Examination of axonal injury and regeneration in micropatterned neuronal culture using pulsed laser microbeam dissection

Abstract

We describe the integrated use of pulsed laser microbeams and microfluidic cell culture to examine the dynamics of axonal injury and regeneration in vitro. Microfabrication methods are used to place high purity dissociated central nervous system neurons in specific regions that allow the axons to interact with permissive and inhibitory substrates. Acute injury to neuron bundles is produced via the delivery of single 180 ps duration, λ=532 nm laser pulses. Laser pulse energies of 400 nJ and 800 nJ produce partial and complete transection of the axons, respectively, resulting in elliptical lesions 25 μm and 50 μm in size. The dynamics of the resulting degeneration and regrowth of proximal and distal axonal segments are examined for up to 8 h using time-lapse microscopy. We find the proximal and distal dieback distances from the site of laser microbeam irradiation to be roughly equal for both partial and complete transection of the axons. In addition, distinct growth cones emerge from the proximal neurite segments within 1-2 h post-injury, followed by a uniform front of regenerating axons that originate from the proximal segment and traverse the injury site within 8 h. We also examine the use of EGTA to chelate the extracellular calcium and potentially reduce the severity of the axonal degeneration following injury. While we find the addition of EGTA to reduce the severity of the initial dieback, it also hampers neurite repair and interfere with the formation of neuronal growth cones to traverse the injury site. This integrated use of laser microbeam dissection within a microfluidic cell culture system to produce precise zones of neuronal injury shows potential for high-throughput screening of agents to promote neuronal regeneration.

Integrated Microfluidics Platforms for Investigating Injury and Regeneration of CNS Axons

“…13 Pulsed femtosecond laser microbeams have been used to produce axonal injury in vivo in animal models, which are attractive tools for cell microsurgery in vivo due to their ability to generate extremely localized damage zones in single cells. 1,32,35 However, the high cost and complex operation of these femtosecond laser systems makes them impractical for many researchers.…”

Neuronal growth cones respond to laser-induced axonal damage

“…Through precise control of the laser parameters, it is possible to cause either complete transection of neurites extending from neurons in culture or localized subaxotomy damage from which the neurites recover [8]. In a recent study, axons from embryonic rat cortical neurons were grown as oriented bundles using a patterned substrate and were completely severed using a 532 nm 180 ps laser microbeam [9]. Subsequent dieback and regeneration of these axons were studied.…”

Acute and Chronic Neural Stimulation via Mechano-Sensitive Ion Channels

“…44 Pulsed laser excitation has also been integrated with micro-channels to induce acute axonal injuries and such systems can offer high throughput results for drug screening to accelerate axonal recovery. 45 Co-culture microfluidic platforms when combined with micro-technologies to exert mechanical forces on seeded neurons, astrocytes and oligodendrocytes can also be developed to investigate the role of different neural cells in neuronal development, injury and recovery. 46 Potential tools: Progress in droplet microfluidics has enabled the generation of droplets having different sizes, composition and shapes.…”

“…108 Using surface patterning and fluidic resistance, axons can be preferentially separated from the dendrites, hence creating a model to study axonal growth, injury and recovery. 45,109,110 Nguyen et al utilized a vacuum-supplemented microfluidic channel array that models an array of 25 micropipette tips to apply mechanical tension to seeded neurons. 111 It was found that applying tension to the neurites enhanced their typical growth rates by 2-12x.…”