Lactate pro device

Seven weeks after vehicle or streptozotocin injection, rats (referred to as non-diabetic “n,” and diabetic “d,” respectively) were randomly assigned to the control (CON) or IR group. The control groups (nCON, dCON, 8 rats per group) underwent 5 h of isoflurane anesthesia and similar surgical manipulation to the IR groups, except for hindlimb ischemia (sham-operated). The ischemia–reperfusion groups (nIR and dIR, 10 rats per group) underwent 3 h of ischemia induced by infra-renal aortic occlusion and collateral vessel ligation, followed by 2 h of reperfusion. Ischemia was clinically characterized by cyanosis and lack of an arterial pulse distal to the clamp, and biochemically by an increase in capillary blood lactate measured in the right hindlimb (Lactate Pro device, LT1710; Arkray, KGK, Japan).
After reperfusion, gastrocnemius muscles, that are considered more sensitive to IR (Charles et al., 2017 (link)), were harvested and either analyzed immediately (mitochondrial respiration) or kept in ice or in liquid nitrogen-cooled isopentane. Animals were sacrificed by heart retrieval under deep anesthesia (5% isoflurane).

WEx and CEx animals were familiarized with running on a motor-driven animal treadmill (KN-73; Natsume, Tokyo, Japan) for approximately 10–20 m/min for 5 min per day beginning 1 week before the experiment. After 5 h of fasting, they were subjected to run on the treadmill at 20 m/min without grade for 30 minutes in a climate-controlled room at 25°C or 4°C, respectively. Electric shocks were rarely used to motivate animals to run. To verify an elevation in body temperature, rectal temperature was measured using a calibrated thermistor probe (LT-8; Gram Corporation, Saitama, Japan) inserted approximately 6–7 cm past the anal sphincter into the colon before and immediately after exercise. Muscle temperature was also measured immediately after exercise using a needle thermistor probe inserted into one gastrocnemius muscle. After measuring rectal temperature, the animals were anesthetized using isoflurane, and blood glucose and lactate concentrations were measured from the tail vein using the Glutest Neo Super device (Sanwa Kagaku Kenkyusho, Aichi, Japan) and the Lactate Pro device (Arkray, Kyoto, Japan), respectively. Next, blood and gastrocnemius muscles were removed, rapidly frozen in liquid nitrogen, and stored at −80°C until analysis. In this study, the needle-inserted leg was not used in the analyses.