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.

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⁺/CD45^med-high cells after injury, as determined by multi-color flow cytometry. tMCAO increased expression of αV and β₃ integrin subunits in CD11b⁺/CD45^low microglial cells and cells of non-monocyte lineage (CD11b^-/CD45^-), but not in CD11b⁺/CD45^med-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.

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-1β 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-1β 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.

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.