Histological outcome evaluation of selected brain preparation protocols for white fiber dissection.

BACKGROUND: White fiber dissection is essential for studying brain connections.　However, preparation protocols have not been validated. METHODS: We microstructurally analyzed Klingler's brain preparation method and freezing process and assessed changes under two protocols:freeze-only and freeze-thaw.　The microstructure changes of these protocols were evaluated by measuring the ratio of the total gap area to the white matter area and determining the mean eccentricity value to assess the degree of anisotropy. RESULTS: Sixty hemispheres were allocated to ten different freezing protocols.　In the freeze-only protocols, the total gap area ratio was significantly higher compared to that of specimens fixed with only formaldehyde, particularly after continuous freezing for 3-4 weeks;however, the difference in eccentricity was not significant.　In the freeze-thaw protocols, both the area ratio and eccentricity were significantly higher compared to the freeze-only.　The optimum degree of fiber separation in the freeze-thaw protocols reached its peak with four cycles of 1-week freezing periods interrupted by six hours of thawing. CONCLUSION: The Klingler method assists in the separation of the white fibers through the gaps formed by ice crystals, but an appropriate degree of anisotropy is reached when the freezing protocol is interrupted by at least four thawing cycles.

Overlapping stimulation of subthalamic nucleus and dentato-rubro-thalamic tract in Parkinson's disease after deep brain stimulation.

BACKGROUND: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) reduces tremor, rigidity, and akinesia. According to the literature, the dentato-rubro-thalamic tract (DRTt) is verified target for DBS in essential tremor; however, its role in the treatment of Parkinson's disease is only vaguely described. The aim of our study was to identify the relationship between symptom alleviation in PD patients and the distance of the DBS electrode electric field (EF) to the DRTt. METHODS: A single-center retrospective analysis of patients (N = 30) with idiopathic Parkinson's disease (PD) who underwent DBS between November 2018 and January 2020 was performed. DRTt and STN were visualized using diffusion-weighted imaging (DWI) and tractography protocol of magnetic resonance (MR). The EF was calculated and compared with STN and course of DRTt. Evaluation of patients before and after surgery was performed with use of UPDRS-III scale. The association between distance from EF to DRTt and clinical outcomes was examined. To confirm the anatomical variation between DRTt and STN observed in tractography, white matter dissection was performed with the Klingler technique on ten human brains. RESULTS: Patients with EF overlapping STN and DRTt benefited from significant motor symptoms improvement. Anatomical findings confirmed the presence of population differences in variability of the DRTt course and were consistent with the DRTt visualized by MR. CONCLUSIONS: DRTt proximity to STN, the main target in PD DBS surgery, confirmed by DWI with tractography protocol of MR combined with proper predefined stimulation parameters may improve efficacy of DBS-STN.

Educational stereoscopic representation of a step-by-step brain white fiber dissection according to Klingler's method.

BACKGROUND: Understanding and teaching the three-dimensional architecture of the brain remains difficult because of the intricate arrangement of grey nuclei within white matter tracts. Although cortical area functions have been well studied, educational and three-dimensional descriptions of the organization of deep nuclei and white matter tracts are still missing. OBJECTIVE: We propose herein a detailed step-by-step dissection of the lateral aspect of a left hemisphere using the Klingler method and provide high-quality stereoscopic views with the aim to help teach medical students or surgeons the three-dimensional anatomy of the brain. METHODS: Three left hemispheres were extracted and prepared. Then, according to the Klingler method, dissections were carried out from the lateral aspect. Photographs were taken at each step and were modified to provide stereoscopic three-dimensional views. RESULTS: Gray and white structures were described: cortex, claustrum, putamen, pallidum, caudate nucleus, amygdala; U-fibers, external and internal capsules, superior longitudinal fasciculus, frontal aslant fasciculus, uncinate fasciculus, inferior fronto-occipital fasciculus, inferior longitudinal fasciculus, corticospinal fasciculus, corona radiata, anterior commissure, and optic radiations. CONCLUSION: This educational stereoscopic presentation of an expert dissection of brain white fibers and basal ganglia would be of value for theoretical or hands-on teaching of brain anatomy; labeling and stereoscopy could, moreover, improve the teaching, understanding, and memorizing of brain anatomy. In addition, this could be also used for the creation of a mental map by neurosurgeons for the preoperative planning of brain tumor surgery.

Fixation and staining methods for macroscopical investigation of the brain.

The proper preservation of human brain tissue is an indispensable requirement for post-mortem investigations. Neuroanatomical teaching, neuropathological examination, neurosurgical training, basic and clinical neuroscientific research are some of the possible downstream applications of brain specimens and, although much apart from one another, proper tissue fixation and preservation is a common denominator to all of them. In this review, the most relevant procedures to fixate brain tissue are described. In situ and immersion fixation approaches have been so far the most widespread ways to deliver the fixatives inside the skull. Although most of them rely on the use of formalin, alternative fixative solutions containing lower amounts of this compound mixed with other preservative agents, have been attempted. The combination of fixation and freezing paved the way for fiber dissection, particularly relevant for the neurosurgical practice and clinical neuroscience. Moreover, special techniques have been developed in neuropathology to tackle extraordinary problems, such as the examination of highly infective specimens, as in the case of the Creutzfeldt-Jakob encephalopathy, or fetal brains. Fixation is a fundamental prerequisite for further staining of brain specimens. Although several staining techniques have been developed for the microscopical investigation of the central nervous system, numerous approaches are also available for staining macroscopic brain specimens. They are mostly relevant for neuroanatomical and neuropathological teaching and can be divided in white and gray matter staining techniques. Altogether, brain fixation and staining techniques are rooted in the origins of neuroscience and continue to arouse interest in both preclinical and clinical neuroscientists also nowadays.

Frontal aslant tract: Anatomy and tractography description in the Mexican population.

Background: The aim of the study was to describe the origin, course, and termination of frontal aslant tract (FAT) in the Mexican population of neurosurgical referral centers. Methods: From January 2018 to May 2019, we analyzed 50 magnetic resonance imaging (MRI) studies in diffusion tensor imaging sequences of patients of the National Institute of Neurology and Neurosurgery "Manuel Velasco Suárez." Five brains were fixed by the Klingler method and dissected in the neurosurgery laboratory of the Hospital Civil de Guadalajara to identify the origin, trajectory, and ending of the FAT. Results: FAT was identified in 100% of the MRI and brain dissections. The origin of the FAT was observed in 63% from the supplementary premotor area, 24% from the supplementary motor area, and 13% in both areas. Its ending was observed in the pars opercularis in 81%, pars triangularis in 9%, and in both pars opercularis and ventral premotor area in 10% in the magnetic resonance images, with a left side predominance. In the hemispheres dissections, the origin of FAT was identified in 60% from the supplementary premotor area, 20% from the supplementary motor area, and 20% in both areas. Its ending was observed in the pars opercularis in 80% and the pars triangularis in 20%. It was not identified as an individual fascicle connected with the contralateral FAT. Conclusion: In the Mexican population, FAT has a left predominance; it is originated more frequently in the supplementary premotor area, passes dorsal to the superior longitudinal fascicle II and the superior periinsular sulcus, and ends more commonly in the pars opercularis.

A stepwise laboratory manual for the dissection and illustration of limbic and paralimbic structures: lessons learned from the Klingler's technique.

BACKGROUND: Three-dimensional relationships within the limbic and paralimbic areas are often hard to grasp. Relevant anatomical structures exhibit a complicated architecture and connectivity and therefore surgical approaches targeting lesions or functional resections in this area pose a distinct challenge. PURPOSE: To provide an educational, comprehensive, systematic and stepwise manual for the dissection and illustration of major limbic structures since there is a gap in the pertinent literature. Further, we aim to offer a thorough yet simplified roadmap for laboratory and intraoperative dissections. METHODS: Twenty (20) normal adult, formalin-fixed cerebral hemispheres were studied through the fiber dissection technique and under the microscope. Stepwise and in tandem medial to lateral and lateral to medial dissections were performed in all specimens aiming to reveal the morphology and spatial relationships of major limbic and paralimbic areas. RESULTS: Fourteen (14) consecutive, discrete and easily reproducible laboratory anatomical steps are systematically described to reveal the intricate anatomy of the limbic and paralimbic structures and their main connections. CONCLUSION: This study offers for the first time in the pertinent literature a focused, step-by-step laboratory manual for the dissection and illustration of the limbic and paralimbic structures. The overreaching goal is to supplement the novice and experienced anatomist and neurosurgeon with a thorough and systematic reference to facilitate laboratory or intraoperative dissections.

Corpus Callosum and Its Connections: A Fiber Dissection Study.

OBJECTIVE: We performed a fine white fiber dissection to demonstrate the extensive connections of the corpus callosum. MATERIAL AND METHODS: Three formalin-fixed frozen cadaveric human brain specimens were dissected using Klingler's technique. On one half of each hemisphere, the superior connections of the corpus callosum were dissected and in the other half the inferior connections of the corpus callosum were dissected. RESULTS: The mean length of the corpus callosum from the rostrum to the splenium was 7.8 cm. The fibers of the corpus callosum were classified as superior/dorsal radiations, inferior/ventral radiations, anterior radiations, and posterior radiations. The entire transverse length of the dorsal callosal radiation from one hemisphere to the other was 8.1 cm. For anterior interhemispheric approaches, an incision in the anterior part of the body not extending beyond 3.5 cm from the genu was found to be safe with regards to crossing motor fibers. CONCLUSIONS: The corpus callosum was found to have intricate connections with all the lobes of the cerebral hemispheres, including the insular region and the limbic and paralimbic areas. Based on the course and traverse of the callosal fibers, a transverse incision in the corpus callosum should be preferred when performing a callosotomy to access intraventricular lesions as this splits the callosal fibers instead of transecting them. The analysis of the course of the callosal radiations enhances understanding of the growth pattern of primary corpus callosal gliomas and helps to design a safe surgical strategy.

White matter dissection with the Klingler technique: a literature review.

The aim of this literature review is to present a summary of the published literature relating the details of the different modifications of specimen preparation for white matter dissection with the Klingler technique. For this review, 3 independent investigators performed an electronic literature search that was carried out in the Pubmed, Scopus and Web of Science databses up to December 2019. Furthermore, we performed citation tracking for the articles missed in the initial search. Studies were eligible for inclusion when they reported details of at least the first 2 main steps of Klingler's technique: fixation and freezing. A total of 37 full-text articles were included in the analysis. We included original anatomical studies in which human white matter dissection was performed for study purposes. The main three steps of preparation are the same in each laboratory, but the details of each vary between studies. Ten percent formalin is the most commonly used (34 studies) solution for fixation. The freezing time varied between 8 h and a month, and the temperature varied from - 5 to - 80 °C. After thawing and during dissections, the specimens were most often kept in formalin solution (13), and the concentration varied from 4 to 10%. Klingler's preparation technique involves three main steps: fixation, freezing and thawing. Even though the details of the technique are different in most of the studies, all provide subjectively good quality specimens for anatomical dissections and studies.

The ansa peduncularis in the human brain: A tractography and fiber dissection study.

INTRODUCTION: The ansa peduncularis is a composite of white matter fiber bundles closely packed together that sweeps around the cerebral peduncle. The exact components of the ansa peduncularis and their anatomical trajectories are still not established firmly in the literature. OBJECTIVE: The aim of this study was to examine the topographical anatomy of the ansa peduncularis and its subcomponents using the fiber dissection and tractography techniques. METHODS: Ten formalin-fixed brains were prepared according to Klingler's method and dissected by the fiber dissection technique from the lateral, medial and inferior surfaces. The ansa peduncularis was also traced using high definition fiber tracking (HDFT) from the MRI data of twenty healthy adults and a 1021-subject template from the Human Connectome Project. RESULTS: The ventral amygdalofugal pathway system includes white matter fiber bundles with a topographically close relation as they sweep around the cerebral peduncle and contribute to form the ansa peduncularis: amygdaloseptal fibers connect the amygdala and anterior temporal cortex to the septal region and amygdalohypothalamic fibers project from the amygdala to the hypothalamus. Additionally, from the amygdala and anterior temporal cortex, amygdalothalamic fibers project to the medial thalamic region. The ansa lenticularis, which connects the globus pallidus to the thalamus, was not shown in our study. CONCLUSION: The study demonstrated the trajectory of the ansa peduncularis and its subcomponents, based on fiber dissection and tractography, improving our understanding of human brain anatomical connectivity.

Dissection of white matter association fasciculi in bovine (Bos taurus), pig (Sus scrofa domesticus) and rabbit (Oryctolagus cuniculus) brains.

The cerebral fasciculi (association, commissural and projection) pass through the cerebral white matter in organized groups connecting regions, hemispheres, gyri, areas and brain lobes to each other. The study can be done in vivo through diffusion tensor imaging (DTI) but presenting some technical problems. The post-mortem study by dissection allows to have a clearer view of its location, path and connections. In this work, we dissect, identify and compare the fasciculi of association of the white matter of the dorsolateral face of bovine hemispheres (Bos taurus), pig hemispheres (Sus scrofa domesticus) and rabbit hemispheres (Oryctolagus cuniculus), applying the Klingler´s technique. In 30 cerebral hemispheres (10 of each species, five right and five left), we applied the Klingler technique to identify and isolate the occipitofrontal fasciculus, uncinate fasciculus, inferior longitudinal fasciculus and superior longitudinal fasciculus; we established its location by fixing landmarks, and determined the difference in its length and width between the right and left hemispheres as well as between species using the statistical tests of t-student and one-way ANOVA. We identify the gyri, sulci and fasciculi of the dorsolateral surface of the cerebral hemispheres of the three species. We found statistically significant differences in several fasciculi between species principally in the occipitofrontal fasciculus. The preparation of the brains through the modified Klingler technique allowed a successful identification of the fascicules of association of the dorsolateral face of the cerebral hemispheres and the empowerment of these animal models for future research work in this field.

Neural and vascular architecture of the septum pellucidum: an anatomical study and considerations for safe endoscopic septum pellucidotomy.

OBJECTIVE: The septum pellucidum is a bilateral thin membranous structure representing the border between the frontal horns of the lateral ventricles. Its most examined components are the septal veins due to their surgical importance during endoscopic septum pellucidotomy (ESP), which is a well-accepted method for surgical treatment of unilateral hydrocephalus. It is widely accepted that the septum pellucidum contains nerve fibers as well, but interestingly, no anatomical study has been addressed to its neural components before. The aim of the present study was to identify these elements as well as their relations to the septal veins and to define major landmarks within the ventricular system for neurosurgical use. METHODS: Nine formalin-fixed human cadaveric brains (18 septa pellucida) were involved in this study. A central block containing both septa pellucida was removed and frozen at -30°C for 2 weeks in 7 cases. The fibers of the septum pellucidum and the adjacent areas including the venous elements were dissected under magnification by using homemade wooden spatulas and microsurgical instruments. In 2 cases a histological technique was used to validate the findings of the dissections. The blocks were sliced, embedded in paraffin, cut in 7-µm-thick slices, and then stained as follows: 1) with H & E, 2) with Luxol fast blue combined with cresyl violet, and 3) with Luxol fast blue combined with Sirius red. RESULTS: The septum pellucidum and the subjacent septum verum form the medial wall of the frontal horn of the lateral ventricle. Both structures contain nerve fibers that were organized in 3 groups: 1) the precommissural fibers of the fornix; 2) the inferior fascicle; and 3) the superior fascicle of the septum pellucidum. The area directly rostral to the postcommissural column of the fornix consisted of macroscopically identifiable gray matter corresponding to the septal nuclei. The histological examinations validated the findings of the authors' fiber dissections. CONCLUSIONS: The nerve elements of the septum pellucidum as well as the subjacent septum verum were identified with fiber dissection and verified with histology for the first time. The septal nuclei located just anterior to the fornix and the precommissural fibers of the fornix should be preserved during ESP. Considering the venous anatomy as well as the neural architecture of the septum pellucidum, the fenestration should ideally be placed above the superior edge of the fornix and preferably dorsal to the interventricular foramen.

Uncovering the inferior fronto-occipital fascicle and its topological organization in non-human primates: the missing connection for language evolution.

Whether brain networks underlying the multimodal processing of language in humans are present in non-human primates is an unresolved question in primate evolution. Conceptual awareness in humans, which is the backbone of verbal and non-verbal semantic elaboration, involves intracerebral connectivity via the inferior fronto-occipital fascicle (IFOF). While non-human primates can communicate through visual information channels, there has been no formal demonstration that they possess a functional homologue of the human IFOF. Therefore, we undertook a post-mortem diffusion MRI tractography study in conjunction with Klingler micro-dissection to search for IFOF fiber tracts in brain of Old-World (vervet) monkeys. We found clear and concordant evidence from both techniques for the existence of bilateral fiber tracts connecting the frontal and occipital lobes. These tracts closely resembled the human IFOF with respect to trajectory, topological organization, and cortical terminal fields. Moreover, these fibers are clearly distinct from other bundles previously described in this region of monkey brain, i.e., the inferior longitudinal and uncinate fascicles, and the external and extreme capsules. This demonstration of an IFOF in brain of a species that diverged from the human lineage some 22 millions years ago enhances our comprehension about the evolution of language and social behavior.

Neural Circuitry: Architecture and Function-A Fiber Dissection Study.

OBJECTIVE: White fiber dissection using a gyrus-based approach was performed to study the various associations, commissural, and projection fiber bundles of the brain. METHODS: Ten previously frozen and formalin-fixed cadaveric human brains were included. The fiber dissection techniques described by Klingler were used. The primary dissection tools were thin handmade wooden spatulas and curved metallic spatulas with tips of various sizes. The fibers were studied by the naked eye and with the use of magnification. The various fiber bundles were studied using a gyrus-based approach. The dissection was performed through each named gyrus, and the fiber tracts encountered during dissection were identified, and their relationship to other adjacent fiber bundles was studied. RESULTS: From our dissections, the white fibers of the brain were divided architecturally into 5 groups-4 horizontal groups and 1 vertical group. The 4 horizontal groups were the superficial, middle, deep, and central groups. The association fibers constituted the superficial, middle, and deep groups. The commissural fibers formed the central group, and the projection fibers formed the vertical group. The course of the fiber bundles and their functional co-relationship were determined. CONCLUSIONS: When planning the surgical trajectory, knowledge of the location of white matter tracts is essential to help minimize the occurrence of postoperative deficits. Fiber dissection using the Klingler technique is useful in gaining an understanding of the complex 3-dimensional nature of these white matter tracts and can provide a valuable resource in neurosurgical training.

Klingler's method of brain dissection: review of the technique including its usefulness in practical neuroanatomy teaching, neurosurgery and neuroimaging.

Klingler's technique was discovered in the 1930s. It is a modified method of brain fixation and dissection, based on freezing and thawing of the brain tissue, subsequent peeling away of white matter fibres and the gradual exposure of white matter tracts. The added value of this technique is that it is carried out in a stratigraphic manner. This fact makes it an invaluable tool for an in-depth understanding of the complex anatomical organisation of the cerebral hemispheres. The purpose of this paper is to provide a review of Klingler's method while taking into account the original description of the technique and its value for medical training. The historical background, the concise outline of white matter organisation, as well as our own experience in using this procedure for research and teaching activities were also included. The fibre dissection technique may still be considered an excellent complementary research tool for neuroanatomical studies. Numerous detailed observations about the white matter topography and spatial organisation have been recently made by applying this method. Using this technique may also improve understanding of the three-dimensional intrinsic structure of the brain, which is particularly important both in under- and postgraduate training in the field of neuroanatomy.

A White Matter Fiber Microdissection Study of the Anterior Perforated Substance and the Basal Forebrain: A Gateway to the Basal Ganglia?

BACKGROUND: Studies detailing the anatomy of the basal forebrain (BF) from a neurosurgical perspective are missing. OBJECTIVE: To describe the anatomy of the BF and of the anterior perforated substance (APS), the BF emphasizing surgical useful anatomical relationship between surface landmarks and deep structures. METHODS: White matter fiber microdissection was performed on 5 brain specimens to analyze the topographic anatomy of the APS and expose layer-by-layer fiber tracts and nuclei of the BF. RESULTS: The APS, as identified anatomically, surgically, and neuroradiologically, has clear borders measured 23.3 ± 3.4 mm (19-27) in the mediolateral and 12.5 ± 1.2 mm (11-14) in the anteroposterior directions. A detailed stratigraphy of the BF was performed from the APS up to basal ganglia and thalamus allowing identification and dissection of the main components of the BF (septum, nucleus accumbens, amygdala, innominate substance) and its white matter tracts (band of Broca, extracapsular thalamic peduncle, ventral amygdalohypothalamic fibers). The olfactory trigone together with diagonal gyrus and the APS proper is a relevant superficial landmark for the basal ganglia (inferior to the nucleus accumbens, lateral to the caudate head, and medial to the lentiform nucleus). CONCLUSION: The findings in our study supplement available anatomic knowledge of APS and BF, providing reliable landmarks for precise topographic diagnosis of BF lesions and for intraoperative orientation. Surgically relevant relationships between surface and deep anatomic structures are highlighted offering thus a contribution to neurosurgeons willing to perform surgery in this delicate area.

Vertical bundles of the white matter fibers in the pons revisited: preliminary study utilizing the Klingler technique.

The inner structure of the pons contains several layers of transverse and vertical fibers and many nuclei. The vertical bundles are described as fibers of the corticospinal tract, corticonuclear tract, frontopontine tract and parieto-temporopontine tract organized in three layers. The aim of this study was to investigate the structure of the vertical bundles in the ventral pons using the modified Klingler method. Ten brain stem specimens were investigated. Specimens were fixed in 10% formalin, frozen for 24 h to separate nerve fibers by ice crystals, and then unfrozen again in 10% formalin solution. Afterwards, the specimens were dissected using a sharpened spatula. Results point to the existence of three main layers of vertical bundles and a small, constant, and superficial fourth fascicle that is yet to be described in the literature. We propose the name fasciculus longitudinalis superficialis (superficial longitudinal fascicle) for this group of vertical fibers of the pons.

White matter tract anatomy in the rhesus monkey: a fiber dissection study.

Brain connectivity in non-human primates (NHPs) has been mainly investigated using tracer techniques and functional connectivity studies. Data on structural connections are scarce and come from diffusion tensor imaging (DTI), since gross anatomical white matter dissection studies in the NHP are lacking. The current study aims to illustrate the course and topography of the major white matter tracts in the macaque using Klingler's fiber dissection. 10 hemispheres obtained from 5 primate brains (Macaca mulatta) were studied according to Klingler's fiber dissection technique. Dissection was performed in a stepwise mesial and lateral fashion exposing the course and topography of the major white matter bundles. Major white matter tracts in the NHP include the corona radiata, tracts of the sagittal stratum, the uncinate fasciculus, the cingulum and the fornix. Callosal fiber topography was homologous to the human brain with leg motor fibers running in the posterior half of the corpus callosum. The relative size of the anterior commissure was larger in the NHP. NHPs and humans share striking homologies with regard to the course and topography of the major white matter tracts.

Posterior Quadrant Disconnection: A Fiber Dissection Study.

BACKGROUND: Posterior quadrant disconnection can be highly effective in the surgical treatment of selected cases of refractory epilepsy. The technique aims to deafferent extensive areas of epileptogenic posterior cortex from the rest of the brain by isolating the temporoparietooccipital cortex. OBJECTIVE: To describe this procedure and relevant white matter tracts with a specific emphasis on the extent of callosotomy in an anatomic study. METHODS: Twenty hemispheres were dissected according to Klingler's fiber dissection technique illustrating the peri-insular (temporal stem, superior longitudinal fasciculus, corona radiata) and mesial disconnection (mesiotemporal cortex, cingulum, and corpus callosum). RESULTS: Extensive white matter tract disconnection is obtained after posterior quadrant disconnection. Callosal fibers connecting the anterior most part of the parietal cortex invariably ran through the isthmus of the corpus callosum and need to be disconnected, while frontal lobe connections including the corticospinal tract and the anterior two-thirds of the corpus callosum are spared during the procedure. CONCLUSION: Our findings suggest the involvement of both the splenium and the isthmus in interhemispheric propagation in posterior cortex epilepsies. Sectioning the total extent of the posterior one-third of the corpus callosum might therefore be necessary to achieve optimal outcomes in posterior quadrant epilepsy surgery.

Alzheimer's senile plaque as shown by microcryodissection, a new technique for dissociating tissue structures.

Extracellular accumulation of Aß peptides and intracellular aggregation of hyperphosphorylated tau proteins are the two hallmark lesions of Alzheimer disease (AD). The senile plaque is made of a core of extracellular Aß surrounded by phospho-tau positive neurites. It includes multiple components such as axons, synapses, glial fibers and microglia. To visualize the relationships of those elements, an original technique was developed, based on the dilation of interstitial water during freezing. Samples of neocortex, hippocampus and striatum were taken from formalin-fixed brains (one control case; three cases with severe Alzheimer disease). The samples were subjected to various numbers of freezing/thawing cycles (from 0 to 320) with an automated system we devised. The samples were embedded in paraffin, cut and stained with haematoxylin-eosin or immunostained against Aß, phospho-tau, and antigens enriched in axons, synapses, macrophages or astrocytes. Microcryodissection induced the dissociation of tissue components, especially in the grey matter where the neuropil formed an oriented "mesh". The size of the empty spaces separating the fiber bundles and cells increased with the number of cycles. The amyloid core of the senile plaque separated from its neuritic crown at around 300 freezing/thawing cycles. The dissected core remained associated with macrophages containing Aß in their cytoplasm. Phospho-tau positive axons were distinctly seen projecting from the neuritic crown to the isolated amyloid core, where they ended in large synapses. The microcryodissection showed astrocytic processes stuck directly to the core. The original method we developed-microcryodissection-helped understanding how histological components were assembled in the tissue.

Neurosurgical relevance of the dissection of the diencephalic white matter tracts using the Klingler technique.

OBJECTIVE: The Klingler fiber dissection technique is a relevant and reliable method for neurosurgery to identify with accuracy the fine structure of the brain anatomy highlighting white matter tracts. In order to demonstrate the significance of the application of this technique, we aimed to observe the course and relations of the mammillothalamic and habenulo-interpeduncular tracts as there are very few papers showing these important diencephalic tracts. MATERIAL AND METHODS: Twelve formalin-fixed brains were dissected using the Klingler technique in order to expose the medial diencephalic surface. Diencephalic white matter tracts, particularly the mammillothalamic and habenulo-interpeduncular tracts, were dissected using wooden spatulas and metallic dissectors with different sizes and tips. Several measurements were performed in both dissected hemispheres relative to the mammillothalamic and habenulo-interpeduncular tracts. RESULTS: The course and length of these two tracts were visualized and the relations with other fiber systems and with the neighboring gray matter structures quantified and registered. The mammillothalamic tract approximately marks the anteroposterior coordinate of the anterior pole of the subthalamic nucleus in the anterior commissure - posterior commissure plane. CONCLUSION: The present study helps to understand the three-dimensional architecture of the white matter systems of tracts when the Klingler technique is used. The numerical data obtained may be helpful to neurosurgeons while approaching brain paraventricular and ventricular lesions and deep brain stimulation. Finally, the anatomical knowledge can lower surgical complications and improve patient care particularly in the field of neurosurgery.