) and a draft-free location to facilitate this process. The abdomen is opened with a midline incision; approximately 1?.5 cm. Using a wound spreader the intestines are carefully pushed aside and the left kidney is exposed, however not exteriorized to avoid rigorous cooling of the kidney and ischemic preconditioning during manipulation of the kidney. The renal pedicle is carefully dissected with fine-point tweezers to remove the perihilar adipose tissue, exposing the blood vessels for renal pedicle clamping. It is important that the blood supply to the adrenal gland remains unaffected. The renal pedicle is clamped with an atraumatic vascular clip (Scanlan, Saint Paul, Minnesota; see study set-up for the indicated LY2510924 site ischemia times and body temperature used in the experiments described in this manuscript) using holding forceps, ensuring minimal vascular damage, and as little as possible perihilar fat in the clamp. Successful ischemia is characterized by a gradual colour change of the kidney from red to dark purple within 1? minutes. The right kidney is left undisturbed. The abdomen is temporarily closed with a suture and the animal is transferred to an infant incubator, kept at the temperature that allows the body temperature of the animal to remain stable at the setpoint temperature for the duration of ischemia (see study set-up). Temperature of the animal is monitored continuously with a rectal thermometer (Bioseb, AUY922MedChemExpress AUY922 Vitrolles, France). Variations in body temperature during ischemia should be kept less than 0.5 , as this will increase variability to the degree of renal injury. The vascular clip is released at the desired time to start reperfusion. After verification of kidney colour to change back to red (roughly within 10 seconds), a Vicryl 4? suture (Ethicon, Norderstedt, Germany) is used to first close the muscle layer, followed by closing of the skin. Sham-operated animals are subjected to the exact same surgical procedure, aside from clamp placement. Post-operative care: Immediately after surgery, 1 ml saline or Plasma-Lyte (Baxter, Lessen, Belgium; i.e. buffered low chloride saline solution) is given intraperitoneally to compensate for the fluid loss during surgery. Even though the superiority of Plasma-Lyte over saline is not proven, clinical reports allow assuming that high chloride solutions are associated with worse AKI as compared to low-chloride solutions [48]. The animals are kept on a warm water mat (water temperature at 37 ) until awakening after which they are placed in an fpsyg.2017.00209 open grid recovery cage under a heating lamp until full consciousness is regained. Heating lamp and cage are placed in such a way that one end of the cage maintains room temperature whilst the distance between lamp and animals is held large enough to avoid overheating. After 24 hours the animals are transferred to their housing cage. During the first 3 days after the surgery, the animals, in addition to the standard chow, are supplemented with DietGel Recovery Purified Soft Diet for Rodents (Clear H2O, Portland, Maine), rich in water and sugars, to reduce the post-operative weight loss and allow faster recovery after surgery. Weekly follow up of the body weight SART.S23503 and behaviour is performed as measures of good health. Real-time PCR. Total mRNA is extracted from a pole section of the ischemic kidney (PureLink RNA Mini Kit; Life Technologies, Gent, Belgium) and converted to cDNA (High Capacity cDNA archive kit; Life Technologies, Gent, Belgium). To quantify gene.) and a draft-free location to facilitate this process. The abdomen is opened with a midline incision; approximately 1?.5 cm. Using a wound spreader the intestines are carefully pushed aside and the left kidney is exposed, however not exteriorized to avoid rigorous cooling of the kidney and ischemic preconditioning during manipulation of the kidney. The renal pedicle is carefully dissected with fine-point tweezers to remove the perihilar adipose tissue, exposing the blood vessels for renal pedicle clamping. It is important that the blood supply to the adrenal gland remains unaffected. The renal pedicle is clamped with an atraumatic vascular clip (Scanlan, Saint Paul, Minnesota; see study set-up for the indicated ischemia times and body temperature used in the experiments described in this manuscript) using holding forceps, ensuring minimal vascular damage, and as little as possible perihilar fat in the clamp. Successful ischemia is characterized by a gradual colour change of the kidney from red to dark purple within 1? minutes. The right kidney is left undisturbed. The abdomen is temporarily closed with a suture and the animal is transferred to an infant incubator, kept at the temperature that allows the body temperature of the animal to remain stable at the setpoint temperature for the duration of ischemia (see study set-up). Temperature of the animal is monitored continuously with a rectal thermometer (Bioseb, Vitrolles, France). Variations in body temperature during ischemia should be kept less than 0.5 , as this will increase variability to the degree of renal injury. The vascular clip is released at the desired time to start reperfusion. After verification of kidney colour to change back to red (roughly within 10 seconds), a Vicryl 4? suture (Ethicon, Norderstedt, Germany) is used to first close the muscle layer, followed by closing of the skin. Sham-operated animals are subjected to the exact same surgical procedure, aside from clamp placement. Post-operative care: Immediately after surgery, 1 ml saline or Plasma-Lyte (Baxter, Lessen, Belgium; i.e. buffered low chloride saline solution) is given intraperitoneally to compensate for the fluid loss during surgery. Even though the superiority of Plasma-Lyte over saline is not proven, clinical reports allow assuming that high chloride solutions are associated with worse AKI as compared to low-chloride solutions [48]. The animals are kept on a warm water mat (water temperature at 37 ) until awakening after which they are placed in an fpsyg.2017.00209 open grid recovery cage under a heating lamp until full consciousness is regained. Heating lamp and cage are placed in such a way that one end of the cage maintains room temperature whilst the distance between lamp and animals is held large enough to avoid overheating. After 24 hours the animals are transferred to their housing cage. During the first 3 days after the surgery, the animals, in addition to the standard chow, are supplemented with DietGel Recovery Purified Soft Diet for Rodents (Clear H2O, Portland, Maine), rich in water and sugars, to reduce the post-operative weight loss and allow faster recovery after surgery. Weekly follow up of the body weight SART.S23503 and behaviour is performed as measures of good health. Real-time PCR. Total mRNA is extracted from a pole section of the ischemic kidney (PureLink RNA Mini Kit; Life Technologies, Gent, Belgium) and converted to cDNA (High Capacity cDNA archive kit; Life Technologies, Gent, Belgium). To quantify gene.
