Three healthy female beagles weighing 8–10 kg were included in this study. All procedures were approved by the Animal Care and Experimentation Committee, Gunma University, Maebashi, Japan. Our experimental plan is presented in Figure 1A. The dogs fasted overnight. Then, they were anesthetized with a single intravenous injection of thiopental sodium (Ravonal; Tanabe Pharmaceutical, Osaka, Japan) at a dose of 20 mg/kg. General anesthesia was maintained via intratracheal inhalation of halothane (Fluothane; Takeda Chemical Industries, Osaka, Japan) and oxygen. Under aseptic conditions, the Silastic tube (model 602–205; Dow Corning, Midland, MI) was inserted into the superior vena cava via a branch of the right external jugular vein (jugular catheter). Furthermore, it was used to withdraw blood samples and administer medications. The jugular catheter was exteriorized via a skin incision on the neck and was fixed to the adjacent skin with silk sutures. After the abdominal cavity was opened via a middle incision, force transducers [52,53] were implanted onto the serosal surfaces of the fornix, gastric body, gastric antrum, midduodenum, and jejunums 1 and 2 (20 and 40 cm distal to the Treitz’s fascia, respectively) to detect circular muscle contraction.
The lead wires of the force transducers and the Silastic tubes were taken out of the abdominal cavity via a subcutaneous tunnel and were exteriorized via a skin incision made between the scapulae. After closure of the abdominal cavity, a jacket-type protector was placed on each dog to prevent the lead wires and tubes from being damaged when scratched by dogs themselves. The dogs were housed in individual experimental cages, maintained with intravenous drip infusions of Lactec G (Otsuka Pharmaceutical, Tokyo, Japan) for 5 days after surgery, and gradually returned to a normal chow diet (15 g/kg per day; Funabashi Farm, Funabashi, Japan). We waited 2 weeks for the dogs to recover and to present with IMC after the surgeries. Then, total thyroidectomy was performed. The anesthesia method described above was used. A 5-cm skin incision was made in the anterior neck. The parathyroid gland was preserved, and both the thyroid lobes were resected. Day 0 was defined as the day when total thyroidectomy was performed. Next, the dogs returned to a normal chow diet at day 1. The half-life of thyroid hormone in dogs is 10–16 h . Hence, we performed an evaluation from days 1 to 4 in a hypothyroid state.
The production rate of total thyroxine (T4) in dogs is approximately 8 μg/kg/day [55,56]. Then, l-thyroxine sodium salt pentahydrate (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) was administered at a dose of 200 μg/kg/day after the patients were evaluated for hypothyroid state. The medication was continuously administered, and the evaluation of hyperthyroid state was again performed.
L-thyroxine sodium salt pentahydrate was prepared on each day, and it was dissolved in 200 μl of 1 N sodium hydroxide solution and diluted with 600 μl of water.
Evaluation of thyroid function
We collected blood samples, as shown in Figure 1A. In total, 1 mL of blood was collected in one blood collection. Blood samples were immediately transferred into test tubes containing a serum separating agent and were centrifuged at 4°C at 3,000 rpm for 5 min. The plasma samples were sent to outsourced laboratory (FUJIFILM VET Systems Company Limited, Tokyo, Japan). The concentration of TSH, T4, and calcium (Ca) was then assessed. Ca was measured to confirm if the parathyroid gland was preserved. The results were confirmed within 1 day after sending, and thyroid function was confirmed each time.
Monitoring of gastrointestinal motility contractions
The motility index (MI) was also assessed, as shown in Figure 1A. The wires from the transducer were connected to a telemeter, and the data were transmitted to a recording system (Eight Star System, Star Medical, Tokyo, Japan). The recorded signals were used to identify each phase of the contractile activity and the MI. The MI was the integrated area between the baseline (zero level) and the contractile wave on the Eight Star System.
We compared the first IMC that appeared after a meal within 5 h with GI according to thyroid state. Moreover, the MI 2 h before and after thyroid hormone was administered in a hyperthyroid state was calculated.
Evaluation of transmission speed
The distance between jejunums 1 and 2 was 20 cm. The time difference between the start of IMC phase III in jejunums 1 and 2 was evaluated, and the transmission speed (cm/second) was calculated.
Measurement of active ghrelin, GLP-1, and CCK levels
We collected blood samples, as shown in Figure 1A. The samples were obtained during phases III and I. In total, 2 mL of blood was collected in one blood collection. All blood samples were placed into chilled tubes containing ethylenediaminetetraacetic acid-2Na and 500 U apoprotein and were centrifuged at 4°C at 3,000 rpm. Two plasma samples were immediately collected. One was used for active ghrelin measurement, and the other for GLP-1 and CCK measurement. To measure active ghrelin using the enzyme-linked immunosorbent assay (ELISA), 0.1 mL of 1 N hydrochloric acid was added to per 1 mL of the samples. All samples were stored at −80℃ until hormone concentration analyses. The plasma active ghrelin concentrations were measured using the ELISA kit (Life Science Institute, Inc., Tokyo, Japan). The plasma GLP-1 concentrations were evaluated using the canine ELISA kit (MyBioSource, Inc., San Diego, California, the USA). Moreover, the plasma CCK concentrations were assessed using the canine ELISA kit (Antibodies.com., Cambridge, the UK).
The results were expressed as mean ± standard error. The data were compared using the paired t-test (Tukey’s test). P values < 0.05 were considered statistically significant. All statistical analyses were performed using the JMP software (SAS Institute Inc., Cary, NC, the USA).
Because the data were consistent, the experiment was performed on three conscious dogs. The study was performed in accordance with both the Animal Welfare and the International Guiding Principles for Biomedical Research Involving Animals . All procedures were approved by the Animal Care and Experimentation Committee, Gunma University, Maebashi, Japan (approval no. 17-045). The study was carried out in compliance with the ARRIVE guidelines.