Ratiometric Mass Spectrometry Imaging for Stain-Free Delineation of Ischemic Tissue and Spatial Profiling of Ischemia-Related Molecular Signatures

Mass spectrometry imaging (MSI) serves as an emerging tool for spatial profiling of metabolic dysfunction in ischemic tissue. Prior to MSI data analysis, commonly used staining methods, e.g., triphenyltetrazole chloride (TTC) staining, need to be implemented on the adjacent tissue for delineating lesion area and evaluating infarction, resulting in extra consumption of the tissue sample as well as morphological mismatch. Here, we propose an in situ ratiometric MSI method for simultaneous demarcation of lesion border and spatial annotation of metabolic and enzymatic signatures in ischemic tissue on identical tissue sections. In this method, the ion abundance ratio of a reactant pair in the TCA cycle, e.g., fumarate to malate, is extracted pixel-by-pixel from an ambient MSI dataset of ischemic tissue and used as a surrogate indicator for metabolic activity of mitochondria to delineate lesion area as if the tissue has been chemically stained. This method is shown to be precise and robust in identifying lesions in brain tissues and tissue samples from different ischemic models including heart, liver, and kidney. Furthermore, the proposed method allows screening and predicting metabolic and enzymatic alterations which are related to mitochondrial dysfunction. Being capable of concurrent lesion identification, in situ metabolomics analysis, and screening of enzymatic alterations, the ratiometric MSI method bears great potential to explore ischemic damages at both metabolic and enzymatic levels in biological research.

Recent Advances in Nanomaterials for Therapy and Diagnosis of Cardiovascular Disease

Cardiovascular diseases (CVDs) are among the world’s widely affected disorders, including ischemia and stroke. Acute Myocardial ischemia (AMI) is a deadly disease caused by irreversible damage to the left ventricular heart tissues. The thromboembolic plaque stops the oxygen supply to the main blood vessels and ventricles. During chronic inflammation, myocardial infarction and free radicals damage stable myocardium, smooth muscles cell, and epithelial cells caused by outer membrane loss and ventricular wall smoothing and dilation. Specially constructed scaffolds made of biological and nanoparticles have been created to shield the left ventricle from further injury and recover ischemic endothelial cells. Preclinical experiments have demonstrated that scaffolds containing growth factors and cells will regenerate ischemic tissue into a stable pericardium in good working order. Various medicinal approaches that treat cardiovascular disease conditions at different stages are discussed in this review article, with biomaterials receiving special attention. This review further addresses the manipulation and manufacturing of biomedical implantable devices using nanomedicine methods and drug delivery principles. The use of graphene and exosomal nanovesicle in cardiovascular therapeutics recently progressed in research studies.

Nanobody-Mediated Inhibition of P2X7 on Microglia Improves Stroke Outcome

BackgroundPrevious studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple brain disease models. However, tools for the selective and efficient targeting of these receptors are scarce. The new development of P2X7-specific nanobodies (nbs) enables us to effectively block the P2X7-channel.MethodsTemporary middle cerebral artery occlusion (tMCAO) in wildtype and P2X7-transgenic mice was used as a model for ischemic stroke. ATP release was assessed in transgenic ATP sensor mice. Stroke size was measured without treatment and after injection of P2X7-specific nbs i.v. and i.c.v. directly before tMCAO-surgery. P2X7-GFP expressing transgenic mice were used to show immunhistochemically P2X7 distribution in the brain. In vitro cultured microglia were used to investigate calcium-influx, pore-formation via DAPI uptake, caspase 1 activation and IL-1b release after incubation with P2X7-specific nbs. ResultsATP sensor mice showed an increase of ATP-release in the ischemic hemisphere compared to the contralateral hemisphere or sham mice up to 24 h after stroke. We could further verify the role of the ATP-P2X7 axis in P2X7-overexpressing mice, which showed significantly greater stroke volumes after 24 h. In vitro experiments with primary microglia cells showed that P2X7-specific nanobodies were capable of dampening the ATP-trigged calcium-influx and formation of membrane pores measured by Fluo4 fluorescence or DAPI uptake. We found a lower caspase 1 activity and a subsequently lower IL-1b release. However, the intravenous (i.v.) injection of P2X7-specific nanobodies compared to isotype controls before the tMCAO-surgery did not result in smaller stroke size compared to isotype controls. As demonstrated by FACS, nbs had only reached brain infiltrating macrophages but not microglia. To reach microglia, we injected the P2X7-spezific nbs or the isotype directly intraventricularly (icv). 30 mg of P2X7-specific nbs proved efficient for microglial targeting, reducing post-stroke microglia activation and stroke size significantly.ConclusionHere, we demonstrate the importance of locally produced ATP for the tissue damage observed in ischemic stroke and we show the potential of icv injected P2X7-specific nbs to reduce ischemic tissue damage.

Infarct Progression in the Early and Late Phases of Acute Ischemic Stroke

Purpose of ReviewTo explore factors associated with infarct progression in the early and late phase of acute ischemic stroke in patients undergoing endovascular therapy.Recent FindingsFollowing ischemic stroke, brain injury can progress at a variable rate, at the expense of “penumbral tissue,” which is the ischemic tissue at risk of infarction. Despite dramatic advances in endovascular stroke therapies with early revascularization in more than 80% of cases, nearly half of patients do not achieve functional independence despite successful recanalization. This is largely attributed to the irreversible damage that is already extensive at the time of revascularization.SummaryThe underlying pathophysiology and determinants of the core infarct progression are complex and multifactorial, depending on a balance between brain energy consumption and collateral perfusion supply. It is crucial to develop creative and individualized theranostics to predict infarct progression and to “freeze” the tissue at risk prior to recanalization.

Self-renewing tissue-resident endothelial-macrophage progenitor cells originate from yolk sac and are a local source of inflammation and neovascularization in postnatal aorta

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these yolk sac-derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic, self-renewing endothelial-macrophage (EndoMac) progenitor cells in postnatal mouse aorta, heart and lung, that are independent of definitive hematopoiesis and derive from a CX3CR1+ and CSF1R+ yolk sac source. These bipotent progenitors are highly proliferative and vasculogenic, contributing to adventitial neovascularization in the aortic wall and forming perfused blood vessels after adoptive transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic, self-renewal and differentiation properties. Our findings demonstrate that tissue-resident EndoMac progenitors participate in local inflammatory and vasculogenic responses by contributing to the renewal and expansion of yolk sac-derived macrophages and endothelial cells postnatally.

Exploring cell membrane water exchange in aquaporin-4-deficient ischemic mouse brain using diffusion-weighted MRI

Background Aquaporin-4 is a membrane channel protein that is highly expressed in brain astrocytes and facilitates the transport of water molecules. It has been suggested that suppression of aquaporin-4 function may be an effective treatment for reducing cellular edema after cerebral infarction. It is therefore important to develop clinically applicable measurement systems to evaluate and better understand the effects of aquaporin-4 suppression on the living body. Methods Animal models of focal cerebral ischemia were created by surgically occluding the middle cerebral artery of wild-type and aquaporin-4 knockout mice, after which multi-b-value multi-diffusion-time diffusion-weighted imaging measurements were performed. Data were analyzed with both the apparent diffusion coefficient (ADC) model and a compartmental water-exchange model. Results ADCs were estimated for five different b value ranges. The ADC of aquaporin-4 knockout mice in the contralateral region was significantly higher than that of wild-type mice for each range. In contrast, aquaporin-4 knockout mice had significantly lower ADC than wild-type mice in ischemic tissue for each b-value range. Genotype-dependent differences in the ADC were particularly significant for the lowest ranges in normal tissue and for the highest ranges in ischemic tissue. The ADCs measured at different diffusion times were significantly different for both genotypes. Fitting of the water-exchange model to the ischemic region data found that the water-exchange time in aquaporin-4 knockout mice was approximately 2.5 times longer than that in wild-type mice. Conclusions Multi-b-value multi-diffusion-time diffusion-weighted imaging may be useful for in vivo research and clinical diagnosis of aquaporin-4-related diseases.

Can immature granulocytes indicate mortality in patients with acute ischemic stroke?

Background & Objective: Stroke is the most common cause of permanent disability and the most important cause of mortality. Acute ischemic stroke (AIS) reveals inflammation in the ischemic brain tissue. Ischemic tissue causes proinflammatory cytokine release and aggregation of immune cells. Therefore in this study, we aimed to investigate the role of immature granulocyte (IG) in showing 30-day mortality in patients with AİS. Methods: This study was designed as a single-centered, retrospective cohort study. Patients aged >18 years who were diagnosed with AIS in the tertiary emergency department were included in this study. Patients were divided into two groups as low (<0.6%) and high (≥0.6%) by IG values. Demographic and laboratory parameters were compared between the groups at admission to the emergency department. Results: Our study consisted of 172 patients diagnosed with AIS, who met the inclusion criteria. The average age of the study group was 69.19 ± 14.34 years, and 94 (54.7%) of the patients were male. 98 (56.9%) patients were in the low IG group, and 74 (43.1%) of them were in the poor outcome group. IG at the cut-off value of 1.3 was shown to predict mortality in patients with AIS with 80.5% sensitivity and 93.2% specificity (area under the curve: 0.715 95% CI: 0.623-0.807, p <0.001) Conclusion: The results of our study showed that IG is a new and simple predictor to predict 30-day prognosis in patients with AIS.

FGF2-primed 3D spheroids producing IL-8 promote therapeutic angiogenesis in murine hindlimb ischemia

Peripheral artery disease is a progressive, devastating disease that leads to critical limb ischemia (CLI). Therapeutic angiogenesis using stem cell therapy has emerged as a promising approach for its treatment; however, adapting cell-based therapy has been limited by poor cell survival and low treatment efficiency. To overcome unmet clinical needs, we developed a fibroblast growth factor 2 (FGF2)-immobilized matrix that enabled control of cell adhesion to the surface and exerted a priming effect on the cell. Human adipose-derived stem cells (hASCs) grown in this matrix formed a functionally enhanced cells spheroid (FECS-Ad) that secreted various angiogenic factors including interleukin-8 (IL-8). We demonstrated that IL-8 was upregulated by the FGF2-mediated priming effect during FECS-Ad formation. Immobilized FGF2 substrate induced stronger IL-8 expression than soluble FGF2 ligands, presumably through the FGFR1/JNK/NF-κB signaling cascade. In IL-8-silenced FECS-Ad, vascular endothelial growth factor (VEGF) expression was decreased and angiogenic potential was reduced. Intramuscular injection of FECS-Ad promoted angiogenesis and muscle regeneration in mouse ischemic tissue, while IL-8 silencing in FECS-Ad inhibited these effects. Taken together, our data demonstrate that IL-8 contributes to therapeutic angiogenesis and suggest that FECS-Ad generated using the MBP-FGF2 matrix might provide a reliable platform for developing therapeutic agents to treat CLI.