Original article
Ultrastructure of meningiomas: autophagy is involved in the pathogenesis of “intranuclear vacuoles”

Electron Microscopy – “the Big Eye of the 20th century in decline” [28] – is a complex, time-consuming technology no longer widely used in the field of surgical neuropathology, as it has been almost totally replaced by immunohistochemistry with ever-growing numbers of more or less specific commercially available antibodies. However, it is still a powerful technique if diligently used, especially if applied to small brain tumour biopsy specimens. Following personal experience, lasting for some quarter of a century, we decided to produce a series of papers comprising ultrastructural studies of diverse tumour entities. In the past, we published several reviews [19-21,23] and books [18,24,25]. In this paper, we report the first group of such systematically examined brain tumour specimens, namely meningiomas.

Meningioma [7], formerly “fungus of the dura mater” is one of the most common mostly benign tumour of the central nervous system (CNS), originating from the meningoendothelial tissue [31]. A plethora of different types of meningiomas is known.

Conventional variants, all WHO grade I:

• meningothelial,

• fibrous/fibroblastic,

• transitional,

• psammomatous,

• secretory,

• microcystic,

• metaplastic,

• lymphoplasmocyte-rich.

Aggressive variants:

• atypical (WHO grade II),

• chordoid (WHO grade II),

• clear cell (WHO grade II),

• anaplastic with sarcomatous, carcinomatous or melanoma-like pictures [31] (WHO grade III),

• papillary (WHO grade III),

• rhabdoid (WHO grade III).

Material and methods

We used 15 samples of meningiomas recorded on files from the Department of Molecular Pathology and Neuropathology, Chair of Oncology, Medical University Lodz (Table I). They have been collected for over 25 years, immediately fixed at the operation theatre in 2.5% buffered glutaraldehyde, postfixed in 1% osmium tetroxide and embedded in Epon. The semithin sections were stained with toluidine blue and grids were examined first in Zeiss 109 and then in Jeol 1100 transmission electron microscopes.

Results

Irrespective of the meningioma category, the ultrastructural picture was virtually the same and the findings will be presented here divided into ultrastructural categories.

1. General view. At the lower power magnification, a tumour consisted of elongated or round cells and innumerable cellular processes connected with diverse intercellular junctions (Fig. 1). Nuclei presented no specific features, nucleoli were infrequently seen and heterochromatin was clumped beneath the nuclear membranes. In a case of clear cell meningioma, cells were of “watery” cytoplasm (Fig. 2).

Occasionally, immobile cilia, completely ensheathed by the cytoplasm and anchored by blepharoplasts were seen; as we did not encounter those rare cilia in cross-sections, no further insight into their inner microtubular-doublet structure was possible. The cytoplasm of the cells and the processes were filled with the intermediate filaments (Fig. 4). In the intercellular space, collagen fibrils and electron-dense material was occasionally observed.

2. Intercellular junctions. The majority of the tumour samples were filled with processes. Several types of junctional complexes were observed (Fig. 5).

The most frequent were desmosomes and in the proper plane of section their whole pentalaminar structure was readily discernible (Fig. 6). However, robust tonofilaments, as seen in epithelial neoplasms, were not observed. Those desmosomal junctions were either completely symmetric (Fig. 7) or asymmetric, but the exact symmetry could not be judged without the assistance of a goniometer. Some junctional complexes were more elaborate, with desmosomal junctions separated by a tight apposition of membranes, which suggests tight junctions (Fig. 8).

3. “Intranuclear vacuoles”. Those structures, well-visible even at low power (Fig. 9), were defined as indentation of the cytoplasm into the nucleus. Within these vacuoles, autophagic vacuoles and lysosomal bodies were seen, suggesting an active macroautophagy process (Fig. 10A,B).

4. In 2 cases, severe lipidization of meningioma cell cytoplasm was observed (Fig. 11).

5. In a case of anaplastic meningioma, a mitotic figure was found (Fig. 12).

6. In another case, empty rectangular spaces in the cytoplasm, suggestive of pre-existing crystalloid structures, were seen (Fig. 13).

Discussion

The ultrastructural findings as reported here are, in a sense, similar irrespective of the histological type of meningiomas. Numerous cytoplasmic processes connected by long and tortuous zipper-like adhesive plaque junctions are common. Other typical findings include “intranuclear inclusions” (vacuoles) containing autophagic vacuoles and other subcellular organelles. Intracytoplasmic intermediate filaments composed of vimentin are also typical.

The fine structural studies of meningiomas are almost as old as the whole field of neurosurgical electron microscopy [14]. The first to present the fine structure of meningiomas was Lewenthal in 1961 followed by Luse [26], Kepes [13], Gusek [10], Napolitano et al. [29], Cervós-Navarro et al. [3,4], Castaigne et al. [2], Robertson [32], Woyke et al. [37,38], and Szymaś et al. [33]. In meningothelial meningioma, amianthoid fibers, i.e. disorderly patterns of collagen fibres, were reported [5]. In secretory meningiomas, numerous microvilli are seen. In rhabdoid meningiomas, round to oval

rhabdoid cells filled with whorls of intermediate filaments are, as in other rhabdoid tumours, typical [1].

In chordoid meningioma, chordoma-like cells are encountered; by electron microscopy, these cells contain abundant mitochondria and intracytoplasmic vacuoles [11]. The reports of these earlier findings were elegantly summarized by Kepes [14].

Analogously to Kepes [14], our data support the notion that the fine structure of hoard of meningioma subtypes are reduced at the ultrastructural level to the same basic pattern. The exceptions are, and also to a certain degree only, clear cell meningioma, chordoid meningioma and rhabdoid meningioma. In the anaplastic meningiomas, mitotic figures may be found as expected, and as illustrated in Fig. 12.

Several features may need additional comments. The intercellular junctions are either well-developed desmosomal junctions, albeit without robust tonofilaments, or desmosomal junctions interspaced with adhesive plaque (tight) junctions as shown by Tani et al. [34]. The latter investigators supplemented transmission electron microscopy by freeze-fracturing technique to demonstrate particles at the fractured sites of the junctions. The tight junction on freeze-fractured faces revealed stretches of complex ridges and furrows. Copeland et al. [6] reported on another type of junctions composed of cisternal dilatation of intercellular space filled with electron-dense structureless material. Xanthochromic changes were already reported by Matyja et al. and Taraszewska et al. [27,35] in cases of anaplastic meningiomas and we observed lipid-laden vacuoles in a case of angiomatous meningioma. Thus, those vacuoles may not be specific to any particular type of meningioma.

The most interesting ultrastructural finding in meningiomas is the presence of “intranuclear vacuoles”, well-visible at the light microscopy level and described for the first time by Wolf and Orton in 1932 [36]. Those structures were first recognized by Gusek et al. [10] followed by Robertson [32]. The latter investigator noticed “dense and granular membrane-bound bodies” within those vacuoles, but did not relate those structures to the autophagy process, as the latter was first discovered a year later [30]; Kepes [15] was the first to demonstrate autophagic vacuoles in meningiomas. The autophagy or “eaten alive” process [16,40] is a complex mechanism involved in bulk removal of intracellular organelles and its role in diverse cellular pathologies from prion diseases to cancer is recently well recognized [22]. According to the recommendations of the Nomenclature Committee on Cell Death [8,17], three major types of programmed cell death (PCD) can be discriminated. The first type is “apoptosis” and this is unrelated to the observations we report here. The second type – involving macroautophagy (called “autophagy” in abbreviation) – is characterized by the presence of numerous autophagosomes that subsequently fuse with lysosomes to form autolysosomes. The molecular mechanism of this process differs from that of apoptosis and consists of a complex interplay of numerous proteins including the mTOR (mammalian target of rapamycin) kinase [39]. The third type is similar to the second type, except for the negligible or absent involvement of lysosomes. Electron-microscopically, type 3 cell death is characterized by swelling of intracellular organelles resulting in the formation of large empty spaces within the cytoplasm and this leads to necrosis. The electron microscopic features of the contents of “intranuclear vacuoles” in meningiomas resemble those of macroautophagy. Thus, unequivocally, autophagy is taking place there, but it is doubtful if it exerts any significant role in meningioma pathogenesis. Of note, intranuclear inclusions are diversely labelled by anti-p62 antibodies [12], a scaffolding protein involved in the ubiquitination process [for review, 9]. However, the exact role of the p62 in meningioma pathogenesis and its putative linkage to autophagy is lacking.

It is hard to explain why the autophagic vacuoles and autophagosomes are evident only within the “intranuclear vacuoles” in meningiomas, but it is entirely possible that the cytoplasm that is entrapped within this cytoplasmic indentation is “strangulated” by the nuclear mass and dies by autophagy.

References

 1. Buccoliero AM, Castiglione F, Degl’Innocenti DR, Franchi A, Sanzo M, Cetica V, Giunti L, Sardi I, Mussa F, Giordano F, Genitori L, Taddei GL. Pediatric rhabdoid meningioma: a morphological, immunohistochemical, ultrastructural and molecular study. Neuropathology 2011; 31: 59-65.

 2. Castaigne P, Escourolle R, Poirier J. Ultrastructure of meningiomas. Electron microscopic study of 4 cases. Rev Neurol (Paris) 1966; 114: 249-261.

 3. Cervós-Navarro J, Vazquez JJ. An electron microscopic study of meningiomas. Acta Neuropathol 1969; 13: 301-323.

 4. Cervós-Navarro J, Vázquez J. Electron microscopy studies on the occurrence of cilia in meningiomas. Virchows Arch Pathol Anat Physiol Klin Med 1966; 341: 280-290.

 5. Chuaqui R, Gonzales S, Torrealba G. Meningothelial meningioma with “amianthoid” fibers. Case report with ultrastructural study. Pathol Res Pract 1992; 188: 890-893.

 6. Copeland DD, Bell SW, Shelburne JD. Hemidesmosome-like intercellular specializations in human meningiomas. Cancer 1978; 41: 2242-2249.

 7. Cushing H. The meningiomas (dural endotheliomas): their source and favoured seats of origin (Cavendish Lecture). Brain 1922; 45: 282-316.

 8. Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS, Fulda S, Gottlieb E, Green DR, Hengartner MO, Kepp O, Knight RA, Kumar S, Lipton SA, Lu X, Madeo F, Malorni W, Mehlen P, Nun~ez G, Peter ME, Piacentini M, Rubinsztein DC, Shi Y, Simon HU, Vandenabeele P, White E, Yuan J, Zhivotovsky B, Melino G, Kroemer G. Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ 2012; 19: 107-120.

 9. Geetha T, Vishwaprakash N, Sycheva M, Babu JR. Sequestosome 1/p62: across diseases. Biomarkers 2012; 17: 99-103.

10. Gusek W. Submikroskopische Untersuchungen als Beitrag zur Struktur Und Onkologie der „Meningiome.” Beitr Pathol Anat 1962; 127: 274-326.

11. Kano T, Nakazato Y, Tamura M, Ohye C, Zama A, Saito F, Tomizawa S. Ultrastructural and immunohistochemical study of an adult case of choroid meningioma. Brain Tumor Pathol 2009; 26: 37-42.

12. Kärjä V, Alafuzoff I. Protein p62 common in invaginations in benign meningiomas – a possible predictor of malignancy. Clin Neuropathol 2006; 25: 37-43.

13. Kepes JJ Electron microscopic studies of meningiomas. Am J Pathol 1961; 39: 499-510.

14. Kepes JJ (ed) Meningiomas. Biology, pathology and differential diagnosis. Masson Publishing USA, Inc. 1982.

15. Kepes JJ. The fine structure of hyaline inclusions (pseudopsammoma bodies) in meningiomas. J Neuropathol Exp Neurol 1975; 34: 282-294.

16. Klionsky et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2012; 8: 1-100.

17. Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA, Malorni W, Nun~ez G, Peter ME, Tschopp J, Yuan J, Piacentini M, Zhivotovsky B, Melino G; Nomenclature Committee on Cell Death 2009. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ 2009; 16: 3-11.

18. Liberski PP (ed.). Nowotwory mózgu u dzieci. Pol J Pathol 2001; 53 (Suppl): 157-172.

19. Liberski PP. The ultrastructure of glial tumors of astrocytic lineage: a review. Folia Neuropathol 1998; 36: 161-177.

20. Liberski PP. The ultrastructure of oligodendroglioma: personal experience and the review of the literature. Folia Neuropathol 1996a; 34: 206-211.

21. Liberski PP. The ultrastructure of ependymoma: personal experience and the review of the literature. Folia Neuropathol 1996b; 34: 212-220.

22. Liberski PP, Brown DR, Sikorska B, Caughey B, Brown P. Cell death and autophagy in prion diseases (transmissible spongiform encephalopathies). Folia Neuropathol 2008; 46: 1-25.

23. Liberski PP, Kordek R. Ultrastructural pathology of glial brain tumors revisited: a review. Ultrastruct Pathol 1997; 21: 1-31.

24. Liberski PP, Kozubski W, Biernat W, Kordek R (eds.). Neuroonkologia kliniczna. Wydawnictwo Czelej, Lublin 2011.

25. Liberski PP, Papierz W (eds.). Neuropatologia Mossakowskiego. Wydawnictwo Czelej, Lublin 2005.

26. Luse SA. Electron microscopic studies of brain tumors. Neurology 1960; 10: 881-905.

27. Matyja E, Naganska E, Zabek M, Jagielski J. Meningioma with unique coexistence of secretory and lipomatous components: a case report with immunohistochemical and ultrastructural study. Clin Neuropathol 2005; 24: 257-261.

28. Mrak RE. The Big Eye in the 21st century: the role of electron microscopy in modern diagnostic neuropathology. J Neuropathol Exp Neurol 2002; 61: 1027-1039.

29. Napolitano L, Kyle R, Fisher ER. Ultrastructure of meningiomas and the derivation and nature of their cellular components. Cancer 1964; 17: 233-241.

30. Omodei Zorini A. Considerations on primary carcinomatous caverns of the lung. Possibility of the intervention of a phenomenon of “autophagy of the neoplastic cells”. Lotta Tuberc 1965; 35: 946-968.

31. Perry A. Meningiomas. In: McLendon RE, Rosenblum MK, Bigner DD (eds.). Russell & Rubinsten’s Pathology of Tumors of the Nervous System, Hodder Arnold, London 2006.

32. Robertson DM. Electron microscopic studies of nuclear inclusion in meningiomas. Am J Pathol 1964; 45: 835-848.

33. Szymaś J, Biczysko W, Gabryel P, Prokopanow H. Histological features of meningiomas with their ultrastructural aspects. Neuropatol Pol 1982; 20: 155-168.

34. Tani E, Ikeda K, Yamagata S, Nishiura M, Higashi N. Specialized junctional complexes in human meningioma. Acta Neuropathol 1974; 28: 305-315.

35. Taraszewska A, Matyja E, Bogucki J. Xanthomatous chan ges In atypical and anaplastic meningiomas. Light and electron microscopic investigations. Folia Neuropathol 2000; 38: 125-134.

36. Wolf A, Orton ST. Intranuclear inclusions in brain tumors. Bull Neurol Inst N Y 1933; 3: 113-123.

37. Woyke S, Domagala W, Olszewski W. Some particular features of the ultrastructure of meningiomas. Neuropatol Pol 1971; 9: 53-70.

38. Woyke S, Domagala W, Olszewski W. Some peculiar ultrastructural features of meningiomas. Pol Med J 1971; 10: 975-1005.

39. Yang Z, Klionsky DJ. An overview of the molecular mechanism of autophagy. Curr Top Microbiol Immunol 2009; 335: 1-32.

40. Yang Z, Klionsky DJ. Eaten alive: a history of macroautophagy. Nat Cell Biol 2010; 12: 814-822.