The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

Abstract

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.

Fig. 1

Localization of PMNs in the ischemic brain following 60 min of tMCAO and 18 h and 24 h reperfusion. a Inflammatory cells were isolated from the ipsilateral ischemic and contralateral hemisphere of 6 mice following 60 min tMCAO and 24 h reperfusion by enzymatic digestion and density gradient centrifugation, and analyzed by flow cytometry. Using Forward (FSC) and Side Scatter (SSC) profiles inflammatory cells were separated according to size (FSC) and granularity (SSC), respectively, and displayed in a dot blot. PMNs are characterized by a high SSC signal due to their high content of granules. Such a population was only found in the ipsilateral and not in the contralateral hemisphere. Positive Ly6G immunoreactivity of the scatter gated population, as depicted in the histogram (right side), confirmed their identity as PMNs. b Schematic representation of the two planes of the brain examined (X Bregma 0.50 mm, Y Bregma −2.46 mm) and corresponding coronal sections showing areas analyzed. Immunohistochemistry of sections from 60 min tMCAO and 24 h perfusion for CD45 and Ly6G reveal distribution of total leukocytes and PMNs, respectively, in the meninges (Mng), motor–sensory cortex (Ctx), and striatum (Str). The majority of cells were detected either intra- or perivascular in the meninges (arrows) and the penumbral cortex (arrowheads) and the cells comprised mainly Ly6G⁺ PMNs. Bar is 50 μm

Fig. 2

Localization of neutrophils in the neurovascular unit. a Confocal microscopy of a thick section (100 μm) double stained for laminin α5, showing the inner endothelial BM, and laminin α2, showing the outer parenchymal BM, and b schematic representation of the constituents of the NVU. Relative sizes and numbers of the NVU constituents are not to scale. MØ refers to macrophages and DC is dendritic cells. c Schematic representation of the plane of the brain examined (X Bregma 0.50 mm) and corresponding coronal section showing areas analyzed (striatum Str, meninges Mng and motor–sensory cortex Ctx). Corresponding confocal microscopy of thick sections from plane X of 60 min tMCAO, 24 h reperfusion, double stained with antibodies to pan-laminin, to mark all BMs, and CD45, or to laminin α5, as a marker of the endothelial BM, and Ly6G, reveal localization of total leukocytes and PMNs mainly in association with arteries or large arterioles (outer panels) and veins (middle panel). Images shown are from area Ctx. d Analyses of individual Z stacks permitted localization of Ly6G⁺ PMNs within vessel lumina or between the endothelial and parenchymal BM (vessel associated), or intra-parenchymally. Staining shown is for an arteriole. Graphs to the right show total Ly6G⁺ PMN numbers/0.001 mm³ brain volume normalized to the proportion of the brain volume occupied by vessels in Str striatum, Mng meninges and Ctx cortex. Normalized Ly6G⁺ PMN numbers associated with vessels in the different areas are expressed as percentages of the normalized total Ly6G⁺ cell numbers and show no statistically significant differences from 100 % cells associated with vessels (P values 0.56–0.86). Data shown are mean ± SEM from 2 to 3 mice. NS is no statistically significant difference. Bars in c and d are 40 μm

Fig. 3

Endothelial adhesion molecules or platelet accumulation are not sufficient for Ly6G⁺ PMN extravasation in vivo. Data shown are for 60 min tMCAO and 24 h reperfusion (16 μm sections). Double immunofluorescence staining for Ly6G and intercellular adhesion molecule-1 (ICAM-1) and 2 (ICAM-2), vascular endothelial cell adhesion molecule (VCAM-1), or P-selectin showed some upregulation of adhesion molecules in cortical and meningeal microvessels of the ischemic hemisphere but no strict spatial correlation with Ly6G⁺ PMNs which localize intraluminally (arrows), perivascularly (arrowhead) or in the meningeal compartment (asterisk). Immunofluorescence staining for CD41⁺ platelets and Ly6G revealed that platelet accumulation in vessels was not associated with the localization of PMNs. Bars are 40 μm

Fig. 4

PMN interaction with endothelium in an in vitro blood–brain barrier model. PMN interaction with pMBMECs under normoxic, ischemic (OGD + reox) or IL-1β stimulated conditions under physiological flow (1.5 dyn/cm²) was recorded for 20 min and the dynamic behaviour of arrested PMNs was analyzed. a The number of PMNs arrested on pMBMECs counted per field of view (FOV). b Proportions of PMNs that were stationary, crawling, undergoing diapedesis or detachment from pMBMECs. PMNs that remained immobile on the monolayer were defined as ‘Stationary’, PMNs that polarized and crawled on the monolayer but did not diapedese across the endothelial monolayer were described as ‘Crawling’, PMNs that crawled until they found a suitable site for diapedesis were defined as undergoing ‘Diapedsis’, and PMNs that detached during the video acquisition time were termed ‘Detachment’. Data in a and b are mean ± SD, n = 3. c Immunofluorescence staining of pMBMECs for ICAM-1 and ICAM-2 under normoxic, ischemic, and IL-1β stimulated conditions shows upregulation of ICAM-1 under both, ischemic and IL-1β stimulation, ICAM-2 staining remains unaffected. Endothelial cells are counter-stained with Hoechst dye to show the cell nuclei. Bar 50 μm

Fig. 5

Histopathology of human acute stroke specimens (Stage I). a H&E staining of human stage I stroke specimen demonstrating demarcation of the ischemic core from the penumbra (arrows); b H&E staining (high magnification of a) showing the presence of eosinophilic neurons indicating an early ischemic neuronal damage; c At the infarct border zone, eosinophilic neurons (arrowhead, as depicted in Fig. 5b) are surrounded by glial cells showing severe hypoxic changes (arrows) as indicated by the strong expression of hypoxia-inducible factor-1 alpha (Hif1alpha); d CD15 immunohistochemistry indicating that PMNs are primarily located within blood vessels in acute human stroke lesions; e characterization of cells with histomorphological features of PMNs within the CNS parenchyma: while intravascular cells with PMN-like morphology (as seen in Fig. 5d) were strongly CD15-positive, intraparenchymally located cells exhibiting PMN-like morphology and being CD15-negative are strongly positive for cleaved caspase-3 (arrow) indicating that these cells undergo apoptosis; f CD68-positive cells of the monocytic lineage are mainly located in the perivascular space or within the brain parenchyma. Data shown are from a 75-year-old male patient suffering from an acute right parietal ischemic infarct (for details see Supplementary Table 1)