After 60 minutes of shock, shed blood was retransfused within 10 minutes. Animals were continuously monitored for HR, MAP and CBF during 300 minutes. At the end of the experiment, the rats were sacrificed and blood samples were collected for measurement of arterial lactate levels. Aorta and hearts were harvested and www.selleckchem.com/products/Y-27632.html maintained in liquid nitrogen for further in vitro analyses (Western blotting, superoxide anion and NO production) (Figure (Figure11).Figure 1Design of the protocol (in case of hemorrhagic shock).Two additional groups of rats were managed in the same manner as the other animals but were not bled. One group (control-NaHS, n = 5) received a single bolus of NaHS (0.2 mg/kg body weight) while the other group received the vehicle (0.9% NaCl 0.

2 mg/kg body weight) (control-saline n = 5) in order to assess the hemodynamic effects of NaHS in normal rats.Maintenance of fluid was performed with a perfusion of 1.2 ml per hour of 0.9% NaCl in all groups.Hydrogen sulfide donor preparationThe dehydrated NaHS powder (sodium hydrogen sulfide, anhydrous, 2 g, Alpha Aesar GmbH & Co, UK) was dissolved in isotonic saline under argon gas bubbling, until a concentration of 40 mM was achieved. Intravenous (i.v.) administration was preferred to the inhaled form of H2S, as it represented an easier route whilst avoiding side effects such as airway irritation. In accordance with pilot experimentations in our laboratory and a previous study [19], a single intravenous bolus of NaHS (0.2 mg/kg) was infused.Monitoring and measurementsArterial blood gases were controlled after the stabilization period in order to adjust mechanical ventilation.

Blood gases, acid-base status and blood glucose were recorded at baseline (t = 0 minute), at the end of retransfusion (t = 70 minutes) and at the end of the experiment (t = 300 minutes). MAP, HR, CBF and temperature were recorded during the stabilization period (baseline) and every 10 minutes during the observation period.In vitro measurementsDetermination by electron paramagnetic resonance (EPR) NO spin trappingAorta and heart samples were incubated for 30 minutes in Krebs-Hepes buffer containing: BSA (20.5 g/L), CaCl2 (3 mM) and L-Arginine (0.8 mM). N, N D-Ethyldithiocarbamate and Fe3+ citrate complex (FeDETC) (3.6 mg) and FeSO4.7H2O (2.25 mg) were separately dissolved under N2 gas bubbling in 10 ml volumes of ice-cold Krebs-Hepes buffer.

These compounds were rapidly mixed to obtain a pale yellow-brown opalescent colloid Fe(DETC)2 solution (0.4 mM), which was used immediately. The colloid Fe(DETC)2 solution was added to the organs and incubated for 45 minutes at 37��C. Thereafter, the organs were snap frozen in plastic tubes using liquid N2. NO measurement Carfilzomib was performed on a table-top x-band spectrometer Miniscope (Magnettech, MS200, Berlin, Germany). Recordings were performed at 77��K, using a Dewar flask.