|Year : 2017 | Volume
| Issue : 2 | Page : 66-69
Surgical technique for orthotopic small bowel transplantation in a rat
Department of General Surgery, Near East University, Nicosia, Cyprus
|Date of Web Publication||12-Sep-2017|
Department of General Surgery, Near East University, Nicosia
Source of Support: None, Conflict of Interest: None
Objectives: Presently, the results of small bowel transplantation (SBT) procedures are far from ideal. Problems such as rejection, sepsis and graft-versus host disease are still encountered after SBT. Here, we describe a refined rat model of SBT for researchers who wish to investigate these complications. Materials and Methods: Sixty male outbred Sprague Dawley (SD) rats were used for the study. Orthotopic small bowel transplantations were performed. An immunosuppressive regimen was not implemented and the recipients were monitored for seven days. If the recipient survived until the experimental end point, the transplantation was deemed to be successful. Recipient survival shorter than seven days was considered to be a technical failure. Results: The mean operation times for donor and recipients were 40 minutes (40 ± 5 min) and 65 minutes (61 ± 10), respectively. Cold and warm ischemia times were 32.5 ± 5 min and 25 ± 7 min, respectively. End-to side venous and arterial anastomosis times were approximately 20 minutes (20 ± 7 min). Four recipients died within the first three days after transplantation due to technical failure [bleeding (n:3), microvascular anastomosis thrombosis (n:1)]. The rest of the recipients (n: 26) reached the experimental end point in a healthy condition (success rate was 86%). Conclusion: This model is easy to perform and offers various advantages. The principle advantages of our surgical model are that it minimizes the mechanical injury of the graft and provides a high quality intestinal graft. It also allows us to make rapid and safe anastomosis in the recipient operation.
Keywords: Organ transplantation research, rat model, small bowel transplantation
|How to cite this article:|
Özçay N. Surgical technique for orthotopic small bowel transplantation in a rat. Indian J Transplant 2017;11:66-9
| Introduction|| |
Animal models have lost their popularity in organ transplantation research, mainly because most of the technical and immunologic problems have already been solved. Furthermore, animal models are difficult to perform, require fine equipment, qualified personnel as well as expensive animals. They are also time-consuming. Therefore, most researchers now mainly prefer molecular-based studies for their transplantation research.
Solid organ transplantation is a clinical reality for liver, kidney, and heart transplantations in particular. The short- and long-term results of these organ transplantations are satisfactory. A decrease in the number of animal-based studies for these organ transplantations is therefore inevitable. Small bowel transplantation (SBT), however, is still in its experimental phase and unfortunately has not become a clinical reality. Both the short- and long-term results are far from ideal. Problems such as acute and chronic rejection, infection, and graft versus host disease are frequently observed after SBT.,, The mechanisms of these complications are not completely understood and treatments are not standardized. Therefore, unlike other solid organ transplantations, animal studies are required in the field of SBT.
The rat SBT model was first developed by Kort et al. with a relatively high mortality rate and was later modified and improved by Schroeder et al. and Zhong et al. These models have long been used globally in various SBT studies.,,,, Here, we describe the surgical technique of our refine rat model of orthotopic SBT.
| Materials and Methods|| |
This study was approved by the Baskent University Ethical Committee for Experimental Research on Animals (project no: DA13/02) and supported by the Baskent University Research Fund.
Sixty male outbred Sprague-Dawley rats weighing 250–300 g were purchased from the experimental animals breeding center at Baskent University in Turkey. The animals were housed in the BaskentX University Experimental Research Center (Turkey) in accordance with the guidelines established by the Turkish Government. The donor animals received 5% glucose and 0.9% saline orally ad libitum for 6 h before surgery. The recipients' access to food and water was not restricted. The rats were anesthetized with an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg).
A surgical model of orthotopic (incontinuity) SBT was performed. The intestinal graft was anastomosed to the remaining section of the native small bowel after resection of the recipient's jejunum. Host survival is dependent on the graft function in this technique.
Preoperative care and anesthesia
The donor animals received 5% glucose and 0.9% saline orally ad libitum for 6 h before surgery. Access to food and water was not restricted.
The abdomen was opened through a midline incision. An operating microscope (Carl Zeiss OPMI 9-FC, Germany) was used to mobilize the graft. Direct manipulation of the intestinal graft was minimized to avoid graft damage. A wet cotton swab was used when necessary to handle the graft.
Removing the colon and duodenum
Donor operation was initiated by colon dissection. After the abdomen was opened, the left and transverse colon was dissected to free them from their peritoneal attachment. Dissection continued to the right side, mobilizing the caecum along with the terminal ileum. The rectum was left intact. The colon and ileum were cut above from the rectum distally and from the jejunum proximally. Then, the entire colon along with the terminal ileum was removed from the abdomen. After colectomy, the duodenum dissection began. The whole duodenum and 2 cm of the proximal jejunum's mesentery were disconnected from the surrounding pancreas tissues and from the portal vein. The jejunum was cut close to the duodenum and the whole duodenum was removed from the abdomen. The graft small bowel was then covered with warm wet gauze throughout the operation.
Preparing the artery and the vein
The superior mesenteric artery along with a section of the aorta was used for arterial access. A long segment of the aorta containing the superior mesenteric artery was mobilized by ligating and dividing the right and left renal and lumbar arteries. It was not necessary to ligate the left renal artery if its origin was far from the superior mesenteric artery. The lumbar arteries from the aorta were meticulously ligated with 8-0 silk sutures. The connective tissue between the celiac artery and the superior mesenteric artery is avascular and could be directly cut with a pair of fine scissors. In this way, a long segment of aorta was obtained without ligating the celiac artery. The portal vein was prepared for venous access. Two branches of the portal vein, the pyloric and splenic veins, were ligated and divided, respectively, with 7/0 silk sutures. These two knots were used as a guide to correctly position the graft intestine during the recipient operation. It is important to ligate those two branches at the end to maintain blood pressure in a normal range. Finally, the portal vein was dissected up to the bifurcation of the vein.
In situ perfusion of the graft
After arterial and venous dissections, 1 ml cold heparin was given through dorsal penile vein to heparinize the animal. A 30-gauge needle was then introduced into the aorta and the graft was perfused in situ with 10 ml of cold, University of Wisconsin (UW) solution. After dividing the portal vein close to the hilum of the liver and the aorta, the graft was removed en bloc from the abdomen and stored in lactated Ringer's solution at 4°C.
Revascularization of the graft
The abdomen was opened through a midline incision. The native intestine was left intact. The recipient's infrarenal aorta and inferior vena cava (IVC) were carefully mobilized and isolated using a vascular bulldog clamp. First, a longitudinal venotomy was made by puncture with a 30-gauge needle. Two 9-0 nylon stay sutures were placed at both corners of the venotomy. The donor small intestine was then removed from the ice and placed in the right side of the rat's abdomen. After ensuring that the donor portal vein was not twisted, an end-to-side anastomosis was performed using a continuous 9-0 nylon suture. The posterior wall was anastomosed from within the vessel lumen without repositioning the graft. The anterior wall of the portal vein was anastomosed externally using the same suture. Saline irrigation was used to keep the vessel walls apart during the anastomosis. Before tying the sutures, the vein was gently pulled apart to avoid any collapse or narrowing of the vessel at the anastomotic site.
It took approximately 10 min to complete this anastomosis since only 5–6 stitches were required for each side. Immediately below the venotomy, the aorta was punctured using a 30-gauge needle and opened through a longitudinal arteriotomy. The lumen was flushed with a heparinized saline solution. Two 9-0 nylon stay sutures were placed at both apices of the aortotomy. An 8-0 nylon stay suture was also placed at the midpoint of the aortotomy on the left side to act as a self-retaining retractor. The posterior wall was firstly anastomosed from within the vessel lumen using a continuous 9-0 nylon suture. The anterior wall of the aorta was anastomosed externally using the same suture. In general, four to five stitches were needed for each side. It was important at this point to ensure that each stitch passed through all layers of the arterial wall. The graft was rinsed with cold saline solution several times during the anastomosis. The clamp was slowly released after completion of the anastomosis. Absorbable SURGICEL ® (Johnson and Johnson Medical Ltd, UK) was placed around the anastomosis before releasing the clamps. Gentle pressure was applied to the anastomosis with a dry cotton swab for 1–2 min after revascularization.
Orthotopic replacement of the intestinal graft
The intestinal graft was anastomosed to the remaining section of the native small bowel after resection of the recipient's jejunum. Host survival is dependent on the graft function in this technique. The recipient's jejunum was removed leaving almost 10 cm of the native jejunum to be anastomosed with the graft. The intestinal anastomoses were performed using one layer of full thickness 7-0 Prolen ® continuous suture. Since most of the native intestine was removed, the length of the graft should be longer.
Postoperative care and follow-up
The recipient received a total of 6 ml of normal saline by intermittent injection in the dorsal penile vein during the procedure, including 3 ml of saline that was given before and after vascular clamping. After closing the abdomen, 15 ml of saline was given subcutaneously. Postoperatively, the rats were kept on a warm blanket under a heat lamp for the first night. They usually recovered from anesthesia within 1 h of the operation. The rats were given regular water and food ad libitum. Clinical examinations were performed daily for 7 days. Before sacrifice, the animals were anesthetized with ketamine (60 mg/kg ip), and laparotomies were performed to evaluate the abdominal cavity. The gross findings from the animals were evaluated macroscopically during necropsy.
During necropsy, 1 cm tissue segments from the middle of the intestinal grafts were dissected and fixed in 10% formalin at room temperature. The tissue specimens were embedded in paraffin and cut into 3 μm sections. The tissue sections were mounted onto glass slides and stained with hematoxylin and eosin. The graft histology was examined by light microscopy by an experienced pathologist under blinded conditions. The lamina propria, epithelium, and villus architecture of the grafts were carefully evaluated for the following features: mitosis, lymphocytic infiltration, cryptitis, loss of goblet cells, and sloughing of villus tips.
| Results|| |
Thirty orthotopic SBTs were performed for this study. The mean operation times for donor and recipients were 40 min (40 ± 5 min) and 65 min (61 ± 10), respectively. During both operations, approximately 6 ml of normal saline was given intravenously through dorsal penile vein to maintain normal blood pressure. The cold and warm ischemia times were 32.5 ± 5 min and 25 ± 7 min, respectively. End-to-side venous and arterial anastomosis times were approximately 20 min (20 ± 7 min).
Clinical course and survival
Since any technical problems such as bleeding or thrombosis occurred within hours of the operations, these animals usually died on early postoperative days. Therefore, we observed the recipients for 7 days to evaluate whether the procedure was technically successful or not. During this time, all animals were closely followed up in terms of their general condition and weight change. All of the surviving recipients lost 10%–20% of their body weight by the fourth postoperative day. They began to gain weight after this point. Four recipients died within the first 3 days after transplantation due to technical failure (bleeding [n: 3] and microvascular anastomosis thrombosis [n: 1]). The rest of the recipients (n: 26) reached the experimental end point in a healthy condition (success rate was 86%).
Gross findings at necropsy
There was no necrosis, adhesion, or inflammation in the grafts. Most of the grafts appeared normal in color or slightly pale. The walls of the grafts were relatively thin and lumens were minimally distended. The graft lumens were filled with intestinal fluid. Mesenteric lymph nodes of the grafts were normal in size or moderately enlarged.
There was a diversity in the histological changes of the allografts in our study. Analyzing all the criteria, 6 of the 26 surviving animals' grafts had no evidence of rejection. The rest of the grafts mainly demonstrated mild to moderate infiltration of lymphocytes and polymorphs. Mild cryptitis and goblet cell loss were other common features of these grafts. The villi structures of these grafts were generally well preserved although some had minimal mucosal destruction.
| Discussion|| |
SBT in rats is a complex procedure that requires a high level of microsurgery and animal handling experience. However, it could be performed with some practice using a good animal model. The small bowel is a vulnerable organ; it can easily be damaged by hypovolemia, hypothermia, or ischemia during the procurement process. Therefore, it is important to pay attention to prevent these situations to obtain a high-quality intestinal graft. A good quality donor intestine is critical for a successful SBT.
Care must be taken to control the animal's general condition, hypothermia, hypovolemia, and the handling of the graft intestine during the donor operation. Mechanical injury or ischemic injury could damage the graft. To prevent these complications, intravenous fluid replacement and minimal direct manipulation of the graft are necessary during donor operation. The No-Touch Technique was used to minimize the mechanical injury in our model. After the colon and duodenum are removed from the abdomen, the intestinal graft should be covered with a moisturized cotton gauze and should not be touched directly throughout the donor operation.
Procurement of the intestinal graft from the rat is a time-consuming procedure. It usually takes approximately 1 h and animals can easily be hypovolemic during the operation as the abdominal cavity is open and the colon and duodenum are removed. Hypovolemia has to be prevented because it impairs the graft quality. To maintain the health of the intestinal graft, we recommend that intravenous fluid is given during the operation. The animals should receive 10 ml/h of intravenous physiological serum solution. The solution should be given intermittently through the dorsal penile vein of the animal. After vascular clamping, another 2–3 ml of serum physiologic solution should be administered intravenously.
Animals should also receive saline solution subcutaneously after abdominal closure. Hypovolemic shock is the most frequent cause of mortality after SBT in rats. Intra- and extra-luminal isotonic fluid loss from the transplanted gut is the main reason for this complication. To avoid this complication, a large volume of fluid should be given during the surgery.
Early ligation of the pyloric and splenic veins during portal vein dissection causes splanchnic venous congestion, leading to shock, and ischemic injury to the intestinal graft. To avoid this complication, they should be ligated immediately before the in situ perfusion. It was also found to be beneficial not to ligate the celiac artery during the arterial dissection. In this way, the vital organs such as the liver remain viable, which helps the graft quality.
Intraluminal irrigation of the graft is usually not recommended since it can sometimes damage the microcirculation of the graft. However, we believe that intraluminal irrigation is necessary to prevent septic complications after SBT. Therefore, we recommend that the intestinal graft should be gently irrigated with 2–3 ml of cold physiological solution. The irrigation volume should be limited because large volumes could damage the graft's mucosa.
The small bowel graft is a large organ, and therefore, we increased the volume of the intravascular perfusate to 10 ml instead of 3-4 ml which was previously described. A large volume of perfusate has no adverse effect on the graft quality. We also chose to use the UW solution as a perfusate instead of Ringer's lactate since the UW solution keeps the graft viable for a longer period.
Vascular anastomosis of the intestinal graft is an important step for a successful intestinal transplantation. It should be performed as rapidly as possible due to cross-clamping of the aorta and IVC. Direct manipulation of the vascular intima should also be avoided to prevent thrombosis. We used a continuous vascular anastomosis technique with 9/0 Prolen ® sutures without changing the graft position. It allowed us to complete the anastomosis in a shorter time. With this technique, both anastomoses can be completed in <30 min. During the anastomosis, we did not touch the vascular intima to avoid postoperative thrombosis. The meticulous anastomotic technique with the aid of an operating microscope and 9-0 nylon sutures minimizes vascular complications.
Intravenous fluid replacement after the anastomosis is completed and the cross-clamp is released is necessary to prevent hypovolemia. It should be given through the dorsal penile vein of the animal. We recommend that a large volume (10–15 ml) of saline is given subcutaneously once the operation is completed.
Either heterotopic or orthotopic SBT models could be effective, depending on the nature of the study. Orthotopic placement is better than heterotopic placement for physiological studies since this technique provides a normal environment for mucosal enterocytes; however, it is technically more complex. Furthermore, graft failure leads to the animal's death, providing a well-defined, objective marker of impaired gut barrier and absorptive functions. Heterotopic placement is better for immunologic studies. It is technically easy and allows a biopsy to be taken from the intestinal graft.
In summary, we described here a refined SBT model in a rat with technical details. We believe it would be beneficial to those who would like to investigate SBT.
The author thanks to Prof. Dr. Handan Özdemir from Baskent University, Department of Pathology for reading the pathology slides. The author would also like to thank Mr. Simon Thompson from Near East University, for language editing and proofreading.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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