Three female domestic pigs with a mean weight of 48 kg were used. The animals were fasted from solid food for 24 h before ESD but were allowed full access to water. For bowel preparation, each animal received 3 L of polyethylene glycol via orogastric tube 24 h prior to the procedure.

General anesthesia was provided by a veterinarian and performed by using ketamine and isoflurane, nitrous oxide, and O₂ after endotracheal intubation. Continuous pulse oximetry and electrocardiograph findings were monitored throughout the procedure.

Each animal was assigned a different treatment substance (either adrenaline, TXA, or the control group with no hemostatic agent).

For the first animal (Pig1), the standard 9 mL submucosal injectate comprising succinylated gelatin (Gelofusine; B. Braun, Crissier, Switzerland), 1:100000 adrenaline (epinephrine) and indigo carmine, with 1 mL of saline 0.9% was used.

For the second pig (Pig2), 9 mL submucosal injectate comprising succinylated gelatin and indigo carmine with 1 mL of TXA (100 mg/mL) was used.

For the third pig (Pig3), 10 mL of submucosal injectate comprising succinylated gelatin and indigo carmine.

ESD procedures were performed by two attending advanced endoscopists. The endoscopists were blinded to the randomization of the injection substrates until after all study procedures were performed. The syringes for submucosal injection were prepared outside of the endoscopy room. The same volume (20 mL) of injectate was used for each ESD resection.

All procedures were performed by using a commercially available endoscope (Pentax EC-380LKp). Before resection areas median size 3 cm in diameter each were marked at 4 sites in the body of the stomach and 4 in the rectum, mimicking lesions and were easily accessible by the endoscope. Standard ESD using a 2 mm flush-knife (Fuji Flush Knife BTS, Fujifilm, Tokyo, Japan) was used to resect each lesion. Circumferential incisions with standard dissection were applied for gastric lesions, while tunneling techniques were applied for colorectal lesions. At the end of the procedure, all visible vessels were coagulated. No antacids or proton pump inhibitors were given to the pigs.

For the week following the ESD, daily assessments of general health parameters including monitoring for signs of overt bleeding were performed on the animals. Complete blood counts were taken before the procedure and on day 3 after procedure. Diagnostic gastroscopy and colonoscopy were performed after 1 week of follow up (day 7); afterwards, the animals were euthanized by a veterinarian. The 1 week length of time was chosen as it would ensure that ulcers were still visible at each ESD site, while also falling within the time that post-procedure complications such as bleeding often occur.

Samples from the gastric and rectal ESD sites (two samples for each ESD site) from each of the 3 pigs were harvested and fixed in 4% formaldehyde. The tissues were trimmed, placed in embedding cassettes, and processed routinely for paraffin embedding. A total of 48 cassettes were prepared per animal. Paraffin sections (4 microns thick) were cut, placed on glass slides, and stained with Hematoxylin & Eosin (H&E).

During the ESD procedures, an assessment of intraprocedural bleeding was performed. Bleeding less than 1 min was defined as minor, lasting more than 1 min with partial interference in visualization or in which coagulation treatment was needed was defined as moderate, and pulsatile bleeding or complete interference with visualization was defined as severe.

The slides were subjected to histopathological evaluation by two expert veterinary pathologists who were blinded to the study groups. Additionally, each pathologist graded the histopathology slides while blinded from the scores given by the other pathologist. The histological slides were examined, described, and scored using a semi-quantitative grading scale. This established 5-point scale graded the severity of the histopathological changes in terms of inflammation, necrosis, and granulation (13).

Grade 1—Minimal inflammation/necrosis/granulation tissue. Findings (<10 inflammatory cells per ×20 magnification, up to 25% necrosis/granulation tissue).

Grade 2—Mild inflammation/necrosis/granulation tissue. Findings (>10 <25 inflammatory cells per ×20 magnification, 25–50% necrosis/granulation tissue).

Grade 3—Moderate inflammation/necrosis/granulation tissue. Findings (>25 <50 inflammatory cells per ×20 magnification, 50–75% necrosis/granulation tissue).

Grade 4—Severe inflammation/necrosis/granulation tissue. Findings (>50 inflammatory cells per ×20 magnification, 75–100% necrosis/granulation tissue).

Finally, a global score (from 0 to 4) was given to the stomach and rectum of each animal as a summary of the severity of the histopathological findings.

In the event of a difference in any of the scores between the two blinded pathologists, the pathologists would discuss the findings until a consensus score was agreed upon.

Given the small number of pigs used, the study was not powered for formal statistical comparisons between the groups.

Eight ESDs were performed on each animal, 4 in the proximal stomach body and 4 in the rectum. Details of the procedures can be found in Table 1.

Endoscopic examinations were performed after 1 week of follow-up. There was no significant difference in the gross appearance of the wound defects between the groups. All defects had the appearance of clean-based ulcers.

Within each animal, there were no significant histopathological changes noted between the defects. However, differences were noted in histopathological changes between the animals. In the stomach of first 2 animals (adrenaline and TXA), pathological examination revealed a clear ulcer in the mucosa and submucosa with an abundant amount of granulation tissue and mild inflammation, while the muscular layer was unaffected and intact (Figures 1a,b). There was a marked edema in the junction between the lesion and the intact mucosa and submucosa. In the third animal’s stomach (control group), the lesions were deeper, transmurally distributed, with inflammation of the muscular and adventitia layers accompanied by severe inflammation and necrosis (Figure 1c). Thus, in the stomach, Pig3 had higher inflammation and necrosis scores, and lower granulation score, than the other animals (Table 2).

The differences between the lesions in the rectum were less observable and prominent between the animals compared to the stomach lesions. Overall, there was more severe inflammation and necrosis in Pig1 (adrenaline) (Figure 1d).

There was good agreement between the two blinded pathologists in terms of these findings. Of the 18 scores given in Table 2, only two scores differed between the pathologists, and these only differed by 1 grade.

The summary semi-quantitative histological score is shown in Figure 2. There were no differences between the two pathologists in terms of these scores.

The intact tissue surrounding the lesions appeared normal in all samples. Some edema was noted on the edges of the lesion in some samples. There was no evidence of small vessel thrombosis in any specimens.

There were no episodes of severe intraprocedural bleeding, but several moderate bleeding episodes occurred (Table 1). There was a similar number between the groups in the stomach, but in the rectum they only occurred in the adrenaline group. All intraprocedural bleeding either stopped spontaneously or with the use of coagulation from the ESD knife.

The coagulation hemostatic grasper was not required. There was no evidence of overt GI bleeding from any of the animals during the 7 days of follow-up, nor was there any difference in general health parameters. Hemoglobin and WBC increased in all animals on day 3 of follow-up.