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.

Live imaging of the innate immune response in neonates reveals differential TLR2 dependent activation patterns in sterile inflammation and infection.

Activation of microglial cells in response to brain injury and/or immune stimuli is associated with a marked induction of Toll-like receptors (TLRs). While in adult brain, the contribution of individual TLRs, including TLR2, in pathophysiological cascades has been well established, their role and spatial and temporal induction patterns in immature brain are far less understood. To examine whether infectious stimuli and sterile inflammatory stimuli trigger distinct TLR2-mediated innate immune responses, we used three models in postnatal day 9 (P9) mice, a model of infection induced by systemic endotoxin injection and two models of sterile inflammation, intra-cortical IL-1ß injection and transient middle cerebral artery occlusion (tMCAO). We took advantage of a transgenic mouse model bearing the dual reporter system luciferase/GFP under transcriptional control of a murine TLR2 promoter (TLR2-luc-GFP) to visualize the TLR2 response in the living neonatal brain and then determined neuroinflammation, microglial activation and leukocyte infiltration. We show that in physiological postnatal brain development the in vivo TLR2-luc signal undergoes a marked ∼30-fold decline and temporal-spatial changes during the second and third postnatal weeks. We then show that while endotoxin robustly induces the in vivo TLR2-luc signal in the living brain and increases levels of several inflammatory cytokines and chemokines, the in vivo TLR2-luc signal is reduced after both IL-1ß and tMCAO and the inflammatory response is muted. Immunofluorescence revealed that microglial cells are the predominant source of TLR2 production during postnatal brain development and in all three neonatal models studied. Flow cytometry revealed developmental changes in CD11b+/CD45+ and CD11b+/Ly6C+ cell populations, involvement of cells of the monocyte lineage, but lack of Ly6G+ neutrophils or CD3+ cells in acutely injured neonatal brains. Cumulatively, our results suggest distinct TLR2 induction patterns following PAMP and DAMP - mediated inflammation in immature brain.

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 MIP1ß 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 ß3 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.

Claudin-1, -2 and -3 Are Selectively Expressed in the Epithelia of the Choroid Plexus of the Mouse from Early Development and into Adulthood While Claudin-5 is Restricted to Endothelial Cells.

A primary function of epithelial and endothelial monolayers is the formation of barriers that separate tissues into functional compartments. Tight junctions (TJs) seal the intercellular space between the single cells of a monolayer. TJs thus contribute importantly to the homeostasis of the cerebrospinal fluid as they help in maintaining the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (CSF). The composition of TJs differs by its localization as well as the stage of development according to its respective function. Claudin-3 is typically present in the epithelia and has been claimed to be a constituent of the BBB. It is, however, notoriously difficult to demonstrate its expression in endothelial cells of the brain vasculature at the morphological level by means of immunohistochemical techniques. Using an improved fixation strategy (4% paraformaldehyde at pH 11, in the presence of EDTA) and the sensitive alkaline phosphatase as a detection system, we show that claudin-3 is present in mouse epithelia from embryonic day 14 onwards. In brain, it is restricted to the anlage of choroid plexus in the ventricles, together with claudin-1 and -2. In adult mice, it is clearly delineating the epithelium of the choroid plexus in the lateral and fourth ventricles. In contrast, in cerebral blood vessels claudin-3 as well as claudin-1 and -2 are absent in cerebral blood vessels during all developmental stages up to adulthood. Rather, the BBB is characterized by the presence of claudin-5, ZO-1 and occludin. Thus, in mice claudin-3 is an important constituent of TJ in the embryonic and in the adult choroid plexus.

The analysis of neurovascular remodeling in entorhino-hippocampal organotypic slice cultures.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

Barrier mechanisms in neonatal stroke.

Clinical data continue to reveal that the incidence of perinatal stroke is high, similar to that in the elderly. Perinatal stroke leads to significant morbidity and severe long-term neurological and cognitive deficits, including cerebral palsy. Experimental models of cerebral ischemia in neonatal rodents have shown that the pathophysiology of perinatal brain damage is multifactorial. Cerebral vasculature undergoes substantial structural and functional changes during early postnatal brain development. Thus, the state of the vasculature could affect susceptibility of the neonatal brain to cerebral ischemia. In this review, we discuss some of the most recent findings regarding the neurovascular responses of the immature brain to focal arterial stroke in relation to neuroinflammation. We also discuss a possible role of the neonatal blood-CSF barrier in modulating inflammation and the long-term effects of early neurovascular integrity after neonatal stroke on angiogenesis and neurogenesis.

Subfield-specific neurovascular remodeling in the entorhino-hippocampal-organotypic slice culture as a response to oxygen-glucose deprivation and excitotoxic cell death.

Transient ischemia causes delayed neurodegeneration in selective brain areas, particularly in the CA1 field of the hippocampus. This is accompanied by neurovascular impairment. It is unknown whether neurodegeneration is the cause or consequence of vascular changes. In an entorhino-hippocampal-organotypic slice culture system with well-preserved blood vessels, we studied the interplay between neurodegeneration and neurovasculature. Short-term oxygen and glucose deprivation (OGD) resulted in upregulation of hypoxic markers and with a delay of 24 to 48 hours in selective nerve cell death in CA1. In parallel, local vessel density decreased as detected by markers of endothelial cells and of the extracellular matrix. Claudin-5, a tight junction protein and marker of the blood-brain barrier was reduced. Preventing neuronal death with tetrodotoxin or 6-cyano-7-nitroquinoxaline-2,3-dione rescued blood vessels, suggesting that vessel loss is not due to OGD per se but a consequence of neuronal death. Induction of excitotoxic neuronal death with AMPA caused widespread neurodegeneration, but vessel reduction was confined to CA1. In dentate gyrus without neuronal loss, vessel density increased. We propose that neuronal stress and death influence maintenance, loss and remodeling of the neurovasculature and that the type of vascular response is in addition determined by local factors within the hippocampus.

The RNA-binding protein RBM3 is involved in hypothermia induced neuroprotection.

Induced hypothermia is the only therapy with proven efficacy to reduce brain damage after perinatal asphyxia. While hypothermia down-regulates global protein synthesis and cell metabolism, low temperature induces a small subset of proteins that includes the RNA-binding protein RBM3 (RNA-binding motif protein 3), which has recently been implicated in cell survival. Here, immunohistochemistry of the developing postnatal murine brain revealed a spatio-temporal neuronal RBM3 expression pattern very similar to that of doublecortin, a marker of neuronal precursor cells. Mild hypothermia (32°C) profoundly promoted RBM3 expression and rescued neuronal cells from forced apoptosis as studied in primary neurons, PC12 cells, and cortical organotypic slice cultures. Blocking RBM3 expression in neuronal cells by specific siRNAs significantly diminished the neuroprotective effect of hypothermia while vector-driven RBM3 over-expression reduced cleavage of PARP, prevented internucleosomal DNA fragmentation, and LDH release also in the absence of hypothermia. Together, neuronal RBM3 up-regulation in response to hypothermia apparently accounts for a substantial proportion of hypothermia-induced neuroprotection.

Roles of heat-shock protein 90 in maintaining and facilitating the neurodegenerative phenotype in tauopathies.

Neurodegeneration, a result of multiple dysregulatory events, is a lengthy multistep process manifested by accrual of mutant variants and abnormal expression, posttranslational modification, and processing of certain proteins. Accumulation of these dysregulated processes requires a mechanism that maintains their functional stability and allows the evolution of the neurodegenerative phenotype. In malignant cells, the capacity to buffer transformation has been attributed to heat-shock protein 90 (Hsp90). Although normal proteins seem to require limited assistance from the chaperone, their aberrant counterparts seem to be highly dependent on Hsp90. Whereas enhanced Hsp90 affinity for mutated or functionally deregulated client proteins has been observed for several oncoproteins, it is unknown whether Hsp90 plays a similar role for neuronal proteins and thus maintains and facilitates the transformed phenotype in neurodegenerative diseases. Tauopathies are neurodegenerative diseases characterized by aberrant phosphorylation and/or expression of Tau protein, leading to a time-dependent accumulation of Tau aggregates and subsequent neuronal death. Here, we show that the stability of p35, a neuronal protein that activates cyclin-dependent protein kinase 5 through complex formation leading to aberrant Tau phosphorylation, and that of mutant but not WT Tau protein is maintained in tauopathies by Hsp90. Inhibition of Hsp90 in cellular and mouse models of tauopathies leads to a reduction of the pathogenic activity of these proteins and results in elimination of aggregated Tau. The results identify important roles played by Hsp90 in maintaining and facilitating the degenerative phenotype in these diseases and provide a common principle governing cancer and neurodegenerative diseases.