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CANCE R RESEARCH Experimental Brain Tumors CANCE R RESEARCH A MONTHLY JOURNAL OF ARTICLES AND ABSTRACTS REPORTING CANCER RESEARCH VOLUME 1 DECEMBER, 1941 NUMBER 12 Experimental Brain Tumors I. Tumors Produced with Methylcholanthrene* H. M. Zimmerman, M.D., and Hildegarde Arnold, M.D. (Frorn the Laboratory o/Pathology, Yale University School o/ Medicine, New Haven, Conn.) (Received for publication October 9, I941) Efforts to produce intracranial neoplasia by various of 4 in pyrex glass jars having wire mesh covers. The chemical carcinogens have been attended with scant jars were sterilized weekly. Each group of animals was success prior to the work of Seligman and Shear (2). inspected at least twice daily for evidences of tumor By intracerebral implantation of pellets of 2o-methyl- development. cholanthrene, these workers produced ii gliomas and The diet consisted of Purina Fox Chow and oats. 2 fibrosarcomas in a series of 2o male mice of the C3H This and tap water were available to the animals at all strain. Seligman and Shear reported also successful times ad libitum. subcutaneous transplantation of several of these tumors, Carcinogen.--The carcinogen employed for intra- one of which was stated to be a glioma. cranial implantation was 2o-methylcholanthrene (Hoff- Utilizing the same technic, the present writers re- man-LaRoche, Inc., Nutley, N. J.) which was purified ported a preliminary experiment (3) in which they by chromatographic adsorption on A120:~.1 The speci- found u6 intracranial tumors in 51 C3H mice. These men used had a corrected melting point of i79.8- tumors, occurring during the first io months of the I8o.4 ~ C. Cylindrical pellets of this hydrocarbon were experiment, consisted of oligodendroglioma, glioblas- prepared with a diameter of about I mm. and a length toma multiforme, medulloblastoma, unclassified gli- of about i. 5 mm., the average weight of each pellet oma, and meningeal sarcoma. being i. 5 mgm. More recently Peers (1) implanted cholesterol pellets Operation.--Anesthesia was accomplished by the containing lO per cent methylcholanthrene in the subcutaneous injection of 0.25 cc. of a solution con- brains of 99 mice, of which 87 survived into the tumor- taining ioo mgm. nembutal in x5 cc. of o. 9 per cent bearing period. In all, 32 intracranial tumors were sodium chloride solution. The top of the head was produced--i 7 sarcomas and 15 gliomas. shaved and washed with 7 ~ per cent alcohol. For intra- These observations were significant in that they sug- cerebral and subdural implantations a right paramed- gested the future possibility of studying the incidence, Jan incision 5 mm. in length was made in the skin; the histiogenesis, and growth behavior of experimentally periosteum was scraped off the right parietal bone; a induced primary brain tumors. From such a study it hole about 2 ram. in diameter and anterior to the oc- was felt that certain deductions could be drawn regard- cipital suture was made in this bone with a dental burr. ing the human counterparts of these neoplasms. The For intracerebellar implantations the scalp incision was present investigation was thus undertaken for a three- made in the midline over the occipital bone and the fold purpose; namely, the determination of the inci- burr hole in the area between the occipital suture and dence, the histiogenetic origin, and the growth be- the attachments of the occipitalis muscles. Muscle havior of experimental brain tumors. bleeding, which sometimes occurred in this location as the result of trauma, was readily controlled with hot MATERIALS AND METHOD wet sponges. With fine forceps the pellets were pushed through the craniotomy opening and dura for about Care o[ animals.--All the animals employed in these 3 mm. into the right parietal lobe subcortex or into the experiments were male mice of the C3H strain between cerebellum. Those intended for subdural implantation 3 and 4 months of age. They were housed in groups 1 The purified methylcholanthrene was kindly prepared for us * This investigation was aided by a grant from The Jane Coffin in the laboratory of Dr. M. J. Shear of the United States Public Childs Memorial Fund for Medical Research. Health Service. 919 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1941 American Association for Cancer Research. 920 Cancer Research were left in the longitudinal fissure in contact with the chrome stain and with the Wilder silver carbonate meninges between the two cerebral hemispheres. The method for reticulin. Where it seemed indicated, skin margins were then approximated and a drop of preparations were stained with Heidenhain's iron- collodion was applied over the wound. This never re- hematoxylin and with phosphotungstic acid-hematoxy- quired any further attention, healing occurring lin. Frozen sections impregnated with silver and gold promptly and without infection. Until full recovery salts offered such little additional aid in histologic study from the anesthetic, which usually occurred in from that their preparation was abandoned early. 2 to 3 hours, the animals were kept warm on a padded hot plate which was set at about 4 ~ o C. When recovery RESULTS was complete, the mice were returned to their cages in the specially ventilated and heated animal room. For purposes of convenience in presentation, the data Subcutaneous transplantation o/ tumors.--To study of this study will be presented in two parts; I. the certain phases of the growth behavior of the cerebral results of the intracranial implantation of the methyl- neoplasms more effectively, subcutaneous transplanta- cholanthrene pellets and, II. the subcutaneous trans- tion of a number of these tumors was made into male plantation of the intracranial neoplasms thus induced. and female mice of the C3H strain. The animals used were 2 to 3 months of age and the sexes were repre- I. INTRACRANIAL IMPLANTATION OF METHYLCHOLANTHRENE sented in about equal numbers. Mating was prevented by strict segregation. As a rule, 8 mice received trans- The pellets of methylcholanthrene were implanted plants from each tumor, although in some instances 4 in three different locations in groups of mice as fol- mice were used. Subtransplants were made from the lows: the right parietal subcortex, 57 mice; the cere- subcutaneous growths when the latter attained sizes of i cm. or more in diameter, subtransplantation being TABLE I: INCIDENCE OF BRAIN TUMORS INDUCED WITH METHYL- carried out through 9 to 14 passages. These mice re- CHOLANTHRENE ACCORDING TO SITE OF PELLET ceived the same general care and food as those in which I M PLANTATION Group I Group II Group III the pellets of methylcholanthrene were implanted. (Cerebrum) (Cerebellum) (Meninges) The material used for subcutaneous'transplantation Total number of animals... 57 3o consisted of a piece of tissue removed from the main Negative for tumor ........ 3I 17 tumor mass, with careful avoidance of obviously ne- Total number of tumors .... 26 I3 Unclassified tumor ........ t 2 crotic and hemorrhagic areas. This was cut in sterile Gliomas ................ I5 8 saline into fragments about x mm. in diameter. The Astrocytoma ........... I fragment of tumor tissue was deposited in the sub- Glioblastoma multiforme. 4 cutis of the right axillary region or in the right flank Medulloblastoma ....... l Oligodendroglioma ..... 3 by means of a trocar introduced into the right groin Spongioblastoma polare .... through skin washed with alcohol. Unclassified glioma ..... 5 Necropsy technic.--Mice which were moribund were Multiple gliomas ....... i invariably killed by sectioning the cervical spinal cord Sarcomas ................ 5 i Rhabdomyosarcoma ..... 2 and a complete necropsy was performed immediately Meningeal sarcoma ..... 2 thereafter. The brain was removed with sterile pre- Cerebral sarcoma ....... I cautions and, when a tumor was encountered, a piece Mixed sarcoma and glioma. 5 2 was usually excised for subcutaneous transplantation. The brain was then fixed in neutral formalin (U.S.P. bellum, 3 ~ mice; and the subdural space, i6 mice. The formaldehyde I :Io). Animals which were found dead incidence of the various types of neoplasms as they were necropsied immediately on discovery and the occurred at the different sites of pellet implantation in brains fixed in the same manner. It proved expedient the three groups of the io 3 mice of this experiment is in a few instances, because of onsetting post-mortem shown in Table I. softening, to harden the contents of the partially opened Extreme conservativism was employed in the classi- crania in the fixative for :z4 hours before removing the fication of the neoplasms, which accounts for the fact brains. Since tumors were not found in any of the that such noncommittal designations as unclassified other viscera of these animals the organs were not tumor and unclassified glioma appear in Table I. The saved. three tumors under the first designation, although gen- With a few exceptions the brains were embedded in uine neoplasms as indicated by their invasiveness and paraffin and sectioned serially. Hematoxylin-eosin was the presence of cells in mitosis, were nevertheless too the stain employed routinely, but at regularly spaced small to permit more detailed study and classification. intervals sections were prepared with the Masson tri- The ten tumors designated as unclassified glioma were Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1941 American Association for Cancer Research. Ztmmerman and Arnold--Experimental Brain Tumors 921 large enough to permit special study. None of them after the intracerebellar implantation of methylcholanthrene and contained reticulin fibers and, although glial elements after a course which was characterized by disturbances of balance and paralysis of the left hind leg. At necropsy, the brain was were identified in all, they failed to present such char- found adherent to the skull. The cerebral hemispheres were acteristic architectural patterns as are requisite for the uninvolved, but the left half of the cerebellum was bulging and classification of tumors of the glioma group. As an brown in color.
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