RIPK3 mediates pathogenesis of experimental ventilator-induced lung injury.

In patients requiring ventilator support, mechanical ventilation (MV) may induce acute lung injury (ventilator-induced lung injury [VILI]). VILI is associated with substantial morbidity and mortality in mechanically ventilated patients with and without acute respiratory distress syndrome. At the cellular level, VILI induces necrotic cell death. However, the contribution of necroptosis, a programmed form of necrotic cell death regulated by receptor-interacting protein-3 kinase (RIPK3) and mixed-lineage kinase domain-like pseudokinase (MLKL), to the development of VILI remains unexplored. Here, we show that plasma levels of RIPK3, but not MLKL, were higher in patients with MV (i.e., those prone to VILI) than in patients without MV (i.e., those less likely to have VILI) in two large intensive care unit cohorts. In mice, RIPK3 deficiency, but not MLKL deficiency, ameliorated VILI. In both humans and mice, VILI was associated with impaired fatty acid oxidation (FAO), but in mice this association was not observed under conditions of RIPK3 deficiency. These findings suggest that FAO-dependent RIPK3 mediates pathogenesis of acute lung injury.

A novel triple immunoenzyme staining enables simultaneous identification of all muscle fiber types on a single skeletal muscle cryosection from normal, denervated or reinnervated rats.

Triple immunofluorescence staining has recently been developed to simultaneously identify all muscle fibers on a single cryosection which is helpful for clinical and basic research, but it has disadvantages such as fast photobleaching and unclear outlines of muscle fibers. Triple immunoenzyme staining (TIE) is likely to avoid these disadvantages. In this study, we aimed to establish a sensitive and specific TIE technique to identify fiber types in normal, denervated, and reinnervated rat muscles, and to develop a systematic sampling method for muscle fiber quantification. Tibialis anterior and soleus from normal, denervated, and reinnervated Lewis rat hind limbs were used. Five consecutive cryosections were cut from each muscle, including one for TIE and four for single immunoenzyme staining (SIE). The TIE was performed using the polymerized reporter enzyme staining system for the first two antigens (A4.74 for MyHC-IIA, BA-F8 for MyHC-I) and alkaline phosphatase staining system for the third antigen (BF-F3 for MyHC-IIB), followed by corresponding detective systems and respective chromogens. The type of muscle fibers was quantified by systematic sampling at 12.5%, 25%, 33% and 50% of all muscle fibers, and was compared with that acquired from counting all the fibers (100%). All muscle fiber phenotypes, including pure and hybrid, could be simultaneously identified on a single TIE cryosection with clear outlines. The fiber types on TIE slides matched well with their respective counterpart on the consecutive SIE slides with a 95% match rate. Systematic sampling of 12.5% fibers could represent the true fiber type distribution of the entire muscle section. Our results suggest that novel TIE can effectively visualize fiber types in normal, denervated or reinnervated rat muscles.