Jackson Levine – Ultrasound-Induced Growth of Cortical Neuronal Axons

Prepared by Jackson Levine, Audrey Schreiner, and Eli Tsakiris

With the semester coming to a close, I am relatively happy with the developments we have made, which were made possible by the Newcomb-Tulane College Grant for which we applied in the fall. At that point, we were in the planning phase of some really exciting experiments to directly measure linear axonal growth. Throughout this past semester, our primary focus has been on designing these channels. Specific to our experiment, there are no commercially available models which would allow us to effectively measure the growth like we wanted to. In engineering when something does not exist, you have to design it yourself and this has been our primary job this semester. We began by reading many papers on microfluidic channels, hydrogels, Campenot chambers, and many other methods researchers use to quantify axonal growth. After synthesizing all of the different methods and isolating the most effective methods, we began our design. First, we drew many potential prototypes and presented them to the entire lab to gain their feedback on the ideas. This ultimately led to a prototype which incorporated microscopic slits at the level of about 5 micrometers wide. This was chosen because the diameter of the neuronal cell body, or soma, is on average larger than 5 micrometers. Thus, the cell bodies would remain located in the loading area and the axons will be able to travel through the slits and down the channel. We hypothesized that this would be facilitated via the use of chemoattractant, which stimulates the growth and/or movement of cells in a specified direction. The image on the right is a SolidWorks model of the potential channel design. This was then 3D printed at Tulane’s MakerSpace. Several iterations were designed and printed. These 3D-printed models were then inverted to create molds which could be used to create the polydimethylsiloxane (PDMS) channels.

We then moved onto a second design which we believed would be more successful. Slits would have allowed for axons to travel up the slits. This could potentially cause the calculations of axon length to be incorrect. Instead, we hypothesize that a porous membrane would be more effective in facilitating the measurement of axon growth. This porous membrane would have micropores of diameter 5 micrometers. In the same way, this would isolate the cell bodies to the loading area and allow for axons to grow down channels with the use of chemoattractant. We have been working with the Tulane Micro/Nano Fabrication Facility (MNFF) to develop these designs. They have been instrumental in providing constructive feedback on designs and outlining the path forward for actually implementing these designs. The next logical step would be to create a mask for the channels using the SolidWorks model we created. Then, using this mask, we would use photolithography in the MNFF to engrave the design into a metal mold. This process uses ultraviolet rays focused through the holes in the mask onto a piece of metal, thus etching a fine design into the mold. We cannot 3D print the designs because the limitations of the 3D printer will not allow for printing at the microscopic level.

Next semester, we hope to implement these channels in neuronal axon growth. We have been working on optimizing the ultrasound parameters to induce growth. The main parameter we varied was input voltage, which translates to intensity. Once the channels are developed and the parameters optimized for neurons in plates, we will be able to effectively model linear axonal growth and visualize the effects of ultrasound.

Written by Jackson Levine, project group leader and recipient of a combined Dean’s Grant and Cummings Grant, 2018-2019