Prospective study on the effect of abdominal hypertension on intestinal mucosal barrier injury during laparoscopic surgery

Objective Prolonged and high intraperitoneal pressure may lead to impaired intestinal mucosal blood perfusion, increase the risk of surgery and complications, and affect the postoperative recovery of patients. However, the literature reports on the effect of abdominal hypertension on gastrointestinal function mainly focus on animal experiments, and there are few clinical reports. Our study intends to explore the effect of increased CO2 pneumoperitoneum pressure during laparoscopy on intestinal mucosal barrier injury. Methods A prospective study was conducted on 180 patients who underwent laparoscopic cholecystectomy in the First People's Hospital of Shuangliu District, Chengdu from October 2017 to March 2018. A randomizedsingle-blindcontrolled study was performed in the 180 patients who were allocated into the 10 mmHg group(1 mmHg=0.133 kPa)12 mmHg group and 15 mmHg group based on a random number table and setting value of intraoperative CO2 pneumoperitoneum pressure (10 mmHg12 mmHg and 15 mmHg)Main observation indexes such as intraoperative conditions and postoperative recovery were recorded, and the results of serum tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), D-lactic acid, blood endotoxin levelsplasma diamine oxidase (DAO) activity were detected. The measurement data of normal distribution were expressed as mean ± standard deviation (x+S), and one-way analysis of variance was used for comparison between groups. The measurement data with non-normal distribution are represented by M (QR) and non-parametric test is adopted, count data were presented as the n(%), and comparison among groups was analyzed using the chi-square test. Results 180 patients were screened out, 60 patients in each group. Eight patients dropped out during the study (2 in 10 mmHg group, 1 in 12 mmHg group and 5 in 15 mmHg group). All patients in the three groups were cured and discharged without bleeding, secondary bile duct stones, bile leakage and reoperation. There was no significant difference in serum TNF-a, IL-1, D-lactic acid, endotoxin level and plasma diamine oxidase

Intestinal mucosa is not only an important place for human body to digest and absorb nutrients, but also the largest storage organ of bacteria and the main portal for pathogenic microorganisms and toxins to enter the body. Intestinal mucosal barrier is composed of intestinal mucosal mechanical barrier, chemical barrier, biological barrier and immune barrier [1,2] , which can effectively prevent the excessive growth of bacteria in the small intestine and the occurrence of endotoxemia. However, under the influence of ischemia and hypoxia, hypoperfusion, severe trauma, surgery, stress, abdominal hypertension, nutritional disorders, bacterial endotoxin and long-term application of broadspectrum antibiotics, all of these factors can lead to intestinal barrier function damage, bacterial imbalance, excessive growth of intestinal bacteria and endotoxin, induce and aggravate systemic inflammatory response and multiple organ dysfunction MODS , and eventually form a vicious cycle [3,4] . With the advent of the era of minimally invasive surgery, laparoscopic technology has been widely used in various clinical fields. Laparoscopic surgery requires CO 2 gas injection in the abdominal cavity to establish pneumoperitoneum to expand the surgical field, which directly leads to the increase of intra-abdominal pressure. Prolonged and high intraperitoneal pressure may lead to impaired intestinal mucosal blood perfusion, increase the risk of surgery and complications, and affect patients' postoperative recovery. However, the literatures about the effect of abdominal hypertension on gastrointestinal function mainly focus on animal experimental study [5][6][7] , but rarely on clinical report [8] , our study prospectively analyzed the patients who underwent laparoscopic cholecystectomy in our hospital from October 2017 to March 2018, during which different CO 2 pneumoperitoneum pressure was used for surgery, to explore the influence of different abdominal pressure on intestinal mucosal barrier, and the report is as follows:

General information
A prospective study was performed in 180 patients with gallstones admitted to our hospital from October 2017 to March 2018. The patients were 20-81 years old, with an average of (62.5+7.7) years, including 64 males and 116 females. Inclusion criteria :(1) age range from 20 to 81 years; (2) the American society of anesthesiologists (ASA) grade I ~ Ⅱ; (3) the imaging examination (color ultrasound, CT or MRI) was clearly cholecystolithiasis; (4) the patient agreed to undergo laparoscopic cholecystectomy. Exclusion criteria :(1) contraindication of laparoscopic surgery (such as extensive intraperitoneal adhesion, etc.); (2) emergency surgery; (3) preoperatively complicated with obvious intestinal obstruction or intestinal paralysis; (4) preoperative systemic inflammatory response syndrome and other diseases; (5) conversion to open surgery; (6) persistent uncorrected water electrolyte disturbance (e.g., hypokalemia, hypomagnesemia, etc ). Our study was a clinical randomized controlled trial, which was approved by the medical ethics committee of our hospital.
Patients and their families were informed of the test method and operation method before operation, and informed consent was obtained and signed. The research project was registered in the Chinese clinical research registry with the registration number ChiCTR1900023936, which met the requirements of the Helsinki declaration in 1964.

grouping
A randomized single-blind controlled study was performed in the 180 patients who were allocated into the 10 mmHg group(1 mmHg=0.133 kPa) 12 mmHg group and 15 mmHg group based on a random number table and setting value of intraoperative CO 2 pneumoperitoneum pressure (10 mmHg 12 mmHg and 15 mmHg) Clinical data including age, sex, combined disease, ASA anesthesia stage and body mass index were collected. Laparoscopic cholecystectomy was performed by the same group of experienced surgeons, and the anesthesia was endotracheal intubation under general anesthesia. 2) Operation methods: preoperative preparation as routine fasting for 6 hours before surgery, water deprivation for 4 hours, routine skin preparation at the operation area, general anesthesia with endotracheal intubation. Procedure as patients were placed in supine position with head high and foot low and left semi-horizontal position (inclined 30°). The abdominal skin was routinely disinfected with iodine volts, and sterile towels were laid. After skin incision at the lower edge of umbilicus, a disposable puncture instrument was used to puncture to establish CO 2 pneumoperitoneum. The abdominal cavity was routinely probed during the operation, and the relationship between gallbladder morphology and adjacent tissues was observed, the gallbladder triangle structure was dissected clearly, and the gallbladder duct and gallbladder artery were separated. After clipping the gallbladder duct and gallbladder artery with absorbable clip, and which were cut off, the gallbladder was removed, the gallbladder bed electric coagulation hemostasis. According to the situation of hemorrhage and gallbladder inflammation to decide whether to place drainage tube, finally close the trocar hole.

Operation method:
3) Postoperative treatment: on the day after the operation, patients were given fasting water, oxygen, electrocardiogram monitoring, observation of vital signs, abdominal abnormal signs and drainage, and appropriate fluid supplementation to maintain the balance of water and electrolytes.
On the first day after the operation, a liquid or semi-liquid diet was taken, review of color doppler ultrasound found no abnormality in the abdominal cavity, after second or third days postoperation, the drainage was pulled out. as mean ± standard deviation `x+s , and one-way analysis of variance was used for comparison between groups. The measurement data with non-normal distribution are represented by M (QR) and non-parametric test is adopted. The counting data were represented by n (%), and comparison among groups was analyzed using the chi-square test, P<0.05 was considered statistically significant.

Analysis of general data of three groups of patients
Gender, age, combined diseases (hypertension, diabetes, etc.), ASA classification of anesthesia, and body mass index of the three groups were not statistically significant (P > 0.05), and the data were comparable (table 1). In our study, 8 patients withdrew from the study (2 patients in the 10mmHg group had severe inflammation, pneumoperitoneum depression, which could not expose the visual field, and the operation after raising the pneumoperitoneum pressure was excluded from the study. In the 12mmHg group, 1 patient transferred to laparotomy and was excluded from this study. In the 15mmHg group, 3 patients were removed due to high pneumoperitoneum pressure and anesthesia failure to tolerate the reduced pneumoperitoneum pressure to 12mmHg; 1 patient was transferred to laparotomy due to intraoperative peritoneal adhesion and difficulty in visual field exposure; another patient was found to have intraoperative complicated bile duct abnormality, and was removed into the group after changing the operation mode); the remaining 172 patients completed the operation according to the plan. There was no statistically significant difference among the three groups (P>0.05).

Postoperative indicators of the three groups
All patients in the three groups had no postoperative wound bleeding, no bile duct secondary calculi, bile leakage or reoperation, and no significant fever. All patients were cured and discharged, and the difference was not statistically significant. There were no significant differences in serum TNF-a, IL-1, D-lactic acid, endotoxin level and plasma diamine oxidase (DAO) activity among the three groups (P > 0.05) ( Table 2). Due to the fact that intestinal mucosal barrier function injury is a common pathophysiological process of various severe stress reactions, the current understanding is not comprehensive, the diagnostic means are lacking, the diagnostic criteria are uncertain [12] , and the detection method directly reflecting the intestinal mucosal barrier function is still lacking. In clinical practice, most indirect methods are adopted for detection. The main detection methods [13][14]  Its activity is closely related to nucleic acid and protein synthesis of intestinal mucosa cells. It is mainly distributed in the upper mucosa or villi of the intestinal mucosa, and other organs are much lower than small intestine. When intestinal mucosal cells die, the enzyme releases human blood, or enters the intestinal tract with intestinal mucosal cells, leading to increased DAO activity in the blood and intestinal tract. Studies have found that [15] , DAO is a landmark enzyme of intestinal injury, which can indirectly reflect the integrity and damage degree of intestinal mucosa.
The results of our study showed that different intraperitoneal pressures did not significantly change the activity of endotoxin, blood D-lactic acid and plasma diamine oxidase, with no statistically significant. The possible reason was that the establishment time of laparoscopic pneumoperitoneum in this study lasted only 0.5-2 hours, and the intestinal mucosal barrier injury could not be caused.
Although Kozlik [16] found that the increase of pneumoperitoneal pressure during laparoscopic surgery for more than 30 minutes would lead to the occurrence of gastrointestinal peroxidation stress, the possible gastrointestinal injury was confirmed from the perspective of cytology. However, in our study showed that the transient intraperitoneal hypertension was not enough to change the intestinal mucosal barrier based on the analysis of laboratory indicators.

The relationship between intra-abdominal pressure and inflammatory mediators
Under the condition of abdominal hypertension, the local immune environment of abdominal cavity will be changed, inflammatory factors play an important role in the acute phase reaction and their receptors regulate cytokine activity, which reflect the severity of inflammatory. The release of a large number of inflammatory factors in patients will cause damage to intestinal mucosal epithelial cells and damage to the tight junctions between cells, and also cause bacterial migration, release of endotoxin and aggravate inflammatory reaction. Inflammatory factors closely related to the intestinal mucosal barrier include TNF-a, IL-1 [17][18][19] , etc. The results of our study showed that different intraperitoneal pressure did not significantly change the level of inflammatory mediators, but this result was limited by the detection time, because existing research results showed that there was a time-effect relationship between elevated intraperitoneal pressure and serum levels of inflammatory mediators [8] . Therefore, monitoring the level of inflammatory mediators at different time points will help better reflect the true immune status of patients after surgery.

Credibility and limitations of the study
The negative results obtained in our study from the perspective of intestinal mucosal barrier injury confirmed that it was safe and reasonable to set CO 2 pneumoperitoneum pressure at or below 15mmHg in laparoscopic cholecystectomy, and provided a theoretical basis for setting pneumoperitoneum pressure in other laparoscopic surgeries. Its credibility was mainly reflected in the following aspects: 1) of the 180 patients enrolled patients, 8 patients (4.4%) withdrew from the study due to non-compliance with the inclusion criteria, which was consistent with the expected 10% followup loss rate. 2) the blind method was maintained throughout the whole process to eliminate possible bias as far as possible in our study. 3) common confounders such as pneumoperitoneum duration and operation time were eliminated by random grouping.
However, there are still some limitations in our study: 1) the operation time and CO 2 pneumoperitoneum establishment time are short, and the intra-abdominal pressure cannot be continuously monitored after surgery, which limits the reliability of the results. 2)This study only analyzes the indirect indicators related to intestinal mucosal barrier injury, lack of pathological examination. Since the detection time was 24 hours after surgery, slight intestinal mucosal barrier injury could be repaired by itself, so increasing the frequency of laboratory detection indicators would make the results more reliable.
In conclusion, the increased intra-abdominal pressure caused by the transient CO 2 pneumoperitoneum during the operation will not cause the intestinal mucosal barrier injury of patients after laparoscopic surgery, and has no impact on the systemic immune function and stress response of the body. It is safe and reliable to perform laparoscopic surgery when the CO 2 pneumoperitoneum pressure is set at or below 15mmHg. with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Annotation: a is for non-parametric test, BMI is for body mass index.