Aesth Plast Surg (2015) 39:134–140
DOI 10.1007/s00266-014-0418-z

ORIGINAL ARTICLE

EXPERIMENTAL/SPECIAL TOPICS

The Inhibitory Effect of Platelet-Rich Plasma on Botulinum Toxin
Type-A: An Experimental Study in Rabbits
Hakan Bulam • Suhan Ayhan • Billur Sezgin • Murat Zinnuroglu •Ece Konac • Nuray Varol • Kemal Findikcioglu • Serhan Tuncer •Seyhan Cenetoglu

Received: 16 July 2014 / Accepted: 16 October 2014 / Published online: 21 November 2014
Ó Springer Science+Business Media New York and International Society of Aesthetic Plastic Surgery 2014

M. Zinnuroglu
Department of Physical Therapy and Rehabilitation, Gazi
University Faculty of Medicine, Ankara, Turkey

therapy was performed in Group I while Group II received
saline mesotherapy. Group III was designed as the in vitro
mixture group in which onabotulinumtoxinA and plateletrich plasma were mixed and then administered intramuscularly. Group IV received saline within the mixture
instead of platelet-rich plasma. The contralateral ears of all
the rabbits served as control and were only treated withonabotulinumtoxinA. Visual evaluation of ear positions
and electroneuromyographic studies were done prior to allprocedures and at day 14. Anterior auricular muscles were
harvested at day 14 and were evaluated with quantitative
real-time PCR.
Results Visual and electroneuromyographic studies
revealed less onabotulinumtoxinA activity in Groups I and
III. When platelet-rich plasma was administered throughskin mesotherapy, onabotulinumtoxinA activity failure wasmore severe in comparison with direct contact. No significant difference in SNAP-25 mRNA expression throughquantitative real-time PCR was observed between groups.
Conclusion Although we could not explain the exact
mechanism underlying this interaction, platelet-rich plasma
applications result in less onabotulinumtoxinA muscle
paralysis activity.
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E. Konac  N. Varol
Department of Medical Biology and Genetics, Gazi University
Faculty of Medicine, Ankara, Turkey

Keywords Botulinum toxin type-A  Inhibitors 
Metalloproteases  Platelet-rich plasma  SNAP-25 protein

Abstract
Background Combination treatments of botulinum toxin
type-A and other rejuvenation agents or instruments aregradually becoming more popular. After observing a highincidence of therapy failure following simultaneous applications of botulinum toxin type-A and platelet-rich plasma
mesotherapy, we aimed to investigate whether PRP has an
inhibitory effect on botulinum toxin type-A.
Methods Twenty-four New Zealand white rabbits weredivided into 4 groups, and the anterior auricular muscle andoverlying skin were used for injections. Groups I and II
both received onabotulinumtoxinA intramuscular injections. In addition, autologous platelet-rich plasma meso-This study was presented at the 2nd European Association of Plastic
Surgeons Research Council Meeting, Antalya, Turkey, 22–23 May
2013 and was awarded second place in the ‘‘Scientific Competition of
Residents-Experimental Studies’’ at the 35th Congress of the TurkishSociety of Plastic Reconstructive and Aesthetic Surgery, October
28–31, 2013 Istanbul, Turkey.
H. Bulam  S. Ayhan (&)  B. Sezgin  K. Findikcioglu 
S. Tuncer  S. Cenetoglu
Department of Plastic Reconstructive and Aesthetic Surgery,
Gazi University Faculty of Medicine, Ankara, Turkey
e-mail: suayhan@gmail.com

123Aesth Plast Surg (2015) 39:134–140

IntroductionMore than six million botulinum toxin type-A (BoNT-A)
applications were reported in the United States during theyear 2013 [1]. In recent years, combination therapies of
BoNT-A applications together with injectable fillers,mesotherapy agents, chemical peeling agents, radiofrequency, and lasers have been widely performed to reachmore favorable results in facial rejuvenation. Among these,
platelet-rich plasma (PRP) mesotherapy particularly has
the advantage of stimulating proliferation and angiogenesis. The contribution of PRP to cell proliferation is thought
to be based on the growth factors (PDGF, VEGF, TGF-b,
EGF, and IGF-1) present within it [2].With technicalinnovations in preparation and increased availability, thefrequency of PRP mesotherapy procedures has gradually
increased.
The mechanism of botulinum toxin has four criticalstages: binding to presynaptic membranes, internalization
with endocytosis, translocation to the cytoplasm, andproteolysis of SNARE (soluble N-ethylmaleimide
attachment protein receptor) proteins. By breaking the
SNAP-25 (synaptosome-associated protein of 25 kDa)
protein with the zinc-metalloproteinase activity of its
light chain, BoNT-A prevents acetylcholine release to theneuromuscular junction. To achieve the muscle paralysis
effect, all stages must be accomplished. Therapeutic
failure of BoNT-A in cosmetic use is very uncommon[3]. Although it has yet to be proven, in most cases, it
has generally been assumed that the reason for therapeutic failure was the neutralizing antibodies against the
BoNT-A.
Platelets consist of three different types of granules, andfollowing degranulation, plenty of molecules, growth factors, coagulation factors, and enzymes are released into themedium. The therapeutic effects of PRP regarding growth
factors are known but the effect of the others enzymes andmolecules are still under assessment.
In our practice, we encountered therapeutic failure or ashorter duration of muscle paralysis following BoNT-A
injections, only in such cases where PRP mesotherapy was
performed at the same session. This experimental study
was designed following this clinical observation, and the
purpose was to investigate whether PRP has an inhibitory
effect on BoNT-A.

Materials and Methods
The study was approved by the Institutional Ethics Committee for Animal Studies. The study was carried out on 24
male New Zealand white rabbits weighing between 1.5 and2 kg. For surgical procedures, rabbits were anesthetized

135

with an anesthetic cocktail consisting of ketamine (1.5 mL,
100 mg/mL), acepromazine (0.5 mL, 10 mg/mL), andxylazine (0.5 mL, 20 mg/mL). Each animal received
0.5 mL/kg of the anesthetic cocktail intramuscularly. The
periauricular area and inguinal area were shaved before
electroneuromyographic studies, blood sampling, and each
injection.
The anterior auricular muscle model was selected for the
study, since it was a suitable model for visual, electroneuromyographic, and muscle biopsy evaluations. As apilot study, an anatomical dissection of the anterior auricular muscle was performed in a rabbit cadaver, and anatomical details of the muscle were recorded. The anteriorauricular muscle origin was located at the frontal bone,about 2 cm cranial to the medial canthus, and it inserted
along the ventromedial border of the ipsilateral ear, about
3 cm from the base of the ear. Injection points and set-upof electrodes were designed on the basis of these anatomical details.
OnabotulinumtoxinA (Botulinum toxin type-A, BotoxÒ;Allergan, Abdi Ibrahim, Turkey) was used for botulinum
toxin injections following reconstitution with 2 mL ofsterile saline. In the present study, we used 2.5 U(0.05 mL) BoNT-A for botulinum toxin injections.
Autogenous PRP was prepared for each rabbit. Ten cc
blood samples were obtained through the inguinal artery.
The Regen-ACR (Regenlab, Switzerland) PRP preparation
kit was used to obtain PRP. Blood was processed according
to the recommended protocol of the PRP kit. The blood
was administered to the kit’s tube, and the tube was centrifuged at 3100 rpm/min for 8 min with the JK-LC-802C(Jingke, China) centrifuge. One cc of PRP was obtained at
the end of the procedure.
All BoNT-A and PRP injections were performed by thesame researcher and the same technique. Botulinum toxin
was injected intramuscularly to the middle of the muscle
belly, and PRP was injected subcutaneously through multiple and small volume punctures to simulate the mesotherapy procedure. Thirty-gage syringes were used for all
types of injections.
The animals were divided into four groups:
Group I (PRP Mesotherapy Group) (n = 6): Animals
received an injection of 2.5 U (0.05 mL) BoNT-A into
both right and left anterior auricular muscles. The
2 9 1 cm sized skin overlying the right anterior auricular
muscle was treated with 1 mL PRP intradermally bymultiple serial small volume punctures.
Group II (Mesotherapy Group) (n = 6): Animals
received an injection of 2.5 U (0.05 mL) BoNT-A into
both right and left anterior auricular muscles. The
2 9 1 cm sized skin overlying the right anterior auricular
muscle was treated with 1 mL %0.9 NaCl intradermally bymultiple serial small volume punctures.

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Fig. 1 Grading system for the rabbit auricle. All left ears completely
dropped following the BoNT-A injection. Grading was performed
according to level of right ears. Grade 1: Completely paralytic rightear, dropping more than 60° (high paralytic activity of BoNT-A).

Aesth Plast Surg (2015) 39:134–140

Grade 2: Moderately paralytic right ear, dropping between 30° and
60° (moderate paralytic activity of BoNT-A). Grade 3: Mildly
paralytic right ear, right ear can rise more than 60° (low paralytic
activity of BoNT-A)

Group III (PRP and BoNT-A in vivo mixture Group)(n = 6): Animals received an injection of 2.5 U (0.05 mL)
BoNT-A plus 0.05 mL PRP into the right anterior auricular
muscles and 2.5 U (0.05 mL) BoNT-A into the left anteriorauricular muscles.
Group IV (Control in vivo mixture Group) (n = 6):
Animals received an injection of 2.5 U (0.05 mL) BoNT-A
plus 0.05 mL NaCl into the right anterior auricular musclesand 2.5 U (0.05 mL) BoNT-A into the left anterior auricular muscles.
Following visual and electroneuromyographic studies,
the anterior auricular muscles were excised bilaterally
through a bitemporal incision on post-injection day 14 andwere processed by quantitative real-time PCR analysis.
Visual Evaluation
All rabbits had healthy, mobile ears in upright positions
before the injections. With the effect of the BoNT-A, theaction of the anterior auricular muscle was paralyzed andears dropped. The ear positions of the rabbits were evaluated blindly on day 14 after the injections and were gradedwith a three-grade evaluation system (Fig. 1). Dropping of
more than 60° was observed in the left ear of all rabbits,
and left ears were used as controls. This finding showedthat the dose of injected BoNT-A was sufficient to paralyze
the anterior auricular muscles. The positions of the rightears were used as indicators of efficiency of the BoNT-A.
Grade 1 indicated high BoNT-A activity, Grade 2 moderate, and Grade 3 low BoNT activity.
Electroneuromyographic Studies
The Medelec Synergy EMG system (VIASYS HealthCare,UK) was used for the electroneuromyographic studies. Allassessments were performed in standard motor nerve
conduction settings [low-frequency filter, 2 Hz; high-frequency filter, 10 kHz; stimulus intensity was increased in a
stepwise manner until a supramaximal motor response waselicited (usually between 5 and 50 mA), and stimulus

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Fig. 2 Anatomical dissection of the anterior auricular muscle. The
origin of the muscle is indicated with white arrows, and the insertionis indicated as black arrow. The intramuscular injection is also
demonstrated

duration was 0.2 ms]. Three surface disk electrodes wereused to measure the compound muscle action potential.
Before the study, the anatomical dissection of the anteriorauricular muscle was done in a rabbit cadaver (Fig. 2). The
anterior auricular muscle originated from the frontal bone,about 2 cm cranial to the medial canthus, and inserted
along the ventromedial border of the ipsilateral ear, about
3 cm from the base of the ear. The electrodes were placed
considering the muscle belly and tendon principle. The
active recording electrode was placed at the center (muscle
belly) of the anterior auricular muscle, the reference
recording was placed at the insertion (tendon), and theground electrode was placed on the posterior surface of theear. These three locations were marked with a permanentmarker and used for repeated measurements. A supramaximal stimulation was given to the rostral branches ofAesth Plast Surg (2015) 39:134–140

137

the facial nerve which was stimulated through the overlying masseter muscle at the cheek between the lower eyelid
and jaw at lateral decubitus position for the stimulation of
the motor nerve of the anterior auricular muscle.
Compound muscle action potentials (CMAP) wererecorded by supramaximal stimulation of the facial nerve,
and the amplitude of the elicited CMAPs was measured.
This procedure was repeated three times, and one of thethree CMAPs which had the maximum amplitude value
was used for statistical evaluation.
Total RNA Extraction and cDNA Preparation
Total RNA was extracted using Tripure Reagent (Roche,
Invitrogen, Darmstadt, Germany) according to the manufacturer’s protocol. To avoid DNA contamination, we
performed some modifications to the manufacturer’s
instructions. Tissues were homogenized and incubated in
Trizol (100 mg/mL Trizol) for 5 min, and the RNA wasseparated using chloroform (0.2 mL/1 mL Trizol). Aftercentrifugation at 12,000g for 15 min at 4 °C, the aqueous phase of the sample was transferred to a fresh tube
for RNA precipitation by isopropyl alcohol (0.5 mL/
1 mL Trizol). After repeating incubation and centrifugation as above, the pellet was washed with 75 % ethanol. The mixture was centrifuged at 7500g for 5 min at
4 °C. Finally, the air-dried pellet was re-dissolved with
DEPC-treated water. The yield and quality of the RNAof each sample were determined by measuring theabsorbance at 260 and 280 nm using the ‘‘Nano Drop
Spectrophotometer’’ (NanoDrop ND-1000, Montchanin,
DE, USA). The ratio of sample absorbance A260/280 \2.0 and A260/230 in the range of 1.8–2.2 was consideredan acceptable measure of RNA purity. Total RNA (1 lg)
was reverse-transcribed in a 20 lL reaction mixture
using random hexamers and a Transcriptor First Strand
cDNA synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s
instructions.

Fig. 3 Rate of CMAP Reduction in right ears. CAMP reduction was
significantly lower in the PRP mesotherapy-treated group than othergroups. (*P \ 0.05)

TCC TTT TC-30 (reverse primer). The 10 lL reaction
mixture prepared in a LightCycler Ò 480 Multiwell
Plate 96 contained LightCyclerÒ 480 Probes Master reaction mixture (Roche Diagnostics, GmbH, Mannheim,
Germany), 2.5 pmol of each primer, 1 pmol of UPL probe,
4 mM MgCl2, and 1 lL cDNA. The real-time PCR assay
included a no-template control. All PCR reactions were
performed in the LightCycler Ò 480 instrument (Roche
Diagnostics, GmbH, Mannheim, Germany) using the following program conditions: 95 °C for 10 min followed by
45 cycles at 95 °C for 10 s, 60 °C for 20 s, and finally a
cooling step to 40 °C. We analyzed the expression of the
HPRT housekeeping gene to normalize the results obtained
by QRT-PCR. Each sample was tested in triplicate. PCR
efficiency for each gene was tested by serial dilutions, andamplification efficiencies of target gene and HPRT for eachgene were approximately equal.
Statistical Analysis

Quantitative Real-Time PCR (QRT-PCR) Analysis
SNAP25 mRNA expression levels were measured using
the real-time PCR method. Probe and primer sets for
SNAP25 gene were designed at the Universal Probe
Library Assay Design Center (URL: www.roche-appliedscience.com and UPL probe number for SNAP25 was 58).
Primers used in this section were shown as follows:
SNAP25: 50 -AAA AAG CCT GGG GCA ATA AT-30(forward primer), 50 -CTT TCG TCC ACC ACA CGA G-30
(reverse primer); HPRT: 50 -GCT TTC CTT GGT CAAGCA GT-30 (forward primer), 50 -CAC TTC GAG GGG

Data of visual and electroneuromyographic studies wereanalyzed by the Chi-square test, Wilcoxon signed test, andKruskal–Wallis tests, using SPSS 11.0 for Windows. Statistical significance levels of differences in mRNA
expressions were analyzed by pair-wise fixed reallocation
randomization test. The REST software Tool 2009 version
2.013 was used for group-wise comparison and statisticalanalysis of relative expression results [4]. P \ 0.05
(Mann–Whitney U test) was considered statistically significant. The results were expressed in form ofmean ± SD.

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Aesth Plast Surg (2015) 39:134–140

Table 1 Electroneuromyographic results 24 h after injection
GroupsSubjects

Compound muscle action potentials (CMAP)(mV)
R0Group IPGroup II

R14

L0

L14

N1

19.9

5.6

15.7

1.3

N2

15.6

7.9

12.5

4.6

N3
N4

7.24
19.0

4.3
5.7

13.4
14.3

0.2
1.1

N5

12.2

4.8

19.1

4.0

N6

8.9

3.0

14.4

0.9

0.028
N1

10.3

0.6

11.8

1.2

N2

12.2

1.1

11.3

1.2

N3

9.0

1.2

16.6

1.0

N4

9.0

0.9

10.4

0.8

N5

12.9

1.0

9.6

0.8

N6

9.3

1.5

11.5

1.5

P

0.60

Group III

N1

9.1

2.5

9.5

1.2

N2

13.8

1.4

14.6

0.8

N3

12.9

0.2

14.7

0.1

N4
N5

11.8
9.6

0.3
1.2

11.9
8.8

0.3
0.7

N6

8.8

1.4

11.3

0.5

P
Group IV

P

0.038
N1

11.7

0.9

11.9

0.8

N2

13.6

2.2

13.5

2.1

N3

12.5

0.3

12.2

0.3

N4

7.4

1.2

5.6

0.4

N5

9.7

0.4

12.7

0.8

N6

6.6

0.5

8.7

1.2

0.91

0

R Right ear anterior auricular muscle compound muscle action
potential (CMAP) before injections, R14 Right ear anterior auricular
CMAP 14 days after injections, L0 Left ear anterior auricular CMAP
before injections, L14 Left ear anterior auricular muscle CMAP
14 days after injections

Fig. 4 mRNA expression levels of the SNAP25 gene in the left andright ear anterior auricular muscles of all groups (G1, G2, G3, andG4)

rates of decrease in the amplitude of the compound muscle
action potential (CMAP) were calculated with the formulation ofRate of CMAP amplitude decrease
CMAP0  CMAP14
¼
 100
CMAP0
(CMAP0: CMAP before injections, CMAP14: 14 days
after injections)
As seen in Fig. 3, the mean CMAP amplitude reduction
rates in the right ears of Group 1 were significantly lower
than other groups (P = 0.004). No significant difference
was found regarding the left ears in the subjects of allgroups.
When we compared the left ear anterior auricular muscle groups with the right ear groups, we found no significant difference in the mRNA expression levels of the
SNAP25 gene (Fig. 4).

Discussion
Results
According to visual evaluation studies, 83 % of the rabbits
in Group I were classified as Grade 2 and Grade 3 ears
(Fig. 3). In the groups II, III, and IV, the number of thesubjects with Grade 2 and Grade 3 ears was 17, 33, and
17 %, respectively. When compared with the other groups,
right ears of the rabbits in Group 1 were in a significantly
higher position (P \ 0.05).
According to the results of the electroneuromyographic
studies, muscle action potential amplitudes at day 14 weresignificantly higher compared to the contralateral side
measurements in Group I and Group III (Table 1). The123

PRP applications have been performed in various areas anddifferent specialties of medicine especially for wound,bone, nerve and joint cartilage healing, skin rejuvenation,and fat graft survival [5–8]. Despite more recent clinicaluse, the proliferative effect of PRP was shown by Kim
et al. in 2011 [9]. They stated that PRP has a proliferative
effect on fibroblasts, which leads to an increase in type-1
collagen expression. However, the efficiency and reliabilityof mesotherapy applications are controversial [10,11].Taieb et al. reported high patient satisfaction threemonths after hyaluronic acid and mannitol mesotherapy[12]. Similarly, Lacarrubbba et al. found that hyaluronic
acid mesotherapy can positively affect the skin photo-aging

Aesth Plast Surg (2015) 39:134–140

process [13]. However, there are also contrary results insome other studies. El-Domyati et al. stated that no significant changes in skin were detected in clinical and histopathological evaluations following mesotherapy sessions[14]. Amin et al. reported ineffective results of hyaluronic
acid mesotherapy for skin rejuvenation [15].
Although mesotherapy is a cutaneous procedure, theoretically injected drugs or molecules have the potential toreach deeper structures such as muscles or joints. Therefore, mixtures of piroxicam, ketorolac, baclofen, diazepam,
buflomedil, and pentoxifylline have been used through themesotherapy route as a treatment option in osteoarthritis,
fibromyalgia, tendinitis, bursitis, and back pain [16]. So
theoretically intradermal injections of PRP can impact
underlying tissues or muscles. Therefore, intramuscularbotulinum toxin injections and intracutaneous PRP therapy
are able to interact in a combined skin rejuvenation session.
Platelets have three different types of granules: alpha,delta, and lambda granules. The alpha granules consist of
coagulation factors, growth factors, glycoproteins (fibronectin, vWF), proteoglycans, albumin, immunoglobulins,
and protease inhibitors (alpha-2 macroglobulin and alpha-2
antiplasmin). The content of the granules spreads to thesurrounding media when platelets are activated by matrix
components (collagen, laminin, and fibronectin), hormones(epinephrine and vasopressin), thrombin, thromboxane A2,
and calcium. The primary role of alpha-2 macroglobulin
and alpha-2 antiplasmin is inhibition of fibrinolysis throughthe inhibition of plasmin. Beside this major function,alpha-2 macroglobulin is also known as a general proteaseinhibitor and has an inhibitory effect on serine, cysteine,treonin, and aspartic proteases as well as metalloprotease[17]. When we consider that the active light chain portionof the BoNT-A has zinc metalloprotease activity on the
SNAP-25 protein, theoretically we can speculate that thisenzymatic interaction is a possible explanation for the
inhibitory effect of PRP on BoNT-A. There are few studies
about the inhibitory effect of alpha-2 macroglobulin on
different toxins. Vibrio vulnificus, an opportunistic humanpathogen, produces a 45-kDa zinc metalloprotease andcauses a typical hemorrhagic skin lesion. Miyoshi et al.
found that the toxin of V. vulnificus can be quickly inactivated by alpha-2 macroglobulin [18]. In another study,
Saidi et al. stated that alpha-2 macroglobulin could interact
with snake venom called lebetase and inhibit its fibrinolytic
metalloprotease activity [19].
PRP has been shown to enhance nerve healing due to its
growth factor content. Especially IGF, VEGF, TGF-b,
PDGF, and FGF- II play important roles in nerve regeneration by stimulation of axonal outgrowth and Schwann
cell proliferation and protein and lipid synthesis, which arenecessary for nerve regeneration [8, 20, 21]. The action of
BoNT-A ends by axonal sprouting, formation of new

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neuromuscular junctions, and recovery of the affected
neuromuscular junctions. Therefore, the growth factor
content of PRP may also interact with this regeneration
process and may lead to a decrease in the duration of
BoNT-A activity. A long-term study will be more appropriate to evaluate this effect of PRP on BoNT-A activity.
The anterior auricular muscle model was first introduced
by Jabor et al. in 2003 [22].They compared efficiency offreshly reconstituted and stored botulinum toxin. Before
the main study, they conducted a pilot study to determine
the BoNT-A dose for paralysis of the anterior auricular
muscle. They found that 2.5 U BoNT-A was the safest
minimum dose that could yield complete paralysis of themuscle. This model has also been used in several studies
after the initial study, yielding the advantages to perform
visual, electroneuromyographic, and muscle biopsy-based
studies [23, 24].
Toxin inhibitors target a variety of intoxication steps.
Binding, internalization, translocation, and enzymatic toxicity have been targets of candidate botulinum toxin inhibitors. Antibody-based treatments have been known as the
only antitoxin option until the early 2000 s. They caninactivate the toxin in circulation but the circulation intervalof the toxin is very short, and once the toxin is bound to thenerve terminal, antibodies were ineffective. The toxic
reaction step involving the Zn-metalloprotease activity on
one of the SNARE proteins has been the major focus ofattention over the last decade. The first irreversible inhibitor,
based on the benzylidenecyclopentenedione structure that
leads to covalent modification of the active site of botulinum
toxin type-A, was discovered by Capcova et al. in 2010 [25].
But still there are two important handicaps that need to besolved in the production of a botulinum antitoxin. One ofthese is the toxicity of the antitoxin itself and different level
efficacies of the antitoxins in in vivo and cellular-based
mediums. Toxicity and efficacy studies regarding botulinum
antitoxin are still ongoing.
Botulinum toxin injections can cause unintended local
side effects either from technical errors or from diffusion of
the botulinum toxin to muscles other than the targeted.
Overdose treatments can more likely result in lid ptosis,
ectropion, strabismus, lip dysfunction, dysphagia, browmalposition, or mask-face depending on the injection area
[26]. Solutions for accidental overdose BoNT-A treatments
are generally based on symptomatic treatments and they
aim to gain time until the effects of the injection decrease.
In our opinion, PRP can be used as an autologous andeasily accessible option for the treatment of botulinum
toxin overdose.
SNAP-25 mRNA expression was observed in all muscle
samples. No significant difference was found between
groups and the right/left side anterior auricular muscles ofrabbits. It can be assumed that maybe there was a

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difference in post-transcriptional or translational modification levels.

Conclusion
In conclusion, it seems that PRP has an inhibitory effect on
BoNT-A, when applied simultaneously. We suggest performing these two applications in separate sessions. We
could not define the exact mechanism behind the inhibitory
effect but alpha-2 macroglobulin may lead to this interaction between BoNT-A and PRP. More detailed molecularstudies can explain the mechanism of interaction andwhether PRP can decrease the duration of effect once theparalytic effect of botulinum toxin is totally achieved.
Acknowledgments This study was approved by the Gazi University
Ethical Committee for Experimental Research on Animals (Project
no: G.U. ET-43-6865) and was financially supported by the Gazi
University Research Fund (Project no: 01/2012-60).
Conflict of interest The authors declare that they have no conflictsof interest to disclose within the blinded manuscript.
Statement of Animal Rights All applicable institutional and/or
national guidelines for the care and use of animals were followed.

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