Fibrin-Targeted Nanoparticles for Finding, Visualizing and Characterizing Blood Clots in Acute Ischemic Stroke.

Recanalization of the occluded artery is the gold standard treatment for acute ischemic stroke, which includes enzymatic fibrinolytic treatment with the use of recombinant tissue plasminogen activators (rtPAs) to disrupt the occluding clot, the use of mechanical thrombectomy to physically remove the clot, or a combination of both. Fibrin is one of the main components of blood clots causing ischemic stroke and is the target of rtPA upon activation of plasminogen in the clot. In addition, fibrin content also influences the efficacy of mechanical thrombectomy. Current imaging methods can successfully identify occlusions in large vessels; however, there is still a need for contrast agents capable of visualizing small thrombi in ischemic stroke patients. In this work, we describe the synthesis and the in vitro characterization of a new diagnostic nanoparticle, as well as the in vivo evaluation in an animal model of thromboembolic stroke. Gd-labeled KCREKA peptides were synthesized and attached onto the surface of PEGylated superparamagnetic nanoparticles. Magnetic resonance imaging (MRI) of blood clots was performed in vitro and in vivo in animal models of thromboembolic stroke. KCREKA-NPs were synthesized by attaching the peptide to the amino (N) termini of the PEG-NPs. The sizes of the nanoparticles, measured via DLS, were similar for both KCREKA-NPs and PEG-NPs (23 ± 4 nm, PDI = 0.11 and 25 ± 8 nm, PDI = 0.24, respectively). In the same line, r2 relaxivities were also similar for the nanoparticles (149 ± 2 mM Fe s−1 and 151 ± 5 mM Fe s−1), whereas the r1 relaxivity was higher for KCREKA-NPs (1.68 ± 0.29 mM Fe s−1 vs. 0.69 ± 0.3 mM Fe s−1). In vitro studies showed that blood clots with low coagulation times were disrupted by rtPA, whereas aged clots were almost insensitive to the presence of rtPA. MRI in vitro studies showed a sharp decrease in the T1 × T2 signals measured for aged clots incubated with KCREKA-NPs compared with fresh clots (47% [22, 80] to 26% [15, 51]). Furthermore, the control blood showed a higher value of the T1 × T2 signal (39% [20, 61]), being the blood clots with low coagulation times the samples with the lowest values measured by MRI. In vivo studies showed a significant T1 × T2 signal loss in the clot region of 24% after i.v. injection of KCREKA-NPs. The thrombus age (2.5% ± 6.1% vs. 81.3% ± 19.8%, p < 0.01) confirmed our ability to identify in vivo fresh blood clots. In this study, we developed and tested a dual MRI nanoparticle, acting as T1 and T2 contrast agents in MRI analyses. The developed KCREKA-NPs showed affinity for the fibrin content of blood clots, and the MRI signals provided by the nanoparticles showed significant differences depending on the clot age. The developed KCREKA-NPs could be used as a tool to predict the efficacy of a recanalization treatment and improve the triage of ischemic stroke patients.

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.

Associations between RNA-Binding Motif Protein 3, Fibroblast Growth Factor 21, and Clinical Outcome in Patients with Stroke.

BACKGROUND: RNA-binding motif protein 3 (RBM3) is a cold-induced marker of good functional outcome of ischemic stroke that is promising as a protective target. Fibroblast growth factor 21 (FGF21) is an obesity- and temperature-related hormone that upregulates the expression of RBM3, which is beneficial as a recombinant treatment and has been tested under different experimental pathological conditions, including stroke. However, the interaction between RBM3 and FGF21 has not yet been tested for clinical stroke conditions. METHODS: In a sample of 66 stroke patients, we analyzed the associations between the FGF21 and RBM3 serum concentrations on admission and at 72 h, body weight, maximum temperature during the first 24 h, and the outcome of patients at 3 months. We also analyzed their association with biomarkers of obesity (adiponectin and leptin) and inflammation (interleukin-6 (IL-6) and interleukin (IL-10)). RESULTS: Higher concentrations of FGF21 on admission and RBM3 at 72 h were associated with good outcomes. Serum FGF21 and RBM3 were directly related to body mass index and inversely related to the maximum temperature during the first 24 h. We found a positive association between the FGF21 concentrations in obese patients with leptin and a negative correlation with adiponectin in non-obese participants. CONCLUSIONS: This clinical study demonstrates the association between RBM3 and FGF21 levels and the outcome of stroke patients. Although further investigations are required, these data support the pharmacological induction of RBM3 as a promising protective therapy.

sTWEAK is a leukoaraiosis biomarker associated with neurovascular angiopathy.

OBJECTIVE: Leukoaraiosis (LA) refers to white matter lesions of undetermined etiology associated with the appearance and worsening of vascular pathologies. The aim is to confirm an increased frequency and intensity of LA in symptomatic patients with neurovascular pathology compared with asymptomatic subjects, and its association with circulating serum levels of soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK). METHODS: An observational study was conducted in which two groups of patients were compared. Group I (N = 242) comprised of asymptomatic subjects with arterial hypertension and/or diabetes or with a history of transient ischemic attacks, and Group II (N = 382) comprised patients with lacunar stroke or deep hemispheric intracerebral hemorrhage (ICH) of hypertensive origin. Serum levels of sTWEAK were analyzed and correlated with prevalence and intensity of LA according to the Fazekas scale. RESULTS: The prevalence of LA was higher in symptomatic (85.1%) versus asymptomatic patients (62.0%). Logistic regression model showed a significant relation of LA with neurovascular pathologies (OR: 2.69, IC 95%: 1.10-6.59, p = 0.003). When stratified according to the Fazekas scale, LA of grade II (OR: 3.53, IC 95%: 1.10-6.59, p = 0.003) and specially grade III (OR: 4.66, 95% CI: 1.09-19.84, p = 0.037) showed correlation with neurovascular pathologies. Increased sTWEAK levels were found in the symptomatic group in all LA grades (p < 0.0001), and associated with 5.06 times more risk of presenting clinical symptoms (OR: 5.06, 95% CI: 2.66-9.75, p < 0.0001). INTERPRETATION: LA showed a higher prevalence in patients with symptomatic lacunar stroke or deep hemispheric ICH. There is an association between sTWEAK levels and LA degree.

Neurological Instability in Ischemic Stroke: Relation with Outcome, Latency Time, and Molecular Markers.

The National Institutes of Health Stroke Scale (NIHSS) is commonly used to evaluate stroke neurological deficits and to predict the patient's outcome. Neurological instability (NI), defined as the variation of the NIHSS in the first 48 h, is a simple clinical metric that reflects dynamic changes in the area of the brain affected by the ischemia. We hypothesize that NI may represent areas of cerebral instability known as penumbra, which could expand or reduce brain injury and its associated neurological sequels. In this work, our aim was to analyze the association of NI with the functional outcome at 3 months and to study clinical biomarkers associated to NI as surrogate biomarkers of ischemic and inflammatory penumbrae in ischemic stroke (IS) patients. We included 663 IS patients in a retrospective observational study. Neutral NI was defined as a variation in the NI scale between - 5 and 5% (37.1%). Positive NI is attributed to patients with an improvement of > 5% NI after 48 h (48.9%), while negative NI is assigned to patients values lower than - 5% (14.0%). Poor outcome was assigned to patients with mRS ≥ 3 at 3 months. We observed an inverse association of poor outcome with positive NI (OR, 0.35; 95%CI, 0.18-0.67; p = 0.002) and a direct association with negative NI (OR, 6.30; 95%CI, 2.12-18.65; p = 0.001). Negative NI showed a higher association with poor outcome than most clinical markers. Regarding good functional outcome, positive NI was the marker with the higher association (19.31; CI 95%, 9.03-41.28; p < 0.0001) and with the highest percentage of identified patients with good functional outcome (17.6%). Patients with negative NI have higher glutamate levels compared with patients with neutral and positive NI (p < 0.0001). IL6 levels are significantly lower in patients with positive NI compared with neutral NI (p < 0.0001), while patients with negative NI showed the highest IL6 values (p < 0.0001). High glutamate levels were associated with negative NI at short latency times, decreasing at higher latency times. An opposite trend was observed for inflammation, and IL6 levels were similar in patients with positive and negative NI in the first 6 h and then higher in patients with negative NI. These results support NI as a prognosis factor in IS and the hypothesis of the existence of a delayed inflammatory penumbra, opening up the possibility of extending the therapeutic window for IS.

Tear Proteomics in Keratoconus: A Quantitative SWATH-MS Analysis.

Purpose: To elucidate dysregulated proteins in keratoconus (KC) to provide a better understanding of the molecular mechanisms that lead to the development of the disease using sequential window acquisition of all theoretical mass spectra (SWATH-MS) as a protein quantification tool of the tear proteomic profile. Methods: Prospective cross-sectional study that includes 25 keratoconic eyes and 25 healthy eyes. All participants underwent a clinical, tomographic, and aberrometric exam. Tear sample was collected using Schirmer strips and analyzed by liquid chromatography with tandem mass spectrometry. SWATH-MS was used as a quantification tool of the tear proteomic profile. The expression of the quantified proteins was compared between groups, and the biological and molecular functions of the dysregulated proteins as well as their functional relationships were studied by in silico analysis. Results: A total of 203 proteins were quantified in tear samples of patients with KC and control participants, of which 18 showed differential expression between groups (P < 0.05). An increase in the expression of 7 proteins and a decrease in the expression of 11 proteins were observed. Protein-protein interactions and gene ontology analysis showed the involvement of these dysregulated proteins in structural, inflammatory-immune, iron homeostasis, oxidative stress, and extracellular matrix proteolysis processes. Conclusions: Tear protein quantification has revealed the dysregulation of proteins involved in biological processes previously associated with KC. Among them, iron homeostasis should be highlighted as a relevant pathway in the KC pathophysiology, and it should be taken into account in the development of therapeutic targets to cope with tissue damage derived from iron accumulation and toxicity.

Characterization of a Temporal Profile of Biomarkers as an Index for Ischemic Stroke Onset Definition.

BACKGROUND AND PURPOSE: Stroke is a dynamic process in terms of molecular mechanisms, with prominent glutamate-mediated excitotoxicity at the onset of symptoms followed by IL-6-mediated inflammation. Our aim was to study a serum glutamate/IL-6 ratio as an index for stroke onset definition. METHODS: A total of 4408 ischemic stroke patients were recruited and then subdivided into four quartiles according to latency time in minutes (0-121, 121-185, 185-277 and >277). Latency time is defined as the time between stroke onset and treatment at the neurological unit. The primary endpoint of the study was the association of early latency times with different clinical aspects and serum markers. Serum glutamate and interleukin-6 (IL-6) levels at admission were selected as the main markers for excitotoxicity and inflammation, respectively. RESULTS: Glutamate serum levels were significantly higher in the earlier latency time compared with the higher latency times (p < 0.0001). IL-6 levels were lower in early latency times (p < 0.0001). Patients with a glutamate/IL-6 index on admission of >5 were associated with a latency time of <121 min from the onset of symptoms with a sensitivity of 88% and a specificity of 80%. CONCLUSIONS: The glutamate/IL-6 index allows the development of a ratio for an easy, non-invasive early identification of the onset of ischemic stroke symptoms, thus offering a new tool for selecting early stroke patient candidates for reperfusion therapies.

sTWEAK is a marker of early haematoma growth and leukoaraiosis in intracerebral haemorrhage.

OBJECTIVE: To study the association between early growth of haematoma with biomarkers of endothelial dysfunction such as leukoaraiosis (LA) and the soluble tumour necrosis factor-like weak inducer of apoptosis (sTWEAK) in patients with intracerebral haemorrhage (ICH). METHODS: This is a retrospective observational study of patients with nontraumatic ICH. Clinical and biochemical parameters were analysed. sTWEAK levels were measured by ELISA. LA was analysed in the hemisphere without haemorrhage to avoid interference with the acute injury. The main endpoint was the haematoma growth evaluated by the difference in volume between the second and the initial neuroimage. Poor functional outcome, defined as a modified Rankin Scale >2 at 3 months, was considered as secondary endpoint. Receiver operating characteristic curve analysis was performed to stablish the best cut-off for sTWEAK levels associated with haematoma growth. RESULTS: We included 653 patients with ICH in our analysis (71.1±11.9 years, 44% women). Haematoma growth was observed in 188 patients (28.8%). sTWEAK levels ≥5600 pg/mL predicted ICH growth with a sensitivity of 84% and a specificity of 87%. sTWEAK levels ≥5600 pg/mL and the presence of LA were associated with haematoma growth (OR: 42.46; (CI 95% 22.67 to 79.52) and OR: 2.73 (CI 95% 1.39 to 5.34), respectively). Also, the presence of LA (OR: 4.31 (CI 95% 2.89 to 6.42)) and the interaction between ICH growth and sTWEAK (OR: 2.23 (CI 95% 1.40 to 3.55)) were associated with poor functional outcome at 3 months. CONCLUSION: sTWEAKs, together with the presence and grade of LA, are biomarkers able to predict ICH growth and poor functional outcome in patients with ICH.

Mild systemic inflammation enhances response to OnabotulinumtoxinA in chronic migraineurs.

The anti-inflammatory effect of OnabotulinumtoxinA (OnabotA) has been a matter of discussion for many years. In chronic migraine, however, increased pro-inflammatory state is associated with good response to OnabotA. We aimed to investigate whether a mild systemic inflammatory state elicited by a common oral infection (periodontitis) could enhance treatment response to OnabotA. In this study, we included 61 chronic migraineurs otherwise healthy treated with OnabotA of which 7 were poor responders and 54 good responders. Before receiving OnabotA therapy, all participants underwent a full-mouth periodontal examination and blood samples were collected to determine serum levels of calcitonin gene-related peptide (CGRP), interleukin 6 (IL-6), IL-10 and high sensitivity C-reactive protein (hs-CRP). Periodontitis was present in 70.4% of responders and 28.6% of non-responders (P = 0.042). Responders showed greater levels of inflammation than non-responders (IL-6: 15.3 ± 8.7 vs. 9.2 ± 4.7 ng/mL, P = 0.016; CGRP: 18.8 ± 7.6 vs. 13.0 ± 3.1 pg/mL, P = 0.002; and hs-CRP: 3.9 ± 6.6 vs. 0.9 ± 0.8 mg/L, P = 0.003). A linear positive correlation was found between the amount of periodontal tissue inflamed in the oral cavity and markers of inflammation (IL-6: r = 0.270, P = 0.035; CGRP: r = 0.325, P = 0.011; and hs-CRP: r = 0.370, P = 0.003). This report shows that in presence of elevated systemic inflammatory markers related to periodontitis, OnabotA seems to reduce migraine attacks. The changes of scheduled inflammatory parameters after treatment and subsequent assessment during an adequate period still need to be done.

The presence of leukoaraiosis enhances the association between sTWEAK and hemorrhagic transformation.

OBJECTIVE: To investigate whether elevated serum levels of sTWEAK (soluble tumor necrosis factor-like inducer of apoptosis) might be involved in a higher frequency of symptomatic hemorrhagic transformation (HT) through the presence of leukoaraiosis (LA) in patients with acute ischemic stroke (IS) undergoing reperfusion therapies. METHODS: This is a retrospective observational study. The primary endpoint was to study the sTWEAK-LA-HT relationship by comparing results with biomarkers associated to HT and evaluating functional outcome at 3-months. Clinical factors, neuroimaging variables and biomarkers associated to inflammation, endothelial/atrial dysfunction or blood-brain barrier damage were also investigated. RESULTS: We enrolled 875 patients (mean age 72.3 ± 12.2 years; 46.0% women); 710 individuals underwent intravenous thrombolysis, 87 endovascular therapy and 78 both. HT incidence was 32%; LA presence was 75.4%. Patients with poor functional outcome at 3-months showed higher sTWEAK levels at admission (9844.2 [7460.4-12,542.0] vs. 2717.3 [1489.7-5852.3] pg/mL, P < 0.0001). By means of logistic regression models, PDGF-CC and sTWEAK were associated with mechanisms linked simultaneously to HT and LA. Serum sTWEAK levels at admission ≥6700 pg/mL were associated with an odds ratio of 13 for poor outcome at 3-months (OR: 13.6; CI 95%: 8.2-22.6, P < 0.0001). CONCLUSIONS: Higher sTWEAK levels are independently associated with HT and poor functional outcome in patients with IS undergoing reperfusion therapies through the presence of LA. sTWEAK could become a therapeutic target to reduce HT incidence in patients with IS.

Temperature-Induced Changes in Reperfused Stroke: Inflammatory and Thrombolytic Biomarkers.

Although hyperthermia is associated with poor outcomes in ischaemic stroke (IS), some studies indicate that high body temperature may benefit reperfusion therapies. We assessed the association of temperature with effective reperfusion (defined as a reduction of ≥8 points in the National Institute of Health Stroke Scale (NIHSS) within the first 24 h) and poor outcome (modified Rankin Scale (mRS) > 2) in 875 retrospectively-included IS patients. We also studied the influence of temperature on thrombolytic (cellular fibronectin (cFn); matrix metalloproteinase 9 (MMP-9)) and inflammatory biomarkers (tumour necrosis factor-alpha (TNF-α), interleukin 6 (IL-6)) and their relationship with effective reperfusion. Our results showed that a higher temperature at 24 but not 6 h after stroke was associated with failed reperfusion (OR: 0.373, p = 0.001), poor outcome (OR: 2.190, p = 0.005) and higher IL-6 levels (OR: 0.958, p < 0.0001). Temperature at 6 h was associated with higher MMP-9 levels (R = 0.697; p < 0.0001) and effective reperfusion, although this last association disappeared after adjusting for confounding factors (OR: 1.178, p = 0.166). Our results suggest that body temperature > 37.5 °C at 24 h, but not at 6 h after stroke, is correlated with reperfusion failure, poor clinical outcome, and infarct size. Mild hyperthermia (36.5-37.5 °C) in the first 6 h window might benefit drug reperfusion therapies by promoting clot lysis.

Antihyperthermic treatment decreases perihematomal hypodensity.

OBJECTIVE: To investigate the effect on perihematomal hypodensity and outcome of a decrease in body temperature in the first 24 hours in patients with intracerebral hemorrhage (ICH). METHODS: In this retrospective study on a prospectively registered database, among the 1,100 patients, 795 met all the inclusion criteria. Temperature variations in the first 24 hours and perihematomal hypodensity (PHHD) were recorded. Patients ≥37.5°C were treated with antihyperthermic drugs for at least 48 hours. The main objective was to determine the association among temperature variation, PHHD, and outcome at 3 months. RESULTS: The decrease in temperature in the first 24 hours increased the possibility of good outcome 11-fold. Temperature decrease, lower PHHD volume, and a good outcome were observed in 31.8% of the patients who received antihyperthermic treatment. CONCLUSION: The administration of early antihyperthermic treatment in patients with spontaneous ICH with a basal axillary temperature ≥37.5°C resulted in good outcome in a third of the treated patients.

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.

Severe periodontitis is linked with increased peripheral levels of sTWEAK and PTX3 in chronic migraineurs.

OBJECTIVES: Periodontitis (PD) and chronic migraine (CM) have been recently linked, and inflammatory processes and vascular endothelial changes are hypothesized as potential mediators of this relationship. The aim of this cross-sectional analysis was to investigate the potential association of PD with vascular systemic inflammation and complement activation in patients with CM. MATERIALS AND METHODS: Ninety-four chronic migraineurs underwent a full-mouth periodontal evaluation and a measure of PD activity and severity, namely the periodontal inflamed surface area (PISA) was calculated for each patient. We divided CM patients according to their periodontal status: mild PD (N = 14), moderate PD (N = 22), severe PD (N = 19), and non-PD (N = 39). Serum levels of C-reactive protein (CRP), pentraxin 3 (PTX3), soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK), and complements C3 and C4 were measured outside of migraine attacks. RESULTS: We found that severe periodontal patients had significantly higher circulating levels of PTX3 and sTWEAK compared with those without PD (2475.3 ± 1646.8 pg/mL vs. 516.6 ± 1193.8 pg/mL, P < 0.0001 and 672.4 ± 118.2 pg/mL vs. 485.7 ± 112.2 pg/mL, P < 0.0001; respectively). For the remaining biomarkers, no significant differences were found between groups. Severe PD was independently associated with higher levels of PTX3 (ß = 1997.6, P < 0.0001) and sTWEAK (ß = 187.1, P < 0.0001) but not with CRP, C3, and C4. PISA positively correlated to PTX3 (r = 0.475, P < 0.0001) and sTWEAK (r = 0.386, P < 0.0001). CONCLUSIONS: Based on these preliminary results, severe PD was linked with vascular systemic inflammation in patients with CM. However, further longitudinal studies should be performed to confirm these findings. CLINICAL RELEVANCE: sTWEAK and PTX3 measured in serum could be used as biomarkers in the PD-CM link.

Periodontal inflammation is related to increased serum calcitonin gene-related peptide levels in patients with chronic migraine.

BACKGROUND: Recently, a relationship was found between periodontitis and chronic migraine. Calcitonin gene-related peptide (CGRP) is a key element in migraine pathophysiology. However, no information exists of the potential association between periodontal inflammation and CGRP in chronic migraine. The aim of the study was, therefore, to investigate whether there is a link between periodontitis and peripheral levels of CGRP in a cohort of patients with chronic migraine. METHODS: We included 102 chronic migraineurs and 77 age- and sex-matched individuals free of headache/migraine. Full-mouth periodontal parameters were recorded and the periodontal inflamed surface area (PISA) was calculated to quantify the periodontal inflammatory status for each participant. Sociodemographic data and comorbidities were assessed by means of a standard questionnaire. We collected blood samples and serum concentrations were done for CGRP, interleukin (IL)-6 and IL-10. RESULTS: In the chronic migraine group, patients with periodontitis had greater levels of serum CGRP (19.7 ± 6.5 versus 15.3 ± 6.2 pg/mL, P < 0.0001) and IL-6 (15.1 ± 9.2 versus 9.6 ± 6.3 pg/mL, P < 0.0001) while non-significant differences were observed with IL-10 (2.0 ± 1.0 versus 2.8 ± 1.5 pg/mL, P = 0.675) concentrations than those without periodontitis. PISA was independently associated with CGRP in patients with chronic migraine (ß = 0.003; 95% confidence interval: 0.001 to 0.006, P = 0.031). PISA correlated positively with CGRP (r = 0.236; P = 0.017) and IL-6 (r = 0.262; P = 0.008) in chronic migraine. CONCLUSIONS: Periodontal inflammation is associated with increased circulating levels of CGRP in chronic migraineurs. Elucidating the exact mechanisms through which periodontitis and CGRP are linked in these patients deserves further investigation.

Blood glutamate EAAT2-cell grabbing therapy in cerebral ischemia.

BACKGROUND: Excitatory amino acid transporter 2 (EAAT2) plays a pivotal role in glutamate clearance in the adult brain, thereby preventing excitotoxic effects. Considering the high efficacy of EAAT2 for glutamate uptake, we hypothesized that the expression of this transporter in mesenchymal stem cells (MSCs) for systemic administration could yield a cell-based glutamate-grabbing therapy, combining the intrinsic properties of these cells with excitotoxic protection. METHODS: To address this hypothesis, EAAT2-encoding cDNA was introduced into MSCs and human embryonic kidney 293 cells (HEK cells) as the control cell line. EAAT2 expression and functionality were evaluated by in vitro assays. Blood glutamate-grabbing activity was tested in healthy and ischemic rat models treated with 3 × 106 and 9 × 106 cells/animal. FINDINGS: The expression of EAAT2 in both cell types conferred the expected glutamate-grabbing activity in in vitro and in vivo studies. The functional improvement observed in ischemic rats treated with EAAT2-HEK at low dose, confirmed that this effect was indeed mediated by the glutamate-grabbing activity associated with EAAT2 functionality. Unexpectedly, both cell doses of non-transfected MSCs induced higher protection than transfected EAAT2-MSCs by another mechanism independent of the glutamate-grabbing capacity. INTERPRETATION: Although the transfection procedure most likely interferes with some of the intrinsic protective mechanisms of mesenchymal cells, the results show that the induced expression of EAAT2 in cells represents a novel alternative to mitigate the excitotoxic effects of glutamate and paves the way to combine this strategy with current cell therapies for cerebral ischemia.

Heads and Tails of Natriuretic Peptides: Neuroprotective Role of Brain Natriuretic Peptide.

BACKGROUND: Besides the relevant role of brain-type natriuretic peptide (BNP) as biomarker of cardioembolic strokes, new experimental evidences suggest that this peptide may mediate neuroprotective effects. In this study, we have evaluated for the first time the clinical association between BNP (by means of proBNP) and good outcome in ischemic stroke patients, and analyzed the effect of blood BNP increase in an ischemic animal model. METHODS AND RESULTS: A retrospective study with 2 different cohorts (262 patients in cohort I and 610 in cohort II) from the same prospective stroke registry was performed. proBNP concentration was analyzed within the first 12 hours from stroke onset. The primary predictor variable was functional outcome evaluated by modified Rankin Scale at 3 months. For the experimental study, BNP pretreatment was tested in an ischemic animal model subjected to a transient occlusion of the cerebral artery, and the infarct volume and sensorimotor deficit were evaluated for 14 days. Cardioembolic strokes presented a positive correlation between proBNP concentration and modified Rankin Scale at 3 months; however, noncardioembolic strokes presented a negative correlation. In the logistic regression analysis, noncardioembolic strokes with concentrations of proBNP ≥340 pg/mL were associated with a good outcome. In line with these clinical findings, the experimental study revealed that those BNP pretreated animals presented a reduction on infarct volumes at 24 hours and functional recovery at days 7 and 14 compared with the control groups. CONCLUSIONS: These clinical and experimental evidences support the potential role of BNP as a protective factor against cerebral ischemia.