Impact on Prehospital Delay of a Stroke Preparedness Campaign: A SW-RCT (Stepped-Wedge Cluster Randomized Controlled Trial)

Background and Purpose—Public campaigns to increase stroke preparedness have been tested in different contexts, showing contradictory results. We evaluated the effectiveness of a stroke campaign, designed specifically for the Italian population in reducing prehospital delay. Methods—According to an SW-RCT (Stepped-Wedge Cluster Randomized Controlled Trial) design, the campaign was launched in 4 provinces in the northern part of the region Emilia Romagna at 3-month intervals in randomized sequence. The units of analysis were the patients admitted to hospital, with stroke and transient ischemic attack, over a time period of 15 months, beginning 3 months before the intervention was launched in the first province to allow for baseline data collection. The proportion of early arrivals (within 2 hours of symptom onset) was the primary outcome. Thrombolysis rate and some behavioral end points were the secondary outcomes. Data were analyzed using a fixed-effect model, adjusting for cluster and time trends. Results—We enrolled 1622 patients, 912 exposed and 710 nonexposed to the campaign. The proportion of early access was nonsignificantly lower in exposed patients (354 [38.8%] versus 315 [44.4%]; adjusted odds ratio, 0.81; 95% confidence interval, 0.60–1.08; P=0.15). As for secondary end points, an increase was found for stroke recognition, which approximated but did not reach statistical significance (P=0.07). Conclusions—Our campaign was not effective in reducing prehospital delay. Even if some limitations of the intervention, mainly in terms of duration, are taken into account, our study demonstrates that new communication strategies should be tested before large-scale implementation. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT01881152

Molecular detection of bacterial RNA in atheromatous plaques of patients with stroke

Aim: Infections and chronic inflammatory conditions such as periodontitis, have been associated with the development and progression of atherosclerosic process and susceptibility to plaque rupture. The chronicity of periodontal disease provides a rich source of subgingival bacteria, mostly gram negative, which are frequently spread in the blood as a result of the periodontal lesion. DNA from periodontal pathogens has been detected in atherosclerotic lesions, but viable bacteria have not yet been isolated. This study was carried out to detect, by molecular techniques, the presence of RNA of bacteria in atheromatous plaques collected from both symptomatic and asymptomatic carotid stenosis patients. Methods: Total RNA extracts, derived from eighty-one atheromatous plaques collected during carotid endartrectomy (36 from symptomatic patients and 45 from asymptomatic patients) underwent reverse transcription to cDNA using random primers. All cDNA samples obtained were tested by PCR using universal primers for global bacterial detection (16S rRNA). Results: Of eighty-one sample tested, seven were found positive (8.6%), six out of the thirty-six (16.6%) of symptomatic patients and only one out forty-five (2.2%) from the asymptomatic patients group. Conclusion: This pilot study suggests the preferential presence of bacterial RNA in recently symptomatic atherosclerotic plaques. These results are of interest because the presence of viable bacteria may be presumed into the atheromatous plaques and, therefore, indicates a possible bacterial role in activation of several inflammatory factors and in atherosclerotic plaque vulnerability

e-NIHSS: an Expanded National Institutes of Health Stroke Scale Weighted for Anterior and Posterior Circulation Strokes

Background The National Institutes of Health Stroke Scale (NIHSS) is the most widespread clinical scale used in patients presenting with acute stroke. The merits of the NIHSS include simplicity, quickness, and agreement between clinicians. The clinical evaluation on posterior circulation stroke remains still a limit of NIHSS. Methods We assessed the application of a new version of NIHSS, the e-NIHSS (expanded NIHSS), adding specific elements in existing items to explore signs/symptoms of a posterior circulation stroke. A total of 22 consecutive patients with suspected vertebrobasilar stroke were compared with 25 patients with anterior circulation stroke using NIHSS and e-NIHSS. Results We compared the NIHSS and e-NIHSS scores obtained by the 2 examiners, in patients with posterior circulation infarct (POCI), using the Wilcoxon test. Patients with POCI evaluated with e-NIHSS had an average of 2 points higher than patients evaluated with classical NIHSS. The difference was statistically significant (P < .05), weighted by the new expanded items. Conclusions The NIHSS is a practical scale model, with high reproducibility between trained, different examiners, focused on posterior circulation strokes, with the same total score and number of items of the existing NIHSS. The e-NHISS could improve the sensitivity of NIHSS in posterior circulation stroke and could have an impact on clinical trials, as well as on outcomes. Further studies are needed to investigate a larger number of patients and the correlation between the e-NIHSS score and neuroimaging findings

High levels of COX-2 gene expression in peripheral blood of cardioembolic and atherothrombotic stroke patients

Aim: To determine if COX-2 and TLR4 gene expression in atherothrombotic strokes represents a marker of atherosclerotic plaque destabilization and rupture, or it rather is related to the inflammatory response caused by stroke itself. Secondly, to determine if COX-2 and TLR4 gene expression could be specific for stroke subtype. Methods: Total RNA was extracted from peripheral blood samples from atherosclerotic (N=30) and cardioembolic stroke (N=25) patients, and controls (N=27). Gene expression was analysed by real time RT-PCR. Results: Expression of COX-2 gene was increased in cardioembolic compared to atherothrombotic stroke patients or healthy controls (p<0.0001). TLR4 expression was higher cardioembolic stroke patients compared to controls (p=0.0001). Conclusions: Cardioembolic show higher levels of COX-2 expression compared to atherothrombotic stroke patients independent of clinical severity. Additional studies are warrant to establish if high level of COX-2 gene expression could be a marker of cardioembolic stroke

HDAC9, TWIST1 and FERD3L gene expression in asymptomatic stable and unstable carotid plaques

BACKGROUND AND OBJECTIVES: A variant located at the end of HDAC9 gene within clusters of DNAse I sensitivity zones and histone modification hotspots has been associated with large vessel stroke and could be linked to plaque instability. The aim of the study is to define if an altered expression of HDAC9, TWIST1 and FERD3L genes could be involved in plaque vulnerability.METHODS: Histological classification and gene expression analysis were performed in 6 stable and 16 unstable plaques obtained from asymptomatic patients undergoing endarterectomy. Gene expression was analysed by real-time PCR.RESULTS AND CONCLUSIONS: TWIST1 gene expression resulted higher in stable plaques (P &lt; 0.02). HDAC9 gene expression followed a similar trend (P = 0.11). These results highlighting the significant correlation between TWIST and HDAC9 gene expression suggest that both genes may contribute to plaque stability in a coordinated way

TLR4 -2604G>A variant confers differential DNA binding capacity to transcription factors of the GATA family and alters gene expression in peripheral blood of atherosclerotic patients.

Aim: Toll-like receptor-4 (TLR4) is a primary receptor of the innate immune reaction and compelling evidence demonstrate its envelopment in the pathogenesis of atherosclerosis. TLR4 is constitutively expressed on monocytes and endothelial cells, and it has been found to be highly expressed in atherosclerotic plaques, and in peripheral blood of patients after ischemic stroke. Polymorphisms in the promoter region of this gene may represent genetic risk factors involved in the predisposition to atherosclerotic disease. In this study we investigated the effect on TLR4 gene expression of three polymorphisms located in the 5\u2019 upstream region of the gene in peripheral blood of atherosclerotic patients. Methods: Polymorphisms -1607T>C (rs10759932) and -2026A>G (rs1927914) were genotyped by PCR-RFLP, and gene expression analysis was performed by Real Time PCR using Sybr Green in 62 atherosclerotic patients. Results: RNA from patients carrying the allele -2604A in homozygosis showed a lower expression of the gene when compared to patients carrying the counterparts GG + GA (P<0.02). EMSA assays demonstrated that allele -2604A confers a higher DNA binding capacity to transcription factors of the GATA family. Conclusions: The presence of allele -2604A is associated to a diminished level of gene expression in peripheral blood of patients with advanced carotid atherosclerotic plaques, possibly by creating a transcription factor binding site for a negative modulator(s) of transcription. Case/control studies in larger populations are worthy to determine if variants of polymorphism -2604A>G of TLR4 might represent a genetic risk factor for susceptibility to atherosclerotic plaque development and progression

Correlations between gene expression highlight a different activation of ACE/TLR4/PTGS2 signaling in symptomatic and asymptomatic plaques in atherosclerotic patients

Inflammation has a key role and translates the effects of many known risk factors for the disease in atherosclerotic vulnerable plaques. Aiming to look into the elements that induce the development of either a vulnerable or stable atherosclerotic plaque, and considering that inflammation has a central role in the progression of lesions, we analyzed the expression of genes involved in the ACE/TLR4/PTGS2 signaling in carotid plaques of symptomatic and asymptomatic patients. Patients with internal carotid artery stenosis undergoing carotid endarterectomy at Verona University Hospital were included in this study. A total of 71 patients was considered for gene expression analysis (29 atherothrombotic stroke patients and 42 asymptomatic patients). Total RNA was extracted from the excised plaques and expression of PTGS2, ACE, TLR4, PTGER4, PTGER3, EPRAP and ACSL4 genes was analyzed by real-time PCR. The correlation between the pair of genes was studied by Spearman coefficient. From the analyzed genes, we did not observe any individual difference in gene expression but the network of co-expressed genes suggests a different activation of pathways in the two groups of plaques

AUMENTATA ESPRESSIONE DEL GENE COX-2 NEL SANGUE PERIFERICO DI PAZIENTI CON ICTUS ISCHEMICO

Obiettivo: Numerosi studi hanno riportato uno stato infiammatorio a seguito di un evento ischemico cerebrale. L’aumentata espressione di mediatori della cascata attivata dalla COX-2 è stata osservata in placche carotidee sintomatiche, ipotizzando quindi un coinvolgimento di tale via infiammatoria nell’evoluzione verso l’instabilità della placca. Scopo del presente studio è investigare se l’attivazione della cascata infiammatoria dopo un evento ischemico cerebrale sia collegato alla destabilizzazione del processo aterosclerotico sottostante o all’evento ischemico cerebrale in sé. Materiali e Metodi: E’ stato analizzato l’RNA estratto da sangue periferico, ottenuto da: 1) pazienti sottoposti ad endoarterectomia carotidea asintomatica (60 pazienti): 2) pazienti sottoposti ad endoarterectomia per stenosi carotidea recentemente sintomatica (40 pazienti); 3) pazienti con recente ictus cardioembolico (35 pazienti); 4) 27 controlli sani. E’ stata quindi studiata l’espressione di Toll-like Receptor 4 e COX-2 tramite real time PCR su sangue periferico a 24 h, 72 h e a 7 giorni dall’evento ischemico. La gravità clinica dei pazienti sintomatici è stata classificata secondo la scala NIHSS; l’entità della stenosi dei pazienti con stenosi carotidea secondo metodo NASCET.Risultati: L’espressione di COX-2 è risultata significativamente aumentata in entrambi i gruppi di pazienti con recente ictus a confronto con i gruppi di controllo (stenosi carotidea asintomatica e controlli sani) a 24 ore (p 0.02) e a 7 giorni (p 0.001). Si evidenzia inoltre una sovra espressione statisticamente significativa di COX-2 nei pazienti con ictus cardioembolico rispetto a quelli con stenosi sintomatica (p 1.847^10-6 a 7 giorni).Conclusioni: I livelli di COX-2 nei pazienti con recente ictus risultano elevati fino a 7 giorni dopo l’evento, indipendentemente da età, sesso, gravità clinica, e fattori di rischio. Rimane da chiarire la possibile causa della differente espressione tra i due gruppi affetti. Benché i pazienti con ictus cardioembolico abbiano mediamente gravità superiore a quelli con stenosi carotidea, non è stata verificata una correlazione fra espressione di COX2 e TLR4 con la gravità dell’evento. Si potrebbe ipotizzare un effetto sull’espressione di COX2 da parte di classi di terapia nei due gruppi di pazienti sintomatici, in particolare l’aspirina, che era significativamente più utilizzata nei pazienti con ictus aterotrombotico rispetto ai pazienti con ictus cardioembolico (p 0,00007)

Polymorphism -2604G>A variants in TLR4 promoter are associated with different gene expression level in peripheral blood of atherosclerotic patients.

Toll-like receptor-4 (TLR4) is a primary receptor of the innate immune reaction and compelling evidence demonstrates itsinvolvement in the pathogenesis of atherosclerosis and stroke. TLR4 is constitutively expressed on monocytes and endothelialcells; it is highly expressed in atherosclerotic plaques and in peripheral blood of patients after ischemic stroke. Polymorphismsin the promoter region that alter the transcriptional regulation of this gene may represent genetic risk factors involved in thepredisposition to atherosclerotic disease. In this study we investigated the effect on TLR4 gene expression of threepolymorphisms in the upstream regulatory region at positions 1607T4C/rs10759932, 2026A4G/rs1927914 and2604G4A/rs10759931 in peripheral blood of atherosclerotic patients. RNA from individuals homozygous for the 2604Aallele showed a lower expression of the gene when compared to patients carrying the counterparts GG\ufeGA. Electrophoreticmobility shift assays showed differences in the electrophoretic mobility of the DNA\u2013nuclear protein complexes formed by theG4A variants, suggesting that the two alleles differ in their binding affinity to transcriptional factors