Figure 1.
High speed stretch model of neuronal cultures indicates a strain dependent injury response identified by focal swelling of the neurites without porating the membrane.
(A) Neurons were cultured on elastomer membranes that were quickly stretched, transferring injurious forces to neurons. (B) Beta-3-Tubulin immunofluorescence imaging showed that prior to stretch, neurons exhibited a highly branched, smooth neurite morphology. After stretch, many neurons developed widespread focal swellings along their neurites (red arrows) (Scale Bar = 20 µm). (C) Quantification of neuronal injury showed an initial significant response between 0% and 10% strains (n≥4). Neuron loss due to stretch also increased with strain magnitude. (D) The percentage of neurons exhibiting signs of membrane poration, as indicated by the uptake of a membrane impermeable dye, following stretch showed an initial significant increase between 25% and 40% strain (n≥3). All bars SEM for all panels, * p<0.05.
Figure 2.
Substrate coating influences neuronal FAC formation and injury progression.
(A) Neurons are mechanically coupled to the substrate via FACs that couple the intracellular cytoskeleton to the ECM. (B) Immunofluorescent imaging of vinculin puncta indicated the presence of FACs. Scale bars correspond to 8 and 10 µm, for PLL and FN respectively. Quantification of (C) total vinculin puntca area (n = 8) (D) indicated that a FN coated substrate induced FAC formation over a larger area and with greater average cluster size compared to a PLL coated substrate (n = 5). (E) The percentage of neurons that exhibited widespread focal swelling following stretch injury was greater on a FN coated substrate compared to a PLL coated substrate at 10 minutes (n≥4 for PLL and n≥8 for FN). All bars SEM for all panels, * p<0.05.
Figure 3.
Role of integrins in adhesion strengthening and injury.
(A) Paramagnetic beads, as shown by SEM, were bound to neurons. (B) The failure strength of neuron/substrate adhesions was measured using either FN-coated (red) or PLL-coated (blue) substrates. The beads were pulled with an ascending ramp in force as indicated by the inset. (C) The speed at which neurons detached from the substrate (Peeling Speed) during the ascending pull was plotted as a function of the applied force for PLL-coated (blue) and FN-coated substrates (red) (n≥4). (D) The maximum force required for complete detachment (Mean Unbinding Force) for soma (dashed) and neurite (plain) was plotted for PLL-coated substrates (blue) and FN-coated substrates (red) (n≥4). (E) Mean unbinding forces for the soma (circles) and neurites (triangles) of cells on PLL or FN coated substrates was plotted as a function of mean vinculin area (n≥4). (F) Magnetic Tweezers were used to deliver a 100 ms pulse (inset) to neurons with either FN (red) or PLL (blue) coated beads. (G) FN-coated beads were used to establish an injury dose response curve. (H) FN-coated beads were able to injure cells more often than PLL-coated beads and the extent of injury (I) depended upon bead coating. (J) FN-coated beads always caused global cellular injury (focal swellings indicated by black arrows extended throughout the cell), while (K) PLL-coated beads tended to injure locally to the bead-pull site (n = 13 for FN-coated beads and n = 12 for PLL-coated beads). Inverted fluorescence images from neurons loaded with Fluo-4 calcium dye. All bars SEM for all panels, * p<0.05.
Figure 4.
Pharmacological inhibition of secondary injury pathways may reduce neuronal injury.
(A) Immediate administration of a Calpain inhibitor MDL 28170 following 10% stretch of neurons seeded on PLL substrates was unable to reduce the percentage of injured neurons 10 minutes later (n≥4). (B) However, immediate application of a ROCK inhibitor, HA-1077, was able to reduce neuronal injury in a dose dependent manner (n≥5). (C) Decreases in injury were observed at both 5% and 10% strain magnitude (n≥5). All bars SEM for all panels, * p<0.05.