EXPERIMENTAL
NEUROLOGY
64, 225-230
(1979)
RESEARCH
NOTE
The Effect of Triiodothyronine on Axonal Transport Regenerating Peripheral Nerves
in
M. FRIZELL AND W. G. MCLEAN’ Department of Neurology and Institute of Neurobiology, University of Gteborg, G&eborg. Sweden Received
October
9, 1978
Recently it was indicated that triiodothyronine (T3) may accelerate the elongation of regenerating rat sciatic nerve fibers (2,lO). The mechanism of this effect is unknown, but it was suggested that thyroid hormones may increase the synthesis of axoplasmic proteins in regenerating neurons (9). In the central nervous system these hormones can improve nerve regeneration during certain conditions (4, 8). Also, clinically, a positive effect of thyroid hormones on human peripheral nerve regeneration was suggested (12). The axonal transport of labeled proteins was previously studied in regenerating peripheral motor and sensory rabbit nerves (5, 6, 1 l), and it was shown that the progression of the peak of rapidly migrating radiolabeled proteins accumulating at the tips of the sprouting axons along a regenerating nerve could be used to calculate the regeneration rate (5). The aim of the present study was to investigate if T3 had any promoting effect on axonal transport of labeled proteins and on regeneration rate during the initial period of regeneration after a crush of rabbit hypoglossal and vagus nerves. In a first group of experiments the axonal transport of Abbreviations: T3-triiodothyronine; TCA-trichloracetic acid. ’ Supported by the Swedish Medical Research Council (grant B78-12X-0226-12A); Science Research Council, Great Britain; and European Molecular Biology Organisation (W.G.McL). We thank Ann-Katrin Jakobsson for her technical help. Dr. McLean’s present address is Department of Pharmacology, School of Pharmacy, Liverpool Polytechnic, Byrom Street, Liverpool L3 3 AF, U.K. 225 0014-4886/79/040225-06$02.00/O Copyright 0 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.
226
FRIZELL
AND
MCLEAN
rapidly and slowly migrating, labeled proteins was studied in regenerating motor fibers of rabbit hypoglossal and vagus nerves. Rabbits (1.5 to 2.0 kg) were anesthetized with pentobarbital, and the nerves were exposed and crushed unilaterally with a silk thread against a glass rod. The thread was left in position around the nerve to mark the site of crush. The animals received daily subcutaneous injections of T3 (L-triiodothyronine, Sigma, 1 &kg) during the regeneration period. One week after the crush the proteins of the hypoglossal and dorsal vagus nuclei were labeled bilaterally with 30 &i L-[4,53-H]leucine (38 Wmmol, The Radiochemical Centre, Amersham) as previously described (5, 6). Forty-eight hours later, when the fronts of slowly migrating, axonally transported, labeled proteins were in the proximal parts of the nerves, and the rapidly migrating labeled proteins had reached and accumulated at the distal parts of the regenerating axons, the nerves were taken out for analysis of radioactivity. In a second group of experiments, the axonal transport of rapidly migrating labeled proteins was studied in normal and regenerating rabbit sensory vagus fibers in vitro. In the regeneration experiments the vagus nerves had been crushed 4 weeks before labeling, 2 to 4 cm distal to the nodose ganglion as described above. The nerves with the attached ganglia were dissected out, ligated 6 cm distal to the nodose ganglion, and were placed in a two-compartment Perspex chamber for incubation as previously described (11). The proteins in the nodose ganglia were labeled by the addition of 15 &i L-[4,53-H]leucine to the ganglion compartment, and the medium surrounding the axons in the nerve compartment contained 10 rig/ml T3. Twenty-four hours after labeling the incubation was stopped, and the nerves were analyzed for radioactivity. For analysis of radioactivity the nerves were cut into 2.5- or 5-mm pieces; the pieces were soaked 24 h in 10% trichloracetic acid (TCA), washed once more with TCA, and then solubilized in Soluene-100. The radioactivity was measured in a Packard Tri-carb liquid scintillation spectrometer, and corrections were made for quenching. In the in vivo experiments fronts of radiolabeled proteins appeared in the proximal parts of regenerating and contralateral hypoglossal and vagus nerves 48 h after labeling, representing the slow phase of axonal transport. In the hypoglossal nerves the amount of radioactivity in the fronts, and the extension of the fronts from the nerve cell bodies was greater on the regenerating side than on the contralateral side, as previously found (Fig. la) (5). The pattern of distribution of radioactivity in the regenerating and contralateral nerves, however, was identical to that previously found (5) without T, treatment. Similarly (Fig. lb) in the vagus nerves the 48-h profiles of radiolabeled proteins were identical to those previously found without T3 treatment: that is, the amount of radioactivity and the extension
TRIIODOTHYRONINE
AND AXONAL a
TRANSPORT
227
t ::
3ooa
: E E 2ooc E v! N I
1001
10
20
t
mm
30 from
bulb
b
FIG. 1. The front of slowly migrating 3H-labeled proteins in proximal parts of regenerating ( - - - ) and contralateral ( ) hypoglossal (a) and vagus (b) nerves 48 h after labeling. The nerves were crushed 1 week before labeling as indicated by the arrow. Rapidly migrating 3H-labeled proteins show an accumulation distal to (a), or at the crush zone (b). One experiment typical of three is shown. The rabbits received daily injections of T3 (1 &kg) during the regenerating period.
228
FRIZELL
AND
MCLEAN
of the fronts were less on the regenerating side. In these experiments the rapidly migrating radiolabeled proteins accumulated distal to the fronts of slowly migrating proteins: in the hypoglossal nerves 15 to 20 mm distal to the crush zone and in the vagus nerves at the crush zone (Figs. la, b). The site of these accumulation zones, probably representing the sprouting zones of regenerating axons (5,7), was identical to that previously found in regenerating nerves without T3 treatment (5). When rapid axonal transport of radiolabeled proteins was studied in normal vagus sensory fibers in vitro in the presence of T3 in the nerve compartment, an accumulation of these proteins occured at the distal ligature (Fig. 2). The magnitude of this accumulation was not significantly different from the control nerves. In the regenerating sensory vagus fibers an accumulation of rapidly migrating radiolabeled proteins occured at the crush zone 4 weeks after the crush, and there was no evidence of transport beyond it (Fig. 3), similar to previous experiments without T3 treatment (11). The present results show that T3 in the dose used here had no promoting effect on slow axonal transport in normal or regenerating hypoglossal and vagus nerves of the rabbit during the initial period of regeneration. There was no evidence for increased regeneration rate during the 1st week of
I A
t-1-j 6
40
60 mm
from
ganglion
FIG. 2. The profile of 3H-labeled proteins in rabbit vagus nerves incubated 24 h in vitro. The TCA-precipitable radioactivity in 5-mm nerve segments is expressed as a percentage of the total radioactivity in the nerves between points A and B (the ligature). Dotted lines indicate nerves incubated in the presence of 10 rig/ml T,. Each value is a mean of four nerves + SE.
TRIIODOTHYRONINE
AND AXONAL
TRANSPORT
229
1
40
10
1 A
20
B
t
40
60 mm
from
ganglion
FIG. 3. The profile of 3H-labeled proteins in a rabbit vagus nerve subjected to a crush at a point marked (t) 4 weeks previously and incubated 24 h in vitro in the presence of 10 @ml Ts. TCA-precipitable radioactivity in S-mm nerve segments is expressed as in Fig. 2. One experiment of two is shown.
regeneration in hypoglossal or vagus motor fibers after T3 administration. In vitro, T3 had no effect on rapid axonal transport in normal sensory vagus fibers, nor could this hormone support axonal transport into regenerating sensory vagus fibers in vitro 4 weeks after a nerve crush. These findings are in agreement with previous results where it was reported that T3 treatment did not accelerate the regeneration rate of rat sciatic motor and sensory axons (1) or rat facial nerve (13). It is suggested that the beneficial effect of T3 on nerve regeneration previously reported probably was not due to a generally increased synthesis or transport of axoplasmic proteins. Other mechanisms such as an accelerated maturation of the regenerating fibers (1) might be responsible. Although the doses of T3 used in the present study were similar to those previously reported (3,9, lo), it has been claimed that a dose of 1 &kg in the rat does not represent a hyperthyroid state (l), and the possibility cannot be excluded that T, affects nerve regeneration differently when
230
FRIZELL
AND MCLEAN
different doses are used, especially in other nerves (13) and in species other than those used in the present experiments. REFERENCES 1. BERENBERG, R. A., D. S. FORMAN, D. K. WOOD, A. DESILVA, AND J. DEMARRE. 1977. Recovery of peripheral nerve function after axotomy: effect of triiodothyronine. Exp. Neural.
51: 349-363.
2. COCKETT, S. A., AND J. A. KIERNAN. 1973. Acceleration of peripheral nervous regeneration in the rat by exogeneous triiodothyronine. Exp. Neurol. 39: 389-394. 3. COOK. R. A., AND J. A. KIERNAN. 1976. Effects of triiodothyronine on protein synthesis in regenerating peripheral neurons. Exp. Neural. 52: 515-524. 4. FERTIG, A., J. A. KIERNAN, AND S. S. A. S. SEYAN. 1971. Enhancement of axonal regeneration in the brain of the rat by corticotrophin and triiodothyronine. Exp. Neurol. 33: 372-385. 5. FRIZELL, M., AND J. SJ~~STXAND. 1974. The axonal transport of slowly migrating (3H) leucine-labelled proteins and the regeneration rate in regenerating hypoglossal and vagus nerves of the rabbit. Bruin Res. 81: 267-283. 6. FRIZELL, M., AND J. SJ~STRAND. 1974. The axonal transport of (3H) fucose labelled glycoproteins in normal and regenerating peripheral nerves. Brain Res. 78: 109-123. 7. GRIFFIN, J. W., D. B. DRACHMAN, AND D. L. PRICE. 1976. Fast axonal transportinmotor nerve regeneration. J. Neurobiol. 7: 355-370. 8. HARVEY, J. E., AND H. H. SREBNIK. 1967. Locomotor activity and axon regeneration following spinal cord compression in rats treated with t-thyroxine. J. Neuropathol. Exp Neurol. 26: 661-668. 9. KIERNAN, J. A., AND P. M. RAWCLIFFE. 1971. Effects of triiodothyronine on the cerebellar cortex of the new-born rat in tissue culture. Experientia 27: 678-679. 10. MCISAAC, G., AND J. A. KIERNAN. 1975. Acceleration of neuromuscular re-innervation by triiodothyronine. J. Anat. 120: 551-560. 11. MCLEAN, W. G., M. FRIZELL, AND J. SJGSTRAND. 1976. Rapid axonal transport of labeled proteins in regenerating sensory and motor fibers of rabbit vagus nerve. Exp. Neurol.
52: 242-249.
12. MCQUARRIE, I. G. 1975. Nerve regeneration and thyroid hormone treatment. J. Neurol. Sci. 26: 499-502.
13. STELMACK, B. M., AND J. A. KIERNAN. 1977. Effects of triiodothyrouine on the normal and regenerating facial nerve of the rat. Acta Neuropathol. (Berlin) 40: 151- 155.
NEUROLOGY
64, 225-230
(1979)
RESEARCH
NOTE
The Effect of Triiodothyronine on Axonal Transport Regenerating Peripheral Nerves
in
M. FRIZELL AND W. G. MCLEAN’ Department of Neurology and Institute of Neurobiology, University of Gteborg, G&eborg. Sweden Received
October
9, 1978
Recently it was indicated that triiodothyronine (T3) may accelerate the elongation of regenerating rat sciatic nerve fibers (2,lO). The mechanism of this effect is unknown, but it was suggested that thyroid hormones may increase the synthesis of axoplasmic proteins in regenerating neurons (9). In the central nervous system these hormones can improve nerve regeneration during certain conditions (4, 8). Also, clinically, a positive effect of thyroid hormones on human peripheral nerve regeneration was suggested (12). The axonal transport of labeled proteins was previously studied in regenerating peripheral motor and sensory rabbit nerves (5, 6, 1 l), and it was shown that the progression of the peak of rapidly migrating radiolabeled proteins accumulating at the tips of the sprouting axons along a regenerating nerve could be used to calculate the regeneration rate (5). The aim of the present study was to investigate if T3 had any promoting effect on axonal transport of labeled proteins and on regeneration rate during the initial period of regeneration after a crush of rabbit hypoglossal and vagus nerves. In a first group of experiments the axonal transport of Abbreviations: T3-triiodothyronine; TCA-trichloracetic acid. ’ Supported by the Swedish Medical Research Council (grant B78-12X-0226-12A); Science Research Council, Great Britain; and European Molecular Biology Organisation (W.G.McL). We thank Ann-Katrin Jakobsson for her technical help. Dr. McLean’s present address is Department of Pharmacology, School of Pharmacy, Liverpool Polytechnic, Byrom Street, Liverpool L3 3 AF, U.K. 225 0014-4886/79/040225-06$02.00/O Copyright 0 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.
226
FRIZELL
AND
MCLEAN
rapidly and slowly migrating, labeled proteins was studied in regenerating motor fibers of rabbit hypoglossal and vagus nerves. Rabbits (1.5 to 2.0 kg) were anesthetized with pentobarbital, and the nerves were exposed and crushed unilaterally with a silk thread against a glass rod. The thread was left in position around the nerve to mark the site of crush. The animals received daily subcutaneous injections of T3 (L-triiodothyronine, Sigma, 1 &kg) during the regeneration period. One week after the crush the proteins of the hypoglossal and dorsal vagus nuclei were labeled bilaterally with 30 &i L-[4,53-H]leucine (38 Wmmol, The Radiochemical Centre, Amersham) as previously described (5, 6). Forty-eight hours later, when the fronts of slowly migrating, axonally transported, labeled proteins were in the proximal parts of the nerves, and the rapidly migrating labeled proteins had reached and accumulated at the distal parts of the regenerating axons, the nerves were taken out for analysis of radioactivity. In a second group of experiments, the axonal transport of rapidly migrating labeled proteins was studied in normal and regenerating rabbit sensory vagus fibers in vitro. In the regeneration experiments the vagus nerves had been crushed 4 weeks before labeling, 2 to 4 cm distal to the nodose ganglion as described above. The nerves with the attached ganglia were dissected out, ligated 6 cm distal to the nodose ganglion, and were placed in a two-compartment Perspex chamber for incubation as previously described (11). The proteins in the nodose ganglia were labeled by the addition of 15 &i L-[4,53-H]leucine to the ganglion compartment, and the medium surrounding the axons in the nerve compartment contained 10 rig/ml T3. Twenty-four hours after labeling the incubation was stopped, and the nerves were analyzed for radioactivity. For analysis of radioactivity the nerves were cut into 2.5- or 5-mm pieces; the pieces were soaked 24 h in 10% trichloracetic acid (TCA), washed once more with TCA, and then solubilized in Soluene-100. The radioactivity was measured in a Packard Tri-carb liquid scintillation spectrometer, and corrections were made for quenching. In the in vivo experiments fronts of radiolabeled proteins appeared in the proximal parts of regenerating and contralateral hypoglossal and vagus nerves 48 h after labeling, representing the slow phase of axonal transport. In the hypoglossal nerves the amount of radioactivity in the fronts, and the extension of the fronts from the nerve cell bodies was greater on the regenerating side than on the contralateral side, as previously found (Fig. la) (5). The pattern of distribution of radioactivity in the regenerating and contralateral nerves, however, was identical to that previously found (5) without T, treatment. Similarly (Fig. lb) in the vagus nerves the 48-h profiles of radiolabeled proteins were identical to those previously found without T3 treatment: that is, the amount of radioactivity and the extension
TRIIODOTHYRONINE
AND AXONAL a
TRANSPORT
227
t ::
3ooa
: E E 2ooc E v! N I
1001
10
20
t
mm
30 from
bulb
b
FIG. 1. The front of slowly migrating 3H-labeled proteins in proximal parts of regenerating ( - - - ) and contralateral ( ) hypoglossal (a) and vagus (b) nerves 48 h after labeling. The nerves were crushed 1 week before labeling as indicated by the arrow. Rapidly migrating 3H-labeled proteins show an accumulation distal to (a), or at the crush zone (b). One experiment typical of three is shown. The rabbits received daily injections of T3 (1 &kg) during the regenerating period.
228
FRIZELL
AND
MCLEAN
of the fronts were less on the regenerating side. In these experiments the rapidly migrating radiolabeled proteins accumulated distal to the fronts of slowly migrating proteins: in the hypoglossal nerves 15 to 20 mm distal to the crush zone and in the vagus nerves at the crush zone (Figs. la, b). The site of these accumulation zones, probably representing the sprouting zones of regenerating axons (5,7), was identical to that previously found in regenerating nerves without T3 treatment (5). When rapid axonal transport of radiolabeled proteins was studied in normal vagus sensory fibers in vitro in the presence of T3 in the nerve compartment, an accumulation of these proteins occured at the distal ligature (Fig. 2). The magnitude of this accumulation was not significantly different from the control nerves. In the regenerating sensory vagus fibers an accumulation of rapidly migrating radiolabeled proteins occured at the crush zone 4 weeks after the crush, and there was no evidence of transport beyond it (Fig. 3), similar to previous experiments without T3 treatment (11). The present results show that T3 in the dose used here had no promoting effect on slow axonal transport in normal or regenerating hypoglossal and vagus nerves of the rabbit during the initial period of regeneration. There was no evidence for increased regeneration rate during the 1st week of
I A
t-1-j 6
40
60 mm
from
ganglion
FIG. 2. The profile of 3H-labeled proteins in rabbit vagus nerves incubated 24 h in vitro. The TCA-precipitable radioactivity in 5-mm nerve segments is expressed as a percentage of the total radioactivity in the nerves between points A and B (the ligature). Dotted lines indicate nerves incubated in the presence of 10 rig/ml T,. Each value is a mean of four nerves + SE.
TRIIODOTHYRONINE
AND AXONAL
TRANSPORT
229
1
40
10
1 A
20
B
t
40
60 mm
from
ganglion
FIG. 3. The profile of 3H-labeled proteins in a rabbit vagus nerve subjected to a crush at a point marked (t) 4 weeks previously and incubated 24 h in vitro in the presence of 10 @ml Ts. TCA-precipitable radioactivity in S-mm nerve segments is expressed as in Fig. 2. One experiment of two is shown.
regeneration in hypoglossal or vagus motor fibers after T3 administration. In vitro, T3 had no effect on rapid axonal transport in normal sensory vagus fibers, nor could this hormone support axonal transport into regenerating sensory vagus fibers in vitro 4 weeks after a nerve crush. These findings are in agreement with previous results where it was reported that T3 treatment did not accelerate the regeneration rate of rat sciatic motor and sensory axons (1) or rat facial nerve (13). It is suggested that the beneficial effect of T3 on nerve regeneration previously reported probably was not due to a generally increased synthesis or transport of axoplasmic proteins. Other mechanisms such as an accelerated maturation of the regenerating fibers (1) might be responsible. Although the doses of T3 used in the present study were similar to those previously reported (3,9, lo), it has been claimed that a dose of 1 &kg in the rat does not represent a hyperthyroid state (l), and the possibility cannot be excluded that T, affects nerve regeneration differently when
230
FRIZELL
AND MCLEAN
different doses are used, especially in other nerves (13) and in species other than those used in the present experiments. REFERENCES 1. BERENBERG, R. A., D. S. FORMAN, D. K. WOOD, A. DESILVA, AND J. DEMARRE. 1977. Recovery of peripheral nerve function after axotomy: effect of triiodothyronine. Exp. Neural.
51: 349-363.
2. COCKETT, S. A., AND J. A. KIERNAN. 1973. Acceleration of peripheral nervous regeneration in the rat by exogeneous triiodothyronine. Exp. Neurol. 39: 389-394. 3. COOK. R. A., AND J. A. KIERNAN. 1976. Effects of triiodothyronine on protein synthesis in regenerating peripheral neurons. Exp. Neural. 52: 515-524. 4. FERTIG, A., J. A. KIERNAN, AND S. S. A. S. SEYAN. 1971. Enhancement of axonal regeneration in the brain of the rat by corticotrophin and triiodothyronine. Exp. Neurol. 33: 372-385. 5. FRIZELL, M., AND J. SJ~~STXAND. 1974. The axonal transport of slowly migrating (3H) leucine-labelled proteins and the regeneration rate in regenerating hypoglossal and vagus nerves of the rabbit. Bruin Res. 81: 267-283. 6. FRIZELL, M., AND J. SJ~STRAND. 1974. The axonal transport of (3H) fucose labelled glycoproteins in normal and regenerating peripheral nerves. Brain Res. 78: 109-123. 7. GRIFFIN, J. W., D. B. DRACHMAN, AND D. L. PRICE. 1976. Fast axonal transportinmotor nerve regeneration. J. Neurobiol. 7: 355-370. 8. HARVEY, J. E., AND H. H. SREBNIK. 1967. Locomotor activity and axon regeneration following spinal cord compression in rats treated with t-thyroxine. J. Neuropathol. Exp Neurol. 26: 661-668. 9. KIERNAN, J. A., AND P. M. RAWCLIFFE. 1971. Effects of triiodothyronine on the cerebellar cortex of the new-born rat in tissue culture. Experientia 27: 678-679. 10. MCISAAC, G., AND J. A. KIERNAN. 1975. Acceleration of neuromuscular re-innervation by triiodothyronine. J. Anat. 120: 551-560. 11. MCLEAN, W. G., M. FRIZELL, AND J. SJGSTRAND. 1976. Rapid axonal transport of labeled proteins in regenerating sensory and motor fibers of rabbit vagus nerve. Exp. Neurol.
52: 242-249.
12. MCQUARRIE, I. G. 1975. Nerve regeneration and thyroid hormone treatment. J. Neurol. Sci. 26: 499-502.
13. STELMACK, B. M., AND J. A. KIERNAN. 1977. Effects of triiodothyrouine on the normal and regenerating facial nerve of the rat. Acta Neuropathol. (Berlin) 40: 151- 155.
