Injected Evans Blue Dye Research Articles

Protection of the Vascular System by Polyethylene Glycol Reduces Secondary Injury Following Spinal Cord Injury in Rats.

Polyethylene glycol (PEG) is a hydrophilic polymer, which has been known to have a neuroprotective effect by sealing the ruptured cell membrane, but PEG effects on the vascular systems and its underlying mechanisms remain unclear. Here, we showed the neuroprotective effect of PEG by preventing damage to the vascular system. A spinal contusion was made at the T11 segment in male Sprague-Dawley rats. PEG was injected into the subdural space immediately after SCI. Vascular permeability was assessed for 24h after SCI using intraperitoneally injected Evans blue dye. Junctional complexes were stained with CD31 and ZO-1. Infarct size was analyzed using triphenyltetrazolium chloride, and blood vessels were counted in the epicenter. Behavioral tests for motor and sensory function were performed for 6weeks. And then the tissue-sparing area was assessed. Immediately applied PEG significantly reduced the vascular permeability at 6, 12, and 24h after SCI when it compared to saline, and infarct size was also reduced at 0, 6, and 24h after SCI. In addition, a great number of blood vessels were observed in PEG group at 6 and 24h after SCI compared to those of the saline group. The PEG group also showed a significant improvement in motor function. And tissue-sparing areas in the PEG were greater than those of the saline group. The present results provide preclinical evidence for the neuroprotective effects of PEG as a promising therapeutic agent for reducing secondary injury following SCI through vascular protection.

Abstract 15392: Radiation-Induced Coronary Microvascular Leakage is Mediated by Loss of Endothelial Tight Junction Protein Claudin-1

Background: Ionizing radiation (IR) exposure to the heart during cancer therapy induces coronary microvascular leakage. Increased microvascular porosity allows inflammatory cell extravasation into the perivascular and myocardial interstitial space leading to fibrotic end organ damage. Hypothesis: Radiation-induced coronary microvascular leakage is mediated by a loss of a constitutively expressed tight-junction protein, claudin-1 (cldn1). Methods: We studied the function of cldn1 in human coronary vascular endothelial cells (HMVECs) and in mice using oligonucleotide-based cldn1 gene deletion. For the gain of function, we developed vascular-specific (VE-cadherin promotor-linked) claudin-1 overexpressing transgenic (TG) mice. We studied the effect of IR in cldn1 loss and gain of function models. Capillary permeability was assessed by FITC-dextran permeability and Evans blue dye injection, in vitro and in vivo, respectively . The extent of end-organ damage was examined by analyzing myocardial and perivascular fibrosis. Results: In HMVECs, cldn1 gene silencing (control, 1.0±0.34; siRNA, 0.026±0.03; p<0.001) led to increased permeability (control, 1.0±0.01, siRNA, 1.3±0.01; p<001). In mice, genetically engineered anti-cldn1 siRNA administration knocked down myocardial cldn1 expression (control, 1.0±0.18; siRNA, 0.51±0.11; p=0.04). Functionally, cldn1 knockdown led to increased microvascular extravasation (control, 1.0±0.23, cldn1-siRNA 0.38±0.27 p=0.002). Cardiac morphometry in these mice showed myocardial fibrosis (control, 1.0±0.19; siRNA, 1.29±0.35; p=0.048). Cardiac irradiation significantly decreased myocardial cldn1 (control 1.0±0.27, IR 0.45±0.31; p=0.001) and dye leakage (control 1.0±0.18, IR 1.28±0.22; p=0.007). Conversely, endothelium-targeted cldn1 overexpression in mice prevented abnormal microvasculature permeability (WT 1.0±0.23, TG 0.38±0.27 p=0.002) and conferred protection from IR-induced fibroinflammatory changes. Conclusions: Our study shows that cldn1 is crucial for maintaining coronary microvascular permeability. Prevention of cldn1 degradation or cldn1 reconstitution using novel pharmacotherapeutic approaches can prevent cardiac end-organ damage induced by cardiac IR.

Influence of Acoustic Parameters and Sonication Schemes on Transcranial Blood–Brain Barrier Disruption Induced by Pulsed Weakly Focused Ultrasound

Pulsed ultrasound combined with microbubbles use can disrupt the blood–brain barrier (BBB) temporarily; this technique opens a temporal window to deliver large therapeutic molecules into brain tissue. There are published studies to discuss the efficacy and safety of the different ultrasound parameters, microbubble dosages and sizes, and sonication schemes on BBB disruption, but optimal the paradigm is still under investigation. Our study is aimed to investigate how different sonication parameters, time, and microbubble dose can affect BBB disruption, the dynamics of BBB disruption, and the efficacy of different sonication schemes on BBB disruption. Method: We used pulsed weakly focused ultrasound to open the BBB of C57/B6 mice. Evans blue dye (EBD) was used to determine the degree of BBB disruption. With a given acoustic pressure of 0.56 MPa and pulse repetitive frequency of 1 Hz, burst lengths of 10 ms to 50 ms, microbubbles of 100 μL/kg to 300 μL/kg, and sonication times of 60 s to 150 s were used to open the BBB for parameter study. Brain EBD accumulation was measured at 1, 4, and 24 h after sonication for the time–response relationship study; EBD of 100 mg/kg to 200 mg/kg was administered for the dose–response relationship study; EBD injection 0 to 6 h after sonication was performed for the BBB disruption dynamic study; brain EBD accumulation induced by one sonication and two sonications was investigated to study the effectiveness on BBB disruption; and a histology study was performed for brain tissue damage evaluation. Results: Pulsed weakly focused ultrasound opens the BBB extensively. Longer burst lengths and a larger microbubble dose result in a higher degree of BBB disruption; a sonication time longer than 60 s did not increase BBB disruption; brain EBD accumulation peaks 1 h after sonication and remains 81% of the peak level 24 h after sonication; the EBD dose administered correlates with brain EBD accumulation; BBB disruption decreases as time goes on after sonication and lasts for 6 h at least; and brain EBD accumulation induced by two sonication increases 74.8% of that induced by one sonication. There was limited adverse effects associated with sonication, including petechial hemorrhages and mild neuronal degeneration. Conclusions: BBB can be opened extensively and reversibly by pulsed weakly focused ultrasound with limited brain tissue damage. Since EBD combines with albumin in plasma to form a conjugate of 83 kDa, these results may simulate ultrasound-induced brain delivery of therapeutic molecules of this size scale. The result of our study may contribute to finding the optimal paradigm of focused ultrasound-induced BBB disruption.

Protection of Vasoactive Intestinal Peptide on the Blood-Brain Barrier Dysfunction Induced by Focal Cerebral Ischemia in Rats

ObjectiveTo investigate the effects of vasoactive intestinal peptide on the blood brain barrier function after focal cerebral ischemia in rats. Materials and methodsRats were intracerebroventricular injected with vasoactive intestinal peptide after a two hours middle cerebral artery occlusion. Functional outcome was studied with the neurological severity score. The brain edema and the infarction were evaluated via histology. The blood brain barrier permeability was assessed using Evans Blue dye injection method. We also measure the apoptosis of brain microvascular endothelial cells and brain levels of B-cell leukemia-2 protein by immunohistochemical analysis and western blotting, respectively. ResultsIn contrast to the cases treated with vehicle at 72 h after middle cerebral artery occlusion, the treatment with vasoactive intestinal peptide significantly (P < 0.05) reduced the neurological severity score, the brain edema and infarct volume. The Evans Blue leakage and brain water content were obviously reduced (P < 0.05) in vasoactive intestinal peptide-treated rats compared with those of control rats at 72 and 96 h after stroke. In addition, vasoactive intestinal peptide decreased the numbers of terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling positive endothelial cells and increased the protein levels of B-cell leukemia-2 in the ischemic hemisphere at 72 h after ischemia. ConclusionsOur data suggest that treatment with vasoactive intestinal peptide ameliorates the blood brain barrier function, contributing to reduce in brain damage both morphologically and functionally in the ischemic rat. This amelioration may be associated with attenuation in apoptosis of brain microvascular endothelial cells by increased B-cell leukemia-2 expression.

ABNORMAL LYMPHATIC PHENOTYPE IN A CRISPR MOUSE MODEL OF THE HUMAN LYMPHEDEMA-CAUSING CONNEXIN47 R260C POINT MUTATION

Connexin proteins form gap junctions controlling exchange of ions and small molecules between cells and play an important role in movement of lymph within lymphatic vessels. Connexin47 (CX47) is highly expressed in lymphatic endothelial cells and CX47 missense mutations, i.e., R260C, cosegregate with primary lymphedema in humans. However, studies utilizing CX47 knockout mice have failed to demonstrate any lymphatic anomalies. To unravel the lymphatic consequences of expressing a mutant CX47 protein, we used CRISPR technology to create a mouse carrying a Cx47 missense mutation (Cx47R259C) equivalent to the human CX47R260C missense mutation associated with human primary lymphedema. Intradermal Evans Blue dye injection identified a 2-fold increase in regional lymph nodes in homozygous Cx47R259C mice compared to wildtype, particularly in the jugular region (4.8 ± 0.4 and 2.0 ± 0.0, respectively, p<0.01). Associated lymphatic channels were increased in Cx47R259C mice and mesenteric lymph reflux occurred in homozygous Cx47R259C mice but not in wildtype. Contractility of superficial cervical lymphatics, assessed by pressure myography, was reduced in homozygous Cx47R259C mice compared to wildtype. In conclusion, our data are the first to demonstrate a role for the Cx47 protein in lymphatic anatomy and function. This phenotype is similar to that found with other valve deficient mouse mutants, e.g., in Foxc2. Of significance, this study is the first to use CRISPR technology to develop a pre-clinical model of primary lymphedema and demonstrates the importance of distinguishing between lack of and presence of mutant protein when developing clinically relevant animal models for translation of pre-clinical findings.

Endothelial CCR6 expression due to FLI1 deficiency contributes to vasculopathy associated with systemic sclerosis

BackgroundWe have recently demonstrated that serum CCL20 levels positively correlate with mean pulmonary arterial pressure in patients with systemic sclerosis (SSc). Considering a proangiogenic effect of CCL20 on endothelial cells via CCR6, the CCL20/CCR6 axis may contribute to the development of SSc vasculopathy. Therefore, we explored this hypothesis using clinical samples, cultured cells, and murine SSc models.MethodsThe expression levels of CCL20 and CCR6 in the skin, mRNA levels of target genes, and the binding of transcription factor FLI1 to the target gene promoter were evaluated by immunostaining, quantitative reverse transcription PCR, and chromatin immunoprecipitation, respectively. Vascular permeability was evaluated by Evans blue dye injection in bleomycin-treated mice. Angiogenic activity of endothelial cells was assessed by in vitro angiogenesis assay.ResultsCCL20 expression was significantly elevated in dermal fibroblasts of patients with early diffuse cutaneous SSc, while CCR6 was significantly up-regulated in dermal small vessels of SSc patients irrespective of disease subtypes and disease duration. In human dermal microvascular endothelial cells, FLI1 siRNA induced the expression of CCR6, but not CCL20, and FLI1 bound to the CCR6 promoter. Importantly, vascular permeability, a representative SSc-like vascular feature of bleomycin-treated mice, was attenuated by Ccr6 siRNA treatment, and CCR6 siRNA suppressed the angiogenic activity of human dermal microvascular endothelial cells assayed by in vitro tube formation.ConclusionsThe increased expression of endothelial CCR6 due to FLI1 deficiency may contribute to the development of SSc vasculopathy.

Stimulator of Interferon Genes (STING) regulates Re‐endothelialization Following Vascular Injury

Percutaneous coronary interventions (PCI) are commonly used to improve blood flow in Peripheral Artery Disease. Complications of PCI are delayed re-endothelialization and endothelial cell (EC) dysfunction leading to adverse clinical outcomes. Stimulator of Interferon Genes (STING), canonically activated downstream of the cGAS enzyme, is a signaling hub molecule modulating immune responses triggered by self-derived and/or foreign DNA. We hypothesized that EC-intrinsic STING regulates re-endothelialization in response to vascular injury. We modeled PCI in WT, STING-/-, and in a newly generated inducible vascular EC- specific mouse (VECad5Cre-ERT2STINGfl/fl mice: EC-STING-/-) using the wire induced carotid injury model. We used Evans Blue Dye injection post injury to identify the areas denuded of EC and quantified the % of non-blue (re-endothelialized) area. We found that WT mice had complete re-endothelialization five days post injury (95.16 ± 2.77 %). In contrast, injured carotid arteries in STING-/- mice exhibited a decrease in re-endothelialization at the same timepoint (43.57 ± 9.79%). Moreover, EC-STING-/- mice recapitulated the delayed re-endothelialization (10.45 ± 1.56% in Tamoxifen treated mice to induce excision of STING in EC, compared to 40 ± 1.28% observed in vehicle treated VECad5Cre-ERT2STINGfl/fl control mice 5 days post scratch) in the injured arteries, supporting a role of EC STING in re-endothelialization post injury. To further investigate this EC-specific effect of STING mechanistically in vitro, we performed scratch assays on WT and STING mouse heart EC (MHEC) monolayers, as well as on human EC monolayers lacking STING after CRISPR knock down and imaged wound healing in real time by IncuCyte. WT MHEC and HUVEC completely covered the scratch area within 24 hours, and 4 hours, respectively, whereas both EC lacking STING were delayed healing the scratch. Additionally, we found that the healing response was independent of cGAS, since cGAS KD HUVEC had similar post- scratch recovery time as control HUVEC. Ongoing experiments investigate the mechanisms involved in this novel STING dependent, cGAS-independent pathway of EC response to injury, as well as the consequences of EC-STING in adverse chronic vascular remodeling.

Acute cerebrovascular effects in juvenile coho salmon exposed to roadway runoff

Urban stormwater is responsible for recurrent mortality events of coho salmon (Oncorhynchus kisutch) documented in the US Pacific Northwest. Currently, the toxic mode of action is unknown; however, mortality is often associated with large increases in hematocrit (Hct). We found that hemoconcentration and red cell swelling may offer a partial explanation for increased Hct, based on increased total hemoglobin and reduced mean cell hemoglobin concentration of the blood in runoff-exposed juvenile coho salmon, relative to controls. No significant changes were seen in plasma total thiols or antioxidant power, discounting osmoregulatory dysfunction and plasma dehydration as a driver of hemoconcentration. To test for plasma leakage, we injected Evans blue dye complexed with bovine serum albumin (EBD–BSA) intracardially and circulated for 5 min, followed by a 15 min washout with saline. Coho salmon exposed to roadway runoff showed significant accumulation of EBD–BSA in brain and olfactory rosette regions compared with controls, demonstrating plasma leakage from the cerebrovasculature. Our results suggest blood–brain barrier disruption underlies symptom development in coho salmon exposed to roadway runoff.

Lymphangiogenesis near the Cribriform Plate after Cerebral Ischemia

Abstract Recently, it has been hypothesized that antigens, antigen-presenting cells (APC), and cerebrospinal fluid (CSF) may drain from the central nervous system (CNS) into lymphatics near the cribriform plate or dura to provide fluid homeostasis and antigen drainage. However, the role of lymphatic vessels during neuroinflammation is not well understood yet. Our lab has previously shown that lymphatic vessels near cribriform plate undergo lymphangiogenesis during experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis. Here, we looked to see whether lymphangiogenesis also occurs in a stroke mouse model of neuroinflammation. We established the transient middle cerebral artery occlusion (tMCAO) model of stroke by placing a filament for 60 minutes into the middle cerebral artery, that was followed by reperfusion after removing the filament. We show that lymphangiogenesis occurs near the cribriform plate, peaks at day 7 and decreases after 14 days following tMCAO. To confirm that the newly formed lymphoid vessels at the cribriform plate could connect with the CSF, we injected Evans Blue dye through the cisterna magna and confirmed the presence of the dye in these lymphoid vessels. Flow cytometry analysis showed that robust leukocyte infiltration such as microglia (CD45int, CD11b+), macrophage (CD45hi, CD11b+), dendritic cells (CD45hi, CD11b+, CD11c+), CD8 T cells (CD45hi, CD8+), CD4 T cells (CD45hi, CD4+), and B cells (CD45hi, B220+) near the cribriform plate after 7 days of tMCAO correlates with lymphangiogenesis (CD45−, Podoplanin+, CD31+). The manipulation of inflammation-induced lymphangiogenesis may lead to therapeutics for neuroinflammatory diseases.

TMAO Activates Carotid Endothelial Inflammasomes Leading to Enhanced Neointimal Formation in Nlrp3 Mice

Recent studies have shown that gut microbial metabolite, trimethylamine‐N‐Oxide (TMAO), is a biomarker for atherosclerosis. However, the mechanisms of how TMAO induces atherosclerosis are still unclear. The present study tested whether TMAO induces Nlrp3 inflammasome activation and consequent endothelial injury leading to atherosclerotic lesion development in the carotid arterial wall. Confocal microscopy showed that colocalization of Nlrp3 with ASC significantly increased in the intima of the carotid arteries of TMAO‐treated Nlrp3+/+ mice, suggesting the formation of Nlrp3 inflammasomes in the endothelium of these arteries. In Nlrp3−/− mice, however, such colocalization was much less detected. In consistent with inflammasome formation, TMAO‐induced IL‐1β production in the intima was abolished in Nlrp3−/− mice. Furthermore, we found that TMAO treatment decreased ZO2, VE‐cadherin and occuludin expression in carotid arteries of Nlrp3+/+ mice but not in Nlrp3−/− mice, signifying tight junction disruption in the carotid arteries of Nlrp3+/+ mice. In addition, we found that TMAO treatment had significantly increased vascular permeability to intravenously injected Evans blue dye compared to vehicle treated Nlrp3+/+ mice. However, such TMAO‐induced vascular permeability was attenuated in Nlrp3−/− mice, suggesting that Nlrp3 inflammasome activation plays an important role in TMAO‐induced vascular leakage in mouse hearts. Furthermore, we also found that TMAO markedly increased neointimal formation in partially ligated left carotid arteries (PLCA) of Nlrp3+/+ mice. However, such TMAO‐enhanced neointimal formation and increased I/M ratio in PLCA was markedly attenuated in Nlrp3−/− mice. These results suggest that the formation and activation of NLRP3 inflammasomes by TMAO may be an important mechanism in initiating endothelial inflammatory response leading to arterial inflammation and endothelial dysfunction and consequent atherosclerosis in mice.Support or Funding InformationR01DK104031 and R56HL143809

Deficiency of TREK-1 potassium channel exacerbates blood-brain barrier damage and neuroinflammation after intracerebral hemorrhage in mice

BackgroundIntracerebral hemorrhage (ICH) is a devastating medical emergency with high mortality and severe neurological deficit. ICH-related poor outcomes are due to a combination of pathological processes that could be complicated by secondary insults. TWIK-related K+ channel 1 (TREK-1) is a two-pore-domain potassium channel that is highly expressed in the mammalian nervous system. Previous studies have shown that TREK-1 channels play important roles in various central nervous system diseases. However, its role in the secondary injuries after intracerebral hemorrhage remains unknown. In this study, we explored the function of TREK-1 in secondary blood-brain barrier injuries and neuroinflammation after intracerebral hemorrhage in mice.MethodsAdult male TREK-1−/− mice and WT mice were subjected to a collagenase-induced ICH model. Immunostaining, western blot, and enzyme-linked immunosorbent assay were used to assess inflammatory infiltration and neuronal death. Blood-brain barrier compromise was assessed using electron microscopy and Evans Blue dye injection on days 1 and 3 after intracerebral hemorrhage. Magnetic resonance imaging and behavioral assessments were conducted to evaluate the neurologic damage and recovery after intracerebral hemorrhage.ResultsGenetic deficiency of TREK-1 channel exacerbated blood-brain barrier impairment and promoted cerebral edema after intracerebral hemorrhage. Meanwhile, TREK-1 deficiency aggravated focal inflammatory featured by the increased recruitment of microglia and neutrophils, the enhanced secretion of proinflammatory factors interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and cell adhesion molecules (CAMs). Furthermore, TREK-1 deficiency promoted neuronal injury and neurological impairment.ConclusionsThese results establish the first in vivo evidence for the protective role of TREK-1 in blood-brain barrier injury and neuroinflammation after intracerebral hemorrhage. TREK-1 may thereby be harnessed to a potential therapeutical target for the treatment of intracerebral hemorrhage.

NG291: A Stable B2 Kinin Receptor Agonist that Increases the Permeability of Normal Blood Brain Barrier: Possible Role in Organophosphate Poisoning Treatment

Organophosphates are phosphorus containing compounds that inhibit acetylcholinesterase and generate a cholinergic crisis that is fatal if not treated. The classical antidotes prescribed are atropine and pralidoxime. They can protect peripherally, but cannot protect the central nervous system. Pralidoxime is the only FDA approved drug that re‐activates the acetylcholinesterase inhibited by organophosphate. Pralidoxime has limited neuroprotective potential because it is not able to cross the blood brain barrier (BBB) effectively. In order to increase this potential, our group is using a stable B2 kinin agonist NG291. This peptide can transiently open the normal blood brain barrier and increase the pralidoxime delivery to the brain after organophosphate poisoning. Sprague‐Dawley (SD) rats were subjected to NG291 treatment (50μg/Kg &amp; 100μg/Kg) via intravenous (IV) route followed by 2% Evans Blue dye injection. 24 hours after the peptide injection the animals were perfused, and the amount of albumin‐Evans blue that reached the brain was qualitatively analyzed. To verify if the transient opening of blood brain barrier causes increase in the brain water content, controls (saline) and rats injected with the peptide NG291 were euthanized 24 hours after the peptide injection and the percentage of water content was measured. To assure that this transient opening is not causing damage to the brain, control and tests were submitted to analysis by histology using Fluoro Jade to verify the presence of dead neurons and glial fibrillary acidic protein (GFAP) to evaluate the astrocyte activation 72 hours after the injection of NG291. NG291‐treated animals have shown infiltration of Evans Blue to the brain parenchyma and accumulation in the ventricles showing increase of blood brain barrier permeation 24 hours after the injection. The water content of the brain did not increase with NG291 treatment. 72 hours after the treatment the histology using Fluoro Jade and GFAP did not show significant changes when compared to controls. We also determined for how long the BBB remains permeable following NG291 administration. In future studies, this peptide will be used to facilitate the delivery of pralidoxime into the brain and therefore increase the overall effect of this drug. This is done in hope to provide those currently exposed to organophosphates with an immediate treatment strategy.Support or Funding InformationUniversity of Puerto Rico Medical Sciences Campus RCMI‐8G12MD007600 Dr. Pedro Ferchmin and Dr. Vesna Eterovic, Universidad Central del CaribeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Los neuropéptidos SP y CGRP subyacen en las propiedades eléctricas de los puntos de acupuntura

Electrical skin measurements at acupuncture points (acupoints) have been utilized as a diagnostic and therapeutic aid for more than 50 years. Although acupoints are described as having distinct electrical properties, such as high conductance and low impedance, the underlying mechanisms are currently unknown. The present study investigated in a rat model of hypertension whether the high conductance at acupoints is a result of the release of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) during neurogenic inflammation in the referred pain area. When plasma extravasation from neurogenic inflammation was examined by exploring the leakage of intravenously injected Evans blue dye (EBD) to the skin, extravasated EBD was found most frequently in acupoints on the wrist. The increased conductance and temperature at these acupoints occurred during the development of hypertension. The increase in conductance and plasma extravasation at acupoints in hypertensive rats was ablated by cutting median and ulnar nerves, blocking small diameter afferent fibers with resiniferatoxin (RTX) injection into median and ulnar nerves, or antagonizing SP or CGRP receptors in acupoints. In turn, intradermal injection of SP or CGRP resulted in increased conductance and plasma extravasation in naïve rats. Elevated levels of SP and CGRP were found in the acupoints of hypertensive rats. These findings suggest that the high conductance at acupoints is due to vascular leakage following local release of SP and CGRP during neurogenic inflammation.

Neuropeptides SP and CGRP Underlie the Electrical Properties of Acupoints.

Electrical skin measurements at acupuncture points (acupoints) have been utilized as a diagnostic and therapeutic aid for more than 50 years. Although acupoints are described as having distinct electrical properties, such as high conductance and low impedance, the underlying mechanisms are currently unknown. The present study investigated in a rat model of hypertension whether the high conductance at acupoints is a result of the release of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) during neurogenic inflammation in the referred pain area. When plasma extravasation from neurogenic inflammation was examined by exploring the leakage of intravenously injected Evans blue dye (EBD) to the skin, extravasated EBD was found most frequently in acupoints on the wrist. The increased conductance and temperature at these acupoints occurred during the development of hypertension. The increase in conductance and plasma extravasation at acupoints in hypertensive rats was ablated by cutting median and ulnar nerves, blocking small diameter afferent fibers with resiniferatoxin (RTX) injection into median and ulnar nerves, or antagonizing SP or CGRP receptors in acupoints. In turn, intradermal injection of SP or CGRP resulted in increased conductance and plasma extravasation in naïve rats. Elevated levels of SP and CGRP were found in the acupoints of hypertensive rats. These findings suggest that the high conductance at acupoints is due to vascular leakage following local release of SP and CGRP during neurogenic inflammation.

Water intake increases mesenteric lymph flow and the total flux of albumin, long-chain fatty acids, and IL-22 in rats: new concept of absorption in jejunum.

The traditional Japanese health care custom recommends that a suitable volume of water is consumed. However, physiological and immunological mechanisms in support of this practice are unknown. Therefore, we conducted rat and rabbit in vivo experiments to investigate the effects of intragastric administration of distilled water on the jejunal-originated lymph flow and the concentrations and total flux of cells, albumin, long-chain fatty acids, and innate lymphoid cell 3 (ILC-3)-secreted interleukin-22 (IL-22) through mesenteric lymph vessels. The distribution and activity of ILC-3 in rat small intestine by water intake were evaluated using flow cytometry and RT-PCR. The intragastric administration of distilled water caused significant increases in rat mesenteric lymph flow and in the total flux of cells, albumin, long-chain fatty acids, and IL-22 through the lymph vessels. Intravenously injected Evans blue dye was rapidly transported into rabbit mesenteric lymph vessel and cisterna chyli. The distribution of ILC-3 and the expression of IL-22 mRNA were maximal in the lamina propria cells of the rat jejunum. No significant presence of ILC-3 in the lymph was observed in the control and under water intake conditions. In conclusion, the absorbed water in the jejunum is transported through mesenteric lymph vessels. The higher permeability of albumin in the jejunal microcirculation may play key roles in the transport of consumed water and the reservoir and transporter of long-chain fatty acids. Water intake also accelerates the transfer of IL-22 to the mesenteric lymph, which may contribute, in part, to maintaining and promoting the innate immunity in the body. NEW & NOTEWORTHY The higher permeability of albumin-mediated transport of water-soluble substances in mesenteric lymph vessels of the jejunum may have a large impact on the classic concept suggesting that water-soluble small molecules travel to the liver via the portal vein. ILC-3 is mainly housed in the lamina propria of the jejunum, especially its upper part. IL-22 released from the ILC-3 is also transported through mesenteric lymph in collaboration with the albumin-mediated movement of consumed water.

Vascular delivery of intraperitoneal Evans blue dye into the blood-brain barrier-intact and disrupted rat brains.

Blood-brain barrier (BBB) integrity can be determined by tracer infusion into the circulation followed by measurements of its penetration into the brain parenchyma. Tracer injection through the intraperitoneal (i.p.) route (rather than intravascular injection) avoids confounding effects of animal anesthesia or immobilization/surgical stress. Evans blue dye (EBD) can be administered by i.p. injection, and once in circulation, it binds to plasma albumin to become an endogenous protein tracer. Here, we investigated whether a similar level of EBD is extravasated into the brain following i.p. versus intravenous (i.v.) injection in rats. In comparison with i.v. EBD injection, i.p. EBD injection resulted in much of the tracer residing in the peritoneal cavity. Accordingly, comparatively less EBD was found in the blood, liver, or brain of BBB-intact rat. In addition, following unilateral osmotic BBB disruption, i.v. but not i.p. EBD stained the ipsilateral hemisphere blue. Nevertheless, following either route of tracer administration in these rats, spectrophotometric quantification detected more EBD in the ipsilateral (BBB-disrupted) than in the contralateral hemisphere. Taken together, in contrast to a recent report, we found that i.p. EBD resulted in less tracer in circulation and in peripheral/central organs than EBD delivered i.v. We nevertheless conclude that i.p. EBD delivered sufficient tracer for the detection of regional BBB disruption.

Visualization of endothelial barrier damage prior to formation of atherosclerotic plaques.

En-face fat staining is frequently used to visualize atherosclerotic lesions. This method, however, is not suitable to visualize endothelial barrier damage prior to microscopically detectable morphological alterations of the arterial wall such as sub-endothelial lipid deposition. To enable the investigation of early endothelial barrier damage and in particular the initial steps of atherosclerosis, a new method has to fulfill three requirements: (i) easy and fast to perform, (ii) low cost of applicability without requirement for highly sophisticated technical equipment, and (iii) reliable reproducibility of valid results. To this end, we used intracardial Evans blue dye injection after washout of blood and measured dye deposition within the aortic wall as a parameter of endothelial barrier leakiness, which is recognized as one of the earliest signs of atherosclerotic plaque formation. These analyses were performed in ApoE -/-, LDL receptor -/- and Cc1 -/- mouse models which have been reported to develop aortic plaques with or without high cholesterol diet. Our data show that sub-endothelial dye deposition is a reliable and reproducible readout parameter to assess endothelial barrier damage. Along these lines, measurements of aortic intima areas with Evans blue deposition in relation to total intima circumference enabled quantitative assessments of the results. Our technique enables the imaging of endothelial barrier damage prior to detectable aortic lipid deposition and plaque development. Thus, it will facilitate the detection of the initial vascular pathogenetic processes that lead to cardiovascular diseases. It will also enable the testing of new drugs and therapeutic procedures to prevent these disorders.

Effects of GSM modulated radio-frequency electromagnetic radiation on permeability of blood–brain barrier in male & female rats

With the increased use of mobile phones, their biological and health effects have become more important. Usage of mobile phones near the head increases the possibility of effects on brain tissue. This study was designed to investigate the possible effects of pulse modulated 900MHz and 1800MHz radio-frequency radiation on the permeability of blood–brain barrier of rats. Study was performed with 6 groups of young adult male and female wistar albino rats. The permeability of blood-brain barrier to intravenously injected evans blue dye was quantitatively examined for both control and radio-frequency radiarion exposed groups. For male groups; Evans blue content in the whole brain was found to be 0.08±0.01mg% in the control, 0.13±0.03mg% in 900MHz exposed and 0.26±0.05mg% in 1800MHz exposed animals. In both male radio-frequency radiation exposed groups, the permeability of blood–brain barrier found to be increased with respect to the controls (p<0.01). 1800MHz pulse modulated radio-frequency radiation exposure was found more effective on the male animals (p<0.01). For female groups; dye contents in the whole brains were 0.14±0.01mg% in the control, 0.24±0.03mg% in 900MHz exposed and 0.14±0.02mg% in 1800MHz exposed animals. No statistical variance found between the control and 1800MHz exposed animals (p>0.01). However 900MHz pulse modulated radio-frequency exposure was found effective on the permeability of blood-brain barrier of female animals. Results have shown that 20min pulse modulated radio-frequency radiation exposure of 900MHz and 1800MHz induces an effect and increases the permeability of blood-brain barrier of male rats. For females, 900MHz was found effective and it could be concluded that this result may due to the physiological differences between female and male animals. The results of this study suggest that mobile phone radation could lead to increase the permeability of blood-brain barrier under non-thermal exposure levels. More studies are needed to demonstrate the mechanisms of that breakdown.

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye.

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.