**2. Materials and methods**

The materials used in this study were Japanese oculomotor nerves, ophthalmic nerves, inferior alveolar nerves, abducent nerves, facial nerves, vestibular nerves, cochlear nerves, and vagus nerves, recurrent laryngeal nerves in the cranial nerve group; Japanese femoral nerves and tibial nerves in the spinal nerve group; and Japanese greater splanchnic nerves and lesser splanchnic nerves in the autonomic nerve group. All the cadavers were donated with the individual's consent. We proceeded to perform this research in accordance with the law concerning autopsy and preservation of corpses, and concerning donation for medical and dental education. In no case was there a history of peripheral nerve disorders such as neuroparalysis or schwannoma, or of treatment with toxic agents or irradiation therapy. The causes of death did not directly or indirectly influence the nervous system, so the peripheral nerves were considered to be normal. I used right side specimens of right-handed persons to avoid any interaction between the effects of sex and side. Moreover, the age of specimens showed no significant difference between female and male specimens. The data on the above pairs of groups were thus independent of the aging process. The methods for preparation of sections, also described in our previous report [1], were as follows:

© 2013 Moriyama, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2.1. Fixation**

The fixation involved a two-step process. For the first step, all the cadavers were fixed with a 10% solution of formalin (3.7% formaldehyde) within 24 h postmortem. After resecting the peripheral nerves, a 10% solution of formalin (3.7% formaldehyde) was used for immersion for at least a week. The solution was changed once in the first 30–60 min and again, later if desired.

The Relationship Between Sexually Dimorphic Peripheral Nerves and Diseases 127

6. Differentiate in 2% saturated lithium carbonate (= 0.03% Li2CO3) for 60 min

between myelin sheaths and surrounding structures.

15. Immerse sections for around 5 min in Mayer's hematoxylin solution

18. Dehydrate sections in *n*-butyl alcohol (three changes)

16. Wash sections in distilled water (several changes) until sections turn bluish.

8. Finish differentiation by rinsing in 95% ethanol

9. Wash in distilled water (two changes) 10. Oxidize for 5 min in 0.5% periodic acid 11. Wash in distilled water (several changes) 12. Immerse for 15 min in Schiff's reagent

changing the solution three times 14. Wash in distilled water (several changes)

19. Clear sections in xylene (three changes)

17. Rinse in 90–95% ethanol

20. Mount in balsam

**2.5. Morphometry** 

perimeter in mm).

allowing quantitative comparison of round figures.

7. Continue differentiation with one or two changes of 70% ethanol until myelin sheaths can be distinguished. If necessary, repeat steps 5 through 7 until there is sharp contrast

13. Immediately transfer to 5% sodium hydrogen sulfite and leave for about 5 min,

I observed the fascicles at low power (Fig. 1). I covered the entire area of the distributed myelinated axons in the peripheral nerve by moving the eyepiece grid vertically and horizontally. I confirmed that I could distinguish myelinated structures from vessels in the tissue with a computer or grouped unmyelinated axons with the naked eye in each grid. I counted the myelinated axons and measured the transverse area and perimeter of the myelinated axons in a square eyepiece grid at high power (Fig. 2). To avoid duplicate counts, I counted and measured all axons on the side of the grid that did not come into contact with the other grids. In the case of grids adjacent to the other grids, I counted and measured only the axons on the lower right side of the grid, not those on the upper left side. I used a microscope in transmitted light mode (BX50, Olympus, Tokyo, Japan) equipped with a high-resolution digital camera (ColorView12, Soft Imaging System, Münster, Germany), a motorized XYZ stage (Märzhäuser, Wetzlar-Steindorf, Germany), a stage controller (Märzhäuser, Wetzlar-Steindorf, Germany), and a computer (Precision 530, Dell, Austin, TX, USA) with analyzing system software (analySIS 3.0, Soft Imaging System, Münster, Germany) to store data on-line, do calculations, and perform statistical analyses. Circularity ratios (CR) were calculated as follows: CR = 4*π*A/L2 (A = area in mm2; L =

If a circle is regular, the ratio has a maximum value of 1.0, and if it is irregular, the value is less than 1. This indicates how near or far each irregularity is from a regular round figure,

The formalin-fixed materials were then transferred without washing to the secondary fixative (1:4 mixture of 5% K2Cr2O7 and 5% K2CrO4) and maintained at room temperature for 2 weeks. If the solution became turbid or precipitated it was changed. After this, the fixation was continued at 37°C for an additional week. The volume of fixative used was at least ten times the volume of the specimens.

#### **2.2. Washing**

The fixed materials were washed in running water for around 24 h. We used a siphonoperated automatic pipette washer with the materials packed in a small plastic basket.

### **2.3. Dehydration and celloidin embedding**


The alcohol in steps 1 and 2 was changed if it became yellow.


#### **2.4. Staining procedures**

Modified luxol fast blue-periodic acid Schiff-hematoxylin (LPH) triple stain.


desired.

**2.2. Washing** 

times the volume of the specimens.

1. 50% ethanol, for several days 2. 70% ethanol, for several days

5. Pure ethanol, one night or more 6. Ether/ethanol, 1:1 overnight

7. 1% celloidin in ether/ethanol, several days

9. 14% celloidin for embedding, several weeks

1. Cut sections 15 μm thick and place in 90% ethanol

4. Immerse in 95% ethanol and wash off excess stain

3. 90% ethanol, overnight 4. 95% ethanol, overnight

8. 7% celloidin, several days

**2.4. Staining procedures** 

5. Wash in distilled water

2. Rinse sections in 95% ethanol

**2.3. Dehydration and celloidin embedding** 

The alcohol in steps 1 and 2 was changed if it became yellow.

11. Maintained celloidin blocks in 70% ethanol prior to sectioning

Modified luxol fast blue-periodic acid Schiff-hematoxylin (LPH) triple stain.

**2.1. Fixation** 

The fixation involved a two-step process. For the first step, all the cadavers were fixed with a 10% solution of formalin (3.7% formaldehyde) within 24 h postmortem. After resecting the peripheral nerves, a 10% solution of formalin (3.7% formaldehyde) was used for immersion for at least a week. The solution was changed once in the first 30–60 min and again, later if

The formalin-fixed materials were then transferred without washing to the secondary fixative (1:4 mixture of 5% K2Cr2O7 and 5% K2CrO4) and maintained at room temperature for 2 weeks. If the solution became turbid or precipitated it was changed. After this, the fixation was continued at 37°C for an additional week. The volume of fixative used was at least ten

The fixed materials were washed in running water for around 24 h. We used a siphonoperated automatic pipette washer with the materials packed in a small plastic basket.

10. Immerse hardened celloidin embedded blocks in 90–95% ethanol for several hours

3. Keep at 58°C overnight in LFB solution (0.1% solution of luxol fast blue by dissolving 1.0 g of the substance in 1,000 ml of 95% ethanol) placed in a shallow sealed jar.

#### **2.5. Morphometry**

I observed the fascicles at low power (Fig. 1). I covered the entire area of the distributed myelinated axons in the peripheral nerve by moving the eyepiece grid vertically and horizontally. I confirmed that I could distinguish myelinated structures from vessels in the tissue with a computer or grouped unmyelinated axons with the naked eye in each grid. I counted the myelinated axons and measured the transverse area and perimeter of the myelinated axons in a square eyepiece grid at high power (Fig. 2). To avoid duplicate counts, I counted and measured all axons on the side of the grid that did not come into contact with the other grids. In the case of grids adjacent to the other grids, I counted and measured only the axons on the lower right side of the grid, not those on the upper left side. I used a microscope in transmitted light mode (BX50, Olympus, Tokyo, Japan) equipped with a high-resolution digital camera (ColorView12, Soft Imaging System, Münster, Germany), a motorized XYZ stage (Märzhäuser, Wetzlar-Steindorf, Germany), a stage controller (Märzhäuser, Wetzlar-Steindorf, Germany), and a computer (Precision 530, Dell, Austin, TX, USA) with analyzing system software (analySIS 3.0, Soft Imaging System, Münster, Germany) to store data on-line, do calculations, and perform statistical analyses. Circularity ratios (CR) were calculated as follows: CR = 4*π*A/L2 (A = area in mm2; L = perimeter in mm).

If a circle is regular, the ratio has a maximum value of 1.0, and if it is irregular, the value is less than 1. This indicates how near or far each irregularity is from a regular round figure, allowing quantitative comparison of round figures.

The Relationship Between Sexually Dimorphic Peripheral Nerves and Diseases 129

All statistical analyses were performed using JMP statistical software version 9.0.3 (SAS

Researchers have studied shrinkage of embedding materials, and found that celloidin and plastination embedding exhibit less shrinkage (around 10%) than paraffin and other embeddings [2]. Therefore, although I measured every myelinated axon, I calculated the average transverse area and perimeter of myelinated axons after excluding data far from the

Morphological differences between female and male specimens were analyzed by applying a parametric unpaired *t* test (where data were normally distributed with equal variance) to the total number of myelinated axons and the average transverse area, perimeter and CR. A

We estimated the total number or number per unit area of myelinated axons (NM) in the peripheral nerves (Table 1). The myelinated nerve fibers appeared as a blue-green myelin sheath surrounding a dark purple or black axon (Fig. 2). According to the data, there was no statistically significant difference in the total number of myelinated axons between the female and male specimens of the peripheral nerves, except for the vestibular nerve (*P* <

The average transverse area of myelinated axons (ATA) in the peripheral nerves was calculated (Table 1). According to the data, there was no statistically significant difference in the average transverse area of myelinated axons between the female and male specimens of

The average CR of myelinated axons (ACR) in the peripheral nerves was calculated (Table 1) and there was no statistically significant difference in the average CR of myelinated axons between the female and male specimens of the peripheral nerves, except for the vagus nerve

Researchers have reported that a decrease in the number and size of myelinated axons influences the occurrence of peripheral nerve palsy or neuropathy [3-6], and a lower CR of myelinated axons has been partly implicated in the degeneration of nerve fibers [7]. A

*p* value of <0.05 was considered to indicate a statistically significant difference.

Institute Inc. Cary, NC, USA) on a Macintosh personal computer.

**2.6. Statistical analyses** 

median (15%) due to shrinkage.

**3.1. Number of myelinated axons** 

**3.2. Average transverse area of myelinated axons** 

all calculated peripheral nerves (*P* < 0.05; Table 1).

**3.3. CR of myelinated axons** 

(*P* < 0.05; Table 1).

**4. Discussion** 

**3. Results** 

0.05; Table 1).

**Figure 1.** A low-power view of the inferior alveolar nerve from a 65-year-old woman, modified LPH stain. *Scale bar* 200 μm

**Figure 2.** A high-power view of the facial nerve from a 44-year-old woman, modified LPH stain. Axons were stained *dark purple or black*, and surrounded by a myelin sheath stained *deep green*. Scale bar 50 μm

#### **2.6. Statistical analyses**

128 Sexual Dimorphism

stain. *Scale bar* 200 μm

**Figure 1.** A low-power view of the inferior alveolar nerve from a 65-year-old woman, modified LPH

**Figure 2.** A high-power view of the facial nerve from a 44-year-old woman, modified LPH stain. Axons were stained *dark purple or black*, and surrounded by a myelin sheath stained *deep green*. Scale bar 50 μm

All statistical analyses were performed using JMP statistical software version 9.0.3 (SAS Institute Inc. Cary, NC, USA) on a Macintosh personal computer.

Researchers have studied shrinkage of embedding materials, and found that celloidin and plastination embedding exhibit less shrinkage (around 10%) than paraffin and other embeddings [2]. Therefore, although I measured every myelinated axon, I calculated the average transverse area and perimeter of myelinated axons after excluding data far from the median (15%) due to shrinkage.

Morphological differences between female and male specimens were analyzed by applying a parametric unpaired *t* test (where data were normally distributed with equal variance) to the total number of myelinated axons and the average transverse area, perimeter and CR. A *p* value of <0.05 was considered to indicate a statistically significant difference.
