magnetic-isolation-microglial-cells-from-neonate-mouse-for-primary

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Microglia of the developing brain have unique functional properties but how their activation states are regulated is poorly understood. Inflammatory activation of microglia in the still-developing brain of preterm-born infants is associated with permanent neurological sequelae in 9 million infants every year. Investigating the regulators of microglial activation in the developing brain across models of neuroinflammation-mediated injury (mouse, zebrafish) and primary human and mouse microglia we found using analysis of genes and proteins that a reduction in Wnt/β-catenin signalling is necessary and sufficient to drive a microglial phenotype causing hypomyelination. We validated in a cohort of preterm-born infants that genomic variation in the Wnt pathway is associated with the levels of connectivity found in their brains. Using a Wnt agonist delivered by a blood-brain barrier penetrant microglia-specific targeting nanocarrier we prevented in our animal model the pro-inflammatory microglial activation, white matter injury and behavioural deficits. Collectively, these data validate that the Wnt pathway regulates microglial activation, is critical in the evolution of an important form of human brain injury and is a viable therapeutic target.

Decreased microglial Wnt/β-catenin signalling drives microglial pro-inflammatory activation in the developing brain.

Perinatal brain injuries, including encephalopathy related to fetal growth restriction, encephalopathy of prematurity, neonatal encephalopathy of the term neonate, and neonatal stroke, are a major cause of neurodevelopmental disorders. They trigger cellular and molecular cascades that lead in many cases to permanent motor, cognitive, and/or behavioral deficits. Damage includes neuronal degeneration, selective loss of subclasses of interneurons, blocked maturation of oligodendrocyte progenitor cells leading to dysmyelination, axonopathy and very likely synaptopathy, leading to impaired connectivity. The nature and severity of changes vary according to the type and severity of insult and maturation stage of the brain. Microglial activation has been demonstrated almost ubiquitously in perinatal brain injuries and these responses are key cell orchestrators of brain pathology but also attempts at repair. These divergent roles are facilitated by a diverse suite of transcriptional profiles and through a complex dialogue with other brain cell types. Adding to the complexity of understanding microglia and how to modulate them to protect the brain is that these cells have their own developmental stages, enabling them to be key participants in brain building. Of note, not only do microglia help build the brain and respond to brain injury, but they are a key cell in the transduction of systemic inflammation into neuroinflammation. Systemic inflammatory exposure is a key risk factor for poor neurodevelopmental outcomes in preterm born infants. Based on these observations, microglia appear as a key cell target for neuroprotection in perinatal brain injuries. Numerous strategies have been developed experimentally to modulate microglia and attenuate brain injury based on these strong supporting data and we will summarize these.

Microglia-Mediated Neurodegeneration in Perinatal Brain Injuries.

A leading cause of preterm birth is the exposure to systemic inflammation (maternal/fetal infection), which leads to neuroinflammation and white matter injury (WMI). A wide range of cytokines and chemokines are expressed and upregulated in oligodendrocytes (OLs) in response to inflammation and numerous reports show that OLs express several receptors for immune related molecules, which enable them to sense inflammation and to react. However, the role of OL immune response in WMI is unclear. Here, we focus our study on toll-like receptor-3 (TLR3) that is activated by double-strand RNA (dsRNA) and promotes neuroinflammation. Despite its importance, its expression and role in OLs remain unclear. We used an in vivo mouse model, which mimics inflammation-mediated WMI of preterm born infants consisting of intraperitoneal injection of IL-1β from P1 to P5. In the IL-1β-treated animals, we observed the upregulation of Tlr3, IL-1β, IFN-β, Ccl2, and Cxcl10 in both PDGFRα+ and O4+ sorted cells. This upregulation was higher in O4+ immature OLs (immOLs) as compared to PDGFRα+ OL precursor cells (OPCs), suggesting a different sensitivity to neuroinflammation. These observations were confirmed in OL primary cultures: cells treated with TLR3 agonist Poly(I:C) during differentiation showed a stronger upregulation of Ccl2 and Cxcl10 compared to cells treated during proliferation and led to decreased expression of myelin genes. Finally, OLs were able to modulate microglia phenotype and function depending on their maturation state as assessed by qPCR using validated markers for immunomodulatory, proinflammatory, and anti-inflammatory phenotypes and by phagocytosis and morphological analysis. These results show that during inflammation the response of OLs can play an autonomous role in blocking their own differentiation: in addition, the immune activation of OLs may play an important role in shaping the response of microglia during inflammation.

The immune-inflammatory response of oligodendrocytes in a murine model of preterm white matter injury: the role of TLR3 activation.

Acquired perinatal brain injuries are a set of conditions that remains a key challenge for neonatologists and that have significant social, emotional and financial implications for our communities. In our perspective article, we will introduce perinatal brain injury focusing specifically on the events leading to brain damage in preterm born infants and outcomes for these infants. Then we will summarize and discuss the preclinical and clinical studies testing the efficacy of stem cells as neuroprotectants in the last ten years in perinatal brain injury. There are no therapies to treat brain damage in preterm born infants and a primary finding from this review is that there is a scarcity of stem cell trials focused on overcoming brain injuries in these infants. Overall, across all forms of perinatal brain injury there is a remarkable heterogeneity in previous and on-going preclinical and clinical studies in terms of the stem cell type, animal models/patient selection, route and time of administration. Despite the quality of many of the studies this variation makes it difficult to reach a valid consensus for future developments. However, it is clear that stem cells (and stem cell derived exosomes) can reduce perinatal brain injury and our field needs to work collectively to refine an effective protocol for each type of injury. The use of standardized stem cell products and testing these products across multiple models of injury will provide a stronger framework for clinical trials development.

Therapeutic potential of stem cells for preterm infant brain damage: Can we move from the heterogeneity of preclinical and clinical studies to established therapeutics?

Microglial activation during critical phases of brain development can result in short- and long-term consequences for neurological and psychiatric health. Several studies in humans and rodents have shown that microglial activation, leading to a transition from the homeostatic state toward a proinflammatory phenotype, has adverse effects on the developing brain and neurodevelopmental disorders. Targeting proinflammatory microglia may be an effective strategy for protecting the brain and attenuating neurodevelopmental disorders induced by inflammation. In this review we focus on the role of inflammation and the activation of immature microglia (pre-microglia) soon after birth in prematurity-associated neurodevelopmental disorders, and the specific features of pre-microglia during development. We also highlight the relevance of immunomodulatory strategies for regulating activated microglia in a rodent model of perinatal brain injury. An original neuroprotective approach involving a nanoparticle-based therapy and targeting microglia, with the aim of improving myelination and protecting the developing brain, is also addressed.

Targeting Microglial Disturbances to Protect the Brain From Neurodevelopmental Disorders Associated With Prematurity.

In the premature newborn, perinatal inflammation mediated by microglia contributes significantly to neurodevelopmental injuries including white matter injury (WMI). Brain inflammation alters development through neuroinflammatory processes mediated by activation of homeostatic microglia toward a pro-inflammatory and neurotoxic phenotype. Investigating immune regulators of microglial activation is crucial to find effective strategies to prevent and treat WMI.

miR-146b Protects the Perinatal Brain against Microglia-Induced Hypomyelination.

Preterm birth (PTB) represents 15 million births every year worldwide and is frequently associated with maternal/fetal infections and inflammation, inducing neuroinflammation. This neuroinflammation is mediated by microglial cells, which are brain-resident macrophages that release cytotoxic molecules that block oligodendrocyte differentiation, leading to hypomyelination. Some preterm survivors can face lifetime motor and/or cognitive disabilities linked to periventricular white matter injuries (PWMIs). There is currently no recommendation concerning the mode of delivery in the case of PTB and its impact on brain development. Many animal models of induced-PTB based on LPS injections exist, but with a low survival rate. There is a lack of information regarding clinically used pharmacological substances to induce PTB and their consequences on brain development. Mifepristone (RU-486) is a drug used clinically to induce preterm labor. This study aims to elaborate and characterize a new model of induced-PTB and PWMIs by the gestational injection of RU-486 and the perinatal injection of pups with IL-1beta. A RU-486 single subcutaneous (s.c.) injection at embryonic day (E)18.5 induced PTB at E19.5 in pregnant OF1 mice. All pups were born alive and were adopted directly after birth. IL-1beta was injected intraperitoneally from postnatal day (P)1 to P5. Animals exposed to both RU-486 and IL-1beta demonstrated microglial reactivity and subsequent PWMIs. In conclusion, the s.c. administration of RU-486 induced labor within 24 h with a high survival rate for pups. In the context of perinatal inflammation, RU-486 labor induction significantly decreases microglial reactivity in vivo but did not prevent subsequent PWMIs.

The Impact of Mouse Preterm Birth Induction by RU-486 on Microglial Activation and Subsequent Hypomyelination.

Preterm infants often show pathologies of the cerebellum, which are associated with impaired motor performance, lower IQ and poor language skills at school ages. Using a mouse model of inflammation-induced encephalopathy of prematurity driven by systemic administration of pro-inflammatory IL-1β, we sought to uncover causes of cerebellar damage. In this model, IL-1β is administered between postnatal day (P) 1 to day 5, a timing equivalent to the last trimester for brain development in humans. Structural MRI analysis revealed that systemic IL-1β treatment induced specific reductions in gray and white matter volumes of the mouse cerebellar lobules I and II (5% false discovery rate [FDR]) from P15 onwards. Preceding these MRI-detectable cerebellar volume changes, we observed damage to oligodendroglia, with reduced proliferation of OLIG2+ cells at P10 and reduced levels of the myelin proteins myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) at P10 and P15. Increased density of IBA1+ cerebellar microglia were observed both at P5 and P45, with evidence for increased microglial proliferation at P5 and P10. Comparison of the transcriptome of microglia isolated from P5 cerebellums and cerebrums revealed significant enrichment of pro-inflammatory markers in microglia from both regions, but cerebellar microglia displayed a unique type I interferon signaling dysregulation. Collectively, these data suggest that perinatal inflammation driven by systemic IL-1β leads to specific cerebellar volume deficits, which likely reflect oligodendrocyte pathology downstream of microglial activation. Further studies are now required to confirm the potential of protective strategies aimed at preventing sustained type I interferon signaling driven by cerebellar microglia as an important therapeutic target.

Article	Year
Decreased microglial Wnt/β-catenin signalling drives microglial pro-inflammatory activation in the developing brain. Brain : a journal of neurology\| PubMed ID: 31665242	2019
Microglia-Mediated Neurodegeneration in Perinatal Brain Injuries. Biomolecules\| PubMed ID: 33451166	2021
The immune-inflammatory response of oligodendrocytes in a murine model of preterm white matter injury: the role of TLR3 activation. Cell death & disease\| PubMed ID: 33558485	2021
Therapeutic potential of stem cells for preterm infant brain damage: Can we move from the heterogeneity of preclinical and clinical studies to established therapeutics? Biochemical pharmacology\| PubMed ID: 33571501	2021
Targeting Microglial Disturbances to Protect the Brain From Neurodevelopmental Disorders Associated With Prematurity. Journal of neuropathology and experimental neurology\| PubMed ID: 34363661	2021
miR-146b Protects the Perinatal Brain against Microglia-Induced Hypomyelination. Annals of neurology\| PubMed ID: 34741343	2022
The Impact of Mouse Preterm Birth Induction by RU-486 on Microglial Activation and Subsequent Hypomyelination. International journal of molecular sciences\| PubMed ID: 35563258	2022
A unique cerebellar pattern of microglia activation in a mouse model of encephalopathy of prematurity. Glia\| PubMed ID: 35579329	2022

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