Assessment of the Relaxation-Enhancing Properties of a Nitroxide-Based Contrast Agent TEEPO-Glc with In Vivo Magnetic Resonance Imaging

Magnetic resonance imaging examinations are frequently carried out using contrast agents to improve the image quality. Practically all clinically used contrast agents are based on paramagnetic metals and lack in selectivity and specificity. A group of stable organic radicals, nitroxides, has raised interest as new metal-free contrast agents for MRI. Their structures can easily be modified to incorporate different functionalities. In the present study, a stable nitroxide TEEPO (2,2,6,6-tetraethylpiperidin-1-oxyl) was linked to a glucose moiety (Glc) to construct a water-soluble, potentially tumor-targeting compound with contrast-enhancing ability. The ability was assessed with in vivo MRI experiments. The constructed TEEPO-Glc agent proved to shorten the T-1 relaxation time in tumor, while the T-1 time in healthy brain tissue remained the same. The results indicate the potential of TEEPO-Glc as a valuable addition to the growing field of metal-free contrast enhancement in MRI-based diagnostics.Peer reviewe

Human Vascular Endothelial Growth Factor A165 Expression Induces the Mouse Model of Neovascular Age-Related Macular Degeneration

Vascular endothelial growth factor (VEGF) expression induces age-related macular degeneration (AMD), which is a common vision-threatening disease due to choroidal neovascularization and a fibrovascular membrane. We describe a mouse model of neovascular AMD with the local expression of human VEGF-A165 in the eye. We use a transgenic mouse in which human VEGF-A165 has been silenced with the loxP-STOP fragment. The choroidal neovascularization and human VEGF-A165 expression in the mouse are induced by subretinal adenoviral Cre gene delivery. Cre gene transfer is compared with adenoviral LacZ gene transfer control. We characterize the AMD phenotype and changes in the vasculature by using fluorescein angiography, optical coherence tomography, and immunohistochemistry. At early time points, mice exhibit increases in retinal thickness (348 ± 114 µm vs. 231 ± 32 µm) and choroidal neovascularization area (12000 ± 15174 µm2 vs. 2169 ± 3495 µm2) compared with the control. At later time points, choroidal neovascularization develops into subretinal fibrovascular membrane. Human VEGF-A165 expression lasts several weeks. In conclusion, the retinas display vascular abnormalities consistent with choroidal neovascularization. Together with immunohistochemical findings, these changes resemble clinical AMD-like ocular pathologies. We conclude that this mouse model of Cre-induced choroidal neovascularization is useful for mimicking the pathogenesis of AMD, studying the effects of human VEGF-A165 in the retina, and evaluating anti-VEGF treatments for choroidal neovascularization

Cyclodextrin‐Based Organic Radical Contrast Agents for In Vivo Imaging of Gliomas

Cyclodextrins (CDs), a class of cyclic oligosaccharides formed by α-(1,4) linked glucopyranose units, were functionalized with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) radicals to prepare water soluble supramolecular organic radical contrast agents (ORCAs) for the in vivo detection of glioma tumor in animal models. A first set of molecules (CDn1, n=6,7,8 is the number of both TEMPO and glucopyranose units) was studied by Superconducting Quantum Interference Devices (SQUID) magnetometry defining the role of the CD macrocycle on the effective magnetic moment (μeff). It increased from 3.982 μB(CD61) to 4.522 μB (CD81) but was limited by intra-molecular antiferromagnetic (AF) interactions. A set of water soluble ORCAs (CDn8, n=6,7,8) was prepared by a sequence of thiol-ene and Cu(I)-catalyzed alkyne-azide “click reactions”. Their 1H water relaxivities r1 were between 0.739 mM-1 s-1 (CD68) to 1.047 mM-1 s-1 (CD88) in D2O/H2O 9:1 (v:v) at 300 K. One of them (CD78) was tested on glioma-bearing rats with reduced side effects and good relaxivity in vivo

Lentivirus vector-mediated genetic manipulation of oncogenic pathways induces tumor formation in rabbit brain

Translation of promising experimental therapies from rodent models to clinical success has been complicated as the novel therapies often fail in clinical trials. Existing rodent glioma models generally do not allow for preclinical evaluation of the efficiency of novel therapies in combination with surgical resection. Therefore, the aim of the present study was to develop a larger animal model utilizing lentivirus vector-mediated oncogenic transformation in the rabbit brain. Lentiviruses carrying constitutively active AKT and H-Ras oncogenes, and p53 small interfering (si)RNA were introduced into newborn rabbit neural stem cells (NSCs) and intracranially implanted into rabbits' brains to initiate tumor formation. In one of the ten rabbits a tumor was detected 48 days after the implantation of transduced NSCs. Histological features of the tumor mimic was similar to a benign Grade II ganglioglioma. Immunostaining demonstrated that the tissues were positive for AKT and H-Ras. Strong expression of GFAP and Ki-67 was also detected. Additionally, p53 expression was notably lower in the tumor area. The implantation of AKT, H-Ras and p53 siRNA transduced NSCs for tumor induction resulted in ganglioglioma formation. Despite the low frequency of tumor formation, this preliminary data provided a proof of principle that lentivirus vectors carrying oncogenes can be used for the generation of brain tumors in rabbits. Moreover, these results offer noteworthy insights into the pathogenesis of a rare brain tumor, ganglioglioma.Peer reviewe

Redox properties and in vivo magnetic resonance imaging of cyclodextrin-polynitroxides contrast agents

This paper reports the synthesis, characterization and in vivo application of water-soluble supramolecular contrast agents (Mw:5-5.6 kDa) for MRI obtained from β-cyclodextrin functionalized with different kinds of nitroxide radicals, both with piperidine structure (CD2 and CD3) and with pyrrolidine structure (CD4 and CD5). As to the stability of the radicals in presence of ascorbic acid, CD4 and CD5 have low second order kinetic constants (≤0.06 M-1 s-1) compared to CD2 (3.5 M-1 s-1) and CD3 (0.73 M-1s-1). Relaxivity (r1) measurements on compounds CD3-CD5 were carried out at different magnetic field strength (0.7, 3, 7 and 9.4 T). At 0.7 T, r1 values comprised between 1.5 mM-1s-1 and 1.9 mM-1s-1 were found while a significant reduction was observed at higher fields (r1≈0.6-0.8 mM-1 s-1 at 9.4 T). Tests in vitro on HEK293 human embryonic kidney cells, L929 mouse fibroblasts and U87 glioblastoma cells indicated that all compounds were non-cytotoxic at concentrations below 1 µmol mL-1. MRI in vivo was carried out at 9.4 T on glioma-bearing rats and using the compounds CD3-CD5. The experiments showed a good lowering of T1 relaxation in tumor with a retention of the contrast for at least 60 mins confirming improved stability also in vivo conditions