Effect of transgastric peritoneal access on peritoneal innatecellular immunity: experimental study in swine

Rodrigo Rodrigues • Marcelo Rezende • Gustavo Gomes • Fernando Souza •

Maiara Blagitz • Alice Della Libera • Murched Taha • Angelo Ferrari •

Ermelindo Della Libera Jr.

Received: 23 February 2012 / Accepted: 4 August 2012 / Published online: 13 December 2012

� Springer Science+Business Media, LLC 2012

Abstract

Background One of the main concerns of natural orifice

surgery is the local and systemic impact on physiology.

Few studies have compared natural orifice transluminal

endoscopic surgery (NOTES) with other surgical modali-

ties. Most studies are based on systemic variables such as

postoperative serum cytokines, with conflicting results.

Surgical trauma induces an early inflammatory response,

release of cytokines, and local leukocyte activation and

oxidative burst. Major surgical trauma is related to

impairment of phagocytic function and an increase in

production of active oxygen species by phagocytes. The

aim of this study was to evaluate the impact of transgastric

peritoneoscopy on peritoneal innate immune response

compared with laparoscopy and laparotomy in swine.

Methods Thirty-four male Sus scrofa domesticus swine

were assigned to four groups: transgastric peritoneoscopy

(13), laparoscopy (7), laparotomy (7), and sham procedure

(7). Twenty-four hours after the procedure, peritoneal fluid

cells were harvested by peritoneal washing after necropsy.

Flow cytometry analysis of labeled S. aureus and E. coli

phagocytosis by peritoneal neutrophils and macrophages

was blindly performed. Oxidative burst activity measured

by H2O2 production under different challenges was also

evaluated.

Results Total operative time varied between all groups.

The transgastric, laparoscopy, and laparotomy groups

required 56, 17.2, and 40.3 min of mean operative time,

respectively (p \ 0.05). Even though the mean percentage

and intensity of phagocytosis by peritoneal phagocytes

were higher in the sham, transgastric, and laparoscopy

groups, there was no significant difference between these

groups and laparotomy. Macrophage production of H2O2

has been shown to be similar among the transgastric, lap-

aroscopy, and sham groups, and smaller than that in lapa-

rotomy (p \ 0.05), either under basal conditions, while

performing E. coli phagocytosis, or challenged by the

presence of E. coli membrane lipopolysaccharide.

Conclusion Under the conditions of this study, transga-

stric peritoneoscopy has been shown to have minimal

impact on peritoneal innate immune response.

Keywords Natural orifice endoscopic surgery �Laparoscopy � Immunity � Innate � Flow cytometry �Free radicals � Peritoneum

R. Rodrigues � M. Rezende � G. Gomes � A. Ferrari �E. D. Libera Jr. (&)

Clinical Gastroenterology Division, Department of Medicine,

Sao Paulo Federal University (UNIFESP), Sao Paulo, Brazil

e-mail: edellaliberajr@uol.com.br

R. Rodrigues

e-mail: rodrigo.ar@terra.com.br

R. Rodrigues � E. D. Libera Jr.

Endoscopy Unit, Fleury Medicina e Saude, Sao Paulo, Brazil

F. Souza � M. Blagitz � A. D. Libera

Department of Clinical Science, Faculty of Veterinary Medicine

and Animal Sciences, University of Sao Paulo (USP), Sao Paulo,

Brazil

M. Taha

Experimental Surgery Division, Surgery Department, Sao Paulo

Federal University (UNIFESP), Sao Paulo, Brazil

A. Ferrari

Endoscopy Unit, Hospital Israelita Albert Einstein, Sao Paulo,

Brazil

E. D. Libera Jr.

Rua Itapimirum, 326 Ap 121b, Sao Paulo, SP CEP 05716-090,

Brazil

123

Surg Endosc (2013) 27:964–970

DOI 10.1007/s00464-012-2541-8

and Other Interventional Techniques

Natural orifice transluminal endoscopic surgery (NOTES)

was introduced as a potentially less invasive route for

peritoneal access [1]. Eliminating abdominal wounds might

lessen the impact on local and systemic homeostasis

induced by surgical trauma. On the other hand, contami-

nated material carried into the peritoneal cavity by a non-

sterile instrument, among other difficulties, could override

the protective benefits of NOTES. Since the NOSCAR

consortium directions for natural orifice surgery investi-

gations [2], few reports have studied the effects of NOTES

on systemic or peritoneal inflammatory response [3–6].

Most of those reports are based on plasmatic or peritoneal

cytokine assays to indirectly compare the inflammatory

impact of different surgical modalities. Surgical trauma

triggers a complex sequence of inflammatory response,

beginning with first-line defense cell activation such as

macrophages and neutrophils, reactive oxygen species

production, chemotactic stimuli, and proinflammatory

cytokine release [7, 8]. The purpose of the present study

was to evaluate the inflammatory response after transga-

stric peritoneoscopy based on the innate immune response

of peritoneal fluid cells.

Methods

This experimental nonsurvival study was conducted in the

research facility of Federal University of Sao Paulo. After

approval by our Institutional Ethics Research Committee,

34 Sus scrofa domesticus male pigs (C-76 Agroceres�, Sao

Paulo, Brazil), around 12 weeks of age and weighing

between 30 and 40 kg, were used in this study. The pigs

were transferred to the research facility 48 h before the

procedures for acclimation and were individually caged in

fenestrated bedding stables in order to ensure appropriate

fasting. Water was offered ad libitum until 12 h before the

procedure.

Animals were assigned to one of four groups: sham

(anesthesia only) (7), transgastric peritoneoscopy (13),

laparoscopy (7), and laparotomy (7).

Animal preparation and postoperative care

In order to avoid unwanted variation among the groups, the

study’s steps were the same for each animal, except the

main intervention. Animals were fed with the same diet

and then fasted for 48 h, with free access to water during

fasting until 12 h before the procedure. Ketamine

(5–7.5 mg/kg) and midazolam (0.25–0.35 mg/kg) for

sedation were administered intramuscularly (IM). An

intravenous catheter was placed in a marginal ear vein.

Propofol (5 mg/kg) was administered for endotracheal

intubation and anesthesia was maintained with 2.5 %

isoflurane. A veterinary anesthesiologist controlled the

anesthesia and recovery of all animals. In the first few

minutes of anesthesia, the right internal jugular vein was

accessed through a 3-cm vertical incision. An indwelling

catheter was placed and flushed with heparin after aspira-

tion. The catheter was tunneled to the lateral neck and

secured to the skin using 3-0 mononylon and a bandage

cover. Duration of anesthesia was preset at 90 min irre-

spective of the study group. No antibiotic prophylaxis was

used. Surgical procedures were performed under sterile

conditions, including for the operators, the surgical

equipment, and the laparoscopes. Flexible endoscopes and

over-tubes were used after high-level disinfection. After

recovery, all animals were transferred to stables with free

access to water. As standard postoperative analgesia, ani-

mals received 100 mg of tramadol IM by the end of sur-

gery and 8 h later.

Transgastric peritoneoscopy

After oral decontamination with chlorhexidine, the endo-

scope (EG-250WR5, Fujinon Corp., Japan) was inserted

through an over-tube. A first gastric irrigation with saline

was performed to remove residual food, followed by 1 L

of a 1-g cephalotin–sodium solution. Peritoneal access

was obtained using a modification of the submucosal

tunnel technique described by Yoshizumi et al. [9]. A few

centimeters below the cardia, on the posterior gastric

wall, submucosal injection of sterile methylene blue

solution was followed by a 1-cm transverse incision on

the proximal end of the cushion (Microknife XL, Boston

Scientific Corp., Natick, MA, USA). The tip of the

endoscope was then passed through this incision into the

submucosal layer. Instead of using an insulated-tip cath-

eter (as previously described), a regular hot biopsy for-

ceps was used for submucosal dissection and coagulation

of submucosal vessels (Radial Jaw� 3 Hot Biopsy for-

ceps, Boston Scientific Corp.). The seromuscular layer

was perforated at the distal end of the tunnel. The

puncture site was then dilated with a 12-mm hydrostatic

balloon over a guidewire (CRETM and HydraJagwire�,

Boston Scientific Corp.). The gastroscope was advanced

into the peritoneal cavity allowing transgastric periton-

eoscopy and recognition of the main abdominal organs.

Peritoneal insufflation was provided by the endoscope,

with special attention paid to avoid respiratory distress.

Once peritoneoscopy was concluded, the cavity was

aspirated, the endoscope was withdrawn, and the proximal

end of the tunnel was closed with metallic clips (EZ Clip,

Olympus Optical do Brazil Ltda.). If closure was con-

sidered ineffective, cyanoacrylate (n-butyl cyanoacrylate,

Histoacryl�, B. Braun, Melsungen, Germany) was injec-

ted inside the submucosal tunnel.

Surg Endosc (2013) 27:964–970 965

123

Laparoscopy and laparotomy

After skin preparation with povidone-iodine and sterile

draping, a two-trocar laparoscopy was performed. Apo-

neurosis was reached at the lower right quadrant of the

abdomen. Pneumoperitoneum was obtained with a Veress

needle. The needle was connected to a CO2 insufflator with

intra-abdominal pressure preset at 12 mmHg. Once peri-

toneoscopy was concluded, the cavity was aspirated and

the laparoscope was withdrawn. Incision sites were closed

with 2-0 mononylon and sterile dressing was applied over

the wounds.

Laparotomy was performed through a 20-cm-long

midline incision after skin preparation and sterile draping.

After abdominal cavity inspection, abdominal wall layers

were sutured with Vicryl 0 and 2-0 cotton sutures, followed

by sterile dressing over the wound.

Sample collection and data analysis

After 24 h, euthanasia with an intravenous bolus of 19.1 %

potassium chloride after propofol sedation was performed

followed by necropsy. The peritoneal cavity was opened

under sterile conditions. About 500 mL of phosphate-

buffered saline solution (4 �C) were instilled into the cavity

followed by gentle manipulation of the abdominal viscera.

Peritoneal fluid was then aspirated and transferred to sterile

propylene tubes containing heparin stored in cool boxes

and sent to the laboratory. Samples were processed in a

maximum of 4 h after euthanasia in order to preserve cell

viability. After fluid collection, the distal esophagus,

stomach, and proximal duodenum were clamped and

removed. An insufflation catheter connected to an air pump

was tightly adapted to the esophagus. The stomach was

gradually filled with room air until it was completely dis-

tended. The specimen was totally immersed into water for

1 min to test the security of endoscopic access closure.

Weight, age, rectal temperature, and pre- and postop-

erative laboratory results (hematocrit, hemoglobin, white

blood cell, and platelet count) were recorded. Peritoneal

fluid was filtrated and centrifuged until a cell suspension

was obtained. Trypan blue exclusion (cat. No. 368-12;

EMD Chemicals Inc., Gibbstown, NJ, USA) in a Newbauer

chamber was used to evaluate cell viability, and cell sus-

pension was adjusted to 2 9 106 viable cells/mL for

functional immune tests.

Simultaneous flow cytometry was used [10] for identi-

fication of macrophages and neutrophils as well as for

evaluation of phagocytic and oxidative burst activity. A

primary monoclonal antibody (mouse IgG1 anti-bovine

CD14, cat. No. CAM36A; VMRD, Pullman, WA, USA)

and a secondary antibody labeled with a fluorochrome

(AlexaFluor� 633 Goat anti-mouse, cat. No. A-21126;

Invitrogen, Carlsbad, CA, USA) were also used. Peritoneal

fluid cells were incubated with Staphylococcus aureus

(ATCC 25923) and Escherichia coli (O98:H28) stained

with propidium iodide (PI) (P4170; Sigma Aldrich,

St. Louis, MO, USA), and Escherichia coli membrane

lipopolysaccharide (LPS) 250 lg/mL (LPS 055:B5, cat.

No. L-3129; Sigma Aldrich). 20,70-Dichlorofluorescein-

diacetate (DCFH-DA, cat. No. C1157; Molecular Probes,

Eugene, OR, USA) was used as a substrate for measuring

intracellular oxidant production in peritoneal fluid leuko-

cytes [11].

Samples were read in a flow cytometry FACSCaliburTM

(Becton Dickinson Immunocytometry System, BD,

Franklin Lakes, NJ, USA) connected to a computer with

CellQuestTM software (Becton Dickinson). Ten thousand

events were acquired from each assay and the data obtained

were analyzed by FlowJo� software (TreeStar, Inc.,

Ashland, OR, USA).

Localization of macrophages and neutrophils was per-

formed by dot plot with size (FSC) and granularity (SSC)

[12, 13] and by CD14 (APC) expression through the

average fluorescence intensity obtained in a logarithmic

scale with wavelength 661 ± 16 nm (FL4). Phagocytosis

was measured by emission of red fluorescence at wave-

length 670 nm (FL3) for PI. Values were analyzed after

compensation of fluorescence to avoid spectral overlap.

The files with flow cytometry records were encrypted,

blinding the data analysis. An independent investigator

read the data regarding peritoneal neutrophil and macro-

phage phagocytosis of labeled S. aureus and E. coli. Oxi-

dative burst activity, measured by H2O2 production under

different challenges, was also evaluated in the same

fashion.

A literature review failed to identify prior NOTES

studies comparing peritoneal innate immune response to

surgical trauma. Therefore, sample size was arbitrarily

established.

Statistical analysis

Comparison among the groups was done using Kruskal-

Wallis test or an analysis of variance (ANOVA), as indi-

cated, followed by post hoc analysis. A significance alpha

level of 0.05 was used. Statistical analysis was performed

with SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA).

Results

Nine of the 34 animals were excluded from the study

because of complications deemed to interfere in peritoneal

immune response or sample reading (Table 1). The

remaining 25 animals were analyzed.

966 Surg Endosc (2013) 27:964–970

123

The four groups were equivalent in regard to weight,

age, and preoperative rectal temperature (p [ 0.05). There

were no differences in pre- and postoperative laboratory

results between the groups (p [ 0.05). Total operative time

varied among the groups. The mean operative times for the

transgastric, laparoscopy, and laparotomy groups were 56,

17.2, and 40.3 min, respectively (p \ 0.05). A lower per-

centage of cell viability was observed in the sham group

than in the other groups (p \ 0.05, Table 2).

Even though the mean values of percentage and inten-

sity of phagocytosis by peritoneal phagocytes were higher

in the sham, transgastric, and laparoscopy groups, there

was no significant difference between these groups and the

laparotomy group, except for a higher percentage of E. coli

macrophage phagocytosis in the sham group compared to

the laparotomy group (p \ 0.05, Table 3).

Macrophage production of H2O2 was shown to be sim-

ilar among the transgastric, laparoscopy, and sham groups,

and smaller than in the laparotomy group (p \ 0.05), either

under basal conditions (Figs. 1, 2), while performing

E. coli phagocytosis, or challenged by the presence of

E. coli membrane lipopolysaccharide. The neutrophil

function showed a different response to surgical trauma.

Overall, the three surgical groups had a higher percentage

of cells producing H2O2 than did the sham group. The

percentages of nonstimulated neutrophils producing H2O2

were similar for transgastric and laparotomy. A higher

percentage was observed in transgastric than in laparos-

copy (p \ 0.05) (Figs. 3, 4). This pattern also was

observed when neutrophils were challenged with E. coli

and LPS. However, during S. aureus phagocytosis, no

difference was observed among the surgical groups.

There was no statistical difference in the mean intensity

of neutrophil H2O2 production among all four groups in

nonstimulated cells and when cells were challenged

with LPS. During S. aureus phagocytosis, sham group

values were significantly lower than those of laparotomy

(p \ 0.05). In neutrophils performing E. coli phagocytosis,

Table 1 Excluded animals and complications

Group Excluded

(n)

Complications

Sham 2 Loss of jugular catheter and hemorrhage

Death during anesthetic induction

Transgastric 4 Iatrogenic enterotomy during gastrotomy

Inadequate gastric cleansing

Iatrogenic gastric bleeding during

gastrotomy

Minor hemoperitoneum

Laparoscopy 2 Loss of jugular catheter, hemorrhagic

shock

Iatrogenic colon perforation

Laparotomy 1 Inadequate peritoneal fluid sample

Table 2 Percentage of viable cells by trypan blue exclusion

Groups Shama Transgastricb Laparoscopyc Laparotomyd p*

Cell viability (%) 77.2 (10.9) 91.3 (3.9) 91.9 (4.6) 89.1 (5.6) 0.003ab, ac, ad

Data expressed as mean (standard deviation)

* One-way ANOVA and Tukey-Kramer post hoc test, p \ 0.05

pxy, Difference between corresponding groups

Table 3 Percentage and intensity of phagocytosis by peritoneal phagocytes

Groups Shama Transgastricb Laparoscopyc Laparotomyd p*

Macrophage S. aureus % 74.2 (23.7) 63.5 (19.6) 74.1 (19.1) 49.8 (28.3) NS

Intensity 42.9 (30.9) 17.2 (11.1) 21.6 (11.6) 17.9 (9.9) NS

E. coli % 50.2 (30.6) 24.1 (17.3) 15.4 (7.5) 14.9 (9.7) 0.028ad

Intensity 14.4 (14.8) 6.4 (3.64) 3.7 (1.6) 5.5 (2.0) NS

Neutrophil S. aureus % 54.0 (31.8) 70.1 (19.9) 73.2 (13.1) 73.6 (19.4) NS

Intensity 92.7 (143.1) 17.2 (11.3) 19.5 (12.0) 20.2 (20.8) NS

E. coli % 37.9 (18.3) 34.2 (22.2) 37.0 (19.0) 38.8 (21.3) NS

Intensity 24.9 (26.8) 5.8 (3.1) 6.3 (2.9) 6.0 (4.9) NS

Data expressed as mean (standard deviation). Intensity is in arbitrary values

NS Not significant

* One-way ANOVA and Tukey-Kramer post hoc test

pxy, Difference between corresponding groups

Surg Endosc (2013) 27:964–970 967

123

sham group values were significantly lower than those of

laparotomy and transgastric (p \ 0.05).

Discussion

In the last decades, minimally invasive techniques brought

major advances to surgical fields. Natural orifice endo-

scopic surgery may represent an extension of the benefits

of lessened surgical trauma. Although it may seem natural

to accredit to NOTES a lower impact on homeostasis,

many issues remain unclear about real benefits of this new

route of peritoneal access. Such data are essential for the

development of this new surgical modality since NOTES

procedures compete with well-established laparoscopic

procedures.

NOTES has been the object of an organized scientific

effort [14]. In this study we intended to evaluate the

peritoneal response induced by different surgical modali-

ties expressed by innate cellular immunity variables.

Serum cytokine levels have been utilized in female swine

models in order to indirectly estimate inflammatory

response, but estrogens may exert an anti-inflammatory

effect by suppressing the expression of nitric oxide syn-

thetase, and progesterone stimulates neutrophil chemo-

tactic activity [15–17]. Therefore, male animals were used

in our study to avoid the eventual influence of individual

hormonal cycles.

The sham procedure (negative control) and laparotomy

(positive control) were considered as providing minimal

and maximal impact on peritoneal inflammatory response,

respectively.

Fig. 1 Boxplot of mean percentage of nonstimulated macrophages

producing H2O2

Fig. 2 Boxplot of mean intensity of fluorescence of nonstimulated

macrophages producing H2O2

Fig. 3 Boxplot of mean percentage of nonstimulated neutrophils

producing H2O2

Fig. 4 Boxplot of mean intensity of fluorescence of nonstimulated

neutrophils producing H2O2

968 Surg Endosc (2013) 27:964–970

123

Peritoneal leukocytes were successfully retrieved by

peritoneal washing and showed a high percentage of cell

viability. The sham group showed a lower mean percentage

of cell viability compared to the other surgical groups. This

difference could be explained by the influx of younger

inflammatory cells caused by the surgical trauma in the

transgastric, laparoscopy, and laparotomy groups.

To avoid potential confounding factors, a homogeneous

study population was used and pre- and postoperative care

were identical. Time of anesthetic exposure was preset as

90 min irrespective of the study group. However, total

operative time varied among the groups: transgastric pro-

cedures lasted approximately 150 % and 300 % longer

than laparotomy and laparoscopy, respectively. McGee

et al. [5] reported similar results. Such differences are

inherent to the nature of each procedure. In addition, Hazey

et al. [18] reported that NOTES peritoneoscopy took

approximately twice as long as diagnostic laparoscopy in

humans.

Previous studies reported impairment in macrophage

and neutrophil phagocytic function after major surgical

trauma [19, 20]. In our study, a higher percentage of

macrophage phagocytosis was found in the sham, trans-

gastric, and laparoscopy groups compared to laparotomy,

although no statistical difference was observed except for

the percentage of E. coli phagocytosis, which was signifi-

cantly higher in the sham group than in the laparotomy

group. Neutrophil evaluation showed no significant dif-

ference among all groups, although the mean intensities of

E. coli and S. aureus phagocytosis were higher in the sham

group.

Regarding macrophage intracellular H2O2 production,

our findings have shown a similar response in animals that

underwent NOTES and those in the laparoscopy and sham

groups. The laparotomy group has shown the highest pro-

duction compared to others. The percentage of H2O2 neu-

trophil production seemed to be highly sensitive, but not

specific, since higher values were found in the surgical

groups. However, the intensity of H2O2 production while

performing phagocytosis was higher in the laparotomy

group.

Although reactive oxygen species (ROS) such as H2O2

have a major role in phagocytosis and immunomediated

defense, these molecules may lead to local lipid peroxi-

dation which would threaten homeostasis and local tissue

integrity if produced in excess or not adequately inacti-

vated [21, 22]. Increased activity of ROS after major sur-

gical trauma has been widely reported and is related to

ischemic conditions and inflammatory cell activation.

Much of the homeostatic imbalance after surgical trauma,

such as increased vascular permeability, adherence for-

mation, and sepsis, is related to increased postsurgical ROS

activity [23–25].

Overall, our data indicate more preserved phagocytic

ability and decreased production of ROS by peritoneal

leukocytes after minimally invasive procedures. However,

many of the expected differences in inflammatory response

among all groups were not observed. This may be due to an

eventual cancelling-out effect of peritoneal contamination,

present in NOTES, versus abdominal wall trauma alone,

observed in laparoscopy and laparotomy. Although this

was not our primary aim, a study comparing gastrostomy

performed by laparoscopy and laparotomy versus transga-

stric peritoneoscopy would better elucidate relative

inflammatory responses in each of the groups. Furthermore,

a larger sample size could magnify those differences,

indicating a more preserved phagocytic function after

minimally invasive procedures, including NOTES.

The use of a swine model does not allow us to transfer

the conclusions of the study to clinical practice in human

beings, as many variables would be added. Nevertheless,

these results emphasize the viability of NOTES, even

considering the potential contamination and the use of

nonsterile accessories.

Disclosures Drs. R. Rodrigues, M. Rezende, G. Gomes, F. Souza,

M. Blagitz, A. Della Libera, M. Taha, A. Ferrari, and E. Della Libera

have no conflicts of interest or financial ties to disclose.

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