In vivo MRI and ex vivo quantification of iron and Kupffer cells demonstrate residual phagocytic activity in mouse liver after a gadolinium chloride injection.

Kupffer cells (KCs), the resident macrophages of the liver, display a phagocytic activity that is not well quantified in animal models. Its experimental invalidation in rodents has been carried out by various means, among which the gadolinium chloride (GdCl3) injection has been widely used, and has been generally monitored by ex vivo techniques. The aim of our study was to determine the KC phagocytic activity induced in mouse liver following a single GdCl3 injection, through Magnetic Resonance Imaging (MRI) measurement of liver uptake of Ferumoxide in vivo, and through ex vivo quantification of Perls positive and F4/80 labeled macrophages. In this study, we showed that 24 h after an IV injection at a dose of 50 mg/kg body weight, GdCl3 did not induce any hepato-cellular damage, nor did it strongly suppress liver phagocytic activity, as demonstrated by the persistent hepatic uptake of the iron-based MRI contrast agent Ferumoxide. In the GdCl3-treated mice, the injection of Ferumoxide produced an increase in the liver proton transverse relaxation rate R2 which averaged 71 ± 24% of that of the control animals. The ex vivo iron and immune phenotypic quantification, performed after the Ferumoxide injection and MRI, confirmed the presence of activated phagocytes in the liver of the GdCl3-treated animals, with a global iron score and F4/80 positive cell count respectively averaging 85 ± 26% and 46 ± 13% of their values in the untreated mice. In vivo MRI evaluation of the liver phagocytic activity using Ferumoxide may further prove useful in the follow up of both experimental and human pathologies.

The influence of skeletal muscle anisotropy on electroporation: in vivo study and numerical modeling.

The aim of this study was to theoretically and experimentally investigate electroporation of mouse tibialis cranialis and to determine the reversible electroporation threshold values needed for parallel and perpendicular orientation of the applied electric field with respect to the muscle fibers. Our study was based on local electric field calculated with three-dimensional realistic numerical models, that we built, and in vivo visualization of electroporated muscle tissue. We established that electroporation of muscle cells in tissue depends on the orientation of the applied electric field; the local electric field threshold values were determined (pulse parameters: 8 x 100 micros, 1 Hz) to be 80 V/cm and 200 V/cm for parallel and perpendicular orientation, respectively. Our results could be useful electric field parameters in the control of skeletal muscle electroporation, which can be used in treatment planning of electroporation based therapies such as gene therapy, genetic vaccination, and electrochemotherapy.

Selective iron chelation in Friedreich ataxia: biologic and clinical implications.

Genetic disorders of iron metabolism and chronic inflammation often evoke local iron accumulation. In Friedreich ataxia, decreased iron-sulphur cluster and heme formation leads to mitochondrial iron accumulation and ensuing oxidative damage that primarily affects sensory neurons, the myocardium, and endocrine glands. We assessed the possibility of reducing brain iron accumulation in Friedreich ataxia patients with a membrane-permeant chelator capable of shuttling chelated iron from cells to transferrin, using regimens suitable for patients with no systemic iron overload. Brain magnetic resonance imaging (MRI) of Friedreich ataxia patients compared with age-matched controls revealed smaller and irregularly shaped dentate nuclei with significantly (P < .027) higher H-relaxation rates R2*, indicating regional iron accumulation. A 6-month treatment with 20 to 30 mg/kg/d deferiprone of 9 adolescent patients with no overt cardiomyopathy reduced R2* from 18.3 s(-1) (+/- 1.6 s(-1)) to 15.7 s(-1) (+/- 0.7 s(-1); P < .002), specifically in dentate nuclei and proportionally to the initial R2* (r = 0.90). Chelator treatment caused no apparent hematologic or neurologic side effects while reducing neuropathy and ataxic gait in the youngest patients. To our knowledge, this is the first clinical demonstration of chelation removing labile iron accumulated in a specific brain area implicated in a neurodegenerative disease. The use of moderate chelation for relocating iron from areas of deposition to areas of deprivation has clinical implications for various neurodegenerative and hematologic disorders.

An automated image-processing strategy to analyze dynamic arterial spin labeling perfusion studies. Application to human skeletal muscle under stress.

Arterial spin labeling (ASL) perfusion measurements allow the follow-up of muscle perfusion with high temporal resolution during a stress test. Automated image processing is proposed to estimate perfusion maps from ASL images. It is based on two successive analyses: at first, automated rejection of the image pairs between which a large displacement is detected is performed, followed by factor analysis of the dynamic data and cluster analysis to classify pixels with large signal variation characteristic of vessels. Then, after masking these "vascular" pixels, factor analysis and cluster analysis are further applied to separate the different muscles between low or high perfusion increase, yielding a functional map of the leg. Data from 10 subjects (five normal volunteers and five elite sportsmen) had been analyzed. Resulting time perfusion curves from a region of interest (ROI) in active muscles show a good accordance whether extracted with automated processing or with manual processing. This method of functional segmentation allows automated suppression of vessels and fast visualization of muscles with high, medium or low perfusion, without any a priori knowledge.

New insight into abnormal muscle vasodilatory responses in aged hypertensive rats by in vivo nuclear magnetic resonance imaging of perfusion.

OBJECTIVE: Increased peripheral arterial resistances and decreased maximum vasodilation are characteristic features of chronic hypertension. However, little data are available in the literature regarding the possible alterations in the temporal patterns of vasodilatory responses elicited by various stimuli. DESIGN: This question was addressed by measuring skeletal muscle perfusion using nuclear magnetic resonance imaging combined with arterial spin labeling. METHODS: Ninety-week-old male spontaneously hypertensive (SHR; n = 7) and normotensive Wistar Kyoto (WKY; n = 8) rats were studied, and calf muscle perfusion was measured at rest and during reactive hyperemia following total ischemia of 5 and 30 min duration. RESULTS: Reactive hyperemia profiles differed according to duration of ischemia. In WKY rats, 5 min of ischemia induced a short peak of hyperemia lasting no more than 63 s, while 30 min of ischemia were followed by a prolonged hyperemic response of 261 s. In SHRs, after 5 min of ischemia, peak muscle arterial conductance was decreased to 0.5 +/- 0.3 versus 0.9 +/- 0.3 ml.min(-1).100 g(-1).mm Hg(-1) in the WKY rats (p < 0.05), as expected. After 30 min of ischemia, there was, in addition, a shortening of the hyperemic response duration. Time to post-ischemic half normalization of arterial conductance was 38 +/- 24 s in the SHRs versus 149 +/- 58 s in the WKY rats (p < 0.001). CONCLUSION: In vivo perfusion measurement not only confirmed the existence of a reduced maximum peripheral vasodilation in chronically hypertensive rats, it revealed a blunted hyperemic response after prolonged ischemia in the SHRs, which might be an important contributing factor to the increased sensitivity to ischemia in hypertension.

Evaluation of muscle glycogen content by 13C NMR spectroscopy in adult-onset acid maltase deficiency.

Muscle glycogen storage was measured by in vivo, natural abundance 13C nuclear magnetic resonance spectroscopy in distal and proximal lower limb segments of patients suffering from adult-onset acid maltase deficiency. Interleaved T1-weighted acquisitions of glycogen and creatine served to quantify glycogen excess. For acid maltase deficient patients (n=11), glycogen:creatine was higher than controls (n=12), (1.20+/-0.39 vs. 0.83+/-0.18, P=0.0005). Glycogen storage was above the normal 95% confidence limits in at least one site for 7/11 patients. The intra-individual coefficient of reproducibility was 12%. This totally atraumatic measurement of glycogen allows repeated measurement at different muscle sites of acid maltase deficient patients, despite selective fatty replacement of tissue. This could provide an additional parameter to follow the development of disease in individual patients, including in the perspective of forthcoming therapeutic trials. It may also offer an appropriate tool to study the role of glycogen accumulation in progression of the pathology.

Electrotransfer at MR imaging: tool for optimization of gene transfer protocols--feasibility study in mice.

PURPOSE: To test, by using an electrotransfer protocol for the transfection of skeletal muscle with naked plasmid complementary DNA, whether in vivo magnetic resonance (MR) imaging can help delineate either the spatial extent of the electric field when contrast agent is injected intraperitoneally or the transfection area when contrast agent is injected locally. MATERIALS AND METHODS: Three groups of five mice each were examined at 4 T. Gadopentetate dimeglumine was injected intraperitoneally before electroporation in group 1 and after electroporation in group 2. In group 3, gadopentetate dimeglumine was coinjected in situ with plasmid pCMV-beta Gal in the gastrocnemius muscle before electroporation. MR imaging and muscle preparation for histologic examination were performed 3 days later. On T1-weighted images, increase of muscle signal intensity was determined in regions of interest (ROIs) of treated legs and compared with contralateral ROIs. Comparison of signal intensity increase between groups 1 and 2 was performed with Kruskal-Wallis test. RESULTS: In groups 1 and 3, T1-weighted images of treated muscle showed zones of strongly increased signal intensity. In corresponding ROIs of groups 1, 2, and 3, the mean T1-weighted signal intensity increase at day 3 was 1.64 +/- 0.20 (SD), 1.16 +/- 0.06, and 1.58 +/- 0.17, respectively. The difference between groups 1 and 2 (ie, gadopentetate dimeglumine injected before and after electrotransfer) was significant (P <.001) both without and with correction for T2 variation (1.47 +/- 0.19 and 1.04 +/- 0.09, respectively). In group 3, after in situ coinjection of gadopentetate dimeglumine and plasmid, the area of increased signal intensity revealed at ex vivo MR imaging of the muscle showed a reasonable concordance with the transfected area revealed with beta-galactosidase on histologic sections. CONCLUSION: In vivo and ex vivo results indicate that atraumatic visualization of the permeabilized and transfected area is possible.

Ultrafast multiplanar determination of left ventricular hypertrophy in spontaneously hypertensive rats with single-shot spin-echo nuclear magnetic resonance imaging.

OBJECTIVE: To determine whether left ventricular hypertrophy can be correctly evaluated in hypertensive rats with a new nuclear magnetic resonance (NMR) imaging modality that is relatively simple to operate and provides results of constant quality while offering a high signal-to-noise ratio. DESIGN Left ventricular mass as calculated from the NMR imaging analysis was compared with the actual left ventricular mass measured by gravimetry. METHODS: Single-shot ultrafast spin-echo (SSFSE) imaging of hearts of Wistar-Kyoto rats and spontaneously hypertensive rats was performed at 4 T. Left ventricular mass was determined by using Simpson's rule on stacks of images acquired in systole and diastole. RESULTS: SSFSE NMR imaging performed in systole or in diastole evaluated and quantified left ventricular hypertrophy in hearts of spontaneously hypertensive rats very similarly to gravimetry. The left ventricular mass as determined by NMR was in good accordance with the actual left ventricular weight (SEE: 30.39 and 35.86 mg for the systolic and diastolic NMR acquisitions, respectively). CONCLUSION: Using an SSFSE sequence, high-quality NMR images of the rat heart can be generated very reliably with sufficient contrast and temporal and spatial resolution, and allow precise, non-invasive and fast characterization of left ventricular hypertrophy in a hypertensive rat model.