Evans Blue Research Articles

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

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

Mechanisms of hypoxia in the hippocampal CA3 region in postoperative cognitive dysfunction after cardiopulmonary bypass

BackgroundPostoperative cognitive dysfunction (POCD) is a complication with high morbidity and mortality, commonly observed in the elderly who underwent anesthesia and surgery. The incidence is much higher in cardiac surgery. However, the reason and the mechanism of POCD remains unclear, but cerebral hypoxia is a common neurological complication after cardiac surgery. This study aims to investigate what role cerebral hypoxia plays in the pathogenesis of POCD.MethodsThe POCD model was established using cardiopulmonary bypass (CPB) surgery. Cognitive function was detected using Y maze and Morris water maze. The hypoxia in central nervous system was assessed using HE staining, western blot, and immunofluorescence. Inflammatory factors in hippocampus and plasma were detected by enzyme-linked immunosorbent assay. Evans blue was used to detect destruction of the blood brain barrier (BBB).ResultsCognitive impairment markedly occurred to rats underwent 2-h CPB operation. Cerebral thrombosis and hypoxia occurred in the hippocampal CA3 region of rats after surgery. In addition, microglia in hippocampal was activated and the expression of inflammatory factors such as IL-1β, IL-6 and TNF-α was upregulated. Moreover, the permeability of BBB increased in rats after CPB.ConclusionHypoxia in hippocampal CA3 region was involved in the occurrence and the mechanism may be associated with neuroinflammation and the damage of BBB.

Maresin 1 improves cognitive decline and ameliorates inflammation and blood-brain barrier damage in rats with chronic cerebral hypoperfusion

BackgroundChronic inflammation and blood–brain barrier destruction are interrelated pathological changes in chronic cerebral hypoperfusion (CCH) that promote vascular cognitive impairment (VCI). Therefore, we discussed the impact of the macrophage mediator in resolving inflammation 1 (Maresin 1) on the CCH-induced cognitive impairment and its underlying mechanisms. Methods66 rats were randomly divided into three groups: Sham (n = 22), 2VO (n = 22), and 2VO + MaR1 (n = 22). Rats in three groups received 2-Vessel Occlusion (2VO) or sham operation and received intrathecal delivery of PBS or MaR1. Hippocampal blood flow and Modified neurological severity scores (mNSS) were used to confirm models’ effect. Blood-brain barrier (BBB) damage was assessed by Evans blue (EB) leakage experiments and spectrophotometry, the BBB ultrastructure was observed with a transmission electron microscope (TEM), and the expression of zonula occluden-1 (ZO-1), claudin-5, and matrix metalloproteinases-9 (MMP-9) were detected with Enzyme-Linked Immunosorbent Assay (ELISA). Morris water maze (MWM) was used to assess cognitive function. Tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and nuclear factor-κB (NF-κB) expression were examined by Western blotting (WB) and ELISA. Immunofluorescence was used to detect microglia, astrocytes and oligodendrocytes. ResultsRats developed obvious cognitive impairment by CCH. BBB showed EB leakage, ultrastructural destruction, degradation of ZO-1, Claudin-5, and up-regulation of MMP-9. Inactivation of oligodendrocytes, activation of microglia and astrocyte and increased expression of NF-κB, TNF-α, and IL-1β has been detected. MaR1 administration significantly reverted these changes. ConclusionMaR1 can improve the CCH-induced cognitive impairment. Inflammatory resolution and BBB protection may be the mechanism of MaR1 to prevent CCH-induced cognitive impairment.

Evans Blue Dye Interferes with Gating for Certain Fluorochrome‐Conjugated Antibodies Using Flow Cytometry in the Ischemic Rat Heart

BackgroundAcute myocardial infarction (AMI) is the leading cause of death in the U.S. Understanding the immune response to AMI has potential therapeutic impact. In particular, the differentiation of monocytes into macrophages post‐AMI is of interest given their role in inflammatory cytokine production and tissue repair. Recent studies suggest that impaired macrophage (Mϕ) polarization may promote a proinflammatory phenotype in the heart post‐AMI. Flow cytometry is a gold standard technique to assess immune cell populations including M1 and M2 Mϕ subtypes, using a wide range of fluorochrome‐conjugated antibodies (FC‐Abs). Evans Blue (EB) dye is widely used to assess the size of AMI, but the feasibility of the combining approaches due to spectral overlap is uncertain. Thus, the objective of this study was to determine whether the cardiac viability dye EB can be used simultaneously with FC‐Abs to quantify M1 and M2 Mϕ in cardiac tissue post‐AMI.MethodsImmune cells were isolated from ischemic rat hearts (31 min ischemia/48 hr reperfusion) with or without EB perfusion and stained with FC‐Abs (1 μg/1 × 106 cells) to the following cell surface markers: FITC‐CD45 (experiment 1) or AF750‐CD45 (experiment 2) to detect leukocytes; Pacific Blue‐CD11b, AF700‐CD68 and PE‐CD206 (experiment 1) or Pacific Blue‐CD11b, AF488‐CD68 and PE‐CD206 (experiment 2) to quantify M1 and M2 Mϕ. A total of 500,000‐1,000,000 events were acquired using a BD LSR‐Fortessa and were analyzed and plotted using FlowJo software v10.ResultsOverlap of EB (emission peak at 680 nm) was detected in the AF700‐CD68 channel (emission peak at 720nm) and the AF750‐CD45 channel (emission peak at 775nm), interfering with gating within these two channels. In experiment 1, the M1 (CD45+CD11b+CD68+) population differed between samples with EB (2.5% of CD45+CD11b+ population, sample size n=2) vs without EB (68.5%, n=2). However, in experiment 2, the M1 population was similar between samples with EB (54.3% of CD45+CD11b+ population, n=2) and without EB (48.7%, n=1). The M2 (CD45+CD11b+CD206+) population did not differ in experiment 1 or 2.ConclusionOur data suggest that the EB perfusion interferes with fluorochromes with emission peaks close to 680nm. Thus, caution must be taken when selecting FC‐Abs for flow cytometry when combined with EB perfusion or other related dyes. Our next steps will be to confirm these observations in a larger sample size.

Timeline of Multi‐organ Plasma Extravasation After Bleomycin‐Induced Acute Lung Injury

Acute lung injury (ALI) is characterized by the abrupt onset of clinically significant hypoxemia with the presence of pulmonary edema. ALI is associated with cytokine release and plasma extravasation (PE) that can cause pulmonary edema and subsequently acute respiratory distress syndrome (ARDS). Therefore, it is critical we understand the relationship between ALI and lung PE. In addition, it is also important to assess PE in the lungs and other organs post‐ALI since ALI/ARDS often causes multi‐organ failure. We hypothesized that ALI induces time‐dependent lung PE, which promotes extravasation in the heart, liver, kidney, spleen, pancreas, and gastrointestinal (GI) tract, in a time‐dependent manner. To test our hypothesis, we administered bleomycin or saline via tracheal intubation in 8‐week‐old Sprague Dawley rats to create ALI. At the terminal experiments, Evans Blue was injected (IV) through the femoral vein to allow for the visualization of PE. Plasma extravasation of desired organs was evaluated at 3‐, 7‐, 14‐, 21‐, and 28‐days after bleomycin or saline treatment by evaluating Evans Blue concentrations calorimetrically at the absorption maximum for Evans blue (620 nm). Data show that ALI induces lung PE beginning at day 3 and peaking between 7 and 21 days. Extravasation can also be seen in all organs at varying degrees beginning at day 3 and peaking between day 7 and 14. Resolution appears to start after day 21 and continues past day 28. We conclude that ALI caused by bleomycin incites a time‐dependent PE of the lungs and multiple other organs.

LPS‐induced endotoxemia promotes blood‐brain barrier permeability via TSP1‐dependent TGFβ1 activation

IntroductionDuring disease states, the blood‐brain barrier (BBB) can become permeable due to endothelial injury or dysregulation of tight junction proteins. Endotoxemia, which induces systemic inflammation, causes BBB dysfunction and neuroinflammation. We have shown that transforming growth factor beta 1 (TGFβ1) is upregulated during systemic inflammation and promotes BBB permeability by disrupting claudin‐5 and occludin. Therefore, we hypothesize that TGFβ1 is upregulated during endotoxemia and induces BBB permeability by disrupting tight junction proteins.MethodsC57Bl/6 mice were administered 3 mg/kg of LPS and monitored for neurological decline. In a second set of mice 6 hours after LPS administration, an antagonist to TGFβ1 activation, LSKL, or control, SLLK, were injected (1 mg/kg/ip). Mice were euthanized 24 hours after LPS injection and tissue was collected. In a subset of animals, 3 hours prior to euthanasia, Evans Blue was injected (8%/ip) to assess BBB function. TGFβ1, claudin‐5, occludin, IL‐1β, IL6 and TNFα expression was assessed by RTPCR, immunoblotting and immunofluorescence. In vitro studies using mouse brain endothelial cells (bEnd.3 cells) were treated with LPS (10 μg/ml) or rTSP1 (1 μg/ml) to activate TGFβ1. TGFβ1, TSP1, claudin‐5 and occludin expression was assessed by immunofluorescence or RTPCR. Barrier function was determined in bEnd.3 cell monolayers using TEER or 10‐kDa FITC‐dextran diffusion. Data were analyzed using GraphPad Prism with results presented as mean +/‐ SEM. Differences between two groups were compared using Student’s t‐test and differences between three groups were determined using ANOVA.ResultsTGFβ1 expression was increased in cortex and cerebellum of LPS‐treated mice. This was associated with decreased claudin‐5 and occludin mRNA expression and immunofluorescence staining. Treatment of bEnd.3 cells with LPS increased TGFβ1 and TSP1 expression, decreased TEER, and increased FITC‐dextran diffusion. Supplementation of bEnd.3 cells with rTSP1 led to decreased claudin‐5 and occludin mRNA expression, decreased TEER, and increased FITC‐dextran diffusion across the transwell. LSKL treatment in LPS‐treated mice reduced sickness behavior, reduced Evan’s blue penetrance into the brain, restored tight junction expression and alleviated neuroinflammation when compared to SLLK‐treated mice.ConclusionsStrategies to target TGFβ1 signaling may prove effective at alleviating endotoxin‐induced BBB dysfunction.

Abstract 362: Endothelial Increase In Autotaxin Regulates Cerebral Vascular Permeability During Ischemic Reperfusion

Introduction: Pathological progression of stroke is aided by ischemic reperfusion during the treatment procedure. Various molecular signals initiated during ischemic reperfusion exacerbates the disease leading to endothelial permeability. However, a detailed molecular therapeutic regimen with r-TPA and mechanical thrombectomy could limit the detrimental effects. Lysophosphatidic acid (LPA) is a bioactive phospholipid regulated in physiological and pathological conditions. LPA is produced by the enzyme autotaxin (ATX). ATX-LPA axis have been observed to deteriorate physiological status in various diseases. In cerebral ischemic-reperfusion, the ATX-LPA axis could regulate the endothelium disruption. Hypothesis: Increased ATX expression in endothelium disrupts the endothelial barrier by increasing LPA during ischemic-reperfusion. Methods: For the in-vitro study, oxygen and glucose deprivation and reperfusion (OGDR) were carried out in mouse brain microvascular endothelial cells (MBMEC).For invivo transient middle cerebral artery occlusion with 90 minutes of ischemia and 24 hours of reperfusion (I/R) was used as a stroke model in mice. Electric Cell Substrate Impedance System (ECIS) and Evan Blue was used for permeability study for invitro and invivo respectively. AR-2 probe fluorescence assay was used to measure ATX activity. Results: ATX expression was increased (P<0.05) in MBMEC with OGDR suggesting ATX regulation in endothelial cells following ischemic-reperfusion. Permeability measured with ECIS in MBMEC showed that LPA treatment increased the permeability in a time and concentration-dependent manner. The protein levels of junctional proteins such as βcatenin, Claudin5, VEcadherin, and Zo1 weresignificantly (P<0.05) decreased with LPA exposure. In invivo stroke model ATX enzymatic activity is prominently raised (P<0.01) in I/R mice compared to sham mice. Increased ATX activity coheres with the Evans Blue permeability in the mice brain. Simultaneously LPA levels are elevated in I/R mice. LPA produced during the process can disrupt the blood-brain barrier, increasing cerebral permeability. Conclusion: Inhibition of the ATX-LPA axis prospects to be a better therapeutic avenue for cerebral ischemic reperfusion.

Minocycline Pretreatment Prevents Blood-Brain Barrier Disruption in Septic Rats.

The aim of the study was to explore the mechanism by which minocycline protects the blood-brain barrier (BBB) in septic rats. A sepsis rat model was generated in healthy, male Sprague-Dawley rats by cecal ligation and puncture (CLP). The rats were randomly divided into four groups and treated as follows: sham-operated plus normal saline (Sham+S group), CLP plus normal saline (CLP+S group), CLP plus minocycline pretreatment (CLP+M1 group), and CLP plus minocycline treatment (CLP+M2 group). Rats in the CLP+M1 group received 45mg/kg minocycline by intraperitoneal injection every 12h for 72h. Rats in the Sham+S and CLP+S groups were injected with the same volume of normal saline every 12h for 72h. Rats in the CLP+M2 group were intraperitoneally injected with 45mg/kg minocycline immediately after CLP and once every 12h for 72h. All rats were sacrificed at 72h after operation. Tumor necrosis factor α and interleukin 6 levels, the expression of ionized calcium-binding adaptor molecule-1 (Iba-1), and the permeability of the BBB were measured. The expression of matrix metalloproteinases-9 (MMP-9) and the tight junction proteins zonula occludens-1 (ZO-1) and occludin was detected by Western blot. In addition, Evans blue (EB) staining, immunohistochemistry, and ELISA analysis were carried out. Minocycline pretreatment significantly inhibited microglial activation, decreased the sepsis-induced expression of MMP-9, increased the expression of ZO-1 and occludin, and improved the permeability of the BBB. Minocycline treatment failed to inhibit microglial activation, decrease the sepsis-induced expression of MMP-9, increase the expression of ZO-1 or occluding, or improve the permeability of the BBB. Minocycline pretreatment can effectively improve the altered permeability of the BBB caused by sepsis. The mechanism may be related to the inhibition of microglial activation and MMP-9 expression and increased expression of ZO-1 and occludin.

MP07-14 DIABETIC BLADDER DYSFUNCTION IN MALE AKITA MICE PROGRESSES DIRECTLY TO AN UNDERACTIVE PHENOTYPE

You have accessJournal of UrologyCME1 May 2022MP07-14 DIABETIC BLADDER DYSFUNCTION IN MALE AKITA MICE PROGRESSES DIRECTLY TO AN UNDERACTIVE PHENOTYPE Armand Allkanjari, Francis Hughes, and J. Todd Purves Armand AllkanjariArmand Allkanjari More articles by this author , Francis HughesFrancis Hughes More articles by this author , and J. Todd PurvesJ. Todd Purves More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002529.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Diabetic Bladder Dysfunction (DBD) is a common complication of diabetes that may present with urinary frequency and urgency (i.e. overactive bladder (OAB)), detrusor underactivity and bladder decompensation (underactive bladder (UAB)), or some combination thereof. In an effort to define the etiology of this complication and develop models of its development, we have characterized the appearance of DBD in Akita Type 1 diabetic mice. Beginning with the female, which develop less severe diabetes, we demonstrated a transition from OAB at 15 weeks to UAB at 30. The purpose of this study is to explore the progression of DBD in the male mice of this diabetic model. We also explore a role for inflammation in this progression. METHODS: Four groups of mice (non-diab/NLRP3+/+, non-diab/ NLRP3-/-, diab/NLRP3+/+, and diab/NLRP3-/-) were evaluated at four different ages, 7, 10, 15 and 30 weeks. Awake, restrained cystometry was performed 7 to 9 days after suprapubic tubes (SPT) were implanted in the bladder. Blood glucose levels were measured prior to SPT placement. The extravasation of Evans blue dye assessed inflammation in the bladder. All parameters were assessed by ANOVA followed by Student-Newman-Keul’s post-hoc using GraphPad InStat software (La Jolla, CA). Statistical significance was defined as p<0.05. RESULTS: Male mice at 7 weeks showed dramatically increased blood sugar at levels >500 mg/dL which were sustained through 30 weeks of age. In contrast, blood glucose in female diabetic mice are ≈280 mg/dL during this time. At 7 weeks we found there was no difference in void volume or urinary frequency between diabetic and non-diabetic mice (n=8,9). Beginning at 10 weeks the diabetic mice develop signs of UAB with a 55% decrease in urinary frequency and a 240% increase in void volume (n=9/11). At 15 (n=7/6) and 30 weeks (n=10/4) the decrease in frequency was similar (62% and 67%) as was the increase in void volume (305% and 312%). Surprisingly, unlike the females, deletion of the NLRP3 gene did not significantly affect any cystometric parameters. Using Evans blue we confirmed that inflammation was triggered in the bladder by diabetes and was blocked by deletion of NLRP3. In summary, DBD in male Akita mice presents initially as UAB and this pathophysiology does not change over time. While direct development of UAB is correlated with severity of hyperglycemia, NLRP3-dependent inflammation does not seem to play a dominant role. CONCLUSIONS: Development of DBD in male Akita takes a significantly different trajectory from the female suggesting, besides NLRP3-mediated inflammation, alternative pathways can mediate the development of DBD. Source of Funding: Funding: NIDDK (R01DK117890) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e120 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Armand Allkanjari More articles by this author Francis Hughes More articles by this author J. Todd Purves More articles by this author Expand All Advertisement PDF DownloadLoading ...

Nonselective Cation Channels Maintain Alveolar Fluid Clearance After Exposure to Toxins from Respiratory Pathogens

Normal gas exchange in the alveoli is facilitated by a thin fluid layer. This layer is carefully regulated by two ion channels: highly selective channels (HSC or ENaC) and nonselective cation channels (NSC). Previous research found that both channels contribute about equally to alveolar fluid clearance (AFC). Since the molecular composition of NSC is a recent discovery, not much is known about NSC’s response to various respiratory pathogens. NSC are composed of an alpha‐ENaC subunit and at least one acid‐sensing ion channel 1a (ASIC1a) subunit. Our study aims are to understand how NSC contribute to alveolar epithelial permeability and how the lungs respond to toxins from respiratory pathogens. We hypothesize that AFC will be lower in the absence of ASIC1a (ASIC1 KO) when exposed to toxins from respiratory pathogens: Lipopolysaccharide (LPS) from E. coli and Pneumolysin (PLY) from P. pneumoniae. Additionally, we hypothesize that ASIC1 KO animals will have increased baseline alveolar epithelium permeability. All animal experiments were approved by Emory’s IACUC.We introduced pathogen toxins via intratracheal instillation to ASIC1 KO (KO) and wildtype (WT) mouse models (both on a C57Bl6 background). After a four‐hour incubation period, we euthanized the mice by isoflurane overdose. We determined AFC by instilling their lungs with 0.7ml of a 2.0% Evans Blue (EB) or Fluorescein isothiocyanate–dextran (FITC) solution via the trachea. We took samples at t=0 and t=30 minutes after instillation. We determined the EB/ FITC concentration of both samples using spectrophotometry and calculated AFC from the change in dye concentration.Our preliminary findings suggest that LPS and PLY inhibit all AFC associated with HSC leaving only AFC associated with NSC. The AFC of LPS exposed KO animals is low compared to AFC for LPS exposed WT animals (6.04 ± 3.02, n= 15 vs 15.8 ± 5.61, n= 11, p&lt; 0.001). Similarly, AFC of PLY exposed KO mice is lower than AFC for PLY exposed WT mice (2.78 ± 0.96, n= 10 vs 12.4 ± 4.36, n= 4, p&lt; 0.001). In the toxin exposed KO groups, neither HSC nor NSC are fully functioning or contributing to AFC. We are still collecting data on the permeability of the alveolar epithelium.There is evidence to suggest that the regulation of HSC and NSC are drastically different. Given the critical nature of the alveoli fluid layer, it would be logical that NSC channels serve as a compensatory mechanism when HSC are inhibited by pathogens. These findings can help guide treatment for alveolar flooding caused by toxins from different respiratory pathogens.

Tumor-associated macrophage heterogeneity is driven by tissue territories in breast cancer.

Tissue-resident macrophages adapt to local signals within tissues to acquire specific functions. Neoplasia transforms the tissue, raising the question as to how the environmental perturbations contribute to tumor-associated macrophage (TAM) identity and functions. Combining single-cell RNA sequencing (scRNA-seq) with spatial localization of distinct TAM subsets by imaging, we discover that TAM transcriptomic programs follow two main differentiation paths according to their localization in the stroma or in the neoplastic epithelium of the mammary duct. Furthermore, this diversity is exclusively detected in a spontaneous tumor model and tracks the different tissue territories as well as the type of tumor lesion. These TAM subsets harbor distinct capacity to activate CD8+ Tcells and phagocyte tumor cells, supporting that specific tumor regions, rather than defined activation states, are the major drivers of TAM plasticity and heterogeneity. The distinctions created here provide a framework to design cancer treatment targeting specific TAM niches.

Glioma-derived exosomes hijack the blood-brain barrier to facilitate nanocapsule delivery via LCN2.

Exosomes are small extracellular vehicles which could transport genetic materials and proteins between cells. Although there are reports about exosomes crossing the blood-brain barrier (BBB), the underlying mechanisms still need further study. We found that exosomes from primary brain tumors could upregulate the expression of Lipocalin-2 (LCN2) in bEnd.3 brain microvascular endothelial cells (BMVECs). Furthermore, exosomes increased the membrane fluidity of bEnd.3 cells in an LCN2 dependent manner. Both intraperitoneal injection and caudal vein injection of LCN2 increased the number of nanocapsules crossing the BBB. Evans Blue staining revealed that LCN2 does not interrupt the integrity of the BBB, as observed in the traumatic brain injury model. Tandem mass tags quantitative proteomics and bioinformatics analysis revealed that LCN2 is upregulated by exosomes via the JAK-STAT3 pathway, but not delivered from exosomes. Knocking down LCN2 in bEnd.3 cells significantly abrogated the effect of exosomes on BMVEC membrane fluidity. Previously, we have reported that 2-methacryloyloxyethyl phosphorylcholine (MPC) and a peptide crosslinker could encapsulate mAbs to achieve nanocapsules. The nanocapsules containing choline analogs could effectively penetrate the BBB to deliver therapeutic monoclonal antibodies (tAbs) to the glioma. However, the delivered tAbs could be significantly reduced by blocking the release of exosomes from the gliomas. Application of tAb nanocapsules prior to treatment with MK2206, an AKT pathway inhibitor that has been shown to inhibit the production of exosomes, resulted in a better combination. Insights from this study provide a mechanistic framework with regard to how glioblastomas hijack BMVECs using exosomes. In addition, we provide a strategy for maximizing the effect of the choline-containing nanocapsules and MK2206 combination. These results also demonstrate the therapeutic role of tAbs in glioblastoma and brain tumor metastasis, by shedding new light on strategies that can be used for BBB-penetrating therapies.

P-018 - Comparison of Evans Blue vs 4-(p-iodophenyl)butyric acid albumin binding moieties for lutetium-177-labeled LLP2A derivatives as theranostic agents for metastatic melanoma

P-018 - Comparison of Evans Blue vs 4-(p-iodophenyl)butyric acid albumin binding moieties for lutetium-177-labeled LLP2A derivatives as theranostic agents for metastatic melanoma

Ablation of PDE4A Exacerbates Pseudomonas aeruginosa‐Induced Acute Lung Injury in Mice

Type 4 cyclic nucleotide phosphodiesterases (PDE4s) comprise a family of four isoenzymes, PDE4A to D, that hydrolyze the second messenger cAMP. Three of these enzymes, PDE4A, PDE4B and PDE4D, are widely expressed throughout pulmonary and immune cell types, and their concurrent inhibition with non/PAN‐selective PDE4 inhibitors is well‐known to exert potent anti‐inflammatory benefits. To determine if PDE4 inhibition may represent a therapeutic approach for acute lung injury resulting from severe pneumonia, we explored the effect of genetic ablation of individual PDE4 subtypes, or their concurrent inhibition with PAN‐PDE4 inhibitors, in a model of acute intra‐tracheal Pseudomonas aeruginosa (PA) infection in mice. We have shown recently that selective ablation of PDE4B is sufficient to protect mice from lung injury in this model by reducing pulmonary and systemic levels of pro‐inflammatory cytokines, as well as pulmonary vascular leakage and mortality. With the present study we explored whether selective inactivation of PDE4A or treatment with PAN‐PDE4 inhibitors may provide additional therapeutic benefits.MethodsMice deficient in PDE4A (PDE4A‐KO) and their wildtype (WT) littermate controls were infected intratracheally with the PA lab strain PA01 and subjected 16 h later to bronchoalveolar lavage (BAL), cardiac blood draws, and tissue extractions. Alternatively, vascular/alveolar leakage was assessed by measuring Evans Blue Dye extravasation. To test the effect of PAN‐PDE4 inhibition, mice were treated with the PDE4 inhibitor Piclamilast (5 mg/kg, i.p.) at 2 h prior to PA01 infection.ResultsCompared to WT controls, PDE4A‐KO mice displayed a hyper‐inflammatory phenotype characterized by increased levels of pro‐inflammatory cytokines, immune cells (neutrophils) in BAL fluid, and vascular leakage. Despite these hyperinflammatory responses, the bacterial load in lungs was also increased in PDE4A‐KO mice compared to WT controls. Treatment with a PAN‐PDE4 inhibitor produced incongruent effects, with an increased bacterial load mirroring the effect of PDE4A ablation, whereas reduced levels of inflammatory cytokines such as TNFα mimicked the effect of PDE4B ablation.ConclusionsAblation of PDE4A produces detrimental effects in this model of acute PA‐lung infection. Current studies aim to determine whether an impaired bacterial killing, and the resulting increase in bacterial load, are responsible for the hyperinflammation, or whether these are independent effects of PDE4A ablation. PAN‐PDE4 inhibition mimics some of the beneficial effects of selective PDE4B ablation, but also some of the detrimental effects of PDE4A ablation. These findings suggest that selective inactivation of PDE4B may be a more effective therapeutic approach, compared to PAN‐PDE4 inhibition, in settings of acute PA‐lung infection.

Cerebral Ischemic Reperfusion Increases Autotaxin Expression Elevating Brain Endothelial Permeability

IntroductionIschemic reperfusion during stroke treatment increases the pathological progression of stroke. Reperfusion with r‐TPA and mechanical thrombectomy applied to stroke patients increases the risk of intracerebral hemorrhage. In addition, various molecular signals initiated during ischemic reperfusion exacerbate the disease due to increased endothelial permeability. However, a detailed molecular therapeutic regimen with r‐TPA and mechanical thrombectomy could limit the detrimental effects and increase the survival rate. Lysophosphatidic acid (LPA) is a bioactive phospholipid regulated in physiological and pathological conditions. LPA is produced by the enzyme autotaxin (ATX). ATX and LPA have been observed to deteriorate physiological status in various diseases such as myocardial infarction, Alzheimer’s disease, liver fibrosis, neuropathic pain etc. In cerebral ischemic‐reperfusion, the ATX‐LPA axis could initiate the endothelium disruption.HypothesisIncreased ATX expression in endothelium disrupts the endothelial barrier by increasing LPA during ischemic‐reperfusion.MethodsTransient middle cerebral artery occlusion with 90 minutes of ischemia and 24 hours of reperfusion (I/R) was used as a stroke model in mice. LPA was analyzed with mass spectrometry and immunohistochemistry. AR‐2 probe fluorescence assay was used to measure ATX activity. Evans Blue Permeability in vivo was measured with an infrared imager. For the in‐vitro study, oxygen and glucose deprivation and reperfusion (OGDR) were carried out in mouse brain microvascular endothelial cells (MBMEC).ResultsATX enzymatic activity is prominently raised (P&lt;0.01) in I/R mice compared to sham mice. Increased ATX activity coheres with the Evans Blue permeability in the mice brain. Simultaneously LPA levels are elevated (P&lt;0.01) in I/R mice. LPA was increased in brain tissue along the vasculature, indicating an unfavorable role of ATX in maintaining endothelial integrity. LPA receptors expression was regulated following stroke with upregulation of LPAR1, LPAR2, LPAR5, and LPAR6 following I/R. Permeability measured in MBMEC showed that LPA treatment increased the permeability in a time and concentration‐dependent manner. ATX expression was increased (P&lt;0.05) in MBMEC with OGDR suggesting ATX regulation in endothelial cells following ischemic‐reperfusion. LPA produced during the process can disrupt the blood‐brain barrier, increasing cerebral permeability.ConclusionAfter a stroke, enhanced LPA production by ATX can disrupt the blood‐brain barrier, leading to brain edema and neural cell damage followed by irreversible brain injury. Inhibition of the ATX‐LPA axis prospects to be a better therapeutic avenue for cerebral ischemic reperfusion.

Geniposide attenuates early brain injury by inhibiting oxidative stress and neurocyte apoptosis after subarachnoid hemorrhage in rats.

Oxidative stress and neurocyte apoptosis are crucial pathophysiological process in early brain injury (EBI) after subarachnoid hemorrhage (SAH). Geniposide (GNP) has been reported to exert neuroprotective effects by reducing oxidative injury and neurocyte apoptosis. However, the effect of GNP has not been clarified in EBI after SAH. The study was performed to evaluate the neuroprotective effects and mechanisms of GNP in EBI after SAH. A total of 60 male Wistar rats were randomly divided into five groups. The prechiasmatic cistern SAH model was used in this study. SAH grade was evaluated using a grading system. Neurological function was evaluated using the Garcia scores. Brain edema was measured by the wet-dry method. Blood-brain barrier (BBB) permeability was measured by the extravasation of Evans Blue (EB). The neurocyte apoptosis was observed using TUNEL assay. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD), as well as the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), glutathione S-transferase (GST) and quinone oxidoreductase-1 (NQO-1) were performed. The results showed that GNP reduced brain edema, attenuated BBB permeability, inhibited neurocyte apoptosis and improved neurological function. Moreover, GNP also decreased the levels of ROS and MDA, elevated Nrf2 expression in the temporal cortex and up-regulated the expression of NQO-1, HO-1 and GST after SAH. GNP could ameliorate oxidative stress and neurocyte apoptosis to exert neuroprotective effects by Nrf2 pathway.

The influence of electronic acupuncture at a specific frequency in facilitating the passage of NGF through the blood-brain barrier and its effect on learning and memory in MCAO/R rats.

The blood-brain barrier (BBB) maintains the balance of the internal environment of the brain and strictly controls substance exchange between the brain and blood dynamically but stably. Transient increases in the permeability of the BBB plays an important role in helping macromolecular drugs enter the brain to exert their pharmacological effects. Previous research has revealed that electronic acupuncture (EA) stimulation connecting Baihui (GV20) and Shuigou (GV26) at a specific frequency can enhance the permeability of the BBB at 8 minutes after the intervention and induce the entry of 20 kDa fluorescein isothiocyanate-dextran (FITC-dextran) into the cerebral cortex, but whether it can also allow drugs to pass the BBB remains unknown. We hypothesized that EA at a specific frequency could open the BBB and induce the entry of nerve growth factor (NGF) into the brain to exert its therapeutic effect. First, the middle cerebral artery occlusion (MCAO) model is adopted and changes in the permeability and structure of the BBB are assessed by measuring both the intensity of Evans blue (EB) staining and the cerebral infarction volume, and by evaluating the ultrastructure of the BBB. Then, a laser spectrometer and immunofluorescence are used to observe entry of NGF into the brain. Finally, the learning and memory ability of rats are assessed and the DeadEndTM Fluorometric TUNEL System is applied to assess apoptosis in the hippocampus. Our results showed that, in the first, the BBB was essentially repaired three weeks after MCAO operation. Secondly, Electronic Acupuncture (EA) stimulation at a specific frequency can enhance BBB permeability in the prefrontal cortex and induce NGF uptake by prefrontal neurons. Finally, in the presence of EA stimulation, entry of NGF into the brain promoted learning and memory in rats and inhibited the apoptosis of neurons in the hippocampus. In this study, the timing of BBB repair in the MCAO model was determined under pathological conditions and the EA stimulation can induce the entry of NGF into the brain to exert its therapeutic effect. EA could serve as a new strategy for delivering therapeutics to the central nervous system (CNS), given that EA stimulation at a specific frequency was shown to increase the permeability of the BBB. Further study of the mechanism underlying the opening of the BBB and its timing is needed.

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

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

64Cu]Cu-Albumin Clearance Imaging to Evaluate Lymphatic Efflux of Cerebrospinal Space Fluid in Mouse Model.

Clearance of brain waste in the cerebrospinal fluid (CSF) through the meningeal lymphatic vessels (mLV) has been evaluated mostly through the fluorescent imaging which has inherent limitations in the context of animal physiology and clinical translatability. The study aimed to establish molecular imaging for the evaluation of mLV clearance function. Radionuclide imaging after intrathecal (IT) injection was acquired in C57BL/6 mice of 2-9 months. The distribution of [99mTc]Tc-diethylenetriamine pentaacetate (DTPA) and [64Cu]Cu-human serum albumin (HSA) was comparatively evaluated. Evans Blue and [64Cu]Cu-HSA were used to evaluate the distribution of tracer under various speed and volume conditions. [99mTc]Tc-DTPA is not a suitable tracer for evaluation of CSF clearance via mLV as no cervical lymph node uptake was observed while it was cleared from the body. A total volume of 3 to 9 μL at an infusion rate of 300 to 500 nL/min was not sufficient for the tracer to reach the cranial subarachnoid space and clear throughout the mLV. As a result, whole-body positron emission tomography imaging using [64Cu]Cu-HSA at 700 nL/min, to deliver 6 μL of injected volume, was set for characterization of the CSF to mLV clearance. Through this protocol, the mean terminal CSF clearance half-life was measured to be 123.6 min (range 117.0-135.0) in normal mice. We established molecular imaging to evaluate CSF drainage through mLV using [64Cu]Cu-HSA. This imaging method is expected to be extended in animal models of dysfunctional meningeal lymphatic clearance and translational research for disease-modifying therapeutic approaches. The online version contains supplementary material available at 10.1007/s13139-022-00746-6.

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study.

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.