This method can help answer key questions in the field of Alzheimer disease research, especially the early events involved in growth cone collapse. The main advantage of this technique is to make it possible to visualize and analyze axonal growth cones immediately after amyloid beta treatment. Start by using micro-scissors to mince freshly isolated cerebral cortices from embryonic day-14 mice in neuron culture medium without antibiotics.
Collect the tissues and centrifuge the tissues at 87G for three minutes. Following centrifugation, remove the supernatant and add two milliliters of 0.05%trypsin to the pellet. Incubate for 15 minutes at 37 degrees Celsius and mix by tapping every five minutes.
After 15 minutes, add four milliliters of medium A to neutralize the trypsin, and mix by tapping. Then centrifuge the tissues at 178 times G for three minutes. After removing the supernatant, add DNAse and soybean trypsin inhibitor solution and incubate for 15 minutes at 37 degrees Celsius with mixing every five minutes as before.
When the incubation time is elapsed, add four milliliters of medium A.Mix and centrifuge as before. After removing the supernatant, add four milliliters of medium A and triturate the tissues with a polished Pasteur pipette until no debris is observed. Next, filter the triturated tissues with 70-micron pore-sized mesh.
After filtration, count the cells with a hemocytometer and calculate the cell density. Pipette 0.8 times 10 to the fourth cells in medium A into each well of an eight-well culture slide. Then incubate in a humidified atmosphere of 10%CO2 at 37 degrees Celsius.
After four hours of culturing, change the culture medium to medium B.The purity of neurons using this method is approximately 75%Begin this assay at least a week in advance by opening a fresh vial of commercially obtained full-length A beta 42 and dissolving the contents in sterile, distilled water to a concentration of 0.5 millimolar. Incubate at 37 degrees Celsius for seven days to induce aggregation and toxicity. Prepare aliquots of aggregated A beta 1-42 and store at minus 80 degrees Celsius until use.
On day four of the neuronal culture, treat the wells with 100 microliters of 0.5 micromolar aggregated A beta 1-42 or vehicle solution in medium B for one hour as previously demonstrated. After the hour has elapsed, remove the culture medium and immediately fix the neurons with 4%paraformaldehyde containing 4%sucrose in PBS for one hour at 37 degrees Celsius on a hot plate. Fixation is most important as maintaining the shape of growth cones is critical for this protocol.
After fixation, wash the neurons three times with PBS. Then after removing the chamber, mount the neurons with an aqueous mounting medium. Dry the mounting medium at four degrees Celsius for two to four days.
Capture the entire area of each well with a 20X dry objective lens on an inverted microscope. Capturing and analyzing the entire area in each well is important for avoiding subjectivity. Classify the longest neurites of each neuron in stage three or four as axons.
Axonal growth cones lacking lamellipodia or possessing fewer than three filopodia are considered collapsed growth cones. A beta 1-42 oligomers, shown here before incubation, aggregate after seven days of incubation at 37 degrees Celsius. After treatment with aggregated A beta 1-42, immunostaining with an antibody for the toxic oligomer of A beta shows positive staining in red on A beta 1-42 treated neurons, but not vehicle treated neurons.
The early phenomena induced by A beta 1-42 treatment were analyzed after four days in culture. Identified axons were confirmed as such by positive immunostaining for the axonal marker tau-1 shown in red and negative immunostaining for the dendritic marker, MAP-2, shown in green. After one hour of vehicle treatment, growth cones had spread lamellipodia and several filopodia.
These were identified as healthy growth cones. Conversely, one hour of A beta 1-42 treatment led to shrunken growth cones, which developed no lamellipodia or filopodia. These were identified as collapsed growth cones.
While attempting this procedure, it's important to remember to fix the cells with 4%PFA and 4%sucrose at 37 degrees Celsius to my-nor-idge this fixation protocol in the best. Following this protocol, other method like live-cell imaging and gene transfection can be performed to answer additional questions like when and how the axon growth cones are collapsed and how the collapsed growth cones are recovered.
