Heidemann Martina, Streit Jürg, Tscherter Anne
Department of Physiology, University of Bern.
Department of Physiology, University of Bern;
J Vis Exp. 2015 Sep 23(103):53121. doi: 10.3791/53121.
Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.
成年高等脊椎动物从脊髓损伤中恢复的潜力有限。最近，有证据表明脊髓固有连接是改善功能再生干预的一个有前景的靶点。到目前为止，还没有体外模型能够研究脊髓固有纤维的功能恢复情况。因此，开发了一种基于胚胎大鼠的两个器官型脊髓切片的代表性模型，将它们在多电极阵列（MEA）上彼此相邻培养。这些切片生长，并在体外培养几天后，沿着彼此相对的侧面融合。所使用的MEA的设计允许用手术刀刀片通过这个融合部位进行损伤，而不会对MEA造成损害。切片显示出自发活动，通常组织成网络活动爆发，并且可以表征空间和时间活动参数，如爆发起源的位置、其传播的速度和方向以及爆发之间的潜伏期。利用这些特征，还可以通过计算两侧同步爆发的数量来评估切片的功能连接。此外，在记录后通过进行免疫组织化学染色可以对切片进行形态学分析。该模型结合了所使用技术的几个优点：切片在很大程度上保留了具有完整局部突触回路的原始组织结构，组织易于且可重复获取，并且可以在高时间分辨率下同时且非侵入性地在大量位点检测神经元活动。这些特征使得能够以复杂而有效的方式在体外单独研究脊髓内连接的功能再生。
