In Vitro Pancreatic Neuroplasticity Assay: Studying Changes in GI Tract Neurons Exposed to Healthy and Cancerous Human Pancreatic Tissue Extracts

- The dorsal root ganglion or DRG are sensory neurons that relay messages from the periphery to the central nervous system. In contrast, myenteric plexus neurons are involved in peristaltic movements of the bowels.

In cancer conditions such as pancreatic cancer, these neurons alter morphologically and functionally. To study the neuronal morphology changes, seed the neural cells on coverslips pre-coated with laminin protein, placed in a multiwell plate, and top the cells with the neural basal medium.

Laminin protein promotes neurite outgrowth. Incubate the cells overnight. Next, add the pancreatic cancer tissue extract media to the first three wells and healthy pancreatic tissue extract media to the subsequent three wells. Incubate the plate. Now, perform immunohistochemical staining using neuron-specific antibody markers, which bind to neurons. Place the coverslip on the glass slide and place the slide under an inverted microscope. Measure neurite density using the software.

The DRG neurons cultured in pancreatic cancer tissue extract usually show greater neurite density than in healthy pancreatic tissue extract. In contrast, myenteric plexus neurons form longer neurites than in healthy pancreatic tissue extracts. In the following protocol, we will perform in vitro neuroplasticity assay on a newborn mouse's DRG and myenteric plexus neurons.

- To prepare the cells for culture, use a hemocytometer to estimate the total number of neurons and glia. Then, seed the cells on 13 millimeter pre-coated coverslips in a 24-well plate. The coating used depends on the experimental aims. Next, top the wells with neural basal medium and allow the cells to attach to the wells, overnight. The next day, defrost the tissue extract supplemented media, or the cell line supernatant supplemented media.

Next, aspirate the seeding medium from the DRGs and perform an optional, very gentle wash with PBS. Then, slowly pipette the tissue extract supplemented media or the cell line supernatant supplemented media onto the cells to yield 100 micrograms per milliliter. Now, let the cells grow for 48 hours at 37 degrees Celsius in the tissue extract or cell line supernatant supplied media. After 48 hours, aspirate the media and fix in 4% paraformaldehyde for immunostaining.

For double immunofluorescent staining, use neuron-specific and glia-specific markers. For morphometry, use an inverted light microscope equipped with a CCD camera in combination with automated software to measure neurite density. Using 200X magnification, measure the neurite density of neuronal cultures by overlaying a 50 X 50 micron grid and counting the fiber density per square, measured in the intersecting fibers. Repeat this analysis for four different regions of densest growth on four to five representative photomicrographs on each coverslip.

Measure the neurite outgrowth, mean number of branches per neuron, mean branch length, and perikaryonal size from 30 randomly selected solitary neurons from each coverslip by marking the neurites and perikarya.