Search

[Background]: Second-generation antipsychotics (SGAs) are a mainstay therapy for schizophrenia. SGA-treated patients present higher risk for weight gain, dyslipidemia and hyperglycemia. Herein, we evaluated the effects of olanzapine (OLA), widely prescribed SGA, in mice focusing on changes in body weight and energy balance. We further explored OLA effects in protein tyrosine phosphatase-1B deficient (PTP1B-KO) mice, a preclinical model of leptin hypersensitivity protected against obesity., [Methods]: Wild-type (WT) and PTP1B-KO mice were fed an OLA-supplemented diet (5 mg/kg/day, 7 months) or treated with OLA via intraperitoneal (i.p.) injection or by oral gavage (10 mg/kg/day, 8 weeks). Readouts of the crosstalk between hypothalamus and brown or subcutaneous white adipose tissue (BAT and iWAT, respectively) were assessed. The effects of intrahypothalamic administration of OLA with adenoviruses expressing constitutive active AMPKα1 in mice were also analyzed., [Results]: Both WT and PTP1B-KO mice receiving OLA-supplemented diet presented hyperphagia, but weight gain was enhanced only in WT mice. Unexpectedly, all mice receiving OLA via i.p. lost weight without changes in food intake, but with increased energy expenditure (EE). In these mice, reduced hypothalamic AMPK phosphorylation concurred with elevations in UCP-1 and temperature in BAT. These effects were also found by intrahypothalamic OLA injection and were abolished by constitutive activation of AMPK in the hypothalamus. Additionally, OLA i.p. treatment was associated with enhanced Tyrosine Hydroxylase (TH)-positive innervation and less sympathetic neuron-associated macrophages in iWAT. Both central and i.p. OLA injections increased UCP-1 and TH in iWAT, an effect also prevented by hypothalamic AMPK activation. By contrast, in mice fed an OLA-supplemented diet, BAT thermogenesis was only enhanced in those lacking PTP1B. Our results shed light for the first time that a threshold of OLA levels reaching the hypothalamus is required to activate the hypothalamus BAT/iWAT axis and, therefore, avoid weight gain., [Conclusion]: Our results have unraveled an unexpected metabolic rewiring controlled by hypothalamic AMPK that avoids weight gain in male mice treated i.p. with OLA by activating BAT thermogenesis and iWAT browning and a potential benefit of PTP1B inhibition against OLA-induced weight gain upon oral treatment., This work was funded by grants PID-2021-122766OB-100 (to AMV) and PID2019-104399RB-I00 (to GS) funded by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación /10.13039/501100011033 and “ERDF A way of making Europe” by the European Union. We also acknowledge grants H2020 Marie Sklodowska-Curie ITN-TREATMENT (Grant Agreement 721236, European Commission), S2017/BMD-3684 (Comunidad de Madrid, Spain), Fundación Ramón Areces (Spain) and CIBERdem (ISCIII, Spain) to AMV. JWE was funded by the Swedish Diabetes Foundation and the Novo Nordisk Foundation (NNF20OC0063864). VF was a recipient of a contract from ITN-TREATMENT and is currently a PhD fellow from the Portuguese Foundation for Science and Technology (FCT, Portugal)/ERDF (2020.08388.BD). CF was awarded with Sara Borrell contract (CD19/00078, ISCIII, Spain).

Chemotherapy-induced neuropathy (CIN) is one of the most common complications of cancer treatment with sensory dysfunctions which frequently include pain. The mechanisms underlying pain during CIN are starting to be uncovered. Neuroimaging allows the identification of brain circuitry involved in pain processing and modulation and has recently been used to unravel the disruptions of that circuitry by neuropathic pain. The present study evaluates the effects of paclitaxel, a cytostatic drug frequently used in cancer treatment, at the neuronal function in the anterior cingulate cortex (ACC), hypothalamus and periaqueductal gray (PAG) using manganese-enhanced magnetic resonance imaging (MEMRI). We also studied the metabolic profile at the prefrontal cortex (PFC) and hypothalamus using ex vivo spectroscopy. Wistar male rats were intraperitoneal injected with paclitaxel or vehicle solution (DMSO). The evaluation of mechanical sensitivity using von Frey test at baseline (BL), 21 (T21), 28 (T28), 49 (T49) and 56 days (T56) after CIN induction showed that paclitaxel-injected rats presented mechanical hypersensitivity from T21 until T56 after CIN induction. The evaluation of the locomotor activity and exploratory behaviors using open-field test at T28 and T56 after the first injection of paclitaxel revealed that paclitaxel-injected rats walked higher distance with higher velocity at late point of CIN accompanied with a sustained exhibition of anxiety-like behaviors. Imaging studies performed using MEMRI at T28 and T56 showed that paclitaxel treatment increased the neuronal activation in the hypothalamus and PAG at T56 in comparison with the control group. The analysis of data from ex vivo spectroscopy demonstrated that at T28 paclitaxel-injected rats presented an increase of N-acetyl aspartate (NAA) levels in the PFC and an increase of NAA and decrease of lactate (Lac) concentration in the hypothalamus compared to the control group. Furthermore, at T56 the paclitaxel-injected rats presented lower NAA and higher taurine (Tau) levels in the PFC. Together, MEMRI and metabolomic data indicate that CIN is associated with neuroplastic changes in brain areas involved in pain modulation and suggests that other events involving glial cells may be happening.

[Background]: Obesity proceeds with important physiological and microstructural alterations in the brain, but the precise relationships between the diet and feeding status, its physiological responses, and the observed neuroimaging repercussions, remain elusive. Here, we implemented a mouse model of high fat diet (HFD) feeding to explore specific associations between diet, feeding status, phenotypic and endocrine repercussions, and the resulting microstructural and metabolic alterations in the brain, as detected by diffusion tensor imaging (DTI) and neurochemical metabolic profiling. [Methods]: Brain DTI images were acquired from adult male C57BL6/J mice after 6 weeks of HFD, or standard diet (SD) administrations, both under the fed, and overnight fasted conditions. Metabolomic profiles of the cortex (Ctx), hippocampus (Hipc), and hypothalamus (Hyp) were determined by 1H high-resolution magic angle spinning (HRMAS) spectroscopy, in cerebral biopsies dissected after microwave fixation. Mean diffusivity (MD), fractional anisotropy (FA) maps, and HRMAS profiles were complemented with determinations of phenotypic alterations and plasma levels of appetite-related hormones, measured by indirect calorimetry and multiplex assays, respectively. We used Z-score and alternating least squares scaling (ALSCAL) analysis to investigate specific associations between diet and feeding status, physiological, and imaging parameters. [Results]: HFD induced significant increases in body weight and the plasma levels of glucose and fatty acids in the fed and fasted conditions, as well as higher cerebral MD (Ctx, Hipc, Hyp), FA (Hipc), and mobile saturated fatty acids resonances (Ctx, Hipc, Hyp). Z-score and ASLCAL analysis identified the precise associations between physiological and imaging variables. [Conclusions]: The present study reveals that diet and feeding conditions elicit prominent effects on specific imaging and spectroscopic parameters of the mouse brain that can be associated to the alterations in phenotypic and endocrine variables. Together, present results disclose a neuro-inflammatory response to HFD, characterized primarily by vasogenic edema and compensatory responses in osmolyte concentrations., The authors disclosed receipt of the following financial support for research, authorship, and/or publication of this article; Ministry of Science and Innovation (SAF2017-83043-R), and Community of Madrid (S2017/BMD-3688), both to SC and PLL. IG is a predoctoral fellow of the Ministry of Innovation and Science (BES-2015-075202); BL holds a senior investigator contract from CSIC; PLL, SC, and LB are CSIC staff members.

© 2021 by the authors., Conjugated polymer nanoparticles (CPNs) have emerged as advanced polymeric nanoplatforms in biomedical applications by virtue of extraordinary properties including high fluorescence brightness, large absorption coefficients of one and two-photons, and excellent photostability and colloidal stability in water and physiological medium. In addition, low cytotoxicity, easy functionalization, and the ability to modify CPN photochemical properties by the incorporation of dopants, convert them into excellent theranostic agents with multifunctionality for imaging and treatment. In this work, CPNs were designed and synthesized by incorporating a metal oxide magnetic core (Fe3O4 and NiFe2O4 nanoparticles, 5 nm) into their matrix during the nanoprecipitation method. This modification allowed the in vivo monitoring of nanoparticles in animal models using magnetic resonance imaging (MRI) and intravital fluorescence, techniques widely used for intracranial tumors evaluation. The modified CPNs were assessed in vivo in glioblastoma (GBM) bearing mice, both heterotopic and orthotopic developed models. Biodistribution studies were performed with MRI acquisitions and fluorescence images up to 24 h after the i.v. nanoparticles administration. The resulting IONP-doped CPNs were biocompatible in GBM tumor cells in vitro with an excellent cell incorporation depending on nanoparticle concentration exposure. IONP-doped CPNs were detected in tumor and excretory organs of the heterotopic GBM model after i.v. and i.t. injection. However, in the orthotopic GBM model, the size of the nanoparticles is probably hindering a higher effect on intratumorally T2-weighted images (T2WI) signals and T2 values. The photodynamic therapy (PDT)—cytotoxicity of CPNs was not either affected by the IONPs incorporation into the nanoparticles., This research was funded by grants from Ministerio de Economía y Competitividad (SAF2017-83043-R, MINECO/AEI/FEDER, UE), Comunidad de Madrid (S2017/BMD-3688, CM/FEDER/FSE, UE), Agencia Nacional de Promoción Científica y Tecnológica (PICT) (2676/18, 3577/18), and Secretaría de Ciencia y Técnica Universidad Nacional de Río Cuarto (PPI-SECyT UNRC).

Obesity is a current threat to health care systems, affecting approximately 13% of the world’s adult population, and over 18% children and adolescents. The rise of obesity is fuelled by inadequate life style habits, as consumption of diets rich in fats and sugars which promote, additionally, the development of associated comorbidities. Obesity results from a neuroendocrine imbalance in the cerebral mechanisms controlling food intake and energy expenditure, including the hypothalamus and the reward and motivational centres. Specifically, high-fat diets are known to trigger an early inflammatory response in the hypothalamus that precedes weight gain, is time-dependent, and eventually extends to the remaining appetite regulating regions in the brain. Multiple magnetic resonance imaging (MRI) and spectroscopy (MRS) methods are currently available to characterize different features of cerebral obesity, including diffusion weighted, T2 and volumetric imaging and 1H and 13C spectroscopic evaluations. In particular, consistent evidences have revealed increased water diffusivity and T2 values, decreased grey matter volumes, and altered metabolic profiles and fluxes, in the brain of animal models and in obese humans. This review provides an integrative interpretation of the physio-pathological processes associated with obesity development in the brain, and the MRI and MRS methods implemented to characterize them.

[Aim]: To assess monocyte-based delivery of conjugated polymer nanoparticles (CPNs) for improved photodynamic therapy (PDT) in glioblastoma (GBM)., [Materials and methods]: Human monocyte cells (THP-1) and murine monocytes isolated from bone marrow (mBMDMs) were employed as stealth CPN carriers to penetrate into GBM spheroids and an orthotopic model of the tumor. The success of PDT, using this cell-mediated targeting strategy, was determined by its effect on the spheroids., [Results]: CPNs did not affect monocyte viability in the absence of light and did not show nonspecific release after cell loading. Activated monocytes incorporated CPNs in a higher proportion than monocytes in their naive state, without a loss of cellular functionality. In vitro PDT efficacy using cell-mediated delivery was superior to that using non vehiculized CPNs., [Conclusion]: CPN-loaded monocytes could efficiently deliver CPNs into GBM spheroids and the orthotopic model. Improved PDT in spheroids was confirmed using this delivery strategy.

Transcription factor NRF2 orchestrates a cellular defense against oxidative stress and, so far, has been involved in tumor progression by providing a metabolic adaptation to tumorigenic demands and resistance to chemotherapeutics. In this study, we discover that NRF2 also propels tumorigenesis in gliomas and glioblastomas by inducing the expression of the transcriptional co-activator TAZ, a protein of the Hippo signaling pathway that promotes tumor growth. The expression of the genes encoding NRF2 (NFE2L2) and TAZ (WWTR1) showed a positive correlation in 721 gliomas from The Cancer Genome Atlas database. Moreover, NRF2 and TAZ protein levels also correlated in immunohistochemical tissue arrays of glioblastomas. Genetic knock-down of NRF2 decreased, while NRF2 overexpression or chemical activation with sulforaphane, increased TAZ transcript and protein levels. Mechanistically, we identified several NRF2-regulated functional enhancers in the regulatory region of WWTR1. The relevance of the new NRF2/TAZ axis in tumorigenesis was demonstrated in subcutaneous and intracranial grafts. Thus, intracranial inoculation of NRF2-depleted glioma stem cells did not develop tumors as determined by magnetic resonance imaging. Forced TAZ overexpression partly rescued both stem cell growth in neurospheres and tumorigenicity. Hence, NRF2 not only enables tumor cells to be competent to proliferate but it also propels tumorigenesis by activating the TAZ-mediated Hippo transcriptional program., Graphical abstract Scheme showing the main findings of this study: NRF2 induces target genes such as GCLC, NQO1, HMOX1, etc. that in cancer cells may provide a cytoprotective response to stressful signals including radiotherapy and chemotherapy. In addition, we show here that NRF2 also induces the expression of WWTR1, encoding the transcription factor TAZ, that provides direct input into cancer progression by activating a battery of genes that participate in proliferative responses and cancer stem cell renewal. The combined expression of NRF2 and its target TAZ are characteristic of poor prognosis in glioblastomas.Image 1, Highlights • Expression of NRF2 and TAZ positively correlate in gliomas and glioblastomas. • NRF2 regulates the expression of WWTR1 encoding the transcription co-activator TAZ in glioma stem cells. • TAZ provides a redox-independent mechanism of NRF2 induction of glioblastomas. • Downregulation of the new NRF2/TAZ axis may provide a novel therapy for glioblastomas.

We used 1H, 13C HRMAS and genomic analysis to investigate regionally the transition from oxidative to glycolytic phenotype and its relationship with altered gene expression in adjacent biopsies through the brain of rats bearing C6 gliomas. Tumor-bearing animals were anesthetized and infused with a solution of [1-13C]-glucose, and small adjacent biopsies were obtained spanning transversally from the contralateral hemisphere (regions I and II), the right and left peritumoral areas (regions III and V, respectively), and the tumor core (region IV). These biopsies were analyzed by 1H, 13C HRMAS and by quantitative gene expression techniques. Glycolytic metabolism, as reflected by the [3-13C]-lactate content, increased clearly from regions I to IV, recovering partially to physiological levels in region V. In contrast, oxidative metabolism, as reflected by the [4-13C]-glutamate labeling, decreased in regions I-IV, recovering partially in region V. This metabolic shift from normal to malignant metabolic phenotype paralleled changes in the expression of HIF1α, HIF2α, HIF3α genes, downstream transporters, and regulatory glycolytic, oxidative, and anaplerotic genes in the same regions. Together, our results indicate that genetic and metabolic alterations occurring in the brain of rats bearing C6 gliomas colocalize in situ and the profile of genetic alterations in every region can be inferred from the metabolomic profiles observed in situ by multinuclear HRMAS., The present work was supported by grants SAF2014-53739-R, SAF2017-83043-R, and B2017/BMD-3688 from the Ministry of Economy and Competitiveness and from the Community of Madrid to PL-L and SC, Acciones Integradas IH-HI2006-0101 from the Ministry of Science and Technology to L.S. and S.C. The authors are indebted to Mr. Javier Pérez CSIC for careful drafting of the illustrations and to Mrs. Teresa Navarro CSIC for granting access to the Biomedical NMR Services of the Institute of Biomedical Research “Alberto Sols” CSIC.

© 2019 Lizarbe, Fernández-Pérez, Caz, Largo, Vallejo, López-Larrubia and Cerdán., The hypothalamus is the principal regulator of global energy balance, enclosing additionally essential neuronal centers for glucose-sensing and osmoregulation. Disturbances in these tightly regulated neuronal networks are thought to underlie the development of severe pandemic syndromes, including obesity and diabetes. In this work, we investigate in vivo the response of individual hypothalamic nuclei to the i.p. administration of glucose or vehicle solutions, using two groups of adult male C57BL6/J fasted mice and a combination of non-invasive T2∗-weighted and diffusion-weighted functional magnetic resonance imaging (fMRI) approaches. MRI parameters were assessed in both groups of animals before, during and in a post-stimulus phase, following the administration of glucose or vehicle solutions. Hypothalamic nuclei depicted different patterns of activation characterized by: (i) generalized glucose-induced increases of neuronal activation and perfusion-markers in the lateral hypothalamus, arcuate and dorsomedial nuclei, (ii) cellular shrinking events and decreases in microvascular blood flow in the dorsomedial, ventromedial and lateral hypothalamus, following the administration of vehicle solutions and (iii) increased neuronal activity markers and decreased microperfusion parameters in the ARC nuclei of vehicle-administered animals. Immunohistochemical studies performed after the post-stimulus phase confirmed the presence of c-Fos immunoreactive neurons in the arcuate nucleus (ARC) from both animal groups, with significantly higher numbers in the glucose-treated animals. Together, our results reveal that fMRI methods are able to detect in vivo diverse patterns of glucose or vehicle-induced effects in the different hypothalamic nuclei., This work was supported in part by grants SAF-2014-53739-R, SAF2017-83043-R, and S2017/BMD-3688 to SC and PL-L, and grant BFU2014-52149-R and BFU2017-89336-R to MV.

© 2019 Gandía-González, Cerdán, Barrios, López-Larrubia, Feijoó, Palpan, Roda and Solivera., [Objective]: We assess the efficacy of the metabolomic profile from glioma biopsies in providing estimates of postsurgical Overall Survival in glioma patients., [Methods]: Tumor biopsies from 46 patients bearing gliomas, obtained neurosurgically in the period 1992–1998, were analyzed by high resolution 1H magnetic resonance spectroscopy (HR- 1H MRS), following retrospectively individual postsurgical Overall Survival up to 720 weeks., [Results]: The Overall Survival profile could be resolved in three groups; Short (shorter than 52 weeks, n = 19), Intermediate (between 53 and 364 weeks, n = 19) or Long (longer than 365 weeks, n = 8), respectively. Classical histopathological analysis assigned WHO grades II–IV to every biopsy but notably, some patients with low grade glioma depicted unexpectedly Short Overall Survival, while some patients with high grade glioma, presented unpredictably Long Overall Survival. To explore the reasons underlying these different responses, we analyzed HR-1H MRS spectra from acid extracts of the same biopsies, to characterize the metabolite patterns associated to OS predictions. Poor prognosis was found in biopsies with higher contents of alanine, acetate, glutamate, total choline, phosphorylcholine, and glycine, while more favorable prognosis was achieved in biopsies with larger contents of total creatine, glycerol-phosphorylcholine, and myo-inositol. We then implemented a multivariate analysis to identify hierarchically the influence of metabolomic biomarkers on OS predictions, using a Classification Regression Tree (CRT) approach. The CRT based in metabolomic biomarkers grew up to three branches and split into eight nodes, predicting correctly the outcome of 94.7% of the patients in the Short Overall Survival group, 78.9% of the patients in the Intermediate Overall Survival group, and 75% of the patients in the Long Overall Survival group, respectively., [Conclusion]: Present results indicate that metabolic profiling by HR-1H MRS improves the Overall Survival predictions derived exclusively from classical histopathological gradings, thus favoring more precise therapeutic decisions., This work was supported in part by grants PI2017/00361 from Instituto de Investigación Carlos III to JR, grant B2017/BMD-3688 from the Community of Madrid to JR and SC, and grant PI-0143-2016 from the Regional Ministry of Health of the Regional Government of Andalucía to JS.

© 2019 by the authors., Despite important advances in multimodal therapeutic options, glioblastoma (GBM), the most frequent and aggressive form of all astrocytomas, remains with a median overall survival period of 15 months. A direct correlation between GBM hypoxia and higher aggressiveness, poor prognosis and greater resistance to different treatments has been established. However, because of intratumoral and interindividual heterogeneity, it has not been possible to assess accurately the hypoxia degree from physiopathological parameters or neuroimaging methods. This study aims to develop and evaluate a magnetic resonance imaging (MRI) approach to identify more precisely those tumors that could improve the outcome through an oxygen targeted therapy. Methods: To assess the efficacy of radiotherapy in animals irradiated under air and oxygen breathing, we implemented a GBM animal model obtained by intracranial injection of glioma C6 cells in rats. MRI studies, based on the oxygen-induced contrast in blood (BOLD) and tissues (TOLD), were carried out to evaluate the effect of the modulation in oxygen breathing conditions on the tumors in vivo. The efficacy of the oxygen breathing therapies was determined by the relative tumor volume at the end of the experiment, compared to its size on the day before the treatment. Results: Our results categorized the tumors in responding, non-responding and intermediate behaviors. While BOLD analysis did not show any statistical difference between animals, either breathing air or oxygen, TOLD parameters allowed for the identification of the tumors with higher responses to hyperoxygenic radiotherapy. Conclusions: The non-invasive oxygen enhanced MRI acquisitions proposed here show promising potential to identify those tumors that would generally improve their response to a hypoxia targeted treatment., This work was supported by grants from the Ministry of Economy, Industry and Competitiveness (SAF2017-83043-R), and by the Program MULTITARGET&VIEW-CM from the Community of Madrid (S-BIO-0170-2006), involving contributions from FEDER and FSE funds.

The pharmacological action of the V(IV) compound VO(dmpp)2 was previously evaluated in vivo using obese pre-diabetic Zucker rats treated with this compound for four weeks. Besides the promising results regarding biological parameters indicative of insulin-mimetism, a specific biological activity in mitigating impaired lipid metabolism was observed. This work aims at complementing and reinforcing data formerly attained and it represents an attempt to unveil the effect of this compound at the molecular level, particularly on the metabolic profile of different organs. Ex vivo experiments with tissue samples of the brain, liver and skeletal muscle from treated and non-treated obese Zucker rats, using lean Zucker rats as a control, were carried out by using 1H-HRMAS. The results obtained showed that the lipid liver content, a characteristic of obese rats, significantly decreased after 4 weeks of VO(dmpp)2 treatment, as demonstrated by the quantification of the respective signals in the 1H-HRMAS NMR spectra. In the other analyzed tissues, the differences were not statistically significant, but a trend of a decrease of the abnormal metabolite content in treated obese rats was observed. Importantly, the therapeutic dose used showed no renal toxicity. VO(dmpp)2 was demonstrated to be able to revert the impaired lipid metabolism in vivo and 1H-HRMAS NMR was revealed to be a good tool to simultaneously assess the effects of a drug on the metabolic profile of different organs and tissues., This work was supported by grants from the Ministry of Economy, Industry and Competitivity (SAF2017-83043-R), and by the Program MULTITARGET&VIEW-CM from Community of Madrid, Spain (S-BIO-0170-2006), involving contributions from FEDER and FSE. A. M. M. and M. M. C. A. C. acknowledge Fundação para a Ciência e Tecnologia (FCT), Portugal, for the financial support to the CQC research unit through the project 007630 UID/QUI/00313/2013, co-funded by COMPETE2020-EU.

This article belongs to the Special Issue Targeted Drug Delivery., Gold nanoparticles (AuNPs) are interesting for the design of new cancer theranostic tools, mainly due to their biocompatibility, easy molecular vectorization, and good biological half-life. Herein, we report a gold nanoparticle platform as a bimodal imaging probe, capable of coordinating Gd3+ for Magnetic Resonance Imaging (MRI) and 67Ga3+ for Single Photon Emission Computed Tomography (SPECT) imaging. Our AuNPs carry a bombesin analogue with affinity towards the gastrin releasing peptide receptor (GRPr), overexpressed in a variety of human cancer cells, namely PC3 prostate cancer cells. The potential of these multimodal imaging nanoconstructs was thoroughly investigated by the assessment of their magnetic properties, in vitro cellular uptake, biodistribution, and radiosensitisation assays. The relaxometric properties predict a potential T1- and T2- MRI application. The promising in vitro cellular uptake of 67Ga/Gd-based bombesin containing particles was confirmed through biodistribution studies in tumor bearing mice, indicating their integrity and ability to target the GRPr. Radiosensitization studies revealed the therapeutic potential of the nanoparticles. Moreover, the DOTA chelating unit moiety versatility gives a high theranostic potential through the coordination of other therapeutically interesting radiometals. Altogether, our nanoparticles are interesting nanomaterial for theranostic application and as bimodal T1- and T2- MRI / SPECT imaging probes., This research was funded by FCT (Portuguese Foundation for Science and Technology), grant numbers EXCL/QEQ-MED/0233/2012, UID/Multi/04349/2013 and PTDC/MED-QUI/29649/2017. CFGCG and MMCAC thank FCT and FEDER through the COMPETE Program for funding the CQC (UID/QUI/00313/2013 and PEst-OE/QUI/UI0313/2014). P.L-L. thanks Ministry of Economy, Industry and Competitiviy for SAF2017-83043-R, and Comunity of Madrid, FEDER and FSE for S2017/BMD-3688.

Magnetic resonance imaging (MRI) is a technique based on the contents and relaxation features of water in tissues. In basic MRI sequences, diffusion phenomenon of water molecules is not taken into account although it has a notable influence in the relaxation times, and therefore in the signal intensity of images. In fact, MRI techniques that take advantage of water diffusion have experienced a huge development in last years. Diffusion-weighted imaging (DWI) has spectacularly evolved reaching nowadays a great impact both in clinical and preclinical imaging-especially in the neuroimaging field-and in basic research. We present here a protocol to perform DWI studies in a high-field preclinical setup.

Functional diffusion magnetic resonance imaging (fDMRI) is a noninvasive technique that allows elucidating physiological and anatomical changes at a microscopic scale by detection of water molecular displacements in tissue structures. These displacements likely reflect microstructural changes associated with neuronal or glial cells activation. In this chapter, we will describe the physical and biological concepts of fDMRI and how images of brain activation can be acquired in a preclinical setup.

Oxygen monitoring is a topic of exhaustive research due to its central role in many biological processes, from energy metabolism to gene regulation. The ability to monitor in vivo the physiological distribution and the dynamics of oxygen from subcellular to macroscopic levels is a prerequisite to better understand the mechanisms associated with both normal and disease states (cancer, neurodegeneration, stroke, etc.). This chapter focuses on magnetic resonance imaging (MRI) based techniques to assess oxygenation in vivo. The first methodology uses injected fluorinated agents to provide quantitative pO

The use of magnetic resonance imaging (MRI) for studying the cerebral perfusion mechanisms is well proved and contrasted in the clinical and research setups. This methodology is a promising tool in assessing numerous brain diseases like intracranial tumors, neurodegeneration processes, mental disorders, injuries and so on. In the preclinical environment, perfusion MRI offers a powerful resource for characterizing pathological models and specially identifying biomarkers to monitor the illness and validate the efficacy of therapeutical approaches. This chapter presents the theoretical bases and experimental protocols of dynamic susceptibility contrast MRI acquisitions for developing perfusion MRI studies in small animals.

Magnetic resonance imaging (MRI) is a technique based on the contents and relaxation features of water in tissues. In basic MRI sequences, diffusion phenomenon of water molecules is not taken into account although it has a notable influence in the relaxation times, and therefore in the signal intensity of images. In fact, MRI techniques that take advantage of water diffusion have experienced a huge development in last years. Diffusion-weighted imaging (DWI) has spectacularly evolved reaching nowadays a great impact both in clinical and preclinical imaging—especially in the neuroimaging field—and in basic research. We present here a protocol to perform DWI studies in a high-field preclinical setup., This work was supported by grant SAF2014-53739-R. IG held a predoctoral contract from Ministerio de Economía, Indrustria y Competitividad (MINECO) of Spain. DC held a postdoctoral contract from Consejo Superior de Investigaciones Científicas (CSIC).

The use of magnetic resonance imaging (MRI) for studying the cerebral perfusion mechanisms is well proved and contrasted in the clinical and research setups. This methodology is a promising tool in assessing numerous brain diseases like intracranial tumors, neurodegeneration processes, mental disorders, injuries and so on. In the preclinical environment, perfusion MRI offers a powerful resource for characterizing pathological models and specially identifying biomarkers to monitor the illness and validate the efficacy of therapeutical approaches. This chapter presents the theoretical bases and experimental protocols of dynamic susceptibility contrast MRI acquisitions for developing perfusion MRI studies in small animals., This work was supported by grant SAF2014-53739-R.

Oxygen monitoring is a topic of exhaustive research due to its central role in many biological processes, from energy metabolism to gene regulation. The ability to monitor in vivo the physiological distribution and the dynamics of oxygen from subcellular to macroscopic levels is a prerequisite to better understand the mechanisms associated with both normal and disease states (cancer, neurodegeneration, stroke, etc.). This chapter focuses on magnetic resonance imaging (MRI) based techniques to assess oxygenation in vivo. The first methodology uses injected fluorinated agents to provide quantitative pO2 measurements with high precision and suitable spatial and temporal resolution for many applications. The second method exploits changes in endogenous contrasts, i.e., deoxyhemoglobin and oxygen molecules through measurements of T2* and T1, in response to an intervention to qualitatively evaluate hypoxia and its potential modulation., Method development and application supported in part by CPRIT RP140399, RP120670-03, P30 CA142543, and P41 EB015908.

We design, implement and validate a novel image processing strategy to obtain in vivo maps of hunger stimulation in the brain of mice, rats and humans, combining Diffusion Weighted Magnetic Resonance Imaging (DWI) datasets from fed and fasted subjects. Hunger maps were obtained from axial/coronal (rodents/humans) brain sections containing the hypothalamus and coplanar cortico-limbic structures using Fisher's Discriminant Analysis of the combined voxel ensembles from both feeding situations. These maps were validated against those provided by the classical mono-exponential diffusion model as applied over the same subjects and conditions. Mono-exponential fittings revealed significant Apparent Diffusion Coefficient (ADC) decreases through the brain regions stimulated by hunger, but rigorous parameter estimations imposed the rejection of considerable number of pixels. The proposed approach avoided pixel rejections and provided a representation of the combined DWI dataset as a pixel map of the “Hunger Index” (HI), a parameter revealing the hunger score of every pixel. The new methodology proved to be robust both, by yielding consistent results with classical ADC maps and, by reproducing very similar HI maps when applied to newly acquired rodent datasets. ADC and HI maps demonstrated similar patterns of activation by hunger in hypothalamic and cortico-limbic structures of the brain of rodents and humans, albeit with different relative intensities, rodents showing more intense activations by hunger than humans, for similar fasting periods. The proposed methodology may be easily extended to other feeding paradigms or even to alternative imaging methods., This work was supported in part by Grants, SAF-2014-53739-R and SAF-2017-83043-R to SC and PLL, S2017/BMD-3688 to SC, PLL and MSM and grant CAM/UAM (CCG10-UAM/TIC-5864) to LFLF. AB held a predoctoral fellowship from the Spanish Agency for International Cooperation and Development and predoctoral contracts from CSIC and UAM. BL held predoctoral (BES-2009-02765) and postdoctoral contracts from the Spanish Ministry of Economy and Competitiveness and CSIC. IG held a predoctoral contract from the Spanish contracts from the Ministry of Economy and Competitiveness (BES-2015-075202).

The effects of long-term lead (Pb) exposure producing a blood Pb concentration of lower than 20 μg/dL, i.e. below that associated with overt neurological deficits in occupationally exposed individuals, was studied in adult rats. In order to assess gender differences, we performed parallel behavioral experiments in male and female rats. Exposure to Pb acetate (50 ppm in drinking water) for 6 months induced motor and cognitive alterations, however these effects were gender- and task-dependent. Chronic lead exposure impaired spatial learning assessed in the Morris water maze test (MWM) in both genders, whereas it only induced hyperactivity in the open field and impaired motor coordination in the rotarod test, only in male rats. Hyperactivity in male rats was accompanied by an increase in extracellular level of acetylcholine in the prefrontal cortex. Extracellular dopamine concentration in the prefrontal cortex was unaffected by lead exposure whereas serotonin concentration in the same brain area was significantly decreased in both male and female rats exposed to lead. These results unveil new molecular mechanisms underlying neuropsychiatric alterations induced by chronic lead exposure. © 2013 Published by Elsevier Ltd., This work was supported by a grant from the Ministerio de Ciencia e Innovación CTQ2010-20960-C02-02.

Development of neovasculature is a necessary requirement for tumour growth and it provides additional opportunities for therapeutic intervention. However, current antiangiogenic therapies have limited efficacy, mostly because of the development of resistance. Hence, characterization of new antiangiogenic molecular targets is of considerable clinical interest. We report that a calmodulin-binding domain (CaM-BD) deletion mutant of the growth factor receptor bound protein 7 (Grb7) (denoted Grb7Δ) impairs tumour growth and associated angiogenesis in vivo. We implanted glioma C6 cells in rat brains transfected with an enhanced yellow fluorescent protein (EYFP) chimera of Grb7∆, its EYFP-Grb7 wild type counterpart, and EYFP alone. We systematically followed intracerebral growth of the tumours, their cellularity and the functional performance of tumour-associated microvasculature using magnetic resonance imaging, including anatomical T1W and T2W images and functional diffusion and perfusion parameters. Tumours grown from implanted C6 cells expressing EYFP-Grb7Δ developed slower, became smaller and presented lower apparent cellularity than those derived from cells expressing EYFP-Grb7 and EYFP. Vascular perfusion measurements within tumours derived from EYFP-Grb7∆-expressing cells showed significantly lower cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) values. These findings were independently validated by histological and immunohistochemical techniques. Taken together, these findings confirm that the CaM-BD of Grb7 plays an important role in tumour growth and associated angiogenesis in vivo, thus identifying this region of the protein as a novel target for antiangiogenic treatment.

Hypothalamic appetite regulation is a vital homeostatic process underlying global energy balance in animals and humans, its disturbances resulting in feeding disorders with high morbidity and mortality. The objective evaluation of appetite remains difficult, very often restricted to indirect measurements of food intake and body weight. We report here, the direct, non-invasive visualization of hypothalamic activation by fasting using diffusion weighted magnetic resonance imaging, in the mouse brain as well as in a preliminary study in the human brain. The brain of fed or fasted mice or humans were imaged at 7 or 1.5Tesla, respectively, by diffusion weighted magnetic resonance imaging using a complete range of b values (10, This work was supported in part by grants SAF-2008-01327 and SAF2011-23622 to SC, grant CTQ-2010-20960-C02-02 to PLL, grants S-BIO-2006-0170 and S2010/BMD-2349 to SC, PLL andMSM and grant CAM/UAM (CCG10-UAM/TIC-5864) to LFLF. BL and ABSDC held predoctoral fellowships from the Spanish Ministry of Science and Technology (BES 2009-027615) and the Spanish Agency for International Cooperation and Development.

Type 2 diabetes mellitus has been associated with obesity, metabolic syndrome, cardiovascular diseases and cancer. Attempts have been made for early diagnosis and finding effective drugs to prevent severe consequences and ameliorate the symptoms of this disorder. In this work, the pharmacological properties of VO(dmpp) 2, [bis(1,2-dimethyl-3-hydroxy-4-pyridinonato) oxovanadium(IV)], were in vivo evaluated. For 4 weeks fatty Zucker rats were subjected to a daily dose of VO(dmpp) 2 (44 μmol/kg) and their metabolic profile was followed by assessing different biological parameters at established time points: body weight, subcutaneous fat width and hepatic triglyceride content determined by magnetic resonance imaging and spectroscopy, respectively. A glucose tolerance test was performed at the end of the experiment. After treatment, treated obese rats presented a weight significantly lower than the non-treated obese animals (359.0 ± 11.1 vs. 433.5 ± 6.2 g, P < 0.05), a thinner subcutaneous fat width, and a statistically significant decrease in hepatic triglyceride content (5.41 ± 0.59 vs. 21.03 ± 1.40%, P < 0.0005). Additionally, the glucose intolerant profile of fatty Zucker rats was completely reversed in treated animals (102.3 ± 2.1 vs. 172.4 ± 1.3 mg/100 mL; P < 0.0005). These results reinforce the therapeutic action of VO(dmpp) 2 which shows particular effects on lipid metabolism. © 2012 Elsevier Inc. All rights reserved., This work was supported by the grant CTQ2010-20960-C02-02 from Ministerio de Ciencia e Innovación, Spain, to Pilar López-Larrubia.

et al., Proton magnetic resonance spectroscopic imaging (1H-MRSI) has been advocated as a valuable tool for prostate cancer diagnosis. However, a barrier to widespread clinical use of this technique is the lack of robust quantification methods that yield reproducible results in an institution-independent manner. The main goal of this study was to develop a standardized and fully automated approach (LCModel-based) for quantitative prostate 1H-MRSI. To this end, a dedicated basis set was constructed by the combination of simulated (citrate, Cit; choline, Cho, and creatine, CR) and experimentally acquired (spermine, Spm) spectra. The overlapping Spm, Cho, and Cr could be resolved and quantified individually, thus allowing for the independent assessment of glandular (Cit and Spm) and proliferative (Cho) components. Several metabolite ratios were calculated and compared to the histologic findings of prostatectomy specimens from 10 prostate cancer patients with Gleason scores (3 + 3) and (3 + 4). The Cho mole fraction and the Cho/(Cit + Spm) ratio were found to best discriminate between prostate cancer and healthy tissue. The comparison between the quantitative MRSI results and the histologic findings suggests that no correlation exists between the detected metabolic alterations and the Gleason score of low-grade tumors. © 2010 Wiley-Liss, Inc.

Hepatic triglyceride (HTG) accumulation from peripheral dietary sources and from endogenous de novo lipogenesis (DNL) was quantified in adult Sprague–Dawley rats by combining in vivo localized 1H MRS measurement of total hepatic lipid with a novel ex vivo2H NMR analysis of HTG 2H enrichment from 2H-enriched body water. The methodology for DNL determination needs further validation against standard methodologies. To examine the effect of a high-fat diet on HTG concentrations and sources, animals (n = 5) were given high-fat chow for 35 days. HTG accumulation, measured by in vivo1H MRS, increased significantly after 1 week (3.85 ± 0.60% vs 2.13 ± 0.34% for animals fed on a standard chow diet, P, Funded by: Portuguese Foundation for Science and Technology. Grant Numbers: SFRH/BD/17010/2005, POCTI/QUI/55603/2004 and Portuguese Foundation for Science and Technology/Luso-Espanhola Collaborative Actions. Grant Number: GRICES/CSIC 00620.

et al., The relaxivity displayed by Gd3+ chelates immobilized onto gold nanoparticles is the result of the complex interplay between the nanoparticle size, the water exchange rate and the chelate structure. In this work we study the effect of the length of ω-thioalkyl linkers, anchoring fast water exchanging Gd3+ chelates onto gold nanoparticles, on the relaxivity of the immobilized chelates. Gold nanoparticles functionalized with Gd3+ chelates of mercaptoundecanoyl and lipoyl amide conjugates of the DO3A-N-(α-amino)propionate chelator were prepared and studied as potential CA for MRI. High relaxivities per chelate, of the order of magnitude 28-38 mM-1 s-1 (30 MHz, 25 °C), were attained thanks to simultaneous optimization of the rotational correlation time and of the water exchange rate. Fast local rotational motions of the immobilized chelates around connecting linkers (internal flexibility) still limit the attainable relaxivity. The degree of internal flexibility of the immobilized chelates seems not to be correlated with the length of the connecting linkers. Biodistribution and MRI studies in mice suggest that the in vivo behavior of the gold nanoparticles was determined mainly by size. Small nanoparticles (HD = 3.9 nm) undergo fast renal clearance and avoidance of the RES organs while larger nanoparticles (HD = 4.8 nm) undergo predominantly hepatobiliary excretion. High relaxivities, allied to chelate and nanoparticle stability and fast renal clearance in vivo suggest that functionalized gold nanoparticles hold great potential for further investigation as MRI contrast agents. This study contributes to a better understanding of the effect of linker length on the relaxivity of gold nanoparticles functionalized with Gd3+ complexes. It is a relevant contribution towards >design rules> for nanostructures functionalized with Gd3+ chelates as Contrast Agents for MRI and multimodal imaging., This work was financially supported by Fundação para a Ciência e a Tecnologia, Portugal: PhD grant SFRH/BD/63994/2009 to Miguel Ferreira and Sabbatical Grant SFRH/BSAB/1328/2013 to José Martins at Bath University, UK; and Rede Nacional de NMR (REDE/1517/RMN/2005) for the acquisition of the Varian VNMRS 600 NMR spectrometer in Coimbra. T.B.R. was supported by a Marie Curie Fellowship (FP/- PEOPLE-2009-IEF 254380) and an EMBO Fellowship (ALTF1145-2009). Financial support from Ministerio de Ciencia e Innovación, Spain, projects SAF2011-23622 (S.C.) and CTQ2010-20960-C02-02 (P.L.-L.), and Comunidad de Madrid, Spain, project S2010/BMD-2349 (S.C. and P.L.-L), is also acknowledged. B. Mousavi and L. Helm acknowledge financial support by the Swiss National Science Foundation.

Liposomal drug delivery vehicles are promising nanomedicine tools for bringing cytotoxic drugs to cancerous tissues selectively. However, the triggered cargo release from liposomes in response to a target-specific stimulus has remained elusive. We report on functionalizing stealth-liposomes with an engineered ion channel and using these liposomes in vivo for releasing an imaging agent into a cerebral glioma rodent model. If the ambient pH drops below a threshold value, the channel generates temporary pores on the liposomes, thus allowing leakage of the intraluminal medicines. By using magnetic resonance spectroscopy and imaging, we show that engineered liposomes can detect the mildly acidic pH of the tumor microenvironment with 0.2 pH unit precision and they release their content into C6 glioma tumors selectively, in vivo. A drug delivery system with this level of sensitivity and selectivity to environmental stimuli may well serve as an optimal tool for environmentally-triggered and image-guided drug release., From the Clinical Editor: Cancer remains a leading cause of mortality worldwide. With advances in science, delivery systems of anti-cancer drugs have also become sophisticated. In this article, the authors designed and characterized functionalized liposomal vehicles, which would release the drug payload in a highly sensitive manner in response to a change in pH environment in an animal glioma model. The novel data would enable better future designs of drug delivery systems, This work was supported in part by grants SAF-2008-01327, SAF2011-23622 and S2010/BMD-2349 to SC, grants CTQ-2010-20960 and CTQ-2013-47669-R to P.B. and P.L.L. and, grants European Research Council-Ideas Program Starting Grant 208814, ERC-Proof of concept Grant (MOCHA), and the Netherlands Organization for Scientific Research Grant (NWO-VIDI) 700.57.427 to A.K, MEDITRANS (NMP4-CT-2006-026668) to A.K., S.C., P.B. and N.M-. J.P-T received a predoctoral contract from CSIC.

We evaluate the contribution of leptin-dependent anorexigenic pathways and neuropeptide Y (NPY)-dependent orexigenic pathways to the changes in hypothalamic water diffusion parameters observed in vivo by functional diffusion weighted MRI (fDWI). Mice genetically deficient in leptin (B6.V-Lep/J) or NPY (129S-Npy/J) and the corresponding wild-type controls, were subjected to sequential isocaloric feeding, fasting and recovery regimes. Non-invasive fDWI measurements were performed under these conditions, and complemented with parallel determinations of food and water consumption, respiratory exchange ratio (RER), locomotor activity and endocrine profiles. Control mice showed significant increases in hypothalamic water diffusion parameters upon fasting, returning to normal values in the recovery period. Leptin deficient mice depicted permanently increased water diffusion parameters under all feeding conditions as compared to wild type controls, without important changes upon fasting or recovery. These results paralleled sustained increases in food and water intake, significantly augmented body weight, and decreased RER values or locomotor activity, thus configuring an obese phenotype. NPY-deficient mice showed significantly reduced increases (or even slight decreases) in the water diffusion parameters upon fasting as compared to wild type controls, paralleled by decreased food and water intake during the recovery period. In conclusion, leptin deficiency results in sustained orexigenic stimulation, leading to increased water diffusion parameters, while NPY deficiency lead to reduced orexigenic stimulation and water diffusion parameters. Diffusion changes are proposed to reflect net astrocytic volume changes induced by the balance between the orexigenic and anorexigenic firings of AgRP/NPY and POMC/CART neurons, respectively. Together, our results suggest that fDWI provides an adequate tool to investigate hypothalamic appetite disorders., This work was supported in part by Grants SAF-2008-01327, SAF2011-23622 and IPT-2012-1331-006000 to SC, Grant CTQ-2010-20960-C02-02 to PLL, Grants S-BIO-2006-0170 and S2010/BMD-2349 to S.C. and PLL. BL held a predoctoral fellowship from the Spanish Ministry of Science and Technology (BES 2009-027615).

We report an easy and efficient synthetic approach to the first series of (nitroimidazolyl)succinic esters and diacids involving the Michael-type addition of the nitroimidazole to the α,β-unsaturated ester. Voltammetric measurements revealed similar redox potentials to those of previous nitroimidazoles used in hypoxia detection. Theoretical calculations were implemented to understand the mechanism of the addition. © Georg Thieme Verlag Stuttgart., This work was supported in part by grants from the Community of Madrid GR/SAL/0328/2004 and SAF2003-01810 to PB and SAF 2004-03197 to SC.

The complex [(tacn)6Fe8 (μ3-O) 2(μ2 -OH)12] Br8·9H2O(Fe8) was evaluated in vitro as a new kind of possible MRI contrast agent. Relaxivities were measured at 1.41 and 9.4 T for Fe8 and commercial Gd-DTPA dissolved in PBS. There was significant difference for r1 and r2 values between Fe8 and Gd-DTPA at high field (9.4 T) and for r1 at low field (1.4 T) (p < 0.05). Phantom studies with T1-weighted MRI at 9.4 T suggest T1 contrast potential for Fe8. That is, up to 5.2 times higher intensity enhancement with respect to that of equimolar Gd-DTPA was obtained with an Fe8 concentration, referred to the whole molecule, of 0.2 mM, for which no toxicity on C6 cells could be detected. No toxic effects on cultured C6 cells were observed up to a concentration of 1 mM Fe8. Copyright © 2005 John Wiley & Sons, Ltd., Partial funding from the Spanish Ministry of Science and Technology, MCYT SAF 2002–0440, MCYT MAT2003-01052, CIRIT 2002XT-48 and 2001SGR00335 is also acknowledged.