Accumulation of axonal damages from repeated stretches

Introduction: : Traumatic axonal injury (TAI) is the hallmark of traumatic brain injury (TBI), including mild TBI (mTBI). There is evidence that repeated head impacts, even at relatively low magnitudes, could accumulate to lead to injury. However, the biomechanical mechanism behind such accumulated injury remains elusive, as most studies are empirical without any mechanistic insight. At the microscale, studies on axonal injury have focused on the initial injury triggering mechanisms from a single insult, such as microtubule (MT) rupture, localized strain concentration around the node of Ranvier, and failure of tau proteins and neurofilaments (NFs). However, no study exists that investigates how repetitive strains accumulate to produce axonal cytoskeletal damage across multiple insults. In this study, we investigate the biomechanical effect on cytoskeletal damage resulting from a sequence of multiple rapid stretches. A validated male axonal injury model is used for this task, by subjecting it to repetitive stretches with peak strain magnitudes following a combination of different values to emulate real-world repetitive impacts in representative scenarios. We report the accumulation of failure of tau proteins and NFs as well as peak strains in MTs. This study offers valuable insight into the accumulation of TAI from repeated axonal stretches at the microscale, which has important implications to the mechanisms behind accumulated TBI at the macroscale.

Materials and

Methods: : A male axon injury model was employed. The model development, validation, and extensive parametric sensitivity studies were described previously (Zhang et al., 2023). The model incorporates key axonal substructures including 13 microtubules (MTs) based on the average number of MTs measured from in vitro human corpus callosum axon culture, tau proteins, neurofilaments (NFs), axolemma, and myelin. For MTs, tau and NFs, a 50%, 100% and 100% strain threshold was assigned, respectively, meaning that their corresponding finite elements are removed from the model when they exceed the assigned threshold during simulation. A representative white matter fiber strain profile of 30 ms in duration (Zhang et al., 2024) was parametrically scaled to generate three peak strain magnitudes of 5%, 10% and 30%, referred to as “low”, “mild”, and “high” insult. All strain profiles included a full recovery phase to return to the initial zero strain state, important to reproduce MT undulation behavior found in rapid stretch experiment. We systematically designed five loading protocols by combining the strain profiles in various sequences to emulate real-world scenarios (Table 1). Each protocol was composed of up to five stretch cycles in succession. Cycle 1 was first applied to the intact axonal model. If any component failed, the corresponding elements were removed. Then, the next cycle was applied using the undeformed model but without reinstating the removed elements. The process was repeated to apply all loading cycles. At the end of each cycle, we tracked peak MT strains and the failure of tau and NF elements.

Results, Conclusions, and Discussions:: Results
No structural damage was observed under repeated low stretches (Protocol #1). Results for the remaining four protocols are shown in Figure 1. Tau protein failure showed limited accumulation under mild stretches (0.75% to ~2.24%). However, a high stretch sharply increased tau failure, reaching ~31.6%. When four mild stretches were followed by a high stretch, tau failure accumulated to 35.2%, and the reverse sequence resulted in 34.7%. Two consecutive high stretches yielded the highest tau failure of 52.7%.
NF failures remained much lower than tau failures ( < 2%) but showed accumulation. Even mild stretches caused increases (0.05% to ~0.3%). Mild stretches followed by a high stretch led to slightly lower NF failure than the reverse (0.66% vs. 0.87%). Two high stretches yielded the highest NF failure at 1.88%.
Under repeated mild stretches, peak MPS in MTs dropped after the first cycle (24.2% to 12.1%), then rose slightly (13.2%) before sharply increasing after a high stretch (37.2%). With an initial high stretch, peak strain was 35.1%, then dropped with mild stretches (~16.4%). Two high stretches caused the highest MT MPS (45.0%), near the 50% threshold. MT undulation comparisons are shown in (Figure 1de).
Discussion
Contact sport players sustain repeated head impacts of various severities. We used a male axonal injury model to emulate multiple scenarios. Repeated mild stretches led to increased cytoskeletal damage, but low stretches did not. This suggests a threshold exists beyond which injury accumulation starts to initiate. Repeated mild stretches increased failures of tau proteins and NFs but lowered MT strains, likely due to loss of anchoring structures. Two high stretches greatly increased damages and doubled NF failures, but also raised peak MT strains, showing local strain redistribution. Some MTs lost support, lowering their strain, while others sustain higher strain to satisfy assigned input. These findings highlight the complex interplays of damage and strain among axonal cytoskeletal components. A limitation of the study was that no “healing” was considered to reinstate damaged axonal elements.
Conclusions
Incorporating cytoskeletal failure mechanisms allows the investigation of axonal substructural damage accumulation at the microscale, which may inform understanding of macroscopic injury development.

Acknowledgements and/or References (Optional)::

References:
Zhang C., Ji S., Sex differences in axonal dynamic responses under realistic tension using finite element models, J Neurotrauma 40 (2023) 2217–2232. https://doi.org/10.1089/NEU.2022.0512.
Zhang C., Ji S., Micromechanics of axonal injury in rapid tension and compression, Stapp Car Crash J 68 (2024). https://stapp.org/wp-content/uploads/SC24-02.pdf.