Starlee Lively Odorico - Academia.edu (original) (raw)

Papers by Starlee Lively Odorico

Osteoarthritis and Cartilage, 2020

Objective: MicroRNAs act locally and systemically to impact osteoarthritis (OA) pathophysiology, ... more Objective: MicroRNAs act locally and systemically to impact osteoarthritis (OA) pathophysiology, but comprehensive profiling of the circulating miRNome in early vs late stages of OA has yet to be conducted. Sequencing has emerged as the preferred method for microRNA profiling since it offers high sensitivity and specificity. Our objective is to sequence the miRNome in plasma from 91 patients with early [KellgreneLawrence (KL) grade 0 or 1 (n ¼ 41)] or late [KL grade 3 or 4 (n ¼ 50)] symptomatic radiographic knee OA to identify unique microRNA signatures in each disease state. Design: MicroRNA libraries were prepared using the QIAseq miRNA Library Kit and sequenced on the Illumina NextSeq 550.Counts were produced for microRNAs captured in miRBase and for novel micro-RNAs. Statistical, bioinformatics, and computational biology approaches were used to refine and interpret the final list of microRNAs. Results: From 215 differentially expressed microRNAs (FDR < 0.01), 97 microRNAs showed an increase or decrease in expression in 85% of samples in the early OA group as compared to the median expression in the late OA group. Increasing this threshold to 95%, seven microRNAs were identified: hsa-miR-335-3p, hsa-miR-199a-5p, hsa-miR-671-3p, hsa-miR-1260b, hsa-miR-191-3p, hsa-miR-335-5p, and hsa-miR-543. Four novel microRNAs were present in 50% of early OA samples and had 27 predicted gene targets in common with the prioritized set of predicted gene targets from the 97 microRNAs, suggesting common underlying mechanisms.

Osteoarthritis and Cartilage Open, 2020

Objective: Osteoarthritis (OA) is a degenerative joint disease with no approved disease modifying... more Objective: Osteoarthritis (OA) is a degenerative joint disease with no approved disease modifying therapy. The enzyme autotaxin (ATX) converts lysophoshatidylcholine analogues to lysophosphatidic acid. Systemic inhibition of ATX reduces pain in animal models of OA; however, OA disease-modifying effects associated with ATX inhibition remain unknown. Here, we sought to determine whether local (knee joint) injection of an ATX inhibitor attenuates surgically-induced OA in mice. Methods: ATX expression was evaluated in human knee OA cartilage. Ten-week-old mice were subjected to surgically-induced OA. ATX inhibitor (PF-8380, 2.5ng/joint) was injected intra-articularly either at 3-and 5weeks post-surgery or at 2-, 4-, 6-and 8-weeks post-surgery and knee joints were evaluated by histopathology and immunohistochemistry to study the expression of catabolic and cell death markers. mRNA Sequencing of human OA chondrocytes treated with/without ATX inhibitor was performed to identify differentially expressed transcripts, followed by pathway enrichment analysis. Results: ATX expression was elevated in severely degenerated cartilage compared to less degenerated human OA cartilage. In surgically-induced OA mice, intra-articular injection of ATX inhibitor at 3-and 5-weeks post-surgery partially protected knee joints from cartilage degeneration. Interestingly, earlier and more frequent ATX inhibitor injections did not confer significant protection. Immunohistochemical analysis showed decreased expression of catabolic and apoptotic markers with two ATX inhibitor injections. mRNA sequencing followed by pathway analysis identified pathways related to cholesterol analogue metabolism and cell-cycle that could be modulated by ATX inhibition in human OA chondrocytes. Conclusion: Local delivery of ATX inhibitor partially attenuates surgically-induced OA in mice.

Frontiers in Cellular Neuroscience, 2018

Microglia respond to CNS injuries and diseases with complex reactions, often called "activation."... more 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 proinflammatory 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.

Frontiers in Cellular Neuroscience, 2018

Addressing potential sex differences in pre-clinical studies is crucial for developing therapeuti... more 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.

Frontiers in cellular neuroscience, 2018

The cytokine, transforming growth factor β1 (TGFβ1), is up-regulated after central nervous system... more The cytokine, transforming growth factor β1 (TGFβ1), 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 TGFβ1. 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 TGFβ1 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 TGFβ1. Microglia were isolated from Spra...

Journal of Neuroinflammation, 2017

Background: Acute CNS damage is commonly studied using rat and mouse models, but increasingly, mo... more 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 interferonγ + 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-inwardand 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.

Journal of Neuropathology & Experimental Neurology, 2016

Within hours after stroke, potentially cytotoxic pro-inflammatory mediators are elevated within t... more 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 antiinflammatory 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, PPARc), increased phagocytic (Iba1-positive, CD68-positive) microglia/macrophages, and increased VEGF-Apositive infiltrating neutrophils in the infarcts. At 7 days, there was evidence of sustained, propagating responses. IL-4 increased CD206, CD200R1, IL-4Ra, STAT6, PPARc, 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.

Journal of Neuroinflammation, 2016

Background: Microglia are the "professional" phagocytes of the CNS. Phagocytosis is crucial for n... more 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.

Translational Stroke Research, 2012

In the hours to days after intracerebral hemorrhage (ICH), there is an inflammatory response with... more In the hours to days after intracerebral hemorrhage (ICH), there is an inflammatory response within the brain characterized by the infiltration of peripheral neutrophils and macrophages and the activation of brain-resident microglia and astrocytes. Despite the strong correlation of aging and ICH incidence, and increasing information about cellular responses, little is known about the temporal-and age-related molecular responses of the brain after ICH. Here, we monitored a panel of 27 genes at 6 h and 1, 3, and 7 days after ICH was induced by injecting collagenase into the striatum of young adult and aged rats. Several molecules (CR3, TLR2, TLR4, IL-1β, TNFα, iNOS, IL-6) were selected to reflect the classical activation of innate immune cells (macrophages, microglia) and the potential to exacerbate inflammation and damage brain cells. Most of the others are associated with the resolution of innate inflammation, alternative pathways of macrophage/microglial activation, and the repair phase after acute injury (TGFβ, IL-1ra, IL-1r2, IL-4, IL-13, IL-4Rα, IL-13Rα1, IL-13Rα2, MRC1, ARG1, CD163, CCL22). In young animals, the up-regulation of 26 in 27 genes (not IL-4) was detected within the first week. Differences in timing or levels between young and aged animals were detected for 18 of 27 genes examined (TLR2,

Biophysical Journal, 2015

The Ca 2+-activated K + channel, KCa3.1 (KCNN4/IK1/SK4), contributes to "classical," proinflammat... more The Ca 2+-activated K + channel, KCa3.1 (KCNN4/IK1/SK4), contributes to "classical," proinflammatory activation of microglia, and KCa3.1 blockers have improved the outcome in several rodent models of CNS damage. For instance, blocking KCa3.1 with TRAM-34 rescued retinal ganglion neurons after optic nerve damage in vivo and, reduced p38 MAP kinase activation, production of reactive oxygen and nitrogen species, and neurotoxicity by microglia in vitro. In pursuing the therapeutic potential of KCa3.1 blockers, it is crucial to assess KCa3.1 contributions to other microglial functions and activation states, especially the IL-4-induced "alternative" activation state that can counteract pro-inflammatory states. We recently found that IL-4 increases microglia migration-a crucial function in the healthy and damaged CNS-and that KCa3.1 contributes to P 2 Y 2 receptor-stimulated migration. Here, we discovered that KCa3.1 is greatly increased in alternative-activated rat microglia and then contributes to an enhanced migratory capacity. IL-4 up-regulated KCNN4 mRNA (by 6 h) and greatly increased the KCa3.1 current by 1 day, and this required de novo protein synthesis. The increase in current was sustained for at least 6 days. IL-4 increased microglial migration and this was reversed by blocking KCa3.1 with TRAM-34. A panel of inhibitors of signal-transduction mediators was used to analyze contributions of IL-4-related signaling pathways. Induction of KCNN4 mRNA and KCa3.1 current was mediated specifically through IL-4 binding to the type I receptor and, surprisingly, it required JAK3, Ras/MEK/ERK signaling and the transcription factor, activator protein-1, rather than JAK2, STAT6, or phosphatidylinositol 3-kinase.The same receptor subtype and pathway were required for the enhanced KCa3.1-dependent migration. In providing the first direct signaling link between an IL-4 receptor, expression and roles of an ion channel, this study also highlights the potential importance of KCa3.1 in alternative-activated microglia.

The Journal of Neuroscience, 2011

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/mi... more 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+channelKCNN4(KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes inKCNN4mRNA 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.KCNN4mRNA 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 ...

Journal of Neuroscience Research, 2008

The epileptic brain is characterized by increased susceptibility to neuronal hyperexcitability. T... more The epileptic brain is characterized by increased susceptibility to neuronal hyperexcitability. The rat lithium-pilocarpine model, which mimics many features of temporal lobe epilepsy, has been used to study processes leading to the development of recurrent seizures. After a prolonged seizure episode, termed status epilepticus (SE), neural changes occur during a period known as epileptogenesis and include neuronal cell death, reactive gliosis, axonal sprouting, and synaptogenesis. Extracellular matrix adhesion molecules are important regulators of synaptogenesis and axonal sprouting resulting from SE. SC1, also known as hevin, is an antiadhesive extracellular matrix molecule that localizes to synapses in the mammalian brain. In this study, the distribution of SC1 protein in neurons following SE was examined using the lithium-pilocarpine model. SC1 protein levels in neuronal cell bodies showed a transient decrease at 1 day post-SE, which coincided with an increase of SC1 in the synapse-rich neuropil that was identified with the synaptic marker synaptophysin. Immunoelectron microscopy confirmed the decrease of SC1 signal in neurons at 1 day post-SE and showed that SC1 remained localized to postsynaptic elements throughout the seizure time course. Increased colocalization of SC1 was detected with the excitatory synaptic markers vesicular glutamate transporter 1 (VGLUT1), AMPA receptor subunit GluR1, and N-methyl-D-aspartate receptor subunit NR1, but not with the inhibitory synaptic markers vesicular g-aminobutyric acid (GABA) transporter (VGAT) and GABA A receptor subunit b2 (GABA A b2), which could reflect enhanced association of SC1 with excitatory synapses. These findings suggest that SC1 may be involved in synaptic modifications underlying epileptogenesis. V

Journal of Neuropathology & Experimental Neurology, 2011

SC1 is a member of the SPARC family of glycoproteins that regulate cell-matrix interactions in th... more SC1 is a member of the SPARC family of glycoproteins that regulate cell-matrix interactions in the developing brain. SC1 is expressed in astrocytes, but nothing is known about the expression in the aged or after stroke. We found that after focal striatal ischemic infarction in adult rats, SC1 increased in astrocytes surrounding the infarct and in the glial scar, but in aged rats, SC1 was lower at the lesion edge. Glial fibrillary acidic protein (GFAP) also increased, but it was less prominent in reactive astrocytes further from the lesion in the aged rats. On the basis of their differential expression of several molecules, 2 types of reactive astrocytes with differing spatiotemporal distributions were identified. On Days 3 and 7, SC1 was prevalent in cells expressing markers of classic reactive astrocytes (GFAP, vimentin, nestin, S100A), as well as apoliprotein E (ApoE), interleukin 1A, aggrecanase 1 (ADAMTS4), and heat shock protein 25 (Hsp25). Adjacent to the lesion on Days 1 and 3, astrocytes with low GFAP levels and a ''starburst'' SC1 pattern expressed S100A, ApoE, and Hsp32 but not vimentin, nestin, interleukin 1A, ADAMTS4, or Hsp25. Neither cell type was immunoreactive for NG2, CC-1, CD11b, or ionized calcium-binding adapter-1. Their differing expression of inflammation-related and putatively protective molecules suggests different roles for starburst and classic reactive astrocytes in the early glial responses to ischemia.

Journal of Neuropathology & Experimental Neurology, 2012

The progression of white matter damage after ischemic and hemorrhagic strokes can exacerbate the ... more The progression of white matter damage after ischemic and hemorrhagic strokes can exacerbate the initial injury, but little is known about the processes involved. We show that the antiadhesive matricellular glycoprotein SC1 is a novel early marker of white matter damage in 3 models of acute injury in the rat striatum: transient focal ischemia, intracerebral hemorrhage, and a needle penetration wound. SC1 was restricted to the damaged portions of axon bundles that bordered stroke lesions in young-adult and aged rats. SC1 peaked at 1 and 3 days after intracerebral hemorrhage and at 7 days after ischemia. The SC1-positive bundles usually expressed degraded myelin basic protein and amyloid precursor protein, a marker of axonal injury. At the hematoma edge, SC1 was seen in a few axon bundles that retained myelin basic protein staining. In these bundles, punctate SC1 staining filled individual axons, extended beyond a core of pan-axonal neurofilament and NF200 and was inside or overlapped with myelin basic protein staining when it was present. Aged rats had less SC1 (and amyloid precursor protein) after both types of stroke, suggesting a reduced axonal response. SC1 also labeled amyloid precursor proteinYpositive axon bundles along the needle penetration tract of saline-injected rats; thus, SC1 appears to characterize damaged striatal white matter damage after multiple types of injury.

Journal of Neuroinflammation, 2013

Background: Microglial cells are highly mobile under many circumstances and, after central nervou... more 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 matrixdegrading 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.

Journal of Neuroinflammation, 2012

Background Microglia migrate during brain development and after CNS injury, but it is not known h... more 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 Ca2+ signaling is important for cell migration, cell-substrate adhesion and contraction of the actomyosin network. Here, we hypothesized that podosomes contain Ca2+-signaling machinery, and that podosome expression and function depend on Ca2+ influx and specific ion channels. Methods H...

Journal of Neurochemistry, 2008

Epilepsy is the second most common neurological disorder following stroke (Browne and Holmes 2001... more Epilepsy is the second most common neurological disorder following stroke (Browne and Holmes 2001). Temporal lobe epilepsy (TLE) is the epilepsy syndrome most resistant to pharmacological treatment (Cavazos and Cross 2006). The lithium-pilocarpine model exhibits many symptoms of human TLE (Cavalheiro 1995). Following a prolonged seizure episode, known as status epilepticus (SE), a latent phase ensues during which neurological changes, such as neuronal degeneration, reactive gliosis, axonal sprouting, neurogenesis, and synaptogenesis, lead to the formation of recurrent seizures (Mello et al. 1993; Covolan and Mello 2000; Shan et al. 2002; Druga et al. 2003). Involvement of the hippocampus in the generation of seizure activity is well established (Schwartzkroin 1994). Aberrant expression of neurotransmitter receptors as well as cytoskeletal and synaptic proteins has been found in the hippocampus of patients with TLE (Furtinger et al. 2003; Notenboom et al. 2006; Yang et al. 2006; Perosa et al. 2007). In addition to these cellular modifications, seizure activity has been shown to induce a 'matrix response' that includes alterations of extracellular matrix (ECM) molecules (Hoffman et al. 1998). Changes in the localization and synthesis of ECM molecules such as reelin, fibronectin, and chondroitin sulfate and heparan sulfate glycosaminoglycans following seizure activity have also been reported (Hoffman et al. 1998; Naffah-Mazzacoratti et al. 1999; Perosa et al. 2002; Gong et al. 2007). During neural development, ECM molecules play a role in plastic events such as synaptogenesis and axon outgrowth,

Osteoarthritis and Cartilage, 2020

Objective: MicroRNAs act locally and systemically to impact osteoarthritis (OA) pathophysiology, ... more Objective: MicroRNAs act locally and systemically to impact osteoarthritis (OA) pathophysiology, but comprehensive profiling of the circulating miRNome in early vs late stages of OA has yet to be conducted. Sequencing has emerged as the preferred method for microRNA profiling since it offers high sensitivity and specificity. Our objective is to sequence the miRNome in plasma from 91 patients with early [KellgreneLawrence (KL) grade 0 or 1 (n ¼ 41)] or late [KL grade 3 or 4 (n ¼ 50)] symptomatic radiographic knee OA to identify unique microRNA signatures in each disease state. Design: MicroRNA libraries were prepared using the QIAseq miRNA Library Kit and sequenced on the Illumina NextSeq 550.Counts were produced for microRNAs captured in miRBase and for novel micro-RNAs. Statistical, bioinformatics, and computational biology approaches were used to refine and interpret the final list of microRNAs. Results: From 215 differentially expressed microRNAs (FDR < 0.01), 97 microRNAs showed an increase or decrease in expression in 85% of samples in the early OA group as compared to the median expression in the late OA group. Increasing this threshold to 95%, seven microRNAs were identified: hsa-miR-335-3p, hsa-miR-199a-5p, hsa-miR-671-3p, hsa-miR-1260b, hsa-miR-191-3p, hsa-miR-335-5p, and hsa-miR-543. Four novel microRNAs were present in 50% of early OA samples and had 27 predicted gene targets in common with the prioritized set of predicted gene targets from the 97 microRNAs, suggesting common underlying mechanisms.

Osteoarthritis and Cartilage Open, 2020

Objective: Osteoarthritis (OA) is a degenerative joint disease with no approved disease modifying... more Objective: Osteoarthritis (OA) is a degenerative joint disease with no approved disease modifying therapy. The enzyme autotaxin (ATX) converts lysophoshatidylcholine analogues to lysophosphatidic acid. Systemic inhibition of ATX reduces pain in animal models of OA; however, OA disease-modifying effects associated with ATX inhibition remain unknown. Here, we sought to determine whether local (knee joint) injection of an ATX inhibitor attenuates surgically-induced OA in mice. Methods: ATX expression was evaluated in human knee OA cartilage. Ten-week-old mice were subjected to surgically-induced OA. ATX inhibitor (PF-8380, 2.5ng/joint) was injected intra-articularly either at 3-and 5weeks post-surgery or at 2-, 4-, 6-and 8-weeks post-surgery and knee joints were evaluated by histopathology and immunohistochemistry to study the expression of catabolic and cell death markers. mRNA Sequencing of human OA chondrocytes treated with/without ATX inhibitor was performed to identify differentially expressed transcripts, followed by pathway enrichment analysis. Results: ATX expression was elevated in severely degenerated cartilage compared to less degenerated human OA cartilage. In surgically-induced OA mice, intra-articular injection of ATX inhibitor at 3-and 5-weeks post-surgery partially protected knee joints from cartilage degeneration. Interestingly, earlier and more frequent ATX inhibitor injections did not confer significant protection. Immunohistochemical analysis showed decreased expression of catabolic and apoptotic markers with two ATX inhibitor injections. mRNA sequencing followed by pathway analysis identified pathways related to cholesterol analogue metabolism and cell-cycle that could be modulated by ATX inhibition in human OA chondrocytes. Conclusion: Local delivery of ATX inhibitor partially attenuates surgically-induced OA in mice.

Frontiers in Cellular Neuroscience, 2018

Microglia respond to CNS injuries and diseases with complex reactions, often called "activation."... more 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 proinflammatory 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.

Frontiers in Cellular Neuroscience, 2018

Addressing potential sex differences in pre-clinical studies is crucial for developing therapeuti... more 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.

Frontiers in cellular neuroscience, 2018

The cytokine, transforming growth factor β1 (TGFβ1), is up-regulated after central nervous system... more The cytokine, transforming growth factor β1 (TGFβ1), 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 TGFβ1. 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 TGFβ1 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 TGFβ1. Microglia were isolated from Spra...

Journal of Neuroinflammation, 2017

Background: Acute CNS damage is commonly studied using rat and mouse models, but increasingly, mo... more 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 interferonγ + 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-inwardand 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.

Journal of Neuropathology & Experimental Neurology, 2016

Within hours after stroke, potentially cytotoxic pro-inflammatory mediators are elevated within t... more 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 antiinflammatory 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, PPARc), increased phagocytic (Iba1-positive, CD68-positive) microglia/macrophages, and increased VEGF-Apositive infiltrating neutrophils in the infarcts. At 7 days, there was evidence of sustained, propagating responses. IL-4 increased CD206, CD200R1, IL-4Ra, STAT6, PPARc, 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.

Journal of Neuroinflammation, 2016

Background: Microglia are the "professional" phagocytes of the CNS. Phagocytosis is crucial for n... more 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.

Translational Stroke Research, 2012

In the hours to days after intracerebral hemorrhage (ICH), there is an inflammatory response with... more In the hours to days after intracerebral hemorrhage (ICH), there is an inflammatory response within the brain characterized by the infiltration of peripheral neutrophils and macrophages and the activation of brain-resident microglia and astrocytes. Despite the strong correlation of aging and ICH incidence, and increasing information about cellular responses, little is known about the temporal-and age-related molecular responses of the brain after ICH. Here, we monitored a panel of 27 genes at 6 h and 1, 3, and 7 days after ICH was induced by injecting collagenase into the striatum of young adult and aged rats. Several molecules (CR3, TLR2, TLR4, IL-1β, TNFα, iNOS, IL-6) were selected to reflect the classical activation of innate immune cells (macrophages, microglia) and the potential to exacerbate inflammation and damage brain cells. Most of the others are associated with the resolution of innate inflammation, alternative pathways of macrophage/microglial activation, and the repair phase after acute injury (TGFβ, IL-1ra, IL-1r2, IL-4, IL-13, IL-4Rα, IL-13Rα1, IL-13Rα2, MRC1, ARG1, CD163, CCL22). In young animals, the up-regulation of 26 in 27 genes (not IL-4) was detected within the first week. Differences in timing or levels between young and aged animals were detected for 18 of 27 genes examined (TLR2,

Biophysical Journal, 2015

The Ca 2+-activated K + channel, KCa3.1 (KCNN4/IK1/SK4), contributes to "classical," proinflammat... more The Ca 2+-activated K + channel, KCa3.1 (KCNN4/IK1/SK4), contributes to "classical," proinflammatory activation of microglia, and KCa3.1 blockers have improved the outcome in several rodent models of CNS damage. For instance, blocking KCa3.1 with TRAM-34 rescued retinal ganglion neurons after optic nerve damage in vivo and, reduced p38 MAP kinase activation, production of reactive oxygen and nitrogen species, and neurotoxicity by microglia in vitro. In pursuing the therapeutic potential of KCa3.1 blockers, it is crucial to assess KCa3.1 contributions to other microglial functions and activation states, especially the IL-4-induced "alternative" activation state that can counteract pro-inflammatory states. We recently found that IL-4 increases microglia migration-a crucial function in the healthy and damaged CNS-and that KCa3.1 contributes to P 2 Y 2 receptor-stimulated migration. Here, we discovered that KCa3.1 is greatly increased in alternative-activated rat microglia and then contributes to an enhanced migratory capacity. IL-4 up-regulated KCNN4 mRNA (by 6 h) and greatly increased the KCa3.1 current by 1 day, and this required de novo protein synthesis. The increase in current was sustained for at least 6 days. IL-4 increased microglial migration and this was reversed by blocking KCa3.1 with TRAM-34. A panel of inhibitors of signal-transduction mediators was used to analyze contributions of IL-4-related signaling pathways. Induction of KCNN4 mRNA and KCa3.1 current was mediated specifically through IL-4 binding to the type I receptor and, surprisingly, it required JAK3, Ras/MEK/ERK signaling and the transcription factor, activator protein-1, rather than JAK2, STAT6, or phosphatidylinositol 3-kinase.The same receptor subtype and pathway were required for the enhanced KCa3.1-dependent migration. In providing the first direct signaling link between an IL-4 receptor, expression and roles of an ion channel, this study also highlights the potential importance of KCa3.1 in alternative-activated microglia.

The Journal of Neuroscience, 2011

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/mi... more 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+channelKCNN4(KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes inKCNN4mRNA 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.KCNN4mRNA 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 ...

Journal of Neuroscience Research, 2008

The epileptic brain is characterized by increased susceptibility to neuronal hyperexcitability. T... more The epileptic brain is characterized by increased susceptibility to neuronal hyperexcitability. The rat lithium-pilocarpine model, which mimics many features of temporal lobe epilepsy, has been used to study processes leading to the development of recurrent seizures. After a prolonged seizure episode, termed status epilepticus (SE), neural changes occur during a period known as epileptogenesis and include neuronal cell death, reactive gliosis, axonal sprouting, and synaptogenesis. Extracellular matrix adhesion molecules are important regulators of synaptogenesis and axonal sprouting resulting from SE. SC1, also known as hevin, is an antiadhesive extracellular matrix molecule that localizes to synapses in the mammalian brain. In this study, the distribution of SC1 protein in neurons following SE was examined using the lithium-pilocarpine model. SC1 protein levels in neuronal cell bodies showed a transient decrease at 1 day post-SE, which coincided with an increase of SC1 in the synapse-rich neuropil that was identified with the synaptic marker synaptophysin. Immunoelectron microscopy confirmed the decrease of SC1 signal in neurons at 1 day post-SE and showed that SC1 remained localized to postsynaptic elements throughout the seizure time course. Increased colocalization of SC1 was detected with the excitatory synaptic markers vesicular glutamate transporter 1 (VGLUT1), AMPA receptor subunit GluR1, and N-methyl-D-aspartate receptor subunit NR1, but not with the inhibitory synaptic markers vesicular g-aminobutyric acid (GABA) transporter (VGAT) and GABA A receptor subunit b2 (GABA A b2), which could reflect enhanced association of SC1 with excitatory synapses. These findings suggest that SC1 may be involved in synaptic modifications underlying epileptogenesis. V

Journal of Neuropathology & Experimental Neurology, 2011

SC1 is a member of the SPARC family of glycoproteins that regulate cell-matrix interactions in th... more SC1 is a member of the SPARC family of glycoproteins that regulate cell-matrix interactions in the developing brain. SC1 is expressed in astrocytes, but nothing is known about the expression in the aged or after stroke. We found that after focal striatal ischemic infarction in adult rats, SC1 increased in astrocytes surrounding the infarct and in the glial scar, but in aged rats, SC1 was lower at the lesion edge. Glial fibrillary acidic protein (GFAP) also increased, but it was less prominent in reactive astrocytes further from the lesion in the aged rats. On the basis of their differential expression of several molecules, 2 types of reactive astrocytes with differing spatiotemporal distributions were identified. On Days 3 and 7, SC1 was prevalent in cells expressing markers of classic reactive astrocytes (GFAP, vimentin, nestin, S100A), as well as apoliprotein E (ApoE), interleukin 1A, aggrecanase 1 (ADAMTS4), and heat shock protein 25 (Hsp25). Adjacent to the lesion on Days 1 and 3, astrocytes with low GFAP levels and a ''starburst'' SC1 pattern expressed S100A, ApoE, and Hsp32 but not vimentin, nestin, interleukin 1A, ADAMTS4, or Hsp25. Neither cell type was immunoreactive for NG2, CC-1, CD11b, or ionized calcium-binding adapter-1. Their differing expression of inflammation-related and putatively protective molecules suggests different roles for starburst and classic reactive astrocytes in the early glial responses to ischemia.

Journal of Neuropathology & Experimental Neurology, 2012

The progression of white matter damage after ischemic and hemorrhagic strokes can exacerbate the ... more The progression of white matter damage after ischemic and hemorrhagic strokes can exacerbate the initial injury, but little is known about the processes involved. We show that the antiadhesive matricellular glycoprotein SC1 is a novel early marker of white matter damage in 3 models of acute injury in the rat striatum: transient focal ischemia, intracerebral hemorrhage, and a needle penetration wound. SC1 was restricted to the damaged portions of axon bundles that bordered stroke lesions in young-adult and aged rats. SC1 peaked at 1 and 3 days after intracerebral hemorrhage and at 7 days after ischemia. The SC1-positive bundles usually expressed degraded myelin basic protein and amyloid precursor protein, a marker of axonal injury. At the hematoma edge, SC1 was seen in a few axon bundles that retained myelin basic protein staining. In these bundles, punctate SC1 staining filled individual axons, extended beyond a core of pan-axonal neurofilament and NF200 and was inside or overlapped with myelin basic protein staining when it was present. Aged rats had less SC1 (and amyloid precursor protein) after both types of stroke, suggesting a reduced axonal response. SC1 also labeled amyloid precursor proteinYpositive axon bundles along the needle penetration tract of saline-injected rats; thus, SC1 appears to characterize damaged striatal white matter damage after multiple types of injury.

Journal of Neuroinflammation, 2013

Background: Microglial cells are highly mobile under many circumstances and, after central nervou... more 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 matrixdegrading 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.

Journal of Neuroinflammation, 2012

Background Microglia migrate during brain development and after CNS injury, but it is not known h... more 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 Ca2+ signaling is important for cell migration, cell-substrate adhesion and contraction of the actomyosin network. Here, we hypothesized that podosomes contain Ca2+-signaling machinery, and that podosome expression and function depend on Ca2+ influx and specific ion channels. Methods H...

Journal of Neurochemistry, 2008

Epilepsy is the second most common neurological disorder following stroke (Browne and Holmes 2001... more Epilepsy is the second most common neurological disorder following stroke (Browne and Holmes 2001). Temporal lobe epilepsy (TLE) is the epilepsy syndrome most resistant to pharmacological treatment (Cavazos and Cross 2006). The lithium-pilocarpine model exhibits many symptoms of human TLE (Cavalheiro 1995). Following a prolonged seizure episode, known as status epilepticus (SE), a latent phase ensues during which neurological changes, such as neuronal degeneration, reactive gliosis, axonal sprouting, neurogenesis, and synaptogenesis, lead to the formation of recurrent seizures (Mello et al. 1993; Covolan and Mello 2000; Shan et al. 2002; Druga et al. 2003). Involvement of the hippocampus in the generation of seizure activity is well established (Schwartzkroin 1994). Aberrant expression of neurotransmitter receptors as well as cytoskeletal and synaptic proteins has been found in the hippocampus of patients with TLE (Furtinger et al. 2003; Notenboom et al. 2006; Yang et al. 2006; Perosa et al. 2007). In addition to these cellular modifications, seizure activity has been shown to induce a 'matrix response' that includes alterations of extracellular matrix (ECM) molecules (Hoffman et al. 1998). Changes in the localization and synthesis of ECM molecules such as reelin, fibronectin, and chondroitin sulfate and heparan sulfate glycosaminoglycans following seizure activity have also been reported (Hoffman et al. 1998; Naffah-Mazzacoratti et al. 1999; Perosa et al. 2002; Gong et al. 2007). During neural development, ECM molecules play a role in plastic events such as synaptogenesis and axon outgrowth,