Karin Sävman

Characterization of phenotype markers and neuronotoxic potential of polarised primary microglia in vitro

Highlight ► A unique catalogue of phenotype markers and neuronotoxic effects of polarised primary microglia, as a comparative tool to screen neurotherapies.

Matrix Metalloproteinase-9 Gene Knock-out Protects the Immature Brain after Cerebral Hypoxia–Ischemia

Inhibition of matrix metalloproteinase-9 (MMP-9) protects the adult brain after cerebral ischemia. However, the role of MMP-9 in the immature brain after hypoxia–ischemia (HI) is unknown. We exposed MMP-9(−/−) [MMP-9 knock-out (KO)] and wild-type (WT) mice to HI on postnatal day 9. HI was induced by unilateral ligation of the left carotid artery followed by hypoxia (10% O2; 36°C). Gelatin zymography showed that MMP-9 activity was transiently increased at 24 h after HI in the ipsilateral hemisphere and MMP-9-positive cells were colocalized with activated microglia. Seven days after 50 min of HI, cerebral tissue volume loss was reduced in MMP-9 KO (21.8 ± 1.7 mm3; n = 22) compared with WT (32.3 ± 2.1 mm3; n = 22; p < 0.001) pups, and loss of white-matter components was reduced in MMP-9 KO compared with WT pups (neurofilament: WT, 50.9 ± 5.4%; KO, 18.4 ± 3.1%; p < 0.0001; myelin basic protein: WT, 57.5 ± 5.8%; KO, 23.2 ± 3.5%; p = 0.0001). The neuropathological changes were associated with a delayed and diminished leakage of the blood–brain barrier (BBB) and a decrease in inflammation in MMP-9-deficient animals. In contrast, the neuroprotective effects after HI in MMP-9-deficient animals were not linked to either caspase-dependent (caspase-3 and cytochrome c) or caspase-independent (apoptosis-inducing factor) processes. This study demonstrates that excessive activation of MMP-9 is deleterious to the immature brain, which is associated with the degree of BBB leakage and inflammation. In contrast, apoptosis does not appear to be a major contributing factor.

Cytokine response in cerebrospinal fluid after birth asphyxia

Experimental studies suggest that cytokine-mediated inflammatory reactions are important in the cascade leading to hypoxicischemic brain injury. The purpose was to study the content of pro- and antiinflammatory cytokines in cerebrospinal fluid (CSF) of asphyxiated and control infants. Samples of CSF were obtained from 20 infants who fulfilled the criteria of birth asphyxia and from seven newborn control subjects. The concentrations of IL-1β, IL-8, IL-10, tumor necrosis factor (TNF)-α, and granulocyte/monocyte colony-stimulating factor (GM-CSF) were determined with ELISA and of IL-6 using a bioassay. The concentration of IL-6 (pg/mL) was higher in asphyxiated(250, 35-543; median, interquartile range) than in control (0, 0-18) infants(p = 0.001). There was also a significant relationship between IL-6 and the degree of HIE, and between IL-6 and outcome. In addition, the content of IL-8 (pg/mL) was higher (p = 0.009) in the asphyxia group (170, 70-1440), than in the the control group (10, 0-30) and there was an association between IL-8 and degree of HIE. The levels of IL-10, TNF-α, GM-CSF, and IL-1β did not differ between groups. In conclusion, the proinflammatory cytokines IL-6 and IL-8 were markedly elevated in CSF of asphyxiated infants, and the intrathecal levels of these cytokines corresponded to the degree of HIE.

Sequelae of chorioamnionitis.

Chorioamnionitis correlates to preterm delivery prior to 30 weeks of gestation. In most studies, proxies of intrauterine infection (clinical chorioamnionitis, histological chorioamnionitis, intra-amniotic increase in cytokines) are associated with acute neonatal morbidity and mortality and, at least to some degree, with neurological impairments (periventricular leukomalacia, intraventricular hemorrhage, cerebral palsy, polymicrogyria), chronic lung disease, and involution of the thymus in the preterm infant. The connection to visual impairment and cognitive deficits is uncertain or unknown. Full-term babies exposed to intrauterine infection often present with depressed Apgar scores and neonatal encephalopathy, and are at markedly increased risk of developing cerebral palsy. The infectious/inflammatory mechanisms involved are incompletely understood, and the types of microbes, as well as the genetic characteristics of the host adaptive and innate immune response, need to be better characterized.

Non-Protein-Bound Iron Is Elevated in Cerebrospinal Fluid from Preterm Infants with Posthemorrhagic Ventricular Dilatation

Posthemorrhagic ventricular dilatation (PHVD) is closely associated with white matter injury and neurologic disability in the preterm infant. An important factor in periventricular white matter damage may be the specific vulnerability of iron-rich immature oligodendroglia to reactive oxygen species toxicity. Non-protein-bound iron (NPBI) is a potent catalyst in the generation of hydroxyl radicals (Fenton reaction). Our objective was to determine whether NPBI is increased in cerebrospinal fluid (CSF) from preterm infants with PHVD compared with preterm control infants. Samples of CSF were obtained from 20 infants with PHVD and 10 control subjects. The level of NPBI was determined by a new spectrophotometric method using bathophenanthroline as a chelator. To evaluate the effect of hemolysis, CSF and blood were mixed in different proportions, spun, frozen and thawed, and then analyzed for NPBI. NPBI was found in 75% (15 of 20) of infants with PHVD and in 0% (0 of 10) of control infants (p = 0.0002). Hemolysis induced in vitro did not result in any significant levels of NPBI. Within the group with PHVD, NPBI concentrations in CSF did not correlate with disability, parenchymal brain lesions, or the need for shunt surgery. NPBI was increased in CSF from preterm infants with PHVD, and the increase could not be explained by hemolysis alone. Free iron may help to explain the association between intraventricular hemorrhage and white matter damage.

Galectin-3 contributes to neonatal hypoxic-ischemic brain injury.

Inflammation induced by hypoxia-ischemia (HI) contributes to the development of injury in the newborn brain. In this study, we investigated the role of galectin-3, a novel inflammatory mediator, in the inflammatory response and development of brain injury in a mouse model for neonatal HI. Galectin-3 gene and protein expression was increased after injury and galectin-3 was located in activated microglia/macrophages. Galectin-3-deficient mice (gal3-/-) were protected from injury particularly in hippocampus and striatum. Microglia accumulation was increased in the gal3-/- mice but accompanied by decreased levels of total matrix metalloproteinase (MMP)-9 and nitrotyrosine. The protection and increase in microglial infiltration was more pronounced in male gal3-/- mice. Trophic factors and apoptotic markers did not significantly differ between groups. In conclusion, galectin-3 contributes to neonatal HI injury particularly in male mice. Our results indicate that galectin-3 exerts its effect by modulating the inflammatory response.

Pasteurization of mother's own milk reduces fat absorption and growth in preterm infants

Aim: A randomized study was conducted to evaluate whether pasteurized milk (Holder pasteurization 62.5°C, 30 min) reduces fat absorption and growth in preterm infants.

Galectin 3 aggravates joint inflammation and destruction in antigen-induced arthritis

OBJECTIVE Galectin 3, an endogenous β-galactoside-binding lectin, plays an important role in the modulation of immune responses. The finding that galectin 3 is present in the inflamed synovium in patients with rheumatoid arthritis suggests that the protein is associated with the pathogenesis of this disease. We undertook this study to investigate the influence of galectin 3 deficiency in a murine model of arthritis. METHODS Wild-type (WT) and galectin 3-deficient (galectin 3(-/-) ) mice were subjected to antigen-induced arthritis (AIA) through immunization with methylated bovine serum albumin. The concentration of serum cytokines (interleukin-6 [IL-6] and tumor necrosis factor α [TNFα]) and antigen-specific antibodies was evaluated using a cytometric bead array platform and enzyme-linked immunosorbent assay (ELISA). Cellular IL-17 responses were examined by flow cytometry, ELISA, and enzyme-linked immunospot assay. RESULTS The joint inflammation and bone erosion of AIA were markedly suppressed in galectin 3(-/-) mice as compared with WT mice. The reduced arthritis in galectin 3(-/-) mice was accompanied by decreased levels of antigen-specific IgG and proinflammatory cytokines. The frequency of IL-17-producing cells in the spleen was reduced in galectin 3(-/-) mice as compared with WT mice. Exogenously added recombinant galectin 3 could partially restore the reduced arthritis and cytokines in galectin 3(-/-) mice. CONCLUSION Our findings show that galectin 3 plays a pathogenic role in the development and progression of AIA and that the disease severity is accompanied by alterations of antigen-specific IgG levels, systemic levels of TNFα and IL-6, and frequency of IL-17-producing T cells. To our knowledge, this is the first report of in vivo evidence that galectin 3 plays a crucial role in the development of arthritis.

Effect of lipopolysaccharide on global gene expression in the immature rat brain

To improve the understanding of the molecular mechanisms whereby lipopolysaccharide (LPS) affects the immature brain, global gene expression following LPS exposure was investigated in neonatal rats. Brains (n = 5/time point) were sampled 2, 6, and 72 h after LPS and compared with age-matched controls. The mRNA from each brain was analyzed separately on Affymextrix GeneChip Rat Expression Set 230. The number of genes regulated after LPS were 847 at 2 h, 1564 at 6 h, and 1546 genes at 72 h. Gene ontology analysis demonstrated that, at both 2 and 6 h after LPS, genes associated with protein metabolism, response to external stimuli and stress (immune and inflammatory response, chemotaxis) and cell death were overrepresented. At 72 h, the most strongly regulated genes belonged to secretion of neurotransmitters, transport, synaptic transmission, cell migration, and neurogenesis. Several pathways associated with cell death/survival were identified (caspase-tumor necrosis factor α [TNF-α]-, p53-, and Akt/phosphatidylinositol-3-kinase (PI3 K)–dependent mechanisms). Caspase-3 activity increased and phosphorylation of Akt decreased 8 h after peripheral LPS exposure. These results show a complex cerebral response to peripheral LPS exposure. In addition to the inflammatory response, a significant number of cell death-associated genes were identified, which may contribute to increased vulnerability of the immature brain to hypoxia-ischemia (HI) following LPS exposure.

Expression of cytokines and chemokines in cervical and amniotic fluid: Relationship to histological chorioamnionitis

Objective. To correlate cervical and amniotic fluid cytokines and macrophage-related chemokines to the development of histological chorioamnionitis (HCA) in patients with preterm labor (PTL) and preterm prelabor rupture of the membranes (PPROM). Study design. Cervical and amniotic fluid interleukin (IL)-6, IL-8, IL-18, monocyte chemotactic protein (MCP)-1, MCP-2, and MCP-3 from pregnant women (at ≤34 weeks of gestation) in PTL (N = 42) were analyzed and related to the subsequent occurrence of HCA or inflammatory signs in the placenta. For the patients with PPROM (N = 30) only amniotic fluid proteins were analyzed. Results. Intra-amniotic levels of IL-6, IL-8, IL-18, MCP-1, and MCP-3 were significantly higher in PTL cases with HCA compared to non-HCA controls, whereas no such relationship was obtained in the PPROM group. Cervical IL-8 and IL-6 (but not IL-18, MCP-1, MCP-2, and MCP-3) in PTL patients was associated with HCA, and at a cut-off level of 10.0 ng/mL cervical IL-8 was a strong predictor of HCA in the PTL cases (sensitivity 100%, specificity 67%, positive predictive value 63%, negative predictive value 100%). The cytokine and chemokine levels in the group with inflammatory signs were generally higher than in controls but lower compared to the concentrations in the HCA group. Conclusions. The amniotic levels of IL-6, IL-8, IL-18, and the CC-chemokines MCP-1 and MCP-3 in PTL patients all predicted HCA, whereas only IL-8 was a clinically useful marker of HCA in the cervical fluid. In addition there is indication that the levels of inflammatory proteins are related to the degree of inflammatory infiltration in placental tissue samples.