Sequential changes in expression of long non-coding RNAs THRIL and MALAT1 after ischemic stroke.

Background: Inflammation is the major contributor to the pathophysiology of ischemic stroke (IS). Long non-coding ribonucleic acids (lncRNAs) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and tumor necrosis factor and heterogeneous nuclear ribonucleoprotein L-related immunoregulatory (THRIL) have been demonstrated to be up-regulated in inflammation and atherosclerosis. Therefore, we aimed to study the expression profile of these lncRNAs after IS. Methods: This observational case-control study was conducted in Namazi Hospital, Shiraz, Iran. The real-time polymerase chain reaction (RT-PCR) measured the sequential changes in circulating levels of MALAT1 and THRIL on days 1, 3, and 5 after IS. The receiver operating characteristic (ROC) curve analysis was used to estimate the diagnostic and prognostic potential of lncRNAs with the area under the curve (AUC). Results: In patients with IS, the relative MALAT1 and THRIL expressions were significantly higher than the controls (P < 0.001 and P < 0.01, respectively), on days 1, 3, and 5 after stroke. We showed a significantly increase in lncRNAs expression on day five compared to days 1 and 3 after stroke. Moreover, a positive correlation was detected between MALAT1 expression and time within the first 24 hours after stroke (r = 0.27, P = 0.03). Logistic regression analysis showed a significant positive association between MALAT1 and THRIL and the risk of stroke evolution. We found a potential diagnostic marker for MALAT1 with an AUC of 0.78. Conclusion: We demonstrated the significant sequential upregulation in MALAT1 and THRIL expression on days 1, 3, and 5 after IS with a significant positive association with the risk of stroke. MALAT1 also significantly correlated with time within the first 24 hours after stroke.

Simple methods for cerebrospinal fluid collection in fetal, neonatal, and adult rat.

BACKGROUND: Cerebrospinal fluid (CSF) collection and its analysis are common medical practices useful in the diagnosis, therapy, and prevention of central nervous system (CNS) disorders. In recent years, several types of research have improved our insight into CSF and its role in health and disease. Yet, many characteristics of this fluid remain to be fully understood. NEW METHODS: Here, we describe how to collect CSF from embryonic, postnatal, and adult stages of the rat. In adults, CSF can be collected through simple stereotaxic surgery to expose the membrane overlying the cisterna magna (CM) of an anesthetized rat and collection of CSF through micropipette puncture through the membrane. In embryos and pups, CSF is aspirated, using a fire-polished micro-capillary pipette, from the CM of animals. RESULTS: Application of these methods provides the maximum volume of pure, uncontaminated CSF (embryonic day 19: 10-15 microliter, postnatal day 5: 20-30 microliter, adults: 100-200 microliter) with a success rate of approximately 95% in every age. COMPARISON WITH EXISTING METHODS: Compared to the existing protocols, these methods obtain considerable volumes of CSF, which may accelerate the measurement of biological markers in this fluid. Also, these techniques do not require surgical skills and according to the practical points mentioned during sampling, the procedures can be performed in rapid fashion. CONCLUSION: We describe simple methods for collecting CSF in live rats. These protocols provide clean, uncontaminated CSF for experiments to understand the exact role of this fluid in the development and maintenance of the CNS health.

Increased Serum Levels of IL-1ß after Ischemic Stroke are Inversely Associated with Vitamin D.

Background: The initial inflammatory reaction starts following occlusion in ischemic stroke (IS). Interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine with a crucial role in the pathogenesis of neurodegenerative disorders. Objective: To investigate the levels of IL-1ß and vitamin D (VitD) in patients with IS compared with controls and their correlation. Methods: The serum level of 25-OH VitD and IL-1ß was assessed in 102 IS patients (0-24 h after stroke) and 102 controls with an enzyme-linked immunosorbent assay (ELISA) kit. Results: We found a significant increase in IL-1ß (80.14±6.8 vs. 60.32±4.1 pg/ml, p<0.05) and a decrease in VitD level (24.3±1.4 vs. 29.9±1.5 ng/ml, p<0.01) in the IS patients compared with the controls. There was a significantly positive correlation between the National Institutes of Health Stroke Scale (NIHSS) and IL-1ß according to both the Spearman correlation (r=0.35, p=0.0003) and the linear regression (beta=0.255, p=0.014). Also, a significant negative association between VitD and NIHSS was detected by the Spearman correlation (r=-0.41, p<0.0001) and the linear regression (beta=-0.381, p=0.000). Moreover, we found a significant negative correlation (r=-0.26, p=0.006) between the serum levels of VitD and IL-1ß in the patient group. Conclusion: Ischemic stroke correlates positively with IL-1ß and negatively with VitD levels. The speculated role of VitD deficiency in the evolution and severity of stroke may be justified by its role in the modification of inflammation.

Integrated multi-omics analysis identifies epigenetic alteration related to neurodegeneration development in post-traumatic stress disorder patients.

INTRODUCTION: Post-traumatic stress disorder (PTSD), is associated with an elevated risk of neurodegenerative disorders, but the molecular mechanism was not wholly identified. Aberrant methylation status and miRNA expression pattern have been identified to be associated with PTSD, but their complex regulatory networks remain largely unexplored. METHODS: The purpose of this study was to identify the key genes/pathways related to neurodegenerative disorder development in PTSD by evaluating epigenetic regulatory signature (DNA methylation and miRNA) using an integrative bioinformatic analysis. We integrated DNA expression array data with miRNA and DNA methylation array data - obtained from the GEO database- to evaluate the epigenetic regulatory mechanisms. RESULTS: Our results indicated that target genes of dysregulated miRNAs were significantly related to several neurodegenerative diseases. Several dysregulated genes in the neurodegeneration pathways interacted with some members of the miR-17 and miR-15/107 families. Our analysis indicated that APP/CaN/NFATs signaling pathway was dysregulated in the peripheral blood samples of PTSD. Besides, the DNMT3a and KMT2D genes, as the encoding DNA and histone methyltransferase enzymes, were upregulated, and DNA methylation and miRNA regulators were proposed as critical molecular mechanisms. Our study found dysregulation of circadian rhythm as the CLOCK gene was upregulated and hypomethylated at TSS1500 CpGs S_shores and was also a target of several dysregulated miRNAs. CONCLUSION: In conclusion, we found evidence of a negative feedback loop between stress oxidative, circadian rhythm dysregulation, miR-17 and miR-15/107 families, some essential genes involved in neuronal and brain cell health, and KMT2D/DNMT3a in the peripheral blood samples of PTSD.

Association of Long Non-Coding RNA Malat1 with Serum Levels of Interleukin-1 Beta and Vitamin D in Patients with Ischemic Stroke.

BACKGROUND: Previous studies have demonstrated the strong association of inflammatory cytokines and vitamin D (VitD) deficiency and ischemic stroke (IS) pathogenesis. Due to the negative correlation between long non-coding RNA (lncRNA) Malat1 and pro-inflammatory factors we decided to investigate the associations between Malat1 expression with serum interleukin-1ß (IL-1ß), and VitD levels in IS patients. MATERIALS AND METHODS: In this cross-sectional study, 63 IS patients were included. We used enzyme-linked immunosorbent assays to evaluate the serum levels of VitD and IL-1ß. Malat1 expression was evaluated by the real-time polymerase chain reaction test. The associations between Malat1expression with VitD and IL-1ß were analysed with linear regression (Stepwise model) and Pearson's correlation analysis. RESULTS: The Malat1 expression was inversely correlated with stroke severity (r=-0.25, P=0.043). Stepwise regression analysis showed a significant positive relationship between VitD level and Malat1 expression (Beta=0.28, P=0.02), and also showed a non-significant negative relationship between IL-1ß and stroke severity. VitD level showed a positive Pearson correlation with Malat1 (r=0.28, P=0.023) and a negative correlation with IL-1ß (r=-0.29, P=0.018) while it could not detect a significantly negative correlation with stroke severity. CONCLUSION: For the first time the associations between Malat1 expression with IL-1ß and VitD in IS patients was analyzed. We found a significant positive relationship between VitD and Malat1. This correlation needs to be investigated with a larger sample size to achieve a strong and reliable association between VitD and Malat1.

G Protein Coupled Receptors Potentially Involved in Oligodendrogenesis: A Gene Expression Analysis.

Background: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system characterized by infiltration of inflammatory leukocytes to the CNS followed by oligodendrocyte cell death, myelin sheath destruction, and axonal injury. A logical incidence occurring after demyelination is remyelination. G-protein coupled receptors (GPCRs) activate internal signal transduction cascades through binding to different ligands. This family of receptors are targeted by more than 40% of currently marketed drugs. GPCRs can be successfully targeted for induction of remyelination. GPCRs highly enriched in oligodendrocyte progenitor cells compared to oligodendrocytes are proposed to hamper oligodendrocyte differentiation and therefore their inhibition might induce remyelination. This study aimed to investigate the expression of GPCRs in silico and in vitro. Methods: We performed gene expression analysis using DAVID and Panther websites on a RNA-seq dataset (GSE52564 accession number). Primary embryonic neural stem/progenitor cell isolation and culture were performed and subsequently NSPCs were characterized by Immunocytochemistry with Anti-Nestin antibody. Expression of GPR37L1, EDNRB, PDGFRα, CNPase and GFAP were assessed using real-time PCR. All the experiments were conducted at Shiraz University of Medical Sciences (SUMS), Shiraz, Iran, in the year 2018. Results: The 14 most highly expressed GPCRs in oligodendrocyte progenitor cells (OPCs) compared to Oligodendrocytes were presented in our study. Conclusion: The investigation of the most highly expressed GPCRs in OPCs compared to oligodendrocyte in silico and in vitro presents the significant role of GPCRs in remyelination induction. Among the 14 GPCRs mentioned in this study, GPR37L1 is a potential remyelinating drug target and is suggested for further studies.

A study on the effect of JNJ-10397049 on proliferation and differentiation of neural precursor cells.

The orexin 2 receptor plays a central role in maintaining sleep and wakefulness. Recently, it has been shown that sleep and wakefulness orchestrate the proliferation and differentiation of oligodendrocytes. Here, we explored the role of a selective orexin 2 receptor antagonist (JNJ-10397049) in proliferation and differentiation of neural progenitor cells (NPCs). We evaluated the proliferation potential of NPCs after exposure to different concentrations of JNJ-10397049 by using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide and neurosphere assays. Moreover, the expression of differentiation markers was assessed by immunocytochemistry and real-time polymerase chain reaction. JNJ-10397049 significantly increased the proliferation of NPCs at lower concentrations. In addition, orexin 2 receptor antagonist facilitated progression of differentiation of NPCs towards oligodendroglial lineage by considerable expression of Olig2 and 2',3'-cyclic-nucleotide 3'-phosphodiesterase as well as decreased expression of nestin marker. The results open a new avenue for future investigations in which the production of more oligodendrocytes from NPCs is needed.