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

Overview

This video demonstrates in vitro neuroplasticity assay to study the morphological changes in dorsal root ganglia and myenteric plexus neurons exposed to healthy and cancerous human pancreatic tissue extracts. This assay allows us to study the neurite outgrowth, branching pattern and neuronal size.

Protocol

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).

Representative Results

Figure 1. Schematic protocol of the in vitro neuroplasticity assay. The present assay makes use of newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons to study the impact of pancreatic tissue extracts or cell supernatants upon neuronal and glial morphology. DRG are collected after anterior laminectomy and MP-containing seromuscular layer after resection and mechanical stripping of the small intestine (A, B). After collagenase type II digestion, the neurons are seeded in the needed density (see text for details) and cultivated for 24 hr (C, D). Then, the freshly prepared pancreatic (or from any GI organ of interest) extracts and cell supernatants are added in the growth medium of neurons at previously defined concentrations (E). After 48 hr, the cultures are fixed in 4% paraformaldehyde and double-immunostained against neuronal and glial markers (F, G). The neuronal morphology, including neurite density, neural branching pattern and perikaryonal size are measured by means of a standardized software protocol (H). Please click here to view a larger version of this figure.

Materials

Name	Company	Catalog Number	Comments
Poly-D-lysine hydrobromide	Sigma-Aldrich	P1149
Ornithine/laminin	Sigma-Aldrich	P2533/L4544
13 mm Coverslips	Merck		For use in 24-well plate
Dismembranator S	Sartorius
Anti-Beta-III-tubulin antibody	Millipore	MAB1637	1:200 concentration
Anti-GFAP-antibody	DAKO	M0761	1:400 concentration
RIPA buffer + protease inhibitor			Any supplier
Neurobasal medium	Gibco/Life Sciences	21103-049
B-27 Supplement	Gibco/Life Sciences	17504044	Quality of B-27 is known to depend on the lot number
Gentamicin/Metronidazole			Any supplier
Minimal essential medium	Gibco/Life Sciences	31095-029
Hank’s Balanced Salt Solution (HBSS)	Gibco/Life Sciences	24020133	Improves collagenase activity when containing Ca/Mg
Collagenase type II	Worthington Biochemical	CLS-2	Obtain lots with at least 200 U/mg activity
Trypsin-EDTA 0.25%	Gibco/Life Sciences	25200056
4% Paraformaldehyde			Any supplier
AnalySIS docu software	Olympus