LSR/angulin-1 is a tricellular tight junction protein involved in blood-brain barrier formation.

The blood-brain barrier (BBB) is a term used to describe the unique properties of central nervous system (CNS) blood vessels. One important BBB property is the formation of a paracellular barrier made by tight junctions (TJs) between CNS endothelial cells (ECs). Here, we show that Lipolysis-stimulated lipoprotein receptor (LSR), a component of paracellular junctions at points in which three cell membranes meet, is greatly enriched in CNS ECs compared with ECs in other nonneural tissues. We demonstrate that LSR is specifically expressed at tricellular junctions and that its expression correlates with the onset of BBB formation during embryogenesis. We further demonstrate that the BBB does not seal during embryogenesis in Lsr knockout mice with a leakage to small molecules. Finally, in mouse models in which BBB was disrupted, including an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and a middle cerebral artery occlusion (MCAO) model of stroke, LSR was down-regulated, linking loss of LSR and pathological BBB leakage

Microglial Cells Prevent Hemorrhage in Neonatal Focal Arterial Stroke

Perinatal stroke leads to significant morbidity and long-term neurological and cognitive deficits. The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. To understand whether microglial cells limit injury after neonatal stroke by preserving neurovascular integrity, we subjected postnatal day 7 (P7) rats depleted of microglial cells, rats with inhibited microglial TGFbr2/ALK5 signaling, and corresponding controls, to transient middle cerebral artery occlusion (tMCAO). Microglial depletion by intracerebral injection of liposome-encapsulated clodronate at P5 significantly reduced vessel coverage and triggered hemorrhages in injured regions 24 h after tMCAO. Lack of microglia did not alter expression or intracellular redistribution of several tight junction proteins, did not affect degradation of collagen IV induced by the tMCAO, but altered cell types producing TGFβ1 and the phosphorylation and intracellular distribution of SMAD2/3. Selective inhibition of TGFbr2/ALK5 signaling in microglia via intracerebral liposome-encapsulated SB-431542 delivery triggered hemorrhages after tMCAO, demonstrating that TGFβ1/TGFbr2/ALK5 signaling in microglia protects from hemorrhages. Consistent with observations in neonatal rats, depletion of microglia before tMCAO in P9 Cx3cr1(GFP/+)/Ccr2(RFP/+) mice exacerbated injury and induced hemorrhages at 24 h. The effects were independent of infiltration of Ccr2(RFP/+) monocytes into injured regions. Cumulatively, in two species, we show that microglial cells protect neonatal brain from hemorrhage after acute ischemic stroke. SIGNIFICANCE STATEMENT The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. We assessed whether microglial cells preserve neurovascular integrity after neonatal stroke. In neonatal rats, microglial depletion or pharmacological inhibition of TGFbr2/ALK5 signaling in microglia triggered hemorrhages in injured regions. The effect was not associated with additional changes in expression or intracellular redistribution of several tight junction proteins or collagen IV degradation induced by stroke. Consistent with observations in neonatal rats, microglial depletion in neonatal mice exacerbated stroke injury and induced hemorrhages. The effects were independent of infiltration of monocytes into injured regions. Thus, microglia protect neonatal brain from ischemia-induced hemorrhages, and this effect is consistent across two species

Microglial Cells Prevent Hemorrhage in Neonatal Focal Arterial Stroke

Perinatal stroke leads to significant morbidity and long-term neurological and cognitive deficits. The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. To understand whether microglial cells limit injury after neonatal stroke by preserving neurovascular integrity, we subjected postnatal day 7 (P7) rats depleted of microglial cells, rats with inhibited microglial TGFbr2/ALK5 signaling, and corresponding controls, to transient middle cerebral artery occlusion (tMCAO). Microglial depletion by intracerebral injection of liposome-encapsulated clodronate at P5 significantly reduced vessel coverage and triggered hemorrhages in injured regions 24 h after tMCAO. Lack of microglia did not alter expression or intracellular redistribution of several tight junction proteins, did not affect degradation of collagen IV induced by the tMCAO, but altered cell types producing TGFβ1 and the phosphorylation and intracellular distribution of SMAD2/3. Selective inhibition of TGFbr2/ALK5 signaling in microglia via intracerebral liposome-encapsulated SB-431542 delivery triggered hemorrhages after tMCAO, demonstrating that TGFβ1/TGFbr2/ALK5 signaling in microglia protects from hemorrhages. Consistent with observations in neonatal rats, depletion of microglia before tMCAO in P9 Cx3cr1(GFP/+)/Ccr2(RFP/+) mice exacerbated injury and induced hemorrhages at 24 h. The effects were independent of infiltration of Ccr2(RFP/+) monocytes into injured regions. Cumulatively, in two species, we show that microglial cells protect neonatal brain from hemorrhage after acute ischemic stroke. SIGNIFICANCE STATEMENT The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. We assessed whether microglial cells preserve neurovascular integrity after neonatal stroke. In neonatal rats, microglial depletion or pharmacological inhibition of TGFbr2/ALK5 signaling in microglia triggered hemorrhages in injured regions. The effect was not associated with additional changes in expression or intracellular redistribution of several tight junction proteins or collagen IV degradation induced by stroke. Consistent with observations in neonatal rats, microglial depletion in neonatal mice exacerbated stroke injury and induced hemorrhages. The effects were independent of infiltration of monocytes into injured regions. Thus, microglia protect neonatal brain from ischemia-induced hemorrhages, and this effect is consistent across two species

High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)–Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations

This note is to report how histological preparation techniques influence the extravasation pattern of the different molecular sizes of fluorescein isothiocyanate (FITC)–dextrans, typically used as markers for blood-brain barrier leakage. By using appropriate preparation methods, false negative results can be minimized. Wistar rats underwent a 2-h middle cerebral artery occlusion and magnetic resonance imaging. After the last imaging scan, Evans blue and FITC–dextrans of 4, 40, and 70 kDa molecular weight were injected. Different histological preparation methods were used. Sites of blood-brain barrier leakage were analyzed by fluorescence microscopy. Extravasation of Evans blue and high molecular FITC–dextrans (40 and 70 kDa) in the infarcted region could be detected with all preparation methods used. If exposed directly to saline, the signal intensity of these FITC–dextrans decreased. Extravasation of the 4-kDa low molecular weight FITC–dextran could only be detected using freshly frozen tissue sections. Preparations involving paraformaldehyde and sucrose resulted in the 4-kDa FITC–dextran dissolving in these reactants and being washed out, giving the false negative result of no extravasation. FITC–dextrans represent a valuable tool to characterize altered blood-brain barrier permeability in animal models. Diffusion and washout of low molecular weight FITC–dextran can be avoided by direct immobilization through immediate freezing of the tissue. This pitfall needs to be known to avoid the false impression that there was no extravasation of low molecular weight FITC–dextrans

Reduced brain edema and infarct volume in aquaporin-4 deficient mice after transient focal cerebral ischemia

Aquaporin-4 (AQP4) is a water channel expressed in astrocyte end-feet lining the blood-brain barrier. AQP4 deletion in mice is associated with improved outcomes in global cerebral ischemia produced by transient carotid artery occlusion, and focal cerebral ischemia produced by permanent middle cerebral artery occlusion (MCAO). Here, we investigated the consequences of 1-h transient MCAO produced by intraluminal suture blockade followed by 23 h of reperfusion. In nine AQP4(+/+) and nine AQP4(-/-) mice, infarct volume was significantly reduced by an average of 39 ± 4% at 24h in AQP4(-/-) mice, cerebral hemispheric edema was reduced by 23 ± 3%, and Evans Blue extravasation was reduced by 31 ± 2% (mean ± SEM). Diffusion-weighted magnetic resonance imaging showed greatest reduction in apparent diffusion coefficient around the occlusion site after reperfusion, with remarkably lesser reduction in AQP4(-/-) mice. The reduced infarct volume in AQP4(-/-) mice following transient MCAO supports the potential utility of therapeutic AQP4 inhibition in stroke

Acute and Chronic Vascular Responses to Experimental Focal Arterial Stroke in the Neonate Rat

The presence of active developmental angiogenesis and vascular outgrowth in the postnatal brain may differentially affect vascular responses to stroke in newborns and adults, but very little is known about the dynamics of vascular injury and re-growth after stroke during the neonatal period. In this study we used a clinically relevant animal model of ischemic arterial stroke in neonate rats, a transient middle cerebral artery occlusion (MCAO) in postnatal day 7 (P7), to characterize the effects of injury on vascular density and angiogenesis from acute through the chronic phase. A marked vessel degeneration and suppressed endothelial cell proliferation occur in the ischemic regions early after neonatal stroke. In contrast to what has been described in adult animals, endothelial cell proliferation and vascular density are not increased in the peri-ischemic regions during the first week after MCAO in neonates. By two weeks after injury, endothelial cell proliferation is increased in the cortical peri-ischemic region but these changes are not accompanied by an increased vascular density. Suppressed angiogenesis in injured postnatal brain that we report may limit recovery after neonatal stroke. Thus, enhancement of angiogenesis after neonatal stroke may be a promising strategy for the long-term recovery of the affected newborns

Genetic deletion of galectin-3 enhances neuroinflammation, affects microglial activation and contributes to sub-chronic injury in experimental neonatal focal stroke

The pathophysiology of neonatal stroke and adult stroke are distinct in many aspects, including the inflammatory response. We previously showed endogenously protective functions of microglial cells in acute neonatal stroke. We asked if galectin-3 (Gal3), a pleotropic molecule that mediates interactions between microglia/macrophages and the extracellular matrix (ECM), plays a role in early injury after transient middle cerebral occlusion (tMCAO) in postnatal day 9-10 mice. Compared to wild type (WT) pups, in Gal3 knockout pups injury was worse and cytokine/chemokine production altered, including further increase of MIP1α and MIP1β levels and reduced IL6 levels 72h after tMCAO. Lack of Gal3 did not affect morphological transformation or proliferation of microglia but markedly attenuated accumulation of CD11b+/CD45med-high cells after injury, as determined by multi-color flow cytometry. tMCAO increased expression of αV and β3 integrin subunits in CD11b+/CD45low microglial cells and cells of non-monocyte lineage (CD11b-/CD45-), but not in CD11b+/CD45med-high cells within injured regions of WT mice or Gal3-/- mice. αV upregulated in areas occupied and not occupied by CD68+ cells, most prominently in the ECM, lining blood vessels, with expanded αV coverage in Gal3-/- mice. Cumulatively, these data show that lack of Gal3 worsens subchronic injury after neonatal focal stroke, likely by altering the neuroinflammatory milieu, including an imbalance between pro- and anti-inflammatory molecules, effects on microglial activation, and deregulation of the composition of the ECM

Erythropoietin Increases Neurogenesis and Oligodendrogliosis of Subventricular Zone Precursor Cells After Neonatal Stroke

Background and purposeStroke is a common cause of neonatal brain injury. The subventricular zone is a lifelong source of newly generated cells in rodents, and erythropoietin (EPO) treatment has shown benefit in different animal models of brain injury. The purpose of this study is to investigate the specific role of exogenous EPO on subventricular zone progenitor cell populations in response to neonatal stroke.MethodsIntraventricular injections of green fluorescent protein (GFP)-expressing lentivirus to label subventricular zone precursor cells were made in postnatal day 1 (P1) Long-Evans rats, which then underwent transient middle cerebral artery occlusion on P7. Middle cerebral artery occlusion and sham rats were treated with either vehicle or EPO (1000 U/kg) at reperfusion, 24 hours, and 7 days later. The density of double-labeled DCx+/GFP+, NeuN+/GFP+, O4+/GFP+, GFAP+/GFP+, as well as single-labeled GFP+ and Ki67+ cells, was calculated to determine cell fate outcome in the striatum at 72 hours and 2 weeks after stroke.ResultsThere was a significant increase in DCx+/GFP+ and NeuN+/GFP+ neurons and O4+/GFP+ oligodendrocyte precursors, with decreased GFAP+/GFP+ astrocytes at both time points in EPO-middle cerebral artery occlusion animals. There was also a significant increase in GFP+ cells and Ki67+ proliferating cells in EPO compared with vehicle-middle cerebral artery occlusion animals.ConclusionsThese data suggest that subventricular zone neural progenitor cells proliferate and migrate to the site of injury after neonatal stroke and multiple doses of EPO, with a shift in cell fate toward neurogenesis and oligodendrogliosis at both early and late time points. The contribution of local cell proliferation and neurogenesis remains to be determined

Blood-Brain Barrier Permeability Is Increased After Acute Adult Stroke But Not Neonatal Stroke in the Rat

The immaturity of the CNS at birth greatly affects injury after stroke but the contribution of the blood-brain barrier (BBB) to the differential response to stroke in adults and neonates is poorly understood. We asked if the structure and function of the BBB is disrupted differently in neonatal and adult rats by transient middle cerebral artery occlusion. In adult rats, albumin leakage into injured regions was markedly increased during 2–24 h reperfusion but leakage remained low in the neonates. Functional assays employing intravascular tracers in the neonates showed that BBB permeability to both large (70-kDa dextran) and small (3-kDa dextran, Gd-DTPA) tracers remained largely undisturbed 24h after reperfusion. The profoundly different functional integrity of the BBB was associated with the largely nonoverlapping patterns of regulated genes in endothelial cells purified from injured and uninjured adult and neonatal brain at 24h (endothelial transcriptome, 31,042 total probe sets). Within significantly regulated 1,266 probe sets in injured adults and 361 probe sets in neonates, changes in the gene expression of the basal lamina components, adhesion molecules, the tight junction protein occludin, and MMP-9 were among the key differences. The protein expression of collagen-IV, laminin, claudin-5, occludin and ZO-1 was also better preserved in neonatal rats. Neutrophil infiltration remained low in acutely injured neonates but neutralization of CINC-1 in the systemic circulation enhanced neutrophil infiltration, BBB permeability and injury. The markedly more integrant BBB in neonatal brain than in adult brain after acute stroke may have major implications for the treatment of neonatal stroke

CX3CR1-CCR2-dependent monocyte-microglial signaling modulates neurovascular leakage and acute injury in a mouse model of childhood stroke.

Stroke is among the top 10 causes of death in children. The developmental stage of the brain is central to stroke pathophysiology. The incidence of childhood arterial ischemic stroke (CAIS) is lower than of perinatal arterial ischemic stroke but the rate of recurrence is strikingly high. Vascular inflammation is seen as major contributor to CAIS but the mechanisms that govern structural-functional basis of vascular abnormalities remain poorly understood. To identify the contribution of immune-neurovascular interactions to CAIS, we established stroke model in postnatal day 21 (P21) mice. We demonstrate acute functional deficits and histological injury and chronic MRI-identifiable injury, brain atrophy and marked derangements in the vascular network. In contrast to negligible albumin leakage and neutrophil infiltration following acute perinatal stroke, CAIS leads to significantly increased albumin leakage and neutrophil infiltration in injured regions of wild type mice and mice with functional CX3CR1-CCR2 receptors. In mice with dysfunctional CX3CR1-CCR2 signaling, extravascular albumin leakage is significantly attenuated, infiltration of injurious Ccr2+-monocytes essentially aborted, accumulation of Ly6G+ neutrophils reduced and acute injury attenuated. Unique identifiers of microglia and monocytes revealed phenotypic changes in each cell subtype of the monocyte lineage after CAIS. Taken together, CX3CR1-CCR2-dependent microglia-monocyte signaling contributes to cerebrovascular leakage, inflammation and CAIS injury