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Retrograde transport of protein tracer in the rabbit hypoglossal nerve during regeneration
BRAIN RESEARCH
175
RETROGRADE TRANSPORT OF PROTEIN TRACER IN THE RABBIT HYPOGLOSSAL NERVE DURING REGENERATION
KRISTER KRISTENSSON AND JOHAN SJOSTRAND Neuropathological Laboratory, Department of Pathology I and Institute of Neurobiology, University of Gi~teborg, Gi~teborg (Sweden)
(Accepted April 7th, 1972)
INTRODUCTION In previous studies it was found that horseradish peroxidase, and albumin labelled with Evans blue, after injection into the gastrocnemius muscle or tongue of rats, rabbits and suckling mice, were transferred to the corresponding motor neurones in the spinal cord and the brain stem15-17,19. This phenomenon was interpreted to have resulted from an uptake of proteins into axons at the periphery, followed by axonal transport in a retrograde direction to the nerve cell bodies. The results therefore support the hypothesis that there exists an axonal transport of macromolecules in the retrograde direction, as has been indicated by earlier studies6,13,20-22,27,a3. A lesion of a peripheral nerve, which causes interruption of the axons, leads to certain morphological changes in the axons on both sides of the injury. These changes include an accumulation of axoplasmic constituents in the regions adjacent to the lesion; this has been interpreted to be the result of interrupted axonal transport of materials in the anterogradeao and the retrograde directions~l, 3~. When the nerve fibres regenerate after the lesion the reacting nerve cell bodies increase their production of RNA and protein2,11,z8. Little, however, is known about the changes in axonal transport during the various phases of axonal outgrowth, and about how migration of protein and organelles within the axon affect the protein turnover in the nerve cell perikarya. A change in the retrograde axonal transport can possibly be of importance in eliciting a signal for chromatolysis in the early phase of nerve regeneration5, and during later phases it can allow for peripheral influence of the connected end organs on axonal maturationl,2L The present study was undertaken with the aim of examining whether any changes in the neuronal uptake and retrograde transport of an exogenous macromolecule from the periphery occur after lesion to a peripheral nerve. MATERIALSAND METHODS Animals. Twenty-five adult rabbits, weighing 1.5-2.0 kg, were used. Protein tracer and histological technique. 1 ~ (w/v) Evans blue (Merck AG, Brain Research, 45 (1972) 175-181
176
I<. KRISFENSSON AND J. SJ/JSTRANI)
Darmstadt, Germany) in 5 o/(w/v) albumin (Fraction V, Nutritional Biochemical Co., Cleveland, Ohio, U.S.A.), abbreviated EBA. This complex shows a brilliant red secondary fluorescence under ultraviolet irradiation, and can be located at the cellular level. Evans blue forms a firm complex with serum albumin, but a possibility of some dissociation exists 4. To examine whether a protein is taken up by the neurones we have in previous studies found that another protein tracer, horseradish peroxidase (which has a similar molecular size to that of albumin), is incorporated into the motor neuronal cytoplasm in the same manner after intramuscular injectionla, ~7. The brain stems of the injected rabbits were fixed in 10 }o aqueous formaldehyde solution overnight; frozen sections, 10/zm thick, were cut and mounted in 50~o glycerol in water. They were viewed under a Reichert fluorescence microscope equipped with dark field condenser and an Osram HBO high pressure mercury lamp e6. Experimental procedure. All operations were performed on animals anaesthetised by intravenous injections of pentobarbital (Mebumal), 30 mg/kg body weight. The right hypoglossal nerve was exposed and crushed by pressing the nerve firmly between a silk thread and a needle at a level where the nerve traverses the digastric muscle. At various times thereafter, 0.6 ml EBA was injected into the tongue, half the volume on each side. The animals were usually killed and taken for examination 24 h after the injection of EBA. In this way rabbits were examined 5-26 (6), 35 (8), 48 (2) and 80-120 (4) days after the crush lesion; the numbers in parentheses represent the number of animals examined. On the 35th day after the crush lesion, two of the rabbits were examined 6-10 h after injection of EBA, and two after 14 h. To test whether the uptake of the protein tracer into the hypoglossal nerve cell bodies could be blocked by intracisternal injection of colchicine (Colchicin krist, reins, E. Merck AG), 100/zg of this substance dissolved in distilled water were injected intracisternally into two rabbits as described previously 2a. After 14 h, when the animals showed signs of paresis, EBA was injected into the tongues and the animals were taken for examination 24 h later. Three rabbits that had been injected with 0.6 ml EBA, half the volume on each side of the tongue, served as controls. These were taken for examination 24 h after the injection. In order to permit orientation of one side of the brain stem in the experimental animals, an incision with a razor blade was made on one of the sides by one of the investigators. All sections were read blind by the other investigator under coded numbers. To evaluate Wallerian degeneration and remyelination, the hypoglossal nerve I cm distal to the crush injury was examined. These nerves were fixed in 1 ~o OsO4, embedded in paraffin and cut into 5 t~m thick sections. RESULTS
In the 3 control rabbits most of the nerve cell bodies in the hypoglossal nuclei of the brain stem accumulated numerous granules of a red fluorescent material in their cytoplasm, indicating the presence of EBA. No difference in the amount of accumulated material could be observed between the left and right sides of the hypoglossal nuclei in these 3 animals. No red fluorescence was found in the blood vessels of the
Brain Research, 45 (1972) 175-181
177
PROTEIN TRANSPORT IN REGENERATING NERVES
brain stem, in the surrounding neuropil or in other neurones in the neighbourhood. This selective accumulation of tracer in the perikarya of the hypoglossal neurones is interpreted to result from an uptake of the tracer in axons at the periphery, followed by an intraaxonal transfer in a retrograde direction at a relatively rapid rate. The possibility of a spread of the tracer to the nerve cell bodies via other compartments than the axons, i.e., blood or endoneurial spaces, seems to be ruled out because it could be prevented by acute crush injuries to the hypoglossal nerve. Furthermore, there was no change in the vascular permeability to the protein in the brain stem, and no tracer was seen in the surrounding neuropil (for extensive discussion, see Kristensson and OlssonlS). In the two rabbits that were treated with the mitosis inhibitor colchicine, known to block the fast component ofaxonal transport of labelled proteins
I
Fig. 1. Accumulation of fluorescent protein tracer in hypoglossal nerve cell bodies on the left side (L). On the right side (R) the transfer is inhibited. Fourteen days after crushing of the right hypoglossal nerve, x 250.
Fig. 2. Increase in fluorescence intensity of the hypoglossal neurones on the right side (R) compared with those of the left side (L). Thirty-five days after crushing of the right hypoglossal nerve. C, central canal, x 60.
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Fig. 3. Increase in fluorescence intensity and size of the hypoglossal neurones on the right side (R), ipsilateral to lesion of the hypoglossal nerve 35 days previously, x 250.
Fig. 4. Increase in fluorescence intensity of the ipsilateral hypoglossal neurones 48 days after crushing of the right hypoglossal nerve. L, left side; R, right side. × 400. in the anterograde direction in the rabbit hypoglossal nerve2q the accumulation o f red fluorescent tracer in the perikarya o f the hypoglossal neurones was blocked. The transfer of tracer to the hypoglossal nerve cell bodies was interrupted by acute crush injuries to the hypoglossal nerve, as stated above. In the 6 rabbits examined 5-26 days after the lesion o f the right hypoglossal nerve, the accumulation o f red fluorescent granules was limited to the perikarya o f the neurones on the contralateral, left side, while no tracer was seen in the ipsilateral nerve cell bodies (Fig. 1). O n the other hand, in rabbits examined 35 days after the lesion, the neurones on the ipsilateral side had accumulated a large a m o u n t o f red fluorescent material in their cytoplasm, and showed a markedly more intense red fluorescence than the contralateral neurones (Figs. 2 and 3). The ipsilateral neurone perikarya were also increased in Brain Research, 45 (1972) 175-181
PROTEIN TRANSPORT IN REGENERATING NERVES
179
size. This marked difference in tracer accumulation between the two sides was seen in all 6 rabbits examined 14 and 24 h after the injection of EBA, whereas after 10 h only a few red fluorescent granules could be seen in some hypoglossal neurones on both sides, with no certain difference between the sides. In the rabbits examined 48 days after the crush the ipsilateral neurones also had a higher fluorescence intensity than the contralateral ones (Fig. 4), while in the 4 rabbits examined after 80 and 120 days no difference between the sides could be observed. Fourteen days after the crush injury the hypoglossal nerve distal to the crush showed completely disrupted myelin sheaths, which to a large extent were taken up by macrophages. After 26 days some remaining degenerated myelin sheaths were seen. By 35-48 days after the lesion numerous small axons with clearly visible thin myelin sheaths had appeared 1 cm distal to the nerve lesion. After 80 and 120 days the nerve had an almost normal appearance, with myelinated axons of large calibre. DISCUSSION
During nerve regeneration, changes in axonal transport of proteins in the anterograde direction have previously been reported. Grafstein and Murray 9 have demonstrated an increase in the amount and velocity of radioactively labelled proteins transported after crush injury to the goldfish optic nerve; and Kreutzberg and Schubert 14 observed an increase in the amount of protein transported from the nerve cell bodies to the periphery in regenerating facial nerves of the rat. In the present study, at 35 and 48 days after a crush injury to the right hypoglossal nerve, a marked increase in the fluorescence intensity in the ipsilateral hypoglossal neurones was found after injection of a fluorescent protein tracer into the tongue muscle. This increased intensity of tracer fluorescence probably reflects an even greater increase in the amount of tracer accumulated in the neurones, since Brattg~rd et al. 2 have shown that the volume of the regenerating hypoglossal nerve cell bodies in adult rabbits is increased more than two-fold during the period 4-50 days after an axonal lesion. It is conceivable that this increased accumulation is the result of an enhanced uptake and retrograde axonal transport of the tracer from the periphery. However, since it is not known whether any changes in the breakdown of the tracer occur in the regenerating neurones, the possibility that the increased accumulation is due to a decreased degradation cannot be excluded. In a recent study it was found that the perineurium distal to a nerve crush continues to act as an efficient diffusion barrier, preventing proteins from the surrounding tissues from reaching the endoneurium 2a. This makes it likely that the outgrowing axons first come in contact with the proteins injected into the muscles when they have reached the muscle fibres; at the neuromuscular junction the perineurium is lacking, so that exogenous proteins can diffuse into the synaptic region3,3L The increase in neuronal accumulation of protein was observed on the 35th and 48th days after the crush injury, at which time the regenerating axons should have reached and become reconnected with the muscle fibres of the tongue10, al. The increased accumulation of exogenous proteins coincides with the period of maturation of the Brain Research, 45 (1972) 175-181
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newly established axons, characterized by an increase in diameter and return of function of the reconnected axons. During this period the process of remyelination occurs, axons of large diameter appear in the hypoglossal nerve as in other peripheral nerves1, 29, and the nerve cell bodies increase their synthesis of proteins, with a return to normal conditions between 2 and 3 months after the lesion2, 7. Observations both on developmentS, lz,24 and regeneration1, 29 of the nervous system have shown that the diameter of the axon and the size of the nerve cell body are to a large extent dependent upon the axon establishing contact with its endorgan. Our observation of an increased neuronal accumulation of protein tracer during the regenerating process is therefore of interest, since it might reflect an increased uptake of macromolecules from the periphery, providing a mechanism by which information for the regulation of axonal maturation can be transferred from the end organ to the nerve cell body. SUMMARY Albumin labelled with Evans blue, which forms a red fluorescent complex under ultraviolet irradiation, was injected into the tongues of adult rabbits. Ten to 24 h after the injection there was an accumulation of the red fluorescent tracer in the nerve cell bodies of the hypoglossal neurones, indicating an axonal uptake and retrograde transport of the protein tracer. After crush injury to the right hypoglossal nerve the neuronal uptake of protein tracer was blocked on the ipsilateral side for 26 days. Thirty-five to 48 days after the crush lesion, an increase in the intensity of' the fluorescence was seen in hypoglossal neurones on the ipsilateral side relative to the other side. After 80-120 days the difference between the sides could no longer be detected. This finding indicates that during nerve regeneration there is a transient stage involving an increase in the neuronal accumulation ofmacromolecules taken up and transported by the axons from the periphery to the nerve cell bodies; the timing of this stage coincides with the period of maturation of axons reconnected to the muscle. ACKNOWLEDGEMENT This study was supported by Grants B72-12X-3488-01A and B72-13X-2226-06A from the Swedish Medical Research Council.
REFERENCES 1 AITKEN, J. T., SHARMAN,M., AND YOUNG, J. Z., Maturation of regenerating nerve fibres with various peripheral connexions, J. Anat. (Lond.), 81 (1947) 1-22. 2 BRATTG.~RD,S.-O., EDSTROM,J.-E., AND HYDI~N,H., The chemical changes in regenerating neurons, J. Neurochem., 1 (1957) 316-325. 3 BURKEL,W. E., The histological fine structure of perineurium, Anat. Rec., 158 (1967) 177-190. 4 CLASEN, R. A., PANDOLFI, S., AND HASS, G. M., Vital staining, serum albumin and the bloodbrain barrier, J. Neuropath. exp. Neurol., 29 (1970) 266-284. Brain Research, 45 (1972) 175-181
PROTEIN TRANSPORT IN REGENERATING NERVES
] 81
5 CRAGG,B. G., What is the signal for chromatolysis?, Brain Research, 23 (1970) 1-21. 13 DAHLSTROM,A., Observations on the accumulation of noradrenaline in the proximal and distal parts of peripheral adrenergic nerves after compression, J. Anat. (Lond.), 99 (1965) 677-689. 7 ENGH, CH. A., SCHOFmLD,B. H., DUTY, S. B., AND ROmNSON, R. A., Perikaryal synthetic function following reversible and irreversible peripheral axon injuries as shown by radioautography, J. cutup. Neurol., 142 (1971) 465-480. 8 EVANS,D. H. L., ANDVIZOSO,A. D., Observations on the mode of growth of motor nerve fibers in rabbits during post-natal development, J. comp. Nearol., 95 (1951) 429-462. 9 GRAFSTEIN,B., ANDMURRAY,M., Transport ofprotein in goldfish optic nerve during regeneration, Exp. Neurol., 25 (1969) 494-508. l0 GUTMANN,E., GUTTMAN,L., MEDAWAR,P. B., AND YOUNG, J. Z., The rate of regeneration of nerve, J. exp. Biol., 19 (1942) 14-44. 11 HAMBERGER,A., AND SJ6STRAND,J., Respiratory enzyme activities in neurons and gliat cells of the hypoglossal nucleus during nerve regeneration, Acta physiol, scand., 67 (1966) 76-88. 12 HUGHES,A., A quantitative study of the development of the nerves in the hind-limb of Elentherodactylus martini ceusis, J. Embryol. exp. Morph., 13 (1965) 9-34. 13 KERKUT, G. A., SHAPIRA, A., AND WALKER, R. J., The transport of 14C-labelled material from CNS muscle along a nerve trunk, Cutup. Biochem. Physiol., 23 (1967) 729-748. 14 KREOTZBERG,G. W., AND SCHUBERT,P., Changes in axonal flow during regeneration of mammalian motor nerves, Acta neuropath. (Bed.), Suppl. V (1971) 70-75. 15 KRISTENSSON,K., Transport of fluorescent protein tracer in peripheral nerves, Acta neuropath. (Bed.), 16 (1970) 293-300. 16 KRISTENSSON,K., AND OLSSON,Y., Retrograde axonal transport of protein, Brain Research, 29 (1971) 363-365. 17 KRISTENSSON, K., AND OLSSON, Y., Uptake and retrograde axonal transport of peroxidase in hypoglossal neurones, Acta neuropath. (Bed.), 19 (1971) 1-9. 18 KRISTENSSON,K., AND OLSSON, Y., Retrograde axonal transport of protein tracers in peripheral nerves, Progr. Neurobiol., in press. 19 KR1STENSSON,K., OLSSON, Y., AND SJOSTRAND,J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399-406. 20 LASEK,R. J., Protein transport in neurons, Int. Rev. Neurobiol., 13 (1970) 289-324. 21 LUBII~SKA,L., Axoplasmic streaming in regenerating and in normal nerve fibres. In M. SINGERAND J. P. SCHADt~(Eds.), Mechanisms of Neural Regeneration, Progress in Brain Research, Vol. 13, Elsevier, Amsterdam, 1964, pp. 1-71. 22 LUmr~SKA, L., AND NEMIERKO, S., Velocity and intensity of bidirectional migration of acetylcholinesterase in transected nerves, Brain Research, 27 (1971) 329-342. 23 OLSSON, Y., AND KRISTENSSON,K., The perineurium as a diffusion barrier to protein tracers following trauma to nerves, Acta neuropath. (Berl.), in press. 24 PRESTIGE,M. C., The control of cell number in the lumbar spinal ganglia during the development of Xenopus laevis tadpoles, J. Embryol. exp. Morph., 17 (1967) 453-471. 25 SJOSTRAND,J., FR1ZELL,M., AND HASSELGREN,P.-O., Effects of colchicine on axonal transport in peripheral nerves, J. Neurochem., 17 (1970) 1563-1570. 26 STEINWALL,O., AND KLATZO, I., Double tracer methods in studies on blood-brain barrier dysfunction and brain oedema, Acta neurol, scand., 41 (1965) 591-595. 27 WATSON, W. E., Centripetal passage of labelled molecules along mammalian motor axons, J. Physiol. (Lond.), 196 (1968) 122-123. 28 WATSON, W. E., Observations on the nucleolar and total cell body nucleic acid of injured nerve cells, J. Physiol. (Lond.), 196 (1968) 655-676. 29 WEISS, P., EDDS, JR., MAC. V., AND CAVANAGH,M., The effect of terminal connections on the caliber of nerve fibres, Anat. Rec., 92 (1945) 215-233. 30 WEISS,P. A., AND HISCOE,H., Experiments on the mechanism of nerve growth, J. exp. Zool., 107 (1948) 315-396. 31 YOUNG, J. Z., The functional repair nervous tissue, Physiol. Rev., 22 (1942) 318-374. 32 ZACKS, S. I., AND SAITO, A., Uptake of exogenous horseradish peroxidase by coated vesicles in mouse neuromuscular junctions, J. Histochem. Cytochem., 17 (1969) 161-170. 33 ZELENA,J., Bidirectional movements of mitochondria along axons of an isolated nerve segment, Z. Zellforsch., 92 (1968) 186-196.
Brain Research, 45 (1972) 175-181
175
RETROGRADE TRANSPORT OF PROTEIN TRACER IN THE RABBIT HYPOGLOSSAL NERVE DURING REGENERATION
KRISTER KRISTENSSON AND JOHAN SJOSTRAND Neuropathological Laboratory, Department of Pathology I and Institute of Neurobiology, University of Gi~teborg, Gi~teborg (Sweden)
(Accepted April 7th, 1972)
INTRODUCTION In previous studies it was found that horseradish peroxidase, and albumin labelled with Evans blue, after injection into the gastrocnemius muscle or tongue of rats, rabbits and suckling mice, were transferred to the corresponding motor neurones in the spinal cord and the brain stem15-17,19. This phenomenon was interpreted to have resulted from an uptake of proteins into axons at the periphery, followed by axonal transport in a retrograde direction to the nerve cell bodies. The results therefore support the hypothesis that there exists an axonal transport of macromolecules in the retrograde direction, as has been indicated by earlier studies6,13,20-22,27,a3. A lesion of a peripheral nerve, which causes interruption of the axons, leads to certain morphological changes in the axons on both sides of the injury. These changes include an accumulation of axoplasmic constituents in the regions adjacent to the lesion; this has been interpreted to be the result of interrupted axonal transport of materials in the anterogradeao and the retrograde directions~l, 3~. When the nerve fibres regenerate after the lesion the reacting nerve cell bodies increase their production of RNA and protein2,11,z8. Little, however, is known about the changes in axonal transport during the various phases of axonal outgrowth, and about how migration of protein and organelles within the axon affect the protein turnover in the nerve cell perikarya. A change in the retrograde axonal transport can possibly be of importance in eliciting a signal for chromatolysis in the early phase of nerve regeneration5, and during later phases it can allow for peripheral influence of the connected end organs on axonal maturationl,2L The present study was undertaken with the aim of examining whether any changes in the neuronal uptake and retrograde transport of an exogenous macromolecule from the periphery occur after lesion to a peripheral nerve. MATERIALSAND METHODS Animals. Twenty-five adult rabbits, weighing 1.5-2.0 kg, were used. Protein tracer and histological technique. 1 ~ (w/v) Evans blue (Merck AG, Brain Research, 45 (1972) 175-181
176
I<. KRISFENSSON AND J. SJ/JSTRANI)
Darmstadt, Germany) in 5 o/(w/v) albumin (Fraction V, Nutritional Biochemical Co., Cleveland, Ohio, U.S.A.), abbreviated EBA. This complex shows a brilliant red secondary fluorescence under ultraviolet irradiation, and can be located at the cellular level. Evans blue forms a firm complex with serum albumin, but a possibility of some dissociation exists 4. To examine whether a protein is taken up by the neurones we have in previous studies found that another protein tracer, horseradish peroxidase (which has a similar molecular size to that of albumin), is incorporated into the motor neuronal cytoplasm in the same manner after intramuscular injectionla, ~7. The brain stems of the injected rabbits were fixed in 10 }o aqueous formaldehyde solution overnight; frozen sections, 10/zm thick, were cut and mounted in 50~o glycerol in water. They were viewed under a Reichert fluorescence microscope equipped with dark field condenser and an Osram HBO high pressure mercury lamp e6. Experimental procedure. All operations were performed on animals anaesthetised by intravenous injections of pentobarbital (Mebumal), 30 mg/kg body weight. The right hypoglossal nerve was exposed and crushed by pressing the nerve firmly between a silk thread and a needle at a level where the nerve traverses the digastric muscle. At various times thereafter, 0.6 ml EBA was injected into the tongue, half the volume on each side. The animals were usually killed and taken for examination 24 h after the injection of EBA. In this way rabbits were examined 5-26 (6), 35 (8), 48 (2) and 80-120 (4) days after the crush lesion; the numbers in parentheses represent the number of animals examined. On the 35th day after the crush lesion, two of the rabbits were examined 6-10 h after injection of EBA, and two after 14 h. To test whether the uptake of the protein tracer into the hypoglossal nerve cell bodies could be blocked by intracisternal injection of colchicine (Colchicin krist, reins, E. Merck AG), 100/zg of this substance dissolved in distilled water were injected intracisternally into two rabbits as described previously 2a. After 14 h, when the animals showed signs of paresis, EBA was injected into the tongues and the animals were taken for examination 24 h later. Three rabbits that had been injected with 0.6 ml EBA, half the volume on each side of the tongue, served as controls. These were taken for examination 24 h after the injection. In order to permit orientation of one side of the brain stem in the experimental animals, an incision with a razor blade was made on one of the sides by one of the investigators. All sections were read blind by the other investigator under coded numbers. To evaluate Wallerian degeneration and remyelination, the hypoglossal nerve I cm distal to the crush injury was examined. These nerves were fixed in 1 ~o OsO4, embedded in paraffin and cut into 5 t~m thick sections. RESULTS
In the 3 control rabbits most of the nerve cell bodies in the hypoglossal nuclei of the brain stem accumulated numerous granules of a red fluorescent material in their cytoplasm, indicating the presence of EBA. No difference in the amount of accumulated material could be observed between the left and right sides of the hypoglossal nuclei in these 3 animals. No red fluorescence was found in the blood vessels of the
Brain Research, 45 (1972) 175-181
177
PROTEIN TRANSPORT IN REGENERATING NERVES
brain stem, in the surrounding neuropil or in other neurones in the neighbourhood. This selective accumulation of tracer in the perikarya of the hypoglossal neurones is interpreted to result from an uptake of the tracer in axons at the periphery, followed by an intraaxonal transfer in a retrograde direction at a relatively rapid rate. The possibility of a spread of the tracer to the nerve cell bodies via other compartments than the axons, i.e., blood or endoneurial spaces, seems to be ruled out because it could be prevented by acute crush injuries to the hypoglossal nerve. Furthermore, there was no change in the vascular permeability to the protein in the brain stem, and no tracer was seen in the surrounding neuropil (for extensive discussion, see Kristensson and OlssonlS). In the two rabbits that were treated with the mitosis inhibitor colchicine, known to block the fast component ofaxonal transport of labelled proteins
I
Fig. 1. Accumulation of fluorescent protein tracer in hypoglossal nerve cell bodies on the left side (L). On the right side (R) the transfer is inhibited. Fourteen days after crushing of the right hypoglossal nerve, x 250.
Fig. 2. Increase in fluorescence intensity of the hypoglossal neurones on the right side (R) compared with those of the left side (L). Thirty-five days after crushing of the right hypoglossal nerve. C, central canal, x 60.
Brain Research, 45 (1972) 175-181
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K. KR1STENSSONAND J. SJOSTRANI)
Fig. 3. Increase in fluorescence intensity and size of the hypoglossal neurones on the right side (R), ipsilateral to lesion of the hypoglossal nerve 35 days previously, x 250.
Fig. 4. Increase in fluorescence intensity of the ipsilateral hypoglossal neurones 48 days after crushing of the right hypoglossal nerve. L, left side; R, right side. × 400. in the anterograde direction in the rabbit hypoglossal nerve2q the accumulation o f red fluorescent tracer in the perikarya o f the hypoglossal neurones was blocked. The transfer of tracer to the hypoglossal nerve cell bodies was interrupted by acute crush injuries to the hypoglossal nerve, as stated above. In the 6 rabbits examined 5-26 days after the lesion o f the right hypoglossal nerve, the accumulation o f red fluorescent granules was limited to the perikarya o f the neurones on the contralateral, left side, while no tracer was seen in the ipsilateral nerve cell bodies (Fig. 1). O n the other hand, in rabbits examined 35 days after the lesion, the neurones on the ipsilateral side had accumulated a large a m o u n t o f red fluorescent material in their cytoplasm, and showed a markedly more intense red fluorescence than the contralateral neurones (Figs. 2 and 3). The ipsilateral neurone perikarya were also increased in Brain Research, 45 (1972) 175-181
PROTEIN TRANSPORT IN REGENERATING NERVES
179
size. This marked difference in tracer accumulation between the two sides was seen in all 6 rabbits examined 14 and 24 h after the injection of EBA, whereas after 10 h only a few red fluorescent granules could be seen in some hypoglossal neurones on both sides, with no certain difference between the sides. In the rabbits examined 48 days after the crush the ipsilateral neurones also had a higher fluorescence intensity than the contralateral ones (Fig. 4), while in the 4 rabbits examined after 80 and 120 days no difference between the sides could be observed. Fourteen days after the crush injury the hypoglossal nerve distal to the crush showed completely disrupted myelin sheaths, which to a large extent were taken up by macrophages. After 26 days some remaining degenerated myelin sheaths were seen. By 35-48 days after the lesion numerous small axons with clearly visible thin myelin sheaths had appeared 1 cm distal to the nerve lesion. After 80 and 120 days the nerve had an almost normal appearance, with myelinated axons of large calibre. DISCUSSION
During nerve regeneration, changes in axonal transport of proteins in the anterograde direction have previously been reported. Grafstein and Murray 9 have demonstrated an increase in the amount and velocity of radioactively labelled proteins transported after crush injury to the goldfish optic nerve; and Kreutzberg and Schubert 14 observed an increase in the amount of protein transported from the nerve cell bodies to the periphery in regenerating facial nerves of the rat. In the present study, at 35 and 48 days after a crush injury to the right hypoglossal nerve, a marked increase in the fluorescence intensity in the ipsilateral hypoglossal neurones was found after injection of a fluorescent protein tracer into the tongue muscle. This increased intensity of tracer fluorescence probably reflects an even greater increase in the amount of tracer accumulated in the neurones, since Brattg~rd et al. 2 have shown that the volume of the regenerating hypoglossal nerve cell bodies in adult rabbits is increased more than two-fold during the period 4-50 days after an axonal lesion. It is conceivable that this increased accumulation is the result of an enhanced uptake and retrograde axonal transport of the tracer from the periphery. However, since it is not known whether any changes in the breakdown of the tracer occur in the regenerating neurones, the possibility that the increased accumulation is due to a decreased degradation cannot be excluded. In a recent study it was found that the perineurium distal to a nerve crush continues to act as an efficient diffusion barrier, preventing proteins from the surrounding tissues from reaching the endoneurium 2a. This makes it likely that the outgrowing axons first come in contact with the proteins injected into the muscles when they have reached the muscle fibres; at the neuromuscular junction the perineurium is lacking, so that exogenous proteins can diffuse into the synaptic region3,3L The increase in neuronal accumulation of protein was observed on the 35th and 48th days after the crush injury, at which time the regenerating axons should have reached and become reconnected with the muscle fibres of the tongue10, al. The increased accumulation of exogenous proteins coincides with the period of maturation of the Brain Research, 45 (1972) 175-181
180
K. KRISTENSSON AND J. SJOSTRAND
newly established axons, characterized by an increase in diameter and return of function of the reconnected axons. During this period the process of remyelination occurs, axons of large diameter appear in the hypoglossal nerve as in other peripheral nerves1, 29, and the nerve cell bodies increase their synthesis of proteins, with a return to normal conditions between 2 and 3 months after the lesion2, 7. Observations both on developmentS, lz,24 and regeneration1, 29 of the nervous system have shown that the diameter of the axon and the size of the nerve cell body are to a large extent dependent upon the axon establishing contact with its endorgan. Our observation of an increased neuronal accumulation of protein tracer during the regenerating process is therefore of interest, since it might reflect an increased uptake of macromolecules from the periphery, providing a mechanism by which information for the regulation of axonal maturation can be transferred from the end organ to the nerve cell body. SUMMARY Albumin labelled with Evans blue, which forms a red fluorescent complex under ultraviolet irradiation, was injected into the tongues of adult rabbits. Ten to 24 h after the injection there was an accumulation of the red fluorescent tracer in the nerve cell bodies of the hypoglossal neurones, indicating an axonal uptake and retrograde transport of the protein tracer. After crush injury to the right hypoglossal nerve the neuronal uptake of protein tracer was blocked on the ipsilateral side for 26 days. Thirty-five to 48 days after the crush lesion, an increase in the intensity of' the fluorescence was seen in hypoglossal neurones on the ipsilateral side relative to the other side. After 80-120 days the difference between the sides could no longer be detected. This finding indicates that during nerve regeneration there is a transient stage involving an increase in the neuronal accumulation ofmacromolecules taken up and transported by the axons from the periphery to the nerve cell bodies; the timing of this stage coincides with the period of maturation of axons reconnected to the muscle. ACKNOWLEDGEMENT This study was supported by Grants B72-12X-3488-01A and B72-13X-2226-06A from the Swedish Medical Research Council.
REFERENCES 1 AITKEN, J. T., SHARMAN,M., AND YOUNG, J. Z., Maturation of regenerating nerve fibres with various peripheral connexions, J. Anat. (Lond.), 81 (1947) 1-22. 2 BRATTG.~RD,S.-O., EDSTROM,J.-E., AND HYDI~N,H., The chemical changes in regenerating neurons, J. Neurochem., 1 (1957) 316-325. 3 BURKEL,W. E., The histological fine structure of perineurium, Anat. Rec., 158 (1967) 177-190. 4 CLASEN, R. A., PANDOLFI, S., AND HASS, G. M., Vital staining, serum albumin and the bloodbrain barrier, J. Neuropath. exp. Neurol., 29 (1970) 266-284. Brain Research, 45 (1972) 175-181
PROTEIN TRANSPORT IN REGENERATING NERVES
] 81
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