Mitochondria dysregulation contributes to secondary neurodegeneration progression post-contusion injury in human 3D in vitro triculture brain tissue model

Abstract

Traumatic Brain injury-induced disturbances in mitochondrial fission-and-fusion dynamics have been linked to the onset and propagation of neuroinflammation and neurodegeneration. However, cell-type-specific contributions and crosstalk between neurons, microglia, and astrocytes in mitochondria-driven neurodegeneration after brain injury remain undefined. We developed a human three-dimensional in vitro triculture tissue model of a contusion injury, composed of neurons, microglia, and astrocytes, and examined the contributions of mitochondrial dysregulation to neuroinflammation and progression of injury-induced neurodegeneration. Pharmacological studies presented here suggest that fragmented mitochondria released by microglia are a key contributor to secondary neuronal damage progression after contusion injury, a pathway that requires astrocyte-microglia crosstalk. Controlling mitochondrial dysfunction thus offers an exciting option for the development of therapies for TBI patients.

Development of a novel bioengineered 3D brain‐like tissue for studying primary blast‐induced traumatic brain injury

“…Each scaffold was seeded with 100,000 microglia, 500,000 astrocytes, and 2 million iNSCs (which were harvested through a 40‐μm cell strainer) (Figure 1d). The cell ratios and seeding densities for all three cell types were determined following a previous established protocol (Liaudanskaya et al, 2022). Briefly, these were established by evaluating a range of relative cell ratios and were optimized based on the size of the silk scaffold, cell growth rate, viability, functionality, and maturity (Herculano‐Houzel, 2014; Liaudanskaya et al, 2022; Tang‐Schomer et al, 2014).…”

“…The cell ratios and seeding densities for all three cell types were determined following a previous established protocol (Liaudanskaya et al, 2022). Briefly, these were established by evaluating a range of relative cell ratios and were optimized based on the size of the silk scaffold, cell growth rate, viability, functionality, and maturity (Herculano‐Houzel, 2014; Liaudanskaya et al, 2022; Tang‐Schomer et al, 2014). Each scaffold had a total volume of 40 μl of cells in triculture media seeded (Neurobasal Medium, minus phenol red (ThermoFisher, 12348017), 2% B27 supplement (ThermoFisher, 17504044), 1% Glutamax, 1% antibiotic–antimycotic, and 1% astrocyte growth supplement (ScienCell Research Lab, 1852)).…”

“…including induced neural stem cells (iNSCs), microglia, and astrocytes, embedded in collagen (Liaudanskaya et al, 2022). The porous silk scaffold provides a reliable structural backbone that improves functional neuronal network formation and nutrient diffusion.…”

“…Overall, this tissue model allows for reproducible cell type composition, control over the cellular microenvironment, scalability, and long‐term culture support. Variations in this model system have been successfully used to study controlled impact injury (Liaudanskaya et al, 2020, 2022) and to test therapeutic interventions (Rouleau, Bonzanni, et al, 2020), in addition to modeling Alzheimer's (Cairns et al, 2020), Parkinson's disease (Collins et al, 2019), and glioblastomas (Sood et al, 2019). In our current study, we have utilized the Advanced Blast Simulator (ABS) to expose these human 3D brain‐like tissues to a precisely controlled blast wave.…”

“…To circumvent some of the limitations inherent in these 3D culture systems, we have utilized a porous silk scaffold‐based 3D hydrogel culture system, composed exclusively of human cells, including induced neural stem cells (iNSCs), microglia, and astrocytes, embedded in collagen (Liaudanskaya et al, 2022). The porous silk scaffold provides a reliable structural backbone that improves functional neuronal network formation and nutrient diffusion.…”