- Email: [email protected]
Effects of aging on numbers, sizes and conduction velocities of myelinated and unmyelinated fibers of the pelvic nerve in rats
Journal of the Autonomic Nervous System 69 Ž1998. 148–155
Effects of aging on numbers, sizes and conduction velocities of myelinated and unmyelinated fibers of the pelvic nerve in rats Hitomi Nakayama a , Koichi Noda b, Harumi Hotta a
a,)
, Hideo Ohsawa c , Yasuhiko Hosoya
d
Department of the Autonomic NerÕous System, Tokyo Metropolitan Institute of Gerontology, Tokyo 173, Japan b Department of Ultrastructure Research, Tokyo Metropolitan Institute of Gerontology, Tokyo 173, Japan c Laboratory of Physiology, Tsukuba College of Technology, Tsukuba, Ibaraki 305, Japan d College of Medical Technology and Nursing, UniÕersity of Tsukuba, Tsukuba, Ibaraki 305, Japan Received 28 November 1997; revised 25 December 1997; accepted 25 December 1997
Abstract The effects of aging on the conduction velocities, numbers and sizes of the myelinated and unmyelinated fibers of the pelvic nerve in Wistar rats, aged 3–9 and 30–37 months were examined using electrophysiological and ultrastructural techniques. The myelinated fibers did not show significant age-related changes in the maximum conduction velocities Ž9.7 " 1.1 mrs in adult vs. 11.0 " 1.5 mrs in aged rats., the number of fibers Ž662 " 27 vs. 625 " 56. and distribution of fiber diameters. In aged rats, the unmyelinated fibers did not show a significant change in maximum conduction velocity Ž1.5 " 0.1 mrs vs. 1.5 " 0.1 mrs., but showed a significant decrease in the number of fibers Ž4133 " 114 vs. 3113 " 456., specifically of fibers smaller than 0.7 m m in diameter. It is concluded that myelinated fibers of the pelvic nerve in rats preserve their conduction ability and fiber population during aging, but unmyelinated fibers, particularly fibers with smaller diameters decrease in number. Unmyelinated fibers with larger diameters maintain their conduction ability and numbers with aging. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Pelvic nerve; Myelinated fiber; Unmyelinated fiber; Conduction velocity; Aging; Rat
1. Introduction It has generally been accepted that the peripheral nervous system deteriorates with age, as does the central nervous system. There is an accumulation of electrophysiological and morphological studies on the effects of aging on somatic peripheral nerves. For example, in aged humans and laboratory animals, decline of the conduction velocity of large myelinated peripheral nerve fibers ŽChase et al., 1992; Cruz Martınez ´ et al., 1978; Kanda et al., 1986; Norris et al., 1953; Wagman and Lesse, 1952. and a loss of numbers of myelinated peripheral nerve fibers have been found ŽMittal and Logmani, 1987; Tohgi et al., 1977; Wheeler and Plummer, 1989.. Robertson et al. Ž1993. correlated the slowing of the maximum conduction velocity of the sciatic nerve in aged mice with a loss of large ) Corresponding author. Department of the Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173, Japan. Tel.: q81 3 39643241 ext. 3087; fax: q81 3 35794776; e-mail: [email protected]
0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 1 3 - 7
myelinated fibers and a high incidence of demyelination and atrophy of the nerve axons. In contrast to large myelinated peripheral nerve fibers, small myelinated fibers seem to be resistant to aging in somatic peripheral nerves ŽMittal and Logmani, 1987; Rosenberg et al., 1989; Smith and Rosenheimer, 1984; Tohgi et al., 1977.. For example, the small myelinated fibers of the rat phrenic nerve with conduction velocities of 16–18 mrs do not show age-related changes in conduction velocity, fiber diameter, or myelin sheath width ŽSmith and Rosenheimer, 1984.. The unmyelinated fibers of somatic peripheral nerves in aged humans and laboratory animals do not show age-related changes in the maximum conduction velocity ŽSato et al., 1985., total number ŽRosenberg et al., 1989., density ŽKanda et al., 1991. and diameter ŽRosenberg et al., 1989; Wheeler and Plummer, 1989.. Autonomic nerve fibers consist mainly of small myelinated and unmyelinated fibers ŽPick, 1970.. Thus, as observed in small myelinated and unmyelinated fibers of peripheral somatic nerves, it can be assumed from the past
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
studies of aging effects on peripheral somatic nerves that autonomic nerve fibers are not subject to a large aging effect. Only a few studies have been reported concerning aging effects on conduction velocities ŽSato et al., 1985. or structures ŽAppenzeller and Ogin, 1973; Low and Dyck, 1978; Soltanpour and Santer, 1996. of autonomic nerve fibers. Sato et al. Ž1985. showed that the maximum conduction velocity of myelinated fibers in the rat vagus nerve decreased by about 10%, while the conduction velocity of the unmyelinated fibers was unchanged. Recently, Soltanpour and Santer Ž1996. counted the number of fibers in the vagus nerve, and found no significant difference in the total number of myelinated fibers and a slight decrease in the number of myelinated and unmyelinated fibers per square millimeter in aged rats. Effects of aging on the pelvic nerve, which is distributed widely to the pelvic organs, appear not to have been studied. In view of the major role of the pelvic nerves in micturition and defecation, it is important to examine age-related changes in the pelvic nerve. The present study, combining physiological and morphometric techniques, aimed to investigate age-related changes in the autonomic pelvic nerve of the rat. The first approach to this study was to measure the maximum conduction velocities of both myelinated and unmyelinated pelvic nerve fibers using electrophysiological techniques and the second approach was to measure the number and diameter of both myelinated and unmyelinated pelvic nerve fibers using electron microscopy. 2. Materials and methods The experiments were performed in nine adult Ž3–9 months old. and nine aged Ž30–37 months old. female Wistar rats ŽBW, 140–260 g.. All animals were raised in our institute. Laboratory food ŽCRF1, Charles River, Tokyo. and water were given to animals ad libitum. 2.1. Electrophysiological study Five adult and five aged rats were used. The animals were anesthetized with urethane Ž1.1 grkg i.p.. and the pelvic nerves were dissected out bilaterally under a dissecting microscope, which was essential for manipulating the nerve fibers. A ligature of fine silk thread was tied around the nerve close to the L6-S1 trunk, and another ligature was made at the inferior pole of the major pelvic ganglion. The distance between these ligatures was approximately 10–15 mm. The dissection was made with care to avoid placing any tension on the nerve. Then, the nerve was placed into a glass chamber filled with paraffin oil, heated and stabilized using a heating pad and lamp at 37 " 0.58C, to avoid temperature effects on nerve conduction velocity Žsee the work of Paintal, 1965.. The proximal end of the nerve was placed on a bipolar stimulating electrode with an interpolar distance of about
149
2–3 mm and stimulated by square waves of 0.5–15 V Žpulse duration, 0.1 ms. using an electrical stimulator ŽSEN-7103, Nihon Kohden, Japan.. The compound action potentials were recorded by bipolar recording wire electrodes located at the distal part of the nerve. The stimulus voltage was adjusted to more than that necessary to cause a maximal response; usually 1–2 V for myelinated fibers and 10 V for unmyelinated fibers. The distance between stimulating and recording electrodes was from 5 to 12 mm. Action potentials were amplified by a preamplifier Žtime constant 0.33 s, high cut 10 kHz, S-0476, Nihon Kohden. and displayed on a storage oscilloscope ŽMS-5311, Iwatsu, Japan.. The maximum conduction velocities Žmrs. of myelinated A d and unmyelinated C nerve fibers were calculated by dividing the distance Žmm. between the stimulating Žcathode. and recording electrodes by the onset latency Žms. of each action potentials. The nerve fibers showing conduction velocities slower than 2 mrs were classified as unmyelinated fibers. The conduction velocities were measured on bilateral sides of the pelvic nerve in each animal. Data analysis was carried out by calculating the mean " S.E.M. of each animal. 2.2. Morphometric study Four adult and four aged rats were used. The animals were anesthetized with urethane Ž1.1 grkg i.p... The pelvic nerve, proximal to the major pelvic ganglion, was exposed and covered with a fixative composed of 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.4. After preliminary fixation in situ for about 3 min, the pelvic nerve was removed from surrounding tissues and placed into the fixative on a polytetrafluoroethylene plate. A small segment Žapproximately 3 mm. of the proximal portion of the pelvic nerve was cut under a dissecting microscope, and fixed for 2 h or more at room temperature. At the proximal portion, there is a bifurcation and the pelvic nerve divides into two parts: one is the viscero-cutaneous branch which runs toward the major pelvic ganglion, and the other is the muscular branch which runs toward the perineal muscles ŽPacheco et al., 1989.. We focused on the viscero-cutaneous branch, and the muscular branch was excluded from the present study. The tissues were rinsed with cacodylate buffer, and post-fixed with 2% osmic acid buffered cacodylate for 1.5 h at 0–48C. Then, they were dehydrated through a graded series of ethanols and embedded in Epon-812. Cross-sections of the pelvic nerve were cut on an ultramicrotome ŽLKB Ultratome III, Sweden or Reichert Ultracut E, Austria.. For the light microscopic examination, sections were cut at 1.5 m m and stained with toluidine blue. Tissue samples were taken bilaterally in each rat, and checked for orientation of fibers and preservation of tissues. In each animal, a nerve on one side was selected for further evaluation by electron microscopy. The selected
150
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
nerve tissues were re-trimmed and ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined with an electron microscope ŽH-600, Hitachi, Japan.. Micrographs were obtained at a primary magnification of =3500 or =6000 and printed at 1.5 times the size of the original negatives. Approximately 70 films were exposed for each pelvic nerve, covering its entire area. Images of each photograph were stored in a personal computer. The total numbers and sizes of both myelinated and unmyelinated fibers were measured from the images on the photographs using software designed for this purpose ŽVm 32, version 1.0, Rise, Japan.. Outlines of the cross-sections of myelin sheaths of myelinated fibers were traced manually using either a mouse or a digitizer tablet-pen connected to the computer. The fiber diameter was calculated automatically from the traced outline assuming a circular shape ŽVita et al., 1992; Zimmerman et al., 1980.. Some myelinated fibers were excluded from the measurement of diameter if they were sectioned at the nodes of Ranvier, but these were included among the total number of myelinated fibers. Unmyelinated axons were clearly distinguished from profiles of Schwann cells, because unmyelinated axons showed round or oval profiles, and clearer axoplasmic
Table 1 Maximum conduction velocities of pelvic myelinated A d and unmyelinated C fibers Age group
Animal number
Myelinated fibers Žmrs.
Unmyelinated fibers Žmrs.
Adult
1 2 3 4 5 Average 1 2 3 4 5 Average
6.1 9.8 11.8 8.6 12.3 9.7 8.8 14.6 6.9 14.4 10.2 11.0
1.3 1.4 1.5 1.7 1.5 1.5 1.7 1.5 1.4 1.3 1.4 1.5
Aged
density than Schwann cells ŽHulsebosch and Coggeshall, 1982.. Outlines of unmyelinated axons were traced as described above for the myelinated fibers. A small population of unmyelinated axons Žless than 3% of the total number. were sectioned obliquely. They were excluded from the measurement of diameter, but included among the total number of unmyelinated axons. 2.3. Statistical analysis Data were expressed as mean " S.E.M. Statistical analysis of data was carried out by Mann–Whitney U-test.
Fig. 1. ŽA–D. Typical evoked potentials of pelvic myelinated A d and unmyelinated C fibers from one adult and one aged rat. Mass potentials evoked by single 0.1 ms pulse stimulation Ž2 V: A, B and 10 V: C, D at triangle. recorded from the pelvic nerve, 9 mm peripheral to the stimulation site. Onset Žat arrow. latency of each volley was measured to estimate the maximum conduction velocity for A d and C fibers. ŽE, F. Summary of the maximum conduction velocities of pelvic myelinated A d ŽE. and unmyelinated C ŽF. fibers in adult Ž N s 5, open columns. and aged Ž N s 5, shaded columns. rats. Each column and vertical bar shows mean"S.E.M.
Fig. 2. ŽA, B. Electron micrographs showing typical cross-sections of pelvic nerves in adult ŽA. and aged ŽB. rats. ŽC, D. Columns showing the total numbers of pelvic myelinated ŽC. and unmyelinated ŽD. fibers in adult Ž N s 4, open columns. and aged Ž N s 4, shaded columns. rats. Each column and vertical bar shows mean"S.E.M. ) P - 0.05; statistically significant difference between adult and aged rats by Mann–Whitney U-test.
Age group
Animal number
Total fiber number a
Fiber diameter Ž m m. -1.5 1.5–2.0 2.0–2.5
2.5–3.0
3.0–3.5
3.5–4.0
4.0–4.5
4.5–5.0
5.0–5.5
5.5–6.0
6.0–6.5
6.5–7.0
7.0 F
Adult
6 7 8 9 Average 6 7 8 9 Average
598 737 663 650 662 498 581 660 761 625
29 18 18 73 35 3 20 6 11 10
98 136 186 64 121 171 100 217 238 182
19 36 44 13 28 49 16 88 74 57
15 25 10 8 15 10 6 40 21 19
10 17 5 1 8 5 8 10 4 7
4 9 4 1 5 5 3 7 13 7
6 4 1 1 3 y 2 5 3 3
y 1 1 y 1 3 5 4 3 4
y 1 y y y 1 2 1 1 1
1 y y y y 1 2 2 2 2
1 4 5 4 4 6 2 9 2 5
Aged
a
164 191 97 304 189 66 142 65 108 95
241 287 287 177 248 154 263 196 272 221
Mean diameter
Myelinated fibers which were sectioned at the nodes of Ranvier were included in this number, but excluded from the measurement of diameter.
2.32 2.43 2.46 2.04 2.31 2.57 2.34 2.77 2.68 2.59
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
Table 2 Total number and diameter distribution of myelinated fibers
151
152
Age group
Animal number
Total fiber number a
Fiber diameter Ž m m. - 0.2 0.2–0.3 0.3–0.4
0.4–0.5
0.5–0.6
0.6–0.7
0.7–0.8
0.8–0.9
0.9–1.0
1.0–1.1
1.1–1.2
1.2–1.3
1.3–1.4
1.4–1.5
1.5F
Adult
6 7 8 9 Average 6 7 8 9 Average
4227 4120 4361 3824 4133 1739 3352 3601 3760 3113
98 57 25 76 64 13 8 2 4 7
709 635 639 628 653 211 609 453 551 456
626 645 711 763 686 266 569 515 563 478
546 460 602 449 514 224 437 414 433 377
367 335 411 266 345 249 332 269 368 305
223 280 352 231 272 238 299 375 439 338
125 281 239 114 190 125 169 305 334 233
57 240 225 62 146 69 105 266 238 170
18 136 125 28 77 52 84 224 186 137
6 68 45 9 32 35 32 93 76 59
5 19 16 6 12 16 10 114 60 50
1 6 7 4 5 8 y 41 10 15
y 37 1 6 11 1 2 47 12 16
Aged
a
634 335 397 679 511 70 272 123 148 153
812 586 565 503 617 162 424 260 338 296
Mean diameter
Unmyelinated fibers which were sectioned obliquely were included in this number, but excluded from the measurement of diameter.
0.51 0.63 0.61 0.52 0.57 0.70 0.60 0.75 0.68 0.68
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
Table 3 Total number and diameter distribution of unmyelinated fibers
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
153
3. Results 3.1. Electrophysiological study Typical evoked potentials of pelvic nerves, obtained from two adult and aged rats, are demonstrated in Fig. 1A–D. In both adult and aged rats, we observed evoked potentials of A d myelinated fibers in pelvic nerves stimulated at an intensity of 2 V. These had similar latencies of 0.7–0.9 ms and similar durations of 3.0–3.5 ms. Increasing stimulus intensity to 10 V produced an additional evoked potential of unmyelinated C fibers with a longer latency of 6–6.5 ms and a duration of 14–18 ms, in both adult and aged rats. The maximum conduction velocities of myelinated A d and unmyelinated C fibers obtained from five animals in each of the two groups ŽTable 1. are summarized in Fig. 1E and F. The maximum conduction velocity of A d myelinated fibers was 9.7 " 1.1 mrs in adult and 11.0 " 1.5 mrs in aged rats ŽFig. 1E.. It was slightly, but not statistically higher in aged rats. The maximum conduction velocities of the unmyelinated fibers in adult and aged rats were 1.5 " 0.1 mrs and 1.5 " 0.1 mrs, respectively ŽFig. 1F.. There was no significant difference between the adult and aged groups.
Fig. 3. Total distribution histograms of myelinated ŽA. and unmyelinated ŽB. fibers. The values are derived from averages of pooled data in adult Ž N s 4, open bars with heavy borders. and aged Ž N s 4, shaded bars. rats.
3.2. Morphometric study Electron micrographs ŽFig. 2, B. show representative cross-sections of pelvic nerves of adult and aged rats. There were regional differences in the distributions of nerve fibers with respect to their sizes. Total numbers of nerve fibers and fiber sizes were measured in eight pelvic nerves, obtained from four adult and four aged rats ŽTables 2 and 3.. The total numbers of myelinated and unmyelinated fibers are shown and summarized in Fig. 2C and D, respectively. The means of the total numbers of myelinated fibers were 662 " 27 in adult rats and 625 " 56 in aged rats. There was no statistically significant difference between the numbers of myelinated fibers in adult and aged rats. The means of the total numbers of unmyelinated fibers were 4133 " 114 in adult rats and 3113 " 456 in aged rats. In aged rats, the numbers of unmyelinated fibers were significantly decreased Ž P - 0.05. by approximately 25% of the value in adult rats. The distributions of diameters of myelinated fibers in adult and aged pelvic nerves are shown in Fig. 3A. The myelinated fiber diameters ranged between 1 m m and 13 m m, and were distributed unimodally. More than 80% of the myelinated fibers were smaller than 3 m m in diameter in both adult and aged rats. Only 0.5–0.8% of the myelinated fibers were larger than 7 m m, which is beyond the range of A d Žgroup III. fibers ŽBoyd and Davey, 1968.. The numbers of myelinated fibers in each 13 different diameter range as shown in Table 2 did not show signifi-
cant age-related changes. The mean diameters of myelinated fibers were 2.31 " 0.10 m m and 2.59 " 0.09 m m in adult and aged rats, respectively. There was a tendency to be larger in diameter in aged rats but this was not statistically significant. The distributions of unmyelinated fiber diameters are shown in Fig. 3B. The unmyelinated fiber diameters ranged between 0.1 m m and 1.8 m m. The number of unmyelinated fibers smaller than 0.7 m m was selectively decreased in aged rats, while the numbers of unmyelinated fibers larger than 0.7 m m showed no change. The means of the numbers of unmyelinated fibers smaller than 0.7 m m Ž3045 " 149 in adult rats vs. 1767 " 296 in aged rats. were significantly decreased Ž P - 0.05. in aged rats by approximately 42% of the value in adult rats. The mean of the numbers of unmyelinated fibers larger than 0.7 m m Ž1088 " 188 in adult rats vs. 1321 " 240 in aged rats. did not show a significant difference. The mean diameters of unmyelinated fibers were 0.57 " 0.03 m m and 0.68 " 0.03 m m in adult and aged rats, respectively. There was a tendency to larger diameter in aged rats, but this was not statistically significant.
4. Discussion The present study is the first demonstration in aged rats of a reduction Žby 25%, in this case. of the number of unmyelinated fibers in the pelvic nerve, specifically fibers
154
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
smaller than 0.7 m m in diameter. This study also demonstrated that there were no significant changes in the maximum conduction velocity, total numbers and diameters of myelinated fibers and the maximum conduction velocity of unmyelinated fibers. 4.1. Myelinated fibers The present electrophysiological study showed that the maximum conduction velocity of myelinated fibers of the pelvic nerve is approximately 10 mrs, and the diameters of most fibers were smaller than 7 m m. These results indicate that most myelinated fibers of the pelvic nerve in rats are A d fibers. However, the present histological study showed that there were a small number of myelinated fibers Žless than 1% of the total number. that had larger diameters beyond the range of A d fibers. It is certain that we missed recording the evoked action potentials of the larger myelinated fibers, beyond the A d fibers. This is because the interpolar distances between stimulating and recording electrodes were too short to record action potentials whose conduction velocities were much faster than those of the A d fibers recorded in the present study. As in small peripheral somatic myelinated fibers ŽMittal and Logmani, 1987; Rosenberg et al., 1989; Smith and Rosenheimer, 1984; Tohgi et al., 1977. we could not notice any physiological or morphological changes in myelinated A d fibers of the pelvic nerve. The small myelinated A fibers in both visceral and somatic nerves seem to be resistant to aging, in contrast to large myelinated fibers of somatic peripheral nerves in which demyelination and axonal atrophy have been reported during aging ŽRobertson et al., 1993.. The maximum conduction velocity of peripheral motor fibers has been shown to decrease by about 10–30% in both aged humans ŽBouche et al., 1993; Dorfman and Bosley, 1979; Norris et al., 1953; Wagman and Lesse, 1952. and laboratory animals ŽWayner and Emmers, 1958.. Afferent sensory fibers has been reported to maintain their maximum conduction velocities at normal adult levels until advanced age both in the sural nerve of humans ŽBurke et al., 1974. and in the nerve innervating the flexor hallucis longus muscle of rats ŽWayner and Emmers, 1958.. However, decreased conduction velocity of sensory nerves with age has also been shown in muscle spindles innervating the medial gastrocnemius muscle of rats ŽMiwa et al., 1995. as well as in median nerve and sural nerve of humans ŽBouche et al., 1993; Dorfman and Bosley, 1979.. The major portion of myelinated fibers in the pelvic nerve of the rat are sensory fibers ŽHulsebosch and Coggeshall, 1982; Purinton et al., 1973. and responsiveness of pelvic afferent nerve fibers to increases in intravesical pressure is well maintained in aged rats ŽHotta et al., 1995.. Therefore, it is likely that sensory myelinated fibers in autonomic nerves are resistant to aging.
4.2. Unmyelinated fibers The present study demonstrated that the pelvic nerve was composed primarily of unmyelinated fibers and also that the total number of unmyelinated fibers decreased during aging. It was noticeable that numbers of thin unmyelinated fibers smaller than 0.7 m m in diameter were selectively decreased with aging, while unmyelinated fibers larger than 0.7 m m in diameter did not change with aging. Therefore, it is reasonable that the maximum conduction velocities of unmyelinated fibers, which were calculated from the beginning of the evoked action potential of myelinated fibers and reflect the action potentials of largesized unmyelinated fibers, would not reveal any age-related change. Parasympathetic preganglionic nerves innervating the urinary bladder ŽMallory et al., 1989. and the uterus ŽSato et al., 1996. of the rat consist exclusively of unmyelinated fibers. The major portion of unmyelinated fibers in the pelvic nerve of the rat are parasympathetic preganglionic fibers; only 29% of the unmyelinated fibers are sensory fibers ŽHulsebosch and Coggeshall, 1982.. During aging, the number of preganglionic autonomic neurons has been shown to decrease at the levels of the T6–T8 rami communicantes in humans ŽLow and Dyck, 1978. and in the dorsal motor nucleus of the vagus in mice ŽSturrock, 1990.. Contractility of the urinary bladder induced by electrical stimulation of the pelvic efferent nerves declines with age ŽHotta et al., 1995.. Taken in consideration with these previous findings, the selective loss of small unmyelinated fibers in the pelvic nerve, as found in the present study, may be characteristic in parasympathetic efferent fibers. Although, in 4- and 24-month-old rats, Dering et al. Ž1996. reported no significant age-related differences in any of the parameters they measured, i.e. soma area, number of primary dendrites, number of dendritic branch points and total dendritic length, for the parasympathetic neurons in the lumbosacral spinal cord projecting to the hypogastric ganglion, total numbers of the parasympathetic neurons may decrease during aging. The decrease in the number of thin unmyelinated fibers in the pelvic nerve may be one of various causative factors for the age-related deterioration of micturition function.
Acknowledgements This work was supported by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Sasagawa Medical Research Foundation. The authors would like to thank Ms C. Kanai for excellent technical assistance, and Dr A. Sato and Dr Y. Sato for their advice and encouragement in performing this study.
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
References Appenzeller, O., Ogin, G., 1973. Myelinated fibres in the human paravertebral sympathetic chain; quantitative studies on white rami communicantes. J. Neurol. Neurosurg. Psychiatry 36, 777–785. Bouche, P., Cattelin, F., Saint-Jean, O., Leger, J.M., Queslati, S., Guez, ´ D., Moulonguet, A., Brault, Y., Aquino, J.P., Simunek, P., 1993. Clinical and electrophysiological study of the peripheral nervous system in the elderly. J. Neurol. 240, 263–268. Boyd, I.A., Davey, M.R., 1968. Composition of Peripheral Nerves. Livingstone, Edinburgh, 57 pp. Burke, D., Skuse, N.F., Lethlean, A.K., 1974. Sensory conduction of the sural nerve in polyneuropathy. J. Neurol. Neurosurg. Psychiatry 37, 647–652. Chase, M.H., Engelhardt, J.K., Adinolfi, A.M., Chirwa, S.S., 1992. Age-dependent changes in cat masseter nerve: an electrophysiological and morphological study. Brain Res. 586, 279–288. Cruz Martınez, A., Barrio, M., Perez Conde, M.C., Gutierrez, A.M., ´ ´ ´ 1978. Electrophysiological aspects of sensory conduction velocity in healthy adults. J. Neurol. Neurosurg. Psychiatry 41, 1092–1096. Dering, M.A., Santer, R.M., Watson, A.H., 1996. Age-related changes in the morphology of preganglionic neurons projecting to the rat hypogastric ganglion. J. Neurocytol. 25, 555–563. Dorfman, L.J., Bosley, T.M., 1979. Age-related changes in peripheral and central nerve conduction in man. Neurology 29, 38–44. Hotta, H., Morrison, J.F.B., Sato, A., Uchida, S., 1995. The effects of aging on the rat bladder and its innervation. Jpn. J. Physiol. 45, 823–836. Hulsebosch, C.E., Coggeshall, R.E., 1982. An analysis of the axon populations in the nerves to the pelvic viscera in the rat. J. Comp. Neurol. 211, 1–10. Kanda, K., Hashizume, K., Nomoto, E., Asaki, S., 1986. The effects of aging on physiological properties of fast and slow twitch motor units in the rat gastrocnemius. Neurosci. Res. 3, 242–246. Kanda, T., Tsukagoshi, H., Oda, M., Miyamoto, K., Tanabe, H., 1991. Morphological changes in unmyelinated nerve fibers in the sural nerve with age. Brain 114, 585–599. Low, P.A., Dyck, P.J., 1978. Splanchnic preganglionic neurons in man: III. Morphometry of myelinated fibers of rami communicantes. J. Neuropathol. Exp. Neurol. 37, 734–740. Mallory, B., Steers, W.D., DeGroat, W.C., 1989. Electrophysiological study of micturition reflexes in rats. Am. J. Physiol. 257, R410–R421. Mittal, K.R., Logmani, F.H., 1987. Age-related reduction in 8th cervical ventral nerve root myelinated fiber diameters and numbers in man. J. Gerontol. 42, 8–10. Miwa, T., Miwa, Y., Kanda, K., 1995. Dynamic and static sensitivities of muscle spindle primary endings in aged rats to ramp stretch. Neurosci. Lett. 201, 179–182. Norris, A.H., Shock, N.W., Wagman, I.H., 1953. Age changes in the
155
maximum conduction velocity of motor fibers of human ulnar nerves. J. Appl. Physiol. 5, 589–593. Pacheco, P., Martinez-Gomez, M., Whipple, B., Beyer, C., Komisaruk, B.R., 1989. Somato-motor components of the pelvic and pudendal nerves of the female rat. Brain Res. 490, 85–94. Paintal, A.S., 1965. Effects of temperature on conduction in single vagal and saphenous myelinated nerve fibres of the cat. J. Physiol. 180, 20–49. Pick, J., 1970. The Autonomic Nervous System. Lippincott, Philadelphia, 483 pp. Purinton, P.T., Fletcher, T.F., Bradley, W.E., 1973. Gross and light microscopic features of the pelvic plexus in the rat. Anat. Rec. 175, 697–706. Robertson, A., Day, B., Pollock, M., Collier, P., 1993. The neuropathy of elderly mice. Acta Neuropathol. 86, 163–171. Rosenberg, S.I., Malmgren, L.T., Woo, P., 1989. Age-related changes in the internal branch of the rat superior laryngeal nerve. Arch. Otolaryngol. Head Neck Surg. 115, 78–86. Sato, A., Sato, Y., Suzuki, H., 1985. Aging effects on conduction velocities of myelinated and unmyelinated fibers of peripheral nerves. Neurosci. Lett. 53, 15–20. Sato, Y., Hotta, H., Nakayama, H., Suzuki, H., 1996. Sympathetic and parasympathetic regulation of the uterine blood flow and contraction in the rat. J. Auton. Nerv. Syst. 59, 151–158. Smith, D.O., Rosenheimer, J.L., 1984. Factors governing speed of action potential conduction and neuromuscular transmission in aged rats. Exp. Neurol. 83, 358–366. Soltanpour, N., Santer, R.M., 1996. Preservation of the cervical vagus nerve in aged rats: morphometric and enzyme histochemical evidence. J. Auton. Nerv. Syst. 60, 93–101. Sturrock, R.R., 1990. A comparison of age-related changes in neuron number in the dorsal motor nucleus of the vagus and the nucleus ambiguus of the mouse. J. Anat. 173, 169–176. Tohgi, H., Tsukagoshi, H., Toyokura, Y., 1977. Quantitative changes with age in normal sural nerves. Acta Neuropathol. ŽBerl.. 38, 213–220. Vita, G., Santoro, M., Trombetta, G., Leonardi, L., Messina, C., 1992. A computer-assisted automatic method for myelinated nerve fiber morphometry. Acta Neurol. Scand. 85, 18–22. Wagman, I.H., Lesse, H., 1952. Maximum conduction velocities of motor fibers of ulnar nerve in human subjects of various ages and sizes. J. Neurophysiol. 15, 235–244. Wayner, M.J. Jr., Emmers, R., 1958. Spinal synaptic delay in young and aged rats. Am. J. Physiol. 194, 403–405. Wheeler, S.J., Plummer, J.M., 1989. Age-related changes in the fibre composition of equine peripheral nerve. J. Neurol. Sci. 90, 53–66. Zimmerman, I.R., Karnes, J.L., O’Brien, P.C., Dyck, P.J., 1980. Imaging system for nerve and fiber tract morphometry: components, approaches, performance, and results. J. Neuropathol. Exp. Neurol. 39, 409–419.
Effects of aging on numbers, sizes and conduction velocities of myelinated and unmyelinated fibers of the pelvic nerve in rats Hitomi Nakayama a , Koichi Noda b, Harumi Hotta a
a,)
, Hideo Ohsawa c , Yasuhiko Hosoya
d
Department of the Autonomic NerÕous System, Tokyo Metropolitan Institute of Gerontology, Tokyo 173, Japan b Department of Ultrastructure Research, Tokyo Metropolitan Institute of Gerontology, Tokyo 173, Japan c Laboratory of Physiology, Tsukuba College of Technology, Tsukuba, Ibaraki 305, Japan d College of Medical Technology and Nursing, UniÕersity of Tsukuba, Tsukuba, Ibaraki 305, Japan Received 28 November 1997; revised 25 December 1997; accepted 25 December 1997
Abstract The effects of aging on the conduction velocities, numbers and sizes of the myelinated and unmyelinated fibers of the pelvic nerve in Wistar rats, aged 3–9 and 30–37 months were examined using electrophysiological and ultrastructural techniques. The myelinated fibers did not show significant age-related changes in the maximum conduction velocities Ž9.7 " 1.1 mrs in adult vs. 11.0 " 1.5 mrs in aged rats., the number of fibers Ž662 " 27 vs. 625 " 56. and distribution of fiber diameters. In aged rats, the unmyelinated fibers did not show a significant change in maximum conduction velocity Ž1.5 " 0.1 mrs vs. 1.5 " 0.1 mrs., but showed a significant decrease in the number of fibers Ž4133 " 114 vs. 3113 " 456., specifically of fibers smaller than 0.7 m m in diameter. It is concluded that myelinated fibers of the pelvic nerve in rats preserve their conduction ability and fiber population during aging, but unmyelinated fibers, particularly fibers with smaller diameters decrease in number. Unmyelinated fibers with larger diameters maintain their conduction ability and numbers with aging. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Pelvic nerve; Myelinated fiber; Unmyelinated fiber; Conduction velocity; Aging; Rat
1. Introduction It has generally been accepted that the peripheral nervous system deteriorates with age, as does the central nervous system. There is an accumulation of electrophysiological and morphological studies on the effects of aging on somatic peripheral nerves. For example, in aged humans and laboratory animals, decline of the conduction velocity of large myelinated peripheral nerve fibers ŽChase et al., 1992; Cruz Martınez ´ et al., 1978; Kanda et al., 1986; Norris et al., 1953; Wagman and Lesse, 1952. and a loss of numbers of myelinated peripheral nerve fibers have been found ŽMittal and Logmani, 1987; Tohgi et al., 1977; Wheeler and Plummer, 1989.. Robertson et al. Ž1993. correlated the slowing of the maximum conduction velocity of the sciatic nerve in aged mice with a loss of large ) Corresponding author. Department of the Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173, Japan. Tel.: q81 3 39643241 ext. 3087; fax: q81 3 35794776; e-mail: [email protected]
0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 1 3 - 7
myelinated fibers and a high incidence of demyelination and atrophy of the nerve axons. In contrast to large myelinated peripheral nerve fibers, small myelinated fibers seem to be resistant to aging in somatic peripheral nerves ŽMittal and Logmani, 1987; Rosenberg et al., 1989; Smith and Rosenheimer, 1984; Tohgi et al., 1977.. For example, the small myelinated fibers of the rat phrenic nerve with conduction velocities of 16–18 mrs do not show age-related changes in conduction velocity, fiber diameter, or myelin sheath width ŽSmith and Rosenheimer, 1984.. The unmyelinated fibers of somatic peripheral nerves in aged humans and laboratory animals do not show age-related changes in the maximum conduction velocity ŽSato et al., 1985., total number ŽRosenberg et al., 1989., density ŽKanda et al., 1991. and diameter ŽRosenberg et al., 1989; Wheeler and Plummer, 1989.. Autonomic nerve fibers consist mainly of small myelinated and unmyelinated fibers ŽPick, 1970.. Thus, as observed in small myelinated and unmyelinated fibers of peripheral somatic nerves, it can be assumed from the past
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
studies of aging effects on peripheral somatic nerves that autonomic nerve fibers are not subject to a large aging effect. Only a few studies have been reported concerning aging effects on conduction velocities ŽSato et al., 1985. or structures ŽAppenzeller and Ogin, 1973; Low and Dyck, 1978; Soltanpour and Santer, 1996. of autonomic nerve fibers. Sato et al. Ž1985. showed that the maximum conduction velocity of myelinated fibers in the rat vagus nerve decreased by about 10%, while the conduction velocity of the unmyelinated fibers was unchanged. Recently, Soltanpour and Santer Ž1996. counted the number of fibers in the vagus nerve, and found no significant difference in the total number of myelinated fibers and a slight decrease in the number of myelinated and unmyelinated fibers per square millimeter in aged rats. Effects of aging on the pelvic nerve, which is distributed widely to the pelvic organs, appear not to have been studied. In view of the major role of the pelvic nerves in micturition and defecation, it is important to examine age-related changes in the pelvic nerve. The present study, combining physiological and morphometric techniques, aimed to investigate age-related changes in the autonomic pelvic nerve of the rat. The first approach to this study was to measure the maximum conduction velocities of both myelinated and unmyelinated pelvic nerve fibers using electrophysiological techniques and the second approach was to measure the number and diameter of both myelinated and unmyelinated pelvic nerve fibers using electron microscopy. 2. Materials and methods The experiments were performed in nine adult Ž3–9 months old. and nine aged Ž30–37 months old. female Wistar rats ŽBW, 140–260 g.. All animals were raised in our institute. Laboratory food ŽCRF1, Charles River, Tokyo. and water were given to animals ad libitum. 2.1. Electrophysiological study Five adult and five aged rats were used. The animals were anesthetized with urethane Ž1.1 grkg i.p.. and the pelvic nerves were dissected out bilaterally under a dissecting microscope, which was essential for manipulating the nerve fibers. A ligature of fine silk thread was tied around the nerve close to the L6-S1 trunk, and another ligature was made at the inferior pole of the major pelvic ganglion. The distance between these ligatures was approximately 10–15 mm. The dissection was made with care to avoid placing any tension on the nerve. Then, the nerve was placed into a glass chamber filled with paraffin oil, heated and stabilized using a heating pad and lamp at 37 " 0.58C, to avoid temperature effects on nerve conduction velocity Žsee the work of Paintal, 1965.. The proximal end of the nerve was placed on a bipolar stimulating electrode with an interpolar distance of about
149
2–3 mm and stimulated by square waves of 0.5–15 V Žpulse duration, 0.1 ms. using an electrical stimulator ŽSEN-7103, Nihon Kohden, Japan.. The compound action potentials were recorded by bipolar recording wire electrodes located at the distal part of the nerve. The stimulus voltage was adjusted to more than that necessary to cause a maximal response; usually 1–2 V for myelinated fibers and 10 V for unmyelinated fibers. The distance between stimulating and recording electrodes was from 5 to 12 mm. Action potentials were amplified by a preamplifier Žtime constant 0.33 s, high cut 10 kHz, S-0476, Nihon Kohden. and displayed on a storage oscilloscope ŽMS-5311, Iwatsu, Japan.. The maximum conduction velocities Žmrs. of myelinated A d and unmyelinated C nerve fibers were calculated by dividing the distance Žmm. between the stimulating Žcathode. and recording electrodes by the onset latency Žms. of each action potentials. The nerve fibers showing conduction velocities slower than 2 mrs were classified as unmyelinated fibers. The conduction velocities were measured on bilateral sides of the pelvic nerve in each animal. Data analysis was carried out by calculating the mean " S.E.M. of each animal. 2.2. Morphometric study Four adult and four aged rats were used. The animals were anesthetized with urethane Ž1.1 grkg i.p... The pelvic nerve, proximal to the major pelvic ganglion, was exposed and covered with a fixative composed of 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.4. After preliminary fixation in situ for about 3 min, the pelvic nerve was removed from surrounding tissues and placed into the fixative on a polytetrafluoroethylene plate. A small segment Žapproximately 3 mm. of the proximal portion of the pelvic nerve was cut under a dissecting microscope, and fixed for 2 h or more at room temperature. At the proximal portion, there is a bifurcation and the pelvic nerve divides into two parts: one is the viscero-cutaneous branch which runs toward the major pelvic ganglion, and the other is the muscular branch which runs toward the perineal muscles ŽPacheco et al., 1989.. We focused on the viscero-cutaneous branch, and the muscular branch was excluded from the present study. The tissues were rinsed with cacodylate buffer, and post-fixed with 2% osmic acid buffered cacodylate for 1.5 h at 0–48C. Then, they were dehydrated through a graded series of ethanols and embedded in Epon-812. Cross-sections of the pelvic nerve were cut on an ultramicrotome ŽLKB Ultratome III, Sweden or Reichert Ultracut E, Austria.. For the light microscopic examination, sections were cut at 1.5 m m and stained with toluidine blue. Tissue samples were taken bilaterally in each rat, and checked for orientation of fibers and preservation of tissues. In each animal, a nerve on one side was selected for further evaluation by electron microscopy. The selected
150
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
nerve tissues were re-trimmed and ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined with an electron microscope ŽH-600, Hitachi, Japan.. Micrographs were obtained at a primary magnification of =3500 or =6000 and printed at 1.5 times the size of the original negatives. Approximately 70 films were exposed for each pelvic nerve, covering its entire area. Images of each photograph were stored in a personal computer. The total numbers and sizes of both myelinated and unmyelinated fibers were measured from the images on the photographs using software designed for this purpose ŽVm 32, version 1.0, Rise, Japan.. Outlines of the cross-sections of myelin sheaths of myelinated fibers were traced manually using either a mouse or a digitizer tablet-pen connected to the computer. The fiber diameter was calculated automatically from the traced outline assuming a circular shape ŽVita et al., 1992; Zimmerman et al., 1980.. Some myelinated fibers were excluded from the measurement of diameter if they were sectioned at the nodes of Ranvier, but these were included among the total number of myelinated fibers. Unmyelinated axons were clearly distinguished from profiles of Schwann cells, because unmyelinated axons showed round or oval profiles, and clearer axoplasmic
Table 1 Maximum conduction velocities of pelvic myelinated A d and unmyelinated C fibers Age group
Animal number
Myelinated fibers Žmrs.
Unmyelinated fibers Žmrs.
Adult
1 2 3 4 5 Average 1 2 3 4 5 Average
6.1 9.8 11.8 8.6 12.3 9.7 8.8 14.6 6.9 14.4 10.2 11.0
1.3 1.4 1.5 1.7 1.5 1.5 1.7 1.5 1.4 1.3 1.4 1.5
Aged
density than Schwann cells ŽHulsebosch and Coggeshall, 1982.. Outlines of unmyelinated axons were traced as described above for the myelinated fibers. A small population of unmyelinated axons Žless than 3% of the total number. were sectioned obliquely. They were excluded from the measurement of diameter, but included among the total number of unmyelinated axons. 2.3. Statistical analysis Data were expressed as mean " S.E.M. Statistical analysis of data was carried out by Mann–Whitney U-test.
Fig. 1. ŽA–D. Typical evoked potentials of pelvic myelinated A d and unmyelinated C fibers from one adult and one aged rat. Mass potentials evoked by single 0.1 ms pulse stimulation Ž2 V: A, B and 10 V: C, D at triangle. recorded from the pelvic nerve, 9 mm peripheral to the stimulation site. Onset Žat arrow. latency of each volley was measured to estimate the maximum conduction velocity for A d and C fibers. ŽE, F. Summary of the maximum conduction velocities of pelvic myelinated A d ŽE. and unmyelinated C ŽF. fibers in adult Ž N s 5, open columns. and aged Ž N s 5, shaded columns. rats. Each column and vertical bar shows mean"S.E.M.
Fig. 2. ŽA, B. Electron micrographs showing typical cross-sections of pelvic nerves in adult ŽA. and aged ŽB. rats. ŽC, D. Columns showing the total numbers of pelvic myelinated ŽC. and unmyelinated ŽD. fibers in adult Ž N s 4, open columns. and aged Ž N s 4, shaded columns. rats. Each column and vertical bar shows mean"S.E.M. ) P - 0.05; statistically significant difference between adult and aged rats by Mann–Whitney U-test.
Age group
Animal number
Total fiber number a
Fiber diameter Ž m m. -1.5 1.5–2.0 2.0–2.5
2.5–3.0
3.0–3.5
3.5–4.0
4.0–4.5
4.5–5.0
5.0–5.5
5.5–6.0
6.0–6.5
6.5–7.0
7.0 F
Adult
6 7 8 9 Average 6 7 8 9 Average
598 737 663 650 662 498 581 660 761 625
29 18 18 73 35 3 20 6 11 10
98 136 186 64 121 171 100 217 238 182
19 36 44 13 28 49 16 88 74 57
15 25 10 8 15 10 6 40 21 19
10 17 5 1 8 5 8 10 4 7
4 9 4 1 5 5 3 7 13 7
6 4 1 1 3 y 2 5 3 3
y 1 1 y 1 3 5 4 3 4
y 1 y y y 1 2 1 1 1
1 y y y y 1 2 2 2 2
1 4 5 4 4 6 2 9 2 5
Aged
a
164 191 97 304 189 66 142 65 108 95
241 287 287 177 248 154 263 196 272 221
Mean diameter
Myelinated fibers which were sectioned at the nodes of Ranvier were included in this number, but excluded from the measurement of diameter.
2.32 2.43 2.46 2.04 2.31 2.57 2.34 2.77 2.68 2.59
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
Table 2 Total number and diameter distribution of myelinated fibers
151
152
Age group
Animal number
Total fiber number a
Fiber diameter Ž m m. - 0.2 0.2–0.3 0.3–0.4
0.4–0.5
0.5–0.6
0.6–0.7
0.7–0.8
0.8–0.9
0.9–1.0
1.0–1.1
1.1–1.2
1.2–1.3
1.3–1.4
1.4–1.5
1.5F
Adult
6 7 8 9 Average 6 7 8 9 Average
4227 4120 4361 3824 4133 1739 3352 3601 3760 3113
98 57 25 76 64 13 8 2 4 7
709 635 639 628 653 211 609 453 551 456
626 645 711 763 686 266 569 515 563 478
546 460 602 449 514 224 437 414 433 377
367 335 411 266 345 249 332 269 368 305
223 280 352 231 272 238 299 375 439 338
125 281 239 114 190 125 169 305 334 233
57 240 225 62 146 69 105 266 238 170
18 136 125 28 77 52 84 224 186 137
6 68 45 9 32 35 32 93 76 59
5 19 16 6 12 16 10 114 60 50
1 6 7 4 5 8 y 41 10 15
y 37 1 6 11 1 2 47 12 16
Aged
a
634 335 397 679 511 70 272 123 148 153
812 586 565 503 617 162 424 260 338 296
Mean diameter
Unmyelinated fibers which were sectioned obliquely were included in this number, but excluded from the measurement of diameter.
0.51 0.63 0.61 0.52 0.57 0.70 0.60 0.75 0.68 0.68
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
Table 3 Total number and diameter distribution of unmyelinated fibers
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
153
3. Results 3.1. Electrophysiological study Typical evoked potentials of pelvic nerves, obtained from two adult and aged rats, are demonstrated in Fig. 1A–D. In both adult and aged rats, we observed evoked potentials of A d myelinated fibers in pelvic nerves stimulated at an intensity of 2 V. These had similar latencies of 0.7–0.9 ms and similar durations of 3.0–3.5 ms. Increasing stimulus intensity to 10 V produced an additional evoked potential of unmyelinated C fibers with a longer latency of 6–6.5 ms and a duration of 14–18 ms, in both adult and aged rats. The maximum conduction velocities of myelinated A d and unmyelinated C fibers obtained from five animals in each of the two groups ŽTable 1. are summarized in Fig. 1E and F. The maximum conduction velocity of A d myelinated fibers was 9.7 " 1.1 mrs in adult and 11.0 " 1.5 mrs in aged rats ŽFig. 1E.. It was slightly, but not statistically higher in aged rats. The maximum conduction velocities of the unmyelinated fibers in adult and aged rats were 1.5 " 0.1 mrs and 1.5 " 0.1 mrs, respectively ŽFig. 1F.. There was no significant difference between the adult and aged groups.
Fig. 3. Total distribution histograms of myelinated ŽA. and unmyelinated ŽB. fibers. The values are derived from averages of pooled data in adult Ž N s 4, open bars with heavy borders. and aged Ž N s 4, shaded bars. rats.
3.2. Morphometric study Electron micrographs ŽFig. 2, B. show representative cross-sections of pelvic nerves of adult and aged rats. There were regional differences in the distributions of nerve fibers with respect to their sizes. Total numbers of nerve fibers and fiber sizes were measured in eight pelvic nerves, obtained from four adult and four aged rats ŽTables 2 and 3.. The total numbers of myelinated and unmyelinated fibers are shown and summarized in Fig. 2C and D, respectively. The means of the total numbers of myelinated fibers were 662 " 27 in adult rats and 625 " 56 in aged rats. There was no statistically significant difference between the numbers of myelinated fibers in adult and aged rats. The means of the total numbers of unmyelinated fibers were 4133 " 114 in adult rats and 3113 " 456 in aged rats. In aged rats, the numbers of unmyelinated fibers were significantly decreased Ž P - 0.05. by approximately 25% of the value in adult rats. The distributions of diameters of myelinated fibers in adult and aged pelvic nerves are shown in Fig. 3A. The myelinated fiber diameters ranged between 1 m m and 13 m m, and were distributed unimodally. More than 80% of the myelinated fibers were smaller than 3 m m in diameter in both adult and aged rats. Only 0.5–0.8% of the myelinated fibers were larger than 7 m m, which is beyond the range of A d Žgroup III. fibers ŽBoyd and Davey, 1968.. The numbers of myelinated fibers in each 13 different diameter range as shown in Table 2 did not show signifi-
cant age-related changes. The mean diameters of myelinated fibers were 2.31 " 0.10 m m and 2.59 " 0.09 m m in adult and aged rats, respectively. There was a tendency to be larger in diameter in aged rats but this was not statistically significant. The distributions of unmyelinated fiber diameters are shown in Fig. 3B. The unmyelinated fiber diameters ranged between 0.1 m m and 1.8 m m. The number of unmyelinated fibers smaller than 0.7 m m was selectively decreased in aged rats, while the numbers of unmyelinated fibers larger than 0.7 m m showed no change. The means of the numbers of unmyelinated fibers smaller than 0.7 m m Ž3045 " 149 in adult rats vs. 1767 " 296 in aged rats. were significantly decreased Ž P - 0.05. in aged rats by approximately 42% of the value in adult rats. The mean of the numbers of unmyelinated fibers larger than 0.7 m m Ž1088 " 188 in adult rats vs. 1321 " 240 in aged rats. did not show a significant difference. The mean diameters of unmyelinated fibers were 0.57 " 0.03 m m and 0.68 " 0.03 m m in adult and aged rats, respectively. There was a tendency to larger diameter in aged rats, but this was not statistically significant.
4. Discussion The present study is the first demonstration in aged rats of a reduction Žby 25%, in this case. of the number of unmyelinated fibers in the pelvic nerve, specifically fibers
154
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
smaller than 0.7 m m in diameter. This study also demonstrated that there were no significant changes in the maximum conduction velocity, total numbers and diameters of myelinated fibers and the maximum conduction velocity of unmyelinated fibers. 4.1. Myelinated fibers The present electrophysiological study showed that the maximum conduction velocity of myelinated fibers of the pelvic nerve is approximately 10 mrs, and the diameters of most fibers were smaller than 7 m m. These results indicate that most myelinated fibers of the pelvic nerve in rats are A d fibers. However, the present histological study showed that there were a small number of myelinated fibers Žless than 1% of the total number. that had larger diameters beyond the range of A d fibers. It is certain that we missed recording the evoked action potentials of the larger myelinated fibers, beyond the A d fibers. This is because the interpolar distances between stimulating and recording electrodes were too short to record action potentials whose conduction velocities were much faster than those of the A d fibers recorded in the present study. As in small peripheral somatic myelinated fibers ŽMittal and Logmani, 1987; Rosenberg et al., 1989; Smith and Rosenheimer, 1984; Tohgi et al., 1977. we could not notice any physiological or morphological changes in myelinated A d fibers of the pelvic nerve. The small myelinated A fibers in both visceral and somatic nerves seem to be resistant to aging, in contrast to large myelinated fibers of somatic peripheral nerves in which demyelination and axonal atrophy have been reported during aging ŽRobertson et al., 1993.. The maximum conduction velocity of peripheral motor fibers has been shown to decrease by about 10–30% in both aged humans ŽBouche et al., 1993; Dorfman and Bosley, 1979; Norris et al., 1953; Wagman and Lesse, 1952. and laboratory animals ŽWayner and Emmers, 1958.. Afferent sensory fibers has been reported to maintain their maximum conduction velocities at normal adult levels until advanced age both in the sural nerve of humans ŽBurke et al., 1974. and in the nerve innervating the flexor hallucis longus muscle of rats ŽWayner and Emmers, 1958.. However, decreased conduction velocity of sensory nerves with age has also been shown in muscle spindles innervating the medial gastrocnemius muscle of rats ŽMiwa et al., 1995. as well as in median nerve and sural nerve of humans ŽBouche et al., 1993; Dorfman and Bosley, 1979.. The major portion of myelinated fibers in the pelvic nerve of the rat are sensory fibers ŽHulsebosch and Coggeshall, 1982; Purinton et al., 1973. and responsiveness of pelvic afferent nerve fibers to increases in intravesical pressure is well maintained in aged rats ŽHotta et al., 1995.. Therefore, it is likely that sensory myelinated fibers in autonomic nerves are resistant to aging.
4.2. Unmyelinated fibers The present study demonstrated that the pelvic nerve was composed primarily of unmyelinated fibers and also that the total number of unmyelinated fibers decreased during aging. It was noticeable that numbers of thin unmyelinated fibers smaller than 0.7 m m in diameter were selectively decreased with aging, while unmyelinated fibers larger than 0.7 m m in diameter did not change with aging. Therefore, it is reasonable that the maximum conduction velocities of unmyelinated fibers, which were calculated from the beginning of the evoked action potential of myelinated fibers and reflect the action potentials of largesized unmyelinated fibers, would not reveal any age-related change. Parasympathetic preganglionic nerves innervating the urinary bladder ŽMallory et al., 1989. and the uterus ŽSato et al., 1996. of the rat consist exclusively of unmyelinated fibers. The major portion of unmyelinated fibers in the pelvic nerve of the rat are parasympathetic preganglionic fibers; only 29% of the unmyelinated fibers are sensory fibers ŽHulsebosch and Coggeshall, 1982.. During aging, the number of preganglionic autonomic neurons has been shown to decrease at the levels of the T6–T8 rami communicantes in humans ŽLow and Dyck, 1978. and in the dorsal motor nucleus of the vagus in mice ŽSturrock, 1990.. Contractility of the urinary bladder induced by electrical stimulation of the pelvic efferent nerves declines with age ŽHotta et al., 1995.. Taken in consideration with these previous findings, the selective loss of small unmyelinated fibers in the pelvic nerve, as found in the present study, may be characteristic in parasympathetic efferent fibers. Although, in 4- and 24-month-old rats, Dering et al. Ž1996. reported no significant age-related differences in any of the parameters they measured, i.e. soma area, number of primary dendrites, number of dendritic branch points and total dendritic length, for the parasympathetic neurons in the lumbosacral spinal cord projecting to the hypogastric ganglion, total numbers of the parasympathetic neurons may decrease during aging. The decrease in the number of thin unmyelinated fibers in the pelvic nerve may be one of various causative factors for the age-related deterioration of micturition function.
Acknowledgements This work was supported by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Sasagawa Medical Research Foundation. The authors would like to thank Ms C. Kanai for excellent technical assistance, and Dr A. Sato and Dr Y. Sato for their advice and encouragement in performing this study.
H. Nakayama et al.r Journal of the Autonomic NerÕous System 69 (1998) 148–155
References Appenzeller, O., Ogin, G., 1973. Myelinated fibres in the human paravertebral sympathetic chain; quantitative studies on white rami communicantes. J. Neurol. Neurosurg. Psychiatry 36, 777–785. Bouche, P., Cattelin, F., Saint-Jean, O., Leger, J.M., Queslati, S., Guez, ´ D., Moulonguet, A., Brault, Y., Aquino, J.P., Simunek, P., 1993. Clinical and electrophysiological study of the peripheral nervous system in the elderly. J. Neurol. 240, 263–268. Boyd, I.A., Davey, M.R., 1968. Composition of Peripheral Nerves. Livingstone, Edinburgh, 57 pp. Burke, D., Skuse, N.F., Lethlean, A.K., 1974. Sensory conduction of the sural nerve in polyneuropathy. J. Neurol. Neurosurg. Psychiatry 37, 647–652. Chase, M.H., Engelhardt, J.K., Adinolfi, A.M., Chirwa, S.S., 1992. Age-dependent changes in cat masseter nerve: an electrophysiological and morphological study. Brain Res. 586, 279–288. Cruz Martınez, A., Barrio, M., Perez Conde, M.C., Gutierrez, A.M., ´ ´ ´ 1978. Electrophysiological aspects of sensory conduction velocity in healthy adults. J. Neurol. Neurosurg. Psychiatry 41, 1092–1096. Dering, M.A., Santer, R.M., Watson, A.H., 1996. Age-related changes in the morphology of preganglionic neurons projecting to the rat hypogastric ganglion. J. Neurocytol. 25, 555–563. Dorfman, L.J., Bosley, T.M., 1979. Age-related changes in peripheral and central nerve conduction in man. Neurology 29, 38–44. Hotta, H., Morrison, J.F.B., Sato, A., Uchida, S., 1995. The effects of aging on the rat bladder and its innervation. Jpn. J. Physiol. 45, 823–836. Hulsebosch, C.E., Coggeshall, R.E., 1982. An analysis of the axon populations in the nerves to the pelvic viscera in the rat. J. Comp. Neurol. 211, 1–10. Kanda, K., Hashizume, K., Nomoto, E., Asaki, S., 1986. The effects of aging on physiological properties of fast and slow twitch motor units in the rat gastrocnemius. Neurosci. Res. 3, 242–246. Kanda, T., Tsukagoshi, H., Oda, M., Miyamoto, K., Tanabe, H., 1991. Morphological changes in unmyelinated nerve fibers in the sural nerve with age. Brain 114, 585–599. Low, P.A., Dyck, P.J., 1978. Splanchnic preganglionic neurons in man: III. Morphometry of myelinated fibers of rami communicantes. J. Neuropathol. Exp. Neurol. 37, 734–740. Mallory, B., Steers, W.D., DeGroat, W.C., 1989. Electrophysiological study of micturition reflexes in rats. Am. J. Physiol. 257, R410–R421. Mittal, K.R., Logmani, F.H., 1987. Age-related reduction in 8th cervical ventral nerve root myelinated fiber diameters and numbers in man. J. Gerontol. 42, 8–10. Miwa, T., Miwa, Y., Kanda, K., 1995. Dynamic and static sensitivities of muscle spindle primary endings in aged rats to ramp stretch. Neurosci. Lett. 201, 179–182. Norris, A.H., Shock, N.W., Wagman, I.H., 1953. Age changes in the
155
maximum conduction velocity of motor fibers of human ulnar nerves. J. Appl. Physiol. 5, 589–593. Pacheco, P., Martinez-Gomez, M., Whipple, B., Beyer, C., Komisaruk, B.R., 1989. Somato-motor components of the pelvic and pudendal nerves of the female rat. Brain Res. 490, 85–94. Paintal, A.S., 1965. Effects of temperature on conduction in single vagal and saphenous myelinated nerve fibres of the cat. J. Physiol. 180, 20–49. Pick, J., 1970. The Autonomic Nervous System. Lippincott, Philadelphia, 483 pp. Purinton, P.T., Fletcher, T.F., Bradley, W.E., 1973. Gross and light microscopic features of the pelvic plexus in the rat. Anat. Rec. 175, 697–706. Robertson, A., Day, B., Pollock, M., Collier, P., 1993. The neuropathy of elderly mice. Acta Neuropathol. 86, 163–171. Rosenberg, S.I., Malmgren, L.T., Woo, P., 1989. Age-related changes in the internal branch of the rat superior laryngeal nerve. Arch. Otolaryngol. Head Neck Surg. 115, 78–86. Sato, A., Sato, Y., Suzuki, H., 1985. Aging effects on conduction velocities of myelinated and unmyelinated fibers of peripheral nerves. Neurosci. Lett. 53, 15–20. Sato, Y., Hotta, H., Nakayama, H., Suzuki, H., 1996. Sympathetic and parasympathetic regulation of the uterine blood flow and contraction in the rat. J. Auton. Nerv. Syst. 59, 151–158. Smith, D.O., Rosenheimer, J.L., 1984. Factors governing speed of action potential conduction and neuromuscular transmission in aged rats. Exp. Neurol. 83, 358–366. Soltanpour, N., Santer, R.M., 1996. Preservation of the cervical vagus nerve in aged rats: morphometric and enzyme histochemical evidence. J. Auton. Nerv. Syst. 60, 93–101. Sturrock, R.R., 1990. A comparison of age-related changes in neuron number in the dorsal motor nucleus of the vagus and the nucleus ambiguus of the mouse. J. Anat. 173, 169–176. Tohgi, H., Tsukagoshi, H., Toyokura, Y., 1977. Quantitative changes with age in normal sural nerves. Acta Neuropathol. ŽBerl.. 38, 213–220. Vita, G., Santoro, M., Trombetta, G., Leonardi, L., Messina, C., 1992. A computer-assisted automatic method for myelinated nerve fiber morphometry. Acta Neurol. Scand. 85, 18–22. Wagman, I.H., Lesse, H., 1952. Maximum conduction velocities of motor fibers of ulnar nerve in human subjects of various ages and sizes. J. Neurophysiol. 15, 235–244. Wayner, M.J. Jr., Emmers, R., 1958. Spinal synaptic delay in young and aged rats. Am. J. Physiol. 194, 403–405. Wheeler, S.J., Plummer, J.M., 1989. Age-related changes in the fibre composition of equine peripheral nerve. J. Neurol. Sci. 90, 53–66. Zimmerman, I.R., Karnes, J.L., O’Brien, P.C., Dyck, P.J., 1980. Imaging system for nerve and fiber tract morphometry: components, approaches, performance, and results. J. Neuropathol. Exp. Neurol. 39, 409–419.