Galectin-3 Modulates Microglia Inflammation in vitro but Not Neonatal Brain Injury in vivo under Inflammatory Conditions

Microglia may contribute to injury but may also have neuroprotective properties. Galectin-3 has immunomodulatory properties that may affect the microglia phenotype and subsequent development of injury. Galectin-3 contributes to experimental hypoxic-ischemic (HI) injury in the neonatal brain, but it is unclear if galectin-3 has similar effects on infectious and sterile inflammation. Thus, we investigated the effect of galectin-3 on microglia in vitro under normal as well as infectious and sterile inflammatory conditions, and the effect of galectin-3 on neonatal brain injury following an infectious challenge in vivo. Conditions mimicking infectious or sterile inflammation were evaluated in primary microglia cell cultures from newborn mice, using LPS (10 ng/mL) and TNF-alpha (100 ng/mL). The response to galectin-3 was tested alone or together with LPS or TNF-alpha. Supernatants were collected 24 h after treatment and analyzed for 23 inflammatory mediators including pro- and anti-inflammatory cytokines and chemokines using multiplex protein analysis, as well as ELISA for MCP-1 and insulin-like growth factor (IGF)-1. Phosphorylation of proteins (AKT, ERK1/2, I kappa B-alpha, JNK, and p38) was determined in microglia cells. Neonatal brain injury was induced by a combination of LPS and HI (LPS + HI) in postnatal day 9 transgenic mice lacking functional galectin-3 and wild-type controls. LPS and TNF-alpha induced pro-inflammatory (9/11 vs. 9/10) and anti-inflammatory (6/6 vs. 2/6) cytokines, as well as chemokines (6/6 vs. 4/6) in a similar manner, except generally lower amplitude of the TNF-alpha-induced response. Galectin-3 alone had no effect on any of the proteins analyzed. Galectin-3 reduced the LPS- and TNF-alpha-induced microglia response for cytokines, chemokines, and phosphorylation of I kappa B-alpha. LPS decreased baseline IGF-1 levels, and the levels were restored by galectin-3. Brain injury or microglia response after LPS + HI was not affected by galectin-3 deficiency. Galectin-3 has no independent effect on microglia but modulates inflammatory activation in vitro. The effect was similar under infectious and sterile inflammatory conditions, suggesting that galectin-3 regulates inflammation not just by binding to LPS or toll-like receptor-4. Galectin-3 restores IGF-1 levels reduced by LPS-induced inflammation, suggesting a potential protective effect on infectious injury. However, galectin-3 deficiency did not affect microglia activation and was not beneficial in an injury model encompassing an infectious challenge

The Alpha 7 Nicotinic Acetylcholine Receptor Does Not Affect Neonatal Brain Injury

Inflammation plays a central role in the development of neonatal brain injury. The alpha 7 nicotinic acetylcholine receptor (α7nAChR) can modulate inflammation and has shown promising results as a treatment target in rodent models of adult brain injury. However, little is known about the role of the α7nAChR in neonatal brain injury. Hypoxic-ischemic (HI) brain injury was induced in male and female C57BL/6 mice, α7nAChR knock-out (KO) mice and their littermate controls on postnatal day (PND) 9–10. C57BL/6 pups received i.p. injections of α7nAChR agonist PHA 568487 (8 mg/kg) or saline once daily, with the first dose given directly after HI. Caspase-3 activity and cytokine mRNA expression in the brain was analyzed 24 h after HI. Motor function was assessed 24 and 48 h after HI, and immunohistochemistry was used to assess tissue loss at 24 h and 7 days after HI and microglial activation 7 days after HI. Activation of α7nAChR with the agonist PHA 568487 significantly decreased CCL2/MCP-1, CCL5/RANTES and IL-6 gene expression in the injured brain hemisphere 24 h after HI compared with saline controls in male, but not female, pups. However, α7nAChR activation did not alter caspase-3 activity and TNFα, IL-1β and CD68 mRNA expression. Furthermore, agonist treatment did not affect motor function (24 or 48 h), neuronal tissue loss (24 h or 7 days) or microglia activation (7 days) after HI in either sex. Knock-out of α7nAChR did not influence neuronal tissue loss 7 days after HI. In conclusion, targeting the α7nAChR in neonatal brain injury shows some effect on dampening acute inflammatory responses in male pups. However, this does not lead to an effect on overall injury outcome

Dysmaturation of somatostatin interneurons following umbilical cord occlusion in preterm fetal sheep

IntroductionCerebral white matter injury is the most common neuropathology observed in preterm infants. However, there is increasing evidence that gray matter development also contributes to neurodevelopmental abnormalities. Fetal cerebral ischemia can lead to both neuronal and non-neuronal structural-functional abnormalities, but less is known about the specific effects on interneurons.ObjectiveIn this study we used a well-established animal model of fetal asphyxia in preterm fetal sheep to study neuropathological outcome. We used comprehensive stereological methods to investigate the total number of oligodendrocytes, neurons and somatostatin (STT) positive interneurons as well as 3D morphological analysis of STT cells 14 days following umbilical cord occlusion (UCO) in fetal sheep.Materials and MethodsInduction of asphyxia was performed by 25 min of complete UCO in five preterm fetal sheep (98–100 days gestational age). Seven, non-occluded twins served as controls. Quantification of the number of neurons (NeuN), STT interneurons and oligodendrocytes (Olig2, CNPase) was performed on fetal brain regions by applying optical fractionator method. A 3D morphological analysis of STT interneurons was performed using IMARIS software.ResultsThe number of Olig2, NeuN, and STT positive cells were reduced in IGWM, caudate and putamen in UCO animals compared to controls. There were also fewer STT interneurons in the ventral part of the hippocampus, the subiculum and the entorhinal cortex in UCO group, while other parts of cortex were virtually unaffected (p > 0.05). Morphologically, STT positive interneurons showed a markedly immature structure, with shorter dendritic length and fewer dendritic branches in cortex, caudate, putamen, and subiculum in the UCO group compared with control group (p < 0.05).ConclusionThe significant reduction in the total number of neurons and oligodendrocytes in several brain regions confirm previous studies showing susceptibility of both neuronal and non-neuronal cells following fetal asphyxia. However, in the cerebral cortex significant dysmaturation of STT positive neurons occurred in the absence of cell loss. This suggests an abnormal maturation pattern of GABAergic interneurons in the cerebral cortex, which might contribute to neurodevelopmental impairment in preterm infants and could implicate a novel target for neuroprotective therapies

Inflammation and neuroprotective strategies in the immature brain after hypoxic-ischemic brain injury

Perinatal brain injury, as a result of hypoxia-ischemia (HI) or infection/HI, is a major cause of acute mortality and neurological morbidity in infants and children. The mechanisms of perinatal HI are not fully understood, which makes it difficult to find effective treatment. The aim of the thesis was to investigate the mechanisms of perinatal HI brain injury, and to evaluate different neuroprotective strategies; 1) effects of N-acetylcysteine and melatonin after LPS-sensitized HI, 2) effects of a GPE analogue (G-2mPE) after HI, 3) the involvement of matrix metalloproteinase (MMP) -9 and -12 after HI. An animal model of perinatal brain injury was used in the neonatal rat/mouse, i.e. permanent ligation of the left carotid artery, followed by exposure to a gas mixture with low oxygen content, either alone (HI) or in a combination with infection (LPS/HI). Neuroprotective effects of N-acetylcysteine and melatonin were investigated in neonatal pups after LPS/HI. The drugs were given in multiple doses and brain injury was evaluated 7 days after the HI insult. The neuroprotective effect of post HI administered G-2mPE was investigated in neonatal rats. MMP-9 gene deficient mice were used to evaluate the importance of MMP-9 after perinatal HI. MMP-12 expression after HI was investigated in wild type animals after perinatal brain injury. Marked neuroprotection was found with NAC treatment, which was associated with reduced isoprostane activation and nitrotyrosine formation, increased levels of the antioxidants glutathione and thioredoxin-2 and inhibition of caspase-3, calpain, and caspase-1 activation. A moderate reduction of brain damage was obtained after pre/post treatment with melatonin. Post-HI treatment with G-2mPE attenuated neuronal injury and promoted astrogliosis, as well as blood vessel growth. MMP-9 was shown to play an important role in the development of HI injury in the immature brain, particularly with regard to blood-brain barrier leakage and inflammation. MMP-12 may also be important for the development of brain injury, as the MMP-12 mRNA expression is up-regulated 24 hours after HI and an increased number of cells express MMP-12 in the damaged ipsilateral hemisphere

Growth differentiation factor 15 increases in both cerebrospinal fluid and serum during pregnancy.

AimGrowth differentiation factor 15 (GDF15) increases in serum during pregnancy to levels not seen in any other physiological state and is suggested to be involved in pregnancy-induced nausea, weight regulation and glucose metabolism. The main action of GDF15 is regulated through a receptor of the brainstem, i.e., through exposure of GDF15 in both blood and cerebrospinal fluid (CSF). The aim of the current study was to measure GDF15 in both CSF and serum during pregnancy, and to compare it longitudinally to non-pregnant levels.MethodsWomen were sampled at elective caesarean section (n = 45, BMI = 28.1±5.0) and were followed up 5 years after pregnancy (n = 25). GDF15, insulin and leptin were measured in CSF and serum. Additional measurements included plasma glucose, and serum adiponectin and Hs-CRP.ResultsGDF15 levels were higher during pregnancy compared with follow-up in both CSF (385±128 vs. 115±32 ng/l, PConclusionThis, the first study to measure CSF GDF15 during pregnancy, demonstrated increased GDF15 levels in both serum and CSF during pregnancy. The results suggest that effects of GDF15 during pregnancy can be mediated by increases in both CSF and serum levels

Peripheral myeloid cells contribute to brain injury in male neonatal mice

Abstract 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

Systemic activation of Toll-like receptor 2 suppresses mitochondrial respiration and exacerbates hypoxic-ischemic injury in the developing brain

Infection and inflammation are known risk factors for neonatal brain injury. Mycoplasma and Gram-positive bacteria, for which Toll-like receptor 2 (TLR2) plays a key role in recognition and inflammatory response, are among the most common pathogens in the perinatal period. Here, we report that systemic activation of TLR2 by Pam3CSK4 (P3C) increases neural tissue loss and demyelination induced by subsequent hypoxia–ischemia (HI) in neonatal mice. High-resolution respirometry of brain isolated mitochondria revealed that P3C suppresses ADP-induced oxidative phosphorylation, the main pathway of cellular energy production. The results suggest that infection and inflammation might contribute to HI-induced energy failure. </jats:p

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

Immune cells display a high degree of phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with classical and alternative polarization phenotypes described for macrophages and to identify related cell populations in the brain following neonatal hypoxia-ischemia (HI). HI was induced in 9-day old mice and brain tissue was collected up to 7 d post-insult to investigate expression of genes associated with macrophage activation. Using cell-markers, CD86 (classic activa-tion) and CD206 (alternative activation), we assessed temporal changes of CD11b+ cell populations in the brain and studied the protein expression of the immunomodulatory factor galectin-3 in these cells. HI induced a rapid regulation (6h) of genes associated with both classical and alternative polarization phenotypes in the injured hemisphere. FACS analysis showed a marked increase in the number of CD11+CD86+ positive cells at 24 h after HI (+3,667 %), which was coupled with a relative suppression of CD11+CD206+ cells and cells that did not express either CD86 or CD206. The CD11+CD206+ popula-tion was mixed with some cells also expressing CD86. Confocal microscopy confirmed that a subset of cells expressed both CD86 and CD206, particularly in injured grey and white matter. Protein con-centration of galectin-3 was markedly increased mainly in the cell population lacking CD86 or CD206 in the injured hemisphere. These cells were predominantly resident microglia as very few galectin-3 positive cells co-localized with infiltrating myeloid cells in Lys-EGFP-ki mice after HI.In summary, HI was characterized by an early mixed gene response, but with a large expansion of mainly the CD86 positive population during the first day. However, the injured hemisphere also con-tained a subset of cells expressing both CD86 and CD206 and a large population that expressed nei-ther activation marker CD86 nor CD206. Interestingly, these cells expressed the highest levels of ga-lectin-3 and were found to be predominantly resident microglia. Galectin-3 is a protein involved in chemotaxis and macrophage polarization suggesting a novel role in cell infiltration and immuno-modulation for this cell population after neonatal injury