Cotero Victoria, Miwa Hiromi, Graf John, Ashe Jeffrey, Loghin Evelina, Di Carlo Dino, Puleo Chris
General Electric Global Research Center, Niskayuna, NY, USA.
University of California Los Angeles, Los Angeles, CA, USA.
J Neurosci Methods. 2020 Jul 15;341:108721. doi: 10.1016/j.jneumeth.2020.108721. Epub 2020 May 6.
A fundamental limit to the study of the peripheral nervous system and its effect on organ function is the lack of tools to selectively target and stimulate specific neurons. Traditional implant and electrode-based systems remain too large and invasive for use at the organ or sub-organ level (without stimulating or effecting neighboring organs and tissues). Recent progress in optical and genetic tools (such as optogenetics) has provided a new level of molecular specificity and selectivity to the neurons that are stimulated by bioelectronic devices. However, the modified neurons that result from use of these tools (that can be selectively activated based on expression of light, heat, or stimuli sensitive ion channels) often still require stimulation by implantable devices and face difficult scientific, technical, and regulatory hurdles for clinical translation.
Herein, we present a new tool for selective activation of neuronal pathways using anatomical site-specific, peripheral focused ultrasound neuromodulation (pFUS).
We utilize three experimental models to expand upon and further characterize pFUS beyond data outlined to our initial report (Cotero et al., 2019a), and further demonstrate its importance as a new investigative and translational tool. First, we utilized an interconnected microporous gel scaffold to culture isolated dorsal root ganglion (DRG) neurons in an interconnected, three-dimensional in vitro culture. (Griffin et al., 2015, Tay et al., 2018) Using this system, we directly applied ultrasound (US) stimuli and confirmed US activation of peripheral neurons at pressures consistent with recent in vivo observations. (Cotero et al., 2019a, Zachs, 2019, Gigliotti et al., 2013) Next, we tested the capability of pFUS to activate previously reported nerve pathways at multiple locations within the neural circuit, including primary sensory ganglia (i.e. inferior ganglion of the vagus nerve), peripheral ganglia (i.e. sacral ganglia), and within target end-organs. In addition, we compared selective activation of multiple anatomically overlapping neural pathways (i.e. activation of the cholinergic anti-inflammatory pathway (Tracey, 2009, Pavlov and Tracey, 2012) vs. metabolic sensory pathways (O'Hare and Zsombok, 2015, Roh et al., 2016, Pocai et al., 2005) after stimulation of each separate target site. Finally, we utilized an established model of metabolic dysfunction (the LPS-induced inflammation/hyperglycemia model) to demonstrate pFUS capability to stimulate and assess alternative therapeutic stimulation sites (i.e. liver, pancreas, and intestines) in a simple and clinically relevant manner. This is demonstrated by ultrasound induced attenuation of LPS-induced hyperglycemia by stimulation at all three anatomical targets, and mapping of the effect to a specific molecular product of excitable cell types within each stimulus site.
The ease-of-use and non-invasive nature of pFUS provides a solution to many of the challenges facing traditional toolsets, such as implantable electrodes and genetic/optogenetic nerve stimulation strategies.
The pFUS tool described herein provides a fundamental technology for the future study and manipulation of the peripheral nervous and neuroendocrine systems.
外周神经系统及其对器官功能影响的研究存在一个基本限制，即缺乏选择性靶向和刺激特定神经元的工具。传统的植入式和基于电极的系统对于器官或亚器官水平的使用而言仍然太大且具有侵入性（无法刺激或影响邻近器官和组织）。光学和基因工具（如光遗传学）的最新进展为生物电子设备刺激的神经元提供了新的分子特异性和选择性水平。然而，使用这些工具产生的经过修饰的神经元（可基于光、热或刺激敏感离子通道的表达进行选择性激活）通常仍需要植入式设备进行刺激，并且在临床转化上面临困难的科学、技术和监管障碍。
在此，我们展示了一种使用解剖部位特异性、外周聚焦超声神经调节（pFUS）选择性激活神经元通路的新工具。
我们利用三个实验模型，在我们初步报告（Cotero等人，2019a）所述数据之外扩展并进一步表征pFUS，并进一步证明其作为一种新的研究和转化工具的重要性。首先，我们利用相互连接的微孔凝胶支架在相互连接的三维体外培养中培养分离的背根神经节（DRG）神经元。（Griffin等人，2015年，Tay等人，2018年）使用该系统，我们直接施加超声（US）刺激，并在与最近体内观察结果一致的压力下证实了US对外周神经元的激活。（Cotero等人，2019a，Zachs，2019，Gigliotti等人，2013）接下来，我们测试了pFUS在神经回路内多个位置激活先前报道的神经通路的能力，包括初级感觉神经节（即迷走神经下神经节）、外周神经节（即骶神经节）以及靶终末器官内。此外，我们比较了多个解剖学上重叠的神经通路的选择性激活（即刺激每个单独靶位点后胆碱能抗炎通路（Tracey，2009年，Pavlov和Tracey，2012年）与代谢感觉通路（O'Hare和Zsombok，2015年，Roh等人，2016年，Pocai等人，2005年）的激活）。最后，我们利用一个已建立的代谢功能障碍模型（脂多糖诱导的炎症/高血糖模型），以简单且与临床相关的方式证明pFUS刺激和评估替代治疗刺激位点（即肝脏、胰腺和肠道）的能力。通过在所有三个解剖靶点进行刺激，超声诱导脂多糖诱导的高血糖症减轻，并将该效应映射到每个刺激位点内可兴奋细胞类型的特定分子产物，证明了这一点。
pFUS的易用性和非侵入性为传统工具集（如植入式电极和基因/光遗传学神经刺激策略）面临的许多挑战提供了解决方案。
本文所述的pFUS工具为未来外周神经和神经内分泌系统的研究和操作提供了一项基础技术。
