Ascorbate abolishes vascular leakage in mice and cultured endothelial cell monolayers
Kamenos, George C.
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Objectives . Our in-vitro objective is to investigate the effects of ascorbate (AA) or dehydroascorbate (DHA) as prevention or treatment in cultured endothelial cell monolayer dysfunction caused by septic insult (LPS and INF gamma). Our in-vivo objective is to investigate the effect of 3 hour delayed i.v AA or DHA on plasma protein extravasation in a clinically relevant model of sepsis (CLP). Methods . In-vitro experiments were performed using primary endothelial cell cultures from the hind limb of 4–6-week-old C57BL/6J mice. Cells were treated with AA (500 μM), DHA (500 μM), or DMEM 12 h prior, simultaneously, 12 h delayed, or 24 h delayed 50 ng/ml LPS (10 μl/ml) + 1000 U/ml IFN gamma (10 μl/ml). LPS + INF gamma were administered to induce sepsis. Twenty four hours after treatment with LPS + INF gamma (or 48 h in 24 h delayed), EB coupled albumin was added to each insert. Medium was collected from wells, and spectrophotometrically quantified at 595 nm. Fluid absorbance values serve as a quantitative assessment of EB coupled albumin transfer through the endothelial cell monolayer. In-vivo experiments were performed using 7–10-week-old C57BL/6J mice. CLP was performed to induce sepsis. Mice were treated with i.v AA (200 mg/kg body weight), DHA (200 mg/kg body weight), or normal saline 3 h after CLP. Subcutaneous injections of saline were given as fluid resuscitation in CLP mice. Furthermore, injections of buprenorphine/saline were given subcutaneously immediately before, after, and every 3 h after surgery for 12 h. Twelve hours after CLP or control condition, EB was injected into the circulatory system. The mice were euthanized after 30 min and the heart, lung, liver, and muscle tissue was obtained, minced in formamide, vortexed, incubated at room temperature for 3 days, and spetrophotometrically quantified at 620 nm. Fluid absorbance values serve as a quantitative assessment of EB coupled plasma protein extravasation. Results . Differences between conditions were assessed using a two-way analysis of variance, followed by Tukey's honestly significant difference (HSD) post hoc test. Significance was set at p<0.05. Data were presented as mean ± S.D. In-vitro , AA or DHA significantly prevented leak caused by septic insult at the 12 h pretreatment time point, but not in the 12 h delayed or 24 h delayed time point. Simultaneous administration with DHA, not AA, significantly decreased leak caused by septic insult. In-vivo , delayed i.v AA significantly decreased CLP induced plasma protein extravasation in the heart, liver, and muscle, but not lung. DHA did not decrease CLP induced plasma protein extravasation in any organ. Conclusions . In-vitro models of this project conclude that pharmacological levels of AA or DHA can prevent the formation of septic leak caused by LPS + INF gamma. Therapeutic effects of AA were not beneficial when administered simultaneously, 12 h delayed or 24 h delayed septic insult. However, simultaneous administration of DHA showed a therapeutic effect. In-vivo , pharmacological levels of i.v AA treated the formation of septic induced plasma protein extravasation in the heart, liver, and muscle. However, pharmacological levels of i.v DHA administered did not decrease the formation of septic induced plasma protein extravasation. Previous research suggests that AA exerted its effect by decreasing ROS and RNS. Therefore Vitamin C can be used as treatment for plasma protein extravasation in septic animal models. (Abstract shortened by UMI.)