Modelling central and peripheral nervous system connectivity using compartmentalised microfluidic systems

控制慢性伤害性感觉发展的机制尚不完全清楚。尽管最近取得了进展，但由于细胞培养实验忽略了背根神经节(DRG)神经元令人难以置信的解剖极化，不同类型神经元在伤害感受回路中实现的复杂相互作用在体外还没有得到充分的模拟。在这种背景下，显然有必要建立与生理相关的体外系统，以便能够在多细胞回路复杂的背景下研究细胞和分子机制。具有流体隔离隔间的培养平台允许在更生理相关的环境中对神经元域进行功能分离。我们已经在功能实验中通过测量微流控系统细胞体隔室中的钙信号，证明了复制DRG神经元“假单极”性质的能力。我们将通过按顺序增加网络复杂性级别来进一步开发这些新的文化系统。背角原代神经元和角质形成细胞将被种植到三通道微流控设备的侧室中，这些设备将DRG神经元放置在中间通道中。该系统将模拟DRG神经元回路的“中央”和“外周”区域，但具有更大的实验可访问性。我们的初步工作确定了在痛觉过敏启动中发生变化的关键miRNAs，这是一种研究从急性疼痛到慢性疼痛过渡的体内模型。虽然在痛觉过敏的启动机制中发现了局部蛋白表达，但miRNAs的作用还没有被研究过。使用提出的系统，miRNA抑制剂将被添加到外周和中央隔室，同时将评估神经元的兴奋性和突触标志物的表达。这将使我们能够确定miRNAs在痛觉过程中控制蛋白表达和局部调节神经元回路的潜在作用。

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.