Investigating the mechanisms underlying neuronal death in ischemia using in vitro oxygen-glucose deprivation: potential involvement of protein SUMOylation

Abstract

It is well established that brain ischemia can cause neuronal death via different signaling cascades. The relative importance and interrelationships between these pathways, however, remain poorly understood. Here is presented an overview of studies using oxygen-glucose deprivation of organotypic hippocampal slice cultures to investigate the molecular mechanisms involved in ischemia. The culturing techniques, setup of the oxygen-glucose deprivation model, and analytical tools are reviewed. The authors focus on SUMOylation, a posttranslational protein modification that has recently been implicated in ischemia from whole animal studies as an example of how these powerful tools can be applied and could be of interest to investigate the molecular pathways underlying ischemic cell death.

Fig. 1

Schematic of the SUMOylation pathway. SUMOylation comprises three enzymatic steps that culminate in the formation of an isopeptide bond, between the carboxyl group of the C-terminal glycine of SUMO and the substrate ε-amino group of a specific lysine residue. The initial step in SUMOylation is ATP dependent and involves the activation of the C-terminus of the SUMO protein by the enzyme E1. Once activated, the SUMO protein is transferred to a SUMO-conjugating enzyme (E2) called Ubc9. Ubc9 binds substrate proteins directly and, in conjunction, with one of the several SUMO protein ligases (E3s), subsequently mediates the transfer of the SUMO protein to its target protein. However, it should be noted the involvement of E3 is not always required for efficient SUMOylation. Importantly, despite being a covalent modification substrate, SUMOylation is highly labile and readily reversible by the SUMO-specific family of SENP proteases. This highly dynamic system allows cells to respond rapidly to varying cellular demands. SUMO = small ubiquitin-like modifier.

Fig. 2

Slice preparation and culturing followed by oxygen-glucose deprivation (OGD). (A) Decapitate rats one by one with large scissors and remove the brain quickly. (B) Isolate the hippocampi. (C) Section transversely at 400 μm by McIlwain tissue chopper, and place the slices in chilled Hanks’ balanced salt solution (HBSS). (D) Place six slices on each membrane insert in culture trays with six wells, each well containing 1 mL of culture medium (see composition below). (E) Place culture trays in incubator at 36 °C with 5% CO₂. Change the medium twice a week for two weeks. After 14 days in vitro, expose the cultures to OGD. Transfer the inserts to sterilized petri dishes and rinse twice with OGD medium (see composition below). Incubate the inserts in 1 mL of OGD medium for 10 minutes. Exchange for OGD medium previously bubbled with nitrogen for 15 minutes. (F) Transfer the petri dishes containing the slice cultures to an anaerobic chamber. Close the chamber and inject a mixture of N₂ with 5% CO₂ for 10 minutes at 8 L/min. Keep the chamber in the incubator for 45 minutes at 37 °C. Remove the dishes from the chamber and wash the slices twice with HBSS. Return to culture medium and incubate for 24 hours. Add 7.5 mM propidium iodide (PI) and incubate for 1 hour. Examine the cultures in an inverted fluorescent microscope fitted with a rhodamine filter. Photograph the slices and analyze using Scion Image software. Culture medium: minimum essential medium (50%), horse serum (25%), and HBSS (25%) supplemented with (mM, final): glucose 36, glutamine 2, HEPES 25, NaHCO₃ 4, and penicillin/streptomycin 1% (pH 7.3). OGD medium composed of (mM): CaCl₂ 1.26, KCl 5.36, NaCl 136.89, sucrose 36.08, KH₂PO₄ 0.44, Na₂HPO₄ 0.34, MgCl₂ 0.49, MgSO₄ 0.44, HEPES 25, and penicillin/streptomycin 1% (pH 7.2).

Fig. 3

Propidium iodide (PI) staining indicating cell death following oxygen-glucose deprivation (OGD). After 45 minutes of OGD, cultures were returned to culture medium and incubated in the presence of O₂ to mimic the reperfusion that usually follows an ischemic episode in vivo. (A) Photographs of the slices showing PI staining in control and OGD slices at 6 and 24 hours’ exposure to OGD. (B) Quantification of cellular damage in CA1 performed by using Scion Image software (www.scioncorp.com). The area where PI fluorescence was detectable above background levels was determined using the “density slice” option of the software and compared with total CA1 area to obtain the percentage of damage. Data represent mean ± SEM values, n = 15 slices per group. *indicates significant difference from control cultures; #indicates significant difference from all other groups (one-way ANOVA followed by Duncan’s test, P < .001).

Fig. 4

Expression of GFP Sindbis virus in pyramidal CA1 neurons of long-term organotypic hippocampal slice cultures. Fourteen-day in vitro P8 hippocampal slices were infected overnight by bath applying approximately 2 μL of virus directly onto the slice.

Fig. 5

Changes in protein conjugation by SUMO-2/3 following oxygen-glucose deprivation (OGD). Organotypic hippocampal slice cultures were exposed to 45 minutes of OGD and then permitted 1, 6, or 24 hours of recovery. Protein SUMOylation was evaluated by Western blot analysis. Most strikingly, there is a marked increase in SUMOylation of higher molecular weight proteins that run as a smear on the gel at 1 hour and 6 hours that diminishes by 24 hours. (A) Representative pattern of SUMO-2/3 immunoreactivity detected using anti-SUMO-2/3 antibody from Zymed. (B) Cumulative SUMO-2/3 results showing quantified data from separate immunoblots using slices from four different cultures. The results are presented as percentages of control ± SEM. *indicates significant difference from control cultures (one-way ANOVA followed by Duncan’s test, P < .05).