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Ageing changes in the liver of two Poeciliid fishes, the guppy Poecilia (Lebistes) Reticulata and the amazon molly, P. formosa
Exp. Geront. Vol, 13. pp. 37--45. Pergamon Press 1978. Printed in Great Britain.
AGEING
CHANGES
IN THE
LIVER
OF TWO
POECILIID F I S H E S , T H E G U P P Y POECILIA (LEBISTES) R E T I C U L A T A A N D T H E A M A Z O N M O L L Y , P. F O R M O S A * t A. D. WOODHEAD Brookhaven National Laboratory, Department of Biology, Upton, NY, U.S.A.
(Received 21 May 1977) Abstract--Changes in the anatomy of the liver throughout life are described for two fishes, the guppy and the Amazon molly. There were few consistent and marked changes with age, other than an increase in lipochrome pigment in the guppy. Only in the livers of the two oldest females, which were senile and post-reproductive were there signs of alterations in liver cytology, which might have caused impairment of liver function.
INTRODUCTION AGEING changes in the tissues of mammals, especially of man, have been well documented for many years and there is a wealth of descriptive literature. Very little is known about ageing changes in the lower vertebrates; this lack of information is particularly unsatisfactory, since it has often been asserted that animals such as fish, which continue to grow indefinitely do not age (Bidder, 1932; Andrews, 1971). Comfort (1960) has clearly refuted this idea by direct experiment, showing that fish do age, in spite of continued growth. Anatomical studies could provide much further evidence in support of ageing in fish, but very few have been made. The reports which are available, however, suggest that ageing changes in the tissues of fish conform to the common vertebrate pattern (Rasquin and Hafter, 1954; Woodhead and Ellett, 1966; 1967a, b; 1969). The present study, one of a series on histological aspects of ageing in the guppy, Poecilia (Lebistes) reticulatus, describes the changes in the liver with advancing age. We have also made some initial observations on the ageing changes in the liver of the Amazon molly,
P. formosa. This species, a gynogenetic live-bearer, has been used as a model system for carcinogenic studies by Hart and Setlow (1975). This survey was undertaken to provide a background picture of the normal histology of the Amazon molly throughout its life, against which the results of such oncogenic studies could be evaluated. MATERIALS AND METHODS The guppies, Poecilia (Lebistes) reticulatus used in this study were from a laboratory population which had been kept into extreme old age by Dr. Alex Comfort. The fish had been raised from birth in a temperature controlled room at 23°C and had been fed weekly with live Tubifex worms, supplemented with processed wheat germ ('Bemax'). The collection also included five pairs of guppies, one member of each pair having been adequately fed and the other severely underfed from birth. Three pairs were killed at 6 months of age, one pair at 9½ months and the remaining pair at 19 months. *Research carried out under the auspices of the United States Energy Research and Development Administration. Contract YOL-CP-50202. tBy acceptance of this article, the publishers and/or recipient acknowledges the U.S. Government's right to retain a nonexclusive, royaltyfree license in and to any copyright covering this paper. 37
38
A . D . WOODI-rEAD
The Amazon mollies, P. formosa were obtained from a clone maintained by Hart and Set!ow (1975). The fish had been kept at a constant temperature of 23°C throughout their lives, and under normal daylight conditions, and had been fed daily with a commercially prepared fish food. Details of the keeping conditions for the guppies are given by Comfort (1960) and for the mollies by Hart, Livesey and Setlow (1976). Individual fish were killed at various times throughout the lifespan by plunging them into iced water and then into iced fixative. The livers were examined from 25 female and 15 male guppies ranging in age from 6 days to 62 months. The livers of 30 mollies were surveyed over a period from 2 to 36 months of age. All the females (guppies and mollies) used were virgins and had not borne parthogenetic broods. The entire specimen was fixed in Bouin's fluid, routinely embedded in paraffin wax and serially sectioned at 10 t~m throughout its entire length. The sections were stained with haemotoxylin and eosin. The livers were examined microscopically for changes in morphology with age. Note was made of the architecture of the liver, the amount of connective tissue, the appearance of the parenchymal cells, and also of any parenchymal cells with giant nuclei, polyploid or pycnotic nuclei. The parenchymal cells of the guppy normally contain fat, in the form of small droplets. These are dissolved out in paraffin sections, giving a foamy appearance to the cell cytoplasm. We obtained an estimate of the amount of fat in the liver by measuring the extent of such cells on random fields on every 20th section through the liver; readings were made directly on a video monitor screen. A yellow-brown pigment (which we have not tested for specific type) was found in the livers of many guppies; in appearance this pigment resembled the lipofuscin deposits (lipochrome, 'ageing pigment', 'wear and tear pigment') seen in the livers of mammals. The extent of the pigmented areas was also estimated.
RESULTS
The guppy (a) The liver offish under 12 months. The liver o f the g u p p y was c o m p o s e d o f rather poorly defined lobules, roughly hexagonal in shape, surrounding a central hepatic vein and delimited by portal areas. The septa between the lobules was extremely sparse and difficult to distinguish, so that it was very rare that a 'classical' lobule was seen in which the extent o f the unit was clearly defined by the portal areas. Within the portal areas, however, all o f the c o m p o n e n t s were present that are f o u n d in m a m m a l i a n livers--the portal vein, the hepatic artery, bile duct and several lymphatics, but often these were well separated f r o m each other. The parenchymal cells within the lobules were arranged in chords, two or three cells in width, alternating with n a r r o w sinusoids. Again, the arrangement o f the cells was often irregular. The parenchymal cells were usually large, polygonal in shape and lightly staining, with distinct eosinophilic granules in the cytoplasm. The boundaries o f the ceils were usually distinct, but sometimes small groups o f neighboring cells at the periphery o f the organ had fused to f o r m a syncitium. The degree o f granulation, and the staining reaction varied f r o m fish to fish, but was uniform in any one individual. The nuclei o f the parenchyreal cells were centrally sited, r o u n d to oval in shape, with 2-3 nucleoli present. Binucleate ceils were noted infrequently, generally lying close to the portal system. Kupffer-like cells were occasionally seen. There were a few cells containing a yellow b r o w n pigment present in the livers o f fish up to the age o f 12 months. The pigment containing cells appeared to be similar to those seen in the livers o f mammals, the pigment presumably being derived f r o m the breakdown o f red blood cells, which is carried f r o m the spleen t h r o u g h the hepatic portal vein and its branches, to be deposited in the liver cells. The pigment containing cells in the livers o f y o u n g guppies were usually isolated and located in peripheral regions; values o f less than 5 ~ pigment containing cells in the entire livers were recorded. The pigment cells were approximately the same size and shape as the liver parenchyma, but had rather heavily staining nuclei which were displaced eccentrically. Pigment tended to be more abundant in the liver o f female guppies than males.
FIG. 1. Normal liver of the guppy, aged 6 months, showing hepatic cells in cords about 2 cells thick, separated by narrow sinusoids. × 400. FIG. 2. Section through the fatty 'cap' of a liver of an 18 month old guppy. Note the parenchymal cells are not distorted by the fat. × 150.
FIG. 4. The liver of a senile female guppy (60 months) showing masses of yellow-brown pigment cells, loss of nuclear orientation and nuclear pycnosis, x 150.
AGEING CHANGES IN THE LIVER
41
Fat was present in varying degrees in the liver of these young fish, and the total area occupied by fat filled cells ranged from values of 5 ~o to levels of 35 ~o, but most gave values about 10 ~o. Individual variability of this order was not unexpected since fat stores are very labile, and whilst their extent is frequently associated with nutritional state, infections, poisons and hormonal conditions also have a significant effect upon liver fat content. In most young fish, fat was present as small droplets (which had been dissolved out during fixation) within the cytoplasm, and the parenchymal cells were rarely distorted by fat deposits. In some cells, several droplets had coalesced to form a large drop, and the nucleus had become displaced to the periphery of the cell. Fat containing cells were generally dispersed throughout the liver, but in a few fish, they were localized at the posterior and anterior poles of the organ, almost as 'caps' of tissue. Even within such localized fatty masses the fat containing cells remained separate, and there was no fusion of the cells. The supporting stroma of the liver formed a thin tree-like network of tissue between the cells which could hardly be seen, and there was no evident thickening or fibrosis of the stroma or of the liver capsule in young fish. (b) The liver offish aged one to two years. There were few marked alterations in the liver
Fio. 3(a). A drawing of a part of the normal liver of a guppy, aged 6 months.
42
A . D . WOODHEAD
structure of this older group compared with the younger fish. In restricted peripheral areas of the liver of some guppies, the regularity of the liver lobules had become obscure and the parenchymal cells had fewer granules, poorly defined boundaries and tended to stain more lightly. Otherwise, the basic liver architecture was the same as in younger animals. Binucleate cells occurred rather more frequently, and a few scattered cells containing melanin were also seen. There were also present some degenerate liver cells, which had pycnotic nuclei and deeply staining elongated cytoplasm, localized near the hepatic veins. Deposits of lipochrome pigment in the liver cells became more abundant with age, and were universally present in all fish over one year old. They occurred as small masses of cells or as isolated clumps of about 4-6 cells. Measurements showed that the abundance of pigment increased from about 10~o at 18 months to almost 2 5 ~ in the oldest guppies in our collection, aged 61 and 62 months. There was some tendency for the amount of fat to increase in older fish, and in some individuals, almost one fourth of the cells had fat globules (Figs, 2 and 3b). Any age trend
.~f,~-.'~ -. ~;:
FIG. 3(b). A drawing of a part of the fatty liver of an 18 months old guppy. was not clear because of great individual variability. The fat-containing parenchymal cells of older fish (2 yr) often had become swollen and balloon like, and in some fish, fatty 'cysts' had formed by the coalescence of groups of adjacent cells. The membranes between such cells appeared to have ruptured and the flattened pycnotic nuclei were lined up around the periphery of the many celled 'cyst'. There was no increase in the supporting stroma of the organ, nor were there any significant areas of necrosis; no gross lesions were apparent in any of the fish. (c) The liver of senile fish. Definite histological changes which were associated with ageing were seen in the livers of fish of three years and older. The changes remained small until the fish had reached extreme old age (5 yr). Generally, the structure of the liver lobule still remained intact throughout most of the organ, but the boundaries of the cells became
AGEING CHANGES IN THE LIVER.
43
poorly defined and the staining reaction of the cytoplasm less intense. Some of the parenchymal cells had formed syncytia, with the nuclei clustered together. Atypical spindle like ceils with pycnotic nuclei were now fairly common and some of these cells appeared to have invaded the larger blood vessels within the liver. Cells with giant nuclei also increased with age. There was a rise in the numbers of pigment containing ceils, and many old fish had livers with large masses of these cells (Fig. 4). In the oldest fish (61 and 62 months) there were areas of oedema, and dilated sinusoids in the vicinity of the masses of pigmented cells. Melanin deposits were prominent in old fish, especially in females where as many as 10 % of the cells had melanin granules; only small granules of melanin were found in the cells but there were large granules extracellularly, associated with blood vessels. There was much variability in fat deposition in older fish but fat deposition was not exceptionally great--both of the oldest females had about 20 ~o fat in the liver. Commonly the enlarged fatty ceils had fused to form very large multicellular spherules. In the oldest females, over 60 months, there had been an increase in the connective tissue in the liver, and the supporting stroma was noticeably thickened, both peripherally and around arteries and veins. (d) The effect on undernutrition on liver structure. The histology of the liver was compared in the paired groups of fish on different levels of feeding. The differences found were not unexpected. The livers of the starved fish were composed of closely packed, regular lightly stained parenchymal cells, with very few fat droplets. Fat deposits were plentiful in the fed fish, but there were few fatty 'cysts' at 6 and 9½ months, the fat cells remaining distinct and distributed throughout the liver. The liver of the well fed fish of 19 months contained some fatty cysts, and the cells of the cysts were of irregular shape, some with pycnotic nuclei. In the starved fish of 19 months, there was some increase in the numbers of cells with pycnotic nuclei and the sinusoids tended to be engorged with blood, but there was little evident damage to the liver parenchymal cells. There was a reduction in liver size increasing from 12 to 26~o with time in the starved fish compared with their well fed siblings. (e) Pathological conditions. Amongst the laboratory population of the guppies were a number of young fish which had infections and abscesses of the liver; these were also examined. The most common condition was tuberculosis (mycobacterial infections) and the liver was generally heavily infected. In areas where there were large numbers of tubercles, the normal liver structure had been lost and was replaced by an unorganized grouping of islands of irregular liver parenchyma. There was an increase in connective tissue around the follicles, and occasionally fibrosis of other small areas of the liver where tubercles were absent. The structure of most of the uninfected liver tissue seemed quite normal. Fat deposits in these fish fell within the normal range. The livers of four fish with abscesses were examined, three of these having multiple abscesses present. There was little evident morphological change in the unaffected areas of the liver. Two of the guppies had hyperplastic thyroid glands, and had large numbers of thyroid follicles scattered amongst the liver parenchyma (Woodhead and Ellett, 1966). Both fish had livers with a high fat content, and many of the fat containing cells were swollen and distorted. The Amazon molly The liver of the Amazon molly differs superficially from that of the guppy by the presence of the hepatopancreas, with pancreatic acini surrounding each portal vein in addition to
44
A . D . WOODHEAD
the islets of pancreatic tissue disseminated amongst the abdominal mesentery. In all essentials, however, the livers of both species were very similar. The liver of the molly tended to be less well organized into lobules, although the disposition of the cells into two layered chords, separated by sinusoids was clear. As in the guppy, the cytoplasmic staining reaction and degree of granulation of the liver parenchyma varied between individuals, but was remarkably uniform in the same fish. Mollies younger than one year had very little fat in their livers. Fat was almost always present in the livers of older fish, but the amount was variable, and again, there was no clear relationship between fat content and age. The fat containing cells were often located in the peripheral areas of the liver, and there were few fatty 'cysts' in fish of up to two years, and even after this age they were rare. By contrast with the guppy, in which lipochrome pigments regularly occurred in fish over one year, and increased with age, very few deposits were seen in the Amazon molly at any age. In some old fish, over 2 years, occasional small groups of pigment containing cells were noted, associated with the hepatic veins. No gross lesions were seen in the livers of any mollies we examined. Several fish had infections, but as in the guppy, the changes in the liver were localized and minimal. The overall picture suggested that the livers of the Amazon mollies show fewer changes with age than the liver of guppies. The marked degenerative changes in the guppy liver however only became evident in senile fish; so far we have not kept mollies into such extreme old age. DISCUSSION Studies with mammals have shown that the liver exhibits few consistent and marked anatomical changes with age and it is generally agreed that there is little alteration in liver cytology in the healthy aged mammal. The differences described here between the livers of young and old fish were correspondingly small, and there was no clear, progressive degeneration of the liver as a whole with age. Only in the livers of the two oldest females in our series, which were senile and post reproductive, were there signs of a deterioration in liver tissue which might have caused impairment of liver function. The most conspicuous change was an apparent increase in the deposition of lipochrome pigment with advancing age in the guppy. Similar findings have been reported for mammals (Weinbren, 1961). Pigmentation increases with age in the liver of the amphibian, Ambystoma mexicanum, from extremely small amounts at 7 months, to small groups of cells at 18 months, finally to massive deposits at three years of age (Andrew, 1971). Surprisingly we found very little pigment accumulation in the liver of the Amazon mollies, even in the oldest fish. It is difficult to evaluate this finding. In mammals the relationship between pigmentation and ageing is by no means clear; increased pigmentation is not confined to post-reproductive and senile individuals, and complex metabolic relationships control its deposition. Melanin deposits did seem to be restricted to the livers of old and senile guppies. Liver fat content was highly variable in both species and there was no real increase with age. In young fish, fat deposits rarely altered cellular structure, but in many of the older guppies fat inclusions engorged and distorted the cells and fatty 'cysts' were common. This may not lead to impairment of liver function nor to permanent structural changes; in mammals fat may move readily from extracellular into intracellular locations, the fatty cysts disappear and liver architecture is restored, as before. The liver of mammals shows the least definite ageing changes of any organ and the
AGEINGCHANGESIN THELIVER
45
overall picture obtained here was one of minimal changes with age. The liver is involved intimately in a multitude of metabolic activities, its powers of regeneration are large, and most animals appear to have an enormous reserve of liver tissue; m a n y individuals have lived successfully with only 40 ~ of the original organ still present. In fish the leeway may not be so great and any age related derangement in liver function might present considerable disadvantages. Thus the liver is closely involved in the transfer of large quantities of yolk to developing eggs (Woodhead et al., 1968). But in contrast to mammals, where the peak of reproductive capacity is reached in early adulthood, most teleost fishes not only continue to reproduce throughout their lives but produce more offspring as they get older. In many species, gonad weight increases with increasing body length at a greater rate than body weight. There is therefore an increasingly heavy committment of energy and metabolic reserves to reproduction as the fish ages, and correspondingly, greater mobilization of metabolites through the liver. Acknowledgements--Drs. Alex Comfort and Richard Seflow very generously gave me their fish for this
study. Photomicrographs were taken by W. Marin, Jr. REFERENCES Ar,~REW, W. (1971) The Anatomy of Aging in Man and Animals. Grune & Stratton, New York. BIDt)ER, G. P. (1932) Br. Med. J. ii, 5831. CO,FORT, A. (1960) Gerontologia 4, 177. HART, R. W., LIVESEY,H. R. and SErLOW,R. B. (1976) Drum and Croaker 16, 1. HART, R. W. and SETLOW,R. B. (1974) In Molecular Mechanisms for Repair of DNA, Part B (Edited by P. C. HANAWALTand R. B. SETLOW),pp. 719-724. Plenum Press, New York. PLACXC,P. A., WOODI-mAD,A. D. and WOODrmAD, P. M. J. (1961) J. mar. Biol. Ass. UK21, 617. RASQUn~,R. and HAFTER,E. (1951) J. Morph. 89, 397. WErN~EN, K. (1961) In Structural Aspects of Ageing (Edited by G. H. BOtrRNEand E. M. H. WILSON), pp. 221-226. Hafner, New York. WOODrmAD,A. D. and EhhEa'r, S. (1966) Exp. Geront. 1, 315. WOODrIEAD,A. D. and EhhEa'r, S. (1967) Exp. Geront. 2, 159. WOODHEAD,A. D. and ELLE'rT,S. (1969) Exp. Geront. 4, 17. WOODX-mAD,A. D. and ELLETr, S. (1969) Exp. Geront. 4, 194.
AGEING
CHANGES
IN THE
LIVER
OF TWO
POECILIID F I S H E S , T H E G U P P Y POECILIA (LEBISTES) R E T I C U L A T A A N D T H E A M A Z O N M O L L Y , P. F O R M O S A * t A. D. WOODHEAD Brookhaven National Laboratory, Department of Biology, Upton, NY, U.S.A.
(Received 21 May 1977) Abstract--Changes in the anatomy of the liver throughout life are described for two fishes, the guppy and the Amazon molly. There were few consistent and marked changes with age, other than an increase in lipochrome pigment in the guppy. Only in the livers of the two oldest females, which were senile and post-reproductive were there signs of alterations in liver cytology, which might have caused impairment of liver function.
INTRODUCTION AGEING changes in the tissues of mammals, especially of man, have been well documented for many years and there is a wealth of descriptive literature. Very little is known about ageing changes in the lower vertebrates; this lack of information is particularly unsatisfactory, since it has often been asserted that animals such as fish, which continue to grow indefinitely do not age (Bidder, 1932; Andrews, 1971). Comfort (1960) has clearly refuted this idea by direct experiment, showing that fish do age, in spite of continued growth. Anatomical studies could provide much further evidence in support of ageing in fish, but very few have been made. The reports which are available, however, suggest that ageing changes in the tissues of fish conform to the common vertebrate pattern (Rasquin and Hafter, 1954; Woodhead and Ellett, 1966; 1967a, b; 1969). The present study, one of a series on histological aspects of ageing in the guppy, Poecilia (Lebistes) reticulatus, describes the changes in the liver with advancing age. We have also made some initial observations on the ageing changes in the liver of the Amazon molly,
P. formosa. This species, a gynogenetic live-bearer, has been used as a model system for carcinogenic studies by Hart and Setlow (1975). This survey was undertaken to provide a background picture of the normal histology of the Amazon molly throughout its life, against which the results of such oncogenic studies could be evaluated. MATERIALS AND METHODS The guppies, Poecilia (Lebistes) reticulatus used in this study were from a laboratory population which had been kept into extreme old age by Dr. Alex Comfort. The fish had been raised from birth in a temperature controlled room at 23°C and had been fed weekly with live Tubifex worms, supplemented with processed wheat germ ('Bemax'). The collection also included five pairs of guppies, one member of each pair having been adequately fed and the other severely underfed from birth. Three pairs were killed at 6 months of age, one pair at 9½ months and the remaining pair at 19 months. *Research carried out under the auspices of the United States Energy Research and Development Administration. Contract YOL-CP-50202. tBy acceptance of this article, the publishers and/or recipient acknowledges the U.S. Government's right to retain a nonexclusive, royaltyfree license in and to any copyright covering this paper. 37
38
A . D . WOODI-rEAD
The Amazon mollies, P. formosa were obtained from a clone maintained by Hart and Set!ow (1975). The fish had been kept at a constant temperature of 23°C throughout their lives, and under normal daylight conditions, and had been fed daily with a commercially prepared fish food. Details of the keeping conditions for the guppies are given by Comfort (1960) and for the mollies by Hart, Livesey and Setlow (1976). Individual fish were killed at various times throughout the lifespan by plunging them into iced water and then into iced fixative. The livers were examined from 25 female and 15 male guppies ranging in age from 6 days to 62 months. The livers of 30 mollies were surveyed over a period from 2 to 36 months of age. All the females (guppies and mollies) used were virgins and had not borne parthogenetic broods. The entire specimen was fixed in Bouin's fluid, routinely embedded in paraffin wax and serially sectioned at 10 t~m throughout its entire length. The sections were stained with haemotoxylin and eosin. The livers were examined microscopically for changes in morphology with age. Note was made of the architecture of the liver, the amount of connective tissue, the appearance of the parenchymal cells, and also of any parenchymal cells with giant nuclei, polyploid or pycnotic nuclei. The parenchymal cells of the guppy normally contain fat, in the form of small droplets. These are dissolved out in paraffin sections, giving a foamy appearance to the cell cytoplasm. We obtained an estimate of the amount of fat in the liver by measuring the extent of such cells on random fields on every 20th section through the liver; readings were made directly on a video monitor screen. A yellow-brown pigment (which we have not tested for specific type) was found in the livers of many guppies; in appearance this pigment resembled the lipofuscin deposits (lipochrome, 'ageing pigment', 'wear and tear pigment') seen in the livers of mammals. The extent of the pigmented areas was also estimated.
RESULTS
The guppy (a) The liver offish under 12 months. The liver o f the g u p p y was c o m p o s e d o f rather poorly defined lobules, roughly hexagonal in shape, surrounding a central hepatic vein and delimited by portal areas. The septa between the lobules was extremely sparse and difficult to distinguish, so that it was very rare that a 'classical' lobule was seen in which the extent o f the unit was clearly defined by the portal areas. Within the portal areas, however, all o f the c o m p o n e n t s were present that are f o u n d in m a m m a l i a n livers--the portal vein, the hepatic artery, bile duct and several lymphatics, but often these were well separated f r o m each other. The parenchymal cells within the lobules were arranged in chords, two or three cells in width, alternating with n a r r o w sinusoids. Again, the arrangement o f the cells was often irregular. The parenchymal cells were usually large, polygonal in shape and lightly staining, with distinct eosinophilic granules in the cytoplasm. The boundaries o f the ceils were usually distinct, but sometimes small groups o f neighboring cells at the periphery o f the organ had fused to f o r m a syncitium. The degree o f granulation, and the staining reaction varied f r o m fish to fish, but was uniform in any one individual. The nuclei o f the parenchyreal cells were centrally sited, r o u n d to oval in shape, with 2-3 nucleoli present. Binucleate ceils were noted infrequently, generally lying close to the portal system. Kupffer-like cells were occasionally seen. There were a few cells containing a yellow b r o w n pigment present in the livers o f fish up to the age o f 12 months. The pigment containing cells appeared to be similar to those seen in the livers o f mammals, the pigment presumably being derived f r o m the breakdown o f red blood cells, which is carried f r o m the spleen t h r o u g h the hepatic portal vein and its branches, to be deposited in the liver cells. The pigment containing cells in the livers o f y o u n g guppies were usually isolated and located in peripheral regions; values o f less than 5 ~ pigment containing cells in the entire livers were recorded. The pigment cells were approximately the same size and shape as the liver parenchyma, but had rather heavily staining nuclei which were displaced eccentrically. Pigment tended to be more abundant in the liver o f female guppies than males.
FIG. 1. Normal liver of the guppy, aged 6 months, showing hepatic cells in cords about 2 cells thick, separated by narrow sinusoids. × 400. FIG. 2. Section through the fatty 'cap' of a liver of an 18 month old guppy. Note the parenchymal cells are not distorted by the fat. × 150.
FIG. 4. The liver of a senile female guppy (60 months) showing masses of yellow-brown pigment cells, loss of nuclear orientation and nuclear pycnosis, x 150.
AGEING CHANGES IN THE LIVER
41
Fat was present in varying degrees in the liver of these young fish, and the total area occupied by fat filled cells ranged from values of 5 ~o to levels of 35 ~o, but most gave values about 10 ~o. Individual variability of this order was not unexpected since fat stores are very labile, and whilst their extent is frequently associated with nutritional state, infections, poisons and hormonal conditions also have a significant effect upon liver fat content. In most young fish, fat was present as small droplets (which had been dissolved out during fixation) within the cytoplasm, and the parenchymal cells were rarely distorted by fat deposits. In some cells, several droplets had coalesced to form a large drop, and the nucleus had become displaced to the periphery of the cell. Fat containing cells were generally dispersed throughout the liver, but in a few fish, they were localized at the posterior and anterior poles of the organ, almost as 'caps' of tissue. Even within such localized fatty masses the fat containing cells remained separate, and there was no fusion of the cells. The supporting stroma of the liver formed a thin tree-like network of tissue between the cells which could hardly be seen, and there was no evident thickening or fibrosis of the stroma or of the liver capsule in young fish. (b) The liver offish aged one to two years. There were few marked alterations in the liver
Fio. 3(a). A drawing of a part of the normal liver of a guppy, aged 6 months.
42
A . D . WOODHEAD
structure of this older group compared with the younger fish. In restricted peripheral areas of the liver of some guppies, the regularity of the liver lobules had become obscure and the parenchymal cells had fewer granules, poorly defined boundaries and tended to stain more lightly. Otherwise, the basic liver architecture was the same as in younger animals. Binucleate cells occurred rather more frequently, and a few scattered cells containing melanin were also seen. There were also present some degenerate liver cells, which had pycnotic nuclei and deeply staining elongated cytoplasm, localized near the hepatic veins. Deposits of lipochrome pigment in the liver cells became more abundant with age, and were universally present in all fish over one year old. They occurred as small masses of cells or as isolated clumps of about 4-6 cells. Measurements showed that the abundance of pigment increased from about 10~o at 18 months to almost 2 5 ~ in the oldest guppies in our collection, aged 61 and 62 months. There was some tendency for the amount of fat to increase in older fish, and in some individuals, almost one fourth of the cells had fat globules (Figs, 2 and 3b). Any age trend
.~f,~-.'~ -. ~;:
FIG. 3(b). A drawing of a part of the fatty liver of an 18 months old guppy. was not clear because of great individual variability. The fat-containing parenchymal cells of older fish (2 yr) often had become swollen and balloon like, and in some fish, fatty 'cysts' had formed by the coalescence of groups of adjacent cells. The membranes between such cells appeared to have ruptured and the flattened pycnotic nuclei were lined up around the periphery of the many celled 'cyst'. There was no increase in the supporting stroma of the organ, nor were there any significant areas of necrosis; no gross lesions were apparent in any of the fish. (c) The liver of senile fish. Definite histological changes which were associated with ageing were seen in the livers of fish of three years and older. The changes remained small until the fish had reached extreme old age (5 yr). Generally, the structure of the liver lobule still remained intact throughout most of the organ, but the boundaries of the cells became
AGEING CHANGES IN THE LIVER.
43
poorly defined and the staining reaction of the cytoplasm less intense. Some of the parenchymal cells had formed syncytia, with the nuclei clustered together. Atypical spindle like ceils with pycnotic nuclei were now fairly common and some of these cells appeared to have invaded the larger blood vessels within the liver. Cells with giant nuclei also increased with age. There was a rise in the numbers of pigment containing ceils, and many old fish had livers with large masses of these cells (Fig. 4). In the oldest fish (61 and 62 months) there were areas of oedema, and dilated sinusoids in the vicinity of the masses of pigmented cells. Melanin deposits were prominent in old fish, especially in females where as many as 10 % of the cells had melanin granules; only small granules of melanin were found in the cells but there were large granules extracellularly, associated with blood vessels. There was much variability in fat deposition in older fish but fat deposition was not exceptionally great--both of the oldest females had about 20 ~o fat in the liver. Commonly the enlarged fatty ceils had fused to form very large multicellular spherules. In the oldest females, over 60 months, there had been an increase in the connective tissue in the liver, and the supporting stroma was noticeably thickened, both peripherally and around arteries and veins. (d) The effect on undernutrition on liver structure. The histology of the liver was compared in the paired groups of fish on different levels of feeding. The differences found were not unexpected. The livers of the starved fish were composed of closely packed, regular lightly stained parenchymal cells, with very few fat droplets. Fat deposits were plentiful in the fed fish, but there were few fatty 'cysts' at 6 and 9½ months, the fat cells remaining distinct and distributed throughout the liver. The liver of the well fed fish of 19 months contained some fatty cysts, and the cells of the cysts were of irregular shape, some with pycnotic nuclei. In the starved fish of 19 months, there was some increase in the numbers of cells with pycnotic nuclei and the sinusoids tended to be engorged with blood, but there was little evident damage to the liver parenchymal cells. There was a reduction in liver size increasing from 12 to 26~o with time in the starved fish compared with their well fed siblings. (e) Pathological conditions. Amongst the laboratory population of the guppies were a number of young fish which had infections and abscesses of the liver; these were also examined. The most common condition was tuberculosis (mycobacterial infections) and the liver was generally heavily infected. In areas where there were large numbers of tubercles, the normal liver structure had been lost and was replaced by an unorganized grouping of islands of irregular liver parenchyma. There was an increase in connective tissue around the follicles, and occasionally fibrosis of other small areas of the liver where tubercles were absent. The structure of most of the uninfected liver tissue seemed quite normal. Fat deposits in these fish fell within the normal range. The livers of four fish with abscesses were examined, three of these having multiple abscesses present. There was little evident morphological change in the unaffected areas of the liver. Two of the guppies had hyperplastic thyroid glands, and had large numbers of thyroid follicles scattered amongst the liver parenchyma (Woodhead and Ellett, 1966). Both fish had livers with a high fat content, and many of the fat containing cells were swollen and distorted. The Amazon molly The liver of the Amazon molly differs superficially from that of the guppy by the presence of the hepatopancreas, with pancreatic acini surrounding each portal vein in addition to
44
A . D . WOODHEAD
the islets of pancreatic tissue disseminated amongst the abdominal mesentery. In all essentials, however, the livers of both species were very similar. The liver of the molly tended to be less well organized into lobules, although the disposition of the cells into two layered chords, separated by sinusoids was clear. As in the guppy, the cytoplasmic staining reaction and degree of granulation of the liver parenchyma varied between individuals, but was remarkably uniform in the same fish. Mollies younger than one year had very little fat in their livers. Fat was almost always present in the livers of older fish, but the amount was variable, and again, there was no clear relationship between fat content and age. The fat containing cells were often located in the peripheral areas of the liver, and there were few fatty 'cysts' in fish of up to two years, and even after this age they were rare. By contrast with the guppy, in which lipochrome pigments regularly occurred in fish over one year, and increased with age, very few deposits were seen in the Amazon molly at any age. In some old fish, over 2 years, occasional small groups of pigment containing cells were noted, associated with the hepatic veins. No gross lesions were seen in the livers of any mollies we examined. Several fish had infections, but as in the guppy, the changes in the liver were localized and minimal. The overall picture suggested that the livers of the Amazon mollies show fewer changes with age than the liver of guppies. The marked degenerative changes in the guppy liver however only became evident in senile fish; so far we have not kept mollies into such extreme old age. DISCUSSION Studies with mammals have shown that the liver exhibits few consistent and marked anatomical changes with age and it is generally agreed that there is little alteration in liver cytology in the healthy aged mammal. The differences described here between the livers of young and old fish were correspondingly small, and there was no clear, progressive degeneration of the liver as a whole with age. Only in the livers of the two oldest females in our series, which were senile and post reproductive, were there signs of a deterioration in liver tissue which might have caused impairment of liver function. The most conspicuous change was an apparent increase in the deposition of lipochrome pigment with advancing age in the guppy. Similar findings have been reported for mammals (Weinbren, 1961). Pigmentation increases with age in the liver of the amphibian, Ambystoma mexicanum, from extremely small amounts at 7 months, to small groups of cells at 18 months, finally to massive deposits at three years of age (Andrew, 1971). Surprisingly we found very little pigment accumulation in the liver of the Amazon mollies, even in the oldest fish. It is difficult to evaluate this finding. In mammals the relationship between pigmentation and ageing is by no means clear; increased pigmentation is not confined to post-reproductive and senile individuals, and complex metabolic relationships control its deposition. Melanin deposits did seem to be restricted to the livers of old and senile guppies. Liver fat content was highly variable in both species and there was no real increase with age. In young fish, fat deposits rarely altered cellular structure, but in many of the older guppies fat inclusions engorged and distorted the cells and fatty 'cysts' were common. This may not lead to impairment of liver function nor to permanent structural changes; in mammals fat may move readily from extracellular into intracellular locations, the fatty cysts disappear and liver architecture is restored, as before. The liver of mammals shows the least definite ageing changes of any organ and the
AGEINGCHANGESIN THELIVER
45
overall picture obtained here was one of minimal changes with age. The liver is involved intimately in a multitude of metabolic activities, its powers of regeneration are large, and most animals appear to have an enormous reserve of liver tissue; m a n y individuals have lived successfully with only 40 ~ of the original organ still present. In fish the leeway may not be so great and any age related derangement in liver function might present considerable disadvantages. Thus the liver is closely involved in the transfer of large quantities of yolk to developing eggs (Woodhead et al., 1968). But in contrast to mammals, where the peak of reproductive capacity is reached in early adulthood, most teleost fishes not only continue to reproduce throughout their lives but produce more offspring as they get older. In many species, gonad weight increases with increasing body length at a greater rate than body weight. There is therefore an increasingly heavy committment of energy and metabolic reserves to reproduction as the fish ages, and correspondingly, greater mobilization of metabolites through the liver. Acknowledgements--Drs. Alex Comfort and Richard Seflow very generously gave me their fish for this
study. Photomicrographs were taken by W. Marin, Jr. REFERENCES Ar,~REW, W. (1971) The Anatomy of Aging in Man and Animals. Grune & Stratton, New York. BIDt)ER, G. P. (1932) Br. Med. J. ii, 5831. CO,FORT, A. (1960) Gerontologia 4, 177. HART, R. W., LIVESEY,H. R. and SErLOW,R. B. (1976) Drum and Croaker 16, 1. HART, R. W. and SETLOW,R. B. (1974) In Molecular Mechanisms for Repair of DNA, Part B (Edited by P. C. HANAWALTand R. B. SETLOW),pp. 719-724. Plenum Press, New York. PLACXC,P. A., WOODI-mAD,A. D. and WOODrmAD, P. M. J. (1961) J. mar. Biol. Ass. UK21, 617. RASQUn~,R. and HAFTER,E. (1951) J. Morph. 89, 397. WErN~EN, K. (1961) In Structural Aspects of Ageing (Edited by G. H. BOtrRNEand E. M. H. WILSON), pp. 221-226. Hafner, New York. WOODrmAD,A. D. and EhhEa'r, S. (1966) Exp. Geront. 1, 315. WOODrIEAD,A. D. and EhhEa'r, S. (1967) Exp. Geront. 2, 159. WOODHEAD,A. D. and ELLE'rT,S. (1969) Exp. Geront. 4, 17. WOODX-mAD,A. D. and ELLETr, S. (1969) Exp. Geront. 4, 194.