toxins

Systematic Review
High Precision Use of Botulinum Toxin Type A (BONT-A) in
Aesthetics Based on Muscle Atrophy, Is Muscular Architecture
Reprogramming a Possibility? A Systematic Review of
Literature on Muscle Atrophy after BoNT-A Injections
Alexander D. Nassif 1, * , Ricardo F. Boggio 2 , Sheila Espicalsky 3 and Gladstone E. L. Faria 2

1 Departamento de Pesquisa, Núcleo Nassif—Ensino Médico e Pesquisa, Belo Horizonte 30411-148, Brazil
2 Departamento de Pesquisa, Instituto Boggio—Medicina Ensino e Pesquisa, Sao Paulo 04004-030, Brazil;
[email protected] (R.F.B.); [email protected] (G.E.L.F.)
3 Departamento de Pesquisa, Clínica Sheila Espicalsky, Vila Velha 29101-104, Brazil;
[email protected]
* Correspondence: [email protected]

Abstract: Improvements in Botulinum toxin type-A (BoNT-A) aesthetic treatments have been jeop-
ardized by the simplistic statement: “BoNT-A treats wrinkles”. BoNT-A monotherapy relating to
wrinkles is, at least, questionable. The BoNT-A mechanism of action is presynaptic cholinergic
nerve terminals blockage, causing paralysis and subsequent muscle atrophy. Understanding the real
BoNT-A mechanism of action clarifies misconceptions that impact the way scientific productions on
the subject are designed, the way aesthetics treatments are proposed, and how limited the results are



when the focus is only on wrinkle softening. We designed a systematic review on BoNT-A and muscle


atrophy that could enlighten new approaches for aesthetics purposes. A systematic review, targeting
Citation: Nassif, A.D.; Boggio, R.F.;
articles investigating BoNT-A injection and its correlation to muscle atrophy in animals or humans,
Espicalsky, S.; Faria, G.E.L. High
filtered 30 publications released before 15 May 2020 in accordance with the Preferred Reporting
Precision Use of Botulinum Toxin
Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Histologic analysis and
Type A (BONT-A) in Aesthetics Based
histochemistry showed muscle atrophy with fibrosis, necrosis, and an increase in the number of
on Muscle Atrophy, Is Muscular
Architecture Reprogramming a
perimysial fat cells in animal and human models; this was also confirmed by imaging studies. A
Possibility? A Systematic Review of significant muscle balance reduction of 18% to 60% after single or seriated BoNT-A injections were
Literature on Muscle Atrophy after observed in 9 out of 10 animal studies. Genetic alterations related to muscle atrophy were analyzed by
BoNT-A Injections. Toxins 2022, 14, 81. five studies and showed how much impact a single BoNT-A injection can cause on a molecular basis.
https://doi.org/10.3390/ Seriated or single BoNT-A muscle injections can cause real muscle atrophy on a short or long-term
toxins14020081 basis, in animal models and in humans. Theoretically, muscular architecture reprogramming is a
Received: 3 December 2021 possible new approach in aesthetics.
Accepted: 18 January 2022
Published: 21 January 2022 Keywords: botulinum toxins; type A; botox; muscular atrophy; muscle atrophy; wrinkles; ficial lines;
aesthelics; esthetics; muscular architecture reprogramming
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
Key Contribution: A systematic review of literature on muscle atrophy after BoNT-A injections.
published maps and institutional affil-
iations.

1. Introduction
Copyright: © 2022 by the authors. Botulinum toxin type A (BoNT-A) has been historically used for the aesthetic treatment
Licensee MDPI, Basel, Switzerland. of facial lines. Although there are an increasing number of on-label uses to treat a variety
This article is an open access article of disorders using BoNT-A, when it comes to aesthetics, all the on-label approvals refer to
distributed under the terms and facial lines [1]. Currently BoNT-A is approved by the FDA for the aesthetic treatment of
conditions of the Creative Commons forehead, glabellar, and lateral canthal lines, while in some other countries, such as Brazil,
Attribution (CC BY) license (https://
the on-label aesthetic approval is more generic and permits BoNT-A injections all over the
creativecommons.org/licenses/by/
face to treat facial lines [2,3]. The main point is that all the aesthetic on-label approvals
4.0/).

Toxins 2022, 14, 81. https://doi.org/10.3390/toxins14020081 https://www.mdpi.com/journal/toxins

Toxins 2022, 14, 81 2 of 24

concern facial lines only. Numerous published clinical trials objectify the improvement
of facial lines after treatment with BoNT-A [4]. A multitude of articles aimed to compare
the main brands of BoNT-A available on the market regarding the durability of the effect
of softening wrinkles provided by these toxins [5]. Dose comparisons between BoNT-A
brands generate misleading results because they are all different and are not interchangeable
substances [6–8].
Despite differences in market brands, all currently marketed BoNT-A have one thing
in common: a protein complex of 150 kDa composed of a heavy chain (HC, 100 kDa)
linked via a disulfide bond to a light chain (LC, 50 kDa) [9–11]. After a BoNT-A injection,
the simplified mechanism of action cascade can be described based on its biochemical
structure [12–17] (Figure 1).

Figure 1. BoNT-A injection, the simplified mechanism of action cascade.

The whole cascade takes between 24 to 72 h to be completed after BoNT-A injection,

and it is an irreversible process [18]. Once the SNAP-25 (synaptosomal-associated protein of
25 kDa) protein is inactivated, muscle contraction will only be reestablished after neuronal
repair that depends on nerve sprouting and/or motor plate regeneration [19]. Although
scientific evidence on this statement dates back to the 1970s [20], many still argue today
about BoNT-A “durability” in relation to wrinkle control rather than studying the level of
tissue damage caused by a BoNT-A injection and the time required for neuronal healing,
as concerns aesthetics. The previous sentence is fundamental for the purpose of the new
aesthetic approach of BoNT-A use in aesthetics that we intend to propose based on the real
BoNT-A mechanism of action.
Many studies have demonstrated nerve terminal and nodal sprouting in the para-
lyzed nerves as early as two days after botulinum toxin injection [21,22]. Broadening the
scope, studies on botulism have already provided a substrate to support the idea that the
botulinum toxins durability for practical purposes is approximately 24 to 72 h and that
the actual long-term effect of muscle paralysis depends only on nerve and muscle tissue
regeneration processes. Treatment with antitoxin for patients with botulism, in order to
be effective, should be started within 24 to 48 h of contamination, otherwise the already
established neuronal chemical tissue injury is no longer reversible [23]. Once the disease
is established by neuronal inability to release acetylcholine in the synaptic cleft of the
neuromuscular junction, life support becomes essential, which is normally restricted to
clinical care, with special attention to maintaining respiratory capacity, which requires
mechanical ventilation for 2 to 6 months, until neuronal and muscular healing processes
take place, restoring diaphragmatic and intercostal muscle function [24,25].
Studies addressing counter-terrorism measures suggest the use of antidotes against
BoNT-A in the event of a mass attack using BoNT-A as a chemical weapon. Only 1 g
Toxins 2022, 14, 81 3 of 24

of BoNT-A in natura is capable of decimating 1 million humans, showing that it is a

powerful and lethal toxin. All of the antidotes tested, even those capable of neuronal
internalization, require concern regarding the therapeutic window, which must precede a
chemical neuromuscular junction denervation of 24 to 72 h [24,26].
Understanding BoNT-A’s real mechanism of action makes it possible to identify some
semantic misconceptions that have been repeated historically since its first use for aesthetic
purposes and that directly impact the way scientific productions on the subject are designed,
the way aesthetics treatments are proposed, and how limited the results are when the focus
is only on wrinkles softening. Considering the statements above and the questions raised
below (Table 1), we designed a systematic review on BoNT-A and muscle atrophy that
could enlighten new approaches for aesthetics purposes.

Table 1. Questions that should be answered, based on the evidence, after reading this paper.

Questions Answers
Does the muscular impairment for contraction caused by BoNT-A really
?
treats facial lines or causes muscle atrophy?
What is the relation of BoNT-A muscle injections and muscle atrophy in the
?
long term?
Is it possible to modulate the level of muscle atrophy through time by using
?
BoNT-A?
What if we used muscle atrophy caused by BoNT-A injections to optimize
?
muscle architecture for facial aesthetic purposes?
What would it be like to reinterpret articles written in the last 30 years
focused mainly on facial lines unveiling this concept of muscle atrophy?
?
How many less subjective opportunities would arise? How classic BoNT-A
injections techniques would be impacted?

2. Aims
To conduct a systematic review of the literature regarding BoNT-A treatments and
muscle atrophy that could support new perspectives in facial aesthetics and to propose
a new reading for the aesthetic use of BoNT-A, no longer focusing on simple control of
wrinkles and facial lines, but as a drug capable of selectively reprogramming long-term
muscle strength and tonus through muscle atrophy. We will discuss the proposition that
muscle architecture could be altered by creating areas of real atrophy—hyporesponsive
or even irresponsive to acetylcholine stimuli for muscle contraction. The restoration of
neuronal and muscular function would be based exclusively on the healing processes of
these tissues.

3. Method
The present systematic review, targeting articles that investigate BoNT-A injections
and its correlation to muscle atrophy in animals or humans, was conducted in a stepwise
process for studies published before 15 May 2020 and in accordance with the Preferred
Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [27]. The
search strategy, the flow diagram of study selection, and the data extraction are detailed
below, because the review was not registered. By the time our independent research group
tried to register the review at PROSPERO in 2020, we had already started article extraction.
After October 2019, PROSPERO only accepted earlier registration.
STEP 1—PubMed/MEDLINE and BVS (Biblioteca Virtual em Saúde) databases were
explored using the following Medical Subject Headings (MeSH) entry terms: “Botulinum
Toxin Type A” OR “Botulinum A Toxin” OR “Botulinum Neurotoxin A” OR “Botox” AND
combined with the MeSH entry terms “Muscle Atrophy” OR “Muscular Atrophy” (Table 2).
The overlapping studies were excluded in STEP 1.
Toxins 2022, 14, 81 4 of 24

Table 2. PubMed/MEDLINE and BVS (Biblioteca Virtual em Saúde) databases Search strategies.

Four Search Strategies Used, Initially:

Search 1—PubMed/MEDLINE—((((BOTULINUM TOXIN TYPE A) OR (BOTULINUM
A TOXIN)) OR (BOTULINUM NEUROTOXIN A)) OR (BOTOX)) AND (MUSCLE
ATROPHY).
Search 2—PubMed/MEDLINE—((((BOTULINUM TOXIN TYPE A) OR (BOTULINUM
A TOXIN)) OR (BOTULINUM NEUROTOXIN A)) OR (BOTOX)) AND (MUSCULAR
ATROPHY).
Search 3—BVS—tw:((tw:(botulinum toxin type a)) OR (tw:(botulinum a toxin)) OR
(tw:(botulinum neurotoxin a)) OR (tw:(botox)) AND (tw:(muscle atrophy))).
Search 4—BVS—tw:((tw:(botulinum toxin type a)) OR (tw:(botulinum a toxin)) OR
(tw:(botulinum neurotoxin a)) OR (tw:(botox)) AND (tw:(muscular atrophy))).
To encompass all possible missing studies that could not be retrieved from Searches 1–4,
the preferred MeSH term entries “Botulinum Toxin Type A” and “Muscular Atrophy”
were matched with all their alternative MeSH term entries listed below:
Botulinum toxin type A Muscular atrophy
Clostridium Botulinum Toxin Type A Atrophies, Muscular
Botulinum Toxin Type A Atrophy, Muscular
Botulinum A Toxin Muscular Atrophies
Toxin, Botulinum A Atrophy, Muscle
Clostridium botulinum A Toxin Atrophies, Muscle
Botulinum Neurotoxin A Muscle Atrophies
Neurotoxin A, Botulinum Muscle Atrophy
Meditoxin Neurogenic Muscular Atrophy
Botox Atrophies, Neurogenic Muscular
Neuronox Atrophy, Neurogenic Muscular
Oculinum Muscular Atrophies, Neurogenic
Vistabex Muscular Atrophy, Neurogenic
OnabotulinumtoxinA Neurogenic Muscular Atrophies
Onabotulinumtoxin A Neurotrophic Muscular Atrophy
Vistabel Atrophies, Neurotrophic Muscular
Atrophy, Neurotrophic Muscular
Muscular Atrophies, Neurotrophic
Muscular Atrophy, Neurotrophic
Neurotrophic Muscular Atrophies
All the 15 alternative MeSH term entries for “Botulinum Toxin Type A” and all the 19 alternative MeSH term
entries for “Muscle Atrophy” listed above were individually added to Search 1, Search 2, Search 3, and Search 4,
one at a time, to check if any other study would be retrieved. No other search limits were added.

In STEP 2, the studies obtained in STEP 1 were screened by “title” and “abstract” by
two independent researchers (A.D.N. and R.F.B.). Those not satisfying inclusion criteria or
with exclusion criteria (Table 3) were excluded. The group of articles selected to proceed
to the next step was determined through an interactive consensus process. Discrepancies
were judged by a third reviewer (S.E.).
Toxins 2022, 14, 81 5 of 24

Table 3. Inclusion and exclusion criteria.

A study was considered eligible for data extraction if it fulfilled the criteria bellow:
- Human or animal striated skeletal muscle atrophy analysis after botulinum toxin
type A injection(s), and
- Atrophy analyzed by imaging (ultrasonography (USG), nuclear magnetic
resonance (NMR), computerized tomography (CT)), and/or by histological
analysis and/or by biochemical analysis; and
- Minimal follow-up of 3 months, and
- The full manuscript was published in English.

In STEP 3, the full text of all the potential articles selected in STEP 2 were obtained
and carefully read to screen for those whose purposes were in accordance with the aim of
the present review.
In STEP 4, the eligible studies in STEP 3 were thoroughly read, and data for each study
were extracted and analyzed according to a PICO-like structured reading (Table 4).

Table 4. PICO-like structured reading of the eligible studies and data collection.

PICO-like structured reading of the eligible studies and data collection

Population/Problem (P)
Intervention (I)
Comparison group (C)
Outcomes (O)
The following question was adopted to conduct data collection:
“Are botulinum toxin type A injections (I) related to muscle atrophy (O) of animal or
humans (P), when compared to not injected subjects or muscles (C)?”
Detailed data were collected in two different groups (animal and human) to fulfill
comparative tables, including: presence of a control group, population number,
population age, health condition, muscle systems analyzed, BoNT-A number of
injections and dose, muscle atrophy confirmation or not, muscle atrophy identification
tool and correlated changes, follow-up, and muscle atrophy recovery.

The methodological quality of the articles included in the study was evaluated using a
specific scale developed based on STROBE (Strengthening the Reporting of Observational
studies in Epidemiology) principles [28]. Each item was categorized, and the maximum
global score was set to 26 (Table 5).
Toxins 2022, 14, 81 6 of 24

Table 5. Quality analysis form used in the systematic review.

Quality Analysis form Used in the Systematic Review.

Q1 Is there in the abstract an explanation of what was done and found?
Q2 Is the scientific context clearly explained?
Q3 Are the objectives clearly stated?
Q4 Is the sampling size indicated?
Q5 If yes, is the sampling size statistically justified?
Q6 Are the characteristics of the subjects (height, weight, sex, healthy, or pathologic
subject) described?
Q7 What is the design of the study? (0: retrospective study; 1: case study; 2: prospective
study).
Q8 Is there a control group? (0: no, 1: contralateral member or nonrandomized control
group, 2: randomized control group).
Q9 How long is the follow up? (0: ≥3 and <6 months; 1: ≥6 months and <1 year; 2: ≥1
year)
Q10 Is the reliability of the evaluation method clearly described?
Q11 Are the results interpretable?Q12 Are the limitations of the study discussed?Q13 Is
the conclusion clearly stated?
0: no description; 1: limited description; 2: good description.

4. Reults
4.1. Selection of the Studies
From 191 articles initially identified after removing duplicates, thirty-five were deemed
relevant after reading titles and abstracts. Thirty were included in the review (5 were
excluded because they did not meet the selection criteria). Sixteen were animal studies and
fourteen were human studies. The PRISMA Flow Diagram of Article Selection for Review
is summarized in (Figure 2).

Figure 2. PRISMA—Flow Diagram of Article Selection for Review.

Toxins 2022, 14, 81 7 of 24

4.2. Quality of the Reviewed Articles

The quality of the reviewed articles was highly variable and is summed up in
Table 6 [29–58]. Most studies, 28/30, were prospective ones, with 13 well-controlled and
randomized, but this subgroup was only of animal studies. The descriptive quality of
the experimental protocol results, as well as their interpretations and conclusions, were
adequate in most studies. The follow-up ranged from 3 months to 4 years.

Table 6. Quality assessment. ** maximum global score = 26.

Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Q10 Q11 Q12 Q13 TOTAL **

Borodic (1992) [29] 1 2 2 2 0 1 2 1 2 1 1 0 2 17
Hamjian (1994) [30] 1 1 1 2 0 2 2 1 0 2 1 0 1 14
Ansved (1997) [31] 2 2 2 2 0 2 1 1 2 1 1 0 1 17
Fanucci (2001) [32] 2 2 2 2 0 2 2 1 0 2 2 0 2 19
To (2001) [33] 2 2 2 2 0 2 2 1 2 1 2 0 2 20
Kim (2005) [34] 2 2 1 2 0 2 2 0 2 1 2 0 2 18
Shen (2006) [35] 2 2 2 2 0 2 2 2 1 2 2 0 2 21
Singer (2006) [36] 2 2 2 2 0 2 2 0 1 1 2 2 2 20
Herzog (2007) [37] 2 2 2 2 0 2 2 2 1 2 2 2 2 23
Frick (2007) [38] 2 2 1 2 0 2 2 2 0 1 2 1 2 19
Kwon (2007) [39] 2 2 2 2 0 2 2 2 1 2 2 1 2 22
Lee (2007) [40] 2 2 2 2 0 2 2 0 2 1 2 0 2 19
Schroeder (2009) [41] 2 2 2 2 0 1 2 1 2 1 2 0 2 19
Babuccu (2009) [42] 2 2 2 2 0 2 2 2 0 2 2 0 2 20
Tsai (2010) [43] 2 2 1 2 0 1 2 1 2 1 1 1 2 18
Fortuna (2011) [44] 2 2 2 2 0 2 2 2 1 1 1 1 2 20
Fortuna (2013a) [45] 2 2 2 2 0 1 2 2 1 1 2 1 2 20
Van Campenhout (2013) [46] 2 2 2 2 0 1 2 0 1 2 2 2 2 20
Koerte (2013) [47] 2 2 2 2 0 2 2 1 2 1 2 0 1 19
Fortuna (2013b) [48] 2 2 2 2 0 2 2 2 2 1 2 2 1 22
Mukund (2014) [49] 1 2 2 2 0 2 2 1 2 2 2 1 2 21
Fortuna (2015) [50] 2 2 2 2 0 2 2 2 2 2 2 0 2 22
Caron (2015) [51] 2 2 2 2 0 2 2 2 2 1 2 0 1 20
Valentine (2016) [52] 2 2 2 2 0 2 2 1 2 1 2 1 1 20
Li (2016) [53] 1 1 2 2 0 2 1 0 2 0 1 0 1 13
Kocaelli (2016) [54] 2 2 2 2 0 2 2 2 0 2 2 1 2 21
Hart (2017) [55] 2 2 2 2 0 2 2 2 2 2 1 2 1 22
Han (2018) [56] 2 2 2 2 0 2 2 0 1 1 2 1 1 18
Alexander (2018) [57] 2 2 2 2 0 2 2 1 1 2 2 2 2 22
Lima (2018) [58] 2 2 2 2 0 2 2 2 0 1 2 0 2 19
Toxins 2022, 14, 81 8 of 24

4.3. Literature Analysis

A general overview of the population type of the 30 studies is summarized in Table 7.
All Animal studies had good quality control groups. Human studies, on the other hand,
lacked control groups or had poor quality control groups.
Most animal studies used mature healthy animals. Human studies, on the other hand,
used very heterogeneous subjects in relation to age (varying from children to 91-year-old
adults) and health status.
Overall, there were very few studies regarding the facial mimetic musculature in
humans—only two: Borodic (1992) [29] and Koerte (2013) [47]. The facial masticatory
musculature represented mainly by the masseter muscle were studied in three human
studies: To (2001) [33], Kim (2005) [34], Lee (2007) [40]; and three animal studies: Kwon
(2007) [39], Babuccu (2009) [42], Kocaelli (2016) [54].
Numerical heterogenic population samples (from 1 to 383 subjects) and qualitative
heterogenic samples, more specifically in human studies (healthy and subjects with different
muscle disorders), were observed.
There was also heterogenic BoNT-A dose, BoNT-A brand types used in the studies
and follow-up period, summarized in Table 8.
The methodological variability among the small number of studies made it mandatory
to conduct an extensive evaluation based on the identification of muscle atrophy after
BoNT-A injections registered separately via different tools in animal or human studies.
The general findings are summarized in Section 4.3.1. (Animal Studies) and Section 4.3.2.
(Human Studies), below.

4.3.1. Animal Studies

Muscle Balance
Muscle balance was measured in 10 out of 16 animal studies to evaluate muscle
atrophy. Significant muscle balance reduction after seriated BoNT-A injections and after
one single BoNT-A injection were observed in 9 out of 10 studies. The reduction varied
from 18%, Fortuna (2013b) [48], to 60%, Fortuna (2011) [44], and there was a BoNT-A
dose dependency/interval of injection association identified by Herzog (2007) [37], Frick
(2007) [38], Tsai (2010) [43], Fortuna (2011) [44], Fortuna (2013b) [48], and Caron (2015) [51].
The higher the dose, the higher the muscle balance reduction. Long intervals between
injections permitted partial muscle balance recovery. Only Fortuna (2015) [50] found no
muscle balance alterations after 6 months of injection (Table 9).

Optical and Electron Microscopy

Hystologic (optical and electron microscopy) analysis and histochemistry showed pro-
found muscle structure changes in animal models, such as sarcomere distortion, decrease
in myofibrillar diameters, and myofibrillolysis/myonecrosis—Babuccu (2009) [42], Tsai
(2010) [43], Kocaelli (2016) [54]. Significant reduction of percentage of contractile material—
Frick (2007) [38], Fortuna (2011) [44], Fortuna (2013a) [45], Fortuna (2013b) [48], Fortuna
(2015) [50]. Replacement of contractile fibers with fat, fatty infiltration, and increased
collagen fibers forming perimysium—Herzog (2007) [37], Fortuna (2011) [44], Kocaelli
(2016) [54] (Table 10).

Imaging
Kwon (2007) [39] showed a computed tomography (CT) scan rabbit masseter muscle
volume reduction of up to 18.41% (±3.15) after 6 months of a BoNT-A injection. Magnetic
resonance imaging (MRI) was used in monkeys by Han (2018) [56] and showed significant
paraspinal muscles atrophy after BoNT-A injections (Table 11).

Molecular Biology
Direct and indirect muscle atrophy identification via molecular biology was studied
and is detailed in Tables 12 and 13.
Toxins 2022, 14, 81 9 of 24

Table 7. Systematic review—Summary table of the results (PART 1).

Author (Year) Human/Animal Control Group Age Population (Number) Health Condition

Borodic (1992) [29] Human Yes 56–91 years 14 Blepharospasm/Meige’s disease

Hamjian (1994) [30] Human Contralateral muscle 25–49 years 10 Healthy

Ansved (1997) [31] Human Yes 32–54 years 22 Cervical dystonia

Piriformis muscle syndrome

Fanucci (2001) [32] Human Contraleteral Muscle 29–54 years 30
(PMS)

To (2001) [33] Human Yes 16–32 years 15 Masseteric muscle hypertrophy

Kim (2005) [34] Human No Teenagers—40s 383 Masseteric muscle hypertrophy

Shen (2006) [35] Animal (Sprague-Dawley rats) Yes 1 month 56 Healthy
Chronic anterior knee pain and
Singer (2006) [36] Human No 16–40 years 8 related
disability
Animal (New Zealand white
Herzog (2007) [37] Yes 1 year 25 Healthy
rabbits)
Frick (2007) [38] Animal (Sprague-Dawley rats) Contralateral muscle Mature 39 Healthy
Kwon (2007) [39] Animal (New Zealand rabbits) Yes 4 weeks 21 Healthy
Lee (2007) [40] Human No 20–29 years 10 Healthy (square face)

Schroeder (2009) [41] Human Contralateral muscle 31–47 years 2 Healthy

Babuccu (2009) [42] Animal (Wistar rats) Yes 15-day-old 49 Healthy
Tsai (2010) [43] CD® (SD) IGS rats Contralateral muscle Mature 60 Healthy
Animal
Fortuna (2011) [44] Yes 1 year 20 Healthy
(New Zealand White rabbits)
Animal
Fortuna (2013a) [45] Yes Mature 17 Healthy
(New Zealand White rabbits)
Toxins 2022, 14, 81 10 of 24

Table 7. Cont.

Author (Year) Human/Animal Control Group Age Population (Number) Health Condition
Cerebral palsy (symmetric
Van Campenhout (2013) [46] Human No Children 7
spastic diplegia)

Koerte (2013) [47] Human Yes 34–50 years 4 Healthy

Animal
Fortuna (2013b) [48] Yes 1 year 27 Healthy
(New Zealand White rabbits)
Animal (Harlan Sprague-Dawley
Mukund (2014) [49] Contralateral muscle 3 months 20 Healthy
rats)
Animal
Fortuna (2015) [50] Yes 1 year 23 Healthy
(New Zealand White rabbits)
Caron (2015) [51] Animal (Sprague-Dawley rats) Yes Mature 27 Healthy
Different muscle same
Valentine (2016) [52] Human 6–16 years 10 Cerebral palsy
participant

Li (2016) [53] Human No 40–59 years 3 Strabismus

Kocaelli (2016) [54] Animal (Sprague-Dawley rats) Yes 5–6 months 30 Healthy
Animal
Hart (2017) [55] Yes 1 year 22 Healthy
(New Zealand White rabbits)
Animal (Cynomolgus
Han (2018) [56] No 9 years 1 Healthy
monkey—Macaca fascicularis)
Baseline status same
Alexander (2018) [57] Human 5–13 years 11 Cerebral palsy
participant
Lima (2018) [58] Animal (Wistar rats) Yes 10-week-old 50 Healthy

Systematic review—Summary table of the results (PART 1). Human studies Animal studies .
Toxins 2022, 14, 81 11 of 24

Table 8. Systematic review—Summary table of the results (PART 2).

Author (Year) BoNT-A Number of Injections and Dose Follow-Up

Borodic (1992) [29] 2–19 injections over 1–5.5 years. Dose? 1–52 weeks

Hamjian (1994) [30] 1 injection. Dose 10 units of BoNT-A (Oculinum® ) # 0–100 days

Ansved (1997) [31] Number? 2–4 years of treatment. Mean cumulative dose 2.815 units of BoNT-A 2–4 years

Fanucci (2001) [32] 1 or 2 injections. Dose 200 units of BoNT-A (Botox® ) ## 0–3 months

To (2001) [33] 1 or 2 injections. Dose 100–300 units of BoNT-A (Dysport® ) ### per side 0–1 year

Kim (2005) [34] 1 or 2 injections. Dose 100–140 units of BoNT-A (Dysport® ) ### per side 0–2 years
Shen (2006) [35] 1 injection. Dose 6 units/kg body weight of BoNT-A (Botox® ) ## 0–360 days
Singer (2006) [36] 1 injection. Dose 300–500 units of BoNT-A (Dysport® ) ### 0–24 weeks
Herzog (2007) [37] 1–6 injetions over 6 months. Dose 3,5 units/kg body weight of BoNT-A (Botox® ) #### per injetion 1–6 months
Frick (2007) [38] 1 injection. Dose 0.625 units or 2.5 units or 10 units/kg body weight of BoNT-A (Botox® ) ## 128 days
Kwon (2007) [39] 1 injection. Dose 5–15 units of BoNT-A 4–24 weeks
Lee (2007) [40] 1 injection. Dose 25 units of BoNT-A (Botox® ) ## 0–12 months

Schroeder (2009) [41] 1 injection. Dose 75 units of BoNT-A (Xeomin® ) ##### 3–12 months
Babuccu (2009) [42] 1 injection. Dose 0.4 units BoNT-A (Botox® ) ###### per muscle 4 months
1 or 2 injetions. Dose 2.5 ng of BoNT-A (Botox® ) ##
per side (single injection group) or (two injections group full
Tsai (2010) [43] 1–58 weeks
dose—30 weeks apart) or 1.25 ng (two injections group half dose—30 weeks apart)
Fortuna (2011) [44] 1 or 3 or 6 monthly injections. Dose 3.5 units/Kg of BoNT-A (Botox® ) #### per muscle group, per side, per month 1–6 months
Fortuna (2013a) [45] 6 monthly injections. Dose 3.5 units/Kg of BoNT-A (Botox® ) #### per muscle group, per side, per month 6 months
Van Campenhout (2013) [46] 1 injection. Dose 2 units/Kg/psoas muscle of BoNT-A (Botox® ) ## 0–6 months

Koerte (2013) [47] 1 injection. Dose 20 units of BoNT-A (Botox® ) ## 0–12 months
Fortuna (2013b) [48] 6 monthly injections. Dose 3.5 units/Kg of BoNT-A (Botox® ) #### per muscle group, per side, per month 6–12 months
Toxins 2022, 14, 81 12 of 24

Table 8. Cont.

Author (Year) BoNT-A Number of Injections and Dose Follow-Up

Mukund (2014) [49] 1 injection. Dose 6 units/Kg of BoNT-A (Botox® ) ## per side 1–52 weeks
1, 2, or 3 injections (every 3 months). Dose 3.5 units/Kg of BoNT-A (Botox® ) #### per muscle group, per side, per
Fortuna (2015) [50] 6–12 months
injection
Caron (2015) [51] 1 injection. Dose 15 units/Kg of BoNT-A (Dysport® ) ####### per side 12–400 days
Valentine (2016) [52] 1–15 injections. Dose 2–6 units/Kg of BoNT-A (Botox® ) ## per side 3.5 months–3 years

Li (2016) [53] 1–2 injections. Dose 3.75–7.5 units of BoNT-A (Botox® ) ## per side 6–18 months
Kocaelli (2016) [54] 1 injection. Dose 0.5 units of BoNT-A (Botox® ) ## per muscle, per side 12 weeks
1, 2, or 3 injections (every 3 months). Dose 3.5 units/Kg of BoNT-A (Botox® ) #### per muscle group, unilateral, per
Hart (2017) [55] 6–12 months
injection
Han (2018) [56] 10 (one injection every two weeks). Dose 2 units/Kg of BoNT-A (Nabota® ) ######## 0–21 weeks
Alexander (2018) [57] 1 injection. Dose 1.4–4.8 units/Kg of BoNT-A (Botox® ) ## per side 0–25 weeks
Lima (2018) [58] 1 injection. Dose 5 units of BoNT-A (Dysport® ) ### per side 12 weeks

Human studies Animal studies # (Oculinum® )—Allergan Corp., Irvine, CA. ## (Botox® )—Allergan Corp., Irvine, CA. ### (Dysport® ) Ipsen Ltd., Slough, United
Kingdom. #### (Botox ) Allergan Inc., Toronto, Ont., Canada. ##### (Xeomin® ) Merz Pharma, Germany. ###### (Botox® ) Allergan Pharmaceuticals, Ireland. ####### (Dysport® ) Ipsen
®

Ltd., Boulogne-Billancourt, France. ######## (Nabota® ) Daewoong Pharmaceutical Hwaseong, Korea.

Table 9. Animal studies—Muscle balance.

Herzog (2007) [37] Fortuna (2011) [44] Fortuna (2013a) [45] Fortuna (2013b) [48] Fortuna (2015) [50]
Frick (2007) [38] Babuccu (2009) [42] Tsai (2010) [43] Caron (2015) [51] Lima (2018) [58]
Muscle Atrophy Quadriceps Femoris Quadriceps Femoris Quadriceps Femoris Quadriceps Femoris Quadriceps Femoris
Tibialis Masseter and Temporalis Gastrocnemius Gastrocnemius Gastrocnemius
Identification Tool 25 New Zealand White 20 New Zealand White 20 New Zealand White 27 New Zealand White 23 New Zealand White
39 Sprague-Dawley Rats 49 Wistar Rats 60 CD® (SD) IGS Rats 27 Sprague-Dawley Rats 50 Wistar Rats
Rabbits Rabbits Rabbits Rabbits Rabbits

Wet muscle mass and muscle

Wet muscle mass Wet muscle mass Wet muscle mass Wet muscle mass Wet muscle mass Wet muscle mass Wet muscle mass Wet muscle mass Wet muscle mass
weight/body weight ratio

No alteration at 6 months.
Balance Reduction of 10.7% (±3.8) Reduction of 52% Significantly lower weight
(immediately at 58 weeks after a single Significant atrophy (p < 0.001) at 6 months of (p < 0.001) at 12 days post
post-sacrifice muscle Significant (p < 0.05) decrease BoNT-A injection, 29.7% (p < 0.0001). repeated monthly BoNT-A injection.
Significant atrophy
harvest or muscle Mean percent loss of of 20% in (group 0.625 units), Significantly diminished (±8.2) after repeated Mean quadriceps femoris BoNT-A injections and a Significantly lower weight
(p < 0.001). No alteration at 6 months
harvest under general muscle mass of 36% at 33.4% in (group 2.5 units) p = 0.0001 (masseter) and injections half dose and a muscle mass reduction of sustained reduction of (p < 0.001) at 128.43 ± 7.43 Significant reduction
Mean quadriceps femoris after the last BoNT-A
anesthesia) 1 month and 49% at and 50% in (group 10 units) p = 0.001 (temporalis). reduction of 41.7% (±6.1) 45% (1 month group), 60% 18% (p < 0.001) at days post BoNT-A injection. of 37% (p < 0.001).
muscle mass reduction of injection (p > 0.05).
6 months. at day 128. No recovery at No recovery. at 58 weeks after repeated (3 months group), and 6 months after the last Significantly partial weight
52%. No recovery.
day 128. injections of full dose. 56% (6 months group). BoNT-A injection. Partial recovery (p < 0.001) at 371.83
Partial recovery at No recovery. after 6 months of the last ± 24.82 days post BoNT-A
58 weeks. BoNT-A injection. injection.
No recovery.
Toxins 2022, 14, 81 13 of 24

Table 10. Animal studies—Hystologic (optical and electron microscopy) analysis and histochemistry.
Herzog (2007) [37] Fortuna (2011) [44] Fortuna (2013a) [45] Fortuna (2013b) [48] Fortuna (2015) [50]
Frick (2007) [38] Babuccu (2009) [42] Tsai (2010) [43] Kocaelli (2016) [54]
Muscle Atrophy Quadriceps Femoris Quadriceps Femoris Quadriceps Femoris Quadriceps Femoris Quadriceps Femoris
Tibialis Masseter and Temporalis Gastrocnemius Masseter and Gluteal
Identification Tool 25 New Zealand White 20 New Zealand White 20 New Zealand White 27 New Zealand White 23 New Zealand White
39 Sprague-Dawley Rats 49 Wistar Rats 60 CD® (SD) IGS Rats 30 Sprague-Dawley Rats
Rabbits Rabbits Rabbits Rabbits Rabbits

Muscle structure (qualitative) Muscle structure (qualitative) Muscle structure (qualitative) Muscle structure (qualitative) Muscle structure (qualitative) Muscle structure (qualitative) Muscle structure (qualitative) Muscle structure (qualitative) Muscle structure (qualitative)

Increase in the collagen fibers

Fatty infiltration at 3 and
Replacement of contractile forming perimysium
6 months (increased).
fibers with fat. around the striated muscle
No recovery.
cells at 12 weeks.

Muscle structure (percentage Muscle structure (percentage Muscle structure (percentage Muscle structure (percentage Muscle structure (percentage Muscle structure (percentage Muscle structure (percentage Muscle structure (percentage Muscle structure (percentage
of contractile material) of contractile material) of contractile material) of contractile material) of contractile material) of contractile material) of contractile material) of contractile material) of contractile material)

Reduction of 36.1% (±16.9), Reduction of 40.8% (±6.0), at

Significantly reduced (p < 0.001) at 6 months of 6 months after 1 BoNT-A
(p < 0.05) (6 months group) repeated monthly BoNT-A injection, reduction of 37.5%
Significant (p < 0.05) decrease
for 43% (±9.7) vastus Reduction of 36.1% (±16.9), injections and a sustained (±6.1), at 6 months after 2
at day 128. No recovery at
lateralis, for 70% (±8.0) (p < 0.001). No recovery. reduction of 22.2% (±2.0) at BoNT-A injection, reduction
Histologic analysis (optical day 128.
rectus femoris, for 78% (±4.2) 6 months after the last of 40.1% (±11.8), at 6 months
microscopy)/
vastus medialis. No recovery. BoNT-A injection. Partial after 3 BoNT-A injection.
histochemistry
recovery at 6 months. No recovery.

Muscle structure (atrophy Muscle structure (atrophy Muscle structure (atrophy Muscle structure (atrophy Muscle structure (atrophy Muscle structure (atrophy Muscle structure (atrophy Muscle structure (atrophy Muscle structure (atrophy
scoring/quantitative scoring/quantitative scoring/quantitative scoring/quantitative scoring/quantitative scoring/quantitative scoring/quantitative scoring/quantitative scoring/quantitative
analysis) analysis) analysis) analysis) analysis) analysis) analysis) analysis) analysis)

Stratification degree of the

muscle, nucleus
internalization, Significant (p < 0.001)
multinucleation, myofibril decrease of diameters of
diameter, and myonecrosis muscle fibers in bundles and
compatible with muscle fascicles at 12 weeks.
atrophy. No recovery at
4 months.

Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure

Myofibrils atrophic changes

characterized by: decrease in
myofibrillar diameters and
myofibrillolysis, dilatations
Histologic analysis (electron
Sarcomere distorsion (mild in the terminal cisternae and
microscopy)/
distruction at 8 weeks). T-tubules, disorganized Z
histochemistry
Partial recovery at 26 weeks. bands, vacuolar appearance
as a result of dilatation in the
sarcoplasmic reticulum
cisternae and mitochondrial
swelling.
Toxins 2022, 14, 81 14 of 24

Table 11. Animal studies—Imaging.

Han (2018) [56]

Kwon (2007) [39]
Muscle Atrophy Paraspinal
Masseter
Identification Tool 01 Cynomolgus Monkey—Macaca
21 New Zealand Rabbits
Fascicularis
Muscle cross-sectional areas at T12–L1, Muscle cross-sectional areas at T12–L1,
L1–L2, L2–L3, L3–L4 and L4–L5 levels L1–L2, L2–L3, L3–L4, and L4–L5 levels
Magnetic resonance Significant atrophy with decreased
imaging (MRI) cross-sectional areas by 4%, 2%, 8%, 12%,
and 8%, respectively, at 21 weeks (the peak
was at 11 weeks). Partial recovery at
21 weeks.
Muscle volume Muscle volume
Reduction of 19.72% (±4.80) in Group 2 and
Computed tomography of 21.34% (±5.37) in Group 3 at 8 weeks.
(CT) scan Reduction of 13.76% (±5.34) in Group 2 and
of 18.41% (±3.15) in Group 3 at 24 weeks.
Partial recovery at 24 weeks.

Table 12. Animal studies—Direct and indirect muscle atrophy identification via molecular biology.

Molecular Biology Alterations Articles

Upregulation of proapoptotic: anti-apoptotic protein ratio
((Bax:Bcl-2)ratio) significantly had an 83.3 fold increase, peak at
Tsai (2010) [43].
4 weeks.
p < 0.01
Muscle substitution for adipose tissue determined by
adipocyte-related molecules upregulation of adiponectin (APN),
Leptin, adipocyte binding protein 2 (AP2), and adipogenic
lineage marker upregulation of peroxisome
Hart (2017) [55].
proliferator-activated receptor γ (PPARγ). The APN, Leptin,
AP2, and PPARγ were significantly upregulated after BoNT-A
injections.
p < 0.05
Muscle atrophy inferred via molecular biology in regard to
upregulation of Transforming Growth Factor-beta TGF-β;
upregulation of Nuclear Factor-kappaB (NF-κB); upregulation Mukund (2014) [49].
of p53/Cell cycle control; upregulation of Inhibitor of DNA Fortuna (2015) [50].
binding (ID) proteins—Id1, Id2, Id3, Id4, and muscle
RING-finger protein-1 (MuRF1) upregulation.
Muscle atrophy and muscle atrophy recovery response
indirectly identified via NMJ restoration (muscle-specific
receptor tyrosine kinase (MuSK) upregulation, nicotinic
Shen (2006) [35].
acetylcholine receptor (nAChR) upregulation), protection
Mukund (2014) [49].
against muscle cell apoptosis (P21 protein upregulation),
Fortuna (2015) [50].
myogenesis modulation/muscle regeneration (insulin-like
growth factor-1 (IGF-1) upregulation, myogenin upregulation,
and mitogen-activated protein kinase (MAPK) upregulation).
Toxins 2022, 14, 81 15 of 24

Table 13. Animal studies—Molecular biology.

Shen (2006) [35] Tsai (2010) [43] Mukund (2014) [49] Fortuna (2015) [50] Hart (2017) [55]
Muscle Atrophy Identification Tool Gastrocnemius Gastrocnemius Tibialis Anterior Quadriceps Femoris Quadriceps Femoris
56 Sprague-Dawley Rats 60 CD® (SD) IGS Rats 20 Sprague-Dawley Rats 23 New Zealand White Rabbits 22 New Zealand White Rabbits

Indirect atrophy identification via upregulation of gene and molecule expression signaling neuromuscular junction (NMJ) restoration, protection against muscle cell apoptosis, myogenesis modulation/muscle regeneration.

NMJ restoration NMJ restoration NMJ restoration NMJ restoration NMJ restoration

Muscle-specific receptor tyrosine kinase (MuSK)

significant upregulation (p < 0.05) from day 3 to day 60
Nicotinic acetylcholine receptor (nAChR) significant
upregulation (p < 0.05) from day 3 to day 14

Protection against muscle cell apoptosis Protection against muscle cell apoptosis Protection against muscle cell apoptosis Protection against muscle cell apoptosis Protection against muscle cell apoptosis

P21 protein significant (p < 0.05) upregulation from

day 3 to day 30

Myogenesis modulation/muscle regeneration Myogenesis modulation/muscle regeneration Myogenesis modulation/muscle regeneration Myogenesis modulation/muscle regeneration Myogenesis modulation/muscle regeneration

Insulin-like growth factor-1 (IGF-1) significant

upregulation (p < 0.05) from day 3 to day 60
Myogenin significant upregulation (p < 0.05) from day 3
to day 90

Myogenesis modulation/muscle regeneration Myogenesis modulation/muscle regeneration Myogenesis modulation/muscle regeneration Myogenesis modulation/muscle regeneration Myogenesis modulation/muscle regeneration

Insulin-like growth factor-1 (IGF-1) significant

upregulation (p < 0.05) (at 6 months)
Recovery not evaluated

Direct atrophy identification via upregulation of Direct atrophy identification via upregulation of Direct atrophy identification via upregulation of Direct atrophy identification via upregulation of Direct atrophy identification via upregulation of
proapoptotic: proapoptotic: proapoptotic: proapoptotic: proapoptotic:
anti-apoptotic protein ratio (Bax:Bcl-2) anti-apoptotic protein ratio (Bax:Bcl-2) anti-apoptotic protein ratio (Bax:Bcl-2) anti-apoptotic protein ratio (Bax:Bcl-2) anti-apoptotic protein ratio (Bax:Bcl-2)
Molecular biology
(Real-Time Quantitative Polymerase Chain Ratio significantly 83.3 fold increase (p < 0.01) (peak
Reaction (qPCR), and/or Microarray Data at 4 weeks)
Analysis, and/or Western blot analysis) Recovery at 8 weeks

Direct atrophy identification via upregulation of Direct atrophy identification via upregulation of Direct atrophy identification via upregulation of Direct atrophy identification via upregulation of Direct atrophy identification via upregulation of
Transforming Growth Factor-beta TGF-β Transforming Growth Factor-beta TGF-β Transforming Growth Factor-beta TGF-β Transforming Growth Factor-beta TGF-β Transforming Growth Factor-beta TGF-β

TGF-β significantly upregulated (p < 0.05)

(at 6 months)
Recovery not evaluated

Direct atrophy identification via muscle RING-finger Direct atrophy identification via muscle RING-finger Direct atrophy identification via muscle RING-finger Direct atrophy identification via muscle RING-finger Direct atrophy identification via muscle RING-finger
protein-1 (MuRF1) protein-1 (MuRF1) protein-1 (MuRF1) protein-1 (MuRF1) protein-1 (MuRF1)

MuRF1 significantly upregulated (p < 0.05)

(at 6 months)
Recovery not evaluated

Direct atrophy identification via muscle substitution Direct atrophy identification via muscle substitution Direct atrophy identification via muscle substitution Direct atrophy identification via muscle substitution
Direct atrophy identification via muscle substitution for
for adipose tissue. for adipose tissue. for adipose tissue. for adipose tissue.
adipose tissue.
Adipocyte-related molecules upregulation of Adipocyte-related molecules upregulation of Adipocyte-related molecules upregulation of Adipocyte-related molecules upregulation of
Adipocyte-related molecules upregulation of
adiponectin (APN), adiponectin (APN), adiponectin (APN), adiponectin (APN),
adiponectin (APN),
Leptin, adipocyte binding protein 2 (AP2), and Leptin, adipocyte binding protein 2 (AP2), and Leptin, adipocyte binding protein 2 (AP2), and Leptin, adipocyte binding protein 2 (AP2), and
Leptin, adipocyte binding protein 2 (AP2), and
adipogenic lineage marker upregulation of adipogenic lineage marker upregulation of adipogenic lineage marker upregulation of adipogenic lineage marker upregulation of
adipogenic lineage marker upregulation of peroxisome
peroxisome proliferator-activated receptor γ peroxisome proliferator-activated receptor γ peroxisome proliferator-activated receptor γ peroxisome proliferator-activated receptor γ
proliferator-activated receptor γ (PPARγ)
(PPARγ) (PPARγ) (PPARγ) (PPARγ)

APN, Leptin, AP2, and PPARγ significantly

upregulated (p < 0.05) (at 6 months after 3 BoNT-A
injections every 3 months, except for Leptin, which
had partial recovery after 3 BoNT-A injections)
Toxins 2022, 14, 81 16 of 24

4.3.2. Human Studies

Optical and Electron Microscopy
Histologic (optical and electron microscopy) analysis and histochemistry showed
results in humans similar to those found in animal models. Muscle atrophy (atrophic
muscle fibers, myofibrillar disorganization, fibrosis, necrosis, and increase of the number
of perimysial fat cells) were well-documented by Kim (2005) [34], Schroeder (2009) [41],
Valentine (2016) [52], and Li (2016) [53]. The Orbicularis oculi muscle showed that the
morphometric measurements of muscle fibers reduced, with an irregular diameter at
3 months after BoNT-A injections, (p < 0.05). Ansved (1997) [31] showed a mean diameter
reduction of type IIB striated muscle fibers (Vastus lateralis) of 19.6% after 2–4 years of
BoNT-A treatement (p < 0.05). Partial recovery of the changes described above were seen in
some articles (Table 14).

Table 14. Human studies—Histologic (optical and electron microscopy) analysis and histochemistry.

Ansved (1997) [31] Li (2016) [53]

Borodic (1992) [29] Kim (2005) [34] Schroeder (2009) [41] Valentine (2016) [52]
Muscle Atrophy Vastus Lateralis Medial Rectus (Extraocular
Orbicularis Oculi Masseter Gastrocnemius Gastrocnemius
Identification Tool (Non-Target Muscle) Muscle)
14 383 2 10
22 3

Morphometric Morphometric Morphometric Morphometric Morphometric

Morphometric measurements
measurements of muscle measurements of muscle measurements of muscle measurements of muscle measurements of muscle
of muscle fibers
fibers fibers fibers fibers fibers

Reduced and irregular Mean diameter reduction of

diameter at 3 months type IIB fibers of 19.6% after
(p < 0.05). Partial recovery at 2–4 years of BoNT-A
6 months. treatement, (p < 0.05).
Histologic analysis (optical
microscopy)/ Muscle structure Muscle structure Muscle structure Muscle structure Muscle structure Muscle structure
histochemistry
Muscle atrophy and Mild
increase of the
Muscle atrophy, necrosis, number of perimysial fat
Fibrosis with no identifiable
and hyaline cells. Muscle fiber area Muscle atrophy.
muscle fibers.
degeneration at 4 months. reduction of 24% at
12 months. Partial recovery
at 12 months.

Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure Muscle ultrastructure

Atrophic muscle fibers,

Muscle atrophy of a Myofibrillar
Histologic analysis (electron
considerable disorganization,
microscopy)/
number of muscle fibers at redundant basal lamina,
histochemistry
12 months. Partial recovery cores, and wrinkling of
at 12 months. the sarcolemmal
membrane.

Imaging
All the 10 human studies that evaluated images to measure muscle atrophy after
BoNT-A treatments showed signs of muscle atrophy, irrespective of the technology used:
ultrasound, MRI, CT scan, or cephalometry. Muscle atrophy was registered in the short
term (42 days to 3 months) and in the long term (up to 2 years). No full recovery was
identified (Table 15).
Toxins 2022, 14, 81 17 of 24

Table 15. Human studies—Imaging.

Hamjian (1994) [30] Fanucci (2001) [32] To (2001) [33] Kim (2005) [34] Singer (2006) [36] Lee (2007) [40] Schroeder (2009) [41] Van Campenhout (2013) [46] Koerte (2013) [47] Alexander (2018) [57]
Muscle Atrophy
Extensor Digitorum Piriformis Masseter Masseter Vastus Lateralis Masseter Gastrocnemius Psoas Procerus Gastrocnemius
Identification Tool
10 30 15 383 8 10 2 7 4 11

Muscle thickness Muscle thickness Muscle thickness Muscle thickness Muscle thickness Muscle thickness Muscle thickness Muscle thickness Muscle thickness Muscle thickness

Average decrease of 31%

Decrease of 16% at peak Median decrease of 30.9%
(3 months after BoNT-A
(day 42), (p < 0.03). at peak (3 months) and
injection), (p not
Recovery (Partial? 13.4% (1 year), (p < 0.001).
calculated). Partial
Complete?) 100 days Partial recovery 1 year.
Ultrasound recovery 2 years.

Muscle Volume Muscle Volume Muscle Volume Muscle Volume Muscle Volume Muscle Volume Muscle Volume Muscle Volume Muscle Volume Muscle Volume

Decrease of 40% at peak

(day 42), (p < 0.03).
Recovery (Partial?
Complete?) 100 days.

T2 short tau inversion T2 short tau inversion T2 short tau inversion T2 short tau inversion T2 short tau inversion T2 short tau inversion T2 short tau inversion T2 short tau inversion T2 short tau inversion T2 short tau inversion
recovery (S-TIR) weighted recovery (S-TIR) weighted recovery (S-TIR) weighted recovery (S-TIR) weighted recovery (S-TIR) weighted recovery (S-TIR) weighted recovery (S-TIR) weighted recovery (S-TIR) weighted recovery (S-TIR) weighted recovery (S-TIR) weighted
sequence sequence sequence sequence sequence sequence sequence sequence sequence sequence

Muscular atrophy at
3 months.

Signal Intensity (S.I.) Signal Intensity (S.I.) Signal Intensity (S.I.) Signal Intensity (S.I.) Signal Intensity (S.I.) Signal Intensity (S.I.) Signal Intensity (S.I.) Signal Intensity (S.I.) Signal Intensity (S.I.) Signal Intensity (S.I.)

High intensity High intensity

(compatible with muscle (compatible with muscle
atrophy) at 3 months. atrophy) at 12 months.

Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional
Muscle cross-sectional area
area area area area area area area area area
Magnetic resonance
imaging (MRI) Reduction of 14–19% at
3 months, of 27% at
6 months (peak), and
12–22% at 12 months, (p
not calculated). Partial
recovery at 12 months.

Muscle volume Muscle volume Muscle volume Muscle volume Muscle volume Muscle volume Muscle volume Muscle volume Muscle volume Muscle volume

Reduction of 5.9% at
Reduction of 46% to 48%
Reduction of 20% at 4 weeks, of 9.4% at
at 1 month and sustained
2 months and sustained at 13 weeks (peak reduction),
at 12 months, (p not
6 months, (p = 0.004). No of 6.8% at 25 weeks,
calculated). No recovery
recovery at 6 months. (p < 0.05). Partial recovery
at 12 months.
from 13 to 25 weeks.

Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional Muscle cross-sectional
Muscle cross-sectional area
area area area area area area area area area
Computed
tomography (CT) scan Mean decrease of 12.4%
(+5%) at 12 weeks
(p < 0.05).

Soft-tissue Soft-tissue Soft-tissue Soft-tissue Soft-tissue Soft-tissue Soft-tissue Soft-tissue Soft-tissue Soft-tissue
bigonial distance bigonial distance bigonial distance bigonial distance bigonial distance bigonial distance bigonial distance bigonial distance bigonial distance bigonial distance

Decrease from 131 mm

(±4.9) to 123.5 mm (±3.0)
Cephalometry at 3 months (peak),
(p < 0.05) from months 1
to 7, and sustained
decrease to 130.1 mm
(±4.6) at 12 months.
Toxins 2022, 14, 81 18 of 24

5. Discussion
The use of BoNT-A for cosmetic purposes is a fast-growing procedure, with more than
six million treatments performed by plastic surgeons in the year 2018 alone [59]. Despite
this significant number, we believe that improvements in BoNT-A aesthetic treatments have
been jeopardized by the famous, but simplistic, statement used by the media, patients, and
doctors: “BoNT-A treats wrinkles”. BoNT-A monotherapy relating to wrinkles is, at least,
questionable. The BoNT-A mechanism of action is presynaptic cholinergic nerve terminals
blockage by inhibition of the release of acetylcholine, causing paralysis and subsequent
functional denervated muscle atrophy to some degree [60]. It is important to keep in mind
that wrinkles have a multitude of causes, besides muscle contraction, and that treatments
of wrinkles based only on the use of BTX-A have poor quality results in the long term [61].
Rohrich (2007) [62] brilliantly demonstrated modern topographic anatomic studies proving
the relationship between wrinkles and underlying structures other than muscles, such as
arteries, veins, nerves, and septa of fat compartments [62].
The use of BTX-A was first studied by Scott (1973) [63] for the treatment of strabismus
by pharmacologic weakening the extraocular muscles [33]. The first described use of the
toxin in aesthetic circumstances was by Clark and Berris (1989) [64], but it still carried out
the essence of the BoNT-A mechanism of action based on muscle paralysis and atrophy [64].
At some point during the 1990s, Carruthers and Carruthers [65] began to use botulinum
toxin type A in full-scale treatments for aesthetic purposes. Since then, the aesthetic focus
regarding the use of BoNT-A moved towards removing wrinkles only [65]—a shift in the
medical literature on BoTN-A for aesthetics purposes that has persisted until today. We are
not underestimating the importance of Carruthers and many other authors that previously
studied the use of BoNT-A in aesthetics but, as mentioned above, we intend to provide
the aesthetic use of BoNT-A a new perspective. The real mechanism of actions of BoNT-A
for aesthetic purposes have been forgotten, to a level where recent publications still focus
on the fact that muscle paralysis and muscle atrophy is a complication of the “wrinkle
treatment” capacity of BoNT-A instead of its expected effect [66–68].
This systematic review can shed new light on aesthetic BoNT-A treatments basing
itself on old, but scientifically correct, concepts of striated muscle contraction physiology,
muscle hypertrophy, and muscle atrophy—basic concepts of muscle physiology from
reference physiology medical books such as the Guyton and Hall Textbook of Medical
Physiology [69].
The results of this systematic review showed evidence that seriated or single BoNT-A
muscle injections can cause real atrophy on a short or long-term basis, in animal models
and in humans, in skeletal striated muscles of the limbs, facial masticatory muscles, and
facial mimetic muscles. Due to only limited good quality data being available, we included
animal model studies and human studies, but we know that data extrapolation from animal
model studies to humans are, at least, naïve. The sensitivity of animals to BoNT-A has
been known for many years to be less than that perceived in humans [70]. There are even
differences in sensitivity between rats and mice [71]. On this basis, animal studies must be
carefully designed and carefully analyzed, or they cannot be interpreted with respect to
human effects [72]. Here we will discuss the results of this systematic review, making clear
distinguishment between animal model studies and human studies (Figure 3).
Toxins 2022, 14, 81 19 of 24

Figure 3. Animal model studies results—Discussion overview. * This finding might be of clinical rele-
vance, because muscle volume measured using non-invasive imaging techniques (MRI, ultrasound)
are sometimes used to approximate muscle mass in patient populations to determine progression of a
disease or success of a treatment intervention—Damiano and Moreau (2008) [73]. Structural integrity
and functional properties of muscles, rather than muscle mass or volume, might be more appropriate
outcome measures to determine disease progression or aesthetics intervention effects.
Toxins 2022, 14, 81 20 of 24

Increasing the number of injections did not produce additional loss in muscle strength
and contractile material, as one might have suspected, suggesting that most of the muscle
damage effects of BTX-A injection into muscles are caused by the first injection, or that the
recovery period between injections was sufficient for partial recovery, thereby offsetting
the potential damage induced by each injection.
Genetic alterations related to muscle atrophy/recovery through molecular biology
were analyzed by five studies and showed how much impact a single BoNT-A injection
can cause on a molecular basis. Mukund (2014) [49] realized that the direct action of BTX-A
in skeletal muscle is relatively rapid, inducing dramatic transcriptional adaptation at one
week and activating genes in competing pathways of repair and atrophy by gene-related
impaired mitochondrial biogenesis.
Much like the findings of animal studies, human studies have also clearly shown atro-
phy in different muscle types after BTX-A injections. All six human studies that evaluated
muscle histology showed atrophy, and when muscle recovery was assessed, there was no
full recovery—Borodic (1992) [29] and Schroeder (2009) [41]. Bringing this idea into the
context of facial aesthetics, the treatment of the Orbicularis oculi muscle, for example, with
BTX-A sporadic injections could atrophy this muscle, but serial and controlled treatments
could really maintain a certain degree of atrophy capable of allowing a smile with more
open eyes, less caudal traction vector in the cranial part of this muscle postponing gravi-
tational aging, and even give less contribution to the formation of the famous periorbital
wrinkles, this time, as a secondary effect. Extrapolations of the powerful tool of muscle atro-
phy control through time using BTX-A injections could change completely the way BTX-A
is used for aesthetic purposes. Dosages, injection intervals, and target muscles would be
different from the patterns used nowadays. Instead of planning BTX-A injections to treat
wrinkles, a modern anatomy understanding of the facial mimetic muscles as described by
Boggio (2017) [74] would be of unparallel importance for aesthetic treatment planning [74].
New approaches for facial aesthetic treatments using BoNT-A could be completely based
on mimetic facial muscle interactions and focused on reducing the activity of muscles that
enhance gravitational aging (facial depressor muscles), such as the platysma muscle, for
example, and preserving antigravitational muscles (elevator facial muscles), such as the
frontalis (Figure 4).
After analyzing the results of this paper, we can attempt to answer the questions raised
in the introduction (Table 16).

Table 16. Possible and plausible evidence-based answers for the questions raised in the introduction.

Questions Answers
Does the muscular impairment for contraction caused by Muscle atrophy occurs after BoNT-A injections. Facial
BoNT-A really treat facial lines or cause muscle atrophy? lines are, only in part, treated by BoNT-A injections.
What is the relationship betweenf BoNT-A muscle Muscles tend to maintain atrophy or have partially
injections and muscle atrophy in the long term? recover after BoNT-A injections.
Is it possible to modulate the level of muscle atrophy At least theoretically it is, and further studies could help
through time by using BoNT-A? us master this new frontier in facial aesthetics.
What if we used muscle atrophy caused by BoNT-A It seems smart to use the atrophy after BoNT-A injections
injections to optimize muscle architecture for facial as a tool for aesthetic purposes instead of the old idea of
aesthetic purposes? an adverse event.
What would it be like to reinterpret articles written in the
We are sure that understanding BoNT-A as a muscle
last 30 years that focused mainly on facial lines unveiling
atrophy tool for aesthetic purposes will bring us to new
this concept of muscle atrophy? How many less
readings of previous articles and shed new light on future
subjective opportunities would arise? How would classic
treatments.
BoNT-A injections techniques would be impacted?
Toxins 2022, 14, 81 21 of 24

Figure 4. New approaches for facial aesthetic treatments using BoNT-A. The human imaging studies,
similar to the animal studies, also show muscle atrophy and volume reduction. Koerte (2013) [47]
showed a sustained atrophy and volume loss of approximately 50% in the procerus muscle. New
perspectives on aesthetics BoNT-A treatments should consider not only facial mimetic muscles
and their strength in relation to gravitational or antigravitational contraction vectors, but also their
volume. Muscle volume control is also of aesthetic importance. The understanding that some degree
of muscle volume reduction would bring positive aesthetic aspects for some mimetic muscles, such
as the procerus and corrugators and some masticatory muscles such as the masseter, would also
change the current BoNT-A injections patterns. On the other hand, some muscles should be spared
from volume loss, such as the frontalis and the lateral aspect of the orbicularis oculi, to avoid facial
skeletonization.

6. Conclusions
This systematic review showed evidence that seriated or single BoNT-A muscle injec-
tions can cause real muscle atrophy on a short or long-term basis, in animal models and
in humans, in skeletal striated muscles of the limbs, facial masticatory muscles, and facial
mimetic muscles. Theoretically, muscular architecture reprogramming is a possible new
approach in aesthetics. Depressor facial muscles could be targeted to have some degree
of atrophy with BoNT-A injections, while elevator facial muscles could be spared to some
degree to maintain antigravitational traction forces and facilitate a lift effect.

Author Contributions: Conceptualization, A.D.N.; methodology, A.D.N.; validation, A.D.N.; formal

analysis, A.D.N.; investigation, A.D.N.; data curation, A.D.N.; writing—original draft preparation,
A.D.N.; writing—review and editing, R.F.B., S.E. and G.E.L.F.; All authors have read and agreed to
the published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
Toxins 2022, 14, 81 22 of 24

References
1. Gart, M.S.; Gutowski, K.A. Overview of Botulinum Toxins for Aesthetic Uses. Clin. Plast. Surg. 2016, 43, 459–471. [CrossRef]
2. Cavallini, M.; Cirillo, P.; Fundarò, S.P.; Quartucci, S.; Sciuto, C.; Sito, G.; Tonini, D.; Trocchi, G.; Signorini, M. Safety of botulinum
toxin A in aesthetic treatments: A systematic review of clinical studies. Dermatol. Surg. 2014, 40, 525–536. [CrossRef]
3. Ministério da Saúde Consultoria Jurídica/Advocacia Geral da União 1 Nota Técnica N◦ Nota Técnica N◦ 342/2014. Available
online: https://portalarquivos2.saude.gov.br/images/pdf/2014/setembro/17/Toxina-botul--nica-tipo-A.pdf (accessed on
28 July 2019).
4. Monheit, G. Neurotoxins: Current Concepts in Cosmetic Use on the Face and Neck–Upper Face (Glabella, Forehead, and Crow’s
Feet). Plast. Reconstr. Surg. 2015, 136, 72S–75S. [CrossRef] [PubMed]
5. Bonaparte, J.P.; Ellis, D.; Quinn, J.G.; Rabski, J.; Hutton, B. A Comparative Assessment of Three Formulations of Botulinum Toxin
Type A for Facial Rhytides: A Systematic Review with Meta-Analyses. Plast. Reconstr. Surg. 2016, 137, 1125–1140. [CrossRef]
6. Brin, M.F.; James, C.; Maltman, J. Botulinum toxin type A products are not interchangeable: A review of the evidence. Biologics
2014, 8, 227–241. [CrossRef] [PubMed]
7. Albanese, A. Terminology for preparations of botulinum neurotoxins: What a difference a name makes. JAMA 2011, 305, 89–90,
Erratum in JAMA 2011, 305, 1544. [CrossRef] [PubMed]
8. Wilson, A.J.; Chang, B.; Taglienti, A.J.; Chin, B.C.; Chang, C.S.; Folsom, N.; Percec, I. A Quantitative Analysis of Onabotulinumtox-
inA, AbobotulinumtoxinA, and IncobotulinumtoxinA: A Randomized, Double-Blind, Prospective Clinical Trial of Comparative
Dynamic Strain Reduction. Plast. Reconstr. Surg. 2016, 137, 1424–1433. [CrossRef]
9. Burgen, A.S.; Dickens, F.; Zatman, L.J. The action of botulinum toxin on the neuro-muscular junction. J. Physiol. 1949, 109, 10–24.
[CrossRef] [PubMed]
10. Simpson, L.L. The origin, structure, and pharmacological activity of botulinum toxin. Pharmacol. Rev. 1981, 33, 155–188.
11. Aoki, K.R. Pharmacology and immunology of botulinum toxin type A. Clin. Dermatol. 2003, 21, 476–480. [CrossRef]
12. Pellizzari, R.; Rossetto, O.; Schiavo, G.; Montecucco, C. Tetanus and botulinum neurotoxins: Mechanism of action and therapeutic
uses. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 1999, 354, 259–268. [CrossRef]
13. Schiavo, G.; Matteoli, M.; Montecucco, C. Neurotoxins affecting neuroexocytosis. Physiol. Rev. 2000, 80, 717–766. [CrossRef]
14. Lacy, D.B.; Tepp, W.; Cohen, A.C.; DasGupta, B.R.; Stevens, R.C. Crystal structure of botulinum neurotoxin type A and implications
for toxicity. Nat. Struct. Biol. 1998, 5, 898–902. [CrossRef]
15. Koriazova, L.K.; Montal, M. Translocation of botulinum neurotoxin light chain protease through the heavy chain channel. Nat.
Struct. Biol. 2003, 10, 13–18. [CrossRef] [PubMed]
16. Rizo, J.; Südhof, T.C. Snares and Munc18 in synaptic vesicle fusion. Nat. Rev. Neurosci. 2002, 3, 641–653. [CrossRef]
17. Pingel, J.; Nielsen, M.S.; Lauridsen, T.; Rix, K.; Bech, M.; Alkjaer, T.; Andersen, I.T.; Nielsen, J.B.; Feidenhansl, R. Injection of high
dose botulinum-toxin A leads to impaired skeletal muscle function and damage of the fibrilar and non-fibrilar structures. Sci. Rep.
2017, 7, 14746. [CrossRef] [PubMed]
18. Nigam, P.K.; Nigam, A. Botulinum toxin. Indian J. Dermatol. 2010, 55, 8–14. [CrossRef] [PubMed]
19. De Paiva, A.; Meunier, F.A.; Molgó, J.; Aoki, K.R.; Dolly, J.O. Functional repair of motor endplates after botulinum neurotoxin
type A poisoning: Biphasic switch of synaptic activity between nerve sprouts and their parent terminals. Proc. Natl. Acad. Sci.
USA 1999, 96, 3200–3205. [CrossRef]
20. Holland, R.L.; Brown, M.C. Nerve growth in botulinum toxin poisoned muscles. Neuroscience 1981, 6, 1167–1179. [CrossRef]
21. Hopkins, W.G.; Brown, M.C. The distribution of nodal sprouts in a paralysed or partly denervated mouse muscle. Neuroscience
1982, 7, 37–44. [CrossRef]
22. Pamphlett, R. Early terminal and nodal sprouting of motor axons after botulinum toxin. J. Neurol. Sci. 1989, 92, 181–192.
[CrossRef]
23. Anderson, D.M.; Kumar, V.R.; Arper, D.L.; Kruger, E.; Bilir, S.P.; Richardson, J.S. Cost savings associated with timely treatment of
botulism with botulism antitoxin heptavalent product. PLoS ONE 2019, 14, e0224700. [CrossRef] [PubMed]
24. Arnon, S.S.; Schechter, R.; Inglesby, T.V.; Henderson, D.A.; Bartlett, J.G.; Ascher, M.S.; Eitzen, E.; Fine, A.D.; Hauer, J.; Layton, M.;
et al. Working Group on Civilian Biodefense. Botulinum toxin as a biological weapon: Medical and public health management.
JAMA 2001, 285, 1059–1070, Erratum in JAMA 2001, 285, 2081. [CrossRef]
25. Chalk, C.H.; Benstead, T.J.; Pound, J.D.; Keezer, M.R. Medical treatment for botulism. Cochrane Database Syst. Rev. 2019, 4,
CD008123. [CrossRef]
26. Lin, L.; Olson, M.E.; Eubanks, L.M.; Janda, K.D. Strategies to Counteract Botulinum Neurotoxin A: Nature’s Deadliest Biomolecule.
Acc. Chem. Res. 2019, 52, 2322–2331. [CrossRef]
27. Moher, D.; Liberati, A.; Tetzlaff, J.; Altman, D.G.; PRISMA Group. Preferred reporting items for systematic reviews and
meta-analyses: The PRISMA statement. Version 2. BMJ 2009, 339, b2535. [CrossRef]
28. Vandenbroucke, J.P.; von Elm, E.; Altman, D.G.; Gøtzsche, P.C.; Mulrow, C.D.; Pocock, S.J.; Poole, C.; Schlesselman, J.J.; Egger,
M. STROBE Initiative. Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): Explanation and
elaboration. Int. J. Surg. 2014, 12, 1500–1524. [CrossRef]
29. Borodic, G.E.; Ferrante, R. Effects of repeated botulinum toxin injections on orbicularis oculi muscle. J Clin Neuroophthalmol. 1992,
12, 121–127. [CrossRef]
Toxins 2022, 14, 81 23 of 24

30. Hamjian, J.A.; Walker, F.O. Serial neurophysiological studies of intramuscular botulinum-A toxin in humans. Muscle Nerve. 1994,
17, 1385–1392. [CrossRef] [PubMed]
31. Ansved, T.; Odergren, T.; Borg, K. Muscle fiber atrophy in leg muscles after botulinum toxin type A treatment of cervical dystonia.
Neurology 1997, 48, 1440–1442. [CrossRef]
32. Fanucci, E.; Masala, S.; Sodani, G.; Varrucciu, V.; Romagnoli, A.; Squillaci, E.; Simonetti, G. CT-guided injection of botulinic toxin
for percutaneous therapy of piriformis muscle syndrome with preliminary MRI results about denervative process. Eur. Radiol.
2001, 11, 2543–2548. [CrossRef]
33. To, E.W.; Ahuja, A.T.; Ho, W.S.; King, W.W.; Wong, W.K.; Pang, P.C.; Hui, A.C. A prospective study of the effect of botulinum
toxin A on masseteric muscle hypertrophy with ultrasonographic and electromyographic measurement. Br. J. Plast Surg. 2001, 54,
197–200. [CrossRef]
34. Kim, N.H.; Chung, J.H.; Park, R.H.; Park, J.B. The use of botulinum toxin type A in aesthetic mandibular contouring. Plast.
Reconstr. Surg. 2005, 115, 919–930. [CrossRef]
35. Shen, J.; Ma, J.; Lee, C.; Smith, B.P.; Smith, T.L.; Tan, K.H.; Koman, L.A. How muscles recover from paresis and atrophy after
intramuscular injection of botulinum toxin A: Study in juvenile rats. J. Orthop. Res. 2006, 24, 1128–1135. [CrossRef]
36. Singer, B.J.; Silbert, P.L.; Dunne, J.W.; Song, S.; Singer, K.P. An open label pilot investigation of the efficacy of Botulinum toxin
type A [Dysport] injection in the rehabilitation of chronic anterior knee pain. Disabil. Rehabil. 2006, 28, 707–713. [CrossRef]
37. Herzog, W.; Longino, D. The role of muscles in joint degeneration and osteoarthritis. J. Biomech. 2007, 40 (Suppl. 1), S54–S63,
Erratum in J. Biomech. 2008, 41, 2332–2335. [CrossRef] [PubMed]
38. Frick, C.G.; Richtsfeld, M.; Sahani, N.D.; Kaneki, M.; Blobner, M.; Martyn, J.A. Long-term effects of botulinum toxin on
neuromuscular function. Anesthesiology 2007, 106, 1139–1146. [CrossRef] [PubMed]
39. Kwon, T.G.; Park, H.S.; Lee, S.H.; Park, I.S.; An, C.H. Influence of unilateral masseter muscle atrophy on craniofacial morphology
in growing rabbits. J. Oral Maxillofac. Surg. 2007, 65, 1530–1537. [CrossRef]
40. Lee, C.J.; Kim, S.G.; Kim, Y.J.; Han, J.Y.; Choi, S.H.; Lee, S.I. Electrophysiologic change and facial contour following botulinum
toxin A injection in square faces. Plast Reconstr. Surg. 2007, 120, 769–778. [CrossRef] [PubMed]
41. Schroeder, A.S.; Ertl-Wagner, B.; Britsch, S.; Schröder, J.M.; Nikolin, S.; Weis, J.; Müller-Felber, W.; Koerte, I.; Stehr, M.; Berweck, S.;
et al. 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. Mov. Disord. 2009, 24, 1494–1503. [CrossRef]
42. Babuccu, B.; Babuccu, O.; Yurdakan, G.; Ankarali, H. The effect of the Botulinum toxin-A on craniofacial development: An
experimental study. Ann. Plast Surg. 2009, 63, 449–456. [CrossRef]
43. Tsai, F.C.; Hsieh, M.S.; Chou, C.M. Comparison between neurectomy and botulinum toxin A injection for denervated skeletal
muscle. J. Neurotrauma. 2010, 27, 1509–1516. [CrossRef] [PubMed]
44. Fortuna, R.; Vaz, M.A.; Youssef, A.R.; Longino, D.; Herzog, W. Changes in contractile properties of muscles receiving repeat
injections of botulinum toxin (Botox). J. Biomech. 2011, 44, 39–44. [CrossRef] [PubMed]
45. Fortuna, R.; Horisberger, M.; Vaz, M.A.; Van der Marel, R.; Herzog, W. The effects of electrical stimulation exercise on muscles
injected with botulinum toxin type-A (botox). J. Biomech. 2013, 46, 36–42. [CrossRef]
46. Van Campenhout, A.; Verhaegen, A.; Pans, S.; Molenaers, G. Botulinum toxin type A injections in the psoas muscle of children
with cerebral palsy: Muscle atrophy after motor end plate-targeted injections. Res. Dev. Disabil. 2013, 34, 1052–1058. [CrossRef]
[PubMed]
47. Koerte, I.K.; Schroeder, A.S.; Fietzek, U.M.; Borggraefe, I.; Kerscher, M.; Berweck, S.; Reiser, M.; Ertl-Wagner, B.; Heinen, F. 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. 2013, 39, 761–765. [CrossRef]
48. Fortuna, R.; Horisberger, M.; Vaz, M.A.; Herzog, W. Do skeletal muscle properties recover following repeat onabotulinum toxin A
injections? J. Biomech. 2013, 46, 2426–2433. [CrossRef]
49. Mukund, K.; Mathewson, M.; Minamoto, V.; Ward, S.R.; Subramaniam, S.; Lieber, R.L. Systems analysis of transcriptional data
provides insights into muscle’s biological response to botulinum toxin. Muscle Nerve. 2014, 50, 744–758. [CrossRef] [PubMed]
50. Fortuna, R.; Vaz, M.A.; Sawatsky, A.; Hart, D.A.; Herzog, W. 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. 2015,
48, 1700–1706. [CrossRef] [PubMed]
51. Caron, G.; Marqueste, T.; Decherchi, P. Long-Term Effects of Botulinum Toxin Complex Type A Injection on Mechano- and
Metabo-Sensitive Afferent Fibers Originating from Gastrocnemius Muscle. PLoS ONE. 2015, 10, e0140439. [CrossRef]
52. Valentine, J.; Stannage, K.; Fabian, V.; Ellis, K.; Reid, S.; Pitcher, C.; Elliott, C. Muscle histopathology in children with spastic
cerebral palsy receiving botulinum toxin type A. Muscle Nerve 2016, 53, 407–414. [CrossRef]
53. Li, J.; Allende, A.; Martin, F.; Fraser, C.L. Histopathological changes of fibrosis in human extra-ocular muscle caused by botulinum
toxin A. J. AAPOS 2016, 20, 544–546. [CrossRef]
54. Kocaelli, H.; Yaltirik, M.; Ayhan, M.; Aktar, F.; Atalay, B.; Yalcin, S. Ultrastructural evaluation of intramuscular applied botulinum
toxin type A in striated muscles of rats. Hippokratia 2016, 20, 292–298. [PubMed]
55. Hart, D.A.; Fortuna, R.; Herzog, W. Messenger RNA profiling of rabbit quadriceps femoris after repeat injections of botulinum
toxin: Evidence for a dynamic pattern without further structural alterations. Muscle Nerve 2018, 57, 487–493. [CrossRef] [PubMed]
Toxins 2022, 14, 81 24 of 24

56. Han, S.K.; Lee, Y.; Hong, J.J.; Yeo, H.G.; Seo, J.; Jeon, C.Y.; Jeong, K.J.; Jin, Y.B.; Kang, P.; Lee, S.; et al. In vivo study of paraspinal
muscle weakness using botulinum toxin in one primate model. Clin. Biomech. 2018, 53, 1–6. [CrossRef]
57. Alexander, C.; Elliott, C.; Valentine, J.; Stannage, K.; Bear, N.; Donnelly, C.J.; Shipman, P.; Reid, S. Muscle volume alterations after
first botulinum neurotoxin A treatment in children with cerebral palsy: A 6-month prospective cohort study. Dev. Med. Child.
Neurol. 2018, 60, 1165–1171. [CrossRef]
58. Lima, W.; Salles, A.G.; Faria, J.C.M.; Nepomuceno, A.C.; Salomone, R.; Krunn, P.; Gemperli, R. Contralateral Botulinum Toxin
Improved Functional Recovery after Tibial Nerve Repair in Rats. Plast. Reconstr. Surg. 2018, 142, 1511–1519. [CrossRef] [PubMed]
59. International Society of Aesthetic Plastic Surgery. The International Study on Aesthetic/Cosmetic Procedures Performed in 2018.
International Society of Aesthetic Plastic Surgery. 2018. Available online: https://www.isaps.org/wp-content/uploads/2019/12/
ISAPS-Global-Survey-Results-2018-new.pdf (accessed on 10 July 2020).
60. Hastings-Ison, T.; Graham, H.K. Atrophy and hypertrophy following injections of botulinum toxin in children with cerebral palsy.
Dev. Med. Child. Neurol. 2013, 55, 778–779. [CrossRef]
61. Manríquez, J.J.; Cataldo, K.; Vera-Kellet, C.; Harz-Fresno, I. Wrinkles. BMJ Clin. Evid. 2014, 2014, 1711.
62. Rohrich, R.J.; Pessa, J.E. The fat compartments of the face: Anatomy and clinical implications for cosmetic surgery. Plast. Reconstr.
Surg. 2007, 119, 2219–2227, discussion 2228–2231. [CrossRef]
63. Scott, A.B.; Rosenbaum, A.; Collins, C.C. Pharmacologic weakening of extraocular muscles. Invest. Ophthalmol. 1973, 12, 924–927.
[PubMed]
64. Clark, R.P.; Berris, C.E. Botulinum toxin: A treatment for facial asymmetry caused by facial nerve paralysis. Plast. Reconstr. Surg.
1989, 84, 353–355. [CrossRef]
65. Carruthers, A.; Carruthers, J. History of the cosmetic use of Botulinum A exotoxin. Dermatol. Surg. 1998, 24, 1168–1170. [CrossRef]
[PubMed]
66. Salari, M.; Sharma, S.; Jog, M.S. Botulinum Toxin Induced Atrophy: An Uncharted Territory. Toxins 2018, 10, 313. [CrossRef]
[PubMed]
67. Yiannakopoulou, E. Serious and long-term adverse events associated with the therapeutic and cosmetic use of botulinum toxin.
Pharmacology 2015, 95, 65–69. [CrossRef]
68. Durand, P.D.; Couto, R.A.; Isakov, R.; Yoo, D.B.; Azizzadeh, B.; Guyuron, B.; Zins, J.E. Botulinum Toxin and Muscle Atrophy: A
Wanted or Unwanted Effect. Aesthet. Surg. J. 2016, 36, 482–487. [CrossRef]
69. Khonsary, S.A. Guyton and Hall: Textbook of Medical Physiology. Surg. Neurol. Int. 2017, 8, 275. [CrossRef]
70. Pirazzini, M.; Rossetto, O.; Eleopra, R.; Montecucco, C. Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology. Pharmacol.
Rev. 2017, 69, 200–235. [CrossRef] [PubMed]
71. Rosales, R.L.; Bigalke, H.; Dressler, D. Pharmacology of botulinum toxin: Differences between type A preparations. Eur. J. Neurol.
2006, 13, 2–10. [CrossRef]
72. Pickett, A. Animal studies with botulinum toxins may produce misleading results. Anesth. Analg. 2012, 115, 736–737. [CrossRef]
73. Damiano, D.L.; Moreau, N. Muscle thickness reflects activity in CP but how well does it represent strength? Dev. Med. Child
Neurol. 2008, 50, 88. [CrossRef] [PubMed]
74. Boggio, R.F. Dynamic Model of Applied Facial Anatomy with Emphasis on Teaching of Botulinum Toxin A. Plast. Reconstr. Surg.
Glob. Open 2017, 5, e1525. [CrossRef] [PubMed]