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Jitter measurement by axonal micro-stimulation. Guidelines and technical notes
Electroencephalography and clinical Neurophysiology, 85 (1992) 30-37 © 1992 Elsevier Scientific Publishers Ireland, Ltd. 0924-980X/92/$05.00
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ELMOCO 91530
Jitter measurement by axonal micro-stimulation. Guidelines and techaical notes Jo e V. Trontelj
a
and Erik St lberg b
a University Institute of Clinical Neurophysiology, University Medical Center, 61105 Ljubljana (Slovenia), and b Department of Clinical Neurophysiology, University Hospital, Uppsala (Sweden) (Accepted for publication: 24 July 1991)
Summary Single fiber EMG (SFEMG) with axonal micro-stimulation is a convenient method to study the neuromuscular jitter at the individual motor end-plates. Compared to the original method of jitter measurement in voluntarily activated muscle, it has the advantage of perfect control of the discharge rate, including pauses in activity, useful in quantitative estimation of the neuromuscular transmission defect. It obviates the need to search for muscle fiber pairs. It can be used in young children and in uncooperative patients, as well as those with impaired voluntary motor control. It is useful in animal experiments as well as veterinary medicine. The technique eliminates the possibility of overestimating the jitter due to unrecognized interdischarge interval dependent jitter, as well as that of underestimating it due to unrecognized low jitter in split muscle fibers. The technique has certain pitfalls causing under- or overestimation. The paper gives practical guidelines and hints as to how to avoid some of these, particularly errors due to overlooked threshold stimulation and to unrecognized direct muscle fiber stimulation.
Key words: Single fiber EMG; Jitter; Axonal micro-stimulation; Neuromuscular transmission; Myasthenia gravis; Myasthenic syndrome
Electrical stimulation can be used in conjunction with single fiber EMG to study a number of aspects of the motor unit, ranging from membrane parameters of individual directly stimulated muscle fibers to participation of the motor unit in the various reflex arcs. "Stimulation SFEMG" has recently been adopted by an increasing number of EMG laboratories, mainly as a diagnostic procedure for neuromuscular transmission disorders. However, the technique, though convenient and useful, has some pitfalls which appear to have been overlooked even in some published works (Trontelj 1990). The purpose of this paper is to discuss technical aspects of jitter measurement by axonal micro-stimulation in the light of some more recent studies, emphasizing possible errors. The background and detailed discussion of the subject is available elsewhere (St~lberg and Trontelj 1979; Trontelj et al. 1 9 8 6 , 1988, 1990b, 1991; Trontelj 1987; Mihelin et al. 1991; Trontelj and St~lberg 1991), and a comprehensive review of different applications is in the course of publication (St~lberg et al. 1991). The principle of the technique is that, for best results, not only recording but also activation should be as selective as possible, In the present context this
Correspondence to: Prof. Dr. Jo~.e V. Trontelj, University Institute of Clinical Neurophysioiogy, University Medical Center, Zalo[ka 7,
61105 Ljubljana(Slovenia).
Tel.: +38 (0)61 316-152; Fax.: +38 (0)61 302-771.
means not just activation of a small number of motor axons but particularly ensuring that the stimulation is well controlled and reproducible for axons under study, without simultaneously recruiting any other axons that might interfere with selectivity of the recording. The term micro-stimulation has been used to describe this methodological approach. It is based on using needle stimulating electrodes at least for the cathode, which is insulated to near the tip and approximated as close as possible to the axon to be studied; also on using electrical pulses of short duration (10-50 /xsec) and small amplitude (most often below 30 V or 10 mA at 50 /xsec), finely adjustable. Such low voltage and short duration electrical pulses produce electrical fields whose physiological effect is spatially highly restricted because of a very steep radial decline with distance. Finally, it is helpful to stimulate the motor axons at a point where they are already dispersed, e.g., intramuscularly (St~lberg and Trontelj 1970; Trontelj et al. 1986) rather than in the nerve trunk where microstimulation is more difficult, although not impossible. For reasons given below extramuscular stimulation may be preferred. In that situation the facial nerve is a good choice as nerve fascicles destined for a given muscle are already separate proximal to their points of entry (Trontelj et al. 1988). It should be pointed out that, while with stimulation some advantages are gained, some other advantages inherent in SFEMG with voluntary activation are lost, e.g., t h e p h y s i o l o g i c a l r e c r u i t m e n t o r d e r , t h e i r d e p e n -
J I T r E R MEASUREMENT BY AXONAL STIMULATION
dence on afferent inputs and their relation to fatigue, From the technical point of view it is a disadvantage that the asynchronous activation of different motor units, facilitating their recognition, is lost. The axonal stimulation method is particularly convenient in patients who for any reason are unable to cooperate in a jitter study in voluntarily activated muscle (young children, psychologically disturbed adults, patients with severe muscle weakness due to a central or peripheral cause, those with ataxia, tremor or other involuntary movements, unconscious patients). It may be preferred to the voluntary activation method whenever the discharge rate needs to be carefully controlled, e.g., in some cases of myasthenia gravis, in Lambert-Eaton myasthenic syndrome (LEMS) and in the rare cases of overlap syndrome (Trontelj and St~ilberg 1991). It has also been useful in research (e.g., St~lberg 1966; St~dberg and Trontelj 1979; Trontelj and St~lberg 1983; Hilton-Brown et al. 1985; Trontelj et al. 1986; Jabre et al. 1989; Khuraibet and Trontelj 1990). The following notes are intended to give practical guidelines to the experienced electromyographer interested in starting this technique,
(1) Stimulation
Electrode position
A needle stimulating cathode (e.g., Medelec MF37
Teflon coated monopolar needle), is inserted into the muscle near the motor point. The needle and its lead (taped to skin) should preferably be light to ensure a stable position. The anode may either be a similar needle placed subcutaneously about 2-3 cm away perpendicular to the direction of the muscle fibers, or a surface electrode (e.g., plate or strip such as used for grounding),
Stimulus pulse parameters The use of brief rectangular pulses (10-50 /zsec) increases selectivity of the stimulus and makes fine adjustment of the amplitude easier. It has been shown that with these pulse widths no additional jitter is generated at the stimulated point on the axon, provided that the amplitude of the stimulus is suprathreshold (Trontelj et al. 1986, 1990a). Stimulators with elther constant voltage or constant current output may be used. Amplitudes of 2-100 V (or 1-40 mA in case of constant current output)are sufficient in nearly all cases; however, scope for fine adjustment is essential, It is therefore an important advantage to have a stimulus amplitude control with a 10-turn potentiometer or in several ranges. A position of the stimulating cathode is found from which a relatively weak stimulus elicits
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twitches in a small portion of muscle, visible as fine jerking of the stimulating needle, or as fasciculation-like twitches. Strong jerks (elicited from positions near a major nerve branch) should be avoided as such stimulation activates many motor units and makes selective recording difficult. In addition, it may be painful and the contractions tend to displace both the stimulating and the recording needles. At such sites, the stimulation thresholds of different axons are often very close to each other, so it is usually better to move to another site. With the stimulating cathode in a good position, stimulation is hardly perceived or may not be felt at all. Many patients will actually fall asleep during the procedure. In case of stimulation of a facial nerve branch in the face, a sensation of pain usually indicates the proximity of a trigeminal nerve branch. With a slight change of the cathode position the pain disappears.
Stimulation frequency A stimulation rate of 10 Hz is considered convenient, as it is within the range of the physiological innervation rates at which jitter is estimated in voluntarily activated muscle. Furthermore, it seems to be close to optimum for demonstration of impaired transmission in myasthenia, as the abnormality is less prominent at lower rates and tends to improve at higher rates, the latter due to presynaptic potentiation (Trontelj and St~lberg 1991): Significantly higher rates, e.g., 30 Hz and above, may be less easy to use because of greater likelihood of displacement of the needle electrodes. Moreover, the recording may be disturbed by cumulative local refractoriness of the stimulated axon and an increasing subnormality of the muscle fibers, evident as progressive lengthening of the latency due to slowing of propagation velocity, decreasing action potential amplitude and occasionally even disintegration of its shape. However, assessment of an abnormal motor end-plate at several rates, e.g., 0.5, 5, 10 and 20 Hz, may provide valuable information regarding the type of the neuromuscular transmission disturbance (Trontelj and St~lberg 1991). When blocking is present, stimulation rates above 1 Hz may produce a significant additional jitter due to the effects of velocity recovery function in the muscle fiber (see below).
(2) Recording A recording SFEMG needle is inserted into the twitching part of the muscle (located by palpation) about 20 mm away from the stimulating cathode, either proximally or distally along the presumed direction of the muscle fibers. The position is adjusted until satisfactory records are obtained. When searching for the
32 responding fibers it is helpful to use a rather low setting for the high pass filter w e.g., about 5 or 10 Hz - - which allows one to detect relatively distant responses (as a low amplitude slow hump); the SFEMG needle can be subsequently guided in the correct direction towards the center of activity. During this procedure it is appropriate to use a low stimulation rate, e.g., 3 Hz, at which the twitches are better seen and palpated. Once a position is reached from which a satisfactory record is obtained, the high pass filter is raised to 2 or 3 kHz, the stimulus amplitude is reduced to the threshold, the stimulation rate is increased to 10 Hz and the stimulus amplitude is slowly raised again until one or several muscle fibers are seen to be recruited in the response. At first they appear with a large jitter and intermittent blocking, but a further increase in the stimulus intensity results in some shortening of the latency and reduction of the jitter. In order to make the stimulus well suprathreshold, its amplitude is raised by 15% or 5-15 V (3-5 mA) beyond the value at which no further blocking is seen. A recent study (Trontelj et al. 1990a, 1991) has shown that, compared to well-suprathreshold stimulation, near-liminal stimulation without blocking may result in an additional jitter of about 5/xsec originating in the motor axon; however, when the stimulus is at threshold, with intermittent spurious blocking, a much larger additional jitter may arise in the muscle fiber due to uneven activation rate, as a result of the velocity recovcry function of the muscle fiber (St/tlberg 1966; Trontelj et al. 1990a,b). It is therefore important to keep the stimulus intensity well above threshold, bearing in mind the possibility of'changing efficacy of the stimulus during the acquisition of responses. It is helpful to monitor continuously not only the analog SFEMG record but also the sequential histogram of latencies and particularly the presence and frequency of blocking. Propagation velocity along the muscle fiber changes with its length. Stretching of the studied muscle fiber produced by passive or active joint movement (or even movement of the recording needle) may result in considerable increase of latency, depending on the amount of stretch, the length of the muscle fiber segment between the end-plate and the recording electrode and the conduction velocity in the neutral position. Conversely, the latency shortens with maneuvers resulting in slack and consequent shortening of the muscle fiber (Trontelj 1991). Such changes of muscle fiber length do not usually occur during routine jitter studies, except during the first few discharges after rest or a pause in activity, associated with progressive mechanical contraction of the stimulated muscle fiber and concomitant shortening of the latency. A part of the supernormal muscle propagation velocity (see below) appears to be due to this mechanism,
J.V. TRONTELJ,E. STALBERG (3) Latency m e a s u r e m e n t With this technique, the jitter is defined as variation of latencies of consecutive responses, i.e., the measurement of time is made between the stimulus and a selected point on the single fiber action potential (SFAP). This means that only a single motor end-plate is assessed at a time and pairs or multiple potentials from the same motor unit are not required as in the case of jitter study in voluntarily activated muscle. The latency reading point is selected by using an appropriate time window and voltage level, ideally somewhere on the steep part of the rising slope of the action potential. If the response contains multiple action potentials with short intervals and particularly if these show large jitter and blocking, even resulting in variation of the sequence of neighboring action potentials, it may be impossible to perform reliable measurements. In t h e extensor digitorum communis muscle, for example, the latencies of the individual single fiber responses range from below 2 to over 15 msec, even with the short distance of 20 mm between the stimulating cathode and the recording SFEMG needle. This may be more than the duration of the surface-recorded M wave on radial nerve stimulation. The reason for this long duration is that some muscle fibers are activated through axon reflexes involving shorter or longer loops rather than via the direct route (St~lberg and Trontelj 1970). Such responses can be used for jitter study, since there is no additional jitter in the axon reflex (St~lberg and Trontelj 1970; Trontelj et al. 1986). Responses with latencies over 16-20 msec, however, are normally not used since they may represent an H reflex or F response, although their nature can be identified(Trontelj 1973a,b; Trontelj and Trontelj 1973, 1978).
(4) Q u a l i t y of recording For reliable results, high quality of the recording is essential. A single fiber action potential, undisturbed by other fiber action potentials, is selected. Whenever two or several SFAPs overlap partially or superimpose the jitter will be changed, due to both vertical and horizontal shifts which make jitter measurement inaccurate and unacceptable. It may be noted that such disturbances may result in an error in both directions, i.e., they may either increase or decrease the calculated jitter value (St/tlberg et al. 1991). Also rejected are the poorly shaped slow potentials and monophasic positive waves, both of which fail to fulfil the criteria for single fiber action potentials, as do the low SFAPs with prolonged rise time representing distant fibers (St~tlberg and Trontelj 1979). The superimposing activity of distant fibers as well as mains noise is largely eliminated
JITTER MEASUREMENT BY AXONAL STIMULATION
by the use of high setting of the high pass filter (2-3 kHz), making the baseline highly stable. Whenever relatively high gain must be used (e.g., 200-500 tzV/division), a lower setting of the low pass filter such as 5 kHz or below can be used to cut out the high frequency noise ("white noise") which would otherwise contribute to the measured jitter. However, this changes the action potential shape and cannot be recommended as a routine; if consistently low voltage SFAPs are recorded the SFEMG electrode should be checked for input resistance and cleaned if necessary,
(5) Adequacy of stimulus When the stimulus is at threshold for the studied motor axon, there will be an additional jitter in the axon of the order of 5 ~sec. If intermittent (false) blocking is present, the additional jitter may amount to tens or hundreds of/~sec. When a large jitter with or without blocking is seen, one should always exclude the possibility of inadequate stimulus strength before pronouncing it abnormal. An increase in stimulus strength by 10-15 V or 2-5 mA will distinguish between the two possibilities. Therefore, throughout data acquisition, the stability of recording should be carefully monitored. The efficacy of an initially adequate stimulus may change as a result of minor displacement of the stimulating cathode and either approach the threshold (the jitter and the latency increase and some blocking may appear or become more frequent), or it may become effectively stronger and some new SFAPs may be recruited, possibly interfering with the measured potential. Such series should be discarded and the stimulus readjusted. Occasionally the margin between the adequate stimulus for the studied muscle fiber and the threshold for other interfering muscle fibers may be rather narrow; it is then best to change the position of the stimulating or the recording needle or both. In the case of multiple potential records, the various SFAPs usually belong to more than one motor axon, which can be proved by small changes of stimulus amplitude. Care should be taken to make the stimulus supraliminal for each of the SFAPs before it is analyzed. Indeed, this applies even to different SFAPs belonging to the same motor unit, since some may be activated through another axonal branch, i.e., via an axon reflex.
(6) Data acquisition It is good practice to obtain several MCD (mean consecutive difference, St~lberg and Trontelj 1 9 7 9 ) readings for each SFAP, as this provides a quality control. In our laboratories 2 or more series of 50-100
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responses are acquired for each muscle fiber studied, one or two highest MCD readings are rejected and a mean of the remaining 2-4 values calculated. One may use a smaller or larger number of responses within each series, but in practice larger series may prove more difficult to complete with constant stimulation and recording conditions. It is recommended to obtain MCD values for at least 20-30 different SFAPs (it should be borne in mind that the recommended sample of 20 action potential pairs in the voluntarily activated muscle actually represents 40 motor end-plates). One should have at least 3-4 new insertions of the recording needle electrode and also change the position of the stimulating electrode. It is practical to do the MCD computation on-line (Trontelj et al. 1979; St~lberg and St~lberg 1990; Stewart et al. 1990) and have the results immediately available at the end of the test, which takes about 30 min on average. In ease of inadequate patient cooperation, such as restless children, however, it is useful to store good recording sequences on a magnetic tape or a digital disk for later off-line analysis when they can be replayed as many times as needed to analyze all SFAPs in a multiple potential record. During reanalysis one may eliminate obviously wrong data if these are not too numerous. Another possibility is to use mathematical elimination of all individual consecutive difference values exceeding 4 S.D.s (St[ilberg and Trontelj 1979).
Recordingprotocol At the beginning of a series of stimuli at rates higher than 1 Hz, the first few responses tend to show progressive shortening of the latency due to increasing propagation velocity along the muscle fiber (velocity recovery function, VRF; St~lberg 1966; Trontelj et al. 1990b).The shortening is more pronounced at higher rates, e.g., at 10-50 Hz. This will influence the measured jitter. When studies are focused on motor endplate jitter, data acquisition should be started only after the steady state is achieved, which happens after 1 sec of stimulation. On the other hand, with stimulation rates of 20 H z and above, some SFAPs will show progressive increase in latency and duration associated with a fall in amplitude. This is a result of cumulative muscle fiber subnormality, which may make jitter measurements after prolonged stimulation impossible. In cases of disturbed neuromuscular transmission with dynamically changing balance between transmitter depletion (increase in jitter) and intertetanic facilitation (decreasing jitter) one should define the stage at which the measurements are taken. During the first 5 discharges the transmission rapidly deteriorates, followed later by slight improvement and a more steady state; the repetitive stimulation test makes use of this early part of the train of stimuli to demonstrate block-
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ing in myasthenia. For the reason given above (interference of the VRF) the early part of the train is not used for jitter computation, but could be used to evaluate blocking. Later on, fatigue sets in, after a time which depends on stimulation frequency. The protocol should be designed accordingly, depending on the aim of the study,
(7) Large jitter Large jitter can be due to technical reasons which have to be carefully excluded as discussed above, So far, stimulation SFEMG has mainly been used to study neuromuscular transmission in motor end-plate disorders such as myasthenia and LEMS. Both give rise to increased jitter and intermittent impulse blocking but on changing stimulation rate the degree of abnormality changes in different ways. In myasthenia, the neuromuscular transmission abnormality may become more prominent at higher discharge rates. However, a recent study has shown that in a considerable proportion of the motor end-plates the abnormal jitter and blocking actually improve when the stimulation rate is raised from 10 to 15 or 20 Hz, presumably as a result of presynaptic potentiation. Nevertheless, nearly all of these motor end-plates will also show significant improvement of jitter and less frequent blocking when the stimulation rate is lowered to 1 or 0.5 Hz. This is in contrast to LEMS where the abnormal jitter and blocking are most pronounced at the lowest stimulation rates and improve at higher rates (Trontelj and St~lberg 1991), although not necessarily at all end-plates, Jitter measurement at different stimulation rates and after pauses in activity can be used to assess the relative strengths of intertetanic potentiation and depletion at individual presynaptic terminals and help to distinguish between the presynaptic and postsynaptic abnormality at individual motor end-plates (Trontelj and St~lberg 1991). It must be emphasized that abnormal jitter and blocking are not specific to myasthenia and LEMS but may be seen in a variety of neuromuscular disorders (St~lberg and Trontelj 1979). There are, however, biological causes of increased jitter other than disturbed neuromuscular transmission, Liminal ephaptic driving by another fiber's action potential may be a very rare cause of large jitter (Trontelj et al. 1986). In this case the jitter becomes normal or low when the stimulation rate is increased, Birnodal jitter due to alternation between a direct and axon reflex response may produce a false high MCD value. This is easily seen during data acquisition or in the displayed sequential or distribution histograms (more obviously in the former two). The mechanism can be identified by small changes in stimulus
J.V. TRONTELJ, E. ST,~LBERG
strength; a slightly stronger stimulus will produce exclusively responses with the short latency and weaker with the long latency (St/ilberg and Trontelj 1970). Such series should be discarded; however, separate measurements from each of the two latency positions may be acceptable. Care should be taken in this case to make the stimulus supraliminal for the position used. Another cause of bimodal jitter is the so-called flip-flop phenomenon (Thiele and St/ttberg 1974). Here the latency jumps are small, 100-200 /zsec (St~lberg and Trontelj 1970) and do not depend on stimulus strength. In such cases the jumps might only be seen in a sequential histogram. A third, rare possibility is that the early response results from direct stimulation of the muscle fiber (recognized by its low jitter), while the later response is produced by stimulation through the axon. The presence of intermittent blocking, whether genuine or due to liminal stimulus strength, may introduce additional jitter due to changing propagation velocity in the muscle fiber, as the activation rate becomes irregular (Trontelj et al. 1990b, 1991; Trontelj 1991). The amount of this additional "myogenic" jitter tends to vary considerably, related to the length of the muscle fiber segment between the motor end-plate and the SFEMG electrode and to the strength of the VRF (St~lberg 1966). It may be negligible when the SFEMG electrode happens to be close to the end-plate and may run into hundreds of ~sec with more remote positions. In addition, it obviously depends on the frequency and temporal pattern of blocking. Thus, the magnitude of jitter in a blocking SFAP may, by itself, not reflect the degree of motor end-plate abnormality (unless stimulation rates of less than 1 Hz are used, at which the effect of VRF is eliminated). The proportion of blocks should be used instead to describe the degree of neuromuscular transmission failure. While the pathologies underlying myasthenia gravis and LEMS are different, the abnormal jitter and blocking in both conditions share the same underlying mechanism, i.e., low end-plate potentials (EPPs) of varying amplitude, the variation being due to the variable amount of the released acetylcholine (ACh). In myasthenia the variation in ACh release is normal, but becomes abnormally unmasked because of the reduced effect of acetylcholine and the consequently low EPP developed by the abnormal postsynaptic membrane. In fact, due to decreased postsynaptic sensitivity the EPP amplitude variation is actually smaller than normal and the jitter becomes large only because the amplitude approaches the critical values, the EPP reaching the muscle fiber firing threshold with its variable peak rather than the stable and steep initial portion of its upstroke as in the normal end-plate. In LEMS, the variation in ACh release is itself abnormal, while its postsynaptic effect is normal. The resulting EPP ampli-
JITTER MEASUREMENT BY AXONAL STIMULATION tude variation is thus much larger in LEMS compared to myasthenia, which explains the relatively more abnormal jitter in this condition (Trontelj and St~ilberg 1991).
(8) Low jitter Jitter of less than 4 /zsec is considered to indicate direct stimulation of the muscle fibers, i.e., not via their motor axons and the motor end-plates. Such responses are commonly elicited in the stimulating and recording conditions described above and have to be distinguished from those with normal end-plate jitter (Trontelj et al. 1986). The system used to measure the jitter should therefore have sufficient resolution to identify cases of low jitter, i.e., at least 2 /xsec. Such resolution, present in on-line analysis with digital or analog E M G systems, is usually lost in records on magnetic tapes, since even good quality recorders introduce an additional jitter of this size or larger. Low jitter can be identified in analog records on UV sensitive paper, provided that a sufficiently fast sweep speed is used (50 or 100 /zsec/division in electromyographs with "10 x sweep expand" facility). With some practice, direct muscle stimulation is readily suspected when on suprathreshoid stimulation no jitter is visible by the naked eye at the usual sweep speed of 0.5 or 1 msec/division; however, confirmation by jitter mensurement is nevertheless necessary. The jitter on liminal stimulation tends to be larger than in the case of axonal stimulation, and when the stimulus is gradually raised there is commonly a very large continuous or stepwise latency shortening, though this is not always seen. On increasing the stimulus gradually, additional SFAPs are recruited one by one in the same fashion, initially with a long latency and large jitter, after which they tend to superimpose on the previously recruited SFAPs, producing composite potentials without any visible mutual jitter, often looking like single fiber action potentials. On reducing the stimulus strength, the same characteristic sequence of events is seen to occur in the reverse order. Although such responses tend to have somewhat shorter latencies compared to those to axonal stimulation, the latencies of the two types of response definitely overlap and cannot serve as a distinguishing criterion. Moreover, even the stimulation thresholds of the muscle fibers are similar to that of the motor axons, and both types of response may (infrequently) occur in one and the same tracing, Direct muscle fiber stimulation is particularly easily missed when the recording conditions are not good (due to noise or interfering potentials) and when stimulation is near threshold. The jitter values may then be slightly above 4 ~sec. It is recommended that repeated measurements are made in all cases of MCD values
35 between 4 and 8 /zsec. Failure to recognize low jitter will naturally result in an underestimated mean MCD value for the whole study. Extramuscular nerve stimulation is free from this pitfall. However, selective micro-stimulation of a few axons at supraliminal strength without simultaneous threshold stimulation of a number of other motor axons is less easy to achieve. As suggested above, a practical approach is to use muscles whose nerves divide in small branches proximal to their entry points, such as muscles of the face (Trontelj et al. 1988). The needle stimulation cathode is positioned near such a branch outside the muscle.
(9) Low jitter between action potentials Branches of split fibers have jitter of their parent motor end-plate when stimulated electrically via their motor axon. However, they have low jitter between each other. This can only be recognized if time mensurements are made between the suspected spike components rather than from the stimulus (Hilton-Brown et al. 1985). In this way fiber splitting is more readily detected than with the method in voluntarily contracting muscle where the low jitter may be obscured by the frequently prominent velocity recovery function which adds a "myogenic" jitter due to the (physiologically) uneven firing rates (Trontelj et al. 1990b). Failure to recognize split fibers is not a source of serious error, the only mistake being repeated measurement of the same motor end-plate.
(10) Fiber density by axonal stimulation When stimulus strength is increased smoothly or in very fine steps the motor units are recruited into the response in a discrete succession. It is then possible to count the number of SFAPs joining the recorded response simultaneously in an all-or-none fashion. To be sure that only a single axon is added, the stimulus should be kept at its threshold for a small number of discharges. Superimposing or small spikes are easily missed when joining an already complex response, while those with intermittent blocking may be erroneously counted as belonging to a new axon. Therefore not more than 2-3 different motor units can be easily and reliably sampled from a single stimulation and recording site. The procedure can be made easier by using a computer to control the incrementing and decrementing stimulus and to subtract the previous response from the succeeding one. The usual criteria for accepting a SFAP are adopted. It should be borne in mind that, as in jitter measurement, the results may not be comparable to those obtained in voluntarily activated
o
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muscle, as the recruitment order of the motor units is inevitably different. While small tonic motor units are expected to be almost exclusively activated by the slight contraction used in the voluntary activation study (at least in the normal subject), intramuscular electrical stimulation of the motor axons may be assumed to activate both small and large units in varying and probably unpredictable proportions, depending on their representation in the stimulated nerve fascicle. Whenever voluntary activation is possible, the study is more easily and reliably done in the standard way and the results can be compared to the available normal material (St~tlberg and Trontelj 1979, Ad hoe Committee of the A A E M Special Interest Group on Single Fiber EMG: Single fiber E M G reference values: a collaborative effort. Submitted, 1991).
(11) N o r m a l values
Normal jitter values have so far been collected for the extensor digitorum communis (EDC) and the orbicularis oculi (Trontelj et al. 1986, 1988). In the EDC, the mean MCD for the ages between 15 and 39 years was 17/zsec, S.D. 8 /.~sec, range of individual values 5 - 7 2 /zsec, and the range of mean MCD for different subjects was from 13 to 23 /zsec. The upper normal limits suggested for the E D C by this study are 40/xsec for the individual motor end-plates and 25 tzsec for the m e a n M C D of 2 0 - 3 0 e n d - p l a t e s ,
In the orbicularis oculi muscle, the mean MCD for adults below 40 years was 12.4/zsec, S.D. 5.6/zsec, the range of individual values was 4 - 5 0 /~sec, and the range of mean MCDs for different subjects was 9-17 /zsec. T h e suggested u p p e r n o r m a l limits are 30 /zsec
(for individual end-plates) and 20 /~sec (for a mean M C D of 20 e n d - p l a t e s ) .
Compared to jitter measured in voluntarily activated muscle fiber pairs, the jitter of stimulated single m o t o r end-plates should, theoretically, be lower by a factor of V/2 on average (St/ilberg and Trontelj 1979). This relationship has been confirmed in practice (Trontelj et al. 1986). Therefore, for any muscle where normal jitter has b e e n established for v o l u n t a r y activation, provi-
sional normal limits can be set by dividing that by v~-, at least until proper normal material is collected. AS in jitter studies with voluntary activation, 1 out of 20 r e a d i n g s e x c e e d i n g t h e i n d i v i d u a l u p p e r n o r m a l
limit is acceptable as normal, The report of the test may include: (1) the number of end-plates studied; (2) t h e numbers of those with blocking, of those with abnormal jitter without blocking and of those with normal jitter; (3) the median MCD of all e n d - p l a t e s (the a r i t h m e t i c m e a n could b e m i s l e a d -
ingly high in case of a few far out-lying values); and (4)
J.V. TRONTELJ, E. STALBERG
the mean MCD of end-plates within the normal range for individual values. The latter two values may provide diagnostically relevant information even in the absence of individual abnormal end-plates (Sanders and Howard 1986). It is also helpful to note the power of the muscle (MRC scale) and the presence or absence of clinical fatigability in case of suspected myasthenia and muscle wasting in cases of other neuromuscular disorders. The finding of normal jitter in a muscle that clinically appears weak and fatigable is a Strong argument against myasthenia. Severely abnormal jitter in a clinically minimally affected muscle is not uncommon in LEMS. Technical data such as stimulus parameters (rate), recording protocol and analysis procedure should also be included. This work was supported by the Ministry of Science and Technology of the Republic of Slovenia, Grant No. C3-0178/306, and the Swedish Research Council, Grant 135. We are indebted to N.M.F. Murray, FRCP, for linguistic corrections.
References Hilton-Brown,P., St~lberg, E., Trontelj, J.V. and Mihelin, M. Causes of the increased fiber density in muscular dystrophies studied with single fiber EMG during electrical stimulation. Muscle Nerve, 1985, 8: 383-388.
Jabre, J.F., Chirico-Post, J. and Weiner, M. Stimulation SFEMG in myasthenia gravis. Muscle Nerve, 1989, 12: 38-42. Khuraibet,A.J. and Trontelj, J.V. Jitter in the neurapraxic motor unit. Muscle Nerve, 1990, 13: 978. Mihelin, M., Trontelj, J.V. and St~ilberg, E. Muscle fiber recovery functions studied with double pulse stimulation. Muscle Nerve,
1991, 14: 739-747.
Sanders, D.B. and Howard, Jr., J.F. Single-fiber electromyography in
myastbenia gravis. Muscle Nerve, 1986, 9: 809-819. St~lberg,E. Propagation velocity in human muscle fibers in situ.
Acta Physiol. Stand., 1966, 70: 1-287. St~ilberg, E. and St~iberg, S. The use of small computers in the EMG lab. In: J.E. Desmedt (Ed.), Computer-Aided Electromyography and Expert Systems. Elsevier, Amsterdam, 1989: 1-32. St~lberg,E. and Trontelj, J.V. Demonstration of axon reflexes in human motor nerve fibres. J. Neurol. Neurosurg. Psychiat., 1970, 33: 571-579. St~lberg,E. and Trontelj, J.V. Single Fibre Eiectromyography. MirvaUe Press, Old Woking, Surrey, 1979: 1-244. St~lberg, E., Trontelj, J.V. and Mihelin, M. Electrical micro-stimulation with single fiber EMG: a useful method to study the physiology of the motor unit. J. Clin. Neurophysiol., 1991, in press. Stewart, C.R., Nandedkar, S.D. and Sanders, D.B. Personal-computer-based single-fibre EMG jitter analysis system. Med. Biol. Eng. Comput., 1990, 28: 607-612. Thiele, B. and St~lberg, E. The bimodal jitter: a single fibre electromyographic finding. J. Neurol. Neurosurg. Psychiat., 1974, 38:
881-889. Trontelj, J.V. A study of the F-responses by single fibre electromyography. In: J.E. Desmedt (Ed.), New Developments in Electromyography and Clinical Neurophysiology,Vol. 3. Karger, Basel, 1973a: 318-322. Trontelj, J.V. A study of the H-reflex by single fibre EMG.J. Neurol. Neurosurg. Psychiat., 1973b, 36: 951-959.
JITFER MEASUREMENT BY AXONAL STIMULATION Trontelj, J.V. Guidelines and technical notes for stimulated single fiber jitter studies. VII Int. Single-Fiber EMG Course and Symp., May 1-3, 1987. Office of Continuing Education, School of Medicine, University of North Carolina at Chapel Hill. SFEMG Syllabus, 1987, 6: 1-8. Trontelj, J.V. Stimulation SFEMG in myasthenia gravis (Letter to the Editor). Muscle Nerve, 1990, 13: 458. Trontelj, J.V. Muscle fiber conduction velocity changes with length. Muscle Nerve, 1991, in press. Trontelj, J.V. and St~lberg, E. Responses to electrical stimulation of denervated human muscle fibres recorded with single fibre EMG.J. Neurol. Neurosurg. Psychiat., 1983, 46: 310-316. Trontelj, J.V. and St~lberg, E. Single motor end-plates in myasthenia gravis and LEMS at different firing rates. Muscle Nerve, 1991, 14: 226-232. Trontelj, J.V. and Trontelj, M. F-responses of human facial muscles. A single motoneurone study. J. Neurol. Sci., 1973, 20: 211-222. Trontelj, M.A. and Trontelj, J.V. Reflex arc of the first component
37 of the human blink reflex: a single motoneurone study. J. Neurol. Neurosurg. Psychiat., 1978, 41: 538-547. Tronteij, J.V., Mihelin, M., Pleter~ek, T. and Antoni, L. Jittermeter: a microcomputer-based system for single fibre electromyography. Int. J. Biomed. Comput., 1979, 10: 451-459. Trontelj, J.V., Mihelin, M., Fernandez, J.M. and St~lberg, E. Axonal stimulation for end-plate jitter studies. J. Neurol. Neurosurg. Psychiat., 1986, 49: 677-685. Trontelj, J.V., Khuraibet, A. and Mihelin, M. The jitter in stimulated orbieularis oculi muscle: technique and normal values. J. Neurol. Neurosurg. Psychiat., 1988, 51: 814-819. Trontelj, J.V., Mihelin, M., St~tlberg, E. and Khuraibet, A. The jitter of stimulation site on the motor axon. Muscle Nerve, 1990a, 13: 978. Trontelj, J.V., St~lberg, E. and Mihelin, M. Jitter in the muscle fibre. J. Neurol. Neurosurg. Psychiat., 1990b, 53: 49-54. Trontelj, J.V., St~lberg, E., Mihelin, M. and Khuraibet, A. Jitter of the stimulated motor axon. Muscle Nerve, 1991, in press.
30
ELMOCO 91530
Jitter measurement by axonal micro-stimulation. Guidelines and techaical notes Jo e V. Trontelj
a
and Erik St lberg b
a University Institute of Clinical Neurophysiology, University Medical Center, 61105 Ljubljana (Slovenia), and b Department of Clinical Neurophysiology, University Hospital, Uppsala (Sweden) (Accepted for publication: 24 July 1991)
Summary Single fiber EMG (SFEMG) with axonal micro-stimulation is a convenient method to study the neuromuscular jitter at the individual motor end-plates. Compared to the original method of jitter measurement in voluntarily activated muscle, it has the advantage of perfect control of the discharge rate, including pauses in activity, useful in quantitative estimation of the neuromuscular transmission defect. It obviates the need to search for muscle fiber pairs. It can be used in young children and in uncooperative patients, as well as those with impaired voluntary motor control. It is useful in animal experiments as well as veterinary medicine. The technique eliminates the possibility of overestimating the jitter due to unrecognized interdischarge interval dependent jitter, as well as that of underestimating it due to unrecognized low jitter in split muscle fibers. The technique has certain pitfalls causing under- or overestimation. The paper gives practical guidelines and hints as to how to avoid some of these, particularly errors due to overlooked threshold stimulation and to unrecognized direct muscle fiber stimulation.
Key words: Single fiber EMG; Jitter; Axonal micro-stimulation; Neuromuscular transmission; Myasthenia gravis; Myasthenic syndrome
Electrical stimulation can be used in conjunction with single fiber EMG to study a number of aspects of the motor unit, ranging from membrane parameters of individual directly stimulated muscle fibers to participation of the motor unit in the various reflex arcs. "Stimulation SFEMG" has recently been adopted by an increasing number of EMG laboratories, mainly as a diagnostic procedure for neuromuscular transmission disorders. However, the technique, though convenient and useful, has some pitfalls which appear to have been overlooked even in some published works (Trontelj 1990). The purpose of this paper is to discuss technical aspects of jitter measurement by axonal micro-stimulation in the light of some more recent studies, emphasizing possible errors. The background and detailed discussion of the subject is available elsewhere (St~lberg and Trontelj 1979; Trontelj et al. 1 9 8 6 , 1988, 1990b, 1991; Trontelj 1987; Mihelin et al. 1991; Trontelj and St~lberg 1991), and a comprehensive review of different applications is in the course of publication (St~lberg et al. 1991). The principle of the technique is that, for best results, not only recording but also activation should be as selective as possible, In the present context this
Correspondence to: Prof. Dr. Jo~.e V. Trontelj, University Institute of Clinical Neurophysioiogy, University Medical Center, Zalo[ka 7,
61105 Ljubljana(Slovenia).
Tel.: +38 (0)61 316-152; Fax.: +38 (0)61 302-771.
means not just activation of a small number of motor axons but particularly ensuring that the stimulation is well controlled and reproducible for axons under study, without simultaneously recruiting any other axons that might interfere with selectivity of the recording. The term micro-stimulation has been used to describe this methodological approach. It is based on using needle stimulating electrodes at least for the cathode, which is insulated to near the tip and approximated as close as possible to the axon to be studied; also on using electrical pulses of short duration (10-50 /xsec) and small amplitude (most often below 30 V or 10 mA at 50 /xsec), finely adjustable. Such low voltage and short duration electrical pulses produce electrical fields whose physiological effect is spatially highly restricted because of a very steep radial decline with distance. Finally, it is helpful to stimulate the motor axons at a point where they are already dispersed, e.g., intramuscularly (St~lberg and Trontelj 1970; Trontelj et al. 1986) rather than in the nerve trunk where microstimulation is more difficult, although not impossible. For reasons given below extramuscular stimulation may be preferred. In that situation the facial nerve is a good choice as nerve fascicles destined for a given muscle are already separate proximal to their points of entry (Trontelj et al. 1988). It should be pointed out that, while with stimulation some advantages are gained, some other advantages inherent in SFEMG with voluntary activation are lost, e.g., t h e p h y s i o l o g i c a l r e c r u i t m e n t o r d e r , t h e i r d e p e n -
J I T r E R MEASUREMENT BY AXONAL STIMULATION
dence on afferent inputs and their relation to fatigue, From the technical point of view it is a disadvantage that the asynchronous activation of different motor units, facilitating their recognition, is lost. The axonal stimulation method is particularly convenient in patients who for any reason are unable to cooperate in a jitter study in voluntarily activated muscle (young children, psychologically disturbed adults, patients with severe muscle weakness due to a central or peripheral cause, those with ataxia, tremor or other involuntary movements, unconscious patients). It may be preferred to the voluntary activation method whenever the discharge rate needs to be carefully controlled, e.g., in some cases of myasthenia gravis, in Lambert-Eaton myasthenic syndrome (LEMS) and in the rare cases of overlap syndrome (Trontelj and St~ilberg 1991). It has also been useful in research (e.g., St~lberg 1966; St~dberg and Trontelj 1979; Trontelj and St~lberg 1983; Hilton-Brown et al. 1985; Trontelj et al. 1986; Jabre et al. 1989; Khuraibet and Trontelj 1990). The following notes are intended to give practical guidelines to the experienced electromyographer interested in starting this technique,
(1) Stimulation
Electrode position
A needle stimulating cathode (e.g., Medelec MF37
Teflon coated monopolar needle), is inserted into the muscle near the motor point. The needle and its lead (taped to skin) should preferably be light to ensure a stable position. The anode may either be a similar needle placed subcutaneously about 2-3 cm away perpendicular to the direction of the muscle fibers, or a surface electrode (e.g., plate or strip such as used for grounding),
Stimulus pulse parameters The use of brief rectangular pulses (10-50 /zsec) increases selectivity of the stimulus and makes fine adjustment of the amplitude easier. It has been shown that with these pulse widths no additional jitter is generated at the stimulated point on the axon, provided that the amplitude of the stimulus is suprathreshold (Trontelj et al. 1986, 1990a). Stimulators with elther constant voltage or constant current output may be used. Amplitudes of 2-100 V (or 1-40 mA in case of constant current output)are sufficient in nearly all cases; however, scope for fine adjustment is essential, It is therefore an important advantage to have a stimulus amplitude control with a 10-turn potentiometer or in several ranges. A position of the stimulating cathode is found from which a relatively weak stimulus elicits
31
twitches in a small portion of muscle, visible as fine jerking of the stimulating needle, or as fasciculation-like twitches. Strong jerks (elicited from positions near a major nerve branch) should be avoided as such stimulation activates many motor units and makes selective recording difficult. In addition, it may be painful and the contractions tend to displace both the stimulating and the recording needles. At such sites, the stimulation thresholds of different axons are often very close to each other, so it is usually better to move to another site. With the stimulating cathode in a good position, stimulation is hardly perceived or may not be felt at all. Many patients will actually fall asleep during the procedure. In case of stimulation of a facial nerve branch in the face, a sensation of pain usually indicates the proximity of a trigeminal nerve branch. With a slight change of the cathode position the pain disappears.
Stimulation frequency A stimulation rate of 10 Hz is considered convenient, as it is within the range of the physiological innervation rates at which jitter is estimated in voluntarily activated muscle. Furthermore, it seems to be close to optimum for demonstration of impaired transmission in myasthenia, as the abnormality is less prominent at lower rates and tends to improve at higher rates, the latter due to presynaptic potentiation (Trontelj and St~lberg 1991): Significantly higher rates, e.g., 30 Hz and above, may be less easy to use because of greater likelihood of displacement of the needle electrodes. Moreover, the recording may be disturbed by cumulative local refractoriness of the stimulated axon and an increasing subnormality of the muscle fibers, evident as progressive lengthening of the latency due to slowing of propagation velocity, decreasing action potential amplitude and occasionally even disintegration of its shape. However, assessment of an abnormal motor end-plate at several rates, e.g., 0.5, 5, 10 and 20 Hz, may provide valuable information regarding the type of the neuromuscular transmission disturbance (Trontelj and St~lberg 1991). When blocking is present, stimulation rates above 1 Hz may produce a significant additional jitter due to the effects of velocity recovery function in the muscle fiber (see below).
(2) Recording A recording SFEMG needle is inserted into the twitching part of the muscle (located by palpation) about 20 mm away from the stimulating cathode, either proximally or distally along the presumed direction of the muscle fibers. The position is adjusted until satisfactory records are obtained. When searching for the
32 responding fibers it is helpful to use a rather low setting for the high pass filter w e.g., about 5 or 10 Hz - - which allows one to detect relatively distant responses (as a low amplitude slow hump); the SFEMG needle can be subsequently guided in the correct direction towards the center of activity. During this procedure it is appropriate to use a low stimulation rate, e.g., 3 Hz, at which the twitches are better seen and palpated. Once a position is reached from which a satisfactory record is obtained, the high pass filter is raised to 2 or 3 kHz, the stimulus amplitude is reduced to the threshold, the stimulation rate is increased to 10 Hz and the stimulus amplitude is slowly raised again until one or several muscle fibers are seen to be recruited in the response. At first they appear with a large jitter and intermittent blocking, but a further increase in the stimulus intensity results in some shortening of the latency and reduction of the jitter. In order to make the stimulus well suprathreshold, its amplitude is raised by 15% or 5-15 V (3-5 mA) beyond the value at which no further blocking is seen. A recent study (Trontelj et al. 1990a, 1991) has shown that, compared to well-suprathreshold stimulation, near-liminal stimulation without blocking may result in an additional jitter of about 5/xsec originating in the motor axon; however, when the stimulus is at threshold, with intermittent spurious blocking, a much larger additional jitter may arise in the muscle fiber due to uneven activation rate, as a result of the velocity recovcry function of the muscle fiber (St/tlberg 1966; Trontelj et al. 1990a,b). It is therefore important to keep the stimulus intensity well above threshold, bearing in mind the possibility of'changing efficacy of the stimulus during the acquisition of responses. It is helpful to monitor continuously not only the analog SFEMG record but also the sequential histogram of latencies and particularly the presence and frequency of blocking. Propagation velocity along the muscle fiber changes with its length. Stretching of the studied muscle fiber produced by passive or active joint movement (or even movement of the recording needle) may result in considerable increase of latency, depending on the amount of stretch, the length of the muscle fiber segment between the end-plate and the recording electrode and the conduction velocity in the neutral position. Conversely, the latency shortens with maneuvers resulting in slack and consequent shortening of the muscle fiber (Trontelj 1991). Such changes of muscle fiber length do not usually occur during routine jitter studies, except during the first few discharges after rest or a pause in activity, associated with progressive mechanical contraction of the stimulated muscle fiber and concomitant shortening of the latency. A part of the supernormal muscle propagation velocity (see below) appears to be due to this mechanism,
J.V. TRONTELJ,E. STALBERG (3) Latency m e a s u r e m e n t With this technique, the jitter is defined as variation of latencies of consecutive responses, i.e., the measurement of time is made between the stimulus and a selected point on the single fiber action potential (SFAP). This means that only a single motor end-plate is assessed at a time and pairs or multiple potentials from the same motor unit are not required as in the case of jitter study in voluntarily activated muscle. The latency reading point is selected by using an appropriate time window and voltage level, ideally somewhere on the steep part of the rising slope of the action potential. If the response contains multiple action potentials with short intervals and particularly if these show large jitter and blocking, even resulting in variation of the sequence of neighboring action potentials, it may be impossible to perform reliable measurements. In t h e extensor digitorum communis muscle, for example, the latencies of the individual single fiber responses range from below 2 to over 15 msec, even with the short distance of 20 mm between the stimulating cathode and the recording SFEMG needle. This may be more than the duration of the surface-recorded M wave on radial nerve stimulation. The reason for this long duration is that some muscle fibers are activated through axon reflexes involving shorter or longer loops rather than via the direct route (St~lberg and Trontelj 1970). Such responses can be used for jitter study, since there is no additional jitter in the axon reflex (St~lberg and Trontelj 1970; Trontelj et al. 1986). Responses with latencies over 16-20 msec, however, are normally not used since they may represent an H reflex or F response, although their nature can be identified(Trontelj 1973a,b; Trontelj and Trontelj 1973, 1978).
(4) Q u a l i t y of recording For reliable results, high quality of the recording is essential. A single fiber action potential, undisturbed by other fiber action potentials, is selected. Whenever two or several SFAPs overlap partially or superimpose the jitter will be changed, due to both vertical and horizontal shifts which make jitter measurement inaccurate and unacceptable. It may be noted that such disturbances may result in an error in both directions, i.e., they may either increase or decrease the calculated jitter value (St/tlberg et al. 1991). Also rejected are the poorly shaped slow potentials and monophasic positive waves, both of which fail to fulfil the criteria for single fiber action potentials, as do the low SFAPs with prolonged rise time representing distant fibers (St~tlberg and Trontelj 1979). The superimposing activity of distant fibers as well as mains noise is largely eliminated
JITTER MEASUREMENT BY AXONAL STIMULATION
by the use of high setting of the high pass filter (2-3 kHz), making the baseline highly stable. Whenever relatively high gain must be used (e.g., 200-500 tzV/division), a lower setting of the low pass filter such as 5 kHz or below can be used to cut out the high frequency noise ("white noise") which would otherwise contribute to the measured jitter. However, this changes the action potential shape and cannot be recommended as a routine; if consistently low voltage SFAPs are recorded the SFEMG electrode should be checked for input resistance and cleaned if necessary,
(5) Adequacy of stimulus When the stimulus is at threshold for the studied motor axon, there will be an additional jitter in the axon of the order of 5 ~sec. If intermittent (false) blocking is present, the additional jitter may amount to tens or hundreds of/~sec. When a large jitter with or without blocking is seen, one should always exclude the possibility of inadequate stimulus strength before pronouncing it abnormal. An increase in stimulus strength by 10-15 V or 2-5 mA will distinguish between the two possibilities. Therefore, throughout data acquisition, the stability of recording should be carefully monitored. The efficacy of an initially adequate stimulus may change as a result of minor displacement of the stimulating cathode and either approach the threshold (the jitter and the latency increase and some blocking may appear or become more frequent), or it may become effectively stronger and some new SFAPs may be recruited, possibly interfering with the measured potential. Such series should be discarded and the stimulus readjusted. Occasionally the margin between the adequate stimulus for the studied muscle fiber and the threshold for other interfering muscle fibers may be rather narrow; it is then best to change the position of the stimulating or the recording needle or both. In the case of multiple potential records, the various SFAPs usually belong to more than one motor axon, which can be proved by small changes of stimulus amplitude. Care should be taken to make the stimulus supraliminal for each of the SFAPs before it is analyzed. Indeed, this applies even to different SFAPs belonging to the same motor unit, since some may be activated through another axonal branch, i.e., via an axon reflex.
(6) Data acquisition It is good practice to obtain several MCD (mean consecutive difference, St~lberg and Trontelj 1 9 7 9 ) readings for each SFAP, as this provides a quality control. In our laboratories 2 or more series of 50-100
33
responses are acquired for each muscle fiber studied, one or two highest MCD readings are rejected and a mean of the remaining 2-4 values calculated. One may use a smaller or larger number of responses within each series, but in practice larger series may prove more difficult to complete with constant stimulation and recording conditions. It is recommended to obtain MCD values for at least 20-30 different SFAPs (it should be borne in mind that the recommended sample of 20 action potential pairs in the voluntarily activated muscle actually represents 40 motor end-plates). One should have at least 3-4 new insertions of the recording needle electrode and also change the position of the stimulating electrode. It is practical to do the MCD computation on-line (Trontelj et al. 1979; St~lberg and St~lberg 1990; Stewart et al. 1990) and have the results immediately available at the end of the test, which takes about 30 min on average. In ease of inadequate patient cooperation, such as restless children, however, it is useful to store good recording sequences on a magnetic tape or a digital disk for later off-line analysis when they can be replayed as many times as needed to analyze all SFAPs in a multiple potential record. During reanalysis one may eliminate obviously wrong data if these are not too numerous. Another possibility is to use mathematical elimination of all individual consecutive difference values exceeding 4 S.D.s (St[ilberg and Trontelj 1979).
Recordingprotocol At the beginning of a series of stimuli at rates higher than 1 Hz, the first few responses tend to show progressive shortening of the latency due to increasing propagation velocity along the muscle fiber (velocity recovery function, VRF; St~lberg 1966; Trontelj et al. 1990b).The shortening is more pronounced at higher rates, e.g., at 10-50 Hz. This will influence the measured jitter. When studies are focused on motor endplate jitter, data acquisition should be started only after the steady state is achieved, which happens after 1 sec of stimulation. On the other hand, with stimulation rates of 20 H z and above, some SFAPs will show progressive increase in latency and duration associated with a fall in amplitude. This is a result of cumulative muscle fiber subnormality, which may make jitter measurements after prolonged stimulation impossible. In cases of disturbed neuromuscular transmission with dynamically changing balance between transmitter depletion (increase in jitter) and intertetanic facilitation (decreasing jitter) one should define the stage at which the measurements are taken. During the first 5 discharges the transmission rapidly deteriorates, followed later by slight improvement and a more steady state; the repetitive stimulation test makes use of this early part of the train of stimuli to demonstrate block-
34
ing in myasthenia. For the reason given above (interference of the VRF) the early part of the train is not used for jitter computation, but could be used to evaluate blocking. Later on, fatigue sets in, after a time which depends on stimulation frequency. The protocol should be designed accordingly, depending on the aim of the study,
(7) Large jitter Large jitter can be due to technical reasons which have to be carefully excluded as discussed above, So far, stimulation SFEMG has mainly been used to study neuromuscular transmission in motor end-plate disorders such as myasthenia and LEMS. Both give rise to increased jitter and intermittent impulse blocking but on changing stimulation rate the degree of abnormality changes in different ways. In myasthenia, the neuromuscular transmission abnormality may become more prominent at higher discharge rates. However, a recent study has shown that in a considerable proportion of the motor end-plates the abnormal jitter and blocking actually improve when the stimulation rate is raised from 10 to 15 or 20 Hz, presumably as a result of presynaptic potentiation. Nevertheless, nearly all of these motor end-plates will also show significant improvement of jitter and less frequent blocking when the stimulation rate is lowered to 1 or 0.5 Hz. This is in contrast to LEMS where the abnormal jitter and blocking are most pronounced at the lowest stimulation rates and improve at higher rates (Trontelj and St~lberg 1991), although not necessarily at all end-plates, Jitter measurement at different stimulation rates and after pauses in activity can be used to assess the relative strengths of intertetanic potentiation and depletion at individual presynaptic terminals and help to distinguish between the presynaptic and postsynaptic abnormality at individual motor end-plates (Trontelj and St~lberg 1991). It must be emphasized that abnormal jitter and blocking are not specific to myasthenia and LEMS but may be seen in a variety of neuromuscular disorders (St~lberg and Trontelj 1979). There are, however, biological causes of increased jitter other than disturbed neuromuscular transmission, Liminal ephaptic driving by another fiber's action potential may be a very rare cause of large jitter (Trontelj et al. 1986). In this case the jitter becomes normal or low when the stimulation rate is increased, Birnodal jitter due to alternation between a direct and axon reflex response may produce a false high MCD value. This is easily seen during data acquisition or in the displayed sequential or distribution histograms (more obviously in the former two). The mechanism can be identified by small changes in stimulus
J.V. TRONTELJ, E. ST,~LBERG
strength; a slightly stronger stimulus will produce exclusively responses with the short latency and weaker with the long latency (St/ilberg and Trontelj 1970). Such series should be discarded; however, separate measurements from each of the two latency positions may be acceptable. Care should be taken in this case to make the stimulus supraliminal for the position used. Another cause of bimodal jitter is the so-called flip-flop phenomenon (Thiele and St/ttberg 1974). Here the latency jumps are small, 100-200 /zsec (St~lberg and Trontelj 1970) and do not depend on stimulus strength. In such cases the jumps might only be seen in a sequential histogram. A third, rare possibility is that the early response results from direct stimulation of the muscle fiber (recognized by its low jitter), while the later response is produced by stimulation through the axon. The presence of intermittent blocking, whether genuine or due to liminal stimulus strength, may introduce additional jitter due to changing propagation velocity in the muscle fiber, as the activation rate becomes irregular (Trontelj et al. 1990b, 1991; Trontelj 1991). The amount of this additional "myogenic" jitter tends to vary considerably, related to the length of the muscle fiber segment between the motor end-plate and the SFEMG electrode and to the strength of the VRF (St~lberg 1966). It may be negligible when the SFEMG electrode happens to be close to the end-plate and may run into hundreds of ~sec with more remote positions. In addition, it obviously depends on the frequency and temporal pattern of blocking. Thus, the magnitude of jitter in a blocking SFAP may, by itself, not reflect the degree of motor end-plate abnormality (unless stimulation rates of less than 1 Hz are used, at which the effect of VRF is eliminated). The proportion of blocks should be used instead to describe the degree of neuromuscular transmission failure. While the pathologies underlying myasthenia gravis and LEMS are different, the abnormal jitter and blocking in both conditions share the same underlying mechanism, i.e., low end-plate potentials (EPPs) of varying amplitude, the variation being due to the variable amount of the released acetylcholine (ACh). In myasthenia the variation in ACh release is normal, but becomes abnormally unmasked because of the reduced effect of acetylcholine and the consequently low EPP developed by the abnormal postsynaptic membrane. In fact, due to decreased postsynaptic sensitivity the EPP amplitude variation is actually smaller than normal and the jitter becomes large only because the amplitude approaches the critical values, the EPP reaching the muscle fiber firing threshold with its variable peak rather than the stable and steep initial portion of its upstroke as in the normal end-plate. In LEMS, the variation in ACh release is itself abnormal, while its postsynaptic effect is normal. The resulting EPP ampli-
JITTER MEASUREMENT BY AXONAL STIMULATION tude variation is thus much larger in LEMS compared to myasthenia, which explains the relatively more abnormal jitter in this condition (Trontelj and St~ilberg 1991).
(8) Low jitter Jitter of less than 4 /zsec is considered to indicate direct stimulation of the muscle fibers, i.e., not via their motor axons and the motor end-plates. Such responses are commonly elicited in the stimulating and recording conditions described above and have to be distinguished from those with normal end-plate jitter (Trontelj et al. 1986). The system used to measure the jitter should therefore have sufficient resolution to identify cases of low jitter, i.e., at least 2 /xsec. Such resolution, present in on-line analysis with digital or analog E M G systems, is usually lost in records on magnetic tapes, since even good quality recorders introduce an additional jitter of this size or larger. Low jitter can be identified in analog records on UV sensitive paper, provided that a sufficiently fast sweep speed is used (50 or 100 /zsec/division in electromyographs with "10 x sweep expand" facility). With some practice, direct muscle stimulation is readily suspected when on suprathreshoid stimulation no jitter is visible by the naked eye at the usual sweep speed of 0.5 or 1 msec/division; however, confirmation by jitter mensurement is nevertheless necessary. The jitter on liminal stimulation tends to be larger than in the case of axonal stimulation, and when the stimulus is gradually raised there is commonly a very large continuous or stepwise latency shortening, though this is not always seen. On increasing the stimulus gradually, additional SFAPs are recruited one by one in the same fashion, initially with a long latency and large jitter, after which they tend to superimpose on the previously recruited SFAPs, producing composite potentials without any visible mutual jitter, often looking like single fiber action potentials. On reducing the stimulus strength, the same characteristic sequence of events is seen to occur in the reverse order. Although such responses tend to have somewhat shorter latencies compared to those to axonal stimulation, the latencies of the two types of response definitely overlap and cannot serve as a distinguishing criterion. Moreover, even the stimulation thresholds of the muscle fibers are similar to that of the motor axons, and both types of response may (infrequently) occur in one and the same tracing, Direct muscle fiber stimulation is particularly easily missed when the recording conditions are not good (due to noise or interfering potentials) and when stimulation is near threshold. The jitter values may then be slightly above 4 ~sec. It is recommended that repeated measurements are made in all cases of MCD values
35 between 4 and 8 /zsec. Failure to recognize low jitter will naturally result in an underestimated mean MCD value for the whole study. Extramuscular nerve stimulation is free from this pitfall. However, selective micro-stimulation of a few axons at supraliminal strength without simultaneous threshold stimulation of a number of other motor axons is less easy to achieve. As suggested above, a practical approach is to use muscles whose nerves divide in small branches proximal to their entry points, such as muscles of the face (Trontelj et al. 1988). The needle stimulation cathode is positioned near such a branch outside the muscle.
(9) Low jitter between action potentials Branches of split fibers have jitter of their parent motor end-plate when stimulated electrically via their motor axon. However, they have low jitter between each other. This can only be recognized if time mensurements are made between the suspected spike components rather than from the stimulus (Hilton-Brown et al. 1985). In this way fiber splitting is more readily detected than with the method in voluntarily contracting muscle where the low jitter may be obscured by the frequently prominent velocity recovery function which adds a "myogenic" jitter due to the (physiologically) uneven firing rates (Trontelj et al. 1990b). Failure to recognize split fibers is not a source of serious error, the only mistake being repeated measurement of the same motor end-plate.
(10) Fiber density by axonal stimulation When stimulus strength is increased smoothly or in very fine steps the motor units are recruited into the response in a discrete succession. It is then possible to count the number of SFAPs joining the recorded response simultaneously in an all-or-none fashion. To be sure that only a single axon is added, the stimulus should be kept at its threshold for a small number of discharges. Superimposing or small spikes are easily missed when joining an already complex response, while those with intermittent blocking may be erroneously counted as belonging to a new axon. Therefore not more than 2-3 different motor units can be easily and reliably sampled from a single stimulation and recording site. The procedure can be made easier by using a computer to control the incrementing and decrementing stimulus and to subtract the previous response from the succeeding one. The usual criteria for accepting a SFAP are adopted. It should be borne in mind that, as in jitter measurement, the results may not be comparable to those obtained in voluntarily activated
o
36
muscle, as the recruitment order of the motor units is inevitably different. While small tonic motor units are expected to be almost exclusively activated by the slight contraction used in the voluntary activation study (at least in the normal subject), intramuscular electrical stimulation of the motor axons may be assumed to activate both small and large units in varying and probably unpredictable proportions, depending on their representation in the stimulated nerve fascicle. Whenever voluntary activation is possible, the study is more easily and reliably done in the standard way and the results can be compared to the available normal material (St~tlberg and Trontelj 1979, Ad hoe Committee of the A A E M Special Interest Group on Single Fiber EMG: Single fiber E M G reference values: a collaborative effort. Submitted, 1991).
(11) N o r m a l values
Normal jitter values have so far been collected for the extensor digitorum communis (EDC) and the orbicularis oculi (Trontelj et al. 1986, 1988). In the EDC, the mean MCD for the ages between 15 and 39 years was 17/zsec, S.D. 8 /.~sec, range of individual values 5 - 7 2 /zsec, and the range of mean MCD for different subjects was from 13 to 23 /zsec. The upper normal limits suggested for the E D C by this study are 40/xsec for the individual motor end-plates and 25 tzsec for the m e a n M C D of 2 0 - 3 0 e n d - p l a t e s ,
In the orbicularis oculi muscle, the mean MCD for adults below 40 years was 12.4/zsec, S.D. 5.6/zsec, the range of individual values was 4 - 5 0 /~sec, and the range of mean MCDs for different subjects was 9-17 /zsec. T h e suggested u p p e r n o r m a l limits are 30 /zsec
(for individual end-plates) and 20 /~sec (for a mean M C D of 20 e n d - p l a t e s ) .
Compared to jitter measured in voluntarily activated muscle fiber pairs, the jitter of stimulated single m o t o r end-plates should, theoretically, be lower by a factor of V/2 on average (St/ilberg and Trontelj 1979). This relationship has been confirmed in practice (Trontelj et al. 1986). Therefore, for any muscle where normal jitter has b e e n established for v o l u n t a r y activation, provi-
sional normal limits can be set by dividing that by v~-, at least until proper normal material is collected. AS in jitter studies with voluntary activation, 1 out of 20 r e a d i n g s e x c e e d i n g t h e i n d i v i d u a l u p p e r n o r m a l
limit is acceptable as normal, The report of the test may include: (1) the number of end-plates studied; (2) t h e numbers of those with blocking, of those with abnormal jitter without blocking and of those with normal jitter; (3) the median MCD of all e n d - p l a t e s (the a r i t h m e t i c m e a n could b e m i s l e a d -
ingly high in case of a few far out-lying values); and (4)
J.V. TRONTELJ, E. STALBERG
the mean MCD of end-plates within the normal range for individual values. The latter two values may provide diagnostically relevant information even in the absence of individual abnormal end-plates (Sanders and Howard 1986). It is also helpful to note the power of the muscle (MRC scale) and the presence or absence of clinical fatigability in case of suspected myasthenia and muscle wasting in cases of other neuromuscular disorders. The finding of normal jitter in a muscle that clinically appears weak and fatigable is a Strong argument against myasthenia. Severely abnormal jitter in a clinically minimally affected muscle is not uncommon in LEMS. Technical data such as stimulus parameters (rate), recording protocol and analysis procedure should also be included. This work was supported by the Ministry of Science and Technology of the Republic of Slovenia, Grant No. C3-0178/306, and the Swedish Research Council, Grant 135. We are indebted to N.M.F. Murray, FRCP, for linguistic corrections.
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