Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Summary

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Abstract

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Introduction

Alterations in gastrointestinal (GI) nerve morphology and density have caught the attention of gastroenterologists and pathologists for a long time, but their relevance for the pathophysiology of GI diseases remains unknown^1-3. Indeed, several highly common GI disorders such as gastritis, reflux esophagitis, colitis, diverticulitis, and appendicitis are associated with increased innervation density in inflamed tissue areas¹. However, no genuine attention has so far been paid to the mechanisms and meaning of neuroplasticity in the GI tract. Do morphologically altered GI nerves differ from normal GI nerves, i.e. the normal state of the enteric nervous system, in terms of their function? What are the implications of altered neuropeptide/neurotransmitter content in plastic enteric nerves? Does peripheral neuroplasticity always entail altered signaling to the central nervous system? And where are the central projections of plastic extrinsic GI neural pathways? A long series of such key questions can easily be generated when looking at the paucity of our knowledge on the functional aspects of GI neuroplasticity. 

The study of GI neuroplasticity at functional level requires valid, reproducible and still easily applicable experimental models. In an era of increasing popularity and acceptance of genetically engineered conditional mouse models (GECoMM), such in vivo settings bear the potential to elucidate previously unknown facets of GI neuroplasticity in a realistic fashion¹. However, the design and production of GECoMM remains costly, labor-intensive and, especially, time-consuming. Furthermore, they require the a priori selection of the target to be conditionally modulated in the genetically altered mouse (e.g. transgenic overexpression of nerve growth factor/NGF in enteric epithelial cells). Hence, for the design of a successful GECoMM, researchers need some indicators (e.g. previous experimental data) of a worthwhile target, i.e. that the molecule of interest (here NGF) can at least be expected to exert some biologically relevant effects on GI nerves.

Such indicators can easily be derived from adequate in vitro models in which isolated cell subtypes from the complex microenvironment of an in vivo system can be selectively cocultured in a heterotypic manner^4-7. The modulation of molecular targets in such a heterotypic culture setting is on average technically less cumbersome, faster, and can therefore aid in the prefiltering of worthwhile targets for verification in in vivo studies.

Recently, we presented an in vitro neuroplasticity assay which was designed to simulate the increased neural density and hypertrophy of intrapancreatic nerves in human pancreatic cancer (PCa) and chronic pancreatitis (CP) tissues. Here, neurons derived from newborn rat dorsal root ganglia (DRG) or myenteric plexus (MP) were exposed to tissue extracts from surgically resected PCa or CP tissues specimens and compared to those cultured in normal human pancreas (NP) tissue extracts⁵. Instead of tissue extracts, one can also use cell line supernatants to study the impact of selected cell types on neuroplasticity. When combined with a standardized morphometric measurement, the presented neuroplasticity assay allows valid and reproducible assessment of neuronal plasticity in response to different pancreatic microenvironments. Particularly, it allows the simulation of 1) morphological neuroplasticity, i.e. the changes in the neurite outgrowth, branching pattern and neuronal size, and 2) functional neuroplasticity, i.e. alterations in the excitability of peripheral neurons. Moreover, not only peripheral (i.e. enteric), but also central (e.g. DRG or second order spinal) neurons can be included in the present assay to assess their morphological and functional reaction to different GI tissue contents. In the present video tutorial, we demonstrate the technical protocol for the performance of this assay and discuss its advantages and weaknesses. Moreover, we draw attention to the applicability of the basic notion of this assay to the study of neuroplasticity in any GI organ.

Protocol

All animal experimental procedures in the protocol follow the animal care guidelines of Technische Universität München, Germany.

1. Media/Extract Preparation

1. Tissue homogenization
   The quality of tissue homogenization is critical to the subsequent detection of neuroplastic alterations in the cultured neurons. Here, a homogenizer is recommended which enables tissue dissociation without major increase in the tissue temperature.
   1. Transfer 5 mm x 5 mm x 5 mm cubes of pancreatic tissue directly from -80 °C to liquid nitrogen and place in a solid phase in the tissue homogenator. The ideal homogenator would dissociate the tissue fast enough, without allowing it to defreeze.
   2. Immediately after dissociation, resuspend the solid, powder-like homogenate in 300-500 µl of 0.1x phosphate-buffered saline (PBS). Here, do not use any lysis buffer (such as RIPA) since this may lyse the neurons.
   3. Centrifuge the homogenates for at least 15 min at the maximum speed of your bench centrifuge (e.g. 21,130 x g). Collect the clear supernatant which represents the tissue extract.
2. Cell line supernatants
   1. Let the cells of interest reach at least 70 - 80% confluence in normal growth medium.
   2. Usually, these media contain serum components and may exert uncontrollable effects upon neuronal growth. Therefore, after reaching the desired cell density, wash your cells at least 3x with cell-culture grade PBS and place them in serum-free medium (SFM) for up to 48 hr.
      Note: Here, consider that some cells (like pancreatic stellate cells) may need serum in their growth media in order to maintain their normal state and structure. In such cases, perform serial serum dilutions in the medium of the cell of interest in order to find the lowest possible serum content necessary for intact cell function.
   3. Measure the protein concentration of the tissue homogenate or cell supernatants via Bradford protein assay. While these values vary depending on the tissue and cell type, for pancreatic tissue extracts, one would expect a concentration range between 5-12 µg/µl, and for cell line supernatants between 3-8 µg/µl.

2. Aliquot Extracts and Cell Supernatants

Depending on the concentration to be used in the assay, the final concentration of the extract or supernatant in the neuronal medium should be 100 µg/ml ^5,8.

Note: For an assay with neurons growing in 500 µl medium in each well of a 24-well plate, one needs 50 µg of protein from each extract or supernatant per well. At a typical extract concentration of around 10 µg/µl, one would need 5 µl of extract/supernatant for each well. In every setting performed as a triplicate, this would correspond to 15 µl of extract/supernatant. For different tissue or cell types, perform serial dilutions of the final extract or supernatant concentration and compare the observed neurotrophic effects between different extract/supernatant concentrations.

3. 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.
   1. 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.
2. 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, refer to Schäfer et al.⁹, 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.
3. Collect the DRG and seromuscular layer in ice-cold minimal essential medium (MEM) supplied with gentamicin (20mg in 500ml medium) and metronidazol (2.5 mg in 500 ml medium).
4. Following 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).
5. For MP, collect the net-like MP pieces under stereomicroscope and transfer to ice-cold MEM.
6. Then triturate the DRG and MP through syringes with decreasing diameter.
   Note: Excessive trituration can destroy the neurons, but less the glia cells.
7. 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).
8. 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) trypsination 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.
9. 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 cover slips. 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.
10. Allow the cells attach to the wells overnight. On the next day, prepare the extract-supplemented media (at a final extract/supernatant concentration of 100 µg/ml).
11. Aspirate the seeding medium, and perform an optional, very gentle wash with PBS, and then slowly pipette the extract/supernatant-supplemented media (Figure 1E).
12. Let the cells grow for 48 hr. Aspirate the media and fix in 4% paraformaldehyde for immunostaining (Figure 1F).
13. Perform double immunofluorescence staining using neuron-specific (e.g. beta III-tubulin) and glia-specific (e.g. glial fibriallary acidic protein/GFAP) markers (Figure 1G).
14. 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).
15. 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).

Results

Morphological neuroplasticity

In the indicated age range of newborn rats (P2-12) and the seeding densities, MP and DRG neurons already build dense neuronal networks after 48 hr (Figure 2A). Comparison of neurite density between neurons cultivated in PCa, CP, and NP extracts reveals greater neurite density of DRG neurons in PCa or CP extracts than in NP extracts (Figure 2A)⁵. We particularly prefer MP neurons for measurements on individual n...

Discussion

The present protocol is intended to illustrate the methodology behind the in vitro pancreatic neuroplasticity assay which was recently developed by our group to study the mechanisms of neuroplasticity in PCa and CP⁵. The protocol involves a three-day procedure which can easily be applied once the performer has gained sufficient experience in the isolation and culture of DRG and MP neurons. Furthermore, it represents a valuable tool for studying the concomitant reaction of enteric and DRG-associated gl...

Materials

List of materials used in this article

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 plates
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/Metronidazol			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