CD36 neutralisation blunts TLR2-IRF7 but not IRF3 pathway in neonatal mouse brain and immature human microglia following innate immune challenge.

Innate immune response in neonatal brain is associated with a robust microglial activation and induction of Toll-like Receptors (TLRs). To date, the role of the scavenger receptor CD36 in TLRs modulation, particularly TLR2 signaling, has been well established in adult brain. However, the crosstalk between TLR4, TLR2 and CD36 and its immunogenic influence in the neonatal brain remains unclear. In this study, using a CD36 blocking antibody (anti-CD36) at post-natal day 8, we evaluated the response of neonates to systemic endotoxin (lipopolysaccharide; LPS) challenge. We visualized the TLR2 response by bioluminescence imaging using the transgenic mouse model bearing the dual reporter system luciferase/green fluorescent protein under transcriptional control of a murine TLR2 promoter. The anti-CD36 treatment modified the LPS induced inflammatory profile in neonatal brains, causing a significant decrease in inflammatory cytokine levels and the TLR2 and TLR3 mediated signalling.The interferon regulatory factor 3 (IRF3) pathway remained unaffected. Treatment of the LPS-challenged human immature microglia with anti-CD36 induced a marked decrease in TLR2/TLR3 expression levels while TLR4 and IRF3 expression was not affected, suggesting the shared CD36 regulatory mechanisms in human and mouse microglia. Collectively, our results indicate that blocking CD36 alters LPS-induced inflammatory profile of mouse and human microglia, suggesting its role in fine-tuning of neuroinflammation.

Glucosamine-mediated immunomodulation after stroke is sexually dimorphic.

Growing evidence suggests that galectin-3 (Gal-3) is instrumental in orchestrating innate immune response and microglia activation following different brain pathologies. However, its role remains controversial. We recently showed that a readily available natural product glucosamine may act as a strong modulator of Gal-3. Glucosamine is a naturally occurring sugar and a precursor in the synthesis of glycosylated proteins. It is often used as a supplement to treat symptoms of various inflammatory conditions. Our recent work suggests that by increasing the synthesis and availability of Gal-3 ligands and/or by regulating its expression levels, glucosamine may significantly modulate Gal-3 signaling. Because evidence suggests that Gal-3 might be differentially regulated after ischemic injury in the brains of female mice, here we examined and compared the immunomodulatory potential of glucosamine in male and female stroke. The mice were subjected to transient middle cerebral artery occlusion (MCAO), followed by different reperfusion periods. The short-term 5 days treatment with glucosamine (150 â€‹mg/kg i.p.) was initiated 2 â€‹hrs after stroke. To visualize the effects of glucosamine treatment on post-stroke inflammation, we took advantage of a transgenic mouse model bearing the dual reporter system luciferase/GFP under transcriptional control of a murine TLR2 promoter (TLR2-luc-GFP) allowing in vivo bioluminescence imaging of innate immune response and microglial activation. We report that after stroke, both, male and female mice strongly up-regulate the TLR2 bioluminescence signals from activated microglia, however, the observed in vivo immunomodulatory effects of glucosamine after stroke were sex-dependent. Analysis of cytokine profiles at protein level, in glucosamine-treated male mice 72hsr after stroke, revealed down regulation of pro-inflammatory cytokines, an increase in levels of anti-inflammatory cytokines including IL-4, IL13 and colony stimulating factors MCFC and GM-CSF and a significant decrease in the size of ischemic lesion in male mice. Conversely, in female mice glucosamine markedly increases the pro-inflammatory signaling and exacerbates ischemic injury. Analysis of the downstream signaling target of glucosamine/Gal-3 revealed that glucosamine administration restored PPAR-γ activity in male but not in female mice 3 days following MCAO. Together, our results suggest that glucosamine acts as a fine tuner of post-ischemic inflammation in a sex dependent-manner and may have therapeutic potential after stroke in males. Based on our results propose that targeting immune system after stroke may require adapted sex-specific therapeutic approaches.

Delayed Galectin-3-Mediated Reprogramming of Microglia After Stroke is Protective.

Galectin-3 (Gal-3), a ß-galactoside-binding lectin, has recently emerged as a molecule with immunoregulatory functions. We investigated the effects of Gal-3 on microglia morphology, migration, and secretory profile under physiological conditions and in the context of ischemic injury. We show that in the control conditions, exposure to recombinant Gal-3 increases microglial ramification and motility in vitro and in vivo via an IL-4-dependent mechanism. Importantly, after stroke, Gal-3 exerted marked immune-modulatory properties. Delivery of Gal-3 at 24 h after middle cerebral artery occlusion (MCAO) was associated with an increase in Ym1-positive microglia and decrease in iNOS. Analysis of cytokine profiles at the protein level revealed downregulation of pro-inflammatory cytokines and a marked upregulation of the anti-inflammatory cytokine, IL-4, 24 h after i.c.v. injection of Gal-3. Importantly, the observed shift in cytokines in microglia was associated with a significant decrease in the infarct size. Taken together, our results suggest that when delivered well after ischemic injury, Gal-3 might fine tune innate immunity and induce a therapeutic shift in microglia polarization.

Diverging mRNA and Protein Networks in Activated Microglia Reveal SRSF3 Suppresses Translation of Highly Upregulated Innate Immune Transcripts.

Uncontrolled microglial activation may lead to the development of inflammation-induced brain damage. Here, we uncover a ribosome-based mechanism/checkpoint involved in control of the innate immune response and microglial activation. Using an in vivo model system for analysis of the dynamic translational state of microglial ribosomes, with mRNAs as input and newly synthesized peptides as an output, we find a marked dissociation of microglia mRNA and protein networks following innate immune challenge. Highly upregulated and ribosome-associated mRNAs were not translated, resulting in two distinct microglial molecular signatures, a highly specialized pro-inflammatory mRNA signature and an immunomodulatory/homeostatic protein signature. We find that this is due to specific translational suppression of highly expressed mRNAs through a 3' UTR-mediated mechanism involving the RNA-binding protein SRSF3. This discovery suggests avenues for therapeutic modulation of innate immune response in resident microglia.

Live imaging of the innate immune response in neonates reveals differential TLR2 dependent activation patterns in sterile inflammation and infection.

Activation of microglial cells in response to brain injury and/or immune stimuli is associated with a marked induction of Toll-like receptors (TLRs). While in adult brain, the contribution of individual TLRs, including TLR2, in pathophysiological cascades has been well established, their role and spatial and temporal induction patterns in immature brain are far less understood. To examine whether infectious stimuli and sterile inflammatory stimuli trigger distinct TLR2-mediated innate immune responses, we used three models in postnatal day 9 (P9) mice, a model of infection induced by systemic endotoxin injection and two models of sterile inflammation, intra-cortical IL-1ß injection and transient middle cerebral artery occlusion (tMCAO). We took advantage of a transgenic mouse model bearing the dual reporter system luciferase/GFP under transcriptional control of a murine TLR2 promoter (TLR2-luc-GFP) to visualize the TLR2 response in the living neonatal brain and then determined neuroinflammation, microglial activation and leukocyte infiltration. We show that in physiological postnatal brain development the in vivo TLR2-luc signal undergoes a marked ∼30-fold decline and temporal-spatial changes during the second and third postnatal weeks. We then show that while endotoxin robustly induces the in vivo TLR2-luc signal in the living brain and increases levels of several inflammatory cytokines and chemokines, the in vivo TLR2-luc signal is reduced after both IL-1ß and tMCAO and the inflammatory response is muted. Immunofluorescence revealed that microglial cells are the predominant source of TLR2 production during postnatal brain development and in all three neonatal models studied. Flow cytometry revealed developmental changes in CD11b+/CD45+ and CD11b+/Ly6C+ cell populations, involvement of cells of the monocyte lineage, but lack of Ly6G+ neutrophils or CD3+ cells in acutely injured neonatal brains. Cumulatively, our results suggest distinct TLR2 induction patterns following PAMP and DAMP - mediated inflammation in immature brain.

Molecular imaging of nestin in neuroinflammatory conditions reveals marked signal induction in activated microglia.

BACKGROUND: Nestin is a known marker of neuronal progenitor cells in the adult brain. Following neuro- and gliogenesis, nestin is replaced by cell type-specific intermediate filaments, e.g., neurofilaments for panneuronal expression and glial fibrillary acidic protein as a specific marker of mature astrocytes. While previous work have been mostly focused on the neuronal fate of nestin-positive progenitors, in the present study, we sought to investigate in real time how nestin signals and cellular expression patterns are controlled in the context of neuroinflammatory challenge and ischemic brain injury. METHODS: To visualize effects of neuroinflammation on neurogenesis/gliogenesis, we created a transgenic model bearing the dual reporter system luciferase and GFP under transcriptional control of the murine nestin promoter. In this model, transcriptional activation of nestin was visualized from the brains of living animals using biophotonic/bioluminescence molecular imaging and a high resolution charged coupled device camera. Nestin induction profiles in vivo and in tissue sections were analyzed in two different experimental paradigms: middle cerebral artery occlusion and lipopolysaccharide-induced innate immune stimuli. RESULTS: We report here a context- and injury-dependent induction and cellular expression profile of nestin. While in the baseline conditions the nestin signal and/or GFP expression was restricted to neuronal progenitors, the cellular expression patterns of nestin following innate immune challenge and after stroke markedly differed shifting the cellular expression patterns towards activated microglia/macrophages and astrocytes. CONCLUSIONS: Our results suggest that nestin may serve as a context-dependent biomarker of inflammatory response in glial cells including activated microglia/macrophages.

Gamma motor neurons survive and exacerbate alpha motor neuron degeneration in ALS.

The molecular and cellular basis of selective motor neuron (MN) vulnerability in amyotrophic lateral sclerosis (ALS) is not known. In genetically distinct mouse models of familial ALS expressing mutant superoxide dismutase-1 (SOD1), TAR DNA-binding protein 43 (TDP-43), and fused in sarcoma (FUS), we demonstrate selective degeneration of alpha MNs (α-MNs) and complete sparing of gamma MNs (γ-MNs), which selectively innervate muscle spindles. Resistant γ-MNs are distinct from vulnerable α-MNs in that they lack synaptic contacts from primary afferent (IA) fibers. Elimination of these synapses protects α-MNs in the SOD1 mutant, implicating this excitatory input in MN degeneration. Moreover, reduced IA activation by targeted reduction of γ-MNs in SOD1G93A mutants delays symptom onset and prolongs lifespan, demonstrating a pathogenic role of surviving γ-MNs in ALS. This study establishes the resistance of γ-MNs as a general feature of ALS mouse models and demonstrates that synaptic excitation of MNs within a complex circuit is an important determinant of relative vulnerability in ALS.

Estrogen receptors alpha mediates postischemic inflammation in chronically estrogen-deprived mice.

Estrogens are known to exert neuroprotective and immuneomodulatory effects after stroke. However, at present, little is known about the role of estrogens and its receptors in postischemic inflammation after menopause. Here, we provide important in vivo evidence of a distinct shift in microglial phenotypes in the model of postmenopause brain. Using a model-system for live imaging of microglial activation in the context of chronic estrogen- and ERα-deficiency associated with aging, we observed a marked deregulation of the TLR2 signals and/or microglial activation in ovariectomized and/or ERα knockout mice. Further analysis revealed a 5.7-fold increase in IL-6, a 4.7-fold increase in phospho-Stat3 levels suggesting an overactivation of JAK/STAT3 pathway and significantly larger infarction in ERα knockouts chronically deprived of estrogen. Taken together, our results suggest that in the experimental model of menopause and/or aging, ERα mediates innate immune responses and/or microglial activation, and ischemia-induced production of IL-6. Based on our results, we propose that the loss of functional ERα may lead to deregulation of postischemic inflammatory responses and increased vulnerability to ischemic injury in aging female brains.

FUS-SMN protein interactions link the motor neuron diseases ALS and SMA.

Mutations in the RNA binding protein FUS cause amyotrophic lateral sclerosis (ALS), a fatal adult motor neuron disease. Decreased expression of SMN causes the fatal childhood motor neuron disorder spinal muscular atrophy (SMA). The SMN complex localizes in both the cytoplasm and nuclear Gems, and loss of Gems is a cellular hallmark of fibroblasts in patients with SMA. Here, we report that FUS associates with the SMN complex, mediated by U1 snRNP and by direct interactions between FUS and SMN. Functionally, we show that FUS is required for Gem formation in HeLa cells, and expression of FUS containing a severe ALS-causing mutation (R495X) also results in Gem loss. Strikingly, a reduction in Gems is observed in ALS patient fibroblasts expressing either mutant FUS or TDP-43, another ALS-causing protein that interacts with FUS. The physical and functional interactions among SMN, FUS, TDP-43, and Gems indicate that ALS and SMA share a biochemical pathway, providing strong support for the view that these motor neuron diseases are related.

Toll-like receptor 2 deficiency leads to delayed exacerbation of ischemic injury.

BACKGROUND: Using a live imaging approach, we have previously shown that microglia activation after stroke is characterized by marked and long-term induction of the Toll-like receptor (TLR) 2 biophotonic signals. However, the role of TLR2 (and potentially other TLRs) beyond the acute innate immune response and as early neuroprotection against ischemic injury is not well understood. METHODS: TLR2-/- mice were subjected to transient middle cerebral artery occlusion followed by different reperfusion times. Analyses assessing microglial activation profile/innate immune response were performed using in situ hybridization, immunohistochemistry analysis, flow cytometry and inflammatory cytokine array. The effects of the TLR2 deficiency on the evolution of ischemic brain injury were analyzed using a cresyl violet staining of brain sections with appropriate lesion size estimation. RESULTS: Here we report that TLR2 deficiency markedly affects post-stroke immune response resulting in delayed exacerbation of the ischemic injury. The temporal analysis of the microglia/macrophage activation profiles in TLR2-/- mice and age-matched controls revealed reduced microglia/macrophage activation after stroke, reduced capacity of resident microglia to proliferate as well as decreased levels of monocyte chemotactic protein-1 (MCP-1) and consequently lower levels of CD45(high)/CD11b(+) expressing cells as shown by flow cytometry analysis. Importantly, although acute ischemic lesions (24 to 72 h) were smaller in TLR2-/- mice, the observed alterations in innate immune response were more pronounced at later time points (at day 7) after initial stroke, which finally resulted in delayed exacerbation of ischemic lesion leading to larger chronic infarctions as compared with wild-type mice. Moreover, our results revealed that TLR2 deficiency is associated with significant decrease in the levels of neurotrophic/anti-apoptotic factor Insulin-like growth factor-1 (IGF-1), expressed by microglia in the areas both in and around ischemic lesion. CONCLUSION: Our results clearly suggest that optimal and timely microglial activation/innate immune response is needed to limit neuronal damage after stroke.

Galectin-3 is required for resident microglia activation and proliferation in response to ischemic injury.

Growing evidence suggests that galectin-3 is involved in fine tuning of the inflammatory responses at the periphery, however, its role in injured brain is far less clear. Our previous work demonstrated upregulation and coexpression of galectin-3 and IGF-1 in a subset of activated/proliferating microglial cells after stroke. Here, we tested the hypothesis that galectin-3 plays a pivotal role in mediating injury-induced microglial activation and proliferation. By using a galectin-3 knock-out mouse (Gal-3KO), we demonstrated that targeted disruption of the galectin-3 gene significantly alters microglia activation and induces ∼4-fold decrease in microglia proliferation. Defective microglia activation/proliferation was further associated with significant increase in the size of ischemic lesion, ∼2-fold increase in the number of apoptotic neurons, and a marked deregulation of the IGF-1 levels. Next, our results revealed that contrary to WT cells, the Gal3-KO microglia failed to proliferate in response to IGF-1. Moreover, the IGF-1-mediated mitogenic microglia response was reduced by N-glycosylation inhibitor tunicamycine while coimmunoprecipitation experiments revealed galectin-3 binding to IGF-receptor 1 (R1), thus suggesting that interaction of galectin-3 with the N-linked glycans of receptors for growth factors is involved in IGF-R1 signaling. While the canonical IGF-1 signaling pathways were not affected, we observed an overexpression of IL-6 and SOCS3, suggesting an overactivation of JAK/STAT3, a shared signaling pathway for IGF-1/IL-6. Together, our findings suggest that galectin-3 is required for resident microglia activation and proliferation in response to ischemic injury.

Wnt7A identifies embryonic γ-motor neurons and reveals early postnatal dependence of γ-motor neurons on a muscle spindle-derived signal.

Motor pools comprise a heterogeneous population of motor neurons that innervate distinct intramuscular targets. While the organization of motor neurons into motor pools has been well described, the time course and mechanism of motor pool diversification into functionally distinct classes remains unclear. γ-Motor neurons (γ-MNs) and α-motor neurons (α-MNs) differ in size, molecular identity, synaptic input and peripheral target. While α-MNs innervate extrafusal skeletal muscle fibers to mediate muscle contraction, γ-MNs innervate intrafusal fibers of the muscle spindle, and regulate sensitivity of the muscle spindle in response to stretch. In this study, we find that the secreted signaling molecule Wnt7a is selectively expressed in γ-MNs in the mouse spinal cord by embryonic day 17.5 and continues to molecularly distinguish γ-from α-MNs into the third postnatal week. Our data demonstrate that Wnt7a is the earliest known γ-MN marker, supporting a model of developmental divergence between α- and γ-MNs at embryonic stages. Furthermore, using Wnt7a expression as an early marker of γ-MN identity, we demonstrate a previously unknown dependence of γ-MNs on a muscle spindle-derived, GDNF-independent signal during the first postnatal week.

Accumulation of dietary docosahexaenoic acid in the brain attenuates acute immune response and development of postischemic neuronal damage.

BACKGROUND AND PURPOSE: Consumption of fish has been shown to reduce risk of coronary heart disease and, possibly, of ischemic stroke. Because docosahexaenoic acid (DHA) is the most likely neuroactive component within fish oil, we hypothesized that exposing mice to a DHA-enriched diet may reduce inflammation and protect neurons against ischemic injury. METHODS: To visualize the effects of DHA on neuroinflammation after stroke, TLR2-fluc-GFP transgenic mice were exposed to either a control diet, a diet depleted in n-3 polyunsaturated fatty acid, or a diet enriched in DHA during 3 months. Real-time biophotonic/bioluminescence imaging of the TLR2 response was performed before and after middle cerebral artery occlusion, whereas cytokines concentrations and stroke area analyses were performed at 3 and 7 days after middle cerebral artery occlusion, respectively. RESULTS: We show that a 3-month DHA treatment prevented microglial activation after ischemic injury, reduced the ischemic lesion size, and increased levels of the antiapoptotic molecule Bcl-2 in the brain. Additional analysis revealed a significant decrease in the levels of COX2 and IL-1ß, but not in other proinflammatory cytokines. Importantly, long-term DHA supplementation significantly changed the n-3:n-6 polyunsaturated fatty acid ratio in the brain. CONCLUSIONS: Collectively, these data indicate that diet-induced accumulation of DHA in the brain protects against postischemic inflammation and injury. Because DHA is widely available at low cost and has an excellent safety profile, our data suggest that increased DHA intake may provide protection against acute immune response/brain damage in ischemic stroke.

The endocannabinoid 2-arachidonoyl-glycerol activates human neutrophils: critical role of its hydrolysis and de novo leukotriene B4 biosynthesis.

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.

Lipopolysaccharide-QD micelles induce marked induction of TLR2 and lipid droplet accumulation in olfactory bulb microglia.

The intranasal entry of biological and artificial nanoparticles can induce inflammatory responses both locally and more widely in surrounding tissues. The aim of this study was to assess the microglia activation induced by nanoparticles with different surfaces in (i) a transgenic mouse (Toll-like receptor (TLR)-2-luciferase (Luc) reporter) which allowed the biophotonic imaging of microglial activation/innate immune response after intranasal delivery of nanoparticles and (ii) in microglial dispersed cells in vitro. Cadmium selenide nanoparticles (quantum dots, QD), surface-exchanged with lipopolysaccharide (LPS) to form micelles, were tested to assess microglia activation and lipid droplet formation in both model systems. In vivo imaging revealed a robust increase in the extent of microglial activation/TLR2 response, initially in the olfactory bulb, but also in other more caudal brain regions. The increased TLR2 expression was complemented with enhanced CD68 expression in activated microglia in the same regions. Intense in vitro microglial activation by LPS-QD micelles was accompanied by a significant enhancement of nitric oxide production and formation of large lipid droplets, suggesting the possibility of this organelle acting as an inflammatory biomarker in response to nanoparticles, and not simply as a storage site in fat tissues.

Microglial response to gold nanoparticles.

Given the emergence of nanotherapeutics and nanodiagnostics as key tools in today's medicine, it has become of critical importance to define precisely the interactions of nanomaterials with biological systems and to characterize the resulting cellular response. We report here the interactions of microglia and neurons with gold nanoparticles (GNPs) of three morphologies, spheres, rods, and urchins, coated with poly(ethylene glycol) (PEG) or cetyl trimethylammonium bromide (CTAB). Microglia are the resident immune cells of the brain, primarily involved in surveillance, macrophagy, and production of cytokines and trophic factors. Analysis by dark-field microscopy and by two-photon-induced luminescence (TPL) indicates that the exposure of neural cells to GNPs resulted in (i) GNP internalization by both microglial cells and primary hippocampal neurons, as revealed by dark-field microscopy and by two-photon-induced luminescence (TPL), (ii) transient toll-like receptor 2 (TLR-2) up-regulation in the olfactory bulb, after intranasal administration in transgenic mice, in vivo, in real time, and (iii) differential up-regulation in vitro of TLR-2 together with interleukin 1 alpha (IL-1alpha), granulocyte macrophage colony-stimulating factor (GM-CSF) and nitric oxide (NO) in microglia. The study demonstrates that GNP morphology and surface chemistry strongly influence the microglial activation status and suggests that interactions between GNPs and microglia can be differentially regulated by tuning GNP nanogeometry.

Macrophage colony stimulating factor (M-CSF) exacerbates ALS disease in a mouse model through altered responses of microglia expressing mutant superoxide dismutase.

Macrophage colony stimulating factor (M-CSF) is a cytokine that regulates the survival, proliferation and maturation of microglial cells. Administration of M-CSF can promote neuronal survival in various models of central nervous system (CNS) injury. Here, in an attempt to induce a neuroprotective microglial cell phenotype and enhance motor neuron survival, mutant SOD1(G37R) transgenic mice were treated, weekly, with M-CSF starting at onset of disease. Unexpectedly, M-CSF accelerated disease progression in SOD1(G37R) mouse model of ALS. The shortened survival of M-CSF-treated animals was associated with diminished muscle innervation and enhanced adoption of a macrophage-like phenotype by microglial cells characterised by the upregulation of pro-inflammatory cytokines TNF-alpha and IL-1 beta and of the phagocytic marker CD68.

Inflammation, plasticity and real-time imaging after cerebral ischemia.

With an incidence of approximately 350 in 100,000, stroke is the third leading cause of death and a major cause of disability in industrialized countries. At present, although progress has been made in understanding the molecular pathways that lead to ischemic cell death, the current clinical treatments remain poorly effective. There is mounting evidence that inflammation plays an important role in cerebral ischemia. Experimentally and clinically, brain response to ischemic injury is associated with an acute and prolonged inflammatory process characterized by the activation of resident glial cells, production of inflammatory cytokines as well as leukocyte and monocyte infiltration in the brain, events that may contribute to ischemic brain injury and affect brain recovery and plasticity. However, whether the post-ischemic inflammatory response is deleterious or beneficial to brain recovery is presently a matter of debate and controversies. Here, we summarize the current knowledge on the molecular mechanisms underlying post-ischemic neuronal plasticity and the potential role of inflammation in regenerative processes and functional recovery after stroke. Furthermore, because of the dynamic nature of the brain inflammatory response, we highlight the importance of the development of novel experimental approaches such as real-time imaging. Finally, we discuss the novel transgenic reporter mice models that have allowed us to visualize and to analyze the processes such as neuroinflammation and neuronal repair from the ischemic brains of live animals.

Live imaging of neuroinflammation reveals sex and estrogen effects on astrocyte response to ischemic injury.

BACKGROUND AND PURPOSE: We sought to develop a model system for live analysis of brain inflammatory response in ischemic injury. METHODS: Using a reporter mouse-expressing luciferase gene under transcriptional control of the murine glial fibrillary acidic protein (GFAP) promoter (GFAP-luc mice) and biophotonic/bioluminescent imaging as tools, we developed a model system for in vivo analysis of astrocyte activation/response in cerebral ischemia. RESULTS: Analysis of photon emissions from the brains of living animals revealed marked sex differences in astrocyte response to ischemic injury. The increase in GFAP signals was significantly higher in female mice in the metestrus/diestrus period compared with male transgenic mice (1.71 x 10(7)+/-0.19 x 10(7) vs 0.92 x 10(7)+/-0.15 x 10(7), P<0.001). Similar results were obtained by quantitative immunohistochemistry (males vs females: 13.4+/-0.5 vs 16.96+/-0.64, P<0.0001). However, astrocyte activation/GFAP signals showed cyclic, estrus-dependent variations in response to ischemic injury. Physiologically higher levels of estrogen and application of pharmacologic doses of estrogen during replacement therapy attenuated GFAP upregulation after stroke. Interestingly, contrary to a positive correlation between the intensities of GFAP signals and infarct size in male mice, no such correlation was observed in any of the experimental groups of female GFAP-luc mice. CONCLUSIONS: Our results suggest that GFAP upregulation in ischemic injury may have different functional significance in female and male experimental animals and may not directly reflect the extent of ischemia-induced neuronal damage in female GFAP-luc mice. Using a novel live imaging approach, we demonstrated that the early-phase brain inflammatory response to ischemia may be associated with sex-specific biomarkers of brain damage.

Real-time imaging of astrocyte response to quantum dots: in vivo screening model system for biocompatibility of nanoparticles.

Astrocytes are the principle macroglial brain cells. They are activated by different stressors and brain injuries. Quantum dots (QDs) can cause oxidative stress. This study shows a real-time imaging of primary cortical cultures and assessment of QD-induced activation of astrocytes in the brains of transgenic mice with the luciferase gene driven by the murine astrocyte promoter. This approach may be widely applicable for assessing the astroglia/tissue response to nanoparticles in live animals.