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Updated: Aug 24, 2026
Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology
Published on: July 3, 2018
Methylene blue staining for nerve-sparing operative procedures: an animal model
Christoph Seif1, Francisco J Martínez Portillo, Daniar K Osmonov
1Department of Urology, University Hospital Kiel, Kiel, Germany.
Objectives:
To evaluate methylene blue fiber staining as a method of nerve fiber identification in an animal model, because the maintenance of organ function after surgery depends on exact intraoperative identification of the relevant nerve fibers.
Methods:
Brindley electrodes were implanted bilaterally at S3 for sacral anterior root stimulation in six minipigs. For reference, stimulation-induced detrusor contractions were recorded urodynamically. After exposure of the ureterovesical junction on both sides, a 2:8 methylene blue solution was applied to the right side; the left side remained untreated. Bilateral dissection of the ureter from the surrounding tissue for a distance of 4 cm proximal to the ureterovesical junction was performed. The methylene blue-stained nerve fibers on the right side were spared; no particular attention was paid to the nerves on the left. Again, sacral anterior root stimulation-induced detrusor contractions were monitored urodynamically on both sides. Then, the identified nerve fibers on the right were cut intentionally, and the detrusor pressure was recorded again under stimulation. Finally, the dissected nerve structures were evaluated histologically.
Results:
The reference bladder pressures after unilateral stimulation on the left side before ureter dissection showed a mean detrusor pressure (Pdet) of 19 cm H2O. On the right side, the Pdet was 18 cm H2O. After preparation on both sides, a mean Pdet of 3 cm H2O was recorded after left side stimulation, and a Pdet of 17 cm H2O after right side stimulation. When the stained nerve fibers on the right side were cut, no bladder contractions could be induced. The histomorphology of the stained and dissected structures revealed multiple autonomous nerve fibers and small vessels in connective tissue.
Conclusions:
The identification of minute nerve bundles is a tedious and difficult task. The results from our animal model demonstrated that supravital staining of autonomous nerve fibers with methylene blue is a simple and reliable method of identification.
