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.

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.

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.

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.

Porous materials in catalysis: challenges for mesoporous materials.

The discovery of ordered mesoporous materials has opened great opportunities for new applications in heterogeneous catalysis, thanks to their hitherto unprecedented intrinsic structural features. Evidence shows that, however, these materials have not met the researchers' expectations mainly because of the severe limitations related to the strength of acid sites and to the thermal/hydrothermal stability, significantly lower than those of zeolites and due to the amorphous nature of the mesostructured materials. These features are highlighted in the first part of this review, where the peculiarities of mesostructured materials are compared with those of zeolite catalysts in some reactions of industrial interest. New synthesis strategies, especially designed for preparing materials with improved physico-chemical and textural properties, together with the catalytic features of the resulting materials, are described and discussed in the second part of the review.

Evolution of brain injury and neurological dysfunction after cardiac arrest in the rat - A multimodal and comprehensive model.

Cardiac arrest (CA) is one of the leading causes of death worldwide. Due to hypoxic ischemic brain injury, CA survivors may experience variable degrees of neurological dysfunction. This study, for the first time, describes the progression of CA-induced neuropathology in the rat. CA rats displayed neurological and exploratory deficits. Brain MRI revealed cortical and striatal edema at 3 days (d), white matter (WM) damage in corpus callosum (CC), external capsule (EC), internal capsule (IC) at d7 and d14. At d3 a brain edema significantly correlated with neurological score. Parallel neuropathological studies showed neurodegeneration, reduced neuronal density in CA1 and hilus of hippocampus at d7 and d14, with cells dying at d3 in hilus. Microgliosis increased in cortex (Cx), caudate putamen (Cpu), CA1, CC, and EC up to d14. Astrogliosis increased earlier (d3 to d7) in Cx, Cpu, CC and EC compared to CA1 (d7 to d14). Plasma levels of neurofilament light (NfL) increased at d3 and remained elevated up to d14. NfL levels at d7 correlated with WM damage. The study shows the consequences up to 14d after CA in rats, introducing clinically relevant parameters such as advanced neuroimaging and blood biomarker useful to test therapeutic interventions in this model.

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.

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.

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.

Glycogen synthase kinase-3 inhibition reduces ischemic cerebral damage, restores impaired mitochondrial biogenesis and prevents ROS production.

This study was designed to test the hypothesis that improved mitochondrial biogenesis could help reducing ischemic cerebral injury. We found that levels of proliferator-activated receptor γ coactivator 1α and nuclear respiratory factor-1, mitochondrial DNA content and other markers of mitochondrial biogenesis and function were reduced in primary mouse cortical neurons under oxygen-glucose deprivation (OGD). The glycogen synthase kinase-3 (GSK-3) inhibitor SB216763 activated an efficient mitochondrial biogenesis program in control cortical neurons and counteracted the OGD-mediated mitochondrial biogenesis impairment. This was accompanied by the activation of an antioxidant response that reduced mitochondrial reactive oxygen species generation and ischemic neuronal damage. The in vitro effects of SB216763 were mimicked by two other structurally unrelated GSK-3 inhibitors. The protective effects of SB216763 on OGD-mediated neuronal damage were abolished in the presence of diverse mitochondrial inhibitors. Finally, when systemically administered in vivo, SB216763 reduced the infarct size and recovered the loss of mitochondrial DNA in mice subjected to permanent middle cerebral artery occlusion. We conclude that GSK-3 inhibition by SB216763 might pave the way of novel promising therapies aimed at stimulating the renewal of functional mitochondria and reducing reactive oxygen species-mediated damage in ischemic stroke.

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.

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.

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.

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.

c-Jun N-terminal kinase pathway activation in human and experimental cerebral contusion.

The c-Jun N-terminal kinase (JNK) pathway is involved in cell stress and apoptosis. We tested the hypothesis that this pathway plays a role in traumatic brain injury (TBI) by assessing JNK activation in human brain tissues and in brains of mice subjected to controlled cortical impact brain injury. We also assessed the effects of specific inhibition of the JNK pathway by the cell-permeable JNK inhibitor peptide, D-JNKI1, on neurobehavioral function and posttraumatic cell loss in mice. The inhibitor was administered intraperitoneally 10 minutes after injury. The JNK pathway showed robust activation both in human contusion specimens and in injured cortex and hippocampi of TBI-injured mice, 1, 4, and 48 hours after injury. D-JNKI1 treatment significantly improved motor performance at 48 hours and 7 days after injury and reduced the contusion volume compared with saline treatment; the numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were significantly decreased in the hippocampi of injured mice 48 hours after treatment. Thus, because the JNK pathway is activated after human and experimental TBI and the inhibitor peptide D-JNKI1 affords significant neuroprotection and amelioration of neurobehavioral deficits after experimental TBI, therapeutic targeting of the JNK activation pathway may hold promise for future clinical applications.

C1-inhibitor attenuates neurobehavioral deficits and reduces contusion volume after controlled cortical impact brain injury in mice.

OBJECTIVE: The aim of the study was to evaluate the effects of C1-inhibitor (C1-INH), an endogenous inhibitor of complement and kinin systems, on neurobehavioral and histological outcome following controlled cortical impact brain injury. DESIGN: Experimental prospective randomized study in mice. SETTING: Experimental laboratory. SUBJECTS: Male C57Bl/6 mice (n = 81). INTERVENTIONS: Mice were subjected to controlled cortical impact brain injury followed by an intravenous bolus of either C1-INH (15 U either at 10 minutes or 1 hour postinjury) or saline (equal volume, 150 microl at 10 minutes postinjury). Sham-operated mice received identical surgery and saline injection without brain injury. Neurological motor function was evaluated weekly for 4 weeks using the Composite Neuroscore. Cognitive function was evaluated at 4 weeks postinjury using the Morris Water Maze. Histological outcome was performed by measuring the contusion volume at 1 week and 4 weeks postinjury. MEASUREMENTS AND MAIN RESULTS: Brain-injured mice receiving C1-INH at 10 minutes postinjury showed attenuated motor deficits, cognitive dysfunction and reduced contusion volume compared to brain-injured mice receiving saline. Mice receiving C1-INH at 1 hour postinjury showed reduced motor deficits compared to brain-injured mice receiving saline, but no significantly different cognitive and histological outcome. Immunohistochemical analysis showed that 20 minutes after infusion, C1-INH was localised on endothelial cells and in brain tissue surrounding brain capillaries of the injured hemisphere. CONCLUSION: Our results show that post-traumatic administration of C1-INH attenuates neuro-behavioral deficits and histological damage associated with traumatic brain injury.

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.