Research ReportPost-ischemic blood–brain barrier leakage in rats: One-week follow-up by MRI
Introduction
The blood–brain barrier (BBB), composed of endothelial cells and their connections within the brain microvasculature, is the main safeguard of the central nervous system (CNS), controlling its microenvironment. A secondary barrier is provided by the extracellular matrix and basal lamina of the endothelial cells, which are in close interaction with adjacent cells (i.e., astrocytes, pericytes, and mast cells; Strbian et al., 2006). Cerebral ischemia triggers a complex sequence of pathophysiological events (Durukan and Tatlisumak, 2007). Additional harmful mechanisms may occasionally appear with reperfusion, including BBB disruption, the consequences of which may be manifold: edema formation, hemorrhagic transformation, and access of potentially toxic molecules and pathogens to the CNS. All of these contribute to brain damage in the acute phase and may exacerbate brain injury. On the other hand, a leaky BBB may prove necessary to allow entrance into the CNS of blood-borne cell populations involved in the repair process or of potential neuroprotective and neurorestorative drugs. It is therefore important to know the exact temporal dynamics and the magnitude of (any) post-ischemic BBB leakage.
In animal models of cerebral ischemia–reperfusion, BBB disruption is widely believed to be biphasic, which implies an early first opening followed by a refractory period when the BBB is closed, and a delayed second opening (Kuroiwa et al., 1985, Belayev et al., 1996, Rosenberg et al., 1998, Huang et al., 1999). However, several recent works showed no full recovery after the first opening (Kastrup et al., 1999, Wu et al., 2001, Nagel et al., 2004, Veltkamp et al., 2005), indicating a continuous leakage for days (Nagel et al., 2008) or weeks (Lin et al., 2002, Strbian et al., 2008), with or without alteration in its magnitude. But only very limited quantitative data exist concerning BBB permeability beyond 24 h. Vast differences in methodology (qualitative or quantitative approaches in BBB-permeability analysis, varying size of tracers, different animal models) and comprehensiveness (time-points varying from 3 to 15 h) hinder the achieving of any common conclusion.
BBB evaluation with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is a promising in vivo tool to explore BBB dynamics. DCE-MRI allows quantification of parameters related to BBB permeability by several analytic methods (Tofts, 1997). Among these, the multiple time graphic method, Patlak plotting, is one validated against standardized quantitative autoradiography in the rat model of transient focal cerebral ischemia (Ewing et al., 2003, Knight et al., 2005) and it is increasingly used (Candelario-Jalil et al., 2007, Sood et al., 2008). Because DCE-MRI is minimally invasive, it can be repeated in a single group of animals, eliminating inter-group variability. Hence, such a longitudinal study design with BBB-permeability imaging would provide more robust data than from multiple-group studies.
We therefore aimed to reevaluate the presumed pattern of biphasic BBB opening following transient cerebral ischemia by a quantitative and longitudinal MRI approach. For this, we used the intraluminal suture occlusion of the middle cerebral artery (MCAO, a common method mimicking malignant infarction of the MCA) in the rat (n = 10), which is the most common species subjected to MRI-brain ischemia studies. We repeated DCE-MRI at five distinct time-points after reperfusion (2, 24, 48, 72 h, and 1 week). Analyzing the magnitude of contrast enhancement and the blood-to-brain transfer rate constant (Ki) of Patlak, we characterized post-ischemic BBB permeability to gadolinium.
Section snippets
Physiological parameters
Rectal temperature and mean arterial blood pressure measurements were obtained at baseline, after MCAO, and after reperfusion (Table 1). Rectal temperatures fell within physiological ranges (36.0 to 37.4 °C) at all measurements. Blood pressure values showed no significant change among animals or time-points (p = 0.102, RM-ANOVA).
Ischemic lesion size
All ten animals showed substantial baseline lesions on diffusion-weighted images (DWI) at 2 h, which were similar (p = 0.971, ANOVA). During the experiment, mean ischemic
Discussion
Post-ischemic BBB leakage, a major culprit in the devastating consequences following ischemic stroke such as edema and hemorrhagic transformation, is far from being fully understood. In our earlier study (Strbian et al., 2008), we showed continuous and long-lasting BBB leakage after ischemia–reperfusion, which was in opposition to the generally accepted biphasic BBB opening in the same scenario. That study utilized both invasive and minimally invasive methods; hence, separate sets of rats were
Animals
Adult male Wistar rats (Harlan Nederland, Horst, The Netherlands, n = 10), weighing 290 to 340 g and kept in a 12/12 h light/dark cycle with free access to food and water, were anesthetized by an intraperitoneal injection of ketamine hydrochloride (50 mg/kg, Ketalar, Parke-Davis, Detroit, MI) and a subcutaneous injection of medetomidine hydrochloride (0.5 mg/kg, Domitor, Orion, Espoo, Finland). A PE-50 polyethylene tube was inserted into the left femoral artery for blood pressure monitoring (Olli
Acknowledgments
This study was supported by grants from the Helsinki University Central Hospital (DS, TT), the Sigrid Juselius Foundation (TT), the Maire Taponen Foundation (UAR, DS, TT), and the Finnish Academy of Sciences (TT). We thank Dr. Carol Norris for author-editing the language.
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These two authors contributed equally to this work.