Ascensia breeze 2

Mean arterial blood pressure (MABP) was recorded from animals anesthetized (Ketamine 100 mg/Kg and diazepam 2 mg/Kg i.p.), tracheostomized and ventilated with room air (CL Palmer) (60 cycles min–1 and a positive end-expiratory pressure of 2 cm H₂O) or with hypoxic mixture (10% O₂, and 90% N₂). MABP was continuously monitored with a catheter inserted in the right common carotid artery. The catheter was connected to a pressure transducer (Statham) and signals stored (Power Lab 16SP; AD Instruments Castle Hill, Australia) for later analysis. In these animals, blood gases were obtained in normoxic (air) or hypoxic (10% O₂) breathing conditions from a small (0.3 ml) blood sample (ABL, Radiometer Medical A/S, Denmark). Glucose and lactate content were measured from the same blood samples withdrawn when animals were breathing the different gas mixtures described above and analyzed with glucose (Ascensia Breeze 2, Bayer) and lactate meters (Lactate Pro, Arkray).

Mice were fasted overnight and injected i.p. with a 30% glucose solution (1 μL/g of body weight). The glycemia was measured as described above at 15, 30, 60, 90- and 120-min post-injection. Blood glucose levels were measured in blood drops from the lateral tail vein using an Ascensia Breeze2 glucometer (Bayer Healthcare, Leverkusen, Germany).

Blood glucose levels were measured using a glucometer (Ascensia Breeze 2, Bayer HealthCare, Leverkusen, Germany). Blood was collected from the tail tip using microvette tubes with EDTA (Sarstedt, Nümbrecht, Germany). Plasma was collected after centrifugation at 3000× g for 20 min at 4 °C and kept at −80 °C until further analysis. Plasma insulin and leptin were analyzed by ELISA (Crystal Chem, Elk Grove Village, IL, USA).

All animal care procedures were approved by the Committee on Animal Care at Ritsumeikan University. Female 6‐week‐old NOD/ShiJcl mice, which spontaneously develop a T1D‐like phenotype (Makino et al., 1980 (link)), were purchased from Japan SLC. Female mice were used to study the onset of diabetes symptoms because they exhibit a higher incidence of diabetes than male mice (Amrani et al., 1998 (link); Makino et al., 1980 (link)). Mice were housed under controlled conditions with a 12‐h light–dark cycle and provided food and water ad libitum. Non‐fasting blood glucose levels were monitored using a glucometer (Ascensia Breeze 2, Bayer Healthcare) every morning between 10 and 36 weeks of age to diagnose diabetes. Mice were considered to have T1D when the non‐fasting glucose level was >250 mg/dL for two consecutive days. In contrast, normoglycemic mice were used as nondiabetic controls. After diabetes onset, diabetic mice were maintained on daily insulin (0.6 U, Lantus) to avoid death. At least 4 weeks after onset, overnight‐fasted mice (37 weeks old) were euthanized with isoflurane. Subsequently, the hippocampus and cortex were rapidly excised, frozen in liquid nitrogen, and stored at −80°C until analysis.