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Objective: To investigate the role of Rho/ROCK signaling pathway in mediating restraint stress-induced blood-brain barrier (BBB) injury in the amygdala of rats., Methods: Sixty male SD rats were randomized equally into control group (with food and water deprivation for 6 h per day), restraint stress group (with restraint for 6 h per day), stress + fasudil treatment (administered by intraperitoneal injection at 1 mg/100 g 30 min before the 6-h restraint) group, and fasudil treatment alone group. The elevated plus-maze test was used to detect behavioral changes of the rats, serum corticosterone and S100B levels were determined with ELISA, and Evans Blue leakage in the brain tissue was examined to evaluate the changes in BBB permeability. The changes in expression levels of tight junction proteins in the amygdala were detected using immunofluorescence assay and Western blotting, and Rho/ROCK pathway activation was detected by Pull-down test and Western blotting. Ultrastructural changes of the cerebral microvascular endothelial cells were observed using transmission electron microscopy., Results: Compared with those in the control group, the rats in restrain stress group and stress+fasudil group showed obvious anxiety-like behavior with significantly increased serum corticosterone level ( P <0.001). Compared with those in the control group and stress+fasudil group, the rat models of restrain stress showed more obvious Evans Blue leakage and higher S100B expression ( P <0.01) but lower expressions of tight junction proteins in the amygdala. Pull-down test and Western blotting confirmed that the expression levels of RhoA-GTP, ROCK2 and P-MLC 2 were significantly higher in stress group than in the control group and stress + fasudil group ( P <0.05). Transmission electron microscopy revealed obvious ultrastructural changes in the cerebral microvascular endothelial cells in the rat models of restrain stress., Conclusion: Restraint stress induces BBB injury in the amygdala of rats by activating the Rho/ROCK signaling pathway.

Background: : Acupuncture, practiced for millennia, lacks a clear anatomical definition for acupoints. A prevailing theory suggests that acupoints overlap with skin areas with higher mast cell density. Skin spots stained with intravenously infused Evans blue (EB), indicative of neurogenic inflammation, have recently been posited as acupoints in rats., Objectives: : To demonstrate the concordance between EB-reactive skin spots and mast cell-enriched acupoints., Methods: : We employed staining and RNA-seq analysis to delineate the morphological characteristics and gene expression profiles of EB-reactive skin spots in rats., Results: : EB infusion revealed a novel nodal structure on the rat skin surface, visible to the naked eye, with dimensions of approximately 1 mm in both diameter and height. Around 30 such nodes were identified on one side of the abdominal area, spaced roughly 3 mm apart, excluding the linea alba. RNA-seq analysis indicated that the gene expression patterns within these nodes markedly differed from both non-nodal skin areas and lymph nodes. Histological examination using toluidine blue revealed a significantly greater mast cell count in the nodes than in non-nodal skin regions. Additionally, the nodes stained positively with Alcian blue and Hemacolor, reagents known to mark primo vascular tissues., Conclusion: : Our findings suggest that EB-reactive nodes are indeed rich in mast cells. Further research is warranted to establish these skin nodes as surface primo nodes.

Aim: To explore the potential role of microRNA miR-221-5p on the angiopoietin-1 (Ang-1)/Ang-2/Tie-2 signaling axis after subarachnoid hemorrhage (SAH) in a rat model., Methods: Aspects of the rat's behavior were measured using the Kaoutzanis scoring system to test neurological responses. This included feeding behavior, body contraction, motor, and eye-opening responses. Brain sections were studied using transmission electron microscopy and Evans blue extravasation. Levels of Ang-1, Ang-2, and Tie-2 were determined by Western blot, while miR-221-5p was quantified using stem-loop real-time quantitative PCR (RT-qPCR)., Results: The SAH group responded worse to the neurological response test than the sham-operated group. The intercellular space was widened in the SAH group, but not in the sham-operated group. Evans blue dye leaked significantly more into brain tissue cells of the SAH group. Stem-loop qRT-PCR showed elevated miR-221-5p levels. Additionally, Ang-1 and Tie-2 were reduced but Ang-2 expression was increased after SAH. This led to a significant reduction of the Ang-1/Ang-2 ratio in the brain tissue, which was associated with the destruction of the blood-brain barrier., Conclusion: The data indicate that miR-221-5p might regulate blood-brain barrier dysfunction through the Ang-1/Ang-2/Tie-2 signaling axis, suggesting that it should be further investigated as a potential novel biomarker.

Human chorionic membrane mesenchymal stem cells (hCM-MSCs) have increasingly emerged as an excellent source of transplanted cells for regenerative therapy as they can be isolated via a non-invasive and simple method with high proliferative capabilities. However, the roles and mechanisms of hCM-MSCs on traumatic brain injury (TBI) animal models have not been investigated yet. The aim of this study was to investigate the therapeutic potential and mechanism of hCM-MSCs transplantation in a rat model of TBI. Adult male Sprague-Dawley rats were subjected to moderate lateral fluid percussion-induced TBI. At 2 h after TBI, hCM-MSCs, or PBS were administered intravenously via the tail vein. Neurological function, brain water content, Evans blue dye extravasation, immunofluorescence staining, and enzyme-linked immunosorbent were evaluated. The results showed that transplanted hCM-MSCs were observed in the injured brain. Compared with the PBS group, hCM-MSCs treatment significantly decreased the numbers of M1 macrophages/microglia, MPO + neutrophils and caspase-3 + cells ( P  < 0.01). Meanwhile, hCM-MSCs treatment significantly reduced the expression levels of the pro-inflammatory cytokines (TNF-α, interleukin-(IL)6 and IL-1β) while increasing the numbers of M2 macrophages/microglia and the expression of the anti-inflammatory cytokines IL-10 ( P  < 0.01). In addition, hCM-MSCs treatment significantly reduced brain water content and Evans blue extravasation. Lastly, hCM-MSCs treatment significantly promoted neurogenesis and angiogenesis, and attenuated neurological deficits. Collectively, these findings indicate that hCM-MSCs exhibited effective therapeutic efficacy in a rat TBI model, and its mechanism may be by reducing inflammation, apoptosis and the blood-brain barrier disruption, promoting angiogenesis and neurogenesis., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Ethnopharmacological Relevant: YangXueQingNaoWan (YXQNW), a compound Chinese medicine, has been widely used for dizziness, irritability, insomnia, and dreaminess caused by blood deficiency and liver hyperactivity in China. However, whether YXQNW can inhibit cerebral microvascular exudation and cerebral hemorrhage (CH) caused by blood brain barrier (BBB) damage after tissue plasminogen activator (tPA) still unknown., Aim of the Research: To observe the effect of YXQNW on cerebral microvascular exudation and CH after tPA and investigate its mechanism in protecting BBB., Materials and Methods: Male C57BL/6 N mice suffered from ischemia stroke by mechanical detachment of carotid artery thrombi with the stimulation of ferric chloride. Then mice were treated with tPA (10 mg/kg) and/or YXQNW (0.72 g/kg) at 4.5 h. Cerebral blood flow (CBF), infarct size, survival rate, neurological scores, gait analysis, Evans blue extravasation, cerebral water content, fluorescein isothiocyanate-labeled albumin leakage, hemorrhage, junction and basement membrane proteins expression, leukocyte adhesion and matrix metalloproteinases (MMPs) expression were evaluated 24 h after tPA. Proteomics was used to identify target proteins., Results: YXQNW inhibited cerebral infarction, neurobehavioral deficits, decreased survival, Evans blue leakage, albumin leakage, cerebral water content and CH after tPA thrombolysis; improved CBF, low-expression and degradation of junction proteins, basement membrane proteins, Arhgap21 and its downstream α-catenin and β-catenin proteins expression; and suppressed the increase of adherent leukocytes and the release of MMP-9 derived from macrophage., Conclusion: YXQNW relieved BBB damage and attenuated cerebral microvascular exudation and CH after tPA thrombolysis. The effect of YXQNW on cerebral microvascular exudation was associated with the inhibition of the low-expression of junction proteins, especially AJs mediated by Rho GTPase-activating protein 21 (Arhgap21), while the effect on CH was associated with the inhibition of leukocyte adhesion, the release of MMP-9 derived from macrophage, and low-expression and degradation of collagen IV and laminin in the vascular basement membrane., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Blood-brain barrier (BBB) dysfunction and hyperpermeability have been implicated in a myriad of brain pathologies. The Evans Blue assay is one of the most popular methods for studying BBB integrity and permeability in rodent models of brain disorders. Under normal physiological conditions, the BBB is impermeable to albumin, so Evans Blue when injected intravenously binds to serum albumin and remains restricted within blood vessels. In traumatic and ischemic injuries, and other brain pathologies that result in BBB hyperpermeability, neighboring endothelial cells partially lose their close contacts to each other, and the BBB becomes permeable to proteins such as albumin. This paracellular leak of Evans blue-bound albumin is considered a reliable indicator of BBB dysfunction and hyperpermeability. Here, we describe the procedures for the evaluation of BBB integrity and hyperpermeability using Evans Blue extravasation assay in a mouse model of traumatic brain injury. The method described here focuses on intravenous injection of Evans Blue followed by Evans Blue dye extraction. This is followed by the measurement of fluorescence intensity of Evans Blue to determine the dye extravasation as a direct indicator of BBB hyperpermeability., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

This study investigated the effects of isosakuranetin (5,7-dihydroxy-4'-methoxyflavanone) on cerebral infarction and blood brain barrier (BBB) damage in cerebral ischemia and reperfusion (I/R) in a rat model. The right middle cerebral artery was occluded for 2 h followed by reperfusion. The experimental rats were divided into five groups: a sham, or control group; vehicle group; and 5 mg/kg, 10 mg/kg, and 20 mg/kg bodyweight isosakuranetin-treated I/R groups. After 24 h of reperfusion, the rats were tested using a six-point neurological function score. The percentage of cerebral infarction was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. BBB leakage was determined by Evan Blue injection assay and brain morphology changes were observed under light microscopy following staining with hematoxylin and eosin (H&E). The results of neurological function score revealed that isosakuranetin reduced the severity of neurological damage. A dose of 10 and 20 mg/kg bodyweight of isosakuranetin significantly decreased the infarct volume. All three doses of isosakuranetin significantly decreased Evan Blue leakage. The penumbra area of the I/R brains revealed the characteristics of apoptotic cell death. Therefore, isosakuranetin-treated I/R attenuated the brain damage from cerebral I/R injury and further investigation of the mechanisms warrant further investigation to assist in the development of protective strategies against cerebral I/R injury in clinical trials.Communicated by Ramaswamy H. Sarma.

Gintonin, newly extracted from ginseng, is a glycoprotein that acts as an exogenous lysophosphatidic acid (LPA) receptor ligand. This study aimed to demonstrate the in vivo preventive effects of gintonin on gastric damage. ICR mice were randomly assigned to five groups: a normal group (received saline, 0.1 mL/10 g, p.o.); a control group (administered 0.3 M HCl/ethanol, 0.1 mL/10 g, p.o.) or indomethacin (30 mg/kg, p.o.); gintonin at two different doses (50 mg/kg or 100 mg/kg, p.o.) with either 0.3 M HCl/ethanol or indomethacin; and a positive control (Ranitidine, 40 mg/kg, p.o.). After gastric ulcer induction, the gastric tissue was examined to calculate the ulcer index. The expression of gastric damage markers, such as tumor necrosis factor (TNF)-α, cyclooxygenase 2 (COX-2), and LPA2 and LPA5 receptors, were measured by Western blotting. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) levels were measured by enzyme-linked immunosorbent assay. The platelet endothelial cell adhesion molecule (PECAM-1), Evans blue, and occludin levels in gastric tissues were measured using immunofluorescence analysis. Both HCl/ethanol- and indomethacin-induced gastric ulcers showed increased TNF-α, IL-6, Evans blue permeation, and PECAM-1, and decreased COX-2, PGE2, occludin, and LPA5 receptor expression levels. However, oral administration of gintonin alleviated the gastric ulcer index induced by HCl/ethanol and indomethacin in a dose-dependent manner. Gintonin suppressed TNF-α and IL-6 expression, but increased COX-2 expression and PGE2 levels in mouse gastric tissues. Gintonin intake also increased LPA5 receptor expression in mouse gastric tissues. These results indicate that gintonin can play a role in gastric protection against gastric damage induced by HCl/ethanol or indomethacin.

Aim: Blood-brain barrier (BBB) dysfunction is one of the hallmarks of ischemic stroke. USP14 has been reported to play a detrimental role in ischemic brain injury. However, the role of USP14 in BBB dysfunction after ischemic stroke is unclear., Methods: In this study, we tested the role of USP14 in disrupting BBB integrity after ischemic stroke. The USP14-specific inhibitor IU1 was injected into middle cerebral artery occlusion (MCAO) mice once a day. The Evans blue (EB) assay and IgG staining were used to assess BBB leakage 3 days after MCAO. FITC-detran test was slected to examine the BBB leakage in vitro. Behavior tests were conducted to evaluate recovery from ischemic stroke., Results: Middle cerebral artery occlusion increased endothelial cell USP14 expression in the brain. Furthermore, the EB assay and IgG staining showed that USP14 inhibition through IU1 injection protected against BBB leakage after MCAO. Analysis of protein expression revealed a reduction in the inflammatory response and chemokine release after IU1 treatment. In addition, IU1 treatment was found to rescue neuronal loss resulting from ischemic stroke. Behavior tests showed a positive effect of IU1 in attenuating brain injury and improving motor function recovery. In vitro study showed that IU1 treatment could alleviate endothelial cell leakage induced by OGD in cultured bend.3 cells through modulating ZO-1 expression., Conclusions: Our results demonstrate a role for USP14 in disrupting the integrity of the BBB and promoting neuroinflammation after MCAO., (© 2023 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Growth in chickens, especially meat-type chickens (broilers), is extremely rapid, but studies on the regulatory mechanism of intestinal glucose absorption with growth are few, contradictory, and unclear. Here, we investigated the regulation of intestinal glucose absorption with growth in broiler chickens using oral glucose gavage, intestinal Evans blue transit, intestinal glucose absorption, scanning electron microscopy, and glucose absorption- and cell junction-related gene expression analyses. Peak blood glucose levels after oral glucose gavage occurred at 10 and 50 min in chickens at 1 wk (C1W) and 5 wk (C5W) of age, respectively. The area under the curve for glucose levels was greater for the C5W than the C1W (P = 0.035). The stain ratio in the small intestine in the C5W was lower than that in the C1W (P = 0.01), but there were no differences in the tissue regions stained with Evans blue and the migration distance of Evans blue from Meckel's diverticulum. In everted sac and Ussing chamber experiments, we observed reduced intestinal glucose uptake and electrogenic glucose absorption in the jejunum of the C5W. Phloridzin, an inhibitor of sodium/glucose cotransporter 1 (SGLT1), suppressed the glucose-induced short-circuit current in the C1W (P = 0.016) but not the C5W. Although the addition of NaCl solution stimulated the glucose-induced short-circuit current in the C1W, no differences between the treatments were observed (P = 0.056), which was also the case in the C5W. Additionally, tissue conductance was diminished in the C5W compared with that in the C1W. Moreover, in the C5W, the intestinal tract was more developed and the jejunal villi were enlarged. In conclusion, glucose absorption throughout the intestine could be greater in C5W than in C1W; however, reduced SGLT1 sensitivity, decreased ion permeability, and intestinal overdevelopment lead to decreased local glucose absorption in the jejunum with growth in broiler chickens. These data provide a detailed analysis of intestinal glucose absorption in growing broiler chickens, and can contribute to the development of novel feeds., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Objectives: This study investigated the role and potential involvement of pulmonary vascular glycocalyx degradation in acute lung injury in rats with severe heatstroke (HS). Methods: Rats in an established HS model were exposed to a heated environment for 60 min in an incubator (temperature, 40°C ± 2°C; humidity, 65% ± 5%). Following pretreatment with heparanase III (HPSE III) or heparin, pathological lung injury, arterial blood gas, alveolar barrier disruption, and hemodynamic changes were evaluated. The vascular endothelial structures of the lungs were examined using electron microscopy. The concentration of Evans blue dye in the lungs and arterial blood gas were assessed. An enzyme-linked immunosorbent assay was used to quantify the plasma concentration of heparan sulfate proteoglycan. The expression of glypican-1 and syndecan-1 in pulmonary vessels was measured using immunofluorescence. Western blots were used to detect the expression of TNF-α, IL-6, and vascular endothelial biomarkers in the rat lungs. Pulmonary apoptosis was assessed using a TUNEL (terminal dUTP nick end labeling) assay, and the concentrations of malondialdehyde were measured. Results: Glycocalyx shedding aggravated lung injuries. Severe histopathological damage was observed, and indexes of lung function deviated from abnormal ranges. In addition, pulmonary vascular endothelial cells were disrupted. Compared with the HS group, the plasma concentration of heparan sulfate proteoglycan significantly increased in the HPSE group ( P < 0.05). The expression of glypican-1 and syndecan-1 decreased, and the extravasation of Evans blue dye increased ( P < 0.01). Endothelial biomarker expression increased in the lung tissue, whereas occludin expression decreased. Moreover, TNF-α and IL-6 were overexpressed following heat stress. Furthermore, apoptosis of pulmonary tissues and the concentration of malondialdehyde in rat lungs increased in the HS and HPSE groups. Conclusions : Heatstroke induced pulmonary glycocalyx degradation, which increased vascular permeability and aggravated vascular endothelial dysfunction, contributing to apoptosis, inflammation, and oxidation in the pulmonary tissues., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 by the Shock Society.)

NADPH oxidases (NOXs) are major players in generating reactive oxygen species (ROS) and are implicated in various neurodegenerative ocular pathologies. The aim of this study was to investigate the role of a NOX4 inhibitor (GLX7013114) in two in vivo, experimental streptozotocin (STZ) paradigms depicting the early events of diabetic retinopathy (DR). Animals in the diabetic treated group received GLX7013114 topically (20 μL/eye, 10 mg/mL, once daily) for 14 days (paradigm A: preventive) and 7 days (paradigm B: treated) at 48 h and 4 weeks after STZ injection, respectively. Several methodologies were used (immunohistochemistry, Western blot, real-time PCR, ELISA, pattern electroretinography [PERG]) to assess the diabetes-induced early events of DR, namely oxidative stress, neurodegeneration, and neuroinflammation, and the effect of GLX7013114 on the diabetic insults. GLX7013114, administered as eye drops (paradigms A and B), was beneficial in treating the oxidative nitrative stress, activation of caspase-3 and micro- and macroglia, and attenuation of neuronal markers. It also attenuated the diabetes-induced increase in vascular endothelial growth factor, Evans blue dye leakage, and proinflammatory cytokine (TNF-α protein, IL-1β/IL-6 mRNA) levels. PERG amplitude values suggested that GLX7013114 protected retinal ganglion cell function (paradigm B). This study provides new findings regarding the pharmacological profile of the novel NOX4 inhibitor GLX7013114 as a promising therapeutic candidate for the treatment of the early stage of DR., Article Highlights: NADPH oxidases (NOXs) are implicated in the early pathological events of diabetic retinopathy (DR). The NOX4 inhibitor GLX7013114, topically administered, reduced oxidative damage and apoptosis in the rat streptozotocin model of DR. GLX7013114 protected retinal neurons and retinal ganglion cell function and reduced the expression of pro-inflammatory cytokines in the diabetic retina. GLX7013114 diminished the diabetes-induced increase in vascular endothelial growth factor levels and Evans blue dye leakage in retinal tissue. GLX7013114 exhibits neuroprotective, anti-inflammatory, and vasculoprotective properties that suggest it may have a role as a putative therapeutic for the early events of DR., (© 2023 by the American Diabetes Association.)

Atopic dermatitis (AD) is an allergic disease mediated by Th2 cells. In AD, externally stimulated keratinocytes release inflammatory cytokines, such as IL-33 and TSLP. Inflammatory cells infiltrate skin tissue and increase vascular permeability. Therefore, we hypothesized that imatinib mesylate (IMT), which suppresses vascular permeability, may be a candidate therapeutic agent for AD. A vitamin D3 analog (MC903) was administered daily to both ears of Balb/c mice to create a murine AD model to which IMT was applied. The skin lesions were evaluated histopathologically and by immunostaining. Cytokine expression in the skin was assessed by using real-time polymerase chain reaction (PCR) and immunostaining and was investigated using Evans Blue to determine whether IMT suppressed vascular permeability due to histamine. The suppressive effect of TNF-α/IL-4-induced TSLP expression in primary mouse keratinocytes (MKCs) treated with IMT was then investigated. Tslp gene and protein expression in the lesion was measured using real-time PCR and ELISA. The activation of signal transduction was analysed by western blotting. Topical application of IMT significantly reduced ear thickness, Evans blue leakage, and scratch onset. IMT suppressed the number of infiltrating cells (CD4+ T cells, eosinophils, and basophils), and the expression of IL-13, IL-33, and TSLP in a MC903-induced, murine AD model and inhibited TNF-α/IL-4-induced TSLP expression via downregulation of ERK phosphorylation in MKCs. IMT reduced the skin symptoms in a MC903-induced, murine AD model, suggesting that it may have potential as a new treatment for AD., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We have previously shown that selective inhibition of histone deacetylase 3 (HDAC3) decreases infarct volume and improves long-term functional outcomes after stroke. In this study, we examined the effects of HDAC3 inhibition on cerebral edema and blood-brain barrier (BBB) leakage and explored its underlying mechanisms. Adult male Wistar rats were subjected to 2-h middle cerebral artery occlusion (MCAO) and randomly treated i.p. with either vehicle or a selective HDAC3 inhibitor (RGFP966) at 2 and 24 h after stroke. Modified neurological severity scores (mNSS) were calculated at 2 h, 1 day, and 3 days. H&E, Evans blue dye (EBD) assay, and fluorescein isothiocyanate (FITC)-dextran were employed to assess cerebral edema and BBB leakage. Western blot for matrix metalloproteinase-9 (MMP9), MMP-9 zymography, and immunostaining for HDAC3, GFAP, Iba-1, albumin, aquaporin-4, claudin-5, ZO-1, and NF-kB were performed. Early RGFP966 administration decreased cerebral edema (p = 0.002) and BBB leakage, as measured by EBD assay, FITC-dextran, and albumin extravasation (p < 0.01). RGFP966 significantly increased tight junction proteins (claudin-5 and ZO-1) in the peri-infarct area. RGFP966 also significantly decreased HDAC3 in GFAP + astrocytes, which correlated with better mNSS (r = 0.67, p = 0.03) and decreased cerebral edema (r = 0.64, p = 0.04). RGFP966 decreased aquaporin-4 in GFAP + astrocytes (p = 0.002), as well as, the inflammatory markers Iba-1, NF-kB, and MMP9 in the ischemic brain (p < 0.05). Early HDAC3 inhibition decreases cerebral edema and BBB leakage. BBB protection by RGFP966 is mediated in part by the upregulation of tight junction proteins, downregulation of aquaporin-4 and HDAC3 in astrocytes, and decreased neuroinflammation., (© 2022. The Author(s).)

Background: Focused ultrasound (FUS) combined with microbubbles (MBs) has emerged as a potential approach for opening the blood-brain barrier (BBB) for delivering drugs into the brain. However, MBs range in size of microns and thus can hardly extravasate into the brain parenchyma. Recently, growing attention has been paid to gas vesicles (GVs), which are genetically encoded gas-filled nanostructures with protein shells, due to their potential for extravascular targeting in ultrasound imaging and therapy. However, the use of GVs as agents for BBB opening has not yet been investigated., Methods: In this study, GVs were extracted and purified from Halobacterium NRC-1 . Ultrasound imaging performance of GVs was assessed in vitro and in vivo. Then, FUS/GVs-mediated BBB opening for small molecular Evans blue or large molecular liposome delivery across the BBB was examined., Results: The results showed a good contrast performance of GVs for brain perfusion ultrasound imaging in vivo. At the acoustic negative pressure of 1.5 MPa, FUS/GVs opened the BBB safely, and effectively enhanced Evans blue and 200-nm liposome delivery into the brain parenchyma., Conclusion: Our study suggests that biosynthetic GVs hold great potential to serve as local BBB-opening agents in the development of new targeted drug delivery strategies for central nervous system disorders., Competing Interests: The authors declare no conflicts of interest in this work., (© 2022 Zhang et al.)

Backgroud: Hyperhomocysteinemia (HHcy) is implicated in various neurovascular disorders including vascular dementia, subarachnoid hemorrhage and stroke. Elevated homocysteine (Hcy) levels are associated with increased oxidative stress and compromised blood-brain barrier (BBB) integrity. Hydrogen sulfide (H 2 S) has recently emerged as potent neuroprotective molecule in various neurological conditions including those associated with HHcy. The present study evaluates the protective effect of sodium hydrogen sulfide (NaHS; a source of H 2 S) on HHcy-induced BBB dysfunction and underpin molecular mechanisms. Materials and methods: Supplementation of NaHS restored the increased BBB permeability in the cortex and hippocampus of HHcy animals assessed in terms of diffused sodium fluorescein and Evans blue tracer dyes in the brain. Activity of matrix metalloproteinases (MMPs) assessed by gelatinase activity and in situ gelatinase assay was restored to the normal in the cortex and hippocampus of HHcy animals supplemented with NaHS. Results: Application of gelatin zymography revealed that specifically MMP-9 activity was increased in the cortex and hippocampus of HHcy animals, which was inhibited by NaHS supplementation. Real-time RT-PCR analysis showed that NaHS administration also decreased mRNA expression of MMP-9 in the hippocampus of HHcy animals. NaHS supplementation was further observed to reduce water retention in the brain regions of Hcy treated animals. Conclusion: Taken together, these findings suggest that NaHS supplementation ameliorates HHcy-induced BBB permeability and brain edema by inhibiting the mRNA expression and activity of MMP-9. Therefore, H 2 S and H 2 S releasing drugs may be used as a novel therapeutic approach to treat HHcy-associated neurovascular disorders.

Histamine is a major neurotransmitter and alleviates neuronal damage after ischemic injury via H 2 receptors. Herein, we investigated the effects of H 2 receptor agonists on the blood-brain barrier (BBB) disruption after traumatic brain injury (TBI). Male ddY mice were used to generate the TBI model, in which a fluid percussion injury (FPI) was induced by a hydraulic impact. The BBB disruption was evaluated using Evans blue extravasation. H 2 receptor agonists, amthamine and dimaprit, were administered into the lateral cerebroventricle (i.c.v.) or tail vein (i.v.) from 3 hours to 3 days after FPI. The i.c.v. or i.v. administration of amthamine and dimaprit reduced FPI-induced Evans blue extravasation and promoted mRNA expression of vascular protective factors, including angiopoietin-1 and sonic hedgehog. The co-administration of ranitidine, a H 2 receptor antagonist, inhibited these effects. Expression of the H 2 receptor was observed in astrocytes and brain microvascular endothelial cells (BMECs) in the injured cortex. Treatment with amthamine and dimaprit promoted mRNA expression of vascular protective factors in astrocytes and BMECs. These results suggest that H 2 receptor agonists alleviate TBI-induced BBB disruption by increasing the expression of vascular protective factors in astrocytes and BMECs., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

The α-synuclein (α-syn) is involved in methamphetamine (METH)-induced neurotoxicity. Neurons can transfer excessive α-syn to neighboring neurons and glial cells. The effects of α-syn aggregation in astrocytes after METH exposure on the blood-brain barrier (BBB) remains unclear. Our previous study demonstrated that nuclear receptor-related protein 1 (Nurr1), a member of the nuclear receptor family widely expressed in the brain, was involved in the process of METH-induced α-syn accumulated in astrocytes to activate neuroinflammation. The role Nurr1 plays in astrocyte-mediated neuroinflammation, which results in BBB injury induced by METH, remains uncertain. This study found that METH up-regulated α-syn expression in neurons extended to astrocytes, thereby eliciting astrocyte activation, increasing and decreasing IL-1β, IL-6, TNF-α, and GDNF levels by down-regulating Nurr1 expression, and ultimately damaging the BBB. Specifically, the permeability of BBB to Evans blue and sodium fluorescein (NaF) increased; IgG deposits in the brain parenchyma increased; the Claudin5, Occludin, and PDGFRβ levels decreased. Several ultrastructural pathological changes occurred in the BBB, such as abnormal cerebral microvascular diameter, astrocyte end-foot swelling, decreased pericyte coverage, and loss of tight junctions. However, knockout or inhibition of α-syn or astrocyte-specific overexpression of Nurr1 partially alleviated these symptoms and BBB injury. Moreover, the in vitro experiments confirmed that METH increased α-syn level in the primary cultured neurons, which could be further transferred to primary cultured astrocytes, resulting in decreased Nurr1 levels. The decreased Nurr1 levels mediated the increase of IL-1β, IL-6, and TNF-α, and the decrease of GDNF, thereby changing the permeability to NaF, transendothelial electrical resistance, and Claudin5 and Occludin levels of primary cultured brain microvascular endothelial cells. Based on our findings, we proposed a new mechanism to elucidate METH-induced BBB injury and presented α-syn and Nurr1 as promising drug intervention targets to reduce BBB injury and resulting neurotoxicity in METH abusers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Primary blast lung injury (PBLI) is a major cause of death in military conflict and terrorist attacks on civilian populations. However, the mechanisms of PBLI are not well understood, and a standardized animal model is urgently needed. This study aimed to establish an animal model of PBLI for laboratory study., Methods: The animal model of PBLI was established using a self-made mini shock tube simulation device. In brief, mice were randomly divided into two groups: the control group and the model group, the model group were suffered 0.5 bar shock pressures. Mice were sacrificed at 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours after injury. Lung tissue gross observation, hematoxylin and eosin staining and lung pathology scoring were performed to evaluated lung tissue damage. Evans blue dye leakage and bronchoalveolar lavage fluid examination were performed to evaluated pulmonary edema. The relative expression levels of inflammation factors were measured by real-time quantitative polymerase chain reaction and Western blotting analysis. The release of neutrophil extracellular traps was observed by immunofluorescence stain., Results: In the model group, the gross observation and hematoxylin and eosin staining assay showed the inflammatory cell infiltration, intra-alveolar hemorrhage, and damaged lung tissue structure. The Evans blue dye and bronchoalveolar lavage fluid examination revealed that the lung tissue permeability and edema was significantly increased after injury. Real-time quantitative polymerase chain reaction and Western blotting assays showed that IL-1β, IL-6, TNF-α were upregulated in the model group. Immunofluorescence assay showed that the level of neutrophil extracellular traps in the lung tissue increased significantly in the model group., Conclusion: The self-made mini shock tube simulation device can be used to establish the animal model of PBLI successfully. Pathological changes of PBLI mice were characterized by mechanical damage and inflammatory response in lung tissue., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Surgery of Trauma.)

Background: Nafamostat mesylate (nafamostat, NM) is an FDA-approved serine protease inhibitor that exerts anti-neuroinflammation and neuroprotective effects following rat spinal cord injury (SCI). However, clinical translation of nafamostat has been limited by an unclear administration time window and mechanism of action., Methods: Time to first dose of nafamostat administration was tested on rats after contusive SCI. The optimal time window of nafamostat was screened by evaluating hindlimb locomotion and electrophysiology. As nafamostat is a serine protease inhibitor known to target thrombin, we used argatroban (Arg), a thrombin-specific inhibitor, as a positive control in the time window experiments. Western blot and immunofluorescence of thrombin expression level and its enzymatic activity were assayed at different time points, as well its receptor, the protease activated receptor 1 (PAR1) and downstream protein matrix metalloproteinase-9 (MMP9). Blood-spinal cord barrier (BSCB) permeability leakage indicator Evans Blue and fibrinogen were analyzed along these time points. The infiltration of peripheral inflammatory cell was observed by immunofluorescence., Results: The optimal administration time window of nafamostat was 2-12 h post-injury. Argatroban, the thrombin-specific inhibitor, had a similar pattern. Thrombin expression peaked at 12 h and returned to normal level at 7 days post-SCI. PAR1, the thrombin receptor, and MMP9 were significantly upregulated after SCI. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). Nafamostat and argatroban significantly downregulated thrombin and MMP9 expression as well as thrombin activity in the spinal cord. Nafamostat inhibited thrombin enrichment in endothelial cells. Nafamostat administration at 2-12 h after SCI inhibited the leakage of Evans Blue in the epicenter and upregulated tight junction proteins (TJPs) expression. Nafamostat administration 8 h post-SCI effectively inhibited the infiltration of peripheral macrophages and neutrophils to the injury site., Conclusions: Our study provides preclinical information of nafamostat about the administration time window of 2-12 h post-injury in contusive SCI. We revealed that nafamostat functions through inhibiting the thrombin-mediated BSCB breakdown and subsequent peripheral immune cells infiltration., (© 2022. The Author(s).)

Pseudoallergic reactions are hypersensitivity reactions mediated by an IgE-independent mechanism. Since allantoin (AT)-mediated pseudoallergy has not been studied, in this study, our objective is to investigate the anti-pseudoallergy effect of AT and its underlying mechanism. In vitro, β-hexosaminidase (β-Hex) and histamine (HIS) release assays, inflammatory cytokine assays, toluidine blue staining, and F-actin microfilament staining were used to evaluate the inhibitory effect of AT in RBL-2H3 cells stimulated with Compound 48/80 (C48/80). Western blot analysis is further performed to investigate intracellular calcium fluctuation-related signaling pathways. In vivo, Evans Blue extraction, paw swelling, and the diameter of Evans Blue extravasation were evaluated, and skin tissues are examined for histopathological examination in mice with passive cutaneous anaphylaxis (PCA) induced by C48/80. Body temperature is measured, and the levels of cytokines are further determined by ELISA kits in mice with active systemic anaphylaxis (ASA) induced by C48/80. The results show that AT dose-dependently inhibited degranulation in C48/80-stimulated RBL-2H3 cells by inhibiting β-Hex and HIS release, reducing the levels of TNF-α, IL-8, and MCP-1, inhibiting shape changes due to degranulation and disassembling the F-actin cytoskeleton. Furthermore, AT dose-dependently inhibits the phosphorylation of PLCγ and IP3R. In vivo, AT decreased Evans Blue extravasation, paw swelling, and the diameter of Evans Blue extravasation and significantly ameliorate pathological changes and mast cell degranulation in C48/80-induced PCA. Furthermore, AT help the mice recover from the C48/80-induced decrease in body temperature and decreased the levels of cytokines in C48/80-treated ASA mice. Our results indicate that allantoin inhibits compound 48/80-induced pseudoallergic reactions. AT has the potential to be used in IgE-independent anti-allergic and anti-inflammatory therapies.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), obtained from diet and dietary supplements, have been tested in clinical trials for the prevention or treatment of several diseases. n-3 PUFAs exert their effects by activation of free fatty acid (FFA) receptors. FFA1 receptor, expressed in the pancreas and brain, is activated by medium- to long-chain fatty acids. Despite some beneficial effects on cognition, the effects of n-3 PUFAs on the blood-brain barrier (BBB) are not clearly understood. We examined the effects of FFA1 activation on BBB permeability in vitro, using rat brain microvascular endothelial cells (RBMVEC), and in vivo, by assessing Evans Blue extravasation and by performing live imaging of brain microcirculation in adult rats. AMG837, a synthetic FFA1 agonist, produced a dose-dependent decrease in RBMVEC monolayer resistance assessed with Electric Cell-Substrate Impedance Sensing (ECIS); the effect was attenuated by the FFA1 antagonist, GW1100. Immunofluorescence studies revealed that AMG837 produced a disruption in tight and adherens junction proteins. AMG837 increased Evans Blue content in the rat brain in a dose-dependent manner. Live imaging studies of rat brain microcirculation with miniaturized fluorescence microscopy (miniscope) showed that AMG837 increased extravasation of sodium fluorescein. Taken together, our results demonstrate that FFA1 receptor activation reduced RBMVEC barrier function and produced a transient increase in BBB permeability.

Cerebral edema and elevated intracranial pressure (ICP) are common complications observed following ischemic stroke. Osmotherapy has been used as a foundation to manage ICP induced by cerebral edema, and albumin is one of the most commonly used osmotic agents. The present study aimed to explore whether albumin lowered ICP by reducing cerebral edema when albumin elevated the colloid osmotic pressure (COP) of plasma. Sprague‑Dawley rats that underwent middle cerebral artery occlusion were used to assess COP and ICP. Magnetic resonance imaging measurements were performed to evaluate cerebral edema and infarct size. Evans blue was used to assess the blood‑brain barrier (BBB) permeability. Western blotting was used to determine the expression levels of the tight junction proteins in cerebral vascular endothelial cells. The results showed that 25% albumin treatment (1.25 g/kg) by intravenous injection elevated the COP of plasma but did not reduce the ICP in rats that had undergone ischemic stroke. Additionally, albumin did not reduce the infarct size and instead aggravated cerebral edema. Furthermore, the BBB permeability was increased by albumin. Concomitantly, albumin treatment significantly downregulated the expression of tight junction proteins (ZO‑1, occludin, and claudin‑5) in cerebral vascular endothelial cells. Tight junction protein expression was significantly upregulated when the cells were treated with an MMP‑9 inhibitor (GM6001). These results suggest that albumin aggravates cerebral edema in rats with ischemic stroke by increasing BBB permeability.

Before the endothelial mitogenic activity of the Vascular Endothelial Growth Factor A (VEGF) was described, VEGF had already been identified for its ability to induce vascular leakage. VEGF-induced vascular leakage has been most frequently studied in vivo using the Miles assay, a simple yet invaluable technique that has allowed researchers to unravel the molecular mechanisms underpinning vascular leakage both for VEGF and other permeability inducing agents. In this protocol, a mouse is intravenously injected with Evans Blue dye before VEGF is administered locally via intradermal injection. VEGF promotes vascular leak of serum proteins in the dermis, enabling Evans Blue-labeled albumin extravasation from the circulation and subsequent accumulation in the skin. As the volume of dye extravasation is proportional to the degree of vascular leak, it can be quantified as a proxy measurement of VEGF-induced vascular leakage., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: P17, a peptide isolated from Tetramorium bicarinatum ant venom, is known to induce an alternative phenotype of human monocyte-derived macrophages via activation of an unknown G protein-coupled receptor (GPCR)., Objective: We sought to investigate the mechanism of action and the immunomodulatory effects of P17 mediated through MRGPRX2 (Mas-related G protein-coupled receptor X2)., Methods: To identify the GPCR for P17, we screened 314 GPCRs. Upon identification of MRGPRX2, a battery of in silico, in vitro, ex vivo, and in vivo assays along with the receptor mutation studies were performed. In particular, to investigate the immunomodulatory actions, we used β-hexosaminidase release assay, cytokine releases, quantification of mRNA expression, cell migration and differentiation assays, immunohistochemical labeling, hematoxylin and eosin, and immunofluorescence staining., Results: P17 activated MRGPRX2 in a dose-dependent manner in β-arrestin recruitment assay. In LAD2 cells, P17 induced calcium and β-hexosaminidase release. Quercetin- and short hairpin RNA-mediated knockdown of MRGPRX2 reduced P17-evoked β-hexosaminidase release. In silico and in vitro mutagenesis studies showed that residue Lys 8 of P17 formed a cation-π interaction with the Phe 172 of MRGPRX2 and [Ala 8 ]P17 lost its activity partially. P17 activated LAD2 cells to recruit THP-1 and human monocytes in Transwell migration assay, whereas MRGPRX2-impaired LAD2 cells cannot. In addition, P17-treated LAD2 cells stimulated differentiation of THP-1 and human monocytes, as indicated by the enhanced expression of macrophage markers cluster of differentiation 11b and TNF-α by quantitative RT-PCR. Immunohistochemical and immunofluorescent staining suggested monocyte recruitment in mice ears injected with P17., Conclusions: Our data provide novel structural information regarding the interaction of P17 with MRGPRX2 and intracellular pathways for its immunomodulatory action., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Two morphologies of laccase-mineral hybrid complexes, i.e., laccase-mineral hybrid nanoflowers (La-HNF) and nanopetals (La-HNP), were synthesized via biomineralization using Cu 3 (PO 4 ) 2 ·3H 2 O as the mineral for Evans Blue (EB) dye biodegradation. XRD patterns and FT-IR spectra results revealed the successful immobilization of laccase via in-situ formed Cu 3 (PO 4 ) 2 ·3H 2 O crystals. Compared with free laccase, laccase-mineral hybrid complexes showed higher enzymatic activity due to the activation effect induced by copper ions of Cu 3 (PO 4 ) 2 ·3H 2 O, further, the improved kinetic parameters of laccase-mineral hybrid complexes could be ascribed to nanoscale-dispersed laccase molecules within hybrid complexes. For EB dye biodegradation, the reason why the biodegradation efficiency (94.9%) of La-HNF was higher than that (86.8%) of La-HNP could be synergistic effect of immobilized laccase within 3D hierarchical structure of La-HNF. In addition, the optimized biodegradation conditions (pH 4.6 and 40 °C) of La-HNF were obtained, moreover, 93.2% and 48.1% of EB dye were biodegraded by La-HNF after stored for 30 days and reused for 10 cycles, respectively, demonstrating La-HNF have good practicability., (Copyright © 2021 Elsevier B.V. All rights reserved.)

An increase in the brain endothelial (BEnd) cell permeability of blood albumin is often seen as an early sign of blood-brain barrier (BBB) disruption and can precede increases in the BEnd permeability of small molecules and other plasma proteins in the course of brain disease. Therefore, Evans blue dye (EBD), an albumin-binding fluorescent tracer that is simple to detect and quantify, has been widely utilized for studying BEnd permeability during BBB disruption. Here, we investigated whether EBD is a suitable indicator of albumin permeability across mouse BEnd cell monolayers, alone or cocultured with mouse cortical astrocytes, in an in-vitro permeability assay; given the strong affinity of EBD for albumin, we further asked whether EBD can affect albumin permeability and vice versa. Albumin and EBD readily crossed membrane cell culture inserts with pore diameters of no less than 1 µm in the absence of a cellular barrier, and their permeability was substantially reduced when the membranes were overlaid with a monolayer of BEnd cells. In line with albumin binding, the BEnd permeability of EBD was substantially reduced by the presence of albumin. While EBD at an EBD-to-albumin ratio similar to those typically used in in vivo BBB experiments had little effect on the BEnd permeability of albumin, a much higher concentration of EBD augmented the BEnd permeability of albumin. In conclusion, we investigated the use of EBD as an indicator of albumin permeability in vitro, explored some of its drawbacks and further demonstrated that EBD at the concentration used in vivo does not affect albumin permeability., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Intracellular tension plays a crucial role in the destruction of the blood-brain barrier (BBB) in response to lesion stimuli. Tight junction structure could be primarily affected by tension activity. In this study, we aimed to determine the effects of extracellular BBB damage on intracellular tension activity, and elucidate the mechanism underlying the effects of intracellular protein nanoparticle-related osmotic pressure on BBB permeability., Methods: The intracellular tension for tight junction proteins occludin and ZO1 was evaluated using the fluorescence resonance energy transfer (FRET)-based tension probes and cpstFRET analysis. The changes in mobility ratios of occludin were evaluated via the fluorescence recovery after photobleaching (FRAP) test. The cytoplasmic osmotic pressure (OP) was measured using Osmometer. The count rate of cytoplasmic nanoparticles was detected by Nanosight NS300. The activation of cofilin and stathmin was examined by Western blot analysis. The BBB permeability in vivo was determined via the changes of Evans Blue (EB) injected into SD rats. The tight junction formation was assessed by the measurement of transendothelial electrical resistance (TEER). Intracellular calcium or chloride ions were measured using Fluo-4 AM or MQAE dyes., Results: BBB lesions were accompanied by changes in occludin/ZO1 tension. Increases in intracellular osmotic pressure were involved in alteration of BBB permeability, possibly through the depolymerization of microfilaments or microtubules and mass production of protein nanoparticles according to the Donnan effect. Recovery of protein nanoparticle-related osmotic pressure could effectively reverse the effects of changes in occludin/ZO1 tension under BBB lesions. Outward tension of intracellular osmotic potential also caused upregulation of membrane fluidity, which promoted nonselective drug influx., Conclusion: Our results suggest a crucial mechanical mechanism underlying BBB lesions, and protein nanoparticle-related osmotic pressure could be a novel therapeutic target for BBB lesion-related brain diseases., Competing Interests: The authors declare that they have no competing interests., (© 2021 Li et al.)

Myeloablative therapy was modeled by administration of cyclophosphamide to rats in a dose of 2.1 LD50 for 14 days. Under these conditions, increased accumulation of Evans blue dye given by gavage was observed in the blood, brain, lung, liver, kidney, and ileum (by 34, 1.6, 149, 46, 30, and 6.2 times, respectively). After intravenous injection of the dye, its accumulation was increased only in the lung and ileum. Thus, simulation of myeloablation cytostatic therapy by cyclophosphamide injection to rats is associated with impairment of the intestinal and lung-blood barriers. These alterations predispose to penetration of biologically active substances from the small intestine, gastrointestinal chyme, and lungs to the blood, thus contributing to the development of the early toxic effects of cyclophosphamide.

The blood-brain barrier (BBB) is an active and selective barrier that shields the brain from endogenous and exogenous insults. Different stimuli may lead to the disruption of this barrier, including inflammation and trauma. Several methods are used to evaluate BBB disruption. The most widely used method is Evans blue (EB) dye extravasation. EB cannot normally pass through the BBB and thus its presence in brain tissue indicates alterations in permeability. This protocol details the steps of EB extravasation in rodents. Important aspects regarding critical steps and advantages are also provided. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly ( R 2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores ( R 2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.

The effectiveness of numerous molecular drugs is hampered by their poor pharmacokinetics. Different from previous approaches with limited effectiveness, most recently, emerging high-affinity albumin binding moieties (ABMs) for in vivo hitchhiking of endogenous albumin opens up an avenue to chaperone small molecules for long-acting therapeutics. Although several FDA-approved fatty acids have shown prolonged residence and therapeutic effect, an easily synthesized, water-soluble, and high-efficiency ABM with versatile drug loading ability is urgently needed to improve the therapeutic efficacy of short-lived constructs. We herein identified an ideal bivalent Evans blue derivative, denoted as N(tEB) 2 , as a smart ABM-delivery platform to chaperone short-lived molecules, through both computational modeling screening and efficient synthetic schemes. The optimal N(tEB) 2 could reversibly link two molecules of albumin through its two binding heads with a preferable spacer, resulting in significantly extended circulation half-life of a preloaded cargo and water-soluble. Notably, this in situ dimerization of albumin was able to sandwich peptide therapeutics to protect them from proteolysis. As an application, we conjugated N(tEB) 2 with exendin-4 for long-acting glucose control in a diabetic mouse model, and it was superior to both previously tested NtEB-exendin-4 (Abextide) and the newly FDA-approved semaglutide, which has been arguably the best commercial weekly formula so far. Hence, this novel albumin binder has excellent clinical potential for next-generation biomimetic drug delivery systems.

Bradykinin is a mediator of vasogenic brain edema formation. Recent reports suggest that bradykinin interacts with nitric oxide synthase (NOS) system in the central nervous system (CNS). However, role of bradykinin in spinal cord injury (SCI) induced alterations in the blood-spinal cord barrier (BSCB), spinal cord blood flow (SCBF), edema formation and cell changes are still not well known. Our previous reports showed that SCI induces marked upregulation of neuronal NOS (nNOS) in the cord associated with BSCB disruption, edema formation and cell injury. Thus, a possibility exists that bradykinin participates in SCI induced nNOS upregulation and cord pathology. To explore this idea a potent bradykinin B2 receptor antagonist HOE-140 was used in our rat model of SCI and cord pathology. SCI was inflicted in Equithesin anesthetized rats by making a longitudinal incision (2mm deep and 5mm long) into the right dorsal horn of the T10-11 segment. The animals were allowed to survive 5h after injury. A focal SCI significantly disrupted BSCB to Evans blue and [131] I-sodium in the traumatized and adjacent segments. Interestingly, far remote spinal cord segments C4 and T5 segments also affected within 5h. These spinal cord segments also exhibited pronounced reductions in the SCBF (mean-30%), increased edematous swelling and profound neuronal damages. Upregulation of nNOS expression is seen in both the dorsal and ventral horns of the spinal cord exhibiting cord pathology. At the ultrastructural level, exudation of lanthanum is seen within the endothelial cell cytoplasm and occasionally in the basal lamina. Pretreatment with low doses of HOE-140 (0. 1mg to 1mg/kg, i.v.) 30min prior to SCI significantly enhanced the SCBF and reduced the BSCB disruption, edema formation, nNOS upregulation and cell injury. However, HOE-140 in doses ranging from 2mg to 5mg/kg, i.v. did not induce significant neuroprotection. These observations are the first to suggest that bradykinin B2 receptors play an important role in BSCB permeability, SCBF, edema formation, nNOS upregulation and cell injury following acute SCI, not reported earlier., (© 2019 Elsevier Inc. All rights reserved.)

Several lines of evidences show that anesthetics influence neurotoxicity and neuroprotection. The possibility that different anesthetic agents potentially influence the pathophysiological and functional outcome following neurotrauma was examined in a rat model of concussive head injury (CHI). The CHI was produced by an impact of 0.224N on the right parietal bone by dropping a weight of 114.6g from a 20cm height under different anesthetic agents, e.g., inhaled ether anesthesia or intraperitoneally administered ketamine, pentobarbital, equithesin or urethane anesthesia. Five hour CHI resulted in profound volume swelling and brain edema formation in both hemispheres showing disruption of the blood-brain barrier (BBB) to Evans blue and radioiodine. A marked decrease in the cortical CBF and a profound increase in plasma or brain serotonin levels were seen at this time. Neuronal damages were present in several parts of the brain. These pathological changes were most marked in CHI under ether anesthesia followed by ketamine (35mg/kg, i.p.), pentobarbital (50mg/kg, i.p.), equithesin (3mL/kg, i.p.) and urethane (1g/kg, i.p.). The functional outcome on Rota Rod performances or grid walking tests was also most adversely affected after CHI under ether anesthesia followed by pentobarbital, equithesin and ketamine. Interestingly, the plasma and brain serotonin levels strongly correlated with the development of brain edema in head injured animals in relation to different anesthetic agents used. These observations suggest that anesthetic agents are detrimental to functional and pathological outcomes in CHI probably through influencing the circulating plasma and brain serotonin levels, not reported earlier. Whether anesthetics could also affect the efficacy of different neuroprotective agents in CNS injuries is a new subject that is currently being examined in our laboratory., (© 2019 Elsevier Inc. All rights reserved.)

Background and Aim: Metformin has been shown to have anti-cytokine property. Lipopolysaccharide (LPS)-induced or repeated water avoidance stress (WAS)-induced visceral allodynia and increased gut permeability were pro-inflammatory cytokine-dependent responses, which were considered to be animal models of irritable bowel syndrome (IBS). We hypothesized that metformin improves symptoms in the patients with IBS by attenuating these visceral changes and tested the hypothesis in rats., Methods: The threshold of the visceromotor response induced by colonic balloon distention was measured. Colonic permeability was determined in vivo by quantifying the absorbed Evans blue for 15 min spectrophotometrically., Results: Subcutaneously injected LPS (1 mg/kg) reduced the threshold of visceromotor response, and metformin (5-50 mg/kg for 3 days) intraperitoneally attenuated this response in a dose-dependent manner. Repeated WAS (1 h daily for 3 days) induced visceral allodynia, which was also blocked by metformin. The antinociceptive effect of metformin on the LPS-induced allodynia was reversed by compound C, an adenosine monophosphate-activated protein kinase inhibitor or N G -nitro-L-arginine methyl ester, a nitric oxide synthesis inhibitor but not modified by naloxone. Additionally, it was blocked by sulpiride, a dopamine D2 receptor antagonist, but domperidone, a peripheral dopamine D2 receptor antagonist, did not alter it. Metformin also blocked the LPS-induced or repeated WAS-induced increased colonic permeability., Conclusions: Metformin attenuated the visceral allodynia and increased gut permeability in animal IBS models. These actions may be evoked via activation of adenosine monophosphate-activated protein kinase, nitric oxide, and central dopamine D2 pathways. These results indicate the possibility that metformin can be useful for treating IBS., (© 2018 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Nanoparticles targeting transporters of the blood-brain barrier (BBB) are promising candidates to increase the brain penetration of biopharmacons. Solute carriers (SLC) are expressed at high levels in brain endothelial cells and show a specific pattern at the BBB. The aim of our study was to test glutathione and ligands of SLC transporters as single or dual BBB targeting molecules for nanovesicles. High mRNA expression levels for hexose and neutral amino acid transporting SLCs were found in isolated rat brain microvessels and our rat primary cell based co-culture BBB model. Niosomes were derivatized with glutathione and SLC ligands glucopyranose and alanine. Serum albumin complexed with Evans blue (67 kDa), which has a very low BBB penetration, was selected as a cargo. The presence of targeting ligands on niosomes, especially dual labeling, increased the uptake of the cargo molecule in cultured brain endothelial cells. This cellular uptake was temperature dependent and could be decreased with a metabolic inhibitor and endocytosis blockers filipin and cytochalasin D. Making the negative surface charge of brain endothelial cells more positive with a cationic lipid or digesting the glycocalyx with neuraminidase elevated the uptake of the cargo after treatment with targeted nanocarriers. Treatment with niosomes increased plasma membrane fluidity, suggesting the fusion of nanovesicles with endothelial cell membranes. Targeting ligands elevated the permeability of the cargo across the BBB in the culture model and in mice, and dual-ligand decoration of niosomes was more effective than single ligand labeling. Our data indicate that dual labeling with ligands of multiple SLC transporters can potentially be exploited for BBB targeting of nanoparticles., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The effects of light-driven enhancement of Evans Blue dye complexes with blood plasma proteins were observed for the first time, both in vitro and in vivo. The possible background of the effect concerns the photochemical cis-trans isomerization of the azo dye molecules. The effect was induced in the solution with a red laser with a wavelength of 638 nm, which corresponds to the peak of the dye absorption. The lifetime of the enhanced fluorescence is approximately 1 second and enables its use as an optically tagged molecular flow tracer for blood flow velocity measurements. Utilizing the effect, we performed for the first time the intravital molecular tagging velocimetry of the blood velocity in blood vessels in a living animal. The results of the measurements of the blood flow velocities in the cerebral veins of a group of healthy mice are presented., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The investigation of age-related changes in muscle microstructure between developmental and healthy adult mice may help us to understand the clinical features of early-onset muscle diseases, such as Duchenne muscular dystrophy. We investigated the evolution of mouse hind-limb muscle microstructure using diffusion imaging of in vivo and in vitro samples from both actively growing and mature mice. Mean apparent diffusion coefficients (ADCs) of the gastrocnemius and tibialis anterior muscles were determined as a function of diffusion time (Δ), age (7.5, 22 and 44 weeks) and diffusion gradient direction, applied parallel or transverse to the principal axis of the muscle fibres. We investigated a wide range of diffusion times with the goal of probing a range of diffusion lengths characteristic of muscle microstructure. We compared the diffusion time-dependent ADC of hind-limb muscles with histology. ADC was found to vary as a function of diffusion time in muscles at all stages of maturation. Muscle water diffusivity was higher in younger (7.5 weeks) than in adult (22 and 44 weeks) mice, whereas no differences were observed between the older ages. In vitro data showed the same diffusivity pattern as in vivo data. The highlighted differences in diffusion properties between young and mature muscles suggested differences in underlying muscle microstructure, which were confirmed by histological assessment. In particular, although diffusion was more restricted in older muscle, muscle fibre size increased significantly from young to adult age. The extracellular space decreased with age by only ~1%. This suggests that the observed diffusivity differences between young and adult muscles may be caused by increased membrane permeability in younger muscle associated with properties of the sarcolemma., (Copyright © 2018 John Wiley & Sons, Ltd.)

Silver nanoparticles (AgNPs) enter the central nervous system through the blood-brain barrier (BBB). AgNP exposure can increase amyloid beta (Aβ) deposition in neuronal cells to potentially induce Alzheimer's disease (AD) progression. However, the mechanism through which AgNPs alter BBB permeability in endothelial cells and subsequently lead to AD progression remains unclear. This study investigated whether AgNPs disrupt the tight junction proteins of brain endothelial cells, and alter the proteomic metabolism of neuronal cells underlying AD progression in a triple cell coculture model constructed using mouse brain endothelial (bEnd.3) cells, mouse brain astrocytes (ALT), and mouse neuroblastoma neuro-2a (N2a) cells. The results showed that AgNPs accumulated in ALT and N2a cells because of the disruption of tight junction proteins, claudin-5 and ZO-1, in bEnd.3 cells. The proteomic profiling of N2a cells after AgNP exposure identified 298 differentially expressed proteins related to fatty acid metabolism. Particularly, AgNP-induced palmitic acid production was observed in N2a cells, which might promote Aβ generation. Moreover, AgNP exposure increased the protein expression of amyloid precursor protein (APP) and Aβ generation-related secretases, PSEN1, PSEN2, and β-site APP cleaving enzyme for APP cleavage in ALT and N2a cells, stimulated Aβ40 and Aβ42 secretion in the culture medium, and attenuated the gene expression of Aβ clearance-related receptors, P-gp and LRP-1, in bEnd.3 cells. Increased Aβ might further aggregate on the neuronal cell surface to enhance the secretion of inflammatory cytokines, MCP-1 and IL-6, thus inducing apoptosis in N2a cells. This study suggested that AgNP exposure might cause Aβ deposition and inflammation for subsequent neuronal cell apoptosis to potentially induce AD progression., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Following a central nervous system (CNS) injury, restoration of the blood-brain barrier (BBB) integrity is essential for recovering homeostasis. When this process is delayed or impeded, blood substances and cells enter the CNS parenchyma, initiating an additional inflammatory process that extends the initial injury and causes so-called secondary neuronal loss. Astrocytes and profibrotic mesenchymal cells react to the injury and migrate to the lesion site, creating a new glia limitans that restores the BBB. This process is beneficial for the resolution of the inflammation, neuronal survival, and the initiation of the healing process. Salubrinal is a small molecule with neuroprotective properties in different animal models of stroke and trauma to the CNS. Here, we show that salubrinal increased neuronal survival in the neighbourhood of a cerebral cortex stab injury. Moreover, salubrinal reduced cortical blood leakage into the parenchyma of injured animals compared with injured controls. Adjacent to the site of injury, salubrinal induced immunoreactivity for platelet-derived growth factor subunit B (PDGF-B), a specific mitogenic factor for mesenchymal cells. This effect might be responsible for the increased immunoreactivity for fibronectin and the decreased activation of microglia and macrophages in injured mice treated with salubrinal, compared with injured controls. The immunoreactivity for PDGF-B colocalized with neuronal nuclei (NeuN), suggesting that cortical neurons in the proximity of the injury were the main source of PDGF-B. Our results suggest that after an injury, neurons play an important role in both, the healing process and the restoration of the BBB integrity. J. Cell. Physiol. 232: 1501-1510, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The spiral ganglion neurons (SGNs) located in the Rosenthal's canal of cochlea are essential target for cochlear implant. Previous studies found that the canaliculi perforantes, small pores on the surface of the osseous spiral lamina (OSL) of the scala tympanic (ST) of cochlea, may provide communication between the cochlear perilymph and SGNs. In this study, we found that chronic treatment of aminoglycosides antibiotics, which is well known to cause sensory cell damage in the cochlea, induced significant damage of bone lining cells on the OSLs and increased the permeability of the Rosenthal's canal. The pores among the bone lining cells became significantly wider after chronic treatment of amikacin (100 mg/kg/day for 3-7 days). Injection of Evans Blue in the ST resulted in significant increase in its migration in the modulus in the amikacin-treated cochlea compared to the control ears, suggesting increased permeability of these passages. Treatment of amikacin with oxytetracycline, an inhibitor of matrix metalloproteases (MMPs), significantly reduced the amount of dye migrated from the ST to the modiolus. These results suggest that amikacin enhanced the permeability between the ST and SGNs by increasing MMPs. Aggregating the permeability of the bone lining cells on the OSLs may benefit gene and stem cell delivery to the SGNs in the cochlea.

Objective: The authors assessed the diagnostic accuracy of a new modified Evans blue dye test (MEBDT) as a screening test for aspiration in tracheostomized patients., Design: Monocentric retrospective study performed between October 2013 and December 2015., Setting: Anesthesia and Intensive Care Unit, Second University of Naples., Participants: Among 62 eligible patients, 5 were excluded. The authors' study population included 57 patients., Interventions: Patients underwent both fiberoptic endoscopic examination of the swallow (FEES) and MEBDT to evaluate swallow. The MEBDT results were compared with those of FEES and the diagnostic accuracy of MEBDT was calculated using the FEES as the gold standard., Measurements and Main Results: The authors found that both FEES and MEBDT were positive for aspiration in 40 patients (true-positive MEBDT); FEES and MEBDT were negative in 10 (true-negative MEBDT). On the other hand, FEES was positive with an MEBDT negative in 7 patients (false-negative MEBDT), and there were no FEES negative and MEBDT positive (false-positive MEBDT). MEBDT had a sensitivity, specificity, positive, and negative predicted value of 85%, 100%, 100%, and 58.82%, respectively., Conclusions: MEBDT could be a supplementary diagnostic test for aspiration. Patients with positive MEBDT should not undergo oral feeding, while patients with negative MEBDT should undergo FEES before starting oral feeding., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: In acupuncture practice, the most important step is to confirm the location of a sensitized acupoint which reflects a diagnosis and can be stimulated with a specialized needle to treat the disease. Abnormal symptoms such as hyperalgesia or allodynia at the sensitized acupoints in patients with visceral disorders are considered to be in relation with referred pain and neurogenic inflammation. Yet, limited study has investigated the cutaneous neurochemical changes of the sensitized acuponits., Methods: The resent study developed an animal model of gastric mucosal injury (GMI) by HCl administered into the stomach of the rats. Evans Blue (EB) dye was applied by injection of tail vein after mucosal damage to observe the neurogenic plasma extravasation dots in the skin of the rats. The EB dots extravagated in the skin were compared with locations of acupoints. Immnohistochemistry analysis was used to detect the expression of calcitonin gene-related peptide (CGRP)- or substance P (SP)-labeled nerve fibers, histamine (HA)-, serotonin (5-HT)-, and tryptase-labeled cells in EB dots. Images were recorded and analyzed by Confocal imaging system and Olympus Image Processing Software., Results: The results showed that GMI resulted in neurogenic plasma extravasation in the skin of the acupoints over the back and abdomen, which mostly occurred in the T9-11 dermatomere. The EB extravasation dots appeared after GMI and disappeared gradually during the natural self-recovery of the gastric mucosa. More SP and CGRP positive nerve fibers were distributed in EB dots than that in regions beside EB dots and in the control, mostly distributed in the nerve fibers around both the vessels and root of hair follicle. Mast cells also aggregated and degranulated to release algogenic substances of 5-HT and HA around the vessels in areas of the EB dots., Conclusions: Our results indicates that the mechanism of EB extravasation in the skin of the acupoints induced by GMI are closely related to neurogenic inflammation, and that the high expression of local allergic substances and nociceptive neuropeptides in the local skin including SP, CGRP, HA, 5-HT, and mast cell tryptase may be the underlying mechanism of the acupoint sensitization.

In our previous rodent studies, the paclitaxel (PTX)-incorporating polymeric micellar nanoparticle formulation NK105 had showed significantly stronger antitumor effects and reduced peripheral neurotoxicity than PTX dissolved in Cremophor ® EL and ethanol (PTX/CRE). Thus, to elucidate the mechanisms underlying reduced peripheral neurotoxicity due to NK105, we performed pharmacokinetic analyses of NK105 and PTX/CRE in rats. Among neural tissues, the highest PTX concentrations were found in the dorsal root ganglion (DRG). Moreover, exposure of DRG to PTX ( C max&#95;PTX and AUC 0-inf.&#95;PTX ) in the NK105 group was almost half that in the PTX/CRE group, whereas exposure of sciatic and sural nerves was greater in the NK105 group than in the PTX/CRE group. In histopathological analyses, damage to DRG and both peripheral nerves was less in the NK105 group than in the PTX/CRE group. The consistency of these pharmacokinetic and histopathological data suggests that high levels of PTX in the DRG play an important role in the induction of peripheral neurotoxicity, and reduced distribution of PTX to the DRG of NK105-treated rats limits the ensuing peripheral neurotoxicity. In further analyses of PTX distribution to the DRG, Evans blue (Eb) was injected with BODIPY ® -labeled NK105 into rats, and Eb fluorescence was observed only in the DRG. Following injection, most Eb dye bound to albumin particles of ~8 nm and had penetrated the DRG. In contrast, BODIPY ® -NK105 particles of ~90 nm were not found in the DRG, suggesting differential penetration based on particle size. Because PTX also circulates as PTX-albumin particles of ~8 nm following injection of PTX/CRE, reduced peripheral neurotoxicity of NK105 may reflect exclusion from the DRG due to particle size, leading to reduced PTX levels in rat DRG (275)., Competing Interests: Disclosure All authors are employees of Nippon Kayaku Co., Ltd. The authors report no other conflicts of interest in this work.

Mild hyperthermia has been used in combination with polymer therapeutics to further increase delivery to solid tumors and enhance efficacy. An attractive method for generating heat is through non-invasive high intensity focused ultrasound (HIFU). HIFU is often used for ablative therapies and must be adapted to produce uniform mild hyperthermia in a solid tumor. In this work a magnetic resonance imaging guided HIFU (MRgHIFU) controlled feedback system was developed to produce a spatially uniform 43°C heating pattern in a subcutaneous mouse tumor. MRgHIFU was employed to create hyperthermic conditions that enhance macromolecular delivery. Using a mouse model with two subcutaneous tumors, it was demonstrated that MRgHIFU enhanced delivery of both Evans blue dye (EBD) and Gadolinium-chelated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. The EBD accumulation in the heated tumors increased by nearly 2-fold compared to unheated tumors. The Gadolinium-chelated HPMA copolymers also showed significant enhancement in accumulation over control as evaluated through MRI T1-mapping measurements. Results show the potential of HIFU-mediated hyperthermia for enhanced delivery of polymer therapeutics., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a potentially lethal complication of clinical malaria. Acute lung injury in MA-ARDS shares features with ARDS triggered by other causes, including alveolar inflammation and increased alveolar-capillary permeability, leading to leak of protein-rich pulmonary oedema fluid. Mechanisms and physiologic alterations in MA-ARDS can be examined in murine models of this syndrome. Integrin αDβ2 is a member of the leukocyte, or β2 (CD18), sub-family of integrins, and emerging observations indicate that it has important activities in leukocyte adhesion, accumulation and signalling. The goal was to perform analysis of the lungs of mice wild type C57Bl/6 (a D (+/+) ) and Knockout C57Bl/6 (a D (-/-) ) with malaria-associated acute lung injury to better determine the relevancy of the murine models and investigate the mechanism of disease., Methods: C57BL/6 wild type (a D (+/+) ) and deficient for CD11d sub-unit (a D (-/-) ) mice were monitored after infection with 10(5) Plasmodium berghei ANKA. CD11d subunit expression RNA was measured by real-time polymerase chain reaction, vascular barrier integrity by Evans blue dye (EBD) exclusion and cytokines by ELISA. Protein and leukocytes were measured in bronchoalveolar lavage fluid (BALF) samples. Tissue cellularity was measured by the point-counting technique, F4/80 and VCAM-1 expression by immunohistochemistry. Respiratory function was analysed by non-invasive BUXCO and mechanical ventilation., Results: Alveolar inflammation, vascular and interstitial accumulation of monocytes and macrophages, and disrupted alveolar-capillary barrier function with exudation of protein-rich pulmonary oedema fluid were present in P. berghei-infected wild type mice and were improved in αDβ2-deficient animals. Key pro-inflammatory cytokines were also decreased in lung tissue from α D (-/-) mice, providing a mechanistic explanation for reduced alveolar-capillary inflammation and leak., Conclusions: The results indicate that αDβ2 is an important inflammatory effector molecule in P. berghei-induced MA-ARDS, and that leukocyte integrins regulate critical inflammatory and pathophysiologic events in this model of complicated malaria. Genetic deletion of integrin subunit αD in mice, leading to deficiency of integrin αDβ2, alters lung inflammation and acute lung injury in a mouse model of MA-ARDS caused by P. berghei.

The arcuate nucleus (ARC) regulates body weight in response to blood-borne signals of energy balance. Blood-brain barrier (BBB) permeability in the ARC is determined by capillary endothelial cells (ECs) and tanycytes. Tight junctions between ECs limit paracellular entry of blood-borne molecules into the brain, whereas EC transporters and fenestrations regulate transcellular entry. Tanycytes appear to form a barrier that prevents free diffusion of blood-borne molecules. Here we tested the hypothesis that gestation in an obese mother alters BBB permeability in the ARC of offspring. A maternal high-fat diet model was used to generate offspring from normal-weight (control) and obese dams (OffOb). Evans Blue diffusion into the ARC was higher in OffOb compared with controls, indicating that ARC BBB permeability was altered. Vessels investing the ARC in OffOb had more fenestrations than controls, although the total number of vessels was not changed. A reduced number of tanycytic processes in the ARC of OffOb was also observed. The putative transporters, Lrp1 and dysferlin, were up-regulated and tight junction components were differentially expressed in OffOb compared with controls. These data suggest that maternal obesity during pregnancy can compromise BBB formation in the fetus, leading to altered BBB function in the ARC after birth.

The blood brain barrier (BBB) could be damaged within the thrombolytic time window and is considered to be a precursor to hemorrhagic transformation during reperfusion. Although we have recently reported the association between BBB damage and tissue injury within the thrombolytic time window, our knowledge about this early BBB damage is limited. In this study, rats were subjected to 2-h middle cerebral artery occlusion (MCAO) followed by 10 min reperfusion with Evan's blue as a tracer to detect BBB damage. Rat brain was sliced into 10 consecutive sections and with TTC staining, a macro and full view of the spatial distribution of BBB damage and tissue injury could be clearly seen in the same group of animals. After 2-h MCAO, tissue injury started from 2nd slice and the BBB leakage started from the 5th slice, of note, there is no colocalization between BBB damage and tissue injury. Fluoro Jade B was employed to explore the localization of neuronal degeneration, and our results showed that 2-h MCAO produced greater number of positive cells in ischemic cortex and dorsal striatum than other areas. More important, 2-h MCAO induced occludin but not claudin-5 degradation in the ischemic hemisphere and pretreatment with MMP inhibitor GM6001 significantly reduced occludin degradation as well as BBB damage detected by IgG leakage. Taken together, our findings demonstrated a "mismatch" between ischemic tissue injury and BBB leakage and a differential degradation of occludin and claudin-5 by MMP-2 after 2-h MCAO., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Nitric oxide (NO) is an important bioactive molecule involved in cell wall metabolism, which has been recognized as a major target of aluminium (Al) toxicity. We have investigated the effects of Al-induced NO production on cell wall composition and the subsequent Al-binding capacity in roots of an Al-sensitive cultivar of wheat (Triticum aestivum L. cv. Yang-5). We found that Al exposure induced NO accumulation in the root tips. Eliminating NO production with an NO scavenger (cPTIO) significantly alleviated the Al-induced inhibition of root growth and thus reduced Al accumulation. Elimination of NO, however, did not significantly affect malate efflux or rhizosphere pH changes under Al exposure. Levels of cell wall polysaccharides (pectin, hemicelluloses 1, and hemicelluloses 2) and pectin methylesterase activity, as well as pectin demethylation in the root apex, significantly increased under Al treatment. Exogenous cPTIO application significantly decreased pectin methylesterase activity and increased the degree of methylation of pectin in the root cell wall, thus decreasing the Al-binding capacity of pectin. These results suggest that the Al-induced enhanced production of NO decreases cell wall pectin methylation, thus increasing the Al-binding capacity of pectin and negatively regulating Al tolerance in wheat., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background/aims: Advanced glycation end products (AGEs) could elicit oxidative stress, trigger and aggravate endothelium damage in several ischemic retinopathies including diabetic retinopathy (DR). The leaves of Eucommia ulmoides O., also referred to as Tu-chung or Du-zhong, have been used for the treatment of hypertension and diabetes, showing great antioxidant activity and anti-glycation activity. Lignans is one of the main bioactive components of Eucommia ulmoides. This study mainly investigated the effect of lignans treatment on AGEs-induced endothelium damage., Methods: MTT assay, Hoechst staining, and calcein-AM/ propidium iodide (PI) staining was conducted to determine the effect of lignans treatment on endothelial cell function in vitro. Retinal trypsin digestion, Evans blue assay, isolectin staining, and western blots were conducted to determine the effect of lignans treatment on retinal microvascular function in vivo. Western blot, protein immunoprecipitation (IP), MTT assays, and enzyme activity assay was conducted to detect the effect of ligans treatment on oxidative stress response., Results: Lignans protected retinal endothelial cell against AGEs-induced injury in vitro and diabetes-induced vascular dysfunction in vivo. Lignans treatment could regulate oxidative stress response in retinal endothelial cell line, retina, and liver. Moreover, we showed that NRF2/HO-1 signaling was critical for lignans-mediated oxidative stress regulation., Conclusion: Lignans treatment could protect against endothelial dysfunction in vivo and in vitro via regulating Nrf2/HO-1 signaling. Lignans might be developed as a promising drug for the treatment of diabetes-induced microvascular dysfunction., (© 2016 The Author(s) Published by S. Karger AG, Basel.)