Spinal Cord Injury Results in Chronic Mechanical Stiffening

Abstract

Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is thought to present both molecular and mechanical barriers to neuronal regeneration. The scar consists of a meshwork of reactive glia and deposited, cross-linked, extracellular matrix (ECM) that has long been assumed to present a mechanically "stiff" blockade. However, remarkably little quantitative information is available about the rheological properties of chronically injured spinal tissue. In this study we utilize atomic force microscopy microindentation to provide quantitative evidence of chronic mechanical stiffening after SCI. Using the results of this tissue characterization, we assessed the sensitivity of both mouse and human astrocytes in vitro and determined that they are exquisitely mechanosensitive within the relevant range of substrate stiffness observed in the injured/uninjured spinal cord. We then utilized a novel immune modifying nanoparticle (IMP) treatment as a tool to reveal fibrotic scarring as one of the key drivers of mechanical stiffening after SCI in vivo. We also demonstrate that glial scar-forming astrocytes form a highly aligned, anisotropic network of glial fibers after SCI, and that IMP treatment mitigates this pathological alignment. Taken together, our results identify chronic mechanical stiffening as a critically important aspect of the complex lesion milieu after SCI that must be considered when assessing and developing potential clinical interventions for SCI.

Keywords: fibrotic scar; immune modifying nanoparticles; mechanical properties; spinal cord injury; stiffness.

FIG. 1.

Determination of AFM experiment and data analysis parameters, and sample preparation to characterize mouse spinal cord stiffness. (A) Schematic description of AFM microindentation experiment from the identification of area of interest on mouse spinal cord tissue to the determination of the elastic modulus. (B) Representative force curves performed on mouse spinal cord tissue (from control uninjured mouse spinal cord, 12 weeks post-sham injury) at an applied force corresponding to 10, 25, 50, and 75% of the maximal force threshold. (C) Schematic explanation of the determination of contact point and Hertz's model fitting on a force curve performed on control spinal cord tissue. Mean elastic modulus ± SEM of control uninjured mouse spinal cord samples (white and gray matter areas combined) from AFM measurements performed at 10, 25, 50, and 75% of the force threshold (D) and at 0.5 Hz, 1 Hz, and 2 Hz of loading rate (E). In (D), statistical results are presented considering values at 25% as a reference and with significance at *p < 0.05. All statistical results are shown in Supplementary Table S1 and Supplementary Table S2. (F) There was no significant difference between the elastic moduli of uninjured mouse spinal cords prepared using the “fresh” and “frozen/thawing” processes. In box and whisker plots, the box indicates the 25th and 75th percentiles of the values, the line inside the box is the median value, and the whiskers the smallest and largest values. Markers represent the average of 10 force curves performed per area. In total, two control uninjured mouse spinal cords samples were analyzed with two areas (white and gray matter) per slice and two non-consecutive slices per animal. Statistical analysis of the elastic modulus values using Kruskal-Wallis test (multiple comparisons) (D,E) and Mann-Whitney test (F). AFM, atomic force microscopy; SEM, standard error of the mean.

FIG. 2.

Chronic stiffening of the spinal cord after contusion SCI. (A) Transverse sections from control uninjured (12 weeks post-sham injury) mouse spinal cords. Unstained (top), H&E stain (bottom) (B) There was no significant difference in elastic moduli of white and gray matter in uninjured (12 weeks post-sham injury) mouse spinal cords (p = 0.9774, Mann-Whitney test). (C) Transverse sections from injured (12 weeks post-severe contusion, saline-treated) mouse spinal cord. H&E stain (top), fibronectin and GFAP immunohistochemistry (bottom). (D) No statistical difference between the elastic moduli of lesion core and lesion rim regions of injured, saline-treated, spinal cords (12 WPI). (E) Stiffness maps of white and gray matter on uninjured spinal cord tissue. (F) Chronically injured spinal cord tissue (12 WPI) was significantly stiffer than uninjured tissue, including all regions (white and gray matters, lesion core and rim) (****p < 0.0001, Mann-Whitney test). (G) Stiffness maps of two different lesion core and rim areas on injured, saline-treated, spinal cord tissue (12 WPI). In box and whisker plots, the box indicates the 25th and 75th percentiles of the values, the line inside the box is the median value, and the whiskers the smallest and largest values. The color of each marker (B,D,F) identifies the individual mouse from which the sample was taken. For stiffness maps (E,G) each colored pixel represents the elastic modulus value measured at that point on the corresponding image according to the scale below. 12 WPI, 12 weeks post-injury; GFAP, glial fibrillary acidic protein; H&E, hematoxylin and eosin; SCI, spinal cord injury.

FIG. 3.

Scar-stiffness substrates induce complex morphologies in cultured mouse astrocytes. (A) Representative images of murine astrocytes cultured for 4 days on scar stiffness (2000 Pa: top) or intact-tissue stiffness (200 Pa: bottom) stained for actin (phalloidin) and S100ß. Scale bar = 100 μm. (B) Enlarged images of the outlined areas demonstrate actin stress fibers. Scale bar = 25 μm. Astrocytes cultured on the stiffer 2000 Pa substrate demonstrate (C) increased surface area and (D) decreased circularity (n = 6 culture replicates, two-tailed Student's t test). (E) Stiffer culture substrates result in the extension of more numerous processes or “arms” (n = 6 culture replicates, two-way ANOVA). (F, G) Western blot analysis shows significantly reduced levels of mechanosensitive signaling molecules ß1-integrin and ILK in astrocytes cultured on soft (200 Pa) as compared with stiff (2000 Pa) substrate. GFAP expression is unchanged. (n = 5–7 culture replicates, two-tailed Student's t test) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars are SEM. ANOVA, analysis of variance; GFAP, glial fibrillary acidic protein; ILK, integrin-linked kinase; SEM, standard error of the mean.

FIG. 4.

Cultured human astrocytes respond to substrate-stiffness cues. (A) Representative images of hESC-derived astrocytes cultured for 5 days on scar stiffness (2000 Pa: top) or intact-tissue stiffness (200 Pa: bottom) stained for actin (phalloidin) and S100ß. Scale bar = 100 μm. (B) Enlarged images of the outlined areas demonstrate actin stress fibers. Scale bar = 25 μm. Astrocytes cultured on the stiffer 2000 Pa substrate show (C) no change in surface area but (D) demonstrate decreased circularity (n = 5 culture replicates, two-tailed Student's t test). (E) Stiffer culture substrates result in the extension of more numerous processes or “arms” (n = 5 culture replicates, two-way ANOVA). **p < 0.01, ****p < 0.0001. Error bars are SEM. ANOVA, analysis of variance; hESC, human embryonic stem cell; SEM, standard error of the mean.

FIG. 5.

IMP treatment after SCI rescues chronic tissue stiffening. (A) Mid-sagittal sections from 12 WPI IMP- or saline-treated mice stained for fibronectin and GFAP. Scale bar = 250 μm. (B) Mice who received IMP treatment after injury have significantly softer lesion sites than their saline treated counterparts (*p = 0.0180, Kruskal-Wallis test). There was no significant difference in elastic moduli between uninjured spinal cord and the IMP-treated lesion site at 12 WPI. (C) Spinal lesion rim regions are stiffer than lesion core regions in IMP-treated mice (12 WPI) (*p = 0.0432, Mann-Whitney test). (D) Stiffness maps of lesion core and rim regions on injured, IMP-treated, spinal cord tissue (12 WPI). In box and whisker plots, the box indicates the 25th and 75th percentiles of the values, the line inside the box is the median value, and the whiskers the smallest and largest values. The color of each data point identifies the individual mouse from which the sample was taken. *p < 0.05, ****p < 0.0001. 12 WPI, 12 weeks post-injury; GFAP, glial fibrillary acidic protein; IMP, immune modifying nanoparticle; SCI, spinal cord injury;

FIG. 6.

IMP treatment alters glial fiber alignment in the chronic glial scar. (A) The GFAP-positive astrocytic fiber network is visualized along the glial/fibrotic interface using traditional immunohistochemistry at 12 WPI. Scale bar = 50 μm. (B) Visualization of extracted individual fibers using CurveAlign CT-FIRE mode overlaid on the IHC image. (C) A heatmap view of curvelet alignment. Red coloring indicates areas with high alignment. (D) Curvelet overlay visualization showing the location (red dots) and orientation (green lines) of individual curvelets computed using the CurveAlign curvelet fiber representation mode. Enlarged images illustrate outlined representative ROIs selected along the glial border region adjacent to the fibrotic core. Quantification results from large images and individual ROIs are displayed in the format: “mean angle°/alignment coefficient” at the top of each panel. (E) Quantification of GFAP-positive fiber alignment within ROIs along the glial border zone shows reduced glial alignment in IMP-treated tissue as compared with saline. There is no significant difference in alignment between uninjured and IMP-treated tissue. Each marker represents the mean alignment coefficient of 16 regions of interest from a single experimental animal. (four ROIs per image, four images per mouse) (n = 5 mice per group, one-way ANOVA with Tukey's post hoc test) **p < 0.01. 12 WPI, 12 weeks post-injury; ANOVA, analysis of variance; GFAP, glial fibrillary acidic protein; IMP, immune modifying nanoparticle; ROI, region of interest.