Gerontologia 21: 81-94 (1975)

The Effect of Age on Mitochondrial Ultrastructure P atricia D. W ilson and L. M. F ranks Imperial Cancer Research Fund, London

Key Words. Mouse liver • Mitochondria • Electron microscopy Abstract. The ultrastructure of perfused livers and of mitochondrial fractions from 6-month-old and 30-nionth-o!d C57/BL mice were studied. In old mice the liver cell mitochondria were enlarged and rounded with a light ‘foamy’, vacuolat­ ed matrix, short cristae and a loss of dense granules. Quantitative studies showed a 60% increase in the mean size and an increased proportion of larger mitochon­ dria in intact 30-month-old perfused livers. Endothelial and Kupffer cell mito­ chondria were smaller than those of the parenchymal cells. Mitochondria in pellets prepared from 6- and 30-month-old livers were round­ ed and condensed, although there were a few larger and ‘foamy’ mitochondria in the preparations from old mice. Up to 47% of large mitochondria in the old livers were lost during cell fractionation.


Received: January 2, 1975.

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There is considerable evidence to suggest that there are mitochondri­ al changes in ageing and in tumours [T auchi et al., 1964; C hen et al., 1972; M enzies and G o ld , 1972; T ribe and A shurst . 1972; S iliprandi et al., 1973; W ilson , 1972. 1973; B ernhard , 1969; F rolkis and BoGATSKAYA, 1968; G lew et al., 1973; M artin et al., 1974; W ilson , 1974], but there are few detailed studies. In this preliminary paper we review the problem and describe age-associated changes in mitochondria in the mouse liver in our colony of ageing mice.


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Materials and Methods The livers from 6-month-old and 30-month-old male C57BL/lCRF/al mice were compared. For morphology the livers were perfused with Waymouth’s medi­ um and 2.5% glutaraldehyde in 0.2 M cacodylate buffer. Mitochondrial fractions were prepared from liver homogenates by a modification of Schneider’s method [F alcone and H adler, 1968). The pellets were then fixed in 2.5% glutaraldehyde. All specimens were post-fixed in Palade's fluid, dehydrated in graded alcohols and embedded in Araldite using epoxy propane as transitional solvent. Uranyl acetateand lead citrate-stained sections were viewed under an Hitachi HS7S or Siemens 1 electron microscope. The number and size of mitochondria were measured using a modification of the techniques described by W eibel et al. [1966] and Berger [1973].


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Morphology oj Perfused Young and Old Liver Mitochondria There were obvious morphological differences between the mitochon­ dria of young (6-month) and old (30-month) mouse livers which had been similarly ‘well’ fixed by perfusion with glutaraldehyde and postfixed with osmium tetroxide (fig. 1-4). In the parenchymal cells the mi­ tochondria of young mice appeared to be smaller and perhaps more nu­ merous. Although there was some variation in shape, the majority of mi­ tochondria in young liver were elongated and rod-like (the ’classical' mi­ tochondrial structure), while old liver mitochondria were mostly rounded and enlarged (fig. 1). The degree of enlargement was not uniform but depended on the region of the lobule. The largest mitochondria were found in the central region of the lobules. The arrangement and number of cristae varied, but the cristae in young liver mitochondria tended to extend further into the matrix and showed a more regular arrangement than those in the old mitochondria. In the old mitochondria cristae tend­ ed to be short and irregularly spaced. An obvious difference was found in matrix density. The matrix in young mitochondria was dense and homogeneous, while in the old mitochondria the matrix was light and of­ ten had a ’foamy’ vacuolated appearance. Occasionally, foamy mito­ chondria were found in the 6-month-old liver, but these were very rare (fig. 2a). The most swollen areas of the mitochondria showed the great­ est vacuolation. Although normal mitochondrial dense granules were eommon in young mitochondria, they were rarely seen in the old mito.-hondria (fig. 2). In some mitochondria a separation of the inner and

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Fig. I . a Liver from normal 6-month-old mouse showing rod-shaped mitochon­ dria. X 3,000. b 30-month-old mouse liver showing a sinusoid with a Kupffer cell with many inclusions. The mitochondria of the parenchymal cells are rounded, swollen and pale staining. X 3,000.


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Fig. 2. a A normal mitochondrion (right) and a •foamy' mitochondrion (left) in a 6-month-old mouse liver. Foamy mitochondria are very rarely seen in young an­ imals. Dense bodies are present in the matrix of both mitochondria. X60.000. b A mitochondrion from a 30-month-old mouse liver showing swelling and vacuolation. The whole of a mitochondrion is not affected to the same extent. The great­ er the extent of the vacuolation. the greater the swelling. 60.000.

The Effect of Age on Mitochondrial Ultrastructure


outer membranes was noted, but this seemed to occur in both young and old mitochondria. There was also a considerable difference between the morphology of mitochondrial from different cell types in the liver within one age group. The mitochondria of the Kupffer and endothelial cells were much small­ er than those of the parenchymal cells, although sometimes elongate. The cristal pattern was characteristic. In young liver the cristae often ex­ tended almost the complete width of the mitochondria, sometimes with a wavy outline. Occasionally, the cristae were oblique or longitudinal within the mitochondria (fig. 3). In the old liver the Kupffer and endoth­ elial cell mitochondria were often increased in size, while the matrix density decreased and showed patchy vacuolation. Some endothelial cell cristae showed localised swelling (fig. 4). The age-associated changes did not affect all cells or all mitochondria in a single paranchymal cell to the same extent. In some cells apparently severely affected and apparently normal mitochondria could be found side by side. In the old mouse liver, there was some evidence of fibrosis in the subendothelial space of Disse, which contained an increased amount of collagen and interstitial material (fig. 4a). Lipofuchsin granules were present in many cells.

Quantitation The total profile area in a plane through random particles in space is equal to the total volume occupied by the particles in that space [B er ­ ger , 1973]. The principle was employed by W eibei . et al. [1966] in their development of stereological techniques. The method was em­ ployed on electron micrographs of young and old perfused liver and of isolated mitochondria to give a comparison of size. A transparent sheet

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Morphology of Isolated Mitochondrial Fractions The fractions obtained were relatively pure consisting of mitochon­ dria with some glycogen contamination. There was little, if any, differ­ ence in the structure of mitochondria isolated from young or old mice (fig. 5). The mitochondria were rounded and showed the 'condensed' configuration with dense matrix and swollen cristae characteristic of ac­ tive respiration. The mitochondria from old liver tended to be larger and occasionally, there were distended mitochondria with a light 'foamy' ma­ trix. These were rarely found in young preparations.


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Fig. 3. Parenchymal. Kupffer and endothelial cells from the same liver lobule. a A 6-month-old liver showing a Kupffer cell (centre), sinusoid lined by cndotheli-

The Effect of Age on Mitochondrial Ultrastructure


Table /. Mean size of mitochondria1; mean SD values in parentheses

Perfused liver Isolated mitochondria



3.66 (0.38) 2.47 (0.32)

5.77 (1.03) 2.96 (0.26)

1 Number of dots per mitochondrion at standard magnification.

Table II. Total number of mitochondria1; mean SD values in parentheses 6-month-old


182 (41.5)

145 (31.1)

1 Per 10x8 cm field of perfused liver at standard magnification.

of equally spaced dots was superimposed over electron micrographs of the same magnification, and the number of dots touching any mitochon­ drion was recorded. An estimate of relative numbers of mitochondria was made by counting mitochondria per unit area.

al cell processes and edges of two parenchymal cells. X 3,000. b Elongated mito­ chondrion in an endothelial cell process. X 30,000. c Elongated mitochondrion from the Kupffer cell in a. X 30.000. d A liver cell (lower left) space of Disse and an edge of an endothelial cell (right). The mitochondrion of the endothelial cell is much smaller than the parenchymal cell mitochondria. X 30,000.

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Perfused Mouse Liver Table I shows the results of the determination of the mean size of mitochondria in perfused liver from 6- and 30-month-old mice. This was measured as the mean number of dots per mitochondrion. There was a significant increase of approximately 60°/o in the mean size of mi­ tochondria in the 30-month-old liver as compared with the 6-month-old. The standard deviation (SD) was larger in the 30-month-old livers indi­ cating that there was a wider range of variation in mitochondrial size in the old animals. The size distribution of mitochondria was different in the 6-monthold and 30-month-old liver (fig. 6). The 30-month-old tissue contained

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Fin. 4. a 30-month-old mouse liver showing a Kupffer cell lining a sinusoid (right) with processes of an endothelial cell (left). The space of Disse is enlarged

The Effect of Age on Mitochondrial Ultrastrueture


a lower proportion of small mitochondria and a higher proportion of larger mitochondria than the livers of 6-month-old animals. Table II shows that there was a decrease in the total number of mito­ chondria per unit area of tissue in the 30-month-old liver, but the differ­ ence was not statistically significant. Isolated Mitochondria There was a slight increase in the mean size of mitochondria isolated from 30-month-old livers compared with those from 6-month-old ani­ mals, but this increase was much less than observed for the perfused liv­ ers (table I). Figure 6 shows that the size distributions for mitochondria isolated from 6-month-old and 30-month-old livers were different. Again, the 30-month-old mitochondrial fractions contained a lower pro­ portion of small mitochondria and a higher percentage of large mito­ chondria than those from 6-month-old livers. None of the very large mi­ tochondria was present in either fraction (over 12 dots), and there was a loss of a porportion of all large mitochondria (4 dots and over) com­ pared with the intact livers. This amounted to 28% of the total in the 6-month-old and 47% in the 30-month-old animals.

Discussion The morphological changes we have found in old liver cell mitochon­ dria strongly suggest that there may also be functional changes but the nature of these changes is difficult to define, since there are many prob­ lems involved in any attempt to measure these changes biochemically. Methods involving cell fractionation and preparation of isolated mito­ chondria also have their drawbacks since our results show that the frac­ tionation procedure leads to the loss of abnormal mitochondria. Com­ parative studies using morphological and biochemical methods should help to overcome some of the difficulties. A particular virtue of the morphological studies is that they have demonstrated clearly that the changes do not affect all mitochondria to the same extent. In some in-

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ami contains collagen bundles. X3,000. h. c Enlarged mitochondria of Kupffer cells. The matrix shows patchy vacuolation, and in c there is some breakdown of the cristae. There is a lipofuchsin granule in c (top). X 30.000.

The effect of age on mitochondrial ultrastructure.

The ultrastructure of perfused livers and of mitochondrial fractions from 6-month-old and 30-month-old C57/BL mice were studied. In old mice the liver...
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