Source: Demir, I. E., et al. Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay. J. Vis. Exp. (2014).
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.
1. Isolation of Neurons
Once the extract/supernatant collection is finished, carry on with isolation of neurons.
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.
The authors have nothing to disclose.
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/Metronidazo | 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 |