A microfluidic culture platform for CNS axonal injury, regeneration and transport

Anne M Taylor, Mathew Blurton-Jones, Seog Woo Rhee, David H Cribbs, Carl W Cotman and Noo Li Jeon

Nature Methods 2, 599 - 605 (2005)

Summary:

This group has devised a microfluidic culture system that allows growth of central nervous system (CNS) neurons into compartments that separate axons from its somata and dendrites. Their microfluidic device allows observation and experimentation on the axon region of the neuron. This device was used to observe how the neuron responded to damage resulting from axotomy. Other experimental parameters were investigated to ensure validity of their methods.

The device contains two regions where one houses the somal part of the neuron while the other offers room for axonal growth. Dividing the regions were microgrooves which allowed the axon to grow into the axonal region while the cell bodies stayed in the somal side of the device. A slight volume difference between the two sides caused fluid to constantly flow into the axonal side in order to fluidically isolate the axonal regions of the neuron from the rest of the device. The microgrooves and volume difference served to keep the fluid in the axonal side from diffusing back into the somal side.

Experiments performed include verification that the axonal side was isolated from the somal side and observations on neuronal response to axonal damage. Texas red dextran, a fluorescent dye, was administered in the axonal side. After twenty hours, the somal side did not show traces of this dye, presenting effective isolation of the two regions. [35S] methionine (131 Da) was also successfully confined to the axonal side for over twenty hours to further demonstrate isolation of the axonal side from the rest of the device.

After isolation verification, neurons were grown where their axons were separated from the dendrite regions with barriers in the device. The placement of the barriers was assessed with immunocytochemistry as the neuron matured in order to keep the axons isolated from dendrites at different stages of neuron development. The group also isolated axonal mRNA, and using reverse transcriptase- polymerase chain reaction (RT-PCR), they found that in the mRNA of developing axons, coding for synaptophysin or the presynaptic vesicle protein was present.

Vacuum aspiration was performed on the axonal side for five seconds to damage the axons. Again, RT-PCR was used to observe mRNA activity occurring in the somal side of the device. Upon response to axonal injury, the somal side expressed FBJ murine osteosarcoma viral oncogene homolog or c-fos mRNA in large amounts, demonstrating immediate recovery activity. A final experiment was administration of BDNF and NT-3 for five days to an experimental batch of injured axons while a control batch didn’t receive any neurotrophins. The result was tremendous growth in the axons for the neurotrophin-treated batch compared to the control batch.

Significance:

The methods and devices used prior to this group’s work such as Campenot chambers failed to culture CNS neurons. Campenot chambers were excellent in culturing peripheral neural system neurons using either nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF). However, these devices weren’t able to culture CNS neurons because CNS neurons didn’t mainly depend on neurotrophins for growth and are generally more difficult to culture. The Campenot chambers were also leaky at times.

The microfluidic device that this group created overcame these problems and has cultured rat cortical and hippocampal CNS neurons. This group innovatively cultured CNS neurons, effectively kept axon regions isolated, and successfully experimented on the recovery activity of axons. They are also first to report found that coding for synaptophysin or the presynaptic vesicle protein was present in the mRNA of developing axons.