Mapping of fiber orientation in human internal capsule by means of polarized light and confocal scanning laser microscopy
Introduction
The fiber architecture especially of the white matter in the human brain is presently the focus of interest. Many neurologic diseases affect the cerebral white matter, and modern imaging procedures such as magnetic resonance imaging (MRI) provide detailed visual information about the white matter. Anatomical knowledge about nervous fibers was achieved earlier by fiber preparations (Ebeling and Reulen, 1992), myelogenic studies (Kretschmann, 1988) and by observations after brain injury (Fries et al., 1993).
We were able to demonstrate the orientation and texture of the nervous fibers to influence the value of impedance measurements, and so an online verification of the needle’s position in a stereotactic procedure can be performed (Axer et al., 1998). In addition, the orientation of nervous fibers can be visualized by modern MRI procedures (Curnes et al., 1988, Douek et al., 1991, Peled et al., 1998). Nevertheless, a detailed anatomical mapping of fiber orientations in the adult human brain is difficult to assess. Our aim was to perform a mapping of the orientation of the fibers in the internal capsule. Thus we wanted to find out how anatomically defined fiber tracts are oriented in the white matter. The method should give information about fiber texture, fiber orientation and identification of specific fiber tracts.
Therefore we used the combination of confocal laser microscopy and polarized light microscopy. The confocal laser microscope allows information to be collected from well-defined optical sections. This is done by a sequential illumination which is focused on one volume element of the specimen at a time (Wright et al., 1993). This way stacks of optical sections can be produced, which allow three-dimensional reconstruction of the fiber texture and provide good information about the orientation of the fibers at high resolution.
Polarized light microscopy can selectively visualize anisotropic structures. In polarization microscopy, optically polarized light passes through a sample of tissue and into a second polarizer (analyzer), which polarizes light in a perpendicular plane with respect to the first polarizer. Birefringence is able to twist some of the light so that it can pass through the analyzer and be imaged. The birefringence of the nervous tissue has been well known for a long time (Schmidt, 1923, Schmidt, 1924, Schmitt and Bear, 1936, Kretschmann, 1967, Wolman, 1975). As the presence of anisotropy indicates polarity and order, polarization microscopy can be used to visualize long fiber tracts in the brain (Fraher and MacConnaill, 1970, Miklossy and Van der Loos, 1991). The orientation of the fibers influences the transmission of plane-polarized light at different velocities at different azimuths.
The combination of both techniques allowed us to obtain detailed information of the fiber structure and orientation with confocal microscopy and to collect information about the localization and orientation of long myelinated fiber tracts with polarized light microscopy.
The internal capsule in the adult human brain represents a collection of different systems of fibers closely located in a small space. It is a structure of high clinical importance, since the fibers of the pyramidal tract are located here. Nevertheless the exact location of the pyramidal tract in the internal capsule was a matter of dispute for decades (Maurach and Strian, 1981).
Section snippets
Macroscopic preparation
Four human cadaver brains without macroscopically definable pathology and without a history of neurologic or psychiatric disease were fixed in 4% aqueous formalin solution and macroscopically prepared. The meninges were removed and the brains cut in the median-sagittal plane. Important landmarks were identified on the median surface of the hemispheres: the anterior and posterior commissure, the interventricular foramen of Monro and the internal cerebral vein (or the tela choroidea ventriculi
Results
In polarization microscopy, the detectable signal depends on the order and the inclination of the fibers. In particular, parallel, horizontally cut fibers give a bright signal at a special azimuth whereas the signal decreases after rotation of 45°. The steeper these tracts of fibers are, the less bright the signal is. Thus different fiber tracts of different orientation and order produce areas of different brightness. In low magnification, the polarization picture provides information about the
Methodological considerations
Traditionally the internal capsule is divided into the anterior limb, the genu and the posterior limb (Dejerine, 1901). The areas occupied by the distinct bundles of fibers differ from the borders of the macroscopically defined parcellation of the internal capsule. In his classical description, Vogt (1902) subdivided the posterior limb into three parts according to the intensity of the Weigert staining. This subdivision, however, does not take care of the orientation of the fibers.
The central
Acknowledgements
We would like to thank Petra Ibold and Anita Agbedor for their technical assistance.
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