Media was replaced every 48 hours until experiments were executed

Media was replaced every 48 hours until experiments were executed. adhesion on the neurite. Both systems revealed variations in the rate and nature of neuronal injury as a function of focal adhesion density and direct integrin stimulation without membrane poration. Pharmacological Exemestane inhibition of calpains did not mitigate the injury yet the inhibition of Rho-kinase immediately after injury reduced axonal injury. These data suggest that integrin-mediated activation of Rho may be a contributor to the diffuse axonal injury reported in mild Traumatic Brain Injury. Introduction Blast-induced mild Traumatic Brain Injury (mTBI) is the most frequent wound of the conflicts in Afghanistan and Iraq [1]. Approximately 60% of total combat casualties are associated with blast events generated by improvised explosive devices, and recent studies suggest that nearly 16% of US combatants have been diagnosed with mTBI [2]. Although how blast energy is transmitted to the brain is not well understood, studies and clinical reports have shown that exposure to blast can cause mTBI [2], [3], [4]. Interestingly, the neuronal injury observed in these studies resembles diffuse axonal injury (DAI), a common pathology observed following mTBI models of TBI may not fully recapitulate the complexity of the brain, but they provide unique insight into its cellular pathology. Previous models of mTBI have proposed that a disruption in ion homeostasis initiates a sequence of secondary events ultimately leading to neuronal death, however, membrane poration can only account for a portion of injured neurons [9], [10], and excitotoxicity due to changes in ion channel homeostasis [11] cannot account for observations of axonal retraction. We hypothesized that mechanical perturbation of integrins in the neuronal membrane may represent an injury pathway that would account for DAI in mTBI. Integrins are transmembrane proteins that couple the cytoskeleton in the intracellular space to SAT1 the matrix network in the extracellular space, providing mechanical continuity across the membrane [12]. Mechanical forces propagating through these coupled networks can activate signal transduction pathways, alter ion channel currents, and initiate pathological cascades [13], [14]. In the brain, integrin signaling is implicated in development and memory potentiation [15], [16], [17], [18], [19], [20], however, there are no reports on the role of integrin signaling in mTBI. To test our hypothesis, we built a high velocity tissue stretcher to deliver an abrupt mechanical perturbation to cultured neonatal rat cortical neurons. These experiments demonstrated that neuronal injury is a function of focal adhesion size and density. Using magnetic tweezers and coated paramagnetic beads bound to neurons, we measured the difference in the failure strengths of focal adhesions in the soma versus neurites, and found the latter to have significantly weaker attachments to the substrate. Using the magnetic tweezers, we applied an abrupt force to these neurons and found that with fibronectin (FN)-coated beads neurite focal swelling, including abrupt mechanical failure in neurites, occurred 100s of microns away from the soma, suggesting that injury forces may propagate through the neuronal cytoskeleton. Conversely, poly-L-lysine (PLL)-coated beads attached to neurites induced only a local injury. Membrane poration was only observed at extreme strains in a subset of experiments, whereas at lower strains, integrin-induced focal swelling was observed without membrane poration. The injury was not mitigated with the use of a calpain inhibitor, suggesting a calpain-independent injury mechanism. Treatment with a Rho-kinase Exemestane inhibiter decreased neuronal injury, suggesting a role for downstream integrin-mediated cascade events in neuronal injury. Results High Speed Stretch Induces Strain-Dependent Neuronal Injury The spatio-temporal profile of the mechanical perturbation, Exemestane such as a blast wave, in the brain is likely variable Exemestane and, given the timescale of blast wave propagation, quite rapid. In order to mimic this sudden mechanical stimulus, we designed and built a high speed stretcher (HSS) system to deliver an abrupt strain to a population of neurons cultured on a flexible silicon elastomer substrate coated with PLL (Fig. 1A), similar to previous stretch models [21]. We seeded primary neonatal rat cortical neurons on stretchable membranes five days before experiments to allow dendritic Exemestane and axonal extension. During experiments, the substrates underwent an abrupt, uniaxial stretch (at 1% per ms) to generate.