ATP-P2X7 signaling mediates brain pathology while contributing to viral control in perinatal Zika virus infection.

Zika virus (ZIKV), the causative agent of Zika fever, is a flavivirus transmitted by mosquitoes of the Aedes genus. Zika virus infection has become an international concern due to its association with severe neurological complications such as fetal microcephaly. Viral infection can induce the release of ATP in the extracellular environment, activating receptors sensitized by extracellular nucleotides, such as the P2X7 receptor. This receptor is the primary purinergic receptor involved in neuroinflammation, neurodegeneration, and immunity. In this work, we investigated the role of ATP-P2X7 receptor signaling in Zika-related brain abnormalities. Wild-type mice (WT) and P2X7 receptor-deficient (P2X7-/-) C57BL/6 newborn mice were subcutaneously inoculated with 5 × 106plaque-forming units of ZIKV or mock solution. P2X7 receptor expression increased in the brain of Zika virus-infected mice compared to the mock group. Comparative analyses of the hippocampi from WT and P2X7-/-mice revealed that the P2X7 receptor increased hippocampal damage in CA1/CA2 and CA3 regions. Doublecortin expression decreased significantly in the brains of ZIKV-infected mice. WT ZIKV-infected mice showed impaired motor performance compared to P2X7-/- infected mice. WT ZIKV-infected animals showed increased expression of glial markers GFAP (astrocytes) and IBA-1 (microglia) compared to P2X7-/- infected mice. Although the P2X7 receptor contributes to neuronal loss and neuroinflammation, WT mice were more efficient in controlling the viral load in the brain than P2X7 receptor-deficient mice. This result was associated with higher induction of TNF-α, IFN-ß, and increased interferon-stimulated gene expression in WT mice than P2X7-/-ZIKV-infected. Finally, we found that the P2X7 receptor contributes to inhibiting the neuroprotective signaling pathway AKT/mTOR while stimulating the caspase-3 activation, possibly two distinct pathways contributing to neurodegeneration. These findings suggest that ATP-P2X7 receptor signaling contributes to the antiviral response in the brain of ZIKV-infected mice while increasing neuronal loss, neuroinflammation, and related brain abnormalities.

Long-term recruitment of peripheral immune cells to brain scars after a neonatal insult.

Although brain scars in adults have been extensively studied, there is less data available regarding scar formation during the neonatal period, and the involvement of peripheral immune cells in this process remains unexplored in neonates. Using a murine model of neonatal hypoxic-ischemic encephalopathy (HIE) and confocal microscopy, we characterized the scarring process and examined the recruitment of peripheral immune cells to cortical and hippocampal scars for up to 1 year post-insult. Regional differences in scar formation were observed, including the presence of reticular fibrotic networks in the cortex and perivascular fibrosis in the hippocampus. We identified chemokines with chronically elevated levels in both regions and demonstrated, through a parabiosis-based strategy, the recruitment of lymphocytes, neutrophils, and monocyte-derived macrophages to the scars several weeks after the neonatal insult. After 1 year, however, neutrophils and lymphocytes were absent from the scars. Our data indicate that peripheral immune cells are transient components of HIE-induced brain scars, opening up new possibilities for late therapeutic interventions.

Hyperacute transplantation of umbilical cord mesenchymal stromal cells in a model of severe intracerebral hemorrhage.

Aim: Intracerebral hemorrhage (ICH) has limited therapeutic options. We have shown that an intravenous injection of human umbilical cord-derived mesenchymal stromal cells (hUC-MSC) 24 h after an ICH in rats reduced the residual hematoma volume after a moderate hemorrhage but was inefficient in severe ICH. Here, we investigated whether a treatment in the hyperacute phase would be more effective in severe ICH. Materials & methods: Wistar rats were randomly selected to receive an intravenous injection of hUC-MSC or the vehicle 1 h after a severe ICH. Results: The hyperacute treatment with hUC-MSC did not affect the 22-day survival rate, the motor function or the residual hematoma volume. Conclusion: These results indicate the need for optimization of hUC-MSC-based therapies for severe ICH.

Subacute AMD3100 Treatment Is Not Efficient in Neonatal Hypoxic-Ischemic Rats.

BACKGROUND AND PURPOSE: Despite the advances in treating neonatal hypoxic-ischemic encephalopathy (HIE) with induced hypothermia, the rates of severe disability are still high among survivors. Preclinical studies have indicated that cell therapies with hematopoietic stem/progenitor cells could improve neurological outcomes in HIE. In this study, we investigated whether the administration of AMD3100, a CXCR4 antagonist that mobilizes hematopoietic stem/progenitor cells into the circulation, has therapeutic effects in HIE. METHODS: P10 Wistar rats of both sexes were subjected to right common carotid artery occlusion or sham procedure, and then were exposed to hypoxia for 120 minutes. Two subcutaneous injections of AMD3100 or vehicle were given on the third and fourth day after HIE. We first assessed the interindividual variability in brain atrophy after experimental HIE and vehicle treatment in a small cohort of rats. Based on this exploratory analysis, we designed and conducted an experiment to test the efficacy of AMD3100. Brain atrophy on day 21 after HIE was defined as the primary end point. Secondary efficacy end points were cognitive (T-water maze) and motor function (rotarod) on days 17 and 18 after HIE, respectively. RESULTS: AMD3100 did not decrease the brain atrophy in animals of either sex. Cognitive impairments were not observed in the T-water maze, but male hypoxic-ischemic animals exhibited motor coordination deficits on the rotarod, which were not improved by AMD3100. A separate analysis combining data from animals of both sexes also revealed no evidence of the effectiveness of AMD3100 treatment. CONCLUSIONS: These results indicate that the subacute treatment with AMD3100 does not improve structural and functional outcomes in a rat HIE model.

GD3 synthase deletion alters retinal structure and impairs visual function in mice.

Gangliosides are glycosphingolipids abundantly expressed in the vertebrate nervous system, and are classified into a-, b-, or c-series according to the number of sialic acid residues. The enzyme GD3 synthase converts GM3 (an a-series ganglioside) into GD3, a b-series ganglioside highly expressed in the developing and adult retina. The present study evaluated the visual system of GD3 synthase knockout mice (GD3s-/- ), morphologically and functionally. The absence of b- series gangliosides in the retinas of knockout animals was confirmed by mass spectrometry imaging, which also indicated an accumulation of a-series gangliosides, such as GM3. Retinal ganglion cell (RGC) density was significantly reduced in GD3s-/- mice, with a similar reduction in the number of axons in the optic nerve. Knockout animals also showed a 15% reduction in the number of photoreceptor nuclei, but no difference in the bipolar cells. The area occupied by GFAP-positive glial cells was smaller in GD3s-/- retinas, but the number of microglial cells/macrophages did not change. In addition to the morphological alterations, a 30% reduction in light responsiveness was detected through quantification of pS6-expressing RGC, an indicator of neural activity. Furthermore, electroretinography (ERG) indicated a significant reduction in RGC and photoreceptor electrical activity in GD3s-/- mice, as indicated by scotopic ERG and pattern ERG (PERG) amplitudes. Finally, evaluation of the optomotor response demonstrated that GD3s-/- mice have reduced visual acuity and contrast sensitivity. These results suggest that b-series gangliosides play a critical role in regulating the structure and function of the mouse visual system.

Intracerebral Injection of Heme Induces Lipid Peroxidation, Neuroinflammation, and Sensorimotor Deficits.

Background and Purpose: Heme is a red blood cell component released in the brain parenchyma following intracerebral hemorrhage. However, the study of the pathophysiological mechanisms triggered by heme in the brain is hampered by the lack of well-established in vivo models of intracerebral heme injection. This study aims to optimize and characterize a protocol of intrastriatal heme injection in mice, with a focus on the induction of lipid peroxidation, neuroinflammation and, ultimately, sensorimotor deficits. We also evaluated the involvement of NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), an inflammasome sensor, in the behavior deficits induced by heme in this model. Methods: Mice were injected with heme in the striatum for the evaluation of neuroinflammation and brain damage through histological and biochemical techniques. Immunoblot was used to evaluate the expression of proteins involved in heme/iron metabolism and antioxidant responses and the activation of the MAPK (mitogen-activated protein kinase) signaling pathway. For the assessment of neurological function, we followed-up heme-injected mice for 2 weeks using the rotarod, elevated body swing, and cylinder tests. Mice injected with the vehicle (sham), or autologous blood were used as controls. Results: Heme induced lipid peroxidation and inflammation in the brain. Moreover, heme increased the expression of HO-1 (heme oxygenase-1), ferritin, p62, and superoxide dismutase 2, and activated the MAPK signaling pathway promoting pro-IL (interleukin)-1ß production and its cleavage to the active form. Heme-injected mice exhibited signs of brain damage and reactive astrogliosis around the injection site. Behavior deficits were observed after heme or autologous blood injection in comparison to sham-operated controls. In addition, behavior deficits and IL-1ß production were reduced in Nlrp3 knockout mice in comparison to wild-type mice. Conclusions: Our results show that intracerebral heme injection induces neuroinflammation, and neurological deficits, in an NLRP3-dependent manner, suggesting that this is a feasible model to evaluate the role of heme in neurological disorders.

Human Wharton's jelly mesenchymal stem cells protect neural cells from oxidative stress through paracrine mechanisms.

AIM: Mesenchymal stem cells (MSCs) have neuroprotective and immunomodulatory properties, which are partly mediated by extracellular vesicles (EVs) secretion. We aimed to evaluate the effects of human Wharton's jelly-derived MSCs (WJ-MSCs) and their EVs on rat hippocampal cultures subjected to hydrogen peroxide (H2O2). MATERIALS & METHODS: Hippocampal dissociated cultures were either co-cultured with WJ-MSCs or treated with their EVs prior to H2O2 exposure and reactive oxygen species levels and cell viability were evaluated. RESULTS: Coculture with WJ-MSCs or pre-incubation with EVs prior to the insult reduced reactive oxygen species after H2O2 exposure. Cell viability was improved only when coculture was maintained following the insult, while EVs did not significantly improve cell viability. CONCLUSION: WJ-MSCs have potential antioxidant and neuroprotective effects on hippocampal cultures which might be partially mediated by EVs.

Therapeutic Benefit of the Association of Lodenafil with Mesenchymal Stem Cells on Hypoxia-induced Pulmonary Hypertension in Rats.

Pulmonary arterial hypertension (PAH) is characterized by the remodeling of pulmonary arteries, with an increased pulmonary arterial pressure and right ventricle (RV) overload. This work investigated the benefit of the association of human umbilical cord mesenchymal stem cells (hMSCs) with lodenafil, a phosphodiesterase-5 inhibitor, in an animal model of PAH. Male Wistar rats were exposed to hypoxia (10% O2) for three weeks plus a weekly i.p. injection of a vascular endothelial growth factor receptor inhibitor (SU5416, 20 mg/kg, SuHx). After confirmation of PAH, animals received intravenous injection of 5.105 hMSCs or vehicle, followed by oral treatment with lodenafil carbonate (10 mg/kg/day) for 14 days. The ratio between pulmonary artery acceleration time and RV ejection time reduced from 0.42 ± 0.01 (control) to 0.24 ± 0.01 in the SuHx group, which was not altered by lodenafil alone but was recovered to 0.31 ± 0.01 when administered in association with hMSCs. RV afterload was confirmed in the SuHx group with an increased RV systolic pressure (mmHg) of 52.1 ± 8.8 normalized to 29.6 ± 2.2 after treatment with the association. Treatment with hMSCs + lodenafil reversed RV hypertrophy, fibrosis and interstitial cell infiltration in the SuHx group. Combined therapy of lodenafil and hMSCs may be a strategy for PAH treatment.

Erratum to "Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists".

[This corrects the article DOI: 10.1155/2019/7692973.].

New Benzofuran N-Acylhydrazone Reduces Cardiovascular Dysfunction in Obese Rats by Blocking TNF-Alpha Synthesis.

INTRODUCTION: Diabetic obese patients are susceptible to the development of cardiovascular disease, including hypertension and cardiac dysfunction culminating in diabetic cardiomyopathy (DC), which represents a life-threatening health problem with increased rates of morbidity and mortality. The aim of the study is to characterize the effects of a new benzofuran N-acylhydrazone compound, LASSBio-2090, on metabolic and cardiovascular alterations in Zucker diabetic fatty (ZDF) rats presenting DC. METHODS: Male non-diabetic lean Zucker rats (ZL) and ZDF rats treated with vehicle (dimethylsulfoxide) or LASSBio-2090 were used in this study. Metabolic parameters, cardiovascular function, left ventricle histology and inflammatory protein expression were analyzed in the experimental groups. RESULTS: LASSBio-2090 administration in ZDF rats reduced glucose levels to 85.0 ± 1.7 mg/dL (p < 0.05). LASSBio-2090 also lowered the cholesterol and triglyceride levels from 177.8 ± 31.2 to 104.8 ± 5.3 mg/dL and from 123.0 ± 11.4 to 90.9 ± 4.8 mg/dL, respectively, in obese diabetic rats (p < 0.05). LASSBio-2090 normalized plasma insulin, insulin sensitivity and endothelial function in aortas from diabetic animals (p < 0.05). It also enhanced systolic and diastolic left-ventricular function and reverted myocardial remodeling by blocking the threefold elevation of TNF-α levels in hearts from ZDF rats. CONCLUSION: LASSBio-2090 alleviates metabolic disturbance and cardiomyopathy in an obese and diabetic rat model, thus representing a novel strategy for the treatment of cardiovascular complications in obesity-associated type 2 diabetes mellitus.

Gut Microbiota in Acute Ischemic Stroke: From Pathophysiology to Therapeutic Implications.

The microbiota-gut-brain axis is considered a central regulator of the immune system after acute ischemic stroke (AIS), with a potential role in determining outcome. Several pathways are involved in the evolution of gut microbiota dysbiosis after AIS. Brain-gut and gut-brain signaling pathways involve bidirectional communication between the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, the enteric nervous system, and the immune cells of the gut. Alterations in gut microbiome can be a risk factor and may also lead to AIS. Both risk factors for AIS and gut-microbiome composition are influenced by similar factors, including diabetes, hypertension, hyperlipidemia, obesity, and vascular dysfunction. Furthermore, the systemic inflammatory response after AIS may yield liver, renal, respiratory, gastrointestinal, and cardiovascular impairment, including the multiple organ dysfunction syndrome. This review focus on biochemical, immunological, and neuroanatomical modulation of gut microbiota and its possible systemic harmful effects after AIS, as well as the role of ischemic stroke on microbiota composition. Finally, we highlight the role of gut microbiota as a potential novel therapeutic target in acute ischemic stroke.

Intravenous Human Umbilical Cord-Derived Mesenchymal Stromal Cell Administration in Models of Moderate and Severe Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is as a life-threatening condition that can occur in young adults, often causing long-term disability. Recent preclinical data suggest mesenchymal stromal cell (MSC)-based therapies as promising options to minimize brain damage after ICH. However, therapeutic evidence and mechanistic insights are still limited, particularly when compared with other disorders such as ischemic stroke. Herein, we employed a model of collagenase-induced ICH in young adult rats to investigate the potential therapeutic effects of an intravenous injection of human umbilical cord Wharton's jelly-derived MSCs (hUC-MSCs). Two doses of collagenase were used to cause moderate or severe hemorrhages. Magnetic resonance imaging showed that animals treated with hUC-MSCs after moderate ICH had smaller residual hematoma volumes than vehicle-treated rats, whereas the cell therapy failed to decrease the hematoma volume in animals with a severe ICH. Functional assessments (rotarod and elevated body swing tests) were performed for up to 21 days after ICH. Enduring neurological impairments were seen only in animals subjected to severe ICH, but the cell therapy did not induce statistically significant improvements in the functional recovery. The biodistribution of Technetium-99m-labeled hUC-MSCs was also evaluated, showing that most cells were found in organs such as the spleen and lungs 24 h after transplantation. Nevertheless, it was possible to detect a weak signal in the brain, which was higher in the ipsilateral hemisphere of rats subjected to a severe ICH. These data indicate that hUC-MSCs have moderately beneficial effects in cases of less severe brain hemorrhages in rats by decreasing the residual hematoma volume, and that optimization of the therapy is still necessary.

Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are dynamic cells that can sense the environment, adapting their regulatory functions to different conditions. Accordingly, the therapeutic potential of BM-MSCs can be modulated by preconditioning strategies aimed at modifying their paracrine action. Although rat BM-MSCs (rBM-MSCs) have been widely tested in preclinical research, most preconditioning studies have employed human and mouse BM-MSCs. Herein, we investigated whether rBM-MSCs modify their phenotype and paracrine functions in response to Toll-like receptor (TLR) agonists. The data showed that rBM-MSCs expressed TLR3, TLR4, and MDA5 mRNA and were able to internalize polyinosinic-polycytidylic acid (Poly(I:C)), a TLR3/MDA5 agonist. rBM-MSCs were then stimulated with Poly(I:C) or with lipopolysaccharide (LPS, a TLR4 agonist) for 1 h and were grown under normal culture conditions. LPS or Poly(I:C) stimulation did not affect the viability or the morphology of rBM-MSCs and did not modify the expression pattern of key cell surface markers. Poly(I:C) did not induce statistically significant changes in the release of several inflammatory mediators and VEGF by rBM-MSCs, although it tended to increase IL-6 and MCP-1 secretion, whereas LPS increased the release of IL-6, MCP-1, and VEGF, three factors that were constitutively secreted by unstimulated cells. The neurotrophic activity of the conditioned medium from unstimulated and LPS-preconditioned rBM-MSCs was investigated using dorsal root ganglion explants, showing that soluble factors produced by unstimulated and LPS-preconditioned rBM-MSCs can stimulate neurite outgrowth similarly, in a VEGF-dependent manner. LPS-preconditioned cells, however, were slightly more efficient in increasing the number of regrowing axons in a model of sciatic nerve transection in rats. In conclusion, LPS preconditioning boosted the production of constitutively secreted factors by rBM-MSCs, without changing their mesenchymal identity, an effect that requires further investigation in exploratory preclinical studies.

Review of Preclinical and Clinical Studies of Bone Marrow-Derived Cell Therapies for Intracerebral Hemorrhage.

Stroke is the second leading cause of mortality worldwide, causing millions of deaths annually, and is also a major cause of disability-adjusted life years. Hemorrhagic stroke accounts for approximately 10 to 27% of all cases and has a fatality rate of about 50% in the first 30 days, with limited treatment possibilities. In the past two decades, the therapeutic potential of bone marrow-derived cells (particularly mesenchymal stem cells and mononuclear cells) has been intensively investigated in preclinical models of different neurological diseases, including models of intracerebral hemorrhage and subarachnoid hemorrhage. More recently, clinical studies, most of them small, unblinded, and nonrandomized, have suggested that the therapy with bone marrow-derived cells is safe and feasible in patients with ischemic or hemorrhagic stroke. This review discusses the available evidence on the use of bone marrow-derived cells to treat hemorrhagic strokes. Distinctive properties of animal studies are analyzed, including study design, cell dose, administration route, therapeutic time window, and possible mechanisms of action. Furthermore, clinical trials are also reviewed and discussed, with the objective of improving future studies in the field.

Radiopharmaceutical stem cell tracking for neurological diseases.

Although neurological ailments continue to be some of the main causes of disease burden in the world, current therapies such as pharmacological agents have limited potential in the restoration of neural functions. Cell therapies, firstly applied to treat different hematological diseases, are now being investigated in preclinical and clinical studies for neurological illnesses. However, the potential applications and mechanisms for such treatments are still poorly comprehended and are the focus of permanent research. In this setting, noninvasive in vivo imaging allows better understanding of several aspects of stem cell therapies. Amongst the various methods available, radioisotope cell labeling has become one of the most promising since it permits tracking of cells after injection by different routes to investigate their biodistribution. A significant increase in the number of studies utilizing this method has occurred in the last years. Here, we review the different radiopharmaceuticals, imaging techniques, and findings of the preclinical and clinical reports published up to now. Moreover, we discuss the limitations and future applications of radioisotope cell labeling in the field of cell transplantation for neurological diseases.

The impact of Ly6Clow monocytes after cerebral hypoxia-ischemia in adult mice.

After an ischemic stroke, mononuclear phagocytic cells such as microglia, macrophages, and monocytes migrate to the lesion site and coordinate an immune response. Monocytes, which are recruited from the bloodstream after ischemic brain injury, can be categorized into two subsets in mice: inflammatory and patrolling monocytes. Although inflammatory monocytes (Ly6C(hi)) seem to have a protective role in stroke progression, the impact of patrolling monocytes (Ly6C(low)) is unknown. To address the role of Ly6C(low) monocytes in stroke, we generated bone marrow chimeric mice in which their hematopoietic system was replaced by Nr4a1(-/-) cells, allowing the complete and permanent ablation of Ly6C(low) monocytes without affecting the Ly6C(hi) subset. We then subjected adult mice to cerebral hypoxia-ischemia using the Levine/Vannucci model. Functional outcomes after stroke such as body weight change, neurologic score, motor functions and spatial learning were not affected. Moreover, depletion in Ly6C(low) monocytes did not change significantly the total infarct size, cell loss, atrophy, the number, or the activation state of microglia/macrophages at the lesion site. These data suggest that Ly6C(low) patrolling monocytes are redundant in the progression and recovery of ischemic stroke.

The rise of cell therapy trials for stroke: review of published and registered studies.

Stroke is the second leading cause of death and the third leading cause of disability worldwide. Approximately 16 million first-ever strokes occur each year, leading to nearly 6 million deaths. Nevertheless, currently, very few therapeutic options are available. Cell therapies have been applied successfully in different hematological diseases, and are currently being investigated for treating ischemic heart disease, with promising results. Recent preclinical studies have indicated that cell therapies may provide structural and functional benefits after stroke. However, the effects of these treatments are not yet fully understood and are the subject of continuing investigation. Meanwhile, different clinical trials for stroke, the majority of them small, nonrandomized, and uncontrolled, have been reported, and their results indicate that cell therapy seems safe and feasible in these conditions. In the last 2 years, the number of published and registered trials has dramatically increased. Here, we review the main findings available in the field, with emphasis on the clinical results. Moreover, we address some of the questions that have been raised to date, to improve future studies.