METABOLIC AND MOLECULAR RESPONSE OF ADULT MOUSE BRAIN TISSUE TO CRYOPRESERVATION BY VITRIFICATION

Cryopreservation of brain tissue offers a promising avenue for banking viable CNS samples for research and translational applications. We have recently demonstrated that vitrification of adult mouse brain slices preserves neural architecture and hippocampal neurophysiology, resuming neuronal discharge and synaptic transmission and plasticity after rewarming. To characterize the metabolic and molecular impact of the vitrification process itself, we performed analyses for mitochondrial activity and bulk RNA-sequencing of acute mouse brain slices with and without vitrification. Seahorse analysis of oxygen consumption showed that, while cryoprotectant loading alone produced modest reduction in mitochondrial function, vitrification at -196 ^oC had no further impact on the basal respiration and spare capacity. RNA-sequencing analysis after 3 hours of incubation at 34°C, combining gene expression with weighted gene co-expression network analysis and computational single-cell deconvolution, revealed an immune-stunned state with significant upregulation of IL1B, TNF, CCL9 without vitrification and downregulation CD33 und ADGRE1 post-vitrification. The transcriptomic signature of cryopreservation was further defined by an upregulation of proteotoxic (HSP70/HASPA1A) and excitotoxic (NPAS4) stress response genes, alongside a downregulation of metabolism and water permeability (ATP1A2, AQP4). Moreover, FACS indicated a reduction of the functional microglial pool with a shift toward CD11 and CD44-positive phenotypes. Qualitative assessment of microglia morphology suggested a comparable or higher degree of ramification post-vitrification. Taken together, our findings demonstrate that vitrification induces a stress response involving both cellular metabolism and nuclear transcription which may serve homeostasis. Tentatively, the present results suggest vitrified brain slices as a model with reduced microglia activation.