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

1. Isolation of Neurons

Once the extract/supernatant collection is finished, carry on with isolation of neurons.

1. For DRG neurons, collect the cervical to lumbar DRG of newborn rats between postnatal day (P) 2-12 after decapitation and stereomicroscopic dissection of DRG (Figure 1A). In order to have sufficient DRG neurons for a 24-well plate, collect all cervical to lumbar DRG of one newborn rat (equaling 52 DRG) per plate.
2. Cut away the peripheral (neural) and central projections (roots) of DRG by means of microscissors. Leaving the projections in place impedes trituration and increases the contamination risk of culture by fibroblasts.
3. For MP neurons, cut away the mesentery from the small intestine and manually and carefully strip off the seromuscular layer of the small intestine (for a detailed protocol on MP isolation, Figure 1B). For MP, collect the plexus from two rats per 24-well-plate.
   NOTE: Try to be as gentle as possible in order to avoid tears in the seromuscular layer since they impede its successful separation from the small intestine.
4. Collect the DRG and seromuscular layer in ice-cold minimal essential medium (MEM) supplied with gentamicin (20mg in 500ml medium) and metronidazole (2.5 mg in 500 ml medium).
5. Following the collection of DRG, incubate them in Hank’s Balanced Salt Solution (HBSS) supplied with collagenase Type II for 20-30 min. For MP isolation, incubate in collagenase type between 1-3 hr, depending on the age of the animal (Figure 1C).
6. For MP, collect the net-like MP pieces under a stereomicroscope and transfer to ice-cold MEM.
7. Then triturate the DRG and MP through syringes with decreasing diameter.
   NOTE: Excessive trituration can destroy the neurons, but less the glial cells.
8. Once the medium containing the DRG or MP has become cloudy, centrifuge the suspension at 93.9 x g for 5 min, discard the medium and resuspend in Neurobasal medium (supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.5mM L-glutamine and 2% B-27).
9. Count the total number of cells (i.e. neurons AND glia) by means of a hemocytometer.
   NOTE: The number of needed cells is dependent on the measurement parameter. For the quantification of neurite density, one needs denser cultures and thus a greater number of cells than for the measurement of neurite outgrowth, perikaryonal size and branching pattern of individual neurons. For neurite density measurements, use e.g. 10,000 cells (neuron+glia)/well or 1,500 neurons/well. For morphometry on individual neurons, brief (1 minute-long) trypsinization of cells and seeding of 2,500 cells/well or 400 neurons/well is recommended. The typical yield of cells obtained from one rat is around 300,000-500,000 cells.
10. Seed the cells on 13 mm coverslips which have been coated the night before and top the wells with Neurobasal medium (supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.5 mM L-glutamine and 2% B-27) (Figure 1D).
    NOTE: Use poly-D-lysine (40 mg/m²) OR ornithine- and laminin-coated (1 mg/ml each) coated coverslips. Cells attach to poly-D-lysine extremely fast, thus making it suitable for experiments in which many cells are needed (i.e. neurite density measurements). Attachment to laminin is in general somewhat weaker, but laminin is a strong promoter of neurite outgrowth. Therefore, for measurements on neuronal branching and neurite length, prefer ornithine/laminin-coating.
11. Allow the cells to attach to the wells overnight. On the next day, prepare the extract-supplemented media (at a final extract/supernatant concentration of 100 µg/ml).
12. Aspirate the seeding medium, and perform an optional, very gentle wash with PBS, and then slowly pipette the extract/supernatant-supplemented media (Figure 1E).
13. Let the cells grow for 48 hr. Aspirate the media and fix in 4% paraformaldehyde for immunostaining (Figure 1F).
14. Perform double immunofluorescence staining using neuron-specific (e.g. beta III-tubulin) and glia-specific (e.g. glial fibrillary acidic protein/GFAP) markers (Figure 1G).
15. For morphometry, use an inverted light microscope equipped with a CCD camera in combination with automated software which allows measurement of neurite density (see Table).
16. The neurite density of neuronal cultures is measured on 4-5 representative photomicrographs at 200x magnification from 4 different regions of densest growth on each coverslip by overlaying a 50 µm x 50 µm grid and counting the fiber density per square measured in the intersecting fibers. Neurite outgrowth, mean number of branches per neuron, the mean branch length and perikaryonal size can be measured from randomly selected 30 solitary neurons from each coverslip by marking the neurites and perikarya (Figure 1H).