Peripheral myeloid cells contribute to brain injury in male neonatal mice.

BACKGROUND: Neonatal brain injury is increasingly understood to be linked to inflammatory processes that involve specialised CNS and peripheral immune interactions. However, the role of peripheral myeloid cells in neonatal hypoxic-ischemic (HI) brain injury remains to be fully investigated. METHODS: We employed the Lys-EGFP-ki mouse that allows enhanced green fluorescent protein (EGFP)-positive mature myeloid cells of peripheral origin to be easily identified in the CNS. Using both flow cytometry and confocal microscopy, we investigated the accumulation of total EGFP+ myeloid cells and myeloid cell subtypes: inflammatory monocytes, resident monocytes and granulocytes, in the CNS for several weeks following induction of cerebral HI in postnatal day 9 mice. We used antibody treatment to curb brain infiltration of myeloid cells and subsequently evaluated HI-induced brain injury. RESULTS: We demonstrate a temporally biphasic pattern of inflammatory monocyte and granulocyte infiltration, characterised by peak infiltration at 1 day and 7 days after hypoxia-ischemia. This occurs against a backdrop of continuous low-level resident monocyte infiltration. Antibody-mediated depletion of circulating myeloid cells reduced immune cell accumulation in the brain and reduced neuronal loss in male but not female mice. CONCLUSION: This study offers new insight into sex-dependent central-peripheral immune communication following neonatal brain injury and merits renewed interest in the roles of granulocytes and monocytes in lesion development.

Neonatal microglia: The cornerstone of brain fate.

Microglia, mainly known for their role in innate immunity and modulation of neuroinflammation, play an active role in central nervous system development and homeostasis. Depending on the context and environmental stimuli, microglia adopt a broad spectrum of activation status from pro-inflammatory, associated with neurotoxicity, to anti-inflammatory linked to neuroprotection. Pro-inflammatory microglial activation is a key hallmark of white matter injury in preterm infants and is involved in developmental origin of adult neurological diseases. Characterization of neonatal microglia function in brain development and inflammation has allowed the investigation of promising therapeutic targets with potential long-lasting neuroprotective effects. True prevention of neuro-degenerative diseases might eventually occur as early as the perinatal period.

Temporal Characterization of Microglia/Macrophage Phenotypes in a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury.

Immune cells display a high degree of phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with classical and alternative polarization phenotypes described for macrophages and to identify related cell populations in the brain following neonatal hypoxia-ischemia (HI). HI was induced in 9-day old mice and brain tissue was collected up to 7 days post-insult to investigate expression of genes associated with macrophage activation. Using cell-markers, CD86 (classic activation) and CD206 (alternative activation), we assessed temporal changes of CD11b+ cell populations in the brain and studied the protein expression of the immunomodulatory factor galectin-3 in these cells. HI induced a rapid regulation (6 h) of genes associated with both classical and alternative polarization phenotypes in the injured hemisphere. FACS analysis showed a marked increase in the number of CD11b+CD86+ cells at 24 h after HI (+3667%), which was coupled with a relative suppression of CD11b+CD206+ cells and cells that did not express neither CD86 nor CD206. The CD11b+CD206+ population was mixed with some cells also expressing CD86. Confocal microscopy confirmed that a subset of cells expressed both CD86 and CD206, particularly in injured gray and white matter. Protein concentration of galectin-3 was markedly increased mainly in the cell population lacking CD86 or CD206 in the injured hemisphere. These cells were predominantly resident microglia as very few galectin-3 positive cells co-localized with infiltrating myeloid cells in Lys-EGFP-ki mice after HI. In summary, HI was characterized by an early mixed gene response, but with a large expansion of mainly the CD86 positive population during the first day. However, the injured hemisphere also contained a subset of cells expressing both CD86 and CD206 and a large population that expressed neither activation marker CD86 nor CD206. Interestingly, these cells expressed the highest levels of galectin-3 and were found to be predominantly resident microglia. Galectin-3 is a protein involved in chemotaxis and macrophage polarization suggesting a novel role in cell infiltration and immunomodulation for this cell population after neonatal injury.

Brain barrier properties and cerebral blood flow in neonatal mice exposed to cerebral hypoxia-ischemia.

Insults to the developing brain often result in irreparable damage resulting in long-term deficits in motor and cognitive functions. The only treatment today for hypoxic-ischemic encephalopathy (HIE) in newborns is hypothermia, which has limited clinical benefit. We have studied changes to the blood-brain barriers (BBB) as well as regional cerebral blood flow (rCBF) in a neonatal model of HIE to further understand the underlying pathologic mechanisms. Nine-day old mice pups, brain roughly equivalent to the near-term human fetus, were subjected to hypoxia-ischemia. Hypoxia-ischemia increased BBB permeability to small and large molecules within hours after the insult, which normalized in the following days. The opening of the BBB was associated with changes to BBB protein expression whereas gene transcript levels were increased showing direct molecular damage to the BBB but also suggesting compensatory mechanisms. Brain pathology was closely related to reductions in rCBF during the hypoxia as well as the areas with compromised BBB showing that these are intimately linked. The transient opening of the BBB after the insult is likely to contribute to the pathology but at the same time provides an opportunity for therapeutics to better reach the infarcted areas in the brain.

Neonatal peripheral immune challenge activates microglia and inhibits neurogenesis in the developing murine hippocampus.

The early postnatal period represents an important window in rodent hippocampal development with peak hilar neurogenesis and widespread microgliogenesis occurring in the first week of life. Inflammation occurring during this period may negatively influence development, potentially facilitating or increasing susceptibility to later-life pathology. We administered the Gram-negative bacterial coat protein lipopolysaccharide (LPS) systemically at postnatal day 5 (1 mg/kg i.p.) and assessed potential effects on microgliogenesis, inflammation and neurogenesis in the developing hippocampus. LPS administration led to an acute but transient increase in absolute number and density of ionized calcium-binding adaptor molecule 1-immunoreactive microglia, a change attributable to increased proliferation of central nervous system-resident microglia/microglial precursor cells but not infiltration of peripheral monocyte-derived macrophages. qRT-PCR analysis of hippocampal gene expression showed these LPS-mediated changes to be associated with persistent dysregulation of genes associated with both M1 and M2 microglial phenotypes, indicating prolonged alteration in hippocampal inflammatory status. Further, analysis of progenitor cell regulation in the hippocampal subgranular zone revealed a transient inhibition of the neuronal differentiation pathway up to 2 weeks after LPS administration, a change occurring specifically through effects on type 3 neural progenitor cells and independently of altered cell proliferation or survival of newly born cells. Together, our results show that systemic inflammation occurring during the early neonatal period is sufficient to alter inflammatory status and dysregulate the ongoing process of neurogenesis in the developing hippocampal germinal niche.

Astrocytes negatively regulate neurogenesis through the Jagged1-mediated Notch pathway.

Adult neurogenesis is regulated by a number of cellular players within the neurogenic niche. Astrocytes participate actively in brain development, regulation of the mature central nervous system (CNS), and brain plasticity. They are important regulators of the local environment in adult neurogenic niches through the secretion of diffusible morphogenic factors, such as Wnts. Astrocytes control the neurogenic niche also through membrane-associated factors, however, the identity of these factors and the mechanisms involved are largely unknown. In this study, we sought to determine the mechanisms underlying our earlier finding of increased neuronal differentiation of neural progenitor cells when cocultured with astrocytes lacking glial fibrillary acidic protein (GFAP) and vimentin (GFAP(-/-) Vim(-/-) ). We used primary astrocyte and neurosphere cocultures to demonstrate that astrocytes inhibit neuronal differentiation through a cell-cell contact. GFAP(-/-) Vim(-/-) astrocytes showed reduced endocytosis of Notch ligand Jagged1, reduced Notch signaling, and increased neuronal differentiation of neurosphere cultures. This effect of GFAP(-/-) Vim(-/-) astrocytes was abrogated in the presence of immobilized Jagged1 in a manner dependent on the activity of γ-secretase. Finally, we used GFAP(-/-) Vim(-/-) mice to show that in the absence of GFAP and vimentin, hippocampal neurogenesis under basal conditions as well as after injury is increased. We conclude that astrocytes negatively regulate neurogenesis through the Notch pathway, and endocytosis of Notch ligand Jagged1 in astrocytes and Notch signaling from astrocytes to neural stem/progenitor cells depends on the intermediate filament proteins GFAP and vimentin.

Regulation of toll-like receptor 1 and -2 in neonatal mice brains after hypoxia-ischemia.

BACKGROUND: Hypoxic-ischemic (HI) brain injury remains a major problem in newborns, resulting in increased risk of neurological disorders. Neonatal HI triggers a broad inflammatory reaction in the brain, including activation of the innate immune system. Toll-like receptors (TLRs), which are key components of the innate immune system, are believed to play a role in adult cerebral ischemic injury. The expression of TLRs in the neonatal brain and their regulation after HI is unknown. METHODS: Wild type C57BL/6, TLR 1 knockout (KO) and TLR 2 KO mice were subjected to HI at postnatal day 9 and sacrificed 30 min, 6 h, 24 h or 5 days after HI. TLR mRNA expression was determined by RT-qPCR and protein and cell type localisation by immunohistochemistry (IHC). To evaluate brain injury, infarct volume was measured in the injured hemisphere. RESULTS: mRNA expression was detected for all investigated TLRs (TLR1-9), both in normal and HI exposed brains. After HI, TLR-1 was down-regulated at 30 min and up-regulated at 6 h and 24 h. TLR-2 was up-regulated at 6 h and 24 h, and TLR-7 at 24 h. Both TLR-5 and TLR-8 were down-regulated at 24 h and 30 min respectively. IHC showed an increase of TLR-1 in neurons in the ipsilateral hemisphere after HI. TLR-2 was constitutively expressed in astrocytes and in a population of neurons in the paraventricular nucleus in the hypothalamus. No changes in expression were detected following HI. Following HI, TLR-2 KO mice, but not TLR-1 KO, showed a decreased infarct volume compared to wild type (p = 0.0051). CONCLUSIONS: This study demonstrates that TLRs are regulated after HI in the neonatal brain. TLR-1 protein was up-regulated in injured areas of the brain but TLR-1 KO animals were not protected from HI. In contrast, TLR-2 was constitutively expressed in the brain and TLR-2 deficiency reduced HI injury. These data suggest that TLR-2, but not TLR-1, plays a role in neonatal HI brain injury.

Increased neurogenesis and astrogenesis from neural progenitor cells grafted in the hippocampus of GFAP-/- Vim-/- mice.

After neurotrauma, ischemia, or neurodegenerative disease, astrocytes upregulate their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin (Vim), and nestin. This response, reactive gliosis, is attenuated in GFAP(-/-)Vim(-/-) mice, resulting in the promotion of synaptic regeneration after neurotrauma and improved integration of retinal grafts. Here we assessed whether GFAP(-/-)Vim(-/-) astrocytes affect the differentiation of neural progenitor cells. In coculture with GFAP(-/-)Vim(-/-) astrocytes, neural progenitor cells increased neurogenesis by 65% and astrogenesis by 124%. At 35 days after transplantation of neural progenitor cells into the hippocampus, adult GFAP(-/-)Vim(-/-) mice had more transplant-derived neurons and astrocytes than wild-type controls, as well as increased branching of neurite-like processes on transplanted cells. Wnt3 immunoreactivity was readily detected in hippocampal astrocytes in wild-type but not in GFAP(-/-)Vim(-/-) mice. These findings suggest that GFAP(-/-)Vim(-/-) astrocytes allow more neural progenitor cell-derived neurons and astrocytes to survive weeks after transplantation. Thus, reactive gliosis may adversely affect the integration of transplanted neural progenitor cells in the brain. Disclosure of potential conflicts of interest is found at the end of this article.

Signaling through C5aR is not involved in basal neurogenesis.

The complement system, an important part of the innate immune system, provides protection against invading pathogens, in part through its proinflammatory activities. Although most complement proteins are synthesized locally in the brain and the relevant complement receptors are expressed on resident brain cells, little is known about brain-specific role(s) of the complement system. C3a and C5a, complement-derived peptides with anaphylatoxic properties, have been implicated in noninflammatory functions, such as tissue regeneration and neuroprotection. Recently, we have shown that signaling through C3a receptor (C3aR) is involved in the regulation of neurogenesis. In the present study, we assessed basal neurogenesis in mice lacking C5a receptor (C5aR(-/-)) and mice expressing C3a and C5a, respectively in the CNS under the control of glial fibrillary acidic protein (GFAP) promoter (C3a/GFAP and C5a/GFAP, respectively) and thus without the requirement for complement activation. We did not observe any difference among C5aR(-/-), C3a/GFAP and C5a/GFAP mice and their respective controls in the number of newly formed neuroblasts and newly formed neurons in the subventricular zone (SVZ) of lateral ventricles and hippocampal dentate gyrus, the two neurogenic niches in the adult brain, or the olfactory bulb, the final destination of new neurons formed in the SVZ. Our results indicate that signaling through C5aR is not involved in basal neurogenesis in adult mice and that basal neurogenesis in adult C3a/GFAP and C5a/GFAP mice is not altered.