Blast-induced shockwave model of traumatic brain injury in organotypic brain slices

Introduction: : Blast-induced traumatic brain injury (TBI) has become increasingly prevalent, especially among military personnel exposed to explosive forces during training and on the battlefield. Blast induced shockwave (BIS) injuries trigger a complex cascade of cellular disruptions, including cellular activation, neuroinflammation, oxidative stress, and diffuse axonal injury (DAI), all of which are challenging to mimic comprehensively in existing model systems. Research into BIS injuries faces significant challenges, including the ability to create reproducible blast exposures. In animal models, the interaction of the blast wave with the skull creates significant heterogeneity, introducing challenges in the molecular analysis of tissue. Cell culture models do not recapitulate important features of the brain, including multiple cell types and structural heterogeneity of the brain. Organotypic brain slices provide an ex vivo culture system that preserves 3D-tissue architecture, cellular diversity, and essential cell-cell interactions that may be affected by blast injury but remove the complication of head orientation and interactions of the shockwave with the skull with in vivo experimentation. This work aimed to establish a novel, reproducible model of BIS-induced TBI using organotypic brain slices. To do so, we used an exploding bridgewire shockwave (EBWS) system to generate a physiologically relevant injury model. In EBWS, high electric current is passed over a wire and is vaporized in a matter of microseconds generating a propagating shockwave. Using microscopy, cell viability analysis, RT-qPCR and immunohistochemistry, we demonstrated that this system successfully replicated key in vivo cellular and molecular responses.

Materials and

Methods: : 300 µm organotypic brain slices were collected by dissecting an excised mouse brain with a Stoelting device (Fig. 1A). The slices were then maintained in a culture insert (Millicell) at 37°C and 5% CO2 for 24 hours. Slices were then transferred into a 35 mm culture dish and wrapped with an oxygen permeable membrane (BreathEasyTM). An EBWS was then used to deliver controlled, reproducible blast injuries at defined pressures to these slices by adjusting the platform height (Fig. 1B). Blast pressure profiles were measured by pressure transducers along the pressure pathway with a maximum of 70 kPa or 10 psi (Fig. 1C). Cytotoxicity assays, including lactate dehydrogenase (LDH) release, MTS assay, propidium iodide (PI), and TUNEL staining, assessed slice viability pre- and post-injury. Time-course analyses of cellular activation, neuroinflammation, oxidative stress, and diffuse axonal injury were conducted at 6 and 24 hours post-injury using RT-qPCR. Immunohistochemistry (IHC) provided high-resolution imaging for neuronal and axonal injury, neuroinflammation, and oxidative stress markers.

Results, Conclusions, and Discussions:: We successfully generated reproducible and controllable BIS injuries in organotypic brain slices using the EBWS system (Fig. 1A-1C). Brightfield imaging following trypan blue staining revealed frayed edges on the slice and an increased number of dead cells immediately after injury (Fig. 1D, scale bar = 100 µm). We evaluated multiple viability assays to characterize the injury response of organotypic slices after blast injury. We sampled media from blast-injured slices for LDH release to evaluate membrane integrity and observed rapid LDH release in injured slices compared to naïve and sham controls within 6 hours post-blast that largely stabilized at 24 hours post-injury (Fig. 1E). Live fluorescence imaging using PI staining corroborated these findings, showing a marked increase in PI+ cells after injury (Fig. 1F, scale bar = 100 µm). Quantitative analysis of colocalized PI+/Hoechst+ cells from five cortical regions per slice indicated ~1.8-fold increase in cell death in injured slices compared to sham controls at 1 hour post-injury (Fig. 1G). To further support characterization of the injury response, we will complete RT-qPCR analyses of known molecular responses after injury, such as increased expression of cellular activation markers (Iba1, GFAP), enhanced neuroinflammation (TNF-alpha, IL-1beta), elevated oxidative stress markers (acute NOS2 upregulation, subacute SOD modulation), and diffuse axonal injury (APP, Calpain). Additionally, immunohistochemical staining will be utilized to visualize neuronal and axonal injury (Fluoro-Jade C-positive degenerating neurons, increased APP staining), glial activation (GFAP and Iba1), and oxidative damage (4-HNE staining) at 24 hours post-injury. Collectively, these results highlight the effectiveness and innovation of our organotypic brain slice culture model for studying blast-induced TBI. To date, no established organotypic slice models exist that accurately replicate blast-induced injuries. Therefore, our platform uniquely bridges a critical gap between simplified in vitro cellular models and complex in vivo systems. By replicating essential aspects of blast pathophysiology, this model provides a valuable tool for unraveling the precise cellular and molecular mechanisms underlying blast injuries. In the future, this system can help to advance pharmacological screening efforts and facilitate the discovery of novel therapeutic targets and genes involved in blast-induced neuropathology.

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