CARDIOVASCULAR JOURNAL OF AFRICA • Volume 25, No 3, May/June 2014
AFRICA
101
The surgical procedure was performed sterilely. After a
midline muscle and skin incision was made over the sternum,
the xiphoid process was carefully detached from the sternal part
of the diaphragm. A median sternotomy was then performed; the
median incision went down from the xiphoid process towards
the jugular notch of the sternum exactly along the midline of the
sternum so that injury to the parietal pleura was avoided. Sternal
retractors were used to spread the sternal edges and maintain
surgical exposure. The epicardium and parietal pericardium
related to the right ventricle atrium, and right and left ventricle
were abraded with 10 vertically reciprocal movements of dry
gauze in order to create local inflammation.
13
The rabbits were divided into two groups: the Ankaferd
group was treated with a sponge that had been soaked in a 2-ml
concentration ofABS solution (Ankaferd blood stopper
®
ampoule,
2 ml, Istanbul, Turkey) and applied over the abraded epicardium
for five minutes (
n
=
8). The sponge was then removed. The
abraded areas of the epicardium were irrigated immediately with
enough saline to dispose of the remaining ABS. In the control
group, the sponge was soaked in a 0.9% isotonic NaCl solution
(serum fizyolojik 0.9% NaCl, 5 ml/ampoule, Adeka, Turkey) and
was applied to the surface of the abraded epicardium for five
minutes (
n
=
8). The sponge was then removed. The investigators
were blinded during the application of Ankaferd or saline.
The sternum was closed with three interrupted sutures using
3-0 nylon and a needle with a tapered point. The muscle layers
and skin were then closed with continuous sutures using 4-0
nylon and a cutting needle. The rabbits were allowed to recover.
During the surgical procedure, all rabbits exhibited spontaneous
respiration and loss of the pedal reflex.
The rabbits were sacrificed two weeks after surgery with a
lethal dose of pentobarbital (150 mg/kg) (Nembutol, IE Ulagay,
Istanbul, Turkey). The heart and pericardium were removed
en
bloc
. Specimens were fixed in 10% formaldehyde, embedded in
paraffin and sectioned into 4-
µ
m slices, which were stained with
haematoxylin and eosin to assess the inflammatory reaction and
degree of fibrosis, and to check for remnants of the pericardial
substitute in the two groups.
Macroscopic examination
The heart and pericardium were removed with the anterior
chest wall
en bloc
. The severity of pericardial adhesions and
visibility of coronary vessels were evaluated by the same two
blinded observers and scored. The following qualitative grading
system was used to evaluate the tenacity of the adhesions: 0
=
no adhesions; 1
=
mild adhesions (transparent filmy adhesions
separable by lifting the pericardium from the myocardium
without dissection); 2
=
moderate adhesions (fibrous and easily
separated by blunt dissection); 3
=
severe adhesions (thick,
requiring aggressive blunt dissection); 4
=
very severe adhesions
(multiple thick adhesions requiring aggressive dissection that
damaged adherent tissue).
14
In addition, another grading system
was used to evaluate the visibility of the coronary arteries: 0
=
clearly visible, 1
=
blurred, 2
=
completely obscured.
6
Light microscopic examination
After the macroscopic scoring, the paraffin-embedded heart
tissues (segments of the pericardium and heart from the site of
abrasion, which had been marked with a prolene stitch) were
cut into 4-
µ
m thick sections and stained with haematoxylin
and eosin. Histopathological evaluation was performed by one
pathologist who was blinded to the study groups.
The severity of the inflammatory reaction was based on
quantification of the inflammatory cells (i.e. neutrophils, plasma
cells, lymphocytes) and inflammatory foci. The scoring schemes
of Lu
et al
.
15
were used to grade inflammation (0
=
no cell
infiltration; 1
=
sparse, focal infiltration of lymphocytes and
plasma cells; 2
=
focal infiltration of neutrophils, plasma cells and
lymphocytes; and 3
=
diffuse infiltration of neutrophils, plasma
cells and lymphocytes), and fibrosis (0
=
no fibrous reaction; 1
=
sparse, focal fibrous connective tissue, hyalinisation and fibrin
deposition; 2
=
a thin layer of focal fibrous connective tissue,
hyalinisation and fibrin deposition; and 3
=
a thick layer of focal
fibrous connective tissue, hyalinisation and fibrin deposition).
Statistical analysis
The sample size of our study was calculated with G*Power
(G*Power Ver. 3.00.10, Franz Faul, Üniversität Kiel, Germany,
. de/aap/projects/gpower/)
statistical packages. The required sample size for 80% power,
α
=
0.05 type I error,
β =
0.20 type II error, and
f
=
0.70 effect size
was calculated as 16, including eight New Zealand white rabbits
in each group. To protect the study from potential loss to follow
up, one more rabbit was included in each group and the study
was completed with a sample size of 18.
Data coding and statistical analyses were conducted with
SPSS (version 15; SPSS Inc, Chicago, IL, USA). Following the
entering of the rabbits’ data into the computer, all the necessary
diagnostic checks and corrections were performed. Conformity
of the measured values to normal distribution was examined
graphically and using a Shapiro-Wilks test.
In presenting descriptive statistics, numbers and percentages
were used for categorical variables, and medians and ranges were
used for non-normally distributed variables. The Mann-Whitney
U
-test was used to compare the median values of the groups.
The chi-square test was performed to evaluate the difference in
the proportion of rabbits in the groups. Groups that were close to
each other were combined and a 2 × 2 table chi-square test was
created. The likelihood ratio test was used for the comparison
of these groups. The two-tailed test of
p
≤
0
.
05 was considered
statistically significant.
Results
All animals tolerated the procedurewith no apparent postoperative
complications. Results were analysed in terms of macro- and
microscopic findings.
Macroscopic findings
A total of 16 rabbits were evaluated for grading of pericardial
adhesions by macroscopic findings. In six cases in the control
group and one case in the Ankaferd group, pericardial adhesions
were split by blunt dissection (Fig. 1A). By contrast, seven
of the Ankaferd group and two of the control group were
associated with tight adherences to the sternum and the rest of
the pericardium, requiring sharp dissection (Fig. 1B).
