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Differential effects of aging on motoneurons and peripheral nerves innervating the hindlimb and forelimb muscles of rats
NEUROSCIENCE RESEI]I]CH ELSEVIER
Neuroseience Research 22 (1995) 189-196
Differential effects of aging on motoneurons and peripheral nerves innervating the hindlimb and forelimb muscles of rats K e n H a s h i z u m e 1, K e n r o K a n d a * Department of Central Nervous System, Tokyo Metropolitan Institute of Gerontology, 35-2, Sakaecho, ltabushi-ku, Tokyo 173, Japan Received 30 September 1994; accepted 27 January 1995
Abstract
We examined the number and size of ulnar (forelimb) and medial gastrocnemius (MG, hindlimb) motoneurons in middle-aged (9 months of age) and aged (27 months of age) male Fischer 344 rats. Morphological properties of the ulnar and the MG nerves were also studied. No significant difference was found in the mean number of the ulnar motoneurons between the two age groups, while that of MG motoneurons was significantly less in aged animals. A decrease in the number of myelinated fibers (including both afferent and efferent fibers) in the ulnar nerves was less than that in the MG nerves, although the age difference was not significant in either of the nerves. Soma atrophy of aged motoneurons was found in both MG and ulnar motor nuclei. The mean fascicular areas and myelinated fiber diameters were significantly increased in both the MG and the ulnar nerves in aged rats, but these were less pronounced for the ulnar nerve. The results indicate that most ulnar motoneurons, unlike MG motoneurons, survive at least to the age of 27 months. Morphological changes in the peripheral nerves were also less for the ulnar nerve than for the MG nerve. Thus, we conclude that the effects of aging on motoneurons and peripheral nerves innervating MG muscle of the hindlimb are greater than those innervating forelimb muscles. Keywords: Motoneuron; Cell survival; Peripheral nerve; Horseradish peroxidase methods; Forelimb; Hindlimb; Aging; Rat
1. Introduction Various morphological and physiological changes occur in the nervous system with advancing age. It has been reported that the total number of ventral horn neurons (i.e., presumed motoneurons) in the spinal cord decreases in both humans (Kawamura et al., 1977b; Tomlinson and Irving, 1977; Tsukagoshi et al., 1979) and animals (Wright and Spink, 1959). A decrease in the number of myelinated fibers in the ventral roots and peripheral nerves also suggests a loss of motoneurons in the aged (Samorajski, 1974; Kawamura et al., 1977a; Tohgi et al., 1977; Caccia et al., 1979; Jacobs and Love,
* Corresponding author, Tel.: +81 3 3964 3241 (ext. 3083); Fax: +81 3 3579 4779. I Current address: Faculty of Health and Sport Sciences, Osaka University, 1-17, Machikaneyama, Toyonaka, Osaka 560, Japan. K.H. was at the Department of Kinesiology, TMIG at the time of the experiment.
1985; Mittal and Logmani, 1987; Ansved and Larsson, 1990). In previous experiments, we investigated agerelated changes of motoneurons labeled with retrograde axonal transport of horseradish peroxidase (HRP) (Hashizume et al., 1988; see also Ishihara and Araki, 1988). There was a significant decrease in the number and size of motoneurons innervating the medial gastrocnemius (MG) muscle in the hindlimb of rats with advancing age. On the other hand, it has been reported that agerelated, mechanical and morphological changes of the muscle, including a decrease in maximum voluntary force, elongation of contraction time, fiber-type grouping, and atrophy of muscle fibers, are greater in leg (or hindlimb) muscles than in arm (or forelimb) muscles (Fujisawa, 1974; Grimby et al., 1982; Nygaard and Sanchez, 1982; McDonagh et al., 1984; Oertel, 1986). It has been suggested that some of these alterations seen in aging skeletal muscle are neurogenic (Fujisawa, 1974; Tomonaga, 1977; Kanda and Hashizume, 1989). Fur-
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thermore, St[dberg and Fawcett (1982) reported that an increase in size of the Macro motor unit potential was found in the tibialis anterior and the vastus lateralis muscles in aged humans of 60 years of age or over, but not in the biceps brachii muscle, and suggested that degeneration of motoneuron and peripheral nerves, entrapment at root or more distal sites and repeated nerve traumata, occurring with age, differ between motoneurons and peripheral nerves innervating different muscles, such as leg (or hindlimb) and arm (or forelimb) muscles. To obtain more direct evidence for such differential aging effects on brachial and lumbar motoneurons, we investigated age-related morphological changes in motor nuclei and peripheral nerves innervating forelimb and hindlimb muscles of the rat in the present experiments. Preliminary reports have appeared elsewhere (Hashizume and Kanda, 1990, 1992). 2. Materials and methods
2.1. Experimental animals and surgical procedures Experiments were performed on 15 male Fischer 344 rats, including 7 middle-aged (9 months of age, body weight: 390-485 g) and 8 aged (27 months of age, 320-470 g) animals. They were raised in a specific pathogen-free colony at our institute, in which the 75%, 50% and 25% survival times of the rats were about 25.5, 28 and 31.5 months, respectively. They were kept in conventional housing, with less than four animals/cage, without restrictions on feeding and without any special exercise requirements. The rat was anesthetized with pentobarbital (35 mg/kg; i.p.). Under aseptic conditions, the ulnar and MG nerves were bilaterally freed of surrounding tissues by microdissection. The ulnar nerve was cut and tied with suture material at a point just proximal to the elbow joint, and the MG nerve at a point near the entry to the MG muscle. Glass micropipettes, broken to a tip diameter of 20-80 t~m and filled with a fresh solution of 40% HRP (Sigma type VI or Toyobo I-C) in distilled water, were used to impale the proximal stump of the ulnar and MG nerves under a dissecting microscope. Small volumes (0.4-0.8 #1 for the ulnar nerve, 0.1-0.5 #1 for the MG nerve) of HRP solution were injected using 20-50 brief (15-30 ms) pulses of pressure (200-400 Pa), produced by a pulse-operated valve device (Picospritzer II, General Valve Co.). After HRP injection, the injection site was washed with sterile saline, then the wound was closed in layers. Both forelimbs and hindlimbs were treated identically in all rats. After 2 days, the animal was reanesthetized with pentobarbital and perfused transcardially with 500 ml of warmed saline followed by 700 ml of cold fixative mixture (1.25% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C). Thereafter, the spinal cord was removed and placed in 20% sucrose-
phosphate buffer at 4°C for 15-20 h. The spinal segment boundaries were marked with small holes made by inserting a pin into the dorsal columns at points between the entry zone of adjacent, identified dorsal roots. The cord was then cut into suitable blocks, with the C7-Th2 or L3-L6 segments included in a single block. Serial sections in the horizontal plane were cut at a thickness of 80 #In on a freezing microtome. All sections were saved in serial order and collected individually into 0.1 M phosphate buffer. They were processed for demonstration of HRP with the chromogen 3,3',5,5'-tetramethylbenzidine (TMB) using Mesulam's protocol (Mesulam, 1978). All sections were mounted on gelatinized slides in serial order without counterstaining. Sections containing HRP-labeled cells were photographed on high-contrast film (Kodak Technical Pan), and photomontage maps for cell identification were made of each section by printing on Kodalith Ortho Film. A small piece of the ulnar and MG nerve on either side was excised from the distal cut end just before HRP injection and fixed in 2.5% glutaraldehyde. This was later osmicated in 1% OsO4. Semithin cross-sections were cut at a thickness of about 1/zm from the specimen embedded in Epon and stained with toluidine blue.
2.2. Analytic procedures Individual HRP-labeled neurons were identified and counted under microscopic observation with the aid of photomontage maps. Labeled neurons were considered 'cell bodies' when the entire cell body was included in the section. When a cell body was located at the upper (or lower) surface of a section and a part of the cell body was also found at the lower (or upper) surface of the corresponding position in the adjoining section, we considered them to be parts of a single cell. In many cases, it was possible to confirm this by detecting the nucleus in only one of the divided cell bodies. The cell position was identified on the photomontage map. Using these techniques, it was possible to obtain a direct estimate of the motoneuron number in a given motor nucleus (or a given group of motor nuclei). The cross-sectional area (CSA) of the individual cell body was measured under brightfidd microscopy at x400 magnification. The outline of each cell body was traced with the aid of a digitizing unit connected to a computer (Macintosh IIfx) and the area within the outline boundary was calculated. The fascicular area of each nerve was calculated using the same tools as with the CSA of the cell body at x 250 magnification. In the MG nerve, the total number of myelinated axons was counted from photographic prints made of these sections at a magnification of about × 2070. The myelinated axon density was calculated from the fascicular area and the total number of myelinated axons. The maximum and minimum diameters of each axon and myelin sheath thickness
K. Hashizume, K. Kanda / Neuroscience Research 22 (1995) 189-196
were measured from these photographic prints. Average axon diameters (one-half of the sum of the measured maximum and minimum diameters) were then calculated. The fiber diameter was then calculated from the average axon diameters and myelin sheath thickness. In the ulnar nerve, the total number of myelinated axons was counted from photographic prints at a magnification of about x 690. The average axon diameters and myelin sheath thickness were measured for 650-700 individual myelinated axons located in the center of the fasciculus from photographic prints at a magnification of about x 2070. The average axon diameters were not measured for axons with an irregular or very flattened shape. No correction for shrinkage during fixation was attempted in this study.
191
A) Number of motoneurons Ulnar
MG 1401
310
'
I
!
I
300
130
tt
290 280
120
270
110
260
100
250
B) Soma cross-sectional area ( i.tm2 )
MG 2.3. Statistical analysis For the motoneurons, the mean values obtained, respectively from both sides of the limb were averaged for individual rats, then the means were compared between the middle-aged and aged groups. For the peripheral nerves, data from one side of the leg (either left or right) were considered as the representative values of the individual rats. We used the two-tailed independent t-test to test for differences between two mean values. 3. Results
900 800 700
600 500 r - - i middle-aged *
3.1. Number and size of MG and ulnar motoneurons Fig. 1A shows the mean numbers of MG (left) and ulnar (right) motoneurons labeled with HRP in middleaged and aged rats. The number of MG motoneurons in aged rats (121.0 ± 3.6, n = 8) was significantly smaller than that in middle-aged rats (132.1 -#- 5.7, n = 7). On the contrary, no significant difference was found in the number of ulnar motoneurons in aged (290.9 ± 7.0, n = 7) and middle-aged rats (287.4 4- 15.5, n = 7, P > 0.6). Fig. 1B shows the mean CSA of cell soma of MG (left) and ulnar (right) motoneurons. The CSA of MG motoneurons in aged rats (697.5 4- 126.3 /zm2) was smaller than that in middle-aged rats (777.4 4- 78.4 #m2), although the difference was not significant (P > 0.1). The mean CSA of ulnar motoneurons in aged rats (761.6 ± 28.0 #m 2) was significantly smaller than that in middle-aged rats (845.3 ± 47.5 #m2). 3.2. Morphological changes of corresponding peripheral nerves Fig. 2 shows photomicrographs of transverse semithin sections of the MG nerve of a middle-aged (A) and an aged rat (B). A reduction in the number of myelinated fibers and an increase in the amount of endoneural connective tissue were observed in aged MG nerve. Several fibers, especially those with a large diameter, showed myelin sheath irregularities, including myelin splitting
Ulnar
p<0.01
II
aged
** p < 0 . 0 0 1
Fig. 1. The numbers and sizes of the medial gastrocnemius (MG) and ulnar motoneurons. The MG motor nuclei in aged rats contained a fewer number of motoneurons compared to those of middle-aged rats (A, left). No significant difference was found in the number of ulnar motoneurons between the two age groups (A, right). The soma size of ulnar motoneurons in aged rats was significantly smaller than that in middle-aged rats (B, right). Vertical bars indicate 1 standard deviation from the mean.
with myelin balloon formation, infolded myelin loops and myelin reduplication as reported by other investigators (Knox et al., 1989; Ansved and Larsson, 1990). No cluster of small diameter fibers with a thin myelin sheath was found in the present experiments. Such regenerative sprouts were reported to be observed commonly in the ventral root of aged rats (Knox et al., 1989; Ansved and Larsson, 1990). The mean incidence of these fibers in aged rats (15%, range: 9-17%) was higher than that in middle-aged rats (2%, range: 1-3%). Myelin sheath irregularities were observed commonly in fibers with a large diameter. Fig. 3 shows photomicrographs of transverse semithin sections of the ulnar nerve of a middle-aged (A) and an aged rat (B). Unlike the MG nerve, an obvious reduction in myelinated fibers and an increase in the amount of endoneural connective tissue were not observed in
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,o,
Fig. 2. Photomicrographs of transverse semithin sections of the MG nerve stained with toluidine blue in a middle-aged (A) and an aged rat (B). A reduction in myelinated fibers and increases in fascicular area, axon diameter and the amount of endoneural connective tissue were observed in aged MG nerve. Some fibers in aged nerves showed myelin sheath irregularities, including myelin splitting with myelin balloon formation, infolded myelin loops and myelin reduplication. Calibration bar = 50 ~m.
Fig. 3. Photomicrographs of transverse semithin sections of the ulnar nerve stained with toluidine blue in a middle-aged (A) and an aged rat (B). Unlike the M G nerve, an obvious reduction in myelinated fibers and an increase in the amount of endoneural connective tissue were not observed in aged nerves, although increases in fascicular area and axonal diameter were observed. No cluster of regenerating fibers was found in aged ulnar or MG nerves. Calibration bar = 50 ~m.
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193
Table i Morphologial properties of the medial gastrocnemius and ulnar nerves in middle-aged and aged rats Group
n
Faseicular area (ram 2)
Number of fibers
Axon diameter (tzm)
Myelin sheath thickness (/~m)
Medial gastrocnemius nerve Middle-aged 4 Aged 6
0.04 4- 0.003 0.07 ± 0.01"*
285.3 4- 23.7 257.0 4- 22.0
5.5 4- 0.5 6.5 4- 0.3*
1.2 ± 0.1 1.4 ± 0.2
Ulnar nerve Middle-aged Aged
0.16 + 0.01 0.19 4- 0.01*
2247.8 + 34.0 2202.3 4- 68.3
4.3 4- 0.2 5.1 4- 0.2**
0.92 + 0.08 0.98 + 0.11
6 6
Fiber density (n/mm 2)
7142 ± 965 3787 ± 331'*
13 903 4- l l31 II 543 4- 663*
n, number of myelinated fibers. Values are expressed as group means ± S.D. *P < 0.01, **P < 0.001.
aged nerves. No cluster of presumed regenerating fibers was found, although some fibers showed myelin sheath irregularities as in the MG nerves. The mean incidence of these fibers in aged rats (2%) was lower than that in the MG nerves. Table 1 shows the quantitative data of the MG and ulnar nerves in middle-aged and aged rats. The mean fascicular area of the MG nerve was larger by 75% in aged rats than in middle-aged rats whereas that of the ulnar nerve was larger by only 19% in aged rats than in middle-aged rats. The mean number of myelinated fibers (including both afferent and efferent fibers) in aged rats did not statistically differ from that in middle-aged rats. The myelinated fiber density, calculated from the fascicular area and the total number of myelinated
(%) 7 ~=
6
t-
5
° - -
~" E
4
$
3
e~
E
2
t-
._> II)
re
1 0 0
5
10
15
20
25
Average fiber diameter (~tm) Fig. 4. Comparison of average diameters of MG myelinated fibers in middle-aged (open bars) and aged (shaded bars) rats plotted as percentages. These histograms include all myelinated fibers obtained from four middle-aged (1133 fibers) and six aged nerves (1513 fibers), respectively.
fibers, of the MG nerves was reduced by 47% in aged rats. The ulnar nerves showed a smaller reduction (17%) in the fiber density. The mean axon diameter in aged rats was larger by 19% for the MG nerve and 18% for the ulnar nerve compared to that in middle-aged rats. Age difference in myelin sheath thickness was not found in either the MG or ulnar nerves. The histograms in Fig. 4 show the distribution of diameters of MG myelinated fibers pooled from middleaged (open bars) and aged (shaded bars) rats plotted as percentages. The fiber diameters were distributed bimodally in both groups. The transition point between small and large was at about 7.5 ~m for middle-aged rats and about 9 #m for aged rats. The histograms from aged rats shifted to the right, but fibers with a larger diameter exceeding 25 t~m were not observed (see also Fig. 2B). The count of large fibers with a diameter larger than 9 ~m in aged rats was 130.7 ± 13.4 (n = 6). This value was significantly smaller than that for fibers with a diameter larger than 7.5 /~m in middle-aged rats (154.5 ± 12.7, n = 4, P < 0.05). No significant difference was found in the number of small fibers between the age groups (130.8 ± 13.7 for aged rats, 133.5 ± 30.7 for middle-aged rats, respectively). The range of motion at the knee joint was also measured before sacrificing the animal. The maximally extended position was reduced from 155° for middleaged rats to 135° for aged rats. The wet weight of the MG muscle in aged rats (560 mg) was significantly lighter than that in middle-aged rats (747 mg) (P < 0.001). The flexor carpi ulnaris muscle innervated by the ulnar nerve also had reduced weight with advancing age from 162 mg to 143 mg (P < 0.01). 4. Discussion
4.1. Differential effects of aging on MG and ulnar motoneurons and corresponding peripheral nerves The present experiments demonstrated that the number of ulnar motoneurons did not differ significantly between middle-aged (9 months of age) and aged (27
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months of age) rats, whereas the MG motor nucleus in aged rats contained a smaller number of labeled motoneurons compared to that in middle-aged rats. A decrease in the number of labeled motoneurons could be explained by cell death and/or an increase in the number of unlabeled cells. The latter may be due to a deficit of axonal transport. If the axon does not extend to the injection site, HRP can not be taken up and transported to the cell soma. A decrease in the rate of anterograde fast axonal flow has been observed in the sciatic nerve of aged rats (McMartin and O'Conner, 1979; Frolkis et al., 1985). In previous experiments on middle-aged rats, we have shown that labeled motoneurons can be observed 13 h after HRP injection into the MG muscle and that labeling intensity reached near maximum level at about 20 h after HRP injection (Sato et al., 1989). Forty-eight hours of survival time between HRP injection and animal sacrifice seems to be enough to label all motoneurons existing in the MG nucleus even in the case of aged rats. Furthermore, the number of unlabeled neurons located within and around the MG motor nucleus did not differ between aged and middle-aged rats (Kanda et al., unpublished observation). Thus, the present results indicate that neuronal cell loss occurs in the MG motor nucleus whereas most ulnar motoneurons survive at least to the age of 27 months. The common features of aged nerves observed in the present study were an increase in fascicular area and average axon diameter, and a decrease in myelinated fiber density. The ratios of these changes in the ulnar nerves were 19%, 18% and 17%, respectively, whereas those in the MG nerves were 75%, 19% and 47%, respectively. The mean incidence of fibers with myelin sheath irregularities in aged animals was also lower in the ulnar nerve than in the MG nerve. Thus, alterations in various morphological parameters of the peripheral nerve were also greater in the MG nerve than in the ulnar nerve. These findings in motor nuclei and peripheral nerves suggest that the effects of aging on motoneurons and peripheral nerves innervating the MG muscle in the hindlimb are greater than in those innervating forelimb muscles. The differential age effect on the sensory neurons innervating forelimb and hindlimb regions has also been suggested by the finding that a decrease in vibration sensitivity with age is greater in the foot than in the hand (Kenshalo, 1986). It has also been found that axonal dystrophy occurs consistently and more severely in the gracilis than in the cuneate nuclei of aged humans (Jellinger and Jirasek, 1971). The underlying mechanisms causing the differential extent and/or rate of progress of aging among different motor nuclei and peripheral nerves are not yet clear. The following are some comments on the factors causing the differential age effects on MG and ulnar motoneurons. It is known that neurotrophic factors play an important role in cell survival during development. The effect of nerve growth factor (NGF) on neuronal survival after
axotomy has also been demonstrated in the medial septum of the adult rat (Hefti, 1986; Williams et al., 1986; Kromer, 1987). Thus, difference in survival rate between MG and ulnar motoneurons in aged rats may be due to the differential effect of aging on the supply of trophic substances. A decrease in the rate of axonal transport with age (McMartin and O'Conner, 1979; Komiya, 1980; Frolkis et al., 1985) may cause difficulty in the maintenance structures of axons, and a deficit of trophic factors in the soma. MG motoneurons with a longer axon may be affected more severely than ulnar motoneurons with a relatively short axon. Alternatively, there might be an intrinsic difference in susceptibility to a deficit of trophic substances between MG motoneurons and ulnar motoneurons. Mettling et al. (1993) have shown in the chick embryo that brachial motoneurons die later than lumbar motoneurons during embryonic development, and that the difference is not a result of differing peripheral trophic support, but is intrinsically programmed, at least partially through a higher sensitivity to trophic factors. At present, however, it is not clear whether a deficit in trophic factors caused the motoneuronal death with age seen in the present experiments. Another factor may be neuronal activity. Degenerative changes found in motoneurons in aged rats might be attributable to some extent to disuse of muscles. It has been reported that spontaneous locomotion activity gradually decreases in aging rats (Samorajski and Rolsten, 1975; Holloszy et al., 1985; Mondon et al., 1985; Brown et al., 1992). A decrease in the range of motion at the knee joint found in the present experiments also suggests reduced activity in hindlimb muscles. In contrast, activity of forelimb motoneurons might be high because of maintained movement for grooming and feeding. In fact, the ratio of decrease in the wet weights was larger in the MG muscle (about 25%, P < 0.001) than in the flexor carpi ulnaris muscle (about 12%, P < 0.01). Reduced activity seems to lead to a decline in metabolic activity of motoneurons and eventually to their atrophy. Recently, we demonstrated that age-related changes in MG motoneurons and MG myelinated fibers were retarded by long-term, moderate endurance exercise (Hashizume and Kanda, 1991, 1993). The difference in the amount of activity, therefore, may cause a different degree of age-related changes between MG and ulnar motoneurons. The innervation ratio of MG motoneurons appeared to be greater than that of ulnar motoneurons, which means that MG motoneurons have a greater number of terminal branches than do ulnar motoneurons. In addition, the lengths of MG axons are longer than those of ulnar axons. It seems that the cell soma of the MG motoneurons have to synthesize more protein to maintain their structures than do ulnar motoneurons. Thus, declining synthesis and capability for protein breakdown with increasing age (cf. Ekstrom et al.,
K. Hashizume, K. Kanda/Neuroscience Research 22 (1995) 189-196
1980), if this occurs in spinal motoneurons, may affect MG cells more severely than ulnar cells. 4.2. Alterations of the peripheral nerves We found an increased fascicular area and fiber diameter of the MG muscle nerves in aged rats whereas the majority of studies on human and experimental animals have reported a decreased or unaltered fascicular area and myelinated fiber diameter (Samorajski, 1974; Sharma et al., 1980; Krinke, 1983; Jacobs and Love, 1985; Knox et al., 1989; Chase et al., 1992). Ansved and Larsson (1990) have reported similar findings in the soleus muscle nerve in aged rats to those in the present experiments. Thus, changes in the sizes of peripheral nerves during the aging periods is a matter of controversy. The reason for the difference is not clear at present. Since the quantitative data in the present study were obtained from a single transverse section of nerves, it is not known whether uniform enlargement has occurred along the entire axon. A study on teased fiber preparations revealed that the formation of myelin balloons appeared focally along myelinated fibers (Knox et al., 1989). These were seen commonly in the ventral root and the diameter of these fibers often exceeded more than 50 #m. The distribution of myelinated fiber diameters in aged MG nerve shifted to the right, but none exceeded 25 t~m (Figs. 2 and 4). Myelinated fibers of the ulnar nerves showed similar features to those of the MG nerves (Fig. 3). Hence, these results suggest that an increase in the mean axon diameter seems to be part of a general trend in all axons in aged MG and ulnar nerves rather than to the existence of axons with an extremely expanded diameter. An increased axon diameter in aged rats seems to reflect some sort of failure of metabolism and/or axonal transport rather than an increased activity. We did not see any clusters of small diameter fibers with thin myelin sheaths in the present experiments. Such regenerating fibers have been found frequently in the ventral root of aged rats (Knox et al., 1989; Ansved and Larsson, 1990). Thus, it appears that regeneration reaction occurs preferentially in the proximal portions of the peripheral nerves. Decline in the conduction velocity of individual motor and primary afferent fibers in aged cats and rats has also been demonstrated in other experiments (Chase et al., 1985, 1992; Morales et al., 1987; Kanda and Hashizume, 1989; Ramirez and Ulfhake, 1992; Kanda, unpublished data). Segmental demyelination and remyelination have been found frequently both in ventral roots and peripheral nerves in aged humans and animals (Sharma et al., 1980; Thomas et al., 1980; Grover-Johnson and Spencer, 1981; Braund et al., 1982a,b; Jacobs and Love, 1985; Knox et al., 1989; Ansved and Larsson, 1990). These degenerative changes in myelin sheath structure may reduce the conduction velocity in aged rats (Adinolfi et al., 1991).
195
4.3. Loss of motoneurons and primary sensory neurons Peyronnard and Charron (1983), by using a combination of HRP methods and posterior rhizotomy in middle-aged rats, demonstrated that the number of presumed motor axons was close to that of the tibialis anterior motoneurons labeled with HRP, suggesting that extramuscular branching of motor axons was rather insignificant in total axon counts in adult rats. In the present study, clusters of small-diameter fibers with a thin myelin sheath, suggesting fiber regeneration (Knox et al., 1989; Ansved and Larsson, 1990), were not found in either of the age groups. Thus, the total number of myelinated fibers seemed to correspond to the actual count of their original neurons. If so, we can estimate that 46% (132.1/285.3) of the total number of myelinated fibers in middle-aged MG nerves consist of motor fibers. This value is also comparable to that of the tibialis anterior nerve (Peyronnard and Charron, 1983). The ratio of motor fibers in aged MG nerve can be estimated as 47% (121.0/257.0), which was very close to that in the middleaged nerves. A decrease of sensory neuron with advancing age, therefore, was of the same order of magnitude as that of motoneurons.
Acknowledgements The authors are grateful to Dr. H. Nagasaki for providing K.H. with the opportunity to participate in this study, and Ms. E. Nomoto and Ms. S. Asaki for their technical assistance.
References Adinolfi, A.M., Yamuy, J., Morales, F.R. and Chase, M.H. (1991) Segmental demyelination in peripheral nerves of old cat. Neurobiol. Aging, 12: 175-176. Ansved, T. and Larsson, L. (1990) Quantitative and qualitative morphological properties of the soleus motor nerve and the L5 ventral root in young and old rats. J. Neurol. Sci., 96: 269-282. Braund, K.G., McGuire, J.A. and Lincoln, C.E. (1982a) Age-related changes in peripheral nerves of the dog. I. A morphologic and morphometric study of single-teased fibers. Vet. Pathol., 19: 365-378. Braund, K.G., McGuire, J.A. and Lincoln, C.E. (1982b) Age-related changes in peripheral nerves of the dog. II. A morphologic and morphometric study of cross-sectional nerve. Vet. Pathol., 19: 379-398. Brown, M., Ross, T,P. and Hoiioszy, J.O. (1992) Effects of ageing and exercise on soleus and extensor digitorum Iongus muscles of female rats. Mech. Ageing Dev., 63: 69-77. Caccia, M.C., Harris, J.B. and Johnson, M.A. (1979) Morphology and physiology of skeletal muscle in aging rodents. Muscle Nerve, 2: 202 -212. Chase, M.H., Morales, F.R., Boxer, P.A. and Fung, S.J. (1985) Aging of motoneurons and synaptic processes in the cat. Exp. Neurol., 90: 471-478. Chase, M.H., Engeihardt, J.K., Adinolfi, A.M. and Chirwa, S.S. (1992) Age-dependent changes in cat masseter nerve: An electrophysiological and morphological study, Brain Res., 586: 279-288. Ekstrom, R., Lui, D,S.H. and Richardson, A. (1980) Changes in brain
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protein synthesis during the life-span of male Fischer rats. Gerontology, 26: 121-128. Frolkis, V.V., Tanin, S.A., Marcinko, V.J., Kulchitsky, O.K. and Yasechko, A.V. (1985) Axoplasmic transport of substances in motoneuronal axons of the spinal cord in old age. Mech. Ageing Dev., 29: 19-28. Fujisawa, K. (1974) Some observations on the skeletal musculature of aged rats. Part 1. Histological aspects. J. Neurol. Sci., 22: 353-366. Grimby, G., Danneskiold-Samsoe, B., Hvid, K. and Saltin, B. (1982) Morphology and enzyme capacity in arm and leg muscles in 78-81 year old men and women. Acta Physiol. Stand., 115: 125-134. Grover-Johnson, N. and Spencer, P.S. (1981) Peripheral nerve abnormalities in aging rats. J. Neuropathol. Exp. Neurol., 40: 155-165. Hashizume, K. and Kanda, K. (1990) Neuronal dropout is greater in hindlimb motor nuclei than in forelimb motor nuclei in aged rats. Neurosci. Lett., 113: 267-269. Hashizume, K. and Kanda, K. (1991) Effects of swimming exercise on aging rat motoneuron. Neurosei. Res., Suppl. 14, SI 1. Hashizurne, K. and Kanda, K. (1992) Age-related changes in the peripheral nerves in the rat. Neurosci. Res., Suppl. 17, $205. Hashizume, K. and Kanda, K. (1993) Effects of swimming exercise on age-related changes in motoneurons and peripheral nerves in the rat. Soc. Neurosci. Abstr. 19: 1742. Hashizume, K., Kanda, K, and Burke, R.E. (1988) Medial gastroenemius motor nucleus in the rat: Age-related changes in the number and size of motoneurons. J. Comp. Neurol., 269: 425-430. Hefti, F. (1986) Nerve growth factor (NGF) promotes survival of septal cholinergic neurons after fimbrial transections. J. Neurosci., 6: 2155-2162. HoUoszy, J.O., Smith, E.K., Vining, M. and Adams, S. (1985) Effect of voluntary exercise on longevity of rats. J. Appl. Physiol., 59: 826-831. Ishihara, A. and Araki, H. (1988) Effects of age on the number and histoehemical properties of muscle fibers and motoneurons in the rat extensor digitorum Iongus muscle. Mech. Ageing Dev., 45: 213-221. Jacobs, J.M. and Love, S. (1985) Qualitative and quantitative morphology of human sural nerve at different ages. Brain, 108: 897-924. Jellinger, K. and Jirasek, A. (1971) Neuroaxonal dystrophy in man: Character and natural history. Acta Neuropathol., 5: 3-16. Kanda, K. and Hashizume, K. (1989) Changes in properties of the medial gastrocnemius motor units in aging rat. J. Neurophysiol., 61: 737-746. Kawamura, Y., Okazaki, H., O'Brien, P. and Dyck, P.J. (1977a) Lumbar motoneurons of man I: Number and diameter histogram of alpha and gamma axons of ventral root. J. Neuropathol. Exp. Neurol., 36: 853-860. Kawamura, Y., O'Brien, P., Okazaki, H. and Dyck, P.J. (1977b) Lumbar motoneurons of man II: The number and diameter distribution of large- and intermediate-diameter cytons in 'motoneuron colunms' of spinal cord of man. J. Neuropathoi. Exp. Neurol., 36: 861-870. Kenshalo, D.R. (1986) Somesthetic sensitivity in young and elderly humans. J. G-erontol. 41: 732-742. Knox, C.A., Kokmen, E. and Dyck, P.J. (1989) Morphometric alteration of rat myelinated fibers with aging. J. Neuropathol. Exp. Neurol., 48: i 19-139. Komiya, Y. (1980) Slowing with age of the rate of slow axonal flow in bifurcating axons of rat dorsal root ganglion cells. Brain Res., 183: 477-480. Krinke, G. (1983) Spinal radiculoneuropathy in aging rats: Demyelination secondary to neuronal dwindling? Acta Neuropathol. (Bed.), 59: 63-69. Kromer, L.F. (1987) Nerve growth factor treatment after brain injury prevents neuronal death. Science, 235: 214-216. McDonagh, M.J.N., White, M.L. and Davies, C.T.M. (1984) Different
effects of ageing on the mechanical properties of human arm and leg muscles. Gerontology, 30: 49-54. McMartin, D.N. and O'Conner, J.A., Jr. (1979) Effect of age on axoplasmic transport of cholinesterase in rat sciatic nerves. Mech. Ageing Dev., 10: 241-248. Mesulam, M.-M. (1978) Tetramethyl benzidine for horseradish peroxidase neurohistochemistry; a non-carcinogenic blue reactionproduct with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem., 26:106-117. Mettling, C., Camu, W. and Henderson, C.E. (1993) Embryonic wing and leg motoneurons have intrinsically different survival properties. Development, 118:1149-1156. Mittal, K.R. and Logrnani, F.H. (1987) Age-related reduction in 8th cervical ventral nerve root myelinated fiber diameters and numbers in man. J. Gerontol., 42: 8-10. Mondon, C.E., Dolkas, C.B., Sims, C. and Reaven, G.M. (1985) Spontaneous running activity in male rats: Effect of age. J. Appl. Physiol. 58: 1553-1557. Morales, F.R., Boxer, P.A., Fung, S.J. and Chase, M.H. (1987) Basic electrophysiological properties of spinal cord motoneurons during old age in the cat. J. Neurophysiol., 58: 180-194. Nygaard, E. and Sanchez, J. (1982) Intramuscular variation of fiber types in the brachial biceps and the lateral vastus muscles of elderly men: How representative is a small biopsy sample? Anat. Rec., 203: 451-459. Oertel, G. (1986) Changes in human skeletal muscles due to ageing. Acta Neuropathol. (Bed.), 69: 309-313. Peyronnard, J.M. and Charron, L. (1983) Motoneuronal and motor axon innervation in the rat hindlimb: A comparative study using horseradish peroxidasc. Exp. Brain Res., 50: 125-132. Ramirez, V. and Ulfhake, B. (1992) Anatomy of dendrites in motoneurons supplying the intrinsic muscles of the foot sole in the aged cat: Evidence for dendritic growth and neosynaptogenesis. J. Comp. Neurol., 316: 1-16. Samorajski, T. (1974) Age differences in the morphology of posterior tibial nerves of mice. J. Comp. Neurol., 157: 439-452. Samorajski, T. and Bolsten, C. (1975) Nerve fiber hypertrophy in posterior tibial nerves of mice in response to voluntary running activity during aging. J. Comp. Neurol., 159: 553-558. Sato, H., Kanda, K., Hashizume, K. and Yamada, J. (1989) Nerve stimulation does not necessarily enhance retrograde HRP labeling of rat motoneuron. Neurosci. Lett., 107: 81-84. Sharma, A.K., Bajada, S. and Thomas, P.K. (1980) Age changes in the tibial and plantar nerves of the rat. J. Anat., 130: 417-428. St~lberg, E. and Fawcett, P.R.W. (1982) Macro EMG in healthy subjects of different ages. J. Neurol. Neurosurg. Psychiat., 45: 870-878. Thomas, P.K., King, R.H.M and Sharma, A.K. (1980) Changes with age in the peripheral nerves of the rat. Acta Neuropathol., 52: 1-6. Tohgi, H., Tsukagoshi, H. and Toyokura, Y. (1977) Quantitative changes with age in normal sural nerves. Acta Neuropathol. (Berl.), 38: 213-220. Tomlinson, B.E. and Irving, D. (1977) The numbers of motor neurons in the human lumbosacral cord throughout life. J. Neurol. Sci., 34: 213-219. Tomonaga, M. (1977) Histochemical and ultrastructural changes in senile human skeletal muscle. J. Am. Geriatr. Soc., 25: 125-131. Tsukagoshi, H., Yanagisawa, N., Oguchi, K., Nagashima, K. and Murakami, T. (1979) Morphometric quantification of the cervical limb motor cells in controls and in amyotrophic lateral sclerosis. J. Neurol. SCi., 41: 287-297. Williams, L.R., Varon, S. and Perterson, G.M. (1986) Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection. Proc. Natl. Acad. Sci. USA, 83: 9231-9235. Wright, E.A. and Spink, J.M. (1959) A study of the loss of nerve cells in the central nervous system in relation to age. Gerontologia, 3: 277-287.
Neuroseience Research 22 (1995) 189-196
Differential effects of aging on motoneurons and peripheral nerves innervating the hindlimb and forelimb muscles of rats K e n H a s h i z u m e 1, K e n r o K a n d a * Department of Central Nervous System, Tokyo Metropolitan Institute of Gerontology, 35-2, Sakaecho, ltabushi-ku, Tokyo 173, Japan Received 30 September 1994; accepted 27 January 1995
Abstract
We examined the number and size of ulnar (forelimb) and medial gastrocnemius (MG, hindlimb) motoneurons in middle-aged (9 months of age) and aged (27 months of age) male Fischer 344 rats. Morphological properties of the ulnar and the MG nerves were also studied. No significant difference was found in the mean number of the ulnar motoneurons between the two age groups, while that of MG motoneurons was significantly less in aged animals. A decrease in the number of myelinated fibers (including both afferent and efferent fibers) in the ulnar nerves was less than that in the MG nerves, although the age difference was not significant in either of the nerves. Soma atrophy of aged motoneurons was found in both MG and ulnar motor nuclei. The mean fascicular areas and myelinated fiber diameters were significantly increased in both the MG and the ulnar nerves in aged rats, but these were less pronounced for the ulnar nerve. The results indicate that most ulnar motoneurons, unlike MG motoneurons, survive at least to the age of 27 months. Morphological changes in the peripheral nerves were also less for the ulnar nerve than for the MG nerve. Thus, we conclude that the effects of aging on motoneurons and peripheral nerves innervating MG muscle of the hindlimb are greater than those innervating forelimb muscles. Keywords: Motoneuron; Cell survival; Peripheral nerve; Horseradish peroxidase methods; Forelimb; Hindlimb; Aging; Rat
1. Introduction Various morphological and physiological changes occur in the nervous system with advancing age. It has been reported that the total number of ventral horn neurons (i.e., presumed motoneurons) in the spinal cord decreases in both humans (Kawamura et al., 1977b; Tomlinson and Irving, 1977; Tsukagoshi et al., 1979) and animals (Wright and Spink, 1959). A decrease in the number of myelinated fibers in the ventral roots and peripheral nerves also suggests a loss of motoneurons in the aged (Samorajski, 1974; Kawamura et al., 1977a; Tohgi et al., 1977; Caccia et al., 1979; Jacobs and Love,
* Corresponding author, Tel.: +81 3 3964 3241 (ext. 3083); Fax: +81 3 3579 4779. I Current address: Faculty of Health and Sport Sciences, Osaka University, 1-17, Machikaneyama, Toyonaka, Osaka 560, Japan. K.H. was at the Department of Kinesiology, TMIG at the time of the experiment.
1985; Mittal and Logmani, 1987; Ansved and Larsson, 1990). In previous experiments, we investigated agerelated changes of motoneurons labeled with retrograde axonal transport of horseradish peroxidase (HRP) (Hashizume et al., 1988; see also Ishihara and Araki, 1988). There was a significant decrease in the number and size of motoneurons innervating the medial gastrocnemius (MG) muscle in the hindlimb of rats with advancing age. On the other hand, it has been reported that agerelated, mechanical and morphological changes of the muscle, including a decrease in maximum voluntary force, elongation of contraction time, fiber-type grouping, and atrophy of muscle fibers, are greater in leg (or hindlimb) muscles than in arm (or forelimb) muscles (Fujisawa, 1974; Grimby et al., 1982; Nygaard and Sanchez, 1982; McDonagh et al., 1984; Oertel, 1986). It has been suggested that some of these alterations seen in aging skeletal muscle are neurogenic (Fujisawa, 1974; Tomonaga, 1977; Kanda and Hashizume, 1989). Fur-
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thermore, St[dberg and Fawcett (1982) reported that an increase in size of the Macro motor unit potential was found in the tibialis anterior and the vastus lateralis muscles in aged humans of 60 years of age or over, but not in the biceps brachii muscle, and suggested that degeneration of motoneuron and peripheral nerves, entrapment at root or more distal sites and repeated nerve traumata, occurring with age, differ between motoneurons and peripheral nerves innervating different muscles, such as leg (or hindlimb) and arm (or forelimb) muscles. To obtain more direct evidence for such differential aging effects on brachial and lumbar motoneurons, we investigated age-related morphological changes in motor nuclei and peripheral nerves innervating forelimb and hindlimb muscles of the rat in the present experiments. Preliminary reports have appeared elsewhere (Hashizume and Kanda, 1990, 1992). 2. Materials and methods
2.1. Experimental animals and surgical procedures Experiments were performed on 15 male Fischer 344 rats, including 7 middle-aged (9 months of age, body weight: 390-485 g) and 8 aged (27 months of age, 320-470 g) animals. They were raised in a specific pathogen-free colony at our institute, in which the 75%, 50% and 25% survival times of the rats were about 25.5, 28 and 31.5 months, respectively. They were kept in conventional housing, with less than four animals/cage, without restrictions on feeding and without any special exercise requirements. The rat was anesthetized with pentobarbital (35 mg/kg; i.p.). Under aseptic conditions, the ulnar and MG nerves were bilaterally freed of surrounding tissues by microdissection. The ulnar nerve was cut and tied with suture material at a point just proximal to the elbow joint, and the MG nerve at a point near the entry to the MG muscle. Glass micropipettes, broken to a tip diameter of 20-80 t~m and filled with a fresh solution of 40% HRP (Sigma type VI or Toyobo I-C) in distilled water, were used to impale the proximal stump of the ulnar and MG nerves under a dissecting microscope. Small volumes (0.4-0.8 #1 for the ulnar nerve, 0.1-0.5 #1 for the MG nerve) of HRP solution were injected using 20-50 brief (15-30 ms) pulses of pressure (200-400 Pa), produced by a pulse-operated valve device (Picospritzer II, General Valve Co.). After HRP injection, the injection site was washed with sterile saline, then the wound was closed in layers. Both forelimbs and hindlimbs were treated identically in all rats. After 2 days, the animal was reanesthetized with pentobarbital and perfused transcardially with 500 ml of warmed saline followed by 700 ml of cold fixative mixture (1.25% glutaraldehyde and 1.0% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C). Thereafter, the spinal cord was removed and placed in 20% sucrose-
phosphate buffer at 4°C for 15-20 h. The spinal segment boundaries were marked with small holes made by inserting a pin into the dorsal columns at points between the entry zone of adjacent, identified dorsal roots. The cord was then cut into suitable blocks, with the C7-Th2 or L3-L6 segments included in a single block. Serial sections in the horizontal plane were cut at a thickness of 80 #In on a freezing microtome. All sections were saved in serial order and collected individually into 0.1 M phosphate buffer. They were processed for demonstration of HRP with the chromogen 3,3',5,5'-tetramethylbenzidine (TMB) using Mesulam's protocol (Mesulam, 1978). All sections were mounted on gelatinized slides in serial order without counterstaining. Sections containing HRP-labeled cells were photographed on high-contrast film (Kodak Technical Pan), and photomontage maps for cell identification were made of each section by printing on Kodalith Ortho Film. A small piece of the ulnar and MG nerve on either side was excised from the distal cut end just before HRP injection and fixed in 2.5% glutaraldehyde. This was later osmicated in 1% OsO4. Semithin cross-sections were cut at a thickness of about 1/zm from the specimen embedded in Epon and stained with toluidine blue.
2.2. Analytic procedures Individual HRP-labeled neurons were identified and counted under microscopic observation with the aid of photomontage maps. Labeled neurons were considered 'cell bodies' when the entire cell body was included in the section. When a cell body was located at the upper (or lower) surface of a section and a part of the cell body was also found at the lower (or upper) surface of the corresponding position in the adjoining section, we considered them to be parts of a single cell. In many cases, it was possible to confirm this by detecting the nucleus in only one of the divided cell bodies. The cell position was identified on the photomontage map. Using these techniques, it was possible to obtain a direct estimate of the motoneuron number in a given motor nucleus (or a given group of motor nuclei). The cross-sectional area (CSA) of the individual cell body was measured under brightfidd microscopy at x400 magnification. The outline of each cell body was traced with the aid of a digitizing unit connected to a computer (Macintosh IIfx) and the area within the outline boundary was calculated. The fascicular area of each nerve was calculated using the same tools as with the CSA of the cell body at x 250 magnification. In the MG nerve, the total number of myelinated axons was counted from photographic prints made of these sections at a magnification of about × 2070. The myelinated axon density was calculated from the fascicular area and the total number of myelinated axons. The maximum and minimum diameters of each axon and myelin sheath thickness
K. Hashizume, K. Kanda / Neuroscience Research 22 (1995) 189-196
were measured from these photographic prints. Average axon diameters (one-half of the sum of the measured maximum and minimum diameters) were then calculated. The fiber diameter was then calculated from the average axon diameters and myelin sheath thickness. In the ulnar nerve, the total number of myelinated axons was counted from photographic prints at a magnification of about x 690. The average axon diameters and myelin sheath thickness were measured for 650-700 individual myelinated axons located in the center of the fasciculus from photographic prints at a magnification of about x 2070. The average axon diameters were not measured for axons with an irregular or very flattened shape. No correction for shrinkage during fixation was attempted in this study.
191
A) Number of motoneurons Ulnar
MG 1401
310
'
I
!
I
300
130
tt
290 280
120
270
110
260
100
250
B) Soma cross-sectional area ( i.tm2 )
MG 2.3. Statistical analysis For the motoneurons, the mean values obtained, respectively from both sides of the limb were averaged for individual rats, then the means were compared between the middle-aged and aged groups. For the peripheral nerves, data from one side of the leg (either left or right) were considered as the representative values of the individual rats. We used the two-tailed independent t-test to test for differences between two mean values. 3. Results
900 800 700
600 500 r - - i middle-aged *
3.1. Number and size of MG and ulnar motoneurons Fig. 1A shows the mean numbers of MG (left) and ulnar (right) motoneurons labeled with HRP in middleaged and aged rats. The number of MG motoneurons in aged rats (121.0 ± 3.6, n = 8) was significantly smaller than that in middle-aged rats (132.1 -#- 5.7, n = 7). On the contrary, no significant difference was found in the number of ulnar motoneurons in aged (290.9 ± 7.0, n = 7) and middle-aged rats (287.4 4- 15.5, n = 7, P > 0.6). Fig. 1B shows the mean CSA of cell soma of MG (left) and ulnar (right) motoneurons. The CSA of MG motoneurons in aged rats (697.5 4- 126.3 /zm2) was smaller than that in middle-aged rats (777.4 4- 78.4 #m2), although the difference was not significant (P > 0.1). The mean CSA of ulnar motoneurons in aged rats (761.6 ± 28.0 #m 2) was significantly smaller than that in middle-aged rats (845.3 ± 47.5 #m2). 3.2. Morphological changes of corresponding peripheral nerves Fig. 2 shows photomicrographs of transverse semithin sections of the MG nerve of a middle-aged (A) and an aged rat (B). A reduction in the number of myelinated fibers and an increase in the amount of endoneural connective tissue were observed in aged MG nerve. Several fibers, especially those with a large diameter, showed myelin sheath irregularities, including myelin splitting
Ulnar
p<0.01
II
aged
** p < 0 . 0 0 1
Fig. 1. The numbers and sizes of the medial gastrocnemius (MG) and ulnar motoneurons. The MG motor nuclei in aged rats contained a fewer number of motoneurons compared to those of middle-aged rats (A, left). No significant difference was found in the number of ulnar motoneurons between the two age groups (A, right). The soma size of ulnar motoneurons in aged rats was significantly smaller than that in middle-aged rats (B, right). Vertical bars indicate 1 standard deviation from the mean.
with myelin balloon formation, infolded myelin loops and myelin reduplication as reported by other investigators (Knox et al., 1989; Ansved and Larsson, 1990). No cluster of small diameter fibers with a thin myelin sheath was found in the present experiments. Such regenerative sprouts were reported to be observed commonly in the ventral root of aged rats (Knox et al., 1989; Ansved and Larsson, 1990). The mean incidence of these fibers in aged rats (15%, range: 9-17%) was higher than that in middle-aged rats (2%, range: 1-3%). Myelin sheath irregularities were observed commonly in fibers with a large diameter. Fig. 3 shows photomicrographs of transverse semithin sections of the ulnar nerve of a middle-aged (A) and an aged rat (B). Unlike the MG nerve, an obvious reduction in myelinated fibers and an increase in the amount of endoneural connective tissue were not observed in
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,o,
Fig. 2. Photomicrographs of transverse semithin sections of the MG nerve stained with toluidine blue in a middle-aged (A) and an aged rat (B). A reduction in myelinated fibers and increases in fascicular area, axon diameter and the amount of endoneural connective tissue were observed in aged MG nerve. Some fibers in aged nerves showed myelin sheath irregularities, including myelin splitting with myelin balloon formation, infolded myelin loops and myelin reduplication. Calibration bar = 50 ~m.
Fig. 3. Photomicrographs of transverse semithin sections of the ulnar nerve stained with toluidine blue in a middle-aged (A) and an aged rat (B). Unlike the M G nerve, an obvious reduction in myelinated fibers and an increase in the amount of endoneural connective tissue were not observed in aged nerves, although increases in fascicular area and axonal diameter were observed. No cluster of regenerating fibers was found in aged ulnar or MG nerves. Calibration bar = 50 ~m.
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193
Table i Morphologial properties of the medial gastrocnemius and ulnar nerves in middle-aged and aged rats Group
n
Faseicular area (ram 2)
Number of fibers
Axon diameter (tzm)
Myelin sheath thickness (/~m)
Medial gastrocnemius nerve Middle-aged 4 Aged 6
0.04 4- 0.003 0.07 ± 0.01"*
285.3 4- 23.7 257.0 4- 22.0
5.5 4- 0.5 6.5 4- 0.3*
1.2 ± 0.1 1.4 ± 0.2
Ulnar nerve Middle-aged Aged
0.16 + 0.01 0.19 4- 0.01*
2247.8 + 34.0 2202.3 4- 68.3
4.3 4- 0.2 5.1 4- 0.2**
0.92 + 0.08 0.98 + 0.11
6 6
Fiber density (n/mm 2)
7142 ± 965 3787 ± 331'*
13 903 4- l l31 II 543 4- 663*
n, number of myelinated fibers. Values are expressed as group means ± S.D. *P < 0.01, **P < 0.001.
aged nerves. No cluster of presumed regenerating fibers was found, although some fibers showed myelin sheath irregularities as in the MG nerves. The mean incidence of these fibers in aged rats (2%) was lower than that in the MG nerves. Table 1 shows the quantitative data of the MG and ulnar nerves in middle-aged and aged rats. The mean fascicular area of the MG nerve was larger by 75% in aged rats than in middle-aged rats whereas that of the ulnar nerve was larger by only 19% in aged rats than in middle-aged rats. The mean number of myelinated fibers (including both afferent and efferent fibers) in aged rats did not statistically differ from that in middle-aged rats. The myelinated fiber density, calculated from the fascicular area and the total number of myelinated
(%) 7 ~=
6
t-
5
° - -
~" E
4
$
3
e~
E
2
t-
._> II)
re
1 0 0
5
10
15
20
25
Average fiber diameter (~tm) Fig. 4. Comparison of average diameters of MG myelinated fibers in middle-aged (open bars) and aged (shaded bars) rats plotted as percentages. These histograms include all myelinated fibers obtained from four middle-aged (1133 fibers) and six aged nerves (1513 fibers), respectively.
fibers, of the MG nerves was reduced by 47% in aged rats. The ulnar nerves showed a smaller reduction (17%) in the fiber density. The mean axon diameter in aged rats was larger by 19% for the MG nerve and 18% for the ulnar nerve compared to that in middle-aged rats. Age difference in myelin sheath thickness was not found in either the MG or ulnar nerves. The histograms in Fig. 4 show the distribution of diameters of MG myelinated fibers pooled from middleaged (open bars) and aged (shaded bars) rats plotted as percentages. The fiber diameters were distributed bimodally in both groups. The transition point between small and large was at about 7.5 ~m for middle-aged rats and about 9 #m for aged rats. The histograms from aged rats shifted to the right, but fibers with a larger diameter exceeding 25 t~m were not observed (see also Fig. 2B). The count of large fibers with a diameter larger than 9 ~m in aged rats was 130.7 ± 13.4 (n = 6). This value was significantly smaller than that for fibers with a diameter larger than 7.5 /~m in middle-aged rats (154.5 ± 12.7, n = 4, P < 0.05). No significant difference was found in the number of small fibers between the age groups (130.8 ± 13.7 for aged rats, 133.5 ± 30.7 for middle-aged rats, respectively). The range of motion at the knee joint was also measured before sacrificing the animal. The maximally extended position was reduced from 155° for middleaged rats to 135° for aged rats. The wet weight of the MG muscle in aged rats (560 mg) was significantly lighter than that in middle-aged rats (747 mg) (P < 0.001). The flexor carpi ulnaris muscle innervated by the ulnar nerve also had reduced weight with advancing age from 162 mg to 143 mg (P < 0.01). 4. Discussion
4.1. Differential effects of aging on MG and ulnar motoneurons and corresponding peripheral nerves The present experiments demonstrated that the number of ulnar motoneurons did not differ significantly between middle-aged (9 months of age) and aged (27
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months of age) rats, whereas the MG motor nucleus in aged rats contained a smaller number of labeled motoneurons compared to that in middle-aged rats. A decrease in the number of labeled motoneurons could be explained by cell death and/or an increase in the number of unlabeled cells. The latter may be due to a deficit of axonal transport. If the axon does not extend to the injection site, HRP can not be taken up and transported to the cell soma. A decrease in the rate of anterograde fast axonal flow has been observed in the sciatic nerve of aged rats (McMartin and O'Conner, 1979; Frolkis et al., 1985). In previous experiments on middle-aged rats, we have shown that labeled motoneurons can be observed 13 h after HRP injection into the MG muscle and that labeling intensity reached near maximum level at about 20 h after HRP injection (Sato et al., 1989). Forty-eight hours of survival time between HRP injection and animal sacrifice seems to be enough to label all motoneurons existing in the MG nucleus even in the case of aged rats. Furthermore, the number of unlabeled neurons located within and around the MG motor nucleus did not differ between aged and middle-aged rats (Kanda et al., unpublished observation). Thus, the present results indicate that neuronal cell loss occurs in the MG motor nucleus whereas most ulnar motoneurons survive at least to the age of 27 months. The common features of aged nerves observed in the present study were an increase in fascicular area and average axon diameter, and a decrease in myelinated fiber density. The ratios of these changes in the ulnar nerves were 19%, 18% and 17%, respectively, whereas those in the MG nerves were 75%, 19% and 47%, respectively. The mean incidence of fibers with myelin sheath irregularities in aged animals was also lower in the ulnar nerve than in the MG nerve. Thus, alterations in various morphological parameters of the peripheral nerve were also greater in the MG nerve than in the ulnar nerve. These findings in motor nuclei and peripheral nerves suggest that the effects of aging on motoneurons and peripheral nerves innervating the MG muscle in the hindlimb are greater than in those innervating forelimb muscles. The differential age effect on the sensory neurons innervating forelimb and hindlimb regions has also been suggested by the finding that a decrease in vibration sensitivity with age is greater in the foot than in the hand (Kenshalo, 1986). It has also been found that axonal dystrophy occurs consistently and more severely in the gracilis than in the cuneate nuclei of aged humans (Jellinger and Jirasek, 1971). The underlying mechanisms causing the differential extent and/or rate of progress of aging among different motor nuclei and peripheral nerves are not yet clear. The following are some comments on the factors causing the differential age effects on MG and ulnar motoneurons. It is known that neurotrophic factors play an important role in cell survival during development. The effect of nerve growth factor (NGF) on neuronal survival after
axotomy has also been demonstrated in the medial septum of the adult rat (Hefti, 1986; Williams et al., 1986; Kromer, 1987). Thus, difference in survival rate between MG and ulnar motoneurons in aged rats may be due to the differential effect of aging on the supply of trophic substances. A decrease in the rate of axonal transport with age (McMartin and O'Conner, 1979; Komiya, 1980; Frolkis et al., 1985) may cause difficulty in the maintenance structures of axons, and a deficit of trophic factors in the soma. MG motoneurons with a longer axon may be affected more severely than ulnar motoneurons with a relatively short axon. Alternatively, there might be an intrinsic difference in susceptibility to a deficit of trophic substances between MG motoneurons and ulnar motoneurons. Mettling et al. (1993) have shown in the chick embryo that brachial motoneurons die later than lumbar motoneurons during embryonic development, and that the difference is not a result of differing peripheral trophic support, but is intrinsically programmed, at least partially through a higher sensitivity to trophic factors. At present, however, it is not clear whether a deficit in trophic factors caused the motoneuronal death with age seen in the present experiments. Another factor may be neuronal activity. Degenerative changes found in motoneurons in aged rats might be attributable to some extent to disuse of muscles. It has been reported that spontaneous locomotion activity gradually decreases in aging rats (Samorajski and Rolsten, 1975; Holloszy et al., 1985; Mondon et al., 1985; Brown et al., 1992). A decrease in the range of motion at the knee joint found in the present experiments also suggests reduced activity in hindlimb muscles. In contrast, activity of forelimb motoneurons might be high because of maintained movement for grooming and feeding. In fact, the ratio of decrease in the wet weights was larger in the MG muscle (about 25%, P < 0.001) than in the flexor carpi ulnaris muscle (about 12%, P < 0.01). Reduced activity seems to lead to a decline in metabolic activity of motoneurons and eventually to their atrophy. Recently, we demonstrated that age-related changes in MG motoneurons and MG myelinated fibers were retarded by long-term, moderate endurance exercise (Hashizume and Kanda, 1991, 1993). The difference in the amount of activity, therefore, may cause a different degree of age-related changes between MG and ulnar motoneurons. The innervation ratio of MG motoneurons appeared to be greater than that of ulnar motoneurons, which means that MG motoneurons have a greater number of terminal branches than do ulnar motoneurons. In addition, the lengths of MG axons are longer than those of ulnar axons. It seems that the cell soma of the MG motoneurons have to synthesize more protein to maintain their structures than do ulnar motoneurons. Thus, declining synthesis and capability for protein breakdown with increasing age (cf. Ekstrom et al.,
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1980), if this occurs in spinal motoneurons, may affect MG cells more severely than ulnar cells. 4.2. Alterations of the peripheral nerves We found an increased fascicular area and fiber diameter of the MG muscle nerves in aged rats whereas the majority of studies on human and experimental animals have reported a decreased or unaltered fascicular area and myelinated fiber diameter (Samorajski, 1974; Sharma et al., 1980; Krinke, 1983; Jacobs and Love, 1985; Knox et al., 1989; Chase et al., 1992). Ansved and Larsson (1990) have reported similar findings in the soleus muscle nerve in aged rats to those in the present experiments. Thus, changes in the sizes of peripheral nerves during the aging periods is a matter of controversy. The reason for the difference is not clear at present. Since the quantitative data in the present study were obtained from a single transverse section of nerves, it is not known whether uniform enlargement has occurred along the entire axon. A study on teased fiber preparations revealed that the formation of myelin balloons appeared focally along myelinated fibers (Knox et al., 1989). These were seen commonly in the ventral root and the diameter of these fibers often exceeded more than 50 #m. The distribution of myelinated fiber diameters in aged MG nerve shifted to the right, but none exceeded 25 t~m (Figs. 2 and 4). Myelinated fibers of the ulnar nerves showed similar features to those of the MG nerves (Fig. 3). Hence, these results suggest that an increase in the mean axon diameter seems to be part of a general trend in all axons in aged MG and ulnar nerves rather than to the existence of axons with an extremely expanded diameter. An increased axon diameter in aged rats seems to reflect some sort of failure of metabolism and/or axonal transport rather than an increased activity. We did not see any clusters of small diameter fibers with thin myelin sheaths in the present experiments. Such regenerating fibers have been found frequently in the ventral root of aged rats (Knox et al., 1989; Ansved and Larsson, 1990). Thus, it appears that regeneration reaction occurs preferentially in the proximal portions of the peripheral nerves. Decline in the conduction velocity of individual motor and primary afferent fibers in aged cats and rats has also been demonstrated in other experiments (Chase et al., 1985, 1992; Morales et al., 1987; Kanda and Hashizume, 1989; Ramirez and Ulfhake, 1992; Kanda, unpublished data). Segmental demyelination and remyelination have been found frequently both in ventral roots and peripheral nerves in aged humans and animals (Sharma et al., 1980; Thomas et al., 1980; Grover-Johnson and Spencer, 1981; Braund et al., 1982a,b; Jacobs and Love, 1985; Knox et al., 1989; Ansved and Larsson, 1990). These degenerative changes in myelin sheath structure may reduce the conduction velocity in aged rats (Adinolfi et al., 1991).
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4.3. Loss of motoneurons and primary sensory neurons Peyronnard and Charron (1983), by using a combination of HRP methods and posterior rhizotomy in middle-aged rats, demonstrated that the number of presumed motor axons was close to that of the tibialis anterior motoneurons labeled with HRP, suggesting that extramuscular branching of motor axons was rather insignificant in total axon counts in adult rats. In the present study, clusters of small-diameter fibers with a thin myelin sheath, suggesting fiber regeneration (Knox et al., 1989; Ansved and Larsson, 1990), were not found in either of the age groups. Thus, the total number of myelinated fibers seemed to correspond to the actual count of their original neurons. If so, we can estimate that 46% (132.1/285.3) of the total number of myelinated fibers in middle-aged MG nerves consist of motor fibers. This value is also comparable to that of the tibialis anterior nerve (Peyronnard and Charron, 1983). The ratio of motor fibers in aged MG nerve can be estimated as 47% (121.0/257.0), which was very close to that in the middleaged nerves. A decrease of sensory neuron with advancing age, therefore, was of the same order of magnitude as that of motoneurons.
Acknowledgements The authors are grateful to Dr. H. Nagasaki for providing K.H. with the opportunity to participate in this study, and Ms. E. Nomoto and Ms. S. Asaki for their technical assistance.
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