CNS cell distribution and axon orientation determine local spinal cord mechanical properties

Abstract

Mechanical signaling plays an important role in cell physiology and pathology. Many cell types, including neurons and glial cells, respond to the mechanical properties of their environment. Yet, for spinal cord tissue, data on tissue stiffness are sparse. To investigate the regional and direction-dependent mechanical properties of spinal cord tissue at a spatial resolution relevant to individual cells, we conducted atomic force microscopy (AFM) indentation and tensile measurements on acutely isolated mouse spinal cord tissue sectioned along the three major anatomical planes, and correlated local mechanical properties with the underlying cellular structures. Stiffness maps revealed that gray matter is significantly stiffer than white matter irrespective of directionality (transverse, coronal, and sagittal planes) and force direction (compression or tension) (K(g) = ∼ 130 P(a) vs. K(w) = ∼ 70 Pa); both matters stiffened with increasing strain. When all data were pooled for each plane, gray matter behaved like an isotropic material under compression; however, subregions of the gray matter were rather heterogeneous and anisotropic. For example, in sagittal sections the dorsal horn was significantly stiffer than the ventral horn. In contrast, white matter behaved transversely isotropic, with the elastic stiffness along the craniocaudal (i.e., longitudinal) axis being lower than perpendicular to it. The stiffness distributions we found under compression strongly correlated with the orientation of axons, the areas of cell nuclei, and cellular in plane proximity. Based on these morphological parameters, we developed a phenomenological model to estimate local mechanical properties of central nervous system (CNS) tissue. Our study may thus ultimately help predicting local tissue stiffness, and hence cell behavior in response to mechanical signaling under physiological and pathological conditions, purely based on histological data.

Figure 1

Anatomy of the spinal cord. The three anatomical planes in a schematic drawing of the spinal cord. Sagittal plane (red), transverse plane (blue), and coronal plane (green) are shown with their corresponding representative images of mouse spinal cord slices. White dashed and white solid lines represent the border between gray and white matter and between the ventral and dorsal horn of the gray matter, respectively. Scale bar: 500 μm. To see this figure in color, go online.

Figure 2

Elasticity and immunohistochemistry maps of the spinal cord. Elasticity maps and their corresponding IHC stainings (myelin: green; cell nuclei: blue) of transverse (A and B), coronal (C and D), and sagittal sections (E and F). White dashed and white solid lines represent the border between gray and white matter and between the ventral and dorsal horn, respectively. K is shown for full indentation; the larger K, the stiffer the tissue. Black squares in the elasticity map indicate missing K values due to unsuccessful measurements. Asterisks represent imaging artifact. Scale bars: 500 μm. To see this figure in color, go online.

Figure 3

Mechanical heterogeneity and anisotropy of the spinal cord. Combined box and jittered scatter plots of K from indentation (A) and E from creep experiments (B) of white and gray matter for the three anatomical directions. Red line, blue box, and black dots represent the median, Q₁-Q₃ percentile, and single data points, respectively. (A) Apparent reduced elastic modulus K for full indentation of five coronal (n_g = 1622, n_w = 902), five sagittal (n_g = 2514, n_w = 623), and five transverse sections (n_g = 1261, n_w = 709). (B) The elastic modulus E of white and gray matter for coronal (two sections, n_g = 20, n_w = 24), sagittal (two sections, n_g = 17, n_w = 15) and transverse sections (three sections, n_g = 22, n_w = 30). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. To see this figure in color, go online.

Figure 4

Mechanical heterogeneity of the spinal cord gray matter. Combined box and jittered scatter plots of K for full indentation of the dorsal horn (n = 402), ventral horn (n = 836), and white matter (n = 709) in the transverse plane (A), of the dorsal horn (n = 1021), ventral horn (n = 1535), and white matter (n = 623) in the sagittal plane (B) and the comparison of the dorsal and ventral horn dependent on anatomical plane (C). Red line, blue box, and black dots represent the median, Q₁-Q₃ percentile, and single data points, respectively. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. To see this figure in color, go online.

Figure 5

Morphology of the spinal cord. (A–C) IHC staining for neurofilaments and cell nuclei for coronal (A), sagittal (B), and transverse planes (C). (A and B) Cranial is top; caudal is bottom. White arrows indicate long axons. (A–C) Predominantly craniocaudally orientated neurofilaments in the dorsal horn and white matter; mixed orientation of the neurofilaments in the ventral horn. Scale bar: 50 μm. (D) Combined box and jitter scatter plot of $P_{nuclei}$ for the dorsal horn, ventral horn, and white matter for the coronal, sagittal, and transverse planes. Red line, red box, blue box, and black dots represent the mean, SE, SD, and single data points, respectively. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. To see this figure in color, go online.

Figure 6

Local spinal cord tissue stiffness depends on cell distribution and axon orientation. (A and B) Semi-independent linear correlations; K reduced by the influence of $\Theta$ and $P_{nuclei}$ versus $P_{nuclei}$ and $\Theta$, respectively. Blue crosses and red lines represent single data points and linear fits. The Pearson’s linear correlation coefficient is 0.97 (A) and 0.79 (B). To see this figure in color, go online.