Cellular and subcellular factors related to the initiating pathogenesis of DTBIassociated axonal injury

Appreciating that the above described cascades of axonal perturbation leading to impaired transport and disconnection evolved over a relatively long

Fig. 3. In contrast to the phenomenon of resealing shown in Fig. 2, this figure provides evidence of delayed neuronal plasmalemmal opening. Here confocal images of pre-injury infused dextran (A), post-injury-infused dextran (B) and their overlay (C) demonstrate scattered neurons flooding with the post-injury infused dextran alone (arrows), among with double-flooded neurons (arrowheads). Scale bar: 100 mm. (Adapted with permission from Farkas et al. (2006); Copyright 2006 by the Society for Neuroscience).

Fig. 3. In contrast to the phenomenon of resealing shown in Fig. 2, this figure provides evidence of delayed neuronal plasmalemmal opening. Here confocal images of pre-injury infused dextran (A), post-injury-infused dextran (B) and their overlay (C) demonstrate scattered neurons flooding with the post-injury infused dextran alone (arrows), among with double-flooded neurons (arrowheads). Scale bar: 100 mm. (Adapted with permission from Farkas et al. (2006); Copyright 2006 by the Society for Neuroscience).

post-traumatic course, emphasis has been placed upon identifying the initiating intraaxonal cellular and subcellular factors both to understand better the pathobiology of this axonal injury and to develop therapies targeting these cellular and sub-cellular changes. While immediate physical transsection of the axon cylinder has been ruled out, the potential for focal disturbances in the axo-lemma leading to local ionic dysregulation was evaluated in the experimental setting using extracellular tracers normally excluded by the intact/unaltered axolemma. Through this approach, our laboratory showed that discreet axonal foci scattered throughout the injured brain revealed evidence of altered focal axolemmal permeability to various normally excluded extracellular tracers (Pettus and Povlishock, 1996; Povlishock and Pet-tus, 1996). Moving on the premise that this alteration in axolemmal permeability would be accompanied by local calcium dysregulation, we probed the same axonal segments with antibodies targeting calcium-activated, CMSP. In these studies, CMSP was observed initially in the subaxo-lemmal domain followed by its activation in the axon's core, with a particular predilection for the mitochondria that appeared swollen with disrupted cristae (Buki et al., 1999). Because such mi-tochondrial damage appeared consistent with local calcium overloading and caspase activation, our laboratory probed these same segments with antibodies targeting cytochrome C and caspase-mediated spectrin proteolysis (Buki et al., 2000). Via this approach, we demonstrated that the damaged mitochondria released cytochrome C that, in turn, activated caspase-mediated spectrin degradation (Buki et al., 2000; Buki and Povlishock, 2006). Collectively, both cysteine proteases, calpain and caspase were recognized to participate in the degradation of the axonal cytoskeleton associated with concomitant neurofilament side-arm cleavage, neurofilament compaction and microtubular loss (Buki and Povlishock, 2006). Because of these local cytoskeletal abnormalities, these compacted axonal segments could also be routinely identified by use of antibodies targeting altered neurofilament sub-units (RMO14) that, in our hands, provided a surrogate marker for identifying such sites of injury (Povlishock et al., 1997). Based on these findings in our lab, as well as others, it was assumed that these intraaxonal changes universally led to an upstream impairment of axonal transport leading to swelling and disconnection (Maxwell et al., 1997). However, continued investigation failed to establish a routine correlation between these two axonal events, leading us to question whether neurofilament compaction and axonal swelling, consistently occurred within the same axonal segment. More recent studies, using multiple strategies to qualitatively and

Fig. 4. This figure provides a semiquantitative, graphic representation of those different forms and prognosis of membrane perturbation following DTBI. Here the distribution of neurons with resealed (gray column), enduring (white column) or delayed membrane perturbation (black column) are shown after DTBI-induced membrane perturbation. As membranes remain closed, open or reseal in response to injury, tracer availability determines the type of membrane perturbation assigned categorically to each neuron. The pre-injury administration of Alexa Fluor 488 and post-injury administration of Texas Red conjugated dextrans permit the evaluation of neuronal membrane perturbation distribution at 4 and 8 h after injury. Across time points, the proportion of neurons with delayed membrane perturbation is significantly different from the proportion of resealed neurons and the proportion of enduring membrane perturbation (%2, p<0.016). The change in the proportion of neurons with re-sealed and enduring membrane perturbation over time was not significant (%2, p = 0.19). The redistribution of the types of membrane perturbation between 4 and 8 h post-injury may result from resealed neurons reopening (gray arrow) to become neurons with enduring damage and/or additional neurons (black arrow) suffering delayed membrane perturbation.

quantitatively assess axonal numbers as well as the spatial relationship of axons showing neurofilament compaction and impaired transport have now convincingly shown that the above described sites of altered axonal permeability, cysteine protease activation and cytoskeletal collapse do not routinely correlate with sites of impaired axonal transport (Stone et al., 2004; Marmarou et al., 2005). This suggested that the neurofilament-compacted axonal segments and swollen axonal segments demonstrating impaired axonal transport most likely represent two different populations of injured axons responding to the traumatic episode in different fashions. For those axonal segments showing focally altered axolemmal permeability it appears, as noted previously, that local calcium dysregula-tion with the activation of the cysteine proteases, causes local degradation of the axonal cytoskele-ton, leading to axonal failure and disconnection (Stone et al., 2004; Marmarou et al., 2005). Why these sites of axonal injury do not reveal impaired axonal transport and axonal swelling is unclear, yet, it is conceivable that the suprathreshold calcium uptake occurring at these sites most likely converts anterograde to retrograde transport, thereby precluding the development of reactive axonal swelling (Sahenk and Lasek, 1988; Martz et al., 1989). The validity of the above is supported by the use of various therapeutic strategies targeting calpain inhibition that, as such, significantly reduce the numbers of axonal profiles showing the above described cysteine protease activation and cytoskeletal collapse (Buki et al., 2003; Buki and Povlishock, 2006). In contrast, it appears that those axons showing impaired axonal transport and local swelling do not sustain any alteration in local axolemmal permeability or any activation of the cysteine proteases (Povlishock and Stone, 2001). Rather, it is posited that other mechanisms are at work and that these are linked to more subtle forms of calcium dysregulation. These potentially involve the activation of micromolar calpains to trigger the activation of calcineurin that, in turn, alters the microtubular network to disrupt local axonal transport kinetics and thereby elicit the swellings described above (Povlishock and Stone, 2001). Although limited direct evidence exists to support this pathway, the use of calcineurin antagonists, such as FK506, directly attenuate the numbers of axons showing impaired axonal transport and swelling while having no effect upon those axons showing neurofilament compaction and disconnection (Marmarou and Povlishock, 2006). This supports the premise that calcineurin is integral to the pathogenesis of impaired axonal transport and swelling. Collectively, these studies illustrate the complexity of the pathogenesis of diffuse axonal injury, suggesting at least two differing types of initiating mechanisms, with the caveat that both populations of injured axons will not likely be amenable to one form of therapeutic intervention.

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