Sepsis remains a major therapeutic challenge and its pathophysiology remains poorly understood. In sepsis, the large increase in sympathetic nerve activity (SNA) (1) may modulate not only cardiovascular and renal function, but also the immune response. In particular, increased cardiac SNA appears partly responsible for the tachycardia and increased cardiac output of sepsis (2), while the early decrease in renal SNA causes polyuria (1). In contrast, late increases in renal SNA have no effect on the development of acute kidney injury (3). Moreover, increased SNA may cause down-regulation of myocardial β-adrenergic receptors, thus contributing to septic myocardial dysfunction and failure (4, 5), and have potent anti-inflammatory effects (6). These observations have led to suggestions that “decatecholaminization,” as may result from inhibition of central sympathetic outflow, may be beneficial in patients with septic shock (5).
Centrally acting α2-adrenoceptor agonists, such as clonidine, that inhibit central sympathetic outflow, may be beneficial in sepsis by reducing such high levels of SNA (7, 8). Indeed, α2-adrenoceptor agonists appear to improve renal function and outcome in rodent models of sepsis (9–12) and responsiveness to vasopressor drugs in rats, sheep and, perhaps, humans (13–16). However, these effects of clonidine have been observed with supra-clinical doses of 5, 10, or 75 or even 200 μg/kg, which are far greater than the doses considered safe and given in the clinical setting (0.25–1.0 μg/kg/h). Currently, the effects of clonidine at such clinically relevant doses on the systemic circulation, renal circulation and function and on cytokine release are poorly understood, but highly relevant to the management of sepsis. Accordingly, we studied the effects of clonidine at two clinically relevant doses (0.25 and 1.0 μg/kg/h) in an ovine model of gram negative, hyperdynamic sepsis. We hypothesized that although clonidine is an antihypertensive drug, that in sepsis such doses of clonidine would not worsen the level of hypotension or the degree of kidney injury and would also have an anti-inflammatory effect.
MATERIALS AND METHODS
Experiments were performed on adult Merino ewes housed in individual metabolic cages, fed 800 g of oaten chaff a day and with free access to food and water. The experimental procedures were approved by the Animal Experimental Ethics Committee of the Florey Institute of Neuroscience under guidelines laid down by the National Health and Medical Research Council of Australia.
Sheep underwent two sterile surgical procedures under general anaesthesia. First, a bilateral carotid arterial loop was created to facilitate subsequent arterial cannulation and a transit-time flow probe (Transonics Systems, Ithaca, NY) was placed on the pulmonary artery through a left side 4th intercostal space thoracotomy (2). After 2 weeks, during the second procedure, a transit-time flow probe was placed on the left renal artery. The animals were then allowed at least 2 weeks to recover before the start of experiments. For all the operations, animals were treated with intramuscular antibiotics (900 mg of procaine penicillin, Ilium Propen, Troy Laboratories Ptd Ltd, Smithfield, NSW, Australia or Mavlab, Qld, Australia) at the start of surgery and then for 2 days postoperatively. Post-surgical analgesia was maintained with intramuscular injection of flunixin meglumine (1 mg/kg) (Troy Laboratories or Mavlab, Qld, Australia) at the start of surgery, then 8 and 24 h post-surgery.
The day before the experiment, a Tygon catheter (Cole-Parmers, Boronia, Australia; ID 1.0 mm, OD 1.5 mm) was inserted into the carotid arterial loop to measure arterial pressure and to obtain blood samples. Three polythene catheters (Microtube Extrusions, North Rocks NSW, Australia) were inserted into a jugular vein: one for measurement of central venous pressure (ID 1.19 mm, OD 1.7 mm), one for infusion of normal saline or clonidine (ID 1.19 mm, OD 1.7 mm), and one for infusion of Escherichia coli (ID 0.58 mm, OD 0.96 mm). Analogue signals (mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), and renal blood flow (RBF) were collected on computer using a customized data-acquisition system (Labview, National Instruments, Austin, Tex). The data were recorded at 100 Hz for 10 s every minute during the experiments. Standard formulae were used to calculate total peripheral conductance (TPC = CO/MAP), renal vascular conductance (RVC = RBF/MAP), and stroke volume (SV = CO/HR).
Experimental protocol and sepsis induction
Baseline measurements were collected for 24 h prior to the induction of sepsis. Sepsis was induced with an intravenous bolus of 7.5 × 107 CFU (colony forming units)/kg of live E coli infused over 15 min diluted in 15 mL of normal saline immediately followed by a continuous infusion of live E coli at 3 × 107 CFU kg/h continued for 32 h. Fluid replacement at 1.0 mL/kg/h of normal saline was given throughout the experiment. After 24 h of sepsis, low clinical dose clonidine (LCDC) (0.25 μg/kg/h) or high clinical dose clonidine (HCDC) (1.0 μg/kg/h) or vehicle (normal saline at 1.0 mL/kg/h) was infused for 8 h (from 24 to 32 h of sepsis).
To start treatment, predefined hemodynamic criteria had to be met: an increase in heart rate of at least 50% compared with baseline; a decrease in mean arterial pressure of at least 10 mmHg compared with baseline, and a decrease in urine output of at least 25% compared with baseline. At the end of the baseline period and at 24, 28, 32, 36, 48, and 72 h after the start of E coli infusion, blood samples were taken and respiratory rate and rectal temperature recorded. Urine was collected hourly throughout the experiment from a bladder catheter using an automated fraction collector and sampled in 12-h lots for baseline and 72 h samples, and in 4-h lots for all the other time points. At 32 h, the infusions of E coli and clonidine were stopped and the animals received intramuscular gentamicin (150 mg). The animals were followed for further 40 h into the recovery phase and those that survived were crossed over to one of the other arms of the experiment 2 weeks later.
Blood and urine samples
Creatinine (Cr), urea, sodium, and potassium were measured in the blood and urine samples. Creatinine clearance (CrCl) and fractional excretion of sodium (FENa) and urea (FEUN) were calculated according to standard formulae: CrCl = (UCreat*UO)/(PCreat/time(min)) where UCreat is urine creatinine, P creat plasma creatinine and UO urine volume in 4 h (in mL); FENa and FEUN = Px/Ux*CrCl*100 where P is plasma concentration and U urine concentration of sodium or urea. Plasma and urine osmolality were measured on 10 μL fresh samples with a vapor pressure osmometer (Vapro model 5600, Wescor Biomedical Systems, ELITechGroup, Logan, Utah). Arterial blood gases and lactate were measured (ABL System 625; Radiometer Medical, Copenhagen, Denmark) and oxygen delivery (DO2) and oxygen consumption (VO2) were calculated as per standard formulae: DO2 = 10*CO*Arterial O2 concentration, VO2 = 10*CO*(Arterial − venous O2 concentration).
Assay for plasma arginine vasopressin (AVP)
Arterial blood samples were collected into heparinized tubes on ice, plasma was separated (10 min at 3000 rpm centrifugation) and stored at −80° C. Samples were assayed using an in-house radioimmunoassay using synthetic AVP as a standard (0–500 pg/tube, Bachem), radiolabeled 125I-AVP (Prosearch, Melbourne, Australia) and a specific AVP antibody raised in rabbit (17, 18). The inter- and intra-assay coefficients of variation were less than 8% and the limit of detection was approximately 1.0 pM.
Arterial blood samples were collected into EDTA tubes on ice, centrifuged at 3,000 g at 4°C for 10 min and plasma was aliquoted and frozen at −80°C. Interleukin 6 (IL-6) and interleukin 10 (IL-10) levels were measured using in-house enzyme-linked immunosorbent assays (ELISA), as previously described (3).
All data are presented as mean ± standard error of mean (SEM) or geometric mean (95% confidence interval) as appropriate. Statistics were performed on the baseline values (mean of 24 h of the baseline period) and predefined time points of interest (24, 28, 32, 36, 48, and 72 h) that consisted of a mean of the previous 4 h of data. Statistical analysis was performed using SAS version 9.2 (SAS Institute Inc, Cary, NC). All variables were assessed for normality and log-transformed where appropriate.
Mixed linear modeling was performed with each sheep treated as a random effect. Main effects were fitted for sequence, time, and treatment with an interaction between time and treatment used to determine if treatments behaved differently over time. Specific time point comparisons were performed using post-hoc pairwise t tests. To account for multiple comparisons, a reduced P value of ≤0.01 was considered to be statistically significant.
Data were obtained from eight sheep per group in which the predefined criteria were met at 24 h of sepsis. The weights of the animals were similar in the groups (high clinical dose clonidine (HCDC): 33.3 ± 1.7 kg; low clinical dose clonidine (LCDC): 33.2 ± 1.6 kg; vehicle: 31.2 ± 1.5 kg). Data from four animals (one each in the vehicle and HCDC groups and two in the LCDC group) that died during the development of sepsis were excluded from the analysis. Thus, seven animals in both the vehicle and clonidine HCDC groups and six in the LCDC group completed the study up to the end of the intervention period (32nd hour of sepsis) and their data are presented and statistically analyzed. One animal in the vehicle and one in the HCDC group died at the end of the 8 h of intervention, and their data were used for analysis up to 32 h of sepsis. For the recovery phase, data from the six animals per group that completed the experiment until the end of the recovery period were analyzed.
Cardiovascular responses to clonidine during sepsis and recovery
In the three groups, a similar degree of hypotensive, hyperdynamic sepsis developed within 24 h (Fig. 1), with similar increases in CO, HR and TPC and a similar degree of hypotension, tachypnea, and fever (Fig. 1).
Both doses of clonidine caused similar reductions in HR, but did not cause any sustained decrease in MAP (Fig. 1). The mechanisms by which MAP was preserved were, however, different with the two doses of clonidine. The HCDC reduced CO and MAP was maintained by peripheral vasoconstriction, whereas the LCDC induced peripheral vasodilatation and MAP was maintained by an increase in CO (Fig. 1). During the recovery period, the restoration of MAP to baseline values occurred more rapidly with the HCDC, and at both 48 and 72 h (16 and 40 h of recovery) MAP was significantly higher than in the vehicle group (P < 0.001 Fig. 1).
Changes in renal hemodynamics and function, plasma AVP, and plasma osmolality
The high level of RBF in septic animals was reduced by HCDC, but not LCDC (Fig. 1). Baseline urine flow was similar in the three groups and oliguria developed similarly during sepsis (Fig. 2). However, by the third hour both doses of clonidine had induced a 3-fold increase in urinary output (Fig. 2; HCDC vs. control P = 0.002; LCDC vs. control P = 0.006). This increase slowly attenuated during the infusions. However, 16 h after the end of both treatments (at 48 h), there were large increases in urine output compared with controls (P < 0.001), but there were no substantial differences in FENa (Fig. 2) or FEUN or urea (data not shown). In all groups there were similar increases in plasma creatinine and decreases in creatinine clearance (Fig. 2).
The baseline levels of arginine vasopressin (AVP) were similar in all the groups (Fig. 3). However, there was a progressive increase in plasma osmolality in both clonidine treated groups that reached significance by 8 h of treatment (Fig. 3). This was associated with a dose-dependent increase in plasma AVP that with the HCDC reached significance at 8 h of treatment and at 4 h post-treatment (P = 0.007 and P = 0.006, respectively).
Blood biochemistry, blood gases, systemic oxygenation, and body temperature
In all the three groups, arterial lactate increased by about three-fold during sepsis (Table 1). In addition, in all the groups during sepsis there were similar increases in respiration rate with similar decreases in pO2, but pCO2 was increased in the HCDC group at the 4th h of treatment (Table 1). Calculated DO2 and VO2 were not altered by either dose of clonidine (Table 1). There were also similar changes in all groups during the intervention period in arterial O2 saturation, pH, base excess, HCO3, hemoglobin, plasma urea, and fractional excretion of urea (data not shown). Clonidine caused a dose-related decrease in body temperature at 4 and 8 h of sepsis (Table 1).
Levels of circulating cytokines
There were large increases in the plasma levels of interleukin-6 (IL-6) 6 and IL-10 during sepsis and the levels of IL-6 remained elevated above baseline at 16 h of recovery (Fig. 4). Clonidine did not affect the level of IL-6, but caused a dose-dependent increase in the levels of IL-10 at 4 and 8 h of treatment (P = 0.001).
In a conscious ovine model of hyperdynamic sepsis, the effects of clonidine at clinically relevant doses were complex and dose dependent. Specifically, the high clinical dose (HCDC) attenuated sepsis-related increases in heart rate, cardiac output, and renal blood flow, with little effect on arterial pressure. It also induced a water diuresis with increased plasma osmolarity, stimulated AVP release, reduced body temperature, had an anti-inflammatory effect, and expedited normalization of MAP during recovery from sepsis. Low clinical dose clonidine (LCDC) had similar, but less pronounced effects, except that it induced moderate vasodilatation and an increase in cardiac output.
Comparison with previous studies
We have previously demonstrated that in sepsis treatment with the antihypertensive drug clonidine at a high clinical dose did not reduce blood pressure (14). In the present study, we demonstrate that the mechanisms maintaining pressure vary depending on the dose of clonidine. With the LCDC there was peripheral vasodilatation, which was likely due to a central action of clonidine to reduce sympathetic vasomotor tone (19–22). In this case, blood pressure was maintained by an increase in CO. In contrast, HCDC caused a level of peripheral vasoconstriction that maintained blood pressure in the face of a decrease in CO. The vasoconstrictor effect of HCDC in hyperdynamic sepsis is in contrast to its expected effect to reduce sympathetic vasomotor tone and cause vasodilatation. It is likely that this vasoconstrictor effect of clonidine to preserve blood pressure in sepsis is due to its action to restore pressor sensitivity to vasoconstrictor agents in sepsis, which has been demonstrated both in an ovine model of sepsis (with a dose of clonidine of 1.0 μg/kg/h) and in endotoxic rats (with a 100-fold greater dose of clonidine) (13, 14). Indeed, in ovine hyperdynamic sepsis, we demonstrated that clonidine restored the pressor sensitivity to not only noradrenaline, but also to angiotensin II (14). The different vascular responses with the two dose of clonidine suggest that vascular reactivity is only effectively restored with a high dose of clonidine. In addition, during sepsis clonidine significantly increased plasma vasopressin levels; this may have induced vasoconstriction and thus helped to maintain MAP.
Renal effects of clonidine
The HCDC decreased RBF, due to a combination of non-significant decreases in MAP and RVC. We have previously shown that HCDC (1.0 μg/kg/h) reduced the elevated level of renal sympathetic nerve activity in sepsis to normal levels (14), which would be expected to cause renal vasodilatation. The renal vasoconstriction observed in this study may therefore be due to an action of clonidine to improve vascular reactivity to endogenous vasoconstrictor agents, as described above (14). Both doses of clonidine almost doubled urine output during the first 4 h of treatment. This diuretic response was not due to changes in creatinine clearance or in fractional excretion of sodium or urea. Surprisingly clonidine increased AVP in sepsis, in contrast to the reduction in AVP by clonidine in non-septic animals (23–25). It is therefore likely that the water diuresis and high plasma osmolality in the clonidine treated animals stimulated AVP release (26). It is possible that the diuretic effect of clonidine may depend on a direct renal action (25, 27, 28) since it has been shown that α2-adrenoceptor agonists can increase urine flow through inhibition of AVP receptor sensitivity in the cortical collecting tubules (29). There was also a transient decrease in FENa and urine osmolality, as previously shown in studies in isolated tubules and in conscious rats and dogs (30–33).
Effects on cytokines
Previous studies have reported that in experimental sepsis treatment with the highly selective α2-adrenoceptor agonist dexmedetomidine decreased pro-inflammatory cytokine levels and reduced mortality (9–12). A blunted pro-inflammatory cytokine response was observed when dexmedetomidine treatment was started 1 and 2 h after induction of sepsis in rats (12), although this finding was not reproduced in mice (9). In sheep with established sepsis, we found that clonidine increased plasma levels of the anti-inflammatory cytokine IL-10, but did not change the level of the pro-inflammatory cytokine IL-6. These changes in cytokine levels are similar to those with selective β1-adrenoceptor blockade with atenolol in this model of sepsis (2), suggesting that the effect of clonidine may depend on reduced stimulation of β1-adrenoceptors due to reduced catecholamine release.
Other effects of clonidine were a decrease in body temperature, which has been shown to be due to a central action on α2-adrenoceptors (34, 35). Clonidine given as a high bolus dose has been shown to cause respiratory depression and severe hypoxemia in sheep (36), but this effect was not seen with infusion of clonidine in septic sheep.
Our study findings imply that in vasodilatory hypotensive sepsis, clonidine at a high clinical dose (1.0 μg/kg/h) can be given to modulate α2-adrenoceptors, attenuate tachycardia, diminish the IL-10 response, and decrease fever without inducing hypotension. Moreover, these results imply that such actions will be accompanied by other effects including a decreased cardiac output and renal blood flow. Finally, they imply that increased water diuresis and increased osmolarity also develop and stimulate AVP release, which may contribute to the vasoconstriction seen with HCDC.
Strengths and limitations of the study
This study has several strengths. It used a conscious model of sepsis where there was no confounding impact of anaesthesia on hemodynamics or the sympathetic nervous system. The model carried a mortality rate similar to that in humans with sepsis and similar phenotype to human sepsis. The study assessed the impact of two clinically relevant doses of clonidine (instead of markedly supra-clinical doses of limited clinical relevance) over 8 h after 24 h of sepsis, rather than before or immediately after its induction, thus providing a realistic simulation of what might happen clinically. Our study also carries some limitations. Treatment for a longer period may have revealed further effects of clonidine in sepsis. However, sustaining gram-negative infusion for a longer period in our experimental animals would have markedly increased mortality. We did not study the interaction between vasopressor therapy and clonidine as might happen in a clinical situation, and we did not determine liver function. We only measured two cytokines (one representative of a pro-inflammatory response and the other of an anti-inflammatory response), thus our understanding of the inflammatory impact of clonidine infusion is limited. In this large animal study there were insufficient numbers to determine if clonidine altered mortality and although treatment with α2-adrenoceptor agonists increased survival in rodent models of sepsis (9–12), we are unaware of studies that have examined the effect on mortality in septic patients.
There is increasing interest in the effects of treatment with clonidine in sepsis following both experimental and clinical studies demonstrating beneficial effects, although some of these studies have used supra-clinical doses. The present findings confirm that in hyperdynamic, hypotensive sepsis, treatment with high clinical doses of clonidine decreased heart rate, cardiac output and fever and increased IL-10 blood levels, without worsening hypotension, while expediting recovery of MAP to baseline values. Clonidine also decreased renal blood flow, with a transient, but substantial, increase in urine output, due to a water diuresis, leading to increased plasma osmolality and an increase in circulating AVP. Our findings provide novel and detailed physiological insights that might assist clinicians considering inhibition of the sympathetic nervous system with sympatholytics during severe sepsis.
The authors acknowledge the expert technical assistance of Alan McDonald and Tony Dornom.
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