Effects of 1400W and/or nitroglycerin on renal oxygenation and kidney function during endotoxaemia in anaesthetized rats.

1. The pathogenesis of acute renal failure (ARF) in sepsis is multifactorial. The role of nitric oxide (NO) in septic ARF has been a source of controversy. We hypothesized that endotoxaemia-induced exacerbation of inducible nitric oxide synthase (iNOS)-related NO release impairs renal oxygenation and contributes to ARF in anaesthetized rats. 2. In the present study, rats received lipopolysaccharide (2.5 mg/kg) for 30 min. Two hours later, fluid resuscitation was started (HES130; 5 mL/kg per h after a 5 mL/kg bolus) supplemented either by the NO donor nitroglycerin (NTG; 0.5 µg/kg per min after a 2 µg/kg bolus), the selective iNOS inhibitor 1400 W (3 mg/kg per h after a 3 mg/kg bolus) or both. Systemic haemodynamics and renal microvascular Po2 (µPo(2)) were recorded continuously. Furthermore, creatinine clearance, plasma NO(x) (nitrate + nitrite + S-nitrosothiols) levels and the expression of iNOS mRNA were measured. 3. Endotoxaemia reduced renal blood flow, decreased mean arterial pressure, resulted in anuria and was associated with an increase in plasma NO(x) levels and renal iNOS expression. Renal µPo2 deteriorated gradually during endotoxaemia and there was a significant decrease in renal O(2) delivery and consumption. Manipulation of NO levels had no beneficial effect on systemic haemodynamics, renal µPo(2) or creatinine clearance over standard fluid resuscitation. The application of 1400 W+NTG significantly reduced plasma NO(x) levels compared with fluid resuscitation and NTG alone. 4. Neither iNOS inhibition, NO donation nor a combination of both showed beneficial effects on systemic haemodynamics, renal oxygenation and renal function compared with fluid resuscitation alone. Our results question the proposed key role of NO in the pathogenesis of septic ARF in rats.

Low-dose dexamethasone-supplemented fluid resuscitation reverses endotoxin-induced acute renal failure and prevents cortical microvascular hypoxia.

There is growing evidence that impairment in intrarenal oxygenation and hypoxic injury might contribute to the pathogenesis of septic renal failure. An important molecule known to act on the renal microvascular tone and therefore consequently being involved in the regulation of intrarenal oxygen supply is NO. The main production of NO under septic conditions derives from iNOS, an enzyme that can be blocked by dexamethasone (DEX). In an animal model of endotoxin-induced renal failure, we tested the hypothesis that inhibition of iNOS by low-dose DEX would improve an impaired intrarenal oxygenation and kidney function. Twenty-two male Wistar rats received a 30-min intravenous infusion of LPS (2.5 mg/kg) and consecutively developed endotoxemic shock. Two hours later, in 12 animals, fluid resuscitation was initiated. Six rats did not receive resuscitation; four animals served as time control. In addition to the fluid, six animals received a bolus of low-dose DEX (0.1 mg/kg). In these animals, the renal iNOS mRNA expression was significantly suppressed 3 h later. Dexamethasone prevented the appearance of cortical microcirculatory hypoxic areas, improved renal oxygen delivery, and significantly restored oxygen consumption. Besides a significant increase in MAP and renal blood flow, DEX restored kidney function and tubular sodium reabsorption to baseline values. In conclusion, treatment with low-dose DEX in addition to fluid resuscitation reversed endotoxin-induced renal failure associated by an improvement in intrarenal microvascular oxygenation. Therefore, low-dose DEX might have potential application in the prevention of septic acute renal failure.

A pilot open-label study of the efficacy of subanesthetic isomeric S(+)-ketamine in refractory CRPS patients.

OBJECTIVE: Complex regional pain syndrome (CRPS) is a severe neuropathic pain state that is often disproportionate to the initial trauma. Associated features are autonomic dysregulation, swelling, motor dysfunction, and trophic changes to varying degrees. Despite a multitude of treatment modalities, a subgroup of CRPS patients remain refractory to all standard therapies. In these patients, the disease may spread extraterritorially, which results in severe disability. A critical involvement of N-methyl-D-aspartate receptors (NMDARs) has been demonstrated both clinically and by animal experimentation. NMDA antagonists may be effective in many neuropathic pain states. In long-standing, generalized CRPS, we investigated the effects of S(+)-ketamine on pain relief and somatosensory features, assessed by quantitative sensory testing (QST). METHODS: Four refractory CRPS patients received continous S(+)-ketamine-infusions, gradually titrated (50 mg/day-500 mg/day) over a 10-day period. Pain intensities (average, peak, and least pain) and side effects were rated on visual analogue scales, during a 4-day baseline, over 10 treatment days, and 2 days following treatment. QST (thermo-, mechanical detection, and pain thresholds) was analyzed at baseline and following treatment. RESULTS: Subanesthetic S(+)-ketamine showed no reduction of pain and effected no change in thermo- and mechanical detection or pain thresholds. This procedure caused no relevant side effects. The lack of therapeutic response in the first four patients led to termination of this pilot study. CONCLUSION: S(+)-ketamine can be gradually titrated to large doses (500 mg/day) without clinically relevant side effects. There was no pain relief or change in QST measurements in this series of long-standing severe CRPS patients.

Efficacy of ketamine in anesthetic dosage for the treatment of refractory complex regional pain syndrome: an open-label phase II study.

OBJECTIVE: Advanced complex regional pain syndrome (CRPS) remains very difficult to treat. While subanesthetic low-dose ketamine has shown promise in early localized CRPS, its use in advanced CRPS has not been as effective. Since ketamine's analgesic potency and duration of effect in neuropathic pain are directly dose-dependant, we investigated the efficacy of ketamine in anesthetic dosage in refractory CRPS patients that had failed available standard therapies. METHODS: Twenty ASA I-III patients suffering from refractory CRPS received ketamine in anesthetic dosage over 5 days. Outcome criteria were pain relief, effect on the movement disorder, quality of life, and ability to work at baseline and up to 6 months following treatment. RESULTS: Significant pain relief was observed at 1, 3, and 6 months following treatment (93.5 +/- 11.1%, 89.4 +/- 17.0%, 79.3 +/- 25.3%; P < 0.001). Complete remission from CRPS was observed at 1 month in all patients, at 3 months in 17, and at 6 months in 16 patients. If relapse occurred, significant pain relief was still attained at 3 and 6 months (59.0 +/- 14.7%, P < 0.004; 50.2 +/- 10.6%, P < 0.002). Quality of life, the associated movement disorder, and the ability to work significantly improved in the majority of patients at 3 and 6 months. CONCLUSIONS: This open-label trial suggests benefit in pain reduction, associated CRPS symptoms, improved quality of life and ability to work following anesthetic ketamine in previously refractory CRPS patients. However, a randomized controlled trial will be necessary to prove its efficacy.

Activity of the lipoxygenase inhibitor 1-phenyl-3-pyrazolidinone (phenidone) and derivatives on the inhibition of adhesion molecule expression on human umbilical vascular endothelial cells.

Leukocyte adhesion contributes to perfusion abnormalities and tissue damage during trauma, shock or overwhelming inflammation. This study was performed to determine whether the lipoxygenase inhibitor phenidone and derivatives decrease the expression of adhesion molecules on tumor necrosis factor-alpha (TNF-alpha) stimulated endothelial cells and attenuate leukocyte-endothelial interactions under flow in vitro. TNF-alpha stimulated human umbilical venous endothelial cells (HUVECs) were incubated with phenidone, 4-methyl-phenidone, 4-4-dimethyl-phenidone, 5-methyl-phenidone, 5-phenyl-phenidone, and 5-methyl-1,(2,5-di-chloro-phenyl)-3-pyrazolidone. We tested the inhibition of adhesion molecule expression at different inhibitor concentrations before, during, and after the stimulation of HUVECs. The inhibition of endothelial cell expression on HUVECs was measured by flow cytometry. Rolling and firm adhesion of leukocytes to pretreated endothelium was examined in a parallel plate flow chamber. Phenidone inhibited the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial-leukocyte adhesion molecule-1 on HUVECs when added prior to HUVEC stimulation. The inhibitory effect of phenidone was still observed when added simultaneously, but not when added after HUVEC stimulation. 4-4-dimethyl-phenidone and 5-phenyl-phenidone inhibited the expression of adhesion molecules more effectively than phenidone. The attenuation of leukocyte rolling under flow conditions was also significantly more effective with 4-4-dimethyl-phenidone than with phenidone. Lipoxygenase inhibitors might be of therapeutically interest for the treatment of overwhelming systemic inflammation during shock, trauma, and sepsis.

Effect of hydroxyethyl starch on vascular leak syndrome and neutrophil accumulation during hypoxia.

OBJECTIVE: Several studies have suggested that intravenous hydroxyethyl starch treatment may dampen acute inflammatory responses. It is well documented that limited oxygen delivery to tissues (hypoxia) is common in acute inflammation, and numerous parallels exist between acute responses to hypoxia and to inflammation, including the observation that both are associated with increased vascular leakage and neutrophil infiltration of tissues. Therefore, we compared functional influences of hydroxyethyl starch on normoxic or posthypoxic endothelia. DESIGN: Laboratory study. SETTING: University hospital. SUBJECTS: Cultured human microvascular endothelial cells and mice (C57BL/6/129 svj). INTERVENTIONS: We measured functional influences of hydroxyethyl starch on normoxic or posthypoxic endothelia. MEASUREMENTS AND MAIN RESULTS: Studies to assess endothelial barrier function in vitro indicated that the addition of hydroxyethyl starch promotes endothelial barrier in a dose-dependent fashion and hydroxyethyl starch-barrier effects are increased following endothelial hypoxia exposure (human microvascular endothelial cells, 48 hrs, 2% oxygen). Treatment of human microvascular endothelial cells with hydroxyethyl starch resulted in a dose-dependent increase in 157-phosphorylated vasodilator-stimulated phosphoprotein, a protein responsible for controlling the geometry of actin-filaments. Neutrophil adhesion was decreased in the presence of physiologically relevant concentrations of hydroxyethyl starch in vitro, particularly after endothelial hypoxia exposure. Using a murine model of normobaric hypoxia, increases in vascular leakage and pulmonary edema associated with hypoxia exposure (4 hrs at 8% oxygen) were decreased in animals treated with intravenous hydroxyethyl starch. Increases of tissue neutrophil accumulation following hypoxia exposure were dampened in hydroxyethyl starch-treated mice. CONCLUSIONS: Taken together, these results indicate that hypoxia-induced increases in vascular leakage and acute inflammation are attenuated by hydroxyethyl starch treatment.

Dose-dependent influence of barbiturates but not of propofol on human leukocyte phagocytosis of viable Staphylococcus aureus.

OBJECTIVE: Deep sedation with barbiturates or propofol is a standard therapy for patients with critically elevated intracranial pressure. Such patients are prone to infectious complications, especially to pneumonias, which are most commonly caused by Staphylococcus aureus. Although various immunomodulatory effects of barbiturates have been described in vitro, their influence on the phagocytosis of viable S. aureus has yet to be investigated. Therefore, we examined the effects of thiopentone, methohexitone, and propofol on the phagocytosis of viable S. aureus. DESIGN: Laboratory study. SETTING: University laboratory. PATIENTS: Ten healthy volunteers aged 32.5 +/- 7 yrs. INTERVENTIONS: Blood sampling. MEASUREMENTS AND MAIN RESULTS: Whole blood samples were preincubated with different concentrations of thiopentone, methohexitone, and propofol, which is an isopropylphenol derivate. After viable S. aureus was added, phagocytosis was stopped at different time points. Leukocytes were then stained with monoclonal antibodies for flow cytometric analysis of granulocyte recruitment (ratio of ingesting granulocytes) and phagocytosis activity (fluorescence intensity of ingested bacteria). Both barbiturates inhibited granulocyte recruitment and phagocytosis activity in a dose-dependent manner, whereas propofol did not affect any of the investigated variables. At concentrations higher than 7.6 x 10(-3) M (for thiopentone, p < .008) and 1.1 x 10(-3) M (for methohexitone, p < .04), granulocyte recruitment and phagocytosis activity were significantly inhibited. The calculated inhibitory concentrations (IC50) of thiopentone for granulocyte recruitment and for phagocytosis activity were 1.3 x 10(-2) M and 1.1 x 10(-2) M, respectively. The corresponding values for methohexitone were 3.6 x 10(-3) M and 1.1 x 10(-3) M. CONCLUSIONS: Our in vitro model points at substantially different effects of barbiturates and propofol on phagocytosis of S. aureus, which is one of the most important pathogens in patients who need neuroprotective therapy. The inhibitory effects of both barbiturates demonstrate a strong dose-dependency, with more pronounced effects for methohexitone. Impairment of phagocytosis activity was more pronounced than granulocyte recruitment.

Effect of intravenous hydroxyethyl starch on the accuracy of measuring hemoglobin concentration.

STUDY OBJECTIVE: To determine if intravenous hydroxyethylstarch (HES) affects the accuracy of hemoglobin (Hb) measurements, as artificial colloids are known to increase red blood cell sedimentation rates. DESIGN: Prospective, randomized study. SETTING: Tertiary-care academic medical institution. PATIENTS AND INTERVENTIONS: We randomized 40 surgical American Society of Anesthesiologists (ASA) physical status I and II patients undergoing preoperative autologous blood donation before elective orthopedic surgery. Patients were randomized to receive volume replacement with 500 mL of 6% HES 200,000/0.5 or 500 mL of electrolyte solution. Measurements of Hb concentration and leukocyte count were performed using an analyzer with a suction needle sampling from the bottom of the test tube. Measurements were performed after mixing and repeated after a 10-minute period of upright positioning of the tube (at rest). MAIN RESULTS: In the study group that received HES, Hb levels were increased above baseline after resting (mean increase to 151% of baseline values, P < .01), whereas the leukocyte count was decreased (mean decrease to 39% of baseline values, P < .01). No difference between baseline and resting measurements were observed in patients who received intravenous crystalloids. In addition, we repeatedly measured the Hb concentration in an unstirred tube with and without the addition of HES. In blood samples containing HES, the Hb concentration was increased above baseline after 2.5 minutes of resting, compared with 30 minutes without HES addition (P < .05). CONCLUSIONS: Mixing of test tube contents before sampling is critical for accurate measurement of the Hb concentration in the blood of patients who received intravenous HES.

Effects of intraoperative blood salvage on leukocyte recruitment to the endothelium.

BACKGROUND: The contamination of salvaged wound blood with activated leukocytes has been suspected to play a role in leukocyte-mediated tissue injury by increased adhesion to the endothelium. To verify this hypothesis, the authors performed a clinical study to examine the effects of blood salvage on leukocyte-endothelial interactions. METHODS: Expression of L-selectin, CD18, and CD11b and leukocyte adhesion to activated endothelium from human umbilical veins were measured in 25 patients undergoing major orthopedic surgery. Adhesion of fluorescently labeled leukocytes was examined in a flow chamber at shear rates of 50-1,600 s. Comparisons were made between samples from venous blood and from processed salvaged wound blood (SWB). RESULTS: At 30% hematocrit, SWB contained 2,162 +/- 147 leukocytes/microl. In comparison with venous blood, CD11b was up-regulated in SWB 1.3- to 3.6-fold on monocytes and neutrophils, whereas L-selectin and CD18 decreased on monocytes by 53% and 15%, respectively (P < 0.05). Despite up-regulation of CD11b, firm adhesion was significantly reduced by 74-76% in SWB. Rolling fractions and rolling velocities were significantly higher in SWB, and their relation to shear rate was markedly altered (P < 0.01). In addition, adherent leukocytes from SWB were significantly less resistant to increments of shear rate than leukocytes from venous blood (P < 0.01). CONCLUSIONS: Despite up-regulated CD11b, integrin-mediated adhesion is markedly impaired in salvaged blood. Therefore, the effect of blood salvage cannot be predicted from cell surface expression but rather from functional assays. The former hypothesis, that leukocytes from SWB aggravate leukocyte-mediated tissue injury by increased adhesion, may not be as great a concern as previously suggested.

Influence of long-term volume therapy with hydroxyethyl starch on leukocytes in patients with acute stroke.

A repeated administration of hydroxyethyl starch affects hemostasiological and rheological factors such as the concentration of factor VIII/von Willebrand factor, platelet volume and plasma viscosity. An earlier study showed that HES also lowers the concentration of fibronectin, a molecule important in the reticuloendothelial system (RES). RES has a "clearing function" and is a part of the non-immune-specific defense mechanisms of the body. It is involved in the elimination of HES from the blood. Since leukocytes are another important part of the unspecific defense system, the goal of the present study was to investigate whether HES affects leukocytes. After giving their informed consent, 20 patients with cerebral perfusion disorders were randomized and underwent a double-blind 10-day hypervolemic hemodilution with HES 200/0.5/13 or HES 70/0.5/4. The numbers of leukocytes, percentage of lymphocytes, percentage of neutrophilic granulocytes and hemoglobin concentration were measured. The absolute number of leukocytes did not change significantly, but the share of neutrophilic granulocytes increased. The increase in neutrophilic granulocytes reflects an increase in phagocytic activity. HEs 200/0.5/13, which has the larger in vivo molecular weight (MW = 95 kD), caused a larger increase in neutrophilic granulocytes than HES 70/0.5/4, which has an in vivo MW of 58 kD.

Recent developments in European colloid solutions.

Coagulopathy resulting from hydroxyethyl starch (HES) administration is well documented for high-molecular-weight, highly substituted HES solutions. These are the only starch-based synthetic colloids approved for volume replacement in the United States. Recent developments in European colloid solutions revolve around designing new starch molecules. By increasing the C2:C6 ratio and decreasing the molar substitution to 0.4, a new HES with a molecular weight of 130,000 was introduced. This solution is without significant influence on coagulation. Actual experimental work is concentrating on modulating the inflammatory response of monocytes and endothelial cells after infusion of colloid solutions. This may help prevent or even correct capillary leak, and the intravascular persistence of infused colloidal might be prolonged.

Local anesthetics impair human granulocyte phagocytosis activity, oxidative burst, and CD11b expression in response to Staphylococcus aureus.

BACKGROUND: With invasion of bacteria, the host defense system is activated by a complex cascade of various mechanisms. Local anesthetics previously were shown to interact with diverse components of the immune response, such as leukocyte adherence on endothelial monolayers, oxidative burst, or crosstalk within lymphocyte subset populations. However, effects of newer local anesthetics like bupivacaine and ropivacaine on antibacterial host defense-primarily phagocytosis activity, oxidative burst, or CD11b expression-still remain unclear. METHODS: Whole blood samples were preincubated with local anesthetics (lidocaine, 9.2, 92.2, and 1,846 microm bupivacaine, 6.1, 61, and 770 microm; ropivacaine, 6.4, 64, and 801 microm). For the oxidative burst and CD11b assay, dihydroethidium was added to the probes. After viable Staphylococcus aureus was added in a 5 to 1 ratio following leukocyte count, phagocytosis was stopped at different times, and staining with monoclonal antibodies was performed for subsequent flow cytometric analysis of phagocytosis activity, oxidative burst, and CD11b expression. RESULTS: Granulocyte phagocytosis activity, CD11b expression, and generation of reactive oxygen species were significantly reduced by lidocaine (P < 0.0002) and bupivacaine (P < 0.005) in the highest concentration (1,846 microm and 770 microm, respectively). The capability of granulocytes to ingest bacteria was significantly depressed only by lidocaine (P < 0.003). Ropivacaine had no significant effect on any parameter investigated. CONCLUSIONS: Local anesthetic dose and structure dependently inhibit inflammatory and immunologic parameters of granulocyte functions. Ropivacaine shows low interference with granulocyte immunologic and inflammatory functions.

Penetration of intravenous hydroxyethyl starch into the cerebrospinal fluid in patients with impaired blood-brain barrier function.

UNLABELLED: Hypovolemic patients with impairment of the blood-brain barrier may receive IV hydroxyethyl starch (HES) to stabilize cardiovascular function and to increase cerebral perfusion pressure. It is not known whether HES can penetrate into the cerebrospinal fluid (CSF) under those conditions. We investigated plasma and CSF levels of HES after IV infusion in patients with suspected disturbance of the blood-brain barrier. Eight adult patients were studied who were being treated for head trauma or subarachnoid hemorrhage, with an external CSF drain in place. All patients exhibited radiographic signs of blood-brain barrier impairment diagnosed by cerebral computed tomography. After IV infusion of 500 to 1000 mL of HES 200,000/0.5, plasma HES levels were measured. Additionally, all CSF that was drained within 8 h after the HES infusion was collected, and HES concentrations were measured. All patients had detectable HES plasma concentrations (3.41 to 9.95 mg/mL). In contrast, no HES could be detected in the CSF of any patient. These data indicate that IV HES 200,000/0.5 does not penetrate into the CSF in patients with disturbed blood-brain barrier function after subarachnoid hemorrhage or head trauma. Further study is required to determine whether HES penetrates into the intracranial interstitium, despite the absence of HES in the CSF. IMPLICATIONS: Patients may receive IV hydroxyethyl starch (HES) after head trauma or subarachnoid hemorrhage. The results of the present study indicate that in patients with suspected blood-brain barrier impairment, HES does not penetrate from the plasma into the cerebrospinal fluid.

Influence of a long-term, high-dose volume therapy with 6% hydroxyethyl starch 130/0.4 or crystalloid solution on hemodynamics, rheology and hemostasis in patients with acute ischemic stroke. Results of a randomized, placebo-controlled, double-blind study.

BACKGROUND: This study was performed to investigate the clinical effects of a 4-day volume therapy with a newly developed, 6% hydroxyethyl starch (HES) 130/0.4 versus crystalloid solution, with particular regard to systemic and cerebral hemodynamics, rheology and safety. METHODS: In a randomized, double-blind study, 40 patients suffering from an acute ischemic stroke received either 6% HES 130/0.4 or crystalloid solution as continuous infusion over 4 days with a total dose of 6.5 liters. Efficacy parameters studied included hemodynamics (cardiac output, blood pressure, flow velocity with transcranial Doppler) and rheology (hematocrit and plasma viscosity). Safety parameters examined included laboratory, hemostaseology (including factor VIII) and an adverse event questionnaire (including pruritus). RESULTS: In both groups, a small, but not significant increase in cardiac output was observed. There were no significant changes regarding the remaining efficacy or safety parameters, except for the well-known increase in serum alpha-amylase through the infusion of HES. CONCLUSION: In our study with patients suffering from acute ischemic stroke, continuous infusion (1 ml/min) of HES 130/0.4 or crystalloid solution did not differ regarding safety or hemodynamic efficacy.

Effects of magnetic cell separation on monocyte adhesion to endothelial cells under flow.

Studies on monocyte adhesion are frequently limited by spontaneous changes of CD11b and CD62L during cell purification. Most isolation protocols for flow cytometric analysis that overcome this problem cannot be used when large numbers of living cells are needed for functional adhesion assays. This study investigated whether magnetic cell separation of monocytes with a paramagnetic bead against CD33 is a feasible method combining high yield with a low degree of spontaneous activation. As determined by flow cytometry, isolation of magnetically tagged monocytes at 4 degrees C did not alter the expression of CD11b and CD62L when compared to whole blood controls. Warming the cells slowly to room temperature immediately before starting the adhesion assay in a parallel plate flow chamber at 37 degrees C prevented further upregulation of adhesion molecules due to rewarming. When adhesion of magnetically tagged monocytes was compared with untouched monocytes that had been isolated via depletion of contaminating leukocytes, videomicroscopy showed that labelling CD33 neither affected rolling nor firm adhesion to human umbilical venous endothelial cells under flow. Finally, the subsequent upregulation of tissue factor expression on adherent monocytes indicates that magnetically separated monocytes responded properly to activating stimuli during cell adhesion. We conclude that magnetic cell separation via CD33 represents a feasible method for cell separation whenever large numbers of non-activated monocytes are needed for adhesion assays under flow.