Traumatic axonal injury in the perisomatic domain triggers ultrarapid secondary axotomy and Wallerian degeneration
Introduction
Traumatic brain injury (TBI) remains a leading cause of death and disability, especially in children and young adults (Kraus et al., 1996, Langlois et al., 2004, Sosin et al., 1995). Within this context, diffuse brain injury (DBI) occurs via rapid cranial acceleration–deceleration with or without impact and generates little overt pathology in comparison to focal lesions (Adams, 1992, Hardman and Manoukian, 2002, Meythaler et al., 2001). A significant component of DBI is diffuse axonal injury (DAI) which contributes to the associated morbidity and mortality (Christman et al., 1994, Graham et al., 2002). Traumatic axonal injury (TAI), the experimental counterpart of DAI, is characterized microscopically by focal impairment of axonal transport leading to progressive axonal swelling and disconnection over several hours post-injury (secondary axotomy) due to cytoskeletal misalignment and collapse via neurofilament compaction, microtubule loss, and disruption of the subaxolemmal spectrin network (Okonkwo et al., 1998, Pettus and Povlishock, 1996, Povlishock, 1992, Povlishock and Pettus, 1996). Proposed mechanisms of moderate to severe TAI pathogenesis include traumatically induced transient perturbation of the axonal membrane allowing for massive calcium influx which stimulates pathological cascades targeting the axonal cytoskeleton for degradation (Buki et al., 1999, Buki et al., 2000, Povlishock and Pettus, 1996).
Although considerable information has been generated concerning TAI and its pathogenesis, particularly in long tract axons of the brainstem, it has been difficult to evaluate the consequences of TAI in terms of the related neuronal somatic fate that also must contribute to any ensuing traumatically induced morbidity. To better explore this issue, our laboratory recently utilized a midline/central fluid percussion injury (cFPI) model to study the neuronal somatic response to DBI-mediated axotomy that occurred in the perisomatic domain (Singleton et al., 2002). Using this model, perisomatic axotomized fibers were localized to within 40–60 μm of the sustaining somata and were found within distinct anatomical loci, namely the neocortex, hippocampus, and thalamus. Contrary to expectations based on the existing literature of experimental transection-induced primary axotomy (Barron, 1983, Kreutzberg, 1995), perisomatic traumatic axotomy did not result in acute neuronal cell death. Rather the related somata revealed impaired protein synthesis followed by neuronal cell reorganization and repair (Singleton et al., 2002). Based on these unanticipated, non-lethal neuronal responses, we questioned whether the structural and related subcellular changes associated with perisomatic TAI significantly differed from those TAI changes previously described within brainstem fiber tracts. To this end, we followed the course of this perisomatic axotomy, focusing on its occurrence in discrete nuclei within the thalamus.
Recognizing that axotomy within the long tract axons of the brainstem is typically associated with impaired axonal transport and altered axolemmal permeability (Pettus et al., 1994, Stone et al., 2004), we used immunocytochemical light and electron microscopy as well as confocal microscopy with antibodies to β-amyloid precursor protein (APP), a marker of impaired axonal transport, as well as extracellular tracers (fluorescently conjugated 10-kDa dextrans) normally excluded from intact axons to critically evaluate the spatiotemporal and ultrastructural features of perisomatic TAI following moderate cFPI. Routine LM analysis offered insight into the temporal course of perisomatic axonal injury progression, while EM semiserial image reconstruction permitted the characterization of the axonal subcellular responses at the site of injury in addition to those proximal–distal changes ongoing in the axon cylinder. Parallel confocal microscopy was used to explore post-injury alterations of axolemmal permeability and its relation to focal APP accumulation at the axotomy site. Contrary to expectations, these approaches demonstrated that the thalamic perisomatic TAI was associated with an ultrarapid axotomy followed by rapid initiation of Wallerian degeneration. Further, despite the rapidity of this axotomy, this event was not accompanied by overt alterations in axolemmal permeability.
Section snippets
Animal preparation and injury
To follow the pathogenesis of thalamic perisomatic TAI, animals were subjected to moderate cFPI consistent with methods described previously (Dixon et al., 1987, Singleton et al., 2002). Adult male Sprague–Dawley rats (375–400 g) were anesthetized with 4% isoflurane in 70% N2O and 30% O2, intubated, and maintained on a ventilator with 1–2% isoflurane for injury preparation. Intubated animals were placed on a heating pad connected to a thermostat controlled by a rectal probe (Harvard Apparatus,
Sham-injury—general findings
Macroscopically, sham-injured brains showed no evidence of compression, contusion, or tissue loss. Tissue sections from sham-injured animals processed with APP antibody and examined at the LM level demonstrated limited background staining with the finding of only isolated immunoreactive somata. However, there was no evidence of immunoreactive axons or swellings adjacent to these somata (data not shown).
Injury—LM findings
The injured brains shared identical macroscopic features with sham-injured brains with the
Discussion
The results of this communication reveal, for the first time, the pathogenesis of DBI-mediated perisomatic TAI with concomitant secondary axotomy and Wallerian degeneration following moderate cFPI. Using a well-documented antibody marker of impaired axonal transport (Stone et al., 2000), secondary axotomy and disconnection at the site of injury were seen within the thalamus as early as 15 min post-injury, without evidence of axonal tearing or related parenchymal disruption. Wallerian change
Acknowledgments
The authors wish to thank Susan Walker, Lynn Davis, and Thomas Colburn for their excellent technical assistance. This work was supported by NIH/NINDS Grants NS045824, T32NS007288, and 5P30NS047463.
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This work was supported by NIH/NINDS grants NS045824, T32NS007288, and 5P30NS047463.