BACKGROUND: The membrane components of cardiomyocytes are rich in polyunsaturated fatty acids, which are easily oxidized. Thus, an efficient glutathione-based lipid redox system is essential for maintaining cellular functions. However, the relationship between disruption of the redox system during ischemia-reperfusion (IR), oxidized lipid production, and consequent cell death (ferroptosis) remains unclear. We investigated the mechanisms underlying the disruption of the glutathione-mediated reduction system related to ferroptosis during IR and developed intervention strategies to suppress ferroptosis. METHODS: In vivo fluctuations of both intra- and extracellular metabolite levels during IR were explored via microdialysis and tissue metabolome analysis. Oxidized phosphatidylcholines were assessed using liquid chromatography high-resolution mass spectrometry. The areas at risk following IR were assessed using triphenyl-tetrazolium chloride/Evans blue stain. RESULTS: Metabolomic analysis combined with microdialysis revealed a significant release of glutathione from the ischemic region into extracellular spaces during ischemia and after reperfusion. The release of glutathione into extracellular spaces and a concomitant decrease in intracellular glutathione concentrations were also observed during anoxia-reperfusion in an in vitro cardiomyocyte model. This extracellular glutathione release was prevented by chemical inhibition or genetic suppression of glutathione transporters, mainly MRP1 (multidrug resistance protein 1). Treatment with MRP1 inhibitor reduced the intracellular reactive oxygen species levels and lipid peroxidation, thereby inhibiting cell death. Subsequent in vivo evaluation of endogenously oxidized phospholipids following IR demonstrated the involvement of ferroptosis, as levels of multiple oxidized phosphatidylcholines were significantly elevated in the ischemic region 12 hours after reperfusion. Inhibition of the MRP1 transporter also alleviated intracellular glutathione depletion in vivo and significantly reduced the generation of oxidized phosphatidylcholines. Administration of MRP1 inhibitors significantly attenuated infarct size after IR injury. CONCLUSIONS: Glutathione was released continuously during IR, primarily in an MRP1-dependent manner, and induced ferroptosis. Suppression of glutathione release attenuated ferroptosis and reduced myocardial infarct size following IR.

Strategies for therapeutic cell transplantation have been assessed for use in the treatment of massive peripheral nerve defects. To support safe and efficient cell transplantation, we have focused on the purification of cells using cell surface markers. Our group previously reported low-affinity nerve growth factor receptor (LNGFR)- and thymocyte antigen-1 (THY-1)-positive neural crest-like cells (LT-NCLCs), generated from human induced pluripotent stem cells (hiPSCs). In the present study, we investigated the efficacy of transplantation of hiPSC-derived LT-NCLCs in a murine massive peripheral nerve defect model. Animals with a sciatic nerve defect were treated with a bridging silicone tube prefilled with LT-NCLCs or medium in the transplantation (TP) and negative control (NC) groups, respectively. The grafted LT-NCLCs survived and enhanced myelination and angiogenesis, as compared to the NC group. Behavioral analysis indicated that motor functional recovery in the TP group was superior to that in the NC group, and similar to that in the autograft (Auto) group. LT-NCLCs promoted axonal regrowth and remyelination by Schwann cells. Transplantation of LT-NCLCs is a promising approach for nerve regeneration treatment of massive peripheral nerve defects.

Background: Costal osteochondral grafting is a technique to achieve anatomical and biological repair of articular defects. Some small series of clinical applications of this procedure for advanced osteochondritis dissecans of the humeral capitellum, with short-term follow-up, have been reported; however, longer-term outcomes remain unclear. The purpose of this study was to clarify longer-term clinical outcomes of costal osteochondral autografts in the treatment of advanced osteochondritis dissecans of the humeral capitellum. Methods: Seventy-two patients with an osteochondral defect of the humeral capitellum were treated with costal osteochondral autograft and followed for a minimum of 3 years (mean follow-up, 57 months; range, 36 to 147 months). The mean patient age was 14.3 years. Clinical outcomes, including elbow range of motion, Timmerman and Andrews clinical rating score, donor-site morbidity, responses to a questionnaire regarding a return to sporting activities, and radiographic findings, were evaluated. Results: The mean elbow range of extension/flexion increased significantly, from −21°/122° preoperatively to −4°/136° postoperatively (p < 0.001). The mean clinical rating score improved significantly, from 101 to 190 by the latest follow-up (p < 0.001). The overall clinical score-based assessment was excellent for 60 patients, good for 9, and fair for 3. Seventy of the 72 patients returned to their original sport. The remaining 2 patients had changed sporting activities before surgery and did not return to baseball, despite satisfactory clinical results. Conclusions: Costal osteochondral autograft successfully achieved anatomical and biological reconstruction in the treatment of advanced osteochondritis dissecans of the humeral capitellum. Level of Evidence: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

The nicotinic receptor α7nAchR reportedly regulates vagal nerve targets in brain and cardiac tissue. Here we show thatnAchR7^−/−mice exhibit increased bone mass due to decreased osteoclast formation, accompanied by elevated osteoprotegerin/RANKL ratios in serum. Vagotomy in wild-type mice also significantly increased the serum osteoprotegerin/RANKL ratio, and elevated bone mass seen innAchR7^−/−mice was reversed in α7nAchR/osteoprotegerin-doubly-deficient mice. α7nAchR loss significantly increased TNFα expression in Mac1-positive macrophages, and TNFα increased the osteoprotegerin/RANKL ratio in osteoblasts. Targeting TNFα innAchR7^−/−mice normalized both serum osteoprotegerin/RANKL ratios and bone mass. Administration of nicotine, an α7nAchR ligand, to wild-type mice increased serum RANKL levels. Thus, vagal nerve stimulation of macrophages via α7nAchR regulates bone mass by modulating osteoclast formation.