The effect of electrical stimulation on reinnervation of rat muscle: contractile properties and endplate morphometry

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The Effect of Electrical Stimulation on Reinnervation of Rat Muscle: Contractile Properties and Endplate Morphometry ARTHUR EBERSTEIN and BRUCE R. PACHTER Department of Rehabilitation Medicine, New York Universi O, Medical Center, New York. N Y lO010 ( U ,S.A, )

(Accepted 11 March 1986) Key words: Electrical stimulation - - Reinnervation - - Denervated muscle - - Contractile prope~t~--

Endplate morphometry - - Isometric twitch tension

Denervated extensor digitorum longus muscles of Wistar rats were electrically stimulated in vivo for 4 days (2 h per day) after peroneal nerve crush 1 cm from the muscle. Isometric contractile properties and endplate ultrastructure were measured on days 11 and 18. On day 11, the time to peak (116% of control) and 1/2-relaxation time (136% of control) for the twitch tensions of stimulated muscles measured in vivo were significantly less than those (127% and 157% of controls, respectively) of non-stimulated muscles. Peak twitch and tetanic tensions were not significantly different. The postsynaptic area of endplates for stimulated muscles were closer in size to controls than those for the non-stimulated ones. On day 18, no difference was found in the contractile responses between stimulated and non-stimulated groups. Similarly, the postsynaptic areas were the same for both groups. These results demonstrate that denervated muscle stimulated electrically for 4 days prior to reinnervation can preserve the structure of the endplate as well as accelerate recovery of normal function in reinnervated muscle fibers after 11 days of denervation.

INTRODUCTION The n e u r o m u s c u l a r junction following denervation undergoes a series of degenerative changes

evoked by indirect stimulation is an excellent indicator of functional recovery. MATERIALS AND METHODS

which lead to removal of the nerve terminal and its replacement by Schwann cells. Sebille and BondouxJahan It found that daily electrical stimulation of denervated muscle accelerated reinnervation and suggested that the stimulation preserved the neuromuscular junction and thereby accelerated the recovery of function. Pachter and Eberstein 7 confirmed this hypothesis by demonstrating that stimulation of denervated muscle m a i n t a i n e d the postsynaptic area of clefts and folds in the endplates of type I and II muscle fibers. The present study was designed to extend the latter work with the primary objective to determine if recovery of function is enhanced or in any way affected by the preservation of endplate structure. Sebille and B o n d o u x - J a h a n 11 used the toespreading reflex to determine the onset of recovery, whereas we measured isometric muscle tension developed after reinnervation. Muscle tension as

Female Wistar rats (200-250 g) were denervated by nerve crush of the left peroneal nerve following the method of Hasegawa 5. The site of the nerve crush, approximately 1 cm from the extensor digitorum longus ( E D L ) muscle, was labelled with India ink prior to crushing. The denervated rats were divided into 4 groups, 6 rats per group. Two groups were stimulated for 4 days and tested on day 1 1 and day 18, respectively. Two groups were not stimulated and were tested as denervated controls on days 11 and 18. A n o t h e r group of 4 rats were not denervated and served as normal controls. Treatments began approximately 24 h following denervation. Each rat was placed in a restrainer cage and the left hind limb immobilized. Two stainless steel needle electrodes were inserted into the denervated limb, one near the proximal t e n d o n of the E D L

Correspondence: A. Eberstein, New York University Medical Center, 400 East 34th Street, New York, NY 10016, U.S.A.

0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

305 TABLE 1 Isometric contractile properties of rat ED L muscles after 4 days electrical stimulation prior to reinnervation

Number of animals in group is given in parentheses TPT, time to peak twitch tension; ~/2RT, one-half relaxation time of twitch tension. Values are means + S.D. Differences between non-stimulated and stimulated groups were not significant, with two exceptions (see footnotes). Peak twitch and tetanus are given in g per mg of muscle weight. Time after crush (days)

11 18

Test group

TPT (ms)

I/2 R T (ms)

Peak twitch (g/mgMW)

Peak tetan us g/mgMW

Normal (4) Non-stim (6) Stim (6) Non-stim (6) Stim (6)

22.4 28.4 25.9 26.0 25.0

39.6 62.2 53.8 49.3 48.4

0.40 0.18 0.20 0.32 0.33

1.6(/+_ 0.1 0.43 _+ 0.1 0.41 _+ 0.1 1.00 + 0.2 (I.92 _+ 0.1

_+ 1.1 _+ 1.4" _+ 1.7 + 0.8 _+ 1.6

_+ 2.1 _+ 7.0** _+ 5.2 + 0.6 _+ 3.2

+_ 0.1 +_ 0.06 + 0.06 _+ 0.1 =_+0.1

* Significant difference (P < 0.02) between non-stimulated and stimulated groups. ** Significant difference (P < 0.05) between non-stimulated and stimulated groups.

m u s c l e , o n e n e a r t h e distal t e n d o n . T h e s e e l e c t r o d e s w e r e c o n n e c t e d t h r o u g h s e p a r a t e s t i m u l a t i o n isolation u n i t s to a G r a s s $88 s t i m u l a t o r . T h i s a r r a n g e m e n t was a d j u s t e d to a u t o m a t i c a l l y r e v e r s e p o l a r i t y a f t e r e a c h s t i m u l u s . T h e s t i m u l a t o r was set at 1 m s p u l s e d u r a t i o n , 40 H z f r e q u e n c y f o r a s t i m u l u s t r a i n cycle o f 350 m s ' o n ' , 650 'off'. O n c e it was d e t e r mined that the electrodes were properly placed and t h e E D L m u s c l e was b e i n g s t i m u l a t e d , t h e v o l t a g e was a d j u s t e d f o r m a x i m a l r e s p o n s e ( t o e - s p r e a d ) in t h e s t i m u l a t e d g r o u p . T h e n o n - s t i m u l a t e d g r o u p received identical treatment except that the electrodes w e r e n o t c o n n e c t e d to a s t i m u l a t o r . T h e t r e a t m e n t p e r i o d was 2 h p e r d a y (1 h in t h e m o r n i n g , 1 h in aft e r n o o n ) f o r 4 days. M u s c l e t e n s i o n m e a s u r e m e n t s w e r e p e r f o r m e d 11 a n d 18 days a f t e r d e n e r v a t i o n . T h e r a t s w e r e a n e s -

E Fig. 1. Twitch and tetanus recorded from muscles denervated for 11 days. Top: non-stimulated. Bottom: stimulated for 4 days prereinnervation. Calibration: twitch, 25 ms and 13 g: tetanus, 75 ms and 10g.

Fig. 2. Neuromuscular junction from a normal control muscle. An axonal terminal (AT) is seen to lie within a cup-shaped synaptic cleft, the muscle's sarcolemma of which extends inward and forms primary junctional folds and secondary clefts. x 12,000.

306

Fig. 3. Endplate from a non-stimulated muscle fiber, 1l days post crush. Note flattening of synaptic cleft. × 12,000.

Fig. 4. Non-stimulated endplate, 11 days post crush, exhibiting highly branched postjunctional folds. × 12.(1{}{I.

thetized (sodium pentobarbital, 50 mg/kg) and the left ( d e n e r v a t e d ) E D L muscle exposed, the distal tendon was released and attached to a Grass force displacement transducer. The left sciatic nerve was exposed, and a pair of stimulating electrodes was placed on the nerve proximal to the crush site. The sciatic nerve was transected proximal to the stimulating electrodes, and all branches except the p e r o n e a l nerve were also cut. The E D L muscle and sciatic nerve were b a t h e d with mineral oil to prevent drying. Muscle surface t e m p e r a t u r e was m a i n t a i n e d at 30 °C +_ 0.5 °C. Twitch and tetanic tensions were r e c o r d e d on an F M tape r e c o r d e r ( D C to 10 kHz) and later fed into a D E C P D P 11/34 c o m p u t e r for analysis, The isometric twitch tension amplitude, time to peak tension (TPT), one-half twitch relaxation time (1/2 RT), and tetanic tension amplitude were measured. T P T is the time from the start of the twitch to the peak; 1/2 R T is the time from the p e a k to the point where the

tension decreases to one/half the peak amplitude. Tetanus was o b t a i n e d by stimulating at a frequency of 100 Hz for 500 ms. After the tension m e a s u r e m e n t s were c o m p l e t e d , the p e r o n e a l nerve was exposed along its entire length and m e a s u r e d from its entrance into the E D L muscle to the labelled crush site. The E D L muscles were then dissected free, and the proximal tendon cut, releasing the muscle. Excess fluid was r e m o v e d and the muscles immediately weighed. Following this, the muscles were pinned at their tendon ends and immediately fixed by 4% glutaraldehyde in p h o s p h a t e buffer (0.1 M, pH = 7.2) for 10-15 rain. The muscles were then transferred to fresh cold 4% glutaraldehyde solution overnight, postfixed in l % osmium tetroxide for 2 h, d e h y d r a t e d in graded alcohols, and e m b e d d e d whole in E p o n 812. The fixation technique was the same for all groups, so that any change brought about by the fixation was uniform for

307

"FABLE II Total postsynaptic area (in square microns) of junctional Jblds and clefts of endplates J?om control, non-stimulated and stimulated rat EDL muscles Values are means + SD. n, number of endplates cxamined. n

Postsvnaptic area

Control

65

16.74 -+ 4.48

11 days postcrush Non-stimulated Stimulated

55 58

11.85 _+ 3.67* 17.14 _+ 5.11

18 days postcrush Non-stimulated Stimulated

50 54

15.00 _+ 2.73 16.22 ± 3.36

* Significantly different (P < 0.001) from control and stimulated muscle fibers.

Fig. 6. In this stimulated endplate, 11 days post crush, several axonal sprouts (arrow) are seen within synaptic cleft, x 12,000.

all muscles. The muscles w e r e e m b e d d e d flat and serially sect i o n e d transversally at 15/tm by a steel knife on a sliding m i c r o t o m e . T h e thick E p o n sections w e r e cleared for light m i c r o s c o p y 7s and the muscles then s u r v e y e d by phase contrast for location of the e n d p l a t e zones. Thick

Epon

sections

containing

endplates

were

m o u n t e d on a B e e m capsule and ultrathin sections were cut, stained with uranyl acetate and lead citrate, and e x a m i n e d with a Zeiss e l e c t r o n m i c r o s c o p e . All e n d p l a t e s e n c o u n t e r e d in the e l e c t r o n microscope w e r e p h o t o g r a p h e d

and a n a l y z e d at a final

m a g n i f i c a t i o n of 12,500×. F o r each g r o u p , the postsynaptic area of j u n c t i o n a l folds and clefts associated Fig. 5. Stimulated endplate, 11 days post crush. Note regularly shaped synaptic clefts and junctional folding as well as several axonal sprouts (arrow) isolated from each other by Schwann cell cytoplasm. × 12,000.

with a given muscle fiber was assessed by tracing the area p e r i m e t e r s with a N u m o n i c s e l e c t r o n i c graphics digitizer and e x p r e s s e d in s q u a r e m i c r o n s according

308

Fig. 7. Non-stimulated endplate, 18 days post crush. Note that the junctional folds are still irregularly shaped. Several axonal sprouts (arrow) are seen within synaptic cleft separated from each other by Schwann cell cytoplasm, x 12,000.

Fig. 8. Endplate from a muscle treated with electrical stimulation. 18 days post crush. The junctional folds and synaptic cleft are regularly shaped, x 12,000.

to the m o r p h o m e t r i c principles of Santa and Engel ~ and Engel et al. 2.

with India ink) to the point where the nerve entered the muscle was measured in 18 animals to be 10 +0.8 i n ITI.

RESULTS

Tension measurements First signs of reinnervation In a preliminary e x p e r i m e n t , the first signs of reinnervation were d e t e c t e d by tension m e a s u r e m e n t s in 5 animals d e n e r v a t e d on one side. They a p p e a r e d 7 - 8 days after nerve crush. The peaks of the twitch tension and tetanus at these days were 3 and 2% of control values, respectively, In a similar series of experiments, Yeagle et a1.12 r e p o r t e d the earliest signs of reinnervation in the E D L at 8 - 9 days after nerve crush. The difference in findings were p r o b a b l y due to higher sensitivity of our recording apparatus. The distance from the nerve crush site ( m a r k e d

At day 11 after nerve crush, there was no significant difference between the peak tensions d e v e l o p e d by non-stimulated and stimulated muscles (Table I). The peak twitch tension was 45.0% of control for the non-stimulated group. The peak tetanic tension was 26.2% and 30.0% of control for non-stimulated and stimulated muscles, respectively. Twitch-tetanus ratios were also the same for both groups. Fig. 1 shows typical twitch and tetanic responses for each group. A l t h o u g h muscles of both groups contracted slower than the control muscles, there was a significant difference in twitch time characteristics between the

309 two groups at day 11 post nerve crush (Table I). TPT and 1/2RT were 28.4 + 1.4 ms and 62.2 + 7.0 ms in the non-stimulated group, respectively; whereas. they were 25.9 + 1.7 ms and 53.8 + 5.2 ms for the stimulated group. Thus, time characteristics for the isometric twitches of the stimulated muscles were closer to normal values as compared to the non-stimulated muscles. At day 18 after nerve crush, there was no significant difference in all the measured parameters between the two experimental groups. Twitch amplitudes were now approximately 80% of control and contraction times had decreased to values closer to normal (Table I).

Ultrastructure of endplates The ultrastructure of endplates in rat muscles is generally similar to that delineated by Santa and Engel 9. Axonal terminals were observed to lie within a depression of the muscle fiber surface which tends to be cup-shaped in appearance. The muscle's sarcolemma in this region extended inward and formed an elaborate system of both primary junctional folds and secondary clefts (Fig. 2). Following 11 days post nerve crush, the postsynaptic area of the nonstimulated muscles was found to be significantly reduced as compared to controls (Table If). In the stimulated muscles, the postsynaptic area was observed to be similar in size to controls. Ultrastructurally, the non-stimulated endplates exhibited a flattening of the synaptic clefts (Fig. 3) and atypical branching of the junctional folds (Fig. 4). In contrast, the junctional folding and architecture of the cupshaped synaptic clefts tended to be more regular and preserved in the stimulated group (Fig. 5). In addition, we observed greater numbers of reinnervating axonal terminals and sprouting in the stimulated group (Figs. 5 and 6). The postsynaptic area of endplates after 18 days post nerve crush in non-stimulated and stimulated muscles were found not to be significantly different from each other (Table II). However, the postjunctional folding of the non-stimulated muscle fibers still tended to exhibit somewhat irregularly shaped and branched postjunctional folding (Fig. 7) as compared to that seen in the stimulated muscle fibers (Fig. 8). Overall, both experimental groups contained greater numbers of overlying axonal terminals, indicating

enhanced reinnervation. DISCUSSION It is well known that the time characteristics of an isometric twitch after denervation is prolonged 3. In the present study we found as have other investigatorsl'12that the isometric twitch is also prolonged even after reinnervation, at least during the first 18 days following denervation. The amplitudes of the twitch and tetanic responses at days 11 and 18 post nerve crush were not significantly different between the stimulated and non-stimulated groups, although the stimulated group tended to have higher amplitudes. This was in agreement with the findings of Cole and Gardiner I who found after 8 weeks of stimulation equal twitch and tetanic tensions for both groups when normalized per unit muscle weight. Since twitch amplitude was proportional to the number of active fibers, one can conclude that stimulation of denervated muscle does not affect the number of muscle fibers reinnervated bv the regenerating nerve - - approximately the same number of fibers were active in both stimulated and non-stimulated muscles. We found however, that stimulation does affect the twitch time characteristics at day ll post nerve crush. At this time TPT and 1/2RT of the stimulated group decreased toward more normal values compared to the non-stimulated group. At 18 days post crush, twitch time characteristics of both groups had decreased and were now equal. The positive effect of stimulation at day 11 may be attributed to the influence of activity on the neuromuscular junction. Our ultrastructural data supports this conclusion. As shown in the results section, not only was the postsynaptic area of junctional folds and clefts per stimulated muscle fiber similar to control cndplates, 11 days post crush, but there also appeared to be less degradation of endplate structure as well as increased numbers of axonal sprouts at the stimulated junctions. Even at 18 days post crush, the overall architecture of the stimulated endplates appeared better delineated than endplates in the nonstimulated group. In their study of reinnervation of the rat E D L muscle, Gorio et al. 4 found that as soon as the regenerating neurite reached the endplate the rate of growth dropped about 1000-fold compared to axonal regen-

310 eration, and that the n u m b e r of polyinnervated end-

denervation and then remain fairly constant ~. Stimu-

plates increases with time, reaching a m a x i m u m 7 - 1 0

lation was limited, however, to the period prior to reinnervation to eliminate any effect muscle activity may have on new synapse formatior{' ~o

days after reinnervation. We have demonstrated in a previous paper 7 that stimulation of denervated muscle maintains the structure of the endplate, so that it is possible that the rate of growth of the neurite in the stimulated muscles is faster than in the non-stimu-

ACKNOWLEDGEMENTS

lated muscles. Thus, functional recovery would be accelerated. Stimulation was instituted 24 h after nerve crush to

This study was supported by G r a n t G008300071

attempt to reverse the alterations that develop in the

from the National Institute of Handicapped Research, U.S. D e p a r t m e n t of Education, Washington,

muscle soon after denervation. Major changes in the

DC. The authors wish to acknowledge the technical

twitch response occur during the first 2 - 6 days after

assistance of Ruth Johnston and Barbara Z i m m e r .

REFERENCES

(Ed.), Motor lnnervation qf Muscle, Academic Press, New York, pp. 289-321. 7 Pachter, B.R. and Eberstein, A., Endplate postsynaptic structure dependent upon muscle activity, Neurosci. Lett., 43 (1983) 277-283. 8 Pachter, B.R. and Eberstein, A., Neuromuscular plasticity following limb immobilization, .I. Neuro~ytol.. 13 (1984) 1(/13-1025. 9 Santa, T. and Engel, A.G., Histometric analysis of neuromuscular junction ultrastructure in rat red, white and intermediate muscle fibers. In J.E. Desmedt (Ed.), New Developments in Electromyography and Clinical Neurophysiology, Karger, Basel, 1973, pp. 41-54. 10 Schimrigk, K., McLaughlin, J. and Gruninger, W., The effect of electrical stimulation on the experimentally denervated rat muscle, Scand. J. Rehabil. Med., 9 (1977) 55-60. 11 Sebille, A. and Bondoux-Jahan, M., Effects of electric stimulation and previous nerve injury on motor function recovery in rats, Brain Research, 193 (1980) 562-565. 12 Yeagle, S.P., Mayer, R.F. and Max, S.R., Contractile properties of rat fast-twitch skeletal muscle during reinnervation: effects of testosterone and castration, Exp. Neurol., 82 (1983) 344-357.

1 Cole, B.G. and Gardiner, P.F., Does electrical stimulation of denervated muscle, continued after reinnervation, influence recovery of contractile function?, Exp. Neurol., 85 (1984) 52-62. 2 Engel, A.G., Santa, T., Stonnington, H.H., Jerusalem, F., Tsujihata, M., Brownell, A.K.W., Sahakibara, H., Banker, B.Q., Sahashi, K. and Lambert, E.H., Morphometric study of skeletal muscle ultrastructure, Muscle ,Verve, 3 (1979) 229-236. 3 Finol, H.J., Lewis, D.M. and Owens, R., The effects of denervation on contractile properties of rat skeletal muscle, J. Physiol. (London), 319 (1981) 81-92. 4 Gorio, A., Carmignoto, G., Finesso, M., Polato, P. and Nunzi, M.G., Muscle reinnervation-II. Sprouting, synapse formation and repression, Neuroscience, 8 (1983) 403-416. 5 Hasegawa, K., A new method of measuring functional recovery after crushing the peripheral nerves in unanesthetized and unrestrained rats. Experientia, 34 (1978) 272-273. 6 Le~mo,T., The role of activity in the control of membrane and contractile properties of skeletal muscle. In S. Thesleff