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Occurrence of aging pigment (lipofuscin) in the nuclei and cortices of the canine brain
Exp. Geront. Vol. 8. pp. 1-7. P e r g a m o n Press 1973. Printed in G r e a t Britain.
OCCURRENCE OF AGING PIGMENT THE NUCLEI AND CORTICES OF THE
(LIPOFUSCIN) IN C A N I N E BRAIN*~"
B. S. N,~ND,~:~and R. G~.TTY§
Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State Univer.~ity, Ames, Iowa 50010, U.S.A. (Received 16 June 1972)
INTRODUCTION LIPOFUSCIN pigment has received mucl~ attention in recent years because of its potential relation to the process of aging. The name lipofuscin was first used by Borst (1922). A number of studies have been conducted since over fifty years, especially of man, mouse, guinea pig and rat. Lipofuscin in the nervous tissue of the dog was studied by Dolley (1911); Harms (1924); Sulkin (1955a, b); Whitef~rd (1964); Whiteford and Getty (1966); Few (1966) and Few and Getty (1967). Both these and other workers mention the accumulation of lipofuscin pigment as a consistent cytological age change in neurons (Bensley, 1947; Bondareff, 1957; Samorajski, Keefe and Ordy, 1964; Samorajski, Ordy and Keefe, 1965; Samorajski, Ordy and Radyheimer, 1968; and Brizzee, Cancilla, Sherwood and Timiras, 1969). The present study was designed to observe the occurrence of lipofuscin pigment and its increase with advancing age in clinically healthy dogs. MATERIALS AND METHODS Forty dogs ranging from 1 day to 16 yr of age were studied. The animals were divided into six age groups; birth to 6 months: 6 months to 1 yr; 1 yr to 4 yr; 4 yr to 8 yr; 8 yr to 12 yr; and 12 yr to 16 yr of age. The dogs were whelped in the department of Veterinary Anatomy. A complete history including genetic background and dietary records were kept for all animals. Each brain collected was divided into six segments and embedded in paraplast. Sections of 81a thickness were obtained from each block. The unstained sections were examined for autofluorescence, and Alican blue and P.A.S. staining technique was also employed to confirm the presence of the lipofuscin pigment. Fifteen areas, nucleus olivaris inferioris or nucleus olivaris, nucleus hypoglossus or nucleus motorius n. hypoglossi, dorsal m o t o r nucleus of vagus or nucleus parasympatheticus n. vagi, nucleus cuneatus accessorius or nucleus cuneatus lateralis, nuclei vestibulares, nuclei cochleares, nuclei cerebellares, nucleus rubrum or red nucleus, nucleus oculomotorius or oculomotor nucleus, thalamic area, gyrus parahippocampalis, cortex cerebri (frontal), nucleus caudatus, putamen and globus pallidus, were examined for the study in the dog. This terminology for different parts of the brain has been adopted in agreement with N.A.V. (1968). * This investigation was supported in part by the U.S.P.H.S. research Grants Nos. HE-04487 and HD00041 from the National Institutes of Health, Department of Health, Education and Welfare. ~"Taken in part from the Ph.D. thesis of senior author. * Associate Professor and Head, Department of Veterinary Anatomy and Histology, College of Veterinary Medicine, Punjab Agricultural University, Ludhiana, India. § Deceased. Late Professor and Head, Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50010, U.S.A.
2
B.S.
NANDA AND R. GETTY
RESULTS
Nucleus olivaris inferioris or nucleus olivaris (Fig. 1) Lipofuscin pigment granules were seen by the age of 11 months. The granules were loosely arranged in the perinuclear areas of neurons. The percentage of neurons with lipofuscin pigment was very low but it increased from 1 yr of age onwards, increasing from 35 per cent at 1 yr and 5 months to about 49 per cent at 4 yr of age. The pigment granules were perinuclear and polar in location. The progressive increase in mean percentage of neurons with pigmentation increased steadily to about 82 per cent in the fourth age group, ranging between 4 and 8 yr of age. The amount of pigment increased correspondingly. About 92 per cent of neurons were pigmented in the sixth age group which included animals between 12 and 16 yr of age. The pigment was heavily concentrated in both the perinuclear and polar areas of neurons. Nucleus hypoglossus or nucleus motorius n. hypoglossi (Fig. 2) The pigment was first observed at the age of 11 months when it was loosely distributed in the form of fine granules, but became loosely focal by 4 yr of age. The pattern of accumulation was polar or bipolar. About 56 per cent of neurons were pigmented by 4 yr of age. The increase was not only associated with the amount of the pigment but also in the number of neurons involved. About 75 per cent of neurons were pigmented in the animals ranging between 8 and 12 yr and about 87 per cent between 12 and 16 yr of age. Dorsal motor nucleus of vagus or nucleus parasympatheticus n. vagi (Fig. 3) The pigmentation appeared very late in this nucleus. Few disseminated pigment granules were present at 4 yr. However, the rate of pigment deposition increased with age in the fourth age group, (4-8 yr). The pattern of the focal deposition was perinuclear and polar; some neurons had loose granular distribution also. The pigmentation in the fifth age group was focal and heavy. The increase in the deposition was very conspicuous in the sixth age group in which the pigmentation was almost completely masking the nucleus and cytoplasm in some, whereas in others the granulation covered about one-half or more of the neuronal volume. About 79 per cent of the neurons were pigmented by 16 yr of age. Nucleus cuneatus accessorius or nucleus cuneatus lateralis The pigment appeared as loose granules at 2 yr of age. The pigment was small in amount, but increased in amount and frequency, at 4 yr of age. Seventy-eighty per cent of neurons contained pigment, in the polar and bipolar forms, in the fourth age group. This increasing trend was also evident in the fifth and sixth age groups where the pigmentation occupied more and more of the cytoplasmic volume of the neurons and the number of pigmented neurons increased. In the sixth age group 79-92 per cent of neurons were showing pigment which was present almost throughout their cytoplasmic volume in many cases. Nuclei vestibulares (Fig. 4) The pigment appeared consistently in the form of axonal and bipolar accumulation from one year and seven months onwards. The rate of accumulation increased steadily in the third age group in which _~ 36 per cent of neurons were pigmented. The increase was further evidenced in the fourth age group where approximately 70 per cent of neurons were pigmented.
FIG. I. Nucleus
olivaris Inferior. 250. Alican blue and PAS stain. a. II months b. 16 yr. 250. Alican blue and PAS stain. FIG,. 2. Nucleus hypoglossus, a. 1 yr and 5 months h. I6 yr.
FK;. 3. Nucleus
parasympatheticus n. vagi. 250, Alican blue and PAS stain. b. 16 yr. a. 4 yr. 250. Alican blue and PAS stain. FIG;. 4. Nuclei vestibuiares. a. I yr. and 5 months. h. I6 yr.
250, Alican blue and PAS stain FIG. 5. Nuclei cochleares, b. I6 yr‘. a. 2 yr and 7 months. 100. Alican blue and PAS stain. FIG. 6. Nucleus tuber. b. I6 yr. a. I I months.
FIG. 7. Thalamic area. 250, Alican blue b. I6 a. 2 yr and 7 months. 250, Alican FIG. 8. Cortex cerebri (frontal). b. I6 a. I I Imonths.
and PAS stain. yr. blue and PAS stain yr.
AGING IN CANINE~BRAIN
3
The observations in the age group five and six revealed that the increased deposition was perinuclear, polar, and bipolar in distribution. The frequency of the neurons involved was considerably higher, reaching 91 per cent in the sixth age group. The cytoplasm of some neurons was filled with disseminated pigment granules. Nuclei cochleares (Fig. 5)
The disseminated pigment granules were present by 1 yr and 7 months. The distribution was observable in a very small percentage of cells. The pigment increased to become focal by 2 yr and 7 months. Twenty-five per cent of neurons were pigmented in the third age group. The pattern of distribution was perinuclear and sometimes polar; however discrete fine granules could also be seen in the neuronal cytoplasm. Eighty-two per cent of neurons were pigmented by 16 yr of age. Nuclei cerebellares
The dentate and fastigial nuclei were considered only. The pigmentation was loose and dispersed in the neuronal cytoplasm of the fastigial nucleus : it was loosely focal in the dentate nucleus at 4 yr of age. The granules were perinuclear and polar in location. The increase in the pigmentation was comparatively slower in the fastigial nucleus than in the dentate nucleus; there was more, and more focal pigmentation, in the second of these than the first, in the same specimens and same age grcup. The pigmentation continued to increase slightly with age in the fifth age group, but in the sixth age group there seemed to be a rapid increase both in amount of pigment and frequency of neurons pigmented. This was very evident in both nuclei at the age of 16 yr. Cortex cerebelli No substantial intraneuronal pigmentation was observed in the Purkinje cells of the cerebellum of the dog at any age group; but, pigment accumulations were discernable in the areas surrounding the Purkinje cells beyond 4 yr of age. This accumulation increased steadily with age. Nucleus rubrum or red nucleus (Fig. 6)
The presence of small amounts of lipofuscin pigment in the form of loose focal (perinuclear and axonal) accumulation was present in the second age group. The focal clumping of the pigment was distinct by 4 yr of age, though in some neurons disseminated granules were also present. 21 55 per cent of the neurons were pigmented by 4 yr of age. The pigmentation increased with advancing age so that 2: 75 per cent of neurons in this nucleus were pigmented by the fourth age group. The pigment occupied axonal, perinuclear and bipolar sites which in some cases masked the nucleus of the neurons. The pigmentation was very heavy and occurred in 90-95 per cent of the neurons in the sixth age group. Nucleus oculomotorius or oculomotor nucleus
The pigment was observed as loosely disseminated granules at about two years of age. It appeared to occur in the perinuclear and polar areas of neurons in the third age group. The progressive increase was observed to continue to the fifth age group, with the pigment axonal or perinuclear but rarely bipolar. The cells were heavily pigmented by 12 yr of age, and the pigmentation was densely focal in 60-70 per cent of neurons. This increase
4
B. S. NANDA
AND
R. GETTY
continued in the sixth age group where the pigmentation was very heavy and almost all neurons showed varying degree of accumulation. The pigmentation was mostly axonal, but some neurons also had a perinuclear or bipolar distribution of the pigment. Thalamic area (Fig. 7)
The lipofuscin pigment was observed at the age of 2 yr in the nucleus ventralis caudalis pars medialis, nucleus ventralis caudalis pars lateralis and nucleus ventralis rostralis of the thalamus. Few lipofuscin granules, however, were observed even at 1 yr and 7 months. The pattern of its distribution was axonal or perinuclear and was loosely packed by 2 yr of age. The rate of accumulation was relatively slow; only 40-50 per cent of neurons were pigmented by 8 yr of age. The neuronal accumulation of pigment increased steadily with age, but the increase in the amount of intraneuronal pigment was relatively slow. Seventyeighty per cent of the neurons were pigmented, though the amount of intraneuronal pigment was not significantly increased by 16 yr of age. Gyrus parahippocampalis
The pigmentation of neurons in this gyrus was observed in the form of few disseminated granules at 2 yr and 7 months of age. This became loosely focal in few neurons by 4 yr of age. The progression of deposition and neuronal involvement was very slow but still granular. Thirty-forty per cent of neurons were showing loosely focal pigmentation by 7 yr and 6 months; it became slightly focal, in small masses, in 70-80 per cent of neurons by 12 yr of age. The pigmentation was present in 90 per cent of neurons by the sixth age group. The amount and pattern of the deposition was variable, perinuclear and axonal; a few neurons with disseminated pigment granules were also observed. Cortex cerebri (Frontal) (Fig. 8)
The pigment was observed in the area precentralis gigantopyramidalis. Fine pigment granules were present in the giant pyramidal cells at the age of 10 months but became focal by the end of the second age group (1 yr). The presence of lipofuscin in the pyramidal neurons of the third cortical layer was evident by 2 yr and 7 months. The Betz cells by that age showed further clumping of the pigment and were seen to mask the nucleus at places. The common pattern of distribution was perinuclear and polar. The pigmentation became very heavy in the fourth age group. The pigment occupied about one-third or one-half of neuronal cytoplasm in some cases. A similar increase in the pyramidal neurons of the third layer was observed though it was comparatively less than in the giant pyramidal cells of the fifth cortical layer. The trend continued in the succeeding age groups until almost all Betz cells were heavily pigmented. The pyramidal cells of the third layer had a similar increase though the amount of pigment and the proportion of neurons involved were comparatively less than for the large pyramidal cells. Nucleus caudatus
Pigment was observed in the form of loose polar and perinuclear clumps by 4 yr. Slight granular deposition was present in a few neurons after 3 yr and 2 months of age, and increased steadily. The changes in distribution of lipofuscin pigment in the fifth age group indicated that there was an increase in the amount of the pigment as well as in the number of neurons containing it. The basic pattern of loose focal distribution, however, did not
AGING
IN CANINE
BRAIN
5
change significantly. Focal perinuclear accumulation of the pigment was observed by the age of 16 yr at which 80-90 per cent of neurons were involved. Putamen and globus pallidus
The putamen and globus pallidus showed the presence of the lipofuscin pigment at the age of 2 yr and 7 months. The pigment was in the form of small, loosely packed polar accumulations. This pigment was more in globus pallidus than the putamen. The pigment became more focal in the polar and pcrinuclear areas of the neurons. Forty-fifty per cent of neurons were pigmented in the globus pallidus by 12 yr of age. This increased to 70-80 per cent by 16 yr of age. The pigment was more coherent and focal in globus pallidus than in the putamen, in which loosely placed granules were observed even at 16 yr of age. I)ISCUSSION Our study included fifteen brain areas of the dog, and showed progressive accumulation of lipofuscin pigment in neurons with advancing age. This corresponded with the findings of the earlier workers who regarded this accumulation as the most reliable neurocytological age change (Bondareff, 1957, 1962, 1964; Brody, 1960; Samorajski, Keefe and Ordy, 1964; Samorajski, Ordy and Keefe, 1965; Samorajski, Ordy and Radyheimer, 1968; Whiteford, 1964; Whiteford and Getty, 1966; Few, 1966; Few and Getty, 1967). Various views regarding the origin of the lipofuscin pigment have been advanced from time to time. Dolley (1917) regarded the presence of the pigment as a product of chromatin transformation. Payne (1949, 1952); Hess (1952); Duncan, Nall and Morales (1960) and Gosh, Bern and Nishioka (1960) propounded that the pigment was derived from mitochondrial alterations or degeneration. The introduction of lysosomes into the problem of lipofuscin genesis received considerable attention following the works of de Duve and Wattiaux (1966) ; Essner and Novikoff (1960) and Novikoff (1961). They suggested that the lipofuscin pigment granules were altered lysosomes. Studies of Samorajski, Keefe and Ordy (1964); Samorajski, Ordy and Keefe (1965); Few and Getty (1967) and Munnell and Getty (1968) supported this view, The!{ suggest that the pigment granules appeared to be formed by coalescence of altered lysosomes and other cellular organelles in varying degrees of autolysis. The present study revealed that pigment granules appeared in the perinuclear areas of neurons which increased in amount and clumped focally at different sites with advancing age. The various sites of focal accumulation have been described earlier by Hopker (1951), Whiteford and Getty (1966) and Few a.nd Getty (1967). From the present study it looks as il’ different areas of brain show differences in time of pigment deposition as well as in the rate and degree of deposition with increasing age. The earliest pigment was observed in large pyramidal cells (Betz) of the frontal cortex, nucleus olivaris inferioris or nucleus olivaris, nucleus hypoglossus or nucleus motorius n. hypoglossi and nucleus rubrum or red nucleus. The pigment increased progressively, and the rate of increase was rapid. The neurons became heavily pigmented with advancing age. A cursory examination of neurons of the reticular formation revealed much the same pattern. The lipofuscin pigment accumulation appeared to be somewhat delayed in the nuclei cochleares, nuclei vestibulares, nucleus oculomotorius and nucleus cuneatus lateralis. The increase of the pigment with age in these sites was moderate. The cerebellar nuclei, putamen, globus pallidus, nucleus caudatus, and gyrus parahip-
6
B. S. NANDA
AND R. GE-l-l-Y
pocampalis were pigmented comparatively late (between 2 yr and 4 yr of age). The rate of increase of the pigment varied from moderate to low. The lowest rate of increase of lipofuscin pigment was observed in the thalamic nuclear areas studied. The dorsal motor nucleus of vagus or nucleus parasympatheticus n. Vagi showed the pigment very late but the rate of increase was very high. No pigment was observed intraneuronally in the Purkinje cells of the cerebellar cortex, though pigment granules were present in the granular layer. The times of occurrence of lipofuscin in relation to function of different brain areas have been examined by a number of workers. Sulkin (19%~) observed pigment granules to be more widespread in the efferent than the afferent neurons. This was also seen by Few and Getty (1967) who concluded that the autonomic cells were most resistant to pigmentation and efferent ventral horn cells of the spinal cord were less resistant. These differences were progressively reduced as the animal increased in age. Similar views were expressed by Wilcox (1956, 1959). Nandy (1968) studied the lipofuscin pigment in the nervous system of guinea pig and divided it into four categories based on rhe degree of deposition. Our categorisation, which is based on the time of appearance as well as on the degree of pigmentation, substantially agrees with the views of Nandy (1968) except for some differences which may be due to the functional status of the nuclear areas in different species. We have no direct evidence regarding the progressive accumulation of lipofuscin pigment in the neurons and its effect on neuronal metabolism and physiology. Strehler, Mark, Mildvan and Gee (1959) proposed that the pigment may affect the efficient working of the heart muscle. Similar conclusion has been drawn by Murray and Stout (1947). Samorajski, Keefe and Ordy (1964); Samorajski, Ordy and Keefe (1965); Whiteford (1964) and Few (1966). Samorajski, Ordy and Radyheimer (1968) hold that the accumulation of the lipofuscin material may affect all aspects of neuronal physiology and possibly result in loss of the cell. They also regarded the location of the pigment in the area between the nucleus and apical dendrite as of considerable consequence for neuronal function. We ourselves consider that the presence of the pigment in the neuron, occupying much of its cytoplasmic volume, may well be detrimental to normal cellular metabolism even if the material is a by-product or end-product of cellular metabolism. The physiological and metabolic functions of neurons will probably be affected through organelle loss and also because of the reduction of cell volume with advancing age. Acknowledgements-Sincere appreciation is extended to Dr. D. J. Hillmann for his assistance in photography and Mr. Ralph Lanz and Mr. Lynn Martin in preparing histological preparations. The authors acknowledge the help extended by Dr. J. H. Magilton, Acting Head, Department of Veterinary Anatomy during preparation of this manuscript.
REFERENCES BENSLEY,R. (1947) Amt. Rec. 98, BONDAREFF,W. (1957) J. Gerontol. BONDAREFF,W. (1959) Handbook Press, Chicago, 111. BONDAREFF,W. (1964) Advances New York.
609. 12, 364. of Aging and Individual in Gerontological
(Edited by J. E. Birren), University of Chicago
Research
(Edited by B. L. Strehler), Academic
M. (1922)Pathologische Histologic. Leipzig, Vogel. BRIZZEE,K. R., CANCILLA,A. P., SHERWOOD,N. and TIMIRAS,P. S. (1969) J. Gerontol. 24, 127. BRODY,H. (1960) J. Gerontol. 15, 258. DE DUVE, C. and WATTIAUX,R. (1966) Ann. Rev. Physiol. 28, 435.
BORST,
Press,
AGII\;GIN CANINEBRAIN DOLLEY,D. H. (1911) J. med. Res. 24, 309. NALL, D. M. and MORALES,R. (1960) J. Geronrol. 15, 568. ESSNER,E. and NOVIKOFF,A. B. (1960) J. Ultrastnrct. Res. 3, 341. FEW, A. B. (1966) Unpublished Ph.D. thesis. Iowa State University, Ames, Iowa. FEW, A. B. and GETTY, R. (1967) J. Gerontol. 22, 357. GOSH, A., BERN, J. A., GOSH, 1. and NISHIOKA,R. A. (1960) Anat. Rec. 143, 195. HARMS,J. W. (1924) 2. Anat. Entwicklung. 71, 319. HESS, A. (1955) Anat. Rec. 123, 399. HOPKER, W. (1951) 2. Alternsforchung. 5, 256. MURRAY, M. R. and STOUT,A. P. (1947) Amer. J. Anat. 80, 225. NANDY, K. (1968) J. Geronfol. 23, 82. Nomina Anatomica Veterinaria (1968) Adolf Holzhausen’s Successors, Vienna. NOVIKOFF,A. B., BEAUFY,H. and DE DWE, <:. (1956) J. biophys. biochem. Cytol. 2, 179. PAYNE,F. (1949) J. Gerontol. 4, 195. PAYNE,F. (1952) Cow&y’s Problems of Aging (Edited by A. I. Lansing), Williams and Wilkins, Baltimore. SAMORAJSKI,T., KEEFE,J. R. and ORDY, J. M. (1964) J. Gerontol. 19, 262. SAMORAJSKI, T., ORDY, J. M. and KEEFE, J. R. (1965) J. Cell Biol. 26, 779. SAMORA~KI,T., ORDY, J. M. and RADYHEIMEIX, R. (1968) Anat. Rec. 160, 5.55. STREHLER,B. L., MARK, D. D., MILDVAN,A. S. and GEE, M. B. (1959) J. Gerontol. 14, 430. SULKIN, N. M. (1955a) J. Gerontol. 10, 135. SULKIN, N. M. (19556) J. biophys. biochem. Cvtol. 1, 459. SULKIN, N. M. and KUNTZ, A. (1952) J. Geroqtol. 7, 533. WHITEFORD,R. D. (1964) Unpublished Ph.D. thesis. Iowa State University, Ames, Iowa. WHITEFORD,R. D. and GETTY,R. (1966) J. Gcrontol. 21, 31. WILCOX,H. H. (1956) U.S. Public Health Report. Washington, 71, 1179. WILCOX, H. H. (1959) The Process of Aging 1,~ Nervous System (Edited by J. E. Birren, H. A. Imus and W. F. Windle), Thomas, Springfield, Ill.
Summary-The occurrence of lipofuscill pigment in fifteen nuclear and cortical areas of brain of dogs ranging from one day old to sixteen years of age was studied. Histological and autoflorescence techniques showed that the lipofuscin pigment was present in the brain of young adult and old dogs, increasing in ascending order of age. Localisation of the pigment, in apparently healthy doss, revealed its presence as the only neurocytological change in neurons of different areas. It was observed that the motor nuclei and areas developed the pigment at an early age; the increase was rapid with age when compared with that in the sensory and associative nuclei. The nuclei related with autonomic functions developed the pigment relatively late. RBsumk-On a &udiC l’apparition du pigment de la lipofuscine dans quinze sites nucleaires et corticaux du cerveau chez des chiens BgCs d’un jour B seize ans. Des techniques histologiques et d’auto-fluorescence ont rCv& que le pigment Ctait present dans le cerveau des chiens adultes, jeunes et IgCs, en quantitks croissantes avec l’bge. La localisation du pigment chez des chiens app:rremment bien portants a revel& que sa prksence dtait la seule modification neurocytologique des neurones des diff&ents sites. On a constatt que le pigment apparait t8t dans les zones et ~luclei moteurs; son augmentation avec l’Pge est rapide, comparativement & celle observCe dans les nuclei sensoriels et associatifs. 11apparait relativement tard dans les nuclei associts aux .‘onctions autonomes. Zusammenfassung-Das Auftreten VOIDLipofuszinpigment in fiinfzehn Kern- und Rindenbezirken des Gehirns des Hundes wurde untersucht bei einen Tag bis sechzehn Jahre alten Tieren. Die histologischen und autofluoromctrischen Verfahren zeigten, daR das Lipofuszinpigment im Gehirn junger erwachsener und alter Hunde vorlag und mit dem Alter zunahm. Die Lokalisation des Pigments bei augenschein lich gesunden Hunden ergab die einzige neurocytologische Verznderung in Neuronen unterschiedlichen Alters. Es wurde beobachtet, dal3 die motorischen Kerne und Bezirke das Pigment in friihem Alter entwickelten. Die Zunahme mit dem Alter war schnell im Vergleich mit demjenigen in sensorischen oder assoziativen Kernen. Die Kerne, die mit autonomen Funktionen in Beziehung stehen, entwickelten das Pigment relativ sp8t.
OCCURRENCE OF AGING PIGMENT THE NUCLEI AND CORTICES OF THE
(LIPOFUSCIN) IN C A N I N E BRAIN*~"
B. S. N,~ND,~:~and R. G~.TTY§
Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State Univer.~ity, Ames, Iowa 50010, U.S.A. (Received 16 June 1972)
INTRODUCTION LIPOFUSCIN pigment has received mucl~ attention in recent years because of its potential relation to the process of aging. The name lipofuscin was first used by Borst (1922). A number of studies have been conducted since over fifty years, especially of man, mouse, guinea pig and rat. Lipofuscin in the nervous tissue of the dog was studied by Dolley (1911); Harms (1924); Sulkin (1955a, b); Whitef~rd (1964); Whiteford and Getty (1966); Few (1966) and Few and Getty (1967). Both these and other workers mention the accumulation of lipofuscin pigment as a consistent cytological age change in neurons (Bensley, 1947; Bondareff, 1957; Samorajski, Keefe and Ordy, 1964; Samorajski, Ordy and Keefe, 1965; Samorajski, Ordy and Radyheimer, 1968; and Brizzee, Cancilla, Sherwood and Timiras, 1969). The present study was designed to observe the occurrence of lipofuscin pigment and its increase with advancing age in clinically healthy dogs. MATERIALS AND METHODS Forty dogs ranging from 1 day to 16 yr of age were studied. The animals were divided into six age groups; birth to 6 months: 6 months to 1 yr; 1 yr to 4 yr; 4 yr to 8 yr; 8 yr to 12 yr; and 12 yr to 16 yr of age. The dogs were whelped in the department of Veterinary Anatomy. A complete history including genetic background and dietary records were kept for all animals. Each brain collected was divided into six segments and embedded in paraplast. Sections of 81a thickness were obtained from each block. The unstained sections were examined for autofluorescence, and Alican blue and P.A.S. staining technique was also employed to confirm the presence of the lipofuscin pigment. Fifteen areas, nucleus olivaris inferioris or nucleus olivaris, nucleus hypoglossus or nucleus motorius n. hypoglossi, dorsal m o t o r nucleus of vagus or nucleus parasympatheticus n. vagi, nucleus cuneatus accessorius or nucleus cuneatus lateralis, nuclei vestibulares, nuclei cochleares, nuclei cerebellares, nucleus rubrum or red nucleus, nucleus oculomotorius or oculomotor nucleus, thalamic area, gyrus parahippocampalis, cortex cerebri (frontal), nucleus caudatus, putamen and globus pallidus, were examined for the study in the dog. This terminology for different parts of the brain has been adopted in agreement with N.A.V. (1968). * This investigation was supported in part by the U.S.P.H.S. research Grants Nos. HE-04487 and HD00041 from the National Institutes of Health, Department of Health, Education and Welfare. ~"Taken in part from the Ph.D. thesis of senior author. * Associate Professor and Head, Department of Veterinary Anatomy and Histology, College of Veterinary Medicine, Punjab Agricultural University, Ludhiana, India. § Deceased. Late Professor and Head, Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50010, U.S.A.
2
B.S.
NANDA AND R. GETTY
RESULTS
Nucleus olivaris inferioris or nucleus olivaris (Fig. 1) Lipofuscin pigment granules were seen by the age of 11 months. The granules were loosely arranged in the perinuclear areas of neurons. The percentage of neurons with lipofuscin pigment was very low but it increased from 1 yr of age onwards, increasing from 35 per cent at 1 yr and 5 months to about 49 per cent at 4 yr of age. The pigment granules were perinuclear and polar in location. The progressive increase in mean percentage of neurons with pigmentation increased steadily to about 82 per cent in the fourth age group, ranging between 4 and 8 yr of age. The amount of pigment increased correspondingly. About 92 per cent of neurons were pigmented in the sixth age group which included animals between 12 and 16 yr of age. The pigment was heavily concentrated in both the perinuclear and polar areas of neurons. Nucleus hypoglossus or nucleus motorius n. hypoglossi (Fig. 2) The pigment was first observed at the age of 11 months when it was loosely distributed in the form of fine granules, but became loosely focal by 4 yr of age. The pattern of accumulation was polar or bipolar. About 56 per cent of neurons were pigmented by 4 yr of age. The increase was not only associated with the amount of the pigment but also in the number of neurons involved. About 75 per cent of neurons were pigmented in the animals ranging between 8 and 12 yr and about 87 per cent between 12 and 16 yr of age. Dorsal motor nucleus of vagus or nucleus parasympatheticus n. vagi (Fig. 3) The pigmentation appeared very late in this nucleus. Few disseminated pigment granules were present at 4 yr. However, the rate of pigment deposition increased with age in the fourth age group, (4-8 yr). The pattern of the focal deposition was perinuclear and polar; some neurons had loose granular distribution also. The pigmentation in the fifth age group was focal and heavy. The increase in the deposition was very conspicuous in the sixth age group in which the pigmentation was almost completely masking the nucleus and cytoplasm in some, whereas in others the granulation covered about one-half or more of the neuronal volume. About 79 per cent of the neurons were pigmented by 16 yr of age. Nucleus cuneatus accessorius or nucleus cuneatus lateralis The pigment appeared as loose granules at 2 yr of age. The pigment was small in amount, but increased in amount and frequency, at 4 yr of age. Seventy-eighty per cent of neurons contained pigment, in the polar and bipolar forms, in the fourth age group. This increasing trend was also evident in the fifth and sixth age groups where the pigmentation occupied more and more of the cytoplasmic volume of the neurons and the number of pigmented neurons increased. In the sixth age group 79-92 per cent of neurons were showing pigment which was present almost throughout their cytoplasmic volume in many cases. Nuclei vestibulares (Fig. 4) The pigment appeared consistently in the form of axonal and bipolar accumulation from one year and seven months onwards. The rate of accumulation increased steadily in the third age group in which _~ 36 per cent of neurons were pigmented. The increase was further evidenced in the fourth age group where approximately 70 per cent of neurons were pigmented.
FIG. I. Nucleus
olivaris Inferior. 250. Alican blue and PAS stain. a. II months b. 16 yr. 250. Alican blue and PAS stain. FIG,. 2. Nucleus hypoglossus, a. 1 yr and 5 months h. I6 yr.
FK;. 3. Nucleus
parasympatheticus n. vagi. 250, Alican blue and PAS stain. b. 16 yr. a. 4 yr. 250. Alican blue and PAS stain. FIG;. 4. Nuclei vestibuiares. a. I yr. and 5 months. h. I6 yr.
250, Alican blue and PAS stain FIG. 5. Nuclei cochleares, b. I6 yr‘. a. 2 yr and 7 months. 100. Alican blue and PAS stain. FIG. 6. Nucleus tuber. b. I6 yr. a. I I months.
FIG. 7. Thalamic area. 250, Alican blue b. I6 a. 2 yr and 7 months. 250, Alican FIG. 8. Cortex cerebri (frontal). b. I6 a. I I Imonths.
and PAS stain. yr. blue and PAS stain yr.
AGING IN CANINE~BRAIN
3
The observations in the age group five and six revealed that the increased deposition was perinuclear, polar, and bipolar in distribution. The frequency of the neurons involved was considerably higher, reaching 91 per cent in the sixth age group. The cytoplasm of some neurons was filled with disseminated pigment granules. Nuclei cochleares (Fig. 5)
The disseminated pigment granules were present by 1 yr and 7 months. The distribution was observable in a very small percentage of cells. The pigment increased to become focal by 2 yr and 7 months. Twenty-five per cent of neurons were pigmented in the third age group. The pattern of distribution was perinuclear and sometimes polar; however discrete fine granules could also be seen in the neuronal cytoplasm. Eighty-two per cent of neurons were pigmented by 16 yr of age. Nuclei cerebellares
The dentate and fastigial nuclei were considered only. The pigmentation was loose and dispersed in the neuronal cytoplasm of the fastigial nucleus : it was loosely focal in the dentate nucleus at 4 yr of age. The granules were perinuclear and polar in location. The increase in the pigmentation was comparatively slower in the fastigial nucleus than in the dentate nucleus; there was more, and more focal pigmentation, in the second of these than the first, in the same specimens and same age grcup. The pigmentation continued to increase slightly with age in the fifth age group, but in the sixth age group there seemed to be a rapid increase both in amount of pigment and frequency of neurons pigmented. This was very evident in both nuclei at the age of 16 yr. Cortex cerebelli No substantial intraneuronal pigmentation was observed in the Purkinje cells of the cerebellum of the dog at any age group; but, pigment accumulations were discernable in the areas surrounding the Purkinje cells beyond 4 yr of age. This accumulation increased steadily with age. Nucleus rubrum or red nucleus (Fig. 6)
The presence of small amounts of lipofuscin pigment in the form of loose focal (perinuclear and axonal) accumulation was present in the second age group. The focal clumping of the pigment was distinct by 4 yr of age, though in some neurons disseminated granules were also present. 21 55 per cent of the neurons were pigmented by 4 yr of age. The pigmentation increased with advancing age so that 2: 75 per cent of neurons in this nucleus were pigmented by the fourth age group. The pigment occupied axonal, perinuclear and bipolar sites which in some cases masked the nucleus of the neurons. The pigmentation was very heavy and occurred in 90-95 per cent of the neurons in the sixth age group. Nucleus oculomotorius or oculomotor nucleus
The pigment was observed as loosely disseminated granules at about two years of age. It appeared to occur in the perinuclear and polar areas of neurons in the third age group. The progressive increase was observed to continue to the fifth age group, with the pigment axonal or perinuclear but rarely bipolar. The cells were heavily pigmented by 12 yr of age, and the pigmentation was densely focal in 60-70 per cent of neurons. This increase
4
B. S. NANDA
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R. GETTY
continued in the sixth age group where the pigmentation was very heavy and almost all neurons showed varying degree of accumulation. The pigmentation was mostly axonal, but some neurons also had a perinuclear or bipolar distribution of the pigment. Thalamic area (Fig. 7)
The lipofuscin pigment was observed at the age of 2 yr in the nucleus ventralis caudalis pars medialis, nucleus ventralis caudalis pars lateralis and nucleus ventralis rostralis of the thalamus. Few lipofuscin granules, however, were observed even at 1 yr and 7 months. The pattern of its distribution was axonal or perinuclear and was loosely packed by 2 yr of age. The rate of accumulation was relatively slow; only 40-50 per cent of neurons were pigmented by 8 yr of age. The neuronal accumulation of pigment increased steadily with age, but the increase in the amount of intraneuronal pigment was relatively slow. Seventyeighty per cent of the neurons were pigmented, though the amount of intraneuronal pigment was not significantly increased by 16 yr of age. Gyrus parahippocampalis
The pigmentation of neurons in this gyrus was observed in the form of few disseminated granules at 2 yr and 7 months of age. This became loosely focal in few neurons by 4 yr of age. The progression of deposition and neuronal involvement was very slow but still granular. Thirty-forty per cent of neurons were showing loosely focal pigmentation by 7 yr and 6 months; it became slightly focal, in small masses, in 70-80 per cent of neurons by 12 yr of age. The pigmentation was present in 90 per cent of neurons by the sixth age group. The amount and pattern of the deposition was variable, perinuclear and axonal; a few neurons with disseminated pigment granules were also observed. Cortex cerebri (Frontal) (Fig. 8)
The pigment was observed in the area precentralis gigantopyramidalis. Fine pigment granules were present in the giant pyramidal cells at the age of 10 months but became focal by the end of the second age group (1 yr). The presence of lipofuscin in the pyramidal neurons of the third cortical layer was evident by 2 yr and 7 months. The Betz cells by that age showed further clumping of the pigment and were seen to mask the nucleus at places. The common pattern of distribution was perinuclear and polar. The pigmentation became very heavy in the fourth age group. The pigment occupied about one-third or one-half of neuronal cytoplasm in some cases. A similar increase in the pyramidal neurons of the third layer was observed though it was comparatively less than in the giant pyramidal cells of the fifth cortical layer. The trend continued in the succeeding age groups until almost all Betz cells were heavily pigmented. The pyramidal cells of the third layer had a similar increase though the amount of pigment and the proportion of neurons involved were comparatively less than for the large pyramidal cells. Nucleus caudatus
Pigment was observed in the form of loose polar and perinuclear clumps by 4 yr. Slight granular deposition was present in a few neurons after 3 yr and 2 months of age, and increased steadily. The changes in distribution of lipofuscin pigment in the fifth age group indicated that there was an increase in the amount of the pigment as well as in the number of neurons containing it. The basic pattern of loose focal distribution, however, did not
AGING
IN CANINE
BRAIN
5
change significantly. Focal perinuclear accumulation of the pigment was observed by the age of 16 yr at which 80-90 per cent of neurons were involved. Putamen and globus pallidus
The putamen and globus pallidus showed the presence of the lipofuscin pigment at the age of 2 yr and 7 months. The pigment was in the form of small, loosely packed polar accumulations. This pigment was more in globus pallidus than the putamen. The pigment became more focal in the polar and pcrinuclear areas of the neurons. Forty-fifty per cent of neurons were pigmented in the globus pallidus by 12 yr of age. This increased to 70-80 per cent by 16 yr of age. The pigment was more coherent and focal in globus pallidus than in the putamen, in which loosely placed granules were observed even at 16 yr of age. I)ISCUSSION Our study included fifteen brain areas of the dog, and showed progressive accumulation of lipofuscin pigment in neurons with advancing age. This corresponded with the findings of the earlier workers who regarded this accumulation as the most reliable neurocytological age change (Bondareff, 1957, 1962, 1964; Brody, 1960; Samorajski, Keefe and Ordy, 1964; Samorajski, Ordy and Keefe, 1965; Samorajski, Ordy and Radyheimer, 1968; Whiteford, 1964; Whiteford and Getty, 1966; Few, 1966; Few and Getty, 1967). Various views regarding the origin of the lipofuscin pigment have been advanced from time to time. Dolley (1917) regarded the presence of the pigment as a product of chromatin transformation. Payne (1949, 1952); Hess (1952); Duncan, Nall and Morales (1960) and Gosh, Bern and Nishioka (1960) propounded that the pigment was derived from mitochondrial alterations or degeneration. The introduction of lysosomes into the problem of lipofuscin genesis received considerable attention following the works of de Duve and Wattiaux (1966) ; Essner and Novikoff (1960) and Novikoff (1961). They suggested that the lipofuscin pigment granules were altered lysosomes. Studies of Samorajski, Keefe and Ordy (1964); Samorajski, Ordy and Keefe (1965); Few and Getty (1967) and Munnell and Getty (1968) supported this view, The!{ suggest that the pigment granules appeared to be formed by coalescence of altered lysosomes and other cellular organelles in varying degrees of autolysis. The present study revealed that pigment granules appeared in the perinuclear areas of neurons which increased in amount and clumped focally at different sites with advancing age. The various sites of focal accumulation have been described earlier by Hopker (1951), Whiteford and Getty (1966) and Few a.nd Getty (1967). From the present study it looks as il’ different areas of brain show differences in time of pigment deposition as well as in the rate and degree of deposition with increasing age. The earliest pigment was observed in large pyramidal cells (Betz) of the frontal cortex, nucleus olivaris inferioris or nucleus olivaris, nucleus hypoglossus or nucleus motorius n. hypoglossi and nucleus rubrum or red nucleus. The pigment increased progressively, and the rate of increase was rapid. The neurons became heavily pigmented with advancing age. A cursory examination of neurons of the reticular formation revealed much the same pattern. The lipofuscin pigment accumulation appeared to be somewhat delayed in the nuclei cochleares, nuclei vestibulares, nucleus oculomotorius and nucleus cuneatus lateralis. The increase of the pigment with age in these sites was moderate. The cerebellar nuclei, putamen, globus pallidus, nucleus caudatus, and gyrus parahip-
6
B. S. NANDA
AND R. GE-l-l-Y
pocampalis were pigmented comparatively late (between 2 yr and 4 yr of age). The rate of increase of the pigment varied from moderate to low. The lowest rate of increase of lipofuscin pigment was observed in the thalamic nuclear areas studied. The dorsal motor nucleus of vagus or nucleus parasympatheticus n. Vagi showed the pigment very late but the rate of increase was very high. No pigment was observed intraneuronally in the Purkinje cells of the cerebellar cortex, though pigment granules were present in the granular layer. The times of occurrence of lipofuscin in relation to function of different brain areas have been examined by a number of workers. Sulkin (19%~) observed pigment granules to be more widespread in the efferent than the afferent neurons. This was also seen by Few and Getty (1967) who concluded that the autonomic cells were most resistant to pigmentation and efferent ventral horn cells of the spinal cord were less resistant. These differences were progressively reduced as the animal increased in age. Similar views were expressed by Wilcox (1956, 1959). Nandy (1968) studied the lipofuscin pigment in the nervous system of guinea pig and divided it into four categories based on rhe degree of deposition. Our categorisation, which is based on the time of appearance as well as on the degree of pigmentation, substantially agrees with the views of Nandy (1968) except for some differences which may be due to the functional status of the nuclear areas in different species. We have no direct evidence regarding the progressive accumulation of lipofuscin pigment in the neurons and its effect on neuronal metabolism and physiology. Strehler, Mark, Mildvan and Gee (1959) proposed that the pigment may affect the efficient working of the heart muscle. Similar conclusion has been drawn by Murray and Stout (1947). Samorajski, Keefe and Ordy (1964); Samorajski, Ordy and Keefe (1965); Whiteford (1964) and Few (1966). Samorajski, Ordy and Radyheimer (1968) hold that the accumulation of the lipofuscin material may affect all aspects of neuronal physiology and possibly result in loss of the cell. They also regarded the location of the pigment in the area between the nucleus and apical dendrite as of considerable consequence for neuronal function. We ourselves consider that the presence of the pigment in the neuron, occupying much of its cytoplasmic volume, may well be detrimental to normal cellular metabolism even if the material is a by-product or end-product of cellular metabolism. The physiological and metabolic functions of neurons will probably be affected through organelle loss and also because of the reduction of cell volume with advancing age. Acknowledgements-Sincere appreciation is extended to Dr. D. J. Hillmann for his assistance in photography and Mr. Ralph Lanz and Mr. Lynn Martin in preparing histological preparations. The authors acknowledge the help extended by Dr. J. H. Magilton, Acting Head, Department of Veterinary Anatomy during preparation of this manuscript.
REFERENCES BENSLEY,R. (1947) Amt. Rec. 98, BONDAREFF,W. (1957) J. Gerontol. BONDAREFF,W. (1959) Handbook Press, Chicago, 111. BONDAREFF,W. (1964) Advances New York.
609. 12, 364. of Aging and Individual in Gerontological
(Edited by J. E. Birren), University of Chicago
Research
(Edited by B. L. Strehler), Academic
M. (1922)Pathologische Histologic. Leipzig, Vogel. BRIZZEE,K. R., CANCILLA,A. P., SHERWOOD,N. and TIMIRAS,P. S. (1969) J. Gerontol. 24, 127. BRODY,H. (1960) J. Gerontol. 15, 258. DE DUVE, C. and WATTIAUX,R. (1966) Ann. Rev. Physiol. 28, 435.
BORST,
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AGII\;GIN CANINEBRAIN DOLLEY,D. H. (1911) J. med. Res. 24, 309. NALL, D. M. and MORALES,R. (1960) J. Geronrol. 15, 568. ESSNER,E. and NOVIKOFF,A. B. (1960) J. Ultrastnrct. Res. 3, 341. FEW, A. B. (1966) Unpublished Ph.D. thesis. Iowa State University, Ames, Iowa. FEW, A. B. and GETTY, R. (1967) J. Gerontol. 22, 357. GOSH, A., BERN, J. A., GOSH, 1. and NISHIOKA,R. A. (1960) Anat. Rec. 143, 195. HARMS,J. W. (1924) 2. Anat. Entwicklung. 71, 319. HESS, A. (1955) Anat. Rec. 123, 399. HOPKER, W. (1951) 2. Alternsforchung. 5, 256. MURRAY, M. R. and STOUT,A. P. (1947) Amer. J. Anat. 80, 225. NANDY, K. (1968) J. Geronfol. 23, 82. Nomina Anatomica Veterinaria (1968) Adolf Holzhausen’s Successors, Vienna. NOVIKOFF,A. B., BEAUFY,H. and DE DWE, <:. (1956) J. biophys. biochem. Cytol. 2, 179. PAYNE,F. (1949) J. Gerontol. 4, 195. PAYNE,F. (1952) Cow&y’s Problems of Aging (Edited by A. I. Lansing), Williams and Wilkins, Baltimore. SAMORAJSKI,T., KEEFE,J. R. and ORDY, J. M. (1964) J. Gerontol. 19, 262. SAMORAJSKI, T., ORDY, J. M. and KEEFE, J. R. (1965) J. Cell Biol. 26, 779. SAMORA~KI,T., ORDY, J. M. and RADYHEIMEIX, R. (1968) Anat. Rec. 160, 5.55. STREHLER,B. L., MARK, D. D., MILDVAN,A. S. and GEE, M. B. (1959) J. Gerontol. 14, 430. SULKIN, N. M. (1955a) J. Gerontol. 10, 135. SULKIN, N. M. (19556) J. biophys. biochem. Cvtol. 1, 459. SULKIN, N. M. and KUNTZ, A. (1952) J. Geroqtol. 7, 533. WHITEFORD,R. D. (1964) Unpublished Ph.D. thesis. Iowa State University, Ames, Iowa. WHITEFORD,R. D. and GETTY,R. (1966) J. Gcrontol. 21, 31. WILCOX,H. H. (1956) U.S. Public Health Report. Washington, 71, 1179. WILCOX, H. H. (1959) The Process of Aging 1,~ Nervous System (Edited by J. E. Birren, H. A. Imus and W. F. Windle), Thomas, Springfield, Ill.
Summary-The occurrence of lipofuscill pigment in fifteen nuclear and cortical areas of brain of dogs ranging from one day old to sixteen years of age was studied. Histological and autoflorescence techniques showed that the lipofuscin pigment was present in the brain of young adult and old dogs, increasing in ascending order of age. Localisation of the pigment, in apparently healthy doss, revealed its presence as the only neurocytological change in neurons of different areas. It was observed that the motor nuclei and areas developed the pigment at an early age; the increase was rapid with age when compared with that in the sensory and associative nuclei. The nuclei related with autonomic functions developed the pigment relatively late. RBsumk-On a &udiC l’apparition du pigment de la lipofuscine dans quinze sites nucleaires et corticaux du cerveau chez des chiens BgCs d’un jour B seize ans. Des techniques histologiques et d’auto-fluorescence ont rCv& que le pigment Ctait present dans le cerveau des chiens adultes, jeunes et IgCs, en quantitks croissantes avec l’bge. La localisation du pigment chez des chiens app:rremment bien portants a revel& que sa prksence dtait la seule modification neurocytologique des neurones des diff&ents sites. On a constatt que le pigment apparait t8t dans les zones et ~luclei moteurs; son augmentation avec l’Pge est rapide, comparativement & celle observCe dans les nuclei sensoriels et associatifs. 11apparait relativement tard dans les nuclei associts aux .‘onctions autonomes. Zusammenfassung-Das Auftreten VOIDLipofuszinpigment in fiinfzehn Kern- und Rindenbezirken des Gehirns des Hundes wurde untersucht bei einen Tag bis sechzehn Jahre alten Tieren. Die histologischen und autofluoromctrischen Verfahren zeigten, daR das Lipofuszinpigment im Gehirn junger erwachsener und alter Hunde vorlag und mit dem Alter zunahm. Die Lokalisation des Pigments bei augenschein lich gesunden Hunden ergab die einzige neurocytologische Verznderung in Neuronen unterschiedlichen Alters. Es wurde beobachtet, dal3 die motorischen Kerne und Bezirke das Pigment in friihem Alter entwickelten. Die Zunahme mit dem Alter war schnell im Vergleich mit demjenigen in sensorischen oder assoziativen Kernen. Die Kerne, die mit autonomen Funktionen in Beziehung stehen, entwickelten das Pigment relativ sp8t.