Ethical statement
The ethics committee of Xi’an Jiaotong University approved this study (permit number: 2018-001). The research protocol and all experimental procedures were strictly in accordance with the Guidelines for the Care and Use of Experimental Animals issued by the Xi'an Jiaotong University Medical Center.
Study design
Eighty-four Sprague-Dawley albino rats (weight, 200 – 250 g) were bought from the Experimental Animal Center, College of Medicine, Xi’an Jiaotong University (Xi’an, China). The animals were acclimatized to laboratory conditions (23°C, 12h/12h light/dark, 50% humidity, ad libitum access to food and water) for one week prior to commencing the experiments. The animal protocol was designed to minimize discomfort to the animals. The rats were randomly divided into 14 groups (n = 6 per group). Groups were named according to the postoperative day (POD) on which they were sacrificed (D1, D3, D5, and so on up to D27). All rats underwent colonic MCA and this was an exploratory study. Therefore, all animals were included in the experimental investigations, and there was no control group. Intramuscular injection of pethidine hydrochloride (1 mg/kg) was administered every 12 hours for analgesia for three days after surgery. At the end of the study, all animals were euthanized by a barbiturate overdose (intraperitoneal injection, 150 mg/kg pentobarbital sodium) for tissue collection.
Magnetic anastomosis device
The magnetic anastomosis rings were designed according to the anatomical characteristics of the rat colon. The thickness, external diameter, and internal diameter of the magnetic anastomosis rings were 1 mm, 8 mm, and 5 mm, respectively (Fig. 1). The rings were made of neodymium-iron-boron (grade N45) and were plated with zinc to increase resistance to erosion. Each ring weighed 0.2 g and had a magnetic density of 0.26 T.
Surgical procedures
After being fasted for 1 day, the rats were weighed and then anesthetized using an intraperitoneal injection of pentobarbital sodium solution (0.1 mL/100 g). After confirming that the paw withdrawal reflex was absent, the animal was fixed in the supine position. Sterile surgical instruments were used throughout the procedure. The abdominal region of the rat was shaved, and a 3-cm-long midline abdominal incision was made. The end-to-end colonic anastomosis was performed using the magnetic anastomosis rings. The operation was carried out as described in our previous study (Fig. 2)13.
Postoperative care
All rats were maintained in a single cage after surgery. After recovery from anesthesia, they were allowed only liquids and no standard chow. During the first three PODs, the rats were fed a liquid diet, and thereafter received normal chow. The rats’ general condition, eating habits, and magnet discharge time were observed daily.
The rats were sacrificed on the PODs corresponding to their group name to obtain the anastomotic specimens. For example, rats in the D1 group were sacrificed on POD1, those in the D3 group were sacrificed on POD3, and so on. The specimens were tested for burst pressure. The gross and histological changes at the anastomotic site were also studied.
Measurement of burst pressure
The rats were euthanized using high-dose barbiturates. The burst pressure of the colonic anastomosis was measured using a pressure gauge. An 8-cm colonic segment containing the anastomotic site was resected. The proximal end of the colon was clamped using hemostatic forceps. A catheter was introduced through the anus, and a single silk suture was used to securely ligate the anus around the catheter. The entire colon specimen was completely immersed in 0.9% saline. The colonic pressure was gradually increased by pumping air into the catheter. The pressure at which the first air bubble escaped from the anastomotic site was recorded as the burst pressure.
Specimen collection and histological analysis
All anastomosis-bearing colonic segments having sufficient length on either side of the anastomosis were harvested. After gross examination, all samples were immersed in 10% buffered formalin overnight. The fixed colonic segment was paraffin embedded, sliced into 4-μm-thin sections at the anastomosis site, stained with hematoxylin and eosin or Masson trichrome stain, and examined using bright-field microscopy.
Statistical analysis
Data were analyzed using the SPSS statistical software package (v20.0). All quantitative data are presented as mean and standard deviation. The independent-sample t-test was used to compare the groups. Differences were considered to be statistically significant at P value of < 0.05.