Alloxan monohydrate

All drugs and reagents used were obtained commercially and of analytical grade and products of May and Baker, England; BDH, England; Merck, Darmstadt, Germany; Accu-check active glucometer by Roche Diagnostic, Germany; alloxan monohydrate, Sigma-Aldrich Chemical (St. Louis, MO, USA).

Hyperglycemia was induced in mice by a single i.p. injection of 150 mg/kg·bw alloxan monohydrate (Sigma-Aldrich) dissolved in 0.15 M NaCl after 16 h of fasting [55 (link)]. Blood samples were collected from the tail vein and the glucose level was determined using a clinical glucometer (AccuCheck Active^®, Roche, Indianapolis, IN, USA). The blood glucose level was checked before and 72 h after alloxan injection to confirm the development of diabetes. Animals with blood glucose levels ≥300 mg/dL were selected for the study.

A total of 90 male and female Japanese rabbits (weighing 1.7–2.5 kg at the beginning of the study) were obtained from Beijing Medical Animals Research Institute (Beijing, China). They were randomly and equally divided into the control group (C, n=30), alloxan‐induced group with DM (DM, n=30) and allopurinol‐treated group with DM (ALLO, n=30). Ten rabbits from each group were used for echocardiographic examination, hemodynamic‐, histological‐ and electrophysiological studies, oxidative stress marker measurements, Western blot analyses, I_CaL recordings, and intracellular Ca²⁺ imaging. For the induction of diabetes mellitus, alloxan monohydrate (Sigma, Saint Louis, MO, USA) was dissolved to sterile normal saline to achieve a concentration of 5% (w/v), and a dose of 120 mg/kg was immediately administered intravenously via the marginal ear vein. The presence of diabetes mellitus was confirmed 48 hours later by blood glucose levels ≥14 mmol/L (once) or ≥11 mmol/L (twice). Subsequently, blood glucose level monitoring was performed weekly using the glucometer Optium Xceed (Abbott, Bedford, MA, USA). Rabbits in the ALLO group received allopurinol (60 mg/kg per day) orally for 8 weeks.

Povidone iodine ointment was obtained from the pharmaceutical industry Eskayef Bangladesh Limited. Alloxan monohydrate was obtained from Sigma Aldrich Chemicals, Germany. All other chemicals were obtained from Merck (Darmstadt, Germany) and were of analytical grade.

Alloxan monohydrate was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pentoxifylline, glibenclamide and metformin were from EMS Laboratory (São Paulo, Brazil) and dissolved in distilled water before use. Kits for blood biochemistry were from Lab Test Diagnóstica SA (Minas Gerais, Brazil). All other reagents were of analytical grade.

Diabetes mellitus was induced by intravenous injection of alloxan monohydrate (50 mg/kg; Sigma Chemical Co., St. Louis, MO, USA) dissolved in physiologic saline (SAL, 0.9% NaCl). Control mice were injected with physiologic SAL only. After 10 days, the presence of diabetes was verified by blood glucose concentrations higher than 300 mg/dL, which were determined with a blood glucose monitor (Accu-Chek Advantage II, Roche Diagnostica, São Paulo, São Paulo, Brazil), in blood samples obtained from mouse tails (15 (link)).

The diabetes was induced in the two diabetes groups by administration of 2% alloxan aqueous solution, with a dose of 170 mg/kg of alloxan (Alloxan monohydrate, Sigma-Aldrich, St. Louis, MO, United States) dissolved in 0.2 mL of saline (0.9%) intraperitoneally injected. The animals of two control groups received only 0.2 mL of saline solution. Animals with glycemia above 200 mg/dL (16 mmol/L) were considered diabetic. Confirmation of the diabetes was made 48 h after the induction by an automated process using a glycemic meter (OnCall^® Plus, brand ACON Laboratories, Inc.) through blood collection performed with a slight incision in the animal’s tail after fasting previous 12 h. The body mass and blood glucose for all groups were monitored every 15 days and started in the same day of diabetes induction, continuing during the experiment period in all experimental groups.