Quantitative histological study in the sural nerves of children

Quantitative Histological Study in the Sural Nerves of Children y oshihiro Origuchi, MD

Measurements have been made of the diameters of both myelinated and unmyelinated fibers in the sural nerves obtained at autopsy from 10 children without neurological disorders. The distributions of the myelinated fiber diameters were unimodal, except for two cases in which showed bimodal patterns as seen in adults. The second peak in bimodal histograms may appear around age 2 years. The densities of both myelinated and unmyelinated fibers were calculated in the same nerves. The fascicular areas of the same nerves were also measured. The densities of both myelinated and unmyelinated fibers, and the estimated numbers of both myelinated and unmyelinated fibers showed a tendency toward decrease, but the fascicular areas did a tendency to increase with aging. Although a definite proposal regarding the correlation cannot be made at present, it is interesting that the ratio of unmyelinated to myelinated fibers decreases with age. This tendency may be related to development of myelination. Origuchi Y. Quantitative histological study in the sural nerves of children. Brain Dev 1981;3:395-402

Biopsy of the sural nerve is often done to aid in the diagnosis of various neurological disorders, particularly those related to peripheral nerve diseases. As indicators of pathological processes, changes in the number and diameter of both myelinated and unmyelinated nerve fibers are sought in quantitative histological studies of the nerve fibers. However, studies on normal controls for a comparison are inadequate in the pediatric age range. The available quantitative studies [1-6] have been mainly on materials from adults. The present work was

From the Department of Pediatrics, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto. Received for pUblication: December 27,1980. Accepted for pUblication: August 5, 1981.

Key words: Sural nerves, age changes, nerve fiber diameter, nerve fiber density, quantitative study, electron microscopy. Correspondence address: Dr. Yoshihiro Origuchi, Department of Pediatrics, Nishibeppu National Hospital, 4548 Tsurumi, Beppu, Oita 874, Japan.

undertaken to establish the variation with age of quantitative changes in the sural nerve.

Materials and Methods All sural nerves were obtained at autopsies. Age, sex and anatomic diagnosis of the patients are listed in Table 1. In no patient was there evidence of neurological disease and all samples were obtained within eight hours of death. The nerves were excised about 2-3 cm at the site of usual nerve biopsy, that is, a few cm higher from and behind the lateral malleolus. The nerves were fixed in a solution of 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 20-30 minutes at room temperature, then washed in 0.1 M cacodylate buffer over night and for one day, and post-fixed for two hours in 2% solution of osmium tetroxide in 0.1 M cacodylate buffer at pH 7.4. After dehydration through an ascending series of alcohol and propylene oxide, the specimens were embedded in Bpon 812 by conventional methods. Transverse sections of 1 p.m thick were cut with glass knives on a Reichert ultra-

Table I Clinical summary Case Sex

Age

Clinical and anatomical diagnosis

2 M 3 M

4 days Hypoplastic left heart syndrome 2mos VSD*, TGA** 3 mos Severe diarrhea, ulcerous en terocoli tis

4 M 5 F

4 mos 4 mos

1 F

6 F

4 mos 1 yr 7 M 6 mos 1 yr 8 M 11 mos

9 F 3 yrs 10 M 10 yrs

Vascular ring ASD***, large VSD, TGA VSD,ASD Drowned Hemorrhagic pneumonia Subdural hemorrhage Double outlet right ventricle, VSD

* Ventricular septal defect, ** Transposition of the great arteries, *** Atrial septal defect.

microtome, and stained with 0.1% toluidine blue and observed under a light microscope. The light microscopic photographs of 100 x magnification were taken using Polaroid 4 x 5 Land mm. Total areas of all fascicles were measured using Kontron quantitative digital image analyzer. Using an oil-immersion objective, photographs were taken at a magnification of 1,000 times using the same film. The minimum of the external diameter of the myelinated fibers was measured using the same analyzer. The diameter of more than 2,000 myelinated. fibers was measured in each case. Ultrathin sections were doubly stained with uranyl acetate and lead citrate, and examined under a Hitachi H-300 electron microscope. Ten or more electron microscopic photographs were taken at a magnification of 2,000 times from different regions, then enlarged to a fmal magnification of 10,000 times. Using the same analyzer as above-mentioned, the diameter of 500 or more unmyelinated fibers was measured and the fiber density was calculated by the number of both myelinated and unmyelinated fibers. The estimated number of total myelinated fibers in the fascicles was calculated by multiplying the total areas of fascicles (S) by the density of myelinated fibers per sq mm (M). In the same way, the total areas of fascicles (S)

396 Brain & Development, Vol 3, No 4,1981

multiplied by the density of unmyelinated fibers per sq. mm (U) represents the estimated number of total unmyelinated fibers. The sum of S x M and S x U is the estimated number of total nerve fibers in the sural nerve. Results Light microscopic photographs of transverse sections of sural nerves of some cases are shown at the same magnification in Fig 1. The distributions of diameters of both myelinated and unmyelinated fibers in the sural nerve in each case are shown in Figs 2 and 3. The calibrations in the myelinated fibers are 111m, and those in the unmyelinated are 0.1 11m. All nerves showed a peak of the diameter of the myelinated fibers at 2-3 11m. The mode of distribution of the diameters in the myelinated fibers, as seen in the histograms, was unimodal except for cases 9 and 10 which show a suggestion of a bimodal pattern. Electron microscopic photographs of transverse sections of sural nerves of some cases are shown at the same magnification in Fig 4. Most nerves showed a peak of the diameter of the unmyelinated fibers at 0.30.5 11m except for case 4 which has a peak at 0.2-0.3 11m. Density of both myelinated (M) and unmyelinated fibers (U) and total fascicular areas (S) are shown in Table 2. The estimated numbers of both myelinated and unmyelinated fibers were calculated by multiplying the fascicular areas by the numbers of respective density (Table 2). The total numbers of fibers were estimated as the sum of S x M and S x U. Discussion The peaks of the frequency curves for the diameters of myelinated fibers were 2-3 11m in all specimens. These observations coincide with the findings of Dyck et al [1] and Gutrecht and Dyck [4], but the values are less than those reported by O'Sullivan and Swallow [2], Ochoa and Mair [3], Tohgi et al [5] and Tackmann et al [6]. The discrepancy between the latter and the present data may be the result of the different methods of fixation and measuring. Tackmann et al [6] estimated the diameter of a certain fiber as the mean of the largest and smallest diameter of the fiber. The fiber has a cylindric shape in vivo. If the plane of the sec-

tion is not at a right angle to the fibers , the largest diameter of the fiber is too large [1] . O'Sullivan and Swallow [2] and Tohgi et al [5] measured the diameter in 5 J.Lm sections fixed for light microscopy. If the sections of this thickness are cut at an oblique angle, the same reasoning as the above-mentioned is applicable [1] . The discrepancy between the data reported by Ochoa and Mair [3] and that of the present paper may be related to differences in age, because all children in the present study

were under 10 years and all of their patients were over 15 years. Growth in diameter of the axons occurs during development of the human fetus [9] and may even continue after birth. On the other hand, the distributions of myelinated fiber diameters in all specimens were unimodal, except for two cases aged 3 years and 10 years. This contrasts with findings in the literature [1-6] , where bimodal patterns have been described except for the case of one newborn reported by Gutrecht and Dyck [4].

Fig 1 Transverse sections of sural nerves in cases 1 (lA). 3 (lB). 8 (1 C) . and 10 (lD). (0.1% toluidine· blue. x 800).

Origuchi: Histometry in the sural nerves 397

In the newborn, the histogram of myelinated fibers in the sural nerve is unimodal [10]. The second peaks in the bimodal histograms of the myelinated fiber diameters reported by many authors [1-6], were larger than 6 J-Lm. The second peak in bimodal histograms shifts to a larger size with advancing age [4]. If the sec-

ond peak depends on advancing age i.e., developing with myelination, the discrepancy in the data could be readily understood [1-6]. All patients in the reported cases of bimodal distribution [1-6] were older than the children in the present study. The youngest in the cases with bimodal histograms was 2 years 2 months,

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398 Brain & Development, Vol 3, No 4,1981

Case 10 8103

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Fig 2 Distribution of diameters of the myelinated nerve fibers in the sural nerves. 10 I'

al [13], there is a linear relationship between nerve conduction velocity and diameter of the myelinated nerve fiber. The conduction velocity in younger children is slightly slower than that of adult [14-17]. Minejima [15] and Dunn [16] reported that conduction velocity was close to the value of adults in children 2 years of age. The present data are insufficient for statistical analysis; however, there is a trend

in the report of Gutrecht and Dyck [4]. In the present study case 8 aged 1 year 11 months showed unimodal pattern, but cases 9 and 10, aged 3 years and 10 years respectively, did bimodal. Thus, the age at which a bimodal distribution becomes apparent is probably about 2 years. As shown by Gasser and Grundfest [11], Hursh [12], Tackmann et al [6], and Behse et

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Origuchi: Histometry in the sural nerves 399

in which the appearance of second peaks in distributions of myelinated fibers are related to the development of faster nerve conduction velocity in children. The mode of distribution of the diameter of unmyelinated fibers was unimodal, as has found by others [3, 18]. The peaks of size frequency in unmyelinated fibers in most cases were at 0.3-0.5 11m, but that in unmyelinated fibers of a healthy boy of 15 years, reported by Ochoa et al [3] , was at 1.4 11m. Further studies are required to elucidate the differences.

The densities of the myelinated fibers per sq. rum are similar to the findings of Gutrecht and Dyck [4] and those in children reported by Dyck et al [1] and Tackmann et al [6], but are higher than those in adults reported by Dyck et al [1], O'Sullivan and Swallow [2], Ochoa and Mair [3] and Tackmann et al [6]. There are apparently no reports on unmyelinated fiber densities of healthy human peripheral nerves, based on counts of electron microscope preparations, except for one case reported by Ochoa and Mair [3]. These authors reported that the

Fig 4 Electron microscopical photographs of sural nerves in the same cases as Fig 1. (case 1: 4A, case 3: 4n, case 8: 4c, case 10: 4D, x 3,200).

400 Brain & Development, Vol 3, No 4,1981

Table 2 Total fascicular areas, and density of both myelinated and unmyelinated fibers Total area of fascicles Case (mm2, S) 1 2 3 4 5 6 7 8 9 10

0.138 0.300 0.164 0.248 0.324 0.404 0.439 0.324 0.556 0.407

Density of myelinated fibers/mm 2 (M) 28,840 24,340 24,192 16,162 19,278 17,056 22,296 16,801 16,781 18,215

. Estimated Estimated Density of counts of counts of unmyelinated total mye· 2 total unmye· fibers/mm linated fibers lina ted fibers (U) (SU) (SM) 3,980 7,302 3,967 4,008 6,246 6,891 9,788 5,444 9,330 7,414

densities of unmyelinated fibers per sq. rnm were 21,755-33,859 in the age from 15 years to 32 years. These numbers are much lower than those of the present study. Estimated numbers of total nerve fibers were calculated in an attempt to clarify this discrepancy. Total areas of fascicles showed a tendency to increase in size with advancing age, in contrast to the density of both myelinated and unmyelinated fibers. This tendency was also noted by Gutrecht and Dyck [4] and Tackmann et al [6]. Thus, although the areas of fascicles increase in size with advancing age, the absolute numbers of fibers remain unchanged, independently of age [19]. However, the estimated numbers of both total nerve fibers (SM + SU) decreased with age (Table 2). The ratio of unmyelinated to myelinated fibers was also calculated and was higher than that obtained by Ochoa and Mair [3], except for two cases aged 3 and 10 years (cases 9 and 10). As compared with the data of Ochoa and Mair [3] there was a tendency toward decrease in the ratio with age, and it may probably be close to that of adults at about 2 years (Table 2). It can be deduced that the ratio decreases with age because immature axons devoid myelin decreases as a result of myelination and that myelinated fibers increase, although why the estimated numbers of both myelinated and unmyelinated fibers decrease remain to be ascertained. If higher values of the ratio indicate delay of myelination, then the values in cases 3 and 4 could be explained. Case 3 died with severe diarrhea, and he weighed 2,500 g at

235,868 192,014 209,057 253,199 134,052 116,904 101,975 102,153 59,143 80,318

32,550 57,604 34,285 62,793 43,433 47,229 44,767 33,098 32,884 32,689

Ratio U/M

Estimated counts of total fibers

8.19/1 7.89/1 8.64/1 15.67/1 6.95/1 6.85/1 4.57/1 6.08/1 3.52/1 4.41/1

36,530 64,906 38,252 66,801 49,679 54,120 54,555 38,542 42,214 40,103

death. Case 4 weighed 2,800 g though he was 4 months of age at death. The onset of myelination of peripheral nerves begins during fetal life [9, 20], but because of the poor growth the development of myelination may have been delayed in these two patients. Acknowledgments Gratitude is extended to Prof. I. Matsuda (Department of Pediatrics) and Associate Prof. M. Ohta (Department of Neuropathology, Kyushu University, Fukuoka) for encouragement and advice, and to M. Ohara for assistance with the manuscript. I am grateful to Dr. K. Ishibashi of Kumamoto Red Cross Hospital for making the specimens available.

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Origuchi: Histometry in the sural nerves 401

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