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Further observations on the effect of bilirubin encephalopathy on the Purkinje cell population in Gunn rats
EXPERIMENTAL
NEUROLOGY
69, 408-413 (1980)
RESEARCH Further Observations Encephalopathy Population
on the Effect of Bilirubin on the Purkinje Cell in Gunn Rats
PAMELAJ. MOOREAND Departments
Received
of Anatomy
August
NOTE
B. KARP’
WARREN
and Pediatrics, The Medical Augusta, Georgia 30912 28, 1979; revision
received
College
February
of Georgia,
25, 1980
Cerebella of young weanling Wistar rats and homozygous and heterozygous Gunn rats were prepared for electron microscopy. A number of immatureappearing cells were observed in the vicinity of mature and affected Purkinje cells in jaundiced homozygous Gunn rats suffering from bilirubin encephalopathy. Careful examination of these immature-appearing cells indicated that they were immature Purkinje cells whose maturation process had probably been delayed by excessive levels of bilirubin.
Recent studies in our laboratory (12) confirmed earlier reports (11, 19, 20) that jaundiced, homozygous Gunn rats with bilirubin encephalopathy display mitochondrial hypertrophy with intramitochondrial glycogen deposition and intracytoplasmic membranous inclusions in cerebellar Purkinje cells. We also observed distinct and statistically significant differences in the Purkinje cell populations among Wistar controls, heterozygous Gunn rats, and homozygous Gunn rats. In our study, degenerated and glycogen-containing Purkinje cells significantly increased in numbers as normal Purkinje cells decreased in homozygous Gunn rats. Control I The authors thank Ms. Donna Brown and Ms. Beth McBride for their competent technical assistance and Ms. Peggy King for typing and preparing the manuscript for publication. This project was supported by National Institutes of Health Biomedical Research Support Grant BRS5S07RR05365-18 and by research support from the Department of Pediatrics, Medical College of Georgia. Reprint requests should be addressed to Dr. Karp, Department of Pediatrics.
00144886/80/080408-06$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved
BILIRUBIN
ENCEPHALOPATHY
IN GUNN
RATS
409
Wistar and heterozygous Gunn rats displayed some degree of Purkinje cell degeneration but never to the extent seen in the homozygous rats. The homozygous Gunn rats also displayed significantly higher values of total and free plasma bilirubin compared to those seen in control and heterozygous animals. As we counted and categorized Purkinje cells and quantitatively correlated affected Purkinje cells with bilirubin concentrations (12), we became aware of some large, lightly staining cells in the region of the Purkinje cell layer that bore no resemblance to the cells normally found in this region in 30-day-old rats. We observed these immature-appearing cells only in the homozygous Gunn rats. Our experiments were conducted on weanling heterozygous (N = 12) and homozygous jaundiced (N = 10) male Gunn rats and normal Wistar (N = 10) male rats. All rats were 28 to 30 days old at necropsy (0900 to 1000) when cerebellar Purkinje cell neurogenesis should have been completed (3-5). Animals were killed by systemic perfusion with cold, cacodylatebuffered (7.3 to 7.4 PH) 2% paraformaldehyde-2% gluteraldehyde fixative. One-half of each cerebellum was carefully cubed (1 x 2 x 2 mm) and processed for electron microscopy and embedded in Araldite 501. Thick sections of each block of cerebellum allowed us to localize the Purkinje cell layer for thin sectioning. Ultrathin, glass-cut sections were stained with uranyl acetate and lead citrate and examined on a Phillips 400 transmission microscope. In every instance, the cerebella of the homozygous Gunn rats were noticeably smaller than those of the control or heterozygous Gunn rats. In 9 of the 10 homozygous animals, large immature-appearing cells were found in close proximity to the Purkinje cell layer (Fig. 1). There was no apparent correlation between the number of immature-appearing cells and the free or total bilirubin concentrations. The animal with the highest bilirubin values certainly had no more of these large cells than any of the other homozygous animals. The number of these large cells varied from animal to animal and at that time the proportion of these “immature” cells compared to the mature affected or degenerating Purkinje cells had no clear-cut pattern. However, these large cells usually were found close to large affected Purkinje cells (cells with noticeable intramitochondrial glycogen) and not as close to the totally degenerated Purkinje cells. Careful examination of the literature on cerebellar neurogenesis (1-7, 13, 14, 16-18, 21) with particular scrutiny of the photomicrographs of others led us to believe that these large pale immature-appearing cells are Purkinje cells. The indented nucleus, the organization of the cytoplasmic organelles, the size of the cells (larger than granule or Golgi II
410
MOORE AND KARP
FIG. 1. A portion of a mature Purkinje cell (P) with intramitochondrial glycogen (arrow), a mature granule cell (G), and a large immature-appearing cell (I) which we believe to be a young Purkinje cell are all visible in this micrograph. These affected (glycogen-laden) and immature cells were observed only in homozygous Gunn rats. Note the sparseness of organelles in the immature cell. x5225.
BILIRUBIN
ENCEPHALOPATHY
IN GUNN
RATS
411
cells), and their location in the region of mature Purkinje cells provided the evidence for the identification of these cells (Figs. 1, 2). Additional persuasion came from the fact that these large pale cells often displayed
FIG. 2. A large “young” cell similar in size to that in Fig. 1. The indented nucleus, juxtanuclear ribosomal material, and sparseness of organelles indicate a lack of maturation. The intramitochondrial deposition of glycogen (arrow) was the convincing feature for identifying these cells as immature Purkinje cells. x9200.
412
MOORE
AND
KARP
intramitochondrial glycogen deposition (Fig. 2) and in this region of the cerebellum only Purkinje cells have been reported to be affected with intramitochondrial glycogen deposition in bilirubin encephalopathy. This glycogen deposition was not seen in granule, Golgi II, or glial cells in the vicinity of the Purkinje cell layer. According to other studies on rat and mouse cerebella, Purkinje cells are formed prenatally (2, 15), migrate to a single layer between the molecular and granule layers by day 10 postnatally (16), and are essentially mature by day 21 postnatally (4,6, 15) with an increase in perikaryon size to day 30 (7). Some investigators, however, reported delayed Purkinje cell development (10) and morphologic abnormalities in genetically affected mice. Altman ef al. (8) reported alterations in Purkinje cell structure with exposure to low levels of radiation, so it is easy to believe that bilirubin not only might cause cellular changes (9, 11, 12, 19,20) but also may affect postnatal development. We hope that autoradiographic studies on prenatal and postnatal cerebellar histogenesis in the Gunn rat will lead to a more positive classification of this immature-appearing cell type. REFERENCES I. ALTMAN, J. 1969. Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J. Comp. Neurol. 136: 269-294. 2. ALTMAN, J. 1970. Postnatal neurogenesis and the problem of neural plasticity. Pages 197-237 in W. A. HIMWICH, Ed., Developmental Neurobiology. Charles C Thomas, Springfield, Ill. 3. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol. 145: 353-398. 4. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular level. J. Camp. Neurol. 145: 399-464.
5. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex of the rat. III. Maturation of the components of the granular layer. J. Comp. Neurol. 145: 465-514. 6. ALTMAN, J., AND G. D. DAS. 1966. Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats with special reference to postnatal neurogenesis in some brain regions. J. Comp. Neurol. 126: 337-390. 7. ALTMAN, J., AND W. WINFREE. 1977. Postnatal development of the cerebellar cortex in the rat. V. Spatial organization of Purkinje cell perikarya. J. Camp. Neural. 171: I-16. 8. ALTMAN,J.,
W. J. ANDERSON,
AND K. A. WRIGHT.
1968. Gross morphological
con-
BILIRUBIN
9.
10. 11. 12.
13.
ENCEPHALOPATHY
IN GUNN RATS
413
sequences of irradiation of the cerebellum in infant rats with repeated doses of lowlevel x-ray. Exp. Neural. 21: 69-91. BATTY, H. K., AND 0. E. MILLHOUSE. 1976. Ultrastructure of Gunn rat substantia nigra. Acta Neuropathol. 34: 7- 19. HIRANO, A., AND H. M. DEMBITZER. 1975. The fine structure of staggeres cerebellum. J. Neuropathol. Exp. Neurol. 34: 1- 11. JEW, J. Y., AND D. SANDQUIST. 1979. CNS changes in hyperbilirubinemia. Functional implications. Arch. Neuro/. 36: 149-154. KARP, W. B., P. .I. MOORE, S. B. SUBRAMANYAM, AND D. B. BROWN. 1980. The relationship of plasma total bilirubin, apparent unbound bilirubin and total albumin with cerebellar glycogen and Purkinje ceils in the Gunn rat (submitted for publication). KORNGUTH, S. E., J. W. ANDERSON, AND G. SCOTT. 1967. Observations on the ultrastructure of the developing cerebellus of the Macaca mulatta. .I. Comp. Neurol. 130: l-24.
14. LARRAMENDI, L. M. H. 1969. Analysis of synaptogenesis in the cerebellum of the mouse. Pages 803-843 in R. LLINAS, Ed., Neurobiology of Cerebellar Evolution and Development. American Medical Association, Chicago. 15. MARES, V., B. SCHULTZE, AND W. MAURER. 1974. Stability of DNA in Purkinje cell nuclei of the mouse. An autoradiographic study. J. Cell Biol. 63: 665-674. 16. MELLER, K., AND P. GLEES. 1969. The development of the mouse cerebellum. A Golgi and electron microscopical study. Pages 783-802 in R. LLINAS, Ed., Neurobiology of Cerebellar Evo/ution and Development. American Medical Association, Chicago. 17. MIALE, I. L., AND R. L. SIDMAN 1961. An autoradiographic analysis of histogenesis in the mouse cerebellum. Exp. Neurol. 4: 277-296. 18. MIGNAINI, E. 1969. Ultrastructural studies on cerebellar histogenesis. II. Maturation of nerve cell populations and establishment of synaptic connections in the cerebellar cortex of the chick. Pages 749-782 in R. LLINAS, Ed., Neurobiology of Cerebellar Evolution and Development. American Medical Association, Chicago. 19. SCHUTTA, H. S., AND L. JOHNSON. 1967. Bilirubin encephalopathy in the Gunn rat: a fine structure study of the cerebellar cortex. J. Neuropathol. Exp. Neurol. 26: 377-396.
SCHUTTA, H. S., L. JOHNSON, AND H. E. NEVILLE. 1970. Mitochondrial abnormalities in bilirubin encephalopathy. J. Neuropathol. Exp. Neurol. 29: 296-305. 21. UZMAN, L. L. l%O. The histogenesis of the mouse cerebellum as studied by its tritiated thymidine uptake. J. Comp. Neural. 114: 137-160. 20.
NEUROLOGY
69, 408-413 (1980)
RESEARCH Further Observations Encephalopathy Population
on the Effect of Bilirubin on the Purkinje Cell in Gunn Rats
PAMELAJ. MOOREAND Departments
Received
of Anatomy
August
NOTE
B. KARP’
WARREN
and Pediatrics, The Medical Augusta, Georgia 30912 28, 1979; revision
received
College
February
of Georgia,
25, 1980
Cerebella of young weanling Wistar rats and homozygous and heterozygous Gunn rats were prepared for electron microscopy. A number of immatureappearing cells were observed in the vicinity of mature and affected Purkinje cells in jaundiced homozygous Gunn rats suffering from bilirubin encephalopathy. Careful examination of these immature-appearing cells indicated that they were immature Purkinje cells whose maturation process had probably been delayed by excessive levels of bilirubin.
Recent studies in our laboratory (12) confirmed earlier reports (11, 19, 20) that jaundiced, homozygous Gunn rats with bilirubin encephalopathy display mitochondrial hypertrophy with intramitochondrial glycogen deposition and intracytoplasmic membranous inclusions in cerebellar Purkinje cells. We also observed distinct and statistically significant differences in the Purkinje cell populations among Wistar controls, heterozygous Gunn rats, and homozygous Gunn rats. In our study, degenerated and glycogen-containing Purkinje cells significantly increased in numbers as normal Purkinje cells decreased in homozygous Gunn rats. Control I The authors thank Ms. Donna Brown and Ms. Beth McBride for their competent technical assistance and Ms. Peggy King for typing and preparing the manuscript for publication. This project was supported by National Institutes of Health Biomedical Research Support Grant BRS5S07RR05365-18 and by research support from the Department of Pediatrics, Medical College of Georgia. Reprint requests should be addressed to Dr. Karp, Department of Pediatrics.
00144886/80/080408-06$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved
BILIRUBIN
ENCEPHALOPATHY
IN GUNN
RATS
409
Wistar and heterozygous Gunn rats displayed some degree of Purkinje cell degeneration but never to the extent seen in the homozygous rats. The homozygous Gunn rats also displayed significantly higher values of total and free plasma bilirubin compared to those seen in control and heterozygous animals. As we counted and categorized Purkinje cells and quantitatively correlated affected Purkinje cells with bilirubin concentrations (12), we became aware of some large, lightly staining cells in the region of the Purkinje cell layer that bore no resemblance to the cells normally found in this region in 30-day-old rats. We observed these immature-appearing cells only in the homozygous Gunn rats. Our experiments were conducted on weanling heterozygous (N = 12) and homozygous jaundiced (N = 10) male Gunn rats and normal Wistar (N = 10) male rats. All rats were 28 to 30 days old at necropsy (0900 to 1000) when cerebellar Purkinje cell neurogenesis should have been completed (3-5). Animals were killed by systemic perfusion with cold, cacodylatebuffered (7.3 to 7.4 PH) 2% paraformaldehyde-2% gluteraldehyde fixative. One-half of each cerebellum was carefully cubed (1 x 2 x 2 mm) and processed for electron microscopy and embedded in Araldite 501. Thick sections of each block of cerebellum allowed us to localize the Purkinje cell layer for thin sectioning. Ultrathin, glass-cut sections were stained with uranyl acetate and lead citrate and examined on a Phillips 400 transmission microscope. In every instance, the cerebella of the homozygous Gunn rats were noticeably smaller than those of the control or heterozygous Gunn rats. In 9 of the 10 homozygous animals, large immature-appearing cells were found in close proximity to the Purkinje cell layer (Fig. 1). There was no apparent correlation between the number of immature-appearing cells and the free or total bilirubin concentrations. The animal with the highest bilirubin values certainly had no more of these large cells than any of the other homozygous animals. The number of these large cells varied from animal to animal and at that time the proportion of these “immature” cells compared to the mature affected or degenerating Purkinje cells had no clear-cut pattern. However, these large cells usually were found close to large affected Purkinje cells (cells with noticeable intramitochondrial glycogen) and not as close to the totally degenerated Purkinje cells. Careful examination of the literature on cerebellar neurogenesis (1-7, 13, 14, 16-18, 21) with particular scrutiny of the photomicrographs of others led us to believe that these large pale immature-appearing cells are Purkinje cells. The indented nucleus, the organization of the cytoplasmic organelles, the size of the cells (larger than granule or Golgi II
410
MOORE AND KARP
FIG. 1. A portion of a mature Purkinje cell (P) with intramitochondrial glycogen (arrow), a mature granule cell (G), and a large immature-appearing cell (I) which we believe to be a young Purkinje cell are all visible in this micrograph. These affected (glycogen-laden) and immature cells were observed only in homozygous Gunn rats. Note the sparseness of organelles in the immature cell. x5225.
BILIRUBIN
ENCEPHALOPATHY
IN GUNN
RATS
411
cells), and their location in the region of mature Purkinje cells provided the evidence for the identification of these cells (Figs. 1, 2). Additional persuasion came from the fact that these large pale cells often displayed
FIG. 2. A large “young” cell similar in size to that in Fig. 1. The indented nucleus, juxtanuclear ribosomal material, and sparseness of organelles indicate a lack of maturation. The intramitochondrial deposition of glycogen (arrow) was the convincing feature for identifying these cells as immature Purkinje cells. x9200.
412
MOORE
AND
KARP
intramitochondrial glycogen deposition (Fig. 2) and in this region of the cerebellum only Purkinje cells have been reported to be affected with intramitochondrial glycogen deposition in bilirubin encephalopathy. This glycogen deposition was not seen in granule, Golgi II, or glial cells in the vicinity of the Purkinje cell layer. According to other studies on rat and mouse cerebella, Purkinje cells are formed prenatally (2, 15), migrate to a single layer between the molecular and granule layers by day 10 postnatally (16), and are essentially mature by day 21 postnatally (4,6, 15) with an increase in perikaryon size to day 30 (7). Some investigators, however, reported delayed Purkinje cell development (10) and morphologic abnormalities in genetically affected mice. Altman ef al. (8) reported alterations in Purkinje cell structure with exposure to low levels of radiation, so it is easy to believe that bilirubin not only might cause cellular changes (9, 11, 12, 19,20) but also may affect postnatal development. We hope that autoradiographic studies on prenatal and postnatal cerebellar histogenesis in the Gunn rat will lead to a more positive classification of this immature-appearing cell type. REFERENCES I. ALTMAN, J. 1969. Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J. Comp. Neurol. 136: 269-294. 2. ALTMAN, J. 1970. Postnatal neurogenesis and the problem of neural plasticity. Pages 197-237 in W. A. HIMWICH, Ed., Developmental Neurobiology. Charles C Thomas, Springfield, Ill. 3. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol. 145: 353-398. 4. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular level. J. Camp. Neurol. 145: 399-464.
5. ALTMAN, J. 1972. Postnatal development of the cerebellar cortex of the rat. III. Maturation of the components of the granular layer. J. Comp. Neurol. 145: 465-514. 6. ALTMAN, J., AND G. D. DAS. 1966. Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics, migration and transformation of cells incorporating tritiated thymidine in neonate rats with special reference to postnatal neurogenesis in some brain regions. J. Comp. Neurol. 126: 337-390. 7. ALTMAN, J., AND W. WINFREE. 1977. Postnatal development of the cerebellar cortex in the rat. V. Spatial organization of Purkinje cell perikarya. J. Camp. Neural. 171: I-16. 8. ALTMAN,J.,
W. J. ANDERSON,
AND K. A. WRIGHT.
1968. Gross morphological
con-
BILIRUBIN
9.
10. 11. 12.
13.
ENCEPHALOPATHY
IN GUNN RATS
413
sequences of irradiation of the cerebellum in infant rats with repeated doses of lowlevel x-ray. Exp. Neural. 21: 69-91. BATTY, H. K., AND 0. E. MILLHOUSE. 1976. Ultrastructure of Gunn rat substantia nigra. Acta Neuropathol. 34: 7- 19. HIRANO, A., AND H. M. DEMBITZER. 1975. The fine structure of staggeres cerebellum. J. Neuropathol. Exp. Neurol. 34: 1- 11. JEW, J. Y., AND D. SANDQUIST. 1979. CNS changes in hyperbilirubinemia. Functional implications. Arch. Neuro/. 36: 149-154. KARP, W. B., P. .I. MOORE, S. B. SUBRAMANYAM, AND D. B. BROWN. 1980. The relationship of plasma total bilirubin, apparent unbound bilirubin and total albumin with cerebellar glycogen and Purkinje ceils in the Gunn rat (submitted for publication). KORNGUTH, S. E., J. W. ANDERSON, AND G. SCOTT. 1967. Observations on the ultrastructure of the developing cerebellus of the Macaca mulatta. .I. Comp. Neurol. 130: l-24.
14. LARRAMENDI, L. M. H. 1969. Analysis of synaptogenesis in the cerebellum of the mouse. Pages 803-843 in R. LLINAS, Ed., Neurobiology of Cerebellar Evolution and Development. American Medical Association, Chicago. 15. MARES, V., B. SCHULTZE, AND W. MAURER. 1974. Stability of DNA in Purkinje cell nuclei of the mouse. An autoradiographic study. J. Cell Biol. 63: 665-674. 16. MELLER, K., AND P. GLEES. 1969. The development of the mouse cerebellum. A Golgi and electron microscopical study. Pages 783-802 in R. LLINAS, Ed., Neurobiology of Cerebellar Evo/ution and Development. American Medical Association, Chicago. 17. MIALE, I. L., AND R. L. SIDMAN 1961. An autoradiographic analysis of histogenesis in the mouse cerebellum. Exp. Neurol. 4: 277-296. 18. MIGNAINI, E. 1969. Ultrastructural studies on cerebellar histogenesis. II. Maturation of nerve cell populations and establishment of synaptic connections in the cerebellar cortex of the chick. Pages 749-782 in R. LLINAS, Ed., Neurobiology of Cerebellar Evolution and Development. American Medical Association, Chicago. 19. SCHUTTA, H. S., AND L. JOHNSON. 1967. Bilirubin encephalopathy in the Gunn rat: a fine structure study of the cerebellar cortex. J. Neuropathol. Exp. Neurol. 26: 377-396.
SCHUTTA, H. S., L. JOHNSON, AND H. E. NEVILLE. 1970. Mitochondrial abnormalities in bilirubin encephalopathy. J. Neuropathol. Exp. Neurol. 29: 296-305. 21. UZMAN, L. L. l%O. The histogenesis of the mouse cerebellum as studied by its tritiated thymidine uptake. J. Comp. Neural. 114: 137-160. 20.