Clamping Of Superior Mesenteric Artery Research Articles

Raising Systemic Blood Pressure to Delay Irreversible Intestinal Ischemia in a Swine Model of Proximal Superior Mesenteric Artery Occlusion

IntroductionAcute proximal superior mesenteric artery (SMA) occlusion is highly lethal, and adjuncts are needed to mitigate ischemic injury until definitive therapy. We hypothesized that raising mean arterial pressure (MAP) >90 mmHg with norepinephrine may delay irreversible bowel ischemia by increasing gastroduodenal artery (GDA) flow despite possible pressor-induced vasospasm. Methods12 anesthetized swine underwent laparotomy, GDA flow probe placement, and proximal SMA exposure and clamping. Animals were randomized between conventional therapy (CT) versus targeted MAP >90 mmHg (MAP push; MP) where norepinephrine was titrated after 45 min of SMA occlusion. Animals were followed until bowel death or 4 h. Kaplan–Meier bowel survival, mean normalized GDA flow, and histology were compared. Results12 swine (mean 57.8 ± 7.6 kgs) were included, six per group. Baseline weight, HR, MAP and GDA flows were not different. Within 5 min following SMA clamping, all 12 animals had an increase in MAP without other intervention from 81.7 to 105.5 mmHg (29.1%, P < 0.01) with a concomitant 74.9% increase in GDA flow as compared to baseline (P < 0.01). Beyond 45 min postclamp, MAP was greater in the MP group as intended, as were GDA flows. Median time to irreversibly ischemic bowel was 31% longer for MAP push animals (CT: 178 versus MP: 233 min, P = 0.006), Hazard Ratio of CT 8.85 (95% CI: 1.86-42.06); 3/6 MP animals versus 0/6 CT animals with bowel survived to predetermined end point. ConclusionsIn this swine model of acute complete proximal SMA occlusion, increasing MAP >90 mmHg with norepinephrine was associated with an increase in macrovascular blood flow through the GDA and bowel survival. Norepinephrine was not associated with worse bowel survival and a MAP push may increase the time window where ischemic bowel can be salvaged.

The efficacy of bone marrow mesenchymal stem cells on rat intestinal immune-function injured by ischemia/reperfusion

BackgroundTransplantation of bone marrow mesenchymal stem cells (BMSCs) has a promising therapeutic efficiency for varieties of disorders caused by ischemia or reperfusion impairment. It has been shown that BMSCs can mitigate intestinal ischemia/reperfusion (I/R) injuries, but the underlying mechanism is still unclear. This study aimed at investigating the efficacy of BMSCs on the immune function of intestinal mucosal microenvironment after I/R injuries. MethodsTwenty adult Sprague-Dawley rats were randomly assigned to a treatment or a control group. All the rats underwent superior mesenteric artery clamping and unclamping. In the treatment group, BMSCs were implanted into the intestine of ten rats by direct submucosal injection whereas the other ten rats in the control group were injected with the same volume of saline. On the fourth and seventh day after BMSCs transplantation, intestinal samples were examined for the CD4 (CD4-positive T-lymphocytes)/CD8 (CD8-positive T-lymphocytes) ratio of the bowel mucosa via flow cytometry, and for the level of Interleukin-2 (IL-2), Interleukin-4 (IL-4) and Interleukin-6 (IL-6) via ELISA. Paneth cell counts and Secretory Immunoglobulin A (SIgA) level were examined via immunohistochemical (IHC) analysis. Real time PCR (RT-PCR) was used to detect the expression levels of tumor necrosis factor-α (TNF-α) and trypsinogen (Serine 2) (PRSS2) genes. White blood cell (WBC) count was measured by manual counting under the microscope. ResultsThe CD4/CD8 ratio in the treatment group was significantly lower compared with that in the control group. The concentration of IL-2 and IL-6 was lower in the treatment group compared with the control group, while the level of IL-4 is the reverse between the two groups. The number of Paneth cells in intestinal mucosa increased significantly, while the level of SIgA in intestinal mucosa decreased significantly, after BMSCs transplantation. The gene expression levels of TNF-α and PRSS2 in intestinal mucosa of treatment group were significantly lower than those of control group. The WBC count in the treatment group was significantly lower than that in the control group. ConclusionWe identified immune-relevant molecular changes that may explain the mechanism of BMSCs transplantation efficacy in alleviating rat intestinal immune-barrier after I/R.

Laparoscopic pancreaticoduodenectomy with mesopancreas dissection using artery first approach

Laparoscopic pancreaticoduodenectomy (LPD) is the one of most technically challenging operations of minimally invasive procedures. Furthermore, LPD remains controversial, especially in the treatment of pancreatic cancer. In this article, the aim is to review our experience of LPD in PDAC that can be performed with safety and good results including oncologic outcome, resection margins and survival rates at a single institution. From August 2017 to August 2020, 117 patients underwent LPD in our center. Among 117 patient, 20 patients underwent artery first approach LPD. The patient characteristics, perioperative variables, and post op outcomes were retrospectively collected and analysed. Seven male and 13 female patients were included in this study. The median age of the patients was 66.8 years. Eleven cases were performed by portal vein resection and reconstruction. Open conversion was none. The average operative time was 475.9 minutes (350-685 minutes). The mean time of superior mesenteric artery clamp was 40.6 minutes (30-55 minutes). Thirty day mortality was not observed in this series. Among 20 patient, 19 patient had negative resection margins, and the median number of lymph node harvested was 17.3 (4-38). The median postoperative hospital stay was 14.3 days (7-24 days). The 1 patient suffered from gastric mucosa bleeding. The 1 patient suffered from grade C POPF. The artery first approach has been devised to achieve a complete tumor resection for pancreatic cancer. Artery first approach LPD can be safely and feasibly performed in experienced surgeon. However, more study should be required.

Temporary Mesenteric-Portal Shunt Using Prosthetic Graft Together with Intermittent Superior Mesenteric Artery Clamping (Pringle-Like) during Complex Venous Resction/Reconstruction in Pancreaticoduodenctomy for Locally Advanced Pancreatic Tumours

Introduction: Portal and superior mesenteric vein resection and reconstruction during pancreatic surgery for locally advanced tumors poses a challenging surgical scenario. Potential problems are; major bleeding, prolonged clamping with hepatic ischemia, venous congestion of the gut, and using the more difficult artery first approach. This potentially may increase morbidity and mortality of the procedure. To obviate these potential problems, we propose a pre-emptive temporary SMV-portal venous shunt with intermittent SMA clamping (pringle-like).

The role of mesenteric lymph exosomal lipid mediators following intestinal ischemia-reperfusion injury on activation of inflammation

Intestinal ischemia caused by hemorrhagic shock is known to induce systemic inflammatory responses. Previous studies have shown that mesenteric lymph (ML) plays a crucial role in gut-mediated inflammation. Lipid mediators, such as lysophosphatidylcholines (LPCs), which contain polyunsaturated fatty acids (PUFAs), are present in the postshock ML. Exosomes are also present in the ML and act as transcellular carriers of lipids; however, their role in postshock systemic inflammation has not been revealed. Here, we aimed to identify changes in lipid mediators in ML exosomes after intestinal ischemia. Male Sprague-Dawley rats underwent laparotomy, followed by ML duct cannulation. Animals were subjected to 60 minutes of intestinal ischemia by superior mesenteric artery clamping, followed by 120 minutes of reperfusion. Mesenteric lymph was obtained before and after intestinal ischemia, and exosomes were isolated from ML by ultracentrifugation. The biological activity of ML exosomes was determined using the monocyte nuclear factor κB (NF-κB) activation assay. Lipids of ML exosomes were extracted and quantified by liquid chromatography/electrospray ionization mass spectrometry. Mesenteric lymph exosome-induced NF-κB activation significantly increased after intestinal ischemia, and lipid analysis revealed a significant increase in the concentration of PUFA-containing LPCs. In addition, PUFA-containing LPCs also induced NF-κB activation. Our results suggest that biologically active lipid mediators in ML exosomes may be involved in the inflammatory response after intestinal ischemia.

P3B.18: INT 767 – a novel dual Farnesoid-X Receptor (FXR) and Takeda G-protein-coupled Receptor-5 (TGR5) agonist attenuates intestinal ischemia reperfusion injury

Introduction: Ischemia reperfusion injury (IRI) occurs inevitably during intestinal transplantation and after intestinal infarction. The intestine is especially susceptible to IRI which leads to loss of villi, resulting in systemic translocation contributing to poorer outcomes. The Farnesoid-X receptor (FXR), is a member of the nuclear receptor family. TGR5 is a G-protein-coupled bile activated receptor. Both are abundantly expressed in the gastro-intestinal tract. In pre-clinical models, they have shown to reduce inflammation and improve epithelial permeability when administered before ischemia. The aim of our study was to test the effect of a dual FXR/TGR5-agonist as treatment of intestinal IRI, administered intravenously after onset of ischemia. Material and Methods: In a validated rat model (Sprague-Dawley, male, 300g) of intestinal IRI (laparotomy and clamping of superior mesenteric artery), 3 groups (n=6/group) were investigated: i/ Sham (only laparotomy); ii/ Ischemia 60min + reperfusion 60min (IRI) + intravenous vehicle; iii/ Ischemia 60min + reperfusion 60min + intravenous FXR/TGR5-agonist (IRI+FXR/TGR5). For each group, 10 additional animals were included for a 7-day survival analysis. FXR/TGR5-agonist INT-767 (Intercept Pharma, USA) or vehicle only was administered intravenously in a single dose at 10 mg/kg, 15 minutes after start of ischemia. Analyzed endpoints: 1/ Histology: Park/Chiu score and villus length; 2/ intestinal barrier function (transepithelial electrical resistance (TEER) and FD20 permeability measurements in Ussing chambers); 3/ Inflammatory cytokines: IL-6 (ELISA), IL-1-β and TNFα (qPCR); and 4/ Anti-inflammatory cytokines: IL-10, IL-13 (qPCR). Results: IRI led to pronounced damage resulting in high Park/Chiu scores, increased intestinal permeability and systemic inflammation. Dual FXR/TGR5 treatment dramatically improved intestinal histology (Figure) and all other parameters. Survival was substantially improved after treatment (P< 0.05). Results are summarized in the table. Conclusion: We demonstrated that intravenous treatment with a dual FXR/TGR5 agonist (INT-767) after onset of ischemia significantly decreased intestinal damage caused by IRI. These results show that FXR and TGR5 receptors are promising targets for intestinal graft protection. The ability to administer this substance intravenously greatly enhances the potential applicability for the frequent pathology of intestinal infarction as well as for transplantation.

Protective effect of melatonin‐supported adipose‐derived mesenchymal stem cells against small bowel ischemia‐reperfusion injury in rat

We tested the hypothesis that combined melatonin and autologous adipose-derived mesenchymal stem cells (ADMSC) was superior to either alone against small bowel ischemia-reperfusion (SBIR) injury induced by superior mesenteric artery clamping for 30 min followed by reperfusion for 72 hr. Male adult Sprague Dawley rats (n = 50) were equally categorized into sham-operated controls SC, SBIR, SBIR-ADMSC (1.0 × 10(6) intravenous and 1.0 × 10(6) intrajejunal injection), SBIR-melatonin (intraperitoneal 20 mg/kg at 30 min after SI ischemia and 50 mg/kg at 6 and 18 hr after SI reperfusion), and SBIR-ADMSC-melatonin groups. The results demonstrated that the circulating levels of TNF-α, MPO, LyG6+ cells, CD68+ cells, WBC count, and gut permeability were highest in SBIR and lowest in SC, significantly higher in SBIR-ADMSC group and further increased in SBIR-melatonin group than in the combined therapy group (all P < 0.001). The ischemic mucosal damage score, the protein expressions of inflammation (TNF-α, NF-κB, MMP-9, MPO, and iNOS), oxidative stress (NOX-1, NOX-2, and oxidized protein), apoptosis (APAF-1, mitochondrial Bax, cleaved caspase-3 and PARP), mitochondrial damage (cytosolic cytochrome C) and DNA damage (γ-H2AX) markers, as well as cellular expressions of proliferation (PCNA), apoptosis (caspase-3, TUNEL assay), and DNA damage (γ-H2AX) showed an identical pattern, whereas mitochondrial cytochrome C exhibited an opposite pattern compared to that of inflammation among all groups (all P < 0.001). Besides, antioxidant expressions at protein (NQO-1, GR, and GPx) and cellular (HO-1) levels progressively increased from SC to the combined treatment group (all P < 0.001). In conclusion, combined melatonin-ADMSC treatment offered additive beneficial effect against SBIR injury.

Role of IL-17 in intestinal ischemia-reperfusion injury. (MUC2P.829)

Abstract Background: Ischemia-reperfusion injury (IRI) to the small intestine following clamping of the superior mesenteric artery (SMA) results in an intense local inflammatory response characterized by neutrophil infiltration and villous damage. IL-17A is a cytokine expressed by a variety of cells in response to inflammatory cytokines that are released following tissue injury and/or inflammation. IL-17A induces epithelial cells to secrete neutrophil chemoattractants, such as CXCL2. The cytokine IL-23, which can be produced by epithelial cells, plays an important role in IL-17A production. Objective: To test the hypothesis that IL-17A is critical for intestinal damage following IRI. Materials and Methods: IRI was induced in mice by SMA clamping. IL-17A deficient (Il17a-/-) and IL-23R deficient (Il23r-/-) mice. Intestinal injury was evaluated by Chiu scores of jejunal histological sections. Results: IRI was significantly less in both Il17a-/- mice and Il23r/- mice than in WT controls. We also found that pretreatment of WT mice with neutralizing antibody to IL-17A or IL-23 resulted in significant decrease in IRI compared to mice treated with isotype control antibody. Conclusions: These results indicate that IL-17A plays a critical role in IRI. They also suggest that IL-23 contributes to IRI, possibly by promoting IL-17A production.

Suppression of inflammatory events associated to intestinal ischemia–reperfusion by 5-HT1A blockade in mice

Intestinal ischemia and reperfusion (I/R) is a potentially life-threatening disease, ensuing from various clinical conditions. Experimentally, either protective or detrimental roles have been attributed to 5-HT in the functional and morphological injury caused by mesenteric I/R. Recently, we proved the involvement of 5-HT2A receptors in the intestinal dysmotility and leukocyte recruitment induced by 45min occlusion of the superior mesenteric artery (SMA) followed by 24h reperfusion in mice. Starting from these premises, the aim of our present work was to investigate the role played by endogenous 5-HT in the same experimental model where 45min SMA clamping was followed by 5h reflow. To this end, we first observed that ischemic preconditioning before I/R injury (IPC+I/R) reverted the increase in 5-HT tissue content and in inflammatory parameters induced by I/R in mice. Second, the effects produced by intravenous administration of 5-HT1A ligands (partial agonist buspirone 10mgkg−1, antagonist WAY100135 0.5–5mgkg−1), 5-HT2A antagonist sarpogrelate (10mgkg−1), 5-HT3 antagonist alosetron (0.1mgkg−1), 5-HT4 antagonist GR125487 (5mgkg−1) and 5-HT re-uptake inhibitor fluoxetine (10mgkg−1) on I/R-induced inflammatory response were investigated in I/R mice and compared to those obtained in sham-operated animals (S). Our results confirmed the significant role played by 5-HT2A receptors not only in the late but also in the early I/R-induced microcirculatory dysfunction and showed that blockade of 5-HT1A receptors protected against the intestinal leukocyte recruitment, plasma extravasation and reactive oxygen species formation triggered by SMA occlusion and reflow. The ability of α7 nicotinic receptor (α7nAchR) antagonist methyllycaconitine (5mgkg−1) to counteract the beneficial action provided by buspirone on I/R-induced neutrophil infiltration suggests that the anti-inflammatory effect produced by 5-HT1A receptor antagonism could be partly ascribed to the indirect activation of α7nAch receptors.

Effects and mechanism of osthole on intestinal ischemia-reperfusion injury in mice

To explore the protective role of osthole in intestinal ischemia-reperfusion (I/R) injury in mice and examine its underlying mechanism. A murine model of intestinal I/R injury was established with clamping of superior mesenteric artery for 120 min and then clamping was relieved for 60 min. Forty-five SD male mice weighing 27-31 g were randomly divided into 3 groups (n = 15 each): sham group (S), I/R injury group (I/R) and I/R plus osthole treatment group (Ost+). Intestinal wet/dry weight ratio, superoxide dismutase (SOD), malondialdehyde (MDA) in serum were examined by colorimetric assay and diamine oxidase (DAO) was examined by automatic biochemical analyzer, the levels of tumor necrosis factor (TNF) -α, interleukin (IL)-1β and IL-2 were examined by enzyme-linked immunosorbent assay (ELISA). Intestinal barrier permeability was detected by Evans blue (EB) test. One-way ANOVA was used to analyze all experimental data variance. Intestinal tissues wet/dry weight ratios, Evans blue content and Chiu's score of I/R group mice were significantly higher than those of S and Ost+ groups (0.80% ± 0.03% vs 0.77% ± 0.02% & 0.78% ± 0.02%, (0.11 ± 0.04) vs (0.05 ± 0.02) & (0.06 ± 0.02) µg/mg, 3.42 ± 0.73 vs 0.87 ± 0.35 & 2.63 ± 0.58, P < 0.05 or P < 0.01) . Serum level of DAO, MDA, IL-1β & TNF-α of I/R group mice were significantly higher than those of S and Ost+ groups ( (18.9 ± 4.0) vs (14.5 ± 2.3) & (16.0 ± 2.6) U/L, (8.4 ± 1.2) vs (6.9 ± 1.7) & (6.1 ± 2.4) µmol/L, (93 ± 6) vs (51 ± 4) & (67 ± 5) ng/L, (467 ± 31) vs (235 ± 21) & (323 ± 30) ng/L, P < 0.01 or P < 0.05) . Serum SOD activity and IL-2 level were significantly lower than those of S and Ost+ groups ( (75 ± 7) vs (93 ± 16) & (89 ± 5) U/ml, (95 ± 16) vs (198 ± 14) & (139 ± 11) ng/L, all P < 0.05) . All parameters showed no significant difference between S and Ost+ groups (all P > 0.05). Treatment of osthole may protect murine intestinal tissue against intestinal I/R injury. And the mechanisms may be due to its actions of preventing oxygen stress and inflammatory responses.

Potential Role of Mesenchymal Stem Cells in Alleviating Intestinal Ischemia/Reperfusion Impairment

BackgroundTransplantation of bone marrow mesenchymal stem cells (MSCs) provides a promising therapeutic efficiency for a variety of disorders caused by ischemia or reperfusion impairment. We have previously demonstrated the efficacy of MSCs in mitigating intestinal ischemia/reperfusion (I/R) injuries in rats, but the mechanism by which MSCs engraft ameliorates I/R injuries has largely been unknown. The present study aimed at investigating probable mechanisms by which MSCs exert their function.MethodsMale donor derived rat MSCs were implanted into intestine of female recipient rat by direct submucosal injection after superior mesenteric artery clamping and unclamping. The homed MSCs were detected by Y chromosome in situ hybridization probe, and the tumor necrosis factor-α (TNF-α) content in intestinal mucosa was determined by ELISA. Expression of proliferative cell nuclear antigen (PCNA) in bowel mucosa was assayed by real-time PCR and intestinal mucosa expression of phosphorylation extracellular signal-regulated kinase (pERK1/2) and nuclear factor-κB (NF-κB) were evaluated by western blot.ResultsFour and seven days after MSCs transplantation, the TNF-α content of bowel mucosa in MSCs group was significantly lower than that in saline group. The PCNA in bowel mucosa showed higher expression in MSCs treated group than the saline group, both at 4 and 7 days after cell transplantation. The expression of intestinal mucosal pERK1/2 in MSCs treated group was markedly higher than that in saline group, and the expression of NF-κB in MSCs treated group was noticeably decreased than that in saline group at 4 and 7 days post MSCs transplantation.ConclusionThe present investigation provides novel evidence that MSCs have the potential to reduce intestinal I/R injuries probably due to their ability to accelerate cell proliferation and decrease the inflammatory response within intestinal mucosa after ischemia and reperfusion.

Ischemic preconditioning of the graft for intestinal transplantation in rats

To test the hypothesis that two modalities of IPC should decrease acute rejection and BT after SBTx in rats. Orthotopic allogenic SBTx was performed from Wistar to BN. IPC was performed by 2 ' and 5 ' superior mesenteric artery clamping, following 2-min and 5-min reperfusion before graft cooling and retrieving. Donor-recipient sets were randomly allocated to five groups: IPC2m4d, IPC2m7d, IPC5min7d, and the control groups for the two end points; ctrl4d and ctrl7d. IRI, rejection, and BT were assessed after four or seven days depending on the groups. Measured variables included: histology, leukocyte activation by tissue MPO determination, and proinflammatory cytokines (IL-b and TNF-α) to assess inflammatory response. Leukocyte activation was significantly reduced in IPC2m7d in comparison with Ctrl and IPC5min7d. Rejection tended to be lower in IPC2min7d. Cytokine levels were contradictory and not consistent with histology. Finally, BT was less frequent in IPC2min4d group but this benefit was missed in animals with rejection (7d). Inflammatory response (MPO) was reduced and rejection tended to be lower after in IPC2m7d. Bacterial translocation was reduced in IPC2min4d but the benefit was missed at day 7.

Intestinal ischaemia/reperfusion upregulates β-defensin-2 expression and causes acute lung injury in the rat

Objective Human β-defensin-2 (BD-2) is a positive ion antimicrobial peptide. We investigated the effects of intestinal ischaemia/reperfusion (II/R) on rat BD-2 mRNA and protein expressions in rat lung to address the potential role of BD-2 in acute lung injury (ALI) induced by II/R. Methods Rats were randomly divided into two groups ( n = 36 each). (i) Sham control and (ii) II/R group (1 h superior mesenteric artery clamping, followed by reperfusion of different durations). In II/R group, 6 animals were sacrificed at 0 min, 15 min, 30 min, 60 min, 3 h and 6 h after reperfusion, and serum, lung tissue and bronchoalveolar lavage fluid were harvested. Samples were taken at the corresponding time points in the sham group. Lung histological changes were observed under microscope and the pulmonary permeability index (PPI) was calculated. The lung tissue levels of TNFα were detected by ELISA. BD-2 mRNA and protein expressions were examined by RT-PCR and western blotting techniques, respectively. Results ALI induced by II/R was confirmed by pathological examination and significantly increased PPI ( P < 0.05 or 0.01). II/R significantly increased the lung TNFα levels and upregulated the expressions of BD-2 mRNA and protein expressions ( P < 0.05 or 0.01). BD-2 mRNA expression was significantly positively correlated to the lung TNFα level ( r = 0.823, P < 0.01) and negatively correlated to PPI ( r = −0.615, P < 0.05). Conclusion II/R can upregulate BD-2 mRNA and protein expressions in rat lung. BD-2 could be an innate protective factor against II/R-induced lung injury.

Study of enteral nutrients transport in intestinal hypoperfused rat model

To study the transport of glutamine and glucose, expression of their transporters and tissue morphology in intestinal hypoperfusion. Sprague-Dawley rats were randomized to receive 60 min of intestinal hypoperfusion (superior mesenteric artery clamp) or serve as normoxic controls (celiotomy only). At the same time, jejunal loops were randomized to receive in situ perfusion of mannitol,glucose,or glutamine.Intestinal brush border membrane vesicles (BBMV) were prepared by calcium precipitation. Sodium-dependent uptake of glucose and glutamine into BBMV were quantitated by rapid mixing and filtration. Histologic examination and immunohistochemistry were performed by pathologists blinded to the groups. When compared with the control group, tissue lactate concentration of the hypoperfused group increased significantly (4.9+/-0.3 vs 3.1+/-0.2), especially in the glucose perfused groups (P<0.01). Transport and transporters of glucose in brush border, but not glutamine, decreased during hypoperfusion [(76+/-10) pmol d mg(-1) d 10 s(-1) vs (290+/-13)pmol d mg(-1) d 10 s(-1)]. Tissue structural damage was most severe in glucose perfused groups during hypoperfusion. Transport and expression of transporters of glucose and glutamine in enteral nutrition are differently regulated under conditions of trauma and stress.

Endothelin receptor blockers protect against ischemia/reperfusion impairment of gastrointestinal motility in rats

Intestinal ischemia/reperfusion (I/R) injury remains associated with high morbidity and mortality. The protective efficacy of the following endothelin (ET) receptor blockers: BQ-123 (ET A receptor), BQ-788 (ET B); tezosentan (dual ET blocker) was tested against the inhibition of gastrointestinal (GI) motility induced by intestinal I/R. Intestinal Evans blue transit was measured in untreated (UN) rats and animals subjected to skin incision (SI), I/R (1 h superior mesenteric artery clamping followed by 2–24 h reperfusion) or sham operation (SO). Surgical procedures were conducted under diethyl ether anesthesia. Anesthesia and SI did not affect the GI transit compared to UN rats. In contrast both SO and I/R significantly reduced GI motility, the latter evident at 2–24 h of reperfusion. Tezosentan (1–10 mg/kg), BQ-123 and BQ-788 (0.1–1 mg/kg) protected against I/R-induced inhibition of intestinal motility in a time- and dose-dependent manner at the early and late stages of reperfusion. Furthermore tezosentan alleviated the I/R-induced decrease in the contractile response of the longitudinal jejunal smooth muscle strips to carbachol in vitro. The serum ET(1–21) level was increased at 2 h but not 24 h of reperfusion compared to SO animals and ET(1–21) was higher in tezosentan pretreated rats.

Adrenomedullin and Adrenomedullin Binding Protein-1 Prevent Acute Lung Injury after Gut Ischemia-Reperfusion

Ischemic bowel remains a critical problem, resulting in up to 80% mortality. Acute lung injury, a common complication after intestinal ischemia/reperfusion (I/R), might be responsible for such a high mortality rate. Our previous studies have shown that administration of a novel vasoactive peptide adrenomedullin (AM) and its binding protein (AMBP-1) reduces the systemic inflammatory response in rat models of both hemorrhage and sepsis. It remains unknown whether administration of AM/AMBP-1 has any protective effects on intestinal I/R-induced acute lung injury. We hypothesized that administration of AM/AMBP-1 after intestinal I/R prevents acute lung injury through downregulation of proinflammatory cytokines. Intestinal I/R was induced by placing a microvascular clip across superior mesenteric artery (SMA) for 90 minutes in adult male Sprague-Dawley rats (275 to 325 g). On release of the SMA clamp, the animals were treated with either AM (12 mug/kg body weight) in combination with AMBP-1 (40 microg/kg body weight) or vehicle (1 mL normal saline) during a period of 30 minutes through a femoral vein catheter. Lung samples were collected at 4 hours after treatment or sham operation. Lung injury was assessed by examining lung water content, morphologic changes, and granulocyte myeloperoxidase activity. Tumor necrosis factor-alpha and interleukin-6 gene expression and their protein levels in the lungs were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. In additional groups of animals, AM/AMBP-1 or vehicle was administered at 1 hour after onset of reperfusion. Lung histology was examined at 3 hours after treatment. Intestinal I/R induced considerable lung injury, as characterized by lung edema, histopathologic changes, increased myeloperoxidase activity, and proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) levels in the lungs. Administration of AM/AMBP-1 after ischemia mitigated lung injury and dramatically downregulated proinflammatory cytokines. Lung injury was also ameliorated by delayed AM/AMBP-1 treatment as evidenced by improvement in lung histology. AM/AMBP-1 can be developed as a novel treatment to attenuate acute lung injury after an episode of gut ischemia. The protective effect of AM/AMBP-1 appears to be mediated through downregulation of proinflammatory cytokines.

INTRAPERITONEAL ADMINISTRATION OF HYPERBARICALLY OXYGENATED PERFLUOROCHEMICAL ENHANCES PRESERVATION OF INTESTINAL MUCOSA AGAINST ISCHEMIA/REPERFUSION INJURY

Perfluorochemicals (PFCs) have a high solubility for oxygen. We have previously demonstrated the effect of peritoneal lavage with oxygenated PFC (O2-PFC) on ameliorating ischemia/reperfusion (I/R)-induced intestinal ischemic damage in an animal model. In this study, we applied hyperbarically O2-PFC (HBO-PFC) to investigate whether a larger amount of oxygen carried by PFC could enhance the protective effect of O2-PFC during intestinal malperfusion. Rats were subjected to ischemia by clamping the superior mesenteric artery (SMA) for 90 min. The SMA was then declamped. Rats were divided into four groups. In group A, only anesthesia and abdominal incision were performed. In group B, SMA was clamped without O2-PFC. In group C, during the SMA clamp, 1 atm O2-PFC was injected into the abdominal cavity. In group D, 5 atm O2-PFC (HBO-PFC) was prepared using a custom-made hyperbaric oxygen tank and administered to the abdominal cavity during the SMA clamp. Ileal tissue adenosine triphosphate (ATP) levels 90 min after SMA declamping were determined using luciferase assay. To assess intestinal mucosal barrier function at 90 min after release of the SMA clip, everted gut sacs were prepared to measure the mucosal-to-serosal passage of fluorescein-conjugated dextran (FD4, molecular weight = 4 kDa). Thirty minutes after i.p. administration, partial pressure of oxygen in HBO-PFC remained around 1000 mmHg, whereas partial pressure of oxygen in 1 atm O2-PFC decreased to around 400 mmHg. The intestinal tissue ATP was significantly preserved in group D. Moreover, the mucosal hyperpermeability of the gut sac after I/R was significantly ameliorated in group D. Hyperbarically oxygenated perfluorochemical might supply a larger amount of oxygen to ischemic tissue during SMA clamp, which protected the small intestine from I/R injury, possibly caused by the maintenance of tissue ATP levels during ischemia.

Adrenomedullin and adrenomedullin-binding protein-1 downregulate inflammatory cytokines and attenuate tissue injury after gut ischemia-reperfusion

Recent studies have shown that adrenomedullin (AM) and AM-binding protein-1 (AMBP-1) possess anti-inflammatory properties in sepsis. We hypothesized that administration of AM/AMBP-1 after gut ischemia-reperfusion (I/R) downregulates inflammatory cytokines and attenuates tissue injury. Male Sprague-Dawley rats (275-325 g) were used. Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery (SMA) for 90 minutes. Upon release of the SMA clamp, the animals were treated by AM (12 microg per kilogram of body weight) and AMBP-1 (40 microg per kilogram of body weight) in combination, or vehicle (1 mL 0.9% NaCl) over 30 minutes via a femoral vein catheter. The animals undergoing sham operation or ischemia for 90 minutes only, did not receive AM/AMBP-1 treatment. At 60 minutes after the completion of the treatment (ie, 90 minutes after reperfusion), blood samples were collected. Plasma AM and AMBP-1 were measured by radioimmunoassay and Western blot analysis, respectively. Serum levels of TNF-alpha, interleukin (IL)-1beta, IL-6, IL-10, transaminases (ie, alanine aminotransaminase, aspartate aminotransaminase), lactate, and creatinine were determined with the use of enzyme-linked immunosorbent assay and other standard methods. In additional groups of animals, the 10-day survival rate was recorded after gut I/R. Ischemia alone was sufficient to downregulate both AM and AMBP-1. Unlike AMBP-1 that remained decreased, AM levels increased significantly after reperfusion. I/R but not ischemia alone significantly increased serum levels of inflammatory cytokines. Moreover, I/R-induced tissue injury was evidenced by increased levels of transaminases, lactate, and creatinine. Administration of AM/AMBP-1 after ischemia, however, markedly reduced cytokine levels, attenuated tissue injury, and improved survival. AM/AMBP-1 may be a novel treatment to attenuate the reperfusion injury after gut ischemia.

Role of protein kinase C in intestinal ischemic preconditioning 1,2

Tissue protection by ischemic preconditioning (IPC) has been previously characterized in organs such as the heart and involves at least in part PKC activation. It is not yet clear whether such preconditioning against ischemia/reperfusion (I/R) injury operates in the intestine, and, if so, whether IPC involves protein kinase C (PKC). IPC of the small intestine in male Sprague Dawley rats was induced by 10-min superior mesenteric artery (SMA) clamp followed by 120-min reperfusion. Sham-operated control or IPC rats were then rechallenged with 20-min SMA clamp. Histological injury to jejunal mucosa was assessed by microscopic examination and Parks' injury score (Grade 0-4; 0 = no damage). PKC activity was determined by immunoprecipitation of specific isoforms followed by in vitro kinase assay using mucosal scrapings of the harvested jejunum. Data were expressed as mean +/- SEM and analyzed by one-way ANOVA with multiple comparison tests. Ten-minute SMA clamp led to epithelial damage that was fully reversed by 120-min reperfusion. Activity of several PKC isoforms (PKCalpha, -delta, -epsilon) increased after 10-min ischemia. Epithelial injury associated with 20-min SMA clamp was attenuated by prior IPC. The protective effect of IPC on intestinal mucosa was prevented when animals were pretreated with the conventional (c) and novel (n) PKC inhibitor Go6850, but not with Go6976 (selective cPKC inhibitor), rottlerin (selective PKCdelta inhibitor), or saline control. Brief mesenteric ischemia induces a reversible epithelial injury in rats associated with activation of several PKC isoforms. Injury induced by mesenteric ischemia is reduced by brief ischemic preconditioning, an effect that is abolished by nonselective PKC inhibition but not by a selective inhibitor of cPKC or PKCdelta. The results suggest that activation of nPKC isoform(s), especially PKCepsilon during and following ischemic insults (IPC), may play an important role in protection against I/R injury in the intestine.

Enteral glutamine but not alanine maintains small bowel barrier function after ischemia/reperfusion injury in rats.

We previously demonstrated that glucose and glutamine, solutes metabolized by the gut, replenish ATP and enhance gut function compared with alanine, a solute not metabolized by the gut, following mesenteric ischemia/reperfusion (I/R). The purpose of the present study was to determine if the nonmetabolizable solute alanine differentially modulates cytoskeletal organization and paracellular small intestinal permeability compared with the metabolizable solutes glucose and glutamine following mesenteric I/R. At laparotomy, rats had jejunal sacs filled with 10 mM glucose, glutamine, alanine, or magnesium sulfate (5 mm, osmotic control) followed by superior mesenteric artery clamping for 60 min and 30 min of reperfusion or sham laparotomy. Jejunum was harvested for evaluation by deconvolution microscopy, fluorescent measurement of F:G actin ratio, or mounted in an Ussing chamber for determination of intestinal permeability. Deconvolution microscopy revealed that the actin cytoskeleton was preserved by enteral glutamine, comparable to shams, but disrupted by enteral alanine. Glucose and controls resulted in comparable disruption, which was less than that with alanine. The F:G actin ratio was highest for glutamine and lowest for alanine; glucose was comparable to controls. Intestinal permeability was highest for alanine and lowest for glutamine, which was comparable to shams. Permeability following glucose and controls was higher than that following glutamine but lower than that following alanine. The nonmetabolizable solute alanine resulted in disruption of the actin cytoskeleton and enhanced intestinal permeability under conditions of mesenteric I/R. The metabolizable solute glutamine was protective under these conditions, whereas glucose exerted minimal effect on the integrity of the cytoskeleton and intestinal permeability. The individual components of enteral diets may differentially modulate intestinal barrier function, which could have important implications when administered to critically injured patients.