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Neurophysiological changes after intramuscular injection of botulinum toxin
Clinical Neurophysiology 123 (2012) 54–60
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Neurophysiological changes after intramuscular injection of botulinum toxin Francisco J. Palomar, Pablo Mir ⇑ Unidad de Trastornos del Movimiento, Servicio de Neurología y Neurofisiología Clínica, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain
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Article history: Available online 2 November 2011 Keywords: Clinical neurophysiology Botulinum toxin Brainstem Peripheral nervous system Spinal cord reflexes Central nervous system
h i g h l i g h t s This review is focussed on neurophysiological changes after botulinum toxin (BT) injection. Will help readers to understand better what are the distant effects of BT treatment. Will help readers to know about the different neurophysiological techniques to study functional changes after BT treatment.
a b s t r a c t Botulinum toxin (BT) acts peripherally by inhibiting acetylcholine release from the presynaptic neuromuscular terminals and by weakening muscle contraction. Therefore, its clinical benefit is primarily due to its peripheral action. As a result, local injection of BT has become a successful and safe tool in the treatment of several neurological and non-neurological disorders. Studies in animals have also shown that the toxin can be retrogradely transported and even transcytosed to neurons in the central nervous system (CNS). Further human studies have suggested that BT could alter the functional organisation of the CNS indirectly through peripheral mechanisms. BT can interfere with and modify spinal, brainstem and cortical circuits, including cortical excitability and plasticity/organisation by altering spindle afferent inflow directed to spinal motoneurons or to the various cortical areas. It is well demonstrated that the distant CNS effects of BT treatment parallel the peripheral effect, although there is limited evidence as to the cause of this. Therefore, further studies focussed on central changes after BT treatment is needed for a better understanding of these non-peripheral effects of BT. Ó 2011 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7. 8.
Introduction . . . . . . . . . . . . . . . . . . . . . . . Studies in animals . . . . . . . . . . . . . . . . . . Effects on the peripheral neuromuscular Effects on spinal cord mechanisms . . . . . Effects on brainstem mechanisms. . . . . . Effects on cortical mechanisms . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .
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⇑ Corresponding author at: Unidad de Trastornos del Movimiento, Servicio de Neurología y Neurofisiología Clínica, Hospital Universitario Virgen del Rocío, Avda. Manuel Siurot sn, 41013 Seville, Spain. Tel.: +34 955012593; fax: +34 955012597. E-mail address: [email protected] (P. Mir). 1388-2457/$36.00 Ó 2011 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2011.05.032
F.J. Palomar, P. Mir / Clinical Neurophysiology 123 (2012) 54–60
1. Introduction Botulinum toxin (BT) is a metalloproteinase that, when injected intramuscularly, weakens muscle power and induces denervation for a well-understood time period (Hamjian and Walker, 1994). This biochemical action induces further denervation of extrafusal muscle fibres (Simpson, 1981). The molecular mechanism underlying the local action of BTX-A has been best characterised at the neuromuscular junction, where the toxin acts by cleaving essential proteins in the vesicle re-cycling machinery. Over the past two decades, BT has become a safe and effective therapeutic tool for neurological and non-neurological conditions (Simpson et al., 2008a,b). BT is now widely used to treat a variety of clinical conditions characterised by muscle hyperactivity, such as focal dystonia (blepharospasm, torticollis, spasmodic dysphonia and writer’s cramp (WC)), hemifacial spasm, tremor and spasticity. The use of BT can also improve pain associated with these conditions, probably via central effects rather than the well-established effect on muscle. BT also blocks parasympathetic and postganglionic sympathetic cholinergic nerve synapses in the autonomic nervous system. This collateral action also allows clinicians to use BT to treat overactive smooth muscle (anal fissure and outlet-type constipation and oesophageal achalasia) as well as secretory disorders (focal hyperhidrosis, gustatory sweating and sialorrhoea) (Naumann and Jost, 2004). BT can also be used in cosmetics; however, the effect of BT treatment for this purpose is limited to its direct biochemical action of muscle paralysis. It is generally accepted that the clinical benefits of BT injections depend primarily on the toxin’s peripheral action. Experimental studies in animals and humans have also provided evidence that BT can act centrally and at distant sites from where the injection was performed (Simpson, 1981). These central mechanisms might also help to explain why BT injections sometimes leave the injected muscles disproportionately weak. The aim of this article is to summarise the neurophysiological changes that BT injections cause at different sites in humans.
2. Studies in animals After injecting BT into skeletal muscles, it acts at the intrafusal and the extrafusal neuromuscular junctions. These two effects start within 80 min of the injection. A morphological study compared the effects of BT on extrafusal and intrafusal muscle fibres in rats (Rosales et al., 1996). The toxin caused fibre atrophy and acetylcholine staining to spread into the end plates, indicating a general denervation of extrafusal and intrafusal fibres. This point was also reported by Filippi et al. (1993) by studying the effect of BT injected into the jaw muscles of rats. The final conclusion is that BT acts not only on the alpha motor endings but also on gamma motor endings, leading to a reduction of the spindle input to the alpha motoneurons. The alpha motoneurons are critical in maintaining the tonic myotatic reflex, suggesting that the relief in dystonic patients cannot be solely due to muscle paralysis, but also due to a decrease in the tonic myotatic reflex. The question of central effects depends on the ability of BT to pass through the blood–brain barrier. Labelled toxin was detected by means of autoradiography and fluorescent labelling in the brain parenchyma of mice after high doses of BT marked with 125I were injected intravenously (Boroff and Chen, 1975). The BT dose used in this study was extremely high (1.5 105 U) compared to the low doses used in therapeutic clinical applications. In fact, the animals used survived only between 35 and 38 min. The authors suggested that this short survival time was related to the high doses of labelled BT injected. The method used in this study was able to demonstrate the presence of the labelled BT in the brain blood ves-
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sels and quite far from them in the brain parenchyma. These results were not present in control animals where no BT was injected. However, there is no evidence at the present time that low therapeutic doses of BT could diffuse through the blood stream and cross the blood–brain barrier.
3. Effects on the peripheral neuromuscular system The time course of the effects of intramuscular BT has been described in studies recording the electromyographic (EMG) activity from the injected muscles. Compound muscle action potential (CMAP) amplitude in the injected extensor digitorum brevis (EDB) muscle starts to decrease 48 h after injection and the maximum decrease occurs between days 7 and 21. The CMAP amplitude remains decreased for at least 90 days following a full recovery (Hamjian and Walker, 1994; Eleopra et al., 1998). Because of its easy application and low cost (compared to antibody assays), this effect is currently used in the EDB muscle as an initial investigative tool to observe the responsiveness of a patient before starting BT treatment and/or to verify the cause of secondary non-responsiveness (Cordivari et al., 2006). The CMAP reduction is produced at the same time as a decrease in the mean rectified voltage during maximal voluntary contraction (MVC) (Hamjian and Walker, 1994). Spontaneous denervation follows the reduced EMG recruitment pattern 20 days after BT injection in normal muscles (Van Putten et al., 2002), and muscle atrophy was observed 10–20 days (peaking at day 42) after injection in spastic muscles (Hamjian and Walker, 1994) with motor unit rearrangement. Bertolasi et al. (1997) found an increase in the muscle cramp threshold frequency when measured with repetitive electrical peripheral nerve stimulation in patients with inherited benign fasciculation syndrome. These findings support the idea that BT acts mainly on presynaptic release of acetylcholine in motor nerve terminals. However, this action is not the same in all areas of the injected muscle. Recently, it has been proven that the effect of the injected BT can be increased when it is injected precisely in the end plate zone (Lapatki et al., 2010). The authors suggest that this differential effect of the injection site within the muscle could be a promising strategy for minimising the amount of injected BT and, consequently, the appearance of unwanted side effects such as weakness of adjacent muscles. The toxin’s peripheral action also depends on the state of the muscle in which BT is injected. This was investigated first in animals by Hughes and Whaler (1962) in rat diaphragm preparations with BT, where indirect stimulation of the BT diaphragm preparation anticipates the induced paralysis. This study suggests that the advance in the onset of paralysis observed in electrically stimulated preparations was due to an increase in muscular contraction and in the nerve-ending activity. Recent animal studies confirmed this suggestion using stretching exercises in BT-injected animals (Kim et al., 2003). Further studies in humans have shown similar effects to animal studies with regard to enhanced toxin uptake and acceleration in the onset of the paralytic effect after electrical stimulation (mainly at low frequency), stretching exercise (Glocker et al., 1995; Eleopra et al., 1997) or writing in the case of patients suffering from WC (Chen et al., 1999). These neurophysiologic characteristics of BT’s effectiveness and its mechanisms of action are associated with a better clinical improvement. This was reported in hemiparetic patients with lower (Hesse et al., 1995) and upper (Hesse et al., 1998) limb spasticity. Patients who received a combined treatment of BT injection and repetitive electrical stimulation of injected muscles had a better improvement of gait parameters, such as velocity, stride length, stance and swing symmetry, Ashworth score and in upper limb motor tasks after
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F.J. Palomar, P. Mir / Clinical Neurophysiology 123 (2012) 54–60
the BT injection. This enhanced improvement was paralleled by a greater reduction in the muscle tone of muscles that received the combined treatment, suggesting that this additional electrical stimulation enhances the effectiveness of BT in the treatment of spasticity after stroke. This enhanced effectiveness is the effect of increasing the amount of muscle contraction and nerve-ending activity by electrical stimulation in initially weak spastic muscles after stroke. This probably leads to a better uptake of the injected BT in those muscles (Hesse et al., 1995). This hypothesis is further supported by recent research in patients with upper limb spasticity (Trompetto et al., 2008) where clinical and neurophysiological (tonic vibration reflex (TVR) decrease and M-wave reduction) improvements after BT injection were only present in patients who had some residual motor capacity. Historically, the effectiveness of BT lays in the fact that it did not change the EMG parameters of the non-injected muscles. It has been reported that BT could reduce the CMAP in the non-injected orbicularis oculi muscle, but the possibility of the BT spreading from the injected side to the contralateral side could not be ruled out (Girlanda et al., 1996). In addition, repeated treatments may lead to functional weakening (decrease in turns/amplitude) of non-injected muscles in dystonic patients (Erdal et al., 1999). BT injections can cause abnormal jitter measurements in distant muscles where muscle tone or strength continues to remain unchanged (Girlanda et al., 1992; Lange et al., 1987). 4. Effects on spinal cord mechanisms BT effect on spinal cord mechanisms has been studied by several authors using F-wave parameters (Hamjian and Walker, 1994; Pauri et al., 2000; Wohlfarth et al., 2001), H-reflex parameters and reciprocal inhibition of the H-reflex (Priori et al., 1995;
Girlanda et al., 1997; Modugno et al., 1998; Frascarelli et al., 2011) in patients and healthy subjects. The characteristics of these studies are displayed in Table 1. These studies found contradictory results of the studied parameters after the BT injection. An increase in the F-wave and in the F-wave/M-wave ratio was observed in healthy subjects (Hamjian and Walker, 1994), suggesting an enhanced alpha-motoneuron excitability after the BT injection. This result could not be confirmed some time after in patients with lower limb spasticity (Pauri et al., 2000). However, changes in F-wave parameters have been observed in patients with focal dystonia (Wohlfarth et al., 2001), again suggesting excitability changes in the alpha-motoneurons after BT injection. In light of this mechanism, a further study demonstrates that high doses of BT are needed to produce a central transportation in experimental animals (Dressler and Adib Saberib, 2005). Similar contradictory results have been observed in studies using H-reflex parameters and reciprocal inhibition between agonist and antagonist muscles (Priori et al., 1995; Girlanda et al., 1997; Modugno et al., 1998; Frascarelli et al., 2011). Again, different pathophysiological conditions were studied. The first study involved patients with idiopathic segmental forearm dystonia, and an increase of the second phase of reciprocal inhibition (defective phase in dystonic patients) was observed after BT injection (Priori et al., 1995). Similar results were observed by Modugno et al. (1998) when patients with essential hand tremor were studied. Both studies suggested that BT can transiently restore presynaptic inhibition between forearm antagonist muscles. A different study showed no change in any phase of reciprocal inhibition (Girlanda et al., 1997); although, this time patients with upper limb poststroke spasticity were studied. Differences in the afferent feedback from the affected muscles in these different clinical conditions could be the underlying factor in the different results observed.
Table 1 Research studies of BT effects on spinal cord mechanisms. Study
Subjects/patients
Neurophysiological measures
BT treatment
Hamjian and Walker (1994)
10 Healthy subjects
Pauri et al. (2000)
15 Patients with lower limb spasticity
Wohlfarth et al. (2001)
14 Patients with focal dystonia
Priori et al. (1995)
12 Patients with idiopathic segmental forearm dystonia
Modugno et al. (1998)
10 Patients with essential hand tremor
RI curve H reflex H/M wave ratio
80–100 Units of BT type A
Girlanda et al. (1997)
20 Patients with upper limb poststroke spasticity
RI curve CMAP amplitude H reflex
100 Units of BT type A
Frascarelli et al. (2011)
18 Children with cerebral palsy
Lower limb SEPs latency and amplitude Hmax:Mmax ratio
4–6 Units/kg body weight of BT type A for each lower limb muscle
Results
CMAP amplitude Peroneal NCV F wave amplitude, area and latency TA and Gastrocnemius TMS MEPs F wave + CMAP and H reflex
10 Units of BT type A in EDB muscle
" F wave amplitude ; CMAP amplitude " F/M wave ratio
150–700 Units of BT type A in affected muscles
Shortest and mean F wave latency Distal M wave latency M wave amplitude CMCT RI curve Maximum M and H waves amplitudes H/M wave ratio
105 Units of BT type A in WC patients 175 Units of BT type A in TC patients
" MEPs latency and CMCT ; H wave and CMAP amplitudes No change in F wave, H reflex and CMAP latencies " Shortest and mean F wave latencies ; F wave persistence No change in M wave measures " H reflex of RI at 30 ms ISI ; Second phase of RI between 10–20 ms ISI ; M and H waves, no change in H/M ratio ; Second phase of RI between 10 and 20 ms ISI ; M and H waves, no change in H/M ratio No change in 1st and 2nd phase of RI ; M and H waves, no change in H/M ratio " SEPs amplitude. No change in SEPs latency ; Hmax:Mmax ratio
15–100 (mean 49.5) Units of BT type A
CMAP = compound muscle action potential; NCV = nerve conduction velocity; TA = tibialis anterior; TMS = transcranial magnetic stimulation; MEP = motor evoked potential; CMCT = central motor conduction time; RI = reciprocal inhibition; SEP = somatosensory evoked potential; BT = botulinum toxin; EDB = extensor digitorum brevis; WC = writer’s cramp; TC = torticolli; ISI = interstimulus interval; " = increase; ; = decrease.
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Frascarelli et al. (2011) have recently showed an improvement of Hmax:Mmax ratio in spastic children. Nevertheless, BT seems to indirectly alter the excitability of spinal cord circuitry in humans, by also acting on the presynaptic intrafusal neuromuscular junction and decreasing spindle afferent input to the spinal cord. This effect leads to an increase in the presynaptic inhibition of flexor muscle afferents. An indirect action is in keeping with the experimental evidence of a fusimotor denervation after BT intramuscular injection in animals (Rosales et al., 1996). The possible role of BT injection in reducing muscle afferent feedback has been studied in a group of patients with focal hand dystonia (WC) (Trompetto et al., 2006). In this study, the maximal M-wave (Mmax) ratio before and after BT injections, the MVC and the TVR were measured. The TVR ratio was significantly more depressed and remained depressed for a long time as compared to the Mmax and MVC ratios. This finding could not be explained by the concomitant denervation of extrafusal fibres. Besides acting on extrafusal fibres, BT injection could change the afferent inflow from the vibrated muscle by acting on the fusimotor synapses as well, and changing the reflex activation of intrafusal muscle fibres during vibration. The denervation of intrafusal muscle fibres may have a special sensitivity to suppress the TVR after BT injection, suggesting that this effect may contribute to the clinical benefits of BT injection. Similar results in the decrease of TVR were also obtained in patients with upper limb spasticity after stroke (Trompetto et al., 2008). The significant decrease of the TVR was only observed in those patients who retain some degree of strength and active movement, suggesting that the BT action on the injected muscles is directly related to the reduction of muscle spindles firing after the blockade of intrafusal fibres.
5. Effects on brainstem mechanisms The possible effect of BT injections on brainstem mechanisms has been investigated mainly by studying changes in the excitability of brainstem reflexes in patients with cranial dystonia. Brainstem circuits can be easily studied by the blink reflex technique. After unilateral stimulation of the supraorbital nerve, a first early response (R1), ipsilateral to the stimulated side, and a second bilateral late response (R2) can be recorded from the orbicularis oculi muscle. The R1 component is relayed centrally through an oligosynaptic (trigeminal-facial) arc, whereas the R2 component reflects a polysynaptic bilateral pathway. An increased excitability of brainstem interneurons was observed in patients with blepharospasm; one of the most common cranial dystonia characterised by involuntary recurrent spasms of both eyelids (Berardelli et al., 1985). Later, the recovery cycle of the R2 component was analysed in patients with blepharospasm treated with BT (Valls-Sole et al., 1991). The authors found that the amplitude of the R1 component was reduced; however, the recovery cycle of the R2 response remains abnormally enhanced even at the time of maximal clinical benefit (Valls-Sole et al., 1991). Similar results were confirmed in patients with blepharospasm who were only treated unilaterally (Girlanda et al., 1996, 1997). The recovery curve of the R2 blink reflex component recorded from the untreated orbicularis oculi muscle remained unchanged, although a clinical benefit was present bilaterally possibly due to the toxin spreading. Patients who were affected by cervical dystonia also showed no change in the recovery cycle of the R2 response after BT injection (Valls-Sole et al., 1994). More recently, no changes in the blink reflex recovery cycle have been reported after BT treatment of blepharospasm patients (Conte et al., 2010). In this study, the authors suggested that the lack of BT effects on brainstem circuits provides evidence supporting that BT therapy is safe for patients. Brainstem circuits can also be studied inducing time-specific plasticity in the blink reflex with
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the application of high-frequency stimulation protocols. This protocol has recently been used by Zeuner et al. (2010) in patients with blepharospasm before and after BT treatment. Again, the study results did not show any change in recovery curves of the blink reflex after BT injections, showing that BT has no effect on the enhanced excitability of brainstem interneurons in blepharospasm patients. However, changes in the recovery cycle of R2 response after BT were observed in a patient with hemimasticatory spasm (Mir et al., 2006). After BT injection of the affected masseter muscle, enhanced blink reflex recovery cycle of the R2 component was restored, paralleled by the clinical improvement. It was suggested that hemimasticatory spasm may produce changes in the spindle afferents that were completely restored after BT injections. It is possible that differences in the specific pathophysiology and innervation involved in different types of cranial dystonia and hemimasticatory spasm could be the reason for the different BT effects on the blink reflex recovery cycle of the R2 component. The trigeminal nerve, as part of the blink reflex circuit, can have a major involvement in hemimasticatory spasm pathophysiology. It is possible that BT injections into the masseter muscle could reduce the spindle activity arriving at the trigeminal nucleus, thus reducing or even normalising the alterations observed in the R2 component of the blink reflex recovery cycle. This reduction of the spindle activity after BT injection of facial nerve innervated muscles is unlikely to be relevant in the improvement of blink reflex recovery cycle abnormalities. This issue could explain changes in the blink reflex recovery cycle of the R2 component after BT injection in hemimasticatory spasm while no changes are observed in cranial dystonias, where the trigeminal nerve musculature is not affected. A similar direction of change in brainstem circuitry was observed in a recent study of analysed F waves and F/M ratios from the mentalis muscle before and after BT treatment of the orbicularis oculi muscle in patients with hemifacial spasm (Ishikawa et al., 2010). The authors found that the duration and frequency of F waves and the F/M ratio decreased significantly after BT injection, demonstrating a decrease in the facial motonucleus excitability. The decrease in the trigeminal afferent input caused by BT alters the excitability of the facial motor nucleus, suggesting that the trigeminal afferent input and the cortical control contribute to the hyperexcitability of the facial motor nucleus in the pathophysiology of hemifacial spasm. The facial musculature in humans is a perfect structure to study possible central changes due to interventions on the skeletal musculature. The sensory supply of these muscles is unique. Facial muscles do not act on joints, so they have few or no muscle spindles, and cannot have the axon collaterals needed to receive recurrent inhibition. Further, their motoneurons do not demonstrate reciprocal inhibition such as agonist–antagonist muscles related to a specific joint (Vaughan et al., 1988). Therefore, BT injected into facial muscles cannot alter sensory input, and alternative mechanisms must be responsible for the supposed BTinduced central effects. Transcranial magnetic stimulation (TMS) studies in humans showed that after partial or total axotomy of the facial nerve, the contralateral hemisphere reorganises the motor output by increasing its drive to intact perioral muscles (Yildiz et al., 2007). Brainstem auditory evoked potentials were used to observe changes in its specific circuitry in patients with craniocervical dystonia and hemifacial spasm treated with BT (Ce, 2000). BT injections did not induce changes in brainstem auditory evoked potentials; so the authors suggested that BT injections have no effect on brainstem interneurons. Although the available neurophysiological methods used to test brainstem mechanisms are not sufficiently sensitive, the specific studies performed in this area suggest that BT treatment has little influence on brainstem mechanisms.
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6. Effects on cortical mechanisms Various neurophysiological techniques have been used to investigate possible BT-induced changes to functional cortical organisation. One example of this approach comes from the study of longlatency reflexes (LLRs), which have been successfully used to test afferent sensory and efferent motor pathways travelling via the cortex. After electrical stimulation of the median nerve, two EMG responses can be recorded in thenar muscles: the first (LLR1) is analogous to the stretch reflex, while the second (LLR2, occurring at about 50 ms) is believed to be of cortical origin. LLR behaviour was investigated in a group of patients with focal hand dystonia (Naumann and Reiners, 1997). A significant reduction in the amplitude of the late (LLR2) component was observed on the clinically affected side after BT treatment. LLR2 component is supposed to have a cortical generator (involving the supplementary motor area). These results were compared with healthy controls that presented a normal behaviour of LLR1 and LLR2 components. However, control subjects did not receive BT, so their results on LLR could be only compared with the LLR results of patients before BT injection. This finding suggests that BT injections can modify the afferent input coming from the injected muscles and modulate the abnormal central motor pattern involved in focal dystonia. Another way of studying the influence of afferent peripheral inputs on the cortex is by recording the somatosensory evoked potentials (SEPs) on electrical stimulation of peripheral nerve trunks. SEPs before and after treatment with BT for cervical dystonia were recorded by Kañovsky´ et al. (1998). They observed a significant reduction in the amplitude of the P22/N30 precentral component (recorded contralaterally to the direction of head deviation) paralleling the clinical improvement in head position. The SEP precentral component seems to reflect the activation of a functional cortico–subcortical–cortical loop (including the basal ganglia as well as the premotor and supplementary motor areas); so the authors postulated that their findings might reflect changes in precentral cortex excitability secondary to BT-induced modulation of spindle afferent inputs. Similar results were also reported in patients with hemiplegic cerebral palsy with spasticity after BT injections (Park et al., 2002). An improvement in the cortical SEPs with associated reduction of spasticity was observed, although these results were used to suggest that spasticity by itself could be considered a factor affecting cortical SEPs. This SEP improvement after BT injection has been recently confirmed in a study performed in children with spastic diplegic cerebral palsy (Frascarelli et al., 2011). The improvement in the amplitude of SEPs after BT injection is explained by the authors to be directly related to a central reorganisation of the affected muscles after BT injections. Moreover, it has also been reported that no changes were seen in cortical SEPs in a group of WC patients before and after BT treatment (Contarino et al., 2007). Overall, there is still some debate on whether cortical SEPs are sensitive enough to assess the central sensory effects of BT treatment. Evidence of changes in cortical organisation of different areas after BT injections has also been reported (Byrnes et al., 1998). In this study, TMS was used to map the topography of primary motor cortex projections of upper limb muscles in patients with WC during a sustained isometric contraction. TMS was delivered before and after BT injections in the affected muscles. Corticomotor representation was initially different in patients compared to normal subjects. BT treatment induced a clinical improvement of focal hand dystonia with a temporal reversal of cortical map abnormalities. After cessation of clinical benefit, the cortical maps returned to their original topography. This study suggested that changes in the primary motor cortex of dystonic patients might be secondary to abnormal afferent inputs that injected BTX-A may transiently
modulate. A reversible reorganisation of the hand motor cortical representation after BTX-A injections was also demonstrated by the same group in patients with cervical dystonia (Thickbroom et al., 2003) and primary writing tremor (Byrnes et al., 2005). Using the paired-pulse TMS protocols, alterations in the intracortical inhibitory mechanisms after BT injections were observed in patients with upper limb dystonia (Gilio et al., 2000; Ridding et al., 1995). One month after BT injection, all the patients had fewer dystonic movements in their arms and intracortical inhibition increased and returned to normal values. Three months after treatment, values of intracortical inhibition returned to pre-treatment levels. The authors suggest that BT can modify the excitability of the motor cortical areas by reorganising inhibitory and excitatory intracortical circuits during the clinical improvement time between BT injections. The effect probably results indirectly from the toxin’s peripheral action. The normalisation of cortical excitability after BT was not confirmed in subsequent studies by other authors (Boroojerdi et al., 2003; Allam et al., 2005). One of the last studies to examine the intracortical inhibitory mechanisms was performed on healthy subjects (Kim et al., 2006). Again, changes in the intracortical inhibitory measures were observed after BT injections. Increased intracortical inhibition paired with reduced intracortical facilitation was observed up to 3 months after BT injections in healthy subjects. A novel result was also reported: a reduced cortical silent period and an increase in the MEP/CMAP ratio up to 3 months after the BT injections; both results reflecting central inhibitory and excitability mechanisms, respectively. The reduced cortical silent period in addition to the increased intracortical inhibition provides evidence of a general effect of BT over different inhibitory intracortical circuits associated with short intracortical inhibition and cortical silent period. Therefore, these findings provide evidence of central changes after BT injections not only related to the pathophysiology of different clinical conditions, but also in healthy normal subjects. Positron emission tomography (PET) has been used to investigate possible changes in the pattern of cortical activation induced by BT injections. In dystonia, and during voluntary movements, PET activation studies show an overactivity of striatum and nonprimary motor areas and an underactivity of the primary motor cortex (Ceballos-Baumann et al., 1997). After BT injections, patients with focal hand dystonia had improved writing technique with increased activation in the parietal cortex and caudal supplementary motor area, leaving the pattern of activity in the primary motor cortex unchanged. These results suggest a cortical reorganisation secondary to the deafferentation of alpha-motoneurons or changes in motor strategy. Functional magnetic resonance imaging (fMRI) studies have also shown cortical central changes after the use of BT. A study performed in patients with hemiplegia and chronic distal arm spasticity after ischaemic stroke affecting the motor cortex (Senkárová et al., 2010) showed a significant correlation between the clinical improvement and the decrease of activation of the posterior cingulate/precuneus region after BT injection. This associative area seems to have an increased activation after a motor stroke and the authors suggest that changes in the cortical reorganisation after BT treatment could produce a more focussed activation of areas affected by stroke. This BT effect could lead to the decrease of activation of the previously enhanced areas. Another concept relevant to the potential targets of BT action is brain plasticity. Brain plasticity has been widely demonstrated by functional studies using PET or TMS techniques. In particular, transient deafferentation can result in short-term plasticity (Traversa et al., 1997). Changes in peripheral feedback or its central integration play a major role in the pathophysiology of movement disorders (Abbruzzese and Berardelli, 2003). With these two principle premises, it is possible that long-term clinical benefits of BT treat-
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ment could also reflect plastic changes in motor cortex output after the reorganisation of the whole synaptic circuitry. Changes in cortical plasticity have recently been reported in patients with cervical dystonia after BT treatment (Kojovic et al., 2011). Enhanced typical dystonic response to paired associative stimulation in hand muscles was suppressed 1 month after BT was injected into neck muscles. This effect was correlated with a clinical improvement of cervical dystonia posture and pain, suggesting that changes in plasticity of the cortical hand area are probably due to changes in the afferent input at distant sites of the BT injection. These results also suggest an additional cortical effect of BT. In contrast with these plastic changes in adults, there is no evidence of plastic brain changes after BT treatment in children. No changes in the corticomotor projection to the upper limb were observed by Redman et al., 2008, after BT treatment of children with hemiplegic cerebral palsy. The optimal site of stimulation of the affected and unaffected first dorsal interosseous was analysed before and after BT treatment by using single TMS pulses; therefore, the authors suggest that in children there is no central adaptation to BT injection. However, this assumption seems unlikely considering the greater potential for plasticity in children. Human studies nevertheless support the idea that BT injected at therapeutic doses induces spinal (Wohlfarth et al., 2001; Priori et al., 1995) or cortical (Giladi, 1997; Gilio et al., 2000; Thickbroom et al., 2003; Walsh and Hutchinson, 2007; Trompetto et al., 2006; Kañovsky´ et al., 1998) effects by indirectly inducing brain changes; this occurs by plastic rearrangement subsequent to denervation or alterations in sensory input. An excellent clinical example is that BT injected into the affected muscles relieves pain in patients with cervical dystonia (Jankovic, 2006). BT injected intramuscularly eases dystonic pain not by improving muscle contraction, but probably by acting centrally. This analgesic effect seems to be related to central changes induced by muscle denervation. In addition, muscle denervation could promote central plastic rearrangements or alterations in sensory inputs (Giladi, 1997; Kañovsky´ et al., 1998). Putting this together, BT treatment could be indirectly responsible for the decrease in pain sensation in dystonic patients.
7. Conclusion In conclusion, the primary action of BT is at the neuromuscular junction producing a biochemical denervation. This action becomes crucial to weaken the injected muscle, which is the principal clinical effect of BT. The amount of weakness is directly related to the activity level of the blocked neuromuscular synapses. Therefore, it has been proven that activation of injected muscles immediately after the BT injection increases the weakness and shortens the onset of the clinical effect. Nevertheless, BT also influences the sensory afference to the spinal cord and supraspinal areas (brainstem and cortical areas) by affecting the intrafusal fibres of the injected muscles. This influence on sensory feedback can produce changes in spinal and cortical circuitry (with little effect on the brainstem) and also affect distant muscles to the ones initially injected that can be observed using several neurophysiological studies. Nevertheless, new neurophysiological investigations can be used to clarify peripheral and CNS effects of BT injection. Use of well-known brain plasticity protocols by transcranial (magnetic or direct current) stimulation could lead to a better understanding of plasticity effects of BT over CNS. However, these functional changes in the CNS induced by BT probably originate in the peripheral mechanism of its action. The use of quantitative EMG analysis and quantitative single fibre EMG studies could shed some light on the specific muscle changes after BT injections, so that it would clarify the exact changes produced in the sensitive afferences arriving at the spinal cord, brainstem and brain hemispheres.
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Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Neurophysiological changes after intramuscular injection of botulinum toxin Francisco J. Palomar, Pablo Mir ⇑ Unidad de Trastornos del Movimiento, Servicio de Neurología y Neurofisiología Clínica, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain
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Article history: Available online 2 November 2011 Keywords: Clinical neurophysiology Botulinum toxin Brainstem Peripheral nervous system Spinal cord reflexes Central nervous system
h i g h l i g h t s This review is focussed on neurophysiological changes after botulinum toxin (BT) injection. Will help readers to understand better what are the distant effects of BT treatment. Will help readers to know about the different neurophysiological techniques to study functional changes after BT treatment.
a b s t r a c t Botulinum toxin (BT) acts peripherally by inhibiting acetylcholine release from the presynaptic neuromuscular terminals and by weakening muscle contraction. Therefore, its clinical benefit is primarily due to its peripheral action. As a result, local injection of BT has become a successful and safe tool in the treatment of several neurological and non-neurological disorders. Studies in animals have also shown that the toxin can be retrogradely transported and even transcytosed to neurons in the central nervous system (CNS). Further human studies have suggested that BT could alter the functional organisation of the CNS indirectly through peripheral mechanisms. BT can interfere with and modify spinal, brainstem and cortical circuits, including cortical excitability and plasticity/organisation by altering spindle afferent inflow directed to spinal motoneurons or to the various cortical areas. It is well demonstrated that the distant CNS effects of BT treatment parallel the peripheral effect, although there is limited evidence as to the cause of this. Therefore, further studies focussed on central changes after BT treatment is needed for a better understanding of these non-peripheral effects of BT. Ó 2011 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . Studies in animals . . . . . . . . . . . . . . . . . . Effects on the peripheral neuromuscular Effects on spinal cord mechanisms . . . . . Effects on brainstem mechanisms. . . . . . Effects on cortical mechanisms . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .
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⇑ Corresponding author at: Unidad de Trastornos del Movimiento, Servicio de Neurología y Neurofisiología Clínica, Hospital Universitario Virgen del Rocío, Avda. Manuel Siurot sn, 41013 Seville, Spain. Tel.: +34 955012593; fax: +34 955012597. E-mail address: [email protected] (P. Mir). 1388-2457/$36.00 Ó 2011 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2011.05.032
F.J. Palomar, P. Mir / Clinical Neurophysiology 123 (2012) 54–60
1. Introduction Botulinum toxin (BT) is a metalloproteinase that, when injected intramuscularly, weakens muscle power and induces denervation for a well-understood time period (Hamjian and Walker, 1994). This biochemical action induces further denervation of extrafusal muscle fibres (Simpson, 1981). The molecular mechanism underlying the local action of BTX-A has been best characterised at the neuromuscular junction, where the toxin acts by cleaving essential proteins in the vesicle re-cycling machinery. Over the past two decades, BT has become a safe and effective therapeutic tool for neurological and non-neurological conditions (Simpson et al., 2008a,b). BT is now widely used to treat a variety of clinical conditions characterised by muscle hyperactivity, such as focal dystonia (blepharospasm, torticollis, spasmodic dysphonia and writer’s cramp (WC)), hemifacial spasm, tremor and spasticity. The use of BT can also improve pain associated with these conditions, probably via central effects rather than the well-established effect on muscle. BT also blocks parasympathetic and postganglionic sympathetic cholinergic nerve synapses in the autonomic nervous system. This collateral action also allows clinicians to use BT to treat overactive smooth muscle (anal fissure and outlet-type constipation and oesophageal achalasia) as well as secretory disorders (focal hyperhidrosis, gustatory sweating and sialorrhoea) (Naumann and Jost, 2004). BT can also be used in cosmetics; however, the effect of BT treatment for this purpose is limited to its direct biochemical action of muscle paralysis. It is generally accepted that the clinical benefits of BT injections depend primarily on the toxin’s peripheral action. Experimental studies in animals and humans have also provided evidence that BT can act centrally and at distant sites from where the injection was performed (Simpson, 1981). These central mechanisms might also help to explain why BT injections sometimes leave the injected muscles disproportionately weak. The aim of this article is to summarise the neurophysiological changes that BT injections cause at different sites in humans.
2. Studies in animals After injecting BT into skeletal muscles, it acts at the intrafusal and the extrafusal neuromuscular junctions. These two effects start within 80 min of the injection. A morphological study compared the effects of BT on extrafusal and intrafusal muscle fibres in rats (Rosales et al., 1996). The toxin caused fibre atrophy and acetylcholine staining to spread into the end plates, indicating a general denervation of extrafusal and intrafusal fibres. This point was also reported by Filippi et al. (1993) by studying the effect of BT injected into the jaw muscles of rats. The final conclusion is that BT acts not only on the alpha motor endings but also on gamma motor endings, leading to a reduction of the spindle input to the alpha motoneurons. The alpha motoneurons are critical in maintaining the tonic myotatic reflex, suggesting that the relief in dystonic patients cannot be solely due to muscle paralysis, but also due to a decrease in the tonic myotatic reflex. The question of central effects depends on the ability of BT to pass through the blood–brain barrier. Labelled toxin was detected by means of autoradiography and fluorescent labelling in the brain parenchyma of mice after high doses of BT marked with 125I were injected intravenously (Boroff and Chen, 1975). The BT dose used in this study was extremely high (1.5 105 U) compared to the low doses used in therapeutic clinical applications. In fact, the animals used survived only between 35 and 38 min. The authors suggested that this short survival time was related to the high doses of labelled BT injected. The method used in this study was able to demonstrate the presence of the labelled BT in the brain blood ves-
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sels and quite far from them in the brain parenchyma. These results were not present in control animals where no BT was injected. However, there is no evidence at the present time that low therapeutic doses of BT could diffuse through the blood stream and cross the blood–brain barrier.
3. Effects on the peripheral neuromuscular system The time course of the effects of intramuscular BT has been described in studies recording the electromyographic (EMG) activity from the injected muscles. Compound muscle action potential (CMAP) amplitude in the injected extensor digitorum brevis (EDB) muscle starts to decrease 48 h after injection and the maximum decrease occurs between days 7 and 21. The CMAP amplitude remains decreased for at least 90 days following a full recovery (Hamjian and Walker, 1994; Eleopra et al., 1998). Because of its easy application and low cost (compared to antibody assays), this effect is currently used in the EDB muscle as an initial investigative tool to observe the responsiveness of a patient before starting BT treatment and/or to verify the cause of secondary non-responsiveness (Cordivari et al., 2006). The CMAP reduction is produced at the same time as a decrease in the mean rectified voltage during maximal voluntary contraction (MVC) (Hamjian and Walker, 1994). Spontaneous denervation follows the reduced EMG recruitment pattern 20 days after BT injection in normal muscles (Van Putten et al., 2002), and muscle atrophy was observed 10–20 days (peaking at day 42) after injection in spastic muscles (Hamjian and Walker, 1994) with motor unit rearrangement. Bertolasi et al. (1997) found an increase in the muscle cramp threshold frequency when measured with repetitive electrical peripheral nerve stimulation in patients with inherited benign fasciculation syndrome. These findings support the idea that BT acts mainly on presynaptic release of acetylcholine in motor nerve terminals. However, this action is not the same in all areas of the injected muscle. Recently, it has been proven that the effect of the injected BT can be increased when it is injected precisely in the end plate zone (Lapatki et al., 2010). The authors suggest that this differential effect of the injection site within the muscle could be a promising strategy for minimising the amount of injected BT and, consequently, the appearance of unwanted side effects such as weakness of adjacent muscles. The toxin’s peripheral action also depends on the state of the muscle in which BT is injected. This was investigated first in animals by Hughes and Whaler (1962) in rat diaphragm preparations with BT, where indirect stimulation of the BT diaphragm preparation anticipates the induced paralysis. This study suggests that the advance in the onset of paralysis observed in electrically stimulated preparations was due to an increase in muscular contraction and in the nerve-ending activity. Recent animal studies confirmed this suggestion using stretching exercises in BT-injected animals (Kim et al., 2003). Further studies in humans have shown similar effects to animal studies with regard to enhanced toxin uptake and acceleration in the onset of the paralytic effect after electrical stimulation (mainly at low frequency), stretching exercise (Glocker et al., 1995; Eleopra et al., 1997) or writing in the case of patients suffering from WC (Chen et al., 1999). These neurophysiologic characteristics of BT’s effectiveness and its mechanisms of action are associated with a better clinical improvement. This was reported in hemiparetic patients with lower (Hesse et al., 1995) and upper (Hesse et al., 1998) limb spasticity. Patients who received a combined treatment of BT injection and repetitive electrical stimulation of injected muscles had a better improvement of gait parameters, such as velocity, stride length, stance and swing symmetry, Ashworth score and in upper limb motor tasks after
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the BT injection. This enhanced improvement was paralleled by a greater reduction in the muscle tone of muscles that received the combined treatment, suggesting that this additional electrical stimulation enhances the effectiveness of BT in the treatment of spasticity after stroke. This enhanced effectiveness is the effect of increasing the amount of muscle contraction and nerve-ending activity by electrical stimulation in initially weak spastic muscles after stroke. This probably leads to a better uptake of the injected BT in those muscles (Hesse et al., 1995). This hypothesis is further supported by recent research in patients with upper limb spasticity (Trompetto et al., 2008) where clinical and neurophysiological (tonic vibration reflex (TVR) decrease and M-wave reduction) improvements after BT injection were only present in patients who had some residual motor capacity. Historically, the effectiveness of BT lays in the fact that it did not change the EMG parameters of the non-injected muscles. It has been reported that BT could reduce the CMAP in the non-injected orbicularis oculi muscle, but the possibility of the BT spreading from the injected side to the contralateral side could not be ruled out (Girlanda et al., 1996). In addition, repeated treatments may lead to functional weakening (decrease in turns/amplitude) of non-injected muscles in dystonic patients (Erdal et al., 1999). BT injections can cause abnormal jitter measurements in distant muscles where muscle tone or strength continues to remain unchanged (Girlanda et al., 1992; Lange et al., 1987). 4. Effects on spinal cord mechanisms BT effect on spinal cord mechanisms has been studied by several authors using F-wave parameters (Hamjian and Walker, 1994; Pauri et al., 2000; Wohlfarth et al., 2001), H-reflex parameters and reciprocal inhibition of the H-reflex (Priori et al., 1995;
Girlanda et al., 1997; Modugno et al., 1998; Frascarelli et al., 2011) in patients and healthy subjects. The characteristics of these studies are displayed in Table 1. These studies found contradictory results of the studied parameters after the BT injection. An increase in the F-wave and in the F-wave/M-wave ratio was observed in healthy subjects (Hamjian and Walker, 1994), suggesting an enhanced alpha-motoneuron excitability after the BT injection. This result could not be confirmed some time after in patients with lower limb spasticity (Pauri et al., 2000). However, changes in F-wave parameters have been observed in patients with focal dystonia (Wohlfarth et al., 2001), again suggesting excitability changes in the alpha-motoneurons after BT injection. In light of this mechanism, a further study demonstrates that high doses of BT are needed to produce a central transportation in experimental animals (Dressler and Adib Saberib, 2005). Similar contradictory results have been observed in studies using H-reflex parameters and reciprocal inhibition between agonist and antagonist muscles (Priori et al., 1995; Girlanda et al., 1997; Modugno et al., 1998; Frascarelli et al., 2011). Again, different pathophysiological conditions were studied. The first study involved patients with idiopathic segmental forearm dystonia, and an increase of the second phase of reciprocal inhibition (defective phase in dystonic patients) was observed after BT injection (Priori et al., 1995). Similar results were observed by Modugno et al. (1998) when patients with essential hand tremor were studied. Both studies suggested that BT can transiently restore presynaptic inhibition between forearm antagonist muscles. A different study showed no change in any phase of reciprocal inhibition (Girlanda et al., 1997); although, this time patients with upper limb poststroke spasticity were studied. Differences in the afferent feedback from the affected muscles in these different clinical conditions could be the underlying factor in the different results observed.
Table 1 Research studies of BT effects on spinal cord mechanisms. Study
Subjects/patients
Neurophysiological measures
BT treatment
Hamjian and Walker (1994)
10 Healthy subjects
Pauri et al. (2000)
15 Patients with lower limb spasticity
Wohlfarth et al. (2001)
14 Patients with focal dystonia
Priori et al. (1995)
12 Patients with idiopathic segmental forearm dystonia
Modugno et al. (1998)
10 Patients with essential hand tremor
RI curve H reflex H/M wave ratio
80–100 Units of BT type A
Girlanda et al. (1997)
20 Patients with upper limb poststroke spasticity
RI curve CMAP amplitude H reflex
100 Units of BT type A
Frascarelli et al. (2011)
18 Children with cerebral palsy
Lower limb SEPs latency and amplitude Hmax:Mmax ratio
4–6 Units/kg body weight of BT type A for each lower limb muscle
Results
CMAP amplitude Peroneal NCV F wave amplitude, area and latency TA and Gastrocnemius TMS MEPs F wave + CMAP and H reflex
10 Units of BT type A in EDB muscle
" F wave amplitude ; CMAP amplitude " F/M wave ratio
150–700 Units of BT type A in affected muscles
Shortest and mean F wave latency Distal M wave latency M wave amplitude CMCT RI curve Maximum M and H waves amplitudes H/M wave ratio
105 Units of BT type A in WC patients 175 Units of BT type A in TC patients
" MEPs latency and CMCT ; H wave and CMAP amplitudes No change in F wave, H reflex and CMAP latencies " Shortest and mean F wave latencies ; F wave persistence No change in M wave measures " H reflex of RI at 30 ms ISI ; Second phase of RI between 10–20 ms ISI ; M and H waves, no change in H/M ratio ; Second phase of RI between 10 and 20 ms ISI ; M and H waves, no change in H/M ratio No change in 1st and 2nd phase of RI ; M and H waves, no change in H/M ratio " SEPs amplitude. No change in SEPs latency ; Hmax:Mmax ratio
15–100 (mean 49.5) Units of BT type A
CMAP = compound muscle action potential; NCV = nerve conduction velocity; TA = tibialis anterior; TMS = transcranial magnetic stimulation; MEP = motor evoked potential; CMCT = central motor conduction time; RI = reciprocal inhibition; SEP = somatosensory evoked potential; BT = botulinum toxin; EDB = extensor digitorum brevis; WC = writer’s cramp; TC = torticolli; ISI = interstimulus interval; " = increase; ; = decrease.
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Frascarelli et al. (2011) have recently showed an improvement of Hmax:Mmax ratio in spastic children. Nevertheless, BT seems to indirectly alter the excitability of spinal cord circuitry in humans, by also acting on the presynaptic intrafusal neuromuscular junction and decreasing spindle afferent input to the spinal cord. This effect leads to an increase in the presynaptic inhibition of flexor muscle afferents. An indirect action is in keeping with the experimental evidence of a fusimotor denervation after BT intramuscular injection in animals (Rosales et al., 1996). The possible role of BT injection in reducing muscle afferent feedback has been studied in a group of patients with focal hand dystonia (WC) (Trompetto et al., 2006). In this study, the maximal M-wave (Mmax) ratio before and after BT injections, the MVC and the TVR were measured. The TVR ratio was significantly more depressed and remained depressed for a long time as compared to the Mmax and MVC ratios. This finding could not be explained by the concomitant denervation of extrafusal fibres. Besides acting on extrafusal fibres, BT injection could change the afferent inflow from the vibrated muscle by acting on the fusimotor synapses as well, and changing the reflex activation of intrafusal muscle fibres during vibration. The denervation of intrafusal muscle fibres may have a special sensitivity to suppress the TVR after BT injection, suggesting that this effect may contribute to the clinical benefits of BT injection. Similar results in the decrease of TVR were also obtained in patients with upper limb spasticity after stroke (Trompetto et al., 2008). The significant decrease of the TVR was only observed in those patients who retain some degree of strength and active movement, suggesting that the BT action on the injected muscles is directly related to the reduction of muscle spindles firing after the blockade of intrafusal fibres.
5. Effects on brainstem mechanisms The possible effect of BT injections on brainstem mechanisms has been investigated mainly by studying changes in the excitability of brainstem reflexes in patients with cranial dystonia. Brainstem circuits can be easily studied by the blink reflex technique. After unilateral stimulation of the supraorbital nerve, a first early response (R1), ipsilateral to the stimulated side, and a second bilateral late response (R2) can be recorded from the orbicularis oculi muscle. The R1 component is relayed centrally through an oligosynaptic (trigeminal-facial) arc, whereas the R2 component reflects a polysynaptic bilateral pathway. An increased excitability of brainstem interneurons was observed in patients with blepharospasm; one of the most common cranial dystonia characterised by involuntary recurrent spasms of both eyelids (Berardelli et al., 1985). Later, the recovery cycle of the R2 component was analysed in patients with blepharospasm treated with BT (Valls-Sole et al., 1991). The authors found that the amplitude of the R1 component was reduced; however, the recovery cycle of the R2 response remains abnormally enhanced even at the time of maximal clinical benefit (Valls-Sole et al., 1991). Similar results were confirmed in patients with blepharospasm who were only treated unilaterally (Girlanda et al., 1996, 1997). The recovery curve of the R2 blink reflex component recorded from the untreated orbicularis oculi muscle remained unchanged, although a clinical benefit was present bilaterally possibly due to the toxin spreading. Patients who were affected by cervical dystonia also showed no change in the recovery cycle of the R2 response after BT injection (Valls-Sole et al., 1994). More recently, no changes in the blink reflex recovery cycle have been reported after BT treatment of blepharospasm patients (Conte et al., 2010). In this study, the authors suggested that the lack of BT effects on brainstem circuits provides evidence supporting that BT therapy is safe for patients. Brainstem circuits can also be studied inducing time-specific plasticity in the blink reflex with
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the application of high-frequency stimulation protocols. This protocol has recently been used by Zeuner et al. (2010) in patients with blepharospasm before and after BT treatment. Again, the study results did not show any change in recovery curves of the blink reflex after BT injections, showing that BT has no effect on the enhanced excitability of brainstem interneurons in blepharospasm patients. However, changes in the recovery cycle of R2 response after BT were observed in a patient with hemimasticatory spasm (Mir et al., 2006). After BT injection of the affected masseter muscle, enhanced blink reflex recovery cycle of the R2 component was restored, paralleled by the clinical improvement. It was suggested that hemimasticatory spasm may produce changes in the spindle afferents that were completely restored after BT injections. It is possible that differences in the specific pathophysiology and innervation involved in different types of cranial dystonia and hemimasticatory spasm could be the reason for the different BT effects on the blink reflex recovery cycle of the R2 component. The trigeminal nerve, as part of the blink reflex circuit, can have a major involvement in hemimasticatory spasm pathophysiology. It is possible that BT injections into the masseter muscle could reduce the spindle activity arriving at the trigeminal nucleus, thus reducing or even normalising the alterations observed in the R2 component of the blink reflex recovery cycle. This reduction of the spindle activity after BT injection of facial nerve innervated muscles is unlikely to be relevant in the improvement of blink reflex recovery cycle abnormalities. This issue could explain changes in the blink reflex recovery cycle of the R2 component after BT injection in hemimasticatory spasm while no changes are observed in cranial dystonias, where the trigeminal nerve musculature is not affected. A similar direction of change in brainstem circuitry was observed in a recent study of analysed F waves and F/M ratios from the mentalis muscle before and after BT treatment of the orbicularis oculi muscle in patients with hemifacial spasm (Ishikawa et al., 2010). The authors found that the duration and frequency of F waves and the F/M ratio decreased significantly after BT injection, demonstrating a decrease in the facial motonucleus excitability. The decrease in the trigeminal afferent input caused by BT alters the excitability of the facial motor nucleus, suggesting that the trigeminal afferent input and the cortical control contribute to the hyperexcitability of the facial motor nucleus in the pathophysiology of hemifacial spasm. The facial musculature in humans is a perfect structure to study possible central changes due to interventions on the skeletal musculature. The sensory supply of these muscles is unique. Facial muscles do not act on joints, so they have few or no muscle spindles, and cannot have the axon collaterals needed to receive recurrent inhibition. Further, their motoneurons do not demonstrate reciprocal inhibition such as agonist–antagonist muscles related to a specific joint (Vaughan et al., 1988). Therefore, BT injected into facial muscles cannot alter sensory input, and alternative mechanisms must be responsible for the supposed BTinduced central effects. Transcranial magnetic stimulation (TMS) studies in humans showed that after partial or total axotomy of the facial nerve, the contralateral hemisphere reorganises the motor output by increasing its drive to intact perioral muscles (Yildiz et al., 2007). Brainstem auditory evoked potentials were used to observe changes in its specific circuitry in patients with craniocervical dystonia and hemifacial spasm treated with BT (Ce, 2000). BT injections did not induce changes in brainstem auditory evoked potentials; so the authors suggested that BT injections have no effect on brainstem interneurons. Although the available neurophysiological methods used to test brainstem mechanisms are not sufficiently sensitive, the specific studies performed in this area suggest that BT treatment has little influence on brainstem mechanisms.
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6. Effects on cortical mechanisms Various neurophysiological techniques have been used to investigate possible BT-induced changes to functional cortical organisation. One example of this approach comes from the study of longlatency reflexes (LLRs), which have been successfully used to test afferent sensory and efferent motor pathways travelling via the cortex. After electrical stimulation of the median nerve, two EMG responses can be recorded in thenar muscles: the first (LLR1) is analogous to the stretch reflex, while the second (LLR2, occurring at about 50 ms) is believed to be of cortical origin. LLR behaviour was investigated in a group of patients with focal hand dystonia (Naumann and Reiners, 1997). A significant reduction in the amplitude of the late (LLR2) component was observed on the clinically affected side after BT treatment. LLR2 component is supposed to have a cortical generator (involving the supplementary motor area). These results were compared with healthy controls that presented a normal behaviour of LLR1 and LLR2 components. However, control subjects did not receive BT, so their results on LLR could be only compared with the LLR results of patients before BT injection. This finding suggests that BT injections can modify the afferent input coming from the injected muscles and modulate the abnormal central motor pattern involved in focal dystonia. Another way of studying the influence of afferent peripheral inputs on the cortex is by recording the somatosensory evoked potentials (SEPs) on electrical stimulation of peripheral nerve trunks. SEPs before and after treatment with BT for cervical dystonia were recorded by Kañovsky´ et al. (1998). They observed a significant reduction in the amplitude of the P22/N30 precentral component (recorded contralaterally to the direction of head deviation) paralleling the clinical improvement in head position. The SEP precentral component seems to reflect the activation of a functional cortico–subcortical–cortical loop (including the basal ganglia as well as the premotor and supplementary motor areas); so the authors postulated that their findings might reflect changes in precentral cortex excitability secondary to BT-induced modulation of spindle afferent inputs. Similar results were also reported in patients with hemiplegic cerebral palsy with spasticity after BT injections (Park et al., 2002). An improvement in the cortical SEPs with associated reduction of spasticity was observed, although these results were used to suggest that spasticity by itself could be considered a factor affecting cortical SEPs. This SEP improvement after BT injection has been recently confirmed in a study performed in children with spastic diplegic cerebral palsy (Frascarelli et al., 2011). The improvement in the amplitude of SEPs after BT injection is explained by the authors to be directly related to a central reorganisation of the affected muscles after BT injections. Moreover, it has also been reported that no changes were seen in cortical SEPs in a group of WC patients before and after BT treatment (Contarino et al., 2007). Overall, there is still some debate on whether cortical SEPs are sensitive enough to assess the central sensory effects of BT treatment. Evidence of changes in cortical organisation of different areas after BT injections has also been reported (Byrnes et al., 1998). In this study, TMS was used to map the topography of primary motor cortex projections of upper limb muscles in patients with WC during a sustained isometric contraction. TMS was delivered before and after BT injections in the affected muscles. Corticomotor representation was initially different in patients compared to normal subjects. BT treatment induced a clinical improvement of focal hand dystonia with a temporal reversal of cortical map abnormalities. After cessation of clinical benefit, the cortical maps returned to their original topography. This study suggested that changes in the primary motor cortex of dystonic patients might be secondary to abnormal afferent inputs that injected BTX-A may transiently
modulate. A reversible reorganisation of the hand motor cortical representation after BTX-A injections was also demonstrated by the same group in patients with cervical dystonia (Thickbroom et al., 2003) and primary writing tremor (Byrnes et al., 2005). Using the paired-pulse TMS protocols, alterations in the intracortical inhibitory mechanisms after BT injections were observed in patients with upper limb dystonia (Gilio et al., 2000; Ridding et al., 1995). One month after BT injection, all the patients had fewer dystonic movements in their arms and intracortical inhibition increased and returned to normal values. Three months after treatment, values of intracortical inhibition returned to pre-treatment levels. The authors suggest that BT can modify the excitability of the motor cortical areas by reorganising inhibitory and excitatory intracortical circuits during the clinical improvement time between BT injections. The effect probably results indirectly from the toxin’s peripheral action. The normalisation of cortical excitability after BT was not confirmed in subsequent studies by other authors (Boroojerdi et al., 2003; Allam et al., 2005). One of the last studies to examine the intracortical inhibitory mechanisms was performed on healthy subjects (Kim et al., 2006). Again, changes in the intracortical inhibitory measures were observed after BT injections. Increased intracortical inhibition paired with reduced intracortical facilitation was observed up to 3 months after BT injections in healthy subjects. A novel result was also reported: a reduced cortical silent period and an increase in the MEP/CMAP ratio up to 3 months after the BT injections; both results reflecting central inhibitory and excitability mechanisms, respectively. The reduced cortical silent period in addition to the increased intracortical inhibition provides evidence of a general effect of BT over different inhibitory intracortical circuits associated with short intracortical inhibition and cortical silent period. Therefore, these findings provide evidence of central changes after BT injections not only related to the pathophysiology of different clinical conditions, but also in healthy normal subjects. Positron emission tomography (PET) has been used to investigate possible changes in the pattern of cortical activation induced by BT injections. In dystonia, and during voluntary movements, PET activation studies show an overactivity of striatum and nonprimary motor areas and an underactivity of the primary motor cortex (Ceballos-Baumann et al., 1997). After BT injections, patients with focal hand dystonia had improved writing technique with increased activation in the parietal cortex and caudal supplementary motor area, leaving the pattern of activity in the primary motor cortex unchanged. These results suggest a cortical reorganisation secondary to the deafferentation of alpha-motoneurons or changes in motor strategy. Functional magnetic resonance imaging (fMRI) studies have also shown cortical central changes after the use of BT. A study performed in patients with hemiplegia and chronic distal arm spasticity after ischaemic stroke affecting the motor cortex (Senkárová et al., 2010) showed a significant correlation between the clinical improvement and the decrease of activation of the posterior cingulate/precuneus region after BT injection. This associative area seems to have an increased activation after a motor stroke and the authors suggest that changes in the cortical reorganisation after BT treatment could produce a more focussed activation of areas affected by stroke. This BT effect could lead to the decrease of activation of the previously enhanced areas. Another concept relevant to the potential targets of BT action is brain plasticity. Brain plasticity has been widely demonstrated by functional studies using PET or TMS techniques. In particular, transient deafferentation can result in short-term plasticity (Traversa et al., 1997). Changes in peripheral feedback or its central integration play a major role in the pathophysiology of movement disorders (Abbruzzese and Berardelli, 2003). With these two principle premises, it is possible that long-term clinical benefits of BT treat-
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ment could also reflect plastic changes in motor cortex output after the reorganisation of the whole synaptic circuitry. Changes in cortical plasticity have recently been reported in patients with cervical dystonia after BT treatment (Kojovic et al., 2011). Enhanced typical dystonic response to paired associative stimulation in hand muscles was suppressed 1 month after BT was injected into neck muscles. This effect was correlated with a clinical improvement of cervical dystonia posture and pain, suggesting that changes in plasticity of the cortical hand area are probably due to changes in the afferent input at distant sites of the BT injection. These results also suggest an additional cortical effect of BT. In contrast with these plastic changes in adults, there is no evidence of plastic brain changes after BT treatment in children. No changes in the corticomotor projection to the upper limb were observed by Redman et al., 2008, after BT treatment of children with hemiplegic cerebral palsy. The optimal site of stimulation of the affected and unaffected first dorsal interosseous was analysed before and after BT treatment by using single TMS pulses; therefore, the authors suggest that in children there is no central adaptation to BT injection. However, this assumption seems unlikely considering the greater potential for plasticity in children. Human studies nevertheless support the idea that BT injected at therapeutic doses induces spinal (Wohlfarth et al., 2001; Priori et al., 1995) or cortical (Giladi, 1997; Gilio et al., 2000; Thickbroom et al., 2003; Walsh and Hutchinson, 2007; Trompetto et al., 2006; Kañovsky´ et al., 1998) effects by indirectly inducing brain changes; this occurs by plastic rearrangement subsequent to denervation or alterations in sensory input. An excellent clinical example is that BT injected into the affected muscles relieves pain in patients with cervical dystonia (Jankovic, 2006). BT injected intramuscularly eases dystonic pain not by improving muscle contraction, but probably by acting centrally. This analgesic effect seems to be related to central changes induced by muscle denervation. In addition, muscle denervation could promote central plastic rearrangements or alterations in sensory inputs (Giladi, 1997; Kañovsky´ et al., 1998). Putting this together, BT treatment could be indirectly responsible for the decrease in pain sensation in dystonic patients.
7. Conclusion In conclusion, the primary action of BT is at the neuromuscular junction producing a biochemical denervation. This action becomes crucial to weaken the injected muscle, which is the principal clinical effect of BT. The amount of weakness is directly related to the activity level of the blocked neuromuscular synapses. Therefore, it has been proven that activation of injected muscles immediately after the BT injection increases the weakness and shortens the onset of the clinical effect. Nevertheless, BT also influences the sensory afference to the spinal cord and supraspinal areas (brainstem and cortical areas) by affecting the intrafusal fibres of the injected muscles. This influence on sensory feedback can produce changes in spinal and cortical circuitry (with little effect on the brainstem) and also affect distant muscles to the ones initially injected that can be observed using several neurophysiological studies. Nevertheless, new neurophysiological investigations can be used to clarify peripheral and CNS effects of BT injection. Use of well-known brain plasticity protocols by transcranial (magnetic or direct current) stimulation could lead to a better understanding of plasticity effects of BT over CNS. However, these functional changes in the CNS induced by BT probably originate in the peripheral mechanism of its action. The use of quantitative EMG analysis and quantitative single fibre EMG studies could shed some light on the specific muscle changes after BT injections, so that it would clarify the exact changes produced in the sensitive afferences arriving at the spinal cord, brainstem and brain hemispheres.
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