Muscle fiber recovery functions studied with double pulse stimulation

Direct electrical stimulation with paired pulses at varied intervals was used to study the propagation velocity and action potential amplitude recovery functions (VRF and ARF) of single muscle fibers. Following a subnormal period with slowed conduction, most of the muscle fibers tested in healthy subjects showed a period of supernormal propagation velocity starting at 3 to 12 ms, with a peak between about 5 and 15 ms, a mean increase of 7%, and an approximately logarithmic decay toward 1 second. The onset of supernormality was earlier in muscle fibers from patients with muscular dystrophy and significantly delayed in those from denervated muscles. Dener- vated muscle fibers also had a significantly longer refractory period. Key words: muscle fiber propagation velocity single fiber electromyogra- phy velocity recovery function muscular dystrophy denervation

MUSCLE 81 NERVE 14~739-747 1991

MUSCLE FIBER RECOVERY FUNCTIONS STUDIED WITH DOUBLE PULSE STIMULATION

MARJAN MIHELIN, EE, DSc, JOZE V. TRONTELJ, MD, DSc, and ERIK STALBERG, MD, DSc

Propagation velocity in the muscle fiber changes as a function of the interval from the preceding discharge.5 This function follows different courses in different fibers; it has an initial subnormal part followed, in a majority of fibers, by a supernormal part during which the velocity is increased. At 50 ms after the conditioning discharge, the calculated increase was between 1.6% and 12.3%. Thereaf- ter, the supernormal velocity gradually returned toward the basal value, reaching it about 1 second after the preceding d i~charge .~ These values were collected using measurement of propagation velocity across two surfaces of a multielectrode during voluntary activity. A special algorithm had to be used to account for preceding activity, and the velocity changes for the shortest intervals could not be conveniently collected. Indirect stimulation

From the University Institute of Clinical Neurophysiology. University Med- ical Center, Ljubljana, Yugoslavia (Drs. Mihelin and Trontelj) and Depart- ment of Clinical Neurophysiology, University Hospital, Uppsala, Sweden (Dr Stilberg).

Acknowledgments: We are grateful to Mr. Blai Konec-Pinki for his assis- tance in a number of technical details. The study was supported by the Research Community of Slovenia (Grant C3-0558-306) and by the Swedish Research Council (Grant 135).

Address reprint requests to Dr. Trontelj, University Institute of Clinical Neurophysiology, University Medical Center, ZaloSka 7, 61 105 Ljubljana, Yugoslavia.

Accepted for publication June 29, 1990.

CCC 0148-639X/91/080739-09 $04.00 0 1991 John Wiley & Sons, Inc.

of muscle fibers with paired stimuli has also been reported on a small ample.^ In these experi- ments, a peak supernormal velocity ranging from between 0% and 24% greater than normal was obtained between 8 and 50 ms. The interdischarge interval-dependent velocity changes are of partic- ular importance in the studies of neuromuscular jitter, as they may give rise to an additional time variability,', especially with irregular activation rhythms. 1p,15

The amplitude of the muscle fiber action potential also changes as a function of the interval from the preceding discharges. Immediately after the end of the refractory period, it may be as low as 30% of n ~ r m a l . ~ The drop in amplitude is associated with a longer rise time and total potential duration. Also, this phenomenon is of practical importance in clinical jitter studies as it may ob- scure the real nature of extra-discharges occur- ring at short intervals after the original dis- ~ h a r g e . ~ It has not, so far, been quantitatively studied.

Direct muscle fiber stimulation was considered more suitable than voluntary activation or indirect stimulation through the nerve to study these phe- nomena, as it eliminates any influence of the motor axon and the neuromuscular junction upon conduction time, and offers perfect control of interdischarge intervals, making it also possible to study the recovery functions at very short inter- v a ~ s . ~ , ' ~

Recovery of Muscle Fibers MUSCLE & NERVE August 1991 739

MATERIALS AND METHODS

Stimulation. A pair of 0.3-mm monofilar tung- sten needles, electrolytically sharpened and insu- lated to about 1 mm from the tip, served as stimulating electrodes. The cathode was inserted perpendicularly into the brachioradialis muscle at about 25% of the distance from the lateral epi- condyle of the humerus to the styloid process of the radius. This muscle was chosen as it has a relatively well-defined end-plate zone, which had to be avoided in order to exclude stimulation of the motor axons. The anode was inserted subcutane- ously about 20 mm laterally. The stimuli were pairs of rectangular ~ O - F S electrical pulses (conditioning or S1 and test or S2), of an equal amplitude between 0.6 and 15 V. They were delivered by a two-channel electrical stimulator’ whose tim- ing was computer controlled. Stimulus amplitude was controlled manually with the aid of a 10-turn potentiometer to allow fine adjustment. A position of the needle cathode was sought from which the weak stimulus used produced small twitches, most often only visible as fine jerking of the cathode.

Recording. A single-fiber EMG (SFEMG) electrode with two 2 5 - ~ m surfaces oriented along the needle axis, spaced by about 200 pm was used. Usually the recordings were made between one of the small surfaces and the cannula, however, sometimes bipolar derivations between the two surfaces were used. The electrode was introduced into the twitching portion of the muscle about 20 mm proximally to the stimulating cathode, and a position was found from which a single responding muscle fiber could be recorded with an action potential amplitude higher than 2 mV, undis- turbed by any other action potentials. The finding of such position was often difficult because of the small dimensions of the responding muscle fiber fascicle. The filters on the electromyograph were rather widely opened (32 Hz to 16 kHz) so that the action potential amplitude and shape would be undistorted. When there was a need to improve selectivity and attenuate some low-amplitude action potentials from distant muscle fibers, bipolar recording was used to restrict the uptake area.

The direct muscle fiber stimulation (ie, not through the motor axon) was identified by mea- suring the .jitter, which had to be less than 4 F S , ’ ~ ’ ~ . ’ ~ using a resolution of 0.1 ps.’ The stimulus amplitude was carefully adjusted to a value of at least 25% to 35% above the threshold, and as high as possible without recruiting other muscle fibers. An EMG machine (MS6 by Medelec, Wok-

ing, UK), triggered by the HP 2100s (by Hewlett- Packard, USA) computer was used for recording and monitoring.

Data Acquisition and Analysis. ‘The analog output from the EMG machine was fed to an HP 2313, 12-bit, A/D converter (by Hewlett-Packard, USA) and the digitized data were stored on a hard disk. The sampling rate was 47 kHz, covering a 20-ms epoch per stimulating sequence. When the S1 -S2 interstimulus interval (ISI) exceeded 10 ms, the acquisition was divided in two parts, each covering 10 ms from S1 and S2 pulses, respectively.

Stimulus- response latency was measured off- line from the digitized data stored on the disk. A semiautomatic algorithm was used to recognize the point closest to the negative peaks of the stimulus artifacts and the action potentials for both the conditioning response to S1 and the test response to S2. Because of a somewhat coarse sampling, the exact shape of the peak had to be reconstructed using a mathematical algorithm, which also accu- rately determined the action potential amplitude. The algorithm fitted the second degree polynomial function to the three points closest to the action potential peak. The coordinates of the ex- treme value of this polynomial defined the actual peak latency and amplitude.

The latency measurements were then made between the stimulus artifact and the action oten- tial peak with an actual resolution of 1 ~s.‘*’Such accuracy was considered necessary to make certain that: (1) stimulation was direct rather than through a motor axon, as the latter would add the recovery functions of the terminal axon and the motor end-plate; (2) stimulation was “focal” rather than “diffuse.” If the fiber is activated at one of the low threshold sites, there is little or no latency increase when the effective stimulus strength drops to near threshold, which is in contrast to the very large increase with “diffuse” stimulation; and (3) that stimulation is well supraliminal, to avoid variable delay of the propagated muscle action potential as it arises from the local response. Low jitter (<4 ps ) is a criterion that all three conditions have been fulfilled. The measurement to peak was preferred to measurement to baseline intersection point as it eliminated possible errors due to slight baseline fluctuation, occasionally present due to low setting of the high-pass filter.

For each muscle fiber 20 paired stimulations were obtained at different IS1 values. These followed an exponential function starting at 1 ms and ending at 1000 ms. The steps were fine up to

740 Recovery of Muscle Fibers MUSCLE & NERVE August 1991

5.00 k h-. v

12.6 b h

63.0 c x L 126 L

5 ms

FIGURE 1. A typical example of actual recording of a complete sequence of stimulus and response pairs as stored on hard disk. The IS1 displayed are real until the 11th trace (inclusive), following which a lapse interval was introduced at 10 ms after each S1 up to S2. The actual IS1 values (in ms) are shown at the left of each trace.

16 ms, beyond which only 7 further points were obtained (Fig. 1). The stimulus pairs were pre- sented at a rate of 0.33 Hz, in order to minimize the residual effect of the preceding stimulation.

The measurements were done in 8 normal vol- unteers aged 25 to 35 years, in whom 70 muscle fibers were analyzed, however, the complete series of good quality were obtained in only 16 fibers. Furthermore, 23 muscle fibers were analyzed in 3

patients with muscular dystrophy, 2 with limb gir- dle, and one with Becker's form (complete data were obtained for 13). In addition, 25 muscle fibers of 3 patients with completely denervated muscles 1 to 4 months after nerve injury were studied (14 were fully analyzed). All procedures were approved by the Committee of Medical Eth- ics of the Republic of Slovenia.

RESULTS

The obtained latencies and amplitudes were used to compute the velocity and amplitude recovery functions (VRF and ARF), comparing the respective parameter of the test responses with that of the conditioning responses in each response pair. Both latency and amplitude of the conditioning responses remained remarkably stable throughout the sequence of 20 stimulations.

Figure 1 shows an example of actual recording as stored on the hard disk. The latency changes were converted into propagation velocity changes relative to the latency of the conditioning responses and were plotted against the IS1 values as VRF. The VRF for fibers from the normal, dystrophic and denervated muscles are shown in Fig- ure 2. In most muscle fibers, the VRF followed a course in which certain typical points could be identified (Fig. 3). The statistics of VRF is given in Table 1 and in Figure 4.

Table 1. Characteristic parameters of velocity recovery function (VRF) for the three groups of muscle fibers (see Fig. 3).

1 . 2. 3. 4. 5. 6. Shortest IS1 IS1 at IS1 at IS1 at VRF (%) at VRF,,,

with response VRF = 100% VRF,,, VRF = 101% shortest IS1 (%)

Normal Mean SD Minimum Maximum

Dystrophic Mean SD Minimum Maximum

Denervated Mean SD Minimum Maximum

4.12 1.73 2.69 8.13

2.98* 0.64 2.09 4.17

7.56f 2.59 4.17

12.70

5.99 2.70 2.88

12.40

3.80$ 0.77 2.75 5.93

11.44E 2.16 8.20

15.30

10.19 3.20 4.86

15.70

7.56* 1.48 4.91

10.06

14.76E 1.52

12.70 15.80

365.5 230.6 100.0 990.0

300.5 203.4 24.0

821 .O

328.8 242.9 20.0

906.0

90 7

78 99

92 5

86 105

80* 15 54 97

107 5

99 117

109 3

105 114

103 7

87 111

~~

Note Interstimulus mtervals (IS/) m ms Statisbcal cornpansons to the group of normal muscle fibers (Student's t test) ,001 < P < 005, #0001 < P < 001 f < 0001


VRF IS1 126

100

76

126

100

76

126

100

FIGURE 2. VRF curves for groups of muscle fibers from normal, dystrophic, and denervated muscles. The individual points of each curve were obtained from peak latencies of the test responses relative to those of the conditioning responses. The successive VRF points are connected with straight lines in order to allow visual distinguishing between the individual

76

fie

As can be seen from Figures 2 and 3 and from the Table 1, the shortest IS1 at which the test response appeared ranged between 2.7 and 8.1 ms in the normal muscle fibers. At these intervals, the test propagation velocity was lower than the conditioning velocity by 10% on the average (maximum 22%). The velocity reached that of the conditioning response at IS1 between 2.9 and 12.4 ms (mean 6.0 ms). The maximum increase in velocity was reached between about 8 and 12 ms in most fibers, but occasionally as early as 5 ms. Some curves were obviously shifted to the right, and two muscle fibers failed to ever reach the baseline velocity before the end of 1 s.

Compared to normal muscle fibers, those from

WOO muscle fibers. Notice smoothness of the VRF curves. The IS1 scale is logarithmic.

dystrophic muscle showed significantly shorter IS1 at which the first response occurred and earlier peak of supernormal velocity. The most significant difference was in the VRF baseline crossing point, which was much earlier for the dystrophic muscle fibers. The maximum supernormal velocity increase was insignificantly larger than in the normal fibers.

The denervated muscle fibers differed even more significantly from the normal ones in all three before-mentioned parameters, but the difference was in the opposite direction, ie, the IS1 with the first response was longer, while the VRF baseline crossing point and the peak of supernormal velocity were strongly delayed. In addition,


120

110

100

90

1.00 1.68 2.61 4.00 8.30 10.0 16.8 25.1 40.0 63.0 126 251 500 1000 1st [mr l

FIGURE 3. Characteristic points on the VRF, shown on an actual example: (7) the shortest IS1 at which test response was obtained. (2) IS1 at which VRF crosses the baseline (VRF = l00Y0). (3) IS1 at maximum VRF value. (4) IS1 at 101% baseline VRF value. (5) VRF value at point 7. (6) maximum VRF value. Only points 2 and 4 were determined by linear interpolation between two neighboring points. All other points represent the actual experimentally obtained values.

0 Rango * Mom

1st lmrl VRF 120

100


the slowing of propagation velocity for the response with the shortest IS1 was significantly more prominent. These fibers also showed a rather modest supernormal velocity increase.

Many of the curves showed an ill-defined, very slight second hump with a peak at about 100 ms.

Reproducibility of the obtained results was tested by repeating the complete stimulation sequence up to 4 times in 3 normal muscle fibers. The results were remarkably similar, with the co- efficient of variation for anyone of the VRF data points being less than 1%, except for the first two after the end of the refractory period, when it was less than 3%.

ARF showed less consistent shapes (Fig. 5). The amplitudes of the earliest responses were often con- siderably lower, between 50% and 85% of the conditioning response amplitude for the normal fibers. Supernormal amplitudes were seen in a proportion of fibers in all three groups, but the curves showed a greater variability than those of VRF.

DISCUSSION

The aim of this study was to quantify the time courses of changes in propagation velocity and action potential amplitude of muscle fibers as a function of interval to the preceding discharge. The considerable technical difficulty of' these recordings should be pointed out. It was tricky to find the low threshold sites in a muscle fiber fascicle with the stimulating electrode and then hit the same tiny fascicle with the recording needle 20 mm away, without knowing the exact muscle fiber orientation. (Finding of responses to indirect stimulation would have been much easier because muscle fibers of a single motor unit are repre- sented in a number of fascicles and in a large cross-sectional area of the muscle.) It was even more difficult to maintain two needles exactly in place without moving them by more than a few km, with visual feedback (unchanged conditioning response amplitude) only once every 3 seconds.

High quality recordings of the complete stimulation sequence could only be obtained in 43 of 118 muscle fibers studied; in the rest the sequence was prematurely terminated as the amplitude of the conditioning response changed due to slight needle movement. The fibers with incomplete se- quences were not included in the reported results, although they showed identical VRF patterns as their respective groups.

The method of direct stimulation of the muscle fibers combined with latency measurement eliminated any influence of the recovery functions

of the motor axon and the motor end-plate; however, there was a drawback that propagation velocity was not measured directly as in the origi- nally described method. Propagation velocity could have been determined from the latency and the distance between the stimulating cathode and the recording electrode, but this was considered somewhat inaccurate due to the difficulty of exact distance measurement and the uncertainty as to whether the muscle fiber action potential actually started from the point closest to the tip of the cathode or from an undefined point further along the muscle fiber.' Only later, it became apparent that the very weak stimuli used to ensure selectivity of stimulation must have activated the muscle fibers at one of the discrete low-threshold sites quite close to the electrode tip.14 The obtained latency changes could, therefore, with a reasonable degree of confidence, be converted into corre- sponding velocity changes.

It should be noted that the stimulus threshold for S2 (muscle fiber excitability) changed as a function of ISI. This phenomenon has been called excitability recovery function (ERF).4 There has been concern whether the ERF might introduce an error in estimation of propagation velocity from the response latency. It might be argued that during the period of sub- and supernormality, the stimulation threshold for S2 changed enough to result in a shift of the effective starting point of the action potential in opposite directions; however, because of the focal stimulation at discrete low-threshold sites, as described above, this seems rather unlikely. The threshold immediately out- side the stimulated point was most often consider- ably higher, as proved by stable latency when the stimulus amplitude was varied by up to 50% to 100%. Such sites have also been found in denervated muscles.'3 Whether or not these sites coincide with a membrane injury caused by the needle, or are related to motor end-plate or other structures is unknown. It is believed, therefore, that the conduction distance for both responses in the pair was identical and computation of propagation velocity changes from latency changes is justified. In fact, our values are similar to those obtained by direct measurement of propagation velocity over the multielectrode.5

Another objection against computing velocity from latency and distance between stimulating and recording single fiber electrode might be the failure to account for the local delay in generation of the action potential which may be longer during subnormality. In absolute terms, however, the


ARF Irl 160 . .

! I

ARF I%l 160 . I

1 1000

FIGURE 5. ARF curves for groups of muscle fibers from normal, dystrophic, and denervated muscles, obtained in the same way as the VRF curves. A supernormal part is seen in some ARF curves.

difference is minor compared with the effect of slower velocity. '

The validity of the obtained results is supported by their nearly ideal reproducibility when

repeatedly testing individual muscle fibers. The VRF curves shown in Figure 2, using graphic con- nection with straight lines between the actual data points, are remarkably smooth, suggesting that


the IS1 steps used were fine enough to reflect the real VRF function with satisfactory accuracy.

The actual end of refractory period was not ac- curately determined due to discrete IS1 points used; however, due to the logarithmic IS1 incre- ments by 25%, the error could not have exceeded 25% of the preceding ISI. Thus, if the first response occurred at IS1 of 4 ms, the maximum error of estimated end of refractory period is less than 0.8 ms, as the preceding IS1 was 3.2 ms. It should be noted that this duration of refractoriness is only valid for the stimulus strength used, ie, 25% to 35% above the threshold for the conditioning response. Stronger stimuli might have re- sulted in shorter refractory periods.

The baseline propagation velocity was reached in almost all normal fibers at IS1 shorter than 10 ms (mean about 6, range 3 to 12 ms). In the fibers from dystrophic muscles, this happened even much earlier, in almost all at less than 5 ms (mean below 4, range 3 to 6 nis). Moreover, the maximum supernormality, which took place in the normal fibers at about 5 to 15 ms, mean 10 ms, occurred in the fibers from dystrophic muscles already at 5 to 10 ms, mean 7.5 nis, following the conditioning stimulus. On the other hand, both of these two VRF points were significantly delayed in the denervated fibers, the respective approximate means being 11.5 ms and 15 ms (ranges 8 to 15 ms and 13 to 16 ms). Furthermore, the subnormal propagation velocity of the earliest test responses was relatively much more reduced than in the normal and dystrophic fibers. The denervated muscle fibers also had less pronounced supernormal velocity. On the other hand, even a few muscle fibers from healthy muscles remained subnormal until the end of the testing period, while VRFs of some others were obviously shifted to the right.

The lack of a supernormal period has also been reported in 30% to 50% of normal muscle fibers in an earlier study.5 However, fibers with only prologned subnormality have not been found in normal muscle, except at low temperatures or during ischemia." On the other hand, in dystrophic muscle, a higher proportion of fibers were found to have supernormal VRF,' and, when present, it tended to be more pronounced.6 This emphasizes the fact that dystrophic fibers may ei- ther be electrophysiologically normal or exhibit an exaggerated excitability. The shorter duration of refractoriness is actually an expression of altered ERF, shifted to the left.

In the denervated fibers, there was a prominent slowing of propagation velocity at shortest ISIs. The present study demonstrates that denervated muscle fibers may also have a supernormal VRF. However, the refractory period was significantly prolonged and the baseline intersection point of the VRF even more significantly delayed, the whole curve being shifted to the right. This may suggest that the ERF is less abnormal than VRF, perhaps due to the lower depolarization threshold in the denervated fibers. The recovery functions of denervated fibers presumably change with time after the severance of their axons.

Most fibers from healthy and dystrophic muscles remained slightly supernormal until 200 to 500 ms. It is of interest to note the presence of a slight second hump of supernormality in a proportion of muscle fibers in all three groups at about 100 ms. This may coincide with the con- tracted state of the muscle fibers. If this change of the conduction velocity is really related to change in muscle fiber length it should be expected to be stnaller during a single twitch, but more significant during higher rates of activation which give rise to progressive contraction. Indeed, more prominent latency shortening is seen to occur during trains of stimulation, eg, at 10 and even more at 20 Hz. Whether this mechanism is really re- sponsible for the described second peak of supernormality, and for the pronounced velocity increase during repetitive activity at higher rates, requires further study.

For most of the parameters studied, there was a significant difference between the normal and the denervated muscle fibers. On the other hand, the differences between the normal muscle fibers and those from the dystrophic muscles were less conspicuous and there was a considerable overlap. This may be due to the fact that fibers in the dystrophic muscle tend to show greatly variable de- grees of abnormalities, both histologically and electroph ysiologicall y .'

Although the general shape of the ARF curve was rather irregular, most muscle fibers studied had a rather marked drop of amplitude at the shortest 1%. This was associated with an increase in action potential rise time and total duration. A supernormal part was seen in a proportion of muscle fibers from all three groups. Such increase in amplitude has been noted b e f ~ r e . ~ Both the subnormal and the supernormal amplitudes may be much more prominent in myotonias."


~ ~~

REFERENCES

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7. Stilberg E, Trontelj JV: Single Fiber Electromyugraphy. Old Woking, UK, Mirvalle Press, 1979, pp 1-244.

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11. Trontelj JV, Mihelin M , Stilberg E: Extracellularly recorded single muscle fiber responses to electrical stiniula- tion in niyotonia congenita. Electroenceph Clin Neuropplyiol 1987;66(5):S 106.

12. Trontelj JV, Mihelin M , Stilberg E, Khuraibct A: Jitter in the stimulated motor axon. (Muscle Nerve accepted for publication.)

13. Trontelj J V , Stsiberg E: Responses to electrical stimulation of denervated muscle tibers recorded with single fiber EMG. J Neurul Nmrusurg Psychiatly 1983;46:305-309.

14. Trontelj JV, Stilberg E, Mihelin M: Jitter in the muscle ti- ber. J Neurol Neurosurg fsychiatly, 1YY0; 153:49-54.

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Muscle fiber recovery functions studied with double pulse stimulation