Traumatic axonal injury in the perisomatic domain triggers ultrarapid secondary axotomy and Wallerian degeneration

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Abstract

Traumatic axonal injury (TAI) arising from diffuse brain injury (DBI) results in focally impaired axonal transport with progressive swelling and delayed disconnection over several hours within brainstem axons. Neocortical DBI-mediated perisomatic axotomy does not result in neuronal death, suggesting that a comparably delayed axotomy progression was responsible for this unanticipated response. To evaluate delayed perisomatic axotomy, the current study was initiated. Rats received intracerebroventricular 10-kDa dextran followed by moderate midline/central fluid percussion injury (FPI) or FPI alone. At 15, 30, 60, and 180 min post-injury, light and transmission electron microscopy identified impaired axonal transport via antibodies targeting amyloid precursor protein (APP), while double-label fluorescent microscopy explored concomitant focal axolemmal alterations via dextran-APP co-localization. At 15 min post-injury, perisomatic TAI was identified with LM within dorsolateral and ventral posterior thalamic nuclei. Using TEM, many sustaining somata and related proximal/distal axonal segments revealed normal ultrastructural detail that was continuous with focal axonal swellings characterized by cytoskeletal and organelle pathology. In other cases, axotomy was confirmed by loss of axonal continuity distal to the swelling. By 30 min post-injury, perisomatic axotomy predominated. By 60–180 min, somatic, proximal axonal segment, and swelling ultrastructure were comparable to earlier time points although swelling diameter increased. Distal axonal segment ultrastructure now revealed the initial stages of Wallerian degeneration. The site of perisomatic axotomy did not internalize dextran, suggesting that its pathogenesis occurred independent of altered axolemmal permeability. Collectively, this DBI-mediated ultrarapid perisomatic axotomy and its sequelae further illustrate the varied axonal responses to trauma.

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.

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