Journal of Investigative Surgery, 27, 169–175, 2014 C 2014 Informa Healthcare USA, Inc. Copyright ISSN: 0894-1939 print / 1521-0553 online DOI: 10.3109/08941939.2013.863986
NEW METHODOLOGIES
Does Long Intestinal Tube Splinting Aggravate Intestinal Adhesions? Min Li, MD, Gang Wang, MD, Bo Zhou, MD, Xianfeng Xia, MD, Ning Li, MD, PhD
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Department of General Surgery, Jinling Hospital, Medicine School of Nanjing University, Nanjing, Jiangsu Province, China
ABSTRACT Objective: Long intestinal tube splinting (LITS) is useful for clinically reducing the recurrence of adhesive small bowel obstruction (ASBO). However, a controversy exists whether LITS aggravates intestinal adhesions. This study evaluated the postoperative effects of LITS relative to simple enterolysis on intestinal adhesions in an experimental porcine model. Methods: A porcine model (n = 24) of dense intestinal adhesion was established by abrading the ileal wall with sterile P240 sandpaper. Enterolysis was performed on postoperative day 14. Animals were randomly divided into a group that underwent enterolysis only (control; n = 12) and those who underwent LITS as well as enterolysis (LITS; n = 12). The long intestinal tube was removed on post-LITS day 14, after abdominal radiography. All animals were euthanized on postenterolysis day 28 for assessment of intestinal adhesions using a semiquantitative macroscopic grading scale, hematoxylin-eosin histology, and hydroxyproline assay. Results: Prior to enterolysis, the experimentally induced intestinal adhesions of the two groups were similar in extent and severity. On postenterolysis day 28 the LITS and control groups were comparable with regard to adhesion loop length (p = .440), macroscopic adhesion severity (p = .820), serosal fibrosis grading (p = .450), and hydroxyproline content of the adhesion ileal segment (p = .630). Conclusion: Placement of the long intestinal tube did not aggravate intestinal adhesions over that of simple enterolysis in this intestinal adhesion porcine model. Keywords: splinting; long intestinal tube; adhesion; intestine; severity; swine model
INTRODUCTION
crease risks of iatrogenic intestinal injury and intraabdominal hemorrhage [10]. There is a knowledge gap in the current literature regarding whether LITS aggravates postoperative intestinal adhesions. In this study, we evaluated the effect of LITS on the severity of intestinal adhesions after enterolysis in a porcine model of dense intestinal adhesion with respect to macroscopy and histology. In particular, we further examined hydroxyproline content, a more sensitive, objective measure for assessing tissue adhesions compared to conventional macroscopic grading, in adhesive tissues [11].
Adhesive small bowel obstruction (ASBO) is a major cause of morbidity and hospital readmission after abdominal surgery, and its occurrence places substantial burden on public healthcare systems worldwide [1]. The incidence of ASBO after all laparotomies is approximately 3% [2]. More unfortunate, ASBO is refractory to surgical intervention, and the recurrence rate is 16%–46% among surgically treated ASBO patients [3–6]. Long intestinal tube splinting (LITS), applied surgically, allows intestinal loops to adhere permanently to each other in an orderly pattern, and can effectively limit the recurrence of postoperative ASBO to 5.9%–12.9% [7–9]. However, the use of LITS has raised concerns that the long intestinal tube may exacerbate the severity of intestinal adhesions and complicate any further surgical intervention if obstruction recurs [10]. Aggravation of intestinal adhesions is known to in-
MATERIALS AND METHODS Laboratory Animals The Institutional Animal Care and Use Committee at Jinling Hospital approved the study protocol, which
Received 21 May 2013; accepted 5 November 2013. Address correspondence to Ning Li, MD, PhD, Department of General Surgery, Jinling Hospital, Medicine School of Nanjing University, NO 305 East Zhongshan Road, Nanjing, Jiangsu Province, China. E-mail: [email protected]
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was performed in accordance with the Guide for Care and Use of Laboratory Animals established by the National Institutes of Health of the United States and China’s legal regulations. Thirty female laboratory swine, weighing 24.3 ± 4.2 kg, were housed at the Center for Laboratory Animals of Jinling Hospital. Prior to anesthesia, all animals were deprived of chow for 12 hr and drinking water for 4 hr.
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Anesthetic Procedure Each swine was anesthetized with 40 mg/kg intramuscular ketamine and 0.06 mg/kg intramuscular atropine, and subsequently placed in the supine position. Venous access was established through the auricular vein, and a single dose of 50 mg/kg ceftazidime was given intravenously 15 min before endotracheal intubation. An intravenous bolus of 1 mg/kg propofol emulsion (2%; Astra Zeneca, Wedel, Germany) was given to facilitate endotracheal intubation. An 8-mm cuffed endotracheal tube was inserted into the trachea through the oral route using a direct laryngoscope. The animal was ventilated using a large animal ventilator, and anesthesia was maintained with continuous infusion of intravenous 2% propofol emulsion titrated at 9–20 mg/kg·h combined with ketamine at 0.3–0.6 mg/kg·hand fentanyl at 2–5 μg/kg·h (both: Janssen Cilag, Neuss, Germany). All animals were monitored throughout anesthesia and surgery with an automatic electrocardiography monitoring system. After the surgery, when the animal exhibited motorsensory reflex return and spontaneous breathing, the narcotics were down-titrated and the tracheal tube was removed.
Surgical Procedures All surgical procedures were performed using standard aseptic technique. The entire abdominal skin was routinely shaved, prepared, and draped. Six swine were euthanized with a lethal overdose of intravenous propofol for the baseline assessment. A 5-cm ileal segment, located 50 cm proximal to the ileocecal valve,
TABLE 1
was harvested for routine histology and hydroxyproline content assay. Twenty-four swine were used to create a modification of the experimental intestinal adhesion model described by Yusuf and his colleagues [12]. Sterile P240 sandpaper (rather than gauze) was used to induce dense intestinal adhesions. A 5-cm left paramedian skin incision was made, and a 150-cm-long ileal segment, immediately proximal to the ileocecal valve, was exteriorized. Both surfaces of the ileal wall were abraded with five strokes to create experimental serosal defects 150 cm in length. The exteriorized ileal segment was reduced into the abdominal cavity free of torsion or tension. The fascial and cutaneous layers were separately sutured using continuous 3/0 silk sutures to close the abdominal wall incision. All animals were fasted for three successive days but given intravenous infusions 1,000 ml of 5% warm glucose saline containing 2 g of potassium chloride once daily, 100 ml of 10% sodium chloride solution containing 50 mg/kg of cefazolin sodium twice daily, and 100 ml of 10% sodium chloride solution containing 1 mg/kg of parecoxib sodium twice daily. The animals had free access to swine chow and drinking water after this period. Enterolysis was performed on all 24 swine on postoperative day 14. The abdominal cavity was explored via the right paramedian incision. The extent and severity of intestinal adhesions were evaluated using a semiquantitative macroscopic grading scale as previously reported by Zuhlke et al. [13] (Table 1). The adhesions were completely dissected, and a 5-cm-long ileal segment, located 50 cm proximal to the ileocecal valve, was harvested for routine histology and hydroxyproline content assay. Bowel continuity was restored by one-layer end-to-end anastomosis using 3/0 polydioxanone suture (Johnson & Johnson Medical, Livingston, UK). After enterolysis, dense intestinal adhesion model animals were equally and randomly divided into two groups, namely, an LITS group undergoing LITS (n = 12) and a control group undergoing no further surgical intervention (n = 12). In the LITS group, a 4/0 purse-string silk suture (Johnson & Johnson Medical, Livingston, UK) was used to circle the antimesenteric margin of the ileal segment located 10 cm proximal to
Scoring system for classifying macroscopic intestinal adhesion length and severity [13]
Score
Adhesion length
0 1 2
No adhesions 150 cm
Adhesion severity No adhesions Filmy adhesion, easy to separate by blunt dissection Stronger adhesion; blunt dissection possible, partly sharp dissection necessary; beginning of vascularization Strong adhesion; division possible by sharp dissection; clear vascularization Very strong adhesion between organs; its division by sharp dissection damages organ serosa
Journal of Investigative Surgery
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FIGURE 1 LITS experimental procedure. (A) 150-cm-long 10F silicone catheter; (B) formation of extensive, dense adhesions between the loops of the terminal ileum 14 days after serosal abrasion; (C) insertion of silicone catheter after enterolysis; and (D) histological presence of massive adhesive tissues on the serosal surface (H&E; original magnification, 400×).
the ileocecal valve. A 0.5-cm circumferential incision was then made on the ileal wall. A 150-cm 10F silicone catheter (Figure 1A) was entirely inserted into the intestinal lumen through the ileal wall opening cephalad (Figure 1C). The purse-string suture was subsequently tightened if the catheter and the intestines showed no invagination or torsion. A second purse-string suture was used to enhance the closure of the ileal wall opening. The catheter was brought out 2 cm against the parietal peritoneum and through a 0.5-cm incision in the left flank. The needle of the second purse-string suture was passed through the parietal peritoneum adjacent to the flank incision, and appropriately tightened to secure the position of the catheter stoma. The catheter was further secured using a cutaneous silk suture. The postoperative care regimen was identical to that after the creation of experimental intestinal adhesions described above. LITS swine were sedated with ketamine for plain abdominal radiography to position the long intestinal tube on post-LITS day 14. Thirtymilliliters of liquid paraffin were injected through the catheter, and the catheter was gently retracted and removed. All surviving animals were humanely euthanized on postenterolysis day 28 with a lethal overdose of intravenous bolus of propofol (15–20 mg/kg). The entire abdominal cavity was macroscopically examined through a U-shaped incision, and the extent and severity of intestinal adhesions were determined using the semiquantitative macroscopic grading scale C
2014 Informa Healthcare USA, Inc.
[13]. A 5-cm-long ileal segment that was located 10 cm proximal to the anastomosis was sharply dissected for routine histology and hydroxyproline content assay of the terminal ileal segment.
Macroscopic and Histopathological Examinations The terminal ileal segment specimen was fixed in 10% buffered formaldehyde and embedded in paraffin. Four-micron sections were routinely stained with hematoxylin and eosin (H&E), in triplicate. A boardcertified clinical pathologist, blinded to the treatment group, evaluated the severity of ileal wall fibrosis under a light microscope using a semiquantitative microscopic grading scale [14]: grade 0, none; grade 1, mild; grade 2, moderate; and grade 3, severe.
Hydroxyproline Assay Terminal ileal segments were snap frozen in liq◦ uid nitrogen and stored at –80 C until used for further experiments. Terminal ileal segment samples were initially lyophilized, scaled, pulverized, and repeatedly lyophilized. The samples were further hydrolyzed with 6 N hydrogen chloride and derivatized with phenylisothiocyanate. The hydroxyproline
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content was determined using high-performance liquid chromatography and expressed as μg/mg of dry tissue [11].
Statistical Analyses All data were processed using the statistical software SPSS 18.0 for Windows (SPSS, Chicago, IL, US) and expressed as mean ± standard deviation. Differences in the means of the two groups were compared using the Mann–Whitney U test. A p-value less than .05 was considered statistically significant.
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RESULTS All the six animals euthanized for the baseline assessment showed no macroscopically visible intraabdominal adhesions or histologically significant serosal fibrosis. The mean basal hydroxyproline content of the terminal ilea was 7.5 ± 0.5 μg/mg of dry tissue. All the 24 dense intestinal adhesion model animals survived serosal abrasion, and during enterolysis dense adhesions among terminal ileal loops were observed macroscopically (Figure 1B), with significant serosal fibrosis viewed under the light microscope (Figure 1D). The mean scores for intestinal adhesion length, macroscopic adhesion severity, and microscopic serosal fibrosis prior to enterolysis were 3.3 ± 0.4, 3.4 ± 0.8, and 2.3 ± 0.7, respectively; hydroxyproline content in the terminal ileal segment was 19.5 ± 2.7 μg/mg of dry tissue. All these parameters were comparable between the LITS group and the control group (all p-values > .05; Table 2) and increased significantly in both groups after experimental serosal abrasion compared with the baseline (all p-values < .05). One pig in the LITS group and one in the control group did not survive the enterolysis due to enterocutaneous fistula; 2 in the control group succumbed due to ASBO. The remaining 20 swine survived uneventfully until the completion of this study and were euthanized as scheduled. As observed on plain abdominal radiography, all the long intestinal tubes were wellpositioned in situ in the LITS swine (Figure 2), and all
TABLE 2 Length of intestinal adhesion, macroscopic adhesion severity, serosal fibrosis grading, and ileal hydroxyproline content prior to enterolysis
Intestinal adhesion length Macroscopic adhesion severity Serosal fibrosis grading Hydroxyproline content (μg/mg)
LITS
Control
p-value
3.3 ± 0.5 3.5 ± 0.7
3.2 ± 0.4 3.2 ± 0.8
.800 .530
2.3 ± 0.8 19.7 ± 2.9
2.3 ± 0.7 19.2 ± 2.6
.900 .730
FIGURE 2 Macroscopic photographs (2A and 1A) of ileal loop adhesions and photomicrographs (2B and 1B) of serosal tissue fibrosis (H&E; original magnification, 400×) at necropsy 28 days after LITS or enterolysis alone.
these tubes were smoothly removed 14 days after the LITS operation. All intestinal adhesion model animals had dense adhesions between the loops of the 150-cm-long terminal ileal segment, the extent and severity of which were macroscopically similar between the two groups. However, intestinal loop adhesion in the LITS group was in a relatively orderly pattern (Figure 3, 2A) as compared with that in the control group (Figures 3 and 1A). The percentage of animals with adhesions of proximal intestinal loops to the affected terminal ileal segment was similar in the LITS group (8/11, 72.7%) to that of the control group (5/9, 55.6%; p > .05). Histological examination showed that massive serosal fibrosis was present in all 20 surviving animals and was similar between the two groups (Figure 3, 2B and 1B). Overall, intraabdominal adhesions were comparable between the two groups, in terms of intestinal adhesion length, macroscopic adhesion severity, and microscopic serosal fibrosis (all p-values > .05; Figure 4). The hydroxyproline content of the terminal ileal segment increased significantly in both groups compared with the content prior to enterolysis (both p-values < .05), but remained similar between the two groups (LITS vs. control, 29.4 ± 9.3 μg/mg vs. 27.8 ± 6.6 μg/mg, p > .05; Figure 5).
DISCUSSION Long intestinal tube stenting, splinting the small bowel in a gentle curve, was first proposed by White in 1956 [15]. This surgical technique prevents recurrent ASBO by encouraging the formation of intestinal loop Journal of Investigative Surgery
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FIGURE 3 Length of intestinal adhesions, macroscopic adhesion severity, and serosal fibrosis grading at necropsy 28 days after LITS or enterolysis alone. (3.73 ± 0.47 vs. 3.56 ± 0.53, p = .440; 3.82 ± 0.40 vs. 3.78 ± 0.44, p = .820; 2.82 ± 0.40 vs. 2.67 ± 0.50, p = .450)
adhesions in a nonobstructed configuration. It has been documented that LITS can reduce the risk of ASBO recurrence more effectively than simple enterolysis [7–9]. However, some authors assume that this procedure could aggravate intestinal adhesions, although no follow-up histological evidence after LITS has been available [10]. To the best of our knowledge, the present study is the first to evaluate with macroscopic, microscopic, and biochemical evidence of the extent and severity of intestinal adhesions after LITS in an animal model.
A swine model was used in this study, because the peritoneal cavity of swine is similar to that of humans in terms of size and anatomy. Abrasion of the intestinal serosa is reported to be a reliable technique for the creation of an animal model of intestinal adhesion [12, 16, 17]. However, in most cases, this method has resulted in filmy adhesions [18]. We used P240 sandpaper instead of gauze in our study to damage the serosal surfaces, to induce extensive, dense intestinal adhesions similar to those encountered by surgeons when performing LITS [19–21]. Previous studies showed that extensive
FIGURE 4 Terminal ileum hydroxyproline content at necropsy 28 days after LITS or enterolysis alone.
FIGURE 5 Length of intestinal adhesion, macroscopic adhesion severity, serosal fibrosis grading, and ileal hydroxyproline content prior to enterolysis.
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intestinal adhesions had formed 10 days after abrasion [22, 23]; in our present study, enterolysis was performed on postoperative day 14. During laparotomic explorations before enterolysis, we found that extensive, dense intestinal adhesions were present in all the animals. This indicates that our modified technique was more effective in creating an animal model with dense intestinal adhesions. Our modification was proved to be reliably consistent and replicable, which ensured the validity of comparisons between the LITS and control groups for evaluating the macroscopic, microscopic, and biochemical effects of LITS on intestinal adhesions. This also made it possible to compare the effect of additional LITS on intestinal adhesions with that of enterolysis alone. Our macroscopic analysis of intestinal adhesion length and severity showed that LITS, as compared to simple enterolysis, resulted in neither new adhesions nor aggravated pre-existing adhesions. The formation of intestinal adhesions after laparotomy is resulted from multiple pathological processes, including tissue inflammation, fibrin deposition, fibrin organization, collagen formation, and adhesive tissue maturation. Serosal fibrosis grading is a primary microscopic measure of intestinal adhesions. Our microscopic and biochemical results showed that LITS did not worsen adhesions between the intestinal loops. According to our histological results, terminal ileal wall fibrosis after LITS was not significantly different from that occurring after simple enterolysis. This finding suggests that the local formation of fibroconnective tissue in the affected area were similar in the two groups. Collagen, a long, fibrous structural protein, is the major component of adhesive tissues [24] and enriched with hydroxyproline (up to 14%). Hydroxyproline content has been historically used for the estimation of collagen content in a given tissue [24], and is considered an accurate, objective parameter for the estimation of intestinal loop adhesions [25]. In the present study, the hydroxyproline content was similar between the two groups at the time of euthanasia, although the content increased significantly in both groups after enterolysis. This finding biochemically confirmed that LITS did not further aggravate intraabdominal adhesions compared to simple enterolysis. In conclusion, the placement of a long intestinal tube after enterolysis in a setting of experimental dense intestinal adhesions did not aggravate adhesions between intestinal loops, as proven by macroscopic, microscopic, and biochemical assay results. A long intestinal tube can act as an effective, temporary stent that favors the formation of orderly intestinal loop adhesion and prevents the recurrence of ASBO after surgical intervention, without exacerbating intestinal adhesions. Declaration of interest: The authors have no conflicts of interest. The authors alone are responsible for the content and writing of the article.
REFERENCES [1] Di Saverio S, Catena F, Ansaloni L, et al. Water-soluble contrast medium (gastrografin) value in adhesive small intestine obstruction (ASIO): a prospective, randomized, controlled, clinical trial. World J Surg. 2008;32:2293–2304. [2] Menzies D, Ellis H. Intestinal obstruction from adhesions: how big is the problem? Ann R Coll Surg Engl. 1990;72:60–63. [3] Landercasper J, Cogbill TH, Merry WH, et al. Long-term outcome after hospitalization for small-bowel obstruction. Arch Surg. 1993;128:765–770. [4] Barkan H, Webster S, Ozeran S. Factors predicting the recurrence of adhesive small-bowel obstruction. Am J Surg. 1995;170:361–365. [5] Fevang BT, Fevang J, Stangeland L, et al. Complications and death after surgical treatment of small bowel obstruction: a 35-year institutional experience. Ann Surg. 2000;231:529–537. [6] Duron JJ, Silva NJ, du Montcel ST, et al. Adhesive postoperative small bowel obstruction: incidence and risk factors of recurrence after surgical treatment: a multicenter prospective study. Ann Surg. 2006;244:750–757. [7] Robbins RD, Hayes SR, Thow GB. Long-tube gastrostomy with internal intestinal splinting: ten-year Experience. Dis Colon Rectum. 1980;23:10–16. [8] Sprouse LR, Arnold CI, Thow GB, et al. Twelve-year experience with the Thow long intestinal tube a means of preventing postoperative bowel obstruction. Am Surg. 2000; 67:357–360. [9] Weigelt JA, Snyder WH, Norman JL. Complications and results of 160 Baker tube publications. Am J Surg. 1980;140:810–815. [10] Wei B, Wei HB, Guo WP, et al. Diagnosis and treatment of abdominal cocoon: a report of 24 cases. Am J Surg. 2009;198:348–353. [11] Ikeda M, Sorimachi K, Akimoto K, et al. Reversed-phase high-performance liquid chromatographic analysis of hydroxyproline and proline from collagen by derivatization with dabsyl chloride. J Chromatogr. 1993;621:133–138. ˘ Y, Baykal A, Onat D, et al. An experimental study [12] Ozogul of the effect of aprotinin on intestinal adhesion formation. Am J Surg. 1998;175:137–141. [13] Zuhlke HV, Lorenz EM, Straub EM, et al. Pathophysiology and classification of adhesions. Langenbecks Arch Chir Suppl II Verh Dtsch Ges Chir. 1990;1009–1016. [14] Lalountas M, Ballas KD, Michalakis A, et al. Postoperative adhesion prevention using a statin-containing cellulose film in an experimental model. Br J Surg. 2012; 99:423–429. [15] White RR. Prevention of recurrent small bowel obstruction due to adhesions. Ann Surg. 1956;143:714–719. [16] Freeman ML, Saed GM, Elhammady EF, et al. Expression of transforming growth factor beta isoform mRNA in injured peritoneum that healed with adhesions and without adhesions and in uninjured peritoneum. Fertil Steril. 2003;80:708–713. [17] Polishuk WZ, Aboulafia Y. Dextran in prevention of peritoneal adhesions. Isr J Med Sci. 1967;3:806–808. [18] Rajab TK, Wauschkuhn CA, Smaxwil L, et al., An improved model for the induction of experimental adhesions. J Invest Surg. 2010;23:35–39. [19] Fazel MZ, Jamieson RW, Watson CJ. Long-term follow-up of the use of the Jones intestinal tube in adhesive small bowel obstruction. Ann R Coll Surg Engl. 2009; 91:50–54. [20] Rodriguez-Ruesga R, Meagher AP, Wolff BG. Twelve-year experience with the long intestinal tube. World J Surg. 1995;19:627–630. [21] Meissner K. Effectiveness of intestinal tube splinting: a prospective observational study. Dig Surg. 2000;17:49–56. Journal of Investigative Surgery
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[22] Heydinger DK, Taylor PH, Roettig LC. Recurrent intestinal obstruction. Arch Surg. 1960; 80:670–676. [23] Cheung M, Chapman M, Kovacik M, et al. A method for the consistent creation and quantitative testing of postoperative pelvic adhesions in a porcine model. J Invest Surg. 2009;22:56–62.
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[24] Geles K, Poschl E, Aigner T. Collagens-structure, function, and biosynthesis. Adv Drug Deliv Rev. 2003;55:1531– 1546. ¨ [25] Binnebosel M, Klinge U, Rosch R, et al. Morphology, quality, and composition in mature human peritoneal adhesions. Langenbecks Arch Surg. 2008;393:59–66.
NEW METHODOLOGIES
Does Long Intestinal Tube Splinting Aggravate Intestinal Adhesions? Min Li, MD, Gang Wang, MD, Bo Zhou, MD, Xianfeng Xia, MD, Ning Li, MD, PhD
J Invest Surg Downloaded from informahealthcare.com by Universitat de Girona on 12/19/14 For personal use only.
Department of General Surgery, Jinling Hospital, Medicine School of Nanjing University, Nanjing, Jiangsu Province, China
ABSTRACT Objective: Long intestinal tube splinting (LITS) is useful for clinically reducing the recurrence of adhesive small bowel obstruction (ASBO). However, a controversy exists whether LITS aggravates intestinal adhesions. This study evaluated the postoperative effects of LITS relative to simple enterolysis on intestinal adhesions in an experimental porcine model. Methods: A porcine model (n = 24) of dense intestinal adhesion was established by abrading the ileal wall with sterile P240 sandpaper. Enterolysis was performed on postoperative day 14. Animals were randomly divided into a group that underwent enterolysis only (control; n = 12) and those who underwent LITS as well as enterolysis (LITS; n = 12). The long intestinal tube was removed on post-LITS day 14, after abdominal radiography. All animals were euthanized on postenterolysis day 28 for assessment of intestinal adhesions using a semiquantitative macroscopic grading scale, hematoxylin-eosin histology, and hydroxyproline assay. Results: Prior to enterolysis, the experimentally induced intestinal adhesions of the two groups were similar in extent and severity. On postenterolysis day 28 the LITS and control groups were comparable with regard to adhesion loop length (p = .440), macroscopic adhesion severity (p = .820), serosal fibrosis grading (p = .450), and hydroxyproline content of the adhesion ileal segment (p = .630). Conclusion: Placement of the long intestinal tube did not aggravate intestinal adhesions over that of simple enterolysis in this intestinal adhesion porcine model. Keywords: splinting; long intestinal tube; adhesion; intestine; severity; swine model
INTRODUCTION
crease risks of iatrogenic intestinal injury and intraabdominal hemorrhage [10]. There is a knowledge gap in the current literature regarding whether LITS aggravates postoperative intestinal adhesions. In this study, we evaluated the effect of LITS on the severity of intestinal adhesions after enterolysis in a porcine model of dense intestinal adhesion with respect to macroscopy and histology. In particular, we further examined hydroxyproline content, a more sensitive, objective measure for assessing tissue adhesions compared to conventional macroscopic grading, in adhesive tissues [11].
Adhesive small bowel obstruction (ASBO) is a major cause of morbidity and hospital readmission after abdominal surgery, and its occurrence places substantial burden on public healthcare systems worldwide [1]. The incidence of ASBO after all laparotomies is approximately 3% [2]. More unfortunate, ASBO is refractory to surgical intervention, and the recurrence rate is 16%–46% among surgically treated ASBO patients [3–6]. Long intestinal tube splinting (LITS), applied surgically, allows intestinal loops to adhere permanently to each other in an orderly pattern, and can effectively limit the recurrence of postoperative ASBO to 5.9%–12.9% [7–9]. However, the use of LITS has raised concerns that the long intestinal tube may exacerbate the severity of intestinal adhesions and complicate any further surgical intervention if obstruction recurs [10]. Aggravation of intestinal adhesions is known to in-
MATERIALS AND METHODS Laboratory Animals The Institutional Animal Care and Use Committee at Jinling Hospital approved the study protocol, which
Received 21 May 2013; accepted 5 November 2013. Address correspondence to Ning Li, MD, PhD, Department of General Surgery, Jinling Hospital, Medicine School of Nanjing University, NO 305 East Zhongshan Road, Nanjing, Jiangsu Province, China. E-mail: [email protected]
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was performed in accordance with the Guide for Care and Use of Laboratory Animals established by the National Institutes of Health of the United States and China’s legal regulations. Thirty female laboratory swine, weighing 24.3 ± 4.2 kg, were housed at the Center for Laboratory Animals of Jinling Hospital. Prior to anesthesia, all animals were deprived of chow for 12 hr and drinking water for 4 hr.
J Invest Surg Downloaded from informahealthcare.com by Universitat de Girona on 12/19/14 For personal use only.
Anesthetic Procedure Each swine was anesthetized with 40 mg/kg intramuscular ketamine and 0.06 mg/kg intramuscular atropine, and subsequently placed in the supine position. Venous access was established through the auricular vein, and a single dose of 50 mg/kg ceftazidime was given intravenously 15 min before endotracheal intubation. An intravenous bolus of 1 mg/kg propofol emulsion (2%; Astra Zeneca, Wedel, Germany) was given to facilitate endotracheal intubation. An 8-mm cuffed endotracheal tube was inserted into the trachea through the oral route using a direct laryngoscope. The animal was ventilated using a large animal ventilator, and anesthesia was maintained with continuous infusion of intravenous 2% propofol emulsion titrated at 9–20 mg/kg·h combined with ketamine at 0.3–0.6 mg/kg·hand fentanyl at 2–5 μg/kg·h (both: Janssen Cilag, Neuss, Germany). All animals were monitored throughout anesthesia and surgery with an automatic electrocardiography monitoring system. After the surgery, when the animal exhibited motorsensory reflex return and spontaneous breathing, the narcotics were down-titrated and the tracheal tube was removed.
Surgical Procedures All surgical procedures were performed using standard aseptic technique. The entire abdominal skin was routinely shaved, prepared, and draped. Six swine were euthanized with a lethal overdose of intravenous propofol for the baseline assessment. A 5-cm ileal segment, located 50 cm proximal to the ileocecal valve,
TABLE 1
was harvested for routine histology and hydroxyproline content assay. Twenty-four swine were used to create a modification of the experimental intestinal adhesion model described by Yusuf and his colleagues [12]. Sterile P240 sandpaper (rather than gauze) was used to induce dense intestinal adhesions. A 5-cm left paramedian skin incision was made, and a 150-cm-long ileal segment, immediately proximal to the ileocecal valve, was exteriorized. Both surfaces of the ileal wall were abraded with five strokes to create experimental serosal defects 150 cm in length. The exteriorized ileal segment was reduced into the abdominal cavity free of torsion or tension. The fascial and cutaneous layers were separately sutured using continuous 3/0 silk sutures to close the abdominal wall incision. All animals were fasted for three successive days but given intravenous infusions 1,000 ml of 5% warm glucose saline containing 2 g of potassium chloride once daily, 100 ml of 10% sodium chloride solution containing 50 mg/kg of cefazolin sodium twice daily, and 100 ml of 10% sodium chloride solution containing 1 mg/kg of parecoxib sodium twice daily. The animals had free access to swine chow and drinking water after this period. Enterolysis was performed on all 24 swine on postoperative day 14. The abdominal cavity was explored via the right paramedian incision. The extent and severity of intestinal adhesions were evaluated using a semiquantitative macroscopic grading scale as previously reported by Zuhlke et al. [13] (Table 1). The adhesions were completely dissected, and a 5-cm-long ileal segment, located 50 cm proximal to the ileocecal valve, was harvested for routine histology and hydroxyproline content assay. Bowel continuity was restored by one-layer end-to-end anastomosis using 3/0 polydioxanone suture (Johnson & Johnson Medical, Livingston, UK). After enterolysis, dense intestinal adhesion model animals were equally and randomly divided into two groups, namely, an LITS group undergoing LITS (n = 12) and a control group undergoing no further surgical intervention (n = 12). In the LITS group, a 4/0 purse-string silk suture (Johnson & Johnson Medical, Livingston, UK) was used to circle the antimesenteric margin of the ileal segment located 10 cm proximal to
Scoring system for classifying macroscopic intestinal adhesion length and severity [13]
Score
Adhesion length
0 1 2
No adhesions 150 cm
Adhesion severity No adhesions Filmy adhesion, easy to separate by blunt dissection Stronger adhesion; blunt dissection possible, partly sharp dissection necessary; beginning of vascularization Strong adhesion; division possible by sharp dissection; clear vascularization Very strong adhesion between organs; its division by sharp dissection damages organ serosa
Journal of Investigative Surgery
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FIGURE 1 LITS experimental procedure. (A) 150-cm-long 10F silicone catheter; (B) formation of extensive, dense adhesions between the loops of the terminal ileum 14 days after serosal abrasion; (C) insertion of silicone catheter after enterolysis; and (D) histological presence of massive adhesive tissues on the serosal surface (H&E; original magnification, 400×).
the ileocecal valve. A 0.5-cm circumferential incision was then made on the ileal wall. A 150-cm 10F silicone catheter (Figure 1A) was entirely inserted into the intestinal lumen through the ileal wall opening cephalad (Figure 1C). The purse-string suture was subsequently tightened if the catheter and the intestines showed no invagination or torsion. A second purse-string suture was used to enhance the closure of the ileal wall opening. The catheter was brought out 2 cm against the parietal peritoneum and through a 0.5-cm incision in the left flank. The needle of the second purse-string suture was passed through the parietal peritoneum adjacent to the flank incision, and appropriately tightened to secure the position of the catheter stoma. The catheter was further secured using a cutaneous silk suture. The postoperative care regimen was identical to that after the creation of experimental intestinal adhesions described above. LITS swine were sedated with ketamine for plain abdominal radiography to position the long intestinal tube on post-LITS day 14. Thirtymilliliters of liquid paraffin were injected through the catheter, and the catheter was gently retracted and removed. All surviving animals were humanely euthanized on postenterolysis day 28 with a lethal overdose of intravenous bolus of propofol (15–20 mg/kg). The entire abdominal cavity was macroscopically examined through a U-shaped incision, and the extent and severity of intestinal adhesions were determined using the semiquantitative macroscopic grading scale C
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[13]. A 5-cm-long ileal segment that was located 10 cm proximal to the anastomosis was sharply dissected for routine histology and hydroxyproline content assay of the terminal ileal segment.
Macroscopic and Histopathological Examinations The terminal ileal segment specimen was fixed in 10% buffered formaldehyde and embedded in paraffin. Four-micron sections were routinely stained with hematoxylin and eosin (H&E), in triplicate. A boardcertified clinical pathologist, blinded to the treatment group, evaluated the severity of ileal wall fibrosis under a light microscope using a semiquantitative microscopic grading scale [14]: grade 0, none; grade 1, mild; grade 2, moderate; and grade 3, severe.
Hydroxyproline Assay Terminal ileal segments were snap frozen in liq◦ uid nitrogen and stored at –80 C until used for further experiments. Terminal ileal segment samples were initially lyophilized, scaled, pulverized, and repeatedly lyophilized. The samples were further hydrolyzed with 6 N hydrogen chloride and derivatized with phenylisothiocyanate. The hydroxyproline
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content was determined using high-performance liquid chromatography and expressed as μg/mg of dry tissue [11].
Statistical Analyses All data were processed using the statistical software SPSS 18.0 for Windows (SPSS, Chicago, IL, US) and expressed as mean ± standard deviation. Differences in the means of the two groups were compared using the Mann–Whitney U test. A p-value less than .05 was considered statistically significant.
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RESULTS All the six animals euthanized for the baseline assessment showed no macroscopically visible intraabdominal adhesions or histologically significant serosal fibrosis. The mean basal hydroxyproline content of the terminal ilea was 7.5 ± 0.5 μg/mg of dry tissue. All the 24 dense intestinal adhesion model animals survived serosal abrasion, and during enterolysis dense adhesions among terminal ileal loops were observed macroscopically (Figure 1B), with significant serosal fibrosis viewed under the light microscope (Figure 1D). The mean scores for intestinal adhesion length, macroscopic adhesion severity, and microscopic serosal fibrosis prior to enterolysis were 3.3 ± 0.4, 3.4 ± 0.8, and 2.3 ± 0.7, respectively; hydroxyproline content in the terminal ileal segment was 19.5 ± 2.7 μg/mg of dry tissue. All these parameters were comparable between the LITS group and the control group (all p-values > .05; Table 2) and increased significantly in both groups after experimental serosal abrasion compared with the baseline (all p-values < .05). One pig in the LITS group and one in the control group did not survive the enterolysis due to enterocutaneous fistula; 2 in the control group succumbed due to ASBO. The remaining 20 swine survived uneventfully until the completion of this study and were euthanized as scheduled. As observed on plain abdominal radiography, all the long intestinal tubes were wellpositioned in situ in the LITS swine (Figure 2), and all
TABLE 2 Length of intestinal adhesion, macroscopic adhesion severity, serosal fibrosis grading, and ileal hydroxyproline content prior to enterolysis
Intestinal adhesion length Macroscopic adhesion severity Serosal fibrosis grading Hydroxyproline content (μg/mg)
LITS
Control
p-value
3.3 ± 0.5 3.5 ± 0.7
3.2 ± 0.4 3.2 ± 0.8
.800 .530
2.3 ± 0.8 19.7 ± 2.9
2.3 ± 0.7 19.2 ± 2.6
.900 .730
FIGURE 2 Macroscopic photographs (2A and 1A) of ileal loop adhesions and photomicrographs (2B and 1B) of serosal tissue fibrosis (H&E; original magnification, 400×) at necropsy 28 days after LITS or enterolysis alone.
these tubes were smoothly removed 14 days after the LITS operation. All intestinal adhesion model animals had dense adhesions between the loops of the 150-cm-long terminal ileal segment, the extent and severity of which were macroscopically similar between the two groups. However, intestinal loop adhesion in the LITS group was in a relatively orderly pattern (Figure 3, 2A) as compared with that in the control group (Figures 3 and 1A). The percentage of animals with adhesions of proximal intestinal loops to the affected terminal ileal segment was similar in the LITS group (8/11, 72.7%) to that of the control group (5/9, 55.6%; p > .05). Histological examination showed that massive serosal fibrosis was present in all 20 surviving animals and was similar between the two groups (Figure 3, 2B and 1B). Overall, intraabdominal adhesions were comparable between the two groups, in terms of intestinal adhesion length, macroscopic adhesion severity, and microscopic serosal fibrosis (all p-values > .05; Figure 4). The hydroxyproline content of the terminal ileal segment increased significantly in both groups compared with the content prior to enterolysis (both p-values < .05), but remained similar between the two groups (LITS vs. control, 29.4 ± 9.3 μg/mg vs. 27.8 ± 6.6 μg/mg, p > .05; Figure 5).
DISCUSSION Long intestinal tube stenting, splinting the small bowel in a gentle curve, was first proposed by White in 1956 [15]. This surgical technique prevents recurrent ASBO by encouraging the formation of intestinal loop Journal of Investigative Surgery
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FIGURE 3 Length of intestinal adhesions, macroscopic adhesion severity, and serosal fibrosis grading at necropsy 28 days after LITS or enterolysis alone. (3.73 ± 0.47 vs. 3.56 ± 0.53, p = .440; 3.82 ± 0.40 vs. 3.78 ± 0.44, p = .820; 2.82 ± 0.40 vs. 2.67 ± 0.50, p = .450)
adhesions in a nonobstructed configuration. It has been documented that LITS can reduce the risk of ASBO recurrence more effectively than simple enterolysis [7–9]. However, some authors assume that this procedure could aggravate intestinal adhesions, although no follow-up histological evidence after LITS has been available [10]. To the best of our knowledge, the present study is the first to evaluate with macroscopic, microscopic, and biochemical evidence of the extent and severity of intestinal adhesions after LITS in an animal model.
A swine model was used in this study, because the peritoneal cavity of swine is similar to that of humans in terms of size and anatomy. Abrasion of the intestinal serosa is reported to be a reliable technique for the creation of an animal model of intestinal adhesion [12, 16, 17]. However, in most cases, this method has resulted in filmy adhesions [18]. We used P240 sandpaper instead of gauze in our study to damage the serosal surfaces, to induce extensive, dense intestinal adhesions similar to those encountered by surgeons when performing LITS [19–21]. Previous studies showed that extensive
FIGURE 4 Terminal ileum hydroxyproline content at necropsy 28 days after LITS or enterolysis alone.
FIGURE 5 Length of intestinal adhesion, macroscopic adhesion severity, serosal fibrosis grading, and ileal hydroxyproline content prior to enterolysis.
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intestinal adhesions had formed 10 days after abrasion [22, 23]; in our present study, enterolysis was performed on postoperative day 14. During laparotomic explorations before enterolysis, we found that extensive, dense intestinal adhesions were present in all the animals. This indicates that our modified technique was more effective in creating an animal model with dense intestinal adhesions. Our modification was proved to be reliably consistent and replicable, which ensured the validity of comparisons between the LITS and control groups for evaluating the macroscopic, microscopic, and biochemical effects of LITS on intestinal adhesions. This also made it possible to compare the effect of additional LITS on intestinal adhesions with that of enterolysis alone. Our macroscopic analysis of intestinal adhesion length and severity showed that LITS, as compared to simple enterolysis, resulted in neither new adhesions nor aggravated pre-existing adhesions. The formation of intestinal adhesions after laparotomy is resulted from multiple pathological processes, including tissue inflammation, fibrin deposition, fibrin organization, collagen formation, and adhesive tissue maturation. Serosal fibrosis grading is a primary microscopic measure of intestinal adhesions. Our microscopic and biochemical results showed that LITS did not worsen adhesions between the intestinal loops. According to our histological results, terminal ileal wall fibrosis after LITS was not significantly different from that occurring after simple enterolysis. This finding suggests that the local formation of fibroconnective tissue in the affected area were similar in the two groups. Collagen, a long, fibrous structural protein, is the major component of adhesive tissues [24] and enriched with hydroxyproline (up to 14%). Hydroxyproline content has been historically used for the estimation of collagen content in a given tissue [24], and is considered an accurate, objective parameter for the estimation of intestinal loop adhesions [25]. In the present study, the hydroxyproline content was similar between the two groups at the time of euthanasia, although the content increased significantly in both groups after enterolysis. This finding biochemically confirmed that LITS did not further aggravate intraabdominal adhesions compared to simple enterolysis. In conclusion, the placement of a long intestinal tube after enterolysis in a setting of experimental dense intestinal adhesions did not aggravate adhesions between intestinal loops, as proven by macroscopic, microscopic, and biochemical assay results. A long intestinal tube can act as an effective, temporary stent that favors the formation of orderly intestinal loop adhesion and prevents the recurrence of ASBO after surgical intervention, without exacerbating intestinal adhesions. Declaration of interest: The authors have no conflicts of interest. The authors alone are responsible for the content and writing of the article.
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