Imaging neuroinflammation after stroke: current status of cellular and molecular MRI strategies.

Cellular and molecular magnetic resonance imaging (MRI) strategies for studying the spatiotemporal profile of neuroinflammatory processes after stroke are increasingly being explored since the first reports appeared about a decade ago. These strategies most often employ (super)paramagnetic contrast agents, such as (ultra)small particles of iron oxide and gadolinium chelates, for MRI-based detection of specific leukocyte populations or molecular inflammatory markers that are involved in the pathophysiology of stroke or plasticity. In this review we describe achievements, limitations and prospects in the field of cellular and molecular MRI of neuroinflammation in preclinical and clinical stroke. Several studies in rodent stroke models have demonstrated the application of MRI contrast agents for imaging of monocyte infiltration, which served as the foundation for pilot (small-scale proof-of-concept) cellular MRI studies in stroke patients. This may be achieved with isolated cells that are loaded with contrast agent through in vitro incubation prior to systemic administration. Alternatively, superparamagnetic iron oxide particles may be directly injected into the circulation to allow in vivo uptake by phagocytic cells. Both strategies have been successfully employed to measure the spatiotemporal profile of invasion of monocytes in and around cerebral ischemic lesions in experimental stroke models. Molecular MRI studies with target-specific contrast agents have shown the capability for in vivo detection of molecular markers after experimental stroke. For example, (super)paramagnetic micro- or nanoparticles that are functionalized with a ligand (e.g. an antibody) for specific cell adhesion molecules, such as E-selectin and vascular cell adhesion molecule 1 (VCAM-1), can target inflamed, activated endothelium, whose presence can subsequently be detected with MRI. Present applications remain limited as most of the currently available contrast agents provide relatively poor contrast enhancement, which is not easily discriminated from endogenous sources of tissue contrast. Nevertheless, current developments of more efficient particles, such as biocompatible liposomes, micelles and nanoemulsions that can contain high payloads of (super)paramagnetic material as well as other substances, such as dyes and drugs, may open a window of opportunities for promising translational multimodal imaging strategies that enable in vivo assessment of (neuroinflammatory) disease markers, therapeutic targets as well as drug delivery after stroke.

Magnetic resonance imaging of brain angiogenesis after stroke.

Stroke is a major cause of mortality and long-term disability worldwide. The initial changes in local perfusion and tissue status underlying loss of brain function are increasingly investigated with noninvasive imaging methods. In addition, there is a growing interest in imaging of processes that contribute to post-stroke recovery. In this review, we discuss the application of magnetic resonance imaging (MRI) to assess the formation of new vessels by angiogenesis, which is hypothesized to participate in brain plasticity and functional recovery after stroke. The excellent soft tissue contrast, high spatial and temporal resolution, and versatility render MRI particularly suitable to monitor the dynamic processes involved in vascular remodeling after stroke. Here we review recent advances in the field of MR imaging that are aimed at assessment of tissue perfusion and microvascular characteristics, including cerebral blood flow and volume, vascular density, size and integrity. The potential of MRI to noninvasively monitor the evolution of post-ischemic angiogenic processes is demonstrated from a variety of in vivo studies in experimental stroke models. Finally, we discuss some pitfalls and limitations that may critically affect the accuracy and interpretation of MRI-based measures of (neo)vascularization after stroke.

In Vivo Molecular MRI of ICAM-1 Expression on Endothelium and Leukocytes from Subacute to Chronic Stages After Experimental Stroke.

Molecular MRI allows in vivo detection of vascular cell adhesion molecules expressed on inflamed endothelium, which enables detection of specific targets for anti-neuroinflammatory treatment. We explored to what extent MR contrast agent targeted to intercellular adhesion molecule-1 (ICAM-1) could detect endothelial- and leukocyte-associated ICAM-1 expression at different stages after experimental stroke. Furthermore, we assessed potential interfering effects of ICAM-1-targeted contrast agent on post-stroke lesion growth. Micron-sized particles of iron oxide (MPIO) functionalized with control IgG (IgG-MPIO) or anti-ICAM-1 antibody (αICAM-1-MPIO) were administrated at 1, 2, 3, 7, and 21 days after unilateral transient middle cerebral artery occlusion in mice, followed by in vivo MRI and postmortem immunohistochemistry. αICAM-1-MPIO induced significant contrast effects in the lesion core on post-stroke days 1, 2, and 3, and in the lesion borderzone and contralesional tissue on post-stroke day 2. αICAM-1-MPIO were confined to ICAM-1-positive vessels and occasionally co-localized with leukocytes. On post-stroke day 21, abundant leukocyte-associated αICAM-1-MPIO was immunohistochemically detected in the lesion core. However, MRI-based detection of αICAM-1-MPIO-labeled leukocytes was confounded by pre-contrast MRI hypointensities, presumably caused by phagocytosed blood remains. IgG-MPIO did not induce significant MRI contrast effects at 1 h after injection. Lesion development was not affected by injection of αICAM-1-MPIO or IgG-MPIO. αICAM-1-MPIO are suitable for in vivo MRI of ICAM-1 expression on vascular endothelium and leukocytes at different stages after stroke. Development of clinically applicable MPIO may offer unique opportunities for MRI-based diagnosis of neuroinflammation and identification of anti-inflammatory targets in acute stroke patients.

Uric Acid Is Protective After Cerebral Ischemia/Reperfusion in Hyperglycemic Mice.

Hyperglycemia at stroke onset is associated with poor long-term clinical outcome in numerous studies. Hyperglycemia induces intracellular acidosis, lipid peroxidation, and peroxynitrite production resulting in the generation of oxidative and nitrosative stress in the ischemic tissue. Here, we studied the effects of acute hyperglycemia on in vivo intercellular adhesion molecule-1 (ICAM-1) expression, neutrophil recruitment, and brain damage after ischemia/reperfusion in mice and tested whether the natural antioxidant uric acid was protective. Hyperglycemia was induced by i.p. administration of dextrose 45 min before transient occlusion of the middle cerebral artery. Magnetic resonance imaging (MRI) was performed at 24 h to measure lesion volume. A group of normoglycemic and hyperglycemic mice received an i.v. injection of micron-sized particles of iron oxide (MPIOs), conjugated with either anti-ICAM-1 antibody or control IgG, followed by T2*w MRI. Neutrophil infiltration was studied by immunofluorescence and flow cytometry. A group of hyperglycemic mice received an i.v. infusion of uric acid (16 mg/kg) or the vehicle starting after 45 min of reperfusion. ICAM-1-targeted MPIOs induced significantly larger MRI contrast-enhancing effects in the ischemic brain of hyperglycemic mice, which also showed more infiltrating neutrophils and larger lesions than normoglycemic mice. Uric acid reduced infarct volume in hyperglycemic mice but it did not prevent vascular ICAM-1 upregulation and did not significantly reduce the number of neutrophils in the ischemic brain tissue. In conclusion, hyperglycemia enhances stroke-induced vascular ICAM-1 and neutrophil infiltration and exacerbates the brain lesion. Uric acid reduces the lesion size after ischemia/reperfusion in hyperglycemic mice.

In vivo MR imaging of intercellular adhesion molecule-1 expression in an animal model of multiple sclerosis.

Upregulation of intercellular adhesion molecule 1 (ICAM-1) is an early event in lesion formation in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Monitoring its expression may provide a biomarker for early disease activity and allow validation of anti-inflammatory interventions. Our objective was therefore to explore whether ICAM-1 expression can be visualized in vivo during EAE with magnetic resonance imaging (MRI) using micron-sized particles of iron oxide (MPIO), and to compare accumulation profiles of targeted and untargeted MPIO, and a gadolinium-containing agent. Targeted αICAM-1-MPIO/untargeted IgG-MPIO were injected at two model-characteristic phases of EAE (in myelin oligodendrocyte glycoprotein35-55 -immunized C57BL/6 J mice), that is, at the peak of the acute phase (14 ± 1 days post-immunization) and during the chronic phase (26 ± 1 days post-immunization), followed by T2 *-weighted MRI. Blood-brain barrier (BBB) permeability was measured using gadobutrol-enhanced MRI. Cerebellar microvessels were analyzed for ICAM-1 mRNA expression using quantitative PCR (qPCR). ICAM-1 and iron oxide presence was examined with immunohistochemistry (IHC). During EAE, ICAM-1 was expressed by brain endothelial cells, macrophages and T-cells as shown with qPCR and (fluorescent) IHC. EAE animals injected with αICAM-1-MPIO showed MRI hypointensities, particularly in the subarachnoid space. αICAM-1-MPIO presence did not differ between the phases of EAE and was not associated with BBB dysfunction. αICAM-1-MPIO were associated with endothelial cells or cells located at the luminal side of blood vessels. In conclusion, ICAM-1 expression can be visualized with in vivo molecular MRI during EAE, and provides an early tracer of disease activity.

PECAM-1-targeted micron-sized particles of iron oxide as MRI contrast agent for detection of vascular remodeling after cerebral ischemia.

An increasing amount of studies have provided evidence for vascular remodeling, for example, angiogenesis, after cerebral ischemia, which may play a significant role in post-stroke brain plasticity and recovery. Molecular imaging can provide unique in vivo whole-brain information on alterations in the expression of specific endothelial markers. A possible target for molecular magnetic resonance imaging (MRI) of post-stroke (neo)vascularization is platelet endothelial cell adhesion molecule-1 (PECAM-1). Here we describe significantly increased PECAM-1 mRNA levels in ipsilesional brain tissue at 6 h, 24 h and 3 days after transient middle cerebral artery occlusion in mice, and elevated PECAM-1 staining throughout the lesion at 3, 7 and 21 days post-stroke. The potential of micron-sized particles of iron oxide (MPIO) conjugated with PECAM-1-targeted antibodies, that is, αPECAM-1-MPIO, to expose stroke-induced PECAM-1 upregulation with molecular MRI was assessed. In vitro studies demonstrated that PECAM-1-expressing brain endothelial cells could be effectively labeled with αPECAM-1-MPIO, giving rise to a fourfold increase in MRI relaxation rate R2. Injection of near-infrared fluorescent dye-labeled αPECAM-1 showed target specificity and dose efficiency of the antibody for detection of brain endothelial cells at 3 days post-stroke. However, in vivo molecular MRI at 3 and 7 days after stroke revealed no αPECAM-1-MPIO-based contrast enhancement, which was corroborated by the absence of αPECAM-1-MPIO in post mortem brain tissue. This indicates that this molecular MRI approach, which has been proven successful for in vivo detection of other types of cell adhesion molecules, is not invariably effective for MRI-based assessment of stroke-induced alterations in expression of cerebrovascular markers.

MRI of ICAM-1 upregulation after stroke: the importance of choosing the appropriate target-specific particulate contrast agent.

PURPOSE: Magnetic resonance imaging (MRI) with targeted contrast agents provides a promising means for diagnosis and treatment monitoring after cerebrovascular injury. Our goal was to demonstrate the feasibility of this approach to detect the neuroinflammatory biomarker intercellular adhesion molecule-1 (ICAM-1) after stroke and to establish a most efficient imaging procedure. PROCEDURES: We compared two types of ICAM-1-functionalized contrast agent: T 1-shortening gadolinium chelate-containing liposomes and T2(*)-shortening micron-sized iron oxide particles (MPIO). Binding efficacy and MRI contrast effects were tested in cell cultures and a mouse stroke model. RESULTS: Both ICAM-1-targeted agents bound effectively to activated cerebrovascular cells in vitro, generating significant MRI contrast-enhancing effects. Direct in vivo MRI-based detection after stroke was only achieved with ICAM-1-targeted MPIO, although both contrast agents showed similar target-specific vascular accumulation. CONCLUSIONS: Our study demonstrates the potential of in vivo MRI of post-stroke ICAM-1 upregulation and signifies target-specific MPIO as most suitable contrast agent for molecular MRI of cerebrovascular inflammation.

Tumor angiogenesis phenotyping by nanoparticle-facilitated magnetic resonance and near-infrared fluorescence molecular imaging.

One of the challenges of tailored antiangiogenic therapy is the ability to adequately monitor the angiogenic activity of a malignancy in response to treatment. The α(v)ß(3) integrin, highly overexpressed on newly formed tumor vessels, has been successfully used as a target for Arg-Gly-Asp (RGD)-functionalized nanoparticle contrast agents. In the present study, an RGD-functionalized nanocarrier was used to image ongoing angiogenesis in two different xenograft tumor models with varying intensities of angiogenesis (LS174T > EW7). To that end, iron oxide nanocrystals were included in the core of the nanoparticles to provide contrast for T(2)*-weighted magnetic resonance imaging (MRI), whereas the fluorophore Cy7 was attached to the surface to enable near-infrared fluorescence (NIRF) imaging. The mouse tumor models were used to test the potential of the nanoparticle probe in combination with dual modality imaging for in vivo detection of tumor angiogenesis. Pre-contrast and post-contrast images (4 hours) were acquired at a 9.4-T MRI system and revealed significant differences in the nanoparticle accumulation patterns between the two tumor models. In the case of the highly vascularized LS174T tumors, the accumulation was more confined to the periphery of the tumors, where angiogenesis is predominantly occurring. NIRF imaging revealed significant differences in accumulation kinetics between the models. In conclusion, this technology can serve as an in vivo biomarker for antiangiogenesis treatment and angiogenesis phenotyping.

Multifunctional imaging nanoprobes.

Multifunctional imaging nanoprobes have proven to be of great value in the research of pathological processes, as well as the assessment of the delivery, fate, and therapeutic potential of encapsulated drugs. Moreover, such probes may potentially support therapy schemes by the exploitation of their own physical properties, e.g., through thermal ablation. This review will present four classes of nanoparticulate imaging probes used in this area: multifunctional probes (1) that can be tracked with at least three different and complementary imaging techniques, (2) that carry a drug and have bimodal imaging properties, (3) that are employed for nucleic acid delivery and imaging, and (4) imaging probes with capabilities that can be used for thermal ablation. We will highlight several examples where the suitable combination of different (bio)materials like polymers, inorganic nanocrystals, fluorophores, proteins/peptides, and lipids can be tailored to manufacture multifunctional probes to accomplish nanomaterials of each of the aforementioned classes. Moreover, it will be demonstrated how multimodality imaging approaches improve our understanding of in vivo nanoparticle behavior and efficacy at different levels, ranging from the subcellular level to the whole body.