Magdesian Margaret H, Anthonisen Madeleine, Lopez-Ayon G Monserratt, Chua Xue Ying, Rigby Matthew, Grütter Peter
Department of Physics, McGill University; Department of Neurology and Neurosurgery, Montreal Neurological Institute; Ananda Devices.
Department of Physics, McGill University.
J Vis Exp. 2017 Jun 13(124):55697. doi: 10.3791/55697.
Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h) and 10 times faster than previously reported for the same neuronal type at an earlier stage of development. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.
脑和脊髓损伤可能导致永久性残疾和死亡，因为目前仍无法使神经元进行长距离再生并准确地与合适的靶标重新连接。本文描述了一种可快速启动、延长并精确连接新的长距离功能性神经元回路的方法。所实现的延伸速率超过1.2毫米/小时，比外周神经系统中生长最快的轴突的体内生长速率（0.02至0.04毫米/小时）快30至60倍，比之前报道的相同神经元类型在发育早期的生长速率快10倍。首先，将分离出的大鼠海马神经元群体在微流控装置中培养2至3周，以精确地定位细胞，便于进行显微操作并实现实验的可重复性。接下来，将涂有聚-D-赖氨酸（PDL）的珠子放置在神经突上以形成粘附接触，并使用移液管显微操作来移动由此形成的珠子-神经突复合体。随着珠子的移动，它会拉出一条新的神经突，该神经突可以延伸数百微米，并在不到1小时的时间内与靶细胞建立功能连接。这个过程在绕过诱导神经突生长的较慢化学策略的同时，实现了实验的可重复性和操作的简便性。此处给出的初步测量结果表明，神经元的生长速率远远超过生理速率。将这些创新结合起来，可以以前所未有的控制程度在培养物中精确建立神经元网络。这是一种新颖的方法，为深入了解神经元网络内的信号传递和通信打开了一扇充满大量信息和见解的大门，同时也是一个探索神经元生长极限的平台。其潜在的应用和实验非常广泛，对旨在修复创伤后或神经退行性疾病中神经元回路的治疗方法具有直接影响。
