Intestinal Reperfusion Research Articles

MicroRNA-682-mediated downregulation of PTEN in intestinal epithelial cells ameliorates intestinal ischemia-reperfusion injury.

Intestinal ischemia–reperfusion (I/R) injury causes inflammation and tissue damage and contributes to high morbidity and mortality, but the underlying mechanism remains elusive and effective therapies are still lacking. We report here a critical role of the microRNA 682 (miR-682) as a key regulator and therapeutic target in intestinal I/R injury. MiR-682 was markedly induced in intestinal epithelial cells (IECs) during intestinal ischemia in mice and in the human colonic epithelial cells during hypoxia, but was undetected rapidly after intestinal reperfusion in IEC of mice. MiR-682 induction during hypoxia was modulated by hypoxia-inducible factor-1α (HIF-1α). On lentivirus-mediated miR-682 overexpression in vivo during intestinal reperfusion or miR-682 mimic transfection in vitro during hypoxia, miR-682 decreased the expression of phosphatase and tensin homolog (PTEN) and subsequently activated nuclear translocation of nuclear factor kappa B (NF-κB) p65. Consequently, NF-κB activation by miR-682-mediated PTEN downregulation prevented reactive oxygen species (ROS) induction, inflammatory reaction, mitochondrial-mediated apoptosis and IEC apoptosis. The effect of miR-682-mediated PTEN/NF-κB pathway on IECs resulted in protection against intestinal I/R injury in mice. However, NF-κB chemical inhibitor reversed miR-682-mediated decreased PTEN expression, ROS induction, inflammation and IEC apoptosis. Collectively, these results identify a novel miR-682/PTEN/NF-κBp65 signaling pathway in IEC injury induced by I/R that could be targeted for therapy.

Synergistic effects of glutamine, citrulline, and antioxidant vitamin C and E on modulating splenocytic immune responses in rats with intestinal ischemia and reperfusion

Intestinal ischemia and reperfusion (IIR) is a common event in patients with acute mesenteric ischemia, intestinal obstruction, organ transplantation, and hemorrhagic and septic shock. It has been demonstrated that IIR‐induced intestinal mucosa damage may cause systemic inflammation and multiple organ failure and further lead to death. Our previous study showed that oral glutamine/citrulline and antioxidant vitamins C/E have additive effects on attenuating intestinal damage and systemic inflammation in IIR rats. Herein, we investigated the effects of these supplements on IIR‐induced immune alterations. Male Wistar rats were divided into normal rats and IIR rats that underwent intestinal ischemia for 60 min and fed with placebo, glutamine/citrulline (AA), vitamins C/E (CE), or glutamine/citrulline plus vitamins C/E (AV) at 15 minutes before and 3, 9 and 21 hr after reperfusion. After 24 hr reperfusion, IIR‐increased plasma nitrate/nitrite was significantly reversed in rats with AA and CE and IIR‐increased interleukin‐6 was reversed in rats with AA, CE, and AV. The results of two‐way ANOVA showed that CE was a main factor to reverse IIR‐altered T‐helper leukocytes, B‐leukocytes, and monocytes and to increase tumor necrosis‐factor (TNF)‐alpha production in lipopolysaccharide‐stimulated leukocytes; and AA and CE were main factors to increase granulocytes in the circulation. In concanavalin A‐ and lipopolysaccharide‐stimulated splenocytes, AA was a main factor to increase TNF‐alpha and interleukin‐4 productions, CE was a main factor to increase interleukin‐4 productions, and AA and CE have significant interactions in TNF‐alpha and IL‐4 productions as shown in the significant increases in IIR rats with AV, not with AA or CE alone. In conclusion, oral glutamine/citrulline and vitamins C/E may have synergistic effects on modulating splenocytic immune responses in intestinal ischemia and reperfusion injury. The alleviated intestinal damage and systemic inflammation and the elevated splenocytic activity reveal that oral, combined administration of glutamine, citrulline, and antioxidant vitamins C and E may have beneficial effects in patients with intestinal ischemia and reperfusion.Support or Funding InformationNSC 98‐2320‐B‐030‐003‐MY3

Sa1431 Intestinal Hypoxia and Reperfusion: Role for Eph-ephrin System

Sa1431 Intestinal Hypoxia and Reperfusion: Role for Eph-ephrin System

Acute and Chronic Pretreatment With Atenolol Attenuates Intestinal Ischemia and Reperfusion Injury in Hypercholesterolemic Rats.

To evaluate the protective effects of preinjury atenolol (acute v chronic) on apoptosis, contractility, oxidative stress, and inflammatory markers in hypercholesterolemic rats undergoing intestinal ischemia-reperfusion (I/R) injury. Prospective, experimental animal study. University laboratory. Male Wistar rats (n = 32). Rats were divided into the following 4 groups: 1 group was fed a normal diet (ND) (group ND+NoAT [no atenolol]), and the other 3 groups were fed a high-cholesterol diet (HCD)-group HCD+NoAT, group HCD+ChAT (chronic atenolol, 3 mg/kg/day for 8 weeks), and group HCD+AcAT (acute atenolol, 1.5 mg/kg, given 5 minutes before intestinal clamping). All rats underwent I/R injury. The superior mesenteric artery was clamped for 60 minutes, then opened for 120 minutes (reperfusion). Apoptotic cells and stimulated contractions of ileal segments were examined. Tissue markers of intestinal I/R injury were examined. Intestinal malondialdehyde, superoxide dismutase, and nitrate/nitrite levels were measured. The chronic atenolol group had fewer apoptotic cells and higher superoxide dismutase activity compared with the other groups. Intestinal contraction was higher in both atenolol pretreatment groups compared with the NoAT groups. Chronic and acute atenolol resulted in lower ileal levels of malondialdehyde and immunolabeling-positive cells (intestinal inducible nitric oxide synthase, endothelial nitric oxide synthase, interleukin-1, and interleukin-8) after I/R injury compared with the no atenolol groups. Both chronic and acute pre-I/R injury treatment with atenolol attenuated I/R injury in this hypercholesterolemic rat model. These findings should encourage future studies of atenolol in hypercholesterolemic patients undergoing procedures with a high risk of intestinal ischemia.

Calcitonin Gene-Related Peptide Downregulates Expression of Inducible Nitride Oxide Synthase and Caspase-3 after Intestinal Ischemia-Reperfusion Injury in Rats

Various investigations have demonstrated that calcitonin gene-related peptide (CGRP) plays an important role in mediating ischemic preconditioning. CGRP has been shown to mimic the protective effects of ischemic preconditioning and mitigate ischemia-reperfusion (I/R) injury in the heart, brain, gastrointestinal system, and other tissues. This study aimed to examine whether CGRP, a proven intestinal cytoprotective molecule, exerted its protective effects through modulation of inducible nitride oxide synthase (iNOS) and apoptosis after intestinal I/R injury. This animal study randomly divided 30 rats into the following five groups: (1) the normal control group, (2) the ischemia group with normal saline, (3) the I/R group with normal saline, (4) the ischemia group with CGRP (300μg/kg), and (5) the I/R group with CGRP (300μg/kg). Levels of iNOS messenger RNA (mRNA) and protein, and caspase-3 protein were determined by real-time quantitative polymerase chain reaction and Western blotting analyses, respectively. Statistical analysis was performed using analysis of variance with Dunn test. The mRNA levels of iNOS increased after the intestinal ischemia or intestinal reperfusion phase (p<0.01), and CGRP pretreatment significantly decreased iNOS mRNAs and protein levels (p<0.01). The expression protein levels of caspase-3 increased after the intestinal ischemia or intestinal reperfusion phase. CGRP pretreatment significantly decreased the levels of caspase-3 proteins. CGRP intestinal cytoprotection is mediated, in part, by downregulation of expression of iNOS and caspase-3 after intestinal I/R injury. The study indicates that the cytoprotective role of CGRP (i.e., antiapoptotic effect) after I/R injury could be via downregulation of iNOS, which may relieve I/R tissue damage by blocking iNOS activity.

Human Adipose Stromal Cells Increase Survival and Mesenteric Perfusion Following Intestinal Ischemia and Reperfusion Injury.

Intestinal ischemia can quickly escalate to bowel necrosis and perforation. Transplantation of stem cells presents a novel treatment modality for this problem. We hypothesized that: human adipose-derived stromal cells (hASCs) would increase survival and mesenteric perfusion to a greater degree compared with differentiated cellular controls following ischemic intestinal injury, and improved outcomes with hASC therapy would be associated with preservation of intestinal histological and tight junction architecture, and lower levels of systemic inflammation following intestinal injury. hASCs and keratinocytes (differentiated cellular control) were cultured on polystyrene flasks at 37°C in 5% CO2 in air. Adult male C57Bl6J mice were anesthetized and a midline laparotomy performed. The intestines were eviscerated, the small bowel mesenteric root identified, and intestinal ischemia was established by temporarily occluding the superior mesenteric artery for 60 min with a noncrushing vascular clamp. Following ischemia, the clamp was removed, and the intestines were returned to the abdominal cavity. Before abdominal closure, 2 million hASCs or keratinocytes in 250 μL of phosphate-buffered saline (carrier for cells and control solution) were infused into the peritoneum. Animals were allowed to recover for 12 or 24 h (perfusion, histology, cytokine, and immunofluoresence studies), or 7 days (survival studies). Intestinal perfusion was assessed by laser Doppler imaging. Intestinal tissue segments were stained with hematoxylin and eosin, as well as antibodies for the tight junction protein claudin-1. Separate aliquots of intestine, liver, and lung tissue were homogenized and assessed for inflammatory cytokines via multiplex beaded assay. Animals administered hASCs following intestinal ischemia and reperfusion (I/R) injury had significantly greater 7-day survival and better postischemic recovery of mesenteric perfusion compared with vehicle or keratinocyte therapy. hASCs also abated intestinal mucosal destruction, facilitated preservation of intestinal tight junctions, and decreased the systemic inflammatory response to injury. Human adipose-derived stromal cells improved survival and mesenteric perfusion and attenuated the mucosal damage associated with intestinal I/R injury. hASCs should be considered as a plausible cell source for novel cellular treatment plans following intestinal ischemia.

Reduction of Acute Lung Injury by Administration of Spironolactone After Intestinal Ischemia and Reperfusion in Rats.

Multiple organ failure, including acute lung injury, is a common complication of intestinal ischemia and reperfusion (I/R) injury and contributes to its high mortality rate. Activated polymorphonuclear neutrophils and reactive oxygen species contribute to the lung injury caused by intestinal I/R. Mineralocorticoid receptor antagonist spironolactone has a protective effect against I/R injury in animal models of retina, kidney, heart, and brain. The aim of the present study is to investigate the effect of aldosterone receptor blocker spironolactone on lung injury induced by intestinal I/R. Wistar albino rats were divided into four groups: (1) sham control; (2) intestinal I/R (30 min of ischemia by superior mesenteric artery occlusion followed by 3 h of reperfusion); (3) spironolactone pretreatment (20 mg/kg) + I/R; and, (4) spironolactone pretreatment without I/R. Spironolactone was given orally 3 days prior to intestinal I/R. A marker for lipid peroxidation (malondialdehyde; MDA), an indicator or oxidation state (reduced glutathione; GSH), an index of polymorphonuclear neutrophil sequestration (myeloperoxidase; MPO), inducible nitric oxide synthase (iNOS) immunoreactivity, and the histopathology of the lung tissue were analyzed. Spironolactone pretreatment markedly reduced intestinal I/R-induced lung injury as indicated by histology and MDA and MPO levels. Moreover, the pretreatment decreased the iNOS immunoreactivity. The present study strongly suggests that spironolactone pretreatment decreased neutrophil infiltration, iNOS induction, oxidative stress, and histopathological injury in an experimental model of intestinal I/R induced-lung injury of rats.

Ischemic preconditioning modifies mortality and inflammatory response.

To evaluate the effect of ischemic preconditioning on mortality, inflammatory mediators and oxidative stress after intestinal ischemia and reperfusion. Male Wistar rats were allocated according to the period of ischemia with or without ischemic preconditioning which consist on clamping the superior mesenteric artery for 10 minutes followed by reperfusion for 10 minutes before the sustained ischemia period. Mortality was assessed in Phase 1 study, and the CINC-1, CINC-2 and MDA levels in the lungs were analyzed in Phase 2. Mortality was lower in the ischemic preconditioning group subjected to 90 minutes of ischemia compared to the group without ischemic preconditioning (I-90: 50% and IPC-90: 15%, p=0.018), and it was lower in the ischemic preconditioning group as a whole compared to the groups without ischemic preconditioning (IPC-14% and I=30%, p=0.006). Lower levels of MDA, CINC-1, and CINC-2 were observed in the animals that were subjected to ischemic preconditioning compared to the animals that were not (MDA: I-45=1.23 nmol/mg protein, and IPC-45=0.62 nmol/mg protein, p=0.0333; CINC-1: I-45=0.82 ng/mL and IPC-45=0.67 ng/mL, p=0.041; CINC-2: I-45=0.52 ng/mL and IPC-45=0.35 ng/mL, p=0.032). Ischemic preconditioning reduces mortality, inflammatory process and oxidative stress in rats subjected to intestinal ischemia and reperfusion.

Pretreatment Combination Reduces Remote Organ Damage Secondary to Intestinal Reperfusion Injury in Mice: Follow-up Study

BackgroundIntestinal ischemia-reperfusion injury occurs after different surgical treatments, including intestinal transplantation. This harmful process may have an effect in remote organs, leading to multiple organ dysfunction syndrome and death. Therefore, to establish strategies to attenuate local and remote damage constitutes a challenge for experimental and clinical surgeons in the intestinal surgical field. MethodsWe evaluated the effect of ischemic preconditioning and tacrolimus pretreatment applied alone and in combination against local and remote damage caused by prolonged intestinal ischemia-reperfusion injury in a mouse model of warm ischemia. ResultsIschemic preconditioning applied alone and in combination with tacrolimus decreased histological damage (P < .05), number of apoptotic cells (P < .05), nitrosative stress (P < .01), and serum lactate dehydrogenase activity (P < .05) and lowered uremia (P < .05) compared with untreated post-reperfused intestines. Regarding remote organ damage, combination therapy was the unique condition able to attenuate lung (mainly neutrophil infiltration and hemorrhage), liver (sinusoidal congestion and hepatic vacuolization), and kidney (acute tubular necrosis and hydropic degeneration) histological alterations (P < .05), compared with the untreated group. ConclusionsThese results support the application of these strategies in combination to minimize the impact of ischemia-reperfusion injury in the whole organism as a strategy to prevent multiple organ dysfunction syndromes and minimize the clinical impact.

Oral Citrulline Mitigates Inflammation and Jejunal Damage via the Inactivation of Neuronal Nitric Oxide Synthase and Nuclear Factor–κB in Intestinal Ischemia and Reperfusion

Intestinal ischemia and reperfusion (I/R) is a life-threatening emergency accompanied by inflammation and organ damage. We compared the mechanisms and the effects of arginine, citrulline, and glutamine on inflammation and intestinal damage. Male Wistar rats underwent 60 minutes of superior mesenteric artery occlusion and either 3 (I/R3) or 24 (I/R24) hours of reperfusion and were orally administered vehicle, arginine, citrulline, or glutamine 15 minutes before reperfusion and at 3, 9, and 21 hours of reperfusion. I/R3 rats experienced jejunal damage and apoptosis, and I/R24 rats had liver dysfunction compared with normal rats (one-way ANOVA, P < .05). Arginine and citrulline administrations improved jejunal morphology, and citrulline and glutamine administrations alleviated the loss of jejunal mass in I/R3 rats. I/R3-increased circulating nitrate/nitrite (NOx), tumor necrosis factor-α, and interleukin-6 were significantly decreased by citrulline, glutamine and citrulline, and arginine, glutamine, and citrulline, respectively. These amino acids decreased plasma NOx and interferon-γ in I/R24, decreased jejunal neuronal nitric oxide synthase (NOS) protein in I/R3 rats, and alleviated jejunal apoptosis in I/R3 and I/R24 rats. In addition, the jejunal phosphorylated to total nuclear factor-κB (NF-κB) ratio was decreased by arginine and citrulline in I/R24 rats. Oral administration of arginine, citrulline, and glutamine may alleviate systemic inflammation, jejunal apoptosis, and neuronal NOS in intestinal I/R. Citrulline may further attenuate jejunal damage by preserving jejunal mass, partially via the inactivation of NOS and the NF-κB pathway. In conclusion, oral citrulline may have more benefits than arginine and glutamine in mitigating intestinal ischemia and reperfusion-induced adverse effects.

Direct peritoneal resuscitation improves survival and decreases inflammation after intestinal ischemia and reperfusion injury

BackgroundDirect peritoneal resuscitation (DPR) has previously been shown to alter blood flow in the small bowel mesenteric vessels in models of intestinal ischemia. However, a survival advantage or its effects on local tissue inflammation have not been previously demonstrated. We hypothesized that DPR would increase survival and decrease intestinal tissue inflammation after intestinal ischemia and reperfusion (I/R) injury. MethodsEight-week-old male C57Bl6J mice were anesthetized and underwent midline laparotomy. I/R and DPR groups were exposed to superior mesenteric artery occlusion for 60 min with a nontraumatic clamp. Immediately after removal of the clamp, 1 mL of phosphate-buffered saline, 1 mL of minimal essential media, or 1 mL of minimal essential media supplemented with fetal bovine serum, penicillin and/or streptomycin, and glutamine were placed into the abdominal cavity of DPR groups. Animals were then closed in two layers and allowed to reperfuse for 6 h (cytokine analysis, n = 6 per group) or 7 d (survival analysis, n = 10 per group). After 6 h of reperfusion, animals were euthanized. Intestines were harvested and homogenized. Extracts were quantified for total protein content (Bradford assay), myeloperoxidase activity, tissue inflammatory cytokine, and growth factor production. P < 0.05 was significant. ResultsI/R caused marked intestinal ischemia, significant mortality, and a significant increase in tissue cytokine and growth factor levels (P < 0.05). Seven-day survival was 30% for I/R without treatment and rose to 60% with DPR therapy using phosphate-buffered saline as the dialysate. DPR using plain MEM or MEM with supplements after ischemia increased 7-d survival to 90% (P < 0.05). DPR also significantly decreased intestinal tissue levels of myeloperoxidase, as well as intestinal tissue levels of multiple growth factors and inflammatory cytokines. ConclusionsDPR increases survival and decreases intestinal inflammation after intestinal I/R injury. Translational applications are readily achievable and should be considered for patients with intestinal ischemic pathology.

Vagus Nerve Mediates the Neural Stem Cell Response to Intestinal Injury

Intestinal ischemia and reperfusion injury results in damage to elements critical to maintaining intestinal barrier function, including neurons and glia cells, which are part of the enteric nervous system (ENS). To limit inflammation, the ENS must be restored or replaced, yet the process by which this occurs is poorly understood. Multipotent progenitor cells called enteric nervous stem cells (ENSC) can differentiate into neurons or glia when stimulated. The ability of this cell population to respond to intestinal injury is unknown. In this study, we hypothesized that resolution of intestinal barrier injury would be associated with vagus nerve-mediated expansion of ENSCs. Ischemia and reperfusion injury was reproduced in male mice by occluding the superior mesenteric artery for 30 minutes. Abdominal vagotomy was performed in a separate cohort to study the effects of the vagus nerve. Terminal ileum was harvested at various time points after reperfusion and analyzed with histology, flow cytometry, and immunohistochemistry. Enteric nervous stem cell expansion occurs at 2, 4, and 8 hours after injury compared with sham (4.6% vs 2.1%; p < 0.001) and correlated with increased glial fibrillary acidic protein on immunohistochemistry. Vagotomy prevented both ENSC expansion and increased glial fibrillary acidic protein staining after injury. Intestinal permeability was restored to baseline by 48 hours after injury, but remained elevated in the vagotomy group compared with sham and injury alone at 48 hours (3.25 mg/mL vs 0.57 mg/mL and 0.26 mg/mL, respectively; p < 0.05). Vagal-mediated expansion of ENSCs occurs after ischemia and reperfusion injury and results in improved kinetics of injury resolution.

Effects of Sevoflurane and Propofol on Intestinal Ischemic Reperfusion Injury in Patients Undergoing Elective Open Infrarenal Abdominal Aortic Aneurysm Repair: A Randomized Controlled Trial

Background: We previously demonstrated that propofol had a protective effect on the intestine at risk after an ischemic injury in a rat model. The current study tested the hypothesis that such beneficial effects, so far mainly seen in the laboratory, are reproducible in humans.Methods: Seventy patients undergoing elective open infrarenal abdominal aortic aneurysm repair were randomized to receive general anesthesia with either propofol or sevoflurane. General anesthesia was induced with 3 µg/kg fentanyl, 0.2 mg/kg cisatracurium, and target-controlled infusion of propofol, set at a plasma target concentration of 4-6 µg/ml, or sevoflurane initially started at 8%. Anesthesia was maintained with target-controlled infusion of propofol (2-4 µg/ml) or sevoflurane (0.8-1.0 minimum alveolar concentration). Except for this, anesthetic and surgical management was the same in both groups. Serum intestinal fatty acid binding protein was taken as the primary outcome for evaluating intestinal injury. The secondary outcomes included other intestinal injury variables (serum endotoxin levels, serum diamine oxidase activity and the score of intestinal injury severity) as well as markers reflecting oxidative stress and systemic inflammatory response.Results: The levels of biomarkers reflecting intestinal injury were higher than baseline in both groups (all P<0.05). However, in sevoflurane-anesthetized patients, these variables increased significantly more than those in patients anesthetized with propofol (all P<0.05), whereas the scores of the intestinal injury severity didn't differ between two groups (P=0.53).Conclusions: Patients receiving propofol for abdominal aortic aneurysm repair surgery had less intestinal injury than patients receiving sevoflurane, which may be related to its better anti-oxidative property. Citation: Cai Li, Wu Yan, Jian- Tong Shen, Shi-Hong Wen, Ke-Xuan Liu. Effects of sevoflurane and propofol on intestinal ischemic reperfusion injury in patients undergoing elective open infrarenal abdominal aortic aneurysm repair: a randomized controlled trial. J Anesth Perioper Med 2015; 2: 126-35. Published on April 30, 2015. doi: 10.24015/JAPM.2015.0018This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Higenamine regulates Nrf2-HO-1-Hmgb1 axis and attenuates intestinal ischemia-reperfusion injury in mice.

Intestinal ischemia and reperfusion (IR) syndrome is a life-threatening dilemma caused by diverse events. Higenamine (HG), an active ingredient of Aconiti Lateralis Radix Praeparata, has been traditionally used as a heart stimulant and anti-inflammatory agent in oriental countries. But the function of HG on intestine IR injury has never been investigated. Mice underwent a 2 cm midline laparotomy, and the superior mesenteric artery (SMA) was obstructed by micro-vascular clamp to induce intestinal ischemia. In our current study, HG increases mouse intestinal epithelial (IEC-6) cell viability through induced heme oxygenase-1 (HO-1) production in vitro. In our in vivo murine intestinal IR injury model, the increased HO-1 protein level and activity, decreased intestinal injury score, Myeloperoxidase (MPO) activity, and inflammatory cytokine expression induced by HG were all abolished with additional treatment of HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX). Furthermore, HG reduced high mobility group box-1 (Hmgb1) expression in IR injury-performed intestine which was inhibited by additional administration of ZnPPIX. And HG treatment significantly decreased HO-1 expression in nuclear factor erythroid 2-related factor (Nrf-2) SiRNA-transfected cells but not in control SiRNA-transfected cells. Our study provides evidence HG regulates Nrf2-HO-1-Hmgb1 axis and attenuates intestinal IR injury in mice.

Neutrophilia Delays Equine Neutrophil Apoptosis

Regulation of neutrophil apoptosis has assumed a critical role in inflammation. The objective of this study was to determine the effect of neutrophil concentration on their apoptosis in vitro and ex vivo in horses with experimental intestinal ischemia and reperfusion injury. Neutrophils were isolated from healthy horses randomized to the following treatments: sham celiotomy (CEL, n=4), intestinal ischemia and reperfusion (IR, n=6), intestinal ischemia and reperfusion with gadolinium chloride treatment to deplete pulmonary intravascular macrophages (IRGC, n=6). All horses experienced a significant neutrophilia following surgery without detectable plasma endotoxin concentrations (&lt;0.06 EU/ml). Isolated neutrophils were cultured for 12 or 24 hours at concentrations of 2, 5, or 8 x 106 cells/ml and evaluated for the occurrence of apoptosis using Annexin V and propidium iodide staining. Caspase‐3, ‐8, and ‐9 activities were measured in cell lysates before and after treatment. Delayed neutrophil apoptosis was highly correlated (12 h incubation: r = ‐0.75, 24 h incubation: r = ‐0.57) with increasing neutrophil concentration in vitro. Neutrophil apoptosis was significantly delayed ex vivo after treatment, except in IRGC horses (12 h incubation: CEL: P =0.03, IR: P = 0.05;24 h incubation: CEL:P = 0.001, IR: P = 0.004). Caspase‐3, ‐8, and ‐9 activities were reduced in neutrophils isolated after surgery in all groups. The data show that equine neutrophil apoptosis is delayed in vitro and ex vivo via down‐regulation of caspase activity when neutrophils become more concentrated, which could have implications for the development and progression of systemic inflammatory responses.

P0009 : The effect of glutamine in the liver injury resulting from intestinal ischemia and reperfusion in rats

P0009 : The effect of glutamine in the liver injury resulting from intestinal ischemia and reperfusion in rats

Effects of Arginine, Citrulline and Glutamine in Intestinal Ischemia and Reperfusion‐Induced Immune Disorders

Intestinal ischemia and reperfusion (IIR) commonly results in systemic inflammation, multiple organ failure and death. Previous study showed that arginine, citrulline and glutamine may improve IIR‐induced intestinal damage. Herein, we aimed to compare the effects of arginine, citrulline, and glutamine on immune alteration. Male Wistar rats were divided into a healthy group and 4 IIR groups that underwent intestinal ischemia for 60 min and fed with placebo, arginine, citrulline, or glutamine at 15 minutes before and 3, 9 and 21 hr after reperfusion. After 24 hr reperfusion, arginine, citrulline, or glutamine alleviated IIR‐increased monocytes and IIR‐decreased splenocytic TNF‐α productions (P&lt;0.05, one‐way ANOVA). In leukocytes, arginine decreased T‐helper cells and reversed IIR‐decreased IL‐4 production; citrulline and glutamine decreased monocytic TNF‐α and IL‐6 productions; and glutamine further increased phagocytosis activity. In splenocytes, arginine and citrulline increased phagocytosis and IFN‐γ production and further decreased macrophages. Moreover, arginine reversed IIR‐decreased T cytotoxic cells and IL‐4 production; citrulline reversed IIR‐decreased cytotoxicity activity; and glutamine alleviated IIR‐increased TNF‐α and IL‐6 production in macrophages and further decreased IL‐4 production in splenocytes. In conclusion, arginine, citrulline, and glutamine may alleviate systemic inflammatory response in IIR injury. However, arginine, citrulline and glutamine have potential ability to elevate T cell activity, activate innate immunity and alleviate inflammatory response, respectively, in splenocytes of IIR rats. (NSC 98‐2320‐B‐030‐003‐MY3)

Pentoxifylline and prostaglandin E1 action on ischemia and reperfusion of small intestine tissue in rats. An immunohistochemical study.

To investigate the action of pentoxifylline (PTX) and prostaglandin E1 (PGE1) on ischemia and reperfusion of small intestine tissue in rats, using immunohistochemical analysis. Thirty-five Wistar rats were distributed as follows: group A (n=10): subjected to intestinal ischemia and reperfusion for 60 min, with no drugs; group B (n=10): PTX given during tissue ischemia and reperfusion; group C (n=10): PGE1 given during tissue ischemia and reperfusion; group D (n=5): sham. A segment of the small intestine was excised from each euthanized animal and subjected to immunohistochemical examination. Mean number of cells expressing anti-FAS ligand in the crypts was highest in Group A (78.9 ± 17.3), followed by groups B (16.7 ± 2.8), C (11.3 ± 1.8), and D (2.5 ± 0.9), with very significant differences between groups (p<0.0001). The use of pentoxifylline or prostaglandin E1 proved beneficial during tissue reperfusion. The immunohistochemical results demonstrated a decrease in apoptotic cells, while protecting other intestinal epithelium cells against death after reperfusion, allowing these cells to renew the epithelial tissue.

Effects of ukrain in rats with intestinal ischemia and reperfusion

BackgroundThe purpose of this study is to investigate the potential protective effect of the ukrain on ischemia–reperfusion (IR) injury in rat intestine, which has not previously been studied. MethodsThirty-one male Sprague–Dawley rats were randomly assigned to four groups, each consisting of eight rats as follows: (1) a sham group (S) (laparotomy, but no IR injury); (2) ukrain group (U) (no IR, and ukrain was administered intraperitoneally 1 h before laparotomy); (3) intestinal ischemia–reperfusion (II/R) group (30-min occlusion of the superior mesenteric artery then 2-h reperfusion); and (4) ukrain + II/R group (U + II/R) (30-min occlusion of the superior mesenteric artery then 2-h reperfusion; ukrain was administered intraperitoneally 1 h before IR). ResultsSerum total oxidant status (TOS) and total antioxidant status (TAS) were measured using Erel method. Oxidative stress index was calculated using the TOS/TAS ratio. TAS levels increased and TOS serum levels were also significantly decreased in the ukrain + IR group compared with the IR group (P = 0.000 and P = 0.015). ConclusionsIn this study, we demonstrated for the first time in literature that ukrain helps to prevent intestinal tissue breakdown against II/R injury and that this effect can be achieved by antioxidant activities.

Antagonism of ionotropic glutamate receptors attenuates chemical ischemia-induced injury in rat primary cultured myenteric ganglia.

Alterations of the enteric glutamatergic transmission may underlay changes in the function of myenteric neurons following intestinal ischemia and reperfusion (I/R) contributing to impairment of gastrointestinal motility occurring in these pathological conditions. The aim of the present study was to evaluate whether glutamate receptors of the NMDA and AMPA/kainate type are involved in myenteric neuron cell damage induced by I/R. Primary cultured rat myenteric ganglia were exposed to sodium azide and glucose deprivation (in vitro chemical ischemia). After 6 days of culture, immunoreactivity for NMDA, AMPA and kainate receptors subunits, GluN1 and GluA1–4, GluK1–3 respectively, was found in myenteric neurons. In myenteric cultured ganglia, in normal metabolic conditions, -AP5, an NMDA antagonist, decreased myenteric neuron number and viability, determined by calcein AM/ethidium homodimer-1 assay, and increased reactive oxygen species (ROS) levels, measured with hydroxyphenyl fluorescein. CNQX, an AMPA/kainate antagonist exerted an opposite action on the same parameters. The total number and viability of myenteric neurons significantly decreased after I/R. In these conditions, the number of neurons staining for GluN1 and GluA1–4 subunits remained unchanged, while, the number of GluK1–3-immunopositive neurons increased. After I/R, -AP5 and CNQX, concentration-dependently increased myenteric neuron number and significantly increased the number of living neurons. Both -AP5 and CNQX (100–500 µM) decreased I/R-induced increase of ROS levels in myenteric ganglia. On the whole, the present data provide evidence that, under normal metabolic conditions, the enteric glutamatergic system exerts a dualistic effect on cultured myenteric ganglia, either by improving or reducing neuron survival via NMDA or AMPA/kainate receptor activation, respectively. However, blockade of both receptor pathways may exert a protective role on myenteric neurons following and I/R damage. The neuroprotective effect may depend, at least in part, on the ability of both receptors to increase intraneuronal ROS production.