Animals
Four-week-old male C57BL/6J mice, weighted 15.7 (median)±2.7 g, were purchased from SLC Center Japan. We used male mice to avoid the influence of sextual cycle hormone in the behavioral experiments. The animals were group-housed 3 per plastic cage at our animal experimental center until the behavioral tests in a temperature-(24℃), humidity- and light-controlled room (12-hour light-dark cycle) with ad libitum access to water and rodent chow according to the Kobe University standard for animal welfare.
All animal experiments and procedures were performed in accordance with the national institutional guidelines for proper conduct of animal experiments and complied with the international guiding principles for biomedical research involving animals. This study was approved by the Kobe University Animal Experiment Committee (approved on 23th/October/2017, No. P151004).
Experimental protocol
The aim of the current study was to investigate the effect of surgical stress on T2DM model mice. Our experimental protocol is shown in Fig.1. Eighty-four mice were randomly divided into four groups of mice (n = 21 per group, control group, surgery control group, T2DM group, surgery T2DM group). Sample size was calculated with EZR [35] followed by the results of preliminary behavioral experiment of OF test by using five mice in each T2DM and surgery T2DM group. In each group, fifteen mice were used for the behavioral tests, and six mice were employed to examine the hippocampal NA level. The T2DM groups were fed HFD32 (32% fat content and 56.7% of fat kcal; CLEA Japan, Inc), and the control groups were fed normal rodent chow. Diet intervention was started at 6 weeks of age and lasted for 8 weeks until 14 weeks of age. As biophysical parameters, we investigated body weight every week and fasting blood glucose level every 2 weeks. Glucose tolerance evaluation by an intraperitoneal glucose tolerance test (IPGTT) was performed at 13 weeks of age and hemoglobin A1c (HbA1c) level was examined at 14 weeks of age. At 14 weeks of age, a series of behavioral tests was performed, and surgery was performed for the surgery groups 24 hours before the behavioral tests. After the behavioral tests, mice were euthanized by removing blood transcardially under deep general anesthesia with sevoflurane.
Surgical procedure
Open abdominal surgery was performed in surgical groups (surgery control and surgery T2DM group) according to the previously study [36]. The procedure was conducted at animal raboatory (at 8:00 a.m.). In detail, general anesthesia was induced with 3.5% sevoflurane in 50% oxygen in a plastic chamber and was maintained with 3.5% sevoflurane (covering 1 minimal alveolar concentration (1MAC) of adult C57BL/6J mice [37]) by using mask during the surgical procedure. The concentration of sevoflurane and oxygen in the inhalational anesthesia was continuously monitored by using anesthetic monitoring equipment (Datex, IMI Co., Ltd,). Each mouse was gently restrained to a heating pad (37℃) during the procedure. After shaving the abdominal incision area, sterilization was provided by povidone iodine. A 1-cm vertical incision was made in the middle of the abdomen. During the surgery, the whole small intestine was exteriorized from the peritoneal cavity, covered with moist gauze, and then manipulated with sterile cotton swabs for 1 minute. Afterward, the peritoneum muscle and skin were repaired separately with 5-0 nylon (Natsume Seisakusho Co, Japan). Total anesthesia time was 20 minutes for each procedure. After the closing incision and 8 hours later, EMLA® cream (2.5% lidocaine and 2.5% prilocaine) was applied to treat the surgery-associated pain [38].
Body weight and fasting blood glucose level
After food intervention, body weight was recorded every week and blood glucose level was examined every 2 weeks. The fasting glucose level was measured by Glutest Neo α (Sanwa Kagaku Kenkyusho, Japan) following 6 hours of fasting [39].
Intraperitoneal glucose tolerance test (IPGTT)
At 13 weeks of age, 7 weeks after food intervention, IPGTTs were performed. The animals were fasted for 16 hours prior to the glucose tolerance test. After measuring basal levels of blood glucose (0 minutes), each animal was injected with glucose solution (2 g glucose/kg body weight) intraperitoneally (i.p.), and blood glucose was measured at 15, 30, 60 and 120 minutes after the injection [39].
Hemoglobin A1c (HbA1c)
At 14 weeks of age, blood samples were collected from the tail vein and HbA1c was measured by using a glycated hemoglobin A1c kit (DCA 2000® HbA1c cartridge, Simens Healthineers Japan).
Behavioral tests
We performed a series of sequential behavioral tests to evaluate perioperative behaviors. For the surgical groups, behavioral tests were performed 24 hours after the open abdominal surgery. Every mouse was isolated in an individual cage and transferred to the test room for habituation one hour before the behavioral tests. The orders of mice for the test were in a random manner and the test chamber was cleaned after each session. Every behavioral test was automatically recorded. The behavioral tests were performed at 8:00 AM to 15:00 PM in the light phase. The details of the series of behavioral experiments are as follows.
Open field test (OF test)
An open field test was used for measuring locomotor activity and anxiety [40]. The open field apparatus was constructed of a gray acrylic plate of 60 × 60 cm in size with 40 cm walls. Each mouse was placed close to the wall of the apparatus and exposed to the novel environment, and activity was monitored for 5 minutes twice with a one-hour interval. Activity was automatically recorded by a video tracking system [41] and analyzed by using the SMART® system (Bio Research Center Corporation, Japan). Repeated exposure to the open field apparatus results in acclimation to the field for the novel objective recognition test [42].
Novel objective recognition test (NOR test)
The NOR test was performed to evaluate both explorative activity for objects and cognitive ability. One hour after the open field test, the NOR test was performed in the same apparatus. The test consists of two phases; a familiarization phase and a testing phase. In the familiarization phase, two identical objects, glass beakers (5.5 cm in diameter × 7 cm in height), were placed 20 cm from each side wall of the chamber and 20 cm apart. The familiarization phase was performed for 5 minutes and each animal was returned to its home cage after the familiarization phase. In the testing phase one hour after the familiarization phase, one of the familiar objects was replaced with a novel object, a plastic ball (7 cm in diameter). The testing phase was performed for 5 minutes. The time spent exploring each object was recorded and analyzed by the SMART® system. Cognitive outcome was determined by the “discrimination index”. The discrimination index (%) was calculated as the time spent in the novel object zone × 100 / (time spent in old object zone + time spent in novel object zone) [43].
Light-dark box test (LD test)
The LD test was performed one hour after the NOR test to investigate spontaneous explorative activity and anxiety behavior. The new environment and light are known as mild stressors and an innate aversion response to the light chamber represents explorative and anxiety behavior of rodents [44]. The LD test apparatus consists of bright and dark compartments (length of 18 cm × width of 30 cm × height of 16 cm) connected by a small opening (10 cm in diameter, semicircle shape). To evaluate the naive behavior of mice in the usual state without excessive stress, the light chamber was illuminated (200 lux), whereas the dark chamber was 1-2 lux. The LD test was performed for 5 minutes. The total number of transitions and the time spent in the light chamber were recorded on video.
High-performance liquid chromatography (HPLC)
Hippocampal NA concentration was measured by HPLC. Hippocampal tissues of six mice in each group for evaluating NA were collected after beheading the mice and were homogenized in 0.4M perchloric acid in a ratio of one part tissue to ten parts perchloric acid and centrifuged at 4,000 g for 20 minutes at 4℃. Then the supernatant was collected and preserved at -80℃ until sample analysis. Samples were purified by using MonoSpin® (GL Sciences Inc. Japan). NA level was measured by the HPLC system and 3,4-dihidroxybenxoylamin was used as the internal standard. Respective peak and elution times of the samples were evaluated by relative comparison to standard. Chromatographic separation was performed on an Inertsil ODS-4 column, 5 μm, 250 × 3.0 mm I.D. (GL Sciences Inc, Japan). The column temperature was maintained at 35 ℃. The flow rate was 0.5 mL/min. Injection volume was 20 μL. The mobile phase consisted of acetate-citrate buffer and acetonitrile. The mobile phase was composed of 20 mM citric acid, 20 mM citric acid monohydrate, 4.6 mM 1-octanesulfonic acid sodium and 13.7% acetonitrile. The electrochemical detector ED703, Diamond was used to detect monoamine.
Statistical analysis
Statistical analysis by comparison between two groups (control vs T2DM, surgery control vs surgery T2DM, control vs surgery control and T2DM vs surgery T2DM) was conducted using EZR [35] and JMP Statistical Discovery™ (SAS Institute Inc, Japan). The data for the behavioral test, and for hippocampal NA and HbA1c levels were analyzed by the Mann-Whitney U test using EZR. Body weight, fasting blood glucose level and IPGTT were subjected to two-way repeated measures analysis of variance (ANOVA) by using JMP. P values less than 0.05 were considered significant difference. In the figure, error bar of data is presented as medians ± quartile for the Mann-Whitney U test and means ± SD for ANOVA.