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Lipofuscin pigment accumulation in human brain during aging
Experimental Gerontology, Vol. 17, pp. 481-487, 1982 Printed in the USA. All rights reserved.
0531-5565/S2/060481-07503.00/0 Copyright © 1983 Pergamon Press Ltd
LIPOFUSCIN PIGMENT ACCUMULATION HUMAN BRAIN DURING AGING
IN
V.K. GOYAL Central Forensic Science Laboratory, C.B.I., New Delhi-I I0 066, India
(Received 3 March 1982) Abstract--Histopathologic and autofluorescence investigations were carried out to study lipofuscin pigment accumulation in various age groups of human beings died in accidents (in 100 cases ranging from 1-70 years-of-age). Lipofuscin pigment granules were first observed to appear at 9 years of age. Qualitative studies revealed a progressive increase in the intracellular lipofuscin pigment accumulation with advancing age. These pigment granules were found to congregate in mass in nerve cells of old individuals. An increase in the lipofuscin pigment accumulation and decrease of Nissl substance was observed during aging. The percentage of pigmented nerve ceils and the percentage of cytoplasmic area occupied by lipofuscin pigment granules increased significantly with the advancement of age.
INTRODUCTION THE IMPORTANTcharacteristic of mammalian nervous system is that the nerve cells do not divide after reaching final state of differentiation during early postnatal life in comparison to those where cellular replacement remains continuous by mitosis throughout infancy, maturity and old age. So, obviously the cause of aging may be linked directly with the loss of these post mitotic nerve cells. Brody (1970) examined superior frontal gyrus of eighteen human samples and reported that nerve cells decreased to about half in old age. Lipofuscin pigment accumulation has been reported as one of the most important neurocytological change occurring with age (Brody, 1960; Samorajski et ak, 1964, 1965, 1968; Whiteford and Getty, 1966; Few and Getty, 1967, Nanda and Getty, 1971, 1973 and Brizzee et ak, 1974, 1976). Tappel et al. (1973) did not notice any significant increase of lipofuscin pigment in nerve cells of mouse with advancing age. There ale very few quantitative studies on lipofuscin pigment accumulation (Reichel et al., 1969 and Brizzee et al., 1974) and no significant report exists on intraneuronal lipofuscin pigment accumulation in nerve cells of cerebral cortex of man in different age groups. There are contradictory reports regarding the onset of lipofuscin pigment in the nerve cells. Lipofuscin pigment has been observed to appear in the nerve cells of Children (Obersteiner, 1903 and Chu, 1954) and fetus (Humphrey, 1944). While Brody (1960) denied the presence of lipofuscin pigment in the new born. Later on, Pallis et al. (1967) reported the absence of pigment in nerve cells of persons below 10 years of age. The survey of existing literature indicates that the studies on changes with age in human 4"al
482
V.K. GOYAL
brain o f .different age groups are inadequate. Moreover, no attempt has been made to study changes with age in Indians. The present study has been undertaken to examine age changes in human brain o f different age groups with a view: (1) to determine the earliest onset of lipofuscin pigment in nerve cells o f superior frontal gyrus; (2) to determine an increase in the percentage o f pigmented nerve cells with age; (3) to find out art increase in the percentage of cytoplasmic area occupied by lipofuscin pigments with the advancement o f age; and (4) to find out a link in the accumulation of lipofuscin pigment and a decrease o f Nissl substance during aging. MATERIALS AND METHODS The human post-mortem brain tissue (from 100 accidental cases ranging from 1-70 years-of-age)was collected from the police morgue of N.R.S. Medical College, Calcutta, India. Every care was taken to get the material from healthy normal persons. The superior frontal gyrus from frontal cerebral cortex was immediately removed after the autopsy and fixed in 10e/0neutral buffered formalin within 24 hours after death. The fixed tissues were washed, dehydrated, cleared and embedded in paraffin. Paraffin sections were cut at a thickness of 4 #. The unstained sections were examined under blue light fluorescence microscope. For histologic and histochemical studies, the sections were stained with Sudan black B, Oil red 'O', Long Ziehi Neel~..,I carbol fuscin, and Schmorl methods. For the estimation of percentage of neurons containing pigment granules, the nerve cells were randomly selected from Sudan black B stained sections and examined in the light microscope.The percentage of nerve cells that contained pigment granules was obtained by an examination of 100 cells from each slide. The percentage of cytoplasmicareas occupied by lipofuscin pigment granules was measured by drawing randomly twenty camera lucida diagrams from each Sudan black B stained slide on mm~graph paper under oil immersion field of the microscope. The percentage of area occupied by pigment granules in the cytoplasm was then calculated. OBSERVATIONS Histopathologic and autofluorescence investigations were carried out to study qualitative as well as quantitative intracellular lipofuscin pigment accumulation with advancing age.
Qualitative observations Autofluorescence and histological examination revealed the absence o f lipofuscin pigment in the nerve cells of y o u n g children. Lipofuscin pigment granules first observed in the cytoplasm of nerve cells at 9 years-of-age. These pigment granules were observed to accumulate progressively in the nerve cells with advancing age. In the second decade, the number of pigment granules increased more tfian that observed at 9 years-of-age. By the third decade, more pigment bodies appeared and became prominent. In the fourth decade, the pigment bodies were more closely associated near the nuclear periphery. During the fifth decade, lipofuscin pigment accumulation was intense and occupied a greater part of the cytoplasm. Similarly in the sixth and seventh decades, the pigment accumulation was intense and became concentrated in masses, and in some neurons 50% of the cytoplasmic area was occupied by these pigments. It was also observed that the whole of the nuclear periphery in some nerve cells was surrounded by accumulated lipofuscins.
483
LIPOFUSCIN PIGMENT ACCUMULATION
Lipofuscin pigment granules appeared light yellow in young persons and intense golden yellow in old individuals when unstained sections were examined under ultraviolet light. These pigments were insoluble in lipid solvents as the pigments were stainable by various histochemical stains in paraffin sections. These pigments were positive to lipid stains like Sudan black B and Oil red 'O'. Lipofuscins were stained by alcian blue/periodic acid Schiff reaction which revealed their carbohydrate nature. The proteinous nature o f the pigments was revealed by their positive staining with mercury bromphenol blue. These pigments were observed to be acid fast and positive to Schmorl reaction. When the sections .were examined under ultraviolet light, the fluorescence corresponded only to those areas which were positive to histological reactions in the same series o f sections. In young persons, Nissl substance was seen distributed in the cytoplasm. With the advancement of age, some changes were observed that Nissl granules were pushed towards the cellular periphery during aging since lipofuscin pigments appeared and accumulated above the nucleus. A little RNA or Nissl substance was seen in the nerve cells in which more cytoplasmic area was occupied by lipofuscins in comparison to those cells which contained a few pigment granules or were free f r o m lipofuscins.
Quantitative observations The quantitative increase in the intracellular lipofuscin accumulation was estimated by the increase in the percentage of nerve cells that contained lipofuscin pigment granules and the percentage o f cytoplasmic area occupied by lipofuscin pigment granules in six age decades, i.e., from 11-70 year-old individuals. The percentage of nerve cells that contained pigment granules increased progressively with advancing age (Table 1 ; Fig. 1). The differences in the percentage o f pigmented nerve cells between the second decade and third decade onwards were significant (p < 0.001). The percentage o f cytoplasmic pigmented area increased progressively with advancing age (Table 2; Fig. 2). The differences in the percentage of cytoplasmic pigmented area between the second and the sixth decade, and second and the seventh decade were significant (p < 0.001).
TABLE 1. CHANGES WITH AGE IN THE PERCENTAGE OF NERVE CELLS THAT CONTAIN LIPOFUSClN PIGMENT GRANULES.
Age Group A B C D E F
Age-in-Years 11-20 21-30 31-40 41-50 51-60 61-70
Number of subjects
Percentage of pigmented nerve cells
9 11 12 l0 15 15
32.33 4- 2.05 44.81 4- 2.17 48.42 4- 1.98 51.80 4- 1.86 57.53 4- 1.87 68.47 4- 2.06
This table does not include persons below I 1 years-of-ageas the pigment started appearing at 9 years-of-age. p < 0.001 A and B, A and C, A and D, A and E, A and F, B and E, B and F, C and F, D and F, E and F. p < 0.01 C and E. p < 0.05 B and D, D and E. NS B and C, C and D.
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FIG. 1. Changes with age in the percentage of pigmented nerve ceils.
DISCUSSION A number of workers considered lipofuscin accumulation as the most important neurocytological change with age (Brody, 1960; Samorajski, et al., 1964, 1965, 1968; Whiteford and Getty, 1966, Few and Getty, 1967; Nanda and Getty, 1971, 1973; Brizzee et al., 1974, 1975, 1976). The present study revealed a constant accumulation of lipofuscin pigments in nerve cells with age. A significant increase in the percentage of pigmented nerve cells and the percentage of cytoplasmic area occupied by lipofuscin pigment granules was also observed during aging (Tables 1, 2; Figs. 1, 2). Samorajski et al. (1968) observed an increase in the percentage of cytoplasmic area occupied by lipofuscin pigment granules and percentage of pigmented nerve cells in mice with advancing age. The earliest appearance of lipofuscin pigment in man was observed at different ages by TABLE 2. CHANGESWITH AGE IN THE PERCENTAGEOF AREAOCCUPIED BY LIPOFUSCIN PIGMENTGRANULESIN THE CYTOPLASMOF NERVE CELLS
Age Group
Age-in-Years
Humber of subjects
A B C D E F
11-20 21-30 31-40 41-50 51-60 61-70
9 11 12 10 15 15
Percentage of area occupied by lipofus.cin pigment bodies in the cytoplasm 9.25 13.07 13.97 15.44 18.42 21.08
4- 1.17 4- 0.77 4- 0.86 4- 1.16 4- 0.86 4- 0.95
This table does not include persons below 11 years of age as the onset of pigment started at 9 years of age. p < 0.001 A and E, A and F, B and E, B and F, C and E, C and F. p < 0.01 A and C, A and D, D and F. p < 0.05 A and B, E and F. HS B and C, B and D, C and D, C and E.
LIPOFUSCINPIGMENTACCUMULATION
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AGE IN YEARS
FIG. 2. Changes with age in the percentage of pigmented area occupied in the cytoplasm of nerve cells.
various workers. The pigments were observed in the spinal cord of human fetus by Humphrey (1944). These pigments were not observed in the nerve cells of the new born (Brody, 1960). Lipofuscin pigments were observed in the motor neurons of lumbar spinal cortex in children by Obersteiner (1903) and Chu (1954). Pallis et a/. (1967) reported absence of pigment in normal persons below 10 years-of-age. In the present study, however, lipofuscin pigments were observed as early as 9 years-of-age. Miihlmann (1910) observed that lipofuscin pigment granules, which were distributed homogenously, started accumulating in clusters and finally localized in mass that continued to increase in size with age. The pigment was found to appear in a congregate mass in a polarized pattern deep to the base of the apical dendrites in pyramidal neurons of motor cortex in rat and pyramidal layer of hippocampus in rat and monkey (Brizzee et al., 1969, 1974; and Brizzee and Johnson, 1970). Whiteford and Getty (1966) described four types of distribution patterns of lipofuscins (homogenous, perinuclear, polar and bipolar) in nerve cells of dog. Nanda and Getty (1971) and Nandy (1971) described their appearance at the perinuclear position. The present study showed the early appearance of lipofuscin pigment at the perinuclear position. Thereafter, lipofuscin pigment granules increased with age and started accumulating at the perinuclear position. Later on, these pigments became concentrated in mass which continued to increase in size at the nuclear periphery in old individuals. Hyd6n and Lindstr6m (1950), Pope and Hess (1957) and Mann and Yates (1974) reported that during cellular aging, Nissl substance decreased whereas pigment amount increased. It was found in the present study that Nissl substance was pushed towards the cellular periphery in nerve cells of old individuals as the pigment accumulated at the nuclear periphery. Nissl substance was observed in small quantity in nerve cells which were occupied by more lipofuscins in comparison to those which contained a very small quantity of lipofuscin pigment granules. The present study showed that lipofuscin pigments were positive to histochemical tests
486
V.K. GOYAL
for lipids, glycogens, proteins, acid fast groups and Schmorl reaction. Strehler (1964) also reported that these pigments were positive to similar kinds of histochemical reactions. As the nerve cells do not divide, their lo.ss in the aging nervous system may be associated with intraneuronal lipofuscin accumulation. Samorajski et al. (1968), Zeman (1974) and Brizzee et al. (1975) suggested that intraneuronal lipofuscin accumulation during aging may cause loss of nerve cells. But Brizzee et al. (1968) reported an increase of nerve cells in rat with the advancement of age. No loss of nerve cells was reported in guinea pig (Wilcox, 1956), house fly (Sohal and Sharma, 1972). A considerable loss of nerve cells was reported in mice (Johnson and Erner, 1972) and man (Brody, 1955, 1970 and Ball, 1977). Brody (1970) reported 50o70 cell loss in superior frontal gyrus with advancing age. The present study showed a considerable accumulation of lipofuscin pigment and decrease of Nissl substance in nerve cells of superior frontal gyrus during aging. A constant and progressive accumulation of lipofuscin pigment in nerve cells indicates an aging process. As in the present study, a considerable accumulation of lipofuscin pigment and a decrease of Nissl substance has been observed with age, it seems quite reasonable to assume that the presence of pigment in nerve cells occupying most of the cytoplasmic area may be detrimental to normal cellular metabolism. The physiology and metabolic functions of nerve cells may probably be affecting because of reduction in cytoplasmic area in old age. The reduction in RNA content or Nissl substance of cell may be such that the cell is unable to maintain its metabolic activity and cell death or atrophy may occur. REFERENCES BALL, M.J. (1977)Acta neuropath. 37, 111. B~ZZEE, K.R., and JOHr~SO~,F.A. (1970) Acta neuropath. 16, 205. BRIZZEE, K.R., SHERWOOD,N. and TIMmAS, P.S. 0968) J. Geront. 23, 289. B~ZZEE, K.R., CANCILLA,P.A., SHERWOOD,N. and T~IRAS, P.S. 0969) J. Geront. 24, 127. BRIZZEE, K.R., ORDY, J.M. and KAACK,B. (1974) J. Geront. 29, 366. B~ZZEE, K.R., KAACK,B. and KLARA,P. (1975) In: Neurobiology of Aging (Edited by J.M. ORDY and K.R. B~ZZEE), 463, Plenum Press, New York and London. BPaZZEE, K.R., HARKIN, J.C., ORDY, J.M. and KAACK, B. (1976) In: Clinical, Morphological and NeurochemicalAspects in the Aging Nervous System (Edited by H. BRODY, D. HARMANand J.M. ORDY) p. 39. Excerpta Medica Elsevier/North-Holland Biomedical Press, Amsterdam, New York, and Oxford. BRODY, H. 0955) J. Comp. Neurol. 102, 511. BRODY, H. (1960) J. Geront. 15, 258. BRODY, H. (1970) Interdiscipl. Topics Geront. 77, Basel S. Karger. CHu, L. (1954) J. Comp. Neurol. 100, 381. FEW, A. and GETTY, R. (1967) J. Geront. 22, 357. HANtEY, T. (1974)AgeAging. 3, 133. HUm'rXREY, T. (1944) J. Comp. Neurol. 81, 1. HYDe.N, H. and LrSDSTROM,B. (1950) Discuss. Faraday Soc. 9, 436. Jom~soN, H.A. and ERNER, S. (1972) Exp. Gerontok 7, I II. KONIGSMARK,B.W. and Mtn~,HV, E.A. (1970) Nature. 228, 1335. MANN, D.M. and YATES,P.O. (1974) Brain. 97, 481. MOm.MA~N, M. (1910) Virchows Arch. 211, 155. NANDA, B.S. and G~TTY, R. (1971) Exp. Gerontol. 6, 447. NANDA, B.S. and GETTY, R. (1973) Exp. Gerontol. 8, 1. NANDV, K. (1971) Acta neuropath. 19, 25. OBERSTEI~ER, H. (1903) Arb. Neurol. Inst. Wien. Univ. 10, 245. PALLIS, C.A., DUCKETT,S. and PEARSE, A.G.E. (1967) Neurology 17, 381. POPE, A. and HESS, H.H. (1957) In: Metabolism of Nervous System (Edited by D. RlCHTER)p. 772. Pergamon Press, London.
LIPOFUSCINPIGMENT
ACCUMULATION
REICHEL, W. (1968) J. Gerontol. 23, 145. SAMORAJSKI,T., KEEFE, J.R. and ORDY, J.M. (1964) J. Gerontol, 19, 262. SAmO~SKI, T., ORDY, J.M. and KEEFE, J.R. 0965) J. Cell. Biol. 26, 779. SAMOR~SKI, T., ORDY, J.M. and RADY-REIMER,P. (1968) Anat. Rec. 160, 555. SOHAL, R.S. and SHARMA, S.P. (1972) Exp. Gerontol. 7, 243. STREHLER, B.L. (1964) Adv. Geront. Res. 1, 343. TAPPEL, A.L., FLETCHER, B. and DESMER, D. (1973) J. Gerontol. 28, 415. WHrrEFORD, R. and GETTY, R. (1966) J. Geront. 21, 31. WrLCOX, H.H. (1956) J. Geront. 11, 442. ZEMA~, W. (1974) J. Neuropath. Exp. Neurol. 33, 1.
487
0531-5565/S2/060481-07503.00/0 Copyright © 1983 Pergamon Press Ltd
LIPOFUSCIN PIGMENT ACCUMULATION HUMAN BRAIN DURING AGING
IN
V.K. GOYAL Central Forensic Science Laboratory, C.B.I., New Delhi-I I0 066, India
(Received 3 March 1982) Abstract--Histopathologic and autofluorescence investigations were carried out to study lipofuscin pigment accumulation in various age groups of human beings died in accidents (in 100 cases ranging from 1-70 years-of-age). Lipofuscin pigment granules were first observed to appear at 9 years of age. Qualitative studies revealed a progressive increase in the intracellular lipofuscin pigment accumulation with advancing age. These pigment granules were found to congregate in mass in nerve cells of old individuals. An increase in the lipofuscin pigment accumulation and decrease of Nissl substance was observed during aging. The percentage of pigmented nerve ceils and the percentage of cytoplasmic area occupied by lipofuscin pigment granules increased significantly with the advancement of age.
INTRODUCTION THE IMPORTANTcharacteristic of mammalian nervous system is that the nerve cells do not divide after reaching final state of differentiation during early postnatal life in comparison to those where cellular replacement remains continuous by mitosis throughout infancy, maturity and old age. So, obviously the cause of aging may be linked directly with the loss of these post mitotic nerve cells. Brody (1970) examined superior frontal gyrus of eighteen human samples and reported that nerve cells decreased to about half in old age. Lipofuscin pigment accumulation has been reported as one of the most important neurocytological change occurring with age (Brody, 1960; Samorajski et ak, 1964, 1965, 1968; Whiteford and Getty, 1966; Few and Getty, 1967, Nanda and Getty, 1971, 1973 and Brizzee et ak, 1974, 1976). Tappel et al. (1973) did not notice any significant increase of lipofuscin pigment in nerve cells of mouse with advancing age. There ale very few quantitative studies on lipofuscin pigment accumulation (Reichel et al., 1969 and Brizzee et al., 1974) and no significant report exists on intraneuronal lipofuscin pigment accumulation in nerve cells of cerebral cortex of man in different age groups. There are contradictory reports regarding the onset of lipofuscin pigment in the nerve cells. Lipofuscin pigment has been observed to appear in the nerve cells of Children (Obersteiner, 1903 and Chu, 1954) and fetus (Humphrey, 1944). While Brody (1960) denied the presence of lipofuscin pigment in the new born. Later on, Pallis et al. (1967) reported the absence of pigment in nerve cells of persons below 10 years of age. The survey of existing literature indicates that the studies on changes with age in human 4"al
482
V.K. GOYAL
brain o f .different age groups are inadequate. Moreover, no attempt has been made to study changes with age in Indians. The present study has been undertaken to examine age changes in human brain o f different age groups with a view: (1) to determine the earliest onset of lipofuscin pigment in nerve cells o f superior frontal gyrus; (2) to determine an increase in the percentage o f pigmented nerve cells with age; (3) to find out art increase in the percentage of cytoplasmic area occupied by lipofuscin pigments with the advancement o f age; and (4) to find out a link in the accumulation of lipofuscin pigment and a decrease o f Nissl substance during aging. MATERIALS AND METHODS The human post-mortem brain tissue (from 100 accidental cases ranging from 1-70 years-of-age)was collected from the police morgue of N.R.S. Medical College, Calcutta, India. Every care was taken to get the material from healthy normal persons. The superior frontal gyrus from frontal cerebral cortex was immediately removed after the autopsy and fixed in 10e/0neutral buffered formalin within 24 hours after death. The fixed tissues were washed, dehydrated, cleared and embedded in paraffin. Paraffin sections were cut at a thickness of 4 #. The unstained sections were examined under blue light fluorescence microscope. For histologic and histochemical studies, the sections were stained with Sudan black B, Oil red 'O', Long Ziehi Neel~..,I carbol fuscin, and Schmorl methods. For the estimation of percentage of neurons containing pigment granules, the nerve cells were randomly selected from Sudan black B stained sections and examined in the light microscope.The percentage of nerve cells that contained pigment granules was obtained by an examination of 100 cells from each slide. The percentage of cytoplasmicareas occupied by lipofuscin pigment granules was measured by drawing randomly twenty camera lucida diagrams from each Sudan black B stained slide on mm~graph paper under oil immersion field of the microscope. The percentage of area occupied by pigment granules in the cytoplasm was then calculated. OBSERVATIONS Histopathologic and autofluorescence investigations were carried out to study qualitative as well as quantitative intracellular lipofuscin pigment accumulation with advancing age.
Qualitative observations Autofluorescence and histological examination revealed the absence o f lipofuscin pigment in the nerve cells of y o u n g children. Lipofuscin pigment granules first observed in the cytoplasm of nerve cells at 9 years-of-age. These pigment granules were observed to accumulate progressively in the nerve cells with advancing age. In the second decade, the number of pigment granules increased more tfian that observed at 9 years-of-age. By the third decade, more pigment bodies appeared and became prominent. In the fourth decade, the pigment bodies were more closely associated near the nuclear periphery. During the fifth decade, lipofuscin pigment accumulation was intense and occupied a greater part of the cytoplasm. Similarly in the sixth and seventh decades, the pigment accumulation was intense and became concentrated in masses, and in some neurons 50% of the cytoplasmic area was occupied by these pigments. It was also observed that the whole of the nuclear periphery in some nerve cells was surrounded by accumulated lipofuscins.
483
LIPOFUSCIN PIGMENT ACCUMULATION
Lipofuscin pigment granules appeared light yellow in young persons and intense golden yellow in old individuals when unstained sections were examined under ultraviolet light. These pigments were insoluble in lipid solvents as the pigments were stainable by various histochemical stains in paraffin sections. These pigments were positive to lipid stains like Sudan black B and Oil red 'O'. Lipofuscins were stained by alcian blue/periodic acid Schiff reaction which revealed their carbohydrate nature. The proteinous nature o f the pigments was revealed by their positive staining with mercury bromphenol blue. These pigments were observed to be acid fast and positive to Schmorl reaction. When the sections .were examined under ultraviolet light, the fluorescence corresponded only to those areas which were positive to histological reactions in the same series o f sections. In young persons, Nissl substance was seen distributed in the cytoplasm. With the advancement of age, some changes were observed that Nissl granules were pushed towards the cellular periphery during aging since lipofuscin pigments appeared and accumulated above the nucleus. A little RNA or Nissl substance was seen in the nerve cells in which more cytoplasmic area was occupied by lipofuscins in comparison to those cells which contained a few pigment granules or were free f r o m lipofuscins.
Quantitative observations The quantitative increase in the intracellular lipofuscin accumulation was estimated by the increase in the percentage of nerve cells that contained lipofuscin pigment granules and the percentage o f cytoplasmic area occupied by lipofuscin pigment granules in six age decades, i.e., from 11-70 year-old individuals. The percentage of nerve cells that contained pigment granules increased progressively with advancing age (Table 1 ; Fig. 1). The differences in the percentage o f pigmented nerve cells between the second decade and third decade onwards were significant (p < 0.001). The percentage o f cytoplasmic pigmented area increased progressively with advancing age (Table 2; Fig. 2). The differences in the percentage of cytoplasmic pigmented area between the second and the sixth decade, and second and the seventh decade were significant (p < 0.001).
TABLE 1. CHANGES WITH AGE IN THE PERCENTAGE OF NERVE CELLS THAT CONTAIN LIPOFUSClN PIGMENT GRANULES.
Age Group A B C D E F
Age-in-Years 11-20 21-30 31-40 41-50 51-60 61-70
Number of subjects
Percentage of pigmented nerve cells
9 11 12 l0 15 15
32.33 4- 2.05 44.81 4- 2.17 48.42 4- 1.98 51.80 4- 1.86 57.53 4- 1.87 68.47 4- 2.06
This table does not include persons below I 1 years-of-ageas the pigment started appearing at 9 years-of-age. p < 0.001 A and B, A and C, A and D, A and E, A and F, B and E, B and F, C and F, D and F, E and F. p < 0.01 C and E. p < 0.05 B and D, D and E. NS B and C, C and D.
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I 20
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I 40
I 50
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A G E I N YEARS
FIG. 1. Changes with age in the percentage of pigmented nerve ceils.
DISCUSSION A number of workers considered lipofuscin accumulation as the most important neurocytological change with age (Brody, 1960; Samorajski, et al., 1964, 1965, 1968; Whiteford and Getty, 1966, Few and Getty, 1967; Nanda and Getty, 1971, 1973; Brizzee et al., 1974, 1975, 1976). The present study revealed a constant accumulation of lipofuscin pigments in nerve cells with age. A significant increase in the percentage of pigmented nerve cells and the percentage of cytoplasmic area occupied by lipofuscin pigment granules was also observed during aging (Tables 1, 2; Figs. 1, 2). Samorajski et al. (1968) observed an increase in the percentage of cytoplasmic area occupied by lipofuscin pigment granules and percentage of pigmented nerve cells in mice with advancing age. The earliest appearance of lipofuscin pigment in man was observed at different ages by TABLE 2. CHANGESWITH AGE IN THE PERCENTAGEOF AREAOCCUPIED BY LIPOFUSCIN PIGMENTGRANULESIN THE CYTOPLASMOF NERVE CELLS
Age Group
Age-in-Years
Humber of subjects
A B C D E F
11-20 21-30 31-40 41-50 51-60 61-70
9 11 12 10 15 15
Percentage of area occupied by lipofus.cin pigment bodies in the cytoplasm 9.25 13.07 13.97 15.44 18.42 21.08
4- 1.17 4- 0.77 4- 0.86 4- 1.16 4- 0.86 4- 0.95
This table does not include persons below 11 years of age as the onset of pigment started at 9 years of age. p < 0.001 A and E, A and F, B and E, B and F, C and E, C and F. p < 0.01 A and C, A and D, D and F. p < 0.05 A and B, E and F. HS B and C, B and D, C and D, C and E.
LIPOFUSCINPIGMENTACCUMULATION
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80
AGE IN YEARS
FIG. 2. Changes with age in the percentage of pigmented area occupied in the cytoplasm of nerve cells.
various workers. The pigments were observed in the spinal cord of human fetus by Humphrey (1944). These pigments were not observed in the nerve cells of the new born (Brody, 1960). Lipofuscin pigments were observed in the motor neurons of lumbar spinal cortex in children by Obersteiner (1903) and Chu (1954). Pallis et a/. (1967) reported absence of pigment in normal persons below 10 years-of-age. In the present study, however, lipofuscin pigments were observed as early as 9 years-of-age. Miihlmann (1910) observed that lipofuscin pigment granules, which were distributed homogenously, started accumulating in clusters and finally localized in mass that continued to increase in size with age. The pigment was found to appear in a congregate mass in a polarized pattern deep to the base of the apical dendrites in pyramidal neurons of motor cortex in rat and pyramidal layer of hippocampus in rat and monkey (Brizzee et al., 1969, 1974; and Brizzee and Johnson, 1970). Whiteford and Getty (1966) described four types of distribution patterns of lipofuscins (homogenous, perinuclear, polar and bipolar) in nerve cells of dog. Nanda and Getty (1971) and Nandy (1971) described their appearance at the perinuclear position. The present study showed the early appearance of lipofuscin pigment at the perinuclear position. Thereafter, lipofuscin pigment granules increased with age and started accumulating at the perinuclear position. Later on, these pigments became concentrated in mass which continued to increase in size at the nuclear periphery in old individuals. Hyd6n and Lindstr6m (1950), Pope and Hess (1957) and Mann and Yates (1974) reported that during cellular aging, Nissl substance decreased whereas pigment amount increased. It was found in the present study that Nissl substance was pushed towards the cellular periphery in nerve cells of old individuals as the pigment accumulated at the nuclear periphery. Nissl substance was observed in small quantity in nerve cells which were occupied by more lipofuscins in comparison to those which contained a very small quantity of lipofuscin pigment granules. The present study showed that lipofuscin pigments were positive to histochemical tests
486
V.K. GOYAL
for lipids, glycogens, proteins, acid fast groups and Schmorl reaction. Strehler (1964) also reported that these pigments were positive to similar kinds of histochemical reactions. As the nerve cells do not divide, their lo.ss in the aging nervous system may be associated with intraneuronal lipofuscin accumulation. Samorajski et al. (1968), Zeman (1974) and Brizzee et al. (1975) suggested that intraneuronal lipofuscin accumulation during aging may cause loss of nerve cells. But Brizzee et al. (1968) reported an increase of nerve cells in rat with the advancement of age. No loss of nerve cells was reported in guinea pig (Wilcox, 1956), house fly (Sohal and Sharma, 1972). A considerable loss of nerve cells was reported in mice (Johnson and Erner, 1972) and man (Brody, 1955, 1970 and Ball, 1977). Brody (1970) reported 50o70 cell loss in superior frontal gyrus with advancing age. The present study showed a considerable accumulation of lipofuscin pigment and decrease of Nissl substance in nerve cells of superior frontal gyrus during aging. A constant and progressive accumulation of lipofuscin pigment in nerve cells indicates an aging process. As in the present study, a considerable accumulation of lipofuscin pigment and a decrease of Nissl substance has been observed with age, it seems quite reasonable to assume that the presence of pigment in nerve cells occupying most of the cytoplasmic area may be detrimental to normal cellular metabolism. The physiology and metabolic functions of nerve cells may probably be affecting because of reduction in cytoplasmic area in old age. The reduction in RNA content or Nissl substance of cell may be such that the cell is unable to maintain its metabolic activity and cell death or atrophy may occur. REFERENCES BALL, M.J. (1977)Acta neuropath. 37, 111. B~ZZEE, K.R., and JOHr~SO~,F.A. (1970) Acta neuropath. 16, 205. BRIZZEE, K.R., SHERWOOD,N. and TIMmAS, P.S. 0968) J. Geront. 23, 289. B~ZZEE, K.R., CANCILLA,P.A., SHERWOOD,N. and T~IRAS, P.S. 0969) J. Geront. 24, 127. BRIZZEE, K.R., ORDY, J.M. and KAACK,B. (1974) J. Geront. 29, 366. B~ZZEE, K.R., KAACK,B. and KLARA,P. (1975) In: Neurobiology of Aging (Edited by J.M. ORDY and K.R. B~ZZEE), 463, Plenum Press, New York and London. BPaZZEE, K.R., HARKIN, J.C., ORDY, J.M. and KAACK, B. (1976) In: Clinical, Morphological and NeurochemicalAspects in the Aging Nervous System (Edited by H. BRODY, D. HARMANand J.M. ORDY) p. 39. Excerpta Medica Elsevier/North-Holland Biomedical Press, Amsterdam, New York, and Oxford. BRODY, H. 0955) J. Comp. Neurol. 102, 511. BRODY, H. (1960) J. Geront. 15, 258. BRODY, H. (1970) Interdiscipl. Topics Geront. 77, Basel S. Karger. CHu, L. (1954) J. Comp. Neurol. 100, 381. FEW, A. and GETTY, R. (1967) J. Geront. 22, 357. HANtEY, T. (1974)AgeAging. 3, 133. HUm'rXREY, T. (1944) J. Comp. Neurol. 81, 1. HYDe.N, H. and LrSDSTROM,B. (1950) Discuss. Faraday Soc. 9, 436. Jom~soN, H.A. and ERNER, S. (1972) Exp. Gerontok 7, I II. KONIGSMARK,B.W. and Mtn~,HV, E.A. (1970) Nature. 228, 1335. MANN, D.M. and YATES,P.O. (1974) Brain. 97, 481. MOm.MA~N, M. (1910) Virchows Arch. 211, 155. NANDA, B.S. and G~TTY, R. (1971) Exp. Gerontol. 6, 447. NANDA, B.S. and GETTY, R. (1973) Exp. Gerontol. 8, 1. NANDV, K. (1971) Acta neuropath. 19, 25. OBERSTEI~ER, H. (1903) Arb. Neurol. Inst. Wien. Univ. 10, 245. PALLIS, C.A., DUCKETT,S. and PEARSE, A.G.E. (1967) Neurology 17, 381. POPE, A. and HESS, H.H. (1957) In: Metabolism of Nervous System (Edited by D. RlCHTER)p. 772. Pergamon Press, London.
LIPOFUSCINPIGMENT
ACCUMULATION
REICHEL, W. (1968) J. Gerontol. 23, 145. SAMORAJSKI,T., KEEFE, J.R. and ORDY, J.M. (1964) J. Gerontol, 19, 262. SAmO~SKI, T., ORDY, J.M. and KEEFE, J.R. 0965) J. Cell. Biol. 26, 779. SAMOR~SKI, T., ORDY, J.M. and RADY-REIMER,P. (1968) Anat. Rec. 160, 555. SOHAL, R.S. and SHARMA, S.P. (1972) Exp. Gerontol. 7, 243. STREHLER, B.L. (1964) Adv. Geront. Res. 1, 343. TAPPEL, A.L., FLETCHER, B. and DESMER, D. (1973) J. Gerontol. 28, 415. WHrrEFORD, R. and GETTY, R. (1966) J. Geront. 21, 31. WrLCOX, H.H. (1956) J. Geront. 11, 442. ZEMA~, W. (1974) J. Neuropath. Exp. Neurol. 33, 1.
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