Acta Neuropathol (1991) 81: 546- 551
Heuropathologga O Springer-Verlag 1991
Spontaneous mineralization of the sciatic nerve of senescent rats* E. Terao I, B. Corman 2, and P. van den Bosch de Aguilar I 1 Laboratoire de Biologie Cellulaire, Universit6 Catholique de Louvain, Place Croix du Sud 5, B-1348 Louvain-la-Neuve, Belgium 2 Service de Biologie Cellulaire, D6partement de Biologie, Centre d'Etudes Nucl6aires de Saclay, F-91191 Gif-sur-Yvette, France Received August 9, 1990/Revised, accepted November 27, 1990
Summary. A s p o n t a n e o u s mineralization of the sciatic nerve of senescent specific p a t h o g e n - f r e e - b r e d rats (aged 42 m o n t h s ) is r e p o r t e d . D e p o s i t s were f o u n d in the e n d o n e u r i u m o f different b r a n c h e s o f the nerve at mid-thigh level. T h e y a p p e a r e d as small discrete deposits or as large t u b u l a r - s h a p e d concretions, p r o b a b l y f o r m e d by the g r o w t h and m e r g e r o f the smaller deposits. S o m e of the concretions were f o u n d in close proximity to b l o o d vessels. D e p o s i t s consisted o f dense aggregations of r a n d o m l y e n t a n g l e d spicules spreading within bundles of collagen fibrils. Calcium was d e t e c t e d by histochemistry and X-ray dispersion microanalysis. P h o s p h o r u s was also f o u n d , possibly associated with calcium to f o r m hydroxyapatite. Key words: R a t - Sciatic nerve - Mineralization - A g i n g - X-ray microanalysis
E c t o p i c mineralized deposits have b e e n o b s e r v e d in different regions of the central n e r v o u s system, in various pathological cases, such as lead poisoning [18], L a f o r a ' s disease [11], A l z h e i m e r ' s disease and D o w n ' s s y n d r o m e [8], genetic neurological s y n d r o m e [14], infarcts [4] and as well as in aged mice [3, 9]. T h e r e are fewer reports o n mineralization of the peripheral nervous system. T h e y m a i n l y c o n c e r n alterations of the p e r i n e u r i u m associated with diabetes [7], n e p h r o p a t h y [6, 12, 171, c h l o r o q u i n e t r e a t m e n t [16] and less o f t e n with n o r m a l h u m a n aging [7]. E n d o n e u r i a l small dense calcified bodies have b e e n o b s e r v e d in diabetic h m n a n peripheral nerves b u t are very rare [7]. We describe here an extensive s p o n t a n e o u s mineralization of the e n d o n e u r i u m of the sciatic nerve of * Supported by grants from the FRFC and carried out within the framework of the Commission of the European Communities Concerted Action on Ageing and Diseases (EURAGE). E.T. was supported by a grant from the IRSIA. Offprint requests to: E. Terao (address see above)
senescent specific p a t h o g e n - f r e e (SPF) rats, aged 42 m o n t h s . Ultrastructural studies were carried out at light and electron m i c r o s c o p e level. H i s t o c h e m i s t r y and e n e r g y dispersive X-ray microanalysis were used to detect the p r e s e n c e of calcium in these deposits.
Material and methods Wistar Louvain rats were bred in the SPF husbandry of the C.E.N. (Saclay, Gif-sur-Yvette, France). They were maintained on a 14 h: 10 h light: dark cycle, at 20 ~ and at 50 % humidity. Food and water were provided ad libitum. The mean life span of the colony (50 % survival) was over 35 months for males and 39 months for females. Maximal life span was not determined as the study ended when the animals reached 42 months. Fourteen animals (2 males and 12 females) were killed at the age of 42 months. A study of the kidney was als0 undertaken using the same material. Animals were anesthetized by an intraperitoneal injection of Inactine (100 mg/100 g body weight, Byk Gulden, Constance, FRG), placed on a heated table and fixed by perfusion, according to the method of Kaissling et al. [5]. Briefly, a catheter filled with 250 ~tl of a solution containing 2 g/1 procain, 5 mM CaC12and 5,000 IU heparin (Roche, Neuilly-sur-Seine, France) was introduced in the abdominal aorta. Between 100 to 200 ml of the fixative solution warmed at 37 ~ was then perfused under a pressure of 200 mm Hg. The fixative was composed of 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), supplemented with 5 mM CaC12, 0.5 g/1 picric acid and 4 % (v/v) hydroxyethyl starch solution (HAES, Fresenius, Bad Hamburg, FRG). An incision was made in the abdominal vein to allow blood to flow out. After perfusion, mid-thigh portions of the sciatic nerves were dissected and stored overnight in the fixative solution. They were postfixed in osmium tetroxide (2 % in cacodylate buffer 0.1 M, pH 7.4), dehydrated in increasing concentrations of ethanol and processed for Epon embedding (Glycidether, Roth, Karlsruhe, FRG) through propylene oxide as intermediate. Cross sections of the nerves were cut with glass knives. Semithin (1 am) sections were stained with toluidine blue (0.5 % in 1% sodium borate; ultrathin sections (0.1 ~m) were contrasted with uranyl acetate and lead citrate. Ultrastructural analysis were carried out on a Jeol 100 C and a Jeo1100 SX TEM, at 80 kV. Histochemical detection of calcium by Stoelzner's method was performed on Epon semithin sections. For X-ray microanalysis, 0.1 ~m- and 0.2 9m-thick sections were layed on carbon-coated nylon grids (Agar Aids, Cambridge, UK),
547
Fig. 1. Sciatic nerve at 42 months.The endoneurium is locally devoid of cells and shows a granular aspect. Note the presence of dystrophic and degenerated nerve fibers as well as regenerated fibers forming onion bulb-like clusters. Toluidine blue, • 280 Fig. 2. Mineralized deposits within the endoneurium of the sciatic nerve. Toluidine blue, x 280 Fig. 3. Calcification located in very close proximity to a blood vessel wall. Toluidine blue, • 750 Fig. 4. Detection of calcium by Stoelzner's method, resulting in dark staining of the deposits, x 280
548 coated with 0.1% celloidin(Gurr, Poole, UK; in isoamylacetate). Both stained and unstained specimens were used. Analysiswere performed using the TEM mode of a Jeol 100 C microscope equipped with a Kevex microanalysis system and a graphite specimen holder, at a tilting angle of +25 ~
Results
A mineralization of the endoneurium of the sciatic nerve was observed in six rats (two males and four females) out of fourteen animals examined.
Light microscopy On semithin sections, all nerves display considerable fiber degeneration such as dystrophy, splitting of the myelin sheath and reduced fiber density (Fig. 1). Mineralized deposits are located in either the tibial, peroneal or sural branch of the nerve. Crumbled cores as well as severe damage to the knives suggest their toughness. Sizes vary from small, discrete spots identified at electron microscope level to large, irregularly shaped concretions threading in the surrounding endoneurium (Fig. 2). The latter occupy up to a third of the cross-sectional area of the nerve branch. They are also extended longitudinally in the nerve. Concretions are closely bordered by large fibroblasts or macrophages. Compared to intact areas, the cell density is higher and the diameter of the myelinated fibers is reduced in the vicinity of the deposits. The largest deposits enclose nerve fibers, Schwann cells, fibroblasts, macrophages and blood vessels as well as portions of damaged endoneurium containing only a few non-neuronal cells. Some of the deposits are found in close proximity to blood vessels (Fig. 3). Mineralized areas were not stained with toluidine blue but were positive to alcian blue at pH 2.5. The presence of calcium was detected by Stoelzner's method, which resulted in dark staining of the deposits (Fig. 4).
Electron microscopy Very strong contrasting with uranyl acetate and lead citrate as well as brittleness of the mineralized areas under the electron beam made any analysis of their core impossible. Examination of the periphery and of the small deposits showed that they consist of clumps of randomly entangled fibrils, spreading through the endoneurium (Fig. 5).The mean diameter of these fibrils is approximately 5.5 nm. Small clusters of spicules are scattered in the vicinity of larger ones (Fig. 6), which seem to be formed by the growth and merger of the small deposits, like a patchwork. Deposits always appear homogenously stained and no concentric ring structure has been found. The ultrastructural location of the spicules, whenever discernable, is extracellular. No calcified cell has been
observed. Spicules are spread within dense bundles of collagen fibrils (Fig. 7). Large deposits are bordered by longitudinally oriented fibroblastic and macrophagic cells. These tend to constitute a barrier keeping the nerve fibers from the growing concretions. Portions of endoneurium are frequently enclosed in the extensive deposits. They contain small nerve fibers, non-neuronal cells (Schwann cells, fibroblasts and macrophages) as well as thin-walled dilated blood vessels. Regenerating fibers have also been observed. Some necrotic cells are wedged in dense calcified areas. They display a dark cytoplasm containing degenerated organites and numerous vacuoles including mineralized spicules. The latter represent intrusions of the calcified extracellular matrix within these cells. In close proximity to blood vessels, deposits remain in the adventice. In some rare cases, necrotic endothelial cells have been observed. Evidence of a specific relationship with the endothelium or with the basal lamina has not been found.
Microanalysis X-ray dispersion analysis allowed us to study the chemical composition of the deposits, at electron microscopic level. As contrasting of the sections can modify the calcium/phosphorus ratio [1], unstained samples have also been examined. A typical spectrum is shown in Fig. 8. It confirms the presence of calcium within the concretions and reveals a possible association with phosphorus. This pattern is strictly limited to the mineralized areas, even the small ones. No gradient of calcium is detected in the surroundings. The calcium/phosphorus ratio varies from 1.9 to 2.6, which is similar to the one of hydroxyapatite (Ca/P ratio = 2 to 2.6 [131).
Discussion
Calcifications of the endoneurium of the peripheral nerves have rarely been reported. In diabetic patients, King et al. [7] found very occasional small deposits close to the Schwann cells basal lamina. The concretions observed in the present report ultrastructurally resemble other ectopic calcifications of the nervous system. They are formed by aggregation of spicules which remain in the extracellular space but are not limited to the adventice of the blood vessels. Unlike the endoneurial deposits described by King et al. [7], ring structures are not observed. Histochemical features differ from those of Morgan et al. [9]: they are not stained by toluidine blue but are positive to alcian blue. This could be due to differences in the tissue fixation and processing as well as in the composition of the substrate. Staining of the concretions with alcian blue suggests that calcium deposition occurs on a polysaccharide matrix. The concentration ratio of calcium and phosphorus shows that the deposits probably consist of hydroxyapatite.
549
Fig. 5. Electron micrograph of the periphery of a mineralized deposit. Dense deposits are formed by random aggregation of spicules. Uranyl acetate-lead citrate, x 54,000 Fig. 6. Small clusters of spicules are scattered in the vicinity of larger ones. Uranyl acetate-lead citrate, x 3,200 Fig. 7. Spicules are located in dense collagen bundles. Uranyl acetate-lead citrate, • 39,000
Calcifications of the peripheral nervous system have mainly b e e n attributed to metabolic disturbances. Their incidence increases in cases of diabetes [7] and chronic renal insufficiency [12]. According to a joint study focused on the kidney [2], no sign of n e p h r o p a t h y is
found in the SPF Wistar Louvain rats, even at 42 months. Glucosuria remains stable but renal losses of calcium and phosphorus increase in these rats. This suggests a possible p e r t u r b a t i o n of the homeostasis of calcium.We, therefore, cannot exclude a modification of
550 Ca
P
Ca
Fig. 8. X-ray microanalysis spectrum of a calcified area shows peaks for calcium and phosphorus. P = Phosphorus Ka; Ca = Calcium Kct and K[3. Unstained sample
the ionic concentrations and/or the p H of the endoneurial compartment, which could trigger spontaneous calcification of the tissue [13]. The blood vessels of the sciatic nerve act as an impermeable barrier for large macromolecules [10]. Their permeability is known to increase during experimental Wallerian degeneration [18]. Adventice of the blood vessels are frequently thickened and fiber degeneration is very extensive in the sciatic nerve of our rats. The permeability of the blood-nerve barrier could, therefore, be modified. This alteration could result in transudation of proteinaceous blood components which constitute an organic matrix susceptible to undergo calcification [4]. This is supported by the location of some of the deposits in the vicinity of the blood vessels. Altered blood vessel walls and free erythrocytes have been observed in the damaged endoneurium but whether this is a preliminary step or the result of the mineralization remains unknown. A n o t h e r factor likely to be involved in the mineralization of the extracellular space is the degeneration of the myelinated nerve fibers, which is very frequent in senescent rats. This process results in the breakdown of membranes into free fatty acids. A m o n g these, phosphatidylserine is known to bind spontaneously and strongly to calcium (for review see [13]). Mineralization sites can be created by binding of phosphatidylserine to different components of the extracellular matrix, such as the collagen fibrils, and lead to their calcification. This process seems to be involved in the calcification of the perineurium of peripheral nerves, where the occurence of the deposits have been found to be related to the accumulation of lipids and the breakdown of the nerve fibers. Moreover, the presence of lipids in the perineurium is correlated with age. These could derive from the disintegration of fibroblasts, macrophages or myelin within the endoneurium, which have further been transported into the perineurium [17].This translocation does not seem to take place in the sciatic nerve of the senescent SPF rats studied in this report since calcification has only been detected in the endoneurium.
Animals bred in SPF conditions have a longer mean life-span (35 to 39 months vs 29 months for conventionally bred rats). Although the aging process seems to be slightly delayed, examination of the oldest SPF rats allowed us to describe unusual alterations in the peripheral nervous system. Mineralization of the endoneurium of the sciatic nerve has never been reported in conventionally bred animals of the same strain. This could be explained by the fact that their maximal life-time (about 32-34 months) is much shorter than 42 months and thus does not allow the terminal degeneration pattern to develop. Calcification of the nerve evidently constitutes the result of an extreme degeneration related to the ageing process. Due to their extensive size, mineralized deposits should affect the normal function of the nerve by spatial obstruction and compression, and also by disturbing the homeostasis and the metabolic exchanges between the different compartments of the nerve.
We are indebted to Prof. B. Delmon for the use of the X-ray microanalysis equipment as well as to Dr. L. Daza, Mr. M. Genet and Mr. E. Chain for their kind help and useful advice during these investigations. We wish to thank Mr. E Desneux for his expert technical assistance.
Acknowledgements.
References 1. Arsenault AL, Hunziker EB (1988) Electron microscopic analysis of mineral deposits in the calcifying epiphyseal growth plate. Calcif Tissue Int 42:119-126 2. Dodane V, Chevalier J, Bariety J, Pratz J, Corman B (1990) Longitudinal study of solute excretion and glomerular ultrastructure in an experimental model of aging rats, free of kidney disease. Lab Invest (in press) 3. Fraser H (1968) Bilateral thalamic calcification in the ageing mice. J Pathol Bacteriol 96:220-222 4. Haymaker W, Adams RD (eds) (1982) Histology and histopathology of the nervous system. Charles C. Thomas, Springfield, pp 2597 5. Kaissling B, Bachman S, Kriz W (1985) Structural adaptation of the distal convoluted tubule to prolonged furosemide treatment. Am J Physiol 248:F374 6. Kalimo H, Maki J, Paetau A, Haltia M (1981) Microanalysis of perineurial calcification in diabetic nephropathy. Muscle Nerve 4:228-233 7. King RH, Llewelyn JG, Thomas PK, Gilbey SG, Watkins PJ (1988) Perineurial calcification. Neuropathol Appl Neurobiol 14:105-123 8. Mann DM (1988) Calcification of the basal ganglia in Down's syndrome and Alzheimer's disease. Acta Neuropathol 76: 595-598 9. Morgan KT, Johnson BE Frith CH, Townsend J (1982) An ultrastructural study of spontaneous mineralization in the brains of aging mice. Acta Neuropathol (Berl) 58:120-124 10. OlssonY, Kristensson K, Klatzo I (1971) Permeability of blood vessels and connective tissue sheaths in the peripheral nervous system to exogenous proteins. Acta Neuropathol (Bed) [Suppl] V: 61-69 11. Oster S, Reske-Nielsen E, Bruun I (1988) Lafora's disease and brain calcification. Acta Neuropathol 76:532-538 12. Paetau A, Haltia M (1976) Calcification of the perineurium- a case report. Acta Neuropathol (Berl) 36:185-191
551 13. Posner AS (1987) Bone mineral and the mineralization process. In: Peck WA (ed) Bone and mineral research, vol 5. Elsevier Science, Amsterdam, pp. 65-116 14. Reske-Nielsen E, Jensen PKA, Hein-Soresen O, Abelskov K (1988) Calcification of the central nervous system in a hereditary neurological syndrome. Acta Neuropathol (Berl) 75:590-596 15. Saal JR, Coombe IF, Thomas BW, Tonge JI, Burry AF (1978) Cerebellar calcification - ultrastructure and histochemistry. Pathology 10:351-363
16. Tegner R, Tome FMS, Godeau P, Lhermitte F, Fardeau M (1988) Morphological study of peripheral changes induced by chloroquine treatment. Acta Neuropathol (Berl) 75: 253-260 17. Van Lis JMJ, Jennekens FGI, Veldman H (1979) Calcium deposits in the perineurium and their relation to lipid accumulation. J Neurol Sci 43:367-375 18. Weerasuriya A, Rapoport SI, Taylor RE (1980) Perineurial permeability increases during Wallerian degeneration. Brain Res 192:581-585
Heuropathologga O Springer-Verlag 1991
Spontaneous mineralization of the sciatic nerve of senescent rats* E. Terao I, B. Corman 2, and P. van den Bosch de Aguilar I 1 Laboratoire de Biologie Cellulaire, Universit6 Catholique de Louvain, Place Croix du Sud 5, B-1348 Louvain-la-Neuve, Belgium 2 Service de Biologie Cellulaire, D6partement de Biologie, Centre d'Etudes Nucl6aires de Saclay, F-91191 Gif-sur-Yvette, France Received August 9, 1990/Revised, accepted November 27, 1990
Summary. A s p o n t a n e o u s mineralization of the sciatic nerve of senescent specific p a t h o g e n - f r e e - b r e d rats (aged 42 m o n t h s ) is r e p o r t e d . D e p o s i t s were f o u n d in the e n d o n e u r i u m o f different b r a n c h e s o f the nerve at mid-thigh level. T h e y a p p e a r e d as small discrete deposits or as large t u b u l a r - s h a p e d concretions, p r o b a b l y f o r m e d by the g r o w t h and m e r g e r o f the smaller deposits. S o m e of the concretions were f o u n d in close proximity to b l o o d vessels. D e p o s i t s consisted o f dense aggregations of r a n d o m l y e n t a n g l e d spicules spreading within bundles of collagen fibrils. Calcium was d e t e c t e d by histochemistry and X-ray dispersion microanalysis. P h o s p h o r u s was also f o u n d , possibly associated with calcium to f o r m hydroxyapatite. Key words: R a t - Sciatic nerve - Mineralization - A g i n g - X-ray microanalysis
E c t o p i c mineralized deposits have b e e n o b s e r v e d in different regions of the central n e r v o u s system, in various pathological cases, such as lead poisoning [18], L a f o r a ' s disease [11], A l z h e i m e r ' s disease and D o w n ' s s y n d r o m e [8], genetic neurological s y n d r o m e [14], infarcts [4] and as well as in aged mice [3, 9]. T h e r e are fewer reports o n mineralization of the peripheral nervous system. T h e y m a i n l y c o n c e r n alterations of the p e r i n e u r i u m associated with diabetes [7], n e p h r o p a t h y [6, 12, 171, c h l o r o q u i n e t r e a t m e n t [16] and less o f t e n with n o r m a l h u m a n aging [7]. E n d o n e u r i a l small dense calcified bodies have b e e n o b s e r v e d in diabetic h m n a n peripheral nerves b u t are very rare [7]. We describe here an extensive s p o n t a n e o u s mineralization of the e n d o n e u r i u m of the sciatic nerve of * Supported by grants from the FRFC and carried out within the framework of the Commission of the European Communities Concerted Action on Ageing and Diseases (EURAGE). E.T. was supported by a grant from the IRSIA. Offprint requests to: E. Terao (address see above)
senescent specific p a t h o g e n - f r e e (SPF) rats, aged 42 m o n t h s . Ultrastructural studies were carried out at light and electron m i c r o s c o p e level. H i s t o c h e m i s t r y and e n e r g y dispersive X-ray microanalysis were used to detect the p r e s e n c e of calcium in these deposits.
Material and methods Wistar Louvain rats were bred in the SPF husbandry of the C.E.N. (Saclay, Gif-sur-Yvette, France). They were maintained on a 14 h: 10 h light: dark cycle, at 20 ~ and at 50 % humidity. Food and water were provided ad libitum. The mean life span of the colony (50 % survival) was over 35 months for males and 39 months for females. Maximal life span was not determined as the study ended when the animals reached 42 months. Fourteen animals (2 males and 12 females) were killed at the age of 42 months. A study of the kidney was als0 undertaken using the same material. Animals were anesthetized by an intraperitoneal injection of Inactine (100 mg/100 g body weight, Byk Gulden, Constance, FRG), placed on a heated table and fixed by perfusion, according to the method of Kaissling et al. [5]. Briefly, a catheter filled with 250 ~tl of a solution containing 2 g/1 procain, 5 mM CaC12and 5,000 IU heparin (Roche, Neuilly-sur-Seine, France) was introduced in the abdominal aorta. Between 100 to 200 ml of the fixative solution warmed at 37 ~ was then perfused under a pressure of 200 mm Hg. The fixative was composed of 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), supplemented with 5 mM CaC12, 0.5 g/1 picric acid and 4 % (v/v) hydroxyethyl starch solution (HAES, Fresenius, Bad Hamburg, FRG). An incision was made in the abdominal vein to allow blood to flow out. After perfusion, mid-thigh portions of the sciatic nerves were dissected and stored overnight in the fixative solution. They were postfixed in osmium tetroxide (2 % in cacodylate buffer 0.1 M, pH 7.4), dehydrated in increasing concentrations of ethanol and processed for Epon embedding (Glycidether, Roth, Karlsruhe, FRG) through propylene oxide as intermediate. Cross sections of the nerves were cut with glass knives. Semithin (1 am) sections were stained with toluidine blue (0.5 % in 1% sodium borate; ultrathin sections (0.1 ~m) were contrasted with uranyl acetate and lead citrate. Ultrastructural analysis were carried out on a Jeol 100 C and a Jeo1100 SX TEM, at 80 kV. Histochemical detection of calcium by Stoelzner's method was performed on Epon semithin sections. For X-ray microanalysis, 0.1 ~m- and 0.2 9m-thick sections were layed on carbon-coated nylon grids (Agar Aids, Cambridge, UK),
547
Fig. 1. Sciatic nerve at 42 months.The endoneurium is locally devoid of cells and shows a granular aspect. Note the presence of dystrophic and degenerated nerve fibers as well as regenerated fibers forming onion bulb-like clusters. Toluidine blue, • 280 Fig. 2. Mineralized deposits within the endoneurium of the sciatic nerve. Toluidine blue, x 280 Fig. 3. Calcification located in very close proximity to a blood vessel wall. Toluidine blue, • 750 Fig. 4. Detection of calcium by Stoelzner's method, resulting in dark staining of the deposits, x 280
548 coated with 0.1% celloidin(Gurr, Poole, UK; in isoamylacetate). Both stained and unstained specimens were used. Analysiswere performed using the TEM mode of a Jeol 100 C microscope equipped with a Kevex microanalysis system and a graphite specimen holder, at a tilting angle of +25 ~
Results
A mineralization of the endoneurium of the sciatic nerve was observed in six rats (two males and four females) out of fourteen animals examined.
Light microscopy On semithin sections, all nerves display considerable fiber degeneration such as dystrophy, splitting of the myelin sheath and reduced fiber density (Fig. 1). Mineralized deposits are located in either the tibial, peroneal or sural branch of the nerve. Crumbled cores as well as severe damage to the knives suggest their toughness. Sizes vary from small, discrete spots identified at electron microscope level to large, irregularly shaped concretions threading in the surrounding endoneurium (Fig. 2). The latter occupy up to a third of the cross-sectional area of the nerve branch. They are also extended longitudinally in the nerve. Concretions are closely bordered by large fibroblasts or macrophages. Compared to intact areas, the cell density is higher and the diameter of the myelinated fibers is reduced in the vicinity of the deposits. The largest deposits enclose nerve fibers, Schwann cells, fibroblasts, macrophages and blood vessels as well as portions of damaged endoneurium containing only a few non-neuronal cells. Some of the deposits are found in close proximity to blood vessels (Fig. 3). Mineralized areas were not stained with toluidine blue but were positive to alcian blue at pH 2.5. The presence of calcium was detected by Stoelzner's method, which resulted in dark staining of the deposits (Fig. 4).
Electron microscopy Very strong contrasting with uranyl acetate and lead citrate as well as brittleness of the mineralized areas under the electron beam made any analysis of their core impossible. Examination of the periphery and of the small deposits showed that they consist of clumps of randomly entangled fibrils, spreading through the endoneurium (Fig. 5).The mean diameter of these fibrils is approximately 5.5 nm. Small clusters of spicules are scattered in the vicinity of larger ones (Fig. 6), which seem to be formed by the growth and merger of the small deposits, like a patchwork. Deposits always appear homogenously stained and no concentric ring structure has been found. The ultrastructural location of the spicules, whenever discernable, is extracellular. No calcified cell has been
observed. Spicules are spread within dense bundles of collagen fibrils (Fig. 7). Large deposits are bordered by longitudinally oriented fibroblastic and macrophagic cells. These tend to constitute a barrier keeping the nerve fibers from the growing concretions. Portions of endoneurium are frequently enclosed in the extensive deposits. They contain small nerve fibers, non-neuronal cells (Schwann cells, fibroblasts and macrophages) as well as thin-walled dilated blood vessels. Regenerating fibers have also been observed. Some necrotic cells are wedged in dense calcified areas. They display a dark cytoplasm containing degenerated organites and numerous vacuoles including mineralized spicules. The latter represent intrusions of the calcified extracellular matrix within these cells. In close proximity to blood vessels, deposits remain in the adventice. In some rare cases, necrotic endothelial cells have been observed. Evidence of a specific relationship with the endothelium or with the basal lamina has not been found.
Microanalysis X-ray dispersion analysis allowed us to study the chemical composition of the deposits, at electron microscopic level. As contrasting of the sections can modify the calcium/phosphorus ratio [1], unstained samples have also been examined. A typical spectrum is shown in Fig. 8. It confirms the presence of calcium within the concretions and reveals a possible association with phosphorus. This pattern is strictly limited to the mineralized areas, even the small ones. No gradient of calcium is detected in the surroundings. The calcium/phosphorus ratio varies from 1.9 to 2.6, which is similar to the one of hydroxyapatite (Ca/P ratio = 2 to 2.6 [131).
Discussion
Calcifications of the endoneurium of the peripheral nerves have rarely been reported. In diabetic patients, King et al. [7] found very occasional small deposits close to the Schwann cells basal lamina. The concretions observed in the present report ultrastructurally resemble other ectopic calcifications of the nervous system. They are formed by aggregation of spicules which remain in the extracellular space but are not limited to the adventice of the blood vessels. Unlike the endoneurial deposits described by King et al. [7], ring structures are not observed. Histochemical features differ from those of Morgan et al. [9]: they are not stained by toluidine blue but are positive to alcian blue. This could be due to differences in the tissue fixation and processing as well as in the composition of the substrate. Staining of the concretions with alcian blue suggests that calcium deposition occurs on a polysaccharide matrix. The concentration ratio of calcium and phosphorus shows that the deposits probably consist of hydroxyapatite.
549
Fig. 5. Electron micrograph of the periphery of a mineralized deposit. Dense deposits are formed by random aggregation of spicules. Uranyl acetate-lead citrate, x 54,000 Fig. 6. Small clusters of spicules are scattered in the vicinity of larger ones. Uranyl acetate-lead citrate, x 3,200 Fig. 7. Spicules are located in dense collagen bundles. Uranyl acetate-lead citrate, • 39,000
Calcifications of the peripheral nervous system have mainly b e e n attributed to metabolic disturbances. Their incidence increases in cases of diabetes [7] and chronic renal insufficiency [12]. According to a joint study focused on the kidney [2], no sign of n e p h r o p a t h y is
found in the SPF Wistar Louvain rats, even at 42 months. Glucosuria remains stable but renal losses of calcium and phosphorus increase in these rats. This suggests a possible p e r t u r b a t i o n of the homeostasis of calcium.We, therefore, cannot exclude a modification of
550 Ca
P
Ca
Fig. 8. X-ray microanalysis spectrum of a calcified area shows peaks for calcium and phosphorus. P = Phosphorus Ka; Ca = Calcium Kct and K[3. Unstained sample
the ionic concentrations and/or the p H of the endoneurial compartment, which could trigger spontaneous calcification of the tissue [13]. The blood vessels of the sciatic nerve act as an impermeable barrier for large macromolecules [10]. Their permeability is known to increase during experimental Wallerian degeneration [18]. Adventice of the blood vessels are frequently thickened and fiber degeneration is very extensive in the sciatic nerve of our rats. The permeability of the blood-nerve barrier could, therefore, be modified. This alteration could result in transudation of proteinaceous blood components which constitute an organic matrix susceptible to undergo calcification [4]. This is supported by the location of some of the deposits in the vicinity of the blood vessels. Altered blood vessel walls and free erythrocytes have been observed in the damaged endoneurium but whether this is a preliminary step or the result of the mineralization remains unknown. A n o t h e r factor likely to be involved in the mineralization of the extracellular space is the degeneration of the myelinated nerve fibers, which is very frequent in senescent rats. This process results in the breakdown of membranes into free fatty acids. A m o n g these, phosphatidylserine is known to bind spontaneously and strongly to calcium (for review see [13]). Mineralization sites can be created by binding of phosphatidylserine to different components of the extracellular matrix, such as the collagen fibrils, and lead to their calcification. This process seems to be involved in the calcification of the perineurium of peripheral nerves, where the occurence of the deposits have been found to be related to the accumulation of lipids and the breakdown of the nerve fibers. Moreover, the presence of lipids in the perineurium is correlated with age. These could derive from the disintegration of fibroblasts, macrophages or myelin within the endoneurium, which have further been transported into the perineurium [17].This translocation does not seem to take place in the sciatic nerve of the senescent SPF rats studied in this report since calcification has only been detected in the endoneurium.
Animals bred in SPF conditions have a longer mean life-span (35 to 39 months vs 29 months for conventionally bred rats). Although the aging process seems to be slightly delayed, examination of the oldest SPF rats allowed us to describe unusual alterations in the peripheral nervous system. Mineralization of the endoneurium of the sciatic nerve has never been reported in conventionally bred animals of the same strain. This could be explained by the fact that their maximal life-time (about 32-34 months) is much shorter than 42 months and thus does not allow the terminal degeneration pattern to develop. Calcification of the nerve evidently constitutes the result of an extreme degeneration related to the ageing process. Due to their extensive size, mineralized deposits should affect the normal function of the nerve by spatial obstruction and compression, and also by disturbing the homeostasis and the metabolic exchanges between the different compartments of the nerve.
We are indebted to Prof. B. Delmon for the use of the X-ray microanalysis equipment as well as to Dr. L. Daza, Mr. M. Genet and Mr. E. Chain for their kind help and useful advice during these investigations. We wish to thank Mr. E Desneux for his expert technical assistance.
Acknowledgements.
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