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Faculty Sponsor

Dr. Robert Sweazey

Department/Program

Department of Biology

University Affiliation

Indiana University – Purdue University Fort Wayne

Abstract

Organotypic hippocampal brain slice cultures (OHCs) are widely used in vitro models of ischemic stroke. This model simulates an in vivo environment where neurons retain their cellular connections and functions. Detection of neuronal cell death is a standard procedure to assess the severity of damage in tissue culture models of neurodegenerative diseases. However, information about the time course of injury during an ischemic event is limited; therefore we examined the time course of apoptosis and necrosis in a rat OHC model of ischemia.

We exposed OHCs from postnatal day 6–9 Sprague-Dawley rats to varying periods of oxygen and glucose deprivation (OGD) to mimic conditions following cerebral ischemia. All analytical tests were run immediately after OGD to avoid reperfusion effects. The viability of the hippocampal slices exposed to OGD was determined qualitatively and quantitatively using YO-PRO 1 iodide, which stains apoptotic cells, and propidium iodide (PI), which stains necrotic cells.

Apoptosis in the OHCs increased gradually with increased time of OGD as shown by changes in the YO-PRO 1 iodide fluorescent intensity. Quantitative analysis showed a small number of PI stained cells which could also be detected as early as 1 hour following OGD. The data gathered suggest that the most suitable length of ischemia without reperfusion for the maximization of apoptosis is between 4 - 8 hours. Within this time frame we see a large increase in apoptotic cells which are potentially salvageable and are easily accessible for application and assessment of neuroprotective interventions.

In conclusion, our in vitro model provides us with the unique opportunity (1) to select the severity of the ischemic insult by varying the period of OGD, (2) estimate the amount of damage by evaluating the effects on viability through generation of apoptotic and necrotic cells, and (3) test potential new treatments for hypoxic-ischemic stroke.

Disciplines

Biology | Life Sciences

Apoptotic and Necrotic Processes in a Rat Organotypic Hippocampal Slice Culture Model of Ischemic Stroke without Reperfusion

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