j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e7
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The neurokinin 1 receptor regulates peritoneal fibrinolytic activity and postoperative adhesion formation Michael R. Cassidy, MD, Holly K. Sheldon, MD, Melanie L. Gainsbury, MD, Earl Gillespie, PhD, Hisashi Kosaka, MD, PhD, Stanley Heydrick, PhD, and Arthur F. Stucchi, PhD* Department of Surgery, Boston University School of Medicine, Boston, Massachusetts
article info
abstract
Article history:
Background: Intra-abdominal adhesions are a common source of postoperative morbidity.
Received 8 March 2014
Previous studies in our laboratory have shown that a neurokinin 1 receptor antagonist (NK-
Received in revised form
1RA) reduces abdominal adhesion formation and increases peritoneal fibrinolytic activity.
1 April 2014
However, the cellular pathway by which the antagonist exerts its effects is unclear, as cultured
Accepted 15 April 2014
peritoneal mesothelial cells exposed to the NK-1RA show increases in fibrinolytic activity
Available online xxx
despite having very low expression of neurokinin 1 receptor (NK-1R) messenger RNA and protein. Our aim was to determine whether the NK-1R plays an essential role in the adhesion-
Keywords:
reducing effects of the NK-1RA, or if the NK-1RA is acting independently of the receptor.
Intra-abdominal adhesions
Methods: Homozygous NK-1R knockout mice and age matched wild-type mice underwent
Neurokinin-1 receptor
laparotomy with cecal cautery to induce adhesions. At the time of surgery, mice received a
Neurokinin-1 receptor antagonist
single intraperitoneal dose of either NK-1RA (25 mg/kg) or saline alone. Adhesion severity
Substance P
at the site of cecal cautery was assessed on postoperative day 7. In a separate experiment,
Tissue plasminogen activator
peritoneal fluid was collected from wild type and NK-1R knockout mice 24 h after lapa-
Fibrinolytic activity
rotomy with cecal cautery and administration of either NK-1RA or saline. Tissue plasminogen activator levels, representative of total fibrinolytic activity, were then measured in peritoneal fluid. Results: In wild-type mice, NK-1RA administration significantly decreased adhesion formation compared with saline controls. Among the NK-1R knockout mice, there was no significant reduction in adhesion formation by the NK-1RA. Fibrinolytic activity increased 244% in wild-type mice administered NK-1RA compared with saline controls; however, the NK-1RA did not raise fibrinolytic activity above saline controls in NK-1R knockout mice. Conclusions: These data indicate that the NK-1R mediates the adhesion-reducing effects of the NK-1RA, in part, by the upregulation of peritoneal fibrinolysis, and suggest that the NK1R is a promising therapeutic target for adhesion prevention. ª 2014 Elsevier Inc. All rights reserved.
Presented, in part, at the seventh Academic Surgical Congress, Las Vegas, Nevada, February 14e16, 2012, and published in abstract form in J Surg Res 172(2): 340; 2012. * Corresponding author. Department of Surgery, Boston University School of Medicine, Section of Surgical Research, 700 Albany Street W408E, Boston, MA 02118. Tel.: þ1 617 638 8665; fax: þ1 617 638 8662. E-mail address: [email protected] (A.F. Stucchi). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.04.030
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1.
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e7
Introduction
Although surgeons have recognized postoperative intraperitoneal adhesions for nearly two centuries, their prevalence remains disturbingly high and unacceptable [1]. Nearly 100% of patients who have undergone an open abdominopelvic operation will develop adhesions, classifying them as the most frequent postoperative complication [2]. The consequences of adhesions can cause life-threatening sequelae such as small bowel obstruction, debilitating abdominal and pelvic pain, and secondary infertility in women [1]. The treatment of adhesion-related complications is estimated to be as high as $5 billion annually, incurring a considerable burden on the US health care system [3,4]. Despite the severity and frequency of complications due to adhesions, efforts to curtail their formation to date have emphasized meticulous surgical technique and application of a barrier, such as seprafilm [5,6]. Although physical barriers are effective [7], there are significant limitations to their use, including difficulty of application and limited use in minimally invasive and laparoscopic procedures [8,9]. In addition, we have shown that physical barriers only prevent adhesions where directly applied and have no remote efficacy [10]. There is also evidence suggesting that barrier methods fail to consistently prevent small bowel obstructions, despite their efficacy in adhesion reduction [11e13]. Thus, the current state of adhesion prevention is inadequate in the clinical realm, and novel preventative strategies are needed to reduce the high incidence of morbidity. Only with an understanding of the basic biology of adhesion formation can we hope to reduce the incidence of adhesions and their associated complications [6]. Although the cellular and molecular events that underlie adhesiogenesis are multifactorial, decades of research have firmly established the central role that the peritoneal fibrinolytic system plays in modulating intra-abdominal adhesion formation [14e17]. Peritoneal fibrinolysis is regulated primarily by tissue plasminogen activator (tPA) and its principal inhibitor, plasminogen activator inhibitor 1 (PAI-1) [18]. After an abdominal operation, tissue injury and ischemia prompts a peritoneum-wide inflammatory response that rapidly downregulates the peritoneum’s fibrinolytic capacity by reducing tPA and/or increasing PAI-1 [19]. Many adhesion biologists feel this is a key underlying event in the pathogenesis of adhesion formation [17]. Our laboratory has sought to identify pharmacologic interventions that mediate adhesion formation in an effort to better define the pathophysiology of adhesion biology. Early work in a rat model of adhesion formation implicated the inflammatory neuropeptide substance P (SP) in adhesiogenesis, as messenger RNA expression for SP and its receptor, the neurokinin 1 receptor (NK-1R), was upregulated in peritoneal adhesion tissue shortly after surgery [20]. We subsequently showed that a neurokinin 1 receptor antagonist (NK-1RA) administered as a single intraoperative dose reduces postoperative adhesions by increasing peritoneal fibrinolysis [21]. Although the cellular mechanism(s) by which the NK-1RA increases peritoneal fibrinolytic activity has remained elusive, unpublished in vitro experiments from our laboratory showed that when cultured human mesothelial cells were incubated
with the NK-1RA, fibrinolytic activity increased >5-fold despite the very low expression levels of NK-1R messenger RNA and protein. This finding led us to question whether the NK-1RA increases fibrinolytic activity by binding to the NK-1R and interfering with the proinflammatory signaling of SP, or if the antagonist signals independently of the receptor through an off-target pathway. To address this mechanism, we used an NK-1R knockout mouse model of adhesion formation to definitively establish the role of NK-1R modulating peritoneal fibrinolysis and adhesion prevention.
2.
Materials and methods
2.1.
Materials
The NK-1RA (3R,4S,5S,6S)-6-diphenylmethyl-5-(5-isopropyl-2methoxybenzlyamino)-1-azabicyclo[2,2,2]octane-3-carboxylic acid (CJ-12,255) was obtained from Pfizer (Groton, CT). This antagonist is highly specific for the NK-1R with no affinity for the NK-2 or NK-3 receptors.
2.2.
Animals
Homozygous NK-1R/ mice (25e30 g) lacking the NK-1R gene were generated as described [22] and bred at the Boston University School of Medicine Laboratory Animal Science Center. The original breeding pair was a gift from Dr Norma Gerard (Harvard Medical School, Boston, MA) to Dr Arthur F. Stucchi, a coauthor of this article. Age-matched male wild-type mice (20e25 g) were purchased from Charles River (Wilmington, MA). All animals were housed at constant room temperature and under 12-h lightedark cycles and allowed access to food and water ad libitum. The Institutional Animal Care and Use Committee at the Boston University School of Medicine approved this study, and all procedures described were performed in accordance with recommendations outlined in the Guide for the Care and Use of Laboratory Animals: Eighth Edition (NRC 2011) published by the National Academies of Science.
2.3.
Induction of adhesion formation
Mice underwent laparotomy with cecal cautery (Valleylab Force EZ Instant Response Electrosurgical Generator, Pfizer), a validated model of adhesion formation, to induce intraperitoneal adhesions [23]. Briefly, mice were anesthetized with isoflurane and a 1 cm laparotomy was performed through which the cecum was identified and expressed (Fig. 1A). Using bipolar forceps, a thermal defect (5 W, w7 U for 1 s) was created on the antimesenteric side of the cecum approximately 5 mm from the tip (Fig. 1B). The cautery defect did not perforate the cecum (Fig. 1C). The cecum was returned to the peritoneal cavity and the wound closed in two layers. Mice survived until postoperative day 7, when adhesions were scored as described in the following, respectively. In separate experiments, mice survived for 24 h, at which time they were sacrificed and peritoneal fluid collected for fibrinolytic activity as described in the following, respectively.
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e7
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Fig. 1 e Cecal cauterization mouse model was used to produce quantitative adhesions. (A) A 1 cm laparotomy was performed, after which the cecum was identified and expressed through the incision; (B) Using bipolar forceps, a thermal defect (5 W, w7 U for 1 s) was created on the antimesenteric side of the cecum approximately 5 mm from the tip; and, (C) The resulting cautery injury (arrow) to the cecum did not cause perforation. Standard scoring system of 1e5 based on adhesion severity as described previously [24]. (D) 0, no adhesion; (E) 1, one thin filmy adhesion; (F) 2, more than one thin adhesion; (G) 3, thick adhesion with focal point; (H) 4, thick adhesion with plantar attachment or more than one thick adhesion with focal point; I) 5, very thick vascularized adhesion or more than one plantar adhesion. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.4.
Assessment of NK-1RA on adhesion formation
Wild type and NK-1R knockout mice underwent laparotomy with cecal cautery as described previously (n ¼ 43). Animals were randomized to receive either a single intraoperative dose of 25 mg/kg NK-1RA in 0.5 mL normal saline, or 0.5 mL normal saline alone. Seven days later, adhesions to the site of cecal cautery were assigned a numerical score from 0e5 (Table) based on adhesion number, density, and tenacity as shown in Figure 1 and as previously described [24]. Animals were each assigned an adhesion score by two independent observers, one of whom was blinded to the treatment group. The two scores for each animal were averaged to determine the final adhesion score used for analysis.
2.5.
Fibrinolytic activity in peritoneal fluid
NK-1RA or saline as previously mentioned (n ¼ 24). After 24 h, peritoneal fluid was collected, acetate treated, and frozen at 80 C until use. A tPA activity bioassay was performed as we have described previously [25]. In brief, fluid samples were thawed on wet ice and acidified with 0.2 volumes 0.375 N hydrochloric acid and then diluted 10-fold with distilled water. The fibrinolytic activity of tPA in each sample was
Table e Adhesion scoring scale. Score 0 1 2 3 4 5
To evaluate the fibrinolytic activity in peritoneal fluid, wild type and NK-1R knockout mice underwent laparotomy and cecal cautery with intraoperative administration of 25 mg/kg
Description of adhesion No adhesions One thin filmy adhesion More than one thin adhesion Thick adhesion with focal point Thick adhesion with plantar attachment or more than one thick adhesion with focal point Very thick, vascularized adhesion or more than one plantar adhesion
Adapted from Kennedy et al. [24].
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assayed in duplicate by adding 50 mL of the diluted sample to each well of a 96-well microtiter plate containing 50 mL tPA stimulator (0.6 mg/mL cyanogen bromide-digested fibrinogen; American Diagnostica, Stamford, CT). Next, 150 mL of assay buffer (16.7 mg/mL human plasminogen (Athens Research and Technologies, Athens, GA), 667 mmol/L S-2251 substrate (American Diagnostica), and 20 mmol/L Tris, pH 8.3) were added to each well and gently mixed. Cleavage of the S-2251 substrate by tPA-activated plasmin produces a yellow color that absorbs at 405 and 490 nm (calibration blank). The change in absorbance was measured at 37 C over the course of 10 h using a spectrophotometric microplate reader (SpectraMax 250; Molecular Devices, Sunnyvale, CA). The fibrinolytic activity of tPA in each sample was determined by extrapolation from a tPA (humans; Calbiochem, San Diego, CA) standard curve.
2.6.
Statistical analysis
Data were analyzed either with 1-way analysis of variance or the Student t-test where appropriate using analytical software (Sigma-Stat; SPSS Inc, Chicago, IL). In all analyses, before parametric statistical analyses, if the analyses of the data failed either normality or equivariance testing, log10 transformation was performed. If the data still failed these criteria, the nonparametric Dunn test of analysis of variance by ranks followed by the appropriate nonparametric post hoc test was used. When significant effects (P < 0.05) were detected, the differences between specific multiple means were determined by post hoc analysis with the StudenteNewmaneKeuls test. Groups were deemed significantly different from one another when P < 0.05. All the data are expressed as means standard error of the mean.
3.
Results
3.1. An NK-1RA reduces adhesions in wild type but not in NK-1R knockout mice In wild-type mice, NK-1RA administration at the time of surgery significantly decreased adhesion severity compared with saline controls (Fig. 2). Animals administered saline developed thick, vascularized, tenacious adhesions by postoperative day 7 as shown in Figure 1H and I, with a mean adhesion severity score of 4.8 0.1 (min: 3.5dmax: 5) whereas wild-type animals administered NK-1RA had thin, filmy adhesions as shown in Figure 1E and F, with a mean adhesion score of 2.5 0.5 (min: 0dmax: 5) (P 0.05). In contrast, the administration of an NK-1RA to the NK-1R knockout mice failed to reduce adhesion formation. All knockout animals developed thick, tenacious adhesions, with no statistical differences noted between animals administered saline and those administered NK-1RA. Mean severity scores in these groups were 3.7 0.4 (min: 2dmax: 5) and 4.4 0.3 (min: 3dmax: 5), respectively (Fig. 2). Both surgeons who scored the adhesions (M.R.C. and H.K.) were experienced with the model and there was no statistical difference between the average of the two scores (P ¼ 0.44).
Fig. 2 e Adhesion formation in wild-type (n [ 22) and NK1R knockout (n [ 21) mice exposed to cecal cautery. Wildtype mice have significantly decreased adhesion formation in response to treatment with an NK-1R antagonist; NK-1R knockout mice do not. In addition, untreated NK-1R knockout mice have a slight reduction in adhesion formation compared with wild-type controls. *P £ 0.05 compared with saline treated wild type.
3.2. An NK-1RA increases peritoneal fibrinolytic activity in wild type but not in NK-1R knockout mice In wild-type mice, fibrinolytic activity in peritoneal fluid at 24 h postoperatively was increased 344% in animals administered NK-1RA compared with those administered saline (Fig. 3). tPA concentration was 4.44 0.83 U/mL in NK-1RAtreated mice and 1.29 0.11 in saline-treated mice (P 0.05). In contrast, the administration of an NK-1RA to the NK-1R knockout mice failed to increase fibrinolytic activity above baseline levels in the wild-type saline controls, where tPA concentration was 1.64 0.11 U/mL in NK-1RA-treated mice and 1.28 0.13 U/mL in saline-treated mice.
4.
Discussion
Prior studies from this laboratory have demonstrated that a single intraoperative dose of an NK-1RA significantly reduced intra-abdominal adhesion formation primarily by increasing peritoneal tPA activity in the immediate postoperative period [21,25e30]. Although these earlier findings strongly implicate SP and the NK-1R pathway in adhesiogenesis, we have been unable to definitively establish whether the NK-1R participates in mediating the fibrinolytic actions of the NK-1RA. The data presented herein show that a highly specific NK-1RA significantly reduces adhesions in wild-type mice, but fails to do so in NK-1R knockout mice. Furthermore, the NK-1RA increases peritoneal fibrinolytic activity in wild-type mice, but has no effect in NK-1R knockout mice. Collectively, these data not only indicate that the NK-1R pathway is involved in
j o u r n a l o f s u r g i c a l r e s e a r c h x x x ( 2 0 1 4 ) 1 e7
Fig. 3 e Peritoneal fibrinolytic activity in wild-type (n [ 12) and NK-1R knockout (n [ 12) mice exposed to cecal cautery. Wild-type mice have significantly increased peritoneal fibrinolysis in response to treatment with an NK-1R antagonist. Both treated and untreated NK-1R knockout mice show no change in fibrinolytic activity compared with wild-type controls. *P £ 0.05 compared with saline treated wild type; #£0.05 compared with NK-1RA treated wild type.
adhesion formation, but that the NK-1R is essential in mediating the fibrinolytic effects of the NK-1RA. Moreover, firmly establishing the role of the NK-1R in adhesiogenesis advances our understanding of adhesion biology and provides a basis for our continued efforts in exploring this pathway for adhesion prevention. During abdominal surgery, iatrogenic injuries to the serosal surfaces of the peritoneum such as manual manipulation of organs and the surrounding the viscera, vessel ligation, electrocautery, and vascular clamping disrupts blood flow to the mesothelium inducing localized ischemia, followed by a more widespread inflammatory response. One of the earliest consequences of these surgically-induced injuries is the extravasation of a fibrinous exudate from the underlying vasculature into the peritoneum. Most adhesion biologists concur that this event is the first step in adhesion formation [14]. As the central player in the peritoneal wound-healing cascade, the peritoneal fibrinolytic system would normally resolve the fibrinous exudate to facilitate adhesion-free tissue repair [17]. However, ischemia and inflammation have been shown to reduce tPA and increase PAI-1, the major regulators of peritoneal fibrinolytic activity; thus stabilizing the fibrinrich matrix and facilitating adhesion formation [17,31e34]. Conversely, the intraperitoneal administration of exogenous tPA increases peritoneal fibrinolytic activity and reduces adhesions [35e37], and application of diverse agents such as methylene blue [38], statins [39], and N-acetyl-L-cysteine [34] to peritoneal surfaces at the time of operation all reduce adhesion formation by increasing peritoneal fibrinolytic activity. The results presented herein further underscore the
5
importance of fibrinolysis to adhesion prevention, but also indicate that targeting fibrinolysis alone maybe inadequate for a clinically meaningful reduction in adhesions. In addition to the data we report in this communication that directly links the profibrinolytic actions of an NK-1RA with its respective receptor and a reduction in adhesion formation, there is also indirect evidence that SP and the NK-1R are involved in adhesiogenesis. Compared with their wildtype counterparts, mice that lack neprilysin or neutral endopeptidase, a cell-surface metalloprotease which degrades SP, had more severe intra-abdominal adhesion formation after the induction of colitis, presumably because of increased levels of SP [40]. Additionally, a role for SP and the NK-1R in adhesion formation is supported by data that show that mice lacking the TAC1 gene, which encodes for SP, have reduced adhesion formation compared with wild-type controls [23]. Mice deficient in the tPA gene were also more susceptible to adhesion formation compared with wild-type mice [41] whereas in PAI-1 knockout mice, adhesions were reduced compared with wild-type mice [42]. Taken together with our findings, these data suggest that the persistence of fibrin because of decreased tPA activity after surgery plays a major role in adhesion formation. Because blocking the NK-1R leads to a significant reduction in adhesions in wild-type mice, we might expect NK-1R knockout mice to have low adhesion formation at baseline, regardless of treatment with saline or NK-1RA, because SPmediated adhesion formation cannot occur in this group. Although there was a reduction in adhesion severity in knockout mice administered saline compared with wild-type mice administered saline, knockout mice still had greater adhesion severity than did wild-type mice administered an NK-1RA. This finding was unexpected because although antagonizing the NK-1R clearly reduced adhesion severity in wild-type mice, the fact that a similar reduction was not observed in the NK-1R knockout mice indicates that other key adhesion pathways exist. We now understand that the pathophysiology of adhesiogenesis is multifactorial and involves numerous cellular pathways including those mediated by SP and the NK-1R. Although pharmacologic targeting of the NK1R this pathway significantly reduces adhesion formation, like many successful therapies of other complex diseases, a single therapeutic strategy is unlikely to provide comprehensive clinical efficacy. The NK-1R, therefore, is not absolutely necessary for adhesion formation, but is a key target for adhesion prevention therapies. Although the current experiments establish the role of the NK-1R in mediating adhesion prevention, they also suggest that NK-1R blockade alone is not sufficient for complete resolution of adhesions. The future of adhesion prevention must improve on our current understanding of cellular pathways of adhesiogenesis and novel strategies for prevention. Pharmacologic approaches can improve on the inherent drawbacks of current barrier methods; they will be easier to apply and efficacious throughout the peritoneum. The NK-1RA is a promising treatment for postoperative adhesions, based on our understanding of the role of the NK-1R in fibrinolysis and adhesiogenesis; however, future work should proceed with an emphasis on simultaneously targeting multiple cellular pathways in adhesiogenesis.
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Acknowledgment This research was supported in part, by the CoopereTyler and Smithwick Endowment Funds, from the Department of Surgery, Boston University School of Medicine, Boston, MA. The homozygous NK-1R knockout mice were a generous gift from Dr Norma Gerard, Harvard Medical School, Boston, MA. Authors’ contributions: M.R.C. and A.F.S. contributed to the conception and design. M.R.C., E.G., S.H., and A.F.S. did the analysis and interpretation. M.R.C., H.K.S., M.L.G., and H.K. did the data collection. M.R.C. and A.F.S. did the writing of the article. S.H. did the critical revisions. A.F.S. obtained the funding.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
references
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The neurokinin 1 receptor regulates peritoneal fibrinolytic activity and postoperative adhesion formation Michael R. Cassidy, MD, Holly K. Sheldon, MD, Melanie L. Gainsbury, MD, Earl Gillespie, PhD, Hisashi Kosaka, MD, PhD, Stanley Heydrick, PhD, and Arthur F. Stucchi, PhD* Department of Surgery, Boston University School of Medicine, Boston, Massachusetts
article info
abstract
Article history:
Background: Intra-abdominal adhesions are a common source of postoperative morbidity.
Received 8 March 2014
Previous studies in our laboratory have shown that a neurokinin 1 receptor antagonist (NK-
Received in revised form
1RA) reduces abdominal adhesion formation and increases peritoneal fibrinolytic activity.
1 April 2014
However, the cellular pathway by which the antagonist exerts its effects is unclear, as cultured
Accepted 15 April 2014
peritoneal mesothelial cells exposed to the NK-1RA show increases in fibrinolytic activity
Available online xxx
despite having very low expression of neurokinin 1 receptor (NK-1R) messenger RNA and protein. Our aim was to determine whether the NK-1R plays an essential role in the adhesion-
Keywords:
reducing effects of the NK-1RA, or if the NK-1RA is acting independently of the receptor.
Intra-abdominal adhesions
Methods: Homozygous NK-1R knockout mice and age matched wild-type mice underwent
Neurokinin-1 receptor
laparotomy with cecal cautery to induce adhesions. At the time of surgery, mice received a
Neurokinin-1 receptor antagonist
single intraperitoneal dose of either NK-1RA (25 mg/kg) or saline alone. Adhesion severity
Substance P
at the site of cecal cautery was assessed on postoperative day 7. In a separate experiment,
Tissue plasminogen activator
peritoneal fluid was collected from wild type and NK-1R knockout mice 24 h after lapa-
Fibrinolytic activity
rotomy with cecal cautery and administration of either NK-1RA or saline. Tissue plasminogen activator levels, representative of total fibrinolytic activity, were then measured in peritoneal fluid. Results: In wild-type mice, NK-1RA administration significantly decreased adhesion formation compared with saline controls. Among the NK-1R knockout mice, there was no significant reduction in adhesion formation by the NK-1RA. Fibrinolytic activity increased 244% in wild-type mice administered NK-1RA compared with saline controls; however, the NK-1RA did not raise fibrinolytic activity above saline controls in NK-1R knockout mice. Conclusions: These data indicate that the NK-1R mediates the adhesion-reducing effects of the NK-1RA, in part, by the upregulation of peritoneal fibrinolysis, and suggest that the NK1R is a promising therapeutic target for adhesion prevention. ª 2014 Elsevier Inc. All rights reserved.
Presented, in part, at the seventh Academic Surgical Congress, Las Vegas, Nevada, February 14e16, 2012, and published in abstract form in J Surg Res 172(2): 340; 2012. * Corresponding author. Department of Surgery, Boston University School of Medicine, Section of Surgical Research, 700 Albany Street W408E, Boston, MA 02118. Tel.: þ1 617 638 8665; fax: þ1 617 638 8662. E-mail address: [email protected] (A.F. Stucchi). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.04.030
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1.
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Introduction
Although surgeons have recognized postoperative intraperitoneal adhesions for nearly two centuries, their prevalence remains disturbingly high and unacceptable [1]. Nearly 100% of patients who have undergone an open abdominopelvic operation will develop adhesions, classifying them as the most frequent postoperative complication [2]. The consequences of adhesions can cause life-threatening sequelae such as small bowel obstruction, debilitating abdominal and pelvic pain, and secondary infertility in women [1]. The treatment of adhesion-related complications is estimated to be as high as $5 billion annually, incurring a considerable burden on the US health care system [3,4]. Despite the severity and frequency of complications due to adhesions, efforts to curtail their formation to date have emphasized meticulous surgical technique and application of a barrier, such as seprafilm [5,6]. Although physical barriers are effective [7], there are significant limitations to their use, including difficulty of application and limited use in minimally invasive and laparoscopic procedures [8,9]. In addition, we have shown that physical barriers only prevent adhesions where directly applied and have no remote efficacy [10]. There is also evidence suggesting that barrier methods fail to consistently prevent small bowel obstructions, despite their efficacy in adhesion reduction [11e13]. Thus, the current state of adhesion prevention is inadequate in the clinical realm, and novel preventative strategies are needed to reduce the high incidence of morbidity. Only with an understanding of the basic biology of adhesion formation can we hope to reduce the incidence of adhesions and their associated complications [6]. Although the cellular and molecular events that underlie adhesiogenesis are multifactorial, decades of research have firmly established the central role that the peritoneal fibrinolytic system plays in modulating intra-abdominal adhesion formation [14e17]. Peritoneal fibrinolysis is regulated primarily by tissue plasminogen activator (tPA) and its principal inhibitor, plasminogen activator inhibitor 1 (PAI-1) [18]. After an abdominal operation, tissue injury and ischemia prompts a peritoneum-wide inflammatory response that rapidly downregulates the peritoneum’s fibrinolytic capacity by reducing tPA and/or increasing PAI-1 [19]. Many adhesion biologists feel this is a key underlying event in the pathogenesis of adhesion formation [17]. Our laboratory has sought to identify pharmacologic interventions that mediate adhesion formation in an effort to better define the pathophysiology of adhesion biology. Early work in a rat model of adhesion formation implicated the inflammatory neuropeptide substance P (SP) in adhesiogenesis, as messenger RNA expression for SP and its receptor, the neurokinin 1 receptor (NK-1R), was upregulated in peritoneal adhesion tissue shortly after surgery [20]. We subsequently showed that a neurokinin 1 receptor antagonist (NK-1RA) administered as a single intraoperative dose reduces postoperative adhesions by increasing peritoneal fibrinolysis [21]. Although the cellular mechanism(s) by which the NK-1RA increases peritoneal fibrinolytic activity has remained elusive, unpublished in vitro experiments from our laboratory showed that when cultured human mesothelial cells were incubated
with the NK-1RA, fibrinolytic activity increased >5-fold despite the very low expression levels of NK-1R messenger RNA and protein. This finding led us to question whether the NK-1RA increases fibrinolytic activity by binding to the NK-1R and interfering with the proinflammatory signaling of SP, or if the antagonist signals independently of the receptor through an off-target pathway. To address this mechanism, we used an NK-1R knockout mouse model of adhesion formation to definitively establish the role of NK-1R modulating peritoneal fibrinolysis and adhesion prevention.
2.
Materials and methods
2.1.
Materials
The NK-1RA (3R,4S,5S,6S)-6-diphenylmethyl-5-(5-isopropyl-2methoxybenzlyamino)-1-azabicyclo[2,2,2]octane-3-carboxylic acid (CJ-12,255) was obtained from Pfizer (Groton, CT). This antagonist is highly specific for the NK-1R with no affinity for the NK-2 or NK-3 receptors.
2.2.
Animals
Homozygous NK-1R/ mice (25e30 g) lacking the NK-1R gene were generated as described [22] and bred at the Boston University School of Medicine Laboratory Animal Science Center. The original breeding pair was a gift from Dr Norma Gerard (Harvard Medical School, Boston, MA) to Dr Arthur F. Stucchi, a coauthor of this article. Age-matched male wild-type mice (20e25 g) were purchased from Charles River (Wilmington, MA). All animals were housed at constant room temperature and under 12-h lightedark cycles and allowed access to food and water ad libitum. The Institutional Animal Care and Use Committee at the Boston University School of Medicine approved this study, and all procedures described were performed in accordance with recommendations outlined in the Guide for the Care and Use of Laboratory Animals: Eighth Edition (NRC 2011) published by the National Academies of Science.
2.3.
Induction of adhesion formation
Mice underwent laparotomy with cecal cautery (Valleylab Force EZ Instant Response Electrosurgical Generator, Pfizer), a validated model of adhesion formation, to induce intraperitoneal adhesions [23]. Briefly, mice were anesthetized with isoflurane and a 1 cm laparotomy was performed through which the cecum was identified and expressed (Fig. 1A). Using bipolar forceps, a thermal defect (5 W, w7 U for 1 s) was created on the antimesenteric side of the cecum approximately 5 mm from the tip (Fig. 1B). The cautery defect did not perforate the cecum (Fig. 1C). The cecum was returned to the peritoneal cavity and the wound closed in two layers. Mice survived until postoperative day 7, when adhesions were scored as described in the following, respectively. In separate experiments, mice survived for 24 h, at which time they were sacrificed and peritoneal fluid collected for fibrinolytic activity as described in the following, respectively.
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Fig. 1 e Cecal cauterization mouse model was used to produce quantitative adhesions. (A) A 1 cm laparotomy was performed, after which the cecum was identified and expressed through the incision; (B) Using bipolar forceps, a thermal defect (5 W, w7 U for 1 s) was created on the antimesenteric side of the cecum approximately 5 mm from the tip; and, (C) The resulting cautery injury (arrow) to the cecum did not cause perforation. Standard scoring system of 1e5 based on adhesion severity as described previously [24]. (D) 0, no adhesion; (E) 1, one thin filmy adhesion; (F) 2, more than one thin adhesion; (G) 3, thick adhesion with focal point; (H) 4, thick adhesion with plantar attachment or more than one thick adhesion with focal point; I) 5, very thick vascularized adhesion or more than one plantar adhesion. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.4.
Assessment of NK-1RA on adhesion formation
Wild type and NK-1R knockout mice underwent laparotomy with cecal cautery as described previously (n ¼ 43). Animals were randomized to receive either a single intraoperative dose of 25 mg/kg NK-1RA in 0.5 mL normal saline, or 0.5 mL normal saline alone. Seven days later, adhesions to the site of cecal cautery were assigned a numerical score from 0e5 (Table) based on adhesion number, density, and tenacity as shown in Figure 1 and as previously described [24]. Animals were each assigned an adhesion score by two independent observers, one of whom was blinded to the treatment group. The two scores for each animal were averaged to determine the final adhesion score used for analysis.
2.5.
Fibrinolytic activity in peritoneal fluid
NK-1RA or saline as previously mentioned (n ¼ 24). After 24 h, peritoneal fluid was collected, acetate treated, and frozen at 80 C until use. A tPA activity bioassay was performed as we have described previously [25]. In brief, fluid samples were thawed on wet ice and acidified with 0.2 volumes 0.375 N hydrochloric acid and then diluted 10-fold with distilled water. The fibrinolytic activity of tPA in each sample was
Table e Adhesion scoring scale. Score 0 1 2 3 4 5
To evaluate the fibrinolytic activity in peritoneal fluid, wild type and NK-1R knockout mice underwent laparotomy and cecal cautery with intraoperative administration of 25 mg/kg
Description of adhesion No adhesions One thin filmy adhesion More than one thin adhesion Thick adhesion with focal point Thick adhesion with plantar attachment or more than one thick adhesion with focal point Very thick, vascularized adhesion or more than one plantar adhesion
Adapted from Kennedy et al. [24].
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assayed in duplicate by adding 50 mL of the diluted sample to each well of a 96-well microtiter plate containing 50 mL tPA stimulator (0.6 mg/mL cyanogen bromide-digested fibrinogen; American Diagnostica, Stamford, CT). Next, 150 mL of assay buffer (16.7 mg/mL human plasminogen (Athens Research and Technologies, Athens, GA), 667 mmol/L S-2251 substrate (American Diagnostica), and 20 mmol/L Tris, pH 8.3) were added to each well and gently mixed. Cleavage of the S-2251 substrate by tPA-activated plasmin produces a yellow color that absorbs at 405 and 490 nm (calibration blank). The change in absorbance was measured at 37 C over the course of 10 h using a spectrophotometric microplate reader (SpectraMax 250; Molecular Devices, Sunnyvale, CA). The fibrinolytic activity of tPA in each sample was determined by extrapolation from a tPA (humans; Calbiochem, San Diego, CA) standard curve.
2.6.
Statistical analysis
Data were analyzed either with 1-way analysis of variance or the Student t-test where appropriate using analytical software (Sigma-Stat; SPSS Inc, Chicago, IL). In all analyses, before parametric statistical analyses, if the analyses of the data failed either normality or equivariance testing, log10 transformation was performed. If the data still failed these criteria, the nonparametric Dunn test of analysis of variance by ranks followed by the appropriate nonparametric post hoc test was used. When significant effects (P < 0.05) were detected, the differences between specific multiple means were determined by post hoc analysis with the StudenteNewmaneKeuls test. Groups were deemed significantly different from one another when P < 0.05. All the data are expressed as means standard error of the mean.
3.
Results
3.1. An NK-1RA reduces adhesions in wild type but not in NK-1R knockout mice In wild-type mice, NK-1RA administration at the time of surgery significantly decreased adhesion severity compared with saline controls (Fig. 2). Animals administered saline developed thick, vascularized, tenacious adhesions by postoperative day 7 as shown in Figure 1H and I, with a mean adhesion severity score of 4.8 0.1 (min: 3.5dmax: 5) whereas wild-type animals administered NK-1RA had thin, filmy adhesions as shown in Figure 1E and F, with a mean adhesion score of 2.5 0.5 (min: 0dmax: 5) (P 0.05). In contrast, the administration of an NK-1RA to the NK-1R knockout mice failed to reduce adhesion formation. All knockout animals developed thick, tenacious adhesions, with no statistical differences noted between animals administered saline and those administered NK-1RA. Mean severity scores in these groups were 3.7 0.4 (min: 2dmax: 5) and 4.4 0.3 (min: 3dmax: 5), respectively (Fig. 2). Both surgeons who scored the adhesions (M.R.C. and H.K.) were experienced with the model and there was no statistical difference between the average of the two scores (P ¼ 0.44).
Fig. 2 e Adhesion formation in wild-type (n [ 22) and NK1R knockout (n [ 21) mice exposed to cecal cautery. Wildtype mice have significantly decreased adhesion formation in response to treatment with an NK-1R antagonist; NK-1R knockout mice do not. In addition, untreated NK-1R knockout mice have a slight reduction in adhesion formation compared with wild-type controls. *P £ 0.05 compared with saline treated wild type.
3.2. An NK-1RA increases peritoneal fibrinolytic activity in wild type but not in NK-1R knockout mice In wild-type mice, fibrinolytic activity in peritoneal fluid at 24 h postoperatively was increased 344% in animals administered NK-1RA compared with those administered saline (Fig. 3). tPA concentration was 4.44 0.83 U/mL in NK-1RAtreated mice and 1.29 0.11 in saline-treated mice (P 0.05). In contrast, the administration of an NK-1RA to the NK-1R knockout mice failed to increase fibrinolytic activity above baseline levels in the wild-type saline controls, where tPA concentration was 1.64 0.11 U/mL in NK-1RA-treated mice and 1.28 0.13 U/mL in saline-treated mice.
4.
Discussion
Prior studies from this laboratory have demonstrated that a single intraoperative dose of an NK-1RA significantly reduced intra-abdominal adhesion formation primarily by increasing peritoneal tPA activity in the immediate postoperative period [21,25e30]. Although these earlier findings strongly implicate SP and the NK-1R pathway in adhesiogenesis, we have been unable to definitively establish whether the NK-1R participates in mediating the fibrinolytic actions of the NK-1RA. The data presented herein show that a highly specific NK-1RA significantly reduces adhesions in wild-type mice, but fails to do so in NK-1R knockout mice. Furthermore, the NK-1RA increases peritoneal fibrinolytic activity in wild-type mice, but has no effect in NK-1R knockout mice. Collectively, these data not only indicate that the NK-1R pathway is involved in
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Fig. 3 e Peritoneal fibrinolytic activity in wild-type (n [ 12) and NK-1R knockout (n [ 12) mice exposed to cecal cautery. Wild-type mice have significantly increased peritoneal fibrinolysis in response to treatment with an NK-1R antagonist. Both treated and untreated NK-1R knockout mice show no change in fibrinolytic activity compared with wild-type controls. *P £ 0.05 compared with saline treated wild type; #£0.05 compared with NK-1RA treated wild type.
adhesion formation, but that the NK-1R is essential in mediating the fibrinolytic effects of the NK-1RA. Moreover, firmly establishing the role of the NK-1R in adhesiogenesis advances our understanding of adhesion biology and provides a basis for our continued efforts in exploring this pathway for adhesion prevention. During abdominal surgery, iatrogenic injuries to the serosal surfaces of the peritoneum such as manual manipulation of organs and the surrounding the viscera, vessel ligation, electrocautery, and vascular clamping disrupts blood flow to the mesothelium inducing localized ischemia, followed by a more widespread inflammatory response. One of the earliest consequences of these surgically-induced injuries is the extravasation of a fibrinous exudate from the underlying vasculature into the peritoneum. Most adhesion biologists concur that this event is the first step in adhesion formation [14]. As the central player in the peritoneal wound-healing cascade, the peritoneal fibrinolytic system would normally resolve the fibrinous exudate to facilitate adhesion-free tissue repair [17]. However, ischemia and inflammation have been shown to reduce tPA and increase PAI-1, the major regulators of peritoneal fibrinolytic activity; thus stabilizing the fibrinrich matrix and facilitating adhesion formation [17,31e34]. Conversely, the intraperitoneal administration of exogenous tPA increases peritoneal fibrinolytic activity and reduces adhesions [35e37], and application of diverse agents such as methylene blue [38], statins [39], and N-acetyl-L-cysteine [34] to peritoneal surfaces at the time of operation all reduce adhesion formation by increasing peritoneal fibrinolytic activity. The results presented herein further underscore the
5
importance of fibrinolysis to adhesion prevention, but also indicate that targeting fibrinolysis alone maybe inadequate for a clinically meaningful reduction in adhesions. In addition to the data we report in this communication that directly links the profibrinolytic actions of an NK-1RA with its respective receptor and a reduction in adhesion formation, there is also indirect evidence that SP and the NK-1R are involved in adhesiogenesis. Compared with their wildtype counterparts, mice that lack neprilysin or neutral endopeptidase, a cell-surface metalloprotease which degrades SP, had more severe intra-abdominal adhesion formation after the induction of colitis, presumably because of increased levels of SP [40]. Additionally, a role for SP and the NK-1R in adhesion formation is supported by data that show that mice lacking the TAC1 gene, which encodes for SP, have reduced adhesion formation compared with wild-type controls [23]. Mice deficient in the tPA gene were also more susceptible to adhesion formation compared with wild-type mice [41] whereas in PAI-1 knockout mice, adhesions were reduced compared with wild-type mice [42]. Taken together with our findings, these data suggest that the persistence of fibrin because of decreased tPA activity after surgery plays a major role in adhesion formation. Because blocking the NK-1R leads to a significant reduction in adhesions in wild-type mice, we might expect NK-1R knockout mice to have low adhesion formation at baseline, regardless of treatment with saline or NK-1RA, because SPmediated adhesion formation cannot occur in this group. Although there was a reduction in adhesion severity in knockout mice administered saline compared with wild-type mice administered saline, knockout mice still had greater adhesion severity than did wild-type mice administered an NK-1RA. This finding was unexpected because although antagonizing the NK-1R clearly reduced adhesion severity in wild-type mice, the fact that a similar reduction was not observed in the NK-1R knockout mice indicates that other key adhesion pathways exist. We now understand that the pathophysiology of adhesiogenesis is multifactorial and involves numerous cellular pathways including those mediated by SP and the NK-1R. Although pharmacologic targeting of the NK1R this pathway significantly reduces adhesion formation, like many successful therapies of other complex diseases, a single therapeutic strategy is unlikely to provide comprehensive clinical efficacy. The NK-1R, therefore, is not absolutely necessary for adhesion formation, but is a key target for adhesion prevention therapies. Although the current experiments establish the role of the NK-1R in mediating adhesion prevention, they also suggest that NK-1R blockade alone is not sufficient for complete resolution of adhesions. The future of adhesion prevention must improve on our current understanding of cellular pathways of adhesiogenesis and novel strategies for prevention. Pharmacologic approaches can improve on the inherent drawbacks of current barrier methods; they will be easier to apply and efficacious throughout the peritoneum. The NK-1RA is a promising treatment for postoperative adhesions, based on our understanding of the role of the NK-1R in fibrinolysis and adhesiogenesis; however, future work should proceed with an emphasis on simultaneously targeting multiple cellular pathways in adhesiogenesis.
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Acknowledgment This research was supported in part, by the CoopereTyler and Smithwick Endowment Funds, from the Department of Surgery, Boston University School of Medicine, Boston, MA. The homozygous NK-1R knockout mice were a generous gift from Dr Norma Gerard, Harvard Medical School, Boston, MA. Authors’ contributions: M.R.C. and A.F.S. contributed to the conception and design. M.R.C., E.G., S.H., and A.F.S. did the analysis and interpretation. M.R.C., H.K.S., M.L.G., and H.K. did the data collection. M.R.C. and A.F.S. did the writing of the article. S.H. did the critical revisions. A.F.S. obtained the funding.
Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
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