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Background: Little is known about the prevalence of cerebral microangiopathy (CM), which is related to cognitive impairment, in an asymptomatic population. Pulsatility index (PI) is an easily measurable parameter of cerebral vascular resistance in transcranial duplex of the middle cerebral artery (MCA) study. We aimed to determine the prevalence of CM measured by PI of MCA in low to moderate vascular risk subjects.Methods: We included 3,721 subjects between 45 and 70 years without previous history of vascular disease or diabetes mellitus and with at least one other vascular risk factor from the cross-sectional study ILERVAS (Lleida, Spain). Patients underwent transcranial duplex to determine MCA-PI. Possible CM was defined by MCA-PI >1.1. Carotid and femoral arteries ultrasound registration was done to determine the presence, the number, and the area of atheromatous plaques. Body mass index (BMI), pulse pressure (PP) and laboratory data were also recorded.Results: 439 (11.8%) subjects were excluded due to the low quality of transcranial duplex images. Median age was 57 [IQR 52, 62] years. Possible CM was found in 424 (12.9%) subjects. CM patients had higher prevalence of plaques than non-CM (77.4 vs. 66.4%, p < 0.001). PI showed a positive linear correlation with the number of territories with plaques (r = 0.130, p < 0.001), and the total plaque area (r = 0.082, p < 0.001). The predictors of possible CM were the age, male gender, and PP.Conclusions: In low-to-moderate vascular risk asymptomatic population, the proportion of abnormal brain microvascular bed determined by MCA-PI is not negligible. The planned 10-year follow-up will describe the clinical relevance of these findings.

BackgroundCOVID-19 may predispose to both venous and arterial thromboembolism event (TEE). Reports on the prevalence and prognosis of thrombotic complications are still emerging.ObjectiveTo describe the rate of TEE complications and its influence in the prognosis of hospitalized patients with COVID-19 after a cross-sectional study.MethodsWe evaluated the prevalence of TEE and its relationship with in-hospital death among hospitalized patients with COVID-19 who were admitted between 1st March to 20th April 2020 in a multicentric network of sixteen Hospitals in Spain. TEE was defined by the occurrence of venous thromboembolism (VTE), acute ischemic stroke (AIS), systemic arterial embolism or myocardial infarction (MI).ResultsWe studied 1737 patients with proven COVID-19 infection of whom 276 died (15.9%). TEE were presented in 64 (3.7%) patients: 49 (76.6%) patients had a VTE, 8 (12.5%) patients had MI, 6 (9.4%%) patients had AIS, and one (1.5%) patient a thrombosis of portal vein. TEE patients exhibited a diffuse profile: older, high levels of D-dimer protein and a tendency of lower levels of prothrombin. The multivariate regression models, confirmed the association between in-hospital death and age (odds ratio [OR] 1.12 [95% CI 1.10-1.14], pConclusionsSpecial attention is needed among hospitalized COVID-19 patients with TTE and other comorbidities as they have an increased risk of in-hospital death.

Rnd proteins are a subfamily of Rho GTPases involved in the control of actin cytoskeleton dynamics and other cell functions such as motility, proliferation and survival. Unlike other members of the Rho family, Rnd proteins lack GTPase activity and therefore remain constitutively active. We have recently described that RhoE/Rnd3 is expressed in the Central Nervous System and that it has a role in promoting neurite formation. Despite their possible relevance during development, the role of Rnd proteins in vivo is not known. To get insight into the in vivo function of RhoE we have generated mice lacking RhoE expression by an exon trapping cassette. RhoE null mice (RhoE gt/gt) are smaller at birth, display growth retardation and early postnatal death since only half of RhoE gt/gt mice survive beyond postnatal day (PD) 15 and 100% are dead by PD 29. RhoE gt/gt mice show an abnormal body position with profound motor impairment and impaired performance in most neurobehavioral tests. Null mutant mice are hypoactive, show an immature locomotor pattern and display a significant delay in the appearance of the hindlimb mature responses. Moreover, they perform worse than the control littermates in the wire suspension, vertical climbing and clinging, righting reflex and negative geotaxis tests. Also, RhoE ablation results in a delay of neuromuscular maturation and in a reduction in the number of spinal motor neurons. Finally, RhoE gt/gt mice lack the common peroneal nerve and, consequently, show a complete atrophy of the target muscles. This is the first model to study the in vivo functions of a member of the Rnd subfamily of proteins, revealing the important role of Rnd3/RhoE in the normal development and suggesting the possible involvement of this protein in neurological disorders.

DSCR1/ RCAN1 is a chromosome 21 gene found to be overexpressed in the brains of Down syndrome (DS) and postulated as a good candidate to contribute to mental disability. However, even though Rcan1 knockout mice have pronounced spatial learning and memory deficits, the possible deleterious effects of its overexpression in DS are not well understood. We have generated a transgenic mouse model overexpressing DSCR1/RCAN1 in the brain and analyzed the effect of RCAN1 overexpression on cognitive function. TgRCAN1 mice present a marked disruption of the learning process in a visuo-spatial learning task. However, no significant differences were observed in the performance of the memory phase of the test (removal session) nor in a step-down passive avoidance task, thus suggesting that once learning has been established, the animals are able to consolidate the information in the longer term.

Abstract Remote ischemic conditioning (RIC) is a promising therapeutic approach for ischemic stroke patients. It has been proven that RIC reduces infarct size and improves functional outcomes. RIC can be applied either before ischemia (pre-conditioning; RIPreC), during ischemia (per-conditioning; RIPerC) or after ischemia (post-conditioning; RIPostC). Our aim was to systematically determine the efficacy of RIC in reducing infarct volumes and define the cellular pathways involved in preclinical animal models of ischemic stroke. A systematic search in three databases yielded 50 peer-review articles. Data were analyzed using random effects models and results expressed as percentage of reduction in infarct size (95% CI). A meta-regression was also performed to evaluate the effects of covariates on the pooled effect-size. 95.3% of analyzed experiments were carried out in rodents. Thirty-nine out of the 64 experiments studied RIPostC (61%), sixteen examined RIPreC (25%) and nine tested RIPerC (14%). In all studies, RIC was shown to reduce infarct volume (− 38.36%; CI − 42.09 to − 34.62%) when compared to controls. There was a significant interaction caused by species. Short cycles in mice significantly reduces infarct volume while in rats the opposite occurs. RIPreC was shown to be the most effective strategy in mice. The present meta-analysis suggests that RIC is more efficient in transient ischemia, using a smaller number of RIC cycles, applying larger length of limb occlusion, and employing barbiturates anesthetics. There is a preclinical evidence for RIC, it is safe and effective. However, the exact cellular pathways and underlying mechanisms are still not fully determined, and its definition will be crucial for the understanding of RIC mechanism of action.

Abstract Senescent cells are capable of expressing a myriad of inflammatory cytokines and this pro-inflammatory phenomenon is known as senescence-associated secretory phenotype (SASP). The contribution of this phenomenon in brain ischemia was scarce. A mouse model of transient focal cerebral ischemia by compressing the distal middle cerebral artery (tMCAo) for 60 min was used. SASP, pro-inflammatory cytokines and cell cycle mRNAs levels were quantified at 30-min and 72 h post-surgery. Immunohistochemistry in paraffin embedded human brain slides and mouse brain tissue was performed. Our results showed an increase of both p16 and p21 mRNA restricted to the infarct area in the tMCAo brain. Moreover, there was an induction of Il6, Tnfa, Cxc11, and its receptor Cxcr2 mRNA pro-inflammatory cytokines with a high positive correlation with p16/p21 mRNA levels. The p16 was mainly shown in cytoplasm of neurons and cytoplasm/membrane of microglial cells. The p21 was observed in membrane of neurons and also it showed a mixed cytoplasmic and membranous pattern in the microglial cells. In a human stroke patient, an increase of P16 in the perimeter of the MCA infarct area was observed. These suggest a role of SASP in tMCAo mouse model and in human brain tissue. SASP potentially has a physiological role in acute ischemic stroke and neurological function loss.

BackgroundTransient ischemic attack (TIA) provides a unique opportunity to optimize secondary preventive treatments to avoid subsequent ischemic stroke (SIS). Although atrial fibrillation (AF) is the leading cause of cardioembolism in IS and anticoagulation prevents stroke recurrence (SR), limited data exists about the risk of new-diagnosed AF (NDAF) after TIA and the consequences of the diagnostic delay. The aim of our study was to determine this risk in a cohort of TIA patients with long-term follow-up.MethodsWe carried out a prospective cohort study of 723 consecutive TIA patients from January 2006 to June 2010. Median follow-up was 6.5 (5.0–9.6) years. In a subgroup of 204 (28.2%) consecutive patients, a panel of biomarkers was assessed during the first 24 h of the onset of symptoms. Multivariate analyses were performed to find out the associated factors of NDAF. Kaplan-Meier analysis was also performed to analyzed risk of SIS.ResultsNDAF was indentified in 116 (16.0%) patients: 42 (36.2%) during admission, 18 (15.5%) within first year, 29 (25%) between one and five years and 27 (23.3%) beyond 5 years. NDAF was associated with sex (female) [hazard ratio (HR) 1.61 (95% CI, 1.07- 2.41)], age [[HR 1.05 (95% CI, 1.03–1.07)], previous ischemic heart disease (IHD) [HR 1.84, (95% CI 1.15–2.97)] and cortical DWI pattern [HR 2.81 (95% CI, 1.87–4.21)]. In the Kaplan-Meier analysis, NT-proBNP ≥ 218.2 pg/ml (log-rank test P < 0.001) was associated with significant risk of NDAF during the first 5 years of follow-up. Patients with NDAF after admission and before 5 years of follow-up had the highest risk of SIS (P = 0.002).ConclusionThe risk of NDAF after TIA is clinically relevant. We identified clinical and neuroimaging factors of NDAF. In addition, NT-proBNP was related to NDAF. Our results can be used to evaluate the benefit of long-term cardiac monitoring in selected patients.

Abstract Background Remote ischemic conditioning during cerebral ischemia (remote ischemic perconditioning, RIPerC) refers to the application of several cycles of brief ischemia and reperfusion (I/R) commonly to a limb, and it represents a new paradigm in neuroprotection with multiple mechanisms of action in ischemic stroke (IS) patients during acute phase. Some clinical trials just finished, and a few others are still ongoing; gather the current knowledge and pull it down to influence the present and future studies was the goal of this paper. Methods A systematic review of published research papers and/or registered clinical trials since 2000 was performed. Results Nineteen studies were identified and only four studies were completed. All of them have demonstrated that RIPerC is safe, feasible and well tolerated in IS patients. However, a high heterogeneity of clinical trial characteristics was observed: five (26.3%) randomized clinical trials (RCTs) included only thrombolytic-treated patients, three (15.8%) RCTs only thrombectomy-treated patients, and five (26.3%) RCTs required radiological confirmation of IS. Temporal inclusion criteria vary from 4 h to 48 h. Most of the clinical trials used 4 cycles of RIPerC in the upper non-affected limb. Interestingly, only three (16.7%) RCTs applied RIPerC during the transportation in the ambulance. Neuroimaging outputs were the main endpoints when endovascular therapy was applied; functional outcome is also the main endpoint in large-medium size studies. Conclusions This review summarizes the completed and ongoing clinical trials on RIPerC in IS patients, where RIPerC has been used alone or in combination with recanalization therapies. Ongoing clinical trials will provide new information on the best RIPerC intervention strategy and potentially improve the functional outcome of IS patients; definition of new RIPerC strategies would ideally aim at enhancing tissue preservation, promoting neurological recovery, and stratify patients to improve treatment feasibility.

Introduction: Ischemic tolerance (IT) refers to a state where cells are resistant to the damaging effects caused by periods of ischemia. In a clinical scenario, the IT phenomenon would be activated by a recent transient ischemic attack (TIA) before an ischemic stroke (IS). The characterization of inflammatory protein expression patterns will contribute to improved understanding of IT.Methods: A total of 477 IS patients from nine hospitals, recruited between January 2011 and January 2016, were included in the current study and divided in three groups: 438 (91.9%) patients without previous TIA (group 1), 22 (4.6%) patients who suffered TIA 24 h before IS (group 2), and 17 (3.5%) patients who suffered TIA between 24 h and 7 days prior to IS (group 3). An inflammatory biomarker panel (IL-6, NT-proBNP, hsCRP, hs-Troponin, NSE, and S-100b) on plasma and a cytokine antibody array was performed to achieve the preconditioning signature potentially induced by TIA phenomena. Primary outcome was modified rankin scale (mRs) score at 90 days.Results: Recent previous TIA was associated with better clinical outcome at 90 days (median mRS of group 1: 2.0 [1.0–4.0]; group 2: 2.0 [0.0–3.0]; group 3: 1.0 [0–2.5]; p = 0.086) and smaller brain lesion (group 1: 3.7 [0.7–18.3]; group 2: 0.8 [0.3–8.9]; group 3: 0.6 [0.1–5.5] mL; p = 0.006). All inflammation biomarkers were down regulated in the groups of recent TIA prior to IS compared to those who did not suffer a TIA events. Moreover, a cytokine antibody array revealed 30 differentially expressed proteins between the three groups. Among them, HRG1-alpha (Fold change 74.4 between group 1 and 2; 74.2 between group 1 and 3) and MAC-1 (Fold change 0.05 between group 1 and 2; 0.06 between group 1 and 3) expression levels would better stratify patients with TIA 7 days before IS. These two proteins showed an earlier inflammation profile that was not detectable by the biomarker panel.Conclusion: Inflammatory pathways were activated by transient ischemic attack, however the period of time between this event and a further ischemic stroke could be determined by a protein signature that would contribute to define the role of ischemic tolerance induced by TIA.

Rationale: Remote ischemic perconditioning during cerebral ischemia (RIPerC) refers to the application of brief episodes of transient limb ischemia commonly to a limb, it represents a new safe, simple and low-cost paradigm in neuroprotection.Aim and/or Hypothesis: To evaluate the effects of RIPerC on acute ischemic stroke (AIS) patients, applied in the ambulance, to improve functional outcomes compared with standard of care.Sample Size Estimates: A sample size of 286 patients in each arm achieves 80% power to detect treatment differences of 14% in the outcome, using a two-sided binomial test at significance level of 0.05, assuming that 40% of the control patients will experience good outcome and an initial misdiagnosis rate of 29%.Methods and Design: We aim to conduct a multicentre study of pre-hospital RIPerC application in AIS patients. A total of 572 adult patients diagnosed of suspected clinical stroke within 8 h of symptom onset and clinical deficit >0 according to prehospital rapid arterial occlusion evaluation (RACE) scale score will be randomized, in blocks of size 4, to RIPerC or sham. Patients will be stratified by RACE score scale. RIPerC will be started in the ambulance before hospital admission and continued in the hospital if necessary. It will consist of five cycles of electronic tourniquet inflation and deflation (5 min each). The cuff pressure for RIPerC will be 200 mmHg during inflation. Sham will only simulate vibration of the device.Study Outcome(s): The primary outcome will be the difference in the proportion of patients with good outcomes as defined by a mRS score of 2 or less at 90 days. Secondary outcomes to be monitored will include early neurological improvement rate, treatment related serious adverse event rates, size of the infarct volume, symptomatic intracranial hemorrhage, metabolomic and lipidomic response to RIPerC and Neuropsychological evaluation at 90 days.Discussion: Neuroprotective therapies could not only increase the benefits of available reperfusion therapies among AIS patients but also provide an option for patients who are not candidates for these treatments. REMOTE-CAT will investigate the clinical benefit of RIC as a new neuroprotective strategy in AIS.Clinical Trial Registration:www.ClinicalTrials.gov, identifier: NCT03375762.

Dual-specificity tyrosine-regulated kinase 1A (DYRK1A) is a candidate gene for the Down syndrome neurological defects and may be involved in the progression of Alzheimer's disease. Heterozygous mice for Dyrk1A (Dyrk1A+/−) exhibit decreased brain size, motor abnormalities and cognitive deficits in the adult. However, there is no information about the mutant phenotype in old ages. Here we analyze the impact of Dyrk1A dosage reduction on motor performance and hippocampal-dependent learning and memory in aged Dyrk1A+/− mice. Whereas motor tests showed marked alterations in traction ability, prehensile reflex and balance, heterozygous mice only present a slight impairment of visuo-spatial memory even though they show a robust decrease of CA1–CA3 and dentate gyrus cells.

Individuals with Down syndrome (DS) present important motor deficits that derive from altered motor development of infants and young children. DYRK1A, a candidate gene for DS abnormalities has been implicated in motor function due to its expression in motor nuclei in the adult brain, and its overexpression in DS mouse models leads to hyperactivity and altered motor learning. However, its precise role in the adult motor system, or its possible involvement in postnatal locomotor development has not yet been clarified. During the postnatal period we observed time-specific expression of Dyrk1A in discrete subsets of brainstem nuclei and spinal cord motor neurons. Interestingly, we describe for the first time the presence of Dyrk1A in the presynaptic terminal of the neuromuscular junctions and its axonal transport from the facial nucleus, suggesting a function for Dyrk1A in these structures. Relevant to DS, Dyrk1A overexpression in transgenic mice (TgDyrk1A) produces motor developmental alterations possibly contributing to DS motor phenotypes and modifies the numbers of motor cholinergic neurons, suggesting that the kinase may have a role in the development of the brainstem and spinal cord motor system.

BackgroundPathogenic aneuploidies involve the concept of dosage-sensitive genes leading to over- and underexpression phenotypes. Monosomy 21 in human leads to mental retardation and skeletal, immune and respiratory function disturbances. Most of the human condition corresponds to partial monosomies suggesting that critical haploinsufficient genes may be responsible for the phenotypes. The DYRK1A gene is localized on the human chromosome 21q22.2 region, and has been proposed to participate in monosomy 21 phenotypes. It encodes a dual-specificity kinase involved in neuronal development and in adult brain physiology, but its possible role as critical haploinsufficient gene in cognitive function has not been explored.Methodology/principal findingsWe used mice heterozygous for a Dyrk1A targeted mutation (Dyrk1A+/-) to investigate the implication of this gene in the cognitive phenotypes of monosomy 21. Performance of Dyrk1A+/- mice was assayed 1/ in a navigational task using the standard hippocampally related version of the Morris water maze, 2/ in a swimming test designed to reveal potential kinesthetic and stress-related behavioral differences between control and heterozygous mice under two levels of aversiveness (25 degrees C and 17 degrees C) and 3/ in a long-term novel object recognition task, sensitive to hippocampal damage. Dyrk1A+/- mice showed impairment in the development of spatial learning strategies in a hippocampally-dependent memory task, they were impaired in their novel object recognition ability and were more sensitive to aversive conditions in the swimming test than euploid control animals.Conclusions/significanceThe present results are clear examples where removal of a single gene has a profound effect on phenotype and indicate that haploinsufficiency of DYRK1A might contribute to an impairment of cognitive functions and stress coping behavior in human monosomy 21.