Early Toll-like receptor 4 inhibition improves immune dysfunction in the hippocampus after hypoxic-ischemic brain damage.

Background: Toll-like receptor 4 (TLR4) is implicated in neonatal hypoxic-ischemic brain damage (HIBD), but the underlying mechanism is unclear. Hypothesis: We hypothesized that TLR4 mediates brain damage after hypoxic ischemia (HI) by inducing abnormal neuroimmune responses, including activation of immune cells and expression disorder of immune factors, while early inhibition of TLR4 can alleviate the neuroimmune dysfunction. Method: Postnatal day 7 rats were randomized into control, HI, and HI+TAK-242 (TAK-242) groups. The HIBD model was developed using the Rice-Vannucci method (the left side was the ipsilateral side of HI). TAK-242 (0.5 mg/kg) was given to rat pups in the TAK-242 group at 30 min before modeling. Immunofluorescence, immunohistochemistry, and western blotting were used to determine the TLR4 expression; the number of Iba-1+, GFAP+, CD161+, MPO+, and CD3+ cells; ICAM-1 and C3a expression; and interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-10 expression in the hippocampal CA1 region. Result: Significantly increased TLR4 expression was observed in the left hippocampus, and was alleviated by TAK-242. The significant increases in Iba-1+, MPO+, and CD161+ cells at 24 h and 7 days after HI and in GFAP+ and CD3+ T cells at 7 days after HI were also counteracted by TAK-242, but no significant differences were observed among groups at 24 h after HI. ICAM-1 expression increased 24 h after HI, while C3a expression decreased; TAK-242 also alleviated these changes. TNF-α and IL-1ß expression increased, while IL-10 expression decreased at 24 h and 7 days after HI; TAK-242 counteracted the increased TNF-α and IL-1ß expression at 24 h and the changes in IL-1ß and IL-10 at 7 days, but induced no significant differences in IL-10 expression at 24 h and TNF-α expression at 7 days. Conclusion: Early TLR4 inhibition can alleviate hippocampal immune dysfunction after neonatal HIBD.

Comprehensive Analysis of RNA Expression Profile Identifies Hub miRNA-circRNA Interaction Networks in the Hypoxic Ischemic Encephalopathy.

Hypoxic ischemic encephalopathy (HIE) is classified as a sort of serious nervous system syndrome that occurs in the early life period. Noncoding RNAs had been confirmed to have crucial roles in human diseases. So far, there were few systematical and comprehensive studies towards the expression profile of RNAs in the brain after hypoxia ischemia. In this study, 31 differentially expressed microRNAs (miRNAs) with upregulation were identified. In addition, 5512 differentially expressed mRNAs, long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) were identified in HIE groups. Bioinformatics analysis showed these circRNAs and mRNAs were significantly enriched in regulation of leukocyte activation, response to virus, and neutrophil degranulation. Pathway and its related gene network analysis indicated that HLA - DPA1, HLA - DQA2, HLA - DQB1, and HLA - DRB4 have a more crucial role in HIE. Finally, miRNA-circRNA-mRNA interaction network analysis was also performed to identify hub miRNAs and circRNAs. We found that miR-592 potentially targeting 5 circRNAs, thus affecting 15 mRNA expressions in HIR. hsa_circ_0068397 and hsa_circ_0045698 were identified as hub circRNAs in HIE. Collectively, using RNA-seq, bioinformatics analysis, and circRNA/miRNA interaction prediction, we systematically investigated the differentially expressed RNAs in HIE, which could give a new hint of understanding the pathogenesis of HIE.

H2S prevents peripheral immune cell invasion, increasing [Ca2+]i and excessive phagocytosis following hypoxia-ischemia injury in neonatal mice.

We previously reported that L-Cysteine, H2S donor, remarkably attenuated neuroinflammation following hypoxia-ischemia (HI) brain injury in neonatal mice. However, its anti-inflammatory mechanism for HI insult is still unknown. The study focus on the effects of L-Cysteine on immune cell populations, Ca2+ mobilization and phagocytosis after neonatal HI. We found that L-Cysteine treatment skewed CD11b+/CD45low microglia and CD11b+/CD45high brain monocytes/macrophages towards a more anti-inflammatory property 72 h after HI-injured brain. Moreover, L-Cysteine treatment reduced cerebral infiltration of CD4 T cells 7 days following HI insult. Furthermore, CD4 T cell subset analysis revealed that L-Cysteine treatment decreased Th1 and Th2 counts, while increased Th17/Th2 ratio. Moreover, L-Cysteine treatment suppressed LPS-induced cytosolic Ca2+ and LPS-stimulated phagocytosis in primary microglia. The anti-inflammatory effect of L-Cysteine was associated with improving neurobehavioral impairment following HI insult. Our results demonstrate L-Cysteine treatment suppressed the invasion of peripheral immune cells, increasing [Ca2+]i and excessive phagocytosis to improve neurobehavioral deficits following hypoxia-ischemia injury in neonatal mice by H2S release.

Serum cytokine profiling in neonates with hypoxic ischemic encephalopathy.

BACKGROUND: The fetal brain is vulnerable to severe and sustained hypoxia during and after birth, which can lead to hypoxic-ischemic encephalopathy (HIE). HIE is characterized by clinical and laboratory evidence of acute or subacute brain injury. The role of cytokines in the pathogenesis of brain injury and their relation to neurological outcomes of asphyxiated neonates are not fully understood. In this study, we investigated cytokine profile related to cerebral palsy (CP) with neonatal hypoxic ischemic encephalopathy (HIE) and HIE severity. METHODS: Eligible subjects were HIE newborns with a gestational age between 36 and 42 weeks. We included newborns who was born at our NICU and did not admit to NICU as healthy controls. The study comprised 52 newborns, including 13 with mild to severe HIE and 39 healthy control. Serum cytokine profiles were performed using a LUMINEX cytokine kit (R&D Systems). RESULTS: VEGF, MCP-1, IL-15, IL-12p70, IL-12p40, IL-1Ra, IL-2, IL-6, IL-7, IL-8, IL-10, IFN-γ, G-CSF and eotaxin in the HIE patients were significantly increased compared with the healthy neonates. In the subgroup analysis, IL-6 and G-CSF were significantly increased in CP infants (n = 5) compared with non-CP infants (n = 8). Five and eight HIE patients were classified into the mild HIE and moderate-severe HIE groups, respectively. IL-6, 10, 1Ra, and G-CSF in the moderate-severe HIE group were significantly higher than those in the mild HIE group. CONCLUSION: We demonstrated that higher serum IL-6 and G-CSF at birth in HIE patients were associated with CP and moderate-severe HIE.

Staphylococcus epidermidis Sensitizes Perinatal Hypoxic-Ischemic Brain Injury in Male but Not Female Mice.

Background:Staphylococcus epidermidis is the most common nosocomial infection and the predominant pathogen in late-onset sepsis in preterm infants. Infection and inflammation are linked to neurological and developmental sequelae and bacterial infections increase the vulnerability of the brain to hypoxia-ischemia (HI). We thus tested the hypothesis that S. epidermidis exacerbates HI neuropathology in neonatal mice. Methods: Male and female C57Bl/6 mice were injected intraperitoneally with sterile saline or 3.5 × 107 colony-forming units of S. epidermidis on postnatal day (PND) 4 and then subjected to HI on PND5 (24 h after injection) or PND9 (5 d after injection) by left carotid artery ligation and exposure to 10% O2. White and gray matter injury was assessed on PND14-16. In an additional group of animals, the plasma, brain, and liver were collected on PND5 or PND9 after infection to evaluate cytokine and chemokine profiles, C5a levels and C5 signaling. Results: HI induced 24 h after injection of S. epidermidis resulted in greater gray and white matter injury compared to saline injected controls in males, but not in females. Specifically, males demonstrated increased gray matter injury in the cortex and striatum, and white matter loss in the subcortical region, hippocampal fimbria and striatum. In contrast, there was no potentiation of brain injury when HI occurred 5 d after infection in either sex. In the plasma, S. epidermidis-injected mice demonstrated increased levels of pro- and anti-inflammatory cytokines and chemokines and a reduction of C5a at 24 h, but not 5 d after infection. Brain CCL2 levels were increased in both sexes 24 h after infection, but increased only in males at 5 d post infection. Conclusion: Ongoing S. epidermidis infection combined with neonatal HI increases the vulnerability of the developing brain in male but not in female mice. These sex-dependent effects were to a large extent independent of expression of systemic cytokines or brain CCL2 expression. Overall, we provide new insights into how systemic S. epidermidis infection affects the developing brain and show that the time interval between infection and HI is a critical sensitizing factor in males.

Effects of xenon gas on human airway epithelial cells during hyperoxia and hypothermia.

BACKGROUND: Hypothermia with xenon gas has been used to reduce brain injury and disability rate after perinatal hypoxia-ischemia. We evaluated xenon gas therapy effects in an in vitro model with or without hypothermia on cultured human airway epithelial cells (Calu-3). METHODS: Calu-3 monolayers were grown at an air-liquid interface and exposed to one of the following conditions: 1) 21% FiO2 at 37°C (control); 2) 45% FiO2 and 50% xenon at 37°C; 3) 21% FiO2 and 50% xenon at 32°C; 4) 45% FiO2 and 50% xenon at 32°C for 24 hours. Transepithelial resistance (TER) measurements were performed and apical surface fluids were collected and assayed for total protein, IL-6, and IL-8. Three monolayers were used for immunofluorescence localization of zonula occludens-1 (ZO-1). The data were analyzed by one-way ANOVA. RESULTS: TER decreased at 24 hours in all treatment groups. Xenon with hyperoxia and hypothermia resulted in greatest decrease in TER compared with other groups. Immunofluorescence localization of ZO-1 (XY) showed reduced density of ZO-1 rings and incomplete ring-like staining in the 45% FiO2- 50% xenon group at 32°C compared with other groups. Secretion of total protein was not different among groups. Secretion of IL-6 in 21% FiO2 with xenon group at 32°C was less than that of the control group. The secretion of IL-8 in 45% FiO2 with xenon at 32°C was greater than that of other groups. CONCLUSION: Hyperoxia and hypothermia result in detrimental epithelial cell function and inflammation over 24-hour exposure. Xenon gas did not affect cell function or reduce inflammation.

δ-Opioid Receptors, microRNAs, and Neuroinflammation in Cerebral Ischemia/Hypoxia.

Hypoxia and ischemia are the main underlying pathogenesis of stroke and other neurological disorders. Cerebral hypoxia and/or ischemia (e.g., stroke) can lead to neuronal injury/death and eventually cause serious neurological disorders or even death in the patients. Despite knowing these serious consequences, there are limited neuroprotective strategies against hypoxic and ischemic insults in clinical settings. Recent studies indicate that microRNAs (miRNAs) are of great importance in regulating cerebral responses to hypoxic/ischemic stress in addition to the neuroprotective effect of the δ-opioid receptor (DOR). Moreover, new discovery shows that DOR can regulate miRNA expression and inhibit inflammatory responses to hypoxia/ischemia. We, therefore, summarize available data in current literature regarding the role of DOR and miRNAs in regulating the neuroinflammatory responses in this article. In particular, we focus on microglia activation, cytokine production, and the relevant signaling pathways triggered by cerebral hypoxia/ischemia. The intent of this review article is to provide a novel clue for developing new strategies against neuroinflammatory injury resulting from cerebral hypoxia/ischemia.

Properdin: A Novel Target for Neuroprotection in Neonatal Hypoxic-Ischemic Brain Injury.

Background: Hypoxic-ischemic (HI) encephalopathy is a major cause of neonatal mortality and morbidity, with a global incidence of 3 per 1,000 live births. Intrauterine or perinatal complications, including maternal infection, constitute a major risk for the development of neonatal HI brain damage. During HI, inflammatory response and oxidative stress occur, causing subsequent cell death. The presence of an infection sensitizes the neonatal brain, making it more vulnerable to the HI damage. Currently, therapeutic hypothermia is the only clinically approved treatment available for HI encephalopathy, however it is only partially effective in HI alone and its application in infection-sensitized HI is debatable. Therefore, there is an unmet clinical need for the development of novel therapeutic interventions for the treatment of HI. Such an alternative is targeting the complement system. Properdin, which is involved in stabilization of the alternative pathway convertases, is the only known positive regulator of alternative complement activation. Absence of the classical pathway in the neonatal HI brain is neuroprotective. However, there is a paucity of data on the participation of the alternative pathway and in particular the role of properdin in HI brain damage. Objectives: Our study aimed to validate the effect of global properdin deletion in two mouse models: HI alone and LPS-sensitized HI, thus addressing two different clinical scenarios. Results: Our results indicate that global properdin deletion in a Rice-Vannucci model of neonatal HI and LPS-sensitized HI brain damage, in the short term, clearly reduced forebrain cell death and microglial activation, as well as tissue loss. In HI alone, deletion of properdin reduced TUNEL+ cell death and microglial post-HI response at 48 h post insult. Under the conditions of LPS-sensitized HI, properdin deletion diminished TUNEL+ cell death, tissue loss and microglial activation at 48 h post-HI. Conclusion: Overall, our data suggests a critical role for properdin, and possibly also a contribution in neonatal HI alone and in infection-sensitized HI brain damage. Thus, properdin can be considered a novel target for treatment of neonatal HI brain damage.

SIRT1-regulated HMGB1 release is partially involved in TLR4 signal transduction: A possible anti-neuroinflammatory mechanism of resveratrol in neonatal hypoxic-ischemic brain injury.

Neonatal hypoxic-ischemic brain injury (HIBI) is a knotty disease that lacks appropriate treatment. Inflammation is an important contributor to brain damage, and microglia are responsible for eliciting early and pronounced inflammatory reactions in the immature brain after hypoxic-ischemic (HI) insult. Acetylated HMGB1 can be released from immune cells into the extracellular space, where it acts as a danger-associated molecular pattern molecule to activate TLR4 signalling-mediated inflammatory responses. Resveratrol has neuroprotective and anti-inflammatory effects against HIBI, but whether these effects involve the regulation of the TLR4 signalling pathway and whether HMGB1 participates in this process is still unclear. We investigated the anti-inflammatory effects of resveratrol in HIBI and the molecular mechanisms potentially involved in the effect. The in vivo and in vitro results indicated that the level of cytoplasmic HMGB1 in microglia increased after insult and that treating experimental animals or mouse BV2 microglial cells with resveratrol attenuated HI insult-induced neuroinflammation, which was characterized by improved behavioural defects, reduced microglial activation and TLR4/MyD88/NF-κB signalling, and attenuated primary neuronal damage; this was accompanied by the inhibition of HMGB1 nucleoplasmic transfer and extracellular release. EX527 pretreatment reversed these effects. In addition, co-immunoprecipitation confirmed that SIRT1 was directly involved in the HMGB1 acetylation process in BV2 cells after oxygen glucose deprivation. These data demonstrate that resveratrol plays a neuroprotective role in neonatal HIBI by activating SIRT1 to inhibit HMGB1/TLR4/MyD88/NF-κB signalling and subsequent neuroinflammatory responses.

Intranasal Delivery of Mesenchymal Stromal Cells Protects against Neonatal Hypoxic⁻Ischemic Brain Injury.

Cerebral palsy (CP) is a permanent motor disorder that results from brain injury and neuroinflammation during the perinatal period. Mesenchymal stromal cells (MSCs) have been explored as a therapy in multiple adult neuroinflammatory conditions. Our study examined the therapeutic benefits of intranasal delivery of human umbilical cord tissue (UC) derived-MSCs in a rat model of neonatal hypoxic-ischemic (HI) brain injury. To do this, HI was performed on postnatal day 10 Sprague-Dawley rat pups via permanent ligation of the left carotid artery, followed by a hypoxic challenge of 8% oxygen for 90 min. A total of 200,000 UC-MSCs (10 million/kg) were administered intranasally 24 h post-HI. Motor control was assessed after seven days, followed by post-mortem. Analysis included brain immunohistochemistry, gene analysis and serum cytokine measurement. Neonatal HI resulted in brain injury with significant loss of neurons, particularly in the hippocampus. Intranasal administration of UC-MSCs significantly reduced the loss of brain tissue and increased the number of hippocampal neurons. HI significantly upregulated brain inflammation and expression of pro-inflammatory cytokines, while intranasal UC-MSCs significantly reduced markers of neuroinflammation. This study demonstrated that a clinically relevant dose (10 million/kg) of UC-MSCs was neuroprotective following HI by restoring neuronal cell numbers and reducing brain inflammation. Therefore, intranasal delivery of UC-MSCs may be an effective therapy for neonatal brain injury.

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.

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.

Identification of a panel of cytokines in neonates with hypoxic ischemic encephalopathy treated with hypothermia.

PURPOSE: Inflammation is a crucial but understudied mechanism of neuronal injury after hypoxia-ischemia. The aim was to identify a panel of cytokines involved in brain injury in neonates with hypoxic ischemic encephalopathy (HIE). METHODS: Ten newborns with HIE undergoing to therapeutic hypothermia (TH, HIE Group) and 8 healthy newborns (CTRL Group) were enrolled. For the HIE group, 5 samples were collected: between 0 and 6 h of life (time 1), 12 h (time 2), 24 h (time 3), 48 h (time 4) and 96 h of life (time 5). For the CTRL group, one sample was collected. A panel of 48 inflammatory cytokines was determined in all samples. Data were analyzed using multivariate statistical analysis (Principal component analysis, PCA) RESULTS: 17 cytokines, among 48 analyzed, were found to be significantly different, initially, between the CTRL and HIE groups: 12 with reported pro-inflammatory effects and 5 with reported anti-inflammatory effects. In the HIE group cytokines showed a decreasing trend during the TH and at the end of treatment comparable to the CTRL group. IL-18 did demonstrate a slight increase at time 3 during HT but decreased steadily at sampling times, 4 and 5. CONCLUSIONS: Our data demonstrates that many pathways of the inflammatory cascade are activated following hypoxic-ischemic injury. This information will increase our understanding of changes in cytokines over time in neonates with HIE undergoing TH.

Inflammatory Responses are Sex Specific in Chronic Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxic-ischemic encephalopathy (HIE) is increasingly recognized as a sexually dimorphic disease. Male infants are not only more vulnerable to ischemic insult; they also suffer more long-term cognitive deficits compared with females with comparable brain damage. The innate immune response plays a fundamental role in mediating acute neonatal HIE injury. However, the mechanism underlying the sex difference in chronic HIE is still elusive. The present study investigated the sex difference in HIE outcomes and inflammatory response in the chronic stage (30 days after HIE). Postnatal day 10 (P10) male and female C57BL/6 pups were subjected to 60-min Rice-Vanucci model (RVM) to induce HIE. Brain atrophy and behavioral deficits were analyzed to measure stroke outcomes at 30 days of HIE. Flow cytometry (FC) was performed to examine central (microglial activation) and peripheral immune responses. Serum levels of cytokines and sex hormones were determined by enzyme-linked immunosorbent assay (ELISA). Neurogenesis was quantified by 5-Bromo-2'-deoxyuridine (BrdU) incorporation with neurons. Results showed males had worse HIE outcomes than females at the endpoint. Female microglia exhibited a more robust anti-inflammatory response that was corresponding to an enhanced expression of CX3C chemokine receptor 1 (CX3CR1) than males. More infiltration of peripheral lymphocytes was seen in male vs. female HIE brains. Cytokine levels of tumor necrosis factor (TNF)-α and interleukin (IL)-10 were more upregulated in males and females respectively than their counterparts. Neurogenesis was more highly induced in females vs. males. No significant difference in circulating hormonal level was found between males and females after HIE. We conclude that a sex dichotomy in pro- and anti-inflammatory response underlies the sex-specific chronic HIE outcomes, and an enhanced neurogenesis in females also contribute to the sex difference.

Chronic inflammation and impaired development of the preterm brain.

The preterm newborn is at significant risk of neural injury and impaired neurodevelopment. Infants with mild or no evidence of injury may also be at risk of altered brain development, with evidence impaired cell maturation. The underlying causes are multifactorial and include exposure of both the fetus and newborn to hypoxia-ischemia, inflammation (chorioamnionitis) and infection, adverse maternal lifestyle choices (smoking, drug and alcohol use, diet) and obesity, as well as the significant demand that adaptation to post-natal life places on immature organs. Further, many fetuses and infants may have combinations of these events, and repeated (multi-hit) events that may induce tolerance to injury or sensitize to greater injury. Currently there are no treatments to prevent preterm injury or impaired neurodevelopment. However, inflammation is a common pathway for many of these insults, and clinical and experimental evidence demonstrates that acute and chronic inflammation is associated with impaired brain development. This review examines our current knowledge about the relationship between inflammation and preterm brain development, and the potential for stem cell therapy to provide neuroprotection and neurorepair through reducing inflammation and release of trophic factors, which promote cell maturation and repair.

Brain-immune interactions in perinatal hypoxic-ischemic brain injury.

Perinatal hypoxia-ischemia remains the primary cause of acute neonatal brain injury, leading to a high mortality rate and long-term neurological deficits, such as behavioral, social, attentional, cognitive and functional motor deficits. An ever-increasing body of evidence shows that the immune response to acute cerebral hypoxia-ischemia is a major contributor to the pathophysiology of neonatal brain injury. Hypoxia-ischemia provokes an intravascular inflammatory cascade that is further augmented by the activation of resident immune cells and the cerebral infiltration of peripheral immune cells response to cellular damages in the brain parenchyma. This prolonged and/or inappropriate neuroinflammation leads to secondary brain tissue injury. Yet, the long-term effects of immune activation, especially the adaptive immune response, on the hypoxic-ischemic brain still remain unclear. The focus of this review is to summarize recent advances in the understanding of post-hypoxic-ischemic neuroinflammation triggered by the innate and adaptive immune responses and to discuss how these mechanisms modulate the brain vulnerability to injury. A greater understanding of the reciprocal interactions between the hypoxic-ischemic brain and the immune system will open new avenues for potential immunomodulatory therapy in the treatment of neonatal brain injury.

Insights Into the Neuroinflammatory Responses After Neonatal Hypoxia-Ischemia.

Neonatal hypoxia-ischemia (HI) is one of the major causes of death and/or lifelong neurobehavioral and cognitive dysfunction. Undoubtedly, brain damage following HI insult is a complex process with multiple contributing mechanisms and pathways resulting in both early and delayed injury. It is increasingly recognized that one of the leading pathogenic factors of neonatal brain damage is inflammation, induced by activation of the central and peripheral immune system. Immune responses are induced within minutes and can expand for weeks and even months after the insult. Both activated intrinsic (glia) and infiltrating cells (mast cells, monocytes/macrophages) produce soluble inflammatory molecules such as cytokines, chemokines, reactive oxygen, and nitrogen species, which are thought to be pivotal mediators of persistent neuronal injury. This manuscript provides a brief summary of the current knowledge concerning the specific contribution of different cell types and soluble factors to injury of the developing brain caused by neonatal HI. Finally, we discuss the potential forthcoming treatments aimed at targeting inflammation and then attenuation of damaging effects caused by neonatal HI.

Cord Blood IL-16 Is Associated with 3-Year Neurodevelopmental Outcomes in Perinatal Asphyxia and Hypoxic-Ischaemic Encephalopathy.

Activation of the inflammatory pathway is increasingly recognized as an important mechanism of injury following neonatal asphyxia and encephalopathy. This process may contribute to the poor prognosis seen in some cases, despite therapeutic hypothermia. Our group has previously identified raised interleukin (IL)-6 and IL-16, measured in umbilical cord blood at birth, to be predictive of grade of hypoxic-ischaemic encephalopathy (HIE). Our aim in this study was to examine the ability of these cytokines to predict the 3-year neurodevelopmental outcome in the same cohort. As part of a prospective, longitudinal cohort study set in a single tertiary maternity unit, term infants with biochemical and clinical evidence of perinatal asphyxia were recruited at birth. Umbilical cord blood was collected and analyzed for IL-6 and IL-16 using a Luminex assay. The neurodevelopmental outcome of these infants was assessed at 3 years using the Bayley Scales of Infant and Toddler Development (Edition 3). Early cord blood measurement of IL-6 and IL-16 and long-term outcome were available in 33/69 infants. Median (IQR) IL-16 differentiated infants with a severely abnormal outcome (n = 6) compared to all others (n = 27), (646 [466-1,085] vs. 383.5 [284-494] pg/mL; p = 0.012). IL-16 levels were able to predict a severe outcome with an area under the receiver-operating characteristic (ROC) curve of 0.827 (95% CI 0.628-1.000; p = 0.014). Levels ≥514 pg/mL predicted a severe outcome with a sensitivity of 83% and a specificity of 81%. IL-16 also outperformed other routine biochemical markers available at birth for the prediction of severe outcome. APGAR scores at 1 and 10 min were also predictive of a severe outcome (p = 0.022 and p = 0.036, respectively). A combination of IL-16 with these clinical markers did not improve predictive value, but IL-16 combined with electroencephalogram grading increased the area under the ROC curve. IL-6 did not show any association with 3-year outcome. This is the first report studying the association of IL-16 measured at birth with long-term outcome in a cohort of neonates with perinatal asphyxia. IL-16 may be an early biomarker of severe injury and aid in the long-term prognostication in infants with HIE.

Neutralizing anti-interleukin-1ß antibodies reduce ischemia-related interleukin-1ß transport across the blood-brain barrier in fetal sheep.

Hypoxic ischemic insults predispose to perinatal brain injury. Pro-inflammatory cytokines are important in the evolution of this injury. Interleukin-1ß (IL-1ß) is a key mediator of inflammatory responses and elevated IL-1ß levels in brain correlate with adverse neurodevelopmental outcomes after brain injury. Impaired blood-brain barrier (BBB) function represents an important component of hypoxic-ischemic brain injury in the fetus. In addition, ischemia-reperfusion increases cytokine transport across the BBB of the ovine fetus. Reducing pro-inflammatory cytokine entry into brain could represent a novel approach to attenuate ischemia-related brain injury. We hypothesized that infusions of neutralizing IL-1ß monoclonal antibody (mAb) reduce IL-1ß transport across the BBB after ischemia in the fetus. Fetal sheep were studied 24-h after 30-min of carotid artery occlusion. Fetuses were treated with placebo- or anti-IL-1ß mAb intravenously 15-min and 4-h after ischemia. Ovine IL-1ß protein expressed from IL-1ß pGEX-2T vectors in Escherichia coli (E. coli) BL-21 cells was produced, purified, and radiolabeled with 125I. BBB permeability was quantified using the blood-to-brain transfer constant (Ki) with 125I-radiolabeled-IL-1ß. Increases in anti-IL-1ß mAb were observed in the brain of the mAb-treated group (P<0.001). Blood-to-brain transport of 125I-IL-1ß was lower (P<0.04) across brain regions in the anti-IL-1ß mAb-treated than placebo-treated ischemic fetuses. Plasma 125I-IL-1ß counts were higher (P<0.001) in the anti-IL-1ß mAb- than placebo-treated ischemic fetuses. Systemic infusions of anti-IL-1ß mAb reduce IL-1ß transport across the BBB after ischemia in the ovine fetus. Our findings suggest that conditions associated with increases in systemic pro-inflammatory cytokines and neurodevelopmental impairment could benefit from an anti-cytokine therapeutic strategy.

Osteopontin Is a Blood Biomarker for Microglial Activation and Brain Injury in Experimental Hypoxic-Ischemic Encephalopathy.

Clinical management of neonatal hypoxic-ischemic encephalopathy (HIE) suffers from the lack of reliable surrogate marker tests. Proteomic analysis may identify such biomarkers in blood, but there has been no proof-of-principle evidence to support this approach. Here we performed in-gel trypsin digestion of plasma proteins from four groups of 10-d-old mice [untouched and 24 h after low-dose lipopolysaccharide (LPS) exposure, hypoxia-ischemia (HI), or LPS/HI injury; n = 3 in each group) followed by liquid chromatography-tandem mass spectrometry and bioinformatics analysis to search for HI- and LPS/HI-associated brain injury biomarkers. This analysis suggested the induction of plasma osteopontin (OPN) by HI and LPS/HI, but not by sham and injury-free LPS exposure. Immunoblot confirmed post-HI induction of OPN protein in brain and blood, whereas Opn mRNA was induced in brain but not in blood. This disparity suggests brain-derived plasma OPN after HI injury. Similarly, immunostaining showed the expression of OPN by Iba1+ microglia/macrophages in HI-injured brains. Further, intracerebroventricular injection of LPS activated microglia and up-regulated plasma OPN protein. Importantly, the induction of plasma OPN after HI was greater than that of matrix metalloproteinase 9 or glial fibrillary acid protein. Plasma OPN levels at 48 h post-HI also parallel the severity of brain damage at 7-d recovery. Together, these results suggest that OPN may be a prognostic blood biomarker in HIE through monitoring brain microglial activation.