Simple rat model for esophagogastric anastomosis wound healing after esophageal transection: surgical technique and postoperative care

Simple rat model for esophagogastric anastomosis wound healing after esophageal transection: surgical technique and postoperative care | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Simple rat model for esophagogastric anastomosis wound healing after esophageal transection: surgical technique and postoperative care Ivan Kováč, Monika Miklóšová, Jozef Belák, Dominika Matiová, Ján Gajdoš, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6397105/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Use of experimental animal models is crucial for advancing our understanding of pathophysiological aspects and nature of diseases. Esophageal resection is only curative treatment for esophageal cancer. Disruption of esophagogastric anastomosis results in high morbidity and mortality. Presence of simple reproducible rat model is essential for better understanding of anastomosis healing and further investigations. Here a novel easily reusable technique of esophagogastric anastomosis formation using rat model is presented. Hand sewn end-to-end esophagogastric anastomoses were performed in rat model after esophageal dissection. 4 mm silicone catheter was inserted from gastrotomy to distal esophagus and anastomoses were sewn with continuous nonabsorbable suture. Standardized anastomoses were carried out in 10 male Sprague Dawley rats and physiological healing was observed by histological analysis 5 days after procedure. Normal anastomotic healing was found in 9 rats. Failure of the esophagogastric anastomosis caused by anastomosis insufficiency and leakage was observed in 1 animal. One animal died on third postoperative day on septic complications. Histologically, the healing of an anastomosis in physiological conditions was observed. This study presents a simple reproducible rodent model for esophagogastric anastomosis healing, so the gap between basic research and clinical applications could be bridged. Health sciences/Gastroenterology Health sciences/Medical research esophagogastric anastomosis rat Sprague-Dawley wound healing rodent model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Esophagogastric anastomosis (EGA) is a surgical connection between the esophagus and the stomach, commonly performed after esophagectomy in procedures for esophageal cancer or benign esophageal diseases, such as strictures, or esophageal motility disorders ( 1 ). Some aspects of the esophagogastric anastomosis make it more vulnerable to early and late postoperative complications than other anastomosis in gastrointestinal tract. The anatomic characteristics and the technical originalities of esophageal surgery have a leading role in these surgical complications ( 2 ). Advancing a rodent model for esophagogastric anastomosis healing is essential for investigating surgical outcomes and is crucial for studying post-surgical complications such as leakage, stricture formation, and delayed healing. Easily reproducible rat model for esophagogastric anastomosis wound healing warrants further research on how to study and improve the anastomotic healing process and improve outcomes for patients after esophagectomy. Earlier rodent model for esophagogastric anastomosis healing was described by Cui and Drescher ( 3 , 4 ). Our aim was to develop a new reusable and reliable rat model to facilitate experimental investigations into esophagogastric anastomosis healing, including gastric ischemic conditioning statements. Ethical statement: The experimental conditions met the requirements of the European rules for ethical standards of animal treatment and welfare. The study was approved by the Ethics Committee of the Faculty of Medicine of Pavol Jozef Šafarik University and by the State Veterinary and Food Administration of Slovakia (4893- 5/2021-220) on May 17th, 2021 and is reported in accordance with ARRIVE guidelines. Materials and Methods All methods performed during the present study were in accordance with the relevant guidelines and regulations, following the recommendations of the State Veterinary and Food Administration of Slovakia. Animal model: A total of 10 male Sprague‑Dawley rats (weight, 400±50 g) were obtained from the Animal Facility of P. J. Šafarik University and included in the present study. Animals were individually housed in plexiglass cages (22‑24˚C, 55±5% relative humidity, 12/12 h light/dark cycles) allowed free access to water and standard laboratory diet ad libitum. All rats were fasted for 12 hours (with free access to water) before the experiment. Surgical procedure: For general intramuscular anesthesia combination of Zoletil 20 mg/kg i.m. (Zoletil, Tiletamine, Zolazepam, Virbac, Westlake, Texas) and mabron 5 mg/kg (Tramadoliumchlorid, Medochemie, Limassol, Cyprus) was administered to the rats and standardized operative procedures were performed under aseptic conditions. Rats were placed in supine position to achieve the optimal access to abdominal cavity. Proper preparation of surgical field was achieved using standard methods (clipper used to remove hair and surgical site was cleaned with an antiseptic solution [Kodan antiseptic tincture, Schulke, Norderstedt, Germany]). An upper median laparotomy from the xiphoid process downward toward the umbilicus followed by mobilization and exploration of the stomach was performed ( Fig 1,2 ). Following, distal part of esophagus, gastroesophageal junction, cardia, left gastric artery and short gastric arteries were identified ( Fig 3 ). Procedure continued with gastrotomy in anterior aspect of stomach body and placement of 4mm silicone catheter to intubate abdominal esophagus (Fig 4 ). Transection of esophagus was executed followed by hand sewn end to end esophagogastric anastomosis formation using continues nonabsorbable sutures (Chiraflon 6/0 running suture, Chirmax, Prague, Czech Republic) ( Fig 5,6 ). Catheter was gently removed, and stomach opening was closed with interrupted suture (Chiraflon 5/0 Chirmax, Prague, Czech Republic). Unit of 10ml 0.9% NaCl as perioperative infusion was admitted intraperitoneally. Layered closure was used for laparotomy closing using continuous sutures for the musculofascial layer and peritoneum (3-0 Polysorb suture Covidien) and intradermal running sutures for skin closure (Chiraflon 5/0, Prague, Czech Republic). In addition, 10 ml 0.9% NaCl was administered subcutaneously to prevent postoperative dehydration of the animals. Postoperatively, the animals were allowed free access to water and milk (Nutrilon Comfort, Nutricia, Holand). Postoperative analgesia was performed every day with administration of mabron i.m. (2.5 mg/kg Tramadoliumchlorid, Medochemie, Limassol, Cyprus) and extra 5ml 0.9% NaCl infusion was applied subcutaneously. On the fifth postoperative day, in above-described general anesthesia, samples (the abdominal esophagus and whole stomach en-block) were harvested, and all animals were euthanized by an anesthetic overdose described by intramuscular administration of Zoletil (Zoletil, Tiletamine, Zolazepam, Virbac, Westlake, Texas) following AVMA guidelines of euthanasia in already anesthetized animals. Macroscopic photographs of the esophagogastric anastomosis mucosa were taken, and samples were stained for histological evaluation. Macroscopic assay: The whole stomach, gastroesophageal junction and the abdominal esophagus were opened along the greater curvature. Esophagogastric anastomosis was photographed with a scale after dissection and cleaning of samples with sterile solution. Signs of esophagogastric anastomosis failure by means of anastomotic dehiscence were documented. Microscopic assay: All samples were processed by using routine techniques for light microscopy, i.e. fixation in 4% buffered formaldehyde, clearing in xylene, dehydration with increasing concentrations of alcohol, embedding in paraffin, cutting, and staining with hematoxylin-eosin. Two sections (5-μm thick) were obtained from the area of esophagogastric anastomosis and evaluated by an experienced pathologist. Results Healing of an esophagogastric anastomosis followed by a predictable physiological process was observed in 9 animals, which were verified on macroscopic and microscopic levels ( Fig 7 ). One animal died on third postoperative day on septic complications. Leakage with dehiscence of esophagogastric anastomosis was documented during autopsy. High vulnerability of esophagogastric anastomosis to ischemia was estimated as a leading factor of anastomosis failure. DISCUSSION Esophageal cancer is the seventh most common cancer worldwide and the sixth in cancer mortality (5). Esophagectomy with two-field lymphadenectomy is the golden standard and only curative treatment for patients with esophageal cancer. Reconstruction of gastrointestinal tract is undertaken mainly by creating gastric conduit with an esophagogastric anastomosis (6). Despite significant progress in perioperative management, esophagectomy remains a procedure with high rates of morbidity and mortality. The spectrum of early perioperative complications varies from anastomotic insufficiency, bleeding, recurrent laryngeal nerve injury, chyle leak to respiratory complications. Anastomotic leakage is well recognized, yet poorly understood, one of the most severe complications after esophagectomy leading to a sequence of fatal aberrances, and increased risk of mortality (7). Moniek reports the 19.7% anastomotic leakage rate after transhiatal esophagectomy, 16.9% after Ivor Lewis esophagectomy and 22.2% after McKeown esophagectomy (8). The incidence of the anastomotic dehiscence and leakage varies from 5-20% in other studies (9). Various strategies aim at prevention of anastomotic leakage, including proper patient selection, preparation and prehabilitation programs, techniques to improve and control vascularization of the gastric conduit, and many surgical and anesthesiological details (7). Gastric ischemic conditioning (GIC) is a preconditioning technique that involves a temporary, controlled reduction of blood flow to the stomach to enhance its resistance to ischemic injury (10). The concept of gastric ischemic conditioning (GIC) involves laparoscopic ligation or percutaneous embolization of arteries supplying stomach in adequate time interval before esophagectomy (11). Experimental and clinical studies have demonstrated improved tissue perfusion and neovascularization of the gastric submucosa following laparoscopic or arteriographic GIC, with peak effects observed after 14 days (12-14). On the other hand, Hanna reported a similar leak rate after GIC via laparoscopic ligation prior to a hybrid McKeown oesophagectomy for malignancy to previously published data for a McKeown oesophagectomy without GIC (15). Few animal models were determined in the past for the purpose of esophagogastric anastomosis healing including GIC statement (3,4). The rat model is widely used in esophageal surgery research due to its cost-effectiveness, reproducibility, and feasibility for histological and molecular studies. However, technical challenges arise due to the small size of the esophagus and stomach. Main strength of our work is presence of new simple rat model of esophagogastric anastomosis healing. Conclusion Physiological anastomotic healing was observed in 9 rats, while one animal died on a septic complication related to anastomotic leakage observed during autopsy. Adequate perfusion of anastomosis is crucial for the healing process and ischemia with subsequent necrosis and anastomotic dehiscence was verified as reason of anastomotic failure in mentioned case. The primary objective of this study was to develop a novel, easily reproducible rat model of esophagogastric anastomosis healing focusing on technique and postoperative care and to settle a base for further investigations by means of verification of GIC statement on esophagogastric anastomosis healing. From this point of view, the discrepancy between protagonist/antagonist of GIC concept and optimal time interval of GIC could be verified in future using our present model. Secondly, our model could be used also in other indications like testing effects of substances to improve esophagogastric anastomosis healing administrated perorally or intravenously. This study has some limitations. Foremost, the sample size is small. Nevertheless, the safety and effectiveness of our model is presented, which lays the groundwork for future investigations. Declarations Author Contributions All authors whose names appear on the submission made substantial contributions to the conception or design of the work. All authors contributed equally to the creation of the manuscript. I.K. and M.K then reviewed and approved the version to be sent. Acknowledgments Authors would like to thank Darina Petrášová, head of Laboratory of Research Bio-models Pavol Jozef Šafárik University for participating in animal care. Conflict of interest The authors declare no competing interests. Data availability statements All data generated or analyzed during this study are included in this published article. References Bonavina L. Progress in the esophagogastric anastomosis and the challenges of minimally invasive thoracoscopic surgery. Ann Transl Med. 2021 May;9(10):907. doi: 10.21037/atm.2020.03.66. Mingol-Navarro F, Ballester-Pla N, Jimenez-Rosellon R. Ischaemic conditioning of the stomach previous to esophageal surgery. J Thorac Dis. 2019 Apr;11(Suppl 5):S663-S674. doi: 10.21037/jtd.2019.01.43. Cui Y, Urschel JD, Petrelli NJ. Esophagogastric anastomoses in rats--an experimental model. J Invest Surg. 1999 Sep-Oct;12(5):295-8. doi: 10.1080/089419399272412. PMID: 10599005. Drescher DG, et al. Model of wound healing for esophagogastric anastomoses in rats. Eur Surg Res. 2012;48(4):194-9. doi: 10.1159/000338625. Obermannová R, et al. Oesophageal cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up. Ann Oncol. 2022 Oct;33(10):992-1004. doi: 10.1016/j.annonc.2022.07.003. Tagkalos E, et al. Robot-assisted minimally invasive thoraco-laparoscopic esophagectomy versus minimally invasive esophagectomy for resectable esophageal adenocarcinoma, a randomized controlled trial (ROBOT-2 trial). BMC Cancer. 2021 Sep 26;21(1):1060. doi: 10.1186/s12885-021-08780-x. Vetter D, Gutschow CA. Strategies to prevent anastomotic leakage after esophagectomy and gastric conduit reconstruction. Langenbecks Arch Surg. 2020 Dec;405(8):1069-1077. doi: 10.1007/s00423-020-01926-8. Verstegen MHP, et al. Outcomes of Patients with Anastomotic Leakage After Transhiatal, McKeown or Ivor Lewis Esophagectomy: A Nationwide Cohort Study. World J Surg. 2021 Nov;45(11):3341-3349. doi: 10.1007/s00268-021-06250-w van Workum F, et al. Intrathoracic vs Cervical Anastomosis After Totally or Hybrid Minimally Invasive Esophagectomy for Esophageal Cancer: A Randomized Clinical Trial. JAMA Surg. 2021 Jul 1;156(7):601-610. doi: 10.1001/jamasurg.2021.1555. Jogiat UM, et al. Gastric ischemic conditioning prior to esophagectomy reduces anastomotic leaks and strictures: a systematic review and meta-analysis. Surg Endosc. 2022 Jul;36(7):5398-5407. doi: 10.1007/s00464-021-08866-4. Fernández-Moreno MC, et al. Pilot Trial on Ischemic Conditioning of the Gastric Conduit in Esophageal Cancer: Feasibility and Impact on Anastomotic Leakage (TIGOAL-I). Ann Surg Open. 2024 Feb 5;5(1):e379. doi: 10.1097/AS9.0000000000000379. Mittermair C, et al. Functional capillary density in ischemic conditioning: implications for esophageal resection with the gastric conduit. Am J Surg. 2008 Jul;196(1):88-92. doi: 10.1016/j.amjsurg.2007.07.025. Pham TH, et al. Laparoscopic ischemic conditioning of the stomach increases neovascularization of the gastric conduit in patients undergoing esophagectomy for cancer. J Surg Oncol. 2017 Sep;116(3):391-397. doi: 10.1002/jso.24668. Epub 2017 May 29. Akiyama S, et al. Preoperative embolization of gastric arteries for esophageal cancer. Surgery. 1996 Sep;120(3):542-6. doi: 10.1016/s0039-6060(96)80075-4. Hanna N, et al. Laparoscopic ischaemic conditioning of the gastric conduit prior to a hybrid mckeown oesophagectomy may not decrease the risk of anastomotic leak. Wideochir Inne Tech Maloinwazyjne. 2021 Dec;16(4):669-677. doi: 10.5114/wiitm.2021.105529. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 18 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 05 Sep, 2025 Reviews received at journal 28 Aug, 2025 Reviewers agreed at journal 17 Aug, 2025 Reviews received at journal 16 Jul, 2025 Reviewers agreed at journal 14 Jul, 2025 Reviewers invited by journal 06 May, 2025 Editor assigned by journal 06 May, 2025 Editor invited by journal 16 Apr, 2025 Submission checks completed at journal 15 Apr, 2025 First submitted to journal 07 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6397105","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":453637936,"identity":"961504e3-00b8-464e-9aaf-8c60715ae58d","order_by":0,"name":"Ivan Kováč","email":"","orcid":"","institution":"Pavol Jozef Šafárik University","correspondingAuthor":false,"prefix":"","firstName":"Ivan","middleName":"","lastName":"Kováč","suffix":""},{"id":453637937,"identity":"2d26b03a-b211-4667-8276-7461a5d9ee12","order_by":1,"name":"Monika Miklóšová","email":"","orcid":"","institution":"Pavol Jozef Šafárik 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2","display":"","copyAsset":false,"role":"figure","size":707946,"visible":true,"origin":"","legend":"\u003cp\u003eStomach mobilization\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6397105/v1/0b8019b3f2ca1e147664afa9.jpg"},{"id":82582775,"identity":"f0160e3f-8fd1-4e82-a30f-9ad8d177283a","added_by":"auto","created_at":"2025-05-13 06:47:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1571082,"visible":true,"origin":"","legend":"\u003cp\u003eExploration of peritoneal cavity (LGA – left gastric artery, GEJ – gastroesophageal junction, AE – abdominal esophagus, SGV – short gastric vessels)\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6397105/v1/89182cecbdc53a87324fb67a.jpg"},{"id":82584640,"identity":"350f2d41-0f27-42af-b742-f1e8723b5fc4","added_by":"auto","created_at":"2025-05-13 06:55:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3060899,"visible":true,"origin":"","legend":"\u003cp\u003eA circular suture placed on anterior wall of stomach, B gastrotomy, C placement of 4mm silicone catheter D intubation of distal aspect of esophagus\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6397105/v1/922f4b94c6732d716da27e6e.jpg"},{"id":82582778,"identity":"2dff21e7-5968-4f78-8de6-dbde8baa89d8","added_by":"auto","created_at":"2025-05-13 06:47:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":739612,"visible":true,"origin":"","legend":"\u003cp\u003ehand sewn end to end esophagogastric anastomosis formation over 4mm silicone catheter (silicone catheter is shown by arrow)\u003c/p\u003e","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6397105/v1/c756ac807d710abf2ae15194.jpg"},{"id":82582784,"identity":"3ff7574a-22ec-4e9d-87cf-0dcf1b9c75dc","added_by":"auto","created_at":"2025-05-13 06:47:04","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":503025,"visible":true,"origin":"","legend":"\u003cp\u003eEsophagogastric anastomosis (shown by arrow)\u003c/p\u003e","description":"","filename":"figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6397105/v1/d7a83d1f330ed1d7c2333104.jpg"},{"id":82585119,"identity":"546f39fd-c868-4231-b601-0f92c8378ea1","added_by":"auto","created_at":"2025-05-13 07:03:04","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":325280,"visible":true,"origin":"","legend":"\u003cp\u003eBrief histological view of esophagogastric anastomosis shown by arrow (H\u0026amp;E-stained sections of gastroesophageal junction, *mild angiogenesis along anastomosis, ** inflammation consists of leukocytes and macrophages)\u003c/p\u003e","description":"","filename":"figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6397105/v1/41027efb25f3031180fea277.jpg"},{"id":96651370,"identity":"71887f5a-cb01-4103-a79f-6774dbbe1d41","added_by":"auto","created_at":"2025-11-24 16:14:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7604314,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6397105/v1/c669bc05-a9cd-422a-afe9-4cd065c77d04.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Simple rat model for esophagogastric anastomosis wound healing after esophageal transection: surgical technique and postoperative care","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophagogastric anastomosis (EGA) is a surgical connection between the esophagus and the stomach, commonly performed after esophagectomy in procedures for esophageal cancer or benign esophageal diseases, such as strictures, or esophageal motility disorders (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome aspects of the esophagogastric anastomosis make it more vulnerable to early and late postoperative complications than other anastomosis in gastrointestinal tract. The anatomic characteristics and the technical originalities of esophageal surgery have a leading role in these surgical complications (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdvancing a rodent model for esophagogastric anastomosis healing is essential for investigating surgical outcomes and is crucial for studying post-surgical complications such as leakage, stricture formation, and delayed healing.\u003c/p\u003e \u003cp\u003eEasily reproducible rat model for esophagogastric anastomosis wound healing warrants further research on how to study and improve the anastomotic healing process and improve outcomes for patients after esophagectomy. Earlier rodent model for esophagogastric anastomosis healing was described by Cui and Drescher (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Our aim was to develop a new reusable and reliable rat model to facilitate experimental investigations into esophagogastric anastomosis healing, including gastric ischemic conditioning statements.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthical statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental conditions met the requirements of the European rules for ethical standards of animal treatment and welfare. The study was approved by the Ethics Committee of the Faculty of Medicine of Pavol Jozef \u0026Scaron;afarik University and by the State Veterinary and Food Administration of Slovakia (4893- 5/2021-220) on May 17th, 2021 and is reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n"},{"header":"Materials and Methods","content":"\u003cp\u003eAll methods performed during the present study were in accordance with the relevant guidelines and regulations, following the recommendations of the State Veterinary and Food Administration of Slovakia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimal model:\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA total of 10 male Sprague‑Dawley rats (weight, 400\u0026plusmn;50 g) were obtained from the Animal Facility of P. J. \u0026Scaron;afarik University and included in the present study. Animals were individually housed in plexiglass cages (22‑24˚C, 55\u0026plusmn;5% relative humidity, 12/12 h light/dark cycles) allowed free access to water and standard laboratory diet ad libitum. All rats were fasted for 12 hours (with free access to water) before the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSurgical procedure:\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor general intramuscular anesthesia combination of Zoletil 20 mg/kg i.m. (Zoletil, Tiletamine, Zolazepam, Virbac, Westlake, Texas) and mabron 5 mg/kg (Tramadoliumchlorid, Medochemie, Limassol, Cyprus) was administered to the rats and standardized operative procedures were performed under aseptic conditions. Rats were placed in supine position to achieve the optimal access to abdominal cavity. Proper preparation of surgical field was achieved using standard methods (clipper used to remove hair and surgical site was cleaned with an antiseptic solution [Kodan antiseptic tincture, Schulke, Norderstedt, Germany]). An upper median laparotomy from the xiphoid process downward toward the umbilicus followed by mobilization and exploration of the stomach was performed (\u003cstrong\u003eFig 1,2\u003c/strong\u003e). Following, distal part of esophagus, gastroesophageal junction, cardia, left gastric artery and short gastric arteries were identified (\u003cstrong\u003eFig 3\u003c/strong\u003e). Procedure continued with gastrotomy in anterior aspect of stomach body and placement of 4mm silicone catheter to intubate abdominal esophagus \u003cstrong\u003e(Fig 4\u003c/strong\u003e). Transection of esophagus was executed followed by hand sewn end to end esophagogastric anastomosis formation using continues nonabsorbable sutures (Chiraflon 6/0 running suture, Chirmax, Prague, Czech Republic) (\u003cstrong\u003eFig 5,6\u003c/strong\u003e). \u0026nbsp; Catheter was gently removed, and stomach opening was closed with interrupted suture (Chiraflon 5/0 Chirmax, Prague, Czech Republic). Unit of 10ml 0.9% NaCl as perioperative infusion was admitted intraperitoneally. Layered closure was used for laparotomy closing using continuous sutures for the musculofascial layer and peritoneum (3-0 Polysorb suture Covidien) and intradermal running sutures for skin closure (Chiraflon 5/0, Prague, Czech Republic). In addition, 10 ml 0.9% NaCl was administered subcutaneously to prevent postoperative dehydration of the animals.\u0026nbsp;Postoperatively, the animals were allowed free access to water and milk (Nutrilon Comfort, Nutricia, Holand). Postoperative analgesia was performed every day with administration of mabron i.m. (2.5 mg/kg Tramadoliumchlorid, Medochemie, Limassol, Cyprus) and extra 5ml 0.9% NaCl infusion was applied subcutaneously. On the fifth postoperative day, in above-described general anesthesia, samples (the abdominal esophagus and whole stomach en-block) were harvested, and all animals were euthanized by an anesthetic overdose described by intramuscular administration of Zoletil \u0026nbsp;(Zoletil, Tiletamine, Zolazepam, Virbac, Westlake, Texas) following AVMA guidelines of euthanasia in already anesthetized animals. Macroscopic photographs of the esophagogastric anastomosis mucosa were taken, and samples were stained for histological evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMacroscopic assay:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe whole stomach, gastroesophageal junction and the abdominal esophagus were opened along the greater curvature. Esophagogastric anastomosis was photographed with a scale after dissection and cleaning of samples with sterile solution. Signs of esophagogastric anastomosis failure by means of anastomotic dehiscence were documented.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicroscopic assay:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll samples were processed by using routine techniques for light microscopy, i.e. fixation in 4% buffered formaldehyde, clearing in xylene, dehydration with increasing concentrations of alcohol, embedding in paraffin, cutting, and staining with hematoxylin-eosin. Two sections (5-\u0026mu;m thick) were obtained from the area of esophagogastric anastomosis and evaluated by an experienced pathologist. \u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eHealing of an esophagogastric anastomosis followed by a predictable physiological process was observed in 9 animals, which were verified on macroscopic and microscopic levels (\u003cstrong\u003eFig 7\u003c/strong\u003e). One animal died on third postoperative day on septic complications. Leakage with dehiscence of esophagogastric anastomosis was documented during autopsy. High vulnerability of esophagogastric anastomosis to ischemia was estimated as a leading factor of anastomosis failure. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n"},{"header":"DISCUSSION","content":"\u003cp\u003eEsophageal cancer is the seventh most common cancer worldwide and the sixth in cancer mortality (5). Esophagectomy with two-field lymphadenectomy is the golden standard and only curative treatment for patients with esophageal cancer. Reconstruction of gastrointestinal tract is undertaken mainly by creating gastric conduit with an esophagogastric anastomosis (6). Despite significant progress in perioperative management, esophagectomy remains a procedure with high rates of morbidity and mortality. The spectrum of early perioperative complications varies from anastomotic insufficiency, bleeding, recurrent laryngeal nerve injury, chyle leak to respiratory complications. Anastomotic leakage is well recognized, yet poorly understood, one of the most severe complications after esophagectomy leading to a sequence of fatal aberrances, and increased risk of mortality (7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoniek reports the 19.7% anastomotic leakage rate after transhiatal esophagectomy, 16.9% after Ivor Lewis esophagectomy and 22.2% after McKeown esophagectomy (8). The incidence of the anastomotic dehiscence and leakage varies from 5-20% in other studies (9). Various strategies aim at prevention of anastomotic leakage, including proper patient selection, preparation and prehabilitation programs, techniques to improve and control vascularization of the gastric conduit, and many surgical and anesthesiological details (7).\u003c/p\u003e\n\u003cp\u003eGastric ischemic conditioning (GIC) is a preconditioning technique that involves a temporary, controlled reduction of blood flow to the stomach to enhance its resistance to ischemic injury (10). The concept of gastric ischemic conditioning (GIC) involves laparoscopic ligation or percutaneous embolization of arteries supplying stomach in adequate time interval before esophagectomy (11). Experimental and clinical studies have demonstrated improved tissue perfusion and neovascularization of the gastric submucosa following laparoscopic or arteriographic GIC, with peak effects observed after 14 days (12-14). On the other hand, Hanna reported a similar leak rate after GIC via laparoscopic ligation prior to a\u0026nbsp;hybrid McKeown oesophagectomy for malignancy to previously published data for a\u0026nbsp;McKeown oesophagectomy without GIC (15).\u003c/p\u003e\n\u003cp\u003eFew animal models were determined in the past for the purpose of esophagogastric anastomosis healing including GIC statement (3,4). The rat model is widely used in esophageal surgery research due to its cost-effectiveness, reproducibility, and feasibility for histological and molecular studies. However, technical challenges arise due to the small size of the esophagus and stomach. Main strength of our work is presence of new simple rat model of esophagogastric anastomosis healing. \u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003ePhysiological anastomotic healing was observed in 9 rats, while one animal died on a septic complication related to anastomotic leakage observed during autopsy. Adequate perfusion of anastomosis is crucial for the healing process and ischemia with subsequent necrosis and anastomotic dehiscence was verified as reason of anastomotic failure in mentioned case. The primary objective of this study was to develop a novel, easily reproducible rat model of esophagogastric anastomosis healing focusing on technique and postoperative care and to settle a base for further investigations by means of verification of GIC statement on esophagogastric anastomosis healing. From this point of view, the discrepancy between protagonist/antagonist of GIC concept and optimal time interval of GIC could be verified in future using our present model. Secondly, our model could be used also in other indications like testing effects of substances to improve esophagogastric anastomosis healing administrated perorally or intravenously. This study has some limitations. Foremost, the sample size is small. Nevertheless, the safety and effectiveness of our model is presented, which lays the groundwork for future investigations. \u0026nbsp;\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors whose names appear on the submission made substantial contributions to the conception or design of the work. All authors contributed equally to the creation of the manuscript. I.K. and M.K then reviewed and approved the version to be sent. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors would like to thank Darina Petr\u0026aacute;\u0026scaron;ov\u0026aacute;, head of Laboratory of Research Bio-models Pavol Jozef \u0026Scaron;af\u0026aacute;rik University for participating in animal care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBonavina L. Progress in the esophagogastric anastomosis and the challenges of minimally invasive thoracoscopic surgery. Ann Transl Med. 2021 May;9(10):907. doi: 10.21037/atm.2020.03.66. \u003c/li\u003e\n\u003cli\u003eMingol-Navarro F, Ballester-Pla N, Jimenez-Rosellon R. Ischaemic conditioning of the stomach previous to esophageal surgery. J Thorac Dis. 2019 Apr;11(Suppl 5):S663-S674. doi: 10.21037/jtd.2019.01.43. \u003c/li\u003e\n\u003cli\u003eCui Y, Urschel JD, Petrelli NJ. Esophagogastric anastomoses in rats--an experimental model. J Invest Surg. 1999 Sep-Oct;12(5):295-8. doi: 10.1080/089419399272412. PMID: 10599005.\u003c/li\u003e\n\u003cli\u003eDrescher DG, et al. Model of wound healing for esophagogastric anastomoses in rats. Eur Surg Res. 2012;48(4):194-9. doi: 10.1159/000338625. \u003c/li\u003e\n\u003cli\u003eObermannov\u0026aacute; R, et al. Oesophageal cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up. Ann Oncol. 2022 Oct;33(10):992-1004. doi: 10.1016/j.annonc.2022.07.003. \u003c/li\u003e\n\u003cli\u003eTagkalos E, et al. Robot-assisted minimally invasive thoraco-laparoscopic esophagectomy versus minimally invasive esophagectomy for resectable esophageal adenocarcinoma, a randomized controlled trial (ROBOT-2 trial). BMC Cancer. 2021 Sep 26;21(1):1060. doi: 10.1186/s12885-021-08780-x. \u003c/li\u003e\n\u003cli\u003eVetter D, Gutschow CA. Strategies to prevent anastomotic leakage after esophagectomy and gastric conduit reconstruction. Langenbecks Arch Surg. 2020 Dec;405(8):1069-1077. doi: 10.1007/s00423-020-01926-8. \u003c/li\u003e\n\u003cli\u003eVerstegen MHP, et al. Outcomes of Patients with Anastomotic Leakage After Transhiatal, McKeown or Ivor Lewis Esophagectomy: A Nationwide Cohort Study. World J Surg. 2021 Nov;45(11):3341-3349. doi: 10.1007/s00268-021-06250-w\u003c/li\u003e\n\u003cli\u003evan Workum F, et al. Intrathoracic vs Cervical Anastomosis After Totally or Hybrid Minimally Invasive Esophagectomy for Esophageal Cancer: A Randomized Clinical Trial. JAMA Surg. 2021 Jul 1;156(7):601-610. doi: 10.1001/jamasurg.2021.1555.\u003c/li\u003e\n\u003cli\u003eJogiat UM, et al. Gastric ischemic conditioning prior to esophagectomy reduces anastomotic leaks and strictures: a systematic review and meta-analysis. Surg Endosc. 2022 Jul;36(7):5398-5407. doi: 10.1007/s00464-021-08866-4. \u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-Moreno MC, et al. Pilot Trial on Ischemic Conditioning of the Gastric Conduit in Esophageal Cancer: Feasibility and Impact on Anastomotic Leakage (TIGOAL-I). Ann Surg Open. 2024 Feb 5;5(1):e379. doi: 10.1097/AS9.0000000000000379. \u003c/li\u003e\n\u003cli\u003eMittermair C, et al. Functional capillary density in ischemic conditioning: implications for esophageal resection with the gastric conduit. Am J Surg. 2008 Jul;196(1):88-92. doi: 10.1016/j.amjsurg.2007.07.025. \u003c/li\u003e\n\u003cli\u003ePham TH, et al. Laparoscopic ischemic conditioning of the stomach increases neovascularization of the gastric conduit in patients undergoing esophagectomy for cancer. J Surg Oncol. 2017 Sep;116(3):391-397. doi: 10.1002/jso.24668. Epub 2017 May 29. \u003c/li\u003e\n\u003cli\u003eAkiyama S, et al. Preoperative embolization of gastric arteries for esophageal cancer. Surgery. 1996 Sep;120(3):542-6. doi: 10.1016/s0039-6060(96)80075-4. \u003c/li\u003e\n\u003cli\u003eHanna N, et al. Laparoscopic ischaemic conditioning of the gastric conduit prior to a hybrid mckeown oesophagectomy may not decrease the risk of anastomotic leak. Wideochir Inne Tech Maloinwazyjne. 2021 Dec;16(4):669-677. doi: 10.5114/wiitm.2021.105529. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"esophagogastric anastomosis, rat, Sprague-Dawley, wound healing, rodent model","lastPublishedDoi":"10.21203/rs.3.rs-6397105/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6397105/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUse of experimental animal models is crucial for advancing our understanding of pathophysiological aspects and nature of diseases. Esophageal resection is only curative treatment for esophageal cancer. Disruption of esophagogastric anastomosis results in high morbidity and mortality. Presence of simple reproducible rat model is essential for better understanding of anastomosis healing and further investigations. Here a novel easily reusable technique of esophagogastric anastomosis formation using rat model is presented.\u003c/p\u003e \u003cp\u003eHand sewn end-to-end esophagogastric anastomoses were performed in rat model after esophageal dissection. 4 mm silicone catheter was inserted from gastrotomy to distal esophagus and anastomoses were sewn with continuous nonabsorbable suture. Standardized anastomoses were carried out in 10 male Sprague Dawley rats and physiological healing was observed by histological analysis 5 days after procedure. Normal anastomotic healing was found in 9 rats. Failure of the esophagogastric anastomosis caused by anastomosis insufficiency and leakage was observed in 1 animal. One animal died on third postoperative day on septic complications. Histologically, the healing of an anastomosis in physiological conditions was observed. This study presents a simple reproducible rodent model for esophagogastric anastomosis healing, so the gap between basic research and clinical applications could be bridged.\u003c/p\u003e","manuscriptTitle":"Simple rat model for esophagogastric anastomosis wound healing after esophageal transection: surgical technique and postoperative care","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 06:46:59","doi":"10.21203/rs.3.rs-6397105/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-05T06:41:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-28T16:37:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141580444356891103156012050791880444876","date":"2025-08-17T07:36:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-16T06:30:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153730776071327132273516370001846569421","date":"2025-07-14T09:40:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-06T16:50:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-06T16:41:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-16T12:35:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-15T13:47:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-07T19:37:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d4104e6f-53b0-4b0f-8213-75b277a839af","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48239967,"name":"Health sciences/Gastroenterology"},{"id":48239968,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-11-24T16:13:47+00:00","versionOfRecord":{"articleIdentity":"rs-6397105","link":"https://doi.org/10.1038/s41598-025-24163-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-18 15:57:41","publishedOnDateReadable":"November 18th, 2025"},"versionCreatedAt":"2025-05-13 06:46:59","video":"","vorDoi":"10.1038/s41598-025-24163-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-24163-9","workflowStages":[]},"version":"v1","identity":"rs-6397105","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6397105","identity":"rs-6397105","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}