Electron Microscopical Observations on Mediastinal Neurolemmoma, Neurofibroma, and Ganglioneuroma Maruf A. Razzuk, M.D., Harold C. Urschel, Jr., M.D., James A. Martin, Ph.D., William B. Kingsley, M.D., and Donald L. Paulson, M.D. ABSTRACT Observations on the ultrastructural morphology of benign mediastinal neurolemmoma, neurofibroma, and ganglioneuroma are presented. Distinguishing features of each tumor are described as observed in this study and reported in the literature. In neurolemmoma, the Antoni type A cells exhibit a cellular differentiation totally different from that of the Antoni B cells. The Antoni type A cells are characterized by numerous thin cytoplasmic processes emanating from a cell body with a narrow rim of cytoplasm. The Antoni type B cells lack these processes and have abundant cytoplasm which possesses a complex system of organelles. The extracellular compartment of Antoni type A tissue is composed of lacunae outlined by lamina similar in appearance to the basement membrane surrounding the principal tumor cells. Neurofibroma is distinguished by elongated cells with a few thick cytoplasmic processes interspersed with occasional myelinated and unmyelinated axons in an extensive collagenous stroma. The principal tumor cells are distinguished from fibroblasts by the basement membrane coating their plasmalemma. Ganglioneuroma is characterized by the presence of large ganglion cells and their satellites and a vast number of myelinated and unmyelinated axons. A well- defined basement membrane coats the tumor cells. The small, membrane-bound osmiophilic granules encountered in the cytoplasm of ganglion cells have been observed in other tumors arising in sympathetic ganglia and paraganglionic cells. eurolemmoma, neurofibroma, and ganglioneuroma are neoplasms of the peripheral nervous system. Neurolemmoma and neurofibroma N arise in peripheral nerves, whereas ganglioneuroma arises in the sympathetic ganglia. The application of electron microscopy to the study of these tumors has revealed a number of diagnostic characteristics of importance in the identification of these lesions [7, 171. Although they usually exhibit distinctive light microscopical patterns which enable them to be identified with reasonable certainty, diffic~lties , in classification may nonetheless be prompted in occasional cases of neurolemmoma and neurofibroma because some attributes of one tumor may be present in the From the Baylor University Medical Center and the University of Texas Southwestern Aided in part by National Institutes of Health General Research Support Grant FR-5577. Accepted for publication May 23, 1972. Address reprint requests to Dr. Razzuk, 3810 Swiss Ave., Dallas, Tex. 75204. Medical School, Dallas, Tex. VOL. 15, NO. 1, JANUARY, 1973 73
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Electron Microscopical Observations on Mediastinal Neurolemmoma, Neurofibroma, and GanglioneuromaElectron Microscopical Observations on Mediastinal Neurolemmoma, Neurofibroma, and Ganglioneuroma Maruf A. Razzuk, M.D., Harold C. Urschel, Jr., M.D., James A. Martin, Ph.D., William B. Kingsley, M.D., and Donald L. Paulson, M.D.
ABSTRACT Observations on the ultrastructural morphology of benign mediastinal neurolemmoma, neurofibroma, and ganglioneuroma are presented. Distinguishing features of each tumor are described as observed in this study and reported in the literature. In neurolemmoma, the Antoni type A cells exhibit a cellular differentiation totally different from that of the Antoni B cells. The Antoni type A cells are characterized by numerous thin cytoplasmic processes emanating from a cell body with a narrow rim of cytoplasm. The Antoni type B cells lack these processes and have abundant cytoplasm which possesses a complex system of organelles. The extracellular compartment of Antoni type A tissue is composed of lacunae outlined by lamina similar in appearance to the basement membrane surrounding the principal tumor cells.
Neurofibroma is distinguished by elongated cells with a few thick cytoplasmic processes interspersed with occasional myelinated and unmyelinated axons in an extensive collagenous stroma. The principal tumor cells are distinguished from fibroblasts by the basement membrane coating their plasmalemma.
Ganglioneuroma is characterized by the presence of large ganglion cells and their satellites and a vast number of myelinated and unmyelinated axons. A well- defined basement membrane coats the tumor cells. The small, membrane-bound osmiophilic granules encountered in the cytoplasm of ganglion cells have been observed in other tumors arising in sympathetic ganglia and paraganglionic cells.
eurolemmoma, neurofibroma, and ganglioneuroma are neoplasms of the peripheral nervous system. Neurolemmoma and neurofibroma N arise in peripheral nerves, whereas ganglioneuroma arises in the
sympathetic ganglia. The application of electron microscopy to the study of these tumors has revealed a number of diagnostic characteristics of importance in the identification of these lesions [7, 171. Although they usually exhibit distinctive light microscopical patterns which enable them to be identified with reasonable certainty, diffic~lties , in classification may nonetheless be prompted in occasional cases of neurolemmoma and neurofibroma because some attributes of one tumor may be present in the
From the Baylor University Medical Center and the University of Texas Southwestern
Aided in part by National Institutes of Health General Research Support Grant FR-5577. Accepted for publication May 23, 1972. Address reprint requests to Dr. Razzuk, 3810 Swiss Ave., Dallas, Tex. 75204.
Medical School, Dallas, Tex.
RAZZUK E T AL.
other. In such instances, histological distinctions can be aided by electron microscopical evaluation, since each neoplasm displays a distinct ultrastruc- tural pattern.
The purpose of this communication is to present observations on the ultrastructural morphology of neurolemmoma, neurofibroma, and ganglio- neuroma, with special emphasis on the dominant and distinguishing features of each tumor.
Materials and Methods Tissues included in this study were obtained from 9 surgically excised
benign neurogenic tumors of the mediastinum. Histological classification as made by light microscopy included 4 neurolemmomas, 2 neurofibromas, and 3 ganglioneuromas. The neurolemmoma and neurofibroma tumors presented as solitary, round, encapsulated, and solid masses. Occasional cystic changes were noticed in some of the neurolemmomas. The tumors varied from 4 x 2 x 2 to 9 x 4 x 2 cm. in diameter and were located in the posterior mediastinurn except for 1 neurofibroma, which was located farther forward at the level of the midaxillary line. A definite relation to an intercostal nerve was noticed in each case. The ganglioneuromas were elongated and encapsulated masses measuring 7 x 5 x 4 to 32 x 5 x 3 cm. in diameter. Their consistency varied from firm to cystic. They were located in the posterior mediastinum, and each tumor showed a definite relation to a sympathetic ganglion. None of these neurogenic tumors extended into the spinal canal.
Directly after excision, tissues from each specimen were fixed in neutral formalin for routine paraffin sections, as well as in paraformaldehyde fixative for electron microscopy with subsequent postfixation in osmium followed by dehydration, embedding in Epon 812 resin [S], and staining with uranyl acetate [18] and lead citrate [13]. The electron microscopical study of each group was guided by the light microscopical features characteristic of each category.
Results NEUROLEMMOMA
Histologically, this tumor demonstrated the two typical patterns of Antoni type A and Antorii type B cells. The Antoni type A pattern comprised spindle-shaped cells with the nuclei frequently aligned in parallel rows (Fig. 1A) , and the Antoni type B pattern consisted of stellate-shaped cells which showed small areas of degeneration (Fig. 1B). Hyalin deposits surrounding blood vessels were encountered in some areas.
By electron microscopy the cells of the Antoni type A tissue possessed relatively large nuclei surrounded by narrow bands of cytoplasm. The nuclei contained pale- to dark-staining chromatin which was dispersed evenly
74 THE ANNALS OF THORACIC SURGERY
Electron Microscopy of Thoracic Neurogenic Tumors
F I G . 1 . Light micrographs of a neurolemmoma. ( A ) Antoni type A tissue showing pali- sading of nuclei. (H&E; x 200.) (B) Antoni type B tissue with microcystic degeneration. Hyalin deposits surrounding blood vessels are seen. (H&E; x 200.)
F I G . 2. Electron micrograph of a neurolemmoma, showing an Antoni type A cell with its numerous thin cytoplasmic processes (Cp) , basement membrane ( B M ) , lacunae (L), banded structures (B) , and a fibroblast ( F b ) . (X 10,000.)
VOL. 15, NO. 1, JANUARY, 1973 75
F U Z Z U K E T AL.
except for thin, marginal condensation and occasional clump formation. The scanty cytoplasm contained a small amount of granular endoplasmic reticulum, ribosome rosettes, and a few mitochondria. Emanating from the cell bodies were numerous thin, interdigitating cytoplasmic processes which often subdivided into secondary branches that, in some areas, assumed a parallel arrangement (Fig. 2) .
The cells of the Antoni type B tissue had relatively small nuclei surrounded by abundant cytoplasm. The chromatin was dispersed evenly inside the nucleus and stained from pale to dark. The abundant cytoplasm contained numerous mitochondria, a well-developed Golgi network with secondary vesicles in various stages of maturation, several profiles of granular endoplasmic reticulum, free ribosomes, and several lysosomes (Fig. 3). The typical cytoplasmic processes encountered in the Antoni type A cells were lacking in these cells.
A constant ultrastructural feature of neurolemmoma was the presence of a basement membrane coating the plasmalemma of both Antoni A and Antoni B cell types and their cytoplasmic processes (see Figs. 2, 3) . The matrix of these tumors consisted of fibroblasts, bundles of collagen, and banded structures which were present in both Antoni A and Antoni B
FIG. 3 . Electron micrograph of a neurolemmoma, showing an Antoni type B cell with abundant cytoplasm and increased organelles: mitochondria (M), Golgi network (G), granular endoplasmic reticulum (GER), free ribosomes (FR), and lysosomes (L). Note basement membrane (BM) coating plasmalemma. (x12,300.)
76 THE ANNALS OF THORACIC SURGERY
Electron Microscopy of Thoracic Neurogenic Tumors
tissues. The collagen fibers were not distributed uniformly in the Antoni type A tissue, however. Lacunar spaces bounded by lamina which resembled the basement membrane in appearance and seemed to be directly continuous with it were encountered only in areas of Antoni type A cells (see Fig. 2) . No axons were seen in these tumors.
NEUROFIBROMA
By light microscopy, this tumor showed the usual components: elongated cells, neurites, fibrous tissue, and fibroblasts. The fibroblasts were not readily distinguishabIe from tumor cells by Iight microscopy (Fig. 4) .
By electron microscopy, the principal tumor cells exhibited some variation in shape and size, but the majority were elongated. The nuclei possessed pale- to dark-staining chromatin. The cytoplasm contained aggregates of microfibrils, a few mitochondria, Golgi apparatus, a few profiles of granular endoplasmic reticulum, and free ribosomes. Few and fairly thick cytoplasmic processes emanated from the cell bodies (Fig. 5) . A well-defined basement membrane coated the plasmalemma of these cells and their cytoplasmic processes. This membrane distinguished the principal tumor cells from fibroblasts, which were scattered in a stroma consisting of bundles of collagen fibers. Occasional myelinated and unmyelinated axons showing a definite relation to the tumor cells were observed (Fig. 6) .
GANGLIONEUROMA
By light microscopy, this lesion demonstrated the usual components:
FIG. 4 . Light micrograph of a neuro- fibroma consisting of tumor cells, neurites, bundles of coflagen, and fibroblasts. (H&E; ~ 3 6 . )
VOL. 15, NO. 1 , JANUARY, 1973 77
RAZZUK E T AL.
FIG. 5 . Electron micrograph of a neurofibroma, showing an elongated tumor cell with a few wide cytoplasmic processes (Cp), neurofibrils (NF) dispersed in the cytoplasm, and a basement membrane (BM) coating its plasmalemma. Areas of loose and dense collagen fibers (CF) are present. (X8,SOO.)
ganglion cells and vast numbers of nerve fibers ensheathed by Schwann’s cells. Most of the ganglion cells were surrounded by satellite cells. Sheath cells of Schwann, arranged in bundles, made up the bulk of the stroma (Fig.
By electron microscopy the ganglion cells were large in size, round or oval in shape, and had occasional irregular indentations. The nuclei were fairly large and possessed prominent nucleoli and evenly distributed, pale- staining chromatin. Electron-dense granules were seen near aggregates of chromatin, which possibly represented perichromatin granules (Fig. 8) . The cytoplasm was abundant and contained several mitochondria, numerous vesicles of various sizes containing fibrillar material, a few profiles of granular endoplasmic reticulum, free ribosomes, Golgi apparatus, and microfibrils (Fig. 8) . Numerous small osmiophilic granules, often membrane bound, were scattered in the cytoplasm of ganglion cells and neural processes (Fig. 9) . Satellite cells with elongated and fairly large nuclei surrounded by a narrow rim of cytoplasm were in close contact with the ganglion cells. A thin basement membrane coated the plasmalemma of both the ganglion cells and their satellites (Fig. 8). Numerous unmyelinated and a few myelinated
7) .
78 THE ANNALS OF THORACIC SURGERY
Electron Microscopy of Thoracic Neurogenic Tumors
FIG. 6 . Electron micrographs of a neurofibroma. ( A ) An unmyelinated axon (UA) en- sheathed by a Schwann’s cell (SC). (~10,300.) ( B ) A myelinated axon (MA) is wrapped with a myelin sheath (MS). (~6 ,100 . ) Cytoplasmic processes (Cp) of tumor cells are in close proximity of both axons.
FIG. 7. Light micrograph of a ganglio- neuroma, showing ganglion cells with their satellite cells (SC) and bundles 01 Schwann’s cells. (HQE; ~ 2 0 0 . )
VOL. 15, NO. 1, JANUARY, 19’73 79
RAZZUK E T AL.
axons were dispersed in a loose, collagenous stroma. Schwann’s cells with fairly large nuclei and abundant cytoplasm ensheathed the axons (Fig. 10).
Comment Neurogenic tumors are the most frequently encountered primary
neoplasms of the mediastinum [14]. They are classified into tumors of peripheral nerves-neurolemmoma, neurofibroma, and malignant Schwan- noma; tumors of sympathetic ganglia-ganglioneuroma, ganglioneuroblas- toma, and sympathicoblastoma (neuroblastoma) ; and tumors of paragangli- onic cells-benign and malignant pheochromocytoma and benign and malignant paraganglioma [6, 141.
The largest group of these tumors are the neurolemmoma, neurofibro- ma, and ganglioneuroma. The first and second arise in peripheral nerves and are derived from Schwann’s cells [7, 10-121, whereas the third arises in sympathetic ganglia from immature neuroblasts [l]. The possibility that the ganglion cell is the cell of origin of ganglioneuroma has been postulated upon demonstration in vitro of proliferation of explanted ganglion cells which reproduced themselves by mitosis [ 161. Despite their different cells of origin, the three tumors have one constant ultrastructural feature in
FIG. 8. Electron micrograph of a ganglioneuroma, showing a portion of a ganglion cell and satellite cell (SC). Basement membrane (BM) coats plasmalemma of both cells. Peri- chromatin granules (PG) are seen inside the nucleus. (x11,400.)
80 THE ANNALS OF THORACIC SURGERY
Electron Microscopy of Thoracic Neurogenic Tumors
FIG. 9. Electron micrograph of a ganglioneuroma, showing osmiophilic membrane-bound granules (G) and clear vesicles (V) containing fibrillar material. (~16,000.)
common: the presence of a basement membrane coating the plasmalemma of the principal tumor cells. The presence of this membrane has been a major factor in excluding the fibroblast as the stem cell, particularly in neurolemmoma and neurofibroma [ 171.
By electron microscopy it is readily apparent that each tumor exhibits a distinct ultrastructural pattern. Observations made in this study are in conformity with findings reported in the literature for similar but nonmediastinal tumors [5, 7, 171.
In neurolemmoma, the ultrastructural features vary according to the tissue pattern. The most characteristic features of Antoni type A tissue are the numerous extremely attenuated and complexly interdigitated cytoplas- mic processes that emanate from cells possessing narrow rims of cytoplasm, together with an extracellular compartment composed predominantly of lacunae and clefts that represent basement membrane coating the plasmalemma of the cytoplasmic processes [7, 171. The Antoni type B tissue is distinguished by the presence of round or oval cells with abundant cytoplasm containing a well-developed Golgi network, endoplasmic reticu- lum, and numerous mitochondria and lysosomes. This complex organelle system suggests that the cells are capable of a high degree of metabolic activity [ 171. Observations on tissue culture preparations revealed a greater
VOL. 15, NO. 1 , JANUARY, 1973 81
KAZZUK E T AL.
capacity for lysis of fibrin by Antoni type B cells than by type A cells [lo]. Neurofibroma is distinguished by elongated tumor cells possessing a
limited number of fairly thick cytoplasmic processes. The cells and occasional axons, both myelinated and unmyelinated, are interspersed in an extensive collagenous stroma. A basement membrane coats the plasmalemma of the principal tumor cells and their processes [7, 171.
In ganglioneuroma, the most prominent features are large ganglion cells and their satellites and a vast number of axons which are predominantly unmyelinated. As in neurolemmoma and neurofibroma, a basement membrane coats the plasmalemma of the tumor cells. The small osmiophilic, often membrane-bound granules observed in the cytoplasm of ganglion cells and their neural processes have been reported in a patient with peripheral ganglioneuroma and in several patients with neuroblastoma, ganglioneuro- blastoma, or pheochromocytoma [2, 5, 9, 151. These granules are morphologically similar to catecholamine granules encountered in the adrenal medulla and are presumed to contain the necessary enzymes for catecholamine synthesis [4, 5, 201.
The electron-dense granules seen in the cell nuclei in ganglioneuroma possibly represent perichromatin granules. These granules have been observed in a variety of tissues in man [3] and experimental animals [19].
FIG. 10. Electron micrograph of a ganglioneuroma, showing a few unmyelinated axons (UA) and a single myelinated axon (MA) wrapped by myelin sheath (MS) formed by the investing membranes of Schwann’s cell (SC). (xI0,QOQ.)
82 THE ANNALS OF THORACIC SURGERY
Electron Microscopy of Thoracic Neurogenic Tumors
T h e shape and size of the granules and the possibility that they may contain DNA and perhaps RNA suggest that they may be virus particles [19].
References 1. Abell, M. R., Hart, W. R., and Olson, J. R. Tumors of the peripheral
nervous system. Hum. Pathol. 1:503, 1970. 2. Beltran, G., Leiderman, E., Stuckey, W. J., Jr., Ferrans, V. J., and
Mogabgab, W. J. Metastatic ganglioneuroblastoma: Ultrastructural, histo- chemical, and virological studies in a case. Cancer 24:552, 1969.
3. Bloom, W., and Fawcett, F. A Textbook of Histology (9th ed.) . Philadel- phia: Saunders, 1968.
4. De Robertis, E., and Ferreira, A. V. Electron microscope study of the excretion of catechol-containing droplets in the adrenal medulla. Exp. Cell. Res. 12568, 1957.
5. Greenberg, R., Rosenthal, I., and Falk, G. S. Electron microscopy of human tumors secreting catecholamines: Correlation with biochemical data. J. Neuropathol. Exp. Neurol. 28:475, 1969.
6. Leigh, T. F., and Weens, H. S. The Mediastinum. Springfield, Ill: Thomas, 1959. Pp. 83-102.
7. Luse, S. A. Electron microscopic studies of brain tumors. Neurology 10:881, 1960.
8. Lynn, J. A., Martin, J. H., and Race, G. J. Recent improvements of histologic technics for the combined light and electron microscopic examination of surgical specimens. Am. J . Clin. Pathol. 45:704, 1966.
Fine structural study of neuroblastoma, ganglioneuroblastoma, and pheochromocytoma. Arch. Pathol. 86: 160, 1968.
10. Murray, M. R., Stout, A. P., and Bradley, C. F. Schwann cell versus fibroblast as the origin of the specific nerve sheath tumor. Am. J . Pathol. 16:41, 1940.
11. Pineda, A. Submicroscopic structure of acoustic tumors. Neurology 14:171, 1964.
12. Raimondi, A. J., Mullan, S., and Evans, J. P. Human brain tumors-an electron microscopic study. J . Neurosurg. 19: 73 1, 1962.
13. Reynolds, E. S. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J . Cell. Riol. 17:208, 1963.
14. Schlumberger, H. G. Tumors of the Mediastinum. In Atlas of Tumor Pathology, Fasc. 18. Washingtdn, D.C.: Armed Forces Institute of Pathology,
15. Staley, N. A., Polesky, H. F., and Bensch, K. G. Fine structural and biochemical studies on the malignant ganglioneuroma. J . Neurofmthol. Exp. Neurol. 26:634, 1967.
Tumors of the Peripheral Nervous System. In Atlas of Tumor Pathology, Fasc. 6. Washington, D.C.: Armed Forces Institute of Pathology,
17. Waggener, J. D. Ultrastructure of benign peripheral nerve sheath tumors. Cancer 19:699. 1966.
18. Watson, M. L. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4:475, 1958.
19. Watson, M. L. Observations on a granule associated with chromatin in the nuclei of cells of rat and mouse. J. Cell. Biol. 13:162, 1962.
20. Wood, J. F., and Barnett, R. J. Histochemical demonstration of norepinephrine at a fine structural level. J . Histochem. Cytochem. 12: 197, 1964.
9. Misugi, K., Misugi, N., and Newton, W. A.
1951. Pp. 13-33.
ABSTRACT Observations on the ultrastructural morphology of benign mediastinal neurolemmoma, neurofibroma, and ganglioneuroma are presented. Distinguishing features of each tumor are described as observed in this study and reported in the literature. In neurolemmoma, the Antoni type A cells exhibit a cellular differentiation totally different from that of the Antoni B cells. The Antoni type A cells are characterized by numerous thin cytoplasmic processes emanating from a cell body with a narrow rim of cytoplasm. The Antoni type B cells lack these processes and have abundant cytoplasm which possesses a complex system of organelles. The extracellular compartment of Antoni type A tissue is composed of lacunae outlined by lamina similar in appearance to the basement membrane surrounding the principal tumor cells.
Neurofibroma is distinguished by elongated cells with a few thick cytoplasmic processes interspersed with occasional myelinated and unmyelinated axons in an extensive collagenous stroma. The principal tumor cells are distinguished from fibroblasts by the basement membrane coating their plasmalemma.
Ganglioneuroma is characterized by the presence of large ganglion cells and their satellites and a vast number of myelinated and unmyelinated axons. A well- defined basement membrane coats the tumor cells. The small, membrane-bound osmiophilic granules encountered in the cytoplasm of ganglion cells have been observed in other tumors arising in sympathetic ganglia and paraganglionic cells.
eurolemmoma, neurofibroma, and ganglioneuroma are neoplasms of the peripheral nervous system. Neurolemmoma and neurofibroma N arise in peripheral nerves, whereas ganglioneuroma arises in the
sympathetic ganglia. The application of electron microscopy to the study of these tumors has revealed a number of diagnostic characteristics of importance in the identification of these lesions [7, 171. Although they usually exhibit distinctive light microscopical patterns which enable them to be identified with reasonable certainty, diffic~lties , in classification may nonetheless be prompted in occasional cases of neurolemmoma and neurofibroma because some attributes of one tumor may be present in the
From the Baylor University Medical Center and the University of Texas Southwestern
Aided in part by National Institutes of Health General Research Support Grant FR-5577. Accepted for publication May 23, 1972. Address reprint requests to Dr. Razzuk, 3810 Swiss Ave., Dallas, Tex. 75204.
Medical School, Dallas, Tex.
RAZZUK E T AL.
other. In such instances, histological distinctions can be aided by electron microscopical evaluation, since each neoplasm displays a distinct ultrastruc- tural pattern.
The purpose of this communication is to present observations on the ultrastructural morphology of neurolemmoma, neurofibroma, and ganglio- neuroma, with special emphasis on the dominant and distinguishing features of each tumor.
Materials and Methods Tissues included in this study were obtained from 9 surgically excised
benign neurogenic tumors of the mediastinum. Histological classification as made by light microscopy included 4 neurolemmomas, 2 neurofibromas, and 3 ganglioneuromas. The neurolemmoma and neurofibroma tumors presented as solitary, round, encapsulated, and solid masses. Occasional cystic changes were noticed in some of the neurolemmomas. The tumors varied from 4 x 2 x 2 to 9 x 4 x 2 cm. in diameter and were located in the posterior mediastinurn except for 1 neurofibroma, which was located farther forward at the level of the midaxillary line. A definite relation to an intercostal nerve was noticed in each case. The ganglioneuromas were elongated and encapsulated masses measuring 7 x 5 x 4 to 32 x 5 x 3 cm. in diameter. Their consistency varied from firm to cystic. They were located in the posterior mediastinum, and each tumor showed a definite relation to a sympathetic ganglion. None of these neurogenic tumors extended into the spinal canal.
Directly after excision, tissues from each specimen were fixed in neutral formalin for routine paraffin sections, as well as in paraformaldehyde fixative for electron microscopy with subsequent postfixation in osmium followed by dehydration, embedding in Epon 812 resin [S], and staining with uranyl acetate [18] and lead citrate [13]. The electron microscopical study of each group was guided by the light microscopical features characteristic of each category.
Results NEUROLEMMOMA
Histologically, this tumor demonstrated the two typical patterns of Antoni type A and Antorii type B cells. The Antoni type A pattern comprised spindle-shaped cells with the nuclei frequently aligned in parallel rows (Fig. 1A) , and the Antoni type B pattern consisted of stellate-shaped cells which showed small areas of degeneration (Fig. 1B). Hyalin deposits surrounding blood vessels were encountered in some areas.
By electron microscopy the cells of the Antoni type A tissue possessed relatively large nuclei surrounded by narrow bands of cytoplasm. The nuclei contained pale- to dark-staining chromatin which was dispersed evenly
74 THE ANNALS OF THORACIC SURGERY
Electron Microscopy of Thoracic Neurogenic Tumors
F I G . 1 . Light micrographs of a neurolemmoma. ( A ) Antoni type A tissue showing pali- sading of nuclei. (H&E; x 200.) (B) Antoni type B tissue with microcystic degeneration. Hyalin deposits surrounding blood vessels are seen. (H&E; x 200.)
F I G . 2. Electron micrograph of a neurolemmoma, showing an Antoni type A cell with its numerous thin cytoplasmic processes (Cp) , basement membrane ( B M ) , lacunae (L), banded structures (B) , and a fibroblast ( F b ) . (X 10,000.)
VOL. 15, NO. 1, JANUARY, 1973 75
F U Z Z U K E T AL.
except for thin, marginal condensation and occasional clump formation. The scanty cytoplasm contained a small amount of granular endoplasmic reticulum, ribosome rosettes, and a few mitochondria. Emanating from the cell bodies were numerous thin, interdigitating cytoplasmic processes which often subdivided into secondary branches that, in some areas, assumed a parallel arrangement (Fig. 2) .
The cells of the Antoni type B tissue had relatively small nuclei surrounded by abundant cytoplasm. The chromatin was dispersed evenly inside the nucleus and stained from pale to dark. The abundant cytoplasm contained numerous mitochondria, a well-developed Golgi network with secondary vesicles in various stages of maturation, several profiles of granular endoplasmic reticulum, free ribosomes, and several lysosomes (Fig. 3). The typical cytoplasmic processes encountered in the Antoni type A cells were lacking in these cells.
A constant ultrastructural feature of neurolemmoma was the presence of a basement membrane coating the plasmalemma of both Antoni A and Antoni B cell types and their cytoplasmic processes (see Figs. 2, 3) . The matrix of these tumors consisted of fibroblasts, bundles of collagen, and banded structures which were present in both Antoni A and Antoni B
FIG. 3 . Electron micrograph of a neurolemmoma, showing an Antoni type B cell with abundant cytoplasm and increased organelles: mitochondria (M), Golgi network (G), granular endoplasmic reticulum (GER), free ribosomes (FR), and lysosomes (L). Note basement membrane (BM) coating plasmalemma. (x12,300.)
76 THE ANNALS OF THORACIC SURGERY
Electron Microscopy of Thoracic Neurogenic Tumors
tissues. The collagen fibers were not distributed uniformly in the Antoni type A tissue, however. Lacunar spaces bounded by lamina which resembled the basement membrane in appearance and seemed to be directly continuous with it were encountered only in areas of Antoni type A cells (see Fig. 2) . No axons were seen in these tumors.
NEUROFIBROMA
By light microscopy, this tumor showed the usual components: elongated cells, neurites, fibrous tissue, and fibroblasts. The fibroblasts were not readily distinguishabIe from tumor cells by Iight microscopy (Fig. 4) .
By electron microscopy, the principal tumor cells exhibited some variation in shape and size, but the majority were elongated. The nuclei possessed pale- to dark-staining chromatin. The cytoplasm contained aggregates of microfibrils, a few mitochondria, Golgi apparatus, a few profiles of granular endoplasmic reticulum, and free ribosomes. Few and fairly thick cytoplasmic processes emanated from the cell bodies (Fig. 5) . A well-defined basement membrane coated the plasmalemma of these cells and their cytoplasmic processes. This membrane distinguished the principal tumor cells from fibroblasts, which were scattered in a stroma consisting of bundles of collagen fibers. Occasional myelinated and unmyelinated axons showing a definite relation to the tumor cells were observed (Fig. 6) .
GANGLIONEUROMA
By light microscopy, this lesion demonstrated the usual components:
FIG. 4 . Light micrograph of a neuro- fibroma consisting of tumor cells, neurites, bundles of coflagen, and fibroblasts. (H&E; ~ 3 6 . )
VOL. 15, NO. 1 , JANUARY, 1973 77
RAZZUK E T AL.
FIG. 5 . Electron micrograph of a neurofibroma, showing an elongated tumor cell with a few wide cytoplasmic processes (Cp), neurofibrils (NF) dispersed in the cytoplasm, and a basement membrane (BM) coating its plasmalemma. Areas of loose and dense collagen fibers (CF) are present. (X8,SOO.)
ganglion cells and vast numbers of nerve fibers ensheathed by Schwann’s cells. Most of the ganglion cells were surrounded by satellite cells. Sheath cells of Schwann, arranged in bundles, made up the bulk of the stroma (Fig.
By electron microscopy the ganglion cells were large in size, round or oval in shape, and had occasional irregular indentations. The nuclei were fairly large and possessed prominent nucleoli and evenly distributed, pale- staining chromatin. Electron-dense granules were seen near aggregates of chromatin, which possibly represented perichromatin granules (Fig. 8) . The cytoplasm was abundant and contained several mitochondria, numerous vesicles of various sizes containing fibrillar material, a few profiles of granular endoplasmic reticulum, free ribosomes, Golgi apparatus, and microfibrils (Fig. 8) . Numerous small osmiophilic granules, often membrane bound, were scattered in the cytoplasm of ganglion cells and neural processes (Fig. 9) . Satellite cells with elongated and fairly large nuclei surrounded by a narrow rim of cytoplasm were in close contact with the ganglion cells. A thin basement membrane coated the plasmalemma of both the ganglion cells and their satellites (Fig. 8). Numerous unmyelinated and a few myelinated
7) .
78 THE ANNALS OF THORACIC SURGERY
Electron Microscopy of Thoracic Neurogenic Tumors
FIG. 6 . Electron micrographs of a neurofibroma. ( A ) An unmyelinated axon (UA) en- sheathed by a Schwann’s cell (SC). (~10,300.) ( B ) A myelinated axon (MA) is wrapped with a myelin sheath (MS). (~6 ,100 . ) Cytoplasmic processes (Cp) of tumor cells are in close proximity of both axons.
FIG. 7. Light micrograph of a ganglio- neuroma, showing ganglion cells with their satellite cells (SC) and bundles 01 Schwann’s cells. (HQE; ~ 2 0 0 . )
VOL. 15, NO. 1, JANUARY, 19’73 79
RAZZUK E T AL.
axons were dispersed in a loose, collagenous stroma. Schwann’s cells with fairly large nuclei and abundant cytoplasm ensheathed the axons (Fig. 10).
Comment Neurogenic tumors are the most frequently encountered primary
neoplasms of the mediastinum [14]. They are classified into tumors of peripheral nerves-neurolemmoma, neurofibroma, and malignant Schwan- noma; tumors of sympathetic ganglia-ganglioneuroma, ganglioneuroblas- toma, and sympathicoblastoma (neuroblastoma) ; and tumors of paragangli- onic cells-benign and malignant pheochromocytoma and benign and malignant paraganglioma [6, 141.
The largest group of these tumors are the neurolemmoma, neurofibro- ma, and ganglioneuroma. The first and second arise in peripheral nerves and are derived from Schwann’s cells [7, 10-121, whereas the third arises in sympathetic ganglia from immature neuroblasts [l]. The possibility that the ganglion cell is the cell of origin of ganglioneuroma has been postulated upon demonstration in vitro of proliferation of explanted ganglion cells which reproduced themselves by mitosis [ 161. Despite their different cells of origin, the three tumors have one constant ultrastructural feature in
FIG. 8. Electron micrograph of a ganglioneuroma, showing a portion of a ganglion cell and satellite cell (SC). Basement membrane (BM) coats plasmalemma of both cells. Peri- chromatin granules (PG) are seen inside the nucleus. (x11,400.)
80 THE ANNALS OF THORACIC SURGERY
Electron Microscopy of Thoracic Neurogenic Tumors
FIG. 9. Electron micrograph of a ganglioneuroma, showing osmiophilic membrane-bound granules (G) and clear vesicles (V) containing fibrillar material. (~16,000.)
common: the presence of a basement membrane coating the plasmalemma of the principal tumor cells. The presence of this membrane has been a major factor in excluding the fibroblast as the stem cell, particularly in neurolemmoma and neurofibroma [ 171.
By electron microscopy it is readily apparent that each tumor exhibits a distinct ultrastructural pattern. Observations made in this study are in conformity with findings reported in the literature for similar but nonmediastinal tumors [5, 7, 171.
In neurolemmoma, the ultrastructural features vary according to the tissue pattern. The most characteristic features of Antoni type A tissue are the numerous extremely attenuated and complexly interdigitated cytoplas- mic processes that emanate from cells possessing narrow rims of cytoplasm, together with an extracellular compartment composed predominantly of lacunae and clefts that represent basement membrane coating the plasmalemma of the cytoplasmic processes [7, 171. The Antoni type B tissue is distinguished by the presence of round or oval cells with abundant cytoplasm containing a well-developed Golgi network, endoplasmic reticu- lum, and numerous mitochondria and lysosomes. This complex organelle system suggests that the cells are capable of a high degree of metabolic activity [ 171. Observations on tissue culture preparations revealed a greater
VOL. 15, NO. 1 , JANUARY, 1973 81
KAZZUK E T AL.
capacity for lysis of fibrin by Antoni type B cells than by type A cells [lo]. Neurofibroma is distinguished by elongated tumor cells possessing a
limited number of fairly thick cytoplasmic processes. The cells and occasional axons, both myelinated and unmyelinated, are interspersed in an extensive collagenous stroma. A basement membrane coats the plasmalemma of the principal tumor cells and their processes [7, 171.
In ganglioneuroma, the most prominent features are large ganglion cells and their satellites and a vast number of axons which are predominantly unmyelinated. As in neurolemmoma and neurofibroma, a basement membrane coats the plasmalemma of the tumor cells. The small osmiophilic, often membrane-bound granules observed in the cytoplasm of ganglion cells and their neural processes have been reported in a patient with peripheral ganglioneuroma and in several patients with neuroblastoma, ganglioneuro- blastoma, or pheochromocytoma [2, 5, 9, 151. These granules are morphologically similar to catecholamine granules encountered in the adrenal medulla and are presumed to contain the necessary enzymes for catecholamine synthesis [4, 5, 201.
The electron-dense granules seen in the cell nuclei in ganglioneuroma possibly represent perichromatin granules. These granules have been observed in a variety of tissues in man [3] and experimental animals [19].
FIG. 10. Electron micrograph of a ganglioneuroma, showing a few unmyelinated axons (UA) and a single myelinated axon (MA) wrapped by myelin sheath (MS) formed by the investing membranes of Schwann’s cell (SC). (xI0,QOQ.)
82 THE ANNALS OF THORACIC SURGERY
Electron Microscopy of Thoracic Neurogenic Tumors
T h e shape and size of the granules and the possibility that they may contain DNA and perhaps RNA suggest that they may be virus particles [19].
References 1. Abell, M. R., Hart, W. R., and Olson, J. R. Tumors of the peripheral
nervous system. Hum. Pathol. 1:503, 1970. 2. Beltran, G., Leiderman, E., Stuckey, W. J., Jr., Ferrans, V. J., and
Mogabgab, W. J. Metastatic ganglioneuroblastoma: Ultrastructural, histo- chemical, and virological studies in a case. Cancer 24:552, 1969.
3. Bloom, W., and Fawcett, F. A Textbook of Histology (9th ed.) . Philadel- phia: Saunders, 1968.
4. De Robertis, E., and Ferreira, A. V. Electron microscope study of the excretion of catechol-containing droplets in the adrenal medulla. Exp. Cell. Res. 12568, 1957.
5. Greenberg, R., Rosenthal, I., and Falk, G. S. Electron microscopy of human tumors secreting catecholamines: Correlation with biochemical data. J. Neuropathol. Exp. Neurol. 28:475, 1969.
6. Leigh, T. F., and Weens, H. S. The Mediastinum. Springfield, Ill: Thomas, 1959. Pp. 83-102.
7. Luse, S. A. Electron microscopic studies of brain tumors. Neurology 10:881, 1960.
8. Lynn, J. A., Martin, J. H., and Race, G. J. Recent improvements of histologic technics for the combined light and electron microscopic examination of surgical specimens. Am. J . Clin. Pathol. 45:704, 1966.
Fine structural study of neuroblastoma, ganglioneuroblastoma, and pheochromocytoma. Arch. Pathol. 86: 160, 1968.
10. Murray, M. R., Stout, A. P., and Bradley, C. F. Schwann cell versus fibroblast as the origin of the specific nerve sheath tumor. Am. J . Pathol. 16:41, 1940.
11. Pineda, A. Submicroscopic structure of acoustic tumors. Neurology 14:171, 1964.
12. Raimondi, A. J., Mullan, S., and Evans, J. P. Human brain tumors-an electron microscopic study. J . Neurosurg. 19: 73 1, 1962.
13. Reynolds, E. S. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J . Cell. Riol. 17:208, 1963.
14. Schlumberger, H. G. Tumors of the Mediastinum. In Atlas of Tumor Pathology, Fasc. 18. Washingtdn, D.C.: Armed Forces Institute of Pathology,
15. Staley, N. A., Polesky, H. F., and Bensch, K. G. Fine structural and biochemical studies on the malignant ganglioneuroma. J . Neurofmthol. Exp. Neurol. 26:634, 1967.
Tumors of the Peripheral Nervous System. In Atlas of Tumor Pathology, Fasc. 6. Washington, D.C.: Armed Forces Institute of Pathology,
17. Waggener, J. D. Ultrastructure of benign peripheral nerve sheath tumors. Cancer 19:699. 1966.
18. Watson, M. L. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4:475, 1958.
19. Watson, M. L. Observations on a granule associated with chromatin in the nuclei of cells of rat and mouse. J. Cell. Biol. 13:162, 1962.
20. Wood, J. F., and Barnett, R. J. Histochemical demonstration of norepinephrine at a fine structural level. J . Histochem. Cytochem. 12: 197, 1964.
9. Misugi, K., Misugi, N., and Newton, W. A.
1951. Pp. 13-33.
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