Methods to Study the Myenteric Plexus of Rat Small Intestine

Abstract

The close interaction between the enteric nervous system, microbiome, and brain in vertebrates is an emerging topic of recent studies. Different species such as rat, mouse, and human are currently being used for this purpose, among others. The transferability of protocols for tissue isolation and sample collection is not always straightforward. Thus, the present work presents a new protocol for isolation and sample collection of rat myenteric plexus cells for in vivo as well as in vitro studies. With the methods and chemicals described in detail, a wide variety of investigations can be performed with regard to normal physiological as well as pathological processes in the postnatal developing enteric nervous system. The fast and efficient preparation of the intestine as the first step is particularly important. We have developed and described a LIENS chamber to obtain optimal tissue quality during intestinal freezing. Cryosections of the flat, snap-frozen intestine can then be prepared for histological examination of the various wall layers of the intestine, e.g. by immunohistochemistry. In addition, these cryosections are suitable for the preparation of defined regions, as shown here using the ganglia of the mesenteric plexus. This specific tissue was obtained by laser microdissection, making the presented methodology also suitable for subsequent analyses that require high quality (specificity) of the samples. Furthermore, we present here a fully modernized protocol for the cultivation of myenteric neurons from the rat intestine, which is suitable for a variety of in vitro studies.

Progesterone: A Neuroprotective Steroid of the Intestine

“…Intestines of Wistar rats at the postnatal (p) day 9, 15 and 30 were used for the methods previously described by Hecking et al [ 19 ]. All solutions were prepared with DEPC-treated water (#D5758-50ML, Sigma Aldrich, St. Louis, MO, USA).…”

Neuroprotective Effects of VEGF in the Enteric Nervous System

“…Cryosections of the myenteric plexus were obtained using the flat frozen small intestine as described before [ 51 ]. For RNase-free conditions, removable parts of the cryostat (CryoStar NX50, Thermo Scientific) were cleaned with NaOH-EDTA (0.1 M NaOH, 1 mM EDTA).…”

“…After 20–30 min acclimation time, a small piece was attached to the object holder of the cryostat with the serosa now facing up. In this way, the intestine was cut layer by layer until the myenteric plexus could be seen between the circular muscle layer and the longitudinal muscle layer as already described before [ 51 , 52 ]. Then, 10 μm cryosections for immunohistochemistry were mounted on Superfrost-Plus Adhesion Slides (J1800AMNZ, Thermo Scientific) and stored at 4 °C until further use.…”

“…Laser microdissection was performed as described previously [ 51 ]. Leica LMD software (LMD650, Leica Application Suite (LAS), Leica Microsystems, Wetzlar, Germany) was used to mark the myenteric ganglia.…”

“…Myentericus plexus cell cultures from p15 Wistar rats were prepared as described before [ 51 , 54 ]. Succinctly, the small intestine from three rats was removed as explained in 4.1, stored in cooled MEM-P/S (MEM-Hepes (M7278, Sigma-Aldrich) containing 1% penicillin-streptomycin (p4333, Sigma-Aldrich) and cut into 3 cm long pieces.…”