Evans Blue Extravasation Research Articles

Dexmedetomidine postconditioning alleviates spinal cord ischemia-reperfusion injury in rats via inhibiting neutrophil infiltration, microglia activation, reactive gliosis and CXCL13/CXCR5 axis activation

Purpose Spinal cord ischemia-reperfusion (I/R) injury is an unresolved complication and its mechanisms are still not completely understood. Here, we studied the neuroprotective effects of dexmedetomidine (DEX) postconditioning against spinal cord I/R injury in rats and explored the possible mechanisms. Materials and methods In the study, rats were randomly divided into five groups: sham group, I/R group, DEX0.5 group, DEX2.5 group, and DEX5 group. I/R injury was induced in experimental rats; 0.5 μg/kg, 2.5 μg/kg, 5 μg/kg DEX were intravenously injected upon reperfusion respectively. Neurological function, histological assessment, and the disruption of blood-spinal cord barrier (BSCB) were evaluated via the BBB scoring, hematoxylin and eosin staining, Evans Blue (EB) extravasation and spinal cord edema, respectively. Neutrophil infiltration was evaluated via Myeloperoxidase (MPO) activity. Microglia activation and reactive gliosis was evaluated via ionized calcium-binding adapter molecule-1(IBA-1) and glial fibrillary acidic protein (GFAP) immunofluorescence, respectively. The expression of C-X-C motif ligand 13 (CXCL13), C-X-C chemokine receptor type 5(CXCR5), caspase-3 was determined by western blotting. The expression levels of interleukin 6(IL-6), tumor necrosis factor-α(TNF-α), IL-1β were determined by ELISA assay. Results DEX postconditioning preserved neurological assessment scores, improved histological assessment scores, attenuated BSCB leakage after spinal cord I/R injury. Neutrophil infiltration, microglia activation and reactive gliosis were also inhibited by DEX postconditioning. The expression of CXCL13, CXCR5, caspase-3, IL-6, TNF-α, IL-1β were reduced by DEX postconditioning. Conclusions DEX postconditioning alleviated spinal cord I/R injury, which might be mediated via inhibition of neutrophil infiltration, microglia activation, reactive gliosis and CXCL13/CXCR5 axis activation.

Ac-FLTD-CMK inhibits pyroptosis and exerts neuroprotective effect in a mice model of traumatic brain injury.

Pyroptosis has been reported to contribute to the traumatic brain injury (TBI) process. Ac-FLTD-CMK is a newly synthesized pyroptosis inhibitor. However, whether Ac-FLTD-CMK inhibits pyroptosis and plays a neuroprotective role after TBI is unknown. The present study aimed to determine the effects of Ac-FLTD-CMK on TBI in a mouse model. Male C57BL/6 mice were randomly divided into sham, TBI + vehicle, and TBI + Ac-FLTD-CMK groups. TBI was induced using a weight-drop apparatus. Intraventricular injection of Ac-FLTD-CMK was performed 30 min after TBI. Caspase-1, caspase-11, gasdermin-D (GSDMD), and caspase-3 expression in the peri-contusional cortex were assessed by western blotting. Interleukin-1β (IL-1β) and interleukin-18 (IL-18) expression in the peri-contusional cortex were measured using ELISA. Behavioral experiments, brain water content, Evans blue extravasation, lactate dehydrogenase (LDH) release, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining were also performed. The results showed that Ac-FLTD-CMK administration significantly downregulated caspase-1 p20, caspase-11 p20, GSDMD N-terminal, IL-1β, and IL-18 expression; reduced LDH release; alleviated neuronal death; attenuated brain edema and blood-brain barrier damage; and improved neurobehavioral function. These findings indicate that Ac-FLTD-CMK treatment suppresses pyroptosis and protects mice against TBI.

Anti-Inflammatory Potential Exhibited by Amomum subulatum Fruits Mitigates Experimentally Induced Acute and Chronic Inflammation in Mice: Evaluation of Antioxidant Parameters, Pro-Inflammatory Mediators and HO-1 Pathway

Objective Conventional anti-inflammatory drugs are associated with serious adverse effects which bring about an ever-increasing demand to supersede them with natural and safe anti-inflammatory agents. Hence, the prime objective of this study was to evaluate the anti- inflammatory potential of an underutilized culinary spice “Amomum subulatum”. Methods To assess anti-inflammatory activity of MEAS, acute and chronic inflammation studies were carried out in carrageenan and formalin induced mice paw edema models respectively. Paw volume was measured by vernier caliper. Status of antioxidant enzymes and oxidative stress markers were determined in paw tissue homogenates following standard protocols. Histopathology and immunohistochemistry analysis of paw tissue samples were also performed. Levels of proinflammatory cytokines in serum were quantified by ELISA. Effect of MEAS on vascular permeability was evaluated by evans blue dye extravasation assay. Involvement of heme oxygenase (HO)-1 pathway in anti-inflammatory action of MEAS was investigated by pretreating mice with zinc protoporphyrin (ZnPP) IX, a specific inhibitor of HO-1. Results MEAS administration significantly reduced paw edema, as evidenced by paw volume measurement and histopathology analysis. Additionally, pretreatment with MEAS markedly reduced vascular permeability, serum proinflammatory cytokine levels, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Further, the anti-inflammatory mechanism of MEAS showed the involvement of HO-1 pathway when HO-1 was inhibited by ZnPPIX. Conclusion Our results manifested strong anti-inflammatory activity of MEAS, suggesting its potential use as a therapeutic alternative for treating inflammatory disorders.

17β-Estradiol Attenuates Intracerebral Hemorrhage-Induced Blood-Brain Barrier Injury and Oxidative Stress Through SRC3-Mediated PI3K/Akt Signaling Pathway in a Mouse Model.

Estrogen is neuroprotective in brain injury models, and steroid receptor cofactor 3 (SRC3) mediates estrogen signaling. We aimed to investigate whether and how SRC3 is involved in the neuroprotective effects of 17ß-estradiol (E2) in a mouse model of intracerebral hemorrhage (ICH). Ovariectomized female mice were treated with E2 after autologous blood injection-induced ICH. Brain damage was assessed by neurological deficit score, brain water content, and oxidative stress levels. Blood–brain barrier (BBB) integrity was evaluated by Evan's blue extravasation and claudin-5, ZO-1, and occludin levels. SRC3 expression and PI3K/Akt signaling pathway were examined in ICH mice treated with E2. The effect of SRC3 on E2-mediated neuroprotection was determined by examining neurological outcomes in SRC3-deficient mice undergone ICH and E2 treatment. We found that E2 alleviated ICH-induced brain edema and neurological deficits, protected BBB integrity, and suppressed oxidative stress. E2 enhanced SRC3 expression and PI3K-/Akt signaling pathway. SRC3 deficiency abolished the protective effects of E2 on ICH-induced neurological deficits, brain edema, and BBB integrity. Our results suggest that E2 suppresses ICH-induced brain injury and SRC3 plays a critical role in E2-mediated neuroprotection.

MicroRNA miR-126 attenuates brain injury in septic rats via NF-κB signaling pathway

ABSTRACT The purpose of this study was to investigate the impact and mechanism of microRNA miR-126 on brain injury induced by blood-brain barrier (BBB) damage in septic rats. We used cecal ligation and perforation (CLP) to create a rat model of sepsis. The experimental rats were randomly divided into Control group, CLP group, CLP + miR-NC group, CLP + miR-126 group and CLP + miR-126 + NF-κB pathway agonist (PMA) group. MiR-126 expressed in the brain tissue of CLP rats was down-regulated by qRT-PCR. Upregulation of miR-126 in CLP rats could improve brain injury and BBB marker protein level, reduce brain water content, Evans blue extravasation, inflammation, and excessive oxidative stress. This could also result in an inhibition of NF-κB signaling pathway activity. In conclusion, miR-126 overexpression can prevent brain injury caused by BBB damage via the inhibition of NF-κB signaling pathway activity.

The neuroprotection of cerebrolysin after spontaneous intracerebral hemorrhage through regulates necroptosis via Akt/ GSK3β signaling pathway.

ABSTRACTPurpose:Spontaneous intracerebral hemorrhage (ICH) is a major cause of death and disability with a huge economic burden worldwide. Cerebrolysin (CBL) has been previously used as a nootropic drug. Necroptosis is a programmed cell death mechanism that plays a vital role in neuronal cell death after ICH. However, the precise role of necroptosis in CBL neuroprotection following ICH has not been confirmed.Methods:In the present study, we aimed to investigate the neuroprotective effects and potential molecular mechanisms of CBL in ICH-induced early brain injury (EBI) by regulating neural necroptosis in the C57BL/6 mice model. Mortality, neurological score, brain water content, and neuronal death were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, Evans blue extravasation, Western blotting, and quantitative real-time polymerase chain reaction (PCR).Results:The results show that CBL treatment markedly increased the survival rate, neurological score, and neuron survival, and downregulated the protein expression of RIP1 and RIP3, which indicated that CBL-mediated inhibition of necroptosis, and ameliorated neuronal death after ICH. The neuroprotective capacity of CBL is partly dependent on the Akt/GSK3β signaling pathway.Conclusions:CBL improves neurological outcomes in mice and reduces neuronal death by protecting against neural necroptosis.

Irisin Rescues Blood-Brain Barrier Permeability following Traumatic Brain Injury and Contributes to the Neuroprotection of Exercise in Traumatic Brain Injury.

Traumatic brain injury (TBI) has a high incidence, mortality, and morbidity all over the world. One important reason for its poor clinical prognosis is brain edema caused by blood-brain barrier (BBB) dysfunction after TBI. The mechanism may be related to the disorder of mitochondrial morphology and function of neurons in damaged brain tissue, the decrease of uncoupling protein 2 (UCP2) activity, and the increase of inflammatory reaction and oxidative stress. In this study, we aimed to investigate the effects of exogenous irisin on BBB dysfunction after TBI and its role in the neuroprotective effects of endurance exercise (EE) in mice. The concentrations of irisin in cerebrospinal fluid (CSF) and plasma of patients with mild to severe TBI were measured by ELISA. Then, male C57BL/6J mice and UCP2 knockout mice with C57BL/6J background were used to establish the TBI model. The BBB structure and permeability were examined by transmission electron microscopy and Evans blue extravasation, respectively. The protein expressions of irisin, occludin, claudin-5, zonula occludens-1 (ZO-1), nuclear factor E2-related factor 2(Nrf2), quinine oxidoreductase (NQO-1), hemeoxygenase-1 (HO-1), cytochrome C (Cyt-C), cytochrome C oxidase (COX IV), BCL2-associated X protein (Bax), cleaved caspase-3, and UCP2 were detected by western blot. The production of reactive oxygen species (ROS) was evaluated by the dihydroethidium (DHE) staining. The levels of inflammatory factors were detected by ELISA. In this study, we found that the CSF irisin levels were positively correlated with the severity of disease in patients with TBI and both EE and exogenous irisin could reduce BBB damage in a mouse model of TBI. In addition, we used UCP2−/− mice and further found that irisin could improve the dysfunction of BBB after TBI by promoting the expression of UCP2 on the mitochondrial membrane of neurons, reducing the damage of mitochondrial structure and function, thus alleviating the inflammatory response and oxidative stress. In conclusion, the results of this study suggested that irisin might alleviate brain edema after TBI by promoting the expression of UCP2 on the mitochondrial membrane of neurons and contribute to the neuroprotection of EE against TBI.

Inhibitory effect of paeoniflorin on IgE-dependent and IgE-independent mast cell degranulation in vitro and vivo.

The incidence of allergic diseases has increased to such a point that they have become common and have reached epidemic levels. However, their pathogenesis is not fully understood. Paeoniae Radix Rubra is a traditional Chinese medicine that is also used as a dietary supplement. Its main active ingredient is paeoniflorin. Paeoniflorin has good anti-inflammatory, immunomodulation, and antitumor effects. It is utilized in the treatment of various diseases in clinical settings. However, its effects on type I allergies and pseudoallergic reactions have not been comprehensively studied. In this study, we aimed to use DNP-IgE/DNP-BSA and C48/80 to simulate type I allergies and pseudoallergic reactions to evaluate the therapeutic effects of paeoniflorin to these diseases and identify its molecular mechanisms in cell degranulation both in vivo and in vitro. Results showed that paeoniflorin inhibited the degranulation of RBL-2H3 cells induced by these two stimuli (IgE-dependent and IgE-independent stimuli) in a dose-dependent manner. Moreover, qPCR and western blot analyses indicated that paeoniflorin may regulate the IgE/FcεR I, MRGPRB3, and downstream signal transduction pathways to exert its therapeutic effects on type I allergies and pseudoallergic reactions. In addition, DNP-IgE/DNP-BSA and compound 48/80 were used to induce the establishment of a passive cutaneous anaphylaxis mouse model. Paeoniflorin was found to suppress the extravasation of Evans Blue and tissue edema in the ears, back skin, and paws of the mice. This result further confirmed that paeoniflorin has a notable therapeutic effect on type I allergies and pseudoallergic reactions. Therefore, paeoniflorin could potentially be used as a drug for the treatment of type I allergies and pseudoallergic reactions. This study provides new insights into expanding the treatment range of paeoniflorin and its pharmacological mechanism.

The Probable Protective Effect of Photobiomodulation on the Inflammation of the Airway and Lung in COVID-19 Treatment: A Preclinical and Clinical Meta-Analysis.

Preliminary studies also show that many of the fatalities of COVID-19 are due to over-activity of the immune system, and photobiomodulation (PBM) therapy mainly accelerates wound healing and reduces pain and inflammation. Therefore, this systematic review and meta-analysis was conducted to evaluate the probable effect of the PBM therapy on the lung inflammation or ARDS and accelerate the regeneration of the damaged tissue. We systematically searched major indexing databases, including PubMed/Medline, ISI web of science (WOS), Scopus, Embase, and Cochrane central, using standard terms without any language, study region, or type restrictions. Of the 438 studies found through initial searches, 13 met the inclusion criteria. After applying the exclusion criteria, the main properties of 13 articles on 384 animals included in this meta-analysis with a wide range of species include rat (n=10) and rabbit (n=3). The analysis revealed that PBM therapy reduced TNFα (SMD:-3.75, 95% CI: -4.49, -3.02, P<0.00001, I2=10%), IL-1β (SMD:-4.65, 95% CI: -6.15, -3.16, P<0.00001, I2=62%), and IL-6 (SMD:-4.20, 95% CI: -6.42, -1.97, P=0.0002, I2=88%) significantly compared with the model controls. Hence, PBM therapy increased IL-10 significantly compared with the model controls (SMD:-4.65, 95% CI: -6.15, -3.16, P<0.00001, I2=62%). PBM therapy also reduced MPO activity (SMD:-2.13, 95% CI: -3.38, -0.87, P=0.0009, I2=64%) and vascular permeability (SMD:-2.59, 95% CI: -4.40, -0.77, P=0.0052, I2=71%) in the lung using the Evans blue extravasation technique significantly compared with the model controls. This systematic review and meta-analysis revealed that the PBM therapy does utilize beneficial anti-inflammatory effect, modulation of the immune system, lung permeability, or bronchoalveolar lavage on lung damage in both animal models and clinical studies. However, animal model and clinical studies appear limited considering the quality of the included evidences; therefore, large clinical trials are still required.

MiR-128-3p Alleviates Spinal Cord Ischemia/Reperfusion Injury Associated Neuroinflammation and Cellular Apoptosis via SP1 Suppression in Rat.

BackgroundNeuroinflammation and cellular apoptosis caused by spinal cord ischemia/reperfusion (I/R) injury result in neurological dysfunction. MicroRNAs (miRs) have crucial functions in spinal cord I/R injury pathogenesis according to previous evidences. Herein, whether miR-128-3p contributes to spinal cord I/R injury by regulating specificity protein 1 (SP1) was assessed.MethodsA rat model of spinal cord I/R injury was established by occluding the aortic arch for 14 min. Then, miR-128-3p’s interaction with SP1 was detected by dual-luciferase reporter assays. Next, miR-128-3p mimic and inhibitor, as well as adenovirus-delivered shRNA specific for SP1 were injected intrathecally for assessing the effects of miR-128-3p and SP1 on rats with spinal cord I/R injury. SP1, Bax and Bcl-2 expression levels in I/R injured spinal cord tissues were evaluated by Western blotting, while IL-1β, TNF-α, and IL-6 were quantitated by ELISA. Tarlov scores were obtained to detect hind-limb motor function. Evans blue (EB) dye extravasation was utilized to examine blood–spinal cord barrier (BSCB) permeability. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining was performed for neuronal apoptosis assessment.ResultsMiR-128-3p expression was decreased, while SP1 amounts were increased in rat spinal cord tissue specimens following I/R. SP1 was identified as a miR-128-3p target and downregulated by miR-128-3p. MiR-128-3p overexpression or SP1 silencing alleviated I/R-induced neuroinflammation and cell apoptosis, and improved Tarlov scores, whereas pretreatment with miR-128-3p inhibitor aggravated the above injuries.ConclusionOverexpression of miR-128-3p protects neurons from neuroinflammation and apoptosis during spinal cord I/R injury partially by downregulating SP1.

Neural Progenitor Cell-Derived Extracellular Vesicles Enhance Blood-Brain Barrier Integrity by NF-κB (Nuclear Factor-κB)-Dependent Regulation of ABCB1 (ATP-Binding Cassette Transporter B1) in Stroke Mice.

Extracellular vesicles (EVs) derived from neural progenitor cells enhance poststroke neurological recovery, albeit the underlying mechanisms remain elusive. Since previous research described an enhanced poststroke integrity of the blood-brain barrier (BBB) upon systemic transplantation of neural progenitor cells, we examined if neural progenitor cell-derived EVs affect BBB integrity and which cellular mechanisms are involved in the process. Approach and Results: Using in vitro models of primary brain endothelial cell (EC) cultures as well as co-cultures of brain ECs (ECs) and astrocytes exposed to oxygen glucose deprivation, we examined the effects of EVs or vehicle on microvascular integrity. In vitro data were confirmed using a mouse transient middle cerebral artery occlusion model. Cultured ECs displayed increased ABCB1 (ATP-binding cassette transporter B1) levels when exposed to oxygen glucose deprivation, which was reversed by treatment with EVs. The latter was due to an EV-induced inhibition of the NF-κB (nuclear factor-κB) pathway. Using a BBB co-culture model of ECs and astrocytes exposed to oxygen glucose deprivation, EVs stabilized the BBB and ABCB1 levels without affecting the transcellular electrical resistance of ECs. Likewise, EVs yielded reduced Evans blue extravasation, decreased ABCB1 expression as well as an inhibition of the NF-κB pathway, and downstream matrix metalloproteinase 9 (MMP-9) activity in stroke mice. The EV-induced inhibition of the NF-κB pathway resulted in a poststroke modulation of immune responses. Our findings suggest that EVs enhance poststroke BBB integrity via ABCB1 and MMP-9 regulation, attenuating inflammatory cell recruitment by inhibition of the NF-κB pathway. Graphic Abstract: A graphic abstract is available for this article.

Escin ameliorates the impairments of neurological function and blood brain barrier by inhibiting systemic inflammation in intracerebral hemorrhagic mice

This study aims to investigate whether escin ameliorates the impairments of neurological function by ameliorating systemic inflammation instead of targeting the brain directly in intracerebral hemorrhage (ICH) mice. It showed that escin did not cross the blood brain barrier (BBB). Compared with the ICH group, the Garcia test scores in the escin groups were significantly increased. Brain water contents and Evans blue extravasation of the right basal ganglia in the ICH group were augmented, and significantly reduced by escin. Escin abated the increases of monocyte counts and serum IL-1β levels induced by ICH. IL-1β administration reversed the effect of escin on Garcia test scores, the brain water contents, and the Evans blue extravasation. Escin ameliorated the increasing levels of RhoA, ROCK1, nuclear NF-κB and the decreasing expression of IκBα, cytosolic NF-κB, occludin, claudin-5 in the ICH group. IL-1β administration blocked not only escin-mediated increases of IκBα, cytosolic NF-κB, occludin, and claudin-5, but also escin-caused decreases of RhoA, ROCK1, and nuclear NF-κB. The results indicate that escin improves neurological outcomes and the BBB function in ICH mice, which is associated with attenuating ICH-induced peripheral system inflammation, and therefore, inhibiting IL-1β/RhoA/NF-κB signaling pathway in BBB, at least in part. These findings suggest that it may be useful to ameliorate brain injury by inhibiting systemic inflammation instead of aiming to target the brain directly after ICH.

Sectm1a Facilitates Protection against Inflammation-Induced Organ Damage through Promoting TRM Self-Renewal

Tissue-resident macrophages (TRMs) are sentinel cells for maintaining tissue homeostasis and organ function. In this study, we discovered that lipopolysaccharide (LPS) administration dramatically reduced TRM populations and suppressed their self-renewal capacities in multiple organs. Using loss- and gain-of-function approaches, we define Sectm1a as a novel regulator of TRM self-renewal. Specifically, at the earlier stage of endotoxemia, Sectm1a deficiency exaggerated acute inflammation-induced reduction of TRM numbers in multiple organs by suppressing their proliferation, which was associated with more infiltrations of inflammatory monocytes/neutrophils and more serious organ damage. By contrast, administration of recombinant Sectm1a enhanced TRM populations and improved animal survival upon endotoxin challenge. Mechanistically, we identified that Sectm1a-induced upregulation in the self-renewal capacity of TRM is dependent on GITR-activated T helper cell expansion and cytokine production. Meanwhile, we found that TRMs may play an important role in protecting local vascular integrity during endotoxemia. Our study demonstrates that Sectm1a contributes to stabling TRM populations through maintaining their self-renewal capacities, which benefits the host immune response to acute inflammation. Therefore, Sectm1a may serve as a new therapeutic agent for the treatment of inflammatory diseases.

Abstract 15575: The Clonal Hematopoiesis Mutation Jak2 V617F Aggravates NETosis and Endothelial Injury in Mouse Arteries With Erosion-like Intimas

Introduction: In the current era of intense LDL lowering, superficial erosion may be on the rise as a cause of ACS (25-30%). Experimental data on human atheromata support a role for neutrophils and the formation of neutrophil extracellular traps (NETs) in the pathogenesis of superficial erosion and subsequent thrombosis. The common mutation of Janus kinase 2 ( Jak2 V617F ) borne by clones of leukocytes derived from bone marrow stem cells sensitizes neutrophils to undergo NETosis and linked NET formation to increased thrombosis. Jak2 V617F associates with accelerated atherosclerosis and venous thrombosis in patients with myeloproliferative neoplasms (MPN) and in individuals harboring this mutation but without a demonstrable MPN. Hypothesis We hypothesized that i) the Jak2 V617F associated with clonal hematopoiesis and increased atherosclerotic and thrombotic risk in humans, predisposes to NETosis and subsequent endothelial injury and thrombosis at sites of flow disturbance in arteries with erosion-like intimas; and ii) the clinically approved Jak-1,2 inhibitor, ruxolitinib (Rux), can preserve endothelial integrity and reduce thrombosis in mice with myeloid Jak2 V617F . Methods: We generated cohorts of mice harboring Jak2 V617F or wild-type Jak2 . We used a surgical preparation recapitulating features of superficial erosion and assessed endothelial integrity, thrombosis, and NET formation in relation to genotype and in vivo Rux treatment. Results and Conclusions: Evans blue extravasation following introduction of flow disturbance at sites of erosion-prone intimas showed significant impairment of endothelial barrier in the group of mice bearing the Jak2 V617F mutation compared to control (WT) mice ( ** p&lt;0.005). We further observed more thrombosis in mice bearing the Jak2 V617F mutation. Immunohistochemical evaluation of the regions of experimental superficial erosion revealed reduced endothelial continuity and increased NET accumulation in Jak2 V617F vs. WT mice. Rux treatment mitigated the adverse effects of Jak2 V617F on both NET accumulation and endothelial integrity, supporting the translational potential of these observations in individuals with clonal hematopoiesis due to Jak2 V617F and acute coronary syndromes.

Abstract 13614: Nck1 Regulates Endothelial Activation and Blood Brain Integrity After Ischemia Reperfusion Injury

Introduction: Stroke remains a leading cause of disease disability worldwide. Rapid restoration of blood flow after occlusion is required to minimize brain damage. However, with the reperfusion there is upregulation to oxidative stress pathways that stimulates interleukin-1 activates kinase type I (IRAK-1) within endothelial cells leading to proinflammatory genes expression (ICAM-1/VCAM-1). We previously showed that the Nck family of adaptor proteins promotes oxidative stress-induced inflammation following ischemia reperfusion (IRI). Nck1 and Nck2 are two highly similar proteins but have inherent functional differences. Hypothesis: We hypothesized that Nck1 is required for IRAK-1 activation in response to IRI. Methods: Human brain endothelial cells (ECs) were genetically silenced for Nck1, Nck2, or interleukin-1 receptor type I kinase-1 (IRAK-1) and exposed to hydrogen peroxide (exogenous oxidative stress) or hypoxia re-oxygenation (endogenous oxidative stress). Additionally, adult male mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) followed by 24h reperfusion. Neurological deficits were assessed using the 24 sensorimotor scoring system. Infarct volume, blood brain barrier (BBB) integrity, and brain inflammatory profiles were assessed using immunostaining and immunoblotting. Results: Knock down of endothelial Nck1 or Nck2 of cells markedly reduced VCAM-1 and ICAM-1 expression and NF-κB activation in response to H 2 O 2 and hypoxia re-oxygenation. In ECs treated with the proinflammatory mediator interleukin-1 (IL-1β), there was upregulation of IRAK-1 and NF-κB activation. However, deletion of Nck1, but not Nck2, significantly decreased IRAK-1/ NF-κB activation by IL-1β. Moreover, Nck1 inhibition decreased endothelial permeability-induced by oxidative stress. In response to oxidative stress, Nck1 directly binds to IRAK-1. Nck1 KO mice had significantly less infarct volume and neurological deficits after MCAO/reperfusion. BBB disruption measured by Evans Blue extravasation and Fibrinogen staining was remarkably reduced in Nck1 KO mice. Conclusions: Nck1 promotes ischemia/reperfusion-induced inflammation, and brain injury, therefore, Nck1 inhibition is a promising therapy to improve IRI.

Mibefradil and Flunarizine, Two T-Type Calcium Channel Inhibitors, Protect Mice against Lipopolysaccharide-Induced Acute Lung Injury.

Recent studies have illuminated that blocking Ca2+ influx into effector cells is an attractive therapeutic strategy for lung injury. We hypothesize that T-type calcium channel may be a potential therapeutic target for acute lung injury (ALI). In this study, the pharmacological activity of mibefradil (a classical T-type calcium channel inhibitor) was assessed in a mouse model of lipopolysaccharide- (LPS-) induced ALI. In LPS challenged mice, mibefradil (20 and 40 mg/kg) dramatically decreased the total cell number, as well as the productions of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF). Mibefradil also suppressed total protein concentration in BALF, attenuated Evans blue extravasation, MPO activity, and NF-κB activation in lung tissue. Furthermore, flunarizine, a widely prescripted antimigraine agent with potent inhibition on T-type channel, was also found to protect mice against lung injury. These data demonstrated that T-type calcium channel inhibitors may be beneficial for treating acute lung injury. The important role of T-type calcium channel in the acute lung injury is encouraged to be further investigated.

Molecular hydrogen alleviates brain injury and cognitive impairment in a chronic sequelae model of murine polymicrobial sepsis.

Sepsis-related encephalopathy (SAE), which causes a series of brain injuries and long-term, potentially irreversible cognitive dysfunction, is closely associated with increased morbidity and mortality. Hydrogen (H2) is a new type of medical gas molecule that has been widely used in the treatment of various diseases in recent years. The aim of the present study was to explore the protective effects of H2 inhalation on brain injury and long-term cognitive impairment in an improved chronic septic mouse model. Male C57BL/6J mice were randomized into four groups: Control, Control + H2, SAE and SAE + H2. The SAE and Control models were established by intraperitoneal injection of human stool suspension or saline in mice. H2 (2%) was inhaled for 60min at 1h and 6h after SAE or Control treatment. The survival rates were recorded for 14days (days 1-14) and the Morris Water Maze was performed for 7 days (days 8-14). To assess the severity of the brain injury, hematoxylin and eosin staining, Nissl staining, Evans blue (EB) extravasation and the wet/dry weight ratio of brain tissue were detected 24h after SAE or Control treatment. In addition, inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin 6 (IL-6), high-mobility group box 1 (HMGB1), as well as the protein levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), zonula occludens-1 (ZO-1) and Occludin, were measured 6, 12 and 24h after SAE or Control treatment. The results showed that H2 treatment increased survival rates, mitigated cognitive impairment, reduced hippocampal histological damage, decreased EB and water content, and decreased the levels of TNF-α, IL-6, HMGB1, Nrf2, HO-1, ZO-1 and Occludin, as compared with the SAE group. These data revealed that 2% H2 could suppress brain damage and improve cognitive function in septic mice by inhibiting oxidative stress, inflammatory response and the sepsis-induced blood-brain barrier (BBB) disruption.

Lithium enhances post-stroke blood-brain barrier integrity, activates the MAPK/ERK1/2 pathway and alters immune cell migration in mice

Lithium induces neuroprotection against cerebral ischemia, although the underlying mechanisms remain elusive. We have previously suggested a role for lithium in calcium regulation and (extra)cerebral vessel relaxation under non-ischemic conditions. Herein, we aimed to investigate whether or not lithium contributes to post-stroke stabilization of the blood-brain barrier (BBB) in mice. Using an oxygen-glucose-deprivation (OGD) model, we first analyzed the impact of lithium treatment on endothelial cells (EC) in vitro. Indeed, such treatment of EC exposed to OGD resulted in increased cell survival as well as in enhanced expression of tight junction proteins and P-glycoprotein. Additional in vivo studies demonstrated an increased stabilization of the BBB upon lithium treatment in stroke mice, as shown by a reduced Evans blue extravasation and an elevation of tight junction protein expression. Furthermore, stabilization of the BBB as a consequence of lithium treatment was associated with an inhibition of matrix metalloproteinase-9 activity, independent of calveolin-1 regulation. In line with this, flow cytometry analysis revealed that lithium treatment led to a decreased neutrophil invasion and an increased T cell extravasation from the blood compartment towards the brain parenchyma. We finally identified the pro-survival MAPK/ERK1/2 pathway as the key regulator of the impact of lithium on the BBB. In conclusion, we demonstrate for the first time that lithium is able to enhance post-stroke BBB integrity. Importantly, our work delivers novel insights into the exact mechanism of lithium-induced acute neuroprotection, providing critical information for future clinical trials involving lithium treatment in stroke patients.

Calcium imaging in population of dorsal root ganglion neurons unravels novel mechanisms of visceral pain sensitization and referred somatic hypersensitivity.

Mechanisms of visceral pain sensitization and referred somatic hypersensitivity remain unclear. We conducted calcium imaging in Pirt-GCaMP6s mice to gauge responses of dorsal root ganglion (DRG) neurons to visceral and somatic stimulation in vivo. Intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colonic inflammation and increased the percentage of L6 DRG neurons that responded to colorectal distension above that of controls at day 7. Colorectal distension did not activate L4 DRG neurons. TNBS-treated mice exhibited more Evans blue extravasation than did control mice and developed mechanical hypersensitivity in low-back skin and hind paws, which are innervated by L6 and L4 DRG neurons, respectively, suggesting that colonic inflammation induced mechanical hypersensitivity in both homosegmental and heterosegmental somatic regions. Importantly, the percentage of L4 DRG neurons activated by hind paw pinch and brush stimulation and calcium responses of L6 DRG neurons to low-back brush stimulation were higher at day 7 after TNBS than those in control mice. Visceral irritation from intracolonic capsaicin instillation also increased Evans blue extravasation in hind paws and low-back skin and acutely increased the percentage of L4 DRG neurons responding to hind paw pinch and the response of L6 DRG neurons to low-back brush stimulation. These findings suggest that TNBS-induced colitis and capsaicin-induced visceral irritation may sensitize L6 DRG neurons to colorectal and somatic inputs and also increase the excitability of L4 DRG neurons that do not receive colorectal inputs. These changes may represent a potential peripheral neuronal mechanism for visceral pain sensitization and referred somatic hypersensitivity.

Anti-hemorrhoidal activity of Lian-Zhi-San, a traditional Chinese medicine, in an experimental hemorrhoidal model in rats

ObjectiveHemorrhoidal disease (HD) is the most common proctological disease, with an estimated prevalence rate of 4.4%, and a peak in individuals between 45 and 65 years of age. This study was done to evaluate whether Lian-Zhi-San (LZS), a clinically used anti-hemorrhoidal ointment could alleviate the inflammatory injury, with its associated changes of inflammatory cytokines and morphology of anorectal tissues, in an experimental model of HD in rats. MethodsHD was induced by croton oil preparation (COP) applied to the anorectal region. Rats were then treated with cotton swabs soaked in LZS ointment, water or white vaseline, twice a day for 7 d. At the end of the experiment, HD was evaluated by measuring hemorrhoidal and biochemical parameters along with histopathological observations. ResultsIn this study, COP induced a significant increase in the macroscopic severity score, anorectal coefficient and Evans blue extravasation, compared to normal rats. Additionally, it greatly enhanced the expression and secretion levels of some important inflammation-related cytokines along with marked histological damage, compared to normal rats. Rats treated with LZS ointment experienced significantly ameliorated Evans blue extravasation (P < 0.05), decreased macroscopic severity score (0.86 ± 0.14 vs. 1.65 ± 0.16) and the anorectal coefficient (P < 0.01); its use also attenuated tissue damage and inhibited the expression and secretion levels of inflammation-related cytokines (interleukin-1β, interleukin-6 and tumor necrosis factor-α). ConclusionThis study validates a preliminary understanding of the use of LZS ointment to treat inflammatory factors and tissue damage in an experimental model of HD in rats.