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Changes in fast axonal transport during experimental nerve compression at low pressures
EXPERIMENTAL
NEUROLOGY
Changes
84,29-36
(1984)
in Fast Axonal Transport during Experimental Nerve Compression at Low Pressures
LARS B. DAHLIN,
BJ~RN RYDEVIK, W. GRAHAM AND JOHAN SJ~STBAND’
MCLEAN,
Laboratory of Experimental Biology, Department of Anatomy; Institute of Neurobiology, Department of Histology, University of Giiteborg; Department of Orthopaedic Surgery I; Department of Ophthalmology, Sahlgrens Hospital, Gothenburg, Sweden; and Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, United Kingdom Received April 27, 1983; revision received November 29, 1983 The minimal pressure for impairment of fast anterograde axonal transport was determined in rabbit vagus nerve. Proteins, transported by fast anterograde axonal transport, were labeled by a microinjection of [3H]1eucine into the nodose ganglion, and a small compression chamber was applied around the cervical vagus nerve. In this way the nerve was subjected to acute, graded compression. Compression at 20 mm Hg for 2 h as well as sham compression did not induce accumulation of axonally transported proteins at the level of compression. However, a pressure of 30 mm Hg for 2 h induced a block of axonal transport at the site of compression. The causes of the axonal transport block are discussed as well as the minimal pressure level in relation to pressures found in clinical nerve compression lesions. INTRODUCTION
The axonal transport systems transfer, for example, proteins and organelles from the nerve cell body to the axon terminals (anterograde axonal transport) as well as in the opposite direction (retrograde axonal transport). The anterograde transport includes slow components (0.1 to 8 and 25 mm/day) and fast components (34 to 68 and 240 to 400 mm/day) (2, 6, 14). The latter are known to be blocked by &hernia and compression (7, 8, 28) as well as Abbreviations: TCA-trichloroacetic acid, EFP-endoneurial fluid pressure. ’ This work was supported by grants from the Swedish Medical Research Council (5 188 and 2226), the Giiteborg Medical Society, the Swedish Work Environment Fund, and the University of Giiteborg, Sweden. Please send correspondence to Lam B. Dahlin, M.D., Laboratory of Experimental Biology, Department of Anatomy, University of Giiteborg, Box 33031, S-400 33 Giitehorg, Sweden. 29 0014-4886/84 $3.00 Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any form lcsened
30
DAHLIN
ET
AL.
by toxic substances (29). Different experimental methods have been described to study the impairment of axonal transport induced by local compression, for example by tourniquet and in vitro compression (7, 8, 20). Rydevik et al. (28) demonstrated that fast anterograde axonal transport in the rabbit vagus nerve was blocked by local compression in vivo at 50 mm Hg for 2 h, and this block was reversible within 24 h. Blockage of axonal transport in optic nerves of monkeys has also been induced by experimental elevation of intraocular pressure (15, 24, 25). Block of axonal transport may be one of several important factors contributing to the development of nerve dysfunction in compression lesions of peripheral nerves, nerve roots, or optic nerves. The pathophysiology of compression-induced nerve lesions, including mechanisms behind, and consequences of, blockage of axonal transport, is not fully clarified. However, both &hernia and mechanical deformation of nerve fibers are believed to be of importance (3, 23, 26, 31). Our purpose was to identify the minimal pressure necessary for blockage of fast anterograde axonal transport in a defined experimental model. METHODS The method of application of pressure to the vagus nerve and the method of measuring axonal transport were identical to those used previously by Rydevik et al. (28). Eighteen albino rabbits of either sex, weighing 2.0 to 2.5 kg, were anesthetized by an i.m. injection of 0.75 ml/kg body weight of Hypnorm (fluanisone 10 mg and fentanyl 0.2 mg/ml) followed by 1 mg/kg Valium (diazepam 5 mg/ml). Anesthesia was maintained with 0.25 ml/kg bodyweight Hypnorm at 30-min intervals. The fast axonal transport of 3H-labeled proteins in the sensory fibers of the vagus nerve was measured in the following way. The nodose ganglion was carefully exposed and 100 &i [3H]leucine (L-4,5[3H]leucine, 53 Ci/ mmol, Radiochemical Centre, Amersham, England) in 20 ~10.9% NaCl were injected subepineurially into the nodose ganglion via a 30-gauge stainlesssteel needle. Two hours later a 30 to 40-mm length of the vagus nerve was exposed in the region 10 to 50 mm from the nodose ganglion and a small compression chamber, consisting of two Plexiglas halves onto which thin rubber membranes were glued, was applied around the nerve. The compressed area was 10 mm. The chamber was secured around the nerve in such a way that the nerve could be subjected to acute, graded compression by inflation of the rubber membranes. The surrounding muscle and skin were loosely sutured in place and the chamber was then inflated with air at a known constant pressure. This pressure was automatically maintained at the desired
NERVE
COMPRESSION
AND
AXONAL
TRANSPORT
31
level throughout the experiments by a special ah-pump which compensated for any leakage in the system. In five sham experiments, the chamber was applied around the nerve trunk but was not inflated. In the rmnaining experiments the nerves were compressed either at 20 (N = 5) or 30 mm Hg (N = 8) for 2 h. At the end of the 2-h period, the animals were killed by an overdose of barbiturate and the vagus nerve and the nodose ganglion were quickly removed and transferred to ice The nerve was then cut into 2.5mm pieces and the pieces were soaked 24 h individually in 2 ml 10% trichloroacetic acid (TCA) at 4°C. The TCA was discarded and the nerve pieces washed in a further 2 ml cold TCA and dissolved overnight at room temperature in 0.5 ml Soluene (Packard). Radioactivity in the dissolved nerve pieces was measured in 2 ml Permablend (Packard) scintillation fluid in a Packard Tricarb liquid scintillation counter with automatic quench correction. RESULTS The TCA-insoluble radioactivity in each nerve piece was plotted for each nerve against the distance of the piece from the nodose ganglion (Fig. 1). Previous studies showed that the prolile of radiolabeled proteins in untreated nerves exhibited a front of radiolabeled proteins about 60 mm from the ganglion, indicating a rate of fast axonal transport greater than 360 mm/day (14, 28). In five nerves subjected to sham compression there was no difference in the prolile from that in untreated nerves (Fig. la). Therefore, the application of the compression chamber per se did not lead to any block of axonal transport. In five nerves subjected to compression at 20 mm Hg for 2 h, there was again no difference in the profile from that in untreated or shamcompressed nerves (Fig. 1b). Three of eight nerves subjected to a compression of 30 mm Hg for 2 h showed a profile of radioactive proteins as seen in Fig. 1c. In these cases there was increased radioactivity in those pieces immediately proximal to the region of the nerve to which the compression chamber was applied. There was, however, also a signi@cant wave of radioactive proteins the front of which had moved to the same position as that in untreated nerves. Thus the compression in those nerves produced a partial inhibition of axonal transport. One of the eight nerves compressed at 30 mm Hg showed no block of axonal transport. The remaining four nerves compressed at this pressure showed a more complete inhibition of axonal transport, in which there were only very low amounts of radioactive proteins distal to the site of the compression as seen in Fig. Id. The results are summarized in Table 1.
DAHLIN
32
ET AL.
c,
mm
from
nodose
ganglo~n
mm
30mm 2h acute
tlg
‘mm
nodose
ganghon
FIG. 1. Summary of the typical findings. The applied pressure and the time of compression are shown in each diagram. The black bar indicates the site of the compression chamber. Shamcompression (a) as well as compression at 20 mm Hg (b) induced no block of axonal transport. In some of the nerves compressed by 30 mm Hg a wave of radioactive proteins was seen distal to the accumulation at the compression cuff, i.e., the block was partial (c). In other experiments, compression at 30 mm Hg induced a more complete block (d).
DISCUSSION This study showed that the minimal pressure that blocks the fast anterograde axonal transport in sensory fibers of rabbit vagus nerves is 30 mm Hg applied for 2 h. Consistently, compression at 20 mm Hg for 2 h did not impair the fast axonal transport. It is of course possible that a lower pressure may inhibit fast axonal transport when applied for longer than 2 h or over a longer segment of nerve. The exact mechanism behind accumulation of axomdly transported material induced by 30 mm Hg is not known. Generally, axonal transport block induced by compression may be an effect of(i) ischemia and/or (ii) mechanical deformation of axons. The details of the basic mechanisms behind normal axonal transport were extensively investigated by Ochs and co-workers (19-
NERVE COMPRESSION
AND AXONAL
33
TRANSPORT
TABLE I Effect of Compression on Fast Axonal Transport in Vagus Nerve of Rabbits PressUE
No block
Partial block
Complete block
$5)
5
0
0
(N2f 5)
5
0
0
(N3i 8)
1
3
4
(mm
Nz)
22). Leone and Ochs (8) studied the impairment of axonal transport in cat sciatic nerves induced by anoxia in vitro and by a tourniquet in vivo. Those authors demonstrated that axonal transport is blocked within 15 min of onset of anoxia in vitro and by compression at about 300 mm Hg in vim, induced by tourniquets. After 1.5 h of anoxia there was a good recovery but with clear evidence of failure of recovery after 2 h of anoxia. The block induced by tourniquet-compression gradually recovered if sufficient recovery time was allowed and if the compression time had not exceeded 6 to 7 h. They suggested that ischemia was the major cause of the block. It seems reasonable to assume that the circulatory disturbances in a nerve segment subjected to compression at low levels, i.e., 30 to 50 mm Hg, play an important role in blocking axonal transport (28). Direct compression of a rabbit tibiaI nerve at 20 to 30 mm Hg induces a marked reduction of intraneural venular blood flow and the compressed nerve segment is completely ischemic at 60 to 80 mm Hg cuff pressure (27). Disturbances in venular blood flow may give retrograde effects on the capillary flow in the endoneurial space and can induce endoneurial edema formation (3 1). Due to factors such as the unyielding properties of the perineurium and the lack of lymphatic vessels in the endoneurial space, an endoneurial edema will lead to an increase of the endoneurial fluid pressure. It recently was shown that local compression at 30 mm Hg for 8 h induces an increase in endoneurial fluid pressure (EFP) to about three times the original value and persisting at least 24 h (13). An increase in EFP has been interpreted as the probable cause of the nerve fiber lesions seen in different neuropathies ( 10, 18). There is evidence that the nerve fiber lesions in these cases are based on impaired capillary perfusion secondary to increased endoneurial fluid pressure (17), although it was suggested that a rise in EFP will not cause complete collapse
34
DAHLIN
ET
AL.
of the capillaries (9). However, one has to consider the special arrangement of the anastomoses between the epineurial and endoneurial vessels, often piercing the perineurium obliquely thereby constituting a possible valve mechanism when the EFP increases (11). An increase in EFP may also affect axonal transport due to the disturbances in microcirculation. An endonemial edema may also alter for example the local ionic balance around the nerve fibers, which may contribute to the inhibition of axonal transport and deterioration of nerve function. Deviation from isotonicity toward a hypo- or hypertonic milieu is known to inhibit partially axonal transport (4). Although it seems justified to assume that block of axonal transport induced by low pressure, i.e., 30 to 50 mm Hg, is caused by circulatory disturbances, one cannot disregard that some degree of mechanical nerve fiber deformation might contribute to blockage of axonal transport even at these low pressures (7, 23, 28). The direct mechanical deformation of nerve fibers is however probably more important when the nerves are subjected to compression at high pressures e.g., 200 to 400 mm Hg (28). Some experiments, at the 30 mm Hg pressure, showed a partial block of fast axonal transport. This may indicate that only part of the fibers are affected and could be explained on the basis of an incomplete &hernia of the compressed nerve segment at this pressure level (27). Further, the superficial nerve fibers in a nerve are considered to be more deformed during compression than the deeply situated fibers (30). Such a mechanism could allow axonal transport to continue undisturbed in the deeply situated fibers in the nerve trunk through the compressed nerve segment. Clinical Implications. Our findings are interesting considering recent investigations by Gelberman et al. (5) on patients with carpal tunnel syndrome. Those authors found that patients with the carpal tunnel syndrome had a pressure of about 32 mm Hg in the carpal tunnel with the wrist in neutral position. The mean pressure in an asymptomatic control group was 2.5 mm Hg. Intracarpal canal pressure has also been measured in one case with acute carpal tunnel syndrome occurring subsequent to a comminute fracture of the wrist (1). The carpal tunnel pressure, in neutral wrist position, was 34 mm Hg preoperatively and 10 mm Hg immediately after surgical decompression, compared with the mean pressure of 2 mm Hg in the control group. Furthermore, external pressure, experimentally applied to the median nerve in the carpal tunnel of human volunteers, induced mild neurophysiologic and clinical signs of nerve dysfunction including paresthesia in the hand when the pressure in the carpal canal was 30 mm Hg (12). Experimental elevation of the tissue fluid pressure to 30 mm Hg in muscle compartments in dogs was found to be the critical pressure at which the first signs of nerve dysfunction occurred ( 16).
NERVE
COMPRESSION
AND
AXONAL
TRANSPORT
35
Thus, experimental and clinical data indicate that pressures of about 30 mm Hg may occur in association with nerve entrapment syndromes and some acute nerve injuries. These pressures are high enough to all&t, e.g., axonal transport and intraneural blood flow. Long-standing compression of a peripheral nerve may, via reduced axonal transport, lead to changes in the structure and function of the distal part of the axon. Furthermore, the axons may thereby possibly become more sensitive to additional compression distally (32). REFERENCES I. BAUMAN, T. D., R. H. GELBERMAN, S. J. MUBARAK, AND S. R. GARFIN. 1981. The acute csrpal tunnel syndrome. Clin. Orthop. 156: 151-156. 2. BRADY, S. T., AND R. J. LASEK. 1982. The slow components of axonal transportz movements, compositions and organization. Pa8es 206-217 in D. G. WEISS,Ed., Axoplasmic Transport. Springer-Ver&, Berlin/Heidelberg. 3. DAHLIN, L. B., B. RYDEVIK, AND G. LUND~ORG. 1984. The biological basis of nerve entrapments and nerve compression injuries. In A. R. HARGENS, Ed., Ef&Ys of Mechanical Stress on Tissue Transport and Viability. Springer-Verla& New York, in press. 4. EDSTROM, A. 1975. Ionic requirements for rapid axonal transport in vitro in frog sciatic nerves. Acta Physiol. &and. 93: 104-I 12. 5. GELBERMAN, R. H., P. T. HERGENROEDER,A. R. HARGENS, G. LUNDBORG, AND W. H. &CESON. 1981. The carpal tunnel syndrome. A study of carpal canal pressure. J. Bone Joint Surg. 63A: 380-383. 6. GRA!=STEIN,B., AND D. S. FORMAN. 1980. Intmcellular transport in neurons. Physiol. Rev. 60: 1167-1283. 7. HAHNENBERGER,R. W. 1978. Effects of pressure on fast axoplasmic flow. An in vitro study in the vagus nerve of rabbits. Acta Physiol. Stand. 104: 299-308. 8. LEONE, J., AND S. GCHS. 1978. Anoxic block and recovery of axoplasmic tmnsport and electrical excitability of nerve. J. Neurobiol. 9: 229-245. 9. Low, P. A., P. J. DYCK, AND J. D. SCHMELZER. 1980. Mammalian peripheral nerve sheath has unique responses to chronic elevations of endoneurial fluid pressure. Exp. Neural. 70: 300-306. 10. Low, P. A., P. J. DYCK, AND J. D. SCHMELZER. 1982. Chronic elevation of endoneurial fluid pressure is associated with low-grade fiber pathology. Muscle Nerve 5: 162-165. 11. LUNDBORG, G. 1975. Structure and function of the intraneural microvessels as related to trauma, edema formation and nerve function. J. Bone Joint Surg. 57A: 938-948. 12. LUNDBORG, G., R. H. GELBERMAN, M. A. MINTEER-CONYERY, Y. F. LEE, AND A. R. HARGENS. 1982. Median nerve compression in the carpal tunnel--functional response to experimentally induced controlled pressure. J. Hand Surg. 7: 252-259. 13. LUNDLIORG, G., R. R. MYERS, AND H. C. POWELL. 1983. Nerve compression injury and increase in endonemial fluid pressure: A “miniature compartment syndrome.” .I. Neurol. Neurosurg. Psychiatry 46: 1119-I 124. 14. MCLEAN, W. G., M. FRIZELL, AND J. SJ&TRAND. 1976. Slow axonal transport of labeled proteins in sensory fibers of rabbit vagus nerve. .I. Neurochem. w 1213-1216. 15. MINCKLER, D. S., M. 0. M. Tso, AND L. E. ZIMMERMAN. 1976. A light micmscopic, autoradiographic study of sxoplasmic transport in the optic nerve head during ocular
36
16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
DAHLIN
ET
AL.
hypotony, increased intraocular pressure, and papilledema. Am. J. Ophthalmol. 82: 741757. MUBARAK, S. J., ANO A. R. HARGENS. 1981. Compartment Syndromes and Volkmanns Contracture. Saunders, Philadelphia/London/Toronto. MYERS, R. R., A. P. MIZIS~N, H. C. POWELL, AND P. W. LAMPERT. 1982. Reduced nerve blood flow in hexachlorophene neuropathy. Relationship to elevated endoneurial fluid pressure. J. Neuropathol. Exp. Neurol. 41: 391-399. MYERS, R. R., AND H. C. POWELL. 198 1. Endoneurird fluid pressure in peripheral neuropathies. Pages 193-207 in A. R. HARGENS, Ed., Tissue Fluid Pressure and Composition. Williams & Wilkins, Baltimore. GCHS, S. 1974. Energy metabolism and supply of -P to the fast axoplasmic transport mechanism in nerve. Fed. Proc. 33: 1049-1058. QCHS, S. 1975. Axoplasmic transport-a basis for neural pathology. Pages 213-230 in P. J. DYCK, P. K. THOMAS, AND E. H. LAMPERT, Eds., Peripheral Neuropathy, Vol. 1. Saunders, Philadelphia/London/Toronto. QCHS, S. 1982. Calcium and the mechanism of axoplasmic transport. Fed. Proc. 41: 23012306. GCHS, S., AND R. M. WORTH. 1978. Axoplasmic transport in normal and pathological systems.Pages 25 l-264 in S. G. WAXMAN, Ed., Physiology and Pathobiology of Axons. Raven Press, New York. QCHOA, J., T. J. FOWLER, AND R. W. GILLIATT. 1972. Anatomical changes in peripheral nerves compressed by a pneumatic tourniquet. J. Anat. 113: 433-455. QUIGLEY, H. A., AND D. R. PERSON. 1976. The dynamics and location of axonal transport blockade by acute intraocular pressure elevation in primate optic nerve. Invest. Ophthalmol. 15: 606-616. QUIGLEY, H. A., R. W. FLOWER, E. M. ADDICKS, AND D. S. MCLEOD. 1980. The mechanism of optic nerve damage in experimental acute intraocular pressure elevation. Invest. Ophthalmol. Vis. Sci. 19: 505-517. RYDEVIK, B., AND G. LUNDBORG. 1977. Permeability of intraneural microvessels and perineurium following acute, graded experimental nerve compression. Stand. J. Plast. Reconstr. Surg. 11: 179-187. RYDEVIK, B., G. LUNDBORG, AND U. BAGCE. 1981. Effects of graded compression on intraneural blood tlow. An in vivo study on rabbit tibial nerve. J. Hand Surg. 6: 3-12. RYDEVIK, B., W. G. MCLEAN, J. SJ~STRAND, AND G. LUNDBORG. 1980. Blockage of axonal transport induced by acute, graded compression of the rabbit vagus nerve. J. Neurol. Neurosurg.
Psychiatry
43: 690-698.
29. SJ&TRAND, J., M. FRIZELL, AND B. RYDEVIK. 1978. Changes in axonal transport in various experimental neuropathies. Pages 147-l 57 in N. CANAL AND G. POZZA, Eds., Peripheral Neuropathies, Vol. 1. Elsevier/North-Holland, Amsterdam. 30. SPINNER,M., AND P. S. SPENCER.1974. Nerve compression lesions of the upper extremity. A clinical and experimental review. Clin. Orthop. 104: 46-67. 31. SUNDERLAND,S. 1976. The nerve lesion in the carpal tunnel syndrome. J, Neural. Neurosurg. Psychiatry 39: 6 15-626. 32. UPTON, A. R. M., AND A. J. MCCOMAS. 1973. The double crush in nerve-entrapment syndromes. Lancet ii: 359-362.
NEUROLOGY
Changes
84,29-36
(1984)
in Fast Axonal Transport during Experimental Nerve Compression at Low Pressures
LARS B. DAHLIN,
BJ~RN RYDEVIK, W. GRAHAM AND JOHAN SJ~STBAND’
MCLEAN,
Laboratory of Experimental Biology, Department of Anatomy; Institute of Neurobiology, Department of Histology, University of Giiteborg; Department of Orthopaedic Surgery I; Department of Ophthalmology, Sahlgrens Hospital, Gothenburg, Sweden; and Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, United Kingdom Received April 27, 1983; revision received November 29, 1983 The minimal pressure for impairment of fast anterograde axonal transport was determined in rabbit vagus nerve. Proteins, transported by fast anterograde axonal transport, were labeled by a microinjection of [3H]1eucine into the nodose ganglion, and a small compression chamber was applied around the cervical vagus nerve. In this way the nerve was subjected to acute, graded compression. Compression at 20 mm Hg for 2 h as well as sham compression did not induce accumulation of axonally transported proteins at the level of compression. However, a pressure of 30 mm Hg for 2 h induced a block of axonal transport at the site of compression. The causes of the axonal transport block are discussed as well as the minimal pressure level in relation to pressures found in clinical nerve compression lesions. INTRODUCTION
The axonal transport systems transfer, for example, proteins and organelles from the nerve cell body to the axon terminals (anterograde axonal transport) as well as in the opposite direction (retrograde axonal transport). The anterograde transport includes slow components (0.1 to 8 and 25 mm/day) and fast components (34 to 68 and 240 to 400 mm/day) (2, 6, 14). The latter are known to be blocked by &hernia and compression (7, 8, 28) as well as Abbreviations: TCA-trichloroacetic acid, EFP-endoneurial fluid pressure. ’ This work was supported by grants from the Swedish Medical Research Council (5 188 and 2226), the Giiteborg Medical Society, the Swedish Work Environment Fund, and the University of Giiteborg, Sweden. Please send correspondence to Lam B. Dahlin, M.D., Laboratory of Experimental Biology, Department of Anatomy, University of Giiteborg, Box 33031, S-400 33 Giitehorg, Sweden. 29 0014-4886/84 $3.00 Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any form lcsened
30
DAHLIN
ET
AL.
by toxic substances (29). Different experimental methods have been described to study the impairment of axonal transport induced by local compression, for example by tourniquet and in vitro compression (7, 8, 20). Rydevik et al. (28) demonstrated that fast anterograde axonal transport in the rabbit vagus nerve was blocked by local compression in vivo at 50 mm Hg for 2 h, and this block was reversible within 24 h. Blockage of axonal transport in optic nerves of monkeys has also been induced by experimental elevation of intraocular pressure (15, 24, 25). Block of axonal transport may be one of several important factors contributing to the development of nerve dysfunction in compression lesions of peripheral nerves, nerve roots, or optic nerves. The pathophysiology of compression-induced nerve lesions, including mechanisms behind, and consequences of, blockage of axonal transport, is not fully clarified. However, both &hernia and mechanical deformation of nerve fibers are believed to be of importance (3, 23, 26, 31). Our purpose was to identify the minimal pressure necessary for blockage of fast anterograde axonal transport in a defined experimental model. METHODS The method of application of pressure to the vagus nerve and the method of measuring axonal transport were identical to those used previously by Rydevik et al. (28). Eighteen albino rabbits of either sex, weighing 2.0 to 2.5 kg, were anesthetized by an i.m. injection of 0.75 ml/kg body weight of Hypnorm (fluanisone 10 mg and fentanyl 0.2 mg/ml) followed by 1 mg/kg Valium (diazepam 5 mg/ml). Anesthesia was maintained with 0.25 ml/kg bodyweight Hypnorm at 30-min intervals. The fast axonal transport of 3H-labeled proteins in the sensory fibers of the vagus nerve was measured in the following way. The nodose ganglion was carefully exposed and 100 &i [3H]leucine (L-4,5[3H]leucine, 53 Ci/ mmol, Radiochemical Centre, Amersham, England) in 20 ~10.9% NaCl were injected subepineurially into the nodose ganglion via a 30-gauge stainlesssteel needle. Two hours later a 30 to 40-mm length of the vagus nerve was exposed in the region 10 to 50 mm from the nodose ganglion and a small compression chamber, consisting of two Plexiglas halves onto which thin rubber membranes were glued, was applied around the nerve. The compressed area was 10 mm. The chamber was secured around the nerve in such a way that the nerve could be subjected to acute, graded compression by inflation of the rubber membranes. The surrounding muscle and skin were loosely sutured in place and the chamber was then inflated with air at a known constant pressure. This pressure was automatically maintained at the desired
NERVE
COMPRESSION
AND
AXONAL
TRANSPORT
31
level throughout the experiments by a special ah-pump which compensated for any leakage in the system. In five sham experiments, the chamber was applied around the nerve trunk but was not inflated. In the rmnaining experiments the nerves were compressed either at 20 (N = 5) or 30 mm Hg (N = 8) for 2 h. At the end of the 2-h period, the animals were killed by an overdose of barbiturate and the vagus nerve and the nodose ganglion were quickly removed and transferred to ice The nerve was then cut into 2.5mm pieces and the pieces were soaked 24 h individually in 2 ml 10% trichloroacetic acid (TCA) at 4°C. The TCA was discarded and the nerve pieces washed in a further 2 ml cold TCA and dissolved overnight at room temperature in 0.5 ml Soluene (Packard). Radioactivity in the dissolved nerve pieces was measured in 2 ml Permablend (Packard) scintillation fluid in a Packard Tricarb liquid scintillation counter with automatic quench correction. RESULTS The TCA-insoluble radioactivity in each nerve piece was plotted for each nerve against the distance of the piece from the nodose ganglion (Fig. 1). Previous studies showed that the prolile of radiolabeled proteins in untreated nerves exhibited a front of radiolabeled proteins about 60 mm from the ganglion, indicating a rate of fast axonal transport greater than 360 mm/day (14, 28). In five nerves subjected to sham compression there was no difference in the prolile from that in untreated nerves (Fig. la). Therefore, the application of the compression chamber per se did not lead to any block of axonal transport. In five nerves subjected to compression at 20 mm Hg for 2 h, there was again no difference in the profile from that in untreated or shamcompressed nerves (Fig. 1b). Three of eight nerves subjected to a compression of 30 mm Hg for 2 h showed a profile of radioactive proteins as seen in Fig. 1c. In these cases there was increased radioactivity in those pieces immediately proximal to the region of the nerve to which the compression chamber was applied. There was, however, also a signi@cant wave of radioactive proteins the front of which had moved to the same position as that in untreated nerves. Thus the compression in those nerves produced a partial inhibition of axonal transport. One of the eight nerves compressed at 30 mm Hg showed no block of axonal transport. The remaining four nerves compressed at this pressure showed a more complete inhibition of axonal transport, in which there were only very low amounts of radioactive proteins distal to the site of the compression as seen in Fig. Id. The results are summarized in Table 1.
DAHLIN
32
ET AL.
c,
mm
from
nodose
ganglo~n
mm
30mm 2h acute
tlg
‘mm
nodose
ganghon
FIG. 1. Summary of the typical findings. The applied pressure and the time of compression are shown in each diagram. The black bar indicates the site of the compression chamber. Shamcompression (a) as well as compression at 20 mm Hg (b) induced no block of axonal transport. In some of the nerves compressed by 30 mm Hg a wave of radioactive proteins was seen distal to the accumulation at the compression cuff, i.e., the block was partial (c). In other experiments, compression at 30 mm Hg induced a more complete block (d).
DISCUSSION This study showed that the minimal pressure that blocks the fast anterograde axonal transport in sensory fibers of rabbit vagus nerves is 30 mm Hg applied for 2 h. Consistently, compression at 20 mm Hg for 2 h did not impair the fast axonal transport. It is of course possible that a lower pressure may inhibit fast axonal transport when applied for longer than 2 h or over a longer segment of nerve. The exact mechanism behind accumulation of axomdly transported material induced by 30 mm Hg is not known. Generally, axonal transport block induced by compression may be an effect of(i) ischemia and/or (ii) mechanical deformation of axons. The details of the basic mechanisms behind normal axonal transport were extensively investigated by Ochs and co-workers (19-
NERVE COMPRESSION
AND AXONAL
33
TRANSPORT
TABLE I Effect of Compression on Fast Axonal Transport in Vagus Nerve of Rabbits PressUE
No block
Partial block
Complete block
$5)
5
0
0
(N2f 5)
5
0
0
(N3i 8)
1
3
4
(mm
Nz)
22). Leone and Ochs (8) studied the impairment of axonal transport in cat sciatic nerves induced by anoxia in vitro and by a tourniquet in vivo. Those authors demonstrated that axonal transport is blocked within 15 min of onset of anoxia in vitro and by compression at about 300 mm Hg in vim, induced by tourniquets. After 1.5 h of anoxia there was a good recovery but with clear evidence of failure of recovery after 2 h of anoxia. The block induced by tourniquet-compression gradually recovered if sufficient recovery time was allowed and if the compression time had not exceeded 6 to 7 h. They suggested that ischemia was the major cause of the block. It seems reasonable to assume that the circulatory disturbances in a nerve segment subjected to compression at low levels, i.e., 30 to 50 mm Hg, play an important role in blocking axonal transport (28). Direct compression of a rabbit tibiaI nerve at 20 to 30 mm Hg induces a marked reduction of intraneural venular blood flow and the compressed nerve segment is completely ischemic at 60 to 80 mm Hg cuff pressure (27). Disturbances in venular blood flow may give retrograde effects on the capillary flow in the endoneurial space and can induce endoneurial edema formation (3 1). Due to factors such as the unyielding properties of the perineurium and the lack of lymphatic vessels in the endoneurial space, an endoneurial edema will lead to an increase of the endoneurial fluid pressure. It recently was shown that local compression at 30 mm Hg for 8 h induces an increase in endoneurial fluid pressure (EFP) to about three times the original value and persisting at least 24 h (13). An increase in EFP has been interpreted as the probable cause of the nerve fiber lesions seen in different neuropathies ( 10, 18). There is evidence that the nerve fiber lesions in these cases are based on impaired capillary perfusion secondary to increased endoneurial fluid pressure (17), although it was suggested that a rise in EFP will not cause complete collapse
34
DAHLIN
ET
AL.
of the capillaries (9). However, one has to consider the special arrangement of the anastomoses between the epineurial and endoneurial vessels, often piercing the perineurium obliquely thereby constituting a possible valve mechanism when the EFP increases (11). An increase in EFP may also affect axonal transport due to the disturbances in microcirculation. An endonemial edema may also alter for example the local ionic balance around the nerve fibers, which may contribute to the inhibition of axonal transport and deterioration of nerve function. Deviation from isotonicity toward a hypo- or hypertonic milieu is known to inhibit partially axonal transport (4). Although it seems justified to assume that block of axonal transport induced by low pressure, i.e., 30 to 50 mm Hg, is caused by circulatory disturbances, one cannot disregard that some degree of mechanical nerve fiber deformation might contribute to blockage of axonal transport even at these low pressures (7, 23, 28). The direct mechanical deformation of nerve fibers is however probably more important when the nerves are subjected to compression at high pressures e.g., 200 to 400 mm Hg (28). Some experiments, at the 30 mm Hg pressure, showed a partial block of fast axonal transport. This may indicate that only part of the fibers are affected and could be explained on the basis of an incomplete &hernia of the compressed nerve segment at this pressure level (27). Further, the superficial nerve fibers in a nerve are considered to be more deformed during compression than the deeply situated fibers (30). Such a mechanism could allow axonal transport to continue undisturbed in the deeply situated fibers in the nerve trunk through the compressed nerve segment. Clinical Implications. Our findings are interesting considering recent investigations by Gelberman et al. (5) on patients with carpal tunnel syndrome. Those authors found that patients with the carpal tunnel syndrome had a pressure of about 32 mm Hg in the carpal tunnel with the wrist in neutral position. The mean pressure in an asymptomatic control group was 2.5 mm Hg. Intracarpal canal pressure has also been measured in one case with acute carpal tunnel syndrome occurring subsequent to a comminute fracture of the wrist (1). The carpal tunnel pressure, in neutral wrist position, was 34 mm Hg preoperatively and 10 mm Hg immediately after surgical decompression, compared with the mean pressure of 2 mm Hg in the control group. Furthermore, external pressure, experimentally applied to the median nerve in the carpal tunnel of human volunteers, induced mild neurophysiologic and clinical signs of nerve dysfunction including paresthesia in the hand when the pressure in the carpal canal was 30 mm Hg (12). Experimental elevation of the tissue fluid pressure to 30 mm Hg in muscle compartments in dogs was found to be the critical pressure at which the first signs of nerve dysfunction occurred ( 16).
NERVE
COMPRESSION
AND
AXONAL
TRANSPORT
35
Thus, experimental and clinical data indicate that pressures of about 30 mm Hg may occur in association with nerve entrapment syndromes and some acute nerve injuries. These pressures are high enough to all&t, e.g., axonal transport and intraneural blood flow. Long-standing compression of a peripheral nerve may, via reduced axonal transport, lead to changes in the structure and function of the distal part of the axon. Furthermore, the axons may thereby possibly become more sensitive to additional compression distally (32). REFERENCES I. BAUMAN, T. D., R. H. GELBERMAN, S. J. MUBARAK, AND S. R. GARFIN. 1981. The acute csrpal tunnel syndrome. Clin. Orthop. 156: 151-156. 2. BRADY, S. T., AND R. J. LASEK. 1982. The slow components of axonal transportz movements, compositions and organization. Pa8es 206-217 in D. G. WEISS,Ed., Axoplasmic Transport. Springer-Ver&, Berlin/Heidelberg. 3. DAHLIN, L. B., B. RYDEVIK, AND G. LUND~ORG. 1984. The biological basis of nerve entrapments and nerve compression injuries. In A. R. HARGENS, Ed., Ef&Ys of Mechanical Stress on Tissue Transport and Viability. Springer-Verla& New York, in press. 4. EDSTROM, A. 1975. Ionic requirements for rapid axonal transport in vitro in frog sciatic nerves. Acta Physiol. &and. 93: 104-I 12. 5. GELBERMAN, R. H., P. T. HERGENROEDER,A. R. HARGENS, G. LUNDBORG, AND W. H. &CESON. 1981. The carpal tunnel syndrome. A study of carpal canal pressure. J. Bone Joint Surg. 63A: 380-383. 6. GRA!=STEIN,B., AND D. S. FORMAN. 1980. Intmcellular transport in neurons. Physiol. Rev. 60: 1167-1283. 7. HAHNENBERGER,R. W. 1978. Effects of pressure on fast axoplasmic flow. An in vitro study in the vagus nerve of rabbits. Acta Physiol. Stand. 104: 299-308. 8. LEONE, J., AND S. GCHS. 1978. Anoxic block and recovery of axoplasmic tmnsport and electrical excitability of nerve. J. Neurobiol. 9: 229-245. 9. Low, P. A., P. J. DYCK, AND J. D. SCHMELZER. 1980. Mammalian peripheral nerve sheath has unique responses to chronic elevations of endoneurial fluid pressure. Exp. Neural. 70: 300-306. 10. Low, P. A., P. J. DYCK, AND J. D. SCHMELZER. 1982. Chronic elevation of endoneurial fluid pressure is associated with low-grade fiber pathology. Muscle Nerve 5: 162-165. 11. LUNDBORG, G. 1975. Structure and function of the intraneural microvessels as related to trauma, edema formation and nerve function. J. Bone Joint Surg. 57A: 938-948. 12. LUNDBORG, G., R. H. GELBERMAN, M. A. MINTEER-CONYERY, Y. F. LEE, AND A. R. HARGENS. 1982. Median nerve compression in the carpal tunnel--functional response to experimentally induced controlled pressure. J. Hand Surg. 7: 252-259. 13. LUNDLIORG, G., R. R. MYERS, AND H. C. POWELL. 1983. Nerve compression injury and increase in endonemial fluid pressure: A “miniature compartment syndrome.” .I. Neurol. Neurosurg. Psychiatry 46: 1119-I 124. 14. MCLEAN, W. G., M. FRIZELL, AND J. SJ&TRAND. 1976. Slow axonal transport of labeled proteins in sensory fibers of rabbit vagus nerve. .I. Neurochem. w 1213-1216. 15. MINCKLER, D. S., M. 0. M. Tso, AND L. E. ZIMMERMAN. 1976. A light micmscopic, autoradiographic study of sxoplasmic transport in the optic nerve head during ocular
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hypotony, increased intraocular pressure, and papilledema. Am. J. Ophthalmol. 82: 741757. MUBARAK, S. J., ANO A. R. HARGENS. 1981. Compartment Syndromes and Volkmanns Contracture. Saunders, Philadelphia/London/Toronto. MYERS, R. R., A. P. MIZIS~N, H. C. POWELL, AND P. W. LAMPERT. 1982. Reduced nerve blood flow in hexachlorophene neuropathy. Relationship to elevated endoneurial fluid pressure. J. Neuropathol. Exp. Neurol. 41: 391-399. MYERS, R. R., AND H. C. POWELL. 198 1. Endoneurird fluid pressure in peripheral neuropathies. Pages 193-207 in A. R. HARGENS, Ed., Tissue Fluid Pressure and Composition. Williams & Wilkins, Baltimore. GCHS, S. 1974. Energy metabolism and supply of -P to the fast axoplasmic transport mechanism in nerve. Fed. Proc. 33: 1049-1058. QCHS, S. 1975. Axoplasmic transport-a basis for neural pathology. Pages 213-230 in P. J. DYCK, P. K. THOMAS, AND E. H. LAMPERT, Eds., Peripheral Neuropathy, Vol. 1. Saunders, Philadelphia/London/Toronto. QCHS, S. 1982. Calcium and the mechanism of axoplasmic transport. Fed. Proc. 41: 23012306. GCHS, S., AND R. M. WORTH. 1978. Axoplasmic transport in normal and pathological systems.Pages 25 l-264 in S. G. WAXMAN, Ed., Physiology and Pathobiology of Axons. Raven Press, New York. QCHOA, J., T. J. FOWLER, AND R. W. GILLIATT. 1972. Anatomical changes in peripheral nerves compressed by a pneumatic tourniquet. J. Anat. 113: 433-455. QUIGLEY, H. A., AND D. R. PERSON. 1976. The dynamics and location of axonal transport blockade by acute intraocular pressure elevation in primate optic nerve. Invest. Ophthalmol. 15: 606-616. QUIGLEY, H. A., R. W. FLOWER, E. M. ADDICKS, AND D. S. MCLEOD. 1980. The mechanism of optic nerve damage in experimental acute intraocular pressure elevation. Invest. Ophthalmol. Vis. Sci. 19: 505-517. RYDEVIK, B., AND G. LUNDBORG. 1977. Permeability of intraneural microvessels and perineurium following acute, graded experimental nerve compression. Stand. J. Plast. Reconstr. Surg. 11: 179-187. RYDEVIK, B., G. LUNDBORG, AND U. BAGCE. 1981. Effects of graded compression on intraneural blood tlow. An in vivo study on rabbit tibial nerve. J. Hand Surg. 6: 3-12. RYDEVIK, B., W. G. MCLEAN, J. SJ~STRAND, AND G. LUNDBORG. 1980. Blockage of axonal transport induced by acute, graded compression of the rabbit vagus nerve. J. Neurol. Neurosurg.
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29. SJ&TRAND, J., M. FRIZELL, AND B. RYDEVIK. 1978. Changes in axonal transport in various experimental neuropathies. Pages 147-l 57 in N. CANAL AND G. POZZA, Eds., Peripheral Neuropathies, Vol. 1. Elsevier/North-Holland, Amsterdam. 30. SPINNER,M., AND P. S. SPENCER.1974. Nerve compression lesions of the upper extremity. A clinical and experimental review. Clin. Orthop. 104: 46-67. 31. SUNDERLAND,S. 1976. The nerve lesion in the carpal tunnel syndrome. J, Neural. Neurosurg. Psychiatry 39: 6 15-626. 32. UPTON, A. R. M., AND A. J. MCCOMAS. 1973. The double crush in nerve-entrapment syndromes. Lancet ii: 359-362.