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

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.