Intraperitoneal Mesalamine Does Not Restores Mesenteric Perfusion or Prevent Mucosal Injury Following Intestinal Ischemia and Reperfusion

Background and Hypothesis: Acute Mesenteric Ischemia (AMI) is characterized by a sudden decrease in blood flow to varying segments of the small intestine. It can lead to cellular damage, life-threatening intestinal necrosis and, if corrected, subsequent reperfusion injury. Reducing inflammation is key to preventing further cell damage. We therefore hypothesized that administration of mesalamine prior to intestinal ischemia would reduce epithelial cell damage by I/R and restore mesenteric perfusion. Project Methods: C57Bl6J wild type (WT) mice were assigned to mesalamine or vehicle treatment groups (N=8/group). Prior to surgery, mice underwent intraperitoneal injection of treatment. Midline laparotomy was performed. Intestines were eviscerated, superior mesenteric artery (SMA) located, and baseline intestinal perfusion determined using Laser Doppler. SMA was then occluded to induce intestinal ischemia for sixty minutes, thereafter the occlusion was removed. Mesenteric reperfusion was then determined by Laser Doppler. Midline incisions were reapproximated with suture and animals were allowed to recover. After twentyfour hours, animals were re-anesthetized and underwent final assessment of mesenteric perfusion by Laser-Doppler. Animals were then euthanized, and intestines explanted. A portion of tissue was snap frozen for assessment for proinflammatory cytokines by ELISA. Another portion of tissue was stained with H&E and scored for intestinal mucosal injury. Data were assessed for normalcy and compared by Mann-Whitney-U test. P<.05 was significant. Results: Preliminary data suggests mesalamine treated mice show no significant change in mortality compared to vehicle. Mesalamine treated mice also show an insignificant increase in histological damage score. Despite this, they show an insignificant improvement in oxygen perfusion. Conclusion: Intraperitoneal mesalamine administration does not appear to be a useful method for limiting cell damage in GI diseases associated with AMI such as necrotizing enterocolitis. A larger sample size is needed to further elucidate treatment effects.

SILDENAFIL IS NOT PROTECTIVE TO THE BOWEL FOLLOWING INTESTINAL ISCHEMIA AND REPERFUSION INJURY

Background and Hypothesis: Acute Mesenteric Ischemia (AMI) occurs when blood supply to the intestine is decreased. This can lead to intestinal ischemia, cellular damage, necrosis, and if corrected, subsequent reperfusion injury. There are currently no medical treatments to help reverse ischemia and reperfusion injury (I/R) and, therefore novel treatments are necessary. Sildenafil, a compound that increases endogenous nitric oxide (NO) by blocking the phosphodiesterase-5 induced breakdown of cGMP, may function as a potent mesenteric vasodilator. We therefore hypothesized that sildenafil would improve mesenteric perfusion during a mouse model of intestinal I/R. Experimental Design or Project Methods: Adult male C57Bl6J mice were anesthetized with isoflurane and a midline laparotomy performed. The base of the superior mesenteric artery was occluded with a non-crushing vascular clamp for 60 minutes. At the end of ischemia, sildenafil (1mg/kg, 10mg/kg, or 100mg/kg) or a PBS vehicle control were administered via intraperitoneal injection. Animals were then allowed to recover. Twenty-four hours after ischemia, animals underwent assessment of mesenteric perfusion by Laser-Doppler imaging. Animals were then euthanized. Perfusion was expressed as a percentage of baseline, depicted as mean +/- SEM, and analyzed by ANOVA. P<0.05 was significant. Results: There were no significant differences in mesenteric perfusion between the vehicle group or any of the therapeutic groups. (PBS: 53.03±11.35%; Sildenafil-low: 59.59±7.55%; Sildenafil-medium: 70.51±8.49; Sildenafil-high: 66.4±10.2, p=0.61). Conclusion: Sildenafil does not appear to be an effective treatment for improving mesenteric perfusion following intestinal ischemia. Further studies are required to determine the reasons for the ineffectiveness of sildenafil. One possible explanation for these observations could be a lack of PGE5 in the intestinal vascular endothelium.

Infliximab alleviates the mortality, mesenteric hypoperfusion, aortic dysfunction, and multiple organ damage in septic rats

Tumor necrosis factor-alpha (TNF-α) is a pivotal mediator that triggers inflammatory process, oxidative stress, and multiple organ injury in sepsis. We investigated the effects of infliximab on survival, mesenteric artery blood flow (MBF), vascular reactivity, and oxidative and inflammatory injuries in cecal ligation and puncture (CLP)-induced sepsis. Wistar rats were divided into Sham, CLP, Sham+infliximab, and CLP+infliximab subgroups. Twenty-four hours before the operations, rats were injected intraperitoneally with infliximab (7 mg/kg) or vehicle (saline; 1 mL/kg). Twenty hours after the operations, MBF and phenylephrine responses of isolated aortic rings were measured. Tissue damages were examined biochemically and histopathologically. Furthermore, survival rates were monitored throughout 96 h. Infliximab improved survival, mesenteric perfusion, and aortic function after CLP. Increases of serum AST, ALT, LDH, BUN, Cr, and inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6) induced by CLP were blocked by infliximab. Infliximab prevented malondialdehyde elevations in septic liver, lung, spleen, and kidney tissues, as well as glutathione reductions in septic liver, spleen, and kidney tissues. Protective effects of infliximab on multiple organ damage were also observed histopathologically. Infliximab showed protective effects in sepsis due to its improvement effects on mesenteric perfusion, aortic function, and its anti-inflammatory and antioxidative effects.

Hydrogen sulfide improves intestinal recovery following ischemia by endothelial nitric oxide-dependent mechanisms

Hydrogen sulfide (H2S) is an endogenous gasotransmitter that has vasodilatory properties. It may be a novel therapy for intestinal ischemia-reperfusion (I/R) injury. We hypothesized that 1) H2S would improve postischemic survival, mesenteric perfusion, mucosal injury, and inflammation compared with vehicle and 2) the benefits of H2S would be mediated through endothelial nitric oxide. C57BL/6J wild-type and endothelial nitric oxide synthase knockout (eNOS KO) mice were anesthetized, and a midline laparotomy was performed. Intestines were eviscerated, the small bowel mesenteric root identified, and baseline intestinal perfusion was determined using laser Doppler. Intestinal ischemia was established by temporarily occluding the superior mesenteric artery. Following ischemia, the clamp was removed, and the intestines were allowed to recover. Either sodium hydrosulfide (2 nmol/kg or 2 µmol/kg NaHS) in PBS vehicle or vehicle only was injected into the peritoneum. Animals were allowed to recover and were assessed for mesenteric perfusion, mucosal injury, and intestinal cytokines. P values < 0.05 were significant. H2S improved mesenteric perfusion and mucosal injury scores following I/R injury. However, in the setting of eNOS ablation, there was no improvement in these parameters with H2S therapy. Application of H2S also resulted in lower levels of intestinal cytokine production following I/R. Intraperitoneal H2S therapy can improve mesenteric perfusion, intestinal mucosal injury, and intestinal inflammation following I/R. The benefits of H2S appear to be mediated through endothelial nitric oxide-dependent pathways. NEW & NOTEWORTHY H2S is a gaseous mediator that acts as an anti-inflammatory agent contributing to gastrointestinal mucosal defense. It promotes vascular dilation, mucosal repair, and resolution of inflammation following intestinal ischemia and may be exploited as a novel therapeutic agent. It is unclear whether H2S works through nitric oxide-dependent pathways in the intestine. We appreciate that H2S was able to improve postischemic recovery of mesenteric perfusion, mucosal integrity, and inflammation. The beneficial effects of H2S appear to be mediated through endothelial nitric oxide-dependent pathways.

The human milk oligosaccharide 2′-fucosyllactose attenuates the severity of experimental necrotising enterocolitis by enhancing mesenteric perfusion in the neonatal intestine

Necrotising enterocolitis (NEC) is a common disease in premature infants characterised by intestinal ischaemia and necrosis. The only effective preventative strategy against NEC is the administration of breast milk, although the protective mechanisms remain unknown. We hypothesise that an abundant human milk oligosaccharide (HMO) in breast milk, 2′-fucosyllactose (2′FL), protects against NEC by enhancing intestinal mucosal blood flow, and we sought to determine the mechanisms underlying this protection. Administration of HMO-2′FL protected against NEC in neonatal wild-type mice, resulted in a decrease in pro-inflammatory markers and preserved the small intestinal mucosal architecture. These protective effects occurred via restoration of intestinal perfusion through up-regulation of the vasodilatory molecule endothelial nitric oxide synthase (eNOS), as administration of HMO-2′FL toeNOS-deficient mice or to mice that received eNOS inhibitors did not protect against NEC, and by 16S analysis HMO-2′FL affected the microbiota of the neonatal mouse gut, although these changes do not seem to be the primary mechanism of protection. Induction of eNOS by HMO-2′FL was also observed in cultured endothelial cells, providing a link between eNOS and HMO in the endothelium. These data demonstrate that HMO-2′FL protects against NEC in part through maintaining mesenteric perfusion via increased eNOS expression, and suggest that the 2′FL found in human milk may be mediating some of the protective benefits of breast milk in the clinical setting against NEC.