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Clonidine Restores Pressor Responsiveness to Phenylephrine and Angiotensin II in Ovine Sepsis* Yugeesh R. Lankadeva, PhD1,2; Lindsea C. Booth, PhD1; Junko Kosaka, MD, PhD1; Roger G. Evans, PhD2; Luc Quintin, MD, PhD3; Rinaldo Bellomo, MD, PhD4,5,6; Clive N. May, PhD1 Objectives: In sepsis, prolonged, sympathetic overstimulation may lead to vasopressor-refractory hypotension. We therefore examined the effects of the α2-adrenergic agonist clonidine on mean arterial pressure, renal sympathetic nerve activity, and pressor responsiveness to phenylephrine and angiotensin II during hypotensive sepsis in conscious sheep. Design: Interventional study. Setting: Research institute. Subjects: Twelve adult Merino ewes (n = 6 per group). Interventions: Sepsis was induced by IV infusion of Escherichia coli for 32 hours. Pressor responses to increasing doses of phenylephrine and angiotensin II were measured at baseline and at 24, *See also p. 1548. 1 Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, VIC, Australia. 2 Department of Physiology, Monash University, VIC, Australia. 3 Department of Physiology, University of Lyon, Lyon, France. 4 Department of Intensive Care, Austin Health, Heidelberg, VIC, Australia. 5 Department of Medicine, Austin Health, Heidelberg, VIC, Australia. 6 The Australian and New Zealand Intensive Care Research Centre, Monash University, Melbourne, VIC, Australia. Supported, in part, by a grant from the National Health and Medical Research Council of Australia (1050672) and by funding from the Victorian Government Operational Infrastructure Support Grant. Dr. Lankadeva received grant support and disclosed employment. Dr. Booth is employed by the Florey Institute; received grant support from the University of Melbourne; and received support for travel from the Harold Mitchell Foundation, American Heart Association, and Federation of American Societies for Experimental Biology. Her institution received grant support from the National Health and Medical Research Council (NHMRC) of Australia. Dr. Kosaka’s institution received grant support. Dr. Evans is employed by Monash University. His institution received grant support from the NHMRC of Australia. Dr. Quintin received support for article research from Centre National de la Recherche Scientifique Projet Exploratoire Premier Soutien grant and has patent (U.S. Patent issued to Dr. Quintin). His institution received grant support from CNRS. Dr. May was supported by a NHMRC Research Fellowship. Dr. May received grant support from the NHMRC of Australia, is employed by the Florey Institute of Neuroscience, lectured (honoraria and travel support for presentations from Medtronic), has a patent (patent for use of flavonols for cardiac injury), and received support for article research from NHMRC. His institution received grant support from the NHMRC. Dr. Bellomo has disclosed that he does not have any potential conflicts of interest. For information regarding this article, E-mail: [email protected] Copyright © 2015 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved. DOI: 10.1097/CCM.0000000000000963
Critical Care Medicine
28, and 32 hours of sepsis. Sheep were treated with clonidine (1 μg/kg/hr) or saline-vehicle from 24 to 32 hours of sepsis. Measurements and Main Results: Sepsis was characterized by hypotension (~12 mm Hg), increased heart rate (~80 beats/min), increased renal sympathetic nerve activity (~70%), and blunted pressor responses to phenylephrine and angiotensin II. In vehicletreated sheep, mean arterial pressure progressively declined from 25 to 32 hours of sepsis (73 ± 3 to 66 ± 3 mm Hg; p = 0.013) while the elevations in heart rate and renal sympathetic nerve activity and reduced pressor responsiveness to vasopressors persisted. Clonidine treatment prevented the further decline in mean arterial pressure, substantially reduced heart rate and renal sympathetic nerve activity and restored pressor responsiveness to both phenylephrine and angiotensin II toward preseptic levels. Conclusions: Administration of clonidine during hypotensive sepsis reduced renal sympathetic nerve activity, restored vascular sensitivity to both phenylephrine and angiotensin II, and resulted in better preservation of arterial pressure. Considering these findings, a clinical trial for the use of clonidine in the treatment of persistent vasopressor-refractory hypotension in patients with septic shock would be worthwhile. (Crit Care Med 2015; 43:e221–e229) Key Words: α2-adrenergic agonist; Escherichia coli; hyperdynamic sepsis; sympathetic nerve activity; vascular sensitivity
S
epsis remains the main cause of death in ICUs, with mortality rates of 30–70% when associated with multiple organ dysfunction (1, 2). A major problem encountered during treatment of sepsis is reduced vascular responsiveness, causing vasopressor-refractory hypotension, which can eventually lead to death (3–5). Decreased pressor responsiveness to phenylephrine and norepinephrine has been reported in patients with septic shock (3) and during Escherichia coli endotoxin–induced experimental sepsis in humans (6). Similarly, we observed decreased pressor responsiveness to norepinephrine and angiotensin II (Ang II) in ovine hypotensive, hyperdynamic sepsis (7, 8). The mechanisms underlying vascular hyporesponsiveness in sepsis are not fully understood, and currently, there are no effective treatments. During sepsis, increased circulating levels of norepinephrine and Ang II may lead to down-regulation of vascular α1-adrenergic receptors (9, 10) and Ang II type 1 receptors www.ccmjournal.org
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(11–13), causing vascular hyporesponsiveness, hypotension, tissue hypoperfusion, and potentially multiple organ dysfunction (14, 15). This provided the impetus for studies of the effects of α2-adrenergic agonists (e.g., clonidine and dexmedetomidine) as sympatholytic agents in rodent models of sepsis (16–18). These studies indicate that in sepsis α2-adrenergic agonists increase arterial pressure without administration of vasopressors, improve survival, and reduce levels of inflammatory mediators (16–18). Recently, it has been proposed that α2-agonists may reverse vascular hyporesponsiveness induced by high levels of sympathetic nerve activity (SNA) in sepsis by reducing the release of endogenous norepinephrine from central and peripheral nerve terminals (19). Indeed, pretreatment with high doses of clonidine (200 μg/kg) or dexmedetomidine (100 μg/kg) restored pressor responsiveness to norepinephrine in lipopolysaccharide-challenged anesthetized rats (20). Despite these encouraging findings, there remains a paucity of information on the effects of α2-adrenergic agonists, at clinically relevant doses, on vascular sensitivity and SNA in conscious large animal models of hypotensive sepsis that more closely mimic human sepsis (21). We have established a model of hyperdynamic, hypotensive sepsis in conscious sheep, characterized by peripheral vasodilatation, tachycardia, increased cardiac output (22), and profoundly elevated renal and cardiac SNA (23). This hemodynamic profile is similar to that commonly seen in patients with sepsis (24). In the present study, we examined the effects of sympathoinhibition, induced by a clinically relevant dose of clonidine (1 μg/kg/hr) (19, 25), on systemic and renal hemodynamics and renal sympathetic nerve activity (RSNA) in conscious sheep following administration of live E. coli. We also determined whether clonidine treatment altered arterial pressure responsiveness to increasing doses of the selective α1-adrenergic agonist phenylephrine in septic sheep. To assess whether vascular sensitivity to another vasoconstrictor was also altered by clonidine, arterial pressure responses to Ang II were examined. We hypothesized that in hypotensive sepsis, clonidine would reduce the high levels of SNA resulting in improvement of basal vasomotor tone and would improve vascular sensitivity to phenylephrine, but not to Ang II.
MATERIALS AND METHODS Animal Preparation Female merino ewes (28–38 kg body weight) were housed in individual metabolic cages with free access to water and food daily. All experiments were approved by the Animal Ethics Committee of the Florey Institute of Neuroscience and Mental Health under guidelines laid down by the National Health and Medical Research Council of Australia. All animals underwent two aseptic surgical procedures. For all procedures, anesthesia was induced with IV sodium thiopentone (10–15 mg/kg) and following intubation was maintained with oxygen/air/isoflurane (end-tidal isoflurane, 1.5–2.0% v/v). First, the left carotid artery was exteriorized into a skin fold to form a carotid arterial loop, allowing easy access for arterial e222
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cannulation. Three weeks later, a transit-time flow probe was placed around the left renal artery (4 mm; Transonic Systems, Ithaca, NY) for measurement of renal blood flow (RBF) (26). At the same time, five to six intrafascicular electrodes were inserted into the left renal nerves for measurement of RSNA (27). In addition, a Tygon catheter (Cole-Parmer, Boronia, Australia; inner diameter [ID], 1.0 mm; outer diameter [OD], 1.5 mm) was inserted 20 cm into the carotid artery and connected to a pressure transducer (ITL Healthcare, Chelsea Heights, VIC, Australia) to allow measurement of mean arterial pressure (MAP) and heart rate (HR) and for collection of blood samples. Three polythene catheters were inserted into the right jugular vein: one for clonidine/saline infusion (Portex, Smiths Medical International, Hythe, Kent, United Kingdom; ID, 1.19 mm; OD, 1.7 mm), one for administering E. coli, and one for infusing vasopressors (Portex, Smiths Medical International; ID, 0.58 mm; OD, 0.96 mm). To maintain the patency of the arterial and venous catheters, they were continuously infused with heparinized saline (10 U heparin/mL at 3 mL/hr, Pfizer, West Ryde, NSW, Australia). For all surgical procedures, animals were treated with intramuscular antibiotics (900 mg procaine penicillin, Ilium Propen, Troy Laboratories, Smithfield, NSW, or Mavlab, QLD, Australia, at surgery and at 24 and 48 hr postsurgery). Postsurgical analgesia was maintained with intramuscular injection of flunixin (1 mg/kg; Troy Laboratories or Mavlab) administered at the start of surgery and then 8 and 24 hours postsurgery. Before experiments, animals were allowed at least 5 days of recovery following the second surgical procedure in order to minimize any effects of surgical stress. Analog signals (MAP, HR, and RBF) were recorded continuously at 100 Hz for 10 seconds every minute during experiments using a data acquisition system (Labview, National Instruments, Austin, TX). Renal vascular conductance (RVC) was calculated as RBF/MAP. RSNA was recorded differentially between the pair of electrodes with the best signal-to-noise ratio. The signal was amplified (×20,000) and filtered (band pass, 300–1,000 Hz), displayed on an oscilloscope, and passed through an audio amplifier and a loud speaker. In addition to recordings using Labview, RSNA (5,000 Hz) and MAP, HR, and RBF (100 Hz) were recorded on computer using a CED 1401 interface and Spike 2 Software (Cambridge Electronic Design, Cambridge, United Kingdom). RSNA was analyzed on a beatto-beat basis using custom-written routines in Spike 2 software, as previously reported (23). This program determines diastolic pressure, systolic pressure, MAP, heart period, and the number of discriminated spikes of RSNA above threshold between successive diastoles, which is a measure of burst size. The background noise in the RSNA signal was determined as the spikes/s during the highest dose of phenylephrine during the baseline period when SNA was abolished. The threshold was set just above the background noise level so that spikes from even small bursts were included in the analysis. Experimental Protocol Following a 24-hour baseline period, sepsis was induced in conscious sheep with an IV dose of live E. coli (2.8 × 109 July 2015 • Volume 43 • Number 7
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colony-forming units [CFUs] over 30 min) as a bolus, followed by a continuous infusion (1.26 × 109 CFU/hr up to 32 hr of sepsis). A continuous IV infusion of 0.9% w/v NaCl (1 mL/kg/hr) was administered to sheep from the start of the baseline period until the end of the intervention period (56 hr). Twentyfour hours after commencing E. coli infusion, when HR had increased by ~80% and MAP had decreased by ~10 mm Hg compared with baseline levels, the animals received a continuous IV infusion of either clonidine (1 μg/kg/hr; Boehringer Ingelheim, North Ryde, NSW, Australia) or vehicle (1 mL/kg/ hr 0.9% w/v NaCl) for 8 hours. Pressor responses of arterial pressure to phenylephrine and Ang II were examined at baseline, at 24 hours of sepsis, and at 28 and 32 hours of sepsis, which corresponded to 4 and 8 hours of treatment with clonidine or vehicle. Increasing bolus doses of phenylephrine (0.2, 0.4 0.8, 1, 1.5 μg/kg; Sigma Aldrich, Castle Hill, NSW, Australia) followed by Ang II (1, 2, 5, 8, 10 ng/kg; Auspep, Parkville, VIC, Australia) were administered IV (in 3 mL of 0.9% w/v NaCl) to evoke changes in arterial pressure. The highest dose of vasopressor was chosen to obtain an increase in MAP of approximately 20–25 mm Hg. Following each dose, adequate time (5 min for low doses and 10 min for the higher doses) was allowed for MAP to return to its respective control levels before the next dose was administered. In preseptic animals, MAP at baseline and 10 minutes after the highest dose of phenylephrine was similar in the clonidine (84.6 ± 2.5 vs 84.8 ± 2.5 mm Hg, respectively; p = 0.93) and vehicle (85.4 ± 2.4 vs 85.2 ± 2.4 mm Hg, respectively; p = 0.92) groups. Similarly, at the 32-hour sepsis time point, MAP at baseline and 10 minutes after the highest dose of phenylephrine was similar in the clonidine (74.9 ± 5.2 vs 75.1 ± 5.1 mm Hg; p = 0.95) and vehicle (66.8 ± 2.8 vs 66.4 ± 0.91 mm Hg, respectively; p = 0.92) groups.
Arterial blood samples were collected at baseline, just before the infusion of E. coli commenced, and then 4, 8, 24, 28, and 32 hours later for measurement of blood gases, electrolytes, and lactate (ABL System 625; Radiometer Medical, Copenhagen, Denmark). At the end of the protocol, the infusions of E. coli and clonidine/saline were terminated and the animals were treated with intramuscular gentamycin (150 mg, Troy Laboratories, Glendenning, NSW, Australia). Statistical Analysis All variables are reported as mean ± sem or as geometric mean (95% CI) as appropriate. MAP, HR, RBF, RVC, RSNA, and biochemical markers are reported as the average over the 24th hour of the baseline period and as hourly averages from the 24 to 32 hours after commencing the infusion of E. coli. Data were analyzed using repeated-measures analysis of variance (ANOVA) with factors treatment (ptreatment: saline or clonidine), time (ptime), and their interaction (pinteraction). Specific post hoc comparisons were made using Student t test. The Bonferroni correction was applied to protect against the risk of type 1 error. The absolute changes in MAP in response to each dose of vasopressor after induction of sepsis were compared with responses at baseline, prior to induction of sepsis, and following 4 and 8 hours of subsequent treatment with clonidine or saline, using repeated-measures ANOVA. Factors in the analysis were time (ptime), dose (pdose), and their interaction (pinteraction). p Values from within-subjects factors were conservatively adjusted using the Greenhouse-Geisser method (28). Statistical analysis was performed using GraphPAD PRISM 6.0 (Graphpad Software, La Jolla, CA) for Windows. All variables were assessed for normality and log-transformed where appropriate. Two-sided p value less than or equal to 0.05 was considered statistically significant.
Table 1. Cardiovascular, Respiratory, and Biochemical Variables During the Baseline Period and at 24 Hours of Sepsis in the Groups of Sheep to be Treated With Vehicle or Clonidine Vehicle Group Variables
Baseline
Clonidine Group
Sepsis 24 Hr
Baseline
Sepsis 24 Hr
Mean arterial pressure (mm Hg)
83 ± 3
72 ± 3a
85 ± 2
72 ± 3a
Heart rate (beats/min)
74 ± 4
162 ± 7b
79 ± 2
153 ± 8b
Renal blood flow (mL/min)
200 ± 10
329 ± 8b
188 ± 20
292 ± 25a
Renal vascular conductance (mL/min/mm Hg)
2.4 ± 0.1
4.4 ± 0.4b
2.3 ± 0.3
4.1 ± 0.4a
100 (99–101)
166 (128–205)a
100 (99–102)
175 (150–201)b
97 ± 1.4
83 ± 3a
95 ± 0.8
79 ± 4a
0.28 ± 0.05
1.23 ± 0.21a
0.31 ± 0.05
1.11 ± 0.14b
Renal sympathetic nerve activity (% baseline) Arterial Po2 (mm Hg) Blood lactate (mmol/L)
< 0.01 indicates significant differences between variables at baseline compared with variables at 24 hr of sepsis in the groups of sheep to be treated with p vehicle or clonidine from a two-tailed unpaired Student t test. b p < 0.001 indicates significant differences between variables at baseline compared with variables at 24 hr of sepsis in the groups of sheep to be treated with vehicle or clonidine from a two-tailed unpaired Student t test. Values are mean ± sem for normally distributed variables and geometric mean (95% CI) for those not normally distributed. a
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RESULTS The body weight of sheep treated with clonidine (32 ± 2 kg; n = 6) was similar to that of those treated with saline (31 ± 1 kg; n = 6). There was no significant time effect on cardiovascular and renal variables during the 24-hour baseline period (ptime always > 0.05), and the basal levels were similar in the two groups of sheep (Table 1). Two animals from each treatment group died during the period 10–24 hours after the infusion of E. coli commenced and were excluded from analysis. Development of Sepsis Over 24 Hours By 24 hours of sepsis, there were similar changes in both groups that were consistent with our previous findings in this ovine model of hypotensive, hyperdynamic sepsis (22, 23). In both groups, there were similar reductions in MAP, accompanied by similar increases in HR, RBF, RVC, and RSNA (Table 1 and Figs. 1–3). The decrease in arterial Po2 and increase in arterial lactate were also similar in each group. Effects of Clonidine and Saline Infusion During Sepsis During treatment with vehicle from 24 to 32 hours of sepsis, MAP progressively declined from 73 ± 3 to 66 ± 3 mm Hg (–8% ± 2%) (Fig. 1A). The fall in MAP was accompanied by increases in RVC (4.6 ± 0.4 to 5.6 ± 0.7 mL/min/mm Hg; +21% ± 8%)
(Fig. 1D) and RBF (329 ± 25 to 371 ± 46 mL/min; +11% ± 5%) (Fig. 1B). Both HR (160 ± 14 to 150 ± 11 beats/min) (Fig. 1C) and RSNA (166% [128%; 205%] to 175% [136%; 210%] of baseline) (Fig. 2A) remained elevated. By contrast, in sheep treated with clonidine, there was no decrease in MAP (72 ± 3 to 75 ± 5 mm Hg) (Fig. 1A) and no increase in RVC (4.1 ± 0.4 to 3.7 ± 0.7 mL/min/mm Hg) (Fig. 1D), so that the increase in RBF seen in the vehicle group did not occur (292 ± 25 to 273 ± 38 mL/min) (Fig. 1B) during the 24- to 32-hour period of sepsis. The profile of changes in MAP, RBF, and RVC differed significantly between clonidine and vehicle-treated sheep (all p < 0.01) (Fig. 1, A, B, and D). In sheep treated with clonidine, HR decreased (from 153 ± 8 to 116 ± 10 beats/min; –23% ± 6%) (Fig. 1C) and RSNA decreased to baseline levels (from 166% [150%; 201%] to 103% [85%; 212%] of baseline) (Fig. 2A). As sepsis developed, there were similar progressive decreases in arterial Po2 in both the vehicle (–22 ± 2 mm Hg) and clonidine (–24 ± 5 mm Hg) groups (Fig. 3A). Arterial Pco2 fell by 3.5 ± 1 mm Hg in both groups, over the first 8 hours after commencing the infusion of E. coli, but increased thereafter. The rate of increase of arterial Pco2 was greater in clonidine than vehicletreated sheep (Fig. 3B). Blood lactate increased during the first 24 hours after commencing the infusion of E. coli and thereafter remained stable; the pattern of change was similar in the two groups of sheep (Fig. 3C). There was a progressive reduction in plasma K+ concentration during sepsis, with the degree of hypokalemia being augmented by clonidine treatment (Fig. 3D).
Figure 1. Mean arterial pressure (A), renal blood flow (B), heart rate (C), and renal vascular conductance (D) during infusion of Escherichia coli from 0 to 32 hr and subsequent treatment with clonidine (n = 5; closed circles) or saline (n = 5; open circles) from 24 to 32 hr in conscious sheep. Time 0 is the mean of the 24 hr baseline period, data are mean ± sem, and p values represent treatment-time interactions from two-way repeated-measures analysis of variance.
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Effects of Clonidine on Arterial Pressure Responses to Vasopressors During Sepsis Administration of phenylephrine and Ang II evoked dosedependent increases in MAP (Figs. 4 and 5). By 24 hours of sepsis, the pressor responses to phenylephrine (Fig. 4) and Ang II (Fig. 5) were both significantly less than during the baseline (preseptic) period. For example, the highest dose of phenylephrine (1.5 μg/kg) increased MAP by 21 ± 2 mm Hg at baseline but only by 14 ± 2 mm Hg at 24 hours after commencing the infusion of E. coli (Fig. 4, A and B). Similarly, the highest dose of Ang II (10 ng/kg) increased MAP by 25 ± 2 mm Hg at baseline but only by 14 ± 2 mm Hg at 24 hours of sepsis (Fig. 5, A and B). July 2015 • Volume 43 • Number 7
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Figure 2. Renal sympathetic nerve activity (RSNA) during infusion of Escherichia coli from 0 to 32 hr and subsequent treatment with clonidine (n = 5; closed circles) or saline (n = 5; open circles) from 24 to 32 hr in conscious sheep (A). Time 0 is the mean of the 24 hr baseline period, data are mean ± sem, and p values represent treatment-time interactions from two-way repeated-measures analysis of variance. Raw traces (10 s) from a conscious sheep of arterial pressure (AP) and RSNA during the baseline period (B), 24 hr after commencing the infusion of E. coli (C), and after 8 hr of clonidine infusion (32 hr of sepsis) (D).
In vehicle-treated sheep, the pressor responses to the highest dose of phenylephrine and Ang II, respectively, remained diminished at 28 hours (14 ± 2 and 15 ± 2 mm Hg) (Figs. 4C and 5C) and 32 hours (13 ± 2 and 16 ± 2 mm Hg) (Figs. 4E and 5E) of sepsis compared with the responses before sepsis. By contrast, in clonidine-treated sheep, the pressor responses to phenylephrine and Ang II at 28 hours (19 ± 1 and 21 ± 4 mm Hg) (Figs. 4D and 5D) and 32 hours (21 ± 4 and 21 ± 3 mm Hg) (Figs. 4F and 5F) of sepsis were indistinguishable from those observed at baseline (21 ± 2 and 25 ± 2 mm Hg).
DISCUSSION The main findings are that a clinically relevant dose of clonidine given to conscious sheep with established hypotensive sepsis, at a clinically appropriate time, prevented the continual decline in MAP, reduced the high levels of HR and RSNA toward baseline levels, and prevented further increases in RVC and RBF. We also found a large reduction in the pressor responsiveness to both phenylephrine and Ang II at 24 hours of sepsis. Interestingly, clonidine treatment restored the pressor responsiveness to not only phenylephrine but also Ang II. These findings suggest that sympathoinhibition induced by Critical Care Medicine
clonidine improves vascular sensitivity to both endogenous and exogenously infused vasoconstrictors, indicating that it may be a useful adjunctive treatment for patients with established sepsis who are resistant to vasopressor therapy. Effect of Clonidine on Vascular Hyporesponsiveness in Sepsis In critical care units, α2-adrenergic agonists are now often used as sedatives (29), and a recent case report indicated that treatment with clonidine resulted in an increased pressor response to norepinephrine in a patient with septic shock (25). The use of sympatholytics in a hypotensive state such as sepsis is, however, counterintuitive and raises concerns because their actions to reduce SNA would be expected to enhance the degree of hypotension and promote multiple organ failure. In the current study, treatment with clonidine in ovine hyperdynamic, hypotensive sepsis prevented the continuous decline in MAP seen in the vehicle-treated group. This occurred despite the decrease in RSNA and HR, which would be expected to lead to a further decrease in MAP. Indeed, we have shown that renal denervation results in an enhanced decrease in blood pressure in septic sheep (22). This indicates the important role elevated RSNA plays in maintaining MAP www.ccmjournal.org
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up-regulation of vascular α1adrenergic receptors that were down-regulated in sepsis (19). Interestingly, however, clonidine also restored the pressor sensitivity to Ang II, suggesting that its actions to improve vasopressor responsiveness may result from mechanisms that are either downstream or independent of α1-adrenergic receptors. There are other mechanisms by which clonidine may improve vascular reactivity during sepsis. First, proinflammatory cytokines, which are abundantly generated during sepsis, have been shown to mediate a down-regulation of both α1-adrenergic and Ang II type 1 receptor expression in mice and rats (9–11, 13). Furthermore, blockade of these Figure 3. Arterial partial pressure o2 (A), arterial partial pressure co2 (B), arterial blood lactate (C), and plasma proinflammatory cytokines K+ (D) during infusion of Escherichia coli from 0 to 32 hr and subsequent treatment with clonidine (n = 6; prevents and/or attenuates the closed circles) or saline (n = 6; open circles) from 24 to 32 hr in conscious sheep. Data (mean ± sem) are sepsis-induced down-regureported at time 0 (24th hr of the baseline period) and at 4, 8, 24, 28, and 32 hr after commencing the infusion of E. coli. p values represent treatment-time interactions from two-way repeated-measures analysis of variance. lation of these receptors (10, 13). It is well accepted that α2at least during the early phases of sepsis, most likely by causing adrenergic agonists lead to a significant reduction in inflamrenal vasoconstriction and renin release. Indeed, Ang II levmatory cytokines during sepsis in the experimental setting els are increased in sepsis (30) and blockade of angiotensin(16–18), after cardiopulmonary bypass surgery (32), and in the converting enzyme in anesthetized septic pigs exacerbated the critical care unit (33). Second, it is known that hyperpolarizaseverity of the hypotension (31). Our finding that in septic tion of vascular smooth muscle cells via excessive opening of sheep clonidine prevented a further decline in arterial pressure K+ATP channels plays a critical role in the development of vascular hyporesponsiveness during sepsis (14). The K+ATP chandespite a reduction in RSNA suggests that arterial pressure was maintained by increased peripheral vascular tone due to resto- nel is mainly composed of two subunits: the sulphonylurea receptor and a pore-forming subunit belonging to the Kir6.0 ration of vascular sensitivity to endogenous vasoconstrictors. family (34). There is evidence that the K+ATP channel sulphonylurea receptor may become dysfunctional or uncoupled from Mechanisms by Which Clonidine May Improve the pore-forming subunit during sepsis (35). This may explain Vascular Sensitivity to Vasopressors the ineffectiveness of the non-selective sulfonylurea drug glibThe present findings indicate that septic sheep developed a enclamide in clinical trials on patients with sepsis (36, 37). large reduction in pressor responsiveness to both phenylephBy contrast, selective inhibition of the vascular K+ATP channel rine and Ang II, which would result in a lack of responsiveness pore-forming Kir6.0 subunit leads to effective restoration of the to endogenous norepinephrine and Ang II and enhance the sepsis-induced hypotension. Treatment with clonidine for 4–8 pressor responses in isolated vessels incubated with lipopolyhours completely restored the pressor responsiveness to phen- sacchride (34, 35). Importantly, clonidine at clinically relevant concentrations has been shown to directly bind and inhibit ylephrine, which probably accounted for the maintained blood vascular K+ATP channels, primarily through its effects on the pressure due to increased vascular responsiveness to endogpore-forming subunit Kir6.0, independent of any interactions enous norepinephrine. The 4- to 8-hour time interval in our with adrenergic receptors (38). Thus, the relative hypokalemia study was similar to the interval reported between the start of clonidine administration and the observed increase in blood observed in septic sheep following clonidine treatment may result from inhibition of K+ATP channels, which may also play pressure response in a patient with established septic shock (25). It has been proposed that the ability of clonidine to restore a role in restoring vascular sensitivity to both endogenous and exogenously infused vasoconstrictors. Finally, it is feasible that pressor sensitivity to catecholamines is due to the reduction in sympathetic outflow and norepinephrine release leading to clonidine acting directly on extrasynaptic α2b- and postsynaptic e226
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Figure 4. Effects of treatment with saline-vehicle or clonidine on the changes in mean arterial pressure induced by bolus doses of phenylephrine in conscious sheep. Responses in preseptic sheep in the vehicle group (open boxes, A, C, and E) and clonidine group (open circles, B, D, and F) and at 24 hr of sepsis in the vehicle group (closed boxes, A) and clonidine group (closed circles, B). Pressor responsiveness after 4 hr infusion of saline (closed boxes, C) or clonidine (closed circles, D) and after 8 hr of saline (closed boxes, E) or clonidine (closed circles, F). Dose-response curves were compared using two-way repeated-measures analysis of variance, and p values represent time-dose interactions. Data are mean ± sem (n = 6/group).
Figure 5. Effects of treatment with saline-vehicle or clonidine on the changes in mean arterial pressure induced by bolus doses of angiotensin II in conscious sheep. Responses in preseptic sheep in the vehicle group (open boxes, A, C, and E) and clonidine group (open circles, B, D, and F) and at 24 hr of sepsis in the vehicle group (closed boxes, A) and clonidine group (closed circles, B). Pressor responsiveness after 4 hr infusion of saline (closed boxes, C) or clonidine (closed circles, D) and after 8 hr of saline (closed boxes, E) or clonidine (closed circles, F). Dose-response curves were compared using two-way repeated-measures analysis of variance, and p values represent time-dose interactions. Data are mean ± sem (n = 6/group).
α1-adrenergic receptors on the vascular smooth muscle could be partly responsible for the enhanced vasoconstriction observed in response to phenylephrine and Ang II (39, 40).
exercised when extrapolating our findings to the clinical situation. Nevertheless, since the hemodynamic profile reported in septic sheep is similar to that reported in human sepsis, this model has clinical relevance. Indeed, the progressive decline in arterial pressure along with increased blood lactate and peripheral vasodilatation we observed fulfills the consensus criteria for sepsis in humans (24). Importantly, all observations were made in conscious animals, thus ruling out the potentially confounding effects of anesthesia on SNA and arterial pressure responses to vasopressors. The ~33% mortality rate observed in our study is comparable to the ~30% mortality rate reported in humans with severe sepsis. Limitations of our current study include the fact that we did not separately assess the central and peripheral actions of clonidine and we did not investigate the effects
Strengths and Limitations To our knowledge, this is the first report of the effects of a clinically relevant dose of the α2-adrenergic agonist clonidine (1 μg/kg/hr) on resting arterial pressure, RSNA, as well as the responsiveness of arterial pressure to increasing doses of the selective α1-adrenergic agonist phenylephrine in a conscious large animal model of hypotensive, hyperdynamic sepsis. In addition, we demonstrate that clonidine restored pressor responsiveness to another vasopressor system (Ang II). The study was randomized and vehicle-controlled, but as with any experimental animal studies, caution must be Critical Care Medicine
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of other α2-adrenergic agonists such as dexmedetomidine or an imidazoline receptor agonist. In contrast to patients commonly diagnosed with sepsis, the sheep studied in our experiment did not have comorbidities, such as old age, vascular disease, hypertension, or diabetes. We only assessed a single dose of clonidine, but we used a dose that is commonly used in critical care units. Finally, we did not assess whether clonidine also restores vascular responsiveness to vasopressin and terlipressin. In a large animal model of hypotensive hyperdynamic sepsis, an 8-hour infusion of a dose of clonidine commonly used in critical care units prevented the progressive decline in arterial pressure and improved pressor responsiveness to phenylephrine and Ang II. Clonidine reduced the elevated levels of RSNA, RBF, and RVC and had no observed deleterious effects. Considering these findings, a feasibility and safety clinical trial for the use of clonidine in the treatment of persistent vasopressor-refractory hypotension in patients with established septic shock would be worthwhile.
ACKNOWLEDGMENTS We thank Alan McDonald and Tony Dorman for their excellent technical assistance.
REFERENCES
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15. Dünser MW, Hasibeder WR: Sympathetic overstimulation during critical illness: Adverse effects of adrenergic stress. J Intensive Care Med 2009; 24:293–316 16. Taniguchi T, Kidani Y, Kanakura H, et al: Effects of dexmedetomidine on mortality rate and inflammatory responses to endotoxin-induced shock in rats. Crit Care Med 2004; 32:1322–1326 17. Taniguchi T, Kurita A, Kobayashi K, et al: Dose- and time-related effects of dexmedetomidine on mortality and inflammatory responses to endotoxin-induced shock in rats. J Anesth 2008; 22:221–228 18. Hofer S, Steppan J, Wagner T, et al: Central sympatholytics prolong survival in experimental sepsis. Crit Care 2009; 13:R11 19. Pichot C, Géloën A, Ghignone M, et al: Alpha-2 agonists to reduce vasopressor requirements in septic shock? Med Hypotheses 2010; 75:652–656 20. Geloen A, Chapelier K, Cividjian A, et al: Clonidine and dexmedetomidine increase the pressor response to norepinephrine in experimental sepsis: A pilot study. Crit Care Med 2013; 41:e431–e438 21. Dünser MW, Gradwohl-Matis I: α(2)-agonists to restore adrenergic vasoconstrictor responsiveness in septic shock: Thinking outside of the box or fishing in the wrong pond? Crit Care Med 2013; 41:2838–2840 22. Calzavacca P, Bailey M, Velkoska E, et al: Effects of renal denervation on regional hemodynamics and kidney function in experimental hyperdynamic sepsis. Crit Care Med 2014; 42:e401–e409 23. Ramchandra R, Wan L, Hood SG, et al: Septic shock induces distinct changes in sympathetic nerve activity to the heart and kidney in conscious sheep. Am J Physiol Regul Integr Comp Physiol 2009; 297:R1247–R1253 24. Dellinger RP, Levy MM, Rhodes A, et al; Surviving Sepsis Campaign Guidelines Committee including The Pediatric Subgroup: Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Med 2013; 39:165–228 25. Pichot C, Mathern P, Khettab F, et al: Increased pressor response to noradrenaline during septic shock following clonidine? Anaesth Intensive Care 2010; 38:784–785 26. May CN, Bednarik JA: Effects of infusion of combinations of adrenocorticosteroids on systemic and regional haemodynamics in conscious sheep. J Hypertens 1996; 14:475–482 27. May CN, McAllen RM: Baroreceptor-independent renal nerve inhibition by intracerebroventricular angiotensin II in conscious sheep. Am J Physiol 1997; 273:R560–R567 28. Ludbrook J: Repeated measurements and multiple comparisons in cardiovascular research. Cardiovasc Res 1994; 28:303–311 29. Pichot C, Ghignone M, Quintin L: Dexmedetomidine and clonidine: From second- to first-line sedative agents in the critical care setting? J Intensive Care Med 2012; 27:219–237 3 0. Doerschug KC, Delsing AS, Schmidt GA, et al: Renin-angiotensin system activation correlates with microvascular dysfunction in a prospective cohort study of clinical sepsis. Crit Care 2010; 14:R24 31. Corrêa TD, Jeger V, Pereira AJ, et al: Angiotensin II in septic shock: Effects on tissue perfusion, organ function, and mitochondrial respiration in a porcine model of fecal peritonitis. Crit Care Med 2014; 42:e550–e559 32. von Dossow V, Baehr N, Moshirzadeh M, et al: Clonidine attenuated early proinflammatory response in T-cell subsets after cardiac surgery. Anesth Analg 2006; 103:809–814 33. Venn RM, Bryant A, Hall GM, et al: Effects of dexmedetomidine on adrenocortical function, and the cardiovascular, endocrine and inflammatory responses in post-operative patients needing sedation in the intensive care unit. Br J Anaesth 2001; 86:650–656 34. Buckley JF, Singer M, Clapp LH: Role of KATP channels in sepsis. Cardiovasc Res 2006; 72:220–230 35. O’Brien AJ, Thakur G, Buckley JF, et al: The pore-forming subunit of the K(ATP) channel is an important molecular target for LPSinduced vascular hyporeactivity in vitro. Br J Pharmacol 2005; 144:367–375 36. Warrillow S, Egi M, Bellomo R: Randomized, double-blind, placebocontrolled crossover pilot study of a potassium channel blocker in patients with septic shock. Crit Care Med 2006; 34:980–985 July 2015 • Volume 43 • Number 7
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39. Inomata S, Nishikawa T, Kihara S, et al: Enhancement of pressor response to intravenous phenylephrine following oral clonidine medication in awake and anaesthetized patients. Can J Anaesth 1995; 42:119–125 40. Watanabe Y, Iida H, Tanabe K, et al: Clonidine premedication modifies responses to adrenoceptor agonists and baroreflex sensitivity. Can J Anaesth 1998; 45:1084–1090
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Clonidine Restores Pressor Responsiveness to Phenylephrine and Angiotensin II in Ovine Sepsis* Yugeesh R. Lankadeva, PhD1,2; Lindsea C. Booth, PhD1; Junko Kosaka, MD, PhD1; Roger G. Evans, PhD2; Luc Quintin, MD, PhD3; Rinaldo Bellomo, MD, PhD4,5,6; Clive N. May, PhD1 Objectives: In sepsis, prolonged, sympathetic overstimulation may lead to vasopressor-refractory hypotension. We therefore examined the effects of the α2-adrenergic agonist clonidine on mean arterial pressure, renal sympathetic nerve activity, and pressor responsiveness to phenylephrine and angiotensin II during hypotensive sepsis in conscious sheep. Design: Interventional study. Setting: Research institute. Subjects: Twelve adult Merino ewes (n = 6 per group). Interventions: Sepsis was induced by IV infusion of Escherichia coli for 32 hours. Pressor responses to increasing doses of phenylephrine and angiotensin II were measured at baseline and at 24, *See also p. 1548. 1 Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, VIC, Australia. 2 Department of Physiology, Monash University, VIC, Australia. 3 Department of Physiology, University of Lyon, Lyon, France. 4 Department of Intensive Care, Austin Health, Heidelberg, VIC, Australia. 5 Department of Medicine, Austin Health, Heidelberg, VIC, Australia. 6 The Australian and New Zealand Intensive Care Research Centre, Monash University, Melbourne, VIC, Australia. Supported, in part, by a grant from the National Health and Medical Research Council of Australia (1050672) and by funding from the Victorian Government Operational Infrastructure Support Grant. Dr. Lankadeva received grant support and disclosed employment. Dr. Booth is employed by the Florey Institute; received grant support from the University of Melbourne; and received support for travel from the Harold Mitchell Foundation, American Heart Association, and Federation of American Societies for Experimental Biology. Her institution received grant support from the National Health and Medical Research Council (NHMRC) of Australia. Dr. Kosaka’s institution received grant support. Dr. Evans is employed by Monash University. His institution received grant support from the NHMRC of Australia. Dr. Quintin received support for article research from Centre National de la Recherche Scientifique Projet Exploratoire Premier Soutien grant and has patent (U.S. Patent issued to Dr. Quintin). His institution received grant support from CNRS. Dr. May was supported by a NHMRC Research Fellowship. Dr. May received grant support from the NHMRC of Australia, is employed by the Florey Institute of Neuroscience, lectured (honoraria and travel support for presentations from Medtronic), has a patent (patent for use of flavonols for cardiac injury), and received support for article research from NHMRC. His institution received grant support from the NHMRC. Dr. Bellomo has disclosed that he does not have any potential conflicts of interest. For information regarding this article, E-mail: [email protected] Copyright © 2015 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved. DOI: 10.1097/CCM.0000000000000963
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28, and 32 hours of sepsis. Sheep were treated with clonidine (1 μg/kg/hr) or saline-vehicle from 24 to 32 hours of sepsis. Measurements and Main Results: Sepsis was characterized by hypotension (~12 mm Hg), increased heart rate (~80 beats/min), increased renal sympathetic nerve activity (~70%), and blunted pressor responses to phenylephrine and angiotensin II. In vehicletreated sheep, mean arterial pressure progressively declined from 25 to 32 hours of sepsis (73 ± 3 to 66 ± 3 mm Hg; p = 0.013) while the elevations in heart rate and renal sympathetic nerve activity and reduced pressor responsiveness to vasopressors persisted. Clonidine treatment prevented the further decline in mean arterial pressure, substantially reduced heart rate and renal sympathetic nerve activity and restored pressor responsiveness to both phenylephrine and angiotensin II toward preseptic levels. Conclusions: Administration of clonidine during hypotensive sepsis reduced renal sympathetic nerve activity, restored vascular sensitivity to both phenylephrine and angiotensin II, and resulted in better preservation of arterial pressure. Considering these findings, a clinical trial for the use of clonidine in the treatment of persistent vasopressor-refractory hypotension in patients with septic shock would be worthwhile. (Crit Care Med 2015; 43:e221–e229) Key Words: α2-adrenergic agonist; Escherichia coli; hyperdynamic sepsis; sympathetic nerve activity; vascular sensitivity
S
epsis remains the main cause of death in ICUs, with mortality rates of 30–70% when associated with multiple organ dysfunction (1, 2). A major problem encountered during treatment of sepsis is reduced vascular responsiveness, causing vasopressor-refractory hypotension, which can eventually lead to death (3–5). Decreased pressor responsiveness to phenylephrine and norepinephrine has been reported in patients with septic shock (3) and during Escherichia coli endotoxin–induced experimental sepsis in humans (6). Similarly, we observed decreased pressor responsiveness to norepinephrine and angiotensin II (Ang II) in ovine hypotensive, hyperdynamic sepsis (7, 8). The mechanisms underlying vascular hyporesponsiveness in sepsis are not fully understood, and currently, there are no effective treatments. During sepsis, increased circulating levels of norepinephrine and Ang II may lead to down-regulation of vascular α1-adrenergic receptors (9, 10) and Ang II type 1 receptors www.ccmjournal.org
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(11–13), causing vascular hyporesponsiveness, hypotension, tissue hypoperfusion, and potentially multiple organ dysfunction (14, 15). This provided the impetus for studies of the effects of α2-adrenergic agonists (e.g., clonidine and dexmedetomidine) as sympatholytic agents in rodent models of sepsis (16–18). These studies indicate that in sepsis α2-adrenergic agonists increase arterial pressure without administration of vasopressors, improve survival, and reduce levels of inflammatory mediators (16–18). Recently, it has been proposed that α2-agonists may reverse vascular hyporesponsiveness induced by high levels of sympathetic nerve activity (SNA) in sepsis by reducing the release of endogenous norepinephrine from central and peripheral nerve terminals (19). Indeed, pretreatment with high doses of clonidine (200 μg/kg) or dexmedetomidine (100 μg/kg) restored pressor responsiveness to norepinephrine in lipopolysaccharide-challenged anesthetized rats (20). Despite these encouraging findings, there remains a paucity of information on the effects of α2-adrenergic agonists, at clinically relevant doses, on vascular sensitivity and SNA in conscious large animal models of hypotensive sepsis that more closely mimic human sepsis (21). We have established a model of hyperdynamic, hypotensive sepsis in conscious sheep, characterized by peripheral vasodilatation, tachycardia, increased cardiac output (22), and profoundly elevated renal and cardiac SNA (23). This hemodynamic profile is similar to that commonly seen in patients with sepsis (24). In the present study, we examined the effects of sympathoinhibition, induced by a clinically relevant dose of clonidine (1 μg/kg/hr) (19, 25), on systemic and renal hemodynamics and renal sympathetic nerve activity (RSNA) in conscious sheep following administration of live E. coli. We also determined whether clonidine treatment altered arterial pressure responsiveness to increasing doses of the selective α1-adrenergic agonist phenylephrine in septic sheep. To assess whether vascular sensitivity to another vasoconstrictor was also altered by clonidine, arterial pressure responses to Ang II were examined. We hypothesized that in hypotensive sepsis, clonidine would reduce the high levels of SNA resulting in improvement of basal vasomotor tone and would improve vascular sensitivity to phenylephrine, but not to Ang II.
MATERIALS AND METHODS Animal Preparation Female merino ewes (28–38 kg body weight) were housed in individual metabolic cages with free access to water and food daily. All experiments were approved by the Animal Ethics Committee of the Florey Institute of Neuroscience and Mental Health under guidelines laid down by the National Health and Medical Research Council of Australia. All animals underwent two aseptic surgical procedures. For all procedures, anesthesia was induced with IV sodium thiopentone (10–15 mg/kg) and following intubation was maintained with oxygen/air/isoflurane (end-tidal isoflurane, 1.5–2.0% v/v). First, the left carotid artery was exteriorized into a skin fold to form a carotid arterial loop, allowing easy access for arterial e222
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cannulation. Three weeks later, a transit-time flow probe was placed around the left renal artery (4 mm; Transonic Systems, Ithaca, NY) for measurement of renal blood flow (RBF) (26). At the same time, five to six intrafascicular electrodes were inserted into the left renal nerves for measurement of RSNA (27). In addition, a Tygon catheter (Cole-Parmer, Boronia, Australia; inner diameter [ID], 1.0 mm; outer diameter [OD], 1.5 mm) was inserted 20 cm into the carotid artery and connected to a pressure transducer (ITL Healthcare, Chelsea Heights, VIC, Australia) to allow measurement of mean arterial pressure (MAP) and heart rate (HR) and for collection of blood samples. Three polythene catheters were inserted into the right jugular vein: one for clonidine/saline infusion (Portex, Smiths Medical International, Hythe, Kent, United Kingdom; ID, 1.19 mm; OD, 1.7 mm), one for administering E. coli, and one for infusing vasopressors (Portex, Smiths Medical International; ID, 0.58 mm; OD, 0.96 mm). To maintain the patency of the arterial and venous catheters, they were continuously infused with heparinized saline (10 U heparin/mL at 3 mL/hr, Pfizer, West Ryde, NSW, Australia). For all surgical procedures, animals were treated with intramuscular antibiotics (900 mg procaine penicillin, Ilium Propen, Troy Laboratories, Smithfield, NSW, or Mavlab, QLD, Australia, at surgery and at 24 and 48 hr postsurgery). Postsurgical analgesia was maintained with intramuscular injection of flunixin (1 mg/kg; Troy Laboratories or Mavlab) administered at the start of surgery and then 8 and 24 hours postsurgery. Before experiments, animals were allowed at least 5 days of recovery following the second surgical procedure in order to minimize any effects of surgical stress. Analog signals (MAP, HR, and RBF) were recorded continuously at 100 Hz for 10 seconds every minute during experiments using a data acquisition system (Labview, National Instruments, Austin, TX). Renal vascular conductance (RVC) was calculated as RBF/MAP. RSNA was recorded differentially between the pair of electrodes with the best signal-to-noise ratio. The signal was amplified (×20,000) and filtered (band pass, 300–1,000 Hz), displayed on an oscilloscope, and passed through an audio amplifier and a loud speaker. In addition to recordings using Labview, RSNA (5,000 Hz) and MAP, HR, and RBF (100 Hz) were recorded on computer using a CED 1401 interface and Spike 2 Software (Cambridge Electronic Design, Cambridge, United Kingdom). RSNA was analyzed on a beatto-beat basis using custom-written routines in Spike 2 software, as previously reported (23). This program determines diastolic pressure, systolic pressure, MAP, heart period, and the number of discriminated spikes of RSNA above threshold between successive diastoles, which is a measure of burst size. The background noise in the RSNA signal was determined as the spikes/s during the highest dose of phenylephrine during the baseline period when SNA was abolished. The threshold was set just above the background noise level so that spikes from even small bursts were included in the analysis. Experimental Protocol Following a 24-hour baseline period, sepsis was induced in conscious sheep with an IV dose of live E. coli (2.8 × 109 July 2015 • Volume 43 • Number 7
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colony-forming units [CFUs] over 30 min) as a bolus, followed by a continuous infusion (1.26 × 109 CFU/hr up to 32 hr of sepsis). A continuous IV infusion of 0.9% w/v NaCl (1 mL/kg/hr) was administered to sheep from the start of the baseline period until the end of the intervention period (56 hr). Twentyfour hours after commencing E. coli infusion, when HR had increased by ~80% and MAP had decreased by ~10 mm Hg compared with baseline levels, the animals received a continuous IV infusion of either clonidine (1 μg/kg/hr; Boehringer Ingelheim, North Ryde, NSW, Australia) or vehicle (1 mL/kg/ hr 0.9% w/v NaCl) for 8 hours. Pressor responses of arterial pressure to phenylephrine and Ang II were examined at baseline, at 24 hours of sepsis, and at 28 and 32 hours of sepsis, which corresponded to 4 and 8 hours of treatment with clonidine or vehicle. Increasing bolus doses of phenylephrine (0.2, 0.4 0.8, 1, 1.5 μg/kg; Sigma Aldrich, Castle Hill, NSW, Australia) followed by Ang II (1, 2, 5, 8, 10 ng/kg; Auspep, Parkville, VIC, Australia) were administered IV (in 3 mL of 0.9% w/v NaCl) to evoke changes in arterial pressure. The highest dose of vasopressor was chosen to obtain an increase in MAP of approximately 20–25 mm Hg. Following each dose, adequate time (5 min for low doses and 10 min for the higher doses) was allowed for MAP to return to its respective control levels before the next dose was administered. In preseptic animals, MAP at baseline and 10 minutes after the highest dose of phenylephrine was similar in the clonidine (84.6 ± 2.5 vs 84.8 ± 2.5 mm Hg, respectively; p = 0.93) and vehicle (85.4 ± 2.4 vs 85.2 ± 2.4 mm Hg, respectively; p = 0.92) groups. Similarly, at the 32-hour sepsis time point, MAP at baseline and 10 minutes after the highest dose of phenylephrine was similar in the clonidine (74.9 ± 5.2 vs 75.1 ± 5.1 mm Hg; p = 0.95) and vehicle (66.8 ± 2.8 vs 66.4 ± 0.91 mm Hg, respectively; p = 0.92) groups.
Arterial blood samples were collected at baseline, just before the infusion of E. coli commenced, and then 4, 8, 24, 28, and 32 hours later for measurement of blood gases, electrolytes, and lactate (ABL System 625; Radiometer Medical, Copenhagen, Denmark). At the end of the protocol, the infusions of E. coli and clonidine/saline were terminated and the animals were treated with intramuscular gentamycin (150 mg, Troy Laboratories, Glendenning, NSW, Australia). Statistical Analysis All variables are reported as mean ± sem or as geometric mean (95% CI) as appropriate. MAP, HR, RBF, RVC, RSNA, and biochemical markers are reported as the average over the 24th hour of the baseline period and as hourly averages from the 24 to 32 hours after commencing the infusion of E. coli. Data were analyzed using repeated-measures analysis of variance (ANOVA) with factors treatment (ptreatment: saline or clonidine), time (ptime), and their interaction (pinteraction). Specific post hoc comparisons were made using Student t test. The Bonferroni correction was applied to protect against the risk of type 1 error. The absolute changes in MAP in response to each dose of vasopressor after induction of sepsis were compared with responses at baseline, prior to induction of sepsis, and following 4 and 8 hours of subsequent treatment with clonidine or saline, using repeated-measures ANOVA. Factors in the analysis were time (ptime), dose (pdose), and their interaction (pinteraction). p Values from within-subjects factors were conservatively adjusted using the Greenhouse-Geisser method (28). Statistical analysis was performed using GraphPAD PRISM 6.0 (Graphpad Software, La Jolla, CA) for Windows. All variables were assessed for normality and log-transformed where appropriate. Two-sided p value less than or equal to 0.05 was considered statistically significant.
Table 1. Cardiovascular, Respiratory, and Biochemical Variables During the Baseline Period and at 24 Hours of Sepsis in the Groups of Sheep to be Treated With Vehicle or Clonidine Vehicle Group Variables
Baseline
Clonidine Group
Sepsis 24 Hr
Baseline
Sepsis 24 Hr
Mean arterial pressure (mm Hg)
83 ± 3
72 ± 3a
85 ± 2
72 ± 3a
Heart rate (beats/min)
74 ± 4
162 ± 7b
79 ± 2
153 ± 8b
Renal blood flow (mL/min)
200 ± 10
329 ± 8b
188 ± 20
292 ± 25a
Renal vascular conductance (mL/min/mm Hg)
2.4 ± 0.1
4.4 ± 0.4b
2.3 ± 0.3
4.1 ± 0.4a
100 (99–101)
166 (128–205)a
100 (99–102)
175 (150–201)b
97 ± 1.4
83 ± 3a
95 ± 0.8
79 ± 4a
0.28 ± 0.05
1.23 ± 0.21a
0.31 ± 0.05
1.11 ± 0.14b
Renal sympathetic nerve activity (% baseline) Arterial Po2 (mm Hg) Blood lactate (mmol/L)
< 0.01 indicates significant differences between variables at baseline compared with variables at 24 hr of sepsis in the groups of sheep to be treated with p vehicle or clonidine from a two-tailed unpaired Student t test. b p < 0.001 indicates significant differences between variables at baseline compared with variables at 24 hr of sepsis in the groups of sheep to be treated with vehicle or clonidine from a two-tailed unpaired Student t test. Values are mean ± sem for normally distributed variables and geometric mean (95% CI) for those not normally distributed. a
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RESULTS The body weight of sheep treated with clonidine (32 ± 2 kg; n = 6) was similar to that of those treated with saline (31 ± 1 kg; n = 6). There was no significant time effect on cardiovascular and renal variables during the 24-hour baseline period (ptime always > 0.05), and the basal levels were similar in the two groups of sheep (Table 1). Two animals from each treatment group died during the period 10–24 hours after the infusion of E. coli commenced and were excluded from analysis. Development of Sepsis Over 24 Hours By 24 hours of sepsis, there were similar changes in both groups that were consistent with our previous findings in this ovine model of hypotensive, hyperdynamic sepsis (22, 23). In both groups, there were similar reductions in MAP, accompanied by similar increases in HR, RBF, RVC, and RSNA (Table 1 and Figs. 1–3). The decrease in arterial Po2 and increase in arterial lactate were also similar in each group. Effects of Clonidine and Saline Infusion During Sepsis During treatment with vehicle from 24 to 32 hours of sepsis, MAP progressively declined from 73 ± 3 to 66 ± 3 mm Hg (–8% ± 2%) (Fig. 1A). The fall in MAP was accompanied by increases in RVC (4.6 ± 0.4 to 5.6 ± 0.7 mL/min/mm Hg; +21% ± 8%)
(Fig. 1D) and RBF (329 ± 25 to 371 ± 46 mL/min; +11% ± 5%) (Fig. 1B). Both HR (160 ± 14 to 150 ± 11 beats/min) (Fig. 1C) and RSNA (166% [128%; 205%] to 175% [136%; 210%] of baseline) (Fig. 2A) remained elevated. By contrast, in sheep treated with clonidine, there was no decrease in MAP (72 ± 3 to 75 ± 5 mm Hg) (Fig. 1A) and no increase in RVC (4.1 ± 0.4 to 3.7 ± 0.7 mL/min/mm Hg) (Fig. 1D), so that the increase in RBF seen in the vehicle group did not occur (292 ± 25 to 273 ± 38 mL/min) (Fig. 1B) during the 24- to 32-hour period of sepsis. The profile of changes in MAP, RBF, and RVC differed significantly between clonidine and vehicle-treated sheep (all p < 0.01) (Fig. 1, A, B, and D). In sheep treated with clonidine, HR decreased (from 153 ± 8 to 116 ± 10 beats/min; –23% ± 6%) (Fig. 1C) and RSNA decreased to baseline levels (from 166% [150%; 201%] to 103% [85%; 212%] of baseline) (Fig. 2A). As sepsis developed, there were similar progressive decreases in arterial Po2 in both the vehicle (–22 ± 2 mm Hg) and clonidine (–24 ± 5 mm Hg) groups (Fig. 3A). Arterial Pco2 fell by 3.5 ± 1 mm Hg in both groups, over the first 8 hours after commencing the infusion of E. coli, but increased thereafter. The rate of increase of arterial Pco2 was greater in clonidine than vehicletreated sheep (Fig. 3B). Blood lactate increased during the first 24 hours after commencing the infusion of E. coli and thereafter remained stable; the pattern of change was similar in the two groups of sheep (Fig. 3C). There was a progressive reduction in plasma K+ concentration during sepsis, with the degree of hypokalemia being augmented by clonidine treatment (Fig. 3D).
Figure 1. Mean arterial pressure (A), renal blood flow (B), heart rate (C), and renal vascular conductance (D) during infusion of Escherichia coli from 0 to 32 hr and subsequent treatment with clonidine (n = 5; closed circles) or saline (n = 5; open circles) from 24 to 32 hr in conscious sheep. Time 0 is the mean of the 24 hr baseline period, data are mean ± sem, and p values represent treatment-time interactions from two-way repeated-measures analysis of variance.
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Effects of Clonidine on Arterial Pressure Responses to Vasopressors During Sepsis Administration of phenylephrine and Ang II evoked dosedependent increases in MAP (Figs. 4 and 5). By 24 hours of sepsis, the pressor responses to phenylephrine (Fig. 4) and Ang II (Fig. 5) were both significantly less than during the baseline (preseptic) period. For example, the highest dose of phenylephrine (1.5 μg/kg) increased MAP by 21 ± 2 mm Hg at baseline but only by 14 ± 2 mm Hg at 24 hours after commencing the infusion of E. coli (Fig. 4, A and B). Similarly, the highest dose of Ang II (10 ng/kg) increased MAP by 25 ± 2 mm Hg at baseline but only by 14 ± 2 mm Hg at 24 hours of sepsis (Fig. 5, A and B). July 2015 • Volume 43 • Number 7
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Figure 2. Renal sympathetic nerve activity (RSNA) during infusion of Escherichia coli from 0 to 32 hr and subsequent treatment with clonidine (n = 5; closed circles) or saline (n = 5; open circles) from 24 to 32 hr in conscious sheep (A). Time 0 is the mean of the 24 hr baseline period, data are mean ± sem, and p values represent treatment-time interactions from two-way repeated-measures analysis of variance. Raw traces (10 s) from a conscious sheep of arterial pressure (AP) and RSNA during the baseline period (B), 24 hr after commencing the infusion of E. coli (C), and after 8 hr of clonidine infusion (32 hr of sepsis) (D).
In vehicle-treated sheep, the pressor responses to the highest dose of phenylephrine and Ang II, respectively, remained diminished at 28 hours (14 ± 2 and 15 ± 2 mm Hg) (Figs. 4C and 5C) and 32 hours (13 ± 2 and 16 ± 2 mm Hg) (Figs. 4E and 5E) of sepsis compared with the responses before sepsis. By contrast, in clonidine-treated sheep, the pressor responses to phenylephrine and Ang II at 28 hours (19 ± 1 and 21 ± 4 mm Hg) (Figs. 4D and 5D) and 32 hours (21 ± 4 and 21 ± 3 mm Hg) (Figs. 4F and 5F) of sepsis were indistinguishable from those observed at baseline (21 ± 2 and 25 ± 2 mm Hg).
DISCUSSION The main findings are that a clinically relevant dose of clonidine given to conscious sheep with established hypotensive sepsis, at a clinically appropriate time, prevented the continual decline in MAP, reduced the high levels of HR and RSNA toward baseline levels, and prevented further increases in RVC and RBF. We also found a large reduction in the pressor responsiveness to both phenylephrine and Ang II at 24 hours of sepsis. Interestingly, clonidine treatment restored the pressor responsiveness to not only phenylephrine but also Ang II. These findings suggest that sympathoinhibition induced by Critical Care Medicine
clonidine improves vascular sensitivity to both endogenous and exogenously infused vasoconstrictors, indicating that it may be a useful adjunctive treatment for patients with established sepsis who are resistant to vasopressor therapy. Effect of Clonidine on Vascular Hyporesponsiveness in Sepsis In critical care units, α2-adrenergic agonists are now often used as sedatives (29), and a recent case report indicated that treatment with clonidine resulted in an increased pressor response to norepinephrine in a patient with septic shock (25). The use of sympatholytics in a hypotensive state such as sepsis is, however, counterintuitive and raises concerns because their actions to reduce SNA would be expected to enhance the degree of hypotension and promote multiple organ failure. In the current study, treatment with clonidine in ovine hyperdynamic, hypotensive sepsis prevented the continuous decline in MAP seen in the vehicle-treated group. This occurred despite the decrease in RSNA and HR, which would be expected to lead to a further decrease in MAP. Indeed, we have shown that renal denervation results in an enhanced decrease in blood pressure in septic sheep (22). This indicates the important role elevated RSNA plays in maintaining MAP www.ccmjournal.org
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up-regulation of vascular α1adrenergic receptors that were down-regulated in sepsis (19). Interestingly, however, clonidine also restored the pressor sensitivity to Ang II, suggesting that its actions to improve vasopressor responsiveness may result from mechanisms that are either downstream or independent of α1-adrenergic receptors. There are other mechanisms by which clonidine may improve vascular reactivity during sepsis. First, proinflammatory cytokines, which are abundantly generated during sepsis, have been shown to mediate a down-regulation of both α1-adrenergic and Ang II type 1 receptor expression in mice and rats (9–11, 13). Furthermore, blockade of these Figure 3. Arterial partial pressure o2 (A), arterial partial pressure co2 (B), arterial blood lactate (C), and plasma proinflammatory cytokines K+ (D) during infusion of Escherichia coli from 0 to 32 hr and subsequent treatment with clonidine (n = 6; prevents and/or attenuates the closed circles) or saline (n = 6; open circles) from 24 to 32 hr in conscious sheep. Data (mean ± sem) are sepsis-induced down-regureported at time 0 (24th hr of the baseline period) and at 4, 8, 24, 28, and 32 hr after commencing the infusion of E. coli. p values represent treatment-time interactions from two-way repeated-measures analysis of variance. lation of these receptors (10, 13). It is well accepted that α2at least during the early phases of sepsis, most likely by causing adrenergic agonists lead to a significant reduction in inflamrenal vasoconstriction and renin release. Indeed, Ang II levmatory cytokines during sepsis in the experimental setting els are increased in sepsis (30) and blockade of angiotensin(16–18), after cardiopulmonary bypass surgery (32), and in the converting enzyme in anesthetized septic pigs exacerbated the critical care unit (33). Second, it is known that hyperpolarizaseverity of the hypotension (31). Our finding that in septic tion of vascular smooth muscle cells via excessive opening of sheep clonidine prevented a further decline in arterial pressure K+ATP channels plays a critical role in the development of vascular hyporesponsiveness during sepsis (14). The K+ATP chandespite a reduction in RSNA suggests that arterial pressure was maintained by increased peripheral vascular tone due to resto- nel is mainly composed of two subunits: the sulphonylurea receptor and a pore-forming subunit belonging to the Kir6.0 ration of vascular sensitivity to endogenous vasoconstrictors. family (34). There is evidence that the K+ATP channel sulphonylurea receptor may become dysfunctional or uncoupled from Mechanisms by Which Clonidine May Improve the pore-forming subunit during sepsis (35). This may explain Vascular Sensitivity to Vasopressors the ineffectiveness of the non-selective sulfonylurea drug glibThe present findings indicate that septic sheep developed a enclamide in clinical trials on patients with sepsis (36, 37). large reduction in pressor responsiveness to both phenylephBy contrast, selective inhibition of the vascular K+ATP channel rine and Ang II, which would result in a lack of responsiveness pore-forming Kir6.0 subunit leads to effective restoration of the to endogenous norepinephrine and Ang II and enhance the sepsis-induced hypotension. Treatment with clonidine for 4–8 pressor responses in isolated vessels incubated with lipopolyhours completely restored the pressor responsiveness to phen- sacchride (34, 35). Importantly, clonidine at clinically relevant concentrations has been shown to directly bind and inhibit ylephrine, which probably accounted for the maintained blood vascular K+ATP channels, primarily through its effects on the pressure due to increased vascular responsiveness to endogpore-forming subunit Kir6.0, independent of any interactions enous norepinephrine. The 4- to 8-hour time interval in our with adrenergic receptors (38). Thus, the relative hypokalemia study was similar to the interval reported between the start of clonidine administration and the observed increase in blood observed in septic sheep following clonidine treatment may result from inhibition of K+ATP channels, which may also play pressure response in a patient with established septic shock (25). It has been proposed that the ability of clonidine to restore a role in restoring vascular sensitivity to both endogenous and exogenously infused vasoconstrictors. Finally, it is feasible that pressor sensitivity to catecholamines is due to the reduction in sympathetic outflow and norepinephrine release leading to clonidine acting directly on extrasynaptic α2b- and postsynaptic e226
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Figure 4. Effects of treatment with saline-vehicle or clonidine on the changes in mean arterial pressure induced by bolus doses of phenylephrine in conscious sheep. Responses in preseptic sheep in the vehicle group (open boxes, A, C, and E) and clonidine group (open circles, B, D, and F) and at 24 hr of sepsis in the vehicle group (closed boxes, A) and clonidine group (closed circles, B). Pressor responsiveness after 4 hr infusion of saline (closed boxes, C) or clonidine (closed circles, D) and after 8 hr of saline (closed boxes, E) or clonidine (closed circles, F). Dose-response curves were compared using two-way repeated-measures analysis of variance, and p values represent time-dose interactions. Data are mean ± sem (n = 6/group).
Figure 5. Effects of treatment with saline-vehicle or clonidine on the changes in mean arterial pressure induced by bolus doses of angiotensin II in conscious sheep. Responses in preseptic sheep in the vehicle group (open boxes, A, C, and E) and clonidine group (open circles, B, D, and F) and at 24 hr of sepsis in the vehicle group (closed boxes, A) and clonidine group (closed circles, B). Pressor responsiveness after 4 hr infusion of saline (closed boxes, C) or clonidine (closed circles, D) and after 8 hr of saline (closed boxes, E) or clonidine (closed circles, F). Dose-response curves were compared using two-way repeated-measures analysis of variance, and p values represent time-dose interactions. Data are mean ± sem (n = 6/group).
α1-adrenergic receptors on the vascular smooth muscle could be partly responsible for the enhanced vasoconstriction observed in response to phenylephrine and Ang II (39, 40).
exercised when extrapolating our findings to the clinical situation. Nevertheless, since the hemodynamic profile reported in septic sheep is similar to that reported in human sepsis, this model has clinical relevance. Indeed, the progressive decline in arterial pressure along with increased blood lactate and peripheral vasodilatation we observed fulfills the consensus criteria for sepsis in humans (24). Importantly, all observations were made in conscious animals, thus ruling out the potentially confounding effects of anesthesia on SNA and arterial pressure responses to vasopressors. The ~33% mortality rate observed in our study is comparable to the ~30% mortality rate reported in humans with severe sepsis. Limitations of our current study include the fact that we did not separately assess the central and peripheral actions of clonidine and we did not investigate the effects
Strengths and Limitations To our knowledge, this is the first report of the effects of a clinically relevant dose of the α2-adrenergic agonist clonidine (1 μg/kg/hr) on resting arterial pressure, RSNA, as well as the responsiveness of arterial pressure to increasing doses of the selective α1-adrenergic agonist phenylephrine in a conscious large animal model of hypotensive, hyperdynamic sepsis. In addition, we demonstrate that clonidine restored pressor responsiveness to another vasopressor system (Ang II). The study was randomized and vehicle-controlled, but as with any experimental animal studies, caution must be Critical Care Medicine
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of other α2-adrenergic agonists such as dexmedetomidine or an imidazoline receptor agonist. In contrast to patients commonly diagnosed with sepsis, the sheep studied in our experiment did not have comorbidities, such as old age, vascular disease, hypertension, or diabetes. We only assessed a single dose of clonidine, but we used a dose that is commonly used in critical care units. Finally, we did not assess whether clonidine also restores vascular responsiveness to vasopressin and terlipressin. In a large animal model of hypotensive hyperdynamic sepsis, an 8-hour infusion of a dose of clonidine commonly used in critical care units prevented the progressive decline in arterial pressure and improved pressor responsiveness to phenylephrine and Ang II. Clonidine reduced the elevated levels of RSNA, RBF, and RVC and had no observed deleterious effects. Considering these findings, a feasibility and safety clinical trial for the use of clonidine in the treatment of persistent vasopressor-refractory hypotension in patients with established septic shock would be worthwhile.
ACKNOWLEDGMENTS We thank Alan McDonald and Tony Dorman for their excellent technical assistance.
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