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. 2020 Feb 12;236(6):1101–1111. doi: 10.1111/joa.13168

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

Masahiro Sakita 1,✉, Shinichiro Murakami 2, Koji Nonaka 3, Ryuji Sakamoto 4, Takafumi Saito 5, Wataru Isobe 6, Shuzo Kumagai 7
PMCID: PMC7219619  PMID: 32052433

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

Age‐related regression in the tibial nerve and capillary was investigated. In young, middle‐aged, and old rats, myelinated fiber (MF) and capillary parameters were compared. MFs and capillaries started to regress in old and middle‐aged rats, respectively. Preservation of capillaries in the adult age may prevent subsequent MF regression.

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1. INTRODUCTION

Morphological and functional regressions of peripheral nerves have been associated with aging processes (Ceballos et al., 1999; Verdu et al., 2000; Tamaki et al., 2014; Pannerec et al., 2016; Sakita et al., 2016). Age‐related neural dysfunctions are caused by structural, biochemical, and molecular biological alterations in neurons and their surrounding tissue (Ceballos et al., 1999; Verdu et al., 2000; Shokouhi et al., 2008; Shen et al., 2011; Takagishi et al., 2016; Krause Neto et al., 2017). The decline in balance performance and consecutive falls in the elderly are associated with the regressive loss of skeletal muscle mass and atrophy (sarcopenia) (Cruz‐Jentoft et al., 2010; Landi et al., 2012). A previous study has revealed that age‐associated sarcopenia is accompanied by regression of myelinated fibers (MFs) (Verdu et al., 2000).

Morphological and histological evidence in peripheral nerves demonstrate that MFs of both proximal (sciatic nerve) (Ceballos et al., 1999) and distal (tibial or sural nerve) (Jeronimo et al., 2005; Sakita et al., 2016) sites show age‐related progressive degeneration and dysfunction. This is evidenced by several rodent and human studies establishing that the number, size, and density of MFs in the spinal cord and dorsal root ganglia decrease with age (Bergman and Ulfhake, 1998; Fogarty et al., 2018). Furthermore, a recent animal study revealed that during aging, peripheral nerve regression precedes sarcopenia onset (Liu et al., 2013). Based on these findings, we suggest that balance instabilities and the tendency to falls in the elderly are affected not only by sarcopenia but also by age‐related MF regression in the peripheral nervous system.

Similar to MFs, peripheral nerve capillaries also regress with age (Sakita et al., 2016). This regression is characterized by morphological changes such as decreases in both capillary diameter and number of microvascular branch points (Sakita et al., 2014; 2016). Morphological and structural alterations of capillaries that supply glucose and oxygen to MFs for their energy metabolism may lead to neurodegeneration (Verdu et al., 2000; Girach and Vignati, 2006). Additionally, reduction or abnormalities in the cerebral microvasculature precede age‐related neural regression (Brown et al., 2009). Thus, we suggest that preservation of capillary structure and function can potentially prevent age‐related MF regression. In fact, age‐related reductions in the levels of glucose and oxygen provided from microvessels for the required neural oxidative glycolysis are evoked by a decreased blood flow in the peripheral nerves of rats (Low et al., 1986). Thus, in peripheral nerves, reduction of capillaries may emerge prior to age‐related MF regression.

Studies have rarely investigated the simultaneous age‐related regression of MFs and reduction of capillaries in peripheral nerves. Previous studies demonstrated that fiber diameter, myelin thickness, and axon diameter of MFs in the tibial nerve of aged rats (Sakita et al., 2016), as well as capillary diameter and number of microvascular branch points (Sakita et al., 2014; 2016), are notably decreased compared with those of young rats. However, it is not clear whether the age‐related reduction of capillaries in peripheral nerves precedes that of MFs, since these studies compared only young and old animals. If capillaries reduce earlier than MFs, morphological signs may appear at an intermediate timepoint.

Thus, we compared age‐related morphological and histological changes of MFs and capillaries in peripheral nerves from groups of young, middle‐aged, and old rats. With this study, we can identify whether, if at all, capillaries reduce or MFs regress earlier.

2. MATERIALS AND METHODS

2.1. Animals

Twenty‐one male Wistar/ST rats (Japan SLC) were acquired at 10 weeks of age (mean body mass ± standard error of the mean: 328.62 ± 1.50 g). Each rat was assigned a letter from A to U and identified. Thereafter, seven rats were selected at 20 weeks (young group) and 70 weeks (middle group) using a random number generator according to the following procedure: a random number was generated for each rat, sorted in numerical order, and the first through seventh selected at each week of age. After 70 weeks of age, the remaining seven animals were reared until age 97 weeks. Rats were housed under a 12‐hr light/dark cycle (lights on at 07:00 hr) at a temperature of 22 ± 2°C with 40–60% humidity. Access to food and water was ad libitum. The present study was approved by the Animal Care and Use Committee of Kyoto Tachibana University (approval no. 16‐07) and was performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (National Research Council, 1996).

2.2. Nerve sample preparation

Rats were intraperitoneally injected with pentobarbital sodium (15 mg/kg) and heparin (10 000 IU/L) and were subsequently anesthetized by isoflurane inhalation (2.0%). The nerve segment between the sciatic L4‐L5 anastomosis and the distal side of the tibial nerve that branches into the medial and lateral plantar nerves was removed from the right hind limb. Following whole‐body perfusion with .9% physiological saline at 37°C, a contrast medium solution consisting of 10% glucose, 1% fluorescent PUSR‐80 (Mitsubishi Pencil), and 8% gelatin was administered at 37°C to fill the vasculature of the left hind limb (Sakita et al., 2014; 2016). Subsequently, rats were euthanized (pentobarbital sodium, 100 mg/kg) and the left hind limb was immediately immersed in cold saline for 20 min before the segment of the tibial nerve was removed as described for the right hind limb. Both nerve samples were immediately frozen in liquid nitrogen and stored at −80°C for further processing.

2.3. Histochemical analysis procedures

2.3.1. Tissue fixation and paraffin embedding

A 5‐mm segment from the distal part of each frozen nerve was prepared as described previously (Di Scipio et al., 2008). Nerve samples were fixed in 4% paraformaldehyde phosphate buffer at pH 7.4 for 3 hr, washed, and stored in .1 m phosphate‐buffered saline containing .2% glycine. On the day of the paraffin embedding, specimens were washed in phosphate‐buffered saline for 3 min and immersed in phosphate‐buffered saline containing 2% osmium tetroxide for 2 hr. After the dehydration process using ethanol, specimens were embedded in paraffin overnight at 60°C.

2.3.2. Paraffin section preparation

Paraffin‐embedded specimens were solidified at room temperature (19–21°C) for 1 day, and then about 10 serial transverse 1.5‐μm semi‐thin sections were sliced using an ultramicrotome (Reichert Ultracut S; Leica Biosystems). These sections were deparaffinized with 100% xylene and subjected to 100%, 90%, and 80% ethanol.

2.4. Staining procedures and observation of myelinated fibers

Deparaffinized sections were immersed in .5% Sudan Black B in 70% ethanol for 30 s. Thereafter, sections were briefly immersed several times in 70% ethanol. To remove the ethanol, sections were immersed in 40%, 30%, and 20% ethanol, and finally in distilled water. After that, the glycerin‐enclosed sections were secured with a coverslip.

Bright‐field images of Sudan Black‐stained sections were acquired using a BX‐53 microscope (Olympus) with a 40× objective, and regions of interest (ROIs) were visualized using a complementary metal‐oxide semiconductor camera (EOS kiss X7i; Canon) via a 5× lens (PE × 5; Olympus). Captured images (145 × 220 μm) were transferred to a computer.

2.5. Quantification of myelinated fibers

About 10 serial sections per specimen were equally aligned on glass slides. There were some sections with staining failure. Sections other than those that failed to stain were arranged from partially to entirely clear staining sections. A few random sections from these stained sections were selected for the quantitative analysis of MFs as determined using a random number generator according to the following procedure: a random number was generated for each section, sorted in numerical order, and the first few sections were selected. From a few sections, the clearly stained ROI was divided into 6–10 areas per specimen and numbered while laterally shifting the specimen without overlapping regions. Approximately six ROIs were then selected using a random number generator and captured. The selected six ROIs were again numbered, and either three or four of the numbered ROIs were selected using the random number generator. The following parameters were assessed in 40–60 MFs per selected image: fiber diameter, axon diameter, myelin thickness, and G‐ratio as described by Jeronimo et al., (2005) and Krause Neto et al., (2017). These parameters were measured using the imagej software (version 1.48; National Institutes of Health) by an observer blinded to the experimental conditions (Chen et al., 2014; Sakita et al., 2016). First, myelin and axon perimeters were measured by tracing the outer edge of the myelin sheath and axon, respectively. Second, the fiber and axon diameters were respectively derived from myelin perimeter/π and axon perimeter/π, assuming that both structures were perfect circles (Sakita et al., 2018). Third, the myelin thickness and G‐ratio were calculated as (fiber diameter – axon diameter)/2 and axon diameter/fiber diameter, respectively. All parameters were collected for 1350 data points per experimental group.

2.6. Sample preparation to observe the capillary architecture

Specimens of the left tibial nerve perfused with the fluorescent contrast medium were cut 5 mm from the distal side at −20°C. These samples were immediately fixed in 4% paraformaldehyde phosphate buffer at pH 7.4 and embedded using Tissue‐Tek O.C.T. Compound (Sakura Finetek, Tokyo, Japan). The embedded samples were frozen in liquid nitrogen‐precooled isopentane. The exact capillary diameter cannot be directly determined in transverse sections because peripheral nerve capillaries are rarely parallel to nerve fibers (Sakita et al., 2014; 2016). Hence, the frozen samples were sliced into 80‐μm‐thick sagittal sections using a microtome (CM‐3050; Leica Biosystems, Mannheim, Germany). Thereafter, they were thawed at room temperature (19–21°C) for about 10 min.

2.7. Quantification of the capillary architecture

The three‐dimensional (3‐D) architecture of tibial nerve capillaries was captured using a confocal laser microscope (TCS‐SP; Leica Biosystems, Mannheim, Germany) with a 20× objective lens in fluorescein isothiocyanate mode using a 488‐nm argon laser (Sakita et al., 2014; 2016). Each ROI (800 × 800 μm) was scanned up to a depth of 50 μm in 1‐μm steps, and 50 images were stacked into a 3‐D image. Luminal diameters of capillaries were defined to be less than 10 μm (Hall et al., 2014; Zong et al., 2017). ROI images were captured while laterally shifting the specimen without overlapping regions and three to four ROI images per specimen were collected (Tata & Anderson, 2002; Sakita et al., 2016; 2018). Age‐related capillary regression is indicated by a reduction in diameter and the number of microvascular branch points (Sakita et al., 2014; 2016; 2018). Therefore, 15 equally spaced horizontal lines were drawn on the ROI, and then the luminal diameter at the intersectional sites of the horizontal line and the fluorescent luminal vessels was measured using imagej software (Figure 1a). Only diameters of < 10 μm were renumbered, and about 30–60 capillary luminal diameters per ROI image were selected using a random number generator. To measure the diameter, the luminal borders of randomly selected capillaries were traced, and the longer side of the rectangular shape (maximum Feret diameter; Figure 1a,b) was determined as the luminal diameter using imagej software (Sakita et al., 2018). Additionally, microvascular branches were counted using imagej software as all visible portions in the same images.

Figure 1.

Figure 1

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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

2.8. Statistical analysis

One‐way analysis of variance (ANOVA) assessed the effect of the age group (young/middle/old) on fiber diameter, axon diameter, myelin thickness, G‐ratio, capillary diameter, and number of microvascular branch points, and the effect size (η2) was calculated. The effect size (η2) was evaluated according to criteria described by Cohen (Cohen, 1988) which is defined as small (η 2 = .01), medium (η 2 = .06), and large (η 2 = .14). Tukey’s honestly significant difference (Tukey’s HSD) post hoc test and effect size (Cohen’s d) were used when a result in a one‐way ANOVA presented a significant main effect. Tukey’s HSD post hoc test is one of the multiple comparison tests that is performed after one‐way ANOVA and is an all combination comparison between two groups. Cohen’s d represents an index of the difference between the two groups that does not depend on the number of samples; regarding the effect size criteria of Cohen’s d, .2, .5, and .8 are defined as small, medium, and large effect sizes, respectively (Chen et al., 2010). This indicates that when the mean difference between the two groups does not differ by more than .2 standard deviations, the difference is slight, even if that difference is statistically significant in Tukey’s HSD post hoc test. All statistical analyses were conducted using IBM SPSS Statistics (version 24.0; Tokyo, Japan), and results having both p‐values < .05 and effect size greater than the median (η2 ≥ .06, Cohen’s d ≥ .5) were considered statistically significant.

3. RESULTS

3.1. Cross‐sectional images of tibial nerves stained with Sudan Black B

The visual evaluation of cross‐sectional images from tibial nerves of the young, middle, and old groups revealed that the myelin sheaths and axons of the MFs in the middle groups (Figure 2b) maintained the regularity seen in the young group (Figure 2a), whereas those in the old group were markedly altered, with many irregularities due to degeneration (Figure 2c).

Figure 2.

Figure 2

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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

3.2. Distribution of MF diameters in young, middle, and old groups

Regarding the relative distributions of the parameter fiber diameter in MFs of the young, middle, and old groups, median and skewness values of the young (Figure 2d), middle (Figure 2e), and old (Figure 2f) groups were 9.44 and .03, 8.85 and .04, and 7.58 and .33, respectively. While the left shift from the young to the middle group was only slight (difference between the medians: .59), the shift from the middle to the old group was notably larger (difference between the medians: 1.27). That is, MFs in the tibial nerves exhibited marked regression with aging between the middle and the old groups.

3.3. Morphometric MF characteristics among young, middle, and old groups

Significant main effects and effect sizes greater than the median with regard to the age group were confirmed for fiber diameter, axon diameter, and myelin thickness (all p < .001, all η2 ≥ .06) in contrast to the G‐ratio (p = .98, η2 = .00; Table 1).

Table 1.

Morphometric measurement features of myelinated fibers among the young (20 weeks), middle‐aged (70 weeks), and old (97 weeks) groups

Morphometric parameters Group F p in ANOVA Effect size in ANOVA (η2) Effect size in post hoc test (Cohen’s d)
Young Middle‐aged Old Total
(n = 1,350) (n = 1,350) (n = 1,350) (n = 4,050)
Fiber diameter (μm) 9.39 ± 2.42* 8.94 ± 2.49** 7.62 ± 2.46*** 8.65 ± 2.57 F (2, 4,047) 188.33 <.001 .09 .19*
.53**
.72***
Axon diameter (μm) 6.47 ± 1.96* 6.18 ± 2.05** 5.27 ± 2.03*** 5.97 ± 2.08 F (2, 4,047) 131.14 <.001 .06 .15*
.50**
.62***
Myelin thickness (μm) 1.46 ± .34* 1.38 ± .36** 1.18 ± .39*** 1.34 ± .38 F (2, 4,047) 218.57 <.001 .1 .24*
.54**
.78***
G‐ratio .68 ± .06 .68 ± .07 .68 ± .08 .68 ± .07 F (2, 4,047) .024 .98 0 –
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Each morphometric parameter value of the young, middle‐aged, and old groups is represented as the mean ± standard error.

F, F‐values (degrees of freedom) of each morphometric factor with respect to the groups. η2, effect size in the one‐way ANOVA. Cohen’s d, effect size in Tukey’s HSD post hoc test.

*

Significant difference between the young and middle‐aged groups with Tukey’s honestly significant difference (Tukey’s HSD) post hoc test (p < .01).

**

Significant difference between the middle‐aged and old groups with Tukey’s HSD post hoc test (p < .01).

***

Significant difference between the young and old groups with Tukey’s HSD post hoc test (p < .01).

Post hoc analyses revealed that the mean fiber diameter, axon diameter, and myelin thickness of the old group were significantly lower than those of the young and middle groups (both p < .01). These parameters were significantly lower in the middle group than in the young group (all p < .01) with an extremely small effect size (Table 1, Figure 2g–i). The post hoc analyses of the G‐ratio revealed no differences between any two groups (Table 1, Figure 2j).

3.4. 3‐D images of the capillary architecture in the tibial nerve

The visual evaluation of the stacked 3‐D fluorescence images of the capillary lumen confirmed that tibial nerve capillaries of young rats indicated a noticeably dense network of microvascular branches (Figure 3a). By contrast, this characteristic was remarkably lacking in middle‐aged and old rats (Figure 3b,c, respectively).

Figure 3.

Figure 3

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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

3.5. Distribution of capillary luminal diameter in the young, middle, and old groups

We also analyzed the distribution of the capillary diameters in the young (Figure 3d), middle (Figure 3e), and old (Figure 3f) groups. The distribution of this parameter was shifted to smaller values with progressing age from young (median: 6.82; skewness: −0.61) to middle (median: 5.18; skewness: 0.10), and old (median: 3.38; skewness: 0.88) animals.

3.6. Age‐dependent changes of capillary luminal diameter and number of microvascular branch points

The one‐way ANOVA of the capillary diameter with respect to the age group (young/middle/old) verified a significant main effect (p < .001) with a notably large effect size (η2 = .26). Subsequent post hoc tests demonstrated that the mean capillary diameters of the old‐ (3.87 ± .06 μm) and middle (5.30 ± .09 μm) groups were significantly lower than those of the middle and young (6.64 ± .08 μm) groups (both p < .01), respectively, both with medium effect sizes (Table 2, Figure 3g).

Table 2.

Morphometric characteristics of capillary diameter and number of microvascular branch points in tibial nerves of the young, middle‐aged, and old groups

Morphometric parameters Group F p in ANOVA Effect size in ANOVA (η2) Effect size in post hoc test (Cohen’s d)
Young (20 weeks) Middle‐aged (70 weeks) Old (97 weeks) Total
Capillary diameter (μm) 6.64 ± .08* 5.30 ± .09** 3.87 ± .08*** 5.28 ± .06 F (2, 1593) 277.61 <.001 .26 .69*
(n = 537) (n = 531) (n = 528) (n = 1596) .74**
1.48***
Number of microvascular branch points 46.05 ± 11.69* 31.09 ± 12.95 28.27 ± 14.1*** 35.07 ± 1.83 F (2, 64) 12.04 <.001 .27 1.21*
(n = 22) (n = 23) (n = 22) (n = 67) 1.38***
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Each morphometric parameter value of the young, middle‐aged, and old groups is shown as the mean ± standard error. F, F‐values (degrees of freedom) of each morphometric factor with respect to the groups. η2, effect size in the one‐way ANOVA. Cohen’s d, effect size in Tukey’s HSD post hoc test.

*

Significant difference between the young and middle‐aged groups with Tukey’s honestly significant difference (Tukey’s HSD) post hoc test (p < .01).

**

Significant difference between the middle‐aged and old groups with Tukey’s HSD post hoc test (p < .01).

***

Significant difference between the young and old groups with Tukey’s HSD post hoc test (p < .01).

The one‐way ANOVA regarding the number of microvascular branch points revealed a significant main effect for age groups (p < .001), with notably large effect size (η2 = .27). Subsequent post hoc tests showed that the mean microvascular branch numbers in the old (28.27 ± 14.06) and middle (31.09 ± 12.95) groups were significantly lower than that in the younger (46.05 ± 11.69) group (both p < .01), with large effect sizes (Table 2, Figure 3h).

4. DISCUSSION

The aim of the present study was to investigate age‐related morphological alterations of MFs and capillaries in the tibial nerve of young‐, middle‐, and old‐aged rats to clarify which of these occurs earlier between MF regression or capillary reduction.

The fiber diameter, axon diameter, and myelin thickness of the MFs in tibial nerves were only slightly smaller in middle‐aged rats (if at all) in comparison with young animals. By contrast, rats of the old group exhibited markedly reduced values with respect to rats of the middle group. Previous longitudinal studies (Ceballos et al., 1999; Verdu et al., 2000) using sciatic nerves of mice between 24 and 104 weeks of age, have shown that the MF morphology is stable until week 48, and indicated only mild changes between weeks 48 and 80. However, from week 80, MFs show a marked fiber loss, decrease in size, and increase in irregular forms (Ceballos et al., 1999; Verdu et al., 2000). In our study, rats of the middle‐ (70 weeks) and old‐aged groups (97 weeks) exhibited similar characteristics; additionally, MFs of the sciatic nerve as well as those of the tibial nerve exhibited no or mild regression in the middle group but remarkable regression in the old group. Thus, age‐related regression of peripheral nerves does not appear to be site‐specific, and MFs throughout the peripheral nervous system may uniformly regress.

Age‐related changes in fiber diameter, axon diameter, and myelin thickness seem to reflect regression processes that develop in parallel (Bergman and Ulfhake, 1998; Sakita et al., 2016). Previous studies demonstrated that the age‐related regression of myelin sheaths and axons in peripheral nerves is associated with failures in the axonal transport of neurofilaments, coexisting with the degeneration of the cytoskeletal framework, as well as the retention and delayed transport of various proteins involved in the composition of axons and myelin sheaths (Bergman and Ulfhake, 1998; Uchida et al., 2001). This impairment of axonal protein transport in aged rodents was particularly induced in MFs at proximal peripheral nerves (Bergman and Ulfhake, 1998; Uchida et al., 2001). According to these findings, if neurofilament regression becomes prominent at very old age, regression of both axons and myelin sheaths might be noticeable in old animals in our study.

Furthermore, previous studies have revealed that oxidative stress or free radicals may be associated with age‐related neuronal regression and death (Wickens, 2001; Thrasivoulou et al., 2006). Concerning spinal neurons, proteasome activity that protects proteins, DNA, and lipids against oxidative stress was markedly decreased in rats after middle age and mediated motor neuron death (Keller et al., 2000). Additionally, model mice with increased oxidative stress showed reduction of motor function by 32 weeks, and the structural deficits in axons, myelin, and the cell body of motor neurons by 80 weeks were similar to the defects associated with normal aging in wild‐type mice at 120 weeks (Sims‐Robinson et al., 2013). Presumably, the reduction in the activity of neuroprotective factors such as proteasomes, and the increase in oxidative stress are involved in the regression of spinal neurons after the middle age; hence, there is a possibility that the regression also affects MFs in peripheral nerves.

Myelinated fibers were visually observed as more irregular shapes in cross‐sectional images of the tibial nerve of old rats compared with those of young and middle‐aged rats. The circularity of MFs was also dependent on the axonal morphology (Verdu et al., 2000), and until 88 weeks of age, the number of irregularly shaped MFs of all sizes increased by 50% in rats (Ceballos et al., 1999). Furthermore, irregular shape alterations of the axons that are of a less circular profile in neurons have been related to a reduction in neurofilament mRNA expression with aging (Parhad et al., 1995), and irregularly shaped MFs are common features of axonal atrophy, along with myelin sheath abnormalities and separation of myelin loops and lamella (Krinke et al., 1988). Additionally, rnRNA expression levels of neurofilaments in the dorsal root ganglia of old rats (> 92 weeks) decreased by approximately 50% compared with those of young (12 weeks) and mature (48 weeks) rats. Morphometric analysis also showed a 50% decrease in neurofilament numbers within the axons of aged rats; this was accompanied by a decrease in cross‐sectional area and circularity of all MFs (Parhad et al., 1995). On the basis of these findings, we speculate that many irregular MFs observed in the old rats in this study could be caused by the progressive failure of neurofilament preservation after middle age; consequently, its functions of providing cytoskeletal framework and protein transport are lost (Uchida et al., 2001).

In this study, the axon diameter and myelin sheath thickness were decreased in the old group. This age‐related decline of myelin thickness may be closely related to expression defect of several myelin‐associated proteins, as revealed by previous investigations comparing young and old rats (Verdu et al., 2000; Shen et al., 2011; Fogarty et al., 2018). The internodal retractions of myelin sheaths in older nerves leave a long exposed axonal segment. The local retractions can be compensated by extension of surviving internodes or by forming new short inserted internodes, producing re‐myelination with the arrival of new Schwann cells (Chase et al., 1992; Ceballos et al., 1999; Gomez‐Sanchez et al., 2017). Compensatory elongation of the myelin sheath in these findings, might have led to the decreased myelin thickness of the old group in this study. Regarding motor neurons, both physiological and histological alterations in the neuromusculature with aging have been reported as potential risk factors for sarcopenia (Punga and Ruegg, 2012; Gonzalez‐Freire et al., 2014). In rodent studies, it has been clarified that the neuromuscular junction is easily fatigued and the number of motor units decreased from middle age prior to old age when the signs of sarcopenia are confirmed (Barns et al., 2014; Tamaki et al., 2014; Pannerec et al., 2016). Considering these findings, it is possible that along with the atrophy and deformation of MFs, the number of neurons was also reduced in old rats in this study.

The metabolism of nerve fibers in peripheral nerves depends on cytoplasmic glycolysis and mitochondrial oxidative phosphorylation that generate adenosine triphosphate (ATP) and creatine phosphate as energy sources required for axonal transport processes (Verdu et al., 2000). The decline in endoneurial ATP and creatine phosphate levels appears to be progressively caused by aging (Low et al., 1986). These findings suggest that dysfunction of energy production in peripheral nerve fibers contributes to the regression of axons and myelin sheaths. Furthermore, preservation and repair of nerve fibers and blood vessels are facilitated by their interaction (Kerschensteiner et al., 2004; Muramatsu et al., 2012; Sakita et al., 2018). However, the results of the current study demonstrate that in contrast to MFs, the middle‐aged microvasculature remarkably exhibited signs of reduction. Although there may be compensatory effects, including axonal transport mechanisms, accounting for the lack of evident MF regression in middle‐aged rats.

Our results also confirmed that there were no noticeable differences in G‐ratios among age groups. The G‐ratio is a quantitative index representing the relative axonal myelin thickness with proven reliability in peripheral nerves. The G‐ratio values in rat tibial nerves range from 0.68 to 0.76 (Chomiak and Hu, 2009; Sakita et al., 2018). In a longitudinal study in mice, the G‐ratios of MFs in sciatic nerves exhibited an unchanged mean value of 0.67 between the age of 24 and 132 weeks (Ceballos et al., 1999). In our study in rats, the mean G‐ratios in the three groups all had values of 0.68, similar to the findings in mice. Thus, the G‐ratio in the tibial nerve seems to be less susceptible to aging, suggesting that both axon diameter and myelin thickness exhibit similar age‐related reduction rates among young, middle, and old animals.

It has been clarified that most peripheral nerve capillaries do not parallel nerve fibers but are tortuous (Sakita et al., 2014; 2016; 2018). A recent study established a method to analyze luminal capillary diameters and numbers of microvascular branch points in confocal 3‐D images with reproducible results (Sakita et al., 2018). Based on this approach, we measured the capillary diameters and microvascular branch numbers in tibial nerves and found that the capillary diameter substantially decreased with age in all three groups. Conversely, a noticeable decrease in the number of microvascular branch points was only observed between young and middle‐aged rats, but no further substantial reduction was detected between the middle and old groups. This indicates that capillaries were reduced after the middle group, although myelin thickness and axon diameter of MFs remarkably decreased in the old group. The mechanisms underlying this phenomenon, that the reduction of capillaries precedes that of MFs in peripheral nerves, remain unclear. However, it has been shown that apoptosis of the cerebral microvasculature precedes neuronal loss in the deep white matter (Yamauchi et al., 1991; Brown et al., 2009). Therefore, it is possible that capillary apoptosis may precede regression of MFs even in the peripheral nerve. In microvascular pathology such as diabetic neuropathy, axon and myelin regression results from microvascular disease that causes ischemia and/or hypoxia, and microangiopathy precedes the progression of diabetic neuropathy (Giannini and Dyck, 1995; Thrainsdottir et al., 2003). Additionally, the microvascular abnormality complicates or precedes nerve regression in cerebral microvascular pathology (van Dijk et al., 2008; Brown et al., 2009). Nerve damage does not always result from conditions with limited blood supply due to microvascular changes, but from endoneurial hypoxia with inefficient oxygen extravasation (Ostergaard et al., 2015; Gonçalves et al., 2017) and reduction of glucose supply from microvessels (Girach and Vignati, 2006). The rats used in this study were sedentary without regular exercise habits, so it was possible that oxygen extravasation from the microvasculature to MFs may have become gradually inefficient with this chronic physical inactivity during aging. Another study showed that brain‐derived neurotrophic factors (BDNFs) that promote the repair and preservation of myelin sheaths and axons remain in the blood, and extravasation of BDNFs from capillaries to nerves reduce when there were no regular exercise habits (Nofuji et al., 2008). Furthermore, an in vitro study demonstrated that the mRNA of neuronal vascular endothelial growth factor (VEGF) (growth factors of endothelial cells essential in capillary sprouting and differentiation) and protein levels increased following BDNF exposure of neural cells (Nakamura et al., 2006). Additionally, the apoptotic cascade of capillaries can be induced when regression or loss of VEGF occurs (LaManna et al., 2004). Hence, these findings suggest that at the initial stage, extravasation oxygen and BDNF from capillaries to peripheral nerves is reduced by chronic physical inactivity, and thereafter regression and apoptosis of capillary is promoted through down‐regulation of neuronal VEGF expression.

Reactive oxygen species (ROS) and free radicals are other factors promoting the apoptosis cascade of vascular endothelial cells. Consequently, increases in ROS and free radicals are associated with the onset of endothelial cell death (Rajendran et al., 2013, Maiuolo et al., 2019). Additionally, a mice study revealed that physical inactivity enhanced vascular ROS production, which contributes to endothelial dysfunction in sedentary as opposed to a physically active lifestyle (Laufs et al., 2005). In fact, middle‐aged sedentary men have markedly high‐risk factors of metabolic syndrome that is a representative indicator of vascular dysfunction (Carroll et al., 2000). Thus, in middle‐aged rats in this study, increased vascular endothelial oxidative stress combined with chronic physical inactivity may cause capillary regression.

The present study has several limitations. First, we defined only three age groups, (young, middle, and old); however, the lifetime morphological changes of MFs and capillaries in peripheral nerves should be carefully observed at more timepoints. Therefore, further studies are required to examine the progressive morphological changes of MFs and capillaries in the tibial nerve at multiple timepoints, and to assay the level of age‐related proteins participating in the preservation, repair, and regression of MFs and capillaries. Furthermore, additional studies should be conducted to biochemically and immunohistochemically investigate increases in oxidative stress and activities of apoptosis signal pathways associated with aging of MFs and capillaries.

5. CONCLUSIONS

In this study, the tibial nerves of young, middle‐aged, and old rats were morphologically and histologically analyzed to clarify if either MFs or capillaries, regressed earlier. Our findings indicated that capillary diameter and microvascular branch points were markedly declined already in middle‐aged rats, whereas MFs showed signs of regression in the old age group. This marked decline of capillaries in middle‐aged animals may result from early increase in oxidative stress and apoptosis cascade activity following chronic hypoxia with physical inactivity. Consequently, it may be a potential suggestion that MF regression occurs gradually.

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

AUTHOR CONTRIBUTIONS

Masahiro Sakita and Shinichiro Murakami contributed to study design and conception. Material preparation was performed by Masahiro Sakita and Shinichiro Murakami. Masahiro Sakita, Shinichiro Murakami, Koji Nonaka, Ryuji Sakamoto, Takafumi Saito, and Wataru Isobe conducted the experiments and collected data. The analysis was performed by all authors. Shuzo Kumagai participated in the sequence alignment and coordination of this study. The first draft of the manuscript was written by Masahiro Sakita. All authors read and approved the final manuscript.

ACKNOWLEDGEMENTS

We would like to thank Editage (http://www.editage.jp/) for English language editing.

Sakita M, Murakami S, Nonaka K, et al. Different patterns in age‐related morphometric alteration of myelinated fibers and capillaries of the tibial nerve: a longitudinal study in normal rats. J. Anat. 2020;236:1101–1111. 10.1111/joa.13168

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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