Experimental animals
We used Forty New Zealand rabbits of either sex, (4 ± 0.5) months of age and weighing (3.0 ± 0.5) kg, provided by the Laboratory Animal Center of Guizhou Medical University, with the license number: SCXK-2018-001. Rabbits were housed in individual cages at a temperature between 17 °C to 19 °C with humidity of 40–60% on a 12-hour light/dark cycle and fed a normal chow diet. The animal model research was reviewed and approved by the Medical Ethics Committee of Guizhou Provincial People's Hospital (Ethical Approval No.: 2017059). All experimental procedures were conducted in accordance with the 1996 revision of the Guide for the Care and Use of Laboratory Animals issued by the National Research Council Laboratory Animal Resources Institute. Rabbits were randomly divided into the calculi group and the sham control group, 20 rabbits in each group.
Experimental procedures
In the calculi group, the experimental rabbits were fasted before surgery but allowed to drink water overnight. After being weighed, each rabbit was anesthetized by intravenous injection of sodium pentobarbital (30 mg/kg, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). The rabbit was placed on the operating table in the supine position. Skin in the left lower abdomen was shaved and disinfected with iodophor. The surgical area was draped with a sterile surgical towel. A 5 cm incision was made in the left lower abdomen and the subcutaneous tissue, muscle, and peritoneum were incised. The colon and small intestine were mobilized to expose the left kidney and ureter. We selected the ureter at a distance of 4 cm from ureteropelvic junction and gently lifted 1 cm ureter with hemostatic forceps. The intravenous infusion needle with a diameter of 0.45 mm (needle tip towards the kidney) was inserted into the ureteric lumen. Then we injected 0.1 ml flowable resin (FiltekTM Z350 XT Flowable Restorative) (Fig. 1A). Care was taken throughout to minimize damage to ureter. Flowable resin in the ureter was light-cured for 40 sec by means of a dental curing light(R&D, IvoclarVivadent AG, Schaan, Principality of Liechtenstein, 2013)operated at 550 mW/cm2 and subsequently hardened to form stones (Fig. 1B). We took photographs and recorded the position of the stones. After ensuring no incidence of urinary leakage, bowel was repositioned and the abdomen was closed with 3-0 resorbable sutures.
The sham control group was injected with the same volume of saline, and the remaining operations were the same as the calculi group. Prophylactic antibiotics were given preoperatively and intramuscular injections of antibiotics were given for 3 days after surgery. All operations were performed by the 2 senior urological Surgeons. The operation time per animal did not exceed 20 minutes. The calculi group was randomly divided into 1st day (A), 3rd day (B), 5th day (C), 7th day (D) postoperatively according to the number of postoperative days. Similarly, the sham control group was randomly divided into 1st day (E), 3rd day (F), 5th day (G), 7th day (H) postoperatively. Each group has 5 rabbits.
Preoperative and postoperative treatment
Before surgery, when the rabbits were anesthetized, the chest X-ray examinations were performed on the lower abdomen by a small animal DR machine. Five from all rabbits were randomly selected to obtain blood from the vein of rabbit ear margin and urine collected by bladder massage by bladder massage. At the end of the surgery, the X-ray examinations were again taken.
At 1, 3, 5 and 7 days after surgery, rabbits in each group were anesthetized by intravenous injection of sodium pentobarbital, and X-ray examinations were conducted in rabbits of the lower abdomen, respectively. We observed whether stone locations had changed by comparing two postoperative imaging results. In Group D, high-resolution computed tomography (CT) was performed on two randomly chosen rabbits to observe the locations of calculi from different angles (axial, coronal, and sagittal). Subsequently, rabbits were euthanized by overdose of intravenous pentobarbital sodium. Kidneys and ureters were dissected and exposed through the median abdominal incision. Then they were photographed and recorded. Blood and urine samples from all rabbits were collected. Kidneys and ureters were removed from the ribbits. We measured the long-diameter of the kidneys bilaterally and renal pelvic width.
Histopathology assessment
The bilateral kidneys and ureters were fixed in 4% paraformaldehyde, dehydrated with alcohol, embedded in paraffin, sectioned, stained with hematoxylin and eosin. Pathological changes were observed under a microscope. The extent of renal tubule damage was recorded using a renal histopathological scoring system as follows: 0, normal; 1, areas of tubular epithelial cell swelling, vacuolar degeneration or necrosis involving < 25% of cortical tubules; 2, similar changes involving > 25% but < 50% of cortical tubules; 3, similar changes involving > 50% but < 75% of cortical tubules; and 4, similar changes involving > 75% of cortical tubules. The mean score of each successive field (magnification × 200) was taken as the renal histopathological score of that kidney section.
Blood and urine routine examination
Blood parameters including white blood cell (WBC), blood urea nitrogen (BUN) and serum creatinine (Scr). Urine parameters including urine red blood cell (URBC) and urine white blood cell (UWBC).
Statistical analysis
All data were expressed as mean ± standard deviation. The statistical analysis of the experimental results was conducted with the software SPSS 19.0(SPSS Inc., Chicago, IL, USA), and presented with GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, United States). The difference between groups was statistically significant (P < 0.05).