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Lipofuscin pigment in the nervous system of aging pig
Exp. Geront. Vol. 6, pp. 447--452. Pergamon Press 1971. Printed in Great Britain
L I P O F U S C I N P I G M E N T IN THE NERVOUS SYSTEM OF A G I N G PIG* t B. S. NANDA~ and R. GETTY§ Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50010, U.S.A.
(Received 16 August 1971) INTRODUCTION A NUMBERof studies on the aging pigment in nervous tissue, with regard to its occurrence, accumulation with age, its origin and significance, have been published. These have been reviewed in detail by Lansing (1952), Birren (1959). Bourne (1961), Barka and Anderson (1963), Whiteford (1964), Few (1966) and Nanda (1970). The available records show that the occurrence of aging pigment in nervous tissue has been studied extensively in rat, mouse and guinea pig. Few studies have been reported on the human and dog, whereas there are only two detailed studies available on the aging of nervous tissue of the pig (Whiteford, 1964; Few, 1966). MATERIALS AND M E T H O D S Thirty pigs, varying from 2 days to 10 yr of age, were included in the study. The pigs were obtained from the Swine Nutrition Farm, Iowa State University. A complete history, including genetic background and diet, was known for all animals. The animals were divided into five age groups: birth to 6 months; 6 months to 1 yr; 1 to 4 yr; 4 to 8 yr; and 8 to 10 yr. The blocks, made from the brains collected, were sectioned at 8 tz and examined with autofluorescence technique as well as with Alican blue and PAS stain. Twelve areas were included in the study. These included the nucleus olivaris inferior or nucleus olivaris, nucleus hypoglossus or nucleus motorius n. hypoglossi, dorsal motor nucleus of vagus or nucleus parasympatheticus n. vagi, nucleus cuneatus accessorius or lateralis, nuclei vestibulares, nuclei cochleares, cortex cerebelli, nucleus ruber or red nucleus, nucleus oculomotorius, thalamus, parahippocampal gyrus or gyrus parahippocampalis and cortex cerebri (frontal). Different brain areas were named in agreement with N.A.V. (1968) as far as possible. * This investigationwas supported in part by the U.S.P.H.S. Research Grants Nos. HE-04487 and HD-00041 from National Institute of Health, Department of Health, Education and Welfare. # Taken in part from the Ph.D. thesis of senior author done under the direction of late Dr. R. Getty. $ Post-Doctoral, Research Associate, N.I.H., Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50010, U.S.A. § Late Professor and Head, Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50010, U.S.A. E 447
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B. S. NANDA AND R. GETTY
OBSERVATIONS Nucleus olivaris inferior nucleus olivaris The neurons were observed to have the pigment at the age of 1 yr and 2 months. This pigment was in the form of fine loose focal masses in the polar and perinuclear areas of neurons. The pigment was sporadic but waswell evident, in many neurons, by the age of 2 yr. The rate of accumulation of pigment at different age groups indicated that this nuclear area had greater tendency for lipofuscin accumulation. About 40 to 50 per cent of neurons were pigmented at the age of 3 yr and 9 months. The magnitude of intraneuronal pigment and the number of pigmented neurons increased with age so that by 10 yr about 90 to 95 per cent of neurons were heavily pigmented. Nucleus hypoglossus or nucleus motorius n. hypoglossi The pigment in the nucleus hypoglossus was observed to be loosely granular at 1 yr and 5 months but was focal in few cells. The pigment was polar in neurons by 2 yr which was evident in 60 to 70 per cent of neurons by 3 yr and 9 months of age. Bipolar and perinuclear distribution of pigment was also evident. The amount of pigment increased with advancing age to fill about one-fourth to one-third of cytoplasmic volume by 10 yr. The percentage of pigmented neurons increased between 85 to 95 per cent in the fifth age group. Dorsal motor nucleus of vagus or nucleus parasympatheticus n. vagi The pigment was distributed in neurons of the above nucleus in granular form at the age of 2 yr and tended to become focal by 2 yr and 5 months. The distribution was focal in the polar and perinuclear area of neurons. About 35 to 40 per cent of neurons were pigmented by 3 yr and 9 months. The pigmentation increased with advancing age so that about 70 to 80 per cent of neurons were pigmented in the fifth age group. The amount of pigment also increased but some loosely arranged granules could still be seen in some neurons. Nucleus cuneatus accessorius or nucleus cuneatus lateralis The lipofuscin granules were present in loose focal form at the age of 2 yr and 9 months. The pigmentation increased in the third and fourth age groups assuming a loose focal form at the perinuclear or polar part of neurons. The pigment was present in about 70 to 80 per cent of neurons by 8 and 10 yr, but its loose focal nature still persisted in some neurons. Nuclei vestibulares The neurons showed sporadic pigment granules at 1 yr and 5 months. The pigment became focal by the age of 2 yr; perinuclear and axonal. The focal accumulation was very conspicuous at 2 yr and 11 months. About 45 to 55 per cent neurons were pigmented by 3 yr and 9 months. The pigment was both polar and perinuclear in distribution. The neurons were heavily pigmented at the age of 7 yr and 3 months. About 75 to 85 per cent of the neurons were pigmented by 10 yr. Nuclei cochleares The pigmentation in the above nuclei was not evident at 2 yr and 5 months of age. Few neurons were pigmented at 2 yr and 11 months, but pigmentation became significant
3 FIG. 1.
Nucleus
olivaris inferior, Fluorescence photomicrograph, no stain. A. 1 yr 5 months, x 250. B. 10 yr, x 250. FIG. 2. Nucleus hypogiossus, Fluorescence photomicrograph, no stain. A. 1 yr 11 months, x 400. B. 10 yr, x 100. FIG. 3. Nucleus parasympatheticus n. vagi, Fluorescence photomicrograph, no stain. A. 2 yr 5 months, x 250. B. 10 yr, x 400.
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FIG. 4. Nuclei vestibulares, Fluorescence photomicrograph, no stain. A. 2 yr 5 months, x 250. B. 10 yr, ~< 400. FIG. 5. Nuclei cochleares, Fluorescence photomicrograph, no stain. A. 2 yr 11 months, :- 250. B. 10 yr, .i 2.50. FIG. 6. Nucleus ruber, Fluorescence photomicrograph, no stain. A. 2 yr, 250. B. 10 pr, ‘,: 400.
THE NnWOUSSYSTEMOF AGINGPiG
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by 3 yr and 9 months of age. The pigmentation was crescentic perinuclear and loosely focal in distribution. It increased with advancing age and retained its characteristic manner of distribution. The rate of intraneuronal pigment increase with age was low. About 75 to 80 per cent of neurons were pigmented by 10 yr of age. Cortex cerebeUi Purkinje cells of the cerebeUar cortex were pigmented with lipofuscin granules at the age of 2 yr. The granules occupied polar areas intraneuronally. The pigment was present in about 40 to 45 per cent of Purkinje cells at 3 yr and 9 months. The rate of increase of the intraneuronal pigment was, however, slower with advancing age. This was evident in the older age groups in which pigment, though increased, was very low when compared with other nuclear areas, except the nuclei eochleares and thalamus. About 80 to 90 per cent of neurons were pigmented at 10 yr of age. The pattern of distribution was polar and sometimes perinuclear. Nucleus tuber or red nucleus The neurons were pigmented at the age of 2 yr. The distribution was perinuclear and polar in 40 to 50 per cent of neurons at 2 yr and 11 months. The pigmentation was heavy in the fourth age group (4 to 8 yr) where most neurons were having bipolar, polar or perinuclear pigment accumulation. About 65 to 76 per cent of neurons were pigmented by 7 yr and 3 months of age. The pigmentation increased in the fifth age group where almost all neurons of the nucleus ruber were heavily pigmented at 10 yr of age. The pattern of accumulation was generally bipolar and polar though some neurons were having perinuclear pigment distribution. Nucleus oculomotorius The pigment was distributed in the form of disseminated granules at 2 yr of age. It was loosely focal at 2 yr and 5 months and was perinuclear and axonal in distribution. The rate of increase with age was moderately high in the fourth age group. The lipofuscin pigment was heavy in most neurons by 10 yr, however, few cells showed diffuse accumulation also. Thalamus Nucleus ventralis caudalis (pars medialis and pars lateralis) and nucleus ventralis rostralis of the thalamus were included in the study. The granular pigmentation was present at 2 yr and 5 months. Few neurons with fine lipofuscin accumulation were evident at 2 yr and 9 months. About 50 to 60 per cent of the neurons were pigmented at 3 yr and 9 months of age. The pigmentation increased in the subsequent age groups, but the amount of intraneuronal pigment progressed comparatively at a slower rate which was comparable with the rate of increase in the nuclei cochleares and cortex cerebelli (Purkinje cells). The pigment was present in the polar and perinuclear areas in most neurons by 10 yr of age. Gyrus paralu'ppocampalis or parahippocampal gyrus The lipofuscin pigment was present in fine granular form at 3 yr and 9 months of age. It was slightly focal in some neurons. The pigment increased with advancing age and occupied perinuclear and polar areas. The pigmentation was loosely focal in about 40 to
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B . S . NANDA AND R. GETTY
45 per cent of the neurons by 4 yr and 6 months of age, which increased to 60 to 70 per cent by 7 yr and 3 months. The pigmentation remained loosely focal at 10 yr of age ; by then about 90 to 95 per cent of neurons were affected. Cortex cerebri The pigment in large pyramidal neurons of the fifth cortical layer of the precentral (frontal) cortex had loosely focal lipofuscin pigment granules at 1 yr and 11 months of age. The pigment became focal and compact by 2 yr and 9 months. The pattern of distribution of the pigment was axonal and perinuclear. In the fourth and fifth age group many large pyramidal cells of the fifth cortical layer showed pigment in moderate amount.
DISCUSSION The study, carried out on twelve areas of aging pig brains, revealed the presence or lipofuscin pigment. The time of appearance of pigment and its subsequent increase varied in different areas. The pigment appeared relatively earlier in the nucleus olivaris inferioris or nucleus olivaris, nucleus hypoglossus or nucleus motorius n. hypoglossi and nucleus rubrum or red nucleus of the pig. This was accompanied by a comparatively higher rate of accumulation with advancing age. The nuclei cochleares, nuclei vestibulares, dorsal motor nucleus of vagus or nucleus parasympatheticus n. vagi, nucleus oculomotorius, cortex cerebri (large pyramidal neurons of the fifth cortical layer of frontal or precentral cortex) and nucleus cuneatus accessorius or lateralis showed pigment later than the forementioned set of areas. The increase in the pigmentation with advancing age was moderate. The only exception in the above nuclei was the nucleus oculomotorius in which pigment appeared late, but the magnitude of pigment increased similar to first set of brain areas. The third category included cortex cerebelli (Purkinje cells), gyrus parahippoeampalis and thalamus. These brain areas indicated low rate of lipofuscin pigment increase with advancing age. A similar categorization of brain areas of the dog indicated many similarities. The main difference was the presence of lipofuscin pigment in the Purkinje cells of cerebellar cortex in the pig, absent in case of dog. This was in agreement with Whiteford and Getty (1966) and Nanda and Getty (1971). Aging of Purkinje cells of cerebellum have been investigated by several workers (Samorajski, Ordy and Radyheimer, 1968, in mice; Nandy, 1969, in guinea pig; Ellis, 1920, in human). Ellis (1920) mentioned that the pigment in the Purkinje cells of dog was rarely seen. He related the absence of pigment to their greater tendency to disintegrate with age. Similar studies by Harms (1924) and Inkuai (1928) on dog and rat also indicated the greater loss of Purkinje cells with age. Sulkin (1955) observed the presence of pigment in the granular cells but not frequently in the Purkinje cells of cerebellum in dog. Friede (1962) mentioned that lipofuscin pigment could be removed by glial cells, however, Issidorides and Shanklin (1961) and Shanklin, Issidorides and Nassar (1957) described such pigment formation to neurosecretory function of the Purkinje cells since they found abundant extracellular lipofusein granules in the synaptic fields. The presence of intraneuronal pigment in Purkinje cells of the pig and its extracellular location in dog may be attributed to higher rate of cell loss and higher neurosecretory functions of Purkinje cells in dog. Aside from the relative differences in time period for the appearance of lipofuscin pigment in dog and pig it was observed that the rate of pigment increase was lower in the
THE NERVOUS SYSTEM OF AGING PIG
451
cortex cerebri (large pyramidal cells of the fifth cortical layer of the precentral or frontal cortex) which may be attributed to difference in functional activity of these cells between species. A similar explanation may hold for the nucleus parasympatheticus n. vagi which showed pigmentation earlier in the pig than in the dog. The authors are of the opinion that in comparing the magnitude or time of occurrence of lipofuscin pigment in similar brain areas consideration may be given to the difference in functional activity of those areas in various species. The present observations indicated that the lipofuscin pigment was present in all brain areas studied. The amount of intraneuronal pigment as well as the percentage of neurons pigmented with lipofuscin increased with age. The consistency of its occurrence in adult pig and its increase with advancing age suggested that the presence of lipofuscin pigment was the only neurocytological age change in the neurons. This was in agreement with the earlier findings of various investigators (Bondareff, 1957, 1962, 1964; Brody, 1960; Samorajski et al., 1968; Whiteford, 1964; Whiteford and Getty, 1966; Few, 1966; Few and Getty, 1967; Nanda, 1970 and Nanda and Getty, 1971). Acknowledgements--The authors wish to acknowledge the cooperation of Drs. V. C. Speer, D. F.
Cox and L. N. Hazel of the Department of Animal Science, Iowa State University, for making available the pigs used in this study. Sincere appreciation for the help in photographic work by Dr. D. J. Hillmann, in biological preparations by Dr. Ralph Lanz, Mr. Lynn Martin and Miss Rose Aspengren is also acknowledged. REFERENCES B~mKA,T. and A_WDmaSON,P. J. (1963) Histochemistry, Theory, Practice and Bibliography. Hoeber Division, Harper and Row, New York. BIRR~, J. E. (Ed.) (1959) Handbook of Aging and the Individual. University of Chicago Press, Chicago, Illinois. BOND~aEFI~,W. (1957)y. Gerontol. 12, 364. B O N D ~ , W. (1959) In Handbook of Aging and Individual, (Edited by J. E. BIRRm~),University of Chicago Press, Chicago, Illinois. BOND~'F, W. (1964) In Advances in Gerontological Research, (Edited by B. L. STaSHLFaQ. Academic Press, New York. BOURNE,G. H. (Ed.) (1961) Structural Aspects of Aging. Hafner Publishing, New York. BRODY,H. (1960)]. Gerontol. 16, 258. ELLIS, M. S. (1920)]. comp. Neurol. 32, 1. FEw, A. B. (1966) Unpublished Ph.D. Thesis, Iowa State University, Ames, Iowa. FEw, A. B. and GETTY,R. (1967)]. Gerontol. 22, 357. FRmDE, R. L. (1962) Acta Neuropath. (Berlin) 2, 113. HARMS,J. W. (1924) Z. Anat. Entwick. 71, 319. INKUAI,T. (1928) ]. comp. Neurol. 45, 1. ISSDORIDES, M. and SHANKLIN, W. M. (1961) ]. Anat. London 95, 151. LANSING, A. I. (Ed.) (1952) Cowdry's Problems of Aging. Williams and Wilkins, Baltimore, Maryland. NANDA,B. S. (1970) Unpublished Ph.D. Thesis, Iowa State University, Ames, Iowa. N~rDA, B. S. and GETTY, R. (1971)]. Gerontol. (In Press). NANDY, K. (1969)J. Gerontol. 23, 82. SAMO~JSKI, T., O~g, J. M. and RAv,e~.-miMma,R. (1968) Anat. Rec. 160, 555. SHANKLIN,W. M., IssmomDES, M. and NASS~m,T. K. (1957)]. comp. NeuroI. 107, 315. SULtaN, N. M. (1955)]. Gerontol. 10, 135. WHn~-a~ORD,R. D. (1964) Unpublished Ph.D. Thesis, Iowa State University, Ames, Iowa. WHIan~FORD,R. and GETTY, R. (1966)]. Gerontol. 21, 31.
452
B. S. NKNDA AND R. G~1-r~/"
S u m m a r y - - T h e localization of the lipofuscin pigment in the twelve brain areas of the pig was included in the present investigation. It was observed that pigment was present in the adult pig brain and increased with advancing age. T h e study was conducted on thirty pigs ranging between the ages of 2 days and 10 yr. The pigment was identified with histologic stains and autoflorescence techniques. The time of appearance of pigment varied in different brain areas, but these differences progressively diminished with advancing age. T h e presence of pigment formed the most consistent neurocytological age associated change in aging nerve cells. R 6 s u m 6 - - L e s recherches d6crites ici impliquaient la localisation de la lipofuscine dans les douze r6gions c6r6brales du pore. On a constat6 que le pigment est pr6sent dans le cerveau du porc adulte et y augmente avec l'age. Ces recherches ont 6t6 effectu6es sur trente pores ag6s de deux jours/l dix ans. Le pigment a 6t6 identifi6 1'aide de colorations histologiques et de techniques d'autofluoroescence. Le moment d'apparition du pigment dans les diff6rentes r6gions c6r6brales varie, mais ces diff6rences s'amenuisent progressivement avec l'age. La pr6sence du pigment constitue la modification neurocytologique associde au vieillissement la plus r6guli~re dans les cellules nerveuses vieillissantes. Z u s a m m e n f a s s u n g - - D i e Lokalisation des Lipofuscinpigments in den zw61f Hirnarealen des Schweins wurde untersucht. Es wurde beobachtet, dab beim erwachsenen Schwein im Gehirn dieses Pigment auftritt u n d mit wachsendem Alter zunimmt. Die Untersuchung wurde an 30 Schweinen im Alter yon 2 Tagen bis 10 Jahren durchgefiihrt. Das Pigment wurde mit histologischer Farbung u n d mit Eigenfluoreszenz nachgewiesen. Der Zeitpunkt des Auftretens des Pigments war bei verschiedenen Hirnarealen unterschiedlich. Diese Unterschiede nahmen jedoch mit zunehmendem Alter ab. Die Gegenwart yon Pigment bildete die einheitlichste altersbezogene neurozytologische Veriinderung in altemden Nervenzellen. Pe3mMe----B HaCTO~meM HCCne~OBaHHH ~r3yqaJzacb HogaJIH3aLn~ HHHOdpyCIIRROBOFO HHFMeHTaB ]IBeHa.n~aTHoT~eHax Mo3ra CBHHbH. O6Hapyxanoc~ npHcyTc'rsxe nRrMeHTa B MO3r'y B3pocHMx CBHHel~,npI~eM KOHIIxleCTBOeFO IIOBbmlaHocbilpH cTapOHHR. I~CCJIe~OBaHHe IIpO~RROCb Ha TpHgR~aTRCBRFI]bRXB BO3paCTe OT ,RByx ]!Hen ,~o ,~ITH JIeT. HHrMeHT onpc)zengac~ np~ noMoum racTonormlccIo~ KpaCOKH MeTe]loB aBTo~bnyopec~em.mH. BpeM~HORBHeI-IH~IIHFMeHTa B pa3nHaH~IX OT~eHax M o 3 r a BapbzpoBa~O, He 3TH pa3~ HpoFpeCCHBHO yMeHsma.rmcs c Bo3pacToM. HpHCyTCTBHermrMewra 6stno H a H 6 O H ~ 3aKOHOMepHO CBR3aHHBIM C Bo3paCTOM HeBpoHHTOHOF~eCKHM H3MeHeHHeM B CTape~oRpdX HepBHMX KHCTKaX.
L I P O F U S C I N P I G M E N T IN THE NERVOUS SYSTEM OF A G I N G PIG* t B. S. NANDA~ and R. GETTY§ Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50010, U.S.A.
(Received 16 August 1971) INTRODUCTION A NUMBERof studies on the aging pigment in nervous tissue, with regard to its occurrence, accumulation with age, its origin and significance, have been published. These have been reviewed in detail by Lansing (1952), Birren (1959). Bourne (1961), Barka and Anderson (1963), Whiteford (1964), Few (1966) and Nanda (1970). The available records show that the occurrence of aging pigment in nervous tissue has been studied extensively in rat, mouse and guinea pig. Few studies have been reported on the human and dog, whereas there are only two detailed studies available on the aging of nervous tissue of the pig (Whiteford, 1964; Few, 1966). MATERIALS AND M E T H O D S Thirty pigs, varying from 2 days to 10 yr of age, were included in the study. The pigs were obtained from the Swine Nutrition Farm, Iowa State University. A complete history, including genetic background and diet, was known for all animals. The animals were divided into five age groups: birth to 6 months; 6 months to 1 yr; 1 to 4 yr; 4 to 8 yr; and 8 to 10 yr. The blocks, made from the brains collected, were sectioned at 8 tz and examined with autofluorescence technique as well as with Alican blue and PAS stain. Twelve areas were included in the study. These included the nucleus olivaris inferior or nucleus olivaris, nucleus hypoglossus or nucleus motorius n. hypoglossi, dorsal motor nucleus of vagus or nucleus parasympatheticus n. vagi, nucleus cuneatus accessorius or lateralis, nuclei vestibulares, nuclei cochleares, cortex cerebelli, nucleus ruber or red nucleus, nucleus oculomotorius, thalamus, parahippocampal gyrus or gyrus parahippocampalis and cortex cerebri (frontal). Different brain areas were named in agreement with N.A.V. (1968) as far as possible. * This investigationwas supported in part by the U.S.P.H.S. Research Grants Nos. HE-04487 and HD-00041 from National Institute of Health, Department of Health, Education and Welfare. # Taken in part from the Ph.D. thesis of senior author done under the direction of late Dr. R. Getty. $ Post-Doctoral, Research Associate, N.I.H., Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50010, U.S.A. § Late Professor and Head, Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50010, U.S.A. E 447
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B. S. NANDA AND R. GETTY
OBSERVATIONS Nucleus olivaris inferior nucleus olivaris The neurons were observed to have the pigment at the age of 1 yr and 2 months. This pigment was in the form of fine loose focal masses in the polar and perinuclear areas of neurons. The pigment was sporadic but waswell evident, in many neurons, by the age of 2 yr. The rate of accumulation of pigment at different age groups indicated that this nuclear area had greater tendency for lipofuscin accumulation. About 40 to 50 per cent of neurons were pigmented at the age of 3 yr and 9 months. The magnitude of intraneuronal pigment and the number of pigmented neurons increased with age so that by 10 yr about 90 to 95 per cent of neurons were heavily pigmented. Nucleus hypoglossus or nucleus motorius n. hypoglossi The pigment in the nucleus hypoglossus was observed to be loosely granular at 1 yr and 5 months but was focal in few cells. The pigment was polar in neurons by 2 yr which was evident in 60 to 70 per cent of neurons by 3 yr and 9 months of age. Bipolar and perinuclear distribution of pigment was also evident. The amount of pigment increased with advancing age to fill about one-fourth to one-third of cytoplasmic volume by 10 yr. The percentage of pigmented neurons increased between 85 to 95 per cent in the fifth age group. Dorsal motor nucleus of vagus or nucleus parasympatheticus n. vagi The pigment was distributed in neurons of the above nucleus in granular form at the age of 2 yr and tended to become focal by 2 yr and 5 months. The distribution was focal in the polar and perinuclear area of neurons. About 35 to 40 per cent of neurons were pigmented by 3 yr and 9 months. The pigmentation increased with advancing age so that about 70 to 80 per cent of neurons were pigmented in the fifth age group. The amount of pigment also increased but some loosely arranged granules could still be seen in some neurons. Nucleus cuneatus accessorius or nucleus cuneatus lateralis The lipofuscin granules were present in loose focal form at the age of 2 yr and 9 months. The pigmentation increased in the third and fourth age groups assuming a loose focal form at the perinuclear or polar part of neurons. The pigment was present in about 70 to 80 per cent of neurons by 8 and 10 yr, but its loose focal nature still persisted in some neurons. Nuclei vestibulares The neurons showed sporadic pigment granules at 1 yr and 5 months. The pigment became focal by the age of 2 yr; perinuclear and axonal. The focal accumulation was very conspicuous at 2 yr and 11 months. About 45 to 55 per cent neurons were pigmented by 3 yr and 9 months. The pigment was both polar and perinuclear in distribution. The neurons were heavily pigmented at the age of 7 yr and 3 months. About 75 to 85 per cent of the neurons were pigmented by 10 yr. Nuclei cochleares The pigmentation in the above nuclei was not evident at 2 yr and 5 months of age. Few neurons were pigmented at 2 yr and 11 months, but pigmentation became significant
3 FIG. 1.
Nucleus
olivaris inferior, Fluorescence photomicrograph, no stain. A. 1 yr 5 months, x 250. B. 10 yr, x 250. FIG. 2. Nucleus hypogiossus, Fluorescence photomicrograph, no stain. A. 1 yr 11 months, x 400. B. 10 yr, x 100. FIG. 3. Nucleus parasympatheticus n. vagi, Fluorescence photomicrograph, no stain. A. 2 yr 5 months, x 250. B. 10 yr, x 400.
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FIG. 4. Nuclei vestibulares, Fluorescence photomicrograph, no stain. A. 2 yr 5 months, x 250. B. 10 yr, ~< 400. FIG. 5. Nuclei cochleares, Fluorescence photomicrograph, no stain. A. 2 yr 11 months, :- 250. B. 10 yr, .i 2.50. FIG. 6. Nucleus ruber, Fluorescence photomicrograph, no stain. A. 2 yr, 250. B. 10 pr, ‘,: 400.
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by 3 yr and 9 months of age. The pigmentation was crescentic perinuclear and loosely focal in distribution. It increased with advancing age and retained its characteristic manner of distribution. The rate of intraneuronal pigment increase with age was low. About 75 to 80 per cent of neurons were pigmented by 10 yr of age. Cortex cerebeUi Purkinje cells of the cerebeUar cortex were pigmented with lipofuscin granules at the age of 2 yr. The granules occupied polar areas intraneuronally. The pigment was present in about 40 to 45 per cent of Purkinje cells at 3 yr and 9 months. The rate of increase of the intraneuronal pigment was, however, slower with advancing age. This was evident in the older age groups in which pigment, though increased, was very low when compared with other nuclear areas, except the nuclei eochleares and thalamus. About 80 to 90 per cent of neurons were pigmented at 10 yr of age. The pattern of distribution was polar and sometimes perinuclear. Nucleus tuber or red nucleus The neurons were pigmented at the age of 2 yr. The distribution was perinuclear and polar in 40 to 50 per cent of neurons at 2 yr and 11 months. The pigmentation was heavy in the fourth age group (4 to 8 yr) where most neurons were having bipolar, polar or perinuclear pigment accumulation. About 65 to 76 per cent of neurons were pigmented by 7 yr and 3 months of age. The pigmentation increased in the fifth age group where almost all neurons of the nucleus ruber were heavily pigmented at 10 yr of age. The pattern of accumulation was generally bipolar and polar though some neurons were having perinuclear pigment distribution. Nucleus oculomotorius The pigment was distributed in the form of disseminated granules at 2 yr of age. It was loosely focal at 2 yr and 5 months and was perinuclear and axonal in distribution. The rate of increase with age was moderately high in the fourth age group. The lipofuscin pigment was heavy in most neurons by 10 yr, however, few cells showed diffuse accumulation also. Thalamus Nucleus ventralis caudalis (pars medialis and pars lateralis) and nucleus ventralis rostralis of the thalamus were included in the study. The granular pigmentation was present at 2 yr and 5 months. Few neurons with fine lipofuscin accumulation were evident at 2 yr and 9 months. About 50 to 60 per cent of the neurons were pigmented at 3 yr and 9 months of age. The pigmentation increased in the subsequent age groups, but the amount of intraneuronal pigment progressed comparatively at a slower rate which was comparable with the rate of increase in the nuclei cochleares and cortex cerebelli (Purkinje cells). The pigment was present in the polar and perinuclear areas in most neurons by 10 yr of age. Gyrus paralu'ppocampalis or parahippocampal gyrus The lipofuscin pigment was present in fine granular form at 3 yr and 9 months of age. It was slightly focal in some neurons. The pigment increased with advancing age and occupied perinuclear and polar areas. The pigmentation was loosely focal in about 40 to
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B . S . NANDA AND R. GETTY
45 per cent of the neurons by 4 yr and 6 months of age, which increased to 60 to 70 per cent by 7 yr and 3 months. The pigmentation remained loosely focal at 10 yr of age ; by then about 90 to 95 per cent of neurons were affected. Cortex cerebri The pigment in large pyramidal neurons of the fifth cortical layer of the precentral (frontal) cortex had loosely focal lipofuscin pigment granules at 1 yr and 11 months of age. The pigment became focal and compact by 2 yr and 9 months. The pattern of distribution of the pigment was axonal and perinuclear. In the fourth and fifth age group many large pyramidal cells of the fifth cortical layer showed pigment in moderate amount.
DISCUSSION The study, carried out on twelve areas of aging pig brains, revealed the presence or lipofuscin pigment. The time of appearance of pigment and its subsequent increase varied in different areas. The pigment appeared relatively earlier in the nucleus olivaris inferioris or nucleus olivaris, nucleus hypoglossus or nucleus motorius n. hypoglossi and nucleus rubrum or red nucleus of the pig. This was accompanied by a comparatively higher rate of accumulation with advancing age. The nuclei cochleares, nuclei vestibulares, dorsal motor nucleus of vagus or nucleus parasympatheticus n. vagi, nucleus oculomotorius, cortex cerebri (large pyramidal neurons of the fifth cortical layer of frontal or precentral cortex) and nucleus cuneatus accessorius or lateralis showed pigment later than the forementioned set of areas. The increase in the pigmentation with advancing age was moderate. The only exception in the above nuclei was the nucleus oculomotorius in which pigment appeared late, but the magnitude of pigment increased similar to first set of brain areas. The third category included cortex cerebelli (Purkinje cells), gyrus parahippoeampalis and thalamus. These brain areas indicated low rate of lipofuscin pigment increase with advancing age. A similar categorization of brain areas of the dog indicated many similarities. The main difference was the presence of lipofuscin pigment in the Purkinje cells of cerebellar cortex in the pig, absent in case of dog. This was in agreement with Whiteford and Getty (1966) and Nanda and Getty (1971). Aging of Purkinje cells of cerebellum have been investigated by several workers (Samorajski, Ordy and Radyheimer, 1968, in mice; Nandy, 1969, in guinea pig; Ellis, 1920, in human). Ellis (1920) mentioned that the pigment in the Purkinje cells of dog was rarely seen. He related the absence of pigment to their greater tendency to disintegrate with age. Similar studies by Harms (1924) and Inkuai (1928) on dog and rat also indicated the greater loss of Purkinje cells with age. Sulkin (1955) observed the presence of pigment in the granular cells but not frequently in the Purkinje cells of cerebellum in dog. Friede (1962) mentioned that lipofuscin pigment could be removed by glial cells, however, Issidorides and Shanklin (1961) and Shanklin, Issidorides and Nassar (1957) described such pigment formation to neurosecretory function of the Purkinje cells since they found abundant extracellular lipofusein granules in the synaptic fields. The presence of intraneuronal pigment in Purkinje cells of the pig and its extracellular location in dog may be attributed to higher rate of cell loss and higher neurosecretory functions of Purkinje cells in dog. Aside from the relative differences in time period for the appearance of lipofuscin pigment in dog and pig it was observed that the rate of pigment increase was lower in the
THE NERVOUS SYSTEM OF AGING PIG
451
cortex cerebri (large pyramidal cells of the fifth cortical layer of the precentral or frontal cortex) which may be attributed to difference in functional activity of these cells between species. A similar explanation may hold for the nucleus parasympatheticus n. vagi which showed pigmentation earlier in the pig than in the dog. The authors are of the opinion that in comparing the magnitude or time of occurrence of lipofuscin pigment in similar brain areas consideration may be given to the difference in functional activity of those areas in various species. The present observations indicated that the lipofuscin pigment was present in all brain areas studied. The amount of intraneuronal pigment as well as the percentage of neurons pigmented with lipofuscin increased with age. The consistency of its occurrence in adult pig and its increase with advancing age suggested that the presence of lipofuscin pigment was the only neurocytological age change in the neurons. This was in agreement with the earlier findings of various investigators (Bondareff, 1957, 1962, 1964; Brody, 1960; Samorajski et al., 1968; Whiteford, 1964; Whiteford and Getty, 1966; Few, 1966; Few and Getty, 1967; Nanda, 1970 and Nanda and Getty, 1971). Acknowledgements--The authors wish to acknowledge the cooperation of Drs. V. C. Speer, D. F.
Cox and L. N. Hazel of the Department of Animal Science, Iowa State University, for making available the pigs used in this study. Sincere appreciation for the help in photographic work by Dr. D. J. Hillmann, in biological preparations by Dr. Ralph Lanz, Mr. Lynn Martin and Miss Rose Aspengren is also acknowledged. REFERENCES B~mKA,T. and A_WDmaSON,P. J. (1963) Histochemistry, Theory, Practice and Bibliography. Hoeber Division, Harper and Row, New York. BIRR~, J. E. (Ed.) (1959) Handbook of Aging and the Individual. University of Chicago Press, Chicago, Illinois. BOND~aEFI~,W. (1957)y. Gerontol. 12, 364. B O N D ~ , W. (1959) In Handbook of Aging and Individual, (Edited by J. E. BIRRm~),University of Chicago Press, Chicago, Illinois. BOND~'F, W. (1964) In Advances in Gerontological Research, (Edited by B. L. STaSHLFaQ. Academic Press, New York. BOURNE,G. H. (Ed.) (1961) Structural Aspects of Aging. Hafner Publishing, New York. BRODY,H. (1960)]. Gerontol. 16, 258. ELLIS, M. S. (1920)]. comp. Neurol. 32, 1. FEw, A. B. (1966) Unpublished Ph.D. Thesis, Iowa State University, Ames, Iowa. FEw, A. B. and GETTY,R. (1967)]. Gerontol. 22, 357. FRmDE, R. L. (1962) Acta Neuropath. (Berlin) 2, 113. HARMS,J. W. (1924) Z. Anat. Entwick. 71, 319. INKUAI,T. (1928) ]. comp. Neurol. 45, 1. ISSDORIDES, M. and SHANKLIN, W. M. (1961) ]. Anat. London 95, 151. LANSING, A. I. (Ed.) (1952) Cowdry's Problems of Aging. Williams and Wilkins, Baltimore, Maryland. NANDA,B. S. (1970) Unpublished Ph.D. Thesis, Iowa State University, Ames, Iowa. N~rDA, B. S. and GETTY, R. (1971)]. Gerontol. (In Press). NANDY, K. (1969)J. Gerontol. 23, 82. SAMO~JSKI, T., O~g, J. M. and RAv,e~.-miMma,R. (1968) Anat. Rec. 160, 555. SHANKLIN,W. M., IssmomDES, M. and NASS~m,T. K. (1957)]. comp. NeuroI. 107, 315. SULtaN, N. M. (1955)]. Gerontol. 10, 135. WHn~-a~ORD,R. D. (1964) Unpublished Ph.D. Thesis, Iowa State University, Ames, Iowa. WHIan~FORD,R. and GETTY, R. (1966)]. Gerontol. 21, 31.
452
B. S. NKNDA AND R. G~1-r~/"
S u m m a r y - - T h e localization of the lipofuscin pigment in the twelve brain areas of the pig was included in the present investigation. It was observed that pigment was present in the adult pig brain and increased with advancing age. T h e study was conducted on thirty pigs ranging between the ages of 2 days and 10 yr. The pigment was identified with histologic stains and autoflorescence techniques. The time of appearance of pigment varied in different brain areas, but these differences progressively diminished with advancing age. T h e presence of pigment formed the most consistent neurocytological age associated change in aging nerve cells. R 6 s u m 6 - - L e s recherches d6crites ici impliquaient la localisation de la lipofuscine dans les douze r6gions c6r6brales du pore. On a constat6 que le pigment est pr6sent dans le cerveau du porc adulte et y augmente avec l'age. Ces recherches ont 6t6 effectu6es sur trente pores ag6s de deux jours/l dix ans. Le pigment a 6t6 identifi6 1'aide de colorations histologiques et de techniques d'autofluoroescence. Le moment d'apparition du pigment dans les diff6rentes r6gions c6r6brales varie, mais ces diff6rences s'amenuisent progressivement avec l'age. La pr6sence du pigment constitue la modification neurocytologique associde au vieillissement la plus r6guli~re dans les cellules nerveuses vieillissantes. Z u s a m m e n f a s s u n g - - D i e Lokalisation des Lipofuscinpigments in den zw61f Hirnarealen des Schweins wurde untersucht. Es wurde beobachtet, dab beim erwachsenen Schwein im Gehirn dieses Pigment auftritt u n d mit wachsendem Alter zunimmt. Die Untersuchung wurde an 30 Schweinen im Alter yon 2 Tagen bis 10 Jahren durchgefiihrt. Das Pigment wurde mit histologischer Farbung u n d mit Eigenfluoreszenz nachgewiesen. Der Zeitpunkt des Auftretens des Pigments war bei verschiedenen Hirnarealen unterschiedlich. Diese Unterschiede nahmen jedoch mit zunehmendem Alter ab. Die Gegenwart yon Pigment bildete die einheitlichste altersbezogene neurozytologische Veriinderung in altemden Nervenzellen. Pe3mMe----B HaCTO~meM HCCne~OBaHHH ~r3yqaJzacb HogaJIH3aLn~ HHHOdpyCIIRROBOFO HHFMeHTaB ]IBeHa.n~aTHoT~eHax Mo3ra CBHHbH. O6Hapyxanoc~ npHcyTc'rsxe nRrMeHTa B MO3r'y B3pocHMx CBHHel~,npI~eM KOHIIxleCTBOeFO IIOBbmlaHocbilpH cTapOHHR. I~CCJIe~OBaHHe IIpO~RROCb Ha TpHgR~aTRCBRFI]bRXB BO3paCTe OT ,RByx ]!Hen ,~o ,~ITH JIeT. HHrMeHT onpc)zengac~ np~ noMoum racTonormlccIo~ KpaCOKH MeTe]loB aBTo~bnyopec~em.mH. BpeM~HORBHeI-IH~IIHFMeHTa B pa3nHaH~IX OT~eHax M o 3 r a BapbzpoBa~O, He 3TH pa3~ HpoFpeCCHBHO yMeHsma.rmcs c Bo3pacToM. HpHCyTCTBHermrMewra 6stno H a H 6 O H ~ 3aKOHOMepHO CBR3aHHBIM C Bo3paCTOM HeBpoHHTOHOF~eCKHM H3MeHeHHeM B CTape~oRpdX HepBHMX KHCTKaX.