Head Down Tilt 15° in Acute Ischemic Stroke with Poor Collaterals: A Randomized Preclinical Trial.

Cerebral collaterals are recruited after arterial occlusion with a protective effect on tissue outcome in acute ischemic stroke. Head down tilt 15° (HDT15) is a simple, low cost and accessible procedure that could be applied as an emergency treatment, before recanalization therapies, with the aim to increase cerebral collateral flow. Spontaneously hypertensive rats have been shown to display anatomical differences in morphology and function of cerebral collaterals, compared to other rat strains, resulting in an overall poor collateral circulation. We investigate the efficacy and safety of HDT15 in spontaneously hypertensive (SHR) rats, which were considered as an animal stroke model with poor collaterals. Cerebral ischemia was induced by 90 minute endovascular occlusion of the middle cerebral artery (MCA). SHR rats were randomized to HDT15 or flat position (n = 19). HDT15 was applied 30 minutes after occlusion and lasted 60 minutes, until reperfusion. HDT15 application increased cerebral perfusion (+16.6% versus +6.1%; p = 0.0040) and resulted in a small reduction of infarct size (83.6 versus 107.1 mm3; - 21.89%; p = 0.0272), but it was not associated with early neurological improvement, compared to flat position. Our study suggests that the response to HDT15 during MCA occlusion is dependent on baseline collaterals. Nonetheless, HDT15 promoted a mild improvement of cerebral hemodynamics even in subjects with poor collaterals, without safety concerns.

Harmonization of sensorimotor deficit assessment in a registered multicentre pre-clinical randomized controlled trial using two models of ischemic stroke.

Multicentre preclinical randomized controlled trials (pRCTs) are a valuable tool to improve experimental stroke research, but are challenging and therefore underused. A common challenge regards the standardization of procedures across centres. We here present the harmonization phase for the quantification of sensorimotor deficits by composite neuroscore, which was the primary outcome of two multicentre pRCTs assessing remote ischemic conditioning in rodent models of ischemic stroke. Ischemic stroke was induced by middle cerebral artery occlusion for 30, 45 or 60 min in mice and 50, 75 or 100 min in rats, allowing sufficient variability. Eleven animals per species were video recorded during neurobehavioural tasks and evaluated with neuroscore by eight independent raters, remotely and blindly. We aimed at reaching an intraclass correlation coefficient (ICC) ≥0.60 as satisfactory interrater agreement. After a first remote training we obtained ICC = 0.50 for mice and ICC = 0.49 for rats. Errors were identified in animal handling and test execution. After a second remote training, we reached the target interrater agreement for mice (ICC = 0.64) and rats (ICC = 0.69). In conclusion, a multi-step, online harmonization phase proved to be feasible, easy to implement and highly effective to align each centre's behavioral evaluations before project's interventional phase.

Head down tilt 15° to preserve salvageable brain tissue in acute ischemic stroke: A pre-clinical pooled analysis, with focus on cerebral hemodynamics.

Neurological outcome after ischemic stroke depends on residual salvageable brain tissue at the time of recanalization. Head down tilt 15° (HDT15) was proven effective in reducing infarct size and improving functional outcome in rats with transient middle cerebral artery occlusion (t-MCAO) by increasing cerebral perfusion within the ischemic penumbra. In this pooled analysis, individual animal-level data from three experimental series were combined in a study population of 104 t-MCAO rats (45 in HDT15 group and 59 in flat position group). Co-primary outcomes were infarct size and functional outcome at 24 h in both groups. The secondary outcome was hemodynamic change induced by HDT15 in ischemic and non-ischemic hemispheres in a subgroup of animals. Infarct size at 24 h was smaller in HDT15 group than in flat position group (absolute mean difference 31.69 mm3 , 95% CI 9.1-54.2, Cohen's d 0.56, p = 0.006). Functional outcome at 24 h was better in HDT15 group than in flat position group (median [IQR]: 13[10-16] vs. 11), with a shift in the distribution of the neurobehavioural scores in favour of HDT15. Mean cerebral perfusion in the ischemic hemisphere was higher during HDT15 than before its application (Perfusion Unit [P.U.], mean ± SD: 52.5 ± 19.52 P.U. vs. 41.25 ± 14.54 P.U., mean of differences 13.36, 95% CI 7.5-19.18, p = 0.0002). Mean cerebral perfusion in the non-ischemic hemisphere before and during HDT15 was unchanged (P.U., mean ± SD: 94.1 ± 33.8 P.U. vs. 100.25 ± 25.34 P.U., mean of differences 3.95, 95%, CI -1.9 to 9.6, p = 0.1576). This study confirmed that HDT15 improves the outcome in t-MCAO rats by promoting cerebral perfusion in the ischemic territory, without disrupting hemodynamics in non-ischemic areas.

Upper Respiratory Microbiome in Pregnant Women: Characterization and Influence of Parity.

During pregnancy, the woman's immune system changes to support fetal development. These immunological modifications can increase the risk of respiratory diseases. Because the respiratory microbiome is involved in airway homeostasis, it is important to investigate how it changes during pregnancy. Additionally, since parity is associated with immune system alterations and cohabitants shared a similar microbiome, we investigated whether having a child may influence the respiratory microbiome of pregnant women. We compared the microbiome of 55 pregnant with 26 non-pregnant women using 16S rRNA gene sequencing and analyzed taxonomy, diversity, and metabolic pathways to evaluate the differences among nulliparous, primiparous, and multiparous women. The microbiome was similar in pregnant and non-pregnant women, but pregnant women had higher alpha diversity (Chao1 p-value = 0.001; Fisher p-value = 0.005) and a lower abundance of several metabolic pathways. Multiparous pregnant women had a higher relative abundance of Moraxella (p-value = 0.003) and a lower abundance of Corynebacterium (p-value = 0.002) compared with primiparous women. Both multiparous (pregnant) and primiparous/multiparous (non-pregnant) women reported a higher abundance of Moraxella compared with primiparous (pregnant) or nulliparous ones (p-value = 0.001). In conclusion, we characterized for the first time the upper airway microbiome of pregnant women and observed the influence of parity on its composition.

Selective Cerebrospinal Fluid Hypothermia: Bioengineering Development and In Vivo Study of an Intraventricular Cooling Device (V-COOL).

Hypothermia is a promising therapeutic strategy for severe vasospasm and other types of non-thrombotic cerebral ischemia, but its clinical application is limited by significant systemic side effects. We aimed to develop an intraventricular device for the controlled cooling of the cerebrospinal fluid, to produce a targeted hypothermia in the affected cerebral hemisphere with a minimal effect on systemic temperature. An intraventricular cooling device (acronym: V-COOL) was developed by in silico modelling, in vitro testing, and in vivo proof-of-concept application in healthy Wistar rats (n = 42). Cerebral cortical temperature, rectal temperature, and intracranial pressure were monitored at increasing flow rate (0.2 to 0.8 mL/min) and duration of application (10 to 60 min). Survival, neurological outcome, and MRI volumetric analysis of the ventricular system were assessed during the first 24 h. The V-COOL prototyping was designed to minimize extra-cranial heat transfer and intra-cranial pressure load. In vivo application of the V-COOL device produced a flow rate-dependent decrease in cerebral cortical temperature, without affecting systemic temperature. The target degree of cerebral cooling (- 3.0 °C) was obtained in 4.48 min at the flow rate of 0.4 mL/min, without significant changes in intracranial pressure. Survival and neurological outcome at 24 h showed no significant difference compared to sham-treated rats. MRI study showed a transient dilation of the ventricular system (+ 38%) in a subset of animals. The V-COOL technology provides an effective, rapid, selective, and safe cerebral cooling to a clinically relevant degree of - 3.0 °C.

Nasopharyngeal Bacterial Microbiota Composition and SARS-CoV-2 IgG Antibody Maintenance in Asymptomatic/Paucisymptomatic Subjects.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19), ranging from asymptomatic conditions to severe/fatal lung injury and multi-organ failure. Growing evidence shows that the nasopharyngeal microbiota composition may predict the severity of respiratory infections and may play a role in the protection from viral entry and the regulation of the immune response to the infection. In the present study, we have characterized the nasopharyngeal bacterial microbiota (BNM) composition and have performed factor analysis in a group of 54 asymptomatic/paucisymptomatic subjects who tested positive for nasopharyngeal swab SARS-CoV-2 RNA and/or showed anti-RBD-IgG positive serology at the enrolment. We investigated whether BNM was associated with SARS-CoV-2 RNA positivity and serum anti-RBD-IgG antibody development/maintenance 20-28 weeks after the enrolment. Shannon's entropy α-diversity index [odds ratio (OR) = 5.75, p = 0.0107] and the BNM Factor1 (OR = 2.64, p = 0.0370) were positively associated with serum anti-RBD-IgG antibody maintenance. The present results suggest that BNM composition may influence the immunological memory against SARS-CoV-2 infections. To the best of our knowledge, this is the first study investigating the link between BNM and specific IgG antibody maintenance. Further studies are needed to unveil the mechanisms through which the BNM influences the adaptive immune response against viral infections.

Particulate Matter Exposure and Allergic Rhinitis: The Role of Plasmatic Extracellular Vesicles and Bacterial Nasal Microbiome.

Particulate matter (PM) exposure is linked to the worsening of respiratory conditions, including allergic rhinitis (AR), as it can trigger nasal and systemic inflammation. To unveil the underlying molecular mechanisms, we investigated the effects of PM exposure on the release of plasmatic extracellular vesicles (EV) and on the complex cross-talk between the host and the nasal microbiome. To this aim, we evaluated the effects of PM10 and PM2.5 exposures on both the bacteria-derived-EV portion (bEV) and the host-derived EVs (hEV), as well as on bacterial nasal microbiome (bNM) features in 26 AR patients and 24 matched healthy subjects (HS). In addition, we assessed the role exerted by the bNM as a modifier of PM effects on the complex EV signaling network in the paradigmatic context of AR. We observed that PM exposure differently affected EV release and bNM composition in HS compared to AR, thus potentially contributing to the molecular mechanisms underlying AR. The obtained results represent the first step towards the understanding of the complex signaling network linking external stimuli, bNM composition, and the immune risponse.

Blood-derived extracellular vesicles isolated from healthy donors exposed to air pollution modulate in vitro endothelial cells behavior.

The release of Extracellular Vesicles (EVs) into the bloodstream is positively associated with Particulate Matter (PM) exposure, which is involved in endothelial dysfunction and related to increased risk of cardiovascular disease. Obesity modifies the effects of PM exposure on heart rate variability and markers of inflammation, oxidative stress, and acute phase response. We isolated and characterized plasmatic EVs from six healthy donors and confirmed a positive association with PM exposure. We stratified for Body Mass Index (BMI) and observed an increased release of CD61+ (platelets) and CD105+ (endothelium) derived-EVs after high PM level exposure in Normal Weight subjects (NW) and no significant variations in Overweight subjects (OW). We then investigated the ability to activate endothelial primary cells by plasmatic EVs after both high and low PM exposure. NW-high-PM EVs showed an increased endothelial activation, measured as CD105+/CD62e+ (activated endothelium) EVs ratio. On the contrary, cells treated with OW-high-PM EVs showed reduced endothelial activation. These results suggest the ability of NW plasmatic EVs to communicate to endothelial cells and promote the crosstalk between activated endothelium and peripheral cells. However, this capacity was lost in OW subjects. Our findings contribute to elucidate the role of EVs in endothelial activation after PM exposure.

Nasal Microbiota Modifies the Effects of Particulate Air Pollution on Plasma Extracellular Vesicles.

Air pollution exposure has been linked to modifications of both extracellular vesicle (EV) concentration and nasal microbiota structure (NMB), which might act as the respiratory health gatekeeper. This study aimed to assess whether an unbalanced NMB could modify the effect of particulate matter (PM) exposure on plasmatic EV levels. Due to two different NMB taxonomical profiles characterized by a widely different relative abundance of the Moraxella genus, the enrolled population was stratified into Mor- (balanced NMB) and Mor+ (unbalanced NMB) groups (Moraxella genus's cut-off ≤25% and >25%, respectively). EV features were assessed by nanoparticle tracking analysis (NTA) and flow-cytometry (FC). Multivariable analyses were applied on EV outcomes to evaluate a possible association between PM10 and PM2.5 and plasmatic EV levels. The Mor- group revealed positive associations between PM levels and plasmatic CD105+ EVs (GMR = 4.39 p = 0.02) as for total EV count (GMR = 1.92 p = 0.02). Conversely, the Mor+ group showed a negative association between exposure and EV outcomes (CD66+ GMR = 0.004 p = 0.01; EpCAM+ GMR = 0.005 p = 0.01). Our findings provide an insight regarding how a balanced NMB may help to counteract PM exposure effects in terms of plasmatic EV concentration. Further research is necessary to understand the relationship between the host and the NMB to disentangle the mechanism exerted by inhaled pollutants in modulating EVs and NMB.

Multicentre translational Trial of Remote Ischaemic Conditioning in Acute Ischaemic Stroke (TRICS): protocol of multicentre, parallel group, randomised, preclinical trial in female and male rat and mouse from the Italian Stroke Organization (ISO) Basic Science network.

INTRODUCTION: Multicentre preclinical randomised controlled trials (pRCT) are emerging as a necessary step to confirm efficacy and improve translation into the clinic. The aim of this project is to perform two multicentre pRCTs (one in rats and one in mice) to investigate the efficacy of remote ischaemic conditioning (RIC) in an experimental model of severe ischaemic stroke. METHODS AND ANALYSIS: Seven research laboratories within the Italian Stroke Organization (ISO) Basic Science network will participate in the study. Transient endovascular occlusion of the proximal right middle cerebral artery will be performed in two species (rats and mice) and in both sexes. Animals will be randomised to receive RIC by transient surgical occlusion of the right femoral artery, or sham surgery, after reperfusion. Blinded outcome assessment will be performed for dichotomised functional neuroscore (primary endpoint) and infarct volume (secondary endpoint) at 48 hours. A sample size of 80 animals per species will yield 82% power to detect a significant difference of 30% in the primary outcome in both pRCTs. Analyses will be performed in a blind status and according to an intention-to-treat paradigm. The results of this study will provide robust, translationally oriented, high-quality evidence on the efficacy of RIC in multiple species of rodents with large ischaemic stroke. ETHICS AND DISSEMINATION: This is approved by the Animal Welfare Regulatory Body of the University of Milano Bicocca, under project license from the Italian Ministry of Health. Trial results will be subject to publication according to the definition of the outcome presented in this protocol. TRIAL REGISTRATION NUMBER: PCTE0000177.

INSIDE Project: Individual Air Pollution Exposure, Extracellular Vesicles Signaling and Hypertensive Disorder Development in Pregnancy.

Hypertensive disorders are common complications during pregnancy (HDP) with substantial public health impact. Acute and chronic particulate matter (PM) exposure during pregnancy increases the risk of HDP, although the underlying molecular mechanisms remain unclear. Extracellular vesicles (EVs) may be the ideal candidates for mediating the effects of PM exposure in pregnancy as they are released in response to environmental stimuli. The INSIDE project aims to investigate this mechanism in pregnancy outcomes. The study population is enrolled at the Fetal Medicine Unit of Fondazione IRCCS Ca'Granda-Ospedale Maggiore Policlinico at 10-14 weeks of gestation. Exposure to PM10 and PM2.5 is assessed using the flexible air quality regional model (FARM) and Bayesian geostatistical models. Each woman provides a blood sample for EV analysis and circulating biomarker assessment. Moreover, a subgroup of recruited women (n = 85) is asked to participate in a cardiovascular screening program including a standard clinical evaluation, a non-invasive assessment of right ventricular function, and pulmonary circulation at rest and during exercise. These subjects are also asked to wear a personal particulate sampler, to measure PM10, PM2.5, and PM1. The INSIDE study is expected to identify the health impacts of PM exposure on pregnancy outcomes.

Inter-generational resemblance of methylation levels at circadian genes and associations with phenology in the barn swallow.

Regulation of gene expression can occur via epigenetic effects as mediated by DNA methylation. The potential for epigenetic effects to be transmitted across generations, thus modulating phenotypic variation and affecting ecological and evolutionary processes, is increasingly appreciated. However, the study of variation in epigenomes and inter-generational transmission of epigenetic alterations in wild populations is at its very infancy. We studied sex- and age-related variation in DNA methylation and parent-offspring resemblance in methylation profiles in the barn swallows. We focused on a class of highly conserved 'clock' genes (clock, cry1, per2, per3, timeless) relevant in the timing of activities of major ecological importance. In addition, we considerably expanded previous analyses on the relationship between methylation at clock genes and breeding date, a key fitness trait in barn swallows. We found positive assortative mating for methylation at one clock locus. Methylation varied between the nestling and the adult stage, and according to sex. Individuals with relatively high methylation as nestlings also had high methylation levels when adults. Extensive parent-nestling resemblance in methylation levels was observed. Occurrence of extra-pair fertilizations allowed to disclose evidence hinting at a prevalence of paternal germline or sperm quality effects over common environment effects in generating father-offspring resemblance in methylation. Finally, we found an association between methylation at the clock poly-Q region, but not at other loci, and breeding date. We thus provided evidence for sex-dependent variation and the first account of parent-offspring resemblance in methylation in any wild vertebrate. We also showed that epigenetics may influence phenotypic plasticity of timing of life cycle events, thus having a major impact on fitness.

PM10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in Lombardy, Italy.

Respiratory syncytial virus (RSV) is the primary cause of acute lower respiratory infections in children, bronchiolitis in particular. Airborne particulate matter (PM) may influence the children's immune system and foster the spread of RSV infection. We aimed to verify whether PM10 exposure is associated with hospitalization due to RSV bronchiolitis. We selected hospital discharge records (HRD) with ICD-9-CM code 466.11 of infants < 1 year of age, occurring in the epidemic seasons of two years (2012-2013) in Lombardy, Italy. Cases were assigned daily PM10 and apparent temperature levels of the capital city of their residential province. Different exposure windows were considered: single days preceding hospitalization (lag 0 to 30), their average estimates (lag 0-1 to 0-30), and the four weeks preceding hospitalization (week 1 to 4). Negative binomial regression models adjusted for apparent temperature and season were applied to the daily counts of hospitalizations in each province. Results were expressed as incidence rate ratios (IRR) and 95% confidence intervals (95%CI) per 10 µg/m3 increase in PM10 concentration. Random effects meta-analyses of province-specific IRR were performed to obtain regional estimates. 2814 HRD met our inclusion criteria; males represented about 55% of the cases. A 6% increased risk of hospitalization (95%CI: 1.03-1.10) was found at lag 0 and an almost overlapping 7% increase at lag 1. IRR ranged from 1.03 to 1.05 between lags 2 and 11. No increased risk was observed from lag 12. When considering averaged daily lags, risk estimates gradually increased in the two weeks preceding hospitalization from 1.08 (1.04-1.12) at lag 0-1 to 1.15 (1.08-1.23) between lags 0-11 and 0-13. Analyses on weekly lags showed a risk increase of 6% (1.01-1.12) during week 1 and of 7% (1.02-1.13) during week 2. Our study found a clear association between short- and medium-term PM10 exposures and increased risk of hospitalization due to RSV bronchiolitis among infants.

Short-term particulate matter exposure influences nasal microbiota in a population of healthy subjects.

BACKGROUND: Exposure to air pollutants, such as particulate matter (PM), represents a growing health problem. The aim of our study was to investigate whether PM could induce a dysbiosis in the nasal microbiota in terms of α-diversity and taxonomic composition. METHODS: We investigated structure and characteristics of the microbiota of 40 healthy subjects through metabarcoding analysis of the V3-V4 regions of the 16s rRNA gene. Exposure to PM10 and PM2.5 was assessed with a personal sampler worn for 24h before sample collection (Day -1) and with measurements from monitoring stations (from Day -2 to Day -7). RESULTS: We found an inverse association between PM10 and PM2.5 levels of the 3rd day preceding sampling (Day -3) and α-diversity indices (Chao1, Shannon and PD_whole_tree). Day -3 PM was inversely associated also with the majority of analyzed taxa, except for Moraxella, which showed a positive association. In addition, subjects showed different structural profiles identifying two groups: one characterized by an even community and another widely dominated by the Moraxella genus. CONCLUSIONS: Our findings support the role of PM exposure in influencing microbiota and altering the normal homeostasis within the bacterial community. Whether these alterations could have a role in disease development and/or exacerbation needs further research.

Effects of metal-rich particulate matter exposure on exogenous and endogenous viral sequence methylation in healthy steel-workers.

BACKGROUND: Inhaled particles have been shown to produce systemic changes in DNA methylation. Global hypomethylation has been associated to viral sequence reactivation, possibly linked to the activation of pro-inflammatory pathways occurring after exposure. This observation provides a rationale to investigate viral sequence (both exogenous and endogenous) methylation in association to metal-rich particulate matter exposure. To verify this hypothesis, we chose the Wp promoter of the Epstein-Barr Virus (EBV-Wp) and the promoter of the human-endogenous-retrovirus w (HERV-w), respectively as a paradigm of an exogenous and an endogenous retroviral sequence, to be investigated by bisulfite PCR Pyrosequencing. We enrolled 63 male workers in an electric furnace steel plant, exposed to high level of metal-rich particulate matter. RESULTS: Comparing samples obtained in the first day of a work week (time 0-baseline, after 2 days off work) and the samples obtained after 3 days of work (time 1-post exposure), the mean methylation of EBV-Wp was significantly higher at baseline compared to post-exposure (meanbaseline = 56.7%5mC; meanpost-exposure = 47.9%5mC; p-value = 0.009), whereas the mean methylation of HERV-w did not significantly differ. Individual exposure to inhalable particles and metals was estimated based on measures in all working areas and time spent by the study subjects in each area. In a regression model adjusted for age, body mass index and smoking, PM and metal components had a positive association with EBV-Wp methylation (i.e. PM10: ß = 5.99, p-value < 0.038; nickel: ß = 17.82, p-value = 0.02; arsenic: ß = 13.59, p-value < 0.015). CONCLUSIONS: The difference observed comparing baseline and post-exposure samples may be suggestive of a rapid change in EBV methylation induced by air particles, while correlation between EBV methylation and PM/metal exposure may represent a more stable adaptive mechanism. Future studies investigating a larger panel of viral sequences could better elucidate possible mechanisms and their role in pro-inflammatory pathways leading to systemic health effects.