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.
Highlights
According to an estimation model based on previous epidemiological studies, traumatic brain injury (TBI) is a major cause of global mortality (Hyder et al, 2007; Rubiano et al, 2015)
In order to ensure that the axons in this study were stretched with deviation as small as possible, the strain fields of Petri dishes under the maximum deformation under different parameters were tested by finite element analysis (FEA) (Figure 1A)
The results showed that the strain field of the Petri dish in the pull direction (x-axis) was terraced, and the maximum strain was obtained in the central region of the Petri dish, which was 0.05, 0.1, 0.2, and 0.5
Summary
According to an estimation model based on previous epidemiological studies, traumatic brain injury (TBI) is a major cause of global mortality (Hyder et al, 2007; Rubiano et al, 2015). In cases of DAI, many axons become overstretched because they have sustained tremendously accelerated motion caused by overwhelming forces during unwanted incidents such as car crashes and sport accidents (Povlishock and Christman, 1995). This inevitably results in axonal degeneration during the phase of secondary axotomy. Since the first report of DAI by Strich (1956), we have made tremendous progress in understanding the mechanisms underlying this condition. Experiments featuring in vivo animal models of DAI involved fixing the heads of animals on a customized rotating device, through which violent rotation was used to cause a subsequent trauma. Previous studies had initially applied these techniques to the skulls of large mammals such as baboons and pigs (Gennarelli et al, 1982; Ross et al, 1994) and were subsequently applied to rats using a modified device (He et al, 2004; Wang et al, 2010; Li et al, 2013). Marmarou et al (1994) established a rat copper-rod strike model, which was relatively easy to construct, and allowed significant progress in the in vivo modeling of DAI
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