In a previous study, we concluded that overproduction of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) in the late phase of sepsis prevents hypothalamic activation, blunts vasopressin secretion and contributes to hypotension, irreversible shock and death. The aim of this follow-up study was to evaluate if the same neuronal activation pattern happens in brain structures related to cardiovascular functions. Male Wistar rats received intraperitoneal injections of aminoguanidine, an iNOS inhibitor, or saline 30 min before cecal ligation and puncture (CLP) or sham surgeries. The animals were perfused 6 or 24h after the surgeries and the brains were removed and processed for Fos immunocytochemistry. We observed an increase (P<0.001) in c-fos expression 6h after CLP in the area postrema (AP), nucleus of the tractus solitarius (NTS), ventral lateral medulla (VLM), locus coeruleus (LC) and parabrachial nucleus (PB). At 24h after CLP, however, c-fos expression was strongly decreased in all these nuclei (P<0.05), except for the VLM. Aminoguanidine reduced c-fos expression in the AP and NTS at 6h after CLP, but showed an opposite effect at 24h, with an increase in the AP, NTS, and also in the VLM. No such effect was observed in the LC and PB at 6 or 24h. In all control animals, c-fos expression was minimal or absent. We conclude that in the early phase of sepsis iNOS-derived NO may be partially responsible for the activation of brain structures related to cardiovascular regulation. During the late phase, however, this activation is reduced or abolished.

The objective of this work was to describe the retention and separation of the major fat soluble and water soluble vitamins in the Ultra High Performance Supercritical Fluid Chromatography (UHPSFC) technique as a function of the main experimental factors involved and to use the concepts of Analytical Quality by Design for the development of analytical methods. Initially, through a screening study, the stationary phases Acquity HSS C18 SB and Acquity Torus 2-PIC were identified as the most promising along with methanol as the organic modifier of the mobile phase and ammonium formate (10 mmol L -1 ), water (5%) and ammonium hydroxide (10 mmol L -1 ) as additives, which were mandatory for the improvement of chromatographic parameters for water soluble vitamins. Then, a Central Composite Design was performed to study the variables that influence the fluid density: percentage of organic modifier at the end of the gradient, counter-pressure and temperature. The evaluated responses were: retention factor (k), number of plates (N), asymmetry (As), area of the peaks and resolution (Rs). The multivariate study showed that the three factors influenced in a different way each analytical response and allowed, by means of desirability functions, the construction of the Design Space, which is the region where robustness and adequacy of the parameters of the chromatographic method are guaranteed. All vitamins presented analytical responses (areas) directly proportional to the concentration, with r > 0.99. In the analysis of a pharmaceutical soft capsule, no interferences of the matrix and the diluent were detected.Palavras-chave: Vitamins, Ultra high performance supercritical fluid chromatography (UHPSFC), Analytical Quality by Design (A-QbD).