Identifying the Phenotypes of Diffuse Axonal Injury Following Traumatic Brain Injury

Abstract

Diffuse axonal injury (DAI) is a significant feature of traumatic brain injury (TBI) across all injury severities and is driven by the primary mechanical insult and secondary biochemical injury phases. Axons comprise an outer cell membrane, the axolemma which is anchored to the cytoskeletal network with spectrin tetramers and actin rings. Neurofilaments act as space-filling structural polymers that surround the central core of microtubules, which facilitate axonal transport. TBI has differential effects on these cytoskeletal components, with axons in the same white matter tract showing a range of different cytoskeletal and axolemma alterations with different patterns of temporal evolution. These require different antibodies for detection in post-mortem tissue. Here, a comprehensive discussion of the evolution of axonal injury within different cytoskeletal elements is provided, alongside the most appropriate methods of detection and their temporal profiles. Accumulation of amyloid precursor protein (APP) as a result of disruption of axonal transport due to microtubule failure remains the most sensitive marker of axonal injury, both acutely and chronically. However, a subset of injured axons demonstrate different pathology, which cannot be detected via APP immunoreactivity, including degradation of spectrin and alterations in neurofilaments. Furthermore, recent work has highlighted the node of Ranvier and the axon initial segment as particularly vulnerable sites to axonal injury, with loss of sodium channels persisting beyond the acute phase post-injury in axons without APP pathology. Given the heterogenous response of axons to TBI, further characterization is required in the chronic phase to understand how axonal injury evolves temporally, which may help inform pharmacological interventions.

Keywords: axonal injury; microtubules; neurofilament; spectrin; traumatic brain injury.

Figure 1

Disruption of different cytoskeletal elements can be visualized with different antibody markers. (A) α-spectrin proteolytic breakdown can be observed through antibodies to target sites of the calpain proteolysis site within the C-terminus (Ab39) or N-terminus (Ab38 or SNTF) [62]. (B) Microtubule disruption is typically examined via accumulation of fast axonally transported proteins, with APP as the gold standard. (C) Neurofilament pathology can be examined via a number of different antibodies that target both normal neurofilament proteins and those revealed only after injury. (D) Within myelinated axons, the nodes of Ranvier may be uniquely vulnerable to injury, with loss of NaV1.6 from the nodal region, or Caspr from the paranode with loss of diffusion of the nodal cytoskeletal proteins βIV-spectrin and ankyrin-G into the paranodal space. This can be visualized as heminodes or void nodes depending on the cytoskeletal pathology. Note RMO* refers to RMO-14, -44, -48, and -194. Created with Biorender.com.

Figure 2

Temporal profile of cytoskeletal damage markers. Lines indicate the detection period for markers based on the current literature, as well as the anticipated peaks in pathology. APP pathology peaks around 24 h with a small amount of pathology present chronically. Calpain-mediated cleavage of α-spectrin peaks earlier at 6 h and then diminishes over time, although chronic pathology has not been examined. Neurofilament pathology, detected by RMO antibodies, peaks earlier than markers of the intact neurofilament proteins, with NF-L pathology evident earlier than NFM and NF-H with evidence that, at least in more severe TBI, neurofilament pathology may persist chronically. Nodal pathology develops early and appears to persist chronically, at least to six weeks post-injury, the latest timepoint examined. Question marks denote pathologies that have not been assessed chronically. Created with Biorender.com.