In Vitro Pancreatic Neuroplasticity Assay: Studying Changes in GI Tract Neurons Exposed to Healthy and Cancerous Human Pancreatic Tissue Extracts
Published: April 30, 2023
Abstract
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.
Protocol
1. Isolation of Neurons Once the extract/supernatant collection is finished, carry on with isolation of neurons. 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. Cut away the p…
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 resect…
Divulgaciones
The authors have nothing to disclose.
Materials
| 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 |
Citar este artículo
In Vitro Pancreatic Neuroplasticity Assay: Studying Changes in GI Tract Neurons Exposed to Healthy and Cancerous Human Pancreatic Tissue Extracts. J. Vis. Exp. (Pending Publication), e20322, doi: (2023).