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

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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).

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