Botulinum Toxin-A Injections for Equinus Deformity:

Evidence of Risks, Adverse Effects, & Disappointments

At the annual meetings of the American Academy of Developmental Medicine and Child Neurology in 2018 and 2019, H. Kerr
Graham, orthopedic surgeon, and world-renowned leader in the field of cerebral palsy care, announced that he is retracting
everything he has said about using botulinum toxin-A (BTX-A or BoNT-A) injections for children with cerebral palsy. In a
broadly detailed review article, he joined Multani et al (2019) in advising that injections be limited to no more than one per
year.1
Botulinum toxin (Botox) is a neurotoxic protein produced by the bacterium clostridium botulinum and related species.2
Clostridium botulinum is the most potent, naturally occurring bacterial toxin known to humankind. It is the source of
botulinum toxin.
Ambulatory children with CP showed that the gastrocnemius muscle is stiffer than that of their typically developing peers after
the age of 12 months.3 And so, the muscles that are targeted for injection are generally transformed – altered physiologically -
and already weak.4,5 Injecting a toxin that imposes more weakness is counter-intuitive to the idea of optimizing muscle
function. Furthermore, if an injected muscle “recovers” to pre-injection strength, then it is still weaker than normal. NO
studies have reported that the injected muscle achieves normal strength at any time.
The building and integrating of mature stability and walking skills requires seven to 10 years in typically developing children
who practice for several hours daily in optimum alignment.6,7 Cerebral palsy is a complex, multifactorial, life-long condition.
Any study that looks at the outcome of an isolated intervention aimed at one muscle after 3 to 6 months dismisses the reality
that the gait pathologies in children with CP are anything but singular in origin. There are no quick fixes for this condition.
Are we ready to redirect the funds spent on repeatedly researching the short-term and temporary effects of BTX-A injections
toward investigating the effects of building fundamental body weight management and movement skills in optimum trunk,
limb, and foot alignment?
I have compiled this list of references and, to save space, attached accompanying abstracts in part or in full. You may copy
and paste the first two or three authors into the search window at www.pubmed.gov or google scholar to find the complete
abstracts and in many cases, free pdfs. By way of annotation, and to help you manage this sizeable piece, I have yellow-
highlighted the papers that I find most significant and colored notable statements in blue.
I invite you to share this document with your colleagues in physical medicine, orthopedics, neurology, orthotics, physical
therapy, and occupational therapy, and to engage each other in discussions of the risks of continued use of BTX-A for children
with CP in your facilities.

Studies and Systematic Reviews Involving Children and Adults with Cerebral Palsy and Stroke.
Ackman JD, Russman BS, Thomas SS, et al. 2005. Comparing botulinum toxin A with casting for treatment of dynamic equinus in children
with cerebral palsy. Dev Med Child Neurol. 47(9): 620-627. [39 children with CP (mean age 5y 10mo, range 3 to 9y) were enrolled in a
multicenter, randomized, double blind, placebo-controlled, 1-year prospective study. Children were randomly assigned to one of 3 treatment
groups: BTX-A only (B), placebo injection plus casting (C), or BTX-A plus casting (B+C). The dosage for the BTX-A injections was 4U/kg per
extremity. Assessments were performed at baseline, 3, 6, 7.5, and 12 months with a total of 3 treatments administered after the evaluations
at baseline, 3, and 6 months. Primary outcome measures were ankle kinematics, velocity, and stride length. Secondary outcome measures
were ankle spasticity, strength, range of motion, and ankle kinetics. Group B made no significant change in any variable at any time. Groups C
and B+C demonstrated significant improvements in ankle kinematics, spasticity, passive range of motion, and dorsiflexor strength. Results
indicate that BTX-A alone provided no improvement in the parameters measured in this study, while casting and BTX-A/casting were effective
in the short- and long-term management of dynamic equinus in children with spastic CP].
Ade-Hall RA, Moore AP. 2000. Botulinum toxin type A in the treatment of lower limb spasticity in cerebral palsy. Cochrane Database Syst Rev.
(2):CD001408. Review. [CONCLUSIONS: This systematic review has not revealed strong controlled evidence to support or refute the use of
BtA for the treatment of leg spasticity in cerebral palsy. Ongoing randomised controlled trials are likely to provide useful data on the short
term effects of BtA for leg spasticity. Future research should also assess the longer term use of BtA.]

1
Multani I, Manji J, Hastings-Ison T, Khot A, Graham K. 2019. Botulinum toxin in the management of children with cerebral palsy. Paediatr Drugs.
21(4):261-281.
2 Montecucco C, Molgó J (2005). Botulinal neurotoxins: revival of an old killer. Curr Opin Pharmacol. 5 (3): 274–9.
3 Willerslev-Olsen M, Lorentzen J, Sinkjaer T, Nielsen JB. Passive muscle properties are altered in children with cerebral palsy before the age of 3 years and

are difficult to distinguish clinically from spasticity. Dev Med Child Neurol. 2013;55(7):617–623.
4 Rose J, Haskell WL, Gamble JG, et al.1994. Muscle pathology and clinical measures of disability in children with cerebral palsy. J Orthop Res.12(6):758-68.
5 Rose J, McGill KC. 1998. The motor unit in cerebral palsy. Devel Med Child Neurol. 40(4):270-7.
6 Ganley KJ, Powers CM. 2005. Gait kinematics and kinetics of 7-year-old children: a comparison to adults using age-specific anthropometric data. Gait

Posture. 21(2):141-5.
7 Schepens B, Bastien GJ, Heglund NC, Willems PA. 2004. Mechanical work and muscular efficiency in walking children. J Exper Biol. 207(4):587-96.

Compiled and annotated by Beverly Cusick PT, MS, NDT, COF/BOC – April 10, 2020 1
Alexander C, Elliott C, Valentine J, et al. 2018. Muscle volume alterations after first botulinum neurotoxin A treatment in children with cerebral
palsy: a 6-month prospective cohort study. Dev Med Child Neurol. 60(11):1165-1171. [AIM: to track alterations in muscle volume for 6
months in children with CP after the first exposure to BoNT-A, a commonly used focal spasticity treatment. METHOD: 11 ambulant children (8
M, 3F) with spastic CP, mean age 8 yrs 10 mos (SD 3y 1mo) participated. Participants received injections to the affected gastrocnemius. The
muscle volume of the gastrocnemius, soleus, tibialis anterior, and hamstrings was measured using MRI. Muscle volume was normalized to
bone length, and changes analysed relative to baseline. Assessments were conducted 1 week before, and 4 weeks, 13 weeks, and 25 weeks
after BoNT-A treatment. RESULTS: All children demonstrated positive clinical and functional gains. Muscle volume of the injected
gastrocnemius was found to be significantly reduced at 4 weeks (-5.9%), 13 weeks (-9.4%), and 25 weeks (-6.8%). Significant increases in
normalized soleus muscle volume were identified at each follow-up, while hamstrings showed significant increase at 4 weeks only.
INTERPRETATION: Absolute and normalized muscle volume of the injected muscle reduces after first BoNT-A exposure and does not return to
baseline volume by 25 weeks. Hypertrophy is seen in the soleus up to 25 weeks; the volume of the plantar flexor compartment is stable.
WHAT THIS PAPER ADDS: Muscle atrophy after first botulinum neurotoxin A (BoNT-A) exposure in children with cerebral palsy is noted. Mild
BoNT-A-induced muscle atrophy is still apparent 6 months after BoNT-A exposure. Hypertrophy is evident in soleus after gastrocnemius BoNT-
A exposure. Total plantarflexor volume is unchanged.]
Alhusaini AA, Crosbie J, Shepherd RB, Dean CM, Scheinberg A. 2011. No change in calf muscle passive stiffness after botulinum toxin injection
in children with cerebral palsy. Devel Med Child Neurol. 53(6):553-8. [AIM: to investigate non-neurally mediated calf-muscle tightness in
children with CP before and after BoNT-A injection. 16 children with spastic CP (7F, 9M; 8 at GMFCS level I, 8 at level II; age range 4–
10y) and calf muscle spasticity were tested before and during the pharmaceutically active phase after injection of BoNT-A. Measures of
passive muscle compliance and viscoelastic responses, hysteresis, and the gradient of the torque–angle curve were computed and
compared before and after injection. RESULTS: After injection, a slight, but significant increase in ankle ROM (2.5o into DF, 4o total)
and a small, significant decrease in the torque required to achieve plantigrade and 5° of DF, no significant difference in myotendinous
stiffness or hysteresis were detected. INTERPRETATION: Despite any effect on neurally mediated responses, the compliance of the calf
muscle was not changed and the muscle continued to offer significant resistance to passive motion of the ankle.]
Antonucci F, Rossi C, Gianfranceschi L, Rossetto O, Caleo M. 2008. Long-distance retrograde effects of botulinum neurotoxin A. J Neurosci.
28(14):3689-96. [It is widely assumed that BoNT/A remains at the synaptic terminal and its effects are confined to the injection site. Here we
demonstrate that catalytically active BoNT/A is retrogradely transported by central neurons and motoneurons and is then transcytosed to
afferent synapses, in which it cleaves SNAP-25. SNAP-25 cleavage by BoNT/A was observed in the contralateral hemisphere after unilateral
BoNT/A delivery to the hippocampus. Appearance of cleaved SNAP-25 resulted in blockade of hippocampal activity in the untreated
hemisphere. Injections of BoNT/A into the optic tectum led to the appearance of BoNT/A-truncated SNAP-25 in synaptic terminals within the
retina. Cleaved SNAP-25 also appeared in the facial nucleus after injection of the toxin into rat whisker muscles. Experiments excluded passive
spread of the toxin and demonstrated axonal migration and neuronal transcytosis of BoNT/A. These findings reveal a novel pathway of
BoNT/A trafficking in neurons and have important implications for the clinical uses of this neurotoxin.] So, it migrates to the brain…
Baird MW, Vargus-Adams J. 2010. Outcome measures used in studies of botulinum toxin in childhood cerebral palsy: a systematic review. J
Child Neurol. 25:721–7. [This literature review uses the International Classification of Functioning, Disability and Health to describe the quality
and variety of the studies of botulinum toxin in children with cerebral palsy since 2001. Articles were identified via electronic query and then
reviewed for strength of evidence and classification of outcome measures. The distribution of levels of evidence for the 63 articles was I (n =
8), II (n = 12), III (n = 21), and IV (n = 22). One or more measurements were used in the International Classification of Functioning,
Disability and Health domains of body structure and function (47 papers), activity (47 papers), participation (12 papers), and
environmental/personal factors (10 papers). A total of 67% of all outcomes demonstrated a significant difference (I 49%, II 39%, III 74%, IV
88%). This review illustrates that few studies provide a high level of evidence and that outcomes focus on arenas such as spasticity or range
of motion rather than activity or participation domains such as walking.]
Barber L, Hastings-Ison T, Baker R, et al. 2013. The effects of botulinum toxin injection frequency on calf muscle growth in young children
with spastic cerebral palsy: a 12-month prospective study. J Child Orthop. 7(5): 425-433. [This study was a 12-month prospective
investigation of changes in the medial gastrocnemius (MG) muscle morphology in children aged 2-5 years with spastic CP who had received
no previous intramuscular injections of BoNT-A and were randomised to receive either single or multiple (three) BoNT-A injections to the
gastrocsoleus. MG morphological changes were compared to age-matched TD peers. METHODS: 13 children with spastic CP, mean age 45
months and 18 TD children, mean age 48 months. The principal outcome measures were MG muscle volume, fascicle length, pennation angle
and physiological cross-sectional area (PCSA), which were obtained using 2D and 3D ultrasound. RESULTS: The single and multiple injection
frequency groups significantly increased MG muscle volume at 12 months relative to the baseline by 13 and 15 %, respectively. There were
no significant differences in the MG muscle volume 28.5 (12.3) versus 30.3 (3.8) ml, fascicle length 48.0 (10.4) versus 44.8 (1.2) mm or PCSA
7.0 (1.2) versus 6.6 (1.7) cm(2) between the single and multiple injection groups, respectively, at 12 months follow-up. The change in MG
muscle volume in the single and multiple injection groups was significantly lower than the TD peers by 66 and 60 %, respectively.
INTERPRETATION: In young children with spastic CP, naive to BoNT-A treatment, MG muscle growth over 12 months does not appear to be
influenced by intramuscular BoNT-A injection frequency. However, MG muscle growth in the spastic CP groups was significantly lower than
the age-matched TD peers. It is unclear whether this is an effect of intramuscular BoNT-A injections or reduced growth rates in children with
spastic CP in general.]
Barclay L. 2008. Respiratory compromise, death may be linked to botulinum toxin. Medscape. Feb: 8. Available from: http://www.medscape.com

Blumetti FC, Belloti JC, Tamaoki MJ, Pinto JA. 2019. Botulinum toxin type A in the treatment of lower limb spasticity in children with cerebral
palsy [published online ahead of print, 2019 Oct 8. Cochrane Database Syst Rev. 10(10):CD001408. [Authors' conclusions: The quality of
the evidence was low or very low for most of the outcomes analysed. We found limited evidence that BoNT-A is more effective than placebo
or a non-placebo control at improving gait, joint range of motion, satisfaction, and lower limb spasticity in children with CP, whereas the
results for function were contradictory. The rate of adverse events with BoNT-A is similar to placebo. BoNT-A is not more effective than
ankle serial casting to treat ankle contractures for any of the assessed outcomes, but is more effective than orthotics at improving range of
motion and spasticity.]
Bradley LJ, Huntley JS. 2014. Is there any long-term benefit from injecting botulinum toxin-A into children with cerebral palsy? Arch Dis
Child. 99:392–4.

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Chaturvedi SK, Rai Y, Chourasia A, et al. 2013. Comparative assessment of therapeutic response to physiotherapy with or without botulinum
toxin injection using diffusion tensor tractography and clinical scores in term diplegic cerebral palsy children. Brain Dev. 35(7):647-53. [The
present study was to compare the effects of combined therapy [BTX plus physiotherapy] with physiotherapy alone using diffusion tensor
imaging (DTI) derived fractional anisotropy (FA) values of motor and sensory fiber bundles and clinical grade of the disability to see the value
of BTX in term children with spastic diplegic CP. Clinically diagnosed 36 children participated in the study. All these children were born at term,
and had no history of seizures. The study was randomly categorized into two groups: group I (n=18) - physiotherapy alone and group II
(n=18) - physiotherapy plus BTX injection. Quantitative diffusion tensor tractography on all these children was performed on motor and
sensory fiber bundles on baseline as well as after 6 months of therapy. Motor function and clinical grades were also measured by gross motor
function measures (GMFM) scale on both occasions. We observed significant change in FA value in motor and sensory fiber bundle as well as
in GMFM scores at 6 months compared to baseline study in both the groups. However, delta change and relative delta change in FA values of
sensory and motor fiber bundle as well as GMFM score between group I and group II were statistically insignificant. We conclude that addition
of BTX to physiotherapy regimen does not influence the outcome at 6months with similar insult in children with term diplegic spastic CP. This
information may influence management of diplegic CP especially in developing countries, where BTX is beyond the reach of these children. ]
Degelaen M, de Borre L, Kerckhofs E, et al. 2013. Influence of botulinum toxin therapy on postural control and lower limb intersegmental
coordination in children with spastic cerebral palsy. Toxins (Basel). 5(1):93-105. [Botulinum toxin injections may significantly improve lower
limb kinematics in gait of children with spastic forms of cerebral palsy. Here we aimed to analyze the effect of lower limb botulinum toxin
injections on trunk postural control and lower limb intralimb (intersegmental) coordination in children with spastic diplegia or spastic
hemiplegia (GMFCS I or II). We recorded tridimensional trunk kinematics and thigh, shank and foot elevation angles in fourteen 3-12 year-old
children with spastic diplegia and 14 with spastic hemiplegia while walking either barefoot or with ankle-foot orthoses (AFO) before and after
botulinum toxin infiltration according to a management protocol. We found significantly greater trunk excursions in the transverse plane
(barefoot condition) and in the frontal plane (AFO condition). Intralimb coordination showed significant differences only in the barefoot
condition, suggesting that reducing the degrees of freedom may limit the emergence of selective coordination. Minimal relative phase analysis
showed differences between the groups (diplegia and hemiplegia) but there were no significant alterations unless the children wore AFO. We
conclude that botulinum toxin injection in lower limb spastic muscles leads to changes in motor planning, including through interference with
trunk stability, but a combination of therapies (orthoses and physical therapy) is needed in order to learn new motor strategies.]
Desloovere K, Schörkhuber V, Fagard K, et al. 2012. Botulinum toxin type A treatment in children with cerebral palsy: evaluation of treatment
success or failure by means of goal attainment scaling. Eur J Paediatr Neurol. 16(3):229-36. [Retrospective cohort study to evaluate the
clinical responsiveness of BTX-A treatment in children with CP and specifically delineate features of treatment success and failure. METHODS:
438 children (251 boys, 187 girls; mean age 8 yrs 2 mos, SD 4 years). Goal Attainment Scaling (GAS) was used to classify and evaluate
treatment efficacy. Two study groups were defined: one group with an excellent response (GAS≥60.0) and one group with a lack of response
(GAS≤40.0) to BTX-A. RESULTS: 75 patients (17.1%) had an excellent response and treatment was found to be unsuccessful for 31 patients
(7.1%). Children with a lack of response to BTX-A were significantly older compared to children with a high responsiveness (p=0.0013). In
the latter group, more children received multi-level injections and fewer children had injections in proximal parts of the lower limb compared
to the low responsiveness group (p=0.0024). Moreover, there was a significant difference in the use of different types of casts between both
study groups (p=0.0263). CONCLUSION: Age, level of treatment and casting seem to be crucial features of BTX-A treatment success or
failure in children with CP.]
Dietz V, Sinkjaer T. 2012. Spasticity. Handb Clin Neurol. 109:197-211. [Antispastic medications that are directed to reduce clinical signs of
spasticity, such as exaggerated reflexes and muscle tone, do not improve the movement disorder. Medication can even increase weakness
which might interfere with functional movements, such as walking. In this chapter we address how spasticity affects mobility and how this
should be taken into account in the treatment of spasticity. In clinical practice, signs of exaggerated tendon tap reflexes associated with
muscle hypertonia are the consequence of spinal cord injury (SCI). They are generally thought to be responsible for spastic movement
disorders. Most antispastic treatments are, therefore, directed at the reduction of reflex activity. In recent years, a discrepancy between
spasticity as measured in the clinic and functional spastic movement disorder was noticed, which is primarily due to the different roles of
reflexes in passive and active states, respectively. We now know that central motor lesions are associated with loss of supraspinal drive and
defective use of afferent input with impaired behavior of short-latency and long-latency reflexes. These changes lead to paresis and
maladaptation of the movement pattern. Secondary changes in mechanical muscle fiber, collagen tissue, and tendon properties (e.g., loss of
sarcomeres, subclinical contractures) result in spastic muscle tone, which in part compensates for paresis and allows functional movements on
a simpler level of organization. Antispastic drugs should primarily be applied in complete SCI. In mobile patients they can accentuate paresis
and therefore should be applied with caution.]
Druschel C, Althuizes HC, Funk JF, Placzek R. 2013. Off label use of botulinum toxin in children under two years of age: a systematic review.
Toxins (Basel). 5:60–72. [The effect of BoNT-A injection on juvenile skeletal muscle especially on neuromuscular junction density,
distribution and morphology is poorly delineated and concerns of irreversible damage to the motor endplates especially in young children
exist. In contrast, earlier treatment could be appropriate to improve the attainment of motor milestones and general motor development.
This review systematically analyzes the evidence regarding this hypothesis. A database search, including PubMed and Medline databases,
was performed and all randomized controlled trials (RCTs) comparing the efficacy of Botulinum Toxin in children younger than two years
were identified. Three RCTs met the inclusion criteria. The results of the analysis revealed an improvement in spasticity of the upper and
lower extremities as well as in the range of motion in the joints of the lower limbs. However, evidence of an improvement of general motor
development could not be found, as the assessment of this area was not completely specified for this patient group. Based on available
evidence it cannot be concluded that Botulinum Toxin treatment in children younger than two years improves the achievement of motor
milestones. However, there is evidence for the reduction of spasticity, avoiding contractures and delaying surgery. Due to some limitations,
the results of this review should be cautiously interpreted. More studies, long-term follow up independent high-quality RCTs with
effectiveness analyses are needed.]
Dunne J, Singer BJ, Silbert PL, Singer KP. 2010. Prolonged vastus lateralis denervation after botulinum toxin type A injection. Mov Disord. 25:
397–401. [Intramuscular injection of BoNT produces reversible blockade of neuromuscular transmission. In animal experimental models,
recovery begins within four weeks and is usually complete by twelve weeks. We present evidence of prolonged denervation following BoNT
injection of the vastus lateralis (VL) muscle to correct quadriceps muscle imbalance in patients with chronic anterior knee pain. Needle EMG
data were obtained from 10 subjects who had received a single BoNT treatment 5 to 19 months earlier as part of a clinical trial. Insertional
and spontaneous activity, recruitment, and motor unit action potentials were examined. Clear differences between the injected and non-

Compiled and annotated by Beverly Cusick PT, MS, NDT, COF/BOC – April 10, 2020 3
injected VL muscles, which correlated with the time since injection, were identified in all subjects. All 10 subjects studied with needle EMG
showed evidence of persisting denervation in the BoNT-A injected VL muscle beyond the period of neuromotor recovery expected from animal
experimental studies.]
Engström P, Bartonek Å, Tedroff K, et al. 2013. Botulinum toxin A does not improve the results of cast treatment for idiopathic toe-walking: a
randomized controlled trial. J Bone Joint Surg Am. 95(5):400-7. [AIM: conduct a randomized controlled trial to test the hypotheses that
combined treatment with casts and botulinum toxin A is more effective than casts alone in reducing toe-walking by patients five to fifteen
years of age, and that the treatment effect correlates with the extent of coexisting neuropsychiatric problems. METHODS: All patients who
had been consecutively admitted to the pediatric orthopaedics department of our institution because of idiopathic toe-walking between
November 2005 and April 2010 were considered for inclusion in the study. Forty-seven children constituted the study population. The children
were randomized to undergo four weeks of treatment with below-the-knee casts either as the sole intervention or to undergo the cast
treatment one to two weeks after receiving injections of botulinum toxin A into the calves. Before treatment and three and twelve months
after cast removal, all children underwent three-dimensional (3-D) gait analysis. The severity of the idiopathic toe-walking was classified on
the basis of the gait analysis, and the parents rated the time that their child spent on his/her toes during barefoot walking. Passive hip, knee,
and ankle motion as well as ankle dorsiflexor strength were measured. Before treatment, all children were evaluated with a screening
questionnaire for neuropsychiatric problems. RESULTS: No differences were found in any outcome parameter between the groups before
treatment or at three or twelve months after cast removal. Several gait-analysis parameters, passive ankle motion, and ankle dorsiflexor
strength were improved at both three and twelve months in both groups, even though many children still demonstrated some degree of toe-
walking. The treatment outcomes were not correlated with coexisting neuropsychiatric problems. CONCLUSION: Adding botulinum toxin-A
injections prior to cast treatment for idiopathic toe-walking does not improve the outcome of cast-only treatment.]
Glanzman AM, Kim H, Swaminathan K, Beck T. 2004. Efficacy of botulinum toxin A, serial casting, and combined treatment for spastic
equinus: a retrospective analysis. Dev Med Child Neurol. 46(12): 807-811. [Chart review. 55 patients.. With cast number controlled, change in
ROM after casting with and without BTX-A was not significantly different. Casting with or without BTX-A improved ROM to a comparable
degree, and to a greater degree than BTX-A alone. Casting demonstrated a significantly more robust impact on range of motion than BTX-A
alone]. No difference in effects? So why add the pain, expense, and risks of Botox?
Gough M, Fairhurst C, Shortland AP. 2005. Botulinum toxin and cerebral palsy: time for reflection? Dev Med Child Neurol. 47: 709–712. [ BTX-
A is increasingly being used in early management of spasticity in ambulant children with cerebral palsy (CP), with the aim of improving
function, promoting muscle growth, and delaying the need for surgical intervention. However, there is a lack of evidence about the long-term
outcome of BTX-A injections. The focus on spasticity as the predominant problem in younger children with spastic CP may not fully consider
the associated muscle weakness. It also raises concern that although BTX-A may improve function in the short term, it has the potential to
affect muscle growth and function adversely in the long term. A cautious approach to the early use of BTX-A, with the use of objective
outcome measures within a specialized multidisciplinary setting, is recommended, particularly in ambulant children with spastic diplegic CP,
until further evidence is available on the long-term outcome of early BTX-A injections in children with CP.]
Graham HK, Boyd R, Carlin JB, Dobson F, et al. 2008. Does botulinum toxin A combined with bracing prevent hip displacement in children
with cerebral palsy and “hips at risk”? A randomized, controlled trial. J Bone Joint Surg. 90:23–33. […progressive hip displacement continued
to occur in the treatment group, and our data do not support recommending this treatment. ]
Hastings-Ison T, Sangeux M, Thomason P, et al. 2018. Onabotulinum toxin-A (Botox) for spastic equinus in cerebral palsy: a prospective
kinematic study. J Child Orthop. 12(4):390-397. [Purpose: Botulinum toxin-A (or Botox) is widely used for the management of equinus gait in
children with cerebral palsy but few recent studies have included instrumented gait analysis. Methods: This was a prospective cohort study.
Gait analysis was performed four weeks before and four weeks after Botulinum toxin-A injection for spastic equinus to detect the maximum
effects on gait kinematics. Outcome measures included the Gait Profile Score (GPS), the Gait Variable Score (GVS) for the ankle, maximal
ankle dorsiflexion and maximal knee extension at midstance. Results: In all, 37 children participated (20 boys); mean age five years seven
months (4 years 1 month to 8 years 2 months); 19 with unilateral and 18 bilateral involvement. At a mean four weeks post-injection, the GPS
and ankle GVS were unchanged. However maximum ankle dorsiflexion increased for the whole group; median 7.7° (confidence interval (CI)
4° to 10.6°) to 11.5° (CI 7.7° to 12.9°), p = 0.02. Maximum midstance knee extension was unchanged for the whole group, but median knee
flexion increased in children with bilateral involvement; 10.9° (CI 7.4° to 20.8°) to 16.5° (CI 8.4° to 19.7°), p = 0.58. Conclusion: Injections
of the gastrocsoleus for spastic equinus did not result in objective improvements in overall gait. Improvements in ankle dorsiflexion for
children with bilateral involvement may be offset by deterioration at the knee.]
Hawamdeh ZM, Ibrahim AI, Al-Qudah AA. Long-term effect of botulinum toxin (A) in the management of calf spasticity in children with
diplegic cerebral palsy. Eura Medicophys. 2007;43:311–8. [Reviewers state: “This study is subject to major bias, sufficient to downgrade the
level of evidence accorded, and the results should be treated/interpreted with major reservation.” – Huntley JS, Bradley LJ 2017, p. 371.
Herrmann J, Geth K, Mall V, et al. 2004. Clinical impact of antibody formation to botulinum toxin A in children. Ann Neurol. 55(5):732-5. [We
studied the clinical impact of neutralizing antibodies to botulinum toxin A that occurred during long-term treatment of children between 1993
and 2001. Antibodies were found in high titers in 35 of 110 (31.8%) samples from individual patients. Antibody formation correlated with
secondary nonresponse (p < 0.001). The most significant risk factors for antibody formation were the frequency of treatments (p = 0.0001)
and the injection of a higher weight-adapted maximum dose per treatment (p = 0.001).]
Hong BY, Chang HJ, Lee SJ, et al. 2017. Efficacy of repeated botulinum toxin type a injections for spastic equinus in children with cerebral
palsy-a secondary analysis of the randomized clinical trial. Toxins (Basel). 9(8):253. [Botulinum toxin A is considered an important tool to
control spasticity in children with cerebral palsy. Several factors are known to affect the efficacy of botulinum toxin, such as dosage,
appropriate muscle selection and application, age, and accompanying therapy. A multicenter, double-blind, randomized, prospective phase III
clinical trial of botulinum toxin A for the treatment of dynamic equinus in 144 children with cerebral palsy was performed to compare the
efficacies of letibotulinumtoxin A and onabotulinumtoxin A. Secondary analyses were performed to evaluate factors that affected the outcome,
focusing on the number of times injections were repeated. Effectiveness was defined as a change of 2 or more in the physician's rating scale.
Multivariate regression analyses were performed with multiple variables. The first injection of botulinum toxin A significantly improved D
subscale of Gross Motor Function Measure-88 scores at 3 months compared to repeated injections (p < 0.05). After 6 months, patients who
had one injection or none before the study showed significantly better outcomes than those who had more than one injection in terms of
observational gait scores.]

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Hong BY, Chang HJ, Lee SJ, et al. 2017. Supplementary materials: Efficacy of repeated botulinum toxin type a injections for spastic equinus in
children with cerebral palsy-a secondary analysis of the randomized clinical trial. Toxins (Basel). 9(8):253. doi:10.3390/toxins9080253, S1-S2.
Howell K, Selber P, Graham HK, Reddihough D. 2007. Botulinum neurotoxin A: an unusual systemic effect. J Paediatrics Child health.
43(6):499-501. [Systemic effects from Botulinum neurotoxin A are uncommon but can have serious consequences. We report the case of a
boy with severe cerebral palsy who developed deterioration in respiratory and oromotor function following repeated injections of Botulinum
neurotoxin A. Caution is needed in using this treatment in children with severe cerebral palsy and pseudobulbar palsy.]
http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/DrugSafetyInformationforHeathcareProfessio
nals/ucm070366.htm US Food and Drug Administration. Response to Citizen’s Petition. April 2009.
http://www.fda.gov/downloads/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/DrugSafetyInformationforHeathc
areProfessionals/UCM143989.pdf
Huntley JS, Bradley LJ. 2017. The evidence base for botulinum toxin injection for the treatment of cerebral palsy–related spasticity in the
lower limb: The long-term effects. In: Paediatric Orthopaedics, pp. 369-373. Springer International Publishing. [Botulinum toxin is a potent
neurotoxin that is widely used (and has been for many years) for the treatment of focal spasticity. Its short-term effects have been well
documented. There remains considerable ambivalence over its long-term effects, and some concern over side-effects. A scoping assessment
of the literature defines the paucity of the evidence base concerning long-term effectiveness. Only three level 1 and two level 2 studies
could be identified, on lower limb effects of botulinum toxin in children with cerebral palsy at a time interval greater than 1 year.
Furthermore, our interpretation of the results of these studies is that there is no evidence of clinical benefit. We hope this analysis and
summary can guide clinicians in counseling parents and patients as regards long-term expectations, and reservations/implications of
management.]
Kay RM, Rethlefsen SA, Fern-Buneo A, Wren TA, Skaggs DL. 2004. Botulinum toxin as an adjunct to serial casting treatment in children with
cerebral palsy. J Bone Joint Surg Am. 86-A(11): 2377-2384. [CONCLUSION: The present study demonstrates the efficacy of serial casting in
the treatment of equinus contractures in children with cerebral palsy who are able to walk. Contrary to our hypothesis, the addition of
botulinum toxin A to a serial casting regimen led to earlier recurrence of spasticity, contracture, and equinus during gait. The results of the
present study suggest that botulinum toxin combined with serial casting for the treatment of fixed contractures will lead to a recurrence of
plantar flexor spasticity and equinus contracture by six months in this patient population. While previous research has indicated that the
injection of botulinum toxin A is superior to casting for the treatment of dynamic equinus, the present study suggests that serial casting alone
is preferable for the treatment of fixed equinus contractures in children with cerebral palsy.]
Koerte IK, Schroeder AS, Fietzek UM, et al. 2013. Muscle atrophy beyond the clinical effect after a single dose of OnabotulinumtoxinA
injected in the procerus muscle: a study with magnetic resonance imaging. Dermatol Surg. 39(5):761-765. [AIM: To investigate the loss and
regain of muscular volume in relation to clinical wrinkle severity as assessed using standardized scales. METHODS: The facial procerus and
corrugator supercilii muscles were injected in two drug-naïve men with 20 U of onabotulinumtoxinA at five injection points (onA). Two men
served as controls (one with the same volume of placebo injection using saline solution, one without any intervention). All subjects
underwent 3 Tesla magnetic resonance imaging before and after the injection and 1, 4, 6, 10, and 12 months after the injection.
Standardized photographs were taken at each test point. RESULTS: Volumetric muscle analysis revealed a 46% to 48% reduction in
procerus muscle volume lasting for 12 months after a single dose of onA; glabellar line severity returned to the drug-naïve status after 6 to
10 months. CONCLUSION: The gap between long-term focal muscular atrophy and regained function remains to be elucidated. Future
studies will be needed to investigate the complex interaction between focal neurogenic atrophy and potential compensatory functional
muscle changes.]
Koog YH, Min BI. 2010. Effects of botulinum toxin on calf muscles in children with cerebral palsy: a systematic review. Clin Rehabil. 24:685–
700. [Objective: To assess the efficacy of botulinum toxin A injection for the management of spastic calf muscles in children with cerebral
palsy. Data sources: We reviewed all relevant literature indexed in MEDLINE, CINAHL, EMBASE, PEDro and the Cochrane Registered Trials,
and also hand reviewed abstracts. Methods: Eligible studies were randomized controlled trials that compared botulinum toxin A injection with
any type of treatment or no treatment with identical conditions. We extracted data on calf muscle tone, passive ankle range of motion, gait
speed, ankle kinematics and Gross Motor Function Measure, and assessed methodological qualities. Results: Fifteen studies met our inclusion
criteria. When botulinum injection was compared with a non-sham control, it was found to be effective at improving calf muscle tone (one
month: —2.73 (confidence interval (CI) —3.42 to —2.04), three months: —1.72 (—2.68 to —0.76)), passive ankle range of motion (one
month: 3.29 (CI 2.52 to 4.05), three months: 1.00 (CI 0.44 to 1.56)) and gait speed (one month: 0.91 (CI 0.29 to 1.53), three months: 0.61
(CI 0.01 to 1.21)) for four months, as well as Gross Motor Function Measure (2.02 (CI 1.30 to 2.75)) for two months. When compared with
sham injection, botulinum injection was only effective on Gross Motor Function Measure (0.98 (CI 0.28 to 1.69)) after four months.
Conclusions: Although we found evidence supporting the efficacy of botulinum toxin A in studies comparing botulinum injection with non-
sham controls, we did not find clear evidence of support in studies comparing botulinum injection with sham injection.]
Lin JP. 2005. Efficacy of botulinum toxin A, serial casting, and combined treatment for spastic equinus: a retrospective analysis. Dev Med Child
Neurol. 47(9):635; author reply 635. A letter to the editor pertaining to Glanzman’s study.
Linder-Lucht M, Kirschner J, Herrmann J et al. 2006. Why do children with cerebral palsy discontinue therapy with botulinum toxin A? Devel
Med Child Neurol. 48(4):319-20. [Letter to the editor] Lots of reasons.
Madsen ES1, Sonne-Holm S, Wong C, Curtis D, Bencke J. 2015. [Treatment of spasticity in children with cerebral palsy with botulinum toxin
A]. Ugeskr Laeger. 177(3):V07140409. [Article in Danish] […both randomized controlled and non-controlled trials have been inconclusive. One
explanation may be that children with cerebral palsy do not always exhibit pure spasticity and/or dystonia of the affected muscles.
Furthermore, the dose, injection volume and injection technique may vary from study to study. The evidence for the effect is so small that
careful consideration on whether to continue this treatment regimen or not is needed.]
Mohamed KA, Moore AP, Rosenbloom L. 2001. Adverse events following repeated injections with botulinum toxin A in children with spasticity.
Dev Med Child Neurol. 43(11):791-2. Awful details of adverse episodes – more significant with increasing levels of severity - and the authors
still say it is safe.

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Moore AP, Ade-Hall RA, Tudur Smith C, et al. 2008. Two-year placebo-controlled trial of botulinum toxin A for leg spasticity in cerebral
palsy. Neurology. 71:122–8. [Background: The controlled evidence favoring botulinum toxin A (BtA) treatment for spasticity in cerebral palsy
is based on short-term studies. Methods: We conducted a randomized, double-blind, placebo-controlled, parallel-group study of BtA (Dysport)
for leg spasticity in 64 children with cerebral palsy. For 2 years, the children received trial injections of up to 30 mu/kg every 3 months if
clinically indicated. Results: For the primary endpoints of Gross Motor Function Measure (GMFM) and Pediatric Evaluation of Disability Index
(PEDI) scaled scores at 2 years (trough rather than peak effect), there were no differences between the mean change scores of each group.
For the GMFM total score, the 95% CI of −4.81 to 1.90 excluded a 5-point difference in either direction, and a 2-point benefit with 95%
confidence. There were no differences in adverse events. Conclusions: There was no evidence of cumulative or persisting benefit from
repeated botulinum toxin A (BtA) at the injection cycle troughs at 1 year or 2 years. The dose was not enough to change spasticity measures
and thus GMFM in this heterogeneous group. Ceiling effects in GMFM and Pediatric Evaluation of Disability Index (PEDI) may have reduced
responsiveness. This finding does not deny the value, individually, of single injection cycles or prove that repeating them is unhelpful. In this
regard, BtA treatment can be viewed in the same light as other temporary measures to relieve spasticity, such as oral or intrathecal agents:
there is no evidence of continuing benefit if the treatment ceases. The study provides long-term, fully controlled adverse event data and has
not revealed any long-term adverse effects. ]
Multani I, Manji J, Hastings-Ison T, Khot A, Graham K. 2019. Botulinum toxin in the management of children with cerebral palsy. Paediatr
Drugs. 21(4):261-281. [During the past 25 years, botulinum toxin type A (BoNT-A) has become the most widely used medical intervention in
children with cerebral palsy. In this review we consider the gaps in our knowledge in the use of BoNT-A and reasons why muscle morphology
and function in children with cerebral palsy are impaired. We review limitations in our knowledge regarding the mechanisms underlying the
development of contractures and the difficulty in preventing them. It is clear from this review that injection of BoNT-A in the large muscles of
both the upper and lower limbs of children with cerebral palsy will result in a predictable decrease in muscle activity, which is usually reported
as a reduction in spasticity, for between 3 and 6 months. These changes are noted by the use of clinical tools such as the Modified Ashworth
Scale and the Modified Tardieu Scale. Decreased muscle over-activity usually results in improved range of motion in distal joints. Injection of
the gastrocnemius muscle for toe-walking in a child with hemiplegia or diplegia usually has the effect of increasing the passive range of
dorsiflexion at the ankle. In our review, we found that this may result in a measurable improvement in gait by the use of observational gait
scales or gait analysis, in some children. However, improvements in gait function are not always achieved and are small in magnitude and
short lived. We found that some of the differences in outcomes in clinical trials may relate to the use of adjunctive interventions such as serial
casting, orthoses, night splints and intensive therapy. We note that the majority of clinical trials of the use of BoNT-A in children with cerebral
palsy have focussed on a single injection cycle and this is insufficient to understand the balance between benefit and harm. Most outcomes
were reported in terms of changes in muscle tone and there were fewer studies with robust methodology that reported improvements in
function. Changes in the domains of activities and participation have rarely been reported in studies to date. There were no clinical reviews to
date that consider the findings of studies in human volunteers and in experimental animals and their relevance to clinical protocols. In this
review we found that studies in human volunteers and in experimental animals show muscle atrophy after an injection of BoNT-A for at least
12 months. Muscle atrophy was accompanied by loss of contractile elements in muscle and replacement with fat and connective tissue. It is
not currently known if these changes, mediated at a molecular level, are reversible. We conclude that there is a need to revise clinical
protocols by using BoNT-A more thoughtfully, less frequently and with greatly enhanced monitoring of the effects on injected muscle for both
short-term and long-term benefits and harms.]
Naidu K, Smith K, Sheedy M, et al. 2010. Systemic adverse events following botulinum toxin A therapy in children with cerebral palsy. Dev
Med Child Neurol. 52(2):139-44. [We studied the incidence of incontinence and respiratory events in children with cerebral palsy who received
injections of botulinum toxin A (BoNT-A). METHOD: We used multivariable logistic regression to investigate relationships between (BoNT-A)
dose, Gross Motor Function Classification System (GMFCS) level, and the incidence of bladder or bowel incontinence, unplanned hospital
admission, emergency department consultation or prescription of antibiotics for respiratory symptoms, and diagnosis of upper respiratory tract
infection. RESULTS: Of 1980 injection episodes in 1147 children (mean age 4y 7mo, SD 1y 10mo, range 9mo-23y), 488 (25%) were in
children with unilateral involvement and 1492 (75%) in children with bilateral involvement. At the time of injection 440 (22.2%) of children
were at GMFCS level I, 611 (30.9%) were at level II, 330 (16.7%) were at level III, 349 (17.6%) were at level IV, and 250 (12.6%) were at
level V. The incidence of serious adverse events was low, with 19 episodes of incontinence (1% of injection episodes) and 25 unplanned
hospital admissions due to respiratory symptoms (1.3%). Incontinence typically resolved spontaneously 1 to 6 weeks after injection. The
incidence of adverse events was associated with GMFCS level and dose of BoNT-A. INTERPRETATION: The incidence of serious adverse
events was low but suggests systemic spread as well as a procedural effect. We recommend reviewing upper dose limits for children at all
GMFCS levels, particularly those at levels IV and V with a history of aspiration and respiratory disease. In these children, alternatives to mask
anaesthesia may be particularly important.]
Naumann M, Albanese A, Heinen F, Molenaers G, Relja M. 2006. Safety and efficacy of botulinum toxin type A following long-term use. Eur J
Neurol 2006; 13: 35–40. [Botulinum toxin serotype A (BoNT-A) has long heritage of use leading to confidence in its safety and efficacy. The
application of BoNT-A does not lead to persistent histological changes in the nerve terminal or the target muscle. [WRONG!] Clinical trials
defined the safety and tolerability profile of BoNT-A across common therapeutic indications and showed an incidence of adverse events of
approximately 25% in the BoNT-A-treated group compared with 15% in the control group. Focal weakness was the only adverse event to
occur more often following BoNT-A treatment.] Since when is weakness OK?
O’Flaherty S, Janakan V, Morrow A, Scheinberg A, Waugh M. 2011. Botulinum toxin A adverse events and health status in children with
cerebral palsy in all GMFCS levels. Devel Med Child Neurol. 53:125–30. [A total of 334 children (190 male; 144 female) aged 1y 6mo to 19y
4mo (mean 9y 2mo, SD 4y) with CP who were undergoing BoNT-A injections (596 injection courses in total) were clinically audited over a 16-
month period. Of the 334 children, 62 were classified at Gross Motor Function Classification System (GMFCS) level I, 52 of whom had
unilateral CP and 10 of whom had bilateral CP. Eighty-six children were classified at GMFCS level II, 39 of whom had unilateral CP and 47 of
whom had bilateral CP. Forty-four children were classified at GMFCS level III, two of whom had unilateral CP and 42 of whom had bilateral
CP. Sixty-six of the 334 children were classified at GMFCS level IV and 76 as level V. All the children classified as level IV or V had bilateral
involvement. The health status of the children in the month before and a prospective audit of health status and adverse events in the month
after BoNT-A injections were examined in order to assess the effects of the toxin. Results: The data gathered for the month before
administration of BoNT-A indicated that children with CP had significant background morbidities. After injection of BoNT-A, adverse events
occurred in 23.2% of children. All adverse events were temporary and there were no deaths.] So, no decision to avoid injecting children at
higher risk?

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Omprakash Hm, Rajendran Sc. 2008. Botulinum toxin deaths: What is the fact? J Cutan Aesthet Surg. 1(2): 95-7. Not much info on CP
population.
Palomar FJ, Mir P. 2012. Neurophysiological changes after intramuscular injection of botulinum toxin. Clin Neurophysiol. 123(1):54-60. This is
about distant effects in the CNS, far from the injection site.
Papavasiliou AS, Nikaina I, Foska K, et al. 2013. Safety of botulinum toxin A in children and adolescents with cerebral palsy in a pragmatic
setting. Toxins (Basel). 5(3):524-36. [This retrospective study aimed to examine the safety of botulinum toxin A (BoNT-A) treatment in a
paediatric multidisciplinary cerebral palsy clinic. In a sample of 454 patients who had 1515 BoNT-A sessions, data on adverse events were
available in 356 patients and 1382 sessions; 51 non-fatal adverse events were reported (3.3% of the total injections number, 8.7% of the
patients). On five occasions, the adverse reactions observed in GMFCS V children were attributed to the sedation used (rectal midazolam plus
pethidine; buccal midazolam) and resulted in prolongation of hospitalization. Of the reactions attributed to the toxin, 23 involved an excessive
reduction of the muscle tone either of the injected limb(s) or generalized; others included local pain, restlessness, lethargy with pallor,
disturbance in swallowing and speech production, seizures, strabismus, excessive sweating, constipation, vomiting, a flu-like syndrome and
emerging hypertonus in adjacent muscles. Their incidence was associated with GMFCS level and with the presence of epilepsy (Odds ratio
(OR) = 2.74 - p = 0.016 and OR = 2.35 - p = 0.046, respectively) but not with BoNT-A dose (either total or per kilogram). In conclusion,
treatment with BoNT-A was safe; adverse reactions were mostly mild even for severely affected patients. Their appearance did not
necessitate major changes in our practice.] So these authors apparently did not swear to “do no harm”…? No decision to avoid injecting
children at higher risk?
Park ES, Sim E, Rha DW, Jung S. 2014. Architectural changes of the gastrocnemius muscle after botulinum toxin type a injection in children
with cerebral palsy. Yonsei Med J. 55(5):1406-12. [Thirteen children with CP who received a BoNT-A injection into their GCM to treat equinus
were recruited (9 males and 4 females). Architectural changes in both the medial and lateral heads of the GCM from a total of 20 legs were
assessed using B-mode, real-time US. Muscle thickness (MT), fascicle length (FL), and fascicle angle (FA) were measured over the middle of
the muscle belly in both a resting and neutral ankle position. Measures at 1 and 3 months after the injection were compared with baseline
data taken before the injection. RESULTS: The mean age of the subjects was 5.8 (±1.6) years….The MT and FA of both the medial and lateral
heads of the GCM were significantly reduced for both neutral and resting ankle positions at 1 and 3 months after the injection. The FL of both
the medial and lateral heads of the GCM were significantly increased in a resting position (p<0.05), but not in a neutral position.]
Petition to the FDA requesting regulatory action concerning the possible spread of botulinum toxin from the site of injection to other parts of
the body (HRG Publication #1834). http://www.citizen.org/publications/publicationredirect.cfm? ID=7559
Pin TW, Elmasry J, Lewis J. 2013. Efficacy of botulinum toxin A in children with cerebral palsy in Gross Motor Function Classification System
levels IV and V: a systematic review. Dev Med Child Neurol. 55(4):304-13. [AIM: Previous studies have shown the efficacy of botulinum toxin
type A (BoNT-A) in the management of ambulant individuals with cerebral palsy (CP). There is little evidence on its use in non-ambulant
children with CP. This review aimed to investigate indications and efficacy for BoNT-A use in managing pain, care, and comfort, and improving
function in children with CP in Gross Motor Function Classification System (GMFCS) levels IV and V. METHOD: Electronic databases were
searched from the earliest available date to June 2012 using a combination of subject headings and free text. Inclusion criteria consisted of
studies with (1) participants aged 18 or under, (2) participants with CP in GMFCS levels IV and V, (3) participants receiving BoNT-A treatment,
and (4) studies published in English-language peer-reviewed journals. RESULTS: The search resulted in a total of 814 studies, of which 19
met the inclusion criteria. Eighteen studies provided level IV or V evidence and one level I evidence according to the American Academy for
Cerebral Palsy and Developmental Medicine guidelines for the development of systematic reviews. Most of the studies were of weak to
moderate methodological quality. INTERPRETATION: The evidence that BoNT-A is effective in reducing postoperative pain in children with CP
in GMCFS levels IV and V is limited, with only one level I study identified. Remaining indications were general pain reduction, maintaining hip
integrity, achieving functional changes, and goal attainment. A high percentage of participants in the studies showed positive changes in
these areas. With the poor level of evidence of the included studies, no definite conclusion could be drawn on the indications for BoNT-A use
in children with CP in GMCFS levels IV and V. Further investigation by rigorous studies is required.]
Rameckers EA, Speth LA, Duysens J, Vles JS, Smits-Engelsman BC. 2009. Botulinum toxin-a in children with congenital spastic hemiplegia
does not improve upper extremity motor-related function over rehabilitation alone: a randomized controlled trial. Neurorehabil Neural Repair.
23(3):218-25.
Ramirez-Castaneda J, Jankovic J, Comella C, et al. 2013. Diffusion, spread, and migration of botulinum toxin. Mov Disord. 28(13): 1775-83.
[The efficacy and safety of BoNT depends on accurate selection and identification of intended targets but also may be determined by other
factors, including physical spread of the molecule from the injection site, passive diffusion, and migration to distal sites via axonal or
hematogenous transport. Most of the local and remote complications of BoNT injections are thought to be due to unwanted spread or
diffusion of the toxin's biologic activity into adjacent and distal muscles. Despite >30 years of widespread therapeutic and cosmetic use of
BoNT, there is a remarkable paucity of published data on the mechanisms of distribution and its effects on clinical outcomes.]
Read FA, Boyd RN, Barber LA. 2017. Longitudinal assessment of gait quality in children with bilateral cerebral palsy following repeated lower
limb intramuscular Botulinum toxin-A injections. Res Dev Disabil. 68:35-41. [Aim: To determine the effect of three consecutive treatment
cycles of lower limb intramuscular BoNT-A injections on gait quality using the EVGS in children with bilateral CP (BCP) by retrospectively
reviewing repeated 2DVGA measures. Methods: 17 children with BCP and dynamic equinus (8 females and 9 males, age mean (SD), 4.0 (2.2)
years, GMFCS I=2 and II=15) were included in the study after a retrospective audit of the records of the Queensland Children's Gait
Laboratory (QCGL), Children's Health Queensland, Brisbane. The medical records of children who attended the QCGL between January 2001
and January 2016 were searched for eligibility. Children who had undertaken pre- and post-treatment 2DVGA for the first three lower limb
BoNT-A treatment cycles (6 assessments) were reviewed using the EVGS. BoNT-A treatments were administered 7.7 (2.3) months apart and
post-BoNT-A reviews occurred 12.6 (6.7) weeks after injection. Mixed-effects linear regression assessed the change from baseline to each
subsequent assessment (p<0.05). Outcomes and results: EVGS reduced significantly by a mean of 2.4 points from pre- to post-BoNT-A in the
first treatment cycle (p=0.001). Compared to baseline, mean total EVGS reduced significantly during the second (pre-BoNT-A -1.7 (p=0.020),
post BoNT-A -2.8 (p<0.001)) and third (pre-BoNT-A -2.6 (p=0.001), post BoNT-A -2.4 (p=0.002)) treatment cycles. There was no difference
in EVGS between post-BoNT-A in the first treatment cycle and scores for the second and third treatment cycles. Conclusions and implications:
Improvements in gait quality were statistically significant, but did not reach the EVGS smallest real difference value of 4 points. Repeated
lower limb intramuscular BoNT-A injections to improve gait quality in children with BCP should be reconsidered.]

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Restani L, Giribaldi F, Manich M, et al. 2012. Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons.
PLoS Pathog 8(12): e1003087. [We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal
retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT,
following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo,
BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but
lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is
composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the
soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system.]
Salari M, Sharma S, Jog MS. 2018. Botulinum toxin induced atrophy: an uncharted territory. Toxins (Basel). 10(8). Review. […the
development of muscle atrophy following chronic exposure to BoNTs has not received sufficient attention. Muscle atrophy is not only
cosmetically distressing, but also has an impact on future injections. An extensive literature search was conducted on atrophy and
mechanisms of atrophy. Five hundred and four relevant articles in the English language were reviewed. This review revealed the
surprising lack of documentation of atrophy within the literature. In addition, as demonstrated in this review, the mechanisms and the
clinical factors that may lead to atrophy have also been poorly studied. …This review highlights the need for further study of atrophy
following BoNT injections]
Schasfoort F, Pangalila R, Sneekes EM, et al. 2018. Intramuscular Botulinum toxin prior to comprehensive rehabilitation has no added value
for improving motor impairments, gait kinematics and goal attainment in walking children with spastic cerebral palsy. J Rehabil Med.
50(8):732–742. [A comparative multi-centre trial in which two groups underwent comprehensive rehabilitation (i.e. high-intensive functional
physiotherapy, and indicated casting/orthoses). One group received intramuscular BoNT-A prior to rehabilitation, and the other group did not
receive BoNT-A. SUBJECTS/PATIENTS: Children with spastic cerebral palsy, Gross Motor Function Classification System (GMFCS) levels I-III,
age range 4-12 years, indicated for BoNT-A treatment regarding mobility problems. METHODS: 65 children participated (37 boys), mean age
7.3 years (+/- 2.3, range 4-12 years), equally distributed across GMFCS levels. 41 children received BoNT-A+ comprehensive rehabilitation
and 24 received comprehensive rehabilitation only. Functional leg muscle strength, passive range of motion, angle of catch, cerebral palsy-
related pain, walking speed, kinematic gait parameters, goal attainment, and proxy-reported general functioning were assessed at baseline,
primary end-point (12 weeks) and 24-week follow-up. Statistical analyses were performed with linear mixed models. RESULTS: At the primary
end-point there were no statistically significant differences in treatment effects between the groups, except for the angle-of-catch of the rectus
femoris, which was in favour of comprehensive rehabilitation without BoNT-A (12° difference, 95% confidence interval (95% CI) 2:23,
p = 0.025). Results at follow-up were similar. CONCLUSION: At the group level, treating with BoNT-A prior to comprehensive rehabilitation did
not add to the clinical effectiveness of rehabilitation. Thus, BoNT-A prescription and use should be critically reconsidered in this cerebral palsy
age- and GMFCS-subgroup.]
Schasfoort F, Dallmeijer A, Pangalila R, et al. 2018. Value of botulinum toxin injections preceding a comprehensive rehabilitation period for
children with spastic cerebral palsy: a cost-effectiveness study. J Rehabil Med. 50(1):22–29. [A pragmatically designed, multi-centre trial,
comparing the effectiveness of botulinum toxin + intensive physiotherapy with intensive physiotherapy alone, including economic evaluation.
SUBJECTS/PATIENTS: Children with spastic cerebral palsy, age range 4-12 years, cerebral palsy-severity Gross Motor Function Classification
System levels I-III, received either botulinum toxin type A + intensive physiotherapy or intensive physiotherapy alone and, if necessary,
ankle-foot orthoses and/or casting. METHODS: Primary outcomes were gross motor function, physical activity levels, and health-related
quality-of-life, assessed at baseline, 12 (primary end-point) and 24 weeks (follow-up). Economic outcomes included healthcare and patient
costs. Intention-to-treat analyses were performed with linear mixed models. RESULTS: There were 65 participants (37 males), with a mean
age of 7.3 years (SD 2.3 years), equally distributed across GMFCS levels. 41 children received botulinum toxin type A plus intensive physio-
therapy and 24 received intensive physiotherapy treatment only. At primary end-point, one statistically significant difference was found in
favour of intensive physiotherapy alone: objectively measured percentage of sedentary behaviour (-3.42, 95% confidence interval 0.20-6.64,
p=0.038). Treatment costs were significantly higher for botulinum toxin type A plus intensive physiotherapy (8,963 vs 6,182 euro, p=0.001).
No statistically significant differences were found between groups at follow-up. CONCLUSION: The addition of botulinum toxin type A to
intensive physiotherapy did not improve the effectiveness of rehabilitation for ambulatory children with spastic cerebral palsy and was also not
cost-effective. Thus botulinum toxin is not recommended for use in improving gross motor function, activity levels or health-related quality-of-
life in this cerebral palsy age- and severity-subgroup.]
Schasfoort F, Dallmeijer A, Pangalila R, et al. 2015. Botulinum toxin has no added therapeutical value or cost-effectiveness for gross motor
function, everyday physical activity or quality of life when combined with intensive functional physiotherapy. Devel Med Child Neurol 57:8-9.
[Since 20-25 years, children with spastic CP are increasingly treated with botulinum toxin type A injections (BtA) followed by a period of
intensive physiotherapy (iPT) as treatment combination. Despite previous research, the added value of BtA remained unclear. In the SPACE
BOP study, we therefore compared the effectiveness of BtA+iPT versus only-iPT treatment on gross motor function, everyday physical activity
and quality of life in children with CP, including economic evaluation. Study Design: Multicenter (partly) randomized controlled trial, single
blinded. Study Participants and Setting: Children with uni- or bilateral lower extremity spastic CP, Gross Motor Function Classification System
levels I-III, aged 4–12 years were eligible. Multilevel BtA treatment was in (university) hospitals (i.e. usual care) and the 12-week iPT period
(3x/wk) with the children’s own therapist (best available evidence guideline 2010). Materials/Methods: Measurements were performed at
baseline, after the iPT period ended (primary endpoint) and 12 weeks thereafter (follow-up). Primary outcome measures were: (1) actual
everyday physical activity levels measured with ambulatory monitoring devices over 7 days (Actigraph), (2) (health related) quality of life
measured with the Health Utility Index, Child Health Questionnaire, DISABKIDS (generic, smileys and CP module), and (3) gross motor
function measured with the Gross Motor Function Measure (GMFM-item sets). We used linear mixed models with random intercept and
statistically corrected for effects of (non-)randomization, GMFCS level and age (using SPSS 21). Economic evaluation was performed according
to Dutch guidelines: societal perspective, including health care and patient costs for time and travel. Results: Sixty-five children (of whom n =
26 randomized) participated: mean age 7.3 _ 2.3yrs, n = 37 boys, n = 14 unilateral CP, n = 19 GMFCS level I, n = 23 level II and n = 23
level III, n = 41 received BtA+iPT and n = 24 received onlyiPT. No statistically significant evidence was found for added value of BtA
injections on primary outcomes. At primary end point, we found two trends towards an intervention effect in favor of only-iPT treatment:
improved gross motor function (effect size 1.12, 95% CI [_0.20:2.44], p = 0.095) and decreased % of sedentary behavior during everyday
physical activity (effect size _2.59, 95% CI [_5.58:0.39], p = 0.087). At follow-up, we found one trend in favor of only-iPT: improved general
quality of life measured with DISABKIDS-smileysproxy (effect size 5.9, 95% CI [_0.40:12.20], p = 0.066). We also found one trend in favor of
BtA+iPT: improved everyday physical activity counts per minute (effect size 91, 95% CI [_6:188], p = 0.064).

Compiled and annotated by Beverly Cusick PT, MS, NDT, COF/BOC – April 10, 2020 8
Average treatment costs (including plasters and AFO’s) were significantly higher in BtA+iPT than in only-iPT (8.963 versus 6.182 euro, p =
0.001). Conclusions/Significance: We conclude that adding BtA to iPT in combined BtA+iPT treatment does not improve effectiveness (at
group level) and that adding BtA to iPT is also not cost-effective when treatment is aimed at improving gross motor function, everyday
physical activity levels or quality of life.]
Scholtes VA, Dallmeijer AJ, Knol DL, et al. 2007. Effect of multilevel botulinum toxin a and comprehensive rehabilitation on gait in cerebral
palsy. Pediatr Neurol. 36(1):30-9. Clinical Trial. [A multicenter randomized trial was performed in 46 children with spastic cerebral palsy who
walk with flexed knees. Their mean age was 8.0 years (range 4 to 11 years). They were randomly allocated to the intervention group
(multilevel botulinum toxin A and comprehensive rehabilitation) or the control group (usual care). After 6 weeks, a significant treatment effect
in the intervention group was observed on: improved knee extension during midstance and terminal swing (7 degrees and 5 degrees , P <
0.01, respectively); hip rotation during terminal swing (4 degrees , P = 0.02); gait score (1.7, P < 0.01); decreased spasticity in hamstrings
(11 degrees , P < 0.01), gastrocnemius (6 degrees , P = 0.01), and soleus (5 degrees , P = 0.02); and increased muscle length in hamstrings
(9 degrees , P < 0.01) and gastrocnemius (5 degrees , P < 0.01). The improved muscle length was maintained up to 24 weeks. This study
demonstrated that multilevel botulinum toxin A and comprehensive rehabilitation improves knee extension during gait, increases muscle
length, and decreases spasticity in injected muscles after 6 weeks in children who walk with flexed knees. Although the effect on muscle
length was maintained after 24 weeks, the effect on gait and spasticity had disappeared.] The control group program does not compare with
the study group program.
Schroeder AS, Koerte I, Berweck S, Ertl-Wagner B, Heinen F. 2010. How doctors think--and treat with botulinum toxin. Dev Med Child Neurol.
52(9):875-6. Letter to the editor. [Recent data on the duration of neurogenic atrophy after injection into healthy human muscle have had little
impact on clinical practice so far and data on spastic muscle are missing. Authors show specialized MRI images of muscle from a male with CP
after one injection of BTX-A. The images show the same high signal intensity pattern as the authors reported in 2009 in healthy volunteers,
indicating that focal neurogenic atrophy at the site of injection is still present 6 months (and to some extent 1 year) after BoNT‐A injection.
“Since its introduction in the 1980s, and encouraged by positive clinical effects, BoNT‐A has been used ahead of a comprehensive
fundamental knowledge about its biological effects in humans. The data on spastic muscle presented here are consistent with earlier
observations in healthy muscle indicating that focal neurogenic atrophy following BoNT‐A persists longer than expected. The discrepancy
between the observed ‘focal long‐term structural alterations’ and the clinically reported ‘short‐term functional efficacy’ remains unclear. This
should induce further investigations and provoke reconsideration of the established regimes of BoNT‐A treatment.”]
Schroeder AS, Ertl‐Wagner B, Britsch S, et al. 2009. Muscle biopsy substantiates long‐term MRI alterations one year after a single dose of
botulinum toxin injected into the lateral gastrocnemius muscle of healthy volunteers. Movement Disorders.24(10):1494-503.[Despite
numerous clinical and experimental studies on botulinum toxin type A (BoNT/A), long-term alterations of muscle texture and fine structure
following BoNT/A treatment have thus far not been studied in normal human skeletal muscle. After obtaining institutional review board
approval, we performed a prospective, placebo-controlled, double-blinded follow-up study on two healthy adults using magnetic resonance
imaging (MRI) and muscle biopsy to visualize long-term alterations after a single BoNT/A injection into the lateral head of the gastrocnemius
muscle. MRI disclosed a high-signal-intensity pattern in short tau inversion recovery sequences, and a reduction of the cross-sectional area in
the BoNT/A-injected, but not in the saline-injected contralateral control muscle (at 6 to 9 months in volunteer A: 73%, in B: 62%; at 12
months in A: 88%, and in B: 78%). Enzyme histochemistry, 12 months after injection, confirmed neurogenic atrophy of muscle fibers only in
the BoNT/A-injected muscle. Electron microscopy revealed additional degenerative changes at the neuromuscular junction.]
Sheean GL. 2001. Botulinum treatment of spasticity: why is it so difficult to show a functional benefit? Curr Opin Neurol. 14(6):771-6. [Clinical
experience seems to indicate that botulinum toxin injections can, in selected patients with upper motor neurone syndrome, reduce spasticity
and improve voluntary movement and active function. However, double-blind placebo-controlled trials have had difficulty showing active
functional improvement, despite the clear ability of botulinum toxin to reduce spasticity. This prompts a re-analysis of the basic assumption
that spasticity impairs voluntary movement and a review of the methodology of the clinical trials. Motor dysfunction is usually caused by
weakness and the other ‘negative’ features of upper motor neurone syndrome, rather than muscle overactivity. Recent research has explored
the pathophysiological basis of the voluntary movement disorder, in particular the role of the various forms of motor overactivity, which might
be amenable to botulinum toxin treatment. The failure of double-blind placebo-controlled clinical trials to show improvement in active function
is, to a large extent, a result of their methodology, especially patient selection, injection protocols, and the choice of outcome measures.
Clinical trials need to be re-designed and based upon expert experience and a better understanding of the pathophysiology of the motor
disorder.]
Swinney CM, Bau K, Burton KLO, et al. 2018. Severity of cerebral palsy and likelihood of adverse events after botulinum toxin A injections.
Dev Med Child Neurol. 60(5):498–504. [RESULTS: In total, 591 children underwent 2219 injection episodes. There were significantly increased
rates of systemic adverse events in injection episodes involving children in GMFCS levels IV and V. Adverse events reported at the time of
botulinum toxin A injection occurred in 6% of injection episodes. Adverse events were reported at follow-up in 22% of injection episodes.]
Tedroff K, Granath F, Forssberg H, Haglund-Akerlind Y. 2009. Long-term effects of botulinum toxin A in children with cerebral palsy. Dev Med
Child Neurol. 51(2): 120-127. [The long-term effects of botulinum toxin A (BoNT-A) treatment in children with CP are still elusive. We studied
a prospective clinical cohort of 94 children with different subtypes (50% spastic diplegic CP, 22% hemiplegic CP, 25% tetraplegic CP, 3%
dyskinetic CP), sex (55% male, 45% female), severity according to Gross Motor Function Classification System (29% Level I, 15% Level II,
16% Level III, 17% Level IV, 23% Level V), and age (median 5y 4mo, range 11mo-17y 8mo). The longest follow-up time was 3 years 7
months (median 1y 6mo) and included a maximum of eight injections per muscle (median two injections to a specific muscle). Outcome
measurements were muscle tone (Modified Ashworth Scale) and joint range of motion (ROM). Assessments were made at a minimum before
and 3 months after each injection. Ninety-five per cent confidence intervals for differences from baseline were used to identify significant
changes. BoNT-A injections induced reduction of long-term spasticity in all muscle-groups examined: the gastrocnemius, hamstring, and
adductor muscles. The reduction in tone was most distinct in the gastrocnemius muscle, and each repeated injection produced an immediate
reduction in muscle tone. However, improvement in ROM was brief and measured only after the first injections, whereupon the ROM declined.
Thus, the results suggest that BoNT-A can be effective in reducing muscle tone over a longer period, but not in preventing development of
contractures in spastic muscles. The dissociation between the effects on muscle tone and ROM indicates that development of contractures is
not coupled to increased muscle tone only, but might be caused by other mechanisms.]

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Tedroff K, Lowing K, Haglund-Akerlind Y, Gutierrez-Farewik E, Forssberg H. 2010. Botulinum toxin A treatment in toddlers with cerebral
palsy. Acta Paediatr. 99:1156–62. [Reviewers state: Several concerns about the study method, small sample size, differences between
study groups, and “difficulty in deriving meaningful values from the data largely negate any value of the study.” – Huntley JS, Bradley LJ
2017, p. 371.
Thacker BE, Tomiya A, Hulst JB, et al. 2012. Passive mechanical properties and related proteins change with botulinum neurotoxin A injection
of normal skeletal muscle. Journal Orthop Res.30(3):497-502. [The effects of botulinum neurotoxin A on the passive mechanical properties of
skeletal muscle have not been investigated, but may have significant impact in the treatment of neuromuscular disorders including spasticity.
Single fiber and fiber bundle passive mechanical testing was performed on rat muscles treated with BTX-A. Neurotoxin‐treated single fiber
passive elastic modulus was reduced compared to control fibers. Fiber stiffness and slack sarcomere length were also reduced compared to
control fibers and myosin heavy chain composition shifted from faster to slower isoforms. Average titin molecular weight increased 1.77%
after treatment. Fiber bundle passive elastic modulus increased following treatment. Bundle stiffness also increased while collagen content per
mass of muscle tissue increased 38%. Injection of botulinum neurotoxin A produces an effect on the passive mechanical properties of normal
muscle that is opposite to the changes observed in spastic muscles.]
Tsai FC, Hsieh MS, Chou CM. 2010. Comparison between neurectomy and botulinum toxin A injection for denervated skeletal muscle. J
Neurotrauma. 27(8):1509-16. [Neurectomy and botulinum toxin A (BoNT-A) injection cause denervated muscle atrophy, but questions remain
about their clinical utility. We investigated time-series alterations of rat muscle weight, functional deficits, signaling pathways, and microscopic
structures, to gain an understanding of the clinical implications. Between 2008 and 2009, the maximal calf circumference of patients for calf
reduction either by neurectomy or BoNT-A injections were recorded for study. A rat skeletal muscle model was established through repeated
or dose-adjusted BoNT-A injections and neurectomy. The survival, apoptosis (natural cell death) pathways, functional deficits, and
microscopic structures were investigated using Western blot, sciatic functional index (SFI), and transmission electron microscopy (TEM),
respectively. The rat muscle weight ratio of the BoNT-A group had recovered to 89.3 +/- 3.8% by week 58, but it never recovered in the
neurectomy group. Muscle weight reduction by BoNT-A not only depended on the dose, but additive effects were also obtained through
repeated injections. Rat SFI demonstrated rapid recovery in both groups. Molecular expressions showed a coherent and biphasic pattern. p-
Akt and apoptosis-inducing factor (AIF) were upregulated significantly, with a peak at 8 weeks in the neurectomy group (p < 0.01), but
cleaved caspase-9 and caspase-3 showed no significant changes in either group. TEM findings showed irreversible and reversible inner-
structure disruption and sarcomere discontinuity in the neurectomy and BoNT-A groups, respectively. We demonstrated that denervation
induced lasting muscle weight and structural changes of different degrees. Muscle weight reduction by BoNT-A was related to frequency and
dose. AIF-mediated caspase-independent apoptosis was significantly different for neurectomy and BoNT-A injection.] Healthy animal muscle,
not stiff, transformed CP muscle.
US Food and Drug Administration. Early Communication about an ongoing safety review of botox and botox cosmetic (Botulinum toxin Type
A) and Myobloc (Botulinum toxin Type B). February 2008.
US Food and Drug Administration. Early Communication about an Ongoing Safety Review of Botox and Botox Cosmetic (Botulinum toxin Type
A) and Myobloc (Botulinum toxin Type B). April 2009.
http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/DrugSafetyInformationforHeathcareProfessio
nals/ucm070366.htm
US Food and Drug Administration. Information for Healthcare Professionals: OnabotulinumtoxinA (marketed as Botox ⁄ Botox Cosmetic),
AbobotulinumtoxinA (marketed as Dysport) and RimabotulinumtoxinB (marketed as Myobloc). August 2009. http://www.fda.gov/Drugs/Drug
Safety/PostmarketDrugSafetyInformationforPatientsandProviders/DrugSafetyInformationforHeathcareProfessionals/ucm174949.htm
Ward SR, Minamoto VB, Suzuki KP, Hulst JB, Bremner SN, Lieber RL. 2018. Recovery of rat muscle size but not function more than 1 year
after a single botulinum toxin injection. Muscle Nerve. 57(3):435-441. [AIM: to measure the functional and structural properties of botulinum
toxin-injected adult rat skeletal muscle over [50% to 75% of the entire lifespan of ~24 months]. Ten groups of animals were subjected to
either neurotoxin injection [Botox, Type A (BT-A) or saline solution injection. Neurotoxin-injected animals (n = 90) were analyzed at different
time-points: 1 week; 1 month; 3 months; 6 months; 12 months; or 18 months. RESULTS: In spite of the recovery of structural features, such
as muscle mass and fiber area, dorsiflexion torque production remained significantly depressed by 25%, even at 12 months after neurotoxin
injection. DISCUSSION: The data demonstrate that, after a single BTX-A injection, although gross muscle morphology recovered over a 12-
month time period, loss of contractile function did not recover.]
Wasiak J, Hoare B, Wallen M. 2004. Botulinum toxin A as an adjunct to treatment in the management of the upper limb in children with
spastic cerebral palsy. Cochrane Database Syst Rev. 4:CD003469. [Selected all RCTs comparing intramuscular BTX-A injections into any
muscle group of the upper limb with placebo, no treatment or other interventions. MAIN RESULTS: Two trials met the inclusion criteria, each
having short-term follow up, a small number of subjects and using a single set of injections. When data were analysed no treatment effect
was found for quality of upper limb function, passive range of motion, muscle tone, grip strength or self-care ability. CONCLUSIONS: This
systematic review has not found sufficient evidence to support or refute the use of intramuscular injections of BTX-A as an adjunct to
managing the upper limb in children with spastic CP.]
Weigl DM, Arbel N, Katz K, Becker T, Bar-On E. 2007. Botulinum toxin for the treatment of spasticity in children: attainment of treatment
goals. J Pediatr Orthop B. 16(4):293-6. [40 patients (mean age 5 years) with a spastic disorder treated by BTX injections (53 sessions) were
evaluated for functional outcome in terms of achievement of their individual predetermined goals of treatment, rated on a modified Goal
Attainment Scale from 1 (worsening function) to 4 (improved gross motor function). At the 2-week follow-up, 40% had a score of 1, 33% a
score of 2, and 13% a score of 3; 12% showed no functional change. Botulinum treatments failed to significantly improve the gross motor
function in this patient group. Treatment goals and expectations should clearly be established beforehand.]
Welham NV, Marriott G, Tateya I, Bless DM.2008. Proteomic changes in rat thyroarytenoid muscle induced by botulinum neurotoxin injection.
Proteomics. 8(9):1933-44.
Williams SA, Reid S, Elliott C, Shipman P, Valentine J. 2013. Muscle volume alterations in spastic muscles immediately following botulinum
toxin type‐A treatment in children with cerebral palsy. Devel Med Child Neurol. 55(9):813-20. [AIM: With evidence for an atrophic effect of
botulinum toxin type A (BoNT‐A) documented in typically developing muscles, this study investigated the immediate morphological alterations
of muscles in children with cerebral palsy (CP) after BoNT‐A treatment. Method: Fifteen children (10 males, five females; age range 5–11y,
mean age 8y 5mo, SD 1y 10mo) with spastic diplegic CP [Gross Motor Function Classification System Levels I (n=9) and II (n=6)] receiving

Compiled and annotated by Beverly Cusick PT, MS, NDT, COF/BOC – April 10, 2020 10
BoNT‐A injections for spasticity management were included. None of the children was a first‐time receiver of BoNT‐A. Magnetic resonance
imaging and Mimics software assessed muscle volume, timed 2 weeks before and 5 weeks after injection. All participants received BoNT‐A
bilaterally to the gastrocnemius muscle, and five participants also received BoNT‐A bilaterally to the medial hamstring muscles. Functional
assessment measures used were the 6‐Minute Walk Test (6‐MWT), the Timed Up and Go (TUG) test, and hand‐held dynamometry. Results:
Whilst total muscle group volume of the injected muscle group remained unchanged, a 4.47% decrease in the injected gastrocnemius muscle
volume (p=0.01) and a 3.96% increase in soleus muscle volume (p=0.02) was evident following BoNT‐A. There were no statistically
significant changes in function after BoNT‐A as assessed by the TUG. There was also no statistically significant change in distance covered in
the 6‐MWT. Muscle strength, as assessed using hand‐held dynamometry was also not statistically different after BoNT‐A treatment.
Interpretation: Muscle volume decreases were observed in the injected muscle (gastrocnemius), with synergistic muscle hypertrophy that
appeared to compensate for this decrement. The 4% to 5% decrease in the volume of BoNT‐A injected muscles are not dramatic in
comparison to reports in recent animal studies, and are a positive indication for BoNT‐A, particularly as it also did not negatively alter
function.] Unlike animal studies of effects on healthy muscle, these were all unhealthy muscle tissue (CP) – had already lost volume due to
transformation - and were already weakened by earlier BTX injections.
Willoughby K, Ang SG, Thomason P, Graham HK. 2012. The impact of botulinum toxin A and abduction bracing on long-term hip development
in children with cerebral palsy. Dev Med Child Neurol. 54(8):743-7. [RESULTS: Forty-six children with bilateral spastic CP (31 males, 15
females; 10 with diplegia, 36 with quadriplegia; mean age at enrolment of 3 y 2 mo, mean age at most recent clinical review 13 y 11 mo
[range 10 y 6 mo-16 y 8 mo]; three children in Gross Motor Function Classification System level II, 11 in level III, 20 in level IV, 12 in level V)
were followed for a mean of 10 years 10 months from recruitment to the trial. Mean migration percentage was 15.9% in the BoNT-A group
and 15.2% in the comparison group (t = 0.26, p = 0.79). Eighty-nine percent of hips in the treatment group and 91% hips in the comparison
group had satisfactory development, using a valid scale (Mann-Whitney U test = 867.50, z = -1.59, p = 0.11). Forty children had preventive
surgery (21 treatment group, 19 comparison group) and 18 children had reconstructive surgery (10 treatment, 8 comparison).
INTERPRETATION: In children with bilateral spastic CP, early treatment with BoNT-A and hip abduction bracing does not reduce the need for
surgery or improve hip development at skeletal maturity.]

Botox and Muscle Pathophysiology/Strength Loss - Studies using healthy animal muscles.
Ateş F, Yucesoy CA. 2014. Effects of botulinum toxin type A on non-injected bi-articular muscle include a narrower length range of force
exertion and increased passive force. Muscle Nerve. 49(6):866-78. [AIM: to test the hypothesis that BTX-A injection in rat tibialis anterior (TA)
muscle affects the mechanics of its bi-articular synergist, both actively and passively. METHODS: Two groups of Wistar rats were tested:
control (no BTX-A) and BTX (0.1 U of BTX-A) animals were injected exclusively to the mid-belly of TA. Extensor digitorum longus (EDL)
muscle isometric forces were measured after proximal and distal lengthening. RESULTS: Five days after injection, BTX-A administration
changed EDL mechanics: (1) active forces decreased (proximal muscle length dependently); (2) length range of active force exertion
decreased both proximally and distally; and (3) passive muscle forces increased. CONCLUSIONS: Effects of BTX-A appear to not be limited to
decreased active muscle tone but may cause also a narrower active range of movement and increased passive resistance.]
Caron G, Rouzi T, Grelot L, et al. 2014. Mechano- and metabosensitive alterations after injection of botulinum toxin into gastrocnemius
muscle. J Neurosci Res. 92(7):904-14. [AIM: to investigate effects of motor denervation by Clostridium botulinum toxin serotype A (BoNT/A)
on the afferent activity of fibers originating from the gastrocnemius muscle of rats. Animals were randomized in two groups, 1) untreated
animals acting as control and 2) treated animals in which the toxin was injected in the left muscle. Locomotor activity was evaluated once per
day during 12 days with a test based on footprint measurements of walking rats (sciatic functional index). At the end of the functional
assessment period, electrophysiological tests were used to measure muscle properties, metabosensitive afferent fiber responses to chemical
(KCl and lactic acid) injections, electrically induced fatigue (EIF), and mechanosensitive responses to tendon vibrations. Additionally,
ventilatory response was recorded during repetitive muscle contractions. Then, rats were sacrificed, and the BoNT/A-injected muscles were
weighed. Twelve days postinjection we observed a complete motor denervation associated with a significant muscle atrophy and loss of force
to direct muscle stimulation. In the BoNT/A group, the metabosensitive responses to KCl injections were unaltered. However, we observed
alterations in responses to EIF and to 1 mM of lactic acid (which induces the greatest activation). The ventilatory adjustments during
repetitive muscle activation were abolished, and the mechanosensitive fiber responses to tendon vibrations were reduced. These results
indicate that BoNT/A alters the sensorimotor loop and may induce insufficient motor and physiological adjustments in patients in whom a
motor denervation with BoNT/A was performed.]
Dodd SL, Selsby J, Payne A, et al. 2005. Botulinum neurotoxin type A causes shifts in myosin heavy chain composition in muscle. Toxicon 46:
196–203. [Botulinum neurotoxin type A has gained widespread use for treatment of a host of neuromuscular conditions. However, the
potential effect of this toxin has on the histological and biochemical properties of skeletal muscle remains largely unexplored. AIM: to
characterize the myosin heavy chain (MHC) distribution of adult rat skeletal muscle treated with botulinum neurotoxin type A. Varying doses
of the toxin were injected into the triceps surae muscle group of one hind limb. Force production was assessed periodically to access the
functional deficit incurred. After 10 weeks, animals were sacrificed, muscles removed, and MHC composition determined. Body weight, muscle
weight and force of the injected leg were significantly reduced in all groups, while loss of muscle weight and force in the contralateral leg was
variable. In the injected plantaris and gastrocnemius muscles, type I MHC increased approximately 100%, while type IIa/x decreased
approximately 50%. In the contralateral gastrocnemius, types I and IIa/x MHC increased approximately 100%, while type IIb decreased
approximately 45%. These data suggest that botulinum neurotoxin causes shifts in MHC composition in injected and contralateral muscles
that are contrary to those seen with denervation and similar to those seen with aging.]
Ellman R, Grasso DJ, van Vliet M, et al. 2014. Combined effects of botulinum toxin injection and hind limb unloading on bone and muscle.
Calcif Tissue Int. 94(3):327-37. [Bone receives mechanical stimulation from two primary sources, muscle contractions and external
gravitational loading; but the relative contribution of each source to skeletal health is not fully understood. Understanding the most effective
loading for maintaining bone health has important clinical implications for prescribing physical activity for the treatment or prevention of
osteoporosis. Therefore, we investigated the relative effects of muscle paralysis and reduced gravitational loading on changes in muscle mass,
bone mineral density, and microarchitecture. Adult female C57Bl/6J mice (n = 10/group) underwent one of the following: unilateral BTX
injection of the hind limb, hind limb unloading (HLU), both unilateral BTX injection and HLU, or no intervention. BTX and HLU each led to
significant muscle and bone loss. The effect of BTX was diminished when combined with HLU, though generally the leg that received the

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combined intervention (HLU+BTX) had the most detrimental changes in bone and muscle. We found an indirect effect of BTX affecting the
uninjected (contralateral) leg that led to significant decreases in bone mineral density and deficits in muscle mass and bone architecture
relative to the untreated controls; the magnitude of this indirect BTX effect was comparable to the direct effect of BTX treatment and HLU.
Thus, while it was difficult to definitively conclude whether muscle force or external gravitational loading contributes more to bone
maintenance, it appears that BTX-induced muscle paralysis is more detrimental to muscle and bone than HLU.]
Fortuna R, Vaz MA, Sawatsky A, Hart DA, Herzog W. 2015. A clinically relevant BTX-A injection protocol leads to persistent weakness,
contractile material loss, and an altered mRNA expression phenotype in rabbit quadriceps muscles. J Biomech. 48(10):1700-6. [Botulinum
toxin type-A (BTX-A) injections have become a common treatment modality for patients suffering from muscle spasticity. Despite its benefits,
BTX-A treatments have been associated with adverse effects on target muscles. Currently, application of BTX-A is largely based on clinical
experience, and research quantifying muscle structure following BTX-A treatment has not been performed systematically. The purpose of this
study was to evaluate strength, muscle mass, and contractile material six months following a single or repeated (2 and 3) BTX-A injections
into the quadriceps femoris of New Zealand white rabbits. 23 skeletally mature rabbits were divided into four groups: experimental group
rabbits received 1, 2, or 3 injections at intervals of 3 months (1-BTX-A, 2-BTX-A, 3-BTX-A, respectively) while control group rabbits received
volume-matched saline injections. Knee extensor strength, quadriceps muscle mass, and quadriceps contractile material of the experimental
group rabbits were expressed as a percentage change relative to the control group rabbits. One-way ANOVA was used to determine group
differences in outcome measures (α=0.05). Muscle strength and contractile material were significantly reduced in experimental compared to
control group rabbits but did not differ between experimental groups. Muscle mass was the same in experimental BTX-A and control group
rabbits. We concluded from these results that muscle strength and contractile material do not fully recover within six months of BTX-A
treatment. [Healthy rabbits, not stiff, transformed gastrocs as in CP.]
Fortuna R, Horisberger M, Vaz MA, Herzog W. 2013. Do skeletal muscle properties recover following repeat botulinum toxin A injections? J
Biomech. 46(14): 2426-2433. [Although considered safe, previous studies have shown that BTX-A injections cause muscle atrophy and
deterioration in target and non-target muscles. Ideally, muscles should fully recover following BTX-A treatments, so that muscle strength and
performance are not affected in the long-term. However, systematic, long-term data on the recovery of muscles exposed to BTX-A treatments
are not available, thus practice guidelines on the frequency and duration of BTX-A injections, and associated recovery protocols, are based on
clinical experience with little evidence-based information. Therefore, the purpose of this study was to investigate muscle recovery following a
six months, monthly BTX-A injection (3.5 U/kg) protocol. Twenty seven skeletally mature NZW rabbits were divided into 5 groups: Control
(n=5), zero month recovery - BTX-A+0M (n=5), one month recovery - BTX-A+1M (n=5), three months recovery - BTX-A+3M (n=5), and six
months recovery - BTX-A+6M (n=7). Knee extensor strength, muscle mass and percent contractile material in injected and contralateral non-
injected muscles was measured at each point of recovery. Strength and muscle mass were partially and completely recovered in injected and
contralateral non-injected muscles for BTX-A+6M group animals, respectively. The percent of contractile material partially recovered in the
injected, but did not recover in the contralateral non-injected muscles. We conclude from these results that neither target nor non-target
muscles fully recover within six months of a BTX-A treatment protocol and that clinical studies on muscle recovery should be pursued.]
[Healthy rabbits, not stiff gastrocs as in CP.]
Fortuna R, Vaz MA, Rehan Youssef A, Longino D, Herzog W. 2011. Changes in contractile properties of muscles receiving repeat injections of
botulinum toxin. J Biomech. 44: 39–44. [ BTX-A is a frequently used therapeutic tool to denervate muscles in the 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 use of BTX-A for neuromuscular disorders, the effects of repeat injections of BTX-A on strength, muscle mass and
structure need to be known. Therefore, the purpose of this study was to investigate the changes in strength, muscle mass and contractile
material in New Zealand White (NZW) rabbits. Twenty NZW rabbits were divided into 4 groups: control and 1, 3 and 6 months of unilateral,
repeat injections of BTX-A into the quadriceps femoris. Outcome measures included knee extensor torque, 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.] [Healthy rabbits, not stiff gastrocs as in CP.]
Frick CG, Fink H, Blobner M, Martyn J. 2012. A single injection of botulinum toxin decreases the margin of safety of neurotransmission at local
and distant sites. Anesth Analg. 114(1):102-9. [We tested the hypothesis that a single injection of botulinum toxin into the tibialis muscle of
anesthetized rats (n = 26) not only has local, but also distant effects on muscle function, biochemistry, and pharmacodynamics of atracurium
(the same drug). The contralateral side with no injection served to study distant effects. Control animals (n = 25) received a saline injection.
Neuromuscular function, pharmacology, and expression of acetylcholine receptors (nAChRs) were evaluated in the tibialis at 0, 4, and 16 days
after injection and in comparison with saline-injected controls. RESULTS: On day 4, botulinum toxin caused complete paralysis of the tibialis,
while its contralateral side showed a decrease in absolute twitch tension. On day 16, muscle weakness was only present on the toxin-injected
side where absolute twitch tension was decreased (0.6 N [0.6, 0.7] vs 3.4 N [3.1, 3.7], P < 0.05). Tibialis mass was decreased on the toxin-
injected side at day 4 (1.46 mg/g [1.43, 1.48] vs 1.74 mg/g [1.72; 1.75], P < 0.05) and on day 16 (0.78 mg/g [0.76, 0.79] vs 1.73 mg/g
[1.69; 1.77], P < 0.05). Effects distant from the site of injection were seen on day 16, when muscle atrophy was also present in the adjacent
gastrocnemius and soleus muscles. Normalized to tibialis mass, specific twitch tension (tension/g muscle) was reduced on the contralateral
side at day 4 and on the toxin-injected side at day 16 in relation to saline controls…CONCLUSIONS: Botulinum toxin has local and distant
effects on muscle. The decrease in specific twitch tension indicates that the muscle atrophy alone cannot explain the functional changes;
neuromuscular transmission is also impaired]
Han N, Kim HD, Eom MJ, et al. 2013. Proteomic changes in rat gastrocnemius muscle after botulinum toxin a injection. Ann Rehabil Med.
37(2):157-66. [AIM: to observe the changes in protein expression induced by botulinum toxin A (BoNT-A) injection and to characterize the
molecular and cellular action of mechanisms of BoNT-A injection on skeletal muscles using proteomic elements as biomarkers. METHODS:
BoNT-A was injected into left gastrocnemius muscles of 12 Sprague-Dawley rats (2 months of age) at a dosage of 5 units/kg body weight. For
the controls the same volume of normal saline was injected to right gastrocnemius muscle of each rat. Muscle samples were obtained at 4
time points (3 rats per time point): 3, 7, 14, and 56 days post-injection. To reveal the alterations in muscle protein, we performed 2-
dimensional electrophoresis (2DE) and compared Botox group and normal saline group at each time point. Altered protein spots in 2DE were

Compiled and annotated by Beverly Cusick PT, MS, NDT, COF/BOC – April 10, 2020 12
identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer (MALDI-TOF MS) proteomics analysis. RESULTS:
Compared with normal saline group, 46 protein spots showed changed protein expression. Twelve protein spots demonstrated increased
volume and 34 protein spots demonstrated decreased volume. Among spots of decreased volume, 17 spots showed statistically significant
differences. Thirty-eight identified proteins were associated with alterations in energy metabolism, muscle contractile function, transcription,
translation, cell proliferation, and cellular stress response. CONCLUSION: BoNT-A gives influences on muscle contractile function and energy
metabolism directly or indirectly besides neurotoxic effects. Proteomic expression provides better understanding about the effect of BoNT-A
on skeletal muscle.]
Hong B, Chen M, Hu XY. 2013. Influence of injection of Chinese botulinum toxin type A on the histomorphology and myosin heavy chain
composition of rat gastrocnemius muscles. J Zhejiang Univ Sci B. 14(11):983-92. [In China, Chinese botulinum toxin type A (CBTX-A), a type
of BoNT/A, is in widespread clinical use. However, the changes in the morphological and biochemical properties of treated muscles and in
remote muscles from the CBTX-A injection site are relatively unknown. Therefore, we investigated the changes in histomorphology and
myosin heavy chain (MyHC) isoform composition and distribution in rat gastrocnemius muscles after intramuscular injection of CBTX-A.
METHODS: The weakness of the injected muscles was assessed periodically to identify their functional deficiency. RESULTS: Our findings
demonstrate that following injection of CBTX-A 5 U into rat gastrocnemius muscles, shifts in MyHC isoform composition emerged on the third
day after injection and peaked in the fourth week. The composition remained distinctly different from that of the control group after the
twelfth week. More specifically, there was a decrease in the proportion of the type IIb isoform and an increase in the proportions of type IIx,
type IIa, and type I isoforms. CONCLUSIONS: Data revealed that CBTX-A led to a shift in MyHC composition towards slower isoforms and that
the MyHC composition remained far from normal six months after a single injection. However, no noticeable remote muscle weakness was
induced.]
Legerlotz K, Matthews KG, McMahon CD, Smith HK. 2009. Botulinum toxin‐induced paralysis leads to slower myosin heavy chain isoform
composition and reduced titin content in juvenile rat gastrocnemius muscle. Muscle Nerve. 39(4):472-479.
Lieber RL, Ward SR. 2013. Cellular mechanisms of tissue fibrosis. 4. Structural and functional consequences of skeletal muscle fibrosis.
American J Physiol-Cell Physiology. 305(3):C241-52. Authors present images of muscle biopsies taken from the anterior tibialis muscle of a rat
before-and- six months after two botox-A injections were administered at 3-months intervals. Fibrosis accumulation is considerable.
Extracellular material is increased from ~5% of the cross section at baseline to ∼20% at follow-up.
Manske SL, Good CA, Zernicke RF, Boyd SK. 2012. High-frequency, low-magnitude vibration does not prevent bone loss resulting from muscle
disuse in mice following botulinum toxin injection. PLoS One. 7(5):e36486.
Manske SL, Boyd SK, Zernicke RF. 2011. Vertical ground reaction forces diminish in mice after botulinum toxin injection. J Biomech. 44(4):
637-43.
Manske SL, Boyd SK, Zernicke RF. 2010. Muscle changes can account for bone loss after botulinum toxin injection. Calcif Tissue Int. 7(6):541-
9.
Marchand-Libouban H, Le Drévo MA, Chappard D. 2013. Disuse induced by botulinum toxin affects the bone marrow expression profile of
bone genes leading to a rapid bone loss. J Musculoskelet Neuronal Interact. 13(1):27-36. [Molecular events occurring in the bone marrow
microenvironment of an immobilized mouse limb after Botulinum toxin (BTX) injection haven't been characterized. BTX injection induces a
localized disuse in which the tissue events have well been characterized. METHODS: BTX injection was performed in the right quadriceps;
saline injection in the left side was used as control. Mice were sacrificed at 0, 7, 14, 21 and 28 days; tibias were used for microCT analysis;
bone marrow from femurs for RT-PCR analysis. RESULTS: MicroCT revealed bone loss and microarchitectural damages on the immobilized
side as from 7d; cortical area tended to be lower on the immobilized limb at 28d. Gene expression of formation factors was altered as from 7
days post-BTX: alkaline phosphatase, Tgfβ1, Lrp5, Sfrp2. Only Sfrp2 and Lrp5 were maintained altered until 28d. Expression of Dkk1
increased from 21d and represented a late inhibitor of formation. Gene expression of resorption markers increased as from 7d (Rankl, Tracp,
Il1α, Il1β and Il6) and was maintained until 28d for Tracp and Il6. CONCLUSION: A localized disuse induces rapid modifications in the bone
marrow gene expression leading to bone loss due to an early decrease of formation associated with an increase in resorption.]
Matak I, Riederer P, Lacković Z. 2012. Botulinum toxin's axonal transport from periphery to the spinal cord. Neurochem Int.; 61(2): 236-9.
[Axonal transport of enzymatically active BTX-A from periphery to the CNS has been described in facial and trigeminal nerve, leading to
cleavage of synaptosomal-associated protein 25 (SNAP-25) in central nuclei. Aim of present study was to examine the existence of axonal
transport of peripherally applied BTX-A to spinal cord via sciatic nerve. We employed BTX-A-cleaved SNAP-25 immunohistochemistry of
lumbar spinal cord after intramuscular and subcutaneous hind limb injections, and intraneural BTX-A sciatic nerve injections. Truncated SNAP-
25 in ipsilateral spinal cord ventral horns and dorsal horns appeared after single peripheral BTX-A administrations, even at low intramuscular
dose applied (5 U/kg). Cleaved SNAP-25 appearance in the spinal cord after BTX-A injection into the sciatic nerve was prevented by proximal
intrasciatic injection of colchicine (5 mM, 2 μl). Cleaved SNAP-25 in ventral horn, using choline-acetyltransferase (ChAT) double labeling, was
localized within cholinergic neurons. These results extend the recent findings on BTX-A retrograde axonal transport in facial and trigeminal
nerve. Appearance of truncated SNAP-25 in spinal cord following low-dose peripheral BTX-A suggest that the axonal transport of BTX-A occurs
commonly following peripheral application.]
Mathevon L, Michel F, Decavel P, et al. 2015. Muscle structure and stiffness assessment after botulinum toxin type A injection. A systematic
review. Ann Phys Rehabil Med. 58(6):343–350. [Results: From the 228 initially identified articles, 21 articles were included. Histological
analyses were performed, especially on animals. A neurogenic atrophy systematically occurred. In humans, one year after a single injection,
the histological recovery remained incomplete. Furthermore, 2D ultrasound analyses showed a reduction of the gastrocnemius thickness and
pennation angle. MRI volumetric analysis evidenced muscular atrophy six months or one year after a single injection. Passive muscle stiffness
depends on these structural changes. On the short term, the biomechanical analysis showed an elastic modulus increase in animals whereas
no change was recorded in humans. On the short term, ultrasound elastography imaging showed a decreased elastic modulus. Discussion: To
date, few data are available, but all show a structural and mechanical muscle impact post injections, specifically muscle atrophy which can
linger over time. Further studies are necessary to validate this element, and the possibility of change must be taken into account particularly
with repeated injections.]
Minamoto VB, Suzuki KP, Bremner SN, Lieber RL, Ward SR. 2015. Dramatic changes in muscle contractile and structural properties after 2
botulinum toxin injections. Muscle & nerve. 52(4):649-57.

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Moon YM, Kim YJ, Kim MK, et al. 2015. Early effect of Botox-A injection into the masseter muscle of rats: functional and histological
evaluation. Maxillofac Plast Reconstr Surg. 37(1):46. Reduced muscle volume persisted after recovery of use. Healthy animal muscle, not stiff,
transformed CP muscle.
Park C, Park K, Kim J. 2015. Growth effects of botulinum toxin type A injected unilaterally into the masseter muscle of developing rats. J
Zhejiang Univ Sci B. 16(1):46-51. [AIM: To evaluate the effects of BTX-A on mandible skeletal development by inducing muscle hypofunction.
METHODS: Four-week-old Sprague-Dawley rats (n=60) were divided into three groups: Group 1 animals served as controls and were injected
with saline; Group 2 animals were injected unilaterally with BTX-A (the contralateral side was injected with saline); and Group 3 animals were
injected bilaterally with BTX-A. In Group 2, the saline-injected side was designated the control side (Group 2-1), whereas the BTX-A-injected
side was designated the experimental side (Group 2-2). After four weeks, the animals were sacrificed, dry skulls were prepared, and
mandibles were measured. RESULTS: In the unilateral group, the experimental side (Group 2-2) had reduced dimensions for all mandible
measurements compared with Group 2-1, suggesting a local effect of BTX-A on mandible growth, likely due to muscle reduction.]
Pingel J, Wienecke J, Lorentzen J, Nielsen JB. 2016. Botulinum toxin injection causes hyper-reflexia and increased muscle stiffness of the
triceps surae muscle in the rat. J Neurophysiol. 116(6):2615-2623. [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.]
Poliachik SL, Bain SD, Threet D, Huber P, Gross TS. 2010. Transient muscle paralysis disrupts bone homeostasis by rapid degradation of bone
morphology. Bone. 46(1):18–23. [We have previously shown that transient paralysis of murine hindlimb muscles causes profound degradation
of both trabecular and cortical bone in the adjacent skeleton within 3 weeks. Morphologically, the acute loss of bone tissue appeared to arise
primarily due to osteoclastic bone resorption. Given that the loss of muscle function in this model is transient, we speculated that the stimulus
for osteoclastic activation would be rapid and morphologic evidence of bone resorption would appear before 21 d. We therefore utilized high-
resolution in vivo serial micro-CT to assess longitudinal alterations in lower hindlimb muscle volume, proximal tibia trabecular and tibia
middiaphysis cortical bone morphology in 16 wk old female C57 mice following transient calf paralysis from a single injection of botulinum
toxin A (BtA; 2U/100g body weight). In an acute study, we evaluated muscle and bone alterations at d 0, 3, 5 and 12 following transient calf
paralysis. In a chronic study, following d 0 imaging, we assessed the recovery of these tissues following the maximum observed trabecular
degradation (d 12) through d 84 post-paralysis. The time course and degree of recovery of muscle, trabecular and cortical bone varied
substantially. Significant atrophy of lower limb muscle was evident by d 5 of paralysis, maximal at d 28 (−34.1 ± 0.9%) and partially
recovered by d 84. Trabecular degradation within the proximal tibia metaphysis occurred more rapidly, with significant reduction in BV/TV by
d 3, maximal loss at d 12 (−76.8 ± 2.9%) with only limited recovery by d 84 (−51.7 ± 5.1% vs. d 0). Significant cortical bone volume
degradation at the tibia mid-diaphysis was first identified at d 12, was maximal at d 28 (−9.6 ± 1.2%), but completely recovered by d 84. The
timing, magnitude and morphology of the observed bone erosion induced by transient muscle paralysis was consistent with a rapid
recruitment and prolific activation of osteoclastic resorption.]
Ramirez‐Castaneda J, Jankovic J, Comella C, et al. 2013. Diffusion, spread, and migration of botulinum toxin. Movement Disorders.
28(13):1775-83. [The efficacy and safety of BoNT depends on accurate selection and identification of intended targets but also may be
determined by other factors, including physical spread of the molecule from the injection site, passive diffusion, and migration to distal sites
via axonal or hematogenous transport. The passive kinetic dispersion of the toxin away from the injection site in a gradient-dependent
manner may also play a role in toxin spread. In addition to unique properties of the various BoNT products, volume and dilution may also
influence local and systemic distribution of BoNT. Most of the local and remote complications of BoNT injections are thought to be due to
unwanted spread or diffusion of the toxin's biologic activity into adjacent and distal muscles. Despite widespread therapeutic and cosmetic use
of BoNT over more than three decades, there is a remarkable paucity of published data on the mechanisms of distribution and its effects on
clinical outcomes. The primary aim of this article is to critically review the available experimental and clinical literature and place it in the
practical context.]
Rauch F, Hamdy R. 2006. Effect of a single botulinum toxin injection on bone development in growing rabbits. J Musculoskelet Neuronal
Interact. 6(3):264-8. [Intramuscular injections with botulinum toxin A (BTX-A) lead to a rapid decrease in muscle mass and force, but the
effect of this drug on bone development is unclear. In the present pilot study we evaluated the effect of a one-time injection of BTX-A in
growing rabbits. Twelve young (weight 1.5 kg) New Zealand rabbits were randomly assigned to receive either BTX-A (total dose 8 units per
kg body weight) or sodium chloride 0.9% injections into the left quadriceps and gastrocnemius muscles. Both groups continued to gain weight
in a similar manner following the injection. However, when the animals were sacrificed at five weeks after the injection, the group receiving
BTX-A had a significant deficit (of 10%) in gastrocnemius muscle mass on the injected side, whereas no significant side-difference was found
for the quadriceps. BTX-A injections did not affect the length of the tibia. Nevertheless, bone mineral content of the whole tibia, as measured
by dual-energy X-ray absorptiometry, was 7% lower in the BTX-A injected side than on the contralateral side. Peripheral quantitative
computed tomography showed that this bone mass deficit was larger in the metaphysis than in the epiphysis or diaphysis. In the diaphysis,
the bone mass deficit was due to a reduction in cross-sectional bone dimensions, which equally affected the cross-section of the entire bone,
the cortical compartment and the marrow space. BTX-A injections did not have a detectable effect on cortical bone mineral density. The bone
mass deficit in the diaphysis thus appeared to be caused by a lack of periosteal bone apposition rather than increased endocortical or
intracortical resorption. These preliminary data suggest that intramuscular BTX-A injections can have a deleterious effect on the development
of bones that are loaded by the injected muscles.]

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Tsai FC, Hsieh MS, Chou CM. 2010. Comparison between neurectomy and botulinum toxin A injection for denervated skeletal muscle. J
Neurotrauma. 27(8):1509-16. [Neurectomy and botulinum toxin A (BoNT-A) injection cause denervated muscle atrophy, but questions remain
about their clinical utility. We investigated time-series alterations of rat muscle weight, functional deficits, signaling pathways, and microscopic
structures, to gain an understanding of the clinical implications. Between 2008 and 2009, the maximal calf circumference of patients for calf
reduction either by neurectomy or BoNT-A injections were recorded for study. A rat skeletal muscle model was established through repeated
or dose-adjusted BoNT-A injections and neurectomy. The survival, apoptosis (natural cell death) pathways, functional deficits, and
microscopic structures were investigated using Western blot, sciatic functional index (SFI), and transmission electron microscopy (TEM),
respectively. The rat muscle weight ratio of the BoNT-A group had recovered to 89.3 +/- 3.8% by week 58, but it never recovered in the
neurectomy group. Muscle weight reduction by BoNT-A not only depended on the dose, but additive effects were also obtained through
repeated injections. Rat SFI demonstrated rapid recovery in both groups. Molecular expressions showed a coherent and biphasic pattern. p-
Akt and apoptosis-inducing factor (AIF) were upregulated significantly, with a peak at 8 weeks in the neurectomy group (p < 0.01), but
cleaved caspase-9 and caspase-3 showed no significant changes in either group. TEM findings showed irreversible and reversible inner-
structure disruption and sarcomere discontinuity in the neurectomy and BoNT-A groups, respectively. We demonstrated that denervation
induced lasting muscle weight and structural changes of different degrees. Muscle weight reduction by BoNT-A was related to frequency and
dose. AIF-mediated caspase-independent apoptosis was significantly different for neurectomy and BoNT-A injection.]
Welham NV, Marriott G, Tateya I, Bless DM. 2008. Proteomic changes in rat thyroarytenoid muscle induced by botulinum neurotoxin injection.
Proteomics. 8(9):1933-44. Free PMC Article [In this study, 12 adult Sprague Dawley rats underwent unilateral TA muscle BoNT serotype A
injection followed by tissue harvest at 72 h, 7 days, 14 days, and 56 days postinjection. Three additional rats were reserved as controls.
Proteomic analysis was performed using 2-D SDS-PAGE followed by MALDI-MS. Vocal fold movement was significantly reduced by 72 h, with
complete return of function by 56 days. Twenty-five protein spots demonstrated significant protein abundance changes following BoNT
injection, and were associated with alterations in energy metabolism, muscle contractile function, cellular stress response, transcription,
translation, and cell proliferation. A number of protein abundance changes persisted beyond the return of gross physiologic TA function.]
Worton LE, Gardiner EM, Kwon RY, et al. 2018. Botulinum toxin A-induced muscle paralysis stimulates Hdac4 and differential miRNA
expression. PLoS One.13(11):e0207354. [At sufficient dose, intramuscular injection of Botulinum toxin A causes muscle wasting that is
physiologically consistent with surgical denervation and other types of neuromuscular dysfunction. The aim of this study was to clarify early
molecular and micro-RNA alterations in skeletal muscle following Botulinum toxin A-induced muscle paralysis. Quadriceps were analyzed for
changes in expression of micro- and messenger RNA and protein levels after a single injection of 0.4, 2 or 4U Botulinum toxin A (/100g body
weight). After injection with 2.0U Botulinum toxin A, quadriceps exhibited significant reduction in muscle weight and increased levels of
ubiquitin ligase proteins at 7, 14 and 28 days. Muscle miR-1 and miR-133a/b levels were decreased at these time points, whereas a dose-
responsive increase in miR-206 expression at day 14 was observed. Expression of the miR-133a/b target genes RhoA, Tgfb1 and Ctfg, and the
miR-1/206 target genes Igf-1 and Hdac4, were upregulated at 28 days after Botulinum toxin A injection. Increased levels of Hdac4 protein
were observed after injection, consistent with anticipated expression changes in direct and indirect Hdac4 target genes, such as Myog. Our
results suggest Botulinum toxin A-induced denervation of muscle shares molecular characteristics with surgical denervation and other types of
neuromuscular dysfunction, and implicates miR-133/Tgf-β1/Ctfg and miR-1/Hdac4/Myog signaling during the resultant muscle atrophy.]
Warner SE, Sanford DA, Becker BA, et al. 2006. Botox induced muscle paralysis rapidly degrades bone. Bone. 38(2):257-64. [We have
developed a novel murine model of unilateral transient hindlimb muscle paralysis using botulinum toxin A (Botox). Female C57BL/6 mice (16
weeks) received IM injections of either saline or Botox (n = 10 each) in both the quadriceps and calf muscles of the right hindleg. Gait
dysfunction was assessed by multi-observer inventory, muscle alterations were determined by wet mass, and bone alterations were assessed
by micro-CT imaging at the distal femur, proximal tibia, and tibia mid-diaphysis. Profound degradation of both muscle and bone was observed
within 21 days despite significant restoration of weight bearing function by 14 days. The muscle mass of the injected quadriceps and calf
muscles was diminished −47.3% and −59.7%, respectively, vs. saline mice (both P < 0.001). The ratio of bone volume to tissue volume
(BV/TV) within the distal femoral epiphysis and proximal tibial metaphysis of Botox injected limbs was reduced −43.2% and −54.3%,
respectively, while tibia cortical bone volume was reduced −14.6% (all P < 0.001). Comparison of the contralateral non-injected limbs
indicated the presence of moderate systemic effects in the model that were most probably associated with diminished activity following
muscle paralysis. Taken as a whole, the micro-CT data implied that trabecular and cortical bone loss was primarily achieved by bone
resorption. These data confirm the decisive role of neuromuscular function in mediating bone homeostasis and establish a model with unique
potential to explore the mechanisms underlying this relation. Given the rapidly expanding use of neuromuscular inhibitors for indications such
as pain reduction, these data also raise the critical need to monitor bone loss in these patients.]
Vegger JB, Bruel A, Brent MB, Thomsen JS. 2018. Disuse osteopenia induced by botulinum toxin is similar in skeletally mature young and
aged female C57BL/6J mice. J Bone Miner Metab. 36(2):170-179.

On Industry-Sponsored Research
Seehusen DA, Koren KG. 2013. Impact of industry sponsorship on research outcomes. Am Fam Physician. 88(11):746. [Drug and medical
device studies sponsored by the manufacturers tend to have more favorable effectiveness and harm findings and more favorable conclusions
than studies with other funding sources.]
Sung Sung KH, Chung CY, Lee KM, et al. 2013. Conflict of interest in the assessment of botulinum toxin A injections in patients with cerebral
palsy: a systematic review. J Pediatr Orthop. 33(5):494-500. [Fifteen (53.6%) of the 28 industry-sponsored studies had favorable conclusions,
whereas only 5 (20%) of the 25 non-industry-sponsored studies had favorable conclusions. The efficacy of using botulinum toxin A injections
in CP is controversial. The financial conflict of interest related to medical research can affect the conclusion of an evidence-based review. This
study was performed to determine as to what proportion of studies on botulinum toxin A injections in patients with CP was sponsored by the
industry and whether the assessments of botulinum toxin injection in CP were associated with industry support. METHODS: Studies were
identified with a search of the PubMed database (January 1991 to November 2011). All prospective, comparative, English language studies on
the use of botulinum toxin A injections in patients with CP were included. A total of 374 articles were screened, 128 potentially eligible full
articles were retrieved, and 66 studies met our inclusion criteria. The funding sources of the articles were reviewed, and qualitative
conclusions regarding the effect of botulinum toxin A injection were classified as being either favorable, neutral, or unfavorable.

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RESULTS: Of 66 eligible articles, 28 were funded by the industry, and 25 were not. The other 13 studies did not include information on the
funding source. A significant association was observed between the funding source and qualitative conclusions (P=0.042). Fifteen (53.6%) of
the 28 industry-sponsored studies had favorable conclusions, whereas only 5 (20%) of the 25 non-industry-sponsored studies had favorable
conclusions. CONCLUSIONS: About half of studies on the effect of botulinum toxin A in CP were sponsored by the industry. This systematic
review revealed that the qualitative conclusions in those studies are more favorable to the use of the botulinum toxin A than the non-industry-
sponsored studies. Clinicians should be aware of an industry-related conflict of interest regarding reports on the efficacy of botulinum toxin A
injections in patients with CP.]

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