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Jens Randel Nyengaard

An unbiased stereological method for efficiently quantifying the innervation of the heart and other organs based on total length estimations

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  • Christian Mühlfeld, Justus Liebig University Giessen, Germany
  • Tamara Papadakis, Justus Liebig University Giessen, Germany
  • Gabriela Krasteva, Justus Liebig University Giessen, Germany
  • Jens Randel Nyengaard
  • Ute Hahn
  • Wolfgang Kummer, Justus Liebig University Giessen, Germany
  • Department of Mathematical Sciences
  • Stereological Research Laboratory

Quantitative information about the innervation is essential to analyze
the structure-function relationships of organs. So
far, there has been no unbiased stereological tool
for this purpose. This study presents a new unbiased and efficient
method
to quantify the total length of axons in a given
reference volume, illustrated on the left ventricle of the mouse heart.
The
method is based on the following steps: 1) estimation of the reference volume; 2) randomization of location and orientation using appropriate sampling techniques; 3) counting of nerve fiber profiles hit by a defined test area within an unbiased counting frame on paraffin sections stained
immunohistochemically for protein gene product 9.5; 4) electron microscopic estimation of the mean number of axon profiles contained in one nerve fiber profile; 5) estimation of the degree of tissue shrinkage of specimens in paraffin; and 6)
calculation of the total axon length within the reference volume,
taking tissue shrinkage into account. In a set of five
mouse hearts, the total length of axons ramifying
between cardiomyocytes ranged between ∼50 and 100 m, with a mean of
75.98
m (SD 23.73). The time required for the
microscopical analysis was ∼8 h/animal for an experienced observer.
Using antibodies
specific for different neuron subtypes and
immunoelectron microscopy, this method is also suited to estimate the
total axon
length of neurons expressing different
transmitters. This new and efficient method is particularly useful when
structural
remodeling takes place and is suspected to involve
gain or loss of axons.

Original languageEnglish
JournalJournal of Applied Physiology
Volume108
Issue5
Pages (from-to)1402-1409
Number of pages8
ISSN8750-7587
DOIs
Publication statusPublished - 2010

    Research areas

  • design-based stereology, electron microscopy, innervation, myocardium

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