the effect of methionine-sulphoximine on nerve and glia cells in vitro

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Zeitschrift ffir Zellforschung 85, 158--164 (1968) The Effect of Methionine-Sulphoximine on Nerve and Glia Cells in vitro Z. LODIN, J. NOV~K, E. HOLE(~KOVX and J. HARTMAH Institute of Physiology, Czechoslovak Academy of Sciences, Prague, and Laboratory for the Study of Vital Processes by Cinematography, Institute of Experimental Botany, Brno, Czechoslovakia Received August 10, 1967 Summary. The effect of methionine-sulphoximine (MSI) on the behaviour of the nerve and glia elements from brain and cerebellum of rat and chick in tissue culture was studied. It was observed that a concentration of MS1-3 M inhibits the growth of early brain explants. Concentrations of 10-4 and 10-5 M did not show this effect. Time lapse cinematographic recordings evaluating continually the behaviour of living glial and neuronal elements in tissue culture showed that MSI in a concentration of 10 -3 caused, very shortly after administration, a conspicuous oedema of cellular elements. The highest degree of swelling occurred in the perikarya and processes of astroeytes. Cell bodies of the oligodendroglia swelled only in the beginningand later shrank again considerably. This shrinkage was accompanied by a retraction of the glial processes, their fragmentation, and destruction. The perikaryon of the oligoden- droglia contracted discontinually. Similarly the volumetric increase of astrocytes was accom- panied by rhythmic contractions. In neuronal as well as oligodendroglial perikarya move- ments of mitoehondria and other cytoplasmic particles first accelerated, later slowed down. Synchronously with cytoplasmic swelling of neurons the formation of many vacuoles is observed. From a comparison of these changes, their time course, and the degree of damage in various cell types it may be concluded that MSI attacks in the first place glial elements, among them most intensely the oligodendroglia. Methionine-sulphoximine (MSI) is a compound with a characteristic neurotoxic effect. Its administration alters conspicuously the bchaviour of animals (LoDIH and KOLOU~EK, 1956; PROLLER and KELLAWAu 1962) their higher nervous activity (LoDIH, 1958), and electrophysiological manifestations (GASTAUT, TOGA and NAQUET, 1959; LOD~N, 1958), and causes a spontaneous spreading depression (JoHNsoN, GOLDRIHr and O'LEARY, 1965). Neuropathological lesions which arise after administration of MSI in the brain are very complex and do not allow identification of the nervous structures primarily affected by this substance. The effect of MSI per se on brain metabolism is accompanied by the effect of seizures elicited by it which are associated with a temporary anoxia of the brain. Therefore, an attempt was made to investigate this problem in tissue culture. Materials and Methods The study was performed on explants of chick and rabbit brain and cerebellum. The animals were 1 to 8 days old and the explants were cultured for 6--14 days in suspended drops in a medium consisting of equal parts of chick plasma and 25 to 50% of embryonic extract (EE). In preliminary experiments carried out in cultures of brain and cerebellum of two-day- old rats the appropriate concentration of MSI was determined that allowed the migration of a sufficient number of cells from the explant but exhibited already a toxic effect when compared with the control explants. The concentration determined was 10-3 M of MSI present in the medium in a ratio of 1:2 (1 part MSIq- 1 part plasmaq- 1 part EE). The reaction of

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Page 1: The effect of methionine-sulphoximine on nerve and glia cells in vitro

Zeitschrift ffir Zellforschung 85, 158--164 (1968)

The Effect of Methionine-Sulphoximine on Nerve and Glia Cells in vitro

Z. LODIN, J. NOV~K, E. HOLE(~KOVX and J. HARTMAH

Institute of Physiology, Czechoslovak Academy of Sciences, Prague, and Laboratory for the Study of Vital Processes by Cinematography, Institute of Experimental Botany,

Brno, Czechoslovakia

Received August 10, 1967

Summary. The effect of methionine-sulphoximine (MSI) on the behaviour of the nerve and glia elements from brain and cerebellum of rat and chick in tissue culture was studied. It was observed that a concentration of MS1-3 M inhibits the growth of early brain explants. Concentrations of 10 -4 and 10 -5 M did not show this effect. Time lapse cinematographic recordings evaluating continually the behaviour of living glial and neuronal elements in tissue culture showed that MSI in a concentration of 10 -3 caused, very shortly after administration, a conspicuous oedema of cellular elements. The highest degree of swelling occurred in the perikarya and processes of astroeytes. Cell bodies of the oligodendroglia swelled only in the beginning and later shrank again considerably. This shrinkage was accompanied by a retraction of the glial processes, their fragmentation, and destruction. The perikaryon of the oligoden- droglia contracted discontinually. Similarly the volumetric increase of astrocytes was accom- panied by rhythmic contractions. In neuronal as well as oligodendroglial perikarya move- ments of mitoehondria and other cytoplasmic particles first accelerated, later slowed down. Synchronously with cytoplasmic swelling of neurons the formation of many vacuoles is observed. From a comparison of these changes, their time course, and the degree of damage in various cell types it may be concluded that MSI attacks in the first place glial elements, among them most intensely the oligodendroglia.

Methionine-sulphoximine (MSI) is a compound with a characteristic neurotoxic effect. I t s admin is t ra t ion alters conspicuously the bchaviour of animals (LoDIH and KOLOU~EK, 1956; PROLLER and KELLAWAu 1962) their higher nervous ac t iv i ty (LoDIH, 1958), and electrophysiological manifestat ions (GASTAUT, TOGA and NAQUET, 1959; LOD~N, 1958), and causes a spontaneous spreading depression (JoHNsoN, GOLDRIHr and O'LEARY, 1965). Neuropathological lesions which arise after adminis t ra t ion of MSI in the bra in are very complex and do not allow identif icat ion of the nervous structures pr imari ly affected by this substance. The effect of MSI per se on bra in metabol ism is accompanied by the effect of seizures elicited by it which are associated with a temporary anoxia of the brain. Therefore, an a t t emp t was made to invest igate this problem in tissue culture.

Materials and Methods The study was performed on explants of chick and rabbit brain and cerebellum. The

animals were 1 to 8 days old and the explants were cultured for 6--14 days in suspended drops in a medium consisting of equal parts of chick plasma and 25 to 50% of embryonic extract (EE). In preliminary experiments carried out in cultures of brain and cerebellum of two-day- old rats the appropriate concentration of MSI was determined that allowed the migration of a sufficient number of cells from the explant but exhibited already a toxic effect when compared with the control explants. The concentration determined was 10 -3 M of MSI present in the medium in a ratio of 1:2 (1 part MSIq- 1 part plasmaq- 1 part EE). The reaction of

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Z. LODII~ et al. : Methionine-Sulphoximine Effects on Neural Tissue 159

the explant to MSI was determined by time lapse cinematography. First a long-term (4 to 5 hours} registration of the culture in the perfusion chamber without MSI was performed, then MSI was added and the reaction of the explant was recorded at shor$ intervals {film 35 ram, rate 16/rain and 32/rain).

In another experimental series the changes in shape and volume of the observed elements were studied with high resolution phase contrast optics. The various cell elements were photographed at approximately 1 min intervals before and after introduction of MSI into the perfusion chamber using phase contrast (Leitz) with an automatic camera Orthomat. The areas of the cells in these microphotographic recordings were evaluated by planimeter.

Results I n cultures of brain and cerebellum of both newborn rats and chicks the

addit ion of MSI to the medium ehcited characteristic cellular changes affecting part ieulary the glial elements. The growth zone in the vicinity of the central f ragment contained two clearly distin- giushable types of glila cells, oligodendro- gha plus microglia, and astrocytes. The small pearshaped oligodendrogha with two thin processes and intensely refrac- tfle particles inside the cell body ap- peared most ly in clusters. Their proces- ses were in perpetual mot ion; in addi- t ion some elements pulsated. The pro- cesses of astrocytes, with undulat ing membranes, were also considerably ac- tive. I n the processes and cell bodies of astrocytcs pinocytosis was repeatedly observed. Isolated or clustered macro- phages showed a t tachement of their pedicles to the processes of astrocytes.

A few minutes after administrat ion of the 10 -2 M solution of MSI into the chamber an intensive reaction set in in both types of glial elements. The move-

~176 t .. "I ' '~

I ./:. / #

1 3 5 8 10 15 25 time/rnin

Fig. l. Changes in size of individual cellular elements from rat brain explant due to the introduction of MSI in a concentration of 10 -a M. �9 . . . . perikaryon of neuron, �9 . . . . . astrocyte, ~ 1 7 6 oligodendroglia, ~ ....... �9 changes in the body of oligodendroglia after

administration of 0.9 % NaC1. Greatest increment in astrocyte

ment of the mitochondria markedly accelerated in the oligodendroglia cells which at the same time increased much in size. The rise in cellular volume was so con- spicuous and disproport ionate tha t in m a n y cells a kind of second, ra ther hydra ted pole developed containing only very few mitochondria. After this pronounced swelling the processes of the ohgodendroglia gradually retracted and often broke off due to the violent movement . The bodies and processes of astrocytes also swelled very quickly after the administrat ion of MSI. The processes spread out, their densi ty and their mobil i ty decreased. At first, the movement of the peripheral parts of the processes ceased and only an irregular movement of the main t runks remained perceptible. The body of astrocytes became more trans- parent, the difference between plasma and nucleus disappeared. The hydra t ion kept increasing for several hours (Fig. 1).

Fig. 1 also demonstrates the characteristic curve of volumetric changes of the ohgodendroglia. M t e r a brief period of hydra t ion the cellular size decreased due

Page 3: The effect of methionine-sulphoximine on nerve and glia cells in vitro

:Fig, 2a and b. Group of oligodendroglia cells, a) Shortly after administration of MSI 10 -~ into perfusion chamber, b) 5 rain after administratioa

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Z. LODIN et al. : Methionine-Sulphoximine Effects on Neural Tissue 161

to contraction and retraction of processes which fragmentated and fell off. The cell body contracted stepwise and not continually. The question whether the ob- served phenomenon was a manifestation of cellular degeneration or of an at least partial transformation to a cell of the macrophage type still remaines unsettled.

The volume of astrocytes did not rise continually either (Figs. 3 and 4), the surface of the cell showed periodic contractions in the course of hydration. The curve seems to suggest that the increment in intracellular fluid and the con- comitant volumetric increase led to a contraction of the cell which was, however, not strong enough to expel the fluid. Then a further volumetric increase followed.

In certain, particularly older cultures it was possible to distinguish cell bodies of neurons, by their considerable size, large nuclei and processes. The recordings showed a movement of intracellular particles, especially mitochondria. In these cells too the movement of particles became considerably accelerated within 10 minutes after administration of MSI, first in all directions, later towards the juxtanuclear region. Due to the agglomeration of mitochondria small vacuoles developed which enlarged, disappeared and recombined. After a certain period (mostly 20--40 rain) the movement of the mitochondria slowed down. In spite of the cell body's gradual increase in size during this period, even the longest recordings (5--6 hours) did not show signs of distortion in shape of these pre- sumed neuronal elements. The volumetric changes of these cells are also recorded in Fig. 1.

A certain swelling and accelerated movement of particles after administration of MSI also occurred in microglial cells.

I t may be concluded from these results tha t MSI in the concentration used has a toxic influence on all types of cells present in our explants, but tha t glial elements were most affected. The reaction of the nerve cells to the toxic effect of MSI occurred later, proceeded more slowly and was much slighter.

Discussion

As stated before, the interpretation of changes induced by the administration of MSI in vivo presents certain difficulties. HICKS and CoY (1958) claimed to have found non-specific changes fundamentally similar to those due to asphyxia or insulin hypoglycemia, or caused by intoxication with inhibitors of cytochrome oxydase. FISCEER and LODI~ (1961) described in the rabbit a picture similar to that observed in other experimental models of epilepsy and to human epilepsy. SILVER (1940) reported on various degrees of chromatolysis, swelling, and pyk- nosis of neurons similar to the results of GASTAUT (1959), who also observed proli- feration of glial elements. HICKS and CoY noticed acute vacuolar changes in the neuropil of the cortex, lymphocyte infiltration of the meninges, and later a necrosis of nerve cells.

In most of the cited studies, lesions of ganglion cells are emphasized and various degrees of these degenerative changes are described. In turn, L~.wv.y (1950) assumed tha t the acute intoxication by the MSI is characterized by a glial reaction resulting in fluid-filled spaces in the gray matter . According to this author 's view, no similar changes can be observed in chronic intoxications where the glial reaction is absent. HARRIS (1964) also described a massive destruction

11 Z. ZeIlforsch., Bd. 85

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162 Z. LODIN, J. NovXK, E. HOLE6KOVX and J. HARTMAN :

Fig. 3a--c. Large plasmatic astrocyte surrounded by a group of macrophages, a) Micro- photograph made prior to administration of MSI. b) 10 rain. c) 45 min after MSI administration.

(All figures were taken by phase contrast, obj. 40, 35 mm movie)

of glial e lements and sha rp ly out l ined lesions in the grey m a t t e r uns ta inab le b y CAJAL'S subl imate method , following a ve ry intense in tox ica t ion b y MSI in r abb i t s and dogs. The e lect ron microscope dis t inguished in these lesions t races

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Methionine-Sulphoximine Effects on Neural Tissue 163

Fig. 3c

of swollen processes of ohgodendroglia. The author did not report any clearly defined changes in the neurons elicited by MSI under these conditions.

These findings agree with our results obtained in tissue cultures where MSI was shown to at tack primarily neuroglial elements, to cause an enormous swelling of the perikarya and of processes of plasmatic astrocytes and to elicit a strong

% ~ ~Astrocyfe

0 5 10 15 20 25 mln 30

Fig. 4. Changes in astrocyte body with periodic contractions during gradual enlargement of the cell

oedema with successive destructions of processes and cell bodies of the oligoden- droglia. The neurons are probably less affected and the more serious lesions described in them may be of secondary nature.

The curve characterizing the gradual volumetric enlargement of the body and the main astrocyte processes is very interesting. I ts course shows clearly tha t the cells have a tendency to reduce by contraction a superfluous par t of their volume but tha t they succeed to do so only for a short, and transient period.

11"

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164 Z. LODIN et al. : Methionine-Sulphoximine Effects on Neural Tissue

References FISCHEIr J., u. Z. LoDI~: Cytochemisehe Ver~nderungen beim epileptischen Anfall. Proc.

of the 5th Neuropath. Congr. Miinchen, 4---8th Sept. 1961. GASTAUT, H., M. TOGA, and R. NAQV~T: Clinical, electrographic and anatomical study of

the epilepsy induced in dogs by the ingestion of agenized proteins. In: M. BALDWI~ and P. BAILEV, Temporal lobe epilepsy, p. 268--285. Springfield, Ill.: Ch. C. Thomas 1959.

HARRIS, B. : Cortical alternations due to methionine-sulphoximine. Arch. Neurol. l l , 388~407 (1964).

Hicks, S. P., and M. A. CoY: Pathologic effect of antimetabolites. Part 2 (Convulsions and brain lesions caused by methionine-sulfoximine, and their variations with genotype). Arch. Path. 65, 378--387 (1958).

JOHNSON, W. L., S. GOLDRING, and J. O'LEARu : Behavioral, unit and slow potential changes in methionine sulfoximine seizures. Eleetroenceph. clin. Neurol. 18, 229--238 (1965).

LEWEu F. H. : Neuropathological changes in nitrogen triehloride intoxication of dogs. J. Neuropath. exp. Neurol. 9, 296--405 (1950).

LODIN, Z. : An electroencephalographie study of the changes produced by the administration of methionine sulfoximine in dogs and rats. Physiol. bohemoslov. 7, 95--101 (1958).

- - Attempt of a functional analysis of the changes in the central nervous system caused by MSI. [Czech.] ~s. Fysiol. 7, 122--128 (1958).

- - , and J. KOLOU~EK: Symptomatology of the epilepsy caused by methionine sulfoximine. Cs. Neurol. 19, 83--88 (1956).

PROLLER, M., and P. KELLAWAY: The methionine sulfoximine syndrome in the cat. Epilepsia (Amsterdam) 3, 117--130 (1962).

SILVEtr M. L. : Canine epilepsy caused by fluor bleached with nitrogen trichloride. J. Neuro- path. exp. Neurol. 8, 441-445 (1949).

Dr. Z. LODIN Czechoslovak Academy of Science Institute of Physiology Bud~jovicka 1083 Prague 4--KrS/Czechoslovakia