Intestinal ischemia-reperfusion (I/R) injury is a devastating complication in the perioperative period. Dexmedetomidine is commonly applied in the perioperative period. The authors aimed to determine the effects of different doses of dexmedetomidine (given before or after intestinal ischemia) on intestinal I/R injury and to explore the underlying mechanisms.
Intestinal I/R injury was produced in rat by clamping the superior mesenteric artery for 1 h followed by 2 h reperfusion. Intravenous infusion of dexmedetomidine was performed at 2.5, 5, and 10 μg · kg(-1) · h(-1) for 1 h before or after ischemic insult. In addition, yohimbine hydrochloride was administered intravenously to investigate the role of α2 adrenoreceptor in the intestinal protection conferred by dexmedetomidine.
Intestinal I/R increased mortality of rats and caused notable intestinal injury, as evidenced by statistically significant increases in Chiu's scores; serum diamine oxidase and tumor necrosis factor-α concentration, accompanied by increases in the intestinal mucosal malondialdehyde concentration; myeloperoxidase activity; and epithelial cell apoptosis (all P < 0.05 vs. Sham). Except malondialdehyde and myeloperoxidase, all changes were improved by the administration of 5 μg · kg(-1) · h(-1) dexmedetomidine before ischemia (all P < 0.05 vs. Injury) but not after ischemia. Infusion of 2.5 μg · kg(-1) · h(-1) dexmedetomidine before or after ischemia produced no beneficial effects, and infusion of 10 μg · kg(-1) · h(-1) dexmedetomidine led to severe hemodynamic suppression. Yohimbine abolished the intestinal protective effect of the 5 μg · kg(-1) · h(-1) dexmedetomidine infusion before ischemia and was accompanied by the disappearance of its antiapoptotic and antiinflammatory effect.
Dexmedetomidine administration before, but not after, ischemia dose-dependently protects against I/R-induced intestinal injury, partly by inhibiting inflammatory response and intestinal mucosal epithelial apoptosis via α2 adrenoreceptor activation.
What We Already Know about This Topic
The mortality caused by intestinal ischemia-reperfusion (I/R)–induced intestinal injury remains high, and few techniques or drugs are effective enough to use clinically.
What This Article Tells Us That Is New
Dexmedetomidine administration before an ischemic insult dose-dependently reduces intestinal I/R injury. However, the initiation of dexmedetomidine administration after ischemia produced no protective effect.
INTESTINAL ischemia-reperfusion (I/R) injury is a potentially serious consequence of acute mesenteric ischemia; hemorrhagic, traumatic, or septic shock; severe burns; or some surgical procedures, including cardiopulmonary bypass, small bowel transplantation, and abdominal aortic surgery.1Intestinal I/R is associated with the exacerbation of intestinal injury and a systemic inflammatory response leading to progressive distal organ impairment, finally resulting in cardiocirculatory, respiratory, hepatic, and renal failure.2,3In the critical care setting, the development of intestinal ischemia contributes to high mortality (67–80%).4
Dexmedetomidine, a potent and highly selective α2adrenoreceptor agonist, is widely used for sedation in intensive care units (ICU).5Dexmedetomidine also offers good perioperative hemodynamic stability and an intraoperative anesthetic-sparing effect.6Thus, dexmedetomidine is used as an anesthetic adjuvant during surgery.7Either in vivo or in vitro , dexmedetomidine has demonstrated a protective effect against the I/R injury of heart, kidney, brain, and testis in animal models.8,–,12Schaak et al. reported that the activation of α2adrenoreceptor increased intestinal epithelial cell proliferation,13which indicates that α2adrenoreceptor agonist might be able to accelerate the wound healing process in the intestine. However, whether or not dexmedetomidine administration can provide protection against intestinal injury induced by intestinal I/R injury remains unclear. Based on the characteristics of dexmedetomidine, we hypothesized that dexmedetomidine could attenuate intestinal mucosal injury and mortality in rats after intestinal I/R.
Therefore, the current experiment was designed to investigate the effects of different doses of dexmedetomidine (given before or after the ischemic phase) on intestinal injury after I/R injury and explore the potential mechanisms involved in the effects of dexmedetomidine.
Materials and Methods
Animals and Operative Procedure
The current study was approved by the Animal Care Committee of Sun Yat-sen University, Guangzhou, China, and was performed in accordance with National Institutes of Health guidelines for the use of experimental animals. Adult pathogen-free male Sprague-Dawley rats weighing between 220 and 270 g were housed in individual cages in a temperature-controlled room with alternating 12-h light–dark cycles, and acclimated for 1 week before the study. Food was removed 8 h before the study, but all animals had free access to water.
All animals were anesthetized with pentobarbital (30 mg/kg body weight, intraperitoneal). Tracheotomy were performed, and 14-gauge intravenous catheters were inserted as tracheotomy tubes. The tubes were connected to small-animal ventilators (DH-150; Medical Instrument Company of Zhe Jiang University, Hangzhou, China). All rats were mechanically ventilated with a standard tidal volume ventilation protocol (tidal volume 10 ml/kg; respiratory rate 50 breaths/min; inspiratory/expiratory ratio of 1:1; 100% oxygen). A polyethylene catheter was inserted into the left carotid artery and connected to a digital data acquisition system (PowerLab/4SP; AD Instruments, Comerio, Italy) for monitoring the heart rate and mean arterial pressure (MAP), as described previously.14The femoral vein was cannulated to draw blood samples and for the drug infusion. The small intestine was exteriorized by midline laparotomy, and the intestinal I/R injury was established by occluding the superior mesenteric artery with a microvessel clip for 1 h, followed by 2 h reperfusion, as described previously.15,16During the study period, the rat rectal temperature was maintained at ∼36°–38°C with the aid of a heating pad.
Groups and Drug Administration
As figure 1A shows, animals were randomly allocated into 10 groups (n = 12 per group), as follows. Sham group (Sham): the rats received continuous intravenous infusion of normal saline and sham surgical preparation, including isolation of the superior mesenteric artery without occlusion. Injury group (Injury): the rats received continuous intravenous infusion of normal saline, and intestinal I/R was induced by clamping the superior mesenteric artery for 1 h followed by declamping (reperfusion) for 2 h. Dexmedetomidine 2.5, 5, and 10 administration before ischemia (ABI) groups: intravenous dexmedetomidine (Orion Pharma, Turku, Finland) was infused continuously at 2.5, 5, and 10 μg · kg−1· h−1for 1 h before the intestinal ischemia was induced. Dexmedetomidine 2.5, 5, and 10 administration after ischemia (AAI) groups: intravenous dexmedetomidine was infused continuously at 2.5, 5, and 10 μg · kg−1· h−1for 1 h after the beginning of reperfusion. The different doses of dexmedetomidine (2.5, 5 and 10 μg · kg−1· h−1) had been used previously to investigate the dose-related effect of dexmedetomidine on mortality and inflammatory responses in endotoxemic rats.17In the current study, all drugs were dissolved in normal saline, and all rats received intravenous fluid at a rate of 0.5 ml/h to ensure the same volume of fluid administration (fig. 1).
Experiment 1: To Investigate the Effects of Different Doses of Dexmedetomidine on Hemodynamics and Blood Gas Analysis
The rats' heart rate and MAP values at various time points were recorded and compared among groups. At 2 h after reperfusion, arterial blood (0.25 ml) was drawn into a heparinized syringe for blood gas analysis (G3+ chips, i-STAT; Abbott Laboratories, East Windsor, NJ).
Experiment 2: To Investigate the Effects of Different Doses of Dexmedetomidine on Intestinal I/R Injury
Based on the results of experiment 1, the appropriate doses of dexmedetomidine were chosen to investigate the effect of dexmedetomidine on intestinal injury induced by intestinal I/R.
An additional 2-ml blood sample was drawn 2 h after reperfusion. The whole blood was centrifuged at 3,500 rpm for 15 min at 4°C and plasma collected and stored at −70°C. The rats were then killed with an overdose of pentobarbital sodium. A segment of intestine, 0.5–1.0 cm, was cut from 5 cm to terminal ileum, fixed in formaldehyde polymerization (4%), and embedded in paraffin for preparation. Another segment of small intestine (9–10 cm) was cut from 10 cm to terminal ileum and washed with cold saline. The intestinal mucosa was scraped off gently, dried with suction paper, and preserved at −70°C.15,16
Histologic Measurement of Intestinal Mucosal Injury.
The segment of small intestine was stained with hematoxylin-eosin. Damage of intestinal mucosa was evaluated using the criteria of Chiu's method18by two independent experienced pathologists who were blinded to the study groups. A minimum of six randomly chosen fields from each rat were evaluated and averaged to determine mucosal damage, and then the results of two pathologists were averaged.
Detection of Diamine Oxidase Concentration in Serum.
Diamine oxidase is an enzyme synthesized primarily in gastrointestinal mucosal cells. The intestinal tissue and serum concentrations of diamine oxidase have been used as an indicator of the integrity and functional mass of the intestinal mucosa.19In this study, serum diamine oxidase was detected using a chemical assay kit (Nanjing Jiancheng Biologic Product, Nanjing, China) with a spectrophotometer according to the provided protocol of the manufacturer. Results were expressed as unit per liter serum.
The rats (n = 15 per group) receiving the same protocols (fig. 1A) were used to detect survival time. From the onset of reperfusion, animals were monitored via video recording for 24 h. Two hours after reperfusion, the survivors were transferred to their individual cages and allowed free access to food and water. Twenty-four hours after reperfusion, all animals were sacrificed by lethal sodium pentobarbital injection.
Experiment 3: To Investigate the Protective Mechanisms of Dexmedetomidine against Intestinal I/R Injury
Based on the results of experiment 2, the optimal regimen with the most effective dose of dexmedetomidine were chosen to investigate the mechanisms of dexmedetomidine protection against intestinal I/R injury.
Detection of Tumor Necrosis Factor-α (TNF-α) in Serum.
Serum concentrations of TNF-α were determined using commercially available ELISA kits (R&D, Minneapolis, MN) according to the manufacturer's procedure, as described previously.14The results were expressed as picograms per liter serum.
Detection of Lactic Acid Concentration in Serum.
Serum concentrations of lactic acid were determined using a chemical assay kit (Nanjing Jiancheng Biologic Product) with a spectrophotometer according to the protocol provided by the manufacturer. Results were expressed as micromoles per liter serum.
Detection of Malondialdehyde Concentration and Myeloperoxidase Activity in Intestinal Mucosa.
Intestinal mucosal tissues were homogenized on ice with normal saline, frozen in a refrigerator at −20°C for 5 min and centrifuged for 15 min at 4,000 rpm. Supernatants were transferred into fresh tubes for the evaluation. The lipid peroxidation product malondialdehyde concentration was measured using chemical assay kits (Nanjing Jiancheng Biologic Product). The results were expressed as nanomoles per 100 mg protein.
The myeloperoxidase activity was used as an index to reflect neutrophil migration into the small intestine.20The intestine assay sample was homogenized and the homogenate was frozen–thawed twice, and then centrifuged at 13,000 rpm for 5 min. The resulting supernatant was assayed spectrophotometrically for myeloperoxidase activity. One unit of myeloperoxidase was defined as that degrading 1 μmol peroxide per minute at 25°C. Results were expressed as unit per gram intestinal tissue.
In Situ Detection of Intestinal Mucosal Epithelial Apoptosis.
The ileal fragments were fixed in formaldehyde polymerization (4%) and embedded in paraffin. The apoptosis of intestinal mucosal epithelial cell was detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method, as described previously.16Cell death was assessed using an assay kit (Roche, Indianapolis, IN). The cells with clear nuclear labeling were defined as positive cells. The apoptotic index was calculated as: (the number of positive cell nuclei/the number of total cell nuclei) × 100.
Detection of Caspase-3 Expression in Intestinal Mucosa.
To further confirm the existence of apoptosis, caspase-3 was detected via western blot. The intestinal mucosal tissues were suspended in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% sodium deoxycholate and 0.05% sodium dodecyl sulfate, pH 7.4), broken into pieces on ice, and subjected to three freeze–thaw cycles. The insoluble cell debris was removed by centrifugation. Supernatants were collected, and the protein concentration was determined using a bicinchoninic acid assay reagent (Pierce Chemical Company, Rockford, IL). Protein samples (30 μg) were subjected to sodium dodecyl sulfate (15%) polyacrylamide gel electrophoresis, then transferred electrophoretically to Hybond ECL Nitrocellulose Membranes (Amersham, Arlington Heights, IL) and incubated with a blocking solution composed of 5% fat dry milk in Tween-containing Tris-buffered saline (pH 8.0, 10 mm Tris, 150 mm NaCl, 0.1% Tween). Membranes were incubated overnight with rabbit monoclonal anticleaved caspase-3 antibody (number 9664; Cell Signaling Technology, Danvers, MA) or rabbit polyclonal antiactin antibody (number 4967; Cell Signaling Technology). After the membranes were washed with Tween-containing Tris-buffered saline three times, they were incubated for 1 h at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody (number 7074; Cell Signaling Technology) directed at the primary antibody. The band density was analyzed densitometrically and normalized with the housekeeping protein β-actin and then presented as percentage of injury.
Experiment 4: To Explore the Role of α2Adrenoceptor in the Intestinal Protective Effect of Dexmedetomidine
Based on the results of experiments 2 and 3, the α2adrenoceptor antagonist yohimbine hydrochloride (YOH; Sigma Aldrich, St. Louis, MO) was used to examine whether the α2-adrenergic receptor is involved in the protective effect of dexmedetomidine. The rats were randomly divided into the YOH + dexmedetomidine and YOH groups (n = 12 per group). Based on the results of a previous study involving dexmedetomidine and YOH,21YOH (1 mg/kg) was administered intravenously 10 min before dexmedetomidine or normal saline infusion (fig. 1B). Blood samples and intestinal tissues were harvested and preserved according to the methods of experiments 1 and 2.
Statistics were analyzed with SPSS 15.0 software (SPSS Inc, Chicago, IL). Hemodynamic data during the study were expressed as mean ± SE and analyzed using two-way ANOVA with repeated measures, followed by Bonferroni posttest. Survival time from the beginning of reperfusion was expressed as median (range), and results were compared by Kaplan–Meier log-rank test. The mortality rate was analyzed by Fisher exact test. The other data were expressed as mean ± SE, and one-way ANOVA (Tukey posttest) was used for comparisons. Biochemical assays for diamine oxidase, lactic acid, malondialdehyde, and myeloperoxidase were performed in triplicate for each specific sample. Therefore, all data points are means of numbers that themselves are means of triplicate measurements for these parameters. P < 0.05 in two-tailed testing was considered statistically significant.
Experiment 1: Effects of Different Doses of Dexmedetomidine on Hemodynamics and Blood Gas Analysis
Changes of Hemodynamic Variables and Blood Gas Parameters.
As shown in figure 2, there were no statistical differences in baseline heart rate or MAP among the groups. Dexmedetomidine in dosages of 2.5 and 5 μg · kg−1· h−1in the ABI and AAI groups did not affect heart rate and MAP (all P > 0.05, vs. Injury). Whereas, 10 μg · kg−1· h−1dexmedetomidine in the ABI and AAI groups statistically significantly reduced heart rate and MAP (all P < 0.05, vs. Injury). As shown in table 1, the arterial PaCO2, and PaO2values did not differ among the groups (all P > 0.05). The arterial pH value was reduced in the dexmedetomidine 10 AAI group (P = 0.003 vs. Injury) but was not reduced in other groups (all P > 0.05 vs. Injury).
Experimental 2: Effects of Different Doses of Dexmedetomidine on Intestinal I/R Injury
Experiment 1 showed that 10 μg · kg−1· h−1dexmedetomidine caused severe hemodynamic instability. Thus, the dexmedetomidine 10 regimens were suspended in the following experiments because hypotension and bradycardia induced by this large dose of dexmedetomidine probably interfered with the investigation of the intestinal protective effect of dexmedetomidine.
Intestinal Mucosal Morphologic Changes.
Representative mucosal morphologic changes are presented in figure 3, A–H. Detailed descriptions of morphologic changes in different groups are provided in the legend of figure 3. The mucosal injury was quantified as Chiu's score in figure 3I. Chiu's scores in the Injury group were higher than in the Sham group (P < 0.001), and were attenuated by 5 μg · kg−1· h−1dexmedetomidine in the ABI group (P < 0.001 vs. Injury) but not by other regimens (all P > 0.05 vs. Injury).
Changes of Serum Diamine Oxidase Concentration.
As shown in figure 4, the diamine oxidase concentration in the Injury group was higher than that in the Sham group (P < 0.001), and was reduced in the dexmedetomidine 5 ABI group (P = 0.005 vs. Injury) but not in the other regimens (all P > 0.05 vs. Injury).
The survival time of the animals in the Injury group was 2.25 h (1.6∼4.5 h). Survival time was prolonged in the dexmedetomidine 2.5 and 5 ABI groups, but not in the dexmedetomidine 2.5 and 5 AAI groups [2.5 ABI: 4 h (1.7∼24 h), P = 0.016; 5 ABI: 4.5 h (2.25∼24 h), P < 0.001 versus Injury]. Mortality rates at 24 h after reperfusion were 6.7%, 100%, 80%, 100%, 66.7%, and 93.3% for the Sham, Injury, Dexmedetomidine 2.5 ABI and AAI, and Dexmedetomidine 5 ABI and AAI groups, respectively (fig. 5). Only dexmedetomidine 5 in the ABI group statistically significantly reduced mortality (P = 0.042 vs. Injury), and mortality rates in other groups were similar to that in the Injury group (all P > 0.05).
Experiment 3: The Mechanisms of Dexmedetomidine Conferring Protection against Intestinal I/R Injury
Experiment 2 showed that dexmedetomidine 2.5 in the ABI and AAI groups produced no protective effects on intestinal I/R injury, and dexmedetomidine 5 μg in the ABI, but not the AAI, group exhibited statistical beneficial effect. Thus, in experiment 3, we performed mechanistic investigations to further detect why dexmedetomidine 5 in the ABI group, rather than the AAI group, could confer intestinal protection.
Changes of Intestinal Mucosal Malondialdehyde Concentration.
As shown in figure 6, the malondialdehyde concentration in the Injury group was higher than in the Sham group (P < 0.001). Dexmedetomidine 5 ABI and AAI did not reduce the malondialdehyde concentration (ABI: P = 0.511 and AAI: P = 0.996 vs. Injury).
Changes of Intestinal Mucosal Myeloperoxidase Activity.
As shown in figure 7, the activity of myeloperoxidase in the Injury group was higher than in the Sham group (P < 0.001). Neither Dexmedetomidine 5 ABI nor AAI reduced myeloperoxidase activity (ABI: P = 0.839 and AAI: P = 0.851 vs. Injury).
Changes of Serum Lactic Acid Concentration.
As shown in figure 8, the concentrations of lactic acid were not statistically significantly different among the groups (all P > 0.05).
Changes of Serum TNF-α Concentration.
As shown in figure 9, the serum concentration of TNF-α in the Injury group was higher than that in the Sham group (P < 0.001), which was statistically significantly reduced by dexmedetomidine 5 ABI (P < 0.001 vs. Injury) but not by AAI (P = 0.269 vs. Injury).
Changes of Intestinal Mucosal Epithelial Apoptosis.
The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling positive epithelial cells at the villus surface stained dark brown in the nuclei (fig. 10). Detailed descriptions of mucosal epithelial apoptosis in different groups are shown in the legend of figure 10. The apoptotic index was statistically significantly higher in the Injury group than in the Sham group (P < 0.001, fig. 10G). As shown in figure 10G, dexmedetomidine 5 ABI reduced the apoptotic index (P = 0.018 vs. Injury). However, dexmedetomidine 5 AAI did not reduce the apoptotic index (P = 0.900 vs. Injury).
Changes of Caspase-3 Expression of Intestinal Mucosa.
Representative bands from the western blotting are presented in figure 11A. As shown in figure 11B, cleaved caspase-3 expression of intestinal mucosa in the Injury group was higher than that in the Sham group (P < 0.001). Dexmedetomidine 5 ABI, but not AAI, reduced caspase-3 expression (ABI: P = 0.002 and AAI: P = 0.545 vs. Injury).
Experiment 4: Effects of α2Adrenoceptor Antagonists
As shown in figures 3I, 4, 9, 10G, and 11B, Chiu's score, serum diamine oxidase activity, TNF-α concentrations, and apoptotic index and caspase-3 expression in the YOH + Dexmedetomidine group were statistically significantly higher than that in the dexmedetomidine 5 ABI group (Chiu's score: P = 0.034; diamine oxidase activity: P = 0.033; TNF-α: P < 0.001; apoptotic index: P = 0.017; and caspase-3: P = 0.048) and similar to that in the Injury group (all P > 0.05), suggesting that the YOH pretreatment abolished the protective effects of dexmedetomidine 5 ABI on intestinal I/R injury. On the other hand, YOH alone (YOH group) produced no effects on the above variables in comparison with the Injury group (all P > 0.05).
Intestinal I/R injury is associated with some critical clinical phenomena, such as severe infection, shock, and some major surgery, including cardiopulmonary bypass, small intestine transplantation, and abdominal aortic artery surgery, which are commonly seen in the operating room and ICU. Dexmedetomidine is often used as a sedative and anesthetic adjuvant for such patients because of its anesthetic-sparing effect and properties of antiinflammation and cardiovascular protection.22,23Thus, we sought to determine whether dexmedetomidine protects against intestinal I/R injury and to find its appropriate dose and administration regimen.
To determine the appropriate dose of dexmedetomidine for intestinal protection, low, median, and large (2.5, 5, and 10 μg · kg−1· h−1) doses of dexmedetomidine were used in the current study. Our data showed that a low dose of dexmedetomidine did not confer intestinal protection and a large dose led to severe hemodynamic instability, especially hypotension. The current data about hemodynamics and serum lactic acid concentration indicated that the median dose of dexmedetomidine (5 μg · kg−1· h−1) did not contribute to tissue hypoperfusion in the current model. In addition, this dose had been used previously to decrease inflammatory responses and mortality in septic rats.24,25In recent years, the body surface area normalization method was recommended by the Food and Drug Administration for the more appropriate conversion of drug doses from animal studies to human studies.26Using this method, the dose of 5 μg · kg−1· h−1dexmedetomidine used in rats is equal to approximately 0.8 μg · kg−1· h−1in humans, which has been demonstrated to be safely applied in cardiovascular surgery and the ICU.27,–,29The human equivalent dose of 10 μg · kg−1· h−1dexmedetomidine far exceeds the clinically licensed dose of 1 μg · kg−1· h−1, which may cause frequent bradycardia and hypotension in general anesthesia.6This information indicates 5 μg · kg−1· h−1dexmedetomidine is the optimal dose in the current study.
We found for the first time that intestinal injury and rat mortality after intestinal I/R were reduced by dexmedetomidine 5 ABI but not by 5 AAI. This finding seems to be similar to previous investigations involved in the effects of dexmedetomidine on myocardial I/R injury in which dexmedetomidine administration before ischemia significantly reduced the infarct size in an isolated rat heart model,8whereas dexmedetomidine administration at the initiation of reperfusion with the same dose in the same experimental model increased the myocardial infarct size.30Our previous studies demonstrated the very early moments of reperfusion (less than 3 min) are critical to intestinal protection in the same model as the current study.14,31These findings all suggested that, although the exact mechanisms are not clear, the timing of performing treatment is of utmost important for conferring protection against I/R injury. In the current study, the dexmedetomidine 5 AAI regimen did not exhibit intestinal protective effect despite immediate infusion at the beginning of reperfusion. We speculated that this ineffectiveness might be attributed to the slow onset of dexmedetomidine, which reaches its effect approximately 15 min after intravenous administration.32To our knowledge, the timing of administration of dexmedetomidine is quite different and empirically decided by physicians in clinical settings.23Although additional research for investigating the optimal timing of dexmedetomidine administration is mandatory, the current results at least indicate that early intervention of dexmedetomidine is critical for its intestinal protection. It may help clinicians to develop a good perioperative administration regimen of dexmedetomidine. In the majority of clinical conditions involving intestinal I/R, the ischemic insult occurs before patient admission to the ICU. Therefore, dexmedetomidine should be used as an anesthetic adjuvant and administered before surgery for patients with potential intestinal ischemia. The initiation of administration of dexmedetomidine after the operation or in the ICU may produce no beneficial effect.
We previously demonstrated that inflammatory response, oxidative injury, and neutrophil infiltration were involved in the pathogenesis of intestinal I/R injury.14,16The current data show that the dexmedetomidine 5 ABI regimen reduced serum TNF-α concentration, suggesting that dexmedetomidine could confer its intestinal protection by inhibiting inflammatory response. Compared with 5 μg · kg−1· h−1dexmedetomidine, 2.5 μg · kg−1· h−1dexmedetomidine failed to attenuate the increases of TNF-α and interleukin-6 in endotoxin-exposed rats,17which might be an important reason the low dose of dexmedetomidine did not produce beneficial effects in the current study. However, the current results showed that dexmedetomidine did not reduce intestinal mucosal malondialdehyde concentration or myeloperoxidase activity after intestinal I/R. That finding is not in accordance with previous reports, in which dexmedetomidine showed a superior ability in inhibiting neutrophil infiltration25,33and lipid peroxidation.34We speculate that two factors could be involved in this finding. First, the median dose of dexmedetomidine was not adequate for suppressing oxidative injury and the neutrophil infiltration induced by severe intestinal I/R in the current model. Second, oxidative stress and neutrophil accumulation could be not involved in the protection of dexmedetomidine against intestinal I/R injury.
Intestinal mucosal epithelial cells are the main component of the intestinal mucosal barrier. Apoptosis is a major mode of cell death in the destruction of rat intestinal epithelial cells induced by ischemia and I/R injury.35,36Several reports have demonstrated that prophylactic antiapoptotic treatment is an effective therapeutic strategy for the prevention of intestinal I/R injury.16,37In the current study, we for the first time showed that dexmedetomidine could attenuate intestinal mucosal epithelial cell apoptosis, as evidenced by decreases in the apoptotic index, caspase-3 protein expression, intestinal injury, and consequently rat mortality. The antiapoptotic effect of dexmedetomidine might be associated with reducing the production of TNF-α and thereby inhibiting the activation of extrinsic apoptotic cascade.38It has also been reported that dexmedetomidine could reduce the proapoptotic protein Bax expression and increase the antiapoptotic protein Bcl-2 expression,10thereby attenuating apoptosis by inhibiting the activation of intrinsic apoptotic cascade. In addition, previous reports, in vivo or in vitro , suggest the antiapoptotic effect of dexmedetomidine on axoneuron is related to the α2-adrenoceptor.39,40Therefore, in the current study, YOH was applied to further explore whether the α2-adrenoceptor is involved in the protective effect of dexmedetomidine. The results show that YOH totally abolished the intestinal protective effect of dexmedetomidine, accompanied by the disappearance of its antiinflammatory and antiapoptotic effects. This is a novel finding, indicating that apoptosis and inflammation mediated by the activation of α2-adrenoreceptor play an important role in the intestinal protection conferred by dexmedetomidine.
There were several possible limitations in this study. We used only three different doses to investigate the dose–effect relationship of the intestinal protection conferred by dexmedetomidine. The results showed that the large dose of dexmedetomidine severely affected hemodynamics and thereby was removed from the dose–effect relationship study, and the median dose of dexmedetomidine was superior to the low dose in intestinal protection. Nevertheless, more doses should be used to further investigate the dose–effect relationship in the future. In addition, α2-adrenoceptor is composed of three subtypes: α2A, B, C. YOH, a nonspecific α2-adrenoceptor antagonist, can not provide an exact profile of which subtype plays the key role in the intestinal protection of dexmedetomidine. Nevertheless, based on the results of Ma et al. ,40we concluded dexmedetomidine produced its intestinal protective effect mainly via the α2A-adrenoceptor subtype.
In summary, this study reports for the first time that dexmedetomidine administration before ischemia, but not after ischemia, dose-dependently attenuates intestinal injury induced by intestinal I/R, in part by suppressing the inflammatory response and intestinal mucosal epithelial apoptosis via the activation of the α2-adrenoceptor. Large doses of dexmedetomidine may contribute to severe hemodynamic inhibition and are not suitable for clinical application. Our results provide a new insight for the clinical use of dexmedetomidine, showing that an appropriate dose of dexmedetomidine can be initiated before ischemic insult occurs in the critical clinical settings involving intestinal I/R to produce effective intestinal protection.
The authors thank Professor Lei Wan Long, M.D., Ph.D. (Department of Anatomy and Brain Research, Sun Yat-sen University, Guang Dong, China), and Professor Wu Wei Kang, M.D., Ph.D. (Department of Pathophysiology, Sun Yat-sen University), for technical support.