The interface between the brain and the skull consists of three fibrous tissue layers, dura mater, arachnoid, and pia mater, known as the meninges, and strands of collagen tissues connecting the arachnoid to the pia mater, known as trabeculae. The space between the arachnoid and the pia mater is filled with cerebrospinal fluid which stabilizes the shape and position of the brain during head movements or impacts. The histology and architecture of the subarachnoid space trabeculae in the brain are not well established in the literature. The only recognized fact about the trabeculae is that they are made of collagen fibers surrounded by fibroblast cells and they have pillar- and veil-like structures. In this work the histology and the architecture of the brain trabeculae were studied, via a series of
Traumatic brain injury (TBI), which is mainly due to vehicular collisions, contact sports, falls, or shock wave blasts from improvised explosive devices (IEDs), is caused by the relative motion between the brain and the skull. Anatomically the interface between the skull and the brain consists of a series of three fibrous tissue layers, dura mater, arachnoid, and pia mater, and arachnoid trabeculae which are strands of collagen tissue (Figure
Meningeal layers, the SAS, the pia mater, and the arachnoid. OpenStax College: Anatomy & Physiology, Connexions Web site.
The SAS itself has a complex geometry due to the fact that there is an abundance of trabeculae which stretch from the arachnoid (subdural) to the pia mater. Also, since the pia mater adheres to the surface of the brain and follows all its contours including the folds of the cerebral and cerebellar cortices, the resulting SAS is highly irregular and the associated distribution of CSF within the SAS is very nonuniform. Consequently, this irregular geometry produces a complex CSF flow around the brain, which results in a solid-fluid interaction that damps and stabilizes the movement of the brain when the head is exposed to external loads.
Unfortunately, the complicated geometry of the SAS and trabeculae makes it impossible to model all the details of the region. Thus, in many studies [
This is borne out by the fact that the subarachnoid space (SAS) trabeculae play an important role in damping and reducing the relative movement of the brain with respect to the skull, thereby reducing traumatic brain injuries (TBI), as was shown by Zoghi-Moghadam and Sadegh [
With regard to material properties various studies [
In addition, there have been a few experimental studies associated with the architecture of the SAS, with one experimental study by Alcolado et al. [
The goal of this present study was therefore to investigate the histology and morphology of the SAS of the brain and in particular the SAS trabeculae, which is needed for sophisticated and accurate modeling of TBI. Specifically, in this paper, the histology and the architecture of the brain trabeculae are presented via cadaveric and animal experimental studies. In the first experimental study of the brain a histological sectioning with florescent and bright field illumination was done. In the second set of experimental studies scanning and transmission electron microscopy were used.
The first set of experiments designed to examine the histology and architecture of the SAS was done, by using fluorescence and bright field microscopy, to determine the structure of the SAS associated with a cadaver brain.
In this experiment an image of the trabecular architecture of a cadaver was acquired using florescent and bright field microscopy. However, since the cadaveric human brain was already fixed by formaldehyde, the arachnoid had been collapsed onto the pia mater and the CSF had been drained. Several techniques were employed to separate the arachnoid from the pia mater and to recreate and restore the subarachnoid space, which is approximately 2 to 3 mm wide in a human head. The techniques involved several steps that included confining a cortical region of the brain and the injection of Microfil (a silicone rubber injection compound) from Flowtech, Inc., into the region between the two layers of the arachnoid and the pia mater. The Microfil solidified quickly and kept the two layers separated. To inject the Microfil solution into the SAS a fixture was designed to confine the Microfil within the cortex, where the fixture consisted of a clear tube with a key-way on one side to allow for the insertion of the syringe needle. Extreme care was taken during this process since it was necessary to ensure that the solution was injected exactly between the pia mater and the arachnoid. The viscosity of the fluid was also an important factor because if the Microfil that was mixed with the solidifier was too thick it was not possible to inject it between the two layers using a small needle; however, if it were diluted too much it would then just be drained out from between the neighboring cortexes and would not cause the subarachnoid space to open. Several tissue samples from different regions of the cadaver’s brain were prepared using this technique. Figure
The brain tissue with Microfil fixed in the formalin.
In addition to the cadaver work, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to study the morphology of trabeculae and to obtain a better understanding of the density and the configuration of the trabeculae network in a rat’s brain. A rat was used since it has been shown that there is a similarity in the morphology of the trabeculae of rats and humans [
To investigate the histology and the architecture of the SAS trabeculae
The basket was then placed in a tube and 30 mL of alcohol at 25%, 50%, 70%, 80%, 90%, and 100% concentrations was added consecutively to the tube for 5–10 minutes each time in a second step to further guarantee that no water remained within the sample. The tube was shaken from time to time to ensure a better penetration of the alcohol within the tissues. The Critical Point Drying (CPD) technique was also used by immersing the samples in a chamber that was then placed in liquid CO2, rinsing them 7 times for 5–10 minutes. After the alcohol was washed out, the samples were dried, and the temperature of the chamber was raised to 40°C, letting the CO2 change from its liquid phase to a dry gas phase. Finally, the samples were coated with gold particles just prior to observing them with the SEM.
The tissue samples for the TEM study were obtained from the same batch of brain tissue prepared for use with the SEM; however, in this process the specimens had to be only 1 or 2 mm in thickness; therefore it was necessary to choose and cut the tissue samples from a specific location of the brain due to the small physical size of the specimen. Consequently, extracting the tissue from the area surrounding the superior sagittal sinus by cutting the brain along a plane parallel and next to the sagittal plane on both sides of the sinus was decided. The sections were then shortened to be about 1 mm from the top of the brain and were cut into approximately 1 mm sized pieces. The samples were subsequently immersed in acrolein for an hour followed by a secondary fixative using an osmium tetroxide 2% solution, and then they were washed with ddH2O three times for 5 minutes. The samples were dehydrated and rinsed twice in propylene oxide for 5 minutes. For each concentration the jar containing the resin and the samples was placed on a shaker for three hours. Eventually each piece was transferred to the bottom of a triangle-shaped beam capsule with a pipette and then each capsule was filled up to three-quarters of the way with resin at 100% concentration. Finally, the capsules were placed in an oven at 60°C to allow the resin to solidify.
The block sample was transferred to the microtome and its position was adjusted with respect to the diamond knife. The thickness of the sections was set to 100 nm. Once enough sections had been obtained, a moist eyelash was used to create groups of six pieces, and they were transferred onto the shiny side of the small round grids. Subsequently, the samples were stained with uranyl acetate and Reynolds lead citrate and studied using a Philips CM-12 transmission electron microscope at an accelerating voltage of 80 kV.
The bright field microscopy image of the subarachnoid space is shown in Figure
Light microscopic view of subarachnoid, (a) the arachnoid mater, (b) SAS, (c) the pia mater, and (d) the brain.
Trabeculae of cadaveric brain tissue under the fluorescent light.
The results obtained from the SEM showed that the SAS was almost fully open and the blood vessels were extremely well preserved, as shown in Figures
SEM micrograph of the dura and arachnoid layer in the rat brain: (a) the brain side, (b) the SAS, and (c) the dura mater.
SEM image of the SAS in the rat with some trabecula (star) surrounding a blood vessel (BV).
Specifically, Figure
The SAS appeared to have a more complicated morphology than is presented in much of the literature [
The TEM images allowed the cellular and subcellular structure of the SAS to be observed. In comparison to the SEM, the TEM images allowed different cell layers to be clearly identified; however, the separation between the brain and the SAS was not as clear as in the SEM case; therefore the myelin sheath of the neurons was taken as a reference to locate the brain. Figures
The results from the TEM provided good information about the cellular arrangement within the SAS. Specifically, the flat cells on the arachnoid side of the SAS were well defined and arranged in layers as shown in Figure
In addition, when these bands were examined using high magnification, they were seen to be composed of an alternating collection of light and dark strips, and it is known that such periodicity is a characteristic of the type of collagen that forms the fibrils. It was also observed that the fibril bundles appeared to have different compositions with some bundles being full of fibrils tightly packed whereas other bundles were half or less full of fibrils. The reason for the inhomogeneity among bundles appeared to be that the arachnoid did not need to be as hard as some other structures containing collagen in the body. The collagen fibrils forming the trabeculae have a filler role rather than a support role.
In the SEM results (Figures
The SEM image of the rat’s brain: (a) the brain, (b) the veil-like network of trabeculae, (c) the blood vessel in the SAS, and (d) the dura mater.
SEM image of a platelike trabecula showing permeable characteristics (stars): (a) the arachnoid mater, (b) the plate trabecula, and (c) the pia mater.
Image of the collagen fibrils (star) that constitute the internal structure of a trabecula.
TEM section of the SAS in the rat’s brain: (a) the arachnoid mater, (b) collagen fibrils cross section, (c) the SAS, (d) nucleus in pia mater, and (e) the axon in the gray mater.
TEM section of the SAS in the rat: (a) the arachnoid mater, (b) the SAS architecture (trabeculae), and (c) the pia mater.
Bundle of collagen fibrils (stars) surrounded by fibroblasts (arrows).
Layers of fibroblast cells in the arachnoid. The flat cells on the arachnoid side were well defined and arranged in layers (stars).
Longitudinal and cross section of collagen fibers in the arachnoid side of the SAS: (a) the brain, (b) the basal lamina, (c) the pia mater, (d) longitudinal section of collagen fibers, (e) cross section of collagen fibers, (f) the arachnoid mater, and (g) periodicity.
It should also be noted that the TEM required a much longer preparation process than did the SEM. The preparation of samples had more steps, and many of those steps were critical for the cleanliness of the sections; however the benefit was that the TEM images provided information about the cellular content of the SAS that was lacking in the SEM. Specifically, it was found from these results that the arachnoid layers were made of fibroblasts and not only of collagen fibrils, that these cells formed a permeable layer, that the pia mater was represented by a single layer of fibroblast cells covering the brain, and that the collagen fibrils were arranged in bundles and scattered within the SAS at the border within the arachnoid layer and around the endothelial sheet of the blood vessels.
A wide range of material properties for the SAS, of up to three orders of magnitude different, have been reported in the literature. In a previous study performed by Saboori [
With regard to the arachnoid mater, the experimental studies revealed that it was composed of about ten layers of fibroblasts cells joined together via tight junctions. In contrast the pia mater appeared to be much thinner than the arachnoid layer since it was composed of only one layer of fibroblasts. Also within the cells of the arachnoid, some collagen fibril bundles could be observed, with the fibrils being produced by the fibroblasts, and thereby provided a structural support to the arachnoid layer, in addition to the support provided by the fibroblasts themselves. The arachnoid layer appeared to be permeable in the TEM results, and some fluid was observed in the spaces between the fibroblasts. At the junction of the arachnoid layer with the SAS the trabeculae branch out to meet the arachnoid mater at some locations and form a descending trabecula from the arachnoid to the pia mater in tree-shaped structures.
In the case of the trabeculae, the experimental studies revealed that the architecture of the trabecular could be quite complex with individual structures being like tree-shaped rods, pillars, and plates and in some cases having an intricate veil-like geometry. Structurally, the trabeculae were found to consist mainly of bundles of collagen fibrils wrapped together by fibroblast cells, with the fibroblasts being found in all connective tissues. In addition, the trabecula were found to be surrounded by an extracellular matrix (ECM) with a thickness of between 50 and 200 nm and composed of collagen fibers, proteoglycans, laminins, and fibronectins.
In addition to the rods, plates, and tree-shaped architecture, it was observed that in some regions complex networks of more randomly oriented trabeculae were found in the SAS. This network architecture was mainly located in the vicinity of the blood vessels and was very complex and inhomogeneous. These trabeculae networks were composed of fibroblasts, collagen fibrils, and extracellular matrix. Some holes/cavities were also seen to exist in the networks. These cavities facilitated the flow of cerebrospinal fluid around the brain. The results of this experimental study were compared and validated with regard to a study by Killer et al. [
Cartoon sketch of the SAS in a rat’s brain.
It was concluded from this work that the trabeculae are collagen based Type I, as was found by Abraham [
The authors declare that there is no conflict of interests regarding the publication of this paper.
This research was supported by grants from PSC-CUNY and the City University of New York.