Modelling central and peripheral nervous system connectivity using compartmentalised microfluidic systems

控制慢性伤害感受发展的机制尚未完全了解。尽管最近取得了进展，但在伤害感受回路中由不同神经元类型实现的复杂相互作用尚未在体外充分建模，细胞培养实验忽略了背根神经节（DRG）神经元的令人难以置信的解剖学极化。显然需要实现生理学相关的体外系统，该系统可以允许在多细胞环境中研究细胞和分子机制，蜂窝电路复杂度。具有流体隔离隔室的培养平台允许在更生理相关的背景下功能分离神经元结构域。我们已经证明了在功能实验中通过测量微流体系统的细胞体区室中的Ca2+信号来复制DRG神经元的“伪单极”性质的能力。我们将通过依次增加网络复杂性的级别来进一步开发这些新的文化系统。将背角初级神经元和角质形成细胞接种到三通道微流体装置的侧隔室中，所述三通道微流体装置具有放置在中间通道中的DRG神经元。该系统将模拟DRG神经元回路的“中枢”和“外周”区域，但具有更大的实验accessibility.Our初步工作确定了在痛觉过敏启动中改变的关键miRNAs，这是一种研究从急性疼痛到慢性疼痛转变的体内模型。虽然局部蛋白质表达已被建议在痛觉过敏启动机制，小RNA的作用还没有被调查。使用所提出的系统，miRNA抑制剂将被添加到外周和中枢隔室中，同时神经元兴奋性和突触标记物的表达将被评估。这将使我们能够确定在伤害感受过程中miRNA在蛋白质表达控制和神经元回路局部调节中的潜在作用。

The mechanisms that control the development of chronic nociception are not yet fully understood. Despite recent advances, the complex interplay achieved by different neuronal types in nociceptive circuits has not been adequately modelled in vitro, with cell culture experiments ignoring the incredible anatomical polarization of dorsal root ganglia (DRG) neurons.In this context, there is a clear need to implement physiologically relevant in vitro systems that could allow the investigation of cellular and molecular mechanisms in a context of multi-cellular circuit complexity. Culture platforms with fluidically isolated compartments allow the functional separation of neuronal domains in more physiologically relevant contexts. We have already demonstrated the ability to replicate the "pseudo-unipolar" nature of DRG neurons in functional experiments by measuring Ca2+ signals in the cell body compartment of microfluidic systems. We will further develop these novel culture systems by sequentially adding levels of network complexity. Dorsal horn primary neurons and keratinocytes will be seeded into the lateral compartments of three-channel microfluidic devices that have DRG neurons placed in the middle channel. This ststem will model the "central" and "peripheral" domains of DRG neuron circuitry, but with greater experimental accesibility.Our preliminary work identified key miRNAs that are changed in hyperalgesic priming, an in vivo model for studying the transition from acute to chronic pain. Although local protein expression has been suggested in hyperalgesic priming mechanisms, the role of miRNAs has not been investigated. Using the proposed system, miRNA inhibitors will be added into the peripheral and central compartments while neuron excitability and expression of synaptic markers will be evaluated. This will allow us to determine the potential role of miRNAs in the control of protein expression and local regulation of neuronal circuits during nociception.