Different patterns in age-related morphometric alteration of myelinated fibers and capillaries of the tibial nerve: a longitudinal study in normal rats

Abstract

Age-related regression of myelinated fibers in peripheral nerves of the lower limbs is strongly influenced by capillaries and results in balance dysfunction and falls. However, the temporal relationships between alteration patterns of myelinated fibers and capillaries have not yet been clarified. This study aimed to investigate age-related morphological and histological changes of both myelinated fibers and capillaries in peripheral nerves to clarify whether myelinated fibers or capillaries change earlier. Seven male Wistar rats each were randomly selected at 20 weeks (young group), 70 weeks (middle group), and 97 weeks (old group) for histological evaluations. The left and right tibial nerves were removed morphologically and histologically to examine myelinated fibers and capillaries. Axon diameter and myelin thickness were almost unaltered in the middle group compared with the young group but were significantly reduced in the old group when compared with the other two groups. However, the capillary diameter and number of microvascular branch points were substantially reduced in the middle group. The current study demonstrates that myelinated fibers of peripheral nerves show signs of regression in elderly rats, whereas capillaries start to reduce in middle-aged animals. In normal aging of the tibial nerve, capillaries may regress before myelinated fibers.

Keywords: capillary; myelinated fiber; regression; three-dimensional image; tibial nerve.

Figure 1

Measurement of capillary luminal diameters in rat tibial nerves using stacked 3‐D images of sagittal sections. An intersectional site between 15 equally spaced horizontal lines and the lumen of the fluorescent vessels, a square (white #), is shown in the left image (a). The left image (A) was converted into a grayscale (256 gradations) image, and the inverted image was expanded (B). A rectangle (white dotted lines) including the intersectional site between the horizontal line and the microvascular lumen was traced within the luminal borders. The longer side (X, maximum Feret diameter) of this rectangle was defined as the luminal diameter of the selected capillary. Scale bars: white, 100 μm; black, 10 μm

Figure 2

Age‐related alteration of myelinated fibers in the tibial nerves. Upper three transverse sectional images indicate myelinated fibers in tibial nerves of young (A), middle‐aged (B), and old (C) rats stained with Sudan Black B. Lower left histograms show the myelinated fiber diameters in the tibial nerves of rats in young (D: open columns, n = 1350), middle‐aged (E: hatched columns, n = 1350), and old (F: dark gray columns, n = 1350) groups. Lower four bar graphs arranged in the center show comparison of the values for fiber diameter (G), axon diameter (H), and myelin thickness (I), and G‐ratio (J) among young, middle‐aged, and old groups, respectively. In upper sectional images, on visual evaluation, myelinated fibers in young (A) and middle‐aged (B) rats have mainly regular morphologies. Conversely, myelinated fibers in old rats (C) exhibit many irregular morphologies due to age‐related degeneration. In histograms, the relative frequency of myelinated fibers above 11 μm in diameter was higher in the young (D) group than in the middle (E) and old (F) groups. Furthermore, the relative frequency of myelinated fibers with a diameter above 10 μm was elevated in the middle (E) group compared with the old (F) group. By contrast, the relative frequency of myelinated fibers below 9 μm in diameter was increased in the old (F) group in comparison with the young (D) and middle‐aged (E) groups. As for bar graphs, the values for fiber diameter (G), axon diameter (H), and myelin thickness (I) were slightly lower in the middle‐aged group than in the young group, with no or small effect sizes (see Cohen’s d in Table 1). The values in the old group were significantly lower than those in the middle‐aged and young groups with medium effect sizes (see Cohen’s d in Table 1). There were no significant differences in the G‐ratio among the young, middle‐aged, and old groups (G). Open (young), hatched (middle‐aged), and dark gray (old, n = 1350 each) columns and error bars indicate mean values and standard errors, respectively (see also Table 1 for numerical values). All values indicate mean and standard errors (see also Table 1). **p < .01, n.s.: not significant, Scale bar: 40 μm

Figure 3

Age‐related alteration of capillary luminal diameters and the number of microvascular branch points in tibial nerves. Upper, stacked 3‐D fluorescence images show capillaries in the tibial nerves of young (A), middle‐aged (B), and old rats (C) acquired using confocal laser microscopy. Each image represents a sagittal view of the tibial nerve. Lower left histograms display the distributions of capillary luminal diameters in the tibial nerves of young (D: open columns, n = 537), middle‐aged (E: hatched columns, n = 531), and old (F: dark gray columns, n = 528) groups. The lower two bar graphs indicate capillary luminal diameters (G) and numbers of microvascular branch points (H) in tibial nerves among the young (open columns), middle‐aged (hatched columns), and old (dark gray columns) groups. In 3‐D fluorescence images of capillaries, the young rat (A) shows dense growth with abundant microvascular branches; however, many capillaries and microvascular branch points in middle‐aged (B) and old (C) rats seemed to have disappeared. In the lower left histograms, the relative frequency of capillaries above 6 μm in luminal diameter was elevated in the young (D) group compared with the middle‐aged (E) group. Moreover, the relative frequency of capillaries above 4 μm in luminal diameter was higher in the middle‐aged (E) group than in the old (F) group. The relative frequency of capillaries with a diameter below 4 μm was higher in the old (F) group than in the middle‐aged (E) group. The capillary luminal diameter (G) substantially decreased with increasing age in the young (n = 537), middle (n = 531), and old (n = 528) groups with medium effect sizes (see Table 2 for numerical values). However, the number of microvascular branch points (B) showed a remarkable decrease between young (n = 22) and middle‐aged (n = 23) animals with a large effect size, whereas, subsequently, the old group (n = 22) exhibited no further decline compared with the middle‐aged group (see Table 2 in terms of numerical values). Open (young), hatched (middle‐aged), and dark gray (old) columns. (G and E) All values indicate mean and standard errors. **p < .01, n.s.: not significant. (A–C) Asterisks and arrowheads indicate capillaries and microvascular branch points, respectively. Scale bar: 100 μm