Traumatic axonal injury induces calcium influx modulated by tetrodotoxin-sensitive sodium channels

Abstract

Diffuse axonal injury (DAI) is one of the most common and important pathologies resulting from the mechanical deformation of the brain during trauma. It has been hypothesized that calcium influx into axons plays a major role in the pathophysiology of DAI. However, there is little direct evidence to support this hypothesis, and mechanisms of potential calcium entry have not been explored. In the present study, we used an in vitro model of axonal stretch injury to evaluate the extent and modulation of calcium entry after trauma. Using a calcium-sensitive dye, we observed a dramatic increase in intra-axonal calcium levels immediately after injury. Axonal injury in a calcium-free extracellular solution resulted in no change in calcium concentration, suggesting an extracellular source for the increased post-traumatic calcium levels. We also found that the post-traumatic change in intra-axonal calcium was completely abolished by the application of the sodium channel blocker tetrodotoxin or by replacement of sodium with N-methyl-d-glucamine. In addition, application of the voltage-gated calcium channel (VGCC) blocker omega-conotoxin MVIIC attenuated the post-traumatic increase in calcium. Furthermore, blockade of the Na(+)-Ca(2+) exchanger with bepridil modestly reduced the calcium influx after injury. In contrast to previously proposed mechanisms of calcium entry after axonal trauma, we found no evidence of calcium entry through mechanically produced pores (mechanoporation). Rather, our results suggest that traumatic deformation of axons induces abnormal sodium influx through mechanically sensitive Na(+) channels, which subsequently triggers an increase in intra-axonal calcium via the opening of VGCCs and reversal of the Na(+)-Ca(2+) exchanger.

Fig. 1.

Schematic illustration of axonal stretch injury.Top, Cutaway of the injury device reveals the culture well placed in a sealed chamber on a microscope stage. Axons grow between two populations of neurons plated on a flexible substrate.Bottom, A pressure pulse deforms only the region of axons, inducing tensile elongation.

Fig. 2.

Changes in intra-axonal calcium fluorescence immediately after stretch injury in control saline solution (CSS; n = 12 wells, 72 axons) compared with those after injury in calcium-free CSS (0 Ca²⁺; n = 3 wells) or Na⁺-free CSS [N-methyl-d-glucamine replacement (NMDG); n = 3 wells] or with pretreatment with tetrodotoxin (TTX;n = 8 wells), ω-conotoxin MVIIC (CTX; n = 4 wells), or bepridil (BEP; n = 9 wells).F/F₀ = change in calcium fluorescence over initial fluorescence (*p < 0.01; **p < 0.001).

Fig. 3.

A–D, Representative photomicrographs of changes in calcium fluorescence before (left) and after (right) axonal stretch injury in control saline solution (CSS) or with pretreatment with tetrodotoxin (TTX), ω-conotoxin MVIIC (CTX), or bepridil (Bep). E, Demonstration of changes in calcium fluorescence after axonal stretch injury in calcium-free CSS with calcium added back 2 min after injury. F, Demonstration of the persistent increase in calcium fluorescence over 20 min after axonal injury.

Fig. 4.

Temporal change in calcium fluorescence over 20 min after axonal stretch injury (n = 3 wells).F/F₀ = change in calcium fluorescence over initial fluorescence (*p < 0.05).

Fig. 5.

An example tracing (mean of 6 axons) from a Ca²⁺-free experiment in which Ca²⁺ is added back 2 min after injury. No data are taken during removal of the top plate after injury to gain access to the well (t, 20–100 sec) or addition of the Ca²⁺ back to the extracellular solution.F/F₀ = change in calcium fluorescence over initial fluorescence.

Fig. 6.

Proposed mechanisms of calcium entry into stretch-injured axons. 1, Strain on the axonal membrane inducing an abnormal influx of Na⁺ through mechanosensitive sodium channels. 2, In response, reversal of axonal Na⁺–Ca²⁺exchangers (a) and activation of voltage-gated calcium channels (b), collectively contributing to a pathological influx of Ca⁺² into the axons.