Submicroscopic Distribution of Ruthenium Red in Human Glioblastoma Multiforme*

I. Neurosurg. / Volume 32 / February, 1970 Submicroscopic Distribution of Ruthenium Red in Human Glioblastoma Multiforme* WILLIAM BONDAREFF, M.D., PH.D. Department of Biostructure,. Northwestern University Medical School, Chicago, Illinois LECTRON microscope studies have forme is well documented,24 and more recent suggested the presence of a poly- electron microscope studies further demon- anionic intercellular substance in strate this complexity.8,11,13,18,19 The ultra- mammalian central nervous tissue. 3-6,~ The structure of the intercellular matrix of glio- composition of this material, which appears blastoma multiforme has not been described to occupy the extracellular space of the previously, but light microscope studies sug- brain, is not known, but chemical studies in- gest an intercellular matrix with staining dicate the presence of an acid mucopolysac- properties similar to those of connective charide. 2~ Its intercellular distribution has tissue. 11 been demonstrated by visualization in the electron microscope of electron-opaque Materials and Methods metal complexes (for example, of phospho- Specimens of nine suspected gliomas were tungstic acid '-'~ and ruthenium redO, but the obtained during operation at the Neurosurgi- relation of these complexes to plasma mem- cal Clinic, Sahlgrenska Hospital, Gothen- branes is obscured in central nervous tissue burg, Sweden. After surgical exposure of the by the sparsity of intercellular space. The di- neoplasm, a specimen was taken by sharp mensions of the extracellular space in brain dissection from an area thought not to be are not known with certainty, and the stain- necrotic. Electrocautery was avoided and able material localized within the intercellu- special care was exercised in the use of lar spaces may be either a plasma mem- clamps and hemostats. Within 2 to 3 min- brane-component (cell coat) or a true inter- utes after excision, the specimen was cut into cellular substance. strips about 3 to 5 mm by 0.3 to 0.5 ram, The electron microscope study of glioblas- which were immersed in one of four fixa- toma multiforme reported here concerns the tives. In the appropriate fixative the speci- relationship of neoplastic cells to their mens were cut into cubes about 0.3 to 0.5 immediate extracellular environment. Al- mm on a side. In each case, other specimens though it is recognized that there is no a were fixed in formalin and processed for priori relationship between the intercellular routine neuropathological diagnosis. substance of the tumor and that described in Specimens of each tumor were processed normal brain, this study represents an at- according to the following procedures. tempt to differentiate plasma membrane and Glutaraldehyde. Initial fixation was ac- intercellular components in the central ner- complished by immersion in 3 % glutaralde- vous system. hyde in sodium cacodylate buffer adjusted to The term "glioblastoma multiforme" con- pH 7.4 at 0~ for 1 hour. Following two 5- notes a heterogeneous group of morphologi- min washes in the cacodylate buffer, the speci- cally complex tumors, which because of sim- mens were additionally fixed in 1% OsO4, ilarities of histology and clinical course may prepared according to Caulfield,7 for 1 hour be defined as a clinical entity. The morpho- at O~ logical complexity of glioblastoma multi- Osmium Tetroxide. Specimens were fixed by immersion in 1% OsO4, prepared accord- Received for publication October 16, 1968. ing to Caulfield7 at O~ for 1 hour. Revision received September 29, 1969. Ruthenium Red. Specimens were fixed in * Supported by USPHS Research Grant NB 07044, Career Development Award 1K3 NB 4929, a solution containing equal parts of 3 % glu- and a Lecture/Research Travel Grant under the taraldehyde in cacodylate buffer (pH 7.3), Fulbright-Hays Program with Sweden. sodium cacodylate buffer adjusted to pH 7.3, 145 146 William Bondareff and 0.25% aqueous ruthenium red, for 90 nature of these pleomorphic structures was min at O~ Following two 5-min washes in not determined. They often appeared to be the buffer, the specimens were transferred to continuous with the membranes of the endo- a solution containing equal parts of 0.25% plasmic reticulum, and contained complex aqueous ruthenium red, cacodylate buffer membranous configurations often resembling adjusted to pH 7.3, and 5% aqueous OsO4 myelin figures. for 3 hours at room temperature in the dark. The cells of glioblastoma multiforme This procedure was described initially by characteristically possessed many tortuous LufW and has been used by Bondareff~ for processes, which apposed one another at the demonstration of extracellular substance varying distances (Fig. 1 The extracel- in rat brain. top). lular space was, therefore, of variable di- Lanthanum Nitrate. Specimens were fixed mensions, being several microns wide in by immersion in a solution containing 1% some places and only 100 to 200 A wide in La(NO3)3.6H~O and 1% KMnO4 as de- others (Fig. 1 top). Apposing plasma mem- scribed by Lesseps 1~ for 1 hour at O~ branes did not appear to fuse, and mem- In each case, following fixation, the tis- brane configurations resembling tight junc- sues were dehydrated in a graded series of tions, close junctions, or desmosomes ~-5 were ethyl alcohol, immersed in propylene oxide not found. and embedded in Araldite 502. Plasma membranes were typically trilami- Of the nine cases biopsied, a pathological nar (Fig. 2 bottom), and the outer surface diagnosis of glioblastoma multiforme was re- of the external leaflet was either smooth or turned for seven. Although viable tumor tis- associated with a variable amount of indis- sue was indicated by gross inspection, it was not always possible to avoid sampling ne- tinct, fibrillar material, which was not uni- crotic or hemorrhagic areas. Because such formly distributed (Figs. 1 bottom and 2 top areas were often of microscopic dimensions, and bottom). The appearance of plasma tissues were sectioned first at 0.5 to 0.1 ~, membranes did not differ noticeably in speci- stained with methylene blue-Azure II, and mens fixed with glutaraldehyde-OsO4 or in examined light microscopically. Selected those fixed with OsO4 only. areas of these were cut on a Sorvall MT-2 The immediate external environment of ultramicrotome or an LKB Ultratome and the parenchymal tumor cells contained examined in a Siemens Elmiskop IA electron many, thin, non-collagenous fibrils (about microscope. Specimens fixed with solutions 20 A in width), which appeared as single containing ruthenium red or lanthanum ni- strands or aggregates of variable dimension trate were examined unstained. Specimens (Fig. 1 bottom), and thicker fibrils (100- fixed in glutaraldehyde or osmium tetroxide 200 A in width), in which two limiting sur- were stained prior to viewing with lead ci- faces were apparent in longitudinal sections trate and uranyl acetate. Images were re- (Fig. 2 top). These different fibrils were in- corded on DuPont Cronar film and devel- termingled and variously associated with one oped in D- 19. another. They appeared to be similar to the microfibrils described in normal and patho- Results logical connective tissues. 1~ Microfibrils in Glutaraldehyde-Os04 and OsO~ Fixation. glioblastoma multiforme showed no particu- The cytoplasma of well-preserved cells were lar distribution except at the cell surface dense with closely-packed organelles (Fig. where they often formed bundles organized 1 top). Many ribosomes occurred free in the with their long axes lying parallel to the cytoplasm, usually arranged in rosettes of 4 plane of section (Fig. 1 bottom). They were, in such cases, always distinct from the to 8 ribosomes, and the nucleus was often plasma membrane and did not appear to be large and irregular in outline. These cells, an integral part of it. and especially their processes, contained Ruthenium Red. A dense, granular reac- many closely-packed fibrils, about 70 A in tion product was found in a zone about 1 diameter. There were many cytoplasmic thick immediately subjacent to the surface of dense bodies (Fig. 1 top), but the specific the specimen, in which the cells were often .

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