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Sevoflurane protects against intestinal ischemia–reperfusion injury partly by phosphatidylinositol 3 kinases/Akt pathway in rats

Published:February 06, 2015DOI:https://doi.org/10.1016/j.surg.2014.12.013

      Background

      Intestinal ischemia–reperfusion injury (IRI) is a clinical challenge with high morbidity and mortality, leading to intestine damage, systemic inflammation, and multiorgan failure. Previous research has shown that the inhaled anesthetic sevoflurane protects various organs from IRI. However, whether sevoflurane protects against intestinal IRI and which application condition is the most effective are not completely clear. Thus, we investigated the effects of sevoflurane on intestinal IRI with sevoflurane given before, during or after intestinal ischemia, and the role of phosphatidylinositol 3 kinases (PI3K)/Akt pathway in these effects.

      Methods

      Rat model of intestinal ischemia–reperfusion (IR) was used in this study. The superior mesenteric artery was clamped for 60 minutes followed by 120 minutes of reperfusion. Sevoflurane at 0.25, 0.5, and 1.0 minimum alveolar concentration (MAC) was inhaled for 30 minutes before, during, or after ischemic insult. LY294002, a PI3K inhibitor, was injected intraperitoneally before sevoflurane inhalation.

      Results

      Intestinal IR caused a significant decrease of mean arterial blood pressure, severe intestinal mucosa injury and epithelial cell apoptosis, downregulation of the levels of phospho-Akt and phospho-Bad proteins. Exposure to 0.5 or 1.0 MAC sevoflurane before or after intestinal ischemia or 0.5 MAC during intestinal ischemia significantly ameliorated IR-induced histopathologic changes and decreased Chiu's scores. Pretreatment with 0.5 MAC sevoflurane also inhibited intestinal IR-induced increase of terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling–positive cells and activation of caspase-3 and restored expression of phospho-Akt and phospho-Bad. LY294002 partly blocked the protective effects induced by 0.5 MAC sevoflurane pretreatment.

      Conclusion

      Our results suggest that sevoflurane inhalation at clinical related concentration before, during, or after ischemia protects against IR-induced intestinal injury. The pretreatment-induced protection was partly mediated by inhibiting intestinal mucosal epithelial apoptosis via activation of the PI3K/Akt pathway.
      Intestinal ischemia–reperfusion injury (IRI) commonly occurs in operative procedures, such as abdominal aortic aneurysm operations, cardiopulmonary bypass, intestinal transplantation, and strangulated hernia operations. Disruption of blood and oxygen supply and the subsequent reperfusion of intestine result in mucosal barrier failure, bacteria translocation, inflammation and even multiorgan dysfunction syndrome.
      • Cerqueira N.F.
      • Hussni C.A.
      • Yoshida W.B.
      Pathophysiology of mesenteric ischemia/reperfusion: a review.
      • Yasuhara H.
      Acute mesenteric ischemia: the challenge of gastroenterology.
      Various treatments have been developed to protect against intestinal IRI. Intestinal ischemic preconditioning, an effective measure against intestinal IRI, may have limited application in clinic owing to its invasive nature and the unpredictability of when intestinal ischemia will occur.
      • Mallick I.H.
      • Yang W.
      • Winslet M.C.
      • Seifalian A.M.
      Ischemia-reperfusion injury of the intestine and protective strategies against injury.
      Although extensive researches have been carried out, a satisfactory therapeutic method with high efficacy and safety is not established yet.
      Inhaled anesthetics, such as isoflurane and sevoflurane, are used extensively during operative procedures. In recent years, studies have found that isoflurane protects various organs against IRI, such as heart, brain, liver, and kidney, via multiple mechanisms.
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      • Schmeling T.J.
      • Pagel P.S.
      • Gross G.J.
      • Warltier D.C.
      Isoflurane mimics ischemic preconditioning via activation of K(ATP) channels: reduction of myocardial infarct size with an acute memory phase.
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      Isoflurane preconditioning and postconditioning in rat hippocampal neurons.
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      Pre-treatment with isoflurane ameliorates renal ischemic-reperfusion injury in mice.
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      Emulsified isoflurane preconditioning protects against liver and lung injury in rat model of hemorrhagic shock.
      Moreover, Kim et al
      • Kim S.I.
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      • et al.
      Activation of NF-kappaB pathway in oral buccal mucosa during small intestinal ischemia-reperfusion injury.
      have identified that isoflurane postconditioning protects against intestinal IRI and multiorgan dysfunction. Sevoflurane is also reported to reduce neuronal damage after cerebral ischemia in rats in either preconditioning or postconditioning fashion.
      • Payne R.S.
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      • Kehl F.
      Sevoflurane-induced preconditioning protects against cerebral ischemic neuronal damage in rats.
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      • Yu J.
      • Yan M.
      • et al.
      Postconditioning with sevoflurane protects against focal cerebral ischemia and reperfusion injury via PI3K/Akt pathway.
      • Ren X.
      • Wang Z.
      • Ma H.
      • Zuo Z.
      Sevoflurane postconditioning provides neuroprotection against brain hypoxia-ischemia in neonatal rats.
      Whether sevoflurane protects against intestinal IRI remains undetermined.
      The phosphatidylinositol 3 kinases (PI3K)/Akt pathway is of great significance in the balance between cell survival and death.
      • Cantley L.C.
      The phosphoinositide 3-kinase pathway.
      Signaling molecules of this pathway are involved in a cascade reaction and provide a precise regulation on cell apoptosis.
      • Cantley L.C.
      The phosphoinositide 3-kinase pathway.
      Recently, accumulating studies have reported that PI3K/Akt pathway plays a pivotal role in IRI.
      • Wang J.K.
      • Yu L.N.
      • Zhang F.J.
      • Yang M.J.
      • Yu J.
      • Yan M.
      • et al.
      Postconditioning with sevoflurane protects against focal cerebral ischemia and reperfusion injury via PI3K/Akt pathway.
      • Huang C.Y.
      • Hsiao J.K.
      • Lu Y.Z.
      • Lee T.C.
      • Yu L.C.
      Anti-apoptotic PI3K/Akt signaling by sodium/glucose transporter 1 reduces epithelial barrier damage and bacterial translocation in intestinal ischemia.
      Sodium/glucose transporter 1 alleviates intestinal IRI-induced mucosa barrier failure via activation of PI3K/Akt pathway.
      • Huang C.Y.
      • Hsiao J.K.
      • Lu Y.Z.
      • Lee T.C.
      • Yu L.C.
      Anti-apoptotic PI3K/Akt signaling by sodium/glucose transporter 1 reduces epithelial barrier damage and bacterial translocation in intestinal ischemia.
      In addition, the PI3K/Akt pathway also plays an important role in sevoflurane postconditioning-induced neuroprotection against cerebral ischemia.
      • Wang J.K.
      • Yu L.N.
      • Zhang F.J.
      • Yang M.J.
      • Yu J.
      • Yan M.
      • et al.
      Postconditioning with sevoflurane protects against focal cerebral ischemia and reperfusion injury via PI3K/Akt pathway.
      Therefore, we presume that activation of PI3K/Akt pathway may be one of the potential mechanisms, if sevoflurane can reduce intestinal IRI.
      In this study, we investigated the effects of different concentrations of sevoflurane on intestinal IRI in rats. Sevoflurane was given before, during, or after ischemia. Furthermore, we tested the hypothesis that activation of PI3K/Akt pathway contributed to sevoflurane preconditioning-induced protection.

      Materials and methods

      All experimental procedures and protocols in this study were approved by the animal care committee at Sun Yat-sen University, Guangzhou, China, and were performed in strict accordance with National Institutes of Health Guidelines for the use of experimental animals. All efforts were made to minimize suffering of the animals.
      Adult, male Sprague-Dawley rats weighing 200–220 g were obtained from Guangdong Medical Laboratory Animal Co, China (Permission number: SCXK 2008-0002). They were housed under standardized conditions of temperature (22°C–25°C), humidity (55–58%), and 12-hour dark–light cycle with free access to food and water. Food was withheld for 12 hour prior to the experimentation, but free access to water was allowed.

      Model of intestinal IRI

      Rats were anesthetized by intraperitoneal injection of 20% urethane (0.6 mL/100 g) and allowed to breathe spontaneously during the surgery. The right common carotid artery was isolated and then a catheter was inserted into the artery. The rat model of intestinal IRI was established according to the described technique previosuly.
      • Liu K.X.
      • Chen S.Q.
      • Huang W.Q.
      • Li Y.S.
      • Irwin M.G.
      • Xia Z.
      Propofol pretreatment reduces ceramide production and attenuates intestinal mucosal apoptosis induced by intestinal ischemia/reperfusion in rats.
      • Liu K.X.
      • Rinne T.
      • He W.
      • Wang F.
      • Xia Z.
      Propofol attenuates intestinal mucosa injury induced by intestinal ischemia-reperfusion in the rat.
      The superior mesenteric artery (SMA) was isolated and occluded with a noninvasive artery clamp. Intestinal ischemia was identified by the loss of mesenteric pulsation and paleness of the intestine. The intestine was put back into the abdominal cavity in situ. After 60 minutes of intestinal ischemia, the clamp was removed from the SMA. Reperfusion lasted for 120 minutes. Reperfusion was verified by recovery of mesenteric pulsation and flush of the intestine. Sham group rats underwent laparotomy without SMA occlusion.

      Anesthesia exposure

      Anesthesia exposure was performed as described previously.
      • Li Y.
      • Liu C.
      • Zhao Y.
      • Hu K.
      • Zhang J.
      • Zeng M.
      • et al.
      Sevoflurane induces short-term changes in proteins in the cerebral cortices of developing rats.
      Rats were placed in a chamber with an inflow hose at the bottom and an outflow hose at the top of the chamber. Sevoflurane in a humidified 30% oxygen carrier gas at 2–3 L/min was delivered to the chamber for 30 minutes using an agent-specific vaporizer (Datex-Ohmeda, Madison, WI). The concentrations of sevoflurane, oxygen, and carbon dioxide in the chamber were measured by a gas analyzer (Datex Cardiocap II, Datex-Ohmeda) via a sensing device placed in the chamber immediately adjacent to the rats. One minimum alveolar concentration (MAC) of sevoflurane is approximately 2.0% in adult rats, as determined by Orliaguet et al.
      • Orliaguet G.
      • Vivien B.
      • Langeron O.
      • Bouhemad B.
      • Coriat P.
      • Riou B.
      Minimum alveolar concentration of volatile anesthetics in rats during postnatal maturation.
      Therefore, 0.25 or 0.5 MAC of sevoflurane is approximately 0.5 or 1%, respectively, in adult rats. The fresh gas flow rate was regulated to maintain CO2 at <1%. Rats were placed on a heating pad and under a warming light to maintain body temperature at approximately 37°C. Rats in the sham operated and IRI groups were exposed only to carrier gas.

      Experimental protocol

      Three experiments were performed. In experiment 1, rats were exposed to 0.25, 0.5, or 1.0 MAC sevoflurane or carrier gas, respectively, for 30 minutes before ischemia, and then underwent intestinal IRI (n = 5 per group). Mean arterial blood pressure (MAP) was monitored continuously via the catheter by a biological signal collecting and processing system (Biolap 98).
      • Liu K.X.
      • Li Y.S.
      • Huang W.Q.
      • Chen S.Q.
      • Wang Z.X.
      • Liu J.X.
      • et al.
      Immediate postconditioning during reperfusion attenuates intestinal injury.
      Blood samples (100 μL) were collected from right common carotid artery via the catheter, and immediately analyzed with a blood gas analyzer (i-STAT; Abbott Laboratories, East Windsor, NJ) before and 30 minutes after sevoflurane exposure (n = 5).
      In experiment 2, rats were randomly assigned to receive 1 of the following treatments (Fig 1; n = 9 per group): (1) sham operated (sham-O), rats were subject to laparotomy and isolation of SMA without occlusion; (2) IRI, rats underwent SMA occlusion for 60 minutes and reperfusion for 120 minutes; (3) IRI + sevoflurane exposure before ischemia (SBI), rats were exposed to sevoflurane at 0.25, 0.5, or 1.0 MAC for 30 minutes before ischemia; (4) IRI + sevoflurane exposure during ischemia (SDI), rats were exposed to 0.25, 0.5, or 1.0 MAC sevoflurane for 30 minutes during ischemia; (5) IRI + sevoflurane exposure after ischemia (SAI), rats were exposed to 0.25, 0.5, or 1.0 MAC sevoflurane for 30 minutes after ischemia; and (6) sham sevoflurane exposure (sham-S), rats were exposed to 1.0 MAC sevoflurane for 30 minutes after isolation of SMA but without occlusion. At the end of the experiment, all rats were humanely killed and the intestine tissues were collected 10 cm away from the ileocecal junction for evaluating histopathologic changes after hematoxylin-eosin (HE) staining.
      Figure thumbnail gr1
      Fig 1Protocol for experiments 2 and 3. Sham-O group, involving isolation of superior mesenteric artery (SMA) without occlusion; ischemia/reperfusion injury (IRI) group, performed SMA occlusion for 60 minutes followed by reperfusion for 120 minutes without any interventions; 0.25 MAC-SBI, 0.5 MAC-SBI, and 1.0 MAC-SBI groups, sevoflurane inhalation at 0.25, 0.5, and 1.0, MAC, respectively, for 30 minutes before ischemia (pretreatment); 0.25 MAC-SDI, 0.5 MAC-SDI, and 1.0 MAC-SDI, sevoflurane inhalation at 0.25, 0.5, and 1.0 MAC, respectively, for 30 minutes during ischemia; 0.25 MAC-SAI, 0.5 MAC-SAI, and 1.0 MAC-SAI groups, sevoflurane inhalation at 0.25, 0.5, and 1.0 MAC, respectively, for 30 minutes after ischemia; sham-S group, sevoflurane inhalation at 1.0 MAC for 30 minutes, followed by isolation of SMA without occlusion; 0.5 MAC-LY, LY294002 (0.3 mg/kg) was administered intraperitoneally 15 minutes before 0.5 MAC sevoflurane inhalation. IRI, Ischemia–reperfusion injury; LY, LY294002; MAC, minimum alveolar concentration; SAI, sevoflurane inhalation after ischemia; SBI, sevoflurane inhalation before ischemia; SDI, sevoflurane inhalation during ischemia; sham-O, sham operation; sham-S, sham sevoflurane; SMA, superior mesenteric artery.
      In experiment 3, rats received intraperitoneal injection of the PI3K inhibitor LY294002 (0.3 mg/kg in a dimethylsulfoxide solution) 15 minutes before preconditioning with 0.5 MAC sevoflurane (Fig 1; n = 9). At the end of the experiment 3, the rats were killed humanely and the intestine tissues were collected 10 cm away from the ileocecal junction for evaluating histopathologic changes after HE staining, detecting apoptosis by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) staining, and determining protein expression of cleaved caspase-3 and Akt by Western blotting.

      Intestinal histopathology

      The samples of ileum were fixed in 4% paraformaldehyde for 12 hours, embedded in paraffin, cut into 4-μm-thick sections and then stained with HE (n = 9 per group). The histopathologic changes were scored from 0 to 9 using the criteria of Chiu's method by 2 independent pathologists who were blinded to the study groups
      • Quaedackers J.S.
      • Beuk R.J.
      • Bennet L.
      • Charlton A.
      • Oude E.M.
      • Gunn A.J.
      • et al.
      An evaluation of methods for grading histologic injury following ischemia/reperfusion of the small bowel.
      : grade 0, normal mucosa; grade 1, subepithelial Gruenhagen's space, capillary congestion; grade 2, moderate intestinal grand damage; grade 3, extension of subepithelial space with moderate epithelial lifting; grade 4, massive epithelial lifting down sides of villi, few tips denuded; grade 5, denuded villi; grade 6, loss (destruction) of villi, hemorrhage; grade 7, injured crypt layer, hemorrhage; grade 8, necrosis of the entire mucosa and submucosa, hemorrhage; and grade 9, transmural necrosis, hemorrhage. A minimum of 6 randomly chosen fields from each rat were evaluated and averaged to determine mucosal damage, and then the results from 2 pathologists were averaged.

      TUNEL staining

      TUNEL staining assay was performed using the Dead End Colorimetric TUNEL System Kit (Promega Corporation, Madison, WI) as described previously.
      • Li Y.
      • Liang G.
      • Wang S.
      • Meng Q.
      • Wang Q.
      • Wei H.
      Effects of fetal exposure to isoflurane on postnatal memory and learning in rats.
      In brief, sections were permeabilized by proteinase K solution (20 mg/mL) for 8 minutes, incubated in equilibration buffer for 10 minutes, and the terminal deoxynucleotidyl transferase and biotinylated nucleotide were added to the section and incubated in a humidified chamber at 37°C for 1 hour. The reaction was then stopped, followed by incubation with horseradish peroxidase–labeled streptavidin, colorized with diaminobenzidine/H2O2 and counterstained with modified hematoxylin. TUNEL-positive cells in the crypt epithelium were counted in ≥6 randomly chosen fields with an original magnification of ×400 from each rat by 2 persons who were blinded to the group assignment and the average number of cells was calculated. The density of TUNEL-positive cells was calculated by dividing the average number of TUNEL-positive cells by the area of a microscopic field.

      Immunoblotting

      Western blotting was performed as described previously.
      • Li Y.
      • Liu C.
      • Zhao Y.
      • Hu K.
      • Zhang J.
      • Zeng M.
      • et al.
      Sevoflurane induces short-term changes in proteins in the cerebral cortices of developing rats.
      • Li Y.
      • Liang G.
      • Wang S.
      • Meng Q.
      • Wang Q.
      • Wei H.
      Effects of fetal exposure to isoflurane on postnatal memory and learning in rats.
      In brief, protein concentrations of samples were determined using the bicinchoninic acid assay reagent (Pierce Chemical Company, Rockford, IL). Thirty micrograms of each protein sample were subjected to Western blot analysis using the following primary antibodies: anti–cleaved caspase-3 at 1:2000 dilution, anti–phospho-Akt at 1:1000 dilution, anti-Akt at 1:2000 dilution, anti–phospho-Bad at 1:1000 dilution, and anti–β-actin at 1:2000 dilution. All antibodies were purchased from Cell Signaling Technology (Beverly, MA). Images were scanned by an Image Master II scanner (GE Healthcare, Milwaukee, WI) and were analyzed using ImageQuant TL software v2003.03 (GE Healthcare). The protein expression of phospho-Akt was normalized to the total Akt. The band signals of other interesting proteins were normalized to those of the corresponding β-actin and then expressed as fractions of control sample from the same gels.

      Statistical analysis

      Data from blood gas analyses, Chiu scores, TUNEL-positive cells, and Western blots were normally distributed and had equal variances. They were expressed as mean values ± the standard error of the mean and analyzed by 1-way analysis of variance (ANOVA), followed by the least significance difference (LSD) posttest. Data of hemodynamics were analyzed using 2-way repeated measures ANOVA. All possible comparisons between groups were made using the LSD posttest. The Graphpad Prism 6.0 software was used to conduct the statistical analyses.

      Results

      Effects of sevoflurane preconditioning on hemodynamics and blood gases

      Intestinal IR led to a significant decrease of MAP compared with sham control animals. The decrease of MAP in the reperfusion period was more severe than that in the ischemia period. Exposure to sevoflurane at both 0.5 and 1.0 MAC before ischemia (pretreatment) alleviated significantly intestinal IR-induced decrease of MAP in the reperfusion period (Fig 2), but not that in the ischemia period. Pretreatment with sevoflurane at 0.25 MAC had no effect on IR-induced decrease of MAP (Fig 2). Pretreatment with sevoflurane at 0.5 MAC slightly, and at 1.0 MAC more severely, decreased pH and arterial oxygen pressure (PaO2) and increased arterial carbon dioxide pressure (PaCO2) of rats (Table), suggesting that pretreatment with 1.0 MAC sevoflurane may lead to more severe respiratory inhibition than that with 0.5 MAC sevoflurane (pH, P < .0001; PaO2, P = .0373; PaCO2, P < .0001, all vs 0.5 MAC-SBI).
      Figure thumbnail gr2
      Fig 2Effect of sevoflurane pretreatment on mean arterial pressure during the study period. Data are expressed as mean values ± standard error of the mean (n = 5). Results were compared by 2-way analysis of variance with repeated measures followed by least significance difference t posttest. *P < .05 versus sham-O group. #P < .05 versus the IRI group. IRI, Ischemia–reperfusion injury; MAC, minimum alveolar concentration; SBI, sevoflurane inhalation before ischemia; sham-O, sham operated.
      TableEffect of different concentration of sevoflurane pretreatment on the blood gas analysis
      GrouppHaPaO2 (mmHg)PaCO2 (mmHg)
      Baseline30 minBaseline30 minBaseline30 min
      Sham operated7.37 ± 0.037.36 ± 0.03132.6 ± 7.0129.3 ± 9.245.7 ± 1.348.6 ± 1.1
      0.25 MAC-SBI7.37 ± 0.027.35 ± 0.02130.1 ± 9.6128.3 ± 10.246.3 ± 1.747.1 ± 1.3
      0.5 MAC-SBI7.37 ± 0.047.31 ± 0.02
      P < .05 versus the sham operated group.
      135.0 ± 8.0110.3 ± 9.8
      P < .05 versus the sham operated group.
      46.1 ± 1.054.4 ± 3.6
      P < .05 versus the sham operated group.
      1.0 MAC-SBI7.38 ± 0.027.20 ± 0.04
      P < .05 versus the sham operated group.
      P < .05 versus the 0.5 MAC-SBI group by 1-way analysis of variance with least significance difference t posttest.
      132.6 ± 6.596.6 ± 8.9
      P < .05 versus the sham operated group.
      P < .05 versus the 0.5 MAC-SBI group by 1-way analysis of variance with least significance difference t posttest.
      46.1 ± 1.167.5 ± 2.2
      P < .05 versus the sham operated group.
      P < .05 versus the 0.5 MAC-SBI group by 1-way analysis of variance with least significance difference t posttest.
      Data are expressed as mean values ± standard error of the mean (n = 5).
      0.25 MAC-SBI, 0.5 MAC-SBI, 1.0 MAC-SBI, Rats exposed to 0.25, 0.5, or 1.0 MAC sevoflurane for 30 minutes before ischemia respectively; MAC, minimum alveolar concentration; pHa, arterial pH; PaO2, arterial oxygen pressure; PaCO2, arterial carbon dioxide pressure.
      P < .05 versus the sham operated group.
      P < .05 versus the 0.5 MAC-SBI group by 1-way analysis of variance with least significance difference t posttest.

      Sevoflurane exposure at 0.5 or 1.0 MAC before or after ischemia reduces small intestinal mucosal injury

      At the end of the experiment, the terminal ileum was collected for evaluating histopathologic changes. The morphology in intestinal villi and glands was normal in the sham operated rats and sham sevoflurane–treated rats (Fig 3, A and B). Intestinal IR led to severe damage to the intestinal mucosa with sloughing of the villous tips and lifting of the epithelium from the lamina propria and the development of subepithelial Gruenhagen's spaces (Fig 3, C). Rats exposed to 0.5 MAC sevoflurane for 30 minutes before, during, or after ischemia (Fig 3, GI), as well as 1.0 MAC sevoflurane before or after ischemia (Fig 3, J, L) were protected from severe intestinal IRI. Sevoflurane exposure at 0.25 MAC did not reduce intestinal IRI (Fig 3, DF). The Chiu scores of the rats were shown in Fig 3, M. Compared with sham operated rats, intestinal IRI remarkably increased Chiu's score (P < .0001), whereas in the 0.5 MAC-SBI, 0.5 MAC-SDI, 0.5 MAC-SAI, 1.0 MAC-SBI, and 1.0 MAC-SAI groups, Chiu's scores of rats were dramatically decreased compared with those in the IRI group (P < .0001, P = .0055, P < .0001, P = .0013, and P = .0003, respectively, vs the IRI group). The Chiu's scores of rats in 0.5 MAC-SBI group were the lowest among the above treatments. In the 0.25 MAC-SBI, 0.25 MAC-SDI, 0.25 MAC-SAI, and 1.0 MAC-SDI groups, Chiu's scores of rats were not different from those in the IRI group (P = .5614, P = .7993, P = .6369, and P = .1123, respectively, vs the IRI group).
      Figure thumbnail gr3
      Fig 3Histopathologic changes of intestinal mucosa and the evaluation of injury with Chiu's scores under light microscopy (original magnification, ×200). Animals underwent various interventions according to the protocol of experiment 2. In the sham-O (A) and sham-S (B) groups, there were no injury in villi and glands. However, severe intestinal glands injury, mucosa villi disintegration or edema, increased gap of epithelial cells and severe hemorrhage were observed in the IRI group (C). In the groups of 0.25 MAC-SBI (D), 0.25 MAC-SDI (E), and 0.25 MAC-SAI (F), the severity of intestinal mucosal injury was similar to that in the IRI group. In the groups of 0.5 MAC-SBI (G), 0.5 MAC-SDI (H), 0.5 MAC-SAI (I), and 1.0 MAC-SBI (J) and 1.0 MAC-SAI (L) groups, the damages of intestinal villi and glands were much more slight than that in the IRI group, the gap between epithelial cells only increased slightly, whereas in the 1.0 MAC-SDI group (K), edema of mucosal villi and denuded villus tips were observed, and a number of capillaries were congested. The data of Chiu's score were expressed as mean values ± standard error of them mean (n = 9; M). Results were compared by 1-way analysis of variance with least significance difference t posttest. ***P < .001 versus sham-O group; ##P < .01 and ###P < .001 versus the IRI group. IRI, Ischemia–reperfusion injury; MAC, minimum alveolar concentration; SAI, sevoflurane inhalation after ischemia; SBI, sevoflurane inhalation before ischemia; SDI, sevoflurane inhalation during ischemia; sham-O, sham operated; sham-S, sham sevoflurane.

      PI3K inhibition reversed protection induced by pretreatment with 0.5 MAC sevoflurane

      Consistent with previous data, intestinal IR led to severe damage to the intestinal mucosa. However, pretreatment with 0.5 MAC sevoflurane alleviated small intestinal mucosal injury and the Chiu scores were significantly decreased compared with rats with intestinal IR (P < .0001). LY294002, a PI3K inhibitor, partly reversed the protection induced by preconditioning with 0.5 MAC sevoflurane. The intestinal mucosal injury and the Chiu scores in the LY294002 plus sevoflurane group were increased significantly compared with rats pretreated with 0.5 MAC sevoflurane (P = .0066; Fig 4, A and C).
      Figure thumbnail gr4
      Fig 4LY partly inhibited protective effect of sevoflurane pretreatment against intestinal IRI. (A) Morphologic changes of intestinal mucosa under light microscopy (original magnification, ×200). (B) Representative images of TUNEL in the intestinal mucosa (original magnification, ×100 and ×400; scan bar = 100 μm). (C) Evaluation of intestinal injury with Chiu's scores. (D) Quantification of TUNEL positive cells in the intestinal mucosal. Apoptotic nuclei are stained dark brown under light microscopy shown by the arrows. Data are expressed as mean values ± standard error of the mean (n = 9). Results were compared by 1-way analysis of variance with least significance difference t posttest. ***P < .001 versus sham-O group; ##P < .01 and ###P < .001 versus the IRI group; P < .05 versus 0.5 MAC-SBI group. IRI, Ischemia–reperfusion injury; LY, LY294002; MAC, minimum alveolar concentration; SBI, sevoflurane inhalation before ischemia; sham-O, sham operated.

      Pretreatment with 0.5 MAC sevoflurane reduces small intestinal apoptosis after IRI

      We evaluated intestinal apoptosis by TUNEL staining and active caspase-3. TUNEL-positive cells per square millimeter in rats after intestinal IR was increased by approximately 23-fold compared with sham operated rats (P < .0001). Preconditioning with 0.5 MAC sevoflurane reduced significantly the number of apoptotic cells compared with rats after intestinal IR (P = .0002; Fig 4, B). The expression of cleaved caspase-3 protein in intestinal tissues detected by Western blots revealed that intestinal IR induced significantly caspase-3 activation (P < .0001). Pretreatment with 0.5 MAC sevoflurane reduced significantly intestinal IRI-induced caspase-3 activation (P = .0012; Fig 5, B).
      Figure thumbnail gr5
      Fig 5LY reversed sevoflurane-induced activity of PI3K/Akt pathway. (A) Representative Western blot of caspase-3, phosphor-Akt, Akt, and phosphor-Bad. (BD) Quantitative analysis of cleaved caspase-3 (B), phospho-Akt (C), and phospho-Bad (D). Data are expressed as mean values ± standard error of the mean (n = 9). Results were compared by 1-way analysis of variance with least significance difference t posttest. *P < .05, **P < .01, and ***P < .001 versus the sham-O group; ##P < .01 and ###P < .001 versus the IRI group; P < .05 and ▲▲▲P < .001 versus the 0.5 MAC-SBI group. IRI, Ischemia–reperfusion injury; LY, LY294002; MAC, minimum alveolar concentration; SBI, sevoflurane inhalation before ischemia; sham-O, sham operated.

      The PI3K/Akt pathway was partly involved in the protection of pretreatment with 0.5 MAC sevoflurane by inhibiting apoptosis

      The PI3K inhibitor LY294002 reversed the protection of 0.5 MAC sevoflurane pretreatment as reflected by increasing significantly TUNEL-positive cells (P = .0222; Fig 4, B and D) and cleaved caspase-3 expression (P = .0274; Fig 5, A and B). Intestinal IR increased slightly the protein expression of phospho-Akt (P = .0006) and phospho-Bad (P = .0066), whereas pretreatment with 0.5 MAC sevoflurane induced a greater increase of the expression of phospho-Akt and phospho-Bad than IR (P = .0006 and P < .0001, respectively, vs the IRI group). LY294002 significantly reversed the 0.5 MAC sevoflurane preconditioning-induced increases in phospho-Akt and phospho-Bad (P < .0001; Fig 5, A, C, and D).

      Discussion

      The present study has demonstrated that exposure to clinically relevant concentrations of sevoflurane at 0.5 or 1.0 MAC before or after intestinal ischemia as well as sevoflurane at 0.5 MAC during intestinal ischemia reduced intestinal IRI. Rats pretreated with 0.5 MAC sevoflurane exhibited the greatest protective effects among all the treatments. In addition, PI3K/Akt signaling pathway activation may be one of the mechanisms for pretreatment with 0.5 MAC sevoflurane to reduce intestinal IR-induced apoptosis.
      Intestinal IRI is a serious clinical problem in the perioperative period and settings, such as intestinal or liver transplantation and all kinds of shock that may result in intestinal ischemia.
      • Cerqueira N.F.
      • Hussni C.A.
      • Yoshida W.B.
      Pathophysiology of mesenteric ischemia/reperfusion: a review.
      • Yasuhara H.
      Acute mesenteric ischemia: the challenge of gastroenterology.
      • Mallick I.H.
      • Yang W.
      • Winslet M.C.
      • Seifalian A.M.
      Ischemia-reperfusion injury of the intestine and protective strategies against injury.
      Furthermore, acute mesenteric ischemia is associated with an exceedingly high mortality rate, despite operativeintervention.
      • Mallick I.H.
      • Yang W.
      • Winslet M.C.
      • Seifalian A.M.
      Ischemia-reperfusion injury of the intestine and protective strategies against injury.
      The ischemia and reperfusion phases have distinct pathophysiological features, with the majority of mucosal injury occurring during the reperfusion phase. Gut-derived factors are known to play an important role in mediating the systemic inflammatory state and multiorgan dysfunction in settings such as trauma and shock.
      • Cerqueira N.F.
      • Hussni C.A.
      • Yoshida W.B.
      Pathophysiology of mesenteric ischemia/reperfusion: a review.
      Therefore, once an ischemic event is inevitable, strategies to inhibit the cascade of events leading to intestinal and multiorgan injury during the reperfusion phase may help to reduce morbidity and mortality.
      Volatile anesthetics have been reported to provide protection against IRI in various organs, including heart, brain, liver, and kidney.
      • Kersten J.R.
      • Schmeling T.J.
      • Pagel P.S.
      • Gross G.J.
      • Warltier D.C.
      Isoflurane mimics ischemic preconditioning via activation of K(ATP) channels: reduction of myocardial infarct size with an acute memory phase.
      • McMurtrey R.J.
      • Zuo Z.
      Isoflurane preconditioning and postconditioning in rat hippocampal neurons.
      • Zhang L.
      • Huang H.
      • Cheng J.
      • Liu J.
      • Zhao H.
      • Vizcaychipi M.P.
      • et al.
      Pre-treatment with isoflurane ameliorates renal ischemic-reperfusion injury in mice.
      • Zhang L.
      • Luo N.
      • Liu J.
      • Duan Z.
      • Du G.
      • Cheng J.
      • et al.
      Emulsified isoflurane preconditioning protects against liver and lung injury in rat model of hemorrhagic shock.
      Because 0.25 MAC is the minimum concentration of isoflurane to ameliorate myocardial infarction
      • Obal D.
      • Weber N.C.
      • Zacharowski K.
      • Toma O.
      • Dettwiler S.
      • Wolter J.I.
      • et al.
      Role of protein kinase C-epsilon (PKCepsilon) in isoflurane-induced cardioprotection.
      and concentrations of 0.5 and 1.0 MAC are clinically relevant,
      • Li Y.
      • Liang G.
      • Wang S.
      • Meng Q.
      • Wang Q.
      • Wei H.
      Effects of fetal exposure to isoflurane on postnatal memory and learning in rats.
      we used these 3 concentrations. Because either preconditioning or postconditioning with sevoflurane protects against focal cerebral ischemia and reperfusion injury,
      • Payne R.S.
      • Akca O.
      • Roewer N.
      • Schurr A.
      • Kehl F.
      Sevoflurane-induced preconditioning protects against cerebral ischemic neuronal damage in rats.
      • Wang J.K.
      • Yu L.N.
      • Zhang F.J.
      • Yang M.J.
      • Yu J.
      • Yan M.
      • et al.
      Postconditioning with sevoflurane protects against focal cerebral ischemia and reperfusion injury via PI3K/Akt pathway.
      we exposed rats before, during, or after intestinal ischemia, respectively. The results of our study showed that sevoflurane exposure at a low concentration (0.25 MAC) provided no protective effect, whereas 0.5 and 1.0 MAC sevoflurane provided effective protection against intestinal IRI as indicated by the significantly lower Chiu scores than those in animals with intestinal IR only. Moreover, it was interesting to note that 0.5 MAC sevoflurane provided the protection when administered during 3 periods of times, whereas 1.0 MAC sevoflurane provided protection only when administered before or after ischemia. It was noted that there was also a slight reduction in MAP in sham rats owing to the vasodilation effect of anesthetic and the lack of liquid supplement during the experiments. These effects did not lead to intestinal mucosal injury. On the contrary, intestinal IR induced dramatic hypotension and severe intestinal mucosal injury. Although pretreatment with sevoflurane at both 0.5 and 1.0 MAC ameliorated intestinal IR-induced hypotension during reperfusion periods, 1.0 MAC sevoflurane led to more severe respiratory inhibition, as shown by a significant increase of PaCO2 and decrease of PH and PaO2 than did 0.5 MAC sevoflurane. The severe acidosis induced by respiratory depression may partially weaken the protection induced by 1.0 MAC sevoflurane, which might be a reason that 1.0 MAC sevoflurane did not provide greater protection than 0.5 MAC. Because our results suggested pretreatment with 0.5 MAC sevoflurane is the most effective method among all the strategies determined in our study, we chose this strategy to study further the possible protective mechanism provided by sevoflurane.
      Apoptosis is regarded as an histopathologic process leading to cell death in ischemia–reperfusion (IR).
      • Broughton B.R.
      • Reutens D.C.
      • Sobey C.G.
      Apoptotic mechanisms after cerebral ischemia.
      • Gottlieb R.A.
      Cell death pathways in acute ischemia/reperfusion injury.
      Recent studies have showed that apoptotic signaling pathways are involved in the mechanism of IRI not only in the myocardium and brain,
      • Broughton B.R.
      • Reutens D.C.
      • Sobey C.G.
      Apoptotic mechanisms after cerebral ischemia.
      • Inamura Y.
      • Miyamae M.
      • Sugioka S.
      • Domae N.
      • Kotani J.
      Sevoflurane postconditioning prevents activation of caspase 3 and 9 through antiapoptotic signaling after myocardial ischemia-reperfusion.
      but also in the intestinal mucosa.
      • Noda T.
      • Iwakiri R.
      • Fujimoto K.
      • Matsuo S.
      • Aw T.Y.
      Programmed cell death induced by ischemia-reperfusion in rat intestinal mucosa.
      • Fujise T.
      • Iwakiri R.
      • Wu B.
      • Amemori S.
      • Kakimoto T.
      • Yokoyama F.
      • et al.
      Apoptotic pathway in the rat small intestinal mucosa is different between fasting and ischemia-reperfusion.
      Ischemic preconditioning, one of the most effective strategies for protection from intestinal IRI, reduces apoptosis by upregulating the antiapoptosis gene bcl-2 and inhibiting activation of caspase-3, one of the most remarkable apoptotic regulators.
      • Taha M.O.
      • Ferreira R.M.
      • Taha N.S.
      • Monteiro H.P.
      • Caricati-Neto A.
      • Oliveira-Junior I.S.
      • et al.
      Ischemic preconditioning and the gene expression of enteric endothelial cell biology of rats submitted to intestinal ischemia and reperfusion.
      Our present results showed 0.5 MAC sevoflurane preconditioning reduced intestinal IR-induced increase of TUNEL-positive cells and activation of caspase-3, suggesting that sevoflurane preconditioning protected against intestinal IRI by inhibiting apoptosis. Our results are consistent with the findings of Kim et al that isoflurane postconditioning also reduces intestinal IRI by decreasing inflammation and apoptosis.
      • Kim S.I.
      • Kim Y.B.
      • Koh K.M.
      • Youn Y.K.
      • Suh G.J.
      • Cho E.S.
      • et al.
      Activation of NF-kappaB pathway in oral buccal mucosa during small intestinal ischemia-reperfusion injury.
      The PI3K/Akt pathway has been shown to play a critical role in regulating apoptosis.
      • Cantley L.C.
      The phosphoinositide 3-kinase pathway.
      PI3K is an important intracellular signaling molecule that activates Akt by phosphorylation. Activation of Akt blocks apoptosis by phosphorylating proapoptotic protein Bad, creating a binding site for 14-3-3 proteins and preventing Bad from binding the antiapoptotic Bcl-2 family members Bcl-2 and Bcl-xL.
      • Cantley L.C.
      The phosphoinositide 3-kinase pathway.
      Bcl-2 and Bcl-xL block the translocation of Bax, a proapoptotic protein, into the mitochondria, maintain mitochondrial membrane potential, and prevent the release of cytochrome C from the mitochondria and the subsequent apoptosis.
      • Hsu S.Y.
      • Kaipia A.
      • Zhu L.
      • Hsueh A.J.
      Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11.
      Ye et al
      • Ye Z.
      • Guo Q.
      • Xia P.
      • Wang N.
      • Wang E.
      • Yuan Y.
      Sevoflurane postconditioning involves an up-regulation of HIF-1alpha and HO-1 expression via PI3K/Akt pathway in a rat model of focal cerebral ischemia.
      have reported that sevoflurane postconditioning reduces cerebral IRI in rats by activating PI3K/Akt pathway.
      • Ye Z.
      • Guo Q.
      • Xia P.
      • Wang N.
      • Wang E.
      • Yuan Y.
      Sevoflurane postconditioning involves an up-regulation of HIF-1alpha and HO-1 expression via PI3K/Akt pathway in a rat model of focal cerebral ischemia.
      Ischemic postconditioning also protects brain from IRI by attenuating endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathway.
      • Yuan Y.
      • Guo Q.
      • Ye Z.
      • Pingping X.
      • Wang N.
      • Song Z.
      Ischemic postconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PI3K-Akt pathway.
      Our previous study has demonstrated that the PI3K/Akt pathway also participates in the neuroprotection of dexmedetomidine pretreatment against anesthetics–induced neuroapoptosis in the developing brain.
      • Li Y.
      • Zeng M.
      • Chen W.
      • Liu C.
      • Wang F.
      • Han X.
      • et al.
      Dexmedetomidine reduces isoflurane-induced neuroapoptosis partly by preserving PI3K/Akt pathway in the hippocampus of neonatal rats.
      Our present results showed that intestinal IR slightly increased the phosphorylation of Akt and Bad, which suggests that some protective mechanisms are activated during intestinal IR. However, preconditioning with 0.5 MAC sevoflurane significantly activated Akt and increased phosphorylation of Bad. The PI3K inhibitor LY294002 inhibited the phosphorylation of Akt and Bad and reversed protection induced by preconditioning with 0.5 MAC sevoflurane. These results suggest that the PI3K/Akt pathway was involved in the protection of sevoflurane preconditioning against intestinal IRI by inhibiting intestinal apoptosis.
      It was notable that LY294002 reversed only partly the protection of sevoflurane preconditioning. The PI3K/Akt pathway may not be the only mechanism involved. Recently, accumulating studies have shown that inflammatory response and oxidative stress may play a critical role in the development of intestinal IRI. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is also activated during intestinal IR. This pathway is associated with increased oxidative stress, neutrophil accumulation and intestinal mucosal apoptosis.
      • Wen S.H.
      • Li Y.
      • Li C.
      • Xia Z.Q.
      • Liu W.F.
      • Zhang X.Y.
      • et al.
      Ischemic postconditioning during reperfusion attenuates intestinal injury and mucosal cell apoptosis by inhibiting JAK/STAT signaling activation.
      In addition, dexmedetomidine attenuates intestinal IRI by decreasing significantly the serum diamine oxidase and inflammatory response.
      • Zhang X.Y.
      • Liu Z.M.
      • Wen S.H.
      • Li Y.S.
      • Li Y.
      • Yao X.
      • et al.
      Dexmedetomidine administration before, but not after, ischemia attenuates intestinal injury induced by intestinal ischemia-reperfusion in rats.
      Moreover, Gan et al
      • Gan X.
      • Su G.
      • Zhao W.
      • Huang P.
      • Luo G.
      • Hei Z.
      The mechanism of sevoflurane preconditioning-induced protections against small intestinal ischemia reperfusion injury is independent of mast cell in rats.
      have reported recently that sevoflurane preconditioning attenuates intestinal IRI and significantly downregulates the myeloperoxidase activities, intercellular cell adhesion molecule-1 expression, and interleukin-6 concentrations, indicating that inhibiting neutrophil sequestration and the subsequent systemic inflammation may be a potential mechanism of attenuating intestinal IRI.
      • Gan X.
      • Su G.
      • Zhao W.
      • Huang P.
      • Luo G.
      • Hei Z.
      The mechanism of sevoflurane preconditioning-induced protections against small intestinal ischemia reperfusion injury is independent of mast cell in rats.
      Mast cell (MC) activation also plays a role in intestinal and lung injuries induced by intestinal IR,
      • Ge M.
      • Gan X.
      • Liu D.
      • Zhang W.
      • Gao W.
      • Huang P.
      • et al.
      Time-course analysis of counts and degranulation of mast cells during early intestinal ischemia-reperfusion injury in mice.
      • Zhao W.
      • Gan X.
      • Su G.
      • Wanling G.
      • Li S.
      • Hei Z.
      • et al.
      The interaction between oxidative stress and mast cell activation plays a role in acute lung injuries induced by intestinal ischemia-reperfusion.
      and ischemic preconditioning protected against intestinal IRI by inhibiting the MC degranulation-mediated release of MC–carboxypeptidase A.
      • Xing D.
      • Zhang R.
      • Li S.
      • Huang P.
      • Luo C.
      • Hei Z.
      • et al.
      Pivotal role of mast cell carboxypeptidase A in mediating protection against small intestinal ischemia-reperfusion injury in rats after ischemic preconditioning.
      Sevoflurane preconditioning-induced protections against intestinal IRI are independent of MCs.
      • Gan X.
      • Su G.
      • Zhao W.
      • Huang P.
      • Luo G.
      • Hei Z.
      The mechanism of sevoflurane preconditioning-induced protections against small intestinal ischemia reperfusion injury is independent of mast cell in rats.
      Apart from inflammation and oxidation, other mechanisms may also participate in the development of intestinal IRI. In a pig model of cardiac arrest and successful cardiopulmonary resuscitation, therapeutic hypothermia and postconditioning with the sevoflurane increased significantly intestinal hypoxia-inducible factor 1α (HIF-1α) expression, suggesting that HIF-1α protein may play a role in the protection of sevoflurane.
      • Albrecht M.
      • Gruenewald M.
      • Zitta K.
      • Zacharowski K.
      • Scholz J.
      • Bein B.
      • et al.
      Hypothermia and anesthetic postconditioning influence the expression and activity of small intestinal proteins possibly involved in ischemia/reperfusion-mediated events following cardiopulmonary resuscitation.
      One clinic study has implied that isoflurane protects the intestine through better preservation of intestinal blood flow and oxygenation.
      • Muller M.
      • Schindler E.
      • Roth S.
      • Schurholz A.
      • Vollerthun M.
      • Hempelmann G.
      Effects of desflurane and isoflurane on intestinal tissue oxygen pressure during colorectal surgery.
      Our results also showed sevoflurane (0.5 and 1.0 MAC) preconditioning attenuated intestinal IRI-induced hypotension, implying that sevoflurane preconditioning improves intestinal blood flow and oxygenation, and that this might contribute to the attenuation of intestinal IR-induced apoptosis. Whether sevoflurane reduced apoptosis through inhibiting neutrophil sequestration and oxidative stress needs to be studied further.
      There are limitations to this study. First, we did not observe the effects of sevoflurane on survival rate of rats after intestinal IRI. Also, we did not examine the expression of inflammatory cytokines and oxidative stress in the intestinal mucosa. Additional experiments are needed to determine the role of sevoflurane-induced antiapoptosis and anti-inflammation properties in the protection against intestinal IRI. Moreover, whether PI3K/Akt pathway is also involved in the sevoflurane postconditioning against intestinal IRI needs further study.
      In conclusion, clinically relevant concentrations of sevoflurane protected against intestinal IRI. Activation of PI3K/Akt pathway participated in the protective effect of sevoflurane pretreatment. Sevoflurane is widely used in clinical anesthesia with fewer adverse effects, although this protective effect of sevoflurane are relatively modest, it may still have great translational potential in the patients at the risk of intestinal IRI.

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