Characterization of a hyperdynamic murine model of resuscitated sepsis using echocardiography.

A small animal model of sepsis that reproduces the vasodilation, hypotension, increased cardiac output, and response to treatment seen in patients with septic shock would be useful for studies of pathophysiology and treatment, but no current models replicate all of these features. Mice were made septic by cecal ligation and puncture and resuscitated with fluids and antibiotics every 6 h. Blood pressure was measured in anesthetized mice with manometric catheters, and echocardiography was performed in these animals every 6 h. Survival in treated septic mice was improved compared with untreated mice (44% versus 0%, p < 0.01). In control mice, heart rate (HR, 420 +/- 31 beats/min), mean arterial pressure (Pa, 100 +/- 8 mm Hg), stroke volume (SV, 26 +/- 4 microl), and cardiac output (12.5 +/- 6.6 ml/min) were unchanged over 48 h. In septic mice Pa was significantly decreased (102 +/- 14 to 65 +/- 19 mm Hg, p < 0.02), starting at 12 h. HR and cardiac output increased significantly (HR, 407 +/- 70 to 524 +/- 76 beats/min, cardiac output, 11.6 +/- 2.0 to 17.1 +/- 1.5 ml/min, p < 0.01). SV (24 +/- 5 microl) remained constant. This fluid-resuscitated, antibiotic-treated model replicates the mortality, hypotension, and hyperdynamic state seen in clinical sepsis. Precise determination of serial hemodynamics in this model may be useful to elucidate pathophysiologic mechanisms and to evaluate new therapies for septic shock.

Evidence of transcellular permeability pathway in microvessels.

We used video fluorescence microscopy of the vascular bed in the cremaster muscle of rat and mouse to study the transfer of plasmalemma vesicles (caveolae) across the microvessel barrier in situ. The water-soluble styryl pyridinium dye RH414, which adsorbs to and fluoresces at the membrane-water interface, was used as a marker for vesicular traffic through endothelial cells. Fluorescein isothiocyanate (FITC), similar in molecular size to the styryl pyridinium probe, was used to mark for dye transfer by the paracellular pathway. Transcellular dye flux was determined by comparing the fluorescence intensities of RH414 and FITC on either side of the vessel wall (i.e., in microvessel lumen and in muscle tissue at various distances from the microvessel wall). We observed that RH414 accumulated in the interstitium more rapidly than FITC. We next studied the role of the 60-kDa albumin-binding glycoprotein gp60, hypothesized to activate transcellular permeability, in stimulating the transcellular vesicle traffic. Introduction of anti-gp60 antibody into the microvessel to cross-link and activate gp60 markedly increased the transvascular flux of RH414. Control isotype-matched antibody had no effect on the RH414 flux. The sterol-binding agent filipin, which disassembles caveolae, inhibited the RH414 flux induced by gp60 cross-linking. The transfer of styryl pyridinium dyes in intact microvessels suggests that plasmalemmal membrane traffic across the skeletal muscle microvessel barrier is a constitutively active process. The results indicate that the gp60-dependent pathway is important in regulating endothelial permeability in situ via a transcellular mechanism.

Nitric oxide synthase inhibition increases venular leukocyte rolling and adhesion in septic rats.

OBJECTIVE: Excess production of nitric oxide (NO) has been implicated in hypotension and blood flow abnormalities in sepsis, but NO is also an important inhibitor of leukocyte rolling and adhesion. Leukocyte adhesion is increased in sepsis despite elevated NO production. We hypothesized that inhibition of NO synthase (NOS) could increase leukocyte adhesion in sepsis. DESIGN: Prospective animal study. SETTING: Experimental animal laboratory. SUBJECTS: Twenty-five male rats, anesthetized with ketamine and acepromazine. INTERVENTIONS: Topical superfusion of the nonselective NOS inhibitor N(G)-monomethyl-L-arginine (NMA) on skeletal muscle postcapillary venules. MEASUREMENTS AND MAIN RESULTS: Rats made septic by cecal ligation and puncture were compared with controls that underwent sham ligation. Leukocyte rolling and adhesion were measured in cremasteric postcapillary venules of septic and control rats using in vivo videomicroscopy. The effects of NOS inhibition on leukocyte rolling and adhesion were also measured. After a stable baseline was reached, 1 microM of the nonselective NOS inhibitor NMA was suffused topically followed by physiologic buffer. The effects of L-arginine on leukocyte rolling and adhesion were also measured, both before and after suffusion of NMA. Leukocyte rolling and adhesion was increased in septic rats as compared with controls (control 5.5+/-0.9 rolling cells/min, 1.0+/-0.3 adherent cells/min; septic 13.7+/-2.0 rolling cells/min, 3.1+/-0.6 adherent cells/min; p < .001), and NOS inhibition further increased leukocyte rolling and adhesion in both septic and control rats (control 14.0+/-1.7 rolling cells/min, 2.8+/-0.5 adherent cells/min; septic 25+/-2.1 rolling cells/min, 5.4+/-0.5 adherent cells/min; both p < .001 vs. baseline). Prior suffusion of excess L-arginine prevented the increase in leukocyte adhesion with NMA in septic rats (2.6+/-0.4 adherent cells/min vs. 3.0+/-0.6 adherent cells/min; n = 3; p > .05). When administered after NMA, excess L-arginine partially reversed leukocyte adhesion in septic rats (5.4+/-0.7 adherent cells/min, with NMA vs. 4.3+/-0.7 adherent cells/min, after L-arginine; n = 5; p < .05). Venular shear did not differ between septic and control rats (600+/-109 (sec(-1)) vs. 620+/-37 (sec(-1)); p > .05). CONCLUSIONS: Although NOS inhibition may ameliorate hypotension in sepsis, such therapy may be deleterious by increasing leukocyte adhesion.

Effects of nitric oxide synthase inhibition on microvascular reactivity in septic mice.

Persistent vasodilation refractory to vasopressor agents is characteristic of septic shock. Induction of nitric oxide synthase (NOS) by sepsis-induced cytokines within the vasculature is one of the primary mediators of this refractory vasodilation. To evaluate the mechanism of vasodilation in sepsis, we used in vivo videomicroscopy to measure microvascular vasoconstrictive responses to topical suffusion of norepinephrine in mice made septic by cecal ligation and puncture, and contrasted the effects of topical superfusion of the nonselective NOS inhibitor N(G)-methyl-L-arginine (L-NMMA) and the selective inducible NOS (iNOS) inhibitor S-methyl-isothiourea (SMT). Mice with sepsis were less sensitive to the vasoconstrictive effects of norepinephrine than controls (EC50, the concentration that produces half-maximal response 2.0+/-0.6 x 10(-6) M vs. 7.9+/-2.2 x 10(-8) M, P=0.01). Selective inhibition of inducible iNOS with topical SMT (100 microM) markedly increased catecholamine reactivity in mice with sepsis but did not affect reactivity in controls (P=0.0007 for sepsis, P=0.24 for controls). Nonselective NOS inhibition with topical L-NMMA produced a similar increase in catecholamine reactivity in mice with sepsis but not controls (P=0.001 for sepsis, P=0.56 for controls). When excess (1 mM) L-arginine, the substrate for NOS, was added to the superfusion buffer along with both SMT and L-NMMA, arteriolar responsiveness to norepinephrine was decreased to the original values. These experiments demonstrate that iNOS inhibition is as effective as nonselective NOS inhibition in reversing decreased catecholamine reactivity in sepsis. This suggests a crucial role for microvascular activation of iNOS in the pathophysiology of hypotension and decreased vasopressor responsiveness in sepsis.

Discordant epicardial and microvascular endothelial responses in heart transplant recipients early after transplantation.

BACKGROUND: Cardiac allograft vasculopathy represents the leading cause of death in heart transplant recipients who survive more than 1 year. Functional endothelial abnormalities are sensitive measures of the early development of cardiac allograft vasculopathy, but the relative importance of large and small coronary vessel abnormalities has not been evaluated. The purpose of the study was to distinguish between large and small coronary endothelial dysfunction in patients early after heart transplantation and to test the hypothesis that microvascular endothelial responses can be preserved in the presence of epicardial endothelial dysfunction. METHODS: Changes in epicardial lumen area and coronary artery blood flow in response to intracoronary administration of adenosine, acetylcholine, and nitroglycerin were measured simultaneously by use of an intravascular ultrasound catheter positioned over a Doppler flow wire in the left anterior descending coronary artery. The combination of these techniques allowed distinction between large and small coronary vascular responses. In 19 patients studied early after transplantation, adenosine (16 and 32 microg), acetylcholine (5.4 and 54 microg), and nitroglycerin (200 microg) were infused, with continuous intravascular ultrasound imaging and Doppler velocity measurements. RESULTS: Acetylcholine induced paradoxical epicardial vasoconstriction in 12 of 19 patients (73% +/- 6% of baseline); vasodilation occurred in 7 (108% +/- 3%). In spite of this constriction, coronary artery flow increased in all 19 patients, to the same extent in patients with constriction and those with dilation (239 +/- 26 vs 193 +/- 20, p = 0.38). Adenosine and nitroglycerin increased area (107% +/- 1% and 112% +/- 3%) and flow (258% +/- 17% and 197% +/- 11%) in all patients. None of the area or flow responses correlated with the degree of intimal thickening. CONCLUSIONS: Acetylcholine increased coronary artery flow early after transplantation, indicating preserved microvascular responses in spite of epicardial vasoconstriction. Simultaneous measurement of area and velocity responses, by permitting evaluation of the relative contribution of epicardial and microvascular vessels, may offer unique insights into coronary endothelial function.

Impaired microvascular vasoconstrictive responses to vasopressin in septic rats.

OBJECTIVE: To evaluate mechanisms of vasodilation in sepsis by comparing responses of resistance arterioles to vasopressin in rat cremaster muscle of septic and control rats. DESIGN: Prospective, experimental study. SETTING: Experimental animal laboratory. SUBJECTS: Twenty male rats, anesthetized with ketamine and acepromazine. INTERVENTIONS: Topical superfusion of vasoactive compounds on skeletal muscle resistance arterioles. MEASUREMENTS AND MAIN RESULTS: The effect of sepsis on responses to local application of vasopressin was investigated using in vivo videomicroscopy. Vasopressin was superfused topically on the cremaster muscle resistance arterioles (15 to 25 microns) of rats made septic by cecal ligation and puncture, and the responses were compared with the responses of controls that underwent sham ligation. Responses to topically suffused vasopressin were also assessed in septic and control rats, before and after superfusion of the muscle with the nitric oxide synthase inhibitor NG-methyl-L-arginine (NMA). Sepsis produced a decrease in the vasoconstrictive effects of vasopressin; the maximal response was lower, and the concentration-response curve was shifted to the right in septic rats (p < .05). Contractions at vasopressin concentrations of 0.01, 1, and 10 nM were 39%, 36%, and 40%, respectively, of sham controls. Superfusion of the muscle with NMA partially restored arteriolar responsiveness in the septic rats, significantly increasing the arteriolar constriction of the septic rats in response to vasopressin. This effect was reversed with superfusion of excess L-arginine (1 mM). CONCLUSIONS: This study illustrates the reduced responsiveness of the resistance arterioles of septic rats in response to vasopressin in vivo, and the partial restoration of responsiveness by concurrent application of NMA. In previous studies using this model, we have shown similar results using norepinephrine and endothelin-1, as well as angiotensin II. These findings, and the findings of this study, suggest a generalized abnormality in responsiveness of resistance arterioles to endogenous vasoconstrictors in sepsis. Partial reversal of this abnormality with NMA supports an important role for nitric oxide in mediating abnormal vasopressor responsiveness in sepsis.

Rickets and protein malnutrition in northern Nigeria.

The aim of the study was to explore the relationship between protein nutritional status and the development of rickets in children living in northern Nigeria. The diagnosis of rickets in 16 children between the ages of 10 months and 7 years was confirmed using established, and recently developed clinical and biochemical parameters. Twenty-seven children devoid of skeletal stigmata were age- and sex-matched to the rachitic patients. A battery of clinical laboratory and anthropometric measurements designed to assess calcium homeostasis, skeletal growth, the extent of bone remodeling or resorption, and protein nutritional status were performed on all subjects. Our central finding was that although the rachitic children were moderately malnourished, their protein nutritional status was significantly better as measured by the serum prealbumin concentration (15.4 v. 12.5 mg/dl, P = 0.0012) when compared with the severely malnourished children who were devoid of any indication of rickets. This may be due, in part, to the fact that actively growing children are more likely to develop rickets than are children whose linear growth is impeded. Unexpectedly, we found that the mean concentrations of serum 1,25-dihydroxyvitamin D in both the rachitic and control group were higher than any values for the active vitamin D metabolite previously reported in the literature.

Serum beta-carotene deficiency in HIV-infected children.

Representative levels of serum micronutrients specifically, beta-carotene and vitamins A and E, were studied in symptomatic human immunodeficiency virus (HIV)-infected children. The nutritional status of 23 symptomatic African-American and Hispanic HIV-infected children were compared with an appropriate control group comprised of 36 uninfected children matched for age and sex, using body mass index. Serum beta-carotene and vitamin A and E levels were randomly determined on 15 of the infected children. Beta-carotene concentration was 4.9-fold reduced in symptomatic HIV-infected children when compared with the control group. There was a 6.5-fold decrease in the serum level for children without acquired immunodeficiency syndrome (AIDS) and a 13-fold reduction in children with AIDS. No differences in the mean values for serum vitamins A and E were observed in the groups studied. Although the nutritional status of the symptomatic HIV-infected children was not different from that of the control population, their serum beta-carotene levels were profoundly deficient. This finding may have immunologic and clinical implications for children with rapidly progressing HIV disease.

Drug interaction with gamma-aminobutyric acid/benzodiazepine receptors at the ventral surface of the medulla results in pronounced changes in cardiorespiratory activity.

Studies were carried out in chloralose-anesthetized cats while monitoring respiratory (tidal volume and respiratory rate) and cardiovascular (arterial pressure and heart rate) activity. Midazolam applied bilaterally to the intermediate area of the ventral surface of the medulla in doses of 0.75, 7.5 and 75 micrograms/side reduced tidal volume by -6 +/- 3, -10 +/- 1 and -11 +/- 1 ml, respectively. A dose of 250 micrograms/side produced apnea in each animal tested. Corresponding changes in arterial pressure were -35 +/- 9, -44 +/- 6, -43 +/- 9 and -64 +/- 17 mm Hg, respectively. Larger doses of chlordiazepoxide (e.g., 1000 micrograms/side) were required to produce similar effects. Intravenous administration of midazolam in doses of 1.5 to 150 micrograms had no significant effect on cardiorespiratory activity. However, larger doses of midazolam given i.v. produced cardiorespiratory depression that was similar to that observed with centrally applied drug. Pretreatment or treatment with centrally applied flumazenil or bicuculline counteracted the cardiorespiratory effects of centrally applied midazolam. Most importantly, ventral surface application of flumazenil counteracted the cardiorespiratory depressant effects of i.v. midazolam. Central administration of ethyl-beta-carboline-3-carboxylate produced cardiorespiratory effects opposite to those seen with midazolam, and these stimulatory effects were also counteracted by centrally applied flumazenil. These results indicate that alterations in cardiorespiratory activity can be produced by drugs interacting with gamma-aminobutyric acid/benzodiazepine receptors at the ventral surface of the medulla.