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J Anat. Jan 2005; 206(1): 93–99.
PMCID: PMC1571453

Microscopic anatomy of the sural nerve in the postnatal developing rat: a longitudinal and lateral symmetry study

Abstract

Rat sural nerve is widely used in experimental studies investigating injury and regeneration of the peripheral nervous system. However, it has not yet been established whether morphological and morphometric parameters differ within corresponding levels of the rat sural nerve. The aims of the present study were to investigate the normal morphological and morphometric aspects of the sural nerve in postnatal developing female rats, with special attention to longitudinal morphology and lateral symmetry. Rats aged 30, 90 and 180 days were killed, and proximal and distal segments of the right and left sural nerves were prepared for light microscopy and morphometric study. No differences were found between the proximal and distal segments or between the right and left sides at the same levels. In addition, postnatal growth continuously and symmetrically affected the sural nerve fascicles and myelinated fibres. Fibre population distribution was also affected by increasing body weight; distribution was unimodal at 30 days, and by 180 days this distribution was established as bimodal. We concluded that the sural nerve is long and constant in its morphology and presents a continuous and symmetrical growth, more pronounced between 30 and 90 days of age, thus providing a good model for experimental neuropathies.

Keywords: lateral symmetry, light microscopy, longitudinal symmetry, morphology, morphometry, nerve maturation, rat, sural nerve

Introduction

Despite the wide utilization of the sural nerve in experiments involving injury and regeneration, studies on the normal pattern of development and postnatal maturation of this nerve in rats are scanty. Interestingly, most of the experimental studies on sural nerve morphology have been carried out on male rats (Nagamatsu et al. 1993; Unger et al. 1998), whereas little recent information is available from female rats (Hu & McLachlan, 2003). Nevertheless, sex-related differences in the outcome of nervous system injuries and disorders have been an important issue in the last decade.

Peripheral nerve function is significantly affected by maturation and aging. However, knowledge regarding differences between the nerves of adult and aged animals has been based on comparisons of only two experimental groups, and it has been pointed out that there is a need for multiple time points in maturation and aging studies (Colleman et al. 1990). Rats described as adult have weights that vary between about 180 and 1000 g or more (Saitua & Alvarez, 1988), with no information about the animals’ ages, which might lead to confusion.

It is well known that degenerative changes in peripheral nerves occur first in the distal portion of the fibres (Thomas et al. 1980; Grover-Johnson & Spencer, 1981; Ceballos et al. 1999), indicating the need for longitudinal studies in experimental neuropathies. Lateral asymmetry of peripheral nerves has been described in the literature (Fraher, 1992; Rodrigues Filho & Fazan, 2004). Some experimental studies involving injury of the sciatic nerve and/or its branches use the same nerve of the opposite side as the control (Polvsen et al. 1993; Hu & McLachlan, 2003). Nevertheless, a systematic study on the lateral symmetry of the sural nerve segments has not been reported.

The aim of the present study was to investigate the normal morphological and morphometric aspects of the sural nerve in the postnatal developing female rat, with special attention to longitudinal morphology and lateral symmetry.

Materials and methods

Experiments were performed on 15 female Wistar rats, randomly divided into three groups of five rats each. Group I animals were aged 30 days, Group II animals were aged 90 days and Group III animals were aged 180 days. Additional animals aged 360 (n = 5) and 640 (n = 4) days were used in order to establish an age/weight curve.

At 30, 90 and 180 days of age, animals were anaesthetized with sodium pentobarbital (Nembutal, 40 mg kg−1, i.p.) and perfused through the left ventricle first with a 0.05 m phosphate-buffered saline solution, pH 7.4, and then with a 2.5% glutaraldehyde solution, in 0.1 m cacodylate buffer, pH 7.2. All procedures adhered to The Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication no. 80-23, revised 1978). Every effort was made to minimize the number of animals used.

Both right and left sural nerves, from their origin in the hip (5–7 mm distal to the greater trochanter) through their distal branching at the lateral malleolus level, were carefully dissected without stretching, removed in one piece and placed in the fixative solution for an additional 12 h. They were washed in cacodylate buffer, pH 7.2, and proximal (close to the origin) and distal (close to terminal branching) segments (of approximately 3 mm each) were excised and processed for epoxy resin embedding (PolyBed 812®, Polysciences Inc., Warrington, PA, USA) as described elsewhere (Fazan et al. 1997, 1999, 2002). Semithin transverse sections of the fascicles were stained with 1% toluidine blue and observed with the aid of an Axiophot photomicroscope (Carl Zeiss, Jena, Germany). The images were sent via a digital camera (TK-1270, JVC, Victor Company of Japan Ltd, Tokyo, Japan) to an IBM/PC where the images were digitized. For study of the myelinated fibres, the endoneural space was observed under an oil-immersion lens (×100) and fully scanned without overlap of the microscopic fields, with the aid of a microscope automatic motor plate (Carl Zeiss). This scanning generated 8–15 microscopic fields of 640 × 470 pixels, which were used to count and to measure automatically the myelinated fibres and respective axons. Fibres at the upper and left edges of the microscopic fields were counted whereas those at the lower and right edges were not counted, in order to avoid counting the same fibre twice. All myelinated fibres present in the endoneural space were counted. The program was able to identify and delete irregular fibres (not oval or round in shape), those cut at the level of a Ranvier node and a Schmidt–Lanterman incision and also those at the edge of the microscopic field under study.

Morphometric parameters of the fascicles and the myelinated fibres of the sural nerve segments were obtained as described previously (Fazan et al. 1997, 1999). The investigator was blind to group identity during the morphometry process. Briefly, the number of fascicles within each segment, the total number of myelinated fibres and the total number of Schwann cell nuclei present in each fascicle were counted. The area and lesser diameter of each fascicle (excluding the perineurium) and of at least 70% of the myelinated fibres were measured with image analysis software (KS 400, Kontron 2.0, Eching Bei München, Germany). The lesser diameter is the one that better represents the diameter of a non-circular fascicle and fibre (López-Plana et al. 1993). The percentage of the total cross-sectional area of the endoneural space occupied by the myelinated fibres (defined by the axon and its respective myelin sheath, not including the Schwann cell perikarya, when present) was calculated and hereafter will be referred as the occupancy percentage of the myelinated fibres. The myelinated fibres and Schwann cell nuclei densities were calculated. For the myelinated fibres, both axonal diameter and total fibre diameter were automatically measured. Therefore, the ratio between the two diameters, the g ratio (a measure of degree of myelination), was obtained (Rushton, 1951; Smith & Koles, 1970). The myelin sheath area was calculated for each myelinated fibre measured. We constructed histograms of the population distribution of the myelinated fibres and axons separated into class intervals increasing by 0.5 µm. Histograms of the g ratio distribution separated into class intervals increasing by 0.1 were also constructed.

Morphometric data were tested for normal distribution by the Kolmogorov–Smirnov normality test. If data presented a normal distribution, comparisons were made between proximal and distal segments in the same group by the paired Student's t-test. Otherwise, comparisons were made by Wilcoxon's non-parametric test for paired samples. For comparisons between right and left segments in the same group, normally distributed data were tested using the unpaired Student's t-test. Otherwise, comparisons were made by the Mann–Whitney non-parametric test. Comparisons between groups were made by one-way analysis of variance (anova) followed by Tukey's post hoc test. Differences were considered significant if P < 0.05. Data are presented as mean ± standard error of the mean (SEM).

Results

Body weight

Mean body weight increased rapidly from 30 (Group I) to 90 (Group II) days, rising from 112 ± 3 to 221 ± 10 g, respectively, with a significant difference between them. A less pronounced, but again significant, increase was observed from 90 to 180 days, with the Group III animals (180 days old) reaching 286 ± 11 g. Thereafter, a continuous but moderate increase occurred and by 360 days the mean weight was 319 ± 6 g, with no appreciable increase observed after this age. Statistical analysis did not show significant differences between Group III and older animals (360 and 640 days old).

Morphological aspects

All nerves included in this study showed good preservation of structures. In 60% of all proximal segments observed, the sural nerve consisted of a single fascicle. Two fascicles were observed in 23% of the proximal segments and more than two fascicles in a smaller number of proximal segments. For the distal segments, 43% of the sural nerves consisted of two fascicles and a smaller proportion of distal segments consisted of three or more fascicles. All sural nerve fascicles were delimited by 3–6 layers of flattened cells that constituted the perineurium. The endoneurium consisted of myelinated fibres of various diameters, intermingled with unmyelinated fibres. The main components of the sural nerves did not differ from those of other peripheral nerves. No morphological differences were observed in the comparisons between segments, sides and groups.

Fascicle morphometry

The mean values for fascicular diameter, and total numbers of myelinated fibres and Schwann cell nuclei are given in Table 1. No differences were observed in the comparisons between sides, segments or groups. No relationship was detected between total numbers and age. Fascicle cross-sectional area, myelinated fibre density, Schwann cell nucleus density and the occupancy percentage of myelinated fibres area are shown in Fig. 1. Only the proximal segments of the right side are represented because no differences were observed between segments and sides. Fascicle cross-sectional area of nerves in Groups II and III increased significantly comparing with Group I. There was a slight, but no significant, increase in this area when comparing Groups II and III. In contrast, myelinated fibre density decreased significantly in Groups II and III as compared with Group I and there was no significant difference between Groups II and III. The same pattern was observed for Schwann cell nucleus density. The occupancy percentage of myelinated fibres increased significantly from Group I to Group II and decreased significantly from Group II to Group III. This finding is in agreement with the constant number of fibres and Schwann cell nuclei among groups and the increase observed for fascicle area.

Fig. 1
Cross-sectional fascicular area, myelinated fibre density, Schwann cell nucleus density, percentage occupancy by the myelinated fibres and mean areas of myelinated fibres and myelin sheath from the right proximal segments of the sural nerves of the different ...
Table 1
Morphometric parameters of the proximal and distal segments of the sural nerves fascicles of both sides. No differences were observed between sides and segments, nor between groups for these parameters. Data are expressed as mean ± SEM

Myelinated fibre morphometry

The mean values for myelinated fibre diameter, myelinated axon diameter and g ratio are given in Table 2. No differences were observed in the comparisons between sides and segments. A relationship was detected between mean diameter values and age, as fibre and axonal diameters were significantly larger in Group III than in Groups I and II and also significantly larger in Group II than in Group I. Few data showed significant differences when comparing fibre and axonal diameters between Groups II and III, with no preference for segments or sides. Although the mean g ratio tended to be greater for Group III fibres, statistical analysis did not detect differences between segments, sides and groups.

Table 2
Myelinated fibre and axon diameters and g ratio of the proximal and distal segments of the sural nerves of both sides. No differences were observed between segments and sides. Asterisks and # symbols indicate significant differences compared with Group ...

The mean myelinated fibre and myelin sheath areas are shown in Fig. 1. Again, only the proximal right segments are represented because no differences were observed between segments and sides. The relationship between area values and age was the same as observed from the Table 2 data; fibre, axon and myelin sheath areas were significantly larger in Group III than in Groups I and II and also significantly larger in Group II than in Group I.

Myelinated fibres population

The size distributions of myelinated fibres, myelinated axons and g ratio are shown in Fig. 2. Representative distributions of the proximal left segments are shown. Group I myelinated fibre diameter ranged between 1.5 and 6.5 µm, and was distributed unimodally. Group II myelinated fibre diameter ranged between 2.0 and 9.5 µm and Group III myelinated fibre diameter ranged between 2.0 and 10.0 µm, both with a bimodal distribution. No differences between sides and segments were observed in all groups, whereas the distributions between groups differed significantly. The size distribution of the myelinated axons in Group I was unimodal (range 0.5–4.0 µm) whereas distributions in Groups II and III tended to bimodality (ranges of 0.5–6.0 and 0.5–7.0, respectively). Again, no differences between sides and segments were observed in all groups whereas the distributions between groups differed significantly. Size distribution of the g ratio was unimodal throughout the periods studied, with Group I peak at 0.5 and Groups II and III peaks at 0.6. No differences between sides and segments were observed in all groups. Interestingly, differences were observed only in Group III distributions, compared with Groups I and II, indicating that most of the sural nerve myelinated fibres reached the best g ratio value (0.6) for the maximal conduction velocity (Smith & Koles, 1970) at the age of 180 days.

Fig. 2
Histograms of myelinated fibre, myelinated axon and g ratio distributions of the proximal left segments. No differences between sides and segments were observed in all groups, whereas the distributions among the groups differed significantly from 30 to ...

Discussion

Postnatal development

Female rats were chosen for this study because they are less susceptible to development of a spontaneous peripheral neuropathy (Majeed, 1992) and also because they live longer and in better condition than males (Van Steenis & Kroes, 1971). The present study showed that in rats there is a postnatal growth spurt between 30 and 90 days, as judged by increases in body weight, which is associated with changes in myelinated fibre diameter in peripheral nerves. Our study also indicates that fibre population distribution changes with increasing body weight; at 30 days, distribution was unimodal and by 180 days this distribution was established as being bimodal. This finding is compatible with the results of Jacobs & Love (1985) obtained in human sural nerves. Body weight continued to increase at a less rapid rate up to the age of 180 days but nerve parameters tended to stabilize between the ages of 90 and 180 days. This is also in agreement with studies showing that morphometric parameters of nerve fibres are stable from 6 months to older ages (Saitua & Alvarez, 1988; Knox et al. 1989). These age-related changes in peripheral nerves related to normal postnatal development are important when using young animals as experimental models of diseases. In some experimental studies the animals show growth retardation, which might lead to difficulties when interpreting differences in nerve fibre size, in comparison with control animals that continued to grow normally. In this situation, information on fascicle size might be used as an indicator of the existence of growth retardation (which would affect fascicle size), associated or otherwise with fibre injury.

Morphology and morphometry

The proximal and distal segments of the sural nerves studied were dissected always at the same levels. One, two or more fascicles were randomly observed, without a definite pattern for segments, sides or ages, which suggests a possible anatomical variation in the branching levels of the sural nerves in rats. Information on the number and size of fascicles of the sural nerve is rare in the literature, either for humans or for experimental animals. The morphological and morphometric parameters usually described are the number and density of myelinated fibres, without mention of the fascicular area. Nerve biopsies are usually restricted to fascicles in order to lessen a patient's neurological deficits. In experimental studies, usually a small endoneural area is studied (Unger et al. 1998; Kovacic et al. 2003). Our data of fascicular area, and number and density of myelinated fibres are compatible with data shown by others, for different ages, segments and rat strains (Knox et al. 1989; Nagamatsu et al. 1993; Saitua & Alvarez, 1988). The differences observed in this study with the values described in the literature might be due to methodological differences, such as regards preparation of the specimens and morphometry technique and also due to differences between rat ages and strains. The increase in the fascicular area observed from 30 to 90 days was largely related to the increase in myelinated fibre size; by contrast, from 90 to 180 days, this increase was related to an increase in the connective tissue components. This is evident when observing that the occupancy percentage of the myelinated fibres increased from 30 to 90 days and decreased from 90 to 180 days.

Schwann cells are important in the regeneration processes that follow nerve injury. In mature nerves, we found approximately 30 Schwann cell nuclei spread at a density of 460 mm−2, with no difference between segments and sides. During the regeneration processes, the endoneural content might be altered due to the Scwann cell reduplication rate being higher than that of fibroblasts (Thomas, 1948) and information regarding the number of Schwann cell nuclei is useful in regenerated nerve studies.

Correlation between the myelin sheath and the diameter of the respective axon has been known since Donaldson & Hok (1905) and may differ significantly between nerves and also between large and small fibre classes within individual nerves (Fraher, 1992). Our results are in accordance with those describing a thicker myelin sheath in large axons (Fraher, 1992; Fazan et al. 1997, 1999).

Our data did not show differences between proximal and distal segments or between the same levels among sides. There is no report regarding a longitudinal and laterality study of the normal sural nerve morphology in rats. Exploring different levels of the same nerve is important because pathological alterations of nerves are more pronounced distally (Thomas et al. 1980; Grover-Johnson & Spencer, 1981) and knowledge of normality in different segments of the same nerve might be useful in the interpretation of pathological findings, especially in experimental models of neuropathy. The laterality study is also important because experimental models of injury of the sciatic nerve and/or its branches use the opposite side nerve as a control (Nagamatsu et al. 1993; Kim et al. 1994; Hu & McLachlan, 2003; Kovacic et al. 2003).

In conclusion, the rat sural nerve is long and of constant morphology, and presents a continuous and symmetrical growth, more pronounced between 30 and 90 days of age, thus providing a good model for experimental neuropathies. When designing a morphometric study of nerves, one should take into account possible longitudinal variation and/or lateral asymmetry, in order to avoid introducing systematic bias and to minimize variance between samples.

Acknowledgments

We thank Maria Cristina Lopes Schiavoni and Antônio Renato Meirelles e Silva, Experimental Neurology Laboratory, School of Medicine of Ribeirão Preto, for excellent technical support. We also thank Dr Maria Cristina de Oliveira Salgado for critically revising the manuscript. Financial support was received from CNPq (Conselho Nacional de Pesquisa e Tecnologia) Grant number: 501230/2003-3 and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) Grant numbers: 02/09406-5 and 04/01390-8. L.S.S. was the recipient of a fellowship from the PIBIC/CNPq (Programa Institucional de Bolsas de Iniciação Científica do Conselho Nacional de Pesquisa e Tecnologia) while the study was carried out.

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