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 -conotoxin MVIIC attenuated the post-traumatic increase in calcium. Furthermore, blockade of the Na ϩ -Ca 2ϩ 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 ϩ channels, which subsequently triggers an increase in intraaxonal calcium via the opening of VGCCs and reversal of the Na ϩ -Ca 2ϩ exchanger. Key words: axon; injury; calcium; sodium channels; diffuse axonal injury; mechanosensitivity; mechanoporation; brain traumaDamage to axons is thought to be the most common pathology associated with traumatic brain injury (Maxwell et al., 1997;Smith and Meaney, 2000). Widely distributed damage to axons in the brain, termed diffuse axonal injury (DAI), results from the inertial forces exerted on the white matter tracts in the brain during traumatic incidents such as automobile accidents and, in some cases, falls and assaults (Gennarelli et al., 1982;Gennarelli, 1993;Graham et al., 1995). DAI has been postulated as the major cause of coma and death as a result of these types of injury (Gennarelli et al., 1982). Although severe inertial brain injury may induce tissue tears in the white matter resulting in immediate disconnection of axons (primary axotomy), most damaged axons undergo secondary disconnection over an extended time course (secondary axotomy) (Povlishock et al., 1983;Povlishock, 1992). Accordingly, much attention has been placed on mechanisms of this delayed response of axonal trauma to elucidate potential therapeutic strategies.Although it has been suggested that elevated intra-axonal calcium ([Ca 2ϩ ] i ) levels play a pivotal role in the secondary damage to axons after mech...

Mild Traumatic Brain Injury and Diffuse Axonal Injury in Swine

“…On the other hand, axons that undergo dynamic deformation but do not swell may nonetheless suffer subtle yet important pathophysiological changes, including an increase in intra-axonal calcium and sodium concentrations or mitochondrial dysfunction, as suggested by previous in vivo and in vitro studies Pettus and Povlishock, 1996;Pettus et al, 1994;Smith et al, 1999b;Staal et al, 2010;Wolf et al, 2001;Yuen et al, 2009). These processes are thought to contribute to physiological dysfunction of white matter pathways, such as the reduction in conduction velocity that has been observed in mTBI (Baker et al, 2002;Kumar et al, 2009;Nuwer et al, 2005).…”

Physiological and Pathological Responses to Head Rotations in Toddler Piglets

“…Other animal models of traumatic brain injury such as fluid percussion (McIntosh et al, 1987(McIntosh et al, , 1989Vink et al, 1988;Zhang et al, 1999), cortical impact (Elliott et al, 2008;Igarashi et al, 2007;Lighthall, 1988;Saatman et al, 2006) and weight drop Marmarou et al, 1994;Ucar et al, 2006) injuries also support our finding of increasing injury severity with increasing mechanical load. Within a single age group, the observed relationship between higher mechanical load and increasing severity of certain brain injuries may be directly related to functional or structural damage to the tissue (Bain and Meaney, 2000;Bain et al, 2001;Lusardi et al, 2003;Meaney et al, 1995;Smith et al, 1999;Wolf et al, 2001). Although the severity of several different types of brain injuries have been shown to correlate positively with magnitude of mechanical load, we have chosen to focus on axonal injury in our study of 5-day-old and 4-week-old piglets because it is an easily quantifiable injury that is a pathological hallmark of moderateto-severe TBI in children.…”

Relationship between Flow-Metabolism Uncoupling and Evolving Axonal Injury after Experimental Traumatic Brain Injury

“…It might be considered that in the nonstimulated injured brain, secondary injury occurs in a similar manner: by periodic and uncontrolled energy demand that outstrips the local blood supply resulting in energy failure, for example, because of increased neuronal activity commonly observed after injury (Kirino et al, 1985;Carmichael and Chesselet, 2002;Sunami et al, 1989;Strong et al, 2002). Given that white matter is reliant on oxidative metabolism, at least in vitro (Stys, 1998;Waxman et al, 1992), white matter regions where glycolysis is already abnormally high relative to the underlying level of local blood flow would be especially vulnerable to this type of stress, which would hasten the onset of an energy crisis resulting in membrane pump failure followed by the ensuing cascade of reactions, most notably caused by calcium entry (Buki et al, 1999;Wolf et al, 2001) and ultimately leading to disruption of the axonal cytoskeleton. Given the importance of axonal injury to outcome after TBI (Strich, 1956;Graham, 1996), therapies designed to stabilize and improve flow and metabolism in the acute time period after injury may confer some degree of neuroprotection translating to significant improvements in long-term functional outcome.…”