Responses of rat and mouse primary microglia to pro- and anti-inflammatory stimuli: molecular profiles, K+ channels and migration.

BACKGROUND: Acute CNS damage is commonly studied using rat and mouse models, but increasingly, molecular analysis is finding species differences that might affect the ability to translate findings to humans. Microglia can undergo complex molecular and functional changes, often studied by in vitro responses to discrete activating stimuli. There is considerable evidence that pro-inflammatory (M1) activation can exacerbate tissue damage, while anti-inflammatory (M2) states help resolve inflammation and promote tissue repair. However, in assessing potential therapeutic targets for controlling inflammation, it is crucial to determine whether rat and mouse microglia respond the same. METHODS: Primary microglia from Sprague-Dawley rats and C57BL/6 mice were cultured, then stimulated with interferon-γ + tumor necrosis factor-α (I + T; M1 activation), interleukin (IL)-4 (M2a, alternative activation), or IL-10 (M2c, acquired deactivation). To profile their activation responses, NanoString was used to monitor messenger RNA (mRNA) expression of numerous pro- and anti-inflammatory mediators, microglial markers, immunomodulators, and other molecules. Western analysis was used to measure selected proteins. Two potential targets for controlling inflammation-inward- and outward-rectifier K+ channels (Kir2.1, Kv1.3)-were examined (mRNA, currents) and specific channel blockers were applied to determine their contributions to microglial migration in the different activation states. RESULTS: Pro-inflammatory molecules increased after I + T treatment but there were several qualitative and quantitative differences between the species (e.g., iNOS and nitric oxide, COX-2). Several molecules commonly associated with an M2a state differed between species or they were induced in additional activation states (e.g., CD206, ARG1). Resting levels and/or responses of several microglial markers (Iba1, CD11b, CD68) differed with the activation state, species, or both. Transcripts for several Kir2 and Kv1 family members were detected in both species. However, the current amplitudes (mainly Kir2.1 and Kv1.3) depended on activation state and species. Treatment-induced changes in morphology and migratory capacity were similar between the species (migration reduced by I + T, increased by IL-4 or IL-10). In both species, Kir2.1 block reduced migration and Kv1.3 block increased it, regardless of activation state; thus, these channels might affect microglial migration to damage sites. CONCLUSIONS: Caution is recommended in generalizing molecular and functional responses of microglia to activating stimuli between species.

Complex molecular and functional outcomes of single versus sequential cytokine stimulation of rat microglia.

BACKGROUND: Microglia are the "professional" phagocytes of the CNS. Phagocytosis is crucial for normal CNS development and maintenance, but it can be either beneficial or detrimental after injury or disease. For instance, white matter damage releases myelin debris that must be cleared by microglia in order for re-myelination to occur. However, phagocytosis can also produce damaging reactive oxygen species (ROS). Furthermore, microglia can acquire pro-inflammatory (M1) or anti-inflammatory (M2) activation states that affect cell functions. Although microglia are exposed to a changing cytokine environment after injury or disease, little is known about the molecular and functional consequences. Therefore, we applied several microglial activation paradigms, with or without myelin debris. We assessed (i) gene expression changes reflecting microglial activation and inflammatory states, and receptors and enzymes related to phagocytosis and ROS production, (ii) myelin phagocytosis and production of ROS, and (iii) expression and contributions of several ion channels that are considered potential targets for regulating microglial behavior. METHODS: Primary rat microglia were exposed to cytokines, individually or sequentially. First, responses to individual M1 or M2 stimuli were compared: IFN-γ plus TNF-α ("I + T"; M1 activation), interleukin-4 (M2a/alternative activation), and interleukin-10 (M2c/acquired deactivation). Second, sequential cytokine addition was used to assess microglia repolarization and cell functions. The paradigms were M2a→M1, M2c→M1, M1→M2a, and M1→M2c. RESULTS: M1 stimulation increased pro-inflammatory genes, phagocytosis, and ROS, as well as expression of Kv1.3, KCa3.1, and Kir2.1 channels. M2a stimulation increased anti-inflammatory genes, ROS production, and Kv1.3 and KCa3.1 expression. Myelin phagocytosis enhanced the M1 profile and dampened the M2a profile, and both phagocytosis and ROS production were dependent on NOX enzymes and Kir2.1 and CRAC channels. Importantly, microglia showed some capacity for re-polarization between M1 and M2a states, based on gene expression changes, myelin phagocytosis, and ROS production. CONCLUSIONS: In response to polarizing and re-polarizing cytokine treatments, microglia display complex changes in gene transcription profiles, phagocytic capacity, NOX-mediated ROS production, and in ion channels involved in microglial activation. Because these changes might affect microglia-mediated CNS inflammation, they should be considered in future experimental, pre-clinical studies.

PKA reduces the rat and human KCa3.1 current, CaM binding, and Ca2+ signaling, which requires Ser332/334 in the CaM-binding C terminus.

The Ca(2+)-dependent K(+) channel, KCa3.1 (KCNN4/IK/SK4), is widely expressed and contributes to cell functions that include volume regulation, migration, membrane potential, and excitability. KCa3.1 is now considered a therapeutic target for several diseases, including CNS disorders involving microglial activation; thus, we need to understand how KCa3.1 function is regulated. KCa3.1 gating and trafficking require calmodulin binding to the two ends of the CaM-binding domain (CaMBD), which also contains three conserved sites for Ser/Thr kinases. Although cAMP protein kinase (PKA) signaling is important in many cells that use KCa3.1, reports of channel regulation by PKA are inconsistent. We first compared regulation by PKA of native rat KCa3.1 channels in microglia (and the microglia cell line, MLS-9) with human KCa3.1 expressed in HEK293 cells. In all three cells, PKA activation with Sp-8-Br-cAMPS decreased the current, and this was prevented by the PKA inhibitor, PKI14-22. Inhibiting PKA with Rp-8-Br-cAMPS increased the current in microglia. Mutating the single PKA site (S334A) in human KCa3.1 abolished the PKA-dependent regulation. CaM-affinity chromatography showed that CaM binding to KCa3.1 was decreased by PKA-dependent phosphorylation of S334, and this regulation was absent in the S334A mutant. Single-channel analysis showed that PKA decreased the open probability in wild-type but not S334A mutant channels. The same decrease in current for native and wild-type expressed KCa3.1 channels (but not S334A) occurred when PKA was activated through the adenosine A2a receptor. Finally, by decreasing the KCa3.1 current, PKA activation reduced Ca(2+)-release-activated Ca(2+) entry following activation of metabotropic purinergic receptors in microglia.

The microglial activation state regulates migration and roles of matrix-dissolving enzymes for invasion.

BACKGROUND: Microglial cells are highly mobile under many circumstances and, after central nervous system (CNS) damage, they must contend with the dense extracellular matrix (ECM) in order to reach their target sites. In response to damage or disease, microglia undergo complex activation processes that can be modulated by environmental cues and culminate in either detrimental or beneficial outcomes. Thus, there is considerable interest in comparing their pro-inflammatory ('classical' activation) and resolving 'alternative' activation states. Almost nothing is known about how these activation states affect the ability of microglia to migrate and degrade ECM, or the enzymes used for substrate degradation. This is the subject of the present study. METHODS: Primary cultured rat microglial cells were exposed to lipopolysaccharide (LPS) to evoke classical activation or IL4 to evoke alternative activation. High-resolution microscopy was used to monitor changes in cell morphology and aspects of the cytoskeleton. We quantified migration in a scratch-wound assay and through open filter holes, and invasion through Matrigel™. A panel of inhibitors was used to analyze contributions of different matrix-degrading enzymes to migration and invasion, and quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to assess changes in their expression. RESULTS: Vinculin- and F-actin-rich lamellae were prominent in untreated and IL4-treated microglia (but not after LPS). IL4 increased the migratory capacity of microglia but eliminated the preferential anterior nuclear-centrosomal axis polarity and location of the microtubule organizing center (MTOC). Microglia degraded fibronectin, regardless of treatment, but LPS-treated cells were relatively immobile and IL4-treated cells invaded much more effectively through Matrigel™. For invasion, untreated microglia primarily used cysteine proteases, but IL4-treated cells used a wider range of enzymes (cysteine proteases, cathepsin S and K, heparanase, and matrix metalloproteases). Untreated microglia expressed MMP2, MMP12, heparanase, and four cathepsins (B, K, L1, and S). Each activation stimulus upregulated a different subset of enzymes. IL4 increased MMP2 and cathepsins S and K; whereas LPS increased MMP9, MMP12, MMP14 (MT1-MMP), heparanase, and cathepsin L1. CONCLUSIONS: Microglial cells migrate during CNS development and after CNS damage or disease. Thus, there are broad implications of the finding that classically and alternatively activated microglia differ in morphology, cytoskeleton, migratory and invasive capacity, and in the usage of ECM-degrading enzymes.

Inhibition of the Ca²âº-dependent K⁺ channel, KCNN4/KCa3.1, improves tissue protection and locomotor recovery after spinal cord injury.

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/microglia and astrocytes and molecules that potentially cause secondary tissue damage and functional impairment. Here, we assessed the contribution of the calcium-dependent K⁺ channel KCNN4 (KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes in KCNN4 mRNA levels (RT-PCR), KCa3.1 protein expression (Western blots), and cellular expression (immunofluorescence) in the mouse spinal cord were monitored between 1 and 28 d after SCI. KCNN4 mRNA and KCa3.1 protein rapidly increased after SCI; double labeling identified astrocytes as the main cellular source accounting for this upregulation. Locomotor function after SCI, evaluated for 28 d in an open-field test using the Basso Mouse Scale, was improved in a dose-dependent manner by treating mice with a selective inhibitor of KCa3.1 channels, TRAM-34 (triarylmethane-34). Improved locomotor function was accompanied by reduced tissue loss at 28 d and increased neuron and axon sparing. The rescue of tissue by TRAM-34 treatment was preceded by reduced expression of the proinflammatory mediators, tumor necrosis factor-α and interleukin-1ß in spinal cord tissue at 12 h after injury, and reduced expression of inducible nitric oxide synthase at 7 d after SCI. In astrocytes in vitro, TRAM-34 inhibited Ca²âº signaling in response to metabotropic purinergic receptor stimulation. These results suggest that blocking the KCa3.1 channel could be a potential therapeutic approach for treating secondary damage after spinal cord injury.

Podosomes in migrating microglia: components and matrix degradation.

BACKGROUND: To perform their functions during development and after central nervous system injury, the brain's immune cells (microglia) must migrate through dense neuropil and extracellular matrix (ECM), but it is not known how they degrade the ECM. In several cancer cell lines and peripheral cells, small multi-molecular complexes (invadopodia in cancer cells, podosomes in nontumor cells) can both adhere to and dissolve the ECM. Podosomes are tiny multi-molecular structures (0.4 to 1 µm) with a core, rich in F-actin and its regulatory molecules, surrounded by a ring containing adhesion and structural proteins. METHODS: Using rat microglia, we performed several functional assays: live cell imaging for chemokinesis, degradation of the ECM component, fibronectin, and chemotactic invasion through Matrigel™, a basement membrane type of ECM. Fluorescent markers were used with high-resolution microscopy to identify podosomes and their components. RESULTS: The fan-shaped lamella at the leading edge of migrating microglia contained a large F-actin-rich superstructure composed of many tiny (<1 µm) punctae that were adjacent to the substrate, as expected for cell-matrix contact points. This superstructure (which we call a podonut) was restricted to cells with lamellae, and conversely almost every lamella contained a podonut. Each podonut comprised hundreds of podosomes, which could also be seen individually adjacent to the podonut. Microglial podosomes contained hallmark components of these structures previously seen in several cell types: the plaque protein talin in the ring, and F-actin and actin-related protein (Arp) 2 in the core. In microglia, podosomes were also enriched in phosphotyrosine residues and three tyrosine-kinase-regulated proteins: tyrosine kinase substrate with five Src homology 3 domains (Tks5), phosphorylated caveolin-1, and Nox1 (nicotinamide adenine dinucleotide phosphate oxidase 1). When microglia expressed podonuts, they were able to degrade the ECM components, fibronectin, and Matrigel™. CONCLUSION: The discovery of functional podosomes in microglia has broad implications, because migration of these innate immune cells is crucial in the developing brain, after damage, and in disease states involving inflammation and matrix remodeling. Based on the roles of invadosomes in peripheral tissues, we propose that microglia use these complex structures to adhere to and degrade the ECM for efficient migration.

Regulation of podosome formation, microglial migration and invasion by Ca(2+)-signaling molecules expressed in podosomes.

BACKGROUND: Microglia migrate during brain development and after CNS injury, but it is not known how they degrade the extracellular matrix (ECM) to accomplish this. Podosomes are tiny structures with the unique ability to adhere to and dissolve ECM. Podosomes have a two-part architecture: a core that is rich in F-actin and actin-regulatory molecules (for example, Arp2/3), surrounded by a ring with adhesion and structural proteins (for example, talin, vinculin). We recently discovered that the lamellum at the leading edge of migrating microglia contains a large F-actin-rich superstructure ('podonut') composed of many podosomes. Microglia that expressed podosomes could degrade ECM molecules. Finely tuned Ca(2+) signaling is important for cell migration, cell-substrate adhesion and contraction of the actomyosin network. Here, we hypothesized that podosomes contain Ca(2+)-signaling machinery, and that podosome expression and function depend on Ca(2+) influx and specific ion channels. METHODS: High-resolution immunocytochemistry was used on rat microglia to identify podosomes and novel molecular components. A pharmacological toolbox was applied to functional assays. We analyzed roles of Ca(2+)-entry pathways and ion channels in podosome expression, microglial migration into a scratch-wound, transmigration through pores in a filter, and invasion through Matrigel™-coated filters. RESULTS: Microglial podosomes were identified using well-known components of the core (F-actin, Arp2) and ring (talin, vinculin). We discovered four novel podosome components related to Ca(2+) signaling. The core contained calcium release activated calcium (CRAC; Orai1) channels, calmodulin, small-conductance Ca(2+)-activated SK3 channels, and ionized Ca(2+) binding adapter molecule 1 (Iba1), which is used to identify microglia in the CNS. The Orai1 accessory molecule, STIM1, was also present in and around podosomes. Podosome formation was inhibited by removing external Ca(2+) or blocking CRAC channels. Blockers of CRAC channels inhibited migration and invasion, and SK3 inhibition reduced invasion. CONCLUSIONS: Microglia podosome formation, migration and/or invasion require Ca(2+) influx, CRAC, and SK3 channels. Both channels were present in microglial podosomes along with the Ca(2+)-regulated molecules, calmodulin, Iba1 and STIM1. These results suggest that the podosome is a hub for sub-cellular Ca(2+)-signaling to regulate ECM degradation and cell migration. The findings have broad implications for understanding migration mechanisms of cells that adhere to, and dissolve ECM.

The Ca2+ activated SK3 channel is expressed in microglia in the rat striatum and contributes to microglia-mediated neurotoxicity in vitro.

BACKGROUND: Small-conductance Ca2+ activated K+ channels are expressed in the CNS, where KCNN2/SK2/KCa2.2 and KCNN3/SK3/KCa2.3 help shape the electrical activity of some neurons. The SK3 channel is considered a potential therapeutic target for diseases and disorders involving neuron hyper-excitability but little is known about its expression and roles in non-neuronal cells in either the healthy or damaged CNS. The purpose of this study was to examine expression of KCNN3/SK3 in CNS microglia in vivo and in vitro, and to use an established in vitro model to determine if this channel contributes to the neurotoxic capacity of activated microglia. METHODS: KCNN3 mRNA (real-time RT-PCR) and SK3 immunoreactivity were examined in rat microglia. Lipopolysaccharide was then used to activate microglia (monitored by iNOS, nitric oxide, activation of NF-kappaB and p38 MAPK) and transform them to a neurotoxic state. Microglia-mediated neuron damage (TUNEL, activated caspase 3) and nitrotyrosine levels were quantified using a two-chamber system that allowed microglia to be treated with channel blockers, washed and then added to neuron/astrocyte cultures. Contributions of SK3 to these processes were discriminated using a subtractive pharmacological approach with apamin and tamapin. ANOVA and post-hoc tests were used to assess the statistical significance of differences between treatment groups. SK3 immunoreactivity was then compared in the normal and damaged adult rat striatum, by injecting collagenase (a hemorrhagic stroke) or endothelin-1 (a transient ischemic stroke). RESULTS: KCNN3 mRNA was prevalent in cultured microglia and increased after lipopolysaccharide-induced activation; SK3 channel blockade inhibited microglial activation and reduced their ability to kill neurons. SK3 immunoreactivity was prevalent in cultured microglia and throughout the adult rat striatum (except white matter tracts). After strokes, SK3 was highly expressed in activated microglia/macrophages within the lesions, but reduced in other cells. CONCLUSIONS: SK3 is expressed in microglia in both the healthy and damaged adult striatum, and mechanistic in vitro studies show it contributes to transformation of microglia to an activated neurotoxic phenotype. Thus, SK3 might be a therapeutic target for reducing inflammation-mediated acute CNS damage. Moreover, its roles in microglia must be considered when targeting this channel for CNS diseases, disorders and reducing neuron hyper-excitability.

Mechanisms of microglia-mediated neurotoxicity in a new model of the stroke penumbra.

After an ischemic stroke, neurons in the core are rapidly committed to die, whereas neuron death in the slowly developing penumbra is more amenable to therapeutic intervention. Microglia activation contributes to delayed inflammation, but because neurotoxic mechanisms in the penumbra are not well understood, we developed an in vitro model of microglia activation and propagated neuron killing. To recapitulate inflammatory triggers in the core, microglia were exposed to oxygen glucose-deprived neurons and astrocytes. To model the developing penumbra, the microglia were washed and allowed to interact with healthy naive neurons and astrocytes. We found that oxygen-glucose deprivation (OGD)-stressed neurons released glutamate, which activated microglia through their group II metabotropic glutamate receptors (mGluRs). Microglia activation involved nuclear factor kappaB (NF-kappaB), a transcription factor that promotes their proinflammatory functions. The activated microglia became neurotoxic, killing naive neurons through an apoptotic mechanism that was mediated by tumor necrosis factor-alpha (TNF-alpha), and involved activation of both caspase-8 and caspase-3. In contrast to some earlier models (e.g., microglia activation by lipopolysaccharide), neurotoxicity was not decreased by an inducible nitric oxide synthase (iNOS) inhibitor (S-methylisothiourea) or a peroxynitrite scavenger [5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphinato iron (III) chloride], and did not require p38 mitogen-activated protein kinase (MAPK) activation. The same microglia neurotoxic behavior was evoked without exposure to OGD-stressed neurons, by directly activating microglial group II mGluRs with (2S,2'R,3'R)-2-(2'3'-dicarboxycyclopropyl) glycine or glutamate, which stimulated production of TNF-alpha (not nitric oxide) and mediated TNF-alpha-dependent neurotoxicity through activation of NF-kappaB (not p38 MAPK). Together, these results support potential therapeutic strategies that target microglial group II mGluRs, TNFalpha overproduction, and NF-kappaB activation to reduce neuron death in the ischemic penumbra.

The Ca2+-activated K+ channel KCNN4/KCa3.1 contributes to microglia activation and nitric oxide-dependent neurodegeneration.

Brain damage and disease involve activation of microglia and production of potentially neurotoxic molecules, but there are no treatments that effectively target their harmful properties. We present evidence that the small-conductance Ca2+/calmodulin-activated K+ channel KCNN4/ KCa3.1/SK4/IK1 is highly expressed in rat microglia and is a potential therapeutic target for acute brain damage. Using a Transwell cell-culture system that allows separate treatment of the microglia or neurons, we show that activated microglia killed neurons, and this was markedly reduced by treating only the microglia with a selective inhibitor of KCa3.1 channels, triarylmethane-34 (TRAM-34). To assess the role of KCa3.1 channels in microglia activation and key signaling pathways involved, we exploited several fluorescence plate-reader-based assays. KCa3.1 channels contributed to microglia activation, inducible nitric oxide synthase upregulation, production of nitric oxide and peroxynitrite, and to consequent neurotoxicity, protein tyrosine nitration, and caspase 3 activation in the target neurons. Microglia activation involved the signaling pathways p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB), which are important for upregulation of numerous proinflammatory molecules, and the KCa3.1 channels were functionally linked to activation of p38 MAPK but not NF-kappaB. These in vitro findings translated into in vivo neuroprotection, because we found that degeneration of retinal ganglion cells after optic nerve transection was reduced by intraocular injection of TRAM-34. This study provides evidence that KCa3.1 channels constitute a therapeutic target in the CNS and that inhibiting this K+ channel might benefit acute and chronic neurodegenerative disorders that are caused by or exacerbated by inflammation.

Glial responses, neuron death and lesion resolution after intracerebral hemorrhage in young vs. aged rats.

Intracerebral hemorrhage (ICH) usually affects older humans but almost no experimental studies have assessed aged animals. We address how aging alters inflammation, neuron death and lesion resolution after a hemorrhage in the rat striatum. In the normal aged brain, microglia displayed a 'dystrophic' phenotype, with shorter cellular processes and large gaps between adjacent cells, and there was more astrocyte reactivity. The ICH injury was monitored as hematoma volume and number of dying neurons at 1 and 3 days, and the volume of the residual lesion, ventricles and lost tissue at 28 days. Inflammation at 1 and 3 days was assessed from densities of microglia with resting vs. activated morphologies, or expressing the lysosomal marker ED1. Despite an initial delay in neuron death in aged animals, by 28 days, there was no difference in neuron density or volume of tissue lost. However, lesion resolution was impaired in aged animals and there was less compensatory ventricular expansion. At 1 day after ICH, there were fewer activated microglia/macrophages in the aged brain, but by 3 days there were more of these cells at the edge of the hematoma and in the surrounding parenchyma. In both age groups a glial limitans had developed by 3 days, but astrocyte reactivity and the spread of activated microglia/macrophages into the surrounding parenchyma was greater in the aged. These findings have important implications for efforts to reduce secondary injury after ICH and to develop anti-inflammatory therapies to treat ICH in aged humans.

Microglia Responses to Pro-inflammatory Stimuli (LPS, IFNγ+TNFα) and Reprogramming by Resolving Cytokines (IL-4, IL-10).

Microglia respond to CNS injuries and diseases with complex reactions, often called "activation." A pro-inflammatory phenotype (also called classical or M1 activation) lies at one extreme of the reactivity spectrum. There were several motivations for this study. First, bacterial endotoxin (lipopolysaccharide, LPS) is the most commonly used pro-inflammatory stimulus for microglia, both in vitro and in vivo; however, pro-inflammatory cytokines (e.g., IFNγ, TNFα) rather than LPS will be encountered with sterile CNS damage and disease. We lack direct comparisons of responses between LPS and such cytokines. Second, while transcriptional profiling is providing substantial data on microglial responses to LPS, these studies mainly use mouse cells and models, and there is increasing evidence that responses of rat microglia can differ. Third, the cytokine milieu is dynamic after acute CNS damage, and an important question in microglial biology is: How malleable are their responses? There are very few studies of effects of resolving cytokines, particularly for rat microglia, and much of the work has focused on pro-inflammatory outcomes. Here, we first exposed primary rat microglia to LPS or to IFNγ+TNFα (I+T) and compared hallmark functional (nitric oxide production, migration) and molecular responses (almost 100 genes), including surface receptors that can be considered part of the sensome. Protein changes for exemplary molecules were also quantified: ARG1, CD206/MRC1, COX-2, iNOS, and PYK2. Despite some similarities, there were notable differences in responses to LPS and I+T. For instance, LPS often evoked higher pro-inflammatory gene expression and also increased several anti-inflammatory genes. Second, we compared the ability of two anti-inflammatory, resolving cytokines (IL-4, IL-10), to counteract responses to LPS and I+T. IL-4 was more effective after I+T than after LPS, and IL-10 was surprisingly ineffective after either stimulus. These results should prove useful in modeling microglial reactivity in vitro; and comparing transcriptional responses to sterile CNS inflammation in vivo.

Comparing Effects of Transforming Growth Factor ß1 on Microglia From Rat and Mouse: Transcriptional Profiles and Potassium Channels.

The cytokine, transforming growth factor ß1 (TGFß1), is up-regulated after central nervous system (CNS) injuries or diseases involving microglial activation, and it has been proposed as a therapeutic agent for treating neuroinflammation. Microglia can produce and respond to TGFß1. While rats and mice are commonly used for studying neuroinflammation, very few reports directly compare them. Such studies are important for improving pre-clinical studies and furthering translational progress in developing therapeutic interventions. After intracerebral hemorrhage (ICH) in the rat striatum, the TGFß1 receptor was highly expressed on microglia/macrophages within the hematoma. We recently found species similarities and differences in response to either a pro-inflammatory (interferon-γ, IFN-γ, +tumor necrosis factor, TNF-α) or anti-inflammatory interleukin-4 (IL-4) stimulus. Here, we assessed whether rat and mouse microglia differ in their responses to TGFß1. Microglia were isolated from Sprague-Dawley rats and C57BL/6 mice and treated with TGFß1. We quantified changes in expression of >50 genes, in their morphology, proliferation, apoptosis and in three potassium channels that are considered therapeutic targets. Many inflammatory mediators, immune receptors and modulators showed species similarities, but notable differences included that, for some genes, only one species responded (e.g., Il4r, Il10, Tgfbr2, colony-stimulating factor receptor (Csf1r), Itgam, suppressor of cytokine signaling 1 (Socs1), toll-like receptors 4 (Tlr4), P2rx7, P2ry12), and opposite responses were seen for others (Tgfb1, Myc, Ifngr1). In rat only, TGFß1 affected microglial morphology and proliferation, but there was no apoptosis in either species. In both species, TGFß1 dramatically increased Kv1.3 channel expression and current (no effects on Kir2.1). KCa3.1 showed opposite species responses: the current was low in unstimulated rat microglia and greatly increased by TGFß1 but higher in control mouse cells and decreased by TGFß1. Finally, we compared TGFß1 and IL10 (often considered similar anti-inflammatory stimuli) and found many different responses in both species. Overall, the numerous species differences should be considered when characterizing neuroinflammation and microglial activation in vitro and in vivo, and when targeting potassium channels.

Sex- and Development-Dependent Responses of Rat Microglia to Pro- and Anti-inflammatory Stimulation.

Addressing potential sex differences in pre-clinical studies is crucial for developing therapeutic interventions. Although sex differences have been reported in epidemiological studies and from clinical experience, most pre-clinical studies of neuroinflammation use male rodents; however, sexual dimorphisms in microglia might affect the CNS inflammatory response. Developmental changes are also important and, in rodents, there is a critical period of sexual brain differentiation in the first 3 weeks after birth. We compared rat microglia from sex-segregated neonates (P1) and at about the time of weaning (P21). To study transitions from a basal homeostatic state (untreated), microglia were subjected to a pro-inflammatory (IFNγ + TNFα) or anti-inflammatory (IL-4) stimulus. Responses were compared by quantifying changes in nitric oxide production, migration, and expression of nearly 70 genes, including inflammatory mediators and receptors, inflammasome molecules, immune modulators, and genes that regulate microglial physiological functions. No sex differences were seen in transcriptional responses in either age group but the IL-4-evoked migration increase was larger in male cells at both ages. Protein changes for the hallmark molecules, NOS2, COX-2, PYK2 and CD206 correlated with mRNA changes. P1 and P21 microglia showed substantial differences, including expression of genes related to developmental roles. That is, P21 microglia had a more mature phenotype, with higher basal and stimulated levels of many inflammatory genes, while P1 cells had higher expression of phagocytosis-related molecules. Nevertheless, cells of both ages responded to IL-4 and IFNγ + TNFα. We examined the Kv1.3 potassium channel (a potential target for modulating neuroinflammation) and the Kir2.1 channel, which regulate several microglia functions. Kv1.3 mRNA (Kcna3) was higher at P21 under all conditions and male P21 cells had higher mRNA and Kv currents in response to IFNγ + TNFα. Overall, numerous transcriptional and functional responses of microglia changed during the first 3 weeks after birth but few sex-dependent changes were seen.

Neuron death and inflammation in a rat model of intracerebral hemorrhage: effects of delayed minocycline treatment.

After intracerebral hemorrhage (ICH), blood entry is followed by neuron death and an inflammatory response, but development of pharmacological therapies has been hampered by an inadequate understanding of the spatial and temporal relationship between neuron death and inflammation. Using a rat model of ICH, we first investigated these relationships at 6 h, and 1, 3 and 7 days. At the edge of the hematoma, no degenerating neurons were observed at 6 h; however, dying neurons were present between 1 and 3 days, with peak neuron death occurring at 1 day. This is apparently the first report of ongoing neuron death at the edge of the hematoma during a time window that is appropriate for human therapy. Neuron death was limited to the edge of the hematoma, with no degenerating neurons in the striatum surrounding the hematoma, despite robust and prolonged microglia activation. Importantly, neuron loss at the edge of the hematoma was spatially and temporally associated with accumulation and activation of microglia/macrophages. We then tested the hypothesis that treatment with the tetracycline derivative, minocycline, after the hematoma had reached a maximal size, will reduce inflammation and neuron damage. Minocycline injection (45 mg/kg i.v. at 6 h, and i.p. at 24, 48 and 72 h) failed to reduce neuron loss outside the hematoma or striatal tissue loss (assessed at 7 days), despite reducing the number of neutrophils and activated microglia/macrophages. Thus, minocycline does not appear to target the mechanisms responsible for cell death in this model of ICH.

Evolution of the inflammatory response in the brain following intracerebral hemorrhage and effects of delayed minocycline treatment.

There are no effective treatments for intracerebral hemorrhage (ICH). Although inflammation is a potential therapeutic target, there is a dearth of information about time-dependent and cell-specific changes in the expression of inflammation-related genes. Using the collagenase-induced ICH model in rats and real-time quantitative RT-PCR we monitored mRNA levels of markers of glial activation, pro- and anti-inflammatory cytokines, enzymes responsible for cytokine activation and several matrix metalloproteases at 6 h and 1, 3 and 7 days after ICH onset. For the most highly up-regulated genes, immunohistochemistry was then used to identify cell-specific protein expression. Finally, minocycline, a drug widely reported to reduce damage in several models of brain injury, was used to test the hypothesis that it can reduce up-regulation of inflammation-related genes when administered using a clinically relevant dosing regime: intraperitoneal injection beginning 6 h after ICH. Our results show a complex inflammatory response, with different brain cell types producing several pro- and anti-inflammatory molecules for at least 7 days after ICH onset. Included is the first demonstration that astrocytes are an important source of interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (IL-1ra), interleukin-6 (IL-6) and MMP-12. Importantly, our results demonstrate that while delayed minocycline treatment effectively reduces early up-regulation of TNFalpha and MMP-12, its efficacy is lost when treatment is extended for up to a week, and it does not reduce several other genes associated with microglia activation. These results suggest caution in extrapolating to ICH the promising results of minocycline treatment in other models of brain injury.

Microglia Kv1.3 channels contribute to their ability to kill neurons.

Many CNS disorders involve an inflammatory response that is orchestrated by cells of the innate immune system: macrophages, neutrophils, and microglia (the endogenous CNS immune cell). Hence, there is considerable interest in anti-inflammatory strategies that target these cells. Microglia express Kv1.3 (KCNA3) channels, which we showed previously are important for their proliferation and the NADPH-mediated respiratory burst. Here, we demonstrate the potential for targeting Kv1.3 channels to control CNS inflammation. Rat microglia express Kv1.2, Kv1.3, and Kv1.5 transcripts and protein, but only a Kv1.3 current was detected. When microglia were activated with lipopolysaccharide or a phorbol ester, only the Kv1.3 transcript (but not protein) expression changed. Using a Transwell cell-culture system that allows separate drug treatment of microglia or neurons, we found that activated microglia killed postnatal hippocampal neurons through a process that requires Kv1.3 channel activity in microglia but not in neurons. A major neurotoxic molecule in this model was peroxynitrite, which is formed from superoxide and nitric oxide; thus, it is significant that Kv1.3 channel blockers reduced the respiratory burst, but not nitric oxide production, by the activated microglia. In addressing the biochemical pathway affected by Kv1.3 channel activity, we found that Kv1.3 acts via a different cellular mechanism from the broad-spectrum drug minocycline, which is often used in animal models of neuroinflammation. That is, the dose-dependent reduction in neuron killing by minocycline corresponded with a reduction in p38 mitogen-activated protein kinase activation in microglia; however, none of the Kv1.3 blockers affected p38 activation.

Molecular and Cellular Responses to Interleukin-4 Treatment in a Rat Model of Transient Ischemia.

Within hours after stroke, potentially cytotoxic pro-inflammatory mediators are elevated within the brain; thus, one potential therapeutic strategy is to reduce them and skew the brain toward an anti-inflammatory state. Because interleukin-4 (IL-4) treatment induces an anti-inflammatory, "alternative-activation" state in microglia and macrophages in vitro, we tested the hypothesis that early supplementation of the brain with IL-4 can shift it toward an anti-inflammatory state and reduce damage after transient focal ischemia. Adult male rat striata were injected with endothelin-1, with or without co-injection of IL-4. Inflammation, glial responses and damage to neurons and white matter were quantified from 1 to 7 days later. At 1 day, IL-4 treatment increased striatal expression of several anti-inflammatory markers (ARG1, CCL22, CD163, PPARγ), increased phagocytic (Iba1-positive, CD68-positive) microglia/macrophages, and increased VEGF-A-positive infiltrating neutrophils in the infarcts. At 7 days, there was evidence of sustained, propagating responses. IL-4 increased CD206, CD200R1, IL-4Rα, STAT6, PPARγ, CD11b, and TLR2 expression and increased microglia/macrophages in the infarct and astrogliosis outside the infarct. Neurodegeneration and myelin damage were not reduced, however. The sustained immune and glial responses when resolution and repair processes have begun warrant further studies of IL-4 treatment regimens and long-term outcomes.

After Intracerebral Hemorrhage, Oligodendrocyte Precursors Proliferate and Differentiate Inside White-Matter Tracts in the Rat Striatum.

Damage to myelinated axons contributes to neurological deficits after acute CNS injury, including ischemic and hemorrhagic stroke. Potential treatments to promote re-myelination will require fully differentiated oligodendrocytes, but almost nothing is known about their fate following intracerebral hemorrhage (ICH). Using a rat model of ICH in the striatum, we quantified survival, proliferation, and differentiation of oligodendrocyte precursor cells (OPCs) (at 1, 3, 7, 14, and 28 days) in the peri-hematoma region, surrounding striatum, and contralateral striatum. In the peri-hematoma, the density of Olig2(+) cells increased dramatically over the first 7 days, and this coincided with disorganization and fragmentation of myelinated axon bundles. Very little proliferation (Ki67(+)) of Olig2(+) cells was seen in the anterior subventricular zone from 1 to 28 days. However, by 3 days, many were proliferating in the peri-hematoma region, suggesting that local proliferation expands their population. By 14 days, the density of Olig2(+) cells declined in the peri-hematoma region, and, by 28 days, it reached the low level seen in the contralateral striatum. At these later times, many surviving axons were aligned into white-matter bundles, which appeared less swollen or fragmented. Oligodendrocyte cell maturation was prevalent over the 28-day period. Densities of immature OPCs (NG2(+)Olig2(+)) and mature (CC-1(+)Olig2(+)) oligodendrocytes in the peri-hematoma increased dramatically over the first week. Regardless of the maturation state, they increased preferentially inside the white-matter bundles. These results provide evidence that endogenous oligodendrocyte precursors proliferate and differentiate in the peri-hematoma region and have the potential to re-myelinate axon tracts after hemorrhagic stroke.

Expression and contributions of the Kir2.1 inward-rectifier K(+) channel to proliferation, migration and chemotaxis of microglia in unstimulated and anti-inflammatory states.

When microglia respond to CNS damage, they can range from pro-inflammatory (classical, M1) to anti-inflammatory, alternative (M2) and acquired deactivation states. It is important to determine how microglial functions are affected by these activation states, and to identify molecules that regulate their behavior. Microglial proliferation and migration are crucial during development and following damage in the adult, and both functions are Ca(2+)-dependent. In many cell types, the membrane potential and driving force for Ca(2+) influx are regulated by inward-rectifier K(+) channels, including Kir2.1, which is prevalent in microglia. However, it is not known whether Kir2.1 expression and contributions are altered in anti-inflammatory states. We tested the hypothesis that Kir2.1 contributes to Ca(2+) entry, proliferation and migration of rat microglia. Kir2.1 (KCNJ2) transcript expression, current amplitude, and proliferation were comparable in unstimulated microglia and following alternative activation (IL-4 stimulated) and acquired deactivation (IL-10 stimulated). To examine functional roles of Kir2.1 in microglia, we first determined that ML133 was more effective than the commonly used blocker, Ba(2+); i.e., ML133 was potent (IC50 = 3.5 µM) and voltage independent. Both blockers slightly increased proliferation in unstimulated or IL-4 (but not IL-10)-stimulated microglia. Stimulation with IL-4 or IL-10 increased migration and ATP-induced chemotaxis, and blocking Kir2.1 greatly reduced both but ML133 was more effective. In all three activation states, blocking Kir2.1 with ML133 dramatically reduced Ca(2+) influx through Ca(2+)-release-activated Ca(2+) (CRAC) channels. Thus, Kir2.1 channel activity is necessary for microglial Ca(2+) signaling and migration under resting and anti-inflammatory states but the channel weakly inhibits proliferation.