Blast-induced phenotypic switching in cerebral vasospasm

Abstract

Vasospasm of the cerebrovasculature is a common manifestation of blast-induced traumatic brain injury (bTBI) reported among combat casualties in the conflicts in Afghanistan and Iraq. Cerebral vasospasm occurs more frequently, and with earlier onset, in bTBI patients than in patients with other TBI injury modes, such as blunt force trauma. Though vasospasm is usually associated with the presence of subarachnoid hemorrhage (SAH), SAH is not required for vasospasm in bTBI, which suggests that the unique mechanics of blast injury could potentiate vasospasm onset, accounting for the increased incidence. Here, using theoretical and in vitro models, we show that a single rapid mechanical insult can induce vascular hypercontractility and remodeling, indicative of vasospasm initiation. We employed high-velocity stretching of engineered arterial lamellae to simulate the mechanical forces of a blast pulse on the vasculature. An hour after a simulated blast, injured tissues displayed altered intracellular calcium dynamics leading to hypersensitivity to contractile stimulus with endothelin-1. One day after simulated blast, tissues exhibited blast force dependent prolonged hypercontraction and vascular smooth muscle phenotype switching, indicative of remodeling. These results suggest that an acute, blast-like injury is sufficient to induce a hypercontraction-induced genetic switch that potentiates vascular remodeling, and cerebral vasospasm, in bTBI patients.

Fig. 1.

In vitro model for bTBI in the vasculature. (A) Experimental model represents a single arterial lamella, isolated from surrounding tissue. (B) Phase contrast image of engineered lamella composed of micropatterned VSMCs. (Scale bar,100 μm). (C) Lamellar tissue is engineered on an elastic membrane, which is acutely stretched to mimic a blast pulse. (D) Lagrange strain during 5% and 10% simulated blast. Solid lines: axial strain. Dashed lines: transverse strain. (E) Simulated blast does not result in acute or delayed structural reorganization. Red: actin, Blue: nuclei. (F) Example single-cell cytosolic Ca²⁺ trace showing two transients, pre-ET-1 spontaneous transient and prolonged ET-1 induced transient. (G) Mean ET-1 induced temporal transients for blasted tissues. Black: control, Gray: 5% strain, Red: 10% strain. Inset: transient peaks (H) Single-cell ET-1 induced transient peak values. Mean + /- standard deviation. (I) Mean pre-ET-1 spontaneous transients.

Fig. 2.

Simulated blast induces dysfunctional contractility, as measured with vMTFs. (A) Immediately prior to contraction experiment, vMTF is released from the membrane. (B) Stress is calculated from the radius of curvature (). (C-D) vMTFs were serially stimulated with ET-1 and HA-1077. (C) Single vMTF during experimental protocol. (). (D) Temporal change in vMTF stress due to serial stimulation, 1 h after simulated blast. (E–F) Normalized ET-1 induced contraction and basal tone 1 h after simulated blast. (G–H) Normalized induced contraction and basal tone 1 h after simulated blast for tissues pretreated with ROCK inhibitor. (I–J) Normalized induced contraction and basal tone 24 h after simulated blast. (K–L) Normalized induced contraction and basal tone 24 h after simulated blast for tissues treated ROCK inhibitor immediately following the blast. All graphs: mean + /-SEM.

Fig. 3.

Theoretical model for stress-induced remodeling and phenotype switching. (A) Blast injury induces increased hypercontractility, characterized by greater stress-free shortening (λ_a). (See Methods for details). Left box: contour plot of temporal evolution of contractile shortening. The y-axis represents tissues with increasing blast injury. The x-axis represents the time after the blast. Right box: Temporal plots of stress-free shortening for the tissues indicated by the white lines in the left box. (B–E) Contour plots of the temporal evolution of tissue tension, induced contraction, basal tone, and phenotype population for varying magnitudes of simulated blast. (B) Temporal stress evolution of remodeling tissue, (C) model predicted contractility, and (D) model predicted basal contractile tone for varying blast-induced hypercontraction. (E) Predicted temporal change in fraction of contractile cells for varying blast-induced hypercontraction and tissue remodeling. (F–H) Time point snapshots of (F) induced contraction, (G) basal tone, and (H) fraction contractile cells at an early time in vasospasm development, indicated by the vertical line in (B), for mild injury, indicated by the lower horizontal line in (B), or a more severe injury, indicated by the upper horizontal line in (B). These graphs correspond with the 24 h contractility experiments in Fig. 3 F and G.

Fig. 4.

Simulated blast induces VSMC phenotype switching. (A) Representative Western blot of contractile phenotype markers, smooth muscle myosin heavy chain and smoothelin, 24 h after simulated blast. (B) Quantified protein expression of contractile markers (mean + /-SEM). (C) Quantified mRNA expression of SM-myosin heavy chain (MYH11) and smoothelin (SMTN) (mean + /-SEM). (D) Representative Western blot of contractile markers for tissues treated with ROCK inhibitor immediately following simulated blast.