Botulinum toxin injection causes hyper-reflexia and increased muscle stiffness of the triceps surae muscle in the rat

This study demonstrates, for the first time, adaptive plastic changes in the stretch reflex circuitry following denervation induced by botulinum toxin injection. The study also demonstrates alterations of the elastic properties of the muscles following botulinum injection.

Abstract

Botulinum toxin is used with the intention of diminishing spasticity and reducing the risk of development of contractures. Here, we investigated changes in muscle stiffness caused by reflex activity or elastic muscle properties following botulinum toxin injection in the triceps surae muscle in rats. Forty-four rats received injection of botulinum toxin in the left triceps surae muscle. Control measurements were performed on the noninjected contralateral side in all rats. Acute experiments were performed, 1, 2, 4, and 8 wk following injection. The triceps surae muscle was dissected free, and the Achilles tendon was cut and attached to a muscle puller. The resistance of the muscle to stretches of different amplitudes and velocities was systematically investigated. Reflex-mediated torque was normalized to the maximal muscle force evoked by supramaximal stimulation of the tibial nerve. Botulinum toxin injection caused severe atrophy of the triceps surae muscle at all time points. The force generated by stretch reflex activity was also strongly diminished but not to the same extent as the maximal muscle force at 2 and 4 wk, signifying a relative reflex hyperexcitability. Passive muscle stiffness was unaltered at 1 wk but increased at 2, 4, and 8 wk (P < 0.01). These data demonstrate that botulinum toxin causes a relative increase in reflex stiffness, which is likely caused by compensatory neuroplastic changes. The stiffness of elastic elements in the muscles also increased. The data are not consistent with the ideas that botulinum toxin is an efficient antispastic medication or that it may prevent development of contractures.

NEW & NOTEWORTHY

This study demonstrates, for the first time, adaptive plastic changes in the stretch reflex circuitry following denervation induced by botulinum toxin injection. The study also demonstrates alterations of the elastic properties of the muscles following botulinum injection.

botulinum toxin was introduced for treatment of spasticity in children with cerebral palsy in the early 1990s by Koman and coworkers (1993, 1994). Since then, it has been documented that injection of botulinum toxin efficiently reduces the resistance to muscle stretch and increases the passive range of movement in a joint (Baker et al. 2002; Chaleat-Valayer et al. 2011; Lukban et al. 2009; Ubhi et al. 2000). It is still not clear whether these changes in the response of muscles to passive manipulation also translate into functional benefits, although some studies point to improved heel strike during gait (Balaban et al. 2012; Bjornson et al. 2007; Sutherland et al. 1999; Ubhi et al. 2000).

Botulinum toxin acts by splicing proteins involved in exocytosis of acetylcholine vesicles at neuromuscular endplates and thereby causes denervation of the affected muscle fibers (Moore and Naumann 2003). This prevents unwanted activation of the muscle, but voluntary control of the muscle is also naturally affected. This is not desirable in patients who, to begin with, usually show some degree of paresis. However, some studies have argued that botulinum toxin may suppress stretch reflex activity to a larger extent than voluntary activation of the muscle and thus potentially diminish spasticity without significantly affecting the ability to perform voluntary movements (Bernuz et al. 2012; Frascarelli et al. 2011; Miscio et al. 2004; Stampacchia et al. 2004). One mechanism that would help to explain this is if intrafusal endplates are more sensitive to botulinum toxin than extrafusal endplates, as has been suggested in some studies (Phadke et al. 2013a, b). This way of thinking is challenged in two ways. First, serious doubt has been raised regarding the significance of stretch reflex hyperexcitability in mediating movement impairments in spastic patients (Dietz and Sinkjaer 2007, 2012; Willerslev-Olsen et al. 2014). It is a common misunderstanding that sustained spastic-muscle overactivity (spastic dystonia) is caused by increased stretch reflex activity and sustained sensory afferent motoneuronal activation, when in fact, studies have documented that central mechanisms are primarily involved (Gracies 2005). Second, plastic changes in the nervous system are likely to occur as an adaptation to the changes in sensory activity caused by botulinum toxin injection. Our current understanding of the mechanisms of neuroplasticity would indicate that compensatory increased central gain of stretch reflex activity may be expected as a response to diminished sensory activity in the spinal network, and this is indeed also what is seen in relation to immobilization and disuse in humans and animals (Anderson et al. 1999; Lundbye-Jensen and Nielsen 2008a, b). The idea that botulinum toxin reduces stretch reflex activity—and in that sense, is an antispastic drug—may therefore not necessarily be correct.

Another rationale for botulinum toxin treatment in children with cerebral palsy is that contractures are commonly believed to develop due to spastic overactivity pulling the joint in an undesirable position (Bakheit 2010; Gibson et al. 2007). Muscle contractures are defined as unique muscle changes that increase the passive stiffness of the muscle and limit the mobility of the joints without any active force production of the muscles (Smith et al. 2011). In children with cerebral palsy, these muscular changes develop during early childhood and are often confused with exaggerated stretch reflex activity (Willerslev-Olsen et al. 2013). Despite initial promising findings from mice (Cosgrove and Graham 1994), there is still no convincing evidence from human clinical studies that botulinum toxin affects the development of contractures (Tedroff et al. 2009). Part of the reason for this may be that recent studies have suggested that development of contractures in children with cerebral palsy may be linked to impaired muscle growth rather than spastic overactivity (Gough et al. 2005; Gough and Shortland 2012). If so, botulinum toxin treatment may do more harm than good (Gough 2009; Gough et al. 2005). This has stirred a growing interest in studying the structural and functional changes induced in the muscle tissue following botulinum toxin injection (Haubruck et al. 2012; Mukund et al. 2014; Thacker et al. 2012). Whereas Haubruck et al. (2012) reported decreased in vivo stiffness of mouse muscles following botulinum toxin injection, Thacker et al. (2012) found increased in vitro stiffness of rat muscle fiber bundles.

There is thus clearly a necessity to clarify further the effect of botulinum toxin on stretch reflex activity and muscle stiffness under controlled experimental conditions to evaluate the clinical rationale for botulinum toxin treatment. This was the purpose of the present study.

In human subjects, passive and reflex-mediated stiffness may be evaluated reliably by biomechanical and electrophysiological measurement of the response to muscle stretch of different velocity and amplitude (Lorentzen et al. 2010; Mirbagheri et al. 2005; Sinkjaer and Magnussen 1994; Toft et al. 1989; Willerslev-Olsen et al. 2013). This approach constitutes the currently most objective and reproducible way of evaluating different contributions to muscle stiffness in spastic muscle (Burridge et al. 2005; Wood et al. 2005). We adapted this protocol for use in rats and used it to investigate changes in muscle stiffness, 1–8 wk following botulinum toxin injection.

METHODS

Experimental animals.

The experiments were performed in 64 male Sprague-Dawley rats (160–457 g). All surgery and experimental protocols were performed according to the European Union Directive 2010/63/EU. Ethics approval was obtained from the Danish Animal Experiments Inspectorate. Efforts were made to minimize the number of animals and their suffering. Animals were injected with either botulinum toxin (n = 44) or sodium chloride (sham; n = 20) in the triceps surae muscle group of the left hind limb. Twelve of the botulinum toxin-injected rats were used for determination of the optimal botulinum toxin dosage. The remaining 32 botulinum toxin-injected rats were randomly selected in groups of 8 for acute electrophysiological and biomechanical evaluation of muscle stiffness, 1, 2, 4, and 8 wk (n = 8) following botulinum toxin injection. However, two of the rats in the 1-wk group and one rat in the 4-wk group died during the experimental procedure, and data were therefore obtained in those groups from only six and seven rats, respectively. All sham rats were evaluated electrophysiologically and biomechanically, 8 wk after sham injection. All of the rats had a 12/12-h light/dark cycle and had access to food and water ad libitum.

Botulinum toxin injection.

Before botulinum injection, animals were lightly anesthetized by inhalation of isoflurane (2%). Botulinum toxin A was, in all cases, dissolved in a volume of 100 μl saline (0.9%). In all rats, injections were made into each of the three heads of the triceps surae muscle of the left hind limb. In the majority of rats (n = 29), two units of botulinum toxin A was injected into each of the three triceps surae muscle heads of the left hindlimb, giving a total dose of 6 units for each rat. In 12 rats, different total doses were given (3 rats for each dose): 3, 6, 15, and 30 units. All three rats receiving 30 units and one of the rats receiving 15 units had to be killed within the first week of injection due to severe general muscle weakness causing inability to drink and eat. In the sham control group (n = 20), injection of 10 μl saline (0.9%) was made with a similar distribution in the three heads of the triceps surae muscle as for the botulinum toxin-injected rats.

Behavioral testing following botulinum toxin injection.

The animals were videotaped every day for the first 2 wk after the injection and once each week for the following 4 wk. The clinical evaluation system by Malmsten (1983) was used to estimate the time course of improvement of motor performance in the hindlimbs following botulinum toxin injections. This is a system that scores the movement ability on a scale from one to eight, where one is no active movement, two is a few involuntary movements during handling, three is a few uncontrolled steps with long breaks, four is that the leg is used for walking, five is that the leg is used for walking with some control, six is that the leg is used for walking with good control, seven is that walking is normal with only minor deficits, and eight is normal walking with no deficits.

Acute electrophysiological experiment.

For the acute electrophysiological experiment, rats were anesthetized by inhalation of isoflurane (2%), followed by an intraperitoneal injection of ketamine/xylazine: initial dose [xylazine dose = body weight (g) × (10/20) + ketamine dose = body weight (g) × (75/100)]. A boost dose (1/2 of the initial dose) was then given every 20 min during the entire experiment.

Before surgery, movement range in the ankle joint was evaluated by manually moving the joint from maximal dorsiflexion to maximal plantarflexion. The movement range was measured using a medical plastic goniometer. A rectal probe and heating lamp were used to maintain the rat's temperature between 36° and 37° during surgery and measurements.

The medial gastrocnemius and lateral gastrocnemius-soleus nerves were freed for stimulation but left in continuity. The calcaneus bone was severed distal to the insertion of the Achilles tendon using a bone cutter, leaving only a small bone chip attached to the Achilles tendon. A rigid, nonelastic nylon cord was tied around the tendon proximal to the bone chip and tied to a muscle puller that was equipped with a strain gauge force transducer and a linearly variable differential transformer (Cambridge Technology, Bedford, MA). In two rats, marks were made on the Achilles tendon and in the surrounding tissue with the joint in 90° flexion before cutting the Achilles tendon. This permitted a translation of different lengths of the muscle and tendon into actual ankle-joint positions in the intact animals.

For measurements, the animal was mounted in an experimental frame with the head fixed by ear bars in a stereotaxic frame. The shank was fixed rigidly by clamps to the frame, with the hip in an ∼150° extension and the knee in an ∼170° extension.

For electromyography (EMG) recording, a pair of silver-chloride electrodes was inserted under the muscle fascia with one electrode over the belly of the lateral gastrocnemius muscle and the other electrode over the belly of the medial gastrocnemius muscle. The EMG signal was band-pass filtered (1-1,000 Hz) and amplified ×500 (Fig. 1A). The EMG recording was used to verify the presence or absence of stretch reflexes, but a quantification of the responses was not possible due to movement of the electrodes with each stretch. The muscle was covered in 37° paraffin oil. Temperature was maintained above 36° using a heating lamp.

Fig. 1.

Experimental setup and measurement of muscle stiffness. A schematic diagram of the experimental setup is shown (A). The Achilles tendon was attached to a muscle puller, which was controlled by a computer to produce specified stretches of the muscle. The muscle puller also measured the tension in the muscle. EMG was recorded by silver-chloride electrodes inserted into the triceps surae muscle. The tibial nerve was stimulated by a pair of silver-chloride electrodes with the cathode distal. When this stimulation was supramaximal for activation of all muscle fibers in the muscle, a maximal M-response could be recorded in the EMG (blue line in B) and a corresponding maximal muscle twitch in the force recording from the muscle puller (black line in B). The size of this twitch was used for normalization of the reflex-mediated stiffness (D). When a slow stretch (500 ms duration of rise time, 2 mm amplitude, 200 ms hold phase) was applied (red line in C), no EMG activity was elicited (blue line in C). The linear increase in force (black line in C) therefore reflects the resistance of passive elements in the muscle, tendon, and connective tissue to the applied lengthening, and the passive stiffness could be calculated as the ratio between the change in force and the change in length. When a faster stretch (10 ms rise time, 6 mm amplitude) was applied (red line in D), a clear stretch reflex was observed in the EMG at a latency of 8 ms (blue line in D indicated by arrow). The force recorded by the muscle puller increased rapidly with the stretch but quickly reached a relatively stable plateau during the hold phase of the stretch. Approximately 30 ms into the hold phase of the stretch, a small increase in the force was observed whenever a stretch reflex was present in the EMG. This small increase in force is shown amplified (inset). The dashed line illustrates the level of force at the same time when the muscle was stretched following nerve cut, thereby abolishing the stretch reflex. The difference between the peak force at this interval before and after the nerve was cut was used as a measure of the reflex-mediated force. It was normalized to the maximal force elicited by supramaximal stimulation of the nerve (B). GS, gastrocnemius-soleus.

Evaluation of passive and reflex-mediated stiffness.

For measurements of passive stiffness, reflex stiffness, and maximal muscle force, the muscle was stretched ∼1 mm from its nonstretched length so that a baseline tension of 0.3 N was obtained in all cases. This corresponded to a muscle length in the intact animal, where the ankle joint would be in ∼90° flexion. This was determined by setting markers in the muscle fascia and in the surrounding tissue before cutting the Achilles tendon. Knee and hip were supported and maintained in the same position throughout experiments.

A protocol similar to what has been used for evaluation of passive and reflex-mediated stiffness in the triceps surae muscle in human subjects was adopted (Lorentzen et al. 2010; Mirbagheri et al. 2005; Sinkjaer and Magnussen 1994; Toft et al. 1989; Willerslev-Olsen et al. 2013).

Twenty-seven different stretches of the triceps surae muscle were applied in a random pattern at an interval of 4 s. Each stretch profile was repeated at least 10 times. Three different amplitudes of stretches (1, 2, and 3 mm) were applied with 9 different durations of the rising phase of the stretch (5, 10, 20, 30, 40, 50, 100, 500, and 1,000 ms), resulting in 27 different velocities, ranging from 1 to 600 mm/s. The hold phase was 200 ms, and the falling phase was 100 ms in all cases. To determine the force elicited by the stretch reflex, measurements were made both before and after the tibial nerve was cut.

Before the nerve was cut, stimulation of the nerve was applied through a pair of silver-chloride electrodes placed directly on the nerve, with a distance of 5 mm between the electrodes and with the cathode distal. This was done to elicit a maximal contraction of the muscle by supramaximal nerve stimulation and to use the resulting maximal twitch to standardize the force elicited by the stretch reflex (Crone et al. 1990; Lorentzen et al. 2010).

In all rats, measurements were made from both legs in the same experimental session. It was randomized whether measurements were made first from the left or the right leg. All rats were euthanized with an intraperitoneal injection of 5 ml pentobarbital (50 mg/ml). The triceps muscle group, on both sides, was dissected free and removed for weighing.

Data analysis.

Passive muscle stiffness was measured from the slowest stretches (1 mm/s; Fig. 1C). This stiffness was the same whether the nerve was cut or not. At this velocity, the force increased linearly with the stretch, and stiffness was calculated as the force increment over the whole stretch and expressed relative to the increment in muscle length. This corresponded to measurements obtained for the first 50% of the force increment, with 2 and 3 mm/s stretches.

Reflex-mediated stiffness was measured as the additional force elicited by a stretch reflex contraction of the muscle (Fig. 1D). This was seen as a clear increase in the force, 40–50 ms following the start of the hold phase of the stretch (Fig. 1D). The delay of this increase in force compared with the latency of stretch reflex measured in the EMG is explained by the electromechanical delay. The size of this increase in force was calculated by subtracting the force measured from a similar stretch applied following nerve cut (Fig. 1D). The reflex-mediated force was normalized to the size of the maximal force elicited by supramaximal stimulation of the tibial nerve (Fig. 1B).

Statistics.

Two-way ANOVA was used to determine the effect of time (1, 2, 4, and 8 wk) and treatment (botulinum-injected limb, contralateral limb) on muscle weight, maximal muscle force, passive stiffness, and reflex-mediated stiffness. Holm-Sidak post hoc test was used to isolate which groups differed from each other. In the case of normal distribution of data, maximal muscle force, passive stiffness, and reflex-mediated stiffness were compared in botulinum- and sham-injected rats using an unpaired t-test. If the normality test failed, then a Mann-Whitney rank sum test was used. Analysis of covariance was used for comparison of the relation between length and tension for the botulinum-injected and noninjected limbs. SigmaPlot 13.0 was used for all graphs (Systat Software, Chicago, IL); SPSS (IBM SPSS Statistics, Armonk, NY) was used for all statistics.

RESULTS

Behavior and general condition of rats following botulinum toxin injection.

All rats receiving 6 units of botulinum toxin injection survived for the entire study period without any overt signs of general affection except for the first week, where a stunted weight gain was observed. The botulinum-injected rats, however, quickly caught up in weight gain, and at 2, 4, and 8 wk, no difference was found in weight between botulinum- and sham-injected rats (P > 0.3). Behaviorally, all botulinum-injected animals were able to move freely around the cage but with severe dragging of the botulinum-injected limb and an inability to walk normally on toes on that side. Instead, the rats placed the entire foot, from heel to toes, with clear outward rotation, in the stance phase. According to Malmsten (1983), clinical scores were, on average, reduced from eight to five within the first week after botulinum injection. Only little recovery to an average score of six was observed over the following weeks.

The ankle joint could be moved manually from 70° plantarflexion to 80° dorsiflexion on the noninjected side but only to 20–30° plantarflexion on the botulinum-injected side in rats, 4 wk after botulinum toxin injection. The range of dorsiflexion was unaffected.

Two-way ANOVA revealed a significant interaction between Time and Treatment (botulinum toxin vs. contralateral muscle) for the triceps surae muscle weight (F = 16.8; P < 0.001). Holm-Sidak post hoc test showed a significantly lower muscle weight of the botulinum toxin-treated muscle than the contralateral muscle at all time points (1.5 vs. 1.9 g at 1 wk, 1.12 vs. 1.8 g at 2 wk, 0.89 vs. 2.12 g at 4 wk, 1.15 vs. 2.54 g at 8 wk; P < 0.001). The weight of the triceps surae muscle group increased significantly with age on the contralateral side and was significantly larger at 4 and 8 wk than at 1 and 2 wk (P < 0.01). In contrast, a significantly smaller muscle size was observed at 2 and 4 wk than at 1 wk after botulinum injection on the injected side (P < 0.01).

Comparison of measurements from the two hindlimbs in sham rats.

No significant difference was found when comparing measurements from the two hindlimbs in the 20 sham rats for the maximal muscle force (P = 0.3), the passive muscle stiffness (P = 0.2), or reflex stiffness (P = 0.6). Measurements from the two sides in the sham rats were therefore pooled to obtain a total of 40 control measures for each of the parameters. These control measures were used for comparison with measurements obtained in botulinum toxin-treated animals at different intervals following injection (see Figs. 2–5).

Fig. 2.

Changes in triceps surae maximal muscle force following botulinum toxin injection. The triceps surae maximal muscle force was measured on both sides in botulinum toxin- and sham-injected animals by supramaximal stimulation of the tibial nerve and measurement of the resulting peak force. The mean maximal muscle force in the sham-injected animals is indicated by the bar to the right in the graphs, and the SD is indicated as the shaded areas. Data from all sham-injected animals were obtained 8 wk after botulinum toxin injection. Bars show the average maximal force (in Newtons) of the left (botulinum toxin injected; A) and right (noninjected; B) triceps surae. The different gray shades of bars indicate measurements from rats at, from left to right, 1 (n = 6), 2 (n = 8), 4 (n = 7), and 8 wk (n = 8) following botulinum injection. Measurements from the individual rats are indicated by open circles.

Fig. 5.

Changes in reflex-mediated stiffness following botulinum toxin injection. Reflex-mediated muscle stiffness was measured on both sides in botulinum toxin- and sham-injected animals by a fast stretch of the muscle, which elicited a reflex activation of the muscle. The resulting increase in muscle force was subtracted from the muscle force elicited by a similar stretch after the tibial nerve was cut to obtain the reflex-mediated force. This force was expressed as a percentage of the maximal muscle force (Mmax; y-axis in A and B). The mean reflex-mediated stiffness is indicated by the bar to the right in the graphs. The SD of the measurements is indicated by the shaded areas in A and B. Data from all sham-injected animals were obtained 8 wk after botulinum toxin injection. Bars show the average reflex-mediated stiffness (in percent of maximal force) of the left (botulinum toxin injected; A) and right (noninjected; B) triceps surae. The different gray shades of bars indicate measurements from rats at, from left to right, 1 (n = 6), 2 (n = 8), 4 (n = 7), and 8 wk (n = 8) following botulinum injection. Measurements from individual rats are indicated by open circles.

Reduction in maximal muscle force by botulinum toxin injection.

Botulinum toxin injection caused a strong reduction of maximal muscle force at all intervals (Fig. 2A) compared with the noninjected contralateral limb (Fig. 2B) and compared with sham rats (Fig. 2). Two-way ANOVA revealed no significant interaction between Time and Treatment (F = 0.9; P = 0.43) but a significant difference between the botulinum-injected and contralateral-noninjected limb (P < 0.001). Measurements at different time points were not different from each other for either the injected or noninjected limb (P > 0.3). Separate unpaired t-tests showed a significant difference between maximal muscle force in the botulinum-injected limb and maximal muscle force in the sham rats at all time points (P < 0.01). Measurements from the sham-injected rats, in contrast, showed no difference compared with the noninjected right hind limb for any of the groups of botulinum-injected rats (P > 0.3).

Increased passive stiffness following botulinum toxin injection.

With a baseline tension adjusted to ∼0.3 N, a 1-mm stretch at a velocity of 1 mm/s induced larger tension in the botulinum toxin-injected muscles than the contralateral muscles, 2, 4, and 8 wk after the injection (Fig. 3). In these rats, a larger passive stiffness was thus seen in the botulinum-injected limb compared with the contralateral limb and the sham-injected rats (Fig. 3). Two-way ANOVA failed to show any significant interaction between Time and Treatment (F = 1.5; P = 0.23), but a significantly larger stiffness was observed for the botulinum-injected limb than the contralateral limb (P < 0.001). Significantly larger stiffness in the botulinum-injected limb was observed at 2, 4, and 8 wk compared with sham-injected rats (P < 0.001 in all cases), whereas no significant difference was observed at 1 wk (P = 0.067).

Fig. 3.

Changes in passive stiffness following botulinum toxin injection. The passive muscle stiffness was measured on both sides in botulinum toxin- and sham-injected animals by a slow stretch of the muscle and measurement of the resulting increase in tension. The mean passive stiffness in the sham-treated rats is indicated by the bar to the right in both of the graphs, and the SD of these measurements is indicated as the shaded areas. Data from all sham-injected animals were obtained 8 wk after botulinum toxin injection. Bars show the average passive stiffness (in Newtons/millimeter) of the left (botulinum toxin injected; A) and right (noninjected; B) triceps surae. The different gray shades of bars indicate measurements from rats at, from left to right, 1 (n = 6), 2 (n = 8), 4 (n = 7), and 8 wk (n = 8) following botulinum injection. Measurements from individual rats are indicated as open circles.

When plotting passive stiffness from the two hind limbs in the sham rats against maximal muscle force, passive stiffness was found to increase with the size of maximal muscle force (Fig. 4; correlation coefficient: 0.8; P < 0.0001). Since maximal muscle force was reduced in the botulinum-injected muscles (Fig. 2), the larger passive stiffness following botulinum toxin injection (shown in Fig. 3) is probably underestimated. As can be seen from Fig. 4, the individual measures of passive stiffness in the botulinum-injected muscles are thus much higher than in the sham rats for a comparable size of maximal muscle force. When normalizing passive stiffness to the corresponding maximal muscle force, a highly significant difference was found between measures at all time intervals after botulinum toxin injection and the measurements in sham rats (P < 0.001).

Fig. 4.

Relation between passive stiffness and maximal muscle force. The size of passive stiffness (in Newtons/millimeter) on both sides in sham-treated rats is shown as a function of the maximal muscle force (in Newtons; closed, black circles). Open circles indicate measurements from rats, 1 wk after botulinum injection, whereas open squares and open and closed triangles indicate measurements, 2, 4, and 8 wk after botulinum toxin injection, respectively. All data are from individual muscles.

Increased reflex-mediated stiffness following botulinum toxin injection.

Two-way ANOVA revealed no significant interaction between Time and Treatment for the reflex-mediated stiffness (F = 2.2; P = 0.08), but botulinum toxin-treated muscles showed significantly larger stretch reflex/maximal muscle force ratios than the control muscles (Fig. 5; P = 0.007). Reflex-mediated stiffness was significantly smaller at 1 wk (P = 0.03) and significantly larger at 2 wk (P < 0.01) compared with sham rats (Fig. 5). There was no statistically significant difference between measurements at 4 and 8 wk and measurements from sham rats (P > 0.2).

Dose-response relation.

Fig. 6 illustrates changes in maximal muscle force (Fig. 6A), passive stiffness (Fig. 6B), and reflex-mediated stiffness (Fig. 6C) as a function of the dose of botulinum toxin injected (3, 6, and 15 units). All measures were made 8 wk after botulinum toxin injection. Due to the low number of rats in the study, measures were expressed as a percentage of measures in the contralateral limb in the individual rat, and a statistical analysis was not attempted. Nevertheless, it is seen that the maximal muscle force was progressively suppressed with increasing botulinum toxin dose (Fig. 6A), whereas the reflex-mediated stiffness showed a progressive increase (Fig. 6C). The passive stiffness appeared to be equally increased at all dosages.

Fig. 6.

Changes in maximal muscle force, passive stiffness, and reflex-mediated stiffness as a function of the dose of botulinum toxin. All measurements were made 8 wk following botulinum toxin injection in the left triceps surae muscle. Bars indicate, from left to right, measurements from muscles in which 3 (n = 3), 6 (n = 3), and 15 units (n = 2) were injected. In all cases, measurements from the left limb (botulinum toxin injected) were expressed as a percentage of measurements from the right limb (noninjected). A: measurements of the maximal muscle force; B: size of the passive muscle stiffness; C: size of reflex-mediated stiffness. The dashed horizontal lines indicate 100%.

DISCUSSION

We have found in this study that denervation and subsequent atrophy of rat triceps surae muscles induced by botulinum toxin are accompanied by increased passive muscle stiffness and a relative increase in stretch reflex-mediated stiffness.

Reduced maximal muscle force.

The very significant decrease in maximal muscle force (to 10–20%) and muscle weight (to 50%) following botulinum toxin injection that we observed is not surprising. Although some clinical studies have suggested that muscle atrophy and reduction in strength of the injected muscle may be minimal following botulinum toxin injection (Williams et al. 2013; Yang et al. 2003), the majority of studies has documented a very significant loss of muscle volume (Barber et al. 2013; Gough 2009; Valentine et al. 2016). In view of the fact that neural activity is the primary signal for growth and maintenance of muscle fiber size (Tavi and Westerblad 2011) and that denervation inevitably leads to muscle atrophy (Carmeli et al. 2015; Cisterna et al. 2014), anything else would have been surprising.

Increased passive stiffness.

Due to the great reduction in muscle size following botulinum toxin injection, it is a challenge to make a direct comparison of stiffness at a comparable length of botulinum toxin-treated and nontreated muscles. We approached this challenge by applying standardized slow stretches of 1 mm at 1 mm/s and at the same baseline tension of 0.3 N in all cases (cf. Fig. 3). It is therefore not likely that the difference in stiffness is simply explained by measuring at a different point of the length-tension relation for the small botulinum-injected muscles compared with the much larger contralateral muscles. The fact that larger stiffness was only observed later than 2 wk after botulinum injection—although the muscles were strongly reduced in size and strength already after 1 wk—also suggests that there was a genuine increase in the resistance to the applied stretch.

Atrophy of muscle fibers is known to be accompanied by infiltration and expansion of connective tissue, which will increase the stiffness of the muscle tissue (Lawler and Hindle 2011; Sato et al. 2014). Denervation of muscle, in particular, causes severe muscle atrophy and irreversible fibrotic changes, including connective tissue accumulation (Borisov et al. 2005a, b; Jergovic et al. 2001). Transforming growth factor-β1 and connective tissue growth factor have been suggested to initiate fibrosis in denervated muscle by inducing differentiation of myoblasts into myofibroblasts (Liu et al. 2016). Furthermore, the cross-sectional area of the connective tissue has been shown to correlate positively with the time postdenervation (Liu et al. 2016).

Connective tissue infiltration has also been shown to occur in rat muscle following botulinum toxin injection (Minamoto et al. 2015; Thacker et al. 2012). Thacker et al. (2012) observed reduced stiffness of the muscle fibers themselves but increased stiffness of muscle fiber bundles, suggesting an accumulation of extracellular material with low flexibility. This is consistent with our findings. Haubruck et al. (2012) reported lower stiffness in mouse triceps surae muscles, 1 wk following botulinum toxin injection. We observed no change in passive muscle stiffness, 1 wk following botulinum injection, so it may be that Haubruck et al. (2012) simply measured too soon following botulinum toxin injection to see any increase in stiffness. However, they also did not take the large reduction in muscle volume and strength induced by botulinum injection into account in their measurements. Normalization to the maximal muscle force, as in our study, or to the cross-sectional area of the muscle, as has been done in other studies (Plate et al. 2013; Toscano et al. 2010), would possibly result in a different conclusion.

The effect of botulinum toxin on passive muscle stiffness, measured biomechanically, has been, to our knowledge, evaluated in only a single study on children with cerebral palsy (Alhusaini et al. 2011). In this study, no significant change in either passive range of movement or passive stiffness of the ankle muscles was detected. Muscle volume or muscle strength was not evaluated in this study, and it may therefore be that significant changes would have been observed if changes in these parameters following botulinum toxin treatment had been taken into account.

In other human studies, significant increases in passive range of movement have been documented following botulinum injection for both the ankle joint and other joints (Baker et al. 2002; Chaleat-Valayer et al. 2011; Lukban et al. 2009; Mirska et al. 2014; Ubhi et al. 2000). It seems likely that these observations are related to the reduced neural activation of the muscle following the botulinum toxin injection rather than any changes in the passive elastic properties of the muscle. Indeed, we observed a clinically manifest reduction of plantarflexion in the rats. This suggests that the rats developed contractures either in the dorsiflexor muscles or in the joint that limited movement of the joint into maximal plantarflexion. This is likely related to the muscle paresis induced by the botulinum toxin injection, which prevented the rats from walking on their toes and performing full plantarflexion during gait. They were therefore forced to walk on their heels with the ankle in a static dorsiflexed position during stance. It is consequently also possible that we would have found much more significantly increased passive muscle stiffness if we had been able to measure the stiffness of the dorsiflexor muscles in these experiments.

Increased reflex-mediated stiffness.

The surprising—and to some extent, counterintuitive—finding in the present study is that the stretch reflex-mediated stiffness was increased significantly at 2 wk following botulinum toxin injection. A tendency for an increase was also observed at 4 and 8 wk following botulinum toxin injection but without reaching statistical significance. It should be noted that this increase was not an absolute change in the force generated by the reflex but only a relative change in relation to the maximal force capacity of the muscle. The force generated by the stretch reflex thus decreased with the denervation of the muscle fibers induced by the botulinum toxin injection, but since a larger reduction of the force evoked by a supramaximal stimulation of the motor nerve supplying the muscle was observed, we may conclude that the stretch reflex activity in the muscle was increased. This is more remarkable, since botulinum toxin also affects the endplates on intrafusal muscle fibers, which would have been expected to cause an even larger decrease of the stretch reflexes than can be explained by the effect of botulinum toxin on the extrafusal endplates alone (Phadke et al. 2013a, b). The most parsimonious explanation of the larger relative stretch reflex stiffness is therefore that plastic changes in the central gain of the reflex have counteracted the reduced motor and sensory activity to and from the muscle following the botulinum toxin injection. These plastic changes may be speculated to involve reduced presynaptic inhibition of Ia afferent synapses, increased motoneuronal excitability due to reduced postsynaptic inhibition, or changes in intrinsic membrane properties in the motoneurons (Wolpaw and Tennissen 2001). This would be consistent with studies showing compensatory increases in reflex gain in relation to altered sensory activity and disuse of muscles (Lundbye-Jensen and Nielsen 2008a, b) but counteracts the usual assumption that botulinum toxin injection reduces reflex activity. There is also evidence from human studies showing plastic changes in spinal circuitries following botulinum toxin treatment (Aymard et al. 2013; Marchand-Pauvert et al. 2013).

Clinical implications.

The experiments in the present study were performed in normal rats, and any implications for the clinical use of botulinum toxin should therefore be made with the caution that the response of spastic human muscles may be different. Botulinum toxin may also have other—potentially clinically beneficial—effects than the changes in muscle strength, passive muscle stiffness, and stretch reflex activity investigated here. The data should also be interpreted with caution, since a direct comparison of the dosages of botulinum toxin used in this study with those used in the clinic is not straightforward. That being said, our findings are difficult to reconcile with the current clinical rationale for botulinum toxin treatment in children with cerebral palsy. Although the increased reflex excitability that we observed following botulinum toxin injection was relative to the reduction in maximal force, it seems a misnomer to classify botulinum toxin as an “antispastic medication.” The primary effect of botulinum toxin treatment is to denervate the muscle and cause atrophy and reduced muscle strength. This may have clinical benefits, but they are unlikely to be related to reduced stretch reflexes and spasticity.

There is also an increasing number of studies that indicate that the dominant functional problem for children and adults with cerebral palsy is not spasticity but rather, muscle weakness and reduced range of joint movement, due to contracture development, for which increased passive stiffness is an indicator (Damiano et al. 2001; Geertsen et al. 2015; Willerslev-Olsen et al. 2013). Our findings suggest that botulinum toxin will only make things worse where these important factors are concerned, as has also been predicted previously (Gough 2009). We consequently recommend that botulinum toxin is used with caution until the implications of the findings have been fully clarified.

GRANTS

Support for this work was provided by grants from the Elsass Foundation and Danish Medical Research Council.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

J.P., J.W., J.L., and J.B.N. conception and design of research; J.P., J.L., and J.B.N. performed experiments; J.P. and J.B.N. analyzed data; J.P., J.W., J.L., and J.B.N. interpreted results of experiments; J.P. and J.B.N. prepared figures; J.P. and J.B.N. drafted manuscript; J.P., J.W., J.L., and J.B.N. edited and revised manuscript; J.P., J.W., J.L., and J.B.N. approved final version of manuscript.