Nervenzupfpräparation (nerve fiber teasing) in der Diagnostik peripherer Neuropathien beim Tier

NERVE FIBER TEASING AS A DIAGNOSTIC AID IN DETECTION OF PERIPHERAL NEUROPATHIES – METHODOLOGY AND INTERPRETATION Nerve biopsies are widely used for the investigation of peripheral neuropathies in man and animals in addition to clinical and electrodiagnostic evaluation . Besides light- and electron microscopy, single nerve fiber teasing provides a convincing method in the approach of a correct diagnosis. It is the only practable method for inspection of myelinated fibers along their path. Moreover, teasing preparates are most indicative for the regenerative capabilities of myelinated fibers. Besides knowledge of pathomorphological alterations, the investigator needs some experience in identifying artefacts and autolytic changes since they can look very alike and, thus, may lead to misdiagnosis. Therefore, it was the aim of this study to provoke pure artificial and autolytic changes in peripheral nerves. For evaluating divergent fixation-protocols, 171 samples were taken to prepare about 51300 single teased fibers. Like this, phenomena of delayed fixation as well as of hyperfixation could be described. To avoid artefacts, we recommend a fixation in 2,5% glutaraldehyde for 1 to 2 hours. Minor autolytic changes could already be observed 1 to 2 hours post mortem. Interpretation became nearly impossible due to desintegration of the myelin sheath after 3 to 4 days . Those results, together with the samples of 57 selected cases (17000 single teased fibers) enabled to establish a list of phenomena seen with pathology, artefacts or autolysis. This categorization substantially increases objective diagnosis when evaluating teased fibers. Additionally, clinical signs were correlated with their neuropathological findings. In order to improve the separation of nerve fibers, the value of collagenolytic agents was investigated. About 15700 single fibers from 72 samples were slided. Teasing of samples that had already been fixed could be improved by incubation in collagenase for 1 hour. These findings proved very helpful for investigating referred biopsies which often arrive in a hyperfixed state. Collagenolytic glycin buffering is not advisable in this case, although it did facilitate the removal of the connective tissue of unfixed nerves, when samples were bathed in it. Moreover the often sparse amount of biopsied material can be the limiting factor of diagnosis. Therefore, post-teasing-light and electron microscopy is a useful approach. Even interstitial components can be visualized, and treatment allows for a correlation of suspicious regions within teased fibers with cross-section investigations. To widen the diagnostic range different methods to stain single components of teased fibers were evaluated. The thus established labelling of myelin sheathing or of the axon, respectively, by different staining protocols for the fluorochrome 5,5`-diphenyl-9-ethyl-oxacarbocyanine is helpful. This procedure for axonal investigation proved superior to whole-mount-immunohistochemical approaches as only established the paraformaldehyde fixation allowed for a selective staining of neurofilaments along the entire axon.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Ludwig-Maximilians-Universität

Year

2002

Author dc:creator

• Wieczorek, Lara Alexa

Repository record source_url

https://edoc.ub.uni-muenchen.de/710/

OAI identifier oai:identifier

oai:edoc.ub.uni-muenchen.de:710