Immunostaining: Formalin-fixed, paraffin wax-embedded lung tissue was immunostained with specific polyclonal rabbit antibodies against NOS-III and NOS-II antigens (Cayman, Ann Arbor, Michigan, USA) by the alkaline phospha-tase-antialkaline phosphatase (Dako, Denmark) method . Incubation with the primary antibody took 1 h. Slides were developed with Fast Red (Sigma).
Survival analysis: The model of endotoxic shock produced by intravenous infusion of LPS (2 mg/kg/min for 10 mins) was used because this dose of LPS produced reproducible hemodynamic (Figure 1) and lethal effects. Survival curves were plotted as the probability of survival (ordinate) and as a function of survival time (abcissa) by the Kaplan-Meier method . LPS infusion caused the death of ratstreated with only LPS within 4 to 6 h (Figure 2, solid thin line). Pretreatment of rats with L-NNA (10mg/kg intravenously, L-NNAplus LPS group) intensely decreased survival time to 20 to 25 mins after LPS infusion (P<0.001 compared with the LPS group) (Figure 2, solid thick line). buy asthma inhaler
Figure 1 Scheme tracing of biphasic changes in mean arterial blood pressure induced by lipopolysaccharide (LPS) (Escherichia coli) infusion in Sprague-Dawley rats. i.v. Intravenous
Figure 2 Probability of survival time (ordinate) plotted as a function of survival time in minutes (abscissa). Survival curves were drawn for six rats within each of four studied groups: rats subjected to lipopolysaccharide (LPS) infusion after NG-nitro-L-arginine (L-NNA) (LNNA plus LPS group); rats that were given LPS alone (LPS group); rats that received L-NNA and LPS, as well as S-nitro-N-acetyl-penicyllamine (SNAP) infusion (L-NNA plus SNAP plus LPS group); and rats pretreated with 1-amino-2-hydroxy-guanidine (AGD) before LPS infusion (AGD plus LPS group)