Baris D. Yildiz
1, Serdar Topaloglu
2, Fatih M. Avsar
1, 3, Ibrahim Sakcak
1,
Barlas Sulu
3, Ayse F. Avsar
4The 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
1 Sixth Department of Surgery, Ankara Numune Training and Research Hospital, Ankara, Turkey 2 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
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 solu-tion 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 otrzewnejza pomocą miejscowopodawanychśrodkówjest popularnym tematem badań. Przypuszczalny wpływtych lekównamacierztkankową może ograniczyć ich zużycie.
Cel pracy.Określenie wpływu hialuronianu sodu o małej masie cząsteczkowej i oktreotyduna wytrzymałość tka-nek na rozciąganieistężeniehydroksyproliny.
Materiał i metody. Szczury szczepu Sprague-Dawley znieczulonoketaminąi wykonano 10nacięćwzdłużnychpo 2–3cm długościnaprawejotrzewnejściennej. Warstwę 2 × 1 cmotrzewnejwycięto zlewejściany jamy brzusznej podczas pierwszejlaparotomii. 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 otwar-to 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 statyczniezmniejszyłintensywność powstawania
Adv Clin Exp Med 2011, 20, 6, 711–715 ISSN 1230-025X
ORIGINAL PAPERS
© Copyright by Wroclaw Medical University
Post-operative peritoneal adhesions are ob-served after abdominal operations in as many as 93% of the cases [1]. Adhesions account for 1% of the pa-tients admitted to general surgery departments and for 3% of the laparotomies in one year [2].
Acquired adhesions secondary to foreign bod-ies left in the peritoneal cavity (such as talc, suture materials, operative dressings) and intestinal leak-age can be minimized or completely eliminated by preventive measures. However, peritoneal injury and the formation of ischemic regions are inevi-table during surgical interventions, and treatment modalities specifically aimed at preventing adhe-sions are therefore required [3].
Agents used for the prevention of post-op-erative 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 oct-reotide and Na-hyaluronate derivatives have been used to prevent adhesion [6, 7]. However, the ef-fects of these agents on tissue tensile strength have not been studied yet.
The aim of this study was to investigate the ef-fects of two well known intraperitoneally adminis-tered 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 tempera-ture 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 experi-ments, but were allowed water ad libitum. They were anesthetized with ketamine HCI (5 mg/kg, intramus-cular, 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 intraperitone-al 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 oc-treotide 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 ex-periment, 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 su-ture line were examined for the presence of adhe-sions. The adhesions detected were graded accord-ing 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 ten-sile 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 stan-dard deviation. The values were analyzed with
zrostów(p = 0,005).Podanie oktreotydunieistotnie statystycznie zahamowałopowstawaniezrostów (p > 0,05). Podanie hialuronianu sodu o małej masie cząsteczkowej ani oktreotydu nie wpłynęło na wytrzymałość na rozcią-ganietkaneki stężenie hydroksyproliny(p > 0,05).
Wnioski. Hialuronian sodu ioktreotyd,które są dobrze znanymi środkami przeciwzrostowymi nie wywierają nega-tywnego wpływu nawytrzymałość na rozciąganietkanek anistężeniehydroksyproliny (Adv Clin Exp Med 2011, 20, 6, 711–715).
a one-way variance analysis. If the differences were significant, Scheffe’s test was used to compare the groups. The values obtained from adhesion grad-ing were expressed as medians and were analyzed with the Kruskal-Wallis test. If the differences were significant, the Mann-Whitney-U test with Bonfer-roni 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-hyal-uronate and octreotide groups. The adhesion grade of the LMW Na-hyaluronate group was signifi-cantly 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 mid-line incision. Insignificant differences between the groups were found when the hydroxyproline levels were compared (p > 0.05) (Table 3).Table 3. Tissue hydroxyproline levels (μg/mg tissue)
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 Control
(Kontrolna) 3.09 ± 0.57 3.03 ± 0.21 2.92 ± 0.47
Na-Hyal-uronate (Hialuro-nian sodu)
2.96 ± 0.36 2.91 ± 0.55 2.87 ± 0.39
Octreotide
(Oktreotyd) 2.91 ± 0.52 2.86 ± 0.85 2.84 ± 0.72 p > 0.05
Table 2. Tissue tensile strengths (g)
Tabela 2. Wytrzymałość na rozciąganie tkanek – g
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
Na-Hyal-uronate (Hialuro-nian sodu)
717 ± 17.68 681 ± 11.52 672 ± 3
Octreotide
(Oktreotyd) 71 8 ± 24.4 701 ± 21.47 679 ± 13.5 p > 0.05
Table 1. Adhesion grades in thr experimental groups
Tabela 1. Stopień zrostówwgrupach doświadczalnych
Control
(Grupa kontrolna) LMW Na-hyaluronate (Hialuronian sodu) (Oktreotyd)Octreotide 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
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. Re-duction 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 acceler-ate 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 pro-voking inflammatory responses [16].Despite pre-ventive measures, adhesions cannot be completely eliminated.Ischemia is a major and inevitable fac-tor 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. De-spite the full-thickness injury at the midline inci-sion site, adheinci-sion 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 addi-tion, 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 ef-fect 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, pos-sibly secondary to excessive tissue ischemia.
No ideal agent to prevent peritoneal adhe-sion 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 os-teoarthritis. Since the development of cross-linked
derivatives of hyaluron, their usage and indica-tions 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 (Gen-zyme Co., Cambridge, MA, USA); it has been used for the prevention of adhesion formation [20, 21]. In the current study, a correlation was noted be-tween 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 lev-els after injury remained unaffected by LMW-Na hyaluronate administration.
Various studies have shown that octreotide can inhibit local synthesis of insulin-like growth fac-tor-1 after inflammatory responses and decrease the responsiveness of T lymphocytes, thereby inhibit-ing glycosaminoglycan synthesis in fibroblasts [22, 23]. The reduction of collagen production by fibro-blasts in turn prevents fibrous band formation. Lai et al. and Alatas et al. showed that octreotide signif-icantly reduced intraperitoneal adhesion formation in groups of subjects that which received octreotide intraperitoneally[24, 25]. In the present study, oc-treotide had no adverse effect on the distribution of tensile strength at different sites. Also, as in the LMW Na-hyaloronate group, tissue hydroxypro-line levels were unaffected in the octreotide treated group. Despite controversies about the administra-tion route and dosing, these results suggest that intraperitoneal administration of octreotide is suc-cessful in reducing the adhesion formation without any unwanted effects on the tissue matrix.
The authors conclude that intraperitoneal ad-ministration of LMW-Na hyaluronate and octreo-tide reduce adhesion formation after peritoneal insult. The effects of these agents on tissue tensile strength and hydroxyproline levels are similar. Fur-ther clinical studies are needed to strengthen the evidence forthe 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
