Thirty days after pulp exposure, the rats (n = 12 per group) were subjected to a 14-hour fast, followed by anesthetization as described above. Next, the abdominal region was disinfected using commercial iodine, and a median laparotomy was performed in 12 animals from each group (CN, 1AP and 4AP), to expose the abdominal cavity. The blood samples were collected via inferior vena cava punctures in heparinized tubes. Plasma was isolated by centrifuging the blood at 3000 × g for 15 min at 4 °C, and was stored as aliquots at −80 °C. Blood glucose was measured using the glucose oxidase method (Enzymatic glucose, Analisa Diagnóstica, Belo Horizonte, MG, Brazil) and insulinemia was evaluated by an enzyme-linked immunosorbent assay (ELISA) using a specific commercial kit (Sensitive Rat Insulin, Millipore, St. Charles, MO, USA). IR was evaluated by the homeostasis model assessment of insulin resistance (HOMA-IR) index, calculated according to the following formula (Bonora, 2000 (link)):
The same blood samples used to measure glucose and IR were used to evaluate serum INF-γ, IL-4, and TGF-β by the enzyme-linked immunosorbent method (ELISA) using specific commercial kits (INF-γ, EK0374; IL-4, EK0406; TGF-β, EK0514, Boster Biological Technology), in accordance with the manufacturer’s instructions.
The same blood samples used to measure glucose and IR were used to evaluate serum INF-γ, IL-4, and TGF-β by the enzyme-linked immunosorbent method (ELISA) using specific commercial kits (INF-γ, EK0374; IL-4, EK0406; TGF-β, EK0514, Boster Biological Technology), in accordance with the manufacturer’s instructions.