ProtocolA new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract
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.
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Removal of the gut: 5–10 min.
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Stripping of the muscle layers: 10 min.
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Incubation in collagenase: 3–4 times, 60 respectively 30 min.
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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
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(A) Remove the gut.
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(B) Separate the muscle layer from the mucosa.
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(C) Incubate in collagenase for 1 h.
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(D) Vortex approximately 15 s.
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(E) Screen the dishes for separated plexus and store it in ice-cold MEM.
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(F) Repeat C (30 min), D and E.
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(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...
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