New Findings
What is the central question of this study?
The central question of this study is to understand whether dietary quercetin enrichment attenuates physiologic, histological, and biochemical ...indices of cardiac pathology.
What is the main finding and its importance?
Novel findings from this investigation, in comparison to prior published studies, suggest that mouse strain‐dependent cardiac outcomes in performance and remodelling exist. Unlike Mdx/Utrn−/+ mice, mdx mice receiving lifelong quercetin treatment did not exhibit improvements cardiac function. Similar to prior work in Mdx/Utrn−/+ mice, histological evidence of remodelling suggests that quercetin consumption may have benefited hearts of mdx mice. Positive outcomes may be related to indirect markers that suggest improved mitochondrial wellbeing and to selected indices of inflammation that were lower in hearts from quercetin‐fed mice.
Duchenne muscular dystrophy causes a decline in cardiac health, resulting in premature mortality. As a potential countermeasure, quercetin is a polyphenol possessing inherent anti‐inflammatory and antioxidant effects that activate proliferator‐activated γ coactivator 1α (PGC‐1α), increasing the abundance of mitochondrial biogenesis proteins. We investigated the extent to which lifelong 0.2% dietary quercetin enrichment attenuates dystrophic cardiopathology in mdx mice. Dystrophic animals were fed a quercetin‐enriched or control diet for 12 months, while control C57 mice were fed a control diet. Cardiac function was assessed via 7 T magnetic resonance imaging at 2, 10 and 14 months. At 14 months, hearts were harvested for histology and Western blotting. The results indicated an mdx strain‐dependent decline in cardiac performance at 14 months and that dietary quercetin enrichment did not attenuate functional losses. In contrast, histological analyses provided evidence that quercetin feeding was associated with decreased fibronectin and indirect damage indices (Haematoxylin and Eosin) compared with untreated mdx mice. Dietary quercetin enrichment increased cardiac protein abundance of PGC‐1α, cytochrome c, electron transport chain complexes I–V, citrate synthase, superoxide dismutase 2 and glutathione peroxidase (GPX) versus untreated mdx mice. The protein abundance of the inflammatory markers nuclear factor‐κB, phosphorylated nuclear factor kappa beta (P‐NFκB) and phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B‐cells inhibitor alpha (P‐IKBα) was decreased by quercetin compared with untreated mdx mice, while preserving nuclear factor of kappa light polypeptide gene enhancer in B‐cells inhibitor alpha( IKBα) compared with mdx mice. Furthermore, quercetin decreased transforming growth factor‐β1, cyclooxygenase‐2 (COX2) and macrophage‐restricted F4/80 protein (F4/80) versus untreated mdx mice. The data suggest that long‐term quercetin enrichment does not impact physiological parameters of cardiac function but improves indices of mitochondrial biogenesis and antioxidant enzymes, facilitates dystrophin‐associated glycoprotein complex (DGC) assembly and decreases inflammation in dystrophic hearts.
The overproduction of reactive oxygen species has been linked to a wide array of health disorders. The ability to noninvasively monitor oxidative stress in vivo could provide substantial insight into ...the progression of these conditions and, in turn, could facilitate the development of better diagnosis and treatment options. A mononuclear Mn(II) complex with the redox-active ligand N,N′-bis(2,5-dihydroxybenzyl)-N,N′-bis(2-pyridinylmethyl)-1,2-ethanediamine (H4qtp2) was made and characterized. A previously prepared Mn(II) complex with a ligand containing a single quinol subunit was found to display a modest T 1-derived relaxivity response to H2O2. The introduction of a second redox-active quinol both substantially improves the relaxivity response of the complex to H2O2 and reduces the cytotoxicity of the sensor but renders the complex more susceptible to transmetalation. The addition of H2O2 partially oxidizes the quinol subunits to para-quinones, concomitantly increasing the r 1 from 5.46 mM–1 s–1 to 7.17 mM–1 s–1. The oxidation of the ligand enables more water molecules to coordinate to the metal ion, providing an explanation for the enhanced relaxivity. That the diquinol complex is only partially oxidized by H2O2 is attributed to its activity as an antioxidant; the complex can both catalytically degrade superoxide and serve as a hydrogen atom donor.
GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted ...consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats—either untreated or AAV treated for more than 5 years—and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment.
GM1 gangliosidosis is a fatal, untreatable neurodegenerative disease of children and adults. Gray-Edwards and colleagues demonstrate >5-year survival in AAV-treated GM1 cats and describe novel minimally invasive biomarkers for use in clinical trials. Analyses of blood, cerebrospinal fluid, electroencephalography, MRI, and MRS are included from GM1 cats and human patients.
The redox-active ligand N-(2-hydroxy-5-methylbenzyl)-N,N′,N′-tris(2-pyridinylmethyl)-1,2-ethanediamine (Hptp1) was prepared and complexed to manganese(II). The isolated Mn(Hptp1)(MeCN)2+ serves ...as a magnetic resonance imaging contrast agent, with an r 1 value comparable to those of other mononuclear gadolinium(III) and manganese(II) complexes. The metal and ligand are stable in aerated aqueous solutions, but the addition of H2O2 causes the complex to oxidatively couple to itself through a bimolecular reaction involving the phenol groups of two Hptp1 ligands. The binuclear product is less paramagnetic per manganese(II) than its mononuclear precursor, lowering the measured r 1 per manganese(II). The manganese(II) complex with Hptp1 can thereby serve as a sensor for oxidative stress.
Using noninvasive in vivo functional magnetic resonance imaging (fMRI), we demonstrate that the enhancement of odorant response of olfactory receptor neurons by zinc nanoparticles leads to increase ...in activity in olfaction-related and higher order areas of the dog brain. To study conscious dogs, we employed behavioral training and optical motion tracking for reducing head motion artifacts. We obtained brain activation maps from dogs in both anesthetized state and fully conscious and unrestrained state. The enhancement effect of zinc nanoparticles was higher in conscious dogs with more activation in higher order areas as compared with anesthetized dogs. In conscious dogs, voxels in the olfactory bulb and hippocampus showed higher activity to odorants mixed with zinc nanoparticles as compared with pure odorants, odorants mixed with gold nanoparticles as well as zinc nanoparticles alone. These regions have been implicated in odor intensity processing in other species including humans. If the enhancement effect of zinc nanoparticles observed in vivo are confirmed by future behavioral studies, zinc nanoparticles may provide a way for enhancing the olfactory sensitivity of canines for detection of target substances such as explosives and contraband substances at very low concentrations, which would otherwise go undetected.
The ligand N,N′-bis(2-pyridylmethyl)-bis(ethylacetate)-1,2-ethanediamine (debpn) coordinates divalent transition metal ions in either a pentadentate or hexadentate fashion. The coordination number ...correlates with the ionic radius of the metal ion, with larger cations being heptacoordinate as assessed by solid-state analysis. With Mn(II), the debpn ligand is hexadentate and remains bound to the oxophilic metal ion, even when dissolved in water. The ligand’s incomplete coordination of the manganous ion allows water molecules to coordinate to the metal center. These two properties, coupled with the high paramagnetism associated with the S = 5/2 metal center, enable Mn(debpn)(H2O)(ClO4)2 to serve as a stable and effective magnetic resonance imaging contrast agent despite the ligand’s lack of both a macrocyclic component and an anionic charge.
To prospectively evaluate a gadolinium-based collagen-targeting contrast agent, EP-3533, for in vivo magnetic resonance (MR) imaging of myocardial fibrosis in a mouse model of healed myocardial ...infarction (MI).
All procedures were performed in accordance with protocols approved by the animal care and use committee. MI was induced in eight mice by means of occlusion of the left anterior descending coronary artery followed by reperfusion. Four MR examinations were performed in each animal: one examination before, one examination 1 day after, and two examinations 6 weeks after the MI. For the latter two examinations, electrocardiographically gated inversion-recovery gradient-echo MR images were acquired before and serially (every 5 minutes) after the intravenous injection of either gadopentetate dimeglumine or EP-3533. The image enhancement kinetic properties of the postinfarction scar, normal myocardium, and blood were compared.
Dynamic T1-weighted MR imaging revealed the washout time constants for EP-3533 to be significantly longer than those for gadopentetate dimeglumine in regions of postinfarction scarring (mean, 194.8 minutes +/-116.8 standard deviation vs 25.5 minutes +/- 4.2; P < .05) and in normal myocardium (mean, 45.4 minutes +/- 16.7 vs 25.1 minutes +/- 9.7; P < .05). Findings on postmortem histologic sections stained for collagen correlated well with EP-3533-enhanced areas seen on inversion-recovery MR images. Fifty minutes after EP-3533 injection, the postinfarction scar tissue samples, as compared with the normal myocardium, had a twofold higher concentration of gadolinium.
Use of the gadolinium-based collagen-targeting contrast agent, EP-3533, enabled in vivo molecular MR imaging of fibrosis in a mouse model of healed postinfarction myocardial scarring.
Despite rapid advances in the stem cell field, the ability to identify and track transplanted or migrating stem cells in vivo is limited. To overcome this limitation, we used magnetic resonance ...imaging (MRI) to detect and follow transplanted stem cells over a period of 28 days in mice using an established myocardial infarction model. Pluripotent mouse embryonic stem (mES) cells were expanded and induced to differentiate into beating cardiomyocytes in vitro. The cardiac‐differentiated mES cells were then loaded with superparamagnetic fluorescent microspheres (1.63 μm in diameter) and transplanted into ischemic myocardium immediately following ligation and subsequent reperfusion of the left anterior descending coronary artery. To identify the transplanted stem cells in vivo, MRI was performed using a Varian Inova 4.7 Tesla scanner. Our results show that (a) the cardiac‐differentiated mES were effectively loaded with superparamagnetic microspheres in vitro, (b) the microsphere‐loaded mES cells continued to beat in culture prior to transplantation, (c) the transplanted mES cells were readily detected in the heart in vivo using noninvasive MRI techniques, (d) the transplanted stem cells were detected in ischemic myocardium for the entire 28‐day duration of the study as confirmed by MRI and post‐mortem histological analyses, and (e) concurrent functional MRI indicated typical loss of cardiac function, although significant amelioration of remodeling was noted after 28 days in hearts that received transplanted stem cells. These results demonstrate that it is feasible to simultaneously track transplanted stem cells and monitor cardiac function in vivo over an extended period using noninvasive MRI techniques.
Disclosure of potential conflicts of interest is found at the end of this article.
GM1 gangliosidosis (GM1) is a fatal neurodegenerative lysosomal storage disease that occurs most commonly in young children, with no effective treatment available. Long-term follow-up of GM1 cats ...treated by bilateral thalamic and deep cerebellar nuclei (DCN) injection of adeno-associated virus (AAV)-mediated gene therapy has increased lifespan to 8 years of age, compared with an untreated lifespan of ~8 months. Due to risks associated with cerebellar injection in humans, the lateral ventricle was tested as a replacement route to deliver an AAVrh8 vector expressing feline β-galactosidase (β-gal), the defective enzyme in GM1. Treatment via the thalamus and lateral ventricle corrected storage, myelination, astrogliosis, and neuronal morphology in areas where β-gal was effectively delivered. Oligodendrocyte number increased, but only in areas where myelination was corrected. Reduced AAV and β-gal distribution were noted in the cerebellum with subsequent increases in storage, demyelination, astrogliosis, and neuronal degeneration. These postmortem findings were correlated with endpoint MRI and magnetic resonance spectroscopy (MRS). Compared with the moderate dose with which most cats were treated, a higher AAV dose produced superior survival, currently 6.5 years. Thus, MRI and MRS can predict therapeutic efficacy of AAV gene therapy and non-invasively monitor cellular events within the GM1 brain.
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