Dynamic MRI of Immune Cell Infiltration in Experimental Stroke

Lay Summary

Can Mesenchymal Stem Cells Confer Protection Against Brain Inflammation Following Stroke?

Yale researchers will characterize the roles of three types of immune cells involved in the inflammatory response to ischemic stroke, using cellular imaging in an animal stroke model.  They then will try to block this immune response to limit damage to brain tissues.

In ischemic stroke, which accounts for nearly 80 percent of all strokes, an artery supplying the brain with vital oxygen and nutrients needed by brain cells becomes partially or totally blocked by a blood clot. The resulting death of brain cells in the area directly affected, and damage to tissue in the outer perimeters of this area (called the “penumbra”), triggers a deleterious inflammatory response by three types of immune cells that invade the brain. Research has not yet clarified the specific roles of each of the three types of immune cells or the timing of when each type infiltrates the brain. They will use cellular MRI to answer these questions, and then explore whether injecting mesenchymal stem cells into the stroke penumbra area will thwart immune cell infiltration and inflammation. This could potentially prevent further damage to brain tissue.

In each laboratory rat receiving experimentally induced ischemic stroke, they will label one of the three types of immune cells—neutrophils, T cells, or macrophages—with fluorescent dyes and iron oxide particles.  Then they will perform MRI every 30 minutes in each of the rats to identify when specific immune cells enter the brain and to track where each cell travels to in the brain. Also, for each type of immune cell, they will measure the total number that infiltrate the brain as a function of time, both during and following the stroke. Thereafter, they will transplant mesenchymal stem cells into the penumbra area (outer perimeter of the stroke area) through image-guided injections. These stem cells have been shown to release “cytokines,” substances that suppress or modulate immune actions. The investigators will determine whether these stem cells prevent the immune cells from infiltrating the brain and initiating inflammation.

Significance:  If mesenchymal stem cells injected into rat brain effectively prevent post-stroke inflammation, the study would lead to development of methods to guide mesenchymal stem cells residing in patients’ bone marrow to travel to the brain to confer this protection.

Abstract

Dynamic MRI of Immune Cell Infiltration in Experimental Stroke

Stroke is the third leading cause of death in the industrialized world. 80% of stroke victims suffer ischemic stroke, where an artery supplying the brain with blood becomes blocked, suddenly limiting or stopping blood flow. The lack of oxygen and nutrients causes brain cell death, triggering an immune response. This immune response, in the form of peripheral leukocyte invasion, exacerbates the pathological condition and can impede or counteract potential therapies aimed at anatomical and functional restoration. Therefore, in an effort to enable better stroke therapies, there exists a critical need to block the immune response in the brain following stroke. Thus, the immune response must be first fully characterized in terms of which cell subtypes arrive when and where.

This grant proposes to use high resolution cellular magnetic resonance imaging (MRI) to measure the infiltration rates and numbers of immune cell subtypes that invade the brain following stroke. This is necessary as the available information on immune cell invasion into the brain is scattered and imprecise and may be a major factor in the lack of success in furthering promising experimental therapies for immunoprotection. The innovation of this work with respect to previous histological based methods for measuring cellular infiltration rates will be the detection of single cells, in vivo, at high temporal resolution—time points every 30 minutes. We have previously demonstrated the capability of MRI to detect single cells in vivo by prelabeling cells with micron sized iron oxide particles (MPIOs). Thus, in Aim 1, the primary leukocytes involved in the stroke-induced immune reaction in the brain—neutrophils, T-lymphocytes and macrophages—will be labeled with MPIOs and assayed for viability, retained phagocytotic function, and cytokine release.

Aim 2 will measure the number of immune cells that infiltrate the brain as a function of time following stroke. The stroke model we will use is the cortical endothelin-1 stroke model. Endothelin-1 is a potent vasoconstrictor, and injection into the cortex reduces blood flow to less than 30% of basal blood flow for several hours, resulting in stroke. This method circumvents some of the complications caused by the MCAO model of stroke, including damage to the blood vessels. Labeled immune cell subtypes will be delivered to animals prior to initiation of stroke and will undergo high resolution MRI every 30 minutes, with imaging parameters that enable single cell detection. Separate experiments using labeled neutrophils, T-lymphocytes, and macrophages will allow for the discrimination and quantification of immune cell infiltration into the brain both during the stroke and after the stroke.

Lastly, in an effort to impede immune cell invasion into the brain, Aim 3 will entail the transplantation of mesenchymal stem cells (MSCs) into the penumbra of stroke lesions by way of image guided injections. MSCs have been demonstrated to have immunomodulatory activity, thus we hypothesize that delivery of MSCs into the brain following stroke will be immunoprotective. Again, high spatial and temporal resolution MRI will be used to investigate the ability of these MSCs to ward off immune cell infiltration.

These studies will not only further basic science of immune cell infiltration following stroke, but will also, for the first time, provide information on immune cell invasion during the stroke. Furthermore, while MSCs have been used for neurogenic purposes in the brain following stroke, these experiments will shed light on whether the preliminary published successes with these cells may in part be due to immuno-suppression or modulation. Lastly, these cellular MRI methods developed here will have far reaching implications in both immunotherapy and stem cell therapy. Clearly, the successful implementation of cell therapies in humans will necessitate the use of a non-invasive method for tracking the migration of cells.