skip to main content

Title: Ex-vivo quantification of ovine pia arachnoid complex biomechanical properties under uniaxial tension
Abstract Background The pia arachnoid complex (PAC) is a cerebrospinal fluid-filled tissue conglomerate that surrounds the brain and spinal cord. Pia mater adheres directly to the surface of the brain while the arachnoid mater adheres to the deep surface of the dura mater. Collagen fibers, known as subarachnoid trabeculae (SAT) fibers, and microvascular structure lie intermediately to the pia and arachnoid meninges. Due to its structural role, alterations to the biomechanical properties of the PAC may change surface stress loading in traumatic brain injury (TBI) caused by sub-concussive hits. The aim of this study was to quantify the mechanical and morphological properties of ovine PAC. Methods Ovine brain samples (n = 10) were removed from the skull and tissue was harvested within 30 min post-mortem. To access the PAC, ovine skulls were split medially from the occipital region down the nasal bone on the superior and inferior aspects of the skull. A template was used to remove arachnoid samples from the left and right sides of the frontal and occipital regions of the brain. 10 ex-vivo samples were tested with uniaxial tension at 2 mm s −1 , average strain rate of 0.59 s −1 , until failure at < 5 h post extraction. The force and displacement data more » were acquired at 100 Hz. PAC tissue collagen fiber microstructure was characterized using second-harmonic generation (SHG) imaging on a subset of n = 4 stained tissue samples. To differentiate transverse blood vessels from SAT by visualization of cell nuclei and endothelial cells, samples were stained with DAPI and anti-von Willebrand Factor, respectively. The Mooney-Rivlin model for average stress–strain curve fit was used to model PAC material properties. Results The elastic modulus, ultimate stress, and ultimate strain were found to be 7.7 ± 3.0, 2.7 ± 0.76 MPa, and 0.60 ± 0.13, respectively. No statistical significance was found across brain dissection locations in terms of biomechanical properties. SHG images were post-processed to obtain average SAT fiber intersection density, concentration, porosity, tortuosity, segment length, orientation, radial counts, and diameter as 0.23, 26.14, 73.86%, 1.07 ± 0.28, 17.33 ± 15.25 µm, 84.66 ± 49.18°, 8.15%, 3.46 ± 1.62 µm, respectively. Conclusion For the sizes, strain, and strain rates tested, our results suggest that ovine PAC mechanical behavior is isotropic, and that the Mooney-Rivlin model is an appropriate curve-fitting constitutive equation for obtaining material parameters of PAC tissues. « less
Authors:
; ; ; ; ; ; ; ; ;
Award ID(s):
1656450
Publication Date:
NSF-PAR ID:
10273995
Journal Name:
Fluids and Barriers of the CNS
Volume:
17
Issue:
1
ISSN:
2045-8118
Sponsoring Org:
National Science Foundation
More Like this
  1. Introduction: Vascular diseases like abdominal aortic aneurysms (AAA) are characterized by a drastic remodeling of the vessel wall, accompanied with changes in the elastin and collagen content. At the macromolecular level, the elastin fibers in AAA have been reported to undergo significant structural alterations. While the undulations (waviness) of the collagen fibers is also reduced in AAA, very little is understood about changes in the collagen fibril at the sub-fiber level in AAA as well as in other vascular pathologies. Materials and Methods: In this study we investigated structural changes in collagen fibrils in human AAA tissue extracted at the time of vascular surgery and in aorta extracted from angiotensin II (AngII) infused ApoE−/− mouse model of AAA. Collagen fibril structure was examined using transmission electron microscopy and atomic force microscopy. Images were analyzed to ascertain length and depth of D-periodicity, fibril diameter and fibril curvature. Tissues were also stained using collagen hybridizing peptide (CHP) and analyzed using fluorescent microscopy and second harmonic generation (SHG) microscopy to locate regions of healthy and degraded collagen. Results: Abnormal collagen fibrils with compromised D-periodic banding were observed in the excised human tissue and in remodeled regions of AAA in AngII infused mice (Figuremore »1). These abnormal fibrils were characterized by statistically significant reduction in depths of D-periods and an increased curvature of collagen fibrils. These features were more pronounced in human AAA as compared to murine samples. Additionally, regions of abnormal collagen were located within the remodeled areas of AAA tissue and were distinct from healthy collagen regions as ascertained using CHP staining and SHG (Figure 1). Thoracic aorta from Ang II-infused mice, abdominal aorta from saline-infused mice, and abdominal aorta from non-AAA human controls did not contain abnormal collagen fibrils. Conclusions: The structural alterations in abnormal collagen fibrils appear similar to those reported for collagen fibrils subjected to mechanical overload or chronic inflammation in other tissues. Detection of abnormal collagen could be utilized to better understand the functional properties of the underlying extracellular matrix in vascular as well as other pathologies.« less
  2. null (Ed.)
    Purpose To develop a novel model composed solely of Col I and Col III with the lower and upper limits set to include the ratios of Col I and Col III at 3:1 and 9:1 in which the structural and mechanical behavior of the resident CM can be studied. Further, the progression of fibrosis due to change in ratios of Col I:Col III was tested. Methods Collagen gels with varying Col I:Col III ratios to represent a healthy (3:1) and diseased myocardial tissue were prepared by manually casting them in wells. Absorbance assay was performed to confirm the gelation of the gels. Rheometric analysis was performed on each of the collagen gels prepared to determine the varying stiffnesses and rheological parameters of the gels made with varying ratios of Col I:Col III. Second Harmonic Generation (SHG) was performed to observe the 3D characterization of the collagen samples. Scanning Electron microscopy was used for acquiring cross sectional images of the lyophilized collagen gels. AC16 CM (human) cell lines were cultured in the prepared gels to study cell morphology and behavior as a result of the varying collagen ratios. Cellular proliferation was studied by performing a Cell Trace Violet Assay and themore »applied force on each cell was measured by means of Finite Element Analysis (FEA) on CM from each sample. Results Second harmonic generation microscopy used to image Col I, displayed a decrease in acquired image intensity with an increase in the non-second harmonic Col III in 3:1 gels. SEM showed a fiber-rich structure in the 3:1 gels with well-distributed pores unlike the 9:1 gels or the 1:0 controls. Rheological analysis showed a decrease in substrate stiffness with an increase of Col III, in comparison with other cases. CM cultured within 3:1 gels exhibited an elongated rod-like morphology with an average end-to-end length of 86 ± 28.8 µm characteristic of healthy CM, accompanied by higher cell growth in comparison with other cases. Finite element analysis used to estimate the forces exerted on CM cultured in the 3:1 gels, showed that the forces were well dispersed, and not concentrated within the center of cells, in comparison with other cases. Conclusion This study model can be adopted to simulate various biomechanical environments in which cells crosstalk with the Collagen-matrix in diseased pathologies to generate insights on strategies for prevention of fibrosis.« less
  3. Mechanical distension beyond a particular threshold evokes visceral pain from distal colon and rectum (colorectum) and thus biomechanics plays a central role in visceral nociception. In this study we focused on the layered structure of the colorectum through the wall thickness and determined the biomechanical properties of layer-separated colorectal tissue. We harvested the distal 30 mm of mouse colorectum and dissected into inner and outer composite layers. The inner composite consists of the mucosa and submucosa while the outer composite includes the muscular layers and serosa. We divided each composite axially into three 10 mm-long segments and conducted biaxial mechanical extension tests and opening-angle measurements for each tissue segment. In addition, we quantified the thickness of the rich collagen network in the submucosa by nonlinear imaging via second harmonic generation (SHG). Our results reveal the inner composite is slightly stiffer in the axial direction while the outer composite is stiffer circumferentially. The stiffness of the inner composite in the axial direction is about twice that in the circumferential direction, consistent with the orientations of collagen fibers in the submucosa approximately ±30 degrees to the axial direction. Submucosal thickness measured by SHG showed no difference from proximal to distal colorectum undermore »load-free condition, which likely contributes to the comparable tension stiffness of the inner composite along the colorectum. This, in turn, strongly indicates the submucosa as the load-bearing structure of the colorectum. This further implies nociceptive roles for the colorectal afferent endings in the submucosa that likely encode tissue-injurious mechanical distension.« less
  4. Ligation of the left anterior descending (LAD) coronary artery has been commonly employed to induce myocardial infarction (MI) in animals; however, it is known to pose setbacks in the form of cardiac arrhythmias and unpredictable areas of necrotic damage. Cryo-infarction is an alternate method that has been adopted to create a reproducible model of a myocardial injury. In this study, Sprague-Dawley rats were subjected to thoracotomy followed by cryo-induced infarction of the heart, while the control-sham group was only subjected to thoracotomy following which the heart was collected from all animals. Tissue sections were stained with hematoxylin and eosin and analyzed to determine cardiac muscle density, fiber length, and fiber curvature. Observations revealed reduced muscle density, cardiac fiber length, and distorted fibers in infarcted tissue sections. Gomori’s Trichrome staining was performed on tissue sections to study the effects of post MI on collagen, which showed enhanced intensity of collagen staining indicating fibrosis for the experimental models as compared to the sham models, an established consequence to myocardial injury. Immunohistochemical staining of the tissue sections with DAPI and connexin-43 (Cx-43) revealed that there was reduced DAPI staining and a less pronounced expression of Cx-43 in the experimental samples as compared tomore »the sham samples. Results implied significant cell damage resulting from the cryo-infarction, subsequently disrupting and disaggregating the functional Cx-43 junction in cardiac myocytes, which is essential for normal and healthy cardiac physiology and function. This quantitative histological study of cryo-induced MI in a rat model can aid others attempting to optimize MI models in rats via cryo-injury, to study cardiac disease progression, and to aid in the construction of engineered cardiac tissues.« less
  5. Collagen quantity and integrity play an important role in understanding diseases such as myelofibrosis (MF). Label-free mid-infrared spectroscopic imaging (MIRSI) has the potential to quantify collagen while minimizing the subjective variance observed with conventional histopathology. Infrared (IR) spectroscopy with polarization sensitivity provides chemical information while also estimating tissue dichroism. This can potentially aid MF grading by revealing the structure and orientation of collagen fibers. Simultaneous measurement of collagen structure and biochemical properties can translate clinically into improved diagnosis and enhance our understanding of disease progression. In this paper, we present the first report of polarization-dependent spectroscopic variations in collagen from human bone marrow samples. We build on prior work with animal models and extend it to human clinical biopsies with a practical method for high-resolution chemical and structural imaging of bone marrow on clinical glass slides. This is done using a new polarization-sensitive photothermal mid-infrared spectroscopic imaging scheme that enables sample and source independent polarization control. This technology provides 0.5 µm spatial resolution, enabling the identification of thin (≈1 µm) collagen fibers that were not separable using Fourier Transform Infrared (FT-IR) imaging in the fingerprint region at diffraction-limited resolution ( ≈ 5 µm). Finally, we propose quantitative metrics to identify fiber orientation frommore »discrete band images (amide I and amide II) measured under three polarizations. Previous studies have used a pair of orthogonal polarization measurements, which is insufficient for clinical samples since human bone biopsies contain collagen fibers with multiple orientations. Here, we address this challenge and demonstrate that three polarization measurements are necessary to resolve orientation ambiguity in clinical bone marrow samples. This is also the first study to demonstrate the ability to spectroscopically identify thin collagen fibers (≈1 µm diameter) and their orientations, which is critical for accurate grading of human bone marrow fibrosis.

    « less