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Electrical stimulation of nerves and their regeneration
121
Bimlectrochemistry and Bioenergetics, 29 (1992) 121-126 Elsevier Sequoia CA., Lausanne
JEC BB 01549 Short communication
Electrical stimulation
of nerves and their regeneration
Betty F. Sisken Center for Biomedical Engineering and Department of Anatomy and Neurobiology, Universiry of Kentucky, Lexington, KY40506 WSA) (Received 20 February 1992)
INTRODUCTION
Historically, electric and electromagnetic fields (EMFs) were employed to speed recovery of hard tissue injuries and non-unions [l-3]. Skin contact electrodes or surgically implanted electrodes were used to administer the current. The use of Helmholtz coils to induce electromagnetic fields for healing represents a more recent technology. The primary advantage of this technology is that it is a non-invasive technique that can easily be adapted for treatment of soft tissue injuries. Most of the electric modalities relating to soft tissue healing discussed below have been tested on various tissue culture and animal preparations [31. This paper presents results of some of the more recent research on nerve tissue injuries in which electric and EMFs have been used to stimulate the healing process. Peripheral nerve regeneration Peripheral nerve regeneration is a complex series of processes involving the nerve cell body, its axonal fibres and the milieu through which it grows. For nerve regeneration to be successful, a number of events must occur: the cell body must recover from the initial trauma, the axonal fibres regrow through and beyond the site of injury, and finally, the regrown fibres must form synaptic contacts with the end or target tissue. Regeneration of peripheral nerves following a crush lesion occurs more rapidly than after a transection injury. Although the same types of problem exist in both situations, the cell body and its distal axonal fibres remain in continuity after a crush. When a transection (axotomy) occurs, distal segments of the nerve are completely isolated from the cell body, and therefore undergo extensive degeneration. Attempts to perform surgical reapproximation of the cut 0302-4598/92/$05.00
0 1992 - Elsevier Sequoia S.A. All rights reserved
122
stumps to exactly realign the fibres is at best partial and often results in a mismatch so that the proximal segments may eventually innervate inappropriate targets. Furthermore, the time needed for regeneration of the axons (l-3 mm/day) into the empty tubular sheaths is relatively long so that atrophy of the target tissue occurs. Regeneration after a crush lesion occurs at a faster rate (3-4 mm/day) since the sheath of the fibres, although crushed, recovers and the axoplasm of the proximal segment regenerates through the original channel; in addition, regrowth to appropriate targets is more precise than after the more severe transection injury. Both electric. and electromagnetic fields have been applied to either transected or crushed peripheral nerve models in an attempt to accelerate regeneration. BASIC STUDIES
Weak dc fields
Winter et al. [4] reported that insertion of Pt/Ag bimetallic electrodes (100 nA) intraluminally stimulated regeneration of transected sciatic nerves. Regeneration was determined by analyzing the compound action potential; only when the cathode was present distally was the dc effective. Studies by other investigators [5-g] using dc fields all report promotion of nerve regeneration using various metal or wick electrodes. An in-depth analysis after applying steady dc electric fields (1.5 mV/mm) to transected rat sciatic nerves using battery implants and wick electrodes was reported by Kerns et al. [9]. These fields had a stimulatory effect on early events associated with regenerating axons and growth cones but no effect on walking behaviour. Kerns et al. [lo] delivered 0.6 PA current with silver/silver chloride fitted into tubing containing a saline-soaked cotton thread to a transection lesion with the negative electrode 5 mm distal to the injury site. One week later a circularly vibrating probe was used to measure the current density along the untreated transected and treated transected nerves. There was a marked increase (69%) in the regenerated distance of the axons on the treated side relative to the untreated side. These results with the vibrating probe were substantiated by electron microscopic observations. Recently, Campbell and Pomeranz [ 111 have found a difference in the response of young and old rats to 10 PA dc applied to a crushed nerve; the direct current did not affect regeneration in young animals but did increase the rate of regeneration in old rats. McGiniss [12] performed somewhat similar experiments to those above and found no difference in the extent of regeneration between dc-treated and untreated sciatic nerve preparations assessed functionally (toe spread reflex) and morphologically (numbers of myelinated and unmyelinated axons). The reasons for the discrepancy between results found by McGiniss and those reported by other laboratories is unknown. Finally, Aebischer et al. [13] employed piezoelectricity using poled polyvinylidene fluoride tubes to accelerate regeneration. The tubes bridged proximal and distal stumps of axotomized nerves and the number of myelinated axons that
123
crossed the gap were compared in control and experimental groups. The increase found in the poled tubes was significant at both 4 and 12 weeks following transection. New studies from his laboratory where these channels were seeded with peripheral glia cells indicate a favourable influence on regeneration of sciatic nerves across a transected gap [14]. Electromagnetic fields One of the early studies was by Wilson and Jagadeesh [151 who explored PEMF effects on nerve and spinal cord regeneration using an RF signal (Diapulse, 5-120 mW/cm*). The transected median-ulnar nerves of rats were treated for 15 min/day for 30-60 days. At 30 days, PEMF-treated animals showed significant restoration .of nerve conduction activity and the presence of large diameter nerve fibres. Ito and Bassett [16] subjected the whole body of rats to pulsed electromagnetic fields after transection of the sciatic nerve. The PEMF was generated in a pair of Helmholtz coils; the signal is used clinically for bone repair (ElectroBiology, Inc.). All rats were treated for 12 h/day with motor function evaluated by a plantar-flexion force produced by stimulation of the nerve proximally. Relative to control animals, motor function with PEMF treatment was restored at the four-week time period rather than at eight weeks. Raji and Bowden [17] extended Wilson’s experiments using the Diapulse machine (RF of lop3 W/cm*) on regeneration of the transected common peroneal nerve in rats. PEMF was administered for 15 min daily for periods of three days to eight weeks. Significant increases in skin, deep-tissue and rectal temperatures occurred; these returned to normal after treatment. The size of the intraneural blood vessels was increased after treatment and the amount of collagenous tissue fibrosis reduced as well as acceleration of regeneration of nerve fibres. Parker et al. [lS] used a different signal on transected rate sciatic nerves; rats were treated with a pulse burst signal (15 Hz, Electrobiology, Inc.) for five days following transection. Treated animals exhibited a faster return of function (compound action potential) and more myelinated axons/mm* in the nerve distal to the transection than untreated control animals. PEMF effects on regeneration of the common peroneal nerve of the cat was assessed by Orgel et al. [19]. Five days after transection, the cats were exposed to PEMF for 10 h/day, 6 days/week for twelve weeks. This study tested two different PEMF signals: a 15 Hz pulse burst signal and a single repetitive pulse (72 Hz). Electrophysiologic data were collected pre- and post-operatively. The nerves were biopsied for fibre counts, and retrograde transport of horseradish peroxidase to the motor neurons in the spinal cord were used for assessing regenerative events. No significant differences were noted between controls and either PEMF signal in: numbers of fibres/mm*, axon fibre calibre, areas of nerve compound action potential or muscle compound action potential. However, the numbers of motor neurons retrogradely labelled in spinal cords of cats treated with the pulse burst
124
signal (15 Hz) were increased significantly (96.8% of the unoperated side) relative to untreated animals. Using a crushed nerve model we reported [20] a significant increase in the rate of peripheral nerve regeneration in animals treated with pulsed electromagnetic fields (PEMF 2 Hz, 3 g), for 4 h/day, for three, four or six days. Extrapolation of the regeneration distance vs. time to zero yielded an estimate of the initial delay period of one to two days due to axonal die-back after injury. We found that this period was not influenced by EMF. The apparent lack of EMF influence on the first or second day may be explained by one of two ways: the fields might not affect cells at the injury site, therefore no changes were seen in the initial delay period or the fields affected the cells in ways not observed or tested thus far. Nevertheless the rate of regeneration after the initial period was enhanced by the EM fields. In addition we found that this response was independent of the orientation of the animal to the Helmholtz coils. More importantly, pre-treatment of the animals before lesioning evoked the same degree of augmented response as that observed when the animal was treated after lesioning [21]. Sintioidal
fields
Sinusoidal fields (50 Hz, 0.2 and 0.4 mT) have been tested on the same crushed nerve model [221. The mechanisms underlying these effects may be interpreted more easily since the signal is much simpler to dissect mathematically. Although the same degree of increase in regeneration rate was obtained with these fields (21%) as compared with that with 2 Hz PEMF, the sinusoidal stimulation apparently affected the early time periods of nerve regeneration, that associated with growth cone formation and axonal sprouting. A study by Zienowicz et al. [23] using the 2 Hz, 3 G PEMF signal (Bietic Research, Lyndhurst, NJ) tested a transection model of the sciatic nerve of adult rats. Immediately, or after a five-day interval following transection and surgical repair, the animals were exposed (whole body) to PEMF. Functional return was assessed using the sciatic function walking test. In the group in which surgical repair and PEMF treatment were delayed for the first five days, significant improvement in gait was found starting at the 140 day time point and continued to increase relative to other groups to the end of the experiment (165 days). Static field3
Cordiero et al. [24] investigated the effects of high-intensity static magnetic fields (1 T) on nerve regeneration. They found no effects of the static fields on the regenerative process when determining axonal counts and neurophysiologic function.
The guinea pig superior cervical ganglion was investigated by Maehlen and Nja [25] looking at the effects of electrical stimulation of pre- and postsynaptic cells on
125
sprouting after denervation. Preganglionic stimulation on the cervical sympathetic trunk for 1 h immediately after denervation (100 pulses at 20 Hz) increased the number of axons innervating each ganglion cell. This effect was abolished with a ganglionic blocker (hexamethonium), indicating modulation by impulse activity of retrograde transsynaptic trophic effects. Chronic electrical stimulation of axotomized sciatic nerves of rabbits with 10 Hz continuous or intermittent stimulation at 100 Hz with a 20% duty cycle for 8 h/day for periods up to 230 days was reported by Gordon et al. [26]. This study indicated that threshold and suprathreshold stimulation of the nerves (via a cuff around portions of the nerve proximal to the transection site) reduced the atrophy of nerve fibres initially, but was damaging over a long period of time. Basic mechanisms
Despite the profusion of studies on electrical stimulation of tissue culture preparations, animal studies and clinical trials, no unifying mechanism that encompasses both electric and electromagnetic fields has been proposed and accepted to explain how electrical stimulation produces its effects, whether it be soft or hard tissue. Once such a mechanism has been revealed, questions concerning specific electrical modality (faradic, capacitive, inductive) and electrical parameters (frequency, intensity, etc.) can be addressed directly. ACKNOWLEDGEMENT
This study was supported in part by the Office of Naval Research, NOOO14-86-K0221 and NIH NS29621.
No.
REFERENCES 1 C.A.L. Bassett, R.J. Pawluck and R.O. Becker, Nature (London), 204 (1964) 652. 2 C. Brighton, J.E. Cronkey and A.L. Osterman, Clin. Orthop., 161 (19811 122. 3 J. Black, Electrical Stimulation: Its Role in Growth, Repair and Remodeling of the Musculoskeletal System, Praeger, New York, 1987. 4 W.G. Winter, R.C. Schutt, B.F. Sisken and SD. Smith, Trans. Orthop. Res. Sot., 6 (1981) 304. 5 B. Pomepanz, M. Mullen and H. Markus, Brain Res., 303 (1984) 331. 6 G.C. Roman, H.K Strahlendorf, P.W. Coates and B.A. Rowley, Exp. Neural., 98 (1987) 222. 7 R.B. Borgens, Prog. Clin. Biol. Res., 10 (1986) 239. 8 M.J. Pohtis, M.F. Zanakis, and B.J. Albala, J. Trauma, 28 (1988) 1375. 9 J.M. Kerns, I.M. Pavkovic, A.J. Fakhouri, K.L. Wickersham and J.A. Freeman, 19 (1987) 217. 10 J.M. Kerns, A.J. Fakhouri, H.P. Weinrib and J.A. Freeman, Accepted Neuroscience, 1990. 11 J.J. Campbell and B. Pomeranz, Sot. Neurosci., 16 (1990) 1156. 12 M.E. McGiniss, Sot. Neurosci., 16 (1990) 1156. 13 P. Aebischer, P.D. Valentini, C. Domenici and P.M. Galletti, Brain Res., 436 (1987) 165. 14 Y. Guenard, T.K. Morrisey, N. Kielman, R.P. Bunge and P. Aebischer, Sot. Neurosci., 17 (1991) 585. 15 D.H. Wilson and P. Jagadeesh, Paraplegia, 14 (1976) 12. 16 H. Ito and C.A.L. Bassett, Clin. Orthop. Relat. Res., 181 (1983) 283.
126 17 A.R. Raji and R.E.M. Bowden, J. Bone Joint Surgery, 65 (1983) 478. 18 B. Parker, C. Bryant, J. Apesos, B.F. Sisken and T. Nickel, Trans. Bioelectr. Repair Growth Sot., 3 (1983) 19. 19 M.G. Orgel, W.J. O’Brien and H.M. Murray, Plastic Reconstruct. Surg., 73 (1984) 173. 20 B.F. Sisken, M. Kanje, G. Lundborg, E. Herbst and W. Kurtz, Brain Res., 485 (1989) 309. 21 B.F. Sisken, M. Kanje, G. Lundborg and W. Kurtz, Restor. Neurosci., 1 (1990) 303. 22 A. Rusovan and M. Kanje, Exp. Neur., 112 (1991) 312. 23 R.J. Zienowicz, B.A. Thomas, W.H. Kurtz and M.G. Orgel, Plast. Reconstr. Surg., 87 (1991) 122-129. 24 P. Cordiero, B.R. Seckel, C.D. Miller, P.T. Gross and R.E. Wise, Plast. Reconstr. Surg., 83 (1989) 301. 25 J. Maehlan and A. Nja, J. Physiol., 322 (1982) 151. 26 T. Gordon, J. Gillespie, R. Orozco and L. Davis, J. Neurosci., 11 (1991) 2157.
Bimlectrochemistry and Bioenergetics, 29 (1992) 121-126 Elsevier Sequoia CA., Lausanne
JEC BB 01549 Short communication
Electrical stimulation
of nerves and their regeneration
Betty F. Sisken Center for Biomedical Engineering and Department of Anatomy and Neurobiology, Universiry of Kentucky, Lexington, KY40506 WSA) (Received 20 February 1992)
INTRODUCTION
Historically, electric and electromagnetic fields (EMFs) were employed to speed recovery of hard tissue injuries and non-unions [l-3]. Skin contact electrodes or surgically implanted electrodes were used to administer the current. The use of Helmholtz coils to induce electromagnetic fields for healing represents a more recent technology. The primary advantage of this technology is that it is a non-invasive technique that can easily be adapted for treatment of soft tissue injuries. Most of the electric modalities relating to soft tissue healing discussed below have been tested on various tissue culture and animal preparations [31. This paper presents results of some of the more recent research on nerve tissue injuries in which electric and EMFs have been used to stimulate the healing process. Peripheral nerve regeneration Peripheral nerve regeneration is a complex series of processes involving the nerve cell body, its axonal fibres and the milieu through which it grows. For nerve regeneration to be successful, a number of events must occur: the cell body must recover from the initial trauma, the axonal fibres regrow through and beyond the site of injury, and finally, the regrown fibres must form synaptic contacts with the end or target tissue. Regeneration of peripheral nerves following a crush lesion occurs more rapidly than after a transection injury. Although the same types of problem exist in both situations, the cell body and its distal axonal fibres remain in continuity after a crush. When a transection (axotomy) occurs, distal segments of the nerve are completely isolated from the cell body, and therefore undergo extensive degeneration. Attempts to perform surgical reapproximation of the cut 0302-4598/92/$05.00
0 1992 - Elsevier Sequoia S.A. All rights reserved
122
stumps to exactly realign the fibres is at best partial and often results in a mismatch so that the proximal segments may eventually innervate inappropriate targets. Furthermore, the time needed for regeneration of the axons (l-3 mm/day) into the empty tubular sheaths is relatively long so that atrophy of the target tissue occurs. Regeneration after a crush lesion occurs at a faster rate (3-4 mm/day) since the sheath of the fibres, although crushed, recovers and the axoplasm of the proximal segment regenerates through the original channel; in addition, regrowth to appropriate targets is more precise than after the more severe transection injury. Both electric. and electromagnetic fields have been applied to either transected or crushed peripheral nerve models in an attempt to accelerate regeneration. BASIC STUDIES
Weak dc fields
Winter et al. [4] reported that insertion of Pt/Ag bimetallic electrodes (100 nA) intraluminally stimulated regeneration of transected sciatic nerves. Regeneration was determined by analyzing the compound action potential; only when the cathode was present distally was the dc effective. Studies by other investigators [5-g] using dc fields all report promotion of nerve regeneration using various metal or wick electrodes. An in-depth analysis after applying steady dc electric fields (1.5 mV/mm) to transected rat sciatic nerves using battery implants and wick electrodes was reported by Kerns et al. [9]. These fields had a stimulatory effect on early events associated with regenerating axons and growth cones but no effect on walking behaviour. Kerns et al. [lo] delivered 0.6 PA current with silver/silver chloride fitted into tubing containing a saline-soaked cotton thread to a transection lesion with the negative electrode 5 mm distal to the injury site. One week later a circularly vibrating probe was used to measure the current density along the untreated transected and treated transected nerves. There was a marked increase (69%) in the regenerated distance of the axons on the treated side relative to the untreated side. These results with the vibrating probe were substantiated by electron microscopic observations. Recently, Campbell and Pomeranz [ 111 have found a difference in the response of young and old rats to 10 PA dc applied to a crushed nerve; the direct current did not affect regeneration in young animals but did increase the rate of regeneration in old rats. McGiniss [12] performed somewhat similar experiments to those above and found no difference in the extent of regeneration between dc-treated and untreated sciatic nerve preparations assessed functionally (toe spread reflex) and morphologically (numbers of myelinated and unmyelinated axons). The reasons for the discrepancy between results found by McGiniss and those reported by other laboratories is unknown. Finally, Aebischer et al. [13] employed piezoelectricity using poled polyvinylidene fluoride tubes to accelerate regeneration. The tubes bridged proximal and distal stumps of axotomized nerves and the number of myelinated axons that
123
crossed the gap were compared in control and experimental groups. The increase found in the poled tubes was significant at both 4 and 12 weeks following transection. New studies from his laboratory where these channels were seeded with peripheral glia cells indicate a favourable influence on regeneration of sciatic nerves across a transected gap [14]. Electromagnetic fields One of the early studies was by Wilson and Jagadeesh [151 who explored PEMF effects on nerve and spinal cord regeneration using an RF signal (Diapulse, 5-120 mW/cm*). The transected median-ulnar nerves of rats were treated for 15 min/day for 30-60 days. At 30 days, PEMF-treated animals showed significant restoration .of nerve conduction activity and the presence of large diameter nerve fibres. Ito and Bassett [16] subjected the whole body of rats to pulsed electromagnetic fields after transection of the sciatic nerve. The PEMF was generated in a pair of Helmholtz coils; the signal is used clinically for bone repair (ElectroBiology, Inc.). All rats were treated for 12 h/day with motor function evaluated by a plantar-flexion force produced by stimulation of the nerve proximally. Relative to control animals, motor function with PEMF treatment was restored at the four-week time period rather than at eight weeks. Raji and Bowden [17] extended Wilson’s experiments using the Diapulse machine (RF of lop3 W/cm*) on regeneration of the transected common peroneal nerve in rats. PEMF was administered for 15 min daily for periods of three days to eight weeks. Significant increases in skin, deep-tissue and rectal temperatures occurred; these returned to normal after treatment. The size of the intraneural blood vessels was increased after treatment and the amount of collagenous tissue fibrosis reduced as well as acceleration of regeneration of nerve fibres. Parker et al. [lS] used a different signal on transected rate sciatic nerves; rats were treated with a pulse burst signal (15 Hz, Electrobiology, Inc.) for five days following transection. Treated animals exhibited a faster return of function (compound action potential) and more myelinated axons/mm* in the nerve distal to the transection than untreated control animals. PEMF effects on regeneration of the common peroneal nerve of the cat was assessed by Orgel et al. [19]. Five days after transection, the cats were exposed to PEMF for 10 h/day, 6 days/week for twelve weeks. This study tested two different PEMF signals: a 15 Hz pulse burst signal and a single repetitive pulse (72 Hz). Electrophysiologic data were collected pre- and post-operatively. The nerves were biopsied for fibre counts, and retrograde transport of horseradish peroxidase to the motor neurons in the spinal cord were used for assessing regenerative events. No significant differences were noted between controls and either PEMF signal in: numbers of fibres/mm*, axon fibre calibre, areas of nerve compound action potential or muscle compound action potential. However, the numbers of motor neurons retrogradely labelled in spinal cords of cats treated with the pulse burst
124
signal (15 Hz) were increased significantly (96.8% of the unoperated side) relative to untreated animals. Using a crushed nerve model we reported [20] a significant increase in the rate of peripheral nerve regeneration in animals treated with pulsed electromagnetic fields (PEMF 2 Hz, 3 g), for 4 h/day, for three, four or six days. Extrapolation of the regeneration distance vs. time to zero yielded an estimate of the initial delay period of one to two days due to axonal die-back after injury. We found that this period was not influenced by EMF. The apparent lack of EMF influence on the first or second day may be explained by one of two ways: the fields might not affect cells at the injury site, therefore no changes were seen in the initial delay period or the fields affected the cells in ways not observed or tested thus far. Nevertheless the rate of regeneration after the initial period was enhanced by the EM fields. In addition we found that this response was independent of the orientation of the animal to the Helmholtz coils. More importantly, pre-treatment of the animals before lesioning evoked the same degree of augmented response as that observed when the animal was treated after lesioning [21]. Sintioidal
fields
Sinusoidal fields (50 Hz, 0.2 and 0.4 mT) have been tested on the same crushed nerve model [221. The mechanisms underlying these effects may be interpreted more easily since the signal is much simpler to dissect mathematically. Although the same degree of increase in regeneration rate was obtained with these fields (21%) as compared with that with 2 Hz PEMF, the sinusoidal stimulation apparently affected the early time periods of nerve regeneration, that associated with growth cone formation and axonal sprouting. A study by Zienowicz et al. [23] using the 2 Hz, 3 G PEMF signal (Bietic Research, Lyndhurst, NJ) tested a transection model of the sciatic nerve of adult rats. Immediately, or after a five-day interval following transection and surgical repair, the animals were exposed (whole body) to PEMF. Functional return was assessed using the sciatic function walking test. In the group in which surgical repair and PEMF treatment were delayed for the first five days, significant improvement in gait was found starting at the 140 day time point and continued to increase relative to other groups to the end of the experiment (165 days). Static field3
Cordiero et al. [24] investigated the effects of high-intensity static magnetic fields (1 T) on nerve regeneration. They found no effects of the static fields on the regenerative process when determining axonal counts and neurophysiologic function.
The guinea pig superior cervical ganglion was investigated by Maehlen and Nja [25] looking at the effects of electrical stimulation of pre- and postsynaptic cells on
125
sprouting after denervation. Preganglionic stimulation on the cervical sympathetic trunk for 1 h immediately after denervation (100 pulses at 20 Hz) increased the number of axons innervating each ganglion cell. This effect was abolished with a ganglionic blocker (hexamethonium), indicating modulation by impulse activity of retrograde transsynaptic trophic effects. Chronic electrical stimulation of axotomized sciatic nerves of rabbits with 10 Hz continuous or intermittent stimulation at 100 Hz with a 20% duty cycle for 8 h/day for periods up to 230 days was reported by Gordon et al. [26]. This study indicated that threshold and suprathreshold stimulation of the nerves (via a cuff around portions of the nerve proximal to the transection site) reduced the atrophy of nerve fibres initially, but was damaging over a long period of time. Basic mechanisms
Despite the profusion of studies on electrical stimulation of tissue culture preparations, animal studies and clinical trials, no unifying mechanism that encompasses both electric and electromagnetic fields has been proposed and accepted to explain how electrical stimulation produces its effects, whether it be soft or hard tissue. Once such a mechanism has been revealed, questions concerning specific electrical modality (faradic, capacitive, inductive) and electrical parameters (frequency, intensity, etc.) can be addressed directly. ACKNOWLEDGEMENT
This study was supported in part by the Office of Naval Research, NOOO14-86-K0221 and NIH NS29621.
No.
REFERENCES 1 C.A.L. Bassett, R.J. Pawluck and R.O. Becker, Nature (London), 204 (1964) 652. 2 C. Brighton, J.E. Cronkey and A.L. Osterman, Clin. Orthop., 161 (19811 122. 3 J. Black, Electrical Stimulation: Its Role in Growth, Repair and Remodeling of the Musculoskeletal System, Praeger, New York, 1987. 4 W.G. Winter, R.C. Schutt, B.F. Sisken and SD. Smith, Trans. Orthop. Res. Sot., 6 (1981) 304. 5 B. Pomepanz, M. Mullen and H. Markus, Brain Res., 303 (1984) 331. 6 G.C. Roman, H.K Strahlendorf, P.W. Coates and B.A. Rowley, Exp. Neural., 98 (1987) 222. 7 R.B. Borgens, Prog. Clin. Biol. Res., 10 (1986) 239. 8 M.J. Pohtis, M.F. Zanakis, and B.J. Albala, J. Trauma, 28 (1988) 1375. 9 J.M. Kerns, I.M. Pavkovic, A.J. Fakhouri, K.L. Wickersham and J.A. Freeman, 19 (1987) 217. 10 J.M. Kerns, A.J. Fakhouri, H.P. Weinrib and J.A. Freeman, Accepted Neuroscience, 1990. 11 J.J. Campbell and B. Pomeranz, Sot. Neurosci., 16 (1990) 1156. 12 M.E. McGiniss, Sot. Neurosci., 16 (1990) 1156. 13 P. Aebischer, P.D. Valentini, C. Domenici and P.M. Galletti, Brain Res., 436 (1987) 165. 14 Y. Guenard, T.K. Morrisey, N. Kielman, R.P. Bunge and P. Aebischer, Sot. Neurosci., 17 (1991) 585. 15 D.H. Wilson and P. Jagadeesh, Paraplegia, 14 (1976) 12. 16 H. Ito and C.A.L. Bassett, Clin. Orthop. Relat. Res., 181 (1983) 283.
126 17 A.R. Raji and R.E.M. Bowden, J. Bone Joint Surgery, 65 (1983) 478. 18 B. Parker, C. Bryant, J. Apesos, B.F. Sisken and T. Nickel, Trans. Bioelectr. Repair Growth Sot., 3 (1983) 19. 19 M.G. Orgel, W.J. O’Brien and H.M. Murray, Plastic Reconstruct. Surg., 73 (1984) 173. 20 B.F. Sisken, M. Kanje, G. Lundborg, E. Herbst and W. Kurtz, Brain Res., 485 (1989) 309. 21 B.F. Sisken, M. Kanje, G. Lundborg and W. Kurtz, Restor. Neurosci., 1 (1990) 303. 22 A. Rusovan and M. Kanje, Exp. Neur., 112 (1991) 312. 23 R.J. Zienowicz, B.A. Thomas, W.H. Kurtz and M.G. Orgel, Plast. Reconstr. Surg., 87 (1991) 122-129. 24 P. Cordiero, B.R. Seckel, C.D. Miller, P.T. Gross and R.E. Wise, Plast. Reconstr. Surg., 83 (1989) 301. 25 J. Maehlan and A. Nja, J. Physiol., 322 (1982) 151. 26 T. Gordon, J. Gillespie, R. Orozco and L. Davis, J. Neurosci., 11 (1991) 2157.