Cell Tiss. Res. 174, 129-137 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
A Combined Scanning and Transmission Electron Microscopic Study and Electron Probe Microanalysis of Human Pineal Acervuli R. Krstid* ** *** Institute of Histology and Embryology, University of Lausanne, Switzerland
Summary. Untreated, decalcified and trypsinized acervuli f r o m h u m a n pineal bodies were studied with the scanning and transmission electron microscope as well as by electron probe microanalysis. The mulberry-like acervuli are composed of a various n u m b e r o f spherical lobes (135-800gm) between which clustered groups of globuli ( 4 - 1 4 g m in diameter) are observed. The acervular lobes are very p r o b a b l y formed by an aggregation of these globuli. Small round particles 125-500 A in diameter are observed on the surface o f the pineal concretions. These are not influenced by either decalcification or trypsin treatment. The acervular mineral corresponds morphologically to hydroxyapatite. The electron probe microanalysis reveals the existence o f calcium and phosphorus as main components of the acervuli. Small quantities of magnesium and strontium were also detected. Key words: Pineal organ, h u m a n - Acervuli - Scanning electron microscopy - Transmission electron microscopy - Electron probe microanalysis.
Introduction The cerebral acervuli, concretions, corpora arenacea or "brain sand" are k n o w n to appear in the choroid plexus and the pineal body o f some animals and humans (for references, see Bargmann, 1943). The probability of finding them in the pineal body increases with age. Radiological investigations have revealed cerebral concretions in more than 3/4 of all examined patients 60 years o f age and older. The material of the cerebral concretions consists mainly of hydroxyapatite, calcium carbonate apatite (Angervall et al., 1958; Earle, 1965) and an organic Send offprint requests to: Dr. R. Krstid, Institut d'Histologie et d'Embryologie, Rue du Bugnon 9, CH-1011 Lausanne, Suisse * Dedicated to Professor Berta Scharrer on the occasion of her 70th birthday ** With the technical assistance of Mr. P.A. Milliquet *** The author wishes to thank Mr. Bauer and Mr. Fryder (Nestec SA, La Tour de Peilz) for the use of the Cambridge Stereoscan electron microscope and Dr. T. Jalanti (C.M.E., Lausanne) for his help with the use of the X-ray microanalyser
130
R. Krstid
Fig. 1. Scanning electron microscopic survey of a h u m a n pineal gland. A great number of acervuli are located near a large pineal cyst (asterisk). (Cameca MEB-07). x 50
matrix composed of acid sulphated mucopolysaccharides (Palladini et al., 1965). Very little is known concerning the origin of brain sand. One hypothesis states that the acervuli are calcification products of an original intracellular organic mass, e.g. nucleoli, while another suggests that they are the result of caclification of dead cells (e.g. erythrocytes or glial cells). Some theories explain the formation of the cerebral concretions as a calcification of the stroma or of
Ultrastructure and Composition of Human Pineal Acervuli
131
Fig. 2. An acervulus in situ. A basket of fibrillar structures surrounds each concretion. (Cambridge Stereoscan $4-10). x 275 a "ground substance" of unknown chemical composition (Bargmann, 1943; Wurtman et al., 1968). D6ring (1944) is of the opinion that small colloidal particles of unknown origin represent the substrate ("nuclei") of mineralisation. The mechanism of acervular formation still remains enigmatic. In the literature very little information is available concerning the submicroscopic organisation of the brain sand. Only Earle (1965), who studied the fine structure of the acervuli by transmission electron microscope, has described fine needle-like crystals at the fractured edges of pineal concretions. The present report deals with the fine structure of the human pineal acervuli, studied with both the scanning (SEM) and transmission (TEM) electron microscopes, as well as with an electron probe microanalysis.
Material and Methods Pineal concretionswere obtained from seven65-75 years old humans of both sexes. In order to observe the concretions in their in situ location in pineal bodies, some organs were fixed in formaldehyde, cut in half, air dried, fixed on an SEM specimen holder and coated with gold.
132
R. Krstid
Fig. 3. An isolated acervulus. Note its lobated surface. (Cambridge Stereoscan $4-10). x 275 Under the dissecting microscope, the acervuli were isolated from the tissue of the remaining pineal bodies and washed in distilled water. Some of the acervuli were treated for 3 h in a 0.1 ~ trypsin solution. Others were decalcified for 3 days in 0.3 M EDTA. Following these treatments the concretions were fixed on SEM specimen holders, coated with gold and observed with Cameca MEB 07, Cambridge Stereoscan S 4-10 and JEOL JSM-35 scanning electron microscopes at 10-25 kv. For transmission electron microscopy, some acervuli were embedded without any treatment in Durcupan, cut with a diamond knife, contrasted with uranyl acetate and lead citrate and observed in a Zeiss EM-9A electron microscope. For electron probe microanalysis, 100 nm thick sections of isolated and unfixed acervuli were cut with a diamond knife, mounted on copper grids and analysed for 10 min at 25 kV in a Hitachi HU-12 transmission electron microscope equipped with a Princetone Gamma-Tech X-ray energy dispersive device. The diameter of the electron spot was 1 gin.
Results A survey scanning electron micrograph of a sectionned pineal body shows a great n u m b e r o f a c e r v u l i l o c a l i s e d in t h e c e n t r a l a r e a o f t h e o r g a n (Fig. 1). T h e y a r e
Ultrastructure and Composition of Human Pineal Acervuli
133
Fig. 4. Higher magnification of an acervular surface. Between the lobes, some clustered groups of globuli (arrowheads) can be seen. Note the existence of a fine "bas relief'' on the lobar surface with dimensions corresponding to that of the globular structures (arrows). (Cameca MEB-07). x 885
predominantly spherical, but vary greatly in size. Acervuli can be found ranging from 400 p m to 3 m m in diameter (the latter corresponds to acervular conglomerates). At higher magnification, a fibrillar basket encloses each acervulus (Fig. 2). Unfortunately, with the SEM it is impossible to distinguish which elements of the fibrillar c o m p o n e n t are collagen or reticular fibers and which are pineal cell or glial cell processes. The concretions consist of various numbers o f lobes measuring 135-800 gm in diameter which aggregate to give a mulberry-shaped acervulus (Fig. 3). Clustered groups of globuli having a diameter of 4-14 gm (Fig. 4) can be observed between the acervular lobes. A " b a s relief'' o f corresponding dimensions is visible on the convexity of the lobes (Fig. 4). In the clustered groups the globuli are bound together and to the lobes by 0.2-1.5 pm thick, irregularly shaped bridges (Figs. 4, 5) or by large contact surfaces (Fig. 5). At higher magni-
134
R. Krstid
Fig. 5. Bridges between the globuli and lobes (arrows). An arrowhead points to a large contact surface between two globuli. Note the granular surface of all the structures. (Cameca MEB-07). x 9,750
fication SEM reveals the existence of round 125-500A large particles on the surface of the globuli as well as on the convexity of the acervular lobes (Fig. 5). These are not influenced by either EDTA decalcification or trypsin treatment. Fractured undecalcified acervuli expose a glass-like surface without any concentric structure. Decalcified concretions show an irregularly arranged material and sometimes concentric lamellae (Fig. 6). In the transmission electron microscope, all acervuli are found to be situated in the extracellular space. A boundary between the pineal cells and the very osmiophilic calcified material is always clearly visible. A small gap of about 350-450 A, probably of artefactual origin, separates the calcified material from the pinealocytes. The acervuli are composed of needle-shaped, randomly oriented crystals, 300-500 A long and about 25-35 A thick. The electron probe microanalysis reveals the existence of Ca and P as main components of the acervuli. A small quantity of S, Mg and Sr were also detected (Fig. 7).
Fig. 6. An acervulus after EDTA decalcification. Concentric sheets (arrows) of an organic material surrounding an acervular lobe are visible. (JEOL JSM-35). • 500 Ca
Ca
Mg
0
1
,
i
4
5
i
6
7
i
8
9
10
11
1'2 keV
Fig. 7. X-ray energy dispersive analysis of the acervular mineral. In addition to the large peaks of Ca and P, the existence of small quantities of Mg, S and Sr can also be detected. All Cu-peaks originate from the specimen grid
136
R. Krsti6
Discusdon
At low magnification in the scanning electron microscope, the acervuli show a morphology and a relationship to pineal tissue which are already well documented (Angervall et al., 1958; Bargmann, 1943; D6ring, 1944; Palladini et al., 1965; Wurtman et al., 1968). The greater resolving power of the scanning electron microscope makes it possible to obtain more information about the mechanism of acervular growth. According to the present results, it seems that the globuli with their cactus-like arrangement (Fig. 5) and their subsequent aggregation might produce acervular lobes. Confirmation of this hypothesis can be seen on the surface of each acervular lobe, where the "bas relief" of the preceding globuli (Fig. 4) is detected. Unfortunately, the beginning of the acervular formation escapes observation and still remains unclarified. Whether the round particles (125-500/k) visible on the surface of every structure composing an acervulus represent the first crystallisation nuclei, this must still be proven. The fact that, after decalcification, the particles of the same dimension remain visible on the acervular surface, speaks in favour of the existense of small organic nuclei (supposed also by D6ring, 1944) which would be incrusted with calcium salts. These particles, after aggregation, would give firstly the globuli and then the concentric lamellar system (Fig. 6) which enlarges the acervuli. The fibrillar baskets surrounding the acervuli correspond presumably to the envelope consisting of argyrophilic fibres ("Gitterfaser") described and depicted by Bargmann (1943). Palladini et al. (1965) have demonstrated the presence of acid, neutral and sulphated mucopolysaccharides in the organic part of the acervuli. They have also found, in the same structures, some proteins rich in indol groups and a few lipids. Of all these substances, the electron probe microanalysis has only confirmed the existence of the S, for which it is difficult to say whether this element is indicative of actual sulfated mucopolysaccharides of the acervulus or of the embedding medium. X-ray crystallographic and transmission electron microscopic (TEM) studies performed by Angervall et al. (1958) and Earle (1965) have proven that the pineal concretion mineral is hydroxyapatite and carbonate apatite. Our TEM analyses and measurements of the acervular crystals showing the same dimensions as bone hydroxyapatite crystals (Glimcher, 1959) confirm the findings of the above-cited authors. Concerning the presence of Ca and P in the concretions, our microprobe analysis is in agreement with Angervall et al. (1958), Earle (1965) and Palladini et al. (1965). However, contrary to these authors, and due to the sensitivity of the electron probe microanalysis, we were able to detect the presence of small quantities of Mg and Sr which frequently accompany calcium in biological deposits (McGee-Russel, 1958).
References Angervall, L., Berger, S., R6ckert, H.: A microradiographic and X-ray crystallographic study of calcium in the pineal body and in intracranial tumours. Acta path. microbiol, scan& 44, 113 119 (1958)
Ultrastructure and Composition of Human Pineal Acervuli
137
Bargmann, W.: Die Epiphysis cerebri. In: Handbuch der mikroskopischen Anatomie des Menschen, Bd. VI/1 (ed. W. v. M61lendorff), pp. 309-502. Berlin: Springer 1943 D6ring, F.: fiber die sog. Involutionsver/inderungen der Epiphysis cerebri. Z. Altersforsch. 5, 66-72 (1944) Earle, K.M.: X-ray diffraction and other studies of the calcareous deposits in human pineal glands. J. Neuropath. exp. Neurol. 24, 108-118 (1965) Glimcher, M.J.: Molecular biology of mineralized tissues with particular reference to bone. Rev. Mod. Phys. 31, 359-393 (1959) McGee-Russel, S.M.: Histochemical methods for calcium. J. Histochem. Cytochem. 6, 22-42 (1958) Palladini, G., Alfei, L., Appicciutoli, L.: Osservazioni istochimiche sui Corpora arenacea dell'epifisi umana. Arch. ital. anat. embriol. 70, 253-270 (1965) Wurtman, R.J., Axelrod, J., Kelly, D.E.: The pineal. New York-San Francisco-London: Academic Press 1968
Accepted March 3, 1976 / in final form June 16, 1976
Cell and Tissue Research 9 by Springer-Verlag 1976
A Combined Scanning and Transmission Electron Microscopic Study and Electron Probe Microanalysis of Human Pineal Acervuli R. Krstid* ** *** Institute of Histology and Embryology, University of Lausanne, Switzerland
Summary. Untreated, decalcified and trypsinized acervuli f r o m h u m a n pineal bodies were studied with the scanning and transmission electron microscope as well as by electron probe microanalysis. The mulberry-like acervuli are composed of a various n u m b e r o f spherical lobes (135-800gm) between which clustered groups of globuli ( 4 - 1 4 g m in diameter) are observed. The acervular lobes are very p r o b a b l y formed by an aggregation of these globuli. Small round particles 125-500 A in diameter are observed on the surface o f the pineal concretions. These are not influenced by either decalcification or trypsin treatment. The acervular mineral corresponds morphologically to hydroxyapatite. The electron probe microanalysis reveals the existence o f calcium and phosphorus as main components of the acervuli. Small quantities of magnesium and strontium were also detected. Key words: Pineal organ, h u m a n - Acervuli - Scanning electron microscopy - Transmission electron microscopy - Electron probe microanalysis.
Introduction The cerebral acervuli, concretions, corpora arenacea or "brain sand" are k n o w n to appear in the choroid plexus and the pineal body o f some animals and humans (for references, see Bargmann, 1943). The probability of finding them in the pineal body increases with age. Radiological investigations have revealed cerebral concretions in more than 3/4 of all examined patients 60 years o f age and older. The material of the cerebral concretions consists mainly of hydroxyapatite, calcium carbonate apatite (Angervall et al., 1958; Earle, 1965) and an organic Send offprint requests to: Dr. R. Krstid, Institut d'Histologie et d'Embryologie, Rue du Bugnon 9, CH-1011 Lausanne, Suisse * Dedicated to Professor Berta Scharrer on the occasion of her 70th birthday ** With the technical assistance of Mr. P.A. Milliquet *** The author wishes to thank Mr. Bauer and Mr. Fryder (Nestec SA, La Tour de Peilz) for the use of the Cambridge Stereoscan electron microscope and Dr. T. Jalanti (C.M.E., Lausanne) for his help with the use of the X-ray microanalyser
130
R. Krstid
Fig. 1. Scanning electron microscopic survey of a h u m a n pineal gland. A great number of acervuli are located near a large pineal cyst (asterisk). (Cameca MEB-07). x 50
matrix composed of acid sulphated mucopolysaccharides (Palladini et al., 1965). Very little is known concerning the origin of brain sand. One hypothesis states that the acervuli are calcification products of an original intracellular organic mass, e.g. nucleoli, while another suggests that they are the result of caclification of dead cells (e.g. erythrocytes or glial cells). Some theories explain the formation of the cerebral concretions as a calcification of the stroma or of
Ultrastructure and Composition of Human Pineal Acervuli
131
Fig. 2. An acervulus in situ. A basket of fibrillar structures surrounds each concretion. (Cambridge Stereoscan $4-10). x 275 a "ground substance" of unknown chemical composition (Bargmann, 1943; Wurtman et al., 1968). D6ring (1944) is of the opinion that small colloidal particles of unknown origin represent the substrate ("nuclei") of mineralisation. The mechanism of acervular formation still remains enigmatic. In the literature very little information is available concerning the submicroscopic organisation of the brain sand. Only Earle (1965), who studied the fine structure of the acervuli by transmission electron microscope, has described fine needle-like crystals at the fractured edges of pineal concretions. The present report deals with the fine structure of the human pineal acervuli, studied with both the scanning (SEM) and transmission (TEM) electron microscopes, as well as with an electron probe microanalysis.
Material and Methods Pineal concretionswere obtained from seven65-75 years old humans of both sexes. In order to observe the concretions in their in situ location in pineal bodies, some organs were fixed in formaldehyde, cut in half, air dried, fixed on an SEM specimen holder and coated with gold.
132
R. Krstid
Fig. 3. An isolated acervulus. Note its lobated surface. (Cambridge Stereoscan $4-10). x 275 Under the dissecting microscope, the acervuli were isolated from the tissue of the remaining pineal bodies and washed in distilled water. Some of the acervuli were treated for 3 h in a 0.1 ~ trypsin solution. Others were decalcified for 3 days in 0.3 M EDTA. Following these treatments the concretions were fixed on SEM specimen holders, coated with gold and observed with Cameca MEB 07, Cambridge Stereoscan S 4-10 and JEOL JSM-35 scanning electron microscopes at 10-25 kv. For transmission electron microscopy, some acervuli were embedded without any treatment in Durcupan, cut with a diamond knife, contrasted with uranyl acetate and lead citrate and observed in a Zeiss EM-9A electron microscope. For electron probe microanalysis, 100 nm thick sections of isolated and unfixed acervuli were cut with a diamond knife, mounted on copper grids and analysed for 10 min at 25 kV in a Hitachi HU-12 transmission electron microscope equipped with a Princetone Gamma-Tech X-ray energy dispersive device. The diameter of the electron spot was 1 gin.
Results A survey scanning electron micrograph of a sectionned pineal body shows a great n u m b e r o f a c e r v u l i l o c a l i s e d in t h e c e n t r a l a r e a o f t h e o r g a n (Fig. 1). T h e y a r e
Ultrastructure and Composition of Human Pineal Acervuli
133
Fig. 4. Higher magnification of an acervular surface. Between the lobes, some clustered groups of globuli (arrowheads) can be seen. Note the existence of a fine "bas relief'' on the lobar surface with dimensions corresponding to that of the globular structures (arrows). (Cameca MEB-07). x 885
predominantly spherical, but vary greatly in size. Acervuli can be found ranging from 400 p m to 3 m m in diameter (the latter corresponds to acervular conglomerates). At higher magnification, a fibrillar basket encloses each acervulus (Fig. 2). Unfortunately, with the SEM it is impossible to distinguish which elements of the fibrillar c o m p o n e n t are collagen or reticular fibers and which are pineal cell or glial cell processes. The concretions consist of various numbers o f lobes measuring 135-800 gm in diameter which aggregate to give a mulberry-shaped acervulus (Fig. 3). Clustered groups of globuli having a diameter of 4-14 gm (Fig. 4) can be observed between the acervular lobes. A " b a s relief'' o f corresponding dimensions is visible on the convexity of the lobes (Fig. 4). In the clustered groups the globuli are bound together and to the lobes by 0.2-1.5 pm thick, irregularly shaped bridges (Figs. 4, 5) or by large contact surfaces (Fig. 5). At higher magni-
134
R. Krstid
Fig. 5. Bridges between the globuli and lobes (arrows). An arrowhead points to a large contact surface between two globuli. Note the granular surface of all the structures. (Cameca MEB-07). x 9,750
fication SEM reveals the existence of round 125-500A large particles on the surface of the globuli as well as on the convexity of the acervular lobes (Fig. 5). These are not influenced by either EDTA decalcification or trypsin treatment. Fractured undecalcified acervuli expose a glass-like surface without any concentric structure. Decalcified concretions show an irregularly arranged material and sometimes concentric lamellae (Fig. 6). In the transmission electron microscope, all acervuli are found to be situated in the extracellular space. A boundary between the pineal cells and the very osmiophilic calcified material is always clearly visible. A small gap of about 350-450 A, probably of artefactual origin, separates the calcified material from the pinealocytes. The acervuli are composed of needle-shaped, randomly oriented crystals, 300-500 A long and about 25-35 A thick. The electron probe microanalysis reveals the existence of Ca and P as main components of the acervuli. A small quantity of S, Mg and Sr were also detected (Fig. 7).
Fig. 6. An acervulus after EDTA decalcification. Concentric sheets (arrows) of an organic material surrounding an acervular lobe are visible. (JEOL JSM-35). • 500 Ca
Ca
Mg
0
1
,
i
4
5
i
6
7
i
8
9
10
11
1'2 keV
Fig. 7. X-ray energy dispersive analysis of the acervular mineral. In addition to the large peaks of Ca and P, the existence of small quantities of Mg, S and Sr can also be detected. All Cu-peaks originate from the specimen grid
136
R. Krsti6
Discusdon
At low magnification in the scanning electron microscope, the acervuli show a morphology and a relationship to pineal tissue which are already well documented (Angervall et al., 1958; Bargmann, 1943; D6ring, 1944; Palladini et al., 1965; Wurtman et al., 1968). The greater resolving power of the scanning electron microscope makes it possible to obtain more information about the mechanism of acervular growth. According to the present results, it seems that the globuli with their cactus-like arrangement (Fig. 5) and their subsequent aggregation might produce acervular lobes. Confirmation of this hypothesis can be seen on the surface of each acervular lobe, where the "bas relief" of the preceding globuli (Fig. 4) is detected. Unfortunately, the beginning of the acervular formation escapes observation and still remains unclarified. Whether the round particles (125-500/k) visible on the surface of every structure composing an acervulus represent the first crystallisation nuclei, this must still be proven. The fact that, after decalcification, the particles of the same dimension remain visible on the acervular surface, speaks in favour of the existense of small organic nuclei (supposed also by D6ring, 1944) which would be incrusted with calcium salts. These particles, after aggregation, would give firstly the globuli and then the concentric lamellar system (Fig. 6) which enlarges the acervuli. The fibrillar baskets surrounding the acervuli correspond presumably to the envelope consisting of argyrophilic fibres ("Gitterfaser") described and depicted by Bargmann (1943). Palladini et al. (1965) have demonstrated the presence of acid, neutral and sulphated mucopolysaccharides in the organic part of the acervuli. They have also found, in the same structures, some proteins rich in indol groups and a few lipids. Of all these substances, the electron probe microanalysis has only confirmed the existence of the S, for which it is difficult to say whether this element is indicative of actual sulfated mucopolysaccharides of the acervulus or of the embedding medium. X-ray crystallographic and transmission electron microscopic (TEM) studies performed by Angervall et al. (1958) and Earle (1965) have proven that the pineal concretion mineral is hydroxyapatite and carbonate apatite. Our TEM analyses and measurements of the acervular crystals showing the same dimensions as bone hydroxyapatite crystals (Glimcher, 1959) confirm the findings of the above-cited authors. Concerning the presence of Ca and P in the concretions, our microprobe analysis is in agreement with Angervall et al. (1958), Earle (1965) and Palladini et al. (1965). However, contrary to these authors, and due to the sensitivity of the electron probe microanalysis, we were able to detect the presence of small quantities of Mg and Sr which frequently accompany calcium in biological deposits (McGee-Russel, 1958).
References Angervall, L., Berger, S., R6ckert, H.: A microradiographic and X-ray crystallographic study of calcium in the pineal body and in intracranial tumours. Acta path. microbiol, scan& 44, 113 119 (1958)
Ultrastructure and Composition of Human Pineal Acervuli
137
Bargmann, W.: Die Epiphysis cerebri. In: Handbuch der mikroskopischen Anatomie des Menschen, Bd. VI/1 (ed. W. v. M61lendorff), pp. 309-502. Berlin: Springer 1943 D6ring, F.: fiber die sog. Involutionsver/inderungen der Epiphysis cerebri. Z. Altersforsch. 5, 66-72 (1944) Earle, K.M.: X-ray diffraction and other studies of the calcareous deposits in human pineal glands. J. Neuropath. exp. Neurol. 24, 108-118 (1965) Glimcher, M.J.: Molecular biology of mineralized tissues with particular reference to bone. Rev. Mod. Phys. 31, 359-393 (1959) McGee-Russel, S.M.: Histochemical methods for calcium. J. Histochem. Cytochem. 6, 22-42 (1958) Palladini, G., Alfei, L., Appicciutoli, L.: Osservazioni istochimiche sui Corpora arenacea dell'epifisi umana. Arch. ital. anat. embriol. 70, 253-270 (1965) Wurtman, R.J., Axelrod, J., Kelly, D.E.: The pineal. New York-San Francisco-London: Academic Press 1968
Accepted March 3, 1976 / in final form June 16, 1976
