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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 by 51% in group B (p = 0.009) and 63% in group C rab-
type A (BTX-A) exerts a lipolytic effect by interfering bits (p = 0.009) compared to control animals.
with acetylcholine transmission at the cholinergic para- CONCLUSION: Our pilot animal study revealed a
sympathetic nerve endings. dose-dependent lipolytic effect of subcutaneous BTX-A
STUDY DESIGN: Fifteen male rabbits were divided into injection. (Anal Quant Cytol Histol 2010;32:186191)
3 equal groups: 1 control group (A) and 2 case groups (B
and C). The abdomens of all rabbits were divided into a Keywords: adipose tissue, botulinum toxin, lipoly-
3 3-square grid. The groups received 9 subcutaneous sis.
injections of 0.9% normal saline, 1 U BTX-A (group B)
and 2 U BTX-A (group C), respectively. Four weeks later Today obesity is one of the most problematic health
the entire grid was excised from the abdominal area. issues, not only in rich countries but also in devel-
Hematoxylin-eosinstained tissue was used for stereo- oping countries.1 Subcutaneous adiposity leads to
logic analysis to estimate cell surface and volume in 100 many important psychologic disturbances, such as
randomly selected cells. embarrassment, societal and occupational discrimi-
RESULTS: Gross thinning of subcutaneous fat and shat- nation, lower levels of self-acceptance2 and conse-
tering and disappearance of fat globules were seen in both quently impaired quality of life.3 Available weight-
case groups. Fat cell volume was reduced by 65% in loss therapies include medications such as lipase
group B (p = 0.009) and 77% in group C (p = 0.009) com- inhibitors and appetite suppressants, in addition to
pared to control animals. Fat cell surface also decreased 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, Shi-
raz 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 Shi-
raz, Iran (borhanihaghighi@yahoo.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

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


186 Analytical and Quantitative Cytology and Histology
ARTICLES
Volume 32, Number 4/August 2010 Lipolytic Effect of BTX 187

limited long-term efficacy have made pharmaco- 3 3-square grid (4.5 4.5 cm) of 9 injection sites.
logic treatment options disappointing.4 Subcuta- The upper injection sites were 2 cm below the di-
neous liposuction causes severe complications, aphragm to avoid diaphragmatic muscle paralysis.
such as thromboembolism, and is associated with In control group rabbits, 0.01 mL 0.9% normal
mortality rates as high as 1 per 5,000.5 saline was injected subcutaneously in each site. The
The autonomic nervous system plays an impor- vial of 500 U BTX-A was reconstituted with 5 mL of
tant role in controlling fat metabolism in adipose 0.9% normal saline, resulting in a dilution of 1 U of
tissue.6 Parasympathetic input has anabolic effects BTX-A in each 0.01 mL. The 2 case group rabbits re-
on adipose tissue by modulating its insulin sensi- ceived 9 subcutaneous injections of 1 U (group B) or
tivity and the metabolism of glucose and free fatty 2 U BTX-A (group C). The lethal dose of BTX-A for
acids,7 whereas sympathetic stimulations facilitates rabbits is 0.5 ng/kg,13 which means 10 U/kg.14 The
lipolysis in adipose tissue.8 total injection of 9 and 18 U BTX-A into the abdom-
Botulinum toxin (BTX), produced by Clostridium inal subcutaneous tissue of rabbits was chosen to be
botulinum, can interfere with acetylcholine release under lethal dose for rabbits weighing 2,500 100 g.
at the neuromuscular junctions and in the choliner- Four weeks later, all of the rabbits were anes-
gic parasympathetic and postganglionic sympa- thetized with ketamine 10% (100 mg/kg) and xyla-
thetic nervous system.9,10 On binding to the cholin- zine 2% (10 mg/kg). The entire grid, including skin,
ergic nerve ending membrane via its heavy chain, the subcutaneous adipose tissue and abdominal
the BTX-receptor complex is internalized by endo- wall muscle, was excised from the abdominal area.
cytosis. The neurotoxin is then translocated to the The animals muscular wall and skin were stitched
cytosol to cleave soluble NSF, N-ethyl maleimide- with chromic and silk sutures, respectively. All of
sensitive factor, attachment receptors (SNARE), the samples were divided into halves. Half of the
proteins essential for acetylcholine release.9-11 specimens were maintained in a fresh state and un-
We investigated the effects of subcutaneous BTX derwent frozen section at the center of the speci-
type A (BTX-A) injection on fat cell volume and sur- mens. They were then stained with oil red O stain-
face in the abdominal subcutaneous tissue of rab- ing. These slides were studied by light microscopy
bits using quantitative stereologic methods. to investigate qualitative changes that happened to
the subcutaneous adipose tissue of the rabbits.
Materials and Methods The other half of the specimens were fixed in 4%
The current study was conducted in the Compara- buffered formalin and then cut into small pieces.
tive Medicine Research Center, affiliated with These parts were randomly oriented and sectioned.
Shiraz University of Medical Sciences, Shiraz, We also cut the tissues in random orientations and
Southern Iran, from June 2008 to March 2009. Ex- positions and embedded them randomly in a paraf-
perimental animals were 15 male albino rabbits ob- fin block to achieve isotropic uniform sections,
tained from the Pasteur Institute of Iran. The rabbits as necessary for further stereologic work.15 Sec-
were homogenous in weight (2,500 100 g) and age tions with a 10-m-thickness were obtained. The
(6 months). BTX used was Dysport (Beaufour- sections were mounted on slides and stained with
Ipsen, Dreux, France). The rabbits were divided hematoxylin-eosin. Microscopic fields were select-
into 3 equal groups: group A (control group), group ed in each section in a random manner. The position
B (experimental group in which a 9-mouse unit of the first area was selected randomly outside of
BTX-A was injected), and group C (experimental the section, and the other areas were selected by
group in which an 18-mouse unit BTX-A was inject- moving the microscope stage in an equal interval
ed). Each group contained 5 rabbits. All investiga- along the X and Y directions of the stage using a
tors, including research assistants, pathologists and stage micrometer. A high numerical aperture, 100
a statistician, were completely blinded to drug and magnification (NA = 1.4) oil immersion lens was
placebo. used. Estimation of fat cell volume and surface was
This study was conducted according to the performed at the final magnification of 1,500 using
World Medical Association Declaration principles the method described by Cruz-Orive.16 This meth-
for animal studies, revised in October 200612 and od is an estimator of the number-weighted mean
was approved by the Ethics Committee of Shiraz particle volume and surface, meaning that cell se-
University of Medical Sciences. lection is done according to numerical density in a
The abdomen of all rabbits was divided into a 3-D space.16 To do this, the cells must be selected
188 Bagheri et al Analytical and Quantitative Cytology and Histology

with uniform random probability using the dis- the images of the tissue sections viewed with a light
sector principle.17 The optical dissector is a microscope. Briefly, 100 cells, whose nuclei did not
method in which the cells are sampled in thick sec- touch the left or inferior borders of each quadran-
tions observed with a light microscope. Using the gular frame, were qualified from each rabbit speci-
dissector, the particles are selected according to men for further estimation of number-weighted
their numerical density, not according to their mean volume and surface. Each sampled nucleus
shape, size and volume. In the sections with 10-m was adopted as the pivotal point. The uniform ran-
thickness, the optical section was set at a random dom test system of quadrangles with a predeter-
depth using a microcator (MT 12, Heidenhain, mined area (a) was laid on the image of the cell.
Traunreut, Germany), which can measure the thick- Through each pivotal point, a test line (bold lines in
ness, or z-axis, of the sections. Briefly, the first 3 m Figure 1) was drawn perpendicular to the axis join-
of the sections were ignored and sampling of the ing the points of the test system to the pivotal point
cells was performed in the next 5 m of the sections. (thin lines in Figure 1). The intercept lengths test
A personal computer and a monitor were connect- lines and the number of intersections (small white
ed to a color video camera mounted on top of the circles in Figure 1) with cells was estimated. The
microscope. By means of a stereologic software (de- equations V = aL and S = 2aI were used to estimate
signed at Shiraz University of Medical Sciences), a the volume (V) and surface (S) of the cells, where a
test system of quadrangles was superimposed on 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 depres-
sion, 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 BTX-
A. Subcutaneous BTX-A injection resulted in quali-
tative and quantitative changes in adipose tissue, as
reported below.

Qualitative Changes
Microscopic evaluation of the specimens under-
went frozen section and oil red O staining showed
that in abdominal subcutaneous adipose tissue of
the BTX-Atreated animals compared with the con-
trol group animals, the subcutaneous fat layer be-
came thinner, fat globules became smaller, frag-
ments of fat globules acquired irregular borders, fat
Figure 1 A test system of quadrangles with a determined area globules became shattered compared with the fat
per point (A) was laid on the image of the sampled fat cell. globules of the control group and fat globules in-
Through the pivotal point (nucleus), test lines (bold lines) were side the fat cells disappeared (Figure 2). The differ-
drawn perpendicular to the axis joining the points of the test
ence between the dose of BTX-A injected into the
system to the pivotal point (thin lines). The intercept lengths (L) of
the test lines and the number of intersections (I) (small white abdominal subcutaneous fat of the rabbits in groups
circles) with the cells were estimated. The volume (V) and surface B and C did not produce significant qualitative dif-
(S) of the cell and nucleus were estimated by V = aL and S = 2aI. ferences in the changes that occurred in the subcu-
Volume 32, Number 4/August 2010 Lipolytic Effect of BTX 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. A wealth of evidence reveals that obesity is asso-
ciated with increased morbidity and mortality.18
Quantitative Changes Obesity also induces severe psychosocial conse-
Fat cell volume was reduced by 65% in group B (p quences such as loss of self-confidence and social
= 0.009) and 77% in group C (p = 0.009) compared to discrimination.2 Current pharmacologic treat-
control animals. Fat cell surface also decreased by ments, such as orlistat and subitramine, and surgi-
51% in group B (p = 0.009) and 63% in group C rab- cal methods, including liposuction and gastric sta-
bits (p = 0.009) compared to control animals. Fat cell pling, have their own shortcomings in terms of
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
Table I Mean SD of Fat Cell Surfaces and Volumes in the
animals (p = 0.03) (Table I). Control and BTX-ATreated Rabbits
Discussion Cell surface Cell volume
Group (m2) (m3)
Our study revealed a dose-dependent lipolytic ef-
fect of subcutaneous BTX-A injection in male albino Control (n = 5) 28,744 2,839 353,684 60.771
Group B (n = 5) 13,978 3,281a 123,545 44,533a
rabbits. Qualitatively, gross thinning of the subcu- Group C (n = 5) 10,434 1,272a,c 81,329 14,739a,b
taneous fat layer and shattering and disappearance
ap 0.009, BTX-Atreated animals (1 or 2 U at each injection site) vs. con-
of fat globules were seen in both case groups. In- trol.
jecting BTX-A also induced a statistically significant bp 0.05, group C (2 U at each injection site) vs. group B (1 U at each in-

jection site).
decrease in the fat cell surface and volume as meas- cp 0.03, group C (2 U at each injection site) vs. group B (1 U at each in-

ured stereologically. jection site).


190 Bagheri et al Analytical and Quantitative Cytology and Histology

safety and efficacy.4,5,19-21 Tumescent liposuction mans. The effects of BTX are transitory, lasting for
has been described as a safe procedure but is asso- variable periods ranging from 3 months in spastic
ciated with several complications, such as postop- muscles up to 2 years for sweat glands.10,30 Eventu-
erative pain, syncope, edema, ecchymoses, panni- ally, nerve terminals sprout and new synaptic con-
culitis and fat necrosis in diabetic patients, seroma tacts are formed, so parasympathetic nerve system
formation and irregularity and asymmetry of the functioning can be expected to recover after BTX in-
operated area.22 The efficacy of mesotherapy, as an jection.9 Nevertheless, the duration of the lipolytic
alternative to liposuction, has also been questioned, effects of subcutaneous BTX-A injection should be
and complications such as bruising, edema, skin investigated in further studies.
necrosis, atypical mycobacterial infections, ecchy- In summary, the potential of subcutaneous BTX-
moses and hematomas have been reported.23,24 A injection as an effective treatment for abdominal
The autonomic nervous system modulates lipol- adiposity merits further research.
ysis and lipogenesis by changing local insulin sen-
sitivity and expression levels of adipokine in adi- Acknowledgments
pose tissue and by regulating fat cell numbers.6 We thank Dr. Tanide and Mr. Alizade of the Com-
-Adrenergic sympathetic fibers have lipolytic ac- parative Medicine Research Center, Shiraz Univer-
tion, and parasympathetic fibers have anabolic ef- sity of Medical Sciences, for their help with this
fects.8,25,26 study. We are also grateful to those who offered en-
The injection of BTX causes transient denervation couragement and additional details, especially in
of cholinergic parasympathetic fibers.10 This effect the Histomorphometry and Stereological Research
has been used to treat sialorrhea.27 The lipolytic ef- Center, Shiraz University of Medical Sciences. We
fect of BTX via inhibition of some parasympathetic also thank K. Shashok (Author AID in the Eastern
anabolic effects was first hypothesized by Lim and Mediterranean) for improving the English in parts
Seet.28 They postulated that BTX can be injected of the manuscript.
into subcutaneous deposits of fat: in the buttocks,
thighs or abdominal apron, for cosmetic purposes. References
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