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How Klingler’s dissection permits exploration of brain structural connectivity? An electron microscopy study of human white matter

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Abstract

The objective of this study is to explore histological and ultrastructural changes induced by Klingler’s method. Five human brains were prepared. First, the effects of freezing–defrosting on white matter were explored with optical microscopy on corpus callosum samples of two brains; one prepared in accordance with the description of Klingler (1956) and the other without freezing–defrosting. Then, the combined effect of formalin fixation and freezing–defrosting was explored with transmission electron microscopy (EM) on samples of cingulum from one brain: samples from one hemisphere were fixed in paraformaldehyde–glutaraldehyde (para/gluta), other samples from the other hemisphere were fixed in formalin; once fixed, half of the samples were frozen–defrosted. Finally, the effect of dissection was explored from three formalin-fixed brains: one hemisphere of each brain was frozen-defrosted; samples of the corpus callosum were dissected before preparation for scanning EM. Optical microscopy showed enlarged extracellular space on frozen samples. Transmission EM showed no significant alteration of white matter ultrastructure after formalin or para/gluta fixation. Freezing–defrosting created extra-axonal lacunas, larger on formalin-fixed than on para/gluta-fixed samples. In all cases, myelin sheaths were preserved, allowing maintenance of axonal integrity. Scanning EM showed the destruction of most of the extra-axonal structures after freezing–defrosting and the preservation of most of the axons after dissection. Our results are the first to highlight the effects of Klingler’s preparation and dissection on white matter ultrastructure. Preservation of myelinated axons is a strong argument to support the reliability of Klingler’s dissection to explore the structure of human white matter.

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Acknowledgments

Our data were obtained with the assistance of the RIO EM facility of François-Rabelais University. We would like to express our gratitude to the donors involved in the body donation program of the Association des dons du corps du Centre Ouest, Tours, who made this study possible by generously giving their bodies to Science. We thank Christine Hayot and Julien Gaillard for technical assistance with EM sections, and Daniel Bourry for the macrophotography of white matter samples. We are grateful to Philippe Roingeard for his careful reading of the manuscript and helpful comments.

Conflict of interest

The authors have no conflicts of interest to declare.

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Authors and Affiliations

  1. INSERM, Imagerie et cerveau UMR U930, Université François-Rabelais de Tours, Tours, France

    Ilyess Zemmoura, Christophe Destrieux & Stéphane Velut

  2. Service de Neurochirurgie, CHRU de Tours, CHRU Bretonneau, 2, Boulevard Tonnellé, 37044, Tours Cedex 9, France

    Ilyess Zemmoura, Christophe Destrieux & Stéphane Velut

  3. INSERM U966, Université François-Rabelais and CHRU de Tours, Tours, France

    Emmanuelle Blanchard & Pierre-Ivan Raynal

  4. Plateforme des microscopies, PPF ASB, Université François-Rabelais and CHRU de Tours, Tours, France

    Emmanuelle Blanchard & Pierre-Ivan Raynal

  5. Service d’Anatomie et Cytologie Pathologique, CHRU de Tours, Tours, France

    Cécilia Rousselot-Denis

Authors
  1. Ilyess Zemmoura
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  2. Emmanuelle Blanchard
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  3. Pierre-Ivan Raynal
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  4. Cécilia Rousselot-Denis
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  5. Christophe Destrieux
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  6. Stéphane Velut
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Correspondence to Ilyess Zemmoura.

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Zemmoura, I., Blanchard, E., Raynal, PI. et al. How Klingler’s dissection permits exploration of brain structural connectivity? An electron microscopy study of human white matter. Brain Struct Funct 221, 2477–2486 (2016). https://doi.org/10.1007/s00429-015-1050-7

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  • DOI: https://doi.org/10.1007/s00429-015-1050-7

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