Hypothermia combined with extracellular vesicles from clonally expanded immortalized mesenchymal stromal cells improves neurodevelopmental impairment in neonatal hypoxic-ischemic brain injury.

BACKGROUND: Neonatal encephalopathy following hypoxia-ischemia (HI) is a leading cause of childhood death and morbidity. Hypothermia (HT), the only available but obligatory therapy is limited due to a short therapeutic window and limited efficacy. An adjuvant therapy overcoming limitations of HT is still missing. Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have shown promising therapeutic effects in various brain injury models. Challenges associated with MSCs' heterogeneity and senescence can be mitigated by the use of EVs from clonally expanded immortalized MSCs (ciMSCs). In the present study, we hypothesized that intranasal ciMSC-EV delivery overcomes limitations of HT. METHODS: Nine-day-old C57BL/6 mice were exposed to HI by occlusion of the right common carotid artery followed by 1 h hypoxia (10% oxygen). HT was initiated immediately after insult for 4 h. Control animals were kept at physiological body core temperatures. ciMSC-EVs or vehicle were administered intranasally 1, 3 and 5 days post HI/HT. Neuronal cell loss, inflammatory and regenerative responses were assessed via immunohistochemistry, western blot and real-time PCR 7 days after insult. Long-term neurodevelopmental outcome was evaluated by analyses of cognitive function, activity and anxiety-related behavior 5 weeks after HI/HT. RESULTS: In contrast to HT monotherapy, the additional intranasal therapy with ciMSC-EVs prevented HI-induced cognitive deficits, hyperactivity and alterations of anxiety-related behavior at adolescence. This was preceded by reduction of striatal neuronal loss, decreased endothelial, microglia and astrocyte activation; reduced expression of pro-inflammatory and increased expression of anti-inflammatory cytokines. Furthermore, the combination of HT with intranasal ciMSC-EV delivery promoted regenerative and neurodevelopmental processes, including endothelial proliferation, neurotrophic growth factor expression and oligodendrocyte maturation, which were not altered by HT monotherapy. CONCLUSION: Intranasal delivery of ciMSC-EVs represents a novel adjunct therapy, overcoming limitations of acute HT thereby offering new possibilities for improving long-term outcomes in neonates with HI-induced brain injury.

Neuropilin-1 identifies a subset of highly activated CD8+ T cells during parasitic and viral infections.

Neuropilin-1 (Nrp-1) expression on CD8+ T cells has been identified in tumor-infiltrating lymphocytes and in persistent murine gamma-herpes virus infections, where it interferes with the development of long-lived memory T cell responses. In parasitic and acute viral infections, the role of Nrp-1 expression on CD8+ T cells remains unclear. Here, we demonstrate a strong induction of Nrp-1 expression on CD8+ T cells in Plasmodium berghei ANKA (PbA)-infected mice that correlated with neurological deficits of experimental cerebral malaria (ECM). Likewise, the frequency of Nrp-1+CD8+ T cells was significantly elevated and correlated with liver damage in the acute phase of lymphocytic choriomeningitis virus (LCMV) infection. Transcriptomic and flow cytometric analyses revealed a highly activated phenotype of Nrp-1+CD8+ T cells from infected mice. Correspondingly, in vitro experiments showed rapid induction of Nrp-1 expression on CD8+ T cells after stimulation in conjunction with increased expression of activation-associated molecules. Strikingly, T cell-specific Nrp-1 ablation resulted in reduced numbers of activated T cells in the brain of PbA-infected mice as well as in spleen and liver of LCMV-infected mice and alleviated the severity of ECM and LCMV-induced liver pathology. Mechanistically, we identified reduced blood-brain barrier leakage associated with reduced parasite sequestration in the brain of PbA-infected mice with T cell-specific Nrp-1 deficiency. In conclusion, Nrp-1 expression on CD8+ T cells represents a very early activation marker that exacerbates deleterious CD8+ T cell responses during both, parasitic PbA and acute LCMV infections.

Extracellular vesicles from immortalized mesenchymal stromal cells protect against neonatal hypoxic-ischemic brain injury.

BACKGROUND: Human mesenchymal stromal cell (MSC)-derived extracellular vesicles (EV) revealed neuroprotective potentials in various brain injury models, including neonatal encephalopathy caused by hypoxia-ischemia (HI). However, for clinical translation of an MSC-EV therapy, scaled manufacturing strategies are required, which is challenging with primary MSCs due to inter- and intra-donor heterogeneities. Therefore, we established a clonally expanded and immortalized human MSC line (ciMSC) and compared the neuroprotective potential of their EVs with EVs from primary MSCs in a murine model of HI-induced brain injury. In vivo activities of ciMSC-EVs were comprehensively characterized according to their proposed multimodal mechanisms of action. METHODS: Nine-day-old C57BL/6 mice were exposed to HI followed by repetitive intranasal delivery of primary MSC-EVs or ciMSC-EVs 1, 3, and 5 days after HI. Sham-operated animals served as healthy controls. To compare neuroprotective effects of both EV preparations, total and regional brain atrophy was assessed by cresyl-violet-staining 7 days after HI. Immunohistochemistry, western blot, and real-time PCR were performed to investigate neuroinflammatory and regenerative processes. The amount of peripheral inflammatory mediators was evaluated by multiplex analyses in serum samples. RESULTS: Intranasal delivery of ciMSC-EVs and primary MSC-EVs comparably protected neonatal mice from HI-induced brain tissue atrophy. Mechanistically, ciMSC-EV application reduced microglia activation and astrogliosis, endothelial activation, and leukocyte infiltration. These effects were associated with a downregulation of the pro-inflammatory cytokine IL-1 beta and an elevated expression of the anti-inflammatory cytokines IL-4 and TGF-beta in the brain, while concentrations of cytokines in the peripheral blood were not affected. ciMSC-EV-mediated anti-inflammatory effects in the brain were accompanied by an increased neural progenitor and endothelial cell proliferation, oligodendrocyte maturation, and neurotrophic growth factor expression. CONCLUSION: Our data demonstrate that ciMSC-EVs conserve neuroprotective effects of primary MSC-EVs via inhibition of neuroinflammation and promotion of neuroregeneration. Since ciMSCs can overcome challenges associated with MSC heterogeneity, they appear as an ideal cell source for the scaled manufacturing of EV-based therapeutics to treat neonatal and possibly also adult brain injury.

A report on the International Society for Cell & Gene Therapy 2022 Scientific Signature Series, "Therapeutic advances with native and engineered human extracellular vesicles".

The International Society for Cell & Gene Therapy Scientific Signature Series event "Therapeutic Advances With Native and Engineered Human EVs" took place as part of the International Society for Cell & Gene Therapy 2022 Annual Meeting, held from May 4 to 7, 2022, in San Francisco, California, USA. This was the first signature series event on extracellular vesicles (EVs) and a timely reflection of the growing interest in EVs, including both native and engineered human EVs, for therapeutic applications. The event successfully gathered academic and industrial key opinion leaders to discuss the current state of the art in developing and understanding native and engineered EVs and applying our knowledge toward advancing EV therapeutics. Latest advancements in understanding the mechanisms by which native and engineered EVs exert their therapeutic effects against different diseases in animal models were presented, with some diseases such as psoriasis and osteoarthritis already reaching clinical testing of EVs. The discussion also covered various aspects relevant to advancing the clinical translation of EV therapies, including EV preparation, manufacturing, consistency, site(s) of action, route(s) of administration, and luminal cargo delivery of RNA and other compounds.

Perinatal Hyperoxia and Developmental Consequences on the Lung-Brain Axis.

Approximately 11.1% of all newborns worldwide are born preterm. Improved neonatal intensive care significantly increased survival rates over the last decades but failed to reduce the risk for the development of chronic lung disease (i.e., bronchopulmonary dysplasia (BPD)) and impaired neurodevelopment (i.e., encephalopathy of prematurity (EoP)), two major long-term sequelae of prematurity. Premature infants are exposed to relative hyperoxia, when compared to physiological in-utero conditions and, if needed to additional therapeutic oxygen supplementation. Both are associated with an increased risk for impaired organ development. Since the detrimental effects of hyperoxia on the immature retina are known for many years, lung and brain have come into focus in the last decade. Hyperoxia-induced excessive production of reactive oxygen species leading to oxidative stress and inflammation contribute to pulmonary growth restriction and abnormal neurodevelopment, including myelination deficits. Despite a large body of studies, which unraveled important pathophysiological mechanisms for both organs at risk, the majority focused exclusively either on lung or on brain injury. However, considering that preterm infants suffering from BPD are at higher risk for poor neurodevelopmental outcome, an interaction between both organs seems plausible. This review summarizes recent findings regarding mechanisms of hyperoxia-induced neonatal lung and brain injury. We will discuss common pathophysiological pathways, which potentially link both injured organ systems. Furthermore, promises and needs of currently suggested therapies, including pharmacological and regenerative cell-based treatments for BPD and EoP, will be emphasized. Limited therapeutic approaches highlight the urgent need for a better understanding of the mechanisms underlying detrimental effects of hyperoxia on the lung-brain axis in order to pave the way for the development of novel multimodal therapies, ideally targeting both severe preterm birth-associated complications.

Regulatory T Cells Contribute to Sexual Dimorphism in Neonatal Hypoxic-Ischemic Brain Injury.

BACKGROUND AND PURPOSE: Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of death and disability in newborns. Clinical and experimental studies suggest a sexual dimorphism in HI-induced brain injury and therapy responses. A major hallmark of HI pathophysiology is the infiltration of peripheral immune cells into the injured brain. However, the specific role of regulatory T cells (Tregs) in neonatal HI is still unknown. METHODS: Nine-day-old mice were exposed to HI by ligation of the right common carotid artery followed by 1 hour hypoxia (10% oxygen). Using immunohistochemistry, flow cytometry, and microarray analyses, Tregs were investigated in the brain, spleen, and blood 24 hours post HI. The functional role of Tregs was evaluated by acute Treg depletion in depletion of regulatory T cells transgenic mice. Brain injury, neuroinflammatory responses, and vascular injury were analyzed via immunohistochemistry and Western blot 48 hours and 7 days after HI. Functional outcome was assessed 3 days and 5 weeks after HI. RESULTS: Female mice revealed an increased cerebral Treg infiltration, coinciding with elevated chemokine receptor expression. Treg depletion in females aggravated HI-induced brain tissue injury, short-term motor deficits, and long-term deficits in exploratory activity, paralleled by an increased microglia and endothelial activation and leukocyte infiltration. Treg depletion in male mice reduced HI-induced brain injury, short-term motor, and long-term cognitive deficits, associated with reduced vascular injury. Ex vivo isolated female Tregs displayed an increased immunosuppressive activity on effector T cell proliferation and an increased gene enrichment in pathways related to enhanced Treg activity. CONCLUSIONS: Tregs from neonatal female mice provide endogenous neuroprotection, whereas Tregs from male mice increase secondary neurodegeneration. As potential mechanisms, we identified intrinsic transcriptional differences associated with enhanced anti-inflammatory activity of female Tregs. Our study emphasizes the urgent need for sex-stratified clinical and preclinical analyses.

Peripheral immune cells and perinatal brain injury: a double-edged sword?

Perinatal brain injury is the leading cause of neurological mortality and morbidity in childhood ranging from motor and cognitive impairment to behavioural and neuropsychiatric disorders. Various noxious stimuli, including perinatal inflammation, chronic and acute hypoxia, hyperoxia, stress and drug exposure contribute to the pathogenesis. Among a variety of pathological phenomena, the unique developing immune system plays an important role in the understanding of mechanisms of injury to the immature brain. Neuroinflammation following a perinatal insult largely contributes to evolution of damage to resident brain cells, but may also be beneficial for repair activities. The present review will focus on the role of peripheral immune cells and discuss processes involved in neuroinflammation under two frequent perinatal conditions, systemic infection/inflammation associated with encephalopathy of prematurity (EoP) and hypoxia/ischaemia in the context of neonatal encephalopathy (NE) and stroke at term. Different immune cell subsets in perinatal brain injury including their infiltration routes will be reviewed and critical aspects such as sex differences and maturational stage will be discussed. Interactions with existing regenerative therapies such as stem cells and also potentials to develop novel immunomodulatory targets are considered. IMPACT: Comprehensive summary of current knowledge on the role of different immune cell subsets in perinatal brain injury including discussion of critical aspects to be considered for development of immunomodulatory therapies.

Postischemic Neuroprotection Associated With Anti-Inflammatory Effects by Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles in Aged Mice.

BACKGROUND AND PURPOSE: Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce ischemic neuroprotection in mice by modulating the brain infiltration of leukocytes and, specifically polymorphonuclear neutrophils. So far, effects of MSC-sEVs were only studied in young ischemic rodents. We herein examined the effects of MSC-sEVs in aged mice. METHODS: Male and female C57Bl6/j mice (8-10 weeks or 15-24 months) were exposed to transient intraluminal middle cerebral artery occlusion. Vehicle or sEVs (equivalent of 2×106 MSCs) were intravenously administered. Neurological deficits, ischemic injury, blood-brain barrier integrity, brain leukocyte infiltration, and blood leukocyte responses were evaluated over up to 7 days. RESULTS: MSC-sEV delivery reduced neurological deficits, infarct volume, brain edema, and neuronal injury in young and aged mice of both sexes, when delivered immediately postreperfusion or with 6 hours delay. MSC-sEVs decreased leukocyte and specifically polymorphonuclear neutrophil, monocyte, and macrophage infiltrates in ischemic brains of aged mice. In peripheral blood, the number of monocytes and activated T cells was significantly reduced by MSC-sEVs. CONCLUSIONS: MSC-sEVs induce postischemic neuroprotection and anti-inflammation in aged mice.

Hypothermia modulates myeloid cell polarization in neonatal hypoxic-ischemic brain injury.

BACKGROUND: Neonatal encephalopathy due to hypoxia-ischemia (HI) is a leading cause of death and disability in term newborns. Therapeutic hypothermia (HT) is the only recommended therapy. However, 30% still suffer from neurological deficits. Inflammation is a major hallmark of HI pathophysiology with myeloid cells being key players, participating either in progression or in resolution of injury-induced inflammation. In the present study, we investigated the impact of HT on the temporal and spatial dynamics of microglia/macrophage polarization after neonatal HI in newborn mice. METHODS: Nine-day-old C57BL/6 mice were exposed to HI through occlusion of the right common carotid artery followed by 1 h hypoxia. Immediately after HI, animals were cooled for 4 h or kept at physiological body core temperature. Analyses were performed at 1, 3 and 7 days post HI. Brain injury, neuronal cell loss, apoptosis and microglia activation were assessed by immunohistochemistry. A broad set of typical genes associated with classical (M1) and alternative (M2) myeloid cell activation was analyzed by real time PCR in ex vivo isolated CD11b+ microglia/macrophages. Purity and composition of isolated cells was determined by flow cytometry. RESULTS: Immediate HT significantly reduced HI-induced brain injury and neuronal loss 7 days post HI, whereas only mild non-significant protection from HI-induced apoptosis and neuronal loss were observed 1 and 3 days after HI. Microglia activation, i.e., Iba-1 immunoreactivity peaked 3 days after HI and was not modulated by HT. However, ex vivo isolated CD11b+ cells revealed a strong upregulation of the majority of M1 but also M2 marker genes at day 1, which was significantly reduced by HT and rapidly declined at day 3. HI induced a significant increase in the frequency of peripheral macrophages in sorted CD11b+ cells at day 1, which deteriorated until day 7 and was significantly decreased by HT. CONCLUSION: Our data demonstrate that HT-induced neuroprotection is preceded by acute suppression of HI-induced upregulation of inflammatory genes in myeloid cells and decreased infiltration of peripheral macrophages, both representing potential important effector mechanisms of HT.

Neutrophil dynamics, plasticity and function in acute neurodegeneration following neonatal hypoxia-ischemia.

Neonatal encephalopathy following hypoxia-ischemia (HI) is a major cause of long-term morbidity and mortality in children. Even though HI-induced neuroinflammation, involving infiltration of peripheral immune cells into the CNS has been associated with disease pathogenesis, the specific role of neutrophils is highly debated. Due to immaturity of the neonatal immune system, it has been assumed that neutrophils are less clinically relevant in neonatal HI-induced brain injury. In the present study, we demonstrate that neutrophils are rapidly activated in the neonatal brain after exposure to experimental HI, revealed by an enhanced proportion of CD86+ cells and an increased expression of CD11b compared to splenic and blood neutrophils. Furthermore, production of reactive oxygen species and the proportion of hyperactivated/aged (CXCR4+CD62L-) cells was enhanced in brain compared to peripheral neutrophils. Delayed neutrophil depletion, initiated 12 h after HI resulted in reduced cellular neurodegeneration, associated with reduced micro- and astroglial activation. In the present study, we uncovered a new complex switch of the phenotype in brain neutrophils, which may offer new possibilities for the development of selective therapeutic approaches by modulation of neutrophils in the early post-hypoxic disease phase.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Reduce Neuroinflammation, Promote Neural Cell Proliferation and Improve Oligodendrocyte Maturation in Neonatal Hypoxic-Ischemic Brain Injury.

Background: Neonatal encephalopathy caused by hypoxia-ischemia (HI) is a major cause of childhood mortality and disability. Stem cell-based regenerative therapies seem promising to prevent long-term neurological deficits. Our previous work in neonatal HI revealed an unexpected interaction between mesenchymal stem/stromal cells (MSCs) and the brains' microenvironment leading to an altered therapeutic efficiency. MSCs are supposed to mediate most of their therapeutic effects in a paracrine mode via extracellular vesicles (EVs), which might be an alternative to cell therapy. In the present study, we investigated the impact of MSC-EVs on neonatal HI-induced brain injury. Methods: Nine-day-old C57BL/6 mice were exposed to HI through ligation of the right common carotid artery followed by 1 h hypoxia (10% oxygen). MSC-EVs were injected intraperitoneally 1, 3, and 5 days after HI. One week after HI, brain injury was evaluated by regional neuropathological scoring, atrophy measurements and immunohistochemistry to assess effects on neuronal, oligodendrocyte and vessel densities, proliferation, oligodendrocyte maturation, myelination, astro-, and microglia activation. Immunohistochemistry analyses were complemented by mRNA expression analyses for a broad set of M1/M2- and A1/A2-associated molecules and neural growth factors. Results: While total neuropathological scores and tissue atrophy were not changed, MSC-EVs significantly protected from HI-induced striatal tissue loss and decreased micro- and astroglia activation. MSC-EVs lead to a significant downregulation of the pro-inflammatory cytokine TNFa, accompanied by a significant upregulation of the M2 marker YM-1 and the anti-inflammatory cytokine TGFb. MSC-EVs significantly decreased astrocytic expression of the A1 marker C3, concomitant with an increased expression of neural growth factors (i.e., BDNF, VEGF, and EGF). These alterations were associated with an increased neuronal and vessel density, coinciding with a significant increase of proliferating cells in the neurogenic sub-ventricular zone juxtaposed to the striatum. MSC-EV-mediated neuroprotection went along with a significant improvement of oligodendrocyte maturation and myelination. Conclusion: The present study demonstrates that MSC-EVs mediate anti-inflammatory effects, promote regenerative responses and improve key developmental processes in the injured neonatal brain. The present results suggest different cellular target mechanisms of MSC-EVs, preventing secondary HI-induced brain injury. MSC-EV treatment may be a promising alternative to risk-associated cell therapies in neonatal brain injury.

Involvement of CXCL1/CXCR2 During Microglia Activation Following Inflammation-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Rats.

Background: Microglia are key mediators of inflammation during perinatal brain injury. As shown experimentally after inflammation-sensitized hypoxic ischemic (HI) brain injury, microglia are activated into a pro-inflammatory status 24 h after HI involving the NLRP3 inflammasome pathway. The chemokine (C-X-C motif) ligand 1 (CXCL1), and its cognate receptor, CXCR2, have been shown to be involved in NLRP3 activation, although their specific role during perinatal brain injury remains unclear. In this study we investigated the involvement of CXCL1/CXCR2 in brain tissue and microglia and brain tissue after inflammation-sensitized HI brain injury of newborn rats. Methods: Seven-day old Wistar rat pups were either injected with vehicle (NaCl 0.9%) or E. coli lipopolysaccharide (LPS), followed by left carotid ligation combined with global hypoxia (8% O2 for 50 min). Pups were randomized into four different treatment groups: (1) Sham group (n = 21), (2) LPS only group (n = 20), (3) Veh/HI group (n = 39), and (4) LPS/HI group (n = 42). Twenty-four hours post hypoxia transcriptome and gene expression analysis were performed on ex vivo isolated microglia cells in our model. Additionally protein expression was analyzed in different brain regions at the same time point. Results: Transcriptome analyses showed a significant microglial upregulation of the chemokine CXCL1 and its receptor CXCR2 in the LPS/HI group compared with the other groups. Gene expression analysis showed a significant upregulation of CXCL1 and NLRP3 in microglia cells after inflammation-sensitized hypoxic-ischemic brain injury. Additionally, protein expression of CXCL1 was significantly upregulated in cortex of male pups from the LPS/HI group. Conclusion: These results indicate that the CXCL1/CXCR2 pathway may be involved during pro-inflammatory microglia activation following inflammation-sensitized hypoxic-ischemic brain injury in neonatal rats. This may lead to new treatment options altering CXCR2 activation early after HI brain injury.

Repetitive Erythropoietin Treatment Improves Long-Term Neurocognitive Outcome by Attenuating Hyperoxia-Induced Hypomyelination in the Developing Brain.

Introduction: Preterm infants born before 28 weeks of gestation are at high risk of neurodevelopmental impairment in later life. Cerebral white and gray matter injury is associated with adverse outcomes. High oxygen levels, often unavoidable in neonatal intensive care, have been identified as one of the main contributing factors to preterm brain injury. Thus, preventive and therapeutic strategies against hyperoxia-induced brain injury are needed. Erythropoietin (Epo) is a promising and also neuroprotective candidate due to its clinical use in infants as erythropoiesis-stimulating agent. Objective: The objective of this study was to investigate the effects of repetitive Epo treatment on the cerebral white matter and long-term motor-cognitive outcome in a neonatal rodent model of hyperoxia-induced brain injury. Methods: Three-day old Wistar rats were exposed to hyperoxia (48 h, 80% oxygen). Four doses of Epo (5,000 IU/kg body weight per day) were applied intraperitoneally from P3-P6 with the first dose at the onset of hyperoxia. Oligodendrocyte maturation and myelination were evaluated via immunohistochemistry and Western blot on P11. Motor-cognitive deficits were assessed in a battery of complex behavior tests (Open Field, Novel Object Recognition, Barnes maze) in adolescent and fully adult animals. Following behavior tests animals underwent post-mortem diffusion tensor imaging to investigate long-lasting microstructural alterations of the white matter. Results: Repetitive treatment with Epo significantly improved myelination deficits following neonatal hyperoxia at P11. Behavioral testing revealed attenuated hyperoxia-induced cognitive deficits in Epo-treated adolescent and adult rats. Conclusion: A multiple Epo dosage regimen protects the developing brain against hyperoxia-induced brain injury by improving myelination and long-term cognitive outcome. Though current clinical studies on short-term outcome of Epo-treated prematurely born children contradict our findings, long-term effects up to adulthood are still lacking. Our data support the essential need for long-term follow-up of preterm infants in current clinical trials.

Detrimental Impact of Energy Drink Compounds on Developing Oligodendrocytes and Neurons.

The consumption of energy drinks is continuously rising, particularly in children and adolescents. While risks for adverse health effects, like arrhythmia, have been described, effects on neural cells remain elusive. Considering that neurodevelopmental processes like myelination and neuronal network formation peak in childhood and adolescence we hypothesized that developing oligodendrocytes and neurons are particularly vulnerable to main energy drink components. Immature oligodendrocytes and hippocampal neurons were isolated from P0-P1 Wistar rats and were incubated with 0.3 mg/mL caffeine and 4 mg/mL taurine alone or in combination for 24 h. Analysis was performed immediately after treatment or after additional three days under differentiating conditions for oligodendrocytes and standard culture for neurons. Oligodendrocyte degeneration, proliferation, and differentiation were assessed via immunocytochemistry and immunoblotting. Neuronal integrity was investigated following immunocytochemistry by analysis of dendrite outgrowth and axonal morphology. Caffeine and taurine induced an increased degeneration and inhibited proliferation of immature oligodendrocytes accompanied by a decreased differentiation capacity. Moreover, dendritic branching and axonal integrity of hippocampal neurons were negatively affected by caffeine and taurine treatment. The negative impact of caffeine and taurine on developing oligodendrocytes and disturbed neuronal morphology indicates a high risk for disturbed neurodevelopment in children and adolescents by excessive energy drink consumption.

Early Pro-inflammatory Microglia Activation After Inflammation-Sensitized Hypoxic-Ischemic Brain Injury in Neonatal Rats.

Background: Perinatal asphyxia, leading to neonatal encephalopathy, is one of the leading causes for child mortality and long-term morbidities. Neonatal encephalopathy rates are significantly increased in newborns with perinatal infection. Therapeutic hypothermia is only neuroprotective in 50% of cooled asphyxiated newborns. As shown experimentally, cooling has failed to be neuroprotective after inflammation-sensitized hypoxic ischemic (HI) brain injury. Microglia are thought to be key players after inflammation-sensitized HI brain injury. We performed this study investigating early microglia phenotype polarization in our newborn animal model of inflammation-sensitized HI brain injury, better understanding the underlying pathophysiological processes. Methods: Seven days old Wistar rat pups were injected with either vehicle (NaCl 0.9%) or E. coli lipopolysaccharide (LPS), followed by left carotid ligation combined with global hypoxia inducing a mild unilateral hypoxic-ischemic injury. Pups were randomized to (1) Sham group (n = 41), (2) LPS only group (n = 37), (3) Veh/HI group (n = 56), and (4) LPS/HI group (n = 79). On postnatal days 8 and 14 gene-expression analysis or immunohistochemistry was performed describing early microglia polarization in our model. Results: We confirmed that LPS pre-sensitization significantly increases brain area loss and induced microglia activation and neuronal injury after mild hypoxia-ischemia. Additionally, we show that microglia upregulate pro-inflammatory genes involving NLRP-3 inflammasome gene expression 24 h after inflammation-sensitized hypoxic-ischemic brain injury. Conclusion: These results demonstrate that microglia are early key mediators of the inflammatory response following inflammation-sensitized HI brain injury and that they polarize into a predominant pro-inflammatory phenotype 24 h post HI. This may lead to new treatment options altering microglia phenotype polarization early after HI brain injury.

Zbtb20 Regulates Developmental Neurogenesis in the Olfactory Bulb and Gliogenesis After Adult Brain Injury.

The transcription factor (TF) Zbtb20 is important for the hippocampal specification and the regulation of neurogenesis of neocortical projection neurons. Herein, we show a critical involvement of the TF Zbtb20 in the neurogenesis of both projection neurons and interneurons of the olfactory bulb during embryonic stages. Our data indicate that the lack of Zbtb20 significantly diminishes the generation of a set of early-born Tbr2+ neurons during embryogenesis. Furthermore, we provide evidence that Zbtb20 regulates the transition between neurogenesis to gliogenesis in cortical radial glial progenitor cells at the perinatal (E18.5) stage. In the adult mammalian brain, Zbtb20 is expressed by GFAP+ neural progenitor cells (NPCs) located in the forebrain neurogenic niche, i.e., the subventricular zone (SVZ) of the lateral ventricles. Upon induction of cerebral ischemia, we found that Zbtb20 expression is upregulated in astrocytic-like cells, whereas diminishing the expression levels of Zbtb20 significantly reduces the ischemia-induced astrocytic reaction as observed in heterozygous Zbtb20 loss-of-function mice. Altogether, these results highlight the important role of the TF Zbtb20 as a temporal regulator of neurogenesis or gliogenesis, depending on the developmental context.

Adverse neuropsychiatric development following perinatal brain injury: from a preclinical perspective.

Perinatal brain injury is a leading cause of death and disability in young children. Recent advances in obstetrics, reproductive medicine and neonatal intensive care have resulted in significantly higher survival rates of preterm or sick born neonates, at the price of increased prevalence of neurological, behavioural and psychiatric problems in later life. Therefore, the current focus of experimental research shifts from immediate injury processes to the consequences for brain function in later life. The aetiology of perinatal brain injury is multi-factorial involving maternal and also labour-associated factors, including not only placental insufficiency and hypoxia-ischaemia but also exposure to high oxygen concentrations, maternal infection yielding excess inflammation, genetic factors and stress as important players, all of them associated with adverse long-term neurological outcome. Several animal models addressing these noxious stimuli have been established in the past to unravel the underlying molecular and cellular mechanisms of altered brain development. In spite of substantial efforts to investigate short-term consequences, preclinical evaluation of the long-term sequelae for the development of cognitive and neuropsychiatric disorders have rarely been addressed. This review will summarise and discuss not only current evidence but also requirements for experimental research providing a causal link between insults to the developing brain and long-lasting neurodevelopmental disorders.

White Matter Brain Development after Exposure to Circulating Cell-Free Hemoglobin and Hyperoxia in a Rat Pup Model.

Neonates born with critical congenital heart defects are at risk of diffuse white matter injuries and neurodevelopmental impairments. This study aimed to determine the impact of circulating cell-free hemoglobin and hyperoxia, both present during cardiopulmonary bypass circulation, on white matter brain development. Postnatal day 6 rat pups were injected intraperitoneally with cell-free Hb or vehicle and exposed to hyperoxia (fiO2 = 0.8) or normoxia (fiO2 = 0.21) for 24 h. We evaluated apoptosis, myelination, and oligodendrocyte maturation with immunohistochemistry, gene and protein analyses, and in vivo diffusion tensor magnetic resonance imaging (MRI). Consistent with previous studies, we found an increase in apoptosis of oligodendrocytes as determined by TUNEL+ staining in Olig2+ cells in white matter, cortex, thalamus, and hippocampus following exposure to hyperoxia with no additional effect of cell-free Hb. A transient increase in the mRNA expression of intercellular adhesion molecule 1 at 6 h was observed following combined exposure to cell-free Hb and hyperoxia. No indications of oligodendrocyte maturational delay or hypomyelination were observed after either insult, delivered separately or combined, as determined by immunohistochemistry, Western blot, and diffusion tensor MRI. In our model, exposure to circulatory cell-free Hb, with or without concomitant hyperoxia, did not significantly alter brain white matter development.

Peripheral T Cell Depletion by FTY720 Exacerbates Hypoxic-Ischemic Brain Injury in Neonatal Mice.

Hypoxic-ischemic injury to the developing brain remains a major cause of significant long-term morbidity and mortality. Emerging evidence from neonatal brain injury models suggests a detrimental role for peripheral lymphocytes. The immunomodulatory substance FTY720, a sphingosine-1-phosphate receptor agonist, was shown to reduce adult ischemia-induced neurodegeneration through its lymphopenic mode of action. In the present study, we hypothesized that FTY720 promotes neuroprotection by reducing peripheral lymphocytes and their infiltration into the injured neonatal brain. Term-born equivalent postnatal day 9 C57BL/6 mice were exposed to hypoxia ischemia (HI) followed by a single injection of 1 mg/kg FTY720 or vehicle (0.9% sodium chloride). Brain injury, microglia, and endothelial activation were assessed 7 days post HI using histology and western blot. Peripheral and cerebral leukocyte subsets were analyzed by multichannel flow cytometry. Whether FTY720s' effects could be attributed to its lymphopenic mode of action was determined in T cell-depleted mice. In contrast to our hypothesis, FTY720 exacerbated HI-induced neuropathology including loss of gray and white matter structures. While microglia and endothelial activation remained unchanged, FTY720 induced a strong and sustained depletion of peripheral T cells resulting in significantly reduced cerebral infiltration of CD4 T cells. CD4 T cell subset analysis revealed that circulating regulatory and effector T cells counts were similarly decreased after FTY720 treatment. However, since neonatal HI per se induces a selective infiltration of Foxp3 positive regulatory T cells compared to Foxp3 negative effector T cells effects of FTY720 on cerebral regulatory T cell infiltration were more pronounced than on effector T cells. Reductions in T lymphocytes, and particularly regulatory T cells coincided with an increased infiltration of innate immune cells, mainly neutrophils and inflammatory macrophages. Importantly anti-CD3-mediated T cell depletion resulted in a similar exacerbation of brain injury, which was not further enhanced by an additional FTY720 treatment. In summary, peripheral T cell depletion by FTY720 resulted in increased infiltration of innate immune cells concomitant to reduced T cell infiltration and exacerbation HI-induced brain injury. This study indicates that neonatal T cells may promote endogenous neuroprotection in the term-born equivalent hypoxic-ischemic brain potentially providing new opportunities for therapeutic intervention.

Protection of Oligodendrocytes Through Neuronal Overexpression of the Small GTPase Ras in Hyperoxia-Induced Neonatal Brain Injury.

Prematurely born infants are highly susceptible to various environmental factors, such as inflammation, drug exposure, and also high environmental oxygen concentrations. Hyperoxia induces perinatal brain injury affecting white and gray matter development. It is well known that mitogen-activated protein kinase signaling is involved in cell survival, proliferation, and differentiation. Therefore, we aim to elucidate cell-specific responses of neuronal overexpression of the small GTPase Ras on hyperoxia-mediated brain injury. Six-day-old (P6) synRas mice (neuronal Ras overexpression under the synapsin promoter) or wild-type littermates were kept under hyperoxia (80% oxygen) or room air (21% oxygen) for 24 h. Apoptosis was analyzed by Western blot of cleaved Caspase-3 and neuronal and oligodendrocyte degeneration via immunohistochemistry. Short-term differentiation capacity of oligodendrocytes was assessed by quantification of myelin basic protein expression at P11. Long-lasting changes of hyperoxia-induced alteration of myelin structures were evaluated via transmission electron microscopy in young adult animals (P42). Western blot analysis of active Caspase-3 demonstrates a significant upregulation in wild-type littermates exposed to hyperoxia whereas synRas mice did not show any marked alteration of cleaved Caspase-3 protein levels. Immunohistochemistry revealed a protective effect of neuronal Ras overexpression on neuron and oligodendrocyte survival. Hyperoxia-induced hypomyelination in wild-type littermates was restored in synRas mice. These short-term protective effects through promotion of neuronal survival translated into long-lasting improvement of ultrastructural alterations of myelin sheaths in mice with neuronal overexpression of Ras compared with hyperoxic wild-type mice. Our data suggest that transgenic increase of neuronal Ras activity in the immature brain results in secondary protection of oligodendrocytes from hyperoxia-induced white matter brain injury.