Electrical stimulation of regenerating nerve and its effect on motor recovery

Brain Research, 272 (1983) 21-25

21

Elsevier

Electrical Stimulation of Regenerating Nerve and its Effect on Motor Recovery WILFRED A. NIX and H. C. HOPF

Department of Neurologv, University of Clinic, 65 Mainz (F. R. G.) (Accepted December 21st, 1982)

Key words: electrical stimulation - nerve regeneration - functional reinnervation - rabbit

The rate of recovery of motor function, after axonotmesis of the motor nerve innervating the soleus muscle in the rabbit, was evaluated. In a chronic study over a period of 4 weeks, contraction parameters and muscle action potentials were recorded. A group of rabbits, whose soleus nerves were stimulated with 4 pps for 24 h daily, was compared with a control group. The electrically stimulated animals showed a faster improvement in motor function and reached their initial values a week earlier than the controls. Electrical stimulation proved to have a positive effect on the regeneration and motor recovery of nerves. INTRODUCTION

Cross-innervation of muscles demonstrated the influence of motoneurones upon the regulation of various contractile and biochemical properties of muscle fibers 3.4.2j. Different impulse patterns, imposed indirectly via the nerve on the muscle, proved to be main mediators of the regulatory mechanism 2°,22. Electrical stimulation of denervated muscle influenced various properties of muscle fibers: resting membrane potential 28, contractile properties of the muscle 16, distribution of acetylcholine receptors ~5 and the terminal sprouting ~3. In stimulating denervated muscle and performing a 'conditioning' lesion at the peripheral nerve, an acceleration of axonal growth could be seen 24, also after nerve crush and electrical muscle stimulation 19. No effect on functional reinnervation had been observed by several authors, while stimulating denervated muscle electrically for shorter periods of time 8.m4. In addition, a retarding effect on reinnervation was reported 23. Because of these different findings, it seemed of interest to investigate whether direct electrical stimulation of a nerve has an effect on axonal growth. MATERIALS AND METHODS

A group of 9 rabbits was stimulated and the 0006-8993/83/$03.00 ,~~ '~ ' 1983 Elsevier Science Publishers B.V.

process of motor function recovery compared to a group of 8 control animals. On both groups aseptic surgery under pentobarbital anaesthesia, 30 mg/kg i.v., was performed. Via an incision on the dorsal aspect of the hind limb, the right sciatic nerve was exposed and the tibial portion mobilized. The motor branches for the gastrocnemius and soleus muscle were identified and separated, and an axonotmesis was performed on the soleus motor branch, 30 mm before entering the muscle. With watchmaker forceps, a firm pressure was applied to the nerve for 5 min. Proximal to the lesion, the nerve was placed in a cuff electrode. The cuffs were made from preformed Silastic tubes (Dow Corning, type 601) which had an internal diameter of 2 mm, about 2 times the diameter of the nerve, and a length of 70-100 mm. The tube was slit longitudinally, and the nerve was placed in the tube. To stimulate the nerve, two isolated stranded steel wires (Medwire, Mt. Vernon, NY) were sewn into the cuff. Our cuffs were a modification of the method of Stein; further details are reported elsewhere~5. To record action potentials, an E M G probe was inserted between the soleus muscle and the tibia. The probe consisted of Silastic sheet, onto which teflon-isolated, stranded-steel wires were sewn. 5 cm of teflon was removed and the wires were threaded through several times to form two parallel contacts, each 1 cm

22 long and 0.5 cm apart. Several coats of medical grade Silastic (Dow Corning, type A, number 891) were applied on one side of the probe to insulate the wires. The exposed wires were brought in close contact with the muscle, so that the wires were at 90° angle to the fibers of the soleus. E M G probe and cuff wires were led subcutaneously to the neck of the animal and externalized. To shield the cables from the animal, the former were run through a light metal tube outside the cage. The tube was fastened to a saddle on the rabbit's back, so that the animal could move about freely. The stimulated animals were connected to a Grass $88 stimulator (Grass Instruments, Quincy, MA). For 4 weeks, rectangle impulses of 0.2 ms duration, with a frequency of 4 pps, were applied (24 h daily). The stimulus intensity was adjusted to 1.5 times the threshold for the initiation of muscle contraction before axonotmesis. Stimulation was started 1 day postoperatively. Eight animals were left unstimulated and served as control. To standardize the stimulating and recording procedure, a large number of preliminary experiments were needed to solve technical problems involved with chronic implants. In test animals, nerve cuffs and muscle electrodes were implanted onto the intact soleus and peroneal nerve. Whereas the muscle electrodes were easy to handle, the nerve cuffs had to be designed in such a way as to avoid nerve compression. For soleus nerve stimulation, those electrode designs were used which resulted in stable contraction and action potential recordings in previous experiments over periods as long as 8 weeks. Twitch-, tetanic-force, and muscle-action potential recordings were done in vivo, while stimulating the nerve supramaximally. The initial values of these parameters were recorded before nerve crush. The measurements were repeated in 7day sequences. For these measurements, the animals were anaesthetized by halothane N20 inhalation, using a face mask. The operated hind limb was fixed in a recording device to measure the tension or torque of ankle extension. The foot was constrained in a metal shoe which was free to rotate about a line coaxial with the ankle joint and was connected to a tension transducer.

The lower limb was stabilized between steel rods in a way which did not occlude blood supply as the studies were chronic. A grading system enabled reproducible positioning of the foot and limb. The ankle was rotated until a maximum twitch tension was recorded. The stimulating cuff, which was used for continuous nerve stimulation and stimulation during the measurements, provided, firstly, an efficient way of stimulating the regenerating nerve, and secondly, a constant condition for recording; yet the stimulus, when applied supramaximally, could not be limited to the soleus nerve only. The design of the cuff permitted some spread to the gastrocnemius nerve, which had to be separated from the soleus nerve and layed close to the implanted cuff; therefore, the values of the extensor tension, taken 7 days after the crush, were high, when expected to be low. With the gastrocnemius branches cut, measurements on the exposed, crushed soleus nerve proved that there was a complete conduction block, and no stimulus spread to the gastrocnemius. The situation changed in situ. The high values, 7 days after the nerve lesion, were a result of costimulation of the gastrocnemius nerve by the cuff. Since the measurements were done under constant conditions for each animal, this spread of the stimulus can be looked at as a systematic error which does not obscure the results. For uncontaminated results, co-denervation ofgastrocnemius would have been advisable; yet our intension was to keep the denervated soleus in a physiological environment, that is, being compressed and massaged by gastrocnemius contractions, as well as the limb movement, and thereby the innervation, specifically the stretch reflex pattern, being unchanged2L As we looked at the effect of direct stimulation on the soleus muscle and its effect on reinnervation too, it was unadvisable to denervate the whole calf. In the rabbit, the muscle mass is too large to be stimulated effectively without using currents which are harmful to the animal. RESULTS

Before axonotmesis was performed, the initial

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statistical analysis, the Wilcoxon-matched, pairsigned rank statistic was used. The experiments were terminated when, in successive 7-day measurements, stable recordings were obtained.

ve ,ctrode

Muscle action potential Supramaximal stimulation of the uncrushed nerve generated a soleus muscle potential of 6.6 ___ 2.1 mV in the control, and of 7.7 ___ 1.8 mV in the stimulated group. The amplitude of the muscle action potential dropped 7 days after axonotmesis to a mean value of39.1% in the stimulated, and 35% in the control group. In the stimulated group, the amplitude rose from 69% to 100%, 3 weeks after the nerve crush. The control group took longer, increasing from 37% in the second week to 100% after the fourth week (see Table I).

muscle electrode

~emius mI

Twitch Force The average initial twitch tension in both groups was 2.74 ± 0.9 N. Seven days after the nerve lesion twitch was reduced to 40% in the stimulated group, and 39.2% in the unstimulated group. Two weeks later, the stimulated group achieved 68.4%, and 3 weeks later 100%. The control group took longer to regain its initial value. After the second week, 50.3% could be measured, then 64%, 95.6%, and after the fifth week, the 100% level was reached (see Table I).

Fig. 1. D i a g r a m o f a n a t o m i c a l sites a n d e x p e r i m e n t a l arr a n g e m e n t used for c o n t i n u o u s nerve s t i m u l a t i o n a n d m u s cle action potential m e a s u r e m e n t with i m p l a n t e d electrodes.

data for twitch force, tetanic tension, and muscle action potential amplitude was recorded. This data was taken as 100% values, while after denervation all measurements were expressed as percentages of the initial values; thus the interindividual differences could be eliminated. For

Tetanic tension Tetanic tension was measured by applying a

TABLE I

Recovery time course, in weeks, for twitch force, tetanic tension, and muscle action potential amplitude, expressed as mean percentage ( 4. S.D.) o f the initial preoperative values, which were taken as 100% T h e Wilcoxon m a t c h e d pair-signed rank test was used for e v a l u a t i o n o f significance (P). S = d e n e r v a t e d , s t i m u l a t e d a n i m a l s (n = 9): C = d e n e r v a t e d , u n s t i m u l a t e d a n i m a l s (n = 8).

Week

0

1

2

3

4

5

S C P

100 100

40.2 ___ 7.4 39.2 ___ 23.7 >0.1

68.4 ± 15.8 50.3 ± 20.0 <0.02

100.1 ± 6,0 64.0 ± 17.5 =0.001

104.5 ___ 2.1 95.6 ___ 16.4 >0.1

100.1 ± 6.8

S C P

100 100

40.0 __. 9.6 27.0 ± 15.1 >0.1

63.7 _ 11.0 38.2 _ 13.8 = 0.0001

98.7 ± 3,4 50.2 __. 22.0

99.7 _ 2.5

=0.00004

100.9 ± 3 83.8 ± 19.1 >0.1

Muscle action potential S C P

100 100

39.1 _ 9.4 35.5 ± 25.4 >0.1

69.3 ± 16.6 37.4 _ 23.5 <0.02

99.6 ± 2,8 61.4 ± 18.1 =0.001

99.3 ± 1.6 94.4 __. 14 >0.1

98

Twitch force

Tetanic tension

-+ 4.8

24 supramaximal stimulus of 100 Hz for 250 ms to the nerve. According to different body weights of the animals, variations in the absolute values of tetanic tension were observed. Before denervation, the control group produced forces of 10.17 _+ 3.3 N, and the stimulated 9.63 + 0.14 N. The first week following the nerve lesion, the stimulated animals produced 26.2~ tension, the controls 44.4%. Whereas the stimulated group had reached 100% after 3 weeks, it took the control group 5 weeks to reach 96.4g (see Table 1). DISCUSSION

Recovery rate of twitch force, tetanic tension, and muscle action potential amplitude shows a significant difference within the stimulated and unstimulated group, between the end of the first and the fourth week; so, functional reinnervation is enhanced within the stimulated group, as these animals reached the initial values one week earlier than the unstimulated. Taking into account that the outgrowth of a crushed rabbit nerve is 4.36 _ 0.24 m m / d a f , the nerves in our experiment must have reached the muscle within the second week. Due to the fact that the stimulated group always shows a faster recovery in the second week, it has to be presumed that the stimulated nerves are growing faster, or can establish faster functional connections with the muscle than unstimulated nerves. The mechanism of interaction between nerve fibers and the periphery is not known. Within the spinal cord, the response to axonotomy is a loss of presynaptic boutons, which are regained when peripheral connections are re-established e6. At the motoneurone, a significant decrease in the duration of the after hyperpolarization is seen, following a conduction block of the soleus nerve, which is prevented by electrical stimulation of the nerve~. For the motor nerves, it is presumed that electrical activity plays a role in the maintenance of large myelinated fibers ~. This aspect can help to explain the findings that increased motoractivity could be shown to enhance peripheral nerve regeneration~.'e. In these cases, activation of the

motoneurone was achieved by central activation. Considering these findings, one must investigate to what extent peripheral nerve stimulation can induce a central or peripheral activation of neurons, resulting in a signal for enhanced regeneration. To our knowledge, until now only one publication dealt with direct nerve stimulation to promote regeneration and showed a retarding el: fect2. In this study, the sciatic nerve was cut and only the perineurium sutured with non-resorbable thread. For these technical reasons, we presume that the direct stimulation of the nerve showed no positive effect. Peripheral nerve stimulation has been shown to be effective in stimulating a cut motoneurone, thereby increasing the protein metabolism in the nerve cell tT. Just as appropriate impulse patterns play a major role in nerve muscle interaction, it seems likely that efficient stimulation of a nerve also needs a defined pattern. It is known that the alpha-motoneurones, supplying fast and slow muscles, have different firing rates ('. As we investigated a nerve supplying a slow muscle, we chose a slow frequency pattern for stimulation. This pattern can induce the characteristics of a slow muscle in last muscles and therefore seemed appropriate for the stimulation of the soleus nerve. Our nerve stimulation induced antidromic motor- and orthodromic-sensitive volleys. By afferent sensitive stimulation reflex activation of motoneutones is possible ~. As denervation is known to diminish synaptic contacts within the spinal cord -~e', it is interesting to see that electrical stimulation of partially deafferented ganglion cells is capable of enhancing their ability to establish contacts Is. The peripheral nerve stimulation proved, in our study, to increase functional muscle innervation. Further studies are needed to clarify the process of nerve muscle and nerve nerve interaction to define patterns of stimulation which can induce signals for regeneration of severed nerves. ACKNOWLEDGEMENT

This study was supported by the Deutsche Forschungsgemeinschaft.

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