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Ultrastructural study of the latent effects of methyl mercury on the nervous system after prenatal exposure
Ultrastructural Study of the Latent Effects of Methyl Mercury on the Nervous System After Prenatal Exposure
Received
October
7. 197.5
Tissue samples from the cerebellum were obtained for electron microscopy from mice which were prenatally exposed to methyl mercury. Lysosomal accumulation was observed in both the Purkinje neurons and granule cells. Osmiophilic lipoid material was evident within these lysosomes. Giant-sized lysosomes were also found occasionally. Irregular tubular structures resembling smooth endoplasmic reticulum were also found within many neurites. Some of these tubular structures appeared to be hypertrophied and contained some electronopaque materials. Segmental incomplete myelination and abnormal synaptic terminals were also observed in the tissue samples. Such defects may contribute to the behavioral deviation and mental deficiencies of these animals.
INTRODUCTION
Since the outbreak of Minamata disease (methyl mercury intoxication) in Japan, mercury, particularly methyl mercury, is recognized as one of the most hazardous environmental pollutants. Several studies have demonstrated the rapid transplacental movement of methyl mercury (Berlin and Ullberg, 1963: Suzuki ef nl., 1967; Garcia et (il., 1974) and increasing interest has been drawn to the teratogenic and pathologic effects of methyl mercury on newborns. Although the effects on reproduction, embryonic development, postnatal growth, and behavioral changes on the neonates have been demonstrated after in rrtrvo exposure to methyl mercuric compounds (Beliles et
172
CHANG,
REUHI.,
MATERIALS
AND
SPYKER
AND METHODS
Young pregnant mice (129 SV/SL) were injected with 0.16 mg of methyl mercuric dicyandiamide per 20 g of body weight on the 9th day of pregnancy. Equal volumes of saline solution were administered to control animals. On Day 18, pregnant mice were put in separate cages and allowed to deliver. The young remained with their mother until weaned at 21 days of age. No apparent differences in size and weight were detected between the mercury-exposed animals and the controls. When the animals were 12-1.5 months of age, behavioral deviations and mental deficiencies, such as prolonged reaction time to open-field tests and abnormal swimming behavior, however, could be demonstrated with scientific tests in the mercuryexposed young mice (Spyker rt crl., 1972). For electron microscopy, six animals which had demonstrated behavioral deviations were anesthetized with Nembutal and were perfused intracardially with Karnovsky’s fixative (0.5% glutaraldehyded% paraformaldehyde). Tissue samples from the cerebellum (being most sensitive to methyl mercury toxicity) were obtained with a sharp blade, cut into l-mm cubes, and further fixed in Karnovsky’s FIG. I. Purkinje neuron, methyl mercury-exposed dense bodies. presumably lysosomes (Ly), was lysosomal bodies contained osmiophilic lipoid (RER). x 14.500 FIG. 2. Purkinje neuron, methyl mercury-exposed some neurons had lost their long. parallel array nuclear pores (NP). x 10.500 FIG. 3. Cerebellum. methyl mercury-exposed Large osmiophilic lipoid material was associated Frc;. 4. Cerebellum. taining membranous FIG. tubular
methyl material
5. Cerebellum. methyl structures resembling
Fro. 6. Cerebellum. methyl smooth endoplasmic reticulum hypertrophied and contained
mouse. Accumulation of large, membrane-limited, observed within the neuronal cytoplasm. Some ofthese material. Nucleus tN). rough endoplasmic reticulum mouse. The rough endoplasmic reticulum (RER) of and became shortened and disoriented. Nucleus (N), mouse. Lysosomal accumulation with these lysosomes (Ly).
mercury-exposed mouse. A giant-sized lysosome was found in a granule cell (G). x 31.000
methyl (tSER)
FIG. 9. Cerebellum. methyl nerve fiber. The myelin sheath myelin sheath (M,) of the other seen. Y 35,000 FIG:. 10. Cerebellum. methyl of postsynaptic density (+). FIG. 11. Cerebellum. methyl synaptic density at all points FIN,. 12. Cerebellum. methyl a reduction (arrowhead) and
cell (G).
(cytophagosome)
mercury-exposed mouse. A large neurite containing smooth endoplasmic reticulum (tSER). x 25.000
large
con-
amounts
of
mercury-exposed mouse. Accumulation of tubular structures resembling (tSER) in a large neurite. Some of these tubular structures appeared to be some electron-opaque material (4). x 45,000
FIG. 7. Cerebellum, methyl mercury-exposed mouse. Cross section many smooth endoplasmic reticulum-like tubular structures (tSER). tures was also observed. x 31,000 FIG. 8. Cerebellum, endoplasmic reticulum
in agranule 22,000
x
mercury-exposed within an axon.
ofa large neurite which contained Dilatation of some of these struc-
mouse. Accumulation X 31.000
of some dilated
tubular
smooth
mercury-exposed mouse. Segmental thinning of the myelin sheath in a (MZ) of one segment of the nerve fiber was significantly thinner than the segment. Degenerative changes (DC) near the node of Ranvier were also mercury-exposed mouse. An abnormal synaptic terminal showing a lack ‘K 45,000 mercury-exposed mouse. A large synaptic terminal showing no postof contact (--). x 42.000 mercury-exposed mouse, A large mossy fiber synaptic terminal showing a total lack of postsynaptic density at synaptic junctions (4). ): 42,000
Frt;. 13. Cerebellum. normal control tic pattern with prominent postsynaptic
mouse. A large mossy fiber terminal densities (4). x 35,000
showing
normal
multisynap-
Ftc.. 14. Cerebellum. methyl mercury-exposed mouse. Swelling of the endothelial cell (SEC) was observed in some capillaries (Cap). Such endothelial cells had an electron lucid appearance. X 21.000
EFFECTS
OF
METHYL
MERCURY
ON
CEREBELLUM
173
174
CHANG,
RF.UHL,
AND
SPYKER
EFFECTS
OF
METHYL
MERCURY
ON
CERERF.L.LUM
EFFECTS
OF
METHYL
MERCURY
ON
CEREBELLUM
177
178
CHANG,
REUHL,
AND
SPYtiER
EFFECTS
OF METHYL
MERCURY
ON
CEREBELLU.M
179
EFFECTS
OF
METHYL
MFRCURY
ON
CEREHELLL!Yl
181
fixative for 2 hours at 4°C. The tissue samples were then postfixed in phosphate buffered 1% osmium tetroxide for 1% hours, dehydrated with graded ethanol. and embedded in Epon. Thin sections were cut with an LKB Ultratome III automatic ultramicrotome, counter stained with lead citrate and many1 acetate, and examined with an RCA EMU-3G electron microscope. RESULTS
An accumulation of electron-dense bodies, presumably lysosomes, were observed in both Purkinje neurons (Fig. 1) and granule cells (Fig. 3). Many of these lysosomal bodies appeared to contain osmiophilic lipoid material. Many Purkinje neurons also showed a disorientation of the rough endoplasmic reticulum which appeared to be shortened and to have lost its regular parallel array (Fig. 2). Formation of giant-sized lysosomes in some neurons (Fig. 4) was also observed. Accumulation of irregular tubular structures which had the profile of smooth endoplasmic reticulum was observed in many neurites (Fig. 5). Some of these tubular structures appeared to be hypertrophied, containing electron-opaque materials (Fig. 6). Dilatation and vacuolation of such structures (Figs. 7, 8) were also evident within many neuronal processes. Segmental atrophy of the axon and thinning of the myelin sheath were found in some nerve fibers (Fig. 9). Degenerative changes at the node of Ranvier of those fibers could also be observed (Fig. 9). Abnormal synaptic complexes, displaying a reduction or lack of postsynaptic densities (Figs. 10, 11, 12) as compared to those in
182
CHANG,
REUHL,
AND
SPYKEK
the normal animals (Fig. 13), were frequently observed. Since such abnormal appearance of the synapses were a consistent and frequent finding in the mercuryexposed animals, it was not believed to be an artifact due to tissue sectioning or other means. Swelling of the endothelial cells of the capillaries (Fig. 14) was occasionally found indicating injuries of the cerebral vasculature. DISCUSSION
Among 111 cases of Minamata disease, 19 were encountered in newborns (Iukayama, 1969). Tejining (1967) demonstrated that methyl mercury crossed the human placenta readily and had a preferential concentration in the fetus over the maternal tissues. Similar findings were observed with mice (Suzuki et al., 1967) where 30% higher mercury concentration may be attained in fetal erythrocytes than in maternal ones. Furthermore, exposing pregnant rats to a single high dose of methyl mercury resulted in a four fold greater concentration of mercury in the fetal brain than in the maternal brain (Matsumato et al., 1967). The facilitated placental transfer and increased fetal tissue binding of methylated mercury raised the possibility that the greater risk for mercury to the newborn may be due, in part, to fetal mercury “trapping.” The vulnerability of the cerebellum towards the toxicity of methyl mercury had been repeatedly demonstrated in human cases (Hunter et al., 1940; Takeuchi et al., 1962) and in experimental animals (Brown and Yoshida, 1965; Miyakawa and Deshimaru, 1969; Chang and Hartmann, 1972a). Wistar rats, when given a single oral dose of methylmercuric chloride at doses of 2 or 20 mg/kg on Day 9, 10, or 11 of pregnancy, showed cerebellar changes (Matsumato et al. q 1967). The specific vulnerability of the developing cerebellum towards mercury toxicity was also demonstrated in newborn cats (Morikawa, 1961; Kheraand Clegg, 1969). By means of electron microscopy, the present investigation further documents the long lasting effects of mercury in the nervous system of behaviorally abnormal offspring from methyl mercury-treated mothers. The increase in lysosomal activities, formation of giant sized lysosomes, and disintegration of the rough endoplasmic reticulum probably denote early signs of cellular degeneration of the neurons. Segmental demyelination has been described in lead intoxication (Schlaepfer, 1969) and in mercury neuropathy (Chang and Hartmann, 1972b). Reduction or thinning of the myelination has also been observed in lead-intoxicated developing rats (Krigman et ul., 1974). In the present investigation, segmental thinning of the myelin sheath in the nerve fibers was observed. Such segmental thinning of the myelin may represent defective myelination or incomplete remyelination of the nerve fiber and might contribute to the eventual malfunctioning of the nerve fiber. It has been postulated that in heavy metal-induced neuropathy, particularly those of lead and mercury, the earliest pathological changes in the nerve fibers are initiated at the node of Ranvier (Schlaepfer, 1969; Miyakawart al., 1970; Chang and Hartmann, 1972b). Although no extensive axonal degeneration was observed, accumulation of tubular structures resembling smooth endoplasmic reticulum were observed in many neurites. The precise significance of the accumulation of these tubular structures, however, is still not understood at the present time. Degenera-
EFFECTS
OF
METHYL
MERCURY
ON
183
CEREBELLUM
tive changes could be demonstrated in the nodes of Ranvier in some of the nerve fibers of the animals in the present investigation. The malformation of some of the synaptic complexes was an interesting observation. Although no morphological abnormality was observed in the presynaptic terminals and synaptic vesicles, reduction or absence of the postsynaptic density was evident in many synaptic complexes. Similar synaptic abnormality was reported in hypothyroid animals (Brown et ~1.. 1974) and halothane-intoxicated neonatal rats (Quimby et al., 1974) which also demonstrated behavioral abnormality and mental deficiency. Therefore, such synaptic abnormality, together with the pathological changes in myelination. may contribute to some of the subtle mental changes in these animals (Spyker et cd., 1972). The swollen endothelial cells had an electron-lucid appearance. Admixture of electron lucent and electron-dense endothelial cell processes form continuous cellular barrier. Similar electron lucent endothelial cells were reported in vasculature of cadmium-intoxicated animals (Schlaepfer, 1971) and was believed to represent a direct toxic effect of the heavy metal on the endothelial cells. Disturbance of the blood-brain barrier by mercuric compounds had been reported by various investigators (Steinwall and Olsson, 1969: Chang and Hartmann, 1972~; Ware et (11.. 1974). It is, therefore, not totally surprising to observe endothelial damages in the present investigation. In view that the blood-brain barrier functions as a regulator of metabolic exchange between the blood and brain (Steinwall, 1961: Lajtha, 1962; Broman and Steinwall, 1967), the impairment of the endothelial cells may denote an important relationship of the blood-brain barrier dysfunctions to the subsequent functional, metabolic, and morphologic changes observed in the central nervous system following methyl mercury intoxication. ACKNOWLEDGMENT We would like to thank Mrs. Fran Simandl supported by NSF Grant BMS 74-17927.
for her excellent
technical
assistance.
This
project
is
REFERENCES Beliles. R. P., Clark, R. S.. and Yuile, C. L. (1968). The effects of exposure to mercury vapor on behavior of rats. To.uic,o/. Appl. Ph).v~wo/. 12, 15-71 Berlin. M., and Ullberg, S. (1963). Accumulation and retention of mercury in the mouse. .4rc,lr. Erri,iror~. Hdtl~
6, 5894
16.
Broman, T., and Steinwall. 0. (1967). Blood-brain barrier. I/I “Pathology of the Nervous System” (.I. Minckler. Ed.), Vol. 1, Chap. 33. Blakiston. New York. Brown. W. J., Akers. M.. and Verity, M. A. t 1974). Biochemical and ultrastructural aspects ofcerebellar synaptogenesis in thyroid deficiency. Anrc~r. J. P~rthol. 74, la-2a. Brown, W. J.. and Yoshida, N. (1965). Organic mercurial encephalopathy-an experimental electron microscopic study. J. Co/q’. Ncrrrol. SC,;. 9, 34-42. Chang. L. W.. and Hartmann. H. A. (l972a). Ultrastructural studies ofthe nervous system after mercury intoxication. I. Pathological changes in the nerve cell bodies. Ac,rtr ,?I~~~rop~~~lwl. 20, 122-138. Chang. L. W.. and Hartmann. H. A. (1972b). Ultrastructural studies of the nervous system after mercury intoxication. II. Pathological changes in the nerve fibers. Acftr Nc~un~ptrtho/. 20, 3 16334. Chang. L. W.. and Hartmann, H. A. (1972~). Blood-brain barrier dysfunction in experimental mercury intoxication. ,A<‘ttr Vrrrropcctltr~l. 21, 179-184. Chang. L. W.. Reuhl. K. R.. and Lee. G. W. (1977). Degenerative changes in the developing nervous system as a result of in utero exposure to methyl mercury. Eui~ir~~n. Res. In press.
184
CHANG,
RECHL,
AND
SPYKER
Gale, T. F.. and Ferm. V. H. (1971). Embryopathic effects of mercuric salts. Lijk Sci. 10 (Part II), 1341-134s. Garcia, J. D.. Yang. M. G.. Wang, J. H. C., and Belo, P. S. (1974). Translocation and fluxes of mercury in neonatal and maternal rats treated with methyl mercuric chloride during gestation, ~roc. 50~. Exp. Biol. ,Mrd. 147, 224-231. Hunter, D., Bomford, R. R. and Russell, D. S. (1940). Poisoning by methyl mercury compounds. punrf. .I. jlfed. 33, 193-213. Iukayama, K. (1969). The pollution of Minamata Bay and Minamata disease. A~Ircln. Po//lrf. &J. 3, 153-159. Khera, K. S.. and Clegg. D. J. (1969). Perinatal toxicity of pesticides. Crrntrd. Med. Assuc. J. 100, 167-172. Khera, K. S., and Tabacova. S. A. (1973). Effects of methylmercuric chloride on the progeny of mice and rats treated before or during gestation. Food Cosmef. Tr,.ricof. 11, 245-254. Krigman, M. R., Druse. M. J., Traylor, T. D., Wilson. M. H.. Newell. L. R., and Hogan, E. L. (1974). Lead encephalopathy in the developing rat: effect upon myelination./. Neuropathol. E.up. Neural. 33, 5873. Lajtha, A. (1962). The blood-brain barrier system. Irr “Neurochemistry” K, A. C. Elliot, I. H. Page, and J. H. Quastel, (Eds.), 2nd ed. pp. 399-l30. Charles C Thomas, Springfield, Ill. Matsumato, H.. Suzuki, A., Monta. C., Nakamura. K., and Salki, S. (1967). Preventive effect of penicillamine on the brain effect of fetal rat poisoned transplacentally with methyl mercury, Life Sci. 6, 2321-2330. Miyakawa. T.. and Deshimaru, M. (1969). Electron microscopic study of experimentally induced poisoning due to organic mercury compound. Actor Nerrroparhol. 14, 126-136. Miyakawa. T., Sumiyoshi, S., Tersoka, A.. Udo. N., Hattori. E., and Tatetsu, L. (1970). Experimental organic mercury poisoning-pathological changes in peripheral nerves. Acttr Neuropafho~. 15, 45-55. Morikawa. H. (1961). Pathological studies on organic mercury poisoning. Kmzamoto Med. J. 14,71-86. Newberne. P. M.. Glaser. 0.. Friedman, L.. and Stillings, B. R. (1972). Chronic exposure of rats to methyl mercury in fish protein. Ntrrrrre (London) 237, 40-41. Nonaka, I. (1969). An electron microscopical study on the experimental congenital Minamata disease in rat. Kl,rr,rrnrofo .Med. J. 22, 2740. Post, E. M., Yang, M. G.. King, J. A., and Sanger, V. L. (1973). Behavioral changes of young rats force-fed methyl mercury chloride. J. E.rp. Biol. .&fed. 143, 1113-I 116. Quimby, K. L., Aschkenase, L. J., Bowman, R. E., Katz, J., and Chang, L. W. (1974). Enduring learning deficits and cerebral synaptic malformation from exposure to IO ppm halothane. Science 185, 625627. Ramel, C. (1969). Methylmercury as a mitosis disturbing agent. J. Jpn. Med. Ass. 61, 1072-1077. Schlaepfer. W. M. (1969). Experimental lead neuropathy: a disease of the supporting cells in the peripheral nervous system. J. Nertroprrthnl. Exp. Neorol. 28, 401418. Schlaepfer, W. M. (1971). Sequential study of endothelial changes in acute cadmium intoxication. Ltrb. Invesr. 25, 556-564. Spyker, J. M. and Smithberg. M. (1972). Effects of methyl mercury on prenatal development in mice. Teratology 5, I8 l-l 90. Spyker. J. M., Sparber, S. B., and Goldberg, A. M. (1972). Subtle consequences of methylmercury exposure: Behavioral deviations in offspring from treated mothers. Science 177, 621622. Steinwall, 0. (1961). Transport mechanisms in certain blood-brain barrier phenomena-a hypothesis. Acta Psychicrt. Nrurol. ScanJ. 36, 3 143 18. Steinwall, O., and Olsson, Y. (1969). Impairment ofthe blood-brain barrier in mercury poisoning. Acta Neural. Sctrnd. 45, 351-361. Suzuki. T.. Matsumoto, H.. Miyama, T.. and Katsunuma. H. (1967). Placental transfer of mercuric chloride. phenyl mercury acetate, and methyl mercury acetate in mice. Itzd. Healrh 5, 149-155. Takeuchi. T., Matsumoto, H., and Shirashi, Y. (1962). A pathological study of Minamata disease in Japan. Actor Neuroputhol. 2, 40-57.
EFFECTS
OF
METHYL
MERCURY
ON
CEREBELLUM
18.5
Tejining. S. (1967). Mercury content and distribution in dead and malformed embryos and in extraembryonic membranes. O&YH Suppl. 8, 60-66. Ware, R. A.. Chang. L. W.. and Burkholder, P. M. (1974). An ultrastructural study on the blood-brain barrier dysfunction following mercury intoxication. Acta Newopc~thol. 30, 21 I-224.
Received
October
7. 197.5
Tissue samples from the cerebellum were obtained for electron microscopy from mice which were prenatally exposed to methyl mercury. Lysosomal accumulation was observed in both the Purkinje neurons and granule cells. Osmiophilic lipoid material was evident within these lysosomes. Giant-sized lysosomes were also found occasionally. Irregular tubular structures resembling smooth endoplasmic reticulum were also found within many neurites. Some of these tubular structures appeared to be hypertrophied and contained some electronopaque materials. Segmental incomplete myelination and abnormal synaptic terminals were also observed in the tissue samples. Such defects may contribute to the behavioral deviation and mental deficiencies of these animals.
INTRODUCTION
Since the outbreak of Minamata disease (methyl mercury intoxication) in Japan, mercury, particularly methyl mercury, is recognized as one of the most hazardous environmental pollutants. Several studies have demonstrated the rapid transplacental movement of methyl mercury (Berlin and Ullberg, 1963: Suzuki ef nl., 1967; Garcia et (il., 1974) and increasing interest has been drawn to the teratogenic and pathologic effects of methyl mercury on newborns. Although the effects on reproduction, embryonic development, postnatal growth, and behavioral changes on the neonates have been demonstrated after in rrtrvo exposure to methyl mercuric compounds (Beliles et
172
CHANG,
REUHI.,
MATERIALS
AND
SPYKER
AND METHODS
Young pregnant mice (129 SV/SL) were injected with 0.16 mg of methyl mercuric dicyandiamide per 20 g of body weight on the 9th day of pregnancy. Equal volumes of saline solution were administered to control animals. On Day 18, pregnant mice were put in separate cages and allowed to deliver. The young remained with their mother until weaned at 21 days of age. No apparent differences in size and weight were detected between the mercury-exposed animals and the controls. When the animals were 12-1.5 months of age, behavioral deviations and mental deficiencies, such as prolonged reaction time to open-field tests and abnormal swimming behavior, however, could be demonstrated with scientific tests in the mercuryexposed young mice (Spyker rt crl., 1972). For electron microscopy, six animals which had demonstrated behavioral deviations were anesthetized with Nembutal and were perfused intracardially with Karnovsky’s fixative (0.5% glutaraldehyded% paraformaldehyde). Tissue samples from the cerebellum (being most sensitive to methyl mercury toxicity) were obtained with a sharp blade, cut into l-mm cubes, and further fixed in Karnovsky’s FIG. I. Purkinje neuron, methyl mercury-exposed dense bodies. presumably lysosomes (Ly), was lysosomal bodies contained osmiophilic lipoid (RER). x 14.500 FIG. 2. Purkinje neuron, methyl mercury-exposed some neurons had lost their long. parallel array nuclear pores (NP). x 10.500 FIG. 3. Cerebellum. methyl mercury-exposed Large osmiophilic lipoid material was associated Frc;. 4. Cerebellum. taining membranous FIG. tubular
methyl material
5. Cerebellum. methyl structures resembling
Fro. 6. Cerebellum. methyl smooth endoplasmic reticulum hypertrophied and contained
mouse. Accumulation of large, membrane-limited, observed within the neuronal cytoplasm. Some ofthese material. Nucleus tN). rough endoplasmic reticulum mouse. The rough endoplasmic reticulum (RER) of and became shortened and disoriented. Nucleus (N), mouse. Lysosomal accumulation with these lysosomes (Ly).
mercury-exposed mouse. A giant-sized lysosome was found in a granule cell (G). x 31.000
methyl (tSER)
FIG. 9. Cerebellum. methyl nerve fiber. The myelin sheath myelin sheath (M,) of the other seen. Y 35,000 FIG:. 10. Cerebellum. methyl of postsynaptic density (+). FIG. 11. Cerebellum. methyl synaptic density at all points FIN,. 12. Cerebellum. methyl a reduction (arrowhead) and
cell (G).
(cytophagosome)
mercury-exposed mouse. A large neurite containing smooth endoplasmic reticulum (tSER). x 25.000
large
con-
amounts
of
mercury-exposed mouse. Accumulation of tubular structures resembling (tSER) in a large neurite. Some of these tubular structures appeared to be some electron-opaque material (4). x 45,000
FIG. 7. Cerebellum, methyl mercury-exposed mouse. Cross section many smooth endoplasmic reticulum-like tubular structures (tSER). tures was also observed. x 31,000 FIG. 8. Cerebellum, endoplasmic reticulum
in agranule 22,000
x
mercury-exposed within an axon.
ofa large neurite which contained Dilatation of some of these struc-
mouse. Accumulation X 31.000
of some dilated
tubular
smooth
mercury-exposed mouse. Segmental thinning of the myelin sheath in a (MZ) of one segment of the nerve fiber was significantly thinner than the segment. Degenerative changes (DC) near the node of Ranvier were also mercury-exposed mouse. An abnormal synaptic terminal showing a lack ‘K 45,000 mercury-exposed mouse. A large synaptic terminal showing no postof contact (--). x 42.000 mercury-exposed mouse, A large mossy fiber synaptic terminal showing a total lack of postsynaptic density at synaptic junctions (4). ): 42,000
Frt;. 13. Cerebellum. normal control tic pattern with prominent postsynaptic
mouse. A large mossy fiber terminal densities (4). x 35,000
showing
normal
multisynap-
Ftc.. 14. Cerebellum. methyl mercury-exposed mouse. Swelling of the endothelial cell (SEC) was observed in some capillaries (Cap). Such endothelial cells had an electron lucid appearance. X 21.000
EFFECTS
OF
METHYL
MERCURY
ON
CEREBELLUM
173
174
CHANG,
RF.UHL,
AND
SPYKER
EFFECTS
OF
METHYL
MERCURY
ON
CERERF.L.LUM
EFFECTS
OF
METHYL
MERCURY
ON
CEREBELLUM
177
178
CHANG,
REUHL,
AND
SPYtiER
EFFECTS
OF METHYL
MERCURY
ON
CEREBELLU.M
179
EFFECTS
OF
METHYL
MFRCURY
ON
CEREHELLL!Yl
181
fixative for 2 hours at 4°C. The tissue samples were then postfixed in phosphate buffered 1% osmium tetroxide for 1% hours, dehydrated with graded ethanol. and embedded in Epon. Thin sections were cut with an LKB Ultratome III automatic ultramicrotome, counter stained with lead citrate and many1 acetate, and examined with an RCA EMU-3G electron microscope. RESULTS
An accumulation of electron-dense bodies, presumably lysosomes, were observed in both Purkinje neurons (Fig. 1) and granule cells (Fig. 3). Many of these lysosomal bodies appeared to contain osmiophilic lipoid material. Many Purkinje neurons also showed a disorientation of the rough endoplasmic reticulum which appeared to be shortened and to have lost its regular parallel array (Fig. 2). Formation of giant-sized lysosomes in some neurons (Fig. 4) was also observed. Accumulation of irregular tubular structures which had the profile of smooth endoplasmic reticulum was observed in many neurites (Fig. 5). Some of these tubular structures appeared to be hypertrophied, containing electron-opaque materials (Fig. 6). Dilatation and vacuolation of such structures (Figs. 7, 8) were also evident within many neuronal processes. Segmental atrophy of the axon and thinning of the myelin sheath were found in some nerve fibers (Fig. 9). Degenerative changes at the node of Ranvier of those fibers could also be observed (Fig. 9). Abnormal synaptic complexes, displaying a reduction or lack of postsynaptic densities (Figs. 10, 11, 12) as compared to those in
182
CHANG,
REUHL,
AND
SPYKEK
the normal animals (Fig. 13), were frequently observed. Since such abnormal appearance of the synapses were a consistent and frequent finding in the mercuryexposed animals, it was not believed to be an artifact due to tissue sectioning or other means. Swelling of the endothelial cells of the capillaries (Fig. 14) was occasionally found indicating injuries of the cerebral vasculature. DISCUSSION
Among 111 cases of Minamata disease, 19 were encountered in newborns (Iukayama, 1969). Tejining (1967) demonstrated that methyl mercury crossed the human placenta readily and had a preferential concentration in the fetus over the maternal tissues. Similar findings were observed with mice (Suzuki et al., 1967) where 30% higher mercury concentration may be attained in fetal erythrocytes than in maternal ones. Furthermore, exposing pregnant rats to a single high dose of methyl mercury resulted in a four fold greater concentration of mercury in the fetal brain than in the maternal brain (Matsumato et al., 1967). The facilitated placental transfer and increased fetal tissue binding of methylated mercury raised the possibility that the greater risk for mercury to the newborn may be due, in part, to fetal mercury “trapping.” The vulnerability of the cerebellum towards the toxicity of methyl mercury had been repeatedly demonstrated in human cases (Hunter et al., 1940; Takeuchi et al., 1962) and in experimental animals (Brown and Yoshida, 1965; Miyakawa and Deshimaru, 1969; Chang and Hartmann, 1972a). Wistar rats, when given a single oral dose of methylmercuric chloride at doses of 2 or 20 mg/kg on Day 9, 10, or 11 of pregnancy, showed cerebellar changes (Matsumato et al. q 1967). The specific vulnerability of the developing cerebellum towards mercury toxicity was also demonstrated in newborn cats (Morikawa, 1961; Kheraand Clegg, 1969). By means of electron microscopy, the present investigation further documents the long lasting effects of mercury in the nervous system of behaviorally abnormal offspring from methyl mercury-treated mothers. The increase in lysosomal activities, formation of giant sized lysosomes, and disintegration of the rough endoplasmic reticulum probably denote early signs of cellular degeneration of the neurons. Segmental demyelination has been described in lead intoxication (Schlaepfer, 1969) and in mercury neuropathy (Chang and Hartmann, 1972b). Reduction or thinning of the myelination has also been observed in lead-intoxicated developing rats (Krigman et ul., 1974). In the present investigation, segmental thinning of the myelin sheath in the nerve fibers was observed. Such segmental thinning of the myelin may represent defective myelination or incomplete remyelination of the nerve fiber and might contribute to the eventual malfunctioning of the nerve fiber. It has been postulated that in heavy metal-induced neuropathy, particularly those of lead and mercury, the earliest pathological changes in the nerve fibers are initiated at the node of Ranvier (Schlaepfer, 1969; Miyakawart al., 1970; Chang and Hartmann, 1972b). Although no extensive axonal degeneration was observed, accumulation of tubular structures resembling smooth endoplasmic reticulum were observed in many neurites. The precise significance of the accumulation of these tubular structures, however, is still not understood at the present time. Degenera-
EFFECTS
OF
METHYL
MERCURY
ON
183
CEREBELLUM
tive changes could be demonstrated in the nodes of Ranvier in some of the nerve fibers of the animals in the present investigation. The malformation of some of the synaptic complexes was an interesting observation. Although no morphological abnormality was observed in the presynaptic terminals and synaptic vesicles, reduction or absence of the postsynaptic density was evident in many synaptic complexes. Similar synaptic abnormality was reported in hypothyroid animals (Brown et ~1.. 1974) and halothane-intoxicated neonatal rats (Quimby et al., 1974) which also demonstrated behavioral abnormality and mental deficiency. Therefore, such synaptic abnormality, together with the pathological changes in myelination. may contribute to some of the subtle mental changes in these animals (Spyker et cd., 1972). The swollen endothelial cells had an electron-lucid appearance. Admixture of electron lucent and electron-dense endothelial cell processes form continuous cellular barrier. Similar electron lucent endothelial cells were reported in vasculature of cadmium-intoxicated animals (Schlaepfer, 1971) and was believed to represent a direct toxic effect of the heavy metal on the endothelial cells. Disturbance of the blood-brain barrier by mercuric compounds had been reported by various investigators (Steinwall and Olsson, 1969: Chang and Hartmann, 1972~; Ware et (11.. 1974). It is, therefore, not totally surprising to observe endothelial damages in the present investigation. In view that the blood-brain barrier functions as a regulator of metabolic exchange between the blood and brain (Steinwall, 1961: Lajtha, 1962; Broman and Steinwall, 1967), the impairment of the endothelial cells may denote an important relationship of the blood-brain barrier dysfunctions to the subsequent functional, metabolic, and morphologic changes observed in the central nervous system following methyl mercury intoxication. ACKNOWLEDGMENT We would like to thank Mrs. Fran Simandl supported by NSF Grant BMS 74-17927.
for her excellent
technical
assistance.
This
project
is
REFERENCES Beliles. R. P., Clark, R. S.. and Yuile, C. L. (1968). The effects of exposure to mercury vapor on behavior of rats. To.uic,o/. Appl. Ph).v~wo/. 12, 15-71 Berlin. M., and Ullberg, S. (1963). Accumulation and retention of mercury in the mouse. .4rc,lr. Erri,iror~. Hdtl~
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ON
CEREBELLUM
18.5
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