Junctional adhesion molecule-A deficient mice are protected from severe experimental autoimmune encephalomyelitis.

In multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), early pathological features include immune cell infiltration into the central nervous system (CNS) and blood-brain barrier (BBB) disruption. We investigated the role of junctional adhesion molecule-A (JAM-A), a tight junction protein, in active EAE (aEAE) pathogenesis. Our study confirms JAM-A expression at the blood-brain barrier and its luminal redistribution during aEAE. JAM-A deficient (JAM-A-/-) C57BL/6J mice exhibited milder aEAE, unrelated to myelin oligodendrocyte glycoprotein-specific CD4+ T-cell priming. While JAM-A absence influenced macrophage behavior on primary mouse brain microvascular endothelial cells (pMBMECs) under flow in vitro, it did not impact T-cell extravasation across primary mouse brain microvascular endothelial cells. At aEAE onset, we observed reduced lymphocyte and CCR2+ macrophage infiltration into the spinal cord of JAM-A-/- mice compared to control littermates. This correlated with increased CD3+ T-cell accumulation in spinal cord perivascular spaces and brain leptomeninges, suggesting JAM-A absence leads to T-cell trapping in central nervous system border compartments. In summary, JAM-A plays a role in immune cell infiltration and clinical disease progression in aEAE.

The choroid plexus acts as an immune cell reservoir and brain entry site in experimental autoimmune encephalomyelitis.

The choroid plexus (ChP) has been suggested as an alternative central nervous system (CNS) entry site for CCR6+ Th17 cells during the initiation of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). To advance our understanding of the importance of the ChP in orchestrating CNS immune cell entry during neuroinflammation, we here directly compared the accumulation of CD45+ immune cell subsets in the ChP, the brain and spinal cord at different stages of EAE by flow cytometry. We found that the ChP harbors high numbers of CD45int resident innate but also of CD45hi adaptive immune cell subsets including CCR6+ Th17 cells. With the exception to tissue-resident myeloid cells and B cells, numbers of CD45+ immune cells and specifically of CD4+ T cells increased in the ChP prior to EAE onset and remained elevated while declining in brain and spinal cord during chronic disease. Increased numbers of ChP immune cells preceded their increase in the cerebrospinal fluid (CSF). Th17 but also other CD4+ effector T-cell subsets could migrate from the basolateral to the apical side of the blood-cerebrospinal fluid barrier (BCSFB) in vitro, however, diapedesis of effector Th cells including that of Th17 cells did not require interaction of CCR6 with BCSFB derived CCL20. Our data underscore the important role of the ChP as CNS immune cell entry site in the context of autoimmune neuroinflammation.

Pro-resolving lipid mediator lipoxin A4 attenuates neuro-inflammation by modulating T cell responses and modifies the spinal cord lipidome.

The chronic neuro-inflammatory character of multiple sclerosis (MS) suggests that the natural process to resolve inflammation is impaired. This protective process is orchestrated by specialized pro-resolving lipid mediators (SPMs), but to date, the role of SPMs in MS remains largely unknown. Here, we provide in vivo evidence that treatment with the SPM lipoxin A4 (LXA4) ameliorates clinical symptoms of experimental autoimmune encephalomyelitis (EAE) and inhibits CD4+ and CD8+ T cell infiltration into the central nervous system (CNS). Moreover, we show that LXA4 potently reduces encephalitogenic Th1 and Th17 effector functions, both in vivo and in isolated human T cells from healthy donors and patients with relapsing-remitting MS. Finally, we demonstrate that LXA4 affects the spinal cord lipidome by significantly reducing the levels of pro-inflammatory lipid mediators during EAE. Collectively, our findings provide mechanistic insight into LXA4-mediated amelioration of neuro-inflammation and highlight the potential clinical application of LXA4 for MS.

Novel compounds with dual S1P receptor agonist and histamine H3 receptor antagonist activities act protective in a mouse model of multiple sclerosis.

The sphingosine 1-phosphate (S1P) receptor 1 (S1P1) has emerged as a therapeutic target for the treatment of multiple sclerosis (MS). Fingolimod (FTY720) is the first functional antagonist of S1P1 that has been approved for oral treatment of MS. Previously, we have developed novel butterfly derivatives of FTY720 that acted similar to FTY720 in reducing disease symptoms in a mouse model of experimental autoimmune encephalomyelitis (EAE). In this study, we have synthesized a piperidine derivative of the oxazolo-oxazole compounds, denoted ST-1505, and its ring-opened analogue ST-1478, and characterised their in-vitro and in-vivo functions. Notably, the 3-piperidinopropyloxy moiety resembles a structural motif of pitolisant, a drug with histamine H3R antagonistic/inverse agonist activity approved for the treatment of narcolepsy. Both novel compounds exerted H3R affinities, and in addition, ST-1505 was characterised as a dual S1P1+3 agonist, whereas ST-1478 was a dual S1P1+5 agonist. Both multitargeting compounds were also active in mice and reduced the lymphocyte numbers as well as diminished disease symptoms in the mouse model of MS. The effect of ST-1478 was dependent on SK-2 activity suggesting that it is a prodrug like FTY720, but with a more selective S1P receptor activation profile, whereas ST-1505 is a fully active drug even in the absence of SK-2. In summary, these data suggest that the well soluble piperidine derivatives ST-1505 and ST-1478 hold promise as novel drugs for the treatment of MS and other autoimmune or inflammatory diseases, and by their H3R antagonist potency, they might additionally improve cognitive impairment during disease.

T Cell-Mediated Transport of Polymer Nanoparticles across the Blood-Brain Barrier.

Delivery of therapeutics to the central nervous system (CNS) is challenging due to the presence of the blood-brain barrier (BBB). Amongst various approaches that have been explored to facilitate drug delivery to the CNS, the use of cells that have the intrinsic ability to cross the BBB is relatively unexplored, yet very attractive. This paper presents a first proof-of-concept that demonstrates the feasibility of activated effector/memory CD4+ helper T cells (CD4+ TEM cells) as carriers for the delivery of polymer nanoparticles across the BBB. This study shows that CD4+ TEM cells can be decorated with poly(ethylene glycol)-modified polystyrene nanoparticles using thiol-maleimide coupling chemistry, resulting in the immobilization of ≈105 nanoparticles per cell as determined by confocal microscopy. The ability of these cells to serve as carriers to transport nanoparticles across the BBB is established in vitro and in vivo. Using in vitro BBB models, CD4+ TEM cells are found to be able to transport nanoparticles across the BBB both under static conditions as well as under physiological flow. Finally, upon systemic administration, nanoparticle-modified T cells are shown to enter the brain parenchyma of mice, demonstrating the brain delivery potential of this T cell subset in allogeneic hosts.

Morpholino Analogues of Fingolimod as Novel and Selective S1P1 Ligands with In Vivo Efficacy in a Mouse Model of Experimental Antigen-Induced Encephalomyelitis.

Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system (CNS) which is associated with lower life expectancy and disability. The experimental antigen-induced encephalomyelitis (EAE) in mice is a useful animal model of MS, which allows exploring the etiopathogenetic mechanisms and testing novel potential therapeutic drugs. A new therapeutic paradigm for the treatment of MS was introduced in 2010 through the sphingosine 1-phosphate (S1P) analogue fingolimod (FTY720, Gilenya®), which acts as a functional S1P1 antagonist on T lymphocytes to deplete these cells from the blood. In this study, we synthesized two novel structures, ST-1893 and ST-1894, which are derived from fingolimod and chemically feature a morpholine ring in the polar head group. These compounds showed a selective S1P1 activation profile and a sustained S1P1 internalization in cultures of S1P1-overexpressing Chinese hamster ovary (CHO)-K1 cells, consistent with a functional antagonism. In vivo, both compounds induced a profound lymphopenia in mice. Finally, these substances showed efficacy in the EAE model, where they reduced clinical symptoms of the disease, and, on the molecular level, they reduced the T-cell infiltration and several inflammatory mediators in the brain and spinal cord. In summary, these data suggest that S1P1-selective compounds may have an advantage over fingolimod and siponimod, not only in MS but also in other autoimmune diseases.

CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens ß-Toxin.

Clostridium perfringens ß-toxin (CPB) is a highly active ß-pore-forming toxin (ß-PFT) and the essential virulence factor for fatal, necro-hemorrhagic enteritis in animals and humans. The molecular mechanisms involved in CPB's action on its target, the endothelium of small intestinal vessels, are poorly understood. Here, we identify platelet endothelial cell adhesion molecule-1 (CD31 or PECAM-1) as the specific membrane receptor for CPB on endothelial cells. CD31 expression corresponds with the cell-type specificity of CPB, and it is essential for toxicity in cultured cells and mice. Ectopic CD31 expression renders resistant cells and liposomes susceptible to CPB-induced membrane damage. Moreover, the extracellular Ig6 domain of mouse, human, and porcine CD31 is essential for the interaction with CPB. Hence, our results explain the cell-type specificity of CPB in vitro and in the natural disease caused by C. perfringens type C.

Claudin-12 is not required for blood-brain barrier tight junction function.

BACKGROUND: The blood-brain barrier (BBB) ensures central nervous system (CNS) homeostasis by strictly controlling the passage of molecules and solutes from the bloodstream into the CNS. Complex and continuous tight junctions (TJs) between brain endothelial cells block uncontrolled paracellular diffusion of molecules across the BBB, with claudin-5 being its dominant TJs protein. However, claudin-5 deficient mice still display ultrastructurally normal TJs, suggesting the contribution of other claudins or tight-junction associated proteins in establishing BBB junctional complexes. Expression of claudin-12 at the BBB has been reported, however the exact function and subcellular localization of this atypical claudin remains unknown. METHODS: We created claudin-12-lacZ-knock-in C57BL/6J mice to explore expression of claudin-12 and its role in establishing BBB TJs function during health and neuroinflammation. We furthermore performed a broad standardized phenotypic check-up of the mouse mutant. RESULTS: Making use of the lacZ reporter allele, we found claudin-12 to be broadly expressed in numerous organs. In the CNS, expression of claudin-12 was detected in many cell types with very low expression in brain endothelium. Claudin-12lacZ/lacZ C57BL/6J mice lacking claudin-12 expression displayed an intact BBB and did not show any signs of BBB dysfunction or aggravated neuroinflammation in an animal model for multiple sclerosis. Determining the precise localization of claudin-12 at the BBB was prohibited by the fact that available anti-claudin-12 antibodies showed comparable detection and staining patterns in tissues from wild-type and claudin-12lacZ/lacZ C57BL/6J mice. CONCLUSIONS: Our present study thus shows that claudin-12 is not essential in establishing or maintaining BBB TJs integrity. Claudin-12 is rather expressed in cells that typically lack TJs suggesting that claudin-12 plays a role other than forming classical TJs. At the same time, in depth phenotypic screening of clinically relevant organ functions of claudin-12lacZ/lacZ C57BL/6J mice suggested the involvement of claudin-12 in some neurological but, more prominently, in cardiovascular functions.

Publisher Correction: Claudin-3-deficient C57BL/6J mice display intact brain barriers.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The Genetic Background of Mice Influences the Effects of Cigarette Smoke on Onset and Severity of Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults leading to severe disability. Besides genetic traits, environmental factors contribute to MS pathogenesis. Cigarette smoking increases the risk of MS in an HLA-dependent fashion, but the underlying mechanisms remain unknown. Here, we explored the effect of cigarette smoke exposure on spontaneous and induced models of experimental autoimmune encephalomyelitis (EAE) by evaluating clinical disease and, when relevant, blood leukocytes and histopathology. In the relapsing-remitting (RR) transgenic model in SJL/J mice, we observed very low incidence in both smoke-exposed and control groups. In the optico-spinal encephalomyelitis (OSE) double transgenic model in C57BL/6 mice, the early onset of EAE prevented a meaningful evaluation of the effects of cigarette smoke. In EAE models induced by immunization, daily exposure to cigarette smoke caused a delayed onset of EAE followed by a protracted disease course in SJL/J mice. In contrast, cigarette smoke exposure ameliorated the EAE clinical score in C57BL/6J mice. Our exploratory studies therefore show that genetic background influences the effects of cigarette smoke on autoimmune neuroinflammation. Importantly, our findings expose the challenge of identifying an animal model for studying the influence of cigarette smoke in MS.

Claudin-3-deficient C57BL/6J mice display intact brain barriers.

The tight junction protein claudin-3 has been identified as a transcriptional target of the Wnt/ß-catenin signaling pathway regulating blood-brain barrier (BBB) maturation. In neurological disorders loss of claudin-3 immunostaining is observed at the compromised BBB and blood-cerebrospinal fluid barrier (BCSFB). Although these observations support a central role of claudin-3 in regulating brain barriers' tight junction integrity, expression of claudin-3 at the brain barriers has remained a matter of debate. This prompted us to establish claudin-3-/- C57BL/6J mice to study the role of claudin-3 in brain barrier integrity in health and neuroinflammation. Bulk and single cell RNA sequencing and direct comparative qRT-PCR analysis of brain microvascular samples from WT and claudin-3-/- mice show beyond doubt that brain endothelial cells do not express claudin-3 mRNA. Detection of claudin-3 protein at the BBB in vivo and in vitro is rather due to junctional reactivity of anti-claudin-3 antibodies to an unknown antigen still detected in claudin-3-/- brain endothelium. We confirm expression and junctional localization of claudin-3 at the BCSFB of the choroid plexus. Our study clarifies that claudin-3 is not expressed at the BBB and shows that absence of claudin-3 does not impair brain barrier function during health and neuroinflammation in C57BL/6J mice.

VLA-4 mediated adhesion of melanoma cells on the blood-brain barrier is the critical cue for melanoma cell intercalation and barrier disruption.

Melanoma is the most aggressive skin cancer in humans. One severe complication is the formation of brain metastasis, which requires extravasation of melanoma cells across the tight blood-brain barrier (BBB). Previously, VLA-4 has been assigned a role for the adhesive interaction of melanoma cells with non-BBB endothelial cells. However, the role of melanoma VLA-4 for breaching the BBB remained unknown. In this study, we used a mouse in vitro BBB model and imaged the shear resistant arrest of melanoma cells on the BBB. Similar to effector T cells, inflammatory conditions of the BBB increased the arrest of melanoma cells followed by a unique post-arrest behavior lacking immediate crawling. However, over time, melanoma cells intercalated into the BBB and compromised its barrier properties. Most importantly, antibody ablation of VLA-4 abrogated melanoma shear resistant arrest on and intercalation into the BBB and protected the BBB from barrier breakdown. A tissue microarray established from human brain metastasis revealed that indeed a majority of 92% of all human melanoma brain metastases stained VLA-4 positive. We propose VLA-4 as a target for the inhibition of brain metastasis formation in the context of personalized medicine identifying metastasizing VLA-4 positive melanoma.

Ischemia-reperfusion injury in stroke: impact of the brain barriers and brain immune privilege on neutrophil function.

Reperfusion injury following ischemic stroke is a complex pathophysiological process involving numerous mechanisms ranging from the release of excitatory amino acids and ion disequilibrium to the induction of apoptosis and necrosis, to oxidative stress and inflammation. The migration of neutrophils into the brain parenchyma and release of their abundant proteases are generally considered the main cause of neuronal cell death and acute reperfusion injury following ischemic stroke. Recent findings in experimental and human stroke have challenged this view, as the majority of neutrophils were rather found to accumulate within the neurovascular unit (NVU) and the subarachnoid space (SAS) where they remain separated from the brain parenchyma by the glia limitans. The brain parenchyma is an immune-privileged site that is not readily accessible to immune cells and does not elicit stereotypic adaptive or innate immune responses. Understanding brain immune privilege requires intimate knowledge of its unique anatomy in which the brain barriers, that include the glia limitans, establish compartments that differ remarkably with regard to their accessibility to the immune system. We here propose that the brain immune privilege also extends to an ischemic insult, where the brain parenchyma does not evoke a rapid infiltration of neutrophils as observed in ischemic events in peripheral organs. Rather, neutrophil accumulation in the NVU and SAS could have a potential impact on cerebrospinal fluid (CSF) drainage from the central nervous system (CNS) and thus on edema formation and reperfusion injury after ischemic stroke. Integrating the anatomical and functional implications of the brain immune privilege with the unquestionable role of neutrophils in reperfusion injury is a prerequisite to exploit appropriate strategies for therapeutic interventions aiming to reduce neuronal cell death after ischemic stroke.

Lack of junctional adhesion molecule (JAM)-B ameliorates experimental autoimmune encephalomyelitis.

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) autoaggressive CD4+ T cells cross the blood-brain barrier (BBB) and cause neuroinflammation. Therapeutic targeting of CD4+ T-cell trafficking into the CNS by blocking α4-integrins has proven beneficial for the treatment of MS but comes with associated risks, probably due to blocking CD8+ T cell mediated CNS immune surveillance. Our recent observations show that CD8+ T cells also rely on α4ß1-integrins to cross the BBB. Besides vascular cell adhesion molecule-1 (VCAM-1), we identified junctional adhesion molecule-B (JAM-B) as a novel vascular α4ß1-integrin ligand involved in CD8+ T-cell migration across the BBB. This prompted us to investigate, if JAM-B also mediates CD4+ T-cell migration across the BBB. We first ensured that encephalitogenic T cells can bind to JAM-B in vitro and next compared EAE pathogenesis in JAM-B-/- C57BL/6J mice and their wild-type littermates. Following immunization with MOGaa35-55 peptide, JAM-B-/- mice developed ameliorated EAE compared to their wild-type littermates. At the same time, we isolated higher numbers of CD45+ infiltrating immune cells from the CNS of JAM-B-/- C57BL/6J mice suffering from EAE. Immunofluorescence staining revealed that the majority of CD45+ inflammatory cells accumulated in the leptomeningeal and perivascular spaces of the CNS behind the BBB but do not gain access to the CNS parenchyma. Trapping of CNS inflammatory cells was not due to increased inflammatory cell proliferation. Neither a loss of BBB integrity or BBB polarity potentially affecting local chemokine gradients nor a lack of focal gelatinase activation required for CNS parenchymal immune cell entry across the glia limitans could be detected in JAM-B-/- mice. Lack of a role for JAM-B in the effector phase of EAE was supported by the observation that we did not detect any role for JAM-B in EAE pathogenesis, when EAE was elicited by in vitro activated MOG aa35-55-specific CD4+ effector T cells. On the other hand, we also failed to demonstrate any role of JAM-B in in vivo priming, proliferation or polarization of MOGaa35-55-specific CD4+ T cells in peripheral immune organs. Finally, our study excludes expression of and thus a role for JAM-B on peripheral and CNS infiltrating myeloid cells. Taken together, although endothelial JAM-B is not required for immune cell trafficking across the BBB in EAE, in its absence accumulation of inflammatory cells mainly in CNS leptomeningeal spaces leads to amelioration of EAE.

ICAM-1null C57BL/6 Mice Are Not Protected from Experimental Ischemic Stroke.

Accumulation of neutrophils in the brain is a hallmark of cerebral ischemia and considered central in exacerbating tissue injury. Intercellular adhesion molecule (ICAM)-1 is upregulated on brain endothelial cells after ischemic stroke and considered pivotal in neutrophil recruitment as ICAM-1-deficient mouse lines were found protected from experimental stroke. Translation of therapeutic inhibition of ICAM-1 into the clinic however failed. This prompted us to investigate stroke pathogenesis in Icam1tm1Alb C57BL/6 mutants, a true ICAM-1null mouse line. Performing transient middle cerebral artery occlusion, we found that absence of ICAM-1 did not ameliorate stroke pathology at acute time points after reperfusion. Near-infrared imaging showed comparable accumulation of neutrophils in the ischemic hemispheres of ICAM-1null and wild type C57BL/6 mice. We also isolated equal numbers of neutrophils from the ischemic brains of ICAM-1null and wild type C57BL/6 mice. Immunostaining of the brains showed neutrophils to equally accumulate in the leptomeninges and brain parenchymal vessels of ICAM-1null and wild type C57BL/6 mice. In addition, the lesion size was comparable in ICAM-1null and wild type mice. Our study demonstrates that absence of ICAM-1 neither inhibits cerebral ischemia-induced accumulation of neutrophils in the brain nor provides protection from ischemic stroke.

The choroid plexus is a key cerebral invasion route for T cells after stroke.

Neuroinflammation contributes substantially to stroke pathophysiology. Cerebral invasion of peripheral leukocytes-particularly T cells-has been shown to be a key event promoting inflammatory tissue damage after stroke. While previous research has focused on the vascular invasion of T cells into the ischemic brain, the choroid plexus (ChP) as an alternative cerebral T-cell invasion route after stroke has not been investigated. We here report specific accumulation of T cells in the peri-infarct cortex and detection of T cells as the predominant population in the ipsilateral ChP in mice as well as in human post-stroke autopsy samples. T-cell migration from the ChP to the peri-infarct cortex was confirmed by in vivo cell tracking of photoactivated T cells. In turn, significantly less T cells invaded the ischemic brain after photothrombotic lesion of the ipsilateral ChP and in a stroke model encompassing ChP ischemia. We detected a gradient of CCR2 ligands as the potential driving force and characterized the neuroanatomical pathway for the intracerebral migration. In summary, our study demonstrates that the ChP is a key invasion route for post-stroke cerebral T-cell invasion and describes a CCR2-ligand gradient between cortex and ChP as the potential driving mechanism for this invasion route.

A Novel Cervical Spinal Cord Window Preparation Allows for Two-Photon Imaging of T-Cell Interactions with the Cervical Spinal Cord Microvasculature during Experimental Autoimmune Encephalomyelitis.

T-cell migration across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Two-photon intravital microscopy (2P-IVM) has been established as a powerful tool to study cell-cell interactions in inflammatory EAE lesions in living animals. In EAE, central nervous system inflammation is strongly pronounced in the spinal cord, an organ in which 2P-IVM imaging is technically very challenging and has been limited to the lumbar spinal cord. Here, we describe a novel spinal cord window preparation allowing to use 2P-IVM to image immune cell interactions with the cervical spinal cord microvascular endothelium during EAE. We describe differences in the angioarchitecture of the cervical spinal cord versus the lumbar spinal cord, which will entail different hemodynamic parameters in these different vascular beds. Using T cells as an example, we demonstrate the suitability of this novel methodology in imaging the post-arrest multistep T-cell extravasation across the cervical spinal cord microvessels. The novel methodology includes an outlook to the analysis of the cellular pathway of T-cell diapedesis across the BBB by establishing visualization of endothelial junctions in this vascular bed.

ALCAM (CD166) is involved in extravasation of monocytes rather than T cells across the blood-brain barrier.

Activated leukocyte cell adhesion molecule (ALCAM) has been proposed to mediate leukocyte migration across the blood-brain barrier (BBB) in multiple sclerosis or experimental autoimmune encephalomyelitis (EAE). Here, we confirmed vascular ALCAM expression in human brain tissue samples in situ and on two different human in vitro BBB models. Antibody-mediated inhibition of ALCAM reduced diapedesis of human CD4+ Th1 but not of Th17 cells across the human BBB in vitro. In accordance to human Th1 cells, mouse Th1 cells showed reduced diapedesis across an ALCAM-/- in vitro BBB model under static but no longer under flow conditions. In contrast to the limited role of ALCAM in T cell extravasation across the BBB, we found a contribution of ALCAM to rolling, adhesion, and diapedesis of human CD14+ monocytes across the human BBB under flow and static conditions. Taken together, our study highlights the potential differences in the CNS expression of ALCAM in mouse and human and supports a prominent role for ALCAM in the multi-step extravasation of monocytes across the BBB.

Non-invasive near-infrared fluorescence imaging of the neutrophil response in a mouse model of transient cerebral ischaemia.

Near-infrared fluorescence (NIRF) imaging enables non-invasive monitoring of molecular and cellular processes in live animals. Here we demonstrate the suitability of NIRF imaging to investigate the neutrophil response in the brain after transient middle cerebral artery occlusion (tMCAO). We established procedures for ex vivo fluorescent labelling of neutrophils without affecting their activation status. Adoptive transfer of labelled neutrophils in C57BL/6 mice before surgery resulted in higher fluorescence intensities over the ischaemic hemisphere in tMCAO mice with NIRF imaging when compared with controls, corroborated by ex vivo detection of labelled neutrophils using fluorescence microscopy. NIRF imaging showed that neutrophils started to accumulate immediately after tMCAO, peaking at 18 h, and were still visible until 48 h after reperfusion. Our data revealed accumulation of neutrophils also in extracranial tissue, indicating damage in the external carotid artery territory in the tMCAO model. Antibody-mediated inhibition of α4-integrins did reduce fluorescence signals at 18 and 24, but not at 48 h after reperfusion, compared with control treatment animals. Antibody treatment reduced cerebral lesion volumes by 19%. In conclusion, the non-invasive nature of NIRF imaging allows studying the dynamics of neutrophil recruitment and its modulation by targeted interventions in the mouse brain after transient experimental cerebral ischaemia.