Magnetic resonance imaging to assess the brain response to fasting in glioblastoma-bearing rats as a model of cancer anorexia.

BACKGROUND: Global energy balance is a vital process tightly regulated by the brain that frequently becomes dysregulated during the development of cancer. Glioblastoma (GBM) is one of the most investigated malignancies, but its appetite-related disorders, like anorexia/cachexia symptoms, remain poorly understood. METHODS: We performed manganese enhanced magnetic resonance imaging (MEMRI) and subsequent diffusion tensor imaging (DTI), in adult male GBM-bearing (n = 13) or control Wistar rats (n = 12). A generalized linear model approach was used to assess the effects of fasting in different brain regions involved in the regulation of the global energy metabolism: cortex, hippocampus, hypothalamus and thalamus. The regions were selected on the contralateral side in tumor-bearing animals, and on the left hemisphere in control rats. An additional DTI-only experiment was completed in two additional GBM (n = 5) or healthy cohorts (n = 6) to assess the effects of manganese infusion on diffusion measurements. RESULTS: MEMRI results showed lower T1 values in the cortex (p-value < 0.001) and thalamus (p-value < 0.05) of the fed ad libitum GBM animals, as compared to the control cohort, consistent with increased Mn2+ accumulation. No MEMRI-detectable differences were reported between fed or fasting rats, either in control or in the GBM group. In the MnCl2-infused cohorts, DTI studies showed no mean diffusivity (MD) variations from the fed to the fasted state in any animal cohort. However, the DTI-only set of acquisitions yielded remarkably decreased MD values after fasting only in the healthy control rats (p-value < 0.001), and in all regions, but thalamus, of GBM compared to control animals in the fed state (p-value < 0.01). Fractional anisotropy (FA) decreased in tumor-bearing rats due to the infiltrate nature of the tumor, which was detected in both diffusion sets, with (p-value < 0.01) and without Mn2+ administration (p-value < 0.001). CONCLUSIONS: Our results revealed that an altered physiological brain response to fasting occurred in hunger related regions in GBM animals, detectable with DTI, but not with MEMRI acquisitions. Furthermore, the present results showed that Mn2+ induces neurotoxic inflammation, which interferes with diffusion MRI to detect appetite-induced responses through MD changes.

Assessment of Pre-operative Measurements of Tumor Size by MRI Methods as Survival Predictors in Wild Type IDH Glioblastoma.

Objective: We evaluate the performance of three MRI methods to determine non-invasively tumor size, as overall survival (OS) and Progression Free Survival (PFS) predictors, in a cohort of wild type, IDH negative, glioblastoma patients. Investigated protocols included bidimensional (2D) diameter measurements, and three-dimensional (3D) estimations by the ellipsoid or semi-automatic segmentation methods. Methods: We investigated OS in a cohort of 44 patients diagnosed with wild type IDH glioblastoma (58.2 ± 11.4 years, 1.9/1 male/female) treated with neurosurgical resection followed by adjuvant chemo and radiotherapy. Pre-operative MRI images were evaluated to determine tumor mass area and volume, gadolinium enhancement volume, necrosis volume, and FLAIR-T2 hyper-intensity area and volume. We implemented then multivariate Cox statistical analysis to select optimal predictors for OS and PFS. Results: Median OS was 16 months (1-42 months), ranging from 9 ± 2.4 months in patients over 65 years, to 18 ± 1.6 months in younger ones. Patients with tumors carrying O6-methylguanin-DNA-methyltransferase (MGMT) methylation survived 30 ± 5.2 vs. 13 ± 2.5 months in non-methylated. Our study evidenced high and positive correlations among the results of the three methods to determine tumor size. FLAIR-T2 hyper-intensity areas (2D) and volumes (3D) were also similar as determined by the three methods. Cox proportional hazards analysis with the 2D and 3D methods indicated that OS was associated to age ≥ 65 years (HR 2.70, 2.94, and 3.16), MGMT methylation (HR 2.98, 3.07, and 2.90), and FLAIR-T2 ≥ 2,000 mm2 or ≥60 cm3 (HR 4.16, 3.93, and 3.72), respectively. Other variables including necrosis, tumor mass, necrosis/tumor ratio, and FLAIR/tumor ratio were not significantly correlated with OS. Conclusion: Our results reveal a high correlation among measurements of tumor size performed with the three methods. Pre-operative FLAIR-T2 hyperintensity area and volumes provided, independently of the measurement method, the optimal neuroimaging features predicting OS in primary glioblastoma patients, followed by age ≥ 65 years and MGMT methylation.

Effects of adult male rat feminization treatments on brain morphology and metabolomic profile.

Body feminization, as part of gender affirmation process of transgender women, decreases the volume of their cortical and subcortical brain structures. In this work, we implement a rat model of adult male feminization which reproduces the results in the human brain and allows for the longitudinal investigation of the underlying structural and metabolic determinants in the brain of adult male rats undergoing feminization treatments. Structural MRI and Diffusion Tensor Imaging (DTI) were used to non-invasively monitor in vivo cortical brain volume and white matter microstructure over 30 days in adult male rats receiving estradiol (E2), estradiol plus cyproterone acetate (CA), an androgen receptor blocker and antigonadotropic agent (E2 + CA), or vehicle (control). Ex vivo cerebral metabolic profiles were assessed by 1H High Resolution Magic Angle Spinning NMR (1H HRMAS) at the end of the treatments in samples from brain regions dissected after focused microwave fixation (5 kW). We found that; a) Groups receiving E2 and E2 + CA showed a generalized bilateral decrease in cortical volume; b) the E2 + CA and, to a lesser extent, the E2 groups maintained fractional anisotropy values over the experiment while these values decreased in the control group; c) E2 treatment produced increases in the relative concentration of brain metabolites, including glutamate and glutamine and d) the glutamine relative concentration and fractional anisotropy were negatively correlated with total cortical volume. These results reveal, for the first time to our knowledge, that the volumetric decreases observed in trans women under cross-sex hormone treatment can be reproduced in a rat model. Estrogens are more potent drivers of brain changes in male rats than anti-androgen treatment.

Metabolic adaptations in spontaneously immortalized PGC-1α knock-out mouse embryonic fibroblasts increase their oncogenic potential.

PGC-1α controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1α in cancer. To investigate how loss of PGC-1α activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1α knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1α KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear ß-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysis of amino acid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1α, restraining metabolic adaptations in cancer.

Assessment of Overall Survival in Glioma Patients as Predicted by Metabolomic Criteria.

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.

Interplay between post-translational cyclooxygenase-2 modifications and the metabolic and proteomic profile in a colorectal cancer cohort.

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of cancer death worldwide. It is broadly described that cyclooxygenase-2 (COX-2) is mainly overexpressed in CRC but less is known regarding post-translational modifications of this enzyme that may regulate its activity, intracellular localization and stability. Since metabolic and proteomic profile analysis is essential for cancer prognosis and diagnosis, our hypothesis is that the analysis of correlations between these specific parameters and COX-2 state in tumors of a high number of CRC patients could be useful for the understanding of the basis of this cancer in humans. AIM: To analyze COX-2 regulation in colorectal cancer and to perform a detailed analysis of their metabolic and proteomic profile. METHODS: Biopsies from both healthy and pathological colorectal tissues were taken under informed consent from patients during standard colonoscopy procedure in the University Hospital of Bellvitge (Barcelona, Spain) and Germans Trias i Pujol University Hospital (Campus Can Ruti) (Barcelona, Spain). Western blot analysis was used to determine COX-2 levels. Deglycosylation assays were performed in both cells and tumor samples incubating each sample with peptide N-glycosidase F (PNGase F). Prostaglandin E2 (PGE2) levels were determined using a specific ELISA. 1H high resolution magic angle spinning (HRMAS) analysis was performed using a Bruker AVIII 500 MHz spectrometer and proteomic analysis was performed in a nano-liquid chromatography-tandem mass spectrometer (nano LC-MS/MS) using a QExactive HF orbitrap MS. RESULTS: Our data show that COX-2 has a differential expression profile in tumor tissue of CRC patients vs the adjacent non-tumor area, which correspond to a glycosylated and less active state of the protein. This fact was associated to a lesser PGE2 production in tumors. These results were corroborated in vitro performing deglycosylation assays in HT29 cell line where COX-2 protein profile was modified after PNGase F incubation, showing higher PGE2 levels. Moreover, HRMAS analysis indicated that tumor tissue has altered metabolic features vs non-tumor counterparts, presenting increased levels of certain metabolites such as taurine and phosphocholine and lower levels of lactate. In proteomic experiments, we detected an enlarged number of proteins in tumors that are mainly implicated in basic biological functions like mitochondrial activity, DNA/RNA processing, vesicular trafficking, metabolism, cytoskeleton and splicing. CONCLUSION: In our colorectal cancer cohort, tumor tissue presents a differential COX-2 expression pattern with lower enzymatic activity that can be related to an altered metabolic and proteomic profile.

Carbonic anhydrase IX is a pH-stat that sets an acidic tumour extracellular pH in vivo.

BACKGROUND: Tumour carbonic anhydrase IX (CAIX), a hypoxia-inducible tumour-associated cell surface enzyme, is thought to acidify the tumour microenvironment by hydrating CO2 to form protons and bicarbonate, but there is no definitive evidence for this in solid tumours in vivo. METHODS: We used 1H magnetic resonance spectroscopic imaging (MRSI) of the extracellular pH probe imidazolyl succinic acid (ISUCA) to measure and spatially map extracellular pH in HCT116 tumours transfected to express CAIX and empty vector controls in SCID mice. We also measured intracellular pH in situ with 31P MRS and measured lactate in freeze-clamped tumours. RESULTS: CAIX-expressing tumours had 0.15 pH-unit lower median extracellular pH than control tumours (pH 6.71 tumour vs pH 6.86 control, P = 0.01). Importantly, CAIX expression imposed an upper limit for tumour extracellular pH at 6.93. Despite the increased lactate concentration in CAIX-expressing tumours, 31P MRS showed no difference in intracellular pH, suggesting that CAIX acidifies only the tumour extracellular space. CONCLUSIONS: CAIX acidifies the tumour microenvironment, and also provides an extracellular pH control mechanism. We propose that CAIX thus acts as an extracellular pH-stat, maintaining an acidic tumour extracellular pH that is tolerated by cancer cells and favours invasion and metastasis.

Spatially Resolved Bioenergetic and Genetic Reprogramming Through the Brain of Rats Bearing Implanted C6 Gliomas As Detected by Multinuclear High-Resolution Magic Angle Spinning and Genomic Analysis.

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.

Advanced Contrast Agents for Multimodal Biomedical Imaging Based on Nanotechnology.

Clinical imaging modalities have reached a prominent role in medical diagnosis and patient management in the last decades. Different image methodologies as Positron Emission Tomography, Single Photon Emission Tomography, X-Rays, or Magnetic Resonance Imaging are in continuous evolution to satisfy the increasing demands of current medical diagnosis. Progress in these methodologies has been favored by the parallel development of increasingly more powerful contrast agents. These are molecules that enhance the intrinsic contrast of the images in the tissues where they accumulate, revealing noninvasively the presence of characteristic molecular targets or differential physiopathological microenvironments. The contrast agent field is currently moving to improve the performance of these molecules by incorporating the advantages that modern nanotechnology offers. These include, mainly, the possibilities to combine imaging and therapeutic capabilities over the same theranostic platform or improve the targeting efficiency in vivo by molecular engineering of the nanostructures. In this review, we provide an introduction to multimodal imaging methods in biomedicine, the sub-nanometric imaging agents previously used and the development of advanced multimodal and theranostic imaging agents based in nanotechnology. We conclude providing some illustrative examples from our own laboratories, including recent progress in theranostic formulations of magnetoliposomes containing ω-3 poly-unsaturated fatty acids to treat inflammatory diseases, or the use of stealth liposomes engineered with a pH-sensitive nanovalve to release their cargo specifically in the acidic extracellular pH microenvironment of tumors.

Oxygenation Imaging by Nuclear Magnetic Resonance Methods.

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 T 2* and T 1, in response to an intervention to qualitatively evaluate hypoxia and its potential modulation.

White matter injury restoration after stem cell administration in subcortical ischemic stroke.

INTRODUCTION: Despite its high incidence, nerve fiber (axon and myelin) damage after cerebral infarct has not yet been extensively investigated. The aim of this study was to investigate white matter repair after adipose-derived mesenchymal stem cell (ADMSC) administration in an experimental model of subcortical stroke. Furthermore, we aimed to analyze the ADMSC secretome and whether this could be implicated in this repair function. METHODS: An animal model of subcortical ischemic stroke with white matter affectation was induced in rats by injection of endothelin-1. At 24 hours, 2 × 10(6) ADMSC were administered intravenously to the treatment group. Functional evaluation, lesion size, fiber tract integrity, cell death, proliferation, white matter repair markers (Olig-2, NF, and MBP) and NogoA were all studied after sacrifice (7 days and 28 days). ADMSC migration and implantation in the brain as well as proteomics analysis and functions of the secretome were also analyzed. RESULTS: Neither ADMSC migration nor implantation to the brain was observed after ADMSC administration. In contrast, ADMSC implantation was detected in peripheral organs. The treatment group showed a smaller functional deficit, smaller lesion area, less cell death, more oligodendrocyte proliferation, more white matter connectivity and higher amounts of myelin formation. The treated animals also showed higher levels of white matter-associated markers in the injured area than the control group. Proteomics analysis of the ADMSC secretome identified 2,416 proteins, not all of them previously described to be involved in brain plasticity. CONCLUSIONS: White matter integrity in subcortical stroke is in part restored by ADMSC treatment; this is mediated by repair molecular factors implicated in axonal sprouting, remyelination and oligodendrogenesis. These findings are associated with improved functional recovery after stroke.

Dynamic oxygen challenge evaluated by NMR T1 and T2*--insights into tumor oxygenation.

There is intense interest in developing non-invasive prognostic biomarkers of tumor response to therapy, particularly with regard to hypoxia. It has been suggested that oxygen sensitive MRI, notably blood oxygen level-dependent (BOLD) and tissue oxygen level-dependent (TOLD) contrast, may provide relevant measurements. This study examined the feasibility of interleaved T2*- and T1-weighted oxygen sensitive MRI, as well as R2* and R1 maps, of rat tumors to assess the relative sensitivity to changes in oxygenation. Investigations used cohorts of Dunning prostate R3327-AT1 and R3327-HI tumors, which are reported to exhibit distinct size-dependent levels of hypoxia and response to hyperoxic gas breathing. Proton MRI R1 and R2* maps were obtained for tumors of anesthetized rats (isoflurane/air) at 4.7 T. Then, interleaved gradient echo T2*- and T1-weighted images were acquired during air breathing and a 10 min challenge with carbogen (95% O2 -5% CO2). Signals were stable during air breathing, and each type of tumor showed a distinct signal response to carbogen. T2* (BOLD) response preceded T1 (TOLD) responses, as expected. Smaller HI tumors (reported to be well oxygenated) showed the largest BOLD and TOLD responses. Larger AT1 tumors (reported to be hypoxic and resist modulation by gas breathing) showed the smallest response. There was a strong correlation between BOLD and TOLD signal responses, but ΔR2* and ΔR1 were only correlated for the HI tumors. The magnitude of BOLD and TOLD signal responses to carbogen breathing reflected expected hypoxic fractions and oxygen dynamics, suggesting potential value of this test as a prognostic biomarker of tumor hypoxia.

Image guided drug release from pH-sensitive Ion channel-functionalized stealth liposomes into an in vivo glioblastoma model.

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.

Intralesional Patterns of MRI ADC Maps Predict Outcome in Experimental Stroke.

BACKGROUND: After acute ischemia, the tissue that is at risk of infarction can be detected by perfusion-weighted imaging/diffusion-weighted imaging (PWI/DWI) mismatch but the time that is needed to process PWI limits its use. As DWI is highly sensitive to acute ischemic tissue damage, we hypothesized that different ADC patterns represent areas with a different potential for recovery. METHODS: In a model of permanent middle cerebral artery occlusion (pMCAO), Sprague-Dawley rats were randomly distributed to sham surgery and pMCAO. We further separated the pMCAO group according to intralesional ADC pattern (homogeneous or heterogeneous). At 24 h after ischemia induction, we analyzed lesion size, functional outcome, cell death expression, and brain protection markers including ROS enzyme NOX-4. MRI included DWI (ADC maps), DTI (tractography), and PWI (CBF, CBV and MTT). RESULTS: The lesion size was similar in pMCAO rats. Animals with a heterogeneous pattern in ADC maps showed better functional outcome in Rotarod test (p = 0.032), less expression of cell death (p = 0.014) and NOX-4 (p = 0.0063), higher intralesional CBF (p = 0.0026) and larger PWI/DWI mismatch (p = 0.007). CONCLUSIONS: In a rodent model for ischemic stroke, intralesional heterogeneity in ADC maps was related to better functional outcome in lesions of similar size and interval after pMCAO. DWI ADC maps may assist in the early identification of ischemic tissue with an increased potential for recovery as higher expression of acute protection markers, lower expression of cell death, increased PWI/DWI mismatch, and higher intralesional CBF were present in animals with a heterogeneous ADC pattern.

Magnetoliposomes loaded with poly-unsaturated fatty acids as novel theranostic anti-inflammatory formulations.

We describe the preparation, physico-chemical characterization and anti-inflammatory properties of liposomes containing the superparamagnetic nanoparticle Nanotex, the fluorescent dye Rhodamine-100 and omega-3 polyunsaturated fatty acid ethyl ester (ω-3 PUFA-EE), as theranostic anti-inflammatory agents. Liposomes were prepared after drying chloroform suspensions of egg phosphatidylcholine, hydration of the lipid film with aqueous phases containing or not Nanotex, Rhodamine-100 dye or ω-3 PUFA-EE, and eleven extrusion steps through nanometric membrane filters. This resulted in uniform preparations of liposomes of approximately 200 nm diameter. Extraliposomal contents were removed from the preparation by gel filtration chromatography. High Resolution Magic Angle Spinning (1)H NMR Spectroscopy of the liposomal preparations containing ω-3 PUFA-EE revealed well resolved (1)H resonances from highly mobile ω-3 PUFA-EE, suggesting the formation of very small (ca. 10 nm) ω-3 PUFA-EE nanogoticules, tumbling fast in the NMR timescale. Chloroform extraction of the liposomal preparations revealed additionally the incorporation of ω-3 PUFA-EE within the membrane domain. Water diffusion weighted spectra, indicated that the goticules of ω-3 PUFA-EE or its insertion in the membrane did not affect the average translational diffusion coefficient of water, suggesting an intraliposomal localization, that was confirmed by ultrafiltration. The therapeutic efficacy of these preparations was tested in two different models of inflammatory disease as inflammatory colitis or the inflammatory component associated to glioma development. Results indicate that the magnetoliposomes loaded with ω-3 PUFA-EE allowed MRI visualization in vivo and improved the outcome of inflammatory disease in both animal models, decreasing significantly colonic inflammation and delaying, or even reversing, glioma development. Together, our results indicate that magnetoliposomes loaded with ω-3 PUFA-EE may become useful anti-inflammatory agents for image guided drug delivery.

Brain-derived neurotrophic factor administration mediated oligodendrocyte differentiation and myelin formation in subcortical ischemic stroke.

BACKGROUND AND PURPOSE: Translational research is beginning to reveal the importance of trophic factors as a therapy for cellular brain repair. The purpose of this study was to analyze whether brain-derived neurotrophic factor (BDNF) administration could mediate oligodendrogenesis and remyelination after white matter injury in subcortical stroke. METHODS: Ischemia was induced in rats by injection of endothelin-1. At 24 hours, 0.4 µg/kg of BDNF or saline was intravenously administered to the treatment and control groups, respectively. Functional evaluation, MRI, and fiber tract integrity on tractography images were analyzed. Proliferation (KI-67) and white matter repair markers (A2B5, 2',3'-cyclic-nucleotide 3'-phosphodiesterase [CNPase], adenomatous polyposis coli [APC], platelet-derived growth factor receptor alpha [PDGFR-α], oligodendrocyte marker O4 [O4], oligodendrocyte transcription factor [Olig-2], and myelin basic protein [MBP]) were analyzed at 7 and 28 days. RESULTS: The BDNF-treated animals showed less functional deficit at 28 days after treatment than the controls (P<0.05). Although T2-MRI did not show differences in lesion size at 7 and 28 days between groups, diffusion tensor imaging tractography analysis revealed significantly better tract connectivity at 28 days in the BDNF group than in the controls (P<0.05). Increased proliferation of oligodendrocyte progenitors was observed in treated animals at 7 days (P<0.05). Finally, the levels of white matter repair markers (A2B5, CNPase, and O4 at 7 days; Olig-2 and MBP at 28 days) were higher in the BDNF group than in the controls (P<0.05). CONCLUSIONS: BDNF administration exerted better functional outcome, oligodendrogenesis, remyelination, and fiber connectivity than controls in rats subjected to subcortical damage in ischemic stroke.

Cerebral oedema is not responsible for motor or cognitive deficits in rats with hepatic encephalopathy.

BACKGROUND & AIMS: Low-grade cytotoxic oedema is considered a main contributor to the neurological (motor and cognitive) alterations in patients with hepatic encephalopathy (HE). This assumption is mainly based on studies with cultured astrocytes treated with very large ammonia concentrations or with animal models of acute liver failure with strong HE. However, the possible contribution of cerebral oedema (vasogenic or cytotoxic) to cognitive or motor alterations in chronic mild HE has not been demonstrated. The aim of this work was to assess whether cerebral oedema contributes to cognitive and/or motor alterations in rats with chronic mild HE. METHODS: Motor activity and coordination and different types of learning and memory were assessed in rats with porta-caval shunts (PCS). Brain oedema was assessed by gravimetry in cerebellum and cortex and apparent diffusion coefficient (ADC) by magnetic resonance in 16 areas. RESULTS: Four weeks after surgery, PCS rats show reduced motor activity and coordination, impaired ability to learn a conditional discrimination task in the Y maze and reduced spatial memory in the Morris water maze. PCS rats did not show increased brain water content at 4 or 10 weeks or changes in ADC at 4 weeks. At 10 weeks, increased ADC in some areas is compatible with vasogenic but not cytotoxic oedema. CONCLUSION: Cerebral oedema is not involved in motor and cognitive alterations in rats (and likely in humans) with mild HE. Proper understanding of the mechanisms responsible for the neurological alterations in HE is necessary to design efficient treatments.

Effects of intravenous administration of allogenic bone marrow- and adipose tissue-derived mesenchymal stem cells on functional recovery and brain repair markers in experimental ischemic stroke.

INTRODUCTION: Stem cell therapy can promote good recovery from stroke. Several studies have demonstrated that mesenchymal stem cells (MSC) are safe and effective. However, more information regarding appropriate cell type is needed from animal model. This study was targeted at analyzing the effects in ischemic stroke of acute intravenous (i.v.) administration of allogenic bone marrow- (BM-MSC) and adipose-derived-stem cells (AD-MSC) on functional evaluation results and brain repair markers. METHODS: Allogenic MSC (2 × 106 cells) were administered intravenously 30 minutes after permanent middle cerebral artery occlusion (pMCAO) to rats. Infarct volume and cell migration and implantation were analyzed by magnetic resonance imaging (MRI) and immunohistochemistry. Function was evaluated by the Rogers and rotarod tests, and cell proliferation and cell-death were also determined. Brain repair markers were analyzed by confocal microscopy and confirmed by western blot. RESULTS: Compared to infarct group, function had significantly improved at 24 h and continued at 14 d after i.v. administration of either BM-MSC or AD-MSC. No reduction in infarct volume or any migration/implantation of cells into the damaged brain were observed. Nevertheless, cell death was reduced and cellular proliferation significantly increased in both treatment groups with respect to the infarct group. At 14 d after MSC administration vascular endothelial growth factor (VEGF), synaptophysin (SYP), oligodendrocyte (Olig-2) and neurofilament (NF) levels were significantly increased while those of glial fiibrillary acid protein (GFAP) were decreased. CONCLUSIONS: i.v. administration of allogenic MSC - whether BM-MSC or AD-MSC, in pMCAO infarct was associated with good functional recovery, and reductions in cell death as well as increases in cellular proliferation, neurogenesis, oligodendrogenesis, synaptogenesis and angiogenesis markers at 14 days post-infarct.

Nuclear magnetic resonance imaging of tumour growth and neovasculature performance in vivo reveals Grb7 as a novel antiangiogenic target.

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.

Imaging tumor hypoxia by magnetic resonance methods.

Tumor hypoxia results from the negative balance between the oxygen demands of the tissue and the capacity of the neovasculature to deliver sufficient oxygen. The resulting oxygen deficit has important consequences with regard to the aggressiveness and malignancy of tumors, as well as their resistance to therapy, endowing the imaging of hypoxia with vital repercussions in tumor prognosis and therapy design. The molecular and cellular events underlying hypoxia are mediated mainly through hypoxia-inducible factor, a transcription factor with pleiotropic effects over a variety of cellular processes, including oncologic transformation, invasion and metastasis. However, few methodologies have been able to monitor noninvasively the oxygen tensions in vivo. MRI and MRS are often used for this purpose. Most MRI approaches are based on the effects of the local oxygen tension on: (i) the relaxation times of (19)F or (1)H indicators, such as perfluorocarbons or their (1)H analogs; (ii) the hemodynamics and magnetic susceptibility effects of oxy- and deoxyhemoglobin; and (iii) the effects of paramagnetic oxygen on the relaxation times of tissue water. (19)F MRS approaches monitor tumor hypoxia through the selective accumulation of reduced nitroimidazole derivatives in hypoxic zones, whereas electron spin resonance methods determine the oxygen level through its influence on the linewidths of appropriate paramagnetic probes in vivo. Finally, Overhauser-enhanced MRI combines the sensitivity of EPR methodology with the resolution of MRI, providing a window into the future use of hyperpolarized oxygen probes.