A Metabolomic Approach (1H HRMAS NMR Spectroscopy) Supported by Histology to Study Early Post-transplantation Responses in Islet-transplanted Livers.

Intrahepatic transplantation of islets requires a lot of islets because more than 50% of the graft is lost during the 24 hours following transplantation. We analyzed, in a rat model, early post-transplantation inflammation using systemic inflammatory markers, or directly in islet-transplanted livers by immunohistochemistry. 1H HRMAS NMR was employed to investigate metabolic responses associated with the transplantation. Inflammatory markers (Interleukin-6, α2-macroglobulin) are not suitable to follow islet reactions as they are not islet specific. To study islet specific inflammatory events, immunohistochemistry was performed on sections of islet transplanted livers for thrombin (indicator of the instant blood-mediated inflammatory reaction (IBMIR)) and granulocytes and macrophages. We observed a specific correlation between IBMIR and granulocyte and macrophage infiltration after 12 h. In parallel, we identified a metabolic response associated with transplantation: after 12 h, glucose, alanine, aspartate, glutamate and glutathione were significantly increased. An increase of glucose is a marker of tissue degradation, and could be explained by immune cell infiltration. Alanine, aspartate and glutamate are inter-connected in a common metabolic pathway known to be activated during hypoxia. An increase of glutathione revealed the presence of antioxidant protection. In this study, IBMIR visualization combined with 1H HRMAS NMR facilitated the characterization of cellular and molecular pathways recruited following islet transplantation.

Enhanced-reality video fluorescence: a real-time assessment of intestinal viability.

OBJECTIVE: Our aim was to evaluate a fluorescence-based enhanced-reality system to assess intestinal viability in a laparoscopic mesenteric ischemia model. MATERIALS AND METHODS: A small bowel loop was exposed, and 3 to 4 mesenteric vessels were clipped in 6 pigs. Indocyanine green (ICG) was administered intravenously 15 minutes later. The bowel was illuminated with an incoherent light source laparoscope (D-light-P, KarlStorz). The ICG fluorescence signal was analyzed with Ad Hoc imaging software (VR-RENDER), which provides a digital perfusion cartography that was superimposed to the intraoperative laparoscopic image [augmented reality (AR) synthesis]. Five regions of interest (ROIs) were marked under AR guidance (1, 2a-2b, 3a-3b corresponding to the ischemic, marginal, and vascularized zones, respectively). One hour later, capillary blood samples were obtained by puncturing the bowel serosa at the identified ROIs and lactates were measured using the EDGE analyzer. A surgical biopsy of each intestinal ROI was sent for mitochondrial respiratory rate assessment and for metabolites quantification. RESULTS: Mean capillary lactate levels were 3.98 (SD = 1.91) versus 1.05 (SD = 0.46) versus 0.74 (SD = 0.34) mmol/L at ROI 1 versus 2a-2b (P = 0.0001) versus 3a-3b (P = 0.0001), respectively. Mean maximal mitochondrial respiratory rate was 104.4 (±21.58) pmolO2/second/mg at the ROI 1 versus 191.1 ± 14.48 (2b, P = 0.03) versus 180.4 ± 16.71 (3a, P = 0.02) versus 199.2 ± 25.21 (3b, P = 0.02). Alanine, choline, ethanolamine, glucose, lactate, myoinositol, phosphocholine, sylloinositol, and valine showed statistically significant different concentrations between ischemic and nonischemic segments. CONCLUSIONS: Fluorescence-based AR may effectively detect the boundary between the ischemic and the vascularized zones in this experimental model.

NMR HRMAS spectroscopy of lung biopsy samples: Comparison study between human, pig, rat, and mouse metabolomics.

PURPOSE: Using the metabolomics by NMR high-resolution magic angle spinning spectroscopy, we assessed the lung metabolome of various animal species in order to identify the animal model that could be substituted to human lung in studies on fresh lung biopsies. METHODS: The experiments were conducted on intact lung biopsy samples of pig, rat, mouse, and human using a Bruker Advance III 500 spectrometer. Thirty-five to 39 metabolites were identified and 23 metabolites were quantified. Principal component analysis, partial least-squares discriminant analysis, and analysis of variance tests were performed in order to compare the metabolic profiles of each animal lung biopsies to those of the human lung. RESULTS: The metabolic composition between human and pig lung was similar. However, human lung was distinguishable from mouse and rat regarding: Trimethylamine N-oxide and betaïne which were present in rodents but not in human lung, carnitine, and glycerophosphocholine which were present in mouse but not in human lung. Conversely, succinic acid was undetected in rat lung. Furthermore, fatty acids concentration was significantly higher in rodent lungs compared to human lung. CONCLUSION: Using the metabolomics by NMR high-resolution magic angle spinning spectroscopy on lung biopsy, samples allowed to highlight that pig lung seems to be close to human lung as regarding its metabolite composition with more similarities than dissimilarities.

The assessment of the quality of the graft in an animal model for lung transplantation using the metabolomics 1H high-resolution magic angle spinning NMR spectroscopy.

Standards are needed to control the quality of the lungs from nonheart-beating donors as potential grafts. This was here assessed using the metabolomics 1H high-resolution magic angle spinning NMR spectroscopy. Selective perfusion of the porcine bilung block was set up 30 min after cardiac arrest with cold Perfadex®. Lung alterations were analyzed at 3, 6, and 8 h of cold ischemia as compared to baseline and to nonperfused lung. Metabolomics analysis of lung biopsies allowed identification of 35 metabolites. Levels of the majority of the metabolites increased over time at 4°C without perfusion, indicating cellular degradation, whereas levels of glutathione decreased. When lung was perfused at 4°C, levels of the majority of the metabolites remained stable, including levels of glutathione. Levels of uracil by contrast showed a reverse profile, as its signal increased over time in the absence of perfusion while being totally absent in perfused samples. Our results showed glutathione and uracil as potential biomarkers for the quality of the lung. The metabolomics 1H high-resolution magic angle spinning NMR spectroscopy can be efficiently applied for the assessment of the quality of the lung as an original technique characterized by a rapid assessment of intact biopsy samples without extraction and can be implemented in hospital environment.