A. Degenerative Changes in Myelin Sheaths

The most common age-related morphological alteration in myelin sheaths is the accumulation of pockets of dense cytoplasm between splits of the lamellae at the major dense line (Figure 5.1). These pockets of dense cytoplasm become more frequent with increasing age (e.g., [12, 13]). Because the major dense line is formed by the apposition of the cytoplasmic faces of successive lamellae of the oligodendroglial cell plasma membrane that forms the sheath, it can be concluded that the dense cytoplasm must belong to the parent oligodendroglial cell. Sometimes, there is only one pocket of dense cytoplasm but it is not uncommon for several pockets of dense cytoplasm to occur in the same segment of the sheath, between adjacent turns of the spiraling myelin lamellae, which results in an obvious bulging of the sheath. Also, several loci containing pockets of dense cytoplasm may occur along an individual internodal length of myelin.

FIGURE 5.1

A transversely sectioned bundle of nerve fibers in layer 4C of the visual cortex of a 29-year-old monkey. Three of the nerve fibers (1) have disrupted myelin sheaths with splits between the lamellae that contain dense, vacuolated cytoplasm. Two other (more...)

Based on the fact that the cytoplasm in the pockets is dense, it is assumed that its presence is a sign of degeneration, and this conclusion is supported by the fact that Cuprizone toxicity can also lead to the formation of dense cytoplasm in the inner tongue process of degenerating sheaths (e.g., [14, 15]), and that similar dense cytoplasm occurs in the sheaths of mice with myelin-associated glycoprotein deficiency (e.g., [16]). It should also be pointed out that anti-ubiquitin antibodies immunostain dense inclusions within focal swellings of myelin sheaths in the white matter of old humans [17] and dogs [18], suggesting that the electron-dense material between the lamellae of sheaths in old animals contains proteins that are not being degraded by proteosomes. Interestingly, Wang et al. [19] have shown that in humans there is no correlation between the amount of soluble and insoluble ubiquitinated material in white matter and the cognitive scores of the humans from which the tissue was derived, but there is a correlation between decreased levels of myelin basic protein and decreased cognitive scores. These results lead the authors to conclude that white matter pathology may contribute to age-associated decline in cognition.

In examining the ultrastructure of degenerative changes in myelin during normal aging, it is important to recognize that myelin in white matter is often poorly preserved, largely due to the fact that white matter has many fewer capillaries than gray matter. Consequently, the nerve fibers in white matter are less accessible to fixatives that are introduced by perfusion fixation, and this can result in defects in the preservation of myelin sheaths, However, the most common change brought about by poor preservation of myelin is focal splitting or shearing of the myelin lamellae, and the frequency of this shearing increases as the quality of preservation of the tissue is diminished. There is no indication that such shearing of myelin lamellae occurs as a result of normal aging, and it does not seem to occur even in myelin sheaths altered by experimental interventions. Nevertheless, fixation defects must not be misconstrued as age changes.

An age change less common than the occurrence of pockets of dense cytoplasm is the formation of myelin balloons (Figure 5.2). These balloons can be spectacular because they can be 10 μm or more in diameter, so that in light microscopic preparations they appear as holes in the neuropil [20]. When the balloons are examined by electron microscopy, it becomes evident that they are fluid-filled cavities that occupy splits in the intraperiod line of the sheath. Although myelin balloons often appear as isolated circular profiles bounded by several lamellae of compact myelin, appropriate sections through balloons show that they bulge from the sides of myelin sheaths, leaving the axon flattened against the opposite side sheath from where the balloon protrudes (Figure 5.2). Consequently, the isolated circular profiles are generated by sections that pass to one side of the connection between the balloon and its parent sheath. Because there is no decrease in the widths of the myelin lamellae surrounding the balloons, and because there is no indication that myelin is elastic in nature, the generation of these balloons must require the parent oligodendrocyte to produce large amounts of additional myelin. It should be added that sometimes small pockets of dense cytoplasm can occur at the base of a balloon, and that the nature of the fluid contained in balloons is not known.

FIGURE 5.2

A ballooned nerve fiber in the anterior commissure of a 25-year-old monkey. Ballooning of the sheath results in a fluid-filled space (asterisk) and causes the axon (Ax) to be pushed to one side of the expanded sheath. (Scale bar = 1 μm.)

In monkeys, alterations to myelin sheaths are not common in animals less than about 10 years old but they become noticeable in middle-aged monkeys; and in monkeys over 25 years of age, as many as 5 to 6% of profiles on myelinated nerve fibers show some morphological changes.

Although we have only examined the monkey central nervous system, similar myelin balloons have been reported by Faddis and McGinn [21] in the cochlear nucleus of normally aging gerbils. It is assumed that ballooning of myelin is a degenerative change because it can occur in the early stages of Wallerian degeneration in the dorsal funiculus of the spinal cord following section of dorsal roots [22] and also can occur in rats with severe diabetes [23]. In addition, myelin balloons can be generated by Cuprizone poisoning [24], by experimental toxicity produced by triethyl tin (e.g., [25]), by chronic copper poisoning [26], and by lysolecithin [27].

It has been shown that with age the composition of myelin changes, as reported by Malone and Szoke [28]. They found that in aging rats there are changes in the cholesterol:phospholipid ratios in myelin and an increased saturation of the long acyl chains of myelin glycosphingolipids, and they suggest that these changes may cause an increased fluidity and decreased stability of myelin. Sloane et al. [29] also found the composition of myelin to alter with age, because there is a decrease in the amount of associated glycoprotein. On the other hand, with increasing age, levels of the oligodendrocyte-specific proteins CNPase and myelin/oligodendrocyte specific protein (MOSP) increase dramatically in white matter homogenates and in myelin, suggesting that there is new formation of myelin by oligodendrocytes, perhaps in response to myelin degradation and injury caused by proteolitic enzymes such as calpain, which increases in white matter with age.

In a study using microarrays to determine alterations in gene expression in the hippocampus of aging rats, Blalock et al. [30] found that the genes for myelin-associated oligodendroglial basic protein (MOSP) and myelin-associated glycoprotein (S-MAG) are among those that are upregulated. And in another study in which the effects of aging on the frontal cortex of the human brain were assessed, Lu et al. [31] found that the genes that are upregulated after the age of 40 include those for proteolipid protein and oligodendrocyte lineage transcription factor 2. Blalock et al. [30] suggest that upregulation of these genes is related to myelin degeneration with increasing age, although the upregulation might also be associated with remyelination, which, as will be shown later, is also known to occur during aging.

B. Degeneration and Loss of Nerve Fibers

For a nerve fiber to completely degenerate, it is necessary for the ensheathed axon to degenerate, and in turn this causes the myelin sheath surrounding the axon to also degenerate. Classically, these events were first described in experimental Wallerian degeneration, in which sectioning of a nerve fiber leads to degeneration of the portion of the nerve fiber isolated from its parent neuron. When nerve fibers caused to degenerate following experimental lesions are examined by electron microscopy, it is found that the cytoplasm of the degenerating axon can show accumulations of mitochondria and lysosomes before it eventually becomes dense. Axons with these features are sometimes encountered in the normally aging brain, but to show that there is nerve fiber degeneration and loss during aging requires quantitative analyses.

Among the earliest quantitative studies to show that there is loss of nerve fibers with age are the stereological analyses carried out by Pakkenberg and Gundersen [32]. They examined a total of 94 normal human brains, from individuals ranging in age from 20 to 95 years, and concluded that there is a 12% decrease in the overall volume of the cerebral hemispheres, accompanied by a 28% decrease in the volume of the white matter. This study was followed by a report by Tang et al. [33] that focused on the nerve fibers of white matter in the cerebral hemispheres and concluded that the loss of white matter volume is due to a 27% overall loss in the total length of nerve fibers in the white matter. Later, Marner et al. [34] extended this study by examining a total of 26 brains and concluded that the loss of white matter from the normally aging human cerebral hemispheres is almost 23% between the ages of 20 and 80 years, and that the overall loss of nerve fiber length is 45%. They suggest that this is produced by a loss of the thinner fibers, and that there is a relative preservation of larger-diameter fibers.

The conclusion from these studies is that there is a an overall loss of some nerve fibers from the human brain during normal aging; a similar conclusion was reached previously by Meier-Ruge et al. [35], who examined autopsied brains from cognitively normal humans. Meier-Ruge et al. [35] examined semithick sections in which nerve fibers were stained for light microscopy and, on the basis of counts, they concluded that with age there is a 16% nerve fiber loss from the white matter of the precentral gyrus and a 10.5% loss of nerve fibers from the corpus callosum.

There is also MRI data from humans (e.g., [36, 37]) and from monkeys ([38]; see also [39]) supporting the conclusion that there is a loss of white matter from the cerebral hemispheres with increasing age, and especially from the frontal lobes (e.g., [40, 41]). In addition, there are MRI studies that show that the signal characteristics of white matter alter in normal human aging. These changes are considered to indicate that white matter is undergoing degenerative changes, which result in disconnections between parts of the brain (e.g., [42, 43]). The study by De Groot et al. [44] indicates that the most common locations for white matter lesions in the aging human brain are the subcortical and periventricular white matter. The subcortical fibers mainly consist of short U-fibers that connect adjacent areas of the cortex, while the periventricular fibers are mainly long association fibers. After analyzing the frequency of occurrence of lesions and correlating the data with the cognitive status of the subjects examined, De Groot et al. [44] conclude that it is the lesions of the long association fibers that play a dominant role in bringing about cognitive decline.

It is suggested by Bartzokis et al. [45] that the deterioration of myelin sheaths with age is related to the sequence in which fiber tracts myelinate during development, such that the tracts that myelinate last are the ones most severely affected during aging. It is those same association fiber tracts that Kemper [11] has shown to exhibit staining pallor with age. Kemper [11] has also shown that, in the human brain, the primary cortices, in which myelination is completed earliest, show little change in myelin staining intensity with increasing age, while the association cortices show a distinct loss of staining intensity. But in contrast to these studies emphasizing white matter, there are other studies on monkeys (e.g., [46]) and humans (e.g., [47–49]) that suggest there is cortical thinning during normal aging, such that gray matter loss exceeds white matter loss (e.g., [50]) and, moreover, that the portion of the brain most affected by aging is the frontal lobes (e.g., [51]).

To obtain direct evidence that nerve fibers are lost from white matter with age, we have examined well-circumscribed fiber tracts in the monkey brain using design-based stereology. One of the first tracts we examined was the optic nerve. The cross-sectional area of the optic nerve does not alter much with age, but it was found that while the average total number of nerve fibers in the optic nerves of young monkeys is 16 × 10⁵, in monkeys over 25 years of age the number of nerve fibers is reduced to an average of 9 × 10⁵. Some old monkeys lose only a small percentage of their nerve fibers, but in extreme cases the number of nerve fibers is reduced to 4 × 10⁵, which represents a 75% loss of nerve fibers [52]. In the optic nerves showing such extreme loss, almost every nerve fiber has myelin sheath defects and, while some nerve fibers have degenerating axons, other myelin sheaths are empty (Figure 5.3). Correlated with the loss of nerve fibers there is hypertrophy of astrocytes, which develop abundant glial filaments and fill spaces vacated by degenerated nerve fibers. Oligodendrocytes and microglial cells also increase in number with age, and many of the microglial cells became engorged with phagocytosed debris, much of which can be recognized as degenerating myelin [53]. Cavallotti et al. [54] also found a loss of nerve fibers from the optic nerve of the aging rat, accompanied by an increase in the numbers of astrocytes and an increase in GFAP reactivity.

FIGURE 5.3

This micrograph from the primary visual cortex of a 13-year-old monkey shows two nerve fibers (asterisks) in which the axon has degenerated, leaving empty myelin sheaths behind. (Scale bar = 1 μm.)

The anterior commissure is another well-circumscribed bundle of white matter in which the total numbers of nerve fibers can be accurately determined [55]. In the anterior commissures of young monkeys, the mean number of nerve fibers is 2.2 × 10⁶, while in monkeys over 25 years of age the mean number is reduced to 1.2 × 10⁶. This loss of fibers is accompanied by a 25% reduction in the cross-sectional area of the anterior commissure. Some middle-aged monkeys, 12 to 20 years of age, also were available for study and it became evident that, in terms of the total numbers of nerve fibers, middle-aged monkeys resemble young ones, so most of the loss of nerve fibers appears to occur after middle age. Nerve fibers with abnormal myelin sheaths are evident at all ages, but there is a progressive, age-related increase in their frequency, such that in young monkeys only 0.4% of profiles of nerve fibers show alterations in myelin, while the number increases to 1.8% in middle-aged monkeys, and reaches 5.4% in old monkeys. Similarly, as would be expected from the loss of nerve fibers, there is a significant increase in the numbers of axons that show degenerative changes with age. Because most of the monkeys used in this study had been behaviorally tested, it was possible to correlate the data with a decline in their cognitive status. A positive correlation was found between the reduction in the total numbers of nerve fibers and cognitive impairment, but there was not a strong correlation between myelin sheath abnormalities and cognitive status.

In rats, Fujisawa [56] examined the effects of age on the nerve fibers in the posterior funiculus of the spinal cord and found that degenerating axons begin to appear long before the posterior funiculus has finished growing and has acquired its full complement of nerve fibers. Fujisawa [56] also showed that axonal degeneration occurs simultaneously at all levels of the spinal cord and that it involves nerve fibers of all sizes.

Our conclusion is that there is a loss of myelinated nerve fibers from white matter with age; and it is probably ubiquitous because we also found loss of nerve fibers from the splenium of the corpus callosum [57], as well as from the fornix of monkeys (unpublished data).

C. Continued Production of Myelin

In the primary visual cortex of the monkey, there are vertical bundles of myelinated nerve fibers that are most prominent in layer 4C. It was noticed that with age some of the myelin sheaths around the larger-diameter fibers in the bundles become appreciably thicker. Consequently, electron micrographs were taken of these bundles of nerve fibers in both young and old monkeys, and the diameters of the axons and the thickness of their myelin sheaths were measured [58]. The analysis indicated that no change is evident in the diameters of the axons with age, and there is no change in the width of individual myelin lamellae. Nevertheless, the mean numbers of lamellae in the myelin sheaths increase from 5.6 in young monkeys to 7.0 in old monkeys, and much of this increase in the mean thickness of myelin sheaths is due to an increase in the numbers of larger nerve fibers that have more than ten lamellae. In young monkeys, few nerve fibers have sheaths with more than 10 lamellae; but in the old monkeys it is not uncommon to encounter sheaths with as many as 20 lamellae, and in many cases such sheaths show circumferential splits, so that the sheaths appear to consist of an inner set of compact lamellae surrounded by an outer, separate set. A consequence of this increase in the thickness of myelin sheaths with age is evident at the paranodes of some of the thickened nerve fibers in the central nervous systems of aging monkeys. In longitudinal sectioned nerve fibers, the paranodal loops of myelin normally terminate in a regular sequence, and all of them are in contact with the underlying membrane of the axon. However, at the paranodes formed by many of these thickened sheaths, the paranodal loops pile up on one another and become disarrayed, so that there is only space enough for some of the loops to reach the underlying axon (unpublished data). Earlier, Sugiyama et al. [59] reported this same phenomenon in the thickened nerve fibers of old rats.

As far as can be determined, there are no other studies of the effects of age on the thickness of myelin sheaths in primates, but there have been a number of such studies in rodents. The authors have reached various conclusions. For example, Sturrock [60] examined the anterior and posterior limbs of the anterior commissure in the brains of 5- and 18-month-old mice and concluded that there is no change in the numbers of lamellae with age. In contrast, Godlewski [61] found that the myelin sheaths in the corpus callosum and optic nerves of 2.5-year-old rats were thicker than those of 4-month-old rats. In the peripheral nervous system of rodents, Caselli et al. [62] found no change in the numbers of lamellae in the sciatic nerves of rats with age, while Cebellos et al. [63] reported that in the tibial nerve of mice, myelin sheaths become thicker between 6 and 33 months of age, with some sheaths becoming very thick, as we have found in monkey visual cortex [58]. With such variations in the data, it is difficult to know the true situation in rodents. Obviously, more studies are necessary.

Another morphological alteration that occurs with age is an increase in the frequency of nerve fibers with redundant myelin, that is, sheaths that are overly large for the size of the enclosed axon, so that in cross sections the axon is at one end of a large loop of myelin (see Figure 5.1). Sheaths with redundant myelin were first described by Rosenbluth [64] in the cerebellum of the toad. In a study of the effects of age on myelin sheath in the white matter of mice, Sturrock [60] found such redundant sheaths to be common in old mice. As far as the monkey is concerned, redundant sheaths can be found even in young monkeys, although their frequency of occurrence increases with age.

On the basis of these observations, it can be concluded that — at least in the monkey — oligodendrocytes continue to produce myelin throughout life, and that this continued myelin production is occurring even as some sheaths are degenerating.

D. Remyelination and Aging

As we continued to examine the effects of aging on nerve fibers, it was noticed that in cross sections of the vertically oriented bundles of nerve fibers in both the primary visual and prefrontal cortices of older monkeys examined by electron microscopy, there is an increase in the frequency of occurrence of profiles of paranodes [65]. Paranodes occur at both ends of a length of myelin, adjacent to the nodes of Ranvier, and they are the sites where the lamellae of myelin gradually terminate. In visual cortex, there is a 57% increase in the frequency of cross-sectioned paranodal profiles with age and in area 46 of prefrontal cortex the increase is of the order of 90%. Such an increase in the frequency of profiles of paranodes could be due to either an increase in the lengths of paranodes with age, or to a real increase in the numbers of paranodes. Examination of the lengths of paranodes in primary visual cortex shows that with age there is an 11% increase in the lengths of paranodes, but this is insufficient to account for the large 57% increase in the frequency of paranodal profiles. This suggests that most of the increase in the frequency of paranodal profiles with age must be due to an increase in the total number of paranodes, and hence in the total number of internodal lengths of myelin. The implication is that some shorter internodal lengths of myelin are generated with increasing age, as would occur if the initially formed long internodal lengths of myelin degenerate and the resulting lengths of bare axons are remyelinated by a series of shorter internodal lengths.

In support of the proposal that remyelination occurs in the cerebral cortices of older monkeys, in the vertical bundles of nerve fibers in the visual cortices of old monkeys, we have found inappropriately thin myelin sheaths around some axons, as well as some short internodes that are only 3 to 6 μm long. Both of these features are considered the hallmarks of remyelination and support the contention that during normal aging some myelin sheaths break down, and that the resulting bare lengths of axons are then remyelinated by shorter lengths of new myelin. To determine that some myelin sheaths are completely degenerated, leaving the axon bare, is difficult to prove. However, in support of the concept that some myelin sheaths degenerate with age, it has been found that astrocytes in the cerebral cortices of old monkeys sometimes contain phagocytosed myelin lamellae and that some of the more amorphous phagocytosed inclusions in astrocytes label with antibodies to myelin basic protein [65].

Because an increase in the frequency of internodes can be expected to slow the rate of conduction along a nerve fiber, the correlation between the frequency of paranodal profiles in the vertical bundles of nerve fibers and the decline in cognition was examined. In prefrontal area 46, there is a significant correlation between these measures, but not in primary visual cortex (area 17). The reason why a correlation only exists for area 46 may be because prefrontal cortex plays a much greater role in cognition than area 17.

In a subsequent study, it was found that there is also an increase in the frequency of paranodal profiles in the anterior commissures of old monkeys [55]. In the anterior commissure, the increase is on the order 60%. However, when the frequency of occurrence of paranodal profiles in the anterior commissures of young monkeys, 5 to 10 years of age, is compared to that of middle-aged monkeys, 12 to 20 years of age, there is no difference between them. The increase in frequency of paranodal profiles only occurs in monkeys over 25 years of age. As stated earlier, there is a loss of nerve fibers from the anterior commissure with age, and it might be suggested that the increase in the frequency of occurrence of paranodal profiles is brought about by a preferential loss of the large-diameter fibers with the longest internodes and paranodes. However, this is not the case because the fiber diameter spectrum of nerve fibers is similar in young and old monkeys. So again, the most logical explanation is that there is some degeneration of internodal lengths of myelin, followed by remyelination of the affected axons by shorter internodal lengths of myelin.

Ibanez et al. [66] have reviewed the affects of aging on the remyelination of nerve fibers and the regenerative capacity of myelin sheaths to be restored in conditions such as multiple sclerosis. They cite data to show that as animals grow older, their capacity for remyelination declines. They suggest that the capacity for remyelination can be partially reversed by steroid hormones and their derivatives.

E. Correlations with Cognition

Most of the monkeys in which we have examined the effects of age on nerve fibers have been behaviorally tested and an index of their cognitive impairment (CII) has been generated (see [4, 12, 67,68]; see also Chapter 2). Consequently, it has been possible to ascertain what age-related alterations in nerve fibers might result in cognitive impairment. In area 17 [12], in prefrontal area 46 [57], and in corpus callosum [57], the increase in the frequency of profiles of altered myelin sheaths correlates significantly with cognitive impairment. In area 46 there is also a correlation with the increased frequency of paranodal profiles, but there is no such correlation in other structures examined. This raises the question of what brings about such behavioral correlations. Myelin is known to provide insulation around nerve fibers, so that saltatory conduction is possible. Consequently, it is likely that defects in myelin might lead to some breakdown in the insulation and affect conduction.

Although correlations between the age-related defects in the structure of the myelin and conduction velocity have not been examined, there have been several studies of the effects of age on conduction velocity in old animals. For example, Aston-Jones [69] examined the conduction velocity along nerve fibers connecting nucleus basalis to frontal cortex in rats and found a significant reduction in conduction velocity in old animals. Similarly, Morales et al. [70] have shown a reduction in conduction velocity of lumbar motor neurons in the spinal cords of cats, and Xi et al. [71] have shown a reduction in conduction velocity along nerve fibers in the pyramidal tracts of old cats. Interestingly, in proteolipid deficient mice, in which there is decompaction of the myelin, there is also a reduction in conduction velocity [72], and a reduction also occurs in demyelinating diseases (see [73, 74]). Attention should also be drawn to a recent study by Wang et al. [75] on the visual system of old monkeys. This study shows that the neurons in layer 4 of primary visual cortex in old monkeys exhibit normal visual response latencies, but in other parts of V1, and throughout secondary visual area V2, hyperactivity (increased firing frequency) of neurons is accompanied by delays in both intracortical and intercortical transfer of information. In part this may be explained by delays in the transfer of information along nerve fibers due to alterations in their myelin sheaths. In addition, Chang et al. [76] recently demonstrated a similar increase in the firing rates of rhesus monkey prefrontal cortical pyramidal cells with normal aging. These workers have hypothesized that this “hyperactivity” may represent a compensatory response to increased action potential conduction failures along the axon due to the extensive myelin dystrophy that occurs with age.

F. Summary of Aging-Related Myelin Changes

In summary, in the monkey, the process of myelin formation appears to continue throughout life in the central nervous system and results in the formation of thicker myelin sheaths and in the formation of redundant myelin. Beginning in middle age, some myelin sheaths begin to degenerate, and subsequently some of the resulting bare axons become remyelinated by shorter internodal lengths. This conclusion is supported by the finding that some short internodes do indeed exist, and that some axons have inappropriately thin myelin sheaths. In addition, it is evident that with age there is an increase in the frequency of occurrence of profiles of paranodes, as would occur with an overall increase in the numbers of internodes with increasing age. At the same time that these changes are taking place in myelin, some axons degenerate; this leads to complete degeneration of the affected nerve fibers and results in a reduction in the total number of nerve fibers in the white matter.

It is proposed that these alterations in the structure of myelin sheaths, coupled with an increase in the numbers of internodal lengths along nerves, bring about a reduction in the conduction velocity of affected nerve fibers. This would result in a change in the timing of sequential events in neuronal circuits, and it is suggested that this change in timing is at least partially responsible for the cognitive decline exhibited by old primates. In addition, the loss of some nerve fibers from white matter tracts with age would lead to some disconnection between groups of neurons in the brain, which could also adversely affect cognition.

A. Oligodendrocytes

Oligodendrocytes are the neuroglial cells that form the myelin sheaths in the central nervous system. As far as is known, this is their only function. As their name suggests, these cells have few visible processes, so that in light microscopic preparations of young animals stained by one of the silver stains or by Perl’s reaction for ferric iron (which is relatively abundant in oligodendrocytes, along with ferritin and transferrin [78, 79]), a few wispy, undulating processes can be seen leaving the cell body. However, when Perl’s stain is used on old monkeys, it becomes apparent that some of the processes of oligodendrocytes have acquired bulbous enlargements (Figure 5.4). These enlargements are of various sizes, with the largest ones reaching diameters of 5 μm. Another age-related change is that, whereas most oligodendrocytes in young monkeys occur individually, in old monkeys it is common to find oligodendrocytes in pairs, groups, or in rows (Figure 5.5). There is a significant correlation between the increased incidence of pairs, groups, and rows of oligodendrocytes and increasing age [80, 81].

FIGURE 5.4

An oligodendrocyte in layer 3 of the primary visual cortex of a 29-year-old monkey. The oligodendrocyte (O) has a long process (p) that expands into a large bulge containing dense inclusions (I). (Scale bar = 1 μm.)

FIGURE 5.5

A nest of three oligodendrocytes (O) lying next to a capillary (cap) in the primary visual cortex of a 28-year-old monkey. One of the oligodendrocytes has an aggregate of dense inclusions (I) in its cytoplasm. (Scale bar = 1 μm.)

These age-related changes in oligodendrocytes are also evident in electron microscopic preparations. In young monkeys, oligodendrocytes are encountered throughout the gray and white matter, and they are recognized by having a dark nucleus with clumped chromatin. In general, the nucleus has a rounded or oval profile, but sometimes it may have a more irregular shape. The nucleus is surrounded by an electron-dense cytoplasm with short and rather dilated cisternae of granular endoplasmic reticulum, polyribosomes, rather stubby mitochondria, and profiles of the Golgi apparatus. Microtubules occur throughout the cytoplasm but they can be difficult to discern due to the density of the cytoplasmic matrix. However, the microtubules become more evident at the bases of processes into which they funnel, and become closely packed.

In old monkeys, the oligodendrocytes have these same basic features but a difference is that many of them have dense inclusions in their perikaryal cytoplasm. These dense inclusions are irregular in shape and come in various sizes [82, 80]. Furthermore, most of the inclusions are composed of both pale and dense components that sometimes appear to form layers. Vaughan and Peters [83] reported the existence of similar inclusions in the oligodendrocytes within the auditory cortex of aging rats, and Rees [84] reported dense inclusions in oligodendrocytes in the cerebral cortex of aged human brains. At present, the origins of these inclusions are not known and their morphology gives no clues as to the sources of their contents. However, because they are not membrane bound, it is assumed that they are not phagocytic inclusions. Rather, they might be derived from the degeneration of some component of the aging myelin sheath attached to the oligodendrocyte. This possibility is reinforced by the fact that, in addition to the ones in the perikarya, other inclusions are present in the swellings that occur along the process of oligodendrocytes in older monkeys. It has been reported that similar swellings also occur along the processes of oligodendrocytes in the twitcher mouse, which is a model for globoid cell leukodystrophy. Using Perl’s reaction, LeVine and Torres [85] reported that in these animals some of the oligodendrocytes have large swellings in the portions of their processes extending from the outsides of the myelin sheaths, and other swellings along the lengths of the processes. As a consequence, LeVine and Torres [85] suggested that the material in the swellings comes from components in the sheaths that are being turned over or replaced, so that the material originates in the myelin sheaths and is then moved to the cell body of the oligodendrocyte, where it forms the dense inclusions found there.

The fact that there is an age-related increase in the numbers of oligodendrocytes in pairs, rows, and groups suggests that oligodendrocytes are proliferating with age and that there is an increase in their numbers [81]. Such an increase was first suggested when a comparison was made between the mean numbers of neuroglial cells in the primary visual cortex of young and old monkeys [82]. It was found that oligodendrocytes comprised 35% of the total population of neuroglial cells in young monkeys and 40% in the cortices of the neuroglia in old monkeys. For this study, only a few monkeys were available; but in a later study that involved seven young, six middle-aged, and eleven old monkeys, counts were made of the numbers of neuroglial cells in layer 4C of the primary visual cortex and, when young and old monkeys are compared, it was found that there is a 50% increase in the numbers of oligodendrocytes with age in this cortical layer. This increase begins in middle age but, interestingly, there seems to be no parallel increase in the numbers of astrocytes or of microglial cells in layer 4C [81].

There is also an increase in the numbers of oligodendrocytes in the monkey optic nerve with age. This is accompanied by an increase in the numbers of microglial cells, but not of astrocytes [53]. In contrast, there is no increase in the total numbers of any of the neuroglial cell types in the aging anterior commissure [55].

When there is an increase in the number of oligodendrocytes with age, questions arise as to where they come from and why they are needed. The answer to the second part of the question is that they are probably needed to form the increased numbers of internodal lengths of myelin that are generated with age [65]. As to the origins of the increased numbers of oligodendrocytes, the age-related increase in the frequency of oligodendrocytes in pairs, rows, and groups in some parts of the brain suggests that some of the oligodendrocytes might be dividing. However, the prevailing view is that there is little evidence that mature oligodendrocytes divide (see [14, 86, 87]) and that new oligodendrocytes are generated from oligodendroglial precursor cells (see [86, 88–90]), which can be visualized using antibodies to NG2 chondroitin proteoglycan and to platelet derived growth factor alpha receptor (e.g., [91–93]). These NG2+ cells are relatively common in the central nervous system and, as considered in a later section of this chapter, there is good reason to consider that they comprise a fourth and distinct type of neuroglial cell.

B. Astrocytes

As their name might suggest, astrocytes are star-shaped neuroglial cells that have numerous processes radiating from their cell bodies. Under the electron microscope, these cells have pale nuclei with very little clumping of the chromatin. The cytoplasm is also pale, which distinguishes these cells from oligodendrocytes and microglial cells, both of which have dark cytoplasm. A unique component of the cytoplasm of astrocytes is the 9-nm-thick intermediate filaments, which occur throughout the perikaryon and aggregate into bundles that pass into the processes. Because of the richness of the bundles of filaments in their cytoplasm, the astrocytes of white matter are often referred to as filamentous astrocytes, while the astrocytes in gray matter, which has fewer filaments, are referred to as protoplasmic astrocytes. Another feature of astrocytes is that they are extensively coupled by gap junctions. The study by Cotrina et al. [94] shows that in mice, gap junction proteins are maintained at high levels during aging, although there is some reorganization in terms of the number and sizes of the junctions labeled by antibodies to various connexins. What effect this has on the function of the aging brain is not known.

Antibodies to the glial fibrillary acid protein (GFAP) often are used to visualize astrocytes in light microscopic preparations. Such preparations show the cell bodies and radiating processes of astrocytes to good advantage, and give the impression that the processes are smooth. Essentially, this is true of the astrocytes in white matter, which have processes that tend to pass at right angles to the orientation of the nerve fibers, but it is not true of the astrocytes in gray matter. The filaments in these protoplasmic astrocytes extend along the axes of the larger-diameter processes but they do not usually enter and reveal the presence of the many thin and irregularly shaped excrescences that emanate from the thicker processes. And indeed, when astrocytes in gray matter are examined under the electron microscope, they are seen to have very irregular shapes and the fine, irregular processes mold themselves to the components of the surrounding neuropil.

As stated in the above section dealing with oligodendrocytes, astrocytes do not appear to increase in number during normal aging of the monkey. Most reports suggest that the same is true for rodents (e.g., [95–98]) and humans [99]. However, Peinado et al. [100] have reported that in the parietal cortex of the aging rat, there is a 20% increase in the numbers of astrocytes with age; and in the frontal cortex, Peinado et al. [101] found a 10 to 20% increase in neuroglial cells with age, depending on the cortical layer being examined. Similarly, Mouton et al. [102] have reported that the hippocampi of young female mice have about 20% more astrocytes and microglia than the hippocampi of old female mice. They also report that in the dentate gyrus and CA1 regions of the hippocampus, female mice have 25 to 40% more astrocytes and microglia than age-matched males. However, as pointed out, in their study of the total numbers of glial cells in human neocortex, Pakkenberg et al. [77] concluded that there is no significant increase in the total numbers of neuroglia with age.

There is general agreement that astrocytes undergo hypertrophy with age. They both increase in size and become more filamentous. Thus, when nerve fibers are lost with age, the astrocytes undergo hypertrophy and fill up the space vacated by the degenerated nerve fibers, as has been seen in the optic nerves [53] and in the corpus callosum (unpublished data). The hypertrophy also is evident in layer 1 of the cerebral cortex. In monkeys, layer 1 becomes thinner with age, largely because of the loss of some of the branches of the apical dendritic tufts of pyramidal cells [6, 103]. This leads to an impressive thickening of the glial limiting membrane on the outside of the cortex. However, there is no increase in the number of astrocytes. The thickening is brought about by an increase in the number of layers of astrocytic processes forming the glial limiting membrane, and this is accompanied by an increase in the numbers of intermediate filaments in these processes. This hypertrophy of astrocytes with age has also been noted by Hansen et al. [99], who found a strong increase in GFAP labeling in layer 1 of aging human brains. Simultaneous with the thickening of the glial limiting membrane, the processes of the astrocytes in layer 1 become more profuse and more filamentous as they undergo hypertrophy to fill the spaces vacated by the loss of apical dendritic branches of neurons [104]. Overall, the effect is somewhat similar to the response of astrocytes to a lesion when they form a glial scar.

Other studies have also recorded an increase in the amount of GFAP and an increase in the intensity of GFAP labeling with age in the brains of rats and mice [105–107) and in monkeys [108–110]. Consistent with this, Nichols et al. [111] have shown that there is an increase in GFAP mRNA with age in rats and humans. Consequently, hypertrophy of astrocytes with age, but not an increase in their numbers, seems to be a ubiquitous event.

In addition to undergoing hypertrophy, astrocytes in both white and gray matter undertake phagocytosis in the aging brain. Indeed, astrocytes with inclusions in their cell bodies (Figure 5.6) have been encountered in all parts of the brain we have examined, including the cerebral cortex of both the rat [83] and monkey [9, 65, 82], as well as the optic nerves [53] and anterior commissure [55] of the monkey. The inclusions can be quite massive and most commonly consist of an electron-dense, sometimes granular, component intermixed with a paler component that appears to derive from lipid. In addition, we have encountered lamellar inclusions that are obviously the remains of phagocytosed myelin sheaths, because they have a periodic structure with major dense and intraperiod lines that match those in normal myelin. The presence of phagocytosed myelin in astrocytes is consistent with the fact that myelin degeneration occurs throughout the central nervous system of the aging monkey. The degenerating myelin that is phagocytosed by astrocytes is probably then degraded by them and incorporated into the more amorphous inclusions because when an antibody to myelin basic protein is used, labeling can be found over some of the amorphous inclusions [65]. The sources of the other material phagocytosed by astrocytes are not known.

FIGURE 5.6

An astrocyte (As) in layer 5 of the primary visual cortex of a 35-year-old monkey. The cytoplasm of the astrocyte contains bundles of filaments (f), as well as inclusions (I) with dense and pale components. (Scale bar = 1 μm.)

C. Microglial Cells

Microglial cells have dark nuclei, similar in appearance to those of oligodendrocytes, although the nuclei tend to be rather smaller and either oval or bean shaped. The cytoplasm of microglial cells is electron dense, but somewhat paler than that of oligodendrocytes. However, the similarities between microglial cells and oligodendrocytes can make it difficult in light microscopic preparations to distinguish between the two cell types. The differences between them are more obvious in electron microscopic preparations because, in contrast to the short stubby cisternae present in oligodendrocytes, microglial cells have long cisternae of granular endoplasmic reticulum. Moreover, microglial cell bodies have more irregular shapes because they tend to mold themselves to the outlines of the components in the surrounding neuropil. Another interesting difference between these two cell types is that when microglial cells are adjacent to the cell bodies of neurons, there is usually a thin astrocytic process interposed between the cell bodies of the microglial cells and the neurons, whereas the plasma membranes of oligodendrocytes lie immediately adjacent to those of neurons without an intervening astrocytic process. Another important difference between these two cell types is that in old animals it is common to find large inclusions of phagocytosed material in the cell bodies of microglial cells (Figure 5.7).

FIGURE 5.7

A microglial cell in the primary visual cortex of a 27-year-old monkey. The microglial cell has a dark, rounded nucleus (N) and its dark cytoplasm contains a large clump of electron-dense debris (D). (Scale bar = 1 μm.)

Microglial cells are generally regarded as the phagocytes of the central nervous system. Immunolabeling with antibodies to HLA-DRm, an MHC class antigen, and other antibodies shows that microglial cells become activated with age. This activation is evident in the white matter of the cerebral hemispheres of old rats [112], old monkeys (e.g., [113, 114]), and humans (e.g., [115]); and it is of interest that Sloane et al. [114] show that the extent of microglial cell activation in white matter is related to the degree of cognitive impairment in old monkeys. In contrast to white matter, gray matter shows few activated microglial cells and yet when tissue from old animals is examined by electron microscopy, microglial cells containing inclusions of phagocytosed material are encountered in both white and gray matter. The appearance of these inclusions is highly variable. In general, there are both electron-dense and pale components and, while some inclusions are small, others may be so large that the cytoplasm of the microglial cell is distended and confined to a thin rim that surrounds the inclusions. As a consequence, the nucleus is flattened against one side of the cell body. Typically, it is not possible to determine the origins of the phagocytosed material in microglial cells. The one exception we have encountered is in the optic nerves of old monkeys, in which some of the microglial cells can be seen to contain phagocytosed myelin sheaths [53]. In the optic nerves of old monkeys, the degeneration of nerve fibers can be very extensive and may require increased numbers of microglial cells to deal with the removal of this debris. This is probably the reason that the optic nerve is the only structure in which we have so far encountered a significant increase in the numbers of microglial cells with age. Thus, in the optic nerves of monkeys, the numbers of microglial cells increase from about 5% of the total population of neuroglial cells in young monkeys to about 10% of the total in old monkeys. In all other parts of the monkey brain that we have examined, the percentage of microglial cells is between about 5 to 7% of the total population of neuroglial cells and increases little, if at all, with age. Long et al. [98] found the same to be true in the hippocampus of the mouse, although Mouton et al. [102] have reported that in female mice there are about 20% more astrocytes and microglia in the dentate gyrus and CA1 regions of the hippocampus in old mice than in young ones. As pointed out in the section on astrocytes, Mouton et al. [102] find that when males and females are compared, there are 25 to 40% more astrocytes and microglia in females than in males in these same regions of the hippocampus. They suggest than because astrocytes and microglial cells are thought to be targets of gonadal hormones, the effects of sex hormones and of reproductive aging may be at the root of this difference.

Although it has not been studied in detail, it can be presumed that as they become phagocytes, the microglia of the brain undergo morphological transformations from the ramified, resting microglial cell with many branching processes to an activated form in which the branches are withdrawn to enable the cell to become motile and to move to locations where it is needed for phagocytosis. These stages of transformation have been nicely shown by Stence et al. [116] in a study in which they cut tissue slices of rat hippocampus, labeled the microglial cells with a fluorescent label, and followed the activation of the microglial cells that is induced by slicing the tissue. However, Streit et al. [117] suggest that with age, microglial cells undergo morphological alterations that are different from activation, and they designate the changes as microglial dystrophy. Streit et al. [117] examined microglial cells that had been labeled with an antibody, LN-3, in the cerebral cortices of two non-demented humans, one 38 years old and the other 68 years old. They found most of the microglial cells in the younger brain to have a typical ramified morphology, although they also encountered other dystrophic microglia with short, gnarled processes. Such dystrophic cells, which had lost their finely branched processes and had some processes that showed beading and the formation of spheroids, were described as being more common in the older brain. Streit et al. [117] suggest that such microglial dystrophy is a sign that the cells are undergoing senescent changes. However, it would be prudent to undertake further studies before accepting this concept.

D. A Fourth Neuroglial Cell Type

As pointed out when oligodendrocytes were considered, there is a fourth type of neuroglial cell present in the central nervous system. Until recently, this type of neuroglial cell has been largely overlooked in most studies of the normal adult nervous system because superficially they resemble protoplasmic astrocytes. When antibodies to NG2 chondroitin sulfate proteoglycan are used to label these cells, it becomes apparent that the NG2-labeled cells make up some 5% of the total number of neuroglial cells in the central nervous system (e.g., [88]). NG2-labeled cells in gray matter have a variety of shapes. They are generally characterized by small cell bodies from which extend irregular, branched processes with thin protrusions decorating them, while the cells in white matter have processes that pass parallel to the nerve fibers (e.g., [92]). There is general agreement that at least some of the NG2-labeled cells in the mature central nervous system are oligodendroglial progenitors (e.g., [88, 118]), but they may also have other functions that are presently unclear. It is also generally agreed that the cells that label with NG2 antibodies are distinct from the three classical types of neuroglial cells because they do not label with antibodies specific for the classical neuroglia. For example, they do not label with antibodies to GFAP, vimentin, or S-100, which are specific markers for astrocytes (e.g., [91, 119–121]), and Ye et al. [122] have shown that laser dissection captured NG2+ cells do not react with antibodies that are specific for astrocytes, microglial cells, or neurons. However, they do express mRNAs for myelin basic protein and for proteolipid protein, showing that at least some of the NG2+ cells can be precursors for oligodendroglia.

In electron microscopic preparations, the NG2+ cells bear a resemblance to astrocytes [123], in that they have pale nuclei and pale cytoplasm (Figure 5.8). However, the NG2+ cells have more irregular nuclei than typical astrocytes. The chromatin is dispersed and the cells have a thin layer of heretochromatin beneath the nuclear envelope, a layer that is generally more marked than that of astrocytes. The cell bodies of the NG2+ cells have a rather thin layer of cytoplasm around the nucleus, but there is usually abundant cytoplasm at the poles of the cells. It is evident that the cytoplasm contains free polyribosomes, a few cisternae of rough endoplasmic reticulum with a concentration of ribosomes on their surfaces, and some profiles of the Golgi apparatus. However, the mitochondria are generally smaller than those of astrocytes and intermediate filaments are not found in the cytoplasm. Other differences are that the NG2+ cells have more regular outlines than astrocytes; they are often seen to contain centrioles; and although the NG2+ cells can be found throughout the central nervous system, when they are adjacent to capillaries, there is always a thin astrocytic process separating the NG2+ cells from the basal lamina of the capillary.

FIGURE 5.8

A β neuroglial cell, the fourth type of neuroglial cell, in the primary visual cortex of a 16-year-old monkey. The neuroglial cell (β) is lying next to a neuron (N). The neuroglial cell has a pale nucleus and sparse cytoplasm that contains (more...)

There seems little doubt that these NG2+ cells are neuroglial cells that were earlier included in the category of protoplasmic astrocytes. The first ones to recognize the existence of this fourth type of glial cell were Reyners et al. [124, 125] and, because of their resemblance to astrocytes, they called them “β astrocytes.” But in the light of the newer information about these cells, it is evident that this name is no longer appropriate and even misleading. Consequently, it is suggested that they might be called “β neuroglial cells” in acknowledgment of the fact that Reyners and colleagues were the first ones to give a clear description of these cells.

To date it is not clear whether these neuroglial cells play any definitive role in aging, although they might be the source of the increased numbers of oligodendrocytes that occur in some parts of the normally aging brain.