CO2 pneumoperitoneum increases secretory IgA levels in the gutcompared with laparotomy in an experimental animal model

Toru Kusano • Tsuyoshi Etoh • Masafumi Inomata •

Norio Shiraishi • Seigo Kitano

Received: 23 August 2013 / Accepted: 22 December 2013

� Springer Science+Business Media New York 2014

Abstract

Background Secretory immunoglobulin A (s-IgA) plays

an important role in both gut and systemic immunity. This

study aimed to investigate the production of s-IgA resulting

from a CO2 pneumoperitoneum compared with a

laparotomy.

Methods Using enzyme-linked immunosorbent assays,

s-IgA in stool, malondialdehyde (MDA), and Toll-like

receptor 4 (TLR4) in the ileal tissue were evaluated as

markers for gut and systemic immune responses in an

animal model. The rats were randomly divided into

(i) anesthesia-only as the control group; (ii) laparotomy-

only as the open group; and (iii) CO2 pneumoperitoneum-

only as the pneumoperitoneum group. To evaluate the gut

immune system in a time-dependent manner, each group

was further divided into short- and long-time subgroups.

Results s-IgA levels did not increase in the open group

but significantly increased in the pneumoperitoneum group

compared with the control group (p \ 0.05). In addition,

s-IgA levels in the long-time subgroup significantly

increased compared with the short-time subgroup of the

pneumoperitoneum group (p \ 0.05). TLR4 levels steeply

and gradually increased in the open and pneumoperito-

neum groups, respectively. MDA levels in the pneumo-

peritoneum group increased during the early phase and

were significantly higher than those in the open group at

24 h (p \ 0.05).

Conclusions This study demonstrated that s-IgA levels in

stool increased in the pneumoperitoneum group compared

with the open group, suggesting that CO2 pneumoperito-

neum may cause transitory damage to the intestinal

mucosa.

Keywords Secretory immunoglobulin A � CO2

pneumoperitoneum � Laparotomy � Malondialdehyde �Toll-like receptor 4

In clinical practice, laparoscopic surgery is accepted

worldwide as a less invasive surgical option for various

diseases; however, there have been several reports of

adverse events, including gut ischemia, following pneu-

moperitoneum during gastroenterological surgery [1, 2].

We have previously reported transient liver dysfunction in

27 patients following laparoscopic gastrectomy using a

CO2 pneumoperitoneum, of which three patients with liver

disease suffered severe enteritis [3].

To date, there are some basic or clinical studies about

the effects of the splanchnic blood flow on CO2 pneumo-

peritoneum. The limited clinical reports have described the

adverse effects of pneumoperitoneum, such as the transi-

tory splanchnic mucosal ischemia [4–6]. Using animal

models, these reports have demonstrated that pneumoper-

itoneum causes a transitory reduction in the splanchnic

blood flow, resulting in a biochemical evidence of oxida-

tive stress in both pressure- and time-dependent manners

T. Kusano � T. Etoh � M. Inomata (&)

Department of Gastroenterological and Pediatric Surgery,

Faculty of Medicine, Oita University, 1-1 Hasama-machi, Yufu,

Oita 879-5593, Japan

e-mail: inomata@oita-u.ac.jp

T. Kusano

e-mail: kusanot@oita-u.ac.jp

N. Shiraishi

Center for Community Medicine, Faculty of Medicine, Oita

University, Oita, Japan

S. Kitano

Oita University, Oita, Japan

123

Surg Endosc

DOI 10.1007/s00464-013-3408-3

and Other Interventional Techniques

[7–9]. We have also reported that a pneumoperitoneum

markedly decreases total hepatic blood flow in cirrhotic

rats because of an impaired hepatic arterial buffer response

[10]. In addition, several reports state that organ reperfu-

sion injury affects the immune system [11, 12].

The gut immune system forms a significant component of

human immunity. Antigens mainly from intestinal bacterial

load are quickly incorporated into the M cells (microfold

cells) of Peyer’s patches in the ileum. In addition, secretory

immunoglobulin A (s-IgA), which plays a critical role in

mucosal immunity, is secreted into the intestinal lumen and

triggers the immune response [13]. Similarly, Toll-like

receptor 4 (TLR4) is important in the activation of the innate

immune system [14], with reports suggesting that TLR4

signaling in intestinal epithelial cells significantly elevates

the production of s-IgA [15]. Thus, s-IgA is produced via

several pathways; however, there are no reports about CO2

pneumoperitoneum influences on gut immunity.

Therefore, this study aimed to assess the influence of

CO2 pneumoperitoneum on the gut immune system in an

early phase after surgical procedure, particularly on s-IgA

production.

Material and methods

Experimental animal protocols

We used 7-week-old male Sprague–Dawley rats (Kyudo,

Fukuoka, Japan), weighing 220–270 g. The rats were

maintained at 25 �C with a 12-h light/dark cycle, and were

provided free access to water and standard laboratory feed.

The study protocol was approved by the Animal Ethics

Review Committee of Oita University, Faculty of

Medicine.

The rats were randomly divided into the following three

groups: (i) anesthesia-only as the control group; (ii) lapa-

rotomy-only as the open group; and (iii) CO2 pneumo-

peritoneum-only as the pneumoperitoneum group. Each

group was subsequently divided into the following two

subgroups: the short-time subgroup (30 min) and the long-

time subgroup (4 h).

All rats were placed on the operating table and main-

tained in the Trendelenburg position. Each rat was anes-

thetized using 3 % sevoflurane (Maruishi Pharmaceutical

Co., Ltd., Osaka, Japan), intubated with a 22-gauge extra

tube (Surflo� I.V. catheter, Terumo�, Japan), and venti-

lated using a mechanical ventilator (Harvard Apparatus

Inspira, El Cajon, CA, USA). Rectal temperature was

maintained between 36 and 38 �C (BSM-8301, Life Scope

9, Nihon Kohden Co., Ltd., Tokyo, Japan). The rats in the

control group were maintained on anesthesia alone,

whereas those in the open group received a 5-cm midline

incision. In the pneumoperitoneum group, CO2 was insuf-

flated using an electronic CO2 insufflator (Surgiflator 9100,

UHI-3, Olympus, Tokyo, Japan) through a 22-gauge extra

tube (Surflo� I.V. catheter, Terumo�, Japan) to reach a

target intraperitoneal pressure of 8 mmHg.

Sample collection

After ketamine/xylazine anesthesia, the abdominal cavity

was opened. Ileal tissue and stool in the ileum were obtained

at 0, 3, and 24 h after the procedures. Five rats per group

were used at each timepoint. To minimize the amount of

background ileal tissue from the remaining non-adherent

intravascular blood, rats were perfused with at least 50 mL

of normal saline (0.9 % NaCl) by inserting a needle into the

beating heart. Aliquots of the ileal tissue as well as stool

samples were stored at -80 �C until analysis. To determine

the protein content of both the ileal tissue and stool, samples

were weighted, thawed, and homogenized in phosphate-

buffered saline and centrifuged at 10,0009g for 10 min.

Supernatant protein concentrations were determined using

the DC Protein Assay Reagent (Bio-Rad Laboratories,

Hercules, CA, USA). Absorption was measured at 450 nm

using a microplate reader (Bio-Rad Laboratories).

After the samples were obtained, the rats were humanely

euthanized according to the institutional animal care

guidelines of Oita University.

Evaluation of markers associated with the immune

system

First, s-IgA levels in stool were measured using a com-

mercial kit containing a chromogenic reagent (Bethyl

Laboratories, Inc., Montgomery, TX, USA). Absorbance

was detected using a microplate reader (Bio-Rad Labora-

tories) at 450 nm. Second, TLR4 levels in the ileal tissue

were measured using a commercial kit containing a chro-

mogenic reagent (USCN Life Science Inc., USA). Absor-

bance was detected using a microplate reader (Bio-Rad

Laboratories) at 450 nm.

Because malondialdehyde (MDA) is used as a bio-

marker of oxidative stress in an organism [16], its levels

were also measured using a commercial kit containing a

chromogenic reagent (Northwest Life Science Specialties,

LLC, Vancouver, WA, USA). Absorbance was detected

using an ultraviolet/visible spectrum microplate and cuv-

ette spectrophotometer (Thermo Fisher Scientific Inc.,

USA) from 400 to 700 nm.

Statistical analysis

Data are expressed as mean ± standard deviation (SD).

Data were analyzed using the unpaired t-test or one-way

Surg Endosc

123

analysis of variance. For all analyses, a p-value of \0.05

was considered to be statistically significant. All statistical

analyses were performed using SPSS 11.0 statistics soft-

ware (SPSS, Inc., Chicago, IL, USA).

Results

s-IgA levels in stool

s-IgA levels were significantly higher in the short-time

subgroup of the pneumoperitoneum group than in the

control group at 3 and 24 h (p \ 0.05; Fig. 1A). At 24 h,

s-IgA levels were significantly higher in the in the long-

time subgroup of the pneumoperitoneum group than in the

control group (p \ 0.05; Fig. 1B). Levels of s-IgA gradu-

ally increased irrespective of the duration of the procedure

in the pneumoperitoneum group. In the pneumoperitoneum

group, s-IgA levels in stool were significantly higher in the

long-time subgroup than in the short-time subgroup

(p \ 0.05; Fig. 2). At 24 h, s-IgA levels were significantly

higher in both the short- and long-time subgroups of the

pneumoperitoneum group than in either the control or the

open groups (p \ 0.05).

Toll-like receptor 4 levels in the ileal tissue

TLR4 levels were significantly higher in both the open

and pneumoperitoneum groups than in the control group

at 3 and 24 h (p \ 0.05; Fig. 3A, B). TLR4 levels

increased between successive readings in the pneumo-

peritoneum group, whereas they decreased in both the

short- and long-time subgroups of the open group between

3 and 24 h.

Malondialdehyde levels in the ileal tissue

At 3 h, MDA levels were significantly higher in the short-

time subgroup of the pneumoperitoneum group than in the

open group (p \ 0.05; Fig. 4A). At 24 h, MDA levels were

significantly higher in the long-time subgroup of the

pneumoperitoneum group than in the control group

(p \ 0.05; Fig. 4B). In particular, MDA levels in the

pneumoperitoneum group rapidly increased in the short-

and long-time subgroups.

Fig. 1 Production of s-IgA following pneumoperitoneum. A In the

short-time subgroup of the pneumoperitoneum group, s-IgA levels

gradually increased. At 3 and 24 h, s-IgA levels were significantly

higher in the pneumoperitoneum group than in the control group

(p \ 0.05). B In the long-time subgroup of the pneumoperitoneum

group, s-IgA levels gradually increased. At 24 h, s-IgA levels were

significantly higher in the pneumoperitoneum group than in the

control group (p \ 0.05). s-IgA secretory immunoglobulin A.

*p \ 0.05

Fig. 2 Comparison of s-IgA levels between the open and pneumo-

peritoneum groups at 24 h. s-IgA levels in stool gradually increased

and were significantly higher in the long-time subgroup than in the

short-time subgroup of the pneumoperitoneum group at 24 h

(p \ 0.05). At 24 h, s-IgA levels were significantly higher in both

the short- and long-time subgroups of the pneumoperitoneum group

compared with those in the open group. At 24 h, s-IgA levels in the

open group remained similar to those in the short- and long-time

subgroups of the control group. s-IgA secretory immunoglobulin A

*p \ 0.05, �p \ 0.05, §p \ 0.05

Surg Endosc

123

Discussion

To the best of our knowledge, this study is the first

experimental report describing the impact of a CO2 pneu-

moperitoneum on s-IgA production. We demonstrated that

s-IgA levels in the pneumoperitoneum group significantly

increased compared with those in the control group,

whereas these levels did not increase in the open group. In

addition, prolonged pneumoperitoneum increased the pro-

duction of s-IgA.

s-IgA is a non-inflammatory immune globulin mainly

reacting to intestinal bacterial load [17]. There are several

recognized pathways through which s-IgA is produced.

TLR4 signaling in intestinal epithelial cells is one such

pathway [15]. Another recognized pathway is via the M

cells of Peyer’s patches. TLR4 detects lipopolysaccharide

(LPS) from gram-negative bacteria and activates the innate

immune system [15]. In addition, it has been reported that

TLR4 is associated with reactive oxygen species and

nuclear factor kappa-light-chain-enhancer of activated B

cells (NF-jB) [18, 19]. After TLR4 is bound to LPS as a

ligand, TLR4 directly activates B cells without the induc-

tion of T-cell proliferation [15]. In our study, TLR4

expression gradually increased in the pneumoperitoneum

group, whereas it steeply increased in an early phase after a

laparotomy procedure. It is interesting that the peak TLR4

expression was higher in the open group than in the group;

however, s-IgA levels did not increase in the open group.

These results suggest that TLR4 is not associated with

s-IgA production in the open group.

On the other hand, after bacteria is bound to an M cell,

s-IgA is produced by the induced B cell, subsequent to

T-cell activation [13]. The intestinal mucosal surface is

covered by a mucus layer primarily composed of water and

mucins, which can reach 100–300 lm in thickness, and is

secreted by goblet cells [20–22]. It is known that this

mucus layer protects the intestinal mucosa from bacteria

[20–22]. Recently, Unsal et al. [23] reported that CO2

pneumoperitoneum caused intestinal ischemia that resulted

in histopathological damage to the ileal tissue. We

hypothesize that CO2 pneumoperitoneum caused intestinal

ischemia, resulting in s-IgA production via bacteria binding

easily to M cells, which in turn decreased the secretion of

mucin and damaged the mucus layer.

Interestingly, TLR4 expression was steeply elevated

only in an early phase after a laparotomy procedure.

However, the mechanisms or clinical implications of ele-

vated TLR4 expression underwent laparotomy have not yet

been clearly elucidated. It has also been reported that TLR4

expression is associated with LPS, inflammation, and

ischemia [15, 24–26]. Since TLR4 exists not only in

intestinal epithelial cells but also in mesothelium cells [27–

29], in this study steeply elevated TLR4 expression in the

open group might be introduced by exposure to bacteria in

room air. Therefore, further studies about the different

conditions of pressure, distention, splanchnic blood flow,

and hypercarbia using by CO2 endoscopy are needed.

In this study, we evaluated MDA levels in the ileal tissue

to confirm the histopathological damage because MDA is a

useful biomarker of oxidative stress in an organism [16].

Fig. 3 Effect of surgical stress on TLR4 expression. A In the short-

time subgroup of the pneumoperitoneum group, TLR4 levels

gradually increased. At 3 and 24 h, TLR4 levels in both the open

and pneumoperitoneum groups were significantly higher than those in

the control group (p \ 0.05). B In the long-time subgroup of the

pneumoperitoneum group, TLR4 levels gradually increased. At 3 and

24 h, TLR4 levels in both the open and pneumoperitoneum groups

were significantly higher than those in the control group (p \ 0.05).

TLR4 Toll-like receptor 4. *p \ 0.05, �p \ 0.05

Surg Endosc

123

Several studies have reported that MDA levels in the ileal

tissue increase by intra-abdominal pressure in a time-

dependent manner because of intestinal ischemia during

pneumoperitoneum [30, 31]. Reports have also stated that

pneumoperitoneum reduces splanchnic and portal venous

blood flow [32, 33]. Moreover, it was reported that higher

ileal tissue MDA levels were associated with higher scores

of histopathological damage to the ileal tissue [23]. In

addition, Evasovich et al. reported that CO2 pneumoperito-

neum increased the incidence of Escherichia coli bacterial

translocation in a rat model [34]. Although mucosal erosion

within the ileal tissue could not be observed in our study,

MDA levels were compared between the pneumoperito-

neum and open groups. It is interesting that MDA levels

increased early in the pneumoperitoneum group. MDA

levels increased in the open group, but there was no sig-

nificant difference between the open and control groups.

Thus, increased MDA levels in the open group were prob-

ably caused by both the surgical stress of a laparotomy and

exposure of the intestine to air. These results are consistent

with those of earlier reports [30, 31].

Although this study demonstrated that CO2 pneumo-

peritoneum affects the gut immune system, it has several

limitations. First, intestinal manipulation was not per-

formed. Although previous reports have demonstrated that

small intestine manipulation can increase cytokine

inflammatory response [35], we did not incorporate an

assessment of intestinal injury through manipulation on the

gut immune system. Second, we conservatively insufflated

the abdomen (8 mmHg) in the pneumoperitoneum group.

According to the report by Unsal et al. [23], the abdomen

was insufflated to 10 mmHg in a rat model and intestinal

erosion was observed. Because our intra-abdominal pres-

sure is lower, mucosal erosion may not have been observed

in our study. However, we believed that the mucosal injury

of the ileum occurred in the pneumoperitoneum group

because of an increase in MDA levels. Third, we examined

s-IgA levels in an early phase after surgical procedure in

this study. It was reported that s-IgA was produced con-

stantly and its production required several days to be

changed, influencing various factors such as gut bacterial

communities, meal, and environment [36, 37]. Therefore,

to elucidate the influence only by surgical stress on gut

immunity, we first examined it in an early phase after

surgical procedure. The influences on gut immunity caused

by surgical stress in the long-term are also important in the

clinical setting. Our data recommend that lower intra-

abdominal pressure and shorter operative time underwent

CO2 pneumoperitoneum from the viewpoints of gut

immunological response by surgical stress. However, this

study is a limited examination in an animal model. It is

necessary to conduct further studies regarding the effect of

surgical stress with different conditions of intra-abdominal

pressure and operative time on the gut immune system.

Conclusions

This is the first report to demonstrate that gut s-IgA levels

increase as a result of CO2 pneumoperitoneum, and

Fig. 4 Effect of surgical stress on MDA expression. A MDA levels

in the ileum gradually increased in the short-time subgroups of all

groups (control, open, and pneumoperitoneum). At 3 h, MDA levels

were significantly higher in the pneumoperitoneum group than in the

control and open groups (p \ 0.05). B In the long-time subgroup,

MDA levels in the ileum gradually increased in all groups. At 24 h,

MDA levels in the pneumoperitoneum group were significantly

higher than those in the control group (p \ 0.05). MDA malondial-

dehyde. *p \ 0.05

Surg Endosc

123

prolonged pneumoperitoneum results in higher s-IgA lev-

els. For less invasive surgery in the clinical setting, further

evaluations of gut immunity after CO2 pneumoperitoneum

are required.

Disclosures Drs. Toru Kusano, Tsuyoshi Etoh, Masafumi Inomata,

Norio Shiraishi, and Seigo Kitano have no conflicts of interest or

financial ties to disclose.

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