Arterial blood pressure measurements
Male C57BL/6J mice (n=4) were implanted with a PA-C10 blood pressure transmitter in the right carotid artery (Data Sciences International, St. Paul, MN), and after recovery from surgery the data was recorded using the DSI PhysioTel system (Data Sciences International, St. Paul, MN). For each experiment the mice were randomly selected for treatment with ISMN mixed in diH20 and 0.1 ml was injected i.p. for a final dose of 300 mg/kg. This was followed by gavage of 0.1 ml of H20 (control), sildenafil, or malonyl-sildenafil at 10 mg/kg, and the mice were returned to the cage for monitoring. The experiment was repeated until each treatment had been reproduced in 5 independent biological trials after which mice were euthanized.
The surgical procedures and implantable telemetry approach for measuring blood pressure in mice have been described in detail previously (Leeet al. , 2004). In brief, all surgical instruments were autoclaved on a stainless steel tray that was used as the sterile surface during surgery. The mouse is anesthetized in an isoflurane-inducing chamber. Anesthesia used a fitted nose cone connected to an isoflurane system running 2% in a stream of 100% oxygen mixed with room air. The incision area was shaved and prepped with betadine and rinsed with 70% ethanol. Anesthesia was assessed continuously during shaving and prepping for surgery, with appropriate adjustments. An approximately 10 mm incision was made in the ventral neck region over the trachea to expose the left carotid artery. A subcutaneous tunnel was made on the right side leading to the midscapular region where a sterile Data Sciences BP transmitter unit was inserted into a pocket, and the catheter tip from the unit was inserted into the carotid artery and secured in place with silk ties. The incision was closed in one layer with sterile, 5-0 or 6-0 silk suture. After closing the incision, the mouse was removed from anesthesia, buprenorphine SR was administered i.p. and the mouse is placed in a clean, warmed cage to recover. Once fully conscious and able to ambulate, mice were transferred to individual plastic shoe box cages and placed on top of Data Sciences receivers that have been matched to the respective transmitters. Approximately 5-7 days were allowed from surgical procedures before beginning experimental procedures and measurements.