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A morphometric study on the longitudinal and lateral symmetry of the sural nerve in mature and aging female rats
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
A morphometric study on the longitudinal and lateral symmetry of the sural nerve in mature and aging female rats André Jeronimo a,b , Cláudia Além Domingues Jeronimo a,b , Omar Andrade Rodrigues Filho a , Luciana Sayuri Sanada c , Valéria Paula Sassoli Fazan d,⁎ a
Department of Biological Sciences, Federal University of Triângulo Mineiro, Uberaba, Minas Gerais, Brazil Physical Therapy Course, University of Uberaba, Uberaba, Minais Gerais, Brazil c Department of Neurology, School of Medicine of Ribeirão Preto, Ribeirão Preto, São Paulo, Brazil d Department of Surgery and Anatomy, School of Medicine of Ribeirão Preto, Ribeirão Preto, São Paulo, Brazil b
A R T I C LE I N FO
AB S T R A C T
Article history:
Aging affects peripheral nerve function and regeneration in experimental models but few
Accepted 21 May 2008
literature reports deal with animals aged more than one year. We investigated
Available online 29 May 2008
morphological and morphometric aspects of the sural nerve in aging rats. Female Wistar rats 360, 640 and 720 days old were killed, proximal and distal segments of the right and left
Keywords:
sural nerves were prepared for light microscopy and computerized morphometry. No
Sural nerve
morphometric differences between proximal and distal segments or between right and left
Female rat
sides at the same levels were found in all experimental groups. No increase in fiber and axon
Aging
sizes was observed from 360 to 720 days. Likewise, no difference in total myelinated fiber
Morphology
number was observed between groups. Myelinated fiber population distribution was
Morphometry
bimodal, being the 720-days old animals’ distribution shifted to the left, indicating a
Myelinated fibers
reduction of the fiber diameters. The g ratio distribution of the 720-days old animals’ myelinated fiber was also shifted to the left, which suggests axonal atrophy. Morphological alterations due to aging were observed, mainly related to the myelin sheath, which suggests demyelination. Large fibers were more affected than the smaller ones. Axon abnormalities were not as common or as obvious as the myelin changes and Wallerian degeneration was rarely found. These alterations were observed in all experimental groups but were much less pronounced in rats 360 days old and their severity increased with aging. In conclusion, the present study indicates that the aging neuropathy present in the sural nerve of female rats is both axonal and demyelinating. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
It is well established that aging affects peripheral nerve function and regeneration, both in humans and experimental models. Also, age related changes to peripheral nerves are not
linearly progressive with age (Verdú et al., 2000). However, in aging studies, differences between adult and old animals have often been based on comparisons of only two experimental groups, whereas the life span and the duration of growth periods should be carefully taken into account to ensure
* Corresponding author. Department of Surgery and Anatomy, School of Medicine of Ribeirão Preto, 14049-900, Ribeirão Preto, São Paulo, Brazil. Fax: +55 16 3633 0017. E-mail addresses: [email protected], [email protected] (V.P.S. Fazan). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.05.055
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specifically that adult and old animals are compared (Ceballos et al, 1999). In the last decades, morphometric investigations have benefit neuropathies understanding but still little information is available on morphometric changes of peripheral nerves in aging. Likewise, few literature reports deal with animals aged more than one year. Rats are widely used in studies of peripheral neuropathies and nerve regeneration but most reports deal with male animals. It is well known that females are less susceptible to development of a spontaneous peripheral neuropathy (Majeed, 1992) and also live longer and in better condition than males (Van Steenis and Kroes, 1971). Moreover, sexrelated differences in the outcome of nervous system injuries
and disorders have been an important issue in the last years (Jeronimo et al., 2005). Among the peripheral nerves of rats, the sciatic nerve is by far the most investigated in regards of models of injuries and regeneration while the sural nerve, a sensory branch of the sciatic nerve is less explored. Despite some descriptions of peripheral nerve morphologic alterations in aged rats, most of the studies used motor or sensory-motor nerves, but information on sensory nerves is scanty. The sural nerve in rats is widely used in experimental studies investigating injury and regeneration of the peripheral nervous system. Nevertheless, information on morphological and morphometric aspects of the sural nerve in aged rats is not common in the literature.
Fig. 1 – Semithin transverse sections of the sural nerve of female Wistar rats aged 360 (A and B), 640 (C and D) and 720 (E and F) days showing typical endoneural structures. Note that, from Group I (360 days old) to Group III (720 days old) there is an increase in the number of myelinated fibers with contorted and infolded myelin sheaths and myelin splitting. In C, the arrow indicates a large myelinated fiber with very thin myelin sheath. In D, the arrow indicates a Wallerian degeneration. The arrowhead shows a ball of the myelin. In E and in F, note the presence of a large number of myelin splitting. Also, the arrows indicate the presence of macrophages in the endoneural space. Arrowhead shows axoplasmatic inclusions in a myelinated axon. * Indicates the perineurium. Toluidine blue stained. Bar = 10 μm.
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We therefore thought it is of interest to examine morphologic and morphometric changes with aging in the sural nerves of female rats, using sophisticated morphometric approaches.
2.
Results
2.1.
Body weight
Mean body weight was similar between Group I and Group II animals (318 ± 9 g and 325 ± 5 g, respectively) and significantly smaller compared to Group III animals (442 ± 8 g). Nevertheless, animals in Group III showed mammary adenomas (that were described previously for aged female rats (Van Steenis and Kroes, 1971)), which were not present on animals of Groups I and II.
2.2.
Morphological aspects
All nerves included in this study showed good preservation of structures. One, two or more fascicles were present in the sections, in the same percentages as described previously (Jeronimo et al., 2005). General morphological characteristics of the sural nerve fascicles observed in the present study were similar to those described for young and adult female rats (Jeronimo et al., 2005). Also, the main components of the sural nerves did not differ from those of other peripheral nerves. Morphological differences were observed between groups, not only on the myelinated fibers but also on the endoneural blood vessels. Morphologic alterations of the myelinated fibers were present in Group I nerves but were most severe in the nerves of the oldest rats (Group III). The main changes were the presence of contorted and infolded myelin sheaths and myelin loops and splitting (Fig. 1). There was an obvious increase in the irregularity of the myelinated fibers, with loss of circularity with aging. Also, large myelinated fibers with thin myelin sheaths were observed and grossly swollen demyelinated axons were sometimes present. Large myelinated fibers were more affected than the small fibers. Axonal abnormalities were not as common or as obvious as the alterations on the myelin sheaths and the presence of Wallerian degeneration was rare. Macrophages in the endoneural space were seen only in Group III nerves (Fig. 1). Blood vessels were patent in Group I nerves, but showed thickening of the wall (Fig. 2). This thickening was severe on Group II and a layer of connective tissue surrounding the vessels could be easily identified. Most of the vessels in Group III nerves were collapsed. No differences in severity between the lesion on proximal and distal segments of the nerves were observed.
2.3.
Fig. 2 – Semithin transverse sections of the sural nerve of female Wistar rats aged 360 (A), 640 (B) and 720 (C) days showing altered endoneural vessels. Most of Group I (360 days old) endoneural vessels showed thickening of the wall (arrow). This thickening was severe on Group II (640 days old) and a layer of connective tissue surrounding the vessels could be easily identified (arrow). Most of the vessels in Group III (720 days old) nerves were collapsed (arrow). Arrowheads indicate infolds of myelin. Note that the myelinated fibers increase the irregularities and lose the circularity with aging. Toluidine blue stained. Bar = 10 μm.
Morphometric aspects
Table 1 shows the mean values for fascicular area and diameter as the percentage of occupancy of the myelinated fibers. No differences were observed in the comparisons between sides, segments or groups. No relationship was detected between total numbers and aging. The mean values for total numbers of myelinated fibers and Schwann cell nuclei as their respective densities are given in Fig. 3. There was a tendency of reduction of the myelinated fiber number in Group
III animals and myelinated fiber density was clearly reduced in Groups II and III, compared to Group I. Schwann cell nuclei number and density increased from Group I to Group II and decreased in Group III. Nevertheless, no significant differences were observed between segments, sides and groups. The mean values for myelinated fiber area and diameter, myelinated axon area and diameter, myelin sheath area and g ratio are given in Table 2. No differences were observed
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Table 1 – Morphometric parameters of the proximal and distal segments of the sural nerve fascicles in both sides
2
Fascicular area (μm ) Fascicular diameter (μm) Percentage of occupancy (%)
Group I (360 days)
Group II (640 days)
Group III (720 days)
Proximal segments
Proximal segments
Proximal segments
Right
Left
Right
Left
Right
Left
81028 ± 7273 247 ± 35 34 ± 1
66492 ± 8301 250 ± 29 42 ± 6
97509 ± 4370 218 ± 45 34 ± 5
96348 ± 20544 207 ± 59 37 ± 4
84556 ± 10242 210 ± 54 32 ± 3
69222 ± 4852 267 ± 13 36 ± 5
Distal segments
2
Fascicular area (μm ) Fascicular diameter (μm) Percentage of occupancy (%)
Distal segments
Distal segments
Right
Left
Right
Left
Right
Left
76887 ± 7029 164 ± 36 34 ± 1
61123 ± 9145 142 ± 25 33 ± 2
84397 ± 8979 191 ± 60 30 ± 2
70456 ± 15573 175 ± 28 34 ± 3
76806 ± 8149 197 ± 30 30 ± 4
61182 ± 3082 154 ± 15 27 ± 1
No differences were observed between sides, segments or groups. Data are expressed as mean ± SEM.
between segments and sides. Likewise, no differences were observed between groups for the myelinated fibers morphometric parameters. Myelinated axons were significantly
smaller in Group III nerves, compared to Groups I and II. The average g ratio values showed the same differences between groups observed for the axons.
Fig. 3 – Average myelinated fiber and Scwann cell nucleus number and density of the sural nerve of female Wistar rats aged 360, 640 and 720 days. Data are expressed as mean ± SEM. No morphometric differences were observed between segments, sides and groups.
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Table 2 – Myelinated fiber (MF), myelin sheath (MS) and myelinated axon (MA) average morphometric parameters of the proximal and distal segments of the sural nerves in both sides Group I (360 days)
Group II (640 days)
Group III (720 days)
Proximal segments
Proximal segments
Proximal segments
Right MF area (μm2) MF diameter (μm) MS area (μm2) MA area (μm2) MA diameter (μm) g ratio
34 ± 5.7 ± 24 ± 10 ± 3.0 ± 0.52 ±
3 0.2 2 1 0.2 0.02
Left 37 ± 5.9 ± 25 ± 11 ± 3.2 ± 0.53 ±
1 0.1 1 1 0.1 0.01
Distal segments Right 2
MF area (μm ) MF diameter (μm) MS area (μm2) MA area (μm2) MA diameter (μm) g ratio
35 ± 5.5 ± 25 ± 10 ± 3.0 ± 0.52 ±
3 0.3 1 1 0.2 0.02
Left
43 ± 3 6.2 ± 0.2 30 ± 2 14 ± 1 3.5 ± 0.1 0.55 ± 0.01
40 ± 4 5.9 ± 0.3 28 ± 3 11 ± 2 3.2 ± 0.2 0.53 ± 0.02
Right 34 5.5 25 9 2.7 0.49
3 0.3 2 1 0.2 0.01
Right
Left
40 ± 3 6.1 ± 0.2 27 ± 2 13 ± 2 3.6 ± 0.2 0.58 ± 0.02
44 ± 5 6.2 ± 0.3 31 ± 3 13 ± 5 3.4 ± 0.2 0.54 ± 0.01
Left
±3 ± 0.2 ±2 ±1 ± 0.2* ± 0.02*
Distal segments
Left 35 ± 5.6 ± 25 ± 11 ± 3.0 ± 0.53 ±
Right
37 ± 5.8 ± 26 ± 10 ± 2.7 ± 0.49 ±
2 0.2 1 1 0.1* 0.01*
Distal segments Right 32 5.4 25 7 2.5 0.46
±2 ± 0.1 ±1 ± 1* ± 0.1* ± 0.01*
Left 30 ± 2 5.3 ± 0.1 23 ± 1 7 ± 1* 2.5 ± 0.1* 0.47 ± 0.01*
No differences were observed between segments and sides. * indicates differences between groups. Data are expressed as mean ± SEM.
The size distributions of myelinated fibers, myelinated axons and g ratio are shown in Figs. 4–6, respectively. Myelinated fiber diameter (Fig. 4) ranged between 1.5 and 12.0 μm, and was distributed bimodally, with peaks of frequency between 3–4 and 5–6 μm in all experimental groups. No differences were observed between segments, sides and groups. Myelinated axon diameter (Fig. 5) ranged between 0.5 and 10.0 μm, with an unimodal distribution. Peaks of frequency between 3–4 μm were observed for Groups I and II, while Group III showed a high peak at 2.0 μm, with a shift of the histogram to the left. A reduction of the frequency of axons between 4–8 μm was also noted in Group III. Groups I and II g ratio distributions (Fig. 6) ranged between 0.2 and 0.9, with a peak of frequency at 0.6 while Group III distribution showed the same range as Groups I and II but the peak of frequency was shifted to the left, at 0.5. No differences between sides and segments were observed in all groups, whereas the distributions between groups differed significantly for the axons and g ratio.
3.
Discussion
The present study showed that the morphology and morphometric parameters of the sural nerves are altered with aging, being diverse elements affected differently. Morphological alterations were found predominantly on the myelin sheaths of large myelinated fibers. These alterations of the sural nerves are similar to those of the ventral root in 30 months old female Fisher rats (Knox et al., 1989) and differ significantly from the observations in the tibial and plantar nerves of male Wistar rats (Sharma et al., 1980). Sharma et al. (1980) described that in the tibial and plantar nerves of male Wistar rats, after 18 months, the proportion of abnormal fibers increased rapidly and Wallerian type axonal degeneration and regeneration predominated. These differences between the present
study and the findings in male rats (Sharma et al., 1980) suggest that despite the peripheral nerves alteration due to aging, males and females might present different types of aging neuropathies. Nevertheless, it is important to take into account that we studied a mainly sensory nerve while Sharma et al. (1980) used sensory-motor nerves. One could speculate that there might be differences between motor and sensory fibers injury due to aging but this issue was not yet investigated. The main morphological alterations found in the present study were infolded myelin sheaths and myelin loops. Myelin loops and splitting are present in normal nerves as the result of changes in the size of the myelinated axons and an appropriate adjustment in their myelin sheath (Ceballos et al., 1999). However, the increased frequency of these alterations with aging has been related to an early response of large fiber myelin sheaths to axonal atrophy (Krinke et al., 1988), as shown by the morphometric approach in the present study. These alterations would have a direct effect on the reduced conduction velocity of the large myelinated fibers, described for other species (Peters, 2002). The alterations observed in the myelinated fibers might be directly related to the morphological alterations of the endoneural blood vessels, which might be affecting the responsiveness of the Schwann cells to aging, as follows. The number of Schwann cells associated with myelinated fibers and sectioned at the level of the nucleus increased from Group I to Group II and decreased latter. This result is similar to that in the tibial nerve of mice (Ceballos et al., 1999) but is being described for the first time in rats. It is well known that Schwann cells are important in the regeneration processes that follow nerve injury. Nerves that are experiencing regeneration will have a larger number of Schwann cells due to their augmented duplication rate under these circumstances (Thomas, 1948). Our results suggest that Group II nerves might have been under regeneration but this process was not sustained in older animals. Since the blood vessels of animals
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Fig. 4 – Frequency distribution histograms of myelinated fibers from proximal and distal segments of the right and left sural nerves in female Wistar rats aged 360, 640 and 720 days. No morphometric differences were observed between segments, sides and groups. from group III were severely damaged, multiplication of the Schwann cells in order to accomplish the regeneration process could be impaired by a poor blood supply of the endoneural space. The number of fibers in a peripheral nerve is known to be constant during the adulthood lifespan (Sharma et al., 1980; Berthold et al., 1984; Schellens et al., 1993; Jeronimo et al., 2005), thus not correlated with age. On the other hand, it is well known that the myelinated fiber density decreases with development and aging, due to the increase in the amount of the endoneural connective tissue (Jacobs and Love, 1985; Schellens et al., 1993; Ceballos et al., 1999; Jeronimo et al., 2005). In the present study, despite the tendency of a decrease on the fiber number from Group I to Group III, differences were not observed. The fact that there was no significant difference in the myelinated fiber numbers between groups may be explicable in terms of the wide range of inter-animal variability which would tent to obscure fiber losses. This observation is similar to those in the tibial nerves
of mice (Ceballos et al., 1999) and male rats (Sharma et al., 1980) and the sural nerve of female Fisher rats (Knox et al., 1989). Despite that Knox et al. (1989) have performed a morphometric study of the peripheral nervous system in aged female rats (Fisher brand), including the sural nerve, the present study adds new information to this field, specially those related to the Schwann cell nuclei, the g ratio, and the distributions of the myelinated fiber and respective axon sizes. The average morphometric parameters of the myelinated fibers and respective axons showed no myelinated fiber changes but reduced axon area and diameter, as well as reduced myelin sheath area and g ratio in Group III nerves, more evident on the distal segments. The myelinated fiber diameter distribution was similar between groups while the axon distribution was significantly shifted to the left in Group III. A similar shift to the left was observed for the g ratio distributions. These data put together is highly indicative of the presence of axonal atrophy in the myelinated fibers of the
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Fig. 5 – Frequency distribution histograms of myelinated axons from proximal and distal segments of the right and left sural nerves in female Wistar rats aged 360, 640 and 720 days. Histograms differed significantly between groups. Note that 720 days old histograms are shifted to the left, with a higher peak at small axons and that there is a reduction on the frequency of larger fibers.
sural nerves of aged female rats. This observation is similar to the description of a moderate number of fine or medium-sized axons with disproportionately thick myelin sheaths in the sural nerve of humans over 60 years of age (Jacobs and Love, 1985). Sharma et al. (1980) showed reduced average diameter of the myelinated fibers in the tibial and plantar nerves of male aged rats, with no differences on the fiber distributions, which also partially corroborate our results. Nevertheless, these authors did not study the axon distributions. Knox et al. (1989) did not show differences in the myelinated fiber and axon diameters average values in the sural nerves of female Fisher rats, aged 10, 20 and 30 months. However, they did not study the distributions of these diameters and no information on the g ratio was given. An important difference between that study (Knox et al. 1989) and the present is that Knox et al. (1989) used a sampling method to study about 600 myelinated fibers while in the present study, the whole nerve was in-
vestigated. It is well established that nerves with a large fiber population may have a heterogeneous fiber distribution, which would introduce a bias with the use of any sampling scheme (Torch et al., 1989; Romero et al., 2000). Measurement of small myelinated fibers is considered difficult because they are generally harder to stain and also because with manual morphometry techniques or sampling schemes, the observers might underestimate them (Ellis et al., 1980; Mezin et al., 1994; Da Silva et al., 2007). In the present study, no underestimate of the small myelinated fibers was done because no sampling method was used. Correlation between the myelin sheath and the diameter of the respective axon has been known since Donaldson and Hoke in 1905 (Donaldson and Hoke, 1905), and may differ significantly between nerves and between large and small fibers within an individual nerve (Fraher, 1992). Rushton (1951) suggested that values between 0.6 and 0.7 would be the
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Fig. 6 – Frequency distribution histograms of myelinated fibers g ratio from proximal and distal segments of the right and left sural nerves in female Wistar rats aged 360, 640 and 720 days. The histograms differed significantly between groups. Note that most of myelinated fibers of the sural nerves in Groups I and II have g ratios of 0.6 wile fibers in Group III g ratios of 0.5.
g ratio for the best and maximum conduction velocity of a myelinated fiber. The present study showed reduced average g ratio and a shift of the histograms to the left in Group III nerves. These results suggest that the myelinated fibers of the oldest animals might have a reduced conduction velocity.
4.
Concluding remarks
In the present study, morphological alterations were more evident in the large myelinated fibers and endoneural blood vessels. These observations suggested the presence of a demyelinating neuropathy due to aging in female rats, which is similar to the aging neuropathy described for male rats. Nevertheless, the sophisticated morphometric approach used in the present study revealed the presence of an axonal neuropathy, mainly associated to the small myelinated fibers, which is being described for the first time. In conclusion, the present
study indicates that the aging neuropathy present in the sural nerve of female rats is both axonal and demyelinating.
5.
Experimental procedure
Experiments were performed on 14 female Wistar rats, randomly divided into three groups. Group I animals were 360 days old (N = 5), Group II animals were 640 days old (N = 5) and Group III animals were 720 days old (N = 4). The animals were born and raised in the animal care facility of the Department of Physiology, Federal University of Triângulo Mineiro, in a controlled environment (room temperature between 21–23 °C, air humidity between 40 and 70% and dark/light cycle of 12 hs), housed in plastic cages (3–4 animals to a cage) with free access to tap water and rat chow throughout the experiments. All experimental procedures adhered to The Guide for the Care and Use of Laboratory Animals
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prepared by the National Academy of Sciences and published by the National Institutes of Health (Copyright © 1996 by the National Academy of Sciences), and were approved by the Ethics Committee for Animal Research of the School of Medicine of Ribeirão Preto – University of São Paulo (CETEA – Comitê de Ética em Experimentação Animal). Effort was made to minimize the number of animals used. At 360, 640 and 720 days of age, animals were anaesthetized with sodium pentobarbital (Nembutal, 40 mg kg, 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. 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 cut and processed for epoxy resin embedding (PolyBed 812®, Polysciences Inc., Warrington, PA, USA) as described elsewhere (Jeronimo et al., 2005; Campos et al., 2007; Alcântara et al., 2008). Samples of all three experimental groups were histologically processed at once so that they were submitted to absolutely the same experimental conditions throughout the experiments. Semithin (0.2 - 0.3 μm thick) transverse sections of the fascicles were stained with 1% toluidine blue and examined with the aid of an Axiophot II photomicroscope (Carl Zeiss, Jena, Germany). The images were sent via a digital camera (TK1270, JVC, Victor Company of Japan Ltd, Tokyo, Japan) to an IBM/PC where they were digitized. For the study of myelinated fibers, the endoneural space was observed with an optical set including an oil immersion lens (100 x), optovar (1.6 x), camera (0.5 x) and an 8 x computerized magnification, which provided images with good resolution for morphometry. The endoneural space was fully scanned without overlap of the microscopic fields, using an automatic motorized stage (Carl Zeiss, Jena, Germany). Scannings generated 9 to 17 microscopic fields of 640 x 470 pixels, which were used to count and automatically measure the myelinated fibers and their respective axons. Fibers at the upper and left edges of the microscopic fields were counted whereas those at the lower and right edges were not counted (“forbidden line”) in order to avoid counting the same fiber twice. All myelinated fibers present in the endoneural space were counted. Morphometric parameters of the fascicles and myelinated fibers of sural nerve segments were obtained as described previously for other nerves (Fazan et al., 1997; Fazan et al., 1999; Rodrigues Filho and Fazan, 2006; Campos et al., 2007; Alcântara et al., 2008). Briefly, the total number of myelinated fibers 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) as well as each myelinated fiber (defined by the axon and its respective myelin sheath excluding the Schwann cell nucleus when present) and respective axon (fiber excluding the myelin sheath) were measured with image analysis software (KS 400, Kontron 2.0, Eching Bei München, Germany). The lesser diameter better represents the diameter of a non-circular fascicle and fiber
59
(Blight and Decrescito, 1986; Auer, 1994; Jeronimo et al., 2005). The percentage of the total cross-sectional area of the endoneural space occupied by the myelinated fibers was calculated, and hereafter referred as the percentage of occupancy of the myelinated fibers (Jeronimo et al., 2005, Campos et al., 2007; Alcântara et al., 2008). The myelinated fibers and Schwann cell nuclei densities were calculated. For myelinated fibers, both axonal diameter and total fiber diameter were automatically measured. The ratio between the two diameters, the g ratio (which indicates the degree of myelination), was obtained (Rushton, 1951; Smith and Koles, 1970). Myelin sheath area was calculated for each myelinated fiber measured. Histograms of population distribution of myelinated fibers and axons, separated into class intervals increasing by 1.0 μm were constructed. Histograms of the g ratio distribution separated into class intervals increasing by 0.1 were also prepared. The investigators were blind to group identities throughout the experiments. Morphometric data were tested for normal distribution by the Kolmogorov–Smirnov normality test followed by the Levene test for variance equivalence. If data presented a normal distribution and equivalent variance, comparisons were made between proximal and distal segments in the same group by 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. Alternatively, comparisons were made by the Mann–Whitney non-parametric test. Comparisons between groups were made by one-way analysis of variance (ANOVA) followed by HolmSidak post hoc test. Comparisons between histograms were made by one-way analysis of variance (ANOVA) on Ranks provided that the distributions did not pass the normality test. Differences were considered significant if p b 0.05. Data are presented as mean ± standard error of the mean (SEM).
Acknowledgments We thank Mr. Antônio Renato Meirelles e Silva, Experimental Neurology Laboratory, and Ms. Maria Tereza Maglia, Electron Microscopy Laboratory, School of Medicine of Ribeirão Preto, for excellent technical support. Grant sponsor: FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo); Grant numbers: 04/09139–2, 04–01390–8 and 06/03200–7; Grant sponsor: CNPq (Conselho Nacional de Pesquisa e Tecnologia); Grant number: 303802/2006–5.
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Mezin, P., Tenaud, C., Bosson, J.L., Stoebner, P., 1994. Morphometric analysis of the peripheral nerve: advantages of the semi-automated interactive method. J. Neurosci. Meth. 51, 163–169. Peters, A., 2002. The effects of normal aging on myelin and nerve fibers: A review. J. Neurocyitol. 31, 581–593. Rodrigues Filho, O.A., Fazan, V.P.S., 2006. Streptozotocin induced diabetes as a model of phrenic nerve neuropathy in rats. J. Neurosci. Meth. 151, 131–138. Romero, E., Cuisenaire, O., Denef, J.F., Delbeke, J., Macq, B., Veraart, C., 2000. Automatic morphometry of nerve histological sections. J. Neurosci. Meth. 97, 111–122. Rushton, W.A.H., 1951. A theory of the effects of fiber size in medullated nerve. J. Physiol. 115, 101–122. Schellens, R.L., Van Veen, B.K., Gabreels-Festen, A.A., Notermans, S.L., Vanhot, M.A., Stegeman, D.F., 1993. A statistical approach to fiber diameter distribution in human sural nerve. Muscle Nerve 16, 132–150. Sharma, A.K., Bajada, S., Thomas, P.K., 1980. Age changes in the tibial and plantar nerves of the rat. J. Anat. 130, 417–428. Smith, R.S., Koles, Z.J., 1970. Myelinated nerve fibers: computed effects of myelin thickness on conduction velocity. Am. J. Physiol. 219, 1256–1258. Thomas, G.A., 1948. Qualitative histology of wallerian degeneration II. Nuclear population in two different fiber spectrum. J. Anat. 82, 135–145. Torch, S., Stoebner, P., Usson, Y., D’Aubigni, G.D., Saxod, R., 1989. There is no simple adequate sampling scheme for estimating the myelinated fiber size distribution in human peripheral nerve: a statistical ultrastructural study. J. Neurosci. Meth. 27, 149–164. Van Steenis, G., Kroes, R., 1971. Changes in the nervous system and musculature of old rats. Vet. Path. 8, 320–332. Verdú, E., Ceballos, D., Vilches, J.J., Navarro, X., 2000. Influence of aging on peripheral nerve function and regeneration. J. Peripheral Nerv. Syst. 5, 191–208.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
A morphometric study on the longitudinal and lateral symmetry of the sural nerve in mature and aging female rats André Jeronimo a,b , Cláudia Além Domingues Jeronimo a,b , Omar Andrade Rodrigues Filho a , Luciana Sayuri Sanada c , Valéria Paula Sassoli Fazan d,⁎ a
Department of Biological Sciences, Federal University of Triângulo Mineiro, Uberaba, Minas Gerais, Brazil Physical Therapy Course, University of Uberaba, Uberaba, Minais Gerais, Brazil c Department of Neurology, School of Medicine of Ribeirão Preto, Ribeirão Preto, São Paulo, Brazil d Department of Surgery and Anatomy, School of Medicine of Ribeirão Preto, Ribeirão Preto, São Paulo, Brazil b
A R T I C LE I N FO
AB S T R A C T
Article history:
Aging affects peripheral nerve function and regeneration in experimental models but few
Accepted 21 May 2008
literature reports deal with animals aged more than one year. We investigated
Available online 29 May 2008
morphological and morphometric aspects of the sural nerve in aging rats. Female Wistar rats 360, 640 and 720 days old were killed, proximal and distal segments of the right and left
Keywords:
sural nerves were prepared for light microscopy and computerized morphometry. No
Sural nerve
morphometric differences between proximal and distal segments or between right and left
Female rat
sides at the same levels were found in all experimental groups. No increase in fiber and axon
Aging
sizes was observed from 360 to 720 days. Likewise, no difference in total myelinated fiber
Morphology
number was observed between groups. Myelinated fiber population distribution was
Morphometry
bimodal, being the 720-days old animals’ distribution shifted to the left, indicating a
Myelinated fibers
reduction of the fiber diameters. The g ratio distribution of the 720-days old animals’ myelinated fiber was also shifted to the left, which suggests axonal atrophy. Morphological alterations due to aging were observed, mainly related to the myelin sheath, which suggests demyelination. Large fibers were more affected than the smaller ones. Axon abnormalities were not as common or as obvious as the myelin changes and Wallerian degeneration was rarely found. These alterations were observed in all experimental groups but were much less pronounced in rats 360 days old and their severity increased with aging. In conclusion, the present study indicates that the aging neuropathy present in the sural nerve of female rats is both axonal and demyelinating. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
It is well established that aging affects peripheral nerve function and regeneration, both in humans and experimental models. Also, age related changes to peripheral nerves are not
linearly progressive with age (Verdú et al., 2000). However, in aging studies, differences between adult and old animals have often been based on comparisons of only two experimental groups, whereas the life span and the duration of growth periods should be carefully taken into account to ensure
* Corresponding author. Department of Surgery and Anatomy, School of Medicine of Ribeirão Preto, 14049-900, Ribeirão Preto, São Paulo, Brazil. Fax: +55 16 3633 0017. E-mail addresses: [email protected], [email protected] (V.P.S. Fazan). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.05.055
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specifically that adult and old animals are compared (Ceballos et al, 1999). In the last decades, morphometric investigations have benefit neuropathies understanding but still little information is available on morphometric changes of peripheral nerves in aging. Likewise, few literature reports deal with animals aged more than one year. Rats are widely used in studies of peripheral neuropathies and nerve regeneration but most reports deal with male animals. It is well known that females are less susceptible to development of a spontaneous peripheral neuropathy (Majeed, 1992) and also live longer and in better condition than males (Van Steenis and Kroes, 1971). Moreover, sexrelated differences in the outcome of nervous system injuries
and disorders have been an important issue in the last years (Jeronimo et al., 2005). Among the peripheral nerves of rats, the sciatic nerve is by far the most investigated in regards of models of injuries and regeneration while the sural nerve, a sensory branch of the sciatic nerve is less explored. Despite some descriptions of peripheral nerve morphologic alterations in aged rats, most of the studies used motor or sensory-motor nerves, but information on sensory nerves is scanty. The sural nerve in rats is widely used in experimental studies investigating injury and regeneration of the peripheral nervous system. Nevertheless, information on morphological and morphometric aspects of the sural nerve in aged rats is not common in the literature.
Fig. 1 – Semithin transverse sections of the sural nerve of female Wistar rats aged 360 (A and B), 640 (C and D) and 720 (E and F) days showing typical endoneural structures. Note that, from Group I (360 days old) to Group III (720 days old) there is an increase in the number of myelinated fibers with contorted and infolded myelin sheaths and myelin splitting. In C, the arrow indicates a large myelinated fiber with very thin myelin sheath. In D, the arrow indicates a Wallerian degeneration. The arrowhead shows a ball of the myelin. In E and in F, note the presence of a large number of myelin splitting. Also, the arrows indicate the presence of macrophages in the endoneural space. Arrowhead shows axoplasmatic inclusions in a myelinated axon. * Indicates the perineurium. Toluidine blue stained. Bar = 10 μm.
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We therefore thought it is of interest to examine morphologic and morphometric changes with aging in the sural nerves of female rats, using sophisticated morphometric approaches.
2.
Results
2.1.
Body weight
Mean body weight was similar between Group I and Group II animals (318 ± 9 g and 325 ± 5 g, respectively) and significantly smaller compared to Group III animals (442 ± 8 g). Nevertheless, animals in Group III showed mammary adenomas (that were described previously for aged female rats (Van Steenis and Kroes, 1971)), which were not present on animals of Groups I and II.
2.2.
Morphological aspects
All nerves included in this study showed good preservation of structures. One, two or more fascicles were present in the sections, in the same percentages as described previously (Jeronimo et al., 2005). General morphological characteristics of the sural nerve fascicles observed in the present study were similar to those described for young and adult female rats (Jeronimo et al., 2005). Also, the main components of the sural nerves did not differ from those of other peripheral nerves. Morphological differences were observed between groups, not only on the myelinated fibers but also on the endoneural blood vessels. Morphologic alterations of the myelinated fibers were present in Group I nerves but were most severe in the nerves of the oldest rats (Group III). The main changes were the presence of contorted and infolded myelin sheaths and myelin loops and splitting (Fig. 1). There was an obvious increase in the irregularity of the myelinated fibers, with loss of circularity with aging. Also, large myelinated fibers with thin myelin sheaths were observed and grossly swollen demyelinated axons were sometimes present. Large myelinated fibers were more affected than the small fibers. Axonal abnormalities were not as common or as obvious as the alterations on the myelin sheaths and the presence of Wallerian degeneration was rare. Macrophages in the endoneural space were seen only in Group III nerves (Fig. 1). Blood vessels were patent in Group I nerves, but showed thickening of the wall (Fig. 2). This thickening was severe on Group II and a layer of connective tissue surrounding the vessels could be easily identified. Most of the vessels in Group III nerves were collapsed. No differences in severity between the lesion on proximal and distal segments of the nerves were observed.
2.3.
Fig. 2 – Semithin transverse sections of the sural nerve of female Wistar rats aged 360 (A), 640 (B) and 720 (C) days showing altered endoneural vessels. Most of Group I (360 days old) endoneural vessels showed thickening of the wall (arrow). This thickening was severe on Group II (640 days old) and a layer of connective tissue surrounding the vessels could be easily identified (arrow). Most of the vessels in Group III (720 days old) nerves were collapsed (arrow). Arrowheads indicate infolds of myelin. Note that the myelinated fibers increase the irregularities and lose the circularity with aging. Toluidine blue stained. Bar = 10 μm.
Morphometric aspects
Table 1 shows the mean values for fascicular area and diameter as the percentage of occupancy of the myelinated fibers. No differences were observed in the comparisons between sides, segments or groups. No relationship was detected between total numbers and aging. The mean values for total numbers of myelinated fibers and Schwann cell nuclei as their respective densities are given in Fig. 3. There was a tendency of reduction of the myelinated fiber number in Group
III animals and myelinated fiber density was clearly reduced in Groups II and III, compared to Group I. Schwann cell nuclei number and density increased from Group I to Group II and decreased in Group III. Nevertheless, no significant differences were observed between segments, sides and groups. The mean values for myelinated fiber area and diameter, myelinated axon area and diameter, myelin sheath area and g ratio are given in Table 2. No differences were observed
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Table 1 – Morphometric parameters of the proximal and distal segments of the sural nerve fascicles in both sides
2
Fascicular area (μm ) Fascicular diameter (μm) Percentage of occupancy (%)
Group I (360 days)
Group II (640 days)
Group III (720 days)
Proximal segments
Proximal segments
Proximal segments
Right
Left
Right
Left
Right
Left
81028 ± 7273 247 ± 35 34 ± 1
66492 ± 8301 250 ± 29 42 ± 6
97509 ± 4370 218 ± 45 34 ± 5
96348 ± 20544 207 ± 59 37 ± 4
84556 ± 10242 210 ± 54 32 ± 3
69222 ± 4852 267 ± 13 36 ± 5
Distal segments
2
Fascicular area (μm ) Fascicular diameter (μm) Percentage of occupancy (%)
Distal segments
Distal segments
Right
Left
Right
Left
Right
Left
76887 ± 7029 164 ± 36 34 ± 1
61123 ± 9145 142 ± 25 33 ± 2
84397 ± 8979 191 ± 60 30 ± 2
70456 ± 15573 175 ± 28 34 ± 3
76806 ± 8149 197 ± 30 30 ± 4
61182 ± 3082 154 ± 15 27 ± 1
No differences were observed between sides, segments or groups. Data are expressed as mean ± SEM.
between segments and sides. Likewise, no differences were observed between groups for the myelinated fibers morphometric parameters. Myelinated axons were significantly
smaller in Group III nerves, compared to Groups I and II. The average g ratio values showed the same differences between groups observed for the axons.
Fig. 3 – Average myelinated fiber and Scwann cell nucleus number and density of the sural nerve of female Wistar rats aged 360, 640 and 720 days. Data are expressed as mean ± SEM. No morphometric differences were observed between segments, sides and groups.
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Table 2 – Myelinated fiber (MF), myelin sheath (MS) and myelinated axon (MA) average morphometric parameters of the proximal and distal segments of the sural nerves in both sides Group I (360 days)
Group II (640 days)
Group III (720 days)
Proximal segments
Proximal segments
Proximal segments
Right MF area (μm2) MF diameter (μm) MS area (μm2) MA area (μm2) MA diameter (μm) g ratio
34 ± 5.7 ± 24 ± 10 ± 3.0 ± 0.52 ±
3 0.2 2 1 0.2 0.02
Left 37 ± 5.9 ± 25 ± 11 ± 3.2 ± 0.53 ±
1 0.1 1 1 0.1 0.01
Distal segments Right 2
MF area (μm ) MF diameter (μm) MS area (μm2) MA area (μm2) MA diameter (μm) g ratio
35 ± 5.5 ± 25 ± 10 ± 3.0 ± 0.52 ±
3 0.3 1 1 0.2 0.02
Left
43 ± 3 6.2 ± 0.2 30 ± 2 14 ± 1 3.5 ± 0.1 0.55 ± 0.01
40 ± 4 5.9 ± 0.3 28 ± 3 11 ± 2 3.2 ± 0.2 0.53 ± 0.02
Right 34 5.5 25 9 2.7 0.49
3 0.3 2 1 0.2 0.01
Right
Left
40 ± 3 6.1 ± 0.2 27 ± 2 13 ± 2 3.6 ± 0.2 0.58 ± 0.02
44 ± 5 6.2 ± 0.3 31 ± 3 13 ± 5 3.4 ± 0.2 0.54 ± 0.01
Left
±3 ± 0.2 ±2 ±1 ± 0.2* ± 0.02*
Distal segments
Left 35 ± 5.6 ± 25 ± 11 ± 3.0 ± 0.53 ±
Right
37 ± 5.8 ± 26 ± 10 ± 2.7 ± 0.49 ±
2 0.2 1 1 0.1* 0.01*
Distal segments Right 32 5.4 25 7 2.5 0.46
±2 ± 0.1 ±1 ± 1* ± 0.1* ± 0.01*
Left 30 ± 2 5.3 ± 0.1 23 ± 1 7 ± 1* 2.5 ± 0.1* 0.47 ± 0.01*
No differences were observed between segments and sides. * indicates differences between groups. Data are expressed as mean ± SEM.
The size distributions of myelinated fibers, myelinated axons and g ratio are shown in Figs. 4–6, respectively. Myelinated fiber diameter (Fig. 4) ranged between 1.5 and 12.0 μm, and was distributed bimodally, with peaks of frequency between 3–4 and 5–6 μm in all experimental groups. No differences were observed between segments, sides and groups. Myelinated axon diameter (Fig. 5) ranged between 0.5 and 10.0 μm, with an unimodal distribution. Peaks of frequency between 3–4 μm were observed for Groups I and II, while Group III showed a high peak at 2.0 μm, with a shift of the histogram to the left. A reduction of the frequency of axons between 4–8 μm was also noted in Group III. Groups I and II g ratio distributions (Fig. 6) ranged between 0.2 and 0.9, with a peak of frequency at 0.6 while Group III distribution showed the same range as Groups I and II but the peak of frequency was shifted to the left, at 0.5. No differences between sides and segments were observed in all groups, whereas the distributions between groups differed significantly for the axons and g ratio.
3.
Discussion
The present study showed that the morphology and morphometric parameters of the sural nerves are altered with aging, being diverse elements affected differently. Morphological alterations were found predominantly on the myelin sheaths of large myelinated fibers. These alterations of the sural nerves are similar to those of the ventral root in 30 months old female Fisher rats (Knox et al., 1989) and differ significantly from the observations in the tibial and plantar nerves of male Wistar rats (Sharma et al., 1980). Sharma et al. (1980) described that in the tibial and plantar nerves of male Wistar rats, after 18 months, the proportion of abnormal fibers increased rapidly and Wallerian type axonal degeneration and regeneration predominated. These differences between the present
study and the findings in male rats (Sharma et al., 1980) suggest that despite the peripheral nerves alteration due to aging, males and females might present different types of aging neuropathies. Nevertheless, it is important to take into account that we studied a mainly sensory nerve while Sharma et al. (1980) used sensory-motor nerves. One could speculate that there might be differences between motor and sensory fibers injury due to aging but this issue was not yet investigated. The main morphological alterations found in the present study were infolded myelin sheaths and myelin loops. Myelin loops and splitting are present in normal nerves as the result of changes in the size of the myelinated axons and an appropriate adjustment in their myelin sheath (Ceballos et al., 1999). However, the increased frequency of these alterations with aging has been related to an early response of large fiber myelin sheaths to axonal atrophy (Krinke et al., 1988), as shown by the morphometric approach in the present study. These alterations would have a direct effect on the reduced conduction velocity of the large myelinated fibers, described for other species (Peters, 2002). The alterations observed in the myelinated fibers might be directly related to the morphological alterations of the endoneural blood vessels, which might be affecting the responsiveness of the Schwann cells to aging, as follows. The number of Schwann cells associated with myelinated fibers and sectioned at the level of the nucleus increased from Group I to Group II and decreased latter. This result is similar to that in the tibial nerve of mice (Ceballos et al., 1999) but is being described for the first time in rats. It is well known that Schwann cells are important in the regeneration processes that follow nerve injury. Nerves that are experiencing regeneration will have a larger number of Schwann cells due to their augmented duplication rate under these circumstances (Thomas, 1948). Our results suggest that Group II nerves might have been under regeneration but this process was not sustained in older animals. Since the blood vessels of animals
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Fig. 4 – Frequency distribution histograms of myelinated fibers from proximal and distal segments of the right and left sural nerves in female Wistar rats aged 360, 640 and 720 days. No morphometric differences were observed between segments, sides and groups. from group III were severely damaged, multiplication of the Schwann cells in order to accomplish the regeneration process could be impaired by a poor blood supply of the endoneural space. The number of fibers in a peripheral nerve is known to be constant during the adulthood lifespan (Sharma et al., 1980; Berthold et al., 1984; Schellens et al., 1993; Jeronimo et al., 2005), thus not correlated with age. On the other hand, it is well known that the myelinated fiber density decreases with development and aging, due to the increase in the amount of the endoneural connective tissue (Jacobs and Love, 1985; Schellens et al., 1993; Ceballos et al., 1999; Jeronimo et al., 2005). In the present study, despite the tendency of a decrease on the fiber number from Group I to Group III, differences were not observed. The fact that there was no significant difference in the myelinated fiber numbers between groups may be explicable in terms of the wide range of inter-animal variability which would tent to obscure fiber losses. This observation is similar to those in the tibial nerves
of mice (Ceballos et al., 1999) and male rats (Sharma et al., 1980) and the sural nerve of female Fisher rats (Knox et al., 1989). Despite that Knox et al. (1989) have performed a morphometric study of the peripheral nervous system in aged female rats (Fisher brand), including the sural nerve, the present study adds new information to this field, specially those related to the Schwann cell nuclei, the g ratio, and the distributions of the myelinated fiber and respective axon sizes. The average morphometric parameters of the myelinated fibers and respective axons showed no myelinated fiber changes but reduced axon area and diameter, as well as reduced myelin sheath area and g ratio in Group III nerves, more evident on the distal segments. The myelinated fiber diameter distribution was similar between groups while the axon distribution was significantly shifted to the left in Group III. A similar shift to the left was observed for the g ratio distributions. These data put together is highly indicative of the presence of axonal atrophy in the myelinated fibers of the
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Fig. 5 – Frequency distribution histograms of myelinated axons from proximal and distal segments of the right and left sural nerves in female Wistar rats aged 360, 640 and 720 days. Histograms differed significantly between groups. Note that 720 days old histograms are shifted to the left, with a higher peak at small axons and that there is a reduction on the frequency of larger fibers.
sural nerves of aged female rats. This observation is similar to the description of a moderate number of fine or medium-sized axons with disproportionately thick myelin sheaths in the sural nerve of humans over 60 years of age (Jacobs and Love, 1985). Sharma et al. (1980) showed reduced average diameter of the myelinated fibers in the tibial and plantar nerves of male aged rats, with no differences on the fiber distributions, which also partially corroborate our results. Nevertheless, these authors did not study the axon distributions. Knox et al. (1989) did not show differences in the myelinated fiber and axon diameters average values in the sural nerves of female Fisher rats, aged 10, 20 and 30 months. However, they did not study the distributions of these diameters and no information on the g ratio was given. An important difference between that study (Knox et al. 1989) and the present is that Knox et al. (1989) used a sampling method to study about 600 myelinated fibers while in the present study, the whole nerve was in-
vestigated. It is well established that nerves with a large fiber population may have a heterogeneous fiber distribution, which would introduce a bias with the use of any sampling scheme (Torch et al., 1989; Romero et al., 2000). Measurement of small myelinated fibers is considered difficult because they are generally harder to stain and also because with manual morphometry techniques or sampling schemes, the observers might underestimate them (Ellis et al., 1980; Mezin et al., 1994; Da Silva et al., 2007). In the present study, no underestimate of the small myelinated fibers was done because no sampling method was used. Correlation between the myelin sheath and the diameter of the respective axon has been known since Donaldson and Hoke in 1905 (Donaldson and Hoke, 1905), and may differ significantly between nerves and between large and small fibers within an individual nerve (Fraher, 1992). Rushton (1951) suggested that values between 0.6 and 0.7 would be the
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Fig. 6 – Frequency distribution histograms of myelinated fibers g ratio from proximal and distal segments of the right and left sural nerves in female Wistar rats aged 360, 640 and 720 days. The histograms differed significantly between groups. Note that most of myelinated fibers of the sural nerves in Groups I and II have g ratios of 0.6 wile fibers in Group III g ratios of 0.5.
g ratio for the best and maximum conduction velocity of a myelinated fiber. The present study showed reduced average g ratio and a shift of the histograms to the left in Group III nerves. These results suggest that the myelinated fibers of the oldest animals might have a reduced conduction velocity.
4.
Concluding remarks
In the present study, morphological alterations were more evident in the large myelinated fibers and endoneural blood vessels. These observations suggested the presence of a demyelinating neuropathy due to aging in female rats, which is similar to the aging neuropathy described for male rats. Nevertheless, the sophisticated morphometric approach used in the present study revealed the presence of an axonal neuropathy, mainly associated to the small myelinated fibers, which is being described for the first time. In conclusion, the present
study indicates that the aging neuropathy present in the sural nerve of female rats is both axonal and demyelinating.
5.
Experimental procedure
Experiments were performed on 14 female Wistar rats, randomly divided into three groups. Group I animals were 360 days old (N = 5), Group II animals were 640 days old (N = 5) and Group III animals were 720 days old (N = 4). The animals were born and raised in the animal care facility of the Department of Physiology, Federal University of Triângulo Mineiro, in a controlled environment (room temperature between 21–23 °C, air humidity between 40 and 70% and dark/light cycle of 12 hs), housed in plastic cages (3–4 animals to a cage) with free access to tap water and rat chow throughout the experiments. All experimental procedures adhered to The Guide for the Care and Use of Laboratory Animals
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prepared by the National Academy of Sciences and published by the National Institutes of Health (Copyright © 1996 by the National Academy of Sciences), and were approved by the Ethics Committee for Animal Research of the School of Medicine of Ribeirão Preto – University of São Paulo (CETEA – Comitê de Ética em Experimentação Animal). Effort was made to minimize the number of animals used. At 360, 640 and 720 days of age, animals were anaesthetized with sodium pentobarbital (Nembutal, 40 mg kg, 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. 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 cut and processed for epoxy resin embedding (PolyBed 812®, Polysciences Inc., Warrington, PA, USA) as described elsewhere (Jeronimo et al., 2005; Campos et al., 2007; Alcântara et al., 2008). Samples of all three experimental groups were histologically processed at once so that they were submitted to absolutely the same experimental conditions throughout the experiments. Semithin (0.2 - 0.3 μm thick) transverse sections of the fascicles were stained with 1% toluidine blue and examined with the aid of an Axiophot II photomicroscope (Carl Zeiss, Jena, Germany). The images were sent via a digital camera (TK1270, JVC, Victor Company of Japan Ltd, Tokyo, Japan) to an IBM/PC where they were digitized. For the study of myelinated fibers, the endoneural space was observed with an optical set including an oil immersion lens (100 x), optovar (1.6 x), camera (0.5 x) and an 8 x computerized magnification, which provided images with good resolution for morphometry. The endoneural space was fully scanned without overlap of the microscopic fields, using an automatic motorized stage (Carl Zeiss, Jena, Germany). Scannings generated 9 to 17 microscopic fields of 640 x 470 pixels, which were used to count and automatically measure the myelinated fibers and their respective axons. Fibers at the upper and left edges of the microscopic fields were counted whereas those at the lower and right edges were not counted (“forbidden line”) in order to avoid counting the same fiber twice. All myelinated fibers present in the endoneural space were counted. Morphometric parameters of the fascicles and myelinated fibers of sural nerve segments were obtained as described previously for other nerves (Fazan et al., 1997; Fazan et al., 1999; Rodrigues Filho and Fazan, 2006; Campos et al., 2007; Alcântara et al., 2008). Briefly, the total number of myelinated fibers 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) as well as each myelinated fiber (defined by the axon and its respective myelin sheath excluding the Schwann cell nucleus when present) and respective axon (fiber excluding the myelin sheath) were measured with image analysis software (KS 400, Kontron 2.0, Eching Bei München, Germany). The lesser diameter better represents the diameter of a non-circular fascicle and fiber
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(Blight and Decrescito, 1986; Auer, 1994; Jeronimo et al., 2005). The percentage of the total cross-sectional area of the endoneural space occupied by the myelinated fibers was calculated, and hereafter referred as the percentage of occupancy of the myelinated fibers (Jeronimo et al., 2005, Campos et al., 2007; Alcântara et al., 2008). The myelinated fibers and Schwann cell nuclei densities were calculated. For myelinated fibers, both axonal diameter and total fiber diameter were automatically measured. The ratio between the two diameters, the g ratio (which indicates the degree of myelination), was obtained (Rushton, 1951; Smith and Koles, 1970). Myelin sheath area was calculated for each myelinated fiber measured. Histograms of population distribution of myelinated fibers and axons, separated into class intervals increasing by 1.0 μm were constructed. Histograms of the g ratio distribution separated into class intervals increasing by 0.1 were also prepared. The investigators were blind to group identities throughout the experiments. Morphometric data were tested for normal distribution by the Kolmogorov–Smirnov normality test followed by the Levene test for variance equivalence. If data presented a normal distribution and equivalent variance, comparisons were made between proximal and distal segments in the same group by 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. Alternatively, comparisons were made by the Mann–Whitney non-parametric test. Comparisons between groups were made by one-way analysis of variance (ANOVA) followed by HolmSidak post hoc test. Comparisons between histograms were made by one-way analysis of variance (ANOVA) on Ranks provided that the distributions did not pass the normality test. Differences were considered significant if p b 0.05. Data are presented as mean ± standard error of the mean (SEM).
Acknowledgments We thank Mr. Antônio Renato Meirelles e Silva, Experimental Neurology Laboratory, and Ms. Maria Tereza Maglia, Electron Microscopy Laboratory, School of Medicine of Ribeirão Preto, for excellent technical support. Grant sponsor: FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo); Grant numbers: 04/09139–2, 04–01390–8 and 06/03200–7; Grant sponsor: CNPq (Conselho Nacional de Pesquisa e Tecnologia); Grant number: 303802/2006–5.
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