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...
Although axonal injury is a common feature of brain trauma, little is known of the immediate morphological responses of individual axons to mechanical injury. Here, we developed an in vitro model system that selectively stretches axons bridging two populations of human neurons derived from the cell line N-Tera2. We found that these axons demonstrated a remarkably high tolerance to dynamic stretch injury, with no primary axotomy at strains Ͻ65%. In addition, the axolemma remained impermeable to small molecules after injury unless axotomy had occurred. We also found that injured axons exhibited the behavior of "delayed elasticity" after injury, going from a straight orientation before injury to developing an undulating course as an immediate response to injury, yet gradually recovering their original orientation. Surprisingly, some portions of the axons were found to be up to 60% longer immediately after injury. Subsequent to returning to their original length, injured axons developed swellings of appearance remarkably similar to that found in brain-injured humans. These findings may offer insight into mechanical-loading conditions leading to traumatic axonal injury and into potential mechanisms of axon reassembly after brain trauma.
Traumatic axonal injury (TAI) is one of the most important pathologies associated with closed head injury, and contributes to ensuing morbidity. The authors evaluated the potential role of calpains in TAI using a new model of optic nerve stretch injury in mice. Male C57BL/6 mice were anesthetized, surgically prepared, and subjected to a 2.0-mm optic nerve stretch injury (n = 34) or sham injury (n = 18). At various intervals up to 2 weeks after injury, optic nerves were examined for neurofilament proteins and calpain-mediated spectrin breakdown products using immunohistochemistry. In addition, fluorescent tracer was injected into the superior colliculi of mice 1 day before they were killed, to investigate the integrity of retrograde axonal transport to the retina. Optic nerve stretch injury resulted in persistent disruption of retrograde axonal transport by day 1, progressive accumulation and dephosphorylation of neurofilament protein in swollen and disconnected axons, and subsequent loss of neurofilament protein in degenerating axons at day 14. Calpains were transiently activated in intact axons in the first minutes to hours after stretch injury. A second stage of calpain-mediated proteolysis was observed at 4 days in axonal swellings, bulbs, and fragments. These data suggest that early calpain activation may contribute to progressive intraaxonal structural damage, whereas delayed calpain activation may be associated with axonal degeneration.
Recent measurements of the material properties of brain tissue allow an examination of the underlying microstructural basis in both physiological and pathophysiological conditions. The purpose of this study is to develop a mathematical relationship between microstructurally based models of the central nervous system (CNS) white matter and equivalent hyperelastic material models. For simplicity, time dependent material behavior is not included in this formulation. The microstructural representation is used to formulate structural property relationships for highly oriented white matter, and is mathematically compared to one isotropic and two anisotropic hyperelastic formulations. For the anisotropic characterizations, the population of axons in the white matter is assumed to align along one preferred direction of the material, yielding a transversely isotropic formulation. Relatively simple strain-energy functions incorporating material anisotropy provide sufficient flexibility to model the nonlinear behavior predicted from structurally based models, although the tangential stiffness of the hyperelastic approaches does not follow completely the behavior of the structurally based formulations. This analysis is an initial step towards linking microstructural aspects of the tissue to material models commonly used for large deformations, and may be an important step in relating predicted tissue deformation to the deformation and stress of cellular and subcellular structures.
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