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E-selectin levels and histopathologic findings in experimental model
Journal of Pediatric Surgery (2012) 47, 2050–2054
www.elsevier.com/locate/jpedsurg
Evaluation of omental inflammatory response with P-/E-selectin levels and histopathologic findings in experimental model Mustafa Kemal Aslan a,⁎, Özlem Boybeyi a , Tutku Soyer a , Mine Fedakar Şenyücel a , Şebnem Ayva b , Ucler Kısa c , Özkan Cesur a , Murat Çakmak d a
Department of Pediatric Surgery, Kırıkkale University School of Medicine, Kırıkkale 71100, Turkey Department of Pathology, Başkent University, School of Medicine, Ankara 06490, Turkey c Department of Biochemistry, Kırıkkale University School of Medicine, Kırıkkale 71100, Turkey d Department of Pediatric Surgery, Ankara University, School of Medicine, Ankara 06590, Turkey b
Received 12 March 2012; revised 6 June 2012; accepted 7 June 2012
Key words: Omentum; Inflammation; P-/E-selectin; Cecal ligation; Puncture
Abstract Aim: The omentum plays a crucial role in abdominal defense mechanism by adhering to sites of inflammation and absorbing bacteria and debris from the peritoneal cavity. An experimental study was conducted to evaluate the inflammatory response of omentum in different abdominal events with omental P-/E-selectin levels and histopathologic findings. Materials and methods: Thirty Wistar rats were placed into 5 groups (n = 6), including a control group (CG), sham group (SG), bladder perforation (BP) group, splenic laceration (SL) group, and cecal ligation and puncture (CLP) group. Omental samples were obtained in CG after median laparotomy. In accordance with described models, BP, SL, and CLP were performed in experimental groups. Twentyfour hours after the first laparotomy, localization of the omental pad was noted, and omental samples were obtained for biochemical analysis of levels and histopathologic findings (no. of vessels in sections, polymorphic nuclear leukocytes [PMLs], lymphocytes). The mean P-/E-selectin levels and histopathologic findings of inflammation were compared between groups. Results: Although omentum was adhered to the cecum in all subjects after CLP, similar findings were not detected in other groups. P-selectin and E-selectin levels and number of PML were significantly increased in the CLP group when compared with other groups (P b .05). The number of vessels in sections was significantly increased in CLP group when compared with SG and BP groups (P b .05), and the BP group had a decreased number of vessels than CG (P b .05). The number of PML was significantly increased in SG and SL and BP groups with respect to CG (P b .05). Conclusion: Among different experimental intraabdominal catastrophes, only CLP caused an inflammatory response and increased levels of adhesion molecules in the omentum. These findings suggest that the nature of the inflammation is the main determining factor for the omental function in intraabdominal events. © 2012 Elsevier Inc. All rights reserved.
⁎ Corresponding author. Tel.: +90 318 2252489/2333. E-mail address: [email protected] (M.K. Aslan). 0022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2012.06.024
Evaluation of omental inflammatory response The omentum is one of the primary peritoneal defense organs and is known as “policeman of the abdomen.” It plays a crucial role in the defense mechanism by adhering to sites of inflammation and absorbs bacteria and debris from the peritoneal cavity [1,2]. The omentum is composed of 2 mesothelial sheets enclosing adipocytes and loose connective tissue with mononuclear phagocyte cell aggregates [3]. These leucocytes aggregate in perivascular areas to form milky spots [4]. The endothelium lining the omental capillaries and the mesothelium overlying milky spots are adapted to facilitate transmigration of leucocytes [1]. When exposed to a foreign substance, inflammatory factors are activated leading to recruitment of inflammatory cells within the peritoneal cavity [3]. Inflammatory stimuli initiate leukocyte activation in a cascade-like fashion. Adhesion molecules have a significant role in the inflammatory process. The selectins are members of the adhesion molecule family and participate in capture, rolling, and slow-rolling steps of leukocyte recruitment. L-, P-, and E-selectins function in different phases of this process [5,6]. The inhibition of selectins leads to a decreased inflammatory response. The detection of selectin levels is an indicator of inflammatory response [5,6]. The omentum does not only activate the inflammatory process at the cellular level but also mechanically protects the peritoneal cavity by adhering to the site of inflammation or foreign bodies [1-3]. Although it is not inherently motile, it has been shown that it adheres to the area of inflammation within the peritoneal cavity [3]. The exact mechanism of omental movement in the area of inflammation may be immunologic and/or inflammatory mediated. However, the triggering factor for omental movement is controversial. In this study, we aimed to evaluate the omental response to intraabdominal inflammation, bleeding, and urine extravasation. An experimental study was conducted to evaluate the response of omentum in different intraabdominal events measuring omental P-/E-selectin levels and noting histopathologic findings.
1. Materials and methods Thirty Wistar albino rats of both sexes, weighing 300 ± 20 g, were enrolled in the study. The rats were randomized into 5 groups each having 6 rats: sham group (SG), bladder perforation (BP) group, splenic laceration (SL) group, cecal ligation and puncture (CLP) group, and control group (CG). After fasting overnight, rats were anesthetized with intramuscular ketamine hydrochloride (80 mg/kg, Ketalar; Eczacıbaşı, Istanbul, Turkey). Omental samples were obtained in CG after median laparotomy. In SG, only median laparotomy was performed, and omental samples were obtained after a 24-hour period. In BP, SL, and CLP groups, BP, SL, and CLP were performed, respectively, in the first laparotomy.
2051
1.1. Surgical interventions for experimental models 1.1.1. Cecal ligation and puncture After 3-cm-length median laparotomy, cecum was ligated and punctured with a 22G needle twice as previously described in the literature [7]. 1.1.2. Bladder perforation After 3-cm-length median laparotomy, bladder dome was punctured with a 22G needle twice. Urine leakage was observed after puncture. 1.1.3. Splenic laceration After 3-cm-length median laparotomy, a laceration that is 1 cm in length and 1 mm in depth with 22G needle was performed on the anterior surface of the spleen. Hemorrhage was observed after the laceration. In the experimental groups, 24 hours after the first laparotomy, a second laparotomy was performed to evaluate the abdominal localization of the omentum and to obtain omental samples. Samples were evaluated with biochemical analysis of P-/E-selectin levels and histopathologic findings. The study was approved by local ethics committee of Kırıkkale University (17.3.2010—10/17), and experiments were performed in adherence to ethical principles of use of laboratory animals. The data obtained from the experiments were analyzed with Kruskal-Wallis test (SPSS 15.0; SPSS, Chicago, IL). P b .05 was considered significant.
1.2. Histopathologic evaluations The samples were inflated and fixed with 10% formalin. Then, all samples were embedded in paraffin. Tissues were sectioned in 4- to 6-μm pieces and stained with routine hematoxylin and eosin stain. The specimens were examined under a light microscope (Olympus CX31; Mason Technology, Dublin, Ireland) by the same pathologist blinded to the study. Five separate areas of 1-mm2 size were examined under original magnification ×400. Total numbers of vessels including erythrocytes in sections (number per square millimeter) and number of lymphocytes (number per square millimeter) and polymorphic nuclear leukocytes (PMLs) (number per square millimeter) were recorded.
1.3. Determination of P-/E-selectin levels After washing with 0.9% NaCl, tissue samples (which had been preserved at −70°C) were homogenized in 1-mL 0.9% NaCl using a tissue homogenizer. The homogenates were centrifuged at 1500g at 4°C for 10 minutes. P-/Eselectin concentration was determined in the tissue homogenate using a commercially available quantitative enzyme-linked immunosorbent assay (ELISA) kit (Rat-E Selectin ELISA Kit and Rat-P Selectin ELISA Kit;
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M.K. Aslan et al.
Fig. 1 Histopathologic evaluation of omental samples. In CG, small number of capillaries embedded in mature fat tissue (A) (hematoxylin and eosin, original magnification ×400). Cecal ligation and puncture samples revealed vascular proliferation, congestion (arrows) (B) (hematoxylin and eosin, original magnification ×400), and PML clusters in mature fat tissue (arrows) (C) (hematoxylin and eosin, original magnification ×400).
USCNLIFE, Wuhan, China). Results were expressed as picograms per milligram protein.
2. Results After the second laparotomy, omentum was adhered to the cecum in all subjects in the CLP group. However, similar findings were not detected in other groups. In histopathologic evaluations, the CLP group had a significantly higher number of vessels in sections than the SG and BP groups (Fig. 1) (P b .05). The BP group had a decreased number of vessels when compared with CG (P b .05). The number of PML was significantly increased in the CLP group when compared with other groups (P b .05). The number of PML was significantly increased in SG and SL and BP groups with respect to CG (P b .05). Bladder perforation and CLP groups had decreased number of lymphocyte levels when compared with SG (P b .05) (Fig. 2). Biochemical examination revealed that P-selectin and E-selectin levels were significantly increased in the CLP group when compared with other groups (P b .05). In addition, P-selectin and E-selectin levels were significantly increased in SG and SL and BP groups when compared with CG (P b .05) (Fig. 3).
3. Discussion We describe the omental response after different experimentally produced intraabdominal pathologies. Although 3 induced abdominal pathologies (CLP, SL, and BP) were all potentially able to initiate an inflammatory process, only in the cecal ligation perforation group (CLP) did omental adhesions occur. The omentum is the primary peritoneal defense organ that rapidly adheres to the sites of inflammation or foreign bodies
Fig. 2 Comparison of groups regarding the number of vessels (A), PML (B), and lymphocytes (C).
Evaluation of omental inflammatory response
Fig. 3 Comparison of groups regarding P-selectin (A) and E-selectin (B) levels.
in the peritoneal cavity [1,2]. Mononuclear phagocytic cell aggregates are found in loose connective tissue of the omentum [3]. In 1814, Ranvier [4] described leukocyte aggregation in perivascular areas to form milky spots. The milky spots consist of mononuclear cells including macrophages, B cells, T cells, and mast cells. Macrophages in the omentum are scavengers that migrate into the peritoneal cavity and absorb and clear the bacteria and debris [1,3]. In the present study, PMLs were found increased in all experimental groups compared with CG, revealing inflammation in all groups. Inflammation was more prominent in the CLP group than other groups. In addition, the number of lymphocytes was decreased in BP and CLP groups when compared with SG (P b .05). These results suggest that the inflammatory response that occurred in omental samples activated PML and decreased lymphocytic infiltration. This suggests that PML activation is the predominant cellular response and may be related to polymicrobial origin of intraabdominal inflammation. The omentum activates the inflammatory process at the cellular level and mechanically protects the peritoneal cavity by its adherence ability [1-3]. It adheres to areas of inflammation within the peritoneal cavity, although the omentum has no active mobility [8]. In experimental models, omentum was shown to adhere rapidly to damaged serosa at a greater frequency than other viscera, especially when hemorrhage, inflammation, or a foreign body was involved [8-10].
2053 The mechanism of omental adherence is related to fibrin formation at the site of injury. The fibrin encourages migration of leukocytes attracted by inflammatory substances such as endothelial growth factor and basic fibroblast growth factor [3]. Omentum also plays an important role in tissue healing with its angiogenic activity and the immune system with its defensive activity and deposit property for lymphocytes [3,11,12]. In the present study, the number of vessels was increased in the CLP group compared with the SG and BP group. This suggests that angiogenic activity of the omentum occurred in the CLP group. Interestingly, in the BP group, the number of vessels was decreased when compared with CG. Despite all these facts about the omentum, research to date has not yet clarified whether the passive movement of omentum (in response to inflammation) would be also seen in response to blood or urine. In addition, the signals initiating the inflammatory process have not been studied in detail. In the present study, among different intraabdominal events, only CLP caused macroscopic adhesion of the omentum. Similar omental movement at the injury site was not detected in rats with SL and BP. The number of vessels in sections and PML numbers were significantly increased in the CLP group, suggesting that inflammation is the determining factor for omental function after intraabdominal events. The leukocyte recruitment during inflammation is regulated be a variety of adhesion molecules and inflammatory factors. The selectins are members of adhesion molecules that play a role in the capture, rolling, and slowrolling steps of PML recruitment [6,13]. The selectins are cell surface glycoproteins and consist of 3 types: L-, E-, and P-selectins [14]. Selectins are crucial for the natural immune response, as demonstrated in selectin-deficient and selectin ligand–deficient patients and in mouse models. P-selectins were expressed on activated platelets and endothelial cells. P-selectin–deficient mice show a 2- to 4-hour delay in neutrophil infiltration in many models [6]. There is evidence that P-selectin plays a major role in initiating neutrophil attachment to the activated endothelium and the earliest phase of spontaneous rolling of neutrophils in rat postcapillary venules [13]. E-selectins are found on endothelial cells and are rapidly expressed in response to the inflammatory cytokines [6,13]. L-selectins are expressed on all granulocytes, monocytes, and on most lymphocytes [6,13,14]. Elevation of selectins is responsible for the progression of the inflammatory process [6]. In the present study, P-selectin and E-selectin levels were significantly increased in the CLP group when compared with other groups. P-selectin and E-selectin levels were also significantly increased in SG and SL and BP groups when compared with CG. These findings suggest that not only bacterial inflammation but also blood and urine in the peritoneal cavity cause adhesion molecule activation. Although inflammation causes higher levels of P-/E-selectin
2054 activation, intraabdominal bleeding and urine leakage may also activate the adherence molecules in omental samples. Therefore, we suggest from the differences in these adhesion molecules that a complex mechanism of inflammatory and immunologic mechanisms may be involved in the passive movement of omentum. Other inflammatory markers and cytokine levels should be further investigated to form a conclusion about the exact mechanism of omental response to intraabdominal inflammation. Among a series of experimental intraabdominal injuries, only CLP caused macroscopic adherence of omentum. Cecal ligation and puncture also caused an inflammatory response associated with increased levels of adhesion molecules (P-/ E-selectin) in omental samples. Intraabdominal events, especially inflammation, cause increased levels of adherence molecules in the omentum. Because CLP results in polymicrobial intraabdominal inflammation, we suggest that inflammation with a microbial origin may cause omental reaction, and the nature of the inflammation is the major determining factor for the omental function in intraabdominal events.
Acknowledgments This study was presented in the 29th National Congress of Turkish Pediatric Surgeons, in 2011, İstanbul, Turkey. The authors thank Dr Özge Soyer for her statistical evaluation and Serkan Sahinoglu for his English language revision.
M.K. Aslan et al.
References [1] Platell C, Cooper D, Papadimitriou JM, et al. The omentum. World J Gastroenterol 2000;6:169-76. [2] Hall J, Heel K, Papadimitriou J, et al. The pathobiology of peritonitis. Gastroenterology 1998;114:185-96. [3] Collins D, Hogan AM, O'Shea D. The omentum: anatomical, metabolic, and surgical aspects. J Gastrointest Surg 2009;13:1138-46. [4] Ranvier L. Recherches sur la formation des milles du grand epiplon. Arch Physiol 1880;1:421-8. [5] Celebi M, Paul AGA. Blocking both E-selectin and P-selectin inhibits neutrophil recruitment into the murine testis after ischemia-reperfusion–induced injury. Acta Vet Brno 2008;77:321-6. [6] Ley K. The role of selectins in inflammation and disease. Trends Mol Med 2003;9:263-8. [7] Rittirsch D, Huber-Lang MS, Flierl MA, et al. Immunodesign of experimental sepsis by caecal ligation and puncture. Nat Protoc 2008;4:31-6. [8] Wilkosz S, Epstein J, Giogio-Miller A, et al. Remodelling of adipose tissue during experimental omental adhesion formation. Br J Surg 2008;95:387-96. [9] Milligan DW, Rafter AT. Observations on the pathogenesis of peritoneal adhesions: a light and electron microscopical study. Br J Surg 1974;61:274-80. [10] Holmdahl L, al-Jabreen M, Risberg B. Experimental models for quantitative studies on adhesion formation in rats and rabbits. Eur Surg Res 1994;26:248-56. [11] Agalar F, Sayek I, Cakmakci M, et al. Effect of omentectomy on peritoneal defence mechanisms in rats. Eur J Surg 1997;163:605-9. [12] Agca B, Paksoy M, Polat E, et al. Influence of omentectomy on peritoneal defense mechanisms in an experimental model of intraabdominal infection. Eur Surg Res 2003;35:35-40. [13] Gao Y, Li N, Fei R, et al. P-selectin mediated acute inflammation can be blocked by chemically modified heparin, RO-heparin. Mol Cells 2005;19:350-5. [14] Guc D. Adhesion molecules. ANKEM Derg 2004;18:158-63.
www.elsevier.com/locate/jpedsurg
Evaluation of omental inflammatory response with P-/E-selectin levels and histopathologic findings in experimental model Mustafa Kemal Aslan a,⁎, Özlem Boybeyi a , Tutku Soyer a , Mine Fedakar Şenyücel a , Şebnem Ayva b , Ucler Kısa c , Özkan Cesur a , Murat Çakmak d a
Department of Pediatric Surgery, Kırıkkale University School of Medicine, Kırıkkale 71100, Turkey Department of Pathology, Başkent University, School of Medicine, Ankara 06490, Turkey c Department of Biochemistry, Kırıkkale University School of Medicine, Kırıkkale 71100, Turkey d Department of Pediatric Surgery, Ankara University, School of Medicine, Ankara 06590, Turkey b
Received 12 March 2012; revised 6 June 2012; accepted 7 June 2012
Key words: Omentum; Inflammation; P-/E-selectin; Cecal ligation; Puncture
Abstract Aim: The omentum plays a crucial role in abdominal defense mechanism by adhering to sites of inflammation and absorbing bacteria and debris from the peritoneal cavity. An experimental study was conducted to evaluate the inflammatory response of omentum in different abdominal events with omental P-/E-selectin levels and histopathologic findings. Materials and methods: Thirty Wistar rats were placed into 5 groups (n = 6), including a control group (CG), sham group (SG), bladder perforation (BP) group, splenic laceration (SL) group, and cecal ligation and puncture (CLP) group. Omental samples were obtained in CG after median laparotomy. In accordance with described models, BP, SL, and CLP were performed in experimental groups. Twentyfour hours after the first laparotomy, localization of the omental pad was noted, and omental samples were obtained for biochemical analysis of levels and histopathologic findings (no. of vessels in sections, polymorphic nuclear leukocytes [PMLs], lymphocytes). The mean P-/E-selectin levels and histopathologic findings of inflammation were compared between groups. Results: Although omentum was adhered to the cecum in all subjects after CLP, similar findings were not detected in other groups. P-selectin and E-selectin levels and number of PML were significantly increased in the CLP group when compared with other groups (P b .05). The number of vessels in sections was significantly increased in CLP group when compared with SG and BP groups (P b .05), and the BP group had a decreased number of vessels than CG (P b .05). The number of PML was significantly increased in SG and SL and BP groups with respect to CG (P b .05). Conclusion: Among different experimental intraabdominal catastrophes, only CLP caused an inflammatory response and increased levels of adhesion molecules in the omentum. These findings suggest that the nature of the inflammation is the main determining factor for the omental function in intraabdominal events. © 2012 Elsevier Inc. All rights reserved.
⁎ Corresponding author. Tel.: +90 318 2252489/2333. E-mail address: [email protected] (M.K. Aslan). 0022-3468/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jpedsurg.2012.06.024
Evaluation of omental inflammatory response The omentum is one of the primary peritoneal defense organs and is known as “policeman of the abdomen.” It plays a crucial role in the defense mechanism by adhering to sites of inflammation and absorbs bacteria and debris from the peritoneal cavity [1,2]. The omentum is composed of 2 mesothelial sheets enclosing adipocytes and loose connective tissue with mononuclear phagocyte cell aggregates [3]. These leucocytes aggregate in perivascular areas to form milky spots [4]. The endothelium lining the omental capillaries and the mesothelium overlying milky spots are adapted to facilitate transmigration of leucocytes [1]. When exposed to a foreign substance, inflammatory factors are activated leading to recruitment of inflammatory cells within the peritoneal cavity [3]. Inflammatory stimuli initiate leukocyte activation in a cascade-like fashion. Adhesion molecules have a significant role in the inflammatory process. The selectins are members of the adhesion molecule family and participate in capture, rolling, and slow-rolling steps of leukocyte recruitment. L-, P-, and E-selectins function in different phases of this process [5,6]. The inhibition of selectins leads to a decreased inflammatory response. The detection of selectin levels is an indicator of inflammatory response [5,6]. The omentum does not only activate the inflammatory process at the cellular level but also mechanically protects the peritoneal cavity by adhering to the site of inflammation or foreign bodies [1-3]. Although it is not inherently motile, it has been shown that it adheres to the area of inflammation within the peritoneal cavity [3]. The exact mechanism of omental movement in the area of inflammation may be immunologic and/or inflammatory mediated. However, the triggering factor for omental movement is controversial. In this study, we aimed to evaluate the omental response to intraabdominal inflammation, bleeding, and urine extravasation. An experimental study was conducted to evaluate the response of omentum in different intraabdominal events measuring omental P-/E-selectin levels and noting histopathologic findings.
1. Materials and methods Thirty Wistar albino rats of both sexes, weighing 300 ± 20 g, were enrolled in the study. The rats were randomized into 5 groups each having 6 rats: sham group (SG), bladder perforation (BP) group, splenic laceration (SL) group, cecal ligation and puncture (CLP) group, and control group (CG). After fasting overnight, rats were anesthetized with intramuscular ketamine hydrochloride (80 mg/kg, Ketalar; Eczacıbaşı, Istanbul, Turkey). Omental samples were obtained in CG after median laparotomy. In SG, only median laparotomy was performed, and omental samples were obtained after a 24-hour period. In BP, SL, and CLP groups, BP, SL, and CLP were performed, respectively, in the first laparotomy.
2051
1.1. Surgical interventions for experimental models 1.1.1. Cecal ligation and puncture After 3-cm-length median laparotomy, cecum was ligated and punctured with a 22G needle twice as previously described in the literature [7]. 1.1.2. Bladder perforation After 3-cm-length median laparotomy, bladder dome was punctured with a 22G needle twice. Urine leakage was observed after puncture. 1.1.3. Splenic laceration After 3-cm-length median laparotomy, a laceration that is 1 cm in length and 1 mm in depth with 22G needle was performed on the anterior surface of the spleen. Hemorrhage was observed after the laceration. In the experimental groups, 24 hours after the first laparotomy, a second laparotomy was performed to evaluate the abdominal localization of the omentum and to obtain omental samples. Samples were evaluated with biochemical analysis of P-/E-selectin levels and histopathologic findings. The study was approved by local ethics committee of Kırıkkale University (17.3.2010—10/17), and experiments were performed in adherence to ethical principles of use of laboratory animals. The data obtained from the experiments were analyzed with Kruskal-Wallis test (SPSS 15.0; SPSS, Chicago, IL). P b .05 was considered significant.
1.2. Histopathologic evaluations The samples were inflated and fixed with 10% formalin. Then, all samples were embedded in paraffin. Tissues were sectioned in 4- to 6-μm pieces and stained with routine hematoxylin and eosin stain. The specimens were examined under a light microscope (Olympus CX31; Mason Technology, Dublin, Ireland) by the same pathologist blinded to the study. Five separate areas of 1-mm2 size were examined under original magnification ×400. Total numbers of vessels including erythrocytes in sections (number per square millimeter) and number of lymphocytes (number per square millimeter) and polymorphic nuclear leukocytes (PMLs) (number per square millimeter) were recorded.
1.3. Determination of P-/E-selectin levels After washing with 0.9% NaCl, tissue samples (which had been preserved at −70°C) were homogenized in 1-mL 0.9% NaCl using a tissue homogenizer. The homogenates were centrifuged at 1500g at 4°C for 10 minutes. P-/Eselectin concentration was determined in the tissue homogenate using a commercially available quantitative enzyme-linked immunosorbent assay (ELISA) kit (Rat-E Selectin ELISA Kit and Rat-P Selectin ELISA Kit;
2052
M.K. Aslan et al.
Fig. 1 Histopathologic evaluation of omental samples. In CG, small number of capillaries embedded in mature fat tissue (A) (hematoxylin and eosin, original magnification ×400). Cecal ligation and puncture samples revealed vascular proliferation, congestion (arrows) (B) (hematoxylin and eosin, original magnification ×400), and PML clusters in mature fat tissue (arrows) (C) (hematoxylin and eosin, original magnification ×400).
USCNLIFE, Wuhan, China). Results were expressed as picograms per milligram protein.
2. Results After the second laparotomy, omentum was adhered to the cecum in all subjects in the CLP group. However, similar findings were not detected in other groups. In histopathologic evaluations, the CLP group had a significantly higher number of vessels in sections than the SG and BP groups (Fig. 1) (P b .05). The BP group had a decreased number of vessels when compared with CG (P b .05). The number of PML was significantly increased in the CLP group when compared with other groups (P b .05). The number of PML was significantly increased in SG and SL and BP groups with respect to CG (P b .05). Bladder perforation and CLP groups had decreased number of lymphocyte levels when compared with SG (P b .05) (Fig. 2). Biochemical examination revealed that P-selectin and E-selectin levels were significantly increased in the CLP group when compared with other groups (P b .05). In addition, P-selectin and E-selectin levels were significantly increased in SG and SL and BP groups when compared with CG (P b .05) (Fig. 3).
3. Discussion We describe the omental response after different experimentally produced intraabdominal pathologies. Although 3 induced abdominal pathologies (CLP, SL, and BP) were all potentially able to initiate an inflammatory process, only in the cecal ligation perforation group (CLP) did omental adhesions occur. The omentum is the primary peritoneal defense organ that rapidly adheres to the sites of inflammation or foreign bodies
Fig. 2 Comparison of groups regarding the number of vessels (A), PML (B), and lymphocytes (C).
Evaluation of omental inflammatory response
Fig. 3 Comparison of groups regarding P-selectin (A) and E-selectin (B) levels.
in the peritoneal cavity [1,2]. Mononuclear phagocytic cell aggregates are found in loose connective tissue of the omentum [3]. In 1814, Ranvier [4] described leukocyte aggregation in perivascular areas to form milky spots. The milky spots consist of mononuclear cells including macrophages, B cells, T cells, and mast cells. Macrophages in the omentum are scavengers that migrate into the peritoneal cavity and absorb and clear the bacteria and debris [1,3]. In the present study, PMLs were found increased in all experimental groups compared with CG, revealing inflammation in all groups. Inflammation was more prominent in the CLP group than other groups. In addition, the number of lymphocytes was decreased in BP and CLP groups when compared with SG (P b .05). These results suggest that the inflammatory response that occurred in omental samples activated PML and decreased lymphocytic infiltration. This suggests that PML activation is the predominant cellular response and may be related to polymicrobial origin of intraabdominal inflammation. The omentum activates the inflammatory process at the cellular level and mechanically protects the peritoneal cavity by its adherence ability [1-3]. It adheres to areas of inflammation within the peritoneal cavity, although the omentum has no active mobility [8]. In experimental models, omentum was shown to adhere rapidly to damaged serosa at a greater frequency than other viscera, especially when hemorrhage, inflammation, or a foreign body was involved [8-10].
2053 The mechanism of omental adherence is related to fibrin formation at the site of injury. The fibrin encourages migration of leukocytes attracted by inflammatory substances such as endothelial growth factor and basic fibroblast growth factor [3]. Omentum also plays an important role in tissue healing with its angiogenic activity and the immune system with its defensive activity and deposit property for lymphocytes [3,11,12]. In the present study, the number of vessels was increased in the CLP group compared with the SG and BP group. This suggests that angiogenic activity of the omentum occurred in the CLP group. Interestingly, in the BP group, the number of vessels was decreased when compared with CG. Despite all these facts about the omentum, research to date has not yet clarified whether the passive movement of omentum (in response to inflammation) would be also seen in response to blood or urine. In addition, the signals initiating the inflammatory process have not been studied in detail. In the present study, among different intraabdominal events, only CLP caused macroscopic adhesion of the omentum. Similar omental movement at the injury site was not detected in rats with SL and BP. The number of vessels in sections and PML numbers were significantly increased in the CLP group, suggesting that inflammation is the determining factor for omental function after intraabdominal events. The leukocyte recruitment during inflammation is regulated be a variety of adhesion molecules and inflammatory factors. The selectins are members of adhesion molecules that play a role in the capture, rolling, and slowrolling steps of PML recruitment [6,13]. The selectins are cell surface glycoproteins and consist of 3 types: L-, E-, and P-selectins [14]. Selectins are crucial for the natural immune response, as demonstrated in selectin-deficient and selectin ligand–deficient patients and in mouse models. P-selectins were expressed on activated platelets and endothelial cells. P-selectin–deficient mice show a 2- to 4-hour delay in neutrophil infiltration in many models [6]. There is evidence that P-selectin plays a major role in initiating neutrophil attachment to the activated endothelium and the earliest phase of spontaneous rolling of neutrophils in rat postcapillary venules [13]. E-selectins are found on endothelial cells and are rapidly expressed in response to the inflammatory cytokines [6,13]. L-selectins are expressed on all granulocytes, monocytes, and on most lymphocytes [6,13,14]. Elevation of selectins is responsible for the progression of the inflammatory process [6]. In the present study, P-selectin and E-selectin levels were significantly increased in the CLP group when compared with other groups. P-selectin and E-selectin levels were also significantly increased in SG and SL and BP groups when compared with CG. These findings suggest that not only bacterial inflammation but also blood and urine in the peritoneal cavity cause adhesion molecule activation. Although inflammation causes higher levels of P-/E-selectin
2054 activation, intraabdominal bleeding and urine leakage may also activate the adherence molecules in omental samples. Therefore, we suggest from the differences in these adhesion molecules that a complex mechanism of inflammatory and immunologic mechanisms may be involved in the passive movement of omentum. Other inflammatory markers and cytokine levels should be further investigated to form a conclusion about the exact mechanism of omental response to intraabdominal inflammation. Among a series of experimental intraabdominal injuries, only CLP caused macroscopic adherence of omentum. Cecal ligation and puncture also caused an inflammatory response associated with increased levels of adhesion molecules (P-/ E-selectin) in omental samples. Intraabdominal events, especially inflammation, cause increased levels of adherence molecules in the omentum. Because CLP results in polymicrobial intraabdominal inflammation, we suggest that inflammation with a microbial origin may cause omental reaction, and the nature of the inflammation is the major determining factor for the omental function in intraabdominal events.
Acknowledgments This study was presented in the 29th National Congress of Turkish Pediatric Surgeons, in 2011, İstanbul, Turkey. The authors thank Dr Özge Soyer for her statistical evaluation and Serkan Sahinoglu for his English language revision.
M.K. Aslan et al.
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