低能量超音波刺激力學模組與神經細胞離子通道之相關性研究

Abstract

非侵入性神經調控技術迅速發展，其中低強度超音波(Low-Intensity Ultrasound, LIUS)因具備高穿透性、聚焦性佳與空間解析度高等優勢，逐漸成為調控深層腦區活動的重要工具。特別是在無需植入光纖或電極，LIUS可與基因工程結合，發展為超音波遺傳學(Sonogenetics)的新興技術，使得特定細胞能對低強度超音波產生選擇性反應。然而，超音波在細胞層級誘發哪些離子通道及其對應的力學模組，目前仍缺乏系統性的研究。

本研究分為兩大部分。第一部分為超音波微管裝置物理性質的驗證，探討不同參數(如輸入電壓、佔空比)下，產生的聲輻射力與剪應力的變化，並量化其力學模組。第二部分為細胞層級實驗，利用小鼠神經母細胞瘤(N2a)與背根神經節細胞(DRG)進行超音波刺激，利用鈣離子螢光染劑(Fura-2)與Metafluor軟體即時量測細胞內鈣離子濃度變化。進一步透過離子通道抑制劑與基因剔除細胞株，釐清特定離子通道在細胞對超音波刺激反應中的作用。

實驗結果發現，N2a細胞中對剪應力特別敏感，其鈣離子反應依賴三個離子通道：ASIC1a, Piezo1與TRPV1，本研究特別利用基因剔除與細胞轉染技術，驗證出ASIC1a扮演特別重要的角色。DRG透過LoxP-Cre技術標記ASIC1a+，對於相同能量下，聲壓聲流主導的刺激皆無鈣離子反應的顯著變化；本體感覺與痛覺神經中，不論是低還是高能量輸出的力學模組，造成的鈣離子反應也無顯著變化。在本體感覺神經中，反應依賴ASIC3與Piezo2；在痛覺神經中，反應除了依賴Piezo2之外，ASIC1a與ASIC3也扮演重要角色。

本研究建立了一套可量化且具重現性的細胞層級超音波刺激系統，透過物理與生物指標的交叉分析，釐清低強度超音波刺激下，力學模組對不同細胞的活化具決定性影響，以及與特定離子通道(ASIC1a、ASIC3、Piezo2)之間的關聯，深化對神經細胞受超音波調控的認識，並為未來發展精準、非侵入性的神經調控技術提供理論基礎。

Non-invasive neuromodulation has advanced rapidly, with low-intensity ultrasound (LIUS) emerging as a promising tool for deep brain modulation due to its high penetration and precise focus. When combined with genetic engineering, LIUS enables sonogenetics—a technique for selectively activating target cells without invasive implants. However, the specific ion channels and mechanical force modalities involved in ultrasound-induced cellular responses remain poorly understood.

This study is divided into two major parts. The first part involves characterizing the physical properties of the micropipette-guided ultrasound system, focusing on how variations in input voltage and duty cycle influence the resulting acoustic pressure and shear stress, thereby defining the mechanical force modalities. The second part consists of cellular-level experiments in which low-intensity ultrasound was applied to mouse neuroblastoma (N2a) cells and dorsal root ganglion (DRG) neurons. Calcium influx was monitored in real-time using the fluorescent calcium indicator Fura-2 and the Metafluor software. Specific ion channels involved in the ultrasound-induced calcium responses were further identified through the use of pharmacological inhibitors and genetically modified knockout cell lines.

Experimental results showed that N2a cells were particularly sensitive to shear stress, with calcium responses primarily mediated by ASIC1a, Piezo1, and TRPV1. Using gene knockout and transfection techniques, ASIC1a was confirmed to play a crucial role. In DRG neurons marked with ASIC1a⁺ via LoxP-Cre recombination, no significant calcium response was observed under either pressure- or shear-dominant conditions with equivalent acoustic energy. Across different DRG subtypes, no significant calcium response was noted under either low or high energy conditions. In proprioceptive neurons, responses were dependent on ASIC3 and Piezo2, while in nociceptive neurons, Piezo2, ASIC1a, and ASIC3 all played important roles.

This study successfully established a quantifiable and reproducible cellular-level ultrasound stimulation platform. Through integrated physical and biological analyses, it clarified how mechanical force modalities decisively influence cellular activation under low-intensity ultrasound and identified key mechanosensitive ion channels–ASIC1a, ASIC3, and Piezo2–in this process. These findings deepen our understanding of how neural cells are modulated by ultrasound and provide a theoretical foundation for developing precise, non-invasive neuromodulation technologies in the future.