Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke.

Vascular endothelial function is challenged during cerebral ischemia and reperfusion. The endothelial responses are involved in inflammatory leukocyte attraction, adhesion and infiltration, blood-brain barrier leakage, and angiogenesis. This study investigated gene expression changes in brain endothelial cells after acute ischemic stroke using transcriptomics and translatomics. We isolated brain endothelial mRNA by: (i) translating ribosome affinity purification, enabling immunoprecipitation of brain endothelial ribosome-attached mRNA for translatome sequencing and (ii) isolating CD31+ endothelial cells by fluorescence-activating cell sorting for classical transcriptomic analysis. Both techniques revealed similar pathways regulated by ischemia but they showed specific differences in some transcripts derived from non-endothelial cells. We defined a gene set characterizing the endothelial response to acute stroke (24h) by selecting the differentially expressed genes common to both techniques, thus corresponding with the translatome and minimizing non-endothelial mRNA contamination. Enriched pathways were related to inflammation and immunoregulation, angiogenesis, extracellular matrix, oxidative stress, and lipid trafficking and storage. We validated, by flow cytometry and immunofluorescence, the protein expression of several genes encoding cell surface proteins. The inflammatory response was associated with the endothelial upregulation of genes related to lipid storage functions and we identified lipid droplet biogenesis in the endothelial cells after ischemia. The study reports a robust translatomic signature of brain endothelial cells after acute stroke and identifies enrichment in novel pathways involved in membrane signaling and lipid storage. Altogether these results highlight the endothelial contribution to the inflammatory response, and identify novel molecules that could be targets to improve vascular function after ischemic stroke.

Transient Middle Cerebral Artery Occlusion Model of Stroke.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models. In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Poststroke Lung Infection by Opportunistic Commensal Bacteria Is Not Mediated by Their Expansion in the Gut Microbiota.

BACKGROUND: Respiratory and urinary tract infections are frequent complications in patients with severe stroke. Stroke-associated infection is mainly due to opportunistic commensal bacteria of the microbiota that may translocate from the gut. We investigated the mechanisms underlying gut dysbiosis and poststroke infection. METHODS: Using a model of transient cerebral ischemia in mice, we explored the relationship between immunometabolic dysregulation, gut barrier dysfunction, gut microbial alterations, and bacterial colonization of organs, and we explored the effect of several drug treatments. RESULTS: Stroke-induced lymphocytopenia and widespread colonization of lung and other organs by opportunistic commensal bacteria. This effect correlated with reduced gut epithelial barrier resistance, and a proinflammatory sway in the gut illustrated by complement and nuclear factor-κB activation, reduced number of gut regulatory T cells, and a shift of gut lymphocytes to γδT cells and T helper 1/T helper 17 phenotypes. Stroke increased conjugated bile acids in the liver but decreased bile acids and short-chain fatty acids in the gut. Gut fermenting anaerobic bacteria decreased while opportunistic facultative anaerobes, notably Enterobacteriaceae, suffered an expansion. Anti-inflammatory treatment with a nuclear factor-κB inhibitor fully abrogated the Enterobacteriaceae overgrowth in the gut microbiota induced by stroke, whereas inhibitors of the neural or humoral arms of the stress response were ineffective at the doses used in this study. Conversely, the anti-inflammatory treatment did not prevent poststroke lung colonization by Enterobacteriaceae. CONCLUSIONS: Stroke perturbs homeostatic neuro-immuno-metabolic networks facilitating a bloom of opportunistic commensals in the gut microbiota. However, this bacterial expansion in the gut does not mediate poststroke infection.

Age-induced alterations of granulopoiesis generate atypical neutrophils that aggravate stroke pathology.

Aging accounts for increased risk and dismal outcome of ischemic stroke. Here, we investigated the impact of age-related changes in the immune system on stroke. Upon experimental stroke, compared with young mice, aged mice had increased neutrophil clogging of the ischemic brain microcirculation, leading to worse no-reflow and outcomes. Aged mice showed an enhanced granulopoietic response to stroke that led to the accumulation of CD101+CD62Llo mature and CD177hiCD101loCD62Llo and CD177loCD101loCD62Lhi immature atypical neutrophils in the blood, endowed with increased oxidative stress, phagocytosis and procoagulant features. Production of CXCL3 by CD62Llo neutrophils of the aged had a key role in the development and pathogenicity of aging-associated neutrophils. Hematopoietic stem cell rejuvenation reverted aging-associated neutropoiesis and improved stroke outcome. In elderly patients with ischemic stroke, single-cell proteome profile of blood leukocytes identified CD62Llo neutrophil subsets associated with worse reperfusion and outcome. Our results unveil how stroke in aging leads to a dysregulated emergency granulopoiesis impacting neurological outcome.

Differences in the post-stroke innate immune response between young and old.

Aging is associated to progressive changes impairing fundamental cellular and tissue functions, and the relationships amongst them through the vascular and immune systems. Aging factors are key to understanding the pathophysiology of stroke since they increase its risk and worsen its functional outcome. Most currently recognised hallmarks of aging are also involved in the cerebral responses to stroke. Notably, age-associated chronic low-grade inflammation is related to innate immune responses highlighted by induction of type-I interferon. The interferon program is prominent in microglia where it interrelates cell damage, danger signals, and phagocytosis with immunometabolic disturbances and inflammation. Microglia engulfment of damaged myelin and cell debris may overwhelm the cellular capacity for waste removal inducing intracellular lipid accumulation. Acute inflammation and interferon-stimulated gene expression are also typical features of acute stroke, where danger signal recognition by microglia trigger immunometabolic alterations underscored by lipid droplet biogenesis. Aging reduces the capacity to control these responses causing increased and persistent inflammation, metabolic dysregulation, and impaired cellular waste disposal. In turn, chronic peripheral inflammation during aging induces immunosenescence further worsening stroke-induced immunodepression, thus increasing the risk of post-stroke infection. Aging also alters gut microbiota composition inducing dysbiosis. These changes are enhanced by age-related diseases, such as atherosclerosis and type-II diabetes, that further promote vascular aging, predispose to stroke, and exacerbate brain inflammation after stroke. Current advances in aging research suggest that some age-associated alterations may be reversed. Future work will unravel whether such evolving anti-aging research may enable designing strategies to improve stroke outcome in the elderly.

Aged lipid-laden microglia display impaired responses to stroke.

Microglial cells of the aged brain manifest signs of dysfunction that could contribute to the worse neurological outcome of stroke in the elderly. Treatment with colony-stimulating factor 1 receptor antagonists enables transient microglia depletion that is followed by microglia repopulation after treatment interruption, causing no known harm to mice. We tested whether this strategy restored microglia function and ameliorated stroke outcome in old mice. Cerebral ischemia/reperfusion induced innate immune responses in microglia highlighted by type I interferon and metabolic changes involving lipid droplet biogenesis. Old microglia accumulated lipids under steady state and displayed exacerbated innate immune responses to stroke. Microglia repopulation in old mice reduced lipid-laden microglia, and the cells exhibited reduced inflammatory responses to ischemia. Moreover, old mice with renewed microglia showed improved motor function 2 weeks after stroke. We conclude that lipid deposits in aged microglia impair the cellular responses to ischemia and worsen functional recovery in old mice.

Dendritic Cells and Microglia Have Non-redundant Functions in the Inflamed Brain with Protective Effects of Type 1 cDCs.

Brain CD11c+ cells share features with microglia and dendritic cells (DCs). Sterile inflammation increases brain CD11c+ cells, but their phenotype, origin, and functions remain largely unknown. We report that, after cerebral ischemia, microglia attract DCs to the inflamed brain, and astroglia produce Flt3 ligand, supporting development and expansion of CD11c+ cells. CD11c+ cells in the inflamed brain are a complex population derived from proliferating microglia and infiltrating DCs, including a major subset of OX40L+ conventional cDC2, and also cDC1, plasmacytoid, and monocyte-derived DCs. Despite sharing certain morphological features and markers, CD11c+ microglia and DCs display differential expression of pattern recognition receptors and chemokine receptors. DCs excel CD11c- and CD11c+ microglia in the capacity to present antigen through MHCI and MHCII. Of note, cDC1s protect from brain injury after ischemia. We thus reveal aspects of the dynamics and functions of brain DCs in the regulation of inflammation and immunity.

Role of the S1P pathway and inhibition by fingolimod in preventing hemorrhagic transformation after stroke.

Hemorrhagic transformation (HT) is a complication of severe ischemic stroke after revascularization. Patients with low platelet counts do not receive reperfusion therapies due to high risk of HT. The immunomodulatory drug fingolimod attenuated HT after tissue plasminogen activator in a thromboembolic stroke model, but the underlying mechanism is unknown. Fingolimod acts on several sphingosine-1-phosphate (S1P) receptors, prevents lymphocyte trafficking to inflamed tissues, and affects brain and vascular cells. This study aimed to investigate changes in S1P-signaling in response to brain ischemia/reperfusion and the effects of the S1P receptor modulator fingolimod on HT. We studied brain expression of S1P signaling components, S1P concentration, and immune cell infiltration after ischemia/reperfusion in mice. We administered fingolimod after ischemia to wild-type mice, lymphocyte-deficient Rag2-/- mice, and mice with low platelet counts. Ischemia increased S1P-generating enzyme SphK1 mRNA, S1P concentration, and S1P receptor-1 (S1P1)+ T-cells in the brain. Fingolimod prevented lymphocyte infiltration, and attenuated the severity of HT in Rag2-/- mice but it was ineffective under thrombocytopenia. Fingolimod prevented ß-catenin degradation but not Evans blue extravasation. Ischemia/reperfusion upregulates brain S1P signaling pathway, and fingolimod exerts local effects that attenuate HT. Although fingolimod seems to act on the brain tissue, it did not prevent blood-brain barrier leakage.

Location of Neutrophils in Different Compartments of the Damaged Mouse Brain After Severe Ischemia/Reperfusion.

Background and Purpose- Ischemia attracts neutrophils to the injured brain. However, neutrophil location and access to the damaged brain tissue is not yet entirely understood. We aimed to investigate neutrophil location in a mouse model of cerebral ischemia/reperfusion. Methods- Adult male C57BL/6 mice (n=52) received 45-minute intraluminal middle cerebral artery occlusion followed by 14, 24, 48, or 96 hours of reperfusion. Sham-operated mice (n=9) were subjected to the entire surgical procedure. We used wild-type mice and CatchupIVM mice expressing a red fluorescent protein in neutrophils. In addition, fluorescent neutrophils obtained from reporter DsRed (discosoma red fluorescent protein) mice were transferred intravenously to wild-type mice after ischemia. Mice received transcardial paraformaldehyde perfusion, the brain was cryoprotected, frozen, and cryostat sections were studied by immunofluorescence and confocal microscopy. Results- Ischemia induced a time-dependent increase in brain neutrophil numbers versus sham operation. We detected neutrophils in the leptomeninges, ventricles, capillary lumen, perivascular spaces, and parenchyma within the infarcted core. Most ischemic mice showed neutrophils in the leptomeninges and perivascular spaces, whereas the presence and number of neutrophils in the parenchyma was variable among ischemic mice. During the first 24 hours, only a few mice showed parenchymal neutrophils, but the frequency of mice showing neutrophils in the parenchyma and neutrophil numbers increased at 48 and 96 hours. We also detected signs of basement membrane disruption and hints of occasional neutrophil degranulation and formation of neutrophil extracellular traps. Conclusions- After ischemia/reperfusion, neutrophils accumulate in the leptomeninges and perivascular spaces, and eventually can reach the infarcted brain parenchyma.

CD69 Plays a Beneficial Role in Ischemic Stroke by Dampening Endothelial Activation.

RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.

Microglial cell loss after ischemic stroke favors brain neutrophil accumulation.

Stroke attracts neutrophils to the injured brain tissue where they can damage the integrity of the blood-brain barrier and exacerbate the lesion. However, the mechanisms involved in neutrophil transmigration, location and accumulation in the ischemic brain are not fully elucidated. Neutrophils can reach the perivascular spaces of brain vessels after crossing the endothelial cell layer and endothelial basal lamina of post-capillary venules, or migrating from the leptomeninges following pial vessel extravasation and/or a suggested translocation from the skull bone marrow. Based on previous observations of microglia phagocytosing neutrophils recruited to the ischemic brain lesion, we hypothesized that microglial cells might control neutrophil accumulation in the injured brain. We studied a model of permanent occlusion of the middle cerebral artery in mice, including microglia- and neutrophil-reporter mice. Using various in vitro and in vivo strategies to impair microglial function or to eliminate microglia by targeting colony stimulating factor 1 receptor (CSF1R), this study demonstrates that microglial phagocytosis of neutrophils has fundamental consequences for the ischemic tissue. We found that reactive microglia engulf neutrophils at the periphery of the ischemic lesion, whereas local microglial cell loss and dystrophy occurring in the ischemic core are associated with the accumulation of neutrophils first in perivascular spaces and later in the parenchyma. Accordingly, microglia depletion by long-term treatment with a CSF1R inhibitor increased the numbers of neutrophils and enlarged the ischemic lesion. Hence, microglial phagocytic function sets a critical line of defense against the vascular and tissue damaging capacity of neutrophils in brain ischemia.

DNGR-1 in dendritic cells limits tissue damage by dampening neutrophil recruitment.

Host injury triggers feedback mechanisms that limit tissue damage. Conventional type 1 dendritic cells (cDC1s) express dendritic cell natural killer lectin group receptor-1 (DNGR-1), encoded by the gene Clec9a, which senses tissue damage and favors cross-presentation of dead-cell material to CD8+ T cells. Here we find that DNGR-1 additionally reduces host-damaging inflammatory responses induced by sterile and infectious tissue injury in mice. DNGR-1 deficiency leads to exacerbated caerulein-induced necrotizing pancreatitis and increased pathology during systemic Candida albicans infection without affecting fungal burden. This effect is B and T cell-independent and attributable to increased neutrophilia in DNGR-1-deficient settings. Mechanistically, DNGR-1 engagement activates SHP-1 and inhibits MIP-2 (encoded by Cxcl2) production by cDC1s during Candida infection. This consequently restrains neutrophil recruitment and promotes disease tolerance. Thus, DNGR-1-mediated sensing of injury by cDC1s serves as a rheostat for the control of tissue damage, innate immunity, and immunopathology.

CNS-border associated macrophages respond to acute ischemic stroke attracting granulocytes and promoting vascular leakage.

The central nervous system (CNS) contains several types of immune cells located in specific anatomic compartments. Macrophages reside at the CNS borders surrounding the brain vessels, in leptomeningeal spaces and the choroid plexus, where they interact with the vasculature and play immunological surveillance and scavenging functions. We investigated the phenotypic changes and role of these macrophages in response to acute ischemic stroke. Given that CD163 expression is a hallmark of perivascular and meningeal macrophages in the rat and human brain, we isolated CD163+ brain macrophages by fluorescence activated cell sorting. We obtained CD163+ cells from control rats and 16 h following transient middle cerebral artery occlusion, after verifying that infiltration of CD163+ peripheral myeloid cells is negligible at this acute time point. Transcriptome analysis of the sorted CD163+ cells identified ischemia-induced upregulation of the hypoxia inducible factor-1 pathway and induction of genes encoding for extracellular matrix components and leukocyte chemoattractants, amongst others. Using a cell depletion strategy, we found that CNS border-associated macrophages participate in granulocyte recruitment, promote the expression of vascular endothelial growth factor (VEGF), increase the permeability of pial and cortical blood vessels, and contribute to neurological dysfunction in the acute phase of ischemia/reperfusion. We detected VEGF expression surrounding blood vessels and in some CD163+ perivascular macrophages in the brain tissue of ischemic stroke patients deceased one day after stroke onset. These findings show ischemia-induced reprogramming of the gene expression profile of CD163+ macrophages that has a rapid impact on leukocyte chemotaxis and blood-brain barrier integrity, and promotes neurological impairment in the acute phase of stroke.

T Cells Prevent Hemorrhagic Transformation in Ischemic Stroke by P-Selectin Binding.

Objective- Hemorrhagic transformation is a serious complication of ischemic stroke after recanalization therapies. This study aims to identify mechanisms underlying hemorrhagic transformation after cerebral ischemia/reperfusion. Approach and Results- We used wild-type mice and Selplg-/- and Fut7-/- mice defective in P-selectin binding and lymphopenic Rag2-/- mice. We induced 30-minute or 45-minute ischemia by intraluminal occlusion of the middle cerebral artery and assessed hemorrhagic transformation at 48 hours with a hemorrhage grading score, histological means, brain hemoglobin content, or magnetic resonance imaging. We depleted platelets and adoptively transferred T cells of the different genotypes to lymphopenic mice. Interactions of T cells with platelets in blood were studied by flow cytometry and image stream technology. We show that platelet depletion increased the bleeding risk only after large infarcts. Lymphopenia predisposed to hemorrhagic transformation after severe stroke, and adoptive transfer of T cells prevented hemorrhagic transformation in lymphopenic mice. CD4+ memory T cells were the subset of T cells binding P-selectin and platelets through functional P-selectin glycoprotein ligand-1. Mice defective in P-selectin binding had a higher hemorrhagic score than wild-type mice. Adoptive transfer of T cells defective in P-selectin binding into lymphopenic mice did not prevent hemorrhagic transformation. Conclusions- The study identifies lymphopenia as a previously unrecognized risk factor for secondary hemorrhagic transformation in mice after severe ischemic stroke. T cells prevent hemorrhagic transformation by their capacity to bind platelets through P-selectin. The results highlight the role of T cells in bridging immunity and hemostasis in ischemic stroke.

IL-23 (Interleukin-23)-Producing Conventional Dendritic Cells Control the Detrimental IL-17 (Interleukin-17) Response in Stroke.

BACKGROUND AND PURPOSE: Inflammatory mechanisms can exacerbate ischemic tissue damage and worsen clinical outcome in patients with stroke. Both αß and γδ T cells are established mediators of tissue damage in stroke, and the role of dendritic cells (DCs) in inducing the early events of T cell activation and differentiation in stroke is not well understood. METHODS: In a murine model of experimental stroke, we defined the immune phenotype of infiltrating DC subsets based on flow cytometry of surface markers, the expression of ontogenetic markers, and cytokine levels. We used conditional DC depletion, bone marrow chimeric mice, and IL-23 (interleukin-23) receptor-deficient mice to further explore the functional role of DCs. RESULTS: We show that the ischemic brain was rapidly infiltrated by IRF4+/CD172a+ conventional type 2 DCs and that conventional type 2 DCs were the most abundant subset in comparison with all other DC subsets. Twenty-four hours after ischemia onset, conventional type 2 DCs became the major source of IL-23, promoting neutrophil infiltration by induction of IL-17 (interleukin-17) in γδ T cells. Functionally, the depletion of CD11c+ cells or the genetic disruption of the IL-23 signaling abrogated both IL-17 production in γδ T cells and neutrophil infiltration. Interruption of the IL-23/IL-17 cascade decreased infarct size and improved neurological outcome after stroke. CONCLUSIONS: Our results suggest a central role for interferon regulatory factor 4-positive IL-23-producing conventional DCs in the IL-17-dependent secondary tissue damage in stroke.

Neural Stem Cell Transplantation Induces Stroke Recovery by Upregulating Glutamate Transporter GLT-1 in Astrocytes.

Ischemic stroke is the leading cause of disability, but effective therapies are currently widely lacking. Recovery from stroke is very much dependent on the possibility to develop treatments able to both halt the neurodegenerative process as well as to foster adaptive tissue plasticity. Here we show that ischemic mice treated with neural precursor cell (NPC) transplantation had on neurophysiological analysis, early after treatment, reduced presynaptic release of glutamate within the ipsilesional corticospinal tract (CST), and an enhanced NMDA-mediated excitatory transmission in the contralesional CST. Concurrently, NPC-treated mice displayed a reduced CST degeneration, increased axonal rewiring, and augmented dendritic arborization, resulting in long-term functional amelioration persisting up to 60 d after ischemia. The enhanced functional and structural plasticity relied on the capacity of transplanted NPCs to localize in the peri-ischemic and ischemic area, to promote the upregulation of the glial glutamate transporter 1 (GLT-1) on astrocytes and to reduce peri-ischemic extracellular glutamate. The upregulation of GLT-1 induced by transplanted NPCs was found to rely on the secretion of VEGF by NPCs. Blocking VEGF during the first week after stroke reduced GLT-1 upregulation as well as long-term behavioral recovery in NPC-treated mice. Our results show that NPC transplantation, by modulating the excitatory-inhibitory balance and stroke microenvironment, is a promising therapy to ameliorate disability, to promote tissue recovery and plasticity processes after stroke. SIGNIFICANCE STATEMENT: Tissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster axonal rewiring and dendritic plasticity and to induce long-term functional recovery. The observed therapeutic effect of neural precursor cells seems to underlie their capacity to upregulate the glial glutamate transporter on astrocytes through the vascular endothelial growth factor inducing favorable changes in the electrical and molecular stroke microenvironment. Cell-based approaches able to influence plasticity seem particularly suited to favor poststroke recovery.

Antigen Presentation After Stroke.

Stroke induces a local inflammatory reaction and a plethora of innate immune responses in the brain where antigen-presenting cells become prominent. However, to date, it is still unclear whether antigen presentation is relevant to the neuropathological and functional outcome of stroke. Stroke does not trigger overt autoimmune reactions, but neural antigens have been found in lymphoid tissues of patient with stroke and it is unknown whether they promote tolerance or immune reactions that under certain conditions might contribute to the functional worsening observed in some patients. Autoantibodies to neural molecules have also been reported in patients with stroke, but the subclass of antibodies is important for their function, and the contribution of such findings to stroke outcome is not yet clear. Notably, stroke induces immunodepression highlighted by a transient lymphopenia, lymphoid organ atrophy, and monocyte deactivation. While these effects might reduce the chances of autoreactivity, they increase the risk of infection in patients with stroke and most frequently in those with severe stroke. Therefore any potential brain protective effect of stroke-induced immunodepression by attenuating or preventing lymphocyte-mediated brain damage is confounded by stroke severity and an increased incidence of infections. Systemic inflammation due to a number of comorbidities that are frequent in patients with stroke is also associated to a poor outcome. Herein, we review some relevant findings regarding the identification of neural antigens in stroke and discuss their potential contribution to the functional outcome of stroke.

Safety and efficacy of transcranial direct current stimulation in acute experimental ischemic stroke.

BACKGROUND AND PURPOSE: Transcranial direct current stimulation is emerging as a promising tool for the treatment of several neurological conditions, including cerebral ischemia. The therapeutic role of this noninvasive treatment is, however, limited to chronic phases of stroke. We thus ought to investigate whether different stimulation protocols could also be beneficial in the acute phase of experimental brain ischemia. METHODS: The influence of both cathodal and anodal transcranial direct current stimulation in modifying brain metabolism of healthy mice was first tested by nuclear magnetic resonance spectroscopy. Then, mice undergoing transient proximal middle cerebral artery occlusion were randomized and treated acutely with anodal, cathodal, or sham transcranial direct current stimulation. Brain metabolism, functional outcomes, and ischemic lesion volume, as well as the inflammatory reaction and blood brain barrier functionality, were analyzed. RESULTS: Cathodal stimulation was able, if applied in the acute phase of stroke, to preserve cortical neurons from the ischemic damage, to reduce inflammation, and to promote a better clinical recovery compared with sham and anodal treatments. This finding was attributable to the significant decrease of cortical glutamate, as indicated by nuclear magnetic resonance spectroscopy. Conversely, anodal stimulation induced an increase in the postischemic lesion volume and augmented blood brain barrier derangement. CONCLUSIONS: Our data indicate that transcranial direct current stimulation exerts a measurable neuroprotective effect in the acute phase of stroke. However, its timing and polarity should be carefully identified on the base of the pathophysiological context to avoid potential harmful side effects.