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Mild therapeutic hypothermia showed potential neuroprotective properties during and after cerebral hypoxia or ischemia in experimental animal studies. However, in clinical trials, where hypothermia is mainly applied after reperfusion, results were divergent and neurophysiological effects unclear. In our current study, we employed human-derived neuronal networks to investigate how treatment with hypothermia during hypoxia influences neuronal functionality and whether it improves post-hypoxic recovery. We differentiated neuronal networks from human induced pluripotent stem cells on micro-electrode arrays (MEAs). We studied the effect of hypothermia (34°C)-as well hyperthermia (39°C) - on neuronal functionality during and after hypoxia using MEAs. We also studied the effects on the number of synaptic puncta and cell viability by immunocytochemistry. In comparison to neuronal networks under normothermia, we found that hypothermia during hypoxia improved functional neuronal network recovery, expressed as enhanced neuronal network activity. This was associated with prevention of synaptic loss during and after the hypoxic phase. Furthermore, hypothermia improved cell viability after the hypoxic phase. Instead, hyperthermia during hypoxia had detrimental effects, with an irreversible loss of neuronal network function, loss of synaptic puncta and decreased cell viability. Our results show potential neuroprotective properties of hypothermia occurring during hypoxia, indicating that administering hypothermia to bridge the time to reperfusion may be beneficial in clinical settings., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Voogd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fragmented network bursts (NBs) are observed as a phenotypic driver in many patient-derived neuronal networks on multi-electrode arrays (MEAs), but the pathophysiological mechanisms underlying this phenomenon are unknown. Here, we used our previously developed biophysically detailed in silico model to investigate these mechanisms. Fragmentation of NBs in our model simulations occurred only when the level of short-term synaptic depression (STD) was enhanced, suggesting that STD is a key player. Experimental validation with Dynasore, an STD enhancer, induced fragmented NBs in healthy neuronal networks in vitro. Additionally, we showed that strong asynchronous neurotransmitter release, NMDA currents, or short-term facilitation (STF) can support the emergence of multiple fragments in NBs by producing excitation that persists after high-frequency firing stops. Our results provide important insights into disease mechanisms and potential pharmaceutical targets for neurological disorders modeled using human induced pluripotent stem cell (hiPSC)-derived neurons., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: In ischemic stroke, treatments to protect neurons from irreversible damage are urgently needed. Studies in animal models have shown that neuroprotective treatments targeting neuronal silencing improve brain recovery, but in clinical trials none of these were effective in patients. This failure of translation poses doubts on the real efficacy of treatments tested and on the validity of animal models for human stroke. Here, we established a human neuronal model of the ischemic penumbra by using human induced pluripotent stem cells and we provided an in-depth characterization of neuronal responses to hypoxia and treatment strategies at the network level., Approach: We generated neurons from induced pluripotent stem cells derived from healthy donor and we cultured them on micro-electrode arrays. We measured the electrophysiological activity of human neuronal networks under controlled hypoxic conditions. We tested the effect of different treatment strategies on neuronal network functionality., Main Results: Human neuronal networks are vulnerable to hypoxia reflected by a decrease in activity and synchronicity under low oxygen conditions. We observe that full, partial or absent recovery depend on the timing of re-oxygenation and we provide a critical time threshold that, if crossed, is associated with irreversible impairments. We found that hypoxic preconditioning improves resistance to a second hypoxic insult. Finally, in contrast to previously tested, ineffective treatments, we show that stimulatory treatments counteracting neuronal silencing during hypoxia, such as optogenetic stimulation, are neuroprotective., Significance: We presented a human neuronal model of the ischemic penumbra and we provided insights that may offer the basis for novel therapeutic approaches for patients after stroke. The use of human neurons might improve drug discovery and translation of findings to patients and might open new perspectives for personalized investigations.