Modeling Controlled Cortical Impact Injury in 3D Brain‐Like Tissue Cultures

Abstract

Traumatic brain injury (TBI) survivors suffer long term from mental illness, neurodegeneration, and neuroinflammation. Studies of 3D tissue models have provided new insights into the pathobiology of many brain diseases. Here, a 3D in vitro contusion model is developed consisting of mouse cortical neurons grown on a silk scaffold embedded in collagen and used outcomes from an in vivo model for benchmarking. Molecular, cellular, and network events are characterized in response to controlled cortical impact (CCI). In this model, CCI induces degradation of neural network structure and function and release of glutamate, which are associated with the expression of programmed necrosis marker phosphorylated Mixed Lineage Kinase Domain Like Pseudokinase (pMLKL). Neurodegeneration is observed first in the directly impacted area and it subsequently spreads over time in 3D space. CCI reduces phosphorylated protein kinase B (pAKT) and Glycogen synthase kinase 3 beta (GSK3β) in neurons in vitro and in vivo, but discordant responses are observed in phosphprylated ribosomal S6 kinase (pS6) and phosphorylated Tau (pTau) expression. In summary, the 3D brain‐like culture system mimicked many aspects of in vivo responses to CCI, providing evidence that the model can be used to study the molecular, cellular, and functional sequelae of TBI, opening up new possibilities for discovery of therapeutics.

Functional Characterization of Three-Dimensional Cortical Cultures for In Vitro Modeling of Brain Networks

“…Confocal z stack images at 1, 2, and 3 weeks of culture showed that neurons formed extensive 3D structural networks ( Figures 2 A and 2B, and Video S1 ). Using a custom MATLAB code to quantify the 3D neurite density (( Liaudanskaya et al, 2020 ) and detailed in Transparent Methods ), we observed an increase in neurite density over the course of development ( Figure 2 C). To provide further evidence of structural network formation, quantitative real-time PCR (qRT-PCR) was used to analyze the expression of genes known to change during neural development.…”

Genetic inhibition of RIPK3 ameliorates functional outcome in controlled cortical impact independent of necroptosis

“…We used three-dimensional human endothelial cell cultures to assess cell autonomous responses to TBI in vitro and draw parallels to the human condition [ 36 ]. We subjected human brain endothelial cells in three-dimensional silk scaffold cultures to trauma using the same CCI device and injury parameters as in vivo [ 32 ]. In human endothelial cells, significant induction of both total (1.4-fold) and phospho-RIPK3 (2.1-fold) was observed at 24 h in injured vs. sham injured cultures (Fig.…”

“…Three-dimensional scaffolds of human brain endothelial cells were placed on a cell culture dish and subjected to CCI using the same apparatus used for in vivo CCI (6 m/s, 1.2 mm depth) or to sham injury (no impact) as previously described [ 32 ]. Cultures were then returned to media and incubated at 37 °C for 24 h.…”

Integrated functional neuronal network analysis of 3D silk-collagen scaffold-based mouse cortical culture

“…We have previously reported several in vitro brain models, with both primary rodent neurons and human induced pluripotent stem cells (iPSCs), using porous scaffolds fabricated with silk fibroin and extracellular matrix composites. Such models recapitulated many key features of the brain tissue ( Cairns et al., 2020 ; Cantley et al., 2018 ; Dingle et al., 2020 ; Liaudanskaya et al., 2020 ; Rouleau et al., 2020 ; Tang-Schomer et al., 2014 ).…”