不定冠詞<em>インビトロ</em成体マウスの筋層間神経叢から分離腸ニューロンとグリアの>準備
Summary
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.
Abstract
腸神経系は、機能的に消化管運動を制御して消化管の長さを実行しているニューロンとグリアの広大なネットワークである。筋層間神経叢からの神経細胞とグリア細胞の混合集団の単離および培養のための手順を説明する。初代培養は、ニューロンとグリアの間で開発の接続で、7日以上維持することができる。添付叢と縦筋ストリップは、マウスの回腸または結腸の基礎となる円形の筋から除去し、酵素消化に供される。無菌状態では、単離された神経細胞とグリア人口はペレット次遠心内に保存され、カバースリップ上に播種。 24〜48時間以内に、神経突起伸長が起こり、ニューロンは汎神経マーカーで識別することができます。パッチクランプ研究によって観察されるような文化の中で2日後、ニューロン火災活動電位を単離した。さらに、腸グリアもidentifできGFAP染色によりiedを。近い同格のニューロンとグリアのネットワークが5内のフォーム – 7日。腸ニューロンは個別になり、直接的に、例えば免疫組織化学、電気生理学、カルシウムイメージング、および単一細胞PCRなどの方法を用いて研究することができる。さらに、この手順は、遺伝的に改変された動物において行うことができる。この方法は、実行する簡単で安価である。我々はより良い、正常および疾患状態におけるENSの機能を発見することができるように全体的に、このプロトコルは、簡単に操作方法で、腸神経系の構成要素を公開します。
Introduction
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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
National Institute of Health Grant DA024009, DK046367 & T32DA007027.
Materials
| 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 |
References
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