Extracellular ferritin contributes to neuronal injury in an in vitro model of ischemic stroke

Journal of Physiology and Biochemistry - Previous clinical and experimental studies have shown that neurological decline and poor functional outcome after acute ischemic stroke in humans are...     

Administration of thiazolidinediones for neuroprotection in ischemic stroke: a pre-clinical systematic review

Thiazolidinediones (TZDs) may prevent or attenuate CNS injury arising from an ischemic event. We performed meta-analysis of experimental studies in which a TZD (either rosiglitazone or pioglitazone) was administered in a rodent model of focal or global cerebral ischemia. Infarct volume was the primary endpoint for analysis of drug efficacy, and neurological outcome was also assessed. We identified 31 studies through the use of PubMed and Embase, 22 of which met our pre-specified inclusion criteria and were analyzed with the Cochrane Review Manager software. Treatment with TZDs decreased infarct volume and improved neurological outcome regardless of study quality, dose timing, or ischemia model (transient or permanent). Rosiglitazone and pioglitazone were similarly effective in reducing infarct volume and protecting neurologic function. Importantly, the collective data suggest that pre-treatment with a TZD is not required for neuroprotection, although additional studies are clearly needed to define the breadth of the therapeutic window. The data warrant further studies into the potential acute use of TZDs for ischemic stroke therapy in the general population.     

Association between microRNA binding site polymorphisms in immunoinflammatory genes and recurrence risk of ischemic stroke

MicroRNA binding site polymorphisms in immunoinflammatory genes have been implicated as candidate biomarkers for prediction of complex human diseases. However, the roles of microRNA binding site polymorphisms in stroke onset and prognosis remain unclear. Thus, for the first time, five potential functional polymorphisms in immunoinflammatory genes (CXCR2 rs1126579, TLR4 rs11536889, ADIPOR2 rs12342, MMP-2 rs7201 and MMP-9 rs1056628) were genotyped in 657 patients with ischemic stroke. These five polymorphisms were not related with age onset of ischemic stroke. However, we found that ADIPOR2 rs12342 was significantly associated with a decreased recurrence risk, especially for the patients with small-vessel disease. Moreover, by using multivariate Cox regression, the variant genotype GG/GA of rs12342 was observed as an independent protective factor for stroke recurrence, even after Bonferroni correction. In addition, after the addition of rs12342 in the model with clinical factors, the new model showed the improved discriminatory ability to predict stroke recurrence. In short, our results suggested that ADIPOR2 rs12342 may be a novel genetic biomarker and therapeutic target for ischemic stroke recurrence. Further studies are required to replicate our findings and clarify the potential biological mechanism.     

Downregulation of microRNA let-7f mediated the Adriamycin resistance in leukemia cell line

Multidrug resistance (MDR) has become the major cause of failure chemotherapy for leukemia and high mortality of leukemia. The study aimed to investigate whether the let-7f mediate the Adriamycin (ADR) resistance of leukemia, and to explore the potential molecular mechanism. Cell proliferation was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the soft agar clone formation assay. Flow cytometry was performed to detected cell cycle and apoptosis. The targeted regulationship was analyzed by dual-luciferase assay. Real-time polymerase chain reaction and Western blot were used to measure the expressions of let-7f, ABCC5, ABCC10, cell cycle-related proteins, and apoptosis-related proteins. The xenograft mouse model was used to conduct the tumor formation assay in vivo. The results demonstrated that the expression of let-7f was lower in multidrug-resistant K562/A02 cell lines compared to that in K562, while ABCC5 and ABCC10 were upregulated. Overexpression of let-7f in K562/A02 cell lines downregulated the ABCC5 and ABCC10 expression, enhanced cell sensitivity to ADR, promoted cell apoptosis, and inhibited cell proliferation. let-7f was proved to negatively regulate ABCC5 and ABCC10. Tumor formation assay further determined that let-7f overexpression increased sensitivity to ADR. Taken together, the let-7f downregulation induced the ADR resistance of leukemia by upregulating ABCC5 and ABCC10 expression. Our study provided a novel perspective to study the mechanism of MDR and a new target for the reversal of MDR.     

No influence of dabigatran anticoagulation on hemorrhagic transformation in an experimental model of ischemic stroke

Background

Dabigatran etexilate (DE) is a new oral direct thrombin inhibitor. Clinical trials point towards a favourable risk-to-benefit profile of DE compared to warfarin. In this study, we evaluated whether hemorrhagic transformation (HT) occurs after experimental stroke under DE treatment as we have shown for warfarin.

Methods

44 male C57BL/6 mice were pretreated orally with 37.5 mg/kg DE, 75 mg/kg DE or saline and diluted thrombin time (dTT) and DE plasma concentrations were monitored. Ischemic stroke was induced by transient middle cerebral artery occlusion (tMCAO) for 1 h or 3 h. We assessed functional outcome and HT blood volume 24 h and 72 h after tMCAO.

Results

After 1 h tMCAO, HT blood volume did not differ significantly between mice pretreated with DE 37.5 mg/kg and controls (1.5±0.5 µl vs. 1.8±0.5 µl, p>0.05). After 3 h tMCAO, DE-anticoagulated mice did also not show an increase in HT, neither at the dose of 37.5 mg/kg equivalent to anticoagulant treatment in the therapeutic range (1.3±0.9 µl vs. control 2.3±0.5 µl, p>0.05) nor at 75 mg/kg, clearly representing supratherapeutic anticoagulation (1.8±0.8 µl, p>0.05). Furthermore, no significant increase in HT under continued anticoagulation with DE 75 mg/kg could be found at 72 h after tMCAO for 1 h (1.7±0.9 µl vs. control 1.6±0.4 µl, p>0.05).

Conclusion

Our experimental data suggest that DE does not significantly increase hemorrhagic transformation after transient focal cerebral ischemia in mice. From a translational viewpoint, this indicates that a continuation of DE anticoagulation in case of an ischemic stroke might be safe, but clearly, clinical data on this question are warranted.     

Genetic susceptibility to ischemic stroke in Russians

The frequencies of alleles and genotypes for ten functionally significant single-nucleotide polymorphisms were determined in the FGA, FGB, APOE, LPL, ACE, and CMA1 genes for Russian ischemic stroke (IS) patients and for a control group of Russians similar in gender and age distribution. The groups showed no significant differences in the frequencies of individual alleles or genotypes for any polymorphism studied. However, complex analysis of genetic susceptibility by the APSampler algorithm demonstrated that carriership of the APOE (−491A) allele predisposed to IS (p = 0.044, OR 3.8, 95% CI 1.0–15.1). Correspondingly, the APOE (−491T/T) genotype was associated with resistance to IS (p = 0.044, OR 0.26, 95% CI 0.07–1.0). The carriership of FGB (−249C) allele together with this genotype enhanced its protective potential, reducing the p value of the combination twofold (OR 0.17, 95% CI 0.04–0.8). Two more protective combinations were identified: biallelic APOE (−427C) + LPL (1595G) and triallelic APOE (−491C) + LPL (1595G) + CMA1 (−1903G). In both cases, p = 0.0052, OR 0.18, and 95% CI 0.05–0.66. Altogether, involvement in the formation of IS risk in Russians was evidenced for alleles of four genes: APOE, FGB, LPL, and CMA1; the APOE involvement was demonstrated for alleles of two polymorphic loci: −491T and −427C. Linkage analysis suggested that these loci were involved in IS resistance independently of each other.     

Inhibition of SNK‐SPAR signaling pathway promotes the restoration of motor function in a rat model of ischemic stroke

This study aimed to investigate the effects of SPAR signaling pathway on the restoration of motor function in ischemic stroke (IS). Sprague‐Dawley male rats were separated into the control and sham groups, as well as the group for middle cerebral artery occlusion (MCAO) model establishment. Successfully established rat ischemic models were randomly divided into model, SNK^MCAO‐del and pcDNA3.1‐SNK groups. The evaluation of motor function among the rats in each group was assessed using a balance beam, a screen test and the Garcia scoring method. CatWalk gait analysis was employed to evaluate the effect of the SNK signaling pathway on rat motor function. Triphenyltetrazolium chloride (TTC) and TUNEL staining were techniques were utilized for cerebral infarction (CI) area as well for hippocampal neuron apoptosis. The quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blotting methods were performed to detect mRNA and protein expressions of SNK and SPAR. When compared with the model group, the SNK^MCAO‐del group displayed decreased motor function score and CI area, while contrasting results were observed in the pcDNA3.1‐SNK group. According to the results obtained from the CatWalk gait analysis, the SNK^MCAO‐del group showed a clear improvement compared to the model group whereas the pcDNA3.1‐SNK group exhibited poorer results than the model group in the objective parameters of the study, such as movement, speed, running duration, print area, maximal contact area, maximal, mean intensity, and stride length. These findings suggested that SNK gene silencing promotes motor function by inhibiting the SNK‐SPAR signaling pathway in rats with ischemic stroke.     

GRM7 polymorphisms are not associated with ischemic stroke in Iranian population

Abstract

Objectives: Ischemic stroke is the main neurological cause of acquired incapability in adults and a prominent cause of mortality. Several association studies have been conducted to explore the role of candidate genes in this neurological condition.

Methods: In the present study, we aimed at identification of association between Glutamate Metabotropic Receptor 7 (GRM7) and risk of ischemic stroke in Iranian population. Two intronic variants within this gene (rs6782011 and rs779867) were genotyped in 318 sporadic ischemic stroke cases and 300 unrelated, healthy controls individuals.

Results: No significant difference was found in allele, genotype or haplotype frequencies of these SNPs between cases and controls after correction for multiple comparisons.

Conclusion: Consequently, the assessed GRM7 variants are not implicated in risk of ischemic stroke in Iranian population.     

Autophagy activation is associated with neuroprotection in a rat model of focal cerebral ischemic preconditioning

Several recent studies have showed that autophagy is involved in ischemic brain damage, but it may also play a pro-survival role in ischemic preconditioning. This study was taken to determine the role of autophagy in an animal model of cerebral ischemic preconditioning (IPC). Focal cerebral IPC was produced in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The rats were pretreated with intracerebral ventricle infusion of the autophagy inhibitors 3-methyladenine (3-MA) and bafliomycin A1 (Baf A1) or the autophagy inducer rapamycin to evaluate the contribution of autophagy to IPC-induced neuroprotection. The results from electron microscopic examinations and immunofluorescence showed that both IPC and PFI induced autophagy activation, but the extent and persistence of autophagy activation were varied. IPC treatment significantly reduced infarct volume, brain edema and motor deficits after subsequent PFI, whereas 3-MA and Baf A1 suppressed the neuroprotection induced by IPC. 3-MA pretreatment also significantly attenuated upregulation of LC3-II, beclin 1 and HSP70 and downregulation of p62. To further determine if autophagy induction is responsible for IPC-induced neuroprotection, rats were treated with rapamycin 24 h before the onset of PFI. The results showed that rapamycin reduced infarct volume, brain edema and motor deficits induced by PFI. Rapamycin pretreatment also increased the protein levels of LC3-II and beclin 1. These results demonstrate that autophagy activation during IPC offers a remarkable tolerance to a subsequent fatal ischemic insult, and IPC's neuroprotective effects can be mimicked by autophagy inducers.     

An effective disease diagnostic model related to pyroptosis in ischemic cardiomyopathy

Pyroptosis is involved in ischemic cardiomyopathy (ICM). The study aimed to investigate the pyroptosis-related genes and clarify their diagnostic value in ICM. The bioinformatics method identified the differential pyroptosis genes between the normal control and ICM samples from online datasets. Then, protein–protein interaction (PPI) and function analysis were carried out to explore the function of these genes. Following, subtype analysis was performed using ConsensusClusterPlus, functions, immune score, stromal score, immune cell proportion and human leukocyte antigen (HLA) genes between subtypes were investigated. Moreover, optimal pyroptosis genes were selected using the least absolute shrinkage and selection operator (LASSO) analysis to construct a diagnostic model and evaluate its effectiveness using receiver operator characteristic (ROC) analysis. Twenty-one differential expressed pyroptosis genes were identified, and these genes were related to immune and pyroptosis. Subtype analysis identified two obvious subtypes: sub-1 and sub-2. And LASSO identified 13 optimal genes used to construct the diagnostic model. The diagnostic model in ICM diagnosis with the area under ROC (AUC) was 0.965. Our results suggested that pyroptosis was tightly associated with ICM.     

Neuroprotective effects of amiodarone in a mouse model of ischemic stroke

Background

Ion channels play a crucial role in the development of ischemic brain injury. Recent studies have reported that the blockade of various types of ion channels improves outcomes in experimental stroke models. Amiodarone, one of the most effective drugs for life-threatening arrhythmia, works as a multiple channel blocker and its characteristics cover all four Vaughan-Williams classes. Although it is known that amiodarone indirectly contributes to preventing ischemic stroke by maintaining sinus rhythm in patients with atrial fibrillation, the direct neuroprotective effect of amiodarone has not been clarified. The purpose of this study was to investigate the direct effect of amiodarone on ischemic stroke in mice.

Methods

Focal cerebral ischemia was induced via distal permanent middle cerebral artery occlusion (MCAO) in adult male mice. The amiodarone pre-treatment group received 50 mg/kg of amiodarone 1 h before MCAO; the amiodarone post-treatment groups received 50 mg/kg of amiodarone immediately after MCAO; the control group received vehicle only. In addition, the sodium channel opener veratrine and selective beta-adrenergic agonist isoprotelenol were used to elucidate the targeted pathway. Heart rate and blood pressure were monitored perioperatively. Infarct volume analysis was conducted 48 h after MCAO. The body asymmetry test and the corner test were used for neurological evaluation.

Results

Amiodarone pre-treatment and post-treatment reduced the heart rate but did not affect the blood pressure. No mice showed arrhythmia. Compared with the control group, the amiodarone pre-treatment group had smaller infarct volumes (8.9?±?2.1% hemisphere [mean?±?SD] vs. 11.2?±?1.4%; P?<?0.05) and improved functional outcomes: lower asymmetric body swing rates (52?±?17% vs. 65?±?18%; P?<?0.05) and fewer left turns (7.1?±?1.2 vs. 8.3?±?1.2; P?<?0.05). In contrast, amiodarone post-treatment did not improve the outcomes after MCAO. The neuroprotective effect of amiodarone pre-treatment was abolished by co-administration of veratrine but not by isoproterenol.

Conclusions

Amiodarone pre-treatment attenuated ischemic brain injury and improved functional outcomes without affecting heart rhythm and blood pressure. The present results showed that amiodarone pre-treatment has neuroprotective effects, at least in part, via blocking the sodium channels.

     

Rare variants in ischemic stroke: an exome pilot study

The genetic architecture of ischemic stroke is complex and is likely to include rare or low frequency variants with high penetrance and large effect sizes. Such variants are likely to provide important insights into disease pathogenesis compared to common variants with small effect sizes. Because a significant portion of human functional variation may derive from the protein-coding portion of genes we undertook a pilot study to identify variation across the human exome (i.e., the coding exons across the entire human genome) in 10 ischemic stroke cases. Our efforts focused on evaluating the feasibility and identifying the difficulties in this type of research as it applies to ischemic stroke. The cases included 8 African-Americans and 2 Caucasians selected on the basis of similar stroke subtypes and by implementing a case selection algorithm that emphasized the genetic contribution of stroke risk. Following construction of paired-end sequencing libraries, all predicted human exons in each sample were captured and sequenced. Sequencing generated an average of 25.5 million read pairs (75 bp×2) and 3.8 Gbp per sample. After passing quality filters, screening the exomes against dbSNP demonstrated an average of 2839 novel SNPs among African-Americans and 1105 among Caucasians. In an aggregate analysis, 48 genes were identified to have at least one rare variant across all stroke cases. One gene, CSN3, identified by screening our prior GWAS results in conjunction with our exome results, was found to contain an interesting coding polymorphism as well as containing excess rare variation as compared with the other genes evaluated. In conclusion, while rare coding variants may predispose to the risk of ischemic stroke, this fact has yet to be definitively proven. Our study demonstrates the complexities of such research and highlights that while exome data can be obtained, the optimal analytical methods have yet to be determined.     

Role of acute-phase protein ORM in a mice model of ischemic stroke

The only Food and Drug Administration-approved treatment for acute ischemic stroke is tissue plasminogen activator, and the discovery of novel therapeutic targets is critical. Here, we found orosomucoid (ORM), an acute-phase protein mainly produced by the liver, might act as a treatment candidate for an ischemic stroke. The results showed that ORM2 is the dominant subtype in mice normal brain tissue. After middle cerebral artery occlusion (MCAO), the level of ORM2 is significantly increased in the ischemic penumbra compared with the contralateral normal brain tissue, whereas ORM1 knockout did not affect the infarct size. Exogenous ORM could significantly decrease infarct size and neurological deficit score. Inspiringly, the best administration time point was at 4.5 and 6 hr after MCAO. ORM could markedly decrease the Evans blue extravasation, and improve blood–brain barrier-associated proteins expression in the ischemic penumbra of MACO mice and oxygen–glucose deprivation (OGD)-treated bEnd3 cells. Meanwhile, ORM could significantly alleviate inflammation by inhibiting the production of interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α), reduce oxidative stress by improving the balance of malondialdehyde (MDA) and superoxide dismutase (SOD), inhibit apoptosis by decreasing caspase-3 activity in ischemic penumbra of MCAO mice and OGD-treated bEnd.3 cells. Because of its protective role at multiple levels, ORM might be a promising therapeutic target for ischemic stroke.     

Immuno-proteasome subunit LMP7 is up-regulated in the ischemic kidney in an experimental model of renovascular hypertension

Immuno-proteasome is thought to be responsible for the processing of intracellular antigens and is induced when cells are treated with the inflammatory cytokines promoting cellular immunity. We tested the possibility that immuno-proteasome can be up-regulated in renal cells exposed to a long-lasting ischemia and inflammation in an experimental model of two-kidney, one-clip renovascular hypertension in the rat. Western blotting showed that immuno-proteasome subunit, LMP7, was up-regulated in the clipped ischemic kidney that was atrophic, but not in the contralateral unclipped kidney that underwent compensatory hypertrophy. Immunohistochemical analysis revealed that LMP7 was highly expressed in cortical epithelial and endothelial cells of the ischemic kidney. Surprisingly, the second immuno-subunit, LMP2, was almost undetectable, indicating that renal ischemia may induce exclusively the LMP7 subunit. We also found that renal ischemia neither reduced the SDS-stimulated proteasomal activity nor affected a high level of the PA28 activator. Thus, the results provide evidence that LMP7 immuno-subunit is induced in renal cells exposed to a long-lasting renal ischemia and inflammation, and that there is a direct link between LMP induction and renal atrophy. This opens an opportunity to study a role for LMP-containing proteasomes in the kidneys and other organs undergoing reduction in mass in diseases accompanied by a long-lasting ischemia and inflammatory responses.