Protective Effect of Oxygen and Isoflurane in Rodent Model of Intestinal Ischemia-Reperfusion Injury

Animal research in intestinal ischemia-reperfusion injury (IRI) is mainly performed in rodent models. Previously, intraperitoneal (I.P.) injections with ketamine–xylazine mixtures were used. Nowadays, volatile anesthetics (isoflurane) are more common. However, the impact of the anesthetic method on intestinal IRI has not been investigated. We aim to analyze the different anesthetic methods and their influence on the extent of intestinal IRI in a rat model. Male Sprague–Dawley rats were used to investigate the effect of I.P. anesthesia on 60 min of intestinal ischemia and 60 min of reperfusion in comparison to hyperoxygenation (100% O2) and volatile isoflurane anesthesia. In comparison to I.P. anesthesia with room air (21% O2), supplying 100% O2 improved 7-day survival by cardiovascular stabilization, reducing lactic acidosis and preventing vascular leakage. However, this had no effect on the intestinal epithelial damage, permeability, and inflammatory response observed after intestinal IRI. In contrast to I.P. + 100% O2, isoflurane anesthesia reduced intestinal IRI by preventing ongoing low-flow reperfusion hypotension, limiting intestinal epithelial damage and permeability, and by having anti-inflammatory effects. When translating the aforementioned results of this study to clinical situations, such as intestinal ischemia or transplantation, the potential protective effects of hyperoxygenation and volatile anesthetics require further research.


Introduction
Intestinal ischemia-reperfusion injury (IRI) is a frequent clinical entity, typically observed in patients suffering from intestinal ischemia but also as an inevitable part of intestinal transplantation (ITx) [1,2].
Ischemia causes the depletion of adenosine triphosphate (ATP) and the accumulation of radical oxygen species (ROS) precursors [3]. When blood flow is restored, additional mucosal injury occurs because reperfusion exacerbates the ischemic insult through an

Figure 2.
The impact of oxygen supplementation on intestinal epithelial injury was scored accor to the Park-Chiu score (A) and villus height (B). Intestinal epithelial permeability was measure TEER in an Ussing chamber setup, which was corrected for the villus height (C). (n = 6/group) intraperitoneal injection anesthesia; O2: oxygen; TEER: transepithelial electrical resistance. * 0.01; *** p < 0.001.

Oxygen Supplementation and Isoflurane Improved Survival following IRI
When 7-day survival was assessed in the rodent model of 60 min of intestinal ischemia, no survival was seen with I.P. anesthesia with 21% oxygen (air) in contrast to 70% survival with oxygen supplementation and even 90% when isoflurane was used instead (p < 0.0001) (Figure 9).

Oxygen Supplementation and Isoflurane Improved Survival Following IRI
When 7-day survival was assessed in the rodent model of 60 min of intestinal ischemia, no survival was seen with I.P. anesthesia with 21% oxygen (air) in contrast to 70% survival with oxygen supplementation and even 90% when isoflurane was used instead (p < 0.0001) (Figure 9).

Discussion
In this experimental rodent study on intestinal IRI, we studied the effect of oxygen supplementation and isoflurane anesthesia (volatile) compared to I.P. ketamine-xylazine anesthesia.
Oxygen supplementation clearly ameliorated the detrimental effects of intestinal IRI with ketamine-xylazine anesthesia, with significantly improved 7-day survival. The protective effects of hyperoxia (100% O2) in intestinal IRI experiments have been shown before. The main effects were attributed to decreased enterocyte apoptosis and decreased neutrophil recruitment by the downregulation of E-selectin production [20,21]. In this study, we show survival benefits after intestinal IRI, which were mainly mediated by improved vascular permeability, as the endothelial glycocalyx (eGC) was better preserved. Endothelial glycocalyx destruction is now considered a cornerstone for the detrimental effects attributed to IRI [9,24]. As fewer constituents of the eGC were detected in the plasma, a better preservation of the eGC can be assumed. This was confirmed by the fact that the vascular permeability/leakage was improved, as shown by reduced hemoglobin concentration, decreased reperfusion edema, and decreased endotoxin translocation. These measurements were not explained by an altered intestinal epithelial permeability (TEER) nor by histopathological alterations. Hemodynamically, oxygen supplementation resulted in a less depressed heart rate, which presumably led to a better cardiac output throughout the whole experiment. This could explain the less pronounced lactic acidosis, which is normally seen after intestinal IRI. However, it could not counteract the hypotensive episode observed at the moment of reperfusion, which could lead to an ongoing, lowflow ischemic episode in this experimental setting. There was no protective effect of oxygen supplementation on inflammation, which might be mediated through increased ROS formation [25]. However, in IRI studies, hyperoxygenation has been interpreted with caution due to the paradoxical idea of increasing ROS formation. On the other hand, in recent IRI studies, it has been shown that hyperoxygenation might actually protect against IRI by a net favorable effect on plasma oxidative status and hence reduce ROS formation. This effect seems to be mediated by the activation of pro-inflammatory cascades by hyperoxia, which includes interference with neutrophils adhesion and free radical production [20,21].
Volatile anesthetics with isoflurane provided an additional survival benefit over injection anesthesia with ketamine-xylazine and hyperoxia. The protective effects of isoflurane on intestinal IRI have only been shown in a treatment setting so far, where mainly protective effects on epithelial injury were seen [26]. However, isoflurane is known as a potent anti-inflammatory agent, such as in renal IRI [27]. This study confirms the potent anti-inflammatory properties of isoflurane as both systemic and local inflammatory cyto-

Discussion
In this experimental rodent study on intestinal IRI, we studied the effect of oxygen supplementation and isoflurane anesthesia (volatile) compared to I.P. ketamine-xylazine anesthesia.
Oxygen supplementation clearly ameliorated the detrimental effects of intestinal IRI with ketamine-xylazine anesthesia, with significantly improved 7-day survival. The protective effects of hyperoxia (100% O 2 ) in intestinal IRI experiments have been shown before. The main effects were attributed to decreased enterocyte apoptosis and decreased neutrophil recruitment by the downregulation of E-selectin production [20,21]. In this study, we show survival benefits after intestinal IRI, which were mainly mediated by improved vascular permeability, as the endothelial glycocalyx (eGC) was better preserved. Endothelial glycocalyx destruction is now considered a cornerstone for the detrimental effects attributed to IRI [9,24]. As fewer constituents of the eGC were detected in the plasma, a better preservation of the eGC can be assumed. This was confirmed by the fact that the vascular permeability/leakage was improved, as shown by reduced hemoglobin concentration, decreased reperfusion edema, and decreased endotoxin translocation. These measurements were not explained by an altered intestinal epithelial permeability (TEER) nor by histopathological alterations. Hemodynamically, oxygen supplementation resulted in a less depressed heart rate, which presumably led to a better cardiac output throughout the whole experiment. This could explain the less pronounced lactic acidosis, which is normally seen after intestinal IRI. However, it could not counteract the hypotensive episode observed at the moment of reperfusion, which could lead to an ongoing, lowflow ischemic episode in this experimental setting. There was no protective effect of oxygen supplementation on inflammation, which might be mediated through increased ROS formation [25]. However, in IRI studies, hyperoxygenation has been interpreted with caution due to the paradoxical idea of increasing ROS formation. On the other hand, in recent IRI studies, it has been shown that hyperoxygenation might actually protect against IRI by a net favorable effect on plasma oxidative status and hence reduce ROS formation. This effect seems to be mediated by the activation of pro-inflammatory cascades by hyperoxia, which includes interference with neutrophils adhesion and free radical production [20,21].
Volatile anesthetics with isoflurane provided an additional survival benefit over injection anesthesia with ketamine-xylazine and hyperoxia. The protective effects of isoflurane on intestinal IRI have only been shown in a treatment setting so far, where mainly protective effects on epithelial injury were seen [26]. However, isoflurane is known as a potent anti-inflammatory agent, such as in renal IRI [27]. This study confirms the potent antiinflammatory properties of isoflurane as both systemic and local inflammatory cytokines were significantly reduced. This anti-inflammatory effect has been described to be mediated by the activation of the peroxisome-proliferator-activated receptor gamma/nuclear factor-kappa B pathway (PPARγ/NF-κB). PPARγ has been reported to ameliorate LPSinduced inflammation through the TLR4 signaling pathway [28]. Such anti-inflammatory response to intestinal IRI has also been seen with opioids (remifentanil), as shown by the study of Cho et al. [29]. Secondly, we confirmed the protective effect of isoflurane on intestinal epithelial damage, as was previously shown in a treatment setting [26]. The destruction of the intestinal epithelial cells seems to be prevented by increased transforming growth factor-beta1 (TGF-β1) production induced by isoflurane exposure [26]. This epithelial protective effect has also been seen with vitamins (folic acid, alphatocopherol), anti-apoptotic drugs (ruboxustaurin and caveolin-1), opioids (remifentanil), and stem cell therapy [11,12,[29][30][31]. In our study, the improved preservation of the intestinal epithelial lining was also confirmed by reduced intestinal epithelial permeability (TEER). As to the vascular permeability, no difference was seen. Hemodynamically, there was the known vasodilatory effect of isoflurane, immediately from the start of the experiment. However, with volatile anesthesia, there was no hypotensive episode at the moment of reperfusion. As such, there was no persisting low-flow ischemia at the moment of reperfusion in the volatile anesthesia group, in contrast to I.P. injection with ketamine-xylazine. Volatile anesthesia, such as isoflurane, has also been shown to reduce ROS formation in intestinal IRI by reducing malondialdehyde (MDA) production [28]. ROS is also known to modulate the JAK/STAT pathway, and protective effects of isoflurane through activating this pathway have been shown in cardiac IRI [32,33].
The study inherently has limitations. First, although animals received I.P. fluid resuscitation, they were not mechanically ventilated and did not receive intravenous fluid resuscitation and vasopressive support to treat hypotension. The animals not receiving oxygen supplementation had mild to moderate hypoxemia. Hence, it is not clear whether the protective effects could be achieved by preventing hypoxemia rather than by achieving hyperoxemia. Likewise, it is not clear if more aggressive treatment of postreperfusion hypotension could have prevented the observed harm. Second, only isoflurane and oxygen administration were studied and no other common anesthetics. For example, pentobarbital has been a common anesthetic in rodent experiments as well. In a study by Kawai et al., it was shown that pentobarbital reaches a lower depth of anesthesia in comparison to ketamine-xylazine. On the other hand, ketamine/xylazine appears to work faster and lasts longer than pentobarbital [34]. More common clinical anesthetics, such as a mixture with nitrous oxide, which might have beneficial effects, are not tested in this study. Nitrous oxide could help in hemodynamic stabilization and has analgesic effects. In contrast, the use of nitrous oxide can have several adverse effects, such as pneumonia, atelectasis, increased skin infection, and sepsis [35,36]. Third, we did not study the impact of isoflurane vs. ketamine-xylazine in the absence of oxygen supplementation. Hence, we do not know whether the observed protection by isoflurane is dependent on oxygen supplementation. However, the study by Wilding et al. showed that using either 21% or 100% of oxygen as a carrier for isoflurane anesthesia revealed no major differences in main physiologic parameters. With 100% oxygen, hypercapnia leading to relative hypertension, decreased respiratory rates, and more pronounced respiratory acidosis was more commonly seen in comparison to 21% [16]. Further, structural changes were seen in the endothelial glycocalyx, in combination with secondary, indirect effects. However, these structural changes do not warrant mechanistic or pathophysiologic advantages per se. Lastly, the current findings are limited to male rats, as in these intestinal IRIs is more pronounced than in their female counterparts [37,38]. This study has unmasked the substantial influence of the anesthetic method in a rodent model of intestinal IRI. Volatile anesthetics resulted in fewer hemodynamical changes compared to ketamine-xylazine anesthesia. Future implications of this can lead to a better choice of anesthetic strategy in more complex rodent experimental models, such as transplant models, in which preserved hemodynamics is crucial for the outcome. When translating the aforementioned results of this study to clinical situations, such as intestinal ischemia, hyperoxygenation might lead to a significant survival benefit and volatile anesthetics might give an additional survival benefit. IRI is inherent to transplant surgery, and according to the results of this study, intestinal transplant recipients might benefit from hyperoxygenation and volatile anesthetics. This beneficial effect of volatile anesthetics over injection anesthetics has already been shown in liver and kidney transplant recipients but warrants further research [22,23].

Animal Model
Male Sprague-Dawley rats (Janvier Labs, Saint Berthevin Cedex, France), weighing 275-350 g, 6 weeks old, were housed in the KU Leuven animal facility under specific pathogen-free conditions, with 2-3 rats per cage. The rats were acclimatized for 5-7 days before any intervention. The animals were kept in 14/10 h light/dark cycles, controlled temperature, and they received rat chow and water ad libitum. The animals were not fasted before surgery. Institutional animal research ethical committee approval-following the EU directive for animal experiments-was granted under the number P122/2019. The reported animal study is in compliance with the ARRIVE guidelines 2.0 [39].

Surgery
IRI was performed with 60 min of ischemia and 60 min of reperfusion. Intestinal IRI was induced, after median laparotomy of 4 cm on the linea alba, by isolated atraumatic clamping of the superior mesenteric artery. Ischemia was checked by pulselessness in the mesentery and discoloration/dysmotility of the bowel. The laparotomy wound was temporarily closed during the experiment. At reperfusion, 1 mL of warmed saline (37 • C) was administered intraperitoneally to compensate for fluid loss by evaporation. Reperfusion was checked by recovery of arterial pulsations in the mesentery, recoloration, and regain of motility. At the end of the experiment, all animals were sacrificed by exsanguination, followed by blood and intestinal tissue collection. The animals were under anesthesia until exsanguination. All experiments were performed by the same researcher (M.C.).
Rats were randomly divided into five groups (n = 6/group) (Table 1, Figure 10): In accordance with animal welfare, rats were monitored at least 3 times daily, a buprenorphine subcutaneous (0.016 mg/kg BW, 0.3 mg/mL, Vetergesic, Ceva, Belgiu was used for analgesia, once preoperatively and twice daily postoperatively, during t first 3 days. At the end of the experiment, rats were anesthetized with pentobarbital before sac fice (65 mg/kg BW, 200 mg/mL, Dolethal, Vetoquinol, Belgium).

Vital Signs
Vital parameters were measured, every 15 min, from 5 min before onset of ischem until awakening/sacrifice/death. Rectal temperature was measured by usage of a clini thermometer (SC19 flex rapid, SCALA, Frankfurt, Germany). Heart rate and saturati were measured on hind paws by usage of OxiPen (EnviteC, Germany). Tail cuff blo pressure measurements were performed by usage of the Coda non-invasive blood pr sure system (Kent Scientific, Torrington, CT, USA). Ten blood pressure measuremen were taken at each time point, and the median was used for analysis.

Blood and Tissue Sampling
Heparinized blood samples were collected after puncture of the aorta for blood g analysis (0.4 mL) in 2 EDTA tubes. The tubes were spun at 3500 rpm for 10 min at 4 ° Plasma was snap frozen in liquid nitrogen and stored at −80 °C. One ileal tissue sample 5 cm was taken just proximally of the ileocaecal valve, kept in glucose buffer on ice, a mounted in the Ussing chambers. Ileal samples were collected proximally of the previo and preserved in 4% buffered formalin for histological evaluation and snap frozen, af feces removal, for molecular analysis.

Histological Evaluation
Full-thickness samples were formalin-fixed, paraffin-embedded, cut into 5 µ coupes, and stained with hematoxylin-eosin. The ischemic injury was scored in a blind fashion by an experienced pathologist (G.D.H.) on four fields per section by usage of t Park-Chiu score [40,41]. For survival analysis, 10 additional animals were included in each IRI group and observed daily for 7 days. The laparotomy wound was closed subcutaneously in 2 layers with Prolène 4-0 (Ethicon, Belgium), and 0.5 mL of ropivacaine (3.16 mg/kg BW, 2 mg/mL, Naropin, Aspen, Ireland) was administered in the wound edges for local analgesia.
In accordance with animal welfare, rats were monitored at least 3 times daily, and buprenorphine subcutaneous (0.016 mg/kg BW, 0.3 mg/mL, Vetergesic, Ceva, Belgium) was used for analgesia, once preoperatively and twice daily postoperatively, during the first 3 days.

Vital Signs
Vital parameters were measured, every 15 min, from 5 min before onset of ischemia until awakening/sacrifice/death. Rectal temperature was measured by usage of a clinical thermometer (SC19 flex rapid, SCALA, Frankfurt, Germany). Heart rate and saturation were measured on hind paws by usage of OxiPen (EnviteC, Germany). Tail cuff blood pressure measurements were performed by usage of the Coda non-invasive blood pressure system (Kent Scientific, Torrington, CT, USA). Ten blood pressure measurements were taken at each time point, and the median was used for analysis.

Blood and Tissue Sampling
Heparinized blood samples were collected after puncture of the aorta for blood gas analysis (0.4 mL) in 2 EDTA tubes. The tubes were spun at 3500 rpm for 10 min at 4 • C. Plasma was snap frozen in liquid nitrogen and stored at −80 • C. One ileal tissue sample of 5 cm was taken just proximally of the ileocaecal valve, kept in glucose buffer on ice, and mounted in the Ussing chambers. Ileal samples were collected proximally of the previous and preserved in 4% buffered formalin for histological evaluation and snap frozen, after feces removal, for molecular analysis.

Histological Evaluation
Full-thickness samples were formalin-fixed, paraffin-embedded, cut into 5 µm coupes, and stained with hematoxylin-eosin. The ischemic injury was scored in a blinded fashion by an experienced pathologist (G.D.H.) on four fields per section by usage of the Park-Chiu score [40,41].
Villus length-defined as the distance between the mouth of the crypts and the tip of the villi-was measured in 4 different fields per tissue section, and the average was calculated to avoid the potential impact of patchy necrosis.

Ussing Chamber Experiments Electrophysiological Parameters
Full-thickness ileal tissue (mucosa, submucosa, muscular layer, and serosa) was mounted, in triplicate, in a standard vertical Ussing chamber (Mussler Scientific Instruments, Aachen, Germany) with an opening of 9.60 mm 2 by a blinded, experienced researcher (A.A.). Each half chamber was filled with 3 mL Krebs solution with 10 mM mannitol at the mucosal side and 10 mM glucose at the serosal side. Both buffers were maintained at 37 • C and continuously oxygenated with 95%/5% O 2 /CO 2 and stirred by gas flow in the chambers. In this setup, data sampling and pulse inductions are computercontrolled using Clamp software (Version 9.00, Mussler Scientific Instruments, Aachen, Germany). Transepithelial electrical resistance (TEER) was measured by averaging 90 min of measurement after initial 30 min of stabilization. All tissue was mounted within 10 min after the exsanguination of the animal.
In our particular setting of intestinal IRI, leading to a diminished/denudated mucosal surface area (as shown by Grootjans et al. [42]), TEER was corrected by multiplying TEER with its corresponding villus length divided by the average villus length of the sham group [13].

Endotoxin Levels
Quantification of plasma endotoxin levels was obtained by the colorimetric limulus amebocyte lysate test (LAL QCL-1000 TM , Lonza, Belgium), according to the manufacturer's instructions. Absorbance was measured spectrophotometrically by FLUOstar Omega (BMG Labtech, Offenburg, Germany) at 410 nm. Corrections were made by the subtraction of the absorbance of the sample without the addition of LAL.

Edema
Tissue water content (edema) was assessed by the ratio between the weight before and after drying. Snap-frozen, whole-thickness ileal tissue samples were weighed just before and immediately after drying them for 3 h at 80 • C in a drying oven with forced convection (VENTI-Line VL 115, VWR, Belgium). The results were expressed as a wet/dry ratio and a percentage of water in the tissues ((1-(Dry weight/Wet weight))*100).
Plasma heparan sulfate (OKEH02552, Aviva Systems Biology, San Diego, CA, USA) concentration was measured by ELISA according to the manufacturer's instructions.

Statistical Analysis
All data were expressed as mean ± standard deviation and represented in scattered plots. The line in the middle is plotted at the mean. The whiskers indicate the standard deviation. Data were checked for outliers by the ROUT method with Q = 1% and subjected to (log)normality testing (Shapiro-Wilk test). Comparisons between multiple groups were performed with one-way ANOVA and post-hoc Tukey's test in the case of normal distribution or the Kruskal-Wallis and post-hoc Dunn tests for non-normal distribution. Survival analysis was performed by the Kaplan-Meier test (log-rank test). A p-value < 0.05 was considered statistically significant (GraphPad Prism version 9.4.0 for Windows, GraphPad Software, San Diego, CA, USA).

Conclusions
In experimental rodent models, different types of anesthesia are commonly used. In this study on intestinal IRI, it was shown that oxygen supplementation had a protective effect on the vascular permeability of the intestine. Isoflurane anesthesia attenuated the detrimental effects of intestinal IRI even more by reducing intestinal epithelial permeability and the inflammatory cascade compared to ketamine-xylazine anesthesia. The type of anesthesia used in these experimental models can influence the outcome parameters analyzed and should be taken into account. The potential clinical implications should be investigated in ischemic and transplant patients.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approval by the KU Leuven animal research ethical committee-following the EU directive for animal experiments-was granted under the number P122/2019. The reported animal study is in compliance with the ARRIVE guidelines 2.0 [39].
Data Availability Statement: All data are available upon request from the corresponding author.