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

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.

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

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.

Magnetic Resonance Imaging of Transplanted Porcine Neonatal Pancreatic Cell Clusters Labeled with Chitosan-Coated Superparamagnetic Iron Oxide Nanoparticles in Mice

Neonatal pancreatic cell clusters (NPCCs) are potential tissues for the treatment of diabetes. Different from adult cells, they continuously proliferate and differentiate after transplantation. In this study, we utilized magnetic resonance imaging (MRI) to detect and monitor implanted NPCCs. NPCCs were isolated from one-day-old neonatal pigs, cultured for three days, and then incubated overnight with the contrast agent chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles. In vitro, Prussian blue staining and MR scans of CSPIO-labeled NPCCs were performed. In vivo, we transplanted 2000 CSPIO-labeled NPCCs under the kidney capsule of nondiabetic nude mice. Recipients were scanned with 7.0T MRI. Grafts were removed for histology with insulin and Prussian blue staining. After being incubated overnight with CSPIO, NPCCs showed positive iron staining and appeared as dark spots on MR scans. After transplantation of CSPIO-labeled NPCCs, persistent hypointense areas were observed at recipients’ implant sites for up to 54 days. Moreover, histology showed colocalization of the insulin and iron staining in 15-, 51- and 55-day NPCC grafts. Our results indicate that transplanted NPCCs survived and differentiated to β cells after transplantation, and that MRI is a useful tool for the detection and monitoring of CSPIO-labeled NPCC grafts.

SHED Cells Labeled with MIRB can be Tracked in Repairing Periodontal Bone Defects in Rats

Background: Chronic periodontitis could lead to alveolar bone resorption and even tooth loss. Stem cells from exfoliated deciduous teeth (SHED) are the proper seed cells for bone regeneration because of their potential in osteogenic differentiation. However tracking the survival, migration and differentiation of the transplanted stem cells is necessary to improve the transplantation success. Methods: Superparamagnetic iron oxide particles (SPIO) Molday ION Rhodamine-BTM (MIRB) were used for labeling and monitoring SHED cells in vivo by magnetic resonance imaging (MRI). Proper labeling concentration of MIRB was determined by cell viability, proliferation, osteogenic differentiation and MRI analysis in vitro after SHED cells were labeled with MIRB at different concentration of 12.5, 25, 50, 100μgFe/mL. MIRB labeled SHED were transplanted to the periodontal bone defect model in rats and tracked by MRI in vivo. The regeneration of periodontal bone were calculated with HE and immunohistochemical analysis. The survival of transplanted SHED cells in vivo was verified with Prussian blue staining.Results: After testing 25μg Fe/mL MIRB was used in vivo cells tracking. After transplanted to the periodontal bone defect model in rats, the MIRB labeled SHED could be tracked in vivo through the artifact of the low intensity signal caused by Fe3+ at 6 and 9 weeks post-surgery. HE and immunohistochemical analysis showed that both SHED labeled and unlabeled with MIRB could promote regeneration of periodontal bone defect. Prussian blue staining further verified the survival of transplanted SHED cells in vivo. Conclusions: Overall, SHED cells could promote the regeneration of periodontal bone in rats and the survival of SHED cells could be tracked by labeling with MIRB in vivo. However the distribution of the positive cells at the edge of the regenerated new bone remind us the SHED cell could promote the regeneration of new bone by factors section.

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

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.

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

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.

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

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.