Different quantification algorithms may lead to different results: a comparison using proton MRS lipid signals.

Proton magnetic resonance spectroscopy (MRS) is a sensitive method for investigating the biochemical compounds in a tissue. The interpretation of the data relies on the quantification algorithms applied to MR spectra. Each of these algorithms has certain underlying assumptions and may allow one to incorporate prior knowledge, which could influence the quality of the fit. The most commonly considered types of prior knowledge include the line-shape model (Lorentzian, Gaussian, Voigt), knowledge of the resonating frequencies, modeling of the baseline, constraints on the damping factors and phase, etc. In this article, we study whether the statistical outcome of a biological investigation can be influenced by the quantification method used. We chose to study lipid signals because of their emerging role in the investigation of metabolic disorders. Lipid spectra, in particular, are characterized by peaks that are in most cases not Lorentzian, because measurements are often performed in difficult body locations, e.g. in visceral fats close to peristaltic movements in humans or very small areas close to different tissues in animals. This leads to spectra with several peak distortions. Linear combination of Model spectra (LCModel), Advanced Method for Accurate Robust and Efficient Spectral fitting (AMARES), quantitation based on QUantum ESTimation (QUEST), Automated Quantification of Short Echo-time MRS (AQSES)-Lineshape and Integration were applied to simulated spectra, and area under the curve (AUC) values, which are proportional to the quantity of the resonating molecules in the tissue, were compared with true values. A comparison between techniques was also carried out on lipid signals from obese and lean Zucker rats, for which the polyunsaturation value expressed in white adipose tissue should be statistically different, as confirmed by high-resolution NMR measurements (considered the gold standard) on the same animals. LCModel, AQSES-Lineshape, QUEST and Integration gave the best results in at least one of the considered groups of simulated or in vivo lipid signals. These outcomes highlight the fact that quantification methods can influence the final result and its statistical significance.

Tumor and peritumoral adipose tissue crosstalk: De-differentiated adipocytes influence spread of colon carcinoma cells.

Colorectal cancer is the second leading cause of cancer and often has a fatal course. There are many studies in the literature that have described a close functional relationship between the tumor mass and surrounding tissue, or tumor stroma, which is affected by the continuous metabolic exchange that occurs at the interface between tumor and tissues in contact with it. There is much evidence that the presence of adipose tissue in stroma plays a fundamental role in modulating the tumor microenvironment and promote tumor development, growth, and angiogenesis due to its endocrine characteristics. In this analysis, we have studied the alterations of adipose tissue surrounding colorectal tumors with MRI and optical imaging in vivo techniques to monitor tumor progression and also performed histological and molecular analysis. We detected differences in the principal adipose markers expressed by adipocytes residing around the rectal colon and observed that peritumoral adipose tissue is exposed to a mesenchymal transition process that leads to the acquisition of a less differentiated phenotype of adipocyte that represents the main cellular type present in tumor stroma. The mesenchymal transition correlated with the acquisition of more aggressive tumor phenotype and could represent a valid target for tumor therapy.

Inhibition of tyrosine kinase receptors by SU6668 promotes abnormal stromal development at the periphery of carcinomas.

Dynamic contrast-enhanced (albumin-Gd-DTPA) magnetic resonance imaging, performed during 2 weeks of daily administration of an inhibitor of tyrosine kinase receptors (SU6668) in an HT-29 colon carcinoma model, revealed the onset of a hyper-enhancing rim, not observed in untreated tumours. To account for tissue heterogeneity in the quantitative analysis, we segmented tumours into three subunits automatically identified by cluster analysis of the enhancement curves using a k-means algorithm. Transendothelial permeability (Kps) and fractional plasma volume (fPV) were calculated in each subunit. An avascular and necrotic region, an intermediate zone and a well-vascularised periphery were reliably identified. During untreated tumour growth, the identified sub-regions did not substantially change their enhancement pattern. Treatment with SU6668 induced major changes at tumour periphery where a significant increase of Kps and fPV was observed with respect to control tumours. Histology revealed a sub-capsular layer composed of hyper-dense viable tumour cells in the periphery of untreated tumours. The rim of viable neoplastic cells was reduced in treated tumours, and replaced by loose connective tissue characterised by numerous vessels, which explains the observed hyper-enhancement. The present data show a peripheral abnormal development of cancer-associated stroma, indicative of an adaptive response to anti-angiogenic treatment.

Experimental protocol for activation-induced manganese-enhanced MRI (AIM-MRI) based on quantitative determination of Mn content in rat brain by fast T1 mapping.

In activation-induced manganese-enhanced MRI (AIM-MRI) experiments, differential accumulation of Mn in activated and silent brain areas is generally assessed using T(1)-weighted images and quantified by the enhancement of signal intensity (SI), calculated with reference to SI before Mn administration or to SI of brain regions unaffected by the specific stimulus. However, SI enhancement can be unreliable when animals are removed from and reinserted into the magnet. We have developed an experimental protocol based on repeated intraperitoneal (i.p.) injections of Mn, quantitative determination of T(1), and coregistration of images to a rat brain atlas that allows absolute quantification of Mn concentration in selected brain areas. Results showed that interanimal variability of postcontrast T(1) values was very low (compared to the experimental error in T(1) determinations) allowing detection of differential regional Mn uptake in stimulated and unstimulated animals. In addition we have determined in vivo relaxivity of Mn in brain tissue and its frequency dependence.

Synthesis and characterization of polyethylenimine-based iron oxide composites as novel contrast agents for MRI.

OBJECT: Use of polyethylenimines (PEIs) of different molecular weight and selected carboxylated-PEI derivatives (PEI-COOH) in the synthesis and stabilization of iron oxide nanoparticles, to obtain possible multifunctional contrast agents. MATERIALS AND METHODS: Oxidation of Fe(II) at slightly elevated pH and temperature resulted in the formation of highly soluble and stable nanocomposites of iron oxides and polymer. Composites were characterized and studied by atomic force microscopy (AFM), transmission electron microscopy (TEM), X-ray diffractometry, AC and DC magnetometry, NMR relaxometry and magnetic resonance imaging (MRI). RESULTS: From AFM the dimensions of the aggregates were found to be in the ~150-250 nm size region; the mean diameter of the magnetic core of the compounds named PEI-25, PEI-500 and PEI-COOH60 resulted d approximately 20 +/- 5 nm for PEI-25, d approximately 9.5 +/- 1.0 nm for PEI-500 and d approximately 6.8 +/- 1.0 nm for PEI-COOH60. In PEI-COOH60 TEM and X-ray diffractometry revealed small assemblies of mineral magnetic cores with clear indications that the main constituents are maghemite and/or magnetite as confirmed by AC and DC SQUID magnetometry. For PEI-COOH60, the study of NMR-dispersion profiles revealed r (1) and r (2) relaxivities comparable to superparamagnetic iron-oxide commercial compounds in the whole investigated frequency range 7 < or = nu < or = 212 MHz. CONCLUSION: PEI-25 was studied as possible MRI contrast agent (CA) to map the cerebral blood volume (CBV) and cerebral blood flow (CBF) in an animal model obtaining promising results. The reported compounds may be further functionalized to afford novel multifunctional systems for biomedical applications.

DCE-MRI data analysis for cancer area classification.

OBJECTIVES: The paper aims at improving the support of medical researchers in the context of in-vivo cancer imaging. Morphological and functional parameters obtained by dynamic contrast-enhanced MRI (DCE-MRI) techniques are analyzed, which aim at investigating the development of tumor microvessels. The main contribution consists in proposing a machine learning methodology to segment automatically these MRI data, by isolating tumor areas with different meaning, in a histological sense. METHODS: The proposed approach is based on a three-step procedure: i) robust feature extraction from raw time-intensity curves, ii) voxel segmentation, and iii) voxel classification based on a learning-by-example approach. In the first step, few robust features that compactly represent the response of the tissue to the DCE-MRI analysis are computed. The second step provides a segmentation based on the mean shift (MS) paradigm, which has recently shown to be robust and useful for different and heterogeneous clustering tasks. Finally, in the third step, a support vector machine (SVM) is trained to classify voxels according to the labels obtained by the clustering phase (i.e., each class corresponds to a cluster). Indeed, the SVM is able to classify new unseen subjects with the same kind of tumor. RESULTS: Experiments on different subjects affected by the same kind of tumor evidence that the extracted regions by both the MS clustering and the SVM classifier exhibit a precise medical meaning, as carefully validated by the medical researchers. Moreover, our approach is more stable and robust than methods based on quantification of DCE-MRI data by means of pharmacokinetic models. CONCLUSIONS: The proposed method allows to analyze the DCE-MRI data more precisely and faster than previous automated or manual approaches.

Fast and Sensitive Detection of Paramagnetic Species Using Coupled Charge and Spin Dynamics in Strongly Fluorescent Nanodiamonds.

Sensing of a few unpaired electron spins, such as in metal ions and radicals, is a useful but difficult task in nanoscale physics, biology, and chemistry. Single negatively charged nitrogen-vacancy (NV-) centers in diamond offer high sensitivity and spatial resolution in the optical detection of weak magnetic fields produced by a spin bath but often require long acquisition times on the order of seconds. Here, we present an approach based on coupled spin and charge dynamics in dense NV ensembles in strongly fluorescent nanodiamonds (NDs) to sense external magnetic dipoles. We apply this approach to various paramagnetic species, including gadolinium complexes, magnetite nanoparticles, and hemoglobin in whole blood. Taking advantage of the high NV density, we demonstrate a dramatic reduction in acquisition time (down to tens of milliseconds) while maintaining high sensitivity to paramagnetic centers. Strong luminescence, high sensitivity, and short acquisition time make dense NV- ensembles in NDs a potentially promising tool for biosensing and bioimaging applications.

In vivo imaging techniques: a new era for histochemical analysis.

In vivo imaging techniques can be integrated with classical histochemistry to create an actual histochemistry of water. In particular, Magnetic Resonance Imaging (MRI), an imaging technique primarily used as diagnostic tool in clinical/preclinical research, has excellent anatomical resolution, unlimited penetration depth and intrinsic soft tissue contrast. Thanks to the technological development, MRI is not only capable to provide morphological information but also and more interestingly functional, biophysical and molecular. In this paper we describe the main features of several advanced imaging techniques, such as MRI microscopy, Magnetic Resonance Spectroscopy, functional MRI, Diffusion Tensor Imaging and MRI with contrast agent as a useful support to classical histochemistry.

Characterization of magnetic nanoparticles from Magnetospirillum Gryphiswaldense as potential theranostics tools.

We investigated the theranostic properties of magnetosomes (MNs) extracted from magnetotactic bacteria, promising for nanomedicine applications. Besides a physico-chemical characterization, their potentiality as mediators for magnetic fluid hyperthermia and contrast agents for magnetic resonance imaging, both in vitro and in vivo, are here singled out. The MNs, constituted by magnetite nanocrystals arranged in chains, show a superparamagnetic behaviour and a clear evidence of Verwey transition, as signature of magnetite presence. The phospholipid membrane provides a good protection against oxidation and the MNs oxidation state is stable over months. Using an alternate magnetic field, the specific absorption rate was measured, resulting among the highest reported in literature. The MRI contrast efficiency was evaluated by means of the acquisition of complete NMRD profiles. The transverse relaxivity resulted as high as the one of a former commercial contrast agent. The MNs were inoculated into an animal model of tumour and their presence was detected by magnetic resonance images two weeks after the injection in the tumour mass.

Activity and conformational changes of alpha-chymotrypsin in reverse micelles studied by spin labeling.

alpha-Chymotrypsin (CT), spin-labeled at the active site by using an acylating label which constitutes a substrate for this protein, has been investigated in reverse micelles formed by AOT in isooctane. The electron spin resonance spectra provided information on conformation, dynamics and deacylation activity. The dynamics of the label bound to CT appears to be more hindered in reverse micelles than in aqueous solution, probably owing to the effect of the micellar environment on protein conformation. The deacylation rate in reverse micelles does not show the characteristic bell-shaped dependence on water content which is generally found for CT enzymatic activity.

The neuroprotective activity of the glycine receptor antagonist GV150526: an in vivo study by magnetic resonance imaging.

The neuroprotective activity of GV150526 (3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt), a selective glycine receptor antagonist of the NMDA receptor, has been evaluated by magnetic resonance imaging (MRI) in a rat model of middle cerebral artery occlusion. The aim of the work was to evaluate, using an in vivo method, whether GV150526 was able to reduce the extent of ischemic brain damage when administered both before and after (6 h) middle cerebral artery occlusion. GV150526 was administered at a dose of 3 mg/kg i.v. T2-weighted (T2W) and diffusion weighted (DW) images were acquired at 6, 24 and 144 h after the establishment of the cerebral ischemia. Substantial neuroprotection was demonstrated at all investigated time points when GV150526 was administered before the ischemic insult. The ischemic volume was reduced by 84% and 72%, compared to control values, when measured from T2W and DW images, acquired 24 h after middle cerebral artery occlusion. Administration of the same dose of GV150526, 6 h post-ischemia, also resulted in a significant (p < 0.05) neuroprotection. The ischemic volume was reduced by 48% from control values when measured from T2W images and by 45% when measured from DW images. No significant difference was found between volumes of brain ischemia obtained by either MRI or triphenyltetrazolium chloride staining. These data confirm the potential neuroprotective activity of the glycine receptor antagonist GV150526 when administered either before or up to 6 h after ischemia.

Evolution strategy optimization for adiabatic pulses in MRI.

We propose a new type of adiabatic pulses for uniform inversion of the magnetization in magnetic resonance imaging. We produced these pulses with an evolution strategy optimization, by which the search of the "best solution" has been made more efficient than by deterministic algorithms. The pulse parametrization takes into account an "offset-independent adiabaticity condition," which guarantees insensitivity to RF inhomogeneities. The RF pulse power (both peak and mean) contributes to the cost to be minimized, as well as the error function does: in this way we obtain solutions that require lower energy than the well-known hyperbolic-secant pulse, with no loss of quality in the response profile.

Effect of dietary supplementation with zinc sulphate on the aging process: a study using high field intensity MRI and chemical shift imaging.

High field intensity magnetic resonance imaging (HF-MRI) has been applied to the in vivo study of age-related processes of organs located in the cervical-thoracic region in mice and to describe the effects of oral zinc supplementation on these processes. Spin-echo (SE) pulse sequence and chemical shift imaging (CSI) techniques have been used. Aging produced a progressive reduction of muscular masses and of thymic area, whereas the HF-MRI appearances of spinal cord and of salivary glands were unchanged. In some aged animals, subcutaneous fat was reduced while visceral fat was well developed. In the group of old animals supplemented with zinc sulphate, muscular masses were more developed than that of the group of untreated old animals. Oral zinc supplementation also produced an enlargement of the adipose tissue and the thymic area showed an increase of about 65% compared with thymic area measured in the group of old animals used as controls. The present study confirms previous data about the effects of Zn supplementation on aging processes and demonstrates that HF-MRI is a powerful technique to study processes of aging, providing information about the effects of drug treatments on these processes.

Bayesian estimation of relaxation times T(1) in MR images of irradiated Fricke-agarose gels.

The authors present a novel method for processing T(1)-weighted images acquired with Inversion-Recovery (IR) sequence. The method, developed within the Bayesian framework, takes into account a priori knowledge about the spatial regularity of the parameters to be estimated. Inference is drawn by means of Markov Chains Monte Carlo algorithms. The method has been applied to the processing of IR images from irradiated Fricke-agarose gels, proposed in the past as relative dosimeter to verify radiotherapeutic treatment planning systems. Comparison with results obtained from a standard approach shows that signal-to noise ratio (SNR) is strongly enhanced when the estimation of the longitudinal relaxation rate (R1) is performed with the newly proposed statistical approach. Furthermore, the method allows the use of more complex models of the signal. Finally, an appreciable reduction of total acquisition time can be obtained due to the possibility of using a reduced number of images. The method can also be applied to T(1) mapping of other systems.

Correlation MRI/ultrastructure in cerebral ischemic lesions: application to the interpretation of cortical layered areas.

The origin and fate of cortical ischemic lesions, showing a stratified appearance at in vivo MRI-examination, was studied on rats in which a focal brain ischemia was induced by occlusion of the middle cerebral artery. One week after ischemia induction, six rats were selected in which three layers of different intensity were visible in the lesioned cortex. Two animals were sacrificed and studied by histology and electron microscopy. The external hyperintense layer was composed of pial and lesioned nervous tissue, the intermediate of degenerating nervous tissue in which an accumulation of macrophages was found, the deepest of edematous nerve tissue without a marked accumulation of macrophages. The remaining rats underwent further MRI examinations showing that, in the lesioned areas, cerebral blood volume was 14-69% lower than the contralateral healthy cortex. At histological and ultrastructural examination, a large part of the lesion was occupied by enlarged pial tissue and marginal glia. A dilatation of the ventricular cavity and cystic structures were also visible. In three animals an increase of the transverse diameter of the caudo-putamen ipsilateral to the lesion was found. The study suggests that the layered appearance is mainly due to an accumulation of macrophages in the intermediate layer and that several processes contribute to the occlusion of the space created by the removal of the necrotic tissue in stratified ischemic lesions (i.e. expansion of the pial tissue, thickening of the marginal glia; expansion of the caudo-putamen, enlargement of the ventricular cavity and development of cystic structures).

Comparison of results of scanning electron microscopy and magnetic resonance imaging before and after administration of a radiographic contrast agent in the tendon of the deep digital flexor muscle obtained from horse cadavers.

OBJECTIVE: To analyze the tendon of the deep digital flexor (TDDF) muscle of the forelimb in horses by use of a contrast radiographic agent (gadopentate dimeglumine [Gd-DTPA/Dimeg]) and magnetic resonance imaging (MRI) and to determine the concentration of water protons in the tendons by use of MRI. SAMPLE POPULATION: 8 TDDF harvested from the forelimbs of 6 horse cadavers. PROCEDURE: Examinations were performed on the same portion of each tendon. Tendons were examined by use of two techniques: MRI before and after treatment with Gd-DTPA/Dimeg as well as scanning electron microscopy. RESULTS: Tendons did not have detectable signal intensity on MRI before treatment with Gd-DTPA/Dimeg; however, intravascular injection of Gd-DTPA/Dimeg allowed evaluation of the internal structure of the tendons Scanning electron microscopy images correlated well with images obtained by use of MRI before and after administration of Gd-DTPA/Dimeg. Localized spectra revealed the concentration of water protons in the TDDF. CONCLUSIONS AND CLINICAL RELEVANCE: The techniques used in this study provided information about internal organization of the TDDF in horses. Analysis of results revealed that the best technique involved vascular injection of contrast medium. Results of MRI correlated well with results for scanning electron microscopy. After administration of Gd-DTPA/Dimeg, MRI provided additional information about tendon morphologic characteristics. This technique may be of value for examination of tendons in lame horses.

Influence of paramagnetic ions bound to human serum albumin on water 1HNMR relaxation times.

The extent to which various paramagnetic ions (Cu2+, Mn2+ and Gd3+) free and bound to human serum albumin alter the water proton relaxation times at two frequencies has been investigated. NMR relaxation parameters, T1 and T2, were measured at 5 and 10 MHz using a saturation recovery (90 degrees-tau-90 degrees) and a spin-echo (90 degrees-tau-180 degrees) sequence respectively. We found that all three ions enhance their effectiveness in inducing water proton magnetic relaxation when they are bound to human serum albumin and that Gd3+ is the most effective in pure water and Mn2+ in the presence of the protein. Cu2+ has a smaller effect, but it presents an interesting behaviour correlated with the existence of two different binding sites, which is also confirmed by electronic paramagnetic resonance spectra. The results indicate the potential usefulness of large molecular paramagnetic complexes as contrast agents in NMR Imaging.

Gd3+-TPPS: a potential paramagnetic contrast agent in NMR imaging.

A water soluble paramagnetic metalloporphyrin containing Gd3+ as metal ion has been investigated by low resolution 1H NMR to evaluate the extent to which it alters the water proton relaxation times. The obtained results seem to indicate a potential usefulness of this complex as a paramagnetic contrast agent for NMR Imaging.

3D printing of rat salivary glands: The submandibular-sublingual complex.

The morphology and the functionality of the murid glandular complex, composed of the submandibular and sublingual salivary glands (SSC), were the object of several studies conducted mainly using magnetic resonance imaging (MRI). Using a 4.7 T scanner and a manganese-based contrast agent, we improved the signal-to-noise ratio of the SSC relating to the surrounding anatomical structures allowing to obtain high-contrast 3D images of the SSC. In the last few years, the large development in resin melting techniques opened the way for printing 3D objects starting from a 3D stack of images. Here, we demonstrate the feasibility of the 3D printing technique of soft tissues such as the SSC in the rat with the aim to improve the visualization of the organs. This approach is useful to preserve the real in vivo morphology of the SCC in living animals avoiding the anatomical shape changes due to the lack of relationships with the surrounding organs in case of extraction. It is also harmless, repeatable and can be applied to explore volumetric changes occurring during body growth, excretory duct obstruction, tumorigenesis and regeneration processes. 3D printing allows to obtain a solid object with the same shape of the organ of interest, which can be observed, freely rotated and manipulated. To increase the visibility of the details, it is possible to print the organs with a selected zoom factor, useful as in case of tiny organs in small mammalia. An immediate application of this technique is represented by educational classes.

Morphogenetic events in the perinodal connective tissue in a metastatic cancer model.

BACKGROUND: The modifications of connective tissue surrounding metastatic lymph nodes in a murine model of rectal cancer are described. METHODS: Athymic nude mice (n=36) were inoculated with 10×10(5) ht-29 cancer cells into the submucosal layer of the rectum. Control mice (n=5) were treated with a sterile buffer. Tumor and the involved lymph nodes were visualized in vivo by magnetic resonance imaging at 1 to 4 weeks after cell injection. After the sacrifice, the excised samples were processed for histology. RESULTS: After one week from cell injection all treated animals developed rectal cancer. Since the first week, neoplastic cells were visible in the nodes. In the surrounding connective tissue, the diameter of the adipocytes was reduced and a mesenchymal-like pattern with stellate cells embedded in an oedematous environment was visible. Since the second week, in the perinodal connective an enlargement of the stroma was present. The tissue was organized in cords and areas with extracellular accumulation of lipids were found. At the fourth week, we observed an enlargement of multilocular areas and lobules of elongated elements almost devoid of lipid droplets. In control animals, in absence of neoplastic masses, pelvic nodes were surrounded by a typical connective tissue characterized by unilocular adipocytes with groups of multilocular adipocytes. CONCLUSIONS: We have developed a model of rectal cancer with nodal metastases. Using this model, the work demonstrates that around secondary lesions, the morphogenetic events follow a standard evolution characterized by an early phase with lipolysis and mesenchymalization and later phases with a brown-like phenotype acquisition.