Elsevier

Brain Research

Volume 784, Issues 1–2, 16 February 1998, Pages 1-6
Brain Research

Research report
Alteration of the neurofilament sidearm and its relation to neurofilament compaction occurring with traumatic axonal injury

https://doi.org/10.1016/S0006-8993(97)01075-5Get rights and content

Abstract

Traumatic injury evokes two characteristic forms of focal axonal injury, one of which involves focal perturbation of axolemmal permeability associated with rapid compaction of the underlying axonal neurofilament lattice and microtubular loss. In this process, the neurofilament sidearms have been the subject of intense scrutiny in relation to their role in this NF compaction, with the suggestion that the sidearms, thought to maintain interfilament distance, are proteolytically cleaved and degraded at the time of injury. The current communication addresses the fate of the NF sidearms in such injured axons. Adult cats were subjected to moderate/severe fluid percussion brain injury after intrathecal administration of horseradish peroxidase (HRP). This tracer, excluded by the intact axolemma of uninjured axons, was used to recognize injured axons via HRP intra-axonal uptake/flooding with HRP. Animals were perfused and processed for light microscopic and electron microscopic study of both HRP-containing and non-HRP-containing axons from the same field. HRP-containing axons consistently displayed evidence of traumatically-induced (NF) cytoskeletal collapse. Electron micrographs of HRP-containing axons as well as uninjured, non-HRP-containing axons from the same fields were videographically captured, digitized, enlarged and analysed for NF sidearm length and NF density. HRP-containing axons were found to have increased NF density. Surprisingly, this increased NF density occurred despite the retention of the NF sidearms, which now, however, were reduced in height in comparison to the non-HRP-containing uninjured axons. These observations are not consistent with previously published reports suggesting that overt proteolytic degradation of sidearms was responsible for NF compaction. Based on our findings, we suggest that the NF compaction associated with traumatically-induced axolemmal permeability changes may have its genesis in more subtle sidearm modification, perhaps involving a change in phosphorylation state.

Introduction

In a recent communication, we have evaluated those intra-axonal events that lead to the genesis of delayed axonal swelling and disconnection associated with traumatically-induced injury 11, 12, 14. In these studies we demonstrated that the traumatic episode evokes two distinct sets of focal intra-axonal cytoskeletal change in the first minutes post injury; one involving rapid focal misalignment of the cytoskeleton, the other involving a focal compaction of the neurofilaments (NF) and concomitant microtubular (MT) loss. In the case of cytoskeletal misalignment, these changes occurred independent of any overt alterations of axolemmal permeability and the pathobiology appeared restricted to the intra-axonal domain. However, in those axons exhibiting focal NF compaction and MT loss, concomitant changes in focal axolemmal permeability occurred, suggesting that an alteration of the axoplasmic milieu via the influx of extracellular agents precipitated proteolytic cleavage of the NF sidearms.

In this scenario, we posited the loss of NF sidearms and their stearic hindrance allowed for the collapse of NF and their rapid compaction [12]. Although this scenario appeared credible and provided insight into potential mediators of the observed cytoskeletal collapse, it is noteworthy that no direct studies were performed on the NF sidearms in relation to this process of NF compaction. This shortcoming was related, in part, to the fact that such studies are labor-intensive and, moreover, rely on ultra-high magnification EM to confirm the morphology of the sidearm and its specific relation to the adjacent NF.

Appreciating that the issue of NF sidearm change should be addressed to generate a comprehensive understanding of the pathobiology of traumatically-induced axonal change, we have initiated, in the present communication, a detailed study of the NF sidearms in foci of NF compaction as well as other non-injured axonal segments. Using digitized electron micrographs generated from those axons previously evaluated for evidence of traumatically-induced axonal change and NF compaction, we reconstructed the NF sidearms via computer-assisted enlargement and assessed their morphology in relation to the observed compaction. Further quantitative analysis of sidearm height was performed to gain a better impression of their change ongoing in relation to the observed abnormalities. In the following passages we report our findings in relation to these NF sidearm changes.

Section snippets

Animal preparation and injury

The materials used in the current communication relied on electron micrographs harvested from sites of NF compaction gathered as a part of our previous communication focusing on those axolemmal and intra-axonal ultrastructural changes occurring with traumatically-induced axonal injury. To identify such injured axons our previous studies utilized extracellular macromolecules such as horseradish peroxidase (HRP) normally excluded by the intact, uninjured axolemma [11]. The premise of this

Control axons

When these images were videographically captured, digitized, and enlarged to a magnification of approximately 800 000×, fields of non-HRP-containing uninjured axons displayed cytoskeletal detail consistent with that previously described in non-injured axons (Fig. 1b). Long, straight NF (Fig. 1b) with prominent sidearms were readily identified. NF were linear with constant interfilament distances and did not move in and out of the plane of section.

Flooded axons

At a magnification of 800 000×, the NF of

Discussion

The results of the current communication show in a convincing fashion that NF compaction is associated with a change in the overall height of the sidearms but, interestingly, does not involve a complete loss or cleavage of sidearms, as we had proposed previously 11, 12. Previously, we had demonstrated that one characteristic feature of traumatically-induced axonal pathology, associated with altered axolemmal permeability, was NF compaction and MT loss. Our working assumption was that, in this

References (17)

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