Spinal cord contusion causes acute plasma membrane damage

Abstract

Spinal cord injury (SCI) launches a complex cascade of events that leads to progressive damage and loss of function. Compromise of plasma membrane integrity due to the mechanical impact is an acute event that may contribute to cellular dysfunction. Therefore, the objective of this study was to better understand the extent of acute plasma membrane damage associated with SCI as a function of injury severity and membrane defect size. Fluorescent cell-impermeant dyes were injected into the cerebrospinal fluid of adult male rats prior to contusion injury, and the anatomical location of cell bodies and axons taking up the dye within 10 min following SCI was quantified. Lucifer yellow uptake was assessed as a function of impact force (experimental groups: sham, 100 kdyn, 150 kdyn, and 200 kdyn force). In a separate group of animals, FITC-conjugated dextran molecules of various sizes (3 kDa and 10 kDa with a 1.6-nm and 2.7-nm radius, respectively) were used to approximate the size of membrane defects following moderate injury (150 kdyn force). Quantification revealed that cellular uptake of lucifer yellow was positively correlated with the force of the mechanical impact, indicating that the severity of injury is related to the degree of acute membrane failure. In addition, after moderate injury, cell bodies and axons (located up to 2 mm and 3 mm from the epicenter, respectively) took up significantly more of the 3-kDa and 10-kDa dextran permeability marker compared to sham controls. Permeable neuronal cell bodies exhibited a morphological appearance characterized by pericellular blebbing, suggesting that plasma membrane compromise is associated with pathophysiological cellular alterations. Collectively, these results enhance our understanding of acute SCI and provide targets for developing novel treatment strategies.

FIG. 1.

Permeability marker uptake following SCI. Histological assessment revealed permeability marker uptake in neuronal cell bodies and axons 10 min following SCI. Images shown here represent uptake of FITC-conjugated dextrans within 1 mm caudal of the injury epicenter (3 kDa, B and C; 10 kDa, E and F). Immunohistochemistry specific for NeuN (A and C) and NF-160 (D and F) verified that dye uptake was within neuronal cell bodies and axons, respectively. Uptake was similar for both dextrans used in the study. However, lucifer yellow was only observed within cell bodies and not axonal projections, presumably due to dye leakage or rapid diffusion of the small molecule along the axon (scale bars = 50 μm (A–C) and 100 μm (D–F).

FIG. 2.

Lucifer yellow uptake as a function of injury severity and distance from the epicenter. A dye exclusion assay was used to characterize plasma membrane damage 10 min following sham surgery (A and E) or contusion SCI with an impact force of either 100 kdyn (B and F), 150 kdyn (C and G), or 200 kdyn (D and H). Images shown here were taken from representative sections 2 mm caudal of the epicenter (n = 4–5 per group). A linear regression of the total number of lucifer yellow–positive cells (with baseline sham values subtracted) revealed a positive correlation between the actual impact force imparted to the spinal cord and the number of permeable cells (I, mean ± standard deviation; r = 0.80, p < 0.001). Further analysis of the data revealed differences between the groups at discrete distances from the epicenter (J; median ± range; *p < 0.05 compared to sham, ^†p < 0.05 compared to 100 kdyn; scale bar = 500 μm [A–D], 50 μm [E–H]).

FIG. 3.

FITC-dextran uptake in cell bodies within gray matter. The number of cells permeable to 3-kDa FITC-conjugated dextran (3dex; B) and 10-kDa FITC-conjugated dextran (10dex; C) was quantified within tissue sections taken at discrete distances from the injury epicenter and compared to sham controls (A) (n = 3–4 per group; mean ± SEM). A comparison of the sum total of the permeable cells in each group (D) demonstrated a significant difference between 3dex and 10dex uptake after injury compared to sham injury, and that uptake of 3dex was greater than 10dex uptake after injury (mean ± standard deviation; *p < 0.05 compared to sham, ^†p < 0.05 compared to 10dex). Analysis of the data at discrete distances from the epicenter (E) revealed significantly greater cellular uptake of both 3dex and 10dex compared to sham controls up to 2 mm from the injury site in both the rostral and caudal directions (median ± range; *p < 0.05). Images shown here are confocal reconstructions at 1 mm caudal of the epicenter (scale bar = 40 μm).

FIG. 4.

Axolemmal permeability marker uptake. Axonal uptake of 3dex (B) and 10dex (C) permeability markers after injury was compared to sham controls (A). The density of axons taking up the permeability marker was quantified at discrete distances from the epicenter out to 3 mm in the rostral and caudal directions (D). The density of dextran-positive axons was significantly greater than the sham controls up to 2 mm in both the rostral and caudal directions. No significant differences between 3dex and 10dex uptake were found, demonstrating that axonal membrane defects are at least 2.7 nm in radius (n = 3–4 per group; median ± range; *p < 0.05 compared to sham animals). Images shown here are confocal reconstructions taken from sections 1 mm caudal to the injury epicenter (scale bar = 40 μm).

FIG. 5.

Membrane blebbing is associated with plasma membrane permeability. High-magnification confocal imaging (63 × ) of 3dex-filled cells within histological sections fixed 10 min after SCI revealed that membrane permeability is associated with SCI-induced cellular blebbing. Images shown here are representative phase (A, D, G, and J), fluorescence (B, E, H, and K), and phase overlaid with fluorescence (C, F, I, and L). A subset of cells near the epicenter (A–C) displayed a necrotic appearance (stars), but did not take up the fluorescent dye, whereas other neighboring cells became 3dex-positive. Most permeable cells were comprised of a dense, dextran-filled cytoplasm surrounded by a less dense layer of fluid that did not fill with the fluorescent dextran molecule (arrows in A–I). The majority of dextran-impermeable cells did not exhibit blebbing (G–L). These results indicate that plasma membrane damage is associated with pathophysiological alterations in cellular morphology.