An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

Podsumowanie

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Streszczenie

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

Wprowadzenie

The enteric nervous system (ENS) is vast network of nerves and glia that runs the entire length of the gastrointestinal (GI) tract. The ENS functionally controls all aspects of digestion, including peristalsis, fluid absorption/secretion, sensation of stimuli, etc (for review see ¹). It contains over 500 million neurons, more than found in the spinal cord, and contains every neurotransmitter class found in the brain. Furthermore, the ENS is unique in that it can function reflexively without input from the central nervous system ². Understanding of the ENS is crucial, not only to understand its normal physiological role, but to understand its involvement in a variety of neuropathies which can be congenital (Hirschsprung's disease), acquired (Chagas), secondary to disease states (diabetic gastroparesis), drug-induced (Opioid bowel syndrome), or due to injury (postoperative ileus) ¹. In addition, enteric neurons can be a reservoir for viral infection (varicella zoster)³. Because of its similarities to the brain and the high levels of serotonin in the gut, medications aimed at treating central nervous system defects often have unwanted side effects on the ENS ². It is also noteworthy that many neuropathies such as Alzheimer's disease and Parkinson's disease show similar cellular changes in the enteric neurons long before their appearance in central neurons, making the ENS an accessible model to study the pathogenesis of these diseases ⁴. Therefore, a thorough understanding of the ENS is a necessity in understanding disease states and preventing/predicting pharmacological side effects.

The neurons of the ENS have been traditionally studied in the guinea pig using wholemount preparations ^5-7 or cultured neurons ⁸. Despite the ease at which neurons can be studied in this large animal, this model has many limitations including lack of genetically modified strains, lack of reagents specific to this species, and the high cost associated with ordering and housing these subjects. The development of a murine enteric nervous system model has the unique advantage of various knock out systems, a vast array of other established methodologies that can be used in conjunction with the cell culture technique, and the ability to provide a validation for the guinea pig model.

The ENS is comprised of three plexi that run the length of the gastrointestinal tract: the outer myenteric plexus (between the longitudinal and circular muscle) which is mainly responsible for the peristaltic actions of the gut, as well as the submucosal and mucosal plexi, (found under and within the mucosa, respectively) which largely controls fluid absorption/secretion and the detection of stimuli ¹. This method begins with the isolation of the longitudinal muscle/ myenteric plexus (LMMP) preparation by peeling off the outer muscle layer of the GI tract. This dramatically cuts down on contamination issues that arise when the mucosal layer is involved in the isolation. As a result, this process is ideal for the study of neuronal control of motility rather than secretory actions of the ENS.

The method presented here results in a mixed culture of enteric neurons and glia. At least two different types of neurons are present based on previous electrophysiological and immunocytochemical observations ⁹. The presence of glia is highly advantageous, as they are not only an important cell type to study in their own right, but they contribute to the survival of the enteric neurons ¹⁰ and maintain native receptor expression on the neuronal cell surface ¹¹. Furthermore, deficiencies of enteric glia may lead to abnormal gastrointestinal motility disease states, coined 'neuro-gliopathies' ¹². Therefore, the ENS culture presented here results in several cell types that are ripe for investigation.

The advantages to this methodology are ease of isolation, inexpensive tool requirements, and a short time to master the technique by experienced lab personnel. Limitations of the methodology include low overall cell yield from high tissue volumes and the exclusion of ENS neurons from mucosal and submucosal plexi. This procedure will be highly advantageous to scholars specializing in electrophysiology, immunohistochemistry, single-cell PCR, and other methodologies.

Protokół

All animal care and experimental procedures were in accordance with and approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.

1. Preparation of Sterile Poly-D-Lysine- and Laminin-Coated Glass Coverslips in 24-Well Plates

1. All procedures for step 1 are performed in sterile conditions; under a hood, and with sterile reagents. Glass coverslips and double deionized water (ddH₂O) should be sterilized in advance. Preparation of plates can be done up to two weeks before neuron isolation.
2. Under the hood, use sterile forceps to place autoclaved glass coverslips in 12 wells of a 24-well plate.
3. Prepare poly-D-lysine stock ahead of time. Add 50 ml of sterile ddH2O to 5 mg poly-D-lysine. Vortex and store in 3 ml aliquots at -20 °C. Thaw aliquots before use.
4. For a final concentration of 1 ml poly-D-lysine stock per 25 cm²: pipette 80 μl of poly-D-lysine stock on top of each glass coverslip (2 cm²). Let solution settle for 10 min.
5. Remove poly-D-lysine using vacuum and rinse coverslips 3x with sterile ddH₂O.
6. Allow plates to dry for at least 2 hr under the hood.
7. Plates may be stored at 4 °C or - 20 °C before proceeding to laminin coating.
8. Prepare laminin stock ahead of time. Thaw laminin on ice and keep on ice during use. Dilute to a concentration of 50 μg/ml; pay attention to lot concentration; each lot will be different. Depending on lot concentration, approximately 20 ml ddH₂O will be added to each vial of laminin. Aliquot (5 ml) and store at -80 °C. Thaw aliquots on ice before use.
9. Coat coverslips with 5 μg/cm² laminin; pipette 200 μl of laminin stock on each coverslip.
10. Incubate laminin solution on coverslips for 1 hr.
11. Aspirate remaining laminin solution and rinse coverslips once with ddH₂O. Avoid scraping surface of coverslips.
12. Store plates at 4 °C for up to two weeks.

2. Advance Preparation of Neuron Isolation Solutions

1. Prepare Krebs solution (in mM: 118 NaCl, 4.6 KCl, 1.3 NaH₂PO₄, 1.2 MgSO₄, 25 NaHCO₃, 11 glucose, and 2.5 CaCl₂). Place 13.79 g NaCl (FW 58.44), 0.686 g KCl (FW 74.55), 0.312 g NaH₂PO₄ (FW 120), .289 g MgSO₄ (FW 120.4), 4.20 g NaHCO₃ (FW 84.01), 3.96 g glucose (FW 180.2), and 0.555 g CaCl₂ (FW 111) in 2 L ddH₂0. Chill to 4 °C.
2. Prepare rinse media (F12 media with 10% fetal bovine serum (FBS) and antibiotic/antimycotic): To a 500 ml bottle of F12 media, add 50 ml FBS and 5 ml of Antibiotic/Antimycotic 100x Liquid (Gibco).
3. Prepare enteric neuron media (Neurobasal A media with B-27, 2 mM L-glutamine, 1% FBS, 10 ng/ml, Glial Derived Neurotrophic Factor (GDNF), and Antibiotic/Antimycotic 100x Liquid). To make GDNF stock, add 1 ml ddH2O to 10 μg GDNF and freeze back in 50 μl aliquots, store at -80 °C. For a 50 ml vial of complete neuron media, combine 47.5 ml Neurobasal A media, 1 ml B-27 (50x), 500 μl FBS, 500 μl 200 mM L-glutamine (Gibco) 50 μl GDNF stock, and 500 μl Antibiotic/Antimycotic 100x Liquid. Media is made in small 50 ml batches to ensure freshness of GDNF and to avoid contamination of large amounts of this expensive cell media mixture.

3. Harvest Longitudinal Muscle/Myenteric Plexus (LMMP) Preparation from Mice

1. Appropriate national and institutional ethics should be in place before performing animal experiments. Adult Swiss Webster mice weighing over 25 g (typically over 8 weeks of age) are housed in groups of 6 prior to experiments. Tissues from two mice are used for the isolation of ileal enteric neurons and glia and three mice are used for isolation of colonic cells.
2. Prepare surgical area. Gather small surgical scissors, angled forceps, cotton swabs, three 200 ml glass beakers, and a glass/plastic rod (paintbrush). Ensure all supplies are thoroughly washed and rinsed before use to minimize contamination. This step can be performed the day before the experiment.
3. Place Krebs solution on ice and bubble with carbogen (95% oxygen, 5% CO₂) for at least 30 min to stabilize pH.
4. Turn on various equipment and warm chemicals to stabilize at desired temperatures; heat water bath(s) to 37 °C, cool centrifuge to 4 °C, place Hank's balanced salt solution (HBSS) and 0.5% trypsin in water bath (37 °C). Complete neuron media and rinse media should remain refrigerated.
5. Place 150 ml ice cold bubbled Krebs in three 200 ml glass beakers labeled 'dirty', 'clean', and 'LMMP'. Bubble beaker labeled 'LMMP' with carbogen.
6. Following approval from ethics committee, euthanize mouse by cervical dislocation or CO₂ asphyxiation.
7. Place mouse in dorsal recumbence on surgical surface, clean skin with 70% EtOH, and lift abdominal skin using forceps. Using scissors, open abdominal cavity and reveal internal digestive organs.
8. Remove the length of the gastrointestinal tract by lifting a section of ileum and revealing its mesentery. Snip through mesentery with scissors to gently remove and unravel ileum and colon, being careful not to pull mesentery from the ileum/colon.
9. After the full length of intestine is unraveled, remove the ileum by cutting through the intestine distal to the stomach and proximal to the cecum. Note: this procedure can also be performed with colonic tissue by removing the colon from distal to the cecum to proximal to the anus.
10. Tissues can be further divided between proximal and distal colon, and jejenum, and ileum. The rest of this procedure will refer to isolation of the ileum; the procedure is the same for the isolation of colon LMMP. Place the entire ileum in a glass container with ice cold Krebs marked 'dirty'.
11. Create a tool to clean the ileum; blunt a large 20 G needle using a filing stone and attach it to a large syringe (10 ml) containing ice cold Krebs.
12. Clean fecal matter from the ileum by sectioning the ileum into three or more large pieces. Remove an ileal section from the beaker and place the blunted needle into the end of an ileal piece.
13. Gently run Krebs through the gut section until all fecal matter is removed into a separate waste container. Place the cleaned section into the container of Krebs marked 'clean'. Repeat until entire ileum is cleaned.
14. To remove the LMMP, cut the ileum into small segments, approximately 2 - 4 cm. Place a segment of ileum on a plastic or glass rod; ileum should fit snuggly but not be loose or taut (~2 mm). Begin removal of the LMMP by gently removing bits of mesentery still attached to the gastrointestinal (GI) tract using a forceps. Prevent the GI tract from rotating around rod by gently pinning the tube to the rod using the thumb.
15. Separate the LMMP from the underlying circular muscle; first gently rub the edge of the forceps along the entire line where the mesentery was attached, from top to bottom of the segment, gently creating a gap in the longitudinal muscle. Then gently tease away the longitudinal muscle using a cotton swab wetted with Krebs.
16. Begin at the top of the gap in the longitudinal muscle and tease away using the lightest horizontal stroke while applying very light pressure until the longitudinal muscle just begins to separate from the circular muscle; do this down the entire strip along the mesentery attachment point.
17. Then gently begin to work around the GI tube; moving from top to bottom and back as the longitudinal muscle is slowly separated from the circular muscle all the way around the tube. When complete, the LMMP will naturally come off the remainder of the GI tube.
18. Place the resulting thin strip of longitudinal muscle in the beaker marked 'LMMP'. Repeat for all the segments.
19. After all the strips of LMMP have been gathered from one mouse, repeat procedure for any other mice being used. Thoroughly rinse the 'dirty' and 'clean' beakers before re-use.
20. Rinse the LMMP 3x to remove biological contamination. Fill three 2 ml Eppendorf tubes with ice cold Krebs. Place the LMMP strips in the first tube and spin for 30 sec at 356 x g in a centrifuge cooled to 4 °C. Carefully remove the supernatant with a pipette and move the LMMP strips to next clean Krebs filled tube. Repeat this procedure for each remaining tube.

4. Digest LMMP

1. Prepare digestion solution by placing 13 mg collagenase type 2 and 3 mg BSA in 10 ml carbogen-bubbled Krebs solution.
2. Place segments of rinsed LMMP into digestion solution and use scissors to snip LMMP into tiny pieces.
3. Digest LMMP for 60 min at 37 °C in water bath with shaker while being gently bubbled with carbogen.
4. After digestion is complete, gather cells by centrifugation for 8 min at 356 x g in centrifuge cooled to 4 ° C.
5. From this point forward all procedures should be done under sterile conditions in a cell culture hood! All reagents and containers should be sterilized before use.
6. During centrifugation, prepare 0.05% trypsin solution by placing 1 ml warmed 0.25% trypsin and 4 ml warmed HBSS into a sterile 50 ml cell culture tube.
7. After centrifugation, remove and discard supernatant. Do not use a vacuum; this will likely suck up the cell pellet due to low centrifugation speeds. Carefully remove the cell pellet and place into clean tube with 5 ml of 0.05% trypsin solution.
8. Digest cells in 0.05% trypsin solution in 37 °C water bath with shaking for 7 min. Do not exceed 7 min of total trypsin treatment or neurons will perish.
9. Neutralize trypsin with 10 ml of cold rinse media after 7 min of trypsin treatment.
10. Centrifuge cells for 8 min at 356 x g. Remove and discard media.
11. During centrifugation, balance a section of sterilized Nitex mesh on top of a sterile 15 ml cell culture tube.
12. After centrifugation, remove and discard supernatant. Gently resuspend cell mixture by triturating in 3 ml complete neuron media. Avoid generating air bubbles during this step and all future steps in which cell mixture is triturated. All triturations should be done very gently. Filter cell solution through Nitex mesh into clean 15 ml cell culture tube.
13. Optional: Cap cell culture tube and place in refrigerated Hula mixer (or any mixer that provides rotation) with gentle rolling and tilting for 30 min. This step is optional but recommended.
14. Optional: After Hula mixer, add 2 ml cold rinse media to wash cells.
15. Gather cells by centrifugation (8 min, 356 x g). Remove and discard supernatant.
16. Resuspend cells in 1,200 μl complete neuron media. Triturate cells gently using 1 ml plastic pipette tip. Do not generate air bubbles. Pipette slowly and gently until most chunks are broken up and cells are suspended into liquid.
17. Add 750 μl of complete media to each of the 12 wells containing a pre-coated glass coverslip and add 100 μl of the triturated cell solution.
18. Incubate neurons in cell culture incubator at 37 °C with 5% CO₂.
19. Change half of the cell media every 2 days.
20. Neurons are ready for electrophysiological recordings after 1 - 2 days in culture.

Wyniki

Immediately following isolation of LMMP-derived cells, neurons and other cell types will not be readily evident. Living, round cells of indistinct phenotype can be seen as well as tissue detritus from incompletely digested tissue fragments and connective tissue. This flotsam is of no concern and will be largely removed with the first media change in two days. Do not attempt to clean the slides before this as the healthy, viable cells will be removed as well.

After one day in culture, neurons w...

Dyskusje

Animals Used

This protocol has been optimized for Swiss Webster mice. However, this method is easily adaptable to other small-sized mammals such as rats and to other strains of mice. We have successfully performed preliminary isolations with C57 mice and μ-opioid receptor knock-outs. However, it is also possible that other strains of mice may be problematic due to morphological variations in the GI tract. Furthermore, there are known differences between mouse strains (C57Bl/6 vs. Balb/c) in th...

Materiały

Name	Company	Catalog Number	Comments
Reagents
Fisherbrand Coverglass for Growth Cover Glasses (12 mm diameter)	Fisher Scientific	12-545-82
Poly-D-lysine	Sigma	P6407- 5 mg
24-well cell culture plate	CELLTREAT	229124	May use any brand
Laminin	BD Biosciences	354 232
ddH2O			Can prepare in lab
15 ml Sterile Centrifuge Tube	Greiner Bio-one	188261	May use any brand
50 ml Sterile Centrifuge Tube	Greiner Bio-one	227261	May use any brand
NaCl	Fisher BioReagents	BP358-212	MW 58.44
KCl	Fisher BioReagents	BP366-500	MW 74.55
NaH2PO4 .2H2O	Fisher Chemicals	S369-3	MW137.99
MgSO4	Sigma Aldrich	M7506-500G	MW 120.4
NaHCO3	Sigma Aldrich	S6014-5KG	MW 84.01
glucose	Fisher Chemicals	D16-1	MW 180.16
CaCl22H2O	Sigma Aldrich	C5080-500G	MW 147.02
F12 media	Gibco	11330
Fetal Bovine Serum	Quality Biological Inc.	110-001-101HI	May use any brand
Antibiotic/antimycotic 100x liquid	Gibco	15240-062
Neurobasal A media	Gibco	10888
200 mM L-glutamine	Gibco	25030164
Glial Derived Neurotrophic Factor (GDNF)	Neuromics	PR27022
Sharp-Pointed Dissecting Scissors	Fisher Scientific	8940	May use any brand
Dissecting Tissue Forceps	Fisher Scientific	13-812-41	May use any brand
Cotton-Tipped Applicators	Fisher Scientific	23-400-101	May use any brand
250 ml Graduated Glass Beaker	Fisher Scientific	FB-100-250	May use any brand
2 L Glass Erlenmyer flask	Fisher Scientific	FB-500-2000	May use any brand
Plastic rod (child's paint brush)	Crayola	05 3516	May use any brand
Carbogen	Airgas	UN 3156	5% CO2
10 ml Leur-lock Syringe	Becton Dickinson	309604	May use any brand
21 G x 1 1/2 in. Hypodermic Needle	Becton Dickinson	305167	May use any brand
Collagenase type 2	Worthington	LS004174
Bovine Serum Albumin	American Bioanalytical	AB00440
2 ml Microcentrifuge Eppendorf tubes	Fisher Scientific	13-864-252	May use any brand
Nitrex Mesh 500 µM	Elko Filtering Co	100560	May use any brand
Pipette Set	Fisher Scientific	21-377-328	May use any brand
Sharpeining Stone	Fisher Scientific	NC9681212	May use any brand
Equipment
LabGard ES 425 Biological Safety Cabinet (cell culture hood)	Nuaire	NU-425-400	May use any brand
10 L Shaking Waterbath	Edvotek	5027	May use any brand
Microcentrifuge 5417R	Eppendorf	5417R	May use a single larger centrifuge with size adapters
Allegra 6 Series Centrifuge	Beckman Coulter	366816	May use any brand
HuluMixer Sample Mixer	Invitrogen	15920D
AutoFlow Water Jacket CO2 Incubator	Nuiare	NU-4750	May use any brand
Analytical Balance Scale	Mettler Toledo	XS104	May use any brand