RESUMEN
Traumatic brain injury (TBI) is caused by a wide range of physical events and can induce an even larger spectrum of short- to long-term pathophysiologies. Neuroscientists have relied on animal models to understand the relationship between mechanical damages and functional alterations of neural cells. These in vivo and animal-based in vitro models represent important approaches to mimic traumas on whole brains or organized brain structures but are not fully representative of pathologies occurring after traumas on human brain parenchyma. To overcome these limitations and to establish a more accurate and comprehensive model of human TBI, we engineered an in vitro platform to induce injuries via the controlled projection of a small drop of liquid onto a 3D neural tissue engineered from human iPS cells. With this platform, biological mechanisms involved in neural cellular injury are recorded through electrophysiology measurements, quantification of biomarkers released, and two imaging methods [confocal laser scanning microscope (CLSM) and optical projection tomography (OPT)]. The results showed drastic changes in tissue electrophysiological activities and significant releases of glial and neuronal biomarkers. Tissue imaging allowed us to reconstruct the injured area spatially in 3D after staining it with specific nuclear dyes and to determine TBI resulting in cell death. In future experiments, we seek to monitor the effects of TBI-induced injuries over a prolonged time and at a higher temporal resolution to better understand the subtleties of the biomarker release kinetics and the cell recovery phases.
RESUMEN
Optical projection tomography (OPT) is a powerful tool for three-dimensional imaging of mesoscopic biological samples with great use for biomedical phenotyping studies. We present a fluorescent OPT platform that enables direct visualization of biological specimens and processes at a centimeter scale with high spatial resolution, as well as fast data throughput and reconstruction. We demonstrate nearly isotropic sub-28 µm resolution over more than 60 mm3 after reconstruction of a single acquisition. Our setup is optimized for imaging the mouse gut at multiple wavelengths. Thanks to a new sample preparation protocol specifically developed for gut specimens, we can observe the spatial arrangement of the intestinal villi and the vasculature network of a 3-cm long healthy mouse gut. Besides the blood vessel network surrounding the gastrointestinal tract, we observe traces of vasculature at the villi ends close to the lumen. The combination of rapid acquisition and a large field of view with high spatial resolution in 3D mesoscopic imaging holds an invaluable potential for gastrointestinal pathology research.