RNA sequencing reveals novel macrophage transcriptome favoring neurovascular plasticity after ischemic stroke

Blood monocytes/macrophages infiltrate the brain after ischemic stroke and critically influence brain injury and regeneration. We investigated stroke-induced transcriptomic changes of monocytes/macrophages by RNA sequencing profiling, using a mouse model of permanent focal cerebral ischemia. Compared to non-ischemic conditions, brain ischemia induced only moderate genomic changes in blood monocytes, but triggered robust genomic reprogramming in monocytes/macrophages invading the brain. Surprisingly, functional enrichment analysis of the transcriptome of brain macrophages revealed significant overrepresentation of biological processes linked to neurovascular remodeling, such as angiogenesis and axonal regeneration, as early as five days after stroke, suggesting a previously underappreciated role for macrophages in initiating post-stroke brain repair. Upstream Regulator analysis predicted peroxisome proliferator-activated receptor gamma (PPARγ) as a master regulator driving the transcriptional reprogramming in post-stroke brain macrophages. Importantly, myeloid cell-specific PPARγ knockout (mKO) mice demonstrated lower post-stroke angiogenesis and neurogenesis than wild-type mice, which correlated significantly with the exacerbation of post-stroke neurological deficits in mKO mice. Collectively, our findings reveal a novel repair-enhancing transcriptome in brain macrophages during post-stroke neurovascular remodeling. As a master switch controlling genomic reprogramming, PPARγ is a rational therapeutic target for promoting and maintaining beneficial macrophage functions, facilitating neurorestoration, and improving long-term functional recovery after ischemic stroke.

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Abstract 110: RNA Sequencing Reveals Novel Macrophage Transcriptome Favoring Neurovascular Plasticity After Ischemic Stroke

Stroke ◽

10.1161/str.51.suppl_1.110 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Yejie Shi ◽

Rongrong Wang ◽

Yaan Liu ◽

Qing Ye ◽

Sulaiman H Hassan ◽

...

Keyword(s):

Ischemic Stroke ◽

Myeloid Cell ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Functional Enrichment ◽

Neurological Deficits ◽

Blood Monocytes ◽

Genomic Changes ◽

Post Stroke ◽

Neurovascular Remodeling

Introduction: Blood monocytes/macrophages (MΦ) infiltrate the brain in massive numbers after ischemic stroke, but their functional roles in the post-stroke brain remain elusive. Methods: We sorted CD11b + CD45 high monocytes/MΦ from mouse blood and brain 5d after distal MCAO (dMCAO) and investigated their transcriptomic profiles by RNA-seq (n=3/group). Results: Robust genomic changes occurred in MΦ invading the post-dMCAO brain compared to their counterparts in the blood (3196 differentially expressed genes with >2 fold changes; FDR<0.05). In addition to their canonical role in immune responses, brain MΦ expressed unique transcriptomic programs which may favor post-stroke neurovascular repair. Functional enrichment analysis of brain MΦ transcriptome revealed significant overrepresentation (p<0.01) of 110 biological processes linked to neurovascular remodeling and plasticity (e.g., angiogenesis and axonal regeneration), suggesting a previously underappreciated role for MΦ in post-stroke brain repair. Upstream Regulator analysis predicted PPARγ as a master regulator (z-score=2.64, p<10 -15 ) that drives the transcriptional reprogramming in post-stroke brain MΦ and dictates their functional phenotype. Accordingly, tamoxifen-induced, myeloid cell-specific PPARγ knockout mice demonstrated less post-stroke angiogenesis (BrdU+CD31 staining) and neurogenesis (BrdU+NeuN) than WT mice (n=9/group, p<0.01), which correlated significantly with the exacerbation of neurological deficits in these mice (n=9/group, p<0.01 vs WT in foot fault and adhesive removal tests) at 35d after dMCAO. Conclusions: Our study reveals a novel repair-enhancing transcriptome in brain MΦ during post-stroke neurovascular remodeling. As a master molecule controlling genomic reprogramming, PPARγ is a rational therapeutic target for promoting beneficial MΦ functions, and facilitating neurorestoration and long term functional recovery after ischemic stroke.

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Abstract TP274: Orosomucoid-1 Protein Increases Following Ischemic Stroke in the Brain and Periphery

Stroke ◽

10.1161/str.47.suppl_1.tp274 ◽

2016 ◽

Vol 47 (suppl_1) ◽

Author(s):

Hetal Mistry ◽

Madeline Levy ◽

Meaghan Roy-O'Reilly ◽

Louise McCullough

Keyword(s):

Sex Differences ◽

Ischemic Stroke ◽

Rna Sequencing ◽

Multiple Testing ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Stroke Patients ◽

Post Stroke ◽

Protein Levels ◽

The Brain

Background and Purpose: Orosomucoid-1 (ORM-1) is an abundant protein with important roles in inflammation and immunosuppression. We utilized RNA sequencing to measure mRNA levels in human ischemic stroke patients, with confirmation by serum ORM-1 protein measurements. A mouse model of ischemic stroke was then used to examine post-stroke changes in ORM-1 within the brain itself. Hypothesis: We tested the hypothesis that ORM-1 levels increase following ischemic stroke, with sex differences in protein dynamics over time. Methods: RNA sequencing was performed on whole blood from ischemic stroke patients (n=23) and controls (n=12), with Benjamini-Hochberg correction for multiple testing. Enzyme-linked immunosorbent assay was performed on serum from ischemic stroke patients (n=28) and controls (n=8), with analysis by T-test. For brain analysis, mice (n=14) were subjected to a 90-minute middle cerebral artery occlusion (MCAO) surgery and sacrificed 6 or 24 hours after stroke. Control mice underwent parallel “sham” surgery without occlusion. Western blotting was used to detect ORM-1 protein levels in whole brain, with analysis by two-way ANOVA. Results: RNA sequencing showed a 2.8-fold increase in human ORM-1 at 24 hours post-stroke (q=.0029), an increase also seen in serum ORM-1 protein levels (p=.011). Western blot analysis of mouse brain revealed that glycosylated (p=0.0003) and naive (p=0.0333) forms of ORM-1 were higher in female mice compared to males 6 hours post-stroke. Interestingly, ORM-1 levels were higher in the brains of stroke mice at 6 hours (p=.0483), while at 24 hours ORM-1 levels in stroke mice were lower than their sham counterparts (p=.0212). In both human and mouse data, no sex differences were seen in ORM-1 levels in the brain or periphery at 24 hours post-stroke. Conclusion: In conclusion, ORM-1 is a sexually dimorphic protein involved in the early (<24 hour) response to ischemic stroke. This research serves as an initial step in determining the mechanism of ORM-1 in the ischemic stroke response and its potential as a future therapeutic target for both sexes.

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Neuroprotection via AT2 receptor agonists in ischemic stroke

Clinical Science ◽

10.1042/cs20171549 ◽

2018 ◽

Vol 132 (10) ◽

pp. 1055-1067 ◽

Cited By ~ 10

Author(s):

Douglas M. Bennion ◽

U. Muscha Steckelings ◽

Colin Sumners

Keyword(s):

Ischemic Stroke ◽

Neuronal Damage ◽

Recombinant Tissue Plasminogen Activator ◽

Neurological Deficits ◽

At2 Receptor ◽

Neuroprotective Effects ◽

Receptor Agonists ◽

Stroke Epidemiology ◽

The Brain

Stroke is a devastating disease that afflicts millions of people each year worldwide. Ischemic stroke, which accounts for ~88% of cases, occurs when blood supply to the brain is decreased, often because of thromboembolism or atherosclerotic occlusion. This deprives the brain of oxygen and nutrients, causing immediate, irreversible necrosis within the core of the ischemic area, but more delayed and potentially reversible neuronal damage in the surrounding brain tissue, the penumbra. The only currently approved therapies for ischemic stroke, the thrombolytic agent recombinant tissue plasminogen activator (rtPA) and the endovascular clot retrieval/destruction processes, are aimed at restoring blood flow to the infarcted area, but are only available for a minority of patients and are not able in most cases to completely restore neurological deficits. Consequently, there remains a need for agents that will protect neurones against death following ischemic stroke. Here, we evaluate angiotensin II (Ang II) type 2 (AT2) receptor agonists as a possible therapeutic target for this disease. We first provide an overview of stroke epidemiology, pathophysiology, and currently approved therapies. We next review the large amount of preclinical evidence, accumulated over the past decade and a half, which indicates that AT2 receptor agonists exert significant neuroprotective effects in various animal models, and discuss the potential mechanisms involved. Finally, after discussing the challenges of delivering blood–brain barrier (BBB) impermeable AT2 receptor agonists to the infarcted areas of the brain, we summarize the evidence for and against the development of these agents as a promising therapeutic strategy for ischemic stroke.

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Abstract TP111: Activation of the Brain Renin-Angiotensin System by Translational Approaches Following Stroke Onset Is Neuroprotective in a Rat Model of Ischemic Stroke

Stroke ◽

10.1161/str.44.suppl_1.atp111 ◽

2013 ◽

Vol 44 (suppl_1) ◽

Author(s):

Douglas M Bennion ◽

Lauren Donnangelo ◽

David Pioquinto ◽

Robert Regenhardt ◽

Mohan K Raizada ◽

...

Keyword(s):

Ischemic Stroke ◽

Infarct Volume ◽

Renin Angiotensin System ◽

Cerebral Infarct ◽

Intraventricular Administration ◽

Angiotensin System ◽

Renin Angiotensin ◽

Post Stroke ◽

Neurologic Deficits ◽

The Brain

Background: Toward discovering novel stroke therapies, recent research has shown that activation of the newly-discovered angiotensin converting enzyme 2/angiotensin-(1-7)/mas (ACE2/Ang-(1-7)/Mas) pathway, a counter-regulatory axis of the brain renin-angiotensin system, is neuroprotective in ischemic stroke in rats. Specifically, intraventricular administration of the novel ACE2 activator diminazine aceturate (DIZE) before and during an ischemic stroke decreases cerebral infarct and neurologic deficits. Efficacy must now be demonstrated using minimally-invasive methods if this therapy is to be translated to the care of human patients. In this study, we assessed the hypothesis that systemic administration of DIZE post ischemic stroke would be neuroprotective. Methods: Adult male Sprague-Dawley rats underwent ischemic stroke by endothelin-1 induced middle cerebral artery occlusion and were randomly divided into 2 groups (n=9-10/set): 1) intraperitoneal (IP) administrations of DIZE (7.5 mg/kg) at 4, 24, and 48 h after stroke; 2) IP administrations of 0.9% saline vehicle at the same time points. At 24 and 72 h after stroke, rats underwent blinded neurologic assessments. Immediately following the 72 h tests, animals were sacrificed, cerebral infarct volumes assessed by TTC staining, and IL-1β expression in the stroke region analyzed by rt-PCR. Data are expressed as mean ± SEM with significance inferred at p<0.05. Results: Mean infarct volume was significantly decreased by IP injections of DIZE (9.4% ± 4.35) as compared to control (22.8%±3.6, p=0.039). At 24 h post stroke, neurologic deficits (Garcia Scale) were significantly improved in the DIZE treated group (16.7±0.40) versus the saline group (15.22±0.57, p=0.037). Although DIZE tended to improve neurologic deficits 72 h post stroke, this trend was not significant. Finally, DIZE treatment significantly reduced mRNA expression of IL-1β (0.43 ± 0.14) in the cerebral cortical stroke region as compared to saline treatment (1.47±0.08, p=0.001). Conclusions: Our findings suggest that targeting the ACE2/Ang-(1-7)/Mas axis post stroke can improve function, decrease inflammation, and reduce infarct volume - a significant translational step in brain renin-angiotensin system research.

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Age-dependent involvement of gut mast cells and histamine in post-stroke inflammation

10.21203/rs.2.21718/v1 ◽

2020 ◽

Author(s):

Maria Pilar Blasco ◽

Anjali Chauhan ◽

Pedram Honarpisheh ◽

Hilda Ahnstedt ◽

John d’Aigle ◽

...

Keyword(s):

Ischemic Stroke ◽

Mast Cells ◽

Receptor Expression ◽

Mast Cell Activation ◽

Low Grade ◽

Gut Inflammation ◽

Aged Mice ◽

Post Stroke ◽

Age Related ◽

The Brain

Abstract Background Risk of stroke-related morbidity and mortality increases significantly with age. Aging is associated with chronic, low-grade inflammation, which is thought to contribute to the poorer outcomes after stroke seen in the elderly. Histamine (HA) is a major molecular mediator of inflammation and mast cells residing in the gut are a primary source of histamine. Methods Stroke was induced in male C57BL/6J mice at 3 months (young) and 20 months (aged) of age. Role of histamine after stroke was examined using young (Yg) and aged (Ag) mice, mice underwent MCAO surgery and were euthanized at 6h, 24h and 7 days post-ischemia; sham mice received the same surgery but no MCAO. In this work, we evaluated whether worsened outcomes after experimental stroke in aged mice was associated with age-related changes in mast cells, histamine levels, and histamine receptor expression in the gut, brain, and plasma. Results We found increased numbers of mast cells in the gut and the brain with aging. Using the middle cerebral artery occlusion (MCAO) model of ischemic stroke, we demonstrate that stroke leads to increased numbers of mast cells and histamine receptors in the gut. These gut-centric changes are associated with elevated levels of HA and other pro-inflammatory cytokines including IL-6, G-CSF, TNF-α, and IFN-γ in the peripheral circulation. Our data also shows that post-stroke gut inflammation led to a significant reduction of mucin-producing goblet cells and a loss of gut barrier integrity. Lastly, gut inflammation after stroke is associated with changes in the composition of the gut microbiota as early as 24 hours post-stroke. Conclusion An important theme emerging from our results is that acute inflammatory events following ischemic insults in the brain persist longer in the aged mice when compared to younger animals. Taken together, our findings implicate mast cell activation and histamine signaling as a part of peripheral inflammatory response after ischemic stroke, which are profound in aged animals. Interfering with histamine signaling orally might provide translational value to improve stroke outcome.

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Abstract WP261: Sex Differences in Circulating Leukocyte Subtype and Activation Following Ischemic Stroke

Stroke ◽

10.1161/str.47.suppl_1.wp261 ◽

2016 ◽

Vol 47 (suppl_1) ◽

Author(s):

Meaghan Roy-O'Reilly ◽

Hetal Mistry ◽

Madeline Levy ◽

Louise McCullough

Keyword(s):

Flow Cytometry ◽

Immune Response ◽

Sex Differences ◽

Ischemic Stroke ◽

Rna Sequencing ◽

Sequencing Analysis ◽

Stroke Patients ◽

Female Patients ◽

Post Stroke ◽

Upregulated Genes

Background and Purpose: Females exhibit a more robust immune response in many disease models, yet sex differences in the inflammatory response to ischemia remain largely unexplored. We conducted flow cytometry and RNA sequencing of blood from ischemic stroke patients, with follow-up studies in mice. Hypothesis: We assessed the hypothesis that there are sex differences in the acute immune response to ischemic stroke. Methods: Patient samples were drawn at 24 hours post-stroke for flow cytometry (n=6) and RNA sequencing Analysis (n=40). For murine studies, male and ovariectomized (OVX) female animals (n=14) were subjected to 90-minute middle cerebral artery occlusion or sham surgery and sacrificed at 24 hours. Murine blood was stained for leukocyte markers and CD62L (L-selectin). Results were analyzed by student T-test and two-way ANOVA. Results: RNA sequencing revealed that 24 hours after ischemic stroke, female patients had 79 significantly upregulated genes, compared to male patients with 6 significantly upregulated genes. Human flow cytometry revealed that male stroke patients had a significantly higher percentage of monocytes (p=.026), while females had a greater percentage of CD8+ T-cells (p=.023). Murine flow cytometry showed a post-stroke increase in peripheral myeloid cells at 24 hours in male mice only (p=.0046), whereas female mice had a higher CD8/CD4 T cell ratio (p=.0027). Neutrophils from male sham animals displayed greater L-selectin positivity, with stroke-induced shedding of L-selectin seen only in males (sex/stroke p=.0266). Male monocytes and lymphocytes also displayed higher L-selectin positivity (p=.0079, p=.0004). Conclusion: These results suggest that the immune response to ischemic stroke is different in male and female patients, a phenomenon that can be recapitulated in a mouse model of experimental stroke. Female immune cells exhibit a higher level of baseline activation (reduced L-selectin expression) and post-stroke activity, which may enable a quicker and more robust response to immune challenges. Understanding sex differences in the acute immune response is crucial to developing future immunomodulatory drugs for the safe and effective treatment of ischemic stroke in both sexes.

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A rare case report of Russell’s viper snakebite with ischemic stroke

Hong Kong Journal of Emergency Medicine ◽

10.1177/1024907917735071 ◽

2017 ◽

Vol 25 (2) ◽

pp. 95-97 ◽

Cited By ~ 1

Author(s):

VK Pothukuchi ◽

VR Chepuri ◽

Kalyani Natta ◽

Nagaraju Madigani ◽

Alok Kumar

Keyword(s):

Computed Tomography ◽

Ischemic Stroke ◽

Endothelial Damage ◽

Neurological Deficits ◽

Computed Tomography Scan ◽

Rare Case Report ◽

Russell’S Viper ◽

The Brain ◽

Underlying Pathology ◽

Tomography Scan

Ischemic stroke following snakebite is rare. We report a case of a 55-year-old male who developed headache with expressive aphasia following a Russell’s viper bite. Computed tomography scan of the brain revealed infarcts in bilateral frontal lobes. The possible mechanisms for cerebral infarction in this scenario are discussed, which include disseminated intravascular coagulation, toxin-induced vasculitis, and endothelial damage. In viper bites, whenever there is any central nervous system manifestations or deficits, immediate computed tomography scan of the brain should be taken to delineate the underlying pathology, whether hemorrhagic or ischemic, as treatments differ in both situations. Although ischemic stroke is rare in viper bites, if the treatment is started early, the neurological deficits can be minimized.

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The Effect of Intravenously and Intra-arterially Delivered Human Umbilical Cord Blood Mononuclear Cell on Cortical Neurogenesis of Post-Ischemic Stroke Rat Brain

Open Access Macedonian Journal of Medical Sciences ◽

10.3889/oamjms.2021.6555 ◽

2021 ◽

Vol 9 (A) ◽

pp. 1245-1251

Author(s):

Sastia Winda Astuti ◽

Isabella Kurnia Liem ◽

Yetty Ramli

Keyword(s):

Ischemic Stroke ◽

Cord Blood ◽

Rat Brain ◽

Cortical Neurons ◽

Mononuclear Cells ◽

Neurological Deficits ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Cortical Neurogenesis ◽

The Brain

BACKGROUND: Stroke is the second most cause of death in the world. There are several treatments but they often end up with disabilities. Recently, cell therapy has become a new hope as an alternative treatment as it could improve the patients neurological deficits and daily living activities. Cord blood mononuclear cells (CB-MNCs) are one of the cell therapies for post-ischemic neurogenesis by intravenous or intra-arterial administration; however, it is not clear which one is better. AIM: This study aims to compare the effects of intra-arterial and intravenous administration of human CB-MNC on cortical neurogenesis of rat brain after ischemic stroke. METHODS: Twenty-four rats were divided into four groups, that is, control, middle cerebral artery obstruction (MCAO) without treatment, MCAO with intra-arterial CB-MNC injection (MCAO-IA), and MCAO with intravenous CB-MNC injection (MCAO-IV). Two weeks after injection, all rats were sacrificed, the brain was harvested, histologically process and stained with hematoxylin eosin (HE) to determine cellular and tissue morphology changes, and immunohistochemical staining, anti-NeuN antibody to determine the number of cortical neurons. The HE showed that MCAO rat brain had gliosis and shrunken cells. RESULTS: The results showed that MCAO-IA and MCAO-IV had fewer areas of gliosis and shrunken cells when compared to the MCAO group. The number of neurons also showed an increase. However, there was no difference between the MCAO-IA and MCAO-IV groups. It was concluded both of them could improve neurogenesis. CONCLUSION: CB-MNC administration can be an alternative for stroke ischemic therapy because it is proven to increase neurogenesis and reduce gliosis areas. However, there was no difference in neurogenesis in the brain tissue of mice injected with CB-MNC intravenously or intra-arterially.

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Age-dependent involvement of gut mast cells and histamine in post-stroke inflammation

10.21203/rs.2.21718/v3 ◽

2020 ◽

Author(s):

Maria Pilar Blasco ◽

Anjali Chauhan ◽

Pedram Honarpisheh ◽

Hilda Ahnstedt ◽

John d’Aigle ◽

...

Keyword(s):

Ischemic Stroke ◽

Mast Cells ◽

Receptor Expression ◽

Mast Cell Activation ◽

Low Grade ◽

Gut Inflammation ◽

Aged Mice ◽

Post Stroke ◽

Age Related ◽

The Brain

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A New NF-κB Inhibitor, MEDS-23, Reduces the Severity of Adverse Post-Ischemic Stroke Outcomes in Rats

Brain Sciences ◽

10.3390/brainsci12010035 ◽

2021 ◽

Vol 12 (1) ◽

pp. 35

Author(s):

Elina Rubin ◽

Agnese C. Pippione ◽

Matthew Boyko ◽

Giacomo Einaudi ◽

Stefano Sainas ◽

...

Keyword(s):

Ischemic Stroke ◽

Artery Occlusion ◽

Brain Inflammation ◽

Neurological Deficits ◽

Stroke Outcomes ◽

Post Stroke ◽

Factor Α ◽

Cerebral Artery Occlusion ◽

First Time

Aim: Nuclear factor kappa B (NF-κB) is known to play an important role in the inflammatory process which takes place after ischemic stroke. The major objective of the present study was to examine the effects of MEDS-23, a potent inhibitor of NF-κB, on clinical outcomes and brain inflammatory markers in post-ischemic stroke rats. Main methods: Initially, a Toxicity Experiment was performed to determine the appropriate dose of MEDS-23 for use in animals, as MEDS-23 was analyzed in vivo for the first time. We used the middle cerebral artery occlusion (MCAO) model for inducing ischemic stroke in rats. The effects of MEDS-23 (at 10 mg/kg, ip) on post-stroke outcomes (brain inflammation, fever, neurological deficits, mortality, and depression- and anxiety-like behaviours) was tested in several efficacy experiments. Key findings: MEDS-23 was found to be safe and significantly reduced the severity of some adverse post-stroke outcomes such as fever and neurological deficits. Moreover, MEDS-23 significantly decreased prostaglandin E2 levels in the hypothalamus and hippocampus of post-stroke rats, but did not prominently alter the levels of interleukin-6 and tumor necrosis factor-α. Significance: These results suggest that NF-κB inhibition is a potential therapeutic strategy for the treatment of ischemic stroke.

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