Structural and molecular myelination deficits occur prior to neuronal loss in the YAC128 and BACHD models of Huntington disease.

White matter (WM) atrophy is a significant feature of Huntington disease (HD), although its aetiology and early pathological manifestations remain poorly defined. In this study, we aimed to characterize WM-related features in the transgenic YAC128 and BACHD models of HD. Using diffusion tensor magnetic resonance imaging (DT-MRI), we demonstrate that microstructural WM abnormalities occur from an early age in YAC128 mice. Similarly, electron microscopy analysis of myelinated fibres of the corpus callosum indicated that myelin sheaths are thinner in YAC128 mice as early as 1.5 months of age, well before any neuronal loss can be detected. Transcript levels of myelin-related genes in striatal and cortical tissues were significantly lower in YAC128 mice from 2 weeks of age, and these findings were replicated in differentiated primary oligodendrocytes from YAC128 mice, suggesting a possible mechanistic explanation for the observed structural deficits. Concordant with these observations, we demonstrate reduced expression of myelin-related genes at 3 months of age and WM microstructural abnormalities using DT-MRI at 12 months of age in the BACHD rats. These findings indicate that WM deficits in HD are an early phenotype associated with cell-intrinsic effects of mutant huntingtin on myelin-related transcripts in oligodendrocytes, and raise the possibility that WM abnormalities may be an early contributing factor to the pathogenesis of HD.

Optimization of arterial spin labeling MRI for quantitative tumor perfusion in a mouse xenograft model.

Perfusion is an important biomarker of tissue function and has been associated with tumor pathophysiology such as angiogenesis and hypoxia. Arterial spin labeling (ASL) MRI allows noninvasive and quantitative imaging of perfusion; however, the application in mouse xenograft tumor models has been challenging due to the low sensitivity and high perfusion heterogeneity. In this study, flow-sensitive alternating inversion recovery (FAIR) ASL was optimized for a mouse xenograft tumor. To assess the sensitivity and reliability for measuring low perfusion, the lumbar muscle was used as a reference region. By optimizing the number of averages and inversion times, muscle perfusion as low as 32.4 ± 4.8 (mean ± standard deviation) ml/100 g/min could be measured in 20 min at 7 T with a quantification error of 14.4 ± 9.1%. Applying the optimized protocol, heterogeneous perfusion ranging from 49.5 to 211.2 ml/100 g/min in a renal carcinoma was observed. To understand the relationship with tumor pathology, global and regional tumor perfusion was compared with histological staining of blood vessels (CD34), hypoxia (CAIX) and apoptosis (TUNEL). No correlation was observed when the global tumor perfusion was compared with these pathological parameters. Regional analysis shows that areas of high perfusion had low microvessel density, which was due to larger vessel area compared with areas of low perfusion. Nonetheless, these were not correlated with hypoxia or apoptosis. The results suggest that tumor perfusion may reflect certain aspect of angiogenesis, but its relationship with other pathologies needs further investigation.

Early detection of antiangiogenic treatment responses in a mouse xenograft tumor model using quantitative perfusion MRI.

Angiogenesis plays a major role in tumor growth and metastasis, with tumor perfusion regarded as a marker for angiogenesis. To evaluate antiangiogenic treatment response in vivo, we investigated arterial spin labeling (ASL) magnetic resonance imaging (MRI) to measure tumor perfusion quantitatively. Chronic and 24-h acute treatment responses to bevacizumab were assessed by ASL and dynamic-contrast-enhanced (DCE) MRI in the A498 xenograft mouse model. After the MRI, tumor vasculature was assessed by CD34 staining. After 39 days of chronic treatment, tumor perfusion decreased to 44.8 ± 16.1 mL/100 g/min (P < 0.05), compared to 92.6 ± 42.9 mL/100 g/min in the control group. In the acute treatment study, tumor perfusion in the treated group decreased from 107.2 ± 32.7 to 73.7 ± 27.8 mL/100 g/min (P < 0.01; two-way analysis of variance), as well as compared with control group post dosing. A significant reduction in vessel density and vessel size was observed after the chronic treatment, while only vessel size was reduced 24 h after acute treatment. The tumor perfusion correlated with vessel size (r = 0.66; P < 0.005) after chronic, but not after acute treatment. The results from DCE-MRI also detected a significant change between treated and control groups in both chronic and acute treatment studies, but not between 0 and 24 h in the acute treatment group. These results indicate that tumor perfusion measured by MRI can detect early vascular responses to antiangiogenic treatment. With its noninvasive and quantitative nature, ASL MRI would be valuable for longitudinal assessment of tumor perfusion and in translation from animal models to human.

A facile synthetic approach to a biodegradable polydisulfide MRI contrast agent.

A polydisulfide MRI contrast agent was obtained by grafting diethylenetriaminepentaacetic (DTPA) to disulfide-containing poly(amido amine)s-graft-poly(ethylene glycol) (PEG) followed by Gd(iii) complexation. Self-assembly of the MRI contrast agent obtained occurs in aqueous solution forming nanosized micelles with PEG shells and ionic complex cores. The chemistry and structures of the MRI contrast agent and assembly were characterized using NMR, GPC and DLS. Thiol-induced degradation of the backbone and the assembly of the MRI contrast agent were investigated using GPC and DLS, respectively, and easy degradation was observed. Poly(BAC-AMPD)-g-PEG-g-Gd-DTPA also shows a low cytotoxicity and a high r1 value, so it is promising to provide better MRI imaging with fewer side effects.

Quantitative mouse renal perfusion using arterial spin labeling.

Information on renal perfusion is essential for the diagnosis and prognosis of kidney function. Quantification using gadolinium chelates is limited as a result of filtration through renal glomeruli and safety concerns in patients with kidney dysfunction. Arterial spin labeling MRI is a noninvasive technique for perfusion quantification that has been applied to humans and animals. However, because of the low sensitivity and vulnerability to motion and susceptibility artifacts, its application to mice has been challenging. In this article, mouse renal perfusion was studied using flow-sensitive alternating inversion recovery at 7 T. Good perfusion image quality was obtained with spin-echo echo-planar imaging after controlling for respiratory, susceptibility and fat artifacts by triggering, high-order shimming and water excitation, respectively. High perfusion was obtained in the renal cortex relative to the medulla, and signal was absent in scans carried out post mortem. Cortical perfusion increased from 397 ± 36 (mean ± standard deviation) to 476 ± 73 mL/100 g/min after switching from 100% oxygen to carbogen with 95% oxygen and 5% carbon dioxide. The perfusion in the medulla was 2.5 times lower than that in the cortex and changed from 166 ± 41 mL/100 g/min under oxygen to 203 ± 40 mL/100 g/min under carbogen. T1 decreased in both the cortex (from 1570 ± 164 to 1377 ± 72 ms, p < 0.05) and medulla (from 1788 ± 107 to 1573 ± 144 ms, p < 0.05) under carbogen relative to 100% oxygen. The results showed the potential of the use of ASL for perfusion quantification in mice and in models of renal diseases.

Consequences of a fat diet in the distribution of minerals within pancreatic tissues of rats and rabbits.

The effects of plasma lipid overload on pancreatic islet function and on mineral imbalance are issues under debate. However, the outcomes may be biased by the different metabolisms of different species. This prospective study evaluated whether a high fat diet intake changed the distribution of physiologically relevant elements within pancreatic endocrine and exocrine tissues of Sprague Dawley rats and New Zealand White rabbits. Nuclear microscopy techniques provided images of the specimen density and structure as well as the elemental distributions and quantification of P, S, Cl, K, Ca, Fe, and Zn using unstained cryosections of pancreas. Our results indicate that pancreatic islets in normal rats and rabbits had lower tissue density and higher Ca, Fe, and Zn content compared to exocrine tissue, and that rabbit islets exhibit the highest Zn content (3,300 µg/g in rabbits versus 510 µg/g in rats). Fat diet intake resulted in large deposits of fat in the pancreas, which modified the density contrast of tissues and also resulted in a twofold decrease of Ca and Zn concentrations in islets of both rats and rabbits. This result indicates that a fat diet leads to a reduction in essential trace element concentrations in pancreas, which in turn may hamper endocrine function.

Does iron inhibit calcification during atherosclerosis?

Oxidative stress has been implicated in the etiology of atherosclerosis and even held responsible for plaque calcification. Transition metals such as iron aggravate oxidative stress. To understand the relation between calcium and iron in atherosclerotic lesions, a sensitive technique is required that is quantitatively accurate and avoids isolation of plaques or staining/fixing tissue, because these processes introduce contaminants and redistribute elements within the tissue. In this study, the three ion-beam techniques of scanning transmission ion microscopy, Rutherford backscattering spectrometry, and particle-induced X-ray emission have been combined in conjunction with a high-energy (MeV) proton microprobe to map the spatial distribution of the elements and quantify them simultaneously in atherosclerotic rabbit arteries. The results show that iron and calcium within the atherosclerotic lesions exhibit a highly significant spatial inverse correlation. It may be that iron accelerates the progression of atherosclerotic lesion development, but suppresses calcification. Alternatively, calcification could be a defense mechanism against atherosclerotic progression by excluding iron.

Nuclear microscopy: a novel technique for quantitative imaging of gadolinium distribution within tissue sections.

All clinically-approved and many novel gadolinium (Gd)-based contrast agents used to enhance signal intensity in magnetic resonance imaging (MRI) are optically silent. To verify MRI results, a "gold standard" that can map and quantify Gd down to the parts per million (ppm) levels is required. Nuclear microscopy is a relatively new technique that has this capability and is composed of a combination of three ion beam techniques: scanning transmission ion microscopy, Rutherford backscattering spectrometry, and particle induced X-ray emission used in conjunction with a high energy proton microprobe. In this proof-of-concept study, we show that in diseased aortic vessel walls obtained at 2 and 4 h after intravenous injection of the myeloperoxidase-sensitive MRI agent, bis-5-hydroxytryptamide-diethylenetriamine-pentaacetate gadolinium, there was a time-dependant Gd clearance (2 h = 18.86 ppm, 4 h = 8.65 ppm). As expected, the control animal, injected with the clinically-approved conventional agent diethylenetriamine-pentaacetate gadolinium and sacrificed 1 week after injection, revealed no significant residual Gd in the tissue. Similar to known in vivo Gd pharmacokinetics, we found that Gd concentration dropped by a factor of 2 in vessel wall tissue in 1.64 h. Further high-resolution studies revealed that Gd was relatively uniformly distributed, consistent with random agent diffusion. We conclude that nuclear microscopy is potentially very useful for validation studies involving Gd-based magnetic resonance contrast agents.

A novel approach to the identification and quantitative elemental analysis of amyloid deposits--insights into the pathology of Alzheimer's disease.

There is considerable interest in the role of metals such as iron, copper, and zinc in amyloid plaque formation in Alzheimer's disease. However to convincingly establish their presence in plaques in vivo, a sensitive technique is required that is both quantitatively accurate and avoids isolation of plaques or staining/fixing brain tissue, since these processes introduce contaminants and redistribute elements within the tissue. Combining the three ion beam techniques of scanning transmission ion microscopy, Rutherford back scattering spectrometry and particle induced X-ray emission in conjunction with a high energy (MeV) proton microprobe we have imaged plaques in freeze-dried unstained brain sections from CRND-8 mice, and simultaneously quantified iron, copper, and zinc. Our results show increased metal concentrations within the amyloid plaques compared with the surrounding tissue: iron (85 ppm compared with 42 ppm), copper (16 ppm compared to 6 ppm), and zinc (87 ppm compared to 34 ppm).

Zinc supplementation inhibits lipid peroxidation and the development of atherosclerosis in rabbits fed a high cholesterol diet.

Developing atherosclerotic lesions in hypercholesterolemic rabbits are depleted in zinc, while iron accumulates. This study examined the influence of zinc supplementation on the development of atherosclerosis and used isotope dilution gas chromatography-mass spectrometry techniques to measure biomarkers of oxidative lipid damage in atherosclerotic rabbit aorta. Our previous method for F(2)-isoprostane measurement was adapted to include the quantitation of cholesterol oxidation products in the same sample. Two groups of New Zealand white rabbits were fed a high cholesterol (1% w/w) diet and one group was also supplemented with zinc (1 g/kg) for 8 weeks. Controls were fed a normal diet. Zinc supplementation did not significantly alter the increase in total plasma cholesterol levels observed in animals fed high cholesterol. However, in cholesterol-fed animals zinc supplementation significantly reduced the accumulation of total cholesterol levels in aorta which was accompanied by a significant reduction in average aortic lesion cross-sectional areas of the animals. Elevated levels of cholesterol oxidation products (5,6-alpha and beta cholesterol epoxides, 7beta-hydroxycholesterol, 7-ketocholesterol) in aorta and total F(2)-isoprostanes in plasma and aorta of rabbits fed a cholesterol diet were significantly decreased by zinc supplementation. Our data indicate that zinc has an antiatherogenic effect, possibly due to a reduction in iron-catalyzed free radical reactions.

Promotion of atherogenesis by copper or iron--which is more likely?

Iron levels increase in atherosclerotic lesions in cholesterol fed-rabbits and play a role in atherosclerosis. We investigated whether copper also rises. Male New Zealand White rabbits were fed high-cholesterol diets for 8 weeks. After sacrifice, lesion sizes were determined, and elemental analyses of the lesion and unaffected artery wall performed using nuclear microscopy. Unlike iron, lesion copper is decreased by about half compared with the unaffected artery wall, and much less copper than iron is present. Our data suggest that iron may be more likely to play a role in the promotion of atherosclerosis than copper.

Zinc supplementation decreases the development of atherosclerosis in rabbits.

Developing atherosclerotic plaques in cholesterol-fed rabbits are enriched in iron but depleted in zinc. In order to examine further the role of zinc, New Zealand White rabbits were fed a high-cholesterol 1% (w/w) diet with zinc (1 g/kg) supplementation for 8 weeks. After the 8-week period, the average atherosclerotic lesion cross-sectional areas in the aortas of the animals fed with the zinc supplement were significantly decreased (1.0 mm2) compared with lesion areas of the animals fed only on the high-cholesterol diet (3.1 mm2). Using nuclear microscopy, a technique for mapping and measuring trace elements in tissue sections, lesion zinc levels (24 ppm) were observed to be unchanged in the zinc-fed rabbits compared to controls. However, average lesion Fe levels in the zinc-fed group were measured at 32 ppm, whereas in the control group the average Fe levels were significantly higher at 43 ppm (P = 0.03). Our data support the concept that zinc may have an antiatherogenic effect by decreasing iron levels in the lesion, possibly leading to inhibition of iron-catalyzed free radical reactions.

The iron chelator desferrioxamine inhibits atherosclerotic lesion development and decreases lesion iron concentrations in the cholesterol-fed rabbit.

Several epidemiological studies have suggested that increased iron stores are associated with increased atherosclerotic events. In order to test the hypothesis that decreasing the vascular level of iron slows lesion growth, we examined the effects of the iron chelator Desferal (72 mg/kg/day, 5 days/week) on atherosclerosis and lesion iron content in cholesterol-fed New Zealand White rabbits. Rabbits were fed with a 1% w/w cholesterol diet for either 8 weeks (and for the last 5 weeks injected daily with Desferal) or 12 weeks (and for the last 9 weeks injected with Desferal). Controls were injected with saline. A significant reduction in average lesion area (p = 0.038) was observed in the 12-week treated animals compared with the 12-week controls. The average lesion iron level of the 12-week treated animals (58 ppm dry wt) was also significantly lower (p = 0.030) than in 12-week control animals (95 ppm dry wt), as measured using nuclear microscopy with the combination of scanning transmission ion microscopy, Rutherford back-scattering spectroscopy, and particle-induced X-ray emission. No reduction in lesion area or iron content was observed in the 8-week treated animals compared with controls, and no change in lesion zinc concentration was observed for either group. Our data strengthen the concept that iron contributes to the early stages of the development of atherosclerosis.