Carcinoembryonic Antigen–Related Cell Adhesion Molecule 1 Inhibits MMP-9–Mediated Blood–Brain–Barrier Breakdown in a Mouse Model for Ischemic Stroke

Abstract

E very year, ischemic stroke accounts for ≈10% of global deaths, leaving 5 million patients permanently disabled, not only in high-income, but also in less-developed countries. 1 Besides the primary hypoxic damage and secondary processes, such as excitotoxicity, recruitment of inflammatory cells following restoration of cerebral perfusion contributes to ischemic brain injury.2,3 Especially, inflammatory events occurring at the blood-brain barrier (BBB) during cerebral ischemia are critical for the pathogenesis of tissue damage in ischemic stroke. Cell adhesion molecules (eg, intercellular adhesion molecule 1 4 and the selectins) 5 are involved in the development of postischemic brain inflammation by promoting cell adhesion, migration, and activation of leukocytes. Although genetic deficiency or antibody blocking of cellular adhesion molecules, such as intercellular adhesion molecule 1 and platelet selectin, demonstrated attenuated cerebral damage in mice, [5][6][7] links between early BBB breakdown and the innate immune response are still not fully understood. Neutrophils are an essential component of the innate immune system and among the first cells to adhere to the cerebral endothelium and infiltrate the ischemic brain. 8 This postischemic inflammation leads to impairment of BBB function and neuronal damage by release of matrix metalloproteinases (MMPs), for example MMP-9 and proinflammatory cytokines, including interleukin (IL)-1β, as well as reactive oxygen species. Objective: We sought to identify and characterize the relevance of CEACAM1-expressing inflammatory cells in BBB breakdown and outcome after ischemic stroke in Ceacam1 −/− and wild-type mice. Methods and Results:Focal ischemia was induced by temporary occlusion of the middle cerebral artery with a microfilament. Using MRI and Evans blue permeability assays, we observed increased stroke volumes, BBB breakdown and edema formation, reduction of cerebral perfusion, and brain atrophy in Ceacam1 −/− mice. This translated into poor performance in neurological scoring and high poststroke-associated mortality. Elevated neutrophil influx, hyperproduction, and release of neutrophil-related matrix metalloproteinase-9 in Ceacam1 −/− mice were confirmed by immune fluorescence, flow cytometry, zymography, and stimulation of neutrophils. Importantly, neutralization of matrix metalloproteinase-9 activity in Ceacam1 −/− mice was sufficient to alleviate stroke sizes and improve survival to the level of CEACAM1-competent animals. Immune histochemistry of murine and human poststroke autoptic brains congruently identified abundance of CEACAM1 + matrix metalloproteinase-9 + neutrophils in the ischemic hemispheres. Conclusions: CEACAM1 controls matrix metalloproteinase-9 secretion by neutrophils in postischemic inflammationat the BBB after stroke. We propose CEACAM1 as an important inhibitory regulator of neutrophil-mediated tissue damage and BBB breakdown in focal cerebral ischemia. Thus, understanding the regulation of neutrophils at the ischemic BBB will provide nov...

Iron Overload Exacerbates the Risk of Hemorrhagic Transformation After tPA (Tissue-Type Plasminogen Activator) Administration in Thromboembolic Stroke Mice

“…On the other hand, neutrophil infiltration, which according to the literature takes places mainly in the infarct core extending from the cortical surface along large vessels, 30 was decreased in tPA 20-minute groups and increased in iron overloaded animals when administering tPA 3 hours after ischemia, likely as a consequence, respectively, of reduced infarct size in early reperfusion and increased reactive oxygen species production in iron overloaded animals. Although some publications point to neutrophils as key players in HT, 31,32 our results support just a secondary role. However, because of an increased oxidative damage in animals with high levels of iron, neutrophil N1 polarization 33 could also contribute to vascular damage.…”

Matrix metalloproteinases in the brain and blood–brain barrier: Versatile breakers and makers

“…While most MMP research in stroke is focused on MMP-2 and MMP-9, 183,184 other MMPs also have detrimental effects. Suzuki et al 185 treated mice after thrombotic MCAO with tissue-type plasminogen activator (tPA) and observed an increased incidence of intra-cranial bleeding compared to mice not treated with tPA.…”

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

“…Cresyl violet or fluoro-jade staining have less rigid time requirements for staining after sacrifice, but these classic stains require very thin sections obtained by cryostat or microtome, and thus require more time for integrative summation and analysis. 14 Furthermore, these stains cannot be used in conjunction with EB quantification of BBB disruption, as developed by others, 15 eliminating the possibility of direct comparison between infarct volume and BBB integrity in a given brain. It should be noted that RHP-treated animals typically exhibited pink as opposed to pure white volumes.…”

“…Others have injected EB immediately following reperfusion and allowed EB to circulate for up to 72 hr, 41 or at 4 hr post-tMCAo to circulate for 4 hr, 15 as alternative approaches. The amount of EB that crosses the disrupted BBB and enters brain tissue represents the integral accumulation of albumin-bound dye from time of injection to time of sacrifice.…”