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Fine structural changes in the lateral vestibular nucleus of aging rats
Mechanisms of Ageing and Development, 3 (1974) 203-224 © Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
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FINE STRUCTURAL CHANGES IN THE LATERAL VESTIBULAR NUCLEUS OF A G I N G RATS
J. E. JOHNSON, Jr. and J. MIQUEL National Aeronautics and Space Administration, Ames Research Center, Neuroscienees Branch, Moffett Field, California 94035 (U.S.A.)
(Received June 6, 1974)
SUMMARY The fine structure of the lateral vestibular nucleus was investigated in SpragueDawley rats, that were sacrificed at 4 weeks, 6-8 weeks, 6-8 months, and 18-20 months of age. In the neuronal perikaria, the following age-associated changes were seen with increasing frequency with advancing age: rodlike nuclear inclusions and nuclear membrane invaginations; cytoplasmic dense bodies with the characteristics of lipofuscin; and moderate disorganization of the granular endoplasmic reticulum. Dense bodies were also seen in glial cells. Rats 18 to 20 months old showed dendritic swellings, axonal degeneration, and an apparent increase in the number of axosomatic synaptic terminals containing flattened vesicles (presumed to be inhibitory in function). In our opinion, the lateral vestibular nucleus of the rat is an excellent model to define aging changes in the nerve cells, as opposed to changes linked to vascular degeneration, which is frequently seen in time-associated degeneration of the human brain.
INTRODUCTION Aging of the brain is of paramount importance to human senescence in view of the integrative influence of this organ on every tissue of the body. Some decline in brain function is apparent in old age, even in healthy individuals; the gradual decrease in digit-symbol and incidental memory from a peak value at 15 to 25 years of a g o suggests that subtle degenerative changes may start rather early in life. This should be expected, since neurons are fixed postmitotic cells, which cannot rejuvenate through cell division. In the human brain, variability of neuropathological findings casts doubts about their being genuine age changes. In particular, the severity of cerebral arteriosclerosis is not necessarily related to chronological age 2, and this makes difficult the definition of "normal aging" of the brain as opposed to the degeneration of brain parenchyma, which is a consequence of vascular alterations. On the other hand, the rodent brain, which is usually free of arteriosclerotic disease, provides an excellent
204 model for the study of time-linked degeneration of nerve cells and glia occurring in the presence of a well preserved vasculature. A recent article 3 has documented fine structural changes in the hypothalamus of aging rats, akin to the findings of previous workers who relied on light and electron microscopy to investigate brain senescence in experimental animals 4-I°. The present report deals with the fine structural changes occurring in another area of the aging rat brain, namely the lateral vestibular nucleus. In our opinion, this nucleus is an excellent model for the study of direct aging changes in the central nervous system, since it is rich in both large and small neurons, as well as many types of glial cells 11,12. The lateral vestibular nucleus also contains abundant synaptic terminals, which may be differentiated On the basis of containing predominantly spherical (S) or flattened (F) vesicles. Aging changes in the vestibular nucleus have been investigated by Nanda and Getty 13,14 who, by light microscopy, revealed a moderate lipofuscin accumulation in the vestibular nucleus of aging pigs and dogs. The focus in our present investigation is on time-associated degeneration at the ultrastructural level. Our ultimate goal is to reveal some common structural features of aging, which may be a part not only o f " n o r m a l aging" of the human brain but also of aging of all kinds of postmitotic cells. That common features may indeed exist is suggested by recent findings of cytoplasmic degeneration in the cells of invertebrates (diptera 15,16 and nematodes~7). This degeneration is similar to that occurring in aging mammalian nerve cells, as brought out in the present study.
MATERIALS AND METHODS Twelve male albino Sprague-Dawley rats were used in the study. They were fed and watered ad libitum until sacrifice. The animals were divided into four groups of three, sacrificed at 4 weeks, 6-8 weeks, 6-8 months, and 18-20 months of age. All were sacrificed by perfusion with a solution consisting of 1 ~o paraformaldehyde and 1.5 ~ glutaraldehyde in 0.1 molar sodium phosphate buffer (pH 7.1-7.3). Following perfusion, the animals were placed in a refrigerator for 2 hours before removing the brains, which were then kept in cold fixative overnight. The lateral vestibular nuclei were removed, rinsed briefly in buffer, and placed in vials containing 1.5 ~ osmium tetroxide in the same buffer. Tissue blocks were dehydrated in ethanol and propylene oxide and then embedded in Epon-Araldite. Thick sections (1/~m) for light microscopy were stained with toluidine blue. Ultrathin sections for electron microscopy were stained with different combinations of uranium, lead, and bismuth solutions. Ultrastructural examinations were made on Jeolco 100-B and Philips 300 electron microscopes. Synaptic terminals were classified as S if they contained primarily spherical vesicles, and as F if they contained primarily flattened vesicles. Classifications were made directly in the electron microscope and double-checked on photographic prints. Only axosomatic synaptic terminals were classified, since the origin of dendrites could not be ascertained.
205 RESULTS Within the nucleus, rodlike inclusions were occasionally seen in the older rats (Fig. 1), whereas they were never present in young (1 month) rats, including a series of about twenty animals previously investigated by Johnson (unpublished). These inclusions, usually in the form of z lattice of small filaments, are from 0.05 to 0.2/~m in width and 1.5 to 4/~m in length. These nuclear inclusions were the rarest of the structures found to increase with age. The only other nuclear change noted was a tendency for the nuclear membranes in some larger neurons of older rats to be deeply invaginated (Fig. 7). One of the most characteristic changes in neurons of aging animals and humans is an accumulation of dense bodies in the perikaria and processes. Neurons of the lateral vestibular nucleus are no exception to this general rule. Light microscopy (Fig. 2 A) illustrates this accumulation, showing few or no dense bodies in 1-month-old neuronal cytoplasm but an abundance of these bodies in older rats (Fig. 2B). Electron microscopy (Figs. 3 and 4) reveals dense bodies to be 1 to 2/~m in diameter. In most instances, a lipid droplet is attached to the dark granule. Mitochrondria, granular (rough) endoplasmic reticulum, and Golgi are usually nearby (Fig. 3). A difference was noted, however, in the appearance of dense bodies found in the glial cells, as compared to those in neurons (Fig. 4). In neurons, rounded patches of dark substance were present in the dark granule portion of the dense bodies (Fig. 4 A); in glial cells, strands were seen (Fig. 4 B). These strands are approximately 60 A in width, running in various directions in groups of five or six, rather than singly. Usually, no lipid droplets were associated with the strands (Fig. 4B), but occasionally they, too, were seen (Fig. 10). These strands may be remnants from degenerating cells and processes that have been partially digested by lysosomal enzymes and then reformed. The dense bodies of both neurons and glial cells fluoresce when examined in near-ultraviolet light, which suggests that they are lipofuscin. Figures 5 through 8 illustrate the cytoplasm of neurons from rats 1 to 18 months old. Figures 5 and 6 show an abundant granular, endoplasmic reticulum, typical of young neurons. In large neurons, patches of reticulum are often 10/~m in diameter. Between the patches of reticulum are the usual arrays of mitochondria, microtubules, lysosomes, and Golgi apparatus (Fig. 5). When the patches of reticulum are smaller (1 to 2/~m), they usually consist of parallel cisternae (Fig. 6). In contrast, the neuronal cytoplasm of older rats contains fewer large patches of granular endoplasmic reticulum (Fig. 7), and the small patches no longer consist of parallel cisternae (Fig. 8). The amount of Golgi appears to increase in older rats (Fig. 7), but we have found no objective criteria for measuring this. No change in the number or structure of the mitochondria is apparent. Other structures in older rats are similar to those in the aging human brain TM. Figure 9 illustrates dendritic processes containing large numbers of mitochondria 0.1 to 0.2/~ in diameter. Synaptic terminals often make contact with the dendritic processes (Fig. 9 B). Figure 10 shows a different process, which contains many of the small mitochondria, along with dark, ringed bodies and multivesicular bodies. Near
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Fig. 1. T w o examples of u n u s u a l inclusions are seen in the nuclei (N) of two n e u r o n s f r o m an 8m o n t h - o l d rat. In 1A, the inclusion (arrow) is short a n d fat, while the e x a m p l e in 1 B (arrow) is long a n d thin. Both inclusions have the f o r m of a filament lattice. ( × 25 000)
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Fig. 2. Two light micrographs of neurons from animals of different ages are shown. In 2 A (age 1 month) the cytoplasm is free of dense bodies (tentatively identified as lipofuscin), while in 2 B (age 8 months) the cytoplasm contains numerous dense bodies (arrows). In the neuron on the right, the dense bodies are aggregated at one pole of the nucleus. (A × 590; B × 480)
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Fig. 3. Numerous dense bodies are distributed in the cytoplasm of this neuron from a rat 18 months old. The bodies have a dark and a lighter staining portion. ( × 29 000)
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Fig. 4. The differences between neuronal and glial dense bodies are illustrated in these two micrographs taken from the same animal (age 18 months). In 4 A the dark portion of this neuronal granule contains many rounded dark patches (arrow), and there is an attached lipid droplet (L). The glial dense bodies in 4 B contain dark strands (arrow). (A × 47 000; B x 114 000)
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Fig. 5. In this micrograph from a 1-month-old rat, the cytoplasm of a neuron contains large patches of granular endoplasmic reticulum as well as the usual array of mitochondria, lysosomes, microtubules and Golgi. ( × 15 250)
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Fig. 6. Smaller patches of granular endoplasmic reticulum (arrows) near the nucleus (N) in this neuron from a young rat consist of parallel cisternae. Age 1 month. ( x 23 000)
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Fig. 7. The cytoplasm of this neuron from an 18-month-old rat does not contain the extensive patches of granular endoplasmic reticulum seen in young rats. Although a larger than normal amount of Golgi is present in this neuron, the numbers of other organelles appear normal. The nucleus of this large cell (N) is invaginated (arrow), which does not usually occur in younger rats. ( x 15 250)
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Fig. 8. In this neuron from an 18-month-old rat, the small patches of parallel cisternae of granular endoplasmic reticulum (which are commonly found in young rats) are not seen. ( × 23 000)
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Fig. 9. Two examples of dendritic processes (Den) are illustrated. Both processes contain large number of small mitochondria. The example in 9 B makes contact with two presynaptic terminals (arrows). Age 18 months. ( x 30 500)
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Fig. 10. In the upper p o r t i o n of the field in this m i c r o g r a p h is a process that contains m a n y small m i t o c h o n d r i a (hollow arrow with dot), multivesicular bodies (hollow arrow), a n d dark ringed structures (solid arrow). A cell tentatively identified as microglial m a y be seen in the lower part of the field. T h e c y t o p l a s m of this cell contains a b u n d a n t dense bodies, in which m a y be seen a lipid droplet (L) a n d s o m e of the dark strands (arrow) similarly s h o w n in Fig. 4 B. Age 8 m o n t h s . ( x 15 000)
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Fig. 11. The u n u s u a l contents of an a x o n from an 8-month-old rat are s h o w n in this illustration. D a r k (large solid arrow) and light (hollow arrow) ringed structures are present. Some of the rings e x p a n d to form vesiculated regions (small solid arrow), a n d s o m e of the structures have dark cores (small thin arrow). ( >,: 23 500)
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Fig. 12. T h e ringed structures in this mJcrograph (arrow) are s o m e w h a t different in their a p p e a r a n c e f r o m the examples s h o w n in Fig. 11. T h e rings s h o w n here are more c o n d e n s e d a n d irregular. T h e y are in, or near, a synaptic terminal (hollow arrow). It is difficult to say whether or not the large a m o u n t of glycogen seen in the center portion of the field is contained in the s a m e process which contains the ringed structures. A g e 18 m o n t h s . ( × 37 500)
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Fig. 13. In 13 A a s p o n t a n e o u s l y degenerating axon (d) from an 8 - m o n t h - o l d rat is adjacent to healthy axons. The contents of the degenerating a x o n are quite dark. In 13 B (from the same animal) s p o n t a neous changes are occurring in this m e m b r a n o u s whorl. T h e cisternae in the center portion of the whorl are wider a n d darker t h a n those in the outer portion. Some of the enlarged dark cisternae (arrowhead) appear to c o n d e n s e to f o r m thin dark bands (arrows). A whorl from a y o u n g (2 m o n t h s ) rat is s h o w n in the inset for c o m p a r i s o n . (A x 15 000; B × 29 500; inset x 15 000)
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Fig. 14. Two examples of axosomatic presynaptic terminals are shown. The terminal in 14 A is classified as S since it contains vesicles that are spherical in shape. The terminal in 14 B is classified as F since it contains flattened vesicles. Age 1 month. (× 29 000) the process lies a cell (probably microglial) containing a large amount of dark substance. A few of the dark strands (similar to those in Fig. 4 B) may be seen in the substance. Figure 11 shows the contents of an axon from an 8-month-old rat. Many multiringed structures (vesiculated in places) are present. Some of the ringed structures have dense cores and are thought to be altered mitochondria. Figure 12 shows ringed structures having a different appearance. These structures are in, or near, a synaptic terminal, surrounded by what appears to be an abundant amount of glycogen. Spontaneously degenerating nerve fibers were often seen in the older rats; they were rarely seen in young rats. Figure 13 A shows the spontaneous degeneration of an axon, the contents of which are much darker than normal axons in the same field. Figure 13 B shows a membranous whorl that is also undergoing a spontaneous change rarely observed in young animals. The cisternae in the center portion of the whorl are about the same density as the degenerating axons shown in Fig. 13A. These cisternae are darker and wider than the cisternae in the outer portion of the whorl. Some parts of the inner dark cisternae appear to condense into thin dark bands (Fig. 13 B). The inset view of Fig. 13 B also shows the whorl of a young rat. This whorl, however, is not changing spontaneously. The lateral vestibular nucleus in rats 1 to 2 months old contains 22 ~ F axosomatic synaptic terminals (containing primarily flattened vesicles), and 6 8 ~ S terminals (containing primarily spherical vesicles). Figure 14 shows the two types of terminals. As the rats aged, the percentage of F terminals increased. Rats 6 to 8 months old, had 2 7 ~ F terminals and 73 ~o S terminals. At 18 to 20 months, F terminals increased to 62 ~ (38 ~o S). Scores overlapped for rats 1 to 2 months old and 6 to 8 months old. However, no overlap occurred between rats 1 to 8 months old and those 18 to 20 months old. DISCUSSION The morphological changes observed in older rats are not striking, which is
220 to be expected since rats 18 to 20 months old are middle-aged rather than senescent. However, some evidence of ultrastructural alterations is found in the neurons, illustrating the relentless character of age-associated deterioration of fixed postmitotic cells. The nuclear changes associated with aging have recently been discussed by Hasan and Glees 3. Of course, these changes could have great significance, since a widely accepted theory of aging holds that senescence is programmed at the genome level. In agreement with previous authors 6,19, Hasan and Glees 3 believe that many nuclear changes described in the gerontological literature could not be attributed to aging alone. One possible exception is nuclear invagination, which apparently increases in the hippocampus of aged rats. Andrew 2° suggests that the nuclear invaginations frequently found in the cells of aging organisms may represent an adaptive response to deteriorated conditions of blood supply and nutrition. In our opinion, nuclear invaginations may be a genuine age change since, in addition to finding them in the neurons of aged rats, we have found them in another fixed postmitotic cell, namely the midgut cell of Drosophila melanogaster. Rodlike inclusions in the nucleus of nerve cells of experimental animals were described by various anatomists at the end of the last century (rewieved by Ramon y Caja121). More recently, using the electron microscope, Siegesmund et al. z2 demonstrated the presence of such inclusions in neurons of the rabbit and squirrel monkey. Further electron microscopic work 13 has shown similar bodies in neurons of normal rats, and of a mouse inoculated with scrapie agent. The nature and significance of these rodlike inclusions remain controversial. Seite et al. 24, on the basis of a quantitative study performed on the neurons of the stellate ganglion of the cat, believe that the rodlets are formed by intranuclear microtubules and microfilaments, and that their number is related to the activity level of the nerve cells. Our own observations suggest that the frequency of the rodlets increases in older animals, which agrees with observations of cells in the parietal cortex of aging mice 25. Degenerative changes in mitochondria of nerve cells have been described 26, but in view of the extreme susceptibility of mitochondria to fixation procedures, such changes should be interpreted with great caution. Most authors believe that because of the rapid in vivo replacement of mitochondria ~5, these organelles should be resistant to aging changes. In accordance with this concept, the mitochondria in neurons ~qd glial ceils of our older rats appear normal in every respect. A considerable number of observations dating back almost a century have shown that intracytoplasmic accumulation oflipofuscin is the most striking subcellular morphological change in aging nerve cells, including those of the vestibular nucleus of the dog and the pig13,a4, 2s. The neurons of our older rats were abundant in osmiophilic dense bodies with the morphological and fluorescence characteristics of lipofuscin. In contrast, dense bodies were rarely seen in the neurons of animals 1 to 2 months old. The dense bodies in glial cells were different from those in neurons. In the neurons, the dense bodies had an amorphous or granular appearance, but in the glial cells they showed straight and irregular curved bands running in many directions within the pigment body. However this morphology is not specific for glial dense
221 bodies; strikingly similar pigment, identified as lipofuscin, has been observed by Samorajski et al. 9 in anterior horn nerve cells of a 15-month-old mouse. A specific relationship between lipofuscin accumulation and some biochemical process involved in aging has not been proven. However, a current view is that lipofuscin is the product of lipid peroxidation damage to the membranes of the endoplasmic reticulum 29-32. This view is supported by the finding that the fluorescence of lipofuscin at near-UV light is due to the presence of 1-amino-3-iminopropenes, which are the condensation products of malonaldehyde with the amino residues of proteins, amino sugars, and amino groups of nucleic acids 33. Since malonaldehyde is formed by the peroxidation of unsaturated fatty acids such as those present in the phospholipids of membranes, it is reasonable to assume that lipid peroxidation plays an essential role in the genesis of lipofuscin. Therefore, though lipofuscin may be quite harmless to the cell, the fact that it accumulates gives incontrovertible proof of the onslaught of lipid peroxidation on the cell organelles. Indeed, morphological and biochemical data support the concept that a disorganization of the endoplasmic reticulum, with disturbances in RNA and protein metabolism, very often coexists with lipofuscin accumulation in the nerve cells of aging animals. In this respect, microscopic observation of the Nissl bodies is of special interest. This substance, which consists of stacks of endoplasmic reticulum and interspersed ribosomes 34, was found in decreased amounts in the neurons of the brains of aged humans and laboratory animals 4,35,56. Moreover, in Andrew's 3v opinion, the amount of Nissl material seemed to decrease as the lipofuscin increased. Our own observations on disorganization of the endoplasmic reticulum in the lateral vestibular nucleus of the rat are in agreement with previous ultrastructural observations on age changes in the rat hippocampus ~. Similar time-associated changes have also been observed in neurons of the housefly 16 and in neurons and midgut cells of Drosophila melanogaster 3s. As could be expected, the changes in the endoplasmic reticulum are accompanied by biochemical disturbances. Recovery of the Nissl pattern after strenuous swimming is very limited in nerve cells of old animals 39. Furthermore in old rats, the RNA content per hippocampal neuron decreases markedly 40. Similar results have been observed in human motor nerve cells of the spinal cord 41 and in various areas of the brains of rhesus monkeys 42. Both quantitative 43 and qualitative changes 44 in protein metabolism have also been described in old age, and the protein synthesis of nervous tissue seems to decline gradually both in vivo 45-4s and in vitro49, 5°. On the basis of these and related findings, Gordon 51 has postulated that the biochemical disturbances that develop in the aging brain may be a consequence of a reduction in the "precision, predictability and functional efficiency" of molecular systems involved in the synthesis of RNA and of protein. In our older rats, the morphological changes are not limited to the perikaria, since some alterations in the dendrites and synapses are also observed. Dendritic swellings similar to those in Fig. 9 have been previously reported 11 with no mention of their frequency in relation to the age of the animals. We have observed them more frequently in rats 18 to 20 months old than in younger groups. The dendritic swellings are possibly "growth cones", such as those described by Ramon y Cajal 5~, in which
222 case they would represent active remodeling o f neuronal processes in the adult rat brain. On the other hand, since these dendritic swellings and the process shown in Fig. 10 are very similar to the structures seen in brain "plaques" in patients suffering from senile dementia 18, they could be the expression o f a progressive dystrophic tendency. M e m b r a n o u s whorls and parallel arrays o f tubules have been shown in axons and axon terminals o f the normal lateral vestibular nucleus ~. Since we have found them as frequently in younger as in aged rats, we cannot concur with Sotelo and Palay ~3 that these structures are the expression o f degenerative changes. On the other hand, axon degeneration (Fig. 13) may be a true age change, since we found it more often in the lateral vestibular nucleus o f old rats. Synaptic terminals, which may be classified on the basis of vesicle shape, have been controversial since U c h i z o n o 54 hypothesized that terminals containing spherical (S) vesicles are excitatory in function, and terminals containing flattened (F) vesicles are inhibitory. This hypothesis was strengthened by the fact that, in the cerebellum, k n o w n excitatory terminals (such as climbing fiber synapses on Purkinje cells) contain spherical vesicles, and k n o w n inhibitory terminals (such as basket cells synapses on Purkinje cells) contain flattened vesicles 5~. As our rats aged from 8 to 20 months, F terminals substantially increased. Therefore, if U c h i z o n o ' s hypothesis holds true for the lateral vestibular nucleus, then the relative number of inhibitory terminals increases as the rats age. It has been shown that, in the aging brain, the neurons become more sensitive to certain neurotransmitters ~6. Therefore, as the brain ages, increased inhibition may be needed to keep neuron systems under control. This would be consistent with an increase in F terminals to compensate for age-associated disturbances in neuron biochemistry. ACKNOWLEDGEMENTS J.E.J. is an N R C Associate. This research was supported by N A S A task No. 970-21-11-11.
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FINE STRUCTURAL CHANGES IN THE LATERAL VESTIBULAR NUCLEUS OF A G I N G RATS
J. E. JOHNSON, Jr. and J. MIQUEL National Aeronautics and Space Administration, Ames Research Center, Neuroscienees Branch, Moffett Field, California 94035 (U.S.A.)
(Received June 6, 1974)
SUMMARY The fine structure of the lateral vestibular nucleus was investigated in SpragueDawley rats, that were sacrificed at 4 weeks, 6-8 weeks, 6-8 months, and 18-20 months of age. In the neuronal perikaria, the following age-associated changes were seen with increasing frequency with advancing age: rodlike nuclear inclusions and nuclear membrane invaginations; cytoplasmic dense bodies with the characteristics of lipofuscin; and moderate disorganization of the granular endoplasmic reticulum. Dense bodies were also seen in glial cells. Rats 18 to 20 months old showed dendritic swellings, axonal degeneration, and an apparent increase in the number of axosomatic synaptic terminals containing flattened vesicles (presumed to be inhibitory in function). In our opinion, the lateral vestibular nucleus of the rat is an excellent model to define aging changes in the nerve cells, as opposed to changes linked to vascular degeneration, which is frequently seen in time-associated degeneration of the human brain.
INTRODUCTION Aging of the brain is of paramount importance to human senescence in view of the integrative influence of this organ on every tissue of the body. Some decline in brain function is apparent in old age, even in healthy individuals; the gradual decrease in digit-symbol and incidental memory from a peak value at 15 to 25 years of a g o suggests that subtle degenerative changes may start rather early in life. This should be expected, since neurons are fixed postmitotic cells, which cannot rejuvenate through cell division. In the human brain, variability of neuropathological findings casts doubts about their being genuine age changes. In particular, the severity of cerebral arteriosclerosis is not necessarily related to chronological age 2, and this makes difficult the definition of "normal aging" of the brain as opposed to the degeneration of brain parenchyma, which is a consequence of vascular alterations. On the other hand, the rodent brain, which is usually free of arteriosclerotic disease, provides an excellent
204 model for the study of time-linked degeneration of nerve cells and glia occurring in the presence of a well preserved vasculature. A recent article 3 has documented fine structural changes in the hypothalamus of aging rats, akin to the findings of previous workers who relied on light and electron microscopy to investigate brain senescence in experimental animals 4-I°. The present report deals with the fine structural changes occurring in another area of the aging rat brain, namely the lateral vestibular nucleus. In our opinion, this nucleus is an excellent model for the study of direct aging changes in the central nervous system, since it is rich in both large and small neurons, as well as many types of glial cells 11,12. The lateral vestibular nucleus also contains abundant synaptic terminals, which may be differentiated On the basis of containing predominantly spherical (S) or flattened (F) vesicles. Aging changes in the vestibular nucleus have been investigated by Nanda and Getty 13,14 who, by light microscopy, revealed a moderate lipofuscin accumulation in the vestibular nucleus of aging pigs and dogs. The focus in our present investigation is on time-associated degeneration at the ultrastructural level. Our ultimate goal is to reveal some common structural features of aging, which may be a part not only o f " n o r m a l aging" of the human brain but also of aging of all kinds of postmitotic cells. That common features may indeed exist is suggested by recent findings of cytoplasmic degeneration in the cells of invertebrates (diptera 15,16 and nematodes~7). This degeneration is similar to that occurring in aging mammalian nerve cells, as brought out in the present study.
MATERIALS AND METHODS Twelve male albino Sprague-Dawley rats were used in the study. They were fed and watered ad libitum until sacrifice. The animals were divided into four groups of three, sacrificed at 4 weeks, 6-8 weeks, 6-8 months, and 18-20 months of age. All were sacrificed by perfusion with a solution consisting of 1 ~o paraformaldehyde and 1.5 ~ glutaraldehyde in 0.1 molar sodium phosphate buffer (pH 7.1-7.3). Following perfusion, the animals were placed in a refrigerator for 2 hours before removing the brains, which were then kept in cold fixative overnight. The lateral vestibular nuclei were removed, rinsed briefly in buffer, and placed in vials containing 1.5 ~ osmium tetroxide in the same buffer. Tissue blocks were dehydrated in ethanol and propylene oxide and then embedded in Epon-Araldite. Thick sections (1/~m) for light microscopy were stained with toluidine blue. Ultrathin sections for electron microscopy were stained with different combinations of uranium, lead, and bismuth solutions. Ultrastructural examinations were made on Jeolco 100-B and Philips 300 electron microscopes. Synaptic terminals were classified as S if they contained primarily spherical vesicles, and as F if they contained primarily flattened vesicles. Classifications were made directly in the electron microscope and double-checked on photographic prints. Only axosomatic synaptic terminals were classified, since the origin of dendrites could not be ascertained.
205 RESULTS Within the nucleus, rodlike inclusions were occasionally seen in the older rats (Fig. 1), whereas they were never present in young (1 month) rats, including a series of about twenty animals previously investigated by Johnson (unpublished). These inclusions, usually in the form of z lattice of small filaments, are from 0.05 to 0.2/~m in width and 1.5 to 4/~m in length. These nuclear inclusions were the rarest of the structures found to increase with age. The only other nuclear change noted was a tendency for the nuclear membranes in some larger neurons of older rats to be deeply invaginated (Fig. 7). One of the most characteristic changes in neurons of aging animals and humans is an accumulation of dense bodies in the perikaria and processes. Neurons of the lateral vestibular nucleus are no exception to this general rule. Light microscopy (Fig. 2 A) illustrates this accumulation, showing few or no dense bodies in 1-month-old neuronal cytoplasm but an abundance of these bodies in older rats (Fig. 2B). Electron microscopy (Figs. 3 and 4) reveals dense bodies to be 1 to 2/~m in diameter. In most instances, a lipid droplet is attached to the dark granule. Mitochrondria, granular (rough) endoplasmic reticulum, and Golgi are usually nearby (Fig. 3). A difference was noted, however, in the appearance of dense bodies found in the glial cells, as compared to those in neurons (Fig. 4). In neurons, rounded patches of dark substance were present in the dark granule portion of the dense bodies (Fig. 4 A); in glial cells, strands were seen (Fig. 4 B). These strands are approximately 60 A in width, running in various directions in groups of five or six, rather than singly. Usually, no lipid droplets were associated with the strands (Fig. 4B), but occasionally they, too, were seen (Fig. 10). These strands may be remnants from degenerating cells and processes that have been partially digested by lysosomal enzymes and then reformed. The dense bodies of both neurons and glial cells fluoresce when examined in near-ultraviolet light, which suggests that they are lipofuscin. Figures 5 through 8 illustrate the cytoplasm of neurons from rats 1 to 18 months old. Figures 5 and 6 show an abundant granular, endoplasmic reticulum, typical of young neurons. In large neurons, patches of reticulum are often 10/~m in diameter. Between the patches of reticulum are the usual arrays of mitochondria, microtubules, lysosomes, and Golgi apparatus (Fig. 5). When the patches of reticulum are smaller (1 to 2/~m), they usually consist of parallel cisternae (Fig. 6). In contrast, the neuronal cytoplasm of older rats contains fewer large patches of granular endoplasmic reticulum (Fig. 7), and the small patches no longer consist of parallel cisternae (Fig. 8). The amount of Golgi appears to increase in older rats (Fig. 7), but we have found no objective criteria for measuring this. No change in the number or structure of the mitochondria is apparent. Other structures in older rats are similar to those in the aging human brain TM. Figure 9 illustrates dendritic processes containing large numbers of mitochondria 0.1 to 0.2/~ in diameter. Synaptic terminals often make contact with the dendritic processes (Fig. 9 B). Figure 10 shows a different process, which contains many of the small mitochondria, along with dark, ringed bodies and multivesicular bodies. Near
206
Fig. 1. T w o examples of u n u s u a l inclusions are seen in the nuclei (N) of two n e u r o n s f r o m an 8m o n t h - o l d rat. In 1A, the inclusion (arrow) is short a n d fat, while the e x a m p l e in 1 B (arrow) is long a n d thin. Both inclusions have the f o r m of a filament lattice. ( × 25 000)
207
Fig. 2. Two light micrographs of neurons from animals of different ages are shown. In 2 A (age 1 month) the cytoplasm is free of dense bodies (tentatively identified as lipofuscin), while in 2 B (age 8 months) the cytoplasm contains numerous dense bodies (arrows). In the neuron on the right, the dense bodies are aggregated at one pole of the nucleus. (A × 590; B × 480)
208
Fig. 3. Numerous dense bodies are distributed in the cytoplasm of this neuron from a rat 18 months old. The bodies have a dark and a lighter staining portion. ( × 29 000)
209
Fig. 4. The differences between neuronal and glial dense bodies are illustrated in these two micrographs taken from the same animal (age 18 months). In 4 A the dark portion of this neuronal granule contains many rounded dark patches (arrow), and there is an attached lipid droplet (L). The glial dense bodies in 4 B contain dark strands (arrow). (A × 47 000; B x 114 000)
210
Fig. 5. In this micrograph from a 1-month-old rat, the cytoplasm of a neuron contains large patches of granular endoplasmic reticulum as well as the usual array of mitochondria, lysosomes, microtubules and Golgi. ( × 15 250)
211
Fig. 6. Smaller patches of granular endoplasmic reticulum (arrows) near the nucleus (N) in this neuron from a young rat consist of parallel cisternae. Age 1 month. ( x 23 000)
212
Fig. 7. The cytoplasm of this neuron from an 18-month-old rat does not contain the extensive patches of granular endoplasmic reticulum seen in young rats. Although a larger than normal amount of Golgi is present in this neuron, the numbers of other organelles appear normal. The nucleus of this large cell (N) is invaginated (arrow), which does not usually occur in younger rats. ( x 15 250)
213
Fig. 8. In this neuron from an 18-month-old rat, the small patches of parallel cisternae of granular endoplasmic reticulum (which are commonly found in young rats) are not seen. ( × 23 000)
214
Fig. 9. Two examples of dendritic processes (Den) are illustrated. Both processes contain large number of small mitochondria. The example in 9 B makes contact with two presynaptic terminals (arrows). Age 18 months. ( x 30 500)
215
Fig. 10. In the upper p o r t i o n of the field in this m i c r o g r a p h is a process that contains m a n y small m i t o c h o n d r i a (hollow arrow with dot), multivesicular bodies (hollow arrow), a n d dark ringed structures (solid arrow). A cell tentatively identified as microglial m a y be seen in the lower part of the field. T h e c y t o p l a s m of this cell contains a b u n d a n t dense bodies, in which m a y be seen a lipid droplet (L) a n d s o m e of the dark strands (arrow) similarly s h o w n in Fig. 4 B. Age 8 m o n t h s . ( x 15 000)
216
Fig. 11. The u n u s u a l contents of an a x o n from an 8-month-old rat are s h o w n in this illustration. D a r k (large solid arrow) and light (hollow arrow) ringed structures are present. Some of the rings e x p a n d to form vesiculated regions (small solid arrow), a n d s o m e of the structures have dark cores (small thin arrow). ( >,: 23 500)
217
Fig. 12. T h e ringed structures in this mJcrograph (arrow) are s o m e w h a t different in their a p p e a r a n c e f r o m the examples s h o w n in Fig. 11. T h e rings s h o w n here are more c o n d e n s e d a n d irregular. T h e y are in, or near, a synaptic terminal (hollow arrow). It is difficult to say whether or not the large a m o u n t of glycogen seen in the center portion of the field is contained in the s a m e process which contains the ringed structures. A g e 18 m o n t h s . ( × 37 500)
218
Fig. 13. In 13 A a s p o n t a n e o u s l y degenerating axon (d) from an 8 - m o n t h - o l d rat is adjacent to healthy axons. The contents of the degenerating a x o n are quite dark. In 13 B (from the same animal) s p o n t a neous changes are occurring in this m e m b r a n o u s whorl. T h e cisternae in the center portion of the whorl are wider a n d darker t h a n those in the outer portion. Some of the enlarged dark cisternae (arrowhead) appear to c o n d e n s e to f o r m thin dark bands (arrows). A whorl from a y o u n g (2 m o n t h s ) rat is s h o w n in the inset for c o m p a r i s o n . (A x 15 000; B × 29 500; inset x 15 000)
219
Fig. 14. Two examples of axosomatic presynaptic terminals are shown. The terminal in 14 A is classified as S since it contains vesicles that are spherical in shape. The terminal in 14 B is classified as F since it contains flattened vesicles. Age 1 month. (× 29 000) the process lies a cell (probably microglial) containing a large amount of dark substance. A few of the dark strands (similar to those in Fig. 4 B) may be seen in the substance. Figure 11 shows the contents of an axon from an 8-month-old rat. Many multiringed structures (vesiculated in places) are present. Some of the ringed structures have dense cores and are thought to be altered mitochondria. Figure 12 shows ringed structures having a different appearance. These structures are in, or near, a synaptic terminal, surrounded by what appears to be an abundant amount of glycogen. Spontaneously degenerating nerve fibers were often seen in the older rats; they were rarely seen in young rats. Figure 13 A shows the spontaneous degeneration of an axon, the contents of which are much darker than normal axons in the same field. Figure 13 B shows a membranous whorl that is also undergoing a spontaneous change rarely observed in young animals. The cisternae in the center portion of the whorl are about the same density as the degenerating axons shown in Fig. 13A. These cisternae are darker and wider than the cisternae in the outer portion of the whorl. Some parts of the inner dark cisternae appear to condense into thin dark bands (Fig. 13 B). The inset view of Fig. 13 B also shows the whorl of a young rat. This whorl, however, is not changing spontaneously. The lateral vestibular nucleus in rats 1 to 2 months old contains 22 ~ F axosomatic synaptic terminals (containing primarily flattened vesicles), and 6 8 ~ S terminals (containing primarily spherical vesicles). Figure 14 shows the two types of terminals. As the rats aged, the percentage of F terminals increased. Rats 6 to 8 months old, had 2 7 ~ F terminals and 73 ~o S terminals. At 18 to 20 months, F terminals increased to 62 ~ (38 ~o S). Scores overlapped for rats 1 to 2 months old and 6 to 8 months old. However, no overlap occurred between rats 1 to 8 months old and those 18 to 20 months old. DISCUSSION The morphological changes observed in older rats are not striking, which is
220 to be expected since rats 18 to 20 months old are middle-aged rather than senescent. However, some evidence of ultrastructural alterations is found in the neurons, illustrating the relentless character of age-associated deterioration of fixed postmitotic cells. The nuclear changes associated with aging have recently been discussed by Hasan and Glees 3. Of course, these changes could have great significance, since a widely accepted theory of aging holds that senescence is programmed at the genome level. In agreement with previous authors 6,19, Hasan and Glees 3 believe that many nuclear changes described in the gerontological literature could not be attributed to aging alone. One possible exception is nuclear invagination, which apparently increases in the hippocampus of aged rats. Andrew 2° suggests that the nuclear invaginations frequently found in the cells of aging organisms may represent an adaptive response to deteriorated conditions of blood supply and nutrition. In our opinion, nuclear invaginations may be a genuine age change since, in addition to finding them in the neurons of aged rats, we have found them in another fixed postmitotic cell, namely the midgut cell of Drosophila melanogaster. Rodlike inclusions in the nucleus of nerve cells of experimental animals were described by various anatomists at the end of the last century (rewieved by Ramon y Caja121). More recently, using the electron microscope, Siegesmund et al. z2 demonstrated the presence of such inclusions in neurons of the rabbit and squirrel monkey. Further electron microscopic work 13 has shown similar bodies in neurons of normal rats, and of a mouse inoculated with scrapie agent. The nature and significance of these rodlike inclusions remain controversial. Seite et al. 24, on the basis of a quantitative study performed on the neurons of the stellate ganglion of the cat, believe that the rodlets are formed by intranuclear microtubules and microfilaments, and that their number is related to the activity level of the nerve cells. Our own observations suggest that the frequency of the rodlets increases in older animals, which agrees with observations of cells in the parietal cortex of aging mice 25. Degenerative changes in mitochondria of nerve cells have been described 26, but in view of the extreme susceptibility of mitochondria to fixation procedures, such changes should be interpreted with great caution. Most authors believe that because of the rapid in vivo replacement of mitochondria ~5, these organelles should be resistant to aging changes. In accordance with this concept, the mitochondria in neurons ~qd glial ceils of our older rats appear normal in every respect. A considerable number of observations dating back almost a century have shown that intracytoplasmic accumulation oflipofuscin is the most striking subcellular morphological change in aging nerve cells, including those of the vestibular nucleus of the dog and the pig13,a4, 2s. The neurons of our older rats were abundant in osmiophilic dense bodies with the morphological and fluorescence characteristics of lipofuscin. In contrast, dense bodies were rarely seen in the neurons of animals 1 to 2 months old. The dense bodies in glial cells were different from those in neurons. In the neurons, the dense bodies had an amorphous or granular appearance, but in the glial cells they showed straight and irregular curved bands running in many directions within the pigment body. However this morphology is not specific for glial dense
221 bodies; strikingly similar pigment, identified as lipofuscin, has been observed by Samorajski et al. 9 in anterior horn nerve cells of a 15-month-old mouse. A specific relationship between lipofuscin accumulation and some biochemical process involved in aging has not been proven. However, a current view is that lipofuscin is the product of lipid peroxidation damage to the membranes of the endoplasmic reticulum 29-32. This view is supported by the finding that the fluorescence of lipofuscin at near-UV light is due to the presence of 1-amino-3-iminopropenes, which are the condensation products of malonaldehyde with the amino residues of proteins, amino sugars, and amino groups of nucleic acids 33. Since malonaldehyde is formed by the peroxidation of unsaturated fatty acids such as those present in the phospholipids of membranes, it is reasonable to assume that lipid peroxidation plays an essential role in the genesis of lipofuscin. Therefore, though lipofuscin may be quite harmless to the cell, the fact that it accumulates gives incontrovertible proof of the onslaught of lipid peroxidation on the cell organelles. Indeed, morphological and biochemical data support the concept that a disorganization of the endoplasmic reticulum, with disturbances in RNA and protein metabolism, very often coexists with lipofuscin accumulation in the nerve cells of aging animals. In this respect, microscopic observation of the Nissl bodies is of special interest. This substance, which consists of stacks of endoplasmic reticulum and interspersed ribosomes 34, was found in decreased amounts in the neurons of the brains of aged humans and laboratory animals 4,35,56. Moreover, in Andrew's 3v opinion, the amount of Nissl material seemed to decrease as the lipofuscin increased. Our own observations on disorganization of the endoplasmic reticulum in the lateral vestibular nucleus of the rat are in agreement with previous ultrastructural observations on age changes in the rat hippocampus ~. Similar time-associated changes have also been observed in neurons of the housefly 16 and in neurons and midgut cells of Drosophila melanogaster 3s. As could be expected, the changes in the endoplasmic reticulum are accompanied by biochemical disturbances. Recovery of the Nissl pattern after strenuous swimming is very limited in nerve cells of old animals 39. Furthermore in old rats, the RNA content per hippocampal neuron decreases markedly 40. Similar results have been observed in human motor nerve cells of the spinal cord 41 and in various areas of the brains of rhesus monkeys 42. Both quantitative 43 and qualitative changes 44 in protein metabolism have also been described in old age, and the protein synthesis of nervous tissue seems to decline gradually both in vivo 45-4s and in vitro49, 5°. On the basis of these and related findings, Gordon 51 has postulated that the biochemical disturbances that develop in the aging brain may be a consequence of a reduction in the "precision, predictability and functional efficiency" of molecular systems involved in the synthesis of RNA and of protein. In our older rats, the morphological changes are not limited to the perikaria, since some alterations in the dendrites and synapses are also observed. Dendritic swellings similar to those in Fig. 9 have been previously reported 11 with no mention of their frequency in relation to the age of the animals. We have observed them more frequently in rats 18 to 20 months old than in younger groups. The dendritic swellings are possibly "growth cones", such as those described by Ramon y Cajal 5~, in which
222 case they would represent active remodeling o f neuronal processes in the adult rat brain. On the other hand, since these dendritic swellings and the process shown in Fig. 10 are very similar to the structures seen in brain "plaques" in patients suffering from senile dementia 18, they could be the expression o f a progressive dystrophic tendency. M e m b r a n o u s whorls and parallel arrays o f tubules have been shown in axons and axon terminals o f the normal lateral vestibular nucleus ~. Since we have found them as frequently in younger as in aged rats, we cannot concur with Sotelo and Palay ~3 that these structures are the expression o f degenerative changes. On the other hand, axon degeneration (Fig. 13) may be a true age change, since we found it more often in the lateral vestibular nucleus o f old rats. Synaptic terminals, which may be classified on the basis of vesicle shape, have been controversial since U c h i z o n o 54 hypothesized that terminals containing spherical (S) vesicles are excitatory in function, and terminals containing flattened (F) vesicles are inhibitory. This hypothesis was strengthened by the fact that, in the cerebellum, k n o w n excitatory terminals (such as climbing fiber synapses on Purkinje cells) contain spherical vesicles, and k n o w n inhibitory terminals (such as basket cells synapses on Purkinje cells) contain flattened vesicles 5~. As our rats aged from 8 to 20 months, F terminals substantially increased. Therefore, if U c h i z o n o ' s hypothesis holds true for the lateral vestibular nucleus, then the relative number of inhibitory terminals increases as the rats age. It has been shown that, in the aging brain, the neurons become more sensitive to certain neurotransmitters ~6. Therefore, as the brain ages, increased inhibition may be needed to keep neuron systems under control. This would be consistent with an increase in F terminals to compensate for age-associated disturbances in neuron biochemistry. ACKNOWLEDGEMENTS J.E.J. is an N R C Associate. This research was supported by N A S A task No. 970-21-11-11.
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