- Email: [email protected]
Age-related botulinum toxin effects on muscle fiber conduction velocity in non-injected muscles
Clinical Neurophysiology 118 (2007) 2398–2403 www.elsevier.com/locate/clinph
Age-related botulinum toxin effects on muscle fiber conduction velocity in non-injected muscles Fiete Lange *, Tiemen W. van Weerden, Johannes H. van der Hoeven Department of Clinical Neurophysiology, University Hospital Groningen, Groningen, The Netherlands Accepted 30 July 2007 Available online 25 September 2007
Abstract Objective: We studied systemic effects of botulinum toxin (BTX) treatment on muscle fiber conduction velocity (MFCV) and possible effects of age. Methods: MFCV was determined by an invasive EMG method in the biceps brachii muscle. Seventeen BTX treated patients and 58 controls were investigated. BTX injections were applied in the neck region or forearm, depending on the indication for treatment. Results: We found an increased ratio between fastest and slowest muscle fiber conduction velocity in BTX treated patients. This suggests systemic BTX effects on MFCV distant from the site of injection, probably fiber atrophy secondary to end-plate dysfunction. Furthermore, we found an increased MFCV in part of the patients, suggesting hypertrophy of some of the muscle fibers. No relation was found between the MFCV disturbances and treatment duration or the cumulative dose of BTX. Conclusions: We found a strong positive correlation between the age and the BTX-induced changes of MFCV in patients, suggesting a BTX related, diminished repair capacity of end-plates or muscle fibers with age. Significance: Our findings suggest a reduced repair capacity of end-plates or muscle fibers in elderly patients. MFCV is a sensitive method to show changes related to damage and compensation of the neuromuscular system. Our finding suggests a decreasing efficiency of repair mechanisms in aging. Ó 2007 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: BTX; MFCV; Aging; Muscle damage; Side effects
1. Introduction Botulinum toxin (BTX) is used in clinical practice to establish a therapeutical end-plate blockade in patients with a spectrum of diseases. Botulinum toxin type A (BTA) (Botox) received Food and Drug Administration (FDA) approval for therapeutic treatment of strabismus and blepharospasm in 1989, cervical dystonia in 2000, and cosmetic treatment of glabellar wrinkles (Botox Cosmetic) in 2002. Most reported adverse events reported to the FDA were lack of effect (63%), injection site reaction * Corresponding author. Address: Department of Clinical Neurophysiology, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands. Tel.: +31 503612425. E-mail address: [email protected] (F. Lange).
(19%) and ptosis (11%) (Cote et al., 2005). BTX acts presynaptic by inhibiting the release of the neurotransmitter acetylcholin (ACh). Local application of BTX is considered safe. Most encountered (side) effects are regional weakness adjacent to the primary site of injection. Possible systemic effects of BTX are general weakness or autonomic complaints. Several authors have shown subclinical systemic effects of locally applied BTX. Lange et al. (1991) showed in a single fiber EMG (SFEMG) study dysfunction of neuromuscular junctions in muscles distant to the site of injection. Ansved et al. (1997) found atrophy of part of the muscle fibers in BTX treated patients using histopathological methods in distant located muscles. Previous studies indicated a linear correlation between muscle fiber diameter and muscle fiber conduction velocity (MFCV) (Blijham et al., 2006; van der Hoeven et al., 1993b). This points to
1388-2457/$32.00 Ó 2007 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2007.07.024
F. Lange et al. / Clinical Neurophysiology 118 (2007) 2398–2403
2399
the use of MFCV determination in detecting (subclinical) systemic effects of BTX. In the present study, we used MFCV determination to detect possible systemic effects of BTX in (non-injected) muscles in a group of patients who received regular treatment for months to years. Additionally we studied the effects of age.
est MFCVs and the ratio between the latter two parameters (fast–slow ratio; F/S ratio). Before or after the measurement we checked for positive sharp waves and fibrillations as signs of recent end-plate loss.
2. Materials and methods
Differences between patients and controls were tested by T-test (unpaired samples after testing for normality) for MFCVmean, MFCVfastest, MCFVslowest and the fast–slow ratio (F/S ratio). Furthermore, we investigated the relation of these variables with, respectively, age, duration of treatment with BTX and total cumulative dose of BTX by Pearson correlation and linear regression analysis. The mean age between patients and controls was significantly different. Therefore, non-parametric analysis (Mann–Whitney) of age versus F/S ratio was added. Statistical significance was accepted at a level of 5%. The MFCVs and the F/S ratio were considered to be abnormal when higher or lower than the mean ± 2 SD of the controls.
The experiments were performed on seventeen patients (aged 20–73 years, mean 52.4, SD 13.7; 6 male, 11 female) who gave their informed consent. Sixteen patients were treated with BTX because of torticollis spasmodicus, one patient for writer’s cramp. The type botulinum toxin was in 10 patients DysportÒ (median dose per injection 410, range 280–560 U) and in seven patients Botox AÒ (median dose per injection 100, range 50–175 U). The median duration of treatment was 64 months (range 3–99). In all cases EMG-guided injection in the target muscles was used. None of the patients had complaints of weakness of muscles other than the target muscles or possibly systemic BTX related complaints. None of the patients had clinical signs or symptoms of dystonia in the investigated muscle. All measurements were performed on the left biceps brachii muscle. The results of the measurements on the patients were compared to 58 healthy controls (aged 21–74 years, mean 38.3 SD 12.4, 34 male, 24 female). 3. EMG recording The experiments were performed in the biceps brachii muscle at rest. We used a modified method of Troni et al. (1983) as described by van der Hoeven et al. (1993a) on a Nicolet EMG apparatus (Viking IV). The patients were lying down with the elbow slightly flexed. A stimulation needle electrode (Dantec 13L64, area of uninsulated tip: 2 mm2) was placed in the distal and superficial part of the muscle. A silver surface electrode (anode) was placed in the antecubital fossa. The muscle was stimulated with gradually increasing strength (suprathreshold) until a clear twitch was palpable. The stimulus ranged between 1 and 2 mA with a duration of 0.2 ms at a fixed frequency of 1 Hz. The recording electrode (concentric needle electrode, recording area 0.02 mm2) was inserted, guided by the palpable twitch about 50–60 mm proximal from the stimulation needle and then manipulated until a polyphasic and reproducible response was obtained. The signal was amplified and bandpass filtered (0.5–10 kHz). We checked for reproducibility by visual inspection of at least four traces. In each patient and control we performed two to four measurements at slightly different locations. The traces with the widest range of MFCVs were used for further analysis to avoid sample errors. Only spikes larger than 20 lV were used for calculations (Fig. 1). All latencies were measured at the positive turning points, resulting in multiple muscle fiber conduction velocities (MFCVs). After that, we calculated the mean MFCV, the fastest and slow-
4. Statistics
5. Results The muscle fiber conduction velocity in our normal subjects was close to the values published in the literature (Blijham et al., 2004; Troni et al., 1983; Zwarts, 1989). In all patients, it was possible to measure the MFCV. The number of spikes was higher in patients than in controls (12.2 SD 0.94 versus 7.5 SD 0.36, respectively). We found significant differences between the BTX patients and the controls for the variables MFCVmean (higher in BTX), MFCVslowest (lower in BTX), MFCVfastest (higher in BTX) and F/S ratio (higher in BTX). Separate analysis of female and male patients revealed the same differences between controls and the BTX patients with one exception: MFCVmean in male BTX patients was higher without reaching the level of significance (for details, see Table 1). Fourteen BTX patients had an abnormal high F/S ratio. In seven BTX patients we found an increased MFCVfastest, in two patients a decreased MFCVslowest and in five patients a combination of increased MFCVfastest and decreased MFCVslowest (see Fig. 2). The F/S ratio did not correlate with the cumulative dose of BTX, the duration of treatment. F/S ratio significantly increased with age in BTX patients (linear regression analysis: p = 0.006, r2 = 0.42; see Fig. 3). The slope of this regression line differs significantly from age-matched controls and all controls (p < 0.0001 for both analysis). F/S ratio in the subgroup analysis (patients versus age-matched controls) was significantly higher in the patient group (p < 0.0001, two tailed, Gaussian approximation). In BTX treated patients, age has a profound influence on MFCVslowest (Fig. 4 shows lower MFCVslowest in older patients). MFCVfastest increased in both controls and BTX patients with age (Fig. 5). The regression lines (patients versus all controls) paralleled each other (p = 0.69), but the Y-intercept was higher in BTX patients (p < 0.0001).
2400
F. Lange et al. / Clinical Neurophysiology 118 (2007) 2398–2403
Fig. 1. Example of determination of MFCV. (a) These traces were rejected because we found much better waveforms after repositioning the needle electrode. (b) Those waveforms were accepted. Note relative high voltage of highest waves, relative high number of waves, high ratio of fastest versus slowest fiber. Black bars above each trace indicate the latency of turning points. Two solid lines indicate the minimum amplitude needed to accept a wave as a potential. The superimposed mode is not presented in this figure but regularly used to get a visual impression of the reproducibility.
6. Discussion The main finding of the present study is a systemic effect of locally applied BTX on MFCV of the biceps brachii muscle. The majority of patients show a clear increase in F/S ratio. This could be the result of the presence of muscle fibers with diminished conduction velocity, the presence of faster than normal conducting fibers or a combination. Differences in muscle fiber conduction velocity are sup-
posed to be related to fiber diameter (Blijham et al., 2006; Ha˚kansson, 1956). In our data, the reason for the elevation of the F/S ratio was mixed (Fig. 2). The patients showed a significant decrease of mean slowest MFCV (see Table 1). Theoretically, several factors can be responsible for the observed decrease in conduction velocity. A decrease of membrane potential (Gruener et al., 1979) as well as muscle fiber atrophy (Ha˚kansson, 1956; van der Hoeven et al., 1993b; van
F. Lange et al. / Clinical Neurophysiology 118 (2007) 2398–2403 Table 1 Muscle fiber conduction velocity: normal values versus BTX patients
3.0 BTX
Normal values
BTX
Female (N) Mean MFCV Slowest MFCV Fastest MFCV F/S ratio
24 3.10 ± 0.21 2.65 ± 029 3.61 ± 0.24 1.38 ± 0.17
11 3.36 ± 0.242* 2.29 ± 0.428* 4.52 ± 0.314*** 2.29 ± 0.454**
Male (N) Mean MFCV Slowest MFCV Fastest MFCV F/S ratio
34 3.26 ± 0.23 2.84 ± 0.25 3.75 ± 0.35 1.33 ± 0.13
6 3.48 ± 0.469ns 2.14 ± 0.386** 4.49 ± 0.486*** 2.13 ± 0.288**
All (N) Mean MFCV Slowest MFCV Fastest MFCV F/S ratio
58 3.19 ± 0.24 2.76 ± 0.28 3.69 ± 0.32 1.35 ± 0.15
16 3.41 ± 0.330* 2.24 ± 0.408*** 4.51 ± 0.368*** 2.07 ± 0.396***
Controls
MFCV values expressed as means ± SD (m/s). Asterisks indicate significant differences: *p < 0.05, **p < 0.01 and ***p < 0.001. ns not significant, p > 0.05. MFCV, muscle fiber conduction velocity; F/S ratio, fastest/ slowest ratio, fastest MFCV slowest MFCV.
2
1
F/S ratio [ ]
2.5
2.0
1.5
1.0 20
3.25
-1
3.00
-2
2.75
-4
-5
40
50
60
70
80
Fig. 3. F/S ratio versus age. The regression lines are dashed for normal subjects and solid for the BTX group. Note the significant difference (p = 0.0001) of slope between controls and BTX patients. The slope of the regression line of the BTX patients deviates significantly from zero (p = 0.006). For the controls the slope does not deviate from zero (p = 0.089).
0
-3
30
Age [years]
MFCVslowest [m/s]
Normalised MFCVslowest []
2401
2.50
2.25
2.00
-6
1.75
0
1
2
3
4
5
BTX
6
Normalised MFCVfastest []
Fig. 2. Patient data expressed as normalized MFCVslowest and MFCVfastest; patient data were normalized by calculating the Z-score based on MFCVslowest and MFCVfastest of the normal subjects in our study. The dashed lines indicate the 2 SD border. The gray filled area visualizes the combined normal MFCVslowest and MFCVfastest. Data outside this area will result in abnormal F/S ratio.
der Hoeven, 1996) will both result in a slowing of conduction velocity. Lange et al. (1991) and Garner et al. (1993) found in a SFEMG study an increased jitter distant from BTX injected muscles, indicating (partial) failure of neuromuscular transmission, probably due to focal end-plate degeneration or dysfunction. We hypothesize that this results in (local) muscle fiber atrophy, and hence in slower conducting fibers. Information about possible fiber type specificity is due to the technique not available.
Controls 1.50 20
30
40
50
60
70
80
Age [years]
Fig. 4. MFCVslowest versus age. The regression lines are dashed for normals and solid for the BTX group. Note the difference of slope between controls and BTX patients. In controls the slope deviates not significantly from zero (linear regression analysis; p = 0.71, r2 = 0.001) whereas in BTX patients the slope differs significantly from zero (p = 0.015, r2 = 0.36).
The patients showed a significant increase of mean fastest MFCV as well. Following the same reasoning, this would suggest hypertrophy of a proportion of the muscle fibers. Slater et al. (2006) found increased fiber diameters in muscle biopsies in patients with impaired neuromuscular transmission (‘limb girdle myasthenia’). Muscle fiber hypertrophy can be the result of relative overuse, as is
2402
F. Lange et al. / Clinical Neurophysiology 118 (2007) 2398–2403 5.5
MFCVfastest [m/s]
5.0
4.5
4.0
3.5
3.0 BTX Controls
2.5 20
30
40
50
60
70
80
Age [years]
Fig. 5. MFCVfastest versus age. Linear regression analysis (dashed for normals, solid for the BTX group). The regression lines parallel each other. Note the higher Y-intercept (p < 0.0001) in BTX patients as well as the slight increase of MFCVfastest with age.
the case in training. However, it can also be found in neuromuscular disease as adaptive response to muscle fiber loss, compensating denervated or otherwise damaged muscle fibers. Our finding of an increased muscle fiber conduction velocity suggests increased fiber diameters as well. The dysfunction of a part of the end-plates could result in a relative overload of the remaining functioning fibers, resulting in secondary hypertrophy of these fibers. However, this is in contrast to the findings of Ansved et al. (1997). They investigated the fiber composition of the vastus lateralis muscle using histochemical methods of patients treated with BTX for cervical dystonia. They found atrophy primarily of IIB fibers, but no clear hypertrophy. An alternative explanation could be relative overuse of the biceps muscle due to subclinical dystonia. In patients with focal dystonias (writers cramp) abnormal muscle activity can be found in proximal shoulder muscles (Deepak and Behari, 1999). In this line of reasoning the hypertrophy of some muscle fibers might be the result of a slight overactivity due to subclinical dystonia of the biceps muscle. However, the lack of clinical signs of distonia argues against this mechanism. Another possible explanation for the increased MFCVfastest is a change in membrane properties (Thesleff et al., 1990). A possible mechanism could be involvement of SNAP-25 protein. Botulinum toxin A cleaves the SNQP-25 protein. This protein is known to inhibit delayed rectifying K-channels and Ca2+ dependent K-channels in smooth muscle of oesophagus (Ji et al., 2002). Those K-channels contribute to repolarisation of membrane potential. Diminished function of SNAP-25 and therefore diminished inhibitory effects on K-channels could possibly lead to hyperpolarisation and secondary to faster conduction velocities. However, the inhibitory effects are not yet described for skeletal muscles.
We found a positive correlation between the F/S ratio and age in patients treated with BTX. There was no clear relation with the cumulative dose of BTX or the duration of treatment. In the control group (Fig. 3), we found no correlation of F/S ratio with age. This suggests that the age effect on the MFCV disturbances is caused by BTX. Generally, age-related changes of motor unit physiology and morphology tend to become detectable beyond the age of 60 years. Stalberg and Fawcett (1982) reported a stable macro EMG signal up to the age of 60 in the biceps brachii muscle. Lexell et al. (1983) found age-related atrophy of the vastus lateralis muscle beyond the age of 70 years, mainly due to atrophy (a decreased diameter of type II fast-twitch fibers). This suggests that healthy motor units are not liable to remodelling processes due to physiological loss of motor units (or motor neurons) up to the age range 60–70. At the level of the neuromuscular junction agerelated changes are found as well. Wokke et al. (1990) found increasing length and branching of the postsynaptic membrane, enlargement of the postsynaptic area and degeneration of junctional folds. Oda (1984) found an increase of the number of preterminal axons entering the end-plates as well as lengthening of the end-plate in older cases. Despite these general age-related morphological changes, we only found MFCV disturbances in the BTX receiving group. This suggests that BTX decreases the efficiency of repair mechanisms with age resulting in neurophysiological (and probably related morphological) changes. This view is supported by the findings of Lange et al. (1991), who found increased fiber density in elderly subjects as a systemic effect of BTX. Meunier et al. (2002) found that BTX has a profound effect on the sprouting of nerve terminals in injected muscles. This sprouting is mainly controlled by muscle derived signalling factors. Welle (1998) found a decreasing efficiency of the capacity of muscle to release signalling factors with age, like insulin-like growth factor-I (IGF-I), protein synthesis and mRNA synthesis. We hypothesize that BTX can enhance age-related function decrease like diminished sprouting capacity, impaired release of signalling factors by the muscle fiber and possibly decreasing recovery speed of muscle fibers. These effects could, alone or in combination, result in the observed relation between MFCV changes and age during BTX therapy. In conclusion, we found clear distant effects of locally applied BTX on muscle fiber conduction velocity, manifesting as slower muscle fiber conduction velocities, faster MFCVs or a combination. The decrease of the slowest MFCV can be explained by partial (disuse) atrophy due to (temporary) end-plate dysfunction. We demonstrated a strong positive correlation between age and fast–slow ratio in the BTX treated patients, suggesting a BTX related decreasing repair capacity with age, such as distal axonal sprouting or muscle fiber regeneration mechanisms. Our finding suggests a decreasing efficiency of repair mechanisms in aging. This could theoretically be an important factor in age-related diseases.
F. Lange et al. / Clinical Neurophysiology 118 (2007) 2398–2403
References Ansved T, Odergren T, Borg K. Muscle fiber atrophy in leg muscles after botulinum toxin type A treatment of cervical dystonia. Neurology 1997;48:1440–2. Blijham PJ, Hengstman GJ, ter Laak HJ, van Engelen BG, Zwarts MJ. Muscle-fiber conduction velocity and electromyography as diagnostic tools in patients with suspected inflammatory myopathy: a prospective study. Muscle Nerve 2004;29:46–50. Blijham PJ, ter Laak HJ, Schelhaas HJ, van Engelen BG, Stegeman DF, Zwarts MJ. Relation between muscle fiber conduction velocity and fiber size in neuromuscular disorders. J Appl Physiol 2006;100:1837–41. Cote TR, Mohan AK, Polder JA, Walton MK, Braun MM. Botulinum toxin type A injections: adverse events reported to the US Food and Drug Administration in therapeutic and cosmetic cases. J Am Acad Dermatol 2005;53:407–15. Deepak KK, Behari M. Specific muscle EMG biofeedback for hand dystonia. Appl Psychophysiol Biofeedback 1999;24:267–80. Garner CG, Straube A, Witt TN, Gasser T, Oertel WH. Time course of distant effects of local injections of botulinum toxin. Mov Disord 1993;8:33–7. Gruener R, Stern LZ, Markovitz D, Gerdes C. Electrophysiologic properties of intercostal muscle fibers in human neuromuscular diseases. Muscle Nerve 1979;2:165–72. Ha˚kansson CH. Conduction velocity and amplitude of the action potential as related to circumference of the isolated fibre in of frog muscle. Acta Physiol Scand 1956;37:14–34. Ji J, Salapatek AM, Lau H, Wang G, Gaisano HY, Diamant NE. SNAP25, a SNARE protein, inhibits two types of K channels in esophageal smooth muscle. Gastroenterology 2002;122:994–1006. Lange DJ, Rubin M, Greene PE, Kang UJ, Moskowitz CB, Brin MF, et al. Distant effects of locally injected botulinum toxin: a doubleblind study of single fiber EMG changes. Muscle Nerve 1991;14: 672–5. Lexell J, Henriksson-Larsen K, Winblad B, Sjostrom M. Distribution of different fiber types in human skeletal muscles: effects of aging
2403
studied in whole muscle cross sections. Muscle Nerve 1983;6:588–95. Meunier FA, Schiavo G, Molgo J. Botulinum neurotoxins: from paralysis to recovery of functional neuromuscular transmission. J Physiol (Paris) 2002;96:105–13. Oda K. Age changes of motor innervation and acetylcholine receptor distribution on human skeletal muscle fibres. J Neurol Sci 1984;66: 327–38. Slater CR, Fawcett PR, Walls TJ, Lyons PR, Bailey SJ, Beeson D, et al. Pre- and post-synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with ‘limb-girdle myasthenia’. Brain 2006;129:2061–76. Stalberg E, Fawcett PR. Macro EMG in healthy subjects of different ages. J Neurol Neurosurg Psychiatry 1982;45:870–8. Thesleff S, Molgo J, Tagerud S. Trophic interrelations at the neuromuscular junction as revealed by the use of botulinal neurotoxins. J Physiol (Paris) 1990;84:167–73. Troni W, Cantello R, Rainero I. Conduction velocity along human muscle fibers in situ. Neurology 1983;33:1453–9. van der Hoeven JH. Decline of muscle fiber conduction velocity during short-term high-dose methylprednisolone therapy. Muscle Nerve 1996;19:100–2. van der Hoeven JH, van Weerden TW, Zwarts MJ. Long-lasting supernormal conduction velocity after sustained maximal isometric contraction in human muscle. Muscle Nerve 1993a;16:312–20. van der Hoeven JH, Zwarts MJ, van Weerden TW. Muscle fiber conduction velocity in amyotrophic lateral sclerosis and traumatic lesions of the plexus brachialis. Electroencephalogr Clin Neurophysiol 1993b;89:304–10. Welle S. Growth hormone and insulin-like growth factor-I as anabolic agents. Curr Opin Clin Nutr Metab Care 1998;1:257–62. Wokke JH, Jennekens FG, van den Oord CJ, Veldman H, Smit LM, Leppink GJ. Morphological changes in the human end plate with age. J Neurol Sci 1990;95:291–310. Zwarts MJ. Evaluation of the estimation of muscle fiber conduction velocity. Surface versus needle method. Electroencephalogr Clin Neurophysiol 1989;73:544–8.
Age-related botulinum toxin effects on muscle fiber conduction velocity in non-injected muscles Fiete Lange *, Tiemen W. van Weerden, Johannes H. van der Hoeven Department of Clinical Neurophysiology, University Hospital Groningen, Groningen, The Netherlands Accepted 30 July 2007 Available online 25 September 2007
Abstract Objective: We studied systemic effects of botulinum toxin (BTX) treatment on muscle fiber conduction velocity (MFCV) and possible effects of age. Methods: MFCV was determined by an invasive EMG method in the biceps brachii muscle. Seventeen BTX treated patients and 58 controls were investigated. BTX injections were applied in the neck region or forearm, depending on the indication for treatment. Results: We found an increased ratio between fastest and slowest muscle fiber conduction velocity in BTX treated patients. This suggests systemic BTX effects on MFCV distant from the site of injection, probably fiber atrophy secondary to end-plate dysfunction. Furthermore, we found an increased MFCV in part of the patients, suggesting hypertrophy of some of the muscle fibers. No relation was found between the MFCV disturbances and treatment duration or the cumulative dose of BTX. Conclusions: We found a strong positive correlation between the age and the BTX-induced changes of MFCV in patients, suggesting a BTX related, diminished repair capacity of end-plates or muscle fibers with age. Significance: Our findings suggest a reduced repair capacity of end-plates or muscle fibers in elderly patients. MFCV is a sensitive method to show changes related to damage and compensation of the neuromuscular system. Our finding suggests a decreasing efficiency of repair mechanisms in aging. Ó 2007 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: BTX; MFCV; Aging; Muscle damage; Side effects
1. Introduction Botulinum toxin (BTX) is used in clinical practice to establish a therapeutical end-plate blockade in patients with a spectrum of diseases. Botulinum toxin type A (BTA) (Botox) received Food and Drug Administration (FDA) approval for therapeutic treatment of strabismus and blepharospasm in 1989, cervical dystonia in 2000, and cosmetic treatment of glabellar wrinkles (Botox Cosmetic) in 2002. Most reported adverse events reported to the FDA were lack of effect (63%), injection site reaction * Corresponding author. Address: Department of Clinical Neurophysiology, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands. Tel.: +31 503612425. E-mail address: [email protected] (F. Lange).
(19%) and ptosis (11%) (Cote et al., 2005). BTX acts presynaptic by inhibiting the release of the neurotransmitter acetylcholin (ACh). Local application of BTX is considered safe. Most encountered (side) effects are regional weakness adjacent to the primary site of injection. Possible systemic effects of BTX are general weakness or autonomic complaints. Several authors have shown subclinical systemic effects of locally applied BTX. Lange et al. (1991) showed in a single fiber EMG (SFEMG) study dysfunction of neuromuscular junctions in muscles distant to the site of injection. Ansved et al. (1997) found atrophy of part of the muscle fibers in BTX treated patients using histopathological methods in distant located muscles. Previous studies indicated a linear correlation between muscle fiber diameter and muscle fiber conduction velocity (MFCV) (Blijham et al., 2006; van der Hoeven et al., 1993b). This points to
1388-2457/$32.00 Ó 2007 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2007.07.024
F. Lange et al. / Clinical Neurophysiology 118 (2007) 2398–2403
2399
the use of MFCV determination in detecting (subclinical) systemic effects of BTX. In the present study, we used MFCV determination to detect possible systemic effects of BTX in (non-injected) muscles in a group of patients who received regular treatment for months to years. Additionally we studied the effects of age.
est MFCVs and the ratio between the latter two parameters (fast–slow ratio; F/S ratio). Before or after the measurement we checked for positive sharp waves and fibrillations as signs of recent end-plate loss.
2. Materials and methods
Differences between patients and controls were tested by T-test (unpaired samples after testing for normality) for MFCVmean, MFCVfastest, MCFVslowest and the fast–slow ratio (F/S ratio). Furthermore, we investigated the relation of these variables with, respectively, age, duration of treatment with BTX and total cumulative dose of BTX by Pearson correlation and linear regression analysis. The mean age between patients and controls was significantly different. Therefore, non-parametric analysis (Mann–Whitney) of age versus F/S ratio was added. Statistical significance was accepted at a level of 5%. The MFCVs and the F/S ratio were considered to be abnormal when higher or lower than the mean ± 2 SD of the controls.
The experiments were performed on seventeen patients (aged 20–73 years, mean 52.4, SD 13.7; 6 male, 11 female) who gave their informed consent. Sixteen patients were treated with BTX because of torticollis spasmodicus, one patient for writer’s cramp. The type botulinum toxin was in 10 patients DysportÒ (median dose per injection 410, range 280–560 U) and in seven patients Botox AÒ (median dose per injection 100, range 50–175 U). The median duration of treatment was 64 months (range 3–99). In all cases EMG-guided injection in the target muscles was used. None of the patients had complaints of weakness of muscles other than the target muscles or possibly systemic BTX related complaints. None of the patients had clinical signs or symptoms of dystonia in the investigated muscle. All measurements were performed on the left biceps brachii muscle. The results of the measurements on the patients were compared to 58 healthy controls (aged 21–74 years, mean 38.3 SD 12.4, 34 male, 24 female). 3. EMG recording The experiments were performed in the biceps brachii muscle at rest. We used a modified method of Troni et al. (1983) as described by van der Hoeven et al. (1993a) on a Nicolet EMG apparatus (Viking IV). The patients were lying down with the elbow slightly flexed. A stimulation needle electrode (Dantec 13L64, area of uninsulated tip: 2 mm2) was placed in the distal and superficial part of the muscle. A silver surface electrode (anode) was placed in the antecubital fossa. The muscle was stimulated with gradually increasing strength (suprathreshold) until a clear twitch was palpable. The stimulus ranged between 1 and 2 mA with a duration of 0.2 ms at a fixed frequency of 1 Hz. The recording electrode (concentric needle electrode, recording area 0.02 mm2) was inserted, guided by the palpable twitch about 50–60 mm proximal from the stimulation needle and then manipulated until a polyphasic and reproducible response was obtained. The signal was amplified and bandpass filtered (0.5–10 kHz). We checked for reproducibility by visual inspection of at least four traces. In each patient and control we performed two to four measurements at slightly different locations. The traces with the widest range of MFCVs were used for further analysis to avoid sample errors. Only spikes larger than 20 lV were used for calculations (Fig. 1). All latencies were measured at the positive turning points, resulting in multiple muscle fiber conduction velocities (MFCVs). After that, we calculated the mean MFCV, the fastest and slow-
4. Statistics
5. Results The muscle fiber conduction velocity in our normal subjects was close to the values published in the literature (Blijham et al., 2004; Troni et al., 1983; Zwarts, 1989). In all patients, it was possible to measure the MFCV. The number of spikes was higher in patients than in controls (12.2 SD 0.94 versus 7.5 SD 0.36, respectively). We found significant differences between the BTX patients and the controls for the variables MFCVmean (higher in BTX), MFCVslowest (lower in BTX), MFCVfastest (higher in BTX) and F/S ratio (higher in BTX). Separate analysis of female and male patients revealed the same differences between controls and the BTX patients with one exception: MFCVmean in male BTX patients was higher without reaching the level of significance (for details, see Table 1). Fourteen BTX patients had an abnormal high F/S ratio. In seven BTX patients we found an increased MFCVfastest, in two patients a decreased MFCVslowest and in five patients a combination of increased MFCVfastest and decreased MFCVslowest (see Fig. 2). The F/S ratio did not correlate with the cumulative dose of BTX, the duration of treatment. F/S ratio significantly increased with age in BTX patients (linear regression analysis: p = 0.006, r2 = 0.42; see Fig. 3). The slope of this regression line differs significantly from age-matched controls and all controls (p < 0.0001 for both analysis). F/S ratio in the subgroup analysis (patients versus age-matched controls) was significantly higher in the patient group (p < 0.0001, two tailed, Gaussian approximation). In BTX treated patients, age has a profound influence on MFCVslowest (Fig. 4 shows lower MFCVslowest in older patients). MFCVfastest increased in both controls and BTX patients with age (Fig. 5). The regression lines (patients versus all controls) paralleled each other (p = 0.69), but the Y-intercept was higher in BTX patients (p < 0.0001).
2400
F. Lange et al. / Clinical Neurophysiology 118 (2007) 2398–2403
Fig. 1. Example of determination of MFCV. (a) These traces were rejected because we found much better waveforms after repositioning the needle electrode. (b) Those waveforms were accepted. Note relative high voltage of highest waves, relative high number of waves, high ratio of fastest versus slowest fiber. Black bars above each trace indicate the latency of turning points. Two solid lines indicate the minimum amplitude needed to accept a wave as a potential. The superimposed mode is not presented in this figure but regularly used to get a visual impression of the reproducibility.
6. Discussion The main finding of the present study is a systemic effect of locally applied BTX on MFCV of the biceps brachii muscle. The majority of patients show a clear increase in F/S ratio. This could be the result of the presence of muscle fibers with diminished conduction velocity, the presence of faster than normal conducting fibers or a combination. Differences in muscle fiber conduction velocity are sup-
posed to be related to fiber diameter (Blijham et al., 2006; Ha˚kansson, 1956). In our data, the reason for the elevation of the F/S ratio was mixed (Fig. 2). The patients showed a significant decrease of mean slowest MFCV (see Table 1). Theoretically, several factors can be responsible for the observed decrease in conduction velocity. A decrease of membrane potential (Gruener et al., 1979) as well as muscle fiber atrophy (Ha˚kansson, 1956; van der Hoeven et al., 1993b; van
F. Lange et al. / Clinical Neurophysiology 118 (2007) 2398–2403 Table 1 Muscle fiber conduction velocity: normal values versus BTX patients
3.0 BTX
Normal values
BTX
Female (N) Mean MFCV Slowest MFCV Fastest MFCV F/S ratio
24 3.10 ± 0.21 2.65 ± 029 3.61 ± 0.24 1.38 ± 0.17
11 3.36 ± 0.242* 2.29 ± 0.428* 4.52 ± 0.314*** 2.29 ± 0.454**
Male (N) Mean MFCV Slowest MFCV Fastest MFCV F/S ratio
34 3.26 ± 0.23 2.84 ± 0.25 3.75 ± 0.35 1.33 ± 0.13
6 3.48 ± 0.469ns 2.14 ± 0.386** 4.49 ± 0.486*** 2.13 ± 0.288**
All (N) Mean MFCV Slowest MFCV Fastest MFCV F/S ratio
58 3.19 ± 0.24 2.76 ± 0.28 3.69 ± 0.32 1.35 ± 0.15
16 3.41 ± 0.330* 2.24 ± 0.408*** 4.51 ± 0.368*** 2.07 ± 0.396***
Controls
MFCV values expressed as means ± SD (m/s). Asterisks indicate significant differences: *p < 0.05, **p < 0.01 and ***p < 0.001. ns not significant, p > 0.05. MFCV, muscle fiber conduction velocity; F/S ratio, fastest/ slowest ratio, fastest MFCV slowest MFCV.
2
1
F/S ratio [ ]
2.5
2.0
1.5
1.0 20
3.25
-1
3.00
-2
2.75
-4
-5
40
50
60
70
80
Fig. 3. F/S ratio versus age. The regression lines are dashed for normal subjects and solid for the BTX group. Note the significant difference (p = 0.0001) of slope between controls and BTX patients. The slope of the regression line of the BTX patients deviates significantly from zero (p = 0.006). For the controls the slope does not deviate from zero (p = 0.089).
0
-3
30
Age [years]
MFCVslowest [m/s]
Normalised MFCVslowest []
2401
2.50
2.25
2.00
-6
1.75
0
1
2
3
4
5
BTX
6
Normalised MFCVfastest []
Fig. 2. Patient data expressed as normalized MFCVslowest and MFCVfastest; patient data were normalized by calculating the Z-score based on MFCVslowest and MFCVfastest of the normal subjects in our study. The dashed lines indicate the 2 SD border. The gray filled area visualizes the combined normal MFCVslowest and MFCVfastest. Data outside this area will result in abnormal F/S ratio.
der Hoeven, 1996) will both result in a slowing of conduction velocity. Lange et al. (1991) and Garner et al. (1993) found in a SFEMG study an increased jitter distant from BTX injected muscles, indicating (partial) failure of neuromuscular transmission, probably due to focal end-plate degeneration or dysfunction. We hypothesize that this results in (local) muscle fiber atrophy, and hence in slower conducting fibers. Information about possible fiber type specificity is due to the technique not available.
Controls 1.50 20
30
40
50
60
70
80
Age [years]
Fig. 4. MFCVslowest versus age. The regression lines are dashed for normals and solid for the BTX group. Note the difference of slope between controls and BTX patients. In controls the slope deviates not significantly from zero (linear regression analysis; p = 0.71, r2 = 0.001) whereas in BTX patients the slope differs significantly from zero (p = 0.015, r2 = 0.36).
The patients showed a significant increase of mean fastest MFCV as well. Following the same reasoning, this would suggest hypertrophy of a proportion of the muscle fibers. Slater et al. (2006) found increased fiber diameters in muscle biopsies in patients with impaired neuromuscular transmission (‘limb girdle myasthenia’). Muscle fiber hypertrophy can be the result of relative overuse, as is
2402
F. Lange et al. / Clinical Neurophysiology 118 (2007) 2398–2403 5.5
MFCVfastest [m/s]
5.0
4.5
4.0
3.5
3.0 BTX Controls
2.5 20
30
40
50
60
70
80
Age [years]
Fig. 5. MFCVfastest versus age. Linear regression analysis (dashed for normals, solid for the BTX group). The regression lines parallel each other. Note the higher Y-intercept (p < 0.0001) in BTX patients as well as the slight increase of MFCVfastest with age.
the case in training. However, it can also be found in neuromuscular disease as adaptive response to muscle fiber loss, compensating denervated or otherwise damaged muscle fibers. Our finding of an increased muscle fiber conduction velocity suggests increased fiber diameters as well. The dysfunction of a part of the end-plates could result in a relative overload of the remaining functioning fibers, resulting in secondary hypertrophy of these fibers. However, this is in contrast to the findings of Ansved et al. (1997). They investigated the fiber composition of the vastus lateralis muscle using histochemical methods of patients treated with BTX for cervical dystonia. They found atrophy primarily of IIB fibers, but no clear hypertrophy. An alternative explanation could be relative overuse of the biceps muscle due to subclinical dystonia. In patients with focal dystonias (writers cramp) abnormal muscle activity can be found in proximal shoulder muscles (Deepak and Behari, 1999). In this line of reasoning the hypertrophy of some muscle fibers might be the result of a slight overactivity due to subclinical dystonia of the biceps muscle. However, the lack of clinical signs of distonia argues against this mechanism. Another possible explanation for the increased MFCVfastest is a change in membrane properties (Thesleff et al., 1990). A possible mechanism could be involvement of SNAP-25 protein. Botulinum toxin A cleaves the SNQP-25 protein. This protein is known to inhibit delayed rectifying K-channels and Ca2+ dependent K-channels in smooth muscle of oesophagus (Ji et al., 2002). Those K-channels contribute to repolarisation of membrane potential. Diminished function of SNAP-25 and therefore diminished inhibitory effects on K-channels could possibly lead to hyperpolarisation and secondary to faster conduction velocities. However, the inhibitory effects are not yet described for skeletal muscles.
We found a positive correlation between the F/S ratio and age in patients treated with BTX. There was no clear relation with the cumulative dose of BTX or the duration of treatment. In the control group (Fig. 3), we found no correlation of F/S ratio with age. This suggests that the age effect on the MFCV disturbances is caused by BTX. Generally, age-related changes of motor unit physiology and morphology tend to become detectable beyond the age of 60 years. Stalberg and Fawcett (1982) reported a stable macro EMG signal up to the age of 60 in the biceps brachii muscle. Lexell et al. (1983) found age-related atrophy of the vastus lateralis muscle beyond the age of 70 years, mainly due to atrophy (a decreased diameter of type II fast-twitch fibers). This suggests that healthy motor units are not liable to remodelling processes due to physiological loss of motor units (or motor neurons) up to the age range 60–70. At the level of the neuromuscular junction agerelated changes are found as well. Wokke et al. (1990) found increasing length and branching of the postsynaptic membrane, enlargement of the postsynaptic area and degeneration of junctional folds. Oda (1984) found an increase of the number of preterminal axons entering the end-plates as well as lengthening of the end-plate in older cases. Despite these general age-related morphological changes, we only found MFCV disturbances in the BTX receiving group. This suggests that BTX decreases the efficiency of repair mechanisms with age resulting in neurophysiological (and probably related morphological) changes. This view is supported by the findings of Lange et al. (1991), who found increased fiber density in elderly subjects as a systemic effect of BTX. Meunier et al. (2002) found that BTX has a profound effect on the sprouting of nerve terminals in injected muscles. This sprouting is mainly controlled by muscle derived signalling factors. Welle (1998) found a decreasing efficiency of the capacity of muscle to release signalling factors with age, like insulin-like growth factor-I (IGF-I), protein synthesis and mRNA synthesis. We hypothesize that BTX can enhance age-related function decrease like diminished sprouting capacity, impaired release of signalling factors by the muscle fiber and possibly decreasing recovery speed of muscle fibers. These effects could, alone or in combination, result in the observed relation between MFCV changes and age during BTX therapy. In conclusion, we found clear distant effects of locally applied BTX on muscle fiber conduction velocity, manifesting as slower muscle fiber conduction velocities, faster MFCVs or a combination. The decrease of the slowest MFCV can be explained by partial (disuse) atrophy due to (temporary) end-plate dysfunction. We demonstrated a strong positive correlation between age and fast–slow ratio in the BTX treated patients, suggesting a BTX related decreasing repair capacity with age, such as distal axonal sprouting or muscle fiber regeneration mechanisms. Our finding suggests a decreasing efficiency of repair mechanisms in aging. This could theoretically be an important factor in age-related diseases.
F. Lange et al. / Clinical Neurophysiology 118 (2007) 2398–2403
References Ansved T, Odergren T, Borg K. Muscle fiber atrophy in leg muscles after botulinum toxin type A treatment of cervical dystonia. Neurology 1997;48:1440–2. Blijham PJ, Hengstman GJ, ter Laak HJ, van Engelen BG, Zwarts MJ. Muscle-fiber conduction velocity and electromyography as diagnostic tools in patients with suspected inflammatory myopathy: a prospective study. Muscle Nerve 2004;29:46–50. Blijham PJ, ter Laak HJ, Schelhaas HJ, van Engelen BG, Stegeman DF, Zwarts MJ. Relation between muscle fiber conduction velocity and fiber size in neuromuscular disorders. J Appl Physiol 2006;100:1837–41. Cote TR, Mohan AK, Polder JA, Walton MK, Braun MM. Botulinum toxin type A injections: adverse events reported to the US Food and Drug Administration in therapeutic and cosmetic cases. J Am Acad Dermatol 2005;53:407–15. Deepak KK, Behari M. Specific muscle EMG biofeedback for hand dystonia. Appl Psychophysiol Biofeedback 1999;24:267–80. Garner CG, Straube A, Witt TN, Gasser T, Oertel WH. Time course of distant effects of local injections of botulinum toxin. Mov Disord 1993;8:33–7. Gruener R, Stern LZ, Markovitz D, Gerdes C. Electrophysiologic properties of intercostal muscle fibers in human neuromuscular diseases. Muscle Nerve 1979;2:165–72. Ha˚kansson CH. Conduction velocity and amplitude of the action potential as related to circumference of the isolated fibre in of frog muscle. Acta Physiol Scand 1956;37:14–34. Ji J, Salapatek AM, Lau H, Wang G, Gaisano HY, Diamant NE. SNAP25, a SNARE protein, inhibits two types of K channels in esophageal smooth muscle. Gastroenterology 2002;122:994–1006. Lange DJ, Rubin M, Greene PE, Kang UJ, Moskowitz CB, Brin MF, et al. Distant effects of locally injected botulinum toxin: a doubleblind study of single fiber EMG changes. Muscle Nerve 1991;14: 672–5. Lexell J, Henriksson-Larsen K, Winblad B, Sjostrom M. Distribution of different fiber types in human skeletal muscles: effects of aging
2403
studied in whole muscle cross sections. Muscle Nerve 1983;6:588–95. Meunier FA, Schiavo G, Molgo J. Botulinum neurotoxins: from paralysis to recovery of functional neuromuscular transmission. J Physiol (Paris) 2002;96:105–13. Oda K. Age changes of motor innervation and acetylcholine receptor distribution on human skeletal muscle fibres. J Neurol Sci 1984;66: 327–38. Slater CR, Fawcett PR, Walls TJ, Lyons PR, Bailey SJ, Beeson D, et al. Pre- and post-synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with ‘limb-girdle myasthenia’. Brain 2006;129:2061–76. Stalberg E, Fawcett PR. Macro EMG in healthy subjects of different ages. J Neurol Neurosurg Psychiatry 1982;45:870–8. Thesleff S, Molgo J, Tagerud S. Trophic interrelations at the neuromuscular junction as revealed by the use of botulinal neurotoxins. J Physiol (Paris) 1990;84:167–73. Troni W, Cantello R, Rainero I. Conduction velocity along human muscle fibers in situ. Neurology 1983;33:1453–9. van der Hoeven JH. Decline of muscle fiber conduction velocity during short-term high-dose methylprednisolone therapy. Muscle Nerve 1996;19:100–2. van der Hoeven JH, van Weerden TW, Zwarts MJ. Long-lasting supernormal conduction velocity after sustained maximal isometric contraction in human muscle. Muscle Nerve 1993a;16:312–20. van der Hoeven JH, Zwarts MJ, van Weerden TW. Muscle fiber conduction velocity in amyotrophic lateral sclerosis and traumatic lesions of the plexus brachialis. Electroencephalogr Clin Neurophysiol 1993b;89:304–10. Welle S. Growth hormone and insulin-like growth factor-I as anabolic agents. Curr Opin Clin Nutr Metab Care 1998;1:257–62. Wokke JH, Jennekens FG, van den Oord CJ, Veldman H, Smit LM, Leppink GJ. Morphological changes in the human end plate with age. J Neurol Sci 1990;95:291–310. Zwarts MJ. Evaluation of the estimation of muscle fiber conduction velocity. Surface versus needle method. Electroencephalogr Clin Neurophysiol 1989;73:544–8.