Abstract A056: A Human Cortical Tissue Platform for High-Throughput Screening of Ischemic Injury Therapeutics

Ischemic reperfusion injury pathophysiology is complex and includes the response of the neurovascular unit. Many studies have attempted to develop novel therapeutics that target single mechanisms of injury such as excitotoxicity, inflammation, oxidative stress, and others. These studies are often resource demanding and limited to cell cultures or rodent models, which reduces clinical translation. IPSC and organoids lack the 3D architecture of intact brain. We developed a high-throughput screening tool that allows the simultaneous assessment of several treatments using human cortical tissue. Human cortical brain tissue samples were acquired during temporal lobectomy. Samples were sliced (300 µm) recovered in NMDG-artificial cerebral spinal fluid (aCSF). In 12-well plates plumbed with PE-10 microbubbling catheters, samples were incubated at 37°C with regular aCSF or oxygen/glucose-free aCSF (ischemia, 60min). Samples were transferred to regular aCSF (reperfusion, 60min). To illustrate a high-throughput screening mode, samples were treated with free radical scavenger, Astaxanthin (AST; 1, 10, 50 mM), excitotoxicity antagonist, MK801 (1, 10, 100 mM), antioxidant enhancer, oltipraz (OLT; 1, 10, 100 mM), and pan-caspase inhibitor, ZVAD-fmk (1, 10, 50 mM). To evaluate cell death, samples were incubated in propidium iodide (PI, 5 µg/ml, 20min), washed, and post-fixed (4% PFA). Sections (30 µm) were stained with cell-type-specific markers (NeuN, GFAP, Iba1, and CD31). Z-stack images were taken using stratified random sampling (MicroBrightfield). Cell death was quantified as a ratio of colocalization of PI and markers using QuPath auto cell detection and object classification. The ratio of PI-positive to DAPI for untreated samples was 0.833. AST reduced the ratio for 1, 10, 50 mM to 0.041, 0.005, 0.094 (all p<0.001), respectively. MK801 reduced the ratio for 1, 10, 100 mM to 0.578, 0.281, 0.480 (p<0.05,<0.001,<0.001), respectively. OLT reduced the ratio for 1, 10, 100 mM to 0.391, 0.105, 0.052 (all p<0.001), respectively. ZVAD-fmk reduced the ratio for 1, 10, 50 mM to 0.123, 0.046, 0.00 (all p<0.001), respectively. This novel method of determining treatment success in a high-throughput mode allowed us to evaluate five treatment groups with three doses per drug treatment using a single human cortical tissue resection specimen. This method can be adapted for screening additional research interests involving the cellular biological response of human cortical tissue.