四氢生物喋呤在脓毒血症中的效果

Crit Care Med 2012 Vol. 40, No. 10 1 Copyright (c) Society of Critical Care Medicine and Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Sepsis, the systemic inflamma-tory response to an infection, induces disturbances in both the macro- and the microcircu- lation. Despite early global hemodynamic resuscitation and optimization of con- ventional macrohemodynamics, patients with severe sepsis often have an impaired microcirculation (1, 2), and these micro- circulatory alterations may contribute to the development of multiple organ failure and death (3–6). Although the microcir- culation can be improved by fluids (7), dobutamine (8), or activated protein C (9), these effects may be quite variable or limited by restrictions to the use of the agents. Accordingly, investigation into the use of other agents needs to be encouraged. Endothelial dysfunction is one factor implicated in the development of microvascular alterations (10–13), and the severity of endothelial dysfunction is associated with organ dysfunction and poor outcome (6, 14). Tetrahydrobiopterin (BH4) is an essential cofactor required for the activity of all nitric oxide synthases (NOS), in particular endothelial NOS (eNOS) (15). Suboptimal concentrations of BH4 in the endothelium cause uncoupling of eNOS and increased eNOS-derived production of reactive oxygen species (ROS) (16, 17), contributing to dysfunction of the microvascular endothelium (18). Exogenous BH4 may exert beneficial effects by restoring coupling of eNOS and attenuating oxidative stress. In experimental models of ischemia/ reperfusion injury, BH4 administration improved microvascular perfusion (19, 20). In patients with coronary artery disease, intracoronary infusion of BH4 improved acetylcholine-induced microvascular flow response (21). In humans challenged with low dose glucose, BH4 restored the forearm blood flow response to acetylcholine (22). In rodent models of sepsis, BH4 improved the sepsis-altered microcirculation, organ function, and survival (23–26). However, the effects of BH4 in septic shock have not yet been defined. We hypothesized that supplementation with BH4 may reduce microvascular endothelial dysfunction to improve outcome from septic shock. The aim of the present pilot study was thus to observe the effects of exogenous BH4 administration on global hemodynamics, oxygen consumption, microcirculation, and survival time in a clinically relevant ovine model of peritonitis-induced septic shock. METHODS Experimental Preparation. The study was approved by our Institutional Review Board for animals. Care and handling of the animals were in accord with the National Institute of Health Guidelines (Institute of Laboratory Copyright © 2012 by the Society of Critical Care Medicine and Lippincott Williams and Wilkins DOI: 10.1097/CCM.0b013e31825b88ba Objective: Supplementation with tetrahydrobiopterin, a nitric oxide synthase cofactor, may reduce microvascular endothelial dysfunction in severe sepsis. We studied whether tetrahydrobiopterin administra- tion exerts beneficial effects in an ovine septic shock model. Design: Randomized animal study. Setting: University hospital animal research laboratory. Subjects: Fourteen adult female sheep. Interventions: Fecal peritonitis was induced, and the sheep were randomized to receive tetrahydrobiopterin (n = 7), given intrave- nously as 20 mg/kg boluses at 4 and 12 hrs after sepsis induction, or placebo (n = 7). All animals were fluid resuscitated. The experi- ment was continued until death or for a maximum of 30 hrs. Measurements and Main Results: In addition to standard hemodynamic assessment, the sublingual microcirculation was evaluated using sidestream dark-field videomicroscopy. The first bolus of tetrahydrobiopterin blunted the increase in heart rate and cardiac index seen in the control group without affecting mean arterial pressure, and the second bolus of tetrahydrobiopterin prevented the decreases in cardiac index and mean arterial pres- sure. The reduction in mixed venous blood oxygen saturation and the increase in blood lactate seen in the control group were also delayed. Tetrahydrobiopterin significantly attenuated the deterio- ration in perfused small vessel proportion and density, microvas- cular flow index, and the increase in microvascular heterogeneity observed in the control group. Tetrahydrobiopterin was associated with better preserved lung compliance and Pao2/Fio2 ratio, which were associated with a lower lung wet/dry weight ratio at the end of the study. Median survival time was significantly prolonged in the tetrahydrobiopterin group (25.0 vs. 17.8 hrs, p < .01). Conclusion: In this clinically relevant model of sepsis, tetra- hydrobiopterin supplementation attenuated the impairment in sublingual microvascular perfusion and permeability, which was accompanied by better preserved gas exchange, renal flow and urine output, and prolonged survival. (Crit Care Med 2012; 40:0–0) Key Words: hemodynamics; microcirculation; pulmonary gas exchange; septic shock; tetrahydrobiopterin Administration of tetrahydrobiopterin improves the microcirculation and outcome in an ovine model of septic shock Xinrong He, MD; Fuhong Su, MD, PhD; Dimitrios Velissaris, MD; Diamantino Ribeiro Salgado, MD; Dalton de Souza Barros, MD; Sophie Lorent, PharmD; Fabio Silvio Taccone, MD; Jean-Louis Vincent, MD, PhD, FCCM; Daniel De Backer, MD, PhD From the Department of Intensive Care, Erasme University Hospital, Université Libre de Bruxelles, Brussels, Belgium. Supported, in part, by institutional funds. The authors have not disclosed any potential conflicts of interest. For information regarding this article, E-mail: jlvincen@ulb.ac.be 2 Crit Care Med 2012 Vol. 40, No. 10 Copyright (c) Society of Critical Care Medicine and Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Animal Resources). Experiments were per- formed on 14 female sheep. Prior to surgery, the animals were intramuscularly premedi- cated with midazolam (Dormicum, Roche SA, Belgium) (0.25 mg/kg) and ketamine hy- drochloride (Imalgine, Merial, Lyon, France) (20 mg/kg). The cephalic vein was then can- nulated with a peripheral venous 18G catheter (Surflo I.V Catheter, Terumo, Belgium). After intravenous injection of fentanyl (Fentanyl, Janssen, Berchem, Belgium) (30 µg/kg) and rocuronium (Esmeron, Organon, Oss, the Netherlands) (0.1 mg/kg), the trachea was intubated (Tracheal Tube, 8.0; Hi-Contour, Mallinckrodt Medical, Ireland) and mechani- cal ventilation started in volume-controlled mode (Servo ventilator 900 C, Siemens-Elema, Sweden) with a tidal volume of 10 mL/kg, a positive end-expiratory pressure of 5 cmH2O, an inspiratory time/expiratory time of 1:2, and a square wave pattern. Thereafter, anesthesia was achieved with a continuous intravenous infusion of the mixture of ketamine (10 mg/ kg/hr), morphine (0.5 mg/kg/hr), midazolam (0.5 mg/kg/hr), and rocuronium (0.1 mg/kg/ hr). The stomach was emptied with a 60-cm plastic tube (inner diameter 1.8 cm), and a Foley catheter (14F, Beiersdorf AG, Germany) was placed in the bladder for urine output (UO) monitoring. The right femoral artery and vein were surgically exposed and an arte- rial catheter (6F Vygon, Cirencester, UK) and vein introducer positioned. A 7F pulmonary artery catheter (Edwards Life Sciences, Baxter, Irvine, CA) was advanced into the pulmonary artery via the introducer guided by monitor- ing pressure waveforms. The catheters were connected to pressure transducers (Edwards Lifescience, CA) zeroed at the midthorax level and the pressures, core temperature, and car- diac output were measured by Sirecust 404 (Siemens, Germany) and a Vigilance monitor (Edwards Critical-Care, CA). A colotomy was performed through a midline laparotomy, for collection of 1.5 g/kg body weight of feces. The cecum was then closed by a pouch suture and replaced in the cavity after disinfection around the cut. Ultrasonic Doppler flow probes were placed around the superior mesenteric ar- tery and the left renal artery to continuously measure the regional blood flow by flowmeter (T208, Transonic Systems, Ithaca, NY). A plastic tube (Beldico, Marche-en-Famenne, Belgium) was inserted through the abdominal wall for later injection of feces, and the abdo- men was then closed in two layers by end-knot continuous sutures. After surgery, animals were placed prone. Ringer’s lactate solution and 6% hydroxyethyl starch solution (Voluven, MW130,000) were initially infused at a rate of 2 mL/kg/hr via the cephalic vein catheter. Fluid resuscitation was titrated to maintain stable pulmonary arterial occlusion pressure and to prevent hypovolemia. When hypotension (defined as mean arterial pressure [MAP] <75 mm Hg) occurred and did not respond to a fluid challenge of 100 mL Ringer’s lactate solu- tion plus 100 mL hydroxyethyl starch solution for 6 mins, the fluid infusion rate was gradu- ally reduced to 2 mL/kg/hr. Vasopressor agents and inotropic agents were not used in this model. Potassium chloride was administered for hypokalemia (<3.5 mEq/L) and glucose for hypoglycemia (<2.21 mmol/L). Experimental Protocol. The sheep were randomized into two groups. The BH4 group (n = 7) received two boluses of BH4 ((6R,S)- 5,6,7,8-tetrahydro-L-biopterin dihydrochloride, Schircks Laboratories, Jona, Switzerland) 4 hrs and 12 hrs after injection of feces into the ab- domen. The control group (n = 7) received the equivalent of 0.9% saline solution instead of BH4 at the same time points. BH4 was prepared as a 20 mg/mL solution by a clinical pharmacist and infused as a 20 mg/kg/bolus (i.e., 1 mL/kg/ bolus) at a rate of 100 mL/hr, via the femoral vein. Animals were observed until death or for a maximum of 30 hrs, when they were sacrificed by potassium chloride injection. After baseline measurements, 1.5 kg/body weight feces was injected into the abdominal cavity to induce peritonitis. Measurements were repeated hourly throughout the experiment. Heart rate, MAP, mean pulmonary artery pres- sure, cardiac output, core temperature, ventila- tor parameters, renal and superior mesenteric arterial blood flow were monitored continu- ously; right atrial pressure, pulmonary arterial occlusion pressure, arterial and mixed-venous blood gases, hemoglobin concentration, blood lactate and electrolyte concentrations (ABL725 and OSM3, Radiometer Medical A/S, Denmark) were measured hourly. Derived variables, such as cardiac index, stroke volume index, systemic and vascular resistance index (pulmonary vas- cular resistance index), left ventricular stroke work index, oxygen delivery index, oxygen con- sumption index, and oxygen extraction index were calculated using standard formulas (27). The body surface area of the sheep was calcu- lated from the equation, body surface area = 0.084 × [body weight (kg)]2/3 (28). UO was mea- sured hourly with a graduated cylinder, and the infused fluid volume (IFV) between two points was recorded. The fluid balance (BL) between two points was estimated using the equation BLab = IFVab – UOab + BLa. Cumulative BL com- bined with hematocrit was used to estimate global capillary leakage. The sublingual microvascular network was visualized using Sidestream Dark-Field video- microscopy (MicroScan, MicroVision Medical, The Netherlands) with a 5x imaging objective giving 380x magnification. Five videos last- ing 20 secs at each time point were acquired at baseline, and after 4, 6, 12, 14, 18, and 24 hrs. The videos were given random numbers for blinded off-line analysis. The proportion of perfused small vessels (PPVs), the perfused small vessel density (an estimate of functional capillary density), microvascular flow index, and the heterogeneity index of PPV (htPPV, calculated as the difference between extreme values of PPV from five recordings divided by the mean value) were evaluated according to the methods described by De Backer et al (29). When the sheep died, the central lobe of the right lung was excised and its wet weight determined. Its dry weight was also measured after the lobe had been dehydrated for 24 hrs in an oven at 200°C. The wet/dry weight ratio was used as an estimate of pulmonary edema severity. Statistical Analysis. Data were checked for normality using the Shapiro–Wilk test. As no deviation from normality was detected, data are reported as mean ± sd. Data were ana- lyzed using linear mixed models with group, time, and group × time interactions as fixed effects and subjects (sheep) as a random effect. Each time point difference between groups was compared with a Bonferroni adjustment in case of a significant group effect and/or of a significant time × group interaction. Each time point difference compared with baseline was performed with a Bonferroni adjustment in case of a significant time effect in each group. Kaplan–Meier survival curves were constructed and analyzed by the log-rank test. Statistical tests were two-tailed, and differ- ences were considered to be statistically sig- nificant at the 5% level. The statistical analyses were performed with IBM SPSS Statistics 19 (IBM Corp, Armonk, NY) software. RESULTS Systemic Hemodynamics. Core tem- perature, heart rate, and cardiac index increased after induction of peritonitis in the control group, whereas MAP and sys- temic vascular resistance index progres- sively decreased. Venous blood oxygen saturation progressively decreased and blood lactate levels increased. In the BH4 group, the initial evolution was similar. The first bolus of BH4 at 4 hrs blunted fur- ther increase in heart rate, cardiac index, stroke volume index, and oxygen delivery index, with subsequent values being lower than those in the control group (although these differences were not statistically significant, except for cardiac index). After the second bolus of BH4 at 12 hrs, cardiac index, MAP, oxygen delivery index, stroke volume index, and left ventricular stroke work index decreased later and more slowly in the BH4 group than in the control group (p = nonsignificant, Fig. 1). The decrease in venous blood oxygen sat- uration and the increase in lactate were delayed and less pronounced after BH4 administration compared with controls, although the group differences were not statistically significant. The increases in oxygen consumption index and oxy- gen extraction index were significantly delayed in the BH4 group compared with the control animals (p < .05). There were no significant differences in the evolu- tions of mean pulmonary artery pressure, Crit Care Med 2012 Vol. 40, No. 10 3 Copyright (c) Society of Critical Care Medicine and Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. systemic vascular resistance index, or pulmonary vascular resistance index between the two groups (Table 1). Regional Perfusion. Mesenteric and renal blood flows increased in the initial 8 hrs and then decreased rapidly in the con- trol group. The decrease in these flows was delayed in the BH4 group (Table 1). Microcirculatory Variables and Capil- lary Leakage. In the control group, PPV, perfused small vessel density, and micro- vascular flow index progressively decreased whereas the htPPV increased. However, after administration of BH4, PPV, perfused small vessel density, and microvascular flow index remained stable and signifi- cantly higher than in the control group. As such, the htPPV increased less in the BH4 group than in the control group (Fig. 2). Even though filling pressures were similar in both groups, cumulative BL was less positive in the BH4 than in the control group, and hematocrit was lower in BH4- treated than in control animals, suggest- ing less capillary leakage (Fig. 3). Organ Function Variables. Pao2/Fio2 ratio and lung compliance progressively decreased in the two groups, but BH4 delayed these alterations and BH4-treated animals had better oxygenation up to the end of the experiment. The changes in pH and Pco2 were not significantly different (Table 1). Autopsy showed that the lung wet/dry weight ratio was lower in the BH4 group than in the control group (5.9 ± 0.9 vs. 11.8 ± 1.4, t = 11.50, p < .05). Renal blood flow and UO were also better pre- served in BH4 than in control animals despite similar cumulative infusion vol- ume and filling pressures (Table 1, Fig. 3). Outcome. The survival time was sig- nificantly longer in the BH4 group than in the control group (median survival time 25.0 [22.5, 28.0] hrs vs. 17.8 [17.1, 19.0] hrs) (Log rank χ2 = 14.51, p < .001) (Fig. 4). DISCUSSION In this sheep model of sepsis, we observed that BH4 administration atten- uated the impairment in sublingual microvascular perfusion and vascular per- meability, and this was accompanied by a better preservation of gas exchange, renal flow, and UO and by prolonged survival. This peritonitis-induced septic model presents many clinical features of human sepsis development, and these were already present at the time of the first BH4 administration. Indeed, already 4 hrs after feces injection, typical sepsis symptoms were present, including fever, tachycardia, and a hyperkinetic state with some decrease in arterial pressure and a substantial increase in cardiac output, reflecting a decrease in systemic vascu- lar resistance. We have also previously demonstrated a significant increase in blood interleukin-6 concentration at that time point in this model (30). Our study revealed that BH4 administration in this septic model blunted the increase in car- diac output before shock development and slowed its later decrease; it also blunted the decline in venous blood oxygen satu- ration and the increase in oxygen extrac- tion index. Interestingly, hypotension did not occur after BH4 administration, which is in line with the observations that BH4 administration did not alter MAP in rats with endotoxic shock (25). Figure 1. Evolution of heart rate (HR), cardiac index (CI), mean arterial pressure (MAP), and blood lactate concentration in tetrahydrobiopterin (BH4)-treated (open circles) and control (solid triangles) animals. Arrows indicate times of bolus doses of BH4. Group effects were nonsignificant, time effects were significant (p < .01, <.05 for HR), and group × time interaction effects were not significant except for CI (p < .05). # p < .05 compared with control group; $ p < .05 compared with baseline in control group; § p < .05 compared with baseline in BH4 group. 4 Crit Care Med 2012 Vol. 40, No. 10 Copyright (c) Society of Critical Care Medicine and Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. In severe sepsis or septic shock, severely disturbed microcirculatory com- ponents and shunted microcirculatory units lead to regional hypoperfusion and regional dysoxia despite aggressive initial global hemodynamic resuscitation, and are associated with progression to organ failure (1, 3, 31). In patients with septic shock, early increases in microcirculatory perfusion during protocol-directed resus- citation were associated with reduction of sepsis-induced multiorgan failure (32). We observed that BH4 administration blunted the decrease in perfused vascular density, PPV and microvascular flow index, and reduced the heterogeneity in microcircu- latory perfusion. BH4 therefore improved both convective and diffusive oxygen transport at the microcirculatory level, indicating alleviation of microvascular shunting and improvement in regional perfusion. This improvement in tissue oxygen availability was also reflected by the bl