Pharmacological Research 53 (2006) 135–141

The acute alterations in biochemistry, morphology, and contractility ofrat-isolated terminal ileum via increased intra-abdominal pressure

Mesut A. Unsala,∗, Mustafa Imamoglub, Mine Kadiogluc, Sevim Aydind, Cunay Ulkuc,Murat Kesimc, Ahmet Alvere, Nuri Ihsan Kalyoncuc, Ersin Yarisc, Hasan Bozkayaa

a Department of Obstetrics & Gynecology, Karadeniz Technical University, Faculty of Medicine, Trabzon 61080, Turkeyb Department of Pediatric Surgery, Karadeniz Technical University, Faculty of Medicine, Trabzon, Turkey

c Department of Pharmacology, Karadeniz Technical University, Faculty of Medicine, Trabzon, Turkeyd Department of Histology & Embryology, Karadeniz Technical University, Faculty of Medicine, Trabzon, Turkey

e Department of Biochemistry, Karadeniz Technical University, Faculty of Medicine, Trabzon, Turkey

Accepted 29 September 2005

Abstract

Aim and scope: To determine the acute effects of increased intra-abdominal pressure (IAP) on the biochemistry, morphology, and contractility oftB ischemia-r ischemia-r f high IAPm epresentstM bjected toa of 60 min.T ialdehyde( e analysis,wR ooth musclel oth IAPgC tions whichm©

K

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upe-t inmay

nal, thecriti-the

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he isolated terminal ileum of rats.ackground: Laparoscopic procedures are used clinically in diagnostic and treatment modalities and experimentally as a model of

eperfusion injury induced by the elevation of IAP. Although some clinical and in vivo experimental studies investigate the results ofeperfusion injury whether induced by elevated IAP or clamping, there is no in vitro study that has investigated the acute effects oimicked by a laparoscopic intervention in any of the intra-abdominal organs (like terminal ileum) on the basis of contractility which r

he motility.ethods: Twenty-four adult with either sex Sprague–Dawley rats were divided into three groups. The control group (Group I) was not suny IAP. In Groups II and III, an IAP of 10 and 20 mmHg, respectively, was established by carbon dioxide pneumoperitoneum for a periodhirty minutes after the desufflation, the terminal ileum was removed for in vitro pharmacological investigation, measurement of malondMDA) values, and histopathological examination. Statistical comparisons among groups were done using the Kruskal–Wallis variancith post hoc comparison performed with the Mann–WhitneyU-test.esults: Tissue MDA value and the damage scores of mucosa and submucosa were significantly increased in both IAP groups. The sm

ayer was significantly damaged only in Group III. The contractions obtained by electrical field stimulation (EFS) were inhibited in broups, and the contractions to acetylcholine were inhibited in Group III when compared to the control group.onclusions: In conclusion, we can say that pneumoperitoneum induced IAP may inhibit contractile responses, cause structural alteraay be related to ischemia-reperfusion injury in rat terminal ileum.2005 Elsevier Ltd. All rights reserved.

eywords: Ischemia; Increased abdominal pressure; Laparoscopy; Reperfusion; Intestine

∗ Corresponding author. Tel.: +90 462 377 54 94; fax: +90 462 377 54 98.E-mail addresses: mesutunsal@meds.ktu.edu.tr, mesut.unsal@gmail.com

M.A. Unsal), imamoglu@meds.ktu.edu.tr (M. Imamoglu),kadioglu@meds.ktu.edu.tr (M. Kadioglu),

aydin@meds.ktu.edu.tr (S. Aydin), cunay73@yahoo.de (C. Ulku),kesim@meds.ktu.edu.tr (M. Kesim), alver61@yahoo.com (A. Alver),

alyoncu@meds.ktu.edu.tr (N.I. Kalyoncu), eyaris@meds.ktu.edu.tr (E. Yaris),bozkaya@meds.ktu.edu.tr (H. Bozkaya).

1. Introduction

When laparoscopy was first introduced to surgeons, its’ sriority to classical surgical techniques was underlined, burecent years many studies have shown that laparoscopycause serious harmful effects both to intra-abdominal[1,2] andextra-abdominal organs[3] because of induced intra-abdomipressure (IAP) and many other complicating factors (e.g.level of IAP, the type and some properties of the gas used,cal values of the patient, etc.), which are directly related tomethod’s own characteristics.

043-6618/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.phrs.2005.09.010

136 M.A. Unsal et al. / Pharmacological Research 53 (2006) 135–141

In the laparoscopic technique, a working space is estab-lished by insufflation of a gas (in most cases carbon diox-ide; CO2) into the abdominal cavity, which is common forall abdominopelvic procedures. In some laparoscopic proce-dures, an IAP of 18–25 mmHg is needed[1,4], but this levelis not reached in routine laparoscopy. Most of the clinicalprocedures are performed at 10–15 mmHg[5]. This pressurerange, although it is higher than the normal portal systemicpressure (7–10 mmHg) maintains the balance between estab-lishment of a working space with adequate visualization andthe untoward effects of increased IAP. In a study dealingwith a pig model, the increased IAP from 10 to 20 mmHgcaused a respective decrease of 20% to 40% in mucosalblood flow in the small intestine[5]. Diebel et al. reportedthat elevation of IAP to 20–25 mmHg caused a decrease of63% in mucosal blood flow in rats with a normal meanarterial pressure of 102–123 mmHg[6]. Van de Casteele etal. measured a mean arterial pressure of 108± 20 mmHgand a portal venous pressure of 3± 1 mmHg in normal rats[7].

Most of the studies have shown that the blood flow to abdom-inal organs is reduced due to high venous resistance during alaparoscopic procedure, but normalized, after desufflation ofthe abdominal cavity. This ischemia/reperfusion (I/R) modelpromotes the generation of various free radicals, which areknown to have deterious effects on cellular function resulting ino ciali an-s tility[ on-t ee

ma-c ages ther g,w dels[

2

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2.2. Experimental groups

The rats were divided into three groups. The animals in thecontrol group (Group I;n = 8) were not subjected to IAP. Butthe angiocatheter was placed into the abdomen like the otheranimals, and this catheter was attached to the laparoflator for60 min even though the IAP was not increased. In the othergroups, CO2 was insufflated into the abdominal cavity until IAPat a level of 10 mmHg (Group II;n = 8) and 20 mmHg (GroupIII; n = 8), respectively. IAP was controlled for 60 min with anelectronic laparoflator (Karl Storz GmbH, Tutlingen, Germany).

After the desufflation was completed Groups II and III, theangiocatheters were removed from the abdomens of all animalsincluding the ones in the control group. Reperfusion periods of30 min were given to all of the animals. Following a midlineabdominal incision, the adjacent side of the ileocecal junctionwas localized, and four centimeters proximal to the junctionwas accepted as terminal ileum and that segment was removedfrom the rats. After the terminal ileum was dissected of fat andconnecting tissue, the most distal parts were separated to use inpharmacological experiments (20-mm long). The other parts ofthe removed segments (each 10-mm long) were sent to the his-tology and biochemistry departments. Longitudinal strips of theileum were immediately placed in organ baths for pharmacolog-ical experiments. All pieces of ileum given to the biochemistrydepartment were stored at−70◦C for the measurement of MDAl tionw tho-l

2

tedl odes1be , thep nsionw 0-A)u lieds l.

inedwf FS)w aceda nd a1 , 32,a ntrac-t f thec upsw

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xidative tissue damage, and then organ dysfunction, espen splanchnic organs[8–14]. Some studies reported that trient hypoxia might induce some disorders in intestinal mo15,16], which may play a role in postoperative ileus and spaneous ileum perforation[17–19] via its contribution to thlevated IAP.

The aim of this study is to evaluate acute pharological (contractility), histopathological (tissue damcores), and biochemical (MDA levels) alterations inat terminal ileum under an IAP of 10 and 20 mmHhich were previously used in laparoscopic animal mo

3,20].

. Materials and methods

.1. Animals

The study protocol was approved by the University Aal Care and Ethics Committee (Approval no.: 2004/

wenty-four adult Sprague–Dawley rats with either sex weng between 300 and 350 g were anaesthetized by intramunjection of ketamine hydrochloride 90 mg kg−1 (Ketalar, Parkeavis) plus xylazine 10 mg kg−1 (Rompun, Bayer, Germanynd were allowed to breathe spontaneously during the exents. The animal’s body temperature was kept constant at◦Cith a heating blanket.The method for the creation of the pneumoperitoneum

imilar to that defined by Yilmaz et. al.[21]. The only differencas the use of 18-gauge angiocatheters for peritoneal cuncture caudal to the sternum.

ly

ar

i-

y

evels. The last pieces obtained for histological investigaere fixed within a formaldehyde (10%) solution for histopa

ogical examination.

.3. Measurement of contractile response

The rat-isolated ileum strips (10-mm long) were mounongitudinally in 30 ml jacketed organ baths containing Tyrolution [(mM): NaCl: 137; KCl: 2.68; CaCl2: 1.8; NaHCO3:1.9; NaH2PO4: 0.42; MgSO4: 1.1; glucose: 11] at 37◦C, bub-led with a gas mixture of 95% O2 and 5% CO2. After anquilibration period of 60 min under a resting tension of 1 gharmacological experiment was started. Changes in the teere recorded by a force displacement transducer (FT 1sing MAY Biopac 100 programmer. Chemicals were appo that the total volume of each drug did not exceed 0.5 m

Non-cumulative dose–response curves were obtaith acethylcholine (ACh; 10−8 to 10−4 M). For the

requency–response curves, electrical field stimulation (Eas produced by bipolar parallel platinum electrodes plround the strips. Pulses of 50 V with a 5 ms duration, ams delay were applied at the frequencies of 1, 2, 4, 8, 16nd 64 Hz. For each curve, the percentage of maximum co

ion was calculated assuming the maximum contraction oontrol group is 100%. The contractions of the other two groere ranked in percentage based on this maximum level.

.4. MDA measurement

To evaluate the changes of oxidative stress, the Mevels were assayed as evidence of lipid peroxidation

M.A. Unsal et al. / Pharmacological Research 53 (2006) 135–141 137

piece of ileum tissue obtained from each animal was usedto measure MDA levels. Tissue MDA levels were deter-mined by the method of Mihara and Uchiyama[22]. Tetram-ethoxypropane was used as standard for MDA measurement.MDA levels were expressed as nanomole per gram of wettissue.

2.5. Histology

Histopathological changes in the ileum were studied undera light microscope. Ten mm segments of terminal ileum werefixed in 10% formalin and embedded in paraffin. Serial sec-tions (5�m) were stained with hematoxylin and eosin (H&E)for each animal block. All specimens were evaluated underan Olimpus BX50 microscope and scored blindly by a histol-ogist according to a scoring system described by Liao et al.[23] and Byrka-Owczarek et al.[24] (Table 1). Edema, leuko-cyte infiltration, hemorrhage, and necrosis were evaluated in alllayers.

Slides were assessed at high magnification (×40), fivehigh power fields were randomly sampled, and the meanleukocyte number was calculated for each animal. All lay-ers were evaluated for leukocyte infiltration. Zero to fiveleukocytes were accepted as normal, 6–10 slight, 11–20moderate, and >21 leukocytes were accepted as severei

2

K isti-c medw asa

TH

S

M

cells,

S

M

Fig. 1. MDA levels of ileum in control group (�), 10 mmHg group ( ),20 mmHg group (�) (mean± S.E.M,* p < 0.0005 compared to control group).

3. Results

3.1. MDA levels

Mean MDA values of all groups are shown inFig. 1. TissueMDA values of both IAP groups were significantly increasedwhen compared to the control group (Kruskal–Wallis chi-square 15.735, d.f. 2,p < 0.0005). The mean MDA value of the20 mmHg IAP group was higher than the 10 mmHg IAP group,but the difference between the two groups was not statisticallysignificant.

3.2. Histopathological findings

Histopathological examination scores and the results of theirstatistical analysis are shown inFig. 2.

3.2.1. In the control groupHistopathological examination of seven ileum samples from

control animals showed a normal structure in mucosa (Fig. 3a),

F laris oft( olg

nfiltration.

.6. Statistical analysis

All the values were expressed as mean± S.E.M.ruskal–Wallis analysis of variance was used for statal comparison of groups, with post hoc comparison perforith the Mann–WhitneyU-test. Ap-value of less than 0.05 wccepted as statistically significant.

able 1istopathologic scoring system[23,24]

core Histopathologic changes

ucosa0 Normal1 Subepithelial edema, partial separation of apical epithelial

and broadening of villi2 Epithelial cell slough from tip of villi3 Progression of slough to base of villi4 Partial mucosal necrosis of lamina propria5 Total mucosal necrosis

ubmucosa0 Normal1 Mild edema and/or hemorrhage2 Progression of edema and/or hemorrhage3 Severe edema and hemorrhage

uscularis0 Normal1 Mild edema and/or hemorrhage2 Progression of edema and/or hemorrhage3 Severe edema, partial muscularis necrosis

ig. 2. Histopathologic score results of mucosa, submucosa and muscuhe terminal ileum in control group (�), 10 mmHg group ( ), 20 mmHg group�) (mean± S.E.M.,* p < 0.0005,#p < 0.0005,§p = 0.004 compared to contrroup).

138 M.A. Unsal et al. / Pharmacological Research 53 (2006) 135–141

Fig. 3. (a) A section through normal ileum in control group (H&E X 10); (b) the 10 mmHg IAP group showed separation of villi, mild subepithelial edema, moderateleukocyte infiltration in all layer (H&E X 10); (c) in the 20 mmHg IAP group, progression of slough to base of villi is seen. In submucosa, and muscularis layersprogression of edema and hemorrhage, and moderate-severe leukocyte infiltration were found in all layers (H&E X 10): (1) separation of villi; (2) edema; (3)hemorrhage; (4) leukocyte infiltration).

but because of a slight edema, the damage score was not zeroin three samples of the submucosa layer. There was not anyhistopathologic damage in the muscularis layer of all animals inthis group.

3.2.2. In 10 mmHg groupSubepithelial edema, partial separation of apical epithelial

cells, and broadening of villi were observed in the mucosa layers.Mild edema or hemorrhaging was observed in the submucosalayers, and moderate leukocyte infiltration was observed in bothlayers in almost all specimens in this group. Histopathologicscores were significant in the mucosa and submucosa layerscompared with the control group (p < 0.0005). In the muscularislayer, mild edema or hemorrhaging was found in two specimens,but these histopathologic changes were not significant (Fig. 3b).

3.2.3. In 20 mmHg groupEpithelial cell slough from the tip of the villi in the

mucosal layers and progression of edema or hemorrhagingwere found in both the submucosal and muscularis lay-ers. Histopathologic scores were significant in the mucosa(p < 0.0005), submucosa (p < 0.0005) and muscularis lay-ers (p = 0.004) compared with the control group. Moderate

to severe leukocyte infiltration were found in all layers(Fig. 3c).

3.3. Ileal longitudinal muscle contractility

For Groups II and III, EFS produced frequency (1–64 Hz)-dependent contractions in isolated ileal strips were obtained.

Fig. 4. Frequency–response curves obtained in rat ileum strips [control group(♦); 10 mmHg group (�); 20 mmHg group (�). Means± S.E.M., * p < 0.05:compared to control group].

M.A. Unsal et al. / Pharmacological Research 53 (2006) 135–141 139

Fig. 5. Contractions of ACh in ileum strips. [control group (♦); 10 mmHg group(�); 20 mmHg group (�). Means± S.E.M., * p < 0.05: compared to controlgroup].

These contractions were significantly inhibited by IAP. Whilethis inhibition was significant for all frequencies in the 20 mmHgIAP group, it was not significant at 32 and 64 Hz in the 10 mmHgIAP group (Fig. 4). Like EFS, ACh-caused concentration-dependent contractions in isolated ileal strips (Fig. 5). In the10 mmHg IAP group, these contractions tended to decrease, butthis decrease did not reach a significant level. The reductionin ACh-induced contractions in the 20 mmHg IAP group wasstatistically significant at concentrations of 10−5 and 10−4 M,when compared to the control group (Fig. 5).

4. Discussion

There are some clinical and experimental studies in the literature investigating the results of IAP in animals and in humanson gastrointestinal system motility and contractility. By usingdifferent methods like recording myoelectrical activity[25–27],solid phase gastric emptying, and radiopaque transit[28], theauthors obtained different results of acute IAP that are not consistent with each other. There is no in vitro study in the currentliterature regarding investigation of the intestinal contractility inrat-isolated intestines exposed to acute IAP by means of classcal pharmacological organ bath techniques. In the present studlaparoscopy, which is widely used in clinical interventions, waspreferred as an experimental model for inducing acute IAP ino ileam

on-t gast ersl lingI repres rks,a neur ACa vent

senti

generally depends on acute iatrogenic IAP created by the gaspumped for surgical visualization, which causes ischemia andreperfusion injury following ischemia, dependent upon the leveland time of IAP and properties of the intra-abdominal organexposed to IAP. Previously, the clinically permitted laparoscopicpressure level was 25 mmHg at the beginning and 18 mmHgduring the intervention[4]. Recently, this range was reduced to10–15 mmHg[14]. But some gynecologic laparoscopic proce-dures require an IAP of 25 mmHg[1]. Madl and Druml reportedthat an IAP not exceeding 10 mmHg did not have any majorconsequences, but an IAP higher than 12 mmHg might causean inflammatory process (like leukocyte migration, release ofcytokines, increased formation of reactive oxygen intermedi-ates, etc.). IAP between 18 and 20 mmHg is defined as “intra-abdominal hypertension,” and when exceeding 20 mmHg, itbecomes the main cause of abdominal compartment syndrome,which must be treated in emergency conditions because of multiorgan impairments[17]. I/R injury was defined as the basic pro-cess underlying these clinical manifestations, which are directlyrelated to disorders in microcirculation induced by IAP withits direct mechanical compression on the abdominal vasculature[29]. Our data showed that while muscularis layer remained rela-tively intact in the 10 mmHg IAP groups, some histopathologicalchanges occurred in the mucosa and submucosa (Fig. 3b). In thehigher (20 mmHg) IAP groups, all of the layers were signifi-cantly more affected (Fig. 3c). The highest damage scores werec ferentt neym riove-n rmenti sedb icalc shortc vor-a mageti fen-s tion ins

f thei edi ularl easer esti-n ac tho-l le tos EFSr al net-w to bes ch ap EFSr ounto thatAm morep . A

rder to evaluate the underlying isolated mechanisms ofotility directly affected by increased IAP, in vitro.Regular gastrointestinal motility is an important factor c

rolling intra-abdominal pressure by means of pushing therointestinal ingredients forward. In case of motility disordike constipation or ileus, some clinical symptoms resembAP can be seen. The contractile response of the intestineenting the motility is controlled by several neuronal netwond the cholinergic mechanism is one of the dominatingotransmitter systems. In the present study, exogenousnd electrical field stimulation, which was previously pro

o release ACh, in addition to some mediators, were used.Laparoscopy is a widely used surgical technique pre

ng both advantages[1] and disadvantages[1,2]. This method

-

-

i-y,

l

-

-

-h

-

alculated in the mucosa and submucosa which have a difype of a vasculature look like in the vasa recta of the kidedulla. This countercurrent mechanism based on an arteous shunt makes these layers more sensitive to the impai

n microcirculation. In case of hypoxia, which can be cauy an impairment in circulation related with some pathologonditions such as shock or, probably, increased IAP, thisircuit for oxygen transfer to the intestinal tissue that is fable under normal conditions may cause some harmful da

o the intestine (especially in villi and mucosa)[30]. Mucosas not responsible for the intestinal motility and plays a deive role against bacterial agents, absorption, or sequestraome pathological conditions.

The neuronal network (Auerbach or myenteric plexus) ontestine, which mainly plays the induction of motility, is placn muscularis layer between longitudinal and circular muscayers[30]. Damage to the neuronal structures, which relelated neurotransmitters responsible for induction of intal contractility and motility, may result in distension withontribution from the injured mucosa. Although our histopaogical evaluation method and scoring system are not abhow directly the neurological damage, the decrease inesponses suggests a possible damage also in the neuronork located in the muscularis layer, which appeared noteriously injured especially in the 10 mmHg IAP group. Suossible histopathological injury can explain the decrease inesponses that is the result of a possible reduction in the amf the neurotransmitter released. It was previously shownCh has been released by EFS in intestinal segments[31]. Asentioned above, endogenous cholinergic mechanisms arerominent than the others in induction of intestinal motility

140 M.A. Unsal et al. / Pharmacological Research 53 (2006) 135–141

significant histopathological injury of the submucosal layer waseven present in the 10 mmHg IAP group, which is less thanthe clinically permitted levels. This must be underlined as helpsexplain our results showing a decrease in EFS responses.

In case of exogenously applied ACh, the contractile responsesof the terminal ileum to this agent were significantly decreasedin the 20 mmHg IAP group but only trended down in the10 mmHg IAP group. When the lack of significant histopatho-logical changes in the muscularis layer in the 10 mmHg groupcompared to the control group and the presence of serioushistopathological changes in the 20 mmHg IAP group, we cansay that serious damage in the muscularis layer, which is the tar-get of exogenously applied ACh, might explain the significantreduction of ACh responses observed in the higher IAP group.The responses to ACh was decreased at almost all ACh con-centrations in both groups but reached a significant level onlyfor the 20 mmHg IAP group and only for 10−5 and 10−4 Mlevels of ACh. Since the significance of the decrease of AChresponses is only present for the highest ACh concentrationstested and only in the 20 mmHg IAP group, we can suggest thatthis situation most likely originated from the great differencebetween the higher degree of maximum contractions obtainedin the control group (non IAP group) and the decreased levelof ACh responses due to histopathological damage caused byIAP. It can also be concluded that this decrease in exogenouslyapplied ACh responses were pressure dependent and may bed

lin-e linec easeb aset atedts osaa mah abovd esti-n singg

opicm tivesp rs oi celm leasa ture[ tiona supp nneA turac a ret seei APg cand

not,i rmi-

nal ileum) motility disorders ranging from non-specific, simpleabdominal symptoms to ileus or spontaneous perforation[19] ofthe terminal ileum. These simple symptoms, which can be theinitial signs of ileus, are frequently ignored and often attributedto the laparoscopic surgical procedure both by the patient andthe surgeon. A total of eight cases of spontaneous perforationof the small bowel due to laparoscopic IAP were reported in theliterature[19].

Even the underlying mechanisms are not completely illumi-nated; clinical, histopathological, biochemical, and functionalfindings like in this study, suggest a strong relationship betweenthe reperfusion period following the impairment in microcircu-lation (ischemia) of intra-abdominal organs, i.e., the terminalileum in this case, induced by an iatrogenic (laparoscopic) IAPat a clinically relevant level. Histopathological and biochemicalmechanisms may also be functional alterations that may triggersome clinical manifestations.

Acknowledgement

The authors acknowledge Ms. Janice O. Vantrease for herEnglish grammar review.

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RD.eart

inal

, etblood

n C,ed.

j P, eteum

bac-auma

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[ s K.vated

[ IS.ion

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rgic receptor mechanisms, the decreasing trend of thereated by exogenously applied or endogenous ACh (rely EFS) and shown in the figures suggests a possible decre

he cholinergic receptor number or affinity which may be relo the acute effects of increased IAP (Figs. 4 and 5). It can bepeculated that the inflammatory infiltration to the submucnd muscularis layers of the ileum induced by increased IAPave been related to the pharmacological action discussede Jonge et al. reported that leukocyte infiltration to the intal muscularis layer could itself decrease gut motility cauastroparesis at the postoperative period[32].

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