Korosoglou Grigorios, Hardt Stefan E, Bekeredjian Raffi, Jenne Juergen, Konstantin Mathias, Hagenmueller Marco, Katus Hugo A, Kuecherer Helmut
Department of Cardiology, University of Heidelberg, Heidelberg, Germany.
Ultrasound Med Biol. 2006 Feb;32(2):297-303. doi: 10.1016/j.ultrasmedbio.2005.11.010.
Activated polymorphonuclear neutrophil (PMN) granulocytes can bind and subsequently phagocytose microbubbles used as ultrasound (US) contrast agents. The purpose of the present study was to assess insonation effects on cell membrane integrity and metabolic activity of activated PMN. Furthermore, we investigated whether or not there is an acoustic threshold at which insonation of PMN results in increase of membrane permeability without causing complete cell destruction. PMN isolated from healthy volunteers were activated with phorbol myristate acetate (PMA) for 15 min to allow phagocytosis of albumin and lipid microbubbles and were subsequently exposed to US with a mechanical index between 0.15 and 1.8. Apoptosis, loss of membrane integrity and formation of cell fragments were evaluated by measurement of lactate dehydrogenase leakage and by double staining with annexin V and propidium iodide, using flow cytometry. Neutrophil superoxide anion generation was measured photometrically. Insonation of activated PMN in the presence of microbubbles amplified apoptosis and lactate dehydrogenase leakage and induced loss of membrane integrity and complete cell destruction with increasing acoustic pressures. The bioeffects observed by insonation with high mechanical indices (1.0 to 1.8), and particularly the formation of cell fragments, were significantly more pronounced in the presence of albumin microbubbles. Insonation in the presence of lipid microbubbles increased cell membrane permeability, but caused significantly less cell destruction and left the metabolic activity of activated PMN uninfluenced. Thus, both albumin and lipid microbubbles induce apoptosis and membrane injury during insonation of activated PMN. However, insonation in the presence of lipid microbubbles seems to influence cell viability to a smaller extent. This could be of advantage in the setting of US-guided local drug delivery. In this setting, increase of membrane permeability may allow bioactive substances to enter into cells, which survive the US treatment, and specifically modify their function.
活化的多形核中性粒细胞（PMN）可结合并随后吞噬用作超声（US）造影剂的微泡。本研究的目的是评估超声照射对活化PMN细胞膜完整性和代谢活性的影响。此外，我们研究了PMN超声照射是否存在一个声学阈值，在此阈值下可导致膜通透性增加而不引起细胞完全破坏。从健康志愿者分离的PMN用佛波酯（PMA）活化15分钟，以使其吞噬白蛋白和脂质微泡，随后暴露于机械指数在0.15至1.8之间的超声下。通过测量乳酸脱氢酶泄漏以及使用流式细胞术进行膜联蛋白V和碘化丙啶双重染色来评估细胞凋亡、膜完整性丧失和细胞碎片形成。通过光度法测量中性粒细胞超氧阴离子的产生。在微泡存在下对活化的PMN进行超声照射，随着声压增加，可放大细胞凋亡和乳酸脱氢酶泄漏，并导致膜完整性丧失和细胞完全破坏。在白蛋白微泡存在下，以高机械指数（1.0至1.8）进行超声照射所观察到的生物效应，尤其是细胞碎片的形成，明显更为显著。在脂质微泡存在下进行超声照射可增加细胞膜通透性，但导致的细胞破坏明显较少，且未影响活化PMN的代谢活性。因此，在活化PMN超声照射期间，白蛋白和脂质微泡均可诱导细胞凋亡和膜损伤。然而，在脂质微泡存在下进行超声照射似乎对细胞活力的影响较小。这在超声引导下局部药物递送的情况下可能具有优势。在这种情况下，膜通透性增加可能使生物活性物质进入在超声治疗后存活的细胞，并特异性地改变其功能。
