Evans Blue Assay Research Articles

Tumor necrosis factor alpha (TNF-α) blockage reduces acute inflammation and delayed wound healing in oral ulcer of rats.

Oral traumatic ulcers (OTU) are common in dental routine, and the control of proinflammatory cytokines, such as the tumor necrosis factor-alpha (TNF-α), may interfere with OTU repair. Our aim was to evaluate the role of TNF-α in the healing process of OTU in rats. Wistar male rats were divided into six groups: a control-group (treated with 0.1mL/kg of saline) and five groups treated with anti-TNF-α infliximab (INF) at 1, 3, 5, 7, and 10mg/kg immediately before OTU production. The animals were weighed (day 0) and euthanized on days 1, 3, 7, 14 and 21 after ulceration. The ulcers were clinically measured, and the mucosa samples were histologically (scores 0-4), histochemically (collagen assay (pircrosirius)), histomorphometrically (cell counting), and immunohistochemically (TNF-α, α-smooth-muscle-actin (α-SMA), monocyte-chemoattractive-protein-1 (MCP-1), interleukin-8 (IL-8), and fibroblast-growth-factor (FGF)) analyzed. The Evans blue assay was used to measure the vascular permeability. ANOVA-1-2-way/Bonferroni, Kruskal-Wallis/Dunn, and correlation analyses were performed (GraphPad Prism 5.0, p < 0.05). High doses of INF reduced the OTU area (p = 0.043), body mass loss (p = 0.023), vascular permeability (p < 0.001), and reduced delayed histologic scores (p < 0.05), polymorphonuclear (p < 0.001) and mononuclear (p < 0.001) cells, blood vessel counting (p = 0.006), and total (p < 0.001), type-I (p = 0.018), and type-III (p < 0.001) collagen. INF treatment reduced TNF-α immunostaining and delayed MPC-1, FGF, and α-SMA expression, with little/none influence in IL-8 immunostaining. TNF-α blockage by INF reduced acute inflammation in OTU but delayed cell migration and wound healing.

Immune activation after intraventricular hemorrhage

ObjectivesIntraventricular hemorrhage (IVH) is a subtype of stroke which has high mortality and morbidity, while comprehensive mechanism investigations and effective therapies are still in great need. Plenty of studies have shown that inflammation after stroke plays a critical role in disease outcomes. However, the inflammation after IVH remains unclear. This study aims to observe the immune response after IVH, thus providing therapeutic targets for IVH treatments. Materials and methodsIVH was induced by autologous blood infusion model in SD rats. Totally 588 rats were assigned either in the sham or IVH group. T2* lesion and hemoglobin quantities, ventricular volume, brain edema, ventricular wall damage, blood-brain-barrier (BBB) continuity and immune response were observed by magnetic resonance image (MRI), hematoxylin-eosin staining (HE), Evans Blue, flow cytometry (FACS), and enzyme-linked immunosorbent assay (ELISA) at baseline, 6 h, 1 d, 3 d, 7 d, 14 d after surgery. ResultsWe found that ventricular volume enlargement occurred hours after IVH and peaked at 3 d after IVH, then mildly reduced till 14 d. Similar changes happened in brain edema, ventricular wall damage and BBB leakage. Immune cells and cytokines in the central nervous system and peripheral blood also increased after IVH and experienced similar trends as ventricular enlargement. T2* lesion and hemoglobin degradation occurred 6 h after IVH and kept decreasing till 14 d after IVH. ConclusionsOur investigation illustrates that immune response exists after IVH, which may have a close relationship with disease outcomes. These results may provide promising immune related clues for mechanism and intervention studies in IVH.

Abstract P3002: Endothelial Trpm2 Deletion Attenuates Ischemic Stroke By Preventing Blood-brain Barrier Disruption

Introduction: Endothelial hyperpermeability is a hallmark of brain damage at the early stage of ischemic stroke. The subsequent blood-brain barrier (BBB) disruption drives the initial pathological changes and aggregates neuronal death. Ca 2+ overload is an important cause of endothelial dysfunction. TRPM2 is a Ca 2+ -permeable ion channel activated by oxidative stress, and oxidative stress is a critical contributor for endothelial hyperpermeability during ischemic stroke. Hypothesis: We hypothesized that TRPM2-mediated Ca 2+ influx plays a key role in causing endothelial hyperpermeability during ischemic stroke. Methods: Specific deletion of Trpm2 in endothelial cells was achieved by crossing Cdh5-cre mice with Trpm2 fl/fl mice. Middle cerebral artery occlusion (MCAO) was performed to simulate ischemic stroke. Tetrazolium chloride and Evans blue assays were used to evaluate infarct size and BBB leakage. Occludin and F4-80 / myeloperoxidase staining were used to detect tight junction degradation and immune cell invasion. Cerebral endothelial cells (CECs) were isolated and cultured. Oxygen-glucose deprivation (OGD) was used to simulate in vitro ischemic conditions. In vitro endothelial leakage assays were used to examine CEC permeability. Rhodamine-123 imaging and Ca 2+ imaging were used to evaluate oxidative stress and Ca 2+ overload in CECs. WB was used to examine CD36 signaling activation and whole-cell current recording was used to detect TRPM2 activation. Results: Specific deletion of Trpm2 in endothelial cells protects mice against MCAO-induced brain injury, which is characterized by reduced infarct size, mitigated plasma extravasation, reduced neutrophil / macrophage invasion and inhibited oxidative stress in the brain. In vitro experiments demonstrated that Trpm2 deletion alleviates ROS production, Ca 2+ overload and endothelial hyperpermeability induced by OGD and CD36 ligand thrombospondin-1 (TSP1). In CECs, activation of CD36 signaling induced by OGD and TSP1 was impaired by TRPM2 knockout. In transfected HEK293T cells, OGD and TSP1 induce the activation of TRPM2 currents in a CD36-dependent manner. Conclusion: Endothelial specific TRPM2 knockout attenuates ischemic stroke by preserving BBB integrity.

High Glucose Aggravates Retinal Endothelial Cell Dysfunction by Activating the RhoA/ROCK1/pMLC/Connexin43 Signaling Pathway.

PurposeThis research aims to explore the mechanism underlying the relationship between RhoA/ROCK signaling and Connexin43 (Cx43) in retinal endothelial cell dysfunction and to evaluate the protective effect of ROCK inhibitors against retinal endothelial cell dysfunction in diabetic retinopathy (DR) models.MethodsTUNEL staining, hematoxylin and eosin staining, a retinal digestion assay, and Evans blue assay were conducted to explore the effect of fasudil in alleviating retinal dysfunction induced by DR. ELISA, the CCK-8 assay, and flow cytometry were conducted to study inflammation, viability, and apoptosis of mouse retinal microvascular endothelial cells treated with high glucose and ROCK inhibitors. The qRT–PCR and Western blotting were used to evaluate the expression of RhoA, ROCK1, ROCK2, MLC, pMLC, and Cx43. Co-immunoprecipitation was used to verify the interaction between pMLC and Cx43. Immunofluorescence and scrape-loading and dye transfer were used to evaluate the expression and function of Cx43.ResultsMarked endothelial cell dysfunction resulting from the activation of RhoA/ROCK1 signaling was found in in vivo and in vitro models of DR. Via interaction with pMLC, which is downstream of RhoA/ROCK1, a significant downregulation of Cx43 was observed in retinal endothelial cells. Treatment with ROCK inhibitors ameliorated retinal endothelial dysfunction in vitro. The ROCK inhibitor, fasudil, significantly alleviated retinal dysfunction as shown by a decrease of retinal acellular capillaries, an improvement of vascular permeability, and a reduction of cell apoptosis in vivo.ConclusionsOur study highlights a novel mechanism that high glucose could activate RhoA/ROCK1/pMLC signaling, which targets the expression and localization of Cx43 and is responsible for cell viability, apoptosis, and inflammation, resulting in retinal endothelial cell injury. ROCK inhibitors markedly ameliorate endothelial cell dysfunction, suggesting their therapeutic potential for diabetic retinopathy.

Ameliorative Effect of a Neoteric Regimen of Catechin plus Cetirizine on Ovalbumin-Induced Allergic Rhinitis in Rats.

Allergic rhinitis (AR) affects 20–50% of the global population. Available treatments are limited by their adverse effects. We investigated the anti-allergic effects of catechin alone and combined with cetirizine against ovalbumin-induced AR. Rats were sensitized with ovalbumin and received catechin (14 days) and then challenged with aerosolized ovalbumin (1%) to determine AR clinical scores. Histamine, histamine release, and histidine decarboxylase (HDC) activity were determined in blood, peritoneal mast cells, and stomachs, respectively. Vascular permeability and safety were assessed using Evans blue leakage and barbiturate-induced sleeping-time assays, respectively. Catechin and cetirizine binding with HDC was investigated by docking and binding energy analyses. The clinical scores of the combination regimen were superior to either drug alone. All treatments reduced vascular leakage, with no effect on barbiturate-induced sleeping time. Only the catechin-treated rats showed reduced histamine levels and HDC activity. Docking studies revealed that catechin has a 1.34-fold higher extra-precision docking score than L-histidine. The binding energy scores for catechin-HDC, L-histidine-HDC, and histamine-HDC were −50.86, −37.64, and −32.27 kcal/mol, respectively. The binding pattern of catechin was comparable to the standard HDC inhibitor, histidine methyl ester, but with higher binding free energy. Catechin binds the catalytic residue S354, unlike cetirizine. The anti-allergic effects of catechin can be explained by HDC inhibition and possible antihistaminic activity.

Protein Profiling of Orthosiphon stamineus Embryogenic Callus using Liquid Chromatography Coupled with Tandem-Mass Spectrometry (LC-MS/MS)

Orthosiphon stamineus is one of the most prominent medicinal herbs widely grown in Southeast Asia. Propagation of O. stamineus using tissue culture technique helps to rapidly produce samples for research purposes, increase the production of secondary metabolites and is considered as one of the strategies in plant genetic improvement. Despite vast information on O. stamineus pharmacological properties, the protein profile of this species is currently underexplored. In the present study, proteins expressed in embryogenic callus developed from O. stamineus leaves in Murashige and Skoog medium supplemented with 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) were identified via Liquid chromatography- tandem mass spectrometry (LC-MS/MS). The presence of embryogenic callus was confirmed with microscope observation and Evan Blue assay. Using this proteomic approach, we identified peptides that represent 22 proteins localized to different cell compartments and involved in various biological and metabolic functions in plants. This finding is useful to improve our understanding of protein functions, thus their future applications could be further explored. Keywords: Embryogenic, Callus, Herb, Orthosiphon stamineus, Proteins

Altered Blood Brain Barrier Permeability and Oxidative Stress in Cntnap2 Knockout Rat Model.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by three core symptoms, specifically impaired social behavior, stereotypic/repetitive behaviors, and sensory/communication deficits. Although the exact pathophysiology of ASD is still unknown, host genetics, oxidative stress, and compromised blood brain barrier (BBB) have been implicated in predisposition to ASD. With regards to genetics, mutations in the genes such as CNTNAP2 have been associated with increased susceptibility of developing ASD. Although some studies observed conflicting results suggesting no association of CNTNAP2 with ASD, other investigations correlated this gene with autism. In addition, CNTNAP2 mediated signaling is generally considered to play a role in neurological disorders due to its critical role in neurodevelopment, neurotransmission, and synaptic plasticity. In this investigation, we studied BBB integrity and oxidative stress in Cntnap2−/− rats. We observed that the BBB permeability was significantly increased in Cntnap2−/− rats compared to littermate wild-type (WT) animals as determined by FITC-dextran and Evans blue assay. High levels of thiobarbituric acid reactive substances and lower amounts of reduced glutathione were observed in brain homogenates of Cntnap2−/− rats, suggesting oxidative stress. Brain sections from Cntnap2−/− rats showed intense inducible nitric oxide synthase immunostaining, which was undetectable in WT animals. Quantification of nitric oxide in brain homogenates revealed significantly high levels in Cntnap2−/− rats compared to the control group. As increased permeability of the BBB and oxidative stress have been observed in ASD individuals, our results suggest that Cntnap2−/− rats have a high construct and face validity and can be explored to develop effective therapeutic modalities.

Loss of Endothelial Cell RIPK3 Leads to Increased Vascular Permeability Following Intestinal Ischemia‐Reperfusion Injury

The receptor‐interacting serine/threonine‐protein kinase 3 (RIPK3) is primarily known for promoting necroptosis, but recent findings suggest that RIPK3 has non‐necroptotic roles as well, particularly in endothelial cells (ECs). Our lab has reported both beneficial and detrimental roles for endothelial RIPK3 during vascular development, which has prompted us to study RIPK3 in the context of different postnatal vascular pathologies. Our current study focuses on roles for endothelial RIPK3 following intestinal ischemia‐reperfusion (I/R) injury in mice. First‐order branches of the superior mesenteric artery that feeds the ileum were clamped for 1 hr to induce ischemia, and clamps were removed subsequently to allow 24 hr reperfusion in tamoxifen‐induced Ripk3;Cdh5(PAC)‐CreERT2 mice (RIPK3ECKO) compared to wildtype littermate control mice (WT) (males and females, 12 weeks old). Unclamped small intestinal regions were considered as sham control in the same mouse. An Evans blue permeability assay was utilized in assessing small intestinal and distal organ vascular permeability. Small intestinal vascular permeability directly downstream of I/R injury was significantly increased in both RIPK3ECKO and WT mice (n=6 mice/group), however permeability was also significantly increased in sham small intestinal regions and in livers of RIPK3ECKO unlike in WT controls (n=6 mice/group). No difference in vascular permeability was observed between RIPK3ECKO and WT mice (n=5 mice/group) at baseline without I/R injury in all organs assessed. However, in uninjured RIPK3ECKO mice we do observe an increase in the CD68+ proinflammatory macrophage marker in the liver and submucosal layer of the small intestine compared to uninjured WT mice (n=5 mice/group). When clodronate liposomes were administered to deplete macrophages and determine whether this CD68+ macrophage population was contributing to the increased permeability in RIPK3ECKO mice following I/R injury, we observed a permeability rescue in the small intestine but not in the liver (n=4‐5 mice/group). This finding suggests that there are organ‐specific differences in macrophage contribution to vascular permeability following I/R injury. Our current studies are evaluating circulating and tissue specific cytokines and endothelial cell adhesion molecules to understand why RIPK3ECKO vasculature becomes more permeable in response to I/R injury. Discovering cellular mechanisms for vascular endothelial RIPK3 following I/R injury could provide novel therapeutic targets to promote vessel recovery after I/R injury.

Remodeling Lymphatic Vessels in Intrinsically Aged Skin on SKH-1 Mouse Using Low Dose 5-aminolevulinic Acid Photodynamic Therapy via VEGF-C/VEGFR3 Pathway

BackgroundDecline of lymphatic vessel (LV) density and function in intrinsically aged skin can lead to harmful substance accumulation and fluid imbalance. Whether it will be improved by low dose ALA-PDT needs to be investigated. AimsTo investigate the effect of low dose ALA-PDT on remodeling LVs in intrinsically aged skin. MethodsLow dose ALA-PDT with 3 sessions were applied on the dorsal skin of intrinsically aged SKH-1 mice (15 months old). Skin biopsies were obtained from young mice (4 months old, Young-control), intrinsically aged mice before PDT (Old-pre-PDT), and after PDT at different time points (Old-PDT-24h, Old-PDT-1w, Old-PDT-4w), and skin phenotypes were evaluated by dermoscopy. The structure of LVs and extracellular matrix were evaluated via immunofluorescence staining and HE. The drainage function of LVs was evaluated by Evans Blue assay in vivo. The expression of Calcium-binding EGF domain-containing protein 1 (CCBE1), VE-cadherin, and the activation of VEGF-C/VEGFR3 signaling pathway were evaluated by ELISA and Western Blot. ResultsThe density of LVs decreased and the lymphatic clearance was significantly delayed in aged skin. Low dose ALA-PDT increased the density of LVs and blood vessels. The clearance of Evans Blue assay showed the drainage function of LVs was improved after PDT treatments in vivo. The VE-cadherin and VEGF-C/VEGFR3 pathway up-regulated in intrinsically aged skin after ALA-PDT treatments. ConclusionsLVs in intrinsically aged skin were remodeled and their function were restored by low dose ALA-PDT via up-regulating the VEGF-C/VEGFR3 pathway and stimulating the expression of VE-cadherin.

Effect of Zhixue Mingmu Formula on Retinal Vasopermeability of Streptozotocin-Induced Diabetic Retinopathy in Rats

Aims: To investigate the efficacy of Zhixue Mingmu Formula (ZXMM), a traditional Chinese herbal compound, for retinal vasopermeability in streptozotocin (STZ)-induced diabetic retinopathy (DR) in rats.&#x0D; Materials and Methods: This experimental study was performed at the Hebei Provincial Key Laboratory of Ophthalmology, Hebei Provincial Eye Hospital within 6 months from January 2021 to June 2021. A total of 70 Sprague-Dawley rats aged 7-8 weeks old and weighing 200-240 g were selected. Animals were categorized into 2 groups:&#x0D; &#x0D; A control group (n = 10): normal rats - receive vehicle.&#x0D; A model group (n = 60): diabetic rats - receive 1% STZ in 0.1 mol/L sodium citrate buffer (45 mg/kg, i.p.).&#x0D; &#x0D; Diabetes was induced via a single intraperitoneal injection of 45 mg/kg STZ. After 72 hours, fasting blood glucose (FBG) levels were measured and FBG ≥16.7 mmol/L was taken as diabetes. After 4 weeks of rearing, 10 normal rats and 50 diabetic rats which were diagnosed with retinopathy by fundus fluorescence angiography were further divided into 6 groups:&#x0D; &#x0D; A normal control group (NG, n=10): normal rats - receive 0.9% normal saline.&#x0D; A model control group (MG, n=10): DR rats - receive 0.9% normal saline.&#x0D; A calcium dobesilate group (CD, n=10): DR rats + calcium dobesilate (135 mg/kg/day, p.o.) daily.&#x0D; A low-dose ZXMM group (LZXMM, n=10): DR rats + low-dose ZXMM (15.3 g/kg/day, p.o.) daily.&#x0D; A moderate-dose ZXMM group (MZXMM, n=10): DR rats + moderate-dose ZXMM (30.6 g/kg/day, p.o.) daily.&#x0D; A high-dose ZXMM group (HZXMM, n=10): DR rats + high-dose ZXMM (45.9 g/kg/day, p.o.) daily.&#x0D; &#x0D; Drug treatments were administered daily for 4 weeks. FBG levels were monitored every 2 weeks. Retinal vasopermeability was measured using an Evans blue assay. Then, serum samples and retinal tissues were harvested to assess the levels of inflammatory factors and the activity of antioxidative enzymes by enzyme-linked immunosorbent assay (ELISA). Retinal hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor-A (VEGF-A) protein levels were evaluated by ELISA.&#x0D; Results: In the MG, FBG levels were significantly (P &lt; 0.001) increased, and retinal vasopermeability was markedly (P &lt; 0.001) elevated compared with those in the NG. After oral administration of ZXMM or calcium dobesilate, the elevated retinal vasopermeability was significantly (P &lt; 0.001) decreased in DR rats. Concurrently, ZXMM treatment significantly (P &lt; 0.05) attenuated the levels of the inflammatory factors interleukin-6 (IL-6) and C-reactive protein (CRP) in serum and ameliorated the activity of the antioxidative enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the retina of DR rats. ZXMM treatment significantly (P &lt; 0.05) decreased HIF-1α and VEGF-A expression in retinal tissues compared with MG treatment. Furthermore, the effects of ZXMM were dose dependent.&#x0D; Conclusion: ZXMM was shown to have a protective effect against retinal vasopermeability in DR comparable to that of calcium dobesilate, and its mechanism may be attributed to inhibition of the downstream pathway of glycaemia-induced hypoxia, inflammation and oxidative stress of retinal tissue.

Sulpiride-induced hyperprolactinaemia increases retinal vasoinhibin and protects against diabetic retinopathy in rats.

Excessive vasopermeability and angiogenesis compromise vision in diabetic macular oedema (DME) and diabetic retinopathy (DR). Vasoinhibin is a fragment of the hormone prolactin (PRL) that inhibits diabetes-induced retinal hypervasopermeability and ischaemia-induced retinal angiogenesis in rodents. Hyperprolactinaemia generated by the dopamine D2 receptor antagonist, levosulpiride, is associated with higher levels of vasoinhibin in the vitreous of patients with DR, implying a beneficial outcome due to vasoinhibin-mediated inhibition of retinal vascular alterations. Here, we tested whether hyperprolactinaemia induced by racemic sulpiride increases intraocular vasoinhibin levels and inhibits retinal hypervasopermeability in diabetic rats. Diabetes was generated with streptozotocin and, 4weeks later, rats were treated for 2weeks with sulpiride or osmotic minipumps delivering PRL. ELISA, Western blot, and Evans blue assay were used to evaluate serum PRL, retinal vasoinhibin, and retinal vasopermeability, respectively. Hyperprolactinaemia in response to sulpiride or exogenous PRL was associated with increased levels of vasoinhibin in the retina and reduced retinal hypervasopermeability. Furthermore, sulpiride decreased retinal haemorrhages in response to the intravitreal administration of vascular endothelial growth factor (VEGF). Neither sulpiride nor exogenous PRL modified blood glucose levels or bodyweight. We conclude that sulpiride-induced hyperprolactinaemia inhibits the diabetes- and VEGF-mediated increase in retinal vasopermeability by promoting the intraocular conversion of endogenous PRL to vasoinhibin. These findings support the therapeutic potential of sulpiride and its levorotatory enantiomer, levosulpiride, against DME and DR.

A new nitronyl nitroxide radical with salicylic acid framework attenuates blood-brain barrier disruption and oxidative stress in a rat model of middle cerebral artery occlusion.

ObjectivesA new nitronyl nitroxide radical with a salicylic acid framework (SANR) has been demonstrated to exert antioxidant effects in the previous study by our team. The current study has assessed the protective effect of SANR on cerebral ischemia and reperfusion (I/R) in rat models.MethodsSprague–Dawley rats were randomly divided into four groups: sham, I/R, 10, and 20 mg/kg SANR + I/R groups. A total of 120 min of middle cerebral artery occlusion (MCAO) caused cerebral ischemia. Survival rates were calculated, and neurological deficits were evaluated by a blinded experimenter. Cerebral infarct area, apoptosis cells, and blood–brain barrier (BBB) leakage were measured by 2,3,5-triphenyltetrazolium chloride staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and Evans blue assay, respectively. Reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and 8-hydroxy-2-deoxyguanosine (8-OHdG) also were detected to assess oxidation damage caused by cerebral I/R.ResultsTreatment with SANR significantly promoted survival of rats with cerebral I/R injury. SANR meliorated neurologic deficit and infarct area, improved BBB permeability, and reduced neuronal apoptosis. SANR also reduced ROS levels and the content of MDA and increased SOD and GSH-Px activity in a dose-dependent manner. Furthermore, SANR could inhibit the expression of 8-OHdG.ConclusionOur results suggested that SANR has a neuroprotective effect against cerebral I/R injury, and its effect mechanism is related to the antioxidant function.

Sufentanil alleviates cerebral ischemia-reperfusion injury by inhibiting inflammation and protecting the blood-brain barrier in rats.

Stroke is a brain system disease with a high fatality rate and disability rate. About 80% of strokes are ischemic strokes. Cerebral ischemia-reperfusion injury (CIRI) caused by ischemic stroke seriously affects the prognosis of stroke patients. The purpose of this study is to investigate the effect of sufentanil (SUF) on CIRI model rats. We used middle cerebral artery occlusion (MCAO) to make the CIRI model in rats and monitored region cerebral blood flow (rCBF) to ensure that blood flow was blocked and recanalized. We used ELISA and RT-PCR to detect the expression of inflammatory factors in rat serum and brain tissue. In addition, we detected the expression of metalloproteinase (MMP) 2, MMP9 and collagen IV in brain tissues and performed Evans blue (EB) assay to determine the permeability of the blood-brain barrier (BBB). Finally, we clarified the apoptosis of brain tissue through the TUNEL staining and the detection of caspase 3, Bcl2 and Bax. Various concentrations of SUF, especially 5, 10 and 25μg/kg of SUF, all alleviated the infarct size, neurological function and brain edema of MCAO rats. SUF pretreatment also effectively reduced the expression of inflammatory cytokines in MCAO rats, including interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF)-α. In addition, SUF also inhibited MMP2 and MMP9 and promoted the expression of collagen IV, indicating that SUF attenuated the destruction of the BBB. SUF also inhibited caspase 3 and Bax rats and promoted Bcl2 in MCAO rats, thus inhibiting cell apoptosis. SUF pretreatment effectively improved the neurological function and cerebral infarction of MCAO rats, inhibited excessive inflammation in rats, protected the BBB, and inhibited cell apoptosis in brain tissue.

Euterpe oleracea fruit (A\xe7ai)-enriched diet suppresses the development of experimental cerebral malaria induced by Plasmodium berghei (ANKA) infection

BackgroundCerebral malaria is one of the most severe complications attributed to protozoal infection by Plasmodium falciparum, gaining prominence in children mortality rates in endemic areas. This condition has a complex pathogenesis associated with behavioral, cognitive and motor sequels in humans and current antimalarial therapies have shown little effect in those aspects. Natural products with antioxidant and anti-inflammatory properties have become a valuable alternative therapeutic option in the treatment of distinct conditions. In this context, this study investigated the neuroprotective effect of Euterpe oleracea (açai) enriched diet during the development of experimental cerebral malaria induced by the inoculation of Swiss albino mice with Plasmodium berghei ANKA strain.MethodsAfter Plasmodium infection, animals were maintained on a feeding with Euterpe oleracea enriched ration and parameters such as survival curve, parasitemia and body weight were routinely monitored. The present study has also evaluated the effect of açai-enriched diet on the blood-brain barrier leakage, histological alterations and neurocognitive impairments in mice developing cerebral malaria.ResultsOur results demonstrate that between 7th–19th day post infection the survival rate of the group treated with açai enriched ration was higher when compared with Plasmodium-infected mice in which 100% of mice died until the 11th days post-infection, demonstrating that açai diet has a protective effect on the survival of infected treated animals. The same was observed in the brain vascular extravasation, where Evans blue dye assays showed significantly less dye extravasation in the brains of Plasmodium-infected mice treated with açai enriched ration, demonstrating more preserved blood-brain barrier integrity. Açai-enriched diet also attenuate the histopathological alterations elicited by Plasmodium berghei infection. We also showed a decrease of the neurological impairments arising from the exposure of cerebral parenchyma in the group treated with açai diet, ameliorating motor and neuropsychiatric changes, analyzed through the SHIRPA protocol.ConclusionWith these results, we conclude that the treatment with açai enriched ration decreased the mortality of infected animals, as well as protected the blood-brain barrier and the neurocognitive deficits in Plasmodium-infected animals.

Noninvasive Measurement of Retinal Microvascular Permeability During Loss of Endothelial Quiescence.

In the retina EC dysfunction and angiogenesis are driven by an altered microenvironment e.g., diabetes, leading to hypoxia and inflammation in the retinal layers, resulting in excessive vascular leakage and growth. The gold standard for measuring blood-retinal barrier permeability in response to disease and or therapy has been the gold standard Evans blue (EB) assay. However, this technique has limitations in vivo, including nonspecific tissue binding and toxicity. Here we describe a novel imaging methodology combining sodium fluorescein fundus angiography (FFA) with mathematical quantification allowing retinal permeability to be noninvasively and accurately measured at multiple time points in the same animal, minimizing animal use in line with the 3Rs framework. In addition, this technique is a nontoxic, high throughput, sensitive, and cost-effective alternative technique to the Evans blue assay. Moreover, this technique can be translated to other species.

RETRACTED ARTICLE: Propofol inhibits oxidative stress injury through the glycogen synthase kinase 3 beta/nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway

Statement of Retraction Ziyin Zhan, BaoFeng Yan, Yuguo Li, Shuo Yang, and Jinfeng Li- 2022, Propofol inhibits oxidative stress injury through the glycogen synthase kinase 3 beta/nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway, Bioengineered 13(1), doi: 10.1080/21655979.2021.2021062 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Reconstituted HDL-apoE3 promotes endothelial cell migration through ID1 and its downstream kinases ERK1/2, AKT and p38 MAPK

IntroductionAtherosclerotic Coronary Artery Disease (ASCAD) is the leading cause of mortality worldwide. Novel therapeutic approaches aiming to improve the atheroprotective functions of High Density Lipoprotein (HDL) include the use of reconstituted HDL forms containing human apolipoprotein A-I (rHDL-apoA-I). Given the strong atheroprotective properties of apolipoprotein E3 (apoE3), rHDL-apoE3 may represent an attractive yet largely unexplored therapeutic agent. ObjectiveTo evaluate the atheroprotective potential of rHDL-apoE3 starting with the unbiased assessment of global transcriptome effects and focusing on endothelial cell (EC) migration as a critical process in re-endothelialization and atherosclerosis prevention. The cellular, molecular and functional effects of rHDL-apoE3 on EC migration-associated pathways were assessed, as well as the potential translatability of these findings in vivo. MethodsHuman Aortic ECs (HAEC) were treated with rHDL-apoE3 and total RNA was analyzed by whole genome microarrays. Expression and phosphorylation changes of key EC migration-associated molecules were validated by qRT-PCR and Western blot analysis in primary HAEC, Human Coronary Artery ECs (HCAEC) and the human EA.hy926 EC line. The capacity of rHDL-apoE3 to stimulate EC migration was assessed by wound healing and transwell migration assays. The contribution of MEK1/2, PI3K and the transcription factor ID1 in rHDL-apoE3-induced EC migration and activation of EC migration-related effectors was assessed using specific inhibitors (PD98059: MEK1/2, LY294002: PI3K) and siRNA-mediated gene silencing, respectively. The capacity of rHDL-apoE3 to improve vascular permeability and hypercholesterolemia in vivo was tested in a mouse model of hypercholesterolemia (apoE KO mice) using Evans Blue assays and lipid/lipoprotein analysis in the serum, respectively. ResultsrHDL-apoE3 induced significant expression changes in 198 genes of HAEC mainly involved in re-endothelialization and atherosclerosis-associated functions. The most pronounced effect was observed for EC migration, with 42/198 genes being involved in the following EC migration-related pathways: 1) MEK/ERK, 2) PI3K/AKT/eNOS-MMP2/9, 3) RHO-GTPases, 4) integrin. rHDL-apoE3 induced changes in 24 representative transcripts of these pathways in HAEC, increasing the expression of their key proteins PIK3CG, EFNB2, ID1 and FLT1 in HCAEC and EA.hy926 cells. In addition, rHDL-apoE3 stimulated migration of HCAEC and EA.hy926 cells, and the migration was markedly attenuated in the presence of PD98059 or LY294002. rHDL-apoE3 also increased the phosphorylation of ERK1/2, AKT, eNOS and p38 MAPK in these cells, while PD98059 and LY294002 inhibited rHDL-apoE3-induced phosphorylation of ERK1/2, AKT and p38 MAPK, respectively. LY had no effect on rHDL-apoE3-mediated eNOS phosphorylation. ID1 siRNA markedly decreased EA.hy926 cell migration by inhibiting rHDL-apoE3-triggered ERK1/2 and AKT phosphorylation. Finally, administration of a single dose of rHDL-apoE3 in apoE KO mice markedly improved vascular permeability as demonstrated by the reduced concentration of Evans Blue dye in tissues such as the stomach, the tongue and the urinary bladder and ameliorated hypercholesterolemia. ConclusionsrHDL-apoE3 significantly enhanced EC migration in vitro, predominantly via overexpression of ID1 and subsequent activation of MEK1/2 and PI3K, and their downstream targets ERK1/2, AKT and p38 MAPK, respectively, and improved vascular permeability in vivo. These novel insights into the rHDL-apoE3 functions suggest a potential clinical use to promote re-endothelialization and retard development of atherosclerosis.

MiR-29a-5p Alleviates Traumatic Brain Injury- (TBI-) Induced Permeability Disruption via Regulating NLRP3 Pathway.

Objective Inactivation of NLRP3 inflammasome plays a role in reducing the permeability of endothelial cells and improving blood-brain barrier (BBB) dysfunction following traumatic brain injury (TBI). However, the mechanism controlling NLRP3 inflammasome activation remains unclear. This study is aimed at defining the role of miR-29a-5p in NLRP3 inflammasome activation and permeability of endothelial cells under TBI. Methods The scratch injury model on brain bEnd.3 microvascular endothelial cells was used as in vitro TBI model cells. Effects of miR-29a mimics and inhibitors on TBI model cells were observed by examining their action on FITC, TEER, and protein contents of ZO-1 and occludin, and cell permeability-associated protein. Luciferase reporter assay evaluated miR-29a-5p targeting to NLRP3. ELISA examined of IL-1β and IL-18 levels. miR-29a-5p mimic was injected into TBI mouse and its effect on BBB, indicated by Evans blue (EB) staining assay and cerebral water content, and NLRP3 activation was examined. Results miR-29a-3p and miR-29a-5p mimics decrease the concentration of FITC, and increase TEER and the protein contents of ZO-1 and occludin in TBI model cells. miR-29a-5p silencing disrupted the permeability of mouse bEnd.3 cells. miR-29a-5p targets to NLRP3 through the binding on its 3′UTR and negatively regulates its expression in TBI model cells. NLRP3 inhibition and miR-29a-5p silencing together caused significantly decreased FITC concentration and increased TEER value and release of IL-1β and IL-18. miR-29a-5p mimic alleviated the BBB and cerebral water content and inactivates NLRP3 in the mouse TBI model. Conclusions miR-29a-5p mimics protect TBI-induced increased endothelial cell permeability and BBB dysfunction via suppressing NLRP3 expression and activation.

Cyanidin-3-o-glucoside (C3G) inhibits vascular leakage regulated by microglial activation in early diabetic retinopathy and neovascularization in advanced diabetic retinopathy

ABSTRACT Cyanidin-3-O-glucoside (C3G) is a kind of anthocyanin which shows strong anti-inflammation, anti-tumor and anti-oxidant properties. This paper was designed to explore the potential effects of C3G on diabetic retinopathy (DR). C57BL/6 mice were administrated with streptozotocin (STZ) or vehicle control for the establishment of diabetic models. To simulate hyperglycemia and hypoxia, D-glucose (30 mM) and CoCl2 (200 μm/l) were utilized to treat HRECs, respectively. The migration, invasion, inflammation and tube formation abilities of cells were evaluated with the adoption of wound healing, transwell, ELISA and tube formation assays, respectively. Besides, immunofluorescence staining was utilized to detect proliferation and retinal vessels. Evans blue permeation assay were performed to evaluate the vascular leakage in DR mice. Moreover, western blot and qPCR were used to quantify the mRNA and protein expressions of ionized calcium-binding adapter molecule (Iba)-1 and tight junction proteins. Results showed that C3G alleviated the inflammation, microglial activation and angiogenesis in DR mice. Moreover, the proliferation and inflammation of BV2 cells induced by high glucose (HG) were suppressed by C3G. Evans blue permeation assay demonstrated the potency of C3G in attenuating vascular leakage. In addition, C3G suppressed the migration, invasion and angiogenesis of human retinal endothelial cells (HRECs) DR model in vitro. By confirming the role of C3G in inhibiting vascular leakage regulated by microglia activation in early DR and angiogenesis in advanced DR, this study pointed out the potential of C3G as a therapeutic drug for DR management.

Dimethyl fumarate prevents acute lung injury related cognitive impairment potentially via reducing inflammation

ObjectiveDimethyl fumarate (DMF) has been reported to exert a protective role against diverse lung diseases and cognitive impairment-related diseases. Thus this study aimed to investigate its role on acute lung injury (ALI) and related cognitive impairment in animal model.MethodsC57BL/6 mice were divided into four groups: control group, DMF group, ALI group, and ALI + DMF group. For ALI group, the ALI mice model was created by airway injection of LPS (50 μL, 1 μg/μL); for ALI + DMF group, DMF (dissolved in 0.08% methylcellulose) was treated twice a day for 2 days, and on the third day, mice were injected with LPS for ALI modeling. Mice pre-administered with methylcellulose or DMF without LPS injection (PBS instead) were used as the control group and DMF group, respectively. Morris water maze test was performed before any treatment (0 h) and 6 h after LPS-induction (54 h) to evaluate the cognitive impairment of mice. Next, the brain edema and blood brain barrier (BBB) permeability of ALI mice were assessed by brain water content, Evans blue extravasation and FITC-Dextran uptake assays. In addition, the effect of DMF on the numbers of total cells and neutrophils, protein content in BALF were quantified; the inflammatory factors in BALF, serum, and brain tissues were examined by ELISA, qRT-PCR, and Western blot assays. The effect of DMF on the cognitive impairment-related factor HIF-1α level in lung and brain tissues was also examined by Western blot.ResultsDMF reduced the numbers of total cells, neutrophils and protein content in BALF of ALI mice, inhibited the levels of IL-6, TNF-α and IL-1β in BALF, serum and brain tissues of ALI mice. The protein expressions of p-NF-κB/NF-κB and p-IKBα/IKBα was also suppressed by DMF in ALI mice. Morris water maze test showed that DMF alleviated the cognitive impairment in ALI mice by reducing the escape latency and path length. Moreover, DMF lessened the BBB permeability by decreasing cerebral water content, Evans blue extravasation and FITC-Dextran uptake in ALI mice. The HIF-1α levels in lung and brain tissues of ALI mice were also lessened by DMF.ConclusionIn conclusion, DME had the ability to alleviate the lung injury and cerebral cognitive impairment in ALI model mice. This protective effect partly associated with the suppression of inflammation by DMF.