Exploring Lysophosphatidylcholine as a Biomarker in Ischemic Stroke: The Plasma-Brain Disjunction.

Lipids and their bioactive metabolites, notably lysophosphatidylcholine (LPC), are increasingly important in ischemic stroke research. Reduced plasma LPC levels have been linked to stroke occurrence and poor outcomes, positioning LPC as a potential prognostic or diagnostic marker. Nonetheless, the connection between plasma LPC levels and stroke severity remains unclear. This study aimed to elucidate this relationship by examining plasma LPC levels in conjunction with brain LPC levels to provide a deeper understanding of the underlying mechanisms. Adult male Sprague-Dawley rats underwent transient middle cerebral artery occlusion and were randomly assigned to different groups (sham-operated, vehicle, LPC supplementation, or LPC inhibition). We measured multiple LPC species in the plasma and brain, alongside assessing sensorimotor dysfunction, cerebral perfusion, lesion volume, and markers of BBB damage, inflammation, apoptosis, and oxidative stress. Among five LPC species, plasma LPC(16:0) and LPC(18:1) showed strong correlations with sensorimotor dysfunction, lesion severity, and mechanistic biomarkers in the rat stroke model. Despite notable discrepancies between plasma and brain LPC levels, both were strongly linked to functional outcomes and mechanistic biomarkers, suggesting that LPC's prognostic value is retained extracranially. This study advances the understanding of LPC as a blood marker in ischemic stroke and highlights directions for future research to further elucidate its association with stroke severity, particularly through investigations in more clinically representative models.

High plasma BNP concentration associates with clinical outcome after mechanical thrombectomy: Post hoc analysis of SKIP.

OBJECTIVES: Heart failure may result in reduced brain perfusion, limiting the blood flow needed to achieve clinical recovery. We investigated whether plasma levels of brain natriuretic peptide (BNP), a biological marker of heart failure, were related to clinical outcomes after mechanical thrombectomy (MT). MATERIALS AND METHODS: Data were analyzed from stroke patients with internal carotid or middle cerebral artery occlusion enrolled in the SKIP trial for whom plasma level of BNP was evaluated on admission. Favorable outcome was defined as a modified Rankin scale score of 0-2 at 3 months. RESULTS: Among 169 patients (median age, 74 years; 62% men, median National Institutes of Health Stroke Scale score, 18), 104 (62%) achieved favorable outcomes. Median plasma BNP level was lower in the favorable outcome group (124.1 pg/mL; interquartile range [IQR], 62.1-215.5 pg/mL) than in the unfavorable outcome group (198.0 pg/mL; IQR, 74.8-334.0 pg/mL; p=0.005). In multivariate regression analysis, the adjusted odds ratio for BNP for favorable outcomes was 0.971 (95% confidence interval, 0.993-0.999; p=0.048). At 3 months after onset, the favorable outcome rate was lower in the ≥186 pg/mL group (45%) than in the <186 pg/mL group (72%; p=0.001). This significant difference remained regardless of the presence of atrial fibrillation (AF), with rates of 47% and 76%, respectively, in AF patients (p=0.003) and 33% and 68%, respectively, in patients without AF (p=0.046). CONCLUSION: High plasma BNP concentration appears associated with unfavorable outcomes after MT.

Cooperation Between Platelet ß1 and ß3 Integrins in the Arrest of Bleeding Under Inflammatory Conditions in Mice-Brief Report.

BACKGROUND: Platelets prevent bleeding in a variety of inflammatory settings, the adhesion receptors and activation pathways involved being highly context-dependent and functionally redundant. In some situations, platelets recruited to inflammatory sites act independently of aggregation. The mechanisms underlying stable platelet adhesion in inflamed microvessels remain incompletely understood, in particular, whether and if so, how ß1 and ß3 integrins are involved. METHODS: The impact of isolated or combined platelet deficiency in ß1 and ß3 integrins on inflammation-associated hemostasis was investigated in 3 models of acute inflammation: immune complex-based cutaneous reverse passive Arthus reaction, intranasal lipopolysaccharide-induced lung inflammation, and cerebral ischemia-reperfusion following transient (2-hour) occlusion of the middle cerebral artery. RESULTS: Mice with platelet-directed inactivation of Itgb1 (PF4Cre-ß1-/-) displayed no bleeding in any of the inflammation models, while mice defective in platelet Itgb3 (PF4Cre-ß3-/-) exhibited bleeding in all 3 models. Remarkably, the bleeding phenotype of PF4Cre-ß3-/- mice was exacerbated in the reverse passive Arthus model by the concomitant deletion of ß1 integrins, PF4Cre-ß1-/-/ß3-/- animals presenting increased bleeding. Intravital microscopy in reverse passive Arthus experiments highlighted a major defect in the adhesion of PF4Cre-ß1-/-/ß3-/- platelets to inflamed microvessels. Unlike PF4Cre-ß1-/- and PF4Cre-ß3-/- mice, PF4Cre-ß1-/-/ß3-/- animals developed early hemorrhagic transformation 6 hours after transient middle cerebral artery occlusion. PF4Cre-ß1-/-/ß3-/- mice displayed no more bleeding in lipopolysaccharide-induced lung inflammation than PF4Cre-ß3-/- animals. CONCLUSIONS: Altogether, these results show that the requirement for and degree of functional redundancy between platelet ß1 and ß3 integrins in inflammation-associated hemostasis vary with the inflammatory situation.

Inflammation-Derived and Clinical Indicator-Based Predictive Model for Ischemic Stroke Recovery.

BACKGROUND: Neuroinflammatory responses are closely associated with poststroke prognosis severity. This study aimed to develop a predictive model, combining inflammation-derived markers and clinical indicators, for distinguishing functional outcomes in patients with subacute ischemic stroke. METHODS AND RESULTS: Based on activities of daily living assessments, ischemic stroke participants were categorized into groups with little effective (LE) recovery and obvious effective (OE) recovery. Initial biocandidates were identified by overlapping differentially expressed proteins from proteomics of clinical serum samples (5 LE, 5 OE, and 6 healthy controls) and differentially expressed genes from an RNA sequence of the ischemic cortex in middle cerebral artery occlusion mice (n=3). Multidimensional validations were conducted in ischemia-reperfusion models and a clinical cohort (15 LE, 11 OE, and 18 healthy controls). Models of robust biocandidates combined with clinical indicators were developed with machine learning in the training data set and prediction in another test data set (15 LE and 11 OE). We identified 194 differentially expressed proteins (LE versus healthy controls) and 174 differentially expressed proteins (OE versus healthy controls) in human serum, and 5121 differentially expressed genes (day 3) and 5906 differentially expressed genes (day 7) in middle cerebral artery occlusion mice cortex. Inflammation-derived biomarkers TIMP1 (tissue inhibitor metalloproteinase-1) and galactosidase-binding protein LGLAS3 (galectin-3) exhibited robust increases under ischemic injury in mice and humans. TIMP1 and LGALS3 coupled with clinical indicators (hemoglobin, low-density lipoprotein cholesterol, and uric acid) were developed into a combined model for differentiating functional outcome with high accuracy (area under the curve, 0.8). CONCLUSIONS: The combined model is a valuable tool for evaluating prognostic outcomes, and the predictive factors can facilitate development of better treatment strategies.

Characterization of the Components and Metabolites of Achyranthes Bidentata in the Plasma and Brain Tissue of Rats Based on Ultrahigh Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HR-MS).

BACKGROUND: Achyranthes bidentata (AR) is a traditional Chinese herb used for the treatment of hypertension and cerebral ischemia, but its pharmacological effects are not known. AIM OF STUDY: We aimed to detect and accurately identify the components and metabolites of AR in the plasma and brain tissue of Sprague Dawley rats. METHODS: We employed ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HR-MS) to detect AR components in the plasma and brain tissue of rats. The absorption and metabolites in the plasma and brain tissue of normal control rats and rats that underwent middle cerebral artery occlusion (MCAO) were characterized and compared. RESULTS: A total of 281 compounds, including alkaloids, flavonoids, terpenoids, phenylpropanes, sugars and glycosides, steroids, triterpenes, amino acids, and peptides, was identified in samples of Achyranthes bidentata (TCM-AR). Four types of absorbable prototype components and 48 kinds of metabolites were identified in rats in the normal control plasma group which were given AR (AR plasma group), and five kinds of metabolites were identified in rats of the normal control brain tissue group which were given AR (AR brain group). Three absorbed prototype components and 13 metabolites were identified in the plasma of rats which underwent MCAO and were given AR (MCAO + AR plasma group). Six absorbed prototype components and two metabolites were identified in the brain tissue of rats who underwent MCAO and were administered AR (MCAO + AR brain group). These results showed that, after the oral administration of AR, the number of identified components in plasma was more than that in brain tissue. The number of prototype components in the AR plasma group was higher than that in the MCAO + AR plasma group, which may indicate that metabolite absorption in rats undergoing MCAO was worse. The number of prototype components in the MCAO + AR brain group was higher than that in the AR brain group, indicating that the blood-brain barrier was destroyed after MCAO, resulting in more compounds entering brain tissue. CONCLUSIONS: UHPLC-HR-MS was used to rapidly analyze the components and metabolites of AR in the blood and brain of rats under normal and pathologic conditions, and to comprehensively characterize the components of TCM-AR. We also analyzed and compared the absorbable components and metabolites of normal rats under cerebral ischemia-reperfusion injury to explore the potential mechanism of action. This method could be applied to various Chinese herbs and disease models, which could promote TCM modernization.

Nuclear magnetic resonance spectroscopy reveals biomarkers of stroke recovery in a mouse model of obesity-associated type 2 diabetes.

Obesity and Type 2 diabetes (T2D) are known to exacerbate cerebral injury caused by stroke. Metabolomics can provide signatures of metabolic disease, and now we explored whether the analysis of plasma metabolites carries biomarkers of how obesity and T2D impact post-stroke recovery. Male mice were fed a high-fat diet (HFD) for 10 months leading to development of obesity with T2D or a standard diet (non-diabetic mice). Then, mice were subjected to either transient middle cerebral artery occlusion (tMCAO) or sham surgery and allowed to recover on standard diet for 2 months before serum samples were collected. Nuclear magnetic resonance (NMR) spectroscopy of serum samples was used to investigate metabolite signals and metabolic pathways that were associated with tMCAO recovery in either T2D or non-diabetic mice. Overall, after post-stroke recovery there were different serum metabolite profiles in T2D and non-diabetic mice. In non-diabetic mice, which show full neurological recovery after stroke, we observed a reduction of isovalerate, and an increase of kynurenate, uridine monophosphate, gluconate and N6-acetyllysine in tMCAO relative to sham mice. In contrast, in mice with T2D, which show impaired stroke recovery, there was a reduction of N,N-dimethylglycine, succinate and proline, and an increase of 2-oxocaproate in serum of tMCAO versus sham mice. Given the inability of T2D mice to recover from stroke, in contrast with non-diabetic mice, we propose that these specific metabolite changes following tMCAO might be used as biomarkers of neurophysiological recovery after stroke in T2D.

Sepsis after middle cerebral artery occlusion exacerbates peripheral oxidative stress in a sex-specific manner.

Ischemic stroke occurs due a blockage in the blood flow to the brain, leading to damage to the nervous system. The prevalent morbidities resulting from stroke include post-stroke infection, as sepsis. Additionally, oxidative stress is recognized for inducing functional deficits in peripheral organs during sepsis. Therefore, sex differences in stroke exist and we aimed to investigate the peripheral oxidative stress caused by sepsis after stroke in male and female rats. Wistar rats (male and female) were divided into sham+sham, middle cerebral artery occlusion (MCAO) + sham, sham+ cecal ligation and perforation (CLP) and MCAO+CLP groups to males and female rats. Animals were subjected to MCAO or sham and after 7 days, were subjected to sepsis by CLP or sham. After 24 h, serum, total brain, lung, liver, heart, and spleen were collected. Brain edema, myeloperoxidase (MPO) activity, nitrite/nitrate (N/N) concentration, oxidative damage to lipids and proteins, and catalase activity were evaluated. Brain edema was observed only in male rats in MCAO+CLP group compared to MCAO+sham. Regarding MPO activity, an increase was verified in male in different organs and serum in MCAO+CLP group. For N/N levels, the increase was more pronounced in females submitted to MCAO+CLP. In general, to oxidative stress, an increase was only observed in animals exposed to MCAO+CLP, or with a greater increase in this group compared to the others. The findings provided the first indication that animals exposed to MCAO exhibit a heightened vulnerability to the harmful impacts of sepsis, as evidenced by brain edema and peripheral oxidative stress, and this susceptibility is dependent of sex.

Chronic vascular pathogenesis results in the reduced serum Metrnl levels in ischemic stroke patients.

Metrnl is a secreted protein involved in neurite outgrowth, insulin sensitivity, immunoinflammatory responses, blood lipids and endothelial protection. In this study, we investigated the role of Metrnl in ischemic stroke. Fifty-eight ischemic stroke patients (28 inpatient patients within 2 weeks of onset and 30 emergency patients within 24 h of onset) and 20 healthy controls were enrolled. Serum Metrnl was measured by enzyme-linked immunosorbent assay. We showed that serum Metrnl levels were significantly reduced in both inpatient and emergency patient groups compared with the controls. Different pathological causes for ischemic stroke such as large artery atherosclerosis and small artery occlusion exhibited similar reduced serum Metrnl levels. Transient ischemic attack caused by large artery atherosclerosis without brain infarction also had lower serum Metrnl levels. Metrnl was correlated with some metabolic, inflammatory and clotting parameters. Reduced serum Metrnl was associated with the severity of intracranial arterial stenosis and the presence of ischemic stroke. In order to elucidate the mechanisms underlying the reduced serum Metrnl levels, we established animal models of ischemic stroke in normal mice, atherosclerotic apolipoprotein E-knockout mice and Metrnl-knockout mice by middle cerebral artery occlusion (MCAO) using intraluminal filament or electrocoagulation. We demonstrated that serum Metrnl levels were significantly lower in atherosclerosis mice than normal mice, whereas acute ischemic stroke injury in normal mice and atherosclerosis mice did not alter serum Metrnl levels. Metrnl knockout did not affect acute ischemic stroke injury and death. We conclude that reduced serum Metrnl levels are attributed to the chronic vascular pathogenesis before the onset of ischemic stroke. Metrnl is a potential target for prevention of ischemic stroke.

Diet-induced weight loss in obese/diabetic mice normalizes glucose metabolism and promotes functional recovery after stroke.

BACKGROUND: Post-stroke functional recovery is severely impaired by type 2 diabetes (T2D). This is an important clinical problem since T2D is one of the most common diseases. Because weight loss-based strategies have been shown to decrease stroke risk in people with T2D, we aimed to investigate whether diet-induced weight loss can also improve post-stroke functional recovery and identify some of the underlying mechanisms. METHODS: T2D/obesity was induced by 6 months of high-fat diet (HFD). Weight loss was achieved by a short- or long-term dietary change, replacing HFD with standard diet for 2 or 4 months, respectively. Stroke was induced by middle cerebral artery occlusion and post-stroke recovery was assessed by sensorimotor tests. Mechanisms involved in neurovascular damage in the post-stroke recovery phase, i.e. neuroinflammation, impaired angiogenesis and cellular atrophy of GABAergic parvalbumin (PV)+ interneurons were assessed by immunohistochemistry/quantitative microscopy. RESULTS: Both short- and long-term dietary change led to similar weight loss. However, only the latter enhanced functional recovery after stroke. This effect was associated with pre-stroke normalization of fasting glucose and insulin resistance, and with the reduction of T2D-induced cellular atrophy of PV+ interneurons. Moreover, stroke recovery was associated with decreased T2D-induced neuroinflammation and reduced astrocyte reactivity in the contralateral striatum. CONCLUSION: The global diabetes epidemic will dramatically increase the number of people in need of post-stroke treatment and care. Our results suggest that diet-induced weight loss leading to pre-stroke normalization of glucose metabolism has great potential to reduce the sequelae of stroke in the diabetic population.

Machine learning enables discovery of Gentianine targeting TLR4/NF-κB pathway to repair ischemic stroke injury.

Inflammatory response is believed to accelerate the development of stroke injury. Gentianine, an alkaloid isolated from Gentiana Scabra Bunge, shows effectiveness in anti-inflammation. In this study, the effect of Gentianine on transient middle cerebral artery occlusion (tMCAO) induced mouse model in vivo and further related mechanism in LPS-injuried microglia BV-2 cells in vitro were explored. Effect of Gentianine on tMCAO mouse demonstrated that Gentianine significantly ameliorated tMCAO induced ischemic injury by decreasing brain infarct volume and increasing the neurological score and upper limb muscle strength. Meanwhile, Gentianine significantly decreased the release of serum inflammatory cytokines. Machine learning enables that Gentianine might had anti-ischemic stroke effect through the TLR4/NF-κB signaling pathway. This was verified in vivo and in vitro. Gentianine significantly decrease the TLR4 and Iba-1 expression in vivo. These results also verified in BV-2 cells. Gentianine significantly decreased TLR4, MyD88 and NF-κB expression, as well as NO production and inflammatory cytokines release. Gentianine co-treatment with TLR4 inhibitor, further decreased TLR4, MyD88 and NF-κB expression, NO production, as well as the inflammatory cytokines. Taken together, Gentianine could be used as a potential anti-ischemic stroke agent by suppressing inflammatory responses via TLR4/NF-κB signaling pathway. This study is expected to provide an integrated traditional Chinese and western medicine solution to find potential anti-ischemic stroke compounds based on machine learning.

Growth arrest and DNA damage-inducible protein 34 (GADD34) contributes to cerebral ischemic injury and can be detected in plasma exosomes.

Growth arrest and DNA damage-inducible protein 34 (GADD34), one of the key effectors of negative feedback loops, is induced by stress and subsequently attempts to restore homeostasis. It plays a critical role in response to DNA damage and endoplasmic reticulum stress. GADD34 has opposing effects on different stimulus-induced cell apoptosis events in many nervous system diseases, but its role in ischemic stroke is unclear. In this study, we evaluated the role of GADD34 and its distribution in a rat cerebral ischemic model. The results showed that GADD34 was increased in the cortex and contributed to brain injury in ischemic rats. Furthermore, treatment with a GADD34 inhibitor reduced the infarct volume, improved functional outcomes, and inhibited neuronal apoptosis in the cortical penumbra after ischemia. The role of GADD34 in ischemic stroke was associated with the dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and phosphorylation of p53. In addition, the GADD34 level was increased in plasma exosomes of cerebral ischemic rats. These findings indicate that GADD34 could be a potential therapeutic target and biomarker for ischemic stroke.

The functions of fluoxetine and identification of fluoxetine-mediated circular RNAs and messenger RNAs in cerebral ischemic stroke.

Fluoxetine is used to improve cognition, exercise ability, depression, and neurological functions in patients with cerebral ischemic stroke. Circular RNAs (circRNAs) play important regulatory roles in multiple diseases. However, studies regarding the fluoxetine-mediated circRNA-microRNA-messenger RNA (mRNA) axis have not been conducted. This study is aim to investigate the functions of fluoxetine and identification of fluoxetine-mediated circRNAs and mRNAs in cerebral ischemic stroke. The middle cerebral artery occlusion (MCAO) rat models were successfully established at fisrt, and then rats were intraperitoneally injected with 10-mg/kg fluoxetine hydrochloride for 14 d. Afterward, the cerebral infarction area was evaluated using triphenyltetrazolium chloride staining. High-throughput sequencing was adopted to screen the differential circRNAs and mRNAs. The candidate circRNAs, mRNAs, and potential microRNAs were verified using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). In addtion, microRNA and circRNA binding was verified using the dual-luciferase reporter assay. Results revealed that fluoxetine markedly diminished the cerebral infarction area in rats after MCAO. The circRNAs and mRNAs were differentially expressed, which includes 879 circRNAs and 815 mRNAs between sham and MCAO groups, respectively, and 958 circRNAs and 838 mRNAs between MCAO and fluoxetine groups, respectively. In which, circMap2k1 and Pidd1 expression was significantly increased in the MCAO group but suppressed after fluoxetine treatment. Moreover, circMap2k1 directly binds with miR-135b-5p. Taken together, we verified that fluoxetine could improve brain injury after cerebral ischemic stroke. Moreover, the circMap2k1/miR-135b-5p/Pidd1 axis is potentially involved in cerebral ischemic stroke.

Integrated 16S rRNA Gene Sequencing and LC-MS Analysis Revealed the Interplay Between Gut Microbiota and Plasma Metabolites in Rats With Ischemic Stroke.

Gut microbiome and plasma metabolome serve a role in the pathogenesis of ischemic stroke (IS). However, the relationship between the microbiota and metabolites remains unclear. This study aimed to reveal the specific asso-ciation between the microbiota and the metabolites in IS using integrated 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS) analysis. Male Sprague Dawley (SD) rats were divided into three groups: normal group (n = 8, Normal), model group (n = 9, IS), and sham-operated group (n = 8, Sham). Rats in the IS group were induced by middle cerebral artery occlusion (MCAO), and rats in the Sham group received an initial anesthesia and neck incision only. A neurological function test and 2,3,5-triphenyltetrazolium chloride (TTC) staining were used to assess the IS rat model. Then, the plasma samples were analyzed using untargeted LC-MS. The cecum samples were collected and analyzed using 16S rRNA sequencing. Pearson correlation analysis was performed to explore the association between the gut microbiota and the plasma metabolites. The 16S rRNA sequencing showed that the composition and diversity of the microbiota in the IS and control rats were significantly different. Compared with the Sham group, the abundance of the Firmicutes phylum was decreased, whereas Proteobacteria and Deferribacteres were increased in the IS group. Ruminococcus_sp_15975 and Lachnospiraceae_UCG_001 might be considered as biomarkers for the IS and Sham groups, respectively. LC-MS analysis revealed that many metabolites, such as L-leucine, L-valine, and L-phenylalanine, displayed different patterns between the IS and Sham groups. Pathway analysis indicated that these metabolites were mainly involved in mineral absorption and cholinergic synapse. Furthermore, integrated analysis correlated IS-related microbes with metabolites. For example, Proteobacteria were positively correlated with L-phenylalanine, while they were negatively correlated with eicosapentaenoic acid (EPA). Our results provided evidence of the relationship between the gut microbiome and plasma metabolome in IS, suggesting that these microflora-related metabolites might serve as potential diagnostic and therapeutic markers.

Region-specific changes in aquaporin 4 induced by hyperglycemia underlie the differences in cell swelling in the cortex and striatum after cerebral ischemia-reperfusion.

Brain edema is a major cause of death in patients who suffer an ischemic stroke. Diabetes has been shown to aggravate brain edema after cerebral ischemia-reperfusion, but few studies have focused on the heterogeneity of this response across different brain regions. Aquaporin 4 plays an important role in the formation and regression of brain edema. Here, we report that hyperglycemia mainly affects the continuity of aquaporin 4 distribution around blood vessels in the cortical penumbra after ischemia-reperfusion; however, in the striatal penumbra, in addition to affecting the continuity of distribution, it also substantially affects the fluorescence intensity and the polarity distribution in astrocytes. Accordingly, hyperglycemia induces a more significant increase in the number of swelling cells in the striatal penumbra than in the cortical penumbra. These results can improve our understanding of the mechanism underlying the effects of diabetes in cerebral ischemic injury and provide a theoretical foundation for identification of appropriate therapeutic modalities.

Metformin prevents stroke damage in non-diabetic female mice with chronic kidney disease.

Chronic kidney disease (CKD) worsens ischemic stroke severity in both patients and animals. In mice, these poorer functional outcomes are associated with decreased brain activity of AMP-activated protein kinase (AMPK), a molecule that recently emerged as a potential therapeutic target for ischemic stroke. The antidiabetic drug metformin, a well-known activator of AMPK, has improved stroke outcomes in diabetic patients with normal renal function. We investigated whether chronic metformin pre-conditioning can rescue AMPK activity and prevent stroke damage in non-diabetic mice with CKD. Eight-week-old female C57BL/6J mice were assigned to CKD or SHAM groups. CKD was induced through right kidney cortical electrocautery, followed by left total nephrectomy. Mice were then allocated to receive metformin (200 mg/kg/day) or vehicle for 5 weeks until stroke induction by transient middle cerebral artery occlusion (tMCAO). The infarct volumes were lower in CKD mice exposed to metformin than in vehicle-treated CKD mice 24 h after tMCAO. Metformin pre-conditioning of CKD mice improved their neurological score, grip strength, and prehensile abilities. It also enhanced AMPK activation, reduced apoptosis, increased neuron survival and decreased microglia/macrophage M1 signature gene expression as well as CKD-induced activation of the canonical NF-κB pathway in the ischemic lesions of CKD mice.

Ischemic-time associated reductions in equol monosulfate plasma levels in a mouse model of ischemic stroke: support the existence of a 'brain-gut axis'.

BACKGROUND: Interest is growing in the role played by intestinal flora in the pathogeneses of diseases and in the possibility of treating disease by altering intestinal flora compositions. Recent studies have focused on the relationship between the intestinal microbiome and brain function as proposed by the brain-gut axis hypothesis. OBJECTIVES: To investigate the relation between ischemic stroke and plasma equol monosulfate levels (a soy isoflavone metabolite) in a middle cerebral artery occlusion (MCAO) mouse model. METHODS: Mice (C57BL/6) were subjected to MCAO for various times (30 min to 24 h), and degrees of cerebral damage were assessed using total infarction volumes, brain edema severities and neurological deficit scores. Hematoxylin and eosin and cresyl violet staining were used to observe morphological changes in ischemic brains. Levels of equol monosulfate in plasma and the relationships between these and degree of brain injury were investigated. RESULTS: Infarction volumes, brain edema severity and neurological deficit scores were significantly correlated with ischemic time, and morphological deteriorations of brain neuronal cells also increased with ischemic duration. Equol monosulfate contents were ischemic-time dependently lower in MCAO treated animals than in sham-operated controls. CONCLUSION: Ischemic stroke may time-dependently reduce plasma levels of equol monosulfate by lowering the metabolic rate of equol in MCAO-induced mice. This study provides indirect support of the brain-gut axis hypothesis.

Serum Levels of B-cell Lymphoma-2 Anti-Apoptotic Protein and Malignant Middle Cerebral Artery Infarction Mortality.

INTRODUCTION AND GOAL: There is scarce and contradictory data on B-cell lymphoma 2 (Bcl2), member of the Bcl-2 antiapoptotic molecules family of intrinsic apoptosis pathway, in ischemic stroke patients. The objective of this study was to determine whether there is an association between blood Bcl2 concentrations and mortality of ischemic stroke patients. MATERIAL AND METHODS: Five Intensive Care Units participated in this prospective and observational study of patients with severe malignant middle cerebral artery infarction (MMCAI). Severe MMCAI was diagnosed when acute infarction was present in 50% or more of said region and with a Glasgow Coma Scale (GCS) score of less than 9 points. Serum samples were collected at the time of MMCAI diagnosis. FINDINGS: Higher serum Bcl2 concentrations (p = 0.001), lower platelet count (p = 0.01) and lower GCS (p = 0.002) were found in non-survivors (n = 28) than in MMCAI survivors (n = 28). Serum Bcl2 levels had an area under the curve for mortality prediction of 75% (95% CI = 62%-88%; p < 0.001). Patients with serum Bcl2 levels > 43.6 ng/mL had higher mortality rate according to Kaplan-Meier analysis (Hazard ratio=10.0; 95% CI = 3.4-29.5; p < 0.001). Multiple logistic regression showed an association between serum Bcl2 and mortality at 30 days (OR = 1.041; 95% CI = 1.006-1.077; p = 0.02) controlling for GCS and platelet count. CONCLUSIONS: This study reports for the first time the higher blood Bcl2 concentrations in non-surviving ischemic stroke patients than in survivors and the association between elevated blood Bcl2 and mortality in ischemic stroke patients.

Mesenchymal Stem Cells: The Potential Therapeutic Cell Therapy to Reduce Brain Stroke Side Effects.

Tissue plasminogen activator (tPA) is the gold standard treatment for ischemic stroke in the time window of 3-4.5 hours after the onset of symptoms. However, tPA administration is associated with inflammation and neurotoxic effects. Mesenchymal stem cells (MSC)-based therapy is emerging as a promising therapeutic strategy to control different inflammatory conditions. This project was designed to examine the protective role of MSC administration alone or in combination with royal jelly (RJ) five hours after stroke onset. The mice model of middle cerebral artery occlusion (MCAO) was established and put to six groups, including intact (healthy mice without stroke), control (untreated stroke), treated with mouse MSC (mMSC), Sup (conditioned medium), RJ and combination of mMSC and RJ (mMSC/RJ). Thereafter, behavioral functions, serum and brain (in both infarcted and non-infarcted tissues) levels of interleukin (IL)-1ß, IL-4, IL-10, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) the sizes of brain infarction have been determined in the groups. Administration of mMSC and mMSC/RJ significantly improved the behavioral functions when compared to the controls. mMSC, RJ and mMSC/RJ significantly decreased the infarcted volumes. RJ and mMSC/RJ, but not mMSC, significantly decreased the brain edema. The infarction increased the serum levels of the cytokines, except TNF-α, and treatment with mMSC, Sup and RJ reduced serum levels of the pro-inflammatory cytokines. mMSC reduced IL-1ß in the non-infarcted brain tissue. To conclude, data revealed that using mMSC/RJ combination significantly reduced stroke side effects, including brain edema and serum levels of pro-inflammatory cytokines, and suggested that combination therapy of MSCs with RJ may be considered as an effective stroke therapeutic strategy.

Platelets and lymphocytes drive progressive penumbral tissue loss during middle cerebral artery occlusion in mice.

BACKGROUND: In acute ischemic stroke, cessation of blood flow causes immediate tissue necrosis within the center of the ischemic brain region accompanied by functional failure in the surrounding brain tissue designated the penumbra. The penumbra can be salvaged by timely thrombolysis/thrombectomy, the only available acute stroke treatment to date, but is progressively destroyed by the expansion of infarction. The underlying mechanisms of progressive infarction are not fully understood. METHODS: To address mechanisms, mice underwent filament occlusion of the middle cerebral artery (MCAO) for up to 4 h. Infarct development was compared between mice treated with antigen-binding fragments (Fab) against the platelet surface molecules GPIb (p0p/B Fab) or rat immunoglobulin G (IgG) Fab as control treatment. Moreover, Rag1-/- mice lacking T-cells underwent the same procedures. Infarct volumes as well as the local inflammatory response were determined during vessel occlusion. RESULTS: We show that blocking of the platelet adhesion receptor, glycoprotein (GP) Ibα in mice, delays cerebral infarct progression already during occlusion and thus before recanalization/reperfusion. This therapeutic effect was accompanied by decreased T-cell infiltration, particularly at the infarct border zone, which during occlusion is supplied by collateral blood flow. Accordingly, mice lacking T-cells were likewise protected from infarct progression under occlusion. CONCLUSIONS: Progressive brain infarction can be delayed by blocking detrimental lymphocyte/platelet responses already during occlusion paving the way for ultra-early treatment strategies in hyper-acute stroke before recanalization.

Serum Exosomal microRNA-27-3p Aggravates Cerebral Injury and Inflammation in Patients with Acute Cerebral Infarction by Targeting PPARγ.

Acute cerebral infarction (ACI) possesses high mortality. Exosomes present in serum have potential application value in ACI diagnosis. This study investigated the mechanism of serum exosomes in ACI. Serum exosomes isolated from ACI patients and normal people were identified and then injected into the established middle cerebral artery occlusion (MCAO) rat model to evaluate cerebral injury and inflammation. Exosomal microRNA (miR)-27-3p expression was detected and interfered to analyze rat cerebral inflammation. The binding relationship between miR-27-3p and PPARγ was predicted and verified. The lipopolysaccharide (LPS)-treated microglia model was established and intervened with miR-27-3p to detect PPARγ, Iba-1, and inflammation-related factor expressions. After overexpressing PPARγ, rat cerebral inflammation was evaluated. The clinical significance of serum exosomal miR-27-3p in ACI was evaluated. Serum exosomes from ACI patients caused exacerbated MCAO rat cerebral injury and poor behavior recovery, as well as promoted cerebral inflammation. Serum exosomal miR-27-3p deepened rat brain inflammation. miR-27-3p targeted PPARγ to promote microglia activation and inflammation-related factor expressions in MCAO rats, and overexpressing PPARγ attenuated MCAO rat cerebral inflammation. Serum exosomal miR-27-3p promised to be a biomarker for ACI. We proved that serum exosomes from ACI patients aggravated ACI patient cerebral inflammation via the miR-27-3p/PPARγ axis.