In Vitro Hippocampal Slice Culture – Neuroprotection Assay

Brief Description

Ability of a test compound to block KA and NMDA-induced neurotoxicity in organotypic hippocampal slice cultures.

Introduction

SE can arise in the context of a CNS insult, resulting in excess glutamate release and thus, excitotoxic neuronal death (1,2). In addition to identifying anticonvulsant compounds that can disrupt SE, compounds should ideally also offer some neuroprotection from the excitotoxic insult. Such neuroprotective therapies may thereby prevent the resultant cognitive decline and future risk of refractory SE (3). Using organotypic hippocampal slice cultures subjected to excitotoxic insult allows for the rapid identification of compounds that may be, in fact, neuroprotective under these circumstances. Furthermore, such in vitro testing can rapidly identify potential neuroprotective agents using minimal animal subjects (approximately 12-16 slices/pup). Such compounds can then be further probed to determine their anticonvulsive potential or be investigated as potential adjunct therapy in the clinical management of SE.

Methods

Slice preparation and culture: Male and female Sprague-Dawley rat pups (P10-P11; 18-25g) are sacrificed under sodium pentobarbital anesthesia, brains removed and hippocampi rapidly dissected under sterile conditions. Four uniform, 400 µm hippocampal sections/well are placed into a 6-well culture dish. Sections are maintained for 14-15 days in 50% MEM with glutamax + Hepes, 25% horse serum (Gibco/Invitrogen, Carlsbad, CA), and 25% Hank’s balanced salt solution (Sigma, St. Louis, MO) supplemented with D-glucose (final conc., 25mM) with culture media exchanged 3 times/week. On the day before testing, culture medium is replaced with serum-free Neurobasal medium with 2% B27 supplement (Gibco/Invitrogen, Carlsbad, CA) and 25mM D-glucose. Propidium iodide staining and live cell imaging: Excitotoxic neurodegeneration in hippocampal slice cultures is monitored by propidium iodide (PI) uptake over three consecutive days at 37°C in the presence of 5% CO2 (4). On Day 1, all hippocampal sections are assessed for health by evaluating basal uptake of propidium iodide (PI) (2µM; 45 min exposure) and only healthy slices are used for the assay. PI uptake is measured by fluorescent imaging (Axiovert TE 200; Carl Zeiss, Inc., Germany). Two wells (4 hippocampal sections/well) of the 6-well plate are treated as controls, receiving either 10 µM NMDA (Sigma, St. Louis, MO) or 20µM kainic acid (KA; Tocris Bioscience, Ellisville, MO) alone. Test wells are exposed to NMDA or KA, then 30 min later, the test compound at either 10 µM or 100 µM is added to each well. Altogether, wells are exposed to NMDA or KA for 4 hrs and test compound (10 µM or 100 µM) for 3.5 hrs. After the initial 4 hr excitotoxin and subsequent 3.5 hr test compound exposure, media is replaced with fresh neurobasal medium containing PI and returned to the incubation chamber for an additional 20 hr. Slices are imaged 24 hrs after the initial excitotoxin exposure to determine the extent of neuronal death. After Day 2 image acquisition, 10 mM glutamate is added to the slice cultures to induce extensive neuronal death, slices are returned to the incubation chamber, and re-imaged 24 hrs later (Day 3 imaging) to determine extent of neuronal death.

Results

All image analyses are automated with Axiovision software (Carl Zeiss). The number of slices used ranges from 8-16 slices per concentration. To determine the extent of neuroprotection from the test compound, the Day 3, glutamate-treated image is used to define a region of interest (ROI) around CA1 and CA3 regions of each hippocampal slice. Background intensity values are subtracted from each image prior to calculation of PI uptake ratio. Brightness and intensity within the ROI for each slice is determined. The individual ROI map for each slice is then applied to the corresponding Day 2 image, and brightness and intensity determined for the Day 2 image. The extent of cell death is assessed by dividing the Day 2 (test compound) ROI intensity by the Day 3 (glutamate) ROI intensity, giving a percent of total fluorescence. The percent neuroprotection (100 – average ratio of the % of total fluorescent intensity) and SEM are calculated for each test compound concentration, with EC50 also determined by curve fitting. For compound and control (i.e. NMDA or KA alone), % protection values are averaged among tissue culture plates containing the same concentration of compound.

Discussion

The organotypic hippocampal slice culture provides rapid assessment of the neuroprotective potential of novel compounds for the clinical management of SE. Importantly, this assay models the glutatmate-induced excitotoxicity found in SE (1, 2), thus is a predictive in vitro screen to identify potentially clinically-relevant compounds. For these reasons, the ETSP has applied this in vitro test as an initial screen to identify compounds that can be used either alone or as adjunct therapies during the treatment of SE. While most standard anti-seizure drugs do not confer neuroprotection in this assay (appendix 1), the conantokin CGX-1007, a NR2B-selective antagonist, shows robust neuroprotection in the organotypic hippocampal slice culture, whereas another NR2B selective antagonist, ifenprodil, did not show such neuroprotection (4). Additionally, the prototypical NMDA receptor antagonist, MK-801, also demonstrates robust neuroprotective properties in the organotypic hippocampal slice model (5). Furthermore, both CGX-1007 (6) and MK-801 (7, 8) possess anticonvulsant activity in kindled rats. Thus, compounds that are found to be neuroprotective in the organotypic hippocampal slice culture would then be subjected to further investigation for their potential anticonvulsant properties and ability to interrupt SE induced by various pharmacological compounds routinely applied at the ETSP