Septic shock usually results in a dramatic fall in systemic vascular resistance, cardiac dysfunction, and generalized blood flow maldistribution. Catecholamines are recommended to restore adequate arterial pressure when fluid expansion has failed (1). However, high doses are required to achieve complete or partial correction of hypotension, an observation that is consistent with reduced sensitivity to catecholamines, but its mechanisms are poorly understood. They could include adrenergic receptors desensitization (2–4), postreceptor abnormalities of signal transduction (5,6), modifications of intracellular Ca2+-dependent myofilament contraction (7,8), or, as recently suggested, inactivation of catecholamines by free radicals (9).
Corticosteroids have been advocated over several decades as an efficient strategy to reverse shock. Nevertheless, based on large controlled studies with high methodological standards, the overall use of a large, single dose of steroids in sepsis or septic shock was shown to be ineffective and is no longer recommended (10,11). However, renewed interest for steroid therapy in sepsis is the result of recent studies that have demonstrated that a relatively prolonged course of supraphysiological doses of hydrocortisone improves the rate of shock reversal and reduces the duration of catecholamine dependency in septic shock patients (12–14). Of interest, most of the investigated patients were dependent on high doses of catecholamines for several days before steroid administration. Hemodynamic data suggest that steroids could act primarily by increasing the vasopressor response to catecholamines, as previously demonstrated in healthy animals (15,16) or volunteers (17). Steroids may be effective when administered later during the evolution of sepsis, a hypothesis that challenges previous experimental evidence where earlier administration was a condition for better results (18–20).
The relevant beneficial mechanisms of steroids in septic shock are unknown, although inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 inhibition, a decrease in proinflammatory cytokines, an increase in anti-inflammatory mediators (21), and upregulation of adrenergic receptors (22) could account for them. In addition, their effects may differ according to the timing of their administration (23). We hypothesized that early as opposed to late glucocorticosteroids administration in an animal model of septic shock may have different hemodynamic effects, perhaps through a reversal of adrenergic receptor down-regulation, a time-dependent process.
The objectives of the present study were, first, to study the in vivo dose-response relationship to catecholamines in an animal model of septic shock with emphasis on heart rate (as an estimate of myocardial β-adrenergic response), arterial pressure (as an estimate of vascular α-adrenergic response), and aortic blood flow (as an estimate of integrated cardiovascular response), and second, to compare early and delayed glucocorticosteroid administration on these parameters.
MATERIALS AND METHODS
Animals and study groups
Adult male Wistar rats (Dépré; St. Doulchard, France) weighing from 260 to 330 g were used in the study. The experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals and the study was approved by our Institutional Animal Care and Use Committee. They were housed in cages with a constant temperature (22°C) and were exposed to a 12-h light-dark cycle for at least one week before use in experimental protocols. All animals were fasted overnight with free access to water.
Animals were randomized to one of the following groups: a control group of healthy animals (n = 14); a sham group where animals underwent laparotomy with cecum manipulation but without ligation or perforation (n = 14); a sepsis group where animals underwent cecal ligation and perforation (CLP; n = 14); and early (n = 14) and late (n = 14) dexamethasone groups where 1 mg/kg dexamethasone (Qualimed, Puteaux, France) was administered intraperitoneally at 0 and 22 h, respectively, after CLP. The day of the study (24 h after surgery), groups were randomly allocated in two subgroups each (n = 7 respectively); one subgroup was used for hemodynamic measurements, and the other one was used for biochemical sampling (see below).
Sepsis was induced by CLP as described by Wichterman et al. (24) with minor modification. Briefly, rats were anesthetized with ketamine (150 mg/kg, intraperitoneally, and additional doses were given when necessary) and a 3- to 4-cm abdominal incision was made to expose the cecum. It was ligated and punctured once with a 21-gauge needle and a small amount of feces extruded. The bowel was returned to the abdomen and the abdominal cavity was closed in two layers. For sham rats, a laparotomy was performed and the cecum was manipulated but was neither ligated nor punctured. All of these animals were resuscitated with 5 mL/100 g body weight of normal saline subcutaneously at the completion of surgery. They were fasted with free access to water. They were carefully observed for 24 h after surgery. For the control group, rats did not undergo any surgical procedure, but were fasted as the other animals were for 24 h before investigations.
In vivo hemodynamic measurements
Twenty-four hours after CLP or sham procedure, baseline arterial pressure (mean, systolic, and diastolic), heart rate, abdominal aortic blood flow, and mesenteric blood flow were recorded using the following procedure:
Animals were anesthetized with sodium thiopental (60 mg/kg body weight intraperitoneally) and additional doses were given when necessary (indicated by presence of interdigital reflexes).
A tracheostomy was performed by using PE-160 tubing, and the animals were ventilated using a rodent ventilator with a tidal volume of 1 mL of room air per 100 g body weight. Temperature was monitored continuously by a rectal probe and was maintained at 37°C. The left carotid artery and the left jugular vein were cannulated with PE 50 tubing. Carotid arterial blood pressure was continuously monitored using a disposable pressure transducer (5265014; Viggo-Spectramed, Bilthoven, The Netherlands) and an amplifier-recorder system (Sirecust 302A; Siemens, Berlin, Germany). Through the left jugular vein, 4.5 mL of normal saline was infused for 45 min just before starting catecholamine infusion. The upper abdominal aorta and the mesenteric artery were carefully dissected and perivascular (1RB) probes (Transonic Systems, Ithaca, NY) were placed around them. The abdominal aortic blood flow and mesenteric blood flow were measured with a Transonic small animal flowmeter (T206; Transonic Systems). The preparation lasted about 30 min.
To establish the catecholamine dose-response curves, phenylephrine, epinephrine, and isoproterenol were infused in a stepwise manner (with each dose being maintained for 10 min). At each dose, hemodynamic values were determined as the mean of values recorded after 5 and 10 min of infusion. Only one catecholamine dose-response curve was established for each animal. Left ventricular myocardial samples were taken in the isoproterenol-treated animals when the maximal infusion rate was achieved (10 μg/kg/min) for cAMP assay (see below).
After the last measurement, the animals were sacrificed by an overdose of sodium thiopental intravenously.
Blood and tissue biochemical measurements were done in each subgroup not used for hemodynamic measurements. Samples were obtained 24 h after initiating the model (CLP or sham procedure).
These animals were anesthetized and tracheostomized with the same procedure as for hemodynamic measurements. Blood was withdrawn from the left carotid artery.
Arterial blood samples were collected and then deproteinized by adding 500 μL of perchloric acid (1 mol/L) to 500 μL of whole blood. Lactate was measured by enzymatic-colorimetric method adapted to a Wako automatic analyser (Biochem Systems, Rungis, France). Normal value range is <2 mmol/L.
Samples (1 mL of whole blood) were deproteinized before analysis by using sulfosalicylic acid, and they were centrifuged and added to a buffer containing 5% NH4Cl and 5% NaOH. Samples were injected into a column filled with copper-plated cadmium fillings to reduce nitrate to nitrite. The column effluent was mixed with Griess reagent. Nitrite concentration was determined by measuring the absorbance at 546 nm and was compared with a standard solution of sodium nitrate (25). Normal values were <40 μmol/L.
Plasma epinephrine and norepinephrine
These levels were measured (2 mL of whole blood) using HPLC-electrochemical detection.
β-Adrenergic receptor analysis
Myocardial cellular membrane preparation
Samples were frozen in liquid nitrogen and were stored at −70°C until analysis. Myocardial membrane suspensions were prepared as described by Kim et al. (26) with minor modifications. Samples (300 mg) were homogenized in 5 mL of ice cold Krebs-phosphate buffer (composition, in millilmoles per liter: NaCl, 119; KCL, 4.8; MgSO4, 1.2; CaCl2, 1.9; glucose, 11.7; NaH2 PO4, 1.3; and Na2H PO4, 8.7; pH 7.4) for 20 s at high speed using a Polytron® homogenizer (Kinematica, Switzerland). The homogenates were then centrifuged for 15 min at 12,000 g and 4°C to remove connective tissue. Supernatants were centrifuged twice for 20 min at 100,000 g. The resulting pellets (membrane preparations) were resuspended in 2 mL of Krebs-phosphate buffer. Protein concentration was determined by the method of Lowry (27). Fresh membranes were prepared before each experiment.
β-Adrenergic receptor radioligand binding assay
Radioligand binding was performed with [125I]-cyanopindolol (ICYP; Amersham, Orsay, France). Fresh membranes (100 μg of total protein) were combined with 300 μL of Krebs-phosphate buffer containing 0.1 mmol/L GTP, 1 mmol/L ascorbic acid, and 0.1 mmol/L EDTA. For saturation binding studies, membranes were incubated with saturating concentrations of ICYP (7.5–500 pmol/L) in a final volume of 500 μL, in the absence (total binding) and presence (nonspecific binding) of 100 μmol/L (±) propranolol for 90 min at 37°C. All samples were assayed in duplicate. Incubation was terminated by the addition of ice-cold Krebs-phosphate buffer followed by vacuum filtration. Filtration was carried out using Millipore fraction collectors (Polylabo, Strasbourg, France) equipped with 3.5 cm GF/C glass microfibers filters (1.2 μm porosity). Filtration was performed using constant and controlled depression (−300 mmHg). After two additional washings, radioactivity retained on the filter was measured using a Cobra II® gamma counter (Packard Bell, Meriden, CT) with an efficiency of 70%. Pooled data on each group derived from saturation binding experiments were analyzed using the nonlinear curve-fitting Kell-Radlig® computer program (Biosoft, Ferguson, Milltown, NJ), which performs Scatchard and Hill analyses.
The frozen myocardial samples were homogenized in 1.07 N perchloric acid for 20 s at high speed using a Polytron® homogenizer. The homogenates were then centrifuged for 1 min at 10,000 g and the pellet was used for the determination of protein concentration by the method of Lowry (27). The supernatant was adjusted to pH 7.0, and the cAMP content was determined by radioimmunoassay (Immunotech, Marseille, France).
After blood and tissue collection, the animals were sacrificed by an intravenous overdose of sodium thiopental.
Results are expressed as mean ± SE. A one-way analysis of variance (ANOVA) was performed to compare baseline and slope parameters between groups. Repeated-measure ANOVA was used to compare repeated measurements across time between groups. When significant, between-group comparisons were performed using t tests with the Bonferroni correction for multiple comparisons (28). A P value of less than 0.05 was considered significant.
Confirmation of the septic model
A few hours after surgical manipulation, CLP-operated rats exhibited signs of sepsis, including piloerection, exudates around the eyes and nose, and decreased spontaneous movement. Sham-operated rats were active in their cages and appeared normal. Examination of the peritoneal cavity of septic rats demonstrated copious amounts of foal-smelling purulent peritoneal fluid, and the ligated portion of the cecum was grossly dilated and gray-black in color. Examination of the abdominal cavity of sham rats showed no noticeable odor, minimal peritoneal fluid, and the bowel was pink in color.
As shown in Table 1, there were significant decreases in mean arterial pressure and aortic blood flow in the sepsis group as compared with the control group. Arterial pressures of control, sham, and septic steroid-treated animals were strictly similar. Compared with the sepsis group, aortic blood flow was higher in both steroid-treated groups, but the result was significant only for late steroid-treated group. Heart rate did not differ among groups. Figures 1 to 3 depict changes in mean arterial pressure and aortic blood flow during administration of stepwise increased catecholamines doses compared with baseline levels. The dose-response relationships were similar among all groups with respect to mean arterial pressure. Conversely, the time course of aortic blood flow changes with increased epinephrine doses was different in septic animals as compared with sham animals (P < 0.05); glucocorticosteroid-treated septic animals displayed changes of aortic blood flow similar to those of sham animals. A similar effect was observed with isoproterenol-induced increase in heart rate (Fig. 4) where septic animals had lower increase than sham, with intermediate patterns for steroid-treated septic animals whose restoration of heart rate response was incomplete.
Plasma concentrations of lactate are summarized in Figure 5 and plasma epinephrine and norepinephrine concentrations are displayed in Figure 6. Serum nitrite/nitrate concentrations are shown in Figure 7.
Figure 8 displays the effects of sepsis and corticosteroids on myocardial β-adrenergic receptor number and affinity, based on ICYP binding studies. The Scatchard analyses for ICYP binding indicate single-component binding characteristic (data not shown). As shown in Figure 8A, the Bmax values (maximal number of binding sites determined by Scatchard plot) did not differ among the studied groups. Sepsis + late dexamethasone exhibited a significant increase of Kd (1.2 ± 0.4 nmol/L vs. 0.3 ± 0.1 nmol/L for sepsis + late dexamethasone group and the sepsis group, respectively) as compared with the other groups, suggesting altered β-adrenergic receptor affinity with dexamethasone administrated 22 h after CLP. Kd was lower in sham as compared with septic animals, but the difference was not significant.
Baseline and isoproterenol-stimulated myocardial tissue cAMP content are displayed in Table 2. Although values were highly increased after isoproterenol stimulation, when compared with baseline values, they were similar among groups.
In this experimental model of hypotensive, hypokinetic sepsis, the pressor response to catecholamines was unchanged as compared with sham and was not altered by corticosteroids. Interestingly, septic animals had a blunted response of both heart rate and aortic blood flow. These findings suggest that, at least in this model, the myocardial response could be more depressed than the vascular response to the septic injury. Glucocorticosteroids were effective on arterial pressure, and only partially on blood flow whether they were administered early or late after the onset of sepsis.
During human or experimental septic shock, plasma catecholamine concentrations are usually increased (2,3,29). This is associated with myocardial depression and peripheral vasodilation or inadequate vasomotor tone (30). However, although several studies on the hemodynamic effects of a given vasopressor agent in humans already treated with catecholamines are available, dose-response studies in untreated patients or in vivo experimental models are scarce and controversial. In patients, a recent study has shown a reduced pressor response to phenylephrine (31). In animal models of peritonitis, two studies have shown either a reduced (32) or a normal pressor response to norepinephrine (33). In the present study, using in vivo dose-response curves to catecholamines, we showed a normal sensitivity to vasopressor catecholamines (epinephrine and phenylephrine) and a slope conservation despite a baseline arterial pressure lower than in nonseptic animals. Although these findings were obtained a relatively long interval after the septic insult, this does not preclude further changes with time because it has been demonstrated that nonspecific catecholamine desensitization could need several days to develop (34).
Conversely, there was a significant reduction in the chronotropic response to β-adrenergic stimulation in septic animals. In ex vivo perfused hearts from a similar septic model, Smith et al. (35) found an increased chronotropic response to isoproterenol. However, animals were normotensive and normokinetic, suggesting an earlier phase or a less severe septic insult than in the present model. Finally, although the hypodynamic state we observed in septic animals is evocative of myocardial depression, changes under catecholamines are related to several factors, including preload and afterload modifications.
Early or late dexamethasone administration was associated with reversal of hypotension and improvement of blood flow. The latter was more substantial when corticosteroids were given late. In addition, lactate concentrations were lower in both steroid-treated groups, consistent with an improvement in tissue oxygenation. Reduction in lactate levels was also more pronounced in the late steroid-treated animals. Although previous experimental evidence has suggested that corticosteroids need to be administered no later than a few hours after creating the septic challenge (18–20), the results presented here support the concept that delayed steroid administration can be effective on hemodynamics. Furthermore, flow improvement with possible subsequent attenuation of dysoxia suggest that late therapy could be more effective than early therapy with steroids. The difference between early and late dexamethasone groups could, as a first approach, be explained by lower circulating concentrations of glucocorticoids at hemodynamic assessment in early-treated animals. However, the long half-life of dexamethasone, exceeding 24 h (36), and the identical pressor effects in both groups do not support this hypothesis. In the present intact model, the respective effects of steroids on vascular beds and myocardial tissue cannot be precisely ascertained. However because arterial pressure was completely restored with only partial improvement in blood flow, a global vasoconstrictive effect was predominant in combination with either improved cardiac inotropism or blood volume redistribution.
An important mechanism of action of steroids in late sepsis could be related to their upregulating effects on adrenergic receptors (22). Desensitization of adrenergic receptors is a time-dependent event related to high circulating catecholamines concentrations (37). In sepsis, the high catecholamine concentrations that are observed whatever the model could theoretically account for an agonist-induced internalization of both β- and α-adrenergic receptors. However, available experimental evidence remains controversial. In acute endotoxinic animal models, some investigators found that myocardial β-adrenergic receptor surface density was decreased (2), whereas others detected no significant change in the receptor density, but only uncoupling of β-adrenergic receptors to adenylyl cyclase (4–6). In CLP rodent models, it has been recently demonstrated an early externalization followed by a late—after 16 h—internalization of both β (3,38) and α (39) myocardial receptors. These findings correlated with the sequence of hyper- and hypocardiodynamic states in the septic model. Although not designed to separate sarcolemmal and cytosolic vesicular fractions, the receptor assays in the present study suggest that the overall number and the binding affinity of β-adrenergic receptors are maintained in late sepsis. Furthermore, left ventricle myocardial cAMP concentration was slightly increased at baseline as compared with sham animals, presumably as a response to increased endogenous catecholamines. The similar increase in myocardial cAMP content in isoproterenol-stimulated animals provides evidence of normal receptor coupling to adenylyl cyclase. Thus, these findings suggest that myocardial dysfunction is probably not related in a significant way to receptor abnormalities and signal transduction to cAMP-dependent protein kinase. In agreement with these findings, protein kinase A activity has been found increased in the rat heart of a similar septic model (40). These data are consistent with a more distal mechanism of myocardial dysfunction at the subcellular level. Recent experimental evidence supports the role of decreased myofilament response to Ca2+ in the septic myocardium, either through a NO-cGMP-mediated mechanism (7) or protein phosphorylation (8). Conceivably, glucocorticosteroids may directly help to restore myofilament response through iNOS inhibition or other mechanisms.
Early and late steroid administration displayed some differences with respect to catecholamines and myocardial β-adrenergic receptors patterns. Unexpectedly, the affinity for receptors (Kd) was significantly reduced in animals given dexamethasone late after CLP when relatively high plasma norepinephrine levels were present. The interpretation of these findings is unclear because hemodynamic improvement was rather better in this group. One possible explanation is the following. Hypothesizing that beneficial effects of steroids on myocardial contractility are independent of adrenergic signalling, steroid-induced improvement in myocardial performance could lead to a feedback regulatory mechanism of adrenergic surface receptor desensitization. The simultaneous high levels of circulating norepinephrine could be consistent with this hypothetical mechanism. In addition, this reduced affinity for β receptors could explain the quasi absence of effects of late steroids on the chronotropic response to isoproterenol. Whatever the cause, the differences between early and delayed administration of steroids underline the fact that timing of their administration is of particular relevance during the course of sepsis.
Nitrite/nitrate concentration was markedly reduced in steroid-treated animals. This is probably related to a decreased NO production through iNOS. These patterns are not unexpected because glucocorticosteroids inhibit the expression of iNOS (41,42). Similar findings have been observed in septic shock patients treated with hydrocortisone in a randomized crossover study (12). This could be a relevant mechanism not only of hypotension reversal in the present study, but also of myocardial performance improvement because it has been recently demonstrated that iNOS expression is required for the development of myocardial dysfunction in murine sepsis (43). Whether or not effects of steroids rely solely on iNOS expression inhibition should be further assessed.
The relevance of these findings to human septic shock deserves further consideration. Although the CLP model is probably closer to human sepsis, i.e., peritonitis, than acute endotoxemia, it should be emphasized that most patients with septic shock remain in a hyperdynamic hemodynamic state, even several days after the onset of sepsis. That steroids do not appear to act through a catecholamine desensitization process in this animal model does not obviously implicate that this mechanism is not relevant for late human septic shock where patients are exposed to high catecholamine concentrations for a longer time than in the present model. However, hemodynamic human data recorded in hyperkinetic patients treated with large doses of both α- and β-adrenergic agents suggest that steroids improve blood pressure mainly through a vasoconstrictor effect (13,14), a picture consistent with the present experiment where pressure is completely restored but not blood flow.
In summary, in this model of late hypokinetic septic shock, dexamethasone, whether administered either early or late, was effective in reversing hypotension. Pressor effects of steroids were presumably related to changes independent of the α-adrenergic signalling pathway, including inhibition of iNOS. Blood flow improvement did not result from a resensitization of the β-adrenergic signal transduction because it was functionally active in treated and untreated animals. Additionally, the present results provide some further evidence for postreceptor abnormalities as the predominant cause of myocardial dysfunction in sepsis.
We thank C. Montemont and E. Vauthier for expert technical assistance. We also thank Dr. M. B. Nicolas (biochemistry laboratory), Dr. M. A. Gelot, and Dr. B. Dousset (chemistry laboratory) for catecholamines, nitrite/nitrate, lactate, and cardiac cAMP analysis.
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