Protocol
A new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract

https://doi.org/10.1016/S1385-299X(96)00017-7Get rights and content

Abstract

The myenteric plexus is not only essential for gastrointestinal functions, but it is also a very interesting model for the study of neuronal circuits 2, 4and neuron–glial interrelationships 1, 5, 9and may be a valuable source of donor tissue, for grafting into different regions of the central nervous system 3, 6, 10. For both grafting and culture procedures it is a great advantage to obtain the maximum amount of tissue. To date, most studies have isolated the myenteric plexus by manual microdissection after collagenase digestion 4, 5. Using this method, it has only been possible to obtain relatively small amounts of the myenteric plexus, mostly from the cecum and proximal colon of the guinea-pig or rat. We present here a new method, which enables much greater quantities of the plexus from the small intestine and colon to be obtained. The myenteric plexus of the entire small intestine can be isolated by a combination of enzymatic digestion and mechanical agitation. The method works from birth up to 3 week old pups, and with some modifications tissue from older or even adult animals can also be processed. Another advantage over the microdissection method is that the myenteric plexuses of the different parts of the intestine can be cultured and studied separately.

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Section snippets

Type of research

The isolated myenteric ganglia obtained using this method can be used for a variety of types of research, including: electrophysiology, enteric glial cell proliferation, grafting, neurite outgrowth, neuronal morphology, scanning electron microscopy, tissue culture, transmission electron microscopy.

Time required

The time required is dependent upon whether different parts of the intestine are processed separately. The average time for one animal (small intestine) is about 3.5 h.

  • Removal of the gut: 5–10 min.

  • Stripping of the muscle layers: 10 min.

  • Incubation in collagenase: 3–4 times, 60 respectively 30 min.

  • Vortexing and screening of the dishes: 3–4 times, 20 min.

Materials

For this study newborn Wistar or Sprague–Dawley rats of different ages (0–21 days) and both sexes were used. The animals were bred under the following conditions: food and water ad libitum, 12 h light. Vaginal plugs were not taken.

Detailed procedure

The animals were killed by decapitation. The abdomen was opened with the small scissors, without injuring the bowel. The intestine was removed by cutting the mesentery as near as possible to the gut. With a little practice, this can be done without a microscope. The pieces from several animals were stored in MEM-HEPES on ice. It is not necessary to cut those into smaller pieces, they will disintegrate after the first digestion step.

The pieces were transferred individually to a 35 mm dish of

Results

Using this procedure it is possible to obtain myenteric plexus from all parts of the newborn rat gastrointestinal tract: stomach, duodenum, jejunum, ileum, cecum, proximal and distal colon (Fig. 2Fig. 3Fig. 4Fig. 5). The pieces of isolated plexus from the different areas of the gut varied in appearance and type of meshwork, as seen in whole-mount preparations. While the meshwork of the plexus is fairly loose in the duodenum and the rest of the small intestine, it appears relatively compact in

Discussion

The manual dissection of the myenteric plexus after enzyme digestion in the newborn rat is a very laborious and time consuming task. The small amount of tissue contrasts to the amount of time which is needed. Furthermore, the small intestine as well as the stomach or distal colon are very difficult to dissect manually. The method described here allows larger amounts of myenteric plexus to be obtained from all parts of the gut.

A crucial aspect of this method is the availability of a suitable

Quick procedure

  • (A) Remove the gut.

  • (B) Separate the muscle layer from the mucosa.

  • (C) Incubate in collagenase for 1 h.

  • (D) Vortex approximately 15 s.

  • (E) Screen the dishes for separated plexus and store it in ice-cold MEM.

  • (F) Repeat C (30 min), D and E.

  • (G) Use the dissected plexus for dissociation, culture or grafting.

Essential literature references

References [3], [4], [8].

References (10)

  • Bannermann, P.G., Analysis of enteric neurons, glia and their interactions using explant cultures of myenteric plexus,...
  • Furness, J.B. and Costa, M., The Enteric Nervous System, Churchill Livingstone, Edinburgh,...
  • Jaeger, C.B., Toombs, J.P. and Borgens, R.B., Grafting in acute spinal cord injury: morphological and immunological...
  • Jessen, K.R. and Burnstock, G., The enteric nervous system in tissue culture: a new mammalian model for the study of...
  • Jessen, K.R., Saffrey, M.J. and Burnstock, G., The enteric nervous system in tissue culture. I. Cell types and their...
There are more references available in the full text version of this article.

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