Development of the Central Nervous System (CNS) is reliant on the proper function of numerous intricately orchestrated mechanisms that mature independently, including constant communication between the CNS and the peripheral immune system. This review summarizes experimental knowledge of how cerebral ischaemia in infants and children alters physiological communication between leucocytes, brain immune cells, microglia and the neurovascular unit (NVU)-the "microglia-leucocyte axis"-and contributes to acute and long-term brain injury. We outline physiological development of CNS barriers in relation to microglial and leucocyte maturation and the plethora of mechanisms by which microglia and peripheral leucocytes communicate during postnatal period, including receptor-mediated and intracellular inflammatory signalling, lipids, soluble factors and extracellular vesicles. We focus on the "microglia-leucocyte axis" in rodent models of most common ischaemic brain diseases in the at-term infants, hypoxic-ischaemic encephalopathy (HIE) and focal arterial stroke and discuss commonalities and distinctions of immune-neurovascular mechanisms in neonatal and childhood stroke compared to stroke in adults. Given that hypoxic and ischaemic brain damage involve Toll-like receptor (TLR) activation, we discuss the modulatory role of viral and bacterial TLR2/3/4-mediated infection in HIE, perinatal and childhood stroke. Furthermore, we provide perspective of the dynamics and contribution of the axis in cerebral ischaemia depending on the CNS maturational stage at the time of insult, and modulation independently and in consort by individual axis components and in a sex dependent ways. Improved understanding on how to modify crosstalk between microglia and leucocytes will aid in developing age-appropriate therapies for infants and children who suffered cerebral ischaemia.

Brain injury in the perinatal period occurs in many clinical settings, e.g. hypoxic-ischemic encephalopathy (HIE) in term infants, neonatal stroke, encephalopathy of prematurity, and infections. These insults often result in life-long disabilities including cerebral palsy, cognitive deficits, visual dysfunction, hearing impairments, and epilepsy. However, the success of clinical implementation of a broad array of potential neuroprotective strategies tested experimentally has been limited with the exception of therapeutic hypothermia (TH) used within hours of birth in term human babies with mild to moderate HIE. There is an extensive search for adjuvant therapeutic approaches to enhance the outcomes. One strategy is to modify susceptibility in the developing CNS by means of preconditioning or postconditioning using sublethal stress. The pre-clinical and clinical literature has shown that CNS immaturity at the time of ischemic insult plays a central role in the response to injury. Thus, better understanding of the molecular regulation of the endogenous vulnerability of the immature brain is needed. Further, the use of sublethal stressors of different origin may help shed light on mechanistic similarities and distinctions beween conditioning strategies. In this review we discuss the mechanisms of protection that are achieved by an interplay of changes on the systemic level and brain level, and via changes of intracellular and mitochondrial signaling. We also discuss the barriers to improving our understanding of how brain immaturity and the type of insult-hypoxic, ischemic or inflammatory-affect the efficacy of conditioning efforts in the immature brain.

Brain injury in the perinatal period occurs in many clinical settings, e.g. hypoxic-ischemic encephalopathy (HIE) in term infants, neonatal stroke, encephalopathy of prematurity, and infections. These insults often result in life-long disabilities including cerebral palsy, cognitive deficits, visual dysfunction, hearing impairments, and epilepsy. However, the success of clinical implementation of a broad array of potential neuroprotective strategies tested experimentally has been limited with the exception of therapeutic hypothermia (TH) used within hours of birth in term human babies with mild to moderate HIE. There is an extensive search for adjuvant therapeutic approaches to enhance the outcomes. One strategy is to modify susceptibility in the developing CNS by means of preconditioning or postconditioning using sublethal stress. The pre-clinical and clinical literature has shown that CNS immaturity at the time of ischemic insult plays a central role in the response to injury. Thus, better understanding of the molecular regulation of the endogenous vulnerability of the immature brain is needed. Further, the use of sublethal stressors of different origin may help shed light on mechanistic similarities and distinctions beween conditioning strategies. In this review we discuss the mechanisms of protection that are achieved by an interplay of changes on the systemic level and brain level, and via changes of intracellular and mitochondrial signaling. We also discuss the barriers to improving our understanding of how brain immaturity and the type of insult-hypoxic, ischemic or inflammatory-affect the efficacy of conditioning efforts in the immature brain.

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 incidence of perinatal stroke is high, similar to that in the elderly, and produces a significant morbidity and severe long-term neurologic and cognitive deficits, including cerebral palsy, epilepsy, neuropsychological impairments, and behavioral disorders. Emerging clinical data and data from experimental models of cerebral ischemia in neonatal rodents have shown that the pathophysiology of perinatal brain damage is multifactorial. These studies have revealed that, far from just being a smaller version of the adult brain, the neonatal brain is unique with a very particular and age-dependent responsiveness to hypoxia-ischemia and focal arterial stroke. In this review, we discuss fundamental clinical aspects of perinatal stroke as well as some of the most recent and relevant findings regarding the susceptibility of specific brain cell populations to injury, the dynamics and the mechanisms of neuronal cell death in injured neonates, the responses of neonatal blood-brain barrier to stroke in relation to systemic and local inflammation, and the long-term effects of stroke on angiogenesis and neurogenesis. Finally, we address translational strategies currently being considered for neonatal stroke as well as treatments that might effectively enhance repair later after injury.

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.

Individuals born preterm have higher rates of neurodevelopmental disorders such as schizophrenia, autistic spectrum, and attention deficit/hyperactivity disorders. These conditions are often sexually dimorphic and with different developmental trajectories. The etiology is likely multifactorial, however, infections both during pregnancy and in childhood have emerged as important risk factors. The association between sex- and age-dependent vulnerability to neuropsychiatric disorders has been suggested to relate to immune activation in the brain, including complex interactions between sex hormones, brain transcriptome, activation of glia cells, and cytokine production. Here, we will review sex-dependent effects on brain development, including glia cells, both under normal physiological conditions and following perinatal inflammation. Emphasis will be given to sex-dependent effects on brain regions which play a role in neuropsychiatric disorders and inflammatory reactions that may underlie early-life programming of neurobehavioral disturbances later in life.

Microglial cells are now recognized as the "gate-keepers" of healthy brain microenvironment with their disrupted functions adversely affecting neurovascular integrity, neuronal homeostasis, and network connectivity. The perception that these cells are purely toxic under neurodegenerative conditions has been challenged by a continuously increasing understanding of their complexity, the existence of a broad array of microglial phenotypes, and their ability to rapidly change in a context-dependent manner to attenuate or exacerbate injuries of different nature. Recent studies have demonstrated that microglial cells exert crucial physiological functions during embryonic and postnatal brain development, some of these functions being unique to particular stages of development, and extending far beyond sensing dangerous signals and serving as antigen presenting cells. In this focused review we cover the roles of microglial cells in regulating embryonic vasculogenesis, neurogenesis, and establishing network connectivity during postnatal brain development. We further discuss context-dependent microglial contribution to neonatal brain injuries associated with prenatal and postnatal infection and inflammation, in relation to neurodevelopmental disorders, as well as perinatal hypoxia-ischemia and arterial focal stroke. We also emphasize microglial phenotypic diversity, notably at the ultrastructural level, and their sex-dependent influence on the pathophysiology of neurodevelopmental disorders.

Neonatal stroke is as frequent as stroke in the elderly, but many pathophysiological injury aspects are distinct in neonates, including immune signaling. While myeloid cells can traffic into the brain via multiple routes, the choroid plexus (CP) has been identified as a uniquely educated gate for immune cell traffic during health and disease. To understand the mechanisms of myeloid cell trafficking via the CP and their influence on neonatal stroke, we characterized the phenotypes of CP-infiltrating myeloid cells after transient middle cerebral artery occlusion (tMCAO) in neonatal mice of both sexes in relation to blood-brain barrier permeability, injury, microglial activation, and CX3CR1-CCR2 signaling, focusing on the dynamics early after reperfusion. We demonstrate rapid recruitment of multiple myeloid phenotypes in the CP ipsilateral to the injury, including inflammatory CD45+CD11b+Ly6chighCD86+, beneficial CD45+CD11b+Ly6clowCD206+, and CD45+CD11b+Ly6clowLy6ghigh cells, but only minor leukocyte infiltration into acutely ischemic-reperfused cortex and negligible vascular albumin leakage. We report that CX3CR1-CCR2-mediated myeloid cell recruitment contributes to stroke injury. Considering the complexity of inflammatory cascades triggered by stroke and a role for TLR2 in injury, we also used direct TLR2 stimulation as an independent injury model. TLR2 agonist rapidly recruited myeloid cells to the CP, increased leukocytosis in the CSF and blood, but infiltration into the cortex remained low over time. While the magnitude and the phenotypes of myeloid cells diverged between tMCAO and TLR2 stimulation, in both models, disruption of CX3CR1-CCR2 signaling attenuated both monocyte and neutrophil trafficking to the CP and cortex.SIGNIFICANCE STATEMENT Stroke during the neonatal period leads to long-term disabilities. The mechanisms of ischemic injury and inflammatory response differ greatly between the immature and adult brain. We examined leukocyte trafficking via the choroid plexus (CP) following neonatal stroke in relation to blood-brain barrier integrity, injury, microglial activation, and signaling via CX3CR1 and CCR2 receptors, or following direct TLR2 stimulation. Ischemia-reperfusion triggered marked unilateral CX3CR1-CCR2 dependent accumulation of diverse leukocyte subpopulations in the CP without inducing extravascular albumin leakage or major leukocyte infiltration into the brain. Disrupted CX3CR1-CCR2 signaling was neuroprotective in part by attenuating monocyte and neutrophil trafficking. Understanding the migratory patterns of CP-infiltrating myeloid cells with intact and disrupted CX3CR1-CCR2 signaling could identify novel therapeutic targets to protect the neonatal brain.

BackgroundTherapeutic hypothermia (TH) is the standard of care for neonates with hypoxic-ischemic encephalopathy, but it is not fully protective in the clinical setting. Hypoxia-ischemia (HI) may cause white matter injury (WMI), leading to neurological and cognitive dysfunction.MethodsP9 mice were subjected to HI as previously described. Pups underwent 3.5 h of systemic hypothermia or normothermia. Cresyl violet and Perl's iron staining for histopathological scoring of brain sections was completed blindly on all brains. Immunocytochemical (ICC) staining for myelin basic protein (MBP), microglia (Iba1), and astrocytes (glia fibrillary acidic protein (GFAP)) was performed on adjacent sections. Volumetric measurements of MBP coverage were used for quantitative analysis of white matter.ResultsTH provided neuroprotection by injury scoring for the entire group (n=44; P<0.0002). ICC analysis of a subset of brains showed that the lateral caudate was protected from WMI (P<0.05). Analysis revealed decreased GFAP and Iba1 staining in hippocampal regions, mostly CA2/CA3. GFAP and Iba1 directly correlated with injury scores of normothermic brains.ConclusionTH reduced injury, and qualitative data suggest that hippocampus and lateral caudate are protected from HI. Mildly injured brains may better show the benefits of TH. Overall, these data indicate regional differences in WMI susceptibility and inflammation in a P9 murine HI model.