Chronic Deferasirox Administration Decreases Hepatic Oxidative Stress and Apoptosis in the Iron Overloaded Gerbil.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1996-1996
Author(s):  
Rabaa AL-Rousan ◽  
Anjaiah Katta ◽  
Satyanarayana Paturi ◽  
Brent Kidd ◽  
Kamran Manzoor ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Iron Overload ◽  
Prussian Blue ◽  
Protein Oxidation ◽  
Lipid Accumulation ◽  
Iron Deposition ◽  
Blue Staining ◽  
Hepatic Iron ◽  
Prussian Blue Staining ◽  
Hepatic Oxidative Stress

Abstract Abstract 1996 Poster Board I-1018 Background: Iron overload occurs under conditions such as primary (hereditary) hemochromatosis and secondary iron overload (hemosiderosis) and is associated with an increased risk of developing liver fibrosis, cirrhosis, and hepatocellular carcinoma. Deferasirox is a novel oral chelator with high iron-binding potency and selectivity. Here we investigate the ability of deferasirox to remove excessive hepatic iron and prevent or reverse iron induced hepatic injury. Methods: Adult male Mongolian Gerbils were randomly divided into three groups: control, iron overload, and iron overload + deferasirox treatment (n = 8 / group). Iron overload animals received iron dextran 100mg/kg i.p /5d for 10 wks while deferasirox was given 100mg/kg/d p.o for 1-,3-, or 9- months. Hepatic iron levels were determined by inductively coupled plasma atomic emission spectrometry and Prussian blue staining was performed to examine iron deposition in the corresponding tissues. Immunoblot and immunohistochemical analyses for markers of oxidative stress were employed to assess effects of deferasirox treatment on hepatic protein oxidation and superoxide levels. TUNEL assay was employed to examine the extent of hepatic apoptosis. Results: Compared to the non-treated iron overload group, deferasirox treatment reduced hepatic iron levels by 21.3%, 43.5%, and 47.4% after 1, 3, and 9 months of treatment, respectively (p<0.05). Prussian blue staining and histological analysis detected frequent iron deposition, evidence of hepatic damage, and lipid accumulation in hepatic tissue of the iron overloaded group. Iron deposition was significantly diminished with deferasirox treatment and no evidence of lipid accumulation was observed. Immunoblotting demonstrated that iron overload caused 2- fold increase in hepatic ferritin expression (p< 0.05) which was reduced by 47.5% following three months of deferasirox treatment (p< 0.05). In addition, deferasirox significantly reduced hepatic protein oxidation and superoxide abundance. The percentage of TUNEL-positive nuclei in the deferasirox treated livers was 41.0% lower than that of iron overloaded group (p<0.05). Conclusions: These findings suggest that chronic deferasirox treatment may decrease iron-induced hepatic oxidative stress and apoptosis. Decrease in ROS accumulation in the liver may be the possible mechanism of this protective effect. Further studies are underway to delineate specific mechanisms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Comparison of Computerized Image Analysis with Traditional Semiquantitative Scoring of Perls’ Prussian Blue Stained Hepatic Iron Deposition

2013 ◽  
Vol 41 (7) ◽  
pp. 992-1000 ◽  
Author(s):  
A. Peter Hall ◽  
Wendy Davies ◽  
Katie Stamp ◽  
Isabel Clamp ◽  
Alison Bigley
Keyword(s):  
Image Analysis ◽  
Prussian Blue ◽  
Iron Deposition ◽  
Hepatic Iron ◽  
Computerized Image Analysis

scholarly journals Lidocaine is a nocebo treatment for trigeminally mediated magnetic orientation in birds

2018 ◽  
Vol 15 (145) ◽  
pp. 20180124 ◽  
Author(s):  
Svenja Engels ◽  
Christoph Daniel Treiber ◽  
Marion Claudia Salzer ◽  
Andreas Michalik ◽  
Lyubov Ushakova ◽  
...  
Keyword(s):  
Magnetic Field ◽  
Prussian Blue ◽  
Trigeminal Nerve ◽  
Brain Regions ◽  
Blue Staining ◽  
Magnetic Orientation ◽  
Neuronal Activation ◽  
Neuronal Marker ◽  
Prussian Blue Staining ◽  
Ophthalmic Branch

Even though previously described iron-containing structures in the upper beak of pigeons were almost certainly macrophages, not magnetosensitive neurons, behavioural and neurobiological evidence still supports the involvement of the ophthalmic branch of the trigeminal nerve (V1) in magnetoreception. In previous behavioural studies, inactivation of putative V1-associated magnetoreceptors involved either application of the surface anaesthetic lidocaine to the upper beak or sectioning of V1. Here, we compared the effects of lidocaine treatment, V1 ablations and sham ablations on magnetic field-driven neuronal activation in V1-recipient brain regions in European robins. V1 sectioning led to significantly fewer Egr-1-expressing neurons in the trigeminal brainstem than in the sham-ablated birds, whereas lidocaine treatment had no effect on neuronal activation. Furthermore, Prussian blue staining showed that nearly all iron-containing cells in the subepidermal layer of the upper beak are nucleated and are thus not part of the trigeminal nerve, and iron-containing cells appeared in highly variable numbers at inconsistent locations between individual robins and showed no systematic colocalization with a neuronal marker. Our data suggest that lidocaine treatment has been a nocebo to the birds and a placebo for the experimenters. Currently, the nature and location of any V1-associated magnetosensor remains elusive.

β-Thalassemia Mouse Model: Macrophages and the Induction of Iron Overload.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2699-2699
Author(s):  
Yelena Z. Ginzburg ◽  
Radma Mahmood ◽  
Steven Brunnert ◽  
Mary E. Fabry ◽  
Ronald L. Nagel
Keyword(s):  
Iron Overload ◽  
Prussian Blue ◽  
Kupffer Cells ◽  
Thalassemia Major ◽  
Blue Staining ◽  
Cardiac Iron ◽  
Iron Administration ◽  
Cardiac Iron Overload ◽  
Rbc Indices ◽  
Parenteral Iron

Abstract Despite the use of transfusion and iron chelation therapy, patients with β-thalassemia major have a shortened life expectancy. Many of those deaths are attributable to cardiac iron overload. Nevertheless, the process by which cardiac iron overload occurs is not well understood. We have used the homozygous βmajor deletion [Hbbth-1] (THL) mouse model to assess hepatic and cardiac iron load. RBC indices for 3 THL mice and 2 C57BL/6 wildtype control mice prior to and post therapy with parenteral iron were evaluated with Advia. Intraperitoneal iron dextran injection at 10mg/25gm body weight daily 5 days per week for 12 days was performed and then switched to 1.25mg/25gm body weight of iron injection for another 10 days for a total of 4 weeks. Histological samples of liver and heart were stained with Prussian blue in mice prior to and post administration of parenteral iron. Immunohistochemistry with antibody to F4/80, specific for macrophages, was performed and counterstained with Prussian blue in livers and hearts of THL and C57 mice. The RBC indices in THL mice reveal an anemia (HCT 29.5±2.3 vs 45±2.1%, P=0.005) and reticulocytosis (2218±501 vs 406±101 x 109 cells, P=0.018) prior to therapy relative to the C57 mice (values presented as mean ± standard deviation). In THL mice after parenteral iron, HCT (41.8±6.8 vs 29.5±2.3%, P=0.04) and reticulocyte counts (2218±501 vs 3760±633 x 109 cells, P=0.03) increased significantly from pre-treatment values while in C57 mice, the HCT (53.8±6 vs 45±2.1%, NS) and reticulocyte count (406±101 vs 210±49 x109 cells, NS) did not change appreciably from baseline. Prior to therapy, the liver of THL mice exhibit 20–25% Kupffer cells staining with Prussian blue, with no Prussian blue staining in hepatocytes. The hearts of THL mice have no macrophages and no iron deposition at baseline. Prior to therapy, the livers of C57 mice had similar numbers of Kupffer cells compared to THL mice though none stain with Prussian blue. After treatment with parenteral iron, the livers of THL and C57 mice became significantly iron loaded (75–80% of Kupffer cells are positive for Prussian blue), the number of Kupffer cells increased 4-fold, and the majority of the Prussian blue staining was limited to Kupffer cells (90–95%). After treatment with parenteral iron, the hearts of THL and C57 mice became significantly iron loaded as well, but unlike the liver, most (90%) of the Prussian blue positive cells were myocytes. Only a small fraction of the myocytes in the heart was involved (5%). THL mice appear to be iron deficient and show bone marrow reserve with reticulocytosis significantly above baseline when excess iron is administered. Iron overload secondary to intraperitoneal iron dextran administration affects THL mice as well as C57 mice. In the liver of THL mice, Kupffer cells normally resident in the liver become laden with iron; little iron is deposited in hepatocytes. In the heart, an organ without resident macrophages and few macrophages migrating into the tissue during parenteral iron administration, both THL and C57 mice reveal myocyte deposition of iron. In conclusion, parenteral iron administration leads to a noticeable increase in RBCs in THL mice. Furthermore, both the livers and hearts of THL mice accumulate iron. Finally, these findings correlate well with the natural history of cardiac iron overload in human β-thalassemia major, leading to the conclusion that THL mice are a suitable model for the study of cardiac iron overload in thalassemia.

In Vivo Disruption Of The Hepcidin−Ferroportin Regulatory Circuitry Causes Fatal Systemic and Exocrine Pancreatic Iron Overload

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 175-175
Author(s):  
Sandro Altamura ◽  
Hermann Josef Gröne ◽  
Regina Kessler ◽  
Bruno Galy ◽  
Matthias W. Hentze ◽  
...  
Keyword(s):  
Iron Overload ◽  
Molecular Mechanisms ◽  
Histological Analysis ◽  
Conflicts Of Interest ◽  
Hereditary Hemochromatosis ◽  
Transferrin Saturation ◽  
Iron Accumulation ◽  
Blue Staining ◽  
Hepatic Iron

Abstract Systemic iron levels are tightly controlled by the hepatic hormone hepcidin in response to iron availability, inflammation, hypoxia or the iron demand for erythropoiesis. Hepcidin binds to the iron export protein ferroportin (FPN1) to regulate iron release from exporting cells. A mutation of cysteine 326 (C326S) of FPN1 was reported in a patient with non−classical ferroportin disease (Sham et al, 2005) and shown to abrogate hepcidin binding in vitro (Fernandes et al, 2009). To study consequences of the disruption of the hepcidin−ferroportin interaction in vivo, we generated the first knock−in mouse model of C326S non−classical ferroportin disease. Mice with either heterozygous or homozygous C326S FPN alleles are viable and fertile. At 8−weeks of age both heterozygous and homozygous mice show profoundly increased transferrin saturation and serum ferritin levels as well as hepatic iron overload. Histological analysis by Perl’s Prussian blue staining revealed that hepatic iron accumulation is restricted to hepatocytes and that Kupffer cells are spared of iron. In addition, splenic macrophages and duodenal enterocytes are iron−depleted. Macroscopically, C326S homozygous mice show progressive, brown discoloration of the pancreas that correlates with profound iron deposition. Histological analysis reveals that iron localizes exclusively to the exocrine pancreas sparing the islets of Langerhans. Consistently, C326S homozygous mice do not show any signs of diabetes. Pancreatic iron accumulation is closely associated with increased reactive oxygen species (ROS), degeneration of exocrine pancreatic cells, increased plasma lipase and exocrine pancreatic failure. Starting at the age of 33 weeks, pancreatic failure is accompanied by progressive wasting and death. We believe that C326S FPN mice represent the first example of fatal iron overload in an animal model, opening avenues to investigate the underlying molecular mechanisms. Sham R, Phatak PD, West C, et al. Autosomal dominant hereditary hemochromatosis associated with a novel ferroportin mutation and unique clinical features. Blood Cells Mol. Dis. 2005; 34:157−61. Fernandes A, Preza GC, Phung Y, et al. The molecular basis of hepcidin−resistant hereditary hemochromatosis. Blood. 2009;114:437−443. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MRI detection of the malignant transformation of stem cells through reporter gene expression driven by a tumor-specific promoter

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jun Sun ◽  
Jie Huang ◽  
Guangcheng Bao ◽  
Helin Zheng ◽  
Cui Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Prussian Blue ◽  
Malignant Transformation ◽  
Reporter Gene ◽  
Reporter Gene Expression ◽  
Blue Staining ◽  
Prussian Blue Staining ◽  
Before And After ◽  
Signal Changes

Abstract Background Existing evidence has shown that mesenchymal stem cells (MSCs) can undergo malignant transformation, which is a serious limitation of MSC-based therapies. Therefore, it is necessary to monitor malignant transformation of MSCs via a noninvasive imaging method. Although reporter gene-based magnetic resonance imaging (MRI) has been successfully applied to longitudinally monitor MSCs, this technique cannot distinguish the cells before and after malignant transformation. Herein, we investigated the feasibility of using a tumor-specific promoter to drive reporter gene expression for MRI detection of the malignant transformation of MSCs. Methods The reporter gene ferritin heavy chain (FTH1) was modified by adding a promoter from the tumor-specific gene progression elevated gene-3 (PEG3) and transduced into MSCs to obtain MSCs-PEG3-FTH1. Cells were induced to undergo malignant transformation via indirect coculture with C6 glioma cells, and these transformed cells were named MTMSCs-PEG3-FTH1. Western blot analysis of FTH1 expression, Prussian blue staining and transmission electron microscopy (TEM) to detect intracellular iron, and MRI to detect signal changes were performed before and after malignant transformation. Then, the cells before and after malignant transformation were inoculated subcutaneously into nude mice, and MRI was performed to observe the signal changes in the xenografts. Results After induction of malignant transformation, MTMSCs demonstrated tumor-like features in morphology, proliferation, migration, and invasion. FTH1 expression was significantly increased in MTMSCs-PEG3-FTH1 compared with MSCs-PEG3-FTH1. Prussian blue staining and TEM showed a large amount of iron particles in MTMSCs-PEG3-FTH1 but a minimal amount in MSCs-PEG3-FTH1. MRI demonstrated that the T2 value was significantly decreased in MTMSCs-PEG3-FTH1 compared with MSCs-PEG3-FTH1. In vivo, mass formation was observed in the MTMSCs-PEG3-FTH1 group but not the MSCs-PEG3-FTH1 group. T2-weighted MRI showed a significant signal decrease, which was correlated with iron accumulation in the tissue mass. Conclusions We developed a novel MRI model based on FTH1 reporter gene expression driven by the tumor-specific PEG3 promoter. This approach could be applied to sensitively detect the occurrence of MSC malignant transformation.

scholarly journals The Effects of Chronic Iron Overload in Rats with Acute Acetaminophen Overdose

2018 ◽  
Vol 46 (5) ◽  
pp. 597-607 ◽  
Author(s):  
Zvi Ackerman ◽  
Galina Skarzinski ◽  
Gabriela Link ◽  
Maya Glazer ◽  
Orit Pappo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Iron Overload ◽  
Cell Damage ◽  
Hepatic Cell ◽  
Hepatic Expression ◽  
Iron Administration ◽  
Hepatic Oxidative Stress ◽  
Hepatic Iron Overload ◽  
Sinusoidal Cell ◽  
Parenteral Iron

Background and Aims: Rats are resistant to acetaminophen (APAP) hepatotoxicity. In this study, we evaluated whether by augmentation of the hepatic oxidative stress, through the induction of hepatic iron overload (IO), it will be feasible to overcome the resistance of rats to the toxic effects of APAP. Method: Rats with no or increased hepatic IO. Results: Providing iron by diet induced hepatocellular IO, while parenteral iron administration induced combined hepatocellular and sinusoidal cell IO. APAP administration to rats with no IO caused an increase in hepatic oxidative stress and a decrease in the hepatic antioxidative markers but no hepatic cell damage. APAP administration to rats with hepatocellular IO further amplified the hepatic oxidative stress but induced only hepatocyte feathery degeneration without any increase in serum aminotransaminases. APAP administration to rats with combined hepatocellular and sinusoidal cell IO caused an unexpected decrease in hepatic oxidative stress and increase in the hepatic antioxidative markers and no hepatic cell damage. No hepatic expression of activated c-jun-N-terminal kinase was detected in any of the rats. Conclusions: The hepatic distribution of iron may affect its oxidative/antioxidative milieu. Augmentation of hepatic oxidative stress did not increase the rats’ vulnerability to APAP.

Novel experimental model of brain arteriovenous malformations using conditional Alk1 gene deletion in transgenic mice

2021 ◽  
pp. 1-12
Author(s):  
Chul Han ◽  
Michael J. Lang ◽  
Candice L. Nguyen ◽  
Ernesto Luna Melendez ◽  
Shwetal Mehta ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Prussian Blue ◽  
Hereditary Hemorrhagic Telangiectasia ◽  
Arteriovenous Malformations ◽  
Gene Deletion ◽  
Blue Staining ◽  
Prussian Blue Staining ◽  
Brain Arteriovenous Malformations ◽  
Group 3 ◽  
Brain Avms

OBJECTIVE Hereditary hemorrhagic telangiectasia is the only condition associated with multiple inherited brain arteriovenous malformations (AVMs). Therefore, a mouse model was developed with a genetics-based approach that conditionally deleted the causative activin receptor-like kinase 1 (Acvrl1 or Alk1) gene. Radiographic and histopathological findings were correlated, and AVM stability and hemorrhagic behavior over time were examined. METHODS Alk1-floxed mice were crossed with deleter mice to generate offspring in which both copies of the Alk1 gene were deleted by Tagln-Cre to form brain AVMs in the mice. AVMs were characterized using MRI, MRA, and DSA. Brain AVMs were characterized histopathologically with latex dye perfusion, immunofluorescence, and Prussian blue staining. RESULTS Brains of 55 Tagln-Cre+;Alk12f/2f mutant mice were categorized into three groups: no detectable vascular lesions (group 1; 23 of 55, 42%), arteriovenous fistulas (AVFs) with no nidus (group 2; 10 of 55, 18%), and nidal AVMs (group 3; 22 of 55, 40%). Microhemorrhage was observed on MRI or MRA in 11 AVMs (50%). AVMs had the angiographic hallmarks of early nidus opacification, a tangle of arteries and dilated draining veins, and rapid shunting of blood flow. Latex dye perfusion confirmed arteriovenous shunting in all AVMs and AVFs. Microhemorrhages were detected adjacent to AVFs and AVMs, visualized by iron deposition, Prussian blue staining, and macrophage infiltration using CD68 immunostaining. Brain AVMs were stable on serial MRI and MRA in group 3 mice (mean age at initial imaging 2.9 months; mean age at last imaging 9.5 months). CONCLUSIONS Approximately 40% of transgenic mice satisfied the requirements of a stable experimental AVM model by replicating nidal anatomy, arteriovenous hemodynamics, and microhemorrhagic behavior. Transgenic mice with AVFs had a recognizable phenotype of hereditary hemorrhagic telangiectasia but were less suitable for experimental modeling. AVM pathogenesis can be understood as the combination of conditional Alk1 gene deletion during embryogenesis and angiogenesis that is hyperactive in developing and newborn mice, which translates to a congenital origin in most patients but an acquired condition in patients with a confluence of genetic and angiogenic events later in life. This study offers a novel experimental brain AVM model for future studies of AVM pathophysiology, growth, rupture, and therapeutic regression.

scholarly journals Mechanisms of Iron Clearance from the Joint in FVIII-Deficient Mice after Induced Hemarthrosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 157-157
Author(s):  
Esther J Cooke ◽  
Chanond A Nasamran ◽  
Kathleen M Fisch ◽  
Annette von Drygalski
Keyword(s):  
Prussian Blue ◽  
Rna Sequencing ◽  
Ferric Iron ◽  
Blue Staining ◽  
Lipocalin 2 ◽  
Post Injury ◽  
Advisory Committees ◽  
Joint Tissue ◽  
Prussian Blue Staining ◽  
Deficient Mice

Introduction Hemarthrosis in hemophilia causes toxic iron accumulation in the joint, which contributes to synovitis. This study aimed to explore mechanisms and timing of iron clearance from the joint space in mouse models of induced hemarthrosis. Methods Hemarthrosis was induced by sub-patellar puncture in FVIII-deficient mice and in hypocoagulable BALB/c (HypoBALB/c) mice treated with 10 µg/ml warfarin for 7 days and 0.25 mg/kg anti-FVIII antibody 2 hours before knee puncture. Warfarin was reversed on day 2 post-injury with 100 IU/Kg 4-factor prothrombinase complex concentrate and the hematocrit determined in all mice as a measure of joint bleeding. Joint tissue was harvested at baseline and 2 or 4 weeks post-injury for analysis by histology. Ferric iron (Fe3+) was detected by Prussian Blue staining either during or after joint decalcification. Macrophages and macrophage-like synoviocytes were detected in joint tissue by immunohistochemistry with an anti-CD68 antibody. Synovial tissue was harvested from FVIII-deficient mice on day 3 and 2 weeks post-injury for gene expression studies by RNA sequencing (single-end; 75 bp) on an Illumina NextSeq500 platform. The limma-voom method (R BioConductor) was used for differential expression analyses. Results Knee injury caused substantial and comparable hemarthrosis in FVIII-deficient and HypoBALB/c mice (mean day 2 hematocrit: 27 % and 28 %, respectively). Post-decalcification Prussian Blue staining detected ferric iron accumulation in FVIII-deficient mice at week 4 only (5.3-fold increase compared to baseline, p=0.003). No ferric iron was detected in HypoBALB/c mice despite similar bleed volumes. In FVIII-deficient mice, Prussian Blue staining during decalcification was more sensitive and preserved detection of extracellular ferric iron, revealing a significant increase in ferric iron at 2 weeks post-injury relative to baseline (38-fold, p=0.005), which persisted at 4 weeks (23-fold, p=0.03). These findings coincided with increased CD68 staining at 2 weeks (36-fold, p=0.0002) and 4 weeks (8-fold, p=0.1). CD68-positive cells were dispersed throughout synovium at 2 weeks but appeared more clustered at 4 weeks and co-localized with iron staining, suggesting migration and iron uptake between 2 and 4 weeks post-injury. In HypoBALB/c mice, CD68 staining increased at 2 weeks (11-fold, p=0.008) but to a lesser extent than in FVIII-deficient mice, and was comparable to baseline at 4 weeks. Together, this suggests an altered mechanism of iron clearance in hemophilia. RNA sequencing revealed differential expression of 11/57 genes relating to iron transport in synovium on day 3, persisting somewhat at 2 weeks. Upregulated genes on day 3 included heme-oxygenase-1 (heme-degrading enzyme; 31-fold, p=3x10-6), lipocalin-2 (iron-binding protein; 10-fold, p=0.001) and solute carrier family (slc) 11 member 1 (macrophage iron transporter; 3-fold, p=0.0004). Down-regulated genes on day 3 included ceruloplasmin (efflux of cellular iron; 17-fold, p=5x10-5) and its homolog hephaestin (5-fold, p=0.002), CD163 (macrophage scavenging receptor for hemoglobin; 5-fold, p=0.03) and slc22 member 17 (lipocalin-2 receptor; 2-fold, p=0.03). Gene expression changes revealed key players involved in scavenging, degradation and transport of iron in synovium after hemarthrosis, and may expose mechanisms of impaired iron clearance in hemophilia pending further studies. Conclusions Iron handling after hemarthrosis, including uptake and transport in synovium and/or delivery to plasma transferrin, may be impaired in hemophilia and contribute to the evolution of hemophilic arthropathy. Unbiased RNA sequencing created several hypotheses that can be tested to further to elucidate mechanisms and timing of aberrant iron handling. Disclosures von Drygalski: Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder; University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy.

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