclusively restricted to the granular and molecular layers of the fascia dentata.

In the hippocampal formation we found a temporal gradient of vulnerability. The earliest and most consistent neuronal alterations were largely restricted to interneurons of the hilar region and to a lesser extent to non-pyramidal neurons of strata oriens and radiatum. The severe necrotic destruction of the pyramidal layer of CA3 is conspicuous at a later age (postnatal day 3S35) and with longer survival times. Our results suggest that: (1) the neurotoxin only induces brain damage once it also causes limbic motor seizures and its associated metabolic activations, notably in the amygdala; (2) the earliest pathological sequelae occur in interneurons of the hilar region and (3) sclerosis of the vulnerable region of the Ammon’s horn-the CA3 region-is only obtained once the dentate granules and their mossy fibres are fully operational, thereby reflecting the crucial role of this axonal connection in eliciting hippocampal damage.

Parenteral administration of kainic acid (KA), one of the most powerful of a group of excitotoxic agents,*’ produces a disseminated pattern of brain damage in limbic structures,“,‘y,66 which is remarkably reminiscent of that seen in human chronic epileptics.43,54.67 This is associated with a limbic seizure syndrome.*,” Electrographic and metabolic studies using the 2-deoxyglucose autoradiographic method have provided direct evidence that the hippocampus, the amygdala and other limbic structures occupy a central position in this syndrome.” On the basis of these and other observations, there is now general agreement that the KA syndrome constitutes a useful tool to study the relationship between paroxysmal discharge and brain damage, and to reproduce experimentally some of the features of human temporal lobe epilepsy.‘,*’ In the two companion papers, we describe the maturation of the clinical and metabolic effects of parenteral KA application as well as the development of specific membrane binding sites for the excitotoxin.“,‘” Here we report the maturation of histopathological sequelae. These results have been reported

briefly

elsewhere.7a.‘2

-___

*Present address: Department of Anatomy, Academy of Medicine, 80210 Gdansk, Poland. tTo whom all correspondence should be addressed. Ahhreciations: GABA. y-aminobutyrate; KA, kainic acid; P. postnatal day.

EXPERIMENTAL

PROCEDURES

The present study is based on the histologIca examination of the brains of 30 immature rats (3-35 days old) after the parenteral administration of KA. The convulsions were observed as described in the first report of this series.‘” After various survival periods the animals were deeply anaesthetized with equithesin (Jensen Salsbury, 4 ml/kg i.p.) and brains fixed by intracardial perfusion. The type and duration of KA-induced seizures, the survival times and respective histological procedures are indicated in Table I. Various staining procedures were used. For animals with short survival periods (14 days), brain sections were alternatively stained with Nissl (cresyl violet) and a silver stain; in most cases we used the Fink-Heimer methodZ8 with minor modifications. In four cases, alternate sections were subjected to Gallyas stain,3’ and in one additional case the Nauta-Gygax methodz4 was used to examine terminal and pre-terminal degeneration. For animals with longer survival period (> 4 days), the brains were embedded in paraffin and sections (10 pm) stained with cresyl violet. In one case (1630) the location of every argyrophylic neuron was depicted in a camera lucida drawing (Fig. 4A). Determination of the structures was made according to the atlas of Sherwood and Timiras for young rats.6’

RESULTS

As is evident from Table 1 (also see the preceding companion paper70), the clinical sequelae after parenteral KA were clearly age dependent. In animals of less than 18 days of age, KA produced tonico-clonic generalized type convulsions, whereas starting from postnatal day 18 (P18) repeated but isolated limbic 1073

1074

L. Nitecka c’t ul. Abbreviations

Ah AC AC Acop AI Am BST CA14

:L GM h H-A

nucleus of the amyqdala central nucleus of the amyqdala anterior commissure posterior cortical nucleus of the amyqdala lateral nucleus of the amyqdala medial nucleus of the amyqdala bed nucleus of the stria terminalis fields of the Ammon’s horn according to Lorente de No cingulate cortex claustrum caudate-putamen granular layer of the dentate gyrus lateral geniculate body medial geniculate body hilus of the dentate gyrus transitionnal amygdalohippocampal area

Im INS

massa intercalata insular cortex

Cing Cl CP

basolateral

used in jigurrs

IP LD LS m MD MEA %B(h)

iIR PI PM RE SG-Li SN so sr sub II, III

interpeduncular nucleus laterodorsal thaiamic nucleus lateral septal nucleus molecular layer medial dorsal thalamic nucleus medial entorhinal area medial geniculate body nucleus of the horizontal limb of the diagonal band pyramidal layer of the hippocampus piriform cortex polymorph layer premamillary nucleus reuniens thalamic nucleus supraqeniculate-limitans substantia nigra stratum oriens stratum radiatum subiculum cortical layers, II, III

Fig. 1. Photomicrographs to illustrate the types of neuronal changes observed in the brains of immature rats following parenteral administration of kainate. (A) Degenerated neurons (arrow-heads) of the dentate polymorph layer; staining of cytoplasm is increased and microvacuoli are present. Size and shape are not conspicuously altered. Also note the proliferation of astroglia (double arrow-heads); age 30 days, survival time 7 days (abbreviated P30 + 7 days), cresyl violet, paraffin section ( x 396). (B) Argyrophylic granular neurons in the temporal pole of the dentate gyrus. The argyrophilia is restricted to the nucleus; P35 + 2 days, Fink-Heimer stain ( x 396). (C) Necrotic (arrow-heads) and ghost (clear arrows) neurons in the CA3 hippocampal subfield. Reacting satellite oligodendrocytes are manifest (arrows), notice their hypertrophic nucleus and light swollen cytoplasm; P35 + 2 days, cresyl violet frozen section ( x 396). (D) Argyrophylic neuron of the dentate polymorph layer. The nucleus, cytoplasm and dendritic processes all show strong silver reaction; P30 + 2 days, Fink-Heimer stain ( x 396). (E) Shrunken neurons of the CA3 hippocampal field with increased staining of cytoplasm. Notice the hyperplasia of reactive microglia (curved arrows) and a few astroglia (double arrow-heads); P30 + 7 days; cresyl violet, paraffin section (x 158). (F) Argyrophylic pyramidal neurons of the CA1 hippocampal field. Note the strong argyrophilia of the nucleus, cytoplasm and dendritic processes; P35 + 2 days, Fink-Heimer stain (x 315). In this and following pictures similar symbols are sued to indicate the various types of neuronal and glial damage. Fig. 2. Photomicrographs to illustrate the types of glia and capillary abnormalities observed. (A) Coagulative necrosis in the granular layer of the fascia dentata; note the hypertrophy of the endothelial elements of the capillary (thick arrows); P30 + 14 days, cresyl violet paraffin section ( x 396). (B), (C) Hypertrophy of satellite oligodendrocytes in the hippocampal CA1 field. Notice the heavily stained nuclei and light cytoplasm of the oligodendroglia. The dark asterisks indicate the areas with conspicuous satellitosis. The rectangle which shows a pyramidal neuron surrounded by microglia is enlarged in (C); P24 + 1 day, cresyl violet, frozen section (B, x 396; C, x 792). (D) Strong satellitosis in the massa intercalata of the amygdala, note the swollen oligodendrocytes (arrows). The neurons do not show conspicuous abnormalities; P24 + 1 day, cresyl violet, frozen section (x 196). (E) Hypertrophy and hyperplasia of microglia in mediodorsal nucleus of thalamus. Note the presence of numerous microglia (curved arrow), neuronaophagy (arrow with double head), a degenerated neuron (arrow-head) and astroglia (double arrow-heads); P24 + 14 days, cresyl violet, paraffin section ( x 196). (F) hypertrophy and hyperplasia of microglia in lateral septum. Note the partial loss of neurons and the presence of a few necrotic cells (arrow-heads). Curved arrows indicate microglia and double arrow-heads astroglia; P24 + 14 days, cresyl violet, paraffin section ( x 196). Fig. 3. Photomicrographs to illustrate the extensive neuronophagy (arrows with double heads) and degenerated neurons (arrow-heads) in stratum radiatum (A) and stratum oriens (B) of the hippocampus Notice the accompanying proliferation of astroglia (double arrow-heads); the micrographs are taken from the same sections (A and B); P24 + 14 day cresyl violet, paraffin section (x 500). Fig. 6. Neuronal alterations in a 24 day old rat after parenteral KA and 1 day survival time. (A) Argyrophylic neurons in the pyramidal layer (p) of CAl; the dendritic arborization in stratum radiatum (sr) also reacts positively to the silver stain; the silver grains in stratum moleculare (m) suggest damage to the terminals of the temporo-ammonic system, Fink-Heimer stain (x 175). (B) and (C) Illustrate the grains (arrows) obtained in the granular layer (g) with a Nissl (B) and Fink-Heimer stain (C); (B) and (C). x 400. (D) “Spotted” argyrophylic cell in the polymorph layer; silver grains also delineate two of its branches (curved arrows). Fink-Heimer stain (x 350). Fig. 7. Hippocampal damage in a 35 day old rat after KA and 2 days survival time. (A)-(C) Neuronal alterations in the hilar region; note the strong argyrophylia in the poiymorph zone adjacent to CA3c. (B) is an enlargement of the boxed area in (A). (C) Depicts necrotic neurons (arrow-head), swollen oligodendrocytes (straight arrows) and “rod” cells (curved arrows). (D)(F) Similar arangement and symbols as in (A)-(C) to illustrate the changes in the polymorph layer at a more caudal level. (A), (B), (D), (F) Fink-Heimer stain; (C), (E), cresyl violet, frozen section; (A), (D), x 60; (B), (C), (E), (F), x 230.

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Fig. 8(A)-(C). Granular layer to illustrate the arrangement of silver grains as revealed with the GaIlya\ method (A) and the pre-terminal degeneration observed with the Nauta--Gygax stain (B. C). The route\ of the degenerated pre-terminals are indicated with dark arrows: the density of grams revealed in the neocortex with the Gallyas method from the same section is shown in (D) for comparison. (A. D. x 250. B, C. x 700). Fig. 9. Hippocampal damage in rats with long survival time after KA: comparison between 24 (A. D), 30 (B, E, F) and 35 (C, G) day old rats. Note the considerable increase in the extent of the damage with maturation. (A)-(C), CA3; (D)-(G), CAI. (F) illustrates with a bigger enlargement ( x 510) neurophagy in stratum oriens. Nissl stain, paraffin section (A, D, x 300; B. C, E, G, x 180). Fig. IO. Neuronal alterations in the boundary zones of frontoparietal cortex 7 days after KA in a 30 day old rat. Note the organization of cortical layers (B) with darkly stained clusters of neurons (arrow-heads). The boxed area is magnified in (A) to show the typical fusiform shape of neurons. Cresyl violet. paraffin section (A, x 700; B, x 8X 5)

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Brain damage after kainate in developing rats

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Table 1. Experimental schedule

(days) l&12

14-16 18

18 24-25

3a-35

KA-induced motor seizures Mean duration (h) Type Tonico-clonic l-2.5 l-2.5 1.5-2.0

Survival (days)

n

Nissl (frozen)

l-2 4-5 l&l4

6 8 2

6 8 2

Histological procedures Nissl FinkHeimer Gallyas (paraffin) 4

I

Tonico-clonic

1.25

2

2

2

Limbic

2.0 1.5

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14

1

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1

2.0 1.5

1 14

1 1

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1

2-3 3 2Z4

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2 3

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Limbic Limbic

Limbic

NautaGyggax

I I

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I 2 1 3

1

4 1 2

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2

2

1

Type and duration of seizures, survival periods and histological procedures used in this study. The number of animals and the age groups are also indicated. In a preliminary study, animals of 3-10 days of age were also treated with KA and the frozen sections stained with Nissl; brain damage was not found.

motor seizures were present. These evolved to a limbic status epilepticus at the end of the fourth week of life. No evidence of damage was found in 3-l 8 day old rats in spite of severe tonico-clonic generalized convulsion for up to 2 h (Table 1). The earliest developmental stage when neuronal and glial abnormalities were observed was P18; however, these changes were fully reversible, i.e. they were absent from sections obtained after longer (l-2 weeks) survival time. In contrast, in 3(r35 day old rats, brain lesions were severe and clearly evolved to necrosis associated with neuronal cell loss. We shall first characterize the type of cytopathological changes induced by i.p. KA in neurons and glia at various ages. Their regional distribution will be described in detail in the second part. Types of histological changes caused by kainic acid Neuronal changes. In Nissl-stained sections, the following abnormalities were observed. (a) Neurons with a dark cytoplasm but no other alteration; these neurons showed normal contours of the perikaryon and nucleus; the latter occupied usually its normal position. Occasionally, microvacuoli were observed (see Fig. lA, arrow-head). (b) Neurons with dark cytoplasm and nucleus; they were always shrunken and their dendritic processes were also revealed by the stain (see Fig. IE). (c) Neurons lacking Nissl substance; these were usually shrunken, the nuclei were irregular in shape and did not react with the stain (Fig. lC, arrowheads); the cytoplasm contained microvacuoli; often the initial segment of dendritic processes was stained (ibid.). (dj “Ghost”

neurons; they were characterized by an ill-defined contour and lacked any positively staining organelles (Fig. 1C): cells were often surrounded by microglia, suggesting an active process of neuronophagy (Fig. 3).

Silver stains revealed the following neuronal changes. (a) Neurons with strong argyrophilia (largely restricted to the nucleus); they were usually found in the granular layer of the fascia dentata (Fig. lB), in the amygdala, the septum and the mediodorsal nucleus of the thalamus. (b) Neurons with strong argyrophilia of nucleus, cytoplasm and dendritic processes. This “Golgi” type of stain was characteristically seen in the hippo-

campal formation and other cortical regions (Fig. 1D-F). (c) Neurons showing “spotted” type of dark impregnation over the cytoplasm (Fig. 6D); they were exclusively restricted to the hilar region and only observed in P18-24 cases. The nucleus as well as the dendritic processes were not stained. (d) Silver-stained sections also suggested preterminal and terminal degeneration. Thus, with the Gallyas and Nauta-Gygax methods, silver granuli were present in the granular layer of the fascia dentata and in the pyramidal layer (Fig. 8), probably resulting from the degeneration of interneurons (see below). Furthermore, the highly packed silver grains stained in the molecular layer of CA1 with the Fink-Heimer method (Fig. 6A) suggest degenerative processes in the terminal fields of the temporoammonic pathway. Since the parent cells in the entorhinal cortex did not show any pathological alteration, this may reflect a direct action of the procedure on the terminals. Glial changes. A large number of pathological conditions including anoxia, viral infections or intoxications are known to produce widespread changes in the morphology and distribution of glial elements. ‘s~‘7~‘8~64 Even though we have not made specific glial stains, it was clear from our sections that glial elements also underwent numerous changes after KA-induced seizures. These changes are character-

I (h-l

L. Nitecka et trl

Fred essentially by swelling of cytoplasm and increased staining of the nucleus (hypertrophy) as well :is proliferation of various types of glial cells. (a) After KA-induced seizures, hypertrophy and swelling of the satellite oligodendroglia was observed. Numerous nuclei of ohgodendrocytes stained more intensely than in control cases (Nissl stain). The oligodendroglia occupied large parts of the extraneuronal space (Fig. 2B-D). This satellitosis” was observed only in animals with short survival time (2448 h) and was present in most of the structures in which neuronal changes were manifest, including CAI and the hilar region of the hippocampal formation. amygdaloid nuclei, lateral septal nucleus, piriform and insular cortices, hypothalamic and medial thalamic nuclei. It is worth stressing, however, that occasionally these changes were also observed in structures in which there was little or no neuronal abnormality such as the massa intercalata of the amygdala (Fig. 2D). (b) Intense proliferation of hypertrophic microglia was consistently observed in brain areas showing atrophic neuronal changes and loss of neurons (Fig. 2E.F). The nuclei of reacting microglia were deeply strained with cresyl violet. Occasionally, we have also observed so-called “rod” cells (“Stabchenzellen”‘5) with clear-cut Nissi stain of nucleus and cytoplasmic processes (Fig. 7C). These were particularly numerous in animals with short survival times (< 2 days) in subhippocampal fields including the dentate polymorph and molecular layers and the strata oriens and radiatum. After longer survival times (l--2 weeks) neuronal cell loss was accompanied by microglia proliferation (Figs 2E, F and 3A, B). (c) With long survival times (> I week), proliferation of astroglia was observed in structures with neuronal damage. On several occasions the histological pictures suggested recent multiplication of the astroglia. Thus. in Figs l(A) and 3(A),(B) one can *The tcrmmology used in this report conforms to that used by Blackstadi4 and more recently Amaral.’ The hippocampal formation includes the Ammon’s horn (hippocampus proper) and the fascia dentata. The latter IS a trilaminar structure consisting of the molecular, granular and polymorph layers. The term “hilus” is used synonymously with the latter. The Ammon’s horn is composed of the CA1 region (regio superior), a rather ill-defined CA2 zone, and the CA3 region (regio infertor), which can be further subdivided into three zones (a, h. c: see Amaral’): CA3c corresponds to the pyramidal cell layer between the two blades of the fascia dentata. The definition of the border between the most medial part of the CA3 pyramidal layer and the adjacent hilar region (polymorph zone) is controversial. According to our present study, the medial most part of CA3c is, together with the polymorph zone, the most vulnerable m the brain to systemic KA and depicts early histopathological manifestations well before any sign of damage is noticeable in the rest of CA3 (including the bulk of CA3c). Therefore, we follow Blackstad14 and Amaral’ and shall not use the term ‘CA4”4’ for this region. but regard it as part of the hilar or polymorph zone (e.g. Fig. 7).

notice a pair and a group of closely packed astrogliii nuclei, respectively. (d) On several occasions. abnormalities in capillary walls including enlarged capillary vessels with hyper-trophic intensely stained endotheliai cells were noticed. These abnormalities developed only after long survival times (> 1 week) and were almost exclusively restricted to the fascia dentata, mainly in the immediate vicinity of the granular and molecular layers (see Fig. 2A). Typically, this was associated with complete neuronal cell loss; glial elements were not present in this necrosis.“,‘? Regional damage

distribution

qf kainic

acid-induced

bruin

Before P18, there was no evidence for brain damage subsequently to KA-induced seizures, with the exception of a few swollen oligodendrocytes in the lateral septal nucleus of the bed nucleus of the stria terminalis. Progressively starting from PI 8 damage was found after KA, but the adult pattern of damage was not reached before P3c-35. The location of every argyrophylic neuron found in serial sections from a 35 day old animal, stained with the Fink-Heimer method, is illustrated in Fig. 4(A) (case 1630, see Table 1). The neuronal damage was almost exclusively restricted to structures which are part of or closely related to the so-called “limbic” system, including the hippocampal subfields, the lateral septum, amygdaloid nuclei, medial and posterior thalamic region, the piriform and retrosplenial cortices and the claustrum. This pattern was bilateral; in contrast, scattered argyrophylic neurons were observed in the frontoparietal cortex unilaterally. The regional distribution of neuronal and gliai changes after short survival times in brains from a 24 and a 35 day old rats is depicted in Fig. 5. The general distribution of both the neuronal (dots) and glial (hatched areas) changes was similar, however more extensive in the older case. The regional distribution of the neuronal changes after longer survival times (14 days) in two cases (also P24 and P34) is illustrated in Fig. 4(B). The dark areas indicate marked neuronal cell loss associated with gliosis (except the dentate granular layer, see below); the hatched areas correspond to moderate damage i.e. partial neuronal loss, and the spotted areas indicate scattered neuronal loss. As a general rule, neuronal damage revealed by silver stains after short survival times proved to be irreversible, since after a longer survival time (7 weeks) neuronal cell loss was observed in the same regions, especially in CA3. In some regions, however, such as the CA1 field, the damage was partly reversible in the 24 but not in the 35 day old case. In the following sections, we describe neuronal and glial abnormalities after KA-induced seizures in the main brain structures at various ages, Hippocampal ,formation*. Postnatal day 18; short survival time (l--2 days). Moderate oedematous changes were apparent in the CA1 pyramidal layer;

Brain damage

after kainate

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Nissl stain revealed numerous satellite oligodendrocytes but no evident neuronal lesion. The main alteration was characterized by the presence of a few “spotted” type argyrophilic neurons in the dentate polymorph layer and scattered necrotic cells in the granular layer of the fascia dentata. The latter were located primarily in the part of the granular layer which is adjacent to the polymorph layer (i.e. the subgranular region); the neurons were conspicuously shrunken and contained one to five large inclusions which deeply stained with cresyl violet and also reacted with silver stains. This type of neuronal damage was only observed in 18 and in 24 day (Fig. 6B, C) old rats. Postnatal day 18; longer survival time (2 weeks). We found no evidence for neuronal or glial damage. Postnatal day 24; short survival time. The abnormalities were similarly distributed as in younger animals but were more widespread and more severe. The most consistent pathological alterations were found in the fascia dentata and the hilar region and included: (1) necrotic cells with dark inclusions in the dentate granular layer (Fig. 6B, C, thick arrows); (2) “spotted” type neurons (Fig. 6D) and neurons with argyrophilic nuclei in the polymorph zone including the region immediately adjacent to CA3c (e.g. Fig. 6C). In alternate Nissl-stained sections, neurons in this region were often swollen, with dark cytoplasm and microvacuoli (Fig. 6B), occasionally encapsulated by microglia. In the Ammon’s horn, neuronal damage was particularly conspicuous in non-pyramidal cells, especially in stratum oriens, but also in stratum radiatum. In the pyramidal layer of CAl, shrunken neurons with heavily stained cytoplasm were only scattered, although numerous swollen oligodendrocytes were present. The Fink-Heimer method revealed strong argyrophilic pyramidal cells, with intense impregnation of nucleus, cytoplasm and dendritic processes (Fig. 6A). As evident from longer survival times, most of the damage in CA1 was reversible (see below). Densely packed silver grains were observed in the CA1 molecular layer (Fig. 6A), reflecting damage of temporo ammonic terminals. There was no evidence for damage in the pyramidal layer of CA3. Postnatal day 24; long survival time (1-2 weeks). The distribution of severe neuronal damage confirmed and extended the observations after short survival times. Neuronal loss, necrotic and degenerating neurons associated with intense hyperplasia and

hypertrophy of microglia and multiplication of astroglial nuclei affected the hilar region and non pyramidal cells in strata oriens and radiatum. Damage was particularly severe in the polymorph zone where a large proportion of the neurons was clearly in a process of degeneration. Moderate neuronal loss accompanied by glial reactions was also found in the polymorph zone (Fig. 4B) throughout the strata oriens and radiatum of the Ammon’s horn and the dentate molecular layer (see below). In the granular layer, dark degenerated neurons were less frequent and largely restricted to the boundary between the infra- and suprapyramidal blade (Fig. 4B). This latter neuronal change was not accompanied by glial alterations. In contrast, there was little damage in the pyramidal layer. No swollen oligodendrocytes were seen in CAI, and microglial reaction was weakly expressed. Neurons with dark cytoplasm were rare (Fig. 9D), demonstrating that early changes in CA1 are largely reversible. However, in the CA3 pyramidal layer, damage was manifest for the first time at that age (whereas no damage had been found after short survival times, see above). Neuronal loss and microglia invasion were observed in CA3, particularly in the boundary zone between CA1 and CA3 and in part of CA3a (Figs 4B and 9A). Postnatal day 30-35; short survival time. The most obvious difference with younger animals (with comparable survival time) was that necrotic alterations were apparent in several hippocampal subregions (Figs 4A and 5). These changes were always associated with a massive glial reaction (Fig. 5) including swelling of oligodendroglia (satellitosis) and strong invasion of reacting microglia. The characteristic “rod” cells were frequently revealed by Nissl stain (Fig. 7C), indicating the severity of the damage. The particular vulnerability of neurons in the polymorph zone is illustrated in Fig. 7. The Gallyas and the Nauta-Gygax methods revealed massive terminal and pre-terminal degeneration of axons ending on dentate granular cells (Fig. BA-C) and moderate changes of afferents supplying CA1 and CA3 pyramidal neurons (not shown). The arrangement of the silver grains as seen with the Gallyas method, the course of degenerating pre-terminal fibres shown m the NautaaGygax stain (Fig. 8B, C) and the clear-cut evidence of irreversible damage to non-pyramidal and non-granular perikarya (aide supra) suggest the degeneration of local interneurons. Postnatal day 30-35; long survival time. Although -

Fig. 4. Schematic diagrams to illustrate the regional distribution of neuronal damage at P35 with two survival periods. (A) The location of every argyrophylic neuron seen in the serial sections (case 1630) is depicted in the camera lucida drawings. The argyrophylic neurons in the vicinity of the insular region (arrows) probably correspond to claustrum insulare. See abbreviations list. Fink-Heimer stain. (B) Neuronal damage after long survival times; the inserts depict selective regions obtained from a 24 day old rat for comparison. Strong neuronal necrosis (dark areas) correspond to regions of almost complete neuronal loss and associated gliosis; moderate damage (hatched areas) corresponds to partial neuronal loss, and weak damage (spotted areas) indicates scattered neuronal alterations. Based on cresyl violet-stained paraffin sections.

Brain damage after kainate in developing rats

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the regional distribution of neuronal damage within the hippocampal formation was reminiscent of that observed in 24 day old rats (Fig. 4B), major yuantitative differences were evident. Thus, patches of necrotic neurons were present in the whole CA3 field (not restricted to the CAl/CA3 boundary zone), and also distributed throughout the dentate granular layer, whereas in 24 day old rats it was only seen in the most medial part. The alterations which were observed after short survival times at P30-35 were evidently irreversible; massive neuronal cell loss was apparent in the same regions in which necrotic cells had been seen earlier. These observations are iliustrated in Fig. 9 for CA3 and CA 1. The massive loss of presumed interneurons in the polymorph layer of the fascia dentata was particularly obvious (see Fig. 4B). Hypertrophy of microglia, neuronophagy and multiplication of astroglial nuclei were manifest throughout the hippocampal formation. In contrast, satellitosis was not found; it is not clear whether this reflects the recovery of oiigodendrocytes or their disappearance. Using Nissl stains, we have frequently observed in the granular layer of the fascia dentata a particular type of lesion which was neither seen in younger animals nor in animals of the same age group with shorter survival periods. These “scars” were characterized by a complete loss of neuronal and glial elements. Usually in the vicinity of the scars, capillary vessels showed deeply stained hypertrophic endothelial cells (Figs 2A and 8C). The characteristics of these scars are strongly suggestive of local infarcts.” To summarize: (a) parenteral KA does not produce any damage to the hippocampal formation up to PI 8. (b) At P18, the changes are reversible and restricted to the polymorph layer of the fascia dentata. (c) At P24, interneurons in the polymorph layer of the fascia dentata, but also in strata oriens, radiatum and dentate molecular layer, depict the most conspicuous alterations. In the pyramidal layer, damage is rather scarce and fully reversible in CAI; irreversible damage was only observed in the CAl/CA3 boundary zone and in the transitional zone after long survival time. (d) At P30-35. necrosis and neuronal cell loss is more conspicuous in the pyramidal layer. In the dentate granular and polymorph layers, patches of severe necrotic changes are observed. Other bruin structures. Amygdaloid

complex.

Both neuronal and glial damage were clearly age dependent in this structure (Fig. 4). Thus, in 18-24 day old rats, the most conspicuous and earliest alteration was the swelling of oligodendroglia restricted to the massa intercalata, the posterior part of

Fig. 5. Schematic diagrams to illustrate short survival times in a 24 and a 35 day ones neuronal abnormalities. Symbols was made from serial sections which

the cortical nucleus and pat-t of the medial nucIcu\ adjacent to the strra terminahs. Neuronal darnagc was weak at that age. At P30-35, vve observed .tftc~ short survival time swelling of oligodendt-ocytcs .inc! necrotic neurons in the lateral and medial nucIcus. the lateral part of central nucleus and posterror parr of cortical nucleus (Figs 4A and 5). Neurons were lost after longer survival ttme and replaced by pro16 erating microglia, in particular in the posterror part of cortical nucleus. Damage in the basal nucleus was scarce and thus difficult to appreciate without per-forming cell counts. Septal region. As mentioned above, there was no alteration in this region up to P18, with the exception of a few swollen oligodendrocytes in the lateral septal nucleus. Beginning with P24, massive unequivocal neuronal and glial damage was already noticeable after short survival times; this included swelling of oligodendrocytes, proliferation of microglia, and necrotic neuronal changes. Silver-staining methods revealed numerous argyrophylic neurons and terminals (Fig. 4A), the latter suggesting direct damage to afferent terminals of the lateral septum. Neurons were lost from these regions after longer survival periods. in particular in the bed nucleus of the stria terminalis: its lateral part appeared completely devoid of neurons, leaving a gliotic scar (Fig. 2F). At no age did we find any indication of damage in the medial septum or in the diagonal band nuclei. Piriform

cortex.

In general, the damage was rather moderate (as compared to the previously described regions) and restricted to its caudal portion. This was characterized by satellitosis in layer II in 24 day old rats without evident neuronal damage; in 30-35 day old animals, the satellitosis was associated with neuronal damage (i.e. necrosis and argyrophylia). Interestingly, this satellitosis was observed also after I week survival time. After longer survival time (I week or more), we observed in addition massive neuronal cell loss and microglia proliferation in the third cortical layer (Figs 4 and 5). Insular c0rte.u. Damage was first manifest in 24 day old rats and was restricted to the posterior part of the insular cortex. The early changes were characterized by an intense swelling of oligodendrocytes in layer I1 and neuronal argyrophylia in layers II and IV; after long survival time neurons were lost (Figs 4 and 5).

the regional distribution of neuronal and glial changes, following old case. The hatched areas indicate glial changes and the spotted (arrows) and abbreviations are identical to Fig. 4. The diagram were alternatively stained according to Nissl or Fink-Heimer.

10x0

Brain damage after kainate in developing rats Claustrum.

Swelling of oligodendroglia as well as neuronal argyrophylia, necrosis and cell loss were conspicuous starting from P24 (Figs 4 and 5). The arrows in these figures indicate the argyrophylic neurons adjacent to the capsula externa which likely correspond to claustrum insulare of higher mammals. Hypothalamic region.

Already in 18 day old rats, extensive swelling of oligodendrocytes, argyrophilic ceils and neuronal necrosis were noted in the premammillary region after a short survival time. After a longer survival time, this was followed by neuronal cell loss and proliferation of microglia (Figs 4B and 5). Starting from P24, weak to moderate neuronal and glial changes were also noted in the ventromedial nucleus and lateral hypothalamic area. In contrast, we have not found any indication of pathological changes in the arcuate nucleus. Thalamic nuclei.

The first evident alterations were seen in medial dorsal and reuniens nuclei by P24. They started with moderate swelling of oligodendroglia, went on to degeneration of neurons, and subsequently to their disappearance. Argyrophylic neurons were present in both nuclei. By P30-35, extensive argyrophilia and damage of neurons was additionally observed in the dorsal lateral nucleus and in the suprageniculate limitans zone. All these thalamic nuclei are known to have axonal connections either with the hippocampal formation or with amygdaloid nuclei (e.g. Ben-Ari et aLx and Refs therein). Neocortex.

Starting from P24, argyrophilic cells were seen adjacent to the external capsula in the frontoparietal cortex. They could belong to layer VI or more likely to the insular claustrum, which is ill-defined in the rat (vi& supra). Usually, other neurons in layers II-VI were stained rather sporadically by Nissl and silver methods. In case 1630 (P35 + 2 days), many cortical cells seemed to be affected; they were swollen and contained microvacuoli, but showed no argyrophilia. In the same case, cells of layer II of the retrosplenial cortex were deeply stained with cresyl violet and shrunken, but also not argyrophilic. Moreover, in P30-35 cases, some “patchy” breakdown of neurons in the retrosplenial cortex was seen after longer survival time. In case 1639 (injection at P30, survival time 1 week), the frontoparietal cortex was severely damaged in only one hemisphere; neurons of all layers were deeply stained with cresyl violet, shrunken, and their dendritic processes also sometimes stained. In addition, in the boundary zone separating areas supplied by the medial and anterior cerebral arteries,” neurons appeared extremely dislocated, which led to profound alteration in their

shape (see Fig. 10). This type of displacement heen described in human pathology.‘7,‘9”

has

DISCUSSION

In adult rats, parenteral kainate induces severe motor seizures of the “limbic” type, associated with paroxysmal activity and a dramatic rise in glucose consumption in limbic structures, with subsequent histopathological alterations essentially in the same In animals of less than 3 weeks of brain regions. 11~59.6h age, there is a remarkable dissociation between the acute reactions to parenteral KA on one side and the consequent neurodegenerative outcome. Although KA administered to immature rats does provoke severe tonico-clonic convulsions and electrographic seizures associated with metabolic activation in the hippocampal formation,” it does not induce the slightest brain damage, even at doses which are subsequently lethal. This early tolerance of the central nervous system to the neurotoxic action of KA has also been observed by others who injected the toxin directly into the striatum” or the hippocampus (C. Kiihler, personal communication). Interestingly. intrastriatal injections of other “glutamate-like” excitotoxins such as ibotenic acid produce damage well before the third week of age.52 This, in keeping with other lines of evidence, further stresses the unique features of the neurotoxicity of KA, which obviously depends on operating afferent inputs.9.““.4” Indeed. perhaps the most intriguing message from the present series of studies is that KA produces damage in the hippocampus and other limbic structures starting from the end of the third week after birth, only at a time when the neurotoxin also causes limbic motor seizures and its associated metabolic changes involving the whole limbic circuitry including the amygdala and related structures.*.” A second message which stems from our study concerns the relationship between the pathological sequelae in hippocampal-presumably using y-aminobutyrate (GABA) as transmitter-interneurons and pyramidal neurons of the vulnerable region of the Ammon’s horn (i.e. CA3). Indeed, damage to the former population, notably in the hilar region, appears at an earlier developmental stage and with shorter survival times than in the latter. This temporal gradient not only suggests a causal relationship between the removal of inhibition and neuronal cell loss in CA3 (see below), but may also provide a mean to develop a hippocampal preparation essentially deprived of its main inhibitory constituents. Maturation of gliotoxic properties of’kainate

With the notable exception of the recent report 01 Sperk and coworkers, 66 but also see Ref. 45, earlier studies of the pathological effects of parenteral administration of KA in adult animals have not sufficiently underlined the importance of nonneuronal damage produced by the toxin. In the study

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of Sperk et al. (ibid.), parenteral KA produced an “incomplete tissue necrosis” associated with loss of oligodendrocytes, astroglial scar formation, small perivenous haemorrhages and extensive vascular sprouting. These changes were particularly conspicuous in the piriform cortex, amygdala and various forebrain structures. They were interpreted as anoxic-ischemic damage resulting from the compression of drainage vessels against the skull by local cytotoxic oedema, notably in the ventral forebrain. The present study shows that starting from P18, KA produces severe non-neuronal abnormalities including hypertrophy and swelling of satellite oligodendrocytes and hypertrophy of endothelial elements, in addition to the well-known proliferation of astrocytes and microglial hyperplasia and hypertrophy. The gliotoxic action of KA was primarily directed towards the satellite ohgodendrocytes which showed swelling and hypertrophy after 1 -2 days survival periods. Similar changes are produced by a variety of toxins such as morphine and related drugs, phosphones, and triethyl-tin.64 Presumably. the pathogenic sequence of events underlying these sequelae includes an overreactivity of vulnerable neurons, leading to alterations in the concentrations of ions and metabolites in the extracellular milieu and to local oedematous changes. However, although by and large the distribution of glial and neuronal abnormalities is similar, a number of differences have been consistently observed. In some brain regions (e.g. CAl, lateral septum and thalamic nuclei), gliotoxicity precedes neuronal cell loss; in others (e.g. the massa intercalata), it is not associated with neuronal abnormalities. Whether this reflects a preferential vulnerability of glial elements to kainate in the latter regions remains to be further investigated. In contrast to the widespread distribution of glial abnormalities, infarct of tissue and hypertrophy of endothelial cells were exclusively restricted to the molecular and granular layers of the fascia dentata starting from P3G-35. In constrast, in adult rats the dentate granules are relatively resistant to paror enteral,“.‘“.” local,“” intracerebroventricular”’ intra-amygdaloid8 injections of KA. Parts of the hilar region’7,‘2 contain boundary zones of blood supply and are, as such, particularly vulnerable to brief ischemic episodes.‘* Since the vessels supplying these zones are terminal vessels, it is possible that in 3@35 day old rats the blood circulation in these regions is less efficient than in adult ones to compensate for local vascular changes by means of collateral and anastomotic branches. Maturation of neuronal susceptibility to kainic acid in the hippocampal formation: early changes Because of its distinct laminar considerable postnatal development its exquisite vulnerability to KA animals and to epileptic conditions warranted to examine temporal

organisation, its (see below), and in experimental in humans, it was gradients in the

maturation of susceptibility to KA of neuronal populations in the hippocampal formation. We noticed the earliest pathological signs at PI8 in the hilar zone (spotted large argyrophilic neurons). At P24, a large proportion of neurons in this region showed severe necrotic changes. Our anatomical methods preclude precise identification of neuronal types involved. However, several observations suggest that GABAergic interneurons constitute a high proportion of this population. Thus, these neurons are located in the hilus, immediately below or within the granular layer, where cell types thought to mediate GABinhibition are located.‘” “.v Aergic recurrent Immunocytochemical studies have shown that a large proportion of hilar cells contain glutamate decarboxylase, the GABA-synthetising enzyme. Outside the fascia dentata, neurons in the strata radiatum and oriens-in particular in the vicinity of the alveus are also particularly vulnerable to KA, again in keeping with the glutamate decarboxylase distribution in this region (ibid.). Observations on adult animals suggest a particular vulnerability of GABAergic neurons to KA and other experimentally induced epileptogenic conditions. Thus, parenteral KA reduces recurrent inhibition in dentate granules” and causes a significant drop of glutamate decarboxylase enzyme activity in the hippocampus’5*66 and a virtually complete destruction of hilar neurons. ” A significant reduction of inhibitory mechanisms is noted in hippocampal slices prepared from KA-treated animals4 Also, sustained electrical stimulation of the perforant path produces acute damage in this region and a reduction of recurrent inhibition.” Locally applied KA destroys GABAergic neurons also in several other and there is a loss of GABAergic structures2’.“.” neurons in epileptic foci.49 However, it remains to be directly demonstrated by immunocytochemistry, that glutamate decarboxylase-containing neurons in the hilus are preferentially destroyed after parenteral KA, in comparison to other non-GABAergic neurons in the same zone (see note added in proof). If so. it is possible that this vulnerability is related to the high metabolic levels of GABAergic interneurons” or (and) to the release by the mossy fibres of a toxic factor (see below). Whatever the mechanism for this vulnerability may be, it is likely that the lability of inhibitory GABAergic mechanisms’ as well as the early destruction of interneurons will contribute to disinhibit the granules of the fascia dentata. and thereby facilitate the propagation of paroxysmal discharge along the mossy fibres; on a long term. this may have deleterious consequences for their CA3 target zone (also see Sloviter” and Ben-Ari’). Maturation qf neuronal susceptibility to kainic acid in the hippocampal fbrmation: lute changes The pyramidal layer of CA3 which in adult rats is particularly vulnerable to local and distant actions of KA (uide supra), is resistant to i.p. KA in young rats,

Brain damage after kainate in developing rats up to a relatively late developmental stage. With short survival times, there was no sign of damage at P24; the full extent of irreversible damage in this region was only reached at P30-35. To consider the significance of these observations, it is essential to appreciate the considerable postnatal development of the hippocampal formation. In contrast to the Ammon’s horn, which is formed prenatally, 8085% of the dentate granules originate in the postnatal period, and 10% are yet to be formed after P18.‘,’ This gradient of maturation is reflected by the development of dendritic arborization and synaptogenesis.20*23~30~42~60 Thus, less than 5% of the total synapses of the dentate molecular layer are formed at P4, and there is a lOO-fold increase in synaptic development between P4 and the adult pattern which is reached around P25.23,42Due to this delayed maturation of the dentate gyrus, the immature hippocampal formation lacks its normal inputs-notably the major cortical afferent originating from the entorhinal cortex.69 Furthermore, the characteristic “thorny excrescences” of the mossy fibres are identifiable for the first time around Pl l-14, and the mature image is reached around P18-213 (also see Bliss er ~1.‘~and Stirling and Bliss6*). Stimulation of the granules do not produce orthodromic response in CA3 before P7, and a mature response is elicited around P15.‘” Therefore, parenteral KA will induce in CA3 only once the entorhinal damage cortex-dentate-mossy fibre system is fully operational. On the mechanism qf kainic acid-induced damage to hippocampal neurons

Since anti-convulsant treatment prevents the damage produced in the CA3 region by intra-amygdaloid KA and not at the site of injection, it has been suggested that this damage is causally related to epileptic discharge per se.” Several observations have confirmed and extended this hypothesis. (1) There is an excellent correlation between the severity of local paroxysmal discharge in CA3 and subsequent damage.’ (2) After intracerebral injections of KA, the pattern of “distant” damage is in some agreement with known neuronal connections.53.59 (3) Destruction of the perforant path’ or the mossy fibres4’j prevents the damage. (4) Electrical stimulation of the perforant path produces a somewhat comparable pathological sequelae.62 More recently, we have measured continuously and quantitatively the p02, pC0, and local blood flow directly in the CA3 region of unanaesthetized rats after parenteral KA, and shown that the typical damage is obtained without any hypoxia or hypercapnia in the vulnerable region.‘@ This provides compelling evidence that a factor associated with the severe activation of this circuitry is causing the damage in the CA3 region. The nature of this factor remains to be established (see Ben-Ari6); experiments such as these recently made by Morris and CO-

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workersa showing that there is a striking rise in the intracellularly-recorded calcium concentration in CA3 pyramidal cells during electrically-induced seizures, are in keeping with the hypothesis that an excessive rise in free calcium in the cell may lead to pathological alterations (also see Refs 26 and 33) e.g. by activation of proteolytic enzymes25 or by interference with Ca*+-proton exchange in mitochondria.j5 It is interesting to note that calciumbinding proteins are virtually absent in CA3 pyramidal cells but enriched in the granule cells and presumably their mossy fibres.36 Additional mechanisms including the release of yet unidentified kainate-like endotoxin or other factors highly concentrated in the mossy fibres such as zinc29,‘4.”could also explain the crucial role of these fibres in inducing damage in the CA3 region. The striking parallelism between vulnerable regions to KA in the hippocampus and the distribution of high affinity IL4 binding sites is discussed in the following article.” Conclusions

The present study further stresses the preferential involvement of limbic structures in the seizure-brain damage actions of KA and demonstrate that these effects are only obtained once the limbic circuitry is mature. In keeping with other recent observations made on adult animals6* our results provide additional evidence that epileptic discharge per se propagating along the mossy fibres produce the damage in the vulnerable pyramidal neurons of CA3. In spite of the paroxysmal discharge produced in CA3 by KA and its metabolic correlates” the toxin does not produce damage before P24. Furthermore, destruction of the presumed GABAergic interneurons in the hilus will only produce pathological sequelae in the Ammon’s horn once the perforant path and mossy fibre systems are operational. It should however be born in mind that this type of mechanism does not necessarily apply to all the pathological sequelae of parenteral KA including those seen in other parts of the hippocampal formation. Thus, hypoxia, hypercapnia and other mechanisms such as the presumably vascular deleterious effects which appear to play a role in the fascia dentata, probably underline some of the pathological sequelae of KA. For example. the damage which is characteristically seen in CA1 (also see Karnushina et aL3*) and over all layers of the piriform and other cortices is probably due to the latter type of deleterious conditions. A discussion on the mechanisms underlying the damage produced in other limbic structures by i.p. KA must await studies similar to these which have been conducted to reveal the crucial role of the mossy fibres in inducing the damage in CA3. With regard to the clinical aspects,27,32.5’ it has been already stressed” that once a scar is created in the vulnerable limbic structures, repeated seizures and their organization as status epilepticus will further induce the progressive appearance of “epileptic”

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brain damage, in keeping with the view that the pathological abnormalities constitute a progressive on-going process.‘” Interestingly, if a second dose of KA is administered to adult rats 30 days after the first one, there is a considerable reduction in the threshold of the seizure-brain damage syndrome; in contrast, if the first

injection

has

been

made

at a still immature

These observations further stress the relevance of the actions of kainic acid to temporal lobe epilepsy and may help to stage,

there

are

no

such

sequelae.69”

e’~ul.

clarify the relationship between damage in limbic structures

the seizures

and t ho

Acknowledgemem-We are highly indebted to Dr. 0. Robain for his remarks and to G. Ghilini for technical assistance. Financial support was provided by M.R.I.. INSERM (C.R.E. No. 1953), and “Fondation pour la Recherche Medicale”. The visit of M. L. Berger was supported by the Austrian Science Research Foundation (project EOOO2) and La Fondation pm la Recherche Medicalc

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I. Nitecka

(at rrl

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Note added in proof In a recent study (Nitecka ef al., in preparation) using an antibody directed against observed a rapid destruction of GABA-containing neurons in the hilar zone after parenteral KA.

GABA.

we have