Cortical spheroids show strain-dependent cell viability loss and neurite disruption following sustained compression injury

Abstract

Sustained compressive injury (SCI) in the brain is observed in numerous injury and pathological scenarios, including tumors, ischemic stroke, and traumatic brain injury-related tissue swelling. Sustained compressive injury is characterized by tissue loading over time, and currently, there are few in vitro models suitable to study neural cell responses to strain-dependent sustained compressive injury. Here, we present an in vitro model of sustained compressive neural injury via centrifugation. Spheroids were made from neonatal rat cortical cells seeded at 4000 cells/spheroid and cultured for 14 days in vitro. A subset of spheroids was centrifuged at 209 or 419 rad/s for 2 minutes. Modeling the physical compression of the spheroids via finite element analyses, we found that spheroids centrifuged at 209 and 419 rad/s experienced pressures of 38 kPa and 149 kPa, respectively, and compressive strains of 18% and 35%, respectively. Quantification of LIVE-DEAD assay and Hoechst 33342 nuclear staining showed that centrifuged spheroids exhibited significantly higher DNA damage than control spheroids at 2, 8, and 24 hours post-injury. Immunohistochemistry of {beta}3-tubulin networks at 2, 8, and 24 hours post-centrifugation injury showed increasing degradation of microtubules over time with increasing compressive strain. Our findings show that cellular injuries occur as a result of specific levels and timings of sustained compressive tissue strains. This experimental compressive injury model provides an in vitro platform to examine cellular injury to gain insights into brain injury that could be targeted with therapeutic strategies.