title: How Klingler's dissection permits exploration of brain structural connectivity? An electron microscopy study of human white matter.
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.
authors.affiliation: INSERM, Imagerie et cerveau UMR U930, Université François-Rabelais de Tours, Tours, France. ilyess.zemmoura@univ-tours.fr.|Service de Neurochirurgie, CHRU de Tours, CHRU Bretonneau, 2, Boulevard Tonnellé, 37044, Tours Cedex 9, France. ilyess.zemmoura@univ-tours.fr.
authors.forename: Ilyess
authors.initials: I
authors.lastname: Zemmoura
authors.affiliation: INSERM U966, Université François-Rabelais and CHRU de Tours, Tours, France.|Plateforme des microscopies, PPF ASB, Université François-Rabelais and CHRU de Tours, Tours, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Blanchard
authors.affiliation: INSERM U966, Université François-Rabelais and CHRU de Tours, Tours, France.|Plateforme des microscopies, PPF ASB, Université François-Rabelais and CHRU de Tours, Tours, France.
authors.forename: Pierre-Ivan
authors.initials: PI
authors.lastname: Raynal
authors.affiliation: Service d'Anatomie et Cytologie Pathologique, CHRU de Tours, Tours, France.
authors.forename: Cécilia
authors.initials: C
authors.lastname: Rousselot-Denis
authors.affiliation: INSERM, Imagerie et cerveau UMR U930, Université François-Rabelais de Tours, Tours, France.|Service de Neurochirurgie, CHRU de Tours, CHRU Bretonneau, 2, Boulevard Tonnellé, 37044, Tours Cedex 9, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Destrieux
authors.affiliation: INSERM, Imagerie et cerveau UMR U930, Université François-Rabelais de Tours, Tours, France.|Service de Neurochirurgie, CHRU de Tours, CHRU Bretonneau, 2, Boulevard Tonnellé, 37044, Tours Cedex 9, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Velut
mesh_terms: D001921:Brain / Q000648:ultrastructure*; D003337:Corpus Callosum / Q000187:drug effects / Q000648:ultrastructure; D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006652:Histological Techniques / Q000379:methods*; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D066127:White Matter / Q000648:ultrastructure*
chemical_list: D005404:Fixatives; D005557:Formaldehyde
keywords: Axons; Diffusion tractography; Dissection; Electron microscopy; Histology; Myelin sheath; Ultrastructure; White matter