Analytical and Quantitative Cytology and Histology

A Pilot Study on Lipolytic Effect of

Subcutaneous Botulinum Toxin Injection in
Rabbits
Mansooreh Bagheri, Behnam Moein Jahromi, Mandana Bagheri, M.D.,
Afshin Borhani Haghighi, M.D., Ali Noorafshan, Ph.D., Perikala Vijai Kumar, M.D.,
and Gholamhossein Ranjbar Omrani, M.D.

OBJECTIVE: To determine whether botulinum toxin

type A (BTX-A) exerts a lipolytic effect by interfering
with acetylcholine transmission at the cholinergic parasympathetic nerve endings.
STUDY DESIGN: Fifteen male rabbits were divided into
3 equal groups: 1 control group (A) and 2 case groups (B
and C). The abdomens of all rabbits were divided into a
3 3-square grid. The groups received 9 subcutaneous
injections of 0.9% normal saline, 1 U BTX-A (group B)
and 2 U BTX-A (group C), respectively. Four weeks later
the entire grid was excised from the abdominal area.
Hematoxylin-eosinstained tissue was used for stereologic analysis to estimate cell surface and volume in 100
randomly selected cells.
RESULTS: Gross thinning of subcutaneous fat and shattering and disappearance of fat globules were seen in both
case groups. Fat cell volume was reduced by 65% in
group B (p = 0.009) and 77% in group C (p = 0.009) compared to control animals. Fat cell surface also decreased

by 51% in group B (p = 0.009) and 63% in group C rabbits (p = 0.009) compared to control animals.
CONCLUSION: Our pilot animal study revealed a
dose-dependent lipolytic effect of subcutaneous BTX-A
injection. (Anal Quant Cytol Histol 2010;32:186191)
Keywords: adipose tissue, botulinum toxin, lipolysis.
Today obesity is one of the most problematic health
issues, not only in rich countries but also in developing countries.1 Subcutaneous adiposity leads to
many important psychologic disturbances, such as
embarrassment, societal and occupational discrimination, lower levels of self-acceptance2 and consequently impaired quality of life.3 Available weightloss therapies include medications such as lipase
inhibitors and appetite suppressants, in addition to
surgical interventions. Unpleasant side effects and

From the Student Research Center, Research Center for Traditional Medicine and History of Medicine, Transgenic Technology Reseach
Center, Endocrine and Metabolism Research Center, and Departments of Pathology, Neurology, Anatomy and Internal Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Ms. Bagheri and Mr. Moein Jahromi are Medical Students, Student Research Center.
Dr. Bagheri is Resident, Research Center for Traditional Medicine and History of Medicine, and Department of Pathology.
Dr. Borhani Haghighi is Associate Professor, Transgenic Technology Research Center and Department of Neurology.
Dr. Noorafshan is Professor, Department of Anatomy.
Dr. Kumar is Professor, Department of Pathology.
Dr. Omrani is Professor, Endocrine and Metabolism Research Center, and Department of Internal Medicine.
This study was conducted with grant No. 3646 of the Research Council of Shiraz University of Medical Sciences, Shiraz, Iran.
Address correspondence to: Afshin Borhani Haghighi, M.D., Department of Neurology, Shiraz University of Medical Sciences, 71345 Shiraz, Iran (borhanihaghighi@yahoo.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

186

0884-6812/10/3204-0186/$18.00/0 Science Printers and Publishers, Inc.

Analytical and Quantitative Cytology and Histology

limited long-term efficacy have made pharmacologic treatment options disappointing.4 Subcutaneous liposuction causes severe complications,
such as thromboembolism, and is associated with
mortality rates as high as 1 per 5,000.5
The autonomic nervous system plays an important role in controlling fat metabolism in adipose
tissue.6 Parasympathetic input has anabolic effects
on adipose tissue by modulating its insulin sensitivity and the metabolism of glucose and free fatty
acids,7 whereas sympathetic stimulations facilitates
lipolysis in adipose tissue.8
Botulinum toxin (BTX), produced by Clostridium
botulinum, can interfere with acetylcholine release
at the neuromuscular junctions and in the cholinergic parasympathetic and postganglionic sympathetic nervous system.9,10 On binding to the cholinergic nerve ending membrane via its heavy chain,
the BTX-receptor complex is internalized by endocytosis. The neurotoxin is then translocated to the
cytosol to cleave soluble NSF, N-ethyl maleimidesensitive factor, attachment receptors (SNARE),
proteins essential for acetylcholine release.9-11
We investigated the effects of subcutaneous BTX
type A (BTX-A) injection on fat cell volume and surface in the abdominal subcutaneous tissue of rabbits using quantitative stereologic methods.
Materials and Methods
The current study was conducted in the Comparative Medicine Research Center, affiliated with
Shiraz University of Medical Sciences, Shiraz,
Southern Iran, from June 2008 to March 2009. Experimental animals were 15 male albino rabbits obtained from the Pasteur Institute of Iran. The rabbits
were homogenous in weight (2,500 100 g) and age
(6 months). BTX used was Dysport (BeaufourIpsen, Dreux, France). The rabbits were divided
into 3 equal groups: group A (control group), group
B (experimental group in which a 9-mouse unit
BTX-A was injected), and group C (experimental
group in which an 18-mouse unit BTX-A was injected). Each group contained 5 rabbits. All investigators, including research assistants, pathologists and
a statistician, were completely blinded to drug and
placebo.
This study was conducted according to the
World Medical Association Declaration principles
for animal studies, revised in October 200612 and
was approved by the Ethics Committee of Shiraz
University of Medical Sciences.
The abdomen of all rabbits was divided into a

Lipolytic Effect of BTX

187

3 3-square grid (4.5 4.5 cm) of 9 injection sites.

The upper injection sites were 2 cm below the diaphragm to avoid diaphragmatic muscle paralysis.
In control group rabbits, 0.01 mL 0.9% normal
saline was injected subcutaneously in each site. The
vial of 500 U BTX-A was reconstituted with 5 mL of
0.9% normal saline, resulting in a dilution of 1 U of
BTX-A in each 0.01 mL. The 2 case group rabbits received 9 subcutaneous injections of 1 U (group B) or
2 U BTX-A (group C). The lethal dose of BTX-A for
rabbits is 0.5 ng/kg,13 which means 10 U/kg.14 The
total injection of 9 and 18 U BTX-A into the abdominal subcutaneous tissue of rabbits was chosen to be
under lethal dose for rabbits weighing 2,500 100 g.
Four weeks later, all of the rabbits were anesthetized with ketamine 10% (100 mg/kg) and xylazine 2% (10 mg/kg). The entire grid, including skin,
the subcutaneous adipose tissue and abdominal
wall muscle, was excised from the abdominal area.
The animals muscular wall and skin were stitched
with chromic and silk sutures, respectively. All of
the samples were divided into halves. Half of the
specimens were maintained in a fresh state and underwent frozen section at the center of the specimens. They were then stained with oil red O staining. These slides were studied by light microscopy
to investigate qualitative changes that happened to
the subcutaneous adipose tissue of the rabbits.
The other half of the specimens were fixed in 4%
buffered formalin and then cut into small pieces.
These parts were randomly oriented and sectioned.
We also cut the tissues in random orientations and
positions and embedded them randomly in a paraffin block to achieve isotropic uniform sections,
as necessary for further stereologic work.15 Sections with a 10-m-thickness were obtained. The
sections were mounted on slides and stained with
hematoxylin-eosin. Microscopic fields were selected in each section in a random manner. The position
of the first area was selected randomly outside of
the section, and the other areas were selected by
moving the microscope stage in an equal interval
along the X and Y directions of the stage using a
stage micrometer. A high numerical aperture, 100
magnification (NA = 1.4) oil immersion lens was
used. Estimation of fat cell volume and surface was
performed at the final magnification of 1,500 using
the method described by Cruz-Orive.16 This method is an estimator of the number-weighted mean
particle volume and surface, meaning that cell selection is done according to numerical density in a
3-D space.16 To do this, the cells must be selected

ARTICLES

Volume 32, Number 4/August 2010

188

Bagheri et al

with uniform random probability using the dissector principle.17 The optical dissector is a
method in which the cells are sampled in thick sections observed with a light microscope. Using the
dissector, the particles are selected according to
their numerical density, not according to their
shape, size and volume. In the sections with 10-m
thickness, the optical section was set at a random
depth using a microcator (MT 12, Heidenhain,
Traunreut, Germany), which can measure the thickness, or z-axis, of the sections. Briefly, the first 3 m
of the sections were ignored and sampling of the
cells was performed in the next 5 m of the sections.
A personal computer and a monitor were connected to a color video camera mounted on top of the
microscope. By means of a stereologic software (designed at Shiraz University of Medical Sciences), a
test system of quadrangles was superimposed on

Analytical and Quantitative Cytology and Histology

the images of the tissue sections viewed with a light

microscope. Briefly, 100 cells, whose nuclei did not
touch the left or inferior borders of each quadrangular frame, were qualified from each rabbit specimen for further estimation of number-weighted
mean volume and surface. Each sampled nucleus
was adopted as the pivotal point. The uniform random test system of quadrangles with a predetermined area (a) was laid on the image of the cell.
Through each pivotal point, a test line (bold lines in
Figure 1) was drawn perpendicular to the axis joining the points of the test system to the pivotal point
(thin lines in Figure 1). The intercept lengths test
lines and the number of intersections (small white
circles in Figure 1) with cells was estimated. The
equations V = aL and S = 2aI were used to estimate
the volume (V) and surface (S) of the cells, where a
is the area per test point (here 665.64 m2), L is the
sum of the intercept lengths in each sampled cell
and I is the sum of the number of intersections of the
vertices with the cell borders.
Statistical Analysis
The data were compared with the Mann-Whitney U
test, and p < 0.05 was considered significant.
Results
None of the animals developed respiratory depression, and none died during the study. The feeding
and daily food intake of both case groups of rabbits
were the same as the control group, and we did not
observe any differences in daily food intake among
groups after injecting case group rabbits with BTXA. Subcutaneous BTX-A injection resulted in qualitative and quantitative changes in adipose tissue, as
reported below.
Qualitative Changes

Figure 1 A test system of quadrangles with a determined area

per point (A) was laid on the image of the sampled fat cell.
Through the pivotal point (nucleus), test lines (bold lines) were
drawn perpendicular to the axis joining the points of the test
system to the pivotal point (thin lines). The intercept lengths (L) of
the test lines and the number of intersections (I) (small white
circles) with the cells were estimated. The volume (V) and surface
(S) of the cell and nucleus were estimated by V = aL and S = 2aI.

Microscopic evaluation of the specimens underwent frozen section and oil red O staining showed
that in abdominal subcutaneous adipose tissue of
the BTX-Atreated animals compared with the control group animals, the subcutaneous fat layer became thinner, fat globules became smaller, fragments of fat globules acquired irregular borders, fat
globules became shattered compared with the fat
globules of the control group and fat globules inside the fat cells disappeared (Figure 2). The difference between the dose of BTX-A injected into the
abdominal subcutaneous fat of the rabbits in groups
B and C did not produce significant qualitative differences in the changes that occurred in the subcu-

Lipolytic Effect of BTX

Volume 32, Number 4/August 2010

189

Figure 2 Sections from abdominal subcutaneous fat tissue of rabbits. (A) Thinning of fat layer in BTX-Atreated rabbits (right) compared
with control (left) in low power light microscopy. (B) Thinning of fat layer in BTX-Atreated rabbits (right) compared with control (left) in
high power light microscopy. (C) Small fat globules in BTX-Atreated rabbits (right) compared with control (left). (D) Irregular fragments of
fat globules in BTX-Atreated rabbits (right) in comparison with regular round fat globules in control group (left). (E) Shattered fat globules
in BTX-Atreated rabbits (right) compared with intact fat globules of control group (left). (F) Disappearance of fat globules inside the fat cell
in BTX-Atreated rabbits (right) comparing with the normal fat cell having good fat globules (left) (oil red O, A, 320; B, C, D, and E 900;
F, 1,200).

taneous fat layer of these 2 groups of animals.

Quantitative Changes
Fat cell volume was reduced by 65% in group B (p
= 0.009) and 77% in group C (p = 0.009) compared to
control animals. Fat cell surface also decreased by
51% in group B (p = 0.009) and 63% in group C rabbits (p = 0.009) compared to control animals. Fat cell
volume in group C was reduced by 34% compared
to group B animals (p = 0.05). The fat cell surface in
group C was reduced by 25% compared to group B
animals (p = 0.03) (Table I).

A wealth of evidence reveals that obesity is associated with increased morbidity and mortality.18
Obesity also induces severe psychosocial consequences such as loss of self-confidence and social
discrimination.2 Current pharmacologic treatments, such as orlistat and subitramine, and surgical methods, including liposuction and gastric stapling, have their own shortcomings in terms of

Table I Mean SD of Fat Cell Surfaces and Volumes in the

Control and BTX-ATreated Rabbits

Discussion
Our study revealed a dose-dependent lipolytic effect of subcutaneous BTX-A injection in male albino
rabbits. Qualitatively, gross thinning of the subcutaneous fat layer and shattering and disappearance
of fat globules were seen in both case groups. Injecting BTX-A also induced a statistically significant
decrease in the fat cell surface and volume as measured stereologically.

Group
Control (n = 5)
Group B (n = 5)
Group C (n = 5)
ap 0.009,

Cell surface
(m2)

Cell volume
(m3)

28,744 2,839
13,978 3,281a
10,434 1,272a,c

353,684 60.771
123,545 44,533a
81,329 14,739a,b

BTX-Atreated animals (1 or 2 U at each injection site) vs. control.

bp 0.05, group C (2 U at each injection site) vs. group B (1 U at each injection site).
cp 0.03, group C (2 U at each injection site) vs. group B (1 U at each injection site).

190

Bagheri et al

safety and efficacy.4,5,19-21 Tumescent liposuction

has been described as a safe procedure but is associated with several complications, such as postoperative pain, syncope, edema, ecchymoses, panniculitis and fat necrosis in diabetic patients, seroma
formation and irregularity and asymmetry of the
operated area.22 The efficacy of mesotherapy, as an
alternative to liposuction, has also been questioned,
and complications such as bruising, edema, skin
necrosis, atypical mycobacterial infections, ecchymoses and hematomas have been reported.23,24
The autonomic nervous system modulates lipolysis and lipogenesis by changing local insulin sensitivity and expression levels of adipokine in adipose tissue and by regulating fat cell numbers.6
-Adrenergic sympathetic fibers have lipolytic action, and parasympathetic fibers have anabolic effects.8,25,26
The injection of BTX causes transient denervation
of cholinergic parasympathetic fibers.10 This effect
has been used to treat sialorrhea.27 The lipolytic effect of BTX via inhibition of some parasympathetic
anabolic effects was first hypothesized by Lim and
Seet.28 They postulated that BTX can be injected
into subcutaneous deposits of fat: in the buttocks,
thighs or abdominal apron, for cosmetic purposes.
There is evidence that lipolysis occurs after
parasympathetic denervation. In a study in rats,
vagotomy was linked to fat degradation in obese
animals.29 It was also shown that fat padspecific
vagotomy strongly reduced the insulin-dependent
uptake of glucose and free fatty acids in adipose tissues, whereas the activity of lipase, a catabolic enzyme sensitive to hormone activity, increased.7
Our animal study provides the first evidence that
a lipolytic effect of subcutaneous injections of BTXA in the abdominal area is probable rather than fat
chance. This lipolytic effect has 2 implications: (1)
a subcutaneous BTX-A injection might be used as a
method of melting fat for body sculpting and (2)
lipoatrophy may be an undesired side effect of BTXA injection for cosmetic or therapeutic purposes, especially for facial treatments. It is also important to
emphasize that although subcutaneous BTX-A injection can be effective for regional lipolysis, it does
not reduce visceral adiposity and therefore cannot
decrease the morbidity and mortality induced by
obesity.
One of the shortcomings of our study was the
small number of animals used, so larger studies in
animal models and research are needed in models
that more accurately reproduce conditions in hu-

Analytical and Quantitative Cytology and Histology

mans. The effects of BTX are transitory, lasting for

variable periods ranging from 3 months in spastic
muscles up to 2 years for sweat glands.10,30 Eventually, nerve terminals sprout and new synaptic contacts are formed, so parasympathetic nerve system
functioning can be expected to recover after BTX injection.9 Nevertheless, the duration of the lipolytic
effects of subcutaneous BTX-A injection should be
investigated in further studies.
In summary, the potential of subcutaneous BTXA injection as an effective treatment for abdominal
adiposity merits further research.
Acknowledgments
We thank Dr. Tanide and Mr. Alizade of the Comparative Medicine Research Center, Shiraz University of Medical Sciences, for their help with this
study. We are also grateful to those who offered encouragement and additional details, especially in
the Histomorphometry and Stereological Research
Center, Shiraz University of Medical Sciences. We
also thank K. Shashok (Author AID in the Eastern
Mediterranean) for improving the English in parts
of the manuscript.
References
1. Prentice AM: The emerging epidemic of obesity in developing countries. Int J Epidemiol 2006;35:9399
2. Carr D, Friedman MA: Is obesity stigmatizing? Body weight,
perceived discrimination, and psychological well-being in
the United States. J Health Soc Behav 2005;46:244259
3. Villareal DT, Apovian CM, Kushner RF, Klein S: Obesity in
older adults: Technical review and position statement of the
American Society for Nutrition and NAASO, The Obesity
Society. Obes Res 2005;13:18491863
4. Sidhaye A, Cheskin LJ: Pharmacologic treatment of obesity.
Adv Psychosom Med 2006;27:4252
5. Grazer FM, de Jong RH: Fatal outcomes from liposuction:
Census survey of cosmetic surgeons. Plast Reconstr Surg
2000;105:436446
6. Romijn JA, Fliers E: Sympathetic and parasympathetic innervation of adipose tissue: Metabolic implications. Curr
Opin Clin Nutr Metab Care 2005;8:440444
7. Kreier F, Fliers E, Voshol PJ, Van Eden CG, Havekes LM,
Kalsbeek A, Van Heijningen CL, Sluiter AA, Mettenleiter TC,
Romijn JA, Sauerwein HP, Buijs RM: Selective parasympathetic innervation of subcutaneous and intra-abdominal fat:
Functional implications. J Clin Invest 2002;110:12431250
8. Fliers E, Romijn JA, Sauerwein HP, Kalsbeek A, Kreier F,
Buijs RM: Adipose tissue: An innervated endocrine gland.
Ned Tijdschr Geneeskd 2002;146:19761979
9. Simpson LL: Identification of the major steps in botulinum
toxin action. Annu Rev Pharmacol Toxicol 2003;44:167193
10. Jankovic J: Botulinum toxin in clinical practice. J Neurol

Volume 32, Number 4/August 2010

Neurosurg Psychiatry 2004;75:951957

11. Fon EA, Edwards RH: Molecular mechanisms of neurotransmitter release. Muscle Nerve 2001;24:581601
12. World Medical Association Statement on Animal Use in Biomedical Research. Available at http://www.wma.net/e/
policy/a18.htm. Adopted in September 1989, revised in October 2006
13. Gill DM: Bacterial toxins: A table of lethal amounts. Microbiol Rev 1982;46:8694
14. Patocka J, Splino M: Botulinum toxin: From poison to medicinal agent. ASA Newslett 2002;88:1419
15. Gundersen HJ, Bagger P, Bendtsen TF, Evans SM, Korbo L,
Marcussen N, Mller A, Nielsen K, Nyengaard JR, Pakkenberg B, Srensen FB, Vesterby A, West MJ: The new stereological tools: Disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and
diagnosis. APMIS 1988;96:857881
16. Cruz-Orive LM: A new stereological principle for test lines
in three-dimensional space. J Microsc 2005;219:1828
17. Gundersen HJ: The nucleator. J Microsc 1988;151:321
18. Ness-Abramof R, Nabriski D, Apovian CM: Medical therapy
for obesity: Present and future. Isr Med Assoc J 2004;6:760
765
19. Flageul G, Elbaz JS, Karcenty B: Complications of plastic surgery of the abdomen. Ann Chir Plast Esthet 1999;44:497505
20. Livingston EH: Complications of bariatric surgery. Surg Clin
North Am 2005;85:853868
21. Benotti PN, Wood GC, Rodriguez H, Carnevale N, Liriano E:
Perioperative outcomes and risk factors in gastric surgery

Lipolytic Effect of BTX

191

for morbid obesity: A 9-year experience. Surgery 2006;139:

340346
22. Jayashree V, Mysore V: Microcannular tumescent liposuction. Indian J Dermatol Venereol Leprol 2007;73:377383
23. Vedamurthy M: Mesotherapy. Indian J Dermatol Venereal
Leprol 2007;73:6062
24. Matarasso A, Pfeifer TM: Mesotherapy for body contouring.
Plast Reconstr Surg 2005;115:14201424
25. Berthoud H, Fox EA, Neuhuber WL: Vagaries of adipose tissue innervations. Am J Physiol Regul Integr Comp Physiol
2006;291:12401242
26. Fliers E, Kreier F, Voshol PJ, Havekes LM, Sauerwein HP,
Kalsbeek A, Buijs RM, Romijn JA: White adipose tissue: Getting nervous. J Neuroendocrinol 2003;15:10051010
27. Giess R, Naumann M, Werner E, Riemann R, Beck M, Puls I,
Reiners C, Toyka KV: Injections of botulinum toxin A into
the salivary glands improve sialorrhea in amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry 2000;69:121123
28. Lim E, Seet R: Botulinum toxin injections to reduce adiposity: Possibility or fat chance? Med Hypotheses 2006;67:1086
1089
29. Balbo SL, Mathias PC, Bonfleur ML, Alves HF, Siroti FJ,
Monteiro OG, Ribeiro FB, Souza AC: Vagotomy reduces obesity in MSG-treated rats. Res Commun Mol Pathol Pharmacol 2000;108:291296
30. Fve A, Decq P, Filipetti P, Kravel Y: Treatment of spasticity with injections of botulinum toxin: Review of the literature. Neurochirurgie 1998;44:192196