original papers - Advances in Clinical and Experimental Medicine

original papers
Adv Clin Exp Med 2011, 20, 6, 711–715
ISSN 1230-025X
© Copyright by Wroclaw Medical University
Baris D. Yildiz1, Serdar Topaloglu2, Fatih M. Avsar1, 3, Ibrahim Sakcak1,
Barlas Sulu3, Ayse F. Avsar4
The Effects of Anti-Adhesive Low Molecular Weight
Na-Hyaluronate and Octreotide on Tissue Strength*
Wpływ środków przeciwzrostowych hialuronianu sodu o małej masie
cząsteczkowej i oktreotydu na wytrzymałość tkanek
Sixth Department of Surgery, Ankara Numune Training and Research Hospital, Ankara, Turkey
Department of General Surgery, Karadeniz Technical University Faculty of Medicine, Trabzon, Turkey
3
Department of General Surgery, Kafkas University Faculty of Medicine, Kars, Turkey
4
Department of Obstetrics and Gynecology, Atatürk Training and Research Hospital, Ankara, Turkey
1
2
Abstract
Background. The use of locally administered agents to prevent intraperitoneal adhesions is a popular research
topic. The presumed effects of these agents on tissue matrix limit their use.
Objectives. To determine the effects of low-molecular-weight Na-hyaluronate and octreotide on tissue tensile
strength and hydroxyproline levels.
Materials and Methods. Sprague-Dawley rats were anesthetized with ketamine and 10 longitudinal incisions, 2 to
3 cm in length each, were made in the right parietal peritoneum. A 2 × 1 cm peritoneal layer was excised from
the left abdominal wall during the first laparotomy. Before closing the abdomen, intraperitoneal irrigation was
performed, either with Na-hyaluronate solution at a concentration of 0.25 mg/kg (n = 10) or with octreotide solution at a dosage of 5 µg/ml (n = 10). On the 14th day after the operation, the abdomen was opened and abdominal
adhesions were examined. Part of the abdominal wall (4 × 4 cm in size, including midline incision) was removed
to measure the tensile strength and hydroxyproline level of the tissue on the suture line.
Results. Low-molecular-weight Na-hyaluronate significantly reduced the grade of adhesion (p = 0.005). Octreotide
administration inhibited adhesion formation, although not to a statistically significant degree (p > 0.05). Tissue
tensile strength and hydroxyproline levels were not affected by treatment with either LMW Na-hyaluronate or
octreotide (p > 0.05).
Conclusions. Na-hyaluronate and octreotide, which are well known anti-adhesive agents, do not exert any negative
effects either on tissue tensile strength or hydroxyproline level (Adv Clin Exp Med 2011, 20, 6, 711–715).
Key words: hyaluronate, octreotide, abdominal surgery, adhesion, tissue strength.
Streszczenie
Wprowadzenie. Zapobieganie zrostom w otrzewnej za pomocą miejscowo podawanych środków jest popularnym
tematem badań. Przypuszczalny wpływ tych leków na macierz tkankową może ograniczyć ich zużycie.
Cel pracy. Określenie wpływu hialuronianu sodu o małej masie cząsteczkowej i oktreotydu na wytrzymałość tkanek na rozciąganie i stężenie hydroksyproliny.
Materiał i metody. Szczury szczepu Sprague-Dawley znieczulono ketaminą i wykonano 10 nacięć wzdłużnych po
2–3 cm długości na prawej otrzewnej ściennej. Warstwę 2 × 1 cm otrzewnej wycięto z lewej ściany jamy brzusznej
podczas pierwszej laparotomii. Przed zamknięciem jamy brzusznej podano dootrzewnowo roztwór hialuronianu
sodu w stężeniu 0,25 mg / kg (n = 10) i roztwór oktreotydu w dawce 5 μg/ ml (n = 10). 14. dnia po operacji otwarto brzuch zwierząt i zbadano obecność zrostów w jamie brzusznej. Pobrano skrawek ściany brzucha o wielkości
4 × 4 cm, w tym nacięcie na linii środkowej, do pomiaru wytrzymałości na rozciąganie i stężenia hydroksyproliny
w tkance w linii szwów.
Wyniki. Hialuronian sodu o małej masie cząsteczkowej istotnie statycznie zmniejszył intensywność powstawania
*This study was carried out without any financial support or grant.
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B. D. Yildiz et al.
zrostów (p = 0,005). Podanie oktreotydu nieistotnie statystycznie zahamowało powstawanie zrostów (p > 0,05).
Podanie hialuronianu sodu o małej masie cząsteczkowej ani oktreotydu nie wpłynęło na wytrzymałość na rozciąganie tkanek i stężenie hydroksyproliny (p > 0,05).
Wnioski. Hialuronian sodu i oktreotyd, które są dobrze znanymi środkami przeciwzrostowymi nie wywierają negatywnego wpływu na wytrzymałość na rozciąganie tkanek ani stężenie hydroksyproliny (Adv Clin Exp Med 2011,
20, 6, 711–715).
Słowa kluczowe: hialuronian, oktreotyd, chirurgia jamy brzusznej, zrost, wytrzymałość tkanki.
Post-operative peritoneal adhesions are observed after abdominal operations in as many as 93%
of the cases [1]. Adhesions account for 1% of the patients admitted to general surgery departments and
for 3% of the laparotomies in one year [2].
Acquired adhesions secondary to foreign bodies left in the peritoneal cavity (such as talc, suture
materials, operative dressings) and intestinal leakage can be minimized or completely eliminated by
preventive measures. However, peritoneal injury
and the formation of ischemic regions are inevitable during surgical interventions, and treatment
modalities specifically aimed at preventing adhesions are therefore required [3].
Agents used for the prevention of post-operative peritoneal adhesions can be administered
either systemically or intraperitoneally [4]. The
main purpose of intraperitoneal application of
these agents is to prevent adhesion by making the
peritoneal surfaces slippery and by detaching them
from each other, or to eliminate formed adhesions
in the early stages [5]. In recent studies, both octreotide and Na-hyaluronate derivatives have been
used to prevent adhesion [6, 7]. However, the effects of these agents on tissue tensile strength have
not been studied yet.
The aim of this study was to investigate the effects of two well known intraperitoneally administered anti-adhesive agents – low-molecular-weight
(LMW) Na-hyaluronate and octreotide – on the
tensile strength of the rat abdominal wall.
Material and Methods
The experiments were conducted with adult
male Sprague-Dawley rats weighing from 200 to
240 grams. The rats were kept at room temperature and provided with free access to standard
chow and tap water. The entire study was carried
out under the guidelines of the Selcuk University
Institutional Animal Ethics Committee.
The Experimental Design
The rats were divided into three groups (n = 10 in
each group) as follows: the control group, the LMW
Na-hyaluronate group and the octreotide group. The
animals were fasted for 12 hours before the experiments, but were allowed water ad libitum. They were
anesthetized with ketamine HCI (5 mg/kg, intramuscular, Ketalar, Parke-Davis Inc., USA). Following
anesthesia induction, a midline abdominal incision
approximately 4 cm in length was performed under
sterile conditions. Ten longitudinal incisions 2–3 cm
in length were made on the right parietal peritoneal
surface. A layer of the parietal peritoneum 2 × 1 cm
in size was removed from the left side of the incision.
The rats in the control group received intraoperative
peritoneal irrigation with 6 ml of normal saline. The
LMW Na-hyaluronate group received intraperitoneal irrigation with orthovisc (Anika Research, USA)
at a concentration of 0.25 mg/kg in a total volume of
6 ml of normal saline solution. The octreotide group
received intraperitoneal irrigation with 6 ml of octreotide solution (5 μg/ml in normal saline).
The rats were kept at an ambient temperature
of 22°C after the surgical procedure described.
They were fed with standard rat pellets for the
next 13 days, and were fasted for 12 hours before
the second operation. On the 14th day of the experiment, the animals were re-anesthetized with
ketamine HCI (5 mg/kg, intramuscular, Ketalar,
Parke-Davis Inc., USA) and the abdominal wall
was opened by a suprapubic transverse incision
perpendicular to the bottom end of the previous
midline incision. The areas of peritoneal injury on
the right and left sides of the abdomen and the suture line were examined for the presence of adhesions. The adhesions detected were graded according to the method described by Mazuji et al. [8].
After this, an abdominal wall segment 4 × 4 cm in
size was removed by extending the incision in a “U”
shape so that the previous suture line remained in
the middle. After removing the sutures, the tensile strength of the suture line and of the right and
left sides of the abdominal wall were determined
according to the method described by Peacock [9,
10]. A 1-gram tissue sample was obtained from the
suture line and from the right and left sides of the
abdominal wall. The hydroxyproline level in the
tissue was determined according to Bergman and
Loxley’s method for each individual site [11, 12].
Tissue hydroxyproline levels and tensile
strength values are expressed as mean and standard deviation. The values were analyzed with
713
Effects of Anti-Adhesives on Tissue Strength
a one-way variance analysis. If the differences were
significant, Scheffe’s test was used to compare the
groups. The values obtained from adhesion grading were expressed as medians and were analyzed
with the Kruskal-Wallis test. If the differences were
significant, the Mann-Whitney-U test with Bonferroni correction was used to compare the groups.
P values less than 0.05 were considered significant.
Results
Adhesion Grades
In the control group, peritoneal adhesions
were found extensively to the right of the midline
incision. A similar distribution of adhesions in the
abdominal cavity was seen in the LMW Na-hyaluronate and octreotide groups. The adhesion grade
of the LMW Na-hyaluronate group was significantly lower than in the control group (p = 0.005).
The reduction in adhesion grade in the octreotide
group was insignificant compared to the control
group (p > 0.05) (Table 1).
Tissue Tensile Strength
In the control group tensile strength was
highest in the midline incision and lowest on the
left side of the midline; values on the right side
were in between. A similar distribution of tensile
strength was observed in the other two groups.
There was no statistically significant difference in
tensile strength between the experimental groups
(p > 0.05) (Table 2).
Tissue Hydroxyproline Levels
In each group, samples obtained from the
midline incision contained higher hydroxyproline
levels than either the right or left side of the midline incision. Insignificant differences between the
groups were found when the hydroxyproline levels
were compared (p > 0.05) (Table 3).
Table 1. Adhesion grades in thr experimental groups
Tabela 1. Stopień zrostów w grupach doświadczalnych
Control
(Grupa kontrolna)
LMW Na-hyaluronate
(Hialuronian sodu)
Octreotide
(Oktreotyd)
midline
right
left
midline
right
left
midline
right
left
Grade 0
n = 1
n = 0
n = 2
n = 7
n = 5
n = 7
n = 5
n = 4
n = 6
Grade I
n = 3
n = 2
n = 3
n = 2
n = 3
n = 3
n = 2
n = 2
n = 3
Grade II
n = 3
n = 4
n = 4
n = 1
n = 2
n = 0
n = 2
n = 3
n = 1
Grade III
n = 2
n = 3
n = 1
n = 0
n = 0
n = 0
n = 1
n = 1
n = 0
Grade IV
n = 1
n = 1
n = 0
n = 0
n = 0
n = 0
n = 0
n = 0
n = 0
p = 0.005
p > 0.05
Table 3. Tissue hydroxyproline levels (μg/mg tissue)
Table 2. Tissue tensile strengths (g)
Tabela 2. Wytrzymałość na rozciąganie tkanek – g
Tabela 3. Stężenie hydroksyproliny w tkankach (μg/mg
tkanki)
Groups
(Grupy)
Midline
(Środek)
mean ± SD
Right side
(Strona
prawa)
mean ± SD
Left side
(Strona
lewa)
mean ± SD
Groups
(Grupy)
Midline
(Środek)
mean ± SD
Right side
(Strona
prawa)
mean ± SD
Left side
(Strona
lewa)
mean ± SD
Control
(Kontrolna)
724 ± 25.58
698 ± 18.6
678 ± 22.42
Control
(Kontrolna)
3.09 ± 0.57
3.03 ± 0.21
2.92 ± 0.47
Na-Hyaluronate
(Hialuronian sodu)
717 ± 17.68
681 ± 11.52
672 ± 3
Na-Hyaluronate
(Hialuronian sodu)
2.96 ± 0.36
2.91 ± 0.55
2.87 ± 0.39
Octreotide
(Oktreotyd)
71 8 ± 24.4
701 ± 21.47
679 ± 13.5
Octreotide
(Oktreotyd)
2.91 ± 0.52
2.86 ± 0.85
2.84 ± 0.72
p > 0.05
p > 0.05
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B. D. Yildiz et al.
Discussion
Adhesions are one of the major factors that
are responsible for increased rates of mortality and
morbidity following abdominal surgery, especially
during relaparotomies [13]. Surgical trauma to the
serosal surfaces is considered to be responsible for
more than 90% of post-operative adhesions. Reduction in tissue oxygenation after surgical trauma
plays a significant role in adhesion formation. The
increased secretion of histamine after peritoneal
injury causes accumulation of protein-rich fluid in
the peritoneal cavity. All of these factors accelerate adhesion formation by blocking or inhibiting
the activation of plasminogen activators [14, 15].
When combined with the above events, infection
aggravates the development of adhesions by provoking inflammatory responses [16]. Despite preventive measures, adhesions cannot be completely
eliminated. Ischemia is a major and inevitable factor in the formation of adhesions in abdominal
surgery [17]. In all three experimental groups in
the current study, adhesions were more observed
in greater numbers on the right side of the midline
incision, where the injury was more extensive. Despite the full-thickness injury at the midline incision site, adhesion development was lower on the
midline. These results indicate that the extent of
peritoneal injury is the most important factor in
the formation of adhesions.
In a study by Miro et al., tissue tensile strength
was found to be increased in injury sites where
ischemia was more pronounced [10]. In addition, hydroxyproline levels in healthy tissues were
reported to be lower than the injured site tissue
samples. The differences between healthy and
injured tissues were explained by the positive effect of an extensive inflammatory reaction on the
re-modeling phase in the injured tissues. In the
current study, tissue tensile strength was found to
be higher around the midline incision than at the
other sites. Similarly, tissue hydroxyproline levels
were increased near the midline incision site, possibly secondary to excessive tissue ischemia.
No ideal agent to prevent peritoneal adhesion has been discovered to date. Several drugs
and materials are applied locally or systemically
for this purpose [7]. Hyaluron and its derivatives
have been used in vitreoretinal surgery and in osteoarthritis. Since the development of cross-linked
derivatives of hyaluron, their usage and indications have expanded, and their rate of success has
increased [18, 19]. The use of hyaluron derivatives
for the prevention of post-operative peritoneal
adhesions seems to have encouraging results. In
various studies, Sepracoat solution (Genzyme Co.,
Cambridge, MA, USA), a derivative of hyaluronic
acid, has both prevented and reduced adhesion
formation on serosal surfaces in animal models
and in gynecologic operations without any side
effects or signs of systemic toxicity [19]. Another
form of hyaluronic acid derivative hardened with
carboxymethylcellulose is called Seprafilm (Genzyme Co., Cambridge, MA, USA); it has been used
for the prevention of adhesion formation [20, 21].
In the current study, a correlation was noted between tissue tensile strength and hydroxyproline
levels and the intensity of the injury. The current
results also indicate that the natural distribution
of tissue tensile strength and hydroxyproline levels after injury remained unaffected by LMW-Na
hyaluronate administration.
Various studies have shown that octreotide can
inhibit local synthesis of insulin-like growth factor-1 after inflammatory responses and decrease the
responsiveness of T lymphocytes, thereby inhibiting glycosaminoglycan synthesis in fibroblasts [22,
23]. The reduction of collagen production by fibroblasts in turn prevents fibrous band formation. Lai
et al. and Alatas et al. showed that octreotide significantly reduced intraperitoneal adhesion formation
in groups of subjects that which received octreotide
intraperitoneally [24, 25]. In the present study, octreotide had no adverse effect on the distribution
of tensile strength at different sites. Also, as in the
LMW Na-hyaloronate group, tissue hydroxyproline levels were unaffected in the octreotide treated
group. Despite controversies about the administration route and dosing, these results suggest that
intraperitoneal administration of octreotide is successful in reducing the adhesion formation without
any unwanted effects on the tissue matrix.
The authors conclude that intraperitoneal administration of LMW-Na hyaluronate and octreotide reduce adhesion formation after peritoneal
insult. The effects of these agents on tissue tensile
strength and hydroxyproline levels are similar. Further clinical studies are needed to strengthen the
evidence for the well-known adhesion-preventing
effects of hyaluron derivatives and octreotide.
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Address for correspondence:
Baris D. Yildiz
Selanik cad 29/2 Kizilay
06650 Ankara
Turkey
Phone: +90-532-445-46-55
E-mail: [email protected]
Conflict of interest: None declared
Received: 18.04.2011
Revised: 16.05.2011
Accepted: 7.12.2011