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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.

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 mm

Maternal exposure to air pollutants has been associated with pregnancy complications and adverse birth outcomes. Endothelial dysfunction, an imbalance in vascular function, during pregnancy is considered a key element in the development of pre-eclampsia. Environmental exposure to particulate matter (PM) during the first trimester of pregnancy might increase maternal inflammatory status thus affecting fetal growth, possibly leading to preterm delivery.The purpose of the study was to evaluate possible effects of PM295 pregnant women were recruited. Individual PM exposure was assigned to each subject by calculating the mean of PMFor long-term exposure, we observed an increase in sVCAM-1 and a decrease of PAI-1 levels for each 10 μg/mThe present study showed that both long- and short-term exposures to PM are associated with changes in circulating levels of biomarkers in pregnant women reflecting systemic inflammation and endothelial dysfunction/activation. Our findings support the hypothesis that inflammation and endothelial dysfunction might have a central role in modulating the detrimental effects of air pollution exposure during pregnancy.

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.

BACKGROUND: Head down tilt 15° (HDT15°), applied before recanalization, increases collateral flow and improves outcome in experimental ischemic stroke. For its simplicity and low cost, HDT15° holds considerable potential to be developed as an emergency treatment of acute stroke in the pre-hospital setting, where hemorrhagic stroke is the major mimic of ischemic stroke. In this study, we assessed safety of HDT15° in the acute phase of experimental intracerebral hemorrhage. METHODS: Intracerebral hemorrhage was produced by stereotaxic injection of collagenase in Wistar rats. A randomized non-inferiority trial design was used to assign rats to HDT15° or flat position (n=64). HDT15° was applied for 1 hour during the time window of hematoma expansion. The primary outcome was hematoma volume at 24 hours. Secondary outcomes were mass effect, mortality and functional deficit in the main study and acute changes of intracranial pressure, hematoma growth and cardiorespiratory parameters in separate sets of randomized animals (n=32). RESULTS: HDT15° achieved the specified criteria of non-inferiority for hematoma volume at 24 hours. Mass effect, mortality and functional deficit at 24 hours showed no difference in the two groups. HDT15° induced a mild increase in intracranial pressure with respect to the pre-treatment values (+ 2.91 +/- 1.76 mmHg). HDT15° had a neutral effect on MRI-based analysis of hematoma growth and cardiorespiratory parameters. CONCLUSIONS: Application of HDT15° in the hyperacute phase of experimental intracerebral hemorrhage does not worsen early outcome. Further research is needed to implement HDT15° as an emergency collateral therapeutic for acute stroke.

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.

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.

Background: Increasing evidence suggests a possible link between the bacterial nasal microbiota (bNM) to allergic diseases such as allergic Rhinitis (AR), as it might modulate the allergic response by acting on the onset and progression of allergic inflammation. In this case-control study, we aimed to compare the bNM between 25 AR patients and 25 matched healthy subjects (HS) as well as to evaluate possible modifications within the host-microbiota cross-talk, investigating the release pattern of both bacterial- and host-Extracellular Vesicles (EVs).Results: bNM analysis showed that Actinobacteria (AR: 2.5%-83.5%; HS: 18.6%-92.7%), Firmicutes (AR: 6.1%-63.5%; HS: 6.4%-72.2%) and Proteobacteria (AR: 0.6%-89.6%; HS 0.7%-38%) were the most abundant phyla in the study population. Diversity reduction in AR patient bNM was pointed out compared to the HS group bNM (Observed OTUs: p =0.018; PD whole tree: p =0.014). Moreover, the distribution of the weighted normalized intra-group UniFrac distances analysis showed a less uniform bacterial community within AR patients rather than HS group (nonparametric test p =0.01). Since EVs may have a central role in the cross-talk between bNM and the host, we evaluated their size and concentration in the plasma of the study subjects. AR patients were characterized by a higher concentration of EVs ranging between 130-231 nm, 257-287 nm and 323-348 nm (p Conclusion: Our results suggest that AR patients are characterized by a less diverse and wide intra-group variable bNM compared to that of HS, as well as an altered host-microbiota EV communication network. Further studies are needed to disentangle the relationship between the host and the nasal bacterial community during pathological conditions and health.

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.

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: β = 5.99, p-value 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.

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.

Introduction Inhaled particulate pollutants 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. We aimed at evaluating the effects of PM exposure on DNA methylation of the Wp promoter of the Epstein-Barr Virus (EBV-Wp) and the promoter of the human-endogenous-retrovirus w (HERV-w), chosen as a paradigm of an exogenous virus and an endogenous retroviral sequence, in workers in an electric furnace steel plant with well-characterised exposure to metal-rich particulate matter. Methods We measured EBV-Wp and HERV-w DNA methylation through bisulfite PCR Pyrosequencing on peripheral blood leukocytes DNA obtained from 63 male healthy workers, on the first day of a work week (baseline, after 2 days off work) and after 3 days of work (post-exposure). We determined individual exposure to inhalable particles and metals for all subjects. Paired t-test was used to compare baseline and post-exposure samples. Linear mixed models were fitted to evaluate the association between metal-rich particle exposure and DNA methylation. Results Comparing samples obtained at baseline and after 3 days of work, the mean methylation of EBV-Wp was significantly higher at baseline compared to post-exposure (baseline=56.7; postexposure=47.9; p-value=0.009), whereas the mean methylation of HERV-w did not significantly differ. In a regression model adjusted for age, body mass index and smoking, nickel, arsenic and lead had a positive association with EBV-Wp methylation (nickel: β=16.16, p-value Conclusions The difference observed comparing baseline and postexposure samples may be suggestive of a rapid change in EBV methylation induced by air particles, while correlation between EBV methylation and metal exposure may represent an adaptive mechanism that should be further characterised in future investigations.

Background: human microbiota plays a highly physiological role and influences human health. Researches on microbiota structure and functions are increasing, but is still largely unknown whether the airways microbiota is influenced by airborne pollution, and if it play a role in human response to inhaled particulate matter (PM). Aim: we evaluated the effects of PM on respiratory microbiota structure and functions in healthy subjects. Methods: microbial community was examined through metabarcoding analysis of the V3-V4 16S rRNA gene regions amplified from upper-airway tracts of 40 individuals to perform α-diversity and taxonomic studies. Individual PM10 and PM2.5 exposure was estimated by personal sampler (worn for 24 hours before sampling) and integrated with nearest ambient monitoring station values (for mid-term exposures). Results: downstream analysis showed that microbiota composition was negatively associated with PM measured 3 days before sampling (PM-3). PM-3 decreased also the concentration of Actinobacteria (PM10 ∆%-2,1 CI95% -3,8;-0,4 PM2,5 ∆%-2,6 CI95% -4,7;-0,4), which is the dominant phylum in a healthy microbiota, while it sensibly increased the relative concentration of Moraxella genus (PM10 ∆%4,9 CI95% 0,4;9,6 PM2,5 ∆%5,8 CI95% 0,3;11,7), that includes airways-pathogen species. Discussion: this is the first study showing an effect of PM on upper-airway microbiota in healthy subjects. Further investigations are needed to explore the role of the microbiota as intermediate and possible cause of individual susceptibility to inhaled pollutants. In fact, microbiota has the potential to modulate host inflammatory response, to compete with pathogen species and finally to impact on human health.

The principal aim of the INSIDE project (INdividual air pollution exposure, extracellular vescicles SIgnaling and hypertensive disorder DEvelopment in pregnancy) is to assess the molecular effects of environmental exposure to airborne particulate matter (PM) of susceptible subject. Different approaches are considered to evaluate these effects, including an exposure-effect study performed on a selected population. The short-term exposure to different pollutants (PM and NO2) was evaluated considering 51 subjects recruited from October 2017 to April 2018. Each subject was asked to carry personal instruments for few hours before a clinical evaluation (blood and cardiological examination) from home to hospital. Instruments used in the study were: (I) CairClip – CairPol (NO2) and (II) Aerocet 831 - Aerosol Mass Monitor, Met One Instruments (size-fractionated PM). Moreover, a (III) smartphone with a GPS application and a (IV) Time Activity Diary (TAD) were used in this study to acquire information about the microenvironments (MEs) visited by subjects during the monitoring sessions. The experimental design of the project allowed to further investigate issues related to the mode of exposure: through the analysis of TADs and GPS data, it was possible to document the time spent by each subject in the different MEs and characterize the average exposure and inhaled dose associated to different MEs. The microenvironmental inhaled dose of pollutants was estimated considering the average exposure to PM and NO2, the time spent across these MEs and the specific ventilation rate of each subject. Moreover, to understand which of these parameters has the major impact of the dose model, a sensitivity analysis was performed, on the total and on the MEs dataset.