Establishing a rabbit sham model of cervical esophageal surgery for circumferential esophageal transplantation

Abstract

Background

This study was performed to establish a rabbit model for esophageal surgery in the treatment of cervical esophageal diseases. The necessary steps for postoperative treatment and esophageal examination were detailed.

Methods

This study aimed to confirm the feasibility of the surgical procedure in rabbits and develop a postoperative management protocol. Six New Zealand White rabbits underwent esophagectomy and end-to-end anastomosis of the cervical esophagus under general anesthesia. Postoperatively, the rabbits were fed a specially formulated therapeutic liquid diet for 3 to 7 days. Recovery was monitored for 8 weeks, after which the rabbits were euthanized. Esophagography and endoscopy were conducted during the first postoperative week to assess healing.

Results

Of the six rabbits that underwent surgery, two died from nonsurgical complications within 7 days after the operation. Of the remaining rabbits, two were diagnosed with esophageal stricture via esophagography and underwent endoscopy after the first postoperative week. The final two rabbits completed the 8-week postoperative period without complications.

Conclusions

Esophageal surgery and postoperative treatment in rabbits require careful attention. We anticipate that this study will provide valuable and practical information for researchers conducting esophageal studies using rabbits.

HIGHLIGHTS
A rabbit model for cervical esophageal surgery appears feasible. Anastomosis should involve minimal dissection to prevent stricture. Attention is required regarding postoperative diet formulation. Postoperative endoscopy and esophagogram were performed to identify complications.

INTRODUCTION

The esophagus is one of the most challenging organs to treat surgically, as repair typically requires a conduit created from the stomach, small intestine, or large intestine following defects caused by congenital anomalies, disease, or trauma [1–3]. Such procedures carry high complication rates and can adversely impact patient quality of life after surgery [4–6]. Consequently, many researchers have conducted translational studies to reconstruct the esophagus, including efforts to develop artificial conduits [7–11]. The present animal study represents a preliminary step toward developing an optimal artificial esophageal conduit, performed as a sham operation.

Animal models previously used for esophageal surgery include pigs, dogs, rats, and rabbits [12–14]. However, pigs and dogs present logistical challenges due to their large size, prolonged surgical time, and high costs. Rats pose difficulties in securing the operative field and postoperative examinations due to their small size. Rabbits, however, have a relatively long cervical esophagus, making surgical access straightforward. Anatomically, the rabbit cervical esophagus closely resembles the human esophagus, with a diameter similar to pediatric patients [15–16]. Thus, rabbits are considered an ideal model for various esophageal procedures and have been used in multiple experimental studies.

We previously published research on developing an artificial esophageal structure, and subsequent in vivo rabbit studies using this structure are currently underway [17]. Nonetheless, several challenges persist in rabbit esophageal surgery, particularly concerning postoperative feeding methods. Prolonged fasting periods can cause health deterioration due to malnutrition, including sustained weight loss and anemia. To minimize fasting during postoperative recovery, liquid feeding is essential, yet detailed references for postoperative liquid diet formulations are scarce.

Endoscopic examinations provide direct information about the anastomosis healing process in humans after esophageal surgery and can similarly be useful in rabbits. In this study, we established a rabbit model for esophageal surgery and detailed the required steps for postoperative management, including a dietary formula and esophageal examination procedures.

METHODS

All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Medicine, The Catholic University of Korea (No. CUMS-2020-0001-08). This study adhered to the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines [18].

Rabbits

We used six specific pathogen-free New Zealand White rabbits (male, 3.4–4.2 kg; Granja San Bernardo, Navarra, Spain). Following ethical principles for animal experiments, we determined the minimal necessary number of animals to achieve valuable results (six), as approved by the IACUC. Animals were individually housed in stainless wire cages that could be automatically washed and watered. Each cage included a triangular polycarbonate resting board to prevent foot issues. A mineral block (Bunny Blocks, Certified; Bio-Serv) and a wood block (Rabbit Brick; TAPVEI) were provided for enrichment. During the 3 to 4 week acclimation period, the rabbits became accustomed to human handling, relieved stress through playroom activities, and were fed organic carrots, celery, and cabbage to prepare for the postoperative recovery diet. The housing environment was maintained on a 12-hour light/dark cycle (lights off at 8 pm), the temperature ranged from 18 °C–26 °C, and relative humidity was maintained at 50%±10%. Rabbits received Lab Rabbit Chow, an antibiotic-free/gamma-ray sterilized diet (38302AF; Purina), and tap water ad libitum.

Preoperative Management and Anesthesia

Rabbits scheduled for surgery were acclimatized to carrot juice or milk powder for 1 to 2 weeks before surgery. The rabbits were fed until the evening before surgery and then fasted through the morning of the surgical procedure. General anesthesia was administered by specially trained veterinarians qualified in laboratory animal science, anesthetic agents, and monitoring techniques. The rabbits were anesthetized using inhaled isoflurane. Because isoflurane induction can cause life-threatening apnea in rabbits, induction was initiated with xylazine and ketamine. Xylazine primarily provided sedation, while ketamine induced dissociative anesthesia.

Before intubation, local anesthetic cream was applied to the marginal ear vein, and glycopyrrolate (0.01 mg/kg) was injected subcutaneously as an anticholinergic agent. After 10 to 15 minutes, xylazine (5 mg/kg) was administered subcutaneously. Once the animal was sedated, preoxygenation was initiated using a face mask, and the marginal ear vein was cannulated with a 24G intravenous catheter. Ketoprofen (5 mg/kg) or tramadol (5 mg/kg) was administered intravenously for analgesia, and cefazolin (33 mg/kg) was given intravenously as an antibiotic. Normal saline (1–2 mL) was slowly administered intravenously between drug injections, and intravenous fluid therapy (4 mg/kg/hr) was maintained during surgery.

After anesthesia induction, the rabbit was positioned ventrodorsally on the operating table (Fig. 1). Lidocaine spray was applied locally to the epiglottis, and the tongue was grasped using wet gauze. Intubation was performed with a 3.0- or 3.5-sized endotracheal tube with a stylet. Once induction was achieved, gas anesthesia was maintained using 1%–2.5% isoflurane supplemented with 1 L/min oxygen via ventilation through the endotracheal tube. Rabbits were monitored by pulse oximetry, and end-tidal carbon dioxide, peripheral oxygen saturation, electrocardiogram, and body temperature were recorded.

Fig. 1.

General anesthesia and preparation for esophageal surgery in a rabbit.

Cervical Esophageal Surgery

Exposing the cervical esophagus

After general anesthesia, the skin was shaved from the lower chin to the upper sternum. A vertical midline incision was made in the neck to expose the superficial investing fascia. At this fascia, the paired sternocleidomastoid and strap muscles were separated at their connecting raphe, forming a bloodless midline plane. The raphe was opened between the thyroid cartilage and sternal notch to expose the trachea. The cervical esophagus, located posteriorly to the trachea, was exposed by dissecting toward the posterior wall beyond the cervical artery and jugular vein on the left side of the trachea. After identifying the cervical esophagus, the skin and neck muscles were sutured and ligated to ensure adequate exposure (Fig. 2). Following dissection from the surrounding tissue, the esophagus at each transection site was immobilized with a pair of vessel clamps.

Fig. 2.

Cervical esophageal exposure in the rabbit neck.

Esophageal anastomosis

The esophageal anastomosis was performed using Vicryl 6-0 (Ethicon) interrupted sutures (Fig. 3). As with all anastomoses, careful suturing of both segments, including all layers, was critical to prevent mucosal eversion. Sutures were placed sparingly to close the gap while minimizing leakage and stricture risk. On average, interrupted sutures were placed at seven or eight sites per anastomosis (Fig. 3). Unnecessary dissection around the esophagus was avoided, and the esophageal tissue was handled delicately to prevent mucosal atrophy and stenosis at the anastomosis site. The strap muscles and investing fascia were closed with Vicryl 4-0.

Fig. 3.

Esophageal anastomosis in the rabbit neck.

Postoperative Management and Feeding

Antibiotics and analgesics were administered to rabbits for 3 to 5 days postoperatively. Rabbits were monitored daily for behavioral abnormalities, body weight, and temperature, and the surgical site was disinfected every 2 to 3 days to detect infection. Only water was provided for 48 hours after surgery. From days 3 to 7, rabbits received a liquid diet (grated carrot juice plus Gatorade, 3–5:1) four times daily. Finely chopped vegetables and finely ground rabbit chow were given on days 7 to 14, progressing to vegetables and roughly ground rabbit chow after day 14. Normal saline (30 mL) was injected subcutaneously to prevent dehydration. During fasting and recovery, fluid therapy, blood analysis, and urinalysis were conducted to monitor the rabbits’ conditions.

Rabbits were regularly given playtime in the playroom 3 to 4 weeks before surgery to adapt to human interaction and reduce stress. After surgery and adequate recovery, regular playtime resumed until the rabbits were euthanized.

Postoperative Evaluation

Endoscopy and esophagography were performed on day 7 after surgery to identify complications such as stenosis or leakage at the anastomosis site. The rabbits were injected subcutaneously with xylazine (1 mg/kg) and ketamine (2 mg/kg). After adequate sedation, each rabbit was laid on its side, and a mouthpiece was placed in its mouth. A 5.9-mm flexible endoscope was used to examine the upper esophagus, surgical anastomosis site, and stomach to assess their internal conditions (Fig. 4).

Fig. 4.

Endoscopic system for postoperative evaluation.

The rabbit was then moved to a room equipped with a mobile C-arm fluoroscopic X-ray system for esophagography. Gastrografin (mixture of sodium amidotrizoate and meglumine amidotrizoate; Bayer Korea) was prepared as a contrast agent for esophagography; this agent is commonly used in humans to inspect the upper gastrointestinal system. Due to its high viscosity, the gastrografin was diluted with saline at a 1:2 ratio. Under mild anesthesia, a 6-Fr orogastric tube was inserted through the rabbit's nostril, and the catheter tip location was confirmed by taking an X-ray at 12 to 15 cm. The catheter tip was positioned at the mid-level of the esophagus, and 5 mL of the gastrografin-saline mixture was slowly administered through the catheter. An X-ray image of the esophageal anastomosis site was obtained using the C-arm system (Fig. 5).

Fig. 5.

(A) Mobile C-arm fluoroscopic X-ray system for esophagogram and (B) postoperative finding with gastrografin. Postoperative findings of esophagus with gastrografin (arrow)

RESULTS

Surgical Outcomes of Esophageal Anastomosis

We conducted preliminary experiments on six rabbits to evaluate the suitability of a rabbit sham model for esophageal surgery. Two rabbits died suddenly on postoperative days 1 and 7, respectively. The rabbit that died on day 7 exhibited right atrial hypertrophy and pericardial effusion, indicating right heart failure as the likely cause of death.

Of the remaining four rabbits, two developed anastomotic stenosis at 1 week postoperatively, while the other two recovered well and completed the 8-week experimental period. Fig. 6 shows the endoscopic findings of anastomotic stenosis and nonstenosis at 1 week after surgery. The rabbits with stenosis did not display specific symptoms while consuming the liquid diet during the first postoperative week; however, they exhibited hiccups and gagging when introduced to finely chopped vegetables. These rabbits were euthanized, and the experiment was terminated after confirming the stricture via endoscopy and esophagography. These two rabbits had undergone surgery at the beginning of the experiment. Based on this experience, two rabbits operated on later, with minimal esophageal dissection, recovered without strictures. Body weight and hemoglobin levels were monitored for all four rabbits until euthanasia. The two rabbits with stenosis (sham 1 and sham 2) were euthanized on postoperative days 12 and 11, respectively. Both rabbits experienced rapid declines in body weight and hemoglobin levels, whereas rabbits without stenosis showed no significant changes (Tables 1 and 2).

Fig. 6.

Postoperative endoscopic findings: (A) stenosis, (B) nonstenosis.

Table 1.

Postoperative body weight (kg) changes

Postoperative day	Sham 1	Sham 2	Sham 3	Sham 4
0	3.43	3.40	3.80	3.90
1	3.17	3.28	-	3.87
3	3.08	3.11	-	3.63
5	2.93	3.06	3.42	3.56
7	-	2.97	-	3.49
10	-	2.73	-	3.45
12	2.86	-	3.34	3.50
20	-	-	3.39	3.61
40	-	-	3.24	3.67
60	-	-	3.26	3.93

Table 2.

Postoperative hemoglobin (g/dL) changes

Postoperative day	Sham 1	Sham 2	Sham 3	Sham 4
0	11.5	14.7	18.8	17.4
1	11.5	-	-	-
3	11.2	-	-	-
5	-	-	11.7	-
7	9.2	10.5	-	15.8
10	-	13.6	-	-
12	-	-	16.6	-
20	-	-	10.9	-
40	-	-	11.2	-
60	-	-	15.6	12.5

Histological Outcomes of Esophageal Anastomosis

Rabbits without strictures or leakages during the experiment were fed vegetables and rabbit chow starting 1 week postoperatively. Eight weeks after surgery, these rabbits were euthanized, and the condition of the esophageal anastomosis was evaluated by necropsy. The surgical site was removed as a specimen, and the lumen was opened to inspect the anastomosis. The retrieved specimens were embedded in paraffin blocks, and complete regeneration of the mucosal and submucosal muscle layers was histologically confirmed using hematoxylin and eosin staining (Fig. 7).

Fig. 7.

(A) Gross and (B) histologic findings with hematoxylin and eosin stain (×10) of the anastomosis site (red arrows) from a necropsy specimen at 8 weeks postoperatively.

DISCUSSION

The purpose of this study was to establish a versatile esophageal sham animal model for translational research using surgical methods applicable to various esophageal diseases. The esophagus is a hollow, muscular tube connecting the throat to the stomach, located behind the trachea and in front of the spine. The adult esophagus is typically 25–33 cm long. Although the esophagus is a digestive organ, most of it is situated in the chest, necessitating thoracic access during surgical interventions. When esophageal defects occur, replacement with conduits from the stomach, small intestine, or large intestine—located in the abdomen—becomes necessary. Consequently, postoperative morbidity and mortality rates are high [5,6]. Various strategies, including the development of an artificial esophagus, are being explored to reduce these risks, necessitating further animal studies [7,11,15].

In this study, rabbits were used as an animal model for esophageal surgery. We selected rabbits because they are easy to handle, relatively inexpensive, and easy to procure. In addition, the size of an adult rabbit is comparable to that of a neonate. Rabbits have been extensively used as surgical models, particularly for pediatric gastrointestinal tract surgery, including procedures involving the esophagus [13,19,20]. Immediately after surgery, the animal faces a high risk of certain complications, such as leakage at the esophageal anastomosis site; thus, it is not possible to feed rabbits the usual solid chow.

Moreover, necrotizing enteritis or sepsis due to bacterial overgrowth can occur in rabbits even during short fasting periods, potentially resulting in sudden death [21,22]. Prolonged fasting causes malnutrition and electrolyte imbalance. To provide nutritional support, a balanced liquid feeding formula is essential to minimize fasting and promote the healing process following esophageal surgery in rabbits.

In previous studies, rabbits were fed parenterally for 5 to 7 days without oral intake [15,23]. Another report described feeding rabbits a liquid diet for the first 3 days postoperatively, but it lacked detailed formula information [16]. In our study, the rabbits were fed a liquid formula (grated carrot juice mixed with Gatorade, ratio 3–5:1) orally after surgery, without leakage. On the seventh day after surgery, endoscopy and a mobile C-arm fluoroscopic X-ray examination were performed to assess complications at the anastomosis site. Solid food was introduced afterward.

The endoscope had a diameter of 5.6 mm and was used to directly inspect the condition of the anastomosis site under intravenous anesthesia without sacrificing the animal. Esophagography has been frequently performed in various studies, typically employing barium as the contrast medium [14,24,25]. Gastrografin is water-soluble and relatively safe in case of leakage compared to barium, but its osmotic pressure (1,900 mOsm/L) is higher than plasma, potentially causing acute pulmonary edema upon aspiration. In this study, these complications were prevented by adjusting the catheter tip position under fluoroscopic X-ray guidance and administering only the appropriate amount of diluted gastrografin.

The strengths of this study are as follows. First, it provides a specific postoperative feeding formula based on the time after esophageal surgery in a rabbit experimental model. Most prior research has only mentioned the type of diet, such as free water, liquid diet, or solid diet, without detailing the composition. Second, this study introduces a method to evaluate the status of the esophageal anastomosis after surgery without euthanizing the rabbit. Using endoscopy and esophagography with a mobile C-arm fluoroscopic X-ray system, the anastomosis site can be quickly, easily, and noninvasively examined. Thus, experiments can continue without euthanasia, reducing the required number of experimental animals. We believe that the detailed description of this novel method will greatly assist researchers planning esophageal experiments in rabbits.

This study had several limitations. First, the number of animals used to establish the sham model was small. Second, the early mortality and stenosis rates were high. Although no leakage was observed, two cases of stricture were noted among surgery-related complications, both at the beginning of the experiment. To prevent stricture, the esophagus should be minimally manipulated during surgery. It is particularly essential to dissect as little as possible for the anastomosis because the esophagus lacks a primary blood vessel, receiving its blood supply from the inferior thyroid artery, a small branch of the thoracic aorta [19]. Therefore, the more small vessels sacrificed in tissues surrounding the esophagus, the greater the tissue atrophy and fibrosis due to decreased blood supply at the surgical site. We proceeded with caution during the later phase of the experiment to reduce the risk of developing strictures. As mentioned earlier, experiments on transplanting artificial esophageal structures using this rabbit model are ongoing. The success of these experiments will mark the beginning of a journey toward artificial esophageal transplantation in humans.

In this report, we have detailed the surgical technique, dietary protocol, and postoperative evaluation methods for rabbits as a sham animal model for cervical esophageal surgery. These methods are considered feasible and reliable.