Journal of Biomechanics 44 (2011) 39–44

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Journal of Biomechanics
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Changes in contractile properties of muscles receiving repeat injections of

botulinum toxin (Botox)
Rafael Fortuna a, Marco Aurélio Vaz b, Aliaa Rehan Youssef a, David Longino a, Walter Herzog a,n
a
Human Performance Laboratory, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4
b
Exercise Research Laboratory, School of Physical Education, Federal University of Rio Grande do Sul, Brazil

a r t i c l e in fo abstract

Article history: Botulinum toxin type A (BTX-A) is a frequently used therapeutic tool to denervate muscles in the
Accepted 13 August 2010 treatment of neuromuscular disorders. Although considered safe by the US Food and Drug
Administration, BTX-A can produce adverse effects in target and non-target muscles. With an increased
Keywords: use of BTX-A for neuromuscular disorders, the effects of repeat injections of BTX-A on strength, muscle
Botulinum toxin type A mass and structure need to be known. Therefore, the purpose of this study was to investigate the
Knee changes in strength, muscle mass and contractile material in New Zealand White (NZW) rabbits.
Muscle weakness Twenty NZW rabbits were divided into 4 groups: control and 1, 3 and 6 months of unilateral, repeat
Muscle paralysis injections of BTX-A into the quadriceps femoris. Outcome measures included knee extensor torque,
Rabbit
muscle mass and the percentage of contractile material in the quadriceps muscles of the target and
non-injected contralateral hindlimbs. Strength in the injected muscles was reduced by 88%, 89% and
95% in the 1, 3 and 6 months BTX-A injected hindlimbs compared to controls. Muscle mass was reduced
by 50%, 42% and 31% for the vastus lateralis (VL), rectus femoris (RF) and vastus medialis (VM),
respectively, at 1 month, by 68%, 51% and 50% at 3 months and by 76%, 44% and 13% at 6 months. The
percentage of contractile material was reduced for the 3 and 6 months animals to 80–64%, respectively,
and was replaced primarily by fat. Similar, but less pronounced results were also observed for the
quadriceps muscles of the contralateral hindlimbs, suggesting that repeat BTX-A injections cause
muscle atrophy and loss of contractile tissue in target muscles and also in non-target muscles that are
far removed from the injection site.
& 2010 Elsevier Ltd. All rights reserved.

1. Introduction application of botulinum toxin is considered to be safe by the US

Botulinum toxin type A (BTX-A) has become an accepted and
frequently used therapeutic tool to chemically denervate muscles
in the treatment of neuromuscular disorders (Antonucci et al.,
2008; Brin, 1997). BTX-A is one of seven neuromuscular blocking
agents produced by the bacterium Clostridium botulinum. It has an
affinity for motor nerve endings where it inhibits acetylcholine
release, exerting its paralytic effect on injected muscles (Anto-
nucci et al., 2008; Brin, 1997; Borodic et al., 1990; Yaraskavitch
et al., 2008).
BTX-A is commonly used to treat disorders characterized by
hyperfunction of cholinergic terminals, such as dystonia and
spasticity (Lange et al., 1987, 2007). Because of its time limited
action, repeat injections are routinely performed at intervals of
3–4 months (Brin, 1997; Dutton, 1996; Inagi et al., 1999; Cichon
et al., 1995; Garner et al., 1993; Girlanda et al., 1992). Although
regional or systemic adverse effects have been reported, local
n
Corresponding author. Tel.: +1 403 220 8525; fax: + 1 403 284 3553.
E-mail address: Walter@kin.ucalgary.ca (W. Herzog).
Food and Drug Administration (FDA; Cote et al., 2005). Regional
adverse effects of toxin application include weakness in neighbor-
ing non-target muscles (Yaraskavitch et al., 2008), while possible
systemic effects include general muscle weakness (Cote et al.,
2005). Other side-effects include dysfunction of non-target
neuromuscular junctions (Lange et al., 1991) and atrophy of
non-target muscles (Ansved et al., 1997; Girlanda et al., 1992).
With an increased use of BTX-A for an ever growing number of
disabilities, including repeat injections into young children with
cerebral palsy, the effect of repeat injections on muscle mass,
strength and muscle structure need to be known.
Although decreases in muscle mass and strength have been
reported for specific time points following BTX-A injections (Dood
et al., 2005; Herzog and Leonard, 2002; Longino et al., 2005a, b, c;
Rehan Youssef et al., 2009), the associated structural changes for
single and repeat injections remain unknown. Therefore, the
primary purpose of this study was to investigate the changes in
muscle mass and strength in BTX-A treated muscles while
simultaneously quantifying cross-sectional histology in order to
determine possible changes in the amount of contractile material.
A secondary purpose was to identify whether changes in the

0021-9290/$ - see front matter & 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jbiomech.2010.08.020
40 R. Fortuna et al. / Journal of Biomechanics 44 (2011) 39–44

outcome measures are muscle specific and affect non-target
muscles; therefore, all mass and histological outcomes were
determined for three target and three non-target muscles
following 1, 3 and 6 monthly injections of BTX-A.
Photographs were taken for each stained section using an Axionstar plus
microscope (Carl Zeiss) using a 5  magnification. A customized MatLab program
(MatLab 7.8, R2009a) was used to calculate the contractile material for at least 30%
of the total cross-sectional area of each muscle.

2.5. Data analysis

2. Methods
2.1. Experimental design
Twenty skeletally mature, 1 year old, female NZW rabbits weighing an average
of 5.5 kg were used with the Animal Care Committee approval of the University of
Calgary. Animals were allowed normal activity in a cage (65  45  30 cm3), and
received a standard diet.
Animals were divided into four study groups as follows:
(1) Control group—saline injection unilaterally (n¼5).
(2) 1-month post-single BTX-A injection unilaterally (n ¼5).
(3) 3-months post-repeated monthly BTX-A injections unilaterally (n¼ 5).
(4) 6-months post-repeated monthly BTX-A injections unilaterally (n¼ 5).
Knee extensor strength and muscle mass of the experimental hindlimbs were
expressed as a percentage of the injected control limbs. The reduction in strength
will hereafter be referred to as muscle weakness while the loss of muscle mass will
be referred to as atrophy. The amount of contractile material for each muscle is
given as the percentage area fraction calculated from the total cross-sectional
areas of the analyzed sections.
A two-way mixed model ANOVA with repeated measures was used to assess
muscle weakness with the main factors leg (injected and contralateral) and groups
(control, 1, 3 and 6 months BTX-A injections). A three-way ANOVA with repeated
measures was used to assess muscle atrophy and contractile material with the
main factors leg, groups and muscle (VL, RF and VM). A post-hoc Tukey test was
performed if indicated. The level of significance was chosen as a ¼0.05 a priori.

3. Results
2.2. Botulinum type A toxin: injection protocol
The injection procedures were well tolerated by all rabbits.
Animals were injected with Clostridium botulinum type A neurotoxin complex Data were analyzed from all animals, except for the percentage of
(BOTOX, Allergan, Inc., Toronto, Ontario, Canada), which was reconstituted with contractile material from the rectus femoris of the control rabbits,
0.9% sodium chloride to a concentration of 20 units/ml. Rabbits received
intramuscular BTX-A injections at a total of 3.5 U/kg. Injections were randomized
as these samples were of bad quality.
to either the right or left quadriceps musculature. The anterior compartment of the
thigh was isolated by manual palpation and the quadriceps was visually divided
into superior and inferior halves. Each half was subdivided into medial, central and 3.1. Muscle weakness
lateral section. One sixth of the total botox dose was injected into each section to
increase botox diffusion and to inject directly into the vastus lateralis, rectus There was no difference in muscle strength between hindlimbs
femoris and vastus medialis muscles (Longino et al., 2005b; Yaraskavitch et al.,
2008).
of the control group. The injected hindlimb of group 2, 3 and 4
Group 1 rabbits served as controls and received intramuscular saline injections rabbits were significantly weaker (90–95%) than the non-injected
randomized to either the right or left quadriceps musculature. The total volume of controls (p o0.05; Fig. 1).
injected saline was the same as the total volume of BTX-A. Group 2 rabbits The contralateral hindlimbs of the experimental animals were
received a one-time intramuscular BTX-A injection, while group 3 and 4 rabbits
consistently weaker than the hindlimbs of the non-injected
received monthly BTX-A injections into the same hindlimb (randomized for the
initial injection) for 3 and 6 months, respectively. control rabbits. However, this difference (13%) was not significant
The primary outcome measures of this study were the knee extensor torque, for group 2 and 3 rabbits (1 and 3 months of BTX-A injections) but
quadriceps muscle mass and the percentage of contractile material for the was significant (85%) for group 4 rabbits (6 months of BTX-A
different quadriceps muscles. injections; Fig. 1). Loss of strength in the contralateral hindlimb of
the group 4 rabbits was so big that the difference between
2.3. Determination of knee extensor force and muscle mass injected and contralateral limbs was not significant (p4 0.05) in
these animals.
Isometric knee extensor strength was measured at the end of 1 month (group
2 rabbits), 3 months (group 3 rabbits) and 6 months (group 4 rabbits)
experimental periods. Knee extensor strength in experimental (injected) and
contralateral (non-injected) hindlimbs was obtained by stimulating the knee
extensor muscles via a femoral nerve cuff electrode that was implanted prior to
testing (Longino et al., 2005a, b, c). Following nerve cuff implantation, rabbits were
secured in a stereotactic frame using bone pins at the pelvis and femoral condyles.
The tibia was restrained with an instrumented bar placed proximal to the
bimalleolar axis. A custom-built force sensor was used to measure the knee
extensor torques at a knee angle of 1001.
Stimulation of the knee extensor muscles was given through a Grass S8800
stimulator (Astro-Med Inc., Longueil, Quebec, Canada) at a voltage three times
higher than the alpha motoneuron threshold, to ensure activation of all motor
units (Herzog and Leonard, 1997). Stimulation duration was 500 ms, pulse
duration 0.1 ms, and the frequency of stimulation was 100 Hz. A one minute rest
period was given between trials.
Following determination of knee extensor strength, animals were sacrificed by
an overdose injection of Euthanyl (MTC Pharmaceuticals; Cambridge, Ontario).
Wet muscle mass for each quadriceps portion was determined using a commercial
scale with a resolution of 0.001 g.

2.4. Determination of the percentage of contractile material

To determine the percentage of contractile material in RF, VL and VM, the
central third of each muscle was embedded in paraffin (automatic paraffin
processor, Leica TP 1020) and cut cross-sectionally with a microtome (Leica RM
2165). Every 20 mm, an 8 mm section was collected for staining with hematoxylin-
eosin (Leica ST5010). A total of 5 slides for each muscle were analyzed and
averaged.
Fig. 1. Muscle weakness after 1, 3 and 6 months BTX-A injections for injected
(dark bar) and contralateral hindlimbs (light bars). A significant difference
(p o 0.0001) was observed for the injected hindlimbs compared to control for all
experimental group animals. Muscle weakness was also observed in the non-
injected contralateral hindlimbs of all experimental group animals but this
difference was only statistically significant for group 4 animals (6 monthly
injections of BTX-A) (p o 0.0001).
 R. Fortuna et al. / Journal of Biomechanics 44 (2011) 39–44
3.2. Muscle atrophy
Muscle mass in the saline injected and non-injected hindlimbs
of the control animals was the same. Muscle atrophy in the
injected hindlimbs of groups 2, 3 and 4 rabbits (1, 3 and 6 month
of BTX-A injections) was significantly greater compared to group
1 rabbits (control group). The mean muscle atrophy averaged
across all muscles was 43%, 60% and 56% for groups 2, 3 and 4
rabbits, respectively (Fig. 2). Muscle atrophy increased signifi-
cantly from group 2 to group 3 rabbits (1 and 3 months of BTX-A
injections) but not from group 3 to group 4 rabbits (from 3 to 6
months of BTX-A injections; Fig. 2).
There was also a loss in mass of 15%, 15% and 37% for the
muscles of the contralateral hindlimbs of groups 2, 3 and 4
rabbits, (1, 3 and 6 months of BTX-A injections) compared to the
group 1 control animals, although this difference only reached
statistical significance for the group 4 rabbits (Fig. 2).
Fig. 2. Muscle atrophy after 1, 3 and 6 months BTX-A injections for injected (dark
bars) and contralateral hindlimbs (light bars). Significant atrophy (p o0.0001) was
observed for the injected hindlimbs for all experimental group animals compared
to control. Muscle atrophy was also observed in the contralateral hindlimbs of all
experimental group animals; however, this atrophy was only significant for the
group 4 animals (6 months BTX-A injections) (p o0.0001).
Fig. 3. Muscle atrophy after 1, 3 and 6 months of BTX-A injection for the different
portions of the quadriceps femoris (VL ¼vastus lateralis; RF ¼ rectus femoris;
VM ¼vastus medialis). Muscle atrophy after 1, 3 and 6 months of BTX-A injection
for the different portions of the quadriceps femoris (VL ¼vastus lateralis;
RF ¼rectus femoris; VM ¼vastus medialis). Significant muscle atrophy (p o 0.05)
was observed for the different portions of the quadriceps femoris of the
experimental groups compared to control.
41
The loss in muscle mass following BTX-A injections was
muscle specific with VL showing the greatest losses (51%, 68% and
76% at 1, 3 and 6 months of BTX-A injections), followed by RF
(43%, 52% and 44%, respectively) and VM (32%, 50% and 14%,
respectively) as shown in Fig. 3.
3.3. Percentage of contractile material
The percentage of contractile material for the VL and VM for
the control group rabbits was 96% ( 70.8%), and 96% ( 72.7%),
respectively. Following a single BTX-A injection, these values
remained the same for VL (94%; 73.0%) and VM (98%; 70.5%),
while RF values (for which no reference values were obtained)
had approximately the same amount of contractile material (94%;
77.7%) as VL and VM. After 3 monthly injections of BTX-A, the
values measured were 72% (74.1%), 97% ( 71.1%), and 73%
( 76.7%) for VL, VM and RF, respectively, which were statistically
smaller for VL and RF (p o0.05) than control group values.
Further, the percentage contractile material for the 6-month
group rabbits was 43% ( 79.7%), 78% (4.2%) and 70% ( 78.0%) for
VL, VM and RF, respectively, which were statistically smaller
(p o0.05) than all previous group values (Fig. 4, left column).
For the contralateral hindlimbs of the group 1 control animals,
the percentage contractile material for VL and VM was 95%
Fig. 4. Contractile material (red) for the injected (left column) and the non-
injected contralateral hindlimbs (right column) for the control group and the
different experimental group animals. A fatty infiltration (white) was observed
after 3 months on the injected side and the effect became more evident after 6
months. On the contralateral hindlimb, a significant change in contractile material
was only observed after 6 months of BTX-A injections and resulted in an increase
in connective tissue (white) rather than fat. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
42 R. Fortuna et al. / Journal of Biomechanics 44 (2011) 39–44
( 73.0%) and 94% ( 71.7%), respectively. Following a single BTX-A
injection, these value remained the same: 97% ( 71.0%), 98%
( 70.4), and 97% (70.8) for VL, VM and RF, respectively, as they
did for the 3 months group (96% ( 71.4%), 97% ( 70.7%), and 96%
( 71.2%) for VL, VM and RF, respectively). However, the values of
contractile material on the contralateral hindlimb for the 6-month
group decreased significantly to 74% ( 76.5%), 82% ( 75.2%), 81%
( 76.5%) for VL, VM and RF, respectively (Fig. 4, right column).
4. Discussion
With the increased use of BTX-A in medical applications, its
mechanism of action has become well understood. However, its
long-term effect on structure and function of target and non-
target muscles has not been studied systematically. Repeated use
of BTX-A, especially in children and adolescents suffering from
neuromuscular disorders, such as cerebral palsy, makes under-
standing of BTX-A’s effects on muscles essential.
Here, we demonstrate that BTX-A not only creates great loss of
strength due to its neuromuscular blockage, but also causes a
substantial loss of muscle mass and a decrease in contractile
material of target and non-target muscles. Surprisingly, not all
quadriceps femoris muscles were affected equally by BTX-A
injections, with the vastus lateralis showing the largest loss in
muscle mass and contractile material, followed by the rectus
femoris and the vastus medialis.
4.1. Knee extensor strength
BTX-A injections caused large decreases in knee extensor
strength. These deficits were maintained through the entire 6-
month study period. Dodd et al. (2005) found a similar loss of
force (88–90%) in rat gastrocnemius as observed here after a
single BTX-A injection, and our results are also consistent with
those published previously on the rabbit knee extensor muscles
(Longino et al., 2005a–c; Herzog and Longino, 2007).
Surprisingly and new to the literature is the result that
strength in the contralateral non-injected hindlimb of the
experimental animals was also reduced. A small loss of strength
that was not statistically significant was observed after 1 and 3
months of BTX-A injection, and a statistically significant loss was
observed in the 6-month group animals. The results of this study
do not allow us to distinguish if this contralateral loss of force is
directly related to Botox or a result of atrophy caused by muscle
disuse. However, this result suggests that repeated BTX-A
injection can lead to general muscle weakness, as reported by
Cote et al. (2005), that affects target and non-target muscles.
4.2. Muscle mass
BTX-A injections caused a dramatic loss in muscle mass,
reaching a peak value of approximately 60% after 3 months
(Figs. 2 and 4), which is comparable to results published in the
literature (Dodd et al., 2005). Interestingly, muscle mass was the
same for the 3 and 6-month group animals. However, at 3 months
of BTX-A injections, muscle mass was primarily comprised of
contractile tissue, while at 6 months, a great percentage of the
muscle mass has to be attributed to fat. Thus, atrophy in terms of
muscle mass stopped at 3 months of BTX-A injections, but further
deteriorated from 3 to 6 months in terms of the contractile mass
of the muscles.
For the knee extensor muscles of the contralateral hindlimbs of
the experimental animals, no loss in muscle mass was observed in
group 2 and 3 rabbits, but a statistically significant loss was
observed in the 6-month group rabbits, reinforcing the idea that
repeated BTX-A injections affect muscles far from the injection
site. Whether this loss in muscle mass of the contralateral knee
extensors was a direct effect of Botox, or an indirect effect
associated with disuse atrophy, cannot be resolved based on the
results of this study.
Muscle mass loss following BTX-A injections was greatest for
the vastus lateralis and smallest for the vastus medialis (Fig. 3).
The injection protocol used in this study was carefully chosen to
minimize the possibility that one of the knee extensor muscles
would receive a disproportionate amount of the toxin (Longino
et al., 2005a, b, c; Rehan Youssef et al., 2009). Therefore, we
conclude that muscle atrophy is not homogeneous in a group of
muscles exposed to BTX-A. Inagi et al. (1999) suggested that the
effects of BTX-A injections are more evident in fast twitch than
slow twitch fibers. No differences were reported in the fiber type
distribution of the quadriceps femoris of NZW rabbits, which is
comprised of 100%, 90% and 97% of fast twitch fibers for VL, RF and
VM, respectively (Rab et al., 2000; Hamalainem and Pette, 1993).
However, a difference is observed in the amount of type IIb/d fast
twitch glycolytic fibers: VL has a higher proportion of type IIb/d
than RF or VM, suggesting that BTX-A has a greater effect on type
IIb/d fibers than other fast or slow type muscle fibers.
4.3. Contractile tissue
Strength of muscles does not only depend on the size of the
muscle but also the amount of contractile material. Although
strength and muscle mass decreased after a single BTX-A
injection, the proportion of contractile material within a mid-
cross section of the muscles remained constant. After 3 months of
BTX-A injections, the proportion of contractile material relative to
control was reduced in the vastus lateralis and rectus femoris, but
not the vastus medialis. This result suggests that BTX-A affects
muscles differently, not only in terms of loss of muscle mass but
also in terms of loss of contractile material. The muscle with the
greatest atrophy (VL) also showed the most pronounced loss of
contractile tissue and vice versa (VM). For the 6 month BTX-A
exposed animals, all portions of the knee extensors showed a
significant loss of contractile tissue (Fig. 4, last row), emphasizing
deterioration over time. Contractile tissue in the experimental
hindlimbs of group 3 and 4 rabbits (3 and 6 months of BTX-A
injections) was replaced to a great degree with fat, thereby
limiting the amount of loss in mass of the tissue contained within
the borders of the muscle. Therefore, using loss of muscle mass as
an indicator of BTX-A induced atrophy may severely under-
estimate the amount of contractile tissue loss.
A loss in the proportion of contractile material was also found
in the contralateral hindlimbs of the group 4 rabbits (6 months
BTX-A). In contrasts to the injected hindlimb musculature, the
loss of contractile material was not compensated by fat invasion,
but was a direct result of an increased amount of connective
tissue (Fig. 4, right column, 6-month results). This result suggests
that the loss of contractile tissue in the contralateral hindlimbs is
qualitatively different from that observed in the experimental
hindlimbs and could be due to an indirect effect of BTX-A, which
may induce general muscle weakness leading to a less active
animal.
The injection doses used in the present study (3.5 U/kg) are
similar to those used in patients (3–6 U/kg), including children
with cerebral palsy (Borodic et al., 1990, 1992; Graham et al.,
2000; Heinen et al., 2009; Koman et al., 1993; Russman et al.,
1997). However, therapeutic injections are typically given at
intervals of 2–6 months (Brin, 1997; Borodic et al., 1990; Delgado
et al., 2010; Dutton, 1996; Graham et al., 2000; Heinen et al.,
 R. Fortuna et al. / Journal of Biomechanics 44 (2011) 39–44
Fig. 5. Estimated loss in knee extensor strength in the BTX-A injected hindlimbs of
group 2 (1 month), group 3 (3 months) and group 4 (6 months) animals associated
with the inhibition caused by BTX-A denervation (BTX-A), the loss of muscle mass
(atrophy) or the loss of contractile tissue (contractile tissue). While the loss in
strength associated with BTX-A injections remained about constant throughout
the experimental period, the loss was associated primarily with BTX-A induced
chemical denervation initially (1 month) and the loss of muscle mass and
contractile material at the end (6 months).
2009; Koman et al., 1993; Morton et al., 2004; Russman et al.,
1997; Wissel et al., 2009), rather than monthly, as used here. In
order to allow for optimal spreading of the toxin, we gave six
injections for every treatment by carefully dividing the quadriceps
into different portions as described in the methods section. This
approach is consistent with clinical practice where multiple
injection points are used for effective distribution of the toxin in
the target musculature (Brin, 1997; Borodic et al., 1992; Graham
et al., 2000; Heinen et al., 2009; Koman et al., 1993; Morton et al.,
2004; Shaari et al., 1991; Wissel et al., 2009). Our treatment
period was between 1 and 6 months which is consistent with
some treatment protocols. However, multiple re-injections every
3–4 months and lasting up to three years have been used
clinically with the average treatment period lasting for approxi-
mately one year (Borodic et al., 1990; Koman et al., 1993).
Therefore, our injection protocols are very much aligned with
common clinical practice with the exception of the frequency of
injection that was greater in our study so as to not allow muscle
recovery during the experimental period. Studies with reduced
injection frequencies are currently pursued.
Throughout the experimental period, strength in the experi-
mental knee extensors was reduced to 90–95% of normal.
Assuming that loss of muscle mass and contractile material relate
linearly to loss of force, this loss in force can be explained in equal
measures with atrophy and BTX-A inhibition for the group 2
animals (one BTX-A injection), (Fig. 5). For the group 3 animals
(three BTX-A injections), most of the force loss can be attributed
to atrophy and the loss of contractile material while the effect of
BTX-A becomes secondary. Finally, for the group 4 animals (six
BTX-A injections), virtually all loss of force can be explained with
atrophy and contractile material loss, while the actual denerva-
tion effect of BTX-A becomes negligible.
5. Conclusions
The results of this study lead to the conclusion that BTX-A
injections cause severe muscle atrophy and loss of contractile
tissue, thereby affecting the strength and structure of the target
muscles, while also causing atrophy and contractile tissue loss in
non-target muscles far removed from the injection site. It is not
known if chronic BTX-A induced atrophy and contractile tissue
loss is reversible and over what time frame a full muscle recovery
might be possible. However, when using BTX-A clinically as a
chemical denervation agent, it must be acknowledged that
43
muscle atrophy and contractile tissue loss are side-effects with
unknown consequences on structure and health of the injected
and non-target muscles.
Conflict of interest statement
All five authors declare that they do not have any financial and
personal relationships with other people and organisations that
could inappropriately influence our work on this manuscript.
Acknowledgements
The authors would like to thank CIHR of Canada, the Canada
Research Chair Programme, CAPES-Brazil and CNPq-Brazil for
financial support (MAV).
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