Histology and transcriptome insights into the early processes of intestinal anastomotic healing: a rat model.

BACKGROUND: Understanding the early processes underlying intestinal anastomotic healing is crucial to comprehend the pathophysiology of anastomotic leakage. The aim of this study was to assess normal intestinal anastomotic healing and disturbed healing in rats to investigate morphological, cellular and intrinsic molecular changes in the anastomotic tissue. METHOD: Anastomoses were created in two groups of Wistar rats, using four sutures or 12 sutures to mimic anastomotic leakage and anastomotic healing respectively. At 6, 12, 24 hours and 2, 3, 5 and 7 days, anastomotic tissue was assessed macroscopically using the anastomotic complication score and histologically using the modified Ehrlich-Hunt score. Transcriptome analysis was performed to assess differences between anastomotic leakage and anastomotic healing at the first three time points to find affected genes and biological processes. RESULTS: Ninety-eight rats were operated on (49 animals in the anastomotic leakage and 49 in the anastomotic healing group) and seven rats analysed at each time point. None of the animals with 12 sutures developed anastomotic leakage macroscopically, whereas 35 of the 49 animals with four sutures developed anastomotic leakage. Histological analysis showed increasing influx of inflammatory cells up to 3 days in anastomotic healing and up to 7 days in anastomotic leakage, and this increase was significantly higher in anastomotic leakage at 5 (P = 0.041) and 7 days (P = 0.003). Transcriptome analyses revealed large differences between anastomotic leakage and anastomotic healing at 6 and 24 hours, mainly driven by an overall downregulation of genes in anastomotic leakage. CONCLUSION: Transcriptomic analyses revealed large differences between normal and disturbed healing at 6 hours after surgery, which might eventually serve as early-onset biomarkers for anastomotic leakage.

Evaluation of the Effect of an Intraperitoneal Cytostatic-Loaded Supramolecular Hydrogel on Intestinal Anastomotic Healing in an Animal Model.

The prognosis of colorectal cancer patients with peritoneal metastases is very poor. Intraperitoneal drug delivery systems, like supramolecular hydrogels, are being developed to improve local delivery and intraperitoneal residence time of a cytostatic such as mitomycin C (MMC). In this study, we evaluate the effect of intraperitoneal hydrogel administration on anastomotic healing. Forty-two healthy Wistar rats received a colonic end-to-end anastomosis, after which 6 animals received an intraperitoneal injection with saline, 18 with unloaded hydrogel and 18 with MMC-loaded hydrogel. After 7 days, animals were euthanized, and the anastomotic adhesion and leakage score were measured as primary outcome. Secondary outcomes were bursting pressure, histological anastomosis evaluation and body weight changes. Twenty-two rats completed the follow-up period (saline: n = 6, unloaded hydrogel: n = 10, MMC-loaded hydrogel: n = 6) and were included in the analysis. A trend towards significance was found for anastomotic leakage score between the rats receiving saline and unloaded hydrogel after multiple-comparison correction (p = 0.020, α = 0.0167). No significant differences were found for all other outcomes. The main reason for drop-out in this study was intestinal blood loss. Although the preliminary results suggest that MMC-loaded or unloaded hydrogel does not influence anastomotic healing, the intestinal blood loss observed in a considerable number of animals receiving unloaded and MMC-loaded hydrogel implies that the injection of the hydrogel under the studied conditions is not safe in the current rodent model and warrants further optimalisation of the hydrogel.

LekCheck: A Prospective Study to Identify Perioperative Modifiable Risk Factors for Anastomotic Leakage in Colorectal Surgery.

OBJECTIVE: To assess potentially modifiable perioperative risk factors for anastomotic leakage in adult patients undergoing colorectal surgery. SUMMARY BACKGROUND DATA: Colorectal anastomotic leakage (CAL) is the single most important denominator of postoperative outcome after colorectal surgery. To lower the risk of CAL, the current research focused on the association of potentially modifiable risk factors, both surgical and anesthesiological. METHODS: A consecutive series of adult patients undergoing colorectal surgery with primary anastomosis was enrolled from January 2016 to December 2018. Fourteen hospitals in Europe and Australia prospectively collected perioperative data by carrying out the LekCheck, a short checklist carried out in the operating theater as a time-out procedure just prior to the creation of the anastomosis to check perioperative values on 1) general condition 2) local perfusion and oxygenation, 3) contamination, and 4) surgery related factors. Univariate and multivariate logistic regression analysis were performed to identify perioperative potentially modifiable risk factors for CAL. RESULTS: There were 1562 patients included in this study. CAL was reported in 132 (8.5%) patients. Low preoperative hemoglobin (OR 5.40, P < 0.001), contamination of the operative field (OR 2.98, P < 0.001), hyperglycemia (OR 2.80, P = 0.003), duration of surgery of more than 3 hours (OR 1.86, P = 0.010), administration of vasopressors (OR 1.80, P = 0.010), inadequate timing of preoperative antibiotic prophylaxis (OR 1.62, P = 0.047), and application of epidural analgesia (OR, 1.81, P = 0. 014) were all associated with CAL. CONCLUSIONS: This study identified 7 perioperative potentially modifiable risk factors for CAL. The results enable the development of a multimodal and multidisciplinary strategy to create an optimal perioperative condition to finally lower CAL rates.

Examination of lipid profiles in abdominal fascial healing using MALDI-TOF to identify potential therapeutic targets.

BACKGROUND: Failure of fascial healing in the abdominal wall can result in incisional hernia, which is one of the most common complications after laparotomy. Understanding the molecular healing process of abdominal fascia may provide lipid markers of incisional hernia or therapeutic targets that allow prevention or treatment of incisional hernias. PURPOSE: This study aims to investigate temporal and in situ changes of lipids during the normal healing process of abdominal fascia in the first postoperative week. METHODS: Open hemicolectomy was performed in a total of 35 Wistar rats. The midline fascia was closed identically for all rats using a single continuous suturing technique. These animals were sacrificed with equal numbers (n = 5) at each of 7-time points (6, 12, 24, 48, 72, 120, and 168 h. The local and temporal changes of lipids were examined with mass spectrometry imaging and correlated to histologically scored changes during healing using hematoxylin and eosin staining. RESULTS: Two phosphatidylcholine lipid species (PC O-38:5 and PC 38:4) and one phosphatidylethanolamine lipid (PE O-16:1_20:4) were found to significantly correlate with temporal changes of inflammation. A phosphatidylcholine (PC 32:0) and a monosialodihexosylganglioside (GM3 34:1;2) were found to correlate with fibroblast cell growth. CONCLUSION: Glycerophospholipids and gangliosides are strongly involved in the normal healing process of abdominal fascia and their locally fluctuating concentrations are considered as potential lipid markers and therapeutic targets of fascial healing.

Consensus on the definition of colorectal anastomotic leakage: A modified Delphi study.

BACKGROUND: Despite the emerging knowledge about colorectal anastomotic leakage (CAL) through the increasing number of clinical and experimental studies, there is no generally accepted definition of CAL. Because of the wide variety of definitions used in literature, comparison of study outcomes and quality of care is complicated. AIM: To reach consensus on the definition of CAL using a modified Delphi method. METHODS: The RAND/UCLA appropriateness method was used. The expert panel consisted of international colorectal surgeons and researchers who had published three or more articles about CAL. The consensus process consisted of two online distributed questionnaires and a third round with a recommendation. In the questionnaires participants were asked to rate the appropriateness of statements using a 1-9 Likert scale. Consensus was defined as a panel median between 1-3 or 7-9 without disagreement. In the final round a recommendation was formed regarding the definition of CAL and the expert panel was asked if they agreed or disagreed. RESULTS: Twenty-three authors participated in the first round and twenty-one finished the second round. After two rounds consensus was reached on 37 items (80%) in nine different categories. The International Study Group of Rectal Cancer definition is the most frequently advised general definition by our panel. Consensus was reached regarding the clinical symptoms of CAL, which serum markers contributes to the suspicion of CAL, which radiological and perioperative findings should be considered as CAL, which grading system is appropriate and if there should be a range of postoperative days in the definition. Eventually, 19 experts completed all three rounds of which 16 (84%) agreed with our final recommendations for the definition of CAL. CONCLUSION: A consensus-based recommendation for the definition of CAL was formed using our modified Delphi method that can be widely incorporated in the field.

Near-infrared fluorescence image-guidance in anastomotic colorectal cancer surgery and its relation to serum markers of anastomotic leakage: a clinical pilot study.

OBJECTIVE: Near-infrared fluorescence (NIRF) imaging using indocyanine green (ICG) might help reduce anastomotic leakage (AL) after colorectal surgery. This pilot study aims to analyze whether a relation exists between measured fluorescence intensity (FI) and postoperative inflammatory markers of AL, C-reactive protein (CRP), Intestinal fatty-acid binding protein (I-FABP), and calprotectin, to AL, in order to evaluate the potential of FI to objectively predict AL. METHODS: Patients scheduled for anastomotic colorectal cancer surgery were eligible for inclusion in this prospective pilot study. During surgery, at three time points (after bowel devascularization; before actual transection; after completion of anastomosis) a bolus of 0.2 mg/kg ICG was administered intravenously for assessment of bowel perfusion. FI was scored in scale from 1 to 5 based on the operating surgeon's judgment (1 = no fluorescence visible, 5 = maximum fluorescent signal). The complete surgical procedure was digitally recorded. These recordings were used to measure FI postoperatively using OsiriX imaging software. Serum CRP, I-FABP, and calprotectin values were determined before surgery and on day 1, 3, and 5 postoperative; furthermore, the occurrence of AL was recorded. RESULTS: Thirty patients (n = 19 males; mean age 67 years; mean BMI 27.2) undergoing either laparoscopic or robotic anastomotic colorectal surgery were included. Indication for surgery was rectal-(n = 10), rectosigmoid-(n = 2), sigmoid-(n = 10), or more proximal colon carcinomas (n = 8). Five patients (16.7%) developed AL (n = 2 (6.6%) grade C according to the definition of the International Study group of Rectal Cancer). In patients with AL, the maximum fluorescence score was given less often (P = 0.02) and a lower FI compared to background FI was measured at 1st assessment (P = 0.039). However, no relation between FI and postoperative inflammatory parameters could be found. CONCLUSION: Both subjective and measured FI seem to be related to AL. In this study, no relation between FI and inflammatory serum markers could yet be found.

Baseline performance of the ischaemic button model for induction of adhesions in laboratory rats.

The ischaemic button model is frequently used for the induction of adhesions in laboratory rats. Male rats are often used because of the common belief that the peritesticular (intra-abdominal) fat in males facilitates adhesion formation, although this theory is not evidence based. Comparing the model's performance in both sexes is an important aspect of refining animal experiments. The aim of this study is to compare baseline performance of the modified ischaemic button model in both male and female rats. Follow-up was 1 week and noted endpoints were intra-abdominal adhesion formation and differences in welfare assessment. A total of 192 ischaemic buttons (96 male/96 female) were created in 24 Wistar Han rats (12 male/12 female). After 1 week of follow-up, 93 buttons survived in the male group (96.9%) compared with 91 in the female group (94.8%) (p = 0.409). In the male group, 85 out of 93 (91.4%) buttons induced adhesions compared with 84 out of 91 (92.3%) in the female group (p = 0.881). All but one animal had a Zühlke score of 3. There were no clinically relevant differences in welfare scores. Male animals increased in weight significantly faster compared to females (p < 0.001), after correcting for physiological growth. The ischaemic button model resulted in equal quality and quantity of intra-abdominal adhesions in both male and female Wistar Han rats. Both male and female Wistar Han rats are suitable for the induction of experimental adhesions in the ischaemic button model.

Pilot Study: Detection of Gastric Cancer From Exhaled Air Analyzed With an Electronic Nose in Chinese Patients.

The aim of this pilot study is to investigate the ability of an electronic nose (e-nose) to distinguish malignant gastric histology from healthy controls in exhaled breath. In a period of 3 weeks, all preoperative gastric carcinoma (GC) patients (n = 16) in the Beijing Oncology Hospital were asked to participate in the study. The control group (n = 28) consisted of family members screened by endoscopy and healthy volunteers. The e-nose consists of 3 sensors with which volatile organic compounds in the exhaled air react. Real-time analysis takes place within the e-nose, and binary data are exported and interpreted by an artificial neuronal network. This is a self-learning computational system. The inclusion rate of the study was 100%. Baseline characteristics differed significantly only for age: the average age of the patient group was 57 years and that of the healthy control group 37 years ( P value = .000). Weight loss was the only significant different symptom ( P value = .040). A total of 16 patients and 28 controls were included; 13 proved to be true positive and 20 proved to be true negative. The receiver operating characteristic curve showed a sensitivity of 81% and a specificity of 71%, with an accuracy of 75%. These results give a positive predictive value of 62% and a negative predictive value of 87%. This pilot study shows that the e-nose has the capability of diagnosing GC based on exhaled air, with promising predictive values for a screening purpose.

Definition of colorectal anastomotic leakage: A consensus survey among Dutch and Chinese colorectal surgeons.

AIM: To determine the level of consensus on the definition of colorectal anastomotic leakage (CAL) among Dutch and Chinese colorectal surgeons. METHODS: Dutch and Chinese colorectal surgeons were asked to partake in an online questionnaire. Consensus in the online questionnaire was defined as > 80% agreement between respondents on various statements regarding a general definition of CAL, and regarding clinical and radiological diagnosis of the complication. RESULTS: Fifty-nine Dutch and 202 Chinese dedicated colorectal surgeons participated in the online survey. Consensus was found on only one of the proposed elements of a general definition of CAL in both countries: 'extravasation of contrast medium after rectal enema on a CT scan'. Another two were found relevant according to Dutch surgeons: 'necrosis of the anastomosis found during reoperation', and 'a radiological collection treated with percutaneous drainage'. No consensus was found for all other proposed elements that may be included in a general definition. CONCLUSION: There is no universally accepted definition of CAL in the Netherlands and China. Diagnosis of CAL based on clinical manifestations remains a point of discussion in both countries. Dutch surgeons are more likely to report 'subclinical' leaks as CAL, which partly explains the higher reported Dutch CAL rates.

Predictive Factors for Anastomotic Leakage After Colorectal Surgery: Study Protocol for a Prospective Observational Study (REVEAL Study).

BACKGROUND: Anastomotic leakage (AL) remains the most important complication following colorectal surgery, and is associated with high morbidity and mortality rates. Previous research has focused on identifying risk factors and potential biomarkers for AL, but the sensitivity of these tests remains poor. OBJECTIVE: This prospective multicenter observational study aims at combining multiple parameters to establish a diagnostic algorithm for colorectal AL. METHODS: This study aims to include 588 patients undergoing surgery for colorectal carcinoma. Patients will be eligible for inclusion when surgery includes the construction of a colorectal anastomosis. Patient characteristics will be collected upon consented inclusion, and buccal swabs, breath, stool, and blood samples will be obtained prior to surgery. These samples will allow for the collection of information regarding patients' inflammatory status, genetic predisposition, and intestinal microbiota. Additionally, breath and blood samples will be taken postoperatively and patients will be strictly observed during their in-hospital stay, and the period shortly thereafter. RESULTS: This study has been open for inclusion since August 2015. CONCLUSIONS: An estimated 8-10% of patients will develop AL following surgery, and they will be compared to non-leakage patients. The objectives of this study are twofold. The primary aim is to establish and validate a diagnostic algorithm for the pre-operative prediction of the risk of AL development using a combination of inflammatory, immune-related, and genetic parameters. Previously established risk factors and novel parameters will be incorporated into this algorithm, which will aid in the recognition of patients who are at risk for AL. Based on these results, recommendations can be made regarding the construction of an anastomosis or deviating stoma, and possible preventive strategies. Furthermore, we aim to develop a new algorithm for the post-operative diagnosis of AL at an earlier stage, which will positively reflect on short-term survival rates. TRIAL REGISTRATION: Clinicaltrials.gov: NCT02347735; https://clinicaltrials.gov/ct2/show/NCT02347735 (archived by WebCite at http://www.webcitation.org/6hm6rxCsA).

Colorectal anastomotic healing: why the biological processes that lead to anastomotic leakage should be revealed prior to conducting intervention studies.

BACKGROUND: Anastomotic leakage (AL) remains the most dreaded complication after colorectal surgery and causes high morbidity and mortality. The pathophysiology of AL remains unclear, despite numerous studies that have been conducted on animals and humans, probably due to the undetermined healing process of colorectal anastomoses. Increasing basic knowledge on this healing process may shed more light on causal factors of AL, and additionally reduce the quantity and accelerate the quality of experimental studies. In this debate article, our aim was to provide different perspectives on what is known about the colorectal healing process in relation to wound healing and AL. DISCUSSION: Since knowledge on anastomotic healing is lacking, it remains difficult to conclude which factors are essential in preventing AL. This is essential information in the framework of humane animal research, where the focus should lie on Replacement, Reduction and Refinement (3Rs). While many researchers compare anastomotic healing with wound healing in the skin, there are substantial recognized differences, e.g. other collagen subtypes and different components involved. Based on our findings in literature as well as discussions with experts, we advocate stop considering anastomotic healing in the gastrointestinal tract and cutaneous healing as a similar process. Furthermore, intervention studies should at least address the anastomotic healing process in terms of histology and certain surrogate markers. Finally, the anastomotic healing process ought to be further elucidated - with modern techniques to achieve 3Rs in animal research--to provide starting points for potential interventions that can prevent AL.