Available online 1 April 2023, 152095

Author links open overlay panel, , , , , , , , AbstractBackground

Gastrointestinal anastomoses are performed in many patients every year. The pathogenesis of aberrant anastomotic healing and the causes of intestinal leakage are not fully understood. The present study gathered and critically evaluated histological quantitative data to deepen current knowledge of anastomotic healing in the small and large intestine and its complications and outline the options for further experimental in vivo research in large porcine animal models.

Methods

Three groups of porcine intestinal anastomoses were compared: small intestine without defect (SI; n=7), small intestine with an additional defect (SID; n=8), and large intestine (LI; n=7). Multilevel sampling (2,112 micrographs) and stereological methods were used for histological quantification of proliferation (Ki-67 immunohistochemistry), neutrophil infiltration (myeloperoxidase staining), vascularity (von Willebrand factor) and type I and type III collagen formation (picrosirius red in polarized light) within the region of anastomosis compared to the region outside of anastomosis.

Results

Quantitative histological evaluation revealed the following results. i) Proliferation, vascularity, and collagen, but not neutrophils, were more highly expressed within the anastomosis than outside of the anastomosis region. ii) Porcine large and small intestine were not interchangeable based on histological evaluation of surgical experiments. The presence or absence of an additional experimental defect strongly affected healing, but the healing seemed complete after 21 days. iii) The microscopic structure of small intestine segments was more affected by their proximity to the anastomosis than the structure of large intestine segments.

Conclusions

Histological quantification was more laborious than the previously used semiquantitative scoring system evaluating the healing rate of intestinal anastomoses, but it provided detailed maps of biological processes within individual intestine layers. The primary data collected in the study are open and available for power sample analyses to calculate the minimum numbers of samples justified in future experiments on porcine intestines. The porcine intestine is a promising animal model with translational potential for human surgery.

Section snippetsGastrointestinal anastomoses and leaks

Gastrointestinal anastomoses are performed in thousands of patients annually. However, we do not fully understand the pathogenesis of aberrant anastomotic healing and the causes of intestinal leakage even after a century of research (Binnebösel et al., 2014, Wu et al., 2013). Failure of a gastrointestinal anastomosis leads to leakage, dehiscence and fistulas, which are major complications after abdominal surgery (Kosmidis et al., 2011). Although the surgical technique has continuously improved,

Collection of porcine small and large intestine segments

All paraffin-embedded archived tissue blocks (n=22) of porcine small and large intestines were obtained from previous studies (Rosendorf et al., 2020, Rosendorf et al., 2021a, Rosendorf et al., 2021b). No animals were sacrificed for the purpose of the present study. Intestinal samples were taken from 12 female and 10 male Prestice Black-Pied pigs aged 12-14 weeks and weighing 24-48 kg. The animal samples were obtained from a project verifying the effect of various nanofibrous structures on the

RESULTS

All primary morphometric data obtained from the histological quantification are provided in Supplement 1.

Proliferation, vascularity, and collagen, but not neutrophils, were more highly expressed in the anastomosis than outside of the anastomosis region

The anastomotic region showed increased proliferation compared to outside of the anastomosis region (Suppl. 2 A), which is similar to Grommes et al. (2012). Neutrophilic infiltration (Suppl. 2B) was comparable in all intestine layers within and outside of the anastomosis regions. Rosendorf et al., 2021a, Rosendorf et al., 2021b also did not find any signs of increased inflammation in the intestinal anastomosis. Our findings suggest that the inflammatory phase of anastomosis healing was complete

CONCLUSIONS

Quantitative histological evaluation of proliferation, neutrophilic infiltration, vascularity, and collagen formation in 22 samples of porcine small and large intestinal anastomoses revealed the following results. i) Proliferation, vascularity, and collagen, but not neutrophils, were more highly expressed within the anastomosis than outside of the anastomosis region. ii) Porcine large and small intestine were not interchangeable based on histological evaluations of surgical experiments. The

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ACKNOWLEDGMENTS

The study was supported by the Charles University Cooperation Program, research area MED/DIAG, and Project No. NU20J-08-00009, Prevention of intestinal anastomotic leakage and postoperative adhesions using nanofibrous biodegradable materials awarded by The Ministry of Health, Czech Republic. M.G. and A.M. were also supported by Charles University Research Project No. SVV 260 536. Skillful technical support from Ms. Jana Dosoudilova and Mr. Jan Javurek is gratefully acknowledged.

Declarations of interest

None.

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