Inhibition of mannan-binding lectin associated serine protease (MASP)-2 reduces the cognitive deficits in a mouse model of severe traumatic brain injury.

The lectin pathway (LP) of complement mediates inflammatory processes linked to tissue damage and loss of function following traumatic brain injury (TBI). LP activation triggers a cascade of proteolytic events initiated by LP specific enzymes called MASPs (for Mannan-binding lectin Associated Serine Proteases). Elevated serum and brain levels of MASP-2, the effector enzyme of the LP, were previously reported to be associated with the severity of tissue injury and poor outcomes in patients with TBI. To evaluate the therapeutic potential of LP inhibition in TBI, we first conducted a pilot study testing the effect of an inhibitory MASP-2 antibody (α-MASP-2), administered systemically at 4 and 24 h post-TBI in a mouse model of controlled cortical impact (CCI). Treatment with α-MASP-2 reduced sensorimotor and cognitive deficits for up to 5 weeks post-TBI. As previous studies by others postulated a critical role of MASP-1 in LP activation, we conducted an additional study that also assessed treatment with an inhibitory MASP-1 antibody (α-MASP-1). A total of 78 mice were treated intraperitoneally with either α-MASP-2, or α-MASP-1, or an isotype control antibody 4 h and 24 h after TBI or sham injury. An amelioration of the cognitive deficits assessed by Barnes Maze, prespecified as the primary study endpoint, was exclusively observed in the α-MASP-2-treated group. The behavioral data were paralleled by a reduction of the lesion size when evaluated histologically and by reduced systemic LP activity. Our data suggest that inhibition of the LP effector enzyme MASP-2 is a promising treatment strategy to limit neurological deficits and tissue loss following TBI. Our work has translational value because a MASP-2 antibody has already completed multiple late-stage clinical trials in other indications and we used a clinically relevant treatment protocol testing the therapeutic mechanism of MASP-2 inhibition in TBI.

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.

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.

Mannose-binding lectin promotes blood-brain barrier breakdown and exacerbates axonal damage after traumatic brain injury in mice.

Leukocyte infiltration and blood-brain barrier breakdown contribute to secondary brain damage after traumatic brain injury (TBI). TBI induces neuroimmune responses triggering pathogenic complement activation through different pathways, including the lectin pathway. We investigated mechanisms underlying mannose-binding lectin (MBL)-mediated brain damage focusing on neutrophil infiltration and blood-brain barrier breakdown in a TBI mouse model. Wild type mice and MBL-/- null mice were subjected to controlled cortical impact. We studied neutrophil infiltration and regional localization by confocal microscopy 1, 4 and 15 days post-trauma, and investigated neutrophil extracellular trap (NET) formation. By immunofluorescence and/or Western blotting in various brain regions we studied the presence of fibrin(ogen), pentraxin-3, albumin and immunoglobulin G. Finally, we studied neurofilament proteins, synaptophysin, and αII-spectrin, and assessed white matter content in the injured tissue. TBI triggered an acute wave of neutrophil infiltration at day 1 followed by a more discrete persistence of neutrophils in the injured tissue at least until day 15. We detected the presence of NETs and pentraxin-3 in the injured tissue, as well as accumulation of fibrin(ogen), increased blood-brain barrier permeability, and neurofilament, synaptophysin and white matter loss, and calpain-mediated αII spectrin breakdown. MBL-/- mice showed reduced number of Ly6G+ neutrophils 4 days after TBI, lower accumulation of pentraxin-3 and fibrin(ogen) in the injured tissue, reduced global plasma protein extravasation, and better preservation of axonal and white matter integrity. These results show that MBL participates in secondary neutrophil accumulation and blood-brain barrier breakdown, and promotes axonal and white matter damage after TBI in mice.

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.

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.

Plasma-derived and recombinant C1 esterase inhibitor: Binding profiles and neuroprotective properties in brain ischemia/reperfusion injury.

C1 esterase inhibitor (C1INH) is known to exert its inhibitory effect by binding to several target proteases of the contact and complement systems. One of C1INH's targets comprise mannose-binding lectin (MBL), a critical player in post-stroke pathophysiology. We therefore explored the effects of recombinant human (rh) and plasma derived (pd) C1INH in C57BL/6J mice subjected to transient occlusion of the middle cerebral artery (tMCAo), receiving 15U/mouse of pd or rhC1INH intravenously, at reperfusion. We analyzed the compounds' (i)neuroprotective effects, (ii) plasma presence, (iii)effects on circulating and brain MBL, (iv)time course of endothelial deposition, and (v) effects on the formation of active complement products. rhC1INH-treated mice had neuroprotective effects, including reduced behavioral deficits and neuronal loss, associated with decreased MBL brain deposition and decreased formation of complement C4b active fragments. In contrast, pdC1INH did not show these neuroprotective effects despite its longer plasma residence time. We also analyzed the response to tMCAo in C1INH-deficient mice, observing a poorer ischemic outcome compared to the wild type mice, which could be partially prevented by rhC1INH administration. In conclusion, we show that rhC1INH exhibits stronger neuroprotective effects than the corresponding plasma-derived protein after experimental ischemia/reperfusion injury in the brain, placing it as a promising drug for stroke. Differential effects are likely related to more effective MBL inhibition which further confirms it as a useful pharmacological target for stroke.

Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy.

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.

Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury-Time-Dependent Changes in the Cortex, Striatum, Thalamus and Hippocampus in a Mouse Model.

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein-GFAP), microglia/macrophages (allograft inflammatory factor 1-IBA-1), and microglia (transmembrane protein 119-TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

Traumatic brain injury in mice induces changes in the expression of the XCL1/XCR1 and XCL1/ITGA9 axes.

BACKGROUND: Every year, millions of people suffer from various forms of traumatic brain injury (TBI), and new approaches with therapeutic potential are required. Although chemokines are known to be involved in brain injury, the importance of X-C motif chemokine ligand 1 (XCL1) and its receptors, X-C motif chemokine receptor 1 (XCR1) and alpha-9 integrin (ITGA9), in the progression of TBI remain unknown. METHODS: Using RT-qPCR/Western blot/ELISA techniques, changes in the mRNA/protein levels of XCL1 and its two receptors, in brain areas at different time points were measured in a mouse model of TBI. Moreover, their cellular origin and possible changes in expression were evaluated in primary glial cell cultures. RESULTS: Studies revealed the spatiotemporal upregulation of the mRNA expression of XCL1, XCR1 and ITGA9 in all the examined brain areas (cortex, thalamus, and hippocampus) and at most of the evaluated stages after brain injury (24 h; 4, 7 days; 2, 5 weeks), except for ITGA9 in the thalamus. Moreover, changes in XCL1 protein levels occurred in all the studied brain structures; the strongest upregulation was observed 24 h after trauma. Our in vitro experiments proved that primary murine microglial and astroglial cells expressed XCR1 and ITGA9, however they seemed not to be a main source of XCL1. CONCLUSIONS: These findings indicate that the XCL1/XCR1 and XCL1/ITGA9 axes may participate in the development of TBI. The XCL1 can be considered as one of the triggers of secondary injury, therefore XCR1 and ITGA9 may be important targets for pharmacological intervention after traumatic brain injury.

The CCL2/CCL7/CCL12/CCR2 pathway is substantially and persistently upregulated in mice after traumatic brain injury, and CCL2 modulates the complement system in microglia.

Traumatic brain injury (TBI) is the leading cause of death in the global population. Disturbed inflammatory processes after TBI exacerbate secondary brain injury and contribute to unfavorable outcomes. Multiple inflammatory events that accompany brain trauma, such as glial activation, chemokine release, or the initiation of the complement system cascade, have been identified as potential targets for TBI treatment. However, the participation of chemokines in the complement activation remains unknown. Our studies sought to determine the changes in the expression of the molecules involved in the CCL2/CCL7/CCL12/CCR2 pathway in the injured brain and the effect of CCL2, CCL7, and CCL12 (10, 100, and 500 ng/mL) on the classic and lectin complement pathways and inflammatory factors in microglial cell cultures. Brain injury in mice was modeled by controlled cortical impact (CCI). Our findings indicate a time-dependent upregulation of CCL2, CCL7, and CCL12 at the mRNA and protein levels within the cortex, striatum, and/or thalamus beginning 24 h after the trauma. The analysis of the expression of the receptor of the tested chemokines, CCR2, revealed its substantial upregulation within the injured brain areas mainly on the mRNA level. Using primary cortical microglial cell cultures, we observed a substantial increase in the expression of CCL2, CCL7, and CCL12 after 24 h of LPS (100 ng/mL) treatment. CCL2 stimulation of microglia increased the level of IL-1ß mRNA but did not influence the expression of IL-18, IL-6, and IL-10. Moreover, CCL2 significantly increased the expression of Iba1, a marker of microglia activation. CCL2 and CCL12 upregulated the expression of C1qa but did not influence the expression of C1ra and C1s1 (classical pathway); moreover, CCL2 increased ficolin A expression and reduced collectin 11 expression (lectin pathway). Additionally, we observed the downregulation of pentraxin 3, a modulator of the complement cascade, after CCL2 and CCL12 treatment. We did not detect the expression of ficolin B, Mbl1, and Mbl2 in microglial cells. Our data identify CCL2 as a modulator of the classical and lectin complement pathways suggesting that CCL2 may be a promising target for pharmacological intervention after brain injury. Moreover, our study provides evidence that CCL2 and two other CCR2 ligands may play a role in the development of changes in TBI.

Changes in macrophage inflammatory protein-1 (MIP-1) family members expression induced by traumatic brain injury in mice.

A deep knowledge of the profound immunological response induced by traumatic brain injury (TBI) raises the possibility of novel therapeutic interventions. Existing studies have highlighted the important roles of C-C motif ligands in the development of neuroinflammation after brain injury; however, the participation of macrophage inflammatory protein-1 (MIP-1) family members in this phenomenon is still undefined. Therefore, the goal of our study was to evaluate changes in macrophage inflammatory protein-1 (MIP-1) family members (CCL3, CCL4, and CCL9) and their receptors (CCR1 and CCR5) in a mouse model of TBI (induced by controlled cortical impact (CCI)). We also investigated the pattern of activation of immunological cells (such as neutrophils, microglia and astroglia), which on one hand express CCR1/CCR5, and on the other hand might be a source of the tested chemokines in the injured brain. We investigated changes in mRNA (RT-qPCR) and/or protein (ELISA and Western blot) expression in brain structures (the cortex, hippocampus, thalamus, and striatum) at different time points (24 h, 4 days, 7 days, 2 weeks, and/or 5 weeks) after trauma. Our time-course studies revealed the upregulation of the mRNA expression of all members of the MIP-1 family (CCL3, CCL4, and CCL9) in all tested brain structures, mainly in the early stages after injury. A similar pattern of activation was observed at the protein level in the cortex and thalamus, where the strongest activation was observed 1 day after CCI; however, we did not observe any change in CCL3 in the thalamus. Analyses of CCR1 and CCR5 demonstrated the upregulation of the mRNA expression of both receptors in all tested cerebral structures, mainly in the early phases post injury (24 h, 4 days and 7 days). Protein analysis showed the upregulation of CCR1 and CCR5 in the thalamus 24 h after TBI, but we did not detect any change in the cortex. We also observed the upregulation of neutrophil marker (MPO) at the early time points (24 h and 7 days) in the cortex, while the profound activation of microglia (IBA-1) and astroglia (GFAP) was observed mainly on day 7. Our findings highlight for the first time that CCL3, CCL4, CCL9 and their receptors offer promising targets for influencing secondary neuronal injury and improving TBI therapy. The results suggest that the MIP-1 family is an important target for pharmacological intervention for brain injury.

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.

Specific contribution of mannose-binding lectin murine isoforms to brain ischemia/reperfusion injury.

Mannose-binding lectin (MBL), an initiator of the lectin pathway (LP) of complement activation, is detrimental in ischemic stroke, as shown in clinical studies and rodent models. Whereas humans have one functional MBL protein, rodents have two isoforms, MBL-A and MBL-C, whose functions relative to that of human MBL are unknown. To permit the clinical translation of preclinical data, we aimed to define the specific contributions of MBL-A and MBL-C to brain ischemia. We subjected mice with double (MBL-/-) or single (MBL-A-/- or MBL-C-/-) MBL isoform depletion to transient middle cerebral artery occlusion (tMCAo). MBL-/- mice had fewer neurological deficits and smaller ischemic lesions than WT mice. MBL-A-/- mice had smaller lesions than WT mice and exhibited no significant behavioral defects, whereas MBL-C-/- mice did not differ from WT mice. The induction of Mbl1 and Mbl2 (the MBL-A and MBL-C genes) expression 48 h after tMCAo was similar across genotypes. The time course of Mbl1 and Mbl2 expression in WT ischemic mice showed that Mbl1 activation occurred earlier (24 h) than Mbl2 activation (48 h). The plasma levels of MBL-A and MBL-C in MBL-C-/- and MBL-A-/- mice, respectively, were similar to those in WT mice both at baseline and at 48 h after tMCAo. At 48 h, MBL-A-/- ischemic mice showed higher MBL-C levels in the brain than WT mice. WT and MBL-C-/- ischemic mice had higher LP activity in plasma and, accordingly, higher levels of C3 deposition in the brain than MBL-A-/- and MBL-/- mice. In conclusion, mice with depletion of both MBL isoforms exhibited strong protection from ischemia/reperfusion injury. MBL-A was the main contributor to injury, likely owing to its earlier activation after ischemia and more efficient activation of the complement system than MBL-C.

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.

Dexamethasone Conjugation to Biodegradable Avidin-Nucleic-Acid-Nano-Assemblies Promotes Selective Liver Targeting and Improves Therapeutic Efficacy in an Autoimmune Hepatitis Murine Model.

Steroids are the standard therapy for autoimmune hepatitis (AIH) but the long-lasting administration is hampered by severe side effects. Methods to improve the tropism of the drug toward the liver are therefore required. Among them, conjugation to nanoparticles represents one possible strategy. In this study, we exploited the natural liver tropism of Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS) to carry dexamethasone selectively to the liver in an AIH animal model. An acid-labile biotin-hydrazone linker was developed for reversible dexamethasone loading onto ANANAS. The biodistribution, pharmacokinetics and efficacy of free and ANANAS-linked dexamethasone (ANANAS-Hz-Dex) in healthy and AIH mice were investigated upon intraperitoneal administration. In ANANAS-treated animals, the free drug was detected only in the liver. Super-resolution microscopy showed that nanoparticles segregate inside lysosomes of liver immunocompetent cells, mainly involved in AIH progression. In agreement with these observational results, chronic low-dose treatment with ANANAS-Hz-Dex reduced the expression of liver inflammation markers and, in contrast to the free drug, also the levels of circulating AIH-specific autoantibodies. These data suggest that the ANANAS carrier attenuates AIH-related liver damage without drug accumulation in off-site tissues. The safety and biodegradability of the ANANAS carrier make this formulation a promising tool for the treatment of autoimmune liver disorders.

SELENON (SEPN1) protects skeletal muscle from saturated fatty acid-induced ER stress and insulin resistance.

Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to a congenital myopathy associated with insulin resistance (SEPN1-related myopathy). The exact cause of the insulin resistance in patients with SELENON loss of function is not known. Skeletal muscle is the main contributor to insulin-mediated glucose uptake, and a defect in this muscle-related mechanism triggers insulin resistance and glucose intolerance. We have studied the chain of events that connect the loss of SELENON with defects in insulin-mediated glucose uptake in muscle cells and the effects of this on muscle performance. Here, we show that saturated fatty acids are more lipotoxic in SELENON-devoid cells, and blunt the insulin-mediated glucose uptake of SELENON-devoid myotubes by increasing ER stress and mounting a maladaptive ER stress response. Furthermore, the hind limb skeletal muscles of SELENON KO mice fed a high-fat diet mirrors the features of saturated fatty acid-treated myotubes, and show signs of myopathy with a compromised force production. These findings suggest that the absence of SELENON together with a high-fat dietary regimen increases susceptibility to insulin resistance by triggering a chronic ER stress in skeletal muscle and muscle weakness. Importantly, our findings suggest that environmental cues eliciting ER stress in skeletal muscle (such as a high-fat diet) affect the pathological phenotype of SEPN1-related myopathy and can therefore contribute to the assessment of prognosis beyond simple genotype-phenotype correlations.