Neuron specific metabolic adaptations following multi-day exposures to oxygen glucose deprivation

Abstract

Prior exposure to sub toxic insults can induce a powerful endogenous neuroprotective program known as ischemic preconditioning. Current models typically rely on a single stress episode to induce neuroprotection whereas the clinical reality is that patients may experience multiple transient ischemic attacks (TIAs) prior to suffering a stroke. We sought to develop a neuron enriched preconditioning model using multiple oxygen glucose deprivation (OGD) episodes to assess the endogenous protective mechanisms neurons implement at the metabolic and cellular level for stress adaptations. We found that neurons exposed to a five minute period of glucose deprivation recovered oxygen utilization and lactate production using novel microphysiometry techniques. Using the non-toxic and energetically favorable five minute exposure, we developed a preconditioning paradigm where neurons are exposed to this brief OGD for three consecutive days. These cells experienced 45% greater survival following an otherwise lethal event and exhibited a longer lasting window of protection in comparison to our previous in vitro preconditioning model using a single stress. As in other models, preconditioned cells exhibited mild caspase activation, an increase in oxidized proteins and a requirement for reactive oxygen species for neuroprotection. Heat shock protein 70 was upregulated during preconditioning, yet the majority of this protein was released extracellularly. We believe coupling this neuron enriched multiday model with microphysiometry will allow us to assess neuronal specific real-time metabolic adaptations necessary for preconditioning.

CHIP Is an Essential Determinant of Neuronal Mitochondrial Stress Signaling

“…OGD is a well-appreciated mitochondrial stress mediated by NMDA receptors, and cell death can be observed after neuron-enriched cultures are exposed to > 30 min of deprivation (61). In these experiments, OGD caused an increase in cell death in all genotypes in a time- dependent manner (one-tailed analysis of variance (ANOVA) p < 0.01; Note: time dependence of this effect is not shown with asterisks so as to highlight gene vs. time effects).…”

“…We have previously shown that 90 min OGD results in membrane rupture and greater than 80% cell death evidenced as lactate dehydrogenase (LDH) release commencing 10 h after the onset of stress (61). Neuronenriched cultures from rats were exposed to 90 min OGD to analyze levels and trafficking of redox-sensitive molecules, including CHIP.…”

Multichamber multipotentiostat system for cellular microphysiometry

“…The OCP module was tested using cortical cultures from day 18 Sprague-Dawley rats prepared as previously described [19]. Oxygen consumption and lactate production were measured with amperometric modules, and acid production was measured with the Cytosensor® and OCP modules.…”

“…This instrument has been an invaluable tool for multianalyte microphysiometry, and has been used to study the detection of insulin secretion [17], pathways affected by cholera toxin exposure [18], glucose deprivation on neuronal metabolism [19, 20], and measurement of immunological responses [21, 22]. …”

Application of multianalyte microphysiometry to characterize macrophage metabolic responses to oxidized LDL and effects of an apoA-1 mimetic

“…The MAMP has allowed determination of the metabolic effects of toxins[9; 10; 11], neural preconditioning[9; 12], islets stimulation[13], and use of inhibitors to study pathways. [14] In the present study, we employed the MAMP to characterize the metabolic response of macrophages to oxLDL and also examined the effects of L-4F alone and complexed with 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) on macrophage respiratory burst.…”