Septic impairment of capillary blood flow requires nicotinamide adenine dinucleotide phosphate oxidase but not nitric oxide synthase and is rapidly reversed by ascorbate through an endothelial nitric oxide synthase-dependent mechanism*

Abstract

Objective To determine the roles of nitric oxide synthase (NOS) and NADPH oxidase in the impairment of capillary blood flow in sepsis and in the reversal of this impairment by ascorbate. Design Prospective, controlled laboratory study. Setting Animal laboratory in research institute. Subjects Adult male wild type (WT), nNOS−/−, iNOS−/−, eNOS−/−, and gp91phox−/−mice. Interventions Sepsis was induced by feces injection into peritoneum (FIP). A bolus of ascorbate or NADPH oxidase inhibitor apocynin was injected intravenously at 6 hrs post-FIP. Alternatively, NOS cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) or nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) was superfused on the surface of extensor digitorum longus muscle. Measurements and Main Results Capillary blood flow impairment and NOS activity in extensor digitorum longus muscle were measured by intravital microscopy and by enzymatic assay, respectively. Sepsis at 6 hrs impaired flow in WT mice. Apocynin, and knockout of gp91phox but not of any NOS isoforms, rescued this impairment. Constitutive NOS activity was unaffected by sepsis, but it was abolished by nNOS knockout (iNOS activity was negligible in all mice). Ascorbate rapidly (10 mins) rescued impaired flow in WT, nNOS−/−, iNOS−/− but not eNOS−/− mice. Ascorbate also improved survival of WT mice after FIP. BH4 and SNAP rescued flow in WT mice, while BH4 failed to rescue it in eNOS−/− mice. Conclusion Capillary blood flow impairment in septic skeletal muscle requires NADPH oxidase but not NOS, and it is rapidly reversed by ascorbate and BH4 through an eNOS-dependent mechanism.

Evaluation of Vitamin C for Adjuvant Sepsis Therapy

“…Ascorbate protects microvascular function and improves survival in septic mice (18,59,64). It is not certain to what extent, if any, this protection is an indirect result of bacteriostasis.…”

“…Further, it is possible that myocardial depression lowers blood pressure in the septic mice despite ascorbate administration (59). It is clear from studies of CLP animals that (i) hypotension is associated with falls in plasma ascorbate concentration and arteriolar reactivity and also with increases in iNOS protein and activity levels; (ii) iNOS knockout blocks the effects of CLP on arteriolar reactivity and baseline arterial blood pressure; and (iii) injection of ascorbate prevents the CLP-induced changes in iNOS, arteriolar reactivity and blood pressure (2,29,40,58,59,64). The identities of the molecular targets of iNOS-derived NO that mediate the effects of septic insult and ascorbate therapy on arteriolar reactivity are unknown.…”

“…Blood coagulation involves platelet aggregation and adhesion to endothelial cells, fibrin deposition, and formation of microthrombi (26). The sensitivity of these phenomena to vitamin C has been studied in endotoxemic, CLP and FIP mice (18,45,51,59).…”

“…Further evidence of the importance of vitamin C is that its parenteral administration decreases mortality in wild-type mice that are ascorbate-replete before exposure to septic insults. For example, bolus injection of ascorbate (10-200 mg/kg) improves survival in mice made septic by either CLP or fecal stem solution injection into peritoneum (FIP) (18,59,64) (Fig. 2).…”

“…Bolus injection i.v. of ascorbate blocks hypotension from developing in endotoxemic rats (53) Ascorbate also prevents hypotension for 24 h in CLP rats (2,58), attenuates the fall in baseline blood pressure for 6 h in CLP mice (63), and fails to protect baseline blood pressure in FIP mice (59). The reasons for this variation between the different septic peritonitis models may be that, compared to salinevehicle injected CLP rats (75% survival at 24 h), sepsis Ascorbate also blocked the increases in iNOS and nNOS activities, and the decrease in eNOS activity (B).…”