Canine glioblastoma-derived extracellular vesicles as precise carriers for glioblastoma imaging: Targeting across the blood-brain barrier.

The treatment of glioblastoma (GBM) faces significant challenges due to the difficulty of delivering drugs through the blood-brain barrier (BBB). Extracellular vesicles (EVs) have emerged as potential carriers for targeted drug delivery to brain tumors. However, their use and distribution in the presence of an intact BBB and their ability to target GBM tissue are still under investigation. This study explored the use of EVs for GBM targeting across the BBB. Canine plasma EVs from healthy dogs and dogs with glioma were isolated, characterized, and loaded with diagnostic agents. Biodistribution studies were conducted in healthy murine models and a novel intranasal model that preserved BBB integrity while initiating early-stage GBM growth. This model assessed EVs' potential for delivering the contrast agent gadoteric acid to intracranial tumors. Imaging techniques, such as bioluminescence and MRI, confirmed EVs' targeting and delivery capabilities thus revealing a selective accumulation of canine glioma-derived EVs in brain tissue under physiological conditions. In the model of brain tumor, MRI experiments demonstrated the ability of EVs to accumulate gadoteric acid within GBM to enhance contrast of the tumoral mass, even when BBB integrity is maintained. This study underscores the potential of EVs derived from glioma for the targeted delivery of drugs to glioblastoma. EVs from dogs with glioma showed capacity to traverse the BBB and selectively accumulate within the brain tumor. Overall, this research represents a foundation for the application of autologous EVs to precision glioblastoma treatment, addressing the challenge of BBB penetration and targeting specificity in brain cancer therapy.

Montelukast, an available and safe anti-asthmatic drug, prevents maladaptive remodelling and maintains cardiac functionality following myocardial infarction.

Preclinical and clinical data indicate that the 5-lipoxygenase pathway becomes activated in cardiovascular diseases suggesting an important role of CysLTs in atherosclerosis and in its ischemic complications. This study aims to investigate the effects of montelukast, a CysLTR-1 antagonist, in a mouse model of myocardial infarction (MI). C57BL/6N female mice were subjected to coronary artery ligation and received montelukast (10 mg/kg/day, intraperitoneal) or vehicle. Montelukast exerted beneficial effects in the infarcted area, decreasing mRNA expression of inflammatory genes, such Il1ß and Ccl2 (p < 0.05), at 48 h after MI, and reducing infarct size and preventing ischemic wall thinning (p < 0.05) at 4 weeks. Furthermore, montelukast counteracted maladaptive remodelling of whole heart. Indeed, montelukast reduced LV mass (p < 0.05) and remote wall thickening (p < 0.05), and improved cardiac pumping function, as evidenced by increased global ejection fraction (p < 0.01), and regional contractility in infarcted (p < 0.05) and in remote non-infarcted (p < 0.05) myocardium. Finally, montelukast prevented cardiomyocytes hypertrophy (p < 0.05) in remote myocardium, reducing the phosphorylation of GSK3ß, a regulator of hypertrophic pathway (p < 0.05). Our data strongly demonstrate the ability of montelukast to contrast the MI-induced maladaptive conditions, thus sustaining cardiac contractility. The results provide evidences for montelukast "repurposing" in cardiovascular diseases and in particular in myocardial infarction.

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.

Neural Correlates of Esophageal Speech: An fMRI Pilot Study.

OBJECTIVES: The esophageal speech is one of the possible alaryngeal voices resulting after total laryngectomy. Its production is made by the regurgitation of the air coming from the esophagus, sonorized through the passage from the walls of the upper esophageal sphincter. The neural correlates of this voice have never been investigated, while the neural control of laryngeal voice has been already documented by different studies. METHODS: Four patients using esophageal speech after total laryngectomy and four healthy controls underwent functional magnetic resonance imaging. The fMRI experiment was carried out using a "Block Design Paradigm." RESULTS: Comparison of the phonation task in the two groups revealed higher brain activities in the cingulate gyrus, the cerebellum and the medulla as well as lower brain activities in the precentral gyrus, the inferior and middle frontal gyrus and the superior temporal gyrus in the laryngectomized group. CONCLUSIONS: The findings in this pilot study provide insight into neural phonation control in laryngectomized patients with esophageal speech. The imaging results demonstrated that in patients with esophageal speech, altered brain activities can be observed. The adaptive changes in the brain following laryngectomy reflect the changes in the body and in the voice modality. In addition, this pilot study establishes that a blocked design fMRI is sensitive enough to define a neural network associated with esophageal voice and lays the foundation for further studies in this field.

Cerebral derailment after myocardial infarct: mechanisms and effects of the signaling from the ischemic heart to brain.

Myocardial infarction (MI) is the leading cause of death among ischemic heart diseases and is associated with several long-term cardiovascular complications, such as angina, re-infarction, arrhythmias, and heart failure. However, MI is frequently accompanied by non-cardiovascular multiple comorbidities, including brain disorders such as stroke, anxiety, depression, and cognitive impairment. Accumulating experimental and clinical evidence suggests a causal relationship between MI and stroke, but the precise underlying mechanisms have not yet been elucidated. Indeed, the risk of stroke remains a current challenge in patients with MI, in spite of the improvement of medical treatment among this patient population has reduced the risk of stroke. In this review, the effects of the signaling from the ischemic heart to the brain, such as neuroinflammation, neuronal apoptosis, and neurogenesis, and the possible actors mediating these effects, such as systemic inflammation, immunoresponse, extracellular vesicles, and microRNAs, are discussed.

Nuclear Receptors in Myocardial and Cerebral Ischemia-Mechanisms of Action and Therapeutic Strategies.

Nearly 18 million people died from cardiovascular diseases in 2019, of these 85% were due to heart attack and stroke. The available therapies although efficacious, have narrow therapeutic window and long list of contraindications. Therefore, there is still an urgent need to find novel molecular targets that could protect the brain and heart against ischemia without evoking major side effects. Nuclear receptors are one of the promising targets for anti-ischemic drugs. Modulation of estrogen receptors (ERs) and peroxisome proliferator-activated receptors (PPARs) by their ligands is known to exert neuro-, and cardioprotective effects through anti-apoptotic, anti-inflammatory or anti-oxidant action. Recently, it has been shown that the expression of aryl hydrocarbon receptor (AhR) is strongly increased after brain or heart ischemia and evokes an activation of apoptosis or inflammation in injury site. We hypothesize that activation of ERs and PPARs and inhibition of AhR signaling pathways could be a promising strategy to protect the heart and the brain against ischemia. In this Review, we will discuss currently available knowledge on the mechanisms of action of ERs, PPARs and AhR in experimental models of stroke and myocardial infarction and future perspectives to use them as novel targets in cardiovascular diseases.

PCSK9 deficiency rewires heart metabolism and drives heart failure with preserved ejection fraction.

AIMS: PCSK9 is secreted into the circulation, mainly by the liver, and interacts with low-density lipoprotein receptor (LDLR) homologous and non-homologous receptors, including CD36, thus favouring their intracellular degradation. As PCSK9 deficiency increases the expression of lipids and lipoprotein receptors, thus contributing to cellular lipid accumulation, we investigated whether this could affect heart metabolism and function. METHODS AND RESULTS: Wild-type (WT), Pcsk9 KO, Liver conditional Pcsk9 KO and Pcsk9/Ldlr double KO male mice were fed for 20 weeks with a standard fat diet and then exercise resistance, muscle strength, and heart characteristics were evaluated. Pcsk9 KO presented reduced running resistance coupled to echocardiographic abnormalities suggestive of heart failure with preserved ejection fraction (HFpEF). Heart mitochondrial activity, following maximal coupled and uncoupled respiration, was reduced in Pcsk9 KO mice compared to WT mice and was coupled to major changes in cardiac metabolism together with increased expression of LDLR and CD36 and with lipid accumulation. A similar phenotype was observed in Pcsk9/Ldlr DKO, thus excluding a contribution for LDLR to cardiac impairment observed in Pcsk9 KO mice. Heart function profiling of the liver selective Pcsk9 KO model further excluded the involvement of circulating PCSK9 in the development of HFpEF, pointing to a possible role locally produced PCSK9. Concordantly, carriers of the R46L loss-of-function variant for PCSK9 presented increased left ventricular mass but similar ejection fraction compared to matched control subjects. CONCLUSION: PCSK9 deficiency impacts cardiac lipid metabolism in an LDLR independent manner and contributes to the development of HFpEF.

Towards bio-compatible magnetic nanoparticles: Immune-related effects, in-vitro internalization, and in-vivo bio-distribution of zwitterionic ferrite nanoparticles with unexpected renal clearance.

HYPOTHESIS: Iron oxide and other ferrite nanoparticles have not yet found widespread application in the medical field since the translation process faces several big hurdles. The incomplete knowledge of the interactions between nanoparticles and living organisms is an unfavorable factor. This complex subject should be made simpler by synthesizing magnetic nanoparticles with good physical (relaxivity) and chemical (colloidal stability, anti-fouling) properties and no biological activity (no immune-related effects, minimal internalization, fast clearance). Such an innocent scaffold is the main aim of the present paper. We systematically searched for it within the class of small-to-medium size ferrite nanoparticles coated by small (zwitter)ionic ligands. Once established, it can be functionalized to achieve targeting, drug delivery, etc. and the observed biological effects will be traced back to the functional molecules only, as the nanosized scaffold is innocent. EXPERIMENTS: We synthesized nine types of magnetic nanoparticles by systematic variation of core composition, size, coating. We investigated their physico-chemical properties and interaction with serum proteins, phagocytic microglial cells, and a human model of inflammation and studied their biodistribution and clearance in healthy mice. The nanoparticles have good magnetic properties and their surface charge is determined by the preferential adsorption of anions. All nanoparticle types can be considered as immunologically safe, an indispensable pre-requisite for medical applications in humans. All but one type display low internalization by microglial BV2 cells, a process strongly affected by the nanoparticle size. Both small (3 nm) and medium size (11 nm) zwitterionic nanoparticles are in part captured by the mononuclear phagocyte system (liver and spleen) and in part rapidly (≈1 h) excreted through the urinary system of mice. FINDINGS: The latter result questions the universality of the accepted size threshold for the renal clearance of nanoparticles (5.5 nm). We suggest that it depends on the nature of the circulating particles. Renal filterability of medium-size magnetic nanoparticles is appealing because they share with small nanoparticles the decreased accumulation-related toxicity while performing better as magnetic diagnostic/therapeutic agents thanks to their larger magnetic moment. In conclusion, many of our nanoparticle types are a bio-compatible innocent scaffold with unexpectedly favorable clearance.

Microglial vesicles improve post-stroke recovery by preventing immune cell senescence and favoring oligodendrogenesis.

Contrasting myelin damage through the generation of new myelinating oligodendrocytes represents a promising approach to promote functional recovery after stroke. Here, we asked whether activation of microglia and monocyte-derived macrophages affects the regenerative process sustained by G protein-coupled receptor 17 (GPR17)-expressing oligodendrocyte precursor cells (OPCs), a subpopulation of OPCs specifically reacting to ischemic injury. GPR17-iCreERT2:CAG-eGFP reporter mice were employed to trace the fate of GPR17-expressing OPCs, labeled by the green fluorescent protein (GFP), after permanent middle cerebral artery occlusion. By microglia/macrophages pharmacological depletion studies, we show that innate immune cells favor GFP+ OPC reaction and limit myelin damage early after injury, whereas they lose their pro-resolving capacity and acquire a dystrophic "senescent-like" phenotype at later stages. Intracerebral infusion of regenerative microglia-derived extracellular vesicles (EVs) restores protective microglia/macrophages functions, limiting their senescence during the post-stroke phase, and enhances the maturation of GFP+ OPCs at lesion borders, resulting in ameliorated neurological functionality. In vitro experiments show that EV-carried transmembrane tumor necrosis factor (tmTNF) mediates the pro-differentiating effects on OPCs, with future implications for regenerative therapies.

Impact of BDNF Val66Met Polymorphism on Myocardial Infarction: Exploring the Macrophage Phenotype.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin growth factor family, well known for its role in the homeostasis of the cardiovascular system. Recently, the human BDNF Val66Met single nucleotide polymorphism has been associated with the increased propensity for arterial thrombosis related to acute myocardial infarction (AMI). Using cardiac magnetic resonance imaging and immunohistochemistry analyses, we showed that homozygous mice carrying the human BDNF Val66Met polymorphism (BDNFMet/Met) undergoing left anterior descending (LAD) coronary artery ligation display an adverse cardiac remodeling compared to wild-type (BDNFVal/Val). Interestingly, we observed a persistent presence of pro-inflammatory M1-like macrophages and a reduced accumulation of reparative-like phenotype macrophages (M2-like) in the infarcted heart of mutant mice. Further qPCR analyses showed that BDNFMet/Met peritoneal macrophages are more pro-inflammatory and have a higher migratory ability compared to BDNFVal/Val ones. Finally, macrophages differentiated from circulating monocytes isolated from BDNFMet/Met patients with coronary heart disease displayed the same pro-inflammatory characteristics of the murine ones. In conclusion, the BDNF Val66Met polymorphism predisposes to adverse cardiac remodeling after myocardial infarction in a mouse model and affects macrophage phenotype in both humans and mice. These results provide a new cellular mechanism by which this human BDNF genetic variant could influence cardiovascular disease.

Repurposing of drugs approved for cardiovascular diseases: Opportunity or mirage?

Drug repurposing is a promising way in drug discovery to identify new therapeutic uses -different from the original medical indication- for existing drugs. It has many advantages over traditional approaches to de novo drug discovery, since it can significantly reduce healthcare costs and development timeline. In this review, we discuss the possible repurposing of drugs approved for cardiovascular diseases, such as ß-blockers, angiotensin converting enzyme inhibitors (ACE-Is), angiotensin II receptor blockers (ARBs), statins, aspirin, cardiac glycosides and low-molecular-weight heparins (LMWHs). Indeed, numerous experimental and epidemiological studies have reported promising anti-cancer activities for these drugs. It is worth mentioning, however, that the results of these studies are often controversial and very few data were obtained by controlled prospective clinical trials. Therefore, no final conclusion has yet been reached in this area and no final recommendations can be made. Moreover, ß-blockers, ARBs and statins showed promising results in randomised controlled trials (RCTs) where pathological conditions other than cancer were considered. The results obtained have led or may lead to new indications for these drugs. For each drug or class of drugs, the potential molecular mechanisms of action justifying repurposing, results obtained in vitro and in animal models and data from epidemiological and randomized studies are described.

Drug repurposing in cardiovascular diseases: Opportunity or hopeless dream?

Cardiovascular disease remains - despite the development of new drugs, devices, and therapeutic strategies - the leading cause of death and disability worldwide. There is therefore a great need to implement the pharmacological armamentarium, considering also the need to balance the therapeutic and the side effects. Furthermore, the best choice among the drug treatment options and reduction of side effects remain urgent problems for studies of cardiovascular disease. In this context, drug repurposing could be an innovative way and opportunity to extend and improve pharmacological tools. Indeed, applying well-established drugs and compounds to new indications, drug repurposing has already been proven efficient and safe in humans. Furthermore, this approach generates lower costs and needs shorter time for approval than the development of a de novo drug. In the current review, we discuss the main evidence for the repurposing in cardiovascular diseases of drugs approved and marketed for other pathologies by reviewing their mechanisms of action and the results reported in observational and then in randomized studies.

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.

Improvement of fiber connectivity and functional recovery after stroke by montelukast, an available and safe anti-asthmatic drug.

Stroke is one of the main causes of death, neurological dysfunctions or disability in elderly. Neuroprotective drugs have been proposed to improve long-term recovery after stroke, but failed to reach clinical effectiveness. Hence, recent studies suggested that restorative therapies should combine neuroprotection and remyelination. Montelukast, an anti-asthmatic drug, was shown to exert neuroprotection in animal models of CNS injuries, but its ability to affect oligodendrocytes, restoring fiber connectivity, remains to be determined. In this study, we evaluated whether montelukast induces long-term repair by promoting fiber connectivity up to 8 weeks after middle cerebral artery occlusion (MCAo), using different experimental approaches such as in vivo diffusion magnetic resonance imaging (MRI), electrophysiological techniques, ex vivo diffusion tensor imaging (DTI)-based fiber tracking and immunohistochemistry. We found that, in parallel with a reduced evolution of ischemic lesion and atrophy, montelukast increased the DTI-derived axial diffusivity and number of myelin fibers, the density of myelin binding protein (MBP) and the number of GSTpi+ mature oligodendrocytes. Together with the rescue of MCAo-induced impairments of local field potentials in ischemic cortex, the data suggest that montelukast may improve fibers reorganization. Thus, to ascertain whether this effect involved changes of oligodendrocyte precursor cells (OPCs) activation and maturation, we used the reporter GPR17iCreERT2:CAG-eGreen florescent protein (GFP) mice that allowed us to trace the fate of OPCs throughout animal's life. Our results showed that montelukast enhanced the OPC recruitment and proliferation at acute phase, and increased their differentiation to mature oligodendrocytes at chronic phase after MCAo. Considering the crosstalk between OPCs and microglia has been widely reported in the context of demyelinating insults, we also assessed microglia activation. We observed that montelukast influenced the phenotype of microglial cells, increasing the number of M2 polarized microglia/macrophages, over the M1 phenotype, at acute phase after MCAo. In conclusion, we demonstrated that montelukast improves fiber re-organization and long-term functional recovery after brain ischemia, enhancing recruitment and maturation of OPCs. The present data suggest that montelukast, an already approved drug, could be "repositioned "as a protective drug in stroke acting also on fiber re-organization.

Plasmatic and chamber-specific modulation of cardiac microRNAs in an acute model of DOX-induced cardiotoxicity.

BACKGROUND: Doxorubicin (DOX) is a chemotherapeutic drug limited in its usefulness by an adverse side effect, cardiotoxicity. The mechanisms leading to this detrimental occurrence are not completely clear, and lately many authors focused their attention on the possible role of microRNAs (miRNAs), small regulators of cardiovascular functions, in this phenomenon. Notably, these molecules recently emerged also as potential circulating biomarkers of several cardiac diseases. Thus, the aim of this study was the simultaneous investigation of circulating and cardiac tissue miRNAs expression upon DOX treatment in vivo. METHODS: Twenty C57BL/6 female mice were administered with 24 mg/Kg cumulative dose of DOX or saline (CTRL) for 2 weeks. Echocardiography was performed at baseline and at the end of treatment (T1). Plasma and heart samples were collected at T1, separating atria from left (LV) and right (RV) ventricles, and miRNAs expression was tested by RT-qPCR-based arrays. All putatively DOX-regulated candidates were then validated by single assays in vivo and then evaluated also in murine immortalized cardiomyocytes (HL-1) treated with 1 µM DOX for 24 h. In the end, bioinformatics target prediction was performed for all DOX-miRNAs. RESULTS: Cardiotoxicity onset was diagnosed upon impairment of six cardiac functional parameters in DOX-treated mice at T1. Samples collection, followed by screening and validation steps, identified eleven miRNAs dysregulated by the drug in plasma, while seven resulted as altered in separate heart chambers. Interestingly, miR-34a-5p and miR-451a showed a dysregulation in both plasma and tissue samples of DOX-administered animals, whereas five additional miRNAs presented chamber specific modulation. Of note, in vitro experiments showed a very modest overlap with in vivo results. Bioinformatics prediction analysis performed on miR-34a-5p and miR-451a identified several putative targets presenting no significant association with cardiotoxicity. Anyhow, the same analyses, conducted by combining all miRNAs regulated by DOX in each heart chamber, evidenced a possible dysregulation of the adherens junctions gene network, known to be involved in the onset and progression of dilated cardiomyopathy, an established detrimental side effect of the drug. CONCLUSIONS: This is the first work investigating miRNAs regulation by DOX both in plasma and heart districts of treated animals. Our results indicate a strong association of miR-34a-5p and miR-451a to DOX-induced cardiotoxicity. In addition, the observed altered expression of diverse miRNAs in separated cardiac chambers hints at a specific response to the drug, implying the existence of different players and pathways leading to dysfunction onset.

Identification of new molecular targets for PET imaging of the microglial anti-inflammatory activation state.

Microglia are potential targets for therapeutic intervention in neurological and neurodegenerative diseases affecting the central nervous system. In order to assess the efficacy of therapies aimed to reduce the tissue damaging activities of microglia and/or to promote the protective potential of these cells, suitable pre-clinical and clinical tools for the in vivo analysis of microglia activities and dynamics are required. The aim of this work was to identify new translational markers of the anti-inflammatory / protective state of microglia for the development of novel PET tracers. Methods: New translational markers of the anti-inflammatory/protective activation state of microglia were selected by bioinformatic approaches and were in vitro and ex vivo validated by qPCR and immunohistochemistry in rodent and human samples. Once a viable marker was identified, a novel PET tracer was developed. This tracer was subsequently confirmed by autoradiography experiments in murine and human brain tissues. Results: Here we provide evidence that P2RY12 expression increases in murine and human microglia following exposure to anti-inflammatory stimuli, and that its expression is modulated in the reparative phase of experimental and clinical stroke. We then synthesized a novel carbon-11 labeled tracer targeting P2RY12, showing increased binding in brain sections of mice treated with IL4, and low binding to brain sections of a murine stroke model and of a stroke patient. Conclusion: This study provides new translational targets for PET tracers for the anti-inflammatory/protective activation state of microglia and shows the potential of a rationale-based approach. It therefore paves the way for the development of novel non-invasive methodologies aimed to monitor the success of therapeutic approaches in various neurological diseases.

Sex-Specific Features of Microglia from Adult Mice.

Sex has a role in the incidence and outcome of neurological illnesses, also influencing the response to treatments. Neuroinflammation is involved in the onset and progression of several neurological diseases, and the fact that estrogens have anti-inflammatory activity suggests that these hormones may be a determinant in the sex-dependent manifestation of brain pathologies. We describe significant differences in the transcriptome of adult male and female microglia, possibly originating from perinatal exposure to sex steroids. Microglia isolated from adult brains maintain the sex-specific features when put in culture or transplanted in the brain of the opposite sex. Female microglia are neuroprotective because they restrict the damage caused by acute focal cerebral ischemia. This study therefore provides insight into a distinct perspective on the mechanisms underscoring a sexual bias in the susceptibility to brain diseases.

Impact of angiotensin-converting enzyme inhibition on platelet tissue factor expression in stroke-prone rats.

OBJECTIVE: Hypertension is a well known risk factor for thrombotic events such as myocardial infarction and stroke. Platelets express tissue factor (TF), the key activator of blood coagulation and thrombus formation. The number of TF-positive platelets increases in pathological conditions characterized by thrombotic complications but whether this occurs in hypertension is unknown. Here we investigated whether platelet TF expression is increased in a hypertensive status through a mechanism acting on megakaryocytes; the phenomenon could be modulated by antihypertensive drug as captopril; angiotensin (AngII) influences platelet TF expression. METHODS: Spontaneously hypertensive stroke prone (SHRSP) rats received standard diet (StD) or a Japanese high-salt permissive diet (JpD). After 3 weeks, JpD animals were randomized to receive captopril or vehicle. Normotensive Wistar Kyoto (WKY) rats were used as controls. Cell-associated TF expression and activity were analyzed by flow cytometry and calibrated automated thrombogram, respectively. RESULTS: Hypertensive StD-SHRSP showed an increased number of TF-positive platelets compared with normotensive WKY. After JpD administration, SHRSP developed severe hypertension and renal damage; the number of TF-positive megakaryocytes significantly increased compared with StD-SHRSP resulting in a higher number of TF-positive platelets with a faster kinetic of thrombin generation. These effects were reverted by captopril. Ex-vivo stimulation of platelets, isolated from normotensive WKY and from healthy individuals, with AngII induced a concentration-dependent increase of surface-associated TF expression. CONCLUSION: The current study shows for the first time that in hypertension the number of TF-positive megakaryocytes increases thus releasing in the circulation more platelets carrying a functionally active TF. AngII stimulates platelets to express TF.

A Specific Circulating MicroRNA Cluster Is Associated to Late Differential Cardiac Response to Doxorubicin-Induced Cardiotoxicity In Vivo.

BACKGROUND: Cardiotoxicity is a detrimental side effect of the anticancer drug doxorubicin (DOX), characterized by progressive heart dysfunction. Circulating microRNAs (miRNAs) are recognized as potential biomarkers of cardiac disease; thus, we aimed to investigate their association with late cardiotoxicity in an animal model of disease. METHODS: Twenty C57BL/6 female mice were administered with 24 mg/kg cumulative dose of DOX or saline during 2 weeks, followed by a recovery period of one month (T42). Echocardiography was performed at baseline and at T42, and plasma samples were collected at T42. The selection of all miRNAs of interest was conducted by literature overview and by screening, followed by RT-qPCR validation. Results. The analysis of cardiac function at T42 evidenced five DOX-treated animals indistinguishable (NoTox) from controls (CTRLs), while four presented heart impairment (Tox). Our analyses identified eight dysfunction-associated plasma miRNAs. In particular, seven miRNAs were found downregulated in comparison to CTRLs, miR-1-3p, miR-122-5p, miR-127-3p, miR-133a-3p, miR-215-5p, miR-455-3-p, and miR-499a-5p. Conversely, miR-34a-5p showed increased levels in Tox plasma samples. Noteworthy, we determined a cluster composed of miR-1-3p, miR-34a-5p, miR-133a-3p, and miR-499a-5p that distinguished with high-accuracy Tox from NoTox mice. CONCLUSION: This is the first study indicating that, similarly to what is observed in patients, DOX-administered animals present a differential cardiac response to treatment. Moreover, our results indicate the presence of specific plasma miRNAs whose expression reflect the presence of cardiac dysfunction in response to drug-induced injury.