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

Significance Statement

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.

 

About The Author

Francesco Latini, M.D.

Dr Latini completed his MD degree in 2000 at University of Bologna (Alma Mater). He completed his residency (cum laude) in Neurosurgery at University of Ferrara, Italy and since 2013 is specialist in Neurosurgery. He worked as junior consultant for the division of Neurosurgery at the Dpt of Neuroscience and Rehabilitation, S.Anna University Hospital of Ferrara Italy until 2014 with a specific research project on brain connectivity and patients with lesions in eloquent areas underwent awake surgery.

He did a research fellowship at International Neuroscience Institute , Hannover (Germany)  and a clinical research fellowship with Dr Ali Krisht at the Arkansas Neuroscience Institute at St Vincent’s Infirmary in Little Rock, Arkansas (USA) in 2012. He is currently working as neurosurgeon and visiting researcher at the Department of Neurosurgery, Uppsala University Hospital, Uppsala, Sweden. His main clinical and research focus are: neuroanatomy, neuroimaging, neuropsychology, brain connectivity and the treatment of intracranial tumours.

His current research projects are  “Studies of the tumor to brain interface in meningiomas – Peritumoral edema and invasiveness” and “Neurosurgical anatomy studies of the white matter in the human brain – post mortem fiber dissection and in vivo tractography of magnetic resonance imaging studies”

He is member of the European Association of Neurosurgical Societies (EANS), of the Human Brain Mapping Organization (HBMO), of the Swedish Society for Neuroscience (SSfN) and of the Federation of European Neuroscience Societies (FENS).

About The Author

Mats Hjortberg  PhD 

Senior Lecturer in Anatomy-  Dept. of Medical Cell Biology, Uppsala University

BSc in Biochemistry (1985), MSc in Anatomy (1989), PhD in Anatomy  (1992). Post Doctoral Fellow at Baylor College of Medicine 1993

Junior Lecturer of in Anatomy at Dept. Physiotherapy, Uppsala University 1994-2005

Junior Lecturer in Anatomy at Dept. Medical Cell Biology, Uppsala University 2005-2008

Senior Lecturer in Anatomy and Head of the Anatomy Unit at Dept. Medical Cell Biology 2009-

Among the current research projects there is “Neurosurgical anatomy studies of the white matter in the human brain – post mortem fiber dissection and in vivo tractography of magnetic resonance imaging studies”.

About The Author

Håkan Aldskogius

MD, Uppsala University, 1971

PhD in Anatomy, Karolinska Institutet, 1974

Postdoctoral Fogarty Fellow, Albany Medical College, Neurology, 1977-78

Assistant Professor of Anatomy, Karolinska Institutet, 1978-1984

University Lecturer in Anatomy, Karolinska Institutet, 1984-1995

Professor of Medical Structural Bology, Uppsala University, 1995-2010

Chairman, Department of Neuroscience, Uppsala University, 2006-2010

Professor Emeritus, Uppsala University, 2010-

Field of research: Spinal cord neural degeneration, regeneration and plasticity

137 original and 19 review publications in PubMed, ca 5 000 citations, h-factor 41

Recent publication: Hoeber J, Trolle C, Konig N, Du Z, Gallo A, Hermans E, Aldskogius H, Shortland P, Zhang SC, Deumens R, Kozlova EN. Human embryonic stem cell-derived progenitors assist functional sensory axon regeneration after dorsal root avulsion injury. Scientific Reports. 2015 Jun 8;5:10666. doi: 10.1038/srep10666.

Editor: Glial Interfaces in the Nervous System, IOS Press, 2002; Animal Models of Spinal Cord Repair, Humana Press, 2013

Field of education: Gross Anatomy, Functional Neuroanatomy, Regenerative Neurobiology

Book Chapter: Structure and Function of the Nervous System in Clinical Neuroscience, Liber, 2015 (Swedish)

Commitments of Trust: Evaluator European Commission, Marie Sklodowska-Curie program, 2012-2015; Member Medical Faculty Executive Committee for Medical Education, 1996-2010; Consultant Medical Faculty Executive Committee for Medical Education, 2010-2015

Professional memberships: American Association of Anatomists, Society for Neuroscience, Federations of European Neuroscience Societies, Swedish Society for Physicians.

About The Author

Mats Ryttlefors, M.D., Ph.D.

Dr Ryttlefors completed his MD degree in 2002 and PhD degree in 2009 at Uppsala University, Uppsala, Sweden. He wrote his thesis for the PhD degree on studies of subarachnoid haemorrhage in elderly patients with Prof Per Enblad as main supervisor. He attended residency in Neurosurgery at the Uppsala University Hospital and is since 2010 specialist in Neurosurgery. He did a clinical research fellowship with Dr Ali Krisht at the Arkansas Neuroscience Institute at St Vincent’s Infirmary in Little Rock, Arkansas in 2012. He is currently working as staff neurosurgeon and researcher at the Department of Neurosurgery, Uppsala University Hospital, Uppsala, Sweden. His main clinical and research focus is the treatment of intracranial tumours. He is currently supervisor of two research projects: “Studies of the tumor to brain interface in meningiomas – Peritumoral edema and invasiveness” and “Neurosurgical anatomy studies of the white matter in the human brain – post mortem fiber dissection and in vivo tractography of magnetic resonance imaging studies”. 

Figure Legend 1. Superficial White matter terminations (SWM).

Examples of the superficial white matter terminations in three different regions (the three colored squares) of the brain convexity identified during dissection of a right hemisphere. The white matter terminations demonstrated with high magnification at the microscope, have a parallel direction and end perpendicular to the pial/cortical surface.

AF: Arcuatus Fasciculus (section); CR: corona radiata; SSS: Sagittal stratum of Sachs; Ins: Insula; M1: primary motor area (precentral gyrus). A: Anterior, P: posterior.

use of cerebral perfusion immersion-fixation process for subsequent white matter dissection. Global Medical Discovery

 

 

 

 

 

 

 

 

 

 

 

 

Figure Legend 2. Major fibre bundles.

  1. A) The frontal aslant tract (FASt) has been removed with an “en bloc” dissection from the frontal lobe of this left hemisphere. This bundle connects the Supplementary motor area (SMA) and the region close to it (Pre-SMA) with the frontal operculum (PoP) and the pars triangularis (PT), considered part of the Broca’s area, crucial for speech production.
  2. B) The cingulum (Ci) has been dissected and removed “en bloc” from the medial surface of this right hemisphere, showing the three main parts of the corpus callosum (genu, Body and splenium) and the Callosal Radiation (Ca radiation). The three major components of the Ci are marked in the frontal region (Fro), in the retrosplenial and temporal parahippocampal region (PHG). The cingulum is involved in several high order functions, such as attention, emotion, memory, visual and spatial skills among others.

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure Legend 3 Deep venous anastomosis.

Example of deep medullary venous system of the brain (called “candelabra” type), which can be identified under high magnification and dissected untils its submillimetric division within the deep white matter layers and periventricular areas.

The use of cerebral perfusion & immersion-fixation process for subsequent white matter dissection.Global Medical Discovery

Journal Reference

J Neurosci Methods. 2015;253:161-9.

Latini F1, Hjortberg M2, Aldskogius H3, Ryttlefors M4.

Show Affiliations
  1. Department of Neuroscience, Neurosurgery, Uppsala University, Akademiska sjukhuset, 75185 Uppsala, Sweden. Electronic address: [email protected]
  2. Department of Medical Cell Biology, Education, Uppsala University, Box 571, 75123 Uppsala, Sweden.
  3. Department of Neuroscience, Regenerative Neurobiology, Uppsala University, Box 593, 75124 Uppsala, Sweden.
  4. Department of Neuroscience, Neurosurgery, Uppsala University, Akademiska sjukhuset, 75185 Uppsala, Sweden.

Abstract

BACKGROUND:

The Klingler’s method for white matter dissection revolutionized the study of deep cerebral anatomy. Although this technique madewhite matter dissection more feasible and widely used, it still presents some intrinsic limitations.

NEW METHOD:

We evaluated the quality of different methods for specimen preparation based on an intra-carotidal formalin perfusion fixationprocess. Ten post-mortem human hemispheres were prepared with this method and dissected in a stepwise manner.

RESULTS:

The homogeneous and rapid fixation of the brain allowed documentation of several fine additional anatomical details. Intra-cortical whitematter terminations were described during the first stage of dissection on each specimen. No limitations were encountered during dissection of the major associative bundles. On the contrary, the quality of the fixation of the specimens made it possible to isolate them en bloc. One of the most complex and deep bundles (accumbo-frontal fasciculus) was dissected without technical limitations. Deep vascular structures were very well preserved and dissected within the white matter until their sub-millimetric terminations.

COMPARISON WITH EXISTING METHOD:

Short time for preparation, a more homogeneous fixation, no technical limitation for a detailed description of superficial and deep white matter anatomy, the possibility to dissect with a single technique the fibre organization and the white mattervascular architecture are the advantages reported with the perfusion fixation.

CONCLUSION:

These results provide encouraging data about the possibility to use a perfusion fixation process, which may help in improving the quality of white matter dissection for research, didactic purposes and surgical training.

Copyright © 2015 Elsevier B.V. All rights reserved.

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