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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. [ABSTRACT FROM AUTHOR]

The understanding of the neurophysiological processes that occur in the areas that surround the core of a brain infarct is crucial for the creation of new therapies and treatments to improve neuronal recovery. The present study aims to demonstrate that both rodent and human neuronal networks lose their activity under low oxygen conditions and that electrical stimulation can increase the probability of recovery. Hypoxia was induced in rodent and human neurons and the effects of electrical stimulation were assessed in the rat cultures. The results obtained show that neuronal activation, in the form of electrical stimulation, has the potential to maintain the networks at higher levels of activity and, therefore, to improve cell survival. This study will open the way for new treatment strategies based on brain-stimulation to enhance neuronal recovery and will be of large relevance for patients, families, and society. [ABSTRACT FROM AUTHOR]

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]

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.

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.

Fibroblastic reticular cells (FRCs), the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs). Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.

Cancer cells are frequently exposed to physiological stress conditions such as hypoxia and nutrient limitation. Escape from stress-induced apoptosis is one of the mechanisms used by malignant cells to survive unfavorable conditions. B-cell Translocation Gene 1 (BTG1) is a tumor suppressor that is frequently deleted in acute lymphoblastic leukemia and recurrently mutated in diffuse large B cell lymphoma. Moreover, low BTG1 expression levels have been linked to poor outcome in several solid tumors. How loss of BTG1 function contributes to tumor progression is not well understood. Here, using Btg1 knockout mice, we demonstrate that loss of Btg1 provides a survival advantage to primary mouse embryonic fibroblasts (MEFs) under stress conditions. This pro-survival effect involves regulation of Activating Transcription Factor 4 (ATF4), a key mediator of cellular stress responses. We show that BTG1 interacts with ATF4 and positively modulates its activity by recruiting the protein arginine methyl transferase PRMT1 to methylate ATF4 on arginine residue 239. We further extend these findings to B-cell progenitors, by showing that loss of Btg1 expression enhances stress adaptation of mouse bone marrow-derived B cell progenitors. In conclusion, we have identified the BTG1/PRMT1 complex as a new modifier of ATF4 mediated stress responses.