Intestinal handling-induced mast cell activation and...

Intestinal handling-induced mast cell activation andinflammation in human postoperative ileus

F O The,1 R J Bennink,2 W M Ankum,3 M R Buist,3 O R C Busch,4 D J Gouma,4 S vander Heide,5 R M van den Wijngaard,1 W J de Jonge,1 G E Boeckxstaens1

1 Department ofGastroenterology andHepatology, Academic MedicalCenter, Amsterdam, TheNetherlands; 2 Department ofNuclear Medicine, AcademicMedical Center, Amsterdam,The Netherlands; 3 Departmentof Gynaecology and Obstetrics,Academic Medical Center,Amsterdam, The Netherlands;4 Department of Surgery,Academic Medical Center,Amsterdam, The Netherlands;5 Department of Allergy,University Medical Center,Groningen, The Netherlands

Correspondence to:G E Boeckxstaens, Departmentof Gastroenterology andHepatology, Academic MedicalCenter, Meibergdreef 9, 1105AZ Amsterdam, TheNetherlands;[email protected]

Revised 1 May 2007Accepted 5 June 2007Published Online First20 June 2007

ABSTRACTBackground: Murine postoperative ileus results fromintestinal inflammation triggered by manipulation-inducedmast cell activation. As its extent depends on the degreeof handling and subsequent inflammation, it is hypothe-sised that the faster recovery after minimal invasivesurgery results from decreased mast cell activation andimpaired intestinal inflammation.Objective: To quantify mast cell activation and inflam-mation in patients undergoing conventional and minimalinvasive surgery.Methods: (1) Mast cell activation (ie, tryptase release)and pro-inflammatory mediator release were determinedin peritoneal lavage fluid obtained at consecutive timepoints during open, laparoscopic and transvaginalgynaecological surgery. (2) Lymphocyte function-asso-ciated antigen-1 (LFA-1), intercellular adhesion molecule-1(ICAM-1) and inducible nitric oxide synthase (iNOS)mRNA as well as leucocyte influx were quantified in non-handled and handled jejunal muscle specimens collectedduring biliary reconstructive surgery. (3) Intestinalleucocyte influx was assessed by 99mTc-labelledleucocyte single photon emission computed tomography(SPECT) – computed tomography (CT) scanning beforeand after abdominal or vaginal hysterectomy.Results: (1) Intestinal handling during abdominalhysterectomy resulted in an immediate release of tryptasefollowed by enhanced interleukin 6 (IL6) and IL8 levels.None of the mediators increased during minimal invasivesurgery except for a slight increase in IL8 duringlaparoscopic surgery. (2) Jejunal ICAM-1 and iNOS mRNAtranscription as well as leucocyte recruitment wereincreased after intestinal handling. (3) Leucocyte scanning24 h after surgery revealed increased intestinal activityafter abdominal but not after vaginal hysterectomy.Conclusions: This study demonstrates that intestinalhandling triggers mast cell activation and inflammationassociated with prolonged postoperative ileus. Theseresults may partly explain the faster recovery afterminimal invasive surgery and encourage future clinicaltrials targeting mast cells to shorten postoperative ileus.

Postoperative ileus, characterised by a lack ofcoordinated motility of the entire gastrointestinaltract, leads to increased morbidity and prolongedhospitalisation,1 2 and represents a substantial socio-economical burden. In the USA alone, the additionalannual healthcare expenses related to this conditionhave been estimated to surpass US$1 billion.3 Atpresent, treatment is rather disappointing andlimited to predominantly supportive measures.4

The introduction of minimal invasive surgicaltechniques (eg, laparoscopy) has speeded up post-operative recovery significantly.5 This major

improvement is believed to result from minimalwound trauma and decreased release of stresshormones.6 7 In addition, it is becoming increasinglyclear that intestinal inflammation is a key event inthe pathogenesis of postoperative ileus. In rats, thedegree of gut paralysis is directly proportional to thedegree of intestinal handling and inflammation.8 Thisinflammation leads to local impaired muscle con-tractility9 and the activation of an adrenergicinhibitory neural pathway.10 Reduction of inflam-matory cell influx accomplished by blockade ofadhesion molecules shortens postoperative ileus10 11

and further underscores the importance of thishandling-induced inflammation. As minimal invasivesurgery implies limited handling of the intestine,faster recovery of motility may result from animpaired influx of inflammatory cells.

Although the exact mechanism remains unclear,we previously showed that mast cells play a pivotalrole in triggering the inflammatory process. Inmice, intestinal handling led to degranulation ofmast cells with increased levels of mouse mast cellprotease-1 in peritoneal lavage fluid. In contrast,W/Wv mice, deficient in mast cells, failed todevelop an intestinal muscle inflammation inresponse to manipulation of a bowel loop.Reconstitution of W/Wv mice with mast cells fromwild-type animals restored the handling-inducedinflammatory response, clearly demonstrating theimportance of mast cells. Activation of residentmacrophages has also been demonstrated, possiblysecondary to influx of luminal bacteria during abrief episode of increased mucosal permeability.12

To what extent mast cell activation is the triggerleading to increased mucosal permeability andmacrophage activation remains to be determined.

At present, the evidence supporting the impor-tance of inflammation in humans is rather scarce,13

and data on the relationship between the degree ofinflammation and clinical outcome are lacking. Inaddition, although we provided convincing evi-dence for a crucial role for mast cell degranulationin mice, no data are available in man. In thepresent study, therefore, we examined whetherintestinal manipulation leads to mast cell degra-nulation and inflammation in patients undergoingconventional or minimal invasive surgery andhypothesised that clinical recovery is determinedby the degree of manipulation-induced mast cellactivation and inflammation.

PATIENTS AND METHODSParticipantsBetween December 2003 and July 2005 a total of 44patients were enrolled in three clinical research

Intestinal inflammation

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protocols. The physical condition and comorbidity of potentialparticipants were assessed during preassessment at the out-patient clinic of the department of anaesthesiology, which ispart of the standard preoperative work-up. The AmericanSociety of Anesthesiologists-Physical Status classification (ASA-PS)14 15 was used and comprises a scale from 1 to 6 in which1 equals a normal healthy patient; 2 equals a patient withmild systemic disease; 3 equals a patient with severe systemicdisease; 4 is a patient with severe systemic disease that is aconstant threat to life; 5 equals a moribund patient who is notexpected to survive without the operation; and 6 equals adeclared brain-dead patient whose organs are being removedfor donor purposes.14 In the present study, only patients cate-gorised as ASA-PS 1–3 were asked to participate. In addition,patients were screened for the following exclusion criteria:intra-abdominal inflammation, preoperative radiation therapyand the use of anti-inflammatory or mast cell-stabilisingdrugs. Patients were included after informed consent wasobtained.

Study designThe activation of mast cells and the inflammatory mediatorresponse to intestinal handling were evaluated in protocol 1 (seebelow). Proinflammatory gene transcription and leucocyterecruitment within the handled intestinal muscle layer werestudied in protocol 2. The occurrence of manipulation-inducedleucocyte recruitment in relation to clinical recovery of bowelfunction and duration of hospital admission was evaluated instudy protocol 3. All protocols were evaluated and approved bythe Medical Ethical Review Board of the Academic MedicalCenter, Amsterdam, The Netherlands.

AnaesthesiaTo correct for the influence of the anaesthetic technique andmedication used, patients were subjected to standardisedperioperative care according to our anaesthesiologist’s protocol.In brief, patients were premedicated with paracetamol 1000 mgand lorazepam 1 mg on the evening before surgery andapproximately 2 h before surgery. Induction of general anaes-thesia was attained with propovol 2–2.5 mg/kg; fentanyl 1.5–3 mg/kg; rocuronium 0.6 mg/kg. Anaesthesia was maintainedusing end-tidal. 0.8% isoflurane. Postoperative pain medicationwas introduced after the last sampling in protocols 1 and 2 andwas administered in a similar way to all patients according toour anaesthesiologist’s pain protocol.

Protocol 1: mast cell activation and inflammatory mediator releaseduring abdominal surgeryPeritoneal lavage fluid samples were collected from 18 patients,undergoing either an abdominal hysterectomy (n = 6), alaparoscopic resection of an adnexum (n = 6) or a transvaginalhysterectomy (n = 6). Three consecutive lavages were per-formed in each individual patient. The first lavage sample wascollected immediately after opening of the peritoneum (basal).A second lavage sample was collected immediately afterabdominal inspection and first gentle small intestinal handling(early). The final lavage sample was collected at the end of theprocedure (late). As the intestine is not handled in thetransvaginal hysterectomy group, only two lavages wereperformed in this group (ie, basal and late sampling). Thesystemic release of mediators in response to abdominal surgerywas assessed in two blood samples (abdominal hysterectomyonly), the first sample taken before induction of general

anaesthesia (1 day prior to surgery) and the second sampletaken at the end of the surgical procedure—that is, just beforeclosure of the abdominal cavity. The harvested fluid and serumwere used to measure the release of tryptase, tumour necrosisfactor a (TNFa), interleukin 1b (IL1b), IL6 and IL8 in relation tosurgical handling. The abdominal lavage was performed using100 ml of warm (42uC) sterile 0.9% NaCl solution, which wassprinkled gently onto the small intestine and its mesentery.After approximately 30 s, peritoneal fluid (between 20 and40 ml) was collected using a 22 French Foley catheter (BardLimited, West Sussex, UK) connected to a 50 ml catheter tipsyringe.

Protocol 2: regulatory gene transcription and leucocyte influx uponintestinal handlingJejunal muscle specimens were used to quantify regulatory genetranscription and assess the degree of inflammation. Full-thickness biopsies were obtained from patients undergoingbiliary reconstructive surgery. This specific procedure waschosen because of its considerable length, providing at leastsufficient time for gene transcription to occur.16 Two con-secutive jejunal tissue samples were collected from 10 patients.The first specimen was collected at the beginning of theprocedure and had not been touched by the surgeon untilresection. The second tissue specimen, exposed to the usualhandling during surgery, was collected approximately 3 h later.Following mucosa removal, both specimens were partitioned(5 mm2 segments) and snap-frozen in liquid nitrogen in theoperating theatre, and stored at 280uC.

Protocol 3: abdominal leucocyte recruitment and clinical recoveryAbdominal leucocyte single photon emission computed tomo-graphy (SPECT) – computed tomography (CT) scans wereperformed in 16 gynaecological patients to quantify theleucocyte recruitment in response to surgical handling. Eightpatients undergoing an abdominal hysterectomy were com-pared with eight patients undergoing a vaginal hysterectomy. Ineach patient, a reference (basal) leucocyte scintigraphy wasperformed on the day of admission, 24 h prior to surgery. Asecond leucocyte scintigraphy was performed on the firstpostoperative day—that is, approximately 24 h after surgery.Clinical recovery was assessed until hospital discharge (seebelow for a detailed description).

MethodsTryptase releaseTryptase concentrations were assessed at the routine clinicallaboratory of the Department of Allergy, University MedicalCenter, Groningen, The Netherlands. The total tryptase (a-protryptase and b-tryptase) concentration was measured inperipheral blood and lavage fluid samples using a commercialfluoro-immunoenzyme assay (FIA) (Pharmacia Uppsala,Sweden).17

Cytokine and chemokine releaseCytokine levels were determined by cytometric bead array(BD PharMingen, San Diego, CA). In brief, 5 ml of each testsample was mixed with 5 ml of mixed capture beads and 5 ml ofhuman phycoerythrin (PE) detection reagents consisting of PE-conjugated anti-human IL1b, TNFa, IL6 and IL8. Thesemixtures were incubated at room temperature in the darkfor 3 h, washed and resuspended in 300 ml of wash buffer.Acquisition was performed on a FACSCalibur using a


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high-throughput sampling interface (BD Biosciences,Sunnyvale, CA). Generated data were analysed using CBAsoftware (BD PharMingen) and interpolated from correspond-ing standard curves generated using the mixed cytokinestandard provided by the supplier.18 19

Real-time reverse transcription-PCRTissue specimens were homogenized and total RNA was extractedusing Trizol (Invitrogen, Carlsbad, CA). The total RNA fractionswere treated with DNase and reverse-transcribed using SuperscriptII (Invitrogen). cDNA (150 ng) was subjected to 45 cycles oflightcycler PCR (FastStartDNA Masterplus SYBR Green; Roche,Basel Switzerland). The following primers were used: lymphocytefunction-associated antigen-1 (LFA-1) antisense 59-GACCCAAGTGCTCTCAGGAA-39 and sense 59-AGGAGCACTCCACTTCATGC-39; intercellular adhesion molecule-1 (ICAM-1) antisense 59-CATAGAGACCCCGTTGCCTA-39 and sense 59-GGGTAAGGTTCTTGCCCACT-39; inducible nitric oxide synthase (iNOS)antisense 59-TGGAAGCGGTAACAAAGGAGA-39 and sense59-CGATGCACAGCTGAGTGAAT-39; and glyceraldehyde phos-phate dehydrogenase (GAPDH) antisense 59-CGACCACTTTGTCAAGCTCA-39 and sense 59-AGGGGAGATTCAGTGTGGTG-39. PCR quantification was performed by a linear regressionmethod using the Log(fluorescence) per cycle number20 andnormalised for GAPDH housekeeping gene expression. In eachindividual patient, the late sample value was expressed as foldincrease of the early control sample value.

ImmunocytochemistryImmunocytochemical staining was performed on peritoneal cellcytospins obtained from the harvested abdominal lavage fluid.In brief, spins containing 16105 cells were fixed in Carnoy’sfixation fluid (60% ethanol, 30% chloroform and 10% glacialacidic acid) for 30 min at room temperature and washed withTris-buffered saline–Tween (TBST; 0.1%). Non-specific bindingof antibody was blocked by incubation with TBS containing10% normal goat serum for 20 min. Spins were incubated withanti-tryptase antibodies (mouse anti-human, 1:250) (Chemicon,Temecula, CA) for 2 h at room temperature. Goat anti-mouseAlexa-488 was used as secondary antibody (Molecular Probes,Invitrogen). After final washing, the spins were mounted usingVectashield mounting medium containing 5 mg/ml 49,6-diami-dino-2-phenylindole (DAPI; Vector Laboratories, Burlingame,CA).

Semi-quantitative evaluation of the degree of intestinal muscleinflammationHandled and non-handled jejunal muscle sections were used toassess the extent of inflammation. Leucocyte infiltration wasvisualised by myeloperoxidase (MPO) staining as describedpreviously.11 After 10 min fixation in ice-cold acetone, trans-verse frozen sections (8 mm) were incubated for 10 min with 3-amino-9-ethyl carbazole (Sigma, St Louis, MO) as a substrate,disolved in sodium acetate buffer (pH 5.0) to which 0.01% H2O2

was added.10

To evaluate the degree of inflammation, unmarked myelo-peroxidase-stained early and late collected sections from 10patients were scored independently by three observers (T.K.,O.W. and R.vd W.). A semi-quantitative scoring scale from 0 to4 was utilised, 0 being non-inflamed, 1, very mildly inflamed; 2,mildly inflamed; 3, inflamed; and 4, clearly inflamed. The meanof three scores, calculated for each segment, was used forstatistical analysis (Wilcoxon signed rank test).

In vivo quantification of leucocyte recruitmentWhite blood cells (WBCs) were labelled using technetium-99mhexamethylpropyleneamine oxime (99mTc-HMPAO) (Ceretec,GE Health, Eindhoven, The Netherlands) according to theconsensus protocol for leucocyte labelling.21 The harvested WBCfraction of 100 ml of blood labelled with an average of 450¡10MBq of 99mTc-HMPAO was reinjected into the patient. Sixtymin later, a SPECT scan of the abdomen was performed (GEMillennium Hawkeye, GE Healthcare, Den Bosch, TheNetherlands) followed by a low-dose CT scan without contraston the same gantry. CT data were used for attenuationcorrection and as an anatomical reference for region of interest(ROI) analysis. After data acquisition, images were processed onan Entegra workstation (GE Healthcare) using attenuation-corrected iterative reconstruction and analysed on a Hermesworkstation (Nuclear Diagnostics, Stockholm, Sweden). Fiveconsecutive abdominal SPECT slices were summed and ROIswere drawn around the small intestine and lumbar spine at thelevel of the ileac crest. Small bowel uptake of leucocytes wascalculated as an uptake ratio expressed as a fraction of bonemarrow activity, similar to analysis of leucocyte uptakeassessment in inflammatory bowel disease.22 The small boweluptake ratio determined prior to surgery was considered as basalleucocyte activity. The relative percentage difference in leuco-cyte activity 24 h after surgery was calculated using thefollowing formula: (postoperative small bowel ratio/preopera-tive small bowel ratio)6100%.

Clinical evaluationAll patients received standard postoperative medical careaccording to the usual ward care protocol. Patients were visitedby the research physician once daily until discharge to assesspostoperative clinical recovery of bowel function (time of firstflatus and time of first defecation). Patients were dischargedwhen the following criteria were met: normal urinary tractfunction; spontaneous defecation; tolerance of oral fluid andsolid food intake; adequate pain relief with oral analgesics; andadequate mobilisation and self-support.

Statistical analysisStatistical analysis was performed using SPSS 12.02 software forWindows. Data were non-parametrically distributed andexpressed as median values and interquartile range or medianincrease compared with basal values. In protocol 1, all serumsamples but only vaginal hysterectomy lavage samples wereanalysed using a Wilcoxon signed rank test for two pairedsamples. For all other lavage sample series (consisting of threesamples), a Friedman’s two-way analysis of variance wasapplied. When a statistical difference was observed, a Mann–Whitney test was used to identify the specific sample(s)showing the significant difference. In protocol 2, the quantita-tive PCR data and the semi-quantitative inflammation datawere analysed using a Wilcoxon signed rank test. In protocol 3,leucocyte recruitment was analysed using the Wilcoxon signedrank test. Clinical data were analysed with a Mann–Whitneytest for independent samples. P-values ,0.05 was consideredstatistically significant.

RESULTS

Patient demographicsEighteen patients participated in study protocol 1, six in eachsurgical intervention group. Overall mean age was 47 years,range 21–70 (transvaginal, 52 years, range 43–70; laparoscopy,


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36 years, range 21–49; laparotomy, 49 years, range 44–53). Theindications for surgery in this patient population wereleiomyomata (n = 8), prolapse (n = 4) or a benign ovariantumour (n = 6). In study protocol 2, jejunal tissue samples werecollected from 10 patients (6 male, mean age 42 years, range 32–53) who underwent biliary reconstructive surgery because ofiatrogenic biliary tract injury. Study protocol 3 involved 16patients; eight patients underwent an abdominal hysterectomy(mean age 50 years, range 42–70) and eight patients underwenta vaginal hysterectomy (mean age 55 years, range 42–66). Theindications for surgery were uterine leiomyomata in theabdominal hysterectomy patient group and uterine prolapse(n = 4), leiomyomata (n = 3) and primary dysmenorrhoea(n = 1) in the vaginal hysterectomy group.

Study protocol 1

Mast cell activation and inflammatory response during abdominalsurgeryTo assess the activation of mast cells in response to intestinalhandling, the expression and release of tryptase, a prestoredmast cell-specific protease,23 was analysed. Peritoneal lavagefluid harvested during abdominal surgery (laparotomy) con-tained a distinct mast cell population, as illustrated by thenumber of tryptase-positive cells in fig 1A. In the basal lavagesample collected immediately after opening of the peritonealcavity, the basal median tryptase concentration was 5.2(interquartile range (IQR) 2.7–11.3) mg/l. Tryptase release wassignificantly increased to a median concentration of 23.1 (IQR15.1–46.9) mg/l, (p = 0.02) in early samples taken after gentlepalpation of the small intestines, necessary to allow inspectionof the pelvic organs. In the late sample taken at the end ofsurgery, tryptase levels had increased even further (late: medianconcentration 51.7 (IQR 25.8–90.2) mg/l, n = 6, p = 0.002)(fig 1B). In contrast, neither laparoscopic nor transvaginal

intraperitoneal surgery (n = 6 in both types of surgery) elicited asignificant mast cell response (fig 1C,D). To evaluate possiblerelease of mast cell mediators in the systemic circulation, wealso determined preoperative and postoperative serum tryptaseconcentrations in the laparotomy group (n = 6 patients). Serumtryptase levels did not increase and remained within the normalrange of 1–11.4 mg/l24 (preoperative median concentration 4.1(IQR 2.8–7.5) mg/l and postoperative 1.6 (IQR 1.3–3.5) mg/l,respectively).

The release of the proinflammatory cytokines TNFa, IL1band IL6, and of the chemokine IL8 was analysed in the sameperitoneal lavage samples. Gentle handling of the intestineduring laparotomy did not lead to an immediate increase in anyof these mediators. However, at the end of the surgicalprocedure, IL6 and IL8 were increased significantly (table 1).In laparoscopically treated patients, intraperitoneal IL8, but notIL6, was increased, but not as profound as in the laparotomygroup (table 1). On the other hand, transvaginal surgery did notaffect any of the measured cytokines and chemokines. TNFaand IL1b levels did not change upon first handling or at the endof any of the types of surgery evaluated.

The serum levels of the studied inflammatory mediatorsremained unaltered (median increase compared with preopera-tively for: TNFa, 0.0 (IQR 0.0–0.0) pg/ml; IL1b, 0.0 (IQR 0.0–35.3) pg/ml; IL6, 4.2 (IQR 0.0–11.7) pg/ml; IL8, 0.9 (IQR 0.0–310.4) pg/ml).

Study protocol 2

Regulatory gene transcription upon intestinal handlingRecruitment of leucocytes to the muscularis propria stronglydepends on the upregulation of adhesion molecules and thesynthesis of proinflammatory proteins. Therefore, ICAM-1,LFA-1 and iNOS gene expression was determined in musclespecimens collected during abdominal surgery. As the synthesis

Figure 1 (A) Peritoneal cells collectedin late lavage fluid stained for tryptase (ingreen). Cell nuclei were counterstainedwith 49,6-diamidino-2-phenylindole (DAPI;blue). Individual patient tryptaseconcentrations during (B) open surgery,(C) laparoscopic surgery and (D)transvaginal surgery, measured in lavagefluid collected immediately after openingof the peritoneal cavity (basal), after firstpalpation of the small intestine duringinspection of pelvic organs (early) and atthe end of the procedure (late). AWilcoxon signed rank test (vaginalsamples) and Friedman two-way analysisof variance (laparoscopic and laparotomysamples) were used to determinestatistical significance. Tryptase levelsincreased significantly in patientsundergoing a laparotomy (n = 6,p = 0.002) in contrast to the laparoscopic(n = 6, p = 0.5) or vaginal (n = 6,p = 0.06) approach. Note that no ‘‘early’’lavage was performed in patientsundergoing transvaginal surgery. Thedotted line represents the median changein tryptase concentration of all sixpatients.


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of functional proteins requires several hours,16 mRNA quanti-fication was used to evaluate the kinetics of these inflammatoryproteins in jejunal muscle tissue. As shown in Table 2, iNOSand ICAM-1 levels were significantly increased after intestinalhandling (table 2). In contrast, LFA-1 remained unchanged.

Degree of intestinal muscle inflammation upon intestinal handlingHistological evaluation of leucocyte recruitment was performedbefore and after surgical handling on the same tissue specimensused for gene transcription analysis. Myeloperoxidase wasstained to visualise leucocyte recruitment in response tointestinal handling in transverse sections of the jejunalmuscularis propria. Non-handled early samples contained onlya small number of leucocytes in the muscle layer (fig 2A upperpanel). In contrast, routinely handled late specimens showed amarked extravasation of inflammatory cells (fig 2A lowerpanel), confirmed by semi-quantitative evaluation (fig 2B).These recruited leucocytes predominantly reside in and aroundthe vasculature of the handled intestinal muscle layers, as isillustrated in fig 2B. This extravasation marks the ongoinginflammatory process.

Study protocol 3

Abdominal leucocyte recruitment 24 h after open and minimalinvasive hysterectomyIn vivo leucocyte recruitment in response to intestinal handlingwas investigated by 99mTc-labelled leucocyte imaging.Abdominal leucocyte influx was assessed on five consecutiveleucocyte SPECT images at the level of the ileac crest and

compared with that of the bone marrow.22 The change inleucocyte activity before compared with after surgery showedno increase in the vaginal hysterectomy group (medianpercentage activity before compared with after surgery, 91%(IQR 84–102), n = 8). In the abdominal hysterectomy group,however, leucocyte recruitment was significantly increased to amedian of 127% of the preoperative abdominal activity ((IQR113–148), n = 8, p = 0.01) (fig 3). To determine the exactanatomical location, plain CT images were made immediatelyafter SPECT imaging. The region in which the enhancedleucocyte activity was observed coincided with small intestinalloops and its mesentery, as shown in fig 4.

Clinical recovery after open and minimal invasive hysterectomyIn conjunction with the assessed leucocyte recruitment, clinicalrecovery was also evaluated. Time until first flatus did not differsignificantly between the two patient groups. First bowelmovement and duration of hospital admission, however, weresignificantly prolonged after abdominal hysterectomies com-pared with the vaginal procedure (table 3).

DISCUSSIONInflammation of the muscularis propria following surgicalmanipulation of the intestine is increasingly recognised to delaythe recovery of gastrointestinal motility. Animal studies haverevealed that prevention of this inflammatory process, by eitherantibodies or antisense oligonucleotides to the adhesion mole-cule ICAM-1, macrophage inactivation or cyclo-oxygenase-2(COX-2) inhibition, enhances gastrointestinal transit andshortens postoperative ileus.11 25–27 Recently, we demonstratedthat mast cell activation plays an important role in this processand may be one of the first steps triggering the inflammatoryresponse. Intestinal manipulation indeed induces the immediateactivation of mast cells, leading to increased levels of the murinemast cell proteinase-1 in the abdominal cavity.25 Three hourslater, inflammatory mediators such as macrophage inflamma-tory protein (MIP)-2, MIP-1a, TNFa and IL6 can be detected,27 28

which in their turn enhance the expression of adhesionmolecules such as ICAM-1,11 recruitment of leucocytes andinflammation of the intestine.

In the present study, we investigated whether this cascade ofevents also plays a role in the pathogenesis of humanpostoperative ileus. We found that gentle palpation of theintestines during first inspection of the pelvic organs resulted inthe instantaneous intraperitoneal release of tryptase, a mastcell-specific protease,23 in patients undergoing a laparotomy.This increase in tryptase increased even further towards the endof the procedure and was accompanied by an increase in IL6 andIL8. As the latter is known to be released in response to mast cellactivation and initiates leucocyte recruitment via ICAM-1,29 wealso determined ICAM-1 and iNOS mRNA in intestinal tissuethat was handled at the start of a surgical procedure, but wasonly removed approximately 3 h later. In addition to anupregulation of ICAM-1 and iNOS, the number of inflamma-tory cells was significantly increased in these late tissue samplescompared with untouched specimen harvested at the beginningof the procedure. Interestingly, leucocytes were localisedpredominantly around blood vessels in both the serosa andthe muscularis propria, partly adhering to the endothelial liningmarking the ongoing recruitment and extravasation in this earlystage of inflammation. Kalff et al also reported an intestinalinflammatory response during abdominal surgery in patients.13

To assess the degree of inflammation in a later stage, we also

Table 1 Inflammatory mediator release during surgery

Treatmentgroup Laparotomy (n = 6) Laparoscopy (n = 6) Transvaginal (n = 6)

TNFa 0.0 (0.0 to 3.4) 0.0 (26.6 to 0.0) 0.0 (0.0 to 0.0)

IL1b 0.0 (21.8 to 18.1) 22.5 (23.3 to 0.0) 0.2 (0.0 to 0.8)

IL6 135.6 (4.2 to 5130.0)* 6.1 (1.3 to 15.2) 1.5 (20.4 to 4.0)

IL8 114.2 (32.9 to 208.7)** 28.9 (1.3–166.5)* 0.8 (22.8 to 6.0)

Proinflammatory mediator release was determined in lavage fluids collectedimmediately after opening of the peritoneal cavity (basal), after first palpation of thesmall intestine during inspection of pelvic organs (early) and at the end of the surgicalprocedure (late). Median increase of mediator concentration in the late versus thebasal lavage sample collected during the indicated type of surgery (pg/ml) are shownwith the interquartile range in parentheses. A Wilcoxon signed rank test (vaginalsamples) and Friedman two-way analysis of variance (laparoscopic and laparotomysamples) were used to determine statistical significance. TNFa and IL1b did notchange during surgery. IL8 increased significantly at the end of laparoscopic as well asopen (laparotomy) surgery. IL6 only increased at the end of a laparotomy. None of theproinflammatory proteins increased in the transvaginal surgery group. Note that no‘‘early’’ lavage was performed in patients undergoing transvaginal surgery.*p = 0.02; **p = 0.006.IL, interleukin; TNF, tumour necrosis factor.

Table 2 Quantitative gene transcription analysis

Gene Median fold increase of gene expression

LFA-1 0.9 (0.3–20.0)

ICAM-1 3.3 (1.3–139.9)*

iNOS 3.3 (0.7–20.0)**

The median fold increase of gene transcription in late (handled) versus early (non-handled) jejunal muscle layer with the interquartile range (IQR) in parentheses (n = 10patients).A Wilcoxon signed rank test was used to determine statistical differences. The relativeincrease was significant for iNOS (median fold increase 3.3 (IQR 0.7–20.0), p = 0.022)and ICAM1 (median fold increase 3.3 (IQR 1.3–139.9), p = 0.017).LFA-1, lymphocyte function-associated antigen-1; ICAM-1, intercellular adhesionmolecule-1; iNOS, inducible nitric oxide synthase.*p = 0.017; **p = 0.022.


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performed 99mTc-labelled leukocyte SPECT scanning 24 h aftersurgery. Using this technique, we showed increased intra-abdominal activity compared with the preoperative baselinescan in patients subjected to an abdominal hysterectomy. Asthe actual resection, performed in the pelvic region, did not

involve any gastrointestinal organs, this observed increase inleucocyte activity cannot be explained by the primary surgicaltrauma. Clearly, leucocytes could reside anywhere in theabdominal cavity and may not be restricted to the intestinalwall. The additional CT scanning, however, showed that theincreased leucocyte activity observed with the SPECT scanscoincided with intestinal loops. When the uterus was resectedtransvaginally, a surgical approach that leaves the intestineslargely untouched, no such increase was observed, indirectlysuggesting that the intestinal inflammation is triggered byintestinal manipulation. From these data, we conclude that alsoin man, manipulation of the intestine during surgery leads tomast cell degranulation and a local inflammatory process,which, as in our animal model, plays an important role inpostoperative hypomotility.

In rodents, the extent of gastrointestinal hypomotility orileus is proportionally related to the degree of intestinalhandling and subsequent inflammation.8 As intestinal manip-ulation is minimal in laparoscopic surgery, one might argue thatthe degree of mast cell degranulation and the subsequentinflammatory response in the intestine will be less and thusmay contribute to the faster clinical recovery observed afterminimal invasive surgery. To test this hypothesis, tryptase andinflammatory mediators were quantified during two minimalinvasive surgical procedures—that is, laparoscopic and transva-ginal hysterectomy. In contrast to gentle handling during opensurgery, no mast cell degranulation or increase of IL6 wasobserved in the peritoneal lavage fluid. Only IL8 levels wereincreased, although to a lesser extent compared with lapar-otomy. Moreover, during transvaginal surgery, leaving theintestines largely untouched, none of the evaluated parametersincreased. These findings underscore that the degree ofintestinal handling to a large extent determines the degree ofmast cell activation and the subsequent inflammatory response.The latter was further confirmed by the 99mTc-labelled leucocyteSPECT scanning 24 h after surgery showing increased intra-abdominal activity in patients subjected to an abdominalhysterectomy but not in patients who underwent a transva-ginal hysterectomy. Finally, clinical recovery in our study wassignificantly delayed after abdominal compared with vaginalhysterectomy, a finding in line with previous clinical studiesshowing faster postoperative gastrointestinal recovery afterminimal invasive surgery.6 30–35 It should be emphasised, how-ever, that differences in postoperative pain medication, espe-cially opioids, may have contributed to the delay in

Figure 2 (A) Handling-induced leucocyte infiltration of the jejunalmuscularis propria, visualised by myeloperoxidase staining in early non-handled (upper panel) and late handled tissue segments (lower panel).Note the ongoing extravasation, illustrated by the predominantperivascular localisation of leucocytes in the handled late tissue sample.Semi-quantitative evaluation of handling-induced leucocyte recruitment(B) (scale 0 = non-inflamed, 1 = very mildly inflamed, 2 = mildlyinflamed, 3 = inflamed, 4 = clearly inflamed). Early non-handled (medianscore: 1 (IQR 1–2)) versus late handled (median score: 3 (IQR 2–4)),paired samples from n = 10 patients, p = 0.005 tested with a Wilcoxonsigned rank test. The dotted line represents the median increase inintestinal muscle inflammation of all 10 patients.

Figure 3 Quantification of postoperative leucocyte recruitment to thesmall intestinal region expressed as a percentage of the preoperativescan. A significant increase (Wilcoxon signed rank test) in intestinalleucocyte activity was observed after an abdominal hysterectomy(median percentage of preoperative scan, 127% (IQR 113–148), n = 8,p = 0.01), but not after a vaginal hysterectomy (median percentage ofpreoperative scan, 91% (IQR 84–102), ns, n = 8).


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normalisation of gastrointestinal motility.36 37 In the currentstudy, however, postoperative analgesia in both patient groupswas provided according to a standardised postoperative painprotocol, making this explanation less likely. Therefore, theobservation that delayed clinical recovery is associated withincreased influx of radiolabelled leucocytes indirectly adds to thehypothesis that the degree of intestinal handling, mast celldegranulation and subsequent inflammation determines theduration of postoperative ileus.

A drawback of this study is that the study protocols wereconducted in different groups of patients. Ideally, the samepatient cohort should have been studied in order to understand

better the causative association between mast cell degranulationand the subsequently observed inflammatory responses uponintestinal handling. In particular, as mRNA levels of ICAM-1and iNOS peak only 2–24 h after stimulation,16 38 a long-lastingsurgical procedure had to be chosen in order to allow thedetection of the upregulation of these inflammatory markers inresponse to intestinal handling. Therefore, patients undergoingbiliary reconstructive surgery were selected instead.

Our current findings may have important clinical implica-tions. First, they clearly illustrate that manipulation of theintestine should be limited whenever possible in order to reducethe release of mast cell mediators and limit postoperativeintestinal inflammation. This knowledge should encouragefurther development of minimal invasive surgical or evenendoscopic techniques to minimise intestinal handling.Secondly, if mast cell degranulation is indeed an importantinitial step in the pathophysiology of postoperative ileus inman, mast cells may represent an important therapeutic target.As we previously showed reduction of postoperative ileus bymast cell stabilisation in our mouse model, our current findingsin humans warrant further studies evaluating the effect of amast cell-stabilising agent in patients.

Acknowledgements: We would like to thank Professor M.P.M. Burger and Dr M. vanBeurden for their support in realising the studies in the Department of Gynaecology,Professor M.W. Hollmann for anaesthesiological support and intellectual input, F. vanHemert, C. Veeris and J. de Jong for the WBC labelling, the Nuclear Medicinetechnicians and staff for the SPECT and CT imaging, and A. Groot for her support in thelaboratory.

Funding: Supported by the Technology Foundation STW, Applied Science Division ofNWO, and the Technology Program of the Ministry of Economic Affairs (NWO-STW,grant AKG 5727 to F.O.T. and R.vd W.).Competing interests: None.

REFERENCES1. Collins TC, Daley J, Henderson WH, et al. Risk factors for prolonged length of stay

after major elective surgery. Ann Surg 1999;230:251–9.2. Longo WE, Virgo KS, Johnson FE, et al. Risk factors for morbidity and mortality after

colectomy for colon cancer. Dis Colon Rectum 2000;43:83–91.3. Prasad M, Matthews JB. Deflating postoperative ileus. Gastroenterology

1999;117:489–92.4. Kehlet H, Holte K. Review of postoperative ileus. Am J Surg 2001;182(5A

Suppl):3S–10S.5. Schwenk W, Haase O, Neudecker J, et al. Short term benefits for laparoscopic

colorectal resection. Cochrane Database Syst Rev 2005;(3):CD003145.6. Chen HH, Wexner SD, Iroatulam AJ, et al. Laparoscopic colectomy compares

favorably with colectomy by laparotomy for reduction of postoperative ileus. Dis ColonRectum 2000;43:61–5.

Figure 4 Representative example of leucocyte SPECT – CT imaging24 h before (left column) and after (right column) an abdominalhysterectomy was performed. (A) Coronary SPECT overview slide withanatomical references: (1) liver, (2) bladder, (3) ileac spine and (4)lumbar vertebral range between which quantification was performed. (B)Transverse CT slide and (C) corresponding SPECT image at the sameposition in the quantification range, visualising increased leucocyteactivity in the abdominal region (arrows). (D) CT and SPECT overlayshowing the specific leucocyte activity in the small intestine(arrowheads).

Table 3 Clinical evaluation of postoperative recovery

Treatment group Abdominal hysterectomy Vaginal hysterectomy

Age (years) 50 (42–70) 55 (42–66)

ASA-PS 2 (1–3) 1 (1–2)

Duration of surgery (min) 182 (130–298) 150 (113–179)

Time until first flatulence(days)

2 (2–3) 1 (1–2)

Time until first defecation(days)

4 (4–5)* 2 (2–3)

Time until discharge (days) 8 (7–8)** 4 (4–5)

Patient demographics and clinical recovery data from patients undergoing a vaginalhysterectomy or an abdominal hysterectomy assessed in protocol 3. To identifypotential confounders in clinical parameters and to test significant difference, a Mann–Whitney test was performed. Age and ASA score did not differ between the twopatient populations. Time till first defecation (*p = 0.02) and time until discharge(**p = 0.001) were both significantly prolonged in patients undergoing an abdominalhysterectomy when compared with those undergoing a vaginal hysterectomy. All dataare given as mean and (range).ASA-PS, American Society of Anaesthesiologists-Physical Status (1 being a normalhealthy patient, and 6 being a patient declared brain-dead); see the Patients andmethods section for a detailed description.


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7. Glaser F, Sannwald GA, Buhr HJ, et al. General stress response to conventional andlaparoscopic cholecystectomy. Ann Surg 1995;221:372–80.

8. Kalff JC, Schraut WH, Simmons RL, et al. Surgical manipulation of the gut elicits anintestinal muscularis inflammatory response resulting in postsurgical ileus. Ann Surg1998;228:652–63.

9. Kalff JC, Carlos TM, Schraut WH, et al. Surgically induced leukocytic infiltrateswithin the rat intestinal muscularis mediate postoperative ileus. Gastroenterology1999;117:378–87.

10. de Jonge WJ, van den Wijngaard RM, The FO, et al. Postoperative ileus ismaintained by intestinal immune infiltrates that activate inhibitory neural pathways inmice. Gastroenterology 2003;125:1137–47.

11. The FO, de Jonge WJ, Bennink RJ, et al. The ICAM-1 antisense oligonucleotide ISIS-3082 prevents the development of postoperative ileus in mice. Br J Pharmacol2005;146:252–8.

12. Schwarz NT, Beer-Stolz D, Simmons RL, et al. Pathogenesis of paralytic ileus:intestinal manipulation opens a transient pathway between the intestinal lumen andthe leukocytic infiltrate of the jejunal muscularis. Ann Surg 2002;235:31–40.

13. Kalff JC, Turler A, Schwarz NT, et al. Intra-abdominal activation of a localinflammatory response within the human muscularis externa during laparotomy. AnnSurg 2003;237:301–15.

14. Saklad M. Grading of patients for surgical procedures. Anesthesiology 1941;2:281–4.

15. American Society of Aesthesiologists. New Classification of Physical Status.American Society of Aesthesiologists, Inc. Anesthesiology 1963;24:111.

16. Yan HC, Juhasz I, Pilewski J, et al. Human/severe combined immunodeficient mousechimeras. An experimental in vivo model system to study the regulation of humanendothelial cell–leukocyte adhesion molecules. J Clin Invest 1993;91:986–96.

17. Schwartz LB, Kepley C. Development of markers for human basophils and mastcells. J Allergy Clin Immunol 1994;94:1231–40.

18. Tarnok A, Hambsch J, Chen R, et al. Cytometric bead array to measure six cytokinesin twenty-five microliters of serum. Clin Chem 2003;49:1000–2.

19. Chen R, Lowe L, Wilson JD, et al. Simultaneous quantification of six human cytokinesin a single sample using microparticle-based flow cytometric technology. Clin Chem1999;45:1693–4.

20. Ramakers C, Ruijter JM, Deprez RH, et al. Assumption-free analysis of quantitativereal-time polymerase chain reaction (PCR) data. Neurosci Lett 2003;339:62–6.

21. Roca M, Martin-Comin J, Becker W, et al. A consensus protocol for white blood cellslabelling with technetium-99m hexamethylpropylene amine oxime. InternationalSociety of Radiolabeled Blood Elements (ISORBE). Eur J Nucl Med 1998;25:797–9.

22. Weldon MJ, Masoomi AM, Britten AJ, et al. Quantification of inflammatory boweldisease activity using technetium-99m HMPAO labelled leucocyte single photonemission computerised tomography (SPECT). Gut 1995;36:243–50.

23. Hogan AD, Schwartz LB. Markers of mast cell degranulation. Methods 1997;13:43–52.

24. Schwartz LB, Bradford TR, Rouse C, et al. Development of a new, more sensitiveimmunoassay for human tryptase: use in systemic anaphylaxis. J Clin Immunol1994;14:190–204.

25. de Jonge WJ, The FO, van der CD, et al. Mast cell degranulation during abdominalsurgery initiates postoperative ileus in mice. Gastroenterology 2004;127:535–45.

26. Schwarz NT, Kalff JC, Turler A, et al. Prostanoid production via COX-2 as acausative mechanism of rodent postoperative ileus. Gastroenterology2001;121:1354–71.

27. de Jonge WJ, van der Zanden EP, The FO, et al. Stimulation of the vagus nerveattenuates macrophage activation by activating the Jak2–STAT3 signaling pathway.Nat Immunol 2005;6:844–51.

28. Wehner S, Behrendt FF, Lyutenski BN, et al. Inhibition of macrophage functionprevents intestinal inflammation and postoperative ileus in rodents. Gut2007;56:176–85.

29. Compton SJ, Cairns JA, Holgate ST, et al. The role of mast cell tryptase in regulatingendothelial cell proliferation, cytokine release, and adhesion molecule expression:tryptase induces expression of mRNA for IL-1 beta and IL-8 and stimulates theselective release of IL-8 from human umbilical vein endothelial cells. J Immunol1998;161:1939–46.

30. Isik-Akbay EF, Harmanli OH, Panganamamula UR, et al. Hysterectomy in obesewomen: a comparison of abdominal and vaginal routes. Obstet Gynecol2004;104:710–4.

31. Veldkamp R, Kuhry E, Hop WC, et al. Laparoscopic surgery versus open surgery forcolon cancer: short-term outcomes of a randomised trial. Lancet Oncol 2005;6:477–84.

32. Graber JN, Schulte WJ, Condon RE, et al. Relationship of duration of postoperativeileus to extent and site of operative dissection. Surgery 1982;92:87–92.

33. Huilgol RL, Wright CM, Solomon MJ. Laparoscopic versus open ileocolic resectionfor Crohn’s disease. J Laparoendosc Adv Surg Tech A 2004;14:61–5.

34. Bohm B, Milsom JW, Fazio VW. Postoperative intestinal motility followingconventional and laparoscopic intestinal surgery. Arch Surg 1995;130:415–9.

35. Milsom JW, Hammerhofer KA, Bohm B, et al. Prospective, randomized trialcomparing laparoscopic vs. conventional surgery for refractory ileocolic Crohn’sdisease. Dis Colon Rectum 2001;44:1–8.

36. Miedema BW, Johnson JO. Methods for decreasing postoperative gut dysmotility.Lancet Oncol 2003;4:365–72.

37. Bauer AJ, Boeckxstaens GE. Mechanisms of postoperative ileus. NeurogastroenterolMotil 2004;16(Suppl 2):54–60.

38. Yoo HS, Rutherford MS, Maheswaran SK, et al. Induction of nitric oxide productionby bovine alveolar macrophages in response to Pasteurella haemolytica A1. MicrobPathog 1996;20:361–75.

ANSWERFrom question on page 4

The sonographic characteristics of the gallbladder content excluded the diagnosis of aneoplastic process, suggesting instead a parasitic infestation. Microscopy of fresh stool samplerevealed non-embryonated eggs of Fasciola hepatica. Such a trematode usually parasitisesherbivorous mammals. Humans are accidentally infected by the ingestion of water or rawvegetables contaminated with the metacercaria. Human fascioliasis occurs in two steps: an acutephase, coinciding with hepatic invasion; and a chronic phase, caused by persistence of the adultform in the biliary tract, where it releases eggs. This latter situation occurred in our patient,presenting recurrent episodes of cholangitis. Anamnesis and specific ultrasound findings1 led tothe correct diagnosis. The patient received triclabendazole in two oral postprandial doses of100 mg, 12 h apart, which resulted in eradication of the parasite.

Gut 2008;57:40. doi:10.1136/gut.2006.113928a

REFERENCE1. Richter J, Freise S, Mull R, et al. Fascioliasis: sonographic abnormalities of the biliary tract and evolution after treatment with

triclabendazole. Trop Med Int Health 1999;4:774–81.

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