Kumon R E, Aehle M, Sabens D, Parikh P, Han Y W, Kourennyi D, Deng C X
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109-2099, USA.
Ultrasound Med Biol. 2009 Mar;35(3):494-506. doi: 10.1016/j.ultrasmedbio.2008.09.003. Epub 2008 Nov 17.
To investigate the effects of sonoporation, spatiotemporal evolution of ultrasound-induced changes in intracellular calcium ion concentration (Ca(2+)) was determined using real-time fura-2AM fluorescence imaging. Monolayers of Chinese hamster ovary (CHO) cells were exposed to a 1-MHz ultrasound tone burst (0.2 s, 0.45 MPa) in the presence of Optison microbubbles. At extracellular Ca(2+) of 0.9 mM, ultrasound application generated both nonoscillating and oscillating (periods 12 to 30 s) transients (changes of Ca(2+) in time) with durations of 100-180 s. Immediate Ca(2+) transients after ultrasound application were induced by ultrasound-mediated microbubble-cell interactions. In some cases, the immediately affected cells did not return to pre-ultrasound equilibrium Ca(2+) levels, thereby indicating irreversible membrane damage. Spatial evolution of Ca(2+) in different cells formed a calcium wave that was observed to propagate outward from the immediately affected cells at 7-20 microm/s over a distance >200 microm, causing delayed transients in cells to occur sometimes 60 s or more after ultrasound application. In calcium-free solution, ultrasound-affected cells did not recover, consistent with the requirement of extracellular Ca(2+) for cell membrane recovery subsequent to sonoporation. In summary, ultrasound application in the presence of Optison microbubbles can generate transient Ca(2+) changes and oscillations at a focal site and in surrounding cells via calcium waves that last longer than the ultrasound duration and spread beyond the focal site. These results demonstrate the complexity of downstream effects of sonoporation beyond the initial pore formation and subsequent diffusion-related transport through the cellular membrane.
为了研究声孔效应，利用实时fura - 2AM荧光成像技术测定了超声诱导的细胞内钙离子浓度（Ca(2+)）变化的时空演变。将中国仓鼠卵巢（CHO）细胞单层在Optison微泡存在的情况下暴露于1 MHz的超声脉冲群（0.2 s，0.45 MPa）。在细胞外Ca(2+)为0.9 mM时，施加超声会产生非振荡和振荡（周期为12至30 s）的瞬变（Ca(2+)随时间的变化），持续时间为100 - 180 s。施加超声后立即出现的Ca(2+)瞬变是由超声介导的微泡 - 细胞相互作用引起的。在某些情况下，立即受到影响的细胞没有恢复到超声处理前的平衡Ca(2+)水平，从而表明存在不可逆的膜损伤。不同细胞中Ca(2+)的空间演变形成了一个钙波，观察到该钙波以7 - 20微米/秒的速度从立即受到影响的细胞向外传播超过200微米的距离，导致有时在施加超声60秒或更长时间后细胞中出现延迟瞬变。在无钙溶液中，受超声影响的细胞无法恢复，这与声孔效应后细胞膜恢复需要细胞外Ca(2+)一致。总之，在Optison微泡存在的情况下施加超声可通过持续时间长于超声持续时间且扩散到焦点部位以外的钙波，在焦点部位和周围细胞中产生瞬时Ca(2+)变化和振荡。这些结果证明了声孔效应下游效应的复杂性，超出了最初的孔形成以及随后通过细胞膜的扩散相关运输。
