EXPERIMENTAL
NEUROLOGY
Accelerated
48, 88-94 (1975)
Recovery from Peripheral Nerve in Experimental Hyperthyroidism G. MCISAAC
Department
AND J. A. KIERNAN
of Anatomy, Health Western Ontario, Received
London,
Sciences
Celztre, Ontario,
February
5, 1975
Injury
1
The University Canada
of
The right sciatic nerve was crushed in the thigh in 48 rats, and an indwelling stimulating electrode was implanted at the site of injury. Four groups of rats received respectively, no treatment, triiodothryonine (T3), 1.0 pg/kg daily from day 15 to 21, T3 from day 1 to 7 and T3 from day 1 to 40. Daily testing for recovery of movement at the ankle joint showed that administration of T3 throughout the postoperative period increased the rate of functional recovery by a factor of 50%. The rats were killed after 40 days and a quantitative histological study revealed no differences in the proportions of innervated motor endplates in the tibialis anterior muscle amongst the four experimental groups. It was concluded that reinnervation of this muscle was completed after 40 days, irrespective of hormonal treatment. In specimens of skin from the calf, the proportions of innervated hair follicles were significantly higher in rats treated for the first 7 days and for all 40 days with T5 than in the untreated rats and in those receiving T3 from day 15 to 21. It is possible, therefore, that sensory neurons are more sensitive than motor neurons to T3 administered in the first postoperative week.
INTRODUCTION The thyroid hormones, which play an important role in the maturation of the nervous system (3, 5, lo), have also been shown to promote the regeneration of axons severed within the brains and spinal cords of adult animals (4, 6). Furthermore, elongation of axons regenerating in the sciatic nerve of the rat is accelerated by treatment with triiodothyronine (T3), an effect whose magnitude increases with the dosage of the hormone (1) . The present study was undertaken in order to determine : (a) whether treatment with T3 reduced the time required for functional recovery in de1 Requests for reprints should be addressed to J. A. K. We are grateful to the J. P. Bickell Foundation, Toronto, for generous financial support, and to Mrs. Wendy Venturin for technical assistance.
88 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
PERII’HERAL
NERVE
REGENERATION
89
nervated muscles, (b) the optimal timing of administration of the hormone, and (c) the effect of T3 upon reinnervation of the skin. MATERIALS
AND
METHODS
Animals and Opera&aJeProcedure. The experimental animaIs were 48 male albino Wistar rats, weighing 250-300 g. All were caged individually and fed a standard cube diet with water ad lib. Each rat was anesthetized with pentobarbitone sodium (36 “g/kg, ip). The right sciatic nerve was exposed in the thigh and crushed for 90 set at a point midway between the hip and knee joints. A tungsten wire (0.25 mm diameter) was attached to the epineurium at the site of crushing and led under the skin of the buttock, back and neck to a bipolar socket which was cemented to two small screws in the parietal bones of the skull. A second tungsten wire was led from the other pole of the socket into the subcutaneous tissue of the interscapular region. Both wires were insulated up to their tips with closely fitting polyethylene tubing. Treatment Groups. The operated rats were divided into four groups of 12 animals: Group l-no treatment; Group Z-subcutaneous injection of T3 (3,5,3’-triiodo-L-thyronine sodium, Sigma Chemical Co., St. Louis, MO., U..S.A.), 1.0 pg/kg body wt, once daily from the 15th to the 2lst postoperative days; Group 3-the same dosage of T3 for the first 7 postoperative days; Group 4-the same dosage every day until autopsy. Functional Testing. All the animals were tested every day for recovery of function of the muscles acting on the ankle joint. The sciatic nerve was stimulated by inserting into the socket on the head a plug connected to a Grass SD9 square-wave stimulator. Monophasic pulses (8 msec duration ; lOO/sec) were used, and the minimum voltage producing detectable movement at the ankle joint was determined. With a maximum stimulus of 20 V and no responseit was assumedthat denervation was complete. Great care was taken not to confuse movements at the ankle with those at the hip or knee. The animals were also examined for spontaneousmovement at the ankle, which was occasionally observed earlier than that due to electrical stimulation. The time required for regeneration was recorded as the number of days elapsed between crushing the sciatic nerve and the detection of any type of muscular movement of the tarsus on the tibia. Necropsy and Histological Procedures. All animals were killed by intraperitoneal overdosage with pentobarbitone 40 days after operation. The right sciatic nerve was exposed and, with the femur at right angles to the lumbosacral spine and the knee in full extension, the distance from the site of crushing to the point of entry of the anterior tibia1 nerve into the tibialis anterior muscle was measured. The right tihialis anerior muscle of each rat was fixed by immersion
90
MCISAAC
AND
TABLE EFFECT
Group
1
OF TRIIODOTHYRONINE
KIERNAN
1 ON RATE
OF REGENERATION
Num- Rate of regeneration ber of (mm/day) rats (mean f standard error of mean) 10
0.98f 0.08
9
1.03f 0.10
11
1.31 f 0.07
12
1.46 f 0.14
Significance” of difference from group : 1 2 3 4
-
N.S. N.S. P < 0.025
(Control)
-
N.S. P < 0.025
(T3, da: 15-21) -
N.S.
(T3, da: l-7) -
(T3, da: 140) o Determined by one-way analysis of variance, followed by the Newman-Keuls multiple range test, as described by Zar (17). N.S.-not significant (P > 0.05).
for 24 hr at 4 C in phosphate-buffered 3% formaldehyde, pH 7.2. Frozen sections 60 pm thick were cut in the coronal plane and stained by the bromoindigo-silver method for neuromuscular innervation (12). The proportions of innervated and non-innervated motor end-plates were determined as described in detail elsewhere (13). A piece of skin, 5 mm2, was excised from the posterior aspect of the right leg, halfway between the knee and the ankle, fixed in ammoniated alcohol and stained in the block by Ranson’s (15) silver-pyridine method. In paraffin sections 20 pm thick, the proportions of innervated and noninnervated hair follicles were determined approximately using the following scoring system : O-no (or exceedingly few) innervated follicles ; l-about one third of follicles innervated ; 2-about two thirds of follicles innervated ; 3-all or almost all follicles innervated. The right sciatic nerves were fixed in Bouin’s fluid and paraffin sections 7 pm thick were stained with Luxol fast blue MBS (11) for myelin and by a urea-silver nitrate method (9) for axons. RESULTS Six of the operated animals either died during the course of the experiment or were found at necropsy to have their electrode assembliesbroken. The following account refers to the 42 rats from which acceptable results were obtained.
PERIPHERAL
NERVE
91
REGENERATION
Functional Recovery. The first movement to occur at the ankle joint of the denervated limb was usually plantar flexion (action of the gastrocnemius, plantaris, soleus, tibialis posterior and flexor digitorum longus muscles), though return of dorsiflexion (action of the tibialis anterior, extensor digitorum longus and peroneus longus muscles) sometimes preceded that of plantar flexion. Most of the above-named muscles are innervated at approximately the same level in the leg. The measured distance from the site of crushing the sciatic nerve to the point of entry of the anterior tibia1 nerve into the tibialis anterior muscle varied among animals from 21 to 32 mm. For purposes of statistical comparison, a rate of regeneration was calculated for each animal by dividing this distance by the time elapsed until the first signs of functional recovery were recorded. The results are presented in Table 1, where it is seen that the mean rate of regeneration in the untreated rats (Group 1) was increased by a factor of about 50% by treatment with T3 for the whole postoperative period (Group 4). Smaller increases in rate were found with Groups 2 and 3, but these did not differ significantly from the controls. TABLE QUANTITATIVE
2
HISTOLOGICAL CRUSHING
OBSERVATIONS SCIATIC NERVE
Group
Number rats
Percentage of innervated endplates observed in tibialis anterior (mean f standard error of mean)
1
10
22.8 f
6.8”
1.27 zt O.ll*
9
19.7 f
4.5=
1.51 f
11
33.8 *
5.1”
2.53 & 0.04*
12
27.8 f
8.1a
2.69 z!z 0.02b
of
40 DAYS
AFTER
Score for proportion of innervated hair follicles in skin specimen (mean f standard error of mean)
(Control)
(T3,
da:
15-21)
(T3,
da:
1-7)
(T3,
da;
l-40)
0.11*
a These percentages do not differ significantly from one another (analysis of variance). * Analysis of variance and Newman-Keuls test (17) show that the scores for Groups 3 and 4 are significantly higher than those for Groups 1 and 2 (P < 0.001). There are no significant differences between the scores for Groups 1 and 2 or between those for Groups 3 and 4 (P > 0.05 and P > 0.1, respectively).
92
MCISAAC
AND
KIERNAN
Histological Observations. The percentages of innervated endplates found 40 days after crushing the sciatic nerve are shown in Table 2. The mean values did not differ significantly among the four groups of animals. It should be noted that in the normal tibialis anterior of the rat, prepared in the same way, the innervating axon is not seen, owing to exclusion from the plane of section in about 35% of the motor endplates (13). Thus the observed value of 26% innervated endplates indicates actual innervation of about 40% of these structures, The scores relating to the innervation of hair follicles are also presented in Table 2. Evidently the proportions of innervated follicles on the 40th postoperative day were considerably higher in Groups 3 and 4 than in Groups 1 and 2. Histological examination of the sciatic nerves revealed the presence, distal to the lesion, of abundant argyrophilic axons, many of which were myelinated. At the site of injury and of attachment of the tungsten wire, the trunk of the nerve was narrowed by the formation of collagenous scar tissue. Many axons had regenerated into the scar. The appearances were alike in all animals, and no qualitative differences were discerned among any of the four experimental groups. DISCUSSION The results presented above show that muscular function, following axonotmesis, recovers more rapidly in animals treated with T3 throughout the postoperative period than in untreated animals or in ones treated with the hormone for 7 days. From inspection of Table 1, it is seen that the mean rate of regeneration in Group 3 (T3 for the first 7 days) is almost as high as that for Group 4 (T3 throughout), though not acceptably significantly different (0.1 > P > 0.05) from that found in the control group. So while it may be stated with reasonable certainty that 1 week of treatment with T3 late in the course of regeneration (Group 2) has no appreciable effect, it is not yet proven whether or not treatment for a limited period of time immediately after crushing the nerve is to some extent beneficial. In this context it of interest to note that the action of exogenous T3 upon regenerating axons in the cerebrum of the rat may be mediated principally in the first week after injury (4). More detailed investigations of the time-course of action of T3 upon regeneration of both central and peripheral axons are in progress. With 1.0 pg/kg body weight of T3 daily, the rate of regeneration, measured functionally, was increased by a factor of 50%. By comparison with the quantitative histological data of Cockett and Kiernan (1)) this dosage would be expected to induce an acceleration of axonal elongation of similar magnitude.
PERIPHERAL
NERVE
REGENERATION
93
In an experiment rather similar to the present one, Isenschmid (7)) in 1932 observed slightly more rapid functional recovery following crushing of the sciatic nerve in young rats (average weight 60 g) treated with thyroxine (800 pg/kg daily) combined with an extract of thymus gland. This author also observed retarded regeneration in thyroidectomized rats, which was reversed by treatment with thyroxine. No observations were made upon previously euthyroid rats treated with thyroxine alone. Isens&mid used small numbers of animals, and his results are statistically unsatisfactory. When thyroxine (“1.75 mg” ; almost certainly an error! Probably 1.75 pg) was administered to rats (200 g) with crushed tibia1 nerves by DiazGuerrero et al. (2), it was found that after 25 days the strength of contraction of the gastrocnemius muscle, elicited by stimulation of the nerve, was increased by a factor of 9% relative to the values recorded in untreated control animals. The strength was reduced by 12% in rats made hypothyroid by treatment with thiouracil. The doses of thyroxine used by Isens&mid and by Diaz-Guerrero IA al. were very high and could be expected to produce severe hyperthyroidism (S). The dose of T3 in the present experiment is comparable on a weight-for-weight basis with doses used clinically in the therapy of human hypothyroidism (16) and is also known to produce only mild toxic symptoms in previously euthyroid subjects (14). The quantitative histological assessment of the reinnervation of the tibialis anterior muscle revealed no differences in the proportion of innervated motor enclplates among the four groups of animals. This probably indicates that neuromuscular reinnervation was virtually completed 40 days after injuring the sciatic nerve. However, only about 40% of the endplates were innervated. The most probable reason for this low percentage is that scarring at the site of attachment of the stimulating electrode prevented the regeneration of many axons in all animals. Such an explanation accords with the histological appearances of the operated nerves. In a sequential histological study of the reinnervation of the rat’s tibialis anterior over a period of 35 days (13) it has been shown that the proportion of innervated endplates rises much more rapidly in T3-treated animals than in untreated controls, this effect being most pronounced from the 15th to the 30th postoperative day. Reinnervation was complete after 30 days in rats treated daily with T3 (1.0 pg/kg) and was 607~ complete in the controls after 3.5 days. No indwelling electrodes were attached to the nerves of these animals. Higher proportions of innervated hair follicles were found in the specimens of skin taken from rats treated with T3 for all 40 or for only the first 7 postoperative days than in untreated rats or those treated with T3 from day 15 to day 21. This observation, which indicates that the process of reinnervation is slower in skin than in muscle, supports the contention
94
MCISAAC
AND
KIERNAN
(tide sup?%) that administration of T3 only during the first week after crushing the sciatic nerve may appreciably accelerate axonal regeneration. It is conceivable that sensory neurons may be more sensitive to the hormone than motor neurons.
REFERENCES 1. COCKETT, S. A., and J. A. KIZRNAN. 1973. Acceleration of peripheral nervous regeneration in the rat by exogenous triiodothyronine. Exp. Neural. 39 : 38%394. 2. DIAZ-GUERRBRO, R., J. D. THOMSON, and H. M. HINES. 1947. Effect of thymectomy, hyperthyroidism and hypothyroidism on neuromuscular atrophy and regeneration. Amer. J. Physiol. 151: 91-95. 3. EAYRS, J. T. 1964. Effects of thyroid hormones on brain differentiation, pp. 60-71. In “Brain-Thyroid Relationships,” CIBA Foundation Study Group No. 18. M. P. Cameron and M. O’Connor [Eds.]. Churchill, London. 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. Newel. 33 : 372-385. 5. HAMBURGH, M. l%P. The role of thyroid and growth hormones in neurogenesis, pp. 109-148. 1% “Current Topics in Developmental Biology.” A. A. Moscona and A. Monroy. [Eds.]. Academic Press, New York and London. 6. HARVEY, J. E., and H H. SFCEBNIK 1967. Locomotor activity and axon regeneration following spinal cord compression in rats treated with L-thyroxine. J. Neuropath. Exp. Neural. 26 : 661-668. 7. ISENSCHMID, R. 1932. Ueber den Einfluss von Thymus und Schilddriise auf die Nerven-Regeneration. Schweiz. Med. Wochensch. 62 : 785-789. 8. KHAMSI, F., and J. T. EAYRS. 1966. A study of the effects of thyroid hormones on growth and development. Growth 30 : 143-156. 9. KIERNAN, J. A. 1971. Pituicytes and the regenerative properties of neurosecretory and other axons in the rat. (Appendix: A technique for staining axons in paraffin sections). J. Amt. (London) 109: 97-114. 10. KIERNAN, J. A., and P. M. RAWCLIFFE. 1971. Effects of triiodothyronine on the cerebellar cortex of the new-born rat in tissue culture. Experientiu 27: 678-679. 11. KL~~VER, H., and E. BARRERA. 1953. A method for the combined staining of cells and fibers in the central nervous system. J. Neuropathol. Exfi. Neural. 12: 400-403. 12. MCISAAC, G., and J. A. KIERNAN. 1974. Complete staining of neuromuscular innervation with bromoindigo and silver. Stain Techrtol. 49: 211-214. 13. MCISAAC, G., and J. KIERNAN. 1975. Acceleration of neuromuscular re-innervation by triiodothyronine. J. Anat. (London) (in press). 14. PRANCE, A. J. (Ed.) 1974. “The Thyroid Axis, Drugs and Behavior.” Raven Press, New York. 15. RANSON, S. W. 1911. Non-medullated nerve fibers in the spinal nerves. Amer. J. Amt. 12 : 67-87. 16. RAPOPORT, B., and S. REFETOFF. 1972. Hypothroidism, pp. 461-464. In “Current Therapy, 1972” H. F. Conn [Ed.] Saunders, Philadelphia, Toronto and London. Englewood, Cliffs, N. J. 17. ZAR, J. H. 1974. “Biostatistical Analysis.” Prentice-Hall,
NEUROLOGY
Accelerated
48, 88-94 (1975)
Recovery from Peripheral Nerve in Experimental Hyperthyroidism G. MCISAAC
Department
AND J. A. KIERNAN
of Anatomy, Health Western Ontario, Received
London,
Sciences
Celztre, Ontario,
February
5, 1975
Injury
1
The University Canada
of
The right sciatic nerve was crushed in the thigh in 48 rats, and an indwelling stimulating electrode was implanted at the site of injury. Four groups of rats received respectively, no treatment, triiodothryonine (T3), 1.0 pg/kg daily from day 15 to 21, T3 from day 1 to 7 and T3 from day 1 to 40. Daily testing for recovery of movement at the ankle joint showed that administration of T3 throughout the postoperative period increased the rate of functional recovery by a factor of 50%. The rats were killed after 40 days and a quantitative histological study revealed no differences in the proportions of innervated motor endplates in the tibialis anterior muscle amongst the four experimental groups. It was concluded that reinnervation of this muscle was completed after 40 days, irrespective of hormonal treatment. In specimens of skin from the calf, the proportions of innervated hair follicles were significantly higher in rats treated for the first 7 days and for all 40 days with T5 than in the untreated rats and in those receiving T3 from day 15 to 21. It is possible, therefore, that sensory neurons are more sensitive than motor neurons to T3 administered in the first postoperative week.
INTRODUCTION The thyroid hormones, which play an important role in the maturation of the nervous system (3, 5, lo), have also been shown to promote the regeneration of axons severed within the brains and spinal cords of adult animals (4, 6). Furthermore, elongation of axons regenerating in the sciatic nerve of the rat is accelerated by treatment with triiodothyronine (T3), an effect whose magnitude increases with the dosage of the hormone (1) . The present study was undertaken in order to determine : (a) whether treatment with T3 reduced the time required for functional recovery in de1 Requests for reprints should be addressed to J. A. K. We are grateful to the J. P. Bickell Foundation, Toronto, for generous financial support, and to Mrs. Wendy Venturin for technical assistance.
88 Copyright All rights
@ 1975 by Academic Press, Inc. of reproduction in any form reserved.
PERII’HERAL
NERVE
REGENERATION
89
nervated muscles, (b) the optimal timing of administration of the hormone, and (c) the effect of T3 upon reinnervation of the skin. MATERIALS
AND
METHODS
Animals and Opera&aJeProcedure. The experimental animaIs were 48 male albino Wistar rats, weighing 250-300 g. All were caged individually and fed a standard cube diet with water ad lib. Each rat was anesthetized with pentobarbitone sodium (36 “g/kg, ip). The right sciatic nerve was exposed in the thigh and crushed for 90 set at a point midway between the hip and knee joints. A tungsten wire (0.25 mm diameter) was attached to the epineurium at the site of crushing and led under the skin of the buttock, back and neck to a bipolar socket which was cemented to two small screws in the parietal bones of the skull. A second tungsten wire was led from the other pole of the socket into the subcutaneous tissue of the interscapular region. Both wires were insulated up to their tips with closely fitting polyethylene tubing. Treatment Groups. The operated rats were divided into four groups of 12 animals: Group l-no treatment; Group Z-subcutaneous injection of T3 (3,5,3’-triiodo-L-thyronine sodium, Sigma Chemical Co., St. Louis, MO., U..S.A.), 1.0 pg/kg body wt, once daily from the 15th to the 2lst postoperative days; Group 3-the same dosage of T3 for the first 7 postoperative days; Group 4-the same dosage every day until autopsy. Functional Testing. All the animals were tested every day for recovery of function of the muscles acting on the ankle joint. The sciatic nerve was stimulated by inserting into the socket on the head a plug connected to a Grass SD9 square-wave stimulator. Monophasic pulses (8 msec duration ; lOO/sec) were used, and the minimum voltage producing detectable movement at the ankle joint was determined. With a maximum stimulus of 20 V and no responseit was assumedthat denervation was complete. Great care was taken not to confuse movements at the ankle with those at the hip or knee. The animals were also examined for spontaneousmovement at the ankle, which was occasionally observed earlier than that due to electrical stimulation. The time required for regeneration was recorded as the number of days elapsed between crushing the sciatic nerve and the detection of any type of muscular movement of the tarsus on the tibia. Necropsy and Histological Procedures. All animals were killed by intraperitoneal overdosage with pentobarbitone 40 days after operation. The right sciatic nerve was exposed and, with the femur at right angles to the lumbosacral spine and the knee in full extension, the distance from the site of crushing to the point of entry of the anterior tibia1 nerve into the tibialis anterior muscle was measured. The right tihialis anerior muscle of each rat was fixed by immersion
90
MCISAAC
AND
TABLE EFFECT
Group
1
OF TRIIODOTHYRONINE
KIERNAN
1 ON RATE
OF REGENERATION
Num- Rate of regeneration ber of (mm/day) rats (mean f standard error of mean) 10
0.98f 0.08
9
1.03f 0.10
11
1.31 f 0.07
12
1.46 f 0.14
Significance” of difference from group : 1 2 3 4
-
N.S. N.S. P < 0.025
(Control)
-
N.S. P < 0.025
(T3, da: 15-21) -
N.S.
(T3, da: l-7) -
(T3, da: 140) o Determined by one-way analysis of variance, followed by the Newman-Keuls multiple range test, as described by Zar (17). N.S.-not significant (P > 0.05).
for 24 hr at 4 C in phosphate-buffered 3% formaldehyde, pH 7.2. Frozen sections 60 pm thick were cut in the coronal plane and stained by the bromoindigo-silver method for neuromuscular innervation (12). The proportions of innervated and non-innervated motor end-plates were determined as described in detail elsewhere (13). A piece of skin, 5 mm2, was excised from the posterior aspect of the right leg, halfway between the knee and the ankle, fixed in ammoniated alcohol and stained in the block by Ranson’s (15) silver-pyridine method. In paraffin sections 20 pm thick, the proportions of innervated and noninnervated hair follicles were determined approximately using the following scoring system : O-no (or exceedingly few) innervated follicles ; l-about one third of follicles innervated ; 2-about two thirds of follicles innervated ; 3-all or almost all follicles innervated. The right sciatic nerves were fixed in Bouin’s fluid and paraffin sections 7 pm thick were stained with Luxol fast blue MBS (11) for myelin and by a urea-silver nitrate method (9) for axons. RESULTS Six of the operated animals either died during the course of the experiment or were found at necropsy to have their electrode assembliesbroken. The following account refers to the 42 rats from which acceptable results were obtained.
PERIPHERAL
NERVE
91
REGENERATION
Functional Recovery. The first movement to occur at the ankle joint of the denervated limb was usually plantar flexion (action of the gastrocnemius, plantaris, soleus, tibialis posterior and flexor digitorum longus muscles), though return of dorsiflexion (action of the tibialis anterior, extensor digitorum longus and peroneus longus muscles) sometimes preceded that of plantar flexion. Most of the above-named muscles are innervated at approximately the same level in the leg. The measured distance from the site of crushing the sciatic nerve to the point of entry of the anterior tibia1 nerve into the tibialis anterior muscle varied among animals from 21 to 32 mm. For purposes of statistical comparison, a rate of regeneration was calculated for each animal by dividing this distance by the time elapsed until the first signs of functional recovery were recorded. The results are presented in Table 1, where it is seen that the mean rate of regeneration in the untreated rats (Group 1) was increased by a factor of about 50% by treatment with T3 for the whole postoperative period (Group 4). Smaller increases in rate were found with Groups 2 and 3, but these did not differ significantly from the controls. TABLE QUANTITATIVE
2
HISTOLOGICAL CRUSHING
OBSERVATIONS SCIATIC NERVE
Group
Number rats
Percentage of innervated endplates observed in tibialis anterior (mean f standard error of mean)
1
10
22.8 f
6.8”
1.27 zt O.ll*
9
19.7 f
4.5=
1.51 f
11
33.8 *
5.1”
2.53 & 0.04*
12
27.8 f
8.1a
2.69 z!z 0.02b
of
40 DAYS
AFTER
Score for proportion of innervated hair follicles in skin specimen (mean f standard error of mean)
(Control)
(T3,
da:
15-21)
(T3,
da:
1-7)
(T3,
da;
l-40)
0.11*
a These percentages do not differ significantly from one another (analysis of variance). * Analysis of variance and Newman-Keuls test (17) show that the scores for Groups 3 and 4 are significantly higher than those for Groups 1 and 2 (P < 0.001). There are no significant differences between the scores for Groups 1 and 2 or between those for Groups 3 and 4 (P > 0.05 and P > 0.1, respectively).
92
MCISAAC
AND
KIERNAN
Histological Observations. The percentages of innervated endplates found 40 days after crushing the sciatic nerve are shown in Table 2. The mean values did not differ significantly among the four groups of animals. It should be noted that in the normal tibialis anterior of the rat, prepared in the same way, the innervating axon is not seen, owing to exclusion from the plane of section in about 35% of the motor endplates (13). Thus the observed value of 26% innervated endplates indicates actual innervation of about 40% of these structures, The scores relating to the innervation of hair follicles are also presented in Table 2. Evidently the proportions of innervated follicles on the 40th postoperative day were considerably higher in Groups 3 and 4 than in Groups 1 and 2. Histological examination of the sciatic nerves revealed the presence, distal to the lesion, of abundant argyrophilic axons, many of which were myelinated. At the site of injury and of attachment of the tungsten wire, the trunk of the nerve was narrowed by the formation of collagenous scar tissue. Many axons had regenerated into the scar. The appearances were alike in all animals, and no qualitative differences were discerned among any of the four experimental groups. DISCUSSION The results presented above show that muscular function, following axonotmesis, recovers more rapidly in animals treated with T3 throughout the postoperative period than in untreated animals or in ones treated with the hormone for 7 days. From inspection of Table 1, it is seen that the mean rate of regeneration in Group 3 (T3 for the first 7 days) is almost as high as that for Group 4 (T3 throughout), though not acceptably significantly different (0.1 > P > 0.05) from that found in the control group. So while it may be stated with reasonable certainty that 1 week of treatment with T3 late in the course of regeneration (Group 2) has no appreciable effect, it is not yet proven whether or not treatment for a limited period of time immediately after crushing the nerve is to some extent beneficial. In this context it of interest to note that the action of exogenous T3 upon regenerating axons in the cerebrum of the rat may be mediated principally in the first week after injury (4). More detailed investigations of the time-course of action of T3 upon regeneration of both central and peripheral axons are in progress. With 1.0 pg/kg body weight of T3 daily, the rate of regeneration, measured functionally, was increased by a factor of 50%. By comparison with the quantitative histological data of Cockett and Kiernan (1)) this dosage would be expected to induce an acceleration of axonal elongation of similar magnitude.
PERIPHERAL
NERVE
REGENERATION
93
In an experiment rather similar to the present one, Isenschmid (7)) in 1932 observed slightly more rapid functional recovery following crushing of the sciatic nerve in young rats (average weight 60 g) treated with thyroxine (800 pg/kg daily) combined with an extract of thymus gland. This author also observed retarded regeneration in thyroidectomized rats, which was reversed by treatment with thyroxine. No observations were made upon previously euthyroid rats treated with thyroxine alone. Isens&mid used small numbers of animals, and his results are statistically unsatisfactory. When thyroxine (“1.75 mg” ; almost certainly an error! Probably 1.75 pg) was administered to rats (200 g) with crushed tibia1 nerves by DiazGuerrero et al. (2), it was found that after 25 days the strength of contraction of the gastrocnemius muscle, elicited by stimulation of the nerve, was increased by a factor of 9% relative to the values recorded in untreated control animals. The strength was reduced by 12% in rats made hypothyroid by treatment with thiouracil. The doses of thyroxine used by Isens&mid and by Diaz-Guerrero IA al. were very high and could be expected to produce severe hyperthyroidism (S). The dose of T3 in the present experiment is comparable on a weight-for-weight basis with doses used clinically in the therapy of human hypothyroidism (16) and is also known to produce only mild toxic symptoms in previously euthyroid subjects (14). The quantitative histological assessment of the reinnervation of the tibialis anterior muscle revealed no differences in the proportion of innervated motor enclplates among the four groups of animals. This probably indicates that neuromuscular reinnervation was virtually completed 40 days after injuring the sciatic nerve. However, only about 40% of the endplates were innervated. The most probable reason for this low percentage is that scarring at the site of attachment of the stimulating electrode prevented the regeneration of many axons in all animals. Such an explanation accords with the histological appearances of the operated nerves. In a sequential histological study of the reinnervation of the rat’s tibialis anterior over a period of 35 days (13) it has been shown that the proportion of innervated endplates rises much more rapidly in T3-treated animals than in untreated controls, this effect being most pronounced from the 15th to the 30th postoperative day. Reinnervation was complete after 30 days in rats treated daily with T3 (1.0 pg/kg) and was 607~ complete in the controls after 3.5 days. No indwelling electrodes were attached to the nerves of these animals. Higher proportions of innervated hair follicles were found in the specimens of skin taken from rats treated with T3 for all 40 or for only the first 7 postoperative days than in untreated rats or those treated with T3 from day 15 to day 21. This observation, which indicates that the process of reinnervation is slower in skin than in muscle, supports the contention
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(tide sup?%) that administration of T3 only during the first week after crushing the sciatic nerve may appreciably accelerate axonal regeneration. It is conceivable that sensory neurons may be more sensitive to the hormone than motor neurons.
REFERENCES 1. COCKETT, S. A., and J. A. KIZRNAN. 1973. Acceleration of peripheral nervous regeneration in the rat by exogenous triiodothyronine. Exp. Neural. 39 : 38%394. 2. DIAZ-GUERRBRO, R., J. D. THOMSON, and H. M. HINES. 1947. Effect of thymectomy, hyperthyroidism and hypothyroidism on neuromuscular atrophy and regeneration. Amer. J. Physiol. 151: 91-95. 3. EAYRS, J. T. 1964. Effects of thyroid hormones on brain differentiation, pp. 60-71. In “Brain-Thyroid Relationships,” CIBA Foundation Study Group No. 18. M. P. Cameron and M. O’Connor [Eds.]. Churchill, London. 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. Newel. 33 : 372-385. 5. HAMBURGH, M. l%P. The role of thyroid and growth hormones in neurogenesis, pp. 109-148. 1% “Current Topics in Developmental Biology.” A. A. Moscona and A. Monroy. [Eds.]. Academic Press, New York and London. 6. HARVEY, J. E., and H H. SFCEBNIK 1967. Locomotor activity and axon regeneration following spinal cord compression in rats treated with L-thyroxine. J. Neuropath. Exp. Neural. 26 : 661-668. 7. ISENSCHMID, R. 1932. Ueber den Einfluss von Thymus und Schilddriise auf die Nerven-Regeneration. Schweiz. Med. Wochensch. 62 : 785-789. 8. KHAMSI, F., and J. T. EAYRS. 1966. A study of the effects of thyroid hormones on growth and development. Growth 30 : 143-156. 9. KIERNAN, J. A. 1971. Pituicytes and the regenerative properties of neurosecretory and other axons in the rat. (Appendix: A technique for staining axons in paraffin sections). J. Amt. (London) 109: 97-114. 10. KIERNAN, J. A., and P. M. RAWCLIFFE. 1971. Effects of triiodothyronine on the cerebellar cortex of the new-born rat in tissue culture. Experientiu 27: 678-679. 11. KL~~VER, H., and E. BARRERA. 1953. A method for the combined staining of cells and fibers in the central nervous system. J. Neuropathol. Exfi. Neural. 12: 400-403. 12. MCISAAC, G., and J. A. KIERNAN. 1974. Complete staining of neuromuscular innervation with bromoindigo and silver. Stain Techrtol. 49: 211-214. 13. MCISAAC, G., and J. KIERNAN. 1975. Acceleration of neuromuscular re-innervation by triiodothyronine. J. Anat. (London) (in press). 14. PRANCE, A. J. (Ed.) 1974. “The Thyroid Axis, Drugs and Behavior.” Raven Press, New York. 15. RANSON, S. W. 1911. Non-medullated nerve fibers in the spinal nerves. Amer. J. Amt. 12 : 67-87. 16. RAPOPORT, B., and S. REFETOFF. 1972. Hypothroidism, pp. 461-464. In “Current Therapy, 1972” H. F. Conn [Ed.] Saunders, Philadelphia, Toronto and London. Englewood, Cliffs, N. J. 17. ZAR, J. H. 1974. “Biostatistical Analysis.” Prentice-Hall,
