The Home Stretch: Finalizing Data and Analysis

Hello again. As the summer approaches its conclusion, I seek to approach a data conclusion of my own. This summer so far has been full of successes and failures alike. From the fiasco when my glass-bottomed dish that had promising results on it broke mid experiment, to improving our cultures to the point where we can consistently grow long, singular axons across most of a well, the spectrum of success has been quite distributive. As I type, I am preparing my grand finale experiment of using a 35 mm dish with five DRG explants on it. The axons have had time to grow for two days and look ready for an axotomy, calcium staining, and imaging. I will, once again, use the live cell chamber on the confocal microscope to simulate physiological conditions in the body. Hopefully, this will more accurately and credibly yield data that mimics what would happen in the body of a zebra finch during axon degeneration.

I carried out an experiment last week on a 24 well plate that was a pretty standard setup: axotomy, stain, image. A fellow researcher in my lab showed that Wallerian degeneration occurs around the 36 hour time point after axotomy. With this in mind, I placed the plate on the microscope after 16 hours. I let the confocal run for 24 more hours and we saw no obvious degeneration. I concluded that the cell bodies must have died beforehand because if the soma is inactive, Wallerian degeneration will not commence. This, yet again, was another disappointing result. If I’ve learned anything this summer, it’s that axons are reasonably difficult to cooperate.

The experiment above was still put to good use though. I ran a program called AxonJudge on Image J that uses an algorithm to subtract the background image and track fragmentation during degeneration. Dr. Buchser is currently working on the analysis and hopefully, we will see some results that the eye couldn’t pick up. Unfortunately, I am not happy with the data on calcium imaging we have collected thus far so hopefully, this present experiment will give me solid data and a great jumping off point for the continuation of my research next semester. In my final post, I will present my data from the finale experiment. Wish me luck!