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

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.