The use of a cerebral perfusion and immersion-fixation process for subsequent white matter dissection

White matter is a fundamental component of the brain architecture which allows the communication and the reciprocal modulation between close or distant cortical territories through a balanced network of axons, fibres and fascicles.

An extensive anatomical knowledge of the major white matter (WM) bundles is mandatory in order to tailor the neurosurgical approach and to optimize the resection of various deep subcortical lesions. Modern techniques such as Magnetic Resonance diffusion tensor tractografy, provided a unique insight into the brain white matter networks, revealing the organization of deep and superficial pathways.

 Because of the intrinsic limitations associated with the diffusion tensor imaging, white matter dissection of previously formalin fixed brains still remains one of the most trustable techniques in order to validate the white matter connectivity and at the same time to acquire a comprehensive three-dimensional orientation during neurosurgical-neuroanatomical training.

Anatomists of every century have described many different techniques for specimen preparation and dissection in order to understand the complex WM architectural organization. However it was Josef Klingler, who in the 1935 revolutionized this field of research. He developed a new method of brain fixation, which consisted of freezing already formalin-fixed brains before dissection. Despite the tremendous impact of this technique for both neurosurgical training and research, according to the literature it still presents some intrinsic limitations. Because based on the immersion into 5% formalin, a long time for specimens preparation is needed. Moreover a very variable level of fixation is often reported and this can affect the quality of the dissection. From the anatomical point of view, difficult demonstration of superficial white matter architecture or thin white matter bundles within the basal ganglia, difficult identification of deep vascular structures during the same dissection are some of the limitations experienced during the white matter dissection with Klingler’s technique.

We evaluated the quality of a different method for specimen preparation based on an intra-carotidal formalin perfusion fixation process before the freezing stage and the dissection. This technique showed several practical advantages: the time needed for a “physiological” fixation is minimized. The brain can be removed intact 48 hours after the perfusion and is already fixed, thus decreasing the risk of accidental gyral deformation during its removal. The quality of the fixed brains can be maintained for a very long time without the need for additional procedures. The homogeneous and rapid fixation of the brain allowed us to document several fine additional anatomical details.

A more systematic organization of the white matter terminations underneath the sulcal or gyral surface can be demonstrated with this technique. This aspect is quite important and a more comprehensive knowledge of the superficial white matter should be mandatory in order to tailor cortical and subcortical resection with the least invasive possible surgical trajectories.

All the major associative bundles were isolated without technical problems and it was even possible to isolate them en bloc. This aspect is a direct signal of the quality of the specimens and at the same time this can open new possibilities for the research on symmetry, lateralization, and biophysical features of each white matter bundle.

Some of the most complex and thin bundles were dissected without any technical limitation on these specimens. This aspect can lead to a better understanding of the white matter organization of deep regions such as the basal ganglia region with the possibility to identify new surgical targets i.e. for deep brain stimulation based on their connectivity.

A very fundamental advantage of our specimens is the use of the physiological intracranial vascular network for the fixation process, which preserves vascular landmarks at each stage of white matter dissection until their sub-millimetric terminations. These fine and constant details allow correlation between vessels and white matter structures with the obvious advantage in three-dimensional orientation that can be employed during any standard neurosurgical procedure.

 These results provided encouraging data about the great potential of this technique to improve the quality of white matter dissection and the deep anatomical orientation within the brain. A shorter time for preparation, a more homogeneous fixation and no technical limitation for a fine description of superficial and deep white matter anatomy are some of the advantages of our technique. We suggests the more widely use of perfusion-fixation process, which may help in improving the quality of white matter dissection for research, didactic purposes and neurosurgical training.