Experimental brain tumours induced in rats by nitrosourea derivatives

Journal of the neurological Sciences Elsevier Publishing Company, Amsterdam - Printed in The Netherlands

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Experimental Brain Tumours induced in Rats by Nitrosourea Derivatives Part 1. Morphological Aspects of Methylnitrosourea Tumours D. SCH[FFER, A. FABIAN[, E. GROSSI-PAOLETT[ AND P. PAOLETTI Clinic of Nervous and Mental Diseases (Director: Prof L. Bergamhti), Unh~ersity of Turin; Institute of Pharmacology (Director: Prof. E. Trabucchi) and Neurosurgieal Clinic (Director: Prof. P. E. Maspes ) , Unirersity of Milan (Italy) (Received 11 December, 1969)

INTRODUCTION

Experimental brain tumours were originally obtained by the topical administration of chemical carcinogens, such as methylcolantrene, benzopyrene and dibenzantracene (SEL1GMAN AND SHEAR 1939; PEERS 1940). Brain implantations have been widely applied, both in rats (WEIL 1938 ; RUSSELL1945a, b; DIMANTAND ABDURASULOV1963) and in mice (ZIMMERMAN AND ARNOLD 1941a, b, 1943a, b ; TANSLEYAND WILSON 1947 ; MOORE 1953; PINTO 1955; SCHIEFER 1958, 1962; PERESEAND MOORE 1960; PAOLETT~ AND GROSSI-PAOLE~'TI1964). Brain tumours have also been induced by injecting intracerebrally high doses of SV40 virus (KIRSCHSTEIN AND GERBER 1962; RABOTT1 et al. 1965; WILKINS AND ODOM 1965). Occasionally brain tumours have been obtained in the rat with carcinogens given systemically, as in the case of 2-acetylaminofluorene (LoPEZ 1945; HOCH-LIGETI AND RUSSELL 1950; SiMEONIDIS 1954), 8-orthohydroxychinoleine (HocH-LIGETI 1957) and 2,7 fluorenbisacetamide (SNELLet al. 1961). More recently DRUCKREYet al. (1965, 1967) demonstrated that some drugs related to nitrosamine and nitrosamide, when administered by the oral or intravenous route to experimental animals, can induce a remarkably high incidence of turnouts of the nervous system (CNS). These authors investigated the cancerogenic activity of about 70 nitroso derivatives. Some dialchylnitrosamines (DRUCKREY et al. 1964) were effective in producing neuroepitheliomas in the olfactory bulb roots. An acylalkylnitrosoamidic derivative, methylnitrosourea (MNU), has shown a selective and intense cancerogenic activity in the CNS (DRUCKREY et al. 1964, 1965, 1967~ [VANKOVICet al. 1965, 1966). The mechanism of action of rnethylnitrosourea is, according to DRUCKREY et al. (1967) related to that of nitrosamide, since both compounds are metabolized to This research was supported in part by Consiglio Nazionale delle Ricerche, Rome (Italy). J. neurol. Sci., 1970, l I : 559 572

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diazomethane and consequently induce, through the formation of free radicals, the methytation of guanine. As is well known, nitrogen methylation of the bases modifies the stereochemistry of these fundamental components of nucleic acids. On the other hand, the specificity of MN U for the nervous system may be explained through a neurotropism of the "tracer" molecule (MNU) which may be converted into the active metabolite within the central nervous system. The chronic intravenous administration of methylnitrosourea to rats produces. after 8 to 12 months, in 80--90~!i, of the surviving animals a large variety of tumours of the central nervous system. THOMASAYD KERSTING(1964) and KERSTING( 1965, 1966) have observed the appearance of malignant neurinomas, ependymomas, spongioblastomas, oligodendrogliomas, astrocytomas, glioblastomas and mixed gliomesodermic turnouts. The administration of the drugs by the oral route (THoMAs et al. 1967; SXROOBANDT AYD BRUCHER 1968) induces a marked reduction in the incidence of tumours of the CNS, while many turnouts appear in the gastrointestinal tract. MAIERIAL AND METHODS Long Evans male rats, initially in the second month of life, received 25 mg/kg methylnitrosourea (MNU), by injection in the tail vein, once a month, for 8 months. Within the critical period when manifestations of tumours in the CNS appeared, the treated animals were observed daily. The body weight was measured weekly. The monthly injection of the drug was toxic: the animals lost hair and weight, and signs of toxicity were evident during the week following each injection; a few animals died without signs of tumour during the treatment, evidently due to the general toxicity of the drug. When clinical signs appeared, the animals were isolated for better observation and for preventing cannibalism. At the first signs of brain tumour development the rats became irritable, while later they became apathetic and stuporous. In the final stages motor and sensory disturbances appeared with, occasionally, monoplegia or hemiplegia; usually the animal then died within a few days. Tumours of the spinal cord produced paresis and later paraplegia. Cystitis was often associated with this syndrome. These animals, in spite of their serious condition, usually survived for several weeks. Tumours of the cranial nerves in the rat mostly originated from the trigeminus and the Gasserian ganglion and were easily recognizable from the loss of the corneal reflex and later on from unilateral exophthalmos. Sometimes there was also an inspiratory stridor. The animals were sacrificed when neurological symptoms worsened except when sudden death occurred. RESULTS Heterogeneous glial tumours have been obtained. They were frequently multiple or diffuse. Twenty-nine (80°'i,) out of the 36 treated animals developed neoplasms of the brain. Fifty-four intracranial or spinal tumours were present in these 29 rats. The J. neurol. Sci.,

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number of turnouts appearing increased steadily the longer the time of administration of the carcinogen. The number and the histological types of the 54 neoplasms diagnosed are shown in Table I. Out of the 29 positive rats 12 had a solitary tumour, and 17 multiple tumours, only 6 out of the 45 glial turnouts were solitary.

TABLE I CENTRAL NERVOUS SYSTEM NEOPLASMS OBTAINED WITH METHYLNITROSOUREA

Spinal tumours

Intracranial turnouts

Oligodendrogliomas [somorphous gliomas Polymorphous gliomas G[ial loci Neurinomas Sarcomas Epithelial cyst Plexus-papilloma Spinocellular carcinoma

17 5 7 I0 2 2 I 1 I

Total

46

Isomorphous gliomas Polymorphous glioma Glial focus Neurinomas

4 1 1 2

8

The 2 Gasserian ganglion neurinomas both showed typical features of histological malignancy and were similar to those obtained by ethylnitrosourea. They were associated with perivascular neoplastic cells in the pons-cerebellar region and with a diffuse subarachnoid or subpial extension of such cells. Neoplastic cells may enter the CNS from these perivascular cuffs. Circumscribed areas, formed by cells with abundant cytoplasm and vesicular nuclei of oligodendroglial aspect, were observed in one of the Gasserian ganglion neurinomas. The 2 spinal neurinomas appeared quite similar to the tumours just described. The isomorphous gliomas of the cerebral hemispheres may be classified as oligodendrogliomas, while those of the brain stem and of the spinal cord are regarded as spongioblastomas or oligodendrogliomas. Multiple loci of origin have often been observed in the same case and sometimes they give a picture of diffuse cerebral "gliomatosis". The polymorphous gliomas may be classified as glioblastoma multiforme or malignant oligodendroglioma and they have the same localizations as the isomorphous gliomas: a polymorphous glioma of one site may be associated with isomorphous gliomas of other sites. The neoplastic proliferation in the cerebral hemispheres usually originates in the white matter, such as corpus callosum, cingulum, alveus hippocampi, commissura of the fornix, tapetum or radiations of the corpus callosum (Figs. 1 and 2). This proliferation proceeds in the direction of the subiculum, hippocampus and entorhinal cortex. As the tumour spreads, perineural satellitosis appears and subpial growths develop from which the nervous system is again invaded. The tumour may occupy the whole hemisphere, or it may grow more or less symmetrically in both hemispheres, J. neurol. Sci.,

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Fig. 1. Topography and distribution of tumours in brain hemispheres (modified schema from KOENIG AND KLIPPEL 1963).

Fig. 2. Topography and distribution of tumours in brain hemispheres and stem (modified schema from KOENIG ANO KLI~PEL t963).

Fig. 3. Tumour proliferation with typical distribution in the midline structures (modified schema from KoE~G AND KLIPP~L 1963). J. neurol. Sci., 1970, I1:559--572

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and be associated with small neoplastic foci of only a few cells. These foci are similar to those observed in rats not having brain tumours or showing neoplasms only in the brain stern and spinal cord ; they are composed of small collections ofperineuronal satellite cells in the cortex or at the border between the cortex and white matter. They may also be formed by circumscribed areas of oligodendroglial activation in the white matter, from areas of perineuronal satellitosis in the cortex or from small perivascular foci. In the brain stem the tumours may affect the lateral, ventral and dorsal parts or may even occupy half of this structure. Solitary foci may often be found between the lemniscus medialis and the crus cerebri or may appear within a gray nucleus in the form of satellitosis. In the spinal cord, the central gray matter or the posterior funiculi are often involved. The different localizations may be variously associated and there may even be tumours of different type such as spinal neurinomas associated with gliomas of the cerebral hemispheres or of the brain stem. A case of symmetrical "gliomatosis" of the midline structures such as the ventral commissure of the fornix, subcommissural organ, and of the paraventricular and anterior hypothalamic nuclei was observed (Fig. 3). In the cerebral hemispheres the histological characteristics of the oligodendroglioma predominated (Fig. 4A) with many mitoses, isomorphous nuclei, cytoplasmatic "halos" and honeycomb-like structures, small vessels and scarce reticulin (Fig. 5B). Regressive phenomena, such as foci of fat-granule cells (Fig. 4B), liquefaction and necrosis (Fig. 4C) may appear. They may be associated with the presence of cells with bulky cytoplasm of astrocytic type and less isomorphous nuclei (Figs. 4D, 5A). In this way the appearance of a malignant oligodendroglioma with circumscribed areas of necrosis and pseudopalisadings may be brought about. If this change is more diffuse and the cells are more polymorphous with atypical mitoses, the picture of glioblastoma multiforme is produced. Sometimes, because of the richness of reticular fibres, the abundance of vascular proliferation and the features of the peripheral part of the tumour, sarcomatous or glio-sarcomatous characteristics may be identified (Figs. 5C, D and 6B). Characteristic is the case showing a typical oligodendroglioma in one hemisphere and an adventitial sarcoma in the other (Fig. 6C). The latter tumour had a consistent reticulin base, which was disposed in concentric rings around the vessels (Fig. 6D). All the neoplasms may grow in the subarachnoid spaces and enter into the normal nervous system (Fig. 7A). Spongioblastic, astrocytomatous, or more seldom oligodendrogliomatous appearances prevailed in mid-brain, brain stern and spinal cord (Fig. 7B, C). When the proliferation involved the gray matter, the appearances of oligodendroglial satellitosis were found. On the other hand a spongioblastic aspect was observed, when the white matter was involved. These loci of cellular proliferation may become polymorphous through the sequences discussed above. Finally, loci of glial hyperplasia or perivascular cells are present in the cortex and in the white matter (Figs. 7D and 8A).

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Fig. 4. Oligodendroglioma; I-I-E, ,7 200. A: typical appearance; B: area with fat-granule cells; C: necrotic area with pseudopalisading; D: cells with large cytoplasmic bodies of gemistocytic appearance. J. neurol. Sci., /gT,), 1 l: 559--572

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Fig. 5. Oligodendroglioma. A : as in Fig. 4 with higher magnification; H - E , x 400. B: scarce reticulin limited to the vessels; Gomori, x 200. C: area with abundant reticulin; Gomori, x 200. D: proliferating vessels in one regressive area; H - E , × 200.

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Fig. 6. A: oligodendroglioma: wall of proliferating vessels; H=EI x 200. B: glio-sarcomatous tumour: area with abundant reticulin at the periphery; Gomori, × 200. C: reticular sarcoma; H - E , × 200. D: reticular sarcoma: abundant reticulin, disposed in concentric rings around the vessels; Gomori, x 200. J. neurol. Sci., 1970, 11 : 559-572

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Fig. 8. A: focus of oligodendroglial hyperplasia in the white matter; H-E, , 200. B: ependymal proliferation in the roof of the third ventricle; H-E, :. 200. DISCUSSION

The incidence of tumours of the CNS developing in rats treated with nitrosomethylurea is very consistent, but smaller than that seen with nitrosoethylurea: about 80°i~ of" the treated animals developed one or more turnouts. The tumours obtained with nitrosomethylurea are more heterogeneous than those obtained with nitrosoethylurea and they are generally of glial origin, more frequently oligodendrogliomas. Our results compare well with those of KERST1NG (1965, 1966) who used the same route of administration, while they differ considerably from those of SI~ROOBANDT AND BRUCHER (1968), who treated the animals by the oral route. In our series we have obtained neither cerebellar turnouts nor gangliocytomas. We observed a positive correlation between the duration of administration of the drug, the onset of the disease and the number of tumours developing, even if this was less evident than with nitrosoethylurea treatment. On the contrary there was no correlation between the length of this interval and the appearance of polymorphous gliomas. However, it must be stressed that different factors, principally the localization of the tumour, could mask the relationship. The terminology used requires some explanation. We classified gliomas as isomorphous and polymorphous, excluding from the first group oligodendrogliomas and including in the second one the malignant or polymorphous oligodendrogliomas. As a matter of fact, there was no doubt about the origin and histological appearance of hemisphere oligodendrogliomas, while gliomas of the brain stem or of the spinal cord spared the pre-existing anatomical structures and underwent considerable modiJ. neurol. Nci., t97qL I~: 559 S-t?

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fication, so that they could be diagnosed sometimes as oligodendrogliomas, sometimes as spongioblastomas or astrocytomas. Oligodendrogliomas are located preferentially in the cerebral hemispheres and originate from the white matter. In comparison with nitrosoethylurea they seem to prefer the posterior regions. Isomorphous gliomas of the brain stem appear in symmetrical anatomical situations and often appear simultaneously in structures even considerably separated from one another. We must emphasize the frequency with which the anterior hypothalamic nuclei, the subfornical region and the anterior commissure of the fornix were involved. Occasionally the associations were so complicated that the final picture was that of a diffuse tumour, appearing as a "gliomatosis" of the whole brain. As KERSVlNG (1966) remarked, one may have the impression of observing a single type of turnout focus to which the cells of the various sites react with very different morphological reactions which are modified by the influence of the contiguous anatomical structures, in 1 case, for instance, there were oligodendrogliomas in each cerebral hemisphere, a diffuse isomorphous glioma in the brain stem and a typical ependymal proliferation in the roof of the third ventricle (Fig. 8B). Frequently we found mitoses in normal structures, even if they were far away from the paraventricular regions. Keeping in mind also the frequency of the glial or oligodendroglial loci, it seems clear that the WILLIS field theory (1953) accounts for the genesis of these tumours. The most remarkable observation is, however, that polymorphous gliomas may originate from the isomorphous ones. When the turnout is formed, its further evolution is still conditioned by the site of origin, while the nucleo-cytoplasmatic polymorphism appears to be a secondary event in any localization, as if the polymorphous tumoural cytotypes could be correlated more closely with an intratumoural phenomenon, than with the primary morphology of the cells. It thus seems understandable that polymorphism is always associated with vascular proliferation, necrosis or regressive phenomena. This interpretation is based both on the comparative observation of various gliomas and on the presence in the same turnout of isomorphous and polymorphous areas. The growth rate of these tumours certainly plays a fundamental role as demonstrated not only by the large number of typical and atypical mitoses, but also by the finding of "lymphocyte-like nuclei" in the sense already discussed (S('HIVF:ER,:'t a/. 1966; COGNAZZOet al. 1967). The interpretation of the polymorphism is confirmed by the histoenzymologic findings which suggest that this is a secondary phenomenon in the turnout biology. A more extensive discussion concerning the other characters of oligodendrogliomas and in particular of their polymorphism and satellitosis as well as of neurinomas is made more extensively in the following paper (GRossI-PAOLETII e t a / . 1970). Of some interest is the appearance of sarcomatous turnouts, as mentioned also by KERSTING (1965). It must be emphasized that some loci in the cortex or in the brain stem consist of perivascular cells, which may be of reticulo-endothelial instead of glial origin. These tumours could be more precisely defined as reticular or adventitial sarcomas. The finding of the simultaneous appearance of glial and sarcomatous turnouts is of undoubted importance for some problems of human neuropathology, such as the concept of the gliosarcoma. J. neurol. Sci., 1970, 11 : 5 5 9 - 5 7 2

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Finally, from the standpoint of pathogenesis, the following features must be remarked, viz. : the existence of a long period of latency, the influence of this on the development of multiple turnouts, the high number of otigodendrogliomas and the high incidence of neurinomas in the Gasserian ganglion. The latency is so long that the existence of a turnover of the glial population, as demonstrated by SMART AND LEBLOND (1961), KOENIG et al. (1962) and NOETZEL (1966), cannot be disregarded. This consideration may account for the high number of oligodendrogliomas and the absence of typical hemisphere astrocytomas.

ACKNOWLEDGEMENTS

The authors wish to thank Prof. H. Druckrey for the kind gift of nitrosomethylurea. The technical help of Mr. Gino Caliari and Mr. Bruno Peres is gratefully acknowledged.

SUMMARY

The authors studied the induction of tumours of the central nervous system in rats by the chronic administration of methylnitrosourea. Within 8 months 29 out of the 36 treated animals developed neoptzsms (80'~i,). Forty-six intracranial and 8 spinal tumours were found in these 29 rats. Among the intracranial tumours oligodendrogliomas, isomorphous and polymorphous gliomas were the most frequent tumours. Two neurinomas, 2 sarcomas, I epithelial cyst, 1 plexus-papilloma and 1 spinocellular carcinoma were also observed. Among the spinal tumours 4 isomorphous gliomas, 2 neurinomas, 1 polymorphous glioma and 1 glial focus were observed. The localization and the histological characteristics of these neoplasms are described and discussed in detail.

REFERENCES

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