Systemic biomarker associated with poor outcome after futile reperfusion.

BACKGROUND: Successful recanalization does not lead to complete tissue reperfusion in a considerable percentage of ischemic stroke patients. This study aimed to identify biomarkers associated with futile recanalization. Leukoaraiosis predicts poor outcomes of this phenomenon. Soluble tumour necrosis factor-like weak inducer of apoptosis (sTWEAK), which is associated with leukoaraiosis degrees, could be a potential biomarker. METHODS: This study includes two cohorts of ischemic stroke patients in a multicentre retrospective observational study. Effective reperfusion, defined as a reduction of ≥8 points in the National Institutes of Health Stroke Scale (NIHSS) within the first 24 h, was used as a clinical marker of effective reperfusion. RESULTS: In the first cohort study, female sex, age, and high NIHSS at admission (44.7% vs. 81.1%, 71.3 ± 13.7 vs. 81.1 ± 6.7; 16 [13, 21] vs. 23 [17, 28] respectively; p < .0001) were confirmed as predictors of futile recanalization. ROC curve analysis showed that leukocyte levels (sensitivity of 99%, specificity of 55%) and sTWEAK level (sensitivity of 92%, specificity of 88%) can discriminate between poor and good outcomes. Both biomarkers simultaneously are higher associated with outcome after effective reperfusion (OR: 2.17; CI 95% 1.63-4.19; p < .0001) than individually (leukocytes OR: 1.38; CI 95% 1.00-1.64, p = .042; sTWEAK OR: 1.00; C I95% 1.00-1.01, p = .019). These results were validated using a second cohort, where leukocytes and sTWEAK showed a sensitivity of 100% and specificity of 66.7% and 75% respectively. CONCLUSIONS: Leukocyte and sTWEAK could be biomarkers of reperfusion failure and subsequent poor outcomes. Further studies will be necessary to explore its role in reperfusion processes.

Thrombolytic therapy based on lyophilized platelet-derived nanocarriers for ischemic stroke.

BACKGROUND: Intravenous administration of fibrinolytic drugs, such as recombinant tissue plasminogen activator (rtPA) is the standard treatment of acute thrombotic diseases. However, current fibrinolytics exhibit limited clinical efficacy because of their short plasma half-lives and risk of hemorrhagic transformations. Platelet membrane-based nanocarriers have received increasing attention for ischemic stroke therapies, as they have natural thrombus-targeting activity, can prolong half-life of the fibrinolytic therapy, and reduce side effects. In this study we have gone further in developing platelet-derived nanocarriers (defined as cellsomes) to encapsulate and protect rtPA from degradation. Following lyophilization and characterization, their formulation properties, biocompatibility, therapeutic effect, and risk of hemorrhages were later investigated in a thromboembolic model of stroke in mice. RESULTS: Cellsomes of 200 nm size and loaded with rtPA were generated from membrane fragments of human platelets. The lyophilization process did not influence the nanocarrier size distribution, morphology, and colloidal stability conferring particle preservation and long-term storage. Encapsulated rtPA in cellsomes and administered as a single bolus showed to be as effective as a continuous clinical perfusion of free rtPA at equal concentration, without increasing the risk of hemorrhagic transformations or provoking an inflammatory response. CONCLUSIONS: This study provides evidence for the safe and effective use of lyophilized biomimetic platelet-derived nanomedicine for precise thrombolytic treatment of acute ischemic stroke. In addition, this new nanoformulation could simplify the clinical use of rtPA as a single bolus, being easier and less time-consuming in an emergency setting than a treatment perfusion, particularly in stroke patients. We have successfully addressed one of the main barriers to drug application and commercialization, the long-term storage of nanomedicines, overcoming the potential chemical and physical instabilities of nanomedicines when stored in an aqueous buffer.

Precision Medicine for Blood Glutamate Grabbing in Ischemic Stroke.

Glutamate grabbers, such as glutamate oxaloacetate transaminase (GOT), have been proposed to prevent excitotoxicity secondary to high glutamate levels in stroke patients. However, the efficacy of blood glutamate grabbing by GOT could be dependent on the extent and severity of the disruption of the blood-brain barrier (BBB). Our purpose was to analyze the relationship between GOT and glutamate concentration with the patient's functional status differentially according to BBB serum markers (soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) and leukoaraiosis based on neuroimaging). This retrospective observational study includes 906 ischemic stroke patients. We studied the presence of leukoaraiosis and the serum levels of glutamate, GOT, and sTWEAK in blood samples. Functional outcome was assessed using the modified Rankin Scale (mRS) at 3 months. A significant negative correlation between GOT and glutamate levels at admission was shown in those patients with sTWEAK levels > 2900 pg/mL (Pearson's correlation coefficient: -0.249; p < 0.0001). This correlation was also observed in patients with and without leukoaraiosis (Pearson's correlation coefficients: -0.299; p < 0.001 vs. -0.116; p = 0.024). The logistic regression model confirmed the association of higher levels of GOT with lower odds of poor outcome at 3 months when sTWEAK levels were >2900 pg/mL (OR: 0.41; CI 95%: 0.28-0.68; p < 0.0001) or with leukoaraiosis (OR: 0.75; CI 95%: 0.69-0.82; p < 0.0001). GOT levels are associated with glutamate levels and functional outcomes at 3 months, but only in those patients with leukoaraiosis and elevated sTWEAK levels. Consequently, therapies targeting glutamate grabbing might be more effective in patients with BBB dysfunction.

C-Reactive Protein, Interleukin-6, and Vascular Recurrence After Stroke: An Individual Participant Data Meta-Analysis.

BACKGROUND: Anti-inflammatory therapies reduce recurrent vascular events in coronary disease. Existing studies have reported highly conflicting findings for the association of blood inflammatory markers with vascular recurrence after stroke leading to uncertainty about the potential of anti-inflammatory therapies after stroke and no consensus about the utility of measurement of inflammatory markers in current guidelines. METHODS: We investigated the association between hsCRP (high-sensitivity C-reactive protein), IL-6 (interluekin-6), and recurrent major adverse cardiovascular events (MACE), and stroke from individual participant data from 8420 patients with ischemic stroke/transient ischemic attack from 10 prospective studies. We did within-study multivariable regression analyses and then combined adjusted risk ratio (RR) by random-effects meta-analysis. RESULTS: During 18 920 person-years of follow-up, 1407 (16.7% [95% CI, 15.9-17.5]) patients had MACE and 1191 (14.1% [95% CI, 13.4-14.9]) patients had recurrent stroke. On bivariate analysis, baseline IL-6 was associated with MACE (RR, 1.26 [95% CI, 1.10-1.43]) and recurrent stroke (RR, 1.18 [95% CI, 1.05-1.32]), per unit increase logeIL-6. Similar associations were observed for hsCRP (MACE RR, 1.19 [95% CI, 1.09-1.29]; recurrent stroke RR, 1.12 [95% CI, 1.04-1.21], per unit increase logehsCRP). After adjustment for vascular risk factors and treatment, independent associations remained with MACE (IL-6, RR, 1.12 [95% CI, 1.04-1.21]; hsCRP, RR, 1.09 [95% CI, 1.04-1.15]) and recurrent stroke (IL-6, RR, 1.09 [95% CI, 1.00-1.19]; hsCRP, RR, 1.05 [95% CI, 1.00-1.11]). Comparing the top with the bottom quarters (Q4 versus Q1), IL-6 (RR, 1.35 [95% CI, 1.09-1.67]) and hsCRP (RR, 1.31 [95% CI, 1.07-1.61]) were associated with MACE after adjustment. Similar results were observed for recurrent stroke for IL-6 (RR, 1.33 [95% CI, 1.08-1.65]) but not hsCRP (RR, 1.16 [95% CI, 0.93-1.43]). CONCLUSIONS: Blood markers of inflammation were independently associated with vascular recurrence after stroke, strengthening the rationale for randomized trials of anti-inflammatory therapies for secondary prevention after ischemic stroke/TIA.

Influence of Temperature Chronobiology on Stroke Outcome.

The circadian system regulates numerous physiological variables, including body temperature. Additionally, a circadian patter has been described in stroke onset. Considering this, we hypothesised that the chronobiology of temperature may have an impact on stroke onset and functional outcomes. We also studied the variation of blood biomarkers according to stroke onset time. This is a retrospective observational study. Of the patients included, 2763 had a stroke between midnight and 8:00 h; 1571 between 8:00-14:00 h; and 655 between 14:00 h and midnight. Axillary temperature was measured at admission. At this time, blood samples were collected for biomarker analysis (TNF-α, IL-1ß, IL-6, IL-10, and glutamate). Temperature was higher in patients admitted from 8:00 h to midnight (p < 0.0001). However, the percentage of poor outcome at 3 months was highest in patients from midnight to 8:00 h (57.7%, p < 0.001). The association between temperature and mortality was highest during night time (OR: 2.79; CI 95%: 2.36-3.28; p < 0.001). These patients exhibited high glutamate (220.2 ± 140.2 µM), IL-6 (32.8 ± 14.3 pg/mL) and low IL-10 (9.7 ± 14.3 pg/mL) levels. Therefore, temperature chronobiology could have a significant impact on stroke onset and functional outcome. Superficial body hyperthermia during sleep seems to be more dangerous than during wakefulness. Further studies will be necessary to confirm our data.

Surrogate biomarkers of outcome for wake-up ischemic stroke.

BACKGROUND: Wake-up ischemic stroke (IS) has been usually excluded from acute stroke therapy options for being outside of the safe treatment window. We identified risk factors, and clinical or molecular biomarkers that could be therapeutic targets for wake-up stroke prevention, thus hopefully leading to a decrease in its mortality and disability in medium to long-term outcome. METHODS: 4251 ischemic stroke (IS) patients from a prospectively registered database were recruited; 3838 (90.3%) had known onset-symptom time, and 413 (9.7%) were wake-up strokes. The main endpoint was to analyze the association between different serum biomarkers with wake-up IS episodes and their progression. Leukocytes count, serum levels of C-reactive protein, fibrinogen, interleukin 6 (IL-6), and vitamin D were analyzed as inflammation biomarkers; N-terminal pro-B-type Natriuretic-Peptide and microalbuminuria, used as atrial/endothelial dysfunction biomarkers; finally, glutamate levels as excitotoxicity biomarker. In addition, demographic, clinical and neuroimaging variables associated with the time-evolution of wake-up IS patients and functional outcome at 3 months were evaluated. Good and poor functional outcome were defined as mRS ≤2 and mRS > 2 at 3 months, respectively. RESULTS: Wake-up IS showed a poorer outcome at 3-months than in patients with known on-set-symptom time (59.1% vs. 48.1%; p < 0.0001). Patients with wake-up IS had higher levels of inflammation biomarkers; IL-6 levels at admission (51.5 ± 15.1 vs. 27.8 ± 18.6 pg/ml; p < 0.0001), and low vitamin D levels at 24 h (5.6 ± 5.8 vs. 19.2 ± 9.4 ng/ml; p < 0.0001) are worthy of attention. In a logistic regression model adjusted for vitamin D, OR was 15.1; CI 95%: 8.6-26.3, p < 0.0001. However, we found no difference in vitamin D levels between patients with or without clinical-DWI mismatch (no: 18.95 ± 9.66; yes: 17.84 ± 11.77 ng/mL, p = 0.394). No difference in DWI volume at admission was found (49.3 ± 96.9 ml in wake-up IS patients vs. 51.7 ± 98.2 ml in awake IS patients; p = 0.895). CONCLUSIONS: Inflammatory biomarkers are the main factors that are strongly associated with wake-up IS episodes. Wake-up IS is associated with lower vitamin D levels. These data indicate that vitamin D deficiency could become a therapeutic target to reduce wake-up IS events.

Sonosensitive capsules for brain thrombolysis increase ischemic damage in a stroke model.

BACKGROUND: Ischemic stroke is the most common cerebrovascular disease and is caused by interruption of blood supply to the brain. To date, recombinant tissue plasminogen activator (rtPA) has been the main pharmacological treatment in the acute phase. However, this treatment has some drawbacks, such as a short half-life, low reperfusion rate, risk of hemorrhagic transformations, and neurotoxic effects. To overcome the limitations of rtPA and improve its effectiveness, we recently designed sonosensitive sub-micrometric capsules (SCs) loaded with rtPA with a size of approximately 600 nm, synthesized using the layer-by-layer (LbL) technique, and coated with gelatine for clot targeting. In this study, we evaluated the rtPA release of ultrasound (US)-responsive SCs in healthy mice and the therapeutic effect in a thromboembolic stroke model. RESULTS: In healthy mice, SCs loaded with rtPA 1 mg/kg responded properly to external US exposure, extending the half-life of the drug in the blood stream more than the group treated with free rtPA solution. The gelatine coating also contributed to stabilizing the encapsulation and maintaining the response to US. When the same particles were administered in the stroke model, these SCs appeared to aggregate in the ischemic brain region, probably generating secondary embolisms and limiting the thrombolytic effect of rtPA. Despite the promising results of these thrombolytic particles, at least under the dose and size conditions used in this study, the administration of these capsules represents a risk factor for stroke. CONCLUSIONS: This is the first study to report the aggregation risk of a drug carrier in neurological pathologies such as stroke. Biocompatibility analysis related to the use of nano-and microparticles should be deeply studied to anticipate the limitations and orientate the design of new nanoparticles for translation to humans.

Need for a Paradigm Shift in the Treatment of Ischemic Stroke: The Blood-Brain Barrier.

Blood-brain barrier (BBB) integrity is essential to maintaining brain health. Aging-related alterations could lead to chronic progressive leakiness of the BBB, which is directly correlated with cerebrovascular diseases. Indeed, the BBB breakdown during acute ischemic stroke is critical. It remains unclear, however, whether BBB dysfunction is one of the first events that leads to brain disease or a down-stream consequence. This review will focus on the BBB dysfunction associated with cerebrovascular disease. An added difficulty is its association with the deleterious or reparative effect, which depends on the stroke phase. We will first outline the BBB structure and function. Then, we will focus on the spatiotemporal chronic, slow, and progressive BBB alteration related to ischemic stroke. Finally, we will propose a new perspective on preventive therapeutic strategies associated with brain aging based on targeting specific components of the BBB. Understanding BBB age-evolutions will be beneficial for new drug development and the identification of the best performance window times. This could have a direct impact on clinical translation and personalised medicine.

Corneal and conjunctival alteration of innate immune expression in first-degree relatives of keratoconus patients.

PURPOSE: The innate immune toll-like receptors 2 (TLR2) and 4 (TLR4) may play a key role in the physiopathology of keratoconus (KC). Therefore, the aim of this study was to compare TLR2/TLR4 expression in corneal and conjunctival epithelial cells between healthy first-degree relatives of patients with KC and healthy controls as well as KC patients. METHODS: Case-control study in 72 healthy eyes of 36 control subjects, 53 eyes of 27 first-degree relatives, and 109 eyes with KC (60 patients). All participants were subjected to a clinical, topographic, aberrometric, and tomographic examination with extraction of corneal and conjunctival epithelial cells through scraping. TLR2/TLR4 expression was measured by flow cytometry, and was compared among controls, first-degree relatives, and KC patients. The relationship between TLR expression and epidemiological-clinical variables or topographic-aberrometric-tomographic parameters was also analyzed. RESULTS: Mean TLR2/TLR4 expression showed a significant gradual increase among groups: controls < first-degree relatives < KC patients. Mean expression of TLR2 in corneal epithelial cells and both TLR2/TLR4 in conjunctival epithelial cells were significantly higher in relatives than in controls (p = 0.026, p < 0.001, and p = 0.031, respectively). Sex, age, allergic disease, eye itching, rubbing, and topographic-aberrometric-tomographic parameters were not associated to TLR2/TLR4 expression in relatives. TLR2 conjunctival expression was independently associated to relatives (OR 1.001; CI 95% 1.000-1.002, p = 0.043) after adjustment by sex, age, and rubbing. CONCLUSION: TLR2 and TLR4 are overexpressed in corneal and conjunctival epithelial cells of KC relatives compared with controls. Both biomarkers may monitor early ocular changes in first-degree relatives who not show any abnormal clinical-topographic-aberrometric-tomographic parameters.

Toll-like receptors as diagnostic targets in pellucid marginal degeneration.

The main purpose of this study is to evaluate the diagnostic role of Toll-like receptors 2 (TLR2) and 4 (TLR4) expression in corneal and conjunctival epithelial cells of eyes with pellucid marginal degeneration (PMD) compared to keratoconus patients (KC) and control subjects. A prospective case-control study in 29 PMD eyes, 109 KC eyes and 72 healthy eyes was done. All participants were subjected to a clinical, topographic, aberrometric and tomographic exam with extraction of corneal and conjunctival epithelial cells through scraping. The TLR2 and TLR4 expression was measured with flow cytometry. Receiver operating characteristic (ROC) curve analysis was used to determine the most appropriate cutoff point for predicting the risk of PMD and KC. Correlations between TLR2/TLR4 expression and the severity of PMD/KC were evaluated. A TLRs follow-up review was made 19 ± 4 months after to the first review. As result, mean expression of TLR2 and TLR4 in both corneal and conjunctival epithelial cells was significantly higher in eyes with corneal ectasia (PMD and KC) than in control eyes (all p < 0.05). Conjunctival TLR4 expression showed the highest capacity to diagnose the existence of PMD (odd ratio 42.84; 95% confidence interval:6.20-296.20; p < 0.0001) after adjusting by eye rubbing and steeper corneal meridian. Moreover, we found an association between the TLR2/TLR4 overexpression with the severity of the PMD and KC measured by corneal topographic, aberrometric and tomographic quantitative parameters (all p < 0.05). Differences on TLR2/TLR4 expression between study groups were maintained during the follow-up period. In conclusion, the TLR2/TLR4 overexpression in corneal and conjunctival epithelial cells of PMD and KC patients compared to healthy control subjects have demonstrated their role as diagnostic target in both corneal ectatic disorders.

Regulatory T cells participate in the recovery of ischemic stroke patients.

BACKGROUND: Recent preclinical studies have shown that regulatory T cells (Treg) play a key role in the immune response after ischemic stroke (IS). However, the role of Treg in human acute IS has been poorly investigated. Our aim was to study the relationship between circulating Treg and outcome in human IS patients. METHODS: A total of 204 IS patients and 22 control subjects were recruited. The main study variable was good functional outcome at 3 months (modified Rankin scale ≤2) considering infarct volume, Early Neurological Deterioration (END) and risk of infections as secondary variables. The percentage of circulating Treg was measured at admission, 48, 72 h and at day 7 after stroke onset. RESULTS: Circulating Treg levels were higher in IS patients compared to control subjects. Treg at 48 h were independently associated with good functional outcome (OR, 3.5; CI: 1.9-7.8) after adjusting by confounding factors. Patients with lower Treg at 48 h showed higher frequency of END and risk of infections. In addition, a negative correlation was found between circulating Treg at 48 h (r = - 0.414) and 72 h (r = - 0.418) and infarct volume. CONCLUSIONS: These findings suggest that Treg may participate in the recovery of IS patients. Therefore, Treg may be considered a potential therapeutic target in acute ischemic stroke.

Trends in stroke outcomes in the last ten years in a European tertiary hospital.

BACKGROUND: Studying the impact of demographic changes and progress in the management of stroke patients is necessary in order to organize care structures for the coming years. Consequently, we analyzed the prognostic trends of patients admitted to the Stroke Unit of a tertiary hospital in the last ten years. METHODS: The University Clinical Hospital of Santiago de Compostela is the referral hospital for stroke in a catchment area that accounts for 16.5% of the population of Galicia. Data from patients admitted to the Stroke Unit were registered prospectively. A multinomial logistic regression was performed to determine the influence of new trends in demographic factors and in the management of patients with acute stroke. For the expected trend of progression, a 2008-2011 and 2012-2017 time series model was made by selecting the most appropriate model. RESULTS: In the last 10 years, the age of stroke onset has only increased in women (from 74.4 ± 2.2 years in 2008 to 78.8 ± 2.1 years in 2017; p = 0.037), and the same happens with the severity of neurological symptoms (ischemic stroke (IS), p < 0.0001; from 14 [10, 19] in 2008 to 19 [15, 26] in 2017), with a higher percentage of cardioembolic strokes (40.7% vs. 32.2% of cardioembolic strokes in women vs. men, p < 0.0001). In a multiple linear regression model, hospital improvement was mainly associated with the use of reperfusion treatment (B 53.11, CI 95% 49.87, 56.36, p < 0.0001). A differentiated multinomial logistic regression analysis conducted for the whole sample with ischemic strokes in the two time periods (2008-2011 and 2012-2017) showed no differences in the influence of factors associated with higher morbidity and mortality. The modeling of time series showed a distinct falling trend in mortality, with a slight increase in good outcome as well as morbidity in both ischemic and hemorrhagic stroke. CONCLUSIONS: Our results showed that mortality decreased in the entire sample; however, although outcome at discharge improved in ischemic stroke, severe disability also increased in these patients. Importantly, this tendency towards increased morbidity seems to be confirmed for the coming years.

Differential blood-based biomarkers of subcortical and deep brain small vessel disease.

Background: Cerebral small vessel disease is the most common cause of lacunar strokes (LS). Understanding LS pathogenesis is vital for predicting disease severity, prognosis, and developing therapies. Objectives: To research molecular profiles that differentiate LS in deep brain structures from those in subcortical white matter. Design: Prospective case-control study involving 120 patients with imaging-confirmed LS and a 120 control group. Methods: We examined the relationship between Alzheimer's disease biomarkers [amyloid beta (Aß1-40, Aß1-42)], serum inflammatory marker (interleukin-6, IL-6), and endothelial dysfunction markers [soluble tumor necrosis factor-like weak inducer of apoptosis, and pentraxin-3 (sTWEAK, PTX3)] with respect to LS occurring in deep brain structures and subcortical white matter. In addition, we investigated links between LS, leukoaraiosis presence (white matter hyperintensities, WMHs), and functional outcomes at 3 months. Poor outcome was defined as a modified Rankin scale >2 at 3 months. Results: Significant differences were observed in levels of IL-6, PTX3, and sTWEAK between patients with deep lacunar infarcts and those with recent small subcortical infarcts (20.8 versus 15.6 pg/mL, p < 0.001; 7221.3 versus 4624.4 pg/mL, p < 0.0001; 2528.5 versus 1660.5 pg/mL, p = 0.001). Patients with poor outcomes at 3 months displayed notably higher concentrations of these biomarkers compared to those with good outcomes. By contrast, Aß1-40 and Aß1-42 were significantly lower in patients with deep LS (p < 0.0001). Aß1-42 levels were significantly higher in patients with LS in subcortical white matter who had poor outcomes. WMH severity only showed a significant association with deep LS and correlated with sTWEAK (p < 0.0001). Conclusion: The pathophysiological mechanisms of lacunar infarcts in deep brain structures seem different from those in the subcortical white matter. As a result, specific therapeutic and preventive strategies should be explored.

Plasma fibrinogen and risk of vascular recurrence after ischaemic stroke: An individual participant and summary-level data meta-analysis of 11 prospective studies.

INTRODUCTION: Inflammation is an emerging target for secondary prevention after stroke and randomised trials of anti-inflammatory therapies are ongoing. Fibrinogen, a putative pro-inflammatory marker, is associated with first stroke, but its association with major adverse cardiovascular events (MACE) after stroke is unclear. MATERIALS AND METHODS: We did a systematic review investigating the association between fibrinogen and post-stroke vascular recurrence. Authors were invited to provide individual-participant data (IPD) and where available we did within-study multivariable analyses with adjustment for cardiovascular risk factors and medications. Adjusted summary-level data was extracted from published reports from studies that did not provide IPD. We pooled risk ratios (RR) by random-effects meta-analysis by comparing supra-median with sub-median fibrinogen levels and performed pre-specified subgroup analysis according to timing of phlebotomy after the index event. RESULTS: Eleven studies were included (14,002 patients, 42,800 follow-up years), of which seven provided IPD. Fibrinogen was associated with recurrent MACE on unadjusted (RR 1.35, 95% CI 1.17-1.57, supra-median vs sub-median) and adjusted models (RR 1.21, 95% CI 1.06-1.38). Fibrinogen was associated with recurrent stroke on univariate analysis (RR 1.19, 95% CI 1.03-1.39), but not after adjustment (RR 1.11, 95% CI 0.94-1.31). The association with recurrent MACE was consistently observed in patients with post-acute (⩾14 days) fibrinogen measures (RR 1.29, 95% CI 1.16-1.45), but not in those with early phlebotomy (<14 days) (RR 0.98, 95% CI 0.82-1.18) (Pinteraction = 0.01). Similar associations were observed for recurrent stroke. DISCUSSION AND CONCLUSION: Fibrinogen was independently associated with recurrence after stroke, but the association was modified by timing of phlebotomy. Fibrinogen measurements might be useful to identify patients who are more likely to derive benefit from anti-inflammatory therapies after stroke.

C-Reactive Protein, Interleukin-6, and Vascular Recurrence According to Stroke Subtype: An Individual Participant Data Meta-Analysis

BACKGROUND AND OBJECTIVES: Anti-inflammatory therapies reduce major adverse cardiovascular events (MACE) in coronary artery disease but remain unproven after stroke. Establishing the subtype-specific association between inflammatory markers and recurrence risk is essential for optimal selection of patients in randomized trials (RCTs) of anti-inflammatory therapies for secondary stroke prevention. METHODS: Using individual participant data (IPD) identified from a systematic review, we analyzed the association between high-sensitivity C-reactive protein, interleukin-6 (IL-6), and vascular recurrence after ischemic stroke or transient ischemic attack. The prespecified coprimary end points were (1) any recurrent MACE (first major coronary event, recurrent stroke, or vascular death) and (2) any recurrent stroke (ischemic, hemorrhagic, or unspecified) after sample measurement. Analyses were performed stratified by stroke mechanism, per quarter and per biomarker unit increase after loge transformation. We then did study-level meta-analysis with comparable published studies not providing IPD. Preferred Reporting Items for Systematic Review and Meta-Analyses IPD guidelines were followed. RESULTS: IPD was obtained from 10 studies (8,420 patients). After adjustment for vascular risk factors and statins/antithrombotic therapy, IL-6 was associated with recurrent MACE in stroke caused by large artery atherosclerosis (LAA) (risk ratio [RR] 2.30, 95% CI 1.21-4.36, p = 0.01), stroke of undetermined cause (UND) (RR 1.78, 1.19-2.66, p = 0.005), and small vessel occlusion (SVO) (RR 1.71, 0.99-2.96, p = 0.053) (quarter 4 [Q4] vs quarter 1 [Q1]). No association was observed for stroke due to cardioembolism or other determined cause. Similar results were seen for recurrent stroke and when analyzed per loge unit increase for MACE (LAA, RR 1.26 [1.06-1.50], p = 0.009; SVO, RR 1.22 [1.01-1.47], p = 0.04; UND, RR 1.18 [1.04-1.34], p = 0.01). High-sensitivity CRP was associated with recurrent MACE in UND stroke only (Q4 vs Q1 RR 1.45 [1.04-2.03], p = 0.03). Findings were consistent on study-level meta-analysis of the IPD results with 2 other comparable studies (20,136 patients). DISCUSSION: Our data provide new evidence for the selection of patients in future RCTs of anti-inflammatory therapy in stroke due to large artery atherosclerosis, small vessel occlusion, and undetermined etiology according to inflammatory marker profile.

Toxicity of silver nanoparticles in macrophages.

Silver nanoparticles (nanosilver) are broadly used today in textiles, food packaging, household devices and bioapplications, prompting a better understanding of their toxicity and biological interactions. In particular, the cytotoxicity of nanosilver with respect to mammalian cells remains unclear, because such investigations can be biased by the nanosilver coatings and the lack of particle size control. Here, nanosilver of well-defined size (5.7 to 20.4 nm) supported on inert nanostructured silica is produced using flame aerosol technology. The cytotoxicity of the prepared nanosilver with respect to murine macrophages is assessed in vitro because these cells are among the first to confront nanosilver upon its intake by mammals. The silica support facilitates the dispersion and stabilization of the prepared nanosilver in biological suspensions, and no other coating or functionalization is applied that could interfere with the biointeractions of nanosilver. Detailed characterization of the particles by X-ray diffraction and electron microscopy reveals that the size of the nanosilver is well controlled. Smaller nanosilver particles release or leach larger fractions of their mass as Ag⁺ ions upon dispersion in water. This strongly influences the cytotoxicity of the nanosilver when incubated with murine macrophages. The size of the nanosilver dictates its mode of cytotoxicity (Ag⁺ ion-specific and/or particle-specific). The toxicity of small nanosilver (<10 nm) is mostly mediated by the released Ag⁺ ions. The influence of such ions on the toxicity of nanosilver decreases with increasing nanosilver size (>10 nm). Direct silver nanoparticle-macrophage interactions dominate the nanosilver toxicity at sizes larger than 10 nm.

Perfusion-weighted software written in Python for DSC-MRI analysis.

Introduction: Dynamic susceptibility-weighted contrast-enhanced (DSC) perfusion studies in magnetic resonance imaging (MRI) provide valuable data for studying vascular cerebral pathophysiology in different rodent models of brain diseases (stroke, tumor grading, and neurodegenerative models). The extraction of these hemodynamic parameters via DSC-MRI is based on tracer kinetic modeling, which can be solved using deconvolution-based methods, among others. Most of the post-processing software used in preclinical studies is home-built and custom-designed. Its use being, in most cases, limited to the institution responsible for the development. In this study, we designed a tool that performs the hemodynamic quantification process quickly and in a reliable way for research purposes. Methods: The DSC-MRI quantification tool, developed as a Python project, performs the basic mathematical steps to generate the parametric maps: cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT), signal recovery (SR), and percentage signal recovery (PSR). For the validation process, a data set composed of MRI rat brain scans was evaluated: i) healthy animals, ii) temporal blood-brain barrier (BBB) dysfunction, iii) cerebral chronic hypoperfusion (CCH), iv) ischemic stroke, and v) glioblastoma multiforme (GBM) models. The resulting perfusion parameters were then compared with data retrieved from the literature. Results: A total of 30 animals were evaluated with our DSC-MRI quantification tool. In all the models, the hemodynamic parameters reported from the literature are reproduced and they are in the same range as our results. The Bland-Altman plot used to describe the agreement between our perfusion quantitative analyses and literature data regarding healthy rats, stroke, and GBM models, determined that the agreement for CBV and MTT is higher than for CBF. Conclusion: An open-source, Python-based DSC post-processing software package that performs key quantitative perfusion parameters has been developed. Regarding the different animal models used, the results obtained are consistent and in good agreement with the physiological patterns and values reported in the literature. Our development has been built in a modular framework to allow code customization or the addition of alternative algorithms not yet implemented.

Shotgun Proteomics for the Identification and Profiling of the Tear Proteome of Keratoconus Patients.

Purpose: The qualitative approach followed in this study aims to obtain an extensive view of the keratoconus (KC) tear proteome, which could highlight proteins previously undetected and enlarge our knowledge of the disease's pathophysiology. Methods: Twenty-five patients diagnosed with KC and 25 control subjects were studied in a prospective, cross-sectional study. KC screening examinations, including clinical and tomographic examinations, were performed on all participants. Tear samples were collected using Schirmer strips and analyzed by liquid chromatography-tandem mass spectrometry in a data-dependent workflow. A spectral count was used as a semiquantification tool. The tear proteomes of both groups were identified and profiled, and the functional interactions and biological characterization of differential proteins were analyzed using in silico tools. Results: We identified a total of 232 proteins, of whom 133 were expressed in both groups' samples; 41 were observed only in control samples and 58 were identified just in tears of patients with KC. A semiquantitative analysis showed the dysregulation of 17 proteins in the KC samples. An in silico analysis linked proteins only expressed in KC samples to oxidative stress, skin development, and apoptosis. The dysregulation of proteins involved in iron transport, inflammation, oxidative stress, and protease inhibition was observed in the semiquantitative results. Conclusions: A shotgun analysis showed that the tear proteome of patients with KC differed from controls by more than one-third of the total proteins identified, highlighting the relationship of the proteins only expressed in KC tears with processes of cell death, oxidative damage, and inflammation. The underexpression of proteins involved in iron pathways might support the iron imbalance as a contributing factor to cellular damage and death in KC disease.

Antihyperthermic Treatment in the Management of Malignant Infarction of the Middle Cerebral Artery.

Malignant infarction of the middle cerebral artery (m-MCA) is a complication of ischemic stroke. Since hyperthermia is a predictor of poor outcome, and antihyperthermic treatment is well tolerated, our main aim was to analyze whether the systemic temperature decrease within the first 24 h was associated with a better outcome. Furthermore, we studied potential biochemical and neuroimaging biomarkers. This is a retrospective observational analysis that included 119 patients. The temperature variations within the first 24 h were recorded. Biochemical laboratory parameters and neuroimaging variables were also analyzed. The temperature increase at the first 24 h (OR: 158.97; CI 95%: 7.29−3465.61; p < 0.001) was independently associated with a higher mortality. Moreover, antihyperthermic treatment (OR: 0.08; CI 95%: 0.02−0.38; p = 0.002) was significantly associated with a good outcome at 3 months. Importantly, antihyperthermic treatment was associated with higher survival at 3 months (78% vs. 50%, p = 0.003). Significant independently associations between the development of m-MCA and both microalbuminuria (OR: 1.01; CI 95%: 1.00−1.02; p = 0.005) and leukoaraiosis (OR: 3.07; CI 1.84−5.13−1.02; p < 0.0001) were observed. Thus, antihyperthermic treatment within the first 24 h was associated with both a better outcome and higher survival. An increased risk of developing m-MCA was associated with leukoaraiosis and an elevated level of microalbuminuria.

Involvement of Ceramide Metabolism in Cerebral Ischemia.

Ischemic stroke, caused by the interruption of blood flow to the brain and subsequent neuronal death, represents one of the main causes of disability in worldwide. Although reperfusion therapies have shown efficacy in a limited number of patients with acute ischemic stroke, neuroprotective drugs and recovery strategies have been widely assessed, but none of them have been successful in clinical practice. Therefore, the search for new therapeutic approaches is still necessary. Sphingolipids consist of a family of lipidic molecules with both structural and cell signaling functions. Regulation of sphingolipid metabolism is crucial for cell fate and homeostasis in the body. Different works have emphasized the implication of its metabolism in different pathologies, such as diabetes, cancer, neurodegeneration, or atherosclerosis. Other studies have shown its implication in the risk of suffering a stroke and its progression. This review will highlight the implications of sphingolipid metabolism enzymes in acute ischemic stroke.