The Protective Mechanisms of Intravenous Immunoglobulin (IVIg) in Ischaemic Stroke

Abstract

Stroke is a devastating disease that is held accountable as the second leading cause of death worldwide. It encompasses two distinctively different forms based on its origin, namely ischaemic stroke and haemorrhagic stroke. Ischaemic stroke is the most common type of stroke, which accounts for 80% of all stroke cases. At the molecular level, the reduction of blood supply and oxygen to the affected brain region triggers a series of dynamic, intricate and interdependent biochemical events, which ultimately lead to cell death. This insult affects not only the brain resident cells but also the brain endothelial cells, which form the blood brain barrier, separating the highly exclusive neuro-environment from the periphery. During ischaemic injury, the blood brain barrier breaks down, allowing the infiltration of leukocytes into the brain, which further exacerbates the injury. Currently, intravenous recombinant tissue plasminogen activator (rtPA) is the only approved therapeutic agent used in stroke treatment as a blood clot buster. However, its therapeutic efficacy and narrow time window forces physicians and clinicians to only allow its administration within 3- 4.5 hours from the onset of stroke. There have been various therapeutic agents shown to be effective in preventing cell death in ischaemic-like conditions in experimental stroke animal models, however human clinical trials have shown disappointing outcomes. Although there are many factors contributing to the failure, these are mainly due to the ability of the drugs to only target a single injury mechanism in a single cell type from a complex series of cellular injuries occurring in multiple cell types during ischaemic stroke. Hence, there is an urgent need to develop a therapeutic agent with a multi targeting capability to aim at multiple injury mechanisms in ischaemic stroke. Intravenous immunoglobulin (IVIg), a purified polyclonal immunoglobulin preparation obtained from several thousands of healthy human plasma donors, has shown to be able to regulate various inflammatory mechanisms. It has been FDA-approved as a treatment for various immune- related conditions including some neurological conditions such Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy and multifocal motor neuropathy. A previous study in our laboratory showed that IVIg has been able to reduce the brain infarct size by 60% in an experimental stroke mouse model. It has also been shown to decrease the production level of cell adhesion molecules and the infiltration of inflammatory cells and also to neutralise complement components. Moreover, IVIg is able to reduce the cleavage of caspase 3, which is a marker of apoptosis in neurons subjected to ischaemic-like condition in vitro. However, the exact mechanism of how IVIg directly protects neuronal cells from undergoing apoptosis and assist in the maintenance of blood brain barrier integrity after stroke still remains unknown. The present studies demonstrate that high concentration of IVIg (5mg/mL) protects neuronal cells from undergoing apoptosis. By using in vitro settings of glucose-deprived condition, immunoblot analysis showed that IVIg inhibits neuronal apoptosis in glucose deprived condition and decreases the expression of the phosphorylated JNK MAP kinase and NFkB p65 which are crucial in inducing apoptosis. Moreover, IVIg is also shown to up regulate Bcl-2, an anti-apoptotic protein that promotes cell survival. In addition, in vivo injection of IVIg (2g/kg body weight), previously shown to reduce the infarct size, is shown here by immunofluorescence staining not only to be able to penetrate the brain parenchyma through the blood brain barrier but also to reduce brain cell death. Blood brain barrier breakdown is a major hallmark in ischaemic stroke and the breakdown of the tight junctions allows the penetration of leukocytes into the affected brain region, exacerbating the injury. FACS analysis showed IVIg is able to reduce leukocyte infiltration after stroke-induced brain injury. To investigate the molecular mechanisms of blood brain barrier damage, an in vitro model of blood brain barrier was studied by culturing cells from the murine brain endothelial cell line bEnd.3. Immunoblot analysis of bEnd.3 brain endothelial cells revealed that IVIg prevents the significant down regulation of expression of tight junction proteins such as claudin 5 and occludin experienced by brain endothelial cells under ischaemic-like conditions. In addition, bEnd.3 cells showed increased FITC-dextran penetration when exposed to ischaemic-like conditions, and this effect was prevented with the addition of IVIg. Moreover, IVIg also protects the endothelial cells subjected to ischaemic-like conditions from undergoing cell death as assessed by LDH release as well as preventing the ischaemia-induced decrease in the expression level of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Thus, these studies demonstrate the protective pleotropic functions of IVIg in the setting of ischaemic stroke. The neuroprotective effects of IVIg are shown through the upregulation of cell survival protein and the modulation of the phosphorylated cell death related kinases. In addition, for the first time, the protective role of IVIg on the brain endothelial cells in ischaemic stroke induced brain injury was addressed and the results demonstrated that IVIg is not only able to decrease leukocyte infiltration but also to maintain the endothelial cell survival and the tight junctions protein levels and hence maintain the blood brain barrier integrity. Overall, these studies enhance the existing knowledge of the protective therapeutic effect of IVIg in ischaemic stroke where it acts to palliate the plethora of insults contributing to the overall disease outcome, hence endorsing IVIg as a more attractive potential candidate for pharmacological intervention in stroke therapy.