Department of Neuroscience and Brain Technologies (NBT), Italian Institute of Technology (IIT), via Morego 30, 16163 Genoa, Italy.
Lab Chip. 2015 Dec 21;15(24):4578-90. doi: 10.1039/c5lc01027f. Epub 2015 Oct 28.
We designed a miniaturized and thin polydimethylsiloxane (PDMS) microchannel device compatible with commercial microelectrode array (MEA) chips. It was optimized for selective axonal ablation by laser microdissection (LMD) to investigate the electrophysiological and morphological responses to a focal injury in distinct network compartments over 45 days in vitro (45 DIV). Low-density cortical or hippocampal networks (<3500 neurons per device) were cultured in quasi-closed somal chambers. Their axons were selectively filtered through neurite cavities and guided into the PDMS microchannels aligned over the recording electrodes. The device geometries amplified extracellularly recorded signals in the somal reservoir and the axonal microchannels to detectable levels. Locally extended areas along the microchannel, so-called working stations, forced axonal bundles to branch out and thereby allowed for their repeatable and controllable local, partial or complete dissections. Proximal and distal changes in the activity and morphology of the dissected axons were monitored and compared to those of their parent networks and of intact axons in the control microchannels. Microscopy images confirmed progressive anterograde degeneration of distal axonal segments over four weeks after surgery. Dissection on cortical and hippocampal axons revealed different cell type- and age-dependent network responses. At 17 DIV, network activity increased in both the somal and proximal microchannel compartments of the dissected hippocampal or cortical axons. At later days (24 DIV), the hippocampal networks were more susceptible to axonal injury. While their activity decreased, that in the cortical cultures actually increased. Subsequent partial dissections of the same axonal bundles led to a stepwise activity reduction in the distal hippocampal or cortical axonal fragments. We anticipate that the MEA-PDMS microchannel device for the combined morphological and electrophysiological study of axonal de- and regeneration can be easily merged with other experimental paradigms like molecular or pharmacological screening studies.
我们设计了一种小型化、超薄的聚二甲基硅氧烷（PDMS）微通道设备，与商用微电极阵列（MEA）芯片兼容。它通过激光微切割（LMD）进行了优化，用于选择性轴突消融，以研究体外 45 天（45 DIV）不同网络隔室中局灶性损伤后的电生理和形态学反应。低密度皮质或海马网络（每个设备<3500 个神经元）在准封闭的胞体腔室中培养。它们的轴突通过神经突腔选择性过滤，并引导到对准记录电极的 PDMS 微通道中。该设备几何形状放大了胞体储层和轴突微通道中记录的细胞外信号，使其达到可检测水平。沿着微通道的局部扩展区域，即所谓的工作站，迫使轴突束分支，从而可以对其进行可重复和可控的局部、部分或完全切割。切割后的轴突的近端和远端活动和形态变化被监测，并与它们的母网络以及对照微通道中完整轴突的变化进行比较。显微镜图像证实，手术后 4 周内，远端轴突段逐渐发生顺行性退变。对皮质和海马轴突的切割揭示了不同的细胞类型和年龄依赖性网络反应。在 17 DIV 时，切割的海马或皮质轴突的胞体和近端微通道隔室中的网络活动均增加。在以后的日子（24 DIV），海马网络更容易受到轴突损伤的影响。虽然它们的活动减少，但皮质培养物中的活动实际上增加了。对同一轴突束的后续部分切割导致远端海马或皮质轴突片段的活动逐渐减少。我们预计，用于轴突去再生的形态学和电生理学联合研究的 MEA-PDMS 微通道设备可以很容易地与其他实验范式（如分子或药理学筛选研究）合并。
