Computed tomography (CT) and magnetic resonance imaging (MRI) have revolutionized ischemic stroke diagnosis and management. Initially restricted to structural imaging to exclude bleeding, these imaging modalities can now detect intracranial vessel occlusion, evaluate the ischemic penumbra to select candidates for thrombectomy, and play a key role in identifying stroke etiology. Molecular imaging has the potential to further expand the information provided by CT and MRI by revealing the biological processes that constitute potential diagnostic or therapeutic targets [12]. The use of nanotechnology to design contrast agents has led to significant advances in the field [13].

The recent development of a new family of contrast agents for MRI based on micro-sized particles of iron oxide (MPIOs) has allowed a large increase in the sensitivity and specificity of this imaging modality, paving the way for clinical application [14]. The use of larger particles with a diameter of at least 1 µm has several advantages compared to the more classically used ultrasmall superparamagnetic iron oxide particles (20–50 nm). Indeed, it prevents the passive leakage of particles in the brain parenchyma and increases the payload of the contrast material per particle [15]. Using MPIOs coupled with monoclonal antibodies, non-invasive detection of specific proteins expressed by the cerebrovasculature has been demonstrated in neurovascular disorders in the last few years [16].

The combination of in vivo MRI with NP contrast agents has allowed the clarification of many aspects of post-stroke inflammation, which is one of the main pathogenic processes occurring in the acute and subacute phases of stroke [17]. Monitoring the temporo-spatial regulation of the inflammatory reaction in the affected brain parenchyma could have significant clinical applications, such as identifying patient subsets that could benefit from immunomodulatory treatments. In this regard, the endothelial cells of the blood–brain barrier (BBB) are key players in post-stroke inflammation by mediating the diapedesis of leukocytes from the blood to the brain through the expression of adhesion molecules. Since these adhesion molecules are easily accessible by large contrast-carrying particles, they constitute interesting targets for molecular imaging. Using MPIOs coupled with monoclonal antibodies targeting vascular cell adhesion molecule-1 (VCAM-1), molecular MRI has provided evidence for the existence of a new concept defined as “inflammatory penumbra” in ischemic stroke [18]. In mice, 24 h after ischemic stroke, this technique unveiled an inflammatory area at risk, surrounding the initial ischemic lesion, which was secondarily infiltrated by lymphocytes and ultimately recruited by the ischemic core [18]. By analogy with the ischemic penumbra, the mismatch between the VCAM-1-overexpressing region (revealed by molecular MRI) and the ischemic core (revealed by diffusion-weighted imaging 24 h post onset) was called the “inflammatory penumbra”. Interestingly, the size of this “inflammatory penumbra” varied according to the duration and subtypes of ischemic stroke in experimental models, suggesting that there could be a wide variability in the intensity and spatial extent of the inflammatory reaction between patients.

Besides VCAM-1, other adhesion molecules are involved in the diapedesis of leukocytes from the blood to the brain. For instance, Deddens and co-workers performed molecular MRI of intercellular adhesion molecule-1 (ICAM-1) in an experimental model of ischemic stroke in mice. They used MPIOs targeting ICAM-1 to reveal the overexpression of ICAM-1 by activated endothelial cells at different time points after ischemic onset [19]. They found that ICAM-1 expression was maximal at 48 h and extended in the peri-infarct area, in line with the “inflammatory penumbra” concept. Whether VCAM-1 and ICAM-1 imaging methods provide differential information on stroke, pathophysiology remains to be explored. Moreover, Quenault et al. demonstrated that molecular MRI of P-selectin using MPIOs could be employed to diagnose a transient ischemic attack (TIA). In a TIA, the duration of ischemia is too short to induce changes on an unenhanced MRI scan. Interestingly, experimental studies have revealed that P-selectin is overexpressed for at least 24 h in the affected vascular territory. Using molecular MRI of P-selectin, it is therefore possible to detect the endothelial activation triggered by the TIA and distinguish TIA from stroke mimics, such as epilepsy or migraine [20].

Molecular MRI can also aid in the etiologic assessment of ischemic stroke. Indeed, it has been previously demonstrated that ruptured atherosclerotic plaques overexpress endothelial activation markers. Using MPIOs targeting P-selectin and VCAM-1, McAteer et al. demonstrated that it is possible to detect the activated endothelium inside atherosclerotic plaques using MRI [21]. Molecular MRI of endothelial activation in stroke patients could thus be utilized to identify the culprit vascular lesion and therefore better characterize stroke etiology.

Even if its sensitivity to detect contrast material is lower than that of MRI, CT can also be used for molecular imaging using NPs. This has been demonstrated in experimental models of carotid artery thrombosis and embolic ischemic stroke [22]. Kim et al. showed that fibrin-targeted gold NPs can reveal cerebral thrombus as a high-density endovascular material on CT images. In acute stroke settings, this would allow the assessment of thrombus burden and monitoring of thrombolytic therapy in a non-invasive manner.

Given the demonstration of the high sensitivity and specificity of molecular imaging of inflammation offered by MPIO-enhanced molecular MRI in experimental models, efforts are ongoing to translate this method to clinical imaging. Indeed, the MPIOs used in preclinical studies are non-biodegradable because of their coating and inner structure. In this context, the development of biocompatible MPIOs is mandatory. To overcome this limitation, Perez-Balderas et al. recently reported the production of multimeric magnetite particles that form large MPIO-like particles that are biodegradable [23]. In an experimental model of neuroinflammation, they demonstrated that this method allowed non-invasive imaging of activated endothelial cells.