The soft mechanical signature of glial scars in the central nervous system

Abstract

Injury to the central nervous system (CNS) alters the molecular and cellular composition of neural tissue and leads to glial scarring, which inhibits the regrowth of damaged axons. Mammalian glial scars supposedly form a chemical and mechanical barrier to neuronal regeneration. While tremendous effort has been devoted to identifying molecular characteristics of the scar, very little is known about its mechanical properties. Here we characterize spatiotemporal changes of the elastic stiffness of the injured rat neocortex and spinal cord at 1.5 and three weeks post-injury using atomic force microscopy. In contrast to scars in other mammalian tissues, CNS tissue significantly softens after injury. Expression levels of glial intermediate filaments (GFAP, vimentin) and extracellular matrix components (laminin, collagen IV) correlate with tissue softening. As tissue stiffness is a regulator of neuronal growth, our results may help to understand why mammalian neurons do not regenerate after injury.

Figure 1. Mechanical properties of the intact cortex.

(a) Schematic showing the location of the stab injury in the rat brain and the areas of the cortex where elasticity measurements were recorded by AFM. CC indicates corpus callosum, Cg cingulum, AGm medial agranular, AGI lateral agranular and ACC anterior cingulate regions of the cortex. An AFM cantilever indents the brain tissue; the applied force is translated to a bending of the cantilever that is readable through changes in the laser light path and detected by a photodiode. (b) The force–indentation curves obtained by AFM micro-indentation tests followed a spherical Hertzian contact model. For each force–indention curve, the indentation depth and force were divided by their respective maximum values. The black line passing the averaged force values serves as a guide to the eye; error bars are s.d. The inset shows the normalized force–indentation curves on a log–log scale; the curves follow a slope of ∼1.5, consistent with the slope of the Hertz contact model. (c) Spatial mapping of the elastic modulus of a healthy rat brain cortex. The elastic modulus at each pixel is measured by AFM indentation and is represented as a rainbow-coloured palette map with blue denoting softer and red corresponding to stiffer regions. The elasticity map and the brightfield image of the tissue are overlaid. Scale bars, 500 μm. (d,e) The elasticity map is symmetric around the midline; three regions (that is, R₁, R₂ and R₃) with significantly different mechanical properties were identified in each brain hemisphere. The AGm that contains larger amounts of myelinated axons (that is, R₂ and R′₂) was significantly softer than the AGl (R₁ and R′₁) and the ACC (R₃ and R′₃). (e) The average percentage drop in elastic modulus was calculated considering the combined average elasticity of regions R₃ and R′₃ as the baseline. Error bars are s.e.m., **for P=0.003 and ***for P<0.001, Tukey–Kramer post hoc test.

Figure 2. Changes in brain tissue stiffness and protein expression 9 days after a stab injury to the cortex.

Cf. Supplementary Fig. 1 for a later time point. (a) A 2 mm stab injury (white arrow in the top brightfield image) was induced in the cortex of the rat brain. The colour maps represent the spatial distribution of elastic moduli in the injured and contralateral hemispheres 9 days PI. Five regions were identified for further quantification, including a rectangular region (dashed box) around the injury site (∼150 μm width centred at the scar). (b) Comparison of the elastic properties of these regions. (c) Representative immunofluorescence images showing that GFAP (green) and vimentin (magenta) are upregulated 9 days PI. Similarly, the ECM proteins laminin (red) and collagen (yellow) are upregulated in and around the site of stab injury. Scale bars, 500 μm. (d) Average relative drop in elastic modulus of the regions indicated in (a) compared to the uninjured contralateral hemisphere. (e) Quantification of immunofluorescence for glial cell and ECM markers. GFAP, vimentin, laminin and collagen IV are all significantly upregulated around the site of injury compared to the contralateral cortical hemisphere. The normalized intensity was derived by comparing the average intensity signal of each marker in a 1.5 × 1.5 mm² square around the scar as indicated in (c) with their respective contralateral regions. Error bars are s.e.m., *P<0.01, ***P<0.001.

Figure 3. Softening of the brain in response to stab injury.

(a,b) Comparison of the drop in elastic modulus of different brain regions relative to the contralateral control for individual animals at (a) ∼1.5 weeks PI and (b) ∼three weeks PI. Spatiotemporal changes in brain tissue stiffness were very similar between different animals (Supplementary Tables 2, 3), allowing the data to be pooled. (c–h) Comparison of the mechanical properties and protein expression of injured and contralateral control tissue at 1.5 weeks PI (c–e) and three weeks PI (f–h) (combined data from three animals for each time point). (c,f) Similar to Fig. 2a, four regions were considered for analysis: region A' (∼500 × 1500 μm² lateral to the scar), region C′+D′ (∼700 × 1500 μm² medial to the scar), region B′ (∼600 × 1500 μm² around the scar excluding the actual scar) and the actual scar region (∼150 × 1500 μm² around the scar). (d,g) Regional average relative drop in elastic modulus. Tissue was significantly softer at the (Scar), around (B′), and medial (C′+D′) to the scar. (e,h) The glial cell markers GFAP and vimentin and the ECM markers laminin and collagen were all significantly upregulated around the site of injury both at 1.5 and three weeks PI. Error bars are s.e.m., *P<0.01, ^**P<0.005; ^***P<0.001.

Figure 4. Crush injuries to the rat spinal cord lead to tissue softening.

(a) Schematic drawing of a dorsal column crush lesion. After a laminectomy at the C5 level, the dura is opened and the spinal cord is penetrated with a pair of sharp forceps (∼1.5 mm deep). Two closures of the forceps for 10 s each crush the dorsal column, creating an injury in both white and grey matter. The dashed rectangle depicts the approximate area in which AFM measurements were performed. (b) Transverse spinal cord section of a sham control depicting the outlines of grey (R₁) and white (R₂) matter in the area of interest (dashed lines). The colour map represents the spatial distribution of elastic moduli in the grey and white matter. (c) Transverse spinal cord section of an animal with a dorsal column crush lesion at 7 days PI. The approximate outlines of the injury are indicated by the red dashed lines. The colour map represents the spatial distribution of elastic moduli in both healthy and injured grey (R₁ and R₃, respectively) and healthy and injured white matter (R₂ and R₄, respectively). Scale bars, 500 μm. (d) Comparison of the elastic properties of grey and white matter in the control animal shown in (b). Grey matter was significantly stiffer than white matter (P<0.001, two-tailed Student t-test). (e) Comparison of the elastic moduli of the different tissue regions between four pooled control animals shown in Supplementary Fig. 5 and the tissue shown in (c). (f) Average relative drop in elastic modulus of the regions indicated in (c) compared to pooled control shams. Error bars are s.e.m., ***P<0.001.

Figure 5. Correlation between brain tissue mechanics, gliosis, and ECM changes at 9 days PI.

Cf. Supplementary Fig. 4 for a later time point. (a) Rainbow-pallet map showing the relative difference in elastic modulus between injured and uninjured cortical hemisphere. Scale bar, 500 μm. The panels in the bottom rows represent vertical projection profiles (mean±s.d.) of the changes in elasticity determined for the regions i and ii indicated by dashed lines in the map . (b,c) The normalized and down-scaled pixel intensity image of vimentin and GFAP expression. The panels in the bottom rows represent vertical projection profiles (mean±s.d.) of the vimentin and GFAP normalized intensity estimated for the regions i and ii indicated by dashed lines in their respective images. (d,e) The normalized and down-scaled pixel intensity image of collagen IV and laminin expression. The panels in the bottom row represent vertical projection profiles (mean±s.d.) of the collagen and laminin normalized intensity in region i of their respective fluorescence images. In (a–e), grey arrows indicate the direct positive correlation between a drop in elasticity and an increase in protein expression. A positive correlation between tissue softening and vimentin and GFAP expression was also observed in regions located far away from the injury site medial to the scar (white arrows). (f) Shown are the calculated linear correlation coefficients for regions i and ii as well as the 2D correlation coefficients derived by linearly correlating the 2D matrix of the change in elasticity and the 2D maps of fluorescence intensity for each marker. (g) Average (mean±s.d.) correlation coefficients of three brains at 9, 21 and 22 days PI as a function of protein type and correlation region.

Figure 6. Schematic summary.

1.5 weeks after injury to the rat cortex, brain tissue has significantly softened. Softening is mostly restricted to the site of injury, where collagen IV, laminin, GFAP and vimentin are upregulated. At later time points (that is, at three weeks PI), scar tissue elasticity somewhat recovers at the stab injury site but mild tissue softening spreads away from the scar, which strongly correlates with the dispersion of an increased number of vimentin-expressing cells.