EXPERIMENTAL
NEUROLOGY
52,
242-249 (1976)
Rapid Axonal Transport of Labeled Proteins in Regenerating Sensory and Motor Fibers of Rabbit Vagus Nerve W. G. MCLEAN,
M. FRIZELL,
Institute of Neurobiology University of Giiteborg, Received
AND J. SJ~STRAND
l
and Department of Ophthalmology, Fack, S-400 33 Giiteborg, Swede% April
5,19?6
The axonal transport [aH]leucine-labeled proteins in sensory fibers of rabbit vagus nerve was studied at various times after the nerves had been crushed in viva. Up to 8 weeks after the crush, proteins transported from the nodose ganglion accumulated at the site of the crush under in vitro incubation conditions. This was in contrast to transport in sensory vagal fibers in vivo, which continued distal to the crush zone into the newlyregenerated fibers. Motor fibers in the vagus nerve behaved similarly; in vitro incubation conditions which support transport in mature nerves were unable to support it in newly-grown fibers. Four or five months after the crush some transport occurred in the regenerated axons in vitro, but accumulation still occurred at the crush zone.
Rapid axonal transport at a rate of about 400 mm per day has been observed in several peripheral nerves (10, 13). Changes in this transport have been reported during development and maturation both of the optic nerve (5, 7) and the sciatic nerve (6). In the hypoglossal and vagus nerves marked changes have deen described during the initial period of nerve regeneration (2,4, 13). The aim of the present study was to investigate the transport within regenerating axons of protein constituents synthesized in the neuronal perikarya in order to characterize the delivery of protein to the growing tips. An in vitro method (8) was chosen to enable us to study the influence 1 Supported by the Swedish Medical Research Council (Grant B76-12X-0226-10) and grants from the Medical Faculty, University of Gijteborg; 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. 242 Copyright All rights
1976 by Academic Press, Inc. o? reproduction in any form reserved.
AXONAL
TRANSPORT
IN
REGENERATING
VAGUS
243
of possible agents which control axonal transport during nerve regeneration. This work indicates that both newly-grown sensory and motor axons are unable to support axonal transport in vitro. MATERIALS
AND
METHODS
Male albino rabbits (weight at time of death 1.8 to 2.3 kg) were used throughout. Vagus nerves were exposed under pentobarbital anesthesia and one nerve crushed with 4/O silk thread against a glass rod at a point between 2 and 4 cm from the nodose ganglion. A loose thread was left in position around the nerve at the crush point to mark the site. The contralateral nerve was sham-operated and a loose thread left in position. At intervals up to 5 months after the crush, either (i) motor fibers were labeled by intraventricular injection of [3H] leucine as described previously (3), or (ii) sensory fibers were labeled by subepineural injection of [3H]leucine into the nodose ganglion in viva. In this method 20 &I of [ 3H] leucine (~-45 [ 3H] leucine, Radiochemical Centre, Amersham, England, 58 Ci/mmole) were injected in a volume of 20 ~1 0.9% NaCl through a 30-gauge stainless-steel needle. Where appropriate in (i) and (ii) a ligature was tied on the crushed nerve at a point about 6 cm from the nodose ganglion at the time of labeling and the animal was killed and the vagus nerves removed 24 hr later. Alternatively, vagus nerves and nodose ganglia were removed from untreated rabbits or from rabbits to which [ 3H]leucine had been applied as in (i) above, 3.5 hr previously and the nerve/ganglia preparations were incubated in vitro for 24 hr at 38.5C according to the method of McLean et al. (8). A two-compartment incubation chamber permitted the labeling of sensory nerve cell bodies in the vagi of untreated rabbits by incubation of the nodose ganglia with [3H] leucine in vitro. Accumulation of labeled proteins transported within the axons occurred either at a ligature 6 cm from the nodose ganglion or at the site of the original crush. The extent of accumulation was estimated and expressed as described previously (8). Normally, values were expressed as an average of several nerves treated in the same way. However, this method could be applied only where two or more nerves had the crush zone and the ligature at an equivalent distance from the nodose ganglia. Where this was not the case, the profile of only one nerve is shown and the number of experiments giving qualitatively similar profiles is noted. RESULTS Axonal Transport from Nodose Ganglion in Control Vagus Nerves. Vagus nerves and nodose ganglia incubated in vitro for 24 hr in the two-
244
MCLEAN,
FRIZELL
AND
S JiiSTRAND
compartment system showed an accumulation of labeled proteins immediately proximal to a ligature tied on the nerves 6 cm from the nodose ganglion (Fig. 1). An average of 38% of the [3H]leucine-labeled proteins present in the nerve 20 to 60 mm from the ganglion was present in the S-mm segment immediately proximal to the ligature after 24 hr. Efect of Nerve Crush on Transport in Sensory Fibers. Transport of 3H-labeled proteins in the motor fibers of vagus nerve beyond a crush zone occurred in vivo after a delay of 1 wk and thereafter the accumulation zone of labeled proteins moved distally at a rate of 3 mm per day (4). At 4 wk after a nerve crush an extension of regenerated fibers about 6 cm below the crush zone could be estimated. To investigate the characteristics of the axonal transport in newly grown fibers, vagus nerves were removed and incubated in vitro with a ligature about 4 cm distal to the crush. As seen in Fig. 2 there was no transport in vitro beyond the crush zone, i.e., all proteins were transported down as far as the crush zone and stopped there. To see if it was perhaps a very slow maturation process in the regenerating sensory fibers which might support axonal transport at a more mature stage, we looked at nerves crushed 8 weeks previously and there was still no transport beyond the crush (Fig. 3). After 4 to 5 months of regeneration, however, there was evidence for a partial return of rapid axonal transport in vitro in the regenerating sensory fibers of the vagus nerves. In these experiments labeled proteins were transported beyond the crush zone to accumulate at a ligature although an accumulation also occurred at the crush zone (Fig. 4).
40* c 30L $ 2-L
IO-
I 0
A
6 t 60
t 40
20 mm
from
ganglion
FIG. 1. Profile of [8H]leucine-labeled proteins in ‘rabbit vagus nerve-controls. Vagus nerves and nodose ganglia were remove d and incubated in vitro’ 24 hr with a ligature at B. Labeled proteins in S-mm segments of nerve were expressed as a percentage of the total labeled proteins between points A (20 mm from the nodose ganglion) and B (the ligature). Average of five experiments f SEM.
ASONAL
TRANSPORT
IN
REGENERATING
FIG. 2. Profile of labeled proteins in vagus nerve 4 wk nerves were crushed at a point indicated by the arrowhead to irr vitro conditions with ligature at B. Labeled proteins expressed as a percentage of the total labeled proteins from the nodose ganglion) and B (the ligature). Average
VACUS
24.5
after crush in r&o. Vagus and 4 weeks later removed in each S-mm segment were between points A (10 mm of four nerves f SEM.
By injection of [3H]leucine into the region of the nodose ganglion in viva, we could show that vagus nerves in viva were able to transport labeled proteins beyond the crush zone to accumulate at a distal ligature 4 weeks after a nerve crush (Fig. 5). Only a small accumulation of radioactivity was found at the crush zone. Effect of Nerve Crush on Transport in Motor Fibers. In agreement with previous work (3), rapidly migrating [3H] leucine labeled proteins were
FIG. 3. Profile of labeled proteins in a vagus nerve 8 weeks after crush in z&o. Nerves were crushed in viva and 8 weeks later removed to ilt vitro conditions. Data were expressed as in Figure 1. Seven such experiments gave qualitatively similar results.
246
MCLEAN,
FRIZELL
AND
SJGSTRAND
FIG. 4. Profile of labeled proteins in vagus nerve 4 months after crush ilz viva. Vagus nerves were crushed at a point indicated by the arrowhead and 4 months later removed to in vitro conditions with a ligature at B. Labeled proteins in each 5- or lo-mm segment were expressed as a percentage of the total labeled proteins between points A (20 mm from the nodose ganglion) and B (the ligature). One typical experiment out of three is shown and the profile of labeled proteins in the contralateral sham-operated vagus nerve is indicated with broken lines.
transported distal ,to the crush zone in vagal motor fibers after labeling in V&O, 4 weeks after a nerve crush. An accumulation could be observed in viva at a distal ligature applied at the time of labeling of the nerve cell bodies of the dorsal motor nucleus of the vagus (results not shown).
FIG. 5. Profile of labeled protein in a vagus nerve 4 weeks after crush ilt z&o. Nerves were crushed and 4 weeks later the proteins were labeled in z&o by injection of [‘Hlleucine into the nodose ganglia. A ligature was applied 55 mm from the ganglion and accumulation continued ilz Z&J for 24 hr. Six such experiments gave similar results.
AXONAL
TRANSPORT
0
IN
20
REGENERATING
40 mm
‘roll7
VAGUS
60
247
80
ganglion
FIG. 6. Profile of labeled proteins in motor fibers of rabbit vagus nerve 4 wk after a crush in V&O. Nerves were crushed as indicated and 4 wk later the motor fibers labelled by injection of (“H) leucine into the dorsal motor nucleus in viva. Nerves were removed 3.5 hr later and incubated k vitro with a ligature 70 mm (control) or 65 mm (crush) from the nodose ganglion for 24 hr. Four such experiments gave similar results.
However, when the vagus nerves were removed and incubated in vitro 3.5 hr after labeling of the cell bodies of the dorsal motor nucleus of the vagus, 4 weeks after a nerve crush, no transport of labeled proteins occurred beyond the crush zone (Fig. 6). Ejject of EGTA and Other Additives on the Rapid AxomJ Transport in Regenerating Fibers in vitro. The presence of 5% rabbit serum in the medium 199 did not permit the progression of the label beyond the crush zone, nor did a low-Ca’+ medium containing 5 mM EGTA (results not shown). DISCUSSION The marked changes in both the rapid and slow phasesof axonal transport during nerve regeneration (24) indicate that profound alterations occur in the transport system of the axon during outgrowth. Using an in vitro system to study possible agents which control or accelerate axonal transport in regenerating fibers, we observed that the rapid axonal transport of both the regenerating sensory and motor fibers of the vagus nerve was blocked at the crush zone with no transport observable beyond the crush zone. There are several possible explanations for the failure of regenerating vagus nerves to permit axonal transport beyond the crush zone in vitro. The axolemma or intra-axonal structure of the newly grown fibers may be more vulnerable to damage in vitro; freeze-fracturing studies indicate that the plasmolemma of an outgrowing nerve is immature with a
248
MCLEAN,
FRIZELL
AND
S JiiSTRAND
low number of intramembraneous particles (12). The perineurial barrier of the regenerated nerve may lack the normal capacity to control the environment of the regenerated axons ; Olsson and Kristensson (11) have shown a breakdown of the perineurial barrier to Evans blue albumin at the site of the crush for several months after nerve injury. However, preliminary studies on the crushed vagus nerve have failed to show any marked alterations in the perineurial barrier (Rydevik et al., in preparation) . Some blood-borne factor might be lacking when the nerve is removed for in vitro incubation. In adult postganglionic sympathetic neurons, treatment with nerve growth factor has demonstrated an increase in the rate of fast axonal transport (1) and for other neurons some unknown growthpromoting substance may exist which is especially important during the regenerative process. Another reason for the blockage of rapid transport beyond the crush zone could be the lack of slow axonal transport in newly grown fibers-studies on slow axonal transport have shown that in vitro conditions are unable to support the slow flow (9). One also must consider the possibility that the scar tissue around the crush zone might have obstructed oxygen diffusion from the medium to the axons, causing a hypoxic block at that site. Further experiments, in which the effects of factors rendering the nerve more resistant to hypoxia are tested with respect to axonal transport in regenerating fibers in vitro, would be valuable to clarify this point. We are still in the process of examing the effects of different agents on axonal transport in regenerating fibers to see what factors can be added to the incubation medium to permit the rapid transport to continue in the newly grown fibers. The experiments reported here indicate that addition of 5% rabbit serum to, or removal of Ca2+ from the medium, were insufficient to induce transport. Preliminary experiments on the crushed hypoglossal nerve, which shows a vigorous and rapid axon regeneration (3), indicate that the transport block at the site of the crush in a regenerating nerve incubated in vitro may be a general phenomenon. REFERENCES 1. ALMON, (NGF)
Brain 2. FRIZELL, 3.
R.
R., and W. 0. upon axoplasmic
MCCLURE. transport
1970. The effect in sympathetic
of nerve neurons
growth of the
factor mouse.
Res. 74: 255-267.
M., W. G. MCLEAN, and transport of rapidly migrating labelled ing peripheral nerves. Bra&z Res., in FRIZELL, M., and J. SJ~STRAND. 1974. cholinergic enzymes in regenerating
845-850.
J.
SJ~STRAND. 1976. Retrograde axonal proteins and glycoproteins in regeneratpress. Transport of proteins, glycoproteins and hypoglossa! neurons. J. Neurochem. 22 :
AXONAL
TRANSPORT
IN
REGENERATING
VAGUS
249
4. FRIZELL, M., and J. SJ~STRAND. 1974. The axonal transport of slowly migrating ‘H-leucine labelled proteins and the regeneration rate in regenerating hypoglossal and vagus nerves of the rabbit. Brain Res. 81: 267-283. 5. HENDRICKSON, A. E., and W. M. COWAN. 1971. Changes in the rate of axoplasmic transport during postnatal development of the rabbit’s optic nerve and tract. Exp.
Neural.
30:
403-422.
6. LASEK, R. J. 1970. Protein transport in neurons. Int. Rev. Neurobiol. 13: 289-324. 7. MARCHISIO, P.-C. J. SJ~STRAND, M. AGLIETTA, and J.-O. KARLSSON. 1973. The development of axonal transport of proteins and glycoproteins in the optic pathway of chick embryo. Brain Res. 63: 273-284. 8. MCLEAN, W. G., M. FRIZELL, and J. SJ~STRAND. 1975. Axonal transport of labelled proteins in sensory fibers of rabbit vagus nerve in vitro. J. Neurochevn.
9.
25:
695698.
W. G., M. FRIZELL, and J. SJ~WRAND. 1976. Slow axonal transport of labelled proteins in sensory fibres of rabbit vagus nerve. J. Neurochem. 26: 1213-1216. 10. OCHS, S. 1972. Rate of axoplasmic transport in mammalian nerve fibres. MCLEAN,
J. Physiol.
11.
12. 13.
(London)
227:
627-645.
Y., and K. KRISTENSSON, 1973. The perineurium as a diffusion barrier to protein tracers following trauma to nerves. Acta Neuropathol. (Berl.) 23: 105-111. PFENNINGER, K. H., and R. P. BUNGE. 1974. Freeze-fracturing of nerve growth cones and young fibres. J. Cell Biol. 63: 180-196. SJ~STRAND, J. 1970. Fast and slow components of axoplasmic transport in the hypoglossal and vagus nerves of the rabbit. Brain Res. 18: 461-467. OLSSON,
NEUROLOGY
52,
242-249 (1976)
Rapid Axonal Transport of Labeled Proteins in Regenerating Sensory and Motor Fibers of Rabbit Vagus Nerve W. G. MCLEAN,
M. FRIZELL,
Institute of Neurobiology University of Giiteborg, Received
AND J. SJ~STRAND
l
and Department of Ophthalmology, Fack, S-400 33 Giiteborg, Swede% April
5,19?6
The axonal transport [aH]leucine-labeled proteins in sensory fibers of rabbit vagus nerve was studied at various times after the nerves had been crushed in viva. Up to 8 weeks after the crush, proteins transported from the nodose ganglion accumulated at the site of the crush under in vitro incubation conditions. This was in contrast to transport in sensory vagal fibers in vivo, which continued distal to the crush zone into the newlyregenerated fibers. Motor fibers in the vagus nerve behaved similarly; in vitro incubation conditions which support transport in mature nerves were unable to support it in newly-grown fibers. Four or five months after the crush some transport occurred in the regenerated axons in vitro, but accumulation still occurred at the crush zone.
Rapid axonal transport at a rate of about 400 mm per day has been observed in several peripheral nerves (10, 13). Changes in this transport have been reported during development and maturation both of the optic nerve (5, 7) and the sciatic nerve (6). In the hypoglossal and vagus nerves marked changes have deen described during the initial period of nerve regeneration (2,4, 13). The aim of the present study was to investigate the transport within regenerating axons of protein constituents synthesized in the neuronal perikarya in order to characterize the delivery of protein to the growing tips. An in vitro method (8) was chosen to enable us to study the influence 1 Supported by the Swedish Medical Research Council (Grant B76-12X-0226-10) and grants from the Medical Faculty, University of Gijteborg; 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. 242 Copyright All rights
1976 by Academic Press, Inc. o? reproduction in any form reserved.
AXONAL
TRANSPORT
IN
REGENERATING
VAGUS
243
of possible agents which control axonal transport during nerve regeneration. This work indicates that both newly-grown sensory and motor axons are unable to support axonal transport in vitro. MATERIALS
AND
METHODS
Male albino rabbits (weight at time of death 1.8 to 2.3 kg) were used throughout. Vagus nerves were exposed under pentobarbital anesthesia and one nerve crushed with 4/O silk thread against a glass rod at a point between 2 and 4 cm from the nodose ganglion. A loose thread was left in position around the nerve at the crush point to mark the site. The contralateral nerve was sham-operated and a loose thread left in position. At intervals up to 5 months after the crush, either (i) motor fibers were labeled by intraventricular injection of [3H] leucine as described previously (3), or (ii) sensory fibers were labeled by subepineural injection of [3H]leucine into the nodose ganglion in viva. In this method 20 &I of [ 3H] leucine (~-45 [ 3H] leucine, Radiochemical Centre, Amersham, England, 58 Ci/mmole) were injected in a volume of 20 ~1 0.9% NaCl through a 30-gauge stainless-steel needle. Where appropriate in (i) and (ii) a ligature was tied on the crushed nerve at a point about 6 cm from the nodose ganglion at the time of labeling and the animal was killed and the vagus nerves removed 24 hr later. Alternatively, vagus nerves and nodose ganglia were removed from untreated rabbits or from rabbits to which [ 3H]leucine had been applied as in (i) above, 3.5 hr previously and the nerve/ganglia preparations were incubated in vitro for 24 hr at 38.5C according to the method of McLean et al. (8). A two-compartment incubation chamber permitted the labeling of sensory nerve cell bodies in the vagi of untreated rabbits by incubation of the nodose ganglia with [3H] leucine in vitro. Accumulation of labeled proteins transported within the axons occurred either at a ligature 6 cm from the nodose ganglion or at the site of the original crush. The extent of accumulation was estimated and expressed as described previously (8). Normally, values were expressed as an average of several nerves treated in the same way. However, this method could be applied only where two or more nerves had the crush zone and the ligature at an equivalent distance from the nodose ganglia. Where this was not the case, the profile of only one nerve is shown and the number of experiments giving qualitatively similar profiles is noted. RESULTS Axonal Transport from Nodose Ganglion in Control Vagus Nerves. Vagus nerves and nodose ganglia incubated in vitro for 24 hr in the two-
244
MCLEAN,
FRIZELL
AND
S JiiSTRAND
compartment system showed an accumulation of labeled proteins immediately proximal to a ligature tied on the nerves 6 cm from the nodose ganglion (Fig. 1). An average of 38% of the [3H]leucine-labeled proteins present in the nerve 20 to 60 mm from the ganglion was present in the S-mm segment immediately proximal to the ligature after 24 hr. Efect of Nerve Crush on Transport in Sensory Fibers. Transport of 3H-labeled proteins in the motor fibers of vagus nerve beyond a crush zone occurred in vivo after a delay of 1 wk and thereafter the accumulation zone of labeled proteins moved distally at a rate of 3 mm per day (4). At 4 wk after a nerve crush an extension of regenerated fibers about 6 cm below the crush zone could be estimated. To investigate the characteristics of the axonal transport in newly grown fibers, vagus nerves were removed and incubated in vitro with a ligature about 4 cm distal to the crush. As seen in Fig. 2 there was no transport in vitro beyond the crush zone, i.e., all proteins were transported down as far as the crush zone and stopped there. To see if it was perhaps a very slow maturation process in the regenerating sensory fibers which might support axonal transport at a more mature stage, we looked at nerves crushed 8 weeks previously and there was still no transport beyond the crush (Fig. 3). After 4 to 5 months of regeneration, however, there was evidence for a partial return of rapid axonal transport in vitro in the regenerating sensory fibers of the vagus nerves. In these experiments labeled proteins were transported beyond the crush zone to accumulate at a ligature although an accumulation also occurred at the crush zone (Fig. 4).
40* c 30L $ 2-L
IO-
I 0
A
6 t 60
t 40
20 mm
from
ganglion
FIG. 1. Profile of [8H]leucine-labeled proteins in ‘rabbit vagus nerve-controls. Vagus nerves and nodose ganglia were remove d and incubated in vitro’ 24 hr with a ligature at B. Labeled proteins in S-mm segments of nerve were expressed as a percentage of the total labeled proteins between points A (20 mm from the nodose ganglion) and B (the ligature). Average of five experiments f SEM.
ASONAL
TRANSPORT
IN
REGENERATING
FIG. 2. Profile of labeled proteins in vagus nerve 4 wk nerves were crushed at a point indicated by the arrowhead to irr vitro conditions with ligature at B. Labeled proteins expressed as a percentage of the total labeled proteins from the nodose ganglion) and B (the ligature). Average
VACUS
24.5
after crush in r&o. Vagus and 4 weeks later removed in each S-mm segment were between points A (10 mm of four nerves f SEM.
By injection of [3H]leucine into the region of the nodose ganglion in viva, we could show that vagus nerves in viva were able to transport labeled proteins beyond the crush zone to accumulate at a distal ligature 4 weeks after a nerve crush (Fig. 5). Only a small accumulation of radioactivity was found at the crush zone. Effect of Nerve Crush on Transport in Motor Fibers. In agreement with previous work (3), rapidly migrating [3H] leucine labeled proteins were
FIG. 3. Profile of labeled proteins in a vagus nerve 8 weeks after crush in z&o. Nerves were crushed in viva and 8 weeks later removed to ilt vitro conditions. Data were expressed as in Figure 1. Seven such experiments gave qualitatively similar results.
246
MCLEAN,
FRIZELL
AND
SJGSTRAND
FIG. 4. Profile of labeled proteins in vagus nerve 4 months after crush ilz viva. Vagus nerves were crushed at a point indicated by the arrowhead and 4 months later removed to in vitro conditions with a ligature at B. Labeled proteins in each 5- or lo-mm segment were expressed as a percentage of the total labeled proteins between points A (20 mm from the nodose ganglion) and B (the ligature). One typical experiment out of three is shown and the profile of labeled proteins in the contralateral sham-operated vagus nerve is indicated with broken lines.
transported distal ,to the crush zone in vagal motor fibers after labeling in V&O, 4 weeks after a nerve crush. An accumulation could be observed in viva at a distal ligature applied at the time of labeling of the nerve cell bodies of the dorsal motor nucleus of the vagus (results not shown).
FIG. 5. Profile of labeled protein in a vagus nerve 4 weeks after crush ilt z&o. Nerves were crushed and 4 weeks later the proteins were labeled in z&o by injection of [‘Hlleucine into the nodose ganglia. A ligature was applied 55 mm from the ganglion and accumulation continued ilz Z&J for 24 hr. Six such experiments gave similar results.
AXONAL
TRANSPORT
0
IN
20
REGENERATING
40 mm
‘roll7
VAGUS
60
247
80
ganglion
FIG. 6. Profile of labeled proteins in motor fibers of rabbit vagus nerve 4 wk after a crush in V&O. Nerves were crushed as indicated and 4 wk later the motor fibers labelled by injection of (“H) leucine into the dorsal motor nucleus in viva. Nerves were removed 3.5 hr later and incubated k vitro with a ligature 70 mm (control) or 65 mm (crush) from the nodose ganglion for 24 hr. Four such experiments gave similar results.
However, when the vagus nerves were removed and incubated in vitro 3.5 hr after labeling of the cell bodies of the dorsal motor nucleus of the vagus, 4 weeks after a nerve crush, no transport of labeled proteins occurred beyond the crush zone (Fig. 6). Ejject of EGTA and Other Additives on the Rapid AxomJ Transport in Regenerating Fibers in vitro. The presence of 5% rabbit serum in the medium 199 did not permit the progression of the label beyond the crush zone, nor did a low-Ca’+ medium containing 5 mM EGTA (results not shown). DISCUSSION The marked changes in both the rapid and slow phasesof axonal transport during nerve regeneration (24) indicate that profound alterations occur in the transport system of the axon during outgrowth. Using an in vitro system to study possible agents which control or accelerate axonal transport in regenerating fibers, we observed that the rapid axonal transport of both the regenerating sensory and motor fibers of the vagus nerve was blocked at the crush zone with no transport observable beyond the crush zone. There are several possible explanations for the failure of regenerating vagus nerves to permit axonal transport beyond the crush zone in vitro. The axolemma or intra-axonal structure of the newly grown fibers may be more vulnerable to damage in vitro; freeze-fracturing studies indicate that the plasmolemma of an outgrowing nerve is immature with a
248
MCLEAN,
FRIZELL
AND
S JiiSTRAND
low number of intramembraneous particles (12). The perineurial barrier of the regenerated nerve may lack the normal capacity to control the environment of the regenerated axons ; Olsson and Kristensson (11) have shown a breakdown of the perineurial barrier to Evans blue albumin at the site of the crush for several months after nerve injury. However, preliminary studies on the crushed vagus nerve have failed to show any marked alterations in the perineurial barrier (Rydevik et al., in preparation) . Some blood-borne factor might be lacking when the nerve is removed for in vitro incubation. In adult postganglionic sympathetic neurons, treatment with nerve growth factor has demonstrated an increase in the rate of fast axonal transport (1) and for other neurons some unknown growthpromoting substance may exist which is especially important during the regenerative process. Another reason for the blockage of rapid transport beyond the crush zone could be the lack of slow axonal transport in newly grown fibers-studies on slow axonal transport have shown that in vitro conditions are unable to support the slow flow (9). One also must consider the possibility that the scar tissue around the crush zone might have obstructed oxygen diffusion from the medium to the axons, causing a hypoxic block at that site. Further experiments, in which the effects of factors rendering the nerve more resistant to hypoxia are tested with respect to axonal transport in regenerating fibers in vitro, would be valuable to clarify this point. We are still in the process of examing the effects of different agents on axonal transport in regenerating fibers to see what factors can be added to the incubation medium to permit the rapid transport to continue in the newly grown fibers. The experiments reported here indicate that addition of 5% rabbit serum to, or removal of Ca2+ from the medium, were insufficient to induce transport. Preliminary experiments on the crushed hypoglossal nerve, which shows a vigorous and rapid axon regeneration (3), indicate that the transport block at the site of the crush in a regenerating nerve incubated in vitro may be a general phenomenon. REFERENCES 1. ALMON, (NGF)
Brain 2. FRIZELL, 3.
R.
R., and W. 0. upon axoplasmic
MCCLURE. transport
1970. The effect in sympathetic
of nerve neurons
growth of the
factor mouse.
Res. 74: 255-267.
M., W. G. MCLEAN, and transport of rapidly migrating labelled ing peripheral nerves. Bra&z Res., in FRIZELL, M., and J. SJ~STRAND. 1974. cholinergic enzymes in regenerating
845-850.
J.
SJ~STRAND. 1976. Retrograde axonal proteins and glycoproteins in regeneratpress. Transport of proteins, glycoproteins and hypoglossa! neurons. J. Neurochem. 22 :
AXONAL
TRANSPORT
IN
REGENERATING
VAGUS
249
4. FRIZELL, M., and J. SJ~STRAND. 1974. The axonal transport of slowly migrating ‘H-leucine labelled proteins and the regeneration rate in regenerating hypoglossal and vagus nerves of the rabbit. Brain Res. 81: 267-283. 5. HENDRICKSON, A. E., and W. M. COWAN. 1971. Changes in the rate of axoplasmic transport during postnatal development of the rabbit’s optic nerve and tract. Exp.
Neural.
30:
403-422.
6. LASEK, R. J. 1970. Protein transport in neurons. Int. Rev. Neurobiol. 13: 289-324. 7. MARCHISIO, P.-C. J. SJ~STRAND, M. AGLIETTA, and J.-O. KARLSSON. 1973. The development of axonal transport of proteins and glycoproteins in the optic pathway of chick embryo. Brain Res. 63: 273-284. 8. MCLEAN, W. G., M. FRIZELL, and J. SJ~STRAND. 1975. Axonal transport of labelled proteins in sensory fibers of rabbit vagus nerve in vitro. J. Neurochevn.
9.
25:
695698.
W. G., M. FRIZELL, and J. SJ~WRAND. 1976. Slow axonal transport of labelled proteins in sensory fibres of rabbit vagus nerve. J. Neurochem. 26: 1213-1216. 10. OCHS, S. 1972. Rate of axoplasmic transport in mammalian nerve fibres. MCLEAN,
J. Physiol.
11.
12. 13.
(London)
227:
627-645.
Y., and K. KRISTENSSON, 1973. The perineurium as a diffusion barrier to protein tracers following trauma to nerves. Acta Neuropathol. (Berl.) 23: 105-111. PFENNINGER, K. H., and R. P. BUNGE. 1974. Freeze-fracturing of nerve growth cones and young fibres. J. Cell Biol. 63: 180-196. SJ~STRAND, J. 1970. Fast and slow components of axoplasmic transport in the hypoglossal and vagus nerves of the rabbit. Brain Res. 18: 461-467. OLSSON,
