Protein Expression of the Microglial Marker Tmem119 Decreases in Association With Morphological Changes and Location in a Mouse Model of Traumatic Brain Injury.

The activation of microglia and the infiltration of macrophages are hallmarks of neuroinflammation after acute brain injuries, including traumatic brain injury (TBI). The two myeloid populations share many features in the post-injury inflammatory response, thus, being antigenically indistinguishable. Recently Tmem119, a type I transmembrane protein specifically expressed by microglia under physiological conditions, was proposed as a tool to differentiate resident microglia from blood-borne macrophages, not expressing it. However, the validity of Tmem119 as a specific marker of resident microglia in the context of acute brain injury, where microglia are activated and macrophages are recruited, needs validation. Our purpose was to investigate Tmem119 expression and distribution in relation to the morphology of brain myeloid cells present in the injured area after TBI. Mice underwent sham surgery or TBI by controlled cortical impact (CCI). Brains from sham-operated, or TBI mice, were analyzed by in situ hybridization to identify the cells expressing Tmem119, and by Western blot and quantitative immunofluorescence to measure Tmem119 protein levels in the entire brain regions and single cells. The morphology of Iba1+ myeloid cells was analyzed at different times (4 and 7 days after TBI) and several distances from the contused edge in order to associate Tmem119 expression with morphological evolution of active microglia. In situ hybridization indicated an increased Tmem119 RNA along with increased microglial complement C1q activation in the contused area and surrounding regions. On the contrary, the biochemical evaluation showed a drop in Tmem119 protein levels in the same areas. The Tmem119 immunoreactivity decreased in Iba1+ myeloid cells found in the contused cortex at both time points, with the cells showing the hypertrophic ameboid morphology having no Tmem119 expression. The Tmem119 was present on ramifications of resident microglia and its presence was decreased as a consequence of microglial activation in cortical areas close to contusion. Based on the data, we conclude that the decrease of Tmem119 in reactive microglia may depend on the process of microglial activation, which involves the retracting of their branchings to acquire an ameboid shape. The Tmem119 immunoreactivity decreases in reactive microglia to similar levels than the blood-borne macrophages, thus, failing to discriminate the two myeloid populations after TBI.

ß2 glycoprotein I participates in phagocytosis of apoptotic neurons and in vascular injury in experimental brain stroke.

Beta-2 Glycoprotein I (ß2-GPI) is the main target of anti-phospholipid antibodies (aPL) in the autoimmune anti-phospholipid syndrome, characterized by increased risk of stroke. We here investigated the antibody independent role of ß2-GPI after ischemia/reperfusion, modeled in vivo by transient middle cerebral artery occlusion (tMCAo) in male C57Bl/6J mice; in vitro by subjecting immortalized human brain microvascular endothelial cells (ihBMEC) to 16 h hypoxia and 4 h re-oxygenation. ApoH (coding for ß2-GPI) was upregulated selectively in the liver at 48 h after tMCAo. At the same time ß2-GPI circulating levels increased. ß2-GPI was detectable in brain parenchyma and endothelium at all time points after tMCAo. Parenchymal ß2-GPI recognized apoptotic neurons (positive for annexin V, C3 and TUNEL) cleared by CD68+ brain macrophages. Hypoxic ihBMEC showed increased release of IL-6, over-expression of thrombomodulin and IL-1α after re-oxygenation with ß2-GPI alone. ß2-GPI interacted with mannose-binding lectin in mouse plasma and ihBMEC medium, potentially involved in formation of thrombi. We show for the first time that brain ischemia triggers the hepatic production of ß2-GPI. ß2-GPI is present in the ischemic endothelium, enhancing vascular inflammation, and extravasates binding stressed neurons before their clearance by phagocytosis. Thus ß2-GPI may be a new mediator of brain injury following ischemic stroke.

Ficolin-2 serum levels predict the occurrence of acute coronary syndrome in patients with severe carotid artery stenosis.

BACKGROUND AND PURPOSE: erosion of vulnerable atherosclerotic plaques may cause life-threatening thromboembolic complications. There is indeed an urgent need to recognize a clear-cut biomarker able to identify vulnerable plaques. Here, we focused on circulating proteins belonging to the lectin pathway (LP) of complement activation. METHODS: we analyzed mannose-binding lectin (MBL), ficolin-1, -2 and -3 (LP initiators) levels by ELISA in sera from n = 240 of an already published cohort of patients undergoing endarterectomy for severe carotid stenosis and followed-up until 18 months after surgery. Immunofluorescence followed by confocal and polarized light microscopy was used to detect LP initiator intraplaque localization. Spearman's rank test was drawn to investigate correlation between serum LP levels and circulating inflammatory proteins or intraplaque components. Survival analyses were then performed to test the predictive role of LP on long-term adverse outcome. RESULTS: ficolins, but not MBL, correlated positively with 1) high circulating levels of inflammatory markers, including MPO, MMP-8, MMP-9, ICAM-1, osteopontin, neutrophil elastase, and; 2) immune cell intraplaque recruitment. Immunofluorescence showed ficolins in calcified plaques and ficolin-2 in cholesterol-enriched plaque regions in association with macrophages. In the multivariate survival analysis, ficolin-2 serum levels predicted a major adverse cardiovascular event during the follow-up, independently of symptomatic status and inflammatory markers (hazard ratio 38.6 [95 % CI 3.9-385.2]). CONCLUSIONS: ficolins support intraplaque immune cell recruitment and inflammatory processes ultimately leading to plaque vulnerability. Especially for ficolin-2 a strong predictive value toward adverse cardiovascular events was demonstrated. This evidence offers potentially new pharmacological target to dampen the inflammatory mechanisms leading to plaque vulnerability.

Multicentre translational Trial of Remote Ischaemic Conditioning in Acute Ischaemic Stroke (TRICS): protocol of multicentre, parallel group, randomised, preclinical trial in female and male rat and mouse from the Italian Stroke Organization (ISO) Basic Science network.

INTRODUCTION: Multicentre preclinical randomised controlled trials (pRCT) are emerging as a necessary step to confirm efficacy and improve translation into the clinic. The aim of this project is to perform two multicentre pRCTs (one in rats and one in mice) to investigate the efficacy of remote ischaemic conditioning (RIC) in an experimental model of severe ischaemic stroke. METHODS AND ANALYSIS: Seven research laboratories within the Italian Stroke Organization (ISO) Basic Science network will participate in the study. Transient endovascular occlusion of the proximal right middle cerebral artery will be performed in two species (rats and mice) and in both sexes. Animals will be randomised to receive RIC by transient surgical occlusion of the right femoral artery, or sham surgery, after reperfusion. Blinded outcome assessment will be performed for dichotomised functional neuroscore (primary endpoint) and infarct volume (secondary endpoint) at 48 hours. A sample size of 80 animals per species will yield 82% power to detect a significant difference of 30% in the primary outcome in both pRCTs. Analyses will be performed in a blind status and according to an intention-to-treat paradigm. The results of this study will provide robust, translationally oriented, high-quality evidence on the efficacy of RIC in multiple species of rodents with large ischaemic stroke. ETHICS AND DISSEMINATION: This is approved by the Animal Welfare Regulatory Body of the University of Milano Bicocca, under project license from the Italian Ministry of Health. Trial results will be subject to publication according to the definition of the outcome presented in this protocol. TRIAL REGISTRATION NUMBER: PCTE0000177.

Combined Genetic Deletion of IL (Interleukin)-4, IL-5, IL-9, and IL-13 Does Not Affect Ischemic Brain Injury in Mice.

Background and Purpose- After ischemic injury, microglia and infiltrated macrophages may acquire different polarization phenotypes promoting inflammation and injury (M1) or repair and protection (M2). There is evidence that immunomodulation, via type 2 helper T-cells (Th2) cytokines, exerts neuroprotection after ischemia. We investigated the consequences of simultaneous genetic deletion of Th2 cytokines (IL [interleukin]-4, IL-5, IL-9, IL-13) on the histopathologic outcome, microglia and infiltrated macrophages markers, and ischemic microenvironment at different time points after ischemic injury in mice subjected to permanent occlusion of the middle cerebral artery. Methods- Wild-type and Th2 cytokine-deficient mice (4KO) were subjected to permanent occlusion of the middle cerebral artery by electrocoagulation and followed up to 5 weeks after permanent occlusion of the middle cerebral artery. Neuropathologic outcome was assessed at 24 hours (n=6), 7 days (n=6), and 5 weeks (n=6-7) by examination of the ischemic lesion, neuronal count, microglia and infiltrated macrophages markers, brain atrophy, collagen deposition, and GFAP (glial fibrillary acidic protein) immunohistochemistry. Selected gene expression was investigated at 7 days (n=6). Results- 4KO mice showed no difference in lesion and neuronal count 7 days and up to 5 weeks after permanent occlusion of the middle cerebral artery compared with wild type. Ischemic 4KO mice had lower CD16/32 expression at 24 hours, lower CD11b and CD16/32 expression at 7 days than wild type. They had higher CD206 expression at 24 hours, higher CD206 and arginase1 at 7 days, and increased mRNA for CXCL9 (chemokine [C-X-C motif] ligand 9) compared with wild type. Additional histopathologic analysis, including brain atrophy, gliotic scar, and collagenous scar confirmed no difference between genotypes at 5 weeks. Conclusions- This study casts light on the proposed neuroprotective function of Th2 cytokines, showing that combined IL-4, IL-5, IL-9, IL-13 deletion does not affect the neuropathologic response to ischemic stroke in the subacute and chronic phases. Our findings indicate that Th2 cytokines are not an essential neuroimmunological cue able to drive the brain's ischemic outcome.

Mannose-Binding Lectin Drives Platelet Inflammatory Phenotype and Vascular Damage After Cerebral Ischemia in Mice via IL (Interleukin)-1α.

Objective- Circulating complement factors are activated by tissue damage and contribute to acute brain injury. The deposition of MBL (mannose-binding lectin), one of the initiators of the lectin complement pathway, on the cerebral endothelium activated by ischemia is a major pathogenic event leading to brain injury. The molecular mechanisms through which MBL influences outcome after ischemia are not understood yet. Approach and Results- Here we show that MBL-deficient (MBL-/-) mice subjected to cerebral ischemia display better flow recovery and less plasma extravasation in the brain than wild-type mice, as assessed by in vivo 2-photon microscopy. This results in reduced vascular dysfunction as shown by the shift from a pro- to an anti-inflammatory vascular phenotype associated with MBL deficiency. We also show that platelets directly bind MBL and that platelets from MBL-/- mice have reduced inflammatory phenotype as indicated by reduced IL-1α (interleukin-1α) content, as early as 6 hours after ischemia. Cultured human brain endothelial cells subjected to oxygen-glucose deprivation and exposed to platelets from MBL-/- mice present less cell death and lower CXCL1 (chemokine [C-X-C motif] ligand 1) release (downstream to IL-1α) than those exposed to wild-type platelets. In turn, MBL deposition on ischemic vessels significantly decreases after ischemia in mice treated with IL-1 receptor antagonist compared with controls, indicating a reciprocal interplay between MBL and IL-1α facilitating endothelial damage. Conclusions- We propose MBL as a hub of pathogenic vascular events. It acts as an early trigger of platelet IL-1α release, which in turn favors MBL deposition on ischemic vessels promoting an endothelial pro-inflammatory phenotype.

Intravenous infusion of human bone marrow mesenchymal stromal cells promotes functional recovery and neuroplasticity after ischemic stroke in mice.

Transplantation of human bone marrow mesenchymal stromal cells (hBM-MSC) promotes functional recovery after stroke in animal models, but the mechanisms underlying these effects remain incompletely understood. We tested the efficacy of Good Manufacturing Practices (GMP) compliant hBM-MSC, injected intravenously 3.5 hours after injury in mice subjected to transient middle cerebral artery occlusion (tMCAo). We addressed whether hBM-MSC are efficacious and if this efficacy is associated with cortical circuit reorganization using neuroanatomical analysis of GABAergic neurons (parvalbumin; PV-positive cells) and perineuronal nets (PNN), a specialized extracellular matrix structure which acts as an inhibitor of neural plasticity. tMCAo mice receiving hBM-MSC, showed early and lasting improvement of sensorimotor and cognitive functions compared to control tMCAo mice. Furthermore, 5 weeks post-tMCAo, hBM-MSC induced a significant rescue of ipsilateral cortical neurons; an increased proportion of PV-positive neurons in the perilesional cortex, suggesting GABAergic interneurons preservation; and a lower percentage of PV-positive cells surrounded by PNN, indicating an enhanced plastic potential of the perilesional cortex. These results show that hBM-MSC improve functional recovery and stimulate neuroprotection after stroke. Moreover, the downregulation of "plasticity brakes" such as PNN suggests that hBM-MSC treatment stimulates plasticity and formation of new connections in the perilesional cortex.

Macrophages are essential for maintaining a M2 protective response early after ischemic brain injury.

Resident microglia and recruited macrophages are major contributors to the post-ischemic inflammatory response. Initially considered functionally homogeneous populations, data now suggest distinct but still controversial roles after brain injury. Using a model of conditional monocyte/macrophage depletion we studied the contribution of these myeloid cells to brain lesion progression after ischemia, and their influence on the ischemic inflammatory environment. Male CD11b-DTR transgenic mice, expressing the human diphtheria toxin receptor under the control of the CD11b promoter, were treated with diphtheria toxin to induce monocyte/macrophage depletion. Twenty four hours later the middle cerebral artery was permanently occluded. The ischemic lesion was measured 24h after injury. At the same time microglia and macrophage activation and polarization were assessed by quantitative immunohistochemistry and confocal microscopy for CD45high, CD11b, CD68, CD16/32, iNOS, Arg1, Ym1, and CD206, and gene expression was investigated on CD11b+ sorted cells. Depletion of monocytes/macrophages worsened the ischemic lesion within 24h after the ischemic insult. This effect was associated with higher M1/M2 polarization ratio in the ischemic lesion. Moreover, depletion increased the expression of M1 phenotypic markers on CD11b positive cells. Gene expression on CD11b+ sorted cells indicated a selective increase of iNOS and lower Arg1 mRNA expression than in non depleted mice. Depletion of monocytes/macrophages increases the ischemic lesion, an effect accompanied by an increase in the M1/M2 polarization ratio of microglia and macrophages in the ischemic area. Thus in ischemic injury recruited monocytes/macrophages may control an excessive M1 pro-inflammatory response, suggesting their ability to drive M2 protective polarization.

Protection of Brain Injury by Amniotic Mesenchymal Stromal Cell-Secreted Metabolites.

OBJECTIVES: To define the features of human amniotic mesenchymal stromal cell secretome and its protective properties in experimental models of acute brain injury. DESIGN: Prospective experimental study. SETTING: Laboratory research. SUBJECTS: C57Bl/6 mice. INTERVENTIONS: Mice subjected to sham or traumatic brain injury by controlled cortical impact received human amniotic mesenchymal stromal cells or phosphate-buffered saline infused intracerebroventricularly or intravenously 24 hours after injury. Organotypic cortical brain slices exposed to ischemic injury by oxygen-glucose deprivation were treated with human amniotic mesenchymal stromal cells or with their secretome (conditioned medium) in a transwell system. MEASUREMENTS AND MAIN RESULTS: Traumatic brain injured mice receiving human amniotic mesenchymal stromal cells intravenously or intracerebroventricularly showed early and lasting functional and anatomical brain protection. cortical slices injured by oxigen-glucose deprivation and treated with human amniotic mesenchymal stromal cells or conditioned medium showed comparable protective effects (neuronal rescue, promotion of M2 microglia polarization, induction of trophic factors) indicating that the exposure of human amniotic mesenchymal stromal cells to the injured tissue is not necessary for the release of bioactive factors. Using sequential size-exclusion and gel-filtration chromatography, we identified a conditioned medium subfraction, which specifically displays these highly protective properties and we found that this fraction was rich in bioactive molecules with molecular weight smaller than 700 Da. Quantitative RNA analysis and mass spectrometry-based peptidomics showed that the active factors are not proteins or RNAs. The metabolomic profiling of six metabolic classes identified a list of molecules whose abundance was selectively elevated in the active conditioned medium fraction. CONCLUSIONS: Human amniotic mesenchymal stromal cell-secreted factors protect the brain after acute injury. Importantly, a fraction rich in metabolites, and containing neither proteic nor ribonucleic molecules was protective. This study indicates the profiling of protective factors that could be useful in cell-free therapeutic approaches for acute brain injury.

Internalization of nanopolymeric tracers does not alter characteristics of placental cells.

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA-NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV-MSC). We report that PMMP-NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP-NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP-NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS-treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP-NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP-NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV-MSC in preclinical models of inflammatory-driven diseases.

Bone marrow mesenchymal stromal cells drive protective M2 microglia polarization after brain trauma.

Microglia/macrophages (M) are major contributors to postinjury inflammation, but they may also promote brain repair in response to specific environmental signals that drive classic (M1) or alternative (M2) polarization. We investigated the activation and functional changes of M in mice with traumatic brain injuries and receiving intracerebroventricular human bone marrow mesenchymal stromal cells (MSCs) or saline infusion. MSCs upregulated Ym1 and Arginase-1 mRNA (p < 0.001), two M2 markers of protective M polarization, at 3 and 7 d postinjury, and increased the number of Ym1(+) cells at 7 d postinjury (p < 0.05). MSCs reduced the presence of the lysosomal activity marker CD68 on the membrane surface of CD11b-positive M (p < 0.05), indicating reduced phagocytosis. MSC-mediated induction of the M2 phenotype in M was associated with early and persistent recovery of neurological functions evaluated up to 35 days postinjury (p < 0.01) and reparative changes of the lesioned microenvironment. In vitro, MSCs directly counteracted the proinflammatory response of primary murine microglia stimulated by tumor necrosis factor-α + interleukin 17 or by tumor necrosis factor-α + interferon-γ and induced M2 proregenerative traits, as indicated by the downregulation of inducible nitric oxide synthase and upregulation of Ym1 and CD206 mRNA (p < 0.01). In conclusion, we found evidence that MSCs can drive the M transcriptional environment and induce the acquisition of an early, persistent M2-beneficial phenotype both in vivo and in vitro. Increased Ym1 expression together with reduced in vivo phagocytosis suggests M selection by MSCs towards the M2a subpopulation, which is involved in growth stimulation and tissue repair.

Mannose-binding lectin is expressed after clinical and experimental traumatic brain injury and its deletion is protective.

OBJECTIVE: Mannose-binding lectin protein is the activator of the lectin complement pathway. Goals were (1) to investigate mannose-binding lectin expression after human and experimental traumatic brain injury induced by controlled cortical impact and (2) to evaluate whether mannose-binding lectin deletion is associated with reduced sequelae after controlled cortical impact. DESIGN: Translational research, combining a human/experimental observational study and a prospective experimental study. SETTING: University hospital/research laboratory. PATIENTS AND SUBJECTS: Brain-injured patients, C57Bl/6 mice, and mannose-binding lectin-A and mannose-binding lectin-C double-knockout (-/-) mice. INTERVENTIONS: Using anti-human mannose-binding lectin antibody, we evaluated mannose-binding lectin expression in tissue samples from six patients who underwent surgery for a cerebral contusion. Immunohistochemistry was also performed on tissues obtained from mice at 30 minutes; 6, 12, 24, 48, and 96 hours; and 1 week after controlled cortical impact using anti-mouse mannose-binding lectin-A and mannose-binding lectin-C antibodies. We evaluated the effects of mannose-binding lectin deletion in wild-type and mannose-binding lectin-A and mannose-binding lectin-C double-knockout mice. Functional outcome was evaluated using the neuroscore and beam walk tests for 4 weeks postinjury (n = 11). Histological injury was evaluated by comparing neuronal cell counts in the cortex adjacent to the contusion (n = 11). MEASUREMENTS AND MAIN RESULTS: Following human traumatic brain injury, we observed mannose-binding lectin-positive immunostaining in the injured cortex as early as few hours and up to 5 days postinjury. Similarly in mice, we observed mannose-binding lectin-C-positive immunoreactivity in the injured cortex beginning 30 minutes and persisting up to 1 week postinjury. The extent of mannose-binding lectin-A expression was lower when compared with that of mannose-binding lectin-C. We observed attenuated sensorimotor deficits in mannose-binding lectin (-/-) mice compared with wild-type mice at 2-4 weeks postinjury. Furthermore, we observed reduced cortical cell loss at 5 weeks postinjury in mannose-binding lectin (-/-) mice compared with wild-type mice. CONCLUSIONS: Mannose-binding lectin expression was documented after traumatic brain injury. The reduced sequelae associated with mannose-binding lectin absence suggest that mannose-binding lectin modulation might be a potential target after traumatic brain injury.

Immunosuppression does not affect human bone marrow mesenchymal stromal cell efficacy after transplantation in traumatized mice brain.

The need for immunosuppression after allo/xenogenic mesenchymal stromal cell (MSC) transplantation is debated. This study compared the long-term effects of human (h) bone marrow MSC transplant in immunocompetent or immunosuppressed traumatic brain injured (TBI) mice. C57Bl/6 male mice were subjected to TBI or sham surgery followed 24 h later by an intracerebroventricular infusion of phosphate buffer saline (PBS, control) or hMSC (150,000/5 µl). Immunocompetent and cyclosporin A immunosuppressed (CsA) mice were analyzed for gene expression at 72 h, functional deficits and histological analysis at five weeks. Gene expression analysis showed the effectiveness of immunosuppression (INFγ reduction in CsA treated groups), with no evidence of early rejection (no changes of MHCII and CD86 in all TBI groups) and selective induction of T-reg (increase of Foxp3) only in the TBI hMSC group. Five weeks after TBI, hMSC had comparable efficacy, with functional recovery (on both sensorimotor and cognitive deficits) and structural protection (contusion volume, vessel rescue effect, gliotic scar reduction, induction of neurogenesis) in immunosuppressed and immunocompetent mice. Therefore, long-term hMSC efficacy in TBI is not dependent on immunosuppressive treatment. These findings could have important clinical implication since immunosuppression in acute TBI patients may increase their risk of infection and not be tolerated.

Three-dimensional confocal analysis of microglia/macrophage markers of polarization in experimental brain injury.

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing. Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 µm z-axis and a 0.23 µm step size, corresponding to a 180 x 135 x 10 µm volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

Six-month ischemic mice show sensorimotor and cognitive deficits associated with brain atrophy and axonal disorganization.

AIMS: To identify long-term sensorimotor and cognitive deficits and to evaluate structural alterations in brain ischemic mice. METHODS: C57Bl/6J male mice were subjected to 30 min transient middle cerebral artery occlusion (tMCAo) or sham surgery. Sensorimotor deficits, exploratory behavior, and cognitive functions were evaluated up to 6 months. Cortical and subcortical damage were analyzed by MRI multiparameter analysis and histopathology. RESULTS: tMCAo mice showed significant sensorimotor deficits in the rotarod, negative geotaxis, neuroscore, and beam walk tests. They also showed impairment in exploratory behavior in the open field test and in spatial learning in the Morris water maze. T2-weighted MRI revealed a volume reduction in injured brain areas at 12 and 24 weeks postinjury. Brain atrophy was shown by MRI and conventional postmortem analysis. Diffusion tensor imaging on the external capsule showed increased values of axial and radial diffusivity. Fiber tracking revealed a reduction in the number and length of ipsilateral fibers. CONCLUSIONS: tMCAo in mice induces sensorimotor and cognitive impairments detectable at least up to 6 months postinjury, associated with brain atrophy, and axonal and myelin damage of the external capsule. These behavioral tests and anatomical investigations may represent important tools in translational studies in cerebral ischemia.

Tumor necrosis factor in traumatic brain injury: effects of genetic deletion of p55 or p75 receptor.

The role of tumor necrosis factor (TNF) and its receptors after traumatic brain injury (TBI) remains unclear. We evaluated the effects of genetic deletion of either p55 or p75 TNF receptor on neurobehavioral outcome, histopathology, DNA damage and apoptosis-related cell death/survival gene expression (bcl-2/bax), and microglia/macrophage (M/M) activation in wild-type (WT) and knockout mice after TBI. Injured p55 (-/-) mice showed a significant attenuation while p75 (-/-) mice showed a significant worsening of sensorimotor deficits compared with WT mice over 4 weeks postinjury. At the same time point, contusion volume in p55 (-/-) mice (11.1±3.3 mm(3)) was significantly reduced compared with WT (19.7±3.4 mm(3)) and p75 (-/-) mice (20.9±3.2 mm(3)). At 4 hours postinjury, bcl-2/bax ratio mRNA expression was increased in p55 (-/-) compared with p75 (-/-) mice and was associated with reduced DNA damage terminal deoxynucleotidyl transferaseYmediated dUTP nick end labeling (TUNEL-positivity), reduced CD11b expression and increased Ym1 expression at 24 hours postinjury in p55 (-/-) compared with p75 (-/-) mice, indicative of a protective M/M response. These data suggest that TNF may exacerbate neurobehavioral deficits and tissue damage via p55 TNF receptor whose inhibition may represent a specific therapeutic target after TBI.

CX3CR1 deficiency induces an early protective inflammatory environment in ischemic mice.

The studies on fractalkine and its unique receptor CX3CR1 in neurological disorders yielded contrasting results. We have explored the consequences of CX3CR1 deletion in ischemic (30' MCAo) mice on: (1) brain infarct size; (2) microglia dynamism and morphology; (3) expression of markers of microglia/macrophages (M/M) activation and polarization. We observed smaller infarcts in cx3cr1(-/-) (26.42 ± 7.41 mm(3) , mean ± sd) compared to wild type (36.29 ± 11.57) and cx3cr1(-/+) (34.49 ± 8.91) mice. We longitudinally analyzed microglia by in vivo two-photon microscopy before, 1 and 24 h after transient ischemia. Microglia were stationary in both cx3cr1(-/-) and cx3cr1(-/+) mice throughout the study. In cx3cr1(-/-) mice, they displayed a significantly higher number of ramifications >10 µm at baseline and at 24 h after ischemia compared to cx3cr1(-/+) mice, indicating that CX3CR1 deficiency impaired the development of microglia hypertrophic/amoeboid morphology. At 24 h after ischemia, we performed post mortem quantitative immunohistochemistry for different M/M markers. In cx3cr1(-/-) immunoreactivity for CD11b (M/M activation) and for CD68 (associated with phagocytosis) were decreased, while that for CD45(high) (macrophage and leukocyte recruitment) was increased. In addition, immunoreactivity for Ym1 (M2 polarization) was enhanced, while that for iNOS (M1) was decreased. Our data show that in cx3cr1(-/-) mice protection from ischemia at early time points after injury is associated with a protective inflammatory milieu, characterized by the promotion of M2 polarization markers.

Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice.

BACKGROUND: Emerging evidence indicates that, similarly to what happens for peripheral macrophages, microglia can express different phenotypes depending on microenvironmental signals. In spite of the large literature on inflammation after ischemia, information on M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is lacking. The present study investigates the presence of microglia/macrophage phenotype markers, their temporal expression, whether they are concomitantly expressed by the same subpopulation, or they are expressed at distinct phases or locations in relation to the ischemic lesion. METHODS: Volume of ischemic lesion, neuronal counts and TUNEL staining were assessed in C57Bl/6 mice at 6-12-24-48 h and 7d after permanent occlusion of the middle cerebral artery. At the same time points, the expression, distribution in the lesioned area, association with a definite morphology and coexpression of the microglia/macrophage markers CD11b, CD45, CD68, Ym1, CD206 were assessed by immunostaining and confocal microscopy. RESULTS: The results show that: 1) the ischemic lesion induces the expression of selected microglia/macrophage markers that develop over time, each with a specific pattern; 2) each marker has a given localization in the lesioned area with no apparent changes during time, with the exception of CD68 that is confined in the border zone of the lesion at early times but it greatly increases and invades the ischemic core at 7d; 3) while CD68 is expressed in both ramified and globular CD11b cells, Ym1 and CD206 are exclusively expressed by globular CD11b cells. CONCLUSIONS: These data show that the ischemic lesion is accompanied by activation of specific microglia/macrophage phenotype that presents distinctive spatial and temporal features. These different states of microglia/macrophages reflect the complexity of these cells and their ability to differentiate towards a multitude of phenotypes depending on the surrounding micro-environmental signals that can change over time. The data presented in this study provide a basis for understanding this complex response and for developing strategies resulting in promotion of a protective inflammatory phenotype.

CX3CL1 is neuroprotective in permanent focal cerebral ischemia in rodents.

The chemokine CX3CL1 and its receptor CX3CR1 are constitutively expressed in the nervous system. In this study, we used in vivo murine models of permanent middle cerebral artery occlusion (pMCAO) to investigate the protective potential of CX3CL1. We report that exogenous CX3CL1 reduced ischemia-induced cerebral infarct size, neurological deficits, and caspase-3 activation. CX3CL1-induced neuroprotective effects were long lasting, being observed up to 50 d after pMCAO in rats. The neuroprotective action of CX3CL1 in different models of brain injuries is mediated by its inhibitory activity on microglia and, in vitro, requires the activation of adenosine receptor 1 (A1R). We show that, in the presence of the A1R antagonist 1,3-dipropyl-8-cyclopentylxanthine and in A1R⁻/⁻ mice, the neuroprotective effect of CX3CL1 on pMCAO was abolished, indicating the critical importance of the adenosine system in CX3CL1 protection also in vivo. In apparent contrast with the above reported data but in agreement with previous findings, cx3cl1⁻/⁻ and cx3cr1(GFP/GFP) mice, respectively, deficient in CX3CL1 or CX3CR1, had less severe brain injury on pMCAO, and the administration of exogenous CX3CL1 increased brain damage in cx3cl1⁻/⁻ ischemic mice. We also report that CX3CL1 induced a different phagocytic activity in wild type and cx3cl1⁻/⁻ microglia in vitro during cotreatment with the medium conditioned by neurons damaged by oxygen-glucose deprivation. Together, these data suggest that acute administration of CX3CL1 reduces ischemic damage via an adenosine-dependent mechanism and that the absence of constitutive CX3CL1-CX3CR1 signaling changes the outcome of microglia-mediated effects during CX3CL1 administration to ischemic brain.

Cytokines and innate inflammation in the pathogenesis of human traumatic brain injury.

There is an increasing recognition that following traumatic brain injury, a cascade of inflammatory mediators is produced, and contributes to the pathological consequences of central nervous system injury. This review summarises the key literature from pre-clinical models that underlies our understanding of innate inflammation following traumatic brain injury before focussing on the growing evidence from human studies. In addition, the underlying molecular mediators responsible for blood brain barrier dysfunction have been discussed. In particular, we have highlighted the different sampling methodologies available and the difficulties in interpreting human data of this sort. Ultimately, understanding the innate inflammatory response to traumatic brain injury may provide a therapeutic avenue in the treatment of central nervous system disease.