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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 the core of a brain infarct, characterized by severely reduced blood supply, loss of neuronal function is rapidly followed by neuronal death. In peripheral areas of the infarct, the penumbra, damage is initially reversible, and neuronal activity is typically reduced due to ischemia-induced synaptic failure. There is limited understanding of factors governing neuronal recovery or the transition to irreversible damage. Neuronal activity has been shown to be crucial for survival. Consequently, hypoxia induced neuronal inactivity may contribute to cell death, and activation of penumbral neurons possibly improves survival. Adversely, activation increases ATP demand, and a balance should be found between the available energy and sufficient activity., Approach: We monitored activity and viability of neurons in an in vitro model of the penumbra, consisting of (rat) neuronal networks on micro electrode arrays (MEAs) under controlled hypoxic conditions. We tested effects of optogenetic and electrical activation during hypoxia., Main Results: Mild stimulation yielded significantly better recovery of activity immediately after re-oxygenation, compared with no stimulation, and a 60%-70% higher survival rate after 5 d. Stronger stimulation was not associated with better recovery than no stimulation, suggesting that beneficial effects depend on a delicate balance between sufficient activity and available energy., Significance: We show that mild activation during hypoxia/ischemia is beneficial for cell survival in an in vitro model of the penumbra. This finding opposes the current common belief that suppression of neuronal activity is the cornerstone of neuroprotection during cerebral ischemia, and may open new possibilities for the treatment of secondary brain damage after stroke.

In the penumbra of a brain infarct, neurons initially remain structurally intact, but perfusion is insufficient to maintain neuronal activity at physiological levels. Improving neuronal recovery in the penumbra has large potential to advance recovery of stroke patients, but penumbral pathology is incompletely understood, and treatments are scarce. We hypothesize that low activity in the penumbra is associated with apoptosis and thus contributes to irreversible neuronal damage. We explored the putative relationship between low neuronal activity and apoptosis in cultured neurons exposed to variable durations of hypoxia or TTX. We combined electrophysiology and live apoptosis staining in 42 cultures, and compared effects of hypoxia and TTX silencing in terms of network activity and apoptosis. Hypoxia rapidly reduced network activity, but cultures showed limited apoptosis during the first 12 h. After 24 h, widespread apoptosis had occurred. This was associated with full activity recovery observed upon reoxygenation within 12 h, but not after 24 h. Similarly, TTX exposure strongly reduced activity, with full recovery upon washout within 12 h, but not after 24 h. Mean temporal evolution of apoptosis in TTX-treated cultures was the same as in hypoxic cultures. These results suggest that prolonged low activity may be a common factor in the pathways towards apoptosis. [ABSTRACT FROM AUTHOR]