A total of 106 female Sprague–Dawley rats (weight of 200±20 g) purchased from the Guangdong Medical Laboratory Animal Center (www.gdmlac.com.cn) were used in this study. The animals were raised in the Laboratory Animal Center of the Guangdong Pharmaceutical University at a temperature of 21ºC±2ºC for a week to adapt to the Specific-pathogen-free(SPF)laboratory environment, and the whole experiment lasted for four months in the same environment. During the study, the animals were fed a standard pellet diet and water. At the end of the study, all rats were anesthetized and decapitated without suffering[21]. The study respected the limited use of animals in line with the “three-R” system (replacement, reduction and refinement) and was approved by the Animal Experiments Ethical Committee of Guangdong Pharmaceutical University (identification no. SPF2017033). All treatments were randomized but did not involve blinding. All animal experiments were established using the ARRIVE guidelines and carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986 and associated guidelines, the European Union Directive 2010/63/EU for animal experiments, and the National Institutes of Health guidelines for the care and use of laboratory animals (NIH publications no. 8023, revised in 1978) where appropriate.
Study design: Female rats were chosen in order to achieve better instillation via the urethra. The whole period of the experiment was divided into two parts: the tumor establishment and treatments (Figure 1).
Sample size: The rats were randomly divided into five groups: (1) control group, where 24 rats were instilled only with 0.9% normal saline (NS) in the bladder; (2) experimental groups, where 82 rats were instilled with N-methyl-N-nitrosourea (MNU; Sigma-Aldrich, St. Louis, MO, USA) in the bladder to establish the orthotopic bladder tumor model. 10 rats of the bladder tumor model were subjected to the positive control, while another 72 rats were treated with EPI (n=24), EPI+BCG (n=24), and EPI +CpG-ODN (n=24) via bladder instillation.
Inclusion and exclusion criteria: One death by hyperanesthesia occurred in the 7th week was exclusion.
Randomisation: 106 female Sprague-Dawley rats, were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and randomly divided into five groups: (1) control group (24 rats); (2) experimental groups: positive control (10 rats), EPI group (24 rats), EPI+BCG group (24 rats), and EPI +CpG-ODN group (24 rats) via bladder instillation.
Blinding
Data were coded prior to analysis during the whole experiment.
Outcome measures
Tissue sections embedded in paraffin blocks were cut with thicknesses of 4 µm and then stained with hematoxylin and eosin. According to the World Health Organization (WHO) criteria[28] and Koss’ Diagnostic Cytology and Its Histopathologic Bases, pathological changes in the bladder tumors were confirmed. The serum IL-2 was assessed using an enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Eiaab Science Co. Ltd., Wuhan, China).
Statistical methods
All data were analyzed by a single-factor variance analysis t test using the Statistical Package for the Social Sciences (SPSS) version 19.0 software program (IBM Corp., Armonk, NY, USA), and p < 0.05 was used to indicate statistical significance.
Experimental animals
106 female 6-week Sprague-Dawley rats were used with weight of 200–250g.
Experimental procedures
All of the rats in experimental groups had tumors induced by MNU in the 1st, 3rd, 5th, and 7th weeks, while the rats in the blank control groups were instilled with 0.9% NS. In the 8th week, all rats in the positive control group were sacrificed to evaluate the success of tumor establishment. In the 9th, 11th, 13th, and 15th weeks, animals were treated with EPI, EPI+BCG, EPI+CpG-ODN, or 0.9% NS as a blank control separately and sacrificed in the 10th, 12th, 14th, and 16th weeks. After all the experiments were completed, the rats were anesthetized with chloral hydrate, and then their necks were severed to death. After completion of blood specimen collection and autopsy, the rat carcass were placed in a designated place and processed by a special department.
After overnight balancing in refrigerator at 4°C, 2 mg of MNU was dissolved in 0.2ml of citric acid buffer (pH: 6.0) for instillation per individual. All of the reagent mixture was used within 40 min. The novel CpG-ODN with 72-base sequence (5’-AACGTTGTCGTCGACGTCGTCGTCAGGCCTGACGTTATCGATGGCGTTGTCGTCAACGTTGTCGTTAACGTT-3’) [14, 22], which was designed by our laboratory and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).
All rats were fasted for 12 hours and then intraperitoneally injected with 10% chloral hydrate for anesthesia. After general anesthesia, the urethral orifices of the rats were sterilized by 75% ethanol application twice, then the disposable epidural catheters were inserted and 0.1ml of MNU was instilled into the bladder using an injection syringe. Metal clamps were used to fix the epidural catheters into the animals to ensure that the reagents were kept in the bladders for one hour. The posture of animals was changed every 30 min. Generally, after one hour of the instillation, spontaneous micturition of the rats was observed. Rats in the experimental groups (n=82) received MNU instillation for four times (on the first day of the 1st, 3rd, 5th, and 7th weeks, respectively). Meanwhile, 24 rats (control group) were instilled with 0.9% NS (Fig. 1). All of the procedures were conducted with reference to prior research[23–27]. On the last day of the 8th week, nearly all rats in the positive control group (n=9; one death by hyperanesthesia occurred in the 7th week) were sacrificed, and the whole blood was collected from the eye sockets, followed by the performance of cystectomy to collect the whole bladders. The whole blood was clotted by centrifuge tubes at 4°C overnight, and the serum samples were separated. The bladders were dissected by longitudinal sectioning to calculate the tumor incidence. All bladder specimens were placed in 4% paraformaldehyde to prepare for hematoxylin and eosin and immunohistochemistry examination. Additionally, on the first day of the 9th week, 72 animals among the experimental groups were instilled with EPI (n=24), EPI+BCG (n=24), or EPI+CpG-ODN (n=24), and the blank control group (n=24) was instilled with 0.9% NS (Fig. 1). On the last day of the 10th week, 18 rats from EPI group (n=6), EPI+BCG group (n=6), and EPI+CpG-ODN group (n=6) and 6 rats in the control group (n=6) were sacrificed to collect blood and bladder samples as described above. Similarly, on the first day of the 11th, 13th, and 15th weeks, remaining animals in the experimental groups were instilled with EPI, EPI+BCG, or EPI+CpG-ODN, respectively, and those in the blank control group were instilled by 0.9% NS. Then, on the last days of the 12th, 14th, and 16th weeks, these rats of the experimental groups and blank control group (n=6 each group) were sacrificed to collect samples as outlined above.
Tissue sections embedded in paraffin blocks were cut with thicknesses of 4 µm and then stained with hematoxylin and eosin. According to the World Health Organization (WHO) criteria[28] and Koss’ Diagnostic Cytology and Its Histopathologic Bases, pathological changes in the bladder tumors were confirmed. The tumor grades were as follows: no carcinoma (grade 0), slight hyperplasia (grade 0.5), light dysplasia (grade 1), moderate hyperplasia (grade 2), high dysplasia (grade 3), papillary carcinoma (grade 4), and invasive cancer (grade 5).
Slices were deparaffinized in xylene, hydrated through a series of graded alcohol, washed in distilled water and 0.01 M phosphate-buffered saline, immersed in citrate buffer (pH: 6.0), and put in a microwave for 5 min at 60°C for antigen retrieval. Then, they were placed in methanol containing 3% H2O2 for 10 min to block endogenous peroxidase activity and incubated with 1% bovine serum albumin for 30 min to block nonspecific antibody-binding sites. p53 antibody (Abcam, Cambridge, UK) was diluted into a working concentration (1:500) with 0.01 M of phosphate-buffered saline (pH: 7.2), and then slices were incubated at 4°C overnight. The slices were incubated with secondary antibody for 1 h, then diaminobenzidine was used as a chromogen, and the slices were counterstained with Mayer’s hematoxylin. p53 resides in the nucleus, and positive staining appears brown. The results of expression were determined by the summation of immunostaining intensity and score in the positive ratio of cells[29] as follows: (1) immunostaining intensity: no staining (grade 0), faint staining (grade 1), medium staining (grade 2), or strong staining (grade 3); (2) positive ratio of cells: ≤ 5% (grade 0), 5–25% (grade 1), 25%−50% (grade 2), 50%−75% (grade 3), or ≥ 75% (grade 4); and (3) the result of summation: grades 0−1 indicate negative (−), grades 2−3 indicate positive (+), grades 4−5 indicate medium positive (++), and grades 6−7 indicate strong positive (+++). The images were collected by BIOQUANTOSTEO 2009 (n = 5 each slice), and the intensity was calculated using the Image-Pro Plus 6.0 program as mean ± standard deviation.
The serum IL-2 was assessed using an enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Eiaab Science Co. Ltd., Wuhan, China) following the manufacturer’s protocol. The intensity was detected as indicated above.