Human Stroke Patients Research Articles

Signaling roles of sphingolipids in the ischemic brain and their potential utility as therapeutic targets

Sphingolipids comprise a class of lipids, which are composed of a sphingoid base backbone and are essential structural components of cell membranes. Beyond their role in maintaining cellular integrity, several sphingolipids are pivotally involved in signaling pathways controlling cell proliferation, differentiation, and death. The brain exhibits a particularly high concentration of sphingolipids and dysregulation of the sphingolipid metabolism due to ischemic injury is implicated in consecutive pathological events. Experimental stroke studies revealed that the stress sphingolipid ceramide accumulates in the ischemic brain post-stroke. Specifically, counteracting ceramide accumulation protects against ischemic damage and promotes brain remodeling, which translates into improved behavioral outcome. Sphingomyelin substantially influences cell membrane fluidity and thereby controls the release of extracellular vesicles, which are important vehicles in cellular communication. By modulating sphingomyelin content, these vesicles were shown to contribute to behavioral recovery in experimental stroke studies. Another important sphingolipid that influences stroke pathology is sphingosine-1-phosphate, which has been attributed a pro-angiogenic function, that is presumably mediated by its effect on endothelial function and/or immune cell trafficking. In experimental and clinical studies, sphingosine-1-phosphate receptor modulators allowed to modify clinically significant stroke recovery. Due to their pivotal roles in cell signaling, pharmacological compounds modulating sphingolipids, their enzymes or receptors hold promise as therapeutics in human stroke patients.

Abstract WMP119: CD13 Facilitates Immune Cell Migration and Aggravates Acute Injury but Promotes Chronic Post-Stroke Recovery

Introduction: Acute stroke leads to the activation of myeloid cells. Activated myeloid cells express CD13, which facilitates their migration into the site of injury. Chronically after stroke, repair processes, including angiogenesis, are activated and enhance post-stroke recovery. However, angiogenic blood vessels which play a role in recovery also express CD13. Overall, the specific contribution of CD13 to acute and chronic stroke outcomes is unknown. Methods: CD13 expression was estimated in mice and humans after the ischemic stroke. Young (8-12 weeks) male wild-type (WT) and global CD13 knockout (KO) mice were used for this study. Mice underwent 60 minutes of middle cerebral artery occlusion (MCAO) followed by reperfusion. For acute studies, the mice were euthanized at either 24- or 72 hours post-stroke. For chronic studies, the Y maze, Barnes maze, and the open field were performed on day 7 and day 28 post-stroke. Mice were euthanized at day 30 post-stroke and the brains were collected for assessment of inflammation, white matter injury, tissue loss, and angiogenesis. Flow cytometry was performed on days 3 and 7 post-stroke to quantify infiltrated monocytes and neutrophils and CXCL12/CXCR4 signaling. Results: Brain CD13 expression and infiltrated CD13 + monocytes and neutrophils increased acutely after the stroke. The brain CD13 + lectin + blood vessels increased on day 15 after the stroke. Similarly, an increase in the percentage area CD13 was observed in human stroke patients at the subacute time after stroke. Deletion of CD13 resulted in reduced infarct volume and improved neurological recovery after acute stroke. However, CD13KO mice had significantly worse memory deficits, amplified gliosis, and white matter damage compared to WT animals at chronic time points. CD13KO mice had an increased percentage of CXCL12 + cells but a reduced percentage of CXCR4 + cells and decreased angiogenesis at day 30 post-stroke. Conclusions: CD13 is involved in the transmigration of monocytes and neutrophils after stroke, and acutely, led to decreased infarct size and improved behavioral outcomes. However, loss of CD13 led to reductions in post-stroke angiogenesis by reducing CXCL12/CXCR4 signaling.

A dietary intervention with conjugated linoleic acid enhances microstructural white matter reorganization in experimental stroke.

A dietary supplementation with conjugated linoleic acid (CLA) was shown to attenuate inflammation and increase the proportions of circulating regulatory T cells (Tregs) and M2-type macrophages in disease models such as autoimmune encephalitis and arteriosclerosis. Since Tregs and anti-inflammatory (M2-type) macrophages were found to enhance stroke recovery, we hypothesized that CLA-supplementation might improve stroke recovery via immune modulatory effects. Functional assessment was performed over 90 days after induction of experimental photothrombotic stroke in wild type mice (n = 37, sham n = 10). Subsequently, immunological characterization of different immunological compartments (n = 16), ex vivo magnetic resonance (MR, n = 12) imaging and immunohistochemical staining (n = 8) was performed. Additionally, we tested the effect of CLA in vitro on peripheral blood mononuclear cells from human stroke patients and healthy controls (n = 12). MR diffusion tensor imaging (DTI) demonstrated enhanced microstructural reorganization of interhemispheric white matter tracts, dependent on lesion size. Functional recovery over 90 days remained unaffected. Detailed immunological analyses across various compartments revealed no significant long-term immunological alterations due to CLA. However, analyses of human blood samples post-stroke showed reduced levels of pro-inflammatory interferon-γ (IFN-γ) and tumor necrosis factor alpha (TNF-α) release by T-lymphocytes following in vitro treatment with CLA. We aimed to explore the efficacy of a dietary intervention with minimal known side effects that could be accessible to human stroke patients, regardless of the degree of disability, and without the risks associated with aggressive immunomodulatory therapies. Our main findings include improved microstructural reorganization in small infarcts and a reduced inflammatory response of human T cells in vitro.

CD13 facilitates immune cell migration and aggravates acute injury but promotes chronic post-stroke recovery

IntroductionAcute stroke leads to the activation of myeloid cells. These cells express adhesion molecules and transmigrate to the brain, thereby aggravating injury. Chronically after stroke, repair processes, including angiogenesis, are activated and enhance post-stroke recovery. Activated myeloid cells express CD13, which facilitates their migration into the site of injury. However, angiogenic blood vessels which play a role in recovery also express CD13. Overall, the specific contribution of CD13 to acute and chronic stroke outcomes is unknown.MethodsCD13 expression was estimated in both mice and humans after the ischemic stroke. Young (8–12 weeks) male wild-type and global CD13 knockout (KO) mice were used for this study. Mice underwent 60 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. For acute studies, the mice were euthanized at either 24- or 72 h post-stroke. For chronic studies, the Y-maze, Barnes maze, and the open field were performed on day 7 and day 28 post-stroke. Mice were euthanized at day 30 post-stroke and the brains were collected for assessment of inflammation, white matter injury, tissue loss, and angiogenesis. Flow cytometry was performed on days 3 and 7 post-stroke to quantify infiltrated monocytes and neutrophils and CXCL12/CXCR4 signaling.ResultsBrain CD13 expression and infiltrated CD13+ monocytes and neutrophils increased acutely after the stroke. The brain CD13+lectin+ blood vessels increased on day 15 after the stroke. Similarly, an increase in the percentage area CD13 was observed in human stroke patients at the subacute time after stroke. Deletion of CD13 resulted in reduced infarct volume and improved neurological recovery after acute stroke. However, CD13KO mice had significantly worse memory deficits, amplified gliosis, and white matter damage compared to wild-type animals at chronic time points. CD13-deficient mice had an increased percentage of CXCL12+cells but a reduced percentage of CXCR4+cells and decreased angiogenesis at day 30 post-stroke.ConclusionsCD13 is involved in the trans-migration of monocytes and neutrophils after stroke, and acutely, led to decreased infarct size and improved behavioral outcomes. However, loss of CD13 led to reductions in post-stroke angiogenesis by reducing CXCL12/CXCR4 signaling.

Abstract TMP77: Iga+ B Cells Are Recruited To The Brain After Intracerebral Hemorrhage

Introduction: There is ample evidence of a B cell response in human stroke patients, and evidence that long-term cognitive decline is linked to ongoing B cell activation in ischemic stroke models in mice. However, the role of B cells in hemorrhagic stroke, particularly at late timepoints, is unknown. Methods: Intracerebral hemorrhage (ICH) was induced in 12-week male C57BL/6 mice by collagenase (or saline for sham controls) injection into the striatum. Mice were perfused 2, 4, or 8 weeks post-ICH or sham, ipsilateral and contralateral hemispheres were harvested and B cell counts analyzed by flow cytometry. Motor deficits were measured via grid walk test and short-term memory was tested by Y maze at 1, 2, 4, 8, 12, and 16 weeks post-ICH or sham. B cell repertoire analysis was conducted by PCR amplification and molecular cloning of B cells in 8 week post-ICH or naïve brain tissue, followed by Sanger sequencing and comparison to the IMGT database. Results: B cells are robustly recruited to the ipsilateral hemisphere as early as 2 weeks post-ICH (2439 +/- 987 cells; n=5) and persist at 8 weeks (14,162 +/- 12,103 cells, n=4). At 2 and 4 weeks a small fraction of B cells in the ipsilateral hemisphere are IgA+ (2 weeks=14.0% +/- 6.8%, n=5; 4 weeks=13.3% +/- 4.6%, n=3). Interestingly, by 8 weeks post-ICH this proportion significantly increased in ICH (41.3% +/- 22.8, n=6) but not sham controls (12.0% +/- 11.8%, n=5). While our work has demonstrated no significant motor deficit after 2 weeks post-ICH, preliminary data suggests a cognitive short-term memory deficit starting 16 weeks post-ICH. Furthermore, we have found that post-ICH brain-infiltrating IgA+ B cells are somatically hypermutated and show diverse V gene usage. Conclusion: Our findings suggest that ICH induces a robust, delayed IgA+ B cell response within the affected brain hemisphere, underscoring the need to better understand how B cells shape post-stroke inflammation and recovery, particularly at late timepoints.

Revisiting Transcranial Light Stimulation as a Stroke Therapeutic-Hurdles and Opportunities.

Near-infrared laser therapy, a special form of transcranial light therapy, has been tested as an acute stroke therapy in three large clinical trials. While the NEST trials failed to show the efficacy of light therapy in human stroke patients, there are many lingering questions and lessons that can be learned. In this review, we summarize the putative mechanism of light stimulation in the setting of stroke, highlight barriers, and challenges during the translational process, and evaluate light stimulation parameters, dosages and safety issues, choice of outcomes, effect size, and patient selection criteria. In the end, we propose potential futureopportunities with transcranial light stimulation as a cerebroprotective or restorative tool for future stroke treatment.

Ischemic stroke can have a T1w hyperintense appearance in absence of intralesional hemorrhage.

Magnetic resonance imaging (MRI) signal changes associated with ischemic stroke are typically described as T2w and FLAIR hyperintense, and T1w isointense lesions. Intralesional T1w hyperintensity is generally attributed to either a hemorrhagic stroke, or an ischemic stroke with hemorrhagic transition, and has an associated signal void on gradient echo (GE) sequences. Cases of ischemic stroke with T1w hyperintense signal in absence of associated signal void on GE sequences have been sporadically demonstrated in human stroke patients, as well as in dogs with experimentally induced ischemia of the middle cerebral artery. This multicenter retrospective descriptive study investigates the presence of T1w hyperintensity in canine stroke without associated signal void on GE sequences. High field (1.5 Tesla) MRI studies of 12 dogs with clinical presentation, MRI features, and cerebrospinal fluid results suggestive of non-hemorrhagic stroke were assessed. The time between the observed onset of clinical signs and MRI assessment was recorded. All 12 patients had an intralesional T1w hyperintense signal compared to gray and white matter, and absence of signal void on T2*w GE or SWI sequences. Intralesional T1w hyperintensities were either homogenously distributed throughout the entire lesion (6/12) or had a rim-like peripheral distribution (6/12). The mean time between the recorded onset of clinical signs and MRI assessment was 3 days; however, the age range of lesions with T1w hyperintense signal observed was 1–21days, suggesting that such signal intensities can be observed in acute, subacute, or chronic stages of ischemic stroke. Follow-up was recorded for 7/12 cases, all of which showed evidence of neurological improvement while in hospital, and survived to discharge. Correlation of the age and MRI appearance of lesions in this study with similar lesions observed in human and experimental studies suggests that these T1w hyperintensities are likely caused by partial tissue infarction or selective neuronal necrosis, providing an alternative differential for these T1w hyperintensities observed.

Amide proton transfer imaging in stroke.

Amide proton transfer (APT) imaging, a variant of chemical exchange saturation transfer MRI, has shown promise in detecting ischemic tissue acidosis following impaired aerobic metabolism in animal models and in human stroke patients due to the sensitivity of the amide proton exchange rate to changes in pH within the physiological range. Recent studies have demonstrated the possibility of using APT-MRI to detect acidosis of the ischemic penumbra, enabling the assessment of stroke severity and risk of progression, monitoring of treatment progress, and prognostication of clinical outcome. This paper reviews current APT imaging methods actively used in ischemic stroke research and explores the clinical aspects of ischemic stroke and future applications for these methods.

Neural Network Models for Spinal Implementation of Muscle Synergies.

Muscle synergies have been proposed as functional modules to simplify the complexity of body motor control; however, their neural implementation is still unclear. Converging evidence suggests that output projections of the spinal premotor interneurons (PreM-INs) underlie the formation of muscle synergies, but they exhibit a substantial variation across neurons and exclude standard models assuming a small number of unitary “modules” in the spinal cord. Here we compared neural network models for muscle synergies to seek a biologically plausible model that reconciles previous clinical and electrophysiological findings. We examined three neural network models: one with random connections (non-synergy model), one with a small number of spinal synergies (simple synergy model), and one with a large number of spinal neurons representing muscle synergies with a certain variation (population synergy model). We found that the simple and population synergy models emulate the robustness of muscle synergies against cortical stroke observed in human stroke patients. Furthermore, the size of the spinal variation of the population synergy matched well with the variation in spinal PreM-INs recorded in monkeys. These results suggest that a spinal population with moderate variation is a biologically plausible model for the neural implementation of muscle synergies.

Abstract TP247: Tanshinone IIA Loaded Nanoparticles Enhance Induced Pluripotent Stem Cell-derived Neural Stem Cell Recovery Responses In A Porcine Ischemic Stroke Model

Induced pluripotent stem cell-derived neural stem cells (iNSCs) have shown significant therapeutic promise in rodent stroke models with neuroprotective, regenerative, and cell replacement effects. However, the therapeutic potency of iNSCs has been largely limited by low iNSC survivability in the cytotoxic stroke environment. In this study, we examined the potential of Tanshinone IIA, an anti-inflammatory and antioxidative agent, loaded nanoparticles (Tan IIA-NPs) to improve iNSC survival, engraftment, and recovery responses in a porcine ischemic stroke model. Eighteen Yucatan pigs underwent middle cerebral artery occlusion (MCAO) surgery and were assigned to PBS+PBS, PBS+iNSC, or Tan IIA-NP+iNSC treatment groups. PBS or Tan IIA-NPs were administered intracisternally 1 hour post-stroke and PBS or iNSCs were transcranially transplanted into the penumbra stroke region 5 days post-stroke. Porcine adapted Modified Rankin Scale (mRS) neurologic scores were recorded pre- and post-stroke. Magnetic resonance imaging (MRI) and immunohistochemistry were performed 12 weeks post-iNSC transplantation. mRS evaluations demonstrated that Tan IIA-NP+iNSC treatment led to faster and improved (p<0.05) recovery at 1, 4, and 12 weeks post-transplantation relative to PBS+PBS and PBS+iNSC treatments. MRI results at 12 weeks showed a stepwise decrease in lesion volume and midline shift in PBS+PBS, PBS+iNSC, and Tan IIA-NP+iNSC groups with the Tan IIA-NP+iNSC group showing a significant decrease (p<0.05) relative to the PBS+PBS group. Immunohistochemistry analysis showed that Tan IIA-NPs resulted in increased (p<0.05) iNSC survival and differentiation into NeuN+ neurons and decreased (p<0.05) differentiation into GFAP+ astrocytes. These promising results indicate that the combined Tan IIA-NP+iNSC therapy leads to improved iNSC survival and engraftment, tissue and functional recovery, and may be a viable treatment for human stroke patients.

Roles of Polymorphonuclear Neutrophils in Ischemic Brain Injury and Post-Ischemic Brain Remodeling.

Following ischemic stroke, polymorphonuclear neutrophils (PMNs) are rapidly recruited to the ischemic brain tissue and exacerbate stroke injury by release of reactive oxygen species (ROS), proteases and proinflammatory cytokines. PMNs may aggravate post-ischemic microvascular injury by obstruction of brain capillaries, contributing to reperfusion deficits in the stroke recovery phase. Thus, experimental studies which specifically depleted PMNs by delivery of anti-Ly6G antibodies or inhibited PMN brain entry, e.g., by CXC chemokine receptor 2 (CXCR2) or very late antigen-4 (VLA-4) blockade in the acute stroke phase consistently reduced neurological deficits and infarct volume. Although elevated PMN responses in peripheral blood are similarly predictive for large infarcts and poor stroke outcome in human stroke patients, randomized controlled clinical studies targeting PMN brain infiltration did not improve stroke outcome or even worsened outcome due to serious complications. More recent studies showed that PMNs have decisive roles in post-ischemic angiogenesis and brain remodeling, most likely by promoting extracellular matrix degradation, thereby amplifying recovery processes in the ischemic brain. In this minireview, recent findings regarding the roles of PMNs in ischemic brain injury and post-ischemic brain remodeling are summarized.

Chapter 20 - Biomarkers of plasticity for stroke recovery

Stroke is the commonest cause of physical disability in the world. Our understanding of the biologic mechanisms involved in recovery and repair has advanced to the point that therapeutic opportunities to promote recovery through manipulation of post-stroke plasticity have never been greater. This work has almost exclusively been carried out in rodent models of stroke with little translation into human studies. The challenge ahead is to develop a mechanistic understanding of recovery from stroke in humans. Advances in neuroimaging techniques can now provide the appropriate intermediate level of description to bridge the gap between a molecular and cellular account of recovery and a behavioral one. Clinical trials can then be designed in a stratified manner taking into account when an intervention should be delivered and who is most likely to benefit. This approach is most likely to lead to the step-change in how restorative therapeutic strategies are delivered in human stroke patients.

Thrombolysis by PLAT/tPA increases serum free IGF1 leading to a decrease of deleterious autophagy following brain ischemia

ABSTRACT Cerebral ischemia is a pathology involving a cascade of cellular mechanisms, leading to the deregulation of proteostasis, including macroautophagy/autophagy, and finally to neuronal death. If it is now accepted that cerebral ischemia induces autophagy, the effect of thrombolysis/energy recovery on proteostasis remains unknown. Here, we investigated the effect of thrombolysis by PLAT/tPA (plasminogen activator, tissue) on autophagy and neuronal death. In two in vitro models of hypoxia reperfusion and an in vivo model of thromboembolic stroke with thrombolysis by PLAT/tPA, we found that ischemia enhances neuronal deleterious autophagy. Interestingly, PLAT/tPA decreases autophagy to mediate neuroprotection by modulating the PI3K-AKT-MTOR pathways both in vitro and in vivo. We identified IGF1R (insulin-like growth factor I receptor; a tyrosine kinase receptor) as the effective receptor and showed in vitro, in vivo and in human stroke patients and that PLAT/tPA is able to degrade IGFBP3 (insulin-like growth factor binding protein 3) to increase IGF1 (insulin-like growth factor 1) bioavailability and thus IGF1R activation. Abbreviations: AKT/protein kinase B: thymoma viral proto-oncogene 1; EGFR: epidermal growth factor receptor; Hx: hypoxia; IGF1: insulin-like growth factor 1; IGF1R: insulin-like growth factor I receptor; IGFBP3: insulin-like growth factor binding protein 3; Ka: Kainate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OGD: oxygen and glucose deprivation; OGDreox: oxygen and glucose deprivation + reoxygentation; PepA: pepstatin A1; PI3K: phosphoinositide 3-kinase; PLAT/tPA: plasminogen activator, tissue; PPP: picropodophyllin; SCH77: SCH772984; ULK1: unc-51 like kinase 1; Wort: wortmannin

Danger-associated molecular patterns are locally released during occlusion in hyper-acute stroke

ObjectiveImmune responses are an integral part of the complex reactions to acute cerebral ischemia and contribute to infarct expansion and tissue remodeling. Among damage-associated molecular patterns (DAMPs) the high-mobility group box 1 protein (HMGB1) and calprotectin (S100A8/A9) are released from dying cells and activate the innate immune system. MethodsTo assess DAMPs concentrations and related leukocytic infiltration directly and locally in human stroke patients we performed microcatheter sampling from within the core of the occluded vascular compartment before recanalization by mechanical thrombectomy. These samples from the core of a sealed cerebral-ischemic arterial compartment were compared with systemic control samples from the internal carotid artery obtained after recanalization. ResultsWe found increased plasma levels of total free HMGB1 (+33%) and increased S100A8/A9 (+8%) locally within the ischemic cerebral compartment vs. systemic levels. Local concentrations of HMGB1 were associated with more extensive structural brain infarction on admission. In addition, local ischemic HMGB1 and S100A8/A9 concentrations were associated with the numbers of leukocytes that infiltrate the occluded compartment by collateral pathways. ConclusionThis is the first direct human observation of a local increase in DAMPs concentrations in a uniquely sealed vascular compartment of the ischemic cerebral circulation. These data provide an important pathophysiological link between ischemia-induced cell death and stroke-related inflammation.

Preclinical animal studies in ischemic stroke: Challenges and some solutions.

Despite the impressive efficacies demonstrated in preclinical research, hundreds of potentially neuroprotective drugs have failed to provide effective neuroprotection for ischemic stroke in human clinical trials. Lack of a powerful animal model for human ischemic stroke could be a major reason for the failure to develop successful neuroprotective drugs for ischemic stroke. This review recapitulates the available cerebral ischemia animal models, provides an anatomical comparison of the circle of Willis of each species, and describes the functional assessment tests used in these ischemic stroke models. The distinct differences between human ischemic stroke and experimental stroke in available animal models is explored. Innovative animal models more closely resembling human strokes, better techniques in functional outcome assessment and better experimental designs generating clearer and stronger evidence may help realise the development of truly neuroprotective drugs that will benefit human ischemic stroke patients. This may involve use of newer molecules or revisiting earlier studies with new experimental designs. Translation of any resultant successes may then be tested in human clinical trials with greater confidence and optimism.

X chromosome escapee genes are involved in ischemic sexual dimorphism through epigenetic modification of inflammatory signals

BackgroundStroke is a sexually dimorphic disease. Previous studies have found that young females are protected against ischemia compared to males, partially due to the protective effect of ovarian hormones, particularly estrogen (E2). However, there are also genetic and epigenetic effects of X chromosome dosage that contribute to stroke sensitivity and neuroinflammation after injury, especially in the aged. Genes that escape from X chromosome inactivation (XCI) contribute to sex-specific phenotypes in many disorders. Kdm5c and kdm6a are X escapee genes that demethylate H3K4me3 and H3K27me3, respectively. We hypothesized that the two demethylases play critical roles in mediating the stroke sensitivity.MethodsTo identify the X escapee genes involved in stroke, we performed RNA-seq in flow-sorted microglia from aged male and female wild type (WT) mice subjected to middle cerebral artery occlusion (MCAO). The expression of these genes (kdm5c/kdm6a) were confirmed in four core genotypes (FCG) mice and in post-mortem human stroke brains by immunohistochemistry (IHC), Western blot, and RT-PCR. Chromatin immunoprecipitation (ChIP) assays were conducted to detect DNA levels of inflammatory interferon regulatory factor (IRF) 4/5 precipitated by histone H3K4 and H3K27 antibodies. Manipulation of kdm5c/kdm6a expression with siRNA or lentivirus was performed in microglial culture, to determine downstream pathways and examine the regulatory roles in inflammatory cytokine production.ResultsKdm5c and kdm6a mRNA levels were significantly higher in aged WT female vs. male microglia, and the sex difference also existed in ischemic brains from FCG mice and human stroke patients. The ChIP assay showed the IRF 4/5 had higher binding levels to demethylated H3K4 or H3K27, respectively, in female vs. male ischemic microglia. Knockdown or over expression of kdm5c/kdm6a with siRNA or lentivirus altered the methylation of H3K4 or H3K27 at the IRF4/5 genes, which in turn, impacted the production of inflammatory cytokines.ConclusionsThe KDM-Histone-IRF pathways are suggested to mediate sex differences in cerebral ischemia. Epigenetic modification of stroke-related genes constitutes an important mechanism underlying the ischemic sexual dimorphism.

IgA natural antibodies are produced following T-cell independent B-cell activation following stroke

Up to 30% of stroke patients experience cognitive decline within one year of their stroke. There are currently no FDA-approved drugs that can prevent post-stroke cognitive decline, in part due to a poor understanding of the mechanisms involved. We have previously demonstrated that a B-lymphocyte response to stroke, marked by IgA + cells, can cause delayed cognitive dysfunction in mice and that a similar adaptive immune response occurs in the brains of some human stroke patients that suffer from vascular dementia. The stimuli which trigger B-lymphocyte activation following stroke, and their target antigens, are still unknown. Therefore, to learn more about the mechanisms by which B-lymphocytes become activated following stroke we first characterized the temporal kinetics of the B-lymphocyte, T-lymphocyte, and plasma cell (PC) response to stroke in the brain by immunohistochemistry (IHC). We discovered that B-lymphocyte, T-lymphocyte, and plasma cell infiltration within the infarct progressively increases between 2 and 7 weeks after stroke. We then compared the B-lymphocyte response to stroke in WT, MHCII-/-, CD4-/-, and MyD88-/- mice to determine if B-lymphocytes mature into IgA + PCs through a T-lymphocyte and MyD88 dependent mechanism. Our data from a combination of IHC and flow cytometry indicate that following stroke, a population of IgA + PCs develops independently of CD4 + helper T-lymphocytes and MyD88 signaling. Subsequent sequencing of immunoglobulin genes of individual IgA + PCs present within the infarct identified a novel population of natural antibodies with few somatic mutations in complementarity-determining regions. These findings indicate that a population of IgA + PCs develops in the infarct following stroke by B-lymphocytes interacting with one or more thymus independent type 2 (TI-2) antigens, and that they produce IgA natural antibodies.

Ponatinib-induced ischemic stroke in larval zebrafish for drug screening

Conventional mammalian ischemic stroke models for drug screening are technically challenging, laborious and time-consuming. In this study, using Ponatinib as an inducer, we developed and characterized a zebrafish ischemic stroke model. This zebrafish ischemic stroke had the cerebral vascular endothelial injury, thrombosis, reduced blood flow, inflammation and apoptosis as well as the reduced motility. The zebrafish ischemic stroke model was validated with 6 known human therapeutic drugs of ischemic stroke (Aspirin, Clopidogrel, Naoxintong capsules, Edaravone, Xingnaojing injection, Shuxuening injection). The mRNA levels of the neovascularization-related gene (vegfaa) and vascular endothelial growth factor receptor gene (VEGFR), neurodevelopment related genes (mbp and α1-tubulin), brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) were significantly downregulated; whereas apoptosis-related genes (caspase-3, caspase-7, caspase-9 and bax/bcl-2), and inflammatory factor genes (IL-1β, IL-6, IL-10, TNF-α and NF-κB) were remarkably upregulated in the model. These results suggest that the pathophysiology of Ponatinib-induced zebrafish ischemic stroke is similar to that of human ischemic stroke patients and this whole animal model could be used to study the complex cellular and molecular pathogenesis of ischemic stroke and to rapidly identify therapeutic agents.

Perlecan Domain-V Enhances Neurogenic Brain Repair After Stroke in Mice

The extracellular matrix fragment perlecan domain V is neuroprotective and functionally restorative following experimental stroke. As neurogenesis is an important component of chronic post-stroke repair, and previous studies have implicated perlecan in developmental neurogenesis, we hypothesized that domain V could have a broad therapeutic window by enhancing neurogenesis after stroke. We demonstrated that domain V is chronically increased in the brains of human stroke patients, suggesting that it is present during post-stroke neurogenic periods. Furthermore, perlecan deficient mice had significantly less neuroblast precursor cells after experimental stroke. Seven-day delayed domain V administration enhanced neurogenesis and restored peri-infarct excitatory synaptic drive to neocortical layer 2/3 pyramidal neurons after experimental stroke. Domain V’s effects were inhibited by blockade of α2β1 integrin, suggesting the importance of α2β1 integrin to neurogenesis and domain V neurogenic effects. Our results demonstrate that perlecan plays a previously unrecognized role in post-stroke neurogenesis and that delayed DV administration after experimental stroke enhances neurogenesis and improves recovery in an α2β1 integrin-mediated fashion. We conclude that domain V is a clinically relevant neuroprotective and neuroreparative novel stroke therapy with a broad therapeutic window.

Triiodothyronine modulates neuronal plasticity mechanisms to enhance functional outcome after stroke

The development of new therapeutic approaches for stroke patients requires a detailed understanding of the mechanisms that enhance recovery of lost neurological functions. The efficacy to enhance homeostatic mechanisms during the first weeks after stroke will influence functional outcome. Thyroid hormones (TH) are essential regulators of neuronal plasticity, however, their role in recovery related mechanisms of neuronal plasticity after stroke remains unknown. This study addresses important findings of 3,5,3′-triiodo-L-thyronine (T3) in the regulation of homeostatic mechanisms that adjust excitability – inhibition ratio in the post-ischemic brain. This is valid during the first 2 weeks after experimental stroke induced by photothrombosis (PT) and in cultured neurons subjected to an in vitro model of acute cerebral ischemia. In the human post-stroke brain, we assessed the expression pattern of TH receptors (TR) protein levels, important for mediating T3 actions.Our results show that T3 modulates several plasticity mechanisms that may operate on different temporal and spatial scales as compensatory mechanisms to assure appropriate synaptic neurotransmission. We have shown in vivo that long-term administration of T3 after PT significantly (1) enhances lost sensorimotor function; (2) increases levels of synaptotagmin 1&2 and levels of the post-synaptic GluR2 subunit in AMPA receptors in the peri-infarct area; (3) increases dendritic spine density in the peri-infarct and contralateral region and (4) decreases tonic GABAergic signaling in the peri-infarct area by a reduced number of parvalbumin+ / c-fos+ neurons and glutamic acid decarboxylase 65/67 levels. In addition, we have shown that T3 modulates in vitro neuron membrane properties with the balance of inward glutamate ligand-gated channels currents and decreases synaptotagmin levels in conditions of deprived oxygen and glucose. Interestingly, we found increased levels of TRβ1 in the infarct core of post-mortem human stroke patients, which mediate T3 actions. Summarizing, our data identify T3 as a potential key therapeutic agent to enhance recovery of lost neurological functions after ischemic stroke.