Cardiovascular studies in healthy rats were performed at Covance Inc. (Greenfield, IN, USA) and initiated in 6 male Sprague-Dawley CD/IGS rats (Charles River, Portage, Michigan, USA). Rats were housed under controlled temperature (22 ± 5°C) and humidity conditions (20 - 80%) at a 12 hour normal dark-light cycle (lights on at 06.00 AM) with free access to water and chow (Rodent 2014, Harlan Teklad Global Diets, Indianapolis, IN, USA). The procedures in this study were designed to avoid or minimize discomfort, distress, and pain to animals and in compliance with the U.S. Department of Agriculture's (USDA) Animal Welfare Act (9 CFR Parts 1, 2, and 3), the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 1996), and the National Institutes of Health, Office of Laboratory Animal Welfare.
At least five days prior to infusion the animals were implanted with a DSI Physiotel® Multiplus C50-PXT transmitter (Data Sciences International, St Paul, MN, USA) and a 6-inch beaded polyurethane intravenous catheter (Strategic Applications Inc., Libertyville, IL, USA) set in the jugular vein using aseptic surgical technique. The animals received buprenorphine hydrochloride (0.1 mg/kg, Reckitt Benckiser Pharmaceuticals Inc., Richmond, VA, USA) prior to surgery and ketoprofen (3 mg/kg, Fort Dodge Animal Health, Fort Dodge, IA, USA) following surgery, and were anesthetized with isoflurane (Abbott Laboratories, Abbott Park, IL, USA) throughout the surgical procedure. The DSI system was connected to a data acquisition and analysis system (PONEMAH, Data Sciences International, St. Paul, MN, USA) and enabled to measure systemic arterial pressure and blood temperature. The arterial pressure signal was used to derive systolic, diastolic, and mean arterial pressure as well as heart and respiratory rate. Although data was continuously acquired, derived parameters were collapsed into mean values computed over repetitive logging periods. Data was recorded from 2 hours before starting the infusion until approximately 6.00 AM the following day. A freshly prepared solution of dihydrocapsaicin (0.8 mg/ml, Lot 63908, Clauson Kaas, Farum, Denmark) dissolved in 2% tween 80 (Spectrum Chemicals and Laboratory Products, Gardena, CA, USA) and saline and vehicle control was prepared and sterile filtered before on-set of the infusion. Following a 2 hour settling period and using a KD scientific pump (KD Scientific Inc, Holliston, MA, USA), vehicle control was infused for 1 hour at a flow rate of 3 ml/kg/hr. Next, DHC was infused in 5 steps each lasting 1 hour at the doses of 1.0, 2.0, 2.2, 2.4, and 3.0 mg/kg/hr corresponding to a flow rate of 1.25, 2.5, 2.75, 3.0, and 3.75 ml/kg/hr. Following the termination of the infusion animals were euthanized by CO2 inhalation followed by cervical dislocation.
Cardiac arrested and resuscitated rats
Studies in resuscitated rats were performed at the Weil Institute of Critical Care (Rancho Mirage, CA, USA) in male Sprague-Dawley rats (Harlan Laboratories Inc., station #237, San Diego, CA, USA) aged 6-8 months and weighing 496-523 grams. Animals were housed in groups of 2-3 per cage in a temperature (23 ± 3°C) and humidity (30 - 40%) controlled room and a 12 hour light-dark cycle (lights on at 06.00 AM). Rat diet (NEWCO Distributors, Inc, Rancho Cucamonga, CA, USA) and water were available ad libitum except that food was deprived 12 hours prior to surgery. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (National Institutes of Health publication 0-309-05337-3, Revised 1996). The protocol was approved by the Institutional Animal Care and Use Committee of the Weil Institute of Critical Care Medicine. The Weil Institute of Critical Care Medicine Laboratories is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Preparation and surgery
The animals were anesthetized by an intraperitoneal injection of pentobarbital (45 mg/kg) and additional doses (10 mg/kg) were administrated as required to maintain anesthesia. Antibiotic treatment with cefazolin (250 mg, Moore Medical Corp., New Britain, CT, USA) was administered by intramuscular injection prior to the start of surgery, and a second dose at the end of the experiment. The trachea was orally intubated with a 14 G cannula mounted on a blunt needle with a 145° angled tip (Abbocath-T, Abbott Hospital, North Chicago, IL, USA) as previously described . During surgery animals were spontaneously breathing air and the temperature of the animal was maintained at 37°C ± 0.2°C with the aid of a heating lamp. Blood temperature was measured with a thermocouple microprobe 10 cm in length and 0.5 mm in diameter (9030-12-D-34, Columbus Instruments, Columbus, OH, USA) that was inserted into the right femoral artery and advanced to the distal ascending aorta. A polyethylene catheter (PE-50; Becton-Dickinson, Franklin Lakes, NJ, USA) was advanced into the descending aorta from the surgically exposed left femoral artery for measurement of arterial blood pressure. Electrocardiography (ECG) was continuously monitored by a conventional lead II and end-tidal CO2 was continuously monitored with a side-stream infrared CO2 analyzer (End-Til IL 200; Instrument Laboratory, Lexington, MA, USA). A polyethylene catheter (PE-50; Becton-Dickinson, Franklin Lakes, NJ, USA) was advanced from the left femoral vein into the inferior cava vein for subsequent infusion of compound. All catheters were flushed intermittently with heparinized saline (2.5 IU/ml of BSA, Western Medical Supple, Arcadia, CA, USA).
Cardiac arrest procedure
The cardiac arrest and resuscitation procedure was performed as previously described [29, 30]. Briefly, fifteen minutes prior to inducing ventricular fibrillation (VF), baseline measurements were obtained and mechanical ventilation was initiated with an inspired O2 fraction (FiO2) of 0.21. Ventricular fibrillation was then induced through a guide wire (model C-PMS-301J, Cook Critical Care, Bloomington, IN, USA) advanced from the right jugular vein into the right ventricle. A progressive increase in 60 Hz current to a maximum of 4 mA was delivered to the right ventricular endocardium and maintained for 3 minutes to prevent spontaneous defibrillation. Mechanical ventilation was stopped at the onset of cardiac arrest. After 6 minutes of untreated VF, cardiopulmonary resuscitation (CPR) including pre-cordial chest compressions and mechanical ventilation with a FiO2 of 1.0 was initiated. Chest compressions were performed with the aid of a pneumatically driven mechanical chest compressor at a rate of 200 per minute and synchronized to provide a compression-to-ventilation ratio of 2-to-1 with equal compression-relaxation. The depth of compressions was initially adjusted to maintain a coronary perfusion pressure above 23 mmHg and with end-tidal CO2 above 11 mmHg. After 6 minutes of CPR, resuscitation was attempted with up to 3 two-joule counter shocks. Return of spontaneous circulation (ROSC) was defined as the return of supraventricular rhythm with a mean aortic pressure above 50 mmHg for at least five minutes. If ROSC was not achieved, a 30 second interval of CPR was performed prior to attempt of a subsequent sequence of up to 3 shocks. The procedure was repeated for a maximum of 3 cycles. If ROSC was not achieved the animal was terminated and excluded from the study.
Following ROSC animals were allowed to stabilize for 30 minutes and stratified into 2 groups (n = 4) receiving a 6 hour infusion of DHC with or without pre-treatment with atropine (5 mg/kg, Moore Medical Corp., New Britain, CT, USA). Atropine was administered as a 4 IV injections of a 0.4 mg/ml solution every 30 seconds starting 10 minutes prior to the infusion of DHC. A freshly prepared solution of dihydrocapsaicin (0.4 mg/ml, Cat. 92355, Cayman Chemical Company, AH Diagnostics, Aarhus, Denmark) dissolved in 2% tween 80 (Sigma-Aldrich, St. Louis, MO, USA) and saline was prepared and sterile filtered before on-set of the infusion. Then, at t = 0 hours (corresponding to 30 minutes after ROSC) a continuous infusion of DHC was initiated using a Micro Macro XL pump (Abbott Laboratories, Chicago, IL, USA) with a flow rate of 0.4 ml/kg from t = 0 to 30 minutes, then 0.8 ml/kg from t = 30 to 60 minutes, and finally 1.6 ml/kg from t = 1 to 6 hours corresponding to doses of 0.16, 0.33 and 0.65 mg/kg/hr, respectively. Blood temperature and arterial blood pressure were recorded 15 minutes before and then every 15 minutes during the 6 hour infusion. ECG was recorded throughout the infusion. The ambient temperature during the infusion was similar to the conditions during surgery. Following the termination of the infusion animals were euthanized by intraperitoneal injection of pentobarbital (150 mg/kg).
Data are expressed as mean ± standard error (SE) and compared by a one-way or two-way ANOVA and appropriate post-test, unless otherwise stated. P < 0.05 was considered significant. All statistical calculations were performed using GraphPad Prism version 4.00 (GraphPad Software, Inc., San Diego, CA).