Intracranial Self-Stimulation Modulates Levels of SIRT1 Protein and Neural Plasticity-Related microRNAs.

Deep brain stimulation (DBS) of reward system brain areas, such as the medial forebrain bundle (MFB), by means of intracranial self-stimulation (ICSS), facilitates learning and memory in rodents. MFB-ICSS has been found capable of modifying different plasticity-related proteins, but its underlying molecular mechanisms require further elucidation. MicroRNAs (miRNAs) and the longevity-associated SIRT1 protein have emerged as important regulatory molecules implicated in neural plasticity. Thus, we aimed to analyze the effects of MFB-ICSS on miRNAs expression and SIRT1 protein levels in hippocampal subfields and serum. We used OpenArray to select miRNA candidates differentially expressed in the dentate gyrus (DG) of ICSS-treated (3 sessions, 45' session/day) and sham rats. We further analyzed the expression of these miRNAs, together with candidates selected after bibliographic screening (miR-132-3p, miR-134-5p, miR-146a-5p, miR-181c-5p) in DG, CA1, and CA3, as well as in serum, by qRT-PCR. We also assessed tissue and serum SIRT1 protein levels by Western Blot and ELISA, respectively. Expression of miR-132-3p, miR-181c-5p, miR-495-3p, and SIRT1 protein was upregulated in DG of ICSS rats (P < 0.05). None of the analyzed molecules was regulated in CA3, while miR-132-3p was also increased in CA1 (P = 0.011) and serum (P = 0.048). This work shows for the first time that a DBS procedure, specifically MFB-ICSS, modulates the levels of plasticity-related miRNAs and SIRT1 in specific hippocampal subfields. The mechanistic role of these molecules could be key to the improvement of memory by MFB-ICSS. Moreover, regarding the proposed clinical applicability of DBS, serum miR-132 is suggested as a potential treatment biomarker.

Evaluation of long-term rt-PA effects on bEnd.3 endothelial cells under ischemic conditions; changes in ZO-1 expression and glycosylation of the bradykinin B2 receptor.

Recombinant tissue plasminogen activator (rt-PA) has proven effective in the treatment of acute ischemic stroke, despite the increased risk of hemorrhagic transformation (HT), its major associated complication. Although it is known that HT is related to blood brain barrier (BBB) disruption, the underlying mechanisms are not well established. We assessed time-dependent effects of rt-PA on the bEnd.3 murine brain endothelial cell line subjected either to normoxia or to 2.5 h of oxygen and glucose deprivation (OGD), evaluating a longer period than has previously been done, beyond 6 h post-reoxygenation. Parameters of cell viability, metabolic activity, ionic and transcellular permeability, as well as levels of claudin-5, zonula occludens-1 (ZO-1) and bradykinin B2 receptor (B2R) protein expression were analyzed at 24, 48 and 72 h post-reoxygenation with or without the administration of rt-PA. rt-PA treatment increased both the ionic and transcellular permeability until 72 h and did not modify cell viability or metabolic activity or the expression of claudin-5, ZO-1 and B2R under normoxia at any analyzed time. Under OGD conditions, rt-PA exacerbated OGD effects on metabolic activity from 48 to 72 h, increased transcellular permeability from 24 to 72 h, significantly decreased ZO-1 protein levels at the plasma membrane and increased B2R glycosylation at 72 h post-reoxygenation. Our findings suggest that a long-term analysis is necessary to elucidate time-dependent molecular mechanisms associated to BBB breakdown due to rt-PA administration under ischemia. Thus, protective BBB therapies after ischemic stroke and rt-PA treatment should be explored at least until 72 h after OGD and rt-PA administration.

Cold stress protein RBM3 responds to hypothermia and is associated with good stroke outcome.

RNA-binding motif protein 3 is a molecular marker of hypothermia that has proved neuroprotective in neurodegenerative disease models. However, its relationship to the well-recognized therapeutic effect of hypothermia in ischaemic stroke had not been studied. In this work, the expression of RNA-binding motif protein 3 was investigated in ischaemic animal models subjected to systemic and focal brain hypothermia, specifically the effects of RNA-binding motif protein 3 silencing and overexpression on ischaemic lesions. Moreover, the association of RNA-binding motif protein 3 levels with body temperature and clinical outcome was evaluated in two independent cohorts of acute ischaemic stroke patients (n = 215); these levels were also determined in a third cohort of 31 patients derived from the phase III EuroHYP-1 trial of therapeutic cooling in ischaemic stroke. The preclinical data confirmed the increase of brain RNA-binding motif protein 3 levels in ischaemic animals subjected to systemic and focal hypothermia; this increase was selectively higher in the cooled hemisphere of animals undergoing focal brain hypothermia, thus confirming the direct effect of hypothermia on RNA-binding motif protein 3 expression, while RNA-binding motif protein 3 up-regulation in ischaemic brain regions led to functional recovery. Clinically, patients with body temperature <37.5°C in the first two cohorts had higher RNA-binding motif protein 3 values at 24 h and good outcome at 3 months post-ischaemic stroke, while RNA-binding motif protein 3 levels in the cooled third cohort tended to exceed those in placebo-treated patients. These results make RNA-binding motif protein 3 a molecular marker associated with the effect of hypothermia in ischaemic stroke and suggest its potential application as a promising protective target.

Evaluation of common housekeeping proteins under ischemic conditions and/or rt-PA treatment in bEnd.3 cells.

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is the only pharmacological approved treatment for ischemic stroke, despite its associated increasing risk of hemorrhagic transformation. Since many of rt-PA effects in blood-brain barrier (BBB) are not well characterized, the study of protein changes in BBB cells after rt-PA administration may help to understand its adverse effects. Our aim was to analyze protein levels of four commonly used housekeeping proteins: ß-Actin, α-Tubulin, GAPDH and HPRT in bEnd.3 endothelial cell line subjected to oxygen and glucose deprivation (OGD) conditions and rt-PA treatment to determine their reliability as Western blot loading controls. bEnd.3 monolayers were subjected to 2.5 h of OGD and reperfusion with/without 20 µg/ml of rt-PA. At 3, 6, 24 and 72 h post-OGD, protein levels were analyzed by Western blot using Stain-Free technology. OGD significantly decreased ß-Actin, α-Tubulin, GAPDH and HPRT protein levels at 3, 6, 24 and 72 h post-OGD without significant rt-PA treatment effects except for the GAPDH levels increase in control condition at 3 h post-OGD. The present study clearly demonstrated that ß-Actin, α-Tubulin, GAPDH and HPRT proteins are not suitable as loading controls for Western Blot analysis in bEnd.3 cells after OGD. SIGNIFICANCE: We reported altered levels of ß-Actin, α-Tubulin, GAPDH and HPRT housekeeping proteins in bEnd.3 endothelial cell line after an ischemic insult. Therefore, we demonstrated that these proteins are not suitable as loading controls for Western Blot analysis in our experimental conditions and we recommended the use of Stain-Free gels as an alternative to traditional housekeeping proteins normalization.

Low Levels of Caveolin-1 Predict Symptomatic Bleeding After Thrombolytic Therapy in Patients With Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Experimental models of cerebral ischemia demonstrate that the decrease in the caveolin-1 membrane protein results in an increase in endothelial permeability. Because this phenomenon is responsible for hemorrhagic transformation (HT) after cerebral ischemia, we aimed to determine whether caveolin-1 levels may predict bleeding after recombinant tissue-type plasminogen activator (r-tPA) administration in patients with acute stroke. METHODS: We studied 133 patients with a first hemispheric stroke treated with r-tPA within 4.5 hours of symptom onset. HT was evaluated and classified on cranial computed tomography at 24 hours and was considered as symptomatic HT (sHT) if associated with neurological deterioration. Serum caveolin-1 levels were analyzed before and at 2, 24, and 72 hours post-r-tPA administration in patients and in 40 healthy controls. RESULTS: Baseline caveolin-1 levels were higher in patients than controls (0.24 [0.17-0.40] versus 0.07 [0.0-0.20] ng/mL; P<0.000). Twenty six (19.5%) patients had HT, which was symptomatic in 7 (5.3%). Patients with parenchymal hemorrhage-2 and sHT had lower baseline caveolin-1 levels than the rest of patients (0.08 [0.04-0.19] versus 0.26 [0.14-0.40]; P=0.019 and 0.08 [0.02-0.17] versus 0.26 [0.13-0.41]; P=0.019, respectively). The levels remained stable in the first 72 hours in patients with parenchymal hemorrhage-2 and sHT, whereas in the rest of patients levels decreased in this time. Caveolin-1 levels ≤0.17 ng/mL had the highest predictive capacity of sHT (86% sensitivity, 65% specificity, 99% negative predictive value, 12% positive predictive value). After adjustment for confounders, caveolin-1 levels ≤0.17 ng/mL independently predicted sHT (odds ratio, 11.6; 95% confidence interval, 11.3-102.8; P=0.027). CONCLUSIONS: Low serum levels of caveolin-1 are an independent predictor of sHT after r-tPA administration. Because of the small sample size, further research is needed to validate these data.

Ethyl-bridged hybrid column as an efficient alternative for HPLC analysis of plasma amino acids by pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

Conventional C18 silica columns have proven to be useful for the analysis of amino acids (AA) from protein hydrolysates but undesirable peak overlapping is usually found when analyzing body fluids given that a large number of AAs are present in the samples. As an alternative to silica packings, an ethyl-bridged packing for reversed-phase liquid chromatography of derivatized AAs with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) has been evaluated. The new packing material improves the separation efficiency allowing better separations when analyzing biological fluids. Moreover, this packing has advantages for routine AA analysis, such as a decrease in the total running time and an increase in the life-time of the columns. The pH of the mobile phase has a significant effect on the elution behavior of the AQC hydrolysis product (AMQ) and on the AA derivatives. It is not possible to elute AMQ before detecting the first AA derivative, which requires an accurate adjustment of the pH in the range of 5.30-5.35 to obtain good separation and resolution for the most polar compounds. Under the conditions proposed, it is possible to separate all AAs except the Gly-Gln pair, which is not a problem when hydrolyzed samples are analyzed. The AMQ-Ser pair requires either the use of a different mobile phase pH for its baseline separation or the use of fluorescence detection. Two different procedures for protein removal from plasma samples have been evaluated, solvent precipitation and ultrafiltration (UF) and it has been found that UF gives better results as no significant losses of AAs were observed. The validation of the proposed method with UV detection gives method detection limits in the range of 8-12µM, with repeatability values<8% (n=6) and inter-day precision in plasma samples ranging from 4 to 13% (n=4).

A polymorphism in the promoter region of the survivin gene is related to hemorrhagic transformation in patients with acute ischemic stroke.

Hemorrhagic transformation (HT) of cerebral infarction is a common and serious occurrence following acute ischemic stroke. The expression of survivin, a member of the inhibitor of apoptosis protein family, has been shown to increase after cerebral ischemia. This protein has been mainly located at the microvasculature within the infarcted and peri-infarcted area, so we aimed to investigate whether survivin gene polymorphisms, also known as BIRC5 gene, were associated with HT of cerebral infarction. Polymorphism screening of the BIRC5 gene was performed in 107 patients with a hemispheric ischemic stroke and 93 controls by polymerase chain reaction, single-strand conformation polymorphism and sequencing analysis. Genotype-phenotype correlation was performed in patients. MRI was carried out within 12 h of symptoms onset and at 72 ± 12 h. The presence of HT was determined on the second DWI sequence and classified according to ECASS II criteria. MMP-9 levels were analyzed at admission. Forty-nine patients (45.8%) had HT. The -241 C/T (rs17878467) polymorphism was identified in the promoter region of the survivin gene. The prevalence of the mutant allele (T) was similar in patients and controls (14 vs. 16%, respectively; P = 0.37). However, 9 (29%) patients with allele T had HT compared to 40 (52.6%) of wild-type (P = 0.021). Logistic regression analysis showed that the polymorphism was associated with a lower risk of HT (OR 0.16; 95% CI 0.04-0.65; P = 0.01). The -241 C/T polymorphism in the promoter region of the survivin gene is associated with a lower risk of HT in patients with ischemic stroke. It has recently been reported that the -241 C/T polymorphism increases survivin promoter activity, reinforcing the hypothesis that patients with the mutant allele may have increased survivin expression in the brain. Different mechanisms, including BBB protection by the inhibition or activation of different angiogenic growth factors and the inhibition of apoptosis during angiogenesis, may explain the protective effect of this polymorphism on HT development in ischemic stroke. Further studies are needed to confirm our results and elucidate the mechanisms explaining this effect.

Oct-2 transcription factor binding activity and expression up-regulation in rat cerebral ischaemia is associated with a diminution of neuronal damage in vitro.

Brain plasticity provides a mechanism to compensate for lesions produced as a result of stroke. The present study aims to identify new transcription factors (TFs) following focal cerebral ischaemia in rat as potential therapeutic targets. A transient focal cerebral ischaemia model was used for TF-binding activity and TF-TF interaction profile analysis. A permanent focal cerebral ischaemia model was used for the transcript gene analysis and for the protein study. The identification of TF variants, mRNA analysis, and protein study was performed using conventional polymerase chain reaction (PCR), qPCR, and Western blot and immunofluorescence, respectively. Rat cortical neurons were transfected with small interfering RNA against the TF in order to study its role. The TF-binding analysis revealed a differential binding activity of the octamer family in ischaemic brain in comparison with the control brain samples both in acute and late phases. In this study, we focused on Oct-2 TF. Five of the six putative Oct-2 transcript variants are expressed in both control and ischaemic rat brain, showing a significant increase in the late phase of ischaemia. Oct-2 protein showed neuronal localisation both in control and ischaemic rat brain cortical slices. Functional studies revealed that Oct-2 interacts with TFs involved in important brain processes (neuronal and vascular development) and basic cellular functions and that Oct-2 knockdown promotes neuronal injury. The present study shows that Oct-2 expression and binding activity increase in the late phase of cerebral ischaemia and finds Oct-2 to be involved in reducing ischaemic-mediated neuronal injury.

miRNA expression is modulated over time after focal ischaemia: up-regulation of miR-347 promotes neuronal apoptosis.

Despite the large number of molecules reported as being over-expressed after ischaemia, little is known regarding their regulation. miRNAs are potent post-transcriptional regulators of gene expression, and reports have shown differentially miRNA expression in response to focal cerebral ischaemia. The present study analysed miRNA expression from acute to late phases of ischaemia to identify specific ischaemia-related miRNAs, elucidate their role, and identify potential targets involved in stroke pathophysiology. Of 112 miRNAs, 32 showed significant changes and different expression profiles. In addition to the previously reported differentially expressed miRNAs, new ischaemia-regulated miRNAs have been found, including miR-347. Forty-seven genes involved in brain functions or related to ischaemia are predicted to be potential targets of the differentially expressed miRNAs after middle cerebral artery occlusion. Analysis of four of these targets (Acsl4, Arf3, Btg2 and Dpysl5) showed them to be differentially regulated by ischaemia at the transcriptional or post-transcriptional level. Acsl4, Bnip3l and Phyhip, potential targets of miR-347, were up-regulated after miR-347 over-expression, inducing neuronal apoptotic death. Our findings suggest that miR-347 plays an important role in regulating neuronal cell death, identify Acsl4 as a new protein requiring study in ischaemia, and provide an important resource for future functional studies of miRNAs after ischaemia.

Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia.

BACKGROUND: Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used. The present study aims to determine the expression stability of ten housekeeping genes (Gapdh, beta2m, Hprt, Ppia, Rpl13a, Oaz1, 18S rRNA, Gusb, Ywhaz and Sdha) to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models. RESULTS: The expression stability of the candidate reference genes was evaluated using the 2-Delta C'T method and ANOVA followed by Dunnett's test. For the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for Rpl13a and Sdha were found to have significantly different levels of mRNA expression. These different levels were also found in the case of the in-vivo model of pMCAO in rats except for Hprt, Sdha and Ywhaz mRNA, where the expression did not vary. Sdha and Ywhaz were identified by geNorm and NormFinder as the two most stable genes. CONCLUSION: We have validated endogenous control genes for qRT-PCR analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. For normalization purposes, Rpl13a and Sdha are found to be the most suitable genes for the in-vitro model and Sdha and Ywhaz for the in-vivo model. Genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup.

The ACAA-insertion/deletion polymorphism at the 3' UTR of the IGF-II receptor gene is associated with type 2 diabetes and surrogate markers of insulin resistance.

OBJECTIVE: The IGF-II receptor gene (IGFIIR) is located at chromosome 6q26, a region that harbors a genetic marker linked to insulin-resistant traits in Mexican-Americans. In the present study conducted in Spaniards, we tested a common polymorphism in IGFIIR for association with type 2 diabetes and insulin-resistant traits. DESIGN: Case-control association study. METHODS: One hundred and forty-five type 2 diabetic patients and 217 non-diabetic controls were genotyped for the ACAA-insertion/deletion polymorphism at the 3' UTR of IGFIIR. Phenotyping included anthropometrics and a metabolic profile, including serum lipid levels and surrogate indexes of insulin resistance whenever possible. RESULTS: Diabetic patients were more frequently homozygous for the wild type 144 bp allele of IGFIIR compared with controls (diabetic patients 77.2%, controls 51.6%, P<0.001) suggesting a potential protective role against type 2 diabetes for the IGFIIR 140 bp variant. Carrying 140 bp alleles was associated with an odds ratio of having diabetes of 0.290 (95% confidence interval 0.109-0.770), and controls homozygous for the wild type 144 bp allele presented with lower insulin and triglyceride levels, which are proxies for insulin resistance. CONCLUSIONS: The ACAA-insertion/deletion polymorphism at the 3' UTR of IGFIIR is associated with type 2 diabetes and influences surrogate markers of insulin resistance in non-diabetic subjects.

Natural antibiotics and insulin sensitivity: the role of bactericidal/permeability-increasing protein.

The innate immune system can immediately respond to microorganism intrusion by helping to prevent further invasion. Bactericidal/permeability-increasing protein (BPI) is a major constituent of neutrophils that possesses anti-inflammatory properties. Inflammation is increasingly recognized as a component of the metabolic syndrome. We hypothesized that the production of BPI could be linked to insulin sensitivity and glucose tolerance. We studied circulating BPI across categories of glucose tolerance. We also studied whether these cross-sectional associations were of functional importance. For this reason, we investigated circulating bioactive lipopolysaccharide and the effects of changing insulin action-after treatment with an insulin sensitizer (metformin)-on circulating BPI in subjects with glucose intolerance. Finally, we tested whether a 3'-untranslated region (UTR) BPI polymorphism led to differences in BPI and insulin action among nondiabetic subjects. Age- and BMI-adjusted circulating BPI was significantly lower among patients with type 2 diabetes. Circulating BPI correlated negatively with fasting and postload glucose and insulin concentrations. In subjects with glucose intolerance, BPI was also linked to BMI, waist-to-hip ratio, and age- and BMI-adjusted insulin sensitivity. Bioactive lipopolysaccharide was negatively correlated with circulating BPI (r = -0.57, P < 0.0001) and positively with plasma lipopolysaccharide-binding protein (r = 0.54, P = 0.002). In parallel to improved insulin sensitivity, plasma BPI significantly increased in the metformin group but not in the placebo group. A 3'-UTR BPI polymorphism was simultaneously associated with plasma BPI concentration, waist-to-hip ratio, fasting and postload insulin concentration, fasting plasma triglycerides, and insulin sensitivity. These findings suggest that this component of the innate immune system is associated with metabolic pathways.