MRSI-based molecular imaging of therapy response to temozolomide in preclinical glioblastoma using source analysis.

Characterization of glioblastoma (GB) response to treatment is a key factor for improving patients' survival and prognosis. MRI and magnetic resonance spectroscopic imaging (MRSI) provide morphologic and metabolic profiles of GB but usually fail to produce unequivocal biomarkers of response. The purpose of this work is to provide proof of concept of the ability of a semi-supervised signal source extraction methodology to produce images with robust recognition of response to temozolomide (TMZ) in a preclinical GB model. A total of 38 female C57BL/6 mice were used in this study. The semi-supervised methodology extracted the required sources from a training set consisting of MRSI grids from eight GL261 GBs treated with TMZ, and six control untreated GBs. Three different sources (normal brain parenchyma, actively proliferating GB and GB responding to treatment) were extracted and used for calculating nosologic maps representing the spatial response to treatment. These results were validated with an independent test set (7 control and 17 treated cases) and correlated with histopathology. Major differences between the responder and non-responder sources were mainly related to the resonances of mobile lipids (MLs) and polyunsaturated fatty acids in MLs (0.9, 1.3 and 2.8 ppm). Responding tumors showed significantly lower mitotic (3.3 ± 2.9 versus 14.1 ± 4.2 mitoses/field) and proliferation rates (29.8 ± 10.3 versus 57.8 ± 5.4%) than control untreated cases. The methodology described in this work is able to produce nosological images of response to TMZ in GL261 preclinical GBs and suitably correlates with the histopathological analysis of tumors. A similar strategy could be devised for monitoring response to treatment in patients. Copyright © 2016 John Wiley & Sons, Ltd.

Dimethyl sulfoxide (DMSO) as a potential contrast agent for brain tumors.

Dimethyl sulfoxide (DMSO) is commonly used in preclinical studies of animal models of high-grade glioma as a solvent for chemotherapeutic agents. A strong DMSO signal was detected by single-voxel MRS in the brain of three C57BL/6 control mice during a pilot study of DMSO tolerance after intragastric administration. This led us to investigate the accumulation and wash-out kinetics of DMSO in both normal brain parenchyma (n=3 control mice) by single-voxel MRS, and in 12 GL261 glioblastomas (GBMs) by single-voxel MRS (n=3) and MRSI (n=9). DMSO accumulated differently in each tissue type, reaching its highest concentration in tumors: 6.18 ± 0.85 µmol/g water, 1.5-fold higher than in control mouse brain (p<0.05). A faster wash-out was detected in normal brain parenchyma with respect to GBM tissue: half-lives of 2.06 ± 0.58 and 4.57 ± 1.15 h, respectively. MRSI maps of time-course DMSO changes revealed clear hotspots of differential spatial accumulation in GL261 tumors. Additional MRSI studies with four mice bearing oligodendrogliomas (ODs) revealed similar results as in GBM tumors. The lack of T(1) contrast enhancement post-gadolinium (gadopentetate dimeglumine, Gd-DTPA) in control mouse brain and mice with ODs suggested that DMSO was fully able to cross the intact blood-brain barrier in both normal brain parenchyma and in low-grade tumors. Our results indicate a potential role for DMSO as a contrast agent for brain tumor detection, even in those tumors 'invisible' to standard gadolinium-enhanced MRI, and possibly for monitoring heterogeneities associated with progression or with therapeutic response.

Multicenter study. Nursing professionals' perception of training needs in pain assessment in pediatric patients with cognitive dysfunction.

INTRODUCTION: Pediatric patients with cognitive dysfunction are at greater risk of pain than typically developing children. Pain assessment in these patients is complex and could generate uncertainty in health professionals about what the key aspects are. AIM: To determine the training needs perceived by nursing professionals regarding acute pain assessment in pediatric patients with cognitive dysfunction. METHODS: A descriptive, cross-sectional, and multicenter study was performed using a survey addressed to nursing professionals who work in pediatrics during the months of August and September 2022. RESULTS: 163 responses were obtained. Most of the professionals who responded were female (92.6%, n = 151), with a mean age of 38.98 ±â€¯10.40 years. The most frequent work unit was the pediatric intensive care unit (PICU), in 36% (n = 58). Most of the participants reported not having previously received training on pain assessment in pediatric patients with cognitive disabilities (85.9%, n = 139). However, 70.4% (n = 114) considered it "very necessary" for the development of their work to receive specific training on this topic. Knowing how to assess acute pain in this population (85.3%, n = 139) and knowing the clinical and behavioral manifestations of pain in this type of patient (84.7%, n = 138) were the aspects that obtained higher scores. CONCLUSION: This research notes more than 90% of participants consider "quite necessary" and "strong necessary" to be training in pediatric cognitive dysfunction patients pain assessment. Furthermore, work experience, academic education and to be pediatric specialist obtain statistical significance data.

Development of robust discriminant equations for assessing subtypes of glioblastoma biopsies.

BACKGROUND: In the preceding decade, various studies on glioblastoma (Gb) demonstrated that signatures obtained from gene expression microarrays correlate better with survival than with histopathological classification. However, there is not a universal consensus formula to predict patient survival. METHODS: We developed a gene signature using the expression profile of 47 Gbs through an unsupervised procedure and two groups were obtained. Subsequent to a training procedure through leave-one-out cross-validation, we fitted a discriminant (linear discriminant analysis (LDA)) equation using the four most discriminant probesets. This was repeated for two other published signatures and the performance of LDA equations was evaluated on an independent test set, which contained status of IDH1 mutation, EGFR amplification, MGMT methylation and gene VEGF expression, among other clinical and molecular information. RESULTS: The unsupervised local signature was composed of 69 probesets and clearly defined two Gb groups, which would agree with primary and secondary Gbs. This hypothesis was confirmed by predicting cases from the independent data set using the equations developed by us. The high survival group predicted by equations based on our local and one of the published signatures contained a significantly higher percentage of cases displaying IDH1 mutation and non-amplification of EGFR. In contrast, only the equation based on the published signature showed in the poor survival group a significant high percentage of cases displaying a hypothesised methylation of MGMT gene promoter and overexpression of gene VEGF. CONCLUSION: We have produced a robust equation to confidently discriminate Gb subtypes based in the normalised expression level of only four genes.

Robust discrimination of glioblastomas from metastatic brain tumors on the basis of single-voxel (1)H MRS.

This article investigates methods for the accurate and robust differentiation of metastases from glioblastomas on the basis of single-voxel (1)H MRS information. Single-voxel (1)H MR spectra from a total of 109 patients (78 glioblastomas and 31 metastases) from the multicenter, international INTERPRET database, plus a test set of 40 patients (30 glioblastomas and 10 metastases) from three different centers in the Barcelona (Spain) metropolitan area, were analyzed using a robust method for feature (spectral frequency) selection coupled with a linear-in-the-parameters single-layer perceptron classifier. For the test set, a parsimonious selection of five frequencies yielded an area under the receiver operating characteristic curve of 0.86, and an area under the convex hull of the receiver operating characteristic curve of 0.91. Moreover, these accurate results for the discrimination between glioblastomas and metastases were obtained using a small number of frequencies that are amenable to metabolic interpretation, which should ease their use as diagnostic markers. Importantly, the prediction can be expressed as a simple formula based on a linear combination of these frequencies. As a result, new cases could be straightforwardly predicted by integrating this formula into a computer-based medical decision support system. This work also shows that the combination of spectra acquired at different TEs (short TE, 20-32 ms; long TE, 135-144 ms) is key to the successful discrimination between glioblastomas and metastases from single-voxel (1)H MRS.

1H-MRSI pattern perturbation in a mouse glioma: the effects of acute hyperglycemia and moderate hypothermia.

MR spectroscopic Imaging (MRSI), with PRESS localization, is used here to monitor the effects of acute hyperglycemia in the spectral pattern of 11 mice bearing GL261 gliomas at normothermia (36.5-37.5 degrees C) and at hypothermia (28.5-29.5 degrees C). These in vivo studies were complemented by ex vivo high resolution magic angle spinning (HR-MAS) analysis of GL261 tumor samples from 6 animals sacrificed by focused microwave irradiation, and blood glucose measurements in 12 control mice. Apparent glucose levels, monitored by in vivo MRSI in brain tumors during acute hyperglycemia, rose to an average of 1.6-fold during hypothermia (p < 0.05), while no significant changes were detected at normothermia, or in control experiments performed at euglycemia, or in normal/peritumoral brain regions. Ex vivo analysis of glioma-bearing mouse brains at hypothermia revealed higher glucose increases in distinct regions during the acute hyperglycemic challenge (up to 6.6-fold at the tumor center), in agreement with maximal in vivo blood glucose changes (5-fold). Phantom studies on taurine plus glucose containing solutions explained the differences between in vivo and ex vivo measurements. Our results also indicate brain tumor heterogeneity in the four animal tumors investigated in response to a defined metabolic challenge.

Dual T1/ T2 Nanoscale Coordination Polymers as Novel Contrast Agents for MRI: A Preclinical Study for Brain Tumor.

In the last years, extensive attention has been paid on designing and developing functional imaging contrast agents for providing accurate noninvasive evaluation of pathology in vivo. However, the issue of false-positives or ambiguous imaging and the lack of a robust strategy for simultaneous dual-mode imaging remain to be fully addressed. One effective strategy for improving it is to rationally design magnetic resonance imaging (MRI) contrast agents (CAs) with intrinsic T1/ T2 dual-mode imaging features. In this work, the development and characterization of one-pot synthesized nanostructured coordination polymers (NCPs) which exhibit dual mode T1/ T2 MRI contrast behavior is described. The resulting material comprises the combination of different paramagnetic ions (Fe3+, Gd3+, Mn2+) with selected organic ligands able to induce the polymerization process and nanostructure stabilization. Among them, the Fe-based NCPs showed the best features in terms of colloidal stability, low toxicity, and dual T1/ T2 MRI contrast performance overcoming the main drawbacks of reported CAs. The dual-mode CA capability was evaluated by different means: in vitro phantoms, ex vivo and in vivo MRI, using a preclinical model of murine glioblastoma. Interestingly, the in vivo MRI of Fe-NCPs show T1 and T2 high contrast potential, allowing simultaneous recording of positive and negative contrast images in a very short period of time while being safer for the mouse. Moreover, the biodistribution assays reveals the persistence of the nanoparticles in the tumor and subsequent gradual clearance denoting their biodegradability. After a comparative study with commercial CAs, the results suggest these nanoplatforms as promising candidates for the development of dual-mode MRI CAs with clear advantages.

The relationship between nuclear magnetic resonance-visible lipids, lipid droplets, and cell proliferation in cultured C6 cells.

There is an ongoing controversy about the subcellular origin of the fatty acyl chains that give rise to the NMR visible mobile lipids (MLs) resonance at approximately 1.24 ppm in the 1H spectra of cells and solid tumors. Some groups have been supporting the hypothesis that triglycerides originating MLs are isotropically tumbling in small membrane microdomains, whereas other authors back the proposal that they are inside cytosolic or extracellular (necrotic areas) lipid droplets. Furthermore, MLs are frequently present in in vivo spectra recorded from human brain tumors, but the meaning of this detection is not fully clear. We have addressed the possible contribution of intracellular droplets to the ML pattern recorded from human brain tumors in vivo by studying cultured C6 rat glioma cells as a model system for astrocytic tumors. We show here that cultured C6 cells display ML resonances in high field (9.4 T) 1H NMR spectra recorded at 136 ms echo time when grown at saturation density conditions, but no MLs are visible for log-phase cells. Fluorescence microscopy analysis of cells stained with the lipophylic dye Nile red shows intracellular spherical yellow-gold droplets containing neutral lipids; cells at saturation density present lipid droplets of diameters about 1.6 microm in most cells (85%), whereas they are almost absent in log-phase cells (only 6% of the cells contain them). Furthermore, log-phase cells can be induced to display MLs and accumulate Nile red-positive droplets by culturing them for 24 h at pH 6.2. This acid pH effect can be fully reversed by 24 h of standard media incubation. Lipid droplet volume calculated from fluorescence microscopy preparations in an average cell is different for both culture conditions (2.2 times higher volume for saturation density than for pH-stressed cells). This difference in lipid droplet volume is reflected by a different ML peak height at 1.24 ppm (about 2 times higher for saturation density than for pH-stressed cells). Flow cytometry analysis shows that both culture conditions result in a slowing down of the proliferation rate of the cells. The fact that MLs are found to originate in lipid droplets inside cells that are growth compromised but still viable suggests that changes in the proliferative state of tumor cells, in the absence of necrosis, may be detected non invasively by in vivo NMR spectroscopy.

Evidence that mobile lipids detected in rat brain glioma by 1H nuclear magnetic resonance correspond to lipid droplets.

Mobile lipids have been detected by proton nuclear magnetic resonance (NMR) in animal and human tumors (cultured cells, biopsies, and in vivo), but their origin and subcellular location are still unclear. They have been associated with malignancy, metastatic ability, drug resistance, and necrosis. We wanted to determine whether these lipids are located within plasma membrane microdomains or in lipid droplets for a C6 cell-induced rat glioma. NMR-visible mobile lipids were found in all subcellular fractions isolated from the rat tumor, except in the cytosolic supernatants. Transmission electron microscopy showed that lipid droplets were present in all subcellular fractions containing NMR-visible lipids and in the necrotic and perinecrotic areas of the tumor. The mean diameter of droplets isolated by flotation in the subcellular fractionation protocol was 0.97 microm (n = 682; droplet profile diameter range between 0.2 and 5.0 microm). The apparent diffusion coefficient for these lipids (46 +/- 17 microm2 s(-1) measured in vivo by proton spectroscopy was four orders of magnitude higher than would be expected if mobile lipids were inside plasma membrane microdomains. The combined results demonstrated that mobile lipids detected in vivo by proton NMR in the C6 rat glioma are located in large lipid droplets, associated with the necrotic process.

Application of high-field 1H-NMR spectroscopy for the study of perifused amphibian and excised mammalian muscles.

Frog sartorius and gastrocnemius muscles were perifused at 20 degrees C, the intracellular pH (pHi) and the concentration of phosphocreatine were determined in the resting muscle by 1H-NMR spectroscopy at 470 MHz; values of pHi = 7.31 +/- 0.05 (n = 7) and concentration of phosphocreatine = 20.4 +/- 1.1 mumol/g wet wt. (n = 6) were found. The hydrolysis of phosphocreatine and the simultaneous increase in lactate upon perifusion with 10 mM caffeine (in Ringer's solution) was followed with a time resolution of 1 min. Lactate increased at a rate of 1.0 mumol/g per min, but no pHi change was recorded during the time monitored. The lower limit for the buffering capacity of the muscle cytosol was estimated to be 16.7 mumol/g muscle per pH unit from the uncertainty in pHi determination (+/- 0.03 pH units) and from the amount of lactate produced and phosphocreatine hydrolyzed. Changes in pHi, lactate concentration and fatty acyl chain intensity were monitored by 1H-NMR spectroscopy at 361 MHz in ischemic rat skeletal muscle, excised and stored at 20 degrees C. The resonances in the 1H-NMR spectrum of a human skeletal muscle perchloric acid extract are reported and tentatively assigned.

The separation of phosphocreatine from creatine, and pH determination in frog muscle by natural abundance 13C-NMR.

Phosphocreatine can be separated from creatine in superfused frog muscle by natural abundance 13C-NMR, based on the difference in resonance frequency of their guanidino carbons. After taking into account the longitudinal relaxation times and nuclear Overhauser enhancement factors, the integrated peak areas of the guanidino carbons could be used for determination of the phosphocreatine-to-creatine ratio in the muscle. The pH dependence of the chemical shift of the C-2 carbon in the histidine ring of carnosine was used for estimation of the intracellular pH in the intact muscle.

1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A.

The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5'AMP, 5'GMP, 5'IMP) and halogenated purine mononucleotides (cl6RMP, br8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in the pK values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p1 site, but the p2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571--579). Lys-7 and/or Arg-10 are proposed as part of the p2 phosphate-binding subsite. The pK values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5'AMP, 5'GMP and 5'IMP takes place not only in the so-called purine-binding site B2R2p1 but also in the primary pyrimidine-binding site B1R1 and p0 of RNAase A.

1H NMR of intact muscle at 11 T.

1H NMR spectra of intact frog, and chicken skeletal muscles, were recorded at 470 MHz with the Plateau and Guéron pulse sequence for the suppression of water [(1982) J. Am. Chem. Soc. 104, 7310]. Only a few transients were required to resolve the resonances from the protons of muscle metabolites. The previously unobserved exchangeable protons of muscles were also recorded and thereby phosphocreatine and creatine could be measured simultaneously. During aging of dissected frog muscle, changes in levels of phosphocreatine, creatine and lactic acid, and the decrease in the intracellular pH were followed by 1H NMR.

Classification of brain tumours using short echo time 1H MR spectra.

The purpose was to objectively compare the application of several techniques and the use of several input features for brain tumour classification using Magnetic Resonance Spectroscopy (MRS). Short echo time 1H MRS signals from patients with glioblastomas (n = 87), meningiomas (n = 57), metastases (n = 39), and astrocytomas grade II (n = 22) were provided by six centres in the European Union funded INTERPRET project. Linear discriminant analysis, least squares support vector machines (LS-SVM) with a linear kernel and LS-SVM with radial basis function kernel were applied and evaluated over 100 stratified random splittings of the dataset into training and test sets. The area under the receiver operating characteristic curve (AUC) was used to measure the performance of binary classifiers, while the percentage of correct classifications was used to evaluate the multiclass classifiers. The influence of several factors on the classification performance has been tested: L2- vs. water normalization, magnitude vs. real spectra and baseline correction. The effect of input feature reduction was also investigated by using only the selected frequency regions containing the most discriminatory information, and peak integrated values. Using L2-normalized complete spectra the automated binary classifiers reached a mean test AUC of more than 0.95, except for glioblastomas vs. metastases. Similar results were obtained for all classification techniques and input features except for water normalized spectra, where classification performance was lower. This indicates that data acquisition and processing can be simplified for classification purposes, excluding the need for separate water signal acquisition, baseline correction or phasing.

Diagnosis of brain abscess by magnetic resonance spectroscopy. Report of two cases.

Two cases of brain abscess were diagnosed by combining magnetic resonance spectroscopy (MRS) and magnetic resonance (MR) imaging. The resonances observed in vivo were assigned by means of an in vitro MRS study of the exudates extracted during surgical aspiration of the abscesses. The technique of MRS was demonstrated to be very powerful in the differential diagnosis of brain abscesses from other brain pathologies such as neoplasms. Amino acids, probably originating from extracellular proteolysis, and other compounds, such as acetate, arising from bacterial metabolism, were visible in the MRS spectra of the abscess, whereas they are not present in spectra of neoplasms. In this sense, MRS complemented the information provided by MR imaging to achieve a correct diagnosis of brain abscesses and could be added to routine MR examinations with only a small increase in cost and time.

Magnetic resonance spectroscopy of brain hemangiopericytomas: high myoinositol concentrations and discrimination from meningiomas.

OBJECT: Hemangiopericytomas are a rare type of brain tumor that are very similar to meningiomas in appearance and symptoms but require different treatment. It is not normally possible to distinguish between them by using magnetic resonance (MR) imaging and computerized tomography studies. However, discrimination may be possible by using in vivo MR spectroscopy (MRS) because the biochemical composition of these two lesions is different. The goal of this study was to describe the use of MRS in discriminating between these similar tumor types. METHODS: In vivo MRS spectra were acquired in 27 patients (three with hemangiopericytomas and 24 with meningiomas) by using a single-voxel proton brain examination system at 1.5 teslas with short- (20-msec) and long- (135-msec) echo times. In addition, brain biopsy specimens obtained by open craniotomy were frozen within 5 minutes of resection and stored in liquid nitrogen until they were used. The specimens were powdered, extracted with perchloric acid, redissolved in 2H2O2 and high-resolution in vitro MRS was used at 9.4 teslas to record their spectra. CONCLUSIONS: In this study the authors show that hemangiopericytomas could be clearly distinguished from meningiomas because they have a larger peak at 3.56 ppm. Measurements of extracts of the tumors and comparison of spectra acquired with MRS at long- (135-msec) and short- (20-msec) echo times established that this was due to the much higher levels of myoinositol in the hemangiopericytomas.

31P-MRS of quadriceps reveals quantitative differences between sprinters and long-distance runners.

Phosphorus-31 nuclear magnetic resonance spectroscopy (31P-MRS) has been used to characterize the spectral pattern of quadriceps muscle at rest for sedentaries (N = 10), sprinters (N = 12), and long-distance runners (N = 10). Intracellular pH (pHi), phosphocreatine (PCr), inorganic phosphate (Pi), phosphodiesters (PDE), adenosine triphosphate (ATP) and total phosphate (Pt), defined as the sum of the areas of PCr, PDE, Pi, and 3 times the beta-ATP resonances, were obtained from all spectra. The pHi and ATP/Pt ratio were the same in the three groups of people studied. The PCr/Pi and PCr/Pt ratios were significantly lower and the Pi/Pt ratio was significantly higher for long-distance runners than for sprinters and sedentaries, while the PDE/Pt ratio was significantly lower for sprinters than for sedentaries and long-distance runners. Furthermore, the PCr concentration for sprinters and sedentaries was significantly higher and the Pi was significantly lower than for long-distance runners. The results obtained in this study can be explained by the accepted differences in fiber type composition between sprint and endurance athletes. We suggest that 31P-MRS at rest could be used to monitor noninvasively the individual adaptive response to training in the metabolic characteristics of the athlete muscle fiber.

Brain tumor classification based on long echo proton MRS signals.

There has been a growing research interest in brain tumor classification based on proton magnetic resonance spectroscopy (1H MRS) signals. Four research centers within the EU funded INTERPRET project have acquired a significant number of long echo 1H MRS signals for brain tumor classification. In this paper, we present an objective comparison of several classification techniques applied to the discrimination of four types of brain tumors: meningiomas, glioblastomas, astrocytomas grade II and metastases. Linear and non-linear classifiers are compared: linear discriminant analysis (LDA), support vector machines (SVM) and least squares SVM (LS-SVM) with a linear kernel as linear techniques and LS-SVM with a radial basis function (RBF) kernel as a non-linear technique. Kernel-based methods can perform well in processing high dimensional data. This motivates the inclusion of SVM and LS-SVM in this study. The analysis includes optimal input variable selection, (hyper-) parameter estimation, followed by performance evaluation. The classification performance is evaluated over 200 stratified random samplings of the dataset into training and test sets. Receiver operating characteristic (ROC) curve analysis measures the performance of binary classification, while for multiclass classification, we consider the accuracy as performance measure. Based on the complete magnitude spectra, automated binary classifiers are able to reach an area under the ROC curve (AUC) of more than 0.9 except for the hard case glioblastomas versus metastases. Although, based on the available long echo 1H MRS data, we did not find any statistically significant difference between the performances of LDA and the kernel-based methods, the latter have the strength that no dimensionality reduction is required to obtain such a high performance.

A prototype decision support system for MR spectroscopy-assisted diagnosis of brain tumours.

Our objective is to develop a decision support system that improves the accuracy of non-invasive brain tumour diagnosis and grading by enabling radiologists to use data from Magnetic Resonance Spectroscopy (MRS). The system, which uses pattern recognition techniques, is trained on a validated database of spectra and associated clinical information to provide automated classification of spectra from brain tumours. An innovative user-interface presents classification results as a two-dimensional overview plot in which points representing cases of different diseases form distinct clusters. Users can inspect any cases in these plots and compare them with the new, unknown spectrum. Hence, the overview plot can both communicate the classification of a case and help provide explanation for that classification in part by supporting human case-based reasoning. This paper describes the development of a prototype system implemented in JAVA.