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.

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.

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.

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.

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.

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.

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.

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.

Perinatal Arterial Ischemic Stroke Is Associated to Materno-Fetal Immune Activation and Intracranial Arteritis.

The medium-size intra-cranial arteries arising from the carotid bifurcation are prone to perinatal arterial ischemic strokes (PAIS). PAIS' physiopathology needs to be better understood to develop preventive and therapeutic interventions that are currently missing. We hypothesized that materno-fetal inflammation leads to a vasculitis affecting selectively the carotidian tree and promoting a focal thrombosis and subsequent stroke. Dams were injected with saline or lipopolysaccharide (LPS) from Escherichia coli. A prothrombotic stress was applied on LPS-exposed vs. saline (S)-exposed middle cerebral arteries (MCA). Immunolabeling detected the inflammatory markers of interest. In S-exposed newborn pups, a constitutive higher density of macrophages combined to higher expressions of tumor necrosis factor-α (TNF-α), and interleukin 1ß (IL-1ß) was observed within the wall of intra- vs. extra-cranial cervicocephalic arteries. LPS-induced maternal and placental inflammatory responses mediated by IL-1ß, TNF-α and monocyte chemotactic protein 1 (MCP-1) were associated with: (i) increased density of pro-inflammatory macrophages (M1 phenotype); and (ii) pro-inflammatory orientation of the IL-1 system (IL-1ß/IL-1 receptor antagonist (IL-1Ra) ratio) within the wall of LPS-, vs. S-exposed, intra-cranial arteries susceptible to PAIS. LPS plus photothrombosis, but not sole photothrombosis, triggered ischemic strokes and subsequent motor impairments. Based on these preclinical results, the combination of pro-thrombotic stress and selective intra-cranial arteritis arising from end gestational maternal immune activation seem to play a role in the pathophysiology of human PAIS.

Resveratrol post-treatment protects against neonatal brain injury after hypoxia-ischemia.

Neonatal hypoxic-ischemic brain injury is a devastating disease with limited treatment options. Preventive treatment with resveratrol has indicated to be well tolerated and has lower toxicity in both experimental models and human patients. However, whether resveratrol administration post-hypoxic-ischemic protects against neonatal hypoxic-ischemic injury is not known. Here we reported that post-treatment with resveratrol significantly reduced brain damage at 7-day after the injury. We found that resveratrol reduced the expression levels of key inflammatory factors at the mRNA and protein levels, and at least partially via inhibiting microglia activation. Moreover, resveratrol exerted an anti-apoptotic effect, as assessed by TUNEL staining, and altered the expression of the apoptosis-related genes Bax, Bcl-2 and caspase3. Our data indicate that post-treatment with resveratrol protects against neonatal hypoxic-ischemic brain injury and suggest a promising therapeutic strategy to this disease.

Loss of PINK1 inhibits apoptosis by upregulating α-synuclein in inflammation-sensitized hypoxic-ischemic injury in the immature brains.

The incidence of preterm birth is rising worldwide. Among preterm infants, many face a lifetime of neurologic impairments. Recent studies have revealed that systemic inflammation can sensitize the immature brain to hypoxic-ischemic (HI) injury. Therefore, it is important to identify the mechanisms involved in inflammation-sensitized HI injury in immature brains. PTEN-induced putative kinase 1 (PINK1) is a regulatory protein that is highly expressed in the brain. We have previously found that PINK1 gene knockout can protect matured brains from HI injury in postnatal day 10 mice. However, the mechanisms are unknown. In this study, we employed an inflammation-sensitized HI injury model using postnatal day 3 mice to study the roles and mechanisms that PINK1 plays in the immature brains. Lipopolysaccharide (LPS) was injected intraperitoneally into the mice before HI treatment to set up the model. We found that PINK1-knockout mice had fewer brain infarcts and less cell apoptosis than did the wild-type mice. Furthermore, we found that α-synuclein was markedly higher in the PINK1-knockout mice than in the wild-type mice, and inhibition of α-synuclein through small interfering RNA (siRNA) reversed the protective effect in the PINK1-knockout mice. Collectively, these findings indicate that loss of PINK1 plays a novel role in the protection of inflammation-sensitized HI brain damage.

PML regulates neuroprotective innate immunity and neuroblast commitment in a hypoxic-ischemic encephalopathy model.

Regulation of innate immune responses and activation of tissue regenerative processes are key elements in the pathophysiology of brain injuries. The promyelocytic leukemia (PML) gene was originally identified on a breakpoint of chromosomal translocation t(15;17) associated with acute PML. We have studied the role of PML protein during acute and regenerative phases after hypoxia-ischemia (HI) in brains of neonatal mice. We found that PML prevents tissue loss and apoptotic cell death selectively in subcortical regions of the brain at early stages after damage. In accordance with this, we revealed that PML is important for microglia activation and production of key inflammatory cytokines such as IL1α, IL1ß, IL1RN, CXCL10, CCL12 and TNFα. During the regenerative phase, PML-depleted mice were found to have impaired transformation of transit-amplifying precursors into migratory progenitors. This was accompanied by increased ratios of symmetric versus asymmetric neural progenitor cell divisions during tissue repair and a specific defect in tissue restoration within the striatum 42 days after HI. The data demonstrate a dual role of PML in protection and recovery after brain injury.

Effect of creatine monohydrate supplementation on relative serum level of IL-6 and IL-18 following neonatal hypoxia ischemia in male albino mouse.

IL-6 has been reported to have neuroprotective effects against cerebral ischemia while IL-8 is a pro inflammatory cytokine structurally related to interleukin-1 family. In the present study, we tried to determine whether 2% Creatine monohydrate supplementation for variable duration influence the IL-6 and 18 concentrations in the serum of male albino mouse following right common carotid artery ligation and hypoxia (8% oxygen) for 25 minutes. Our result revealed that serum concentration of IL6 (P=0.0001) as well as IL-18 (P=0.003) were significantly higher in mice supplemented with creatine monohydrate for 15 weeks than in male albino mice on normal rodent diet following hypoxic ischemic insult indicating that long term creatine monohydrate supplementation up regulates the IL-6 and IL-18 concentrations triggering the neuroinflammatory and neuroprotective responses.

Resident microglia, rather than blood-derived macrophages, contribute to the earlier and more pronounced inflammatory reaction in the immature compared with the adult hippocampus after hypoxia-ischemia.

The mechanisms of neuronal injury after hypoxia-ischemia (HI) are different in the immature and the adult brain, but microglia activation has not been compared. The purpose of this study was to phenotype resident microglia and blood-derived macrophages in the hippocampus after HI in neonatal (postnatal day 9, P9) or adult (3 months of age, 3mo) mice. Unilateral brain injury after HI was induced in Cx3cr1(GFP/+) Ccr2(RFP/+) male mice on P9 (n = 34) or at 3mo (n = 53) using the Vannucci model. Resident microglia (Cx3cr1-GFP+) proliferated and were activated earlier after HI in the P9 (1-3 days) than that in the 3mo hippocampus, but remained longer in the adult brain (3-7 days). Blood-derived macrophages (Ccr2-RFP+) peaked 3 days after HI in both immature (P9) and adult (3mo) hippocampi but were twice as frequent in adult brains, 41% vs. 21% of all microglia/macrophages. CCL2 expression was three times higher in the P9 hippocampi, indicating that the proinflammatory response was more pronounced in the immature brain after HI. This corresponded well with the higher numbers of galectin-3-positive resident microglia in the P9 hippocampi, but did not correlate with CD16/32- or CD206-positive resident microglia or blood-derived macrophages. In conclusion, resident microglia, rather than infiltrating blood-derived macrophages, proliferate and are activated earlier in the immature than in the adult brain, but remain increased longer in the adult brain. The inflammatory response is more pronounced in the immature brain, and this correlate well with galectin-3 expression in resident microglia.

The impact of Ly6Clow monocytes after cerebral hypoxia-ischemia in adult mice.

After an ischemic stroke, mononuclear phagocytic cells such as microglia, macrophages, and monocytes migrate to the lesion site and coordinate an immune response. Monocytes, which are recruited from the bloodstream after ischemic brain injury, can be categorized into two subsets in mice: inflammatory and patrolling monocytes. Although inflammatory monocytes (Ly6C(hi)) seem to have a protective role in stroke progression, the impact of patrolling monocytes (Ly6C(low)) is unknown. To address the role of Ly6C(low) monocytes in stroke, we generated bone marrow chimeric mice in which their hematopoietic system was replaced by Nr4a1(-/-) cells, allowing the complete and permanent ablation of Ly6C(low) monocytes without affecting the Ly6C(hi) subset. We then subjected adult mice to cerebral hypoxia-ischemia using the Levine/Vannucci model. Functional outcomes after stroke such as body weight change, neurologic score, motor functions and spatial learning were not affected. Moreover, depletion in Ly6C(low) monocytes did not change significantly the total infarct size, cell loss, atrophy, the number, or the activation state of microglia/macrophages at the lesion site. These data suggest that Ly6C(low) patrolling monocytes are redundant in the progression and recovery of ischemic stroke.

Regional differences of microglial accumulation within 72 hours of hypoxia-ischemia and the effect of acetylcholine receptor agonist on brain damage and microglial activation in newborn rats.

OBJECTIVE: We examined regional specificity of microglial activation in the developing rat brain for 72 hours after hypoxia-ischemia (HI) and the effect of acetylcholine receptor (AChR) agonist on microglial activation. STUDY DESIGN: Seven-day-old Wistar rats were divided into two groups: one receiving a single dose of AChR agonist just before hypoxia (carbachol; 0.1mg/kg) to investigate the reducing effect on brain damage with decreasing activation of microglia and the other group receiving saline as a control. Rats were subjected to left carotid artery ligation followed by 8% hypoxia. Brains were analyzed immunohistochemically at 24, 48, and 72 hours after HI. TNFα production was measured at respective times after HI. RESULTS: Activation of microglia on the hippocampus of the control group was strong for the first 48 hours and then weakened. In contrast, activation of microglia on white matter and the cortex was weak at 24 hours and then became stronger. A single dose of carbachol significantly reduced brain damage with a marked reduction of microglial activation on the hippocampus, whereas it was less effective regarding microglial activation on white matter and the cortex. TNFα production was low in both groups. CONCLUSION: Regional specificity was observed for both microglial activation and susceptibility to carbachol for the first 72 hours after HI. Our data suggested that timely intervention along with region-specific microglial activation, apart from TNFα production, may be critical for the prevention of further brain damage after HI in the newborn.

Mesenchymal stem cells induce T-cell tolerance and protect the preterm brain after global hypoxia-ischemia.

Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 10(6) MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE.

Toll-like receptor-3 activation increases the vulnerability of the neonatal brain to hypoxia-ischemia.

Susceptibility and progression of brain injury in the newborn is closely associated with an exacerbated innate immune response, but the underlying mechanisms are often unclear. Toll-like receptors (TLRs) are important innate immune sensors that may influence the vulnerability of the developing brain. In the current study, we provide novel data to show that activation of the viral innate immune receptor TLR-3 sensitizes the neonatal brain to subsequent hypoxic-ischemic (HI) damage. Poly inosinic:poly cytidylic acid (Poly I:C), a synthetic ligand for TLR-3, was administered to neonatal mice 14 h before cerebral HI. Activation of TLR-3 before HI increased infarct volume from 3.0 ± 0.5 to 15.4 ± 2.1 mm³ and augmented loss of myelin basic protein from 13.4 ± 6.0 to 70.6 ± 5.3%. The sensitizing effect of Poly I:C was specific for the TLR-3 pathway because mice deficient in the TLR-3 adaptor protein Toll/IL-1R domain-containing adaptor molecule-1 (TRIF) did not develop larger brain damage. The increased vulnerability was associated with a TRIF-dependent heightened inflammatory response, including proinflammatory cytokines, chemokines, and the apoptosis-associated mediator Fas, whereas there was a decrease in reparative M2-like CD11b⁺ microglia and phosphorylation of Akt. Because TLR-3 is activated via double-stranded RNA during most viral infections, the present study provides evidence that viral infections during pregnancy or in the neonate could have great impact on subsequent HI brain injury.

Mast cell isolation from the immature rat brain.

Mast cells are immune cells of hematopoietic origin that circulate as precursor cells prior to migration into vascularized tissues where they mature and undergo terminal differentiation in response to different cytokines within the local environment. Mast cells are well known as important regulators of inflammatory processes in peripheral tissues and recent studies support the involvement of mast cells in mediating the inflammatory response to cerebral hypoxia-ischemia in both the neonatal and adult brain. To better study mast cell function in vivo, it is important to be able to identify their environment-specific phenotype, as well as to study their interaction with other neural cells in vitro. Previous such studies of mast cells have relied on mast cells isolated from gut or bone marrow, or on a number of mast cell lines, all of which may behave differently from brain mast cells. The purpose of this study was to develop a technique for the isolation of mast cells from neonatal rat brain and to characterize these cells following hypoxia and hypoxia-ischemia. We adapted a previously described technique of coupling an antibody to the mast cell-specific FcεR1 receptor to a MACS microbead for the selective removal of intact mast cells from a neonatal brain preparation. We have isolated toluidine blue-positive brain mast cells that provide substrate for both protein analysis and in vitro studies. These cells express proteins previously used to specifically identify microglia in the brain, Iba-1 and coronin-1a. A subpopulation of mast cells in vivo also expresses Iba-1. Thus, we report a novel method for isolation of brain mast cells suitable for the study of mast cell phenotype under a variety of conditions. Further, we suggest that the use of proteins such as Iba-1 for the identification of microglia in the brain includes the caveat that mast cells may also be detected.