I-FABP as biomarker for the early diagnosis of acute mesenteric ischemia and resultant lung injury

Abstract

Acute mesenteric ischemia (AMI) is a life-threatening condition that can result in multiple organ injury and death. A timely diagnosis and treatment would have a significant impact on the morbidity and mortality in high-risk patient population. The purpose of this study was to investigate if intestinal fatty acid binding protein (I-FABP) and α-defensins can be used as biomarkers for early AMI and resultant lung injury. C57BL/6 mice were subjected to intestinal ischemia by occlusion of the superior mesenteric artery. A time course of intestinal ischemia from 0.5 to 3 h was performed and followed by reperfusion for 2 h. Additional mice were treated with N-acetyl-cysteine (NAC) at 300 mg/kg given intraperitoneally prior to reperfusion. AMI resulted in severe intestinal injury characterized by neutrophil infiltrate, myeloperoxidase (MPO) levels, cytokine/chemokine levels, and tissue histopathology. Pathologic signs of ischemia were evident at 1 h, and by 3 h of ischemia, the full thickness of the intestine mucosa had areas of coagulative necrosis. It was noted that the levels of α-defensins in intestinal tissue peaked at 1 h and I-FABP in plasma peaked at 3 h after AMI. Intestinal ischemia also resulted in lung injury in a time-dependent manner. Pretreatment with NAC decreased the levels of intestinal α-defensins and plasma I-FABP, as well as lung MPO and cytokines. In summary, the concentrations of intestinal α-defensins and plasma I-FABP predicted intestinal ischemia prior to pathological evidence of ischemia and I-FABP directly correlated with resultant lung injury. The antioxidant NAC reduced intestinal and lung injury induced by AMI, suggesting a role for oxidants in the mechanism for distant organ injury. I-FABP and α-defensins are promising biomarkers, and may guide the treatment with antioxidant in early intestinal and distal organ injury.

Figure 1. Survival and intestinal injury following acute mesenteric ischemia and reperfusion.

A, There was no statistical difference in mice survival following 0.5, 1, 2, or 3 h (n = 6, 12, 15, 5, respectively) ischemia times with 2 h of reperfusion compared to sham treated mice (n = 5). B, Histological examination of intestinal tissue illustrated that there was no difference between 0.5 h ischemia and sham treated mice; however, tissue injury started to become visible after 1 h ischemia with significance in histological score seen after 2 and 3 h of ischemia (n = 3 for all groups). C, Intestinal myeloperoxidase (MPO) corroborated histological score with increased MPO activity found after 1, 2, and 3 h ischemia compared to sham mice(n = 3 for all groups). D, MPO activity mirrored the number of neutrophils found infiltrating the intestinal tissue, where compared to sham treated mice, significant neutrophil infiltrate was found after 1, 2, and 3 h ischemia (n = 3 for all groups). E, Hematoxylin and eosin staining illustrated that intestinal tissue in sham treated animals was normal (100x). F, After 1 h ischemia, however, we begin to observe denuded villi and increased polymorphonuclear (PMN) infiltrates (400x). G, At 3 h ischemia, the full thickness of the intestinal mucosa had areas of coagulative necrosis and complete mucosal erosions (400x). * p<0.05 compared to sham treated mice.

Figure 2. I-FABP as novel biomarker for ischemia/reperfusion injury.

A, Plasma intestinal (I)-FABP, measured via ELISA, was found to be significantly elevated after ischemia at all time points compared to sham animals (n = 3, 4, 4, 4, 5 for sham, 0.5, 1, 2 and 3 h, respectively). Of note was the detection of I-FABP after 0.5 h (n = 3), well before pathological injury was observed (64±18 versus 6.9±0.9 for shams). B, Detection of defensins in intestinal homogenates illustrated that significant levels after 1 and 2 h ischemia compared to shams (n = 3 for all groups). * p<0.05 compared to sham treated mice.

Figure 3. Distant organ injury – increased lung MPO and injury.

A, MPO on lung homogenates illustrated increased activity after 1, 2, and 3 h intestinal ischemia compared to sham treated mice (n = 4 for all groups). B, Quantification of the number of neutrophils infiltrate in the lungs showed significance also after 1, 2, and 3 h of ischemia (n = 4, 5, 6, 6, 7 for sham, 0.5, 1, 2, 3 respectively). C, Histology of a sham treated mice illustrated normal lung parenchyma (400x). D, However, PMN infiltrates were observed in the lung after 2 h of intestinal ischemia (400x). * p<0.05 compared to sham treated mice.

Figure 4. Increased lung inflammation following intestinal ischemia/reperfusion.

Lung homogenates were used to measure inflammatory chemokines and cytokines. A, There was greater concentration of keratinocyte-derived chemokine (KC) after 1, 2, and 3 h of intestinal ischemia compared to sham controls. B, Meanwhile monocyte chemoattractant protein-1 (MCP-1) showed significance after 2 h. C, Inflammatory cytokine IL-1β was found significantly elevated at 2 h ischemia, while, D, TNF-α was significantly elevated after 1 h of intestinal ischemia. (n = 3 for sham, n = 4 for all ischemia treated mice) * p<0.05 compared to sham treated mice.

Figure 5. NAC reduces oxidative injury.

To determine the mechanism behind distal organ injury, mice were pretreated intraperitoneally with, N-acetyl-cysteine (NAC). A, Compared to mice given vehicle control (No-NAC), NAC treated mice had significantly lower plasma I-FABP after 2 h of intestinal ischemia. B, NAC also affected defensins at 1 h ischemia, though no significance was achieved. C, MPO activity of lung homogenates illustrated a significant reduction in mice that were treated with NAC compared to vehicle controls (No-NAC) at 1 h ischemia. D, There was also significant reduction in TNF-α cytokine in lung homogenates after NAC treatment at 1 h ischemia. E, At 2 h ischemia, there was slight reduction in IL-1β in the NAC treated mice compared to vehicle controls, though significance was not achieved probably due to the short half-life of NAC. (n = 3 for shams, n = 4 for both No-NAC and NAC) ^# p<0.05 compared to vehicle (No-NAC) treated mice.