We hypothesized that normotensive sepsis affects the ability of the microcirculation to appropriately regulate microregional red blood cell (RBC) flux. An extensor digitorum longus muscle preparation for intravital study was used to compare the distribution of RBC flux and the functional hyperemic response in SHAM rats and rats made septic by cecal ligation and perforation (CLP). Using intravital microscopy, we found that sepsis was associated with a 36% reduction in perfused capillary density (from 35.3±+1.5 to 22.5±1.0 capillaries/mm of test line) and a 265% increase in stopped-flow capillaries (from 0.9±0.2 to 3.3±0.4 capillaries/mm); the spatial distribution of perfused capillaries was also 72% more heterogeneous. Mean intercapillary distance (ICD) increased 30% (from 25.7±0.8 to 33.5±1.6 ,um), and the proportion of capillary pairs with intercapillary distances > 33.8 ,um (the 75th percentile of ICDsHAM) was greater with sepsis. Mean capillary RBC velocity increased 17% in CLP rats (391 vs 333 ,um/s). Laser Doppler flowmetry was used to assess the functional hyperemic response of the extensor digitorum longus muscle before and after a period of maximal twitch contraction designed to increase oxygen demand. RBC flux was 36% lower in the CLP rats at rest. After contraction, RBC flux increased in both SHAM and CLP rats; however, the relative increase was less in the CLP group. We concluded that sepsis affects the ability of the skeletal muscle microcirculation to appropriately distribute RBC flux and to respond to increases in oxygen need. (J. Clin. Invest. 1994. 94

Septic patients have low plasma ascorbate concentrations and compromised microvascular perfusion. The purpose of the present experiments was to determine whether ascorbate improves capillary function in volume-resuscitated sepsis. Cecal ligation and perforation (CLP) was performed on male Sprague-Dawley rats. The concentration of ascorbate in plasma and urine, mean arterial blood pressure, and density of continuously perfused capillaries in the extensor digitorum longus muscle were measured 24 h after surgery. CLP caused a 50% decrease (from 56 +/- 4 to 29 +/- 2 microM) in plasma ascorbate concentration, 1,000% increase (from 46 +/- 13 to 450 +/- 93 microM) in urine ascorbate concentration, 20% decrease (from 115 +/- 2 to 91 +/- 2 mmHg) in mean arterial pressure, and 30% decrease (from 24 +/- 1 to 17 +/- 1 capillaries/mm) in the density of perfused capillaries, compared with time-matched controls. A bolus of intravenous ascorbate (7.6 mg/100 g body wt) administered immediately after the CLP procedure increased plasma ascorbate concentration and restored both blood pressure and density of perfused capillaries to control levels. In vitro experiments showed that ascorbate (100 microM) inhibited replication of bacteria and prevented hydrogen peroxide injury to cultured microvascular endothelial cells. These results indicate that ascorbate is lost in the urine during sepsis and that a bolus of ascorbate can prevent microvascular dysfunction in the skeletal muscle of septic animals. Our study supports the view that ascorbate may be beneficial for patients with septic syndrome.

Purpose-Impaired microvascular perfusion in sepsis is not treated effectively because its mechanism is unknown. Since inflammatory and coagulation pathways cross-activate, we tested if stoppage of blood flow in septic capillaries is due to oxidant-dependent adhesion of platelets in these microvessels.Methods-Sepsis was induced in wild type, eNOS −/− , iNOS −/− , and gp91phox −/− mice (n = 14-199) by injection of feces into the peritoneum. Platelet adhesion, fibrin deposition, and blood flow NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript stoppage in capillaries of hindlimb skeletal muscle were assessed by intravital microscopy. Prophylactic treatments at the onset of sepsis were intravenous injection of platelet-depleting antibody, P-selectin blocking antibody, ascorbate, or antithrombin. Therapeutic treatments (delayed until 6 h) were injection of ascorbate or the glycoprotein IIb/IIIa inhibitor eptifibatide, or local superfusion of the muscle with NOS cofactor tetrahydrobiopterin or NO donor S-nitroso-Nacetylpenicillamine (SNAP).Results-Sepsis at 6-7 h markedly increased the number of stopped-flow capillaries and the occurrence of platelet adhesion and fibrin deposition in these capillaries. Platelet depletion, iNOS and gp91phox deficiencies, P-selectin blockade, antithrombin, or prophylactic ascorbate prevented, whereas delayed ascorbate, eptifibatide, tetrahydrobiopterin, or SNAP reversed, septic platelet adhesion and/or flow stoppage. The reversals by ascorbate and tetrahydrobiopterin were absent in eNOS −/− mice. Platelet adhesion predicted 90% of capillary flow stoppage.Conclusion-Impaired perfusion and/or platelet adhesion in septic capillaries requires NADPH oxidase, iNOS, P-selectin, and activated coagulation, and is inhibited by intravenous administration of ascorbate and by local superfusion of tetrahydrobiopterin and NO. Reversal of flow stoppage by ascorbate and tetrahydrobiopterin may depend on local eNOS-derived NO which dislodges platelets from the capillary wall.

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.

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Redox regulation of inducible nitric oxide synthase (iNOS) expression was investigated in lipopolysaccharide and interferon-γ (LPS+IFNγ)-stimulated microvascular endothelial cells from mouse skeletal muscles. Unstimulated endothelial cells produced reactive oxygen species (ROS) sensitive to inhibition of NADPH oxidase (apocynin and DPI), mitochondrial respiration (rotenone) and NOS (L-NAME). LPS+IFNγ caused a marked increase in ROS production; this increase was abolished by inhibition of NADPH oxidase (apocynin, DPI and p47phox deficiency). LPS+IFNγ induced substantial expression of iNOS protein. iNOS expression was prevented by the antioxidant ascorbate, NADPH oxidase inhibition (apocynin, DPI and p47phox deficiency), but not by inhibition of mitochondrial respiration (rotenone) and xanthine oxidase (allopurinol). iNOS expression also was prevented by selective antagonists of ERK, JNK, Jak2, and NFκB activation. LPS+IFNγ stimulated activation/phosphorylation of ERK, JNK, and Jak2 and activation/degradation of IκB, but only the activation of JNK and Jak2 was sensitive to ascorbate, apocynin and p47phox deficiency. Ascorbate, apocynin and p47phox deficiency also inhibited the LPS+IFNγ-induced DNA binding activity of transcription factors IRF1 and AP1 but not NFκB. In conclusion, LPS+IFNγ-induced NFκB activation is necessary for iNOS induction but is not dependent on ROS signaling. LPS+IFNγ-stimulated NADPH oxidase activity produces ROS that activate the JNK-AP1 and Jak2-IRF1 signaling pathways required for iNOS induction. Since blocking either NFκB activation or NADPH oxidase activity is sufficient to prevent iNOS expression, they are separate targets for therapeutic interventions that aim to modulate iNOS expression in sepsis.

Inducible nitric oxide synthase (iNOS) expression in blood vessels contributes to the vascular hyporeactivity characteristic of sepsis. Our previous work demonstrated in vitro that ascorbate inhibits iNOS expression in lipopolysaccharide- and interferon-gamma-stimulated skeletal muscle endothelial cells (ECs) through an antioxidant mechanism. The present study evaluated in vivo the hypothesis that administration of ascorbate decreases oxidative stress, prevents endothelial iNOS expression, and improves vascular reactivity in septic skeletal muscle. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Plasma nitrite and nitrate (NOx) levels were elevated by 6 h after CLP. Prior ascorbate bolus injection (200 mg/kg body wt iv) blocked the elevation of plasma NOx and abolished the expression of iNOS protein and activity in the septic skeletal muscle. We also demonstrated that iNOS mRNA determined by RT-PCR was induced in the microvascular ECs of the muscle at 3 h after CLP. This induction was attenuated by prior ascorbate administration. Ascorbate inhibition of iNOS expression was associated with decreased oxidant levels in the septic muscle. Moreover, ascorbate administration restored partially the baseline arterial pressure and preserved completely the microvascular constriction and arterial pressure responses to norepinephrine in CLP mice. These results suggest that early administration of ascorbate may be a valuable adjunct treatment of sepsis.

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