Intestinal ischemia-reperfusion (I/R) is a potentially fatal clinical entity caused by mechanical obliteration of flow in intestinal blood vessels or by a critical secondary reduction in splanchnic organ blood flow. Arterial ischemia initiates tissue alterations by blocking oxygen supply, thus impeding aerobic energetic metabolism and inducing cellular injury (1, 2). Depending on the duration and intensity of the ischemia, tissue injury can be exacerbated when oxygen is reintroduced to the tissues (3). Intestinal I/R injury can accompany a number of clinical and pathophysiological conditions, such as trauma, hemorrhage, small bowel transplantation, and cardiopulmonary bypass. Despite recent improvements in diagnostic and interventional health care, a high in-hospital mortality rate of 60% to 80% was still reported in patients with acute intestinal ischemia (4, 5).
Helium is an odorless, tasteless, colorless monatomic element and does not interact with other compounds. Recent experimental research has shown that helium exerts protective effects in cardiac (6–12), neuronal (13–15), and liver (16) tissues. In the case of I/R injury, organ protection is achieved using several short helium episodes according to a specific protocol known as helium preconditioning (HPC). A typical HPC protocol consists of three cycles of 5 min of 70% helium/30% oxygen (7:3 heliox) breathing combined with three cycles of 5 min of air breathing (6–12, 15, 16). Compared with other interventions, the advantages of HPC include the favorable characteristics of helium and the lack of anesthetic and hemodynamic adverse effects following its use (17). Therefore, the use of helium as a clinical intestinal salvage strategy in the prevention of I/R merits investigation, including a determination of the specific mechanisms responsible for its beneficial actions (17).
In the present study, we explored the effectiveness of HPC in a rat model of intestinal I/R injury and tested four different HPC protocols to determine their efficacy. To our knowledge, this is the first study to investigate the effects of HPC on experimentally induced intestinal I/R.
MATERIALS AND METHODS
Animals and experimental protocol
The 60 male Sprague-Dawley rats (280–329 g; Shanghai Slac Laboratory Animal Co Ltd, Shanghai, China) used in this study were housed in an air-conditioned (23°C–25°C) room under a 12-h light-dark cycle with free access to pelleted rodent food and water. The rats remained in the care facility for a minimum of 72 h prior to the experiments for acclimatization. They were then randomly divided into six groups (n = 10 in each group): (a) sham: animals were untreated; (b) I/R: animals subjected to intestinal I/R injury only; (c) 2-min He + I/R: before intestinal I/R injury, the rats inhaled 7:3 heliox for three 2-min periods interspersed with three 5-min washout periods during which they breathed room air; (d) 5-min He + I/R: before intestinal ischemia, the rats inhaled 7:3 heliox for three 5-min periods interspersed with three 5-min washout periods during which they breathed room air; (e) 10-min He + I/R: before intestinal I/R injury, the rats inhaled 7:3 heliox for three 10-min periods interspersed with three 5-min washout periods during which they breathed room air; and (f) 15-min He + I/R: before intestinal ischemia, the rats inhaled 7:3 heliox for three 15-min periods interspersed with three 5-min washout periods during which they breathed room air (Fig. 1). This study was approved by the Animal and Ethics Review Committee of the Second Military Medical University (Shanghai, China).
Model of intestinal I/R
The animals were anesthetized with an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (8 mg/kg). No additional anesthesia was administered during the procedure. A median laparotomy was performed with a longitudinal median incision exposing the entire abdominal cavity to allow the identification and isolation of the superior mesenteric artery. Occlusion of the artery was achieved using an atraumatic vascular clamp for 60 min. After the ischemic phase, the vascular clamp was removed, the intestine was repositioned within the abdominal cavity, and the incision was closed. During surgery, the animal's body temperature was controlled by a thermostatically heated pad.
The HPC procedure was described previously (16). Briefly, the animals were placed in a 5.3-L transparent hyperbaric rodent chamber (type RDC 150-300-6; Second Military Medical University). Soda lime was added to the chamber, and fresh gas flow during HPC was maintained to avoid carbon dioxide retention.
Histology and immunohistochemistry
The small intestines were collected 60 min after reperfusion and flushed with ice-cold phosphate-buffered saline (PBS) to remove their intraluminal contents. The tissues were fixed in 4% paraformaldehyde for at least 48 h and then embedded in paraffin. Sections (4 μm thick) were stained with hematoxylin-eosin and evaluated using a computerized image analysis system (Smart Scape; Furi Science & Technology, Shanghai, China) as described previously (18). Briefly, intestinal injury was scored from 0 to 5 according to the following criteria: 0, normal mucosal villous structure; 1, presence of subepithelial space at the villous tips; 2, scattered epithelial denudation of the villous tips; 3, denuded tips with an exposed lamina propria and villous blunting; 4, epithelial shedding from both the apex and the midregion of the villi associated with a shortened and widened villous structure; 5, complete destruction of the villi and disintegration of the lamina propria with ulceration (18). The sections were stained with anti-CD68 and antimyeloperoxidase (anti-MPO) antibodies (Abcam, Cambridge, UK) according to the manufacturer's instructions. All data were evaluated by blinded investigators.
Terminal dUTP nick-end labeling staining
Paraffin-embedded intestinal sections were deparaffinized in xylene and dehydrated through a graded series of alcohols, ending with a 15 min of incubation in PBS. The terminal dUTP nick-end labeling (TUNEL) assay was performed using a commercial kit (Roche) according to the manufacturer's recommended protocol. TUNEL-positive cells were counted blindly.
Intestinal permeability assay
This assay was described previously (19), Briefly, 60 min after reperfusion, the distal ileum was identified, and a 5-cm segment was isolated using a silk suture. A 200-mL solution containing 25 mg of fluorescein isothiocyanate (FITC)–dextran in PBS was injected into the lumen of the isolated distal ileal loop. The laparotomy incision was closed with silk sutures. Thirty minutes after the injection, the blood was collected via cardiac puncture. After separation of the serum fraction, FITC-dextran fluorescence was measured at 520 nm.
MPO activity assay
Myeloperoxidase activity assay was performed according to manufacturer recommendation (Beyotime Chemical, NanJing, China). Briefly, intestinal samples were homogenized and sonicated in 10 volumes of potassium phosphate buffer (50 mM, pH 6.0) containing 0.5% HTAB. Lysates were centrifuged, supernatants were diluted, and the enzyme concentration was determined from the absorbance at 460 nm measured every 30 s over a 5-min period at 60°C. One unit of MPO activity was defined as the quantity of enzyme degrading 1 μmol of H2O2 per min, and MPO activity of the gut was expressed in units per milligram of tissue.
Western blot analysis
The tissues were sampled 60 min after reperfusion and then homogenized in tissue lysate (Beyotime Chemical). The tissues were aliquoted, and the protein concentration was measured using the bicinchoninic acid assay (Beyotime Chemical) according to the manufacturer's instructions. Briefly, samples containing 0.2 to 50 μg protein in microliters were prepared, and each 20-μL sample was added to 1-mL standard working solution. Then the samples were incubated at 60°C for 30 min, cooled, and read at 562 nm. Protein samples (20 μg) were denatured for 4 min at 95°C in sample buffer. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed using a 10% acrylamide gel, followed by protein transfer to a nitrocellulose membrane (Beyotime Chemical). The membrane was blocked overnight at 4°C in 5% nonfat dry milk in Tris-buffered saline and Tween 20, followed by incubation with primary rabbit anti–rat caspase 3 polyclonal antibody (Abcam). After a 1-h incubation with anti–rabbit horseradish peroxidase secondary antibody (Abmart, Shanghai, China) at room temperature, the probed protein was detected using the ECL chemiluminescence system (Bestbio, Shanghai, China). The bands of interest were quantified using the Eppendorf system.
Enzyme-linked immunosorbent assay for tumor necrosis factor α and macrophage inflammatory protein 1α
Scraped jejunal mucosa was homogenized and sonicated in PBS, and the resulting lysate was centrifuged. The protein concentration of the supernatant was quantified using the bicinchoninic acid assay (Beyotime Chemical) as described above. The levels of tumor necrosis factor α (TNF-α) and macrophage inflammatory protein 1α (MIP-1α) in the sampled mucosa were measured using enzyme-linked immunosorbent assay kits (Biosource, Camarillo, Calif) according to the manufacturer's instructions. Absorbance was measured at 405 nm, with the correction set at 650 nm. Cytokine levels were expressed in picograms per milligram of protein.
The data were expressed as the mean ± SD. The overall significance of the data was assessed by two-way analysis of variance using SPSS version 13.0 (SPSS Inc, Chicago, Ill). Differences between groups were considered statistically significant at P < 0.05 with appropriate Bonferroni corrections made for multiple comparisons.
Ten- and 15-min He pretreatment reduced the severity of I/R-induced intestinal injury
The intestines of rats subjected to I/R showed villous blunting and epithelial denudation, unlike in the nonischemic tissues of the sham controls (Fig. 2A). Intestinal injury was reduced by 10- and 15-min 7:3 heliox compared with the I/R group (P < 0.05), whereas neither 2- nor 5-min He conferred significant protection (Fig. 2, A and B).
Ten- and 15-min He pretreatment reduced intestinal permeability and MPO levels after I/R
In agreement with the findings of hematoxylin-eosin staining, intestinal permeability to intraluminally injected FITC-dextran was markedly increased after I/R, but significantly attenuated by pretreatment with 10- and 15-min 7:3 heliox (P < 0.05). Protection was not achieved by either 2- or 5-min He (Fig. 3A). Consistent with the permeability data, the intestinal MPO activities in 10- and 15-min heliox groups were significantly decreased compared with I/R group (P < 0.05), but the 2- and 5-min heliox groups failed to show the protection (Fig. 3B).
Ten- and 15-min He pretreatment reduced intestinal I/R-induced apoptosis
The Western blot shown in Fig. 4A is representative of three replicate experiments, which yield similar results. Caspase 3 levels were increased after intestinal I/R compared with sham animals. This increase was prevented by 10- and 15-min 7:3 heliox but not by the 2- and 5-min treatments. TUNEL staining was used to detect DNA degradation after I/R. As shown in Figure 4, B and C, pretreatment with 10- and 15-min 7:3 heliox significantly (P < 0.05) reduced the number of TUNEL-positive cells (Fig. 4B, dark arrows in TUNEL), which in the intestinal I/R control developed 60 min after I/R. Consistent with the previously described results, the number of TUNEL-positive cells in the 2- and 5-min heliox groups was not significantly different from that in the I/R group.
Ten- and 15-min He pretreatment reduced the numbers of CD68+ and MPO+ cells after intestinal I/R
The effect of HPC on CD68+ cells and MPO+ cells in the intestines was evaluated using immunohistochemistry. As shown in Figure 5, large numbers of CD68+ and MPO+ cells (Fig. 5, A and C, dark arrows in CD68+ and MPO+) were observed 60 min after intestinal I/R but not in the sham untreated animals. However, significant (P < 0.05) reductions in the numbers of the two types were detected in rats pretreated with 10 and 15 min of 7:3 heliox compared with the I/R-alone group. By contrast, 2 and 5 min of 7:3 heliox-induced decreases in the number of inflammatory cells did not reach significance (Fig. 5, B and D).
Ten- and 15-min He pretreatment reduced TNF-α and MIP-1α levels after intestinal I/R
The levels of TNF-α and MIP-1α in the intestinal mucosa were measured. In the 10- and 15-min heliox groups, the reductions in both proinflammatory compounds were significant compared with the I/R group (Fig. 6, A and B; P < 0.05). There were no such changes in rats treated with 2- and 5-min 7:3 heliox.
Helium is an inert element that belongs to the family of noble gases, which are characterized by their filled valence orbitals. Recent studies have shown that helium exerts cellular effects in vitro and in vivo and can reduce I/R damage in cardiac (6–12), neuronal (13–15), and liver tissues (16). These organs can be protected against I/R injury by subjecting them to three cycles of a 5-min exposure to 7:3 heliox combined with three cycles of 5 min of air. However, whether HPC can decrease intestinal I/R injury remains unclear. Therefore, in this study, we investigated the effect of HPC on intestinal I/R injury in rats. Our results showed that three cycles of 10- or 15-min 7:3 heliox breathing combined with a repeated shift to air breathing conferred protection against I/R injury in the small intestine, including a decrease in apoptosis and modulation of the inflammatory response. These effects were not observed after three cycles of 2- or 5-min heliox breathing.
In our study, the most frequently used HPC protocol described in the literature for other tissues and organs (three cycles of 7:3 heliox inhalation for 5 min followed each time by a 5-min washout) did not protect against intestinal I/R injury, and neither did shorter helium episodes (2 min). In a previous study, three cycles of 5-min 7:3 heliox breathing similarly failed to reduce I/R damage in the liver (20). However, in our study, HPC consisting of longer helium episodes (10 or 15 min) protected rat intestine against I/R injury. This phenomenon has not been mentioned and described before, and it was possibly explained by the following: (a) the animal model used in the present experiment (60-min ischemia and 60-min reperfusion) was serious, and it might cover the beneficial effect of HPC consisting of shorter helium episodes; (b) the helium episodes in HPC should increase to a level that can eventually induce protection in the intestine. Whether similar phenomenon can be found in other organs requires further investigation.
Inflammatory pathways play an important role in the pathogenesis of intestinal I/R injury. In our study, HPC reduced both TNF-α and MIP-1α levels and the number of CD68- and MPO-positive cells in intestinal tissues subjected to I/R injury; it also improved intestinal permeability. These results provide the evidence that HPC modulates the inflammatory response. Many other studies have described similar anti-inflammatory effects of helium (15–17, 20–23), even when the overall results were negative (20, 23). In a recent experiment, the inhibition of Kupffer cells did not attenuate HPC-induced liver protection in mice (16). Because the early phase of liver I/R injury is characterized by Kupffer cell activation, the effect of helium on the inflammatory response may involve a reduction in other cellular responses during early injury, at least in the liver. The mechanism underlying the anti-inflammatory properties of helium remains to be determined.
Several molecular pathways, such as those involving glycogen synthase kinase (9), nitric oxide (10), and reactive oxygen species (11), have been possibly implicated in the beneficial effects of HPC. Pagel et al. (12) and Zhang et al. (16) found that phosphatidylinositol-3-kinase (PI3K) inhibitors abolish HPC-induced cardiac and liver protection, suggesting that prosurvival signaling kinases mediate the positive effects of HPC. Thus, like the downstream effectors of the PI3K pathway, helium may regulate apoptosis (16). In agreement with previous studies, we found that the activation of caspase 3 is prevented by 10- and 15-min 7:3 heliox, as evidenced by a reduction in the number of TUNEL-positive cells after intestinal I/R. In addition, a lower concentration of helium (30%) was shown to induce cardiac protection comparable with that achieved in this study, albeit 24 h before I/R (6). Other researches show that preconditioning with neon (three cycles of 5-min 70% neon:30% oxygen breathing combined with three cycles of 5-min air breathing) failed to show the protection, whereas HPC protected liver I/R injury (16). These results seem to indicate that the increased concentration of oxygen used in our study (30%) did not mediate the observed antiapoptotic or anti-inflammatory effects or the intestinal protection.
In summary, preconditioning with HPC profile consists of three cycles of 10- or 15-min helium breathing and 5-min air breathing reduced the severity of I/R-induced intestinal injury, apoptosis, and inflammation in rats. These beneficial effects were not observed with shorter helium episodes. Thus, for some types of surgery, HPC profile consisting of longer helium episodes should be considered rather than using the standard 5-min helium cycles in all cases. Such a flexible HPC profile may be useful in clinical practice.
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