Human Ischemic Stroke Research Articles

Identification of endoplasmic reticulum stress genes in human stroke based on bioinformatics and machine learning

After ischemic stroke (IS), secondary injury is intimately linked to endoplasmic reticulum (ER) stress and body-brain crosstalk. Nonetheless, the underlying mechanism systemic immune disorder mediated ER stress in human IS remains unknown. In this study, 32 candidate ER stress-related genes (ERSRGs) were identified by overlapping MSigDB ER stress pathway genes and DEGs. Three Key ERSRGs (ATF6, DDIT3 and ERP29) were identified using LASSO, random forest, and SVM-RFE. IS patients with different ERSRGs profile were clustered into two groups using consensus clustering and the difference between 2 group was further explored by GSVA. Through immune cell infiltration deconvolution analysis, and middle cerebral artery occlusion (MCAO) mouse scRNA analysis, we found that the expression of 3 key ERSRGs were closely related with peripheral macrophage cell ER stress in IS and this was further confirmed by RT-qPCR experiment. These ERS genes might be helpful to further accurately regulate the central nervous system and systemic immune response through ER stress and have potential application value in clinical practice in IS.

Serum heme oxygenase-1 level predicts clinical outcome after acute ischemic stroke.

The relationship between heme oxygenase-1 (HO-1) and human ischemic stroke outcome remains unclear, which was investigated in this study. Acute ischemic stroke patients admitted within 24 h were enrolled. Serum HO-1 levels at baseline were measured via ELISA. Poor 3-month functional outcome was defined as modified Rankin Scale (mRS) score 3-6. Multivariable-adjusted binary logistic regression and restricted cubic spline models were employed to examine association between serum HO-1 and functional outcome. HO-1's additive prognostic utility was assessed by net reclassification index (NRI) and integrated discrimination improvement (IDI). Of 194 eligible patients, 79 (40.7%) developed poor functional outcomes at 3-month follow-up. The highest quartile of serum HO-1 was independently associated with a lower risk of poor functional outcome (adjusted OR 0.13, 95% CI 0.04-0.45; p = 0.001) compared with the lowest HO-1 category. The relationship between higher HO-1 levels and reduced risk of poor functional outcome was linear and dose responsive (p = 0.002 for linearity). Incorporating HO-1 into the analysis with conventional factors significantly improved reclassification for poor functional outcomes (NRI = 41.2%, p = 0.004; IDI = 5.0%, p = 0.004). Elevated serum HO-1 levels at baseline were independently associated with improved 3-month functional outcomes post-ischemic stroke. Serum HO-1 measurement may enhance outcome prediction beyond conventional clinical factors.

Hyperchloremia and Hypernatremia Decreased Microglial and Neuronal Survival during Oxygen-Glucose Deprivation/Reperfusion.

Hyperchloremia and hypernatremia are associated with higher mortality in ischemic stroke, but it remains unclear whether their influence directly contributes to ischemic injury. We investigated the impact of 0.9% sodium chloride (154 mM NaCl), 0.9% sodium acetate (167 mM CH3COONa), and their different combinations (3:1, 2:1, and 1:1) on microglial (HMC-3) and neuronal (differentiated SH-SY5Y) survival during oxygen-glucose deprivation/reperfusion (OGD/R). Further, we assessed the effect of hyperchloremia and hypernatremia-treated and OGD/R-induced HMC-3-conditioned media on differentiated SH-SY5Y cells under OGD/R conditions. We performed cell viability, cell toxicity, and nitric oxide (NO) release assays and studied the alteration in expression of caspase-1 and caspase-3 in different cell lines when exposed to hyperchloremia and hypernatremia. Cell survival was decreased in 0.9% NaCl, 0.9% CH3COONa, combinations of HMC-3 and differentiated SH-SY5Y, and differentiated SH-SY5Y cells challenged with HMC-3-conditioned media under normal and OGD/R conditions. Under OGD/R conditions, differentiated SH-SY5Y cells were less likely to survive exposure to 0.9% NaCl. Expression of caspase-1 and caspase-3 in HMC-3 and differentiated SH-SY5Y cells was altered when exposed to 0.9% NaCl, 0.9% CH3COONa, and their combinations. A total of 0.9% NaCl and 0.9% CH3COONa and their combinations decreased the NO production in HMC-3 cells under normal and OGD/R conditions. Both hypernatremia and hyperchloremia reduced the survival of HMC-3 and differentiated SH-SY5Y cells under OGD/R conditions. Based on the OGD/R in vitro model that mimics human ischemic stroke conditions, it possibly provides a link for the increased death associated with hyperchloremia or hypernatremia in stroke patients.

MANF protein expression is upregulated in immune cells in the ischemic human brain and systemic recombinant MANF delivery in rat ischemic stroke model demonstrates anti-inflammatory effects

Mesencephalic astrocyte-derived neurotrophic factor (MANF) has cytoprotective effects on various injuries, including cerebral ischemia, and it can promote recovery even when delivered intracranially several days after ischemic stroke. In the uninjured rodent brain, MANF protein is expressed almost exclusively in neurons, but post-ischemic MANF expression has not been characterized. We aimed to investigate how endogenous cerebral MANF protein expression evolves in infarcted human brains and rodent ischemic stroke models. During infarct progression, the cerebral MANF expression pattern both in human and rat brains shifted drastically from neurons to expression in inflammatory cells. Intense MANF immunoreactivity took place in phagocytic microglia/macrophages in the ischemic territory, peaking at two weeks post-stroke in human and one-week post-stroke in rat ischemic cortex. Using double immunofluorescence and mice lacking MANF gene and protein from neuronal stem cells, neurons, astrocytes, and oligodendrocytes, we verified that MANF expression was induced in microglia/macrophage cells in the ischemic hemisphere. Embarking on the drastic expression transition towards inflammatory cells and the impact of blood-borne inflammation in stroke, we hypothesized that exogenously delivered MANF protein can modulate tissue recovery processes. In an attempt to enhance recovery, we designed a set of proof-of-concept studies using systemic delivery of recombinant MANF in a rat model of cortical ischemic stroke. Intranasal recombinant MANF treatment decreased infarct volume and reduced the severity of neurological deficits. Intravenous recombinant MANF treatment decreased the levels of pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokine IL-10 in the infarcted cortex one-day post-stroke. In conclusion, MANF protein expression is induced in activated microglia/macrophage cells in infarcted human and rodent brains, and this could implicate MANF’s involvement in the regulation of post-stroke inflammation in patients and experimental animals. Moreover, systemic delivery of recombinant MANF shows promising immunomodulatory effects and therapeutic potential in experimental ischemic stroke.

Cornuside protects against ischemic stroke in rats by suppressing the IL-17F/TRAF6/NF-κB pathway via the brain-gut axis

Ischemic stroke is a serious neurological disease with limited therapeutic options; thus, it is particularly important to find effective treatments. Restoration of gut microflora diversity is an important factor in the treatment of ischemic stroke, but the mechanism remains unclear. Cornuside is known for its unique anti-inflammatory and circulation-promoting effects; however, whether it can effectively treat ischemic stroke and its therapeutic mechanisms remain unknown. In this study, we used a rat middle cerebral artery occlusion-reperfusion model (MCAO/R) to mimic ischemic stroke in humans and to assess the cerebral protective effects of cornuside in rats with ischemic stroke. Using 16S rRNA sequencing and RNA sequencing, we explored the cornuside mechanism in the brain-gut axis that confers protection against ischemic stroke. In conclusion, cornuside can inhibit the IL-17F/TRAF6/NF-κB pathway by improving the dysregulation of intestinal microflora, and reduce intestinal inflammation and neuroinflammation, which treated ischemic stroke rats.

Tensor-valued diffusion MRI of human acute stroke.

Tensor-valued diffusion encoding can disentangle orientation dispersion and subvoxel anisotropy, potentially offering insight into microstructural changes after cerebral ischemia. The purpose was to evaluate tensor-valued diffusion MRI in human acute ischemic stroke, assess potential confounders from diffusion time dependencies, and compare to Monte Carlo diffusion simulations of axon beading. Linear (LTE) and spherical (STE) b-tensor encoding with inherently different effective diffusion times were acquired in 21 acute ischemic stroke patients between 3 and 57 h post-onset at 3 T in 2.5 min. In an additional 10 patients, STE with 2 LTE yielding different effective diffusion times were acquired for comparison. Diffusional variance decomposition (DIVIDE) was used to estimate microscopic anisotropy (μFA), as well as anisotropic, isotropic, and total diffusional variance (MKA , MKI , MKT ). DIVIDE parameters, and diffusion tensor imaging (DTI)-derived mean diffusivity and fractional anisotropy (FA) were compared in lesion versus contralateral white matter. Monte Carlo diffusion simulations of various cylindrical geometries for all b-tensor protocols were used to interpret parameter measurements. MD was ˜40% lower in lesions for all LTE/STE protocols. The DIVIDE parameters varied with effective diffusion time: higher μFA and MKA in lesion versus contralateral white matter for STE with longer effective diffusion time LTE, whereas the shorter effective diffusion time LTE protocol yielded lower μFA and MKA in lesions. Both protocols, regardless of diffusion time, were consistent with simulations of greater beading amplitude and intracellular volume fraction. DIVIDE parameters depend on diffusion time in acute stroke but consistently indicate neurite beading and larger intracellular volume fraction.

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Associations between 5‐lipoxygenase activating protein (FLAP) genetic variation rs9551963 and Alzheimer’s disease phenotypes

AbstractBackgroundLeukotrienes are inflammatory mediators, synthesized by 5‐lipoxygenase and its activating protein (FLAP), and their activity may play a role in Alzheimer’s disease (AD). In AD mouse models, inhibition of FLAP improved memory and reduced amyloid (Aβ) deposition. Single nucleotide polymorphisms (SNPs) of ALOX5AP, which encodes FLAP, have been implicated in ischemic stroke in humans. However, there is limited research on the relationship between ALOX5AP and AD. This longitudinal study investigated associations between the ALOX5AP SNP rs9551963 A/C and various AD phenotypes.MethodParticipants from the Alzheimer’s Disease Neuroimaging Initiative (phases GO, 2, and 3) diagnosed with AD, mild cognitive impairment (MCI), or clinically normal cognition (CN) were categorized as minor allele carriers (AC, CC) or non‐carriers (AA) of rs9551963. Mini Mental State Exam (MMSE) and Rey Auditory Verbal Learning Test (RAVLT) % forgetting were selected to assess cognition. Cerebrospinal fluid biomarkers and MRI brain volumetric data were also considered. Interactions between rs9551963 genotype and time (baseline, month 24, and month 48) were measured in mixed models. All models controlled for age, sex, and APOE e4 status; education and baseline intracranial volume were additional controls in cognitive and volumetric analyses respectively.ResultMinor allele carriers in the AD‐MCI Aβ‐positive group had worse decline on the MMSE (F1,297 = 5.33, p = 0.022, n = 521), and smaller volumes of whole brain (F1,146 = 7.13, p = 0.008, n = 408) and hippocampus (F1,132 = 6.38, p = 0.013, n = 377) over time that were not seen in other subgroups. In the AD subgroup, Aβ‐positive carriers had greater increases in RAVLT % forgetting scores (F1,135.5 = 6.19, p = 0.014, n = 158) and greater whole brain volume loss (F1,27.6 = 5.67, p = 0.024, n = 132), over time. These associations were not seen in Aβ‐negative participants.ConclusionMinor allele carriers of the ALOX5AP SNP rs9551963 who were Aβ‐positive showed greater loss of cognitive status and memory, and more hippocampal and whole brain atrophy, over time. These findings suggest a possible effect of leukotriene synthesis in the progression of AD.

Diesel exhaust particles exposure exacerbates pro-thrombogenic plasma features ex-vivo after cerebral ischemia and accelerates tPA-induced clot-lysis in hypertensive subjects

The combustion of fossil fuels, mainly by diesel engines, generates Diesel Exhaust Particles (DEP) which are the main source of Particulate Matter (PM), a major air pollutant in urban areas. These particles are a risk factor for stroke with 5.6% of cases attributed to PM exposure. Our aim was to evaluate the effect of DEP exposure on clot formation and lysis in the context of stroke. An ex-vivo clot formation and lysis turbidimetric assay has been conducted in human and mouse plasma samples from ischemic stroke or control subjects exposed to DEP or control conditions. Experimental DEP exposure was achieved by nasal instillation in mice, or by ex-vivo exposure in human plasma. Results show consistent pro-thrombogenic features in plasma after human ischemic stroke and mouse cerebral ischemia (distal MCAo), boosted by the presence of DEP. Otherwise, thrombolysis times were increased after ischemia in chronically exposed mice but not in the DEP exposed group. Finally, subjects living in areas with high PM levels presented accelerated thrombolysis compared to those living in low polluted areas. Overall, our results point at a disbalance of the thrombogenic/lytic system in presence of DEP which could impact on ischemic stroke onset, clot size and thrombolytic treatment.

Longitudinal evolution of diffusion metrics after left hemisphere ischaemic stroke

Abstract White matter is often severely affected after human ischaemic stroke. While animal studies have suggested that various factors may contribute to white matter structural damage after ischaemic stroke, the characterization of damaging processes to the affected hemisphere after human stroke remains poorly understood. Thus, the present study aims to thoroughly describe the longitudinal pattern of evolution of diffusion magnetic resonance imaging metrics in different parts of the ipsilesional white matter after stroke. We acquired diffusion and anatomical images in 17 patients who had suffered from a single left hemisphere ischaemic stroke, at 24–72 h, 8–14 days and 6 months post-stroke. For each patient, we created three regions of interest: (i) the white matter lesion; (ii) the perilesional white matter; and (iii) the remaining white matter of the left hemisphere. We extracted diffusion metrics (fractional anisotropy, mean, axial and radial diffusivities) for each region and conducted two-way repeated measures ANOVAs with stage post-stroke (acute, subacute and chronic) × regions of interest (white matter lesion, perilesional white matter and remaining white matter). Fractional anisotropy values stayed consistent across time-points, with significantly lower values in the white matter lesion compared to the perilesional white matter and remaining white matter tissue. Fractional anisotropy values of the perilesional white matter were also significantly lower than that of the remaining white matter. Mean, axial and radial diffusivities in the white matter lesion were all decreased in the acute stage compared to perilesional white matter and remaining white matter, but significantly increased in both the subacute and chronic stages. Significant increases in mean and radial diffusivities in the perilesional white matter were seen in the later stages of stroke. Our findings suggest that various physiological processes are at play in the acute, subacute and chronic stages following ischaemic stroke, with the infarct territory and perilesional white matter affected by ischaemia at different rates and to different extents throughout the stroke recovery stages. The examination of multiple diffusivity metrics may inform us about the mechanisms occurring at different time-points, i.e. focal swelling, axonal damage or myelin loss.

Abstract TP226: Impact Of Apolipoprotein A-i In The Attenuation Of Stroke And Stroke Symptoms

Apolipoprotein AI is the major protein component of high-density lipoprotein (HDL) particles in plasma. Inflammatory cytokines such as TNF and IL-1β repress the production of ApoA-I from hepatocytes and increase the expression of serum amyloid A (SAA), which becomes the major protein component of HDL in this context. Consequently, lipid-poor ApoA-I is rapidly catabolized in the liver and the kidney. These findings could be meaningful if ApoA-I, in addition to its proimmune features, had anti-inflammatory potential. In this way, removal of ApoA-I could intensify the inflammatory response, resulting in a more robust effect. Recent studies have suggested that serum apolipoprotein A1 maybe a predictor and prognostic biomarker in acute ischemic stroke in humans. To understand the impact of ApoA-I in stroke, we have begun to analyze the role of ApoA-I in mouse models of stroke. Mice with reduced or deficient in ApoA-I and subjected to ischemia and reperfusion injury or permanent ischemia, had increased inflammatory responses, larger infarct volumes, and poorer outcomes than wildtype mice. Mice transgenic for the human ApoA-I gene showed a level of protection not seen in the wildtype mice when given an ischemic injury. Administration of AAV human ApoA-I increased HDL levels in deficient mice and provided protection against ischemic injury. Additionally, drugs to enhance ApoA-I expression showed protection against strokes in the mouse models. These studies, along with subsequent investigations will provide a better understanding and potential therapeutic approaches in the field of stroke.

Abstract TP210: Small Extracellular Vesicle-derived Micrornas Differentiate Ischemic Stroke And Intracerebral Hemorrhage: A Pilot Study

Investigating the short and long-term signaling mechanisms via small extracellular vesicles (sEVs) in peripheral blood following human Ischemic Stroke (IS) and Intracerebral Hemorrhage (ICH) is of great interest. The sEV’s cargo can induce distant responses which can be used to derive potential novel therapeutic targets and biomarkers to differentiate the two brain pathologies. Thus, we sequenced the miRNA cargo derived from peripheral blood sEVs in IS (n=3), ICH (n=3), and Vascular Risk Factor-matched Control (VRFC; n=3) subjects. Subjects were divided into contrast groups (IS vs VRFC, ICH vs VRFC, ICH vs IS), and log2 transformed expression underwent Kruskal-Wallis tests to identify differentially expressed (DE; p<0.05) miRNAs. We found 55 DE miRNAs in IS vs VRFC, 38 in ICH vs VRFC, and 45 in ICH vs IS ( Fig. 1A ). The combination of these miRNAs differentiated the three groups on Principal Components Analysis ( Fig. 1B ). IS associated miRNA included miR-30a, miR-30b, and miR-144. miR-30a is involved in hematopoietic stem cell self-renewal and can impair B cell differentiation. miR-30b may be an immune suppressor via Notch1. In male mice, miR-144 is protective against atherosclerosis. ICH associated miRNA included miR-195, miR-1-3p, and miR-20b-5p. miR-195 can inhibit the pro-inflammatory roles of macrophages. miR-1-3p is involved in cardiomyocyte development, can target TLR1 (Toll-Like Receptor 1), and may regulate autophagy. miR-20b-5p reduces Amyloid Precursor Protein (APP) mRNA and protein levels; vascular accumulation of APP is one cause of Lobar ICH. We show differential expression of sEV-derived miRNAs in peripheral blood of human IS and ICH patients that are involved in relevant signaling processes. sEV cargo profiles pose a largely underexplored intercellular signaling mechanism in IS and ICH with the potential to better characterize long distance signaling from injured brain to peripheral blood leukocytes in these brain disorders.

Photothrombotic Middle Cerebral Artery Occlusion in Mice: A Novel Model of Ischemic Stroke.

Stroke is one of the main causes of death and disability worldwide. Over the past decades, several animal models of focal cerebral ischemia have been developed allowing to investigate pathophysiological mechanisms underlying stroke progression. Despite intense preclinical research efforts, the need for noninvasive mouse models of vascular occlusion targeting the middle cerebral artery yet avoiding mechanical intervention is still pressing. Here, by applying the photothrombotic stroke model to the distal branch of the middle cerebral artery, we developed a novel strategy to induce a targeted occlusion of a large blood vessel in mice. This approach induces unilateral damage encompassing most of the dorsal cortex from the motor up to the visual regions 1 week after stroke. Pronounced limb dystonia one day after the damage is partially recovered after one week. Furthermore, we observe the insurgence of blood vessel leakage and edema formation in the peri-infarct area. Finally, this model elicits a notable inflammatory response revealed as a strong increase in astrocyte density and morphologic complexity in the perilesional region of the cortex compared with both other regions of the ipsilesional and contralesional hemispheres, and in sham-operated mice. To conclude, the stroke model we developed induces in mice the light-mediated occlusion of one of the main targets of human ischemic stroke, the middle cerebral artery, free from the limitations of commonly used preclinical models.

Exosome therapies improve outcome in rodents with ischemic stroke; meta-analysis

ObjectiveExosome therapy has been theorized to be safer, more effective, and less cumbersome in replacing stem cell therapies for tissue repair and regeneration. There remains considerable uncertainty on whether exosome therapy is efficacious and safe for recovery from brain injury due to cerebral infarction. There is growing consensus that systematic reviews of data, from preclinical studies which yielded conflicting and confusing results, can provide valuable directions for novel therapeutic options for several clinical conditions. This study systematically evaluated the efficacy of exosome therapy in ischemic stroke in preclinical studies in rodent models. MethodsWe reviewed existing literature on exosome therapy in rodent stroke models from various databases, and reviewed the interventional measures, and outcome measures systematically, with changes in the infarct volume and functional scores as outcome parameters. Seventeen homogeneous studies were found qualitatively acceptable for meta-analysis. The study used software RevMan 5.3 to conduct the meta-analysis (PROSPERO Register Number: CRD42022314138) ResultsCompared to placebo, exosomes treated ischemic stroke models showed significantly reduced brain infarct volume and improved functional recovery on days 7 and 28. Though there are no safety concerns reported in any preclinical studies, there is insufficient data to make robust conclusions on the therapy's safety. InterpretationTherapy with subcellular exosomes is a promising treatment to be explored further in animal ischemic stroke models to arrive at robust conclusions for its safety and therapeutic dosage. This must precede Phase I and II- human randomized clinical trials to establish the safety and proof of concept of efficacy of exosome therapy in human ischemic stroke.

Chebulic Acid Prevents Hypoxia Insult via Nrf2/ARE Pathway in Ischemic Stroke.

Excessive reactive oxygen species (ROS) production contributes to brain ischemia/reperfusion (I/R) injury through many mechanisms including inflammation, apoptosis, and cellular necrosis. Chebulic acid (CA) isolated from Terminalia chebula has been found to have various biological effects, such as antioxidants. In this study, we investigated the mechanism of the anti-hypoxic neuroprotective effect of CA in vitro and in vivo. The results showed that CA could protect against oxygen-glucose deprivation/reoxygenation (OGD/R) induced neurotoxicity in SH-SY5Y cells, as evidenced by the enhancement of cell viability and improvement of total superoxide dismutase (T-SOD) in SH-SY5Y cells. CA also attenuated OGD/R-induced elevations of malondialdehyde (MDA) and ROS in SH-SY5Y cells. Nuclear factor-E2-related factor 2 (Nrf2) is one of the key regulators of endogenous antioxidant defense. CA acted as antioxidants indirectly by upregulating antioxidant-responsive-element (ARE) and Nrf2 nuclear translocation to relieve OGD/R-induced oxidative damage. Furthermore, the results showed that CA treatment resulted in a significant decrease in ischemic infarct volume and improved performance in the motor ability of mice 24 h after stroke. This study provides a new niche targeting drug to oppose ischemic stroke and reveals the promising potential of CA for the control of ischemic stroke in humans.

A Novel Improved Thromboembolism-Based Rat Stroke Model That Meets the Latest Standards in Preclinical Studies.

The animal thromboembolic model of ischemia perfectly mimics human ischemic stroke which remains the leading cause of disability and mortality in humans. The development of new treatment strategies was therefore imperative. The purpose of this study is to improve the thromboembolic stroke model in rats in order to design experiments that use motor tests, and are in accordance with the 3R principles to prevent complications and maintain the same size of the infarct repeatedly. Tail vein dye application, a protective skull mask and a stress minimization protocol were used as additional modifications to the animal stroke model. These modifications significantly minimized the pain and stress severity of the procedures in this model. In our experimental group of Long-Evans rats, a photo-induced stroke was caused by the application of a photosensitive dye (Rose Bengal) activated with white-light irradiation, thus eliminating the need to perform a craniotomy. The animals' neurological status was evaluated using a runway elevated test. Histological examination of the brain tissue was performed at 12, 24 and 48 h, and seven days post-stroke. Tissue examination revealed necrotic foci in the cortex and the subcortical regions of the ipsilateral hemisphere in all experimental groups. Changes in the area, width and depth of the necrotic focus were observed over time. All the experimental groups showed motor disturbances after stroke survival. In the proposed model, photochemically-induced stroke caused long-term motor deficits, showed high reproducibility and low mortality rates. Consequently, the animals could participate in motor tests which are particularly suitable for assessing the efficacy of neuro-regenerative therapies, while remaining in line with the latest trends in animal experimental design.

Comprehensive Analysis of Blood-Based m6A Methylation in Human Ischemic Stroke.

Alterations of N6-methyladenosine (m6A) methylation have been reported in the cerebral cortices of mouse and rat models of ischemic stroke (IS). However, the role of m6A methylation in human IS is still unknown. We assessed m6A levels in peripheral blood from patients with IS and healthy controls. A transient middle cerebral artery occlusion and reperfusion (tMCAO/R) mouse model, and an oxygen-glucose deprivation/reperfusion (OGD/R) model in A172 cells were established to further assess m6A levels. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing were performed in the peripheral blood of patients with IS and healthy controls. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used to identify underlying biological processes. In this study, we found thatglobal m6A levels were elevated in the peripheral blood of patients with IS, in the cerebral cortex of mice after tMCAO/R treatment and in A172 cells after OGD/R treatment. MeRIP-seq analysis identified 2115 altered m6A peaks in patients with IS, 1052 upregulated and 1063 downregulated. Downregulated methylated mRNAs were enriched in Hippo signaling pathway, cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, etc. Upregulated methylated mRNAs were enriched in calcium signaling pathways, Hedgehog signaling pathway, MAPK signaling pathway, etc. Moreover,a total of84 differentially expressed mRNAs with altered m6A peaks were identified andenriched in EGFR tyrosine kinase inhibitor, Hematopoietic cell lineage, and cytokine-cytokine receptor interactions. This study is the first to profile the transcriptome-wide m6A methylome of peripheral blood in human IS and uncover increased global m6A levels in the peripheral blood of patients with IS.

Translating Animal Models of Ischemic Stroke to the Human Condition.

Ischemic stroke is a leading cause of death and disability. However, very few neuroprotective agents have shown promise for treatment of ischemic stroke in clinical trials, despite showing efficacy in many successful preclinical studies. This may be attributed, at least in part, to the incongruency between experimental animal stroke models used in preclinical studies and the manifestation of ischemic stroke in humans. Most often the human population selected for clinical trials are more diverse than the experimental model used in a preclinical study. For successful translation, it is critical to develop clinical trial designs that match the experimental animal model used in the preclinical study. This review aims to provide a comprehensive summary of commonly used animal models with clear correlates between rodent models used to study ischemic stroke and the clinical stroke pathologies with which they most closely align. By improving the correlation between preclinical studies and clinical trials, new neuroprotective agents and stroke therapies may be more accurately and efficiently identified.

Excitatory Synaptic Transmission in Ischemic Stroke: A New Outlet for Classical Neuroprotective Strategies.

Stroke is one of the leading causes of death and disability in the world, of which ischemia accounts for the majority. There is growing evidence of changes in synaptic connections and neural network functions in the brain of stroke patients. Currently, the studies on these neurobiological alterations mainly focus on the principle of glutamate excitotoxicity, and the corresponding neuroprotective strategies are limited to blocking the overactivation of ionic glutamate receptors. Nevertheless, it is disappointing that these treatments often fail because of the unspecificity and serious side effects of the tested drugs in clinical trials. Thus, in the prevention and treatment of stroke, finding and developing new targets of neuroprotective intervention is still the focus and goal of research in this field. In this review, we focus on the whole processes of glutamatergic synaptic transmission and highlight the pathological changes underlying each link to help develop potential therapeutic strategies for ischemic brain damage. These strategies include: (1) controlling the synaptic or extra-synaptic release of glutamate, (2) selectively blocking the action of the glutamate receptor NMDAR subunit, (3) increasing glutamate metabolism, and reuptake in the brain and blood, and (4) regulating the glutamate system by GABA receptors and the microbiota–gut–brain axis. Based on these latest findings, it is expected to promote a substantial understanding of the complex glutamate signal transduction mechanism, thereby providing excellent neuroprotection research direction for human ischemic stroke (IS).

Identification of circulating immune landscape in ischemic stroke based on bioinformatics methods.

Ischemic stroke (IS) is a high-incidence disease that seriously threatens human life and health. Neuroinflammation and immune responses are key players in the pathophysiological processes of IS. However, the underlying immune mechanisms are not fully understood. In this study, we attempted to identify several immune biomarkers associated with IS. We first retrospectively collected validated human IS immune-related genes (IS-IRGs) as seed genes. Afterward, potential IS-IRGs were discovered by applying random walk with restart on the PPI network and the permutation test as a screening strategy. Doing so, the validated and potential sets of IS-IRGs were merged together as an IS-IRG catalog. Two microarray profiles were subsequently used to explore the expression patterns of the IS-IRG catalog, and only IS-IRGs that were differentially expressed between IS patients and controls in both profiles were retained for biomarker selection by the Random Forest rankings. CLEC4D and CD163 were finally identified as immune biomarkers of IS, and a classification model was constructed and verified based on the weights of two biomarkers obtained from the Neural Network algorithm. Furthermore, the CIBERSORT algorithm helped us determine the proportions of circulating immune cells. Correlation analyses between IS immune biomarkers and immune cell proportions demonstrated that CLEC4D was strongly correlated with the proportion of neutrophils (r = 0.72). These results may provide potential targets for further studies on immuno-neuroprotection therapies against reperfusion injury.