Optimized Model of Cerebral Ischemia In situ for the Long-Lasting Assessment of Hippocampal Cell Death

Abstract

Among all the brain, the hippocampus is the most susceptible region to ischemic lesion, with the highest vulnerability of CA1 pyramidal neurons to ischemic damage. This damage may cause either prompt neuronal death (within hours) or with a delayed appearance (over days), providing a window for applying potential therapies to reduce or prevent ischemic impairments. However, the time course when ischemic damage turns to neuronal death strictly depends on experimental modeling of cerebral ischemia and, up to now, studies were predominantly focused on a short time-window—from hours to up to a few days post-lesion. Using different schemes of oxygen-glucose deprivation (OGD), the conditions taking place upon cerebral ischemia, we optimized a model of mimicking ischemic conditions in organotypical hippocampal slices for the long-lasting assessment of CA1 neuronal death (at least 3 weeks). By combining morphology and electrophysiology, we show that prolonged (30-min duration) OGD results in a massive neuronal death and overwhelmed astrogliosis within a week post-OGD whereas OGD of a shorter duration (10-min) triggered programmed CA1 neuronal death with a significant delay—within 2 weeks—accompanied with drastically impaired CA1 neuron functions. Our results provide a rationale toward optimized modeling of cerebral ischemia for reliable examination of potential treatments for brain neuroprotection, neuro-regeneration, or testing neuroprotective compounds in situ.

Maturation of neural stem cells and integration into hippocampal circuits – a functional study in an in situ model of cerebral ischemia

“…With this idea, we aimed to examine the time course of functional maturation of NSPC-derived neurons in the endogenous (physiological) environment where both tissue architecture and signaling pathways remained preserved. Since we have recently established that mouse organotypic hippocampal slices can be suitable for long-term maintenance (at least 4-5 weeks), with no functional deficit in CA1 neurons over this time window (Rybachuk et al, 2017), here, we focused on the functional (electrophysiological) properties of neurons derived from embryonic NSPCs. These NSPCs were grafted into hippocampal tissue in basic artificial medium (no enriched composites) in order to trace the maturation of NSPCs in a host (endogenous) environment.…”

“…We recently described in detail the ischemic impairments, both morphological and functional, in organotypic hippocampal slices at the later time window (1-3 weeks) following OGD (Rybachuk et al, 2017). Therefore, here, we focused on the maturation of NSPC-derived neurons in situ as a possible NSPC therapy for cell replacement strategies after cerebral ischemia.…”

“…The integration of NSPCs into the impaired host circuits was also delayed compared with that in controlwe rarely detected synaptic events in the NSPC-derived neurons at 1-2 weeks, and the sEPSC parameters remained lower even following 3 weeks in situ. Such functional deficits very likely relate to severe post-ischemic impairments in hippocampal tissue due to glutamate-induced excitotoxicity and overwhelmed depolarization of endogenous neurons (Dzhala et al, 2001;Fujiwara et al, 1987), which persist for 2-3 weeks after OGD (Rybachuk et al, 2017). In contrast to the delayed neuronal maturation, NSPC differentiation to a glial cell type was boosted in the post-ischemic environment.…”

“…an in situ experimental model of cerebral ischemia (Rybachuk et al, 2017), OGD boosted the excitatory drive of CA1 neurons (enhanced sEPSC frequency) and changed postsynaptic glutamate receptors (the parameters of postsynaptic currents) in host CA1 neurons at the later period of time post OGD (1-3 weeks; data not shown). However, we did not observe any boosting of excitatory transmission in the NSPCderived neurons in the post-ischemic tissue.…”

“…To examine the time-dependent maturation of electrophysiological properties of the NSPC-derived neurons in the post-ischemic environment, we exposed organotypic hippocampal slices to oxygen-glucose deprivation (OGD, 10 min duration), thereby mimicking the conditions that take place upon cerebral ischemia. Recently, we have demonstrated that this model of cerebral ischemia in situ triggered CA1 neuronal death within 2 weeks or less, and was accompanied by drastic functional impairments in CA1 neurons (Rybachuk et al, 2017). Therefore, the same experimental model was utilized here, followed by the NSPC therapy (Fig.…”