Investigation of the Spatiotemporal Changes in the Biomechanics of Injured Spinal Cord Using Atomic Force Microscopy

Introduction: Spinal cord injury (SCI) is a severe condition that can leave patients paralyzed and suffering from several secondary complications. Regeneration following spinal cord injury is challenging due to modified tissue organization and composition, resulting in gliosis and fibrosis. Gliosis and fibrosis lead to scar formation after spinal cord injury, presenting mechanical and molecular barriers to neuronal outgrowth, targeting, and tissue regeneration. It is unknown whether changes in mechanical characteristics impact axon regrowth through the scar tissue within spinal lesions. This study's objective is to first evaluate the mechanical properties of scar lesions after spinal cord injury using atomic force microscopy (AFM) and decipher how changes in the modulus of elasticity or stiffness (EY, Young's modulus) of the tissue alter cellular characteristics. We then investigated the time-dependent changes in the viscoelastic characteristics of scar lesions towards developing strategies for tissue recovery post-injury.

Materials and

Methods: The experimental setup involved nanoindentation measurements using a MFP-3D atomic force microscopy (AFM; Asylum Research, Santa Barbara, CA). Cryo-sectioned spinal cord tissues were obtained from both injured and uninjured (control) rats. Four time points were chosen for the injured model: 1 day, one week, one month, and six months. A tipless AFM cantilever affixed with a 20 μm bead was used under contact mode, with an approach/retraction velocity of 2 μm/sec, and an indent force of 1 nN. The Young's moduli of the tissues (EY) were then calculated using a modified Hertz's model from the force-indentation curves. Viscoelasticity experiments were done separately in the time and frequency domains, examining a range of indentation speeds (1, 10, 30, 50, and 100 μm/s), and an optimized optimal loading rate of 10 µm/s. In the time-domain experiments, besides the conventional force curves, the approach phase was conducted with an additional dwell phase (60 s), in which either force or indentation is held constant. The indentation was held constant to obtain continuous decay in the interaction force (stress-relaxation), and the force will be held constant for the creep tests. In the frequency-domain experiments, tissue samples were subjected to oscillatory deformations with controlled frequency, and then instantaneous modulus (E0) was characterized by fitting with the derived viscoelastic equation. All statistical analyses were performed using Minitab, and the ANOVA test was used to determine the statistically significant differences between the groups.

Results, Conclusions, and Discussions: Spatial distribution of stiffness and the interface between white and grey matter was captured with Young's Modulus (EY) mapping. Significant differences in EY were evident at random locations tested in control white and grey matter. The EY of grey matter was nearly 3-times that of white matter (396.7 ± 200 Pa). A similar analysis was performed for chronic hemi-section injuries containing glial and fibrotic scar tissue in injured rat spinal cord tissues. EY mapping was designed to visualize distinct differences compared to the injury site, adjacent to the visible site of injury, and far away from the injury site. The average EY of one-month post-injury tissues in grey and white matter regions was 228.1 ± 255.6 Pa and 76.95 ± 118.8 Pa, respectively. The stiffness of spinal cord glial scar decreased radially outward from the injury core. Stress-relaxation curves were used to determine the relaxation time of deformation (t0), relaxation time of load (tε), and complex modulus (G*(ω) = G'(ω) + i G"(ω)), which was used to calculate instantaneous EY. Injured spinal cord tissues with scar lesions exhibited higher instantaneous EY than uninjured tissues, indicating increased initial stiffness due to denser, fibrotic scar tissue formation. These tissues also show altered viscoelastic properties, with an increased storage modulus reflecting higher stiffness and variable changes in the loss modulus depending on the injury's extent and nature.
Our data highlights significant differences in the spatiotemporal Young's Moduli of the SCI scar compared to control tissues. The changes in viscoelastic properties further underscore the relevance of measuring spatiotemporal biomechanical characteristics of tissues post-injury to gain deeper understanding of the tissue pathology. These results guide us towards the development of strategies to repair the injured tissue. This approach will open more opportunities to leverage mechanical manipulation, revealing new therapeutic possibilities.

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