This study was approved by the Kobe University Animal Experiment Committee (approved on October 23, 2017, No. P151004). Male C57BL/6J mice (4 weeks old; body weight, 16-18 g) were purchased from Japan SLC (Shizuoka, Japan). The animals were maintained in a temperature- and humidity-controlled room (22-25℃, 50-60%) on a 12-hour light-dark cycle. They had free access to normal water and normal diet. The study flow is summarized in Figure 1.
Mice with streptozotocin-induced chronic diabetes
We prepared streptozotocin (STZ) (Wako Pure Chemical Industries, Osaka, Japan) dissolved in sodium citrate buffer (pH 4.5). At the age of 5 weeks, mice were intraperitoneally administrated STZ (50 mg/kg body weight) for 5 consecutive days to induce diabetes according to prior studies by Frisch et al. and Jackson et al. [11, 12]. For non-diabetic controls, mice were administered only the buffer at the age of 5 weeks.
Diabetic mice and non-diabetic mice were fed a normal chow diet for 8 weeks. We measured blood glucose levels in tail vein blood using a glucometer (Glutest Neo alfa®, Sanwa Kagaku Kenkyusho, Japan) every 2 weeks. We defined the development of diabetes as random blood glucose level ≧ 300 mg/dL (17 mM) [13] at the age of 14 weeks. We also measured body weight every 2 weeks.
Insulin therapy
We divided the diabetic mice into three groups (Figure 1). Neutral protamine Hagedorn (NPH) insulin (Humulin N; Eli Lilly, Indiana, USA) was injected subcutaneously to maintain the blood glucose level below 200 mg/dL using an insulin sliding scale (supplemental file 1). Insulin was injected at 6 hours before the operation in the short-term groups, and insulin was injected every 12 hours for 5 days before the operation in the long-term group. Determination of the dose of insulin and measurement of blood glucose level were performed before and 6 hours after insulin administration during the period of insulin therapy.
Daily insulin sensitivity factor (ISF) as a surrogate of insulin sensitivity [14], which is the drop in blood glucose caused by 1 unit of insulin, was calculated by using the following formula [15]: ISF ( mg・dL-1・U-1) = change in blood glucose (mg/dL) / amount of insulin (U). A group without any insulin therapy before the operation was used as a control group.
Surgical procedure
We performed intestinal manipulation in each mouse at 14 weeks of age under general anesthesia with 3.5% sevoflurane and air, as shown Figure 2. Each mouse was placed in the supine position on a heating pad (37℃) during the procedure and shaved the hair (a). After injection of 1% lidocaine (Maruishi Pharmaceutical, Osaka, Japan), a vertical incision of 0.5 cm in length was made in the middle of the abdomen (b-1, b-2). The small bowel luminal contents were moved by using two moist and sterile cotton sticks from the pylorus to the cecum [16] (c). The surgical wound was closed with 5-0 nylon (Natume Seisakusho Co., Tokyo, Japan) (d-1, d-2). After the surgical procedure, EMLA® cream including 2.5% lidocaine and 2.5% prilocaine (Sato Pharmaceutical Co, Tokyo, Japan) was applied to the surgical site for analgesia. The animals were placed under a heating lamp until recovery from anesthesia. After completing the experiment, mice were euthanized by cervical dislocation.
Analysis of neutrophil function
Neutrophil functions were examined before and 24 hours after surgery by using two assays. A neutrophil phagocytosis assay was carried out by using fluorescently labeled microspheres. Briefly, neutrophils were isolated from heparinized peripheral blood by centrifugation after lysis of red blood cells (RBC) with lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 100 μM Na2EDTA). The cells were resuspended with RPMI1640 (Gibco® Carisbad, CA, USA) and incubated with Fluoresbrite® Polychromatic Red Microspheres (particles of 2.0 µm in diameter, 2.0 x 107 particles/mL, Polysciences, Inc. Warrington, PA, USA) in RPMI1640 for 2 hours in 5% CO2 at 37 ℃. After centrifugation and aspiration of microspheres that were not phagocytosed, the cells were resuspended with FACS buffer (PBS (-) including 100 U/ml penicillin, 100 μg/ml sterptomycin, 2% FBS, and 2 mM Na2EDTA). After incubation with purified anti-CD16/CD32 antibody (Biolegend®, SanDiego, CA, USA) for 10 minutes for blocking Fc receptors, cells were incubated with Pacific Blue™ anti-mouse Ly-6G antibody (Biolegend®, SanDiego, CA, USA) for 30 minutes on ice in the dark, and washed with FACS buffer. We analyzed the Ly-6G-positive cells with fluorescence of microspheres by using FACSVerse™ with BD FACSuite™ software (BD Bioscience, San Jose, CA, USA). Neutrophil phagocytosis rate was calculated by using following formula:Phagocytosis rate (%) = number of neutrophils with positive fluorescence of microspheres / total number of neutrophils × 100.
An assay of reactive oxygen species (ROS) generated by neutrophils was carried out according to a previous study [17]. Heparinized peripheral blood was incubated for 30 minutes at 37 ℃ with 5 μM 2’, 7’-dichlorofluorescin-diacetate (DCFH-DA, Merck KGaA, Darmstadt, Germany). Blood samples were incubated with phorbol myristate acetate (PMA: 25 μg/ml, Wako Pure Chemical industries, Osaka, Japan) for 30 minutes at 37 ℃ to stimulate neutrophils. Then samples were placed on ice to stop reactions. After lysis of RBC, neutrophils were isolated from blood samples, and were incubated with purified anti-CD16/CD32 antibody for Fc block for 10 minutes and with Pacific Blue™ anti-mouse Ly-6G antibody for 30 minutes on ice to stain neutrophils. Green fluorescence intensity of 2', 7'-dichlorodihydrofluorescein (DCF) in the Ly-6G-positive cells was measured using a FACSVerse™. The results are shown as mean fluorescence intensity (MFI).
Statistical analysis
Data are shown as median values with interquartile range (IQR). A comparison between two groups was performed using the Mann-Whitney U test for unpaired data and Wilcoxon’s signed rank test for paired data with Prism 8 (GraphPad Software, San Diego, USA).
Our primary null hypothesis was that insulin treatment may not alter the neutrophil functions of diabetic mice. Therefore, we considered the untreated group as a reference for comparisons among 4 groups. Considering the bias of multiple comparisons (3 times), a p-value < 0.0167 was considered to indicate statistical significance for the analysis of blood glucose levels and result of neutrophil function tests. For comparisons of insulin demand and ISF value, we considered the value on the first day in the long-term group as a reference. For this analysis, a p-value < 0.01 was considered to indicate statistical significance for adjusting the bias of multiple comparisons (5 times).
To calculate the sample size for the current study, we considered on absolute difference of 10% in the phagocytosis rate and 500 in ROS to be meaningful. Assuming standard deviations of 8% for phagocytosis rate and 250 for ROS, an α level of 0.0167 and a power of 0.80, approximately 15 mice and 6 mice were required in each cohort.