Sepsis is a major and expensive medical problem throughout the world. It describes a complex clinical syndrome that results from a harmful host response to infection. Endothelial dysfunction, which results in the extravasation of fluids and proteins, and progressive cardiovascular failure that is caused by excessive vasodilatation and vascular hyporesponsiveness to catecholamines are substantial clinical problems in the treatment of sepsis (1). Clinical and experimental studies have reported that high levels of arginine vasopressin (AVP) can be found in plasma during the initial phase of sepsis, and this may help restore blood pressure which tends to decrease because of cytokine and nitric oxide (NO) release. However, low plasma AVP levels have been reported during the late phase of sepsis (i.e., when the observed hypotension would normally stimulate AVP secretion) (1,2). These inappropriately low levels of AVP in the circulation may result from an increase in clearance, the impaired of baroreflex-mediated release, or depleted stores of AVP in the neurohypophysis (2,3) and may contribute to the progression of sepsis, septic shock, multiple organ failure, and death (2,4). This evidence supports the recommendation that AVP should be infused together with norepinephrine for hemodynamic support during sepsis (5).
A multicenter, randomized, double-blind trial that was published in 2008 showed that the 28- and 90-day mortality rates between groups of patients who received AVP or norepinephrine concomitantly with other vasopressors (6) were similar. Conversely, in patients with less severe septic shock, the survival rate was higher in the AVP group than in the norepinephrine group (6). Two more recent meta-analyses of the use of AVP and its synthetic analogue terlipressin in septic shock reported that these treatments did not provide any survival benefit, but they showed that these compounds are safe and may preclude the need for catecholamines (7,8). Therefore, still controversial is whether AVP infusion during sepsis indeed improves the survival rate or is only an alternative treatment in cases of catecholamine hyporesponsiveness.
AVP is synthesized in vasopressinergic magnocellular cells (MNCs) of the supraoptic and paraventricular nucleus of the hypothalamus and stored in the posterior pituitary until it is released into the bloodstream (9). Arginine vasopressin secretion depends on the activity of MNCs. This depends on the efficacy of excitatory synapses, and the modulation of glutamatergic neurotransmission is an important mechanism for adjusting neuroendocrine output (10). In the hypothalamus, numerous neuropeptides modulate the glutamatergic synapses.
Endothelins (ETs) constitute a family of acidic 21-amido-acid peptides that are found in at least three distinct isoforms: ET-1, ET-2, and ET-3. These isoforms act as potent vasoconstrictors and neuromodulators. Endothelin exerts numerous biological effects by acting through two main G-protein coupled receptors: ETA and ETB(11). Many investigators have reported that circulating ET levels are significantly increased in septic humans and animals, and these levels are correlated with mortality (12).
Endocannabinoids (eCBs) such as anandamide and 2-arachidonoyl-glycerol are lipidic messengers derived from arachidonic acid present in cell membranes. Once released they can act on two types of G-protein receptors, CB1 and CB2(13). eCBs can be released as retrograde messengers by many neurons, including hypothalamic MNCs, suggesting that eCBs play a role in the neural network of these cells, including AVP cells (14).
Previous studies from our group showed that ET-1 reduced the frequency but not amplitude of spontaneous postsynaptic excitatory currents in vasopressinergic MNCs in rats by activating ETA receptors, suggesting a presynaptic effect. The CB1 receptor antagonist AM251 abolished this effect, suggesting the involvement of eCBs that act retrogradely (14). However, no in vivo evidence has been reported that shows that ET-1 can modulate the release of AVP in the central nervous system through the release of eCBs, particularly under pathological conditions. Also unknown are the consequences of blocking this pathway under such conditions.
Therefore, the present study investigated whether the blockade of ETA or CB1 receptors during severe sepsis, during the phase of reported augmented ET-1 levels, increases the survival rate of rats concomitantly with an increase in plasma AVP levels. A better understanding of these mechanisms may be useful for elucidating the molecular basis of sepsis, and may contribute to the development of new therapeutic strategies.
MATERIALS AND METHODS
The experiments were conducted in male Wistar rats (180–200 g) that were obtained from the animal facility of Federal University of Paraná. The animals were housed five per cage in a temperature-controlled room at 22 ± 1°C under a 12 h/12 h light/dark cycle (lights on at 7:00 AM) with food and water available ad libitum. All of the procedures were approved by the institution's Ethical Committee for Animal Use and were in accordance with Brazilian and EU Directive 2010/63/EU Guidelines for Animal Care. All efforts were made to minimize the number of animals used and their suffering.
Cecal ligation and puncture
Sepsis was induced by cecal ligation and puncture (CLP), as described previously (15). Briefly, the rats were anesthetized with ketamine (90 mg/kg, i.p.; Vetnil Veterinary Products, Louveira, Brazil) plus xylazine (10 mg/kg, i.p.; Syntec Laboratory, Cotia, Brazil), and a 2 cm midline incision was made on the anterior abdomen. The cecum was exposed and ligated below the ileocecal valve. The cecum was then punctured with a 16-gauge needle, and squeezed to allow its contents to be extracted through the punctures. The cecum was placed back in the abdominal cavity, and the incision was sutured. For the survival curve, one, three, and nine punctures were made, and three punctures were chosen for the subsequent experiments. Sham-operated animals were subjected to identical laparotomy without cecal puncture and were used as controls. All of the animals received 3 mL of saline subcutaneously immediately after surgery. The animals were allowed to recover in their cages with free access to food and water. All of the rats that were subjected to CLP developed early clinical signs of sepsis, including lethargy, piloerection, and tachypnea.
Intracerebral cannula implantation and microinjection
For intracerebroventricular (i.c.v.) administration of antagonists, a 22-gauge stainless-steel guide cannula (0.8-mm outer diameter, 12-mm length) was stereotaxically implanted into the right lateral ventricle under the same anesthesia described above. The stereotaxic coordinates were the following: 0.8 mm lateral to midline, 1.5 mm posterior to bregma, and 2.5 mm below the brain surface, with the incisor bar lowered 3.3 mm below the horizontal zero (16). Cannulas were fixed to the skull with jeweler's screws that were embedded in dental acrylic cement. The animals were allowed to recover for at least 5 days before CLP or sham surgery. After the experiment, each rat was microinjected with Evans blue (2.5% in saline) into the lateral ventricle. Brains were removed, and animals that showed cannula misplacement, cannula blockage, or abnormal body weight gain patterns after surgery were excluded from the study.
Core temperature measurement
Abdominal core temperature (Tc) was measured in conscious unrestrained rats using data loggers (Subcue, Calgary, Canada). Briefly, data loggers were implanted in the peritoneal cavity at least 5 days before CLP under the same anesthesia described above. On the day of CLP induction, Tc was continuously monitored at 30-min intervals at least 2 h before surgery until 12 h after.
Effect of CB1, ETA, and ETB receptor blockade on survival rate
Animals that were subjected to CLP and sham surgery were orally treated with the CB1 receptor antagonist rimonabant (Rim; 10 and 20 mg/kg, Cayman Chemical Company, Ann Arbor, MI), or the same volume of vehicle (carboxymethylcellulose [CMC], 10% w/v) 4 h after CLP. The ETA receptor antagonist BQ123 (100 pmol, 2 μL, i.c.v., Sigma Chemicals, São Paulo, Brazil) or vehicle (saline, 2 μL, i.c.v.) was administered using three different regimens: 2 and 4 h after CLP, 4 and 8 h after CLP, or only 8 h after CLP. The ETB receptor antagonist BQ788 (100 pmol, i.c.v., Sigma Chemicals, São Paulo, Brazil) or Rim (2 μg, i.c.v.) or vehicle (saline or 3.5% DMSO, respectively, 2 μL, i.c.v.) were administered using only one regimen: 4 and 8 h after CLP. Only intracerebroventricular route was done for the ETA and ETB receptor antagonists because of the peptidergic nature of these antagonists. In all the experiments, the survival rate was monitored for 7 days.
Effect of CB1 blockade on core temperature (Tc)
The experiment was conducted in rats that were housed in a room that was maintained at 28 ± 1°C (i.e., within the thermoneutral range for rats) (17). Each animal was allowed to adapt to this environment for 12 h before any measurements were taken. Tc was measured at 30-min intervals, 2 h before CLP until 24 h after CLP. Animals that were subjected to CLP were orally treated with 10% CMC or 10 mg/kg Rim 4 h after the induction of CLP. Sham-operated animals received the same volume of vehicle.
Cellular migration and bacterial count into the peritoneal cavity
Sham-operated and CLP animals were orally treated with 10% CMC (vehicle) or 10 mg/kg Rim 4 h after surgery. Cellular migration was assessed 8 h after CLP or sham surgery. Peritoneal fluid was collected by introducing 10 mL of phosphate-buffered saline (PBS) that contained 0.03% (w/v) bovine serum albumin and 5 U/mL heparin. Total leukocyte counts were performed under light microscopy, and the results are expressed as the number of cells per milliliter of peritoneal fluid. The samples were centrifuged at 1000 × g for 10 min at 4°C. Cells were suspended in 3% (w/v) bovine serum albumin and placed onto prepared glass slides. Cells were stained with May-Grunwald-Giemsa (Rosenfeld), and differential cell counts were performed under light microscopy. The results are expressed as the number of neutrophils per milliliter.
For bacterial counts in the peritoneal exudates, animals were anaesthetized with halothane 8 h after CLP or sham surgery. Ten milliliters of sterile and heparinized PBS was injected into the peritoneal cavity. After disinfection, the skin of the abdomen was opened along the midline without injury to the muscle and approximately 5 mL of lavage fluid was collected. Aliquots of serial dilutions of the peritoneal lavage fluid (1:10,000) were plated onto Mueller–Hinton agar dishes (Laborclin, Pinhais, PR, Brazil) and allowed to grow for 24 h at 37° C. After this period, colony-forming units (CFUs) were counted and the results expressed as log CFUs per milliliter (CFU/mL) of peritoneal fluid.
Blood leukocyte number and plasma interleukin-6 and arginine vasopressin determination
For blood leukocyte number and plasma interleukin-6 (IL-6) determination, naive, sham-operated, and CLP animals were orally treated with 10% CMC or 10 mg/kg Rim 4 h after surgery. The animals were anesthetized 2, 4, 6, or 8 h after surgery as described above. Blood samples were collected by cardiac puncture using heparinized syringes and transferred to chilled plastic tubes. A sample was collected for total leukocyte analysis. The blood samples were centrifuged at 1000 × g for 10 min at 4°C, and the plasma samples were stored at −80°C. IL-6 levels were measured in blood only 6 and 8 h after CLP using a specific enzyme-linked immunosorbent assay kit (Thermo Scientific, Rockford, IL).
For AVP determination, naive, sham-operated, and CLP animals were orally treated with 10% CMC or 10 mg/kg Rim 4 h after surgery or intracerebroventricularly treated with 100 pmol BQ123 (2 μL) 4 and 8 h after surgery. The animals were anesthetized 8 or 12 h after surgery as described above. Blood samples were collected and prepared as described above. The assay for AVP was performed using a specific enzyme-linked immunosorbent assay kit (Arginine Vasopressin EIA, Cayman Chemical Company, Ann Arbor, MI).
The survival rate is expressed as a percentage, and a log-rank (Mantel–Cox) test was used to determine differences in survival curves. The temperature data are reported as mean ± standard error of the mean (SEM) of Tc. The statistical analyses were performed using two-way repeated-measures analysis of variance followed by Bonferroni post hoc test. Bacterial and cell counts and IL-6 and AVP levels are expressed as mean ± SEM and were analyzed using one-way analysis of variance followed by Bonferroni post hoc test. Values of P <0.05 were considered statistically significant.
Survival curve and effect of systemic CB1 receptor blockade on survival rate after cecal ligation and puncture
Sham-operated animals had a survival rate of 100% in all the experiments. On the survival curve, the animals had survival rates of 84%, 26%, and 0% with one, three, and nine punctures, respectively, and most of the deaths occurred within the first 48 h (Fig. 1A). Three punctures were selected for the subsequent experiments. Oral treatment with the CB1 receptor antagonist Rim (10 and 20 mg/kg) 4 h after CLP increased the survival rate (Fig. 1B). The animals that were subjected to CLP and received Rim orally 4 h after CLP had a survival rate of 73% at both doses, which was significantly different from the CLP per vehicle group, which had a survival rate of 34%.
Effect of central ETA and ETB receptor blockade on survival rate after cecal ligation and puncture
The animals that were intracerebroventricularly treated with BQ123 2 and 4 or 8 h after CLP (data not shown) did not exhibit a significant improvement in survival rate (44% and 20% in the CLP/BQ123 group compared with 58% and 20% in the CLP per vehicle group, respectively). However, treatment with BQ123 4 and 8 h after CLP (Fig. 2A) significantly improved the survival rate (71% in the CLP/BQ123 group compared with 14% in the CLP per vehicle group). The ETB receptor antagonist BQ788 was administered using the same protocol (Fig. 2B) but did not significantly improve the survival rate (40% in the CLP/BQ788 group compared with 22% in the CLP per vehicle group).
Effect of CB1 receptor blockade on core temperature after cecal ligation and puncture
All of the groups exhibited a decrease in Tc with ketamine/xylazine-induced anesthesia, which lasted approximately 2 h (Fig. 3). Tc returned to normal levels after 2 h, and then all of the groups exhibited an increase in Tc after surgery that peaked at 4 h (sham group: 1.75 ± 0.4°C; CLP group: 1.74 ± 0.4°C). At this time point, CLP animals were treated with Rim or 10% CMC, and sham-operated animals were treated with vehicle. Six hours after CLP, sham-operated animals and CLP/Rim animals had significantly lower Tc 0.56 ± 0.2°C and 0.35 ± 0.4°C, respectively) than the CLP per vehicle group (1.64 ± 0.4°C; Fig. 3). The same pattern was observed until 8 h. After this time point, Tc was similar in all of the groups.
Effect of systemic CB1 receptor blockade on cellular migration, bacterial count, blood leukocytes, and IL-6 and arginine vasopressin levels after cecal ligation and puncture
To evaluate the possible peripheral effects of Rim that could contribute to the reduction of mortality after CLP, we assessed bacterial counts and cellular migration to the peritoneal cavity, blood leukocyte counts, and plasma IL-6 levels. Cecal ligation and puncture increased total leukocyte migration, specifically neutrophil migration to the peritoneal cavity, after 8 h (Fig. 4A and B, respectively). Treatment with Rim 4 h after CLP at the same dose that reduced mortality did not change CLP-induced total leukocytes and neutrophil migration to the peritoneal cavity (Fig. 4A and B, respectively). The number of bacteria was evaluated in peritoneal exudates at 8 h after CLP. CLP increased bacterial count in peritoneal exudates after 8 h compared with sham-operated animals, and the treatment with Rim did not attenuate CLP-induced increase in the number of bacteria in the peritoneal exudates (Fig. 4C). An increase in plasma IL-6 levels was observed in vehicle-treated CLP animals compared with sham-operated (Fig. 5A) and naive animals (0.15 ± 0.03 ng/mL) 6 and 8 h after CLP. No changes in blood leukocyte counts were observed 2 h after CLP (data not shown). In contrast, significant leukopenia was observed 4 h after surgery (data not shown). Leukopenia was also observed in animals that were subjected to CLP compared with naive (2.98 ± 0.57 × 106 cells/mL) and sham-operated animals 6 h after CLP but not 8 h after surgery (Fig. 5B). Treatment with Rim did not change leukopenia or the increase in plasma IL-6 levels 6 or 8 h after CLP (Fig. 5).
Effect of central CB1 receptor blockade on survival rate after cecal ligation and puncture
The animals that were intracerebroventricularly treated with Rim 4 and 8 h after CLP exhibited a significant improvement in survival rate (69% compared with 31% in the CLP per vehicle group) (Fig. 6).
Effect of CB1 receptor antagonist on arginine vasopressin secretion after cecal ligation and puncture
Arginine vasopressin levels remained constant in sham-operated animals 8 and 12 h after surgery (Fig. 7). After 8 h, both the vehicle- and Rim-treated CLP groups exhibited significantly lower AVP levels compared with the sham-operated group (Fig. 7). After 12 h, treatment with either Rim or BQ123 in the CLP group significantly increased plasma AVP levels compared with the CLP per vehicle group (Fig. 7).
The present study showed that the blockade of central CB1 receptors or ETA receptors prevented mortality induced by CLP in rats. The blockade of CB1 receptors 4 h after CLP did not affect peripheral responses, such as neutrophil migration to the peritoneal cavity, bacterial count in the peritoneal exudate, leukopenia, or IL-6 levels, but reduced the febrile response and changed plasma AVP levels, suggesting a central action of this antagonist.
The involvement of eCBs in hypotensive states was described previously by some studies that demonstrated that SR141716A, a selective CB1 receptor antagonist, increased blood pressure in rats that were subjected to hemorrhagic shock (18) and septic shock after a high dose of lipopolysaccharide (LPS) (19). In both studies this effect was attributable to the peripheral blockade of eCBs. Kadoi et al. also showed that the peripheral injection or infusion of another selective CB1 receptor antagonist AM281 concomitantly with or immediately after CLP or a high dose of LPS prevented blood pressure changes, reduced body temperature and circulating levels of IL-1β and TNF-α, and increased survival rate (20–22). Similarly, Caraceni et al. (23) showed that the treatment of animals with Rim in a model of ischaemia-reperfusion injury complicated by endotoxaemia improved hepatic damage, reduced neutrophil migration to the liver, and the levels of TNF-α, restored blood pressure and liver perfusion. Kianian et al. (24) showed that AM251 also reduced the leukocyte migration observed in the intestinal wall 2 h after the injection of endotoxin. Altogether, these results suggest that the blockage of CB1 receptors at the beginning of the sepsis reduces hemodynamics deterioration and the inflammatory condition that may contribute to the increased survival. In fact, the modulation of the endocanabinoid system seems to be effective in several diseases with an inflammatory component by inhibiting inflammation-related pathways and oxidative stress (for reviews see (13,25)).
The intracerebroventricular administration of Rim prevented the initial decrease (10 min to 3 h) in blood pressure induced by LPS in rats suggesting the involvement of central CB1 receptors (26). In the present study, we found that oral administration of Rim significantly increased the survival rate after CLP-induced sepsis. One difference between the present study and previous studies is that Rim was administered 4 h after the induction of sepsis, a protocol that is more closely related to clinical situations. The rationale for administering Rim at this time point was based on an attempt to relate eCB release and ETs. Circulating levels of ET-1 substantially increase in various states of shock (27) which can lead to the excessive vasoconstriction of peripheral vascular beds and may contribute to multiple organ dysfunction (28). It has been suggested that the release of endogenous ET-1 during endotoxemia can help alleviate severe hypotension, especially by inhibiting NO synthase (27). Plasma ET-1 levels are elevated between 8 and 12 h after the induction of sepsis in rats (29,30). Additionally, previous studies have shown that LPS injection increased ET-1 levels cerebrospinal fluid concomitantly with a decrease in its immediate precursor (31). These results suggest that LPS enhances ET-1 levels in the central nervous system by increasing the expression or activity of converting enzymes or preproendothelin-1 mRNA stabilization.
Rim was administered in this study at the moment when ET-1 levels were reported to be elevated in rats, and this treatment improved the survival rate. Based on this, one should expect that the blockade of central endothelinergic receptors at similar time points evidenced by other studies would have similar effects and confirm that the central levels of ETs are increased at this time point.
Administration of the ETA receptor antagonist BQ123 also increased the survival rate compared with the group that was subjected to CLP when the antagonist was administered 4 and 8 h after CLP, but it had no effect on survival when it was administered at earlier time points (2 and 4 h). These results suggest that central ET-1 levels are not high during the first hours after CLP and/or are not important for the survival rate at this time point. Conversely, central ET-1 levels increased at later times points after CLP may be a determining factor for the increase in death rate within the first 24 h, suggesting that the central levels of this peptide reached critical level at these time points. These results may corroborate a previous study by Iskit and Guc (27), who demonstrated that the use of bosentan, nonselective ET receptor antagonist, in a model of severe sepsis improved the survival rate in mice when it was administered during the late phase of sepsis. However, the present study showed that this effect is at least partially attributable to a central effect of the ET-1 receptor antagonist and involves ETA receptors.
Administration of the ETB receptor antagonist BQ788, unlike the ETA receptor antagonist, did not significantly increase the survival rate. Fabricio et al. (32) suggested that LPS-induced fever in rats is attenuated by both bosentan and BQ788 but not by BQ123. Therefore, the increase in the survival rate after ET receptor blockade does not appear to be related to blockade of the febrile response per se. In fact, some studies have shown that the attenuation of fever by antipyretics reduces the survival rate during sepsis (33). Figueiredo et al. (34) reported that the febrile response that is induced by CLP in rats occurred around 6 and 12 h after the procedure and involved some of the already known endogenous pyrogens, such as IL-1β, IL-6, and prostaglandins.
In the present study, the same febrile response pattern was observed. Additionally, Rim, at the same dose that improved the survival rate of the animals, reduced but did not completely abolish the febrile response to levels that were similar to the sham-operated group that received only vehicle. However, neutrophil migration to the peritoneal cavity, leucopenia, and the enhanced levels of IL-6 observed 6 to 8 h after CLP were unchanged by Rim treatment 4 h after CLP. The lack of an effect of Rim on these peripheral hallmarks of CLP-induced sepsis (i.e., neutrophil migration, IL-6 levels, and leucopenia) and the reduction of the febrile response (i.e., a response that is controlled by the hypothalamus) suggest a central effect of Rim.
To confirm this assumption, Rim was injected i.c.v. using the same protocol as used for the ETA receptor antagonist. Rim administered 4 and 8 h after CLP also increased the survival rate confirming that the blockade of central CB1 receptors may be beneficial. Therefore, besides the beneficial effects on hemodynamics and inflammatory parameters when administered at the initial phase of sepsis, the present study suggests that later central effects of rimonabant may contribute to the increase in the survival rate after sepsis.
Strong evidence showed that AVP infusion during the first hours of sepsis in patients may be as effective as other vasopressors or even more effective in cases of less severe septic shock when mortality was evaluated 28 or 90 days later (6). Moreover, the synthetic AVP analog terlipressin was also safe and may spare the need for catecholamines as support treatment for hemodynamic changes during sepsis (7,8). Therefore, we evaluated whether the treatments that effectively reduced the mortality rate can also promote changes in AVP levels in blood. Circulating AVP levels were reduced 8 h after CLP and returned to normal levels at 12 h compared with sham-operated animals. Treatment with both Rim and BQ123 significantly increased AVP levels after 12 h. Altogether, these data suggest that an increase in blood AVP levels during the first 12 h after sepsis as a result of AVP infusion or Rim administration may contribute to the long-term possibility of survival after sepsis similarly to the effects observed in septic patients (6).
In summary, the present results showed that the blockade of central ETA or CB1 receptors increased the survival rate and AVP levels during the late phase of CLP-induced sepsis. Although more studies are needed to better understand the consequences of the activation and blockade of these receptors during different phases of sepsis, the present study may provide important information for development therapeutics strategies for this condition. Rim was previously approved in Europe as an anti-obesity drug, but it has been associated with serious side effects that are related to psychiatric disorders, including the induction of suicidal tendencies (35). However, the use of this drug in controlled intensive care units or the development of safer analogs in conjunction with conventional therapeutic approaches may aid sepsis treatment.
The authors thank Dr Patrícia do Rocio Dalzoto for the help with bacterial counts.
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Keywords:© 2016 by the Shock Society
Endocannabinoids; endothelin-1; sepsis; septic shock; vasopressin