A Precise, Controllable in vitro Model for Diffuse Axonal Injury Through Uniaxial Stretch Injury

Abstract

Regarding the determination of the biomechanical parameters in a reliable in vitro cell model for diffuse axonal injury (DAI), our study aimed to demonstrate connections between those parameters and secondary axotomy through examination of morphological alterations under a variety of traumatic conditions. An in vitro cell model for DAI was established in primary cultured mouse neurons by uniaxial mechanical stretching of non-myelinated axons under various traumatic conditions: strain (ε) = 5, 10, 20, and 50%; strain time (t) = 500, 100, and 20 ms; strain rate ranging between 0.1 and 25 s–1. Axonal real strains (strainaxon) were measured as 4.53 ± 0.27, 9.02 ± 0.91, 17.75 ± 1.65, and 41.8 ± 4.4%. Axonal real strain rates (SRaxon) ranged between 0.096 ± 0.0054 and 20.9 ± 2.2 s–1. Results showed there was no obvious abnormality of axons with a lower strain condition (strainaxon < 17.75 ± 1.65%) during the acute phase within 30 min after injury. In contrast, acute axonal degeneration (AAD) was observed in the axons following injury with a higher strain condition (SRaxon > 17.75 ± 1.65%). In addition, the incidence and degree of AAD were closely correlated with strain rate. Specifically, AAD occurred to all axons that were examined, when ε = 50% (strainaxon = 41.8 ± 4.4%) for 20 ms, while no spontaneous rupture was observed in those axons. Besides, the concentration of Ca2+ within the axonal process was significantly increased under such traumatic conditions. Moreover, the continuity of axon cytoskeleton was interrupted, eventually resulting in neuronal death during subacute stage following injury. In this study, we found that there is a minimum strain threshold for the occurrence of AAD in non-myelinated axons of primary cultured mouse neurons, which ranges between 9.02 ± 0.91 and 17.75 ± 1.65%. Basically, the severity of axonal secondary axotomy post DAI is strain rate dependent under a higher strain above the threshold. Hence, a reliable and reproducible in vitro cell model for DAI was established, when ε = 50% (strainaxon = 41.8 ± 4.4%) for 20 ms.

A model of traumatic brain injury using human iPSC-derived cortical brain organoids

“…22,23 Previous efforts to study TBI in vitro have used stretch and shear forces to either rodent organotypic slices 24 or rudimentary single-or two-cell type cultures. 25,26 While these models are useful for studying axonal injury, they have limited translational relevance with regards to the extracellular matrix, stiffness and cell-cell interactions that substantially influence the biophysical properties of mechanical injury in vivo. [27][28][29] To address these concerns, we adapted a 3-dimensional induced pluripotent stem cell (iPSC)-cortical organoid model 30 coupled with mechanical injury via highintensity focused ultrasound (HIFU) with which we can precisely deliver mild/moderate (e.g.…”

Acute and Delayed Effects of Mechanical Injury on Calcium Homeostasis and Mitochondrial Potential of Primary Neuroglial Cell Culture: Potential Causal Contributions to Post-Traumatic Syndrome

“…There is a limited number of models that allow the assessment of the effect of mechanical trauma on Ca 2+ homeostasis and the viability of primary cortical neurons [ 37 ]. Moreover, there are still no studies that have examined the acute (first seconds, minutes) and delayed (several days after mechanical injury) effects of mechanical trauma on cellular viability and the dynamic of changes in [Ca 2+ ] i and mitochondrial potential (ΔΨm) of neurons.…”

Brain strain rate response: addressing computational ambiguity and experimental data for model validation

“…Many in vivo, ex vivo, and in vitro models of TBI have reported a strain rate dependency of morphological or functional alterations of experimental tissues secondary to mechanical insults with precisely controlled loading parameters [6,24,[60][61][62][63][64][65][66][67][68][69][70]. Several computational studies have also reported strain rate as an appropriate predictor for brain injury [27,29,32,36,71].…”