探討超音波介導壓電刺激下神經細胞之活化響應

Abstract

低強度超音波(Low-intensity ultrasound, LIUS)因具備非侵入性與高空間解析度的優勢，已成為新興的神經調控技術。然而，單純超音波刺激主要仰賴聲壓與聲流等機械效應，其神經活化效率可能受到組織內源性機械敏感通道表現差異影響，進而限制刺激反應的一致性。為突破此限制，本研究使用實驗室開發的超音波玻璃微管裝置，搭配具壓電特性的石英底材作為壓電刺激組，並以不具壓電特性的玻璃底材作為單純超音波刺激組。透過Fura-2鈣離子螢光影像分析，本研究於小鼠神經母細胞瘤細胞(N2a)與背根神經節(DRG)感覺神經元中，量化不同底材、聲壓/聲流參數組合與離子通道條件下的鈣離子反應差異。 本研究實驗結果有三項主要發現。第一，在N2a細胞中，石英底材組在相同超音波參數下呈現較高的鈣離子反應，顯示超音波作用於石英底材後，可能產生額外壓電效應並增強細胞活化反應，且此壓電增益效果可受到輸入電壓與佔空比等超音波參數調控。第二，在DRG感覺神經元中觀察到亞型特異性反應。本體感覺神經元在較高聲壓條件下的壓電刺激，呈現較明顯的增益效果；相較之下，痛覺感覺神經元主要在低聲壓與低聲流條件下的壓電刺激，呈現較明顯的增益效果。第三，透過離子通道剔除實驗，結果顯示ASIC2a可能參與本體感覺神經元的壓電刺激反應，而ASIC3可能參與痛覺感覺神經元的壓電刺激反應。此外，使用電壓門控鈣離子通道抑制劑後，壓電刺激下的鈣離子反應顯著下降，支持電壓門控鈣離子通道，尤其是CaV3.2，可能參與痛覺感覺神經元的壓電刺激反應調控。 綜合上述，本研究建立的超音波介導壓電刺激系統可作為機-電耦合神經調控的體外研究平台。研究結果顯示，壓電刺激可增強神經細胞鈣離子反應，且其作用受到細胞亞型、聲壓/聲流參數組合與離子通道組成共同調控，為未來壓電材料應用於精準神經調控提供實驗依據以及機制參考。

Low-intensity ultrasound (LIUS) has emerged as a promising neuromodulation technique owing to its noninvasiveness and high spatial resolution. However, conventional ultrasound stimulation mainly relies on mechanical effects, such as acoustic pressure and acoustic streaming, and its neural activation efficiency may be influenced by the heterogeneous expression of endogenous mechanosensitive channels in tissue. To address this limitation, this study used a laboratory-developed glass micropipette-guided ultrasound device in combination with a piezoelectric quartz substrate as the piezoelectric stimulation group, while a non-piezoelectric glass substrate served as the ultrasound-only stimulation group. Using Fura-2-based calcium fluorescence imaging, this study quantified calcium responses in mouse neuroblastoma (N2a) cells and dorsal root ganglion (DRG) sensory neurons under different substrate conditions, acoustic pressure/acoustic streaming parameter combinations, and ion channel conditions. The results revealed three major findings. First, N2a cells cultured on quartz exhibited higher calcium responses than those cultured on glass under identical ultrasound parameters, suggesting that the quartz substrate enhanced ultrasound-induced cellular activation. This piezoelectric enhancement was further modulated by input voltage and duty factor. Second, DRG sensory neurons showed subtype-specific responses. Proprioceptive neurons exhibited a more pronounced piezoelectric enhancement of calcium responses under higher acoustic pressure conditions, whereas nociceptive neurons showed a more pronounced enhancement mainly under low acoustic pressure and low acoustic streaming conditions. Third, ion channel knockout experiments suggested that ASIC2a may participate in the piezoelectric response of proprioceptive neurons, whereas ASIC3 may participate in the piezoelectric response of nociceptive neurons. Pharmacological results further supported that voltage-gated calcium channels, particularly CaV3.2, may be involved in regulating the piezoelectric response of nociceptive neurons. Overall, this study establishes an in vitro ultrasound-mediated piezoelectric stimulation platform for investigating mechano-electrical coupling neuromodulation. The findings demonstrate that piezoelectric stimulation enhances neural calcium responses and that this effect is regulated by cell subtype, acoustic pressure/acoustic streaming parameter combinations, and ion channel composition.