Indoleamine 2,3-Dioxygenase Deletion to Modulate Kynurenine Pathway and to Prevent Brain Injury after Cardiac Arrest in Mice.

BACKGROUND: The catabolism of the essential amino acid tryptophan to kynurenine is emerging as a potential key pathway involved in post-cardiac arrest brain injury. The aim of this study was to evaluate the effects of the modulation of kynurenine pathway on cardiac arrest outcome through genetic deletion of the rate-limiting enzyme of the pathway, indoleamine 2,3-dioxygenase. METHODS: Wild-type and indoleamine 2,3-dioxygenase-deleted (IDO-/-) mice were subjected to 8-min cardiac arrest. Survival, neurologic outcome, and locomotor activity were evaluated after resuscitation. Brain magnetic resonance imaging with diffusion tensor and diffusion-weighted imaging sequences was performed, together with microglia and macrophage activation and neurofilament light chain measurements. RESULTS: IDO-/- mice showed higher survival compared to wild-type mice (IDO-/- 11 of 16, wild-type 6 of 16, log-rank P = 0.036). Neurologic function was higher in IDO-/- mice than in wild-type mice after cardiac arrest (IDO-/- 9 ± 1, wild-type 7 ± 1, P = 0.012, n = 16). Indoleamine 2,3-dioxygenase deletion preserved locomotor function while maintaining physiologic circadian rhythm after cardiac arrest. Brain magnetic resonance imaging with diffusion tensor imaging showed an increase in mean fractional anisotropy in the corpus callosum (IDO-/- 0.68 ± 0.01, wild-type 0.65 ± 0.01, P = 0.010, n = 4 to 5) and in the external capsule (IDO-/- 0.47 ± 0.01, wild-type 0.45 ± 0.01, P = 0.006, n = 4 to 5) in IDO-/- mice compared with wild-type ones. Increased release of neurofilament light chain was observed in wild-type mice compared to IDO-/- (median concentrations [interquartile range], pg/mL: wild-type 1,138 [678 to 1,384]; IDO-/- 267 [157 to 550]; P < 0.001, n = 3 to 4). Brain magnetic resonance imaging with diffusion-weighted imaging revealed restriction of water diffusivity 24 h after cardiac arrest in wild-type mice; indoleamine 2,3-dioxygenase deletion prevented water diffusion abnormalities, which was reverted in IDO-/- mice receiving l-kynurenine (apparent diffusion coefficient, µm2/ms: wild-type, 0.48 ± 0.07; IDO-/-, 0.59 ± 0.02; IDO-/- and l-kynurenine, 0.47 ± 0.08; P = 0.007, n = 6). CONCLUSIONS: The kynurenine pathway represents a novel target to prevent post-cardiac arrest brain injury. The neuroprotective effects of indoleamine 2,3-dioxygenase deletion were associated with preservation of brain white matter microintegrity and with reduction of cerebral cytotoxic edema.

Harmonization of sensorimotor deficit assessment in a registered multicentre pre-clinical randomized controlled trial using two models of ischemic stroke.

Multicentre preclinical randomized controlled trials (pRCTs) are a valuable tool to improve experimental stroke research, but are challenging and therefore underused. A common challenge regards the standardization of procedures across centres. We here present the harmonization phase for the quantification of sensorimotor deficits by composite neuroscore, which was the primary outcome of two multicentre pRCTs assessing remote ischemic conditioning in rodent models of ischemic stroke. Ischemic stroke was induced by middle cerebral artery occlusion for 30, 45 or 60 min in mice and 50, 75 or 100 min in rats, allowing sufficient variability. Eleven animals per species were video recorded during neurobehavioural tasks and evaluated with neuroscore by eight independent raters, remotely and blindly. We aimed at reaching an intraclass correlation coefficient (ICC) ≥0.60 as satisfactory interrater agreement. After a first remote training we obtained ICC = 0.50 for mice and ICC = 0.49 for rats. Errors were identified in animal handling and test execution. After a second remote training, we reached the target interrater agreement for mice (ICC = 0.64) and rats (ICC = 0.69). In conclusion, a multi-step, online harmonization phase proved to be feasible, easy to implement and highly effective to align each centre's behavioral evaluations before project's interventional phase.

Brain Kynurenine Pathway and Functional Outcome of Rats Resuscitated From Cardiac Arrest.

Background Brain injury and neurological deficit are consequences of cardiac arrest (CA), leading to high morbidity and mortality. Peripheral activation of the kynurenine pathway (KP), the main catabolic route of tryptophan metabolized at first into kynurenine, predicts poor neurological outcome in patients resuscitated after out-of-hospital CA. Here, we investigated KP activation in hippocampus and plasma of rats resuscitated from CA, evaluating the effect of KP modulation in preventing CA-induced neurological deficit. Methods and Results Early KP activation was first demonstrated in 28 rats subjected to electrically induced CA followed by cardiopulmonary resuscitation. Hippocampal levels of the neuroactive metabolites kynurenine, 3-hydroxy-anthranilic acid, and kynurenic acid were higher 2 hours after CA, as in plasma. Further, 36 rats were randomized to receive the inhibitor of the first step of KP, 1-methyl-DL-tryptophan, or vehicle, before CA. No differences were observed in hemodynamics and myocardial function. The CA-induced KP activation, sustained up to 96 hours in hippocampus (and plasma) of vehicle-treated rats, was counteracted by the inhibitor as indicated by lower hippocampal (and plasmatic) kynurenine/tryptophan ratio and kynurenine levels. 1-Methyl-DL-tryptophan reduced the CA-induced neurological deficits, with a significant correlation between the neurological score and the individual kynurenine levels, as well as the kynurenine/tryptophan ratio, in plasma and hippocampus. Conclusions These data demonstrate the CA-induced lasting activation of the first step of the KP in hippocampus, showing that this activation was involved in the evolving neurological deficit. The degree of peripheral activation of KP may predict neurological function after CA.

Long pentraxin PTX3 is upregulated systemically and centrally after experimental neurotrauma, but its depletion leaves unaltered sensorimotor deficits or histopathology.

Long pentraxin PTX3, a pattern recognition molecule involved in innate immune responses, is upregulated by pro-inflammatory stimuli, contributors to secondary damage in traumatic brain injury (TBI). We analyzed PTX3 involvement in mice subjected to controlled cortical impact, a clinically relevant TBI mouse model. We measured PTX3 mRNA and protein in the brain and its circulating levels at different time point post-injury, and assessed behavioral deficits and brain damage progression in PTX3 KO mice. PTX3 circulating levels significantly increased 1-3 weeks after injury. In the brain, PTX3 mRNA was upregulated in different brain areas starting from 24 h and up to 5 weeks post-injury. PTX3 protein significantly increased in the brain cortex up to 3 weeks post-injury. Immunohistochemical analysis showed that, 48 h after TBI, PTX3 was localized in proximity of neutrophils, likely on neutrophils extracellular traps (NETs), while 1- and 2- weeks post-injury PTX3 co-localized with fibrin deposits. Genetic depletion of PTX3 did not affect sensorimotor deficits up to 5 weeks post-injury. At this time-point lesion volume and neuronal count, axonal damage, collagen deposition, astrogliosis, microglia activation and phagocytosis were not different in KO compared to WT mice. Members of the long pentraxin family, neuronal pentraxin 1 (nPTX1) and pentraxin 4 (PTX4) were also over-expressed in the traumatized brain, but not neuronal pentraxin 2 (nPTX2) or short pentraxins C-reactive protein (CRP) and serum amyloid P-component (SAP). The long-lasting pattern of activation of PTX3 in brain and blood supports its specific involvement in TBI. The lack of a clear-cut phenotype in PTX3 KO mice may depend on the different roles of this protein, possibly involved in inflammation early after injury and in repair processes later on, suggesting distinct functions in acute phases versus sub-acute or chronic phases. Brain long pentraxins, such as PTX4-shown here to be overexpressed in the brain after TBI-may compensate for PTX3 absence.

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.

Mannose-binding lectin has a direct deleterious effect on ischemic brain microvascular endothelial cells.

Mannose-binding lectin (MBL), an initiator of the lectin pathway, is detrimental in ischemic stroke. MBL deposition on the ischemic endothelium indicates the beginning of its actions, but downstream mechanisms are not clear yet.We investigated MBL interactions with the ischemic endothelium by exposing human brain microvascular endothelial cells (hBMECs) to protocols of ischemia. Cells were exposed to hypoxia or oxygen-glucose deprivation (OGD), and re-oxygenated with human serum (HS) or recombinant MBL (rhMBL). Hypoxic hBMECs re-oxygenated with HS showed increased complement system activation (C3c deposition, +59%) and MBL deposition (+93%) than normoxic cells. Super-resolution microscopy showed MBL internalization in hypoxic cells and altered cytoskeletal organization, indicating a potential MBL action on the endothelial structure. To isolate MBL effect, hBMECs were re-oxygenated with rhMBL after hypoxia/OGD. In both conditions, MBL reduced viability (hypoxia: -25%, OGD: -34%) compared to conditions without MBL, showing a direct toxic effect. Ischemic cells also showed greater MBL deposition (hypoxia: +143%, OGD: +126%) than normoxic cells. These results were confirmed with primary hBMECs exposed to OGD (increased MBL-induced cell death: +226%, and MBL deposition: +104%). The present findings demonstrate that MBL can exert a direct deleterious effect on ischemic brain endothelial cells in vitro, independently from complement activation.

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.

The phagocytic state of brain myeloid cells after ischemia revealed by superresolution structured illumination microscopy.

BACKGROUND: Phagocytosis is a key function of myeloid cells and is highly involved in brain ischemic injury. It has been scarcely studied in vivo, thus preventing a deep knowledge of the processes occurring in the ischemic environment. Structured illumination microscopy (SIM) is a superresolution technique which helps study phagocytosis, a process involving the recruitment of vesicles sized below the resolution limits of standard confocal microscopy. METHODS: Mice underwent permanent occlusion of the middle cerebral artery and were sacrificed at 48 h or 7 days after insult. Immunofluorescence for CD11b, myeloid cell membrane marker, and CD68, lysosomal marker was done in the ischemic area. Images were acquired using a SIM system and verified with SIM check. Lysosomal distribution was measured in the ischemic area by the gray level co-occurrence matrix (GLCM). SIM dataset was compared with transmission electron microscopy images of macrophages in the ischemic tissue at the same time points. Cultured microglia were stimulated with LPS to uptake 100 nm fluorescent beads and imaged by time-lapse SIM. GLCM was used to analyze bead distribution over the cytoplasm. RESULTS: SIM images reached a resolution of 130 nm and passed the quality control diagnose, ruling out possible artifacts. After ischemia, GLCM applied to the CD68 images showed that myeloid cells at 48 h had higher angular second moment (ASM), inverse difference moment (IDM), and lower entropy than myeloid cells at 7 days indicating higher lysosomal clustering at 48 h. At this time point, lysosomal clustering was proximal (< 700 nm) to the cell membrane indicating active target internalization, while at 7 days, it was perinuclear, consistent with final stages of phagocytosis or autophagy. Electron microscopy images indicated a similar pattern of lysosomal distribution thus validating the SIM dataset. GLCM on time-lapse SIM from phagocytic microglia cultures revealed a temporal decrease in ASM and IDM and increase in entropy, as beads were uptaken, indicating that GLCM informs on the progression of phagocytosis. CONCLUSIONS: GLCM analysis on SIM dataset quantitatively described different phases of macrophage phagocytic behavior revealing the dynamics of lysosomal movements in the ischemic brain indicating initial active internalization vs. final digestion/autophagy.

Human brain trauma severity is associated with lectin complement pathway activation.

We explored the involvement of the lectin pathway of complement in post-traumatic brain injury (TBI) pathophysiology in humans. Brain samples were obtained from 28 patients who had undergone therapeutic contusion removal, within 12 h (early) or from >12 h until five days (late) from injury, and from five non-TBI patients. Imaging analysis indicated that lectin pathway initiator molecules (MBL, ficolin-1, ficolin-2 and ficolin-3), the key enzymes MASP-2 and MASP-3, and the downstream complement components (C3 fragments and TCC) were present inside and outside brain vessels in all contusions. Only ficolin-1 was found in the parenchyma of non-TBI tissues. Immunoassays in brain homogenates showed that MBL, ficolin-2 and ficolin-3 increased in TBI compared to non-TBI (2.0, 2.2 and 6.0-times) samples. MASP-2 increased with subarachnoid hemorrhage and abnormal pupil reactivity, two indicators of structural and functional damage. C3 fragments and TCC increased, respectively, by 3.5 - and 4.0-fold in TBI compared to non-TBI tissue and significantly correlated with MBL, ficolin-2, ficolin-3, MASP-2 and MASP-3 levels in the homogenates. In conclusion, we show for the first time the direct presence of lectin pathway components in human cerebral contusions and their association with injury severity, suggesting a central role for the lectin pathway in the post-traumatic pathophysiology of human TBI.

C-Reactive Protein Binds to Cholesterol Crystals and Co-Localizes with the Terminal Complement Complex in Human Atherosclerotic Plaques.

Inflammation is a part of the initial process leading to atherosclerosis and cholesterol crystals (CC), found in atherosclerotic plaques, which are known to induce complement activation. The pentraxins C-reactive protein (CRP), long pentraxin 3 (PTX3), and serum amyloid P component (SAP) are serum proteins associated with increased risk of cardiovascular events and these proteins have been shown to interact with the complement system. Whether the pentraxins binds to CC and mediate downstream complement-dependent inflammatory processes remains unknown. Binding of CRP, PTX3, and SAP to CC was investigated in vitro by flow cytometry and fluorescence microscopy. CRP, PTX3, and SAP bound to CC in a concentration-dependent manner. CRP and PTX3 interacted with the complement pattern recognition molecule C1q on CC by increasing the binding of both purified C1q and C1q in plasma. However, CRP was the strongest mediator of C1q binding and also the pentraxin that most potently elevated C1q-mediated complement activation. In a phagocytic assay using whole blood, we confirmed that phagocytosis of CC is complement dependent and initiated by C1q-mediated activation. The pathophysiological relevance of the in vitro observations was examined in vivo in human atherosclerotic plaques. CRP, PTX3, and SAP were all found in atherosclerotic plaques and were located mainly in the cholesterol-rich necrotic core, but co-localization with the terminal C5b-9 complement complex was only found for CRP. In conclusion, this study identifies CRP as a strong C1q recruiter and complement facilitator on CC, which may be highly relevant for the development of atherosclerosis.

Lectin Pathway of Complement Activation Is Associated with Vulnerability of Atherosclerotic Plaques.

Inflammatory mechanisms may be involved in atherosclerotic plaque rupture. By using a novel histology-based method to quantify plaque instability here, we assess whether lectin pathway (LP) of complement activation, a major inflammation arm, could represent an index of plaque instability. Plaques from 42 consecutive patients undergoing carotid endarterectomy were stained with hematoxylin-eosin and the lipid core, cholesterol clefts, hemorrhagic content, thickness of tunica media, and intima, including or not infiltration of cellular debris and cholesterol, were determined. The presence of ficolin-1, -2, and -3 and mannose-binding lectin (MBL), LP initiators, was assessed in the plaques by immunofluorescence and in plasma by ELISA. LP activation was assessed in plasma by functional in vitro assays. Patients presenting low stenosis (≤75%) had higher hemorrhagic content than those with high stenosis (>75%), indicating increased erosion. Increased hemorrhagic content and tunica media thickness, as well as decreased lipid core and infiltrated content were associated with vulnerable plaques and therefore used to establish a plaque vulnerability score that allowed to classify patients according to plaque vulnerability. Ficolins and MBL were found both in plaques' necrotic core and tunica media. Patients with vulnerable plaques showed decreased plasma levels and intraplaque deposition of ficolin-2. Symptomatic patients experiencing a transient ischemic attack had lower plasma levels of ficolin-1. We show that the LP initiators are present within the plaques and their circulating levels change in atherosclerotic patients. In particular, we show that decreased ficolin-2 levels are associated with rupture-prone vulnerable plaques, indicating its potential use as marker for cardiovascular risk assessment in atherosclerotic patients.

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.

A New Surface Plasmon Resonance Assay for In Vitro Screening of Mannose-Binding Lectin Inhibitors.

Mannose-binding lectin (MBL) is a circulating protein that acts as a soluble pattern recognition molecule of the innate immunity. It binds to carbohydrate patterns on the surface of pathogens or of altered self-cells, with activation of the lectin pathway of the complement system. Recent evidence indicates that MBL contributes to the pathophysiology of ischemia-reperfusion injury and other conditions. Thus, MBL inhibitors offer promising therapeutic strategies, since they prevent the interaction of MBL with its target sugar arrays. We developed and characterized a novel assay based on surface plasmon resonance for in vitro screening of these compounds, which may be useful before the more expensive and time-consuming in vivo studies. The assay measures the inhibitor's ability to interfere with the binding of murine MBL-A or MBL-C, or of human recombinant MBL, to mannose residues immobilized on the sensor chip surface. We have applied the assay to measure the IC50 of synthetic glycodendrimers, two of them with neuroprotective properties in animal models of MBL-mediated injuries.

Fractalkine Receptor Deficiency Is Associated with Early Protection but Late Worsening of Outcome following Brain Trauma in Mice.

An impaired ability to regulate microglia activation by fractalkine (CX3CL1) leads to microglia chronic sub-activation. How this condition affects outcome after acute brain injury is still debated, with studies showing contrasting results depending on the timing and the brain pathology. Here, we investigated the early and delayed consequences of fractalkine receptor (CX3CR1) deletion on neurological outcome and on the phenotypical features of the myeloid cells present in the lesions of mice with traumatic brain injury (TBI). Wild type (WT) and CX3CR1(-/-) C57Bl/6 mice were subjected to sham or controlled cortical impact brain injury. Outcome was assessed at 4 days and 5 weeks after TBI by neuroscore, neuronal count, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Compared with WT mice, CX3CR1(-/-) TBI mice showed a significant reduction of sensorimotor deficits and lower cellular damage in the injured cortex 4 days post-TBI. Conversely, at 5 weeks, they showed a worsening of sensorimotor deficits and pericontusional cell death. Microglia (M) and macrophage (µ) activation and polarization were assessed by quantitative immunohistochemistry for CD11b, CD68, Ym1, and inducible nitric oxide synthase (iNOS)-markers of M/µ activation, phagocytosis, M2, and M1 phenotypes, respectively. Morphological analysis revealed a decreased area and perimeter of CD11b(+) cells in CX3CR1(-/-) mice at 4 days post-TBI, whereas, at 5 weeks, both parameters were significantly higher, compared with WT mice. At 4 days, CX3CR1(-/-) mice showed significantly decreased CD68 and iNOS immunoreactivity, while at 5 weeks post-injury, they showed a selective increase of iNOS. Gene expression on CD11b(+) sorted cells revealed an increase of interleukin 10 and insulin-like growth factor 1 (IGF1) at 1 day and a decrease of IGF1 4 days and 5 weeks post-TBI in CX3CR1(-/-), compared with WT mice. These data show an early protection followed by a chronic exacerbation of TBI outcome in the absence of CX3CR1. Thus, longitudinal effects of myeloid cell manipulation at different stages of pathology should be investigated to understand how and when their modulation may offer therapeutic chances.

Results of a preclinical randomized controlled multicenter trial (pRCT): Anti-CD49d treatment for acute brain ischemia.

Numerous treatments have been reported to provide a beneficial outcome in experimental animal stroke models; however, these treatments (with the exception of tissue plasminogen activator) have failed in clinical trials. To improve the translation of treatment efficacy from bench to bedside, we have performed a preclinical randomized controlled multicenter trial (pRCT) to test a potential stroke therapy under circumstances closer to the design and rigor of a clinical randomized control trial. Anti-CD49d antibodies, which inhibit the migration of leukocytes into the brain, were previously investigated in experimental stroke models by individual laboratories. Despite the conflicting results from four positive and one inconclusive preclinical studies, a clinical trial was initiated. To confirm the preclinical results and to test the feasibility of conducting a pRCT, six independent European research centers investigated the efficacy of anti-CD49d antibodies in two distinct mouse models of stroke in a centrally coordinated, randomized, and blinded approach. The results pooled from all research centers revealed that treatment with CD49d-specific antibodies significantly reduced both leukocyte invasion and infarct volume after the permanent distal occlusion of the middle cerebral artery, which causes a small cortical infarction. In contrast, anti-CD49d treatment did not reduce lesion size or affect leukocyte invasion after transient proximal occlusion of the middle cerebral artery, which induces large lesions. These results suggest that the benefits of immune-targeted approaches may depend on infarct severity and localization. This study supports the feasibility of performing pRCTs.

Shape descriptors of the "never resting" microglia in three different acute brain injury models in mice.

BACKGROUND: The study of microglia and macrophage (M/M) morphology represents a key tool to understand the functional activation state and the pattern of distribution of these cells in acute brain injury. The identification of reliable quantitative morphological parameters is urgently needed to understand these cell roles in brain injury and to explore strategies aimed at therapeutically manipulating the inflammatory response. METHODS: We used three different clinically relevant murine models of focal injury, namely, controlled cortical impact brain injury (traumatic brain injury (TBI)) and transient and permanent occlusion of middle cerebral artery (tMCAo and pMCAo, respectively). Twenty-four hours after injury, M/M cells were labeled by CD11b, and ×40 photomicrographs were acquired by unbiased sampling of the lesion core using a motorized stage microscope. Images were processed with Fiji software to obtain shape descriptors. RESULTS: We validated several parameters, including area, perimeter, Feret's diameter (caliper), circularity, aspect ratio, and solidity, providing quantitative information on M/M morphology over wide tissue portions. We showed that the shape descriptors that best represent M/M ramification/elongation are area and perimeter, while circularity and solidity provide information on the ameboid shape. We also provide evidence of the involvement of different populations in local inflammatory events, with macrophages replacing microglia into the lesion core when reperfusion does not occur. Analysis of CD45(high)+ cell morphology, whose shape does not change, did not yield any difference, thus confirming the reliability of the approach. CONCLUSIONS: We have defined specific morphological features that M/M acquire in response to different acute insults by applying a sensitive and readily applicable approach to cell morphological analysis in the brain tissue. Potential application of this method can be extended to all cell types able to change shape following activation, e.g., astrocytes, or to different disease states, including chronic pathologies.

The ischemic environment drives microglia and macrophage function.

Cells of myeloid origin, such as microglia and macrophages, act at the crossroads of several inflammatory mechanisms during pathophysiology. Besides pro-inflammatory activity (M1 polarization), myeloid cells acquire protective functions (M2) and participate in the neuroprotective innate mechanisms after brain injury. Experimental research is making considerable efforts to understand the rules that regulate the balance between toxic and protective brain innate immunity. Environmental changes affect microglia/macrophage functions. Hypoxia can affect myeloid cell distribution, activity, and phenotype. With their intrinsic differences, microglia and macrophages respond differently to hypoxia, the former depending on ATP to activate and the latter switching to anaerobic metabolism and adapting to hypoxia. Myeloid cell functions include homeostasis control, damage-sensing activity, chemotaxis, and phagocytosis, all distinctive features of these cells. Specific markers and morphologies enable to recognize each functional state. To ensure homeostasis and activate when needed, microglia/macrophage physiology is finely tuned. Microglia are controlled by several neuron-derived components, including contact-dependent inhibitory signals and soluble molecules. Changes in this control can cause chronic activation or priming with specific functional consequences. Strategies, such as stem cell treatment, may enhance microglia protective polarization. This review presents data from the literature that has greatly advanced our understanding of myeloid cell action in brain injury. We discuss the selective responses of microglia and macrophages to hypoxia after stroke and review relevant markers with the aim of defining the different subpopulations of myeloid cells that are recruited to the injured site. We also cover the functional consequences of chronically active microglia and review pivotal works on microglia regulation that offer new therapeutic possibilities for acute brain injury.