Maj Hedtjärn

The Suitability of BV2 Cells as Alternative Model System for Primary Microglia Cultures or for Animal Experiments Examining Brain Inflammation

The role of microglia in neurodegeneration, toxicology and immunity is an expanding area of biomedical research requiring large numbers of animals. Use of a microglia-like cell line would accelerate many research programmes and reduce the necessity of continuous cell preparations and animal experimentation, provided that the cell line reproduces the in vivo situation or primary microglia (PM) with high fidelity. The immortalised murine microglial cell line BV-2 has been used frequently as a substitute for PM, but recently doubts were raised as to their suitability. Here, we re-evaluated strengths and potential short-comings of BV-2 cells. Their response to lipopolysaccharide was compared with the response of microglia in vitro and in vivo. Transcriptome (480 genes) and proteome analyses after stimulation with lipopolysaccharide indicated a reaction pattern of BV-2 with many similarities to that of PM, although the average upregulation of genes was less pronounced. The cells showed a normal regulation of NO production and a functional response to IFN-gamma, important parameters for appropriate interaction with T cells and neurons. BV-2 were also able to stimulate other glial cells. They triggered the translocation of NF-kappaB, and a subsequent production of IL-6 in astrocytes. Thus, BV-2 cells appear to be a valid substitute for PM in many experimental settings, incuding complex cell-cell interaction studies.

The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions

Overall, the inflammatory potential of lipopolysaccharide (LPS) in vitro and in vivo was investigated using different omics technologies. We investigated the hippocampal response to intracerebroventricular (i.c.v) LPS in vivo, at both the transcriptional and protein level. Here, a time course analysis of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) showed a sharp peak at 4 h and a return to baseline at 16 h. The expression of inflammatory mediators was not temporally correlated with expression of the microglia marker F4/80, which did not peak until 2 days after LPS injection. Of 480 inflammation-related genes present on a microarray, 29 transcripts were robustly up-regulated and 90% of them were also detected in LPS stimulated primary microglia (PM) cultures. Further in vitro to in vivo comparison showed that the counter regulation response observed in vivo was less evident in vitro, as transcript levels in PM decreased relatively little over 16 h. This apparent deficiency of homeostatic control of the innate immune response in cultures may also explain why a group of genes comprising tnf receptor associated factor-1, endothelin-1 and schlafen-1 were regulated strongly in vitro, but not in vivo. When the overall LPS-induced transcriptional response of PM was examined on a large Affymetrix chip, chemokines and cytokines constituted the most strongly regulated and largest groups. Interesting new microglia markers included interferon-induced protein with tetratricopeptide repeat (ifit), immune responsive gene-1 (irg-1) and thymidylate kinase family LPS-inducible member (tyki). The regulation of the former two was confirmed on the protein level in a proteomics study. Furthermore, conspicuous regulation of several gene clusters was identified, for instance that of genes pertaining to the extra-cellular matrix and enzymatic regulation thereof. Although most inflammatory genes induced in vitro were transferable to our in vivo model, the observed discrepancy for some genes potentially represents regulatory factors present in the central nervous system (CNS) but not in vitro.

IGF-I neuroprotection in the immature brain after hypoxia-ischemia, involvement of Akt and GSK3β?

Insulin‐like growth factor I (IGF‐I) is a neurotrophic factor that promotes neuronal growth, differentiation and survival. Neuroprotective effects of IGF‐I have previously been shown in adult and juvenile rat models of brain injury. We wanted to investigate the neuroprotective effect of IGF‐I after hypoxia‐ischemia (HI) in 7‐day‐old neonatal rats and the mechanisms of IGF‐I actions in vivo. We also wanted to study effects of HI and/or IGF‐I on the serine/threonine kinases Akt and glycogen synthase kinase 3β (GSK3β) in the phophatidylinositol‐3 kinase (PI3K) pathway. Immediately after HI, phosphorylated Akt (pAkt) and phosphorylated GSK3β (pGSK3β) immunoreactivity was lost in the ipsilateral and reduced in the contralateral hemisphere. After 45 min, pAkt levels were restored to control values, whereas pGSK3β remained low 4 h after HI. Administration of IGF‐I (50 µg i.c.v.) after HI resulted in a 40% reduction in brain damage (loss of microtubule‐associated protein) compared with vehicle‐treated animals. IGF‐I treatment without HI was shown to increase pAkt whereas pGSK3β decreased in the cytosol, but increased in the nuclear fraction. IGF‐I treatment after HI increased pAkt in the cytosol and pGSK3β in both the cytosol and the nuclear fraction in the ipsilateral hemisphere compared with vehicle‐treated rats, concomitant with a reduced caspase‐3‐ and caspase‐9‐like activity. In conclusion, IGF‐I induces activation of Akt during recovery after HI which, in combination with inactivation of GSK3β, may explain the attenuated activation of caspases and reduction of injury in the immature brain.

Inflammatory Gene Profiling in the Developing Mouse Brain after Hypoxia-Ischemia

Brain ischemia triggers an inflammatory reaction that progresses for days to weeks and seems to have a role in secondary progression of injury. Inflammation induces a complex pattern of signaling molecules with partly contradictory actions, and the responses may be different in the immature and adult brain. The authors characterized the global inflammatory gene expression in the developing brain as a first step toward understanding the protective and deleterious effects of inflammation after hypoxia-ischemia. Oligonucleotide arrays were used to investigate inflammatory genes in cortex, hippocampus, thalamus, and striatum at 2, 8, 24, and 72 hours after hypoxia-ischemia, which was induced in 9-day-old mice by left carotid artery ligation followed by hypoxia. After hypoxia-ischemia, 148 inflammatory genes were differentially expressed. More than 97% of the genes were upregulated and 93% had not previously been reported after hypoxia-ischemia in the immature brain. The results indicate that microglia/macrophages, T-and B-cells, NK-cells, mast cells, dendritic cells, and polymorphonuclear leukocytes may participate in the response to hypoxia-ischemia. In addition, novel cytokines/chemokines, complement-related, interferon-regulated, components of the TIR/nuclear factor-κB pathway, and a number of immunomodulatory genes were induced. Several of these genes may be of pathophysiologic significance after neonatal hypoxia-ischemia.

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.

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.

Pharmacological and genetic inhibition of NADPH oxidase does not reduce brain damage in different models of perinatal brain injury in newborn mice

BACKGROUND Inflammation and reactive oxygen species (ROS) are important in the development of perinatal brain injury. The ROS-generating enzyme NADPH oxidase (Nox2) is present in inflammatory cells and contributes to brain injury in adult animal models. HYPOTHESIS NADPH oxidase contributes to ROS formation and development of injury in the immature brain and inhibition of NADPH oxidase attenuates perinatal brain injury. METHODS We used animal models of term hypoxia-ischemia (HI) (P9 mice) as well as ibotenate-induced excitotoxic injury (P5 mice) mimicking features of periventricular leukomalacia in preterm infants. In vitro microglia cell cultures were used to investigate NADPH oxidase-dependent ROS formation. In vivo we determined the impact 1) of HI on NADPH oxidase gene expression 2) of genetic (gp91-phox/Nox2 knock-out) and 3) of pharmacological NADPH oxidase inhibition on HI-induced injury and NMDA receptor-mediated excitotoxic injury, respectively. Endpoints were ROS formation, oxidative stress, apoptosis, inflammation and extent of injury. RESULTS Hypoxia-ischemia increased NADPH oxidase subunits mRNA expression in total brain tissue in vivo. In vitro ibotenate increased NADPH oxidase-dependent formation of reactive oxygen species in microglia. In vivo the inhibition of NADPH oxidase did not reduce the extent of brain injury in any of the animal models. In contrast, the injury was increased by inhibition of NADPH oxidase and genetic inhibition was associated with an increased level of galectin-3 and IL-1beta. CONCLUSION NADPH oxidase is upregulated after hypoxia-ischemia and activated microglia cells are a possible source of Nox2-derived ROS. In contrast to findings in adult brain, NADPH oxidase does not significantly contribute to the pathogenesis of perinatal brain injury. Results obtained in adult animals cannot be transferred to newborns and inhibition of NADPH oxidase should not be used in attempts to attenuate injury.

White matter injury in the immature brain: role of interleukin-18

Inflammation is likely to be important in the pathophysiology of white matter damage in the immature brain. In order to investigate the involvement of interleukin (IL)-18, we subjected 9-day-old IL-18-deficient and wild-type (WT) mice to hypoxia-ischemia (HI) (unilateral carotid ligation and exposure to 10% oxygen) and white matter injury was evaluated after 3 days by immunostaining for myelin basic protein (MBP) and neurofilament (NF). The immunoreactivity of MBP was significantly higher by 92, 49 and 21%, respectively, in subcortical white matter, striatum and thalamus in IL-18-deficient mice versus WT mice following HI. Similarly, there was a more pronounced immunoreactivity of NF by 78% in the subcortical white matter in IL-18 KO versus WT mice. IL-18 was expressed by astrocytes and microglia, whereas the IL-18 receptor was mainly found in astrocytes localized in and around the subventricular white matter. Taken together, these results indicate that release of IL-18 may play an important role in the development of white matter injury in the neonatal brain.

Global Gene Expression in the Immature Brain after Hypoxia-Ischemia

Ischemia induces a complex response of differentially expressed genes in the brain. In order to understand the specific mechanisms of injury in the developing brain, it is important to obtain information on global changes in the transcriptome after neonatal hypoxia-ischemia. In this study, oligonucleotide arrays were used to investigate genomic changes at 2, 8, 24, and 72 hours after neonatal hypoxia-ischemia, which was induced in 9-day-old mice by left carotid artery ligation followed by hypoxia (10% O2). In total, 343 genes were differentially expressed in cortex, hippocampus, thalamus, and striatum 2 to 72 hours after hypoxia-ischemia, when comparing ipsilateral with contralateral hemispheres and with controls, using the significance analysis for microarrays. A total of 283 genes were upregulated and 60 were downregulated, and 94% of the genes had not previously been shown after neonatal hypoxia-ischemia. Genes related to transcription factors and metabolism had mostly upregulated transcripts, whereas most downregulated genes belonged to the categories of ion and vesicular transport and signal transduction. Genes involved in transcription, stress, and apoptosis were induced early after the insult, and many new genes that may play important roles in the pathophysiology of neonatal hypoxiaischemia were identified.

Combined Deficiency of IL-1β18, but Not IL-1αβ, Reduces Susceptibility to Hypoxia-Ischemia in the Immature Brain

Interleukin (IL)-1 and IL-18 belong to the IL-1 family. IL-18 deficiency has been shown to confer moderate protection after hypoxia-ischemia (HI) in the immature brain, while the contribution of the two isoforms of IL-1 (IL-1α and IL-1β) in neonatal HI brain injury has not been investigated previously. The aim of this study was to examine the contribution of the different members of the IL-1 family to neonatal HI damage. Unilateral HI was induced at postnatal day 9 in IL-1β, IL-1β18, and IL-1αβ knockout and wild-type mice and brain injury was evaluated 1 week later. IL-1β18-deficient mice showed 17% reduction in brain injury, while no significant reduction in injury was detected between any of the other groups. These results indicate that IL-18, but not IL-1β, or the combination of IL-1α and IL-1β, is a contributor to HI injury in the immature brain.