探究不同力學模組的超音波刺激對機械力敏感通道所引發的鈣離子反應之影響

Abstract

多項文獻的實驗結果已經證實，低強度超音波具備高空間解析度與深度傳播的能力，進而促進神經調節。雖然低強度超音波已為神經刺激的臨床診斷帶來新的方法；但是對於超音波在分子生物層面作用的力學機制，尚未有明確的解釋。本研究開發一種特殊的超音波刺激裝置：超音波微管，刺激背根神經元和小鼠神經纖維瘤細胞；並詳細研究細胞受到超音波刺激後，產生鈣離子反應相關的離子通道。玻璃微管的針尖呈現閉口的狀態；作為一個波傳導的裝置，將超音波傳感器發射的超音波傳導至針尖，再經由針尖釋放機械力刺激。當超音波被釋放時，會產生兩種力學刺激：第一種是聲壓，其會對行進方向上的細胞產生擠壓的效應；第二種是聲流，其會對細胞膜表面施加橫向的作用力，稱為剪力。這兩種力量的量值可以經由超音波參數：輸入電壓與佔空比的組合(模組)進行調控。

本研究的實驗結果證實，酸敏感離子通道一型1a(Acid-sensing ion channels 1a, 簡稱ASIC1a)在背根神經元和小鼠神經纖維瘤受超音波刺激所產生的鈣離子反應中，扮演了機械力敏感通道的角色；另外本研究特別使用ASIC1a基因修飾的老鼠進行實驗，也證實了同一個實驗結果。除此之外，本研究剔除本體感覺神經元上的酸敏感離子通道三型基因(ASIC3)，發現鈣離子反應的程度會受到影響，證實ASIC3可能是本體感覺神經元上的機械力敏感通道。最後，本研究發現比起聲壓主導的超音波刺激，聲流主導刺激更容易造成細胞的鈣離子濃度變化。

Lines of compelling evidence have shown that low-intensity ultrasound has the capability of neuromodulation modality with exquisite spatial specificity and depth penetration. Although low-intensity focused ultrasound holds great promise as a novel approach to the potential clinical applications of neuron stimulation, the underlying mechanism at the cellular and molecular level remain unclear. In this study, we utilized a device of micropipette-guided ultrasound to dissect the involved channels in ultrasound-induced activation of somatosensory neurons and neuroblastoma Neuro-2a cells. The glass micropipette with an end-closed tip is a developed wave-guide device to transport the force of ultrasound. Ultrasound from the tip generates two forms of force: one is the acoustic pressure into cell solution with acoustic pressure exerting on cells placed along its path, and the other is the streaming flow applying shear force on cells. These two force modalities could be fine-tuned by the input voltage and the duty factor. Our results demonstrated ASIC1a has been identified as a vital mechanosensitive channel to mediate low-intensity ultrasound-induced activation of dorsal root ganglion neurons and Neuro-2a cells. We further generated a transgenic mice and probe the role of ASIC1a in ultrasound-sensitive calcium response. In addition, ASIC3 is expressed in proprioceptors so that we generated transgenic mice with a floxed allele of ASIC3 (ASIC3f/f) and probed ASIC3 also has been a mechanoreceptor. Lastly, our results confirmed streaming force dominantly modulate intracellular calcium concentration compared to compressional predominant force.