Ischemic stroke, one of the most devastating of diseases, is caused by blood clotting in the cerebral arteries leading to oxygen deprivation and cerebral infarction. Within three hours (h) of stroke onset, intravenous administration of recombinant tissue plasminogen activator (tPA)—a “clot busting” drug that induces thrombolysis, promotes intravascular fibrinolysis and quickly restores circulation—can improve clinical outcomes. However, because tPA can increase the risk of hemorrhage and induce neurotoxicity, its usage is limited to fewer than 2% of stroke patients. A contributing factor to the deleterious effects of stroke is the pathological activation of matrix metalloproteinases (MMPs), particularly MMP-9, which normally regulate the integrity of cell structures.
Our recently published discoveries indicate that abnormal proteolysis by MMP causes degradation of extracellular matrix components and initiates signaling resulting in neuronal cell death in acute ischemic stroke. Moreover, disruption of neurovascular integrity via MMP signaling can cause blood-brain barrier (BBB) leakage and hemorrhagic transformation (HT). Current studies of brain proteolysis mediated by MMPs have largely relied on in vitro and/or ex vivo assays by zymography, which do not yield information regarding spatial and temporal enzymatic activity in neurovascular units (comprising neurons, astroglia, and endothelium).
We have implemented a new approach to image brain proteolysis using activatable cell-penetrating peptides (CPP). We hypothesize that proteolysis-activatable CPP conjugated with a clinically-proven safe contrast-enhanced magnetic resonance imaging (MRI) agent super paramagnetic iron oxide (SPIO), will serve as a “target-specific” in vivo MRI contrast agent to evaluate dynamics of brain proteolysis of MMP after stroke. We will test this hypothesis using our well-established cerebral ischemia model in mice, which closely resembles that of acute stroke. We will investigate spatiotemporal activity of brain proteolysis in vivo using a 7-Tesla high-performance microMRI system, which may be adapted for clinical application for early diagnosis of acute stroke and/or to evaluate stroke treatment.
The Specific Aims are to determine: (1) whether the novel strategy using activatable CPPs is effective in the analysis of spatiotemporal proteolysis by MMPs in the ischemic brain and (2) whether analysis of brain proteolysis in vivo using a “target-specific,” biocompatible, contrast-enhanced MRI agent (CPP-SPIO) in conjunction with a high-resolution MRI system, can predict neurotoxicity and hemorrhage after cerebral ischemia in mice. Support from the Dana Foundation will launch this study and enable the junior investigators serving as PI and co-PI to develop an integrative research program in brain-immuno imaging. It will also allow us to generate pilot data crucial to obtain further funding. In summary, we seek to accelerate development of an innovative in vivo imaging strategy to investigate brain abnormal proteolysis after ischemic stroke.