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Keywords:

  • myelinated fibers;
  • unmyelinated fibers;
  • white matter;
  • aging;
  • stereology

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

To determine the exact reason for the age-related decline of the myelinated fiber length in white matter, we performed this study. In middle-aged rats, there was age-related loss of the unmyelinated fibers with large diameters. The demyelination of the myelinated fibers with small diameters in middle-aged rat white matter might make the age-related decrease of the unmyelinated fibers with small diameters in the white matter unnoticeable. However, in old-aged female rats, the unmyelinated fibers with large and small diameters significantly degenerated together and that the unmyelinated fibers formed from the demyelination of the myelinated fibers could not replenish the age-related loss of the unmyelinated fibers in the white matter. In conclusion, this study suggested that demyelination of myelinated fibers with small diameters in aged white matter might be the key mechanism of the significant decline of the myelinated fiber length in aged white matter. Anat Rec, 292:528–535, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

In 1955, Brody concluded that there was a significant and progressive loss of cortical neurons during normal aging. The loss reported by Brody amounted to as much as 50% (Brody,1955). This report received a lot of attention, and was probably the seed for the common saying that “she/he is losing her/his neurons” to explain why an aging person is a little forgetful. Brody's study was followed by a lot of other studies in which similar conclusions were made (Colon,1972; Shefer,1973; Devaney and Johnson,1980; Henderson et al.,1980; Anderson et al.,1983). It was not until the 1980s that this concept was seriously challenged. Some investigators presented evidences for the opposite conclusion, that is, there was no neuron loss during normal aging (Haug,1985; Terry et al.,1987). Since 1984, new stereological methods have been developed (Sterio,1984; Gundersen et al.,1988a,b; Moller et al.,1990). With the new designed-based stereological methods, Pakkenberg and Gundersen (1997) investigated a total of 94 normal human brains, ranging in age from 20 to 90 years. They found that there was no significant loss of the neocortical neurons with aging and that the primary structural change in the aged brain was atrophy of the white matter (Pakkenberg and Gundersen,1997). In vivo studies using MRI also revealed a significant age-related loss of white matter volume and a much smaller decline of gray matter volume (Albert,1993; Christiansen et al.,1994; Guttmann et al.,1998; Jernigan et al.,2001). What is the cellular component that induces marked loss of white matter with age? The white matter consists of myelinated fibers, unmyelinated fibers, neuroglial cells, and other tissues. Tang et al., (1997) investigated five young and five old female human brains and found that the total length of the myelinated fibers in the white matter of aged human brains was significantly decreased when compared with that of young brains (Tang et al.,1997). More recently, this group studied 36 normal human brains with an age ranging between 18 and 93 years. They found that the total length of the myelinated fibers in the white matter was significantly decreased between the ages of 20 and 80 years (Marner et al.,2003). To exclude the cases of undiagnosed Alzheimer's disease that confounds the issue of normal aging in human brains, we investigated the age-related changes of the myelinated fibers in the white matter of Long-Evans rats that do not develop Alzheimer's disease. We found that the difference in the total length of the myelinated fibers in white matter between young female rats and middle-aged female rats was not statistically significant. Given that we did not find significant change of the myelinated fibers in the white matter of middle-aged female rats, we investigated the myelinated fibers of the white matter in old-aged female rats. We found that the total length of the myelinated fibers in the white matter of old-aged female rats was significantly decreased when compared with that of young female rats. What is the reason for the loss of the myelinated fiber length in the white matter of aged brain? The demyelination of the myelinated fibers and/or the loss of axons and subsequent demyelination could lead to the decrease of the total length of the myelinated fibers in white matter. To detect which cellular structure is primarily affected so that leads to the decline of the myelinated fiber length in the white matter of aged brain, we investigated the age-related changes of the unmyelinated fibers in the white matter of Long-Evans rats by means of the transmission electron microscopy and stereological techniques. The results of the age-related changes of the unmyelinated fibers and the myelinated fibers in the white matter suggested that the demyelination of the myelinated fibers with small diameters led to the decline of the myelinated fiber length in aged white matter.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Animals

Four young female Long-Evans rats (6–8 month old), four middle-aged female Long-Evans rats (18 month old) and eight old-aged female Long-Evans rats (27–28 month old) were studied. The rats were housed 3–4 per cage at a temperature of 22°C ± 1°C. They were kept under a constant 12 hr light-12 hr dark cycle. Food and water were available ad libitum. The colony was certified specific pathogen free for the following: mouse pneumonia virus, sendia virus, hepatitis virus, reovirus, lymphocytic choriomenigtis, Theiler Martin encephalomyelitis virus, ectromelia, minute virus of rats, and mucoplasma pulmonis. Animal care and treatment followed the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996.

Specimen Fixation

The rats were anaesthetized by an intraperitoneal injection of 4% chloral hydrate. Then, they were perfusion-fixed with a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.4 M phosphate buffered saline (pH 7.4). After perfusion, the cerebellum, brain stem, and cranial nerves under the pavimentum cerebri were cut, and the cerebral hemispheres excised.

Estimation of the White Matter Volume

The two hemispheres were separated from each other along the cerebral longitudinal fissure. The two hemispheres were embedded in 6% agar and were coronally cut into 2-mm-thick slabs, starting randomly at the rostral pole. A transparent counting grid with an area of 0.39 mm2 associated with each point was placed at random on the caudal surface of each slab. Using an anatomical microscope, the grid points hitting the white matter were counted. The white matter volume was calculated according to Cavalieri's principle (Gundersen et al.,1988a; Tang et al.,1997,2003; Tang and Nyengaard,1997,2004; Marner et al.,2003):

  • equation image(1)

where VWM is the total volume of the white matter, t equals the slice thickness (2 mm), a(p) equals the area associated with each grid point (0.39 mm2), and ∑PWM is the total number of grid points hitting the white matter per rat.

Sampling of the White Matter

The right or left hemisphere was selected at random. From the slabs of the selected hemisphere, every third slab was sampled systematically, the first slab being sampled randomly. A plastic sheet with equally spaced points was placed randomly on the caudal cut surface of the sampled slabs. One micrometer cubes were sampled from white matter where the grid points hit the white matter. Four of these blocks per hemisphere were randomly sampled for analysis.

The tissue blocks were fixed in 4% glutaraldehyde for at least 2 hr at 4°C, rinsed in 0.1 M phosphate buffered saline (pH 7.2) three times, then osmicated in 1% 0.1 M phosphate buffered osmium tetroxide (OSO4) at 4°C for 2 hr. The blocks were gradually dehydrated through a series of 50%, 70%, 90% ethanol, 90% ethanol and 90% acetone mixture, and 100% acetone. The blocks were then infiltrated with epoxy resin 618 (ChenGuang Chemical Industry, Sichuan, China). The infiltration steps were acetone: resin 1: 1 (3 hr at room temperature), and absolute resin (2 hr at 37°C). The tissue was embedded in 5 mm diameter Epon spheres. The spheres were rotated randomly before being re-embedded in an oven at 37°C (16 hr), 45°C (12 hr), and 60°C (14 hr). This procedure, known as the isector assures that isotropic, uniform, and random (IUR) sections are obtained so that the fibers of each direction in 3-dimensional space have the same probability to be sampled (Nyenggard and Gundersen,1992).

Estimation of the Length Density and Volume Density of the Myelinated Fibers and the Unmyelinated Fibers in the White Matter

One 60 nm-thick section was cut from each epon block using an ultramicrotome and viewed in a transmission electron microscope (TEM) (Hitachi-7500, made by Hitachi, Japan). For the myelinated fibers, from each section, four fields of view were randomly sampled and photographed at a magnification of 6,000. For the unmyelinated fibers, from each section, eight to twelve fields of view were randomly sampled and photographed at a magnification of 20,000. Using the high magnification photographs, we identified the unmyelinated fiber profiles by the thin cytomembrane and the neurofibrilla in the profiles according to Peters et al. (Peters et al.,1991). An unbiased counting frame (Gundersen,1977) was superimposed on the randomly captured photographs. The myelinated fiber profiles and the unmyelinated fiber profiles completely inside the counting frame or partly inside the counting frame but only touching the top and right lines (inclusion lines) were counted. The myelinated fiber profiles and the unmyelinated fiber profiles touching the bottom line, left line and its extension, and the extension of right lines (exclusion lines) were excluded from counting (Fig. 1).

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Figure 1. An unbiased counting frame is randomly placed over a captured TEM photograph. The myelinated fibers and the unmyelinated fibers were counted if they are completely inside the counting frame or partly inside the counting frame but only touching the counting lines (dotted lines), as indicated by arrows. The myelinated fibers and the unmeylinated fibers were excluded for the counting if they touch the exclusion lines (solid lines), as indicated by the star. The terminals of the myelinated fibers that formed synapses are also excluded for the counting, as indicated in the figure.

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The length density of the myelinated fibers and the unmyelinated fibers in white matter, LV(nf/wm), was estimated using the following formula (Gundersen,1988a; Tang et al.,1997,2003; Tang and Nyengaard,1997,2004; Marner et al.,2003):

  • equation image(2)

where ∑Q(nf) is the total number of the myelinated fiber profiles and the total number of the unmyelinated fiber profiles counted per rat, respectively, a(frame) equals the area associated with a frame, ∑frames is the total number of frames counted, and 2 is a constant that pertains to isotropic, uniform random sections.

A transparent counting grid was placed on the randomly captured photographs. The points hitting the myelinated fibers, unmyelinated fibers, and white matter were counted, respectively (Fig. 2).

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Figure 2. The point grid is randomly put on the randomly captured image. The points hitting the myelinated fibers, unmeylinated fibers, and white matter are counted, respectively.

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The volume density of the myelinated fibers and the unmyelinated fibers in white matter, VV (nf/wm), was estimated using the following formula (Tang et al.,1997,2003; Tang and Nyengaard,1997,2004; Marner et al.,2003):

  • equation image(3)

where ∑P (nf) is the total number of points hitting the myelinated fibers and the total number of points hitting the unmyelinated fibers within white matter per rat, respectively, and ∑P (wm) is the total number of points hitting white matter per rat.

Estimation of the Total Length and Total Volume of the Myelinated Fibers and the Unmyelinated Fibers

The total length of the myelinated fibers and the unmyelinated fibers in white matter was estimated by multiplying the length density of the myelinated fibers and the unmyelinated fibers in white matter by the total volume of the white matter (Tang et al.,1997,2003; Tang and Nyengaard,1997,2004; Marner et al.,2003), respectively. The total volume of the myelinated fibers and the unmyelinated fibers in white matter was estimated by multiplying the volume density of the myelinated fibers and the unmyelinated fibers in white matter by the total volume of the white matter (Tang et al.,1997,2003; Tang and Nyengaard,1997,2004; Marner et al.,2003), respectively.

Estimation of the Mean Diameter of the Myelinated Fibers in White Matter

The diameter for each of the myelinated fibers counted was estimated by measuring the longest profile diameter perpendicular to its longest axis (Tang and Nyengaard,1997,2004) and measurements were obtained from the outer limit of the myelin sheath. The myelinated fibers with diameter less than 0.7 μm were defined as the myelinated fibers with small diameter.

Statistics

Analysis of variance (ANOVA) was performed. The analysis was conducted using the SPSS15.0. A significant difference was considered when P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

White Matter Volume

The mean volume of the white matter in the middle-aged female group was not significantly different from that in the young female group (P > 0.05; Fig. 3). However, when compared with the volume of the white matter in the young female group and that in the middle-aged female group, the total volume of the white matter in the old-aged female group was decreased significantly by 42.5% and 35.4%, respectively (P < 0.01; Fig. 3).

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Figure 3. Comparisons of the white matter volume between the young female rats, the middle-aged female rats, and the old-aged female rats. ** indicates P < 0.01.

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Myelinated Fibers

The total length of the myelinated fibers in the white matter in the middle-aged female group was not significantly different than the young female group (P > 0.05; Fig. 4). However, the total length of the myelinated fibers in the white matter of the old-aged female rats was decreased significantly by 48.6% compared to that of the middle-aged female rats (P < 0.05) and decreased significantly by 52.5% when compared with that of the young female rats (P < 0.05) (Fig. 4). The absolute distributions of the total length of the myelinated fibers in the white matter of three group rats were shown in Fig. 5.

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Figure 4. Comparisons of the total length of the myelinated fibers in the white matter between the young female rats, the middle-aged female rats, and the old-aged female rats. * indicates P < 0.05 ** indicates P < 0.01.

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Figure 5. The absolute distributions of the total length of the myelinated nerve fibers in white matter (a) the fiber length distributions in young and middle-aged female rats, (b) the fiber length distributions in middle-aged and aged female rats, (c) the fiber length distributions in young and aged female rats.

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Unmyelinated Fibers

The total volume of the unmyelinated fibers in the white matter of the middle-aged female group was decreased significantly by 43.3% compared to that in the young female group (P < 0.05; Fig. 6). The total volume of the unmyelinated fibers in the white matter of the old-aged female group was decreased significantly by 72.2% compared to that in the young female group (P < 0.01; Fig. 6). The total volume of the unmyelinated fiber in the white matter of the old-aged female group was decreased significantly by 51.0% compared to that in the middle-aged female group (P < 0.05; Fig. 6).

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Figure 6. Comparisons of the total volume of the unmyelinated fibers in the white matter between the young female rats, the middle-aged female rats, and the old-aged female rats. * indicates P < 0.05 ** indicates P < 0.01.

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On an average, female rats had a total length of 409 ± 130 km unmyelinated fibers at age 6–8 months, 350 ± 171 km at the age of 18 months and 128 ± 48 km at age of 27–28 months. The loss of the total length of the unmyelinated fibers in the white matter of the female rats was 59 km from 6 to 8 months to 18 months and 280 km from 6 to 8 months to 27–28 months. There were no significant differences in the total length of the unmyelinated fibers in the white matter between the young female group and the middle-aged female group (P > 0.05; Fig. 7). However, the total length of the unmyelinated fibers in the white matter of the old-aged female rats was decreased significantly by 68.6% and 63.4%, respectively, when compared with that of the young female rats and the middle-aged female rats (P < 0.01; Fig. 7).

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Figure 7. Comparisons of the total length of the unmeylinated fibers in the white matter between the young female rats, the middle-aged female rats, and the old-aged female rats. ** indicates P < 0.01.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

The sampling strategy of earlier studies on the nerve fibers of white matter was to select a certain typical portion of a region of interest. Partadiredja et al., (2003) examined hemisphere differences in the myelinated fiber density and unmyelinated fiber density of the rat white matter. They did not sample the white matter uniformly but selected three regions (frontal, parietal, and occipital) subcortical white matter for investigation. The conclusions that can be drawn from the analysis of a certain typical portion of the region of interest, a standard section or sections in which the objects of interest are best identified can only apply to that part of tissue and those sections. The “ideal tissue” and “ideal fields” are not representative of the entire tissue. In stereology, the fundamental principle for sampling tissue is that every part inside the region of interest has the same probability of being sampled. Without uniform random sampling, it is essentially impossible to obtain an unbiased estimate. In this study, the described sampling scheme ensures a uniformly random distribution of the samples and provides an equal sampling probability for all parts of the white matter. Therefore, all myelinated fibers and unmyelinated fibers in the entire white matter had the same probability of being sampled.

The white matter consists of myelinated fibers, unmyelinated fibers, neuroglia cells and interstitial substance. Tang et al., (1997) used 10 female brains obtained from autopsy and estimated the volume of white matter, the total volume and total length of the myelinated fibers in white matter. The total volume of the white matter and the total volume of the myelinated fibers in the white matter of aged brain were decreased nonsignificantly by 15% and 17%, respectively, when compared with those of young brain. However, the total length of the myelinated fibers in the white matter of aged females was decreased significantly by 27% compared to that of young females. Moreover, the decline of the myelinated fiber length was mainly due to the loss of the myelinated fibers with small diameter. To exclude the cases of early Alzheimer's disease that confounds the issue of normal aging, we investigated the age-related changes of the myelinated fibers in the white matter of Long-Evans rats which do not develop Alzheimer's disease (Erickson and Barnes,2003). We found that the myelinated fibers with small diameter were pronouncedly lost in the aged rat white matter, which confirmed the previous findings of Tang et al. The demyelination of the myelinated fibers and/or axon degeneration and subsequent myelin breakdown can lead to the decrease of the myelinated fiber length in the white matter of aged brain. To find the exact factor for the myelinated fiber loss in white matter of aged brain, we need to simultaneously investigate the changes of the myelinated fibers and unmyelinated fibers in white matter with age. Therefore, we investigated the unmyelinated fibers in the white matter using the same specimens that we used when investigating the age-related changes of the myelinated fibers in the white matter. In this study, we found that the total length of the unmyelinated fibers in the white matter of 18 month female rats was nonsignificantly decreased by 14%. Regarding the total volume of the unmylinated fibers in the white matter, we found that the total volume of the unmylinated fibers in the white matter of 18 month female rats was decreased significantly by 43% when compared with that of 6–8 month female rats. These results indicated that the age-related loss of the total length of the unmyelinated fibers in white matter was mainly due to the degeneration of the unmyelinated fibers with larger diameters. What are the factors for the fact that we do not find the age-related changes of the unmyelinated fibers with small diameters in rat white matter but find the age-related changes of the myelinated fibers with small diameters in rat white matter? After analyzing the changes of the myelinated fibers and unmyelinated fibers in the aged white matter together, we concluded that there was a decrease of the unmyelianted fibers with small diameters in the white matter of middle-aged rats, but this decrease was masked by the demyelination of the myelinated fibers with small diameters in the white matter of middle-aged rats. The demyelination of the myelinated fibers with small diameters in the white matter of the middle-aged rats became unmyelinated fibers so that the change of the unmyelinated fibers with small diameter during aging was not statistically significant. Because the myelinated fibers with small diameters were demyelinated to become unmyelinated fibers, the total volume of the unmyelinated fibers formed from the demyelination of the myelinated fibers was small. Therefore, the loss of the unmyelinated fibers with larger diameters in the white matter of the middle-aged rats led to the decrease of the total volume of the unmyelinated fibers in the white matter of the middle-aged rats.

We found that the total length of the unmyelinated fibers in the old-aged female group was decreased significantly by 68.6% compared to the young female group, and it was decreased significantly by 63.4% compared to the middle-aged female group. Furthermore, we found that the total volume of the unmyelinated fibers in the white matter of the old-aged female group was decreased significantly by 72.2% compared to the young female group, and it was decreased significantly by 51.0% compared to the middle-aged female group. These results indicated that from young age to the early days of senectitude, the major change of the unmyelinated fibers in the white matter was the loss of the fibers with large diameter and that the age-related change of the unmyelinated fibers with small diameter was masked by the demyelination of the myelinated fibers with small diameter in the white matter. However, after entering senectitude, the unmyelinated fibers with large and small diameters in the white matter significantly degenerated together so that the total length and total volume of the unmyelinated fibers in the old-aged rat white matter were significantly decreased. Moreover, our results showed that both the total length and total volume of the myelinated fibers in the white matter of the old-aged female rats were significantly decreased when compared with those in the middle-aged female rats and in the young female rats. These results demonstrated that the myelinated fibers in the white matter of the old-aged female rats degenerated. We presumed that the myelinated fibers demyelinated and became unmyelinated fibers in the white matter of the old-aged female rats. Because in the old-aged rat white matter, the amount of the unmyelinated fibers formed from the demyelination of the myelinated fibers with small diameter might not retrieve the loss of the unmyelinated fibers with small diameter with age, the total volume and the total length of the unmyelinated fibers were significantly decreased in the old-aged rat white matter.

In conclusion, the demyelination of the myelinated fibers with small diameter in white matter might be the main factor leading to the decrease of the total length of the myelinated fibers with age. The myelin defects might lead to a decrease in the conduction velocity along axons, which might underlie some of the age-related cognitive deficits.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

The authors thank all the staff in the Department of the Electron Microscope, Chongqing Medical University, People's Republic of China for providing assistance in the TEM technique.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
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