Ex Vivo Perfusion With Methylprednisolone Attenuates Brain Death-induced Lung Injury in Rats.

The onset of brain death (BD) leads to the deterioration of potential donor lungs. Methylprednisolone is considered to increase lung oxygenation capacity and enhance the procurement yield of donor lungs, when applied in situ, during donor management. However, whether BD-induced lung damage is ameliorated upon treatment with methylprednisolone during acellular ex vivo lung perfusion (EVLP), remains unknown. We aimed to investigate whether the quality of lungs from brain-dead donors improves upon methylprednisolone treatment during EVLP. METHODS: Rat lungs were randomly assigned to 1 of 3 experimental groups (n = 8/group): (1) healthy, directly procured lungs subjected to EVLP; (2) lungs from brain-dead rats subjected to cold storage and EVLP; and (3) lungs from brain-dead rats subjected to cold storage and EVLP with 40 mg methylprednisolone added to the perfusate. Ventilation and perfusion parameters, histology, edema formation, metabolic profile, and inflammatory status of lungs were investigated. RESULTS: Methylprednisolone treated lungs from brain-dead donors improved positive inspiratory pressures needed to maintain tidal volumes of 7 mL/kg of body weight, which was 25.6 ± 5.8 cm H2O in untreated lungs and 18.0 ± 3.0 cm H2O in methylprednisolone treated lungs, after 6 h EVLP. Furthermore, dynamic lung compliance increased upon methylprednisolone treatment, with values of 0.11 ± 0.05 mL/cm H2O versus 0.18 ± 0.04 mL/cm H2O after 6 h of EVLP. Methylprednisolone treatment ameliorated the amount of lung edema, as corroborated by a reduction of 0.7 in the wet/dry ratio. Although glucose consumption levels were comparable, the BD-induced cumulative lactate production decreased from 0.44 ± 0.26 to 0.11 ± 0.16 mmol/L upon methylprednisolone treatment. Finally, BD-induced inflammatory status was reduced upon methylprednisolone treatment compared to untreated lungs from brain-dead donors, as reflected by lower proinflammatory gene expression levels of IL-1ß, IL-6 and MCP-1, and IL-6 perfusate levels. CONCLUSIONS: We showed that methylprednisolone treatment during EVLP attenuates BD-induced lung injury.

Brain death-induced lung injury is complement dependent, with a primary role for the classical/lectin pathway.

In brain-dead donors immunological activation occurs, which deteriorates donor lung quality. Whether the complement system is activated and which pathways are herein involved, remain unknown. We aimed to investigate whether brain death (BD)-induced lung injury is complement dependent and dissected the contribution of the complement activation pathways. BD was induced and sustained for 3 hours in wild-type (WT) and complement deficient mice. C3-/- mice represented total complement deficiency, C4-/- mice represented deficiency of the classical and lectin pathway, and factor properdin (P)-/- mice represented alternative pathway deficiency. Systemic and local complement levels, histological lung injury, and pulmonary inflammation were assessed. Systemic and local complement levels were reduced in C3-/- mice. In addition, histological lung injury and inflammation were attenuated, as corroborated by influx of neutrophils and gene expressions of interleukin (IL)-6, IL-8-like KC, TNF-α, E-selectin, and MCP-1. In C4-/- mice, complement was reduced on both systemic and local levels and histological lung injury and inflammatory status were ameliorated. In P-/- mice, histological lung injury was attenuated, though systemic and local complement levels, IL-6 and KC gene expressions, and neutrophil influx were not affected. We demonstrated that BD-induced lung injury is complement dependent, with a primary role for the classical/lectin activation pathway.

Critical role for complement receptor C5aR2 in the pathogenesis of renal ischemia-reperfusion injury.

The complement system, and specifically C5a, is involved in renal ischemia-reperfusion (IR) injury. The 2 receptors for complement anaphylatoxin C5a (C5aR1 and C5aR2) are expressed on leukocytes as well as on renal epithelium. Extensive evidence shows that C5aR1 inhibition protects kidneys from IR injury; however, the role of C5aR2 in IR injury is less clear as initial studies proposed the hypothesis that C5aR2 functions as a decoy receptor. By Using wild-type, C5aR1-/-, and C5aR2-/- mice in a model of renal IR injury, we found that a deficiency of either of these receptors protected mice from renal IR injury. Surprisingly, C5aR2-/- mice were most protected and had lower creatinine levels and reduced acute tubular necrosis. Next, an in vivo migration study demonstrated that leukocyte chemotaxis was unaffected in C5aR2-/- mice, whereas neutrophil activation was reduced by C5aR2 deficiency. To further investigate the contribution of renal cell-expressed C5aR2 vs leukocyte-expressed C5aR2 to renal IR injury, bone marrow chimeras were created. Our data show that both renal cell-expressed C5aR2 and leukocyte-expressed C5aR2 mediate IR-induced renal dysfunction. These studies reveal the importance of C5aR2 in renal IR injury. They further show that C5aR2 is a functional receptor, rather than a decoy receptor, and may provide a new target for intervention.-Poppelaars, F., van Werkhoven, M. B., Kotimaa, J., Veldhuis, Z. J., Ausema, A., Broeren, S. G. M., Damman, J., Hempel, J. C., Leuvenink, H. G. D., Daha, M. R., van Son, W. J., van Kooten, C., van Os, R. P., Hillebrands, J.-L., Seelen, M. A. Critical role for complement receptor C5aR2 in the pathogenesis of renal ischemia-reperfusion injury.

Transient von Willebrand factor-mediated platelet influx stimulates liver regeneration after partial hepatectomy in mice.

BACKGROUND & AIMS: In addition to their function in thrombosis and haemostasis, platelets play an important role in the stimulation of liver regeneration. It has been suggested that platelets deliver mitogenic cargo to the regenerating liver, and accumulation of platelets in the regenerating liver has been demonstrated. We studied kinetics of platelet influx in the regenerating liver and investigated the signal that initiates platelet influx. METHODS: We visualized platelets in the liver remnant after partial hepatectomy in mice using intravital microscopy and assessed liver regeneration by examination of liver/body weight ratio and the number of proliferating hepatocytes examined by immunohistochemistry. RESULTS: We demonstrated rapid but transient platelet influx into the liver remnant after a partial liver resection. Liver regeneration in thrombocytopenic mice was substantially impaired as evidenced by a reduced liver-to-body weight ratio and decreased numbers of proliferating hepatocytes at day 3 compared to mice with normal platelet counts. In contrast, liver regeneration was only mildly impaired when thrombocytopaenia was induced 2 hours after partial liver resection. Platelet influx into the liver remnant was virtually absent in the presence of an antibody to von Willebrand factor (VWF) suggesting that VWF release from liver sinusoidal endothelial cells mediates platelet influx. Additionally, liver regeneration in mice deficient in VWF was markedly impaired. CONCLUSIONS: A rapid but transient VWF-dependent platelet influx into the liver remnant drives platelet-mediated liver regeneration.

Vitamin E Attenuates the Progression of Non-Alcoholic Fatty Liver Disease Caused by Partial Hepatectomy in Mice.

BACKGROUND AND AIM: The progression of non-alcoholic fatty liver disease (NAFLD) likely involves a 'multiple hit' mechanism. We hypothesized that partial hepatectomy, a procedure performed frequently in patients with NAFLD, would accelerate the progression of disease. METHODS: C57BL/6JolaHsd mice were fed a choline-deficient L-amino acid-defined diet (CD-AA) or a choline-sufficient L-amino acid-defined control diet (CS-AA). Part of the mice in the CD-AA group received a diet enriched in vitamin E (~20 mg /day). Two weeks after the start of the diet, mice underwent a partial hepatectomy or a sham operation. RESULTS: In the CD-AA group, NAFLD activity scores were significantly higher at 7 days after partial hepatectomy compared to the sham operated mice (3.7 ± 1.3 vs. 1.8 ± 0.7; P<0.05). In addition, TBARS, a measure for oxidative stress, in liver tissue of the CD-AA group were significantly higher at day 1, 3 and 7 after partial hepatectomy compared to the sham operated mice (P<0.05). Vitamin E therapy significantly reduced TBARS level at day 7 after partial hepatectomy compared to the CD-AA diet group (P< 0.05). Vitamin E suppletion reduced NAFLD activity score at day 7 after partial hepatectomy compared to the CD-AA group (2.3 ± 0.8 vs. 3.8 ± 1.0; P<0.05). CONCLUSION: Partial hepatectomy accelerates the progression of NAFLD. Disease progression induced by partial hepatectomy is substantially attenuated by vitamin E.

Diffuse reflectance spectroscopy accurately quantifies various degrees of liver steatosis in murine models of fatty liver disease.

BACKGROUND: A real-time objective evaluation for the extent of liver steatosis during liver transplantation is currently not available. Diffuse reflectance spectroscopy (DRS) rapidly and accurately assesses the extent of steatosis in human livers with mild steatosis. However, it is yet unknown whether DRS accurately quantifies moderate/severe steatosis and is able to distinguish between micro- and macrovesicular steatosis. METHODS: C57BL/6JolaHsd mice were fed wit a choline-deficient L-amino acid-defined diet (CD-AA) or a choline-sufficient L-amino acid-defined control diet (CS-AA) for 3, 8, and 20 weeks. In addition B6.V-Lepob/OlaHsd (ob/ob) mice and their lean controls were studied. A total of 104 DRS measurements were performed in liver tissue ex vivo. The degree of steatosis was quantified from the DRS data and compared with histopathological analysis. RESULTS: When assessed by histology, livers of mice fed with a CD-AA and CS-AA diet displayed macrovesicular steatosis (range 0-74 %), ob/ob mice revealed only microvesicular steatosis (range 75-80 %), and their lean controls showed no steatosis. The quantification of steatosis by DRS correlated well with pathology (correlation of 0.76 in CD-AA/CS-AA fed mice and a correlation of 0.75 in ob/ob mice). DRS spectra did not distinguish between micro- and macrovesicular steatosis. In samples from CD-AA/CS-AA fed mice, the DRS was able to distinguish between mild and moderate/severe steatosis with a sensitivity and specificity of 86 and 81 %, respectively. CONCLUSION: DRS can quantify steatosis with good agreement to histopathological analysis. DRS may be useful for real-time objective evaluation of liver steatosis during liver transplantation, especially to differentiate between mild and moderate/severe steatosis.