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Abstract
Introduction. The purpose of this study was to examine the effects of AM281, a cannabinoid receptor antagonist, on systemic haemodynamics, internal carotid artery blood flow and mortality during septic shock in rats.
Methods. The study included three sets of experiments: measurements of changes in systemic haemodynamics and left internal carotid artery flow (30 animals divided into three groups of 10); measurements of biochemical variables (n=30); assessment of mortality (n=30). Male Wistar rats (7 weeks old) were randomly divided into three groups: group 1, control; group 2, lipopolysaccharide (LPS) i.v., Escherichia coli endotoxin 10.0 mg kg−1 i.v., bolus; group 3, LPS 10.0 mg kg−1 i.v.+AM281 1 mg kg−1 i.v. Systemic haemodynamics, carotid artery flow changes and biochemical variables were assessed at pretreatment and 1, 2 and 3 h after the treatment was performed.
Results. Administration of AM281 could prevent the haemodynamic changes induced by sepsis. Tumour necrosis factor-α and interleukin 1-β increased in the LPS i.v. and LPS i.v.+AM281 groups at 1, 2 and 3 h after treatment; significant differences were observed in these levels in the two groups at these times. Internal carotid artery blood flow remained fairly constant in the control and LPS i.v.+AM281 groups compared with baseline values. In the LPS i.v. group, it decreased at 2 and 3 h after the treatment compared with baseline values [at 2 h: control 12.7 (SD 0.9) ml min−1, LPS i.v. 8.7 (1.4) ml min−1 (P<0.05), LPS i.v.+AM281 11.5 (0.9) ml min−1; at 3 h: control 12.7 (0.4) ml min−1, LPS i.v. 7.7 (1.3) ml min−1 (P<0.05), LPS i.v.+AM281 11.6 (1.0) ml min−1]. Significant differences in mortality within 6 and 12 h were found between the LPS i.v. and LPS i.v.+AM281 groups [6 h mortality: LPS i.v. 5/10 (50%), LPS i.v.+AM281 2/10 (20%), P<0.05; 12 h mortality: LPS i.v. group 10/10 (100%), LPS i.v.+AM281 5/10 (50%), P<0.05].
Conclusions. Administration of AM281 prevented changes in systemic haemodynamic and internal carotid artery blood flow and could improve mortality in experimentally induced septic shock in rats. These findings may have significant therapeutic implications in the treatment of septic shock.
The central nervous system (CNS) is one of the first organs to be affected by sepsis, but the mechanism by which this dysfunction occurs has not been resolved.1—6 Several proposed mechanisms that might account for the CNS dysfunction induced by sepsis include an alteration in the blood—brain barrier, amino acid disruption and brain ischaemia resulting from a global or regional reduction in the cerebral blood flow (CBF).2—5,7,8
It is still unclear whether CBF remains intact during septic shock.7—9 Booke and colleagues9 reported that CBF did not change in sheep with Pseudomonas aeruginosa sepsis. Pollard and colleagues8 reported that a dose of endotoxin sufficient to induce systemic vasodilation in healthy subjects does not influence CBF. In contrast, Bowton and colleagues7 reported that CBF is reduced in patients with sepsis syndrome.
Recently, anandamide, which is generated by endotoxin-induced platelets and macrophages, and has been identified as an endogenous cannabinoid ligand, has been found to be expressed in abundance in the brain.10—13 Sarker and colleagues12 reported that anandamide is a possible causative factor for the hypotension induced by endotoxic shock. Anandamide has a vasodilatory effect and induces a shock-like state via CB1 receptor activation.14 Wang and colleagues15 showed that removing endogenous cannabinoids by polymyxin-B-selective adsorption was effective in improving haemodynamics in patients with septic shock. Yamaji and colleagues16 showed that anandamide induces apoptosis and a marked increase in the activity of the IL-1β-converting enzyme CED-3 (caspase-3) which is a member of the protease family. Thus we hypothesized that administration of cannabinoid antagonists could be effective in minimizing changes in haemodynamics, and possibly CBF, during sepsis. However, there have been few reports regarding the effects of cannabinoid antagonists on haemodynamics and CBF changes during sepsis.10,11
The purpose of this study was to examine the effects of AM281, a cannabinoid receptor antagonist, on systemic haemodynamics, carotid artery blood flow and mortality in septic shock in rats.
Materials and methods
This study proceeded in accordance with the ethical principles provided by the Experimental Animal Laboratory of Gunma University School of Medicine.
Male Wistar rats (7 weeks old), weighing 250—350 g, were maintained in wire mesh cages with standard laboratory feed and water ad libitum under a 12 h light—dark cycle at 22°C.
Experimental protocol
The study was designed to include three sets of experiments: measurements of changes in systemic haemodynamics and left internal carotid artery flow (n=30); measurements of biochemical variables (n=30); assessment of mortality (n=30). A total of 90 animals were used in our experiments.
Haemodynamics and carotid artery flow
To evaluate changes in systemic haemodynamic flow and internal carotid artery flow rats were randomly divided into three groups: group 1, control (n=10); group 2, lipopolysaccharide (LPS) i.v. (n=10), Escherichia coli endotoxin 10.0 mg kg−1 i.v., bolus; group 3 (n=10), LPS 10.0 mg kg−1 i.v.+AM281 1 mg kg−1 i.v. The 10.0 mg kg−1 dosage of endotoxin is capable of causing 50% lethality within 6 h.3,17
The most common septic shock model is the single-bolus injection of endotoxin.2,3 This is because this endotoxin model is simple to prepare and is reproducible. In addition, it is a useful tool for examining the effects of therapeutic drugs on haemodynamic changes induced by sepsis.3 In this study, we chose the cannabinoid receptor antagonist (N-morpholin-4-yl)-5-(2,4-yl)-5-(2,4-dichlorophenyl)-4-methyl 1H-pyrazole-3-carboxamide (AM281). Gifford and colleagues18 reported that AM281 is a less lipophilic analogue of the cannabinoid receptor antagonist SR141716A and has slightly greater potency than SR141716A in hippocampal slices.
Rats were anaesthetized with pentobarbital 10 mg kg−1 intraperitoneally (i.p.). After tracheotomy, the animals were connected to a volume-cycled ventilator (model SN-480-7, Shinano Manufacturing Co., Japan) with 30% oxygen, 70% nitrous oxide and 1% isoflurane. The rectal temperature was monitored using a CMA/150® temperature controller, and maintained at 35.5—36.5°C using a blanket. Saline 2 ml was injected subcutaneously as a single bolus to maintain fluid balance. Haemodynamic variables were measured by inserting a 2 Fr high-fidelity micromanometer catheter into the right carotid artery and then advancing it into the left ventricle, where it was secured. The position was confirmed by a characteristic decrease in diastolic pressure that occurred when the catheter was passed across the aortic valve into the left ventricular cavity. To measure the mean arterial pressure simultaneously, the right femoral artery was also cannulated with a 2 Fr high-fidelity micromanometer catheter. Drugs were administered intravenously through a polyethylene catheter (PE50) placed in the right jugular vein. The free ends of the catheter were tunnelled subcutaneously into the dorsal aspect of the neck. The catheter in the carotid artery was connected to a haemodynamic monitoring system (Power Lab®, BioRes. Co., Nagoya, Japan). All three catheters were flushed with heparinized saline before use. We measured heart rate (HR), mean arterial pressure (MAP), left ventricular end-diastolic pressure (LVEDP) and maximum rate of change in left ventricular pressure (±dp/dtmax) at pretreatment and 1, 2 and 3 h after i.v. injection. We simultaneously measured changes in left internal carotid artery flow using ultrasonic flow probes (Transonic Systems Inc., New York, USA) at pretreatment and 1, 2 and 3 h after i.v. injection. This method allowed us to measure blood flow in the carotid artery. Three hours after E.coli administration, the rats were killed by injection of an overdose of pentobarbital via the right jugular vein.
Biochemical measurements
To exclude the influence of blood sampling on the flow of haemodynamic variables (30 animals divided into three groups of 10 each), we also measured biochemical parameters in another series of animals (n=10 per group). These animals were grouped and treated to represent the control (n=10), LPS i.v. (n=10) and LPS i.v.+AM281 (n=10) groups described above. To extrapolate data from these to the experimental set of animals that were evaluated for haemodynamic variables, the rats were exposed to identical experimental conditions for systemic measurements. We measured the plasma concentrations of lactate, glucose, epinephrine, norepinephrine, tumour necrosis factor-α (TNF-α), IL-1β and the partial pressure of arterial blood gases at pretreatment and 2 and 3 h after i.v. injection in blood (total 2.0 ml) collected from the carotid artery. Partial pressure of the arterial blood gases was analysed using a laboratory acid—base machine (ABL3, Radiometer, Copenhagen, Denmark). Plasma epinephrine and norepinephrine concentrations were measured by high-performance liquid chromatography. Plasma TNF-α activity was quantified by measuring cytotoxicity against L929 cells in rabbit serum. IL-1β was measured using commercial ELISA kits (IL-1, R&D Systems, Tokyo, Japan; IL-8, Biosource, Tokyo, Japan). After the haemodynamic and biochemical parameters had been measured, the rats were killed by pentobarbital overdose.
Mortality
To assess the mortality in the three groups, 30 animals divided into three groups of 10 were treated to represent control, LPS i.v. and LPS i.v.+AM281 animals as described above. Rats were anaesthetized with pentobarbital 10 mg kg−1 i.p., and injected with saline, E.coli endotoxin 10.0 mg kg−1 i.v. bolus or LPS 10.0 mg kg−1 i.v.+AM281 1 mg kg−1 i.v. via the dorsal vein. After treatment, animals were allowed to emerge from anaesthesia to assess mortality in the three groups.
Statistical analysis
All data are presented as arithmetic mean (SD). After confirmation of equal variance among groups using the Bartlett test, analysis of variance (ANOVA) multiple comparisons were performed. Means were compared using Scheffe's method. Comparisons of mortality between groups were made using the Kaplan—Meier and Mantel—Cox methods. Statistical significance was established at P<0.05. All statistical analyses were performed using the Software Stat ViewR 5.0 (Abacus Concepts, Berkeley, CA).
Results
Table 1 shows the time courses of haemodynamic change in the three groups studied in the first set of animals. There were no significant changes in HR, MAP, +dp/dtmax, −dp/dtmax or LVEDP in the control group throughout the study. In the LPS i.v. group, MAP, HR, +dp/dtmax, −dp/dtmax and LVEDP had changed 2 and 3 h after administration of LPS. Co-administration of AM281 appeared to prevent the changes in MAP, +dp/dtmax, −dp/dtmax, and LVEDP induced by LPS. However, HR increased despite concurrent administration of AM281.
Table 2 shows the time course of chemical variables in the three groups studied in the second set of animals. These variables remained fairly constant in the control group throughout the study. No significant changes in , or hematocrit were observed in the LPS i.v. and LPS i.v.+AM281 groups were found. Plasma glucose increased 3 h after treatment in both the LPS i.v. and the LPS i.v.+AM281 groups, with a significant difference in glucose between these two groups. Plasma lactate increased 2 and 3 h after treatment in both the LPS i.v. and LPS i.v.+AM281 groups, with a significant difference in lactate in the two groups 3 h after treatment. Plasma epinephrine and norepinephrine increased in the LPS i.v. and LPS i.v.+AM281 groups 2 and 3 h after treatment; significant differences in these levels were found between the two groups at these times. The values of TNF-α and IL-1β in the control group did not change throughout the study. However, TNF-α and IL-1β increased in the LPS i.v. and LPS i.v.+AM281 groups 1, 2 and 3 h after treatment, with significant differences being observed in these levels in the two groups at these times.
Table 3 shows the time course of changes in internal carotid artery blood flow, measured by ultrasonic flow probes, in the three groups. It remained fairly constant in the control and LPS i.v.+AM281 groups compared with baseline values. In the LPS i.v. group, flow decreased 2 and 3 h after treatment compared with baseline values [at 2 h: control 12.7 (0.9) ml min−1, LPS i.v. 8.7 (1.4) ml min−1 (P<0.05), LPS i.v.+AM281 11.5 (0.9) ml min−1; at 3 h: control 12.7 (0.4) ml min−1, LPS i.v. 7.7 (1.3) ml min−1 (P<0.05), LPS i.v.+AM281 11.6 (1.0) ml min−1].
Significant differences in mortality at 6 and 12 h were found between LPS i.v. and LPS i.v.+AM281 groups [6 h mortality: LPS i.v. 5/10 (50%), LPS i.v.+AM281 2/10 (20%), P<0.05; 12 h mortality: LPS i.v. group 10/10 (100%), LPS i.v.+AM281 5/10 (50%), P<0.05].
Discussion
We found that administration of AM281 prevented changes in systemic haemodynamics and internal carotid artery blood flow and improved the mortality in experimentally induced septic shock in rats.
There have been some reports indicating that cannabinoids such as anandamide or 2-arachidonyl glyceride (2-AG) play a pivotal role in the pathogenesis and development of sepsis.11,14,15 Wang and colleagues15 found an increase in anandamide and 2-AG in the sera of patients with endotoxic shock, and suggested that removal of endogenous cannabinoids by polymyxin-B-selective adsorption was effective in improving haemodynamics in patients with septic shock. Varga and colleagues11 reported that anandamide and 2-AG derived from macrophages and platelets in response to endotoxin could cause hypotension. This haemodynamic change can be prevented by pretreatment with the cannabinoid 1 (CB1) receptor antagonist SR141716A. These authors showed that anandamide is a mediator of endotoxin-induced hypotension via activation of vascular CB1 receptors. These reports imply that anandamide plays an important role in the pathogenesis and development of septic shock. Although we did not measure plasma anandamide or 2-AG concentrations, preventing the increase in plasma anandamide or 2-AG levels induced by endotoxin by administration of AM281 appeared to be beneficial therapy in our experimental animals, and therefore could be beneficial in septic shock states.
We found that internal carotid artery blood flow, measured by ultrasonic flowprobes, decreased 2 and 3 h after treatment, and that this decrease could be prevented by the administration of AM281. Since changes in internal carotid artery blood flow are thought to parallel changes in CBF, decreased internal carotid artery blood flow in this study is indicative of decreased CBF. However, it is still not known whether CBF remains intact during septic shock.7—9,19 Booke and colleagues9 reported that CBF, measured using coloured microspheres, did not change in sheep with P.aeruginosa sepsis. Pollard and colleagues8 reported that administration of endotoxin 4 mg kg−1 induced a decrease in MAP and systemic vascular resistance, while CBF and the cerebral metabolic rate for oxygen remained unchanged at 3 and 5 h in healthy subjects. However, this dosage (4 mg/kg i.v.) did not induce neurologic dysfunction in healthy subjects. In contrast, Ekstrom-Jodal and colleagues19 reported that CBF decreased after the administration of endotoxin in dogs. Bowton and colleagues7 reported that mean CBF in nine patients with neurological dysfunction was significantly lower than the normal age-matched value, and that this decrease did not correlate with changes in MAP. These discrepancies might in part be attributable to differences in the measurement techniques used for CBF and the septic shock models. In this study, we used ultrasonic flow probes in the carotid artery as an indicator of CBF, whereas Pollard and colleagues8 measured CBF using the Kety—Schmidt technique. Booke and colleagues9 induced sepsis by a continuous infusion of P.aeruginosa, whereas we induced septic shock by bolus injection of endotoxin. In addition, there may be some differential estimation between ultrasonic flow probe and microsphere methods, as noted by Booke and colleagues.9 Pollard and colleagues8 reported that a dose of endotoxin sufficient to induce systemic vasodilatation in healthy subjects does not influence CBF. Papadopoulos and colleagues1 noted that although CBF is reduced to ∼62% of normal in septic patients, this decrease does not appear to be a threat to neuronal viability or cause electroencephalographic changes. Although we believe that the amelioration of carotid artery blood flow in this study is not solely due to the recovery of haemodynamics, further study is needed to clarify the effect of improved systemic haemodynamics associated with the administration of AM281 on CBF changes.
In our study, administration of AM281 improved mortality associated with septic shock. Several mechanisms should be considered. Our study showed that administration of AM281 could prevent increases in plasma TNF-α and IL-1β, 2 and 3 h after the endotoxin infusion. Varga and colleagues11 reported that pretreatment of animals with the CB1 antagonist SR141716A not only prevented LPS-induced hypotension, but also improved survival. This observation is consistent with our results. In addition, Yamaji and colleagues16 reported that anandamide induces apoptosis in human endothelial cells. Attenuation of the exaggeration in pro-inflammatory cytokines and apoptotic cell death by administration of cannabinoid antagonists might be responsible for the improvement in mortality in septic shock in rats.
Study limitations
Our study showed that administration of AM281 decreased plasma cytokine levels. However, the effect of cannabinoid ligands on the cytokine system during sepsis remains controversial.20 Molina-Holgado and colleagues21 reported that anandamide inhibited the LPS-induced production of TNF-α by astrocyte cultures. Other cannabinoid ligands, such as HU-211 or THC, have been shown to have similar inhibitory effects to anandamide on TNF-α production. THC also has an inhibitory effect on the LPS-induced secretion of TNF-α in in vitro studies.22 In addition, HU-211 markedly suppressed in vitro TNF-α production by macrophage cells. These reports suggest that administration of AM281 should induce an increase in plasma cytokines, which is inconsistent with our findings. Klein and colleagues20 reviewed the effects of the cannabinoid system on the cytokine network, and noted that cannabinoids have a potentially wide-ranging role in immunomodulation. In contrast, Sarker and colleagues23 showed that anandamide induces cell death independent of cannabinoid receptors or vanilloid receptor 1. Thus further studies are needed to determine whether administration of AM281 could be effective in the reduction of plasma anandamide or 2-AG, the reduction of plasma cytokine levels and the prevention of apoptotic cell death during sepsis.
In this study, LPS and AM281 were injected simultaneously. Therefore it can be concluded that AM281 will protect against the initial insult of LPS. In a hypothetical clinical situation, treatment with AM281 would probably commence only after the establishment of sepsis. Hence further studies are needed to clarify the efficacy of cannabinoid antagonists in established cases of experimental sepsis.
It is well known that sepsis causes major damage to the lungs.1 We did not examine the effects of sepsis on the lung in this study, and hence we cannot comment on whether the attenuation of plasma cytokine levels led to an improvement in lung function and therefore an improvement in mortality.
In conclusion, administration of AM281 prevented the systemic haemodynamic and carotid artery blood flow changes, and could improve the mortality, in experimentally induced septic shock in rats.
Source, Graphs and Figures: Effects of AM281, a cannabinoid antagonist, on systemic haemodynamics, internal carotid artery blood flow and mortality in septic shock in rats
Introduction. The purpose of this study was to examine the effects of AM281, a cannabinoid receptor antagonist, on systemic haemodynamics, internal carotid artery blood flow and mortality during septic shock in rats.
Methods. The study included three sets of experiments: measurements of changes in systemic haemodynamics and left internal carotid artery flow (30 animals divided into three groups of 10); measurements of biochemical variables (n=30); assessment of mortality (n=30). Male Wistar rats (7 weeks old) were randomly divided into three groups: group 1, control; group 2, lipopolysaccharide (LPS) i.v., Escherichia coli endotoxin 10.0 mg kg−1 i.v., bolus; group 3, LPS 10.0 mg kg−1 i.v.+AM281 1 mg kg−1 i.v. Systemic haemodynamics, carotid artery flow changes and biochemical variables were assessed at pretreatment and 1, 2 and 3 h after the treatment was performed.
Results. Administration of AM281 could prevent the haemodynamic changes induced by sepsis. Tumour necrosis factor-α and interleukin 1-β increased in the LPS i.v. and LPS i.v.+AM281 groups at 1, 2 and 3 h after treatment; significant differences were observed in these levels in the two groups at these times. Internal carotid artery blood flow remained fairly constant in the control and LPS i.v.+AM281 groups compared with baseline values. In the LPS i.v. group, it decreased at 2 and 3 h after the treatment compared with baseline values [at 2 h: control 12.7 (SD 0.9) ml min−1, LPS i.v. 8.7 (1.4) ml min−1 (P<0.05), LPS i.v.+AM281 11.5 (0.9) ml min−1; at 3 h: control 12.7 (0.4) ml min−1, LPS i.v. 7.7 (1.3) ml min−1 (P<0.05), LPS i.v.+AM281 11.6 (1.0) ml min−1]. Significant differences in mortality within 6 and 12 h were found between the LPS i.v. and LPS i.v.+AM281 groups [6 h mortality: LPS i.v. 5/10 (50%), LPS i.v.+AM281 2/10 (20%), P<0.05; 12 h mortality: LPS i.v. group 10/10 (100%), LPS i.v.+AM281 5/10 (50%), P<0.05].
Conclusions. Administration of AM281 prevented changes in systemic haemodynamic and internal carotid artery blood flow and could improve mortality in experimentally induced septic shock in rats. These findings may have significant therapeutic implications in the treatment of septic shock.
The central nervous system (CNS) is one of the first organs to be affected by sepsis, but the mechanism by which this dysfunction occurs has not been resolved.1—6 Several proposed mechanisms that might account for the CNS dysfunction induced by sepsis include an alteration in the blood—brain barrier, amino acid disruption and brain ischaemia resulting from a global or regional reduction in the cerebral blood flow (CBF).2—5,7,8
It is still unclear whether CBF remains intact during septic shock.7—9 Booke and colleagues9 reported that CBF did not change in sheep with Pseudomonas aeruginosa sepsis. Pollard and colleagues8 reported that a dose of endotoxin sufficient to induce systemic vasodilation in healthy subjects does not influence CBF. In contrast, Bowton and colleagues7 reported that CBF is reduced in patients with sepsis syndrome.
Recently, anandamide, which is generated by endotoxin-induced platelets and macrophages, and has been identified as an endogenous cannabinoid ligand, has been found to be expressed in abundance in the brain.10—13 Sarker and colleagues12 reported that anandamide is a possible causative factor for the hypotension induced by endotoxic shock. Anandamide has a vasodilatory effect and induces a shock-like state via CB1 receptor activation.14 Wang and colleagues15 showed that removing endogenous cannabinoids by polymyxin-B-selective adsorption was effective in improving haemodynamics in patients with septic shock. Yamaji and colleagues16 showed that anandamide induces apoptosis and a marked increase in the activity of the IL-1β-converting enzyme CED-3 (caspase-3) which is a member of the protease family. Thus we hypothesized that administration of cannabinoid antagonists could be effective in minimizing changes in haemodynamics, and possibly CBF, during sepsis. However, there have been few reports regarding the effects of cannabinoid antagonists on haemodynamics and CBF changes during sepsis.10,11
The purpose of this study was to examine the effects of AM281, a cannabinoid receptor antagonist, on systemic haemodynamics, carotid artery blood flow and mortality in septic shock in rats.
Materials and methods
This study proceeded in accordance with the ethical principles provided by the Experimental Animal Laboratory of Gunma University School of Medicine.
Male Wistar rats (7 weeks old), weighing 250—350 g, were maintained in wire mesh cages with standard laboratory feed and water ad libitum under a 12 h light—dark cycle at 22°C.
Experimental protocol
The study was designed to include three sets of experiments: measurements of changes in systemic haemodynamics and left internal carotid artery flow (n=30); measurements of biochemical variables (n=30); assessment of mortality (n=30). A total of 90 animals were used in our experiments.
Haemodynamics and carotid artery flow
To evaluate changes in systemic haemodynamic flow and internal carotid artery flow rats were randomly divided into three groups: group 1, control (n=10); group 2, lipopolysaccharide (LPS) i.v. (n=10), Escherichia coli endotoxin 10.0 mg kg−1 i.v., bolus; group 3 (n=10), LPS 10.0 mg kg−1 i.v.+AM281 1 mg kg−1 i.v. The 10.0 mg kg−1 dosage of endotoxin is capable of causing 50% lethality within 6 h.3,17
The most common septic shock model is the single-bolus injection of endotoxin.2,3 This is because this endotoxin model is simple to prepare and is reproducible. In addition, it is a useful tool for examining the effects of therapeutic drugs on haemodynamic changes induced by sepsis.3 In this study, we chose the cannabinoid receptor antagonist (N-morpholin-4-yl)-5-(2,4-yl)-5-(2,4-dichlorophenyl)-4-methyl 1H-pyrazole-3-carboxamide (AM281). Gifford and colleagues18 reported that AM281 is a less lipophilic analogue of the cannabinoid receptor antagonist SR141716A and has slightly greater potency than SR141716A in hippocampal slices.
Rats were anaesthetized with pentobarbital 10 mg kg−1 intraperitoneally (i.p.). After tracheotomy, the animals were connected to a volume-cycled ventilator (model SN-480-7, Shinano Manufacturing Co., Japan) with 30% oxygen, 70% nitrous oxide and 1% isoflurane. The rectal temperature was monitored using a CMA/150® temperature controller, and maintained at 35.5—36.5°C using a blanket. Saline 2 ml was injected subcutaneously as a single bolus to maintain fluid balance. Haemodynamic variables were measured by inserting a 2 Fr high-fidelity micromanometer catheter into the right carotid artery and then advancing it into the left ventricle, where it was secured. The position was confirmed by a characteristic decrease in diastolic pressure that occurred when the catheter was passed across the aortic valve into the left ventricular cavity. To measure the mean arterial pressure simultaneously, the right femoral artery was also cannulated with a 2 Fr high-fidelity micromanometer catheter. Drugs were administered intravenously through a polyethylene catheter (PE50) placed in the right jugular vein. The free ends of the catheter were tunnelled subcutaneously into the dorsal aspect of the neck. The catheter in the carotid artery was connected to a haemodynamic monitoring system (Power Lab®, BioRes. Co., Nagoya, Japan). All three catheters were flushed with heparinized saline before use. We measured heart rate (HR), mean arterial pressure (MAP), left ventricular end-diastolic pressure (LVEDP) and maximum rate of change in left ventricular pressure (±dp/dtmax) at pretreatment and 1, 2 and 3 h after i.v. injection. We simultaneously measured changes in left internal carotid artery flow using ultrasonic flow probes (Transonic Systems Inc., New York, USA) at pretreatment and 1, 2 and 3 h after i.v. injection. This method allowed us to measure blood flow in the carotid artery. Three hours after E.coli administration, the rats were killed by injection of an overdose of pentobarbital via the right jugular vein.
Biochemical measurements
To exclude the influence of blood sampling on the flow of haemodynamic variables (30 animals divided into three groups of 10 each), we also measured biochemical parameters in another series of animals (n=10 per group). These animals were grouped and treated to represent the control (n=10), LPS i.v. (n=10) and LPS i.v.+AM281 (n=10) groups described above. To extrapolate data from these to the experimental set of animals that were evaluated for haemodynamic variables, the rats were exposed to identical experimental conditions for systemic measurements. We measured the plasma concentrations of lactate, glucose, epinephrine, norepinephrine, tumour necrosis factor-α (TNF-α), IL-1β and the partial pressure of arterial blood gases at pretreatment and 2 and 3 h after i.v. injection in blood (total 2.0 ml) collected from the carotid artery. Partial pressure of the arterial blood gases was analysed using a laboratory acid—base machine (ABL3, Radiometer, Copenhagen, Denmark). Plasma epinephrine and norepinephrine concentrations were measured by high-performance liquid chromatography. Plasma TNF-α activity was quantified by measuring cytotoxicity against L929 cells in rabbit serum. IL-1β was measured using commercial ELISA kits (IL-1, R&D Systems, Tokyo, Japan; IL-8, Biosource, Tokyo, Japan). After the haemodynamic and biochemical parameters had been measured, the rats were killed by pentobarbital overdose.
Mortality
To assess the mortality in the three groups, 30 animals divided into three groups of 10 were treated to represent control, LPS i.v. and LPS i.v.+AM281 animals as described above. Rats were anaesthetized with pentobarbital 10 mg kg−1 i.p., and injected with saline, E.coli endotoxin 10.0 mg kg−1 i.v. bolus or LPS 10.0 mg kg−1 i.v.+AM281 1 mg kg−1 i.v. via the dorsal vein. After treatment, animals were allowed to emerge from anaesthesia to assess mortality in the three groups.
Statistical analysis
All data are presented as arithmetic mean (SD). After confirmation of equal variance among groups using the Bartlett test, analysis of variance (ANOVA) multiple comparisons were performed. Means were compared using Scheffe's method. Comparisons of mortality between groups were made using the Kaplan—Meier and Mantel—Cox methods. Statistical significance was established at P<0.05. All statistical analyses were performed using the Software Stat ViewR 5.0 (Abacus Concepts, Berkeley, CA).
Results
Table 1 shows the time courses of haemodynamic change in the three groups studied in the first set of animals. There were no significant changes in HR, MAP, +dp/dtmax, −dp/dtmax or LVEDP in the control group throughout the study. In the LPS i.v. group, MAP, HR, +dp/dtmax, −dp/dtmax and LVEDP had changed 2 and 3 h after administration of LPS. Co-administration of AM281 appeared to prevent the changes in MAP, +dp/dtmax, −dp/dtmax, and LVEDP induced by LPS. However, HR increased despite concurrent administration of AM281.
Table 2 shows the time course of chemical variables in the three groups studied in the second set of animals. These variables remained fairly constant in the control group throughout the study. No significant changes in , or hematocrit were observed in the LPS i.v. and LPS i.v.+AM281 groups were found. Plasma glucose increased 3 h after treatment in both the LPS i.v. and the LPS i.v.+AM281 groups, with a significant difference in glucose between these two groups. Plasma lactate increased 2 and 3 h after treatment in both the LPS i.v. and LPS i.v.+AM281 groups, with a significant difference in lactate in the two groups 3 h after treatment. Plasma epinephrine and norepinephrine increased in the LPS i.v. and LPS i.v.+AM281 groups 2 and 3 h after treatment; significant differences in these levels were found between the two groups at these times. The values of TNF-α and IL-1β in the control group did not change throughout the study. However, TNF-α and IL-1β increased in the LPS i.v. and LPS i.v.+AM281 groups 1, 2 and 3 h after treatment, with significant differences being observed in these levels in the two groups at these times.
Table 3 shows the time course of changes in internal carotid artery blood flow, measured by ultrasonic flow probes, in the three groups. It remained fairly constant in the control and LPS i.v.+AM281 groups compared with baseline values. In the LPS i.v. group, flow decreased 2 and 3 h after treatment compared with baseline values [at 2 h: control 12.7 (0.9) ml min−1, LPS i.v. 8.7 (1.4) ml min−1 (P<0.05), LPS i.v.+AM281 11.5 (0.9) ml min−1; at 3 h: control 12.7 (0.4) ml min−1, LPS i.v. 7.7 (1.3) ml min−1 (P<0.05), LPS i.v.+AM281 11.6 (1.0) ml min−1].
Significant differences in mortality at 6 and 12 h were found between LPS i.v. and LPS i.v.+AM281 groups [6 h mortality: LPS i.v. 5/10 (50%), LPS i.v.+AM281 2/10 (20%), P<0.05; 12 h mortality: LPS i.v. group 10/10 (100%), LPS i.v.+AM281 5/10 (50%), P<0.05].
Discussion
We found that administration of AM281 prevented changes in systemic haemodynamics and internal carotid artery blood flow and improved the mortality in experimentally induced septic shock in rats.
There have been some reports indicating that cannabinoids such as anandamide or 2-arachidonyl glyceride (2-AG) play a pivotal role in the pathogenesis and development of sepsis.11,14,15 Wang and colleagues15 found an increase in anandamide and 2-AG in the sera of patients with endotoxic shock, and suggested that removal of endogenous cannabinoids by polymyxin-B-selective adsorption was effective in improving haemodynamics in patients with septic shock. Varga and colleagues11 reported that anandamide and 2-AG derived from macrophages and platelets in response to endotoxin could cause hypotension. This haemodynamic change can be prevented by pretreatment with the cannabinoid 1 (CB1) receptor antagonist SR141716A. These authors showed that anandamide is a mediator of endotoxin-induced hypotension via activation of vascular CB1 receptors. These reports imply that anandamide plays an important role in the pathogenesis and development of septic shock. Although we did not measure plasma anandamide or 2-AG concentrations, preventing the increase in plasma anandamide or 2-AG levels induced by endotoxin by administration of AM281 appeared to be beneficial therapy in our experimental animals, and therefore could be beneficial in septic shock states.
We found that internal carotid artery blood flow, measured by ultrasonic flowprobes, decreased 2 and 3 h after treatment, and that this decrease could be prevented by the administration of AM281. Since changes in internal carotid artery blood flow are thought to parallel changes in CBF, decreased internal carotid artery blood flow in this study is indicative of decreased CBF. However, it is still not known whether CBF remains intact during septic shock.7—9,19 Booke and colleagues9 reported that CBF, measured using coloured microspheres, did not change in sheep with P.aeruginosa sepsis. Pollard and colleagues8 reported that administration of endotoxin 4 mg kg−1 induced a decrease in MAP and systemic vascular resistance, while CBF and the cerebral metabolic rate for oxygen remained unchanged at 3 and 5 h in healthy subjects. However, this dosage (4 mg/kg i.v.) did not induce neurologic dysfunction in healthy subjects. In contrast, Ekstrom-Jodal and colleagues19 reported that CBF decreased after the administration of endotoxin in dogs. Bowton and colleagues7 reported that mean CBF in nine patients with neurological dysfunction was significantly lower than the normal age-matched value, and that this decrease did not correlate with changes in MAP. These discrepancies might in part be attributable to differences in the measurement techniques used for CBF and the septic shock models. In this study, we used ultrasonic flow probes in the carotid artery as an indicator of CBF, whereas Pollard and colleagues8 measured CBF using the Kety—Schmidt technique. Booke and colleagues9 induced sepsis by a continuous infusion of P.aeruginosa, whereas we induced septic shock by bolus injection of endotoxin. In addition, there may be some differential estimation between ultrasonic flow probe and microsphere methods, as noted by Booke and colleagues.9 Pollard and colleagues8 reported that a dose of endotoxin sufficient to induce systemic vasodilatation in healthy subjects does not influence CBF. Papadopoulos and colleagues1 noted that although CBF is reduced to ∼62% of normal in septic patients, this decrease does not appear to be a threat to neuronal viability or cause electroencephalographic changes. Although we believe that the amelioration of carotid artery blood flow in this study is not solely due to the recovery of haemodynamics, further study is needed to clarify the effect of improved systemic haemodynamics associated with the administration of AM281 on CBF changes.
In our study, administration of AM281 improved mortality associated with septic shock. Several mechanisms should be considered. Our study showed that administration of AM281 could prevent increases in plasma TNF-α and IL-1β, 2 and 3 h after the endotoxin infusion. Varga and colleagues11 reported that pretreatment of animals with the CB1 antagonist SR141716A not only prevented LPS-induced hypotension, but also improved survival. This observation is consistent with our results. In addition, Yamaji and colleagues16 reported that anandamide induces apoptosis in human endothelial cells. Attenuation of the exaggeration in pro-inflammatory cytokines and apoptotic cell death by administration of cannabinoid antagonists might be responsible for the improvement in mortality in septic shock in rats.
Study limitations
Our study showed that administration of AM281 decreased plasma cytokine levels. However, the effect of cannabinoid ligands on the cytokine system during sepsis remains controversial.20 Molina-Holgado and colleagues21 reported that anandamide inhibited the LPS-induced production of TNF-α by astrocyte cultures. Other cannabinoid ligands, such as HU-211 or THC, have been shown to have similar inhibitory effects to anandamide on TNF-α production. THC also has an inhibitory effect on the LPS-induced secretion of TNF-α in in vitro studies.22 In addition, HU-211 markedly suppressed in vitro TNF-α production by macrophage cells. These reports suggest that administration of AM281 should induce an increase in plasma cytokines, which is inconsistent with our findings. Klein and colleagues20 reviewed the effects of the cannabinoid system on the cytokine network, and noted that cannabinoids have a potentially wide-ranging role in immunomodulation. In contrast, Sarker and colleagues23 showed that anandamide induces cell death independent of cannabinoid receptors or vanilloid receptor 1. Thus further studies are needed to determine whether administration of AM281 could be effective in the reduction of plasma anandamide or 2-AG, the reduction of plasma cytokine levels and the prevention of apoptotic cell death during sepsis.
In this study, LPS and AM281 were injected simultaneously. Therefore it can be concluded that AM281 will protect against the initial insult of LPS. In a hypothetical clinical situation, treatment with AM281 would probably commence only after the establishment of sepsis. Hence further studies are needed to clarify the efficacy of cannabinoid antagonists in established cases of experimental sepsis.
It is well known that sepsis causes major damage to the lungs.1 We did not examine the effects of sepsis on the lung in this study, and hence we cannot comment on whether the attenuation of plasma cytokine levels led to an improvement in lung function and therefore an improvement in mortality.
In conclusion, administration of AM281 prevented the systemic haemodynamic and carotid artery blood flow changes, and could improve the mortality, in experimentally induced septic shock in rats.
Source, Graphs and Figures: Effects of AM281, a cannabinoid antagonist, on systemic haemodynamics, internal carotid artery blood flow and mortality in septic shock in rats
