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Effects of graded experimental compression on slow and fast axonal transport in rabbit vagus nerve
Journal of the Neurological Sciences, 1986, 72:19-30
19
Elsevier
JNS 2586
Effects of Graded Experimental Compression on Slow and Fast Axonal Transport in Rabbit Vagus Nerve Lars B. Dahlin 1, and W. Graham McLean 2 1Laboratory of Experimental Biology, Department of Anatomy, University of Gothenburg, Gothenburg (Sweden) and 2Department of Pharmacology and Therapeutics, University of LiverpooL Liverpool (U.K.) (Received 23 April, 1985) (Revised, received 31 July, 1985) (Accepted 8 August, 1985)
SUMMARY
Effects of compression at low pressures on slow and fast axonal transport was investigated in rabbit vagus nerve. Proteins in the sensory fibres were radiolabelled by injection of [3H]leucine or [35S]methionine into the nodose ganglion. A small compression chamber and/or ligatures were applied around the cervical part of the vagus nerve for 8 h, at an appropriate time for the subsequent analysis of the effects of compression on both slow and fast transport of radiolabelled proteins. In normal nerves there were two waves of slowly transported proteins with rates of about 12-15 and 25-30 mm/day, respectively. SDS-polyacrylamide gel electrophoresis was used and confLrmed that the main proteins which accumulated proximal to the ligatures had a molecular weight of 54 000-56 000. Neither compression of the nerve at 20 mm Hg nor sham-compression induced any statistically significant accumulation of slowly transported proteins at the site of compression. A higher pressure, i.e. 30 mm Hg, induced a marked but incomplete accumulation of slowly transported proteins. Fast transport was partially inhibited in some, but not all, nerves, when 20 mm Hg was applied for 8 h, in contrast to the lack of effect found previously with the same pressure applied for only 2 h. Despite these slight differences, the results indicate that both slow and fast
This work was supported by grants from the Swedish Medical Research Council (no 5188), the Grteborg Medical Society, the University of Gothenburg, the Swedish Society of Medical Sciences, the Swedish Society for Medical Research, and by the Medical Research Council (U.K.). Correspondence to: Lars B. Dahlin, M.D., Laboratory of Experimental Biology, Department of Anatomy, University of GOteborg, Box 33 031, S-400 33 GOteborg, Sweden. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
20 transport are impaired by low pressure levels of around 20-30 mm Hg, which are comparable with those found in human compression neuropathies. The impaired provision of cytoskeletal elements to the distal axon may be of significance in the pathophysiology of nerve entrapment syndromes.
Key words:
A x o n a l transport - N e r v e c o m p r e s s i o n - N e u r o p a t h y - Tubulin
INTRODUCTION The survival and function of the axon are both dependent on a broad spectrum of substances and organdies which are synthesised in the nerve cell body and transported within the axon and to its terminals. This anterograde axonal transport consists of several components with different velocities. Fast axonal transport (34 rnm/day-up to 400 ram/day) consists of membrane constituents (proteins, glycoproteins and lipids), neurotransmitters and various low molecular weight materials, e.g., amino acids (Grafstein and Forman 1980). Fast axonal transport is a temperaturesensitive, energy-requiring process and can be blocked by ischemia and compression (Leone and Ochs 1978; Rydevik et al. 1980) as well as by toxic substances (for review, see McLean et al. 1985). We have recently reported that fast axonal transport is inhibited in vivo by compression at low pressures (Dahlin et al. 1984). This may imply that in compression neuropathies there is a decreased provision of for example neurotransmitter materials to the axon terminal. However, the main bulk flow of substances in the axon are transported at slower rates, i.e. 0.1-8, 12-30 ram/day. This slow axonal transport includes mainly cytoskeletal elements, e.g. actin, tubulin and neurof'flament proteins (Black and Lasek 1980; Grafstein and Forman 1980; Brady and Lasek 1982; McLean et al. 1983; McLean 1985). There is less knowledge about the properties of slow transport than there is of fast transport. Furthermore, little is known about the relationship between fast transport and slow transport or whether slow transport can be maintained while fast transport is inhibited by for example nerve compression (Grafstein and Forman 1980; Griffiths and McLean 1980; Komiya and Kurokawa 1980; SjOstrand 1981). The question was raised as to whether or not compression at low pressures would also impair the provision of structural proteins to the distal part of the axons. We have therefore investigated the effects of experimental nerve compression on slow transport in the sensory fibres of the rabbit vagus nerve, the same model as used in the previous studies on fast transport (Rydevik et al. 1980; Dahlin et al. 1984). In addition to providing a direct comparison betwe~n fast and slow transport in the same system, the sensory fibres of the rabbit vagus nerve are partiedarly useful for slow transport studies because of the relatively rapid rate of the slow component in that nerve (McLean et al. 1983).
21 MATERIALS AND METHODS
Slow axonal transport Albino rabbits of both sexes (2.5-3.0 kg) were anaesthetized with Valium ® (diazepam), 1 mg/kg i.m., and Hypnorm ® (fentanyl 0.2mg/ml and fluanisone 10 mg/ml), 0.5 ml/kg i.m.. Additional doses of the Hypnorm ® were given at 30-min intervals. The right nodose ganglion was carefully exposed and 4 injections each of 25/~Ci L-[4,5-3H]leucine (spec.activity 53 Ci/mmol; Radiochemical Centre, Amersham, U.K.) were made subepineurially into the ganglion through a glass micropipette, tip diameter 15 ~tm under hydrostatic pressure over a period of 10 rain. The wound was sutured and the animals allowed to recover. Forty-eight hours later various experimental procedures were adopted, with the rabbits again under anaesthetic. In one series of experiments (n -- 6), the rabbits were left untreated and killed 8 h after re-anaesthesia, i.e. 56 (48 + 8)h after injection of [3H]leucine into the nodose ganglion. The ganglion and the vagus nerve were removed, frozen and immediately cut into 2.5-mm pieces. The pieces were soaked individually overnight in 2 ml cold 10~ trichloroacetic acid (TCA), washed once in a further 2 ml TCA and then dissolved in 0.4 ml Protosol ® (New England Nuclear) at room temperature. Two ml of toluenebased scintillation fluid were added to each dissolved nerve piece and the radioactivity of each sample was measured by liquid scintillation counting with automatic quench correction. In a second series of experiments (n = 5), the vagus nerve was carefully dissected free from surrounding tissue and two ligatures were made 20 and 40 mm from the ganglion, with 4-0 silk thread. Eight hours later the rabbits were killed, the nerves removed and prepared for liquid scintillation counting as described above. In a third series of experiments (n = 15), a 30-40 mm length of the fight vagus nerve was carefully exposed in the region 10-50 mm from the ganglion and a small compression chamber (see Dahlin et al. 1984), connected to a compressed air system, was applied around the nerve between about 20 and 30 mm from the ganglion (see Fig. 3) and left in position for 8 h. The chamber was either left uninflated (sham compression, n --- 5) or inflated to a pressure of 20 mm Hg (n = 5) or 30 mm Hg (n --- 5). A ligature was made on the vagus nerve 10-15 mm from the distal edge of the chamber in order to prevent retrogradely transported proteins from entering the compressed area, and so contributing to the accumulation of radioactivity. After 8 h the animals were killed, the vagus nerve and the ganglion removed and prepared for liquid scintillation counting as described above.
Electrophoretic analysis of transportedproteins In a separate group of rabbits (n = 3) analyses were performed in order to conf'n'm that it was the slow component of axonal transport which was being investigated. The right nodose ganglion was exposed with the rabbits under general anaesthesia as described above. Four injections of(total 150/~Ci) L-[35S]methionine (1027 Ci/mmol; New England Nuclear) were made into the ganglion. Forty-eight hours later the right vagus nerve was carefully exposed and 2 ligatures were made 20 and 40 mm from the
22 ganglion. Eight hours later the rabbits were killed and the vagus nerve and nodose ganglion removed and cut into 5-mm pieces. Nerve pieces proximal to, between and distal to the double ligatures were homogenised, and applied to 8-15 ~o gradient SDS polyacrylamide gels. After electrophoresis in one dimension, gels were impregnated with fluor, dried and applied to autoradiography film. The complete method is described by McLean et al. (1983). Fast axonal transport Rabbits (n = 5) were anaesthetized as described above and the right vagus nerve was carefully exposed. A compression chamber was applied around the nerve as described above and inflated to 20 mm Hg - a pressure level known not to affect fast axonal transport when applied for 2 hours (Dahlin et al. 1984). A ligature was also tied around the nerve 10-15 mm from the distal edge of the chamber. Four hours later the right nodose ganglion was exposed and injected with [3H]leucine as above. Four hours after the injection, i.e. 8 h after the chamber application, the animal was killed, the nerve was removed and processed for liquid scintillation counting as described above. Calculation of results The radioactivity (d.p.m.) of each 2.5-ram nerve segment was expressed as a percentage of the total radioactivity in the interval 10-50 mm from the nodose ganglion. In order to quantitate the inhibition of slow transport, the accumulation of radiolabelled proteins was calculated by summating the percentage radioactivity in 3 nerve pieces around the proximal edge of the compressed area (2 pieces immediately proximal to the proximal edge and 1 piece in the proximal part of the compressed area). In ligature experiments the value was calculated by summating the percentage radioactivity in three pieces proximal to the proximal ligature. These values, called accumulation value, are presented as mean 4- SD. For comparison the percentage radioactivity in 3 nerve pieces, at an appropriate distance from the nodose ganglion, was summarized in the uninjured nerves (first series). Statistical analysis was performed with the Wilcoxon rank sum test. P-values < 0.05 were considered significant (two-tail).
RESULTS Slow axonal transport The profile of radiolabeUed proteins in the vagus nerve 56 h after injection of [3H]leucine into the nodose ganglion showed 2 waves of radiolabelled proteins (Fig. 1). The 2 waves were calculated by extrapolation to have fronts at about 35 and 67 mm from the nodose ganglion. These fronts corresponded to maximal rates of transport of about 12-15 and 25-30ram/day, confn'ming previous estimates of transport rate (McLean et al. 1983). In the experimvnts where ligatures were applied around the vagus nerve 20, and 40 mm from the ganglion for 8 h, 48 h after radiolabelling, there was a marked accumulation of radiolabelled proteins both proximal to the proximal ligature (accumu-
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Fig. 1. The profile of radiolabelled proteins in normal rabbit vagus nerves 56 hours after injection of [3H]leucine into the nodos¢ ganglion. Results are mean of 6 nerves. The radioactivity in each 2.5-mm nerve piece is expressed as a percentage oftbe total radioactivity present between points 10 and 50 mm from the ganglion. The two distinguishable waves of slow axonal transport were calculated, by extrapolation, to have fronts (arrows) at about 35 and 67 mm from the ganglion. Fig. 2. The profile ofradiolabelled proteins in ligated rabbit vagus nerves 56 h after radiolabelling. Nerves were ligated 8 h before removal. Results are mean of 5 nerves. The arrows indicate the site of ligatures. lation value 29.1 + 2.2; mean + SD) and around the distal ligature, due to interruption o f anterograde and retrograde axonal transport (Fig. 2). The profiles o f radiolabelled proteins 56 h after injection o f isotope and 8 h after the application o f various pressures to the nerves are shown in Figs. 3 A - C . The application o f the chamber without inflation (sham compression) between about 20 and 30 m m from the ganglia caused no accumulation at the compressed area as shown in Fig. 3A. This was reflected in the low accumulation value o f 15.6 + 1.8. When a pressure o f 20 m m H g was applied around the vagus nerve for 8 h there was a small, but not significant, accumulation at the compressed region (Fig. 3B). The accumulation Value was 15.4 + 1.0 which did not differ from the value produced by sham compression. As in the ligated experiments there was an accumulation o f radiolabelled proteins around the distal ligature in these compressed vagus nerves. A higher pressure, i.e. 30 m m H g for 8 h, induced a marked accumulation o f radiolabelled proteins at the
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Fig. 3. The profile of radiolabelled proteins in compressed rabbit vagus nerves 56 h after radiolabcUing. Nerves were compressed at 0 mm Hg (sham compression) (A), 20 mm Hg (B) and 30 ram Hg (C) for the final 8 h. The black bars indicate the site of compression. The arrows indicate the ligature applied to exclude retrograde axonal transport. Results are mean of 5 nerves in each group. Compression at 30 mm Hg induced a marked accumulation of slowly transported proteins at the site of compression.
region of compression (Fig. 3C). The accumulation value was 22.0 + 2.7. Statistical analysis showed that the accumulation of radiolabelled proteins at the site of compression was significantly greater than in sham-compressed nerves (P ~< 0.01) as well as in nerves compressed at 20 ram Hg (P ~< 0.01). The accumulation was however significantly lower (P ~< 0.01) than that in the ligated nerves (see Figs. 2 and 4). As in previous experiments there was also an accumulation around the distal ligature.
Electrophoresis Fluorographs of the radioactive proteins present in the nerve samples proximal to the proximal ligature, distal to the distal ligature, and bctwoen ligatures on the double-ligated nerves are shown in Fig, 5. The main protein band accumulating proximal to the proximal ligature over the 8-h period has a mol~ular ~ t 54000-56000 and co-migrates with pig brain tub,lin (McLean et al. 1983). It is identical to the only protein de~ected in the 12-15 nun/day phase of transport in that nerve.
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Fast axonal transport Compression of the vagus nerve at a pressure of 20 mm Hg for 8 h induced a significant accumulation of radiolabelled fast transported proteins (Fig. 6) when all nerves were analysed together. There was also a significant accumulation proximal to the distal ligature. When each nerve was considered separately there were various degrees of blockage of axonal transport. In one nerve there was a marked accumulation of radiolabelled proteins in the proximal region of the chamber and very low amounts of radioactivity distally, i.e. a complete block of axonal transport. One nerve showed no block. In the remaining 3 nerves there was an accumulation at the proximal part of the chamber and high amounts of radioactivity distally with a marked accumulation proximal to the ligature, i.e. a partial axonal transport block. DISCUSSION
Two distinguishable waves of slowly transported proteins were demonstrated in the rabbit vagus nerve. The fastest wave, travelling at a rate of about 25-30 mm/day, has previously been reported to consist of aetin and the slower wave, with a rate of about 12-15 mm/day, to be composed of tubulin (McLean et al. 1983; McLean 1985)• In other mammalian nerves, it has been reported that these cytoskeletal elements are
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Fig. 4. Accumulation value at site of compression in nerves compressed at 0 mm Fig, 20 mm Hg, 30 mm Hg, and ligated nerves. Values from uninjured nerves are included for comparison. No distal ligatures were applied around those nerves, thereby including retrograde transport into the accumulation value, which explains the slightly higher value in those experiments. Each group contains 5 or 6 experiments. Levels of statistical significance between relevant groups are indicated as ** P ~< 0.01 (Wilcoxon rank sum test; two-tail). For calculation of accumulation values see text.
transported much more slowly (Black and Lasek 1980; Brady and Lasek 1982), Therefore the sensory axons of the rabbit vagus nerve are suitable for investigating the effects of different sorts of trauma on slow axonal transport, e.g. local nerve compression, due to the relatively fast rate of the slow component of axonal transport. Local compression of the rabbit vagus nerve at a low pressure, i.e. 30 mm Hgfor 8 h, impaired slow axonal transport. Electrophoretic analysis of the proteins which accumulated proximal to the ligatures showed them to have an appm~nt molecular weight identical to tubulin (see Fig. 5) and to be identical to the proteins tr~sported in the 12-15 mm/day phase of axonal transport in the vagus nerve (McLean et al. 1983). No significant protein of that molecular weight is present among the 3SS-radiolabetled proteins of fast axonal transport. We therefore consider that the accumulations which
27
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Fig. 5. A fluorograph of the radiolabelled proteins present in 5-mm pieces of rabbit vagus nerve 56 h after radiolabelling with [35S]methionine. Nerves were ligated 8 h before removal. The nerve pieces shown are these just proximal to the proximal ligature (1), between the two ligatures (2), and distal to the distal ligature (3). The main proteins accumulating proximal to the proximal ligatures have apparent molecular weights of 54000-56 000 as indicated by comparison with standard proteins of known molecular weight electrophoresed simultaneously (4). Fig. 6. The profile of radiolabelled proteins in compressed rabbit vagus nerves 4 h after radiolabelling. Nerves were compressed for 8 h at 20 mm Hg (fast axonal transport). The black bar indicates the site of compression. The arrow indicates the ligature. Results are mean of 5 nerves. Compression induced an accumulation of fast transported proteins to a variable extent.
o c c u r between 48 a n d 56 h after radiolabelling are n o t due to fast t r a n s p o r t e d proteins which have been d e l a y e d in their exit from cell bodies. D e s p i t e the m a r k e d accumulation o f radioactivity at the distal ligature in all experiments, there was no c o r r e s p o n d i n g increase in the a p p e a r a n c e on fluorographs o f any particular proteins. It m a y be that a large n u m b e r o f proteins contributed to the retrograde a c c u m u l a t i o n a n d were n o t detected as a quantitative increase by fluorography. The pressure o f 30 m m H g is also the minimal pressure, when applied for 2 h, at which blockage o f fast axonal t r a n s p o r t in the rabbit vagus nerve occurs (Dahlin et al.
28 1984). Previous studies of fast and slow axonal transport in retina and optic nerve have shown that both fast and slow axonal transport can be blocked by papilloedema secondary to raised intracranial pressure (Tso and Hayreh 1977), increased intraocular pressure and in ocular hypotony with papilloedema (Minckler et al. 1976). It has been suggested that the major transport obstruction affects the stow component and that the transport changes of the fast component are minor and may be secondary to the slow component obstruction (for review see SjOstrand 1981). Other neuropathies in which selective inhibition of components of slow axonal transport has been demonstrated experimentally, and where it is believed to be a major contributory factor to the neuropathy, include those produced by the neurotoxins fl,fl'-iminodipropionitrile (Griffin etal. 1978) and 3,4-dimethyl-2,5-hexanedione (Griffin et al. 1984). Thus, studies on axonal transport in drug-induced neuropathies as well as in retina and optic nerve in certain disorders indicate that, while both components of axonal transport may be affected by different sorts of trauma, in some cases obstruction of the slow transport seems to be the main feature. In the rabbit vagus nerve it is obvious that slow axonal transport as well as fast axonai transport can be impaired by compression at similar low pressures. However, it is difficult to make an exact quantitative comparison between inhibition of fast and slow axonal transport. Fast transport is not affected at a pressure of 20 mm Hg for 2 h (Dahlin et al. 1984) and equally slow transport is not significantly impaired at this low pressure for 8 h. On the other hand, a pressure of 20 mm Hg for 8 h produces a significant accumulation of radiolabelled proteins of fast transport in the vagus nerve. This fits in with the known fact that the impairment of fast transport is a gradedeffect, i.e. is proportional to magnitude of pressure (Rydevik et al. 1980; Dahlin et al. 1985): The results reported here provide evidence that inhibition of fast axonal transport is also proportional to duration of pressure as might be expected. The graded effect on fast axonal transport of the magnitude of pressure is also likely to be true for slow axonal transport: in a small number of experiments (results not shown) we found that compression at a pressure of 50 and 200 mm Hg gives an even greater accumulation of slowly transported proteins than does compression at 30 mm Hg. The same may also be true for duration of pressure. However, it is more difficult to compare accurately fast and slow axonal transport in that regard, due to the different time requirements for detecting accumulation of fast and slow transported proteins. The present study indicates that not only does low pressure at a level comparable with that found in compression neuropathies in humans (Gelberman et al. 1981) influence the fast transport of materials believed to be necessary for, for example transmitter function, but it also impairs the provision to the distal axon of elements of the cytoskeleton. One might envisage that over a prolonged period the lack of supply of the structural proteins might lead to dwindling of distal axons as occurs in other distal axonopathies for example in association with diabetes meUitus (Jakobsen 1976), with consequent degeneration and attempts at a reparative response. Whether or not the pressure levels we have used lead to a typical regenerative response of the nerve cell bodies is currently being investigated. Furthermore, blockage of slow axonal transport, caused by for example stretching
29 or compression, might also render the distal part of the axon more susceptible to additional trauma. Such a phenomenon (the "double-crush syndrome" hypothesis) has been reported from an electrophysiological and clinical study where 7 0 ~ of patients with carpal tunnel syndrome and/or lesion of the ulnar nerve at the elbow had an associated neural lesion in the neck (Upton and McComas 1973). The hypothesis was recently supported by an electrophysiological and histological study by Nemoto (1983). The author reported an increased vulnerability to an additional compression in the distal region of the nerve, when the nerve was compressed proximally by a pressure of 27.6 mm Hg (15 g). The author suggested that the changes may be caused by impairment of axonal transport. In "sick" neurones, where axonal transport is impaired generally, for example in diabetes meUitus, the abnormal situation may lead to an increased susceptibility of peripheral nerves to compression trauma (Upton and McComas 1973; Dahlin et al., to be published). ACKNOWLEDGEMENTS
We wish to thank Drs. Johan SjOstrand and BjOrn Rydevik for valuable advice. REFERENCES Black, M.M. and R.J. Lasek (1980) Slow components of axonal transport - - Two cytoskeletal networks, J. Cell Biol., 86: 616-623. Brady, S.T. and R.J. Lasek (1982) The slow components of axonal transport - - Movements, compositions and organization. In: D.G. Weiss (Ed.), Axoplasmic Transport, Springer-Verlag, Berlin, Heidelberg, pp. 206-217. Dahlin, L.B., B. Rydevik, W.G. McLean and J. Sj0strand (1984) Changes in fast axonal transport during experimental nerve compression at low pressures, Exp. Neurol., 84: 29-36. Dahlin, L.B., B. Rydevik and G. Lundborg (1985) Pathophysiology of nerve entrapments and nerve compression injuries. In: A.R. Hargens (Ed.), Tissue Nutrition and Viability, Springer-Verlag, Berlin, Heidelberg, New York, in press. Dahlin, L. B., K. F. Meiri, W. G. McLean, B. Rydevik and J. Sj0strand, Effects of nerve compression on fast axonal transport in streptozotocin-induced diabetes mellitus, Submitted for publication. Gelberman, R.H., P.T. Hergenroeder, A.R. Hargens, G. Lundborg and W.H. Akeson (1981)The carpal tunnel syndrome - - A study of carpal canal pressure, J. Bone Joint Surg., 63A: 380-383. Grafstein, B. and D. S. Forman (1980) Intracellular transport in neurons, Physiol. Rev., 60(4): 1167-1283. Griffin, J.W., P.N. Hoffman, A.W. Clark, P.T. Carroll and D.L. Price (1978) Slow axonal transport of neurofilament proteins - - Impairment by fl,fl'-iminodipropionitrile administration, Science, 202: 633-635. Griffin, J. W., D. C. Anthony, K. E. Fahnestock, P.N. Hoffman and D. G. Graham (1984) 3,4-Dimethyl-2,5hexanedione impairs the axonal transport of neurofilament proteins, J. Neurosci., 4: 1516-1526. Griffiths, K. F. and W. G. McLean (1980) A pharmacological comparison of rapid and slow axonal transport in rabbit vagus nerve, Brit. J. Pharmac., 70: 173P-174P. Jakobsen, J. (1976) Axonal dwindling in early experimental diabetes, Part 2 (A study of isolated nerve fibres), Diabetologia, 12: 547-553. Komiya, Y. and M. Kurokawa (1980) Preferential blockade of the tubulin transport by colchicine, Brain Res., 190: 505-516. Leone, J. and S. Ochs (1978) Anoxic block and recovery of axoplasmic transport and electrical excitability of nerve, J. Neurobiol., 9: 229-245. McLean, W.G. (1985) Axonal transport of actin and regeneration rate in non-myelinated sensory nerve fibres, Brain Res,, 333: 266-274. McLean, W.G., A.L. McKay and J. Sj6strand (1983) Electrophoretic analysis of axonally transported proteins in rabbit vagus nerve, J. Neurobiol., 14(3): 227-236.
30 McLean, W.G., M. Frizell and J. Sj6strand (1985) Pathology of axonal transport. In: A. Lajtha (Ed.). Handbook ofNeurochemistry, Vol. 9, Plenum, New York, pp. 67-86. Minckler, D. S., M.O.M. Tso and L.E. Zimmerman (1976) A light microscopic, autoradiographic study of axoplasmic transport in the optic nerve head during ocular hypotony, increased intraocular pressure. and papilledema, Amer. J. OphthalmoL, 82: 741-757. Nemoto, K. (1983) An experimental study on the vulnerability of the peripheral nerve, J. Jpn. Orthop. Ass., 57: 1773-1786. Rydevik, B., W.G. McLean, J. SjOstrand and G. Lundborg (1980) Blockage of axonal transport induced by acute, graded compression of the rabbit vagus nerve, J. Neurol. Neurosurg. Psychiat., 43: 690-698. Sj6strand, J. (1981) A dynamic view of the retinal ganglion cell and its transport - - A minireview of the role of axonal transport dysfunction in the pathophysiology of optic neuropathies, Acta Ophthalmol.. 59: 785-797. Tso, M. O. M. and S.S. Hayreh (1977) Optic disc edema in raised intracranial pressure, Part 4 (Axoplasmic transport in experimental papilledema), Arch. Ophthalmol., 95: 1458-1462. Upton, A. R. M. and A.J. McComas (1973) The double crush in nerve-entrapment syndromes, Lancet, ii: 359-362.
19
Elsevier
JNS 2586
Effects of Graded Experimental Compression on Slow and Fast Axonal Transport in Rabbit Vagus Nerve Lars B. Dahlin 1, and W. Graham McLean 2 1Laboratory of Experimental Biology, Department of Anatomy, University of Gothenburg, Gothenburg (Sweden) and 2Department of Pharmacology and Therapeutics, University of LiverpooL Liverpool (U.K.) (Received 23 April, 1985) (Revised, received 31 July, 1985) (Accepted 8 August, 1985)
SUMMARY
Effects of compression at low pressures on slow and fast axonal transport was investigated in rabbit vagus nerve. Proteins in the sensory fibres were radiolabelled by injection of [3H]leucine or [35S]methionine into the nodose ganglion. A small compression chamber and/or ligatures were applied around the cervical part of the vagus nerve for 8 h, at an appropriate time for the subsequent analysis of the effects of compression on both slow and fast transport of radiolabelled proteins. In normal nerves there were two waves of slowly transported proteins with rates of about 12-15 and 25-30 mm/day, respectively. SDS-polyacrylamide gel electrophoresis was used and confLrmed that the main proteins which accumulated proximal to the ligatures had a molecular weight of 54 000-56 000. Neither compression of the nerve at 20 mm Hg nor sham-compression induced any statistically significant accumulation of slowly transported proteins at the site of compression. A higher pressure, i.e. 30 mm Hg, induced a marked but incomplete accumulation of slowly transported proteins. Fast transport was partially inhibited in some, but not all, nerves, when 20 mm Hg was applied for 8 h, in contrast to the lack of effect found previously with the same pressure applied for only 2 h. Despite these slight differences, the results indicate that both slow and fast
This work was supported by grants from the Swedish Medical Research Council (no 5188), the Grteborg Medical Society, the University of Gothenburg, the Swedish Society of Medical Sciences, the Swedish Society for Medical Research, and by the Medical Research Council (U.K.). Correspondence to: Lars B. Dahlin, M.D., Laboratory of Experimental Biology, Department of Anatomy, University of GOteborg, Box 33 031, S-400 33 GOteborg, Sweden. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
20 transport are impaired by low pressure levels of around 20-30 mm Hg, which are comparable with those found in human compression neuropathies. The impaired provision of cytoskeletal elements to the distal axon may be of significance in the pathophysiology of nerve entrapment syndromes.
Key words:
A x o n a l transport - N e r v e c o m p r e s s i o n - N e u r o p a t h y - Tubulin
INTRODUCTION The survival and function of the axon are both dependent on a broad spectrum of substances and organdies which are synthesised in the nerve cell body and transported within the axon and to its terminals. This anterograde axonal transport consists of several components with different velocities. Fast axonal transport (34 rnm/day-up to 400 ram/day) consists of membrane constituents (proteins, glycoproteins and lipids), neurotransmitters and various low molecular weight materials, e.g., amino acids (Grafstein and Forman 1980). Fast axonal transport is a temperaturesensitive, energy-requiring process and can be blocked by ischemia and compression (Leone and Ochs 1978; Rydevik et al. 1980) as well as by toxic substances (for review, see McLean et al. 1985). We have recently reported that fast axonal transport is inhibited in vivo by compression at low pressures (Dahlin et al. 1984). This may imply that in compression neuropathies there is a decreased provision of for example neurotransmitter materials to the axon terminal. However, the main bulk flow of substances in the axon are transported at slower rates, i.e. 0.1-8, 12-30 ram/day. This slow axonal transport includes mainly cytoskeletal elements, e.g. actin, tubulin and neurof'flament proteins (Black and Lasek 1980; Grafstein and Forman 1980; Brady and Lasek 1982; McLean et al. 1983; McLean 1985). There is less knowledge about the properties of slow transport than there is of fast transport. Furthermore, little is known about the relationship between fast transport and slow transport or whether slow transport can be maintained while fast transport is inhibited by for example nerve compression (Grafstein and Forman 1980; Griffiths and McLean 1980; Komiya and Kurokawa 1980; SjOstrand 1981). The question was raised as to whether or not compression at low pressures would also impair the provision of structural proteins to the distal part of the axons. We have therefore investigated the effects of experimental nerve compression on slow transport in the sensory fibres of the rabbit vagus nerve, the same model as used in the previous studies on fast transport (Rydevik et al. 1980; Dahlin et al. 1984). In addition to providing a direct comparison betwe~n fast and slow transport in the same system, the sensory fibres of the rabbit vagus nerve are partiedarly useful for slow transport studies because of the relatively rapid rate of the slow component in that nerve (McLean et al. 1983).
21 MATERIALS AND METHODS
Slow axonal transport Albino rabbits of both sexes (2.5-3.0 kg) were anaesthetized with Valium ® (diazepam), 1 mg/kg i.m., and Hypnorm ® (fentanyl 0.2mg/ml and fluanisone 10 mg/ml), 0.5 ml/kg i.m.. Additional doses of the Hypnorm ® were given at 30-min intervals. The right nodose ganglion was carefully exposed and 4 injections each of 25/~Ci L-[4,5-3H]leucine (spec.activity 53 Ci/mmol; Radiochemical Centre, Amersham, U.K.) were made subepineurially into the ganglion through a glass micropipette, tip diameter 15 ~tm under hydrostatic pressure over a period of 10 rain. The wound was sutured and the animals allowed to recover. Forty-eight hours later various experimental procedures were adopted, with the rabbits again under anaesthetic. In one series of experiments (n -- 6), the rabbits were left untreated and killed 8 h after re-anaesthesia, i.e. 56 (48 + 8)h after injection of [3H]leucine into the nodose ganglion. The ganglion and the vagus nerve were removed, frozen and immediately cut into 2.5-mm pieces. The pieces were soaked individually overnight in 2 ml cold 10~ trichloroacetic acid (TCA), washed once in a further 2 ml TCA and then dissolved in 0.4 ml Protosol ® (New England Nuclear) at room temperature. Two ml of toluenebased scintillation fluid were added to each dissolved nerve piece and the radioactivity of each sample was measured by liquid scintillation counting with automatic quench correction. In a second series of experiments (n = 5), the vagus nerve was carefully dissected free from surrounding tissue and two ligatures were made 20 and 40 mm from the ganglion, with 4-0 silk thread. Eight hours later the rabbits were killed, the nerves removed and prepared for liquid scintillation counting as described above. In a third series of experiments (n = 15), a 30-40 mm length of the fight vagus nerve was carefully exposed in the region 10-50 mm from the ganglion and a small compression chamber (see Dahlin et al. 1984), connected to a compressed air system, was applied around the nerve between about 20 and 30 mm from the ganglion (see Fig. 3) and left in position for 8 h. The chamber was either left uninflated (sham compression, n --- 5) or inflated to a pressure of 20 mm Hg (n = 5) or 30 mm Hg (n --- 5). A ligature was made on the vagus nerve 10-15 mm from the distal edge of the chamber in order to prevent retrogradely transported proteins from entering the compressed area, and so contributing to the accumulation of radioactivity. After 8 h the animals were killed, the vagus nerve and the ganglion removed and prepared for liquid scintillation counting as described above.
Electrophoretic analysis of transportedproteins In a separate group of rabbits (n = 3) analyses were performed in order to conf'n'm that it was the slow component of axonal transport which was being investigated. The right nodose ganglion was exposed with the rabbits under general anaesthesia as described above. Four injections of(total 150/~Ci) L-[35S]methionine (1027 Ci/mmol; New England Nuclear) were made into the ganglion. Forty-eight hours later the right vagus nerve was carefully exposed and 2 ligatures were made 20 and 40 mm from the
22 ganglion. Eight hours later the rabbits were killed and the vagus nerve and nodose ganglion removed and cut into 5-mm pieces. Nerve pieces proximal to, between and distal to the double ligatures were homogenised, and applied to 8-15 ~o gradient SDS polyacrylamide gels. After electrophoresis in one dimension, gels were impregnated with fluor, dried and applied to autoradiography film. The complete method is described by McLean et al. (1983). Fast axonal transport Rabbits (n = 5) were anaesthetized as described above and the right vagus nerve was carefully exposed. A compression chamber was applied around the nerve as described above and inflated to 20 mm Hg - a pressure level known not to affect fast axonal transport when applied for 2 hours (Dahlin et al. 1984). A ligature was also tied around the nerve 10-15 mm from the distal edge of the chamber. Four hours later the right nodose ganglion was exposed and injected with [3H]leucine as above. Four hours after the injection, i.e. 8 h after the chamber application, the animal was killed, the nerve was removed and processed for liquid scintillation counting as described above. Calculation of results The radioactivity (d.p.m.) of each 2.5-ram nerve segment was expressed as a percentage of the total radioactivity in the interval 10-50 mm from the nodose ganglion. In order to quantitate the inhibition of slow transport, the accumulation of radiolabelled proteins was calculated by summating the percentage radioactivity in 3 nerve pieces around the proximal edge of the compressed area (2 pieces immediately proximal to the proximal edge and 1 piece in the proximal part of the compressed area). In ligature experiments the value was calculated by summating the percentage radioactivity in three pieces proximal to the proximal ligature. These values, called accumulation value, are presented as mean 4- SD. For comparison the percentage radioactivity in 3 nerve pieces, at an appropriate distance from the nodose ganglion, was summarized in the uninjured nerves (first series). Statistical analysis was performed with the Wilcoxon rank sum test. P-values < 0.05 were considered significant (two-tail).
RESULTS Slow axonal transport The profile of radiolabeUed proteins in the vagus nerve 56 h after injection of [3H]leucine into the nodose ganglion showed 2 waves of radiolabelled proteins (Fig. 1). The 2 waves were calculated by extrapolation to have fronts at about 35 and 67 mm from the nodose ganglion. These fronts corresponded to maximal rates of transport of about 12-15 and 25-30ram/day, confn'ming previous estimates of transport rate (McLean et al. 1983). In the experimvnts where ligatures were applied around the vagus nerve 20, and 40 mm from the ganglion for 8 h, 48 h after radiolabelling, there was a marked accumulation of radiolabelled proteins both proximal to the proximal ligature (accumu-
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Fig. 1. The profile of radiolabelled proteins in normal rabbit vagus nerves 56 hours after injection of [3H]leucine into the nodos¢ ganglion. Results are mean of 6 nerves. The radioactivity in each 2.5-mm nerve piece is expressed as a percentage oftbe total radioactivity present between points 10 and 50 mm from the ganglion. The two distinguishable waves of slow axonal transport were calculated, by extrapolation, to have fronts (arrows) at about 35 and 67 mm from the ganglion. Fig. 2. The profile ofradiolabelled proteins in ligated rabbit vagus nerves 56 h after radiolabelling. Nerves were ligated 8 h before removal. Results are mean of 5 nerves. The arrows indicate the site of ligatures. lation value 29.1 + 2.2; mean + SD) and around the distal ligature, due to interruption o f anterograde and retrograde axonal transport (Fig. 2). The profiles o f radiolabelled proteins 56 h after injection o f isotope and 8 h after the application o f various pressures to the nerves are shown in Figs. 3 A - C . The application o f the chamber without inflation (sham compression) between about 20 and 30 m m from the ganglia caused no accumulation at the compressed area as shown in Fig. 3A. This was reflected in the low accumulation value o f 15.6 + 1.8. When a pressure o f 20 m m H g was applied around the vagus nerve for 8 h there was a small, but not significant, accumulation at the compressed region (Fig. 3B). The accumulation Value was 15.4 + 1.0 which did not differ from the value produced by sham compression. As in the ligated experiments there was an accumulation o f radiolabelled proteins around the distal ligature in these compressed vagus nerves. A higher pressure, i.e. 30 m m H g for 8 h, induced a marked accumulation o f radiolabelled proteins at the
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Fig. 3. The profile of radiolabelled proteins in compressed rabbit vagus nerves 56 h after radiolabcUing. Nerves were compressed at 0 mm Hg (sham compression) (A), 20 mm Hg (B) and 30 ram Hg (C) for the final 8 h. The black bars indicate the site of compression. The arrows indicate the ligature applied to exclude retrograde axonal transport. Results are mean of 5 nerves in each group. Compression at 30 mm Hg induced a marked accumulation of slowly transported proteins at the site of compression.
region of compression (Fig. 3C). The accumulation value was 22.0 + 2.7. Statistical analysis showed that the accumulation of radiolabelled proteins at the site of compression was significantly greater than in sham-compressed nerves (P ~< 0.01) as well as in nerves compressed at 20 ram Hg (P ~< 0.01). The accumulation was however significantly lower (P ~< 0.01) than that in the ligated nerves (see Figs. 2 and 4). As in previous experiments there was also an accumulation around the distal ligature.
Electrophoresis Fluorographs of the radioactive proteins present in the nerve samples proximal to the proximal ligature, distal to the distal ligature, and bctwoen ligatures on the double-ligated nerves are shown in Fig, 5. The main protein band accumulating proximal to the proximal ligature over the 8-h period has a mol~ular ~ t 54000-56000 and co-migrates with pig brain tub,lin (McLean et al. 1983). It is identical to the only protein de~ected in the 12-15 nun/day phase of transport in that nerve.
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Fast axonal transport Compression of the vagus nerve at a pressure of 20 mm Hg for 8 h induced a significant accumulation of radiolabelled fast transported proteins (Fig. 6) when all nerves were analysed together. There was also a significant accumulation proximal to the distal ligature. When each nerve was considered separately there were various degrees of blockage of axonal transport. In one nerve there was a marked accumulation of radiolabelled proteins in the proximal region of the chamber and very low amounts of radioactivity distally, i.e. a complete block of axonal transport. One nerve showed no block. In the remaining 3 nerves there was an accumulation at the proximal part of the chamber and high amounts of radioactivity distally with a marked accumulation proximal to the ligature, i.e. a partial axonal transport block. DISCUSSION
Two distinguishable waves of slowly transported proteins were demonstrated in the rabbit vagus nerve. The fastest wave, travelling at a rate of about 25-30 mm/day, has previously been reported to consist of aetin and the slower wave, with a rate of about 12-15 mm/day, to be composed of tubulin (McLean et al. 1983; McLean 1985)• In other mammalian nerves, it has been reported that these cytoskeletal elements are
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Fig. 4. Accumulation value at site of compression in nerves compressed at 0 mm Fig, 20 mm Hg, 30 mm Hg, and ligated nerves. Values from uninjured nerves are included for comparison. No distal ligatures were applied around those nerves, thereby including retrograde transport into the accumulation value, which explains the slightly higher value in those experiments. Each group contains 5 or 6 experiments. Levels of statistical significance between relevant groups are indicated as ** P ~< 0.01 (Wilcoxon rank sum test; two-tail). For calculation of accumulation values see text.
transported much more slowly (Black and Lasek 1980; Brady and Lasek 1982), Therefore the sensory axons of the rabbit vagus nerve are suitable for investigating the effects of different sorts of trauma on slow axonal transport, e.g. local nerve compression, due to the relatively fast rate of the slow component of axonal transport. Local compression of the rabbit vagus nerve at a low pressure, i.e. 30 mm Hgfor 8 h, impaired slow axonal transport. Electrophoretic analysis of the proteins which accumulated proximal to the ligatures showed them to have an appm~nt molecular weight identical to tubulin (see Fig. 5) and to be identical to the proteins tr~sported in the 12-15 mm/day phase of axonal transport in the vagus nerve (McLean et al. 1983). No significant protein of that molecular weight is present among the 3SS-radiolabetled proteins of fast axonal transport. We therefore consider that the accumulations which
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Fig. 5. A fluorograph of the radiolabelled proteins present in 5-mm pieces of rabbit vagus nerve 56 h after radiolabelling with [35S]methionine. Nerves were ligated 8 h before removal. The nerve pieces shown are these just proximal to the proximal ligature (1), between the two ligatures (2), and distal to the distal ligature (3). The main proteins accumulating proximal to the proximal ligatures have apparent molecular weights of 54000-56 000 as indicated by comparison with standard proteins of known molecular weight electrophoresed simultaneously (4). Fig. 6. The profile of radiolabelled proteins in compressed rabbit vagus nerves 4 h after radiolabelling. Nerves were compressed for 8 h at 20 mm Hg (fast axonal transport). The black bar indicates the site of compression. The arrow indicates the ligature. Results are mean of 5 nerves. Compression induced an accumulation of fast transported proteins to a variable extent.
o c c u r between 48 a n d 56 h after radiolabelling are n o t due to fast t r a n s p o r t e d proteins which have been d e l a y e d in their exit from cell bodies. D e s p i t e the m a r k e d accumulation o f radioactivity at the distal ligature in all experiments, there was no c o r r e s p o n d i n g increase in the a p p e a r a n c e on fluorographs o f any particular proteins. It m a y be that a large n u m b e r o f proteins contributed to the retrograde a c c u m u l a t i o n a n d were n o t detected as a quantitative increase by fluorography. The pressure o f 30 m m H g is also the minimal pressure, when applied for 2 h, at which blockage o f fast axonal t r a n s p o r t in the rabbit vagus nerve occurs (Dahlin et al.
28 1984). Previous studies of fast and slow axonal transport in retina and optic nerve have shown that both fast and slow axonal transport can be blocked by papilloedema secondary to raised intracranial pressure (Tso and Hayreh 1977), increased intraocular pressure and in ocular hypotony with papilloedema (Minckler et al. 1976). It has been suggested that the major transport obstruction affects the stow component and that the transport changes of the fast component are minor and may be secondary to the slow component obstruction (for review see SjOstrand 1981). Other neuropathies in which selective inhibition of components of slow axonal transport has been demonstrated experimentally, and where it is believed to be a major contributory factor to the neuropathy, include those produced by the neurotoxins fl,fl'-iminodipropionitrile (Griffin etal. 1978) and 3,4-dimethyl-2,5-hexanedione (Griffin et al. 1984). Thus, studies on axonal transport in drug-induced neuropathies as well as in retina and optic nerve in certain disorders indicate that, while both components of axonal transport may be affected by different sorts of trauma, in some cases obstruction of the slow transport seems to be the main feature. In the rabbit vagus nerve it is obvious that slow axonal transport as well as fast axonai transport can be impaired by compression at similar low pressures. However, it is difficult to make an exact quantitative comparison between inhibition of fast and slow axonal transport. Fast transport is not affected at a pressure of 20 mm Hg for 2 h (Dahlin et al. 1984) and equally slow transport is not significantly impaired at this low pressure for 8 h. On the other hand, a pressure of 20 mm Hg for 8 h produces a significant accumulation of radiolabelled proteins of fast transport in the vagus nerve. This fits in with the known fact that the impairment of fast transport is a gradedeffect, i.e. is proportional to magnitude of pressure (Rydevik et al. 1980; Dahlin et al. 1985): The results reported here provide evidence that inhibition of fast axonal transport is also proportional to duration of pressure as might be expected. The graded effect on fast axonal transport of the magnitude of pressure is also likely to be true for slow axonal transport: in a small number of experiments (results not shown) we found that compression at a pressure of 50 and 200 mm Hg gives an even greater accumulation of slowly transported proteins than does compression at 30 mm Hg. The same may also be true for duration of pressure. However, it is more difficult to compare accurately fast and slow axonal transport in that regard, due to the different time requirements for detecting accumulation of fast and slow transported proteins. The present study indicates that not only does low pressure at a level comparable with that found in compression neuropathies in humans (Gelberman et al. 1981) influence the fast transport of materials believed to be necessary for, for example transmitter function, but it also impairs the provision to the distal axon of elements of the cytoskeleton. One might envisage that over a prolonged period the lack of supply of the structural proteins might lead to dwindling of distal axons as occurs in other distal axonopathies for example in association with diabetes meUitus (Jakobsen 1976), with consequent degeneration and attempts at a reparative response. Whether or not the pressure levels we have used lead to a typical regenerative response of the nerve cell bodies is currently being investigated. Furthermore, blockage of slow axonal transport, caused by for example stretching
29 or compression, might also render the distal part of the axon more susceptible to additional trauma. Such a phenomenon (the "double-crush syndrome" hypothesis) has been reported from an electrophysiological and clinical study where 7 0 ~ of patients with carpal tunnel syndrome and/or lesion of the ulnar nerve at the elbow had an associated neural lesion in the neck (Upton and McComas 1973). The hypothesis was recently supported by an electrophysiological and histological study by Nemoto (1983). The author reported an increased vulnerability to an additional compression in the distal region of the nerve, when the nerve was compressed proximally by a pressure of 27.6 mm Hg (15 g). The author suggested that the changes may be caused by impairment of axonal transport. In "sick" neurones, where axonal transport is impaired generally, for example in diabetes meUitus, the abnormal situation may lead to an increased susceptibility of peripheral nerves to compression trauma (Upton and McComas 1973; Dahlin et al., to be published). ACKNOWLEDGEMENTS
We wish to thank Drs. Johan SjOstrand and BjOrn Rydevik for valuable advice. REFERENCES Black, M.M. and R.J. Lasek (1980) Slow components of axonal transport - - Two cytoskeletal networks, J. Cell Biol., 86: 616-623. Brady, S.T. and R.J. Lasek (1982) The slow components of axonal transport - - Movements, compositions and organization. In: D.G. Weiss (Ed.), Axoplasmic Transport, Springer-Verlag, Berlin, Heidelberg, pp. 206-217. Dahlin, L.B., B. Rydevik, W.G. McLean and J. Sj0strand (1984) Changes in fast axonal transport during experimental nerve compression at low pressures, Exp. Neurol., 84: 29-36. Dahlin, L.B., B. Rydevik and G. Lundborg (1985) Pathophysiology of nerve entrapments and nerve compression injuries. In: A.R. Hargens (Ed.), Tissue Nutrition and Viability, Springer-Verlag, Berlin, Heidelberg, New York, in press. Dahlin, L. B., K. F. Meiri, W. G. McLean, B. Rydevik and J. Sj0strand, Effects of nerve compression on fast axonal transport in streptozotocin-induced diabetes mellitus, Submitted for publication. Gelberman, R.H., P.T. Hergenroeder, A.R. Hargens, G. Lundborg and W.H. Akeson (1981)The carpal tunnel syndrome - - A study of carpal canal pressure, J. Bone Joint Surg., 63A: 380-383. Grafstein, B. and D. S. Forman (1980) Intracellular transport in neurons, Physiol. Rev., 60(4): 1167-1283. Griffin, J.W., P.N. Hoffman, A.W. Clark, P.T. Carroll and D.L. Price (1978) Slow axonal transport of neurofilament proteins - - Impairment by fl,fl'-iminodipropionitrile administration, Science, 202: 633-635. Griffin, J. W., D. C. Anthony, K. E. Fahnestock, P.N. Hoffman and D. G. Graham (1984) 3,4-Dimethyl-2,5hexanedione impairs the axonal transport of neurofilament proteins, J. Neurosci., 4: 1516-1526. Griffiths, K. F. and W. G. McLean (1980) A pharmacological comparison of rapid and slow axonal transport in rabbit vagus nerve, Brit. J. Pharmac., 70: 173P-174P. Jakobsen, J. (1976) Axonal dwindling in early experimental diabetes, Part 2 (A study of isolated nerve fibres), Diabetologia, 12: 547-553. Komiya, Y. and M. Kurokawa (1980) Preferential blockade of the tubulin transport by colchicine, Brain Res., 190: 505-516. Leone, J. and S. Ochs (1978) Anoxic block and recovery of axoplasmic transport and electrical excitability of nerve, J. Neurobiol., 9: 229-245. McLean, W.G. (1985) Axonal transport of actin and regeneration rate in non-myelinated sensory nerve fibres, Brain Res,, 333: 266-274. McLean, W.G., A.L. McKay and J. Sj6strand (1983) Electrophoretic analysis of axonally transported proteins in rabbit vagus nerve, J. Neurobiol., 14(3): 227-236.
30 McLean, W.G., M. Frizell and J. Sj6strand (1985) Pathology of axonal transport. In: A. Lajtha (Ed.). Handbook ofNeurochemistry, Vol. 9, Plenum, New York, pp. 67-86. Minckler, D. S., M.O.M. Tso and L.E. Zimmerman (1976) A light microscopic, autoradiographic study of axoplasmic transport in the optic nerve head during ocular hypotony, increased intraocular pressure. and papilledema, Amer. J. OphthalmoL, 82: 741-757. Nemoto, K. (1983) An experimental study on the vulnerability of the peripheral nerve, J. Jpn. Orthop. Ass., 57: 1773-1786. Rydevik, B., W.G. McLean, J. SjOstrand and G. Lundborg (1980) Blockage of axonal transport induced by acute, graded compression of the rabbit vagus nerve, J. Neurol. Neurosurg. Psychiat., 43: 690-698. Sj6strand, J. (1981) A dynamic view of the retinal ganglion cell and its transport - - A minireview of the role of axonal transport dysfunction in the pathophysiology of optic neuropathies, Acta Ophthalmol.. 59: 785-797. Tso, M. O. M. and S.S. Hayreh (1977) Optic disc edema in raised intracranial pressure, Part 4 (Axoplasmic transport in experimental papilledema), Arch. Ophthalmol., 95: 1458-1462. Upton, A. R. M. and A.J. McComas (1973) The double crush in nerve-entrapment syndromes, Lancet, ii: 359-362.