Acta neurol. scandinav. 59, 178-187, 1979 Anatomy Department B, University of Copenhagen, University Clinic of Neurosurgery, Rigshospitalet, Copenhagen, Geological Central Institute, University of Copenhagen, and Physics Laboratory 2, H. C. Orsted Institute, University of Copenhagen, Denmark
Calcification in a pineal tumour studied by transmission electron microscopy, electron diffraction and x-ray microanalysis M. M ~ L L E R F., GERRIS,H. J. HANSEN AND E. JOHNSON The calcification in a totally calcified pineal tumour was studied. Transmission electron microscopy indicated that the tumour was a pinealoma. The type of calcification was investigated by bright-field and dark-field transmission electron microscopy in addition to electron diffraction and electron-induced x-ray fluorescence. The calcified material consisted predominantly of amorphous calcium phosphate. This type of calcification differs from the normal calcification present in pineal acervuli which consists of crystalline hydroxypatite. Key words: Calcification - pineal tumour - transmission electron microscopy - electron diffraction - x-ray microanalysis
Tumours in the pineal region are rare. Of intracranial neoplasms only 0.40.7 % arise in the region of the pineal gland in european population (Evans 1966, Behrend 1974, Obrador et al. 1976). Small spherical calcifications (brain sand, acervuli or corpora arenacea) are common in the normal human pineal gland. The calcifications comprise small carbonat-containing hydroxyapatite crystals (Mabie & Wallace 1974). The purpose of this paper was to examine the calcification in a totally calcified human pineal tumour by brightfield and dark-field transmission electron microscopy in addition to electron diffraction and electron-induced x-ray fluorescence in order to clarify whether or not the observed pathological Calcification consisted of hydroxyapatite. The calcified material was found to consist predominantly of amorphous calcium phosphate.
MATERIAL AND METHODS
In a 49-year-old Caucasian man suffering from disturbances of equilibrium and gait and progressing dementia during 3 years, a neoplasm located to the lower part of the pineal region was totally removed through an infratentorial supracerebellar approach. The tumour was about 2.5 x 2.5 X 1 cm, encapsulated, totally calcified and chalklike. 0001-6314/79/040178-10 $0.2.50/0 @ 1979 Munksgaard, Copenhagen
179 Transmission electron microscopy The tumour was cut into small pieces (about 2 X 2 x 2 mm) and fixed by immersion in 2.5 % glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4) for 2 h, stained en bloc in 0.5 % aqueous uranyl acetate for 10 h, dehydrated in a graded ethanol series and embedded via propylene oxide in Epon 812. One-micron-thick sections for orientation were stained with toluidine blue. Silver to gray thin sections were cut with a diamond knife on a Reichert ultratome. The sections were post-stained with lead citrate and examined in a Philips 300 electron microscope operated at 60 kV acceleration with an objective aperture with a diameter of 30 microns. Electron diffraction and dark-field electron microscopy The diffraction patterns were recorded from ultrathin sections without post-staining with lead citrate. The diffraction constant of the electron microscope was calibrated by use of the diffraction patterns from vacuum-deposited polycrystalline gold films as previously described (Nielsen et al. 1977, Johnson & Nielsen 1978). Tilted beam dark-field microscopy was carried out by use of electrons diffracted from the calcified material for imaging (Nielsen et al. 1977, Johnson & Nielsen 1978). Selected area diffraction analysis and tilted beam dark-field analysis were performed with a JEOL lOOU transmission electron microscope operated at 100 kV acceleration. X-ray microanalysis The elemental composition of the calcified material was determined by electron-induced x-ray fluorescence microanalysis in a Cambridge 180 scanning electron microscope equipped with a Si (Li) energy dispersive detector (Kevex 163). A beryllium window covering the detector renders possible only the detection of elements heavier than soduim. X-ray spectra were recorded from utrathin sections mounted on 200 mesh copper grids coated with formvar and carbon. The sections were not post-stained with lead citrate. The analysis was performed in a modified transmission stage (Hansen, to be published). The electron probe had a diameter of 100 mm. An acceleratory voltage of 20 kV was used. Counting times of 100 sec were selected. Control spectra were obtained from areas of the sections containing only embedding material.
RESULTS
Light microscopy on 1 -micron-thick Epon embedded survey section The main part of the tumour consisted of calcified trabeculae (Figure 1) with only a few elongated cells located mainly to the periphery of the tumour. Haemorrhages also occurred in the periphery of the tumour. Transmission electron microscopy
In the calcified trabeculae a few necrotic cells were observed (Figure 3). Some of these cells still contained a fairly well preserved nucleus, some mitochondria and lipid droplets (Figures 2 and 3). No filaments or cell membrane specializations were found. The cytoplasm as well as the nucleus of the tumour cells exhibited numerous small 20-40 nm long and 5 nm wide needle12.
Figure 1 . Light micrograph of a part of the calcified pineal tumour. Many calcified trabeculae are seen. e = erythrocytes. Touluidine blue stain. X 148. Figure 2 . Electron micrograph of parts of necrotic cells containing many electron dense needle-like structures and some lipid droplets (L). X 11,760. Figure 3. Electron micrograph of parts of two necrotic turnour cells. n = nucleus. X 17,930.
181
Figure 4. Bright-field (Figure 4a) and tilted beam dark-field (Figure 46) transmission electron micrographs o f the calcified material. The segment o f the first diffuse diffraction ring used f o r dark-field imaging is indicated in the inset by the position of the objective aperture (a). X 13,650. Figure 5. Selected area electron diffraction pattern f r o m electron-dense calcified masses. The broad diffuse rings indicate an amorphous nature of the deposits.
182
I
I
I
I
I
Figure 6. Microdensitometer scan of selected area diffraction pattern (Figure 5) from calcified masses (a), showing two diffuse rings. The dotted line is an interpolated background. For comparison a selected area diffraction pattern ( b ) with sharp rings from polycrystalline Ca-apatite from juvenile dermatornyositis skin connective tissue is shown. Prominent sets of planes are indicated with Miller-Bravais indices for hexagonal crystals. 2 0 is the scattering angle.
like electron dense structures (Figures 2, 3 and 4). Electron-dense structures with the same appearance were observed in large amounts in the extra-cellular spaces where erythrocytes, fibrin and few blood platelets also were seen. In some areas the electron dense structures were condensed to large electron dense masses (Figures 4a and 4b).
Electron diflraction and dark-field microscopy Figure 5 shows a selected area diffraction pattern obtained from an electron
183
P-K,
3-Ka
2
3
4
5
6
7
8
X-RAY ENERGY (KEV) Figure 7. Electron-induced x-ray fluorescence spectrum from electron dense calcified masses (full line) showing prominent calcium and phosphorus peaks. The chlorine and zinc peaks are probably due to instrumental background. The copper peak originates from the copper support grid, and the uranium peak is due to uranyl acetate staining agents. The integrated number of counts in the phosphorus K,peak is 40,000 (10 channels). The recording time was 100 see for the calcification spectrum as well as for the spectrum from the embedding material.
dense desposit similar to that in Figure 4. The diffraction pattern consists of two broad, diffuse rings which indicate an amorphous nature of the deposits. The atomic distances in the deposits associated with the two rings are obtained from a calibration of the diffraction constant (Nielsen et al. 1977, Johnson & Nielsen 1978), and were found to be 0.30 -t 0.02 nm and 0.21 k 0.02 nm for the first and second diffuse ring, respectively. A microdensitometer tracing of the diffraction pattern is shown in Figure 6 where it is compared with a diffraction pattern from a crystalline Ca-apatite deposit (Johnson & Nielsen 1978) obtained from a calcified aggregate in an elastic fiber from skin connective tissue from a juvenile dermatomyositis patient. In the latter diffraction pattern, distinct diffraction rings from prominent lattice planes are indexed by the Miller-Bravais four index notation (hkil) for hexagonal crystals (Barrett & Mmsalski 1966). Figure 4 shows a bright-field and associated tilted beam dark-field micrograph from a typical electron-dense structure. The beam is tilted in such a
184 way that electrons diffracted into the innermost diffuse ring are used for darkfield image formation. The needle-like structures which are dark in the brightfield micrograph (Figure 4a) appear uniformly bright in the dark-field micrograph (Figure 4b). Similar dark-field contrast is observed from the larger electron dense masses. X-ray microanalysis Figure 7 shows an x-ray fluorescence spectrum obtained from a dense mass similar to that illustrated in Figure 4. The x-ray spectrum from the deposits indicates that the calcified material contained the elements calcium (peaks at 3.7 and 4.0 keV) and phosphorus (peak at 2,l keV). The associated spectrum obtained from the embedding material contained no significant peaks. DISCUSSION
The nomenclature applied to tumours in the pineal gland has not been consistent. The term “pinealoma” was introduced by Krabbe (1923) and later the “pinealomas” were subdivided into “pineocytomas” and “pineoblastomas” depending on the degree of differentiation of the tumour cells. However, the nomenclature adapted by Russell & Rubinstein in 1971 is now generally used. They divided the tumours into four groups 1) teratomas (typical teratomas and geminomas, 2) pinealomas (pineoblastomas and pineocytomas, 3) glial forms and 4) cysts. We have cautiously classified this tumour as a pinealoma of the subtype pineocytoma because the few tumour cells were well differentiated cells containing lipid droplets, which is a characteristic finding in pinealocytes (Meller 1974). No filaments were present in the tumour cells indicating that these were not glial cells or pineal interstitial cells (Mqjller et al. 1978). The pineal gland is known to secrete several factors (melatonin, argininvasotocin) which inhibit the activity of the anterior pituitary gland, probably by influencing the release or synthesis of the releasing or inhibiting factors in the median eminence (Relkin 1978). In this tumour only a few cells were present and the patient had no signs of endocrine disturbances. However, he presented with signs of normal pressure hydrocephalus. Calcification of the human pineal gland is a common phenomenon. X-ray studies have shown that about 70 % of a population has calcifications in the eigth decade (Daramola & Oluwa 1972), and microscopical examinations show calcifications (acervuli or corpora arenacea) in the pineal gland in nearly all autopsy cases. The human acervuli have been studied with scanning electron microscopy and x-ray microanalysis (Angervall 1958, Earle 1965, KrstiC 1976). The acervuli were demonstrated to be 400 nm-3 mm mulberrylike structures with various numbers of spherical globuli on the surface.
185 Electron probe analysis revealed content of calcium and phosphorus in addition to small quantities of magnesium and strontium to be present in the acervuli. Later studies (Mabie & Wallace 1974) by electron diffraction, thermogravimetry, infrared spectroscopy and chemical analysis showed that the acervuli consisted of carbonate-containing hydroxyapatite crystals, 21.8 nm in length and 3.8 nm in width. Under the light microscope the acervuli has an onion like appearance with calcified lamellae. The matrix between the lamellae in the rodents has been shown histochemically to contain acid mucopolysaccharide complexed to protein (Jupha et al. 1976). The calcified material of the tumour studied in this investigation was not confined to acervuli, but was found to occur in the entire tumour. The results of the x-ray microanalysis showed that the content of elements heavier than sodium in these deposits consisted of calcium and phosphorus. The existence of broad diffuse rings in the diffraction patterns from these deposits demonstrated that the calcified materal is amorphous rather than crystalline. In combination, the two results are therefore interpreted as an indentification of the electron-dense material as amorphous calcium phosphate. This interpretation is supported by a comparison of the diffraction patterns with one from crystalline Ca-apatite (Figure 6) yielding a set of well-defined diffraction rings. The position of the diffuse rings in Figure 6 relative to the diffraction rings from crystalline apatite is identical to that given by Eanes (1972) from x-ray diffraction patterns from amorphous calcium phosphate and crystalline apatite in bone material. Finally, the atomic distance in the deposits of 0.30 nm, obtained from the innermost diffuse diffraction ring, corresponds to an interatomic distance approximately similar in range as for a variety of different crystalline calcium phosphates (Brown & Chow 1976). The direct correspondence between the electron-dense needle-like structures and the dense masses is uniquely established from the titled beam darkfield micrograph (Figure 4b). In tilted beam dark-field microscopy, only electrons diffracted through a certain angle and in a given direction, passing through the objective aperture, will contribute to the formation of the image. Hence, regions of the sample predominantly scattering electrons through the objective aperture will appear bright in dark-field micrographs. Figure 4b shows that when a segment of the first diffuse ring in the diffraction pattern from the calcified deposits is used for imaging, all the needle-like structures appear bright on a dark back-ground. This establishes the correspondence between the needle-like structures and the diffuse diffraction rings. Finally, the even brightness and the appearance of all the calcified structures in the dark-field micrographs, i.e. lack of diffraction contrast, as compared with the bright-field micrographs is a further demonstration of the amorphous nature
186 of the deposits (cf. the dark-field appearance of calcified crystalline apatite deposits in the papers by Nielsen et al. 1977 and Johnson & Nielsen 1978). It has been reported that amorphous calcium phosphate may convert to crystalline hydroxyapatite when exposed to aqueous media (Landis et al. 1977). However, no crystalline hydroxyapatite was observed in this tumour, although the tissue was fixed in an aqueous containing fixative. Based on this observation, it is thus concluded that the pathological calcification in the described pineal tumour consisted of amorphous calcium phosphate. This type of calcification is different from the crystalline state that normally occurs in pineal acervuli.
ACKNOWLEDGMENTS The expert technical assistance by Miss M. Andreasen and photographic assistance by Mrs. G. Hahn are gratefully acknowledged. This work was supported in part by the Carlsberg Foundation.
REFERENCES Angervall, L., S. Berger & H. Rijckert (1958): A microradiographic x-ray crystallographic study of calcium in the pineal body and in intracranial tumours. Acta Path. Microbiol. Scand. 44, 113-119. Barrett, C. S. & T. B. Massalski (1966): The structure of metals, 3rd ed., p. 12. McGrawHill, New York. Behrend, R. C. (1974): Epidemiology of brain tumours. In: Handbook of Clinical Neurology. Tumours of the Brain and Skull (ed. P. J. Vinken & G. W. Bruyn) vol. 16, pp. 56-88. North Holland, Amsterdam. Brown, W. E. & L. C. Chow (1976): Chemical properties of bone mineral. Ann. Rev. Mat. Sci. 6, 213-236. Daramola, G. F. & A. 0. Olowu (1972): Physiological and radiological implications of low incidence of pineal calcification in Nigeria. Neuroendocrinol. 9, 41-57. Eanes, E. D. (1972): X-ray diffraction of vertebrate hard tissue. In: Biological Mineralization (ed. I. Zipkin) pp. 227-256. John Wiley & Sons, New York. Earle, K.M. (1965): X-ray diffraction and other studies of the calcareous deposits in human pineal glands. J. Neuropath. exp. Neurol. 24, 108-118. Evans, R. W. (1966): Histological appearances of turnours, 2nd. ed., p. 441. Williams & Wilkins, Baltimore. Japha, J. L., T. J. Eder & E. D. Goldsmith (1976): Calcified inclusion in the superficial pineal gland of the mongolian gerbil. Meriones unguiculatus. Acta Anat. 94, 533-544. Johnson, E. & A. 0. Nielsen (1978): Identification and description of inorganic particles in a biological matrix (calcification in human skin). Submitted for publication. Available from Physics Laboratory 2, University of Copenhagen, Denmark, as internal report, F L 2, report no. 78-09. Krabbe, K.H. (1923): The pineal gland, especially in relation to the problem of its supposed significance in sexual development. Endocrinol. 7, 379-414. KrstiL, R. (1976): A combined scanning and transmission electron microscopic study
187 and electron probe microanalysis of human pineal acervuli. Cell Tiss. Res. 174, 129-137. Landis, W. J., M. C. Paine & M. J. Glimcher (1977): Electron microscopic observations of bone tissue prepared anhydrously in organic solvents. J. Ultrastruct. Res. 59, 1-30. Mabie, C. P. & B. M. Wallace (1974): Optical and chemical properties of pineal gland calcifications. Calc. Tiss. Res. 16, 59-71. M@ller,M. (1974): The ultrastructure of the human fetal pineal gland. 1. Cell Types and blood vessels. Cell. Tiss. Res. 152, 13-30. M@ller,M., A. Inghild & E. Bock (1978): Immunohistochemical demonstration of S-100 protein and GFA protein in interstitial cells of rat pineal gland. Brain Res. 140, 1-13. Nielsen, A. O., E. Johnson, B. Hentzer, L. Danielsen & F. Carlsen (1977): Apatite crystals in pseudoxanthoma elasticum. A combined study using electron microscopy and selected area diffraction analysis. J. Invest. Derm. 69, 376-378. Obrador, S . , M. Soto & J. A. Gutierrez-Diaz (1976): Surgical management of turnours in the pineal region. Acta Neurochirurgica 34, 159-171. Relkin, R. (1978): Use of melatonin and synthetic TRH to determine site of pineal inhibition of TSH secretion. Neuroendocrinol. 25, 310-318. Russell, D. & L. Rubenstein (1971): Pathology of Turnours of the Nervous Sysfem, pp. 208-220. E. Arnold, London. Received March 4, accepted March 8, 1979
Morten Miller, M.D. Anatomy Department B University of Copenhagen 1, Universitetsparken DK-2100 Copenhagen @ Denmark
Calcification in a pineal tumour studied by transmission electron microscopy, electron diffraction and x-ray microanalysis M. M ~ L L E R F., GERRIS,H. J. HANSEN AND E. JOHNSON The calcification in a totally calcified pineal tumour was studied. Transmission electron microscopy indicated that the tumour was a pinealoma. The type of calcification was investigated by bright-field and dark-field transmission electron microscopy in addition to electron diffraction and electron-induced x-ray fluorescence. The calcified material consisted predominantly of amorphous calcium phosphate. This type of calcification differs from the normal calcification present in pineal acervuli which consists of crystalline hydroxypatite. Key words: Calcification - pineal tumour - transmission electron microscopy - electron diffraction - x-ray microanalysis
Tumours in the pineal region are rare. Of intracranial neoplasms only 0.40.7 % arise in the region of the pineal gland in european population (Evans 1966, Behrend 1974, Obrador et al. 1976). Small spherical calcifications (brain sand, acervuli or corpora arenacea) are common in the normal human pineal gland. The calcifications comprise small carbonat-containing hydroxyapatite crystals (Mabie & Wallace 1974). The purpose of this paper was to examine the calcification in a totally calcified human pineal tumour by brightfield and dark-field transmission electron microscopy in addition to electron diffraction and electron-induced x-ray fluorescence in order to clarify whether or not the observed pathological Calcification consisted of hydroxyapatite. The calcified material was found to consist predominantly of amorphous calcium phosphate.
MATERIAL AND METHODS
In a 49-year-old Caucasian man suffering from disturbances of equilibrium and gait and progressing dementia during 3 years, a neoplasm located to the lower part of the pineal region was totally removed through an infratentorial supracerebellar approach. The tumour was about 2.5 x 2.5 X 1 cm, encapsulated, totally calcified and chalklike. 0001-6314/79/040178-10 $0.2.50/0 @ 1979 Munksgaard, Copenhagen
179 Transmission electron microscopy The tumour was cut into small pieces (about 2 X 2 x 2 mm) and fixed by immersion in 2.5 % glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4) for 2 h, stained en bloc in 0.5 % aqueous uranyl acetate for 10 h, dehydrated in a graded ethanol series and embedded via propylene oxide in Epon 812. One-micron-thick sections for orientation were stained with toluidine blue. Silver to gray thin sections were cut with a diamond knife on a Reichert ultratome. The sections were post-stained with lead citrate and examined in a Philips 300 electron microscope operated at 60 kV acceleration with an objective aperture with a diameter of 30 microns. Electron diffraction and dark-field electron microscopy The diffraction patterns were recorded from ultrathin sections without post-staining with lead citrate. The diffraction constant of the electron microscope was calibrated by use of the diffraction patterns from vacuum-deposited polycrystalline gold films as previously described (Nielsen et al. 1977, Johnson & Nielsen 1978). Tilted beam dark-field microscopy was carried out by use of electrons diffracted from the calcified material for imaging (Nielsen et al. 1977, Johnson & Nielsen 1978). Selected area diffraction analysis and tilted beam dark-field analysis were performed with a JEOL lOOU transmission electron microscope operated at 100 kV acceleration. X-ray microanalysis The elemental composition of the calcified material was determined by electron-induced x-ray fluorescence microanalysis in a Cambridge 180 scanning electron microscope equipped with a Si (Li) energy dispersive detector (Kevex 163). A beryllium window covering the detector renders possible only the detection of elements heavier than soduim. X-ray spectra were recorded from utrathin sections mounted on 200 mesh copper grids coated with formvar and carbon. The sections were not post-stained with lead citrate. The analysis was performed in a modified transmission stage (Hansen, to be published). The electron probe had a diameter of 100 mm. An acceleratory voltage of 20 kV was used. Counting times of 100 sec were selected. Control spectra were obtained from areas of the sections containing only embedding material.
RESULTS
Light microscopy on 1 -micron-thick Epon embedded survey section The main part of the tumour consisted of calcified trabeculae (Figure 1) with only a few elongated cells located mainly to the periphery of the tumour. Haemorrhages also occurred in the periphery of the tumour. Transmission electron microscopy
In the calcified trabeculae a few necrotic cells were observed (Figure 3). Some of these cells still contained a fairly well preserved nucleus, some mitochondria and lipid droplets (Figures 2 and 3). No filaments or cell membrane specializations were found. The cytoplasm as well as the nucleus of the tumour cells exhibited numerous small 20-40 nm long and 5 nm wide needle12.
Figure 1 . Light micrograph of a part of the calcified pineal tumour. Many calcified trabeculae are seen. e = erythrocytes. Touluidine blue stain. X 148. Figure 2 . Electron micrograph of parts of necrotic cells containing many electron dense needle-like structures and some lipid droplets (L). X 11,760. Figure 3. Electron micrograph of parts of two necrotic turnour cells. n = nucleus. X 17,930.
181
Figure 4. Bright-field (Figure 4a) and tilted beam dark-field (Figure 46) transmission electron micrographs o f the calcified material. The segment o f the first diffuse diffraction ring used f o r dark-field imaging is indicated in the inset by the position of the objective aperture (a). X 13,650. Figure 5. Selected area electron diffraction pattern f r o m electron-dense calcified masses. The broad diffuse rings indicate an amorphous nature of the deposits.
182
I
I
I
I
I
Figure 6. Microdensitometer scan of selected area diffraction pattern (Figure 5) from calcified masses (a), showing two diffuse rings. The dotted line is an interpolated background. For comparison a selected area diffraction pattern ( b ) with sharp rings from polycrystalline Ca-apatite from juvenile dermatornyositis skin connective tissue is shown. Prominent sets of planes are indicated with Miller-Bravais indices for hexagonal crystals. 2 0 is the scattering angle.
like electron dense structures (Figures 2, 3 and 4). Electron-dense structures with the same appearance were observed in large amounts in the extra-cellular spaces where erythrocytes, fibrin and few blood platelets also were seen. In some areas the electron dense structures were condensed to large electron dense masses (Figures 4a and 4b).
Electron diflraction and dark-field microscopy Figure 5 shows a selected area diffraction pattern obtained from an electron
183
P-K,
3-Ka
2
3
4
5
6
7
8
X-RAY ENERGY (KEV) Figure 7. Electron-induced x-ray fluorescence spectrum from electron dense calcified masses (full line) showing prominent calcium and phosphorus peaks. The chlorine and zinc peaks are probably due to instrumental background. The copper peak originates from the copper support grid, and the uranium peak is due to uranyl acetate staining agents. The integrated number of counts in the phosphorus K,peak is 40,000 (10 channels). The recording time was 100 see for the calcification spectrum as well as for the spectrum from the embedding material.
dense desposit similar to that in Figure 4. The diffraction pattern consists of two broad, diffuse rings which indicate an amorphous nature of the deposits. The atomic distances in the deposits associated with the two rings are obtained from a calibration of the diffraction constant (Nielsen et al. 1977, Johnson & Nielsen 1978), and were found to be 0.30 -t 0.02 nm and 0.21 k 0.02 nm for the first and second diffuse ring, respectively. A microdensitometer tracing of the diffraction pattern is shown in Figure 6 where it is compared with a diffraction pattern from a crystalline Ca-apatite deposit (Johnson & Nielsen 1978) obtained from a calcified aggregate in an elastic fiber from skin connective tissue from a juvenile dermatomyositis patient. In the latter diffraction pattern, distinct diffraction rings from prominent lattice planes are indexed by the Miller-Bravais four index notation (hkil) for hexagonal crystals (Barrett & Mmsalski 1966). Figure 4 shows a bright-field and associated tilted beam dark-field micrograph from a typical electron-dense structure. The beam is tilted in such a
184 way that electrons diffracted into the innermost diffuse ring are used for darkfield image formation. The needle-like structures which are dark in the brightfield micrograph (Figure 4a) appear uniformly bright in the dark-field micrograph (Figure 4b). Similar dark-field contrast is observed from the larger electron dense masses. X-ray microanalysis Figure 7 shows an x-ray fluorescence spectrum obtained from a dense mass similar to that illustrated in Figure 4. The x-ray spectrum from the deposits indicates that the calcified material contained the elements calcium (peaks at 3.7 and 4.0 keV) and phosphorus (peak at 2,l keV). The associated spectrum obtained from the embedding material contained no significant peaks. DISCUSSION
The nomenclature applied to tumours in the pineal gland has not been consistent. The term “pinealoma” was introduced by Krabbe (1923) and later the “pinealomas” were subdivided into “pineocytomas” and “pineoblastomas” depending on the degree of differentiation of the tumour cells. However, the nomenclature adapted by Russell & Rubinstein in 1971 is now generally used. They divided the tumours into four groups 1) teratomas (typical teratomas and geminomas, 2) pinealomas (pineoblastomas and pineocytomas, 3) glial forms and 4) cysts. We have cautiously classified this tumour as a pinealoma of the subtype pineocytoma because the few tumour cells were well differentiated cells containing lipid droplets, which is a characteristic finding in pinealocytes (Meller 1974). No filaments were present in the tumour cells indicating that these were not glial cells or pineal interstitial cells (Mqjller et al. 1978). The pineal gland is known to secrete several factors (melatonin, argininvasotocin) which inhibit the activity of the anterior pituitary gland, probably by influencing the release or synthesis of the releasing or inhibiting factors in the median eminence (Relkin 1978). In this tumour only a few cells were present and the patient had no signs of endocrine disturbances. However, he presented with signs of normal pressure hydrocephalus. Calcification of the human pineal gland is a common phenomenon. X-ray studies have shown that about 70 % of a population has calcifications in the eigth decade (Daramola & Oluwa 1972), and microscopical examinations show calcifications (acervuli or corpora arenacea) in the pineal gland in nearly all autopsy cases. The human acervuli have been studied with scanning electron microscopy and x-ray microanalysis (Angervall 1958, Earle 1965, KrstiC 1976). The acervuli were demonstrated to be 400 nm-3 mm mulberrylike structures with various numbers of spherical globuli on the surface.
185 Electron probe analysis revealed content of calcium and phosphorus in addition to small quantities of magnesium and strontium to be present in the acervuli. Later studies (Mabie & Wallace 1974) by electron diffraction, thermogravimetry, infrared spectroscopy and chemical analysis showed that the acervuli consisted of carbonate-containing hydroxyapatite crystals, 21.8 nm in length and 3.8 nm in width. Under the light microscope the acervuli has an onion like appearance with calcified lamellae. The matrix between the lamellae in the rodents has been shown histochemically to contain acid mucopolysaccharide complexed to protein (Jupha et al. 1976). The calcified material of the tumour studied in this investigation was not confined to acervuli, but was found to occur in the entire tumour. The results of the x-ray microanalysis showed that the content of elements heavier than sodium in these deposits consisted of calcium and phosphorus. The existence of broad diffuse rings in the diffraction patterns from these deposits demonstrated that the calcified materal is amorphous rather than crystalline. In combination, the two results are therefore interpreted as an indentification of the electron-dense material as amorphous calcium phosphate. This interpretation is supported by a comparison of the diffraction patterns with one from crystalline Ca-apatite (Figure 6) yielding a set of well-defined diffraction rings. The position of the diffuse rings in Figure 6 relative to the diffraction rings from crystalline apatite is identical to that given by Eanes (1972) from x-ray diffraction patterns from amorphous calcium phosphate and crystalline apatite in bone material. Finally, the atomic distance in the deposits of 0.30 nm, obtained from the innermost diffuse diffraction ring, corresponds to an interatomic distance approximately similar in range as for a variety of different crystalline calcium phosphates (Brown & Chow 1976). The direct correspondence between the electron-dense needle-like structures and the dense masses is uniquely established from the titled beam darkfield micrograph (Figure 4b). In tilted beam dark-field microscopy, only electrons diffracted through a certain angle and in a given direction, passing through the objective aperture, will contribute to the formation of the image. Hence, regions of the sample predominantly scattering electrons through the objective aperture will appear bright in dark-field micrographs. Figure 4b shows that when a segment of the first diffuse ring in the diffraction pattern from the calcified deposits is used for imaging, all the needle-like structures appear bright on a dark back-ground. This establishes the correspondence between the needle-like structures and the diffuse diffraction rings. Finally, the even brightness and the appearance of all the calcified structures in the dark-field micrographs, i.e. lack of diffraction contrast, as compared with the bright-field micrographs is a further demonstration of the amorphous nature
186 of the deposits (cf. the dark-field appearance of calcified crystalline apatite deposits in the papers by Nielsen et al. 1977 and Johnson & Nielsen 1978). It has been reported that amorphous calcium phosphate may convert to crystalline hydroxyapatite when exposed to aqueous media (Landis et al. 1977). However, no crystalline hydroxyapatite was observed in this tumour, although the tissue was fixed in an aqueous containing fixative. Based on this observation, it is thus concluded that the pathological calcification in the described pineal tumour consisted of amorphous calcium phosphate. This type of calcification is different from the crystalline state that normally occurs in pineal acervuli.
ACKNOWLEDGMENTS The expert technical assistance by Miss M. Andreasen and photographic assistance by Mrs. G. Hahn are gratefully acknowledged. This work was supported in part by the Carlsberg Foundation.
REFERENCES Angervall, L., S. Berger & H. Rijckert (1958): A microradiographic x-ray crystallographic study of calcium in the pineal body and in intracranial tumours. Acta Path. Microbiol. Scand. 44, 113-119. Barrett, C. S. & T. B. Massalski (1966): The structure of metals, 3rd ed., p. 12. McGrawHill, New York. Behrend, R. C. (1974): Epidemiology of brain tumours. In: Handbook of Clinical Neurology. Tumours of the Brain and Skull (ed. P. J. Vinken & G. W. Bruyn) vol. 16, pp. 56-88. North Holland, Amsterdam. Brown, W. E. & L. C. Chow (1976): Chemical properties of bone mineral. Ann. Rev. Mat. Sci. 6, 213-236. Daramola, G. F. & A. 0. Olowu (1972): Physiological and radiological implications of low incidence of pineal calcification in Nigeria. Neuroendocrinol. 9, 41-57. Eanes, E. D. (1972): X-ray diffraction of vertebrate hard tissue. In: Biological Mineralization (ed. I. Zipkin) pp. 227-256. John Wiley & Sons, New York. Earle, K.M. (1965): X-ray diffraction and other studies of the calcareous deposits in human pineal glands. J. Neuropath. exp. Neurol. 24, 108-118. Evans, R. W. (1966): Histological appearances of turnours, 2nd. ed., p. 441. Williams & Wilkins, Baltimore. Japha, J. L., T. J. Eder & E. D. Goldsmith (1976): Calcified inclusion in the superficial pineal gland of the mongolian gerbil. Meriones unguiculatus. Acta Anat. 94, 533-544. Johnson, E. & A. 0. Nielsen (1978): Identification and description of inorganic particles in a biological matrix (calcification in human skin). Submitted for publication. Available from Physics Laboratory 2, University of Copenhagen, Denmark, as internal report, F L 2, report no. 78-09. Krabbe, K.H. (1923): The pineal gland, especially in relation to the problem of its supposed significance in sexual development. Endocrinol. 7, 379-414. KrstiL, R. (1976): A combined scanning and transmission electron microscopic study
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Morten Miller, M.D. Anatomy Department B University of Copenhagen 1, Universitetsparken DK-2100 Copenhagen @ Denmark
