Microglial TNFR2 signaling regulates the inflammatory response after CNS injury in a sex-specific fashion.

Microglia, the resident immune cells of the central nervous system (CNS), play a major role in damage progression and tissue remodeling after acute CNS injury, including ischemic stroke (IS) and spinal cord injury (SCI). Understanding the molecular mechanisms regulating microglial responses to injury may thus reveal novel therapeutic targets to promote CNS repair. Here, we investigated the role of microglial tumor necrosis factor receptor 2 (TNFR2), a transmembrane receptor previously associated with pro-survival and neuroprotective responses, in shaping the neuroinflammatory environment after CNS injury. By inducing experimental IS and SCI in Cx3cr1CreER:Tnfrsf1bfl/fl mice, selectively lacking TNFR2 in microglia, and corresponding Tnfrsf1bfl/fl littermate controls, we found that ablation of microglial TNFR2 significantly reduces lesion size and pro-inflammatory cytokine levels, and favors infiltration of leukocytes after injury. Interestingly, these effects were paralleled by opposite sex-specific modifications of microglial reactivity, which was found to be limited in female TNFR2-ablated mice compared to controls, whereas it was enhanced in males. In addition, we show that TNFR2 protein levels in the cerebrospinal fluid (CSF) of human subjects affected by IS and SCI, as well as healthy donors, significantly correlate with disease stage and severity, representing a valuable tool to monitor the inflammatory response after acute CNS injury. Hence, these results advance our understanding of the mechanisms regulating microglia reactivity after acute CNS injury, aiding the development of sex- and microglia-specific, personalized neuroregenerative strategies.

A preclinical randomized controlled multi-centre trial of anti-interleukin-17A treatment for acute ischaemic stroke.

Multiple consensus statements have called for preclinical randomized controlled trials to improve translation in stroke research. We investigated the efficacy of an interleukin-17A neutralizing antibody in a multi-centre preclinical randomized controlled trial using a murine ischaemia reperfusion stroke model. Twelve-week-old male C57BL/6 mice were subjected to 45 min of transient middle cerebral artery occlusion in four centres. Mice were randomly assigned (1:1) to receive either an anti-interleukin-17A (500 µg) or isotype antibody (500 µg) intravenously 1 h after reperfusion. The primary endpoint was infarct volume measured by magnetic resonance imaging three days after transient middle cerebral artery occlusion. Secondary analysis included mortality, neurological score, neutrophil infiltration and the impact of the gut microbiome on treatment effects. Out of 136 mice, 109 mice were included in the analysis of the primary endpoint. Mixed model analysis revealed that interleukin-17A neutralization significantly reduced infarct sizes (anti-interleukin-17A: 61.77 ± 31.04 mm3; IgG control: 75.66 ± 34.79 mm3; P = 0.01). Secondary outcome measures showed a decrease in mortality (hazard ratio = 3.43, 95% confidence interval = 1.157-10.18; P = 0.04) and neutrophil invasion into ischaemic cortices (anti-interleukin-17A: 7222 ± 6108 cells; IgG control: 28 153 ± 23 206 cells; P < 0.01). There was no difference in Bederson score. The analysis of the gut microbiome showed significant heterogeneity between centres (R = 0.78, P < 0.001, n = 40). Taken together, neutralization of interleukin-17A in a therapeutic time window resulted in a significant reduction of infarct sizes and mortality compared with isotype control. It suggests interleukin-17A neutralization as a potential therapeutic target in stroke.

The GHB analogue HOCPCA improves deficits in cognition and sensorimotor function after MCAO via CaMKIIα.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented γ-hydroxybutyrate (GHB) ligands as the first small molecules selectively targeting and stabilizing the CaMKIIα hub domain. Here, we report that the cyclic GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), improves sensorimotor function after experimental stroke in mice when administered at a clinically relevant time and in combination with alteplase. Further, we observed improved hippocampal neuronal activity and working memory after stroke. On the biochemical level, we observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated ischemia-specific expression of a constitutively active CaMKII kinase proteolytic fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. Similarly, HOCPCA's effects on dampening inflammatory changes require further investigation as an underlying protective mechanism. HOCPCA's selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy.

Interleukin-10 improves stroke outcome by controlling the detrimental Interleukin-17A response.

BACKGROUND: Lymphocytes have dichotomous functions in ischemic stroke. Regulatory T cells are protective, while IL-17A from innate lymphocytes promotes the infarct growth. With recent advances of T cell-subtype specific transgenic mouse models it now has become possible to study the complex interplay of T cell subpopulations in ischemic stroke. METHODS: In a murine model of experimental stroke we analyzed the effects of IL-10 on the functional outcome for up to 14 days post-ischemia and defined the source of IL-10 in ischemic brains based on immunohistochemistry, flow cytometry, and bone-marrow chimeric mice. We used neutralizing IL-17A antibodies, intrathecal IL-10 injections, and transgenic mouse models which harbor a deletion of the IL-10R on distinct T cell subpopulations to further explore the interplay between IL-10 and IL-17A pathways in the ischemic brain. RESULTS: We demonstrate that IL-10 deficient mice exhibit significantly increased infarct sizes on days 3 and 7 and enlarged brain atrophy and impaired neurological outcome on day 14 following tMCAO. In ischemic brains IL-10 producing immune cells included regulatory T cells, macrophages, and microglia. Neutralization of IL-17A following stroke reversed the worse outcome in IL-10 deficient mice and intracerebral treatment with recombinant IL-10 revealed that IL-10 controlled IL-17A positive lymphocytes in ischemic brains. Importantly, IL-10 acted differentially on αß and γδ T cells. IL-17A producing CD4+ αß T cells were directly controlled via their IL-10-receptor (IL-10R), whereas IL-10 by itself had no direct effect on the IL-17A production in γδ T cells. The control of the IL-17A production in γδ T cells depended on an intact IL10R signaling in regulatory T cells (Tregs). CONCLUSIONS: Taken together, our data indicate a key function of IL-10 in restricting the detrimental IL-17A-signaling in stroke and further supports that IL-17A is a therapeutic opportunity for stroke treatment.

Characterization of the TNF and IL-1 systems in human brain and blood after ischemic stroke.

Preclinical and clinical proof-of-concept studies have suggested the effectiveness of pharmacological modulation of inflammatory cytokines in ischemic stroke. Experimental evidence shows that targeting tumor necrosis factor (TNF) and interleukin (IL)-1 holds promise, and these cytokines are considered prime targets in the development of new stroke therapies. So far, however, information on the cellular expression of TNF and IL-1 in the human ischemic brain is sparse.We studied 14 cases of human post-mortem ischemic stroke, representing 21 specimens of infarcts aged 1 to > 8 days. We characterized glial and leukocyte reactions in the infarct/peri-infarct (I/PI) and normal-appearing tissue (NAT) and the cellular location of TNF, TNF receptor (TNFR)1 and TNFR2, IL-1α, IL-1ß, and IL-1 receptor antagonist (IL-1Ra). The immunohistochemically stained tissue sections received a score reflecting the number of immunoreactive cells and the intensity of the immunoreactivity (IR) in individual cells where 0 = no immunoreactive cells, 1 = many intermediately to strongly immunoreactive cells, and 2 = numerous and intensively immunoreactive cells. Additionally, we measured blood TNF, TNFR, and IL-1 levels in surviving ischemic stroke patients within the first 8 h and again at 72 h after symptom onset and compared levels to healthy controls.We observed IL-1α and IL-1ß IR in neurons, glia, and macrophages in all specimens. IL-1Ra IR was found in glia, in addition to macrophages. TNF IR was initially found in neurons located in I/PI and NAT but increased in glia in older infarcts. TNF IR increased in macrophages in all specimens. TNFR1 IR was found in neurons and glia and macrophages, while TNFR2 was expressed only by glia in I/PI and NAT, and by macrophages in I/PI. Our results suggest that TNF and IL-1 are expressed by subsets of cells and that TNFR2 is expressed in areas with increased astrocytic reactivity. In ischemic stroke patients, we demonstrate that plasma TNFR1 and TNFR2 levels increased in the acute phase after symptom onset compared to healthy controls, whereas TNF, IL-1α, IL-1ß, and IL-1Ra did not change.Our findings of increased brain cytokines and plasma TNFR1 and TNFR2 support the hypothesis that targeting post-stroke inflammation could be a promising add-on therapy in ischemic stroke patients.

Acute Neurofilament Light Chain Plasma Levels Correlate With Stroke Severity and Clinical Outcome in Ischemic Stroke Patients.

Background: Ischemic stroke causes increased blood-brain barrier permeability and release of markers of axonal damage and inflammation. To investigate diagnostic and prognostic roles of neurofilament light chain (NF-L), we assessed levels of NF-L, S100B, interleukin-6 (IL-6), E-selectin, vascular endothelial growth factor-A (VEGF-A), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in patients with acute ischemic stroke or transient ischemic attack (TIA) and healthy controls. Methods: We studied neurofilament (NF) expression in 2 cases of human postmortem ischemic stroke, representing infarcts aged 3- to >7-days. In a prospective study, we measured plasma NF-L and inflammatory markers <8 h of symptom onset and at 72 h in acute ischemic stroke (n = 31), TIA (n = 9), and healthy controls (n = 29). We assessed whether NF-L, S100B, and IL-6 were associated with clinical severity on admission (Scandinavian Stroke Scale, SSS), diagnosis of ischemic stroke vs. TIA, and functional outcome at 3 months (modified Rankin Scale, mRS). Results: NF expression increased in ischemic neurons and in the infarcted brain parenchyma after stroke. Plasma NF-L levels were higher in stroke patients than in TIA patients and healthy controls, but IL-6 levels were similar. Higher acute NF-L levels were associated with lower SSS scores at admission and higher mRS scores at 3 months. No correlation was observed between NF-L and S100B, NF-L and IL-6, nor between S100B or IL-6 and SSS or mRS. Compared to controls, stroke patients had significantly higher VEGF-A and VCAM-1 at <8 h that remained elevated at 72 h, with significantly higher VEGF-A at <8 h; ICAM-1 was significantly increased at <8 h, while S100B and E-selectin were unchanged. Conclusions: Plasma NF-L levels, but not IL-6 and S100B, were significant predictors of clinical severity on admission and functional outcome at 3 months. Plasma NF-L is a promising biomarker of functional outcome after ischemic stroke.

Oligodendrocytes modulate the immune-inflammatory response in EAE via TNFR2 signaling.

The pleotropic cytokine tumor necrosis factor (TNF) is involved in the pathophysiology of multiple sclerosis (MS). In various models of MS, including experimental autoimmune encephalomyelitis (EAE), the membrane-bound form of TNF (tmTNF), which signals primarily via TNFR2, mediates protective and reparative effects, whereas the soluble form (solTNF), which signals primarily via TNFR1, promotes pro-inflammatory and detrimental functions. In this study, we investigated the role of TNFR2 expressed in oligodendrocytes in the early phase of EAE pathogenesis. We demonstrated that mice with specific ablation of oligodendroglial TNFR2 displayed early onset and higher peak of motor dysfunction when subjected to EAE, in advance of which accelerated infiltration of immune cells was observed as early as 10 days post EAE induction. The immune cell influx was preceded by microglial activation and increased blood brain barrier permeability. Lack of oligodendroglial TNFR2 accelerated the expression of inflammatory cytokines as well as expression and activation of the inflammasome. Gene expression profiling of oligodendrocytes sorted from the spinal cord 14 days post EAE induction showed robust upregulation of inflammatory genes, some of which were elevated in cells lacking TNFR2 compared to controls. Together, our data demonstrate that oligodendrocytes are directly involved in inflammation and immune modulation in CNS disease and this function is regulated, at least in part, by TNFR2.

Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor.

Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials.

Cortical Morphogenesis during Embryonic Development Is Regulated by miR-34c and miR-204.

The porcine brain closely resembles the human brain in aspects such as development and morphology. Temporal miRNA profiling in the developing embryonic porcine cortex revealed a distinct set of miRNAs, including miR-34c and miR-204, which exhibited a highly specific expression profile across the time of cortical folding. These miRNAs were found to target Doublecortin (DCX), known to be involved in neuron migration during cortical folding of gyrencephalic brains. In vivo modulation of miRNA expression in mouse embryos confirmed that miR-34c and miR-204 can control neuronal migration and cortical morphogenesis, presumably by posttranscriptional regulation of DCX.

Mass spectrometry imaging of biomarker lipids for phagocytosis and signalling during focal cerebral ischaemia.

Focal cerebral ischaemia has an initial phase of inflammation and tissue injury followed by a later phase of resolution and repair. Mass spectrometry imaging (desorption electrospray ionization and matrix assisted laser desorption ionization) was applied on brain sections from mice 2 h, 24 h, 5d, 7d, and 20d after permanent focal cerebral ischaemia. Within 24 h, N-acyl-phosphatidylethanolamines, lysophosphatidylcholine, and ceramide accumulated, while sphingomyelin disappeared. At the later resolution stages, bis(monoacylglycero)phosphate (BMP(22:6/22:6)), 2-arachidonoyl-glycerol, ceramide-phosphate, sphingosine-1-phosphate, lysophosphatidylserine, and cholesteryl ester appeared. At day 5 to 7, dihydroxy derivates of docosahexaenoic and docosapentaenoic acid, some of which may be pro-resolving mediators, e.g. resolvins, were found in the injured area, and BMP(22:6/22:6) co-localized with the macrophage biomarker CD11b, and probably with cholesteryl ester. Mass spectrometry imaging can visualize spatiotemporal changes in the lipidome during the progression and resolution of focal cerebral inflammation and suggests that BMP(22:6/22:6) and N-acyl-phosphatidylethanolamines can be used as biomarkers for phagocytizing macrophages/microglia cells and dead neurones, respectively.

Angiotensin AT2-receptor stimulation improves survival and neurological outcome after experimental stroke in mice.

This study investigated the effect of post-stroke, direct AT2-receptor (AT2R) stimulation with the non-peptide AT2R-agonist compound 21 (C21) on infarct size, survival and neurological outcome after middle cerebral artery occlusion (MCAO) in mice and looked for potential underlying mechanisms. C57/BL6J or AT2R-knockout mice (AT2-KO) underwent MCAO for 30 min followed by reperfusion. Starting 45 min after MCAO, mice were treated once daily for 4 days with either vehicle or C21 (0.03 mg/kg ip). Neurological deficits were scored daily. Infarct volumes were measured 96 h post-stroke by MRI. C21 significantly improved survival after MCAO when compared to vehicle-treated mice. C21 treatment had no impact on infarct size, but significantly attenuated neurological deficits. Expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB) (receptor for BDNF) and growth-associated protein 43 (GAP-43) were significantly increased in the peri-infarct cortex of C21-treated mice when compared to vehicle-treated mice. Furthermore, the number of apoptotic neurons was significantly decreased in the peri-infarct cortex in mice treated with C21 compared to controls. There were no effects of C21 on neurological outcome, infarct size and expression of BDNF or GAP-43 in AT2-KO mice. From these data, it can be concluded that AT2R stimulation attenuates early mortality and neurological deficits after experimental stroke through neuroprotective mechanisms in an AT2R-specific way. Key message • AT2R stimulation after MCAO in mice reduces mortality and neurological deficits.• AT2R stimulation increases BDNF synthesis and protects neurons from apoptosis.• The AT2R-agonist C21 acts protectively when applied post-stroke and peripherally.

Glutamate-system defects behind psychiatric manifestations in a familial hemiplegic migraine type 2 disease-mutation mouse model.

Migraine is a complex brain disorder, and understanding the complexity of this prevalent disease could improve quality of life for millions of people. Familial Hemiplegic Migraine type 2 (FHM2) is a subtype of migraine with aura and co-morbidities like epilepsy/seizures, cognitive impairments and psychiatric manifestations, such as obsessive-compulsive disorder (OCD). FHM2 disease-mutations locate to the ATP1A2 gene encoding the astrocyte-located α2-isoform of the sodium-potassium pump (α2Na(+)/K(+)-ATPase). We show that knock-in mice heterozygous for the FHM2-associated G301R-mutation (α2(+/G301R)) phenocopy several FHM2-relevant disease traits e.g., by mimicking mood depression and OCD. In vitro studies showed impaired glutamate uptake in hippocampal mixed astrocyte-neuron cultures from α2(G301R/G301R) E17 embryonic mice, and moreover, induction of cortical spreading depression (CSD) resulted in reduced recovery in α2(+/G301R) male mice. Moreover, NMDA-type glutamate receptor antagonists or progestin-only treatment reverted specific α2(+/G301R) behavioral phenotypes. Our findings demonstrate that studies of an in vivo relevant FHM2 disease knock-in mouse model provide a link between the female sex hormone cycle and the glutamate system and a link to co-morbid psychiatric manifestations of FHM2.

Myelin-specific T cells induce interleukin-1beta expression in lesion-reactive microglial-like cells in zones of axonal degeneration.

Infiltration of myelin-specific T cells into the central nervous system induces the expression of proinflammatory cytokines in patients with multiple sclerosis (MS). We have previously shown that myelin-specific T cells are recruited into zones of axonal degeneration, where they stimulate lesion-reactive microglia. To gain mechanistic insight, we used RNA microarray analysis to compare the transcript profile in hippocampi from perforant pathway axonal-lesioned mice with and without adoptively transferred myelin-specific T cells 2 days postlesion, when microglia are clearly lesion reactive. Pathway analysis revealed that, among the 1,447 differently expressed transcripts, the interleukin (IL)-1 pathway including all IL-1 receptor ligands was upregulated in the presence of myelin-specific T cells. Quantitative polymerase chain reaction showed increased mRNA levels of IL-1ß, IL-1α, and IL-1 receptor antagonist in the T-cell-infiltrated hippocampi from axonal-lesioned mice. In situ hybridization and immunohistochemistry showed a T-cell-enhanced lesion-specific expression of IL-1ß mRNA and protein, respectively, and induction of the apoptosis-associated speck-like protein, ASC, in CD11b(+) cells. Double in situ hybridization showed colocalization of IL-1ß mRNA in a subset of CD11b mRNA(+) cells, of which many were part of cellular doublets or clusters, characteristic of proliferating, lesion-reactive microglia. Double-immunofluorescence showed a T-cell-enhanced colocalization of IL-1ß to CD11b(+) cells, including lesion-reactive CD11b(+) ramified microglia. These results suggest that myelin-specific T cells stimulate lesion-reactive microglial-like cells to produce IL-1ß. These findings are relevant to understand the consequences of T-cell infiltration in white and gray matter lesions in patients with MS.

Genetic ablation of soluble tumor necrosis factor with preservation of membrane tumor necrosis factor is associated with neuroprotection after focal cerebral ischemia.

Microglia respond to focal cerebral ischemia by increasing their production of the neuromodulatory cytokine tumor necrosis factor, which exists both as membrane-anchored tumor necrosis factor and as cleaved soluble tumor necrosis factor forms. We previously demonstrated that tumor necrosis factor knockout mice display increased lesion volume after focal cerebral ischemia, suggesting that tumor necrosis factor is neuroprotective in experimental stroke. Here, we extend our studies to show that mice with intact membrane-anchored tumor necrosis factor, but no soluble tumor necrosis factor, display reduced infarct volumes at one and five days after stroke. This was associated with improved functional outcome after experimental stroke. No changes were found in the mRNA levels of tumor necrosis factor and tumor necrosis factor-related genes (TNFR1, TNFR2, TACE), pro-inflammatory cytokines (IL-1ß, IL-6) or chemokines (CXCL1, CXCL10, CCL2); however, protein expression of TNF, IL-1ß, IL-6 and CXCL1 was reduced in membrane-anchored tumor necrosis factor(Δ/Δ) compared to membrane-anchored tumor necrosis factor(wt/wt) mice one day after experimental stroke. This was paralleled by reduced MHCII expression and a reduction in macrophage infiltration in the ipsilateral cortex of membrane-anchored tumor necrosis factor(Δ/Δ) mice. Collectively, these findings indicate that membrane-anchored tumor necrosis factor mediates the protective effects of tumor necrosis factor signaling in experimental stroke, and therapeutic strategies specifically targeting soluble tumor necrosis factor could be beneficial in clinical stroke therapy.

Spatio-temporal regulation of circular RNA expression during porcine embryonic brain development.

BACKGROUND: Recently, thousands of circular RNAs (circRNAs) have been discovered in various tissues and cell types from human, mouse, fruit fly and nematodes. However, expression of circRNAs across mammalian brain development has never been examined. RESULTS: Here we profile the expression of circRNA in five brain tissues at up to six time-points during fetal porcine development, constituting the first report of circRNA in the brain development of a large animal. An unbiased analysis reveals a highly complex regulation pattern of thousands of circular RNAs, with a distinct spatio-temporal expression profile. The amount and complexity of circRNA expression was most pronounced in cortex at day 60 of gestation. At this time-point we find 4634 unique circRNAs expressed from 2195 genes out of a total of 13,854 expressed genes. Approximately 20 % of the porcine splice sites involved in circRNA production are functionally conserved between mouse and human. Furthermore, we observe that "hot-spot" genes produce multiple circRNA isoforms, which are often differentially expressed across porcine brain development. A global comparison of porcine circRNAs reveals that introns flanking circularized exons are longer than average and more frequently contain proximal complementary SINEs, which potentially can facilitate base pairing between the flanking introns. Finally, we report the first use of RNase R treatment in combination with in situ hybridization to show dynamic subcellular localization of circRNA during development. CONCLUSIONS: These data demonstrate that circRNAs are highly abundant and dynamically expressed in a spatio-temporal manner in porcine fetal brain, suggesting important functions during mammalian brain development.

Endogenous IFN-ß signaling exerts anti-inflammatory actions in experimentally induced focal cerebral ischemia.

BACKGROUND: Interferon (IFN)-ß exerts anti-inflammatory effects, coupled to remarkable neurological improvements in multiple sclerosis, a neuroinflammatory condition of the central nervous system. Analogously, it has been hypothesized that IFN-ß, by limiting inflammation, decreases neuronal death and promotes functional recovery after stroke. However, the core actions of endogenous IFN-ß signaling in stroke are unclear. METHODS: To address this question, we used two clinically relevant models of focal cerebral ischemia, transient and permanent middle cerebral artery occlusion, and two genetically modified mouse lines, lacking either IFN-ß or its receptor, the IFN-α/ß receptor. Subsets of inflammatory and immune cells isolated from the brain, blood, and spleen were studied using flow cytometry. Sensorimotor deficits were assessed by a modified composite neuroscore, the rotating pole and grip strength tests, and cerebral infarct volumes were given by lack of neuronal nuclei immunoreactivity. RESULTS: Here, we report alterations in local and systemic inflammation in IFN-ß knockout (IFN-ßKO) mice over 8 days after induction of focal cerebral ischemia. Notably, IFN-ßKO mice showed a higher number of infiltrating leukocytes in the brain 2 days after stroke. Concomitantly, in the blood of IFN-ßKO mice, we found a higher percentage of total B cells but a similar percentage of mature and activated B cells, collectively indicating a higher proliferation rate. The additional differential regulation of circulating cytokines and splenic immune cell populations in wild-type and IFN-ßKO mice further supports an important immunoregulatory function of IFN-ß in stroke. Moreover, we observed a significant weight loss 2-3 days and a reduction in grip strength 2 days after stroke in the IFN-ßKO group, while endogenous IFN-ß signaling did not affect the infarct volume. CONCLUSIONS: We conclude that endogenous IFN-ß signaling attenuates local inflammation, regulates peripheral immune cells, and, thereby, may contribute positively to stroke outcome.

Preserved Cerebral Microcirculation After Cardiac Arrest in a Rat Model.

OBJECTIVE: Recent studies show that sublingual microcirculation is altered in patients resuscitated from CA. The objective of this study was to investigate whether the cerebral microcirculation is disturbed in the early post-resuscitation period. METHODS: Male Sprague-Dawley rats were randomized to either 10 minutes of CA or uninterrupted circulation, and observed to 120 or 360 minutes after ROSC. At 120 and 360 minutes, cerebral microcirculation was evaluated by SDF microscopy through a craniectomy. Plasma samples were analyzed for endothelial adhesion molecules and inflammatory markers, and brains were fixated for histological analysis. RESULTS: Cerebral microcirculation, evaluated by TVD, PVD, PPV, and MFI did not differ between groups (p > 0.16). Plasma samples drawn 360 minutes after ROSC displayed a significant increase in sE-selectin, sL-selectin, sI-CAM1, IL-1ß, IL-6, IL-10, and elastase compared to controls. In the CA animals, sE-selectin and elastase increased between 120 and 360 minutes after resuscitation (p < 0.007). Histological analysis revealed neuronal death in hippocampus layer CA1 360 min after resuscitation. CONCLUSION: When evaluated by SDF, the cerebral microcirculation appears unaffected in the early post-CA period despite hypotension, systemic inflammation, endothelial activation, and neuronal injury.

Cytokine-producing microglia have an altered beta-amyloid load in aged APP/PS1 Tg mice.

Beta-amyloid (Aß) plaques and chronic neuroinflammation are significant neuropathological features of Alzheimer's disease. Microglial cells in aged brains have potential to produce cytokines such as TNF and IL-1 family members (IL-1α, IL-1ß, and IL-1Ra) and to phagocytose Aß in Alzheimer's disease, however the inter-relationship between these processes is poorly understood. Here we show that % Aß plaque load followed a sigmoidal trajectory with age in the neocortex of APPswe/PS1ΔE9 Tg mice, and correlated positively with soluble Aß40 and Aß42. Aß measures were moderately correlated with mRNA levels of CD11b, TNF, and IL-1Ra. Cytokine production and Aß load were assessed in neocortical CD11b(+)(CD45(+)) microglia by flow cytometry. Whereas most microglia in aged mice produced IL-1Ra, relatively low proportions of microglia produced TNF, IL-1α, and IL-1ß. However, microglial production of these latter cytokines was generally increased in APP/PS1 Tg mice. Microglia that phagocytosed endogenously-produced Aß were only observed in APP/PS1 Tg mice. Differences in phagocytic index and total Aß load were observed in microglia with specific cytokine profiles. Both phagocytic index and total Aß load were higher in IL-1α(+) and IL-1Ra(+) microglia, than microglia that did not produce these cytokines. In contrast, total Aß load was lower in IL-1ß(+) and TNF(+) microglia, compared to IL-1ß(-) and TNF(-) microglia, and TNF(+) microglia also had a lower phagocytic index. Using GFP bone marrow chimeric mice, we confirmed that the majority of neocortical CD11b(+)(CD45(+)) microglia were resident cells (GFP(-)) in APP/PS1 Tg mice, even after selectively analysing CD11b(+)CD45(high) cells, which are typically considered to be infiltrating cells. Together, our data demonstrate that cytokine expression is selectively correlated with age and Aß pathology, and is associated with an altered Aß load in phagocytic microglia from APP/PS1 Tg mice. These findings have implications for understanding the regulation of microglial cytokine production and phagocytosis of Aß in Alzheimer's disease.

No effect of ablation of surfactant protein-D on acute cerebral infarction in mice.

BACKGROUND: Crosstalk between the immune system in the brain and the periphery may contribute to the long-term outcome both in experimental and clinical stroke. Although, the immune defense collectin surfactant protein-D (SP-D) is best known for its role in pulmonary innate immunity, SP-D is also known to be involved in extrapulmonary modulation of inflammation in mice. We investigated whether SP-D affected cerebral ischemic infarction and ischemia-induced inflammatory responses in mice. METHODS: The effect of SP-D was studied by comparing the size of ischemic infarction and the inflammatory and astroglial responses in SP-D knock out (KO) and wild type (WT) mice subjected to permanent middle cerebral artery occlusion. SP-D mRNA production was assessed in isolated cerebral arteries and in the whole brain by PCR, and SP-D protein in normal appearing and ischemic human brain by immunohistochemistry. Changes in plasma SP-D and TNF were assessed by ELISA and proximity ligation assay, respectively. RESULTS: Infarct volumetric analysis showed that ablation of SP-D had no effect on ischemic infarction one and five days after induction of ischemia. Further, ablation of SP-D had no effect on the ischemia-induced increase in TNF mRNA production one day after induction of ischemia; however the TNF response to the ischemic insult was affected at five days. SP-D mRNA was not detected in parenchymal brain cells in either naïve mice or in mice subjected to focal cerebral ischemia. However, SP-D mRNA was detected in middle cerebral artery cells in WT mice and SP-D protein in vascular cells both in normal appearing and ischemic human brain tissue. Measurements of the levels of SP-D and TNF in plasma in mice suggested that levels were unaffected by the ischemic insult. Microglial-leukocyte and astroglial responses were comparable in SP-D KO and WT mice. CONCLUSIONS: SP-D synthesis in middle cerebral artery cells is consistent with SP-D conceivably leaking into the infarcted area and affecting local cytokine production. However, there was no SP-D synthesis in parenchymal brain cells and ablation of SP-D had no effect on ischemic cerebral infarction.