Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Summary

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Abstract

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.

Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.

Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Introduction

Ischemia-Reperfusion (IR) injury is found to be the frequent cause of various debilitating complications and deaths associated with stroke, myocardial infarction, trauma, peripheral vascular disease and traumatic brain injury^1,2. IR injury in cerebral vessels is an early secondary injury leading to inflammation and edema³. One of the serious complications that occurs as a result of oxidative and metabolic stress following inflammation is loss of homeostatic balance leading to free radical formation, alterations in the blood-brain barrier (BBB) tight junctions (TJs) and microvascular permeability^4,5.

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Protocol

1. Seeding of Endothelial Cells

1. Obtain primary cultures of RBMEC’s from adult Sprague Dawley rats (or obtain them commercially).
2. Cultivate RBMECs in 100 cm fibronectin (50 µg/ml) coated petri dishes using the rat brain endothelial cell growth medium. Change the medium every two days, until confluency is reached.
3. On reaching 80-90% confluency, gently wash the cells in 5 ml phosphate buffered saline (PBS) by swirling. The cells are then detached by exposing them to 1 ml of warm 0.25% trypsin- ethylenediaminetetraacetic acid (EDTA) solution, equilibrated to 37 °C.
4. Incubate the cells at 37 °C for 2-5....

Results

Cells cultured on fibronectin precoated Nunc II chamber slides were subjected to OGD-R by placing in a Biospherix ProOx model 110 chamber. After subjecting cells to OGD-R, they were processed for ZO-1 junctional staining using immunofluorescence technique as shown in Figure 2 and cytoskeletal assembly indicating F-actin stress fiber formation using rhodamine phalloidin stain label as shown in Figure 3. Control cells that were not subjected to OGD-R showed continual junctional integrity w.......

Discussion

OGD-R as an in vitro model for ischemia-reperfusion injury has been well established for studying neurons^10,11. There are also studies showing the effect of OGD on brain endothelial cells and alterations in permeability and TJ integrity⁹. However, our study shows the effect of OGD as well as reoxygenation, which is a closer representation of ischemic reperfusion injury in in vivo conditions that occur following ischemic stroke.

Hypoxic-ischemic condition.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Proox model 110	Biospherix	Model 110
DMEM, no glucose	Gibco, Life technologies	11966-025
Rhodamine Phalloidin	Life technologies	R415
ZO-1 Rabbit Polyclonal Antibody	Life technologies	617300
Nunc Lab Tek II-CC 8 well sterile, glass slides	Thermo scientific	177402
FITC-tagged anti-rabbit secondary antibody	Santa cruz	sc-2090
DPBS 1X	Thermo scientific	SH 30028.03	Any other PBS available can be used