The entry of most plasma-borne materials into the brain is normally blocked by a blood-brain barrier (BBB) that protects the brain cells from the untoward effects of such substances. Injury to the BBB in ischemic stroke often leads to the leakage of ions, water, amino acids, and plasma proteins into the brain and as a result in large strokes significant brain swelling may occur before treatment is initiated. Over time this decay in barrier function can worsen such that red blood cells extravasate and form hemorrhages, a process referred to as hemorrhagic transformation (HT). Thrombolytic therapy with tissue plasminogen activator (tPA) acts to increase blood flow to the ischemic tissue and is the only approved treatment for acute ischemic stroke. But its usage increases the risk of symptomatic HT ten-fold. At present, the only criterion for tPA treatment of ischemic stroke is time (3 hr post-ictus and CT exam negative for bleeding, as no diagnostic imaging indicators are available for excluding high-risk stroke patients. Our previous stroke studies indicate that regions with acute BBB injury that show leakage of magnetic resonance contrast agents (MRCAs) and elevated T1sat, a magnetization transfer parameter, often develop HT at later times. From this work, two quantitative magnetic resonance imaging (MRI) methods have been identified as possible predictors of BBB injury and HT: 1) magnetization transfer MRI (MT-MRI), particularly the T1sat parameter;and 2) MRCA enhanced MRI of the blood-to-brain distribution of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA), with the influx rate assayed by Patlak plots. Neither the degree of acute BBB opening, predictive of severe vasogenic edema and HT, nor the physiological changes in the BBB components that contribute to these altered MRI signals are known. This proposal aims to test if the size of the MT- and/or MRCA-MRI parameter changes at an early stage of stroke can be used to establish a threshold of BBB damage that portends subsequent HT and if changes of this magnitude or larger can be used to identify subjects that are likely to develop HT after thrombolytic therapy. Two rat middle cerebral artery (MCA) occlusion models, intraluminal filament and embolic, will be used.
Aim 1 is a translational aim;its purpose is to develop a predictive model that links the degree of abnormality of the MT- and/or MRCA-MRI parameters during the first several hours of ischemia and reperfusion to the probability of developing subsequent HT following tPA treatment. If successful, this predictive model would lay the groundwork for clinical trials and might be useful for selecting or rejecting patients for tPA treatment.
Aim 2 is mechanistic in the sense that we will investigate physiological factors that may contribute to these changes in the MT and MRCA-MRI parameters and to acute BBB injury. The long-term goals of this proposal are to develop MRI signatures that can identify tissue at risk for HT, provide criteria for selecting patients for tPA treatment and to investigate the basic mechanisms of BBB opening and edema formation in stroke. Modified Specific Section Specific Aim 1: To quantify: 1) MT-MRI parameters at approximately 2, 5 and 48 hr after ischemia onset;2) the influx of Gd-DTPA across the BBB (a measure of permeability) at approximately 5 and 48 hr after ischemia onset;and 3) ischemic injury at 48 hr as determined by histological assessment and tissue hemoglobin content (a quantitative assay of HT) in both intraluminal suture and embolic stroke model in the rat with half of the animals receiving tPA and the other half placebo 3 hr after MCA occlusion (i.e. shortly after the acute imaging studies and reperfusion;n=20 per group). These data will be used to develop a predictive model that links the degree of MRI abnormality after several hours of ischemia to the incidence of HT at 48 hr and the probability of tPA treatment inducing HT. Hypothesis:
This aim tests the hypothesis that the probability of tPA inducing HT can be accessed from MRI measures of T1sat and MRCA influx during the first few hours of cerebral ischemia and reperfusion.
Specific Aim 2 : To quantify the same set of MRI and histological parameters as in Aim 1, plus estimates of brain swelling by MRI and histology and the expression of various BBB-associated proteins by immunohistochemistry (IHC), in selected regions of interest (ROIs) in animals subjected to 3 hrs of MCA occlusion with half of the animals treated with tPA and half without. MRI studies will be performed at time points during ischemia (~ 2 hr post MCA occlusion) and at approximately 7, 11 and 24 hrs post-occlusion after which the brains will be taken for histological and IHC analysis (n=10 per time and treatment group). Hypothesis:
This aim will test the hypothesis that: 1) changes in the MT parameters will be proportional to Gd-DTPA influx (i.e., BBB opening) and both will be functions of the variations in BBB associated proteins and/or brain swelling;and 2) the size of these changes are correlated over time, ROI, and treatment.
In this proposal we will use MRI, histology and immunohistochemical methods to study the progression of brain tissue injury in ischemia stroke from acute vascular injury to hemorrhagic transformation in two animal stroke models with reperfusion and treatment. We expect that this study will establish MRI-based guidelines for choosing thrombolytic therapy for the treatment of ischemia in rats and will lead to the development of a similar risk assessment model for stroke patients. The observations on the BBB-associated proteins with and without tPA treatment should be useful as we search for therapies to seal the leaky BBB and block the untoward effects of that pathology in stroke.
